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Cbl-b inhibits P-gp transporter function by preventing its translocation into caveolae in multiple drug-resistant gastric and breast cancers.

Zhang Y, Qu X, Teng Y, Li Z, Xu L, Liu J, Ma Y, Fan Y, Li C, Liu S, Wang Z, Hu X, Zhang J, Liu Y - Oncotarget (2015)

Bottom Line: The transport function of P-glycoprotein (P-gp) requires its efficient localization to caveolae, a subset of lipid rafts, and disruption of caveolae suppresses P-gp transport function.However, the regulatory molecules involved in the translocation of P-gp into caveolae remain unknown.In a negative feedback loop, the translocation of Cbl-b from the nucleus to the cytoplasm prevented the localization of P-gp to caveolae resulting in the reversal of MDR through the ubiquitination and degradation of c-Src.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, the First Hospital of China Medical University, Shenyang 110001, China.

ABSTRACT
The transport function of P-glycoprotein (P-gp) requires its efficient localization to caveolae, a subset of lipid rafts, and disruption of caveolae suppresses P-gp transport function. However, the regulatory molecules involved in the translocation of P-gp into caveolae remain unknown. In the present study, we showed that c-Src dependent Caveolin-1 phosphorylation promoted the translocation of P-gp into caveolae, resulting in multidrug resistance in adriamycin resistant gastric cancer SGC7901/Adr and breast cancer MCF-7/Adr cells. In a negative feedback loop, the translocation of Cbl-b from the nucleus to the cytoplasm prevented the localization of P-gp to caveolae resulting in the reversal of MDR through the ubiquitination and degradation of c-Src. Clinical data showed a significant positive relationship between Cbl-b expression and survival in P-gp positive breast cancer patients who received anthracycline-based chemotherapy. Our findings identified a new regulatory mechanism of P-gp transport function in multiple drug-resistant gastric and breast cancers.

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P-gp transport function is dependent on its translocation into caveolae(A) SGC7901, SGC7901/Adr, MCF-7 and MCF-7/Adr cells were treated with or without 20 μg/ml Dox for 5 min, lysed and fractionated by ultracentrifugation. P-gp was detected by western blotting. (B) Cells were incubated with 50 μg/ml nystatin for 2 h, then treated with 20 μg/ml Dox for 5 min, stained with anti-cholera toxin B subunit or anti-P-gp antibody, and analyzed by confocal fluorescence microscopy. Original magnification, × 40. (C) Cells were treated with nystatin (50 μg/ml) and R-123 uptake and efflux were examined in cells treated as in (A). (D)In situ PLA in SGC7901/Adr and MCF-7/Adr cells treated with or without 20 μg/ml Dox for 5 min. Primary antibodies against P-gp and Cav-1 were combined with secondary PLA probes. (E) SGC7901/Adr cells were transiently transfected with Cav-1 siRNA (si-Cav-1) for 48 h, followed by 20 μg/ml Dox and measurement of Dox uptake and efflux.
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Figure 1: P-gp transport function is dependent on its translocation into caveolae(A) SGC7901, SGC7901/Adr, MCF-7 and MCF-7/Adr cells were treated with or without 20 μg/ml Dox for 5 min, lysed and fractionated by ultracentrifugation. P-gp was detected by western blotting. (B) Cells were incubated with 50 μg/ml nystatin for 2 h, then treated with 20 μg/ml Dox for 5 min, stained with anti-cholera toxin B subunit or anti-P-gp antibody, and analyzed by confocal fluorescence microscopy. Original magnification, × 40. (C) Cells were treated with nystatin (50 μg/ml) and R-123 uptake and efflux were examined in cells treated as in (A). (D)In situ PLA in SGC7901/Adr and MCF-7/Adr cells treated with or without 20 μg/ml Dox for 5 min. Primary antibodies against P-gp and Cav-1 were combined with secondary PLA probes. (E) SGC7901/Adr cells were transiently transfected with Cav-1 siRNA (si-Cav-1) for 48 h, followed by 20 μg/ml Dox and measurement of Dox uptake and efflux.

Mentions: To determine the role of lipid rafts in P-gp-mediated MDR, lipid raft and non-lipid raft components were separated by sucrose density gradient centrifugation. The results showed that 20 μg/ml Dox increased the levels of P-gp in lipid rafts from 30% to 63% in SGC7901/Adr gastric cancer cells (Figure 1A). Similar results were obtained in MCF-7/Adr breast cancer cells (Figure 1A). Immunofluorescence analysis confirmed the Dox induced translocation of P-gp to aggregated lipid rafts, and this was inhibited by treatment with 50 μg/ml nystatin, a cholesterol-sequestering agent that disrupts lipid rafts (Figure 1B). P-gp function was assessed by measuring Dox uptake and retention, which showed that pretreatment with 50 μg/ml nystatin increased retention and inhibited the release of Dox and sensitized SGC7901/Adr and MCF7/Adr cells to Dox (Figure 1C). These findings suggest that P-gp transport function is dependent on its localization to lipid rafts.


Cbl-b inhibits P-gp transporter function by preventing its translocation into caveolae in multiple drug-resistant gastric and breast cancers.

Zhang Y, Qu X, Teng Y, Li Z, Xu L, Liu J, Ma Y, Fan Y, Li C, Liu S, Wang Z, Hu X, Zhang J, Liu Y - Oncotarget (2015)

P-gp transport function is dependent on its translocation into caveolae(A) SGC7901, SGC7901/Adr, MCF-7 and MCF-7/Adr cells were treated with or without 20 μg/ml Dox for 5 min, lysed and fractionated by ultracentrifugation. P-gp was detected by western blotting. (B) Cells were incubated with 50 μg/ml nystatin for 2 h, then treated with 20 μg/ml Dox for 5 min, stained with anti-cholera toxin B subunit or anti-P-gp antibody, and analyzed by confocal fluorescence microscopy. Original magnification, × 40. (C) Cells were treated with nystatin (50 μg/ml) and R-123 uptake and efflux were examined in cells treated as in (A). (D)In situ PLA in SGC7901/Adr and MCF-7/Adr cells treated with or without 20 μg/ml Dox for 5 min. Primary antibodies against P-gp and Cav-1 were combined with secondary PLA probes. (E) SGC7901/Adr cells were transiently transfected with Cav-1 siRNA (si-Cav-1) for 48 h, followed by 20 μg/ml Dox and measurement of Dox uptake and efflux.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4466646&req=5

Figure 1: P-gp transport function is dependent on its translocation into caveolae(A) SGC7901, SGC7901/Adr, MCF-7 and MCF-7/Adr cells were treated with or without 20 μg/ml Dox for 5 min, lysed and fractionated by ultracentrifugation. P-gp was detected by western blotting. (B) Cells were incubated with 50 μg/ml nystatin for 2 h, then treated with 20 μg/ml Dox for 5 min, stained with anti-cholera toxin B subunit or anti-P-gp antibody, and analyzed by confocal fluorescence microscopy. Original magnification, × 40. (C) Cells were treated with nystatin (50 μg/ml) and R-123 uptake and efflux were examined in cells treated as in (A). (D)In situ PLA in SGC7901/Adr and MCF-7/Adr cells treated with or without 20 μg/ml Dox for 5 min. Primary antibodies against P-gp and Cav-1 were combined with secondary PLA probes. (E) SGC7901/Adr cells were transiently transfected with Cav-1 siRNA (si-Cav-1) for 48 h, followed by 20 μg/ml Dox and measurement of Dox uptake and efflux.
Mentions: To determine the role of lipid rafts in P-gp-mediated MDR, lipid raft and non-lipid raft components were separated by sucrose density gradient centrifugation. The results showed that 20 μg/ml Dox increased the levels of P-gp in lipid rafts from 30% to 63% in SGC7901/Adr gastric cancer cells (Figure 1A). Similar results were obtained in MCF-7/Adr breast cancer cells (Figure 1A). Immunofluorescence analysis confirmed the Dox induced translocation of P-gp to aggregated lipid rafts, and this was inhibited by treatment with 50 μg/ml nystatin, a cholesterol-sequestering agent that disrupts lipid rafts (Figure 1B). P-gp function was assessed by measuring Dox uptake and retention, which showed that pretreatment with 50 μg/ml nystatin increased retention and inhibited the release of Dox and sensitized SGC7901/Adr and MCF7/Adr cells to Dox (Figure 1C). These findings suggest that P-gp transport function is dependent on its localization to lipid rafts.

Bottom Line: The transport function of P-glycoprotein (P-gp) requires its efficient localization to caveolae, a subset of lipid rafts, and disruption of caveolae suppresses P-gp transport function.However, the regulatory molecules involved in the translocation of P-gp into caveolae remain unknown.In a negative feedback loop, the translocation of Cbl-b from the nucleus to the cytoplasm prevented the localization of P-gp to caveolae resulting in the reversal of MDR through the ubiquitination and degradation of c-Src.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, the First Hospital of China Medical University, Shenyang 110001, China.

ABSTRACT
The transport function of P-glycoprotein (P-gp) requires its efficient localization to caveolae, a subset of lipid rafts, and disruption of caveolae suppresses P-gp transport function. However, the regulatory molecules involved in the translocation of P-gp into caveolae remain unknown. In the present study, we showed that c-Src dependent Caveolin-1 phosphorylation promoted the translocation of P-gp into caveolae, resulting in multidrug resistance in adriamycin resistant gastric cancer SGC7901/Adr and breast cancer MCF-7/Adr cells. In a negative feedback loop, the translocation of Cbl-b from the nucleus to the cytoplasm prevented the localization of P-gp to caveolae resulting in the reversal of MDR through the ubiquitination and degradation of c-Src. Clinical data showed a significant positive relationship between Cbl-b expression and survival in P-gp positive breast cancer patients who received anthracycline-based chemotherapy. Our findings identified a new regulatory mechanism of P-gp transport function in multiple drug-resistant gastric and breast cancers.

Show MeSH
Related in: MedlinePlus