The Role Of Classic Members Of NFAT Family In The Growth Inhibition Effect Of As₄s₄ On Colon Cancer

[OBJECTIVE]To investigate the mechanisms of growth inhibition of Arsenic sulfide（As₄S₄）in colon cancer cells and exploration the role of all four classic NFAT proteins in this course,and enrich the theoretical basis of arsenic sulfide on colon cancer treatment,which will provide a scientific basis for its clinical application.[METHODS]HCT116 were treated with As₄S₄ and/or CsA in various concentrations for different durations and its inhibitive effect on cell proliferation was detected by MTT method.To determine the possible mechanisms of the effects of As₄S₄ on cell growth inhibition,the protein levels of all four classic NFAT,PML,P53,c-Myc were measured by Western blotting analysis.To investigate the correlation of all four classic NFAT proteins with colon cancer,we performed an immunohisto-chemical staining for NFAT proteins in 90 human colon cancer and paired normal tissues.NFATc3 was knocked down in colon cancer cell lines,and the effects on clone formation and growth of xenograft tumors in nude mice were assessed.To investigate the molecular mechanisms about the regulation effects of As₄S₄ on all four classic NFAT,PML and P53 were knocked down and P53 was overexpressed in colon cancer cell lines,and the effects on the expression of associated members were assessed by western blotting and RT-PCR.We analyzed ragulation of NFATc2 by P53 in chromatin immunoprecipitation and Dual-luciferase reporter gene assay studies.[RESULTS]As₄S₄ and CsA synergistically inhibited colon cancer cell growth and As₄S₄ reduced the transcription of NFATc1,NFATc3,and NFATc4 while dramatically stimulated the transcription of NFATc2 by regulating PML and P53.Tumor expression of nuclear NFATc1 was associated with inferior survival while cytoplasmic NFATc2with superior survival in patients with colon cancer.NFATc3 knockdown inhibitedcolon cancer cell growth in vitro and in xenograft.PML knockdown reduced NFATc1,NFATc2,NFATc3,NFATc4.P53 differentially regulates the expression of NFATc2,NFATc3 and NFATc4 by stimulating NFATc2 while repressing NFATc3 and NFATc4.Reciprocally,NFATc2 positively while NFATc3 negatively regulates P53,forming a positive and a negative feedback loop.[CONCLUSION]These results indicate that As₄S₄ differentially regulates NFAT pathway through PML and P53 and reveal an intricate reciprocal regulatory relationship between NFAT proteins and P53 pathway.NFAT play important role in colon cancer and As₄S₄ might inhibit proliferation of colon cancer by regulating NFAT.