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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

Effect of ascorbic acid (1 mM) on intracellular Ca2+ changes in response to treatment of UVB-irradiated B16F10 cells with quercetin.A, B16F10 cells were pre-treated with Qu for 24 h and supplemented with ascorbic acid 1 h before UVB irradiation. Following UVB irradiation, cells were loaded with Fluo-3 AM and analyzed on Olympus Flowview FV1000. B, represents the densitometric measure of the effect of ascorbic acid (AA) on intracellular Ca2+ levels in response to treatment of UVB-irradiated B16F10 cells with Qu.
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pone.0131253.g006: Effect of ascorbic acid (1 mM) on intracellular Ca2+ changes in response to treatment of UVB-irradiated B16F10 cells with quercetin.A, B16F10 cells were pre-treated with Qu for 24 h and supplemented with ascorbic acid 1 h before UVB irradiation. Following UVB irradiation, cells were loaded with Fluo-3 AM and analyzed on Olympus Flowview FV1000. B, represents the densitometric measure of the effect of ascorbic acid (AA) on intracellular Ca2+ levels in response to treatment of UVB-irradiated B16F10 cells with Qu.

Mentions: In skin cells, UVB irradiation stimulates the production of reactive oxygen species [38]. In agreement with this, we noted that when B16F10 cells were irradiated with 5 mJ/cm2 of UVB, a substantial increase in ROS generation was observed (9.3%; Fig 5A). Next we investigated the contribution of quercetin to UVB-induced ROS generation. UVB irradiation documented 2.5-fold increase in ROS formation. Quercetin actually increased UVB-induced ROS generation. 5, 10, 20, and 40 μM quercetin caused 2.6-, 4.9-, 6.9-, 19.6-fold increase in UVB-induced ROS formation respectively (Fig 5A and 5B). Addition of ascorbic acid (1 mM) 1 h prior and subsequent to co-treatment of UVB and quercetin markedly reduced the pro-apoptotic effects of quercetin in UVB–irradiated B16F10 cells (Fig 5C). These results indicate that this pro-oxidant behaviour of quercetin in UVB–irradiated B16F10 cells could be one of the factors responsible for growth inhibitory effects of quercetin in B16F10 cells. We next studied the effect of quercetin on intracellular free calcium. Changes in intracellular free calcium have been reported to initiate ROS formation in UVB-irradiated human HaCaT keratinocytes [39]. UVB irradiation caused 1.3-fold increase in the percentage of cells with high level of intracellular free calcium (5.8%, p<0.01). However, in UVB-irradiated B16F10 cells, quercetin produced a marked increase in intracellular free calcium. 5, 10, 20 and 40 μM quercetin added subsequent to UVB irradiation caused 4.1-, 4.6-, 6.7-, and 4.7-fold increase in the percentage of cells with high levels of intracellular free calcium (Fig 5D and 5F). Treatment of cells with H2O2 (1.25 mM) for 10 minutes enhanced the level of intracellular free calcium, indicating that ROS may be responsible for intracellular calcium elevation (Fig 6A and 6B). However, preloading cells with BAPTA-AM, a specific cell-permeable chelating agent of Ca2+, decreased the subsequent UVB–induced elevation of intracellular free calcium, indicating that intracellular free calcium elevates in response to UVB irradiation of B16F10 cells (Fig 5E). Blocking ROS using 1 mM ascorbic acid as an anti-oxidant reduced the intracellular calcium changes (Fig 6A and 6B). In addition, ascorbic acid could also reduce the pro-apoptotic effects of quercetin in UVB–irradiated B16F10 cells (Fig 5C). Overall, these results indicated that ROS generated by the combination use of UVB and quercetin may be responsible for the intracellular calcium changes and subsequent cell death.


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)

Effect of ascorbic acid (1 mM) on intracellular Ca2+ changes in response to treatment of UVB-irradiated B16F10 cells with quercetin.A, B16F10 cells were pre-treated with Qu for 24 h and supplemented with ascorbic acid 1 h before UVB irradiation. Following UVB irradiation, cells were loaded with Fluo-3 AM and analyzed on Olympus Flowview FV1000. B, represents the densitometric measure of the effect of ascorbic acid (AA) on intracellular Ca2+ levels in response to treatment of UVB-irradiated B16F10 cells with Qu.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493061&req=5

pone.0131253.g006: Effect of ascorbic acid (1 mM) on intracellular Ca2+ changes in response to treatment of UVB-irradiated B16F10 cells with quercetin.A, B16F10 cells were pre-treated with Qu for 24 h and supplemented with ascorbic acid 1 h before UVB irradiation. Following UVB irradiation, cells were loaded with Fluo-3 AM and analyzed on Olympus Flowview FV1000. B, represents the densitometric measure of the effect of ascorbic acid (AA) on intracellular Ca2+ levels in response to treatment of UVB-irradiated B16F10 cells with Qu.
Mentions: In skin cells, UVB irradiation stimulates the production of reactive oxygen species [38]. In agreement with this, we noted that when B16F10 cells were irradiated with 5 mJ/cm2 of UVB, a substantial increase in ROS generation was observed (9.3%; Fig 5A). Next we investigated the contribution of quercetin to UVB-induced ROS generation. UVB irradiation documented 2.5-fold increase in ROS formation. Quercetin actually increased UVB-induced ROS generation. 5, 10, 20, and 40 μM quercetin caused 2.6-, 4.9-, 6.9-, 19.6-fold increase in UVB-induced ROS formation respectively (Fig 5A and 5B). Addition of ascorbic acid (1 mM) 1 h prior and subsequent to co-treatment of UVB and quercetin markedly reduced the pro-apoptotic effects of quercetin in UVB–irradiated B16F10 cells (Fig 5C). These results indicate that this pro-oxidant behaviour of quercetin in UVB–irradiated B16F10 cells could be one of the factors responsible for growth inhibitory effects of quercetin in B16F10 cells. We next studied the effect of quercetin on intracellular free calcium. Changes in intracellular free calcium have been reported to initiate ROS formation in UVB-irradiated human HaCaT keratinocytes [39]. UVB irradiation caused 1.3-fold increase in the percentage of cells with high level of intracellular free calcium (5.8%, p<0.01). However, in UVB-irradiated B16F10 cells, quercetin produced a marked increase in intracellular free calcium. 5, 10, 20 and 40 μM quercetin added subsequent to UVB irradiation caused 4.1-, 4.6-, 6.7-, and 4.7-fold increase in the percentage of cells with high levels of intracellular free calcium (Fig 5D and 5F). Treatment of cells with H2O2 (1.25 mM) for 10 minutes enhanced the level of intracellular free calcium, indicating that ROS may be responsible for intracellular calcium elevation (Fig 6A and 6B). However, preloading cells with BAPTA-AM, a specific cell-permeable chelating agent of Ca2+, decreased the subsequent UVB–induced elevation of intracellular free calcium, indicating that intracellular free calcium elevates in response to UVB irradiation of B16F10 cells (Fig 5E). Blocking ROS using 1 mM ascorbic acid as an anti-oxidant reduced the intracellular calcium changes (Fig 6A and 6B). In addition, ascorbic acid could also reduce the pro-apoptotic effects of quercetin in UVB–irradiated B16F10 cells (Fig 5C). Overall, these results indicated that ROS generated by the combination use of UVB and quercetin may be responsible for the intracellular calcium changes and subsequent cell death.

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