Chemopreventive and chemotherapeutic mechanisms of sphingolipid metabolites in human colon cancer cells and breast stem, normal, and tumorigenic cells

The effects of the sphingolipid metabolites (sphingosine, sphinganine, ceramide, and dihydroceramide) on proliferation and differentiation in human normal epithelial cells versus tumorigenic cells and stem cells have not previously been compared. This dissertation research addressed these questions using following cell culture models: HT-29 and HCT-116 human colon cancer epithelial cells, human breast epithelial cells (HBEC) developed from tissues of healthy women obtained during reduction mammoplasty (type I HBEC with stem cell characteristics as target cells for breast carcinogenesis and type II HBEC with basal epithelial cell phenotypes as breast normal cells) and transformed tumorigenic type I HBEC as breast cancer cells.; The objectives of the present study were to: (1) investigate the effects of sphingoid bases (sphingosine and sphinganine), ceramide, and dihydroceramide on proliferation, cell cycle, and apoptosis of HT-29 and HCT-116 cells; (2) determine the effects of sphinganine on major isoforms of mitogen activated protein kinases (ERK, JNK, and p38) and AKT (protein kinase B) in HT-29 cells; (3) evaluate the effects of sphingoid bases on proliferation, cell cycle, and apoptosis of tumorigenic type I HBEC and type I HBEC compared to type II HBEC; and (4) determine the effects of sphingoid bases on the ability of type I HBEC to differentiate to type II HBEC.; Data from HT-29 and HCT-116 cells indicate that sphingoid bases and C ₂-ceramide at 20–50 μM inhibited the growth, arrested cell cycle at G₂/M phase, and induced apoptosis, with sphinganine being the most potent of the metabolites studied. In contrast, C₂-dihydroceramide had no effect, suggesting that the 4,5-trans double bond is necessary for the inhibitory effects of C₂-ceramide, but not for inhibitory effects of sphingoid bases. Sphinganine at an apoptosis-inducing concentration (35 μM) caused early and strong activation of JNK2/JNK1 and p38 and early inhibition of AKT, with minimal effects on activation of ERK1/ERK2 in HT-29 cells.; Results from HBEC show that sphinganine more potently inhibited the growth and induced apoptosis of tumorigenic type I HBEC than sphingosine (IC ₅₀ for sphinganine 4 μM; sphingosine 6.4 μM). Sphingoid bases (8–10 μM) arrested cell cycle at G₂/M with reductions in S and G₀/G₁ phases. Sphinganine also more potently inhibited the growth and caused death of type I HBEC than sphingosine, while the same concentrations of both sphinganine and sphingosine had minor effects on type II HBEC. At concentrations (0.05–0.5μM), which are below the growth inhibitory range, sphingoid bases induced differentiation of type I HBEC to type II HBEC, as detected with concomitant expression of the novel tumor suppressor protein, maspin, in type II HBEC.; In conclusion, sphingoid bases and ceramide might possess chemotherapeutic properties against colon cancer, with sphinganine being the most potent. Activation of JNK and p38 and inhibition of AKT might mediate sphinganine-induced apoptosis of colon cancer cells. Data indicate the potential for sphingoid bases to be employed as chemotherapeutic and chemopreventive agents against human breast cancer.