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

• Published in: Enzyme and microbial technology Vol. 41; no. 1; pp. 5 - 12
• Main Authors: Tseng, Hui-Chun, Chow, Song-Nan, Huang, Li-Ying, Chien, Chin-Hsiang
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Inc 02.07.2007; Elsevier Science
• Subjects: Apoptosis; Biological and medical sciences; Biotechnology; Clostridium perfringens; Clostridium perfringens ATCC 10543; Desialylation; Fundamental and applied biological sciences. Psychology; Invasion; NanI; Ovarian cancer; TUNEL; Neu5Ac; Desialylation; PARP; NanI; 4-MU-Neu5Ac; Clostridium perfringens ATCC 10543; Invasion; DANA; Apoptosis; Ovarian cancer; Human; Clostridiales; Malignant tumor; Clostridium perfringens; Ovary; Treatment; Clostridiaceae; Bacteria; Tumor cell
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2006.11.020

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.