Nuclear factor-kB signaling pathway constitutively activated in esophageal squamous cell carcinoma cell lines and inhibition of growth of cells by small interfering RNA.
Although constitutive nuclear factor (NF)-kappaB activation has been reported in many human tumors, the role of the NF-kappaB pathway in esophageal squamous cell carcinoma (ESCC) has not been known. In this study, NF-kappaB pathway in two ESCC cell lines was investigated using immunocytochemistry, Western blot and reverse transcription-polymerase chain reaction. The activation of NF-kappaB DNA binding was determined by electrophoretic mobility-shift assay. RNA interference was used to specifically inhibit the expression of p65. Growth of cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results showed that p50, p65, IkappaBalpha p-IkappaBalpha and IkappaB kinase beta were expressed and mainly localized in the cytoplasm. Reverse transcription-polymerase chain reaction results showed the constitutive expressions of p50, p65 and IkappaBalpha mRNA in the two ESCC cell lines. Furthermore, the nuclear extracts revealed that p50 and p65 translocated to the nucleus had DNA-binding activity. Finally, small interfering RNA of p65 decreased the expression of p65, and the viability of cells transfected with p65 small interfering RNA was significantly suppressed at the same concentration of 5-fluorouracil (P < 0.05) compared to untransfected cells. The results of this study showed that there was the constitutively activated NF-kB signaling pathway in the ESCC cell lines. RNA interference targeting at p65 increased the sensitivity of the ESCC cell lines to 5-fluorouracil, suggesting that NF-kappaB might be a good target for cancer treatment.
Tian
Fang
F
Laboratory for Cell Biology, Medical College, Zhengzhou University, Zhengzhou 450052, China.
Zang
Wei-Dong
WD
Hou
Wei-Hong
WH
Liu
Hong-Tao
HT
Xue
Le-Xun
LX