TRAIL Enhances Apoptosis of Human Hepatocellular Carcinoma Cells Sensitized by Hepatitis C Virus Infection: Therapeutic Implications

Hepatitis C virus (HCV) infection causes chronic liver diseases leading to hepatocellular carcinoma (HCC) and liver failure. We have previously shown that HCV sensitizes hepatocytes to mitochondrial apoptosis via the TRAIL death receptors DR4 and DR5. Although TRAIL and its receptors are selective targets for cancer therapy, their potential against HCC with chronic HCV infection has not been explored yet. Here we show that HCV induces DR4/DR5-dependent activation of caspase-8 leading to elevation of apoptotic signaling in infected cells and also present TRAIL effect in HCV-induced apoptotic signaling. HCV induced proteolytic cleavage of caspase-9 by stimulating DR4 and DR5, resulting in subsequent cleavage of caspase-3. Further, HCV-induced proteolytic cleavage in caspase-8, caspase-9, and caspase-3 was enhanced in the presence of recombinant TRAIL. HCV-induced cleavage in caspase-9 and increase in caspase-3/7 activity was completely suppressed by silencing of either DR4 or DR5. Perturbing DR4/DR5-caspase-8 signaling complex by silencing DR4 and DR5 or by chemical inhibitor specific to caspase-8 led to decrease of HCV-induced cleavage of poly(ADP-ribose) polymerase (PARP), a substrate for caspase-3 during apoptosis, indicating the functional role of caspase-8 in HCV-induced apoptotic signaling network. Furthermore, TRAIL enhanced PARP cleavage in apoptotic response induced by HCV infection, indicating the effect of TRAIL for the induction of selective apoptosis of HCC cells infected with HCV. Given the importance of apoptosis in HCC development, our data suggest that HCV-induced DR4 and DR5 may be considered as an attractive target for TRAIL therapy against HCC with chronic HCV infection.


Introduction
Hepatitis C virus (HCV) infects about 170 million people worldwide [1][2][3][4]. In the course of persistent infection, inflammation forms the pathogenetic basis of chronic hepatitis that can lead to fibrosis, which can progress to cirrhosis and, eventually, hepatocellular carcinoma [5]. It has been suggested that dysregulation of the apoptotic process in infected cells might be crucial for establishing chronicity, the failure of antiviral treatment, fibrosis progression, and neoplastic transformation [6].
In addition to inducing apoptosis, these death receptors initiate independent intracellular signaling cascades through mitochondrial dysfunction. Mitochondrial dysfunction plays a critical role in integrating death receptor-initiated apoptosis into a final common pathway by activating a caspase-dependent apoptosis [13][14][15][16]. TRAIL binding causes the formation of death-inducing signaling complex, resulting in the activation of caspase-8. Active caspase-8 can trigger direct activation of caspase-3 and cleavage of Bid, followed by mitochondria-dependent activation of caspase-9 via cytochrome C release and apoptotic protease activating factor-1 (Apaf-1) activation [17]. However, the mechanism by which caspases are activated is still not well defined in hepatitis C virus infection.
We recently demonstrated that co-infection with HCV and HIV stimulates DR4 and DR5 gene expression and subsequently activates caspase-3 signaling pathway, resulting in induction of mitochondrial apoptosis of human hepatocytes [18]. In this study, we demonstrate the significant contribution of caspase-8 activation to HCV-induced DR4/DR5-dependent hepatocyte apoptosis.

Real-time qRT-PCR
To analyze the expression levels of DR4 and DR5, total cellular RNA was extracted from Huh7.5.1 cells using RNeasy Mini kit (Qiagen) and subsequently, complementary DNA was synthesized by using GeneAmp RNA PCR kit (Applied Biosystems) according to the manufacturer's instructions. Power SYBR Green PCR Master Mix (Applied Biosystems) was used to quantify the cellular RNA levels. The following primer sets were used for RT-PCR: DR4 forward, 59-CTGAGCAACGCAGACTCGCTGTCCAC; DR4 reverse, 59-TCAAAGGACACGGCAGAGCCTGTGCCA; DR5 forward, 59-GGGAGCCGCTCATGAGGAAGTTGG; DR5 reverse, 59-GGCAAGTCTCTCTCCCAGCGTCTC; GNB2L1 forward, 59-GACCATCATCATGTGGAAACTGA; GNB2L1 reverse, 59-CCGTTGTGAGATCCCAGAGG. GNB2L1 was used as an internal control. Real-time qPCR was conducted by using a StepOnePlus Real-Time PCR System (Applied Biosystems).

Statistical analysis
Statistical analysis was performed using a paired student's t-test. Results were expressed as means 6 standard deviation of at least three sample replicates.

HCV infection stimulates DR4 and DR5 gene expression
We have recently shown that HCV infection activates DR4 and DR5-mediated intrinsic apoptotic signaling in human hepatocytes [18], consistent with other reports [19][20][21]. To evaluate critical determinants on DR4/DR5-dependent apoptotic signaling network induced by HCV infection, we first examined DR4 and DR5 gene expression. As shown in Figs. 1A and B, HCV infection stimulated the level of both DR4 and DR5 mRNA expression, consistent with previous reports [18,19], and induced consistent increase in DR4 and DR5 protein expression as evidenced by Western blotting with antibodies specific to DR4 and DR5 (Fig. 1C). These results indicate that HCV infection stimulates DR4 and DR5 gene expression to promote DR4/DR5-dependent apoptotic signaling.

HCV infection elevates DR4/DR5-mediated PARP cleavage via caspase-8 activation
PARP cleavage by caspase activation is a hallmark of apoptosis [27,28]. Thus, we investigated proteolytic cleavage of PARP via DR4/DR5-mediated caspase activation in HCV-infected Huh7.5.1 cells. As shown in Fig. 3A, a notable increase in cleaved PARP level was detected in HCV-infected cells compared to uninfected cells (see lane 3). This increase was further augmented by treatment with recombinant TRAIL (Fig. 3A, lane 6), suggesting that TRAIL may induce enhanced apoptosis via DR4 and DR5 receptors of HCV-infected cells. Interference of caspase-8-mediated apoptotic signaling network by silencing either DR4 or DR5 prevented HCV-induced PARP cleavage (Fig. 3B). Next, we examined whether HCV-induced PARP cleavage is mediated by caspase-8 activation using caspase inhibitors specific to caspase-1/ 3, caspase-8, and caspase-9, respectively. As shown in Fig. 3C, HCV-induced increase in cleaved PARP was suppressed by inhibiting caspase activity. Here, we note that caspase-8 activation, an upstream effector of caspase-9 and caspase-3, mediates DR4/ DR5-mediated PARP cleavage in HCV-infected cells. We also analyzed that HCV-induced PARP cleavage is dose-dependently promoted by TRAIL treatment (Fig. 3D). By inhibiting caspase-8 activity, the functional significance of caspase-8 for DR4/DR5dependent apoptotic signaling in HCV-infected cells was revealed through suppression of HCV-induced Bid cleavage and subsequent caspase-9 cleavage (Fig 4). Caspase-8 activation promotes cleavage of Bid and subsequent translocation of truncated Bid to  . HCV-induced caspase-8 activity modulates caspase-9 cleavage. Huh7.5.1 cells were infected with HCVcc in the absence or presence of caspase-8 inhibitor (20 mM) for 72 h before harvest. The expression levels of truncated Bid (t-Bid) and cleaved caspase-9 were analyzed by Western blotting with antibodies specific to t-Bid and cleaved caspase-9. a-tubulin was used as an internal loading control. The relative intensity of t-Bid and cleaved caspase-9 normalized to atubulin was analyzed by ImageJ. Samples: MC, media control. doi:10.1371/journal.pone.0098171.g004 Caspase-8 Is Required for Hepatocyte Apoptosis in HCV PLOS ONE | www.plosone.org the mitochondria, eventually resulting in apoptosis via induction of cytochrome C release [17,29]. Together, these results demonstrate that HCV infection promotes DR4/DR5-mediated PARP cleavage via translocation of caspase 8-truncated Bid.

Discussion
Apoptosis, or programmed cell death, is a process in which cells activate intracellular death pathways to terminate themselves systematically in response to a wide variety of stimuli [30,31]. Previous studies have shown that the severity of liver damage in chronic HCV is associated with the degree of hepatocyte apoptosis [32,33]. However, the contribution of apoptosis or the molecular mechanisms that cause liver cell damage during HCV infection have not yet been clearly defined. Recently, death receptor DR4/ DR5 has been recognized as a specific mediator of HCV-induced hepatocyte apoptosis [34][35][36]. Thus, we sought to evaluate critical determinants, particularly caspase-8 activation, of the DR4/DR5dependent apoptotic signaling pathway induced by HCV infection.
A therapeutic potential of TRAIL for liver cancer with persistent HCV infection has been overlooked. TRAIL receptors including DR4 and DR5 are very attractive targets for cancer therapy [37]. Hence, in this work, our research purpose was (1) to examine the role of caspase-8, one of important determinants of DR4/DR5-mediated apoptosis signaling network during HCV infection as well as (2) to illuminate the possibility of TRAIL use for therapy of hepatocellular carcinoma caused by HCV infection.
It is commonly assumed that virus clearance requires the destruction of infected cells by virus-specific cytotoxic T-lymphocytes via the CD95 receptor, TNF-receptor-1 and TRAIL receptors DR4 and DR5. If death receptor-mediated apoptosis works adequately, it is beneficial for the organism and may lead to elimination of virus-infected cells. However, dysregulation of this system might result in liver damage. Therefore, TRAIL receptors, which selectively kill virus-infected cells while leaving uninfected cells intact, have evolved as a prime physiological mechanism in order to selectively eliminate virus-infected cells [38][39][40]. We and others have previously shown the stimulation of DR4 and DR5 in the liver of patients with chronic HCV as well as in HCV-infected cells [11,18,21,39,41]. In contrast, few reports have suggested that HCV infection does not affect DR4 or DR5 expression [20,42]. In this study, we clarify that HCV infection stimulates DR4 and DR5 gene expression at both the transcriptional and translational levels in Huh7.5.1 cells, as evidenced by qRT-PCR and Western blot analysis (Fig. 1). Consistent with our results, Zhu et al have shown that HCV infection upregulates DR4 and DR5 expression levels and triggers TRAIL-mediated apoptosis in human hepatocellular carcinoma LH86 cells [21]. They also demonstrated that TRAIL expression is correlated with the number of apoptotic LH86 cells [21].
A number of liver diseases are characterized by elevated caspase activation and apoptosis. Caspase is a cysteine protease which plays a critical role in initiating and executing the apoptotic signaling by degrading several cellular components, resulting in the apoptotic phenotype [43]. In death receptor-mediated apoptosis, CD95L-, TNF-and TRAIL-induced signal transduction leads to the formation of a death-inducing signaling complex which results in the proteolytic processing of caspase-8 and activation of downstream effector caspases such as caspase-3, caspase-6, and caspase-7, known as an extrinsic pathway [38,44,45]. The intrinsic apoptotic pathway regulated by the B cell lymphoma 2 (Bcl-2) family proteins results in activation of caspase-9, which in turn activates the downstream effector caspases [43]. Effector caspases are a class of enzymes responsible for the execution of apoptosis and their activation was observed in a considerable percentage of hepatocytes in liver biopsies from chronic HCV infection [46]. Lan et al have shown that HCV infection induces TRAIL-mediated mitochondrial apoptosis via caspase-9 activation [20], however, the precise mechanism still remains to be clarified. In Fig. 2, we show that HCV infection induces the activation of caspase-8 and subsequent downstream effectors caspase-9 and caspase-3 in response to TRAIL. The functional significance of DR4 and DR5 in activation of caspasedependent apoptotic signaling induced by HCV infection was also clarified by reduction of caspase-3/7 activity by DR4 and DR5 silencing (Figs. 2C and D). We propose that HCV infection promotes caspase-8 activity followed by activation of caspase-9 and caspase-3, as evidenced by suppression of DR4/DR5mediated PARP cleavage via translocation of caspase 8-truncated Bid. Taken together, our data demonstrate the functional significance of DR4/DR5-mediated caspase-8 activation as a critical mediator of HCV-induced apoptosis. Efforts to inhibit the TRAIL apoptotic program may be warranted to retard the progression of HCV-related advanced liver disease.