here - Wake Forest College

Examining the combinatorial effects of plant-derived substances on lifespan and tumor growth of Sarcoma 180 cells in mice Dineth Bandarage, Andy Vuong

1

Introduction The basis of this project stemmed from the book Anticancer: A New Way Of Life by David Servan-Schreiber. In the book, Dr. Servan-Schreiber describes multiple different mechanisms in which certain natural or plant-derived substances combat cancer cells. While there is a moderate amount of research on the effectiveness of these natural remedies independent of one another, there is little to no research on the effects of their combination. The three compounds we chose to analyze were curcumin, epigallocatechin gallate, and proanthocyanidins of grape seeds. Curcumin is a natural phenol found in the root of the plant, turmeric. According to studies, curcumin inhibits the spread and survival of almost all types of tumor cells. Evidence suggests that curcumin activates the programmed cell death process of cancer cells, which leads to apoptosis. Cancer cell death is also mediated by curcumin affecting the pathway in which a tumor proliferates. Curcumin inhibits the growth of tumors by binding to as many as 33 different proteins, all of which modulate the growth factor, cell proliferation, and apoptosis of cancer cells [1]. In one study, a daily dosage of 20mg/kg of curcumin led to a 52% decrease in the tumor growth of pancreatic cancer cells when compared to the control group [2]. Epigallocatechin Gallate is an anti-oxidant polyphenol found mostly in green tea. It may have health benefits as a nutritional supplement for cancer, atherosclerosis, blood sugar control, and neurodegenerative diseases. EGCG induces apoptosis in human cancer cell lines; it thwarts the growth of several cancer cells including breast, prostate, lungs, ovaries, and liver. EGCG has also been found to inhibit telomerase and DNA methyltransferase, two enzymes involved in cancer gene expression and cellular immortality. EGCG is also known to bind and inhibit the anti-apoptotic protein Bcl-xl which has been implicated in both cancer cell and normal cell survival. Strong evidence continues to emerge for the direct synergistic benefit of EGCG with common cancer therapeutics. A daily 15mg/kg dose of EGCG has been shown to cause 43% inhibition of S180 cells in mice when given for seven days prior to the S180 injection and for seven days post injection when compared to the control group [3]. Proanthocyanidins are found in various fruits, vegetables, nuts, seeds, flowers and bark. Specifically, proanthocyanidins from grape seeds have been shown to have a variety of benefits including anti-tumor, anti-oxidative, and anti-inflammatory effects [4]. A daily 10mg/kg dose of PA from grape seeds has been shown to cause 37.75% inhibition of S180 cells in mice over a 10 day period when compared to the control group [4]. Grape seed extract has also been shown to inhibit human prostate tumor growth and angiogenesis [5]. Despite the plethora of research regarding the individual effects of each plant-based compound, current research does not examine their interactions or their influence on lifespan. Realistically, people do not only consume turmeric, grape seeds, or grape seeds individually, but rather consume them together. Consequently, this project aims to examine the interactions between curcumin, epigallocatechin gallate, and proanthocyanidins in order to determine whether the compounds interact synergistically or antagonistically as measured by tumor growth and lifespan. Materials Healthy female BALB/c mice, 17-18g, 6-8 weeks, were purchased from Charles River Laboratories. Mice with Sarcoma 180 were provided by the Department of Pathology, Wake Forest University School of Medicine. Curcumin (molecular formula C21H20O6, molecular weight 348.4g/mol, purity ≥ 95%) were purchased from Fischer Scientific. 2



Epigallocatechin Gallate (molecular formula C22H18O11, molecular weight 458.4g/mol, purity ≥ 98%) was purchased from Cayman Chem. Grape seed extract proanthocyanidins (purity ≥ 95%) were purchased from HerbstoreUSA. Hybri-Max Dimethyl sulfoxide (molecular formula (CH3)2SO, molecular weight 78.13g/mol, purity ≥ 99.7%) was purchased from Sigma-Aldrich. Ethylenediaminetetraacetic acid disodium salt dehydrate ACS reagent (molecular formula C10H14N2Na2O8 · 2H2O, molecular weight 372.24 g/mol, purity ≥ 99.0%) was purchased from Sigma-Aldrich. L-ascorbic acid (molecular formula C6H8O6, molecular weight 176.12 g/mol, purity ≥ 99.0%) was purchased from Sigma-Aldrich. Methods General procedure. Mice were acclimated for 13 days and inoculated with Sarcoma 180 on the 14th day of acclimation. Mice were inoculated in the intraperitoneal cavity with 1mL of 5 x 106 S180 cells/mL diluted in phosphate buffered saline, obtained from the peritoneal fluid of a mouse with sarcoma at day 0. Starting the following day, mice were injected intraperitoneally with a 1mL solution of their respective treatments daily. DMSO was used to ensure the curcumin was dissolved and homogenous throughout the solution. All solutions were made fresh daily and mixed by vortex and sonication to ensure solutions were homogenous. DMSO was necessary in order to dissolve curcumin and thus was added to all other solutions except the saline control to ensure accurate comparisons. Mice were euthanized by cervical dislocation once a humane endpoint was reached. Saline Control. Mice (n=3) were injected intraperitoneally daily with 1ml of 0.9% saline solution (by weight). Saline with DMSO Control. Mice (n=3) were injected intraperitoneally daily with a 1mL solution of 0.9% saline (by weight) and 2.86% DMSO (by volume). Curcumin. Curcumin was dissolved in 1mL DMSO then mixed by vortex and sonication. The necessary amount of dissolved curcumin and additional DMSO was added to create a solution of 0.9% saline, 2.86% DMSO, and 20mg/kg curcumin based on cage weight averages. Mice (n=5) then received 1mL of the solution intraperitoneally, daily. Epigallocatechin Gallate. A standard stock solution of EGCG (2.86g/mL) was prepared in 0.2% ascorbic acid, 0.005% EDTA and stored at -80oC for analytical and cost reduction purposes [7]. The stock was defrosted daily and the necessary amount stock EGCG was added to create a solution of 0.9% saline, 2.86% DMSO, and 15mg/kg EGCG based on cage weight averages. Mice (n=5) then received 1mL of the solution intraperitoneally, daily. Proanthocyanidins. Proanthocyanidins were dissolved in 1mL DMSO, 9mL distilled water then mixed by vortex and sonication. The necessary amount of dissolved proanthocyanidins and additional DMSO was added to create a solution of 0.9% saline, 2.86% DMSO, and 10mg/kg proanthocyanidins based on cage weight averages. Mice (n=5) then received 1mL of the solution intraperitoneally, daily. Curcumin and Epigallocatechin Gallate. Curcumin and EGCG were prepared as described above. The necessary amount of curcumin, EGCG, and additional DMSO were added to create a solution of 0.9% saline, 2.86% DMSO, 20mg/kg curcumin, and 15mg/kg EGCG based on cage weight averages. Mice (n=5) then received 1mL of the solution intraperitoneally, daily. Curcumin and Proanthocyanidins. Curcumin and proanthocyanidins were prepared as described above. The necessary amount of curcumin, proanthocyanidins, and additional DMSO were added to create a solution of 0.9% saline, 2.86% DMSO, 20mg/kg curcumin, and 10mg/kg 3

proanthocyanidins based on cage weight averages. Mice (n=5) then received 1mL of the solution intraperitoneally, daily. Epigallocatechin Gallate and Proanthocyanidins. EGCG and proanthocyanidins were prepared as described above. The necessary amount of EGCG, proanthocyanidins, and additional DMSO were added to create a solution of 0.9% saline, 2.86% DMSO, 15mg/kg EGCG and 10mg/kg proanthocyanidins based on cage weight averages. Mice (n=5) then received 1mL of the solution intraperitoneally, daily. Curcumin, Epigallocatechin Gallate and Proanthocyanidins. Curcumin, EGCG, and proanthocyanidins were prepared as described above. The necessary amount of curcumin, EGCG, proanthocyanidins, and additional DMSO were added to create a solution of 0.9% saline, 2.86% DMSO, 20mg/kg curcumin, 15mg/kg EGCG and 10mg/kg proanthocyanidins based on cage weight averages. Mice (n=5) then received 1mL of the solution intraperitoneally, daily.

Results The mean lifespan of saline control (n=3) was 13.7 days, while the mean lifespan of the DMSO control (n=3) was 12.3 days. Since there was no significant difference between the saline control and the DMSO control, p=.173, we will use our DMSO control as our baseline for all subsequent comparisons. The curcumin group had one outlier which lived for 69 days. Excluding this outlier, the curcumin group (n=4) had a mean lifespan of 19.0 days, which is a 54.1% increase in lifespan over the DMSO control (p< .001). The proanthocyanidins group also had one outlier, which lived for 56 days. Excluding this outlier, the proanthocyanidins group (n=4) had a mean lifespan of 16.25 days, which is a 31.8% increase in lifespan over the DMSO control (p= .069). The EGCG group (n=5) had a mean lifespan of 13.2 days, which is a 7.0% increase in lifespan over the DMSO control (p= .291). The combination of curcumin and proanthocyanidins group (n=5) had a mean lifespan of 14.2 days, which is a 15.4% increase in lifespan over the DMSO control (p= .393). Comparatively, the combination of curcumin and proanthocyanidins group had a 25.3% decrease in lifespan (p= .061) compared to curcumin individually and a 12.6% decrease in lifespan (p= .071) compared to proanthocyanidins individually. The combination of EGCG and proanthocyanidins group had one outlier that lived for 28 days since it lost most of its ascites after an injection. Excluding this outlier, the combination of EGCG and proanthocyanidins group (n=4) had a mean lifespan of 14.0 days, which is a 13.5% increase over the DMSO control (p= .398). Comparatively, the combination of EGCG and proanthocyanidins group had a 6.1% increase in lifespan (p= .681) compared to EGCG individually and a 13.8% decrease in lifespan (p= .349) compared to proanthocyanidins individually. The combination of curcumin and EGCG (n=5) has shown much more promising results. As of day 42 after inoculation (9/10/13), only two mice died, while the other three did show any signs of cancer growth. So far, this is a 60% survival rate of at least a 200% increase in lifespan, however, our study is not over and thus, we cannot provide further statistics regarding our results. Similarly, the combination of curcumin, EGCG and proanthocyanidins (n=5) has shown extremely promising results. As of day 42 after inoculation (9/10/13), only one mouse died, while the other four signs no signs of cancer growth. So far, this is an 80% survival rate of at

4

least a 200% increase in lifespan. Again, since our study has not been completed, we are unable to provide further statistics regarding this group. A summary of all the data regarding average lifespan are shown in Figure 1 and Figure 2. We also measured tumor growth by change in weight of the mice for the first twelve days after inoculation as shown in Figure 3. Figure 4 demonstrates this same concept, as the picture of the mice with the weight closest to the average is shown after 12 days after inoculation. Discussion: As described in the results above, the only significant increase in life span of the compounds individually was curcumin. Although, proanthocyanidins or EGCG may have a significant effect on lifespan, we needed a larger sample size in both the DMSO control and treatment groups for an adequate comparison. Although the combination of curcumin and proanthocyanidins group provides an increase in lifespan relative to the control, the data suggests that there is an antagonistic effect between the proanthocyanidins and curcumin since the mean lifespan of the combination is lower than each compound individually. Likewise, the combination of EGCG and proanthocyanidins group also provides an increase in lifespan relative to the control, but the data suggests that there may be an antagonistic effect between EGCG and the proanthocyanidins, since the mean lifespan of the combination is lower than the proanthocyanidins group, but slightly greater than the EGCG group. Despite these interesting results, the data is not significant, but warrants further studies regarding the potential antagonistic effects. Surprisingly, we found that that the combination of curcumin and EGCG, and the combination of all three compounds (i.e., curcumin, EGCG, and proanthocyanidins) potentially have synergistic effects. So far, the mean lifespan of both groups are significantly greater than each of the compounds individually. As of day 42 (9/10/13) there were no signs of tumor growth for the remaining three mice in the curcumin and EGCG group and for the remaining four mice in the combination of three group. After discussing the astounding results with Dr. Cui, we decided to test whether the mice were actually cured or the treatment was stunting the growth of the cancer cells or keeping the cancer dormant. Consequently, we decided to stop injections on day 43 (9/11/13), at which point mice had been injected for six weeks with their respective treatments. Figure 3 shows that only the combination of all three compounds and the combination of curcumin and EGCG maintain a relatively low change in weight since inoculation, indicating that the tumor is not growing as fast as the other groups. We only show the first twelve days in this figure as this is a critical period in determining whether the tumor has grown or not since most mice enter a moribund state between days 12-18. Again, the antagonistic effects between proanthocyanidins and curcumin can be seen very clearly in this figure. Figure 4 shows images of the mice of each group on day 12 whose weight was closest to the average, in which the tumor is visible to the naked eye. The further research regarding the combinatorial effects of natural substances are important, especially, as the combination of proanthocyanidins and curcumin group and the combination of all three compounds have indicated. Further research could validate the antagonistic effects between curcumin and proanthocyanidins, which could influence the diet of many cancer patients around the world to avoid eating the two together. Conversely, with the conclusion of our experiment or further research, cancer patients who choose not to go through chemotherapy could decide to consume the combination of proanthocyanidins, Epigallocatechin 5

Gallate, and curcumin as an alternative treatment. This type of combinatorial research also better reflects the diet of real world cancer patients than studies that compare the effects of each compound individually. Most patients do not consume one compound individually, but rather consume them in combinations as they think they are doing as much they can to fight cancer.

Figure Legend:

Average Lifespan per Treatment E+P

Treatment

C+P EGCG Proanthocyanidins Curcumin DMSO Control Saline Control

0

5

10

15

20

25

Time (days) Figure 1. Graph showing the effect of each treatment group on the average lifespan of the mice. Outliers of curcumin (69 days) and the proanthocyanidins (56 days) have been excluded. Additionally, the combination of curcumin and EGCG group and the combination of all three compounds group are not shown since our experiment is incomplete. Error bars represent ± 1 SD. * represents significant difference from the DMSO control.

6

Kaplan-Meier Survival Curve 100 90

KM Survival %

80

70 60 50 40 30 20 10 0

0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69

Time after injection of Sarcoma 180 cells (days) Saline Control Proanthocyanidins E+P

DMSO Control EGCG C+E

Curcumin C+P C+E+P

Figure 2. Kaplan-Meier survival curve demonstrating the effects of each treatment on lifespan. Data has been updated as of day 46 (9/14/13), however treatment for C+E and C+E+P was ceased as of 9/11/13 or day 43.

7

Average change in mice weights after innoculation (grams)

Measuring Tumor Growth by Change in Weight 8 7 6

5 4 3 2 1 0 -1 0

1

2

3

4

5

6

7

8

9

10

11

12

Time after injection of Sarcoma 180 cells (days) Saline Control

DMSO Control

Curcumin

Proanthocyanidin

EGCG

C+P

E+P

C+E+P

C+E

Figure 3. Graph showing the effects of each compound on change in weight. Since tumor growth cannot be measured without euthanizing the mice, we defined the change or increase in weight from day zero as due to tumor growth. Cage weight averages were taken daily and reported as above. As shown by the graph, the combination of curcumin and EGCG, and the combination of curcumin, EGCG, and proanthocyanidins maintains a relatively stable weight over the first twelve days after inoculation. The graph was chosen to only display the first twelve days as most mice enter a moribund state between days 12-18, thus altering the cage weight averages. One mice from the EGCG group died on day 11, which explains its slight drop in average weight between day 11 and day 12.

8

Figure 4. Images displaying the tumor growth twelve days after inoculation of S180 cells. Mice shown were selected from each treatment group based on which was closest to the mean weight of the cage. Each image says day 26, however this day count includes the 13 day acclimation period and thus the images represent 12 days after initial inoculation of five million sarcoma 180 cells. The larger and darkened bellies of the mice represent tumor growth. Acknowledgements: We would like to thank the Atlantic Coast Conference Inter-institutional Academic Collaborative and Dr. Caryl Guth for funding our project. We would also like to especially thank Dr. Zheng Cui for his funding and continual guidance throughout the project.

9

References: [1] Jayaraj Ravindran, Sahdeo Prasad, Bharat B. Aggarwal, Curcumin and Cancer Cells: How Many Ways Can Curry Kill Tumor Cells Selectively?, AAPS J. 2009 September; 11(3): 495– 510. Published online 2009 July 10. doi: 10.1208/s12248-009-9128-x. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2758121/) [2] Claire M. Mach, Lata Mathew, Scott A. Mosley, Razelle Kurzrock, Judith A. Smith. Determination of minimum effective dose and optimal dosing schedule for liposomal curcumin in a xenograft human pancreatic cancer model. Anticancer Res. 2009 June; 29(6): 1895–1899. (http://ar.iiarjournals.org/content/29/6/1895.long) [3] Manna S, Banerjee S, Mukherjee S, Das S, Panda CK. Epigallocatechin gallate induced apoptosis in Sarcoma180 cells in vivo: mediated by p53 pathway and inhibition in U1B, U4-U6 UsnRNAs expression. Apoptosis. 2006 Dec;11(12):2267-76. (http://www.ncbi.nlm.nih.gov/pubmed/17041754) [4] Xiao-Yu Zhang, Wen-Guang Li, Yong-Jie Wu, Tian-Zhen Zheng, Wei Li, Song-Yi Qu, NaiFa Liu, Proanthocyanidin from grape seeds potentiates anti-tumor activity of doxorubicin via immunomodulatory mechanism, International Immunopharmacology, Volume 5, Issues 7–8, July 2005, Pages 1247-1257, ISSN 1567-5769, 10.1016/j.intimp.2005.03.004. (http://www.sciencedirect.com/science/article/pii/S1567576905000743) [5] Singh, R. P., Tyagi, A. K., Dhanalakshmi, S., Agarwal, R. and Agarwal, C. (2004), Grape seed extract inhibits advanced human prostate tumor growth and angiogenesis and upregulates insulin-like growth factor binding protein-3. Int. J. Cancer, 108: 733–740. doi: 10.1002/ijc.11620. (http://onlinelibrary.wiley.com/doi/10.1002/ijc.11620/abstract) [6] Dong-li ZHAO, Ming-zhong LI, Shu-wen WANG, Ju-zhan SHANG, Experimental study on the anticancer effect of curcumin on mouse of S180 in vivo, Journal of Medical Colleges of PLA, Volume 22, Issue 2, April 2007, Pages 77-81, ISSN 1000-1948, 10.1016/S10001948(07)60017-3.(http://www.sciencedirect.com/science/article/pii/S1000194807600173) [7] Lambert, J. D. et al. Dose-dependent levels of epigallocatechin-3-gallate in human colon cancer cells and mouse plasma and tissues. Drug Metab. Dispos. 34, 8–11 (2006). (http://dmd.aspetjournals.org/content/34/1/8.long?related-urls=yes&legid=dmd;34/1/8) A note to the ACCIAC: Due to the inherent nature of research and the need to adapt to a new laboratory setting, our summer research project was not completed within its expected time frame. In regards to our experiment, we found it necessary to extend the time frame from which we kept our mice alive. While we had originally planned to euthanize mice within 2 weeks of injecting Sarcoma 180 cells to measure tumor size growth, after talking to Dr. Cui, we changed our procedure so that we would allow each mouse to live until it reached a moribund state. By doing this, we offered a new statistical set of data that most other scientific papers had not offered. By allowing the mice to live for longer than 2 weeks, we were able to compare average survival times due to the different treatments. As it happened almost miraculously, we saw that many mice lived much longer than our control groups. We were able to see that one of our compounds had a significant impact on survival time, and that the combination of curcumin, EGCG, and proanthocyanidins was even more effective. Had it not been for the alteration of our experimental procedure, we would have never truly observed the impacts from injecting our selected compounds. While we would like to thank the ACCIAC for their funding as this project would not have been possible without them, we would like to speak about the possible need for extending 10

research projects. When entering a lab as a new researcher, we have learned, it takes quite a while to get used to the equipment, complete training, and fill out animal protocols that need to be completed to conduct animal research. In addition, experiments and planned procedures are always susceptible to change according to new data and research. Due to these unforeseen circumstances, which have actually benefitted us as we have learned the ins and outs of doing independent lab research, we would like to point out that, in general, it is very difficult to adhere to strict end dates for lab research projects. We hope that you consider this when looking at future proposals and that, if necessary, extensions may be given to those who would like to continue their projects."

11