| Background and Aim: Hepatocellular carcinoma (HCC) is one of the most common tumors worldwide, and is currently the second leading cause of cancer death among males in China. Now it is also increasing in United States and Europe. Despite diverse para-operative treatment and the clinical progress achieved in recent years, the overall outcome of HCC remains very poor, which is largely the result of a high ratio of recurrence or metastasis after operation. Tumor markers for HCC, such asα-fetoprotein levels and des-γ-carboxy prothrombin levels, are reported to be additional indicators of tumor progression associated with patient survival. But these factors are not sufficient to accurately discriminate the tumor progression of HCC patients. Moreover, although much is known about the development and causes of HCC, there is no effective chemotherapy for the vast majority of HCC patients. Therefore, other indicators for the prediction of patient outcome and novel approaches to treat HCC are urgently needed. Na+/H+ exchanger 1 (NHE1), a regulator of intracellular pH (pHi) and extracellular pH (pHe), plays a significant role in regulating tumor cell growth, apoptosis, invasion and migration. However, there is no data on its role in HCC, and there are no exact mechanisms on its role in tumor progression. In the present study, we determined its role in HCC.Methods: The intratumoral expression level of NHE1 was determined and compared with that in adjacent non-cancerous hepatic tissue using RT-PCR and Western blotting in 100 patients with HCC. NHE1 protein expression was confirmed by immunohistochemistry. We analyzed the correlations between NHE1 expression level and clinicopathological factors and studied its prognosis role in HCC. NHE1-siRNA or 5- (N-ethyl-N-isopropyl) amiloride (EIPA, highly specific inhibitor of NHE1) were used to assess the function of NHE1 in HCC cells by using gene transfection, MTT, flow cytometry, ELISA assay, western blotting, matrigel-coated transwell cell culture chambers, fluorescence spectroscopy and nude mouse xenograft assays.Results:1. NHE1 at both messenger RNA and protein levels was over-expressed in the majority of HCC tissues compared with matched non-cancerous hepatic tissue using RT-PCR and Western blot (P < 0.05). NHE1 immunostaining was strong in malignant tissue whereas it was minimal in benign tissue. In addition, the increased expression level of NHE1 correlated with tumor size, venous invasion and advanced pTNM stage (P < 0.05).2. The suppression of NHE1 expression by using NHE1-siRNA and EIPA reduced growth of but induced apoptosis of HCC cells. In addition, we also found that inhibition of NHE1 could down-regulate CyclinD1 and up-regulate p27, whereas down-regulate Bcl-2 and up-regulate Bax in HepG2 cells.3. The inhibition of NHE1 by 5-(N-ethyl-N-isopropyl) amiloride (EIPA) is able to suppress migration and invasion of HepG2 cells under hypoxic conditions. In addition, hypoxia activated ERK1/2, which in turn promoted the production of MMP-2, MMP-9 and VEGF. EIPA's suppressive role was determined to act through down-regulation of MMP-2, MMP-9 and VEGF in an ERK1/2 dependent manner.4. In nude mouse xenograft experiment. Tumor growth was significantly inhibited at doses of 3 and 9 mg/kg/day compared to the control. The treatment also significantly reduced mouse body weight at a dose of 9 mg/kg/day. However, EIPA at a dose of 3 mg/kg/day did not induce pathological alterations in the mice.Conclusions: This is the first demonstration that the expression level of NHE1 is correlated with tumor progression and metastasis in HCC. NHE1 in HCC tissue may be considered to be a candidate tumor suppressor gene and its significantly increased expression in HCC may lead to an unfavorable prognosis.
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