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.

Alpha-lipoic acid modulates the diabetes mellitus-mediated neuropathic pain via inhibition of the TRPV1 channel, apoptosis, and oxidative stress in rats.

Diabetes mellitus (DM) is a chronic syndrome involving neuropathic pain. Increased oxidative stress in DM is assumed to increase free reactive oxygen radicals (ROS) and causes diabetic damage. The sciatic nerve (ScN) and dorsal root ganglion (DRG) both contain high levels of the TRPV1 channel, which is triggered by capsaicin and ROSs and results in increased Ca2+ entry into the neurons. Alpha-lipoic acid (ALA) is considered an important part of the antioxidant system. To better characterize the protective effects of ALA on the DM-induced neuronal through TRPV1 modulation, we investigated the role of ALA on DM-induced neuropathic pain, oxidative ScN, and DRG damage in diabetic rats. Forty adult Wistar albino female rats were divided into four groups as control, ALA (50 mg/kg for 14 days), streptozotocin (STZ and 45 mg/kg and single dose), and STZ + ALA. Rats were used for the pain tests. After obtaining the DRGs and ScN, they were used for plate reader, patch-clamp, and laser confocal microscope analyses. We observed the modulator role of ALA on the thresholds of mechanical withdrawal pain (von Frey test) and hot sensitivity pain (hot plate test) in the STZ + ALA group. The treatment of ALA decreased STZ-induced increase of TRPV1 current densities, intracellular free Ca2+ concentrations (Fura-2 and Fluo - 3/AM), ROS, caspase 3, caspase 9, mitochondrial membrane potential, and apoptosis values in the ScN and DRG neurons, although its treatment induced the increase of cell viability and body weight gain. The treatment of ALA acted a neuroprotective role on the TRPV1 channel stimulation-mediated Ca2+ influx, neuropathic pain, and neuronal damage in diabetic rats. The neuroprotective role of ALA treatment can be explained by its modulating the TRPV1 channel activity, intracellular Ca2+ increase-induced oxidative stress, 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.

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.

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.

Low molecular weight heparin treatment reduced apoptosis and oxidative cytotoxicity in the thrombocytes of patients with recurrent pregnancy loss and thrombophilia: Involvements of TRPM2 and TRPV1 channels.

AIM: Recurrent pregnancy loss (RPL) is known to be associated with increased thrombophilia and oxidative toxicity. However, the mechanism of thrombophilia apoptosis and oxidative toxicity is still unclear. In addition, the treatment of heparin induced regulator roles on intracellular free Ca2+ ([Ca2+ ]i ) and cytosolic reactive oxygen species (cytROS) concentrations in several diseases. TRPM2 and TRPV1 channels are activated by different stimuli, including oxidative toxicity. The aim of this study was to investigate the effects of low molecular weight heparin (LMWH) via modulation of TRPM2 and TRPV1 on calcium signaling, oxidative toxicity, and apoptosis in the thrombocytes of RPL patients. STUDY DESIGN: Thrombocyte and plasma samples collected from 10 patients with RPL and 10 healthy controls were used in the current study. MAIN FINDINGS: The [Ca2+ ]i concentration, cytROS (DCFH-DA), mitochondrial membrane potential (JC-1), apoptosis, caspase-3, and caspase-9 levels were high in the plasma and thrombocytes of RPL patients, although they were diminished by the treatments of LMWH, TRPM2 (N-(p-amylcinnamoyl)anthranilic acid) and TRPV1 (capsazepine) channel blockers. CONCLUSIONS: The current study results suggest that the treatment of LMWH is useful against apoptotic cell death and oxidative toxicity in the thrombocytes of patients with RPL, which seem to be dependent on increased levels of [Ca2+ ]i concentration via the activation of TRPM2 and TRPV1.

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.

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.

A novel antagonist of TRPM2 and TRPV4 channels: Carvacrol.

The overload cytosolic free Ca2+ (cCa2+) influx-mediated excessive generation of oxidative stress in the pathophysiological conditions induces neuronal and cellular injury via the activation of cation channels. TRPM2 and TRPV4 channels are activated by oxidative stress, and their specific antagonists have not been discovered yet. The antioxidant and anti-Covid-19 properties of carvacrol (CARV) were recently reported. Hence, I suspected possible antagonist properties of CARV against oxidative stress (OS)/ADP-ribose (ADPR)-induced TRPM2 and GSK1016790A (GSK)-mediated TRPV4 activations in neuronal and kidney cells. I investigated the antagonist role of CARV on the activations of TRPM2 and TRPV4 in SH-SY5Y neuronal, BV-2 microglial, and HEK293 cells. The OS/ADPR and GSK in the cells caused to increase of TRPM2/TRPV4 current densities and overload cytosolic free Ca2+ (cCa2+) influx with an increase of mitochondrial membrane potential, cytosolic (cROS), and mitochondrial (mROS) ROS. The changes were not observed in the absence of TRPM2 and TRPV4 or the presence of Ca2+ free extracellular buffer and PARP-1 inhibitors (PJ34 and DPQ). When OS-induced TRPM2 and GSK-induced TRPV4 activations were inhibited by the treatment of CARV, the increase of cROS, mROS, lipid peroxidation, apoptosis, cell death, cCa2+ concentration, caspase -3, and caspase -9 levels were restored via upregulation of glutathione and glutathione peroxidase. In conclusion, the treatment of CARV modulated the TRPM2 and TRPV4-mediated overload Ca2+ influx and may provide an avenue for protecting TRPM2 and TRPV4-mediated neurodegenerative diseases associated with the increase of mROS and cCa2+. The possible TRPM2 and TRPV4 blocker action of carvacrol (CARV) via the modulation oxidative stress and apoptosis in the SH-SY5Y neuronal cells. TRPM2 is activated by DNA damage-induced (via PARP-1 activation) ADP-ribose (ADPR) and reactive oxygen species (ROS) (H2O2), although it is inhibited by nonspecific inhibitors (ACA and 2-APB). TRPV4 is activated by the treatments of GSK1016790A (GSK), although it is inhibited by a nonspecific inhibitor (ruthenium red, RuRe). The treatment of GSK induces excessive generation of ROS. The accumulation of free cytosolic Ca2+ (cCa2+) via the activations of TRPM2 and TRPV4 in the mitochondria causes the increase of mitochondrial membrane depolarization (ΔΨm). In turn, the increase of ΔΨm causes the excessive generation of ROS. The TRPM2 and TRPV4-induced the excessive generations of ROS result in the increase of apoptosis and cell death via the activations of caspase -3 (Casp-3) and caspase -9 (Casp-9) in the neuronal cells, although their oxidant actions decrease the glutathione (GSH) and glutathione peroxidase (GSHPx) levels. The oxidant and apoptotic adverse actions of TRPM2 and TRPV4 are modulated by the treatment of CARV.

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.

Effects of homocysteine and memantine on oxidative stress related TRP cation channels in in-vitro model of Alzheimer's disease.

Memantine (MEM) has been used to treat patients with Alzheimer' disease though inhibition of reactive oxygen species (ROS), Ca2+ entry and glutamate receptor. The Ca2+ permeable TRPA1, TRPM2 and TRPV1 channels are activated in the hippocampus by ROS, and antioxidant MEM as a potent TRPA1, TRPM2 and TRPV1 channel antagonist may reduce Aß-induced oxidative stress and apoptosis in the neurons. In the current study, we investigated the neuroprotective properties of MEM in Aß-induced hippocampal neuron cultures. Freshly isolated hippocampal neurons of mice were divided into eight groups as control, Aß, Hcy, MEM, Aß + Hcy, Aß + Hcy + MEM, Aß + MEM and Hcy + MEM. The neurons were exposed to incubated with Aß (20 µM for 24 h), Hcy (250 µM for 30 min) and MEM (10 µM for 24 h). TRPA1, TRPM2 and TRPV1 of the eight groups were further stimulated by cinnamaldehyde, cumene hydyroperoxide and capsaicin, respectively although they were further inhibited by AP-18, N-(p-Amylcinnamoyl) anthranilic acid (ACA) and capsazepine (CPZ). The [Ca2+] concentration, apoptosis, caspase 3, caspase 9 activations, mitochondrial membrane depolarization and intracellular ROS production values in the neurons were higher in Aß and Hcy groups than in control although they were lower in the MEM group than in Aß and Hcy groups. The values were further decreased by MEM + AP-18, MEM + CPZ and MEM + ACA treatments as compared to MEM only. Aß and Hcy-induced decrease of cell viability level was increased by MEM treatment although Aß and Hcy-induced increase of caspase 3, caspase 9, PARP1, TRPA1, TRPM2 and TRPV1 expression levels were decreased by MEM treatments. In conclusion, TRPA1, TRPM2 and TRPV1 channels are involved in Aß and Hcy-induced neuronal death, and modulation of the activity of these channels by MEM treatment may account for their neuroprotective activity against apoptosis, excessive ROS production, and Ca2+ entry.

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.

Selenium prevents interferon-gamma induced activation of TRPM2 channel and inhibits inflammation, mitochondrial oxidative stress, and apoptosis in microglia.

Microglia as the primary immune cells of brain act protective effects against injuries and infections in the central nervous system. Inflammation via excessive Ca2+ influx and oxygen radical species (ROS) generation is a known factor in many neurodegenerative disorders. Importantly, the Ca2+ permeable TRPM2 channel is activated by oxidative stress. Thus, TRPM2 could provide the excessive Ca2+ influx in the microglia. Although TRPM2 expression level is high in inflammatory cells, the interplay between mouse microglia and TRPM2 channel during inflammation is not fully identified. Thus, it is important to understand the mechanisms and factors involved in order to enhance neuronal regeneration and repair. The data presented here indicate that TRPM2 channels were activated in microglia cells by interferon-gamma (IFNγ). The IFNγ treatment further increased apoptosis (early and late) and cytokine productions (TNF-α, IL-1ß, and IL-6) which were due to increased lipid peroxidation and ROS generations as well as increased activations of caspase -3 (Casp-3) and - 9 (Casp-9). However, selenium treatment diminished activations of TRPM2, cytokine, Casp-3, and Casp-9, and levels of lipid peroxidation and mitochondrial ROS production in the microglia that were treated with IFNγ. Moreover, addition of either PARP1 inhibitors (PJ34 or DPQ) or TRPM2 blockers (2-APB or ACA) potentiated the modulator effects of selenium. These results clearly suggest that IFNγ leads to TRPM2 activation in microglia cells; whereas, selenium prevents IFNγ-mediated TRPM2 activation and cytokine generation. Together the interplay between IFNγ released from microglia cells is importance in brain inflammation and may affect oxidative cytotoxicity in the microglia. Graphical abstract Summary of pathways involved in IFNγ-induced TRPM2 activation and microglia death through excessive reactive oxygen species (ROS): Modulator role of selenium (Se). The IFNγ causes the microglia activation. Nudix box domain of TRPM2 is sensitive to ROS. The ROS induces DNA damage and ADPR-ribose (ADPR) production in the nucleus via PARP1 enzyme activation. ADPR and ROS-induced TRPM2 activation stimulates excessive Ca2+ influx. ROS are produced in the mitochondria through the increase of free cytosolic Ca2+ (via TRPM2 activation) by the IFNγ treatment, although they are diminished by the TRPM2 channel blocker (ACA and 2-APB) and PARP1 inhibitor treatments. The main mechanism in the cell death and inflammatory effects of IFNγ is mediated by stimulation of ROS-mediated caspase (caspase -3 and - 9) activations and cytokine production (TNF-α, IL-1ß, and IL-6) via TRPM2 activation, respectively. The apoptotic, inflammatory, and oxidant actions of IFNγ are modulated through TRPM2 inhibition by the Se treatment.

Protective effect of cabergoline on mitochondrial oxidative stress-induced apoptosis is mediated by modulations of TRPM2 in neutrophils of patients with endometriosis.

Calcium ion (Ca2+) signaling in endometriosis (ENDO) is associated with increased neutrophil activation and oxidative stress. A Ca2+ signaling modulator and antioxidant actions of cabergoline (CBG) in some cells were recently reported. TRPM2 cation channel is activated by reactive oxygen species (ROS). Antioxidant action of CGB via inhibition of ROS may modulate the channel. We aimed to investigate the effect of CBG on TRPM2 inhibition in serum and neutrophils of patients with ENDO. The serum and neutrophil samples were grouped into healthy samples (no treatment), ENDO and ENDO + CBG treated groups (n = 10 in each). In some experiments, the neutrophils were also incubated with TRPM2 (ACA) and PARP-1 (PJ34) blockers. The values of intracellular ROS, Ca2+ concentration, mitochondrial membrane depolarization, lipid peroxidation, apoptosis, and caspase - 3, caspase - 9, PARP-1 and TRPM2 expressions were high in the neutrophils of patients with ENDO, although antioxidant levels (reduced glutathione, glutathione peroxidase, vitamin A, and vitamin E) were low in the neutrophils and serum from these patients. However, markers for apoptosis, oxidative stress, and mitochondrial dysfunction were reduced with CBG, ACA and PJ34 treatments, although the antioxidant levels were increased in the serum and neutrophils following treatment with CBG. Taken together, our current results suggest that CBG are useful antagonists against apoptosis and mitochondrial oxidative stress via inhibition of TRPM2 in neutrophils of patients with ENDO.

Curcumin diminishes cisplatin-induced apoptosis and mitochondrial oxidative stress through inhibition of TRPM2 channel signaling pathway in mouse optic nerve.

Background: Cisplatin (CiSP), a chemotherapeutic agent, is widely used to treat several types of cancers. However, its clinical use is limited due to adverse side effects caused by excessive production of reactive oxygen species (ROS) and death of neurons. The transient receptor potential (TRP) melastatin 2 (TRPM2) cation channel is activated by ADP-ribose (ADPR) and ROS. The protective effect of curcumin (CURCU) against CiSP-induced apoptosis and mitochondrial ROS through inhibition of TRP channels in several types of neuron except optic nerve, was recently reported. The aim of the current study is to clarify the protective effect of CURCU on CiSP-induced mitochondrial oxidative injury and TRPM2 activation in the mice optic nerve and SH-SY5Y human derived neuronal cells.Material and methods: The SH-SY5Y cells and mice were divided into four groups: Control, CURCU, CiSP, and CURCU + CiSP. The mice were treated for 14 days and the cells were incubated with CiSP and CURCU for 24 h.Results: CURCU and PARP-1 inhibitor (PJ34) treatments ameliorated CiSP-induced mitochondrial membrane depolarization, mitochondrial and cytosolic ROS levels and neuronal death in the optic nerve. In the patch-clamp of SH-SY5Y cells and laser confocal microscopy experiments of optic nerve, CURCU and TRPM2 blocker treatments also decreased ADPR-induced TRPM2 currents and cytosolic free calcium ion (Ca2+) concentration, suggesting a suppression of Ca2+ influx and neuronal death.Conclusion: CURCU prevents CiSP-induced optic nerve oxidative injury and cell death by suppressing mitochondrial ROS production via regulating TRPM2 signaling pathways. CURCU may serve as a potential therapeutic target against CiSP-induced toxicity in the optic nerve of CiSP-treated patients.

Clostridium botulinum neurotoxin A induces apoptosis and mitochondrial oxidative stress via activation of TRPM2 channel signaling pathway in neuroblastoma and glioblastoma tumor cells.

BACKGROUND: The Clostridium botulinum neurotoxin A (BTX) is a polypeptide produced by the bacterium Clostridium botulinum. In addition to the therapeutic actions of BTX against pain and neuromuscular disorders, it is acted as anticancerogenic effect through excessive mitochondria reactive oxygen species (ROS) production, apoptosis, and caspase activations. The TRPM2 cation channel is activated by ROS and ADP-ribose and it is inhibited by 2-aminoethyl diphenylborinate (2-APB) and N-(p-amylcinnamoyl) anthranilic acid (ACA). The aim of this study was an investigation of involvement BTX-induced TRPM2 activation on the mitochondria ROS production and apoptosis levels in the DBTRG glioblastoma and SH-SY5Y neuroblastoma tumor cells. MATERIAL AND METHODS: The DBTRG and SH-SY5Y cells were divided into four groups as control, BTX (5 IU for 24 h), BTX + ACA (25 µM for 30 min), and BTX + 2-APB (100 µM for 30 min). RESULTS: BTX treatment increased mitochondrial membrane depolarization (JC-1), mitochondrial (MitROS), and cytosolic (DHR123 and DCFH-DA) ROS levels, neuronal death (propidium iodide/Hoechst) rate, caspase -3, and -9 levels in the BTX group, although their levels were diminished in the BTX + ACA and BTX + 2-APB groups. The ACA and 2-APB treatments also decreased BTX-induced increase of TRPM2 cytosolic free Ca2+ concentration in the glioblastoma and neuroblastoma cell death. CONCLUSIONS: BTX caused neuroblastoma and glioblastoma tumor cell death by activating the mitochondria ROS production via stimulating TRPM2 signaling pathways. BTX may serve as a potential therapeutic target via activation of TRPM2 for treating glioblastoma and neuroblastoma cells.

The potential protective roles of zinc, selenium and glutathione on hypoxia-induced TRPM2 channel activation in transfected HEK293 cells.

Hypoxia induces cell death through excessive production of reactive oxygen species (ROS) and calcium (Ca2+) influx in cells and TRPM2 cation channel is activated by oxidative stress. Zinc (Zn), selenium (Se), and glutathione (GSH) have antioxidant properties in several cells and hypoxia-induced TRPM2 channel activity, ROS and cell death may be inhibited by the Zn, Se, and GSH treatments. We investigated effects of Zn, Se, and GSH on lipid peroxidation (LPO), cell cytotoxicity and death through inhibition of TRPM2 channel activity in transfected HEK293 cells exposed to hypoxia defined as oxygen deficiency.We induced four groups as normoxia 30 and 60 min evaluated as control groups, hypoxia 30 and 60 min in the HEK293 cells. The cells were separately pre-incubated with extracellular Zn (100 µM), Se (150 nM) and GSH (5 mM). Cytotoxicity was evaluated by lactate dehydrogenase (LDH) release and the LDH and LPO levels were significantly higher in the hypoxia-30 and 60 min-exposed cells according to normoxia 30 and 60 min groups. Furthermore, we found that the LPO and LDH were decreased in the hypoxia-exposed cells after being treated with Zn, Se, and GSH according to the hypoxia groups. Compared to the normoxia groups, the current densities of TRPM2 channel were increased in the hypoxia-exposed cells by the hypoxia applications, while the same values were decreased in the treatment of Zn, Se, and GSH according to hypoxia group. In conclusion, hypoxia-induced TRPM2 channel activity, ROS and cell death were recovered by the Se, Zn and GSH treatments.

Inhibitions of anandamide transport and FAAH synthesis decrease apoptosis and oxidative stress through inhibition of TRPV1 channel in an in vitro seizure model.

The expression level of TRPV1 is high in hippocampus which is a main epileptic area in the brain. In addition to the actions of capsaicin (CAP) and reactive oxygen species (ROS), the TRPV1 channel is activated in neurons by endogenous cannabinoid, anandamide (AEA). In the current study, we investigated the role of inhibitors of TRPV1 (capsazepine, CPZ), AEA transport (AM404), and FAAH (URB597) on the modulation of Ca2+ entry, apoptosis, and oxidative stress in in vitro seizure-induced rat hippocampus and human glioblastoma (DBTRG) cell line. The seizure was induced in the hippocampal and DBTRG neurons using in vitro 4-aminopyridine (4-AP) to trigger a seizure-like activity model. CPZ and AM404 were fully effective in reversing 4-AP-induced intracellular free Ca2+ concentration of the hippocampus and TRPV1 current density of DBTRG. However, AEA and CAP did not activate TRPV1 in the URB597-treated neurons. Hence, we observed TRPV1 blocker effects of URB597 in the DBTRG neurons. In addition, the AM404 and CPZ treatments decreased intracellular ROS production, mitochondrial membrane depolarization, apoptosis, caspases 3 and 9 values in the hippocampus. In conclusion, the results indicate that inhibition of AEA transport, FAAH synthesis, and TRPV1 activity can result in remarkable neuroprotective effects in the epileptic neurons. Possible molecular pathways of involvement of capsazepine (CPZ) and AM4040 in anandamide and capsaicin (CAP)-induced apoptosis, oxidative stress, and Ca2+ accumulation through TRPV1 channel in the seizure-induced rat hippocampus and human glioblastoma neurons. The TRPV1 channel is activated by different stimuli including reactive oxygen species (ROS), anandamide (AEA), and CAP and it is blocked by capsazepine (CPZ). Cannabinoid receptor type 1 (CB1) is also activated by AEA. The AEA levels in cytosol are decreased by fatty acid amide hydrolase (FAAH) enzyme. Inhibition of FAAH through URB597 induces stimulation of CB1 receptor through accumulation AEA. URB597 acts antiepileptic effects through inhibition of TRPV1. Overloaded Ca2+ concentration of mitochondria can induce an apoptotic program by stimulating the release of apoptosis-promoting factors such as caspases 3 and caspase 9 by generating ROS due to respiratory chain damage. AM404 and CPZ reduce TRPV1 channel activation and Ca2+ entry in the in vitro 4-AP seizure model-induced hippocampal and glioblastoma neurons.

Selenium attenuates docetaxel-induced apoptosis and mitochondrial oxidative stress in kidney cells.

Docetaxel (DTX) is a chemotherapeutic agent, and it is used for the treatment of several cancers including prostate and glioblastoma, but it results in many adverse effects in normal tissues, including kidney. The cytoprotective properties of selenium (Se) against adverse effects of DTX were reported in several normal cells, except kidney cell lines. The purpose of this study was to investigate the effects of Se on DTX-induced nephrotoxicity in normal kidney cell lines. The human embryonic kidney 293 (HEK293) cells were divided into four groups as control, Se (200 nmol/l for 10 h), DTX (10 nmol/l for 48 h), and DTX+Se. Laser confocal microscope fluorescence intensity of apoptosis (annexin V and propidium iodide), mitochondrial membrane depolarization, reactive oxygen species production, and lipid peroxidation levels were increased in the cells by the DTX treatments, although cell number, cell viability, reduced glutathione and glutathione peroxidase values were decreased by the treatments. The fluorescence intensities and values were recovered in the DTX+Se group of the cells by Se treatment. In conclusion, DTX-induced adverse effects were recovered through inhibition of apoptosis and mitochondrial oxidative stress through upregulation of reduced glutathione and glutathione peroxidase in the normal kidney (HEK293) cells. Combination therapy of DTX and Se could be used as an effective strategy for protection of kidney cells against adverse effects of DTX.

Mitochondrial oxidative stress-induced brain and hippocampus apoptosis decrease through modulation of caspase activity, Ca2+ influx and inflammatory cytokine molecular pathways in the docetaxel-treated mice by melatonin and selenium treatments.

Docetaxel (DOCE) is widely used to treat several types of glioblastoma. Adverse effects DOCE seriously limit its clinical use in several tissues. Its side effects on brain cortex and hippocampus have not been clarified yet. Limited data indicated a protective effect of melatonin (MLT) and selenium (SELEN) on DOCE-induced apoptosis, Ca2+ influx and mitochondrial reactive oxygen species (ROS) in several tissues except brain and hippocampus. The purpose of this study is to discover the protective effect of MLT and SELEN on DOCE-induced brain and hippocampus oxidative toxicity in mice. MLT and SELEN pretreatments significantly ameliorated acute DOCE-induced mitochondrial ROS production in the hippocampus and brain tissues by reducing levels of lipid peroxidation, intracellular ROS production and mitochondrial membrane depolarization, while increasing levels of total antioxidant status, glutathione, glutathione peroxidase, MLT, α-tocopherol, γ-tocopherol, vitamin A, vitamin C and ß-carotene in the tissues. Furthermore, MLT and SELEN pretreatments increased cell viability and TRPM2 channel activation in the hippocampus and brain followed by decreased activations of TNF-α, IL-1ß, IL-6, and caspase -3 and - 9, suggesting a suppression of calcium ion influx, apoptosis and inflammation responses. However, modulator role of SELEN on the values in the tissues is more significant than in the MLT treatment. MLT and SELEN prevent DOCE-induced hippocampus and brain injury by inhibiting mitochondrial ROS and cellular apoptosis through regulating caspase -3 and - 9 activation signaling pathways. MLT and SELEN may serve as potential therapeutic targets against DOCE-induced toxicity in the hippocampus and brain.