Reversal of multidrug resistance and inhibition of phosphorylation of AKT in human ovarian cancer cell line by wild-type PTEN gene.
The reversing effect of wild-type PTEN gene on resistance of C13K cells to cisplatin and its inhibitory effect on the phosphorylation of protein kinase B (AKT) were studied. The expression of PTEN mRNA and protein in OV2008 cells and C13K cells were semi-quantitatively detected by using RT-PCR and Western blotting. Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine2000. The expression of PTEN mRNA was monitored by RT-PCR and the expression of PTEN, Akt, p-Akt protein were analyzed by Western blotting in PTEN-transfected and non-transfected C13K cells. Proliferation and chemosensitivity of cells to DDP were measured by MTT, and cell apoptosis was detected by flow cytometry after treatment with cisplatin. The expression of PTEN mRNA and protein in OV2008 cells were significantly higher than those in C13K cells. After transfection with PTEN gene for 48 h, the expression of PTEN mRNA and protein in C13K cells were 2.04 +/- 0.10, 0.94 +/- 0.04 respectively and the expression of p-Akt protein (0.94 +/- 0.07) was lower than those in control groups (1.68 +/- 0.14, 1.66 +/- 0.10) (P < 0.05). The IC(50) of DDP to C13K cells transfected with PTEN (7.2 +/- 0.3 micromol/L) was obviously lower than those of empty-vector transfected cells and non-transfected cells (12.7 +/- 0.4 micromol/l, 13.0 +/- 0.3 micromol/L) (P<0.05). The apopototis ratio of wild-type PTEN-transfected, empty vector transfected and non-transfected C13K cells were (41.65 +/- 0.87)%, (18.61 +/- 0.70)% and (15.28 +/- 0.80)% respectively, and the difference was statistically significant (P<0.05). PTEN gene plays an important role in ovarian cancer multidrug resistance. Transfection of PTEN could increase the expression of PTEN and restore drug sensitivity to cisplatin in human ovarian cancer cell line C13K with multidrug-resistance by decreasing the expression of p-Akt.
Wu
Huijuan
H
Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China. huijuanwu_2000_2000@126.com
Weng
Danhui
D
Xing
Hui
H
D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology*; D018432:Drug Resistance, Multiple / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005260:Female; D006801:Humans; D010051:Ovarian Neoplasms / Q000378:metabolism* / Q000473:pathology; D051059:PTEN Phosphohydrolase / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D012333:RNA, Messenger; D051057:Proto-Oncogene Proteins c-akt; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human; D002945:Cisplatin