Anti-Tumor Effects And Mechanism Of 1,2:5,6-Dianhydrogalactitol On Tumor In Nude Mice Transplanted Human Lung Cancer Cell NCI-H460

ObjectiveThe aim of this study is to culture human lung cancer cell NCI-H460 in vitro,establish a nude mice transplanted tumor model of lung cancer cell NCI-H460 and investigate the apoptosis and its related mechanism in nude mice transplanted tumor on human lung cancer cell NCI-H460 induced by 1,2:5,6-dianhydrogalactitol (DAG).Methods1.Human H460 tumor models were established using 4-6 weeks old athymic BABL/c nude mice (SPF grade).Athymic nude mice were inoculated with H460 cells/0.2ml(1.0×107) into right neck s.c.After treatment for 7 days,the mice were randomly assigned into each group (n=6 mice/group) and given i.p. with NS,DAG-D (3mg/kg),DAG-Z (6mg/kg),DAG-G (9mg/kg) and DDP(3mg/kg) of desired drug and dosage for 7 days. After 7 days,the nude mice were put to death,and the tumor was ready to use.2.The tumor tissues were stained with HE to study the pathological changes after treatment of different doses of DAG.3.TUNEL method was used to investigate the degree of apoptosis tumor tissue after DAG treatment.4.The expression of Bax,Bcl-2,Caspase-3,Caspase-9,P53,Survivin,Cytm mRNA levels after treatment of different doses of DAG were detected by real-time PCR.5.The protein expressions of Bcl-2,P53 and Caspase-3 in tumor tissues were determined by immunohistochemistry after treatment of different doses of DAG.6.Protein western blotting method was used to detect the apoptosis-related protein expressions of Bcl-2,P53 and Caspase-3 after treatment of different doses of DAG.Results1.Prior to the nude mice were sacrificed, none in both groups was dead. Before delivery,the growth speed, tumors of nude mice had no difference between the volume(P> 0.05). But But after the treatment had obviously difference(P<0.05).2.The HE staining results showed that in the DAG and DDP groups,cancer cell population decreased and necrosis cells increased.The apoptosis tumor cells showed contraction,karyopyknosis and blue-dyeing morphology changes.3.TUNEL assay results showed that DAG and DDP could significantly increase the apoptotic ratio of tumor tissue in H460 transplanted nude mice. Effect of different treating group of NS,DAG-D,DAG-Z,DAG-G and DDP apoptosis index(AI) respectively were 0.00%,6.34%,41.03%,75.13%,18.60%.4. After different treatment,the mRNA expression variation of gene Bax,Caspase-3,Caspase-9,P53 of lung cancer cell NCI-H460 transplanted tumor in nude mice were up-regulated and the mRNA expression variation of gene Bcl-2 and Cytm were down-regulated by DAG, the Survivin mRNA had no significant variation.5.The immunohistochemistry assay showed that DAG and DDP could up-regulate P53 protein expression in tumor tissues,down-regulate Bcl-2 protein expression and the Caspase-3 protein had no significant variation.6.The protein western blotting assay showed that DAG and DDP could increase the expression of Bax/Bcl-2 and P53 proteins in tumor tissues,decrease Caspase-3 protein expression.ConclusionTransplanted tumor model of non-small cell lung cancer cell NCI-H460 in nude mice is successfully established. The study preliminary verified that DAG could induce the apoptosis on human lung cancer cell NCI-H460 transplanted tumor tissues in nude mice.The study at the molecular level evaluated that the apoptosis mechanisms may be related to the mitochondrial pathway by DAG.