Value Of Mesothelin In Diagnosis And Monitoring Of Epithelial Ovarian Cancer And Its Mechanism Of Action In Cisplatin Resistance

Epithelial ovarian cancer (EOC) has become an extremely important disease in recent years because it is the leading cause for death in gynaecological malignancies. Only 30% patients with EOC can survive for 5 years after initial diagnosis. Because it is difficult to diagnose earlier, around 70% patients are diagnosed with advanced stage, after surgical tumor debulking followed by chemotherapy, the patients response mostly, but around 60% patients relapse ultimately. As resistance to platinum-based chemotherapeutic drugs plays a major role in tumor progression. Consequently, new markers and new way to resolve cisplatin-resistance are in need.Mesothelin is a newly identified tumor-cell surface antigen, it is present on normal mesothelin cells lining the body cavities. It is highly expressed in several cancers , including mesotheliomas , ovarian cancer and pancreatic cancers. Mesothelin protein encoded by mesothelin gene is a glycosyl- phosphatidylinositol (GPI)-anchored cell-surface glycoprotein. Full length mesothelin (69KDa) can be cleaved by the protease furin to yield a 33 KDa soluble protein called megakaryocyte potentiating factor (MPF) and a 40KDa cell membrane bound protein called mesothelin. Soluble mesothelin-related protein (SMRP) is a 42-45 KDa protein which may be a proteolytically cleaved fragment of membrane-bound mesothelin. SMRP can be released into blood and molecular weight of SMRP is lower than that of CA₁₂₅, so SMRP can give higher sensitivity than CA₁₂₅, it is a promising marker for ovarian cancer diagnosis.So far some reports on mesothelin suggested mesothelin could facilitate cells to proliferate and cell-cell binding, it is important in the peritoneal metastasis of ovarian tumors. But the biologic functions of mesothelin remain speculative . A report showed that in patients with EOC, mesothelin expression in cisplatin-resistant tissues were higher than in cisplatin-sensitive tissues and was associated with poor prognosis. Thus we suppose that mesothelin is probably to play a role in cisplatin resistance and become a promising target for cisplatin-resistance therapy.First of all, the study is to compare mesothelin with CA₁₂₅ (a common mareker)and HE₄(a promising marker), evaluate the diagnostic and prognostic performance of three markers either alone or combination and assess clinical significance of SMRP. Then mesothelin expression in EOC resistant cell lines was assessed from gene level and protein level. After that, taking SKOV₃/cDDP for example, mesothelin expression was slilenced using RNA interference technique. The sensitivity of resistant cells to cisplatin was evaluated through MTT drug sensitivity test and colony formation test. The possible mechanism of action was investigated from two aspects: change of resistant protein expression and intracellular GSH level. At the last, human ovarian cancer resistant cell-bearing nude mice models were developed , nude mice were treated by mesothelin-silencing lentivirus solution and cisplatin, the sensitivity of xenograft tissues after mesothelin-sliencing to cisplatin was evaluated further in vivo, the possibility of mesothelin-silencing in reversing cisplatin resistance was investigated and a new thinking was provided for reversing cisplatin resistance.Partâ… SMRP and HE₄—Potential Markers for Diagnosis and Prognosis in Epithelial Ovarian CancerObjective: To compare serum SMRP and HE₄ levels in ovarian benign and malignant tumors and healthy volunteers, analyze their diagnostic performance and evaluate the utility of novel serum tumor markers, HE₄ and SMRP either alone or in combination with CA₁₂₅ in diagnosis and prognosis in patients with EOC.Methods: We analyzed serum CA₁₂₅, SMRP and HE₄ levels in a sample of 44 patients with EOC , 56 patients with benign epithelial ovarian tumors and 40 healthy volunteers. Serum SMRP and HE₄ concentrations were determined using ELISA and serum CA₁₂₅ concentrations were determined using chemiluminescence technique. Statistical comparison of serum SMRP, HE₄ and CA₁₂₅ in all groups, in different pathology types , in different grades and different stages was done. Serum SMRP, HE₄ and CA₁₂₅ levels in preoperative group were compared with in postoperative group. Receiver operating characteristic curves (ROC) were constructed to evaluate the performance of the three markers. The diagnostic values in combination of the three markers were analyzed.Results:1 SMRP, HE₄ and CA₁₂₅ levels were significantly higher in patients with EOC than in patients with benign epithelial ovarian tumors and healthy volunteers respectively(P<0.05).2 SMRP, HE₄ and CA₁₂₅ levels in serous carcinoma were higher than in mucous carcinoma significantly(P<0.05).3 In EOC, serum SMRP, HE₄ and CA₁₂₅ levels differed significantly between early and late stages(P<0.05).4 Serum SMRP, HE₄ and CA₁₂₅ levels in G3 were higher than in G2 and G1 significantly(P<0.05).5 Difference in SMRP, HE₄ and CA₁₂₅ levels in serous cystadenoma and mucinous cystadenoma were not statistically significant(P>0.05).6 Serum SMRP, HE₄ and CA₁₂₅ levels in 22 patients with EOC after ideal cytoreductive surgery decreased significantly, which were lower than in preoperative group significantly(P<0.05).7 The area under receiver operating characteristic curve(AUC) for serum SMRP was 0.861(95% confidence interval, 0.778-0.944), The AUC for serum HE₄ was 0.905(95% confidence interval, 0.830-0.930), The AUC for serum CA₁₂₅ was 0.984(95% confidence interval, 0.969-0.999).8 Based upon Receiver operating characteristic curves analysis, HE₄ had the highest specificity as a single marker in detecting ovarian malignancy (1), followed by SMRP(97.92%), then CA₁₂₅(89.58%). CA₁₂₅ had the highest sensitivity as a single marker(90.91%), followed by HE₄(86.36%), then SMRP(77.27%).9 In Kappa consistency check, the Kappa value of HE₄ was 0.897(P=0.000), the Kappa value of SMRP was 0.791(P=0.000), the Kappa value of CA₁₂₅ was 0.776(P=0.000).10 The combination in parallel of CA₁₂₅ and HE₄ or SMRP gave the highest sensitivity in detecting ovarian canrcinoma(95.45%), the combination in parallel of SMRP and HE₄ gave the highest specificity (97.92%), addition of CA₁₂₅ to this combination did not show any improvement in the specificity. The optional combination in series of the three markers gave the highest specificity(1).Conclusion:1 SMRP levels were significantly higher in patients with EOC than in patients with benign epithelial ovarian tumors and healthy volunteers, SMRP level in serous carcinoma was higher than in mucous carcinoma significantly. Following grades decreased and stages increased, SMRP levels became higher and higher.2 HE₄ levels were significantly higher in patients with EOC than in patients with benign epithelial ovarian tumors and healthy volunteers, HE₄ level in serous carcinoma was higher than in mucous carcinoma significantly. Following grades decreased and stages increased, HE₄ levels became higher and higher.3 Serum SMRP and HE₄ levels decreased significantly postoperatively, which were potential markers for prognosis in EOC.4 As a single marker, serum SMRP, HE₄ and CA₁₂₅ gave higher specificity. At the aspect of consistency with clinical diagnosis, HE₄ was superior to SMRP and CA₁₂₅ significantly, SMRP was nearly equal or superior to CA₁₂₅ slightly. The combination of CA₁₂₅ and HE₄ or SMRP improved sensitivity and specificity, the combination of the three markers was not in need. SMRP and HE₄ is shown to improve the diagnostic and prognostic performance of CA₁₂₅.Partâ…¡siRNA-mediated MSLN silencing can reverse cisplatin resistance in epithelial ovarian cancer Objective: To detect mesothelin expression in EOC resistant cell lines, observe the sensitivity of resistant cell lines to cisplatin after MSLN-silencing, evaluate if MSLN-silencing can reverse cisplatin resistance, investigate its possible mechanism of action in cisplatin resistance and deepen comprehension for resistant mechanism of cisplatin in EOC.Methods: SKOV₃ and 3AO cell lines and corresponding resistant cell lines SKOV₃/cDDP and 3AO/cDDP were cultivated. 50% inhibiting concentration (IC50) of the cells were determined by MTT assay, resistance index(RI) was calculated using a formula: RI= IC50resistance /IC50parent. Mesothelin expression in four cell lines was inspected from gene level and protein level through real-time fluorescence quantitative PCR and Western Blot technique. Mesothelin expression in SKOV₃/cDDP was silenced using RNA interference, and stably transfected cell lines were set up, which were divided into three groups: interference group (SKOV₃/cDDP-LV-MSLN-RNAi cells), negative group (SKOV₃/cDDP-LV-MSLN-neg cells) and blank group (SKOV₃/cDDP cells). Test groups was made up of three cell lines treated with cisplatin respectively, control groups was consist of three cell lines untreated with cisplatin. Cell viability was determined by MTT assay, cells were treated with increasing concentration of cisplatin for 48h, inhibition rate was calculated using the equation as following: Inhibition rate(%)=(1-ODtest/ODcontrol)×100%, IC50 was calculated using SPSS 13.0 software. Colony inhibition rate following treated with cisplatin was determined by colony formation test, colony inhibition rate(%)=(1-clonetest /clonecontrol)×100%. Cell apoptosis rate treated with cisplatin for 48h was determined by flow cytometry, cell apoptosis rate induced by cisplatin=apoptosis ratetest-apoptosis ratecontrol. Expression of P-gp and GST-Ï€in the three cell lines was inspected using Western Blot technique. Intra-cellular GSH level was determined by colorimetric technique.Results:1 The resistance index of SKOV₃/cDDP and 3AO/cDDP was 4.5, 4.2 respectively, which belonged to low grade resistance. 2 In the results of real-time fluorescent quantitation PCR, RQ-values of SKOV₃ cells, SKOV₃/cDDP cells, 3AO cells and 3AO/cDDP cells were 1.228±0.259, 1.933±0.252, 0.972±0.139 and 1.595±0.106, differences in them were statistically significant (F=26.403,P=0.000). Among them, RQ-value of SKOV₃/cDDP was higher than that of SKOV₃ significantly(P=0.000), RQ-value of 3AO/cDDP was higher than that of 3AO significantly(P=0.000), RQ-value of SKOV₃/cDDP was higher than that of 3AO/cDDP significantly (P=0.008), RQ-value of SKOV₃/cDDP was higher than that of 3AO/cDDP significantly(P=0.039).3 In the results of MSLN expression, the protein levels of 3AO, 3AO/cDDP, SKOV₃ and SKOV₃/cDDP were 0.115±0.078, 0.655±0.121, 0.309±0.130 and 0.902±0.137, there were significant difference in them (F=61.511,P=0.000), among them, protein level of SKOV₃/cDDP was higher than that of SKOV₃ significantly(P=0.000), protein level of 3AO/cDDP was higher than that of 3AO significantly(P=0.000), protein level of SKOV₃/cDDP was higher than that of 3AO/cDDP significantly (P=0.000), protein level of SKOV₃/cDDP was higher than that of 3AO/cDDP significantly(P=0.000).4 SKOV₃/cDDP cells were transfected by MSLN-silencing lentivirus effectively, its transfection rate was up to 80-90% and interference rate was 95.72 %.5 After three cell lines in test groups was exposed to cisplatin for 48h, inhibition rate of each group cells increased following cisplatin concentration raised, among them, cell inhibition rate in interference group increased more significantly than in other two groups. IC50 of blank group, negative group and interference group were (5.365±0.259)mg/L, (5.248±0.422)mg/L and (2.661±0.158) mg/L respectively, there were significant difference in them (F=77.851, P=0.000). Among them, IC50 of interference group was lower than that of other two groups(P=0.000), there was no significant difference in IC50 between blank group and negative group(P=0.651). The sensitivity of interference group cells to cisplatin raised 2.01-fold.6 Colony inhibition rate in blank group, negative group and interference group were (44.683±3.767)%, (45.488±3.303)%, (64.56±2.904)% respectively, there was significant difference in them (F=67.935, P=0.000), Colony inhibition rate in interference group was higher than in other two groups significantly(P<0.05), there was no significant difference in colony inhibition rate between negative group and blank group(P=0.683).7 Apoptosis rate induced by cisplatin of blank group, negative group and interference group were (19.667±5.417)%,(18.733±7.057)%,(39.867±5.463) % respectively, there was significant difference in them(F=11.774,P=0.008). Apoptosis rate induced by cisplatin in interference group was significantly higher than in other two groups(P<0.05), there was no significant differenc in apoptosis rate induced by cisplatin between negative group and blank group (P=0.850).8 The protein level of P-gp in blank group, negative group and interference group were 0.868±0.153, 0.796±0.105, 0.764±0.103, respectively, there was no significant difference in them(F=1.322,P=0.291).9 The protein level of GST-Ï€in blank group, negative group and interference group were 1.266±0.137, 1.248±0.135, 0.485±0.163 respectively, there was significant difference in them (F=65.706, P=0.000). The GST-Ï€protein level in interference group was lower than in other two groups (P=0.000), there was no significant difference between negative group and blank group(P=0.820).10 The GSH level in blank group, negative group and interference group were (45.993±5.726) nmol/mg, (44.971±3.824) nmol/mg, (24.743±4.659) nmol/mg, there was significant difference in them (F=49.876, P=0.000). The GSH level in interference group was lower than in other two groups (P=0.000). There was no significant difference between negative group and blank group (P=0.674).Conclusion:1 It is confirmed that the MSLN expression in resistant cell lines is higher than in sensitive cell lines from gene level and protein level.2 MSLN expression in serous carcinoma cell lines is higher than in mucous carcinoma cell lines. 3 The sensitivity of resistant ovarian cancer cell lines to cisplatin is enhanced obviously after MSLN-silencing, MSLN can reverse cisplatin resistance in some degree.4 The possible mechanism on MSLN-silencing reversing cisplatin resistance: MSLN-silence can down-regulated the expression of GST-Ï€protein and cut the intra-cellular GSH level down, which makes combination of GSH and cisplatin reduce and effect of cisplatin enhance, finally cisplatin resistance is reversed.5 The mechanism of action in MSLN-silencing reversing cisplatin resistance was complex, it is probably that many factors participate the process.Partâ…¢Treatment effect of siRNA-mediated MSLN-silencing combined with cisplatin on nude mice xenografts bearing human ovarian cancer resistant cellsObjective: To determine the sensitivity of nude mice xenografts to cisplatin after MSLN silencing, clarify the role of MSLN in ovarian cancer cisplatin resistance in vivo.Methods: Nude mice xenograft model bearing human ovarian cancer resistant cells was set up. SKOV₃/cDDP cells were inoculated into the abdominal cavity of nude mice, then nude mice were treated by lentivirus solution with MSLN siRNA and cisplatin and divided into six groups: interference group, interference+cDDP group, negative group, negative +cDDP group, blank group and cDDP group. The general status and life span were compared among the six groups. The numbers and weights of xenografts were compared among the six groups after dissecting the nude mice, the apoptosis rates of xenografts were detected by flow cytometry, the interference efficiency of lentivirus was inspected.Results:1 The numbers and weights of xenografts in interference group were lower than in negative group and blank group significantly (P<0.05), the numbers and weights of xenografts in interference+cDDP group were lowest, which were lower than other five groups significantly(P<0.05), there was no significant difference in numbers and weights of xenografts in negative group and blank group (P>0.05).2 The nude mice in interference group, negative group, negative +cDDP group, blank group and cDDP group died totally within 30 days after inoculating tumor cells, their median survival time was 28 days, 26days, 27days, 25 days and 25 days respectively, among them, the median survival time of interference group was longer than other four groups significantly (P<0.05). The median survival time of interference+cDDP group was 38 days, which was longer than other five groups significantly (P=0.000).3 The MSLN protein levels in interference group, interference+cDDP group, negative group, negative+cDDP group, blank group and cDDP group were 0.115, 0.121, 0.855, 0.864, 0.876, 0.869 respectively, the interference efficiency in interference group and interference+cDDP group was 86.87% and 86.08%, respectively.4 In the results of flow cytometry, apoptosis rate in interference group was higher than in negative group and blank group (P=0.000), apoptosis rate in interference+cDDP group was higher than in negative+cDDP group and cDDP group (P=0.000), there was no significant difference in apoptosis rate between negative group and blank group (P=0.907), between negative+cDDP group and cDDP group (P=0.837). In apoptosis rate induced by cisplatin simply, the interference group (40.96±4.746)% was higher than negative group (19.88±4.356)% and blank group(19.26±4.746)% significantly (P=0.000).Conclusion:1 The MSLN on xenografts tissues were silenced by lentivirus with MSLN siRNA successfully, MSLN-silencing can inhibit the growth of tumor cells, enhance the apoptosis of tumor cells and make the growth velocity of tumor slow.2 Cisplatin has almost no treatment effect on cisplatin resistant xenografts, the apoptosis rate is low. Combined with MSLN-silence, its anti-neoplasms effect not only enhance obviously but also exceed the sum of them. It is elucidated that MSLN-silencing can enhance the sensitivity of xenograft tissues to cisplatin and increase the cytotoxic effect of cisplatin.