[Construction of reversed caspase-3 and it's effect on apoptosis induction in human lung adenocarcinoma cell line A549].
OBJECTIVE
To investigate the effect of reversed caspase-3 expression vector on apoptosis of human lung adenocarcinoma cell line A549.


METHODS
Reversed pcDNA 3.1-rev-caspase-3 plasmid and wild type pcDNA3.1-caspase-3 plasmid were constructed by genetic recombination. Three experiments (groups) with A549 cells were carried out: a group of transfection with pcDNA 3.1-rev-caspase plasmid, a group of transfection with pcDNA 3.1-caspase plasmid, and a control group of transfection with pcDNA 3.1 plasmid. Intervention with DEVD-fmk, an inhibitor of caspase-3, was carried out in the first two groups. The activity of caspase-3, the apoptosis and the growth of A549 cells were measured.


RESULTS
(1) The activity of caspase-3 in the pcDNA 3.1-rev-caspase-3 plasmid group and pcDNA 3.1-caspase-3 plasmid group was (11.87 +/- 0.92)% and (5.34 +/- 0.38)%, respectively, significantly higher in the former group (t = 16.02, P < 0.01). After intervention with DEVD-fmk, the activity of caspase-3 in pcDNA 3.1-rev-caspase-3 plasmid group and in pcDNA 3.1-caspase-3 plasmid group was (7.04 +/- 0.48)% and (4.51 +/- 0.20)%, respectively, also significantly higher in the former group (t = 11.86, P < 0.01). (2) Flow cytometry showed that the apoptosis rate of A549 cells in pcDNA 3.1-rev-caspase-3 plasmid group, pcDNA 3.1-caspase-3 plasmid group and pcDNA 3.1 plasmid group was (20.1 +/- 3.5)%, (7.8 +/- 2.8)% and (1.4 +/- 0.3)%, respectively. The difference of the apoptosis rate was significant (F = 44.01, P < 0.01). (3) The survival rates of A549 cells in the pcDNA 3.1-rev-caspase-3 plasmid group, pcDNA 3.1-caspase-3 plasmid group and pcDNA 3.1 plasmid group were (35.7 +/- 1.1)%, (72.8 +/- 2.9)% and (85.4 +/- 4.8)%, respectively. The survival rate of A549 cells in pcDNA 3.1-rev-caspase-3 plasmid group was lower than those in the other two groups (F = 375.07, P < 0.01).


CONCLUSIONS
pcDNA 3.1-rev-caspase-3 in A549 cells has a strong activity and can resist caspase-3 inhibitor in A549 cells. It can also induce A549 cell apoptosis and inhibit the growth of A549 cells.
Zhou
Rui
R
Department of Respiratory Medicine, Second Xiangya Hospital of Central South University, Changsha 410011, China.
Long
Wei
W
Chen
Ping
P
D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology*; D017209:Apoptosis; D053148:Caspase 3 / Q000096:biosynthesis*; D045744:Cell Line, Tumor; D005822:Genetic Vectors; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology*; D010957:Plasmids; D014162:Transfection
D053148:Caspase 3