Mechanistic investigation of cell death and sphingolipid alteration induced by vitamin E forms in human colon cancer cells

Xiayu Rao, Purdue University

Abstract

Colon cancer is one of the leading causes of cancer death in the United States. It is important to develop effective chemoprevention and treatment agents with low toxicity. To that end, various molecules in the vitamin E family may be excellent candidates because of their beneficial properties and well-established safety. Vitamin E consists of eight structurally-related lipophilic antioxidants, &agr;-, β-, γ-, δ-tocopherol and &agr;-, β-, γ-, δ-tocotrienol. Most studies of vitamin E in cancer prevention have mainly focused on &agr;-tocopherol (&agr;T), the predominant form of vitamin E in tissues and multi-vitamin supplements. However, recent studies by us and others indicate that γ-tocopherol (γT), δ-tocopherol (δT) and γ-tocotrienol (γTE) have unique properties that are not shared by &agr;T but appear to be important to human disease prevention and therapy including cancer. Here we systemically investigated the effect and mechanism of different forms of vitamin E on the growth and death of human colon cancer HT29 and HCT116 cells. γT, δT and γTE inhibited the proliferation of HT29 and HCT116 cells in a time- and dose-dependent manner, with the potency of γTE > δT > γT. HCT116 cells appeared to be more sensitive to the treatment than HT29 cells. Studies using annexin V and propidium iodide staining revealed that γT (50 µM) and γTE (20 µM) induced externalization of phosphatidylserine and enhanced permeability of the plasma membrane, indicating apoptosis and necrosis. Interestingly, γT, δT and γTE enhanced expression of microtubule-associated protein light chain-3 II (LC3 II) in both HT29 and HCT116 cells, suggesting that these forms of vitamin E may induce autophagy. Consistently, electron microscopic results showed that γTE (20 µM) induced extensive formation of autophagic vacuoles in HCT116 cells. Because there was little induction of chromatin condensation and nuclear fragmentation, apoptosis seemed to be minimal. Although ER dilation was observed after γTE treatment, ER stress markers, Bip and CHOP, did not change following treatment. Thus, ER stress was not induced by γTE. Moreover, myriocin, a specific inhibitor of serine palmitoyltransferase, a key enzyme in de novo synthesis of sphingolipids, protected HT29 and HCT116 cells from γTE-induced cell death. Importantly, γTE caused marked accumulation of dihydrosphingosine and dihydroceramide but not ceramide or sphingosine as early as after 8-hour incubation. Sphingomyelins also accumulated following γTE treatment. These findings demonstrated that vitamin E may induce apoptosis, necrosis and more specifically, autophagy, in colon cancer cells by modulation of the sphingolipid synthesis pathway. These results provide mechanistic insights into potential chemopreventive activity of these vitamin E forms, and will therefore provide molecular bases for further investigation of in vivo chemopreventive efficacy of these vitamin E forms against colon cancer.

Degree

M.S.

Advisors

Jiang, Purdue University.

Subject Area

Nutrition|Oncology

COinS