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
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
). Tyagi et al.
) 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
). 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
). 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.