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...
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...
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.
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 ...
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.
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...
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...
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.
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 ...
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.
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Papers by Ramona Madalina Babes (Linte)