Dual‑sensitive HRE/Egr1 promoter regulates Smac overexpression and enhances radiation‑induced A549 human lung adenocarcinoma cell death under hypoxia.
The aim of this study was to construct an expression vector carrying the hypoxia/radiation dual‑sensitive chimeric hypoxia response element (HRE)/early growth response 1 (Egr‑1) promoter in order to overexpress the therapeutic second mitochondria‑derived activator of caspases (Smac). Using this expression vector, the present study aimed to explore the molecular mechanism underlying radiotherapy‑induced A549 human lung adenocarcinoma cell death and apoptosis under hypoxia. The plasmids, pcDNA3.1‑Egr1‑Smac (pE‑Smac) and pcDNA3.1‑HRE/Egr-1‑Smac (pH/E‑Smac), were constructed and transfected into A549 human lung adenocarcinoma cells using the liposome method. CoCl2 was used to chemically simulate hypoxia, followed by the administration of 2 Gy X‑ray irradiation. An MTT assay was performed to detect cell proliferation and an Annexin V‑fluorescein isothiocyanate apoptosis detection kit was used to detect apoptosis. Quantitative polymerase chain reaction and western blot analyses were used for the detection of mRNA and protein expression, respectively. Infection with the pE‑Smac and pH/E‑Smac plasmids in combination with radiation and/or hypoxia was observed to enhance the expression of Smac. Furthermore, Smac overexpression was found to enhance the radiation‑induced inhibition of cell proliferation and promotion of cycle arrest and apoptosis. The cytochrome c/caspase‑9/caspase‑3 pathway was identified to be involved in this regulation of apoptosis. Plasmid infection in combination with X‑ray irradiation was found to markedly induce cell death under hypoxia. In conclusion, the hypoxia/radiation dual‑sensitive chimeric HRE/Egr‑1 promoter was observed to enhance the expression of the therapeutic Smac, as well as enhance the radiation‑induced inhibition of cell proliferation and promotion of cycle arrest and apoptosis under hypoxia. This apoptosis was found to involve the mitochondrial pathway.
Endoscopy Center, China‑Japan Union Hospital of Jilin University, Changchun, Jilin 130033, P.R. China.
Chang-Feng
CF
Li
Endoscopy Center, China‑Japan Union Hospital of Jilin University, Changchun, Jilin 130033, P.R. China.
Li-Bo
LB
Chen
Endoscopy Center, China‑Japan Union Hospital of Jilin University, Changchun, Jilin 130033, P.R. China.
Dan-Dan
DD
Li
Endoscopy Center, China‑Japan Union Hospital of Jilin University, Changchun, Jilin 130033, P.R. China.
Lei
L
Yang
Endoscopy Center, China‑Japan Union Hospital of Jilin University, Changchun, Jilin 130033, P.R. China.
Bao-Gang
BG
Zhang
Endoscopy Center, China‑Japan Union Hospital of Jilin University, Changchun, Jilin 130033, P.R. China.
Jing-Peng
JP
Jin
Endoscopy Center, China‑Japan Union Hospital of Jilin University, Changchun, Jilin 130033, P.R. China.
Ying
Y
Zhang
Endoscopy Center, China‑Japan Union Hospital of Jilin University, Changchun, Jilin 130033, P.R. China.
Bin
B
Zhang
D000230:Adenocarcinoma / Q000473:pathology; D000077192:Adenocarcinoma of Lung; D017209:Apoptosis / Q000528:radiation effects*; D051017:Apoptosis Regulatory Proteins; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D053453:Caspase 9 / Q000235:genetics / Q000378:metabolism; D059447:Cell Cycle Checkpoints / Q000528:radiation effects; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000528:radiation effects; D045304:Cytochromes c / Q000235:genetics / Q000378:metabolism; D051766:Early Growth Response Protein 1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008175:Lung Neoplasms / Q000473:pathology; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D011839:Radiation, Ionizing; D020218:Response Elements / Q000235:genetics*; D017422:Sequence Analysis, DNA
D051017:Apoptosis Regulatory Proteins; C412403:DIABLO protein, human; D051766:Early Growth Response Protein 1; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D012333:RNA, Messenger; D045304:Cytochromes c; D053148:Caspase 3; D053453:Caspase 9