Dong et al: Vitamin E analogues as anti-cancer agents treatment, with accumulation of apoptotic cells in sub-G1. In MG63 cells, !-TOS induced cell accumulation in the S/G2 phase, and this was associated with disappearance of cells in G1, similarly as observed in SAOS cells. In order to evaluate the molecular mechanism involved in the cell cycle arrest caused by !-TOS, expression of the cell cycle regulatory proteins that control the S/G2 progression was examined. Treatment of SAOS and U2OS cells with !-TOS did not affect the expression of cyclin A and cyclin E, which was similar in the untreated cells for up to 72 h of incubation. Conversely, treatment of MG63 cells with !-TOS caused a reduction in both cyclin A and cyclin E protein levels. The above results showed that vitamin E analogs, epitomized by !-TOS, exert a potent modulatory activity towards cell cycle progression and that different cell types respond differently to the agent, in particularly as shown by arrest in different stages of cell cycle. Although these differences point to possibly different targets for !-TOS in various cells, the vitamin E analog does affect the cell cycle progression, and this in its own right inhibits cell proliferation resulting in suppression of tumor growth, and/or amplifies the apoptogenic signaling pathways.
A. Inhibition of cell cycle progression by vitamin E analogs Several reports implicated inhibition of the cell cycle progression as a means by which vitamin E analogs may induce apoptosis or inhibit proliferation of cancer cells and/or sensitize them to other anti-cancer drugs. Ni et al, (2003) showed that !-TOS inhibits proliferation of prostate cancer cells by down-regulating expression of several critical cyclins and the cognate cyclin-dependent kinases (CDK), resulting in hypo-phosphorylation of the Rb protein and a G1/S arrest. Cell cycle arrest and apoptosis were also induced by !-TOS in osteosarcoma cells via activation of p53 and reduced expression of the transcription factor E2F1, critical for the G1/S transition (Alleva et al, 2006). Further, exposure of osteosarcoma cells to !-TOS promoted a prolonged arrest at the S/G2 border, sensitizing the cells to methotrexate-induced apoptosis (Alleva et al, 2005). These findings can be reconciled with an earlier report, in which !-TOS suppressed proliferation of breast cancer cells by inhibiting the E2F1-dependent trans-activation via increased binding of cyclin A (Turley et al, 1997). Apoptosis induction and inhibition of proliferation by !-TOS have been shown for malignant mesothelioma cells (Tomasetti et al, 2004), the latter paradigm being due to selective disruption of the FGF-FGFR autocrine signaling loop, most likely affected by modulation of the E2F1 and egr-1 trans-activation activity (Stapelberg et al, 2004, 2005). These are exciting results, since malignant mesotheliomas cannot be treated at this stage and since we found that !-TOS shows a strong anti-mesothelioma effect in animal models (Tomasetti et al, 2004; Stapelberg et al, 2005). Thus, proliferation and apoptosis are intimately coupled, and cell cycle modulators can influence both cell division and apoptosis (Vermeulen et al, 2003). The cell cycle is coordinately controlled by CDKs and their cyclin partners, whose levels fluctuate throughout the cell cycle. The pRb pathway plays a central role in cell proliferation by modulating the activity of the transcription factor E2F (Dimova and Dyson, 2005). E2F1 can signal p53 phosphorylation that is coincident with p53 accumulation and apoptosis (Rogoff et al, 2002). The p53 gene is frequently lost or mutated in many cancers, and lack of functional p53 is accompanied by elevated rates of genomic instability, rapid tumor progression and resistance to anti-cancer drugs and radiation (Weller et al, 1998). Alleva et al, (2006) used three human osteosarcoma cell lines, the SAOS and U2OS cells carrying the wildtype p53 gene, and the mutant p53 cell line MG63. They showed that !-TOS markedly inhibited cell proliferation in MG63 cells without affecting cell growth in both SAOS and U2OS cells. In SAOS cells, !-TOS induced cell accumulation in S/G2 phase coincident with a decrease of cells in G1, which was observed after 24 h of treatment. However, the highest !-TOS concentration was able to induce cell death at prolonged times of drug exposure. The U2OS cell line responded to !-TOS treatment by a transient accumulation of cells in the G1 phase. Higher concentration of !-TOS induced cell death after 48h of
B. The c-Jun pathway as a target for apoptosis induced by vitamin E analogues Several signaling pathways that have been shown to play a role in modulation of apoptotic signaling appear to be affected by vitamin E analogs. Of these the c-Jun pathway has been investigated in more detail due to its tight association with modulation of apoptotic pathways (Liu and Lin, 2005). The effect of !-TOS on the activity of the c-Jun NH2-terminal kinase (JNK) pathway up-stream components was investigated (Zu et al, 2005). The vitamin E analog markedly increased the level of expression of the Ask1, GADD45, Sek1, and phospho-Sek1 proteins, of which Ask1 and GADD45 are associated with the cell membrane. Consistent with these findings, the phosphorylated form of JNK was also noticeably increased, although the expression level of total JNK was not affected. Activated Ask1 and GADD45 phosphorylate the Sek1 protein that then leads to phosphorylation of JNK itself. In relation to this effect, the protein Bim, that is normally in the cytosol, tranlocates during apoptosis to the mitochondrial membrane, where it binds to Bcl-2 and BclxL. Prior to this tranlocation, Bim is phosphorylated by JNK (Kirschnek et al, 2005). Thus, JNK activation leads to antagonization of the anti-apoptotic function of proteins like Bcl-2 and Bcl-xL, a mechanism permitting cytosolic translocation of mitochondrial mediators of apoptosis. Activation of c-Jun NH2-terminal kinase by !-TOS has also been shown for gastric cancer cells (Wu et al. 2004a), and it may amplify the mitochondrial apoptosis signaling pathway, as shown for prostate cancer cells (Zu et al. 2005).
C. Akt, NF"B and other pro-survival pathways as a target for vitamin E analoginduced apoptosis 40