Effects of Smac gene over-expression on the radiotherapeutic sensitivities of cervical cancer cell line HeLa.
BACKGROUND
The second mitochondria-derived activator of caspases (Smac) is a novel proapoptotic gene, which plays an important role in the apoptosis-inducing effects of irradiation on tumor cells. The purpose of this study was to investigate the effects of extrinsic Smac gene transfer and its over-expression in radiotherapeutic sensitivities of cervical cancer cells.


METHODS
After the Smac gene was transferred into the cervical cancer cell line HeLa, subcloned cells were obtained by persistent G418 selection. Cellular Smac gene expression was detected by RT-PCR and Western blot, while in vitro cell viabilities were detected by trypan blue staining assay. After treatment with X-ray irradiation, cellular radiotherapeutic sensitivities were investigated by tetrazolium bromide colorimetry. Cellular apoptosis and its rate were determined by electronic microscopy, annexin V-FITC and propidium iodide staining flow cytometry. The expression and activities of cellular caspase-3 were assayed by Western blot and colorimetry.


RESULTS
Smac mRNA and protein levels in HeLa/Smac cells and the selected subclone cell line of cervical cancer were significantly higher than those of HeLa (P < 0.01). There was no significant difference in cellular viabilities between them (P > 0.05). However, after irradiation with 8 Gy X-ray, growth activities of HeLa/Smac were reduced by 22.42% (P < 0.01). When compared with those of HeLa, partial HeLa/Smac cells presented characteristic morphological changes of apoptosis under electronic microscope, with higher apoptosis rates (16.4% vs. 6.2%, P < 0.01); the caspase-3 expression levels in HeLa/Smac cells were improved significantly (P < 0.01), while its activities were increased by 3.42 times (P < 0.01).


CONCLUSIONS
Stable transfer of the extrinsic Smac gene and its over-expression in cervical cancer cell line could significantly enhance the expression and activities of cellular caspase-3 and ameliorate apoptosis-inducing effects of irradiation on cancer cells, which was a novel strategy to improve radiotherapeutic effects on cervical cancer.
Zheng
Li-Duan
LD
Department of Pathology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. ld_zheng@hotmail.com
Xiong
Zhou-Fang
ZF
Zhu
Jian-Wen
JW
Wang
Ze-Hua
ZH
D017209:Apoptosis / Q000528:radiation effects; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D005260:Female; D022761:Gene Transfer, Horizontal; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins / Q000235:genetics / Q000502:physiology*; D011836:Radiation Tolerance; D002583:Uterine Cervical Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000473:pathology
D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases