Abstract 29: Potent curcumin analog FLLL12 induces apoptosis in lung cancer cells through death receptor-5-dependent pathway

Abstract

Abstract Background: Drug-associated toxicity is one of the major challenges in the management of cancer patients. Unlike chemotherapy drugs, the safety of natural compounds such as curcumin has been well established. However, the potential use of curcumin in cancer treatment has been compromised by its low bioavailability, limited tissue distribution, and rapid biotransformation leading to low efficacy. To circumvent these problems, more potent and bioavailable analogs have been synthesized. In the current study, we investigated the mechanism of anti-tumor effect of one such analog, FLLL12, in lung cancer cells. Methods: A panel of premalignant and malignant lung cancer cell lines was used for the study. SRB assay was used to measure cell growth inhibition and IC50. Annexin-V staining was conducted for apoptosis assay. Expression of mRNAs and proteins were measured by RT-PCR and Western blotting, respectively. Small molecule chemical inhibitors and siRNA-mediated knockdown strategies were used to inactivate and shut down the expression of the relevant proteins, respectively. Results: IC50 values (0.63-1.67 μM for FLLL12 as compared to 6.06-12.4 μM for curcumin, depending on the cell lines) and apoptosis results (annexin V staining and cleavage of PARP and caspase 3) suggest that FLLL12 is 5-10-fold more potent than curcumin against lung cancer cells. Moreover, FLLL12 induced the expression of death receptor-5 (DR5). Ablation of the expression of the components of the extrinsic apoptotic pathway (DR5, caspase 8 and BID) significantly protected cells from FLLL12-induced apoptosis as evidenced by reduced annexin V staining (p = 0.0008, p = 0.0001 and p = 0.0007 for DR5, caspase 8 and BID, respectively) and cleavage of PARP and caspase 3. Analysis of mRNA expression by RT-PCR revealed that FLLL-12 had no significant effect on the expression of DR5 mRNA. Interestingly, inhibition of global phosphatase activity by phosphatase inhibitor cocktail (PIC) completely abolished DR5 expression and significantly inhibited apoptosis (p = 0.0007 and p = 0.001, respectively) and the cleavage of casepase-3 and PARP. Similarly, inhibition of protein tyrosine phosphatases (PTPs) by sodium orthovanadate, but not by the alkaline phosphatase inhibitor imidazole, inhibited DR5 expression, apoptosis (p = 0.006) and cleavage of caspase-3 and PARP, suggesting the involvement of PTPs in the regulation of DR5 expression. FLLL12 also induced the expression of p53 and p73. However, inactivation of these proteins with their dominant negative construct or siRNA had no significant effects on apoptosis induction. Conclusions: Our results strongly suggest that FLLL12 induces apoptosis of lung cancer cell lines by posttranscriptional regulation of DR5 through activation of protein tyrosine phosphatase(s). This study was supported by NCI R03 CA171663, NCI P50 CA128613 and Robbins Scholar Award of Winship Cancer Institute of Emory University. Citation Format: Abedul Haque, Mohammad A. Rahman, James R. Fuchs, Zhuo G. Chen, Fadlo R. Khuri, Dong M. Shin, A.R.M. Ruhul Amin. Potent curcumin analog FLLL12 induces apoptosis in lung cancer cells through death receptor-5-dependent pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 29. doi:10.1158/1538-7445.AM2015-29