OBJECTIVE: To examine expression of PTEN gene in ovarian cancer cisplatin-sensitive cell line OV2008 cells and cisplatin-resistant cell line C13K cells, and evaluate the effect of wild-type PTEN gene on reversing cisplatin-resistance of C13K cells and underlying mechanisms. METHODS: The expression of PTEN mRNA and protein in OV2008 and C13K cells were detected by semi-quantitative RT-PCR and western blot. Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine 2000. The expression of PTEN mRNA was monitored by RT-PCR and the expression of PTEN, protein kinase B (AKT), phospho-AKT (p-AKT) protein were analyzed by western blot in PTEN transfected and untransfected C13K cells. Proliferation and chemosensitivity of cells to cisplatin were measured by methyl thiazolyl tetrazolium (MTT), and cell apoptosis was detected by flow cytometry after treatment with cisplatin. RESULTS: (1) The expression of PTEN mRNA and protein (1.02 +/- 0.05, 1.02 +/- 0.07) in OV2008 cells were significantly higher than those in C13K cells, which were 0.45 +/- 0.03 and 0.55 +/- 0.03 respectively (P < 0.05). (2) After transfected with PTEN gene for 48 hours, the expression of PTEN mRNA and protein in C13K cells were 2.04 +/- 0.10, 0.94 +/- 0.04 respectively. Compared with C13K cells transfected with empty vector (1.04 +/- 0.04, 0.36 +/- 0.03) and untransfected C13K cells (1.03 +/- 0.05, 0.37 +/- 0.03), the difference was significant respectively (P < 0.01). The expression of p-AKT protein (0.94 +/- 0.07) was lower than those in control groups (1.66 +/- 0.10, 1.68 +/- 0.14; P < 0.05). (3) The 50% inhibition concentration (IC(50)) to cisplatin of C13K cells transfected with PTEN [(7.2 +/- 0.3) micromol/L] was obviously lower than those of empty-vector transfected cells and untransfected cells [(12.7 +/- 0.4), (13.0 +/- 0.3) micromol/L; P < 0.05]. (4) The apoptosis ratio of C13K cells with wild-type PTEN transfection, empty vector transfection and untransfected were (41.7 +/- 0.9)%, (18.6 +/- 0.7)% and (15.3 +/- 0.8)% respectively (P < 0.01). CONCLUSIONS: 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 multidrug-resistant human ovarian cancer cell line C13K by decreasing the expression of p-AKT.
OBJECTIVE: To examine expression of PTEN gene in ovarian cancercisplatin-sensitive cell line OV2008 cells and cisplatin-resistant cell line C13K cells, and evaluate the effect of wild-type PTEN gene on reversing cisplatin-resistance of C13K cells and underlying mechanisms. METHODS: The expression of PTEN mRNA and protein in OV2008 and C13K cells were detected by semi-quantitative RT-PCR and western blot. Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine 2000. The expression of PTEN mRNA was monitored by RT-PCR and the expression of PTEN, protein kinase B (AKT), phospho-AKT (p-AKT) protein were analyzed by western blot in PTEN transfected and untransfected C13K cells. Proliferation and chemosensitivity of cells to cisplatin were measured by methyl thiazolyl tetrazolium (MTT), and cell apoptosis was detected by flow cytometry after treatment with cisplatin. RESULTS: (1) The expression of PTEN mRNA and protein (1.02 +/- 0.05, 1.02 +/- 0.07) in OV2008 cells were significantly higher than those in C13K cells, which were 0.45 +/- 0.03 and 0.55 +/- 0.03 respectively (P < 0.05). (2) After transfected with PTEN gene for 48 hours, the expression of PTEN mRNA and protein in C13K cells were 2.04 +/- 0.10, 0.94 +/- 0.04 respectively. Compared with C13K cells transfected with empty vector (1.04 +/- 0.04, 0.36 +/- 0.03) and untransfected C13K cells (1.03 +/- 0.05, 0.37 +/- 0.03), the difference was significant respectively (P < 0.01). The expression of p-AKT protein (0.94 +/- 0.07) was lower than those in control groups (1.66 +/- 0.10, 1.68 +/- 0.14; P < 0.05). (3) The 50% inhibition concentration (IC(50)) to cisplatin of C13K cells transfected with PTEN [(7.2 +/- 0.3) micromol/L] was obviously lower than those of empty-vector transfected cells and untransfected cells [(12.7 +/- 0.4), (13.0 +/- 0.3) micromol/L; P < 0.05]. (4) The apoptosis ratio of C13K cells with wild-type PTEN transfection, empty vector transfection and untransfected were (41.7 +/- 0.9)%, (18.6 +/- 0.7)% and (15.3 +/- 0.8)% respectively (P < 0.01). CONCLUSIONS: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 multidrug-resistant humanovarian cancer cell line C13K by decreasing the expression of p-AKT.
Authors: Tan Min Chin; Margaret P Quinlan; Anurag Singh; Lecia V Sequist; Thomas J Lynch; Daniel A Haber; Sreenath V Sharma; Jeffrey Settleman Journal: Clin Cancer Res Date: 2008-11-01 Impact factor: 12.531