DOI: 10.31038/CST.2017224

Abstract

Aim: The aim of this study was to examine the factors influencing count density in low-dose molecular breast imaging and their impact on image quality.

Methods: One hundred patients scheduled for a diagnostic MBI procedure were imaged using a commercially available MBI system following the SNM Practice Guideline for Breast Scintigraphy, with one modification. Each 20 mCi (740 MBq) diagnostic dose of Tc-99m sestamibi was separated into two syringes, with each containing 10 mCi (370 MBq) or with one containing 5 mCi (185 MBq) and the other containing 15 mCi (555 MBq). Patients were randomly injected with 5 mCi, 10 mCi, or 15 mCi and imaged bilaterally in the craniocaudal (CC) view. The remaining fraction of the 20 mCi was then injected, followed by a standard four-view study.

Results: The two sets of CC view images were analyzed to determine the average count density for a given acquisition time and injected activity, after application of an effective half-life correction factor to account for the time-related combined effect of radioactive decay and cellular washout. The average count density of the MBI images was reduced by radiopharmaceutical decay and washout and imaging time should be adjusted accordingly in low dose imaging.

Conclusions : It was observed that the patient weight was one physical characteristic that should be considered for optimal image quality, with the dose increasing in proportion to the patient weight.

Keywords:

Breast Cancer, Breast Specific Gamma Imaging, Molecular Breast Imaging, Low Dose Imaging, Dense Breasts

Introduction

Imaging procedures can be classified into two groups: screening exams and diagnostic exams. The term “screening” is applied to imaging examinations performed on patients without signs or symptoms of disease while “diagnostic” examinations are conducted in response to clinical signs and symptoms present in a particular patient. The majority of medical imaging procedures are performed as diagnostic examinations, however the use of imaging to screen for disease, as is the case in mammography, has significantly increased in recent decades [1]. Mammography does have limitations in its ability to detect breast cancer in women with dense breast tissue [3]. The use of breast MRI has been proposed as an alternative screening examination [4]. Due to the high cost per procedure, its use continues to be limited to high-risk screening populations for whom costeffectiveness has been established [5]. Studies have suggested wholebreast ultrasound as a means to improve cancer detection in patients with dense breast and as a screening examination in women with high risk. Because of its low positive-predictive value and lower sensitivity than MRI, its use has not been broadly accepted [6].

There has been significant interest in the use of nuclear medicine imaging to detect breast malignancies [7, 8] with numerous peerreviewed published articles examining various aspects of its use. Molecular breast imaging (MBI), is a relatively new method for breast cancer detection employing gamma cameras specifically designed to image the breast, primarily using the radiotracer Tc-99m sestamibi [9, 10]. These studies indicate that its sensitivity for the detection of breast malignancies, especially in dense breasts, is comparable to that of MRI and is much higher than that of mammography and ultrasound. The primary limitation to this effort has been the radiation dose to patients (6.7 mSv from the 20 mCi of Tc-99m sestamibi). This dose is acceptable in a diagnostic population where the likelihood of malignancy is significantly higher. However, dose reductions should be achieved if it is to be used for screening; an effort that is being investigated [11]. The purpose of this prospective trial was to evaluate the hypothesis that the count density of MBI breast images scales linearly with the administered dose and to examine patient characteristics that influence the count density when performing low-dose MBI studies to maintain sufficient image quality.

Materials and methods

Study Design

A total of 100 patients scheduled for a routine diagnostic MBI procedure were invited to participate in the prospective dosereduction protocol. The study has been approved by the institutional review board and all subjects signed an informed consent form. Patients enrolling were imaged using a commercially available MBI system (Dilon Technologies, Newport News, Virginia) and imaging was conducted following the SNM Practice Guideline for Breast Scintigraphy with one modification [12]. Each 20 mCi diagnostic dose was separated into 2 syringes containing either two 10 mCi doses or a 5 and a 15 mCi dose. Patients were randomized into initially receiving doses of 5, 10 or 15 mCi, followed by bilateral craniocaudal (CC) acquisitions of 10 minutes. The remaining fraction of the full 20 mCi dose was then delivered and a normal 4-view imaging procedure consisting of bilateral CC and mediolateral oblique (MLO) images was conducted with an acquisition time of 10 minutes for each view. This protocol allowed for a direct comparison between low-dose and normal-dose CC images for each breast of each patient.

Prior to each injection, the activity in each syringe was measured in a dose calibrator. The activity of each dose, the time of each injection and the time between injections were recorded. For the last 60 patients (patients 41-100) the activity remaining in the syringe after the injection was also measured. The patients were typically injected in the arm contralateral to the side of clinical concern, if any, and imaging was performed on the suspicious breast first. Imaging was typically initiated about 10 minutes after injection of the radiopharmaceutical.

Image Analysis

A region-of-interest (ROI) encompassing the breast was drawn on each low-dose and normal-dose image. The total counts and the number of pixels in the ROI were recorded. The average count/pixel was determined and that value was divided by the pixel area (0.1024 cm2/pixel) to determine the average count density (in counts/cm2). Most images were taken for a total of 10 minutes (600 seconds). For images taken for times other than the nominal 10 minutes, the average count density was normalized to a 10-minute acquisition for comparison purposes. The low-dose photon density was compared to the normal-dose photon density for each patient, with each patient serving as her own control. An example set of images showing the region-of-interest and the corresponding average count/pixel value for both the low-dose and normal-dose images is shown in Figure 1.

A dose-normalized count density (counts/cm2/mCi) was calculated for the images by dividing the count density by the injected dose. The injected dose was determined by a measurement of the activity immediately before the injection and in some cases that dose was reduced by a measurement of the dose remaining in the syringe after the injection. Only the last 60 patients had post-injection measurements taken. The injected dose for the normal-dose image was the sum of the injected dose for the low-dose image plus the injected dose from the remaining fraction of the full 20 mCi dose.

Six images were typically recorded for each patient; two at low dose (left and right CC) and four at the normal dose (bilateral CC and MLO). The relationship between the count density in the image and the injected dose, which is typically assumed in nuclear medicine and fundamental to any effort to lower the radiation dose to the patient, was explored using two comparisons. First, the ratio of the count density in the low-dose image to the count density in the normaldose image was compared with the ratio of the measured injected low dose to the measured injected normal dose. Second, the ratio of the count density to the measured injected dose (dose normalized count density) was compared for the low dose and normal dose cases. In each case the comparison was done on a patient-by-patient basis such that each normal-dose image could serve as a control for the low-dose image (Figure 2).

Dose Corrections

To account for known time-related changes to the injected dose due to both physical effects (radioactive half-life) and physiological effects (radiotracer washout), radioactive decay and tissue washout corrections must be applied to determine the actual activity of tracer remaining in the breast tissue at the time of imaging. The decay correction is based on the well-known half-life of Tc-99m (361.2 minutes):

Decay Correction = D × 0.8909^T

where T = time in hours and D = Dose

The washout correction is based on modeling of Sestamibi washout from breast tissue as measured in previously published literature [13], assuming a half-life of Sestamibi in the breast tissue of 3 hours:

Washout Correction = D × exp(-T/3)

When used together, these corrections permit the direct comparison of images take at various time points.

Results

Dose Measurements

One hundred patients were enrolled in the study. The activity in the syringe prior to injection of the patient was recorded for both portions of the divided dose. The protocol was modified after the first 40 patients, to record the dose remaining in the syringe after each injection. This remaining dose was subtracted from the dose measured in the syringe prior to the injection to determine the actual injected dose. The total dose administered to the 100 patients is shown in the graph in Figure 3, for both the first 40 patients where no postinjection measurement was made and the last 60 patients where the dose remaining in the syringes was subtracted from the pre-injection dose. For the last 60 patients, the average actual dose injected for the 5 mCi, 10 mCi, and 15 mCi low dose groups was 3.4 mCi (SD=.5 mCi), 8.2 mCi (SD=1.1 mCi), and 12.5 mCi (SD=1.6 mCi), respectively. The average actual total dose injected for all groups in the last 60 patients was 15.3 mCi (SD=2.1 mCi). The percentage of the dose administered to the various dose groups was: 66% (SD= 10%) for the 5 mCi group, 78% (SD=9%) for the 10 mCi group, 80% (SD=9%) for the 15 mCi group and 74% (SD = 9%) for the normal-dose (20 mCi) group. These results indicated that the dose remaining in the syringe after the injection was a significant fraction of the initial dose prior to injection for this group of 60 patients and are consistent with previously reported values [14] where 20% (+- 8%) was retained in syringes of various types.

Figure 3. Graph showing the dose administered to the patients. Diamonds indicate the low dose and crosses indicate the normal dose. For the first 40 patients, the dose remaining in the syringe was not recorded and therefore the initial dose measured in the syringe prior to the injection was used for these patients. For the last 60 patients, the dose remaining in the syringe was subtracted from the initial dose and the actual delivered dose was calculated.

Image Count Density

The count density for the low-dose and normal-dose images were determined for the right CC image for each patient as described earlier. The ratio of the low-dose and normal-dose count densities was then compared with the ratio of the doses. A plot of this relationship is shown in Figure 4a for the last 60 patients, for which post-injection measurements of the dose remaining in the syringe were made. The quantitative analysis was restricted to these patients with complete information. A linear fit to these results indicates a coefficient of 1.11 (R2=0.96), i.e. slightly higher than 1. This implies that as the dose is increased, there is an 11% larger increase in the uptake. This represents a result that is contrary to what is typically assumed in nuclear medicine imaging and warrants further study. As discussed previously, both physical and physiological corrections were used to adjust the injected low dose for that dose remaining at the time of the second dose. After applying the corrections discussed earlier, a linear coefficient of 0.98 (R2=0.94) was determined, significantly close to a coefficient of 1 (see Figure 4b).

Figure 4. Comparison of the dose normalized count density for the low dose and normal dose images. The results show the relationship a) before and b) after the normal dose was corrected for the decay of the isotope and washout of the radiopharmaceutical. The dashed line represents a slope of 1 and the solid line is a fit to the data (see text).

For the second comparison, the count density (as determined from the average counts per cm2 in the 10-minute breast image) was divided by the dose to determine the dose normalized count density (in counts/cm2/mCi). This value is independent of dose and should be the same for both the low-dose and normal dose-images, for any given patient. This relationship is shown in Figure 5a for the last 60 patients in the study. A linear fit to these data indicated a coefficient of 0.90 (R2 = 0.89). This deviation from a linear slope of 1 again indicates the fact that low-dose and high-dose injections give different count densities in the resulting images. After applying the corrections to the normal dose calculation, a linear coefficient of 0.98 (R2 = 0.87) is obtained for the same patient results (see Figure 5b).

Figure 5. Comparison of the ratio of the low and normal uptake to the ratio of the low and normal dose. The results show the relationship a) before and b) after the normal dose was corrected for the decay of the isotope and washout of the radiopharmaceutical. The dashed line represents a slope of 1 and the solid line is a fit to the data (see text).

Discussion

The data suggest that after correcting for the decay and washout of the radiopharmaceutical there is a linear relationship between the injected dose and the count density in the image.There was, however, a variation of uptake among patients given the approximate same doses. This can be seen in Figure 4b, where the average dose-normalized count density was 64.5 counts/cm2/ mCi, but with standard deviation of 21.5 counts/cm2/mCi. This represents a +- 33% variation in the uptake.

Various factors potentially have an influence upon this uptake, some related to patient metabolism and others to the clinical protocol used to administer the dose and perform the imaging. Recent papers have shown that patient fasting and peripheral warming increases the count density in the images and exercise causes a drop in count density in the images [15]. Another study indicated that menopausal status and postmenopausal hormone therapy increased the count density in the images [16] and there is a weak dependence of the count density on breast density and phase of the patient’s menstrual cycle when the imaging is performed [17]. These factors were not recorded as part of this study and potentially contributed to the wide variation in the measured photon density in the images.

Figure 6. The dose normalized count density for various patients’ weights. The dose values for the first 40 patients (hollow diamonds) have been corrected for the dose remaining in the syringe after the injections, such that they can be compared to the last 60 patient (filled diamonds). The results indicate that there is an upper limit on the count density that decreases with patient weight which is shown by the dashed line in the graph.

The dose-normalized count density measured in the right MLO images versus patient weight for all patients in the study is shown in Figure 6.The count density measurements for the first 40 patients were reduced by 25%, which was the mean fraction of activity remaining in the syringe after injection for the last 60 patients for whom postinjection activity measurements were made. This was done to estimate the actual injected dose for those patients. Of particular note was the fact that there appeared to be an upper limit on the dose-normalized count density in the images, which decreased with patient weight. A line indicating this approximate upper limit has been included in the graph. The equation representing this line is given by:

Uptake (counts/cm²/mCi) = 150 – weight (kg)

This allows us to derive an equation that can be used to adjust the dose given to patients so that image quality can be maintained for heavier patients:

Weighted Dose = Base Dose * (100 / (150-Weight (kg) ) ) for [50< Weight < 125]

Conclusion

The relationship between the count density in the image and the injected dose is typically assumed in nuclear medicine and fundamental to any effort to lower the radiation dose to the patient. Quantitative measurements of the count density (counts/cm2) in gamma camera images of the breast acquired in 100 patients after two subsequent injections of Sestamibi confirm that there is a linear relationship between the image count density and the injected dose only after the decay of the isotope and washout of the agent were considered. This indicates that the decay of the isotope and the washout of the agent between the two injections have a measurable effect on quantitative measurements of the count density. The half-life of the Tc99m isotope is approximately 6 hours, which results in an 11% decrease in the activity over a typical 1-hour imaging sequence. The washout of the Sestamibi varies with the patients’ metabolism, but is typically more rapid than the decay of the isotope. A conservative estimate of 3 hours was used for the calculations in this study. This means that up to 20% of the Sestamibi could washout during a one-hour imaging sequence. In consideration of this decay and washout, the imaging time for each image in a sequence should be adjusted such that all the images in the sequence have approximately the same count density.

Also important in quantifying the actual dose delivered to the patient is a measurement of the dose remaining in the syringe after the injection. In the 60 patients for whom a post-injection measurement was made, an average of 25% of the initial measured dose remained in the syringe after the injection. Therefore, in a typical 20 mCi administered dose, only 15 mCi is actually received by the patient.

While a direct correlation between observed count density and patient weight was not observed, the appearance of an upper limit in the measured count density for a given weight was observed. This upper limit was described by the relationship:

Uptake (counts/cm²/mCi) = 150 – weight (kg)

Consideration of this relationship should dictate that the administered dose be increased as patient weight increases, so that a more constant count density can be achieved for all patient weights. Low-dose imaging can also be hampered by the fact that certain patients exhibit low uptake of tracer compared with other patients of the same weight. As previously discussed, this may be related to various metabolic or physiologic factors. A potential solution would be to adjust the acquisition time to achieve a standard count density in the image on a real-time basis during the acquisition. This requires monitoring the average pixel value during the acquisition and having the acquisition terminate based on that value.Alternatively, efforts have been made to increase the count density in the images by increasing the sensitivity of the MBI camera system through collimator design [18] or by using two detector heads, one on either side of the breast [19]. In this geometry, the reduction in spatial resolution with distance is compensated by the proximity of any area of interest to one of the two detectors [20]. Image combination method can also be used with a two-camera system to enhance the detectability of lesions either by enhancement of the signal to background [18].

An alternative to increase the performance of the molecular breast imaging procedure is to transition from two-dimensional imaging to three-dimensional imaging. Previously suggested concepts have shown increased performance for three-dimensional dedicated SPECT systems and quasi-three-dimensional tomosynthesis systems [21]. These systems have demonstrated superior performance but have yet to be widely implemented. A simple but elegant design that may gain acceptance is the concept of a variable-angle slanthole (VASH) collimator [22]. This type of concept could be easily retrofitted to existing MBI systems, could be used for molecular breast tomosynthesis (MBT), and be easily adapted to perform gammaguided biopsies. Analogous to the leap in diagnostic value seen by the recent transition from digital mammography to digital breast tomosynthesis, the transition from planar molecular breast imaging to 3-dimensional MBT may be a similarly important advancement in diagnosing cancers in the underserved population of high-risk women with dense breast tissue.

This study of 100 patients indicated that the average count density of the MBI images was reduced by radiopharmaceutical decay and washout and imaging time should be adjusted accordingly in low dose imaging. It was also observed that the patient weight was one physical characteristic that should be considered for optimal image quality, with the dose increasing in proportion to the patient weight.

Disclosures:

Benjamin Welch is an employee of Dilon Technologies and possess Dilon stock

Douglas Kieper is a previous employee of Dilon Technology and retains Dilon stock

Marcela Böhm-Vélez has nothing to disclose

Thomas Chang has nothing to disclose

Antoinette Cockroft has nothing to disclose

IRB statement:

This study has been approved by an institutional review board and all subjects signed an informed consent form.

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DOI: 10.31038/CST.2017223

Abstract

Background: HDAC inhibition is known to modulate expression of tumour suppressor genes and induce cell differentiation, growth arrest and apoptosis. The aim of this study was to evaluate the efficacy of a novel series of hydroxamic acid based HDAC inhibitors in cell based assays and tumour xenograft models.

Material and Methods: A series of novel hydroxamate derivatives were synthesized and evaluated. HDAC enzyme inhibitory activity was measured using Hela nuclear extracts. Anti-proliferative activity was assessed in a panel of cancer cell lines. Anti-apoptotic activity was evaluated by caspase-3 activation. In vivo efficacy was evaluated in lung adenocarcinoma xenograft model.

Results: The compounds showed potent HDAC inhibitory activity and anti-proliferative activity in several cancer cell lines. In an in vivo A549 lung xenograft model, the compounds exhibited significant tumor growth inhibition.

Conclusion: The novel HDAC inhibitors showed anti-proliferative activity against several human cancer cell lines and also anti-tumor activity in a Mouse xenograft model.

Keywords:

Histone deacetylation, HDAC inhibitor, Vorinostat

Introduction

Histone acetylation/deacetylation is mediated by a class of enzymes known as Histone acetyl transferase (HATs) and histone deactylases (HDACs). HDACs (histone deacetylases) are important enzymes in the regulation of gene expression in eukaryotic cells [1]. HDACs are key enzymes involved in the regulation of histone and non-histone proteins [2]. Increased levels of HDACs in tumor cells are known to be closely associated with tumor initiation, progression and metastasis [3-4]. Inhibition of Histone deacetylases is known to play an important role in epigenetic regulation by inducing cell death, apoptosis, and cell cycle arrest in cancer cells.

Histone deacetylase (HDAC) inhibitors are a diverse group of small molecule drugs that induce a broad range of effects on cancer cells, including cell cycle arrest, apoptosis, cell differentiation, autophagy and anti-angiogenic effects [5]. Histone deacetylase inhibitors (HDACi) have emerged as a class of therapeutic agents that induce tumor cell cytostasis, differentiation and apoptosis in various hematologic and solid malignancies [6-7]. These drugs inhibit HDAC, and several of them have been developed as anti-cancer agents as they have a significant effect specifically on tumor-cell proliferation compared to non-malignant cells.

HDACs inhibitors can be divided into four major structural classes: (1) small molecular weight carboxylates; (2) hydroxamic acids; (3) benzamides; and (4) cyclic peptides [8-9]. Vorinostat (Zolinza) and Romidepsin (Istodax) are the only HDACs inhibitors currently approved by the U.S. Food and Drug Administration (FDA) for the treatment of refractory cutaneous T-cell lymphoma (CTCL) [10-11].

Most of the HDAC inhibitors that have entered clinical trials have limitations, including low bioavailability, low potency, cardiovascular safety issues, and potential for drug-drug interactions through cytochrome P450 inhibition [12]. Therefore, there is still a clinical opportunity for novel, orally available efficacious HDAC inhibitors with a wider safety margin. HDAC inhibitors exhibit anti-proliferative activity in both in vitro and in vivo pre-clinical models of cancer, with many of them being evaluated as anticancer therapeutics in the clinic. However, keeping in mind the poor bioavailability and efficacy in solid tumors, there still remains an unmet medical need to discover newer HDAC inhibitors derived from novel structural classes of compounds.

In the present paper, we report the identification of novel hydroxamic acid based small-molecule HDAC inhibitors by mediumthroughput screening of a compound library using a fluorescence based assay with Hela Nuclear extracts as the enzyme source. A focused library of about 100 compounds was designed and synthesized, among which several compounds showed equivalent or higher potencies against HDAC as compared to Vorinostat. The hit compounds from the primary screening were evaluated for their effects on cellular proliferation in a panel of human cancer cell lines. We identified four compounds which showed a potent GI50 in the low micromolar range. Several of these novel HDAC inhibitors could be promising new lead structures for further development as improved anticancer drugs. In conclusion, the screening of a library of compounds for HDAC inhibitory activity and anti-proliferative effect in cancer cells has identified several promising new leads for further development.

Materials and methods:

Synthesis of HDAC inhibitors

The compound library was obtained from the Medicinal chemistry department at Anthem Biosciences.

Based on general formula 1, about 100 hydroxamic acid derivatives were designed and synthesized.

These compounds were synthesized as described in below synthetic scheme 1.

Reactions of the compound 1 with sodium azide in dimethylformamide at 40 oC resulted compound 2. Then the compound 3 was synthesized by standard click chemistry by reacting the compound 2 with appropriate alkyne in the presence of copper iodide and Hunig’s base in dimethylformamide [13]. Reaction of the compound 3 with hydroxyl amine in the presence of suitable bases such as sodium methoxide in methanol yielded the compounds of general formula I.

Out of nearly 100 compounds synthesized, 4 compounds i.e. PAT-1101, PAT-1103, PAT-1118 and PAT-1125 were identified as hit molecules from the primary screening. Suitable salts of the four compounds were prepared to improve their drug like properties.

Preparation of Hela Nuclear extracts:

Nuclear fractions prepared from Hela cells as per established protocols were used as a source of HDAC enzyme. Hela cells obtained from ATCC were cultured in complete growth medium containing 10% fetal bovine serum supplemented with antibiotics. Subconfluent cells were harvested and washed in phosphate buffered saline (PBS). 1×107 cells were resuspended in 1 mL of cold lysis buffer containing 10 mM Tris HCl (pH 7.5), 10 mM NaCl, 15 mM MgCl2, 250 mM Sucrose, 0.1 mM EGTA and 0.5% NP-40. The cell lysate was then maintained on ice for 15 min. To the lysate, 4 mL of Sucrose buffer containing 30% sucrose, 10 mM Tris HCl (pH 7.5), 10 mM NaCl and 3 mM MgCl2 was added and the resultant mixture was centrifuged at 1500 rpm for 10 min at 4 deg C. The resultant pellet was resuspended in 1 mL of Tris-HCl buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl) and recentrifuged at 1500 rpm for 10 min at 4 deg C. The resultant supernatant was discarded and the isolated nuclei was resuspended in 100 µL of cold extraction buffer containing 50 mM HEPES pH 7.5, 420 mM NaCl, 5 mM EDTA, 1mM EGTA, and 10% glycerol. The solution was sonicated for 30 sec and incubated on ice for 30 min following which it was centrifuged at 10000 rpm for 10 min at 4 deg C. The supernatant was collected and used as the enzyme source for HDAC assay.

In vitro HDAC inhibition Assay:

HDAC inhibition assay was performed using a fluorescence based assay with a fluorescent substrate (Boc-Lys (Ac)-AMC Substrate) as reported previously [14-15]. Stock solutions of the compounds were prepared in 100% DMSO. 3 µg of the nuclear extracts was preincubated with the compounds for 10 min at 30 deg C. The substrate was diluted in 50 µL of assay buffer (25 mM Tris HCl pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1mM MgCl2) and added to a 96-well plate. The plate was incubated for a further 45 min at 30 deg C. The reaction was terminated by the addition of 50 µL of developer and incubated for 15 min at 30 deg C. The fluorescent deacetylated substrate was detected at lexc of 340/40 and lemi of 460/40 using a Microplate Reader (BioTek Instrument Inc.). The fluorescent signal was compared with the DMSO treated wells and the percentage inhibition was determined. IC50 (50% HDAC inhibitory concentration) was determined by testing in a wide concentration range of 0.001, 0.01, 0.1, 1 and 10μM.

Cell proliferation assays:

Anti-proliferative activity of the compounds was tested against a panel of cancer cell lines including Lung, Cervix, Colon, Brain, Renal, Leukemia, Prostate, Pancreas, Skin, Bone, Breast, Ovary cancer by using a standard MTT assay. Human cancer cell lines (American Type Culture Collection) were cultured in complete media containing 10% heat inactivated fetal bovine serum and 100 U/ml Penicillin, 100 µg/ml Streptomycin in a 37°C, 5% CO2 humidified incubator and passaged twice weekly. Cells were seeded in 96-well plates at a density of 3X103 cells per well in 100 µL and were allowed to attach for 24 h. Stock concentrations of the compounds were made in DMSO. 100 µL of media containing various concentrations of compounds (1, 10 and 100 µM) were added to the cells and were incubated for 48 hours. Vorinostat was tested as a reference compound in the assay. On the day of termination, 50 µL of MTT (3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl-2H-tetrazolium bromide) (Sigma, St Louis, MO, USA) solution (5mg/mL) was added to the medium and the cells were incubated for 3 hours. The medium was then aspirated and 100% DMSO was added to solubilize the violet MTT-formazan product. The absorbance at 570 nm was measured on a 96-well plate reader by spectrophotometry (Biotek Synergy HT). Assays were performed in duplicates for each concentration. Results are expressed as percentage of growth inhibition with respect to the DMSO treated control wells. A dose response curve was generated and GI50 values were interpolated from the growth curves using GraphPad Prism software.

Apoptosis assay (Caspase-3 activation):

Caspase-3 activity was measured in HT-29 cells using a commercially available kit (Sigma- Aldrich). Briefly, HT-29 cells were cultured in McCoy’s 5a medium containing 10% FCS and antibiotics. On the day of the study 10,000 cells were seeded into each well of a 96 well plate and incubated for 12-16 h. The compounds were added at concentrations ranging from 0.1 µM to 30 µM and incubated for 48 h. The cells were then lysed in lysis buffer and the lysates were used to perform the assay according to the manufacturer’s instruction. The assay is based on the hydrolysis of acetyl Asp-Glu-Val-Asp 7-amido- 4-methylcoumarin (Ac-DEVD-AMC) by caspase 3, resulting in the release of the fluorescent 7-amino-4-methylcoumarin (AMC) which is measured at an excitation and emission wavelength of 360 nm and 460 nm respectively.

In vivo anti-tumor activity in A549 lung adenocarcinoma xenograft model:

All experimental procedures involving animals were approved by the Institutional Animal Ethics Committee of Anthem Biosciences. In vivo anti-tumor activity of the HDAC inhibitors was assessed in 6 week old Athymic Nude mice. Animals were purchased from Harlan Laboratories, Indianapolis IN (presently Envigo) and housed in individually ventilated cages under controlled conditions and maintained on a 12-h light/12-h dark cycle, with food and water supplied ad libitum. A549 (lung adenocarcinoma) obtained from ATCC were cultured in RPMI-1640 growth medium containing 10% FBS and antibiotics. Sub-confluent monolayers were harvested and a cell suspension of >90% viability was prepared in 1X HBSS, pH 7.4 (Hank’s Balanced Salts Solution, Sigma) and mixed with an equal volume (1: 1) of ice cold Matrigel® (Corning Life Sciences). 0.1 mL of the cell suspension containing 1×106 cells was injected into the flank region of the animals under isoflurane anesthesia. Animals were monitored daily during the period between inoculation and palpable tumor growth. Tumor volume was calculated using the formula, Tumor volume = (length × width2)/2. Tumor bearing mice were randomized into control and treatment groups (n=8) when the tumor volume reached ~100 mm3. The compounds were formulated in a vehicle containing 0.5% Carboxy methyl cellulose and 0.1% Tween 80 in water and administered by oral gavage to tumor bearing mice once daily for 21 days. The compounds were tested at 150 mg/kg. The Control group received the vehicle alone. Clinical signs were observed daily and tumor volume and was body weight was measured twice weekly during the study.

Data Analysis:

The terminal tumor volumes from in vivo xenograft studies were subjected to one-way ANOVA analysis followed by Dunnett’s test when there were multiple treatment groups. Results were considered statistically significant when P < 0.05.

Results

HDAC enzyme inhibition:

The biological activity of the HDAC inhibitors was assessed in vitro using a cell free HDAC enzymatic assay. Several compounds exhibited potent HDAC-inhibitory activity with IC50 values of in the nanomolar range (Table 1). Our results indicate that the in vitro HDAC inhibition potency is higher than the reference compound Vorinostat (Figure 1).

Table I. Characterization of hit compounds

Compound	M. Wt. (g/mol)	Molecular Formula	HDAC IC50 (nM)
PAT-1101	403.49	C23H25N5O2.HCl	4
PAT-1103	419.49	C23H25N5O3.HCl	1
PAT-1118	393.45	C21H23N5O3.HCl	23
PAT-1125	377.45	C21H23N5O2.HCl	4
Vorinostat	264.32	C14 H20 N2 O3	78

HDAC inhibitory activity of synthesized compounds was measured using Hela cell nuclear extract as the enzyme source by a fluorescence based assay as described under the Materials and Methods section. IC50 was calculated from concentration versus percentage inhibition plotted using Graph Pad Prism software.

Anti-proliferative activity in cancer cells:

The growth-inhibitory activity of 4 compounds identified through the primary HDAC inhibitory screening was assessed in vitro in a panel of Human cancer cell lines. Cells were treated with the HDAC inhibitors at various concentrations and GI50 was determined. All the 4 compounds identified resulted in a dose-dependent inhibition of cellular proliferation at low micro molar concentrations in most of the cell lines tested (Table 2). The inhibitory effect of PAT-1101, PAT- 1103, PAT-1118, PAT-1125 on the proliferation of cancer cells was comparable or superior to that of Vorinostat against several cell lines under our experimental conditions.

Table 2. Anti-proliferative activity of hit compounds expressed as Mean Growth inhibitory concentration (GI₅₀ in µM) in a panel of cancer cell lines

Tissue	Cell line	Growth inhibition: GI50 concentration (µM)
		PAT-1101	PAT-1103	PAT-1118	PAT-1125	Vorinostat
Colon	Colo-205	0.31 ± 0.06	0.2 ± 0.03	0.3 ± 0.071	0.4 ± 0.04	1.6 ± 0.1
	HCT-116	0.10 ± 0.11	0.2 ± 0.05	1.2 ± 0.018	0.2 ± 0.04	2.2 ± 0.0
	HT-29	0.15 ± 0.13	0.4 ± 0.13	1.5 ± 0.65	2.1 ± 0.46	3.6 ± 2.6
Lung	A549	1.58 ± 1.1	2.0 ± 1.52	4.5 ± 2.33	2.2 ± 2.22	5.9 ± 2.4
	NCI-H23	0.52 ± 0.04	0.4 ± 0.09	2.3 ± 1.14	0.5 ± 0.24	3.2 ± 1.0
	NCI-H460	0.3 ± 0.04	0.45 ± 0.28	2.4 ± 0.85	1.9 ± 0.74	4.4 ± 1.4
Prostate	DU-145	0.079 ± 0.04	0.1 ± 0.03	0.3 ± 0.124	0.1 ± 0.04	1.3 ± 0.0
	PC-3	3.5 ± 2.7	1.7 ± 1.02	13.4 ± 3.3	4.8 ± 1.4	8.3 ± 0.6
Ovary	SK-OV-3	0.04 ± 0.016	0.5 ± 1.2	1.8 ± 0.20	1.7 ± 0.16	4.4 ± 1.5
	PA-1	0.08 ± 0.04	0.04 ± 0.01	0.2 ± 0.037	0.1 ± 0.01	0.3 ± 0.05
Cervix	Ca Ski	0.42 ± 0.15	0.4 ± 0.28	1.2 ± 0.22	1.1 ± 0.73	6.0 ± 5.3
	Hela-229	1.0 ± 0.27	0.6 ± 0.38	1.4 ± 0.26	0.7 ± 0.47	4.7 ± 3.2
	Hela-S3	0.23 ± 0.12	0.2 ± 0.02	0.6 ± 0.16	0.2 ± 0.08	2.9 ± 0.5
Brain	IMR-32	0.25 ± 0.07	0.2 ± 0.02	0.9 ± 0.201	0.2 ± 0.04	1.8 ± 0.2
	U-87-MG	0.76 ± 0.66	1.1 ± 0.73	2.7 ± 0.74	1.0 ± 0.33	6.7 ± 1.1
	SH-SY-5Y	0.34 ± 0.033	0.2 ± 0.03	0.2 ± 0.06	0.1 ± 0.0	0.8 ± 0.4
Breast	MCF-7	3.5 ± 2.6	2.1 ± 2.63	5.7 ± 1.62	5.5 ± 1.2	5.5 ± 1.2
Renal	ACHN	0.09 ± 0.02	0.2 ± 0.08	0.7 ± 0.11	0.1 ± 0.04	1.6 ± 0.5
	786-O	2.65 ± 0.08	1.5 ± 0.70	2.5 ± 1.01	2.0 ± 1.1	4.4 ± 2.0
Leukemia	RPMI-8226	0.15 ± 0.05	0.2 ± 0.25	0.5 ± 0.43	0.3 ± 0.16	2.2 ± 2.3
	K562	0.19 ± 0.05	0.2 ± 0.08	0.3 ± 0.074	0.2 ± 0.04	2.2 ± 0.7
Pancreas	PANC-1	1.03	4	6.6	1.4	21.9
Skin	A431	0.28 ± 0.05	0.2 ± 0.03	1.2 ± 0.533	0.4 ± 0.08	1.9 ± 1.0
Bone	KHOS	11.7 ± 1.1	4.6 ± 0.55	2.8 ± 0.36	10.3 ± 3.26	29.3 ± 7.2

Anti-proliferative effect of the compounds in a panel of cancer cell lines obtained from ATCC using MTT reagent as described under Materials and Methods. Results are represented as GI50 or the concentration of the compound which inhibits 50% of cell growth. GI50 was calculated using GraphPad Prism software. The Mean GI50 was derived from individual assays performed in triplicates

Induction of Apoptosis in Human Cancer Cells:

We further investigated the compounds’ ability to induce apoptosis in cancer cells and to determine if the apoptosis was caspase dependent. In this regard, compound induced Caspase-3 activation in HT-29 cells was measured using a fluorescence based assay. The compounds significantly activated caspase-3 enzyme with an EC50 which was similar to that of Vorinostat (Table 3).

Table 3.Caspase-3 activation assay

Compound	EC50 (µM)
PAT-1101	6.59
PAT-1103	3.62
PAT-1118	1.50
PAT-1125	4.09
Vorinostat	4.52

Anti-tumor Activity in Human Tumor Xenograft Models:

To assess the tumor growth inhibitory activity of the HDAC inhibitors in vivo, we evaluated their effect in a subcutaneous human xenograft model in Athymic Nude mice. The anti-tumor efficacy was evaluated in mice engrafted with A549 lung adenocarcinoma cells. Once daily oral administration of PAT-1103, PAT-1118 and PAT-1125 resulted in a significant tumor growth inhibition (TGI) after 21 days (Figure 2). The tumor growth inhibition (TGI) achieved as a result of treatment was between 67 and 69% at 150 mg/Kg dose. The efficacy was superior to that of Vorinostat which produced 54% tumor growth inhibition at the same dose (Table 4). Furthermore, there were no adverse clinical signs and no significant reduction in body weight in the treated mice compared to the vehicle control group.

Table 4.Tumor growth Inhibition (TGI) in subcutaneous A549 lung tumor xenograft models established in Nude mice

Compound	Dose (mg/Kg, p.o)	Mean Tumor volume (mm3)  on Day 21	% TGI
Control (Vehicle)	0	598.5 ± 72.9	–
PAT-1101	150	611.8 ± 125.4	3
PAT-1103	150	282.0 ± 34.7**	68
PAT-1118	150	272.4 ± 42.8**	67
PAT-1125	150	346.4 ± 67.9**	69
Vorinostat	150	342.10 ± 42.80*	54

TGI: Tumor growth inhibition was calculated with respect to the Vehicle treated Control group on Day 21. *P<0.05, **P<0.001, One way ANOVA followed by Dunnett’s test compared to Control

Discussion

We have synthesized and identified a series of hydroxamic acid derivatives designed to inhibit HDAC resulting in anti-cancer activity. In vitro mechanism of action studies demonstrate that the compounds are able to inhibit HDAC with nanomolar potency and activate apoptosis pathways such as caspaze-3 enzyme activation and cause cell death in a wide range of cancer cell lines. Four compounds PAT-1101, PAT-1103, PAT-1118 and PAT-1125 that were identified from the primary screening were tested against a panel of cancer cell lines. All the compounds exhibited anti-proliferative activity against the cancer cell types, with greater or similar potency than that of leading HDAC inhibitors in development. The compounds being novel HDAC inhibitors with good cytotoxic activity against a variety of Human tumor cell lines. Studies to evaluate the drug-likeness of the compounds such as pharmacokinetic profiling and in vivo safety studies would help in developing them as anti-cancer drugs. Improved bioavailability and safety profile of these compounds would help in determining their potential to be more effective in clinical trials than other HDAC inhibitors with poor pharmacokinetic properties and dose limiting side effects.

Acknowledgements

The Authors sincerely thank the management of Anthem Biosciences for their constant support and encouragement in carrying out this study.

Conflict of interest: None

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DOI: 10.31038/CST.2017231

Abstract

Background: Evidence of cancer cure or improvement of quality of life in cancer patients has been reported using prescriptions based on traditional knowledge, but not scientifically investigated. The objectives of the current study is to evaluate the anti-proliferative action of a poly herbal drug which is currently used in traditional medicine name “Le Pana Guliya (LPG)”

Methods: The aqueous extract of LPG was freeze dried. Total phenolic content was assayed using Folin Ciocalteu reagent. Antioxidant activity of the extract was evaluated using DPPH radical scavenging assay in vitro. Brine shrimp assay was conducted to determine the cytotoxicity (LD50) of the LPG. The cell viability was determined by MTT assay and cytotoxicity by LDH leakage assay after 24 and 48 hours incubation with LPG. Morphological Changes after the treatment of the drug was observed using an inverted light microscope. RD, MCF-7 cells and CC1 cells were used in all experiments.

Results: The TPC of the LPG was 5.31 ± 0.14 W/W% of GAE and antioxidant capacity is comparable to ascorbic acid. LPG exhibited strong cytotoxic activity against RD and MCF-7 cell lines with MTT assay. A 50% leakage of LDH was observed at concentrations less than 30μg/mL and 10 μg/mL for both RD and MCF-7 cells respectively after 24 hour exposure. While, LPG exhibited strong cytotoxic activity against RD and MCF-7 cells the brine shrimp and CC1 cells results (EC50>100μg/mL) suggest that the LPG have minimum cytotoxicity towards the normal healthy cells.

Conclusion: The present study provides evidence for potent anti cancer activity of LPG on RD and MCF-7 cells. The brine shrimp bio-assay and CC1 cells results suggests that the extract have minimum cytotoxicity towards the normal healthy cells.

Keywords

Anti-proliferative activity, Cytotoxicity, MTT assays, LDH assay, Brine shrimp assay, Light microscopy, RD cells, MCF-7, CC1, DPPH

Introduction

Natural products have played an imperative role in the lead-finding of candidates for the development of present-day cancer chemotherapy due to their low toxicity and side effects. They offer a valuable source of a wide variety of chemical structures with biological activities (lead molecules) for the development of novel drugs [1]. Approximately 60% of all drugs currently undergoing clinical trials for cancer treatment are natural products or compounds derived from natural products [2, 3]. These substances embrace some of the most exciting new chemotherapeutic agents currently available for use in a clinical setting [3].

Sri Lanka is gifted with many plants which have vast medicinal value. Use of plants for medicinal preparations is a primary part of the Sri Lankan cultural life and this is implausible to change in the years to come. The Sri Lankan traditional medicine system shows extensive use of plant products in cancer treatment. A significant surge of interest in chemoprevention research has thus led to the discovery of many phytochemicals as successful chemo preventive agents.

Many traditional healers and folklore medical practitioners in the country have been treating cancer patients for many years using various medicinal plant species. There is evidence of case reports on cancer cure using traditional knowledge, but they are not scientifically investigated. In addition to use of a single plant, poly herbal formulations of drugs are intensively used in Sri Lanka. The poly herbal drug ‘Le Pana Guliya’ (LPG) found in Ola leaf inscriptions is prescribed to treat different types of cancers by traditional doctors.This study was aimed at investigating of its cytotoxic effect against two different cancer cell lines compared with healthy CC1 cells.

Materials And Methods

Chemicals and equipment

Chemicals needed for cell culture and cytotoxicity studies were purchased from Sigma –Aldrich (St Louis, MO63178, USA). 1-Diphenyl-2-picrylhydrazyl (DPPH), TritonX-100, was purchased from Fluka. Tris base was purchased from Promega (Madison, WI 53711–5399, USA). All chemicals used were of analytical grade.
Shimadzu UV 1601 UV visible spectrophotometer (Kyoto, Japan) was used to measure the absorbance. LFT 600 EC freeze dryer was used to obtain the freeze dried powder of the poly herbal drug. Cells were incubated at 37°C in a humidified CO2 incubator (SHEL LAB/Sheldon manufacturing Inc. Cornelius, OR 97113, USA). Inverted fluorescence microscope (Olympus Optical Co. Ltd. 1X70-S1F2, Japan) for observation of cells, and photographs were taken using microscope digital camera (MDC200 2M PIXELS, 2.0 USB). Deionized water from UV ultra-filtered water system (Waterproplus LABCONCO Corporation, Kansas city, Missouri 64132–2696) and distilled water was used in all experiments.

Cell cultures

Human Rhabdomyosarcoma cell line (RD), and human breast adenocarcinoma cell line (MCF-7) were cultured in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% heat inactivated fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 U/mL). The cells were maintained in 25 cm2 plastic tissue culture flasks at 37oC in a humidified atmosphere containing 5% CO2 in air. Exponentially growing cells were used in all experiments. The normal rat fibroblast (CC1) cell line was employed as the control.

Preparation of poly-herbal extract

The poly herbal drug (5 g) soaked in distilled water (100 mL) was kept in the rotary shaker for 48 hours in an air tight dark bottle. The extract was then filtered through a layer of muslin cloth and filtrate was centrifuged at 3,000 rpm for 15 minutes at 4oC to remove any debris.

The supernatant was freeze dried, and stored at -20°C in an air tight vial until used. The freeze dried extract was reconstituted with distilled water for experimental purposes.

Antioxidant activity

The DPPH assay was used to determine the radical scavenging activity of the extract, as reported previously by Perera (2008) [4]. Briefly different concentrations of plant extract (100 µL) was mixed with DPPH reagent prepared in absolute ethanol (100 µM, 900 µL) and incubated for 30 minutes in darkness at 37oC, in a water bath. The percentage of de-colorization was obtained spectro-photometrically at 517 nm. The control was prepared as above without adding extract while L-ascorbic acid was used as the positive control.

At least three independent tests were performed for each sample. Percentage inhibition was calculated from the following formula:

The effective concentration of the sample required to scavenge the respective radical by 50% (EC50) was calculated using the linear segment of the curve obtained with percentage inhibition against concentration.

Total phenolic content (TPC)

The total phenolic content of the lyophilized sample (n = 6) of the poly herbal drug was determined using the Folin-Ciocalteu method [5].

Brine shrimp bioassay

Brine shrimp assay was conducted to determine the cytotoxicity (LD50) of the poly herbal drug as explained previously by Soysa (2014) and Middleton (2005) [6, 7].

Briefly, dried cysts of brine shrimps (10 mg) were allowed to hatch at room temperature in sterile sea water. After 24 hours the free nauplii were selected and 10 were added to each petridish containing different concentrations of the extract in sea water. After 24 and 48 hours, the petridishes were observed using a magnifying glass and the number of survived nauplii in each petridish was counted.

The mortality end-point of this bioassay was defined as the absence of controlled forward motion during 30 seconds of observation [7].

The percentage lethality was determined by comparing the surviving larvae of the test and the control. The percentage survival was calculated as:

Cell viability assay

The effect of aqueous extract of the drug on the cell viability was determined by MTT assay. The live cells reduce yellow MTT to purple formazan crystals by mitochondrial dehydrogenase enzyme [8]. Cells were suspended in the growth medium and seeded in 24-well plates at 2 x 105 cells/well for overnight. The cells were then treated with different concentrations of the drug at 37oC for 24 and 48 hours. A positive control with cyclohexamide (50 g/mL) and a negative control without the drug were simultaneously conducted.

Percentage viability of the cells treated with the drug was calculated comparing the viability of the un-treated cells.

After 24/48 hours, the utilized growth media was subsequently replaced with 1.0 mL of minimum essential media (MEM), and 100 µL of MTT (5 mg/mL in PBS). The cells were incubated at 37o C for 4 hours and the medium was carefully removed. The formazan product was dissolved in acidified isopropanol (0.05 M HCl in isopropyl alcohol (IPA); 750 µL) and absorbance was read at 570 nm. Cell survival was expressed as a percentage of viable cells of treated samples to that of untreated cells (negative control). The drug extracts were prepared in triplicate and each experiment was performed in triplicates to each preparation.

Lactate dehydrogenase (LDH) activity

Cytotoxicity induced by the LPG evaluated by lactate dehydrogenase (LDH) leakage into the culture medium, as explained in Fotakis and Timbrell (2006) [9]. Cells seeded in 24 well plates (2 x 105 cells/well) were exposed to different concentrations of the drug. After 24/48 hours incubation the culture medium was aspirated and centrifuged at 4000 rpm for 5 min in order to obtain a cell free supernatant. The cell lysate was prepared by treating the cells with Triton X 100 (0.1%; 1 mL) and sonicating the contents for 20 seconds. The final suspension was centrifuged at 4000 rpm for 5 minutes. Medium and the lysate were subjected to LDH assay. The activity of LDH in the medium was determined using a commercially available, LDH assay kit (HUMAN).

Negative control and positive control with cyclohexamide (50g/mL) were also carried out along with the experiment to measure the percentage LDH leakage.

The absorbance was measured at 340 nm at intervals of 15 seconds for 1.5 minutes using an air blank. The rate of decline (gradients of the graphs) of NADH concentration was used to calculate the LDH activity in the supernatant and the lysate.

The percentage LDH leakage to the medium was calculated using the following equation.

Where total LDH activity = LDH activity of supernatant + LDH activity of the lysate.

Light microscopy

RD, MCF-7 and CC1 cells grown at 70% confluence were treated with different concentrations of aqueous drug extracts for 24, 48 and 72 hours and observed under phase-contrast inverted fluorescence microscope (40X). The changes in morphology were compared with positive and negative controls.

Statistical analysis

All the results of the experiments were expressed as the mean ± standard deviation (Mean ± SD). The measurements were performed in triplicate and values shown are representatives of at least three independent experiments. Least square linear regression analysis was applied using Microsoft excel to determine the EC50/LD50 values and for the calibration curves.

R² > 0.99 was considered as linear for the calibration curves.
Significant differences of each test result were statistically analyzed using “Mann-Whitney U” test significances with 95% significance using SPSS version 16.

Results

Anti-oxidant capacity and total Phenolic content

The aqueous extracts of the poly herbal drug showed moderate free radical scavenging activity with a value of EC₅₀ = 68.0 ± 2.8 g/mL with compared to the positive control, L- ascorbic acid (3.4±0.9g/mL; n=6).
The percentage total phenolic content of the aqueous extract of the poly herbal drug was 5.3 ± 0.1 W/W % of Gallic Acid Equivalence (GAE).

Brine shrimp bioassay

LPG shows no lethality towards the brine shrimp even after 48 hours exposure up to a concentration of 10 to 2000 μg/mL. This results show that the aqueous extract either does not possess any significant (p>0.05) alterations in the cellular functions or no significant cytotoxity.

Cell viability assay

The effect of LPG on cell viability of RD, MCF-7 and CC1 cells was evaluated by tetrazolium assay (MTT).
Multiple concentrations of the aqueous extract of the poly herbal drug were used and effective concentrations were calculated for each cell line (EC₅₀) from dose response curve. The percentage cell viability of RD, MCF-7 and CC1 are shown in Figure 1.

Figure 1. The percentage cell viability of on RD, CC1 and MCF-7 cell lines as determined by MTT assay, (a) after 24 hours treatment (b) 48 hours and (c) 72 hours with aqueous extract of the poly herbal drug. The data are presented as mean ± SD of six independent experiments.

The aqueous extract of the LPG exhibited no significant cytotoxic activity (p>0.05) against CC1 cell line, achieving an EC₅₀ of 85.3 ± 9.8 µg/mL after 72 hour incubation.

On the contrary, the aqueous extract of the poly herbal drug exhibited significant cytotoxic activity (p<0.05) against RD and MCF-7 cell lines compared to the CC1 cells as shown in Table I.

Table I. EC₅₀ values obtained for MTT assay for the three different cell lines.

^*All results were mean of 6 independent measurements ± standard deviation. “Mann-Whitney U” test at 95% confidence level showed a significant difference (p < 0.05) in both RD and MCF-7 cells compared to CC1 cells.

After 24 and 48 hour incubation with Cyclohexamide (Positive control) at a concentration of 50µg/mL, the percentage viability showed by tested cells depicted in Table 2. The percentage cell viability obtained for MTT assay at a concentration of 50µg/mL of LPG was calculated using respective regression equations to compare the capacity to inhibit cell proliferation, with that of the positive control. The values are shown in Table 2.These results exhibited the significant cytotoxicity (p< 0.05) exert by the aqueous extract of LPG against cancer cells compared to CC1 cells as determined by MTT assay.

Table 2. Percentage cell viability obtained by MTT assay at 50 µg/mL Cyclohexamide (+ve Control) and 50 µg/mL LPG after 24 hours and 48 hours exposure

^*All results were mean n=6 measurements ± standard deviation in three independent experiments.

Lactate dehydrogenase (LDH) assay

Measurement of LDH activity is also an indicator of cell viability through evaluation of the cell membrane permeability. The enzyme activity is measured externally, as it leaks from dead cells which lose their membrane integrity. The LDH release curves for RD, MCF-7 and CC1 cell lines treated with different concentrations of the LPG suggested that the cytotoxic effect of the extract was concentration dependent (Figure 2).

Figure 2. Lactate Dehydrogenase (LDH) release from RD, MCF-7 and CC1 cells with aqueous extract of the poly herbal drug, (a) after 24 hour, (b) after 48 hour incubation. The data are presented as mean ± SD of six independent experiments.

Fifty percent leakage of LDH to the media was observed at 26.9± 1.6 and 30.5 ±2.8μg/mL for RD and MCF-7 cells after 24 hour incubation respectively. After 48 hours of incubation with LPG the EC₅₀ values further decreased and 50% LDH leakage arise at 8.2± 0.2 and 6.0± 0.2 μg/mL for RD and MCF-7 cells respectively. In contrast to RD and MCF-7 cells the CC1 cells showed 50% leakage of LDH at 159.3± 3.1 and 103.1 ±5.2μg/mL LPG concentrations after 24 and 48 hours respectively.

Light microscopy

Morphological alterations of RD, MCF-7 and CC1 cell lines upon exposure to the aqueous extract of the poly herbal drug was observed under the phase contrast inverted microscope. The microscopic observations revealed that the aqueous extract has a significant cytotoxic effect on RD and MCF-7 cells compared to the negative control. However CC1 cells treated with the drug did not show significant cell death (Figure 3).

Figure 3. Light microscopy images of RD, MCF-7 and CC1 cells treated with their respective EC50 values of LPG, with magnification of 40X. (Black arrow – indicates healthy spindle shape cells; Red arrow – dead and shrinkage cells due to the LPG treatment).

Discussion

Cancer has become the most leading cause of death worldwide. It has become an emerging health problem affecting both developing and developed countries. According to the World Health Organization reports published in 2014, 8.2 million patients have died from cancer in 2012 and the number of annual cancer cases reported in 2012 was 14 million. It has been also estimated that the number of annual cancer cases would have increased from 14 million in 2012 to 22 million within the next two decades [10]. The most effective treatment approaches in cancer are chemotherapy and radiotherapy. Nevertheless, the higher incidence in side effects, have made investigators engage in finding novel anticancer compounds with less adverse effects. As a result of this, plants and other natural sources have provided nearly 60% of anti cancer agents which are currently in use [11]. Especially the most novel chemotherapeutic agents more than ever before are botanical in kind [2].

Traditional folklore medicine which involves the use of natural elements uses either single or multiple herbs as a mixture (polyherbs) for treatment of diseases. The use of polyherbal formulation is found to be more effective than the use of a single herb as the active phytochemical constituents of the individual plants are insufficient to achieve the desirable therapeutic effect. But when combining the herbs in a particular ratio the synergistic effect produced by the active phytochemicals of several plants give a better therapeutic effect [12].

Polyherbal preparations are used worldwide for treatment of various disease conditions including cancer. As the polyherbal formulations have gained more attention, many studies have been conducted to identify the mechanism of action and the efficacy of these polyherbal preparations. A latest study [13] which gives a global perspective on polyherbal formulations reports that for the past six years, only 2 publications have been made regarding polyherbal formulations that show/have anticancer activity.

Zyflamend® used in China [14], Sho-saiko-to® used in Japan [15] and Arbudhcure® used in India [16] are few of the polyherbal preparations used for cancer treatment where attempts have been made to validate scientifically for their mechanism of action and therapeutic efficacy.

With compared to the aforementioned poly herbal drugs the polyherbal LPG is not commercialized and no scientific research has been carried out up to this study to identify its mechanism of action or evaluate its effectiveness on treatment of cancer even though it is being used to treat 32 different types of cancer in traditional medicinal system in Sri Lanka.

Secondary metabolites of plants including phenolic compounds are very important for their essential functions in reproduction, growth and in defense mechanisms [17]. Phenolic compounds also provide natural antioxidants for protection against many diseases including cancers. It has been reported that antioxidant effects of plants are mainly attributed to the radical scavenging activity of phenolic compounds such as flavonoids, polyphenols, tannins, and phenolic terpenes. The polyphenols have the capability to scavenge ROS as well as oxidatively generated free radicals which derive from bio-molecules [18].

As depicted in results the quantitative measurement of poly phenolic content obtained in the present study showed that the water extract of LPG has moderate levels of polyphenols. Similarly the EC50 values obtained for the different samples of LPG for DPPH assay showed higher values compared to EC50 of ascorbic acid which evidently indicate that the LPG has a lower anti-oxidant capacity.

It was reported by several authors, that the proliferation of cancer cells are inhibited by the high antioxidant and poly phenolic content present in plant extracts [14, 19]. This probable fact that presence of low polyphenolic content and low antioxidant potential was explained in Hamberger and Hastettman 1991[20], as possible changes occurring in the chemical composition of the plant materials during the processes of the drug manufacturing that could result in a reduced pharmacological activity in mixtures. Thus it can be suggested that even though the LPG exerts an effective antiproliferative activity and cytotoxicity, the mechanism of action must be in a different pathway compared to most of the other traditional medicinal drugs which exert antiproliferative activity via the phenolics and antioxidants.

Cytotoxic action in brine shrimp assay is brought about by the active ingredients present in the drug by disturbing the fundamental mechanisms associated with cell growth, mitotic activity, differentiation and function [21]. According to previous reports a crude plant extract is toxic (active) if it has an LD50< 1000 µg/mL while non-toxic (inactive) if LD50> 1000 µg/mL [22]. The present study evidently indicates that the aqueous extract of LPG shows no lethality towards the brine shrimp even after 48 hours of exposure within the concentrations tested ranging 10 to 2000 µg/mL. This results show that the aqueous extract either does not possess any significant (p>0.05) alterations in the cellular functions or no significant cytotoxicity. Similar results were observed in a previous study which shows anticancer activity of the plant extract of Flueggea leucopyrus against HEp 2 cells but no toxicity for brine shrimps [6].

In this study the cell viability of the tested cell lines were determined by the MTT reduction assay. Each cell type was treated with different concentration of LPG extract and incubated for 24 and 48 hours. The EC50 value was obtained from the graph between the percentages of cell viability versus concentrations of LPG extract (Figure 1). The values were used to describe the degree of cytotoxicity of the extract towards cell lines. The cell viability of CC1 cells were further determined after 72 hours due to the high viability of the cells at 48 hours of incubation (187.58 ± 9.59 µg/mL).

It was established by the American National Cancer Institute (NCI), that if a crude extract shows EC50< 30 µg/mL after an incubation of 72 hours it is considered as cytotoxic [23]. However crude extract with EC50 less than 20µg/mL after 48 hours incubation is supposed to be highly cytotoxic [24]. Present study shows a potent cytotoxic effect on RD and MCF-7 cells with the aqueous extract of the LPG even at 24 hours which is much lower than the value specified by the NCI. However, the extract was less cytotoxic towards normal mouse fibroblast cell line CC1.

After 24 and 48 hours of incubation with Cyclohexamide (Positive control) at a concentration of 50μg/mL, the percentage viabilities of the tested cells are showed in Table 2. In contrast, the percentage viability obtained for MTT assay at the same concentration of LPG (50 μg/mL) treatment was markedly lower for RD and MCF-7 cells (Table 2).The EC50values obtained for MTT assay indicate that the cytotoxicity of the LPG extract against the cancer cell lines is within the limit of cytotoxicity (EC50 < 30 μg/mL), as reported by the American National Cancer Institute (NCI) over 72 hour post exposure and it is beyond the limits for CC1 cells (Table 1) Thus LPG shows significant anti-proliferative activity on cancer cells studied compared to the standard anti cancer drug. Cyclohexamide showed a % cell viability of 69.93 ± 9.26% and 49.31 ± 3.88% for 24 and 48 hours respectively for CC1 cells, suggesting its high toxicity towards the normal cells compared to the LPG (Table 2).

It is reported that the poly herbal anticancer drug Zyflamend® shows an EC50 value of 200 µg/mL against prostate cancer cell line (PCC) after an incubation period of 96 hours [14]. Japanese poly herbal drug Sho-saiko-to® shows an EC50 value of 353.5 µg/mL against human hepatocellular carcinoma (HCC-KIM) at 72 hour incubation [15], while Arbudhcure® shows an EC50 of 33.0 µg/mL against HeLa cells for 48 hours post exposure to the respective drug [16]. The current study demonstrates that LPG shows higher anti-proliferative activity on four different cancer cell lines investigated in the present study in comparison with aforementioned polyherbal drugs.

Measurement of LDH activity is another indicator of cell viability through evaluation of the cell membrane permeability. Previous studies suggest that LDH is a more reliable and accurate marker of cytotoxicity, since damaged cells is fragmented completely during the course of prolonged incubation with substances [25]. The intracellular LDH release to the medium is a measure of irreversible cell death. Xia (2007) [26] reported that there is a correlation between the direct involvement of LDH up regulation and subsequent induction of apoptosis, which is ideal for an effective anti cancer drug.

Fifty percent leakage of LDH to the media was observed at 26.9± 1.6 and 30.5 ±2.8μg/mL for RD and MCF-7 cells after 24 hour incubation respectively. After 48 hours of incubation with LPG the EC50 values further decreased and 50% LDH leakage arise at 8.2± 0.2 and 6.0± 0.2 μg/mL for RD and MCF-7 cells respectively. In contrast to RD and MCF-7 cells the CC1 cells showed 50% leakage of LDH at 159.3± 3.1 and 103.1 ±5.2μg/mL LPG concentrations after 24 and 48 hours respectively. These results indicate that the LPG is having minimal cytotoxic effect on control cells while it possesses a remarkable cytotoxicity on two cancer cell types studied.

Morphological alterations of RD, MCF-7 and CC1 cell lines upon exposure to the aqueous extract of the LPG revealed that even at low concentrations the morphology of the RD and MCF-7 cells changed into an irregular shape with extremely condensed nuclear contents, signs of membrane blebbing, suggesting an autophagic mechanism of cell death. The untreated cells (negative control)of RD and MCF-7 cell lines show cells with normal morphology (Spindle shape/ elongated cells) that adhered to the culture plate with no signs of cells death and growth disorders.

The positive control also shows the typical morphological changes which indicate the cellular damage in RD and MCF-7 cells. In contrast to the RD and MCF-7 cells the CC1 cells do not show any significant morphological changes even after 72 hour incubation with the extract.

Conclusions

The present study provides strong evidence for potent anti-cancer activity of the poly herbal drug “Le Pana Guliya” on RD and MCF-7 cells compared to controls. The brine shrimp and CC1 cells results suggest that the extract has minimum cytotoxicity towards the normal healthy cells. This calls for further studies on the active components and the mechanism of action involved in cytotoxicity of the chemotherapeutic action of LPG.

Acknowledement: The financial support by World Class University Research Grant No. AP/3/2012/CG/10is highly acknowledged.

Conflict of Interest: No conflict of interest between authors.

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DOI: 10.31038/CST.2017222

Abstract

Background: Breast cancer (BC) and its treatments lead to numerous side effects that affect a person’s life for years after treatment has ended. Research shows that regular exercise limits many of these side effects.However, less than 30% of BC survivors regularly exercise due to many barriers at both the patient and health care professional level. The purpose of this pilot trial is to assess the feasibility and effectiveness of conducting a novel KT intervention using exercise and self-management versus usual care among BC survivors.

Methods: Study Design: Pilot randomized controlled trial. Eligibility: Women older than 18 years who are currently undergoing chemotherapy treatment for BC. Intervention: The intervention group includes an 8-session multi-component intervention with a structured aerobic exercise program plus SM supervised by a physiotherapist. Randomization: Participants will be randomly allocated using a 1: 1 allocation ratio to receive the intervention of structured exercise plus SM program or usual care. Outcomes: The primary feasibility outcomes include recruitment rate, retention rate, and adherence rate. The secondary outcomes include exercise knowledge and behavior, HRQoL and resource utilization. Analysis: A blinded assessor will assess outcomes at baseline, post intervention, at 2- and 4-month follow up. Intervention feasibility and effectiveness will be assessed using descriptive statistics and analysis of covariance for continuous outcomes.

Discussion: This study aims to assess the feasibility of a novel KT intervention to close the current KT gap and increase exercise awareness for women with BC. This project will assess process and resource variables before implementation of a larger scale intervention. The overall project goal is to promote sustainable exercise behaviour to help manage the burden of BC.

Trial Registration: This trial was registered on ClinicalTrials.gov on March 21, 2017 (Identifier: NCT03087461).

Keywords:

Breast neoplasms, exercise, translational medical research, pilot study, rehabilitation

Introduction

Breast cancer (BC) and its treatments lead to numerous side effects that affect a person’s quality of life (QOL) for years after treatment has ended [1-5]. Research has shown that regular exercise limits many of these side effects and can prevent disease recurrence [5-10]. However, less than 30% of survivors participate in regular exercise [11-13]. Previous research conducted by our team has shown that over 80% of BC survivors in southwestern Ontario are unaware of the benefits of exercise and are not educated on the need to stay physically active [11], health professionals face many institutional, personal, and patient-related barriers to promoting exercise [14], and there is a need for novel knowledge translation (KT) strategies within cancer institutions that focus on easy-to-access exercise interventions and education by physiotherapists (PTs) [15].

Moreover, the societal burden of this disease is projected to increase substantially over the next two decades. The Canadian Cancer Society’s 2015 Statistics report [16] suggests that the number of new cancer cases in women will increase by 74% by the year 2032 due to the aging Canadian population. The number of new cases of BC is projected to increase by more than 10,000 in this same time period [16]. Fortunately, due to improved screening and treatment techniques, survival rates for BC are increasing [3]. However, physical and functional sequelae prevent survivors from returning to their activities associated with work, leisure, and domestic roles [3]. Exercise is an effective, safe, and cost-efficient way to manage this burden and return women to their pre-cancer activity levels. Therefore KT research is needed to determine how to best translate and integrate this research knowledge into clinical practice in order to elicit sustainable behaviour change for this population. Pilot work completed for this project has shown that in order to change clinical practice, implementation strategies using accessible exercise options and education are needed within the institution to maximize women’s engagement with exercise information provided and to partake in this behaviour [15]. Along with this, self-management (SM) programs have been shown to improve QOL and physical side effects in BC survivors, however, the implementation of these programs in clinical practice is scarce [17]. The current knowledge to practice gap in the field of BC rehabilitation shows that novel KT strategies are needed.

A pilot study is an, “investigation designed to test the feasibility of methods and procedures for later use on a large scale or to search for possible effects and associations that may be worth following up in a subsequent larger study” [18]. For this project, a pilot study is needed as the first step in order to assess process and resource variables before implementation of a large scale intervention [19]. Process variables include measuring recruitment rate, retention rate, and adherence rates to the intervention provided [19]. Resource variables include determining the centers willingness and capacity to house a specific intervention, equipment availability, intervention location, and budget concerns [19]. There is currently a lack of pilot trials for a novel KT intervention of this sort and therefore this pilot study will aid in shaping and guiding a larger, phase III trial.

Methods & materials

Study Purpose:

The purpose of this study is to determine whether KT strategies, focusing on accessible exercise locations and SM education by PTs using technology, are feasible and impact exercise knowledge and behaviour, QOL, and need for additional health care services among women with BC. Specifically, the objectives of this project are to: (1) Determine the feasibility (through recruitment, retention and adherence rates) of providing a complex KT intervention designed specifically for women with BC using technology, and (2) Determine preliminary estimates of effects of the KT intervention on levels of exercise knowledge and behaviour, health related quality of life and, resource utilization, among BC survivors over a four month period.

Study Design & Participants:

This study is a pilot randomized controlled trial. Eligible participants will include community-dwelling, English-speaking women, over 18 years, who are currently undergoing chemotherapy for Stage 1-3 BC and have been cleared by their oncologist to participate in moderate intensity aerobic exercise. Participants will be excluded from the study if they have another chronic disease, cognitive impairment or injury that prevents them from participating independently in moderate intensity exercise.

Recruitment:

Medical oncologists and Primary Care Nurses at the Juravinski Cancer Centre (JCC) will recognize possible participants for this study within their patient caseload. For those they think are eligible, the health care professional will briefly discuss the study with their patient and get consent for the patient to be contacted by a member of the research team. Possible participants will be contacted by phone to discuss eligibility and potential study enrollment. The Hamilton Integrated Research Board approved this study (reference #: 3124) in April 2017. All participants will provide written informed consent on the approved consent forms prior to enrollment in this study.

Intervention:

This pilot project will implement a multi-dimensional KT intervention including an exercise and SM program. Refer to Figure 1 for study flow chart.

The exercise intervention will involve an evidence-based moderate intensity aerobic exercise program, using recumbent bikes, delivered within the cancer institution. Results from participants in our focus group run during the pilot work for this project suggest that exercise programs should be delivered in the institution where women are waiting for their chemotherapy. Delivering the program in this environment will increase the accessibility of the services and we anticipate this approach will increase exercise awareness. Participants will take part in the 30-minute, moderate intensity (50-70%HRmax or 4-6/10 on Rate of Perceived Exertion scale) aerobic exercise program for 8 sessions. The intervention will be supervised by a PT educated in cancer rehabilitation and who has been trained in the specific protocol used and in working with women with BC.

The SM component will include educational modules created by a PT. Participants will view these 30 minute modules prior to each exercise intervention, over the same 8 sessions. Content provided within the program will include information on the benefits of exercise during and after BC treatment, safe exercise prescription, how to self-monitor exercise levels, action planning for specific exercise strategies, and precautions related to exercise and BC. Refer to Table 1 for details of SM content for each week of the program. A variety of tools will be used in the SM program, including a mobile app and e-health resources for BC. Having numerous sessions will allow the PT to provide consultation in respect to exercise adaptation, parameters, and programming in order to facilitate long-term exercise engagement and participation. Adherence and fidelity to the specified exercise and self-management protocol will be monitored through random observation by study investigators. (Figure 1, Table 1).

Table 1. Self-Management Content

Session	Content
1	Introductions. What are the side effects of treatment? Why do they happen? Benefits of exercise for women with breast cancer. Types of exercise and safety precautions during exercise (how to monitor BP, HR, RPE) What is self-management? How to participate in effective self-management. Self-management and breast cancer. Introduction to goal setting/action planning.
2	Review of previous week goal/action plan. The importance of posture for women with breast cancer (common postural issues, how to assess posture, how to ensure optimal posture). Relaxation and breathing techniques to manage anxiety and stress. Set goal/action plan for week.
3	Review of previous week goal/action plan. Appropriate exercise techniques to maintain/increase endurance: –                      Description of aerobic exercise –                      Types of aerobic exercise –                      Parameters for aerobic exercise Set goal/action plan for week.
4	Review of previous week goal/action plan Appropriate exercise techniques to maintain/increase strength –                      Description of strengthening exercise –                      Types of strengthening exercise –                      Parameters for strengthening exercise Set goal/action plan for week.
5	Review of previous week goal/action plan Other forms of Exercise (flexibility, yoga, Tia Chi, etc) Appropriate exercise techniques to maintain/increase flexibility: –                      Description of flexibility exercises –                      Types of flexibility exercises –                      Parameters for flexibility exercise Description of other forms of exercise: –                      Types of other forms of exercise –                      Parameters of other forms exercise Set goal/action plan for week.
6	Review of previous week goal/action plan. Self-monitoring physical activity levels: –                      Introduction to Breast Cancer Physio Guide (App) –                      Introduction to Stanford Action Planning App –                      Other techniques to monitor physical activity levels Set goal/action plan for week.
7	Review of goal/action plan. Communicating with others (family, health professionals) about exercise and physical activity. Available exercise programs in the community. How to move forward: how to evaluate progress. Set goal/action plan for week.
8	Review of goal/action plan. Summary of self-management program. How did you use self-management information? Questions/comments.

Outcomes:

Primary Outcomes: The feasibility and effectiveness of the KT intervention will be assessed using quantitative outcomes. The primary outcomes of feasibility variables will be assessed at baseline and post intervention, where applicable. Feasibility will be assessed by measuring recruitment (percentage (%) of eligible patients recruited), retention (% of consented patients who complete the intervention), and adherence rates (% of sessions attended) to the intervention).

Secondary Outcomes: Secondary effectiveness outcomes will be assessed at four time points: baseline, post intervention, and at 2 and 4 month follow up. At baseline, participants will be instructed on how to complete each self-report measure by an assessor blinded to participant group allocation. All post-intervention, 2 and 4 month follow up, assessments will be mailed to participants to complete and return to study investigators using pre-paid postage envelops. No identifiers will be used on these assessments and therefore assessors performing the analysis of data will be blinded to participant group allocation. Hard copies of the completed outcome measures will be stored in a locked filing cabinet only accessible by the study investigators at McMaster University and data entered into statistical analysis software will be stored on a password protected computer.

Level of exercise knowledge and behaviour will be assessed using a Theory of Planned Behaviour (TPB) [20] based questionnaire. The TPB has been used extensively to determine levels of intention and behaviour for various health behaviours, including exercise [21,22].

Quality of life will be measured using the FACT-B [23], a selfreport measure designed to assess multi-dimensional QOL specifically for women with BC.

Need for additional health care services will be measured using the EQ-5D [24] and a piloted self-report questionnaire assessing health care facility visits, doctor visits, procedures received, support services used, loss of work, and prescription medications used.

Sample Size:

Debate exists as to whether sample size calculations for pilot studies are necessary. Some authors suggest that no calculation is needed as long as the pilot study is large enough to provide useful information about the aspects that are being examined for feasibility [19]. However other authors suggest using a percentage of the sample required for a full study [25,26], or to use a confidence interval (CI) to establish feasibility [19,26]. For this project we have decided to calculate sample size based on the proportion of success of the primary outcome of feasibility (using estimates for adherence rates) [19,25,26].

Therefore, using a Z value from the standard normal distribution to reflect a 95% confidence interval (1.96), E as the desired margin of error (0.2), and p as the proportion of successes in the population (0.75-estimated adherence rates), the sample size for this pilot study will be at least 18 participants (9/group). With an expected drop out rate of 25%, based on previous exercise based literature with this population, the final sample size for this project should be 23 participants. In order to ensure an even number of individuals can be randomized to each group, this will be rounded up to a total of 24 participants (12/group).

Randomization-Sequence Generation:

Prior to participant randomizations, all eligible participants will complete the following forms: (a) patient information form, (b) Godin leisure time exercise questionnaire, (c) consent form, and (d) baseline measures. All participants will be informed verbally in person and in writing that they have equal chance of being randomized into the intervention or control group. They will not be made aware of the study hypotheses. Randomization to intervention or control group will be completed by a member of the research team who is independent of the intervention on a record by record basis using a computer software program (STATA/MP v14). This researcher will remain blind to the identity of each treatment group (by randomizing only to group A or B) during the randomization process. Participants may withdraw from the study at any time. Investigators may withdraw a participant from the research study if circumstances arise which warrant doing so (for example, safety).

Allocation Concealment & Implementation:

Allocation of participant randomization will be concealed in sequentially numbered, opaque, sealed envelops. The envelops will be opened sequentially by the researchers only after participant details have been written on the envelop by the researcher who completed randomization. If the participant is allocated to the intervention group, they will receive a phone call to organize details of the first intervention session (such as time and location).

Blinding:

Due to the nature of this knowledge translation study, participants and persons administering the intervention will not be blinded to group assignment. However, the assessor receiving the self-reported outcome measures will be blind to group allocation and will not be involved in running of the intervention. A researcher blinded to the group allocation of the participants will conduct all statistical analysis.

Statistical Methods:

Participant characteristics will be analyzed at baseline to ensure no significant differences exist between groups. Means and standard deviations (SD) will be used to report continuous variables and t-tests will be used to assess differences between the two groups for these variables. Frequencies will be used to report categorical variables and Pearsons X2 test will be used to assess differences between groups for these variables. All statistical analysis will be completed using STATA/ MP 14. Refer to Table 2 for a summary table of study objective and methodology.

Research Questions 1: Descriptive statistics will be used to measure feasibility (recruitment rate, retention rate, and adherence rates). Recruitment rates will be calculated by determining the percentage of eligible patients that were actually enrolled in the study. A recruitment log will be kept, detailing reasons for non-participation of eligible patients. Retention rate will be defined by calculating the percentage of enrolled patients who complete the intervention. Adherence rates will be calculated as a percentage of total sessions attended. Attendance will be tracked on the feasibility data collection sheet and reasons for non-participation on scheduled intervention days will be documented using an adherence log.

Table 2. Summary Table

Objective	Hypothesis	Outcome	Analysis Method
Determine the feasibility (through recruitment, retention and adherence rates) of providing a complex KT intervention including accessible exercise programs delivered within the cancer institution and a SM program designed specifically for women with BC using technology	Implementing and providing a complex KT intervention for women with BC is feasible (recruitment, retention, and adherence rates are 50%, 75%, and 75% respectively).	Recruitment Rate	Descriptive statistics (percentage (%) of eligible patients recruited). Recruitment logs will be kept, detailing reasons for non-participation of eligible patients.
		Retention Rate	Descriptive statistics (% of consented patients who complete the intervention).
		Adherence Rate	Descriptive statistics (% of sessions attended). Adherence rates will be tracked using the data collection sheet. Reasons for non-participation on scheduled intervention days will be documented using adherence logs.
Determine preliminary estimates of effects of the KT intervention of exercise plus SM versus usual care among BC survivors over a four-month period.	Compared to the control group, BC survivors participating in the KT intervention will have higher levels of exercise knowledge and behaviours and QOL, and less need for additional health care services.	Levels of exercise knowledge and behaviour (using a Theory of Planned Behaviour  based questionnaire)	An analysis of covariance will be used to determine within and between group differences. An intention to treat analysis will be used for these analyses.
		Health related quality of life (using the FACT-B)
		Resource utilization (using the EQ-5D and a piloted self-report questionnaire assessing health care facility visits, doctor visits, procedures received, support services used, loss of work, and prescription medications used)

Research Question 2: Effectiveness outcomes will be assessed at four time points: baseline, 12 weeks (post intervention), and at 2 and 4 month follow. An analysis of covariance will be used to determine within and between group differences. An intention to treat analysis will be used for these analyses. (Table 2)

Discussion

There is a small risk of participant injury during the exercise intervention. While it has been well documented that exercise is safe for this population if proper screening and precautions are followed, minor injuries have the potential to occur in any active intervention. In order to ensure safety, all participants will need medical clearance to participate in moderate intensity exercise from their medical oncologists and will be supervised by a physiotherapist during each session. Heart rate, blood pressure, rate of perceived exertion and oxygen saturation measurement tools will be present and used during each exercise session. While uncommon, all side effects secondary to exercise will be tracked using Exercise logs. Type, intensity, duration, and management of any side effect will be documented using these logs.

The findings of this KT pilot study will help to determine the feasibility and preliminary effectiveness of a novel implementation strategy. This project will inform a larger intervention trial which has the potential to change the way rehabilitation services are provided in clinical practice and impact all levels of BC prevention; secondary and tertiary prevention of treatment-related side effects, and primary prevention of disease recurrence through sustained behaviour change. The results from this pilot project should be interpreted with an understanding of the potential threats to the generalizability of the results. Specifically, the results of this pilot study will only be relevant to the implementation of a larger intervention at sites comparable to the JCC and will be specific to the unique characteristics of women with BC. As the intervention process and management is more extensive with larger numbers of participants, the researcher team will have to take into consideration additional time and resource needs when implement the larger scale project.

Trial status

Protocol Version Number: 3. Date: April 11, 2017. Approximate recruitment start date: June, 2017. Approximate recruitment end date: August, 2017. Any trial modifications will be updated in a timely manner on ClinicalTrials.gov and sent via email to appropriate parties.

Funding

The Hamilton Division of the Ontario Physiotherapy Association funds this project. This funding body has no role in the design of the study, collection, analysis or interpretation of data, or in writing of this or future manuscripts.

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DOI: 10.31038/CST.2017221

Abstract

The objective of this study was to obtain preliminary information on which non-occupational risk factors are responsible for the excess of lung cancer deaths seen in a cohort of workers in poultry slaughtering & processing plants, and to investigate whether established non-occupational risk factors for lung cancer mortality can be replicated. We conducted a pilot case-cohort study within a cohort of 43,904 poultry and non-poultry plant workers alive in 1990 and followed up to the end of 2003. Cases were 125 lung cancer deaths that occurred between 1990-2003 for whom interviews were successfully obtained. Controls (N=152) were derived from a random sample of the cohort in 1990. Statistical analysis was by logistic and Cox proportional hazards regression. The study successfully identified many of the established risk factors for lung cancer, and a few new ones were identified. The study demonstrates that valid case-cohort studies in this occupational group are feasible, and also successfully identified non-occupational risk factors that may need to be adjusted for in future planned large-scale studies of this occupational group.

Keywords

Chickens; meat; lung cancer; diet; non-occupational

Introduction

Workers in poultry slaughtering/processing plants have high exposures to oncogenic viruses that naturally infect and cause cancer in poultry. They also have exposures to chemical carcinogens at work. We initially performed three cohort mortality studies of 30,411 poultry workers and 16,405 non-poultry workers who were members of the United Food and Commercial Workers unions in the United States (N=46,816). An excess of lung cancer was consistently observed in the poultry workers [1-4]. We next conducted a pilot case-cohort study of lung cancer within a subset of 43,904 subjects of the 46,816 subjects that were alive in 1990, and followed them up to the end of 2003. The findings for occupational exposures have been published, [5] and the corresponding literature reviewed [6]. Here we present the results for non-occupational exposures. The study provides a unique opportunity for examining whether established non-occupational risk factors for lung cancer can be replicated in this group of workers who are among the lowest paid workers in industry. Also, if established risk factors for lung cancers are confirmed in this study, this will be strong evidence that future planned large full-scale studies to investigate lung cancer occurrence in this occupational group will give valid results.

Material & Methods

Cases were the first 125 lung cancer deaths out of 552 (23%) that occurred in the cohort between 1990 and 2003, for whom telephone interviews were obtained. Controls were similarly the first 152 subjects (10%) for whom telephone interviews were obtained. They originate from a random sample of 1,516 persons in the cohort that were alive in 1990. A telephone questionnaire was administered to the next-of-kin of study subjects if they were deceased (all cases, and 13% of controls) or to live control subjects themselves. Statistical analyses were conducted using both logistic regression and Cox proportional hazards regression methods as previously described [5].

Ethics: The study was approved by the University of North Texas Health Science Center’s Institutional Review Board

Results

The results are summarized in Tables 1&2.

Table 1. Poultry associated non-occupational exposures: Associations with lung cancer mortality, 1990-2003

			Adjusted Logistic Regression ORs†	Adjusted Cox Proportional HRs‡
	Cases (n=125)	Controls (n=152)	OR (95% CI)	HR (95% CI)
LIFESTYLE
Ever smoked tobacco	113	135	7.1 (2.8-18.0)	3.7 (1.8-7.4)
Mostly prepared own food	118	147	0.5 (0.2-1.1)	0.5 (0.3-0.9)
Ever drunk wine	113	146	0.4 (0.2-0.9)	0.6 (0.4-0.9)
Swam at least once a month for more than one year	115	147	0.4 (0.1-0.9)	0.5 (0.3-0.9)
Regularly performed any exercise	117	147	0.3 (0.1-0.5)	0.5 (0.3-0.7)
MEDICAL HISTORY
Ever treated with radiation therapy	106	141	16.6 (6.8-40.8)	5.3 (3.5-8.1)
Ever treated with radiation therapy for cancer	67	21	1.6 (0.5-5.4)	1.1 (0.6-1.9)
Ever treated with radiation therapy for skin/scalp conditions	68	20	1.0 (0.1-11.9)	1.5 (0.6-3.9)
Ever treated with radiation therapy for arthritis	70	20	0.5 (0.1-1.9)	0.8 (0.4-1.5)
Tuberculosis	102	144	9.0 (0.3-304.9)	2.2 (0.9-5.4)
Cirrhosis	112	147	7.8 (0.7-82.6)	2.0 (0.8-5.0)
Pancreatic inflammation	111	146	6.2 (0.7-55.5)	1.2 (0.5-2.8)
Infectious mononucleosis	109	147	3.6 (0.3-51.5)	1.7 (0.4- 6.9)
Cold sores on lip	114	145	0.6 (0.3-1.2)	0.6 (0.4-0.9)
Diabetes	114	148	0.3 (0.1-0.8)	0.4 (0.2-0.9)
Allergic to pollen	113	145	0.2 (0.1-0.5)	0.3 (0.2-0.7)
Allergy to drug medications	110	144	0.1 (0.0-0.4)	0.2 (0.1-0.5)
FOOD CONSUMPTION
Ate beef once every two weeks for most of life	114	144	14.9 (1.8-123.0)	9.6 (1.3-69.0)
Ate bacon every week for most of life	112	141	12.1 (4.2-35.1)	5.1 (2.4-11.2)
Ate uncooked fish/shellfish every week for most of life	111	142	2.0 (0.4-10.0)	1.4 (0.5-3.8)
Ate spicy foods every week for most of life	110	142	1.7 (0.9-3.4)	1.3 (0.9-1.9)
Ate chicken once every two weeks for most of life	116	144	1.7 (0.4-7.4)	1.5 (0.6-3.6)
Ate pork once every two weeks for most of life	114	144	1.6 (0.7-3.6)	1.4 (0.8-2.4)
Ate lamb once every two weeks for most of life	115	144	1.6 0.5-5.2)	1.1 (0.6-2.2)
Ate turkey once every two weeks for most of life	115	144	1.0 (0.5-1.9)	1.0 (0.7-1.5)
Ate fruits every week for most of life	108	143	0.8 (0.3-2.0)	0.9 (0.5-1.5)
Ate freshwater fish at least once a month	115	142	0.7 (0.4-1.4)	0.8 (0.5-1.2)
Ate cheese every week for most of life	109	142	0.7 (0.3-1.7)	0.8 (0.5-1.4)
Ate raw eggs at least once every two weeks for most of life	114	143	0.6 (0.1-3.9)	0.7 (0.3-2.1)
Ate veggies every week for most of life	111	143	0.5 (0.3-1.1)	0.6 (0.2-1.7)
Ate seafood once every two weeks for most of life	111	144	0.5 (0.2-0.9)	0.7 (0.4-1.0)
Ever ingested herbal leaves, drinks, medications at least once a week for >1yr	111	140	0.3 (0.1-0.9)	0.4 (0.2-1.0)
Ever adhered to a vegetarian diet for more than a year	114	143	0.2 (0.0-3.8)	0.3 (0.0-2.1)
Method of cooking meats
Ate meat fried at least once every two weeks for most of life	113	144	2.6 (1.0-6.6)	1.9 (1.0-3.8)
Ate meat salted at least once every two weeks for most of life	109	144	2.4 (1.2-4.8)	1.5 (1.0-2.2)
Ate meat raw at least once every two weeks for most of life	111	144	1.6 (0.2-11.3)	1.9 (0.7-5.3)
Ate meat barbequed at least once every two weeks for most of life	112	144	1.5 (0.8-2.9)	1.1 (0.7-1.6)
Ate meat smoked at least once every two weeks for most of life	110	144	1.3 (0.6-2.8)	1.0 (0.6-1.6)
DRUG USE
Use vitamins at least every week for more than a year	103	140	0.4 (0.2-0.8)	0.6 (0.4-1.0)
Ever had general anesthesia during surgery	108	140	0.4 (0.2-0.7)	0.6 (0.4-0.9)
Ever used hormone replacement therapy continuously for at least a year	24	57	0.2 (0.0-0.8)	0.3 (0.1-0.9)
FAMILY HISTORY OF CONDITIONS
Reported cancer in children	114	139	1.7 (0.5-6.4)	1.1 (0.6-1.9)
Reported cancer in parents	107	135	0.8 (0.4-1.6)	0.8 (0.5-1.2)
Reported cancer in spouse	113	139	0.8 (0.3-2.0)	1.1 (0.7-1.8)
IMMUNIZATIONS
Typhoid	46	127	2.1 (0.8-5.5)	1.4 (0.7-2.7)
Pneumococcal infections	48	127	1.7 (0.6-4.3)	1.4 (0.7-2.7)
Yellow fever	41	127	1.7 (0.5-5.6)	0.9 (0.4-2.2)
Ever received gamma globulin	56	131	1.6 (0.3-8.7)	1.8 (0.6-6.0)
Measles	42	124	1.5 (0.6-3.5)	1.0 (0.5-2.0)
Small pox	64	128	1.4 (0.6-3.1)	1.0 (0.6-1.7)
Diphtheria	55	120	1.3 (0.6-3.2)	1.0 (0.5-1.8)
Mumps	43	123	1.3 (0.5-3.0)	0.9 (0.5-1.7)
OTHER
Ever owned a cell phone	115	141	0.2 (0.1-0.4)	0.3 (0.1-0.6)

† Odds ratios (OR) were adjusted for smoking, gender, and age by the Logistic Regression Method
‡ Hazard ratios (HR) were adjusted for smoking, gender, and”> age by the Cox Proportional Hazard Method
@ For ever smoked tobacco, adjustment was for age and gender.

Table 2. Lung cancer mortality associated with frequent consumption of Beef and Bacon, adjusted for occupational exposures, meat preparation type, tobacco smoking, age, gender, and union site – (1990-2003)

	Ate a lot of Beef		Ate a lot of Bacon
	Case/control	HR (95% CI)	Case/control	HR (95% CI)
OCCUPATIONAL EXPOSURES
History of working in stockyard	56/99	—	56/98	2.7 (1.1-6.7)
Ever killed chickens at work	105/143	8.9 (1.2-64.6)	103/140	4.7 (2.1-10.3)
History of working in deli department	66/99	—	66/98	3.1 (1.3-7.4)
History of working in meat department	66/99	—	66/98	3.1 (1.3-7.1)
MEAT PREPARATION
Ate a lot of meat raw	109/144	9.8 (1.3-71.1)	108/141	4.8 (2.2-10.5)
Ate a lot of meat fried	111/144	8.9 (1.2-64.8)	110/141	4.7 (2.1-10.3)
Ate a lot of meat BBQ	111/144	9.7 (1.3-70.4)	109/141	5.0 (2.3-10.9)
Ate a lot of meat smoked	109/144	9.5 (1.3-69.0)	109/141	5.1 (2.3-11.2)
Ate a lot of meat salted	107/144	8.5 (1.2-62.0)	106/141	4.5 (2.0-10.1)

*HR = hazard ratio; CI = confidence interval; NOTE: Questionnaire defined a lot as “once every two weeks for most of life”

Discussion

With regard to lifestyle, established associations in the literature with cigarette smoking, drinking of wine and exercise were confirmed [7-9]. The significant association with radiation exposure may be real and consistent with well-documented reports of this association [7,10]; but it is also likely that it reflects using radiation treatment for the lung cancer, since no significant associations were seen for treating other conditions with radiation. The elevated but not statistically significant risk for tuberculosis seen is consistent with the findings of a comprehensive review [11]. Protective effects were seen for history of cold sores, diabetes, and allergy to pollen and medications. These are consistent with reports of allergies being protective risk factors for the disease [12-14].

Frequent consumption of beef and bacon were significantly associated with increased lung cancer risk, and the risks persisted after adjusting for occupational exposures that were associated with increased risks [5] Table 2. The risks also persisted irrespective of whether the beef or bacon was eaten raw, fried, barbecued, smoked, or salted – Table 2. The method of preparation of any of the different meats (beef, pork, poultry, etc.) was not an independent risk factor for lung cancer (data not shown) except for salted chicken, turkey and lamb for which the hazard ratios were 1.4 (95% CI, 1.0-2.2), 1.5 (95% CI, 1.0-2.2) and 1.5 (95% CI, 1.0-2.2), respectively. These associations with beef and bacon and salted meats are well documented in the literature [7,15-18], and mere consumption of these meats seem to be the most important factor.

Risk estimates for eating of seafood, ingestion of herbal leaves, drinks or medications, adherence to vegetarian diet, vitamin intake, hormone replacement therapy, and history of diabetes and general anesthesia were all below the null. These protective associations have been previously reported [7,19-21], except for history of diabetes and general anesthesia for which we have no explanation. The results for cellphone use are likely due to systematic bias resulting from cases (all deceased) dying during earlier periods when cellphone use was not in existence or infrequent while controls most of whom were alive, lived long enough into the more recent period of popular use; moreover, this reduced risk was also seen for the other cancers (liver, pancreas, brain) investigated [22,23].

Conclusion

The findings in this study are important for three reasons: 1) in spite of its small size, the study remarkably was able to confirm many of the reported non-occupational risk factors in the literature for lung cancer in this low-income population. This indicates that future planned case-cohort studies nested within occupational cohorts of workers in poultry slaughtering and processing plants that are assembled to investigate cancer occurrence in this industry, are feasible and capable of giving valid results. Such studies can provide valuable information on both occupational and non-occupational risk factors for various cancers. Secondly, this study has successfully identified non-occupational exposures that are risk factors for lung cancer in this specific population of poultry workers. These constitute some important potential confounding factors that may need to be adjusted for when investigating the role of occupational carcinogenic exposures (especially oncogenic viruses) in the occurrence of lung cancer in poultry slaughtering & processing plant workers in future full-scale large case-cohort studies. Finally, this small study indicates that established risk factors for lung cancer are equally applicable for this group of minimum-wage workers who belong to the lowest socioeconomic group.

Acknowledgements

Our appreciation and thanks go to the United Food & Commercial Workers International Union for their continuing support and collaboration over the years without which this research would not have been possible.

Funding Source

The study was funded by a grant 1 RO1 OH008071 from the National Institute for Occupational Safety & Health.

Competing interests: None

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