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Phytochemicals are dietary phytoestrogens that may play a role in prostate cancer prevention. Forty percent of Americans use complementary and alternative medicines (CAM) for disease prevention and therapy. Ashwagandha (Withania somnifera) contains flavonoids and active ingredients like alkaloids and steroidal lactones which are called ‘Withanolides’. We hypothesize that the immunomodulatory and anti-inflammatory properties of Ashwagandha might contribute to its overall effectiveness as an anti-carcinogenic agent. The goal of our study was gain insight into the general biological and molecular functions and immunomodulatory processes that are altered as a result of Ashwagandha treatment in prostate cancer cells, and to identify the key signaling mechanisms that are involved in the regulation of these physiological effects using genomic microarray analysis in conjunction with quantitative real-time PCR and western blot analysis. Ashwagandha treatment significantly downregulated the gene and protein expression of proinflammatory cytokines IL-6, IL-1β, chemokine IL-8, Hsp70 and STAT-2, while a reciprocal upregulation was observed in gene and protein expression of p38 MAPK, PI3K, caspase 6, Cyclin D and c-myc. Furthermore, Ashwagandha treatment significantly modulated the JAK-STAT pathway which regulates both the apoptosis process as well as the MAP kinase signaling. These studies outline several functionally important classes of genes, which are associated with immune response, signal transduction, cell signaling, transcriptional regulation, apoptosis and cell cycle regulation and provide insight into the molecular signaling mechanisms that are modulated by Ashwagandha, thereby highlighting the use of this bioflavanoid as effective chemopreventive agent relevant to prostate cancer progression.
The latest statistics from the American Cancer Society estimates that a total of 218 890 new cases of prostate cancer and 27 050 deaths from the disease are anticipated in the United States in 2007, making it the most frequent nondermatologic cancer among US males (1,2). Prostate cancer is about twice as common among African-American men as it is among white American men and exhibits tremendous differences in incidence among populations worldwide (1–3). This profound geographic and racial variation in prostate cancer risk has stimulated investigations into how diet affects the development and progression of prostate cancer (4,5).
Phytochemicals are dietary phytoestrogens that may play a role in prostate cancer prevention (6–10). Plant compounds inhibit carcinogenesis via several mechanisms including antioxidant activity, changes in carcinogen metabolism, modulation of the cell cycle, alterations in intracellular signaling, and inhibition of angiogenesis (10–15). Approximately 40% of Americans use complementary and alternative medicines (CAM) for disease prevention and therapy. Over 5000 flavonoids, the largest class of phenolics, have been described (16–19). Polyphenols that are effective when given during the post-initiation period, inhibiting tumor promotion and progression, appear to be more useful in preventing cancer in humans than polyphenols that are effective only when given before and during carcinogen exposure (20).
Ashwagandha (Withania somnifera)—known as Indian ginseng is cultivated in India and North America, and whose roots have been used for thousands of years by Ayurvedic practitioners (21,22). Ashwagandha's root contains flavonoids and many active ingredients like alkaloids and steroidal lactones which are commonly called Withanolides (16). The chemicals constituents of Ashwagandha include three natural powerful antioxidants-superoxide dismutase, catalase and glutathione peroxidase (17–19). Ashwagandha may have chemopreventive properties that make it a potentially useful adjunct for patients undergoing radiation and chemotherapy (15, 20).
Our previous studies have evaluated the antiproliferative properties of various flavonoids (23,24), and have indicated that flavonoids can modulate cell cycle regulation making them potential anti-cancer agents.
To obtain a comprehensive view of the possible molecular and biological processes that are modulated by Ashwagandha treatment to prostate cells, we performed genomic analysis using a cDNA microarray. The use of the microarray technology allowed us to rapidly screen large numbers of potentially relevant genes that are involved in the controlling of certain key biological processes, this gives us an insight into the general physiological and immunomodulatory processes that are altered as a result of Ashwagandha treatment of prostate cancer cells. In the present study, we have attempted to outline specific groups of genes whose expression is significantly regulated by Ashwagandha in a highly invasive prostate cancer cell line PC-3. These studies show that several functionally important classes of genes, which are associated with immune response, signal transduction, cell signaling and transcriptional regulation, apoptosis and cell cycle regulation, can be important targets in bioflavonoid therapy.
The human prostate cancer cell line, PC-3 is obtained from the American Type Culture Collection (Manassas, VA). Approximately 3 × 106 cells per 60 mm dish are cultured at 37°C in a humidified atmosphere of 5% CO2 and 95% air for 48 h. Only cell suspensions >90% viable are used for experiments.
The alcoholic extract of Ashwagandha was a gift from Sami Labs Limited (Bangalore, India). In brief, dried root powder was extracted with ethyl alcohol at 60–70°C three times and all the three extracts combined. The combined extract was concentrated by distillation of the solvent in Rota vapor under reduced pressure. This extract was stored at –20°C and was stable for several months. For each assay, the extract was freshly solubilized in DMSO and utilized at a 0.5–1 μg/ml concentration.
Cytoplasmic RNA is extracted by an acid guanidinium-thiocyanate-phenol-chloroform method as described (25) using Trizol reagent and stored at –80°C until used.
The gene microarrays (Roswell Park Cancer Institute; Microarray and Genomics Core Facility) consisted of ~5043 cDNA clones (Invitrogen, Carlsbad, CA) which were selected based on their association with cancer progression. A total of six RNA samples that included the three sets of Ashwagandha-treated (1 μg/ml) PC-3 cells and three sets of untreated controls. cDNA was synthesized from each of those RNA samples and the Ashwagandha-treated samples were labeled with Cy5, while the corresponding untreated controls were labeled with the Cy3 dye. To minimize intra-array variations, two hybridizations for each sample were performed, and additionally the labeling with the Cy dyes was inter-changed, so as to have technical replicates of the matched pairs of Ashwagandha-treated and untreated samples.
Expression data extracted from image files were first checked by an M (log2(Cy3/Cy5)) versus A((log2 (Cy3) + log2 (Cy5))/2) plot to see whether intensity-dependent expression bias existed between spots (genes) labeled with Cy3 and Cy5 on each individual slide. Data normalization was done to correct the intensity-dependent expression bias. After data normalization, the average intensity of individual gene from multiple spots on each slide was computed using an in-house developed PERL script. A total of 5043 average expression values were obtained. Paired t-test on normalized intensity with low P-values was used to generate a list of genes with significant change in expression between normal and Ashwagandha-treated samples. The false positive rate (FDR) of the significant genes was estimated by using the SAM algorithm. Detailed normalization approaches have been described in our previous publication (26–28).
Gene expression was quantitated using real-time PCR. Relative expression of mRNA species was calculated using the comparative CT method (29). All data were controlled for quantity of RNA input by performing measurements on an endogenous reference gene, β-actin.
PC-3 cells were cultured with Ashwagandha (1 μg/ml) for 48 h, cells lysed and 30 µg of total extracted protein was separated on 4–20% gradient polyacrylamide gel (30) and electroblotted to PVDF membranes which were further probed with specific primary antibodies against the proteins of interest.
PC-3 cells are plated at a density of ~1000 cells/well in 96-well plates and incubated with varying concentrations of Ashwagandha (0.5–1 μg/ml) in complete medium for 24 h. Cell viability is quantified using the MTS assay [Promega CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (Cat.# G5421); Promega, Madison, WI] as described previously (31,32).
PC3 cells (1000 cells/well) were plated in a 96-well plate and treated with Ashwagandha extract at concentrations ranging from 0.5 to 1 μg/ml for 24 h. The results of our cell proliferation assay show that Ashwagandha treatment significantly inhibited cell proliferation in a dose-dependent manner (Fig. 1). The percentage inhibition at 0.5 and 1 μg/ml Ashwagandha concentrations was 39% and 68%, respectively. Supplementation of medium with 0.2% DMSO, which was used as the solvent had no detectable effect on cell proliferation or on the cell morphology.
To compare gene expression levels between Ashwagandha-treated PC-3 cells and their respective untreated controls. Treatment of PC-3 cells with 0.2% DMSO had no detectable effect on PC-3 cells and consequently this group was not used in subsequent studies. We used the regularized paired t-test (28) to detect differentially expressed genes, which resulted in 266 significant genes with low P-values. For multiple test correction to control the FDR, these genes were further subjected to SAM analysis, which resulted in a shorter list of 249 genes with an estimated <1% FDR. Out of these 249 differentially expressed genes, 137 (55.02%) were up-regulated and 112 (44.57%) were down-regulated with Ashwagandha treatment, as shown in Fig. 2 in black and gray color, respectively. These results indicate that based on statistical analysis a large number of genes were differentially expressed between Ashwagandha-treated and untreated cells. The differentially expressed genes have been subdivided into various functional categories. Table 1 outlines the fold change in the gene expression of significantly modulated genes that are involved in cell cycle regulation, regulation of apoptosis, modulation of stress proteins, cytokine and chemokine regulation, regulation of signal transduction, regulation of structural proteins, and oncogene regulation.
To enhance biological interpretation of the differentially expressed genes from our cDNA microsarray studies, we sought to see if any of the biological processes or molecular function were over-represented by the differentially expressed genes. We therefore performed a function enrichment analyses for these genes using a functional classification tool. DAVID 2.0 (33). Our results indicated that, although there were many over-represented biological function categories as can been seen from Table 2, majority of them are related to a few major function categories. The most relevant function categories include immune response, protein kinase cascade, regulation of apoptosis, and regulation of cell cycle.
We further analyzed our microarray data using functional annotation clustering for our differentially expressed genes to see if any of these genes could be clustered together, thus establishing biological links between genes. The resulting gene clusters are shown in Table 3, in which the group enrichment scores which rank the genes based on their biological significance are also listed. The results of this analysis outline, two groups of genes that are tightly coupled. The first group of genes included caspase recruitment domain family, member 4; receptor-interacting serine-threonine kinase 2; casp8 and fadd-like apoptosis regulator; mitogen-activated protein kinase kinase 5; cell division cycle 2-like 5, while the second group of genes displaying tight coupling included signal transducer and activator of transcription 2 and c-myc binding protein.
Biological implications can also be deduced from biochemical pathways triggered by the list of genes modulated by Ashwagandha treatment. By looking at the over-representation of genes in pathways, we could obtain direct or indirect insight into the physiological functions of these significantly modulated genes. Using yet another feature of the DAVID functional annotation tool called DAVID Pathway Viewer, a short list of significant pathways (Table 4) was obtained which displays key functional genes from the entire list of significantly modulated genes (249 in this case) on pathway maps thus facilitating biological interpretation in a network context. Our results show that our genes were significantly enriched along three distinct pathways. They are JAK-STAT pathway, the apoptosis pathway and the MAPK signaling pathway. Among these the JAK-STAT pathway appears to be the key because it not only modulates both the apoptosis process and the MAPK signaling but also is the signaling pathway that is most significantly modulated as indicated by the P-value <0.0037 (Table 4 and Fig. 3).
Based on the functional annotation analysis using DAVID as described above, a group of genes that modulate the JAK-STAT signaling pathway and regulate key biological and molecular functions were selected. They included IL-6, IL-8, IL-1β, STAT 2, p38 MAPK, PI3K, Caspase 6, Hsp70, Cyclin D, and c-myc. These genes are significantly modulated by Ashwagandha treatment as shown by gene array analysis. To further validate our findings, we performed quantitative real-time PCR using specific PCR primers and western blot analysis for protein expression for these key regulatory genes. Our results presented in Fig. 4 show that PC-3 cells treated with 1 μg/ml Ashwagandha displayed down-regulation of gene expression of the proinflammatory cytokines IL-6 (55% down-regulation, P < 0.002), IL-1β (41% down-regulation, P < 0.05), chemokine IL-8 (32% down-regulation, P < 0.04) and stress heat shock protein Hsp70 (23% down-regulation, P < 0.02). Ashwagandha treatment also down-regulated Signal Transducer and Activator of Transcription 2 (STAT-2) (34% down-regulation, P < 0.01), a dual function protein that regulates signal transduction and activates transcription as part of a phosphorylation cascade. The genes that were significantly upregulated with Ashwagandha treatment were p38 MAPK (69% upregulation, P < 0.013); PI3K (51% upregulation, P < 0.04) caspase 6 (44% upregulation, P < 0.03); Cyclin D (71% upregulation, P < 0.006) and c-myc (29% upregulation, P < 0.043). The respective percent increases in protein expression for each these genes are shown in Fig. 5. Our results show that PC-3 cells treated with 1 μg/ml Ashwagandha downregulated the protein expression of the proinflammatory cytokines IL-6 (72% downregulation, P < 0.01), IL-1β (39% downregulation, P < 0.05), chemokine IL-8 (52% downregulation, P < 0.03), Hsp70 (63% downregulation, P < 0.01) and STAT-2 (38% downregulation, P < 0.05). The proteins that were significantly upregulated with Ashwagandha treatment were p38 MAPK (49% upregulation, P < 0.03); PI3K (56% upregulation, P < 0.02) caspase 6 (44% upregulation, P < 0.05); Cyclin D (81% upregulation, P < 0.01) and c-myc (23% upregulation, P = 0.06). We observed concordance between gene expression and protein expression levels in the Ashwagandha-treated PC-3 cells. Furthermore, these results showed similar trends in gene expression as those obtained by our microarray analysis thus validating our gene microarray results presented in Table 1.
All statistical analysis for the above data was performed using analysis of variance (ANOVA), following the Mann–Whitney U-test to determine the statistical difference between control and treated groups. P < 0.05 was considered significant.
Withania somnifera Dunal, which is commonly known as Ashwagandha is reported to have antitumor, anti-stress, immunomodulatory, anti-inflammatory and antibacterial effects (11–15). The roots of W. somnifera contain several alkaloids, withanolides, a few flavanoids and reducing sugars (14–19). Several studies support the role of ashwagandha as an effective cancer chemopreventive agent, however limited information is available regarding the mechanism of regulation of these chemopreventive effects (20). The goal of our study was gain insight into the general biological and molecular functions and immunomodulatory processes that are altered as a result of Ashwagandha treatment in prostate cancer cells, and to identify the key signaling mechanisms that are involved in the regulation of these physiological effects. Since the alcohol extract of Ashwagandha can contain numerous molecules that are antiproliferative the biological effect that we observed could be due to one or many compounds. The effect could also be additive in nature. However, on initial reverse phase HPLC analysis of the alcohol extract we observed that the anti-proliferative effect was confined to a compound that eluted at 10.7 min, giving a peak at m/z of 567 (data not given). Therefore, it is our conviction that the majority of the antiproliferative and the effects on gene expression that we observed in this study is due to this particular compound; further characterization of the compound is ongoing in our laboratory.
Our results showed that Ashwagandha treatment modulated several functionally important classes of genes, which are associated with cell cycle regulation, regulation of apoptosis, modulation of stress proteins, cytokine and chemokine regulation, regulation of signal transduction, and oncogene regulation in prostate cancer cells (PC-3). Additionally, a good concordance was observed between our microarray data, and the results obtained using both our validation methodologies, namely the real time PCR analysis and the Western blot analysis, however some of the protein expression levels did not show the extent of changes as seen in the gene expression level. This could possibly be due to the post-transcriptional or post-translational modification associated with protein expression. Analysis of our microarray data using functional annotation clustering showed significantly modulated gene clusters and provides group enrichment scores which rank the genes based on their biological significance. These analyses further confirm the important role played by genes that regulate apoptosis, cell signaling and signal transduction in the Ashwagandha-mediated immunomodulation.
Using the function annotation tool DAVID Pathway Viewer, we were able to highlight key signaling pathways via which the immunomodulatory effects of Ashwagandha are mediated. Our results show three distinct pathways that are significantly modulated; they are JAK-STAT pathway, the apoptosis pathway and the MAPK signaling pathway. Among these the JAK-STAT pathway appears to be key because it also modulates both the apoptosis process and the MAPK signaling.
Ashwagandha treatment significantly downregulated the gene and protein expression of proinflammatory cytokines IL-6, IL-1β, chemokine IL-8, Hsp70 and STAT-2, while a reciprocal upregulation was observed in gene and protein expression of p38 MAPK, PI3K, caspase 6, Cyclin D and c-myc. Several in vitro studies show that polyphenols can modulate oncogenes, tumor suppressor genes, cell cycling, apoptosis, angiogenesis and associated signal transduction pathways (34,35). Tyagi et al. (36) showed that the anticancer effects of grape seed extract (GSE) on moderately aggressive DU-145 prostate cancer cells were due to activation of the JNK pathway, resulting in growth inhibition and apoptosis. Recent studies demonstrated that flavonoids can inhibit proliferation of other cancers by interfering with tubulin binding and subsequent microtubule assembly (37), among the 249 genes that were significantly modulated by Ashwagandha, several were structural proteins, like filamin A, laminin and tight junction protein-zona occludens 2. The significant anti-tumor effects of flavonoids are attributed to the modulation of protein tyrosine phosphorylation (38). Suppression of carcinogenesis by tumor suppressor genes is mediated by constraining tumor cell proliferation and colony formation. The tumor suppressor genes, suppresses tumor growth by modulating the phosphatidylinositol 3-kinase (PI3K) and causes G1 cell cycle arrest and cell death (39–41). The cyclin D gene encodes a regulatory subunit of the CDK4 and CDK6 holoenzyme complex that phosphorylates and inactivates the tumor suppressor protein, pRB, thereby modulating cell cycle progression into late G1 and S phases (41–43).
Ashwangandha treatment down regulates the expression of the proinflammatory cytokines IL-6, IL-8 and IL-1β hereby acting as a immunomodulator, via the dynamic regulation of these cytokines. These cytokines are crucial to innate and adaptive inflammatory responses, cell growth and differentiation, cell death, angiogenesis and developmental as well as repair processes. The janus kinase/signal transducer and activator of transduction (JAK/STAT) pathway is a intracellular signal-transducing pathway that is activated by oxygen radicals, various cytokines, and growth factors in various disease states (44,45). Originally described as the regulator for cytokine signaling, JAK/STAT pathway is now recognized as an important membrane-to-nucleus signaling pathway for a variety of stress responses and oxidative stress. STAT proteins have the dual function of signal transduction and activation of transcription as part of a phosphorylation cascade (44). Ashwagandha downregulates the expression of STAT2 which is a potent transactivator and forms stable homodimers which complex with adaptor protein p48 and bind to the interferon-stimulated response element (ISRE) thereby modulating cell proliferation and immunity.
Stress-inducible heat-shock protein 70 (Hsp70) has gained plenty of attention as a putative target for tumor therapy since it is an important regulator of cancer cell growth. Expression of Hsp70 in tumor cells has been proposed to enhance their immunogenicity and prevent tumor cell death (46). Ashwagandha treatment significantly decreases the gene and protein expression of Hsp70, which may result in the generation of a specific immune response by promoting apoptosis. The oncogene c-myc gene participates in most aspects of cellular function, including replication, growth, metabolism, differentiation, and apoptosis (34,35). Our studies highlight that treatment of PC-3 cells with Ashwagandha results in the activation of c-Jun N-terminal kinase (JNK) signaling, indicating that it may be the key signaling pathway involved in the process leading to activation of apoptosis in these cancer cells. Thus, plant-derived antioxidants can modulate biochemical and physiological processes that may affect cancer progression. Based on its pro-apoptotic and its immunomodulatory effects, Ashwagandha can be considered to be useful as novel complementary therapy for integrative oncology care. Furthermore, based on this preliminary finding we would like to characterize the active ingredient using reverse phase HPLC separation. The eluted compound which shows the presence of active ingredients would be further characterized using the NMR and the mass spectroscopy to elucidate the structure of the molecule.