Kenzo Tamai

Studies on the Antitumor Activity of Fusobacterium nucleatum Strain KO-31

Fusobacterium nucleatum is part of the normal flora of the mouth, upper respiratory tract, and intestinal tract of man. It has been frequently isolated from dental calculus in patients with gingivitis and parodontitis, and also from blood of patients receiving tooth extraction. Although the pathogenic role of this organism has not been clearly elucidated, it might play at least some role in pathological processes in man. While extending our studies on the problem of opportunistic infection by gram-negative anaerobic organisms, I by chance observed in 1976 the survival of Ehrlich ascites carcinoma-bearing mice that had received an intraperitoneal injection of a culture supernatant fluid of F. nucleatum, quite unlike untreated control mice. Subsequent experiments proved this observation to be correct, as described below. The KO-31 strain of F. nucleatum used in this study was isolated from dental calculus of a patient with leukoplakia, and identified according to the method outlined by Bergeys Manual (1); biochemical properties were examined by using the API anaerobic system (La Balme les Grottes, Montalieu, France). A 500-ml volume of TF medium (2) inoculated with 25 ml of a 24-hr culture of F. nucleatum in TF medium was incubated at 37 C for 48 hr. The culture fluid was then centrifuged at 1,300 X g for 20 min at 4 C, and separated into a culture supernatant fluid fraction and a sediment fraction. The culture supernatant fluid fraction, after being sterilized by filtration through a membrane filter with a 0.45-,um pore size (Japan-Millipore, Tokyo), was tested for antitumor activity against Ehrlich ascites carcinoma in mice weighing 18-20g. The sediment fraction was washed once with physiological saline solution and resuspended in 150 ml of physiological saline solution. The cell suspension was then sonicated for 20 min with a Tomy ultrasonic vibrator (model UR-200 p, 20 kilohertz Tomy, Tokyo). The bacterial debris was removed by centrifugation at 14,500xg for 20 min at 4 C, and the supernatant (the cell extract) was tested for its in vivo antitumor activity against Ehrlich ascites carcinoma. One-half-ml quantities of the carcinoma cell suspension containing 8.0-12.0 X 106 cells/ml in physiological saline solution were intraperitoneally inoculated into ICR mice weighing 18-20 g.