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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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HMGB1 altered the sensitivity of LAD cells to docetaxel in vitro. Parental and docetaxel-resistant LAD cells were transfected with HMGB1 shRNA or pretreated with EP before incubation with indicated concentrations of docetaxel for 48 h. (A, B) Cell viability was analyzed by MTT assay. (C, D) Cell proliferation was evaluated by colony formation assay. (E, F) Apoptosis was analyzed by flow cytometric analysis of Annexin-V/PI staining. (G) SPC-A1 cells transfected with pcDNA3.1 control or pcDNA3.1-HMGB1 were exposed to indicated doses of docetaxel for 48 h in the presence or absence of 3-MA (5 mM) or Atg5 siRNA. Cell viability was analyzed by MTT assay. The results shown are the representative of three identical experiments, and the bars are the mean ± S.D. *P < 0.05, **P < 0.01.
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Figure 4: HMGB1 altered the sensitivity of LAD cells to docetaxel in vitro. Parental and docetaxel-resistant LAD cells were transfected with HMGB1 shRNA or pretreated with EP before incubation with indicated concentrations of docetaxel for 48 h. (A, B) Cell viability was analyzed by MTT assay. (C, D) Cell proliferation was evaluated by colony formation assay. (E, F) Apoptosis was analyzed by flow cytometric analysis of Annexin-V/PI staining. (G) SPC-A1 cells transfected with pcDNA3.1 control or pcDNA3.1-HMGB1 were exposed to indicated doses of docetaxel for 48 h in the presence or absence of 3-MA (5 mM) or Atg5 siRNA. Cell viability was analyzed by MTT assay. The results shown are the representative of three identical experiments, and the bars are the mean ± S.D. *P < 0.05, **P < 0.01.

Mentions: To clarify the role of HMGB1 in LAD cells following chemotherapy, we analyzed the responses of SPC-A1 and H1299 cells to docetaxel treatment with depleted for HMGB1 expression. Knockdown of HMGB1 or limiting HMGB1 cytosolic translocation in SPC-A1 and H1299 cells enhanced the cellular response to docetaxel (Figure 4A) and inhibited cell proliferation (Figure 4C). Docetaxel induced apoptotic cell death to a great extent after HMGB1 knockdown, as shown by an increase of Annexin-V positive cells by 15.59 ± 1.01% and 12.79 ± 0.88% in SPC-A1 and H1299 cells, respectively (Figure 4E), as well as increased level of activated caspase3 and PARP cleavage (Additional file3: Figure S3A). Moreover, treatment with an apoptosis inhibitor, Z-VAD-fmk[17], reduced the activation of caspase3 (Additional file3: Figure S3A). Conversely, pretreatment with HMGB1 increased drug resistance in both parental LAD cells, as displayed by an elevated cell survival rate. Nevertheless, blockade of autophagy by 3-MA or Atg5 siRNA reversed HMGB1-induced protection against docetaxel (Figure 4G).


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)

HMGB1 altered the sensitivity of LAD cells to docetaxel in vitro. Parental and docetaxel-resistant LAD cells were transfected with HMGB1 shRNA or pretreated with EP before incubation with indicated concentrations of docetaxel for 48 h. (A, B) Cell viability was analyzed by MTT assay. (C, D) Cell proliferation was evaluated by colony formation assay. (E, F) Apoptosis was analyzed by flow cytometric analysis of Annexin-V/PI staining. (G) SPC-A1 cells transfected with pcDNA3.1 control or pcDNA3.1-HMGB1 were exposed to indicated doses of docetaxel for 48 h in the presence or absence of 3-MA (5 mM) or Atg5 siRNA. Cell viability was analyzed by MTT assay. The results shown are the representative of three identical experiments, and the bars are the mean ± S.D. *P < 0.05, **P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: HMGB1 altered the sensitivity of LAD cells to docetaxel in vitro. Parental and docetaxel-resistant LAD cells were transfected with HMGB1 shRNA or pretreated with EP before incubation with indicated concentrations of docetaxel for 48 h. (A, B) Cell viability was analyzed by MTT assay. (C, D) Cell proliferation was evaluated by colony formation assay. (E, F) Apoptosis was analyzed by flow cytometric analysis of Annexin-V/PI staining. (G) SPC-A1 cells transfected with pcDNA3.1 control or pcDNA3.1-HMGB1 were exposed to indicated doses of docetaxel for 48 h in the presence or absence of 3-MA (5 mM) or Atg5 siRNA. Cell viability was analyzed by MTT assay. The results shown are the representative of three identical experiments, and the bars are the mean ± S.D. *P < 0.05, **P < 0.01.
Mentions: To clarify the role of HMGB1 in LAD cells following chemotherapy, we analyzed the responses of SPC-A1 and H1299 cells to docetaxel treatment with depleted for HMGB1 expression. Knockdown of HMGB1 or limiting HMGB1 cytosolic translocation in SPC-A1 and H1299 cells enhanced the cellular response to docetaxel (Figure 4A) and inhibited cell proliferation (Figure 4C). Docetaxel induced apoptotic cell death to a great extent after HMGB1 knockdown, as shown by an increase of Annexin-V positive cells by 15.59 ± 1.01% and 12.79 ± 0.88% in SPC-A1 and H1299 cells, respectively (Figure 4E), as well as increased level of activated caspase3 and PARP cleavage (Additional file3: Figure S3A). Moreover, treatment with an apoptosis inhibitor, Z-VAD-fmk[17], reduced the activation of caspase3 (Additional file3: Figure S3A). Conversely, pretreatment with HMGB1 increased drug resistance in both parental LAD cells, as displayed by an elevated cell survival rate. Nevertheless, blockade of autophagy by 3-MA or Atg5 siRNA reversed HMGB1-induced protection against docetaxel (Figure 4G).

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