Study On The Effects Of Platelet Type 12-lipoxygenase Inhibitor On The Proliferation And Migration Of Gastric Cancer Cells

Objective:To investigate the effects and mechanism of platelet type 12-lipoxygenase (p12-LOX) inhibitor baicalein (BAI) on the proliferation and migration of gastric cancer MKN-28 cells.Methods:The expressions of p12-LOX mRNA in gastric cancer MKN-28 cells and normal gastric mucosa GES-1 cells were detected by reverse transcription PCR (RT-PCR). After GES-1 and MKN-28 cells were treated with different concentrations of BAI, the abilities of proliferation and horizontal migration were detected by MTT, wound-healing and Transwell cell migration assay, respectively. The cell cycle was arrested in G0/G1 phase and the apoptosis was induced after 10μM BAI treatment of gastric cancer MKN-28 cell were detected by Flow cytometry. Hoechst staining to observe the apoptosis of gastric cancer MKN-28 cells. The expressions of E-cadherin and Snail proteins were detected by immunofluorescence staining method. The expression lever ofp12-LOX and Ezrin protein, apoptosis related protein, EMT marker protein, TGF-β/Smad and MAPK/ERK singnaling related protein in MKN-28 cells were detected by Western blot assay following BAI treatment. Results:1. The expression level of p12-LOX and Ezrin genes in MKN-28 cells was higher than in normal gastric mucosa GES-1 cells (P<0.01); 2. MTT results showed that after treatment with different concentration of BAI, the proliferation of MKN-28 cells was significantly inhibited in a dose-and time-dependent manner (P<0.05), but has no significant effect on the proliferation of GES-1 cells; 3. Flow cytometry detection results showed that 10μM BAI can arrest the cell cycle in G0/G1 phase of MKN-28 cells (P<0.05); Hoechst 33342staining and flow cytometry detection results showed that BAI can induce apoptosis of MKN-28 cells; 4. Wound healing assay and Transwell migration assay showed that after treatment with BAI the migration ability of MKN-28 cells was inhibited (P<0.05); Immunofluorescence results also show that following BAI treatment MKN-28 cells the expression of E-cadherin protein was significantly increased (P<0.05), but the expression of Snail protein was decreased (P<0.01); 5. Western blot results showed that MKN-28 cells treatment with BAI, the expression of p12-LOX, Ezrin, GSK-3β, Vimentin, Snail, p-MEKK1, p-Erk1/2, TGF-(31, p-Smad3 proteins were decreased (P<0.01) and Cleaved-Caspases-3 and E-cadherin protein were decresed (P<0.05 or P<0.01).Conclusion:1. The expression of p12-LOX and Ezrin gene were over-expressed in gastric cancer cells MKN-28; 2. BAI inhibited the proliferation, migration and EMT, and induces apoptosis in MKN-28 cells; 3. MAPK/ERK and TGF-p/Smad signaling pathway may be involved in the molecular mechanism of BAI inhibit cell proliferation and migration in gastric cancer MKN-28 cells.