Protein prenylation is a post-translational addition of a lipophilic farnesyl or geranylgeranyl moiety derived from the pyrophosphate substrates, intermediates of the mevalonate pathway. Prenylation of Ras-related small GTP-binding proteins and heterotrimeric G proteins constitute protein activation events associated with cellular proliferation. The monoterpenes limonene and perillyl alcohol inhibit protein isoprenylation resulting in cell cycle arrest and induction of apoptosis. Clinical trials of d-limonene and perillyl alcohol resulted in dose limiting toxicity. Reducing the dosage of isoprenoids, while maintaining the antiproliferative effect is a challenge. Interestingly, the limiting factor of the mevalonate pathway, HMG CoA reductase, is controlled at translation level and hence sensitive to mTOR activity. The purpose of this study was to investigate the dose response using a combinatory treatment of isoprenoids and rapamycin (mTOR inhibitor), on various solid tumour cell line models. 3 solid tumour cell lines, namely PC3 (prostate) C32 (melanoma) and A549 (lung), were chosen on the basis of sensitivity to the mTOR inhibitor, rapamycin. Cytotoxicity assays using XTT were performed on the cell lines treated with Limonene, Perillyl alcohol and α-Pinene. In this study XTT assays were used to quantify viable cells after treatment with Isoprenoids and Rapamycin alone or in combination. Dosages at IC50s were used to measure apoptosis by Annexin V staining and flow cytometry. The C32 and PC3 cell lines were sensitive to rapamycin treatment, resulting in a decrease in cell viability by more than 20%, but retaining a constant growth curve thereafter. A549 was not sensitive to rapamycin at all concentrations tested. For all combinatory treatments, 50ng/ml rapamycin was selected. The viability of PC3 and C32 cell lines decreased significantly by the combinatory effect of 50ng/ml rapamycin and isoprenoids. Although cell death was enhanced after sensitisation with rapamycin, other mechanisms of loss of cellular viability, other than apoptosis, have a major role. Our results show a statistically significant reduction in IC50 of various isoprenoids, following pre-sensitization with 50ng/ml of rapamycin in the prostate cell line (PC3) and the melanoma cell line (C32). Hence this novel combination of drugs targeting two mechanisms that converge on a common target, provide a higher efficacy compared to using either drug on its own. This merits further investigation to characterise the mechanism/s of viability suppression in the solid tumour cellular models.
Buhagiar et al. (Sun,) studied this question.