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The reversal effects of 3-bromopyruvate on multidrug resistance in vitro and in vivo derived from human breast MCF-7/ADR cells.

Wu L, Xu J, Yuan W, Wu B, Wang H, Liu G, Wang X, Du J, Cai S - PLoS ONE (2014)

Bottom Line: The intracellular level of ATP decreased 44%, 46% in the presence of 12.5.25 µM 3-Bromopyruvate, whereas the accumulation of rhodamine 123 and epirubicin (two typical P-glycoprotein substrates) in cells was significantly increased.Furthermore, we found that the mRNA and the total protein level of P-glycoprotein were slightly altered by 3-Bromopyruvate.Multidrug resistance reversal by 3-Bromopyruvate occurred through at least three approaches, namely, a decrease in the intracellular level of ATP and HK-II bioactivity, the inhibition of ATPase activity, and the slight decrease in P-glycoprotein expression in MCF-7/ADR cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Pharmacology, College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China.

ABSTRACT

Purpose: P-glycoprotein mediated efflux is one of the main mechanisms for multidrug resistance in cancers, and 3-Bromopyruvate acts as a promising multidrug resistance reversal compound in our study. To test the ability of 3-Bromopyruvate to overcome P-glycoprotein-mediated multidrug resistance and to explore its mechanisms of multidrug resistance reversal in MCF-7/ADR cells, we evaluate the in vitro and in vivo modulatory activity of this compound.

Methods: The in vitro and in vivo activity was determined using the MTT assay and human breast cancer xenograft models. The gene and protein expression of P-glycoprotein were determined using real-time polymerase chain reaction and the Western blotting technique, respectively. ABCB-1 bioactivity was tested by fluorescence microscopy, multi-mode microplate reader, and flow cytometry. The intracellular levels of ATP, HK-II, and ATPase activity were based on an assay kit according to the manufacturer's instructions.

Results: 3-Bromopyruvate treatment led to marked decreases in the IC50 values of selected chemotherapeutic drugs [e.g., doxorubicin (283 folds), paclitaxel (85 folds), daunorubicin (201 folds), and epirubicin (171 folds)] in MCF-7/ADR cells. 3-Bromopyruvate was found also to potentiate significantly the antitumor activity of epirubicin against MCF-7/ADR xenografts. The intracellular level of ATP decreased 44%, 46% in the presence of 12.5.25 µM 3-Bromopyruvate, whereas the accumulation of rhodamine 123 and epirubicin (two typical P-glycoprotein substrates) in cells was significantly increased. Furthermore, we found that the mRNA and the total protein level of P-glycoprotein were slightly altered by 3-Bromopyruvate. Moreover, the ATPase activity was significantly inhibited when 3-Bromopyruvate was applied.

Conclusion: We demonstrated that 3-Bromopyruvate can reverse P-glycoprotein-mediated efflux in MCF-7/ADR cells. Multidrug resistance reversal by 3-Bromopyruvate occurred through at least three approaches, namely, a decrease in the intracellular level of ATP and HK-II bioactivity, the inhibition of ATPase activity, and the slight decrease in P-glycoprotein expression in MCF-7/ADR cells.

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Inhibition of Rh123 efflux from MCF-7/ADR cells by 3-BrPA and VRP. 3-BrPA and VRP increase the accumulation of EPI in cells.a,b: After incubation 1 h in the presence of Rh123 (5 µM) and 3-BrPA (12.5, 25 µM) and VRP (10 µM),the cells were washed and incubated in fresh medium for indicated times. Each point represents the mean ± SD (n = 6). Each experiment was performed three times. c,d: These cells were incubated with 3-BrPA (12.5, 25 µM) at 37°C for 4 h, then 10 µM EPI was added for another 1 h incubation. Intracellular fluorescence was analyzed by flow cytometry. Control cells that were not exposed to any 3-BrPA, and VRP (10 µM) were used as positive control. The change of intracellular fluorescence in MCF-7 and MCF-7/ADR ***P<0.01 compared with the control.
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pone-0112132-g005: Inhibition of Rh123 efflux from MCF-7/ADR cells by 3-BrPA and VRP. 3-BrPA and VRP increase the accumulation of EPI in cells.a,b: After incubation 1 h in the presence of Rh123 (5 µM) and 3-BrPA (12.5, 25 µM) and VRP (10 µM),the cells were washed and incubated in fresh medium for indicated times. Each point represents the mean ± SD (n = 6). Each experiment was performed three times. c,d: These cells were incubated with 3-BrPA (12.5, 25 µM) at 37°C for 4 h, then 10 µM EPI was added for another 1 h incubation. Intracellular fluorescence was analyzed by flow cytometry. Control cells that were not exposed to any 3-BrPA, and VRP (10 µM) were used as positive control. The change of intracellular fluorescence in MCF-7 and MCF-7/ADR ***P<0.01 compared with the control.

Mentions: The accumulation of Rh123 was greatly enhanced by 3-BrPA in the resistant cells, evidenced by the increased fluorescent intensity in the presence of the compound (Fig. 4a). The extent of enhancement was 1.8 times, as revealed by the quantitative analysis (Fig. 4b). To be specific, the percentage of remaining intracellular Rh123 at 30, 60, 90, and 120 min in MCF-7/ADR is 88%, 83%, 81%, and 37% of control, respectively, after incubation with 25 µM 3-BrPA (Fig. 5b). These results suggested that 3-BrPA can modify the transport property of Rh123, which may result from the inhibition of ABCB-1/P-gp functioning.


The reversal effects of 3-bromopyruvate on multidrug resistance in vitro and in vivo derived from human breast MCF-7/ADR cells.

Wu L, Xu J, Yuan W, Wu B, Wang H, Liu G, Wang X, Du J, Cai S - PLoS ONE (2014)

Inhibition of Rh123 efflux from MCF-7/ADR cells by 3-BrPA and VRP. 3-BrPA and VRP increase the accumulation of EPI in cells.a,b: After incubation 1 h in the presence of Rh123 (5 µM) and 3-BrPA (12.5, 25 µM) and VRP (10 µM),the cells were washed and incubated in fresh medium for indicated times. Each point represents the mean ± SD (n = 6). Each experiment was performed three times. c,d: These cells were incubated with 3-BrPA (12.5, 25 µM) at 37°C for 4 h, then 10 µM EPI was added for another 1 h incubation. Intracellular fluorescence was analyzed by flow cytometry. Control cells that were not exposed to any 3-BrPA, and VRP (10 µM) were used as positive control. The change of intracellular fluorescence in MCF-7 and MCF-7/ADR ***P<0.01 compared with the control.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4221289&req=5

pone-0112132-g005: Inhibition of Rh123 efflux from MCF-7/ADR cells by 3-BrPA and VRP. 3-BrPA and VRP increase the accumulation of EPI in cells.a,b: After incubation 1 h in the presence of Rh123 (5 µM) and 3-BrPA (12.5, 25 µM) and VRP (10 µM),the cells were washed and incubated in fresh medium for indicated times. Each point represents the mean ± SD (n = 6). Each experiment was performed three times. c,d: These cells were incubated with 3-BrPA (12.5, 25 µM) at 37°C for 4 h, then 10 µM EPI was added for another 1 h incubation. Intracellular fluorescence was analyzed by flow cytometry. Control cells that were not exposed to any 3-BrPA, and VRP (10 µM) were used as positive control. The change of intracellular fluorescence in MCF-7 and MCF-7/ADR ***P<0.01 compared with the control.
Mentions: The accumulation of Rh123 was greatly enhanced by 3-BrPA in the resistant cells, evidenced by the increased fluorescent intensity in the presence of the compound (Fig. 4a). The extent of enhancement was 1.8 times, as revealed by the quantitative analysis (Fig. 4b). To be specific, the percentage of remaining intracellular Rh123 at 30, 60, 90, and 120 min in MCF-7/ADR is 88%, 83%, 81%, and 37% of control, respectively, after incubation with 25 µM 3-BrPA (Fig. 5b). These results suggested that 3-BrPA can modify the transport property of Rh123, which may result from the inhibition of ABCB-1/P-gp functioning.

Bottom Line: The intracellular level of ATP decreased 44%, 46% in the presence of 12.5.25 µM 3-Bromopyruvate, whereas the accumulation of rhodamine 123 and epirubicin (two typical P-glycoprotein substrates) in cells was significantly increased.Furthermore, we found that the mRNA and the total protein level of P-glycoprotein were slightly altered by 3-Bromopyruvate.Multidrug resistance reversal by 3-Bromopyruvate occurred through at least three approaches, namely, a decrease in the intracellular level of ATP and HK-II bioactivity, the inhibition of ATPase activity, and the slight decrease in P-glycoprotein expression in MCF-7/ADR cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Pharmacology, College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China.

ABSTRACT

Purpose: P-glycoprotein mediated efflux is one of the main mechanisms for multidrug resistance in cancers, and 3-Bromopyruvate acts as a promising multidrug resistance reversal compound in our study. To test the ability of 3-Bromopyruvate to overcome P-glycoprotein-mediated multidrug resistance and to explore its mechanisms of multidrug resistance reversal in MCF-7/ADR cells, we evaluate the in vitro and in vivo modulatory activity of this compound.

Methods: The in vitro and in vivo activity was determined using the MTT assay and human breast cancer xenograft models. The gene and protein expression of P-glycoprotein were determined using real-time polymerase chain reaction and the Western blotting technique, respectively. ABCB-1 bioactivity was tested by fluorescence microscopy, multi-mode microplate reader, and flow cytometry. The intracellular levels of ATP, HK-II, and ATPase activity were based on an assay kit according to the manufacturer's instructions.

Results: 3-Bromopyruvate treatment led to marked decreases in the IC50 values of selected chemotherapeutic drugs [e.g., doxorubicin (283 folds), paclitaxel (85 folds), daunorubicin (201 folds), and epirubicin (171 folds)] in MCF-7/ADR cells. 3-Bromopyruvate was found also to potentiate significantly the antitumor activity of epirubicin against MCF-7/ADR xenografts. The intracellular level of ATP decreased 44%, 46% in the presence of 12.5.25 µM 3-Bromopyruvate, whereas the accumulation of rhodamine 123 and epirubicin (two typical P-glycoprotein substrates) in cells was significantly increased. Furthermore, we found that the mRNA and the total protein level of P-glycoprotein were slightly altered by 3-Bromopyruvate. Moreover, the ATPase activity was significantly inhibited when 3-Bromopyruvate was applied.

Conclusion: We demonstrated that 3-Bromopyruvate can reverse P-glycoprotein-mediated efflux in MCF-7/ADR cells. Multidrug resistance reversal by 3-Bromopyruvate occurred through at least three approaches, namely, a decrease in the intracellular level of ATP and HK-II bioactivity, the inhibition of ATPase activity, and the slight decrease in P-glycoprotein expression in MCF-7/ADR cells.

Show MeSH
Related in: MedlinePlus