The Inhibition Effect Of Deuterium Depleted Water On Non-small Lung Cancer A549 And Initial Studies Of Its Underlying Mechanisms

Objective: To study the inhibition effect of deuterium depleted water(DDW) on non-small cell lung cancer A549 and the underlying mechanisms.Methods: In vitro, MTT assay was used to test the growth inhibition effect of deuterium depleted water on A549 cells. In vivo, subcutaneous transplanted tumor model and orthotopic transplanted tumor model were established in nude mice with A549 cells, which were used to observe the effects of DDW on the tumors of nude mice. After the transplanted nude mice models were established, the nude mice were sacrificed and xenografted or orthotopic tumors were checked. HE staining was applied to observe the pathological changes of lung tissues. RNA-seq was employed to analyze the differentially expressed genes between A549 cells treated for 5 or 10 days with DDW and the control group. The differentially expressed genes were categorized by functional enrichment using Gene Ontology analysis. Biological process analysis was adopted to find the main biological processes that these genes were involved. At last we applied GC/TOFMS to discover the differential metabolites in the sera of nude mice from different groups.Result: After cultivating A549 cells with DDW for 5 and 10 days, the MTT results revealed significant inhibition effects of DDW on A549 cells comparing with control group. The results of subcutaneous transplanted tumor model showed that tumors of DDW group were much smaller than tumors from control group. In addition, results of orthotopic transplanted tumor model showed that more tumors was found in the lungs of nude mice from control group than from DDW group, and the tumors were densely distributed in lung and heavily pressed the lung tissue which resulted in the malformation of lung tissue. However, fewer tumors showed up in the lungs of nude mice from DDW group and the shapes of lung tissues were nearly normal as healthy ones. HE staining also demonstrated that, the lesions of DDW group were smaller than those of control group. 394 genes were found to be differentially expressed in the 50ppm-5d group and 50ppm-10 d group as compared with control. According to database of GeneCards, we found that, differential expression genes, including KRT18、ANGPTL、 FTH1、EGFR、GREM1、CAV1 are closely related to proliferation and genes including BAX、CDKN1A、 IER3、PEA15、DNAJB9、EGFR are closely related to apoptosis. Biological Process analysis of Gene Ontology analysis revealed that, 23 differential expression genes belong to the biological process of angiogenesis(p=2.8×10-12). In which, ITGAV、COL4A2、ID1、ANGPTL4、SERPINE、EPHA2 are closely related to angiogenesis. To characterize the metabolic changes incurred by DDW treatment, GC/TOFMS was employed to analyze the alteration of metabolites in sera of the nude mice from different groups. The score plots of PCA showed that the metabolites of C vs. D group do not have significant differences. But there are obvious differences in the groups of C vs. LC, D vs. LD. The PCA result of LC vs. LD showed that there are partial differences between them. PLS-DA helped to show significant differences between them and 18 differential metabolites were found contribute to the differences. The 18 metabolites mainly included amino-acids and carbohydrates.Conclusion: DDW can inhibit the growth of A549 cells in vitro. And in vivo, it also inhibits the growth of tumors from subcutaneous transplanted xenograft model and orthotopic transplanted tumor model in nude mice. RNA-seq shows that DDW may inhibit the proliferation of A549 cells by changing the expression of several proliferation-related genes, at the same time DDW also inhibits the angiogenesis of A549 transplanted tumors. In addition, the study of metabonomics showed a lower level of carbohydrates in sera of the nude mice bearing orthotopically transplanted tumor after DDW treatment as compared with ordinary water treatment.