| IntroductionHepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, especially in China. Surgical resection of tumor is a main method possibly to cure some HCC patients. Although some advances in management of HCC have been made, the over survival is still not satisfactory. Intrahepatic recurrence and metastasis are two main factors that will affect surgical outcome of these patients, which remain a major obstacle to further improvement on the long-term survival after curative HCC resection. The recurrence and metastasis of liver cancer refer to a series of molecular processes such as the activation of oncogenes and metastatic gene, the inactivation of tumor suppressor genes, the over expression of growth, angiogenic factors and their receptors, alteration of adhesion molecule, extracellular matrix, the human immunity to tumors, and et al. Biotherapy, such as cytokines, differentiation inducers, anti-angiogenic agents, gene therapy and tumor vaccine will probably play an important role in the prevention of tumor recurrence and metastasis.Hepatocyte growth factor (HGF) and its receptor, c-met may play an important role in tumor migration and metastasis. HGF was first purified from human and rat platelets on the basis of its ability to stimulate mitogenesis of rat hepatocytes. The c-met protein is a tyrosine kinase receptor with an extracellular 50-kD α-subunit and a transmembrane 145-kD β-subunit. HGF stimulates proliferation, migration and morphogenesis of epithelial cells by specific binding to its receptor c-met~J. In patients with partial hepatectomy, elevated HGF level resulting from hepatocyte proliferation may last for a period. Persistent over-expression of HGF leads to its excessive reaction with c-met, providing malignant tumor cells with kinetic and aggressive traits, which may promote the growth of remnant tumor and hepatocarcinogenesis of normal liver. After partial hepatectomy, the increased tyrosine phosphorylation of c-met is seen in the hepatocytes. HGF stimulates the growth and migration of HCC cells through activation of c-Met and MAPK. As a member of MAPK family, extracellular signal regulated kinase (ERK) canbe activated by growth factor stimuli, and play a important role in cell division, migration and survival. Sustained c-Met-dependent ERK signaling is essential for promoting DNA proliferation and for disassembly of adherents junctions, one of the early steps in cell dissociation. The system of HGF/c-met-ERK has an important effect on the progression and metastasis of primary liver cancer. Inhibition of HGF/c-met-ERK signal transduction systems may be a key measure to prevent the tumor growth and metastasis.1,25-Dihydroxyvitamin D (\,2S-(OH)JC>3, Clairol), the most active metabolite of vitamin D, has a significant anti-tumor activity in preclinical models. Numerous studies in vitro and in vivo have shown that vitamin D potently inhibits cell proliferation, induces cell differentiation and apoptosis, reduces angiogenesis, and prevents invasion and metastases in many types of tumors. These preclinical data suggest that vitamin D has potential values of application in cancer prevention and treatment. Up to now, the molecular mechanisms of anti-tumor have not yet been clearly elucidated.Vitamin D exerts its activity mainly by binding to the VDR, a member of the steroid hormone super-family. The existence of the nuclear vitamin D receptor (VDR) has been found in numerous tissues in different organs, which are the so-called 'non-classical' targets of this second-steroid hormone. VDR expression was observed in most of the human hepatocellular carcinoma cell lines. By binding to the VDR, vitamin D regulates target gene transcription via a serious of signing pathways. However, the anti-tumor mechanism of l,25-(OH)2^>3 may vary in different tumor cells. Preclinical and clinical studies have showed that vitamin D has a potent inhibition role to hepatocellular carcinoma cells (HCC), and affects varied processes relating to tumor growth, proliferation. The anti-tumor mechanism of 1,25-(OH)2D3 possible relates to regulate some gene transcription and signing pathways in HCC. According to reports, 1,25(OH)2D3 attenuates an HGF-mediated cell proliferation, migration, and significantly reduces the extent of constitutive phosphorylation of Erkl/2.So, we analyzed that the clinical value of hepatocyte growth factor and its receptor for liver cancer patients with hepatectomy in part one, and studied that the effect of 1,25-(OH)2D3 on HGF/c-met and ERK expression in MHCC-97 heptocellular cells in part two.Part OneThe clinical value of hepatocyte growth factor and its receptor for livercancer patients with hepatectomyIntrahepatic recurrence and metastasis are two main factors that will affect surgical outcome in patients with liver cancer. Hepatocyte growth factor (HGF) and its receptor, c-met play an important role in tumor migration and metastasis. HGF was first purified from human and rat platelets on the basis of its ability to stimulate mitogenesis of rat hepatocytes. HGF stimulates proliferation, migration and morphogenesis of epithelial cells by specific binding to its receptor c-met.Hepatocytes are highly differentiated cells that rarely divide under physiological conditions;however, they will have proliferative capacity to adapt to varying metabolic demands. Hepatocytes may be induced to proliferate following toxic damage and surgical resection1. Hepatectomy initiates the release of a cascade of growth factors that results in proliferation of all hepatic elements1. The rapid metabolic adaptations occur in early stage after partial hepatectomy or injury. HGF is a primary agent promoting the proliferation of mature hepatocytes. It has been suggested that HGF plays a bi-functional role in the invasive behavior of various tumors, and also in tissue repair and regeneration in reaction to tissue damage. Persistent over-expression of HGF leads to its excessive reaction with c-met, providing malignant tumor cells with kinetic and aggressive traits, which may promote the growth of remnant tumor and hepatocarcinogenesis of normal liver. In this study, we observed the dynamic change of HGF after hepatectomy in patients with primary liver cancer, and analyzed the prognostic value of HGF and c-met for these patients. Materials and MethodsThirty-one patients underwent surgical resection for from January 2004 to January 2005 in our hospital. The mean age of these patients was 51 (range 36-72) years, including 25 males and 6 females. Pathologically, these lesions matched for the standards of primary liver cancer. The classification of these tumor tissues were: hepatocellular carcinoma (HCC) (in 24), cholangiocarcinoma (cholangiocellular carcinoma. CC) (in 2)and hepatocellular- cholangiocellular carcinoma (combined HCC)( in 5). According to Edmondson grade, of the 24 HCC, tumors were found well and moderately differentiated in 17, while poorly differentiated in the other 7. According to Child-Paugh score of liver function, grade A was found in 24 patients and grade B in 7 patients. Surgical methods were: major resection for 18 patients (left hemi-hepatectomy for 3 and lobectomy for 15 patients), and local resection (including segmentectomy) for 13 patients.Cancerous and the paracancerous tissues (2 cm away from the carcinoma) were obtained through hepatectomy. The specimens were immediately frozen in liquid nitrogen and then kept at a temperature of -80°C until being examined. Blood samples of all the patients were collected on one day before operation, and 3> 7% 10 days after operation respectively . Blood samples of 20 healthy adults, 22 chronic hepatitis B (Child-Paugh A) and 22 cirrhotic patients (Child-Paugh B/C) were collected as controls. The serum was separated in 30 minutes after coagulation at room temperature and was centrifuged at 3000 r/min for 15 minutes. The separated serums were stored at a temperature of -80°C until being assayed. Serum HGF level was determined by using ELISA kit before and after operation respectively. C-met protein and mRNA expressions in cancerous and paracancerous tissues were examined by immunohistochemical and RT-PCR methods respectively.These patients had been followed up for 6—18 months. Six patients had intrahepatic tumor recurrence and 2 had distant metastasis including 1 lung and 1-bone metastases. The clinical-pathologic factors were: preoperative a-fetoprotein(AFP) level, Child-Paugh score, cirrhosis status, tumor size (>5cm vs. < 5cm), portal vein tumor thrombi (PVTT), pathological classification and postoperative tumor recurrence or metastasis. The correlations between the factors mentioned above and HGF level in serum, c-met expression in cancer tissue were analyzed respectively.All the data were expressed as mean±SD and analyzed by using SPSS 10.0 for Windows. ANOVA, Student t, non-parameter and Chi-Square tests were used to compare the differences between corresponding groups. P<0.05 was regarded as statistically significant.ResultsLiver cancer patients had a significantly higher level of serum HGF than normalcontrols (1.0424 +/- 0.498 vs. 0.685 +/- 0.115ng/mL, p = 0.008). Serum HGF level were positively affected by tumor size, node cirrhosis, portal vein tumor thrombi (PVTT), cholangiocarcinoma (including combined HCC), poorly differentiated HCC and tumor recurrence or metastases. After hepatectomy, serum HGF level peaked on the third postoperative day (POD), and then declined, but did not return to normal level on the POD 10. From the preoperative day to POD 10, the level of serum HGF had a decrease of percent (85.33±10.2%) in the group with large tumors (>5cm), but an elevation of percent (121.9±10.3%) in the group with small tumors < 5cm). From the preoperative day to POD 3, the level of serum HGF had a higher elevation in the group with major resection than in the group with local resection (p=0.016). Moderately or strongly positive expression of c-met protein was observed in 27 cancerous regions (27/31), and only in 5 paracancerous regions. The intensive expression of c-met mRNA was 100% (31/31) detectable in the cancer tissues, but only 22.6% (7/31) in the paracancerous tissues. C-met protein expression in cancerous tissues was correlated with PVTT, cholangiocarcinoma and tumor recurrence or metastases, and the expression in paracancerous tissues was correlated with node cirrhosis. No significant correlation was observed between HGF in serum and c-met in cancerous tissue.Conclusion1. The over-expressions of HGF and c-met indicates an adverse prognosis for patients with liver cancer.2. Liver regeneration may be a main factor leading to high level of serum HGF in early postoperative stage.3. The sustained high level of serum HGF after hepatectomy may be a factor related to early tumor recurrence and metastasis.Part Two Calcitriol inhibits the growth of human HCC cell lines by down-regulating c-met and ERK expressionsThe HGF/c-Met exerts control on cell proliferation, morphogenesis and motility in cancer. Increased mitogenesis is achieved by an active process whereby c-Met and subsequently the mitogen-activated protein kinase (MAPK) pathway are constitutively activated. As a member of MAPK family, extracellular signal regulated kinase (ERK) can be activated by growth factor stimuli, and play a important role in cell division, migration and survival. So, inhibition of HGF/c-met-ERK signaling pathway has a key role to reduce invasion, metastasis of tumor cells.Except for inducing apoptosis, modulating cell cycles, Calcitriol (l,25-(OH)2Dj) has a pleiotropic regulatory role involved in some gene expression which are related to transcription, cell adhesion, DNA synthesis, intracellular signing transduction. According to reports, 1,25(OH)2D3 inhibits proliferation of MG-63 osteosarcoma cell, and HL-60 leukemia cell by inhibiting HGF synthesis. Moreover, 1,25(OH)2D3 attenuates an HGF-mediated cell proliferation > migration. 1,25(OH)2D3 significantly reduces the extent of constitutive phosphorylation of ERK 1/2. It deserve to been studied whether 1,25(OH)2D3 regulate HGF/c-met and ERK signaling pathways in human heptocellular cell or not. In this paper, we study the signaling pathway mediated by HGF/c-met and ERK, and investigate the possible mechanism that 1,25-(OH)2D3 affects MHCC-97 human heptocellular cell lines.Materials and methodsMHCC97-H and MHCC97-L cells originating from human hepatocellular carcinoma were cultured with RPMI 1640 medium supplemented with penicillin, streptomycin, and 10% fetal calf serum (FCS) and incubated at 37°C in a humidified atmosphere containing 5% CO2.These cells growing in logarithmic phase were used for experiments, which were divided six groups (1) control cultures-no vehicle was added to medium;(2)treatment with 1,25(OH)2D3 of a final concentration oflO^ I0"\ 10'8> 10"9M respectively;(3) Treatment with 1,25(OH>2D3 of a 10"6 M concentration in lipiodol ultra-fluid (LUF) of a0.5% concentration;(4) treatment with LUF of 0.5% concentration;(5) treatment with HGF of a final concentration of 40ng/ml;(6) treatment with Human HGF Polyclonal antibody of a final concentration of 80ng/ml. In 48, 72, 96 hours after culture in different medium respectively, the cells were incubated with MTT for 4 hours. Absorbance was read with Bio-Rad instruments at a wavelength of 490 nm. The conditions of cell proliferation were denoted by inhibitive rate (IR). IR=[( A value of control group-A value of treatment group)/ A value of control group] X100%.The morphological character of the cultured cells was observed and took a picture under inverted phase contrast microscope.The cell apoptosis and cycle of treatment group with 1,25(OH)2D3 (10"6 M) and control group were analyzed using a flow cytometer (Coulter XL, USA).Seventy-four hours after the cells were incubated with varied mediums: 1,25(OH)2D3, (lOi^M) 1,25(OH)2D3 (lO"6^ in LUF (0.5%), and control, HGF concentration in the cell supernate was measured by using ELISA method;c-met and VDR mRNAs in cells were detected by using RT-PCR methods;VDR and ERK1/2 proteins were determined by using Western Blotting method.All the data were expressed as mean±SD and analyzed by using SPSS 10.0 for Windows. ANOVA, Student t and Chi-Square tests were used to compare the differences between corresponding groups. PO.05 was regarded as statistically significant.Results1. The effect of 1,25(OH)2D3 ^ HGF and anti-HGF antibody on the proliferation of MHCC97 hepatocellular carcinoma cells1^5(OH)2D3 inhibited the proliferation of MHCC97 cell lines. The inhibitive rate (IR) of the cell proliferation was highest in a concentration of lO^M than in any other concentration and the IR reaches a height in 72h after treatment with 1^5(OH)2D3 (H:50.45±4.67%, L: 65.72±4.04%). Lipid ultra-fluid (LUF) of a concentration of 0.5% has no significant effect on the cells, however, which combined with 1,25(OH)2D3 has a better and longer inhibitive effect on the HCC cells than 1,25(OH)2D3 alone. Anti-HGF antibody has also inhibitive effect on the HCC cells to a certain extent, but the effect is not satisfactory. HGF promoted the proliferation of the HCC cells (Table 1)2. Cell apoptosis and cycle analysisExposure of MHCC97 cells tol,25-(OH)2D3 for 48h induced apoptosis;the apoptosis rates of MHCC97-H and MHCC97-L cells were 7.95%( control: 1.16%) and 5.21%( control: 0.12%) respectively. 1,25-(OH)2D3 caused an accumulation of cells in the G0/G1 phase (H: 63.9%-*75.1%, L: 69%-?76.7%) and reduction of cells in S phase. A reductive percentage of cells in the G2-M phase (7.5%->3.9%) were observed in MHCC97-L cell.3. Effect of 1,25(OH)2D3 on HGF level in cell supernate.HGF was detected in supernate of MHCC97 cells, which indicates MHCC-97 cells secreted HGF. Although HGF the concentration in MHCC97-H cell was higher than in MHCC97-L(356.1 ±98.9vs 241.3+136.8 pg/ml), the concentration between them had no significant difference. After treatment with 1,25(OH)2D3, HGF concentration decline to a certain extern, but compared with control, the decline had no significant difference. However, after 1,25(OH)2D3 combined with LUF treated MHCC97-H cell, HGF concentration had a significant reduction (p=0.043).4. The effect of 1,25(OH)2D3 on the expression of related genes, proteins in the cellsThe expression of VDR mRNA and protein was detected in the two MHCC97 cell lines by RT-PCR and Western Blotting methods respectively, and the expression was relatively more intensive in MHCC97-L cell line than in MHCC97-H cell line. The treatment with 1,25(OH)2D3 in LUF on the cells enhanced the their expressions of VDR mRNA and protein.The two MHCC97 cell lines expressed c-met mRNA. 1,25(OH)2D3 with or without LUF remarkably inhibited c-met mRNA expression. Intensive expression of ERK 1 and ERK2 proteins was observed in the two MHCC97 cells by Western Blotting method. After treatment with 1,25(OH)2D3 with or without LUF on the cells, the expression of ERK1 and ERK2 proteins had a down regulation, which indicated that 1,25(OH)2D3 inhibited the expression of ERK1 and ERK2 proteins.Conclusion1. MHCC97 cell lines have the expression of VDR, and 1,25(OH)2D3 exerts its activity possibly by binding to the VDR.2. MHCC97 cell lines could secreted HGF and have an intensive expression of c-met, ERKl/2, which indicates the signaling pathways of HGF/c-met and ERK play animportant role in progression and metastasis of HCC.3. 1,25(OH>2D3 inhibits the proliferation and induces apoptosis of MHCC97 cells. 1,25(OH)2D3 combined with Lipid ultra-fluid (LUF) has a better and longer inhibitive effect on the HCC cells than 1^5(OH)2D3 alone.4. Anti-hepatocarcinoma effect of 1,25(OH)2D3 is related to the inhibition of c-met and ERK1/2 expression.5. HGF promoted the proliferation of MHCC97 cells and anti-HGF antibody has an inhibitive effect on the cells to a certain extent.
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