The Effect And Molecular Mechanism Of TGF-β1on K562Cell Differentiation Induced By Arsenic Trioxide

BackgroundAt the end of the1970s,All-Trans Retinoic Acid（ATRA）was first used to treatacute promyelocytic leukemia（APL） by professor Wang Zhenyi in Shanghaihemopathy institute, which was the most successful case in the history of treatmentof malignancy[1]. APL which had very poor prognosis before became curable.Butthere are still certain defects, such as Retinoic Acid syndrome, high recurrence rateand easily relapse. Arsenic trioxide（As₂O₃） is well known as traditional medicineand used for the treatment of malignant tumor several hundred years ago. In1992,some scholars in our country found that the complete remission rate of APL patientstreated with arsenic trioxide was about65-98%[2]. Arsenic trioxide used in relapsetand ATRA drug resistant APL also got significant clinical effect.As a new-typeanti-leukemic drug, Arsenic trioxide has been widely applied in teeat ofhematopoietic malignancy.Combination of ATRA and As₂O₃has became thestandard treatment for APL. Study on the mechanism of acton indicated that arsenictrioxide presents dual activites on APL cells.Low concentration can induce celldifferentiation and high concentration can induce cell apoptosis. Apoptosis inducedby arsenic trioxide is associated with the insult of mitochondrial and the activation ofcaspase. The differentiation of APL cells is closely related to the modulation of PML-RARα，the key proein in leukemogensis[3]. Though it has significant effect inthe treatment of leukemia, its molecular mechanism is still not completely confirmed.The progress in the differentiation therapy in AML-M6is relatively slow. In thiscase, We establish the induced differentiation model of k562cells to further explorethe mechanism in induction of differentiation and try to find some targets in itsanti-tumor mechanism. Provide the experimental basis for the treanment with targetdrugs to achieve better therapeutic effect.OBJECTIVEAs₂O₃has got remarkable therapeutic achievement in the clinical treatment ofAPL,but the progress in differentiation treatment in AML-M6is rather slow. In thiscase, We establish the induced differentiation model of k562cells by low doseDetect the mRNA expression level of EVI1, WT1and TGF-β1. Then inhibit theexpression of WT1and EGR1detect the alteration of TGF-β1expression. exploremechanism in induction of differentiation in K562cells. Provide the experimentalbasis for the treanment with target drugs to achieve better therapeutic effect.METHODSWe establish the induced differentiation model of k562cells by low doseAs₂O₃.Observe the cell morphology by the oil microscope,detect the alteration ofthe cell proliferation by MTT experiment. Determinate and analysis cell cycle byFCM, The expressions of mRNA of EVI1、WT1、TGF-β1in k562cells afterexposure to As₂O₃were assayed by semi-quantitative RT-PCR.We also useWT1siRNA and EGR1siRNA to konckdown the gene expression of WT1andEGR1，then detect the expression of TGF-β1.RESULTSAfter K562cells induced by As₂O₃（1.0μmol/L）,we can observe the G0/G1time cell percentage ascension, the S time cell percentage drops.0.5μmol/L As₂O₃treatment of K562cells after0h,24h,48h,72h, EVI1and the WT1expressionreduces, the banding changes dark at1d2d3d, the TGF-β1expression ascension, the banding changes bright. The expression of TGF-β1also increased after silencing ofWT1gene by WT1siRNA. The inhibition of the EGR1expression by EGR1siRN didnot affect the expression of TGF-β1。Its concrete molecular mechanism pendingfurther studies.CONCLUSIONSK562cells treated with As₂O₃can inhibit cell proliferation and promot celldifferentiation. EVI1and WT1reduce in the mRNA level expression, TGF-β1inmRNA level expression ascension. The expression of TGF-β1also increased aftersilencing of WT1gene by WT1siRNA. Prove that WT1could be the upstreamsuppressor of TGF-β1gene in K562cells. As₂O₃may supress the expression ofWT1,released its transcription inhibition of TGF-β1,reduce the EVI1expression.Then restore TGF-β1/smads signal pathway to get the therapeutic effect.