Adenovirus-mediated Human ING4Gene Expression Reverses The Multidrug Resistance Of SGC7901/CDDP Gastric Cancer Cells In Vitro And In Vivo

Objective:1. Establish the cisplatin-resistant phenotype gastric cancer cell lineSGC7901/CDDP (2ug/ml) to further explore the tumor MDR;2. Investigate the underlyingmechanisms of ING4gene in the reversion of human gastric cell MDR in vitro and in vivo.Methods and results:1. Establishment of the MDR phenotype gastric cancer cell line SGC7901/CDDP andidentifying its multidrug resistance characteristic.Parental SGC7901gastric cancer cells were cultured in cisplatin-containing mediumfrom0.05ug/ml to2ug/ml to induce tumor cell MDR. To identify MDR phenotype, TheIC50of SGC7901/CDDP and SGC7901cells to ADM,5-FU and CDDP were evaluated byCCK-8assay. The expression of MDR-and apoptosis-related genes of SGC7901/CDDPand SGC7901cells were detected by qRT-PCR. Our results demonstrated that wesuccessfully established the MDR phenotype gastric cancer cell line SGC7901/CDDP after6months induction. Compared to SGC7901cell, the resistance indexes were19.92,7.53and6.68to CDDP,5-FU and ADM respectively. qRT-PCR shown that MDR1, MRP1,Bcl-2and Survivin genes were significantly upregulation as well as Bax gene wasdownregulation.2. Investigating the underlying mechanisms of ING4gene in the reversion of humangastric cancer cell MDR in vitro and in vivo.We use the high purity and titers of replication-defective recombinant adenovirus(Ad-ING4and Ad-GFP)(100MOI) which preserved in our laboratory to infect gastriccancer MDR phenotype cell SGC7901/CDDP. The expression of ING4gene and proteinwas identified by RT-PCR and Western blot analysis. For further investigated the possiblerole of ING4gene in the reversion of human gastric cancer cell MDR and its underlyingmechanisms, in vitro, we determined the IC50of SGC7901/CDDP which infected byAd-ING4to ADM,5-FU and CDDP using CCK-8assay and the change of MDR-related gene MDR1、MRP1as well as apoptosis-related gene Bax、Bcl-2and Survivin usingqRT-PCR. To test the reversion effects of Ad-ING4to SGC7901/CDDP in vivo, weestablish the gastric cancer athymic nude mouse xenograft and further explore theexpression of MDR-and apoptosis-related proteins P-gp、MRP1、Bax、Bcl-2and Survivinafter infected by Ad-ING4using immunohistochemical analysis. We found that ING4geneand protein were successfully expressed in SGC7901/CDDP. CCK-8assay demonstratedthe IC50of SG7901/CDDP cells which over-expressed ING4was conspicuousdownregulation, the reversion index were9.86,4.09and2.23to CDDP,5-FU and ADMrespectively. The expression of MDR1, MRP1, Bcl-2and Survivin genes wasdownregulation and Bax genes was upregulation after SGC7901/CDDP infected byAd-ING4. In vivo, we found that the growth of xenograft was significantly inhibition afterAd-ING4therapy. Further results showed that the expression of P-gp, MRP1, Bcl-2andSurvivin proteins were downregulation as well as Bax protein was upregulation inxenograft tumor tissues.Conclusions：1. We successfully established the2ug/ml MDR phenotype cell line SGC7901/CDDPby continuing induction of parental gastric cancer cell SGC7901and further identified itsMDR characteristic;2. The underlying mechanisms of SGC7901/CDDP MDR are associated withup-regulating the ABC-binding cassette transporters and down-regulating the apoptosispathway;3. Ad-ING4plus CDDP could inhibit the growth of SGC7901/CDDP athymic nudemouse xenograft, expressing synergistic inhibitory effects.4. Ad-ING4reverses SGC7901/CDDP cell MDR through down-regulating theABC-binding cassette transporters as well as up-regulating the apoptosis pathway.