[Effect of survivin antisense oligodeoxynucleotide on proliferation and apoptosis of human malignant melanoma cells].
OBJECTIVE
To investigate the effect of survivin antisense oligodeoxynucleotide (ASODN) on proliferation and apoptosis of human malignant melanoma cells.


METHODS
hMMC A375 colonies in log growth phase were collected and divided into control group (C, without transfection), sense chain group [SC, transfected with 600 nmol/L survivin sense oligodeoxynucleotide (ODN)], mismatch chain group (MC, transfected with 600 nmol/L survivin mismatch sense ODN), liposome group (L, treated with liposome), antisense chain group (AC, transfected with survivin ASODN, and subdivided into AC 200, 400, 600 nmol/L subgroups) according to the random number table. Transfection result was observed under inverted fluorescence microscope. Inhibition rate of cell proliferation was calculated after determination of cell viability with MTT method. Cell cycle and apoptosis rate were detected with bi-variable flow cytometry. Expression of survivin protein was determined with Western blot. Activity of caspase-3 was assessed with kinase method. Data were processed with analysis of variance.


RESULTS
(1) Cell transfection rates in SC, MC, AC 600 nmol/L groups were all above 80%. (2) Compared with those in SC group [(5.23 +/- 0.25)%], MC group [(5.09 +/- 0.13)%] and L group [(4.70 +/- 0.45)%], inhibition rates of cell proliferation in AC 200, 400, 600 nmol/L groups 24 hours after transfection [(10.30 +/- 0.56)%, (16.69 +/- 0.58)%, (24.67 +/- 0.67)%] were significantly increased (F = 746.91, and P values all below 0.05). As time after transfection went on, proliferation inhibition rate was increased obviously. (3) Apoptosis rate in AC 200, 400, 600 nmol/L groups 24 hours after transfection was respectively (13.5 +/- 1.9)%, (20.1 +/- 1.5)%, (32.1 +/- 2.9)%, which were significantly higher than those in C, SC, MC, and L groups [(6.5 +/- 0.6)%, (5.6 +/- 0.7)%, (6.4 +/- 1.0)%, (6.5 +/- 1.3)%, F = 139.9, P values all below 0.05]. Cells in AC group were blocked in G2/M stage. (4) Compared with those in C group, expression amount of survivin protein decreased, and caspase-3 activity obviously increased (F = 63.1, P values all below 0.05) in AC group. No significant difference in caspase-3 activity between SC, MC, L groups and C group was observed (F = 0.512, P values all above 0.05).


CONCLUSIONS
Survivin ASODN can inhibit the proliferation of hMMC A375 in a concentration-time dependent manner, and it induces G2/M stage block and promotes its apoptosis.
Cao
Yong-qian
YQ
Department of Burns and Plastic Surgery, Provincial Hospital Affiliated to Shandong University, Ji'nan 250021, China.
Wang
Fa-gang
FG
Huo
Ran
R
Cai
Jing-long
JL
Feng
Yong-qiang
YQ
Li
Qiang
Q
Wang
Yi-bing
YB
D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D008545:Melanoma / Q000378:metabolism / Q000473:pathology*; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000494:pharmacology*; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology*; D000077022:Survivin; D014162:Transfection
C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D000077022:Survivin; D053148:Caspase 3