Experimental Study Of Anti-proliferation And Anti-migration Of GSPP On Hepatoma Cells

Objective: Clinically, the majority of malignant tissue stiffness is higher than normal tissue. Ruanjiansanjie is one of the important methods of traditional Chinese medicine treatment of cancer. Gekko-sulfated Glycopeptide(GSPP) derived from Gekko, an anticancer drug in traditional Chinese medicine, is the main effective ingredient of antitumor effect for Gekko. Ourcurrent study is aimed to discussthe effect of GSPP on liver cancer cell proliferation and migration, and tentative exploration the possible mechanism in vitro.Methods:1.Selection human liver cancer Hep G2 cells and seedingthem in culture plates. Set up different concentration groups of GSPP: GSPP low-dosegroup(20μg/m L)、GSPP middle-dose group(100μg/m L)、GSPP high-dose group(200μg/m L) and negative control group.We use MTS assays to explore the effects of GSPP on proliferation of Hep G2 cell, and draw the cell growth curves according to the cell number on different time points.2.Section human liver cancer Hep G2 cells andseedingthem in culture plates. Set up different concentrations groups of GSPP: GSPP low-dose group(20μg/m L)、GSPP middle-dose group(100μg/m L)、GSPP high-dose group(200μg/m L) and blank control group. We use both Wound-healing assay and Transwell assay explorethe effect of GSPP on the migration of Hep G2 cells. Meanwhile, immunofluorescence method was used to observe the expression of E-cadherin、Vimentin and α-SMA.3. Human liver cancer Hep G2 cells were seeded in culture plates. The experimental grouping was the same as above. Flow cytometry was used to detect the expression of integrin β1 on the surface of Hep G2 cells. P-ERK and E-cadherin expression in cells were detected through Western blot assay. The expression status of Rho, Rac, cdc42, Arp3 protein on integrin signal transduction pathway were observed by RT-PCR method.Results:1. MTS assay explore the influence of GSPP on HepG2 cells proliferation: wemeasure the OD value of cells in each group after treat with GSPP at the time of 12h、24h、36h and 48 h. Cells in groups treat with GSPP was significantly less than negative control group(P<0.05), and the high-dose group(200 μg/m L) has the strongest inhibition effect.2.(1) Wound-healing assay: From the images and cell migration distance, compared with blank control group, three dose group treat with GSPP significantly down regulated Hep G2 cell migration(P<0.01).(2) Transwell assay: From the number of cells through the film, we can see that treatment GSPP can inhibit the migration of Hep G2 cells compared with negative control group. And the inhibitory effect of migration is dose dependent.(3) Immunofluorescence method showed that GSPP can significantly up regulated the expression of E-cadherin, and down regulated the expression of Vimentin and α-SMA.3.(1) Flow cytometry showed the expression of integrin β1 on the surface of HepG2 cells were decreased significantly by GSPP treatment compared with the control group.(2) Western blot analysis showed that GSPP significantly inhibition the expression of P-ERK and increased the expression of E-cadherin in Hep G2 cells.(3) RT-PCR method indicated that the expression status of Rho, Rac, cdc42, Arp3 protein on integrin signal transduction pathway were all inhibition by GSPP(P<0.05).Conclusions:1.GSPP can inhibit proliferation of HepG2 cells, and the high-dose group(200 μg/mL) has the strongest inhibition effect.2. GSPP can inhibit migration of Hep G2 cells, and the inhibitory effect of migration is dose dependent.3. GSPP can inhibited HepG2 cells occur EMT and ERK phosphorylation.4. GSPP inhibited Hep G2 cell proliferation and migration, probably by inhibiting the integrin signal transduction pathway.