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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 NF-κB pathwayin vitro. (A) ChIP analysis between NF-κB protein and ABCB1 gene. As a control, mouse monoclonal IgG was used. The blank group was the PCR results with no cDNA, and the input group was the cDNA from cell lysates without RIP procedure. (B) ChIP analysis was carried out to detect the effect of ABCB1 gene in HCT116/L-OHP cells treated with ZJW (50 μg/mL, 48 h). As a control, mouse monoclonal IgG was used. Input group was the cDNA from cell lysates without RIP procedure, and anti-NF-κB group was the cDNA from cell lysates after treatment with NF-κB antibody. (C) Western blotting assay was carried out to detect the level of P-gp, p-Akt (Ser473), p-IκB and p65 between HCT116 cell and HCT116/L-OHP cell. (D) The activity of the ABCB1 promoter 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), was analyzed by a dual-luciferase assay kit. The results are the firefly luciferase/renilla luciferase ratio from different groups. Data are means ± standard deviation of values from triplicate experiments. **P < 0.01 vs. control group.
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Fig5: ZJW suppresses P-gp mediated MDR by inhibiting activation of NF-κB pathwayin vitro. (A) ChIP analysis between NF-κB protein and ABCB1 gene. As a control, mouse monoclonal IgG was used. The blank group was the PCR results with no cDNA, and the input group was the cDNA from cell lysates without RIP procedure. (B) ChIP analysis was carried out to detect the effect of ABCB1 gene in HCT116/L-OHP cells treated with ZJW (50 μg/mL, 48 h). As a control, mouse monoclonal IgG was used. Input group was the cDNA from cell lysates without RIP procedure, and anti-NF-κB group was the cDNA from cell lysates after treatment with NF-κB antibody. (C) Western blotting assay was carried out to detect the level of P-gp, p-Akt (Ser473), p-IκB and p65 between HCT116 cell and HCT116/L-OHP cell. (D) The activity of the ABCB1 promoter 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), was analyzed by a dual-luciferase assay kit. The results are the firefly luciferase/renilla luciferase ratio from different groups. Data are means ± standard deviation 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 NF-κB pathwayin vitro. (A) ChIP analysis between NF-κB protein and ABCB1 gene. As a control, mouse monoclonal IgG was used. The blank group was the PCR results with no cDNA, and the input group was the cDNA from cell lysates without RIP procedure. (B) ChIP analysis was carried out to detect the effect of ABCB1 gene in HCT116/L-OHP cells treated with ZJW (50 μg/mL, 48 h). As a control, mouse monoclonal IgG was used. Input group was the cDNA from cell lysates without RIP procedure, and anti-NF-κB group was the cDNA from cell lysates after treatment with NF-κB antibody. (C) Western blotting assay was carried out to detect the level of P-gp, p-Akt (Ser473), p-IκB and p65 between HCT116 cell and HCT116/L-OHP cell. (D) The activity of the ABCB1 promoter 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), was analyzed by a dual-luciferase assay kit. The results are the firefly luciferase/renilla luciferase ratio from different groups. Data are means ± standard deviation of values from triplicate experiments. **P < 0.01 vs. control group.
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Related In: Results  -  Collection

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Fig5: ZJW suppresses P-gp mediated MDR by inhibiting activation of NF-κB pathwayin vitro. (A) ChIP analysis between NF-κB protein and ABCB1 gene. As a control, mouse monoclonal IgG was used. The blank group was the PCR results with no cDNA, and the input group was the cDNA from cell lysates without RIP procedure. (B) ChIP analysis was carried out to detect the effect of ABCB1 gene in HCT116/L-OHP cells treated with ZJW (50 μg/mL, 48 h). As a control, mouse monoclonal IgG was used. Input group was the cDNA from cell lysates without RIP procedure, and anti-NF-κB group was the cDNA from cell lysates after treatment with NF-κB antibody. (C) Western blotting assay was carried out to detect the level of P-gp, p-Akt (Ser473), p-IκB and p65 between HCT116 cell and HCT116/L-OHP cell. (D) The activity of the ABCB1 promoter 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), was analyzed by a dual-luciferase assay kit. The results are the firefly luciferase/renilla luciferase ratio from different groups. Data are means ± standard deviation 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