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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 regulated docetaxel-induced autophagy in docetaxel-resistant LAD cells. (A) Whole cell lysates, nuclear exacts and cytoplasmic fractions from SPC-A1/DTX and H1299/DTX cells were treated with indicated doses of docetaxel in the presence or absence of EP(10 mM, 1 h) were subjected to western blot analysis for LC3, p62 and HMGB1. (B) Both docetaxel-resistant cells were transfected with HMGB1 shRNA before treatment with the indicated doses of docetaxel for 48 h. Total cell lysates, nuclear extracts, cytoplasmic fractions were subjected to western blot analysis for LC3, p62 and HMGB1. (C) Both docetaxel-resistant cells were co-transfected with either control or HMGB1 shRNA and GFP-LC3 plasmid followed by treatment with docetaxel (100 μg/l) for an additional 24 h. Bar = 50 μm. Values are reported as mean ± S.D. of three independent experiments. *P < 0.05, **P < 0.01.
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Figure 6: HMGB1 regulated docetaxel-induced autophagy in docetaxel-resistant LAD cells. (A) Whole cell lysates, nuclear exacts and cytoplasmic fractions from SPC-A1/DTX and H1299/DTX cells were treated with indicated doses of docetaxel in the presence or absence of EP(10 mM, 1 h) were subjected to western blot analysis for LC3, p62 and HMGB1. (B) Both docetaxel-resistant cells were transfected with HMGB1 shRNA before treatment with the indicated doses of docetaxel for 48 h. Total cell lysates, nuclear extracts, cytoplasmic fractions were subjected to western blot analysis for LC3, p62 and HMGB1. (C) Both docetaxel-resistant cells were co-transfected with either control or HMGB1 shRNA and GFP-LC3 plasmid followed by treatment with docetaxel (100 μg/l) for an additional 24 h. Bar = 50 μm. Values are reported as mean ± S.D. of three independent experiments. *P < 0.05, **P < 0.01.

Mentions: To investigate whether HMGB1 is a direct activator of autophagy, we evaluated the LC3-I to LC3-II conversion and LC3 punctate formation by fluorescent analysis as described above. Knockdown of HMGB1 by transfecting SPC-A1 and H1299 cells with HMGB1 shRNA prevented the appearance of LC3-II and the degradation of p62 after docetaxel treatment (Figure 5A). Similarly, downregulation of HMGB1 expression or inhibition of cytosolic translocation of HMGB1 with EP decreased the level of LC3-II in SPC-A1/DTX and H1299/DTX cells, and a high dose of docetaxel failed to induce LC3-II turnover to a greater degree (Figure 6A and6B). In contrast, overexpression of HMGB1 significantly increased LC3-II accumulation and autophagic p62 degradation. However, this elevated conversion of LC3-I to LC3-II could be abolished by suppression of autophagy with 3-MA (Figure 5B). Fluorescence micrographs showed a punctate pattern of GFP-LC3 in HMGB1-overexpressing SPC-A1 and H1299 cells, which could be greatly attenuated by application of 3-MA (Figure 5C). Meanwhile, compared with the vector control groups, SPC-A1/DTX and H1299/DTX cells transfected with HMGB1 shRNA exhibited reduced formation of GFP-LC3 punctate staining after docetaxel treatment (Figure 6C). These data support a vital role for HMGB1 in the regulation of autophagy in LAD cells.


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 regulated docetaxel-induced autophagy in docetaxel-resistant LAD cells. (A) Whole cell lysates, nuclear exacts and cytoplasmic fractions from SPC-A1/DTX and H1299/DTX cells were treated with indicated doses of docetaxel in the presence or absence of EP(10 mM, 1 h) were subjected to western blot analysis for LC3, p62 and HMGB1. (B) Both docetaxel-resistant cells were transfected with HMGB1 shRNA before treatment with the indicated doses of docetaxel for 48 h. Total cell lysates, nuclear extracts, cytoplasmic fractions were subjected to western blot analysis for LC3, p62 and HMGB1. (C) Both docetaxel-resistant cells were co-transfected with either control or HMGB1 shRNA and GFP-LC3 plasmid followed by treatment with docetaxel (100 μg/l) for an additional 24 h. Bar = 50 μm. Values are reported as mean ± S.D. of three independent experiments. *P < 0.05, **P < 0.01.
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Figure 6: HMGB1 regulated docetaxel-induced autophagy in docetaxel-resistant LAD cells. (A) Whole cell lysates, nuclear exacts and cytoplasmic fractions from SPC-A1/DTX and H1299/DTX cells were treated with indicated doses of docetaxel in the presence or absence of EP(10 mM, 1 h) were subjected to western blot analysis for LC3, p62 and HMGB1. (B) Both docetaxel-resistant cells were transfected with HMGB1 shRNA before treatment with the indicated doses of docetaxel for 48 h. Total cell lysates, nuclear extracts, cytoplasmic fractions were subjected to western blot analysis for LC3, p62 and HMGB1. (C) Both docetaxel-resistant cells were co-transfected with either control or HMGB1 shRNA and GFP-LC3 plasmid followed by treatment with docetaxel (100 μg/l) for an additional 24 h. Bar = 50 μm. Values are reported as mean ± S.D. of three independent experiments. *P < 0.05, **P < 0.01.
Mentions: To investigate whether HMGB1 is a direct activator of autophagy, we evaluated the LC3-I to LC3-II conversion and LC3 punctate formation by fluorescent analysis as described above. Knockdown of HMGB1 by transfecting SPC-A1 and H1299 cells with HMGB1 shRNA prevented the appearance of LC3-II and the degradation of p62 after docetaxel treatment (Figure 5A). Similarly, downregulation of HMGB1 expression or inhibition of cytosolic translocation of HMGB1 with EP decreased the level of LC3-II in SPC-A1/DTX and H1299/DTX cells, and a high dose of docetaxel failed to induce LC3-II turnover to a greater degree (Figure 6A and6B). In contrast, overexpression of HMGB1 significantly increased LC3-II accumulation and autophagic p62 degradation. However, this elevated conversion of LC3-I to LC3-II could be abolished by suppression of autophagy with 3-MA (Figure 5B). Fluorescence micrographs showed a punctate pattern of GFP-LC3 in HMGB1-overexpressing SPC-A1 and H1299 cells, which could be greatly attenuated by application of 3-MA (Figure 5C). Meanwhile, compared with the vector control groups, SPC-A1/DTX and H1299/DTX cells transfected with HMGB1 shRNA exhibited reduced formation of GFP-LC3 punctate staining after docetaxel treatment (Figure 6C). These data support a vital role for HMGB1 in the regulation of autophagy in LAD cells.

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