Date of Award

8-1-2013

Degree Name

Doctor of Philosophy

Department

Pharmacology

First Advisor

Ramkumar, Vickram

Abstract

Prostate cancer is the most commonly diagnosed cancer and the second most leading cause of cancer deaths in American men (www.cancer.org). Most prostate cancer-related deaths are due to the metastatic form of the disease. The 5-year relative survival rate in patient's diagnosed with metastatic prostate cancer is just 28%, as compared to 100% in patient's diagnosed with localized prostate cancer. This clearly indicates the lack of effective treatment available for metastatic prostate cancer. MicroRNAs (miRNAs) are small (18~23 nucleotide long) non-coding RNAs that can influence gene expression by binding to the 3'-untranslated region of coding RNAs at the post-transcriptional level. Some miRNAs has been termed as oncomirs due to their role in promoting tumor growth, invasion and metastasis. One such oncomir is microRNA-21 (miR-21) whose levels are often up-regulated in a number of cancers, including prostate cancer. MiR-21 increases the survival and invasiveness of cancer cells by suppressing its target tumor suppressor genes, namely programmed cell death 4 (PDCD4) and maspin. Thus, drugs which target miR-21 for inhibition could provide novel treatment options for metastatic prostate cancer. Resveratrol (3,5,4'-trihydroxystilbene) is a polyphenolic phytoalexin found in high quantities in various dietary sources, such as grapes, red wine, berries and peanuts. Various reports have demonstrated a significant role of resveratrol in the management of several old age diseases including cancer. The efficacy of resveratrol as an anti-cancer agent resides in its ability to interfere with cell proliferation and metastasis and enhancement of apoptosis. Resveratrol has been shown to act on several intracellular targets to exert these effects. However, the exact mechanism by which resveratrol mediates its beneficial cancer chemotherapeutic actions are not clear and is the focus of this study. Based on the reported data, we hypothesized that resveratrol mediates its anti-cancer action against metastatic prostate cancer by inhibiting the signaling pathway which involves miR-21 expression and function. To address this hypothesis, we show that resveratrol decreased cell viability, migration and invasiveness of androgen-receptor negative and highly aggressive human prostate cancer cells, PC-3M-MM2. These effects of resveratrol were associated with the inhibition of miR-21, since over-expression of miR- 21 with pre-miR-21 oligonucleotides attenuated resveratrol's effect on these cells. Additionally, resveratrol increased the expression of tumor suppressors, PDCD4 and maspin, which are negatively regulated by miR-21 and knockdown of PDCD4 by short interfering (si) RNA reversed the resveratrol's effect on prostate cancer cells. PC-3M-MM2 cells also exhibits high levels of phospho-Akt (pAkt), which were reduced by both resveratrol and LY294002, a known PI3-kinase inhibitor. MiR-21 expression in these cells appears to be dependent on Akt, as LY294002 reduced the levels of miR-21 along with a concurrent increase in PDCD4 expression. These in vitro findings were further corroborated in a severe combined immunodeficient (SCID) mouse xenograft model of prostate cancer. Oral administration of resveratrol not only inhibits the tumor growth but also decreased the incidence and number of metastatic lung lesions. These tumor- and metastatic-suppressive effects of resveratrol were associated with reduced miR-21 and pAkt, and elevated PDCD4 levels. Future investigation into the molecular mechanisms revealed that resveratrol suppressed prostate cancer growth by decreasing the levels of insulin-like growth factor-1 (IGF-1) and its receptor (R). Previous studies had associated elevated levels of serum IGF-1 with high risk of prostate cancer. IGF-1, after binding to its receptors, acts as a potent mitogen which stimulates cancer cell growth and proliferation mainly by activating Akt signaling pathway. Interestingly, this effect of resveratrol on IGF-1/IGF-1R was independent of its effect on miR-21. In summary, our data show that resveratrol exerts its anti-cancer effect on metastatic prostate cancer cells, at least in part, by targeting Akt/miR-21 pathway. These data highlight a potential molecular mechanism for resveratrol's anti-cancer action for the treatment of metastatic prostate cancer and suggest that inhibition of the IGF-1/Akt/miR-21 pathway is a rationale approach for the treatment prostate cancer metastasis.

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