Published in Cancer Detection and Prevention 2000; 24(Supplement 1).

Purified intestinal alkaline sphingomyelinase inhibits proliferation in HT-29 colon carcinoma cells without inducing apoptosis

E Hertervig PhD 1, A Nilsson PhD 1, Y Cheng MD 2, RD Duan PhD 2

1 Dept Medicine,, 2 Dept Cell Biology, Lund University, Lund, Sweden,

AIMS: Sphingomyelin (SM) hydrolysis by sphingomyelinase (SMase) has become an important signaling pathway in the regulation of cell growth, differentiation and apoptosis. An alkaline SMase is specifically located to the intestinal tract. Marked reductions of the enzyme activity have been found in sporadic colorectal carcinomas and in both adenomas and flat mucosa of patients with familial adenomatous polyposis. In the present study, we examined the effects of a purified alkaline SMase on cell growth in HT-29 colon carcinoma cells. METHODS: Alkaline SMase was purified from rat intestinal mucosa and the effects on proliferation and apoptosis was examined in HT-29 cells and compared to a neutral bacterial SMase. SM hydrolysis and ceramide generation was measured. RESULTS: Alkaline SMase inhibited proliferation of HT-29 cells in a both dose- and time-dependent manner. The threshold concentration of the enzyme was about 2.5 microU/ml, and the maximal effect was obtained at about 20 microU/ml, which inhibited the cell growth by 50%. The inhibition occurred rapidly and maximal effect was reached after 12 hours of incubation. Accordingly, a dose-dependent inhibition of DNA synthesis was also demonstrated. The effect of alkaline SMase were preceded and accompanied by an increased hydrolysis of SM and production of ceramide. Neutral SMase with equivalent hydrolytic capacity did not inhibit cell growth. Alkaline SMase did not inhibit growth of IEC-6 cells. Neither alkaline nor neutral SMase induced apoptosis in HT-29 cells. CONCLUSIONS: We conclude that alkaline SMase at doses that induce SM hydrolysis, inhibit growth of colon cancer cells. The inhibition is attributed to an antiproliferative effect rather than an apoptotic effect.

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Paper presented at the International Symposium on Impact of Biotechnology on Cancer Diagnostic & Prognostic Indicators; Geneva, Switzerland; October 28 - 31, 2000; in the section on cell cycle inhibitors.