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. 2005 Feb 7;11(5):726-8.
doi: 10.3748/wjg.v11.i5.726.

Effect of mutated IkappaBalpha transfection on multidrug resistance in hilar cholangiocarcinoma cell lines

Ru-Fu Chen  1 , Zhi-Hua Li, Xian-He Kong, Ji-Sheng Chen
Affiliations

Effect of mutated IkappaBalpha transfection on multidrug resistance in hilar cholangiocarcinoma cell lines

Ru-Fu Chen et al. World J Gastroenterol. .

Abstract

Aim: 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.

Conclusion: 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.

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Figures

Figure 1
Figure 1
Macrorestriction map of mutated IκBα Plasmi. Lane 1: Plasmid DNA; lane 2: Plasmid DNA cut by Xbal and HandIII; lane 3: DNA marker.
Figure 2
Figure 2
EMSA of QBC939 and QBC939HCVC +cells transfected with mutated IκBα plasmid. Lane 1: Control group; lane 2: QBC939HCVC +cells without transfected with mutated IκBα; lane 3: QBC939 cells not transfected with mutated IκBα; lanes 4 and 5: QBC939HCVC + cells transfected with mutated IκBα; lane 6: QBC939 cells transfected with mutated IκBα.
Figure 3
Figure 3
Expression of MDR-1mRNA in the QBC939 cells transfected with mutated IκBα. M: DNA marker. Lane 1: The QBC939HCVC+ cells transfected with mutated IκBα, lane 2: The QBC939HCVC + cells not transfected with mutated IκBα; lane 3: The QBC939 cells not transfected with mutated IκBα; lane 4: The QBC939 cells transfected with mutated IκBα.
Figure 4
Figure 4
Expression intensity of P-GP protein in QBC939 and QBC939HCVC+ cells transfected with mutated IκBα. A: Expression of P-GP in the non-transfection Group, LSAB ×400; B: Expression of P-GP after transfection of mutated IκBα, LSAB ×400.
See this image and copyright information in PMC

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