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3',4',5',5,7-pentamethoxyflavone sensitizes Cisplatin-resistant A549 cells to Cisplatin by inhibition of Nrf2 pathway.

Hou X, Bai X, Gou X, Zeng H, Xia C, Zhuang W, Chen X, Zhao Z, Huang M, Jin J - Mol. Cells (2015)

Bottom Line: In this study, we found that the expression levels of Nrf2 and its target genes GCLC, HO-1, NQO1 were significantly higher in cisplatin-resistant A549 (A549/CDDP) cells than those in A549 cells, and this resistance was partially reversed by Nrf2 siRNA. 3',4',5',5,7-Pentamethoxyflavone (PMF), a natural flavonoid extracted from Rutaceae plants, sensitized A549/CDDP to CDDP and substantially induced apoptosis compared with that of CDDP alone treated group, and this reversal effect decreased when Nrf2 was downregulated by siRNA.Mechanistically, PMF reduced Nrf2 expression leading to a reduction of Nrf2 downstream genes, and in contrast, this effect was decreased by blocking Nrf2 with siRNA.Taken together, these results demonstrated that PMF could be used as an effective adjuvant sensitizer to increase the efficacy of chemotherapeutic drugs by downregulating Nrf2 signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmaceutical Science, Sun Yat-sen University, Guangzhou 510006, China.

ABSTRACT
Nuclear factor erythroid 2-related factor 2 (Nrf2) is an important redox-sensitive transcription factor that regulates the expression of several cytoprotective genes. More recently, genetic analyses of human tumors have indicated that Nrf2 may cause resistance to chemotherapy. In this study, we found that the expression levels of Nrf2 and its target genes GCLC, HO-1, NQO1 were significantly higher in cisplatin-resistant A549 (A549/CDDP) cells than those in A549 cells, and this resistance was partially reversed by Nrf2 siRNA. 3',4',5',5,7-Pentamethoxyflavone (PMF), a natural flavonoid extracted from Rutaceae plants, sensitized A549/CDDP to CDDP and substantially induced apoptosis compared with that of CDDP alone treated group, and this reversal effect decreased when Nrf2 was downregulated by siRNA. Mechanistically, PMF reduced Nrf2 expression leading to a reduction of Nrf2 downstream genes, and in contrast, this effect was decreased by blocking Nrf2 with siRNA. Taken together, these results demonstrated that PMF could be used as an effective adjuvant sensitizer to increase the efficacy of chemotherapeutic drugs by downregulating Nrf2 signaling pathway.

No MeSH data available.


Related in: MedlinePlus

PMF sensitized A549/CDDP cells to CDDP. (A) A549/CDDP cells were exposed to PMF (10, 25, and 50 μM) and were incubated with increasing concentration of CDDP (5 – 400 μM) in culture for 48 h and SRB assay were taken. (B) Comparison of the suppression of PMF (25 μM) combined with different concentration of CDDP on A549 cells and A549/CDDP cells. (C–D) Apoptosis ratios were determined through flow cytometry. (E) The expression of cleaved Caspase 3 and PARP1were determined by Western blot. Data are presented as means ± SD of three independent experiments and significant differences are indicated as *P < 0.05.
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f3-molce-38-5-396: PMF sensitized A549/CDDP cells to CDDP. (A) A549/CDDP cells were exposed to PMF (10, 25, and 50 μM) and were incubated with increasing concentration of CDDP (5 – 400 μM) in culture for 48 h and SRB assay were taken. (B) Comparison of the suppression of PMF (25 μM) combined with different concentration of CDDP on A549 cells and A549/CDDP cells. (C–D) Apoptosis ratios were determined through flow cytometry. (E) The expression of cleaved Caspase 3 and PARP1were determined by Western blot. Data are presented as means ± SD of three independent experiments and significant differences are indicated as *P < 0.05.

Mentions: To determine whether PMF can enhance the sensitivity of A549/DDP cells to CDDP, SRB assay was carried out and IC50 was calculated. As shown in Fig. 3A, IC50 of PMF+CDDP treated group was significantly lower than that of the CDDP alone treated group, indicating that PMF reduced the resistance of A549/CDDP cells to CDDP. The effect of PMF on the cytotoxicity of CDDP was also investigated in A549 cells, which expressed lower Nrf2 than A549/DDP cells. The results showed that the effect of PMF was diminished in A549 cells compared with that in A549/CDDP cells, indicating that PMF might sensitize A549/CDDP cells to cisplatin through inhibition of Nrf2 (Fig. 3B). To investigate whether combination of PMF and CDDP would promote apoptosis of cells, A549/CDDP cells were exposed to CDDP (25 μM) or PMF (25 μM) or in combination for 24 h. Apoptotic cells were determined by PI/Annexin V staining and flow cytometric analysis. Figures 3C–3D showed that the ratio of apoptotic cells in the combined treatment group was significantly increased compared with that in the CDDP alone treated group (P < 0.05). Furthermore, apoptosis-related proteins were assayed by Western blotting. We found that the cleaved PARP1 and caspase3 levels induced by CDDP were significantly enhanced by PMF treatment (Fig. 3E). Taken together, these results illustrated that PMF enhanced the sensitivity of A549/CDDP cells to CDDP and promoted apoptosis.


3',4',5',5,7-pentamethoxyflavone sensitizes Cisplatin-resistant A549 cells to Cisplatin by inhibition of Nrf2 pathway.

Hou X, Bai X, Gou X, Zeng H, Xia C, Zhuang W, Chen X, Zhao Z, Huang M, Jin J - Mol. Cells (2015)

PMF sensitized A549/CDDP cells to CDDP. (A) A549/CDDP cells were exposed to PMF (10, 25, and 50 μM) and were incubated with increasing concentration of CDDP (5 – 400 μM) in culture for 48 h and SRB assay were taken. (B) Comparison of the suppression of PMF (25 μM) combined with different concentration of CDDP on A549 cells and A549/CDDP cells. (C–D) Apoptosis ratios were determined through flow cytometry. (E) The expression of cleaved Caspase 3 and PARP1were determined by Western blot. Data are presented as means ± SD of three independent experiments and significant differences are indicated as *P < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4443280&req=5

f3-molce-38-5-396: PMF sensitized A549/CDDP cells to CDDP. (A) A549/CDDP cells were exposed to PMF (10, 25, and 50 μM) and were incubated with increasing concentration of CDDP (5 – 400 μM) in culture for 48 h and SRB assay were taken. (B) Comparison of the suppression of PMF (25 μM) combined with different concentration of CDDP on A549 cells and A549/CDDP cells. (C–D) Apoptosis ratios were determined through flow cytometry. (E) The expression of cleaved Caspase 3 and PARP1were determined by Western blot. Data are presented as means ± SD of three independent experiments and significant differences are indicated as *P < 0.05.
Mentions: To determine whether PMF can enhance the sensitivity of A549/DDP cells to CDDP, SRB assay was carried out and IC50 was calculated. As shown in Fig. 3A, IC50 of PMF+CDDP treated group was significantly lower than that of the CDDP alone treated group, indicating that PMF reduced the resistance of A549/CDDP cells to CDDP. The effect of PMF on the cytotoxicity of CDDP was also investigated in A549 cells, which expressed lower Nrf2 than A549/DDP cells. The results showed that the effect of PMF was diminished in A549 cells compared with that in A549/CDDP cells, indicating that PMF might sensitize A549/CDDP cells to cisplatin through inhibition of Nrf2 (Fig. 3B). To investigate whether combination of PMF and CDDP would promote apoptosis of cells, A549/CDDP cells were exposed to CDDP (25 μM) or PMF (25 μM) or in combination for 24 h. Apoptotic cells were determined by PI/Annexin V staining and flow cytometric analysis. Figures 3C–3D showed that the ratio of apoptotic cells in the combined treatment group was significantly increased compared with that in the CDDP alone treated group (P < 0.05). Furthermore, apoptosis-related proteins were assayed by Western blotting. We found that the cleaved PARP1 and caspase3 levels induced by CDDP were significantly enhanced by PMF treatment (Fig. 3E). Taken together, these results illustrated that PMF enhanced the sensitivity of A549/CDDP cells to CDDP and promoted apoptosis.

Bottom Line: In this study, we found that the expression levels of Nrf2 and its target genes GCLC, HO-1, NQO1 were significantly higher in cisplatin-resistant A549 (A549/CDDP) cells than those in A549 cells, and this resistance was partially reversed by Nrf2 siRNA. 3',4',5',5,7-Pentamethoxyflavone (PMF), a natural flavonoid extracted from Rutaceae plants, sensitized A549/CDDP to CDDP and substantially induced apoptosis compared with that of CDDP alone treated group, and this reversal effect decreased when Nrf2 was downregulated by siRNA.Mechanistically, PMF reduced Nrf2 expression leading to a reduction of Nrf2 downstream genes, and in contrast, this effect was decreased by blocking Nrf2 with siRNA.Taken together, these results demonstrated that PMF could be used as an effective adjuvant sensitizer to increase the efficacy of chemotherapeutic drugs by downregulating Nrf2 signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmaceutical Science, Sun Yat-sen University, Guangzhou 510006, China.

ABSTRACT
Nuclear factor erythroid 2-related factor 2 (Nrf2) is an important redox-sensitive transcription factor that regulates the expression of several cytoprotective genes. More recently, genetic analyses of human tumors have indicated that Nrf2 may cause resistance to chemotherapy. In this study, we found that the expression levels of Nrf2 and its target genes GCLC, HO-1, NQO1 were significantly higher in cisplatin-resistant A549 (A549/CDDP) cells than those in A549 cells, and this resistance was partially reversed by Nrf2 siRNA. 3',4',5',5,7-Pentamethoxyflavone (PMF), a natural flavonoid extracted from Rutaceae plants, sensitized A549/CDDP to CDDP and substantially induced apoptosis compared with that of CDDP alone treated group, and this reversal effect decreased when Nrf2 was downregulated by siRNA. Mechanistically, PMF reduced Nrf2 expression leading to a reduction of Nrf2 downstream genes, and in contrast, this effect was decreased by blocking Nrf2 with siRNA. Taken together, these results demonstrated that PMF could be used as an effective adjuvant sensitizer to increase the efficacy of chemotherapeutic drugs by downregulating Nrf2 signaling pathway.

No MeSH data available.


Related in: MedlinePlus