Identification Of Taxol Resistance - Related Proteins Mimitin And 14-3-3 ξ / δ In Ovarian Cancer And Their Related Studies

BackgroundOvarian cancer is the commonest cause of gynecological cancer-associated death. Over 70% of the patients had advanced stage disease when diagnosed. The median overall survival time is 57 months for patients receiving optimal cytoreductive surgery, but within 2 years for those receiving suboptimal operation. The standard treatment for patients with epithelial ovarian cancer at stage IC-IV is cytoreductive surgery, followed with adjuvant chemotherapy. In the recent two decades, platinum plus paclitaxel remain the standard regimen, which have been proved by plenty of clinical trials. However, over 75% of patients would develop chemoresistance and relapse. Chemoresistance could be grouped into primary and acquired resistance. Primary resistance is based on the constitution of spontaneous resistant tumor cell clones, which leads to initial presence of insensitivity to chemotherapy. Acquired resistance consists of stochastic events in the individual cancer cells predisposing for cell survival, generation of minimal chemoresistant disease, and finally clinical recurrence.Paclitaxel (Taxol) is a cytotoxic alkaloid that is efficacious in the treatment of ovarian cancer. Paclitaxel exerts its cytotoxic effects mostly by microtubule stabilizing mechanisms. Classically it binds to tubulin in microtubules, causing the formation of unusually stable microtubules and inhibiting the depolymerization of microtubules. The ensuing mitotic arrest triggers the mitotic spindle checkpoint. Paclitaxel can disrupt the normal structure and function of microtubules in interphase, It results in disrupting growth and metabolism and leading to cell death.As far as concerned, the evaluation of chemoresistance in ovarian cancer is based on the platinum-free interval. Accordingly, most studies relevant to ovarian cancer chemoresistance concentrate on platinum resistance but not paclitaxel resistance. Fewer studies work on the paclitaxel resistance. However, the chemoresistance of ovarian cancer is a heterogenic and multfactorial disease. Paclitaxel resistance is not uncommon to see in patients receiving single taxol regimen. The commonest proteins include P-glycoprotein, tubulin and Glutathione S-transferase P1. However, the experience mostly comes from the studies in other solid tumors and the effects of overcoming chemoresistance in ovarian cancer are limited. Comparative proteomic analysis between the ovarian cancer cells and their paclitaxel-resistant sublines would surely bring us more reliable markers for paclitaxel-resistance.Our research group has been supported by the National Science Foundation. Our previous work obtained two paclitaxel-sensitive human ovarian cancer cell lines (SKOV3 and A2780) and their corresponding resistant cell lines (SKOV3-TR and A2780-TR). Guanidine-modified acetyl-stable isotope labelling and liquid chromatography-hybrid linear ion trap Fourier-transform ion cyclotron resonance mass spectrometry (LC-FTICR MS) were performed to find the expressed differential proteinsin mitochondria. Comparative proteomic analysis revealed that 8 differentially expressed proteins in the ovarian cancer cells and their paclitaxel-resistant sublines. Among them, mimitin and 14-3-3 ζ/δ were selected for further research. Both of these two proteins were downregulated in paclitaxel-resistant sublines. The ratio of mimitin was 7.4 for SKOV3/SKOV3-TR and 12 A2780/A2780-TR. The ratio of 14-3-ζ/δ was 4.3 for SKOV3/SKOV3-TR and 3.4 A2780/A2780-TR.The present study mainly identified the effects of paclitaxel-resistance-associtaed proteins, mimitin and 14-3-3ζ/δ in ovarian cancer cells based on the previous study of proteomics. The expression of mimitin and 14-3-3 ζ/δ in paclitaxel-resistant ovarian cancer cells was identified by western blotting. Then the resistance index to paclitaxel was detected by MTT assays after downregulation of mimitin and 14-3-3 ζ/δ by si-RNA transfection in ovarian cancer cells (SKOV3 and TR-SKOV3). Relevant chemoresistant genes were verified by RT-PCR. Moreover, the expression of mimitin and 14-3-3 ζ/δ human tissue specimens was detected by immunohistochemistry. Multivariated analyses were performed for exploration of risk factors respectively.Materials and methods1. The resistance index of TR-SKOV3 to paclitaxel was investigated by MTT assays. The comparison of cell morphology of TR-SKOV3 and SKOV3 was performed via Giemsa-Wright’s staining. The cell growth curve of TR-SKOV3 was calculated by cell counting. Cell cycles of TR-SKOV3 were also analyzed by flow cytometry.2. The expression of mimitin and 14-3-3 ζ/δ in TR-SKOV3 and SKOV3 was detected by western blot. The resistance indices of SKOV3 and TR-SKOV3 to paclitaxel were investigated by MTT assays after siRNA transfection, in order to explore the functions of these two proteins. The changes of relevant drug-resistant genes were detected by RT-PCR and possible mechanisms were explored.3. Seventy-one patients were enrolled into the study cohort and follow-up data were completed. The expression of mimitin and 14-3-3 ζ/δ in human tissue specimens was explored by immunohistochemistry. The relationship between mimitin, 14-3-3 ζ/δ and chemoresistance was analyzed. Risk factors in ovarian cancer patients were investigated using multivariated analysis.Results1. The resistance index of TR-SKOV3 to paclitaxel was 5.59±1.48. The cell morphology of TR-SKOV3 and SKOV3 was similar. The cell growth curve of TR-SKOV3 showed the cell doubling time was (38.9±5.4) h,1.62 times of that of SKOV3 cells, with significant difference. Flow cytometry revealed the proportion of phase G0/G1 cells of TR-SKOV3 was 60.18±1.55%, more than that of SKOV3 (P< 0.001), and the proportion of phase S cells of TR-SKOV3 was 23.02±3.05%, less than that of SKOV3 (P=0.003). The proportion of phase G2/M cells of TR-SKOV3 was 16.80±1.51%, without significant difference from SKOV3.2. The expression of mimitin and 14-3-3 ζ/δ was underexpressed in TR-SKOV3 compared to that of SKOV3. After mimitin or 14-3-3 ζ/δ siRNA transfection,。 the IC50 of SKOV3 to paclitaxel were 7.78±0.87 μg/ml、10.70±0.43 μg/ml while the IC50 of TR-SKOV3 to paclitaxel were 9.22±2.08 μg/ml、13.34±3.43 μg/ml. The resistance indices for SKOV3 cells were 4.03±0.79 and 6.25±1.79, respectively (P=0.01 and P< 0.001, respectively). The resistance indices for TR-SKOV3 cells were 1.93±0.4 and 2.42±0.51, respectively (P=0.043 and P =0.022, respectively). RT-PCR showed the gene amplification of MDR/TAP、 GSTP1 was augmented in TR-SKOV3, compared to SKOV3. The amplification of GSTP1 was further increased after siRNA transfection.3. The expression of mimitin and 14-3-3ζ/δ in human tissue specimens of primary ovarian cancer was explored by immunohistochemistry. The relationship between mimitin,14-3-3 ζ/δ and chemoresistance was analyzed. Risk factors in ovarian cancer patients were investigated using multivariated analysis. Ten of 15 chemo-resistant patients (66.7%) presented with low expression of mimitin, whereas 9 of 56 participants (16.1%) presented with low expression of mimitin in the chemo-sensitive group (χ2=15.453, P< 0.001). Meanwhile,11 of 15 paclitaxel-resistant patients presented with low expression of 14-3-3 ζ/δ (73.3%) compared to 7 of 56 cases (12.5%) with low expression of 14-3-3 ζ/δ in the paclitaxel-sensitive group (χ2=23.135, P=0.001). According to the Kaplan-Meier survival curve, the median PFS for patients with higher mimitin expression and lower expression were 17 and 6 months, and the OS were 48 and 15 months respectively. The median PFS for patients with higher 14-3-3 ζ/δ expression and lower expression were 17 and 10 months, and the OS were 52 and 30 months respectively. Mimitin and 14-3-3 ζ/δ protein expression levels had a significant impact on PFS and OS (mimitin:PFS:P= 0.002, OS:P= 0.001; 14-3-3 ζ/δ :PFS:P=0.028; OS:P=0.014). The effects of mimitin and 14-3-3 ζ/δ were explored using specific siRNAs interference in ovarian cancer cell lines and immunohistochemistry in human tissue specimens. The down-regulation of mimitin and 14-3-3 ζ/δ using specific siRNAs in paclitaxel-resistant ovarian cancer cells led to increasing the resistance index to paclitaxel (P=0.043 and P=0.022, respectively). Multivariate analyses demonstrated that lower expression levels of the mimitin and 14-3-3 ζ/δ proteins were positively associated with shorter progression-free survival and overall survival in patients with primary ovarian cancer (mimitin:PFS:P= 0.041, OS:P =0.003; 14-3-3 ζ/δ:PFS:P=0.031, OS:P=0.011).Conclusions1. The cell line TR-SKOV3 was still resistant to paclitaxel after ten-year interval from induction. Its morphology was similar to SKOV3, with more proportion of G0/G1 phase and less proportion of S phase. It doubling time was prolonged. It could be a good model in vitro to investigate the mechanism of paclitaxel resistance in ovarian cancer.2. Downregulation of mimitin and 14-3-3 ζ/δ could increase the resistance to paclitaxel in paclitaxel-resistant ovarian cancer cells. They were related to paclitaxel resistance and the mechanism might be related to GSTP1.3. The expression of mimitin and 14-3-3 ζ/δ in human tissue specimens was lower in chemoresistant patients compared to chemosensitive participants. The underexpression of mimitin and 14-3-3 ζ/δ was correlated to adverse PFS and OS, which could be independently prognostic factors in ovarian cancer.