Hypoxia-induced autophagy contributes to radioresistance via c-Jun-mediated Beclin1 expression in lung cancer cells

Abstract

Reduced radiosensitivity of lung cancer cells represents a pivotal obstacle in clinical oncology. The hypoxia-inducible factor (HIF)-1α plays a crucial role in radiosensitivity, but the detailed mechanisms remain elusive. A relationship has been suggested to exist between hypoxia and autophagy recently. In the current study, we studied the effect of hypoxia-induced autophagy on radioresistance in lung cancer cell lines. A549 and H1299 cells were cultured under normoxia or hypoxia, followed by irradiation at dosage ranging from 0 to 8 Gy. Clonogenic assay was performed to calculate surviving fraction. EGFP-LC3 plasmid was stably transfected into cells to monitor autophagic processes. Western blotting was used to evaluate the protein expression levels of HIF-1α, c-Jun, phosphorylated c-Jun, Beclin 1, LC3 and p62. The mRNA levels of Beclin 1 were detected by qRT-PCR. We found that under hypoxia, both A549 and H1299 cells were radio-resistant compared with normoxia. Hypoxia-induced elevated HIF-1α protein expression preferentially triggered autophagy, accompanied by LC3 induction, EGFP-LC3 puncta and p62 degradation. In the meantime, HIF-1α increased downstream c-Jun phosphorylation, which in turn upregulated Beclin 1 mRNA and protein expression. The upregulation of Beclin 1 expression, instead of HIF-1α, could be blocked by SP600125 (a specific inhibitor of c-Jun NH2-terminal kinase), followed by suppression of autophagy. Under hypoxia, combined treatment of irradiation and chloroquine (a potent autophagy inhibitor) significantly decreased the survival potential of lung cancer cells in vitro and in vivo. In conclusion, hypoxia-induced autophagy through evaluating Beclin1 expression may be considered as a target to reverse the radioresistance in cancer cells.

Curcumin inhibits hypoxia-induced proliferation and invasion of MG-63 osteosarcoma cells via downregulating Notch1

“…Evidence from experimental and clinical studies have indicated a critical role for hypoxia in the malignant progression of solid tumors (27). Hypoxia has become a central issue in tumor physiology and cancer treatment due to its multiple roles in cancer cell genomic instability (29), angiogenesis (30), invasiveness (31), metastasis (32), resistance to cell death (33), metabolism reprogramming (34), chemoresistance (35) and radioresistance (36). A hypoxic microenvironment has been confirmed by histological studies of osteosarcoma specimens (37).…”

“…The cells were cultured under normoxic or hypoxic conditions. At the indicated time points (12,24,36 or 48 h), cell viability was assessed by the MTT assay according to the manufacturer's protocol, and the absorbance was measured at a wavelength of 490 nm using a multiwell microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA).…”

“…Cells were subsequently cultured in normoxic or hypoxic (3% O 2 ) conditions. At the indicated time points(12,24,36 or 48 h), cell viability in each group was assessed by an MTT assay. Data are expressed as the mean ± standard deviation (n=3).…”

Cervical cancer stem cells manifest radioresistance: Association with upregulated AP-1 activity

“…Recently, our group also showed pivotal role of c-Jun and Fra-2/c-Fos in aggressive tumorigenesis, metastasis and chemo radioresistance of tongue cancer 18 . Further studies demonstrated c-Jun phosphorylation through HIF-1α upregulate Beclin-1 mRNA and protein expression that contributed to radioresistance in lung cancer 19 . Similarly, RNAi-mediated knockdown of the c-Jun gene sensitized human nasopharyngeal carcinoma cells to radiation 20 .…”

Hypoxia ameliorates intestinal inflammation through NLRP3/mTOR downregulation and autophagy activation

“…Hypoxia activates further innate immune protective mechanisms such as autophagy. Thus, HIF-1α induces the expression of several autophagosome markers, including LC3, autophagy related gene (ATG)5 and beclin-1, as well as the conversion of LC3-I to LC3-II 14 .…”