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.

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 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.

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 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⁺.

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.

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).

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.

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.

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).

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.

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.

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).

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.