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Introduction

Malignant melanoma (MM) and osteosarcoma (OS) are the representatives of aggressive tumors that are highly resistant to multidisciplinary treatment including chemo-, radio-, and immunotherapy (1,2). Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is a member of the tumor necrosis factor superfamily. It has emerged as a promising cancer-selective anticancer drug since it exhibits potent cytotoxicity toward various cancer cell types with minimal cytotoxicity toward normal cells (3–5). Binding of TRAIL to two death receptors (DRs), TRAIL receptor (TRAIL-R)1/DR4 and TRAIL-R2/DR5 triggers the extrinsic and intrinsic apoptotic pathways (6,7). It also triggers pathways leading to other modes of cell death such as autophagy (8,9) and necroptosis (10,11). However, MM and OS are resistant to TRAIL-induced cytotoxicity, despite expressing DRs. In addition to their inherent resistance, the acquired resistance of MM and OS cells to the drug dampens TRAIL treatment (12). Consequently, the combined application of medicines that enable to reduce the TRAIL resistance is necessary for effective TRAIL therapy of these cancers.

Ca2+ regulates many complicated cellular processes such as cell activation, proliferation, and death. Recently, Ca2+ is emerging as a new target for cancer treatment. Various cancer cell types exhibit tumor-specific traits in Ca2+ dynamics, which contribute to tumorigenesis, malignant phenotypes, drug resistance, increased proliferation, and survival (13–15). Growing body of evidence suggests that a variety of Ca2+-permeable channels regulate Ca2+ remodeling and survival in cancer cells (16). However, Ca2+ promotes not only survival but also different modalities of cell death including apoptosis, necrosis, autophagy, and anoikis in cancer cells (17). Intracellular Ca2+ overload was early thought to be a critical mediator of necrotic cell death by leading to the increase in the permeability of the mitochondrial membrane (mitochondrial permeability transition) and the resulting dysfunction. Ca2+/calpain, an intracellular Ca2+-dependent cysteine protease, is activated by the rise in the cytosolic Ca2+ concentration ([Ca2+]cyt) and critically involved in cancer cell apoptosis through the processing of the mitochondria-localized pro-apoptotic molecule, apoptosis-inducing factor (18,19). An excess, persistent rise in mitochondrial Ca2+ concentration ([Ca2+]mit) increases the permeability of the inner membrane, thereby leading to release of pro-apoptotic proteins, the collapse of mitochondrial integrity, and activation of the intrinsic apoptotic pathway. Although the dual effect of Ca2+ remodeling is thought to be due to the differences in the magnitude, timing, duration, and the space of the Ca2+ surge generated (13), at present, no model can depict the dual role for Ca2+ remodeling. Thus, drugs targeting overall Ca2+ signals may modulate both pro-death and pro-survival pathways non-specifically, thereby compromising the antitumor effect. Therefore, it is necessary to characterize the cellular parameters and machinery that decide the two types of Ca2+ signal and minimize the onset of the pro-survival Ca2+ pathway by the therapy. To date, Ca2+ remodeling in melanoma and osteosarcoma is poorly characterized, and the role for Ca2+ in their malignant phenotypes and survival remains unclear.

In this study, we analyzed the impact of TRAIL on Ca2+ remodeling in MM and OS cells and the possible role of Ca2+ in their survival and TRAIL resistance. The results showed that acute TRAIL treatment modulates Ca2+ dynamics and that Ca2+ protects these tumor cells to TRAIL-induced apoptotic and non-apoptotic cell death. We also found that Ca2+ remodeling in the mitochondria through mitochondrial uniporter (MCU), mitochondrial permeability transition pore (MPTP), and a Ca2+ transport pathway sensitive to capsazepine and AMG9810 play a vital role in the protection. The findings suggest that mitochondrial Ca2+ removal facilitates non-apoptotic cell death induction by TRAIL and may have therapeutic potential in the treatment of these TRAIL-resistant cancers.

Materials and methods

Materials

Soluble recombinant human TRAIL was obtained from Enzo Life Sciences (San Diego, CA, USA). AMG9810, capsazepine, CGP-37157, atractyloside, thapsigargin (Tg), necrostatin-1, and the pan-caspase-inhibitor z-VAD-fluorometheylketone (z-VAD-FMK) were obtained from Sigma-Aldrich (St. Louis, MO, USA). All insoluble reagents were dissolved in dimethylsulfoxide and diluted with high glucose-containing Dulbecco's modified Eagle's medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma-Aldrich; FBS/DMEM) or Hank's balanced salt solution (HBSS) (pH 7.4) to a final concentration of <0.1% before use.

Cell culture

Human MM (A375, A2058) and OS (MG63, SAOS-2, HOS) cell lines were obtained from Health Science Research Resource Bank (Osaka, Japan) and cultured in FBS/DMEM in a 5% CO2 incubator. Cells were harvested by incubating with 0.25% trypsin-EDTA (Thermo Fisher Scientific, Rochester, NY, USA) for 5 min at 37°C.

Cell growth and apoptosis measurements

Cell growth was measured by WST-8 assay using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan), a colorimetric assay based on the formation of a water-soluble formazan product as previously described (20) with minor modifications. Briefly, cells (8×103/well) were seeded in 96-well plates and cultured with the agents to be tested for 72 h at 37°C in a 5% CO2 incubator. Then 1/10 volume of WST-8 reagent was added, incubated for 1 h at 37°C and absorbance at 450 nm was measured using a microplate reader (ARVO MX, Perkin-Elmer Japan, Tokyo, Japan). Apoptotic cell death was quantitatively assessed by double-staining with fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (PI) as previously described (21). Briefly, cells (2×105/well) in 24-well plates were incubated with the agents to be tested for 24 h in 10% FBS-containing medium at 37°C. The cells were then stained with FITC-conjugated Annexin V and PI using a commercially available kit (Annexin V FITC Apoptosis Detection kit I; BD Biosciences, Tokyo, Japan). The stained cells were evaluated in the FACSCalibur and analyzed using CellQuest software (BD Biosciences). Four cellular subpopulations were assessed: viable cells (Annexin V−/PI−); early apoptotic cells (Annexin V+/PI-); late apoptotic cells (Annexin V+/PI+); and necrotic/damaged cells (Annexin V−/PI+). Annexin V+ cells were considered to be apoptotic cells.

Ca2+ measurements

Changes in [Ca2+]cyt and [Ca2+]mit were measured using the cytosol Ca2+-reactive fluorescence probe Fluo 4-AM and mitochondrial Ca2+-reactive fluorescence probe rhod 2-AM (both were obtained from Dojindo), respectively as previously described (22). To improve its mitochondrial localization, rhod 2-AM was reduced to the colorless, nonfluorescent dihydrorhod 2-AM by sodium borohydride, according to the manufacturer's protocol. Cells were loaded with 4 μM each of Fluo 4-AM or dihydrorhod 2-AM for 40 min at 37°C and washed with HBSS. Then, the cells (1×106/ml) were resuspended in HBSS in 96-well plates. The cells were manually added with the agents to be tested. Then, the cells were measured for fluorescence at 5 sec intervals up to 3 min in a microplate reader (Fluoroskan Ascent, Thermo Fisher Scientific) with excitation and emission at 485 and 538 nm (for Fluo 4-AM), respectively and 542 and 592 nm (for rhod 2-AM), respectively. For Ca2+-independent experiments, cells were suspended in HBSS supplemented with 1 mM EGTA in place of 1 mM CaCl2.

Caspase-3/7 activation, membrane integrity, and cell death assay

Caspase-3/7 activation, membrane integrity, and cell death were simultaneously measured by Muse™ Cell Analyzer (Merck Millipore, Darmstadt, Germany) using Muse Caspase-3/7 kit. Briefly, cells (1×105/ml) in 24-well plates were treated with the agents to be tested for 24 h in 10% FBS/DMEM at 37°C and then stained with a novel Caspase-3/7 reagent NucView™ and 7-amino-actinomycin D (7-AAD), a dead cell marker in the kit. 7-AAD is excluded from healthy and early apoptotic cells, while permeates late apoptotic and dead cells. Consequently, four populations of cells can be distinguished by the kit; Live cells: Caspase−/7-AAD−; early apoptotic cells: Caspase+/7-AAD−; late apoptotic/dead cells: Caspase+/7-AAD+; necrotic cells: Caspase−/7-AAD+.

Statistical analysis

Data were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey's post-hoc test using an add-in software for Excel 2016 for Windows (SSRI, Tokyo, Japan). All values are expressed as means ± SD, and P<0.05 was considered to be significant.

Results

Analyses of Ca2+ dynamics in MM and OS cells stimulated with TRAIL

To determine the impact of TRAIL on Ca2+ dynamics in tumor cells, we measured the effect of TRAIL on [Ca2+]cyt and [Ca2+]mit in osteosarcoma cells in parallel. Treatment with soluble recombinant human TRAIL resulted in a robust increase in [Ca2+]cyt in HOS cells in a dose-dependent manner (Fig. 1A and B). The increase occurred rapidly (within minutes) and persistently (lasted at least for 10 min). TRAIL at concentrations of ≥50 ng/ml had a significant effect in parallel with the cytotoxic effect. In parallel, [Ca2+]mit was increased in a dose-dependent manner (Fig. 1C and D). Depending on the cellular conditions, [Ca2+]mit was elevated maximally at 50 ng/ml, and higher concentrations of TRAIL had a smaller effect. We observed similar results in an array of MM and OS cells including SAOS-2, MG63, A375 and A2058 cells (not shown).

Figure 1 TRAIL modulates Ca2+ dynamics in malignant cells. HOS cells were loaded with 4 μM Fluo 4-AM (A and B) and dihydrorhod 2-AM (C and D), respectively, for 40 min at 37°C, washed with HBSS. The dye-loaded cells (1×106/ml) were resuspended in the Ca2+-containing medium in 96-well plates. After addition of 25, 50, or 100 ng/ml TRAIL to the cells, fluorescence was immediately measured in triplicate in a microplate reader at 0, 1,2,3,5,10 min with excitation and emission at 485 and 538 nm (for Fluo 4-AM), respectively and 542 and 592 nm (for dihydrorhod 2-AM), respectively. The data show means ± SD in a representative experiment (N=3). (B and D) Data were analyzed by ANOVA followed by the Tukey's post-hoc test. *P<0.05; **P<0.01; ***P<0.001 vs. control (CTRL). The number of parentheses represents the data analyzed.

The MCU inhibitor Ruthenium 360 (Ru360) suppresses mitochondrial Ca2+ load in MM and OS cells

MCU is a major molecular machinery responsible for the physiological Ca2+ load into the mitochondrial matrix (23). The role of MCU in mitochondrial Ca2+ remodeling has been studied in few tumor cells including breast cancer cells (24) and neuroblastoma cells (25). Since the role of MCU in mitochondrial Ca2+ remodeling in MM and OS cells is unknown, we examined the impact of MCU-specific agents on their mitochondrial Ca2+ dynamics. The MCU inhibitor Ru360 caused a significant decrease in [Ca2+]mit, while the mitochondrial Na+-Ca2+ exchanger (NCLX) inhibitor CGP-37157 increased [Ca2+]mit in HOS and SAOS-2 cells (Fig. 2A and B). EGTA and the mitochondrial permeability transition pore (MPTP) inhibitor cyclosporine A (CysA) significantly decreased [Ca2+]mit in HOS cells, but not in SAOS-2 cells (Fig. 2A and B). On the other hand, atractyloside, an MPTP opener, significantly reduced [Ca2+]mit in both MM and OS cells (Fig. 2C and D), indicating that Ca2+ extrusion through the MPTP participates in regulating [Ca2+]mit.

Figure 2 The mitochondrial Ca2+ dynamics is regulated by MCU and MPTP. Dihydrorhod 2-AM-loaded HOS (A and D), SAOS-2 (B), and A375 cells (C) were resuspended in the Ca2+-containing medium in 96-well plates. The cells were added with 10 μM CGP-37157 (CGP), 1 μM cyclosporine A (CysA), 1 μM Ruthenium 360 (Ru360), or 0.5 mM EGTA (A and B) or 10 μM CGP or 5 μM atractyloside (ATR) (C and D). Then, fluorescence was immediately monitored in triplicate at 5 sec intervals up to 3 min. The data show means ± SD in a representative experiment (N=3). Data were analyzed by ANOVA followed by the Tukey's post-hoc test. **P<0.01; ***P<0.001; NS, not significant vs. non-treated control (NT). The number of parentheses represents the data analyzed.

Capsazepine and AMG9810 reduce [Ca2+]mit cooperatively with TRAIL in MM and OS cells

We found that capsazepine and AMG9810 modulated mitochondrial Ca2+ dynamics in MM and OS cells. AMG9810 alone at concentrations ranging from 1 to 10 μM decreased [Ca2+]mit in A2058 cells in a dose-dependent manner (Fig. 3A). Capsazepine alone reduced [Ca2+]mit maximally at 1–3 μM (Fig. 3B). When used with TRAIL and AMG9810 together, [Ca2+]mit was dropped to the level lower than that observed with each agent alone (Fig. 3A). Meanwhile, capsazepine enhanced the effects of TRAIL on [Ca2+]mit with a maximal effect at 1–3 μM (Fig. 3B). Essentially similar results were obtained for SAOS-2 cells (Fig. 3C and D). These results show that capsazepine and AMG9810 reduce [Ca2+]mit cooperatively with TRAIL in MM and OS cells.

Figure 3 AMG9810 and capsazepine reduce [Ca2+]mit cooperatively with TRAIL. Dihydrorhod 2-AM-loaded A2058 (A and B) and SAOS-2 cells (C and D) were suspended in the Ca2+-containing medium in 96-well plates. The cells were added with 100 ng/ml TRAIL and AMG9810 or capsazepine at the indicated concentrations alone or in combination and measured for fluorescence at 5 sec intervals up to 3 min. The data show means ± SD in a representative experiment (N=3). Data were analyzed by ANOVA followed by the Tukey's post-hoc test. ***P<0.001; NS, not significant vs. control (CTRL). ###P<0.001 vs. TRAIL, AMG9810 or capsazepine alone. The number of parentheses represents the data analyzed.

Ca2+ removal decreases MM and OS cell viability and potentiates TRAIL cytotoxicity

To determine the role of Ca2+ remodeling in TRAIL cytotoxicity toward MM and OS cells, we examined the effect of Ca2+ removal on the cytotoxicity. Treatment with TRAIL up to 100 ng/ml for 24 h minimally decreased (4–6% decrease) the viability of A2058 and MG63 cells. Treatment with the intracellular Ca2+-chelator BAPTA (30 μM) moderately decreased the viability of A2058 cells (maximum of 30% reduction), while the extracellular Ca2+-chelator EGTA (0.2–0.5 mM) had minimal effect (Fig. 4A), and both Ca2+-chelators decreased the viability of MG63 cells only modestly (<10%) (Fig. 4B). BAPTA and EGTA sensitized both cells to TRAIL, and this effect became pronounced as the concentration was increased, although their effects varied depending on the cell lines tested (Fig. 4A and B).

Figure 4 Ca2+ removal decreases MM and OS cell viability and potentiates TRAIL cytotoxicity. A2058 (A) and MG63 (B) cells (8×104/well) were plated in 96-well plates and treated with 25 or 100 ng/ml TRAIL in the absence or presence of 30 μM BAPTA-AM (BAPTA) or 0.2 mM EGTA for 24 h and analyzed for viability using WST-8 assay in triplicates. The data show means ± SD for a representative experiment (N=3-5). (C) A375 cells were treated with 100 ng/ml TRAIL in the absence or presence of 0.2 mM EGTA, 10, 30 μM BAPTA, or 50 mM KCl for 24 h, and then analyzed for Annexin V/propidium iodide (PI) staining by flow cytometry. Annexin V+ cells were considered to be apoptotic cells while Annexin V−/PI+ cells were regarded as necrotic cells. The data represent means ± SD from 3 independent experiments. A2058 (D) and SAOS-2 cells (E) were treated with 100 ng/ml TRAIL in the absence or presence of 30 μM BAPTA, 0.5 mM EGTA, 10 μM z-VAD-FMK (ZVAD), or 30 μM necrostatin-1 (NS-1) for 72 h and analyzed for viability using WST-8 assay in triplicates. The data show means ± SD for a representative experiment (N=3-5). Data were analyzed by ANOVA followed by the Tukey's post-hoc test. *P<0.05; **P<0.01; ***P<0.001; vs. control. ###P<0.001 vs. TRAIL.

To determine the cell death modality, we performed flow cytometric analyses using Annexin V/PI double staining. Likewise, KCl, a potent TRAIL-sensitizer (21), BAPTA or EGTA remarkably increased apoptotic (Annexin V+) cells compared with TRAIL or either agent alone at 24 h (Fig. 4C). Small but significantly higher levels of necrotic (Annexin V−/PI+) cells were observed in TRAIL + EGTA-treated cells compared with TRAIL or EGTA alone (Fig. 4C). BAPTA and EGTA enhanced Tg-induced apoptosis, while had no significant effect on Tg-induced necrotic cell death. TRAIL toxicity, as well as TRAIL sensitization by the Ca2+-chelators, became pronounced as the incubation period was prolonged. As a result, TRAIL treatment for 72 h substantially decreased the viability of A2058 and SAOS-2 cells (56.4 and 54.8% reduction, respectively), while EGTA treatment alone reduced them moderately (30 and 32.2% reduction). When used together, TRAIL and EGTA considerably decreased cell viability (maximum of 90%) (Fig. 4D and E). The TRAIL cytotoxicity was entirely blocked by the pan-caspase-inhibitor z-VAD-FMK, while necrostatin-1, a specific inhibitor of necroptosis, had only a modest inhibitory effect, indicating that the TRAIL primarily induces apoptosis in these cells. Collectively, these results show that Ca2+ removal sensitizes MM and OS cells to TRAIL-induced apoptosis, although the effect varied considerably depending on the cell line tested.

Ca2+ removal sensitizes MM and OS cells to TRAIL-induced non-apoptotic cell death

The ability of TRAIL to kill MM and OS cells varied considerably in different experiments. Under certain conditions, TRAIL had the minimal cytotoxic effect toward SAOS-2 and HOS cells (Fig. 5A and B). These cells were resistant to the cytotoxic and TRAIL-sensitizing effects of the Ca2+ chelators. As a result, TRAIL and chelator alone or in combination had the minimal cytotoxic effect except for that TRAIL + EGTA significantly reduced the viability of SAOS-2 cells (43.6% reduction). Also, z-VAD-FMK did not inhibit the effect of TRAIL + EGTA. Treatment with Ru360 (5–30 μM) for 24 h had the minimal cytotoxic and TRAIL-sensitizing effect (not shown). However, during another 48 h, Ru360 alone significantly decreased the viability of SAOS-2 and HOS cells (Fig. 5C and D). When Ru360 and TRAIL applied together, only a small increase of cell killing was observed compared with that induced by Ru360 alone. The cell death induced by Ru360 (not shown) or TRAIL + Ru360 was enhanced rather than inhibited by z-VAD-FMK (Fig. 5C and D). Although 5 μM CysA substantially decreased the viability of SAOS-2 cells, but not HOS cells, this cytotoxic effect was entirely counteracted by TRAIL. Moreover, atractyloside also enhanced TRAIL cytotoxicity in these apoptosis-resistant cells (Fig. 5E). On the other hand, consistent with our previous study with A375 cells (26), H2O2 markedly sensitized the cells to TRAIL cytotoxicity, and this effect was completely blocked by z-VAD-FMK, indicating that H2O2 amplifies TRAIL-induced apoptosis. These results show that agents that reduce [Ca2+]mit with different mechanisms of action sensitize these cells to TRAIL-induced non-apoptotic cell death.

Figure 5 The potentiation of TRAIL cytotoxicity by Ca2+ removal is caspase-independent. SAOS-2 (A, C and E) and HOS cells (B, D and F) were treated with 100 ng/ml TRAIL and 30 μM BAPTA, and 0.5 mM EGTA (A and B), 30 μM ruthenium 360 (Ru360) and 1 μM cyclosporin A (CysA) (C and D), 30 μM BAPTA and 5 μM atractyloside (ATR) (E), or 100 μM H2O2 (F) alone or in combination in the presence or absence of 10 μM z-VAD-FMK (ZVAD) for 72 h, and analyzed for viability using WST-8 assay in triplicates. The data show means ± SD in a representative experiment (N=3). Data were analyzed by ANOVA followed by the Tukey's post-hoc test. **P<0.01; ***P<0.001 vs. non-treated control (NT).

Capsazepine and AMG9810 kill or sensitize MM and OS cells in a caspase-independent manner

To further explore the possible relationship between mitochondrial Ca2+ removal and TRAIL sensitization, we assessed the impact of capsazepine and AMG9810 (3–30 μM) alone or in combination with TRAIL on tumor cell survival. Both AMG9810 and capsazepine had a minimal cytotoxic effect for 24 h (not shown). Treatment with AMG9810 at concentrations of ≥3 μM for 72 h reduced the viability of A2058 cells and at concentrations of ≥10 μM potentiated TRAIL cytotoxicity toward them in a dose-dependent manner (Fig. 6A). SAOS-2 cells were more resistant to AMG9810 treatment so that only the highest concentration of the agent exhibited substantial cytotoxic effect and enhanced TRAIL cytotoxicity (Fig. 6C). The effect of capsazepine seemed to be complicated and dependent on the cell lines tested. Capsazepine (10 μM) was more cytotoxic and more efficient in potentiating TRAIL cytotoxicity than 30 μM capsazepine in A2058 cells (Fig. 6B) while exhibiting no significant cytotoxicity nor TRAIL-sensitizing effect in SAOS-2 cells (Fig. 6D). Usually, HOS cells were highly resistant to TRAIL and AMG9810 alone or in combination, and the cytotoxic effect of TRAIL + AMG9810 was significantly augmented by z-VAD-FMK (Fig. 6E). On the other hand, capsazepine alone decreased their viability remarkably (82.9% reduction), and the effect was comparable to that of TRAIL + capsazepine. z-VAD-FMK also enhanced the cytotoxic effect of TRAIL + AMG9810 in SAOS-2 cells (Fig. 6F).

Figure 6 AMG9810 and capsazepine kill and sensitize MM and OS cells to a caspase-independent cell death. A2058 (A and B) and SAOS-2 cells (C and D) were treated with 100 ng/ml TRAIL and AMG9810 (A and C) or capsazepine (B and D) at the indicated concentrations alone or in combination for 72 h. HOS (E) and SAOS-2 cells (F) were treated with 100 ng/ml TRAIL and 10 μM each of AMG9810 or capsazepine alone or in combination in the presence or absence of 10 μM z-VAD-FMK (ZVAD) for 72 h. Then, the cells were analyzed for viability using WST-8 assay in triplicates. The data show means ± SD for a representative experiment (N=3). Data were analyzed by ANOVA followed by the Tukey's post-hoc test. **P<0.01; ***P<0.001.

Capsazepine and AMG9810 initially amplify TRAIL-induced caspase-3/7 activation and cell membrane damage in MM and OS cells

The results presented so far suggested that capsazepine and AMG9810 potentiate TRAIL cytotoxicity in a caspase-independent manner. To determine whether these two agents indeed modulate cell death independently of apoptosis, we examined their effect on caspase-3/7 activation. We simultaneously assessed caspase-3/7 activation and cell membrane damage/death by using a caspase-3/7-specific substrate and 7-AAD, respectively. The latter is a nucleus-staining dye, which is excluded by healthy cells, while it can penetrate cell membranes of dying or dead cells. Results showed that in A375 cells, capsazepine and AMG9810 alone minimally increased caspase-3/7 activated (caspase+) cells and damaged (7-AAD+) cells at 24 h (Fig. 7A). TRAIL treatment modestly increased both caspase+/7-AAD− and caspase+/7-AAD+ cells in A375 cells, and z-VAD-FMK blocked this effect. Capsazepine, and to a lesser extent, AMG9810 potentiated the effect of TRAIL, and z-VAD-FMK also abrogated the amplification (Fig. 7A). Necrostatin-1 inhibited the effect of TRAIL only modestly, while reducing the increase in caspase+/7-AAD− cells, but not caspase+/7-AAD+ cells by TRAIL + AMG9810. Strikingly, necrostain-1 enhanced the increase in caspase+/7-AAD+ cells by TRAIL + capsazepine (Fig. 7A). On the other hand, in HOS cells, AMG9810 was more potent than capsazepine in potentiating the effect of TRAIL, and z-VAD-FMK blocked the effect of capsazepine and AMG9810 (Fig. 7B). Unlike A375 cells, necrostatin-1 alone moderately increased caspase+/7-AAD+ cells while blunting the rise in such cell population by TRAIL, TRAIL + AMG9810, or TRAIL + capsazepine (Fig. 7B). These results indicate that capsazepine and AMG9810 initially amplify TRAIL-induced caspase-3/7 activation, cell membrane damage, and caspase-dependent cell death depending on the cell types.

Figure 7 AMG9810 and capsazepine potentiate TRAIL-induced caspase-3/7 activation and cell membrane damage in the early stage. A375 (A) and HOS cells (B) (1×105/ml) in 24-well plates were treated with 100 ng/ml TRAIL, 30 μM each of AMG9810 and capsazepine alone or in combination the absence or presence of 10 μM z-VAD-FMK (ZVAD) or 30 μM necrostatin-1 (Nec) for 24 h. Then, the cells were stained with a caspase-3/7 reagent NucView™ and 7-amino-actinomycin D (7-AAD) and analyzed for caspase-3/7 activation, membrane integrity, and cell death using Muse™ Cell Analyzer. Four populations of cells can be distinguished by this method; Live cells: caspase-3/7 (CASP)−7-AAD−; early apoptotic cells: CASP+/7-AAD−; late apoptotic/dead cells: CASP+/7-AAD+; necrotic cells: CASP−/7-AAD+. The data show means ± SD for a representative experiment (N=3).

Discussion

In the present study, we analyzed the effect of TRAIL on Ca2+ dynamics in MM and OS cells and the possible role of Ca2+ in the control of their survival and TRAIL sensitivity. Our results revealed that acute TRAIL treatment modulates Ca2+ remodeling in an array of MM and OS cell lines, as indicated by a rapid and persistent increase in [Ca2+]cyt and [Ca2+]mit (Fig. 1A–D). In parallel with its cytotoxicity, TRAIL increased Ca2+ levels at the two intracellular sites in a dose-dependent manner. The mitochondria take up or release Ca2+ depending on [Ca2+]cyt, thereby serving as a critical intracellular Ca2+ reservoir that maintains [Ca2+]cyt. According to this paradigm, the increases in [Ca2+]cyt and [Ca2+]mit may occur in parallel. Strikingly, however, the rise in [Ca2+]cyt was usually dose-dependent while depending on the cellular conditions, the elevation in [Ca2+]mit was maximum at 50 ng/ml, and higher concentrations of TRAIL had a smaller effect. These findings indicate that the mitochondrial Ca2+ responses involve both [Ca2+]cyt-dependent and [Ca2+]cyt-independent processes. Mitochondrial Ca2+ homeostasis is maintained by a well-balanced mitochondrial Ca2+ uptake and efflux. The MCU complex consists of the channel-forming subunit of the uniporter MCU and multiple components such as MICU1/2, MCUb, MCUR1, and EMRE. These complex molecules are proven to be an essential mitochondrial Ca2+ uptake machinery in different cell types including cancer cells (26–28).

The mitochondrion releases Ca2+ through several different pathways including NCLX, Ca2+/H+ antiporter (29) and MPTP (30–33). However, the role of MPTP is still a matter of debate, because other observations suggest its minimal contribution to mitochondrial Ca2+ extrusion (34). In this study, we showed that the MCU inhibitor Ru360 decreased [Ca2+]mit, while the NCLX antagonist CGP-37157 increased it in MM and OS cells (Fig. 2A and B). The results indicate that Ca2+ uptake through MCU and Ca2+ extrusion through NCLX are key regulators of [Ca2+]mit in our cell systems. Cyclosporine A, which targets cyclophilin D, a critical component of the MPTP opening (32,33), affected [Ca2+]mit in some but not all cell types. Whereas, atractyloside, which opens MPTP by modulating adenine nucleotide translocator (34), reduced [Ca2+]mit in different cell types (Fig. 2C and D). These findings suggest that Ca2+ extrusion through MPTP is also necessary for the control of [Ca2+]mit, yet cyclophilin D plays a dispensable role in the MPTP opening in our cell systems as previously reported by other groups (35,36).

It is noteworthy that capsazepine and AMG9810 markedly reduce [Ca2+]mit and potentiate TRAIL-induced drop in [Ca2+]mit in MM and OS cells (Fig. 3A–D). These findings indicate that a Ca2+ transport pathway sensitive to these agents plays a pivotal role in regulating [Ca2+]mit in them. The two agents are known to act as potent antagonists of TRPV1 (37,38), a molecule which localizes to the plasma membrane and serves as a non-selective cation channel. Recently, TRPV1 was shown to also exist in the ER and mitochondria in non-transformed cells and cancer cells. The intracellular TRPV1 contributes to Ca2+ release and ER stress (39–41). These facts suggest a close functional relationship among this channel, ER, and mitochondria in the regulation of Ca2+ signaling and survival of cancer cells. It is now widely accepted that ROS and Ca2+ mutually regulate one another and cooperatively control cell survival and death (13). Several groups, including us, have previously demonstrated that ROS plays a critical role in TRAIL cytotoxicity toward different malignant cell types (42–44).

Moreover, TRPV1 is one of TRP channels that are activated by ROS (45). Collectively, TRPV1 might play a role in the regulation of Ca2+ dynamics, survival, and death of MM and OS cells. However, to date, the role of TRPV1 in the control of Ca2+ in MM and OS is poorly documented. Mergler et al (46) reported the expression of TRPV1 in human uveal melanoma cells and Ca2+ regulation by it. The TRPV1 agonist capsaicin is shown to induce an increase in [Ca2+]mit in G292 human OS cells independently of the extracellular Ca2+ and depletion of intracellular Ca2+ (47). Since the extracellular Ca2+ entry appears to be dispensable for increasing [Ca2+]mit (Fig. 2A and B), an intracellular TRPV1 might play a role in the regulation of [Ca2+]mit homeostasis. However, at present, we failed to detect any TRPV1 in the intracellular sites in these cells (data not shown). Thus, the occurrence and the role of TRPV1 remain to be studied.

Another significant finding in this study was that depletion of Ca2+ potentiated TRAIL cytotoxicity toward MM and OS cells (Figs. 4 and 5). The finding strongly suggests that Ca2+ protects them from cell death. Both intracellular and extracellular Ca2+ seemed to play a role in this pro-survival function while the position of the two Ca2+ varied depending on cell lines. Depletion of Ca2+ enhanced apoptotic, but not necrotic cell death induced by TRAIL and Tg (Fig. 4C), indicating that Ca2+ primarily prevents apoptosis. It is noteworthy that the effect of Ca2+ removal was more pronounced in TRAIL-sensitive cells than in TRAIL-resistant cells (compare Fig. 4 with Fig. 5), and that z-VAD-FMK blocked the effect in the TRAIL-sensitive cells, but not in TRAIL-resistant cells. Collectively, MM and OS cells may each have distinct cellular statuse with different TRAIL sensitivity. One is relatively TRAIL-sensitive status, where they readily undergo apoptosis in response to TRAIL. The other is TRAIL-resistant status where another non-apoptotic cell death is necessary for efficient cell killing because only a small cell population undergo apoptosis. Ca2+ removal also potentiated TRAIL-induced non-apoptotic cell death (Fig. 5), indicating that Ca2+ also prevents this cell death modality.

The data presented in this study revealed the critical role of mitochondrial Ca2+ in the prevention of cell death. We found that the reduction in [Ca2+]mit by inhibiting Ca2+ uptake through MCU sensitized MM and OS cells to TRAIL-induced non-apoptotic cell death (Fig. 5). The finding is consistent with several recent studies in other cancer cell types. Curry and colleagues (27) reported that MCU silencing potentiates caspase-independent cell death in MDA-MB-231 breast cancer cells. The authors demonstrated that caspase-independent cell death induced by the Ca2+-ionophore ionomycin is potentiated by MCU silencing whereas caspase-dependent cell death caused by Bcl-2 inhibition is unaffected. Moreover, the potentiation of caspase-independent cell death occurs independently of overall [Ca2+]cyt changes.

Marchi et al (48) reported that the in silico microRNA miR-25 downregulates MCU expression and reduces mitochondrial Ca2+ uptake in HeLa cells and colon cancer cells and that this downregulation correlates with resistance to apoptotic stimuli. In this case, this MCU manipulation does not affect [Ca2+]cyt. Moreover, we found that the inhibition of a Ca2+ transport pathway by capsazepine and AMG9810 led to the decrease in [Ca2+]mit (Fig. 3) and sensitized MM and OS cells to TRAIL-induced non-apoptotic cell death. Strikingly, capsazepine and AMG9810 eventually amplified cell killing in a caspase-independent manner (Fig. 6) while initially (within 24 h) potentiated TRAIL-induced caspase-3/7 activation and apoptosis (Fig. 8). The reduction in [Ca2+]mit by promoting Ca2+ extrusion through MPTP also amplified TRAIL cytotoxicity (Fig. 5), providing further support for the pro-survival role of mitochondrial Ca2+. TRAIL was recently shown to induce necroptosis, the programmed necrotic cell death (10,11). However, the Ca2+ modulation had the minimal effect on necrosis (Fig. 4) and necrostatin-1, a specific inhibitor of necroptosis, had no or only a modest inhibitory effect on the cell death induced by TRAIL (Fig. 4), TRAIL + capsazepine, and TRAIL + AMG9810 (Fig. 7). These findings suggest that necroptosis plays a minor role in the cell killing yet the silencing of an essential molecular component in this cell death modality such as receptor-interacting protein 1/3 may be necessary to verify this view.

In conclusion, we demonstrate in this study that mitochondrial Ca2+ acts as a pro-survival factor in MM and OS cells by preventing apoptosis and non-apoptotic cell death. The findings suggest that mitochondrial Ca2+ may serve as a promising target for overcoming the resistance of these cancers to TRAIL.

Acknowledgments

The authors thank Drs T. Ito, T. Tokunaga, and A. Onoe for their technical assistance. This work was supported in part by JSPS KAKENHI grant no. 15K09750 to Y.S-K.).

References