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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 promoted HMGB1 expression and cytosolic translocation. (A) SPC-A1 cells were treated with docetaxel (10 μg/l) for the indicated periods. Total cell lysates, nuclear extracts, cytoplasmic fractions and extracellular medium were prepared and HMGB1 levels were analyzed by western blot. (B) SPC-A1 cells were pretreated with or without ethyl pyruvate (EP, 10 mM, 1 h) before addition of docetaxel (10 μg/l) for 48 h. Whole cell lysates, nuclear extracts and cytoplasmic fractions were analyzed by western blot for HMGB1. (C) SPC-A1 cells transfected with pcDNA3.1-HMGB1 or control vector were treated with EP (10 mM, 1 h). Total cell lysates, nuclear extracts, cytoplasmic fractions were analyzed by western blot for HMGB1. (D) Total cell lysates, nuclear extracts, and cytoplasmic fractions from parental and docetaxel-resistance LAD cells were analyzed by western blot for HMGB1. (E) Western blot analysis of HMGB1 in the presence or absence of EP (10 mM, 1 h) in SPC-A1/DTX and H1299/DTX cells. GAPDH was used as a loading control for whole cell lysates, extracellular medium and cytoplasmic extracts, and H2A was used as a loading control for nuclear extracts. The experiments were performed in triplicate. H1299 cells were subjected to the analyses as above and the results are shown in Additional file 2: Figure S2.
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Figure 3: Docetaxel promoted HMGB1 expression and cytosolic translocation. (A) SPC-A1 cells were treated with docetaxel (10 μg/l) for the indicated periods. Total cell lysates, nuclear extracts, cytoplasmic fractions and extracellular medium were prepared and HMGB1 levels were analyzed by western blot. (B) SPC-A1 cells were pretreated with or without ethyl pyruvate (EP, 10 mM, 1 h) before addition of docetaxel (10 μg/l) for 48 h. Whole cell lysates, nuclear extracts and cytoplasmic fractions were analyzed by western blot for HMGB1. (C) SPC-A1 cells transfected with pcDNA3.1-HMGB1 or control vector were treated with EP (10 mM, 1 h). Total cell lysates, nuclear extracts, cytoplasmic fractions were analyzed by western blot for HMGB1. (D) Total cell lysates, nuclear extracts, and cytoplasmic fractions from parental and docetaxel-resistance LAD cells were analyzed by western blot for HMGB1. (E) Western blot analysis of HMGB1 in the presence or absence of EP (10 mM, 1 h) in SPC-A1/DTX and H1299/DTX cells. GAPDH was used as a loading control for whole cell lysates, extracellular medium and cytoplasmic extracts, and H2A was used as a loading control for nuclear extracts. The experiments were performed in triplicate. H1299 cells were subjected to the analyses as above and the results are shown in Additional file 2: Figure S2.

Mentions: To explore the potential role of HMGB1 in the regulation of docetaxel-induced autophagy, we analyzed HMGB1 protein expression and location in LAD cells exposed to docetaxel (10 μg/l) for indicated periods of time. Western blot analysis of nuclear and cytosolic fractions of SPC-A1 and H1299 cells indicated that in the absence of treatment, HMGB1 was mainly located in the nucleus, with very low levels in the cytoplasm. Docetaxel markedly enhanced total levels of HMGB1 in both parental cells in a time-dependent manner. Moreover, following exposure to docetaxel, cytosolic levels were elevated at 6 h and kept rising until 48 h, while the nuclear levels were reduced at the same time. Accumulation of LC3-II showed an obvious elevation after treatment with docetaxel for 6 h, and continued to increase until 48 h (Figure 3A and Additional file2: Figure S2A). Notably, ethyl pyruvate (EP), a pharmacological inhibitor of HMGB1 cytoplasmic translocation[7], attenuated docetaxel-induced autophagy, as shown in Figure 3B and Additional file2: Figure S2B. Furthermore, diminished cytosolic HMGB1 also limited the activation of autophagy even in cells transfected with cDNA encoding full-length human HMGB1 (Figure 3C and Additional file2: Figure S2C).The level of cytosolic HMGB1 exhibited a positive correlation with docetaxel-induced autophagy. Meanwhile, SPC-A1/DTX and H1299/DTX cells also showed relatively higher total level of HMGB1 which included a higher cytosolic level and a lower nuclear level under basal conditions compared to parental cells (Figure 3D). However, application of EP decreased LC3-II accumulation (Figure 3E).


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 promoted HMGB1 expression and cytosolic translocation. (A) SPC-A1 cells were treated with docetaxel (10 μg/l) for the indicated periods. Total cell lysates, nuclear extracts, cytoplasmic fractions and extracellular medium were prepared and HMGB1 levels were analyzed by western blot. (B) SPC-A1 cells were pretreated with or without ethyl pyruvate (EP, 10 mM, 1 h) before addition of docetaxel (10 μg/l) for 48 h. Whole cell lysates, nuclear extracts and cytoplasmic fractions were analyzed by western blot for HMGB1. (C) SPC-A1 cells transfected with pcDNA3.1-HMGB1 or control vector were treated with EP (10 mM, 1 h). Total cell lysates, nuclear extracts, cytoplasmic fractions were analyzed by western blot for HMGB1. (D) Total cell lysates, nuclear extracts, and cytoplasmic fractions from parental and docetaxel-resistance LAD cells were analyzed by western blot for HMGB1. (E) Western blot analysis of HMGB1 in the presence or absence of EP (10 mM, 1 h) in SPC-A1/DTX and H1299/DTX cells. GAPDH was used as a loading control for whole cell lysates, extracellular medium and cytoplasmic extracts, and H2A was used as a loading control for nuclear extracts. The experiments were performed in triplicate. H1299 cells were subjected to the analyses as above and the results are shown in Additional file 2: Figure S2.
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Related In: Results  -  Collection

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Figure 3: Docetaxel promoted HMGB1 expression and cytosolic translocation. (A) SPC-A1 cells were treated with docetaxel (10 μg/l) for the indicated periods. Total cell lysates, nuclear extracts, cytoplasmic fractions and extracellular medium were prepared and HMGB1 levels were analyzed by western blot. (B) SPC-A1 cells were pretreated with or without ethyl pyruvate (EP, 10 mM, 1 h) before addition of docetaxel (10 μg/l) for 48 h. Whole cell lysates, nuclear extracts and cytoplasmic fractions were analyzed by western blot for HMGB1. (C) SPC-A1 cells transfected with pcDNA3.1-HMGB1 or control vector were treated with EP (10 mM, 1 h). Total cell lysates, nuclear extracts, cytoplasmic fractions were analyzed by western blot for HMGB1. (D) Total cell lysates, nuclear extracts, and cytoplasmic fractions from parental and docetaxel-resistance LAD cells were analyzed by western blot for HMGB1. (E) Western blot analysis of HMGB1 in the presence or absence of EP (10 mM, 1 h) in SPC-A1/DTX and H1299/DTX cells. GAPDH was used as a loading control for whole cell lysates, extracellular medium and cytoplasmic extracts, and H2A was used as a loading control for nuclear extracts. The experiments were performed in triplicate. H1299 cells were subjected to the analyses as above and the results are shown in Additional file 2: Figure S2.
Mentions: To explore the potential role of HMGB1 in the regulation of docetaxel-induced autophagy, we analyzed HMGB1 protein expression and location in LAD cells exposed to docetaxel (10 μg/l) for indicated periods of time. Western blot analysis of nuclear and cytosolic fractions of SPC-A1 and H1299 cells indicated that in the absence of treatment, HMGB1 was mainly located in the nucleus, with very low levels in the cytoplasm. Docetaxel markedly enhanced total levels of HMGB1 in both parental cells in a time-dependent manner. Moreover, following exposure to docetaxel, cytosolic levels were elevated at 6 h and kept rising until 48 h, while the nuclear levels were reduced at the same time. Accumulation of LC3-II showed an obvious elevation after treatment with docetaxel for 6 h, and continued to increase until 48 h (Figure 3A and Additional file2: Figure S2A). Notably, ethyl pyruvate (EP), a pharmacological inhibitor of HMGB1 cytoplasmic translocation[7], attenuated docetaxel-induced autophagy, as shown in Figure 3B and Additional file2: Figure S2B. Furthermore, diminished cytosolic HMGB1 also limited the activation of autophagy even in cells transfected with cDNA encoding full-length human HMGB1 (Figure 3C and Additional file2: Figure S2C).The level of cytosolic HMGB1 exhibited a positive correlation with docetaxel-induced autophagy. Meanwhile, SPC-A1/DTX and H1299/DTX cells also showed relatively higher total level of HMGB1 which included a higher cytosolic level and a lower nuclear level under basal conditions compared to parental cells (Figure 3D). However, application of EP decreased LC3-II accumulation (Figure 3E).

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