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

Abstract

Sphingomyelin (SM) hydrolysis by sphingomyelinase (SMase) has become an important signalling pathway, with the product ceramide implicated in regulation of cell growth, differentiation and apoptosis. Alkaline SMase is specifically located in 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, indicating an anti-proliferative role in colonic cell growth. We examined the effects of a purified alkaline SMase from rat intestine and a bacterial neutral SMase on cell growth parameters in HT-29 colonic carcinoma cells. Alkaline SMase was found to inhibit proliferation of HT-29 cells in both dose-dependent and time-dependent manners. The threshold concentration of the enzyme was approximately 2.5 microU/ml, and the maximum effect was obtained at approximately 20 microU/ml, which inhibited the cell growth by 50%. The inhibition occurred rapidly, and maximum effect was reached after 12 h of incubation. Dose-dependent inhibition of DNA synthesis was also demonstrated. The effect of alkaline SMase was preceded and accompanied by increased hydrolysis of SM and production of ceramide. Neutral SMase with equivalent hydrolytic capacity did not inhibit cell growth. Alkaline SMase did not induce apoptosis in HT-29 cells. Alkaline SMase did not inhibit growth of IEC-6 cells. Alkaline SMase, at doses that induce SM hydrolysis, inhibits growth of colon cancer cells. The inhibition is attributed to an anti-proliferative effect rather than an apoptotic effect.