Berry 1 Emily Berry Biology Pre- Medicine with a Chemistry minor Dr. P Caffrey, Dr. Zuchelkowski, Dr. Aune, Dr. Fox Ovarian Cancer, Glutathione S-Transferase, Resveratrol Berry 2 Abstract Ovarian cancer cells have the ability to become resistant to different chemotherapy drugs, including one of the most commonly prescribed drugs Doxorubicin. This drug specifically is focused on during our research. Many previous research studies have investigated causes of ovarian cancer cell resistance, such as the protective enzyme glutathione S-transferase (GST). Studies have also been performed on methods of inhibiting this resistance, possibly through the use of resveratrol, a component found in grapes and red wine. However, some doses of resveratrol have been found to result in an increase in resistance. In order to further investigate the cell’s sensitivity or resistance, this experiment examined a range of doses of doxorubicin and resveratrol administered to ovarian cancer cells. The samples were later analyzed to see if they were preventing or promoting chemotherapy resistance. Doxorubicin and resveratrol pretreatment were administered to triplicate cultures of ovarian cancer cells, either together or separately, and were left in this pretreatment for two days. At this time, replicate dishes of the cells were tested for their GST activity. The GST activity data was collected from a spectrophotometer, measuring the amount of product that was produced via a colorimeteric reaction. We recognized that GST activity may be proportional to doxorubicin resistance in these pretreated ovarian cancer cells. Data was taken for control groups, resveratrol alone, doxorubicin alone, and a combination of doxorubicin and resveratrol treatments at various dosages. Questions aimed to be answered during this study were as follows: In cases where doxorubicin pretreatment results in resistance to doxorubicin, is it accompanied with increased GST levels or are they unrelated. Additionally, if resveratrol causes resistance to doxorubicin alone does it do so by increasing GST levels or through an alternative mechanism. In other words, can the Berry 3 varying degrees of doxorubicin resistance be caused by doxorubicin and/or resveratrol pretreatment. Also, will doxorubicin resistance reflect an increase in GST activity (ie. low GST activity relays low resistance, high GST activity relays high resistance). Further studies will be needed to confirm if resistance was formed by the ovarian cancer cells due to receiving the resveratrol and doxorubicin pretreatments. Index Terms—Ovarian Cancer Cells, Chemotherapy Resistance, Doxorubicin, Resveratrol, Glutathione Stransferase Berry 4 Introduction Ovarian cancer accounts for 5% of all deaths due to cancer in the United States, which in part is assumed to be due to its tendency to become resistant to multiple chemotherapy regimens (1). Ovarian cancer is the most life threatening gynecological cancer and continues to take women’s lives at an alarming rate. The common treatments of ovarian cancer include platinum-based drugs such as cisplatin, as well as doxorubicin which is an anthracyclin. Anthracyclins are also referred to as anthracycline topoisomerase inhibitors. This chemotherapy drug acts by embedding itself in between the base pairs of the DNA helix, therefore preventing the progression of replication and eventually halting gene expression (2). However, cancer cells are able to prevent doxorubicin from having an effect by removing it from the cell itself. The challenge of overcoming chemotherapy resistance is an ongoing battle and continues to become more pivotal in saving the lives of the many women affected by this deadly cancer. Ovarian tumors are more commonly prone to developing resistance to their chemotherapy over time, therefore ovarian cancer research for chemotherapy resistant cells is at the forefront of new treatment research. Although the use of platinum-based agents has increased the survival rates of patients, data supports that the majority who relapse after the first round of chemotherapy will eventually be overtaken by the resistant tumor cells, even when treated with other therapies (3). Thus far, the various mechanisms of resistance are not entirely understood. Of the mechanisms studied previously, the one specifically analyzed in this experiment is Glutathione S-transferase activity. Glutathione S-transferases (GSTs) are metabolic enzymes that in conjunction with their substrate, glutathione (GSH), are present Berry 5 throughout the body with the primary purpose of detoxifying foreign substances. The GST enzyme conjugates GSH to toxic compounds such as chemotherapy drugs. By attaching GSH to the drug, the cell recognizes them as foreign and harmful and is then able to remove the GST/GSH/drug complex from the cytoplasm through drug transporters, such as a multi-drug resistance associated protein (6). After being removed, the drug is incapable of harming the ovarian cancer cell and in turn, GST has begun to create resistance to the chemotherapy drug. Although the GST mechanism of resistance is the main focus of this study, other forms of resistance to chemotherapy have been found. Resistance may also be induced from p-glycoprotein, an efflux of drug from transporters, detoxifying enzyme inhibition, as well as altered gene expression of apoptotic signaling for tumor suppressors and proteins (7). Since GST has previously been observed to be affected by resveratrol, we chose to investigate the effect of resveratrol in combination with doxorubicin on GST activity. Most studies of drug resistance are performed on cells that have already developed resistant because they were previously exposed to a chemotherapy drug in the patient. These resistant cells are then compared to non-resistant cells from patients who never received chemotherapy. However, some research studies have produced methods of reproducing resistance over a few days in vitro, allowing researchers to observe the development of resistance as it occurs (4,5). Using these methods, researchers reported the increase in GST activity in ovarian cancer cells during the development of resistance to the drug cisplatin (5). These methods encourage the study of new treatments using natural compounds that could lower GST activity, such as resveratrol. Berry 6 Resveratrol (3,5,4’-Trihydroxystilbene) is a natural polyphenol, which is found in grapes and in red wine. Previous studies have shown that resveratrol may aide in inducing autophagocytosis, disrupting angiogenesis, and/or inhibiting insulin or seruminduced growth of ovarian cancer (8). Other data has shown that resveratrol may have an adverse effect and may ultimately create resistance, or have no effects towards the chemotherapy (9,10). One study also noted that resveratrol showed promising anti-tumor growth capabilities when in vitro, but did not show any positive results in an in vivo setting (11), which may be an indication that resveratrol’s effects are dependent on its environment. This factor will be the main focus throughout this study and will be investigated at multiple doses with doxorubicin, as well as by itself. The purpose of this study is to test the effect of resveratrol on doxorubicin resistance via GST activity in ovarian cancer cells. A range of doses of resveratrol alone, doxorubicin alone and the two in combination will be studied. This study investigates GST activity as a mechanism for the development of chemotherapy drug resistance. By identifying the enzyme activity in ovarian cancer cells following pretreatments intended to cause or prevent resistance, it may be possible to determine whether GST activity reflects the development of resistance to the drug doxorubicin. A spectrophotometer is used to measure the production of the conjugate product that is formed by the substrates 1-Chloro-2,4-dinitrobenzene (CDNB) and GSH by GST activity (12). This colorimetric enzyme reaction is monitored for the change of absorbance readings that occur from the reaction’s color change over time. The change in absorbance readings over a period of time, directly correlates to an increase in GST activity and provides data supporting there is drug resistance occurring at that time. Berry 7 Figure 1: Chemical structure of Doxorubicin chemotherapy drug. Figure 2: Resveratrol (3,5,4’-Trihydroxystilbene) chemical structure Materials and Methods General Procedure This study was conducted with a line of high grade serous ovarian cancer cells (A2780) purchased from AddexBio Technologies that are known to be initially sensitive to chemotherapy drugs and of low GST starting levels. These cells had been maintained in a nutrient medium of F12/DMEM plus 7% FBS from Life Technologies/ThermoFischer alone or with a pretreatment dose of resveratrol or doxorubicin at 37℃ and at 5% CO2. Replicate cultures were first pretreated with a range of doses of doxorubicin alone, resveratrol alone or a combination of the two. For each of these pretreatments, duplicate culture dishes were set aside for GST analysis. Cells of each culture and dosage were counted throughout each trial to determine the change of proliferation in response to treatments. GST Assay Procedure Berry 8 Cultures of cells were scraped off of the bottom of the plates using a sterile pipet and 1 ml PBS mix to dislodge the cells after excess growth medium was poured off into waste. Cells were placed in centrifuge tubes and centrifuged in an International Clinical Centrifuge at setting 4 for 5 minutes. Pellets were preserved and the supernatant was poured out as waste. The pellet was then resuspended in 1 ml of PBS homogenizing buffer (Life Technologies/ThermoFischer). If necessary, the solutions were frozen at 20℃ until ready to test. Cell samples, 1-Chloro-2,4-dinitrobenzene (CDNB), and glutathione (GSH) were consistently kept on ice when not being used. The GST activity assay measures the conjugation of 1-chloro, 2,4-dinitrobenzene (CDNB, Sigma Aldrich) with reduced glutathione (GSH, Sigma Aldrich). The reaction produces a dinitrophenyl thioether which is detected by a spectrophotometer at 340 nm. Based on the levels of GST, a specific amount of the reaction product is conjugated per minute. The assay cocktail formula for each of the cultures tested were as follows: 4.8 ml of PBS mix, 0.60 ml of CDNB, and 0.60 ml of GSH. For each of the individual cell samples from the various trials and dosages, 0.60 ml of their specific cell sample were placed into the assay cocktail for testing. The blank test tube received this assay cocktail and received 0.60 ml of PBS instead of a cell sample. The spectrophotometer was zeroed with a blank test tube at a wavelength of 340 nm and an absorbance of zero. In some cases, wavelengths were adjusted to compensate for spectrophotometer drift in its readings of the blank test tube. The change of absorbance over time was calculated by taking the final absorbance reading subtracted by the starting absorbance reading, shown in the equation below. The GST activity was also calculated by the following equation. Berry 9 ∆ absorbance=(A340 at Time 1) - (A340 at Time 2) GST activity (Units/cell)=(∆ absorbance)x(0.0096 µM-1/cm)×(0.3 ml) Statistics Means and standard deviations were calculated using Xcel. Statistical significance was calculated by an unpaired 2-tail t test. Asterisks will be used to indicate statistical significance compared to control GST activity for a P value less than 0.05. Results The effect of resveratrol, doxorubicin, or both in combination on GST activity was measured by the production of dinitrophenyl-thioether, resulting in a color change that was detected by a spectrophotometer at approximately 340 nm. In all cases, the numbers represent the change in absorbance over 60 seconds of three replicate cell cultures in each of the indicated treatment doses. There are not replicate readings of the same sample, only replicate readings of different samples with the same cells and doses. For each of the cell treatments, the cells were counted, extracts were made from the cell cultures and the extracts were tested for GST activity, as described in Materials and Methods. Control Cells The control cells were pretreated with only culture medium instead of either drug. The change in absorbance over 60 seconds showed an average of 0.012 change in absorbance with a standard deviation of 0.0067. This data can be found in Figures 3 and 4. The GST Berry 10 activity equation was used to calculate the GST activity for the control sample and found to be 7.32 x 10-12units/cell. This data can be found in Figure 5. An average GST activity value was calculated to be 6.45 x 10-12units/cell with a standard deviation of 3.95 x 1012. This data can be found in Figure 6. Resveratrol Pretreated Cells Two different doses of resveratrol were administered to the cells as a pretreatment. The average change of absorbance for the 11µm dose was .0023 with a standard deviation of 0.0137. The average change of absorbance for the 17µm dose was .0018 with a standard deviation of 0.004. This data can be found in Figures 3 and 4. Using the GST activity equation, the calculated GST activities, found in Figure 5, showed that cells with the 11µm dose GST activity was 1.07 x 10-11units/cell, and at the 17µm dose it was calculated to be 2.11 x 10-11units/cell. An average GST activity was also calculated for each dose. The 11µm dose had an average of 1.09 x 10-11units/cell with a standard deviation of 6.37 x 10-12. The 17µm dose had an average of 2.07 x 10-11units/cell with a standard deviation of 4.75 x 10-12. This data can be found in Figure 6. Using an unpaired 2 tail t test, the only statistically significant GST activity increase throughout the study was the 17µm dose of resveratrol, which is noted by an asterisk in Figure 6. Doxorubicin Pretreated Cells Three different doses of doxorubicin cells were administered to the cells before they were counted and tested for their GST activity. The average change in absorbance for the 2µm doxorubicin sample was 0.003 with a standard deviation of 0.0012, the 4µm sample was 0.005 with a standard deviation of 0.002, and the 8µm sample was 0.004 with a Berry 11 standard deviation of 0.003. This data can be found in Figures 3 and 4. The GST activity of each dosage was calculated using the GST activity equation and found to be 2.09 x 1012units/cell for the 2µm sample, 2.67 x 10-12units/cell for the 4µm sample, and 4.5 x 1012units/cell for the 8µm sample. This data can be found in Figure 5. An average GST activity was then calculated for each of the sample doses. The 2µm dose showed an average of 1.85 x 10-12units/cell with a standard deviation of 8.02 x 10-13. The 4µm dose showed an average of 2.67 x 10-12units/cell with a standard deviation of 1.41 x 1012. Lastly, the 8µm dose showed an average of 4.88 x 10-12units/cell with a standard deviation of 3.44 x 10-12. This data can be found in Figure 6. Doxorubicin Plus Resveratrol Pretreated Cells The combination of doxorubicin and resveratrol given to cells at different concentrations, were counted and tested for their GST activity by their change in absorbance over time. The average change in absorbance for each sample was as follows: 0.005 with a standard deviation of 0.002 for DoxRes 2/11µm sample, 0.010 with a standard deviation of 0.003 for DoxRes 4/11µm sample, 0.007 with a standard deviation of 0.004 for the DoxRes 4/17µm sample, and 0.010 with a standard deviation of 0.003 for the DoxRes 8/17µm sample. This data can be found on Figures 3 and 4. The GST of each dosage was calculated using the GST activity equation and was found to be as follows: 3.78 x 10-12units/cell for the DoxRes 2/11µm sample, 1.09 x 10-11units/cell for the DoxRes 4/11µm sample, 5.69 x 10-12units/cell for the DoxRes 4/17µm sample, and lastly 1.15 x 10-11units/cell for the DoxRes 8/17µm sample. This data can be found in Figure 5. An average GST activity was also calculated for each of the combined doses. The DoxRes 2/11µm sample showed an average of 3.53 x 10-12units/cell with a standard Berry 12 deviation of 1.57 x 10-12. The DoxRes 4/11µm sample showed an average of 1.09 x 1011units/cell with a standard deviation of 3.91 x 10-12. The DoxRes 4/17µm sample showed an average of 5.70 x 10-12units/cell with a standard deviation of 3.73 x 10-12. Finally, the DoxRes 8/17µm sample showed an average of 1.19 x 10-11units/cell with a standard deviation of 3.68 x 10-12. This data can be found in Figure 6. Change in absorbance at over 60 seconds Sample Control Res 11µm Res 17µm Dox 2 µm Dox 4 µm Dox 8 µm DoxRes 2/11 µm DoxRes 4/11 µm DoxRes 4/17 µm DoxRes 8/17 µm Culture Dish A 0.018 0.039 0.014 0.002 0.004 0.001 0.004 0.009 0.011 0.008 Culture Dish B 0.006 0.014 0.022 0.004 0.003 0.005 0.007 0.014 0.008 0.009 Culture Dish C 0.007 0.017 0.017 0.002 0.008 0.007 0.003 0.007 0.002 0.014 Figure 3: Change in absorbance at 340 nm over 60 seconds. The effect of resveratrol or doxorubicin, alone or in combination, on the GST-induced production of dinitrophenyl thioether, was detected by absorbance at 340 nm. The numbers in the three columns were produced by extracts from three replicate cell cultures in each of the indicated treatments. They are not replicate readings of the same sample. Figure 4: This figure shows average change in absorbance over 60 seconds for the triplicate culture dishes for each sample and dosage. Averages of the triplicate cultures Berry 13 were calculated as well as the standard deviation noted by the black bar on each data bar. Figure 5: This figure shows the calculated GST activity in each of the culture dishes tested for each sample using the GST activity equation noted in the methods and materials section. Different colors in the graph indicate the different culture dishes noted in the legend at the top of the graph. Figure 6: This figure shows the average GST activity per sample and as well as their standard deviations noted by the black bar on top of each data bar. * Res 17 µm was the only statistically significant sample found. Discussion Resveratrol The purpose of calculating GST activity for each cell sample and dose was to determine if GST activity increased due to the resveratrol, doxorubicin, or combination of the two. The increase in GST activity may represent an indication that resistance to the chemotherapy drug could be forming. These findings could be used in further studies to Berry 14 determine the cause of resistance to doxorubicin specifically. Significant GST Activity was noted for only resveratrol 17µm, the highest dose of resveratrol administered in this study. Other notable increases were found in doxorubicin and resveratrol combined doses as well, but were not statistically significant. Since the doxorubicin and resveratrol combined samples also showed small amounts of increased GST activity, it provoked questions to be asked about resveratrol since the doxorubicin samples showed little increase in activity on their own. This data may indicate that resveratrol has an activating effect on the GST enzyme even in the presence of doxorubicin. From the GST activity data, it can be hypothesized that resveratrol may have some inducing or activating effects on the GST enzyme and therefore consistently showed increases in GST activity in the samples it was present in. Although the high dose of resveratrol alone showed activating effects of GST, this may not be true at other doses. Although resveratrol has shown increased GST at high doses in this study, it cannot be stated that resveratrol itself will always increase GST levels. Lower doses of resveratrol must be administered to ovarian cancer cells alongside a doxorubicin chemotherapy treatment, and GST activity must then be analyzed in order to make a concluding statement about resveratrol’s effect on GST activity overall. Previous studies have shown that resveratrol may have inhibitory effects on drug resistance, while others show that resveratrol may actually aid in resistance to chemotherapy drugs such as doxorubicin and cisplatin (13). After analyzing our results, our data seems to show that resveratrol may indeed aid in the formation of resistance to the chemotherapy drug doxorubicin by ovarian cancer cells using a mechanism of increased GST levels. Berry 15 Doxorubicin The lowest GST activity was consistently found in the samples of doxorubicin alone. Interestingly, previous studies of ovarian cancer cells have been found to create resistance to doxorubicin at doses 2µm and 4µm consistently, therefore the data found in this study was surprising (14). The low GST activity in each of the doxorubicin samples may indicate that these ovarian cancer cells are not becoming resistant to doxorubicin alone. Another possibility could be if there is resistance then it must be by another mechanism other than GST activity. The mechanism of resistance to doxorubicin is not always clear, but in order to identify what resistance forming mechanism would be found in these samples a further study should be done on p-glycoprotein to investigate it as an alternate pathway of resistance that is common for doxorubicin (15). Other variable factors A factor that should be noted in this study is the type of ovarian cancer cell line used. Ovarian cancer cell line A2780 was purchased and used throughout the study due to its un-elevated starting GST levels (16). The low starting GST levels of this specific cell line were important, otherwise the increased GST activity from the resveratrol and other samples would not have been able to be collected and instead would have been masked by the cells starting GST levels. The A2780 cell line comes from an ovarian endometroid adenocarcinoma patient’s untreated tumour. This line specifically is commonly used for research as a model to test for possible methods of treatment for ovarian cancer. A2780 is the parent line to the cisplatin resistant cell line A2780 cis (Sigma catalogue no. 93112517) and the adriamycin (doxorubicin) resistant cell line A2780 ADR (Sigma catalogue no. 93112520) (17). Berry 16 Another factor that should be noted in this study is resveratrol’s capability of increasing GST levels of healthy individual’s when given supplements. According to a study found, resveratrol has been given an increased amount of attention over the past two decades because of its abilities to inhibit cancer initiation, promotion, and progression in preclinical models. Noting these health benefits, researchers wanted to use healthy participants in a study to be given supplements of resveratrol to compare its effects on cancer cells to healthy patient cells. Therefore, the polyphenol was studied for its effects and was found to increase GST levels by 20% in human keratinocyte cells. These findings coincide with the results found in this study, notwithstanding the fact that our cells were ovarian cancer cells (18). Lastly, since resveratrol seemed to consistently show increased GST levels, it may provoke the question: should cancer patients be advised to stay away from grapes and red wine. during their chemotherapy treatment. After calculating the dose of resveratrol in a single grape, we found that one grape is approximately equal to 2000µg of resveratrol per grape. The dose of resveratrol in this study was only 17µm but when consuming resveratrol in grapes and wine, they must first be gestated and digested before having the possibility of entering into the blood stream. At this point, the amount of resveratrol left over would be little to none. Even though these amounts are overall very minuscule, in the future it may still be advised that chemotherapy receiving patients stay away from red wines and grapes during their treatment as a precaution. Conclusion The samples of resveratrol alone showed the highest increase in GST activity alongside the doxorubicin and resveratrol combined samples. treatment or not. The Berry 17 change of absorbance and GST activity can only show whether or not GST levels are increasing, decreasing, or staying the same. Even though increased GST activity correlates with ovarian cancer cell resistance to doxorubicin, we cannot be certain that this mechanism of resistance is exclusively creating resistance at this time. The other mechanisms of resistance may prove to be as responsible for resistance as GST activity in this study. Further studies of other resistance mechanisms, such as p-gylcoprotein, should be investigated to determine its effect with resveratrol and doxorubicin resistance. Acknowledgements Funding provided by the Department of Biological and Environmental Sciences at California University of Pennsylvania. I would like to thank Dr. P. Caffrey for her assistance and guidance throughout the project. Berry 18 Works Cited (1) Sherman-Baust C, Becker K, Wood Iii W, Zhang Y, Morin P. Gene expression and pathway analysis of ovarian cancer cells selected for resistance to cisplatin, paclitaxel, or doxorubicin. Journal Of Ovarian Research [serial online]. December 5, 2011;4(1):21. Available from: MEDLINE Complete, Ipswich, MA. Accessed April 10, 2018. (2) Doxorubicin. National Center for Biotechnology Information. PubChem Compound Database. https://pubchem.ncbi.nlm.nih.gov/compound/doxorubicin#section=Top. Accessed April 11, 2018. (3) Borley J, Brown R. Epigenetic mechanisms and therapeutic targets of chemotherapy resistance in epithelial ovarian cancer. Annals Of Medicine[serial online]. 2015;47(5):359-369. Available from: MEDLINE Complete, Ipswich, MA. Accessed April 9, 2018. (4) Caffrey, P., Frenkel, G. (2013). Design of a model for the development of cisplatin resistance in H526 human small cell lung cancer xenografts. Abstract #985, Proceedings of the Annual Meeting of the AACR, Washington, DC, April 7, 2013. (5) Caffrey, PB and Kenneth Marks. Prevention of the development of GST-dependent cisplatin resistance in a model of human H526 small cell lung cancer (SCLC) by ethacrynic acid and selenite. Am. Soc. of Cell Biol. Abstracts, Philadelphia, PA. 2014. (6) Sheratt PJ, Hayes JD. 9 Glutathione S-transferases. In: Enzyme Systems That Metabolize Drugs and Other Xenobiotics. John Wiley & Sons, Inc; 2012:323-345. (7) Pereira D, Assis J, Gomes M, Nogueira A, Medeiros R. Improvement of a predictive model in ovarian cancer patients submitted to platinum-based chemotherapy: implications of a GST activity profile. European Journal Of Clinical Pharmacology [serial online]. May 2016;72(5):545-553. Available from: MEDLINE Complete, Ipswich, MA. Accessed April 9, 2018. (8) Lee M-H, Choi BY, Kundu JK, Shin YK, Na H-K, Surh Y-J. Resveratrol Suppresses Growth of Human Ovarian Cancer Cells in Culture and in a Murine Xenograft Model: Eukaryotic Elongation Factor 1A2 as a Potential Target. Cancer Research. 2009;69(18):7449-7458. doi:10.1158/0008-5472.can-09-1266. (9) Bocetti, J. Lincoski, B. and P. B. Caffrey “The effect of Grape Extracts and their component, Resveratrol, on the population growth of A2780 human ovarian tumor cells in culture” at the Annual Meeting of the PA Academy of Science in Doylestown, PA, April 1, 2016. (10) Marchitello, M. “Chemotherapy Cocktail: The Effects of Cisplatin and Resveratrol on Small Cell Lung Cancer” at the 2017 Strike a Spark Conference, California University of PA, April 19, 2017. Berry 19 (11) Stakleff KS, Sloan T, Blanco D, Marcanthony S, Booth TD, Bishayee A. Resveratrol Exerts Differential Effects in Vitro and in Vivo against Ovarian Cancer Cells. Asian Pacific Journal of Cancer Prevention. 2012;13(4):1333-1340. doi:10.7314/apjcp.2012.13.4.1333. (12) Drozd E, Krzysztoń-Russjan J, Marczewska J, et al. Up-regulation of glutathionerelated genes, enzyme activities and transport proteins in human cervical cancer cells treated with doxorubicin. Biomedicine & Pharmacotherapy. 2016;83:397-406. doi:10.1016/j.biopha.2016.06.051. (13) Chow H-HS, Garland L, Hsu C-H, et al. Abstract A57: High-dose resveratrol modulates drug and carcinogen metabolizing enzymes in a healthy volunteer study. Cancer Prevention Research. 2010;3(1 Supplement). doi:10.1158/1940-6207.prev-09a57. (14) Gasper L, Caffrey PB. The study of Ovarian Cancer Cell Resistance to Doxorubicin and Cisplatin . 2016. (15) Khaleel SA, Al-Abd AM, Ali AA, Abdel-Naim AB. Didox and resveratrol sensitize colorectal cancer cells to doxorubicin via activating apoptosis and ameliorating Pglycoprotein activity.Scientific Reports. 2016;6(1). doi:10.1038/srep36855. (16) Godwin AK, Meister A, Odwyer PJ, Huang CS, Hamilton TC, Anderson ME. High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis. Proceedings of the National Academy of Sciences. 1992;89(7):3070-3074. doi:10.1073/pnas.89.7.3070. (17) A2780 Cell Line human CB_93112519. Sigma-Aldrich. https://www.sigmaaldrich.com/catalog/product/sigma/cb_93112519?lang=en®ion=US. Accessed April 19, 2018. (18) Guthrie AR, Chow H-HS, Martinez JA. Effects of resveratrol on drug- and carcinogen-metabolizing enzymes, implications for cancer prevention. Pharmacology Research & Perspectives. 2017;5(1). doi:10.1002/prp2.294.