The Relationship Between Mad2 And Survivin And Molecular Mechanisms Inducing Drug Resisitance Of Gastric Cancer

The cell cycle control is very important to maintain normal bionomics of mammalian cells and the failure of cell cycle regulatory checkpoints is a common event in human cancer.The spindle checkpoint plays an important role in ensuring the fidelity of the cell cycle processing,monitors the interactions between chromosomes and microtubules,and delays mitotic progression to allow extra time to correct some defects.Defects in the checkpoint signaling machinery are associated with increased rates of cell death and aging,and may promote the development and/or progression of cancers in humans.Spindle inhibitors and DNA damaging agents are extensively used in the treatment of human cancers,but their clinical efficacy has been hampered due to the development of drug resistance.Thus,it is an urgent mission to identify and overcome the drug resistance.Recent studies suggested that the mitotic checkpoint defects play a role in aneuploidy and drug resistance mechanisms. Survivin and Mad2 and ATM are three of the mitotic checkpoint components,the inactivation of survivin and Mad2 are correlated with checkpoint impairment and drug resistance.ATM plays a key role in DNA damage-mediated cell apoptosis.These studies indicated that the status of checkpoint proteins might be very important for cancer prevention and diagnosis and therapy.【Purpose】The purposes of present study are as following:1.To investigate the expression of Mad2 and Survivin in human normal tissues and gastric carcinoma; 2.To further validate the effects of Mad2 on drug resistance of gastric cancer cells and explore the mechanisms;3.To study the roles of spindle checkpoint in maintaining the sensitivities of gastric cancer cells to chemodrugs;4.To explore the mechanisms of by which Mad2 and Survivin regulated drug resistance of gastric cancer cells;5.To study the mechanisms of ATM expression status on signaling pathway and apoptosis mechanisms.【Methods】1.The expressions of Mad2 and Survivin in gastric cancer were determined by RT-PCR and Western blot and immunohistochemistry.2.Two pairs of the RNAi expression vectors of Mad2 were transfected into SGC7901,and stable clones were selected by G418 screening.3.The transfection efficiency in different stable clones was dentified by RT-PCR and Western blot.4.The sensitivity of transfected cells to chemodrugs were tested by MTT assay and cloning forming assay.5.Cell scattering assay and Trans-well migration assay were performed to investigate the proliferation and migration of these cells.6.Flow cytometry(FCM) was used to identify the cell cycle distribution and apoptosis of these cells.7.The sensitivity of transfected SGC7901 cells to chemodugs was determined by tumour formation in nude mice in vivo.8.Intracellular adriamycin (ADR) concentration analys was based on the fluorescence of ADR,the intracellular accumulation and retention of ADR was analyzed by fluorescence-activated cell sorting.The efflux rate of ADR was calculated.8.The capacity of cells to undergo apoptosis induced by drugs was detected by Annexin V/PI and Hochest 33258 dye in vitro apoptosis assay.9.The relationship between Mad2 and apoptosis-related molecules such as Bcl-2,Fas L,cytochrome C,cleaved caspase 9 and Survivin were examined by Western blot.10.The interaction of Mad2 and Survivin was studied with co-immunoprecipitation.11.The function of G2 phase checkpoint in MKN28 cells was identified by testing the epressions of ATM,pchkl, p-chk2 and CDC25C by Western blot.The apotosis was tested by DNA Ladder and Hochest 33258/PI staining.【Results】1.Mad2 and Survivin was significantly overexpressed in gastric cancer compared with the matched adjacent tissues,and its expression was related to the clinical parameters of cancer.2.It was showed that by Western blot that Mad2 protein expressed in SGC7901.The RNAi expression vectors of Mad2 were transfected into SGC7901 and stable clones were selected and identified.3.It was showed by drug sensitivity assay of cells transfected with Mad2-siRNA was increassed.The results indicated that Mad2-siRNA promotes drug resistance of gastric cancer through downregulating the expression of Mad2 and upregulating Survivin.4.The RNAi vector of Mad2 was transfected into SGC7901 and stable clones were selected and identified.5.FCM indicated that the cells transfected with Mad2siRNA could not be arrested in G2/M phase after chemodrugs treatment.6. Drug sensitivity assay showed that down regulation of Mad2 promotes the drug resistance of gastric cancer.7.ADR accumulation and retetion was decreased in siMad2/SGC7901.8.The apoptosis index induced by chemodrugs was reduced in siMad2/SGC7901.9.Western blot revealed that the Bcl-2 was increased in RNAi transfectants.Other apoptosis associated molecules,such as caspase 9 and cytochrome c also decreased.But FasL were not affected by the siMad2.10. Western blot indicated that in siMad2 transfectants,the expression of Survivin was increased.11.The G2 phase checkpoint of MKN28 cells was deficient because the expressions of ATM,p-chkl and p-chk2 were weak after treated with DNA damage agent cisplatin. 【Conclusion】1.Mad2 and Survivin was significantly overexpressed in gastric cancer compared with the matched adjacent tissues,and its expression was related to the clinical parameters of cancer.2.Spindle checkpoint plays a vital role in the chemodrugs treatment because it could detect DNA damage and spindle abnormality and induce tumor cells apoptosis.3.Mad2 may regulate the drag resistance phenotype of the gastric cancer by interacting with Survivin and depressing the activity of Survivin proteins.4.ATM deficiency may lead to apoptosis and sensitiveness to DNA damage.