Function Of Transcription Factor Slug In The Actions Of Propofol On The Invasion Of Ovarian Cancer Cells And On The Apoptosis Of Ovarian Cancer Cells Induced By Paclitaxel

Background and AimsOvarian cancer (OVCA) is the most fatal gynecological malignancy that has frustrated both clinicians and researchers for several decades. It is the fifth leading cause of cancer-related death among women. This is primarily due to the lack of methods for an early detection of the disease leading to rapid, aggressive peritoneal dissemination of cells from the primary tumor, which results in an adverse prognosis for most patients. The exact molecular events leading to metastases of ovarian tumor cells have not as yet been well elucidated, although it is recognized that the acquisition of capacity for migration and invasiveness would be a necessary prerequisite.Chemotherapeutic drug resistance is a critical problem in cancer therapy as many tumors are intrinsically tolerant to some of the cytotoxic agents used, while others, although they are initially sensitive, recur and eventually acquire resistance to subsequent treatment with antineoplastic agents. In the ovarian cancer, after optimal surgical debulking of the tumor and standard chemotherapy, patients with advanced disease experience 5-year survival rate. Despite the relative sensitivity of ovarian cancer to chemotherapy, clinical chemotherapeutic treatment often encounters drug resistance. Development of this acquired resistance represents the major limitation to successful treatment. Consequently, there is a pressing need to identify the mechanisms underlying resistance in order to develop novel drugs to re-sensitize tumor cells to primary chemotherapy.Recent work in esophageal squamous cell carcinoma, bladder cancer pancreatic cancer and ovarian cancer, suggests that the transcriptional factor Slug are important effectors of the process of invasiveness and tumorigenecity. In cholangiocarcinoma cells, Slug silencing could increase cell sensitivity to cisplatin and radiation. In the ovarian cancer, Slug could also mediate radioresistance and chemoresistance. We, therefore, suggested that Slug may be a effective gene target.Propofol (2,6-diisopropylphenol), one of the most commonly used intravenous anesthetic agents producing smooth induction and rapid recovery from anesthesia, has gained wide acceptance since its introduction in the late 80s 13.Apart from its multiple anesthetic advantages, propofol exerts a number of non-anesthetic effects. In lung cancer cells, propofol inhibitsMMP-2 and -9 mRNA and protein expressions, resulting in suppression of invasion and migration in vitro 15. Propofol could inhibit the invasion ability of cancer cells by modulating Rho A and suggested that this agent might be an ideal anesthetic for cancer surgery 16. In the colon carcinoma cells, propofol stimulation inhibits cancer cell invasion and that the effect is partly due to ERK1/2- dependent down-regulation of MMPs. However, in gallbladder cancer, propofol induces proliferation and promotes invasion through activation of Nrf2. We have recently reported inactivation of NF-κB signaling pathway by propofol could abrogate gemcitabine-induced active activation of NF-κB resulting in the chemosensitization of pancreatic tumors to gemcitabine.In the present study, we examined the effect of propofol on cell invasion and apoptosis induction in vitro using paclitaxel-sensitive and -resistant ovarian cancer cell lines. At the same time,we observed correlation between slug level and the effect, in order to ascertain whether propofol has inhibitory effect on ovarian cancer cell invasion, whether can it promote ovarian cancer cell apoptosis induced by Paclitaxel, whether the transcription factor Slug is an effective gene target of propofol to inhibit ovarian cancer cell invasion and promote apoptosis of ovarian cancer cells induced by paclitaxel.Materials and MethodsCells and cell cultureHuman ovarian cancer cell lines HO-8910PM, HO-8910, SKOV-3, OVCAR-3, COC1 and ES-2 were purchased from American Type Culture Collection (ATCC, Shanghai China). SKOV-3 and OVCAR-3 cells were grown in DME medium (Sigma-Aldrich, Shanghai China) supplemented with 10% heat-inactivated fetal bovine serum,50 μg/mL gentamicin and 1mmol/L sodium pyruvate.HO-8910PM, HO-8910, COC1 and ES-2 were purchased from American Type Culture Collection (ATCC, Shanghai China) and cultured in RPMI with 10% FBS (fetal bovine serum),1% penicillin/streptomycin and 1% L-glutamine. All the cells was at 37℃ in a humidified atmosphere containing 5% CO2.Drug exposureCells were plated in 96-well plates,5 x 103 cells/well. After 24 h in cubation, the medium was aspirated and replaced with MEM with 5% FBS containing a range of concentrations of paclitaxel (0.01-10 μM) or propofol (0.1-10 μg/ml) and incubated for an additional 72 h for does-dependent assay. For time-dependent assay,5 x 103 cells/well was incubated with 10 μM paclitaxel or 5 μg/ml propofol for 24-72h. Relative numbers of viable cells remaining were determined using the MTT assay. Apoptotic cells were detected by TUNEL staining assay.Chemosensitivity AssaysCells were plated in 96-well plates,5 x 103 cells/well. After 24 h incubation, the medium was aspirated and replaced with MEM with 5% FBS containing 5 μg/ml propofol for an additional 24 h, then the cells were exposed to 0.1 μM paclitaxel for 48 h. Relative numbers of viable cells remaining were determined using the MTT assay. Apoptotic cells were detected by TUNEL staining assay.MTT assays5 x 103 cells/well were treated above for indicating time, after which 10 μl MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well and cells were left for 4 hours. After incubation,150 μl MTT solvent (0.1 N HCl in anhydrous isopropanol) was added to each well and mixed thoroughly by pipetting until all formazan crystals were dissolved. Colorimetric change was measured at 570 nm and background absorbance at 690 nm. Final values were obtained by subtracting OD690 nm from OD570 nm.TUNEL assay1 x 104 cells above in the indicated time were cultured on chamber slides for 24 h. Apoptosis of the cells was evaluated on the basis of the TUNEL assay according to the manufacturer’s instructions. All assays were performed in quadruplicate.Cell invasion assayFor invasion assay, BD BioCoatTM MatrigelTM invasion chambers was used. Briefly, HO-8910PM, HO-8910, SKOV-3, OVCAR-3, COC1 and ES-2 cells were treated with 0.1-10 μg/ml propofol for 72 hours, or 5μg/ml propofol for 24-72 h. Then the 2 x 103 cells above in serum-free media were added to the interior of each insert. Plates were incubated for 24 hours at 37℃ in 5% CO2, and media removed from the insert, which was then washed with phosphate buffered saline (PBS). Insert membranes were fixed with cold methanol for 10 minutes, stained with 0.5% Crystal Violet in 25% methanol for 10 mins and rinsed with water to remove excess dye. Membranes were removed from the insert, placed under a microscope and the number of cells that migrated through the porous membrane was counted.Western blot analysisFor Western blot analysis, cells were rinsed in ice-cold PBS twice and lysed in cell lysis buffer for 30 min. Samples were sonicated for 30 s and centrifuged for 20 min at 12,000 x g at 4℃. Samples were electrophoresed on a 12% SDS-polyacrylamide gel and electrophoretically blotted on nitrocellulose membrane. Membranes were blocked in TBS/Tween-20 with 5% milk and incubated with primary Abs (antibodies) diluted in TBS/Tween-20/BSA (bovine serum albumin) overnight at 4℃. The following primary was anti-slug. All blots were incubated with secondary Abs conjugated to horseradish peroxidase (HRP) (1/2000) and developed using the enhanced chemiluminescence (ECL) method. Protein concentration was determined using bicinchoninic acid assay.Statistical AnalysisStatistically significant differences were determined by two-tailed unpaired Student’s t test and one-way ANOVA and were defined as p < 0.05. All experiments were repeated independently at least three times.ResultsOvarian cancer (OC) cell lines vary in resistance to paclitaxelWe examined the relative sensitivity of six OC cell lines (HO-8910PM, HO-8910, SKOV-3,OVCAR-3, COC1 and ES-2) to paclitaxel in vitro. Cells were treated with different concentrations of paclitaxel for 72 hours and the number of surviving cells was analyzed by MTT assay. Whereas the paclitaxel LD50 was>10 μM for the HO-8910PM, OVCAR-3 and SKOV-3 cells, the LD50 was around 0.1 μM for HO-8910, COC1 and ES-2 cells. The same sensitivities were obtained when the effects of paclitaxel were analyzed on apoptosis using TUNEL assay (data not shown). These data supported the classification of the HO-8910PM, OVCAR-3 and SKOV-3 cell lines as paclitaxel-resistant and the HO-8910, COC1 and ES-2 cell lines as paclitaxel-sensitive.Slug levels in OC cell linesTo test the hypothesis that Slug basal levels could predict paclitaxel sensitivity, we initially evaluated slug levels in the OC cell lines by western blot assay. Highest levels of slug protein were observed with HO-8910PM, OVCAR-3 and SKOV-3 cells, and lowest slug protein level was observed with H0-8910,COC1 and ES-2 cell. Thus, OC cells exhibited elevated slug, and there was consistent pattern of slug in the resistant versus sensitive cell lines. Consistent difference was noted between slug in paclitaxel-sensitive versus paclitaxel-resistant cells.Paclitaxel treatment increases Slug expression in sensitive but not resistant cellsHO-8910PM, HO-8910, SKOV-3, OVCAR-3,COC1 and ES-2 cells were treated with 0.001,0.01,0.1,1,10 μ.M paclitaxel for 24 hours, and slug were measured using western blot assay. Paclitaxel treatment for 24 hours with 0.001-10 μM had no significant effect on slug levels in the paclitaxel-resistant HO-8910PM, OVCAR-3, SKOV-3 cells. However, in the paclitaxel-sensitive HO-8910, COC1 and ES-2 cells, paclitaxel treatment for 24 hours with 0.1 μM had significant increase in slug levels.Propofol treatment inhibits cell invasion in resistant but not sensitive cellsThe target plasma concentrations of propofol for general anesthesia are between 3 and 8 μg/ml. In the present study, HO-8910PM, HO-8910, SKOV-3, OVCAR-3, COC1 and ES-2 cells were treated with 0.1-10μg/ml propofol for 72 hours, or 5μg/ml propofol for 24-72 h. Propofol inhibits invasion in dose- and time-dependent manners in the paclitaxe-lresistant HO-8910PM, OVCAR-3, SKOV-3 cells,5-10μg/ml concentrations of propofol were sufficient to suppress the invasion ability of cancer cells. No significant effect was found in the paclitaxel-sensitive HO-8910, COC1 and ES-2 cells.Propofol treatment inhibits cell proliferation and promotes apoptosis in resistant but not sensitive cellsHO-8910PM, HO-8910, SKOV-3, OVCAR-3, COC1 and ES-2 cells were treated with 0.1-10 μg/ml propofol for 24,48 and 72 hours. Propofol decreased paclitaxel-resistant HO-8910PM, OVCAR-3,SKOV-3 cells proliferation in a time-and dose-dependent manner by MTT assay. Apoptosis analysis using TUNEL, propofol promoted the apoptosis in the paclitaxel-sensitive OVCAR-3, COC1 and ES-2 cells. No significant effect was found in the paclitaxel-sensitive HO-8910, COC1 and ES-2 cells.Propofol inhibits slug in both resistant and sensitive cells after paclitaxel treatmentBecause 5 μg/ml propofol treatment for 48 hours could significantly inhibits invasion and promotes apoptosis in the OC cells. Therefore, we used 5 μg/ml propofol for 48 hours treatment for further study. HO-8910PM, HO-8910, SKOV-3, OVCAR-3, COC1 and ES-2 cells were treated with 5 μg/ml propofol for 48 hours. Slug protein was significantly or completely inhibited cells. Though paclitaxel treatment for 24 hours with 0.1 μM in the paclitaxel-sensitive HO-8910, COC1 and ES-2 cells significantly increased the slug levels, pretreatment with 5 μg/ml propofol completely inhibited Slug protein after 0.1 μM paclitaxel treatment for 48 hours.Propofol enhances paclitaxel-induced apoptosis in both resistant and sensitive cellsTo test the hypothesis that propofol treatment would increase the sensitivity of OC lines to paclitaxel-mediated apoptosis, HO-8910PM, HO-8910, SKOV-3, OVCAR-3, COC1 and ES-2 cells were pertreated with 5 μg/ml propofol for 24 hours, then examined the effects alone and with 0.1 concentrations of paclitaxel. Propofol combined with 0.1 concentrations of paclitaxel treatment significantly reduced cell numbers and increased apoptosis in vitro of all the OC cells.ConclusionsOur findings indicate that propofol inhibits invasion and enhances paclitaxel-induced apoptosis in ovarian cancer cells by effectively inhibiting Slug expression. These results suggest that propofol might be an ideal anesthetic for OC surgery and is potentially useful as a sensitizer in OC therapy.