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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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Suppression of HMGB1 resensitized docetaxel-resistant SPC-A1/DTX cells to docetaxel in vivo. (A) NOD/SCID mice were inoculated with 5.0 × 106 SPC-A1/DTX cells following transfection of control or HMGB1 shRNA and treated with docetaxel (1 mg/kg) beginning at day 6. Tumor volumes were calculated for 14 days. (n = 5; *P < 0.05). (B) Representative photographs of tumors formed at 2 weeks after subcutaneous transplantation are shown. (C) Hematoxylin and eosin (H&E)-stained, proliferating cell nuclear antigen (PCNA)-stained and TUNEL assay stained sections of the transplanted tumors are shown (original magnification, ×400). (D) HMGB1 and LC3 expression in the transplanted tumors were determined by western blot analysis.
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Figure 8: Suppression of HMGB1 resensitized docetaxel-resistant SPC-A1/DTX cells to docetaxel in vivo. (A) NOD/SCID mice were inoculated with 5.0 × 106 SPC-A1/DTX cells following transfection of control or HMGB1 shRNA and treated with docetaxel (1 mg/kg) beginning at day 6. Tumor volumes were calculated for 14 days. (n = 5; *P < 0.05). (B) Representative photographs of tumors formed at 2 weeks after subcutaneous transplantation are shown. (C) Hematoxylin and eosin (H&E)-stained, proliferating cell nuclear antigen (PCNA)-stained and TUNEL assay stained sections of the transplanted tumors are shown (original magnification, ×400). (D) HMGB1 and LC3 expression in the transplanted tumors were determined by western blot analysis.

Mentions: To test the effects of targeted downregulation of HMGB1 on chemosensitivity of LAD cells in vivo, we inoculated NOD/SCID mice with SPC-A1/DTX cells transfected with HMGB1 shRNA. Tumors derived from HMGB1 shRNA-transfected SPC-A1/DTX cells grew more slowly compared with those derived from control shRNA-transfected cells after being treated with docetaxel (Figure 8A and B). Immunohistochemistry analysis showed that the positive rate of PCNA in the HMGB1 shRNA-transfected group was greatly diminished (Figure 8C). TUNEL staining of resected tumor tissues also revealed even lower apoptosis in the HMGB1 shRNA group than the control group. We also observed a decreased level of LC3-II expression in HMGB1 shRNA-transfected tumors in response to docetaxel in comparison with control shRNA-transfected tumors (Figure 8D). These results support the critical role of HMGB1 in modulating the chemosensitivity in docetaxel-resistant LAD cells in vivo.


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)

Suppression of HMGB1 resensitized docetaxel-resistant SPC-A1/DTX cells to docetaxel in vivo. (A) NOD/SCID mice were inoculated with 5.0 × 106 SPC-A1/DTX cells following transfection of control or HMGB1 shRNA and treated with docetaxel (1 mg/kg) beginning at day 6. Tumor volumes were calculated for 14 days. (n = 5; *P < 0.05). (B) Representative photographs of tumors formed at 2 weeks after subcutaneous transplantation are shown. (C) Hematoxylin and eosin (H&E)-stained, proliferating cell nuclear antigen (PCNA)-stained and TUNEL assay stained sections of the transplanted tumors are shown (original magnification, ×400). (D) HMGB1 and LC3 expression in the transplanted tumors were determined by western blot analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4125709&req=5

Figure 8: Suppression of HMGB1 resensitized docetaxel-resistant SPC-A1/DTX cells to docetaxel in vivo. (A) NOD/SCID mice were inoculated with 5.0 × 106 SPC-A1/DTX cells following transfection of control or HMGB1 shRNA and treated with docetaxel (1 mg/kg) beginning at day 6. Tumor volumes were calculated for 14 days. (n = 5; *P < 0.05). (B) Representative photographs of tumors formed at 2 weeks after subcutaneous transplantation are shown. (C) Hematoxylin and eosin (H&E)-stained, proliferating cell nuclear antigen (PCNA)-stained and TUNEL assay stained sections of the transplanted tumors are shown (original magnification, ×400). (D) HMGB1 and LC3 expression in the transplanted tumors were determined by western blot analysis.
Mentions: To test the effects of targeted downregulation of HMGB1 on chemosensitivity of LAD cells in vivo, we inoculated NOD/SCID mice with SPC-A1/DTX cells transfected with HMGB1 shRNA. Tumors derived from HMGB1 shRNA-transfected SPC-A1/DTX cells grew more slowly compared with those derived from control shRNA-transfected cells after being treated with docetaxel (Figure 8A and B). Immunohistochemistry analysis showed that the positive rate of PCNA in the HMGB1 shRNA-transfected group was greatly diminished (Figure 8C). TUNEL staining of resected tumor tissues also revealed even lower apoptosis in the HMGB1 shRNA group than the control group. We also observed a decreased level of LC3-II expression in HMGB1 shRNA-transfected tumors in response to docetaxel in comparison with control shRNA-transfected tumors (Figure 8D). These results support the critical role of HMGB1 in modulating the chemosensitivity in docetaxel-resistant LAD cells in vivo.

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