The role of NF-kappaB in hepatocellular carcinoma cell.
OBJECTIVE
To evaluate the role of nuclear factor-kappa B (NF-kappaB) and inhibitory kappaB alpha (IkappaBalpha) in hepatocellular cacinoma (HCC) SMMC7721 cells, the consequence of NF-kappaB inhibition in SMMC7721 cells transfected with mutated IkappaBalpha (mIkappaBalpha) plasmid and the effect of stable inhibition of NF-kappaB activity in combination with Doxorubicin.


METHODS
Western blot was used to determine the expression of NF-kappaB and IkappaBalpha in SMMC7721 cells and normal liver cells. Nuclear protein was used to evaluate the binding of the (32)P-labeled tandem kappaB sequence using electrophoretic mobility shift assay and the expression of NF-kappaB using Western blot between SMMC7721 cells transfected with mIkappaBalpha plasmid (SMMC7721-MT) and control cells. Furthermore, cell viability was plotted between SMMC7721-MT and control cells. The binding of kappaB sequence and cell viability between SMMC7721-MT and control cells at different concentrations of Doxorubicin were also investigated.


RESULTS
Western blot analysis for nuclear extract showed more P50 (NF-kappaB1) and P65 (RelA) expression in SMMC7721 cells compared with normal liver cells. The expression of cytosolic IkappaBalpha protein in SMMC7721 cells was less than that in normal cells. SMMC7721-MT cells inhibited NF-kappaB nuclear translocation at 0, 24, 48 and 96 hours. Furthermore, NF-kappaB cannot be detected in the nuclear protein of SMMC7721-MT cells by Western blot. By calculating cell viability, the proliferation of SMMC7721-MT cells was shown to be suppressed more significantly than that of control cells. NF-kappaB in untransfected cells was activated by Doxorubicin in a dose-dependent manner, but that in SMMC7721-MT cells was not induced at low concentrations of Doxorubicin. Compared with untransfected cells, the viability of SMMC7721-MT cells was significantly suppressed at the same concentration of Doxorubicin (P < 0.01).


CONCLUSIONS
The present study demonstrates that upregulation of NF-kappaB and downregulation of inhibitory kappa B (IkappaBalpha) in SMMC7721 cells are related with the growth of hepatocellular cacinoma cells. Stable expression of mIkappaBalpha in SMMC7721-MT cells can inhibit NF-kappaB nuclear translocation and suppress cell growth. Furthermore, stable inhibition of NF-kappaB activity in combination with Doxorubicin can significantly inhibit cell proliferation in SMMC7721-MT cells. Thus, modulation of NF-kappaB may represent an improvement in the efficacy of HCC therapies and be worthy of further research and investigation.
Wang
Jianhong
J
Department of Gastroenterology, First Affiliated Hospital, Wenzhou Medical College, Wenzhou 325000, China. wang730221@vip.sina.com
Huang
Qingke
Q
Chen
Minxin
M
D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D002455:Cell Division; D004317:Doxorubicin / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D020672:I-kappa B Proteins / Q000096:biosynthesis; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D016328:NF-kappa B / Q000096:biosynthesis / Q000502:physiology*; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D020672:I-kappa B Proteins; D016328:NF-kappa B; D004317:Doxorubicin