The Effects Of Epigenetic Regulation Of MAL On Gefitinib Sensitivity In NSCLC And Potential Mechanisms

In recent years, lung cancer has become the leading cause of malignant cancer deaths with increasing incidence and mortality in our country, while nonsmall-cell lung cancer(NSCLC) being the most common tumor types. Because of limited efficacy and adverse effects for traditional drugs, molecular targeted agents such as gefitinib(Gef), seems to become new strategy for advanced NSCLC treatment. However, acquired resistance was also serious, which became the main reason for limiting its clinical effects. Epigenetic mechanisms have been focused recently, with its prevalence in acquired resistance for varieties of anticancer drugs. This study started from Gene Chip results, explored the role and mechanisms of Myelin and lymphocyte protein(MAL) in Gef resistance of NSCLC.Searching for epigenetic mechanisms of Gef resistance, and thus overcome resistance or increase sensitivity becomes the critical objective of this study.Part I: The Effects of Epigenetic Regulation of MAL on Gefitinib Sensitivity in NSCLC.Objectives: To investigate the relationship between MAL expression and sensitivity to Gef in various NSCLC cell lines; and the potential mechanisms of MAL downregulation and the effect of its reversal on Gef sensitivity. Methods: MTT assay verified the IC50 values of Gef in NSCLC cells; RT-PCR and Western Blotting detected MAL expression in these cells; MSP detected methylation status of MAL promoter region; Measured the effects of DAC treated alone or in combination with SAHA on MAL expression and Gef sensitivity. Results: MAL expression showed significant differences between resistant and sensitive NSCLC cell lines, while 4/5 resistant cell lines showed reduced or absent of MAL expression, which may be related to Gef sensitivity; Hypermethylation of promoter region closed to the transcription started site was the main reason for MAL silencing;DAC could reverse hypermethylation status, restore MAL m RNA expression, and more obvious effects were observed when combined with SAHA treatment; The cells which restored MAL expression showed increased sensitivity to Gef therapy. Conclusion: Gef resistant NSCLC cells showed frequently declined or absent of MAL expression, with the transcription started region hypermethylated status; Reversing the hypermethylation and thus restoring MAL expression could increase Gef sensitivity in resistant cells.Part II: The Potential Mechanisms of Increased Gef Sensitivity by Restoration of MAL Expression.Objectives: The PC9/G、H1975 cells were used in this study to investigate the potential mechanisms of increased sensitivity to Gef therapy by promoted MAL expression, which induced by epigenetic drugs. Methods: MTT assays were used to measure the effects of elevated MAL protein expression on Gef sensitivity; CCK-8 assay, flow cytometry and wound-healing assay was used to detect the effects of elevated MAL expression on cell morphology, proliferation, apoptosis and cell cycles, colony formation and migration;The effects of elevated MAL expression on EGFR signaling pathway were detected by Western Blotting; Some methods above were used to study the effects of four polyphenol compounds on MAL expression and Gef sensitivity. Results: Treatment of DAC alone or combined with SAHA significantly promoted MAL expression, increased sensitivity to Gef in PC9/G and H1975 cells; Elevated MAL expression by DAC plus SAHA treatment made morphology of PC9/G cells more spreading, significantly inhibited abilities of cell proliferation, colony formation and invasion, while it also induced early apoptosis and G2/M phase arrest slightly; Elevated MAL reduced baseline levels of phosphorylation of EGFR and its downstream signalings, and accelerated activation and degradation of the related signals induced by EGF or Gef stimulation in resistant cells; DAC plus SAHA treatment upregulated the expression of E-Cadherin in these cells as well; Resveratrol,apigenin, myricetin and luteolin, all the compounds above could increase Gef sensitivity in varying degrees, reverse MAL hypermethylation and increase MAL transcription in PC9/G cells. Conclusion: Restoration of MAL expression by DAC and SAHA increased Gef sensitivity might be related to the inhibition of cell proliferation, colony formation and invasion, while morphologic changes were also observed. Reducing the baseline levels, changing the phosphorylation patterns of EGFR pathways signalings may be the potential mechanisms. MAL expression levels hold the potential to be a predictor of Gef sensitivity in NSCLC.