Nitric oxide-dependent cell death in glioblastoma and squamous cell carcinoma via prodeath mitochondrial clustering.

Besides the fission-fusion dynamics, the cellular distribution of mitochondria has recently emerged as a critical biological parameter in regulating mitochondrial function and cell survival. We previously found that mitochondrial clustering on the nuclear periphery, or monopolar perinuclear mitochondrial clustering (MPMC), accompanies the anticancer activity of air plasma-activated medium (APAM) against glioblastoma and human squamous cell carcinoma, which is closely associated with oxidant-dependent tubulin remodeling and mitochondrial fragmentation. Accordingly, this study investigated the regulatory roles of nitric oxide (NO) in the anticancer activity of APAM. Time-lapse analysis revealed a time-dependent increase in NO accompanied by MPMC. In contrast, APAM caused minimal increases in MPMC and NO levels in nontransformed cells. NO, hydroxyl radicals, and lipid peroxide levels increased near the damaged nuclear periphery, possibly within mitochondria. NO scavenging prevented tubulin remodeling, MPMC, perinuclear oxidant production, nuclear damage, and cell death. Conversely, synthetic NO donors augmented all the prodeath events and acted synergistically with APAM. Salinomycin, an emerging drug against multidrug-resistant cancers, had similar NO-dependent effects. These results suggest that APAM and salinomycin induce NO-dependent cell death, where MPMC and oxidative mitochondria play critical roles. Our findings encourage further investigations on MPMC as a potential target for NO-driven anticancer agents against drug-resistant cancers.

Ozone mediates the anticancer effect of air plasma by triggering oxidative cell death caused by H2O2 and iron.

Cold atmospheric plasmas and plasma-treated solutions (PTSs) have emerged as promising approaches in cancer treatment because of their tumor-selective actions. While oxidative stress is critical for their effects, the precise mechanisms, including chemical mediators, remain obscure. Previously, we reported that air plasma-activated medium (APAM) exhibited tumor-selective anticancer activity. The fragmentation of mitochondria and their asymmetrical assembly around the peripheral regions of the damaged nucleus, namely, monopolar perinuclear mitochondrial clustering (MPMC), proceed to the effect. Subsequently, we found that APAM had a substantial amount of O3 in addition to hydrogen peroxide (H2O2), nitrile (NO2-), and nitrate (NO3-). In the present study, we investigated the possible role of O3 in the anticancer effect. For this purpose, we created a nitrogen oxide-free ozonated medium ODM. ODM exhibited potent cytotoxicity against various cancer but not nonmalignant cells. ODM also increased MPMC, hydroxyl radicals, lipid peroxides, and their shifts to perinuclear sites in cancer cells. Catalase and iron chelation prevented these events and cytotoxicity. ODM also decreases the intracellular labile irons while increasing those within mitochondria. ODM had substantial H2O2, but this oxidant failed to cause MPMC and cytotoxicity. These results show that ODM can mimic the effects of APAM, including MPMC and tumor-selective anticancer effects. The findings suggest that O3 is critical in mediating the anticancer effects of APAM by triggering oxidative cell death caused by H2O2 and iron.

Air Plasma-Activated Medium Evokes a Death-Associated Perinuclear Mitochondrial Clustering.

Intractable cancers such as osteosarcoma (OS) and oral cancer (OC) are highly refractory, recurrent, and metastatic once developed, and their prognosis is still disappointing. Tumor-targeted therapy, which eliminates cancers effectively and safely, is the current clinical choice. Since aggressive tumors are substantially resistant to multidisciplinary therapies that target apoptosis, tumor-specific activation of another cell death modality is a promising avenue for meeting this goal. Here, we report that a cold atmospheric air plasma-activated medium (APAM) can kill OS and OC by causing a unique mitochondrial clustering. This event was named monopolar perinuclear mitochondrial clustering (MPMC) based on its characteristic unipolar mitochondrial perinuclear accumulation. The APAM caused apoptotic and nonapoptotic cell death. The APAM increased mitochondrial ROS (mROS) and cell death, and the antioxidants such as N-acetylcysteine (NAC) prevented them. MPMC occurred following mitochondrial fragmentation, which coincided with nuclear damages. MPMC was accompanied by mitochondrial lipid peroxide (mLPO) accumulation and prevented by NAC, Ferrostatin-1, and Nocodazole. In contrast, the APAM induced minimal cell death, mROS generation, mLPO accumulation, and MPMC in fibroblasts. These results suggest that MPMC occurs in a tumor-specific manner via mitochondrial oxidative stress and microtubule-driven mitochondrial motility. MPMC induction might serve as a promising target for exerting tumor-specific cytotoxicity.

Combined Anticancer Effect of Plasma-Activated Infusion and Salinomycin by Targeting Autophagy and Mitochondrial Morphology.

Non-thermal atmospheric pressure plasma (NTAPP)-activated liquids have emerged as new promising anticancer agents because they preferentially injure malignant cells. Here, we report plasma-activated infusion (PAI) as a novel NTAPP-based anti-neoplastic agent. PAI was prepared by irradiating helium NTAP to form a clinically approved infusion fluid. PAI dose-dependently killed malignant melanoma and osteosarcoma cell lines while showing much lower cytotoxic effects on dermal and lung fibroblasts. We found that PAI and salinomycin (Sal), an emerging anticancer stem cell agent, mutually operated as adjuvants. The combined administration of PAI and Sal was much more effective than single-agent application in reducing the growth and lung metastasis of osteosarcoma allografts with minimal adverse effects. Mechanistically, PAI explicitly induced necroptosis and increased the phosphorylation of receptor-interacting protein 1/3 rapidly and transiently. PAI also suppressed the ambient autophagic flux by activating the mammalian target of the rapamycin pathway. PAI increased the phosphorylation of Raptor, Rictor, and p70-S6 kinase, along with decreased LC3-I/II expression. In contrast, Sal promoted autophagy. Moreover, Sal exacerbated the mitochondrial network collapse caused by PAI, resulting in aberrant clustering of fragmented mitochondrial in a tumor-specific manner. Our findings suggest that combined administration of PAI and Sal is a promising approach for treating these apoptosis-resistant cancers.

Aspirin Induces Mitochondrial Ca2+ Remodeling in Tumor Cells via ROS‒Depolarization‒Voltage-Gated Ca2+ Entry.

Aspirin (acetylsalicylic acid) and its metabolite salicylate, have an anti-melanoma effect by evoking mitochondrial dysfunction through poorly understood mechanisms. Depolarization of the plasma membrane potential leads to voltage-gated Ca2+ entry (VGCE) and caspase-3 activation. In the present study, we investigated the role of depolarization and VGCE in aspirin's anti-melanoma effect. Aspirin and to a lesser extent, salicylate (≥2.5 mM) induced a rapid (within seconds) depolarization, while they caused comparable levels of depolarization with a lag of 2~4 h. Reactive oxygen species (ROS) generation also occurred in the two-time points, and antioxidants abolished the early ROS generation and depolarization. At the same concentrations, the two drugs induced apoptotic and necrotic cell death in a caspase-independent manner, and antioxidants and Ca2+ channel blockers prevented cell death. Besides ROS generation, reduced mitochondrial Ca2+ (Ca2+m) and mitochondrial membrane potential preceded cell death. Moreover, the cells expressed the Cav1.2 isoform of l-type Ca2+ channel, and knockdown of Cav1.2 abolished the decrease in Ca2+m. Our findings suggest that aspirin and salicylate induce Ca2+m remodeling, mitochondrial dysfunction, and cell death via ROS-dependent depolarization and VGCE activation.

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.

Autophagy inhibitors regulate TRAIL sensitivity in human malignant cells by targeting the mitochondrial network and calcium dynamics.

In a variety of cancer cell types, the pharmacological and genetic blockade of autophagy increases apoptosis induced by various anticancer drugs. These observations suggest that autophagy counteracts drug­induced apoptosis. We previously reported that in human melanoma and osteosarcoma cells, autophagy inhibitors, such as 3­methyladenine and chloroquine increased the sensitivity to apoptosis induced by tumor necrosis factor­related apoptosis­inducing ligand (TRAIL). In the present study, we report that different autophagy inhibitors regulate the mitochondrial network and calcium (Ca2+) dynamics in these cells. We found that compared to tumor cells, normal fibroblasts were more resistant to the cytotoxicity of TRAIL and autophagy inhibitors used either alone or in combination. Notably, TRAIL increased the autophagic flux in the tumor cells, but not in the fibroblasts. Live­cell imaging revealed that in tumor cells, TRAIL evoked modest mitochondrial fragmentation, while subtoxic concentrations of the autophagy inhibitors led to mitochondrial fusion. Co­treatment with TRAIL and subtoxic concentrations of the autophagy inhibitors resulted in severe mitochondrial fragmentation, swelling and clustering, similar to what was observed with autophagy inhibitors at toxic concentrations. The enhanced aberration of the mitochondrial network was preceded by a reduction in mitochondrial Ca2+ loading and store­operated Ca2+ entry. On the whole, the findings of this study indicate that co­treatment with TRAIL and autophagy inhibitors leads to increased mitochondrial Ca2+ and network dysfunction in a tumor­selective manner. Therefore, the co­administration of TRAIL and autophagy inhibitors may prove to be a promising tumor­targeting approach for the treatment of TRAIL­resistant cancer cells.

Cold PSM, but not TRAIL, triggers autophagic cell death: A therapeutic advantage of PSM over TRAIL.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and cold plasma-stimulated medium (PSM) are promising novel anticancer tools due to their strong anticancer activities and high tumor-selectivity. The present study demonstrated that PSM and TRAIL may trigger autophagy in human malignant melanoma and osteosarcoma cells. Live-cell imaging revealed that even under nutritional and stress-free conditions, these cells possessed a substantial level of autophagosomes, which were localized in the cytoplasm separately from tubular mitochondria. In response to cytotoxic levels of PSM, the mitochondria became highly fragmented, and aggregated and colocalized with the autophagosomes. The cytotoxic effects of PSM were suppressed in response to various pharmacological autophagy inhibitors, including 3-methyladenine (3-MA) and bafilomycin A1, thus indicating the induction of autophagic cell death (ACD). Lethal levels of PSM also resulted in non-apoptotic, non-autophagic cell death in a reactive oxygen species-dependent manner under certain circumstances. Furthermore, TRAIL exhibited only a modest cytotoxicity toward these tumor cells, and did not induce ACD and mitochondrial aberration. The combined use of TRAIL and subtoxic concentrations of 3-MA resulted in decreased basal autophagy, increased mitochondrial aberration, colocalization with autophagosomes and apoptosis. These results indicated that PSM may induce ACD, whereas TRAIL may trigger cytoprotective autophagy that compromises apoptosis. To the best of our knowledge, the present study is the first to demonstrate that PSM can induce ACD in human cancer cells. These findings provide a rationale for the advantage of PSM over TRAIL in the destruction of apoptosis-resistant melanoma and osteosarcoma cells.

Plasma-stimulated medium kills TRAIL-resistant human malignant cells by promoting caspase-independent cell death via membrane potential and calcium dynamics modulation.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and cold plasma-stimulated medium (PSM) have been shown to exhibit tumor-selective cytotoxicity and have emerged as promising new tools for cancer treatment. However, to date, at least to the best of our knowledge, no data are available as to which substance is more potent in killing cancer cells. Thus, in this study, we systematically compared their abilities to kill human malignant cells from different origins. We found that PSM dose-dependently killed TRAIL-resistant melanoma, osteosarcoma and neuroblastoma cells. Moreover, PSM had little cytotoxicity toward osteoblasts. PSM was more potent than TRAIL in inducing caspase-3/7 activation, mitochondrial network aberration and caspase-independent cell death. We also found that PSM was more potent in inducing plasma membrane depolarization (PMD) and disrupting endoplasmic-mitochondrial Ca2+ homeostasis. Moreover, persistent PMD was caused by different membrane-depolarizing agents; the use of the anti-type II diabetes drug, glibenclamide, alone caused mitochondrial fragmentation and enhanced TRAIL-induced Ca2+ modulation, mitochondrial network abnormalities and caspase-independent cell killing. These results demonstrate that PSM has a therapeutic advantage over TRAIL owing to its greater capacity to evoke caspase-independent cell death via mitochondrial network aberration by disrupting membrane potential and Ca2+ homeostasis. These findings may provide a strong rationale for developing PSM as a novel approach for the treatment of TRAIL-resistant malignant cells.

Disrupting mitochondrial Ca2+ homeostasis causes tumor-selective TRAIL sensitization through mitochondrial network abnormalities.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has emerged as a promising anticancer agent with high tumor-selective cytotoxicity. The congenital and acquired resistance of some cancer types including malignant melanoma and osteosarcoma impede the current TRAIL therapy of these cancers. Since fine tuning of the intracellular Ca2+ level is essential for cell function and survival, Ca2+ dynamics could be a promising target for cancer treatment. Recently, we demonstrated that mitochondrial Ca2+ removal increased TRAIL efficacy toward malignant melanoma and osteosarcoma cells. Here we report that mitochondrial Ca2+ overload leads to tumor-selective sensitization to TRAIL cytotoxicity. Treatment with the mitochondrial Na+/Ca2+ exchanger inhibitor CGP-37157 and oxidative phosphorylation inhibitor antimycin A and FCCP resulted in a rapid and persistent mitochondrial Ca2+ rise. These agents also increased TRAIL sensitivity in a tumor-selective manner with a switching from apoptosis to a nonapoptotic cell death. Moreover, we found that mitochondrial Ca2+ overload led to increased mitochondrial fragmentation, while mitochondrial Ca2+ removal resulted in mitochondrial hyperfusion. Regardless of their reciprocal actions on the mitochondrial dynamics, both interventions commonly exacerbated TRAIL-induced mitochondrial network abnormalities. These results expand our previous study and suggest that an appropriate level of mitochondrial Ca2+ is essential for maintaining the mitochondrial dynamics and the survival of these cells. Thus, disturbing mitochondrial Ca2+ homeostasis may serve as a promising approach to overcome the TRAIL resistance of these cancers with minimally compromising the tumor-selectivity.

Mitochondrial Ca2+ removal amplifies TRAIL cytotoxicity toward apoptosis-resistant tumor cells via promotion of multiple cell death modalities.

Ca2+ has emerged as a new target for cancer treatment since tumor-specific traits in Ca2+ dynamics contributes to tumorigenesis, malignant phenotypes, drug resistance, and survival in different tumor types. However, Ca2+ has a dual (pro-death and pro-survival) function in tumor cells depending on the experimental conditions. Therefore, it is necessary to minimize the onset of the pro-survival Ca2+ signals caused by the therapy. For this purpose, a better understanding of pro-survival Ca2+ pathways in cancer cells is critical. Here we report that Ca2+ protects malignant melanoma (MM) and osteosarcoma (OS) cells from tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) cytotoxicity. Simultaneous measurements using the site-specific Ca2+ probes showed that acute TRAIL treatment rapidly and dose-dependently increased the cytosolic Ca2+ concentration ([Ca2+]cyt) and mitochondrial Ca2+ concentration ([Ca2+]mit) Pharmacological analyses revealed that the [Ca2+]mit remodeling was under control of mitochondrial Ca2+ uniporter (MCU), mitochondrial permeability transition pore (MPTP), and a Ca2+ transport pathway sensitive to capsazepine and AMG9810. Ca2+ chelators and the MCU inhibitor ruthenium 360, an MPTP opener atractyloside, capsazepine, and AMG9810 all decreased [Ca2+]mit and sensitized these tumor cells to TRAIL cytotoxicity. The Ca2+ modulation enhanced both apoptotic and non-apoptotic cell death. Although the [Ca2+]mit reduction potentiated TRAIL-induced caspase-3/7 activation and cell membrane damage within 24 h, this potentiation of cell death became pronounced at 72 h, and not blocked by caspase inhibition. Our findings suggest that in MM and OS cells mitochondrial Ca2+ removal can promote apoptosis and non-apoptotic cell death induction by TRAIL. Therefore, mitochondrial Ca2+ removal can be exploited to overcome the resistance of these cancers to TRAIL.

Tumor-selective mitochondrial network collapse induced by atmospheric gas plasma-activated medium.

Non-thermal atmospheric gas plasma (AGP) exhibits cytotoxicity against malignant cells with minimal cytotoxicity toward normal cells. However, the mechanisms of its tumor-selective cytotoxicity remain unclear. Here we report that AGP-activated medium increases caspase-independent cell death and mitochondrial network collapse in a panel of human cancer cells, but not in non-transformed cells. AGP irradiation stimulated reactive oxygen species (ROS) generation in AGP-activated medium, and in turn the resulting stable ROS, most likely hydrogen peroxide (H2O2), activated intracellular ROS generation and mitochondrial ROS (mROS) accumulation. Culture in AGP-activated medium resulted in cell death and excessive mitochondrial fragmentation and clustering, and these responses were inhibited by ROS scavengers. AGP-activated medium also increased dynamin-related protein 1-dependent mitochondrial fission in a tumor-specific manner, and H2O2 administration showed similar effects. Moreover, the vulnerability of tumor cells to mitochondrial network collapse appeared to result from their higher sensitivity to mROS accumulation induced by AGP-activated medium or H2O2. The present findings expand our previous observations on death receptor-mediated tumor-selective cell killing and reinforce the importance of mitochondrial network remodeling as a powerful target for tumor-selective cancer treatment.

Distinct effects of TRAIL on the mitochondrial network in human cancer cells and normal cells: role of plasma membrane depolarization.

Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is a promising anticancer drug due to its tumor-selective cytotoxicity. Here we report that TRAIL exhibits distinct effects on the mitochondrial networks in malignant cells and normal cells. Live-cell imaging revealed that multiple human cancer cell lines and normal cells exhibited two different modes of mitochondrial responses in response to TRAIL and death receptor agonists. Mitochondria within tumor cells became fragmented into punctate and clustered in response to toxic stimuli. The mitochondrial fragmentation was observed at 4 h, then became more pronounced over time, and associated with apoptotic cell death. In contrast, mitochondria within normal cells such as melanocytes and fibroblasts became only modestly truncated, even when they were treated with toxic stimuli. Although TRAIL activated dynamin-related protein 1 (Drp1)-dependent mitochondrial fission, inhibition of this process by Drp1 knockdown or with the Drp1 inhibitor mdivi-1, potentiated TRAIL-induced apoptosis, mitochondrial fragmentation, and clustering. Moreover, mitochondrial reactive oxygen species (ROS)-mediated depolarization accelerated mitochondrial network abnormalities in tumor cells, but not in normal cells, and TRAIL caused higher levels of mitochondrial ROS accumulation and depolarization in malignant cells than in normal cells. Our findings suggest that tumor cells are more prone than normal cells to oxidative stress and depolarization, thereby being more vulnerable to mitochondrial network abnormalities and that this vulnerability may be relevant to the tumor-targeting killing by TRAIL.

Mitochondrial division inhibitor-1 induces mitochondrial hyperfusion and sensitizes human cancer cells to TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising candidate for cancer treatment, but some cancer cell types are resistant to TRAIL cytotoxicity. Therefore, overcoming this resistance is necessary for effective TRAIL therapy. Mitochondrial morphology is important for the maintenance of cell function and survival, and is regulated by the delicate balance between fission and fusion. However, the role of mitochondrial morphology dynamics in TRAIL-induced apoptosis is unknown. Here we show that mitochondrial division inhibitor-1 (mdivi-1), an inhibitor of dynamin-related protein1 (Drp1), modulates mitochondrial morphology and TRAIL-induced apoptosis in human cancer cells. mdivi-1 treatment (≥12.5 µM) caused dose- and time­dependent cell death in malignant melanoma, lung cancer and osteosarcoma cells, while sparing normal cells. mdivi-1 also sensitized cancer cells to TRAIL-induced apoptosis. This potentiation of apoptosis occurred through a caspase-depependent mechanism including the mitochondrial and endoplasmic reticulum (ER) stress pathways. Mdivi-1 potentiated mitochondrial oxidative stress, a major cause of mitochondrial and ER stresses, as evidenced by increases in mitochondrial reactive oxygen species levels, mitochondrial mass, and cardiolipin oxidation. Live cell fluorescence imaging using MitoTracker Red CMXRos revealed that Mdivi-1 caused substantial mitochondrial hyperfusion. Moreover, silencing of Drp1 expression also caused mitochondrial hyperfusion and sensitized cancer cells to TRAIL-induced apoptosis. Our results suggest that cancer cells are more vulnerable than normal cells to a perturbation in mitochondrial morphology dynamics and that this higher susceptibility can be exploited to selectively kill cancer cells and sensitize to TRAIL.

Depolarization Controls TRAIL-Sensitization and Tumor-Selective Killing of Cancer Cells: Crosstalk with ROS.

Conventional genotoxic anti-cancer drugs target the proliferative advantage of tumor cells over normal cells. This kind of approach lacks the selectivity of treatment to cancer cells, because most of the targeted pathways are essential for the survival of normal cells. As a result, traditional cancer treatments are often limited by undesirable damage to normal cells (side-effects). Ideal anti-cancer drugs are expected to be highly effective against malignant tumor cells with minimal cytotoxicity toward normal cells. Such selective killing can be achieved by targeting pathways essential for the survival of cancer cells, but not normal cells. As cancer cells are characterized by their resistance to apoptosis, selective apoptosis induction is a promising approach for selective killing of cancer cells. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising tumor-selective anti-cancer drug. However, the congenital and acquired resistance of some cancer cell types, including malignant melanoma cells, currently impedes effective TRAIL therapy, and an innovative approach that can override TRAIL resistance is urgently required. Apoptosis is characterized by cell shrinkage caused by disruption of the maintenance of the normal physiological concentrations of K(+) and Na(+) and intracellular ion homeostasis. The disrupted ion homeostasis leads to depolarization and apoptosis. Recent evidence suggests that depolarization is an early and prerequisite event during TRAIL-induced apoptosis. Moreover, diverse natural products and synthetic chemicals capable of depolarizing the cell membrane exhibit tumor-selective killing and TRAIL-sensitizing effects. Here, we discuss the role of depolarization in selective killing of cancer cells in connection with the emerging concept that oxidative stress is a critical mediator of mitochondrial and endoplasmic reticulum dysfunctions and serves as a tumor-selective target in cancer treatment.

Crosstalk between mitochondrial ROS and depolarization in the potentiation of TRAIL-induced apoptosis in human tumor cells.

We previously showed that membrane-depolarizing agents such as K+ and ATP-sensitive potassium (KATP) channel inhibitors potentiate tumor necrosis factor-related apoptosis­inducing ligand (TRAIL)-induced apoptosis in human melanoma cells, but not in normal melanocytes. In this study, we investigated whether the tumor-selective effect of depolarization was observed among different tumor cell types and the mechanisms by which depolarization potentiates death pathways. We found that K+ and KATP channel inhibitors elicited similar apoptosis-potentiating effects in human tumor cells with different origins, including leukemia, melanoma and lung cancer cells. In contrast, minimal potentiation of apoptosis was observed in non-transformed lung cells. The potentiation was associated with increased mitochondrial and endoplasmic reticulum stress death pathways. Upregulation of surface TRAIL receptor-2 expression and modulation of the caspase-3 activation pathway seemed to play roles in the enhancement of death signaling. Moreover, the results showed that depolarization and mitochondria­derived reactive oxygen species (mROS) mutually regulated one another. Depolarization potentiated TRAIL-induced mROS accumulation. Conversely, scavenging of mROS by the antioxidant MnTBaP reduced depolarization, whereas mROS accumulation caused by metabolic inhibitors potentiated the depolarization. These findings suggest a positive loop between depolarization and mROS accumulation. This may provide a rationale for the tumor-selective cytotoxicity and/or potentiation of TRAIL cytotoxicity of a wide variety of ROS-producing substances in different types of tumor cells.

Mitochondrial superoxide mediates mitochondrial and endoplasmic reticulum dysfunctions in TRAIL-induced apoptosis in Jurkat cells.

Reactive oxygen species (ROS), such as superoxide (O2(•-)) and hydrogen peroxide (H2O2), have been reported to be important mediators of the apoptosis induced by death ligands, including Fas, tumor necrosis factor-α, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Conversely, there is evidence that H2O2 and prooxidative conditions are protective. Therefore, the roles of ROS in death ligand-induced apoptosis are a matter of debate. In this study, we attempted to define the oxidant species mediating TRAIL-induced apoptosis in human tumor cells. The generation of intracellular O2(•-), but not H2O2, was correlated with apoptosis in the cells. TRAIL treatment resulted in increased mitochondrial O2(•-) generation and the oxidation of cardiolipin. The O2(•-)-selective scavenger MnTBaP [Mn(III) tetrakis (4-benzoic acid) porphyrin chloride] specifically blocked TRAIL-induced apoptosis and proapoptotic events including mitochondrial membrane collapse and caspase-3/7 activation. TRAIL also induced endoplasmic reticulum (ER) stress responses including caspase-12 activation, while inhibition of caspase-12 prevented the apoptosis. In addition, increased mitochondrial O2(•-) generation by uncoupling of oxidative phosphorylation or inhibition of the electron transport chain amplified the TRAIL-induced apoptosis and proapoptotic events. This amplification was also significantly abolished by MnTBaP treatment. Our data indicate that mitochondrial O2(•-) mediates mitochondrial and ER dysfunctions during TRAIL-induced apoptosis in Jurkat cells. The present findings suggest that pharmacological agents increasing mitochondrial O2(•-) may serve as clinical drugs that amplify TRAIL effectiveness toward cancer cells.

Hydrogen peroxide induces cell death in human TRAIL-resistant melanoma through intracellular superoxide generation.

Intracellular reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O2()) are thought to mediate apoptosis induced by death receptor ligands, including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). However, the role of H(2)O(2) is controversial, since some evidence suggests that H(2)O(2) acts as an anti-apoptotic factor. Here, we show that exogenously applied H(2)O(2) (30-100 µM) induces cell death in TRAIL-resistant human melanoma cells via intracellular superoxide (O(2)-) generation. H(2)O(2) induced apoptotic or necrotic cell death, depending on the concentration of the oxidant applied; low concentrations of H(2)O(2) preferentially activated the caspase-dependent apoptotic pathway, while high concentrations of H(2)O(2) induced apoptotic and necrotic cell death in a caspase-independent manner. The H(2)O(2)-induced cell death was associated with increased mitochondrial membrane potential collapse and caspase-3/7 activation and ER stress responses including caspase-12 and X-box-binding protein-1 (XBP-1) activation. H(2)O(2) induced intracellular O2- generation even within the mitochondria, while TRAIL did not. The superoxide dismutase mimetic antioxidant MnTBaP [Mn (III) tetrakis (4-benzonic acid) porphyrin chloride] inhibited the H(2)O(2)-induced O(2)- generation, apoptosis and XBP-1 and caspase-12 activation at comparable concentrations. Importantly, H(2)O(2) treatment caused minimal O(2)- generation and apoptosis in normal primary melanocytes. These data show that H(2)O(2) induces endoplasmic reticulum-associated cell death via intracellular O(2)- generation and that malignant melanoma cells are more susceptible than normal cells to this oxidative cell death. The findings suggest that H(2)O(2) has therapeutic potential in the treatment of TRAIL-resistant melanoma.

Diallyl trisulfide sensitizes human melanoma cells to TRAIL-induced cell death by promoting endoplasmic reticulum-mediated apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is promising for cancer treatment because of its selective cytotoxicity toward tumor cells. However, some cancer cell types including malignant melanoma cells are resistant to TRAIL cytotoxicity. Here, we show that diallyl trisulfide (DATS), a garlic organosulfur compound, sensitizes melanoma cells to TRAIL-induced apoptosis while sparing normal cells. DATS also potentiates apoptosis induced by agonistic antibodies against death receptors (DR) 4 and DR5. The amplification of DR-mediated apoptosis was associated with increased mitochondrial membrane potential collapse and caspase-3/7 activation. However, these events were not sufficient for full sensitization. TRAIL also induced endoplasmic reticulum (ER) stress, as indicated by the activation of X-box-binding protein 1 and caspase-12 and DATS poten-tiated both events. Moreover, inhibition of caspase-12, but not caspase-4, abolished the amplification of apoptosis, indicating that ER stress plays a crucial role. On the other hand, DATS and/or TRAIL induced minimal apoptosis and caspase-12 activation in melanocytes despite their substantial expression of DR4 and DR5 on the cell surface. Our data suggest that DATS amplifies death ligand-induced melanoma cell death by disrupting their adaptation to ER-mediated death pathway. The present findings raise the possibility that DATS may be combined with death ligands to treat TRAIL-resistance melanoma cells without impairing its tumor selectivity.