Effect Of Ca～(2+) Signal On Expression Of Heparanase In Hepatocellular Carcinoma Cells

Objective:The hepatocellular carcinoma (HCC) is one of the most common malignant tumors in our country, which is frequently associated with metastasis in the early stage and high rate of recurrence after operation or intervention treatment. To identify the principium of invasion and metastasis and to search effective approaches to prevent the recurrence and metastasis of HCC remain a great challenge to modern medical science. Heparanase (HPA) is an endo-β-D-glucuronidase and it is high expression in many kinds of carcinomas including the hepatocellular carcinoma. It is the only enzyme that we know by now that degrades the heparin sulphate (HS) chain of heparan sulphate proteoglycans (HSPG), and then degrades the extracellular matrix (ECM) and basement membrane (BM), and releases active materials, and speeps up angiogenesis, in many ways, HPA promotes tumor growth and invasion and metastasis. Studies indicate that there is store-operated calciumion (Ca²⁺) entry in hepatocarcinoma cells, which is NO→cGMP sensitive (negativity regulation) Ca²⁺ regulation path ways. Intracellular stable high level of Ca²⁺ significantly enhances the ability of metastasis and adhersion of hepatocarcinoma cell in vitro. The aim of this experiment was to determine the effect of Ca²⁺ in the process of expression and secretion of HPA by drawing intracellular Ca²⁺ regulation path ways into the process. It will provide an experimental foundation and theoretical rational in investigating therapy of inhibiting the HCC invasion and metastasis.Methods: SMMC-7721 cells were divided as control group and experiment group. The former were cultured with normal condition, the later were treated by Ca²⁺ and Thapsigargin (TG) or S-nitro-N-acetylpenicillamine (SNAP). All of the cells were collected on time. The expression of HPA mRNA level in cultured SMMC-7721 was evaluated by RT-PCR. The OD value of electrophoresis strip of DNA product or that of HPA protein was analyzed by gel scanner. Beta-actin was used as an internal standard. The relative expression level of HPA was represented with the ratio between produces of HPA and that of beta-actin. The HPA protein level in cytoplasm and culture supernatant was evaluated by Western blot. Data were analyzed by using SPSS12.0 statistical software.Results: 1 The expression of HPA mRNA level in SMMC-7721 cell in all experiment groups: The photometric brightness of electrophoresis strip and relative expression of HPA mRNA had no significant difference among any groups with addition of CaCl2 into culture system. The same there were no significant differences among any groups with addition of TG or SNAP. It was thus clear that they had no effect on expression of HPA on the level of mRNA.2 The effect of HPA protein expression and secretion level in SMMC-7721 cell culture system with various Ca²⁺ concentration: Addition of 0.2, 0.6, 0.8, 1.0, 1.2mmol/ L CaCl2 into culture system resulted in an enhanced HPA protein expression level in cytoplasm and culture supernatant (P<0.05). At doses of 0.2mmol/ L to 1.0mmol/ L CaCl2, the expression of HPA protein was in a dose dependent manner. Intracellular Ca²⁺ significantly induced the HPA protein expression and secretion in hepatoma carcinoma cell.3 The effect of HPA protein expression and secretion in SMMC-7721 cell by releasing of intracellular Ca²⁺ stores: At a dose of 4μmol/ L TG, the expression of HPA protein was (2.596±0.187) in cytoplasm and it was (1.403±0.023) in culture supernatant compared to the control, which was (0.930±0.031) in cytoplasm and it was (0.610±0.012) in culture supernatant increased significantly (P<0.05). It was thus clear that TG significantly enhanced the expression and secretion of HPA protein in SMMC-7721 cell.4 The relationship between intracellular Ca²⁺ regulation path ways and HPA protein expression and secretion in SMMC-7721 cell: At a dose of 200μmol/ L SNAP and 4μmol/ L TG, the expression level of HPA protein was (0.951±0.056) in cytoplasm and it was (0.594±0.018) in culture supernatant compared to at the dose of 4μmol/L TG which was (2.596±0.187) in cytoplasm and it was (1.403±0.023) in culture supernatant degraded significantly (P<0.05). But there were no significant difference between these and the control which was (0.930±0.031) in cytoplasm and it was (0.610±0.012) in culture supernatant. At the same time, there were no significant difference between these and at a dose of 200μmol/ L SNAP which was (0.957±0.023) in cytoplasm and it was (0.603±0.014) in culture supernatant. It was thus clear that SNAP significantly inhabited the inducing effect of TG on HPA protein expression and secretion, but there was no significantly inhabiting effect on HPA protein expression in normal cell without TG.Conclusions: 1 Intracellular Ca²⁺ have no effect on the expression of HPA on the level of mRNA, while it significantly induces the HPA protein expression and secretion in hepatoma carcinoma SMMC-7721 cell on the level of protein.2 Thapsigargin (TG), an inducer of intracellular Ca²⁺ stores depletion, significantly enhances the expression and secretion of HPA, while the inducing effect of TG on HPA expression and secretion is significantly inhibited by S-nitro-N-acetylpenicillamine (SNAP).3 Intracellular Ca²⁺ enhances the expression and secretion of HPA on the level of protein by Ca²⁺ regulation path ways which is sensitived to NO→cGMP in hepatoma carcinoma SMMC-7721 cell. And then HPA degrades the extracellular matrix and vascular basement membrane and induces angiogenesis to promote the process of invasion and metastasis of tumor.