The Effect Of Mesothelin On Growth Characteristic Of Human Epithelial Ovarian Cancer And Transplanted Tumor Of Nude Mice

Objective:Most (90%) ovarian cancers are epithelial in origin . There are two unique features of ovarian cancers. irst:it is difficult for early diagnosis and 75% of patients are diagnosed at an advanced stage.The five-year survival rate of patients in advanced stage is only 20%, while in early stage is 85%-90%[1]. The second: ovarian cancer has a unique pattern of progression. The other solid tumors metastasize to distant place by hematological system and lymphatic system, but ovarian cance is limited in abdominal cavity for a long time and the cancer cells adhere and plant in peritoneal mesothelium cells to form metastasis. Finally, the metastasis invasion into the adjacent tissues, such as the epiploon, gastrointestinal tract, liver or spleen. The patients have to be faced with death.Thus, the need to diagnose early and control the generally metastasis of advanced stage cases for treatment of ovarian cancer is self-evident. Mesothelin (MSLN) is a new tumor marker that is closely related with the diagnosis and pathogenetic condition of ovarian cance. Our study group had previously reported that human ovarian cancer contains high levels of MSLN. The previously study also demonstrated that there was significant difference between SKOV3- MLSN-cDNA and SKOV3-MLSN-shRNA, the mesothelin gene of which was up-regulated and down-regulated in adhesion, migration and invasion. MSLN is a glycoprotein that can be shed into the serum and body fluid(peritoneal fluid)and can specifically bind to CA125. Although it is the main composition of microenvironment in abdominal cavity, there is little information on the pathogenesis of MSLN, which is in microenvironment, on the growth, apoptosis and metastasis of ovarian cancer cells. The purpose of my study:1. To investigate the role of mesothelin (MSLN) in growth, metastasis and invasion ability of ovarian cancer cell lines SKOV3. 2. To investigate the effect of mesothelin on anti- Anoikis.3. To investigate the effect of mesothelin on the rate of formed tumor, tumor weight and cell cycle in transplanted tumors of nude mice.Methods:1 Previously, Raffit Hassan study group had reported that 20 of the 21 ovarian cancer patients had serum mesothelin levels between 9 and 49ng/ml, and one patient had a mesothelin level >50 ng/ml. Thus, my preliminary experiment cultured ovarian cancer cell lines SKOV3 in RPMI 1640, mixture containing 10% fetal bovine serum (FBS) and 50ng/ml mesothelin. While, the control group was cultured in RPMI 1640, which contained 10% fetal bovine serum (FBS) and 0ng/ml mesothelin. MTT assay was used to evaluate the proliferation ability of the two groups. The result was that there was no statistical difference between the two group. Then, the ovarian cancer cell line SKOV3 was cultured and incubated with MSLN at different concentration -100ng/ml, 150ng/ml, 200ng/ml, 250ng/ml and 300ng/ml for 12, 24, 36 and 48 h. The control group was incubated with PBS instead of MSLN. The effect of MSLN on cell proliferation was detected by MTT assay. Finally, my study found that the lowest and effective concentration of MSLN on ovarian cancer cell line SKOV3 was 200ng/ml. The best action time was 36 h.2 Plating clone forming test was used to furtherly investigate the effect of 200ng/ml concentration MSLN on ovarian cancer cell line SKOV3 .3 The Matrigel as an adhesion medium was used to investigate wethter 200mg/ml MSLN stimulated adhesion of ovarian cacer cells SKOV3. The rate of adhesion was detected by MTT assay.4 The invasion and migration ability of ovarian cancer cells cultured in 200ng/ml MSLN was evaluated by the Transwell chamber test in vitro .5 Anchorage-independent growth was assessed by monitoring colony formation in soft agar.Suspension culture was used to simulate Anoikis and using flow cytometer (FCM) determined apoptosis rate. To use Western Blot detected the expression of proapoptotic protein Bim.6 My study was designed to investigate effect of MSLN on growth of ovarian cancer cells SKOV3 Xenograft in BALB/c nude mice. To observe the rate of forming tumor, tumor weight, the number of transplanted tumor and the large diameter of the transplanted tumor. Then, the transplanted tumor was detected cell cycle by FCM.6.1 Ovarian cancer cells SKOV3 Xenograft in BALB/c nude mice was established.Femal BALB/c nude mice, 4-6weeks old and 13.5-16.5g weight, live in cleaning environment (SPF). The nude mice was made in two groups in random.It was: Test group A: they were injected with SKOV3 cell, Test group B: they were injected with SKOV3 cells cultured in RPMI 1640 contained 200ng/mL MSLN protein for 36h.Every group contained five nude mice. Two types cell suspension were collected and were injected at the same time. The concentration of every type cell suspension was 2×107/ml. Every nude mouse was injected 0.2ml.6.2 To investigate the correlated indexThe experiment observed life habit of the nude mice for two weeks.The nude mice was executed after two weeks. To compare the rate of forming tumor, tumor weight, the number of transplanted tumor and the large diameter of the transplanted tumor between the two groups. Then, the transplanted tumor was detected cell cycle by FCM.7 The statistical analysis of the data was done using SSPS version 13.0 for windows. A p value less than 0.05 resulting from a two-sided test was considered as statistically significant.Results:1 Mesothelin may promote the growth ability of ovarian cancer cells. The lowest and effective concentration of MSLN on ovarian cancer cell line SKOV3 was 200ng/ml. The best action time was 36 h.2 The clone formation rate of ovarian cancer lines SKOV3 which was cultured in RPMI 1640 contained 200ng/ml MSLN protein was more increased than the control group.3 The adhesion rate of control group (the ovarian cancer cells SKOV3 cultured in RPMI 1640 mixture no MSLN ) was (37.83±3.67) %. The test group(the ovarian cancer cells SKOV3 cultured in RPMI 1640 mixture 200ng/ml MSLN) was (70.75±4.22) %. There was statistical difference between the test and control group (P<0.05).4 The counts of the control group cells that migrated in the under of the filter membrane was (50.33±4.72) and the test group(the ovarian cancer cells SKOV3 cultured in RPMI 1640 mixture 200ng/ml MSLN) was (71.25±6.45). There was statistical difference between the test and control group (P<0.05).5 The counts of the control group cells that invaded in the under of the filter membrane was (19.67±2.57) and the test group(the ovarian cancer cells SKOV3 cultured in RPMI 1640 mixture 200ng/ml MSLN)was (32.08±3.02). There was statistical difference between the test and control group (P<0.05).6 The soft clone formation test: The ability of clone formation could significantly elevate when the culture medium contained 200ng/ml MSLN protein. There was statistical difference between the test and control group (P<0.01). Suspension culture was used to simulate Anoikis: The apoptosis rate of the test group SKOV3 cells was lower than the control group. There was statistical difference between the two groups (P<0.05). The apoptosis rate of the test group SKOV3 was (23.89±3.48)%, while when increased the concentration of MSLN protein to 200ng/ml in the culture medium,the rate was decreased to (12.11±2.7)%.7 When inoculated SKOV3 cells after 5 days, the nude mice of B group (the nude mice injected SKOV3 cells cultured in RPMI 1640 contained 200ng/ml MSLN protein for 36h) started to act slowly.Although the group A (the nude mice injected SKOV3 cells cultured in RPMI 1640 contained no MSLN protein for 36h ) started to appearance like group B after 8 days. After 12 days, there was one nude mice naturally death in group B.The others were alive to the 14 days. For the every index: There was statistical difference between the group A and B (P<0.05). There was no statistical difference between the group A and B in the large diameter of the transplanted tumor (P>0.05). The result of FCM: The cell population of the test group B in S cell stage was significantly more then the control group. The cell population of the test group B in G1 cell stage was significantly lower than the control group. There was statistics difference between the two groups (p<0.05).Conclusion:1 MSLN may stimulate proliferation, adhesion, migration, and invasion of ovarian cancer cells SKOV3 in vitro.2 MSLN enabled ovarian cancer cells SKOV3 to survive under anchorage-independent conditions by suppressing Bim. SKOV3 cells cultured in high levels of MSLN exhibited resistance to anoikis.3 MSLN may enhance the growth and metastasis of the transpalnted tumor in nude mice abdominal cavity.MSLN may accelerate the cell cycle of the xenograft tumor in nude mice.