The Cytotoxic Mechanism of RL112 in Ovarian Cancer Cells

Abstract

Background: Ovarian cancer is the most lethal gynecological malignancy among women, and there is limited treatment. The search for effective therapies for ovarian cancer has led to the emergence of natural compounds such as curcumin (diferuloylmethane), the primary bioactive compound of turmeric (Curcuma longa). Although curcumin’s anticancer activity in various in vitro and in vivo models of ovarian cancer is promising, its clinical application is limited by low solubility and bioavailability. Therefore, developing novel curcumin analogues circumventing the limitations of curcumin have been the focus of research groups. This lab has previously developed a cyclohexanone curcumin analogue 1-methyl-3, 5-bis(3’-nitrobenzylidene)-4-piperidone (RL112), which exhibited enhanced anticancer activity towards ovarian cancer cells compared to curcumin. This project was designed to elucidate the cytotoxic mechanism of RL112 in SKOV3 ovarian cancer cells.

Methods: SKOV3 cells were used as a model of ovarian cancer. An SRB assay determined the IC50 value for RL112. Flow cytometry measured the effect of RL112 (1 M) on cell cycle progression and apoptosis induction. To further clarify the cytotoxic mechanism of RL112, the expression of key proteins involved in cell signalling were determined using Western blotting at 12 and 24 h time points. Results: RL112 exhibited potent anticancer activity in SKOV3 cells, with an IC50 value of 1 M. Mechanistic studies demonstrated that RL112 (1 M) induced cell cycle arrest in the G2/M phase. Moreover, RL112 (1 M) caused apoptosis of SKOV3 cells in a time-dependent manner. Specifically, there was a 2- and 4-fold increase of apoptotic cells at 24 and 48 h, in treated cells compared to DMSO (0.1%) controls, respectively. Western blotting revealed an increase in the phosphorylation status of p38 MAPK at 12 (36 ± 5%) and 24 (61 ± 5%) h, compared to control. Additionally, RL112 (1 M) modulated components of the PI3K/Akt/mTOR pathway in SKOV3 cells. Notably, at 24 h the expression of p-AKT and p-mTOR were 2-fold lower than in controls. RL112 also suppressed the transcriptional activity of NFB and -catenin. Conclusions: The findings provide evidence that RL112 is cytotoxic to cisplatin-resistant (CR) SKOV3 cells. Moreover, its mechanism of action appears to involve the suppression of PI3K/AKT/mTOR signalling, activation of p38 MAPK and downregulation of transcription factors (NFB and -catenin).