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Src/STAT3-dependent heme oxygenase-1 induction mediates chemoresistance of breast cancer cells to doxorubicin by promoting autophagy.

Tan Q, Wang H, Hu Y, Hu M, Li X - Cancer Sci. (2015)

Bottom Line: In the present study, we found an induction of HO-1 expression in doxorubicin (DOX)-treated MDA-MB-231 human breast adenocarcinoma cells, which showed insensitivity to DOX treatment.Knockdown HO-1 expression dramatically upregulated the incidence of MDA-MB-231 cell death under DOX treatment, indicating that HO-1 functions as a critical contributor to drug resistance in MDA-MB-231 cells.Moreover, upregulation of HO-1 expression required the activation of both signal transducer and activator of transcription (STAT)3 and its upstream regulator, protein kinase Src.

View Article: PubMed Central - PubMed

Affiliation: Department of Stress Medicine, Beijing Institute of Basic Medical Sciences, Beijing, China.

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Doxorubicin (DOX)-induced autophagy protected MDA-MB-231 cells from apoptosis. (a) MDA-MB-231 and MDA-MB-453 cells were left untreated or treated with DOX (0.2 μM) for 24 h and then autophagy was detected under confocal microscopy after staining the cells with Cyto-ID Green Autophagy Detection Reagent (ENZO Biotechnology). (b) The cells in (a) were trypsinized, collected and subjected to flow cytometric analysis to obtain the quantitative data indicating the autophagic flux inside the cells. (c) MDA-MB-231 and MDA-MB-453 cells were treated with DOX as described in Figure1(b) and then the expressions of Beclin-1 and LC3BI/II were detected. (d) MDA-MB-231 cells were transfected with ATG5 siRNA or control siRNA and then treated with DOX (0.2 μM) 36 h after transfection. Then the cells were collected and subjected to flow cytometric analysis to test the autophagic flux inside the cells. To avoid the off-target effect, two different siRNA against ATG5 were purchased and tested. Data shown here were obtained from the most effective one. (e) MDA-MB-231 cells were transfected and treated with DOX as described in (d) and then cell death incidence was detected 48 h after DOX treatment. (f) MDA-MB-231 cells were pretreated with chloroquine and then exposed to DOX (0.2 μM). The cell death incidence was detected 48 h after DOX treatment. (g) MDA-MB-231 cells were pretreated with 3-MA and then exposed to DOX (0.2 μM). The cell death incidence was detected 48 h after DOX treatment.
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fig02: Doxorubicin (DOX)-induced autophagy protected MDA-MB-231 cells from apoptosis. (a) MDA-MB-231 and MDA-MB-453 cells were left untreated or treated with DOX (0.2 μM) for 24 h and then autophagy was detected under confocal microscopy after staining the cells with Cyto-ID Green Autophagy Detection Reagent (ENZO Biotechnology). (b) The cells in (a) were trypsinized, collected and subjected to flow cytometric analysis to obtain the quantitative data indicating the autophagic flux inside the cells. (c) MDA-MB-231 and MDA-MB-453 cells were treated with DOX as described in Figure1(b) and then the expressions of Beclin-1 and LC3BI/II were detected. (d) MDA-MB-231 cells were transfected with ATG5 siRNA or control siRNA and then treated with DOX (0.2 μM) 36 h after transfection. Then the cells were collected and subjected to flow cytometric analysis to test the autophagic flux inside the cells. To avoid the off-target effect, two different siRNA against ATG5 were purchased and tested. Data shown here were obtained from the most effective one. (e) MDA-MB-231 cells were transfected and treated with DOX as described in (d) and then cell death incidence was detected 48 h after DOX treatment. (f) MDA-MB-231 cells were pretreated with chloroquine and then exposed to DOX (0.2 μM). The cell death incidence was detected 48 h after DOX treatment. (g) MDA-MB-231 cells were pretreated with 3-MA and then exposed to DOX (0.2 μM). The cell death incidence was detected 48 h after DOX treatment.

Mentions: In the following analysis, we attempted to determine the downstream signaling events following HO-1 induction in mediating DOX resistance in MDA-MB-231 cells. Autophagy is a metabolic process which can exert either an anti-apoptotic or a pro-apoptotic role under different stress conditions.23 One of the novel findings regarding autophagy regulation is that HO-1 has been illustrated to function as an upstream regulator by promoting or antagonizing autophagy.24,25 Therefore, we next investigated whether autophagy was induced upon DOX exposure and was involved in regulating DOX-induced apoptosis in MDA-MB-231 cells. As shown in Figure2(a), when DOX-treated MDA-MB-231 and MDA-MB-453 cells were stained with the Cyto-ID Green Autophagy Detection Reagent, we observed an obvious induction of autophagic flux in MDA-MB-231 cells, evidenced by a green fluorescence signal accumulated in spherical vacuoles in the perinuclear region of the cells; in contrast, no signal was detected in the MDA-MB-453 cells under the same DOX exposure conditions. To further confirm the above results, we next performed flow cytometry-based quantitative analysis of the cell populations loaded with Cyto-ID Green Autophagy Detection Reagent. As shown in Figure2(b), control MDA-MB-231 and MDA-MB-453 cells were stained only faintly, revealing low fluorescence signal intensity. After treatment with DOX for 24 h, the Cyto-ID Green Reagent signal increased dramatically in MDA-MB-231 cells. No similar phenomena were observed in the MDA-MB-453 cells under the same conditions, indicating that DOX caused an increase in autophagic vesicles only in MDA-MB-231 cells. In the following test, we observed a time-dependent increase of LC3B-I/II and Beclin-1 expression levels in the DOX-treated MDA-MB-231 cells, but not in MDA-MB-453 cells, further confirming that drug treatment enhanced autophagy activation in MDA-MB-231 cells.


Src/STAT3-dependent heme oxygenase-1 induction mediates chemoresistance of breast cancer cells to doxorubicin by promoting autophagy.

Tan Q, Wang H, Hu Y, Hu M, Li X - Cancer Sci. (2015)

Doxorubicin (DOX)-induced autophagy protected MDA-MB-231 cells from apoptosis. (a) MDA-MB-231 and MDA-MB-453 cells were left untreated or treated with DOX (0.2 μM) for 24 h and then autophagy was detected under confocal microscopy after staining the cells with Cyto-ID Green Autophagy Detection Reagent (ENZO Biotechnology). (b) The cells in (a) were trypsinized, collected and subjected to flow cytometric analysis to obtain the quantitative data indicating the autophagic flux inside the cells. (c) MDA-MB-231 and MDA-MB-453 cells were treated with DOX as described in Figure1(b) and then the expressions of Beclin-1 and LC3BI/II were detected. (d) MDA-MB-231 cells were transfected with ATG5 siRNA or control siRNA and then treated with DOX (0.2 μM) 36 h after transfection. Then the cells were collected and subjected to flow cytometric analysis to test the autophagic flux inside the cells. To avoid the off-target effect, two different siRNA against ATG5 were purchased and tested. Data shown here were obtained from the most effective one. (e) MDA-MB-231 cells were transfected and treated with DOX as described in (d) and then cell death incidence was detected 48 h after DOX treatment. (f) MDA-MB-231 cells were pretreated with chloroquine and then exposed to DOX (0.2 μM). The cell death incidence was detected 48 h after DOX treatment. (g) MDA-MB-231 cells were pretreated with 3-MA and then exposed to DOX (0.2 μM). The cell death incidence was detected 48 h after DOX treatment.
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fig02: Doxorubicin (DOX)-induced autophagy protected MDA-MB-231 cells from apoptosis. (a) MDA-MB-231 and MDA-MB-453 cells were left untreated or treated with DOX (0.2 μM) for 24 h and then autophagy was detected under confocal microscopy after staining the cells with Cyto-ID Green Autophagy Detection Reagent (ENZO Biotechnology). (b) The cells in (a) were trypsinized, collected and subjected to flow cytometric analysis to obtain the quantitative data indicating the autophagic flux inside the cells. (c) MDA-MB-231 and MDA-MB-453 cells were treated with DOX as described in Figure1(b) and then the expressions of Beclin-1 and LC3BI/II were detected. (d) MDA-MB-231 cells were transfected with ATG5 siRNA or control siRNA and then treated with DOX (0.2 μM) 36 h after transfection. Then the cells were collected and subjected to flow cytometric analysis to test the autophagic flux inside the cells. To avoid the off-target effect, two different siRNA against ATG5 were purchased and tested. Data shown here were obtained from the most effective one. (e) MDA-MB-231 cells were transfected and treated with DOX as described in (d) and then cell death incidence was detected 48 h after DOX treatment. (f) MDA-MB-231 cells were pretreated with chloroquine and then exposed to DOX (0.2 μM). The cell death incidence was detected 48 h after DOX treatment. (g) MDA-MB-231 cells were pretreated with 3-MA and then exposed to DOX (0.2 μM). The cell death incidence was detected 48 h after DOX treatment.
Mentions: In the following analysis, we attempted to determine the downstream signaling events following HO-1 induction in mediating DOX resistance in MDA-MB-231 cells. Autophagy is a metabolic process which can exert either an anti-apoptotic or a pro-apoptotic role under different stress conditions.23 One of the novel findings regarding autophagy regulation is that HO-1 has been illustrated to function as an upstream regulator by promoting or antagonizing autophagy.24,25 Therefore, we next investigated whether autophagy was induced upon DOX exposure and was involved in regulating DOX-induced apoptosis in MDA-MB-231 cells. As shown in Figure2(a), when DOX-treated MDA-MB-231 and MDA-MB-453 cells were stained with the Cyto-ID Green Autophagy Detection Reagent, we observed an obvious induction of autophagic flux in MDA-MB-231 cells, evidenced by a green fluorescence signal accumulated in spherical vacuoles in the perinuclear region of the cells; in contrast, no signal was detected in the MDA-MB-453 cells under the same DOX exposure conditions. To further confirm the above results, we next performed flow cytometry-based quantitative analysis of the cell populations loaded with Cyto-ID Green Autophagy Detection Reagent. As shown in Figure2(b), control MDA-MB-231 and MDA-MB-453 cells were stained only faintly, revealing low fluorescence signal intensity. After treatment with DOX for 24 h, the Cyto-ID Green Reagent signal increased dramatically in MDA-MB-231 cells. No similar phenomena were observed in the MDA-MB-453 cells under the same conditions, indicating that DOX caused an increase in autophagic vesicles only in MDA-MB-231 cells. In the following test, we observed a time-dependent increase of LC3B-I/II and Beclin-1 expression levels in the DOX-treated MDA-MB-231 cells, but not in MDA-MB-453 cells, further confirming that drug treatment enhanced autophagy activation in MDA-MB-231 cells.

Bottom Line: In the present study, we found an induction of HO-1 expression in doxorubicin (DOX)-treated MDA-MB-231 human breast adenocarcinoma cells, which showed insensitivity to DOX treatment.Knockdown HO-1 expression dramatically upregulated the incidence of MDA-MB-231 cell death under DOX treatment, indicating that HO-1 functions as a critical contributor to drug resistance in MDA-MB-231 cells.Moreover, upregulation of HO-1 expression required the activation of both signal transducer and activator of transcription (STAT)3 and its upstream regulator, protein kinase Src.

View Article: PubMed Central - PubMed

Affiliation: Department of Stress Medicine, Beijing Institute of Basic Medical Sciences, Beijing, China.

Show MeSH
Related in: MedlinePlus