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

Abstract

Aims: The rate limiting enzyme for mevalonate synthesis in eukaryotic cells is 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase (Hmgcr) and products of mevalonate synthesis play a key role in tumorigenesis (Li et al., 2002). Isoprenoids have the potential of inducing cancer cell death (apoptosis) by inhibiting Hmgcr. Although isoprenoids gave promising results in the treatment of cancer in pre-clinical trials (Bifulco, 2005), effective doses were poorly tolerated in cancer patients due to toxicity. The study aims to establish a combinatory treatment of isoprenoids with Rapamycin, an mTOR (mammalian target of Rapamycin) inhibitor, suggested to be a potential chemotherapeutic sensitizer (Shi et al., 1995). The combinatory treatment will serve to sensitize tumour cells for induction of apoptosis by isoprenoids, enhancing the therapeutic index of isoprenoids. Methods: Cell culturing and pharmacological inhibitors: 3 cell lines (A549, PC3, C32) were chosen from a panel of cell lines. Pharmacological inhibitors used were Rapamycin, Limonene, Perillyl alcohol, alpha-pinene. Cytotoxicity assays (XTT) were performed on all cell lines alone or in combination and read at 490nm on microplate reader. Annexin V assay was used for apoptotic quantification by flow cytometry. Western blot analysis measures phosphorylation and activation of proteins using 4EBP and p70 S6 kinase antibodies. Results: To evaluate the effect of growth inhibition and apoptosis, cells were cultured in the presence of Rapamycin and Isoprenoids. Preliminary data show that exposure of Rapamycin triggered response in PC3 and A549 whilst response was not observed for C32. All cell lines were differently sensitive to Isoprenoid exposure. Combinatory treatment with both drugs was performed, lowering the dose of Isoprenoids needed for apoptosis. Western blot analysis was carried out and kinase activity was shown to be abrogated in presence of Rapamycin. Conclusions: We propose the synergistic combination of isoprenoids with the mTOR inhibitor, rapamycin (CCI-779) to enhance apoptosis and reduce toxicity. Hence this combinatory treatment might be an effective treatment option in patients with specific solid tumours. The identification of sensitive cell lines will allow further evaluation of molecular mechanisms that initiate apoptosis.