Recent evidence has suggested that Akt2 plays an important role in the protection of cells from paclitaxel (PTX)-induced apoptosis and control of the cell cycle. In addition, some scholars suggested that the PTX sen...Recent evidence has suggested that Akt2 plays an important role in the protection of cells from paclitaxel (PTX)-induced apoptosis and control of the cell cycle. In addition, some scholars suggested that the PTX sensitivity depends on a functional spindle assembly checkpoint. In the present study, we investigated the role of the Akt2/Bubl cross-talking in apoptosis and cell cycle after exposure of the A2780 ovarian cancer cells to paclitaxel (PTX). Recombinant expression plasmid WT-Akt2 was transfected into A2780 cells by lipofectamine2000, and then the expression level of Akt2 gene was detected by using RT-PCR and Western blotting. Cell apoptosis and cell cycle were detected by flow cytometry and Hoechst 33342 staining after treatment with PTX. Moreover, we compared the expression level of Bubl in different groups by Western blotting. Our study showed that up-regulation of Akt2 contributed to A2780 ovarian cancer cells overriding PTX-induced G2/M arrest, and inhibited Bubl expression. Our findings might shed light on the molecular mechanism of PTX-induced resistance in ovarian cancer and help develop novel anti-neoplastic strategies.展开更多
A chemosensitivity test for ovarian cancer using tritiated thymidine incorporation assay was carried out. A dose-response relationship for cisplatin and potentiation of verapamil in increasing vincristine inhibition t...A chemosensitivity test for ovarian cancer using tritiated thymidine incorporation assay was carried out. A dose-response relationship for cisplatin and potentiation of verapamil in increasing vincristine inhibition to ovarian cancer were investigated. A 5- fold increase of cisplatin density converted the tumors which were initially resistance to standard-dose cisplatin Into drug-sensitive ones. Vera-pamil was found to be able to overcome vincristine-resistance of some tumors in vitro. These results suggest that using high dose cisplatin therapy or increasing local drug concentration by using other administration way, we could expect some ovarian cancers that had failed to standard dose cisplatin therapy to be effective. Combination of vincristine with verapamll may be helpful in treating some vincristineresistant cases.展开更多
The reversing effect of wild-type PTEN gene on resistance of C 13K cells to cisplatin and its inhibitory effect on the phosphorylation of protein kinase B (AKT) were studied. The expression of PTEN mRNA and protein ...The reversing effect of wild-type PTEN gene on resistance of C 13K 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 ana- lyzed 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 C 13K 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 IC50 of DDP to C 13 K cells transfected with PTEN (7.2± 0.3 la mol/L) was obviously lower than those of empty-vector transfected cells and non-transfected cells (12.7±0.4 lamol/1, 13.0±0.3 lamol/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 C 13K with multidrug-resistance by decreasing the expression of p-Akt.展开更多
Transforming growth factor beta (TGF β) may cause cell cycle arrest, terminal differentiation, or apoptosis in most normal epithelial cells, whereas most malignant cell lines are resistant to TGF β. Mechanisms of...Transforming growth factor beta (TGF β) may cause cell cycle arrest, terminal differentiation, or apoptosis in most normal epithelial cells, whereas most malignant cell lines are resistant to TGF β. Mechanisms of resistance to TGF β caused by modulation of cell cycle regulators and/or inactivation of components of the TGF β signaling transduction pathway such as C myc and Smad4 are not well understood. To investigate the potential association between loss of sensitivity to TGF β and expression status of transforming growth factor receptor Ⅱ (TβRⅡ), Smad4, CDC25A and C myc in 14 cell lines derived from ovarian cancer, the expression levels of these genes were detected by semi quantitative RT PCR. Normal ovarian surface tissues were used as controls. The expression of TβRⅡ was detectable in all of 14 cell lines. The expression of Smad4 was decreased in 10 cell lines and 9 cell lines overexpressed CDC25A, as compared to normal controls. CDC25A gene was overexpressed with 88 % (8/9) in tumorigenic cell lines as determined by xenografts in nude mice, and only in 20 % (1/5) of non tumorigenic cell lines ( P <0.05). C myc was not overexpressed in any of these cell lines. The loss of sensitivity to TGF β of cell lines derived from ovarian cancers may be related to a decreased expression of Smad4, which mediates TGF β induced growth inhibition, and/or an overexpression of CDC25A. This overexpression of CDC25A correlates with increased tumorigenicity of ovarian cancer cell lines. The loss of sensitivity to TGF β is not associated with a lack of TβRⅡ.展开更多
基金supported by Joint Research Fund for Young Scholars Abroad (No. 30528012)National Key Basic Research Program (Program 973) Foundation of China (No. 2009CB521800)
文摘Recent evidence has suggested that Akt2 plays an important role in the protection of cells from paclitaxel (PTX)-induced apoptosis and control of the cell cycle. In addition, some scholars suggested that the PTX sensitivity depends on a functional spindle assembly checkpoint. In the present study, we investigated the role of the Akt2/Bubl cross-talking in apoptosis and cell cycle after exposure of the A2780 ovarian cancer cells to paclitaxel (PTX). Recombinant expression plasmid WT-Akt2 was transfected into A2780 cells by lipofectamine2000, and then the expression level of Akt2 gene was detected by using RT-PCR and Western blotting. Cell apoptosis and cell cycle were detected by flow cytometry and Hoechst 33342 staining after treatment with PTX. Moreover, we compared the expression level of Bubl in different groups by Western blotting. Our study showed that up-regulation of Akt2 contributed to A2780 ovarian cancer cells overriding PTX-induced G2/M arrest, and inhibited Bubl expression. Our findings might shed light on the molecular mechanism of PTX-induced resistance in ovarian cancer and help develop novel anti-neoplastic strategies.
文摘A chemosensitivity test for ovarian cancer using tritiated thymidine incorporation assay was carried out. A dose-response relationship for cisplatin and potentiation of verapamil in increasing vincristine inhibition to ovarian cancer were investigated. A 5- fold increase of cisplatin density converted the tumors which were initially resistance to standard-dose cisplatin Into drug-sensitive ones. Vera-pamil was found to be able to overcome vincristine-resistance of some tumors in vitro. These results suggest that using high dose cisplatin therapy or increasing local drug concentration by using other administration way, we could expect some ovarian cancers that had failed to standard dose cisplatin therapy to be effective. Combination of vincristine with verapamll may be helpful in treating some vincristineresistant cases.
基金a grant from the National Natural Sciences Foundation of China (No. 30571950)National Key Basic Research Program Foundation (N0.2002CB513107).
文摘The reversing effect of wild-type PTEN gene on resistance of C 13K 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 ana- lyzed 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 C 13K 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 IC50 of DDP to C 13 K cells transfected with PTEN (7.2± 0.3 la mol/L) was obviously lower than those of empty-vector transfected cells and non-transfected cells (12.7±0.4 lamol/1, 13.0±0.3 lamol/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 C 13K with multidrug-resistance by decreasing the expression of p-Akt.
文摘Transforming growth factor beta (TGF β) may cause cell cycle arrest, terminal differentiation, or apoptosis in most normal epithelial cells, whereas most malignant cell lines are resistant to TGF β. Mechanisms of resistance to TGF β caused by modulation of cell cycle regulators and/or inactivation of components of the TGF β signaling transduction pathway such as C myc and Smad4 are not well understood. To investigate the potential association between loss of sensitivity to TGF β and expression status of transforming growth factor receptor Ⅱ (TβRⅡ), Smad4, CDC25A and C myc in 14 cell lines derived from ovarian cancer, the expression levels of these genes were detected by semi quantitative RT PCR. Normal ovarian surface tissues were used as controls. The expression of TβRⅡ was detectable in all of 14 cell lines. The expression of Smad4 was decreased in 10 cell lines and 9 cell lines overexpressed CDC25A, as compared to normal controls. CDC25A gene was overexpressed with 88 % (8/9) in tumorigenic cell lines as determined by xenografts in nude mice, and only in 20 % (1/5) of non tumorigenic cell lines ( P <0.05). C myc was not overexpressed in any of these cell lines. The loss of sensitivity to TGF β of cell lines derived from ovarian cancers may be related to a decreased expression of Smad4, which mediates TGF β induced growth inhibition, and/or an overexpression of CDC25A. This overexpression of CDC25A correlates with increased tumorigenicity of ovarian cancer cell lines. The loss of sensitivity to TGF β is not associated with a lack of TβRⅡ.