Abstract A49: Inhibition of adriamycin-induced 5-lipoxygenase in DU-145 and PC3 prostate cancer cells confers cell death
2009-12-01

Abstract Background: The anthracyclin adriamycin (ADR) is a major antitumor agent known to cause cellular damage via a number of mechanisms including free radical formation and inhibition of topoisomerase II. However, this antitumor agent appears not to be successful in triggering apoptotic cell death in prostate cancer cell lines with high metastatic potential. In this study we investigated the effect of ADR on the 5-lipoxygenase (5-LO) pathway and examined whether the activation of 5-LO, the key determinant in biosynthesis of proinflammatory leukotrienes (LTs), is involved in development of prostate cancer cell resistance. Effects of ADR on the activation of the 5-LO pathway were also studied in regard to p53 status and expression levels of pro- and anti-apoptotic members of the Bcl-2 family in two human, prostate cancer cell lines, DU145 and PC3 (both derived from metastases). Methods: Enzymatic activity and modulations of the 5-LO product profile were analyzed by high pressure liquid chromatography. The relationship between enzymes of the 5-LO pathway and members of Bcl-2 family, before and upon the ADR treatment, were investigated by Western blot analysis and Q-PCR. Cytotoxic effect of ADR was assessed by MTT, while apoptosis was determined by caspase activation. Results: In DU145 and PC3 cells, clinically relevant concentrations of ADR were not found to induce apoptosis. Interestingly, after 24h of ADR treatment the expression of 5-LO was strongly up-regulated at the mRNA and protein levels in both cell lines, independently of their p53 status. This consequently resulted in a high ionophore-induced formation of 5-hydroxyeicosatetraenoic acid (5-HETE). The production of LTB4 was also increased, particularly in DU145 cells, although ADR treatment did not alter expression of LTA4 hydrolase. In DU145 cells ADR furthermore induced overexpression of coactosin-like protein (CLP), a small cytoplasmic 5-LO chaperone, which functions as a scaffold during Ca2+-induced 5-LO activation and up-regulates LTA4 formation in in vitro assays. In PC3 cells, ADR did not alter relatively high constitutive expression of CLP, which appeared to be critical for LTB4 production in this cell line, since CLP knock-down by short hairpin RNA completely abolished it. Increased formation of the 5-LO products observed after 24h treatment with ADR resulted in resistance to apoptosis. No changes in Bax and Bcl-2 expression levels were observed upon genotoxic stress induction. Pre-incubation of the cells with the 5-LO inhibitors, BW-4-AC and particularly Hyperforin (a natural compound isolated from the plant Hypericum perforatum (St John's wort) in clinical use) led to inhibition of 5-LO products' formation and resulted in an extensive programmed cell death engaging caspase-3 activation. Conclusions: Together, these results support a novel role of the 5-LO products in cancer cell chemo-resistance, and provide a rationale for the therapeutic use of 5-LO inhibitors to enhance cancer chemosensitivity. This may lead to development of novel pharmacological approaches for combating prostate cancer. Citation Information: Cancer Res 2009;69(23 Suppl):A49.