Abstract 3694: Selective killing of cancer cells by the styryl lactone (R)- goniothalamin is mediated through glutathione conjugation, oxidative stress and a marked reactivation of the R175H mutant p53 protein

Abstract

Abstract Goniothalamin (GTN) is a secondary metabolite styryl lactone isolated from several species of the tropical medicinal tree Goniothalamus. GTN has been shown to be cytotoxic and induce apoptosis in many cancer cell lines. This study sought to define the molecular basis underlying the antiproliferative actions of GTN. We synthesized the R and S enantiomers of GTN and found the R form to be more cytotoxic against a panel of breast cancer and lung cancer cell lines. The IC50 of GTN against breast cancer cell lines was in the range of 10-20 μM and interestingly, the SKBR3 cells, which harbor a R175H mutation in p53 were more sensitive to the drug. In contrast, the normal breast epithelial MCF10A cells were not killed by GTN, and N-acetylcysteine prevented cell-killing, indicating the ROS involvement. Indeed, the flow cytometry and cell staining with DCF-DA showed high levels of ROS generation, and this was accompanied by significant S-glutathionylation of bulk proteins. Other studies showed that GTN forms conjugate with glutathione with ease and deplete GSH levels in cells. Because p53 is a redox-sensitive protein, we hypothesized that the redox imbalance induced by GTN may affect the structure of the R175H mutant p53 protein, and account for greater cytotoxicity. We also engineered the p53-null H1299 lung cancer cells to stably express the R175H mutant protein by lentiviral technology as an isogenic model. The conformation-specific antibodies for p53, namely Pab1620 that recognize the wt-p53 and pab420 that detects the mutant p53 were used for validating the p53 restoration. Immunoprecipitation and immunostaining using confocal microscopy showed that GTN treatment of R175H p53-containing cells results in a reciprocal loss of mutant protein and increase of wt-like protein. Further, the EMSA revealed a time-dependent restoration of DNA-binding for the mutant p53, which was accompanied by the induction of p53 target genes. The changes were associated with a G2/M arrest and significant apoptosis. Increased levels of apoptotic markers suggested a priming action of GTN on cell death pathways. GTN also suppressed the SKBR3 cell migration and invasion at 5-10 μM. In SKBR3 xenografts developed in nude mice, there was a marked tumor growth delay with either GTN alone or in combination with cisplatin. Our results shed light on the multiple mechanisms, including glutathione depletion, generation of redox imbalance, protein glutathionylation and p53 reactivation in the GTN cytotoxicity. We suggest that GTN-induced oxidative milieu facilitates a functional restoration of the mutant p53 through a thiolation of the redox-sensitive cysteines present in the DNA-binding domain. Our study will help to establish redox-perturbation as a paradigm for reactivation of the defective tumor suppressor (supported by a CPRIT grant [RP130266] to KSS). Citation Format: Surendra R. Punganuru, Hanumantha Rao Madala, Debasish Basak, Kalkunte S. Srivenugopal. Selective killing of cancer cells by the styryl lactone (R)- goniothalamin is mediated through glutathione conjugation, oxidative stress and a marked reactivation of the R175H mutant p53 protein. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3694.