Doxorubicin is a widely used chemotherapeutic drug, effective on patients with acute lymphoblasti... more Doxorubicin is a widely used chemotherapeutic drug, effective on patients with acute lymphoblastic leukemia but associated with significant long term cardio-toxicity. Menadione (vitamine K3) and the flavonoid quercetin are known as strong apoptogens in human leukemia Jurkat T cells. We explored the potential synergic cytotoxic effects of doxorubicin in association with quercetin and Menadione in this cellular model for acute lymphoblastic leukemia. Cellular viability, apoptosis, necrosis oxidative stress and cellular cycle were determined by flow cytometry utilizing Jurkat lymphoblasts labeled with Annexin V-FITC/7-AAD, CM-H2DCFDA/7-AAD and propidium iodide respectively. Results indicate a dose-dependent oxidative-stress generation, cell cycle arrest and apoptosis induction by doxorubicin alone, correlated with a decrease of the required doses when the anticancer drug was associated with quercetin and menadione, hence supporting the theory of an additive cytotoxic effect on leukemia...
Heat-activated ion channels from the vanilloid-type TRP group (TRPV1-4) seem to be central for he... more Heat-activated ion channels from the vanilloid-type TRP group (TRPV1-4) seem to be central for heat-sensitivity of nociceptive sensory neurons. Displaying a high-threshold (> 52°C) for activation, TRPV2 was proposed to act as a sensor for intense noxious heat in mammalian sensory neurons. However, although TRPV2 is expressed in a distinct population of thinly myelinated primary afferents, a widespread expression in a variety of neuronal and non-neuronal tissues suggests a more diverse physiological role of TRPV2. In its role as a heat-sensor, TRPV2 has not been thoroughly characterized in terms of biophysical and pharmacological properties. In the present study, we demonstrate that the features of heterologously expressed rat TRPV2 closely resemble those of high-threshold heat-evoked currents in medium-and large-sized capsaicin-insensitive rat dorsal root ganglion (DRG) neurons. Both in TRPV2-expressing human embryonic kidney (HEK)293t cells and in DRGs, high-threshold heat-currents were sensitized by repeated activation and by the TRPV1-3 agonist, 2-aminoethoxydiphenyl borate (2-APB). In addition to a previously described block by ruthenium red, we identified the trivalent cations, lanthanum (La 3+) and gadolinium (Gd 3+) as potent blockers of TRPV2. Thus, we present a new pharmacological tool to distinguish between heat responses of TRPV2 and the closely related capsaicin-receptor, TRPV1, which is strongly sensitized by trivalent cations. We demonstrate that self-sensitization of heat-evoked currents through TRPV2 does not require extracellular calcium and that TRPV2 can be activated in cell-free membrane patches in the outside-out configuration. Taken together our results provide new evidence for a role of TRPV2 in mediating high-threshold heat responses in a subpopulation of mammalian sensory neurons.
Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using... more Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using the Annexin V/propidium iodide (PI) standard assay, and a simple PI staining in Triton X-100/digitonin-enriched PI/RNase buffer, respectively. Cells treated with doxorubicin or menadione displayed a very strong correlation between the apoptotic cell fraction measured by the Annexin V/PI assay, and the weight of a secondary cell population that emerged on the forward scatter (FS)/PI plot, as well as on the side scatter (SS)/PI and FL1/PI plots generated from parallel cell cycle recordings. In both cases, the Pearson correlation coefficients were >0.99. In cell cycle determinations, PI fluorescence was detected on FL3 (620/30 nm), and control samples exhibited the expected linear dependence of FL3 on FL1 (525/40 nm) signals. However, increasing doses of doxorubicin or menadione generated a growing subpopulation of cells displaying a definite right-shift on the FS/FL3, SS/FL3 and FL1/FL...
Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using... more Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using the Annexin V/propidium iodide (PI) standard assay, and a simple PI staining in Triton X-100/digitonin-enriched PI/RNase buffer, respectively. Cells treated with doxorubicin or menadione displayed a very strong correlation between the apoptotic cell fraction measured by the Annexin V/PI assay, and the weight of a secondary cell population that emerged on the forward scatter (FS)/PI plot, as well as on the side scatter (SS)/PI and FL1/PI plots generated from parallel cell cycle recordings. In both cases, the Pearson correlation coefficients were >0.99. In cell cycle determinations, PI fluorescence was detected on FL3 (620/30 nm), and control samples exhibited the expected linear dependence of FL3 on FL1 (525/40 nm) signals. However, increasing doses of doxorubicin or menadione generated a growing subpopulation of cells displaying a definite right-shift on the FS/FL3, SS/FL3 and FL1/FL...
Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using... more Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using the Annexin V/propidium iodide (PI) standard assay, and a simple PI staining in Triton X-100/digitonin-enriched PI/RNase buffer, respectively. Cells treated with doxorubicin or menadione displayed a very strong correlation between the apoptotic cell fraction measured by the Annexin V/PI assay, and the weight of a secondary cell population that emerged on the forward scatter (FS)/PI plot, as well as on the side scatter (SS)/PI and FL1/PI plots generated from parallel cell cycle recordings. In both cases, the Pearson correlation coefficients were >0.99. In cell cycle determinations, PI fluorescence was detected on FL3 (620/30 nm), and control samples exhibited the expected linear dependence of FL3 on FL1 (525/40 nm) signals. However, increasing doses of doxorubicin or menadione generated a growing subpopulation of cells displaying a definite right-shift on the FS/FL3, SS/FL3 and FL1/FL...
Journal of Pharmacology and Experimental Therapeutics, 2013
High concentrations of nicotine, as in the saliva of oral tobacco consumers or in smoking cessati... more High concentrations of nicotine, as in the saliva of oral tobacco consumers or in smoking cessation aids, have been shown to sensitize/activate recombinant transient receptor potential vanilloid type 1 (rTRPV1) and mouse TRPA1 (mTRPA1) channels. By measuring stimulated calcitonin gene-related peptide (CGRP) release from the isolated mouse trachea, we established a bimodal concentration-response relationship with a threshold below 10 mM (2)-nicotine, a maximum at 100 mM, an apparent nadir between 0.5 and 10 mM, and a renewed increase at 20 mM. The first peak was unchanged in TRPV1/A1 double-null mutants as compared with wild-types and was abolished by specific nicotinic acetylcholine receptor (nAChR) inhibitors and by camphor, discovered to act as nicotinic antagonist. The nicotine response at 20 mM was strongly pH e -dependent,five times greater at pH 9.0 than 7.4, indicating that intracellular permeation of the (uncharged) alkaloid was required to reach the TRPV1/A1 binding sites. The response was strongly reduced in both null mutants, and more so in double-null mutants. Upon measuring calcium transients in nodose/jugular and dorsal root ganglion neurons in response to 100 mM nicotine, 48% of the vagal (but only 14% of the somatic) sensory neurons were activated, the latter very weakly. However, nicotine 20 mM at pH 9.0 repeatedly activated almost every single cultured neuron, partly by releasing intracellular calcium and independent of TRPV1/A1 and nAChRs. In conclusion, in mouse tracheal sensory nerves nAChRs are 200fold more sensitive to nicotine than TRPV1/A1; they are widely coexpressed with the capsaicin receptor among vagal sensory neurons and twice as abundant as TRPA1. Nicotine is the major stimulant in tobacco, and its sensory impact through nAChRs should not be disregarded. interstitial fluid; TRPA1, transient receptor potential ankyrin 1; TRPV1, transient receptor potential vanilloid type 1.
Temperature sensing is a crucial feature of the nervous system, enabling organisms to avoid physi... more Temperature sensing is a crucial feature of the nervous system, enabling organisms to avoid physical danger and choose optimal environments for survival. TRPM8 (Transient Receptor Potential Melastatin type 8) belongs to a select group of ion channels which are gated by changes in temperature, are expressed in sensory nerves and/or skin cells and may be involved in temperature sensing. This channel is activated by a moderate decrease in temperature, with a threshold of approximately 25 °C in heterologous expression systems, and by a variety of natural and synthetic compounds, including menthol. While the physiological role of TRPM8 as a transducer of gentle cooling is widely accepted, its involvement in acute noxious cold sensing in healthy tissues is still under debate. Although accumulating evidence indicates that TRPM8 is involved in neuropathic cold allodynia, in some animal models of nerve injury peripheral and central activation of TRPM8 is followed by analgesia. A variety of inflammatory mediators, including bradykinin and prostaglandin E(2), modulate TRPM8 by inhibiting the channel and shifting its activation threshold to colder temperatures, most likely counteracting the analgesic action of TRPM8. While important progress has been made in unraveling the biophysical features of TRPM8, including the revelation of its voltage dependence, the precise mechanism involved in temperature sensing by this channel is still not completely understood. This article will review the current status of knowledge regarding the (patho)physiological role(s) of TRPM8, its modulation by inflammatory mediators, the signaling pathways involved in this regulation, and the biophysical properties of the channel.
Specify what is your 1st choice of session: CSIV-01_Cancer signalling Abstract: Doxorubicin, an e... more Specify what is your 1st choice of session: CSIV-01_Cancer signalling Abstract: Doxorubicin, an effective anticancer drug, has benn reported to induce either apoptosis or necrosis in various cancer cell lines, in function of dose and treatment duration. We investigated the antiproliferative effects of doxorubicin at clinical doses (50-100 nM) and duration (24h, 72h) in human leukemia JUrkat cells. Doxorubicin induced G 2 /M arrest and significant apoptosis (up to 54% after 72h of exposure), as assessed by flow cytometry analysis of the DNA content. However, cellular viability determined by flow cytometry on propidium iodide labeled cells was 1.6% after the 72h treatment with 100 nM doxorubicin, indicating a significant fraction of necrotic cells as well. Neither quercetin nor menadione or their combination at clinical concentrations (10-15 µM) enhanced doxorubicin-induced apoptosis, despite their apoptigenic effects reported in the Jurkat cell line [1, 2]. Fluorimetric measurements on fura-2 loaded cells showed that doxorubicin (7.5 µM) induced Ca 2+ release at slow rate and elicited a three-fold, progressive increase in cytosolic Ca 2+ concentration over the next 55 min. from drug addition. However, since the fluorescence of internalized doxorubicin displayed a considerable and rapid increase after extracellular addition, this result indicates a sustained but indirect activation of the Ca 2+ release channels. The observed Ca 2+ increase was partly mediated by ryanodine receptors, so that the presence of the specific inhibitor, ryanodine (200 µM), consistently inhibited the doxorubicin-evoked Ca 2+ signal. We also found by spectrofluorimetry that doxorubicin at clinical doses and dose rates generates oxidative stress, which is enhanced by menadione (10-15 µM), but is alleviated by both quercetin (10-15 µM) and the quercetin-menadione combination. However, these agents did not improve cellular viability following the doxorubicin treatment. In addition, quercetin did not affect the cell cycle distribution of doxorubicin treated-cells, whereas menadione and the quercetin-menadione concentration apparently abolished the doxorubicininduced without further progression through the cell cycle phases.
Objectives. The goal of this study was to explore the chemotherapeutic potential of Epigallocatec... more Objectives. The goal of this study was to explore the chemotherapeutic potential of Epigallocatechine-3-gallate (EGCG) and menadione (vitamin K3; MD), and to determine whether a synergic interaction exists between the two agents that could enhance significantly their antitumoral effect in a cellular model for acute lymphoblastic leukemia (ALL). To this end, we investigated the antiproliferative effect of EGCG and MD, applied alone or in combination on human leukemia Jurkat lymphoblasts. Some underlying cellular mechanisms were also scrutinized. Materials and methods. Cell suspensions of Jurkat lymphoblasts were treated at various concentrations of EGCG and/or MD. Cell cycle and apoptosis/necrosis were determined by flow cytometry, using the fluorescent indicators propidium iodide and Annexin V-FITC/7-AAD, respectively. Clonogenic survival was evaluated as the colony forming capacity in 96-well plates. Determination of oxidative stress and mitochondrial polarization was performed by ...
Please cite this article in press as: Baran I, et al. Novel insights into the antiproliferative e... more Please cite this article in press as: Baran I, et al. Novel insights into the antiproliferative effects and synergism of quercetin and menadione in human leukemia Jurkat T cells. Leuk Res (2014), http://dx.
The interaction between cold sensitivity and inflammation in mammals is not entirely understood. ... more The interaction between cold sensitivity and inflammation in mammals is not entirely understood. We have used adult rat dorsal root ganglion neurones in primary culture together with calcium microfluorimetry to assess the effects of selected inflammatory mediators on cold responses of cold- and menthol-sensitive (most likely TRPM8-expressing) neurones. We observed a high degree of functional co-expression of TRPM8, the receptors for the inflammatory agents bradykinin, prostaglandin E2 and histamine, and TRPA1 in cultured sensory neurones. Treatment with either bradykinin or prostaglandin E2 led to a reduction in the amplitude of the response to cooling and shifted the threshold temperature to colder values, and we provide evidence for a role of protein kinases C and A, respectively, in mediating these effects. In both cases the effects were mainly restricted to the subgroups of cold- and menthol-sensitive cells which had responded to the application of the inflammatory agents at basal temperature. This desensitization of cold-sensitive neurones may enhance inflammatory pain by removing the analgesic effects of gentle cooling.
The interaction between cold sensitivity and inflammation in mammals is not entirely understood. ... more The interaction between cold sensitivity and inflammation in mammals is not entirely understood. We have used adult rat dorsal root ganglion neurones in primary culture together with calcium microfluorimetry to assess the effects of selected inflammatory mediators on cold responses of cold- and menthol-sensitive (most likely TRPM8-expressing) neurones. We observed a high degree of functional co-expression of TRPM8, the receptors for the inflammatory agents bradykinin, prostaglandin E2 and histamine, and TRPA1 in cultured sensory neurones. Treatment with either bradykinin or prostaglandin E2 led to a reduction in the amplitude of the response to cooling and shifted the threshold temperature to colder values, and we provide evidence for a role of protein kinases C and A, respectively, in mediating these effects. In both cases the effects were mainly restricted to the subgroups of cold- and menthol-sensitive cells which had responded to the application of the inflammatory agents at basal temperature. This desensitization of cold-sensitive neurones may enhance inflammatory pain by removing the analgesic effects of gentle cooling.
Heat-activated ion channels from the vanilloid-type TRP group (TRPV1-4) seem to be central for he... more Heat-activated ion channels from the vanilloid-type TRP group (TRPV1-4) seem to be central for heat-sensitivity of nociceptive sensory neurons. Displaying a high-threshold (> 52°C) for activation, TRPV2 was proposed to act as a sensor for intense noxious heat in mammalian sensory neurons. However, although TRPV2 is expressed in a distinct population of thinly myelinated primary afferents, a widespread expression in a variety of neuronal and non-neuronal tissues suggests a more diverse physiological role of TRPV2. In its role as a heat-sensor, TRPV2 has not been thoroughly characterized in terms of biophysical and pharmacological properties. In the present study, we demonstrate that the features of heterologously expressed rat TRPV2 closely resemble those of high-threshold heat-evoked currents in medium-and large-sized capsaicin-insensitive rat dorsal root ganglion (DRG) neurons. Both in TRPV2-expressing human embryonic kidney (HEK)293t cells and in DRGs, high-threshold heat-currents were sensitized by repeated activation and by the TRPV1-3 agonist, 2-aminoethoxydiphenyl borate (2-APB). In addition to a previously described block by ruthenium red, we identified the trivalent cations, lanthanum (La 3+ ) and gadolinium (Gd 3+ ) as potent blockers of TRPV2. Thus, we present a new pharmacological tool to distinguish between heat responses of TRPV2 and the closely related capsaicin-receptor, TRPV1, which is strongly sensitized by trivalent cations. We demonstrate that self-sensitization of heat-evoked currents through TRPV2 does not require extracellular calcium and that TRPV2 can be activated in cell-free membrane patches in the outside-out configuration. Taken together our results provide new evidence for a role of TRPV2 in mediating high-threshold heat responses in a subpopulation of mammalian sensory neurons.
Doxorubicin is a widely used chemotherapeutic drug, effective on patients with acute lymphoblasti... more Doxorubicin is a widely used chemotherapeutic drug, effective on patients with acute lymphoblastic leukemia but associated with significant long term cardio-toxicity. Menadione (vitamine K3) and the flavonoid quercetin are known as strong apoptogens in human leukemia Jurkat T cells. We explored the potential synergic cytotoxic effects of doxorubicin in association with quercetin and Menadione in this cellular model for acute lymphoblastic leukemia. Cellular viability, apoptosis, necrosis oxidative stress and cellular cycle were determined by flow cytometry utilizing Jurkat lymphoblasts labeled with Annexin V-FITC/7-AAD, CM-H2DCFDA/7-AAD and propidium iodide respectively. Results indicate a dose-dependent oxidative-stress generation, cell cycle arrest and apoptosis induction by doxorubicin alone, correlated with a decrease of the required doses when the anticancer drug was associated with quercetin and menadione, hence supporting the theory of an additive cytotoxic effect on leukemia...
Heat-activated ion channels from the vanilloid-type TRP group (TRPV1-4) seem to be central for he... more Heat-activated ion channels from the vanilloid-type TRP group (TRPV1-4) seem to be central for heat-sensitivity of nociceptive sensory neurons. Displaying a high-threshold (> 52°C) for activation, TRPV2 was proposed to act as a sensor for intense noxious heat in mammalian sensory neurons. However, although TRPV2 is expressed in a distinct population of thinly myelinated primary afferents, a widespread expression in a variety of neuronal and non-neuronal tissues suggests a more diverse physiological role of TRPV2. In its role as a heat-sensor, TRPV2 has not been thoroughly characterized in terms of biophysical and pharmacological properties. In the present study, we demonstrate that the features of heterologously expressed rat TRPV2 closely resemble those of high-threshold heat-evoked currents in medium-and large-sized capsaicin-insensitive rat dorsal root ganglion (DRG) neurons. Both in TRPV2-expressing human embryonic kidney (HEK)293t cells and in DRGs, high-threshold heat-currents were sensitized by repeated activation and by the TRPV1-3 agonist, 2-aminoethoxydiphenyl borate (2-APB). In addition to a previously described block by ruthenium red, we identified the trivalent cations, lanthanum (La 3+) and gadolinium (Gd 3+) as potent blockers of TRPV2. Thus, we present a new pharmacological tool to distinguish between heat responses of TRPV2 and the closely related capsaicin-receptor, TRPV1, which is strongly sensitized by trivalent cations. We demonstrate that self-sensitization of heat-evoked currents through TRPV2 does not require extracellular calcium and that TRPV2 can be activated in cell-free membrane patches in the outside-out configuration. Taken together our results provide new evidence for a role of TRPV2 in mediating high-threshold heat responses in a subpopulation of mammalian sensory neurons.
Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using... more Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using the Annexin V/propidium iodide (PI) standard assay, and a simple PI staining in Triton X-100/digitonin-enriched PI/RNase buffer, respectively. Cells treated with doxorubicin or menadione displayed a very strong correlation between the apoptotic cell fraction measured by the Annexin V/PI assay, and the weight of a secondary cell population that emerged on the forward scatter (FS)/PI plot, as well as on the side scatter (SS)/PI and FL1/PI plots generated from parallel cell cycle recordings. In both cases, the Pearson correlation coefficients were >0.99. In cell cycle determinations, PI fluorescence was detected on FL3 (620/30 nm), and control samples exhibited the expected linear dependence of FL3 on FL1 (525/40 nm) signals. However, increasing doses of doxorubicin or menadione generated a growing subpopulation of cells displaying a definite right-shift on the FS/FL3, SS/FL3 and FL1/FL...
Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using... more Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using the Annexin V/propidium iodide (PI) standard assay, and a simple PI staining in Triton X-100/digitonin-enriched PI/RNase buffer, respectively. Cells treated with doxorubicin or menadione displayed a very strong correlation between the apoptotic cell fraction measured by the Annexin V/PI assay, and the weight of a secondary cell population that emerged on the forward scatter (FS)/PI plot, as well as on the side scatter (SS)/PI and FL1/PI plots generated from parallel cell cycle recordings. In both cases, the Pearson correlation coefficients were >0.99. In cell cycle determinations, PI fluorescence was detected on FL3 (620/30 nm), and control samples exhibited the expected linear dependence of FL3 on FL1 (525/40 nm) signals. However, increasing doses of doxorubicin or menadione generated a growing subpopulation of cells displaying a definite right-shift on the FS/FL3, SS/FL3 and FL1/FL...
Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using... more Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using the Annexin V/propidium iodide (PI) standard assay, and a simple PI staining in Triton X-100/digitonin-enriched PI/RNase buffer, respectively. Cells treated with doxorubicin or menadione displayed a very strong correlation between the apoptotic cell fraction measured by the Annexin V/PI assay, and the weight of a secondary cell population that emerged on the forward scatter (FS)/PI plot, as well as on the side scatter (SS)/PI and FL1/PI plots generated from parallel cell cycle recordings. In both cases, the Pearson correlation coefficients were >0.99. In cell cycle determinations, PI fluorescence was detected on FL3 (620/30 nm), and control samples exhibited the expected linear dependence of FL3 on FL1 (525/40 nm) signals. However, increasing doses of doxorubicin or menadione generated a growing subpopulation of cells displaying a definite right-shift on the FS/FL3, SS/FL3 and FL1/FL...
Journal of Pharmacology and Experimental Therapeutics, 2013
High concentrations of nicotine, as in the saliva of oral tobacco consumers or in smoking cessati... more High concentrations of nicotine, as in the saliva of oral tobacco consumers or in smoking cessation aids, have been shown to sensitize/activate recombinant transient receptor potential vanilloid type 1 (rTRPV1) and mouse TRPA1 (mTRPA1) channels. By measuring stimulated calcitonin gene-related peptide (CGRP) release from the isolated mouse trachea, we established a bimodal concentration-response relationship with a threshold below 10 mM (2)-nicotine, a maximum at 100 mM, an apparent nadir between 0.5 and 10 mM, and a renewed increase at 20 mM. The first peak was unchanged in TRPV1/A1 double-null mutants as compared with wild-types and was abolished by specific nicotinic acetylcholine receptor (nAChR) inhibitors and by camphor, discovered to act as nicotinic antagonist. The nicotine response at 20 mM was strongly pH e -dependent,five times greater at pH 9.0 than 7.4, indicating that intracellular permeation of the (uncharged) alkaloid was required to reach the TRPV1/A1 binding sites. The response was strongly reduced in both null mutants, and more so in double-null mutants. Upon measuring calcium transients in nodose/jugular and dorsal root ganglion neurons in response to 100 mM nicotine, 48% of the vagal (but only 14% of the somatic) sensory neurons were activated, the latter very weakly. However, nicotine 20 mM at pH 9.0 repeatedly activated almost every single cultured neuron, partly by releasing intracellular calcium and independent of TRPV1/A1 and nAChRs. In conclusion, in mouse tracheal sensory nerves nAChRs are 200fold more sensitive to nicotine than TRPV1/A1; they are widely coexpressed with the capsaicin receptor among vagal sensory neurons and twice as abundant as TRPA1. Nicotine is the major stimulant in tobacco, and its sensory impact through nAChRs should not be disregarded. interstitial fluid; TRPA1, transient receptor potential ankyrin 1; TRPV1, transient receptor potential vanilloid type 1.
Temperature sensing is a crucial feature of the nervous system, enabling organisms to avoid physi... more Temperature sensing is a crucial feature of the nervous system, enabling organisms to avoid physical danger and choose optimal environments for survival. TRPM8 (Transient Receptor Potential Melastatin type 8) belongs to a select group of ion channels which are gated by changes in temperature, are expressed in sensory nerves and/or skin cells and may be involved in temperature sensing. This channel is activated by a moderate decrease in temperature, with a threshold of approximately 25 °C in heterologous expression systems, and by a variety of natural and synthetic compounds, including menthol. While the physiological role of TRPM8 as a transducer of gentle cooling is widely accepted, its involvement in acute noxious cold sensing in healthy tissues is still under debate. Although accumulating evidence indicates that TRPM8 is involved in neuropathic cold allodynia, in some animal models of nerve injury peripheral and central activation of TRPM8 is followed by analgesia. A variety of inflammatory mediators, including bradykinin and prostaglandin E(2), modulate TRPM8 by inhibiting the channel and shifting its activation threshold to colder temperatures, most likely counteracting the analgesic action of TRPM8. While important progress has been made in unraveling the biophysical features of TRPM8, including the revelation of its voltage dependence, the precise mechanism involved in temperature sensing by this channel is still not completely understood. This article will review the current status of knowledge regarding the (patho)physiological role(s) of TRPM8, its modulation by inflammatory mediators, the signaling pathways involved in this regulation, and the biophysical properties of the channel.
Specify what is your 1st choice of session: CSIV-01_Cancer signalling Abstract: Doxorubicin, an e... more Specify what is your 1st choice of session: CSIV-01_Cancer signalling Abstract: Doxorubicin, an effective anticancer drug, has benn reported to induce either apoptosis or necrosis in various cancer cell lines, in function of dose and treatment duration. We investigated the antiproliferative effects of doxorubicin at clinical doses (50-100 nM) and duration (24h, 72h) in human leukemia JUrkat cells. Doxorubicin induced G 2 /M arrest and significant apoptosis (up to 54% after 72h of exposure), as assessed by flow cytometry analysis of the DNA content. However, cellular viability determined by flow cytometry on propidium iodide labeled cells was 1.6% after the 72h treatment with 100 nM doxorubicin, indicating a significant fraction of necrotic cells as well. Neither quercetin nor menadione or their combination at clinical concentrations (10-15 µM) enhanced doxorubicin-induced apoptosis, despite their apoptigenic effects reported in the Jurkat cell line [1, 2]. Fluorimetric measurements on fura-2 loaded cells showed that doxorubicin (7.5 µM) induced Ca 2+ release at slow rate and elicited a three-fold, progressive increase in cytosolic Ca 2+ concentration over the next 55 min. from drug addition. However, since the fluorescence of internalized doxorubicin displayed a considerable and rapid increase after extracellular addition, this result indicates a sustained but indirect activation of the Ca 2+ release channels. The observed Ca 2+ increase was partly mediated by ryanodine receptors, so that the presence of the specific inhibitor, ryanodine (200 µM), consistently inhibited the doxorubicin-evoked Ca 2+ signal. We also found by spectrofluorimetry that doxorubicin at clinical doses and dose rates generates oxidative stress, which is enhanced by menadione (10-15 µM), but is alleviated by both quercetin (10-15 µM) and the quercetin-menadione combination. However, these agents did not improve cellular viability following the doxorubicin treatment. In addition, quercetin did not affect the cell cycle distribution of doxorubicin treated-cells, whereas menadione and the quercetin-menadione concentration apparently abolished the doxorubicininduced without further progression through the cell cycle phases.
Objectives. The goal of this study was to explore the chemotherapeutic potential of Epigallocatec... more Objectives. The goal of this study was to explore the chemotherapeutic potential of Epigallocatechine-3-gallate (EGCG) and menadione (vitamin K3; MD), and to determine whether a synergic interaction exists between the two agents that could enhance significantly their antitumoral effect in a cellular model for acute lymphoblastic leukemia (ALL). To this end, we investigated the antiproliferative effect of EGCG and MD, applied alone or in combination on human leukemia Jurkat lymphoblasts. Some underlying cellular mechanisms were also scrutinized. Materials and methods. Cell suspensions of Jurkat lymphoblasts were treated at various concentrations of EGCG and/or MD. Cell cycle and apoptosis/necrosis were determined by flow cytometry, using the fluorescent indicators propidium iodide and Annexin V-FITC/7-AAD, respectively. Clonogenic survival was evaluated as the colony forming capacity in 96-well plates. Determination of oxidative stress and mitochondrial polarization was performed by ...
Please cite this article in press as: Baran I, et al. Novel insights into the antiproliferative e... more Please cite this article in press as: Baran I, et al. Novel insights into the antiproliferative effects and synergism of quercetin and menadione in human leukemia Jurkat T cells. Leuk Res (2014), http://dx.
The interaction between cold sensitivity and inflammation in mammals is not entirely understood. ... more The interaction between cold sensitivity and inflammation in mammals is not entirely understood. We have used adult rat dorsal root ganglion neurones in primary culture together with calcium microfluorimetry to assess the effects of selected inflammatory mediators on cold responses of cold- and menthol-sensitive (most likely TRPM8-expressing) neurones. We observed a high degree of functional co-expression of TRPM8, the receptors for the inflammatory agents bradykinin, prostaglandin E2 and histamine, and TRPA1 in cultured sensory neurones. Treatment with either bradykinin or prostaglandin E2 led to a reduction in the amplitude of the response to cooling and shifted the threshold temperature to colder values, and we provide evidence for a role of protein kinases C and A, respectively, in mediating these effects. In both cases the effects were mainly restricted to the subgroups of cold- and menthol-sensitive cells which had responded to the application of the inflammatory agents at basal temperature. This desensitization of cold-sensitive neurones may enhance inflammatory pain by removing the analgesic effects of gentle cooling.
The interaction between cold sensitivity and inflammation in mammals is not entirely understood. ... more The interaction between cold sensitivity and inflammation in mammals is not entirely understood. We have used adult rat dorsal root ganglion neurones in primary culture together with calcium microfluorimetry to assess the effects of selected inflammatory mediators on cold responses of cold- and menthol-sensitive (most likely TRPM8-expressing) neurones. We observed a high degree of functional co-expression of TRPM8, the receptors for the inflammatory agents bradykinin, prostaglandin E2 and histamine, and TRPA1 in cultured sensory neurones. Treatment with either bradykinin or prostaglandin E2 led to a reduction in the amplitude of the response to cooling and shifted the threshold temperature to colder values, and we provide evidence for a role of protein kinases C and A, respectively, in mediating these effects. In both cases the effects were mainly restricted to the subgroups of cold- and menthol-sensitive cells which had responded to the application of the inflammatory agents at basal temperature. This desensitization of cold-sensitive neurones may enhance inflammatory pain by removing the analgesic effects of gentle cooling.
Heat-activated ion channels from the vanilloid-type TRP group (TRPV1-4) seem to be central for he... more Heat-activated ion channels from the vanilloid-type TRP group (TRPV1-4) seem to be central for heat-sensitivity of nociceptive sensory neurons. Displaying a high-threshold (> 52°C) for activation, TRPV2 was proposed to act as a sensor for intense noxious heat in mammalian sensory neurons. However, although TRPV2 is expressed in a distinct population of thinly myelinated primary afferents, a widespread expression in a variety of neuronal and non-neuronal tissues suggests a more diverse physiological role of TRPV2. In its role as a heat-sensor, TRPV2 has not been thoroughly characterized in terms of biophysical and pharmacological properties. In the present study, we demonstrate that the features of heterologously expressed rat TRPV2 closely resemble those of high-threshold heat-evoked currents in medium-and large-sized capsaicin-insensitive rat dorsal root ganglion (DRG) neurons. Both in TRPV2-expressing human embryonic kidney (HEK)293t cells and in DRGs, high-threshold heat-currents were sensitized by repeated activation and by the TRPV1-3 agonist, 2-aminoethoxydiphenyl borate (2-APB). In addition to a previously described block by ruthenium red, we identified the trivalent cations, lanthanum (La 3+ ) and gadolinium (Gd 3+ ) as potent blockers of TRPV2. Thus, we present a new pharmacological tool to distinguish between heat responses of TRPV2 and the closely related capsaicin-receptor, TRPV1, which is strongly sensitized by trivalent cations. We demonstrate that self-sensitization of heat-evoked currents through TRPV2 does not require extracellular calcium and that TRPV2 can be activated in cell-free membrane patches in the outside-out configuration. Taken together our results provide new evidence for a role of TRPV2 in mediating high-threshold heat responses in a subpopulation of mammalian sensory neurons.
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Papers by Ramona Madalina Babes (Linte)