Effects Of Altered ABCG2 Expression On Oxidative Stress Indexes And Inflammatory Factors In Cancer Tissue Of Colorectal Cancer Model

Background and ObjectColorectal cancer (CRC) is one of the most common cancers in our country, ranked fifth in common malignant tumor mortality. The National health development planning commission pointed that the incidence and mortality rate of CRC were 23.03/10million and 11.11/10 in 2015. The tumorigenesis of CRC is a complicated process, involved multiple genes and many steps. Epidemiological and tumor molecular biology studies have shown that there is a closely link between inflammation and CRC. Inflammation facilitates tumorigenesis by generating free radicals and releasing cytokines and chemokines, which accelerating proliferation, DNA damage and angiogenesis. ABCG2 is an ATP-binding cassette (ABC) transporter affiliated to ABC gene family, which locates in human chromosome 4q22 and encodes 655 amino acid residues in the transmembrane glycoprotein. Numerous reports demonstrated that ABCG2 acts as a drug transporter in colorectal cancer chemotherapy, with other functions remained unknown. Our previous works have demonstrated that ABCG2 is capable of protecting cells from ROS-mediated damage and death, closely related to the inhibition of ABCG2 on the NF-κB signaling pathway and the expression of inflammation-associated cytokines. Based on the previous study, we establish of human colorectal cancer in nude mice to observe the effect of ABCG2 on the oxidative stress index and the level of inflammatory factors in vivo which could provide a novel theory of neoplasm and therapeutic target for colorectal cancer.Methods:1. The ABCG2 protein expression in different routine cultured human colon cancer cells (LoVo、HCT-116、HT-29) was measured by Western blotting. Human colon cancer cell line HT-29 of ABCG2 high expression was selected in the follow-up study.2. The shABCG2 group and shcont group were transfected by ABCG2-shRNA lentiviral vector and shRNA negative lentiviral vector. ABCG2 expression in protein level were detected by Western blotting. Selecting appropriate multiplicity of infection (MOI) and transfection time for subsequent research.3. Superoxide dismutase (SOD) kit and total glutathione peroxidase kit and glutathione reductase (GR) kit were chosen to detected intracellular anti oxidative stress induced by H2O2 in two group of cells and detected the apoptosis rate of two group of cells.4. Shcont group of cells and shABCG2 groups of cells were injected subcutaneously into nude mice, establishing of mouse model of human colorectal cancer. (shcont group of mice and shABCG2 group of mice)5. The changes of ROS expression level were detected by immunofluorescence.6. The changes of total glutathione peroxidase and glutathione reductase levels were detected by some kits.7. To detected the content of Tumor necrosis factor-α(TNF-α) and Interleukin-6(IL-6) of fresh tissue and serum of nude mouse transplantation tumor by enzyme-linked immuno sorbent assay(ELISA).Detecting the mRNA expression of Interleukin-8 (IL-8), and Growth regulated oncogene-β (GRO-β) in fresh tissue of nude mouse transplantation tumor by RT-PCR.8. Detecting the expression of IλB-α and phospho-IκB-α in fresh tissue of mice transplantation tumor by western blotting.Results:1. ABCG2 protein in different human colon cancer cell lines have different degrees of expression, but expressing higher in HT29 cells,so that we choose HT29 cells for the further research.2. The transfection efficiency rate after the HT-29 cell infected by ABCG2-shRNA lentiviral vector was above 90%.In comparison with shcont group by western blotting, the expression of ABCG2 in shABCG2 group was lower obviously, and MOI value and transfection time were set as 20 and 24h.3. Compare with 0 mM H2O2, The detected by SOD and total glutathione peroxidase showed that H2O2(0.5 and 1 mM) can obviously decrease the capability of SOD and total glutathione peroxidase stress, and compared with the shcont group, the shABCG2 group with 0.5 and lmM can distinctly decrease the level of anti-oxidant stress. Apoptosis experiment showed that with the increase of concentration of H2O2, apoptotic cells significantly increase, but the further research found that with the same concentrations of H2O2, the apoptosis rate in shABCG2 group was lower than shcont group.4. Compared with shcont group, the level of ROS in shABCG2 mice transplanted tumor tissue group was higher.5. Compared with shcont group, the level of total glutathione peroxidase and GR in shABCG2 mice transplanted tumor tissue group was lower.6. Compared with shcont group, the level of IL-8、TNF-α in shABCG2 mice transplanted tumor tissue group was higher, while the level of IL-6 in shABCG2 mice surem was higher,too.7. Compared with shcont group, the mRNA level of IL-8 in shABCG2 group had no significant change, while the mRNA level of GRO-β showed higher than it.8. Detecting the protein of IκB-α and phospho-IicB-a via Western blotting, the results showed that compared with shcont group, the protein of IκB-α was lower and the protein of p-IκB-α was higher in shABCG2 mice transplanted tumor tissue group.Conclusion1. ABCG2 can decrease the level of oxidant stress in HT-29 cells and transplanted tumor tissue in mice.2. ABCG2 can inhibit the inflammatory factors in HT-29 cells and transplanted tumor tissue and serum in mice.3. ABCG2 can inhibit NF-κB signal pathway of HT-29 cells transplanted tumor tissue in mice.4. ABCG2 can promote the apoptotic ability of colorectal cancer cell line HT-29.