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

ARTICLE IN PRESS Pathology – Research and Practice 205 (2009) 828–837

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Original article

Hepatocyte growth factor suppresses tumor cell apoptosis in nasopharyngeal carcinoma by upregulating Bcl-2 protein expression$ Bian Li-juan a,b, Liao Bing a, Li Zhi a,, Li Yang a,b, Liang Ying-jie a a b

Department of Pathology, 1st Affiliated Hospital, Sun Yat-sen University, 58#, Zhongshan Road II, Guangzhou 510080, China Department of Pathology, 2nd Affiliated Hospital, Sun Yat-sen University, 107#, Yanjiangxi Road, Guangzhou 510120, China

a r t i c l e in f o

a b s t r a c t

Article history: Received 26 March 2009 Received in revised form 18 May 2009 Accepted 17 June 2009

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. Crown Copyright & 2009 Published by Elsevier GmbH. All rights reserved.

Keywords: Nasopharyngeal carcinoma (NPC) Hepatocyte growth factor (HGF) Apoptosis Antiapoptotic Bcl-2 protein

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 pathogen-

$ NOTE: Bian LJ and Liao B is co-first author, they have an equal contribution to this study work.  Corresponding author. Tel.: +86 20 8733 0890; fax: +86 20 8733 1780. E-mail address: [email protected] (Z. Li).

esis 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

0344-0338/$ - see front matter Crown Copyright & 2009 Published by Elsevier GmbH. All rights reserved. doi:10.1016/j.prp.2009.06.016

ARTICLE IN PRESS L.-j. Bian et al. / Pathology – Research and Practice 205 (2009) 828–837

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 calcineurinmediated 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 HGFinduced 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.

Materials and methods Specimens of NPC and clinicopathological findings Archived formalin-fixed, paraffin-embedded specimens were recruited from 147 primary NPC patients at the 1st Affiliated Hospital and Cancer Center of Sun Yat-sen University (Guangzhou, China) before treatment in the period 2000–2005. The patients consisted of 104 males and 43 females, their age ranging from 23 to 79 years (median age 48). A hundred and twenty-four patients were diagnosed as having undifferentiated carcinoma (WHO type

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III), and 23 were diagnosed to have differentiated non-keratinized carcinoma (WHO type II). The patients were assigned to disease stage according to the criteria of the China NPC 92 staging system [12]: 7 to stage I, 31 to stage II, 71 to stage III and 38 to stage IV. Cell line and chemicals In this study, we used the human NPC cell lines CNE-1 (a highly differentiated NPC cell line), CNE-2 (a poorly differentiated NPC cell line) and the lymphoblastoid cell line B95-8 (an EBV-infected lymphocyte cell line as positive control for the detection of EBV infection). All of the three cell lines were maintained in RPMI 1640 culture medium with 10% heat-inactivated fetal bovine serum and antibiotics (50 U/ml penicillin and 100 mg/ml streptomycin, Gibco/ Invitrogen) at 37 1C in a humidified incubator with 5% CO2. Human recombined hepatocyte growth factor, purchased from Sigma Chemical, was dissolved in the culture medium for in vitro experiments. In situ detection of apoptosis in NPC tissues and cell lines Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining (Boehringer Mannheim, Germany) was performed on 147 tissue sections of NPC. The detection process was carried out according to the manufacturer’s instructions. TUNEL labels were visualized using diaminobenzidine (DAB) (Dako, Carpinteria, CA) as peroxidase substrates and TUNELpositive nuclei stained brown. The apoptotic index (AI) of NPC tissue was determined according to a previous description [14]. Briefly, the slides were independently reviewed and recorded by two skilled pathologists using a microscope. The total number of apoptotic cells or apoptotic bodies in 100 high-power fields (HPF) was evaluated. The AI of each case was the mean of two evaluations of TUNEL-positive signals in 100 HPFs. In an in vitro study, equal amounts of CNE1 and CNE-2 cells were seeded onto 24-well plates on cover glasses. When 70% confluent cells were observed on the cover glasses, medium was changed to RPMI with 1% FCS (V/V) for 48–72 h to serum starve. The HGF cytokine was added to experiment groups with a final concentration of 25 ng/ml according to our previous study [39]. After treatment, the cover glasses with adherent cells were collected and fixed with freshly prepared 4% paraformaldehyde in phosphate-buffered saline (pH 7.4) for 30 min at room temperature. The cells were then pretreated by blocking endogenous peroxidases, and were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate on ice for 2 min. TUNEL reaction was performed on these cover glasses overnight at 4 1C, followed by DAB visualization. Positive controls were prepared by pretreating cells with DNase according to the manufacturer’s instructions prior to TUNEL staining. Terminal transferase enzyme was omitted from negative controls. All experimental treatments were replicated in triplicates. Apoptotic cells were counted by a single researcher blinded to the study groups. Under the microscope, cells with both dark brown nuclear stain and apoptotic morphology were interpreted as positive, but mitotic figures that occasionally stained positive were excluded. A total of 100 high-power fields were counted on each cover glass, and the AI of each group was the average of all independent experimental repeats. MTT assay NPC cell viability influenced by HGF was measured by 3-[4,5dimethylthiazol-2-thiazolyl]-2,5-diphenyltetrazolium bromide (MTT) assay. Detailed methods have been described previously [40].

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Briefly, NPC cells with a density of 20,000 cells/mL were seeded onto 96-well plates and treated with different concentrations of HGF, respectively, 24 h later. Three wells were tested for each group. Four hours before desired time points, 10 mL of 10 mg/mL MTT was added. After incubation for additive 4 h, the plates were depleted, and 100 mL of DMSO was added. Results represented the OD ratio between the HGF-treated and untreated cells at the same indicated time points. Polymerase chain reaction (PCR) for the detection of EBV infection in NPC cell lines In this study, we used the PCR method to determine EBV infection in NPC cell lines, using primers to detect the Bam HI-W fragment of EBV as described previously [37]. Briefly, CNE1, CNE2 and B95-8 cell lines were routinely cultured, and the genomic DNA was isolated using TIANamp Genomic DNA Kit (Tiangen Biotech, Beijing, China). Template DNA (0.1 mg) was added to a 50 mL reaction mixture containing polymerase chain reaction buffer, dNTP mixture, primers for EBV Bam HI-W (50 -ctttagaggcgaatgggcgc-30 and 50 -aggaccactttataccaggg-30 ) and 5 U Taq polymerase. PCR was performed in an automatic thermal cycler (Perkin-Elmer-Cetus, Norwalk, CT). Samples were amplified through 30 consecutive cycles with an annealing temperature of 45 1C. A 10 mL volume of each PCR product was analyzed by electrophoresis on 1.2% agarose gel containing 0.5 mg/mL ethidium bromide, and the bands were visualized under ultraviolet light. The B95-8 cell line was detected in an EBV-positive control. In a negative control, DNA was replaced by Tris-HCL/EDTA (TE) buffer.

assigned to four levels according to the percentage of cytoplasmic positive tumor cells in 10 high-power fields as follows: (–): less than 5%, (+): 5–25%, (++): 25–50%, (+++): more than 50%. Strong expression of Bcl-2 or Bax was defined as more than 25% tumor cells with positive staining, whereas weak expression of Bcl-2 or Bax was less than 25%. HGF staining results were scored both in tumor cells and stromal cells according to the percentage of cytoplasmic positive cells in 10 high-power fields as follows: (–): less than 10%, (+): 11–20%, (++): 21–50%, (+++): more than 50%. Strong HGF expression was defined as more than 20% in either tumor cells or stromal cells with positive staining, whereas low HGF expression was less than 20%. Western blot analysis for Bcl-2 and Bax expression in an in vitro study CNE-1 and CNE-2 cells were cultured in RPMI medium with 10% FCS until 70% of the cells were confluent. Medium was changed to RPMI with 1% FCS (V/V), and the cells were treated with a final concentration of 25 ng/ml HGF for 48 h. Cell lysates were prepared as described previously [26]. Equal protein samples were subjected to 12% SDS-PAGE electrophoresis, followed by the transfer to Polyvinylidene fluoride (PVDF) membrane, blocking in 5% fat-free milk, and incubation with Bcl-2, Bax (same antibodies as used in immunohistochemistry) or Actin antibody (SigmaAldrich) at 4 1C overnight. Detection was performed using horse radish peroxidase-conjugated secondary antibody and enhanced chemiluminescence reagents from Amersham (Amersham Life Sciences, GB). The relative optical density (ROD, ratio to actin) of each blot band was quantified by NIH Image software (Image J 1.36b).

Immunohistochemistry and scoring Statistical analysis Serial paraffin-embedded tissue sections (4 mm thick) of an NPC biopsy were subjected to immunostaining using a ChemMate Envision/HRP kit (DAKO Co., Denmark). Slides were deparaffnized in xylene and rehydrated in decreasing concentrations of ethanol, and were rinsed in phosphate-buffered saline. The slides were incubated with hydrogen peroxide for 10 min followed by microwave treatment with 10 mM citrate buffer (pH 6.0; SigmaAldrich, Germany) at 5-min intervals for a total of 10 min. After blocking with goat normal serum for 10 min, the slides were incubated with a 1:100 dilution of anti-HGF rabbit polyclonal antibody (Santa Cruz biotechnology, USA), a 1:50 dilution of the mouse anti-human monoclonal Bcl-2 antibody (DAKO Co., Denmark) or a 1:200 dilution of rabbit anti-human polyclonal Bax antibody (DAKO Co., Denmark) for 60 min, respectively. Slides were detected by ChemMate Envision/HRP Kit applied for 30 min at room temperature, followed by development with diaminobenzidine for visualization. Negative controls were carried out by substituting non-immune rabbit serum for the primary antibodies. For in vitro study, HGF-treated and untreated CNE-1 or CNE-2 cells with serum starve seeded on cover glasses were fixed with freshly prepared 4% paraformaldehyde in phosphate-buffered saline (pH 7.4) for 30 min at room temperature. The cells were then pretreated using blocking endogenous peroxidases. They were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min. The primary antibody incubation of Bcl-2 or Bax and following the detection was performed in the same way as for immunohistochemical staining on the slides of NPC tissues. The immunostaining results were evaluated and scored independently by two pathologists without the knowledge of the clinicopathological features and treatment process of NPC cell lines. Evaluation of the immunostaining results was performed as previously described [21]. Bcl-2 and Bax staining results were

Results were given as the mean7SD as indicated. Data were analyzed using SPSS 13.0 software by one-way ANOVA with Dunnett’s post hoc test and Turkey’s post hoc test for multigroup comparisons. The non-parametric test of Mann–Whitney’s test and the Chi-square test was used to analyze the relationship between AI or protein expression and the clinicopathological features of NPC. Differences were considered statistically significant at Po0.05.

Results Protein expression of HGF, Bcl-2 and Bax in NPC and their relation to clinicopathological features A cytoplasmic-positive signal for HGF staining was observed in tumor cells and, in part, in stromal cells (Fig. 1A and B) as well as in normal adjacent nasopharyngeal epithelium with weak expression. Regarding tumor cells, 31 (21.1%) cases were negative (–), 41 (27.9%) cases were (+); 53 (36.1%) cases were (++), and 22 (15.0%) were (+++). Expression of HGF in tumor cells was strong in 51% (75/147). In stromal cells, 6 (4.1%) cases were negative (), whereas 141 cases were positive, of which 25 (17.0%) were (+), 63 (42.9%) were (++), and 53 (36.1%) were (+++). There was strong expression of HGF in stromal cells in NPC in 78.9% (116/147). A cytoplasmic-positive signal for Bcl-2 and Bax staining was detected in tumor cells in NPC. Strong expression of Bcl-2 was observed in 109 cases (74.1%) (Fig. 1C), and Bax expression was strong in 103 (70.0%) out of 147 cases (Fig. 1D), respectively. On serial section, HGF and Bcl-2 could be detected in the same area of tumor cells, at least in a part of the tumor cells (Fig. 1E). Strong expression of HGF in tumor cells or stromal cells was significantly associated with advanced clinical stage (stage III and IV) and

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Fig. 1. Immunohistochemical staining of NPC tissues. A. Cytoplasmic-positive staining for HGF in the tumor cells and stromal cells, including fibroblast and lymphoid cells; B. In some cases, there was strong positive staining of HGF in stromal cells but negative staining in tumor cells; C. Diffuse strong cytoplasmic-positive staining of Bcl-2 in the tumor cells and weak staining of individual stromal cells; D. Strong cytoplasmic-positive staining of Bax in tumor cells was observed and E. HGF and Bcl-2-positive tumor cells in the same area of tumor in serial section. HGF-positive cells on the left, Bcl-2 positive cells on the right. (A–D, immunohistochemical staining, original magnification of 400  ; E, immunohistochemical staining with 200  ).

lymph node metastasis. However, no significant relationship between strong expression of HGF and age, gender, histological type or T-stage was observed. Strong expression of Bcl-2 was significantly associated with advanced clinical stage (P ¼ 0.026), whereas no correlation was observed between strongly expressed Bcl-2 and other clinicopathological parameters. Strong expression of Bax protein was found not to be related to all of the clinicopathological parameters. Furthermore, strong expression of HGF in tumor cells and stromal cells exhibited a positive correlation with strong expression of Bcl-2 in NPC tumors (Table 1). Correlation between apoptosis and protein expression in NPC Nuclear-positive signals with intense dark brown staining for apoptotic cells and apoptotic bodies were observed in NPC tissues

labeled by the TUNEL method (Fig. 2A). The correlation between AI and protein expression in NPC is shown in Fig. 2B. The mean AI values were 32.67720.17 and 35.56722.11 in cases with strong expression of HGF in tumor cells and stromal cells, which were significantly lower than those in cases with weak expression of HGF, respectively, (44.28729.03 and 48.80733.88, P values were less than 0.05). The apoptotic cells were more easily observed in 39 Bcl-2 cases with weak expression (47.10733.45) than in 109 tumor with strong Bcl-2 expression (35.31721.43, P ¼ 0.025). However, the AI value showed no correlation with Bax expression in tumor (P ¼ 0.562), and there was a significant correlation between AI value and the clinicopathological parameters, including age, gender, tumor size, lymph node metastasis and clinical stage (P values were more than 0.05, Mann–Whitney test) (Table 2).

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Table 1 Correlation between expression of proteins and clinicopathological parameters of NPC patients (Chi-square test). Case (n ¼ 147)

Age o48 448

72 75

HGF in tumor cells

HGF in stromal cells

104 43

Low exp. (n ¼ 72)

High exp. (n ¼ 116)

Low exp. (n ¼ 31)

High exp. (n ¼ 109)

Low exp. (n ¼ 38)

High exp. (n ¼ 103)

Low exp. (n ¼ 44)

40 35

32 40

59 57

13 18

51 58

21 17

28 55

24 20

51 24

P ¼ 0.663 53 19

89 27

P ¼ 0.495 WHO Histotype Type II Type III

23 124

13 62

59 88

10 62

32 43

38 109

12 63

22 94

P ¼ 0.006 Bcl-2 expression Low expression High expression

18 20

24 85

16 15

34 69

25 19 P ¼ 0.086

12 26

21 88

9 35 P ¼ 0.340

27 76

P ¼ 0.405

P ¼ 0.020

9 35 P ¼ 0.558

17 21

24 79

P ¼ 0.026

14 30 P ¼ 0.439

38

13

25

23

15

25

13

109

62

47

93

16

78

31

P ¼ 0.016 Bax expression Low expression High expression

14 89

P ¼ 0.508 19 12

29 15 P ¼ 0.550

7 31

41 68

P ¼ 0.001 26 46

75 28

P ¼ 0.678 20 11

17 99

21 17

16 93

P ¼ 0.076 27 45

P ¼ 0.622

P ¼ 0.091 8 23

39 77

P ¼ 0.001 Clincal stage Stage I–II Stage III–IV

83 26

P ¼ 0.185 27 45

9 66

15 16

15 101

P ¼ 0.622 Lymph node status N0 36 111 N13

P ¼ 0.622

P ¼ 0.060

P ¼ 0.499 T-stage T12 T34

Bax

High exp. (n ¼ 75)

P ¼ 0.375 Gender Male Female

Bcl-2

P ¼ 0.047

P ¼ 0.610

44

21

23

32

12

103

54

49

84

19

P ¼ 0.615

P ¼ 0.470

Detection of EBV presence in NPC cells using the polymerase chain reaction method

Prevention of serum starve-induced apoptosis by exogenous HGF in vitro

Using polymerase chain reaction, the Bam HI-W fragment of EBV was detected in B95-8 cells. However, CNE-1 and CNE-2 cells showed no detectable EBV infection (Fig. 3).

CNE-1 and CNE-2 NPC cells were treated with a final concentration of 25 ng/ml of exogenous HGF for periods of 48–72 h. The cells were then labeled with TUNEL reagents. Only cells with both dark brown stain and apoptotic morphology were interpreted as positive. The effect of HGF on the prevention of cells from apoptosis in vitro is shown in Fig. 5. The AI value was significantly decreased in exogenous HGF-treated CNE-1 cells at 48 and 72 h (P value was 0.023 and 0.033, respectively). Similarly, HGF-effective protection was also found in CNE-2 cells after 48 and 72 h of treatment (P value was 0.021 and 0.011, respectively, T test).

Exogenous HGF-induced growth promotion of NPC cells in vitro The effect of exogenous HGF on cell viability was evaluated by MTT assay. The NPC cell lines CNE-1 CNE-2 were exposed to 0–100 ng/ml HGF for 48 and 72 h. As shown in Fig. 4, exposure to HGF resulted in dose- (5–25 ng/ml) and time-dependent enhancement of NPC cell viability. At a concentration of 25 ng/ml, HGF most effectively improved NPC cell growth. Compared to untreated cells, about 20% proliferated cells at 48 h and 40% proliferated cells at 72-h could be observed in CNE-1 and CNE-2 cells, respectively. When the concentration was greater than 50 ng/ml, the effect of HGF on promoting cell growth showed no significant difference compared to a concentration of 25 ng/ml.

Detection of Bcl-2 and Bax in NPC cell lines with HGF treatment HGF-mediated antiapoptotic effects could be found in CNE-1 and CNE-2 NPC cells. Bcl-2 and Bax were the important factors to regulate cell apoptosis. Therefore, we investigated the status of both proteins in NPC tumor lines with exogenous HGF treatment

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Fig. 3. The presence of EBV infections in NPC cell lines using the polymerase chain reaction (PCR) method. Positive band represented the PCR product of EBV Bam HI W fragment. Lanes: M, marker; 1, negative control; 2, B95-8 cell line (EBV positive control); 3, CNE-1 NPC cell line; 4 and CNE-2 NPC cell line.

for 48 h by immunostaining and Western blot. Bcl-2 and Bax protein were readily detected by Western blot in untreated CNE-1 and CNE-2 NPC cells. The amount of Bcl-2 was significantly increased in two NPC cells after 48 h of HGF treatment. However, the amount of Bax protein was not remarkably changed by HGF stimulation in both cells. Similarly, immunostaining for Bcl-2 and Bax in two NPC cells also showed that the Bcl-2-positive signal, but not Bax, could be distinguished more easily in CNE-1 and CNE-2 cells treated with HGF when compared to the untreated cells (Fig. 6).

Discussion

Fig. 2. Apoptosis staining of NPC tissues. A. TUNEL-positive apoptotic cells and apoptotic bodies were observed in NPC tissues (TUNEL method with original magnification of 200  ); B. Correlation between apoptosis and protein expression in NPC; the mean AI in the strongly expressed HGF and Bcl-2 cases was significantly decreased compared to that of low-expressed HGF and Bcl-2 cases (*: Po0.05). However, Bax expression was not significantly correlated with AI.

Table 2 Apoptotic index in NPC tissues in relation to expression of proteins and clinical characteristics.

HGF in tumor cell High expression Low expression HGF in stromal cell High expression Low expression Bcl-2 High expression Low expression Bax High expression Low expression T-stage T12 T34 Lymph node metastasis N0 N13 Clinical stage Stage I–II Stage III–IV

Apoptotic index (mean7SD)

P values*

32.67720.17 44.28729.03

0.001

35.56722.11 48.80733.88

0.002

35.31721.43 47.10733.45

0.025

36.79724.75 42.02727.10

0.562

37.81725.31 38.75725.75

0.865

38.41732.03 38.34723.16

0.377

39.51727.08 37.95727.03

0.628

*, statistical analysis by Mann–Whitney test.

Although there are certain prognostic factors, such as clinical stage, lymph node status and distance metastasis, which have been well-established as reliable indicators of NPC, the cell apoptosis responsible for prognosis in NPC has not been identified. Among the multiple genetic pathways regulating apoptosis, HGF may partly contribute to modulating cell apoptosis by its specific pathway, but the potential mechanism in relation to apoptosis in NPC has not yet been clarified. In this study, we found that the strong expression of HGF protein in NPC tumor cells was not only positively correlated with decreased tumor cells apoptosis, but also with lymph node metastasis and advanced clinical stage of the patients. Strong expression of HGF may not only contribute to the pathogenesis of NPC, but it may also play some roles in NPC progression. HGF is a multipotent growth factor, and a variety of biological effects of HGF have been defined, including mitogenic, angiogenic and antiapoptotic effects [25]. Our results confirm that tumor cells with strong expression of HGF tend to be related to decreased cell apoptosis in NPC tissue, and exogenous HGF indeed protects NPC cells from apoptosis induced by serum starvation in vitro. However, there was no correlation between cell apoptosis and tumor size, lymph node metastasis and clinical stage in this study. These results indicate that cell apoptosis is not a prognostic factor. At least, it is not an independent prognostic factor influencing the prognosis of NPC patients, although a few studies have reported that apoptosis is related to the histopathological characteristics and clinical stage of NPC [14]. As the HGF/c-Met signaling pathway plays an important role in angiogenesis and tumor growth [29], neovascularization and tumor cell proliferation have been identified as prognostic markers for NPC patients [8,28]. Therefore, a possible explanation is that HGF-induced improved survival in NPC has a compositional effect of HGF on tumorigenesis and progression of tumor. HGF-related cell apoptosis inhibition might be a more effective response to the

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development of NPC because a disturbed balance between cell proliferation and apoptosis may be closely related to cancer development [2]. In addition, in our study, we found that HGFinduced cell growth of NPC cells after HGF treatment in vitro. It is difficult to explain the marked improvement in cell survival only by the antiapoptotic effect of HGF on NPC cells. We presume that the antiapoptotic and mitogenic effect of HGF cooperatively improves cell survival in the early phase of NPC development. Once the tumor has developed, we speculate that tumor cells might convert response to the mitogenic effects of HGF and might further influence the progression of NPC. The dual role of HGF in apoptosis resistance and cell proliferation might be a critical pathway of tumor growth promotion in NPC, and consequently might affect the outcome of NPC patients. Of course, further

investigations are necessary to reveal whether tumor cell apoptosis combined with cell proliferation could more accurately predict the overall survival of NPC in a larger case study with unabridged followed-up data. As a potent antiapoptotic factor, several molecular pathways have been found to be responsible for the antiapoptotic action of HGF, including MEK/ERK-dependent phosphorylation of GATA-4 [18], PI3K-dependent activation of Akt [38] and Akt/protein kinase B signaling-mediated calcineurin pathway [36]. Recent studies also suggest that HGF suppresses cell apoptosis by upregulating the expression of Bcl-xl, an antiapoptotic protein [24]. In the present study, we demonstrated that HGF could protect NPC cells from apoptosis. Furthermore, we detected the levels of multiple proteins known to be related to apoptosis in vivo and in vitro.

Fig. 4. Growth promotion effect of HGF on NPC cells. MTT assays were performed in 2 representative NPC cell lines, CNE-1 and CNE-2. Cells were cultured at 20,000 cells/ml in a 96-well plate, exposed to a final concentration of 0–100 ng/ml HGF and incubated for 48 and 72 h points, an average of 3 independent experiments. *, a significant increase was found when compared with control cells (0 ng/ml HGF) (P values were less than 0.05, one way ANOVA).

Fig. 5. Inhibition of apoptosis in NPC cells by HGF. A. Detection by TUNEL showed representative apoptotic cells and apoptotic bodies in non HGF-exposed CNE-1 cells for 48 h serum starvation; B. CNE-1 cells were treated with serum starvation combined with additive 25 ng/ml HGF for 48 h. Thereafter, cells were detected by TUNEL. (A–B, TUNEL method, original magnification of 400  ); C. The apoptotic index was evaluated in NPC cells with or without HGF treatment. *, a significantly decreased AI was observed in NPC cells after 48-h treatment; **, a significantly decreased AI was observed in NPC cells after 72-h treatment.

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Fig. 6. Up-regulation of Bcl-2 protein in NPC cells by HGF. A. Staining by immunocytochemistry for Bcl-2 protein exhibited cytoplasmic positive staining in CNE-2 NPC cells without HGF exposed to serum starvation for 48 h. However, Bcl-2 positive staining could be more easily distinguished in cytoplasm of CNE-2 cells when given HGF treatment (B) (Immunostaining, original magnification of 400  ); C. Detection by Western blot assay for Bcl-2 and Bax protein in CNE-1 and CNE-2 cells showed Bcl-2 protein increased in both HGF-exposed CNE-1 and CNE-2 cells when compared with HGF-untreated cells. However, we failed to observe an altered Bax protein in both the cells regardless of HGF treatment.

We found that HGF enhanced the expression of Bcl-2 in NPC cells in vitro, and there was a tendency of tumor cells to show a strong expression of HGF in tumor cells related to strong expression of Bcl-2 in NPC tissue. However, the expression of the proapoptotic protein Bax was not altered when the NPC cells were subjected to stimulation of HGF. There was also no significant correlation between the HGF expression status and Bax protein in NPC tissues. Our findings, to our knowledge, are the first to indicate that the antiapoptotic effect of HGF in NPC might be mediated by upregulating the expression of Bcl-2. The Bcl-2 and Bax proteins can form heterodimers in cells [33]. The localization of Bcl-2 protein to mitochondria has been proved to associate with the inhibition of mitochondrial permeability transition (MPT) and release of mitochondrial cytochrome C, both of which are central modulators of apoptosis [9]. In this study, we found that Bcl-2 was upregulated in HGF-treated NPC cells, and Bax expression remained to control levels. It is suggested that high-expression of Bcl-2 may increase the Bcl-2/Bax ratio in NPC cells. Therefore, an altered balance between antiapoptotic and proapoptotic protein expression in HGF-treated cells may lead to inhibiting the activation of the whole mitochondrial apoptotic pathway, including the release of cytochrome C, activation of caspase-3 and caspase-9, and finally apoptotic death of NPC cells. A similar result was also achieved by Yamamoto et al. [19] in their study on HGF inhibiting hypoxia-induced endothelial cell death. As NPC tumors cells consistently harbor EBV DNA and some EBV-encoded proteins, previous evidence has shown that Bcl-2 protein could be upregulated by EBV proteins, such as latent membrane protein 1 and BARF1, to protect host cells from apoptosis [31,34]. Before we confirmed the presence of EBV in NCP cells in our study, we could not determine whether or not the upregulated expression of Bcl-2 was induced by EBV-encoded protein or by HGF independently. In the present study, we demonstrated that there was no use of EBV infection in two NPC cells. HGF treatment alone could induce up-regulation of Bcl-2 and might suppress cell apoptosis. These results suggest that

HGF-induced Bcl-2 upregulating expression in NPC is EBVindependent. The mechanism by which HGF upregulates the expression of antiapoptotic protein, such as Bcl-xl or Bcl-2, is still largely unknown. However, recent studies have shown that BAG-1, originally identified as a Bcl-2 binding protein, appears to inhibit cell death by binding to Bcl-2, the raf-1 protein kinase, and HGF/ c-met receptor, and eventually to cooperate with Bcl-2 protein to suppress apoptosis [1,13]. Although the mechanism of inhibition remains unclear, previous results imply that BAG-1 might act as a bridge directly or indirectly linking the HGF receptors to intracellular targets involved in the control of apoptosis. Regrettably, we failed to demonstrate the relationship between LMP-1 expression and apoptosis or bcl-2 expression in NPC tissues, and did not correlate the cooperative effect of HGF on inhibiting cell apoptosis in EBV-infected NPC cells in vitro, which might be different from that of HGF stimulation alone. However, based on the above previous results, we postulate that regulation of cell apoptosis in NPC may be a complex process, and multiple apoptotic factors, including EBV-encoded protein and HGF, are involved in this process through their own specific pathways. Interestingly, in the present study, we found that not only tumor cells with strong expression of HGF, but also stromal cells with strong expression of HGF in stromal cells were significantly correlated with the strong expression of Bcl-2 and decreased apoptotic index in NPC tissues. These results indicate that besides autocrine, the paracrine pathway of HGF can also regulate the expression of Bcl-2 and modulate cell apoptosis through activation of Met receptor in NPC. In HGF-Met pathway, both of them were indispensable molecules to execute their multifunctions. The co-expression of HGF and Met in tumor cells suggests the involvement of an autocrine HGF-Met signaling loop. However, in the present study, only half of the cases showed highly endogenous HGF in tumor cells, whereas nearly 80% cases showed an exogenously strong expression of HGF in stromal cells. Moreover, our pervious study has revealed that both CNE-1 and CNE-2 NPC cells had no endogenous HGF regardless of HGF

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stimulation, but strongly expressed Met protein [39]. To sum up, we presume that in NPC, the paracrine HGF-Met pathway might be more important as far as the influence of tumor cell proliferation, migration and apoptosis, as well as the prognosis of NPC patients are concerned, although the autocrine pathway might be involved in carcinogenesis. It has been documented that paracrine interactions between stromal and cancer cells are crucial for determining the malignant behavior of a tumor [20], and induction of HGF by fibroblast directly promotes tumor cell invasiveness [10]. Therefore, in NPC, it is reasonable to believe that HGF derived from stromal cells plays important roles in the tumorigenesis and progression of NPC by the paracrine pathway. However, the precise role and the activation mechanism involved, as well as the cooperative effect on the HGF autocrine pathway remain to be elucidated. In summary, our data indicate that HGF participates in the modulation of cell apoptosis in NPC by influencing Bcl-2 expression through autocrine and paracrine pathways. Exogenous HGF can protect NPC cells from apoptosis by upregulating the expression of Bcl-2 through EBV-independent pathways. HGF could influence lymph node metastasis of tumor cells and the clinical stage of NPC patients. Therefore, HGF is a potent inhibitor of cell apoptosis, and might be a potential marker for the prognosis of NPC. From this standpoint, diminishing the expression of HGF or interfering with the activation of its receptor in tumor appears to be a promising strategy for developing new therapeutic approaches for the treatment of this malignant tumor.

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