ABSTRACT
Objective:To investigate the effects and mechanisms of copper transporter 1 (CTR1) in radiation induced intestinal injury in vitro. Methods:Human small intestinal epithelial cells (HIEC) were irradiated with 2, 4, 6, 8 Gy of X-rays and rat intestinal epithelial cells (IEC-6) were irradiated with 5, 10, 15, 20 Gy of X-rays. At 2, 4, 8, 24, and 48 h after irradiation, the expression of CTR1 was detected by Western blot assay. In some experiments, HIEC and IEC-6 cells were transfected with CTR1 shRNA and then exposed to X-rays. Copper levels were detected by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The radiosensitivity of cells was verified by colonogenic assay, the cellular reactive oxygen species (ROS) level and DNA damage were detected to further explore the related mechanism. In addition, Western blot was applied to detect the expressions of antioxidants and cuproptosis associated proteins in enterocytes after silencing CTR1 or irradiation.Results:The expression of CTR1 was increased by X-ray irradiation in a dose-dependent manner ( t=3.53, 3.45, 6.37, 11.11, 11.13, P<0.05). CTR1 expression was successfully diminished by CTR1 shRNA adenovirus vectors. According to the survival curves, the enhancement ratios of the radiosensitivity of HIEC and IEC-6 cells with CTR1 knocking-down were 1.146 and 1.201, respectively. Radiation-induced copper accumulation was alleviated after CTR1 silencing in IEC-6 cells ( t=3.10, P<0.05). At 0.5 h after irradiation, the ROS production in the CTR1 knockdown group was significantly lower than that in the control group ( t=5.23, 2.96, P<0.05). At 1 h after irradiation, the protein expression of γ-H2AX in the CTR1 knockdown group was obviously lower than that in the control group ( t=7.50, 4.29, P<0.05). The expressions of Nrf2 and HO-1 were increased after irradiation, which could be further increased after CTR1 silencing. In addition, cuproptosis associated protein DLAT, LIAS and FDX1 were reduced post-irradiation, which were recovered after CTR1 silencing. Conclusions:The radioresistance of HIEC and IEC-6 cells was enhanced after CTR1 silencing, possibly through the intracellular ROS and cuproptosis pathway.
ABSTRACT
OBJECTIVE To study the roles of copper transporter 1 (CTR1 )and Cu2 + transporting ATPase αpolypeptide (ATP7A)in lead exposure-induced copper accu mulation and oxidative stress in rat C6 glio ma cells.METHODS Methyl thiazolyl tetrazoliu m (MTT)assay was performed to determine the proper Pb doses (without affecting cell viability)by treated the cells with lead acetate 0 -100 μmol·L -1 for 24 h and 48 h.Superoxide dis mutase (SOD)activity or malondialdehyde(MDA)level were detected respectively by xanthine oxidase technique and thiobarbituric acid (TBA) method.Ato mic absorption spectrophoto metry was e mployed to determine the intracellular levels of Pb and Cu ions.Real-ti me quan-titative PCR and Western blot were used to detect the mRNA and protein levels of CTR1 and ATP7A, respectively.RESULTS The cell viability significantly decreased when the doses of Pb treat ment was higher than 10 μmol·L -1 ,so 10 μmol·L -1 was chosen as a working concentration of Pb exposure in this study.Co mpared with those in the normal controls,a moderately decreased T-SOD activity and an increased MDA level was determined in the cells treated with Pb 10 μmol·L -1 or Cu 5 μmol·L -1 alone, while a significant drop of T-SOD activity and a re markable increase of MDA level was found in the cells co-exposed to Pb and Cu (P<0.01 ).Pb exposure for 24 and 48 h increased the cellular Cu uptake by 1 .2 and 2.5 fold,respectively (P <0.01 ).Evidences fro m RT-PCR showed that Pb exposure for 24 and 48 h upregulated the CTR1 mRNA level by 23.2% and 58.7%,and downregulated the ATP7A mRNA level by 58.1 % and 50.0%,respectively.Results fro m Western blot confirmed that Pb exposure also resulted in an increased CTR1 expression and a decreased ATP7A expression at protein level (P<0.01 ).CONCLUSION Pb exposure lead to Cu accu mulation,by affecting the expression levels of CTR1 and ATP7A,and increased oxidative stress in C6 cells.