[Effect of hypoxia on the chemotherapeutic sensitivity of human ovarian cancer cells to paclitaxel and its mechanism].
OBJECTIVE
To explore the effect of hypoxia and hypoxia-inducible factor-1alpha (HIF-1alpha) on the expression of multidrug resistance gene-1 (mdr-1) and its coded p-glyeoprotein (P-gp) as well as the chemotherapeutic sensitivity of human ovarian cancer cells to paclitaxel and its mechanism.


METHODS
The mRNA and protein levels of HIF-1alpha, mdr-1 and p-gp were studied by immunocytochemistry, semiquantitative reverse transcription-ploymerase chain reaction (RT-PCR) and Western blot analysis in human ovarian cancer cells (A2780) in 5% CO2 + 1% O2 hypoxic culture and 21% O2 normoxic culture, respectively. Methyl thiazolyl tetrazolium (MIT) was used to evaluate the chemotherapeutic sensitivity of A2780 cells to paclitaxel by inhibition rate. RNA interference technique was used and small hairpin RNAs (shRNAs) eukaryotic expression vector targeting HIF-1alpha was constructed as pSiHIF-1alpha, and transfected into A2780 cells. RT-PCR and Western blot were used to detect gene silencing effect on HIF-1alpha, the expressions of mdr-1 and p-gp. The inhibition rate was observed after HIF-1alpha gene silence.


RESULTS
HIF-1alpha at both mRNA and protein levels was induced significantly under hypoxia. The HIF-1alpha expression at mRNA level was oxygen gradient-independent, while HIF-1alpha expression at protein level was oxygen gradient-dependent. The inhibition rate of paclitaxel to hypoxic A2780 cells in 5% CO2 + 1% O2 was significantly lower than that in normoxic A2780 cells (P <0.05). The shRNAs plasmid targeting HIF-1alpha was constructed successfully and HIF-1alpha gene was silenced in A2780 cells efficiently followed by mdr-1 and p-gp down-regulation. The inhibition rate was greatly increased in hypoxic A2780/siHIF-1alpha cells.


CONCLUSIONS
Hypoxia can decrease the chemotherapeutic sensitivity of human ovarian cancer A2780 cells to paclitaxel through HIF-1alpha regulating the expression of mdr-1 and p-gp.
Huang
Lei
L
Department of Obstetrics & Gynecology, Central Hospital of Wuhan, Wuhan 430014, China.
Zhang
Qing-Hua
QH
Ao
Qi-Lin
QL
Xing
Hui
H
Lu
Yun-Ping
YP
Ma
Ding
D
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis* / Q000235:genetics; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D015153:Blotting, Western; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics; D005260:Female; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis* / Q000235:genetics; D010051:Ovarian Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D017239:Paclitaxel / Q000494:pharmacology*; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000972:Antineoplastic Agents, Phytogenic; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017239:Paclitaxel