Bilal Çiğ

Melatonin potentiates chemotherapy-induced cytotoxicity and apoptosis in rat pancreatic tumor cells

Abstract:  Melatonin has antitumor activity via several mechanisms including its antiproliferative and proapoptotic effects in addition to its potent antioxidant action. Thus, melatonin has proven useful in the treatment of tumors in association with chemotherapeutic drugs. This study was performed to evaluate the effect of melatonin on the cytotoxicity and apoptosis induced by three different chemotherapeutic agents, namely 5‐fluorouracil (5‐FU), cisplatin, and doxorubicin in the rat pancreatic tumor cell line AR42J. We found that both melatonin and the three chemotherapeutic drugs induce a time‐dependent decrease in AR42J cell viability, reaching the highest cytotoxic effect after 48 hr of incubation. Furthermore, melatonin significantly augmented the cytotoxicity of the chemotherapeutic agents. Consistently, cotreatment of AR42J cells with each of the chemotherapeutic agents in the presence of melatonin increased the population of apoptotic cells, elevated mitochondrial membrane depolarization, and augmented intracellular reactive oxygen species (ROS) production compared to treatment with each chemotherapeutic agent alone. These results provide evidence that in vitro melatonin enhances chemotherapy‐induced cytotoxicity and apoptosis in rat pancreatic tumor AR42J cells and, therefore, melatonin may be potentially applied to pancreatic tumor treatment as a powerful synergistic agent in combination with chemotherapeutic drugs.

Melatonin modulates wireless (2.45 GHz)-induced oxidative injury through TRPM2 and voltage gated Ca 2+ channels in brain and dorsal root ganglion in rat

We aimed to investigate the protective effects of melatonin and 2.45 GHz electromagnetic radiation (EMR) on brain and dorsal root ganglion (DRG) neuron antioxidant redox system, Ca(2+) influx, cell viability and electroencephalography (EEG) records in the rat. Thirty two rats were equally divided into four different groups namely group A1: Cage control, group A2: Sham control, group B: 2.45 GHz EMR, group C: 2.45 GHz EMR+melatonin. Groups B and C were exposed to 2.45 GHz EMR during 60 min/day for 30 days. End of the experiments, EEG records and the brain cortex and DRG samples were taken. Lipid peroxidation (LP), cell viability and cytosolic Ca(2+) values in DRG neurons were higher in group B than in groups A1 and A2 although their concentrations were increased by melatonin, 2-aminoethyldiphenyl borinate (2-APB), diltiazem and verapamil supplementation. Spike numbers of EEG records in group C were lower than in group B. Brain cortex vitamin E concentration was higher in group C than in group B. In conclusion, Melatonin supplementation in DRG neurons and brain seems to have protective effects on the 2.45 GHz-induced increase Ca(2+) influx, EEG records and cell viability of the hormone through TRPM2 and voltage gated Ca(2+) channels.

Aminoethoxydiphenyl borate and flufenamic acid inhibit Ca2+ influx through TRPM2 channels in rat dorsal root ganglion neurons activated by ADP-ribose and rotenone.

Exposure to oxidative stress causes health problems, including sensory neuron neuropathy and pain. Rotenone is a toxin used to generate intracellular oxidative stress in neurons. However, the mechanism of toxicity in dorsal root ganglion (DRG) neurons has not been characterized. Melastatin-like transient receptor potential 2 (TRPM2) channel activation and inhibition in response to oxidative stress, ADP-ribose (ADPR), flufenamic acid (FFA) and 2-aminoethoxydiphenyl borate (2-APB) in DRG neurons are also not clear. We tested the effects of FFA and 2-APB on ADPR and rotenone-induced TRPM2 cation channel activation in DRG neurons of rats. DRG neurons were freshly isolated from rats and studied with the conventional whole-cell patch-clamp technique. Rotenone, FFA and 2-APB were extracellularly added through the patch chamber, and ADPR was applied intracellularly through the patch pipette. TRPM2 cation currents were consistently induced by ADPR and rotenone. Current densities of the neurons were higher in the ADPR and rotenone groups than in control. The time courses (gating times) in the neurons were longer in the rotenone than in the ADPR group. ADPR and rotenone-induced TRPM2 currents were totally blocked by 2-APB and partially blocked by FFA. In conclusion, TRPM2 channels were constitutively activated by ADPR and rotenone, and 2-APB and FFA induced an inhibitory effect on TRPM2 cation channel currents in rat DRG neurons. Since oxidative stress is a common feature of neuropathic pain and diseases of sensory neurons, the present findings have broad application to the etiology of neuropathic pain and diseases of DRG neurons.

Investigation of the effects of distance from sources on apoptosis, oxidative stress and cytosolic calcium accumulation via TRPV1 channels induced by mobile phones and Wi-Fi in breast cancer cells.

TRPV1 is a Ca2+ permeable channel and gated by noxious heat, oxidative stress and capsaicin (CAP). Some reports have indicated that non-ionized electromagnetic radiation (EMR)-induces heat and oxidative stress effects. We aimed to investigate the effects of distance from sources on calcium signaling, cytosolic ROS production, cell viability, apoptosis, plus caspase-3 and -9 values induced by mobile phones and Wi-Fi in breast cancer cells MCF-7 human breast cancer cell lines were divided into A, B, C and D groups as control, 900, 1800 and 2450 MHz groups, respectively. Cells in Group A were used as control and were kept in cell culture conditions without EMR exposure. Groups B, C and D were exposed to the EMR frequencies at different distances (0 cm, 1 cm, 5 cm, 10 cm, 20 cm and 25 cm) for 1h before CAP stimulation. The cytosolic ROS production, Ca2+ concentrations, apoptosis, caspase-3 and caspase-9 values were higher in groups B, C and D than in A group at 0 cm, 1 cm and 5 cm distances although cell viability (MTT) values were increased by the distances. There was no statistically significant difference in the values between control, 20 and 25 cm. Wi-Fi and mobile phone EMR placed within 10 cm of the cells induced excessive oxidative responses and apoptosis via TRPV1-induced cytosolic Ca2+ accumulation in the cancer cells. Using cell phones and Wi-Fi sources which are farther away than 10 cm may provide useful protection against oxidative stress, apoptosis and overload of intracellular Ca2+. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.

2.45-Gz wireless devices induce oxidative stress and proliferation through cytosolic Ca2+ influx in human leukemia cancer cells

Purpose: Electromagnetic radiation from wireless devices may affect biological systems by increasing free radicals. The present study was designed to determine the effects of 2.45 GHz radiation on the antioxidant redox system, calcium ion signaling, cell count and viability in human leukemia 60 cells. Materials and methods: Twelve cell cultures were equally divided into two main groups as controls (n = 6) and irradiated (n = 6) and then subdivided into four different subgroups depending on the duration of exposure, namely 1, 2, 12 and 24 hours. The samples were analyzed immediately after the experimental period. Results: The extent of lipid peroxidation, cytosolic free Ca2+ and cell numbers were higher in 2.45 GHz groups than in the controls. The increase of cytosolic free Ca2+ concentrations was radiation time-dependent and was highest at 24-h exposure. The reduced glutathione, glutathione peroxidase, vitamin C and cell viability values did not show any changes in any of the experimental groups. 2-aminoethyl diphenylborinate inhibits Ca2+ ions influx by blockage of the transient receptor potential melastatin 2. Conclusions: 2.45 GHz electromagnetic radiation appears to induce proliferative effects through oxidative stress and Ca2+ influx although blocking of transient receptor potential melastatin 2 channels by 2-aminoethyl diphenylborinate seems to counteract the effects on Ca2+ ions influx.

Modulation of oxidative stress and Ca2+ mobilization through TRPM2 channels in rat dorsal root ganglion neuron by Hypericum perforatum

A main component of St. Johns Wort (Hypericum perforatum, HP) is hyperforin which has antioxidant properties in dorsal root ganglion (DRG) neurons, due to its ability to modulate NADPH oxidase and protein kinase C. Recent reports indicate that oxidative stress through NADPH oxidase activates TRPM2 channels. HP may be a useful treatment for Ca(2+) entry and oxidative stress through modulation of TRPM2 channels in the DRG. We aimed to investigate the protective role of HP on Ca(2+) entry and oxidative stress through TRPM2 channels in DRG neurons of rats. The native rat DRG neurons were used in whole-cell patch-clamp, Fura-2 and antioxidant experiments. Appropriate, nontoxic concentrations and incubation times for HP were determined in the DRG neurons by assessing cell viability. The H2O2-induced TRPM2 currents were inhibited by 2-aminoethyl diphenylborinate (2-APB) and N-(p-amylcinnamoyl)anthranilic acid (ACA). TRPM2 current densities and cytosolic free Ca(2+) concentration in the neurons were also reduced by HP (2 and 24h). In Fura-2 experiments, cytosolic Ca(2+) mobilization was reduced by voltage-gated calcium channel blockers (verapamil+diltiazem, V+D) and HP. Glutathione peroxidase activity and GSH values in the DRG were high in HP, 2-APB and V+D groups although lipid peroxidation level was low in the groups. In conclusion, we observed a protective role for HP on Ca(2+) entry through a TRPM2 channel in the DRG neurons. Since over-production of oxidative stress and Ca(2+) entry are implicated in the pathophysiology of neuropathic pain and neuronal inflammation, our findings may be relevant to the etiology and treatment of neuropathology in DRG neurons.

Melatonin and selenium reduce plasma cytokine and brain oxidative stress levels in diabetic rats.

Abstract Background: Hyperglycaemia-induced progression of brain and erythrocyte oxidative injuries might be modulated by melatonin and selenium as potent antioxidants. The present study was conducted to explore whether melatonin and selenium protect against diabetic brain and erythrocyte oxidative stress levels in streptozotocin (STZ)-induced diabetic rats. Materials and methods: Seventy rats were equally divided into seven groups. The first and second groups were used as untreated and placebo treated controls. The third group was treated with STZ to induce diabetes. The fourth and sixth groups received 10 mg kg−1 melatonin. The fifth and seventh groups were treated with 1.5 mg kg−1 selenium (sodium selenite). The sixth and seventh groups were treated with STZ administered with melatonin and selenium as described for the fourth and fifth groups. Results: Brain and erythrocyte lipid peroxidation levels and plasma IL-1β and IL-4 levels were high in the STZ group, although they were low in melatonin and selenium treatments. Decreased glutathione peroxidase, reduced glutathione, total antioxidant status, vitamins A and vitamin E values in brain and erythrocyte of STZ group were increased by melatonin and selenium treatments. Discussion: Melatonin and selenium induced protective effects against diabetes-induced brain and erythrocyte oxidative injuries through regulation of the antioxidant level and cytokine production.

Selenium potentiates the anticancer effect of cisplatin against oxidative stress and calcium ion signaling-induced intracellular toxicity in MCF-7 breast cancer cells: involvement of the TRPV1 channel.

Abstract Background: In breast cancers, calcium signaling is a main cause of proliferation and apoptosis of breast cancer cells. Although previous studies have implicated the transient receptor potential vanilloid 1 (TRPV1) cation channel, the synergistic inhibition effects of selenium (Se) and cisplatin in cancer and the suppression of ongoing apoptosis have not yet been investigated in MCF-7 breast cancer cells. This study investigates the anticancer properties of Se through TRPV1 channel activity in MCF-7 breast cancer cell line cultures when given alone or in combination with cisplatin. Materials: The MCF-7 cells were divided into four groups: the control group, the Se-treated group (200 nM), the cisplatin-treated group (40 μM) and the Se + cisplatin-treated group. Results: The intracellular free calcium ion concentration and current densities increased with TRPV1 channel activator capsaicin (0.01 mM), but they decreased with the TRPV1 blocker capsazepine (0.1 mM), Se, cisplatin, and Se + cisplatin incubations. However, mitochondrial membrane depolarization, apoptosis, and the caspase 3, and caspase 9 values increased in the Se-treated group and the cisplatin-treated group, although Western blot (procaspase 3 and 9) results and the cell viability levels decreased with the Se and Se + cisplatin treatments. Apoptosis and caspase-3 were further increased with the Se + cisplatin treatment. Intracellular reactive oxygen species production increased with the cisplatin treatment, but not with the Se treatment. Conclusion: This study’s results report, for the first time, that at a cellular level, Se and cisplatin interact on the same intracellular toxic cascade, and the combination of these two drugs can result in a remarkable anticancer effect through modulation of the TRPV1.

Hypericum perforatum modulates apoptosis and calcium mobilization through voltage-gated and TRPM2 calcium channels in neutrophil of patients with Behcet's disease.

Behcet’s disease (BD) is a chronic, inflammatory, and multisystemic condition although its pathogenesis is uncertain. Main component of St. John’s wort (Hypericum perforatum, HP) is hyperforin and induces antiinflammatory and antioxidant properties. We aimed to investigate effects of HP on oxidative stress, apoptosis, and cytosolic-free Ca2+ [Ca2+]i concentration in neutrophil of BD patients. Nine new-diagnosed active patients with BD and nine control subjects were included in the study. Disease activity was considered by clinical findings. Neutrophil samples were obtained from the patients and controls. The neutrophils from patients were divided into three subgroups and were incubated with HP, voltage-gated calcium channel (VGCC) blockers, (verapamil+dilitiazem) and non-specific TRPM2 channel blocker (2-aminoethyl diphenylborinate, 2-APB), respectively. The neutrophils were stimulated by fMLP as a Ca2+-concentration agonist and oxidative stress former. Caspase-3, caspase-9, apoptosis, lipid peroxidation, and [Ca2+]i values were high in the patient groups, although cell viability, glutathione (GSH), and glutathione peroxidase (GSH-Px) values were low in patient group. However, the [Ca2+]i, caspase-3, and caspase-9 values decreased markedly in patient+HP group although GSH and GSH-Px values increased in the group. The [Ca2+]i concentration was also decreased in the patient group by V+D, 2-APB, and HP incubations. In conclusion, we observed the importance of neutrophil Ca2+ entry, apoptosis, and oxidative stress through gating VGCC and TRPM2 channels in the neutrophils in the pathogenesis and activation of the patients with BD. HP induced protective effects on oxidative stress by modulating Ca2+ influx in BD patients.Graphical Abstract

Role of TRPM2 cation channels in dorsal root ganglion of rats after experimental spinal cord injury.

Introduction: We sought to determine the contribution of oxidative stress–dependent activation of TRPM2 and L‐type voltage‐gated Ca2+ channels (VGCC) in dorsal root ganglion (DRG) neurons of rats after spinal cord injury (SCI). Methods: The rats were divided into 4 groups: control; sham control; SCI; and SCI+nimodipine groups. The neurons of the SCI groups were also incubated with non‐specific TRPM2 channel blockers, 2‐aminoethoxydiphenylborate (2‐APB) and N‐(p‐amylcinnamoyl)anthranilic acid (ACA), before H2O2 stimulation. Results:The [Ca2+]i concentrations were higher in the SCI group than in the control groups, although their concentrations were decreased by nimodipine and 2‐APB. The H2O2‐induced TRPM2 current densities in patch‐clamp experiments were decreased by ACA and 2‐APB incubation. In the nimodipine group, the TRPM2 channels of neurons were not activated by H2O2 or cumene hydroperoxide. Conclusions: Increased Ca2+ influx and currents in DRG neurons after spinal injury indicated TRPM2 and voltage‐gated Ca2+ channel activation. Muscle Nerve 48: 945–950, 2013