Over-activated Notch-1 protects gastric carcinoma BGC-823 cells from TNFalpha-induced apoptosis.
OBJECTIVE
The role of Notch-1 in human gastric carcinoma, one of the most common carcinomas of the human digestive tract, remains poorly characterised. Here, we investigated the effect and mechanism of Notch-1 activation on TNFalpha-induced apoptosis of human gastric carcinoma BGC-823 cells.


METHODS
Cell viabililty was measured by MTT assay. Apoptosis was detected by flow cytometry assay. Notch-1, Hes-1, caspase-3 p20 and NF-kappaB p65 expressions were assayed by Western blotting. NF-kappaB activation was tested by electrophoretic mobility shift assay (EMSA), and caspase-3 activation was tested by colorimetric assay.


RESULTS
BGC-823 cells underwent apoptosis following stimulation with TNFalpha. We found that Notch-1 was over-activated by overexpressing exogenous intracellular domain of Notch (ICN) via retrovirus-mediated gene transfer, and over-activated Notch-1 reduced the TNFalpha-induced growth suppression and apoptosis in BGC-823 cells. Down-regulation of Notch-1 by siRNA targeting Notch-1 enhanced TNFalpha-induced apoptosis in BGC-823 cells. As the molecular mechanism involved, we showed over-activated Notch-1 partially suppressed TNFalpha-induced activation of caspase-3. However, TNFalpha-induced activation of NF-kappaB was not affected by over-activated Notch-1.


CONCLUSIONS
Our data indicate that over-activated Notch-1 significantly protects BGC-823 cells from TNFalpha-induced apoptosis, and this effect is mediated, at least in part, by decreasing activation of caspase-3 independent of NF-kappaB.
Yao
J
J
School of Medicine, Taizhou University, Jiaojiang District, Taizhou, Zhejiang 318000, PR China.
Qian
C
C
D000704:Analysis of Variance; D017209:Apoptosis; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005434:Flow Cytometry; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism; D051881:Receptor, Notch1 / Q000378:metabolism*; D013274:Stomach Neoplasms / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D015854:Up-Regulation
D051792:Basic Helix-Loop-Helix Proteins; D053148:Caspase 3; D016328:NF-kappa B; D051881:Receptor, Notch1; D014409:Tumor Necrosis Factor-alpha; C497761:NOTCH1 protein, human