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Zuo Jin Wan reverses P-gp-mediated drug-resistance by inhibiting activation of the PI3K/Akt/NF-κB pathway.

Sui H, Pan SF, Feng Y, Jin BH, Liu X, Zhou LH, Hou FG, Wang WH, Fu XL, Han ZF, Ren JL, Shi XL, Zhu HR, Li Q - BMC Complement Altern Med (2014)

Bottom Line: We found that ZJW significantly enhanced the sensitivity of chemotherapeutic drugs and increased oxaliplatin (L-OHP)-induced cell apoptosis in a time- and dose-dependent manner.The effect of ZJW in reversing drug-resistance and suppressing phosphorylation of Akt (Ser473) and NF-κB were weakened after treatment with a PI3K/Akt activator in HCT116/L-OHP cells.Our study has provided the first direct evidence that ZJW reverses drug-resistance in human colorectal cancer by blocking the PI3K/Akt/NF-κB signaling pathway, and could be considered as a useful drug for cancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China. Lzwf@hotmail.com.

ABSTRACT

Background: Zuo-Jin-Wan (ZJW), a traditional Chinese medicine formula, has been identified to be effective against drug resistance in cancer. In the present study, we investigated the effect of ZJW on acquired oxaliplatin-resistant and the PI3K/Akt/NF-κB pathway in vitro.

Methods: We tested the dose-response relationship of ZJW on reversing drug-resistance by CCK-8 assay and flow cytometry analysis in vitro. The protein expression of P-gp, MRP-2, LRP, and ABCB1 mRNA expression level were evaluated by Western blot and quantitative RT-PCR. The activities of PI3K/Akt/NF-κB pathway were also examined with or without ZJW, including Akt, IκB, p65 and their phosphorylation expression.

Results: We found that ZJW significantly enhanced the sensitivity of chemotherapeutic drugs and increased oxaliplatin (L-OHP)-induced cell apoptosis in a time- and dose-dependent manner. Moreover, both ZJW and a PI3K specific inhibitor (LY294002) suppressed phosphorylation of Akt (Ser473) and NF-κB, which is necessary in the activation of the PI3K/Akt/NF-κB pathway. The effect of ZJW in reversing drug-resistance and suppressing phosphorylation of Akt (Ser473) and NF-κB were weakened after treatment with a PI3K/Akt activator in HCT116/L-OHP cells.

Conclusions: Our study has provided the first direct evidence that ZJW reverses drug-resistance in human colorectal cancer by blocking the PI3K/Akt/NF-κB signaling pathway, and could be considered as a useful drug for cancer therapy.

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ZJW suppresses P-gp mediated MDR by inhibiting activation of Akt (Ser473)/IκB phosphorylation (Ser473)in vitro. (A&B) Western blotting assay was carried out to detect the level of Akt, phosphorylation of Akt (Thr307/Ser473), IκB, p65, and phosphorylation of IκB in HCT116/L-OHP cells treated with LY294002 (20 μM, 2 h), ZJW (50 μg/mL, 48 h), and a combination of ZJW (50 μg/mL, 48 h) and IGF-1 (100 ng/mL, 48 h). GAPDH was used to ensure equal loading of proteins in each lane. Data are means ± SD of values from triplicate experiments. **P < 0.01 vs. control group.
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Fig4: ZJW suppresses P-gp mediated MDR by inhibiting activation of Akt (Ser473)/IκB phosphorylation (Ser473)in vitro. (A&B) Western blotting assay was carried out to detect the level of Akt, phosphorylation of Akt (Thr307/Ser473), IκB, p65, and phosphorylation of IκB in HCT116/L-OHP cells treated with LY294002 (20 μM, 2 h), ZJW (50 μg/mL, 48 h), and a combination of ZJW (50 μg/mL, 48 h) and IGF-1 (100 ng/mL, 48 h). GAPDH was used to ensure equal loading of proteins in each lane. Data are means ± SD of values from triplicate experiments. **P < 0.01 vs. control group.

Mentions: To determine whether the PI3K/Akt pathways are involved in the P-gp mediated drug-resistance phenotype in colorectal cancer, the expression of Akt and Akt phosphorylation (Thr307 and Ser473) were examined in HCT116/L-OHP cells by western blotting. Notably, ZJW or LY294002 decreased the expression of Akt phosphorylation (Ser473) in HCT116/L-OHP cells (Figure 4A), but did not significantly affect the expression of Akt or p-Akt at Thr307. Additionally, this inhibition was weakened after the addition IGF-1 (Figure 4A). These observations suggest that PI3K/Akt pathway activation could regulate the expression of P-gp, which is involved in controlling the drug-resistance phenotype.Evidence suggests that the PI3K/Akt pathway is involved in the development of chemoresistance, at least in part by the activation of NF-κB. In light of our results, we examined the effect of ZJW on NF-κB and phosphorylation of NF-κB in the cytoplasm and p65 levels in the nucleus. Similar to the effect on Akt and p-Akt, we observed a down-regulation of NF-κB phosphorylation in HCT116/L-OHP cells treated with ZJW or LY294002 (Figure 4B).Since the ABCB1 promoter is shown to bind with NF-κB, we hypothesized that there would be a down-regulation of ABCB1 promoter activity after treatment with ZJW. We found that the activity of the ABCB1 promoter was down-regulated after cells were treated with ZJW (Figure 5D). To further ascertain whether NF-κB protein could bind to the ABCB1 gene in HCT116/L-OHP cells, we tested NF-κB and ABCB1 by ChIP. ChIP assay confirmed that the NF-κB protein could bind to the ABCB1 gene promoter in HCT116/L-OHP cells, but not in HCT116 cells (Figure 5A). As presupposed, it indeed a down-regulation of ABCB1 mRNA expression after treatment with ZJW compared with control group (Figure 5B). Similar as the results in Figure 5A, the level of P-gp, p-Akt (Ser473), p-IκB and p65 were significantly increased in HCT116/L-OHP cell compared with HCT116 cell (Figure 5C). It indicated the difference between MDR cell and sensitive cell, which may be an important mechanism of drug-resistance phenotype. Therefore, ZJW was identified as a PI3K/Akt/NF-κB pathway inhibitor, which inhibits the phosphorylation of Akt and NF-κB and the binding of NF-κB and ABCB1 in MDR cell nuclei.Figure 4


Zuo Jin Wan reverses P-gp-mediated drug-resistance by inhibiting activation of the PI3K/Akt/NF-κB pathway.

Sui H, Pan SF, Feng Y, Jin BH, Liu X, Zhou LH, Hou FG, Wang WH, Fu XL, Han ZF, Ren JL, Shi XL, Zhu HR, Li Q - BMC Complement Altern Med (2014)

ZJW suppresses P-gp mediated MDR by inhibiting activation of Akt (Ser473)/IκB phosphorylation (Ser473)in vitro. (A&B) Western blotting assay was carried out to detect the level of Akt, phosphorylation of Akt (Thr307/Ser473), IκB, p65, and phosphorylation of IκB in HCT116/L-OHP cells treated with LY294002 (20 μM, 2 h), ZJW (50 μg/mL, 48 h), and a combination of ZJW (50 μg/mL, 48 h) and IGF-1 (100 ng/mL, 48 h). GAPDH was used to ensure equal loading of proteins in each lane. Data are means ± SD of values from triplicate experiments. **P < 0.01 vs. control group.
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Related In: Results  -  Collection

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Fig4: ZJW suppresses P-gp mediated MDR by inhibiting activation of Akt (Ser473)/IκB phosphorylation (Ser473)in vitro. (A&B) Western blotting assay was carried out to detect the level of Akt, phosphorylation of Akt (Thr307/Ser473), IκB, p65, and phosphorylation of IκB in HCT116/L-OHP cells treated with LY294002 (20 μM, 2 h), ZJW (50 μg/mL, 48 h), and a combination of ZJW (50 μg/mL, 48 h) and IGF-1 (100 ng/mL, 48 h). GAPDH was used to ensure equal loading of proteins in each lane. Data are means ± SD of values from triplicate experiments. **P < 0.01 vs. control group.
Mentions: To determine whether the PI3K/Akt pathways are involved in the P-gp mediated drug-resistance phenotype in colorectal cancer, the expression of Akt and Akt phosphorylation (Thr307 and Ser473) were examined in HCT116/L-OHP cells by western blotting. Notably, ZJW or LY294002 decreased the expression of Akt phosphorylation (Ser473) in HCT116/L-OHP cells (Figure 4A), but did not significantly affect the expression of Akt or p-Akt at Thr307. Additionally, this inhibition was weakened after the addition IGF-1 (Figure 4A). These observations suggest that PI3K/Akt pathway activation could regulate the expression of P-gp, which is involved in controlling the drug-resistance phenotype.Evidence suggests that the PI3K/Akt pathway is involved in the development of chemoresistance, at least in part by the activation of NF-κB. In light of our results, we examined the effect of ZJW on NF-κB and phosphorylation of NF-κB in the cytoplasm and p65 levels in the nucleus. Similar to the effect on Akt and p-Akt, we observed a down-regulation of NF-κB phosphorylation in HCT116/L-OHP cells treated with ZJW or LY294002 (Figure 4B).Since the ABCB1 promoter is shown to bind with NF-κB, we hypothesized that there would be a down-regulation of ABCB1 promoter activity after treatment with ZJW. We found that the activity of the ABCB1 promoter was down-regulated after cells were treated with ZJW (Figure 5D). To further ascertain whether NF-κB protein could bind to the ABCB1 gene in HCT116/L-OHP cells, we tested NF-κB and ABCB1 by ChIP. ChIP assay confirmed that the NF-κB protein could bind to the ABCB1 gene promoter in HCT116/L-OHP cells, but not in HCT116 cells (Figure 5A). As presupposed, it indeed a down-regulation of ABCB1 mRNA expression after treatment with ZJW compared with control group (Figure 5B). Similar as the results in Figure 5A, the level of P-gp, p-Akt (Ser473), p-IκB and p65 were significantly increased in HCT116/L-OHP cell compared with HCT116 cell (Figure 5C). It indicated the difference between MDR cell and sensitive cell, which may be an important mechanism of drug-resistance phenotype. Therefore, ZJW was identified as a PI3K/Akt/NF-κB pathway inhibitor, which inhibits the phosphorylation of Akt and NF-κB and the binding of NF-κB and ABCB1 in MDR cell nuclei.Figure 4

Bottom Line: We found that ZJW significantly enhanced the sensitivity of chemotherapeutic drugs and increased oxaliplatin (L-OHP)-induced cell apoptosis in a time- and dose-dependent manner.The effect of ZJW in reversing drug-resistance and suppressing phosphorylation of Akt (Ser473) and NF-κB were weakened after treatment with a PI3K/Akt activator in HCT116/L-OHP cells.Our study has provided the first direct evidence that ZJW reverses drug-resistance in human colorectal cancer by blocking the PI3K/Akt/NF-κB signaling pathway, and could be considered as a useful drug for cancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China. Lzwf@hotmail.com.

ABSTRACT

Background: Zuo-Jin-Wan (ZJW), a traditional Chinese medicine formula, has been identified to be effective against drug resistance in cancer. In the present study, we investigated the effect of ZJW on acquired oxaliplatin-resistant and the PI3K/Akt/NF-κB pathway in vitro.

Methods: We tested the dose-response relationship of ZJW on reversing drug-resistance by CCK-8 assay and flow cytometry analysis in vitro. The protein expression of P-gp, MRP-2, LRP, and ABCB1 mRNA expression level were evaluated by Western blot and quantitative RT-PCR. The activities of PI3K/Akt/NF-κB pathway were also examined with or without ZJW, including Akt, IκB, p65 and their phosphorylation expression.

Results: We found that ZJW significantly enhanced the sensitivity of chemotherapeutic drugs and increased oxaliplatin (L-OHP)-induced cell apoptosis in a time- and dose-dependent manner. Moreover, both ZJW and a PI3K specific inhibitor (LY294002) suppressed phosphorylation of Akt (Ser473) and NF-κB, which is necessary in the activation of the PI3K/Akt/NF-κB pathway. The effect of ZJW in reversing drug-resistance and suppressing phosphorylation of Akt (Ser473) and NF-κB were weakened after treatment with a PI3K/Akt activator in HCT116/L-OHP cells.

Conclusions: Our study has provided the first direct evidence that ZJW reverses drug-resistance in human colorectal cancer by blocking the PI3K/Akt/NF-κB signaling pathway, and could be considered as a useful drug for cancer therapy.

Show MeSH
Related in: MedlinePlus