The Up-regulation Of Signal Transducing Molecules Associated With Cytoskeleton In Paclitaxel-resistant Ovarian Cancer SKOV3 Subline, SKOV3-TR30

BackgroundOvarian cancer is still the most cause of mortality among gynecological malignancies. As 75% ovarian cancer is associated with advanced stage at presentation, chemotherapy becomes as important as operation in cancer treatment. Approximately 80% patients response to the first-line regimen (carboplatin -paclitaxel). However, most patients will suffer a relapse within 2 or 3 years of the initial treatment. Success rates for second-line chemotherapy are always disappointing and most patients ultimately die of chemoresistance. Therefore, the emergence of chemoresistance becomes a major obstacle in cancer treatment.The target of paclitaxel (taxol) in cell is cytoskeleton. Its antitumor activity is based on increasing the stability of microtubules and preventing mitosis through activating spindle checkpoint. In cells treated with paclitaxel, the transition between interphase and mitosis is normally arrested and the cells die. Defined molecular mechanisms for acquired tumor cell resistance to paclitaxel include over-expression of the P-glycoproteins (P-gp), alteration expression of tubulin isotypes and microtubule-associated proteins, and also alterations in several signal transduction effector molecules of apoptosis. But the exact mechanism of chemoresistance is not yet known.Chemoresistance evolution is a multistep process involving numerous gene alterations leading to the regulation of multiple relevant proteins expression. Proteomics provides a new high-throughput platform for chemoresistance research. Combination of techniques including two dimensional gel electrophoresis (2-DE), mass spectrometry (MS), and bioinformatics, proteomics can be expected to show the changes in the protein expression profile during tumor development and progression, thus leading to the identification of new molecular markers suitable for predicting and reversing chemoresistance. Now, proteomics has been widely used in research of molecular biology, but it is just in beginning to analyse chemoresistance and there is still no research using proteomics to elucidate the mechanism of paclitaxel-resistance.ObjectiveEstablish differential expression profiles of paclitaxel-resistant ovarian cancer sub-line and matched ovarian cancer cell line in order to detect the proteins whose expressions are associated with paclitaxel-resistance evolution and further analyse the possibility of them to be new molecular markers for predicting and reversing chemoresistance.MethodsThe epithelial ovarian cancer cell line SKOV3 and its paclitaxel-resistant sub-line SKOV3-TR30 were selected. Total proteins from the two cell lines are separated by 2-DE and visualized by silver staining. The digitized images then were analyzed with PDQuest software in order to establish the differential expression profiles between SKOV3-TR30 and SKOV3. Differential expressed protein spots were analysed by a Finnigan LTQ MS coupled with a Surveyor HPLC system. Andabased on the MS/MS raw data, protein identification was performed by application of human non-redundant protein database on IPI(IPI.HUMAN.v3.07.fasta). Then 3 protein (Sorcin, Cathepsin B, HSP27) expressions in paclitaxel-resisant cells were compared with that of parental cell line by Western-blot analysis and immunocytochemistry.Results23 protein spots showed significant changes in SKOV3-TR30 compared to SKOV3. Among them, 16 proteins were found to be up-regulated and 7 proteins were down-regulated in chemoresistant cells (expression regulation more than 3-fold). 12 proteins which were clear and stable analysed later, and 7 of these proteins were identified by tandem mass spectrometry as following: sorcin. cathepsin B precursor, calponin-3 , heat-shock protein 27 , vimentin , nicotinate-nucleotide pyrophosphorylase. and hemoglobin beta chain. Western-blot analysis indicated that Sorcin, cathepsin B and HSP27 were up-regulated in SKOV3-TR30. Immunocytochemistry revealed three proteins all located in cytoplasm,especially perinuclear.ConclusionThe expression of signal transducing molecules associated with cytoskeleton were up-regulated in low level paclitaxel-resistant ovarian cancer subline.The effective of new target for therapy and change on acquired drug resistance of them in vivo should be further studied in ovarian cancer.