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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 sub-G1 cell cycle arrest and cleavage of chromosomal DNA.A, analysis of cell cycle progression in B16F10 cells treated with Qu and/or UVB. Cells were collected (including floating cells), stained with propidium iodide and analyzed for DNA content by BD FACS Calibur Aria. ‘Sub-G1 peak’ contains apoptotic cells. B, represents the fold increase in the percentage of sub-G1 cells relative to control. *, P<0.05; **, P<0.01 for control versus treated; #, P<0.05; ##, P<0.01 for UVB-alone treated versus UVB + Qu treated. C, analysis of DNA fragmentation in B16F10 cells at 24 h post-Qu treatment. D, analysis of DNA fragmentation in B16F10 cells at 24 h post-UVB/Qu treatment.
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pone.0131253.g003: Combined UVB and quercetin treatment enhances sub-G1 cell cycle arrest and cleavage of chromosomal DNA.A, analysis of cell cycle progression in B16F10 cells treated with Qu and/or UVB. Cells were collected (including floating cells), stained with propidium iodide and analyzed for DNA content by BD FACS Calibur Aria. ‘Sub-G1 peak’ contains apoptotic cells. B, represents the fold increase in the percentage of sub-G1 cells relative to control. *, P<0.05; **, P<0.01 for control versus treated; #, P<0.05; ##, P<0.01 for UVB-alone treated versus UVB + Qu treated. C, analysis of DNA fragmentation in B16F10 cells at 24 h post-Qu treatment. D, analysis of DNA fragmentation in B16F10 cells at 24 h post-UVB/Qu treatment.

Mentions: Here, we analyzed the effect of quercetin on induction of sub-G1 cell cycle arrest in UVB-irradiated B16F10. UVB irradiation increased the percentage of hypodiploid sub-G1cells (22%) relative to control cells (3%). Quercetin markedly increased the UVB-induced sub-G1 cell cycle arrest, with nearly 23%, 43%, 57% and 46% of cells found in the sub-G1 phases of cell cycle (Fig 3A and 3B). Sub-G1 cells are known to arise as a result of cleavage of chromosomal DNA during the late stages of apoptotic cell death [34]. Therefore, we evaluated whether the cytotoxic action of quercetin on UVB–irradiated B16F10 cells was associated with cleavage of chromosomal DNA which is a hallmark feature of apoptosis [34]. We first treated the B16F10 cells with quercetin and analyzed the cleavage of chromosomal DNA at 24 h post- quercetin treatment. It was found that quercetin caused no or minimal cleavage of chromosomal DNA upto a concentration of 20 μM. However, quercetin at higher concentrations (30–40 μM) induced detectable and significant DNA fragmentation in B16F10 cells (Fig 3C). Upon UVB irradiation at 5 mJ/cm2, cleavage of chromosomal DNA was detected in B16F10 cells. Interestingly, combined UVB and quercetin treatment induced marked and substantial DNA fragmentation (Fig 3D). Taken together, these results indicated that quercetin causes B16F10 cells to undergo apoptosis as a result of UVB–induced DNA fragmentation.


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 sub-G1 cell cycle arrest and cleavage of chromosomal DNA.A, analysis of cell cycle progression in B16F10 cells treated with Qu and/or UVB. Cells were collected (including floating cells), stained with propidium iodide and analyzed for DNA content by BD FACS Calibur Aria. ‘Sub-G1 peak’ contains apoptotic cells. B, represents the fold increase in the percentage of sub-G1 cells relative to control. *, P<0.05; **, P<0.01 for control versus treated; #, P<0.05; ##, P<0.01 for UVB-alone treated versus UVB + Qu treated. C, analysis of DNA fragmentation in B16F10 cells at 24 h post-Qu treatment. D, analysis of DNA fragmentation in B16F10 cells at 24 h post-UVB/Qu treatment.
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pone.0131253.g003: Combined UVB and quercetin treatment enhances sub-G1 cell cycle arrest and cleavage of chromosomal DNA.A, analysis of cell cycle progression in B16F10 cells treated with Qu and/or UVB. Cells were collected (including floating cells), stained with propidium iodide and analyzed for DNA content by BD FACS Calibur Aria. ‘Sub-G1 peak’ contains apoptotic cells. B, represents the fold increase in the percentage of sub-G1 cells relative to control. *, P<0.05; **, P<0.01 for control versus treated; #, P<0.05; ##, P<0.01 for UVB-alone treated versus UVB + Qu treated. C, analysis of DNA fragmentation in B16F10 cells at 24 h post-Qu treatment. D, analysis of DNA fragmentation in B16F10 cells at 24 h post-UVB/Qu treatment.
Mentions: Here, we analyzed the effect of quercetin on induction of sub-G1 cell cycle arrest in UVB-irradiated B16F10. UVB irradiation increased the percentage of hypodiploid sub-G1cells (22%) relative to control cells (3%). Quercetin markedly increased the UVB-induced sub-G1 cell cycle arrest, with nearly 23%, 43%, 57% and 46% of cells found in the sub-G1 phases of cell cycle (Fig 3A and 3B). Sub-G1 cells are known to arise as a result of cleavage of chromosomal DNA during the late stages of apoptotic cell death [34]. Therefore, we evaluated whether the cytotoxic action of quercetin on UVB–irradiated B16F10 cells was associated with cleavage of chromosomal DNA which is a hallmark feature of apoptosis [34]. We first treated the B16F10 cells with quercetin and analyzed the cleavage of chromosomal DNA at 24 h post- quercetin treatment. It was found that quercetin caused no or minimal cleavage of chromosomal DNA upto a concentration of 20 μM. However, quercetin at higher concentrations (30–40 μM) induced detectable and significant DNA fragmentation in B16F10 cells (Fig 3C). Upon UVB irradiation at 5 mJ/cm2, cleavage of chromosomal DNA was detected in B16F10 cells. Interestingly, combined UVB and quercetin treatment induced marked and substantial DNA fragmentation (Fig 3D). Taken together, these results indicated that quercetin causes B16F10 cells to undergo apoptosis as a result of UVB–induced DNA fragmentation.

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