782 SANGUINARINE INHIBITS STAT3 ACTIVATION AND TUMOR GROWTH IN PROSTATE CANCER CELLS

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782 SANGUINARINE INHIBITS STAT3 ACTIVATION AND TUMOR GROWTH IN PROSTATE CANCER CELLS Meng Sun, Chengfei Liu, Wei Lou, Nagalakshmi Nadiminty, Joy Yang, Christopher Evans, Allen Gao*, Sacramento, CA INTRODUCTION AND OBJECTIVES: Signal Transducer and Activator of Transcription 3 (STAT3) is an oncogenic transcription factor implicated in prostate carcinogenesis and progression. Stat3 activation is associated with prostate cancer development and progression. Constitutively activated Stat3 activates androgen receptor signaling and promotes castration-resistant prostate cancer growth. Targeting Stat3 activation is an effective strategy in prostate cancer therapy. METHODS: DU145, C4-2B and LNCaP cells were treated with sanguinarine for 4 hours and then stimulated with Interleukin-6 (IL-6) or vehicle. The phosphorylation status of STAT3 and related proteins were measured with western blots. Activation of transcription by STAT3 via the STAT3 responsive element was measured with luciferase reporter assay. The effect of sanguinarine on DU145 and LN/S17 cells anchorage-independent growth were examined with soft agar assay. The effect of sanguinarine on cells migration and invasion of DU145 cells were measured with scratch assay and invasion assay respectively. RESULTS: Exposure to micromolar concentrations of sanguinarine resulted in suppression of constitutive as well as IL6-induced phosphorylation of STAT3 at both Tyr705 and Ser727 in DU145 cells. The inhibition of STAT3 phosphorylation correlated with the inhibition of Janus-activated Kinase 2 (JAK2) and src phosphorylation. Sanguinarine inhibited the constitutive and IL6-induced STAT3 responsive element luciferase activities. Several STAT3 regulated genes such as c-myc and survivin were downregulated by sanguinarine. We found that sanguinarine inhibited the migration and invasion of DU145 cells that express constitutively activated STAT3. Furthermore, sanguinarine inhibited the anchorage-independent growth of DU145 and LN/S17 cells and suppressed the growth of DU145 xenografts in nude mice. CONCLUSIONS: These data suggest that sanguinarine targets multiple upstream kinases that mediate STAT3 activity. Sanguinarine is a potent STAT3 inhibitor that could be developed as a therapeutic agent for prostate cancer. Source of Funding: This work was supported in part by grants from VA Merits I01 BX000526, NIH CA 109441 and CA 140468.

783 THE EFFECTS OF HDAC-INHIBITION ON TUMOR GROWTH AND INTEGRIN DRIVEN INVASION PROCESSES ARE AUGMENTED BY LOW DOSED INTERFERON ALPHA IN PROSTATE CANCER MODELS IN VITRO AND IN VIVO Steffen Wedel*, Lukasz Hudak, Igor Tsaur, Jasmina Makarevic; Jens-Michael Seibel, Eva Juengel, Axel Haferkamp, Roman Blaheta, Frankfurt am Main, Germany INTRODUCTION AND OBJECTIVES: HDAC up-regulation plays a crucial role in the genesis of prostate cancer, malignant growth and dissemination of the tumor cells. Therefore we evaluated antitumor properties of the HDAC inhibitor valproic acid (VPA) in prostate cancer cells as well as in a xenograft model. Since there is evidence form other tumors that interferon-␣(IFNa) might enhance HDAC inhibitory effects we combined VPA with IFNa in low concentrations which had no cancer blocking effects when applied alone. METHODS: VPA and IFNa were applied to PC-3, DU-145 and LNCaP cells, either separately or in combination. Tumor cell growth, cell cycle progression and cell cycle regulating proteins were then investigated by the MTT dye reduction assay, flow cytometry and western blotting. Tumor cell adhesion to vascular endothelium or to immobilized extracellular matrix proteins was evaluated. Migration and invasion were explored by a modified Boyden chamber assay. Integrin ␣and ␤ adhesion molecules were analyzed by flow cytometry, western

Vol. 187, No. 4S, Supplement, Monday, May 21, 2012

blotting and RT-PCR as well as alterations of cell signaling pathways were analyzed. Finally, effects of the drug treatment on prostate cancer growth in vivo were determined by the NOD/SCID mice model. RESULTS: VPA, separately applied, reduced tumor cell adhesion, migration and growth in vitro. A much stronger anti-cancer potential was evoked by the VPA-IFNa combination, although IFNa alone did not exert any growth or adhesion blocking activity. The same effect was seen when tumor growth was evaluated in vivo. Molecular analysis revealed distinct elevation of histone H3 acetylation caused by VPA which was further up-regulated by VPA-IFNa, whereas IFNa alone did not alter H3 acetylation. Combinatorial benefit became obvious on Akt phosphorylation, p21 and p27 and integrin ␣1, ␣3 and ␤1 expression. CONCLUSIONS: Application of low-dosed IFNa to a VPA based regimen profoundly boosts the anti-tumor characteristics of VPA. Presumably, IFNa renders prostate cancer cells to become more susceptible to HDAC-inhibition resulting in additive effects on integrin receptors and growth related signaling. We conclude that combined use of VPA and low-dosed IFNa may become an innovative option to treat advanced prostate cancer. Source of Funding: None

784 OVERCOMING RAD001 TRIGGERED RESISTANCE IN PROSTATE CANCER: CDK1-CYCLIN B COMPLEX Igor Tsaur*, Jasmina Makarevic, Michael Reiter, Martin Kurosch, Georg Bartsch, Christoph Wiesner, Steffen Wedel, Axel Haferkamp, Roman Blaheta, Frankfurt, Germany INTRODUCTION AND OBJECTIVES: mTOR targeting is a promising strategy for cancer therapy since the phosphatidyl-inositol3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is aberrantly activated in different tumors. It has been argued that chronic drug exposure may trigger the development of resistance, consequently limiting the efficacy of mTOR-inhibitors. As no published studies previously dealt with this issue, a prostate cancer subline resistant to the mTOR–inhibitor RAD001 (everolimus) was established and the impact of drug resistance on the proliferative potential of the tumor cells and molecular characteristics of cell growth regulation were analyzed. METHODS: The growth and proliferation properties of PC3 prostate cancer cells, sensible (PC3par) or resistant (PC3res) to RAD001, were investigated using MTT and BrdU tests as well as clonogenic assay. Cell cycle and western blot analysis of regulating proteins were performed. Knock-down models were established by transfection of cells with cdk1 or cyclin B siRNA. RESULTS: Cell growth and proliferation rates of PC3res and PC3par were not significantly different. Removal of RAD001 from the culture medium of PC3res cells followed by low-dosed treatment with RAD001 resulted in no growth inhibiting effect. Similar basal apoptosis rate was observed in PC3par and PC3res cells. However, late apoptosis increased in PC3par but decreased in PC3res following treatment with low-dosed RAD001. Accordingly, the number of vital cells was reduced in PC3par but augmented in PC3res cells following RAD001 treatment. PC3res accumulated in the G2/M-phase, accompanied by a significant loss of PC3res in G0/G1, compared to PC3par cells. Particularly, cdk1 and cyclin B were strongly up-regulated in PC3res. Incubation of the prostate cancer cells with siRNA against cdk1 or cyclin B distinctly diminished the protein content in both PC3par and PC3res. The specific knockdown was accompanied by a significant growth blockade of PC3par and, most importantly, of the RAD001 resistant subline PC3res. Interestingly, incubation of PC3res cells with cyclin B siRNA restored the tumor cells⬘ sensitivity to RAD001. Cell growth attenuation was now induced in this cell line by treatment with the drug. CONCLUSIONS: The results indicate for the first time that cdk1-cyclin B overexpression is related to resistance development in PC3 cells under chronic treatment with RAD001, leading to enhanced