Effect of mutated IkappaBalpha transfection on multidrug resistance in hilar cholangiocarcinoma cell lines.
OBJECTIVE
To explore the expression effect of mutated IkappaBalpha transfection on multidrug resistance gene (MDR-1) in hilar cholangiocarcinoma cells by inhibiting the activity of nuclear transcription factor-kappaB (NF-kappaB).


METHODS
We used the mutated IkappaBalpha plasmid to transfect QBC(939)HCVC+ cells and QBC939 cells, and electrophoretic gel mobility shift assay (EMSA) to detect the binding activity of NF-kappaB DNA and the effect of the transfecting mutated IkappaBalpha plasmid on multidrug resistance gene (MDR-1) in hilar cholangiocarcinoma cells and its expression protein (P-GP).


RESULTS
Plasmid DNA was digested by restriction enzymes Xbal and Hand III, and its product after electrophoresis showed two bands with a big difference in molecular weight, with a size of 4.9 kb and 1.55 kb respectively, which indicated that the carrier was successfully constructed and digested with enzymes. The radioactivity accumulation of QBC(939)HCVC+ and QBC939 cells transfected with mutated IkappaBalpha plasmid was significantly lower than that of the control group not transfected with mutated IkappaBalpha plasmid. Double densimeter scanning showed that the relative signal density between the tansfection group and non-transfection group was significantly different, which proved that the mutated IkappaBalpha plasmid could inhibit the binding activity of NF-kappaB DNA in hilar cholangiocarcinoma cells. Compared to control group not transfected with m IkappaBalpha plasmid, the expression level of MDR-1mRNA in the QBC939 and QBC939HCVC+ cells transfected with mutated IkappaBalpha plasmid was lower. The expression intensity of P-GP protein in QBC939 and QBC939HCVC+ cells transfected with mutated IkappaBalpha was significantly lower than that of the control group not transfected with mutated IkappaBalpha plasmid.


CONCLUSIONS
The mutated IkappaBalpha plasmid transfection can markedly reverse the multidrug resistance of hilar cholangiocarcinoma cells. Interruption of NF-kappaB activity may become a new target in gene therapy for hilar cholangiocarcinogenesic carcinoma.
Chen
Ru-Fu
RF
Department of Hepatobiliary Surgery, The Second Affiliated Hospital of Zhongshan University, Guangzhou 510120, Guangdong Province, China. chenrf63@163.com
Li
Zhi-Hua
ZH
Kong
Xian-He
XH
Chen
Ji-Sheng
JS
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D001650:Bile Duct Neoplasms / Q000235:genetics / Q000503:physiopathology* / Q000628:therapy; D001653:Bile Ducts, Intrahepatic; D045744:Cell Line, Tumor; D018281:Cholangiocarcinoma / Q000235:genetics / Q000503:physiopathology* / Q000628:therapy; D015972:Gene Expression Regulation, Neoplastic; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics*; D016296:Mutagenesis; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D010957:Plasmids; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D000072000:NF-KappaB Inhibitor alpha