Enhanced efficacy of bioactive compounds: targeting isoprenylation in cancer cells to mediate apoptosis

Abstract

The study aimed to establish a combinatory treatment of low-dose isoprenoids with Rapamycin, an mTOR (mammalian target of Rapamycin) inhibitor, and a potential chemotherapeutic sensitizer. Isoprenylation of proteins has been involved in survival and transformation of cancer cells. Isoprenoids decrease the pool of isoprene precursor molecules, downregulating signal transduction mechanisms. The combinatory treatment served to sensitize tumour cells for induction of apoptosis by isoprenoids, enhancing the therapeutic index of isoprenoids. Three cell lines, a prostate carcinoma (PC3), melanoma (C32) and non small cell lung carcinoma (AS49) were chosen from a panel of cell lines. Isoprenoids (Limonene, Perillyl alcohol and α-pinene) were added alone or in combination with Rapamycin. Cytotoxicity assays (XTT) showed that Perillyl Alcohol was the most effective isoprenoid on all cell lines and addition of 50ng/ml Rapamycin sensitized PC3 and C32 cell lines resulting in a reduced dosage of α-pinene and Limonene to reach the IC50. The sensitivity of PC3 and C32 to rapamycin is supported by the presence of mutations in PTEN known to increase the activity of PI3K/Akt/mTOR pathway. Annexin V assay was used for apoptotic quantification by flow cytometry. Interestingly PC3 and C32 showed no evidence of apoptosis at the dosages required to reach IC50s, which may be caused by highly expressed anti-apoptotic proteins in such cell lines and could be possibly induced by rapamycin treatment. Increased apoptosis induction was observed in AS49 upon exposure to Perillyl alcohol and Limonene in combination with rapamycin. We hypothesize that the combination promotes an early G 1 block in the cell cycle of AS49 cell-line through Ras/MAPK and PI3K/Akt/mTOR pathways. The results obtained in this study can be utilized in further studies to characterize mechanisms of apoptosis induction. Of interest, the cytotoxic effect of isoprenoids alone and in combination using rapamycin sensitive cell lines shall give insights into other possible mechanisms of cell death such as autophagy.