Abstract B40: The melphalan prodrug melphalan-flufenamide shows cytotoxic effect in bladder cancer cell lines and triggers JNK and caspase-3 mediated apoptotic signaling.

Abstract

Background: Bladder cancer is the 4th most common cancer associated death in men in the western world. Cisplatin-based polychemotherapy (1st line) and vinflunine (2nd line) are the only chemotherapy regimens which show survival benefit in patients with metastatic disease. Hence, there is a major need to identify new effective treatment alternatives for patients with this disease. We have developed a prodrug of melphalan, Melphalan-flufenamide (Mel-flufen) which show enhanced cytotoxic efficacy compared to melphalan in several tumour types. Treatment with Mel-flufen gives 10–20 fold higher intracellular drug levels compared to the same dose of melphalan. Mel-flufen is currently undergoing Phase I/II clinical trials. Here we studied if this drug might be a useful in bladder cancer by analyzing its effects in a panel of bladder cancer cell lines. Material and Methods: The bladder cancer cell lines J82, RT4, 5637 and TCC-SUP were profiled for Mel-flufen and melphalan sensitivity using MTT and FMCA cell viability assay. Mel-flufen signaling was measured as caspase-3 and Bak/Bax activation in flow cytometry and PARP cleavage, JNK phosphorylation, p53 and p21WAF1/Cip1 induction on western blot respectively. Results: Mel-flufen caused a dose and time dependent inhibition of cell growth in all the four bladder cancer cell lines studied. Comparison with the parental drug revealed an enhanced cytotoxic effect of Mel-flufen at equimolar doses. Molecular profiling of the apoptotic response showed that Mel-flufen induced activation of the Bcl-2 proteins Bak and Bax followed by caspase-3 activation and induction of apoptotic morphology. Albeit Mel-flufen induced activation of p53, the cytotoxic effect was not dependent on p53 as it was also observed in p53-mutant bladder cancer cell lines. A prominent activation of JNK1/2 was observed in response to Melflufen. Importantly, this JNK activation preceded induction of Melflufen-induced apoptotic signaling and was critical for the induced cytotoxicity in these bladder cancer cell lines. Conclusion: Our data demonstrate that the melphalan prodrug Mel-flufen significantly inhibits bladder cancer cell growth in vitro. Importantly, we show that Mel-flufen is more efficient in inhibiting bladder cancer growth than the parental drug melphalan and causes a more pronounced apoptotic signaling. Taken together these data suggests that Mel-flufen may be a good candidate for bladder cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B40.