Rhein Regulates Rac1/LIMK1/Cofilin Signaling Pathway And Inhibits The Invasion And Metastasis Of Human Ovarian Cancinoma Cells With Directional High Lymphatic Metastasis

Objective:The aim for this study was to investigate the effects of Rhein on motility ability of ovarian cancer SKOV3-pm4 cell and to observe the change of the cell ultrastructure. Explored the relationship between the expression of key protein molecule in Racl/LIMKl/Cofilin signaling pathway and the invasive ability of ovarian cancer SKOV3-pm4 cells. The results was the foundation for the research of Rhein mechanisms and providing new intervention methods for ovarian cancer metastasis and invasion research.Methods:The cell proliferation of ovarian cancer cells with highly directional lymphatic metastasis (SKOV3-pm4) was tested by MTT assay after treated with different concentrations of Rhein. The invasive ability of cells was measured by Transwell chamber assay. The effect of Rhein on the morphology and cytoskeleton ultrastructure of ovarian cancer cells was observed by laser scanning confocal microscope and scanning electron microscope at different times and different concentrations. The PMA was selected as the Rac1 activator and NSC23766 as Rac1 inhibitors. The total protein of Rac1 and Rac1-GTPase in ovarian cancer SKOV3-pm4 was tested by Racl Pull-down Assay and Western Blot. The genes and protein expression level of Rac1/LIMK1/Cofilin and Racl/IRSp53/WAVE2/Arp2 molecular signaling pathway were detected by qRT-PCR and Western Blot.Results:Rhein inhibited proliferation ability of SKOV3-pm4 cells, the half maximal inhibitory concentration (IC50) of 24h was 121.24μmol·L-1. After treated with 26.40μmol·L-1 of Rhein, the abilities of migration and invasion of SKOV3-PM4 cells were inhibited. The morphology and cytoskeleton ultrastructure of SKOV3-PM4 cells were changed, cellular pseudopod reduced, cell microfilament fractured and its distribution disordered, plasma membrane was uneven and cell gap widened. The results of Racl Pull-down Assay and Western Blot shown that Rhein can down-regulate protein expression of total Rac1 protein and Rac1-GTPase activity at a concentration-dependent manner; Racl inhibitors NSC23766 plus Rhein, compared with Racl inhibitor alone, total Rac1 protein and Rac1-GTPase reactive protein of ovarian cancer were significantly decreased (P<0.05). For Rac1 activator PMA, total Rac1 protein and Racl-GTPase activity protein were increased (P<0.05). When Rac1 activator plus Rhein, total Racl protein and Racl-GTPase activity were reduced compared with Racl activator PMA group alone (P<0.05).The results of qRT-PCR and Western Blot experiments shown that after Rhein, Rhein plus Rac1 inhibitors and Rhein plus Rac1 activators treated, compared with the control group, Rac1 inhibitors and Rac1 activator alone, the mRNA expression level of Racl, PAK1, LIMK1 and cofilin decreased (P<0.05), the expression level of Rac1, P-PAK1, P-LIMK1 and P-cofilin proteins were also down-regulated (P<0.05), but the total protein of PAK1, LIMK1 and cofilin did not change significantly. The expression of key genes and proteins levels in Racl/IRSp53/WAVE2/Arp2 molecule signaling pathway shown that after Rhein, Rhein plus Rac1 inhibitors and Rhein plus Rac1 activators treated, compared with the control group,Rac1 inhibitors and Rac1 activator alone, the mRNA expression and protein levels of IRSp53, WAVE2 and Arp2 were also decreased (P<0.05) with a dose dependent manner.Conclusion:Rhein can inhibit migration and invasion of ovarian cancer cells with highly directional lymphatic metastasis, Its inhibition effect was closely associated with the changes of cell morphology and ultrastructure. Total Rac1 protein and Rac1-GTPase activity of the Rac1 protein is significantly inhibited by Rhein, Rhein is a potential Racl small molecule inhibitor. Racl/ LIMK1/Cofilin and Rac1/IRSp53/WAVE2/Arp2 molecule signaling pathway is closely allied to ovarian cancer cell skeleton, Rac1 protein was the target and modulates the Rac1/LIMK1/Cofilin signaling pathway.