Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibition of autophagy enhanced sensitivity of LAD cells to docetaxel. (A, B) LAD cells were treated with 3-methyladenine (3-MA, 5 mM) or transiently transfected with Atg5 or control siRNA. Cells were exposed to the indicated concentrations of docetaxel for 48 h. Viable cells were determined with an MTT assay as described in Materials and methods. (C, D) LAD cells were incubated with various concentrations of docetaxel for 48 h in the presence or absence of 3-MA. The colonies were stained and counted, and survival curves were constructed from three independent experiments. (E) SPC-A1 and H1299 cells were treated with docetaxel (10 μg/l) in the presence or absence of 3-MA (5 mM, 2 h) or Atg5 siRNA. Apoptosis was determined by flow cytometric analysis of Annexin-V/PI staining. (F) SPC-A1/DTX cells were treated with the indicated doses of docetaxel in the presence or absence of 3-MA or Atg5 siRNA. Apoptosis was determined by flow cytometric analysis of Annexin-V/PI staining. Each point or bar represents mean ± S.D. of triplicate determinations. *P< 0.05, **P< 0.01 compared with docetaxel alone treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4125709&req=5

Figure 2: Inhibition of autophagy enhanced sensitivity of LAD cells to docetaxel. (A, B) LAD cells were treated with 3-methyladenine (3-MA, 5 mM) or transiently transfected with Atg5 or control siRNA. Cells were exposed to the indicated concentrations of docetaxel for 48 h. Viable cells were determined with an MTT assay as described in Materials and methods. (C, D) LAD cells were incubated with various concentrations of docetaxel for 48 h in the presence or absence of 3-MA. The colonies were stained and counted, and survival curves were constructed from three independent experiments. (E) SPC-A1 and H1299 cells were treated with docetaxel (10 μg/l) in the presence or absence of 3-MA (5 mM, 2 h) or Atg5 siRNA. Apoptosis was determined by flow cytometric analysis of Annexin-V/PI staining. (F) SPC-A1/DTX cells were treated with the indicated doses of docetaxel in the presence or absence of 3-MA or Atg5 siRNA. Apoptosis was determined by flow cytometric analysis of Annexin-V/PI staining. Each point or bar represents mean ± S.D. of triplicate determinations. *P< 0.05, **P< 0.01 compared with docetaxel alone treatment.

Mentions: Next, to determine whether docetaxel-induced autophagy played a protective or resistant role in the drug treatment, we treated SPC-A1 and H1299 cells with an autophagy inhibitor, 3-methyladenine (3-MA), or transfected cells with small-interfering RNA (siRNA) specifically targeting the autophagic gene Atg5 before addition of docetaxel. Both 3-MA and Atg5 siRNA efficiently blocked activation of autophagy, which caused an increase of cytotoxicity and apoptosis and decrease in the proliferation rate of SPC-A1 and H1299 cells (Figure 2A,2C and2E). Western blot analysis of c-caspase3 and c-PARP also confirmed the results (Additional file1: Figure S1A and S1B). However, the sensitivity of SPC-A1/DTX and H1299/DTX cells to docetaxel was markedly enhanced (Figure 2B) while the proliferation rate was greatly diminished (Figure 2D) after addition of 3-MA or silencing of Atg5. Moreover, both docetaxel-resistant cell lines showed an increased propensity for apoptosis after inhibition of autophagy (Figure 2F, Additional file1: Figure S1C and S1D).


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)

Inhibition of autophagy enhanced sensitivity of LAD cells to docetaxel. (A, B) LAD cells were treated with 3-methyladenine (3-MA, 5 mM) or transiently transfected with Atg5 or control siRNA. Cells were exposed to the indicated concentrations of docetaxel for 48 h. Viable cells were determined with an MTT assay as described in Materials and methods. (C, D) LAD cells were incubated with various concentrations of docetaxel for 48 h in the presence or absence of 3-MA. The colonies were stained and counted, and survival curves were constructed from three independent experiments. (E) SPC-A1 and H1299 cells were treated with docetaxel (10 μg/l) in the presence or absence of 3-MA (5 mM, 2 h) or Atg5 siRNA. Apoptosis was determined by flow cytometric analysis of Annexin-V/PI staining. (F) SPC-A1/DTX cells were treated with the indicated doses of docetaxel in the presence or absence of 3-MA or Atg5 siRNA. Apoptosis was determined by flow cytometric analysis of Annexin-V/PI staining. Each point or bar represents mean ± S.D. of triplicate determinations. *P< 0.05, **P< 0.01 compared with docetaxel alone treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4125709&req=5

Figure 2: Inhibition of autophagy enhanced sensitivity of LAD cells to docetaxel. (A, B) LAD cells were treated with 3-methyladenine (3-MA, 5 mM) or transiently transfected with Atg5 or control siRNA. Cells were exposed to the indicated concentrations of docetaxel for 48 h. Viable cells were determined with an MTT assay as described in Materials and methods. (C, D) LAD cells were incubated with various concentrations of docetaxel for 48 h in the presence or absence of 3-MA. The colonies were stained and counted, and survival curves were constructed from three independent experiments. (E) SPC-A1 and H1299 cells were treated with docetaxel (10 μg/l) in the presence or absence of 3-MA (5 mM, 2 h) or Atg5 siRNA. Apoptosis was determined by flow cytometric analysis of Annexin-V/PI staining. (F) SPC-A1/DTX cells were treated with the indicated doses of docetaxel in the presence or absence of 3-MA or Atg5 siRNA. Apoptosis was determined by flow cytometric analysis of Annexin-V/PI staining. Each point or bar represents mean ± S.D. of triplicate determinations. *P< 0.05, **P< 0.01 compared with docetaxel alone treatment.
Mentions: Next, to determine whether docetaxel-induced autophagy played a protective or resistant role in the drug treatment, we treated SPC-A1 and H1299 cells with an autophagy inhibitor, 3-methyladenine (3-MA), or transfected cells with small-interfering RNA (siRNA) specifically targeting the autophagic gene Atg5 before addition of docetaxel. Both 3-MA and Atg5 siRNA efficiently blocked activation of autophagy, which caused an increase of cytotoxicity and apoptosis and decrease in the proliferation rate of SPC-A1 and H1299 cells (Figure 2A,2C and2E). Western blot analysis of c-caspase3 and c-PARP also confirmed the results (Additional file1: Figure S1A and S1B). However, the sensitivity of SPC-A1/DTX and H1299/DTX cells to docetaxel was markedly enhanced (Figure 2B) while the proliferation rate was greatly diminished (Figure 2D) after addition of 3-MA or silencing of Atg5. Moreover, both docetaxel-resistant cell lines showed an increased propensity for apoptosis after inhibition of autophagy (Figure 2F, Additional file1: Figure S1C and S1D).

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