1.Effects of pEGFP-Bubl-shRNA plasmid on cell cycle and paclitaxel-sensitivity in human ovarian cancer cell line A2780
Ting ZHOU ; Danhui WENG ; Shixuan WANG ; Yunping LU ; Ding MA
China Oncology 2010;20(3):161-166
Background and purpose:Previous studies have shown that Bubl was a critical component of the spindle checkpoint.Paclitaxel sensitivity was considered to be dependent on the functionality of this spindle checkpoint.This study investigated the effects of pEGFP-Bubl-shRNA plasmid stably transfected into the cell cycle and its sensitivity in human ovarian cancer cell line A2780.Methods:After the pEGFP-Bubl-shRNA plasmid and empty plasmids were constructed.they were transfected into A2780 cells by the Lipofectamine 2000~(TM).The nontransfected cells were the control.RT-PCR and Western blotting were used to determine the target gene and protein expression.The rate of proliferation inhibition was tested by an MTT assay,apoptosis and cell cycles were determined by flow cytometry,and the mitotic index was determined bv Hoechst33342 dye.Results:RT-PCR and Western blotting showed that pEGFP-Bubl-shRNA/A2780 group displayed a low expression of Bubl compared to the A2780 and pEGFP-Cl/A2780 group(P<0.05).The sensitivity of the pEGFP-Bubl-shRNA/A2780 group was significantly lower than the non-transfccted and pEGFP-Cl/A2780 cells(P<0.05).Flow cytometry revealed that the rates of G2/M phase and apoptosis were significantly lower in the pEGFP-Bubl-shRNA/A2780 group than those in the control group (P<0.05).Conclusion:Bubl plays an important role in the paclitaxel treatment.A down-regulation of Bubl could reduce the drug sensitivity and rate of G_2/M cells in human ovarian cancer cell line A2780.
2.MYC suppresses ovarian cancer cell adhesion and invasion ability through downregulation of human integrinβ1
Zongyuan YANG ; Xiaoshui ZHOU ; Xiao WEI ; Yi LIU ; Chen GONG ; Gang CHEN ; Danhui WENG
The Journal of Practical Medicine 2014;(7):1048-1052
Objective To explore the influence of MYC expression level on ovarian cancer cells adhesion and invasion ability,and the involvement of the integrinβ1 in the adhesion and invasion control. Methods Comparative study was done to analyze the relationship between the mRNA level of MYC and ITGB1 among sixty cell lines from the NCI-60 cells bank; Western blot was performed to demonstrate the correlation of MYC and ITGB1 protein level in ovarian cancer cell lines;Immunohistochemistry was adopted to compare the expression level of MYC and ITGB1 in paired primary and metastatic ovarian cancer tissue. Furthermore ,we employed in vitro adhesion test to detect the alteration of ovarian cancer cell adhesion ability to ECM molecular like fibronectin ,collagen I and laminin after fluctuation of MYC and ITGB1 expression; Transwell assay was applied to check the invasion ability variation with the disturbance of MYC and ITGB1 level. Results The mRNA levels of MYC and ITGB1 were dramatically inverse-correlated in NCI-60 cells bank(P<0.01);The protein levels of MYC and ITGB1 in ovarian cancer cell lines were also negative correlated; The primary ovarian cancer tissue showed lower expression of ITGB1 and higher expression of MYC,while the matched metastasis displayed the opposite outcome;Ovarian cancer cells reduced adhesive and invasive capacity in case of ITGB1 being down-regulated ,however, the adhesive and metastatic ability improved tremendously with elevation of ITGB1;Promoted adhesion and invasion ability and rise of ITGB1 expression were observed after downregulation of MYC,meanwhile,and inhibition of ITGB1 reversed the increasement of adhesion and invasion ability. Conclusions MYC,inversely-correlated with ITGB1 in ovarian cancer cells,inhibited ovarian cancer cells adhesion and invasion. Inhibition of MYC could lead to enhanced adhesion and invasion ability of ovarian cancer cells.
3.Effect of spindle checkpoint on Akt2-mediated paclitaxel-resistance in A2780 ovarian cancer cells.
Ting, ZHOU ; Yindi, BAO ; Shuangmei, YE ; Danhui, WENG ; Gang, CHEN ; Yunping, LU ; Ding, MA ; Shixuan, WANG
Journal of Huazhong University of Science and Technology (Medical Sciences) 2010;30(2):206-11
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/Bub1 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 Bub1 in different groups by Western blotting. Our study showed that up-regulation of Akt2 contributed to A2780 ovarian cancer cells overriding PTX-induced G(2)/M arrest, and inhibited Bub1 expression. Our findings might shed light on the molecular mechanism of PTX-induced resistance in ovarian cancer and help develop novel anti-neoplastic strategies.
4.Breast cancer associated fibroblasts promote MCF-7 invasion in vitro by secretion of HGF.
Pingbo, CHEN ; Qingqing, MO ; Beibei, WANG ; Danhui, WENG ; Peng, WU ; Gang, CHEN
Journal of Huazhong University of Science and Technology (Medical Sciences) 2012;32(1):92-6
This study was aimed to explore the influence of breast cancer associated fibroblasts (CAFs) in migration and invasion of breast cancer cell line MCF-7, and investigate whether hepatocyte growth factor (HGF) is involved in this process. Primary breast CAFs and their corresponding normal breast fibroblasts (NFs) were obtained by collagenase digestion. On the basis of the co-culture, the migration and invasion capacity of MCF-7 cells was compared between CAFs and NFs by Transwell. The difference in the HGF expression between them was detected by ELISA. The secretion of HGF was knocked down by using RNA interference technology in CAFs. Then the changes of migration and invasion capacity of MCF-7 cells were investigated by Transwell. Eventually, we isolated high-purity CAFs and NFs, and the CAFs had a stronger ability in promoting MCF-7 migration and invasion than the NFs. ELISA results demonstrated that CAFs secreted higher HGF, and the capacity of MCF-7 migration and invasion was declined after knocking down the secretion of HGF in CAFs by RNA interference. It is suggested that CAFs can promote MCF-7 migration and invasion through HGF in vitro.
5.miR-125b confers resistance of ovarian cancer cells to cisplatin by targeting pro-apoptotic Bcl-2 antagonist killer 1.
Fanfei, KONG ; Chaoyang, SUN ; Zhongxian, WANG ; Lingfei, HAN ; Danhui, WENG ; Yunping, LU ; Gang, CHEN
Journal of Huazhong University of Science and Technology (Medical Sciences) 2011;31(4):543-9
Chemotherapy is the preferred therapeutic approach for advanced ovarian cancer, but a successful long-term treatment is prevented by the development of drug resistance. Recent works have underlined the involvement of non-coding RNAs, microRNAs (miRNAs) in cancer development, with several conjectures regarding their possible involvement in the evolution of drug resistance. This study is to investigate the promoting effects and mechanism of miR-125b involved in the development of chemoresistance in ovarian cancer. The different expression of miR-125b in cisplatin-sensitive ovarian cancer cell line (OV2008) and its resistant variant (C13*) was identified by real-time PCR. An in vitro cytotoxicity assay and apoptosis assay using CCK-8 assay and flow cytometry, were carried out to detect the effect of miR-125b and Bak1 on cisplatin resistance of cells. Real-time PCR, Western blotting and luciferase reporter assay were used to detect whether Bak1 is a target of miR-125b. As compared with OV2008 cells, the expression levels of miR-125b in C13* cells were increased. It was found that the up-regulation of microRNA-125b caused a marked inhibition of cisplatin-induced cytotoxicity and apoptosis and a subsequent increase in the resistance to cisplatin in OV2008 and C13* cells. Moreover, Bak1 was a direct target of miR-125b, and down-regulation of Bak1 suppressed cisplatin-induced apoptosis and led to an increased resistance to cisplatin. Our study indicates that miR-125b has a significantly promoting effect on chemoresistance of C13* cells and up-regulation of miR-125b expression contributes to cisplatin resistance through suppression of Bak1 expression. This finding has important implications in the development of targeted therapeutics for overcoming cisplatin resistance in ovarian cancer.
6.Proteasome inhibitors sensitize ovarian cancer cells to cisplatin
Yan LI ; Danhui WENG ; Fanfei KONG ; Liangsheng FAN ; Yi HU ; Xiaohong SONG ; Hui XING ; Wei WANG ; Ding MA ; Shixuan WANG
Chinese Journal of Obstetrics and Gynecology 2010;45(6):445-448
Objective To explore the sensitivity and the molecular mechanism of cisplatinresistance ovarian cancer cell line C13 to proteasome inhibitors and the combination with cisplatin. Methods After different treatments, methyl thiazolyl tetrazolium (MTT) assay was applied to examine the cell viability, annexin-V/propidium iodide(PI) apoptosis detection kit was used to determine the apoptosis rate of different groups, western blot assay was introduced to evaluate the expression levels of Fas-associated death domain-like interleukin-1 beta converting enzyme inhibitory protein (cFLIPs), and the activity of caspase-8 was examined. Results MTT assay shown that the cell viability ratios of combination group at serial time points from 12, 24, 36, 48, 60, 72 hours were ( 56.0 ± 8.4 ) %, (44.7 ± 7.3 ) %, ( 33.7 ±11.2) %, (27.6 ± 8.0) %, (27. 6 ± 7.6) % and (28.1 ± 2.4) %, which were much lower than those of cisplatin group (P <0.05). After treated for 24 hours, apoptosis rates of cisplatin group, bortezomib group and combination group were ( 16.7 ± 1.7) %, (23.4 ± 2.1 ) % and (26.9 ± 1.6) %, respectively. The rate of combination group was much higher than that of non-treated group and that of cisplatin group or bortezomib group ( P < 0.05 ). Western blot assay showed the changes of expression levels of cFLIPs, which were downregulated seriously after cisplatin, bortezomib or combination treatment [ (43.2 ± 2.3 )% vs( 75.7 ± 3.0)%vs (67.9 ± 2.1 ) %, P < 0.05 ]. The caspase-8 activity of combination group was (5.6 ± 1.6) folds than that of non-treated group, which was higher than those of other two groups [ ( 2.3 ± 1.0) and (4.2 ± 0.9 ) folds,P < 0.05 ]. Conclusions The tumor cell lethal effect of cisplatin could be increase significantly by the combination application of proteasome inhibitors, bortezomib. And the cFLIPs/caspase-8 signaling pathway may be play an important role in the molecular mechanism of the combination treatment.
7.Proteasome inhibitors sensitize ovarian cancer cells to paclitaxel induced apoptosis
Danhui WENG ; Yan LI ; Yanfei KONG ; Liangsheng FAN ; Yi HU ; Xiaohong SONG ; Hui XING ; Shixuan WANG ; Ding MA
Chinese Journal of Obstetrics and Gynecology 2008;43(10):770-773
Objective To explore the semitivity of ovarian cancer cell line SKOV3 to paclitaxel,oroteasome inhibitors,bortezomib,and their combination.Methods The methyl thiazolyl tetrazolitim (MTT)assay was applied to examine the cell viability after treatment.The annexin V-propidium iodide apoptosis detection kit was used to determine the apoptosis rate of different groups.Western blot assay was used to evaluate the expression levels of phosphorylated protein kinase B(AKT)and glycogen synthase kinase-3 beta(GSK-3β).Results In MTT assay,the cell viability ratios of the combination group at serial time points from 12,24,36,48 and 72 hours Were(65.2±5.8)%,(58.3±14.4)%,(35.3±5.0)%,(19.2±1.5)%,and(11.4 ±2.5)%,which were significantly lower than those of the paclitaxel group (P<0.05).After arug treatments,apoptosis rates of paclitaxel group,bortezomib group and the combination group were (14.7±0.5)%,(15.1±0.8)%and(20.5±0.7)%respectively.The rate of the combination group was significantly higher than that of non-treated group and paclitaxel group(P<0.05).Western blot assay showed the changes in expression levels of phosphorylated AKT and GSK-3β,which were decreased significantly after paclitaxd and bortezomib combination treatment [(3.2±0.8)%,(19.3±0.4)%;P<0.05].Conclusions The lethal effect of paclitaxel on tumor cells could be increased significantly by its combination with proteasome inhibitors,bertezomib.The AKT/GSK-3β signaling pathway plays an important role in the molecular mechanism of the combination treatment.
8.Reversal of Multidrug Resistance and Inhibition of Phosphorylation of AKT in Human Ovarian Cancer Cell Line by Wild-type PTEN Gene
Huijuan WU ; Danhui WENG ; Hui XING ; Yunping LU ; Ding MA
Journal of Huazhong University of Science and Technology (Medical Sciences) 2007;27(6):713-716
The reversing effect of wild-type PTEN gene on resistance of CI3K 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 CI3K 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 IC50 of DDP to C13K cells transfected with PTEN (7.2±0.3 μmol/L) was obviously lower than those of empty-vector transfected cells and non-transfected cells (12.7±0.4 μmol/l, 13.0±0.3 μmol/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 ex- pression 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.
9.Breast Cancer Associated Fibroblasts Promote MCF-7 Invasion in vitro by Secretion of HGF
CHEN PINGBO ; MO QINGQING ; WANG BEIBEI ; WENG DANHUI ; WU PENG ; CHEN GANG
Journal of Huazhong University of Science and Technology (Medical Sciences) 2012;32(1):92-96
This study was aimed to explore the influence of breast cancer associated fibroblasts (CAFs) in migration and invasion of breast cancer cell line MCF-7,and investigate whether hepatocyte growth factor (HGF) is involved in this process.Primary breast CAFs and their corresponding normal breast fibroblasts (NFs) were obtained by collagenase digestion.On the basis of the co-culture,the migration and invasion capacity of MCF-7 cells was compared between CAFs and NFs by Transwell.The difference in the HGF expression between them was detected by ELISA.The secretion of HGF was knocked down by using RNA interference technology in CAFs.Then the changes of migration and invasion capacity of MCF-7 cells were investigated by Transwell.Eventually,we isolated high-purity CAFs and NFs,and the CAFs had a stronger ability in promoting MCF-7 migration and invasion than the NFs.ELISA results demonstrated that CAFs secreted higher HGF,and the capacity of MCF-7 migration and invasion was declined after knocking down the secretion of HGF in CAFs by RNA interference.It is suggested that CAFs can promote MCF-7 migration and invasion through HGF in vitro.
10.Effect of Spindle Checkpoint on Akt2-mediated Paclitaxel-resistance in A2780 Ovarian Cancer Cells
ZHOU TING ; BAO YINDI ; YE SHUANGMEI ; WENG DANHUI ; CHEN GANG ; LU YUNPING ; MA DING ; WANG SHIXUAN
Journal of Huazhong University of Science and Technology (Medical Sciences) 2010;30(2):206-211
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/Bub1 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 Bub1 expression.Our findings might shed light on the molecular mechanism of PTX-induced resistance in ovarian cancer and help develop novel anti-neoplastic strategies.