[Effect of down-regulation of survivin gene on apoptosis and cisplatin resistance in cisplatin resistant human lung adenocarcinoma A549/CDDP cells].
OBJECTIVE
To investigate the effects of survivin antisense oligodeoxynucleoties (ASODN) transfection mediated by cytofectin on apoptosis and cisplatin resistance in cisplatin resistant human lung adenocarcinoma A549/CDDP cells in vitro.


METHODS
A549/CDDP cells were cultured routinely in RPMI-1640 medium. Survivin ASODN mediated by cytofectin was transfected into the A549/CDDP cells. Reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry SABC assays were performed to determine the regulation of survivin expression by ASODN. The influence of ASODN transfection on apoptosis was determined by fluoroscence microscopy and Hoechst staining, agarose gel electrophoresis, flow cytometry and caspase-3 colorimetric assay. MTT assay was performed to detect the cell viability, half-maximum inhibitory concentration (IC50) and cisplatin resistance index (RI) were thereby calculated.


RESULTS
Transfected by survivin ASODN for 48 h, down-regulation of survivin expression was measured, of which mRNA and protein expression was significantly down-regulated to 41.56% and 0.864 +/- 0.045, respectively (P < 0.05). Transfection with survivin ASODN caused typical apoptotic changes, including characteristic chromatin condensation, nuclear shrinkage, nuclear cleavage and the cells grew more regularly, and some cells were floating. Typical DNA ladder pattern was observed by agarose gel electrophoresis. Furthermore, apoptotic index and caspase-3 activity was enhanced to 34.03% and 1.1298 +/- 0.2502, respectively (P < 0.05). It was significantly different as compared with the control group. While combination with ASODN and 10 micromol/L cisplatin caused far more distinctive apoptotic alterations, of which AI and caspase-3 activity reached to 65.85% and 1.6805 +/- 0.2758, respectively (P < 0.05), and even compared with the single ASODN group, the difference was still significant (P < 0.05). Transfected with survivin ASODN only or with combination of cisplatin for 48 h, the inhibitory rate of cell growth was enhanced to 59.3% and 83.7% (P < 0.05), respectively, while inversely, the cell viability reduced to a lowest value. The half-maximum inhibitory concentration of cisplatin was reduced from 225.03 +/- 10.59 micromol/L to 158.84 +/- 4.26 micromol/L, and the resistant index was conversely reduced from 11.9 to 8.39. Non-sense oligodeoxynucleotides (NSODN) and liposome had no effect on the cells growth (P > 0.05).


CONCLUSIONS
Transfection with survivin ASODN can to a great extent reverse the cisplatin resistance in human cisplatin resistant lung adenocarcinoma cells A549/CDDP in vitro, and can thereby significantly inhibit the growth of the cells. The mechanism of reversal of resistance to cisplatin by this transfection can be associated with specific down-regulation of survivin expression, which decreases the threshold of apoptosis, induces more pronounced apoptosis,and reverses the resistance to apoptosis induced by cisplatin in A549/CDDP cells in vitro.
Zhang
Mei-Chun
MC
Department of Respiratory Diseases, Xiangya Hospital, Central South University, Changsha 410008, China.
Hu
Cheng-Ping
CP
Chen
Qiong
Q
D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology*; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008869:Microtubule-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D000077022:Survivin; D014162:Transfection
D000970:Antineoplastic Agents; C107489:BIRC5 protein, human; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D000077022:Survivin; D002945:Cisplatin