Elsevier

Journal of Hepatology

Volume 47, Issue 4, October 2007, Pages 546-555
Journal of Hepatology

Abrogation of constitutive STAT3 activity sensitizes human hepatoma cells to TRAIL-mediated apoptosis

https://doi.org/10.1016/j.jhep.2007.04.017Get rights and content

Background/Aims

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated and regulates cell growth and survival of various cancer cells. We investigated the anti-tumor effect of AG490, a Janus kinase 2 specific inhibitor, inhuman hepatoma cells.

Methods

Effects of AG490 on STAT3 activation, on cell-growth and survival, and on the expression of cell-cycle- and apoptosis-related proteins were evaluated in Huh-1, Huh-7, HepG2 and Hep3B cells. Next, whether AG490 renders hepatoma cells susceptible to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was examined in vitro and in vivo.

Results

Constitutively activated STAT3 through tyrosine phosphorylation was detected in all hepatoma cells. AG490 inhibited the phosphorylation of STAT3 and its activity. AG490 induced cell cycle arrest in Huh-1, Huh-7 and HepG2 through cyclin D1 downregulation, and induced marked apoptosis in Hep3B. AG490 downregulated at least one of the anti-apoptotic proteins, Bcl-xL, survivin or XIAP in all hepatoma cells. AG490 sensitized Huh-1, Huh-7 and HepG2 to TRAIL-induced apoptosis in vitro. Intraperitoneal injection of AG490, the combination of AG490 and TRAIL more greatly, repressed the growth of subcutaneous Huh-7 tumors in athymic mice.

Conclusions

Abrogation of constitutive activation of STAT3 by AG490 enhances the anti-tumor activity of TRAIL against human hepatoma cells.

Introduction

Signal transducer and activator of transcription (STAT) proteins become activated by tyrosine phosphorylation in response to cytokines and growth factors, which typically occurs through cytokine receptor-associated kinases, the Janus kinase (JAK) family proteins [1]. Recent studies have demonstrated that constitutively activated STAT signaling, especially STAT3, contributes to oncogenesis [2], [3]. In fact, constitutive activation of STAT3 has been observed in various cancer cells [2], [3], [4], [5], [6], [7], [8], suggesting that STAT3 plays a crucial role in the regulation of cell proliferation and survival in cancer. Furthermore, abrogation of STAT3 signaling by chemical inhibitors [9], [10], a dominant negative STAT3 mutant [11], [12], inhibitory phosphotyrosyl peptides [13], decoy oligonucleotides [14], antisense STAT3 oligonucleotides [15] has resulted in inhibition of cell proliferation and induction of apoptosis in various cancer cells [16].

Hepatitis B virus (HBV) and hepatitis C virus (HCV) are closely linked to the development of hepatocellular carcinoma (HCC) [17], [18]. Recently, it has been reported that suppressor of cytokine signaling (SOCS)-1 and SOCS-3, negative regulators of the JAK2-STAT signaling pathway, are silenced by methylation in human hepatoma cell lines and HCC tissues, which leads to constitutive activation of STAT3 in these cells [19], [20]. It was also reported that STATs including STAT3 were aberrantly activated in HCC tissues compared with surrounding normal liver tissues [21], [22]. In addition, several studies have shown that HCV constitutively activates STAT3 through oxidative stress [23], and that the HCV core protein can activate STAT3, resulting in the cellular transformation [24]. Taken together, it is possible that the constitutive activation of STAT3 is involved in hepatocarcinogenesis.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a novel member of the TNF superfamily, is a promising candidate for cancer therapy since it preferentially induces apoptosis in various cancer cells with little or no effect on normal cells [25], [26]. However, high concentrations of TRAIL are necessary to induce apoptosis in certain cancer cells, including human hepatoma cells [27]. Thus, combinations of TRAIL with several chemotherapeutic agents or radiation have been evaluated for rendering such cells susceptible to TRAIL, and some combinations have successfully enhanced TRAIL-mediated cancer cell death [27], [28], [29].

In the present study, we examined the status of STAT3 activation in human hepatoma cells and the effect of the JAK2 inhibitor, AG490, on STAT3 activation and on cell proliferation and survival. Furthermore, we have evaluated whether abrogation of STAT3 signaling by AG490 sensitizes hepatoma cells to TRAIL-induced apoptosis in vitro and combination of AG490 and TRAIL exhibits anti-tumor effect in vivo.

Section snippets

Cell culture and reagents

The human hepatoma cell lines, Huh-1, HuH-7, HepG2 and Hep3B, were maintained in a chemically defined medium, IS-RPMI [30] containing 10% fetal bovine serum. Normal human hepatocytes (Hc cells) [31] were purchased from the Applied Cell Biology Research Institute (Kirkland, WA) and maintained in CS-C complete medium. A JAK-2 specific inhibitor, AG490, was purchased from Calbiochem (San Diego, CA). Recombinant human TRAIL was purchased from R&D systems (Minneapolis, MN). In some experiments,

AG490 inhibits constitutive activation of STAT3 in human hepatoma cells

Constitutive phosphorylation of STAT3 at tyrosine 705 was clearly detected in all hepatoma cells, however, STAT3 was only faintly phosphorylated in normal hepatocytes (Fig. 1A). Since JAK2 is one of the major upstream activators of STAT3, we examined the effect of AG490, a JAK2 specific inhibitor, on the STAT3 phosphorylation. AG490 inhibited the phosphorylation of STAT3 in all hepatoma cells, but not in normal hepatocytes. Since SOCS-1 and SOCS-3, negative regulators of the JAK2-STAT3

Discussion

In the present study, SOCS-1 was silenced in Huh-1 and Hep3B, but not in Huh-7 and HepG2, whereas, SOCS-3 was silenced in all hepatoma cells. In contrast, both SOCS-1 and SOCS-3 were detected in normal human hepatocytes in which the level of STAT3 phosphorylation at tyrosine 705 was much lower than that in hepatoma cells. Taken together, it is possible that both SOCS-1 and SOCS-3 are required for negatively regulating the level of STAT3 phosphorylation in hepatocytes.

Recent studies have shown

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    The authors who have taken part in this study declared that they have no relationship with the manufacturers of the materials involved either in the past or present and did not receive funding from the manufacturers to carry out their research. They did not receive funding from any source to carry out this study.

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