ABSTRACT
Objective:To investigate targeted therapy of ovarian cancer with new fusion proteins that were produced by fusing the first 390 amino acids of diphtheria toxin(DT390)to the TMTP1 peptide.Methods:The cisplatin-resistant cell line,C13*,and cisplatin-sensi-tive cell line,OV2008,were selected as models and divided into control,TMTP1,DT390-TMTP1,DT390-biTMTP1,and DT390-triTMTP1 groups.Laser scanning confocal microscopy was used to observe nuclear morphology.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide(MTT)and flow cytometry assays were used to detect cell survival and apoptosis,respectively.The formation of subcu-taneous tumors in nude mice following injection of C13*cells was used to observe the formation and growth of ovarian cancer.Apop-tosis of cells in the subcutaneous tumor tissue was detected by the terminal deoxynucleotidyl transferase dUTP nick-end labeling(TU-NEL)assay.Results:Laser scanning confocal microscopy showed that DT390-biTMTP1 and DT390-triTMTP1 induced nuclear shrinkage and fragmentation.The MTT assay showed that cell survival was obviously reduced with increasing concentrations of DT390-biTMTP1 and DT390-triTMTP1. Flow cytometry revealed that DT390-biTMTP1 and DT390-triTMTP1 significantly increased cell apoptosis (P<0.05).The apoptosis rates of the DT390-biTMTP1 and DT390-triTMTP1 groups were 66.0%±12.0% and 72.9%±4.6%,respectively.These were higher than the 55.5%±8.9% and 65.1%±9.8% obvserved in OV2008 cells.DT390-biTMTP1 and DT390-triTMTP1 significantly in-hibited the tumor formation (P<0.01) and growth (P<0.05), and increased apoptosis (P<0.05) of subcutaneous tumors. However, DT390-TMTP1 had insignificant effects on C13*and OV2008 cells.Conclusions:DT390-biTMTP1 and DT390-triTMTP1 preferentially tar-geted and inhibited ovarian cancer cells.These fusion proteins may be a promising strategy for clinical therapy of ovarian cancer.
ABSTRACT
In this article, the status of spindle assembly checkpoint and the alteration of its major component, Mad2 protein level were examined in A2780 and SKOV3 ovarian cancer cell lines. Recombinant eukaryotic expression plasmid pEGFP-Mad2 was transfected into paclitaxel-resistant SKOV3 cells and Mad2 protein was knocked down by Mad2-specific siRNA in paclitaxel-sensitive A2780 cells. Then the expression level of Mad2 gene was detected by Western blotting. Flow cytometry revealed that SKOV3 cells were not fully arrested in G(2)/M phase in contrast to A2780 cells in the presence of paclitaxel. However, paclitaxel sensitivity assay showed that sensitivity to paclitaxel was reversed after the transfection in both cell lines in terms of number of cells arrested at G(2)/M phase and the expression of Bcl-2 was significantly changed. These results suggest that weakened spindle checkpoint with reduced expression of Mad2 is associated with resistance to paclitaxel in ovarian cells and Bcl-2 may be involved in this process.
ABSTRACT
The purpose of this study was to pool information in epithelial ovarian cancer by combining studies using Affymetrix expression microarray datasets made at different laboratories to identify novel biomarkers. Epithelial microarray expression information across laboratories was screened and combined after preprocessing raw microarray data, then ANOVA and unpaired T test statistical analysis was performed for identifying differentially expressed genes (DEGs), followed by clustering and pathway analysis for these DEGs. In this work, we performed a combination analysis on microarrays from three different laboratories using gene expression data on ovarian cancer and obtained a list of differential expression profiles identified as potential candidate in aggressiveness of ovarian cancer. The clustering and pathway analysis explored the different molecular basis of different ovarian cancer stages and potential important regulatory pathways in ovarian cancer development. Our results showed that combination of microarray data from different laboratories in the same platforms may overcome biases derived from probe design and technical features, thereby accelerating the identification of trustworthy DEGs, and demonstrating the advantage of integrative analysis in gene expression studies on epithelial ovarian cancer research.
ABSTRACT
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.