Vitamin E metabolite 13'-carboxychromanols inhibit pro-inflammatory enzymes, induce apoptosis and autophagy in human cancer cells by modulating sphingolipids and suppress colon tumor development in mice

Abstract

Vitamin E forms are substantially metabolized to various carboxychromanols including 13'-carboxychromanols (13'-COOHs) that are found at high levels in feces. However, there is limited knowledge about functions of these metabolites. Here we studied δT-13'-COOH and δTE-13'-COOH, which are metabolites of δ-tocopherol and δ-tocotrienol, respectively. δTE-13'-COOH is also a natural constituent of a traditional medicine Garcinia Kola. Both 13'-COOHs are much stronger than tocopherols in inhibition of pro-inflammatory and cancer promoting cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX), and in induction of apoptosis and autophagy in colon cancer cells. The anticancer effects by 13'-COOHs appeared to be partially independent of inhibition of COX-2/5-LOX. Using liquid chromatography tandem mass spectrometry, we found that 13'-COOHs increased intracellular dihydrosphingosine and dihydroceramides after short-time incubation in HCT-116 cells, and enhanced ceramides while decreased sphingomyelins during prolonged treatment. Modulation of sphingolipids by 13'-COOHs was observed prior to or coinciding with biochemical manifestation of cell death. Pharmaceutically blocking the increase of these sphingolipids partially counteracted 13'-COOH-induced cell death. Further, 13'-COOH inhibited dihydroceramide desaturase without affecting the protein expression. In agreement with these mechanistic findings, δTE-13'-COOH significantly suppressed the growth and multiplicity of colon tumor in mice. Our study demonstrates that 13'-COOHs have anti-inflammatory and anticancer activities, may contribute to in vivo anticancer effect of vitamin E forms and are promising novel cancer prevention agents.

Keywords: Apoptosis; Autophagy; Cancer; Sphingolipid; Vitamin E metabolites.

Figure 1. The structures of δT-13’-COOH and δTE-13’-COOH (A); The effect of 13’-COOHs and tocopherols on cell viability of human colon HCT-116 (B) and HT-29 (C) cancer cells; The relative anti-proliferation effect (24-h incubation) of 13’-COOHs on cancer cells vs. human normal CCD841CoN (CCD) colon epithelial cells (D)

Relative cell viability was measured by MTT assay after cells were treated with 13’-COOHs or tocopherols at indicated concentrations and times compared with DMSO controls. IC₅₀s are the concentrations that caused 50% decrease in cell viability after 24-h incubation. The data are mean SD from at least three independent experiments. *p < 0.05 and **p < 0.01 indicate a significant difference between treated and DMSO-control cells. δT-13’-COOH and δTE-13’-COOH are abbreviated as δT-13’ and δTE-13’, respectively.

Figure 2. 13’-COOHs induced apoptosis (A, B) and autophagy (C) in HCT116 cells and AA partially reversed anti-proliferative effects of 13’-COOHs in Caco-2 and HCT-116 cells (D)

HCT-116 cells were treated with δT-13’-COOH or δTE-13’-COOH at 20 μM for 24 h. Apoptosis and necrosis were evaluated by annexin V and PI staining using flow cytometry (A). Western blots were conducted to monitor apoptosis and autophagy as indicated by caspase-9 activation and PARP cleavage (B) and LC3-II (C), respectively. Results of flow cytometry and Western blots are representative of three or more independent experiments. Caco-2 or HCT116 cells were treated with δT-13’-COOH or δTE-13’-COOH with or without 1 μM of AA for 72 h and relative cell viability was measured by MTT assays (D). The data are mean SEM for at least three independent experiments. *p < 0.05 and **p < 0.01 indicate a significant difference between cells treated with 13’-COOHs in the presence vs. absence of AA. δT-13’-COOH and δTE-13’-COOH are abbreviated as δT-13’ and δTE-13’, respectively.

Figure 3. δT-13’-COOH (δT-13’) profoundly modulated sphingolipid metabolism in HCT-116 cells in time- and dose-dependent manners

HCT-116 cells were treated with 10 or 20 μM of δT-13’-COOH for 1, 2, 4, 8, or 16 h. LC-MS/MS was used to measure the sphingolipid levels including total dhCers (A), C_16:0-dhCer (B), dhSph (C), total Cers, C_16:0-Cer, C_18:0-Cer (D) and total SMs (E). Results are shown as mean SEM for at least three independent experiments. *p < 0.05 and **p < 0.01 indicate a significant difference between treated and DMSO control cells at each indicated time.

Figure 4. δT-13’-COOH (δT-13’) inhibited DEGS activity but not protein expression

A: The de novo biosynthesis pathway of sphingolipids (SMS: sphingomyelin synthase; SMase: sphingomyelinase). B: HCT-116 cells were treated with δT-13’-COOH at 10 or 20 μM for 8 or 16 h and the effect on DEGS-1 expression was detected by Western blotting. C: HCT-116 cells were treated with DMSO (control), δT-13’-COOH at 20 μM or C8-CPPC at 1 μM for 1 or 2 h. Cells were collected and homogenized. The homogenates were added with NADH and C_8:0-dhCer as a substrate for DEGS. After 1 h incubation at 37 C, the production of C_8:0-sphingolipids were analyzed by LC-MS/MS. The data are mean SEM of three independent experiments. *p < 0.05 and **p < 0.01 indicate significant differences between DMSO-control cells and 13’-COOH or CPPC-treated cells.

Figure 5. Myriocin and GW48695 partially counteracted 13’-COOHs-induced anticancer activities

A: HCT-116 cells were treated with 13’-COOH at 20 μM with or without myriocin (3 μM), a specific inhibitor of serine palmitoyltransferase to block the de novo sphingolipid synthesis. After 16-h treatment, cells were collected and analyzed for LC3-II by Western blotting. The data are mean SEM of more than 4 independent experiments. *p < 0.05 and **p < 0.01 indicate a significant difference between 13’-COOHs in the presence vs. absence of myriocin. B: HCT-116 cells were treated with δT-13’-COOH (5 or 10 μM) or δTE-13’-COOH (10 μM) with or without GW48695 (5 μM, an inhibitor of neutral SMase) for 24 h. Relative cell viability was measured by MTT assays. The data are mean SEM of three independent experiments. *p < 0.05 and **p < 0.01 indicate a significant difference between 13’-COOHs with vs. without GW48695. δT-13’-COOH and δTE-13’-COOH are abbreviated as δT-13’ and δTE-13’, respectively.

Figure 6. δTE-13’-COOH significantly suppressed colon tumorigenesis induced by AOM with two cycles of DSS (1.5%) in Balb/c mice

A: The study design. B and C: Body weight and food intake of each group during the days after supplementation started. D: The effect of δTE-13’-COOH on colon tumor multiplicity and polyps with sizes of < 2 mm² or > 2 mm² in the AOM/DSS study (mean SEM, n = 15-17). *p < 0.05 and **p < 0.01 differences between the AIN-93G control and δTE-13’-COOH supplement group.