Effects Of Adinbitor On Adhesion And Proliferation In Human Hepatocellular Carcinoma Cell Strain SMMC-7721

Objective: Malignant tumor is one of the main reasons that threaten heavily health and cause death in humans. Toxic side effects of anti-cancer drugs have become one big and main obstacle to different extent. It is very urgent to look for new anti-cancer drugs with high efficiency and low toxicity. The agents inhibiting signal transduction and inducing apoptosis are both new research fields. The adhesion of tumor cells plays an important role in the metastasis of malignant tumor, while the adhesive interaction between cells and extracellular matrix (ECM) is the basis of cell survival and proliferation, which depends on adhesive molecules on cell surface, especially integrin–mediated signal transduction. So the block of tumor cell adhesion is one important strategy to develop anti-cancer drugs. The RGD-containing peptides including disintegrin catch the eyes of reseachers because they can block cell adhesion to ECM and prevent the metastasis of malignant tumors. Soluble RGD peptides can also induce apoptosis of many kinds of cells. To investigate the effects of Adinbitor, a novel RGD peptide obtained by genetic recombination, on the tumor cells , we first employed cell culture technique in vitro, simulated the environment in vivo and stimulated the human hepatocellular carcinoma cell strain SMMC-7721 with fibronectin to explore the effects of Adinbitor on SMMC-7721 cell adhesion. In addition, we studied its effects on the proliferation and apoptosis of SMMC-7721 cells. Our goal was to provide experimental and theoretical basis for Adinbitor in anti-cancer mechanisms.Methods: Our work was composed of two parts: (1) Detecting the effects of Adinbitor on the adhesion of SMMC-7721 cells. Cell adhesion assay was used to observe the adhesion of SMMC-7721 cells to fibronectin. Crystal violet staining was performed to detect the influence of Adinbitor on the adhesion of SMMC-7721 cells. MTT assay was employed to determine the effect of Adinbitor on SMMC-7721 cells that had been adhered. (2) Detecting the effects of Adinbitor on the proliferation of SMMC-7721 cells. SMMC-7721 cells were cultured in media with different concentrations of Adinbitor. The inhibition of proliferation was measured by colorimetric MTT assay. The morphologic changes were observed under light microscope. The apoptosis of SMMC-7721 cells was determined by flow cytometry.Results: 1. The numbers of the adhered cells ware higher in the treated group by fibronectin than the control group (P<0.05). The number of the adhered SMMC-7721 cells had reached to the highest at 20μg/ml, which had no significant difference compared with 40μg/ml and 60μg/ml group. But the numbers of the adhered cells at these three different concentrations are higher than 5μg/ml and 10μg/ml groups(P<0.05). 2. Adinbitor could inhibit the adhesion of SMMC-7721 cells to fibronectin dose-dependently. The higher the concentration was, the stronger the inhibition was. There was significant difference among the groups (P<0.05). 3. The influence of Adinbitor on the adhered SMMC-7721 cells. A few cells became round and smaller and the intercellular space extended at the dosage of 200μg/ml Adinbitor for 24h. After 48h, more detached cells were observed and suspended in the media. But the membranes were in integrity. The result from MTT assay showed that the number of detached cells became increased and the proliferation was strikingly inhibited at higher concentration of Adinbitor (P<0.05). 4. Adinbitor had a strong inhibition on the proliferation of SMMC-7721 cells dosage-dependently (P<0.05). After a 48h exposure, the IC50 value of Adinbitor was 177.83μg/ml. In addition, this result was consistent with the inhibition of Adinbitor on the proliferation of the adhered SMMC-7721 cells when the plates were coated with fibronectin. 5. After exposure of SMMC-7721 cells to Adinbitor at the dosage of 200μg/ml Adinbitor for 36h, the apoptosis rate was 20.68%, significantly higher than that of the control group (2.38%, P<0.05).Conclusions: 1. Fibronectin promotes the ad...