| Hepatocellular carcinoma in the world belong to a high incidence of malignant tumors, after gastric cancer and esophageal cancer mortality in the third place, while 40% of liver cancer patients was mainly concentrated in China. In recent years. HCC incidence rate increased year by year, and there is a small age group goes to the trend, its high rate of recurrence and metastasis to liver cancer patients the treatment more difficult. Looking for a highly efficient low toxicity, and on the proliferation and metastasis of hepatocellular carcinoma cells significantly inhibited from the new drugs have become a hotspot in the current hepatocellular carcinoma research.This study aimed to investigate the water caltrop extracts on human hepatocellular carcinoma SMMC-7721 proliferation and metastasis capacity of the basis for the development of efficient low-toxic anti-tumor proliferation and metastasis of new drugs provide a theoretical basis and experimental basis.Experimental use of the preliminary work task force to extract a variety of water caltrop extract screening, using MTT choice of human hepatocellular carcinoma SMMC-7721 cell proliferation of the most obvious drugs, look for the best action time points, the appropriate subjects drug concentration and cell density; Using phase-contrast microscope to observe the morphology of tumor cells on the changes; Switzerland-Giemsa stain was observed on a single tumor cell colony-forming capacity of the subjects drug; PI staining detected by flow cytometry on tumor cell cycle; AO fluorescence staining was observed on tumor cell apoptosis; At the same time was measured by using HPLC water caltrop extract the main component of the content; MTT assay was used to detect subjects drugs on adhesion ability of tumor cells; Transwell chamber assay used to observe the subjects drugs on tumor cell migration and invasion of capacity.and further determined using gelatin zymography undergo drug test the role of tumor cells after 24h the supernatant MMP-2 and MMP-9 expression.1. Screen activity of water caltrop extractExperimental use of the three kinds of water caltrop extract:tested drugs 1 (polysaccharide 1), and testedd drugs 2(polysaccharide 2), tested drugs 3(trihydroxyb-enzoicacid of water caltrop extract,C1) and tested drugs 4 (trihydroxy benzoicacid standard reference substance, TBA), to observe its human hepatocellular carcinoma SMMC-7721 cells inhibition of proliferation, in order to filter the best subjects drug. Experimental data show that four kinds of drugs in tested drugs 3 and tested drugs 4 significantly inhibited the proliferation of cancer cells, IC50 were 11.0μg/mL and 8.4μg/mL, while the other two subjects tested drugs 1, tested drugs 2's IC50 were 51.5μg/mL and 53.0μg/mL. The results show that, tested drugs 3 and tested drugs 4 can be achieved in smaller concentrations of human liver cancer SMMC-7721 cells in vitro, while the tested drugs 1 and tested drugs 2 will need more than five times the largest the concentration of tested drugs 3 and tested drugs 4 can be played the same inhibitory effect, indicating strong efficacy of tested drugs 3 and tested drugs 4.2. The determination of TBA in Water caltrop extract contentIn view of tested drugs 3 and tested drugs 4 on SMMC-7721 significantly inhibited, and there to promote their role in apoptosis, experimental use of HPLC for tested drugs 3 of trihydroxybenzoic acid (TBA) were determined. According to experimental data, the results of the calculation we can see that tested drugs 3, Water caltrop extract of TBA content of 79.14%, to a more pure product.3. Water caltrop extract on proliferation of SMMC-7721Experiments selected six time points of the subjects drugs on tumor proliferation inhibition were observed, results showed that when the time point to reach 12-24h, the inhibitory rate of change substantially, and in the 24h and reached the peak suppression, while in the 4-1 Oh, the inhibitory rate of change is not obvious, so experiment to select the best time points 24h. When tested drugs 3 and tested drugs 4 concentration increases on human hepatocellular carcinoma SMMC-7721 cells, the inhibition rate increased with increase of the density gradually decreased, and both the cell density of 2×105/mL to 4×105/mL time trend in a smooth, tested drugs 4 inhibition rate of tumor cells were slightly higher than tested drugs 3, Therefore, select the cell density experiments ranged from 2×105/mL and 4×105/mL among the 3×105/mL as the most appropriate cell density for cell metastasis experiments. Based on these experimental data, experiments on the role of 24h by tested drugs 3 and tested drugs 4 after the SMMC-7721, morphological changes were observed. The number of cells tested drugs 3 and tested drugs 4 with increasing concentrations of drugs subjects gradually reduced, and the cell adhesion is not easy to come off strong, irregular cell shape, sizes, shrink the membrane can be seen, indicating tested drugs 3 and tested drugs 4 can be a good change in SMMC-7721 in the normal form, its growth impact. CFU experiments tested drugs 3 and tested drugs 4 on SMMC-7721 single cell colony formation inhibition more pronounced, with the increase of the concentration of colony-forming role in inhibiting the stronger, tested drugs 4 than tested drugs 3 inhibiting cell colony-forming ability, better able to inhibit tumor cell proliferation. Subjects drugs in inhibiting tumor cell proliferation at the same time, often to promote their apoptosis of the phenomenon, experiments by tested drugs 3 and tested drugs 4 through the role of the tumor cells 24h after AO staining, showing that subjects with the increase in drug concentration, cell reduction in the number, the cell size smaller, more common view within the crescent-like, beads-shaped nucleus, nucleus fragmentation or even more visible green debris, showed the typical features of late apoptosis.4. Water caltrop extract on tumor cell cycleExperiment has confirmed that tested drugs 3 and tested drugs 4 on SMMC-7721 significantly inhibited proliferation, based on the impact of its cell cycle studies, with the subjects the increase in drug concentration, cell the number of G2/M phase arrest increased, while PI staining by flow cytometry observations drugs can promote late apoptosis of tumor cells. 5. Water caltrop extract on the ability of tumor cell metastasisThe results show that at lower concentrations, tested drugs 3 on SMMC-7721 cell adhesion inhibitory stronger, but higher concentration, tested drugs 4 effect on cell adhesion is stronger than that of tested drugs 3. In the migration experiment, tested drugs 4 on cell migration of effect were larger than tested drugs 3, and with increasing concentration through the Transwell chamber to the next room of the cells decreased, indicating the role of inhibiting migration significantly, showing a definite dose-effect relationship. For the tested drugs 3 and the effects of tested drugs 4 on cell invasion, experimental results show that two subjects drugs on inhibition of tumor cells were strong through the last room is covered with matrigel (artificial basement membrane) of the cell in subjects with the increase in drug concentration decrease in inhibition rate, between the two subjects drugs no significant differences in the inhibition of tumor cell invasion. MMP-9 in each group tested drugs had no expression; Experimental analysis using Quantity one software quantitative analysis the content of MMP-2, the relative density of the experimental results are explained. The analysis shows, Marker relative density value of 325.7918, the relative density of the negative control group value of 321.6615. The concentration of tested drugs 3 of 2.6μg/mL, the relative density value of 199.9158, the value of the relative density of 210.5933 when The concentration of tested drugs 3 is 5.2μg/mL. Tested drugs 4 concentration is 2.6μg/mL, the relative density value of 211.2246, the relative density value of 244.2673 when The concentration of tested drugs 4 is 5.2μg/mL.Negative control group and Marker expression of MMP-2 is close. All concentrations of the two drugs tested in the expression of MMP-2 were down. In the two lower concentrations tested drugs MMP-2 expression were lower than the high concentration. Low concentrations of two drugs tested expression of MMP-2 content in very high concentration of tested drugs 3 is stronger expression of MMP-2 compared with tested drugs 4. Gelatin zymography can see through the tested drugs 3 and tested drugs 4 are able to significantly down regulation the concentration of MMP-9, MMP-2 in SMMC-7721 and inhibited by a considerable role in providing further evidence that the tested drugs 3 and tested drugs 4 on SMMC-7721 with strong inhibition of metastasis. In conclusion, tested drugs 3 (water caltrop extract C1) on SMMC-7721 proliferation and metastasis were significantly inhibited. HPLC determination of water caltrop extract C1 of trihydroxybenzoic acid content of 79.14%, while the standard reference substance of trihydroxybenzoic acid content of 98%, so we can see water caltrop extract C1 on human hepatocellular carcinoma SMMC-7721 cell proliferation and inhibition of metastasis was stronger than TBA.
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