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A Potent Inhibitor of Phosphoinositide 3-Kinase (PI3K) and Mitogen Activated Protein (MAP) Kinase Signalling, Quercetin (3, 3', 4', 5, 7-Pentahydroxyflavone) Promotes Cell Death in Ultraviolet (UV)-B-Irradiated B16F10 Melanoma Cells.

Rafiq RA, Quadri A, Nazir LA, Peerzada K, Ganai BA, Tasduq SA - PLoS ONE (2015)

Bottom Line: The present study has brought out that the pro-apoptotic effects of quercetin in UVB-irradiated B16F10 cells are mediated through the elevation of intracellular reactive oxygen species (ROS) formation, calcium homeostasis imbalance, modulation of anti-oxidant defence response and depolarization of mitochondrial membrane potential (ΔΨM).Quercetin markedly attenuated MEK-ERK signalling, influenced PI3K/Akt pathway, and potentially enhanced the UVB-induced NF-κB nuclear translocation.Furthermore, combined UVB and quercetin treatment decreased the ratio of Bcl-2 to that of Bax, and upregulated the expression of Bim and apoptosis inducing factor (AIF).

View Article: PubMed Central - PubMed

Affiliation: PK-PD and Toxicology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu Tawi, Jammu and Kashmir, India.

ABSTRACT
Ultraviolet (UV) radiation-induced skin damage contributes strongly to the formation of melanoma, a highly lethal form of skin cancer. Quercetin (Qu), the most widely consumed dietary bioflavonoid and well known inhibitor of phosphoinositide 3-kinase (PI3K) and mitogen activated protein (MAP) kinase signalling, has been reported to be chemopreventive in several forms of non-melanoma skin cancers. Here, we report that the treatment of ultraviolet (UV)-B-irradiated B16F10 melanoma cells with quercetin resulted in a dose dependent reduction in cell viability and increased apoptosis. The present study has brought out that the pro-apoptotic effects of quercetin in UVB-irradiated B16F10 cells are mediated through the elevation of intracellular reactive oxygen species (ROS) formation, calcium homeostasis imbalance, modulation of anti-oxidant defence response and depolarization of mitochondrial membrane potential (ΔΨM). Promotion of UVB-induced cell death by quercetin was further revealed by cleavage of chromosomal DNA, caspase activation, poly (ADP) ribose polymerase (PARP) cleavage, and an increase in sub-G1 cells. Quercetin markedly attenuated MEK-ERK signalling, influenced PI3K/Akt pathway, and potentially enhanced the UVB-induced NF-κB nuclear translocation. Furthermore, combined UVB and quercetin treatment decreased the ratio of Bcl-2 to that of Bax, and upregulated the expression of Bim and apoptosis inducing factor (AIF). Overall, these results suggest the possibility of using quercetin in combination with UVB as a possible treatment option for melanoma in future.

No MeSH data available.


Related in: MedlinePlus

Combined UVB and quercetin treatment enhances apoptotic cell death and induces caspase activation and PARP-1 cleavage.A, Annexin V/propidium iodide assay followed by flow cytometric analysis of apoptosis and necrosis in B16F10 cells at 24 h post-UVB/Qu treatment. B, represents the percentage of apoptotic and necrotic cells. *, P<0.05; **, P<0.01 for control (apoptotic) versus treated; #, P<0.05; ##, P<0.01 for UVB-alone treatment (apoptotic) versus UVB + Qu treatments; *´, P< 0.05 for control (necrotic) versus treated; #', P<0.05 for UVB-alone (necrotic) versus UVB + Qu treatments. C, analysis of lactate dehydrogenase (LDH) leakage in cells treated with Qu and/or UVB. *, P<0.05 for control versus treated; #, P<0.05 for UVB-alone treatment versus UVB + Qu treatment; ns, statistically not significant. D, western blot analysis of caspase-3/caspase-8 activation and PARP-1 cleavage in B16F10 cells at 24 h post-UVB/Qu treatment. The signals for the cleaved forms of caspase-3 (E), caspase-8 (F) and PARP-1 protein (G) were quantified and normalized against β-actin, GAPDH and β-actin respectively using Image Lab software Version 3.0 (BioRad).
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pone.0131253.g002: Combined UVB and quercetin treatment enhances apoptotic cell death and induces caspase activation and PARP-1 cleavage.A, Annexin V/propidium iodide assay followed by flow cytometric analysis of apoptosis and necrosis in B16F10 cells at 24 h post-UVB/Qu treatment. B, represents the percentage of apoptotic and necrotic cells. *, P<0.05; **, P<0.01 for control (apoptotic) versus treated; #, P<0.05; ##, P<0.01 for UVB-alone treatment (apoptotic) versus UVB + Qu treatments; *´, P< 0.05 for control (necrotic) versus treated; #', P<0.05 for UVB-alone (necrotic) versus UVB + Qu treatments. C, analysis of lactate dehydrogenase (LDH) leakage in cells treated with Qu and/or UVB. *, P<0.05 for control versus treated; #, P<0.05 for UVB-alone treatment versus UVB + Qu treatment; ns, statistically not significant. D, western blot analysis of caspase-3/caspase-8 activation and PARP-1 cleavage in B16F10 cells at 24 h post-UVB/Qu treatment. The signals for the cleaved forms of caspase-3 (E), caspase-8 (F) and PARP-1 protein (G) were quantified and normalized against β-actin, GAPDH and β-actin respectively using Image Lab software Version 3.0 (BioRad).

Mentions: To determine whether the decrease in cell viability is caused by enhanced apoptosis, we performed Annexin V/propidium iodide double staining to compare apoptosis and necrosis between control and treated cells. In agreement with the above results, we found that quercetin caused a substantial increase in UVB-induced apoptotic response, with nearly 26%, 32%, 27% and 33% of cells found to be in the early phase of apoptosis (Fig 2A and 2B). No significant increase in necrotic cell death was detected with or without UVB or quercetin (5–10 μM) treatment (Fig 2B). However, subtle but significant increase in necrotic cell death was detected in UVB-irradiated B16F10 cells treated with 20 and 40 μM quercetin relative to control necrotic cells. Next we examined the effect of quercetin on lactate dehydrogenase (LDH) activity in UVB-irradiated B16F10 cells. Treatment of UVB-irradiated B16F10 cells with 5, 10, 20, and 40 μM quercetin induced 1.17-, 1.14-, 1.22-, and 1.46-fold increase in LDH activity respectively relative to un-irradiated control cells (Fig 2C). Western blot analysis also revealed that combined UVB and quercetin treatment enhanced activation of caspases and cleavage of poly(ADP-ribose) polymerase (PARP), which is a hallmark feature of apoptosis (Fig 2D–2G).


A Potent Inhibitor of Phosphoinositide 3-Kinase (PI3K) and Mitogen Activated Protein (MAP) Kinase Signalling, Quercetin (3, 3', 4', 5, 7-Pentahydroxyflavone) Promotes Cell Death in Ultraviolet (UV)-B-Irradiated B16F10 Melanoma Cells.

Rafiq RA, Quadri A, Nazir LA, Peerzada K, Ganai BA, Tasduq SA - PLoS ONE (2015)

Combined UVB and quercetin treatment enhances apoptotic cell death and induces caspase activation and PARP-1 cleavage.A, Annexin V/propidium iodide assay followed by flow cytometric analysis of apoptosis and necrosis in B16F10 cells at 24 h post-UVB/Qu treatment. B, represents the percentage of apoptotic and necrotic cells. *, P<0.05; **, P<0.01 for control (apoptotic) versus treated; #, P<0.05; ##, P<0.01 for UVB-alone treatment (apoptotic) versus UVB + Qu treatments; *´, P< 0.05 for control (necrotic) versus treated; #', P<0.05 for UVB-alone (necrotic) versus UVB + Qu treatments. C, analysis of lactate dehydrogenase (LDH) leakage in cells treated with Qu and/or UVB. *, P<0.05 for control versus treated; #, P<0.05 for UVB-alone treatment versus UVB + Qu treatment; ns, statistically not significant. D, western blot analysis of caspase-3/caspase-8 activation and PARP-1 cleavage in B16F10 cells at 24 h post-UVB/Qu treatment. The signals for the cleaved forms of caspase-3 (E), caspase-8 (F) and PARP-1 protein (G) were quantified and normalized against β-actin, GAPDH and β-actin respectively using Image Lab software Version 3.0 (BioRad).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493061&req=5

pone.0131253.g002: Combined UVB and quercetin treatment enhances apoptotic cell death and induces caspase activation and PARP-1 cleavage.A, Annexin V/propidium iodide assay followed by flow cytometric analysis of apoptosis and necrosis in B16F10 cells at 24 h post-UVB/Qu treatment. B, represents the percentage of apoptotic and necrotic cells. *, P<0.05; **, P<0.01 for control (apoptotic) versus treated; #, P<0.05; ##, P<0.01 for UVB-alone treatment (apoptotic) versus UVB + Qu treatments; *´, P< 0.05 for control (necrotic) versus treated; #', P<0.05 for UVB-alone (necrotic) versus UVB + Qu treatments. C, analysis of lactate dehydrogenase (LDH) leakage in cells treated with Qu and/or UVB. *, P<0.05 for control versus treated; #, P<0.05 for UVB-alone treatment versus UVB + Qu treatment; ns, statistically not significant. D, western blot analysis of caspase-3/caspase-8 activation and PARP-1 cleavage in B16F10 cells at 24 h post-UVB/Qu treatment. The signals for the cleaved forms of caspase-3 (E), caspase-8 (F) and PARP-1 protein (G) were quantified and normalized against β-actin, GAPDH and β-actin respectively using Image Lab software Version 3.0 (BioRad).
Mentions: To determine whether the decrease in cell viability is caused by enhanced apoptosis, we performed Annexin V/propidium iodide double staining to compare apoptosis and necrosis between control and treated cells. In agreement with the above results, we found that quercetin caused a substantial increase in UVB-induced apoptotic response, with nearly 26%, 32%, 27% and 33% of cells found to be in the early phase of apoptosis (Fig 2A and 2B). No significant increase in necrotic cell death was detected with or without UVB or quercetin (5–10 μM) treatment (Fig 2B). However, subtle but significant increase in necrotic cell death was detected in UVB-irradiated B16F10 cells treated with 20 and 40 μM quercetin relative to control necrotic cells. Next we examined the effect of quercetin on lactate dehydrogenase (LDH) activity in UVB-irradiated B16F10 cells. Treatment of UVB-irradiated B16F10 cells with 5, 10, 20, and 40 μM quercetin induced 1.17-, 1.14-, 1.22-, and 1.46-fold increase in LDH activity respectively relative to un-irradiated control cells (Fig 2C). Western blot analysis also revealed that combined UVB and quercetin treatment enhanced activation of caspases and cleavage of poly(ADP-ribose) polymerase (PARP), which is a hallmark feature of apoptosis (Fig 2D–2G).

Bottom Line: The present study has brought out that the pro-apoptotic effects of quercetin in UVB-irradiated B16F10 cells are mediated through the elevation of intracellular reactive oxygen species (ROS) formation, calcium homeostasis imbalance, modulation of anti-oxidant defence response and depolarization of mitochondrial membrane potential (ΔΨM).Quercetin markedly attenuated MEK-ERK signalling, influenced PI3K/Akt pathway, and potentially enhanced the UVB-induced NF-κB nuclear translocation.Furthermore, combined UVB and quercetin treatment decreased the ratio of Bcl-2 to that of Bax, and upregulated the expression of Bim and apoptosis inducing factor (AIF).

View Article: PubMed Central - PubMed

Affiliation: PK-PD and Toxicology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu Tawi, Jammu and Kashmir, India.

ABSTRACT
Ultraviolet (UV) radiation-induced skin damage contributes strongly to the formation of melanoma, a highly lethal form of skin cancer. Quercetin (Qu), the most widely consumed dietary bioflavonoid and well known inhibitor of phosphoinositide 3-kinase (PI3K) and mitogen activated protein (MAP) kinase signalling, has been reported to be chemopreventive in several forms of non-melanoma skin cancers. Here, we report that the treatment of ultraviolet (UV)-B-irradiated B16F10 melanoma cells with quercetin resulted in a dose dependent reduction in cell viability and increased apoptosis. The present study has brought out that the pro-apoptotic effects of quercetin in UVB-irradiated B16F10 cells are mediated through the elevation of intracellular reactive oxygen species (ROS) formation, calcium homeostasis imbalance, modulation of anti-oxidant defence response and depolarization of mitochondrial membrane potential (ΔΨM). Promotion of UVB-induced cell death by quercetin was further revealed by cleavage of chromosomal DNA, caspase activation, poly (ADP) ribose polymerase (PARP) cleavage, and an increase in sub-G1 cells. Quercetin markedly attenuated MEK-ERK signalling, influenced PI3K/Akt pathway, and potentially enhanced the UVB-induced NF-κB nuclear translocation. Furthermore, combined UVB and quercetin treatment decreased the ratio of Bcl-2 to that of Bax, and upregulated the expression of Bim and apoptosis inducing factor (AIF). Overall, these results suggest the possibility of using quercetin in combination with UVB as a possible treatment option for melanoma in future.

No MeSH data available.


Related in: MedlinePlus