Abstract 4454: SRJ09, a semisynthetic anticancer agent, targets Ras-MAPK signaling pathway: assessment in breast and colon cancer cell lines

Abstract

Abstract SRJ09 (3,19-(2-bromobenzylidene)andrographolide) was shown to be more selective towards breast and colon cancers in USA NCI 60 cancer cell line screen, thus conferring it as lead anticancer agent (Jada et al (2008) Br J Pharmacol, 155(5):641-54). The compound was recently reported by us to be a mutant K-Ras binder (Hocker et al (2013), PNAS, 110(25):10201-6). In the present investigation, MCF-7 breast and HCT116 colon cancer cell lines were used to elucidate the mechanism of action of SRJ09. MTT cell viability assay was carried out to determine the growth inhibition by SRJ09 and other analogues of andrographolide (AGP). Cell cycle analysis was performed by flow cytometry technique. Western blot was carried out to determine the expression of cell signaling, cell cycle and apoptosis regulatory proteins. Additionally, reverse transcriptase (RT)-PCR was carried out to determine the expression of cell cycle regulatory genes. SRJ09 was approximately one-fold more potent than AGP and analogues. In terms of selectivity, HCT116 cells were more sensitive towards SRJ09 compared with MCF-7. SRJ09 was found to reduce the phosphorylated c-Raf and ERK1/2 proteins in both cell lines, with HCT116 being more susceptible towards the compound.SRJ09 induced G1 cell cycle arrest in both cell lines through up-regulation of p21 and down-regulation of CDK4 expressions, without affecting cyclin D1. However, HCT116 cells were more prone to undergo cell cycle arrest than MCF-7 cells. This finding was substantiated by western blot and RT-PCR analyses, whereby both the gene and protein expressions implicated in G1 arrest in HCT116 cells were affected at much lower concentration (3 µM) than in MCF-7. Western blot analysis revealed that SRJ09 induces apoptosis in both cell lines via p53-independent extrinsic pathway through activation of caspase 8 and with subsequent activation of Bax, Bid and caspase 9, without affecting Bcl-2. These findings were confirmed using caspase-8 inhibitor (Z-IETD-FMK), whereby HCT116 cells pretreated with the inhibitor failed to undergo apoptosis upon treatment with SRJ09. The differential sensitivity observed might be related to the K-Ras status in both cell lines, with HCT116 harboring mutant K-Ras while MCF-7 with wild type K-Ras. In conclusion, SRJ09 potentially inhibits K-Ras signaling pathway which reduces phosphorylated c-Raf and ERK1/2, thereby leading to G1 cell cycle arrest and apoptosis. Importantly, this study further supports our earlier finding that SRJ09 is a direct binder of K-Ras. This study was funded by Universiti Putra Malaysia Research University Grant Scheme (04-01-09-0713RU; 04-02-12-2017RU). Citation Format: Johnson Stanslas, Charng Choon Wong, Sreenivasa Rao Sagineedu, Shiran Mohd Sidik, Shariful Hasan Sumon, Roger Phillips, Nordin Haji Lajis. SRJ09, a semisynthetic anticancer agent, targets Ras-MAPK signaling pathway: assessment in breast and colon cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4454. doi:10.1158/1538-7445.AM2014-4454