[Pro-apoptotic efficiencies of three reconstructed human caspase-8 on cervical cancer cell line HeLa].
OBJECTIVE
Apoptosis is a kind of evolutional high conservative cell death. Transferring high active pro-apoptotic molecules into cancer cells to induce apoptosis is a potential strategy for cancer gene therapy. Based on our previous generation of reconstructed human caspase-8, which can continuously induce apoptosis of cervical cancer cell line HeLa, by reversing its large and small subunits, this study was designed to investigate the pro-apoptotic efficiencies of 3 reconstructed human caspase-8 (Casp8CD, Rev8, and Rev8L) on HeLa cells, and to explore the feasibility of reconstructed human caspase-8 as potential apoptosis-inducing candidates.


METHODS
The eukaryotic expression vectors pIRES2-EGFP carrying Casp8CD, Rev8, and Rev8L genes were transfected into HeLa cells, and breast cancer MCF-7 cells. Expressions and pro-apoptotic effects of Casp8CD, Rev8, and Rev8L genes were observed under fluorescent microscope, and their pro-apoptotic efficiencies were assessed by MTT assay and cells counting. The flexibilities of linking-peptides between subunits of Rev8 and Rev8L were analyzed by bioinformatics.


RESULTS
Expressions of the 3 reconstructed caspase-8 genes were observed under fluorescent microscope, and the HeLa and MCF-7 cells expressing Rev8 or Rev8L genes displayed typical apoptotic volume decrease (AVD). MTT assay showed that compared with control cells, A(570) values of Rev8- and Rev8L-transfected cells began to decrease 20 h after transfection. Cell counting results indicated that cell death ratio of Casp8CD-, Rev8-, and Rev8L-transfected cells were 16.9%, 52.3%, and 47.7%, respectively, 24 h after transfection; and 12.9%, 51.6%,and 61.2%,respectively,48 h after transfection. Bioinformatics analysis showed that the linking-peptides between subunits of Rev8 and Rev8L were flexible.


CONCLUSIONS
Rev8 and Rev8L molecules have similar pro-apoptotic effects and efficiencies, but over-expressed Casp8CD had no significant pro-apoptotic effects.
Gui
Jun-Hao
JH
Clinical Medical Research Institute, Urumqi General Hospital, Lanzhou Command, Urumqi, Xinjiang, 830000, P. R. China.
Xu
Yan-Ming
YM
Yu
Cui-Juan
CJ
Zhao
Jing
J
Jia
Lin-Tao
LT
Wang
Cheng-Ji
CJ
Yang
An-Gang
AG
Qiu
Dong-Hui
DH
Yu
Wu-Zhong
WZ
He
Jiang
J
D017209:Apoptosis; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D053181:Caspase 8; D020169:Caspases / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D045744:Cell Line, Tumor; D005260:Female; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D011994:Recombinant Proteins; C505530:CASP8 protein, human; D053181:Caspase 8; D020169:Caspases