The Mitochondrial Ca2+ Overload via Voltage-Gated Ca2+ Entry Contributes to an Anti-Melanoma Effect of Diallyl Trisulfide

Allium vegetables such as garlic (Allium sativum L.) are rich in organosulfur compounds that prevent human chronic diseases, including cancer. Of these, diallyl trisulfide (DATS) exhibits anticancer effects against a variety of tumors, including malignant melanoma. Although previous studies have shown that DATS increases intracellular calcium (Ca2+) in different cancer cell types, the role of Ca2+ in the anticancer effect is obscure. In the present study, we investigated the Ca2+ pathways involved in the anti-melanoma effect. We used melittin, the bee venom that can activate a store-operated Ca2+ entry (SOCE) and apoptosis, as a reference. DATS increased apoptosis in human melanoma cell lines in a Ca2+-dependent manner. It also induced mitochondrial Ca2+ (Ca2+mit) overload through intracellular and extracellular Ca2+ fluxes independently of SOCE. Strikingly, acidification augmented Ca2+mit overload, and Ca2+ channel blockers reduced the effect more significantly under acidic pH conditions. On the contrary, acidification mitigated SOCE and Ca2+mit overload caused by melittin. Finally, Ca2+ channel blockers entirely inhibited the anti-melanoma effect of DATS. Our findings suggest that DATS explicitly evokes Ca2+mit overload via a non-SOCE, thereby displaying the anti-melanoma effect.


Introduction
Allium vegetables such as garlic (Allium sativum L.) are rich in allyl sulfides that have been shown to prevent human chronic diseases, including cancer [1]. L-alliin (S-allyl-l-cysteine sulfoxide) is the major allyl sulfide component in garlic, which is converted into 2-propensulfenic acid by the endogenous enzyme alliinase, thereby producing the unstable thiosulfinate compound allicin (S-allyl-2-prop-2-ene-thiosulfinate). Allicin readily breaks down to form organosulfur compounds (OSCs), including allyl sulfides such as diallyl trisulfide (DATS), diallyl disulfide (DADS), and diallyl sulfide (DAS), as well as ajoene and vinyl dithiins [2]. OSCs can modulate the immune system and attenuate inflammation, and these effects contribute to cancer prevention [3]. DATS, DADS, and DAS are known to prevent the well-characterized chemical-induced skin carcinogenesis [4][5][6]. Moreover, the OSCs have anticancer activity in a variety of cancer cell types, including skin cancer such as melanoma and basal cell carcinoma [7]. Of these, DATS is the most widely studied. This compound induces apoptosis in a range of cancer cells, including malignant melanoma, osteosarcoma, and leukemia [8][9][10][11].
V and PI staining revealed that 72-h-treatment with DATS (100 μM) alone resulted in a massive increase in apoptotic (annexin V-positive) cells. TRAIL markedly augmented the effect while the pancaspase inhibitor Z-VAD-FMK entirely blocked it ( Figure 1C,D). We found that Ca 2+ was a critical regulator of drug sensitivity. Treatment with the extracellular Ca 2+ chelator EGTA (≤0.5 mM) or the intracellular Ca 2+ chelator BAPTA had minimal effect on cell viability. However, these chelators significantly reduced the anticancer effect of DATS in A375 and A2058 cells ( Figure 1E,F).

Melittin Exhibits Anti-Melanoma Effect in a Ca 2+ -Dependent Manner
Melittin is known to be a potent inducer of apoptosis in melanoma cells. Consistent with this view, treatment with the compound (≥2.5 µg/mL) for 72 h resulted in a robust increase in apoptotic (annexin V-positive) cells in A375 cells ( Figure 2A). Meanwhile, the treatment minimally increased necrotic (annexin V-negative) cells. The extracellular Ca 2+ removal by EGTA (0.5 mM) augmented the effect of the subtoxic dose (1 µg/mL) of melittin. On the other hand, it mitigated the increase in apoptosis while enhancing the increase in necrosis caused by the toxic concentration (5 µg/mL) of melittin ( Figure 2B). analyzed for viability using the WST-8 assay. *** p < 0.01 vs. untreated control. # p < 0.05; ### p < 0.001 vs. DATS alone. Data represent the mean ± SD (n = 3-6).

Melittin Exhibits Anti-Melanoma Effect in a Ca 2+ -Dependent Manner
Melittin is known to be a potent inducer of apoptosis in melanoma cells. Consistent with this view, treatment with the compound (≥2.5 μg/mL) for 72 h resulted in a robust increase in apoptotic (annexin V-positive) cells in A375 cells ( Figure 2A). Meanwhile, the treatment minimally increased necrotic (annexin V-negative) cells. The extracellular Ca 2+ removal by EGTA (0.5 mM) augmented the effect of the subtoxic dose (1 μg/mL) of melittin. On the other hand, it mitigated the increase in apoptosis while enhancing the increase in necrosis caused by the toxic concentration (5 μg/mL) of melittin ( Figure 2B).

DATS Increases [Ca 2+ ]mit without Increasing [Ca 2+ ]cyt
Next, we determined whether DATS affected the intracellular Ca 2+ level. First, we tested the effect on [Ca 2+ ]cyt. We used the Ca 2+ -ATPase inhibitor, thapsigargin (Tg), as a positive control, because it depletes the ER Ca 2+ stores, thereby stimulating SOCE. Tg substantially increased [Ca 2+ ]cyt, while DATS at the concentration of up to 200 μM minimally increased it ( Figure 3A,B). On the other hand, DATS increased [Ca 2+ ]mit in a dose-dependent manner ( Figure 3C,D). This increase occurred immediately, developed over time, and the effect of DATS (200 μM) was even higher than that induced by the positive control A23187 (5 μM).

DATS Increases [Ca 2+ ] mit without Increasing [Ca 2+ ] cyt
Next, we determined whether DATS affected the intracellular Ca 2+ level. First, we tested the effect on [Ca 2+ ] cyt . We used the Ca 2+ -ATPase inhibitor, thapsigargin (Tg), as a positive control, because it depletes the ER Ca 2+ stores, thereby stimulating SOCE. Tg substantially increased [Ca 2+ ] cyt , while DATS at the concentration of up to 200 µM minimally increased it ( Figure 3A,B). On the other hand, DATS increased [Ca 2+ ] mit in a dose-dependent manner ( Figure 3C,D). This increase occurred immediately, developed over time, and the effect of DATS (200 µM) was even higher than that induced by the positive control A23187 (5 µM).

Melittin Exhibits Anti-Melanoma Effect in a Ca 2+ -Dependent Manner
Melittin is known to be a potent inducer of apoptosis in melanoma cells. Consistent with this view, treatment with the compound (≥2.5 μg/mL) for 72 h resulted in a robust increase in apoptotic (annexin V-positive) cells in A375 cells ( Figure 2A). Meanwhile, the treatment minimally increased necrotic (annexin V-negative) cells. The extracellular Ca 2+ removal by EGTA (0.5 mM) augmented the effect of the subtoxic dose (1 μg/mL) of melittin. On the other hand, it mitigated the increase in apoptosis while enhancing the increase in necrosis caused by the toxic concentration (5 μg/mL) of melittin ( Figure 2B).

DATS Increases [Ca 2+ ]mit without Increasing [Ca 2+ ]cyt
Next, we determined whether DATS affected the intracellular Ca 2+ level. First, we tested the effect on [Ca 2+ ]cyt. We used the Ca 2+ -ATPase inhibitor, thapsigargin (Tg), as a positive control, because it depletes the ER Ca 2+ stores, thereby stimulating SOCE. Tg substantially increased [Ca 2+ ]cyt, while DATS at the concentration of up to 200 μM minimally increased it ( Figure 3A,B). On the other hand, DATS increased [Ca 2+ ]mit in a dose-dependent manner ( Figure 3C,D). This increase occurred immediately, developed over time, and the effect of DATS (200 μM) was even higher than that induced by the positive control A23187 (5 μM).

Melittin Increases Both [Ca 2+ ] cyt and [Ca 2+ ] mit in an Extracellular Ca 2+ -Dependent Manner
The site-specific Ca 2+ measurements showed that melittin (1.3-5 µg/mL) increased both [Ca 2+ ] cyt and [Ca 2+ ] mit in a dose-dependent manner ( Figure 4A,C). Removal of the extracellular Ca 2+ entirely abolished the increase in [Ca 2+ ] cyt but partially reduced the increase in [Ca 2+ ] mit , indicating that these effects depend on the extracellular Ca 2+ influx to different degrees ( Figure 4B,D).

Acidification Has a Reciprocal Effect on Ca 2+ mit Overload Caused by DATS and Melittin
The microenvironments, including the extracellular pH conditions, contribute to the various aspects of malignant phenotypes of cancer cells [27,28]. Therefore, we examined the effect of altered extracellular pH on Ca 2+ mit overload. Cells were suspended in HBSS with different pH 6.8 and 7.4, treated with DATS, and measured for the intracellular Ca 2+ levels. Notably, after DATS treatment, a small but significant increase in [Ca 2+ ] cyt was seen at pH 6.8, but not pH 7.4 ( Figure 5A,B). Similarly, a greater extent of Ca 2+ mit overload occurred at pH 6.8 compared to pH 7.4, and this impact was more pronounced for the higher concentration of the compound ( Figure 5C,D). The effect was apparent for higher concentrations of DATS. Next, we analyzed the effect of the extracellular Ca 2+ removal on Ca 2+ mit overload seen under different pH. Cells were suspended in HBSS (pH 7.4 or 6.8) containing CaCl 2 or EGTA and treated with DATS. At pH 7.4, the Ca 2+ removal almost entirely abolished the increase in [Ca 2+ ] mit , while at pH 6.8, it reduced the increase only a modestly ( Figure 5E,F). In contrast to DATS, acidification significantly mitigated Ca 2+ mit overload caused by melittin. A much smaller increase in [Ca 2+ ] mit occurred at pH 6.8 compared to at pH 7.4 in response to a range of concentrations of melittin (1.3-5 µg/mL). Figure 5G shows the data for melittin (2.5 µg/mL). Notably, we observed a comparable level of [Ca 2+ ] mit increase in the cells placed in HBSS (pH 7.4) and Ca 2+ -containing buffer. The [Ca 2+ ] mit rise seen in HBSS (pH 6.8) and Ca 2+ -free buffer (pH 7.4) after melittin treatment were also comparable ( Figure 5G,H).

Melittin Increases Both [Ca 2+ ]cyt and [Ca 2+ ]mit in an Extracellular Ca 2+ -Dependent Manner
The site-specific Ca 2+ measurements showed that melittin (1.3-5 μg/mL) increased both [Ca 2+ ]cyt and [Ca 2+ ]mit in a dose-dependent manner ( Figure 4A,C). Removal of the extracellular Ca 2+ entirely abolished the increase in [Ca 2+ ]cyt but partially reduced the increase in [Ca 2+ ]mit, indicating that these effects depend on the extracellular Ca 2+ influx to different degrees ( Figure 4B,D).

Acidification Mitigates SOCE
The data presented so far indicated that acidification reduced Ca 2+ mitt overload caused by melittin. Since melittin is known as a potent activator of SOCE, we determined the effect of acidification on SOCE. We activated SOCE by the Ca 2+ depletion-Ca 2+ re-addition technique and examined the impact of acidification on the Ca 2+ entry. At neutral extracellular pH, the addition of Tg to cells in a Ca 2+ -free medium resulted in a small and transient increase in [Ca 2+ ] cyt , indicating the Ca 2+ release from the ER. The re-addition of Ca 2+ to Tg-treated cells led to a higher and persistent increase in [Ca 2+ ] cyt , and the inositol-1,4,5-triphosphate receptor (IP 3 R)/SOCE antagonists 2-aminoethoxydiphenyl borate (2-APB) entirely blocked this Ca 2+ response, verifying the onset of SOCE ( Figure 6A). Even without any Ca 2+ depletion in the ER (i.e., in the absence of Tg), a smaller but significant [Ca 2+ ] cyt rise was seen following Ca 2+ re-addition and was minimally affected by 2-APB ( Figure 6A, Right), indicating the onset of a store-independent Ca 2+ influx (non-SOCE). Meanwhile, at pH 6.8, no substantial SOCE was observed, while the non-SOCE was increased by the acidification ( Figure 6B). increase in [Ca 2+ ]mit occurred at pH 6.8 compared to at pH 7.4 in response to a range of concentrations of melittin (1.3-5 μg/mL). Figure 5G shows the data for melittin (2.5 μg/mL). Notably, we observed a comparable level of [Ca 2+ ]mit increase in the cells placed in HBSS (pH 7.4) and Ca 2+ -containing buffer. The [Ca 2+ ]mit rise seen in HBSS (pH 6.8) and Ca 2+ -free buffer (pH 7.4) after melittin treatment were also comparable ( Figure 5G,H).

Acidification Mitigates SOCE
The data presented so far indicated that acidification reduced Ca 2+ mitt overload caused by melittin. Since melittin is known as a potent activator of SOCE, we determined the effect of acidification on SOCE. We activated SOCE by the Ca 2+ depletion-Ca 2+ re-addition technique and aminoethoxydiphenyl borate (2-APB) entirely blocked this Ca 2+ response, verifying the onset of SOCE ( Figure 6A). Even without any Ca 2+ depletion in the ER (i.e., in the absence of Tg), a smaller but significant [Ca 2+ ]cyt rise was seen following Ca 2+ re-addition and was minimally affected by 2-APB ( Figure 6A, Right), indicating the onset of a store-independent Ca 2+ influx (non-SOCE). Meanwhile, at pH 6.8, no substantial SOCE was observed, while the non-SOCE was increased by the acidification ( Figure 6B). , added with thapsigargin (Tg, 2 μM, red arrow) and incubated for 10 min to deplete intracellular Ca 2+ stores. Then, two mM Ca 2+ was added to the cells (SOCE). After the addition of Tg, the fluorescence was monitored with excitation and emission at 485 and 538 nm, respectively. For validating SOCE, the cells in the Ca 2+ -free buffer were treated as described above in the presence of 2-APB (10 μM). For non-SOCE measurement, the cells in the Ca 2+ -free buffer were treated with the medium and then added with 2 mM Ca 2+ (non-SOCE). Note that at pH 7.4, a higher increase in [Ca 2+ ]cyt was seen in the SOCE trace compared to the non-SOCE trace, and this effect was entirely abolished by 2-APB. Meanwhile, no such effect was seen at pH 6.8.

Ca 2+ Channel Blockers Inhibit Ca 2+ mit Overload and Cell Death Caused by DATS
To gain insight into the transport pathway(s) involved Ca 2+ mitt overload caused by DATS, we look for Ca 2+ channel antagonists affecting the [Ca 2+ ]mit rise. Eventually, we found that the Ca 2+ channel blockers nifedipine, verapamil, and diltiazem significantly inhibited the effect of DATS. The inhibitory effect was seen more pronouncedly at pH 6.8 than at pH 7.4 ( Figure 7A). Under the acidic conditions, nifedipine exhibited the most potent effect (70% reduction), while the other compounds inhibited it around 50% ( Figure 7A). 2-APB had an inhibitory effect comparable to verapamil and diltiazem. Meanwhile, at the neutral pH conditions, these inhibitors mitigated the [Ca 2+ ]mit rise only modestly (20%; Figure 7A). Next, we examined the effect of Ca 2+ blockers on the anti-melanoma effect in highly growing cells. Nifedipine, verapamil, and diltiazem significantly inhibited the antimelanoma effect of DATS (200 μM; Figure 7B). Figure 6. Acidification mitigates store-operated Ca 2+ entry (SOCE). Fluo4-AM-loaded A375 cells were suspended in a Ca 2+ -free HBSS supplemented with 1 mM EGTA at pH 7.4 (A) or pH 6.8 (B), added with thapsigargin (Tg, 2 µM, red arrow) and incubated for 10 min to deplete intracellular Ca 2+ stores. Then, two mM Ca 2+ was added to the cells (SOCE). After the addition of Tg, the fluorescence was monitored with excitation and emission at 485 and 538 nm, respectively. For validating SOCE, the cells in the Ca 2+ -free buffer were treated as described above in the presence of 2-APB (10 µM). For non-SOCE measurement, the cells in the Ca 2+ -free buffer were treated with the medium and then added with 2 mM Ca 2+ (non-SOCE). Note that at pH 7.4, a higher increase in [Ca 2+ ] cyt was seen in the SOCE trace compared to the non-SOCE trace, and this effect was entirely abolished by 2-APB. Meanwhile, no such effect was seen at pH 6.8.

Ca 2+ Channel Blockers Inhibit Ca 2+ mit Overload and Cell Death Caused by DATS
To gain insight into the transport pathway(s) involved Ca 2+ mitt overload caused by DATS, we look for Ca 2+ channel antagonists affecting the [Ca 2+ ] mit rise. Eventually, we found that the Ca 2+ channel blockers nifedipine, verapamil, and diltiazem significantly inhibited the effect of DATS. The inhibitory effect was seen more pronouncedly at pH 6.8 than at pH 7.4 ( Figure 7A). Under the acidic conditions, nifedipine exhibited the most potent effect (70% reduction), while the other compounds inhibited it around 50% ( Figure 7A). 2-APB had an inhibitory effect comparable to verapamil and diltiazem. Meanwhile, at the neutral pH conditions, these inhibitors mitigated the [Ca 2+ ] mit rise only modestly (20%; Figure 7A). Next, we examined the effect of Ca 2+ blockers on the anti-melanoma effect in highly growing cells. Nifedipine, verapamil, and diltiazem significantly inhibited the anti-melanoma effect of DATS (200 µM; Figure 7B).
conditions, nifedipine exhibited the most potent effect (70% reduction), while the other compounds inhibited it around 50% ( Figure 7A). 2-APB had an inhibitory effect comparable to verapamil and diltiazem. Meanwhile, at the neutral pH conditions, these inhibitors mitigated the [Ca 2+ ]mit rise only modestly (20%; Figure 7A). Next, we examined the effect of Ca 2+ blockers on the anti-melanoma effect in highly growing cells. Nifedipine, verapamil, and diltiazem significantly inhibited the antimelanoma effect of DATS (200 μM; Figure 7B).

Discussion
In this study, we elucidated the possible role of Ca 2+ dysregulation in the anti-melanoma effect of DATS with a particular interest in the Ca 2+ pathways involved. We found that DATS induced caspase-dependent apoptosis in melanoma cells, although relatively high concentrations of DATS are required (Figure 1). These results are consistent with previous studies, including our own, which demonstrate that DATS induces apoptosis in a variety of cancer cell types [8][9][10][11]14]. We noticed that cell confluency considerably affected the sensitivity to the compound. Cells growing near confluent tended to be more susceptible than those growing at low confluency. In agreement with the previous reports by others [23,24], melittin also primarily induced apoptosis in the cells (Figure 2A). Moreover, we found that extracellular Ca 2+ entry was necessary for apoptosis caused by DATS and melittin ( Figures 1E,F and 2B). The Ca 2+ mit uptake is generally due to so-called reservoir function of the organelles: the mitochondria take up Ca 2+ (Figure 3), suggesting that the Ca 2+ mit overload is a result of the activation of a specific Ca 2+ transport pathway rather than a simple Ca 2+ mit uptake. Meanwhile, melittin markedly increased [Ca 2+ ] cyt and [Ca 2+ ] mit ( Figure 4). However, extracellular Ca 2+ removal entirely abolished the former, while attenuated the latter partially ( Figure 4B,D). These results suggest that the [Ca 2+ ] cyt rise primarily results from extracellular Ca 2+ entry while the [Ca 2+ ] mit increase is due to Ca 2+ transport of both extracellular and intracellular Ca 2+ . Dysregulation of [Ca 2+ ] mit is a master cause of cell death. A fine-tuned increase in [Ca 2+ ] mit supports energy metabolism, cell activation, and cell survival, while Ca 2+ mitt overload can damage mitochondrial integrity, thereby inducing mitochondrial permeability transition pore (MPTP) opening and the resulting release of apoptogenic proteins [28][29][30]. Collectively, we conclude that different Ca 2+ transport pathways participate in the Ca 2+ mitt overload and apoptosis caused by DATS and melittin (Figure 8). and acidification caused a similar degree of reduction, suggesting that extracellular Ca 2+ entry is specifically sensitive to acidification, as observed with SOCE. Together, our findings strongly suggest that melittin evokes Ca 2+ mit overload via SOCE activation. This view is consistent with other reports demonstrating the requirement of SOCE for active Ca 2+ mit uptake [47,48]. However, further studies, including the examination of the involvement of SOCE components such as STIM1 and ORAI1, are necessary for validation. Figure 8. A schematic summary of the present study. Both DATS and melittin induce mitochondrial Ca 2+ overload and apoptosis in melanoma cells. However, different pathways seem to contribute to the effects, depending on the apoptotic stimuli and the extracellular pH. Specifically, at neutral pH, the extracellular Ca 2+ entry is predominant. Melittin primarily uses store-operated entry (SOCE), while DATS mainly utilizes yet unknown extracellular Ca 2+ entry pathway possibly including voltage-gated Ca 2+ entry (VGCE). Under acidic pH, SOCE is mitigated. In turn, intracellular Ca 2+ entry plays a substantial role in the effects of melittin and DATS. Increased Ca 2+ transport from the intracellular stores (e.g., the endoplasmic reticulum) to the mitochondria and decreased mitochondrial Ca 2+ extrusion could contribute to the event. Under the acidic conditions, DATS, but not melittin, seems to utilize VGCE as the primary route for Ca 2+ entry, leading to mitochondrial Ca 2+ overload and apoptosis. The VGCE could exacerbate SOCE inactivation, and Ca 2+ dysregulation due to the loss of the negative membrane potential, the driving force transporting extracellular Ca 2+ into inner cells through SOCE.
Another important finding in the present study is that Ca 2+ channel blockers significantly mitigate Ca 2+ mit overload and the anti-melanoma effect of DATS at acidic pH ( Figure 7). These findings suggest a role of VGCE in Ca 2+ mit overload and apoptosis. Notably, acidification increased Ca 2+ mit overload caused by DATS ( Figure 5), and sensitivity to Ca 2+ channel blockers was higher at acidic pH than at neutral pH ( Figure 7A). The latter finding seems to provide another evidence for the role of VGCE because Kato and colleagues have shown that an acidic pH (5.4-6.5) increases Ca 2+ influx in mouse B16 melanoma cells through VGCE [49]. The activation of VGCE requires plasma membrane depolarization, which leads to the loss of the negative membrane potential, the driving force transporting extracellular Ca 2+ into inner cells. Accordingly, SOCE could be compromised under these conditions. Thus, acidification activates VGCE while mitigates SOCE, thereby possibly playing a vital role in switching the Ca 2+ entry pathway from SOCE to VGCE. The microenvironments of tumor cells are acidic due to an H + efflux caused by increased vacuolar ATPase activity, and this Figure 8. A schematic summary of the present study. Both DATS and melittin induce mitochondrial Ca 2+ overload and apoptosis in melanoma cells. However, different pathways seem to contribute to the effects, depending on the apoptotic stimuli and the extracellular pH. Specifically, at neutral pH, the extracellular Ca 2+ entry is predominant. Melittin primarily uses store-operated entry (SOCE), while DATS mainly utilizes yet unknown extracellular Ca 2+ entry pathway possibly including voltage-gated Ca 2+ entry (VGCE). Under acidic pH, SOCE is mitigated. In turn, intracellular Ca 2+ entry plays a substantial role in the effects of melittin and DATS. Increased Ca 2+ transport from the intracellular stores (e.g., the endoplasmic reticulum) to the mitochondria and decreased mitochondrial Ca 2+ extrusion could contribute to the event. Under the acidic conditions, DATS, but not melittin, seems to utilize VGCE as the primary route for Ca 2+ entry, leading to mitochondrial Ca 2+ overload and apoptosis. The VGCE could exacerbate SOCE inactivation, and Ca 2+ dysregulation due to the loss of the negative membrane potential, the driving force transporting extracellular Ca 2+ into inner cells through SOCE.
Strikingly, DATS seems to evoke Ca 2+ mit overload through different Ca 2+ pathways, depending on the extracellular pH. At pH 7.4, DATS exhibits the effect mainly via the extracellular Ca 2+ entry ( Figure 5E), while at pH 6.8, DATS seemed to use both extracellular and intracellular Ca 2 entry pathways ( Figure 5F). Ca 2+ released from the IP 3 R can easily reach the mitochondrial matrix via the IP 3 R-voltage-dependent anion channel (VDAC1)-mitochondrial Ca 2+ uniporter (MCU) pathway [31,32]. The ER is the primary mechanism for regulating [Ca 2+ ] mit and tethered to mitochondria via a mitochondria-associated membrane; the physical association allows rapid Ca 2+ transport through specified microdomains [33,34]. The IP 3 R physically links to VDAC1 on the outer mitochondrial membrane and transports Ca 2+ into the mitochondrial matrix [35,36]. We previously showed that Ca 2+ mit remodeling in osteosarcoma cells was under the control of Ca 2+ uptake through the MCU [37]. [Ca 2+ ] mit is also regulated by Ca 2+ mit efflux through several different pathways, including the mitochondrial Na + /Ca 2+ exchanger (NCLX), Ca 2+ /H + antiporter, and MPTP [38][39][40][41][42]. Notably, our previous study showed that the NCLX inhibitor CGP-37157 causes Ca 2+ mit overload and apoptosis in osteosarcoma cells [43]. Therefore, increased Ca 2+ transport via the IP 3 R-VDAC1-MCU pathway and decreased Ca 2+ mitt extrusion could contribute to the Ca 2+ mitt overload caused by DATS. SOCE is the most ubiquitous pathway for Ca 2+ transport from the extracellular space and activated following the depletion of Ca 2+ stores from the ER. Recently, SOCE has emerged as critical machinery for Ca 2+ influx, contributing to various malignant phenotypes in cancer cells [44][45][46]. SOCE is also shown to be responsible for cisplatin-induced apoptosis [20]. It is noteworthy that melittin can activate iPLA 2 to produce arachidonic acid and lysophospholipids and activates SOCE in vascular smooth muscle cells [25,26]. Given that melittin activates SOCE in melanoma cells, too, this action can expect to cause massive Ca 2+ dysregulation, as cisplatin does. A notable property of SOCE is its sensitivity to acidification; SOCE is suppressed by decreasing extracellular pH in several cell types [44,45]. In agreement with these reports, acidification entirely abolished SOCE in melanoma cells ( Figure 6). Notably, the Ca 2+ mit overload induced by melittin was mainly dependent upon extracellular Ca 2+ entry and was suppressed by acidification ( Figure 5). Extracellular Ca 2+ removal and acidification caused a similar degree of reduction, suggesting that extracellular Ca 2+ entry is specifically sensitive to acidification, as observed with SOCE. Together, our findings strongly suggest that melittin evokes Ca 2+ mit overload via SOCE activation. This view is consistent with other reports demonstrating the requirement of SOCE for active Ca 2+ mit uptake [47,48]. However, further studies, including the examination of the involvement of SOCE components such as STIM1 and ORAI1, are necessary for validation.
Another important finding in the present study is that Ca 2+ channel blockers significantly mitigate Ca 2+ mit overload and the anti-melanoma effect of DATS at acidic pH ( Figure 7). These findings suggest a role of VGCE in Ca 2+ mit overload and apoptosis. Notably, acidification increased Ca 2+ mit overload caused by DATS (Figure 5), and sensitivity to Ca 2+ channel blockers was higher at acidic pH than at neutral pH ( Figure 7A). The latter finding seems to provide another evidence for the role of VGCE because Kato and colleagues have shown that an acidic pH (5.4-6.5) increases Ca 2+ influx in mouse B16 melanoma cells through VGCE [49]. The activation of VGCE requires plasma membrane depolarization, which leads to the loss of the negative membrane potential, the driving force transporting extracellular Ca 2+ into inner cells. Accordingly, SOCE could be compromised under these conditions. Thus, acidification activates VGCE while mitigates SOCE, thereby possibly playing a vital role in switching the Ca 2+ entry pathway from SOCE to VGCE. The microenvironments of tumor cells are acidic due to an H + efflux caused by increased vacuolar ATPase activity, and this acidification causes multiple malignant phenotypes, including increased proliferation, drug resistance, and metastasis [27,28]. Therefore, our observations that high confluent cells are relatively high responders of DATS could be explained by the predominant role of VGCE under acidic extracellular microenvironments, because the extracellular pH drops in cells growing at high density.
There remain issues to be resolved in future studies. In particular, the identification of voltage-gated Ca 2+ channels (VGCCs) involved in Ca 2+ mit overload caused by DATS is challenging. Our preliminary experiments revealed that melanoma cells expressed Ca v 1.2 and Ca v 1.3 isoform of l-type VGCCs. Notably, the previous studies demonstrate the occurrence of the coordinate control of STIM1-ORAI1 and Ca v 1.2 [50,51]. Therefore, there is an intriguing possibility that Ca v 1.2 plays a role in Ca 2+ mit overload via a specific interaction with SOCE. Further studies are necessary to clarify the molecular entity of VGCCs involved and to test this hypothesis.
In conclusion, the present study demonstrated that DATS and melittin exhibit the anti-melanoma effects by evoking pro-apoptotic Ca 2+ mit overload through the different pathways, possibly VGCE and SOCE. Our findings suggest the involvement of multiple Ca 2+ entry pathways in Ca 2+ mit remodeling in melanoma cells and switching between them, depending on apoptotic stimuli and pH microenvironments.

Materials
All chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) unless otherwise specified. Alliin and allicin were obtained from Cayman Chemical Co. (Ann Arbor, MI, USA). Soluble recombinant human TRAIL was obtained from Enzo Life Sciences (San Diego, CA, USA). The pan-caspase inhibitor Z-VAD-FMK was purchased from Merck Millipore (Darmstadt, Germany). All insoluble reagents were dissolved in dimethylsulfoxide (DMSO) and diluted with high glucose-containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) or Hank's balanced salt

Statistical Analysis
Data are presented as mean ± standard deviation (SD) or standard error (SE) and were analyzed by one-way analysis of variance followed by Tukey's post hoc test using add-in software for Excel 2016 for Windows (SSRI, Tokyo, Japan). p < 0.05 was considered statistically significant.