Low Oxygen Environment For Melanoma Cells Proliferation And The Influence Of The Expression Of The LDH And Nodal

Abstract:bjective:Melanoma?Malignant Melonoma,MM?is one of the fastest rising incidence,one of the most adverse prognosis of malignant tumor,with high fatality rate.And the metabolism and proliferation mechanism of MM cells are still unknown,but factors like heredity,traumatic,immune and virus infection may involved in the occurrence and development of MM.Local hypoxia is considered as an important phenominon which is existed in the process of common malignant tumor proliferation.The genes and proteins which are related with hypoxia can be caused by low oxygen so that cells can adapt to hypoxic microenvironment.High glycolysis is one of the characteristics of malignant tumor cell's metabolism,the MM will be influenced by intervention degree of malignant tumor cell metabolism.What is the reason and mechanism of MM proliferation,invasion and metastasis?What is the key point of control MM process?With the deepening of the research,under the condition of low oxygen glycolytic pathway of abnormal activation of the tumor and Nodal molecular promotes tumorigenesis.This topic using cobalt chloride?CoCl₂?induced human melanoma cell line A375 cell Hypoxia,build the MM cell Hypoxia model,with LDH mediated role in tumor cell proliferation as the breakthrough point,on A cellular level to explore Nodal molecules?Nodal growth differentiation factor?,HIF?Hypoxia-inducible factor,HIF1-a?,lactate dehydrogenase protein expression and lactate dehydrogenase isozyme spectrum changes,the results can provide theoretical basis for the targeted therapy to the MM.Methods:chemical oxygen revulsant CoCl₂ were choosed to cultivate human melanoma cells with culture medium containing different concentrations of CoCl₂?0,50,100,150,200,250,300,400,uM?,and effect of different time?0,6,12,24,48,72,96 h?,and then MTT were used to observe low oxygen influence on human melanoma cells proliferation ability;human melanoma cells after the suitable concentration of oxygen and time by CoCl₂ induced,HIF1-a,LDH-A and LDH-B,Nodal protein expression and LDH isozyme spectrum of activity was detected by Western Blot and agarose gel electrophoresis technology different concentrations and at different times of human melanoma cells.Results:The method of MTT showed CoCl₂ induced by hypoxia on human melanoma cells proliferation ability as CoCl₂ concentration increment and function as the extension of time its proliferation activity gradually decline.Among them,the role of CoCl₂concentration 50,100,150,100,250 uM no obvious cell proliferation inhibition?P>0.05?.Since 300 um showed obvious toxic effect.The role of CoCl₂ concentration is 50,100,150 uM,cell proliferation reached peak at 48 h,CoCl₂ for 200,250 uM,24 peak cell proliferation?P<0.05?,and the concentration of 200 and 250 um cells in inhibition?P<0.05?.CoCl₂ human melanoma cells induced the suitable concentration of oxygen is 50,100,150,200,250,uM,appropriate time to 6,12,24 h,Western Blot technology test results show that CoCl₂ may increase human melanoma cell line A375 HIF1-a,LDH-A,Nodal protein expression,LDH-B protein expression decreased?P<0.05?,and presents a certain range of concentration and time dependent relationship;Agarose gel electrophoresis test results show that with increasing the concentration of oxygen,LDH isozyme spectrum changes for LDH3,LDH4activity and LDH5,LDH1 and LDH2 decreased?P<0.05?.Conclusion:a moderate amount of CoCl₂ human melanoma cells induced hypoxia model,HIF1-a,LDH-A and Nodal expression,cut and LDH-B expression suggests that LDH isozyme spectrum change,Nodal is associated with the occurrence of tumor development.