Mitochondrial apoptotic pathways

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Mitochondrial dysfunction is one of the crucial events involved in apoptotic cell death. Accumulating evidence suggests that apoptotic pathways converge at the mitochondria; signaling is initiated via a series of molecular events that culminate in the release of death factors (21). Gene polymorphisms in mitochondrial apoptotic pathways usually play a role in the apoptosis of hepatoma cells.

Heat shock protein 70 (HSP70), abundantly expressed in human tumors, protects cells from a wide range of apoptotic stimuli. HSP70 potentially modulates mitochondrial protein apoptosis-inducing factor (AIF)-induced cell death. Furthermore, HSP70 silencing has been shown to dramatically increase AIFDelta1-120 mutant (an active form of AIF after deletion of the mitochondria localization domain)-induced apoptosis (22). On the other hand, the potential pro-apoptotic molecule in HCC cells exhibits a gene polymorphism associated with mitochondrion. Fragile histidine triad (FHIT) is a confirmed tumor suppressor gene in HCC. Yu and colleagues (23) found that transactivator of transcription (TAT)-FHIT robustly inhibits growth and induces apoptosis of HCC cells in vitro; furthermore, they demonstrated that both exogenous and intrinsic apoptotic pathways are involved in TAT-FHIT-mediated apoptosis, and that this effect may be partially attenuated by mitochondrial protector TAT-BH4. These findings indicate that mitochondria play a critical role in TAT-FHIT-mediated pro-apoptotic effects in cancer cells. TAT-FHIT is a potential pro-apoptotic molecule in HCC cells. The tumor suppressive mechanism of the tyrosine aminotransferase gene has also been shown to play a pro-apoptotic role in a mitochondrial-dependent manner by promoting cytochrome-c release and activating caspase-9 and PARP (24). Moreover, a novel gene, homo sapiens longevity assurance homologue 2 (LASS2), has been isolated from a human liver cDNA library, a human homologue of the yeast longevity assurance gene LAG1 (Saccharomyces cerevisiae longevity assurance gene 1) (25). The LASS2 gene may increase intracellular H(+) of HCC cells via interaction with V-ATPase, thereby inducing cell apoptosis through the mitochondrial pathway.

Transforming growth factor-beta (TGF-beta) is a potent inhibitor of hepatocyte growth (26), and is known to induce apoptosis in both hepatocytes and HCC cell lines (27,28). One of the mediators of the TGF-beta pathway is the Smad family of genes, and candidate tumor suppressor genes in Smad2 and Smad4 gene mutations have been identified in HCC; these mutations were suspected to result from oxidative stress, as observed in mtDNA (29). More recently, Zhang and colleagues (30) found that TGF-beta1 treatment of Huh7 cells resulted in increased PDCD4 expression and apoptosis, concomitant with mitochondrial events. The human HCC cell line Huh7 transfected with PDCD4 resulted in increases of cytosolic cytochrome c and mitochondrial Bax, which was accompanied with decreases

Figure 1. Genes in the TGF-beta pathway or downstream of TGF-beta promote apoptosis due to mitochondrial changes. One of the mediators of the TGF-beta pathway is the Smad family of genes, and candidate tumor suppressor genes in Smad2 and Smad4 gene mutations have been identified in HCC; these mutations were suspected to result from oxidative stress, as observed in mtDNA. The human HCC cell line Huh7 transfected with PDCD4 resulted in increases of cytosolic cytochrome c and mitochondrial Bax, which accompanied with decreases of mitochondrial cytochrome c and cytosolic Bax, then inducing apoptosis.

of mitochondrial cytochrome c and cytosolic Bax, then inducing apoptosis. Thus, genes in the TGF-beta pathway or downstream of TGF-beta could promote apoptosis due to mitochondrial changes (Figure 1).

The tumor suppressor p53 gene plays a major role in preventing tumorigenesis by responding to both cellular stress and DNA damage; p53 mutation is frequently associated with oncogenesis (31,32). In some HCC cell lines, its activation leads to the induction of pro-apoptotic Bax expression (33,34), whereas pro-apoptotic Bax also antagonizes anti-apoptotic Bcl-2 and other anti-apoptotic molecules. The mitochondria play a central role in apoptosis, which is regulated by members of the Bcl-2 family through an intrinsic apoptosis pathway (35-37). In p53-deficient Hep3B cells, apoptosis induced by pectenotoxin-2 (PTX-2) is associated with the down-regulation of anti-apoptotic Bcl-2 members (Bcl-2 and Bcl-xL) and IAP family proteins, and the up-regulation of pro-apoptotic Bax protein, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (DR4/DR5), and mitochondrial dysfunction (38).

In contrast, interference with the p53 family network contributes to a gain of oncogenic function of mutant p53 in HCC via mitochondrial apoptotic pathway. Mutant forms of p53, including p53R143A, p53R175D, p53R175H, p53R248W, and p53R273H, can acquire anti-apoptotic gain-of-function in HCC by repressing the activity of genes that regulate both the extrinsic apoptosis pathway initiated by ligation of death receptors and the intrinsic/mitochondrial apoptosis pathway. Furthermore, p53 gain-of-function mutants significantly decrease activation of pro-apoptotic target genes by wild-type p53, TAp63, and TAp73. This contributes to the ability of cancer cells to withstand DNA damage-induced apoptosis (39). TAp63 and TAp73 are the promoters of the Trp63 gene and Trp73 gene, respectively. In general, Trp63 gene and Trp73 gene have two respective promoters that drive the expression of two major p63 and p73 isoform subfamilies: TA and N-terminally truncated (DeltaN). TAp63 and TAp73 isoforms exhibit pro-apoptotic activities, whereas members of DeltaNp63 and DeltaNp73 subfamily exhibit antiapoptotic functions. Müller and colleagues (40) found that TAp73 beta could transactivate the CD95 gene via the p53-binding site in the first intron. Inhibition of CD95 gene transactivation was one mechanism by which DeltaNp73 functionally inactivated the tumor suppressor action of p53 and TAp73 beta. Concomitantly, DeltaNp73 inhibited the apoptosis emanating from mitochondria. Thus, DeltaNp73 expression in tumors selects against both the death receptor and the mitochondrial apoptosis activity of TAp73 beta. In HCC DeltaNp63alpha and DeltaNp73 beta expression interferes

Figure 2. Mfn2 exerts apoptotic effects and inhibits proliferation in HCC cells by the mitochondrial apoptotic pathway. Overexpression of mfn2 also induced cytochrome c release to the cytoplasm by enhancing Bax translocation from the cytoplasm to the mitochondrial membrane. Furthermore, HBx was shown to inhibit p53-mediated up-regulation of mfn2 in HCC cells.

with both the death receptor and the mitochondrial apoptosis activity of the TA isoforms (41,42). DeltaNp63alpha could inhibit the activation of p53 family target genes (41), whereas DeltaNp73beta is oncogenic in HCC by blocking apoptosis signaling via death receptors and mitochondria (42). These findings suggest that DeltaNp63 and DeltaNp73 isoforms repress apoptosis-related genes of mitochondrial apoptosis signaling pathways.

The mitochondrial GTPase mitofusin-2 gene (mfn2), also named hyperplasia suppressor gene (HSG), located on chromosome 1p22.3, encodes a mitochondrial membrane protein that participates in mitochondrial fusion and contributes to the maintenance of the mitochondrial network (43,44). It exhibits a potential apoptotic effect mediated by the mitochondrial apoptotic pathway (45-47). Qu and colleagues (48) reported that heterozygosity (LOH) at mfn2 gene in HCC is associated with clinicopathological features of patients, including tumor size, age, capsule, differentiation, and t HBV infection. Mfn2 was identified as a novel direct target of p53 that could also exert apoptotic effects via Bax signaling and potentially inhibits proliferation in HCC cells (49,50). Overexpression of mfn2 also induced cytochrome c release to the cytoplasm by enhancing Bax translocation from the cytoplasm to the mitochondrial membrane (50). Furthermore, the relationship between mfn2 and HBx was examined. HBx was shown to inhibit p53-mediated up-regulation of mfn2 in HCC cells, though HBx exhibited little effect on the expression of mfn2 or P53. (51) (Figure 2).

Novel genes in the mitochondrial apoptosis signaling pathways have been identified; however, the mechanisms of the p53 family network remain unclear in HCC. The activation of p53 can cause the induction of pro-apoptotic Bax expression, which antagonizes anti-apoptotic Bcl-2. Afterwards, the Bcl-2-mediated apoptotic process is initiated by cytochrome c release from mitochondria and is executed by the caspase family (52,53). Mutant forms of p53 and mfn2 genes play a role in the network