Comparative study on apoptosis induction of SMMC7721 and Vero cells by Clostridium difficile toxin A.

BACKGROUND & OBJECTIVE: Clostridium difficile is recognized as a frequent cause of antibiotic-induced diarrhea. This study was designed to investigate whether Clostridium difficile toxin A might induce apoptosis on human hepatoma cell line SMMC7721 and African green monkey kidney Vero cells.

METHODS

Highly purified toxin A was obtained by bovine thyroglobulin affinity purification followed by ion exchange chromatography on Q sepharose. The apoptosis induction of toxin A was examined on SMMC7721 cells with Vero cells as a control. Inhibition of proliferation was measured by MTT assay. Morphological assessment of apoptosis was performed with fluorescence and electronic microscopy. DNA fragmentation was observed by agarose gel electrophoresis. Cell cycle distribution was analyzed by flow cytometry.

RESULTS

Toxin A (0.293-4.690 mg x L(-1) ) inhibited proliferation of SMMC7721 and Vero cells in a time- and concentration-dependent manner. Morphological changes of typical apoptosis showed that SMMC7721 had a higher percentage of apoptosis than Vero cells. Agarose gel electrophoresis of DNA from SMMC7721 treated with toxin A for 48 hours revealed a "ladder" pattern.

CONCLUSION

Clostridium difficile toxin A induced apoptosis of SMMC7721 and Vero cells. The apoptosis induction on SMMC7721 cells was more effective than that on Vero cells.