Abstract 3189: Potent curcumin analogue FLLL-12 targets protein translational pathways to inhibit EGFR, AKT and Bcl-2: potential role in apoptosis induction.

Curcumin is the major bioactive compound isolated from the rhizome of Curcuma longa (turmeric). Despite its high margin of safety (no dose-limiting toxicity at doses up to 10 g/day in human) and efficacy against various types of cancers, the potential utility of curcumin as a chemopreventive/chemotherapeutic drug is compromised by its low bioavailability and poor selectivity. To circumvent these problems, more potent and selective curcumin analogues have been introduced. In the current study, we investigated one such synthetic analogue, FLLL-12, which was reported to exhibit potent anti-tumor activity against prostate, colon and breast cancer cell lines. However, its mechanism of growth inhibition has yet to be elucidated. Methods: MSK-Leuk1 is an oral premalignant cell line. Other cell lines used in the study are fully transformed head and neck cancer cell lines isolated from cancer patients. Genotyping was conducted to confirm the identity of the cells. SRB assay was used for measuring growth inhibition. Annexin V staining was conducted for apoptosis assay. Expression of mRNAs and proteins were measured by RT-PCR and Western blotting, respectively. Result: IC50 values and apoptosis assay results suggested that FLLL-12 was 10-25-fold more potent than natural curcumin against head and neck cancers. FLLL-12 also strongly inhibited the expression of p-EGFR, p-AKT, and Bcl-2. Overexpression of constitutively active AKT or Bcl-2 significantly inhibited FLLL-12 induced apoptosis. Interestingly, FLLL-12 not only inhibited phosphorylated EGFR and AKT but also total EGFR and AKT proteins. Mechanistic studies revealed that FLLL-12 had an insignificant effect on mRNA expression of EGFR, AKT and Bcl-2 as compared to protein expression. Pretreatment with the proteasome inhibitor MG132 failed to rescue the expression of EGFR, AKT and Bcl-2 proteins inhibited by FLLL-12 treatment. Moreover, FLLL-12 failed to inhibit EGFR and AKT protein expression or induction of apoptosis when global protein synthesis was inhibited by cyclohexamide pretreatment. These results suggest that FLLL-12 inhibited the expression of EGFR, AKT and Bcl-2 at the translational level. Finally, FLLL-12 strongly inhibited phosphorylation of mTOR and its downstream targets pS6 and p-4E-BP1 which are important for cap-dependent protein translation. Conclusion: Our results strongly suggest that FLLL-12 is a potent curcumin analog which induced apoptosis of head and neck cancer cell lines by inhibiting cap-dependent translation of survival proteins including EGFR, AKT and Bcl-2 by inhibiting mTOR-mediated protein translational pathways. Future in vivo studies using appropriate animal models are warranted for further development of this promising compound for cancer prevention and treatment. (Dr. Amin9s research is supported by R03CA159369 and Robbins Scholar Award). Citation Format: A.R.M. Ruhul Amin, Mohammad A. Rahman, Dongsheng Wang, Fadlo R. Khuri, James R. Fuchs, Zhuo G. Chen, Dong M. Shin. Potent curcumin analogue FLLL-12 targets protein translational pathways to inhibit EGFR, AKT and Bcl-2: potential role in apoptosis induction. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3189. doi:10.1158/1538-7445.AM2013-3189