pathologies has not been found thus far, which presents a major obstacle in efficient cancer management (Hopkins, 2008). Cancer treatment requires modulation of concrete targets, which can be compromised by the compensatory mechanisms and/or mutations (Stelling et al, 2004; Kitano, 2007). Overcoming these problems often requires high drug doses that may, on the other hand, promote deleterious effects to non-cancerous tissues (Kassouf et al, 2005). Combinations of two or more agents that exert a synergistic effect can overcome the undesirable toxicity and other side-effects associated with high doses of single drugs, allowing a reduced dosage of each compound.
We therefore tested exposure of prostate cancer cells to three agents, α-TOS, VK3, and AA, widely studied as anti-cancer compounds, alone or in combination (Chen et al, 2005; Ogawa et al, 2007; Neuzil et al, 2007b). α-TOS and VK3 were both highly cytotoxic towards prostate cancer cells, whereas lethal effects of AA were observed only at prolonged times of exposure (c.f. Figure 1). An antagonistic interaction was found for the combination of α-TOS and VK3, whereas AA did not exert any effect when combined with α-TOS. As previously described (De Loecker et al, 1993; Jamison et al, 1996; Verrax et al, 2005; Tareen et al, 2008; Beck et al, 2009) and also observed in this study, an efficient synergistic effect on cell viability was observed for the pro-oxidant mixture containing pharmacological doses of AA and a redox-active compound such as menadione (VK3), (c.f. Figure 2). Indeed, the combination of AA and the redox-cycling quinone VK3 promotes oxidative stress that may kill cancer cells (Taper and Roberfroid, 1992; Taper et al, 2001). Oral administration of the VK3–AA mixture in the ratio 1
:100 (the Apatone preparation) significantly increased the mean survival time of nude mice inoculated i.p. with the DU145 prostate cancer cells and significantly reduced the growth rate of solid tumours without inducing any significant bone marrow toxicity and pathological changes of non-tumour tissues (Jamison et al, 2005). Further, the safety and efficacy of oral Apatone supplementation were demonstrated in patients with prostate cancer resilient to standard therapy (Tareen et al, 2008). However, the potential long-term effect of Apatone on the disease progression and possible secondary side-effects are not known and remain to be investigated.
:100 (the Apatone preparation) significantly increased the mean survival time of nude mice inoculated i.p. with the DU145 prostate cancer cells and significantly reduced the growth rate of solid tumours without inducing any significant bone marrow toxicity and pathological changes of non-tumour tissues (Jamison et al, 2005). Further, the safety and efficacy of oral Apatone supplementation were demonstrated in patients with prostate cancer resilient to standard therapy (Tareen et al, 2008). However, the potential long-term effect of Apatone on the disease progression and possible secondary side-effects are not known and remain to be investigated.To reduce the pharmacological doses of the agents, we combined VK3 plus AA at concentrations that themselves do not induce apoptosis with sub-apoptotic levels of α-TOS. This combination of the three drugs was efficient in induction of cell death in a selective manner that appears to be as autoschizis cell death (c.f. Figures 3 and and4).4). The soft-agar colony-forming assay was carried out to mimic the in vivo situation, where tumour cells grow as masses. Soft-agar assay and phalloidin staining of actin have been employed to reveal a potential effect of the treatment with α-TOS, VK3, and AA, and with VK3–AA–α-TOS on the morphology of the cells. PC3 cells exposed to the combination of VK3, AA and α-TOS displayed blebs and membrane alterations related to cytoskeleton changes (c.f. Figure 4). The cells also significantly decreased their size and changed their shape similarly to those found after a combined VK3–AA treatment (Gilloteaux et al, 2005). The specificity of the anti-tumour activity of VK3 plus AA is related to their ability to induce ROS formation (Venugopal et al, 1996). We observed oxygen consumption only in the presence of both VK3 and AA. Although the sub-apoptotic dose of the combination of VK3 with AA resulted in the appearance of hydroperoxides in the extracellular compartment, intracellular generation of ROS and persistent DNA damage, the cells died only in the presence of α-TOS (c.f. Figure 5). We observed that cell death in cells treated with the combination of α-TOS, VK3, and AA proceeded without caspases activation (c.f. Figure 6). A caspase-3 independent cell death was previously observed in leukaemia cells after VK3–AA treatment (Verrax et al, 2005). This can be reconciled with the notion that peroxidation of the plasma membrane of cells may favour unregulated increase in intracellular levels of calcium as previously observed (Sakagami and Satoh, 1997), which, along with thiol oxidation, may result in mitochondrial destabilisation.
Mitochondria has recently emerged as the effective target for anti-cancer drugs (Fantin and Leder, 2006; Gogvadze et al, 2008). α-TOS, a compound epitomising the ‘mitocan' group of anti-cancer agents (Neuzil et al, 2007a), was found to induce cell death by generation of superoxide anion radicals by targeting complex II of the mitochondria respiratory chain (Dong et al, 2008, 2009). ROS, in turn, promote apoptosis by catalysing the formation of disulfite bridges between monomeric Bax, resulting in the formation of mitochondrial outer membrane channel. ROS also cause oxidation of cardiolipin, triggering the release of cytochrome c and its translocation through the Bax channel (d'Alessio et al, 2005; Neuzil et al, 2006). Mitochondrial channel in response to α-TOS can also be formed by transcriptional upregulation of Noxa, which results in formation of Bak oligomeric structures (Neuzil et al, submitted).
In addition, ROS cause destabilisation of lysosomes, presumably leading to cytosolic translocation of various proteases (Neuzil et al, 1999, 2002). Lysosomal destabilisation as well as cytosolic release of cytochrome c were observed in cells exposed to α-TOS in combination with VK3 plus AA. It was shown earlier that perturbation of lysosomes results in cell death, mostly by autoschizis that is dependant on mitochondria (Boya et al, 2003). We observed in this study that the inhibitor of lysosomal destabilisation did not attenuate mitochondrial ‘leakage', leading to cytochrome c release, and subsequent cell death (c.f. Figures 6 and and7).7). This reinforces the concept that mitochondrial destabilisation constitutes a central event in the programmed cell death such as apoptosis and autoschizis (Verrax et al, 2005). Again, this evokes the possibility that primary thiol oxidation of mitochondrial proteins, derived from an oxidative shift in the cellular redox potential (Venugopal et al, 1996), can induce mitochondrial membrane permeabilisation (Zamzami et al, 1998). Moreover, oxidation of critical thiols within the catalytic centre of caspases annihilates their latent proteolytic potential and, thus, preclude their auto-activation (Mannick et al, 2001).
We conclude that α-TOS synergistically cooperates with VK3 plus AA in the induction of prostate cancer cell apoptosis. The combination of VK3, AA, and α-TOS induces cell death that is selective for cancer cells and proceeds through a caspase-independent pathway. We propose that addition of α-TOS at sub-apoptotic doses may be considered for cancer therapy when high doses of established drugs are required.
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