SB-431542 For Melanoma Cells Proliferation And The Influence Of The Expression Of The LDH And Nodal

Objective:To explore the effects of Hypoxic、Nodal inhibitors（SB-431542）for melanoma cells proliferation and the influence of the expression of the lactate dehydrogenase isozyme subunits（LDH-A,LDH-B）,the Nodal growth differentiation factor and its receptor Activin receptor-like kinase4,Activin receptor-like kinase7 protein expression and LDH isozyme spectrum changes;Preliminary discussion on whether hypoxic environment,SB-431542through influence the expression of LDH,Nodal and its receptors to influence melanoma of the development process,and explore whether LDH and Nodal are related to melanoma energy metabolism,and provide New idea for the prevention,diagnosis and treatment of MM in clinic.Methods:CoCl₂ was used to induce hypoxia in melanoma cell A375 in vitro and a hypoxic model was constructed to simulate its microenvironment in vivo,and treated with SB-431542.Determined by MTT method to detect the influence of hypoxic、SB-431542 in A375 cell proliferation,explore the optimal drug concentration and action time of cell proliferation.Using Western Blot technique to detect Hypoxia-inducible factor protein expression level in order to make clear whether hypoxic model to build success,and detect the expression of LDH-A、LDH-B、Nodal and its receptor ALK4、ALK7;Agarose gel electrophoresis was used to detect the effect of hypoxia and SB-431542 on the activity of lactate dehydrogenase isozymes.Results:1.The method of MTT shows the influence of hypoxic induced by CoCl₂ to the MM cell proliferation:the concentration of CoCl₂ range from 50 to 200uM showed not obvious inhibition for cell proliferation（P>0.05）.When the concentration of more than 250uM,with the increase of concentration of CoCl₂and function of the extension of time,showed obvious inhibitory effect.When the concentration of CoCl₂ is 150 uM,cell proliferation reached peak at48h.The influence of SB-431542 to the MM cell proliferation:With the increase of concentration of SB-431542 and the extension of time,the cell proliferation ability showed a trend of decline.Concentration of less than 10 um weak inhibition;Concentration for 10 to 20 um show some inhibitory effect;More than 30um concentration showed obvious inhibitory effect.Different concentrations of CoCl₂ with SB-431542 effects on cell proliferation:when the concentration of SB-431542 for 10 uM constant,the concentration of CoCl₂range from 50 to 150uM showed not obvious inhibition on cell proliferation（P>0.05）.When the concentration of more than 200uM showed obvious inhibitory effect.When the concentration of SB-431542 for 10uM and the concentration of CoCl₂ for 150u M,cell proliferation reached peak at 48h.2.Western Blot technology test results show that the appropriate concentration of CoCl₂ can induce hypoxia in A375 cells.And then HIF-1αprotein expression decreased,At the same time,the expression of LDH-A,Nodal,ALK4 and ALK7 protein can be upregulated,and the expression of LDH-B protein is downregulated（P<0.05）.The expression of LDH-B protein can be upregulated and the LDH-A,Nodal,ALK4,ALK7 protein can be reduced by SB-431542（P<0.05）.3.The results of agarose gel electrophoresis indicated that LDH5 activity was increased in hypoxic environment,while SB-431542 could reduce LDH5activity（P<0.05）.Conclusion:1.A suitable concentration of CoCl2 could successfully induce melanoma cell hypoxia environment model at appropriate time.Hypoxia could promote the proliferation and energy metabolism of melanoma cells,which may be associated with the high expression of HIF-1α,LDH-A,Nodal and their receptors,and the low expression of LDH-B.2.SB-431542 could inhibit the proliferation and energy metabolism of melanoma cells,which may be related to the expression of LDH-A,Nodal and its receptor protein was downregulated,and the expression of LDH-B protein was upregulated.