Abstract

INTRODUCTION

Hepatocellular carcinoma (HCC) is a highly vascular tumor characterized by a propensity for vascular invasion and distant metastasis. Angiogenesis is a prerequisite for tumor growth and metastasis[1]. In the process of invasion and metastasis of tumor cells, destruction of the extracellular matrix (ECM) is an essential initial step. Among the enzymes responsible for ECM degradation, several studies have shown a critical role played by matrix metalloproteinases (MMPs)[2]. MMP-9 (gelatinase B, 92-ku type IV collagenase) is capable of degrading the main components of the ECM, types IV and V collagen and gelatin[3-6]. Thus, its activities are closely related to the angiogenesis and invasiveness and metastasis of tumor cells[7,8]. However, it is secreted by cells as an inactive form, proMMP-9, which is then activated through an enzymatic cascade involving the generation of plasmin by urokinase-type plasminogen activator (u-PA)[9-12].

Nitric oxide (NO) is an important bioactive agent and a signaling molecule and may contribute to the pathogenesis of cancer[13]. Three distinct isoforms of NOS catalyze the formation of NO. Endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) are constitutively expressed in different tissues, whereas inducible nitric oxide synthase (iNOS) is related to a high-output pathway and is responsible for various pathological processes[14]. Although overexpression of iNOS has been demonstrated in various human neoplasms, such as colorectal cancer[15], pancreatic cancer[16], and breast cancer[17], its exact function in tumor biology is complex and remains to be fully defined. During the initiation of tumor growth, natural killer cells and macrophages kill tumor cells by a NO-mediated mechanism[18]. However, NO may also suppress the antitumor defense, promote tumor angiogenesis and blood flow in the tumor neovasculature, and enhance tumor growth, invasion and metastasis[18]. Recently, investigators have tended to attribute NO-mediated stimulation of invasiveness to an alteration in the balance between the productions of MMPs and TIMPs[19-21]. Since NO has been assessed for the ability to upregulate u-PA in endothelial cells of post capillary venules during the process of NO-mediated stimulation of angiogenesis[22], the u-PA can convert plasminogen to plasmin, which can activate numerous MMPs.

There is evidence that NO produced by iNOS enhances the activity of MMP-9[19-21]. Both iNOS and MMP-9 expression have been reported to be increased in bone and joint disease and cardiovascular disease[23-25]. However, the relationship between them in tumors has been very seldom studied. To determine the role of iNOS and MMP-9 in HCC, we compared their expressions by the immunohistochemical method.

MATERIALS AND METHODS

RESULTS

CD34 expression and its relationship to clinicopathologic parameters in HCC

No CD34 staining was found in the sinusoids of seven healthy liver tissues. In HCC tissues from 32 cases, sinusoid-like tumor vessels reacted intensively with anti-CD34 (mean±SD, 71.53±21.19; range, 31-126), and the positive staining of CD34 distributed with a diffuse pattern or an intensive cluster pattern and was defined as the buffy zonale staining on the cell membrane (Figure ​(Figure1).1). In nonmalignant tissue, anti-CD34 stainings were confined to vessels of the portal triad with weak staining in few of the sinusoids at the periportal area in case of cirrhosis or chronic hepatitis. There was a significant difference in MVD between TNM III stage group (79.25±18.08) and TNM I-II stage group (63.81±21.78, t=2.182, P=0.037). There was also a significant difference in MVD between high-risk group for HCC recurrence (82.56±17.29) and low-risk group (57.36±17.19, t=4.100, P=0.000). But no significant association was noted between MVD and other clinicopathologic features (Table ​(Table11).

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Figure 1

Immunohistochemical staining for CD34 in HCC (×100).

Correlation between MMP-9 and iNOS expression in 32 HCC tissues

There was no MMP-9 staining in the normal liver tissue. MMP-9 immunoreactivity was detected in 25 (78.13%) of 32 HCCs. MMP-9 was mainly localized in cytoplasm and cytoplasmic membranes and its staining was detected in vascular endothelial cells, bile ducts, HCC cells, and stromal fibroblasts (Figures 2A and B). iNOS was expressed weakly in healthy liver tissue and mainly localized in tumor cytoplasm with a diffuse distribution pattern and a granular pattern. It was also found in cell membranes occasionally. There was no expression of iNOS in tumor nuclei or interstitial cells (Figure ​(Figure3A).3A). The common characteristics of MMP-9 and iNOS expression were that their stainings were more frequent in tumor cells localized in the anterior borders of invasion or neighboring capsules of tumor tissues (Figures ​(Figures2C2C and ​and3B)3B) than in those cells in central areas (Figures ​(Figures2D2D and ​and3C)3C) and that they displayed various degrees of intensities and inter-tumor heterogeneity. There was a significant correlation between MMP-9 and iNOS expression (χ²=8.052, P=0.010, Table ​Table33).

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Figure 2

Immunohistochemical staining for MMP-9. A: MMP-9 in cytoplasm and cytoplasmic membranes in HCC (×400); B: MMP-9 in vascular endothelial cells, bile ducts, HCC cells, and stromal fibroblasts (arrow heads from top to bottom) within HCC (×200); C: MMP-9 expression in neighboring capsule of HCC (×200); and D: weak expression of MMP-9 in central areas of HCC tissues (×200).

Table 3

Correlation between MMP-9 and iNOS expression in 32 HCC tissues

iNOS	MMP-9		Total	P
	+	–
+	21	2	23
–	4	5	9	0.010
Total	25	7	32

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Figure 3

Immunohistochemical staining for iNOS. A: iNOS expression in tumor cytoplasm (×100); B: strong expression of iNOS in neighboring capsule of HCC tissues (×400); and C: weak expression of iNOS in central areas of HCC tissues (×400).

DISCUSSION

HCC is the fifth most common malignant disease in the world, causing almost one million deaths annually[28]. Surgical resection is the only proven cure for HCC, yet over 80%[29] of patients with HCC undergoing hepatectomy develop new tumors in the residual livers within 2 years. Further investigation of the pathological and biological factors of HCC in relation to recurrence and metastasis using molecular biology techniques will provide promising strategies for treatment and prevention of cancer invasion.

MMP-9 plays a key role in cancer invasion and metastasis by digesting native type IV collagen, which is a major structural component of basement membranes, since increased MMP-9 levels have been observed in various tumors such as HCC[30], bladder carcinoma[33], head, and neck cancer[19]. There is a trend toward a higher proportion of active MMP-9 with an increasing invasiveness of tumor cells. Our immunohistochemical study showed that the expression of MMP-9 was negative in the 7 normal liver tissues, and positive in 25 (78.13%) of 32 HCC tissues, suggesting that MMP-9 is produced by tumor cells. The strong staining in the marginal areas of tumor tissues suggests the close involvement of MMP-9 in the digestion of ECM. Moreover, positive staining of some fibroblasts and endothelial cells with anti-MMP-9 antibody suggests a participation of these cells in degradation of the ECM. The incidence of MMP-9 expression was significantly higher in high-risk group for HCC recurrence and TNM III stage group than in low-risk group and TNM I-II stage group, suggesting that overexpression of MMP-9 plays an important role in the progress of HCC, and that MMP-9 protein may serve as a marker for invasiveness and metastasis of HCC. These results are similar to the results reported by Arii et al[30] and Hayasaka et al[31]. In contrast to their study, there was also a significant correlation between MMP-9 expression and tumor size and capsule status of HCC. Both MMP-9 mRNA and activation of latent type of MMP-9 are significantly correlated with capsular infiltration of tumor[32]. Durkan et al[33] reported that MMP-9 staining is associated with tumor size. Therefore, the above data further indicate that MMP-9 plays an important role in the dispersion of HCC cells and may accelerate the growth of tumors by enhancing angiogenesis of tumors.

MVD is an important indicator to reflect the level of tumor angiogenesis[34]. The result of our study revealed that there was a positive association between the extent of neovascularization and the clinicopathologic characteristics related to aggressiveness, such as TNM stage and risk of recurrence, indicating that the MVD reflects the potentiality for HCC recurrence and metastasis. We also found that the MVD was statistically different in different MMP-9 immunoreactivity groups. There was a significant positive relationship between MMP-9 and MVD, the higher the MMP-9 expression, the higher the MVD. MMP-9 plays a vital role in the turnover of basement membrane collagen types IV and I, which exert a crucial effect on the formation of new capillary sprouts and neoangiogenesis. Based on the above facts, we speculate that MMP-9 expression may initiate angiogenesis and promote vascularization.

A large body of clinical and experimental data suggest that NO plays a promoting role in tumor progression and metastasis[35-37], though a few reports indicate that the presence of NO in tumor cells or their microenvironment may exert deleterious effects on the tumor cell survival and consequently their metastatic ability[13]. In the current study, we found that iNOS was expressed weakly in healthy liver tissues, suggesting that iNOS is associated with the physiological function of normal liver tissues. The expression of iNOS showed strong cytoplasmic staining in cancerous cells but not in tumor nuclei and interstitial cells, suggesting that NO is produced by HCC cells and directly or indirectly influences the fate of HCC cells. Here, we also demonstrated that the rate of iNOS expression in cases with their tumor capsules being not integral or their tumors without capsules was significantly higher than that in those with their tumor capsules being integral. The expression of iNOS in high recurrence-risk group was significantly higher than that in low recurrence-risk group, indicating that iNOS positivity is upregulated along with the biological aggressiveness of HCC lesions. It seems more likely that iNOS may increase the viability and infiltrative potential of HCC.

In this study, the count of MVD was statistically different in different iNOS immunoreactivity groups, the higher the iNOS expression the higher the MVD. NO has been implicated in all processes of angiogenesis in a manner consistent with a pre-angiogenic phenotype[38]. iNOS-transfected human colon adenocarcinoma DLD-1 cells have a higher vessel density and a higher growth rate in vivo than parental cells in mammary tumor model, suggesting that NO is a key mediator of C3L5 tumor-induced angiogenesis in NOS inhibitor-treated mice[39]. Therefore, the above data further indicate that local NO production by iNOS in HCC cells is able to enhance angiogenesis. It was reported that MMP-9 can also facilitate angiogenesis[40]. In this study, we found that there was a significant association between MMP-9 and iNOS. Both MMP-9 and iNOS positive expressions were more frequent in tumor cells localized in the anterior borders of invasion or neighboring capsules of tumor tissues and were associated with the capsule status and the risk of HCC recurrence, demonstrating that the effect of iNOS on angiogenesis can promote metastatic potential as well as tumor invasiveness by inducing HCC cells to produce MMP-9. To evaluate the mechanism, further investigations are needed.

In conclusion, there is a significant positive correlation between MMP-9 and iNOS expression, suggesting that NO produced by iNOS modulates MMP-9 production and therefore contributes to tumor cell angiogenesis, invasion, and metastasis in HCC.The strong expression of iNOS and MMP-9 in HCC may be helpful in evaluating the recurrence of HCC, predicting poor prognosis. For patients with strong expression of MMP-9 and iNOS, the optimal treatment scheme needs to be selected.

Footnotes

References