NE-AUA 2006 Annual Meeting, September 28 - 30, 2006, The Westin Hotel & Rhode Island Convention Center Providence, Rhode Island
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Role of Apoptosis Inducing Factor in Prostate Cancer Cell Apoptosis Induced by Cisplatin
Wenguang Zhang, PhD1, K.C. Balaji, MD2.
1University of Nebraska Medical Center, Omaha, NE, USA, 2University of Massachusetts Memorial Medical Center, Worcester, MA,

Introduction: Although Cisplatin is an effective chemotherapy drug in treatment of patients with testicular and bladder cancer, it is ineffective against prostate cancer. In this study, we explored alternate mechanisms of Cisplatin induced apoptosis in prostate cancer cells, which may provide novel targets to improve cisplatin sensitivity in prostate cancer. While the role of caspases mediated apoptosis in prostate cancer has been studied, there is currently paucity of data on recently discovered mitochondrial apoptosis-inducing factor (AIF), which induces apoptosis independent of caspases. Therefore, we explored the role of AIF in cisplatin induced cell death in prostate cancer.
Methods: We used LNCaP cells, a well characterized prostate cancer cell line for our experiments. Effect of cisplatin treatment on cell viability was assayed by MTS assay, apoptosis by TUNEL assay, DNA fragmentation, Pulsed Field Gel Electrophoresis, caspase-3 activity assay and subcellular distribution of AIF by immunofluorescence (IF) staining and immunoblot analysis. To further confirm the role of AIF in cisplatin induced prostate cancer cell death, we over expressed AIF by transfecting full length AIF cDNA. The viability experiments were done in triplicate and pairwise comparisons were carried out using Students T-test. P-values < 0.05 considered statistically significant.
Results: Whereas the dose and time dependent decrease in LNCaP cells viability following cisplatin treatment was significantly inhibited by AIF inhibitor N-acetyl L-cysteine (NAC), a non-significant inhibition was noted following treatment with caspase inhibitor ZVAD. Moreover, cisplatin induced large DNA fragmentation characteristic of AIF mediated apoptosis. Although cisplatin treatment did not result in alteration of total cellular AIF protein levels, AIF clearly translocated to the nucleus which was prevented by NAC treatment. Immunoblot analyses of the nuclear fraction of LNCaP cells treated with cisplatin confirmed increased AIF expression. Overexpression of recombinant pcDNA vector coding human AIF gene increased apoptosis compared to pcDNA vector control.
Conclusion: These findings indicate that the cisplatin can lead to AIF translocation and apoptotic cell death in the prostate cancer cells. AIF may become of novel molecule of interest in improving cisplatin sensitivity of prostate cancer cells.


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