Hepatocyte growth factor suppresses tumor cell apoptosis in nasopharyngeal carcinoma by upregulating Bcl-2 protein expression

Abstract

Hepatocyte growth factor (HGF) is a multifunctional cytokine, but cell apoptosis related to HGF in nasopharyngeal carcinoma (NPC) and the potential mechanisms involved have not yet been identified. In this study, we aimed at determining whether HGF is a potent inhibitor of cell apoptosis in NPC, and tried to find out which antiapoptotic or proapoptotic protein is involved in this process.

A hundred and forty-seven cases of NPC and CNE-1, CNE-2 NPC cell line were collected. Expression of HGF, Bcl-2 and Bax in tumor tissues was investigated by immunohistochemical staining, and the apoptotic index was evaluated using the Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) method in all of the NPC cases. NPC cells were treated with HGF (25 ng/ml), followed by an assay for cell viability and apoptosis, as well as by an expression analysis of Bcl-2 and Bax using immunostaining and Western blot. The presence of Epstein-Barr virus (EBV) was also detected in NPC cells using polymerase chain reaction (PCR) for Bam HI W fragment.

In tumor cells, expression of HGF was strong in 51% (75/147) of the NPC cases. In stromal cells, expression of HGF was also strong in 78.9% (116/147) of the cases. Strong expression of HGF in tumor cells and stromal cells was significantly associated with decreased apoptotic index, advanced clinical stage, lymph node metastasis and high-expression of Bcl-2. In vitro, exogenous HGF was found to promote cell growth, to suppress cell apoptosis and to upregulate the expression of Bcl-2 in NPC cells without EBV infection.

HGF is a potent inhibitor of cell apoptosis in NPC by upregulating Bcl-2 through both autocrine and paracrine EBV-independent pathways. HGF might be a potential marker for the prognosis of NPC.

Introduction

Although nasopharyngeal carcinoma (NPC) is rare in the Western world, it is one of the most common squamous cell carcinomas seen in the epithelium lining of nasopharynx in epidemic areas of Southern China, Southeast Asia, Alaska and North Africa [16]. The treatment of NPC has improved significantly over the recent years as the combination of radiotherapy and adjuvant chemotherapy has become the standard treatment for NPC in China [17], but its prognosis remains serious. The 5-year survival rate after treatment amounts to only about 50–60% because of the frequency of distant metastases and local recurrence, and because of the long-term secondary effects of radiotherapy and chemotherapy [5]. The multifactorial pathogenesis identified for NPC relies on germ line genetic susceptibility, acquired cellular genetic and epigenetic alterations, including the influence of diet, carcinogens and Epstein-Barr virus (EBV) infection [6], [7].

The development and progression of NPC result not only from specific oncogene activation, cell cycle checkpoint violation and genetic instability. Inhibition of apoptosis is also considered as a requirement of oncogenesis [3]. Indeed, it has been demonstrated that overexpression of antiapoptotic proteins decreases proapoptotic response and results in resistance of NPC cells to traditional radiation and chemical therapies [4], [35]. Among those antiapoptosis-related factors, the Bcl-2 family proteins, including Bcl-2, Bcl-xL and Mcl-1, are commonly strongly expressed in tumor cells, and are thought to be one of the most important mediators contributing to a variety of human cancers [11]. Recent studies have revealed that Bcl-2 mRNA and protein were detected in most (80%) samples of undifferentiated NPC [27]. Compared to normal or hyperplastic noncancerous nasopharyngeal epithelia, Bcl-2 protein is significantly more strongly expressed in tumor cells of NPC [32]. On the one hand, NPC cells consistently harbor EBV DNA and some EBV proteins. Induction of Bcl-2 expression by latent membrane protein 1 (LMP-1) and BARF1 is suggested to protect host cells from apoptosis [31], [34]. On the other hand, Bcl-2 antisense oligodeoxynucleotide has been shown to have a proapototic effect in NPC cell lines, leading to a regression of NPC xenografts in combination with chemotherapy in an animal model [15]. Hu et al. [40] have recently shown that the inhibitor of the antiapoptotic Bcl-2 family protein, ApoG2, induces apoptosis and suppresses tumor growth in NPC xenografts. Evidence accumulated from these studies suggests that antiapoptotic Bcl-2 protein may be an important mediator to influence the prognosis of patients with NPC by regulating cell apoptosis.

The consistent presence of a massive, infiltrating lymphoid is a histopathological characteristic of the primary tumor of NPC. In the inflammation-like microenvironment, the interaction between stromal and tumor cells may be crucial for the development of NPC. Several cytokines and chemokines derived from stromal cells have been reported to play potent roles in tumorigenesis [22]. Hepatocyte growth factor (HGF), a multifunctional cytokine induced by various cell types, shows various biological activities, including mitogenic, morphogenic, motogenic, angiogenic and antiapoptotic effects [25]. In vitro, HGF has also been found to protect various cell types against apoptosis induced by a variety of stimuli [23], [30]. It has been reported that the antiapoptotic effect of HGF is mediated by the activation of Akt, which is a molecule effective for many cellular functions initiated by growth factors [38]. A recent study suggests that locally overexpressing HGF inhibits apoptosis via the calcineurin-mediated pathway [36]. There is evidence that invasiveness and metastasis of NPC are associated with the activation of HGF receptor, Met, leading to a poor outcome of NPC [8]. Our previous study has also demonstrated that HGF-induced Met overexpression is correlated with the metastasis of NPC [39]. However, the effect of HGF on apoptosis of tumor cells in NPC is still unknown. Since the disturbed balance between cell proliferation and apoptosis may be closely related to cancer development [2], we wonder if HGF-induced apoptosis resistance in NPC can help the clinicians to improve the prognostic prediction and may become a promising strategy for the development of new therapeutic approaches with which this malignant tumor can be treated.

The aim of this study was to evaluate whether HGF is a potent inhibitor of cell apoptosis in NPC, and whether the stromal cells in tumor indeed possess tumor promotion, inducing HGF expression in NPC. We also assessed the related molecules involved in HGF-induced cell survival, and evaluated the relationship between the expression of HGF and apoptotic protein (Bcl-2 and Bax) in 147 biopsy specimens taken from the NPC before radiotherapy and adjuvant chemotherapy. In vivo, we found that HGF arising from either tumor or stromal cells indeed inhibited tumor cell apoptosis, and that it was closely related to strong expression of Bcl-2 in tumor. In vitro, exogenous HGF alone effectively blocked serum starve-induced cell apoptosis by upregulating Bcl-2 expression in NPC cells without EBV infection.