Effects Of Copper On Tumor Growth And Related Proteins Of EGFR Pathway In H22 Tumor Bearing Mice

Objective:To investigate the effect of suitable copper on the growth of tumor in H22tumor bearing mice and the possible mechanism of copper promoting the growth of tumor cells via EGFR signaling pathway.Methods:The mice were feed in the SPF room at temperature 25±2?,humidity60±10%and illumination time 12h.Firstly,one-hundred and sixty mice were randomly assigned for eight groups-water,water plus TM,three Cu²⁺?0.06,0.30,1.50mg/kg?groups and three Cu²⁺plus TM?0.06,0.30,1.50 mg/kg?groups according to weight after adaptive feeding for one week.Secondly,water and Cu²⁺?0.06,0.30,1.50 mg/kg?were administered via gavage daily for 4 weeks to corresponding groups.Then the mice were injected H22 cells on the lower part of the back to establish the mice model of H22hepatocarcinoma xenograft.Lastly,water?TM?1 mg/kg?and Cu²⁺?0.06,0.3,1.5 mg/kg?were administered via gavage daily for 4 weeks according to corresponding groups.After the tumor was inoculated,the size of the tumor was recorded every three days.The blood samples were collected from the eyeballs of mice.Xenograft tumors and corresponding organs,like liver,spleen,kidney and thymus,were collected?weighted and calculated according to formula.Alanine aminotransferase?ALT??aspartate aminotransferase?AST?and albumin?ALB?were measured by automatic biochemical analyzer.Copper concentration in serum of mice was detected by ICP-MS.The mRNA expression of VEGF?bFGF and c-Myc were detected by Real-time PCR.The levels of phosphorylated EGFR?pEGFR?and phosphorylated Erk1/2?pErk1/2?protein were detected by Western blot.The levels of EGFR and c-Myc protein were detected by immunohistochemistry.VEGF?bFGF and c-Myc mRNA were detected by RT-PCR.All data are processed by SPSS software.Results:The incidence of H22 transplanted tumor in mice was 100%.Compared with the group of water and copper+TM,the tumors grew obviously,which also showed in a dose-dependent manner;The result of aspartate aminotransferase?AST?,alanine aminotransferase?ALT?and albumin?ALB?of mice showed that there was no statistical difference between each group?P>0.05?;the spleen index and thymus index showed that there was no significant difference of each group?P>0.05;thymus index spleen index:F=0.175 P>0.05?;the result of serum copper detected by ICP-MS showed that with the increase of different doses of gavage in different groups,serum copper was increased and serum copper of corresponding copper+TM group was lower than copper group,the difference has statistically significant?P<0.05?.The correlation between serum copper andtumorweightshowedthattherewasacorrelationbetweenthem?r=0.316,P<0.05?.The mRNA expression of VEGF?bFGF and c-Myc were increased with copper administration?P<0.05?In addition,,the expression of EGFR,pEGFR,pErk1/2and c-Myc protein were all up-regulated in the cooper group compared with the corresponding copper+TM group in the tumor tissue.Conclusion:Suitable concentration of copper can promote the growth of tumor in H22tumor-bearing mice.TM can chelate copper to reduce the growth of tumor.The dosage of copper in this experiment was within the allowable range of the body and didn't affect the liver and immune system of the body.The altering expression of corresponding proteins suggest us that the mechanism by which copper promotes tumor growth may be via the EGFR/Eek/c-Myc pathway.These evidence provide a basis for the treatment and prevention of HCC.