409 NATURAL COMPOUND ALTERNOL ACTIVATES APOPTOSIS AND AUTOPHAGY IN PROSTATE CANCER CELLS

Vol. 185, No. 4S, Supplement, Sunday, May 15, 2011

408 INTERLEUKIN-6: A POTENTIAL BIOMARKER OF RESISTANCE TO MULTITARGETED RECEPTOR TYROSINE KINASE INHIBITORS IN CASTRATION-RESISTANT PROSTATE CANCER Alexander Kutikov*, Peter Makhov, Konstantin Golovine, Daniel Canter, Mohit Sirohi, Ryan Street, Robert G. Uzzo, Vladimir M. Kolenko, Philadelphia, PA INTRODUCTION AND OBJECTIVES: Clinical experience using tyrosine kinase inhibitors (TKIs) in patients with castration-resistant prostate cancer (CRPC) is starting to mature. In Phase II trials, a heterogeneous response to sunitinib has been noted. PSA levels have proven unreliable for prediction of response to TKIs. IL-6 is a critical mediator of CRPC pathogenesis and has been shown to rise in patients with disease progression. As such, we investigated whether cellular IL-6 production can predict TKI response in both an in-vitro and an in-vivo model. METHODS: IL-6 mRNA levels were examined by RT-PCR. IL-6 protein expression was investigated using ELISA, while apoptosis was examined using the TUNEL assay. For in-vivo studies CRPC xenograft model in C.B17/Icr-scid mice was employed. RESULTS: CRPC cells exhibited heterogeneous response to sunitinib and pazopanib. Dose dependent reduction of IL-6 levels was observed in TKI-sensitive DU-145 cells. In contrast, the TKI-resistant PC-3 cells failed to suppress IL-6 excretion. Instead, in the presence of TNF-␣, IL-6 levels rose significantly upon administration of TKIs. Findings of in-vitro experiments were confirmed in an in-vivo mouse model of CRPC. CONCLUSIONS: Sensitivity of CRPC cells to TKIs is heterogeneous. These findings are consistent with results of recently-published Phase II clinical trials using sunitinib in patients with CRPC. A substantial rise in IL-6 levels occurs both in-vitro and in-vivo in the presence of TKIs in the resistant PC-3 cells but not in the TKI-sensitive DU-145 cells. Source of Funding: National Institutes of Health Grants RO1 CA108890 and CA134463 (VMK); American Institute for Cancer Research Grant (RGU); Department of Defense, Physician Research Training Award (AK)

409 NATURAL COMPOUND ALTERNOL ACTIVATES APOPTOSIS AND AUTOPHAGY IN PROSTATE CANCER CELLS Andrew James*, Benyi Li, Jeffrey Holzbeierlein, Kansas City, KS INTRODUCTION AND OBJECTIVES: Alternol is a natural product derived via fermentation from the microorganism Alternaria Alternata from the bark of a yew tree native of China. In human gastric cancer cell lines and in some prostate cancer cell lines, Alternol has been shown to induce apoptosis. We sought to evaluate the potential for Alternol to induce cell death in prostate cancer cell lines and to more specifically investigate the mechanisms by which Alternol induces apoptosis and cell death. We examined the in vitro effects of Alternol in several different prostate cancer cell lines. METHODS: Three prostate cancer cell lines (LNCaP, C4-2, and PC-3) were treated with Alternol. Cell viability was assessed via trypan blue exclusion assay and the cell cycle distribution of each of these lines was also examined after culturing each of these lines with and without Alternol. Intracellular concentrations of autophagy and apoptotic regulatory proteins were determined via Western blotting by utilizing antibodies against caspase-3, PARP, Bcl-2, and Bax following treatment with Alternol. Cells were also treated with the antimalarial Choloroquine (an inhibitor of autophagy) to further examine the effects of Alternol on autophagy and apoptotic pathways. RESULTS: Alternol, in both androgen dependent and independent cell lines, induced cell arrest at G2/M at 24 hours. Alternol also induced cytotoxicity at 12 hours post-treatment. In all 3 cell lines, apoptosis occurred by caspase-3 processing and PARP cleavage. Additionally, Bcl-2 expression was significantly lowered and Bax pro-

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tein expression was increased in cell lines treated with Alternol, suggesting that Alternol targets expression of these proteins in promoting apoptosis. Bif-1, a regulator of both apoptosis and autophagy, was found to be up-regulated by Alternol. LC-3II levels were also upregulated in Alternol treated cells. Blocking autophagy with Choloroquine enhanced Alternol caspase-3 processing and PARP cleavage and enhanced apoptosis and synergistically promoted cytotoxicity. CONCLUSIONS: Alternol is capable of inducing apoptotic cell death in prostate cancer cells through modulation of pro- and antiapoptotic proteins. This novel natural compound deserves further examination and validation in in-vivo models. Source of Funding: NIH-KU COBRE Grant P20 RR016443

410 ANTI-CANCER EFFICACY OF SIMVASTATIN ON PROSTATE CANCER CELLS AND TUMOR XENOGRAFTS IS MEDIATED THROUGH INHIBITION OF AKT Samith Thomas Kochuparambil, Belal Al-Husein, Anna Goc, Martha Terris*, Payaningal R. Somanath, Augusta, GA INTRODUCTION AND OBJECTIVES: Statins inhibit HMG-CoA reductase, a rate liming enzyme in cholesterol synthesis. Growing body of evidences suggest that statins have the potential to reduce the risk of cancers. In prostate cancer cells, simvastatin is known to induce apoptosis. Our “central hypothesis” is that treatment with simvastatin will inhibit Akt affecting prostate cancer cell function, tumor growth and metastasis. If proven valid, it is likely that prostate tumor growth and metastasis can be down-regulated therapeutically utilizing simvastatin using a novel “Drug-repurposing strategy”. METHODS: In the current study, we sought to investigate the pleiotropic effects of simvastatin on major signaling pathways in prostate cancer cells with respect to the regulation of cellular functions such as migration, proliferation, colony/foci formation and invasion, along with its already known effects on apoptosis. RESULTS: Time- and dose-effects of simvastatin on LNCaP (androgen-dependent) and PC3 (androgen-independent) cells indicated that treatment with as low as 25␮M simvastatin was sufficient to inhibit serum-stimulated activation of Akt-mTOR and cRaf-ERK pathways. Akin to this, treatment with 25␮M simvastatin significantly inhibited serum- and EGF-induced cell migration, invasion, colony formation and proliferation. Simvastatin-mediated effects on cell migration and colony formation was rescued by Adenovirus-mediated expression of constitutively active Akt (myristoylated Akt) in androgen-independent prostate cancer cells lines such as PC3 and LnCAP C4-2. A xenograft model performed in nude mice exhibited reduced PC3 prostate tumor growth with simvastatin treatment (2mg/kg body weight/twice a day for 2 weeks) demonstrating the therapeutic potential of simvastatin for prostate cancer therapy. CONCLUSIONS: Our findings suggest a link between simvastatin and Akt in the regulation of prostate cancer. Further investigation is currently underway in our laboratory to unravel the molecular mechanisms on simvastatin-mediated effects on prostate cancer in vivo using TRAMP⫹ transgenic mice. We also plan to undertake a clinical study on patients with prostate cancer who were on statin treatment prior to and after diagnosis and analyze biopsy specimen from these patients. A prospective study will look at the role of statins in prostate cancer prevention and/or on management. Our ultimate aim is to investigate if statins can be used as an adjuvant drug in the treatment of patients already diagnosed with prostate cancer. Source of Funding: University of Georgia Research Foundation