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HMGB1-mediated autophagy promotes docetaxel resistance in human lung adenocarcinoma.

Pan B, Chen D, Huang J, Wang R, Feng B, Song H, Chen L - Mol. Cancer (2014)

Bottom Line: Mechanistic investigation revealed that HMGB1 promoted the formation of the Beclin-1-PI3K-III complex through activating the mitogen-activated protein kinase (MEK)-extracellular signal-regulated kinase (ERK) signaling pathway, thereby regulating autophagosome formation.Our results demonstrated that HMGB1-regulated autophagy is a significant contributor to docetaxel resistance in LAD cells.Suppression of HMGB1 or limiting HMGB1 cytosolic translocation diminished autophagic protection in response to docetaxel in LAD cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Oncology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002, P,R, China. songhaizhu@163.com.

ABSTRACT

Background: Docetaxel resistance remains a major obstacle in the treatment of non-small cell lung cancer (NSCLC). High-mobility group box 1 (HMGB1) has been shown to promote autophagy protection in response to antitumor therapy, but the exact molecular mechanism underlying HMGB1-mediated autophagy has not been clearly defined.

Methods: Lung adenocarcinoma (LAD) cells were transfected with pcDNA3.1-HMGB1 or HMGB1 shRNA, followed by docetaxel treatment. Cell viability and proliferation were tested by MTT assay and colony formation assay, respectively. Annexin V flow cytometric analysis and western blot analysis of activated caspase3 and cleaved PARP were used to evaluate apoptosis, while immunofluorescence microscopy and transmission electron microscopy were applied to assess autophagy activity. The formation of the Beclin-1-PI3K-III complex was examined by immunoprecipitation analysis. NOD/SCID mice were inoculated with docetaxel-resistant SPC-A1/DTX cells transfected with control or HMGB1 shRNA.

Results: HMGB1 translocated from the nucleus to the cytoplasm in LAD cells exposed to docetaxel and acted as a positive regulator of autophagy, which inhibited apoptosis and increased drug resistance. Suppression of HMGB1 restored the sensitivity of LAD cells to docetaxel both in vivo and in vitro. Mechanistic investigation revealed that HMGB1 promoted the formation of the Beclin-1-PI3K-III complex through activating the mitogen-activated protein kinase (MEK)-extracellular signal-regulated kinase (ERK) signaling pathway, thereby regulating autophagosome formation.

Conclusions: Our results demonstrated that HMGB1-regulated autophagy is a significant contributor to docetaxel resistance in LAD cells. Suppression of HMGB1 or limiting HMGB1 cytosolic translocation diminished autophagic protection in response to docetaxel in LAD cells.

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Docetaxel induced autophagy in LAD cells. (A) SPC-A1 and H1299 cells were treated with either the indicated concentrations of docetaxel for 24 h or 10 μg/l docetaxel for varying periods. Whole cell lysates were subjected to western blot analysis for LC3, p62 and GAPDH expression (as a loading control). (B) Both parental cells (SPC-A1 and H1299) and docetaxel-resistant cells (SPC-A1/DTX and H1299/DTX) were transiently transfected with a GFP-LC3 construct. Twenty-four hours later, parental cells were exposed to docetaxel (10 μg/l) for an additional 24 h. GFP-LC3 dot formation was analyzed as described in Materials and Methods (mean ± S.D. of three independent experiments, *P < 0.05, **P < 0.01). Bar = 50 μm. (C) SPC-A1 and H1299 cells pretreated with or without bafilomycin A1 (20 nM, 2 h) were exposed to docetaxel (10 μg/l) for an additional 24 h. Whole cell lysates were analyzed by western blot. (D, E) Parental cells (SPC-A1 and H1299) and docetaxel-resistant cells (SPC-A1/DTX and H1299/DTX) were analyzed by (D) western blot analysis for LC3 and GAPDH expression and (E) transmission electron microscopy analysis, as described in Materials and methods. A magnified view of the electron photomicrograph showed the characteristic autophagosomes. N: nucleus. The results were obtained from three independent experiments.
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Figure 1: Docetaxel induced autophagy in LAD cells. (A) SPC-A1 and H1299 cells were treated with either the indicated concentrations of docetaxel for 24 h or 10 μg/l docetaxel for varying periods. Whole cell lysates were subjected to western blot analysis for LC3, p62 and GAPDH expression (as a loading control). (B) Both parental cells (SPC-A1 and H1299) and docetaxel-resistant cells (SPC-A1/DTX and H1299/DTX) were transiently transfected with a GFP-LC3 construct. Twenty-four hours later, parental cells were exposed to docetaxel (10 μg/l) for an additional 24 h. GFP-LC3 dot formation was analyzed as described in Materials and Methods (mean ± S.D. of three independent experiments, *P < 0.05, **P < 0.01). Bar = 50 μm. (C) SPC-A1 and H1299 cells pretreated with or without bafilomycin A1 (20 nM, 2 h) were exposed to docetaxel (10 μg/l) for an additional 24 h. Whole cell lysates were analyzed by western blot. (D, E) Parental cells (SPC-A1 and H1299) and docetaxel-resistant cells (SPC-A1/DTX and H1299/DTX) were analyzed by (D) western blot analysis for LC3 and GAPDH expression and (E) transmission electron microscopy analysis, as described in Materials and methods. A magnified view of the electron photomicrograph showed the characteristic autophagosomes. N: nucleus. The results were obtained from three independent experiments.

Mentions: To assess the effect of docetaxel on autophagy and the role of autophagy in determining the sensitivity of LAD cells to docetaxel, first we evaluated autophagy activity in SPC-A1 and H1299 cells after treatment with docetaxel. As shown in Figure 1A, western blot analysis revealed that docetaxel treatment led to a dose- and time-dependent increase in the level of LC3-II and decrease in the amount of p62, two selective markers of autophagy, in both SPC-A1 and H1299 cells. The effect of docetaxel on autophagy was next confirmed by a GFP-LC3 punctate formation assay[15]. After transfecting both cell lines with a GFP-LC3 plasmid, we observed an abundance of punctate fluorescent dots after 24 h docetaxel treatment, which reflects the conversion from cytoplasmic LC3-I to the phosphatidylethanolamine-conjugated form LC3-II (Figure 1B). To further demonstrate the activation of autophagy by docetaxel, we treated cells with an autophagy-lysosomal inhibitor, bafilomycin A1, 2 h prior to docetaxel treatment[15,16]. As displayed in Figure 1C, in the presence of bafilomycin A1, LC3-II levels were increased in both cell lines in comparison with docetaxel alone treatment. We then analyzed autophagy activity in two docetaxel-resistant cell lines, SPC-A1/DTX and H1299/DTX, which were previously established in our lab. The baseline levels of LC3-II were higher in the docetaxel-resistant lines than in the parental cells (Figure 1D). SPC-A1/DTX and H1299/DTX showed increased autophagy activity as measured by analysis of GFP-LC3 punctate formation using confocal microscopy (Figure 1B) and by ultrastructural analysis of characteristic autophagosomes using transmission electron microscopy (Figure 1E).


HMGB1-mediated autophagy promotes docetaxel resistance in human lung adenocarcinoma.

Pan B, Chen D, Huang J, Wang R, Feng B, Song H, Chen L - Mol. Cancer (2014)

Docetaxel induced autophagy in LAD cells. (A) SPC-A1 and H1299 cells were treated with either the indicated concentrations of docetaxel for 24 h or 10 μg/l docetaxel for varying periods. Whole cell lysates were subjected to western blot analysis for LC3, p62 and GAPDH expression (as a loading control). (B) Both parental cells (SPC-A1 and H1299) and docetaxel-resistant cells (SPC-A1/DTX and H1299/DTX) were transiently transfected with a GFP-LC3 construct. Twenty-four hours later, parental cells were exposed to docetaxel (10 μg/l) for an additional 24 h. GFP-LC3 dot formation was analyzed as described in Materials and Methods (mean ± S.D. of three independent experiments, *P < 0.05, **P < 0.01). Bar = 50 μm. (C) SPC-A1 and H1299 cells pretreated with or without bafilomycin A1 (20 nM, 2 h) were exposed to docetaxel (10 μg/l) for an additional 24 h. Whole cell lysates were analyzed by western blot. (D, E) Parental cells (SPC-A1 and H1299) and docetaxel-resistant cells (SPC-A1/DTX and H1299/DTX) were analyzed by (D) western blot analysis for LC3 and GAPDH expression and (E) transmission electron microscopy analysis, as described in Materials and methods. A magnified view of the electron photomicrograph showed the characteristic autophagosomes. N: nucleus. The results were obtained from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 1: Docetaxel induced autophagy in LAD cells. (A) SPC-A1 and H1299 cells were treated with either the indicated concentrations of docetaxel for 24 h or 10 μg/l docetaxel for varying periods. Whole cell lysates were subjected to western blot analysis for LC3, p62 and GAPDH expression (as a loading control). (B) Both parental cells (SPC-A1 and H1299) and docetaxel-resistant cells (SPC-A1/DTX and H1299/DTX) were transiently transfected with a GFP-LC3 construct. Twenty-four hours later, parental cells were exposed to docetaxel (10 μg/l) for an additional 24 h. GFP-LC3 dot formation was analyzed as described in Materials and Methods (mean ± S.D. of three independent experiments, *P < 0.05, **P < 0.01). Bar = 50 μm. (C) SPC-A1 and H1299 cells pretreated with or without bafilomycin A1 (20 nM, 2 h) were exposed to docetaxel (10 μg/l) for an additional 24 h. Whole cell lysates were analyzed by western blot. (D, E) Parental cells (SPC-A1 and H1299) and docetaxel-resistant cells (SPC-A1/DTX and H1299/DTX) were analyzed by (D) western blot analysis for LC3 and GAPDH expression and (E) transmission electron microscopy analysis, as described in Materials and methods. A magnified view of the electron photomicrograph showed the characteristic autophagosomes. N: nucleus. The results were obtained from three independent experiments.
Mentions: To assess the effect of docetaxel on autophagy and the role of autophagy in determining the sensitivity of LAD cells to docetaxel, first we evaluated autophagy activity in SPC-A1 and H1299 cells after treatment with docetaxel. As shown in Figure 1A, western blot analysis revealed that docetaxel treatment led to a dose- and time-dependent increase in the level of LC3-II and decrease in the amount of p62, two selective markers of autophagy, in both SPC-A1 and H1299 cells. The effect of docetaxel on autophagy was next confirmed by a GFP-LC3 punctate formation assay[15]. After transfecting both cell lines with a GFP-LC3 plasmid, we observed an abundance of punctate fluorescent dots after 24 h docetaxel treatment, which reflects the conversion from cytoplasmic LC3-I to the phosphatidylethanolamine-conjugated form LC3-II (Figure 1B). To further demonstrate the activation of autophagy by docetaxel, we treated cells with an autophagy-lysosomal inhibitor, bafilomycin A1, 2 h prior to docetaxel treatment[15,16]. As displayed in Figure 1C, in the presence of bafilomycin A1, LC3-II levels were increased in both cell lines in comparison with docetaxel alone treatment. We then analyzed autophagy activity in two docetaxel-resistant cell lines, SPC-A1/DTX and H1299/DTX, which were previously established in our lab. The baseline levels of LC3-II were higher in the docetaxel-resistant lines than in the parental cells (Figure 1D). SPC-A1/DTX and H1299/DTX showed increased autophagy activity as measured by analysis of GFP-LC3 punctate formation using confocal microscopy (Figure 1B) and by ultrastructural analysis of characteristic autophagosomes using transmission electron microscopy (Figure 1E).

Bottom Line: Mechanistic investigation revealed that HMGB1 promoted the formation of the Beclin-1-PI3K-III complex through activating the mitogen-activated protein kinase (MEK)-extracellular signal-regulated kinase (ERK) signaling pathway, thereby regulating autophagosome formation.Our results demonstrated that HMGB1-regulated autophagy is a significant contributor to docetaxel resistance in LAD cells.Suppression of HMGB1 or limiting HMGB1 cytosolic translocation diminished autophagic protection in response to docetaxel in LAD cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Oncology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002, P,R, China. songhaizhu@163.com.

ABSTRACT

Background: Docetaxel resistance remains a major obstacle in the treatment of non-small cell lung cancer (NSCLC). High-mobility group box 1 (HMGB1) has been shown to promote autophagy protection in response to antitumor therapy, but the exact molecular mechanism underlying HMGB1-mediated autophagy has not been clearly defined.

Methods: Lung adenocarcinoma (LAD) cells were transfected with pcDNA3.1-HMGB1 or HMGB1 shRNA, followed by docetaxel treatment. Cell viability and proliferation were tested by MTT assay and colony formation assay, respectively. Annexin V flow cytometric analysis and western blot analysis of activated caspase3 and cleaved PARP were used to evaluate apoptosis, while immunofluorescence microscopy and transmission electron microscopy were applied to assess autophagy activity. The formation of the Beclin-1-PI3K-III complex was examined by immunoprecipitation analysis. NOD/SCID mice were inoculated with docetaxel-resistant SPC-A1/DTX cells transfected with control or HMGB1 shRNA.

Results: HMGB1 translocated from the nucleus to the cytoplasm in LAD cells exposed to docetaxel and acted as a positive regulator of autophagy, which inhibited apoptosis and increased drug resistance. Suppression of HMGB1 restored the sensitivity of LAD cells to docetaxel both in vivo and in vitro. Mechanistic investigation revealed that HMGB1 promoted the formation of the Beclin-1-PI3K-III complex through activating the mitogen-activated protein kinase (MEK)-extracellular signal-regulated kinase (ERK) signaling pathway, thereby regulating autophagosome formation.

Conclusions: Our results demonstrated that HMGB1-regulated autophagy is a significant contributor to docetaxel resistance in LAD cells. Suppression of HMGB1 or limiting HMGB1 cytosolic translocation diminished autophagic protection in response to docetaxel in LAD cells.

Show MeSH
Related in: MedlinePlus