Clusterin (CLU) is a heterodimeric, disulfide-linked 70-80kDa glycoprotein ubiquitously expressed in many tissues. It has been implicated in several physiologic processes as well as sensitivity to chemotherapy in different types of cancer. Recent observation indicated an association between CLU expression and contradictory functions either cell survival or apoptosis. In this study we show the effect of paclitaxel (TX) on the expression and localization of different isoforms of CLU in TX-sensitive and its resistant ovarian cancer cell lines. Additionally, we studied how CLU might mediate the resistance to TX treatment in the same in vitro experimental model. We analyzed expression differences between the non-resistant (Kf) and TX-resistant (Kf-TX) ovarian cancer cells by microarray. We observed increased levels of CLU expression in the Kf-TX resistant cells compared to the Kf cells. In response to TX treatment, Kf cells responded with an increase in the precursor CLU (60 kDa) in a time and dose dependent manner, while no significant changes were evident in Kf-TX cells over this short treatment period (6, 12 and 24h). The intracellular secretory form of CLU (sCLU; 40 kDa), was upregulated as an early event in both cells, but rapidly decreased with high doses of TX in Kf cells, but not in the Kf-TX cells. Conditioned medium from both cells showed the same expression pattern as that observed in cell lysates. Long term treatment (48h) showed CLU up-regulation in both cells with low doses and finally inhibition of its expression with higher doses indicating a relation between CLU inhibition and TX-induced cell death.

Interestingly, the non-glycosylated intracellular CLU (inCLU; 50KDa) was more detectable in non-treated sensitive cells (Kf) than in resistant (Kf-TX) cells by Western blot analysis. In addition, using the specific inCLU antibody, we showed that the nuclear appearance of CLU was reduced after development of TX-resistance, this was further confirmed by sub-cellular fractionation analysis. Moreover, we observed that the CLU/Ku-70 complex, which was reported to relate to radiation-induced apoptosis, was formed in the cytosol of both cells, and rapidly increased and accumulated in the nucleus of both cells after TX treatment. However, Kf showed earlier nuclear accumulation of that complex than Kf- TX. Finally, SiRNA transfection, specifically knocked down the sCLU but not inCLU, restored the sensitivity of Kf-TX to the drug as confirmed by DNA ladder and FACS analysis. Our data indicate a close relationship between intracellular trafficking of different CLU isoforms and response to TX in ovarian cancer cells. Prevalence of nuclear CLU accumulation appears to relate to TX-induced cell death, while sCLU might be protective. CLU gene products could be a novel target for therapeutic intervention to enhance or restore chemo-sensitivity of ovarian cancer cells.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA