Autophagy has emerged as a powerful mediator of programmed cell death, either opposing or enhancing apoptosis, or acting as an alternative form of programmed cell death, different from apoptosis [14
]. The present study shows that Cas III-ia induces cell death by both autophagy and apoptosis in rat C6 glioma cells. A microscopic analysis of cultured cells 24 h after Cas III-ia administration revealed a significant number of cells showing coexistence of both apoptosis (cell shrinkage, margination and chromatin condensation) and autophagy (autophagic vacuoles and autophagosomes).
Beclin 1 is the mammalian orthologue of the yeast Vps30/Apg6 gene, required for autophagosome formation, and is monoallelically deleted in a high percentage of human carcinomas [28
]. In MCF7 breast carcinoma cells the expression of Beclin 1 protein decreases below detectable levels. Stable transfection of Beclin 1 in MCF7 cells promotes autophagy and reduces tumorigenic capacity, suggesting that autophagic activity is associated with the inhibition of cell proliferation [32
]. Tamoxifen, a drug used to treat breast cancer, may function by activating autophagy, possibly by upregulating Beclin1 in a process mediated by ceramide [33
]. In this study, we observed the inhibition of cell viability and overexpression of the Beclin 1 protein in C6 glioma cells after Cas III-ia treatment. Our results suggest that upregulation of Beclin 1 may contribute to the antineoplastic effect of Cas III-ia.
Recent studies have shown that LC-3, a modifier protein, is processed by a unique protein activation/conjugation system, to form autophagosomal membranes during autophagy; where LC-3 becomes associated with an autophagosomal precursor to form a cup-shaped pre-autophagosome, which finally closes to form autophagosomes that engulf the cytosolic compartment, the autophagosomes fuse with lysosomes to form autolysosomes [34
]. Present results show LC-3-II formation induced by Cas III-ia in glioma C6 cells, by a mechanism which is not yet clearly understood.
LTR is an acidotropic fluorescent probe used to label and track acidic organelles in living cells, including lysosomes, autophagosomes, late endosomes and, to a lesser extent, early endosomes less acidic than other organelles [21
]. An increment in LTR-flouresence represents an increase in autophagosomes and autolysosomes [21
]. In our study, we observed by confocal microscopy a significant increase in the size and number of lysosomal/autophagosomal compartments in response to all doses of Cas III-ia, as compared with controls.
PI3K is a conserved family of lipid kinases that catalyze the phosphorylation of position 3 on the inositol ring of phosphoinositides [35
]. They produce lipids involved in cell proliferation, differentiation, apoptosis, autophagy, cytoskeletal organization, and membrane trafficking. The drug 3-MA commonly used to inhibit the autophagic pathway [36
] interferes with the activity of class III PI3K by interrupting autophagy at the sequestration step [35
]. In our study, 3-MA enhanced cell death induced by Cas III-ia in malignant glioma cells. It seems that autophagy induced by Cas III-ia may antagonize or delay apoptosis; thus, inhibition of autophagy by 3-MA may increase the sensitivity of the cell to cell death signals. Similarly, it has been shown that the inhibition of N-(4- hydroxyphenyl) retinamide-induced autophagy enhances cell death in malignant glioma cells [38
]. Further studies have suggested that inhibition of autophagy induced by radiation/arsenic trioxide/temozolamide decreases survival of glioma cells [28
] and that autophagy antagonizes cell death [41
]. Our results suggest that inhibition of autophagy prevents the removal of damaged mitochondria, promoting loss of
Ψm and subsequent ROS generation, thereby accelerating cell death. When autophagy is inhibited, enhanced cell death may be coupled to an increase in mitochondrial depolarization and ROS generation, thereby increasing mitochondrial damage, and leading to the release of cell death-inducing molecules [38
] such as cyt c, SMAC/Diablo and AIF, which then activate the caspase-dependent or-independent apoptotic pathways.
This work also investigated if Cas III-ia induces apoptosis of C6 glioma cells. Tunnel assay results showed that Cas III-ia induced apoptosis, with most of the cells positive at 10, 15 and 20 μg/ml, and a slight decrease in cell viability at 5 and 10 μg/ml of Cas III-ia, determined by mitochondrial activity and mitochondrial membrane potential. One possible explanation of these results is that the TUNEL assay is not specific for cell death by apoptosis, since it may stain both apoptotic and autophagic cells [44
]. It has been reported that autophagy induced by 4-HPR is associated to slow loss of
Ψm, while apoptosis is associated to rapid loss of
]. Another possible explanation of the decrease in mitochondrial activity and mitochondrial membrane potential at 5 and 10 μg/ml of Cas III-ia is that Cas III-ia may initiate apoptosis by an extrinsic pathway and subsequently activate an intrinsic pathway, since Cas III-ia induces the activation of caspase 8 (the initiating caspase via death receptors) and capase 3, formation of Bidt and Bax; all of these markers initiating at low concentrations. On the other hand, a pronounced fall in mitochondrial membrane potential was detected, and a release of cytosolic cyt-c at high concentrations.
However, abrogation of caspase activation did not prevent cell death; suggesting that the antineoplastic effect of Cas III-ia can be considered as non-apoptotic cell death or caspase-independent cell death. In a previous report we showed that another Casiopeina, the Cas IIgly [Cu(4,7-dimethyl-1,10-phenanthroline)(glycine)(H2
, may induce apoptosis in CH1 cells, with no evidence of DNA laddering and independent of caspase activation [5
]. In C6 glioma cells, Cas IIgly also induces apoptosis by a caspase-independent mechanism, mediated by apoptosis induction factor (AIF) and endonuclease G [45
]. Our findings suggest that Cas III-ia induces autophagy and apoptosis, both processes being caspase-activation independent. Similarly, TNFα, a member of the apoptosis-inducing family, stimulates autophagy and apoptosis of T-lymphoblastic leukemia cells, independent of caspases [46
These results show that neither selective pharmacological inhibition of apoptosis nor of autophagy prevented the antineoplastic effects on glioma cells induced by Cas III-ia, suggesting that both pathways are essential in the cell death process.
Previous studies have shown that ROS may serve as signaling molecules that directly or indirectly activate both autophagy and apoptosis. Overexpression of TrkA diminishes catalase activity, leading to the accumulation of ROS and subsequent autophagy and apoptosis [47
]. Moreover, under starving conditions, ROS oxide cysteine residues of Atg4, induce autophagy and activate the transcription of autophagy-related genes, such as Beclin1 [19
]. In our study, Cas III-ia induced ROS generation, and reduced SOD1, SOD2 and catalase activity. Pretreatment with N
-acety-L- cysteine (NAC) showed a protective effect on Cas III-ia induced cell death. Moreover, overexpression of Beclin 1 and Bax induced by Cas III-ia were almost completely inhibited by NAC, suggesting that Cas III-ia induces autophagy and apoptosis by the generation of ROS.
Various stimuli activate JNK, which participates in the regulation of fundamental cellular pathways such as autophagy and apoptosis [49
]. JNK phosphorylates several proteins of the Bcl-2 family, resulting in inhibition of the antiapoptotic activity of Bcl-2 and Bcl-xL,
and the activation of Bax [50
]. Interestingly, it has been shown that the activation of JNK results in the phosphorylation of Bcl-2 which enhances autophagy and cell survival by disrupting the interaction between Bcl-2 and Beclin1, while prolonged Bcl-2 phosphorylation mediated by JNK promotes apoptosis [51
]. Futhermore, as JNK phosphorylates the c-jun transcription factor it promotes the upregulation of autophagic and apoptotic genes, such as Beclin 1 and Fas [52
]; also, JNK induces the expression of Atg7, a crucial mediator of autophagosome formation [48
]. In agreement with these findings, we demonstrated that Cas III-ia induces JNK activation, phosphorylation of c-jun and expression of Beclin 1, Atg 7 and Bax. Pharmacological inhibition of JNK prevented the antineoplastic effect of Cas III-ia. We also found that ROS generation mediates the activation of JNK in the pathway of Cas III-ia –induced cell death (Figure ).
Figure 9 Suggested pathway initiated by Cas III-ia leading to autophagy and apoptosis in C6 glioma cells. Cas III-ia may cause oxidative stress by JNK activation. JNK can phosphorylate Bcl-2 and release Beclin 1 enhancing autophagy. Alternatively, JNK can induce (more ...)
Most antineoplasic drugs against glioma are highly toxic and have limited efficacy, as they also affect normal cells. Lipopholic cation drugs concentrate into mitochondria due to their negative electric membrane potential; the higher plasma and mitochondrial membrane potentials of tumor cells may enhance the selective targeting by Cas III-ia of tumor cells, particulary inside mitochondria. Such is the case of AS-30D hepatoma mitochondria, which exhibit higher mitochondrial membrane potential values than those from normal hepatocytes. Indeed, AS-30D and HCT-40 cells in culture selectively die within 48 h of exposure to Cas III-ia, co-cultured normal fibroblasts survive [7
] the effect of Cas III-ia (5, 10, 15, and 20 μg/ml). In these experiments, at a 5-10 μg/ml dose of Cas III-ia, cell viability was 100%; when the dose was increased to 15 μg/ml, viability was 90%, and at 20 μg/ml, it fell to 83%, suggesting that the metabolic effect of Cas III-ia at 5-10 μg/ml doses is fairly specific against malignant cells.