| BackgroundEpithelial ovarian cancer(EOC)is the most lethal gynecological genital tract cancer,with more than 240,000 cases emerged and 140,000 deaths per year worldwide[I].In China,approximately 52.1 new patients per 100,000 individuals were diagnosed with ovarian cancer in 2015,with 22.5 annual deaths per 100,000 beings due to this malignancy alone[2].The standard treatment for advanced EOC is primary debulking surgery followed by the first-line platinum-and taxane-based chemotherapy,with the great efforts and based on various clinical research conducted by the gynecologic oncologists in the whole world.However,patients with EOC have extremely poor prognoses,with 45%of patients dying in the following 5 years.To patients with advanced disease,the 5 years overall survival(OS)is only 25%[3]There are three reasons for the high mortality of EOC,the first reason is that there is no specific symptoms when patients is in the early stage,70%of patients has been diagnosed with peritoneum metastasis[4].The second reason is that most of paitents with advanced EOC will relapse in the first two years after the standard treatment and the problem is hard to resolve[5]The third reason is that the disease relapse from time to time and the drug resistance have no effective treatment regimen,which causes patients death by multi-drug resistance.At present,the treatment problem caused by EOC recurrence and drug-resistance comes into attention from the scientist of basic medicine,however,there is no perfect molecular mechanism to clarify the predicament.The theory of stem cell originated in the 1970s.The researchers in the early study found that there was a "rare" "quiescent" cell type in the hematologic system when hematopoietuc stem cells divided into their downstream differentiation hierarchy.And these cells also has the unique capacity to "self-renew"[6].In these pioneering studies,the theory of cancer stem cell was proposed in the disease of human acute myeloid leukemia(AML).It was found that most subtupes of human AML could be engrafted reliably into immune-deficient mice by transplanting a population of leukemic cells that expressed a combination of surface markers(CD34+CD38")characteristic of normal hematopoietic stem cells and the cancer cells expressing CD34+CD38" was identified as cancer stem cells(CSCs)in patients with AML[7-9].The solid tumor CSCs was firsted found in breast cancer.As few as 100 CD44+CD24-breast cancer cells could initiate tumor growth in a serial xenograft setting,whereas tens of thousands of cells with CD44-CD24+ could not[10].Based on this study,similar studies of other solid tumors,such as brain cancer[11],prostate cancer[12],colon cancer[13],pancreatic cancer[14]and lung cancer[15],rapidly followed.And a series of methods to identify CSCs was established.The CSCs theory is interpreted that a tumor is made up by a small proportion of"self-renew" malignant stem cells and a big group of proliferating cells dividied by CSCs.CSCs have the capacity to undergo asymmetric divisions to recreate a tumor with a small number of cells keeping the character of asymmetric division,chemotherapy resistance and the complete original complex pool of tumor cells in immune-suppressed mice,except the capacity of self-renew and serial passages.And other vast majority of cancer cells will lose the character of stem cells[16].Maligant tumor is considered to be a kind of disease caused by genes out-of-control.CSCs accumulate the mutations which maintain tumor growth and generate heterogeneous cells through the multipotential differentiation[16-17].Therefore CSCs maybe the basic reason of cancer recurrence and drug resistance.The research on ovarian cancer stem cells(OCSCs)is still in the exploratory stage.At first,the scientists developed the methods by identifying side population cells(SP)to find OCSCs.Then detection of cancer cell markers has replace SP as new methods to collect OCSCs.The surface markers like CD24,CD44,CD117 and CD133 are widely investeigated as OCSCs[18-22],while ALDH1 is taken for the most studied cytosolic marker[13].However,Ghese markers are not unique to OCSCs,which are derived from the researches on other solid CSCs.And the enrichment of OCSCs is also not ideal.There are more and more studies disagree with these OCSCs markers,such as a number of studyies reported no correlation of CD44 to be associated with patients' outcome[24].In other experiment,CD 117+ OC cells failed to show increased tumor initation with respect to CD 117' cells[25].And another study found that CD133+cells isolated from primary OC cultures failed to prove more spherogenic or tumorigenic than CD 133-counterpart,and in fact they only displayed a slower proliferation rate[26.27].Therefore,it has a great significance for solving the problem of drug resistance and developing target therapy to find new OCSCs marker and to isolate and identify these markers.Our previous study compared the expression of cell membrane protein of side population(SP)and non SP EOC cell lines by isotope labeling method and quantitative proteomic strategy to screen the surface OCSCs marker-cell cycle protein 50A(CDC50A,or protein-transmembrane protein 30A).It was proved that CDC50A might be the surface marker of OCSCs though the standardized CSCs identification of CDC50A positive cells(cells lines and primary cancer cells)in vivo and in vitro.Based on the previous study,we would like to do the research on the specificity of CDC50A as OCSCs in the aspects of chemotherapy resistance,tumor metastasis and cancer recurrence in EOC cell lines and primary cancer cells.And combined with the clinical and prognostic data of patients,we aimed to find its pracrical value.Finally,the mechanism of CDC50A will be studied preliminarily.Methods1.The comparison of cell morphology of SKOV3,A2780,GROV1,OVCAR3 and ES2 was performed by Giemsa staining.The cell growth curve of those five cell lines was sketching by cell counting in 7 days.Cell cycles of those cell lines were analyzed by flow cytometry.The half maximal inhibitory concentration of the five cell lines was compared with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium(MTT)method.Those cell lines were stained by antibody CDC50A,CD44,CD117,CD133 and E-cadherin and analyzed by flow cytometry to understand the expression level of OSCSs candidate markers in different cell lines.2.Forty-eight cases of primary EOC tissues were collected with 15 patients having no neoadjuvent chemotherapy(NAC),17 have beening received NAC and 16 relapsed.After separating and purificating EOC cells,we analyzed the expression difference of CDC50A,CD44,CD117,CD133 and E-cadherin in patients with or without NAC,receiving initial treatment or relapsed and from the primary site or the metastatic site.We would like to know the relationship between those markers and chemotherapy,caner recurrence and metastasis.3.Forty-eight cases of primary EOC tissues and clinical prognostic data were collected.The expression difference of CDC50A,CD44,CD 117,CD 133 and E-cadherin were analyzed by flow cytometry.We would like to know the relationship between those markers and the prognosis of EOC patients.4.To investigate the ability of CDC50A in the process of epithelial mesenchymal transition(EMT)by sphere formation and serial passage test.And we would like to find the role of CDC50A in the process of cancer invasion or metastasis.5.We screened the P4-ATPase family genes closely related to CDC50A by PCR technology to lay the foundation for further research on CDC50A mechanism.Results1.The morphology of SKOV3,A2780,GROV1,OVCAR3 and ES2 were short or long spindle shaped,some were polygonal and adhering to the walls under the light microscope.The population doubling time(PDT)of cell line IGROV1 is shortest for 36.2 hours,while ES2 is longest for 44.8 hours.And PDT of the other three cell lines were between those two.Cell cycle G0/G1 period of OVCAR3 is the shortest accounted for 42.49%of the entire cell cycle,while that of ES2 is the longest accounted for 80.33%.And G0/G1 period of the other three cell lines were between those two.Half maximal inhibitory concentration(IC50)of ES2 was the largest with 0.91 uM under the action of paclitaxel and 28.97uM of cisplatin.And IC50 of other four cell lines were less than that of ES2,and it showed more obvious while cisplatin treated.The expression of five OCSCs candidate markers in five cell lines were as follow:1.1-1.6%of CDC50A+,0.29%-100%of CD44+,0.1%-4.9%of CD117+,0.04%-14%of CD133+ and 0.34%-99%of E-cadherin-.The expression of CDC50A+is low and stable while that of other markers relatively fluctuated.2.The median of five OCSCs candidate markers in primary cells were as follow:7.05%(0.22-67.20%)of CDC50A+,8.71%(0.03-80.90%)of CD133+,0.175%(0-12.00%)of CD117+,42.6%(0.10-98.20%)of CD44+ and 52.3%(9.37-99.30%)of E-cadherin-.T test showed that the mean of CDC50A+ expression in patients without NAC was 7.75%while that in patients with NAC was 18.09%,and the result was statistically significant(P=0.02).Other four OCSCs candidate markers showed difference between patients with or without NAC,however,the result was not statistically significant(P>0.05).The expression of five OCSCs candidate markers have some difference between patients with relapse disease or received initial treatment.The mean of CDC50A+,CD117+,CD44+ and E-cadherin-expression in patients with initial treatment is less than patients with relapse disease,instead,the result is opposite in CDC133+ cells.However,there was no statistical difference(P>0.05).The mean of CDC50A+expression in the primary site was higher than that in the metastatic site,and the result was statistically significant(P=0.04).Other four OCSCs candidate markers showed difference between primary site and metastatic site without trend,and the result was not statistically significant(P>0.05).3.The median age of 48 EOC patients was 50 years(21-72 years),and 43 patients(89.6%)were in stage III-IV.Forty patients(83.3%)had papillary serous histology.Logistic regression analysis showed that there were 20 patients with chemotherapy-sensitive disease and 4 with chemotherapy-resistance disease when CDC50A+expression less than or equal 7.05%,while it showed that there were 14 patients with chemotherapy-sensitive disease and 10 with chemotherapy-resistance disease when CDC50A+ expression mores than 7.05%.Although the result was not statistically significant(P=0.064),it showed there was a tread of chemotherapy resistance in patients with CDC50A+>7.05%.In the univariate Kaplan-Meier analysis,the factors significantly(P<0.05)associated with progress-free survival(PFS)were pathological type,residual disease and the expression difference of CDC50A+.Residual disease,the expression difference of CDC50A+ and CD 117+were significantly(P<0.05)associated with overall survival(OS).Cox regression showed that,pathological type,residual disease and the expression difference of CDC50A+ were the independent prognostic factor to predict PFS.The patients with no papillary serous histology had a two-year PFS rates of 62.5%,as compared with 22.0%for patients with papillary serous histology(P=0.023,HR 0.321,95%CI 0.121-0.856).The patients with visible residual disease(VRD)had a two-year PFS rates of 12.7%,which was significantly lower than 40.7%in patients with no visible residual disease(NVRD)(P=0.010,HR 2.381,95%CI 1.227-4.618).The patients with CDC50A+ ?7.05%had a two-year PFS rates of 36.7%,which was significantly higher than 20.8%in patients with CDC50A+>7.05%(P=0.008,HR 0.418,95%CI 0.219-0.798).Meanwhile,the expression difference of CDC50A+ were the independent prognostic factor to predict OS.Patients with CDC50A+ ?7.05%had 3-year OS rates of 84.8%,as compared to 37.0%for patients with CDC50A+>7.05%(P=0.004,HR 0.030,95%CI 0.003-0.318).4.In 38 cases of primary ovarian cancer cells,the overall expression of E-cadherin-cells was higher than that of CDC50A+E-cadherin-cells,and the results were statistically significant(P=0.0008).In the experiments of sphere formation and serial passage test of ovarian cancer cell lines and primary cells,we found that CDC50A+E-cadherin+ cells can form the most spheres and could have more than three passages,while E-cadherin-cells form the less spheres and could not have more serial passages.The percentage of E-cadherin+N-cadherin+ cells in CDC50A+ cells was higher than that in CDC50A' cells,and the results were statistically significant(P=0.034).E-cadherin+N-cadherin+ cells can form the most spheres,which suggest that ovarian cancer stem cells have a "partial EMT" phenotype.5.In three samples,ATP11A and ATP11B were all positive expression,suggesting that CDC50A may be most closely related to the above two genes.Conclusion1.Among the EOC cells lines SKOV3,A2780,GROV1,OVCAR3 and ES2,ES2 is consistent with the characteristics of ovarian clear cell for it having the longest population double time,the highest rate of G0/G1 stage and the most severe drug resistance of paclitaxel and cisplatin.The expression of CDC50A+ cells is between 1.1%and 1.6%in the five cell lines,which is the best ratio of cancer stem cells.2.The expression of CDC50A+ cells is higher in EOC patients with neoadjuvant chemotherapy(NAC)than that without NAC.And CDC50A positive cells expressed higher in tumor metastatic sites than in the primary site,which showed CDC50A+have the property of CSCs.Otherwise,the other four OCSCs candidate markers CD133+,CD117+,CD44+ and E-cadherin-have not been found possessed the characteristics above.3.The difference of CDC50A expression,pathological type and residual disease are the independent prognostic factors of progression free survival(PFS)in patients with EOC.And the difference of CDC50A expression is the only independent prognostic factors of overall survival(OS).4.OCSCs has a "partial EMT" phenotype,which may be related to the differentiation of cancer stem cells in invasion and metastasis.5.The expression level of ATP11A and ATP11B were the highest in CDC50A+cells.It is the foundation for further research on mechanism. |
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