Ca2+ overload- and ROS-associated mitochondrial dysfunction contributes to δ-tocotrienol-mediated paraptosis in melanoma cells

Abstract

Melanoma is an aggressive tumor with still poor therapy outcomes. δ-tocotrienol (δ-TT) is a vitamin E derivative displaying potent anti-cancer properties. Previously, we demonstrated that δ-TT triggers apoptosis in human melanoma cells. Here, we investigated whether it might also activate paraptosis, a non-canonical programmed cell death. In accordance with the main paraptotic features, δ-TT was shown to promote cytoplasmic vacuolization, associated with endoplasmic reticulum/mitochondrial dilation and protein synthesis, as well as MAPK activation in A375 and BLM cell lines. Moreover, treated cells exhibited a significant reduced expression of OXPHOS complex I and a marked decrease in oxygen consumption and mitochondrial membrane potential, culminating in decreased ATP synthesis and AMPK phosphorylation. This mitochondrial dysfunction resulted in ROS overproduction, found to be responsible for paraptosis induction. Additionally, δ-TT caused Ca²⁺ homeostasis disruption, with endoplasmic reticulum-derived ions accumulating in mitochondria and activating the paraptotic signaling. Interestingly, by using both IP3R and VDAC inhibitors, a close cause-effect relationship between mitochondrial Ca²⁺ overload and ROS generation was evidenced. Collectively, these results provide novel insights into δ-TT anti-melanoma activity, highlighting its ability to induce mitochondrial dysfunction-mediated paraptosis. δ-tocotrienol induces paraptotic cell death in human melanoma cells, causing endoplasmic reticulum dilation and mitochondrial swelling. These alterations induce an impairment of mitochondrial function, ROS production and calcium overload.

Keywords: Ca2+ overload; Melanoma; Mitochondrial impairment; Paraptosis; ROS production; Tocotrienols.

Fig. 1

δ-TT induces cytoplasmic vacuolization in human melanoma cells. a A375 and BLM cells were treated with 15 μg/mL δ-TT for 12 h and then analyzed by light microscopy, showing extensive cytoplasmic vacuolization. Scale bars are 20 µm. b A375 and BLM cells were treated with 15 μg/mL δ-TT for 12 h and then analyzed by electron microscopy. They exhibited swollen mitochondria with rare cristae (m), dilated endoplasmic reticulum (ER) cisternae and enlarged nuclear envelope. Scale bars are 2 µm

Fig. 2

δ-TT induces paraptotic cell death in human melanoma cells. a A375 and BLM cells were incubated with the translation inhibitor cycloheximide (CHX) (20 µM) 4 h before treatment with δ-TT (15 µg/mL, 12 h) and observed under light microscopy. Images show that CHX suppressed δ-TT-induced vacuolization in melanoma cells. Scale bars are 20 µm. b A375 and BLM cells were incubated with 50 μM Z-VAD-FMK, the pan-caspase inhibitor, for 4 h and then treated with 15 μg/mL δ-TT for 24 h. MTT assay was performed to assess melanoma cell viability. Three experiments have been performed. One-way analysis of variance. Post-test: Bonferroni’s test. Mean values  ±  SEM are showed. ***P < 0.001 versus controls. c A375 and BLM cells were treated with 15 µg/mL δ-TT for 6–24 h. To evaluate the expression levels of pJNK, pP38 and pERK1/2, western blot analysis was performed. Tubulin was used as a housekeeping protein control. Three experiments have been performed

Fig. 3

δ-TT treatment affects mitochondrial function in melanoma cells. a A375 and BLM cells were treated with 15 µg/mL δ-TT for 6–24 h. OXPHOS expression levels were evaluated via western blot analysis. Tubulin was used as a housekeeping protein control. Three experiments have been performed. b A375 and BLM cells were treated with 15 µg/mL δ-TT for 12 h. Clark electrode was used to measure oxygen consumption. Analysis was performed in basal respiration conditions, in uncoupled respiration conditions (adding oligomycin, oligo) and in maximal respiration conditions (adding carbonyl cyanide m-chlorophenyl hydrazine, CCCP). Three experiments have been performed. T-test. Mean values  ±  SEM are shown. *P < 0.05 versus controls; **P < 0.01 versus controls; ***P < 0.001 versus controls. c A375 and BLM cells were treated with 15 µg/mL δ-TT for 12 h. Cells were stained with MitoTracker Orange CMTM Ros (10 nM, 30 min) fluorescent probe. Mitochondrial activity was measured by flow cytometry. Three experiments have been performed. T-test. Mean values  ±  SEM are shown. ***P < 0.001 versus controls

Fig. 4

δ-TT causes ATP depletion in human melanoma cells. a A375 and BLM cells were treated with 15 µg/mL δ-TT for 12 h. ATP colorimetric assay was used to measure ATP production levels. Three experiments have been performed. T-test. Mean values  ±  SEM are shown. **P < 0.01 versus controls; ***P < 0.001 versus controls. b A375 and BLM cells were treated with 15 µg/mL δ-TT for 6−24 h. Expression levels of pAMPK were assessed via western blot analysis. Tubulin was used as a housekeeping protein control. Three experiments have been performed

Fig. 5

δ-TT induces mitochondrial ROS overproduction in human melanoma cells. a A375 and BLM cells were treated with 15 µg/mL δ-TT for 12 h and then stained with MitoSOX Red (5 µM, 10 min) fluorescent probe. ROS generation in mitochondria was assessed by flow cytometry. T-test. Mean values ± SEM are shown. ***P < 0.001 versus controls. b A375 and BLM cells were incubated with 5 mM NAC (N-acetyl-l-cysteine), the ROS scavenger, for 2 h and then treated with 15 μg/mL δ-TT for 24 h. MTT assay was performed to assess melanoma cell viability. Three experiments have been performed. One-way analysis of variance. Post-test: Bonferroni’s test. Mean values  ±  SEM are shown. **P < 0.01 versus controls

Fig. 6

δ-TT-induced ROS are involved in paraptotic vacuolization and MAPK activation. a A375 and BLM cells were incubated with 5 mM NAC for 2 h and then treated with 15 μg/mL δ-TT for 12 h. Cell morphology was analyzed by light microscopy. Scale bars are 20 µm. b A375 and BLM cells were incubated with 5 mM NAC for 2 h and then treated with 15 μg/mL δ-TT for 24 h. The expression levels of pJNK, pP38 and pERK1/2 were evaluated via western blot analysis. Tubulin was used as a housekeeping protein control. Three experiments have been performed

Fig. 7

δ-TT causes Ca²⁺ homeostasis dysregulation in human melanoma cells. a A375 and BLM cells were treated with 15 μg/mL δ-TT for 12 h and then stained with 5 µM Fluo-3 AM for 30 min. Cytoplasmic Ca²⁺ levels were assessed by flow cytometry. Three experiments have been performed. T-test. Mean values  ±  SEM are shown. *P < 0.05 versus controls; ***P < 0.001 versus controls. b A375 and BLM cells were treated with 15 μg/mL δ-TT for 12 h and then stained with 5 µM Rhod-2 AM for 30 min. Mitochondrial Ca²⁺ levels were evaluated by flow cytometry. Three experiments have been performed. T-test. Mean values  ±  SEM are shown. *P < 0.05 versus controls; ***P < 0.001 versus controls. c A375 and BLM cells were incubated with the IP3R inhibitor 2APB (20 µM, 2 h) and then treated with 15 μg/mL δ-TT for 24 h. MTT assay was performed to evaluate cell viability. Three experiments have been performed. Mean values  ±  SEM are shown. One-way analysis of variance. Post-test: Bonferroni’s test. *P < 0.05 versus controls; **P < 0.01 versus controls. d A375 and BLM cells were incubated with the VDAC blocker DIDS (75 µM, 2 h) and then treated with 15 μg/mL δ-TT for 24 h. MTT assay was performed to evaluate cell viability. Three experiments have been performed. Mean values  ±  SEM are shown. One-way analysis of variance. Post-test: Bonferroni’s test. **P < 0.01 versus controls; ***P < 0.001 versus controls

Fig. 8

δ-TT-induced Ca²⁺ overload is involved in paraptotic vacuolization and MAPK activation. a A375 and BLM cells were incubated with the IP3R inhibitor 2APB (20 µM, 2 h) before treatment with 15 μg/mL δ-TT for 12 h. Cell morphology was analyzed via light microscopy. Scale bars are 20 µm. b A375 and BLM cells were incubated with the VDAC blocker DIDS (75 µM, 2 h) before treatment with 15 μg/mL δ-TT for 12 h. Cell morphology was analyzed via light microscopy. Scale bars are 20 µm. c A375 and BLM cells were incubated with the IP3R inhibitor 2APB (20 µM, 2 h) before treatment with 15 μg/mL δ-TT for 24 h. The expression levels of pJNK, pP38 and pERK1/2 were assessed via western blot. Tubulin was used as a housekeeping protein control. Three experiments have been performed. d A375 and BLM cells were incubated with DIDS (75 µM, 2 h) before treatment with 15 μg/mL δ-TT for 24 h. The expression levels of pJNK, pP38 and pERK1/2 were assessed via western blot analysis. Tubulin was used as a housekeeping protein control. Three experiments have been performed

Fig. 9

Formation of MAMs is observed after δ-TT treatment. A375 and BLM cells were treated with 15 μg/mL δ-TT for 12 h and subsequently analyzed via electron microscopy. Boxed areas in central panels show the formation of MAMs after δ-TT treatment. Scale bars are 2 μm. Boxed areas are enlarged in the right panels and MAMs are highlighted by arrows. Scale bars are 0,5 μm (upper panel) and 2 μm (lower panel)

Fig. 10

δ-TT-induced Ca²⁺ overload causes mitochondrial ROS overproduction in human melanoma cells. a A375 and BLM cells were incubated with the IP3R inhibitor 2APB (20 µM, 2 h) before treatment with 15 μg/mL δ-TT for 12 h. Then, cells were stained with MitoSOX Red (5 µM, 10 min) fluorescent probe. Mitochondrial ROS generation was measured by flow cytometry. Three experiments have been performed. Mean values  ±  SEM are shown. One-way analysis of variance. Post-test: Bonferroni’s test. *P < 0.05 versus controls; ***P < 0.001 versus controls. b A375 and BLM cells were incubated with the VDAC blocker DIDS (75 µM, 2 h) before treatment with 15 μg/mL δ-TT for 12 h. Then, cells were stained with MitoSOX Red (5 µM, 10 min) fluorescent probe. Mitochondrial ROS generation was measured by flow cytometry. Three experiments have been performed. Mean values ± SEM are shown. One-way analysis of variance. Post-test: Bonferroni’s test. ***P < 0.001 versus controls