| BackgroundLiver cancer refers to the malignant tumor that occurs in the liver with a high mortality rate. Liver cancer could be mainlydivided into primary liver cancer and secondary liver cancer. The primary liver cancer is a major type of liver cancer. The primary liver cancer mainly caused by cells which are part of liver canceration and could be divided into hepatocellular carcinoma, cholangiocellular carcinoma, and mixed hepatocarcinoma. The morbidity and mortality of primary liver cancer are increasing year by year. The hepatocellular carcinoma accounts for about 85.5%. The primary liver cancer is one of the highest malignant tumors in China. There are about 540000 people who died from this disease each year.Therefore, studying the prevention and treatment of hepatocellular carcinoma will be of important scientific significance and clinical value.Hydrogen sulfide is considered to be a poisonous gas with rotten egg smell. In recent years, many studies have shown that hydrogen sulfide can be endogenously synthesized from enzymes in the body, and plays a role in physiological and pathological processes in the body.It has been considered as the third gasotransmitter following nitric oxide and carbon monoxide. It has been shown that hydrogen sulfide plays important roles in cancer biological processes. However, there has been some controversy on therole of H2 S in cancer development and progression. There is a point that hydrogen sulfide could promote the proliferation of liver cancer cells. And another view is that hydrogen sulfide could inhibit the growth of liver cancer cells. In order to clarify the debate, human liver cancer cells have been uesd to determine the effect of H2 S on tumor growth in this study.ObjectiveTo study themechanism of action ofhydrogen sulfide on the growth of human hepatocellular carcinoma(HCC) cells.Methods1. In vitro studies1.1 MTT assayThe human normal hepatocyte cell line L02 and HCC cell lines SMMC-7721 and Huh-7 were treated with NaHS at concentrations of 0, 10, 25, 50, 100, 200, 400, 600, 800, and 1000 ?M for 24 h. 10% MTT was put per hole for 4 h. Then the OD were determined by microplate reader. The aim was to test the effects of hydrogen sulfide on cell proliferation. The higher OD value, the greater the level of cell proliferation.1.2The wound-healing assayThe human normal hepatocyte cell line L02 and HCC cell lines SMMC-7721 and Huh-7 were treated with NaHS at concentrations of 0, 10, 25, 50, 100, 200, 400, 600, 800, and 1000 ?M for 24 h in the wound-healing assay. According to 0 h, 12 h, and 24 h interval, photographs were used to observe the healing condition of scratches. The aim was to assess the effects of hydrogen sulfide on tumor cell migration.1.3Western blottingThe expression of Bax, Bcl-2, Caspase-3, Cleaved Caspase-3, AKT, phosphoated-AKT, ERK, phosphoated-ERK, PTEN, MMP-2, EGF, and phosphoated-EGF in tumor cells which were treated with NaHS at concentrations of 0, 10, 25, 50, 100, 200, 400, 600, 800, and 1000 ?M for 24 h were determined by western blot.1.4TUNEL and DAPI stainingThe TUNEL staining was used to observe apoptosis in cells which were treated with NaHS at concentrations of 0, 10, 25, 50, 100, 200, 400, 600, 800, and 1000 ?M. And at the same time, the DAPI staining was adopted to calculatethe apoptosis rateof each group.2. In vivo studies2.1 Establishing the xenograft model, grouping and administratingEstablishing the xenograft model: SMMC-7721 and Huh-7 cells, at 5 × 105 suspended in 200 ?L of PBS, were implanted by subcutaneous injection into the right flanks of mice. Twenty-four hours after inoculation, tumorigenicity was 100%, which indicated that the xenograft model was established successfully.Grouping and administrating: different concentrations of NaHS(0–1000 ?M) were continuously administrated subcutaneously(near the implanted tumor) for 14 days in the peritumoral tissues after establishing the xenograft model, and the injection of same volume of sterile saline was considered as control group. The length and width of solid tumor were measured every day during the course of experiment. It was used to assess the regulatory effects of H2 S on the growth of solid tumors.2.2 Morphological studyHematoxylin and eosin(HE) staining protocol was used to analyze the morphology of the tumors.It was used to assess the regulatory effects of H2 S on the growth of solid tumors The technology of CD34 immunohistochemistry was applied to count the number of microvessels in solid tumors.2.3 Toxicity assessment testThe toxicity of H2 S on nude mice bearing HCC xenografts was analyzed by measuring theweighs of lung, heart, liver, spleen, kidney, brain, and the whole body, testing the number of white blood cells in each group, and observingthe results of HE staining of the heart, liver, spleen, lung, kidney, and brain.Results1. The MTT and wound-healing assays have shown that 0-1000 ?M NaHS had no obvious effect on the growth of normal liver cells(P> 0.05). However, compared with the negative control group, 10-200 ?M NaHS significantly enhanced the proliferation and migration of human hepatocellular carcinoma cells(P < 0.05), while 600-1000 ?M NaHS inhibited the proliferative activity and migration of human hepatocellular carcinoma cells(P< 0.05).2. The results of TUNEL staining and western blot have shown that 25-100 ?M NaHS significantly reduced the degree of apoptosis in human hepatocellular carcinoma cells(P< 0.05), while 400-1000 ?M NaHS dose-dependently increased the apoptotic level(P< 0.05).3. Compared with the negative control group, low concentration of H2 S could induce phosphorylation of EGFR, ERK, and AKT(P< 0.05), while higher concentration of H2 S could inhibit phosphorylation ofEGFR, ERK, and AKT(P< 0.05).4. After the human hepatocellular carcinoma cells were inoculated on the right flank of nude mice, tumorigenicity was 100%, which indicated that the xenograft model was established successfully.5. The results of HE staining have shown that the effects of 10-1000 ?M NaHS on the growth of human hepatocellular carcinoma cellspresented an earlier increase and later decrease trend.6. Immunohistochemical results show that compared with the negative control group, the microvascular density(MVD) was obviously higher when administrated of 10-100 ?M NaHS(P< 0.05), while 600-1000 ?M NaHS decreased the MVD in a dose-dependent manner(P< 0.05).7. HE staining results showed that there were no obvious morphological changes in heart, liver, spleen, lung, kidney, and brainamong each group. There were no significant differences in both the numbers of the total white blood cells(WBC) and the relative weights of the organs in each group(P> 0.05).Conclusions1. The low concentrations of H2 S could promote proliferation and migration of human hepatocellular carcinoma cells.2. The high concentrations ofH2 S could inhibit the growth and promote apoptosisof human hepatocellular carcinoma cells.3. The low concentrations of H2 S could induce proliferation of human hepatocellular carcinoma cells by enhancing the activity of EGFR and activatingthe expression of ERK and AKT. However, high concentrations of H2 S couldpromoteapoptosis of human hepatocellular carcinoma cells by reducing phosphorylation ofEGFR, ERK and AKT.4. The low concentrations of H2 S could promote the growth of tumors by inducing angiogenesis, while high concentrations of H2 S could accelerate cell death by inhibiing angiogenesis.5. Compared with the control group, 10-1000 ?M NaHS showed no significant toxicity on tumor-burdened nude mice. |
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