Erucic acid increases the potency of cisplatin-induced colorectal cancer cell death and oxidative stress by upregulating the TRPM2 channel.

Erucic acid (ErA) is a source of omega-9 monounsaturated fatty acids. ErA exhibited antitumor effects by causing apoptosis and oxidative stress in tumor cells, with the exception of the HT-29 human colorectal cancer cell line. The apoptotic and Ca2+ signaling pathways in tumor cells are triggered when mitochondrial Ca2+ and Zn2+ accumulation produce reactive free oxygen species (ROS), which in turn activate TRPM2. ErA-induced ROS and TRPM2 stimulation may augment the anticancer action of cisplatin (CSP). We aimed to study the effects of ErA and CSP incubations on ROS, apoptosis, and cell death in the HT-29 cells by activating TRPM2. The cells were divided into five groups: control, ErA (200 µM for 48 h), CSP (25 µM for 24 h), and ErA + CSP + TRPM2 antagonists (200 µM carvacrol and 25 µM N-(p-amylcinnamoyl)anthranilic acid for 24 h). The TRPM2 antagonists reduced ErA plus CSP-induced increases in H2O2-induced intracellular free Ca2+ concentration ([Ca2+]c) and adenosine diphosphate-ribose-caused TRPM2 currents. ErA and CSP were found to cause apoptosis and cell death by raising the intracellular free Zn2+ concentration (Zn2+]c), caspase-3, -8, and -9, mitochondrial membrane dysfunction, and ROS, while lowering reduced glutathione, cell viability, and cell number. The oxidative, apoptotic, and tumor cell death effects of CSP in the cells were enhanced by the increase of ErA-mediated [Ca2+]c and Zn2+]c entering mitochondria through the activation of TRPM2. In conclusion, we observed that the combination of ErA and CSP was synergistic via TRPM2 activation for the treatment of HT-29 tumor cells.

The effects of berberine and curcumin on cardiac, lipid profile and fibrosis markers in cyclophosphamide-induced cardiac damage: The role of the TRPM2 channel.

Cyclophosphamide (CYP) is widely used to treat various types of cancer. In addition to the therapeutic properties of this drug, unfortunately, its side effects are still not fully understood. This study investigated the protective effect of curcumin (CURC) and berberine (BER) on CYP-induced cardiac damage. Thirty-six male rats were equally divided into the control, dimethyl sulfoxide (DMSO), CYP, CYP + CURC, CYP + BER and CYP + BER + CURC groups. Troponin-I, Creatine kinase-myocardial band (CK-MB), total cholesterol, triglyceride levels in serum samples, and reactive oxygen species (ROS), poly(ADP-ribose) polymerase-1 (PARP-1), and transient receptor potential melastatin 2 (TRPM2) channel levels in heart tissue were measured using an enzyme-linked immunoassay (ELISA) kit. In addition, histopathological examination and immunohistochemical investigation of the TRPM2 channel, fibroblast specific protein-1 (FSP1), transforming growth factor-beta- 1 (TGF-ß1) and α-smooth muscle actin (α-SMA) expressions were determined in heart tissue. The CYP group's troponin-I, total cholesterol, triglyceride, CK-MB, ROS, PARP-1 and TRPM2 channel levels were higher than in the other groups in the ELISA measurements (p < 0.05). In contrast, these parameters in the group treated with CURC and BER together with CYP were lower than in the CYP group (p < 0.05). Additionally, CUR and BER reduced CYP-induced pathological damage, TRPM2, FSP1, TGF-ß1 and α-SMA expressions. The data showed that CYP administration can cause cardiac damage by increasing the TRPM2 channel, TGF-ß1, FSP1 and α-SMA expression levels. Therefore, we concluded that CURC and BER administration following CYP application may be used as therapeutic agents to prevent CYP-induced cardiac damage.

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.

Evaluation of TRPM2 Channel-Mediated Autophagic Signaling Pathway in Hippocampus and Cortex Tissues of Rat Offspring Following Prenatal Exposure to Elevated Alcohol Levels.

Fetal alcohol syndrome (FAS) can occur because of high amount of alcohol intake during pregnancy and is characterized by both physical and neurological problems. Children diagnosed with FAS have difficulties in learning, memory, and coordination. Hippocampus has a major role in memory and learning. We aimed to determine whether alcohol exposure during pregnancy had any effect on offspring by evaluating learning ability as well as oxidative stress and autophagy in the hippocampus and cortex tissues of litters. Attention was also paid to sex differences. To do so, TRPM2, Beclin1, p62, LC3B, IBA1, parvalbumin, GAD65, and mGluR5 expression levels were evaluated by immunohistochemistry. Lactate dehydrogenase (LDH), and malondialdehyde (MDA) levels, as well as total oxidant (TOS) and total antioxidant (TAS) status were determined by ELISA. Learning experiments were evaluated by the Morris water maze (MWM) test. Our findings demonstrated that IBA1, LC3B, GAD65, and mGluR5 expression levels were higher in female rats of the chronic alcohol exposure (CAE) model. Our IHC results revealed that TRPM2 expression levels were significantly increased in both males and females in the CAE group. Likewise, TAS was lower, and TOS was higher in CAE animals. Moreover, MWM outcomes supported a learning deficiency in CAE litters compared to controls and indicated that female offspring outperformed males in learning experiments. Therefore, our results revealed the detrimental effects of alcohol exposure during pregnancy on autophagy signaling in the hippocampus and cortex tissue of litters, which could affect the learning ability of animals.

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.

ROS-Activated TRPM2 Channel: Calcium Homeostasis in Cardiovascular/renal System and Speculation in Cardiorenal Syndrome.

The transient receptor potential melastatin 2 (TRPM2) channel is a nonselective calcium channel that is sensitive to oxidative stress (OS), and is widely expressed in multiple organs, such as the heart, kidney, and brain, which is inextricably related to calcium dyshomeostasis and downstream pathological events. Due to the increasing global burden of kidney or cardiovascular diseases (CVDs), safe and efficient drugs specific to novel targets are imperatively needed. Notably, investigation of the possibility to regard the TRPM2 channel as a new therapeutic target in ROS-related CVDs or renal diseases is urgently required because the roles of the TRPM2 channel in heart or kidney diseases have not received enough attention and thus have not been fully elaborated. Therefore, we aimed to review the involvement of the TRPM2 channel in cardiovascular disorders related to kidney or typical renal diseases and attempted to speculate about TRPM2-mediated mechanisms of cardiorenal syndrome (CRS) to provide representative perspectives for future research about novel and effective therapeutic strategies.

Uric acid promotes myocardial infarction injury via activating pyrin domain-containing 3 inflammasome and reactive oxygen species/transient receptor potential melastatin 2/Ca2+pathway.

Cardiomyocytes injury has been considered as a key contributor for myocardial infarction (MI). Uric acid (UA) can induce cardiomyocytes injury, which is closely related to NLRP3 activation and inflammatory factor generation. However, the mechanism how UA modulates cardiomyocytes remains elusive. Western blotting and qRT-PCR were applied for measuring protein and mRNA expression, respectively. ROS production and Ca2+ influx were measured by flow cytometry. Patch clamp technique was used for measuring transient receptor potential melastatin 2 (TRPM2) channel. Ligation of left anterior descending for 2 h was performed to induce MI animal model. The rats were treated by different concentration of uric acid. The artery tissues were stained by HE and collected for measurement of NLRP3 and inflammatory factors. Supplementation of UA significantly promoted apoptosis, and augmented the expression of intercellular adhesion molecule-1, chemoattractant protein-1, vascular cell adhesion molecule-1, and NLRP3 inflammasome. Knockdown of NLRP3 reversed the influence of UA on MI by decreasing collagen deposition, fibrotic area, apoptosis. The expression of NLRP3 inflammasome increased markedly after treatment of UA. UA activated ROS/TRPM2/Ca2+ pathway through targeting NLRP3. UA activated NLRP3 inflammasome and augments inflammatory factor production, which in turn exacerbates cardiomyocytes injury. Knockdown of NLRP3 reversed the influence of UA on apoptosis and cell cycle. UA may promote cardiomyocytes injury through activating NLRP3 inflammasome and ROS/TRPM2 channel/Ca2+ pathway.

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.

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.

TRPM2 contributes to neuroinflammation and cognitive deficits in a cuprizone-induced multiple sclerosis model via NLRP3 inflammasome.

Multiple sclerosis (MS) is a disease of the central nervous system (CNS) that is characterized by demyelination, axonal injury and neurological deterioration. Few medications are available for progressive MS, which is associated with neuroinflammation confined to the CNS compartment. Transient receptor potential melastatin 2 (TRPM2) is a calcium-permeable, non-selective cation channel that plays pathological roles in a wide range of neuroinflammatory diseases; however, the underlying molecular mechanisms of TRPM2 remain elusive. Here, we established a cuprizone model that presents hallmark MS pathologies to investigate the role of TRPM2 in progressive MS. We demonstrated that genetic deletion of TRPM2 yields protection from the cuprizone-induced demyelination, synapse loss, microglial activation, NLRP3 inflammasome activation and proinflammatory cytokines production and ultimately leads to an improvement in cognitive decline. Furthermore, we showed that the pharmacological inhibition of NLRP3 ameliorated the demyelination, neuroinflammation and cognitive impairment in the model with no additive effects on the TRPM2 KO mice. Taken together, these results indicated that TRPM2 plays important roles in regulating neuroinflammation in progressive MS via NLRP3 inflammasome, and the results shed light on TRPM2's potential role as a therapeutic target for MS.

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.

TRPM2 channel: A novel target for alleviating ischaemia-reperfusion, chronic cerebral hypo-perfusion and neonatal hypoxic-ischaemic brain damage.

The transient receptor potential melastatin-related 2 (TRPM2) channel, a reactive oxygen species (ROS)-sensitive cation channel, has been well recognized for being an important and common mechanism that confers the susceptibility to ROS-induced cell death. An elevated level of ROS is a salient feature of ischaemia-reperfusion, chronic cerebral hypo-perfusion and neonatal hypoxia-ischaemia. The TRPM2 channel is expressed in hippocampus, cortex and striatum, the brain regions that are critical for cognitive functions. In this review, we examine the recent studies that combine pharmacological and/or genetic interventions with using in vitro and in vivo models to demonstrate a crucial role of the TRPM2 channel in brain damage by ischaemia-reperfusion, chronic cerebral hypo-perfusion and neonatal hypoxic-ischaemia. We also discuss the current understanding of the underlying TRPM2-dependent cellular and molecular mechanisms. These new findings lead to the hypothesis of targeting the TRPM2 channel as a potential novel therapeutic strategy to alleviate brain damage and cognitive dysfunction caused by these conditions.

TRPM2-mediated extracellular Ca2+ entry promotes acinar cell necrosis in biliary acute pancreatitis.

KEY POINTS: Acute biliary pancreatitis is a significant clinical challenge as currently no specific pharmaceutical treatment exists. Intracellular Ca2+ overload, increased reactive oxygen species (ROS) production, mitochondrial damage and intra-acinar digestive enzyme activation caused by bile acids are hallmarks of acute biliary pancreatitis. Transient receptor potential melastatin 2 (TRPM2) is a non-selective cation channel that has recently emerged as an important contributor to oxidative-stress-induced cellular Ca2+ overload across different diseases. We demonstrated that TRPM2 is expressed in the plasma membrane of mouse pancreatic acinar and ductal cells, which can be activated by increased oxidative stress induced by H2 O2 treatment and contributed to bile acid-induced extracellular Ca2+ influx in acinar cells, which promoted acinar cell necrosis in vitro and in vivo. These results suggest that the inhibition of TRPM2 may be a potential treatment option for biliary pancreatitis. ABSTRACT: Acute biliary pancreatitis poses a significant clinical challenge as currently no specific pharmaceutical treatment exists. Disturbed intracellular Ca2+ signalling caused by bile acids is a hallmark of the disease, which induces increased reactive oxygen species (ROS) production, mitochondrial damage, intra-acinar digestive enzyme activation and cell death. Because of this mechanism of action, prevention of toxic cellular Ca2+ overload is a promising therapeutic target. Transient receptor potential melastatin 2 (TRPM2) is a non-selective cation channel that has recently emerged as an important contributor to oxidative-stress-induced cellular Ca2+ overload across different diseases. However, the expression and possible functions of TRPM2 in the exocrine pancreas remain unknown. Here we found that TRPM2 is expressed in the plasma membrane of mouse pancreatic acinar and ductal cells, which can be activated by increased oxidative stress induced by H2 O2 treatment. TRPM2 activity was found to contribute to bile acid-induced extracellular Ca2+ influx in acinar cells, but did not have the same effect in ductal cells. The generation of intracellular ROS in response to bile acids was remarkably higher in pancreatic acinar cells compared to isolated ducts, which can explain the difference between acinar and ductal cells. This activity promoted acinar cell necrosis in vitro independently from mitochondrial damage or mitochondrial fragmentation. In addition, bile-acid-induced experimental pancreatitis was less severe in TRPM2 knockout mice, whereas the lack of TRPM2 had no protective effect in cerulein-induced acute pancreatitis. Our results suggest that the inhibition of TRPM2 may be a potential treatment option for biliary pancreatitis.

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.

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.

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.

Pyridine Nucleotide Metabolites and Calcium Release from Intracellular Stores.

Ca2+ signals are probably the most common intracellular signaling cellular events, controlling an extensive range of responses in virtually all cells. Many cellular stimuli, often acting at cell surface receptors, evoke Ca2+ signals by mobilizing Ca2+ from intracellular stores. Inositol trisphosphate (IP3) was the first messenger shown to link events at the plasma membrane to release Ca2+ from the endoplasmic reticulum (ER), through the activation of IP3-gated Ca2+ release channels (IP3 receptors). Subsequently, two additional Ca2+ mobilizing messengers were discovered, cADPR and NAADP. Both are metabolites of pyridine nucleotides, and may be produced by the same class of enzymes, ADP-ribosyl cyclases, such as CD38. Whilst cADPR mobilizes Ca2+ from the ER by activation of ryanodine receptors (RyRs), NAADP releases Ca2+ from acidic stores by a mechanism involving the activation of two pore channels (TPCs). In addition, other pyridine nucleotides have emerged as intracellular messengers. ADP-ribose and 2'-deoxy-ADPR both activate TRPM2 channels which are expressed at the plasma membrane and in lysosomes.

A critical role of TRPM2 channel in Aß42 -induced microglial activation and generation of tumor necrosis factor-α.

Amyloid ß (Aß)-induced neuroinflammation plays an important part in Alzheimer's disease (AD). Emerging evidence supports a role for the transient receptor potential melastatin-related 2 (TRPM2) channel in Aß-induced neuroinflammation, but how Aß induces TRPM2 channel activation and this relates to neuroinflammation remained poorly understood. We investigated the mechanisms by which Aß42 activates the TRPM2 channel in microglial cells and the relationships to microglial activation and generation of tumor necrosis factor-α (TNF-α), a key cytokine implicated in AD. Exposure to 10-300 nM Aß42 induced concentration-dependent microglial activation and generation of TNF-α that were ablated by genetically deleting (TRPM2 knockout ;TRPM2-KO) or pharmacologically inhibiting the TRPM2 channel, revealing a critical role of this channel in Aß42 -induced microglial activation and generation of TNF-α. Mechanistically, Aß42 activated the TRPM2 channel via stimulating generation of reactive oxygen species (ROS) and activation of poly(ADPR) polymerase-1 (PARP-1). Aß42 -induced generation of ROS and activation of PARP-1 and TRPM2 channel were suppressed by inhibiting protein kinase C (PKC) and NADPH oxidases (NOX). Aß42 -induced activation of PARP-1 and TRPM2 channel was also reduced by inhibiting PYK2 and MEK/ERK. Aß42 -induced activation of PARP-1 was attenuated by TRPM2-KO and moreover, the remaining PARP-1 activity was eliminated by inhibiting PKC and NOX, but not PYK2 and MEK/ERK. Collectively, our results suggest that PKC/NOX-mediated generation of ROS and subsequent activation of PARP-1 play a role in Aß42 -induced TRPM2 channel activation and TRPM2-dependent activation of the PYK2/MEK/ERK signalling pathway acts as a positive feedback to further facilitate activation of PARP-1 and TRPM2 channel. These findings provide novel insights into the mechanisms underlying Aß-induced AD-related neuroinflammation.