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
Introduction (continued)
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 antioxidative 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 antiproliferative 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
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
Host/Tissue Source
Estrogen Response
HTB-19
Transformed human mammary epithelium
No
HTB-20
Transformed human mammary epithelium
Yes
SKOV-3
Transformed human ovarian epithelium
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 cm2 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 TissueProtein 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 ( 4oC) 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.
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
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
Growth factors
Estrogen
RESV/GSTN
PI3K
PI3P
R
PDK1
ER
?
Akt1
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.
Western blot analysis of SKOV3 protein samples
treated with 100μM 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.
p53
Akt2
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.
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