[Reversal effect of MDR1 and MDR3 gene silencing on resistance of A2780/taxol cells to paclitaxel].
OBJECTIVE
To investigate the reversal effect of MDR1 and MDR3 gene silencing on resistance of A2780/taxol cells to paclitaxel.


METHODS
shRNA plasmid vector specifically targeting MDR1 and MDR3 genes was transfected into A2780/taxol cells. The early stage cell apoptosis and the effect of intracellular rhodamine 123 (Rh123) accumulation were detected by flow cytometry (FCM). The late stage cell apoptosis rate was detected by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL). The 50% inhibition concentration (IC(50)) of paclitaxel on A2780/taxol cells was determined by methyl thiazolyl tetrazolium (MTT) assay. MDR1 and MDR3 mRNA were assessed by RT-PCR, and caspase-3 protein was detected by western blot.


RESULTS
After treatment with MDR1 and MDR3 shRNA plasmid vector, early apoptosis rate of A2780/taxol cells was (20.21 +/- 0.56)% and (10.87 +/- 1.24)%, respectively. MDR1 and MDR3 shRNA could increase cellular Rh123 accumulation (116.6 +/- 8.1 and 98.4 +/- 3.8, respectively). The late stage apoptosis rates detected by TUNEL displayed the same tendency as FCM results did. The IC(50) for paclitaxel of A2780/taxol cells was decreased significantly. The mRNA levels of MDR1 and MDR3 in A2780/taxol cells were decreased by (73.3 +/- 0.8)% and (51.6 +/- 0.4)% of control, and the reduction of MDR1 and MDR3 mRNA was in a time-dependent manner. The expression of caspase-3 protein of MDR1 and MDR3 shRNA vector transfected group in A2780/taxol cells was significantly increased [(80.8 +/- 2.6)% and (72.0 +/- 4.7)%, respectively].


CONCLUSIONS
MDR1 and MDR3 gene silencing could recover sensitivity of A2780/taxol cells to paclitaxel and induce cell apoptosis, thus reversing cell resistance to paclitaxel.
Xiao
Lan
L
Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical School, Huazhong University of Science and Technology, Wuhan 430022, China.
Gao
Rui
R
Lu
Shi
S
Lu
Mei-Song
MS
Liang
Ming-Lin
ML
Ren
Li-Rong
LR
Wang
Ze-Hua
ZH
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics*; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm / Q000187:drug effects* / Q000235:genetics; D005260:Female; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D020287:In Situ Nick-End Labeling; D010051:Ovarian Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D017239:Paclitaxel / Q000494:pharmacology*; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D018435:ATP Binding Cassette Transporter, Subfamily B; D000970:Antineoplastic Agents; D012333:RNA, Messenger; C095122:multidrug resistance protein 3; D053148:Caspase 3; D017239:Paclitaxel