Role of the Fas/FasL pathway in combination therapy with interferon-α and fluorouracil against hepatocellular carcinoma in vitro

Background/Aims

Several studies have reported the efficacy of combination therapy of interferon (IFN) α and 5-fluorouracil (5-FU) for hepatocellular carcinoma (HCC). However, the mechanism underlying the clinical anti-tumor effects of this treatment is not well understood. The aim of this study was to determine the role of Fas/FasL signaling in the anti-tumor effect of this combination therapy.

Methods and Results

We used six human hepatoma cell lines, three of which are known Fas-expressing cells. Growth of Fas-positive hepatoma cell lines was inhibited by an agonistic anti-Fas antibody in a dose-dependent manner, and these effects were enhanced by IFNα or 5-FU alone, but even more so by combination therapy using both agents. Annexin-V assay implicated apoptosis as the main mechanism underlying these growth inhibitory effects, although changes in Fas expression regulated by IFNα and/or 5-FU did not correlate with increased apoptosis. Caspase-3 activation was exclusively increased by IFNα/5-FU combination treatment, which was compatible with enhancement of the synergistic apoptotic effect, and other caspases and apoptotic factors (FLIP, BCL-xl, and Bax) were also regulated by IFNα/5-FU. ⁵¹Cr-release assay revealed that pretreatment with IFN activated cytotoxicity of peripheral blood mononuclear cells (PBMCs) against HCC cells. The largest interaction was observed when both PBMC and HCC cells were pretreated with the combination of IFNα/5-FU. These cytotoxicities were markedly inhibited by a neutralizing anti-Fas antibody.

Conclusions

Our results indicated that IFNα/5-FU combination treatment enhances the induction of apoptosis and the cytotoxic effect of PBMCs via the Fas/FasL pathway. The Fas/FasL pathway seems, at least in part, to contribute to the anti-tumor effects of IFNα/5-FU against HCCs.

Introduction

Hepatocellular carcinoma (HCC) is one of the most common solid tumors [1]. The prognosis for patients with HCC remains poor and most die within several months after diagnosis, particularly in advanced cases with tumor thrombosis in the major portal vein (Vp3-4) [2], [3], [4]. Chemotherapy is the traditional first choice for the treatment of unresectable solid tumors; however, these drugs are not effective in promoting tumor regression and prolonging survival in HCC [5], [6]. In addition, conventional therapeutic modalities such as transcatheter arterial embolization, radiofrequency ablation and microwave coagulation therapy are not recommended when portal vein tumor thrombosis (PVTT) is present because of low efficacy and potential complications [7], [8]. Therefore, a new effective modality is needed to treat advanced HCC, especially in those cases with portal vein involvement.

Interferon (IFN) has a variety of biological properties including immunomodulation and anti-tumor activity. The anti-tumor effect of IFN against HCC was tested in several studies. From a randomized controlled trial, Llovet et al. [9] concluded that IFN used alone provides no clinical benefit for HCC patients with respect to tumor progression rate and survival. However, several other investigators reported a strong anti-tumor activity for IFN in HCC, when used in combination with some other chemotherapeutic agents. Urabe et al. [10] found that treatment with a combination of subcutaneous IFNα injection and intra-arterial infusion of 5-fluorouracil (FU), cisplatin and methotrexate for HCC with PVTT achieved a response rate of 46.7%. In addition, Patt et al. [11] reported that combination treatment with FU and IFN promoted anti-tumor has activity in HCC and could be tolerated even by cirrhotic patients. We also previously reported the beneficial results of subcutaneous IFNα injection and intra-arterial 5-FU infusion against HCC with PVTT [12], [13], [14]. This therapy showed an anti-tumor effect with a response rate approaching 50%, including several complete remissions of the tumor and prolonged survival without major adverse effects. From these results, we proposed that the combination chemotherapy of IFNα and 5-FU should become a standard therapy for advanced HCC.

We have already reported the synergistic effects of IFNα and 5-FU in influencing cell-cycle progression into the S phase via p27^Kip1, inducing apoptosis by downregulating Bcl-xl, and modulating the immune response via the TRAIL/TRAIL-receptor pathway [15], [16], [17]. The present study is an extension of this previous work, to investigate the role of the Fas/FasL pathway in the IFNα/5-FU treatment effect. Fas/FasL signaling participates in an apoptosis-inducing mechanism related to cytotoxic T Lymphocytes (CTL) and natural killer (NK) cells, which was implicated as a major pathway of T-cell-mediated cytotoxicity and a mediator of apoptosis via an IFN-stimulated gene [18]. In addition, we also investigated the mechanism underlying the apoptosis-enhancing effect of IFNα/5-FU that acts via the Fas/FasL pathway.