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12-10-2014

Assessment of the Effects of Caffeine, Gallic Acid,and Epigallocatechin-3-gallate on Cell Inhibition,PIM-3 and E. cadherin Protein Levels in Two Linesof Pancreatic Cancer CellsLena HaddadChapman University, hadda106@mail.chapman.edu

Melissa Rowland-GoldsmithChapman University, rowlandg@chapman.edu

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Recommended CitationHaddad, Lena and Rowland-Goldsmith, Melissa, "Assessment of the Effects of Caffeine, Gallic Acid, and Epigallocatechin-3-gallate onCell Inhibition, PIM-3 and E. cadherin Protein Levels in Two Lines of Pancreatic Cancer Cells" (2014). Student Research Day Abstractsand Posters. Paper 76.http://digitalcommons.chapman.edu/cusrd_abstracts/76

Assessment of the Effects of Caffeine, Gallic Acid, and Epigallocatechin-3-gallateon Cell Inhibition, PIM-3 and E. cadherin Protein Levels in Two Lines of Pancreatic

Cancer CellsHaddad, L., Rowland-Goldsmith, M.

Department of Biological Sciences, Chapman University, Orange, CA

IntroductionAccording to the American Cancer Society, pancreatic cancer iscurrently the fourth leading cause of cancer related deaths in theUnited States. In addition to being an exceptionally aggressive form ofcancer, it is particularly difficult to treat because it is usually diagnosedin late stages after the onset of metastasis (1). Consequently, thecurrent treatments used, including chemotherapy and radiation, havebeen rendered ineffective (2). As a result, focus has been placed onusing dietary alternatives which are known to possesschemopreventive properties (3). Previous studies have indicated thatGallic acid (an important phytochemical in pomegranates) andEpigallocatechin-3-Gallate (the primary catechin in Green Tea) haveinhibited cancer cell growth in a variety of cancer cell types (4-9).Similarly, studies have shown that caffeine, the primary compoundfound in coffee beans, has been successful in stimulating apoptosis inendometrial, liver, skin, breast, prostate, colorectal, oral, and bonecancers (10-20). Although these compounds have individuallydemonstrated great positive effects there has, until now, never been astudy done to compare the effects of these compounds bothindividually and in combination in pancreatic cancer cells. Moreover,there has never been a study done to test the effects of any of thesecompounds on the levels of the PIM-3 and E. cadherin proteins whichhelp cancer cells evade programmed cell death and cause cell-to-celladhesion, respectively.The purpose of this research project is to identify whether caffeine,Gallic Acid (GA), or Epigallocatechin-3-Gallate (EGC3G) is the mosteffective cancer fighting agent. Once we have ascertained the twomost effective compounds, combination studies will be done to seewhat effect the combination treatments have on pancreatic cancercell inhibition and the PIM-3 and E. cadherin protein levels, relativeto the individual treatments.

HypothesisWe hypothesized, due to results from previous experimentation, thatcaffeine and Gallic acid will be the most powerful cancer fightingagents. Moreover, the combination of these two compounds will causegreater increase in cell inhibition, a greater decrease in PIM-3 proteinlevels, and a greater increase in E. cadherin protein levels than eitherof the compounds individually in human pancreatic cancer cells.

Experimental MethodsCell MaintenanceHuman pancreatic cancer cells (COLO-357) were grown in Dulbecco’sModified Eagle’s (DME) complete media. The following componentswere added to the media: 10% fetal bovine serum, 100 µg/ml Penicillinand 100 µg/ml Streptomycin. The cells were maintained in a humidified5% CO2 and 95% air atmosphere at 37°C.

Identifying the Appropriate Epigallocatechin-3-Gallate (EGC3G) Concentration for Cell TreatmentA volume of 8 x105 COLO-357 cells (4 x105 PANC-1 cells for PANC-1experiments) were added to each of 6 wells in the cell-culture 6-wellplates. The cells were grown overnight in DME complete media, thenovernight again in DME serum free media (SFM) with 100 ug/mlpenicillin, 100 ug/ml streptomycin, and 1x ITS before being treated.Cells were treated for 48 hours in DME SFM containing varyingconcentrations of EGC3G (0.046 mg/mL, 0.18 mg/mL, or 0.32 mg/mL).The control cells were treated with DME SFM. Post-treatment cellcounts were determined using a hemocytometer. The cellconcentrations measured were compared to the pre-treatment data

and data was analyzed using analysis of variance (ANOVA).

Experimental Methods (continued)Treatment of Cells (Epigallocatechin-3-Gallate, Caffeine, and the Combination thereof)As in the previous tests, 8 x105 COLO-357 cells (4 x105 PANC-1 cellsfor PANC-1 experiments) cells were added to each of 6 wells in thecell-culture 6-well plates. The cells were grown overnight in DMEComplete media, then overnight again in DME Serum Free Media(SFM). Cells were treated for 48 hours in DME SFM containing either0.32 mg/mL EGC3G, 0.1269 g/mL caffeine, or the combination ofthe two. The control cells were treated with DME SFM. Post-treatment cell counts were determined using a hemocytometer.Data analyzed using analysis of variance (ANOVA).

ResultsCell Inhibition

Western Blot AnalysisWhole cell lysates were generated using Pierce M-PER® Mammalian Protein Extraction Reagent with Protease Inhibitor Cocktail. Proteins were quantified with a Pierce BCA protein assay kit. Equal amounts of protein were added to an SDS-polyacrylamide gel and transferred to a PVDF membrane. The membranes were blocked with 5% non-fat milk in TBS and then probed with indicated primary antibodies with gentle shaking overnight. Primary antibodies against PIM-3 (1:100), E-cadherin (1:100), and the ERK-2 (1:6000) loading control were used in this study. After washing the membranes three times, the immunoblots were incubated with the appropriate secondary antibodies for 1 hour. Antibody-bound proteins were detected using Immobilon Western Chemiluminescent HRP Substrate, and photos taken utilizing Bio-Rad Image Lab Software.

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321-337. Web. 30 Jul. 2013. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3667471/>.

2) Westphalen, CB, KP Olive, et al. "Genetically engineered mouse models of pancreatic cancer.." Cancer J. 18.6 (2012): 502-210. Web. 10 Aug. 2013. <http://www.ncbi.nlm.nih.gov/pubmed/23187836>.

3) Syed D.N.; Afaq F.; Mukhtar H. Pomegranate Derived products for Cancer Chemoprevention. Seminars in Cancer Bio. 2007, 17, 377-385

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<http://www.ncbi.nlm.nih.gov/pubmed/24022184>. 12) Bravi, F, C Bosetti, et al. "Coffee Reduces Risk for Hepatocellular Carcinoma: An Updated Meta-analysis."

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20) Gururajanna, B, AA Al-Katib, et al. "Molecular effects of taxol and caffeine on pancreatic cancer cells.." Int J Mol Med. 4.5 501-7. Web. 22 Sep. 2013. <http://www.ncbi.nlm.nih.gov/pubmed/10534572>.

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Figure 4. Results from Western Blot Analysis of PIM-3 and E. cadherin proteins. Proteinlevels in COLO-357 cells ascertained in cells treated with either nothing, caffeine, EGC3G, orthe combination. A) Western Blot for PIM-3 protein. B) ERK-2 Loading Control for WesternBlot of PIM-3 protein. C) Western Blot for E. cadherin protein. D) ERK-2Loading Control forWestern Blot of E. cadherin protein

.Figure 2. Images of Cell Morphology for Each Treatment 400XMagnification in COLO-357 cells. A) Non-treated cells. B) Cells treated

with Gallic Acid (50 ug/mL). C) Cells treated with Caffeine (0.1269 g/mL). D) Cells treated with EGC3G (0.32 mg/mL).

ConclusionBased on the results from the cell inhibition studies usingEpigallocatechin-3-gallate, it was determined that an increasing dosageof EGC3G resulted in a greater decrease in cell proliferation (Figure 1).The results from the EGC3G, caffeine, and combination cell inhibitionstudies illustrated that the combination treatment of EGC3G and caffeinetogether resulted in a greater degree of cell inhibition than EGC3G orcaffeine alone (Figure’s 5-6). Based on these preliminary results, it isclear that the combination of both caffeine and EGC3G is a potentialviable treatment for inhibiting cancer cell growth. In vivo studies wouldbe required to validate that these compounds would be effective ininhibiting pancreatic cancer cell growth in humans. Although thecombination of Epigallocatechin-3-gallate and caffeine produced thegreatest decrease in the expression of the PIM-3 protein in the COLO-357line of pancreatic cancer cells, the difference relative to the non-treatedcells was not statistically significant (Figure 4A-4B). So, while EGC3G isable to inhibit cancer cell growth, it is predominantly not through theapoptotic PIM-3 pathway. The combination of EGC3G and caffeine didincrease the level of E. cadherin to a great degree, although it was stillnot statistically significant (Figure 4C-4D). These compounds incombination are making cells “stickier,” potentially helping preventmigration (or metastasis of these cells).

Future Research Future research project will focus testing the effects of caffeine and Gallic acid in combination to observe their effects on cell inhibition and apoptosis. Future work would continue to look at other mechanisms of action by which these compounds are able to inhibit cancer cell growth.

AcknowledgmentsI would like to thank the Office of Undergraduate Research for allowingme to be a part of this SURF program. Many thanks to Dr. Murray Korc(Indiana University) for supplying COLO-357 and PANC-1 cells, Dr. MarcoBisoffi, and the Chapman University Faculty Scholarly Research Grant, forwithout them this research would not have been possible.

Figure 1. Dose Response of EGC3G. Results from 3 separate cell inhibition experiments.Number of COLO 357 & PANC-1 cells per well in samples that were either not treated(NT) or treated with EGC3G 1 (0.046 mg/mL), EGC3G 2 (0.18 mg/mL), and EGC3G 3(0.32 mg/mL) for 48 hours . Data shown as mean±SE, n=9. *P<0.05

Western Blot

Figure 3. Results from 3 separate cell inhibition experiments NT, Caffeine, Gallic Acid andEGC3G. Number of cells per well in samples that were either not treated or treated withCaffeine (0.1269 g/mL), Gallic Acid (50 ug/mL), and Epigallocatechin-3-gallate (0.32 mg/mL)Data shown as mean±SE, n=9. *P<0.05

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