Molecular analysis of the effects of resveratrol and genistein on signal transduction in cancer cells Madeleine Hornick, Dr. Deepa Rao*, and Dr. Pramod Mahajan

Department of Pharmaceutical Sciences, Drake University College of Pharmacy and Health Sciences & *Pacific University School of Pharmacy, Oregon Abstract

The overall objective of our research project is to study the effects of genistein and resveratrol on cancer cell proliferation. Resveratrol, a stilbene found in high quantities in red grape skin, and genistein, an isoprenoid found in soy, are both available in supplement form and have exhibited anti oxidative properties. Recent studies have suggested genistein and resveratrol may inhibit growth of certain types of cancer cells in culture, and that diets with higher levels of these two compounds have shown lower incidence of certain types of cancers and inflammatory disorders. However, the precise mechanisms of their antiproliferative effects only on certain types of cancer cells remain poorly defined. Our project focuses on studying the anti proliferative effects of resveratrol and genistein on signal transduction pathways in two human breast cancer cell lines and one human ovarian cancer cell line in culture. Specifically, this poster presents results examining the role of phosphorylation of two signal transduction enzymes, Akt1 and Akt2.

Introduction

Herbal products are one of the most popular dietary supplements in the US health foods market. Specifically, products containing phytoestrogens form a substantial portion of this market because of their apparent benefits to women’s health (1). However, there is considerable debate about their efficacy and the benefit to risk assessments is a topic of ongoing research. Some of this debate is centered around the mechanisms through which these weak estrogenic compounds mediate their multiple physiological actions. We are studying the molecular mechanisms of the antiproliferative actions of phytoestrogens. The two compounds used in this study are genistein (GSTN), an isoprenoid predominantly found in soy, and resveratrol (RESV), a stilbene found in very high quantities in the skin of red grapes.

trans

-Resveratrol Estradiol Genistein As shown in figure 1 above, both of these compounds have some structural similarity to estradiol, the mammalian hormone known to regulate reproductive functions as well as development and various metabolic pathways, including cell proliferation. Both of these compounds also exhibit estrogen-like activity, with GSTN being a more potent ‘phyto-estrogen’ than RESV. Based on epidemiological data, some investigators have concluded that populations consuming diets containing foods with higher levels of GSTN or RESV have lower incidence of certain types of cancers and inflammatory disorders. Additionally, recent studies with both GSTN and RESV have shown to inhibit growth of certain types of cancer cells in culture. Although both compounds are generally believed to inhibit cancer cell growth by inducting DNA damage followed by apoptosis or programmed cell death, the precise mechanisms of their actions are not clear. For example, it is not clear if both compounds activate the same or different cell-signaling pathways as a result of induction of DNA damage/apoptosis. Another important question that remains unanswered is whether the anti-proliferative effects of these compounds are specific to cells expressing estrogen receptors. The estrogen responsive and un-responsive cell lines used in this experiment will be used to address this important question. Copyright Information Here

Introduction (continued)

We hypothesize that genistein (GSTN) and resveratrol (RESV) cause DNA damage through the estrogen receptor. We will analyze this by treating three different cancer cell lines with RESV and GSTN. The cell lines consist of two breast cancer cell lines, HTB-19 and HTB-20, and one ovarian cancer cell line, SKOV-3.

Cell Line

HTB-19

Host/Tissue Source

Transformed human mammary epithelium

Estrogen Response

No HTB-20 SKOV-3 Transformed human mammary epithelium Transformed human ovarian epithelium Yes Yes The enzyme Akt (also known as Protein Kinase B) is a family of closely related proteins found in the cytoplasm of eukaryotic cells ( ) Two members of this family, Akt1 and Akt2, are also involved in the Phosphatidyl-inositol-3 phosphate ( PI3P) kinase pathway. Both Akt1 and Akt2 are serine- threonine class of kinases. The Akt kinases exhibit tight regulation of cell proliferation by phosphorylating a number of target proteins. Furthermore, activation of Akt 2, but not of Akt1, increased incidence of progression and metastases of pulmonary tumors in a transgenic mouse model ( ), indicating a differential biological role for these two members of the Akt kinase family. Recently RESV, the phytoestrogen of our interest, has been shown to inhibit the PI- Kinase pathway in various cancer cell lines ( ). This inhibition occurs in an Akt dependent as well as independent manner. Previous studies have established that the estrogen- like effects of RESV are mediated through estrogen receptor. However, effects of RESV not mediated via the estrogen receptor remain un-elucidated. Recent results from our laboratory show that the breast cancer cell lines HTB19 and HTNB 20 are an excellent model for understanding the differential effects of estrogenic compounds. In this study, we present preliminary observations indicating differential effects of RESV on Akt 1 and Akt 2 levels in SKOV3, the ovarian cancer cells in culture.

Methods

Genistein Administration Cell lines are cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine and grown in 150 cm 2 flasks until 90% confluent. 4 mL of Trypsin are used to detach cells from flask. Trypsin action is stopped by adding 6 mL PBS to each flask and contents of all flasks are pooled into a 50 mL centrifuge tube. Cells are counted to determine volume needed to seed 4, 6-well plates at ~300,000 cells/mL medium. 5 mL of cells is distributed in each well of the plate. Stock solutions of GSTN are prepared to the following dilutions: 10 mM, 5 mM, 2.5 mM, and 1 mM. Each plate of cells is treated with 10 µL of corresponding stock solution at the time point 0 hours, and then the cells are harvested at the corresponding time points of 0.5 hours, 1 hour, 2 hours, and 6 hours. The plate is transferred back to the incubator after each addition. Samples are centrifuged at 5,000 rpm for 7 minutes and the supernatant is discarded. 10 mL of 1x ice-cold PBS is added, and the sample is centrifuges at 5,000 rpm for 7 minutes. This step is then repeated and pellets are stored at -80°C for further analysis.

Resveratrol Administration Procedure is the same as GSTN administration, but with respective stock solutions of RESV rather than GSTN.

Methods (continued)

Protein Extraction Preparation Samples are removed from -80°C freezer and pellets are suspended in 1 mL Tissue Protein Extraction Reagent (T-PER) and vortexed well by pipetting mixture up and down several times. The samples are centrifuged at 14,000 rpm for 10 minutes at 4-6°C. The supernatant is removed carefully and analyzed further. The pellets and supernatant are stored at -80°C when not in use. Polyacrylamide Gel Electrophoresis in presence of SDS Protein extracts were loaded into each well of the gel. Protein samples were combined with an equal volume of 2x SDS-sample buffer for a 1:1 ratio. The prepared samples were heated at 95° C for six minutes. and then were quickly spun after heating and were loaded onto a 4-20% Tris-glycine gel. Two controls, HeLa and Jurkat Cell extract, were loaded along with the samples, in addition to two sets of markers, SeeBlue and SeeBlue-Plus. The gel apparatus was filled with 1% Tris-glycine running buffer containing 0.1 % SDS. The electrophoresis machine was set to deliver 125 volts for 140 minutes. The gel was removed from the plastic casing and the proteins were transferred on o a PVDF membrane using the Western Blot transfer protocol described below. Western Blot The gel was washed with Tris-glycine transfer buffer containing 20% methanol. Six sponge pads were soaked in cold ( 4 o C) 1x transfer buffer containing SDS until the transfer apparatus was assembled. Two filter papers were soaked in 1x transfer buffer. The PVDF membrane was first soaked in methanol due to its hydrophobic nature. Following being soaked in methanol, it was rinsed several times with deionized water, and then place in the 1x transfer buffer. The transfer box was set on a flat surface, and three sponge pads were place in the bottom of the box, and a filter paper was placed on top of these. Next, the gel was carefully placed on the filter paper and its orientation was recorded. The PVDF membrane was then placed carefully on top of the gel in order to ensure there were no air bubbles trapped between the gel and the membrane that would disrupt the protein transfer. The second filter paper was placed on top of the membrane, and the three remaining sponge pads followed. 1x transfer buffer was poured into the transfer box to ensure all contents were saturated. Then, the box was closed and placed into the running apparatus. The inner chamber of the apparatus was filled with 1x transfer buffer, and the outer chamber was filled with cold deionized water. Ice was placed around the running apparatus to ensure that the solutions within the chamber stayed cold. The protein transfer was ran at 25 volts for 120 minutes. Protein Detection The blot was soaked in a non-specific blocking agent, 4% Blotto , for 30 minutes in order to saturate the areas on the blot where there was no protein present so that the protein binding during the transfer process would be specific.. The primary antibody solution was prepared in a centrifuge tube at a 1:1000 dilution (5mL total) primary antibody and 4% Blotto. The PVDF membrane was placed in the centrifuge tube with the protein side facing inward and was placed on a rotator in a 20°C cooler overnight for incubation. Following the primary incubation, the blot was washed 4 times for 15 minutes each in PBST. 5mL of the secondary antibody solution was prepared in centrifuge tubes at a 1:1000 dilution with the corresponding antibody to the primary antibody. The PVDF membrane was placed protein side up in the tube and was incubated on the rotator at room temperature for 1 hour. Following the incubation, the membrane was washed in PBST 4 times at 15 minute increments. After the washings, a 800µL solution was prepared from equal volumes of solution A and solution B of the Western Blot detection kit. The PVDF membrane was placed on saran wrap on a flat surface and the prepared detection solution was pipetted over the top of the membrane. After 5 minutes, the excess solution was dripped off the membrane and the membrane was placed in the chemiluminescence machine for protein detection.

Results and Discussion

In these studies, we examined effect of RESV treatment of the ovarian cancer cell line SKOV3. Cells were treated with RESV concentrations ranging from 0.1 μM to 100 μM for up to six hours. Samples were drawn at 0, 30, 60, 120 and 360 minutes. Cells treated with equal volume of the solvent (DMSO) for six hours served as controls. Protein extracts were prepared and analyzed by SDS-PAGE followed by western blotting as described under materials and methods. Representative results shown in Figure 2 indicate that with increasing exposure of cells to RESV treatment from 0-360 minutes results in a gradual reduction in the amount of Akt proteins. Reduction appears more pronounced in Akt2 than Akt1.

Figure 2. RESV modulates Akt protein levels in SKOV3 cells

M 1 2 3 4 5 M Akt 2 Akt 1

Western blot analysis of SKOV3 protein samples treated with 100 μM RESV for 0 (lane 5) 30 (lane 4) 60 ( lane 3), 120 ( lane 2) and 360 min ( lane 1). M : molecular weight markers. Blots were probed with antibodies specific for Akt1 and Akt2.

Additional experimentation will be needed to confirm these preliminary observations. Further research is underway in our laboratory. Nonetheless, it is tempting to speculate on the possible mechanisms of regulation of Akt1 and Akt 2 by RESV and GSTN. Figure 3 below depicts a model speculating differential effects of GSTN/RESV. This model also forms a template for our future experiments using the HTB-19 and HTB-20 cells.

Figure 3. Differential mechanisms of RESV/GSTN Action A Model

Estrogen RESV/GSTN Growth factors PI3K

ER

PI3P Akt1 PDK1

R

?

Akt2 p53 MDM ?

?

DNA metabolism R= Receptor ER= Estrogen Receptor PI3 = 3phosphoinositol PI3K = PI3- kinase Akt1 = Akt 1 kinase Akt2= Akt2 kinase p53 = Tumor suppressor MDM= p53 regulator protein Red dashed line arrows are speculative steps.

References

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