Synergic actions of botulinum neurotoxin A and oxaliplatin on colorectal tumour cell death through the upregulation of TRPM2 channel-mediated oxidative stress.

Botulinum neurotoxin A (BoNT) is being shown to have anticancer action as a potential adjuvant treatment. The transient receptor potential (TRP) melastatin 2 (TRPM2) stimulator action of BoNT was reported in glioblastoma cells, but not in colorectal cancer (HT29) cells. By activating TRPM2, we evaluated the impacts of BoNT and oxaliplatin (OXA) incubations on oxidant and apoptotic values within the HT29 cells. Control, BoNT (5 IU for 24 h), OXA (50 µM for 24 h) and their combinations were induced. We found that TRPM2 protein is upregulated and mediates enhanced BoNT and OXA-induced Ca2+ entry in cells as compared to control cells. The increase of free reactive oxygen species (ROS), but the decrease of glutathione is the main ROS responsible for TRPM2 activation on H29 exposure to oxidative stress. BoNT and OXA-mediated Ca2+ entry through TRPM2 stimulation in response to H2 O2 results in mitochondrial Ca2+ overload, followed by mitochondrial membrane depolarization, apoptosis and caspase-3/-8/-9, although they were diminished in the TRPM2 antagonist groups (N-(p-amylcinnamoyl)anthranilic acid and carvacrol). In conclusion, by increasing the susceptibility of HT29 tumour cells to oxidative stress and apoptosis, the combined administration of BoNT and OXA via the targeting of TRPM2 may offer a different approach to kill the tumour cells.

Curcumin attenuates hydroxychloroquine-mediated apoptosis and oxidative stress via the inhibition of TRPM2 channel signalling pathways in a retinal pigment epithelium cell line.

PURPOSE: Hydroxychloroquine (HCQ) is used in the treatment of several diseases, such as malaria, Sjögren's disease, Covid-19, and rheumatoid arthritis. However, HCQ induces retinal pigment epithelium death via the excessive increase of cytosolic (cROS) and mitochondrial (mROS) free oxygen radical production. The transient receptor potential melastatin 2 (TRPM2) cation channel is stimulated by ADP-ribose (ADPR), cROS, and mROS, although it is inhibited by curcumin (CRC). We aimed to investigate the modulating action of CRC on HCQ-induced TRPM2 stimulation, cROS, mROS, apoptosis, and death in an adult retinal pigment epithelial 19 (ARPE19) cell line model. MATERIAL AND METHODS: ARPE19 cells were divided into four groups: control (CNT), CRC (5 µM for 24 h), HCQ (60 µM for 48 h), and CRC + HCQ groups. RESULTS: The levels of cell death (propidium iodide positive cell numbers), apoptosis markers (caspases -3, -8, and -9), oxidative stress (cROS and mROS), mitochondria membrane depolarization, TRPM2 current density, and intracellular free Ca2+ and Zn2+ fluorescence intensity were upregulated in the HCQ group after stimulation with hydrogen peroxide and ADPR, but their levels were downregulated by treatments with CRC and TRPM2 blockers (ACA and carvacrol). The HCQ-induced decrease in retinal live cell count and cell viability was counteracted by treatment with CRC. CONCLUSION: HCQ-mediated overload Ca2+ influx and retinal oxidative toxicity were induced in an ARPE19 cell line through the stimulation of TRPM2, although they were attenuated by treatment with CRC. Hence, CRC may be a potential therapeutic antioxidant for TRPM2 activation and HCQ treatment-induced retinal oxidative injury and apoptosis.

NMDA Receptor Activation Stimulates Hypoxia-Induced TRPM2 Channel Activation, Mitochondrial Oxidative Stress, and Apoptosis in Neuronal Cell Line: Modular Role of Memantine.

TRPM2 channel is activated by the increase of hypoxia (HYP)-mediated excessive mitochondrial (mROS) and cytosolic (cROS) free reactive oxygen species generation and intracellular free Ca2+ ([Ca2+]i) influx. The stimulations of the N-methyl-d-aspartate(NMDA) receptor and TRPM2 channel induce mROS and apoptosis in the neurons, although their inhibitions via the treatments of memantine (MEM) and MK-801 decrease mROS and apoptosis. However, the molecular mechanisms underlying MEM treatment and NMDA inhibition' neuroprotection via TRPM2 inhibition in the HYP remain elusive. We investigated the modulator role of MEM and NMDA via the modulation of TRPM2 on oxidative neurodegeneration and apoptosis in SH-SY5Y neuronal cells. Six groups were induced in the SH-SY5Y and HEK293 cells as follows: Control, MEM, NMDA blocker (MK-801), HYP (CoCl2), HYP + MEM, and HYP + MK-801. The HYP caused to the increases of TRPM2 and PARP-1 expressions, and TRPM2 agonist (H2O2 and ADP-ribose)-induced TRPM2 current density and [Ca2+]i concentration via the upregulation of mitochondrial membrane potential, cROS, and mROS generations. The alterations were not observed in the absence of TRPM2 in the HEK293 cells. The increase of cROS, mROS, lipid peroxidation, cell death (propidium iodide/Hoechst) rate, apoptosis, caspase -3, caspase -8, and caspase -9 were restored via upregulation of glutathione and glutathione peroxidase by the treatments of TRPM2 antagonists (ACA or 2-APB), MEM, and MK-801. In conclusion, the inhibition of NMDA receptor via MEM treatment modulated HYP-mediated mROS, apoptosis, and TRPM2-induced excessive [Ca2+]i and may provide an avenue for protecting HYP-mediated neurodegenerative diseases associated with the increase of mROS, [Ca2+]i, and apoptosis.

TRPM2 Channel Inhibition Attenuates Amyloid ß42-Induced Apoptosis and Oxidative Stress in the Hippocampus of Mice.

Alzheimer's disease (AD) is characterized by the increase of hippocampal Ca2+ influx-induced apoptosis and mitochondrial oxidative stress (OS). The OS is a stimulator of TRPM2, although N-(p-amylcinnamoyl)anthranilic acid (ACA), 2-aminoethyl diphenylborinate (2/APB), and glutathione (GSH) are non-specific antagonists of TRPM2. In the present study, we investigated the protective roles of GSH and TRPM2 antagonist treatments on the amyloid ß42 peptide (Aß)-caused oxidative neurotoxicity and apoptosis in the hippocampus of mice with AD model. After the isolation of hippocampal neurons from the newborn mice, they were divided into five incubation groups as follows: control, ACA, Aß, Aß+ACA, and Aß+GSH. The levels of apoptosis, hippocampus death, cytosolic ROS, cytosolic Zn2+, mitochondrial ROS, caspase-3, caspase-9, lipid peroxidation, and cytosolic Ca2+ were increased in the primary hippocampus cultures by treatments of Aß, although their levels were decreased in the neurons by the treatments of GSH, PARP-1 inhibitors (PJ34 and DPQ), and TRPM2 blockers (ACA and 2/APB). The Aß-induced decreases of cell viability, cytosolic GSH, reduced GSH, and GSH peroxidase levels were also increased in the groups of Aß+ACA and Aß+GSH by the treatments of ACA and GSH. However, the Aß-caused changes were not observed in the hippocampus of TRPM2-knockout mice. In conclusion, the present data demonstrate that maintaining the activation of TRPM2 is not only important for the quenching OS and neurotoxicity in the hippocampal neurons of mice with experimental AD but also equally critical to the modulation of Aß-induced apoptosis. The possible positive effects of GSH and TRPM2 antagonist treatments on the amyloid-beta (Aß)-induced oxidative toxicity in the hippocampus of mice. The ADP-ribose (ADPR) is produced via the stimulation of PARP-1 in the nucleus of neurons. The NUT9 in the C terminus of TRPM2 channel acts as a key role for the activation of TRPM2. The antagonists of TRPM2 are glutathione (GSH), ACA, and 2/APB in the hippocampus. The Aß incubation-mediated TRPM2 stimulation increases the concentration of cytosolic-free Ca2+ and Zn2+ in the hippocampus. In turn, the increased concentration causes the increase of mitochondrial membrane potential (ΔΨm), which causes the excessive generations of mitochondria ROS and the decrease of cytosolic GSH and GSH peroxidase (GSH-Px). The ROS production and GSH depletion are two main causes in the neurobiology of Alzheimer's disease. However, the effect of Aß was not shown in the hippocampus of TRPM2-knockout mice. The Aß and TRPM2 stimulation-caused overload Ca2+ entry cause apoptosis and cell death via the activations of caspase-3 (Casp/3) and caspase-9 (Casp/9) in the hippocampus. The actions of Aß-induced oxidative toxicity were modulated in the primary hippocampus by the incubations of ACA, GSH, 2/APB, and PARP-1 inhibitors (PJ34 and DPQ). (↑) Increase. (↓) Decrease.

Honey bee venom melittin increases the oxidant activity of cisplatin and kills human glioblastoma cells by stimulating the TRPM2 channel.

Melittin (MLT) treatment is believed to enhance tumor cell death, apoptotic, and oxidative cytotoxic effects of cisplatin (CSP) via the modulation of Ca2+ channels in several cancer lines. The activation of TRPM2 mediated anticancer and CSP resistance actions via mitochondrial Ca2+ and Zn2+ accumulation-induced mitochondrial reactive free oxygen species (MitSOX) in the glioblastoma cells. The aim was to elucidate the effects of CSP and MLT combination via the TRPM2 stimulation on the tumor cell viability, cell number, cell death (propidium iodide/Hoechst rate), apoptosis, and MitSOX levels in the DBTRG-05MG cells. In the DBTRG-05MG cells, we induced four groups as control, MLT (2.5 µg/ml for 24 h), CSP (25 µM for 24 h), and CSP + MLT. The CSP-induced intracellular Ca2+ influxes to the TRPM2 activation were increased in the cells from coming H2O2 and ADP-Ribose. The influxes were decreased in the cells by the incubations of TRPM2 antagonists (ACA and carvacrol). The incubation of CSP increased the parameters of intracellular Ca2+ responses, mitochondria function, cytosolic free Zn2+ accumulation, apoptosis (caspase -3, -8, and -9), and MitSOX generation in the tumor cells. After the treatment of MLT with/without CSP, the parameters were further increased in the cells. In conclusion, the treatment of MLT increased the anticancer, tumor cell death, apoptotic, and oxidant effects of CSP in the glioblastoma tumor cells via activating the TRPM2. As a result, TRPM2 stimulation by MLT may be utilized as a successful agent in the CSP treatment of glioblastoma tumors.

Transient receptor potential channel stimulation induced oxidative stress and apoptosis in the colon of mice with colitis-associated colon cancer: modulator role of Sambucus ebulus L.

BACKGROUND: Increased Ca2+ entry causes an increase in tumor cell proliferation, apoptosis, cytosolic reactive free oxygen species (cyROS), and mitochondrial ROS (miROS) in tumor cells. The cyROS and miROS stimulate the cation channels, including the TRPA1, TRPM2, and TRPV1. Sambucus ebulus L (SEB) (Dwarf Elder) induced both antioxidant and anticancer effects in the human hepatocarcinoma and human colon carcinoma cancer cell lines. We investigated the etiology of colorectal cancer and the impact of three channels, as well as the protective effects of SEB on apoptosis, cyROS, and miROS in the colon of mice with colitis-associated colon cancer (AOM/DSS). METHODS: A total 28 mice were equally divided into four groups as control, SEB (100 mg/kg/day for 14 days), AOM/DSS, and SEB + AOM/DSS. Azoxymethane/dextran sulfate sodium-induced colon cancer associated with colitis was induced in the AOM/DSS groups within 10 weeks. At the end of the experiments, the colon samples were removed from the mice. RESULTS: The protein bands of caspase - 3, TRPA1, TRPM2, and TRPV1 were increased by the treatments of AOM/DSS. The levels of apoptosis, cyROS, cleaved caspase - 3, and cleaved caspase - 9, as well as the depolarization of the mitochondrial membrane, all increased in the AOM/DSS group. Although they were reduced in the SEB and AOM/DSS + SEB groups by the treatments of SEB, TRPA1 (AP18), TRPM2 (ACA), and TRPV1 (capsazepine) antagonists, the apoptotic and oxidant values were further elevated in the AOM/DSS group by the treatments of TRPA1 (cinnamaldehyde), TRPM2 (H2O2), and TRPV1 (capsaicin) agonists. CONCLUSION: The activations of TRPA1, TRPM2, and TRPV1 channels induced the increase of apoptotic and oxidant actions in the colon cancer cells, although their inhibition via SEB treatment decreased the actions. Hence, TRPA1, TRPM2, and TRPV1 activations could be used as effective agents in the treatment of colon tumors.

Silver nanoparticles potentiate antitumor and oxidant actions of cisplatin via the stimulation of TRPM2 channel in glioblastoma tumor cells.

We investigated the effects of silver nanoparticle (AgNP) and cisplatin (CiSP) exposure via the activation of TRPM2 cation channels in glioblastoma (DBTRG-05MG) cell line. The cells were divided into four groups as control, AgNPs (100 µg/ml for 48 h), CiSP (25 µM for 24 h), and CiSP + AgNPs. We found that the cytotoxic, oxidant and apoptotic actions of CiSP were further stimulated through the activation of TRPM2 (via ADP-ribose and H2O2) in the cells by the treatment of AgNPs. The actions were decreased in the cells by the treatments of TRPM2 antagonists (ACA and 2APB). The apoptotic actions of AgNPs were induced by the stimulation of propidium iodide positive DBTRG-05MG rate, caspase -3, caspase -8, and caspase -9 activations, although their oxidant actions were acted by the increase of mitochondrial membrane depolarization, lipid peroxidation, mitochondrial oxygen free radicals (ROS), and cytosolic ROS, but the decrease of total antioxidant status, glutathione, and glutathione peroxidase. The accumulation of cytosolic free Ca2+ and Zn2+ into mitochondria via the activation of TRPM2 current density and activity accelerated oxidant and apoptotic actions of AgNPs in the cells. We found that the combination of AgNPs and CiSP was synergistic via the stimulation of TRPM2 for treatment of DBTRG-05MG cells. The combination of AgNPs and CiSP showed a favorable action via the stimulation of TRPM2 in the treatment of glioblastoma tumor cells.

TRPV1 stimulation increased oxidative neurotoxicity and apoptosis in the glia cell membrane but not in the perinuclear area: An evidence of TRPV1 subtype.

Glia are essential neurons of the immune system in the central nervous system. The effective mission of glia depends on their activation, release of cytokines, and oxidative cleaning of debris material from neuronal cells. Accumulating evidence indicates that microglia activation-induced oxidative stress via the activation Ca2+ permeable TRPV1 channel has an essential role in the pathophysiology of neurodegenerative diseases. However, there is scarce information on the cytosolic localization of TRPV1 and the induction of oxidative cytotoxicity in the glia. Hence, we investigated the interactions between cytosolic TRPV1 expression levels and oxidative neurotoxicity in the BV2, C8-D1A, N9 glia, and DBTRG glioblastoma cells. We observed TRPV1 expression in the perinuclear area but not in the cell membrane in the BV2, C8-D1A, and N9 cells. Hence, we observed no activation of TRPV1 on the increase of mitochondrial free reactive oxygen species (mROS) and apoptosis in the cells after the capsaicin stimulation. However, we observed TRPV1 channel expression in the positive control (DBTRG) cell membranes. Hence, the Ca2+ influx, TRPV1 current density, apoptosis, and mROS levels were increased in the DBTRG cells after the capsaicin stimulation, although their levels were diminished by the treatment of the TRPV1 blocker (capsazepine). In conclusion, the presence of TRPV1 in the cell membrane of DBTRG cells induced excessive generation of mROS and apoptosis actions, although the presence of TRPV1 in the perinuclear area did not cause the actions. It seems that there is a subtype of TRPV1 in the perinuclear area, and it is not activated by the capsaicin.

Carvacrol protects the ARPE19 retinal pigment epithelial cells against high glucose-induced oxidative stress, apoptosis, and inflammation by suppressing the TRPM2 channel signaling pathways.

PURPOSE: The concentration of plasma high glucose (HGu) in diabetes mellitus (DM) induces the retinal pigment epithelial cell (ARPE19) death via the increase of inflammation, cytosolic (cytROS), and mitochondrial (mitROS) free oxygen radical generations. Transient potential melastatin 2 (TRPM2) cation channel is stimulated by cytROS and mitROS. Hence, the cytROS and mitROS-mediated excessive Ca2+ influxes via the stimulation of TRPM2 channel cause to the induction of DM-mediated retina oxidative cytotoxicity. Because of the antioxidant role of carvacrol (CRV), it may modulate oxidative cytotoxicity via the attenuation of TRPM2 in the ARPE19. We aimed to investigate the modulator action of CRV treatment on the HGu-mediated TRPM2 stimulation, oxidative stress, and apoptosis in the ARPE19 cell model. MATERIAL AND METHODS: The ARPE19 cells were divided into four groups as normal glucose (NGu), NGu + Carv, HGu, and HGu + CRV. RESULTS: The levels of cell death (propidium iodide/Hoechst rate) and apoptosis markers (caspases 3, 8, and 9), cytokine generations (IL-1ß and TNF-α), ROS productions (cytROS, mitROS, and lipid peroxidation), TRPM2 currents, and intracellular free Ca2+ (Fluo/3) were increased in the HGu group after the stimulations of hydrogen peroxide and ADP-ribose, although their levels were diminished via upregulation of glutathione and glutathione peroxidase by the treatments of CRV and TRPM2 blockers. CONCLUSION: Current results confirmed that the HGu-induced overload Ca2+ influx and oxidative retinal toxicity in the ARPE19 cells were induced by the stimulation of TRPM2, although they were modulated via the inhibition of TRPM2 by CRV. CRV may be noted as a potential therapeutic antioxidant to the TRPM2 activation-mediated retinal oxidative injury.

Eicosapentaenoic acid enhanced apoptotic and oxidant effects of cisplatin via activation of TRPM2 channel in brain tumor cells.

Cisplatin (CiSP) induced-overload Ca2+ entry results in the increase of mitochondrial oxidative stress and apoptosis in the cancer cell. TRPM2 cation channel is gated by the cytosolic ADP-ribose (ADPR) and reactive oxygen species (ROS). The high content of polyunsaturated fatty acid (PUFA) in the brain is a main target of ROS. Eicosapentaenoic acid (EPA) induces oxidant action via the enhance of PUFA content in the glioblastoma (DBTRG) cells. We hypothesized that a combination of CiSP and EPA may offer a potential therapy in the DBTRG cell by exerting the antitumor, oxidant, and apoptotic actions and stimulating Ca2+ influx and TRPM2 activity. In the DBTRG cells, we induced four groups as control, EPA (30 µM for 24 h), CiSP (25 µM for 24 h), and CiSP + EPA. The CiSP-induced intracellular Ca2+ responses to the TRPM2 activation were increased in the DBTRG cells from coming H2O2 and ADPR. The responses were decreased in the cells by the inhibitions of TRPM2 (ACA and 2/APB) and PARP/1 (DPQ and PJ34). The incubation of EPA further increased the intracellular Ca2+ responses, mitochondria function, and the generation of ROS in the DBTRGs. After the treatment of EPA, lipid peroxidation, apoptosis, cell death, caspase -3, -8, and -9 levels were further increased in the DBTRG, although the levels of glutathione, glutathione peroxidase, cell numbers, and cell viability were further decreased in the cells. In summary, anticancer, apoptotic, and oxidant actions of CiSP were further increased via the activation of TRPM2 channel in the DBTRGs by the treatment of EPA. Hence, TRPM2 stimulation via EPA could be used as an effective agent in the treatment of glioblastoma tumors with CiSP.

Paclitaxel Promotes Oxidative Stress-Mediated Human Laryngeal Squamous Tumor Cell Death through the Stimulation of Calcium and Zinc Signaling Pathways: No Synergic Action of Melatonin.

The paclitaxel (PAX) and melatonin (MLT)-mediated mitochondria reactive free oxygen radical (miROS) generations via the influx of excessive Ca2+ and Zn2+ induce tumor cell death and apoptosis. However, a presence of resistance was demonstrated against the PAX treatment in the tumor cells. The stimulation of TRPM2 may increase the anticancer action of PAX after the treatment of MLT. We investigated the stimulating role of PAX with/without MLT on the excessive Ca2+ influx and miROS generation-mediated human laryngeal squamous cancer (Hep2) cell death through the stimulation of TRPM2. The Hep2 cells were divided into four groups as control, MLT (1 mM for 2 h), PAX (50 µM for 24 h), and PAX + MLT. In some experiments, we induced additional subgroups such as PAX+ACA and PAX+2APB. The stimulation of TRPM2 induced the increase of TRPM2 current densities, lipid peroxidation, cytosolic ROS, miROS, cytosolic Ca2+, and Zn2+ values in the Hep2 cells after the treatment of PAX, although their values were decreased by the treatment of MLT and TRPM2 antagonists (ACA and 2APB). In addition, the PAX induced apoptosis and cell death via upregulation of caspases and downregulation of antioxidant glutathione peroxidase and glutathione in the cells. The treatment of PAX increased protein band expression values of TRPM2, PARP-1, and caspase 3 and 9 in the Hep2. The increased expression, apoptotic, and cell death values were not affected by the treatment of MLT. In conclusion, PAX induced the increase of Hep2 cell death via upregulations of TRPM2 and Zn2+, although its downregulation via the treatment of MLT did not change the antitumor action of PAX.

Protective role of selenium on MPP+ and homocysteine-induced TRPM2 channel activation in SH-SY5Y cells.

Homocysteine is an intermediate product of biochemical reactions occurring in living organisms. It is known that drugs that increase dopamine synthesis used in Parkinson's disease (PD) cause an increase in the plasma homocysteine level. As the plasma homocysteine level increases, the amount of intracellular free calcium ion ([Ca2+]i) and oxidative stress increase. As a result, it contributes to the excitotoxic effect by causing neurodegeneration. TRPM2 cation channel is activated by high [Ca2+]i and oxidative stress. The role of TRPM2 in the development of neuronal damage due to the increase in homocysteine in PD has not yet been elucidated. In current study, we aimed to investigate the role of the TRPM2 and selenium (Se) in SH-SY5Y neuronal cells treated with homocysteine (HCT) and MPP . SH-SY5Y cells were divided into four groups: control, MPP, MPP + HCT, and MPP + HCT + Se. The results of plate reader assay, confocal microscope imaging, and western blot analyses indicated upregulation of apoptosis, [Ca2+]i, mitochondrial membrane depolarization, caspase activation, and intracellular ROS values in the cells. The MPP + HCT group had considerably higher values than the other groups. The MPP + HCT + Se group had significantly lower values than all the other groups except the control group. In addition, incubation of MPP + HCT and MPP + HCT + Se groups with TRPM2 antagonist 2-APB increased cell viability and reduced intracellular calcium influx and apoptosis levels. It is concluded that the activation of TRPM2 was propagated in HCT and MPP-induced SH-SY5Y cells by the increase of oxidative stress. The antioxidant property of Se regulated the TRPM2 channel activation and neurodegeneration by providing intracellular oxidant/antioxidant balance.

Involvement of TRPM2 in the Neurobiology of Experimental Migraine: Focus on Oxidative Stress and Apoptosis.

Excessive Ca2+ influx and mitochondrial oxidative stress (OS) of trigeminal ganglia (TG) have essential roles in the etiology of migraine headache and aura. The stimulation of TRPM2 channel via the generation of OS and ADP-ribose (ADPR) induces pain, inflammatory, and oxidative neurotoxicity, although its inhibition reduces the intensity of pain and neurotoxicity in several neurons. However, the cellular and molecular effects of TRPM2 in the TG of migraine model (glyceryl trinitrate, GTN) on the induction of pain, OS, apoptosis, and inflammation remain elusive. GTN-mediated increases of pain intensity, apoptosis, death, cytosolic reactive oxygen species (ROS), mitochondrial ROS, caspase -3, caspase -9, cytosolic Ca2+ levels, and cytokine generations (TNF-α, IL-1ß, and IL-6) in the TG of TRPM2 wild-type mouse were further increased by the TRPM2 activation, although they were modulated by the treatments of GSH, PARP-1 inhibitors (PJ34 and DPQ), and TRPM2 blockers (ACA and 2APB). However, the effects of GTN were not observed in the TG of TRPM2 knockout mice. The current data indicate that the maintaining activation of TRPM2 is not only important for the quenching OS, inflammation, and neurotoxicity in the TG neurons of mice with experimental migraine but also equally critical to the modulation of GTN-induced pain.

Interferon Gamma-Mediated Oxidative Stress Induces Apoptosis, Neuroinflammation, Zinc Ion Influx, and TRPM2 Channel Activation in Neuronal Cell Line: Modulator Role of Curcumin.

Host defenses in the brain are modulated by the activation of several factors such as oxygen free radical species (ROS), Ca2+ influx, and TRPM2 activation, and they are well-known adverse factors in neurotoxicity and neurodegenerative diseases. Importantly, recent data indicated a protective action of curcumin (CRC) via inhibition of TRPM2 on the inflammation factors, ROS, and apoptosis in hypoxia-induced SH-SY5Y neuronal cells. However, the relationship between interferon gamma (IFNg) exposure and TRPM2 activation in the SH-SY5Y cells are not fully identified. The SH-SY5Y cells as a neuronal cell line model were used in several neuroinflammation studies. Hence, we used the SH-SY5Y cells in the current study, and they were divided into four main groups as control, CRC, IFNg, and IFNg+CRC. The data presented here indicate that IFNg induced excessive Ca2+ influx via activation of TRPM2. The IFNg treatment further increased cell death, cell debris amount, apoptosis, and cytokine generations (IL-1ß, IL-6, and TNF-α) which were due to increased cytosolic and mitochondrial ROS generations as well as increased activations of caspase-3 and caspase-9. The expression levels of TRPM2, PARP-1, Bax, caspase-3, and caspase-9 were increased in the cells by the IFNg treatment. However, CRC treatment reduced the increase of expression levels, cytokine generations, caspase activations, ROS release, Ca2+ influx, cell death, and apoptosis levels via inhibition of TRPM2 in the SH-SY5Y cells that were treated with IFNg. Moreover, the treatment of TRPM2 blockers (ACA and 2-APB) potentiated the modulator effects of CRC. In conclusion, these results suggest that neuroinflammation via IFNg lead to the TRPM2 activation in the SH-SY5Y cells, whereas CRC prevents IFNg-mediated TRPM2 activation, cell death, and cytokine generations.

Bevacizumab induces oxidative cytotoxicity and apoptosis via TRPM2 channel activation in retinal pigment epithelial cells: Protective role of glutathione.

PURPOSE: Bevacizumab (BEV) is a blocker of circulating VEGF A generation. However, BEV has adverse apoptotic and cytotoxic effects via upregulation of mitochondrial reactive oxygen species (ROS) and TRPM2 activation, and downregulation of cytosolic glutathione (GSH) in neuronal cells. We investigated the possible protective effects of GSH treatment on BEV-induced oxidant and apoptotic adverse actions in the TRPM2 expressing adult retinal pigment epithelial-19 (ARPE-19) and SH-SY5Y neuronal cells. MATERIAL AND METHODS: The ARPE-19 and SH-SY5Y cells were divided into five main groups: Control, GSH (10 mM for 2 h), BEV (0.25 mg/ml for 24 h), BEV+GSH, and BEV+TRPM2 channel blockers (ACA or 2-APB). In the SH-SY5Y cells, the Ca2+ analyses (Fluo-3) were performed only, although Fluo-3 and the remaining analyses were performed in the ARPE-19 cells. RESULTS: The levels of apoptosis, cell death, mitochondrial ROS, lipid peroxidation, caspase-3, caspase-9, ADP-ribose-induced TRPM2 current density, cytosolic-free Zn2+, and Ca2+ were increased by BEV, although their levels were diminished by the treatments of GSH and TRPM2 blockers. The BEV-induced decreases of cell viability, GSH levels, and glutathione peroxidase activities were increased by the treatment of GSH. BEV-induced increase of TRPM2 expression was decreased by the treatment of GSH, although BEV-induced decrease of VEGF A expression was further decreased by the treatment of GSH. CONCLUSION: Our data confirmed that BEV-induced mitochondrial ROS and apoptosis in the human retinal epithelial cells were modulated by GSH and TRPM2 inhibition. The treatment of GSH may be considered as a therapeutic approach to BEV-induced ARPE-19 cell injury.

Involvement of TRPM2 Channel on Hypoxia-Induced Oxidative Injury, Inflammation, and Cell Death in Retinal Pigment Epithelial Cells: Modulator Action of Selenium Nanoparticles.

Hypoxia (HYPX) in several eye diseases such as glaucoma and diabetic retinopathy causes oxidative cell death and inflammation. TRPM2 cation channel is activated by HYPX-induced ADP-ribose (ADPR) and oxidative stress. The protective role of selenium via inhibition of TRPM2 on the HYPX-induced oxidative cytotoxicity and inflammation values in the human kidney cell line was recently reported. However, the protective role of selenium nanoparticles (SeNP) on the values in the retinal pigment epithelial (ARPE-19) cells has not been clarified yet. In the current study, we investigated two subjects. First, we investigated the involvement of TRPM2 channel on the HYPX-induced oxidative injury, inflammation, and apoptosis in the ARPE-19 cells. Second, we investigated the protective role of SeNP via inhibition of TRPM2 channel on the HYPX-induced oxidative injury and apoptosis in the ARPE-19 cells. For the aims, the ARPE-19 cells were divided into four main groups as follows: Control (Ctr), SeNP (2.5 µg/ml for 24 h), HYPX (200 µM CoCl2 for 24 h), and HYPX+SeNP. The TRPM2 current density and Ca2+ fluorescence intensity with an increase of mitochondrial membrane depolarization and oxygen free radical (OFR) generations were increased in the ARPE-19 cells by the treatment of HYPX. There was no increase of Ca2+ fluorescence intensity in the pre-treated cells with PARP-1 inhibitors (DPQ and PJ34) or in the presence of Ca2+-free extracellular buffer. When HYPX-induced TRPM2 activity was treated by SeNP and TRPM2 (2-APB and ACA) blockers, the increases of OFR generation, cytokine (TNF-α and IL-1ß) levels, TRPM2, and PARP-1 expressions were restored. In conclusion, the exposure of HYPX caused mitochondrial oxidative cell cytotoxicity and cell death via TRPM2-mediated Ca2+ signaling and may provide an avenue for treating HYPX-induced retinal diseases associated with the excessive OFR and Ca2+ influx.

Glutathione depletion induces oxidative injury and apoptosis via TRPM2 channel activation in renal collecting duct cells.

Oxidative stress (OS)-induced glutathione (GSH) depletion plays an essential role in several kidney diseases such as chronic kidney disease and nephrotoxicity. The OS-dependent activation of TRPM2 cation channel in several neurons and cells were modulated by the concentration of intracellular GSH. However, the effects of GSH alteration on TRPM2 activation, OS, and apoptosis in the cortical collecting duct (mpkCCDc14) cells still remain elusive. We investigated the effects of GSH supplementation on OS-induced TRPM2 activation, mitochondrial oxidative stress, and apoptosis in the human embryonic kidney 293 (HEK293) and mpkCCDc14 cells treated with buthionine-sulfoximine (BSO), a GSH synthase inhibitor. The HEK293 and mpkCCDc14 cells were divided into five groups as control, GSH (10 mM for 2 h), BSO (0.5 mM for 6 h), BSO + GSH, and BSO + TRPM2 channel blockers. Apoptosis, cell death, mitochondrial OS, caspase -3, caspase -9, cytosolic free Zn2+, and Ca2+ concentrations were increased in the BSO group of the TRPM2 expressing mpkCCDc14 cells, although they were diminished by the treatments of GSH, PARP-1 inhibitors (PJ34 and DPQ), and TRPM2 blockers (ACA and 2-APB). The BSO-induced decreases in the levels of cell viability and cytosolic GSH were increased by the treatments of GSH, ACA, and 2-APB. However, the effects of BSO and GSH were not observed in the non-TRPM2 expressing HEK293 cells. Current results show that maintaining GSH homeostasis is not only important for quenching OS in the cortical collecting duct cells but equally critical to modulate TRPM2 activation. Thus, suppressing apoptosis and mitochondrial OS responses elicited by oxidant action of GSH depletion.

Curcumin Attenuates Hypoxia-Induced Oxidative Neurotoxicity, Apoptosis, Calcium, and Zinc Ion Influxes in a Neuronal Cell Line: Involvement of TRPM2 Channel.

Apoptosis/cell death and reactive oxygen species (ROS) via overload free Ca2+ and Zn2+ uptake into mitochondria are emerging as crucial events in the etiology of hypoxia (HPX)-induced neurodegenerative diseases. The neuroprotective actions of curcumin (CURC) via modulation of oxidative stress and the PARP1-dependent activated TRPM2 cation channel on the ROS generation and cell death in several neurons have been recognized. However, the molecular mechanisms underlying CURC's neuroprotection remain elusive. We investigated the role of CURC via modulation of TRPM2 on cell death and oxidative cytotoxicity in SH-SY5Y neuronal cells. The SH-SY5Y cells were divided into five groups as follows: CURC (10 µM for 24 h), HPX (200 µM CoCl2 for 24 h), CURC + HPX, and HPX + TRPM2 blockers (2-APB-100 µM or ACA-25 µM for 30 min). In some experiments, the cells in the HPX groups were additionally incubated with PARP1 (PJ34) and Zn2+ (TPEN) inhibitors. The exposure of CoCl2 induced increases of TRPM2 current density and Ca2+ fluorescence intensity with an increase of mitochondrial membrane depolarization and ROS generation. When HPX-induced TRPM2 activity was blocked by 2-APB and ACA, or the cells were treated with CURC, the increase of ROS generation, the expression levels of TRPM2 and PARP1 were restored. The levels of apoptosis and cell death in the cells were enriched with increases of caspase-3 and -9 activations, although they were decreased by CURC treatment. HPX-induced increase of cytosolic Zn2+ was attenuated by the TPEN and CURC treatments. In conclusion, CURC attenuates HPX-induced mitochondrial ROS generation, apoptosis, cell death, and TRPM2-mediated Ca2+ signaling and may provide an avenue for treating HPX-induced neurological diseases associated with the ROS, Ca2+, and Zn2+.

Melatonin and Selenium Suppress Docetaxel-Induced TRPV1 Activation, Neuropathic Pain and Oxidative Neurotoxicity in Mice.

Docetaxel (DT) has been reported to positive therapeutic actions in the treatment of glioblastoma, breast tumors, and prostate cancers. However, it can also induce peripheral neuropathic pain and neurotoxicity as adverse effects. Expression level of TRPV1 cation channel is high in dorsal root ganglion (DRG), and its activation via capsaicin and reactive oxygen species (ROS) mediates peripheral neuropathic pain in mice. As cancer is known to increase the levels of ROS, the protective roles of melatonin (MT) and selenium (Se) were evaluated on the TRPV1-mediated neurotoxicity and pain in the DT-treated mice. Mice and TRPV1 expressing SH-SY5Y cells were equally divided into control, MT, Se, DT, DT+MT, and DT+Se groups. In the results of pain tests in the mice, we observed a decrease in DT-mediated mechanical and heat neuropathic pain by MT and Se. The results of plate reader assay and laser confocal microscopy image analyses indicated a protective role of MT and Se on the DT-induced increase of mitochondrial ROS, cytosolic ROS, apoptosis, lipid peroxidation, intracellular free Zn2+, Ca2+, and caspase-3 and -9 levels in the DRG and SH-SY5Y cells. MT and Se modulated DT-induced decreases of total antioxidant status, reduced glutathione and glutathione peroxidase in the DRG. However, the effects of DT were not observed in the non-TRPV1 expressing SH-SY5Y cells. Hence, MT and Se mediated protective effects against DT-induced adverse peripheral oxidative neurotoxicity and peripheral pain. These effects may be attributed to potent antioxidant properties of MT and Se.