Desialylation of human cancer cells leading apoptosis by treatment with purified and overexpressed nanI cloned from Clostridium perfringens ATCC 10543

Abstract

The effect of NanI, overexpressed nanI gene cloned from Clostridium perfringens ATCC 10543, on NIH:OVCAR-3 cancer cells and the mediators relaying the apoptosis induced by NanI were investigated. Overexpressed NanI was purified by Ni-NTA affinity column and eluted by 250 mM imidazole in 10 mM phosphate buffer, pH 7.4. The homogeneously purified NanI was added to the culture medium of OVCAR-3 human ovarian cancer cells at indicated time. The experiments included flow cytometry for glycosidic linkage determinations, in vitro invasion assay for examining cancer cell invasiveness; TUNEL/PI reactions as well as Western blot analysis for cell apoptosis detections. NanI hydrolyzed the terminal sialic acids of glycoconjugates on cancer cell surfaces and predominantly hydrolysed the α 2,6 glycosidic linkages of glycoconjugates on OVCAR-3 cell surfaces. In vitro invasion assays demonstrated that NanI reduced the number of cells; the percentage of invaded cells was reduced to 12.39 ± 3.44% when OVCAR-3 cells were treated with 0.6 U/ml of NanI. NanI suppressed the growth rate of OVCAR-3 cells efficiently. The apoptotic effects were exhibited by positive fluorescence image of TUNEL plus PI assay. Western blot analysis demonstrated down regulation of Bcl-2, releases of cytochrome c, as well as activation of caspase-9 and -3. PARP was found to be cleaved into 85 kDa after the treatment of OVCAR-3 cells with NanI. The effects of 0.6 U/ml NanI were abrogated with the addition of 10 mM of NanI inhibitor DANA. The results delineated that the induction of apoptosis in NanI-treated OVCAR-3 cells is through the mitochondria-dependent pathway. Predominant desialylation of α 2,6 linkages of glycoconjugates on cancer cell surfaces renders cells more susceptible to the membrane damage, which may suppress the invasiveness and trigger apoptosis of OVCAR-3 cells. It is anticipated that potential applications of NanI catalytic domain contained in liposome conjugated with specific monoclonal antibody to target on the cancer cell surfaces may open up a new modality of treating cancers.