γ-Tocotrienol induces apoptosis in pancreatic cancer cells by upregulation of ceramide synthesis and modulation of sphingolipid transport

Abstract

Background: Ceramide synthesis and metabolism is a promising target in cancer drug development. γ-tocotrienol (GT3), a member of the vitamin E family, orchestrates multiple effects that ensure the induction of apoptosis in both, wild-type and RAS-mutated pancreatic cancer cells. Here, we investigated whether these effects involve changes in ceramide synthesis and transport.

Methods: The effects of GT3 on the synthesis of ceramide via the de novo pathway, and the hydrolysis of sphingomyelin were analyzed by the expression levels of the enzymes serine palmitoyl transferase, ceramide synthase-6, and dihydroceramide desaturase, and acid sphingomyelinase in wild-type RAS BxPC3, and RAS-mutated MIA PaCa-2 and Panc 1 pancreatic cancer cells. Quantitative changes in ceramides, dihydroceramides, and sphingomyelin at the cell membrane were detected by LCMS. Modulation of ceramide transport by GT3 was studied by immunochemistry of CERT and ARV-1, and the subsequent effects at the cell membrane was analyzed via immunofluorescence of ceramide, caveolin, and DR5.

Results: GT3 favors the upregulation of ceramide by stimulating synthesis at the ER and the plasma membrane. Additionally, the conversion of newly synthesized ceramide to sphingomyelin and glucosylceramide at the Golgi is prevented by the inhibition of CERT. Modulation ARV1 and previously observed inhibition of the HMG-CoA pathway, contribute to changes in membrane structure and signaling functions, allows the clustering of DR5, effectively initiating apoptosis.

Conclusions: Our results suggest that GT3 targets ceramide synthesis and transport, and that the upregulation of ceramide and modulation of transporters CERT and ARV1 are important contributors to the apoptotic properties demonstrated by GT3 in pancreatic cancer cells.

Keywords: ARV-1; CERT; Ceramide distribution; Ceramide synthesis; Ceramide transport; Free radicals; Lipid transport; Membrane lipid; Pancreatic cancer; Vitamin E; γ-Tocotrienol.

Fig. 1

Time-dependent increase of ceramide synthesis enzymes in GT3 treated MIA-PaCa-2 cells.Ceramide is produced via the de novo pathway (a, c, e) each step is catalized by the following enzymes, SPT(b), CER6 (d), and  DEGS1(f). It is also produced by hydrolysis of sphingomyelin (g) by  ASM (h). Pancreatic cancer MIA PaCa-2 cells were treated with GT3 at a concentration of 40 μM or dissolution vehicle as a control for different time periods (2, 4, 6 h). Cell lysates were analyzed by immunoblot. Membranes were incubated with antibodies against SPT(b), CERS6 (d), DEGS1(f), and ASM(h). The membranes were reprobed with actin as a loading control. All assays were conducted at least three times and blots shown are representative of the results obtained. For quantification band densities from the treatment conditions identified by the lane labels, were calculated as percentages of the value for the treated, untreated cells (100%), and shown averages ± standard deviations from three independent experiments (*p<0.05)

Fig. 2

Time-dependent analysis of enzymes involved in ceramide synthesis in GT3 treated BxPC3, Panc 1 and HPDE-E6E7 cells. Pancreatic cancer cells BxPC3(a-f), Panc 1 (g-l), and non-cancerous ductal pancreatic cells HPDE-E6E7 (m-r) were treated with GT3 at a concentration of 40 μM or dissolution vehicle as a control for different time periods (2, 4, 6 h). Cell lysates were analyzed by immunoblot. Membranes were incubated with antibodies against SPT, CERS6, and  DEGS1. The membranes were reprobed with actin as a loading control (located below each set of enzyme immunoblots, as in Figure 1). For quantification band densities from the treatment conditions identified by the lane labels, were calculated as percentages of the value for the treated, untreated cells (100%). Data shown of representative experiments, (mean ± SE, n = 3) *p<0.05, **p<0.01, significant difference between control and GT3 treated cells analyzed at different time points

Fig. 3

Analysis of the effect of GT3 treatment on cellular levels of ceramide, dihydroceramide, and sphingomyelin. A. LC/MS analysis of sphingolipids present in the plasma membrane for ceramides (a), dihydroceramides (b), and sphingomyelin (c), in MIA Paca-2, and HPDE-E6E7 cells treated with dissolution vehicle (control) or 40 μMGT3. Results are shown as pmol of each indicated sphingolipid / mg of protein in the sample analyzed. The values shown are the averages ± standard deviations obtained from three independent experiments. * p <0.05, significant difference between control and GT3 treated cells

Fig. 4

Analysis of the effect of GT3 treatment on the expression levels of ceramides C16, C24:1 and C24. A. LC/MS of C16, C24:1 and C24 ceramides in MIA PaCa-2 cells (a), and HPDE-E6E7(b) dihydroceramides (c and d), and sphingomyelin (e and f ) treated with dissolution vehicle (control) or 40μM GT3. Results are shown as pmol of each indicated sphingolipid/mg of protein in the sample analyzed. The values shown are the averages ± standard deviations obtained from three independent experiments. * p <0.05, significant difference between control and GT3 treated cells

Fig. 5

Downregulation of ceramide transporter CERT and upregulation of transport protein, ARV-1 in MIA PaCa-2, BxPC3, Panc 1 and HPDE-E6E7 cells treated with GT3. Pancreatic cancer cells MIA PaCa-2, BxPC3, and Panc 1, as well as HPDE-E6E7cells were treated for 4 hours with increasing concentrations of GT3 over a dose range from 0-40μM and probed for CERT and its activated form (a and b for MIA PaCa-2 assays, and g for BxPC3, Panc1, and HPDE-E6E7), ARV-1 (c and d for MIA PaCa-2 assays and f for BxPC3, Panc1, and HPDE-E6E7), and analyzed by western immunoblot. The membranes were reprobed with actin as a loading control. Quantitative RT-PCR using ARV-1 primers was performed on MIA PaCa-2 cells treated with increasing concentrations of GT3 in the same manner as described above (e). All assays were conducted in triplicate and blots shown are representative of the results obtained. For quantification, (graphs) band densities from the treatment conditions identified by the lane labels, were calculated as percentages of the value for the treated, untreated cells (100%). Data shown of representative experiments, (mean ± SE, n = 3). * p <0.05, significant difference between control and GT3 treated cells at different concentrations

Fig. 6

GT3 effect on ARV-1 knockdown MIA PaCa-2 cells. MIA PaCa-2 cells were transduced with ARV-1 shRNA lentiviral particles, and then treated with 40μM GT3 or dissolution vehicle (control). Cell lysates were analyzed by immunoblot with antibodies against ARV-1 (a,b) caveo lin (c,d). The membranes were reprobed with actin as a loading control. Data shown of representative experiments, (mean ± SE, n = 3). * p <0.05, significant difference between control and treated cells in different conditions (One-way ANOVA with Bonferroni’s test). e.  Expression and localization of ceramide and caveolin in ARV-1 knockdown MIA PaCa-2 cells treated with vehicle (control) or 40μM GT3, and processed for immunofluorescence. Single confocal sections are shown in the x-y plane, ceramide (red channel), caveolin (green channel), and DAPI (blue channel)

Fig. 7

Colocalization analysis of ceramide and DR5 after GT3 treatment in MIA PaCa-2 cells. MIA PaCa-2 cells were treated with dissolution vehicle (a top panels ) or 40μM GT3 (a bottom panels), fixed, and processed for immunofluorescence. Single confocal sections shown in the x-y plane, and 3D reconstructions of the confocal stack in the x–z plane perpendicular to the monolayer, apical side up, show localization of DR5 (green channel), ceramide (red channel), and nuclei using DAPI (blue channel). Intensity correlation analysis of 14 fields, each with an average of 12.5 cells from at least three independent experiments, using the Leica Software were run for control cells (b) and (c) and treated cells (d) and were plotted for both sets of cell populations. The values shown are the averages ± standard deviations obtained from at least three independent experiments. Images are representative of fields analyzed. Graphs illustrating the quantification of relative fluorescence for control cells (c) and treated cells (e) show the merge for ceramide and DR5 channels. Images shown are representative of the fields observed

Fig. 8

Fluorescence intensity analysis of ceramide and DR5 after GT3 treatment in MIA PaCa-2 cells. a MIA PaCa-2 cells were treated with dissolution vehicle (A, top panels ) or 40μM GT3 (A, bottom panels), fixed, and processed for immunofluorescence. Single confocal sections shown in the x-y plane, show localization of DR5 (green channel), ceramide (red channel), and nuclei using DAPI (blue channel). b The fluorescence intensities along the white lines indicated in the merged pictures were quantified using the line scan application of Leica software. Data obtained from line scans drawn through the cell were plotted in a graph and expressed as mean ±SE, n= 50 cells, from three independent experiments.*p < 0.05