Exploration To The Roles Of Survivin On Biological Behaviors Of Metastatic Ovarian Cancer Originated From Gastric Carcinoma And Primary Ovarian Cancer

The ovarian cancer is the most common malignant tumor in the gynecologic oncology, which include two types: primary ovarian cancer and metastatic ovarian cancer. The epithelial ovarian cancer is the most common type of primary ovarian cancer ,and it is above 90% of ovarian cancer . On the same time , Ovary is the target organ of metastasis originated from many malignant tumor , and carcinoma of gastrointestinal tract is the most common primary lesion, especially gastric poorly differentiated carcinoma .Up to now, surgery is the main method, but most of patients, who had already undergone radical surgery and chemotherapy, still have poor prognosis because of the tumor's metastasis and recurrence. Five-year survival rate is only 20-30%. There is an problem to differentially diagnose primary ovarian cancer and metastatic ovarian cancer, especially the patients whose first clinical symptoms were metastatic ovarian cancer.The molecular mechanisms involved in the progression of ovarian cancer are unclear, which has hampered the development of an effective prevention and treatment.Now, it become more important to explore the mechanisms.Survivin, a new member of inhibitor of apoptosis protein family, could regulate cell proliferation and cell apoptosis. It has been reported that survivin plays an important role in carcinogenesis, but little is known about the relationship among survivin,metastatic and primary ovarian cancer,neither the roles of survivin on biological behaviors of metastatic and primary ovarian cancer cells. Our experiments investigated the expression of survivin and relatively apoptotic proteins (VEGF,Smac/DIABLO)in normal ovarian epithelium, benign and epithelial ovarian tumor, metastatic ovarian cancer originated from gastric carcinoma, primary and epithelial ovarian cancer. Subsequently, MTT, flow cytometry, RT-PCR, magrition and matrigel invasion assay were applied to explore the roles of survivin on proliferation, apoptosis, migration, invasion, expression of VEGF,Smac/DIABLO in SKOV3 cells (epithelial ovarian cancer cells)and BGC-823 cells(gastric poorly differentiated adenocarcinoma cells). Antisense oligonucleotides aimed to Survivin (Survivin ASODN) which could knock down survivin was used, and the changes of proliferation, apoptosis, migration, invasion, expression of VEGF,Smac/DIABLO in SKOV3 cells and BGC-823 cells were detected in order to analyze the relationship between Survivin and biological behaviors of epithelial ovarian cancer cell and gastric poorly differentiated adenocarcinoma cell, and explore the value and mechanism of Survivin ASODN on treatment.The results of our experiments suggest that abnormal expression of survivin is one of the important factors that affect biological behaviors of metastatic ovarian cancer originated from gastric carcinoma and primary ovarian cancer. Interaction between Survivin and VEGF,Smac/DIABLO might be one of the important factors that affect origins and progression of primary and metastatic ovarian cancer; Survivin ASODN could block origins,progression of primary and metastatic ovarian cancer and might play an important role on treatment.The results are as follows:Part one: Expression and value of survivin,VEGF,Smac/DIABLO in metastatic ovarian cancer originated from gastric carcinomaObjective: To investigate the expressions of Survivin,VEGF,Smac/DIABLO and their correlation in metastatic ovarian cancer originated from gastric carcinoma , and the relationship with origins and development of metastatic ovarian cancer originated from gastric carcinoma.Methods: Immunochemistry and flow cytometry were used to investigate the expressions of Survivin,VEGF,Smac/DIABLO proteins in normal ovarian epithelium,benign and epithelial ovarian tumor,metastatic ovarian cancer originated from gastric carcinoma,primary lesion of gastric carcinoma, analyzed their relationship with clinicobiological characteristics.Results:1 Expression of Survivin,VEGF,Smac/DIABLO in normal ovarian epithelium, benign and epithelial ovarian tumor,metastatic ovarian cancer originated from gastric carcinoma,primary lesion of gastric carcinoma Expression of Survivin,VEGF in normal ovarian epithelium and benign ovarian tumor was negtive or weak, expression of Smac/DIABLO in normal ovarian epithelium is strong. Expression of Survivin,VEGF in metastatic ovarian cancer originated from gastric carcinoma became stronger, and expression of Smac/DIABLO in metastatic ovarian cancer originated from gastric carcinoma became weaker((vs normal ovarian epithelium and benign ovarian tumor P<0.05 or P<0.01). Positive expression of survivin (in nucleus and in cytoplasm),VEGF,Smac/DIABLO were(100.0%,90.3%),77.4%,90.3%; FI were 1.10±0.09,0.84±0.08,1.12±0.11. Expression of cytoplasmic Survivin was higher in metastatic ovarian cancer originated from gastric carcinoma than in primary lesion of gastric carcinoma(P<0.05).2 Correlation of Survivin,VEGF,Smac/DIABLO in metastatic ovarian cancer originated from gastric carcinomaThere was no correlation between nuclear and cytoplasmic Survivin(P>0.05);there was positive correlation between cytoplasmic Survivin and VEGF(r=0.397,P<0.05);there was negative correlation between cytoplasmic Survivin and Smac/DIABLO(r=﹣0.373,P<0.05);there was no correlation between Smac/DIABLO and VEGF(P>0.05).3 Relationship between Survivin,VEGF,Smac/DIABLO and clinicobiological characteristics (age,diameter,differentiation,ascites) of metastatic ovarian cancer originated from gastric carcinomaExpression of cytoplasmic Survivin was higher in diameter≥5cm group (positive rate100.0%; FI=1.16±0.08)than in diameter <5cm group(positive rate66.7%; FI=1.04±0.05), (P<0.05 or P<0.01). There was no relasionship with other clinicobiological characteristics(P>0.05).Expression of Smac/DIABLO was higher in poorly differentiated group (positive rate94.1%; FI = 0.88±0.03 ) than in moderate group(positive rate57.1%; FI=0.78±0.09), (P<0.05). There was no relasionship with other clinicobiological characteristics(P>0.05).Expression of VEGF was higher in diameter≥5cm group (positive rate100.0%; FI=1.18±0.08)than in diameter <5cm group(positive rate66.7%; FI=1.06±0.11), (P<0.05); Expression of VEGF was higher in poorly differentiated group (FI=1.18±0.07) than in moderate group(FI=1.04±0.10), ( P<0.01 ) . There was no relasionship with other clinicobiological characteristics(P>0.05).Conclusions:1 There was strong expression of Survivin in metastatic ovarian cancer originated from gastric carcinoma; there was positive correlation between cytoplasmic Survivin and VEGF;there was negative correlation between cytoplasmic Survivin and Smac/DIABLO, Survivin,VEGF,Smac/DIABLO might be three important factors involved in growth and metastasis of metastatic ovarian cancer originated from gastric carcinoma.2 Survivin was over expressed in both nucleus and cytoplasm of ovarian cancer, Expression of cytoplasmic Survivin might play a critical role on metastasis from gastric carcinoma to ovary because it was higher in metastatic ovarian cancer originated from gastric carcinoma than in primary lesion of gastric carcinoma.3 Expression of Survivin,VEGF,Smac/DIABLO might diagnose and predict the malignancy of metastatic ovarian cancer originated from gastric carcinoma.Part two: Expression and value of survivin,VEGF,Smac/DIABLO in primary and epithelial ovarian cancerObjective: To investigate the expression of Survivin,VEGF,Smac/DIABLO and their correlation in primary and epithelial ovarian cancer, and the relationship with origins and development of primary and epithelial ovarian cancer.Methods: Immunochemistry and flow cytometry were used to investigate the expression of Survivin,VEGF,Smac/DIABLO proteins in normal ovarian epithelium,benign and epithelial ovarian tumor,primary and epithelial ovarian cancer, analyzed their relationship with clinicobiological characteristics and prognosis.Results:1 Expression of Survivin,VEGF,Smac/DIABLO in normal ovarian epithelium, benign and epithelial ovarian tumor,primary and epithelial ovarian cancerExpression of Survivin,VEGF,Smac/DIABLO in normal ovarian epithelium, benign and epithelial ovarian tumor were same as part one. Expression of Survivin,VEGF in epithelial ovarian cancer became stronger, and expression of Smac/DIABLO in primary and epithelial ovarian cancer became weaker((vs normal ovarian epithelium and benign ovarian tumor P<0.05 or P<0.01). Positive expression of survivin (in nucleus and in cytoplasm),VEGF,Smac/DIABLO were(72.3%,87.2%),80.9%,91.5%; Fluorescence Index(FI) were 1.10±0.02,0.88±0.08,1.16±0.10.2 Correlation of Survivin,VEGF,Smac/DIABLO in primary and epithelial ovarian cancerThere was positive correlation between nuclear and cytoplasmic Survivin(r=0.514,P<0.01);there was positive correlation between cytoplasmic Survivin and VEGF(r=0.298,P<0.05);there was negative correlation between cytoplasmic Survivin and Smac/DIABLO(r=﹣0.312,P<0.05);there was negative correlation between Smac/DIABLO and VEGF(r=﹣0.320,P<0.05).3 Relationship between Survivin,VEGF,Smac/DIABLO and clinicobiological characteristics of primary and epithelial ovarian cancer Expression of cytoplasmic Survivin was higher in stageⅢ～Ⅳgroup (positive rate 96.8%; FI=1.10±0.09)than in stageⅠ～Ⅱgroup(positive rate 68.8%; FI=0.93±0.16), (P<0.05). There was no relasionship with other clinicobiological characteristics(P>0.05).Expression of Smac/DIABLO was lower in stageⅢ～Ⅳgroup (positive rate 71.0%)than in stageⅠ～Ⅱgroup(positive rate100.0%),(P<0.05); Expression of Smac/DIABLO was lower in poorly differentiated group (positive rate 61.9%; FI=0.82±0.07)than in moderate and well group(positive rate 96.2%; FI=0.90±0.07), (P<0.01or P<0.05). There was no relasionship with other clinicobiological characteristics(P>0.05).Expression of VEGF was higher in stageⅢ～Ⅳgroup (FI=1.18±0.09)than in stageⅠ～Ⅱgroup(FI=1.03±0.02), (P<0.05). There was no relasionship with other clinicobiological characteristics(P>0.05).4 Relationship between Survivin,VEGF,Smac/DIABLO and prognosis of primary and epithelial ovarian cancerSurvival rate was lower in positive expression than negative expression of cytoplasmic Survivin (P<0.05);Survival rate was higher in positive expression than negative expression of Smac/DIABLO(P<0.01);positive expression of VEGF had no effect on survival rate of epithelial ovarian cancer(P>0.05). but survival rate was lower in 3+ positive expression than negative,weak,moderate expression of VEGF(P<0.05),and 3+ positive expression of VEGF was the only prognostic factor analyzed by Cox model(P<0.05).Conclusions:1 There was strong expression of Survivin in primary and epithelial ovarian cancer; there was positive correlation between cytoplasmic Survivin and VEGF , there was negative correlation between cytoplasmic Survivin and Smac/DIABLO; Survivin,VEGF,Smac/DIABLO might be three important factors during origins and development of primary and epithelial ovarian cancer, and involved in growth and metastasis of primary and epithelial ovarian cancer.2 Survivin was over expressed in both nucleus and cytoplasm of ovarian cancer, expression of cytoplasmic Survivin might be closely related with origins and development of primary and epithelial ovarian cancer.3 Expression of Survivin,VEGF,Smac/DIABLO could predict the malignancy of primary and epithelial ovarian cancer, and 3+ positive expression of VEGF was the independently prognostic index of primary and epithelial ovarian cancer .Part three: Exploration to different expression of survivin,VEGF,Smac/DIABLO in metastatic ovarian cancer originated from gastric carcinoma and primary ovarian cancerObjective: To investigate the different expression and diagnostic value of Survivin,VEGF,Smac/DIABLO and their correlation in metastatic ovarian cancer originated from gastric carcinoma and primary ovarian cancer.Methods: Immunochemistry and flow cytometry were used to investigate the different expression of Survivin,VEGF,Smac/DIABLO proteins in normal ovarian epithelium,benign and epithelial ovarian tumor,primary and epithelial ovarian cancer,metastatic ovarian cancer originated from gastric carcinoma .Results:Expression of Survivin,VEGF,Smac/DIABLO in normal ovarian epithelium, benign and epithelial ovarian tumor was same as part one. Expression of Survivin,VEGF in metastatic ovarian cancer originated from gastric carcinoma became stronger, and expression of Smac/DIABLO in metastatic ovarian cancer originated from gastric carcinoma became weaker((vs normal ovarian epithelium and benign ovarian tumor(P<0.05 or P<0.01). Positive expression of survivin (in nucleus and in cytoplasm),VEGF,Smac/DIABLO were(100.0%,90.3%),77.4%,90.3%; FI were 1.10±0.09,0.84±0.08,1.12±0.11. Expression of Survivin,VEGF in primary and epithelial ovarian cancer became stronger, and expression of Smac/DIABLO in primary and epithelial ovarian cancer became weaker((vs normal ovarian epithelium and benign ovarian tumor P<0.05 or P<0.01).Positive expression of survivin (in nucleus and in cytoplasm),VEGF,Smac/DIABLO were(72.3%,87.2%),80.9%,91.5%; Fluorescence Index(FI) were 1.10±0.02,0.88±0.08,1.16±0.10.Expression of nuclear survivin was higher in metastatic ovarian cancer originated from gastric carcinoma than in primary and epithelial ovarian cancer(P<0.01); There were no different expression of cytoplasmic Survivin,VEGF,Smac/DIABLO between in metastatic ovarian cancer originated from gastric carcinoma and in primary ovarian cancer(P>0.05).Conclusions:1 Survivin,VEGF,Smac/DIABLO might be three important factors during origins and progression of metastatic ovarian cancer originated from gastric carcinoma and primary ovarian cancer. Strong expression of Survivin,VEGF and low expression of Smac/DIABLO involved in development of them at the same time.2 Expression of nuclear survivin was higer in metastatic ovarian cancer originated from gastric carcinoma than in primary and epithelial ovarian cancer, and it might be a biochemical index in differential diagnosis of them. Part four: The roles of Antisense oligonucleotides aimed to survivin (survivin ASODN) on proliferation, apoptosis, magrition, invasion of SKOV3 cells and BGC-823 cellsObjective: To investigate transfection rate and effect of Survivin ASODN on proliferation, apoptosis, migration, invasion of SKOV3 cells and BGC-823 cellsMethods: Survivin ASODN was synthesized by targeting 232-251 region of Survivin mRNA, and transfection was mediated by LipofectamineTM2000. Transfection rate was detected by scanning confocal microscopy and FCM, and proliferation was detected by MTT, and apoptosis was detected by FCM, magrition and invasion was detected by Transwell chamber.Results:1 For SKOV3 cells , Survivin ASODN(ug):LipofectamineTM2000 (ul)was 1:1.5, transfection rate was above 95% detected by FCM.2 The roles of Survivin ASODN on proliferation,apoptosis,migration,invasion of SKOV3 cells2.1The effect of Survivin ASODN on proliferation of SKOV3 cells There was relationship among inhibition of proliferation,Survivin ASODN dose and time(P<0.05 or P<0.01),different dose of Survivin SODN or LipofectamineTM2000 had no effect on cell proliferation(p>0.05),Survival rate of SKOV3 cells trandected by 600nm/L Survivin ASODN for 48 hours was 49.5±20.76%,which closed to the dose of IC50.2.2 The effect of Survivin ASODN on apoptosis and cell cycle of SKOV3 cellsApoptosis index (AI )of SKOV3 cells was higher in Survivin ASODN group(28.67±3.29%) than in control group(5.48±1.55%),(t=6.367, p <0.05). Cellular proportion in G0/G1 period in Survivin ASODN group(42.7±1%)significantly increased compared with control group ( 30.85±0.55 %) , (t=10.383, p <0.01).2.3 The effect of Survivin ASODN on migration and invasion of SKOV3 cells Migration rate of SKOV3 cells was lower in Survivin ASODN group (57.87±1.34%) than in control group(66.57±2.11%),(t =3.474,p<0.05);The cells number of migration was lower in Survivin ASODN group (115.33±6.51) than in control group(189.67±1.53), (t =19.264,p<0.01).Invasion rate of SKOV3 cells was lower in Survivin ASODN group (14.6±1.21%) than in control group(22.27±0.58%),(t = 5.718,p<0.01);The cells number of invasion was lower in Survivin ASODN group (75.67±1.15) than in control group(113.33±2.08), (t =27.416,p<0.01).3 For BGC-823 cells , Survivin ASODN(ug):LipofectamineTM2000 (ul)was 1:2, transfection rate was above 90% detected by FCM.4 The roles of Survivin ASODN on proliferation,apoptosis,migration,invasion of BGC-823 cells4.1The effect of Survivin ASODN on proliferation of BGC-823 cells There was relationship among inhibition of proliferation,Survivin ASODN dose and time(P<0.05 or P<0.01),different dose of Survivin SODN or LipofectamineTM2000 had no effect on cell proliferation(p>0.05),Survival rate of BGC-823 cells trandected by 600nm/L Survivin ASODN for 48 hours was 46.75±4.99%,which closed to the dose of IC50.4.2 The effect of Survivin ASODN on apoptosis and cell cycle of BGC-823 cells AI of BGC-823 cells was higher in Survivin ASODN group(11.31±0.38%) than in control group(1.62±0.36%),((t=18.512, p <0.01). Cellular proportion in G0/G1 period in Survivin ASODN group(72.25±2.95%)significantly increased compared with control group(56.25±0.75%), (t= 5.257, p <0.05); Cellular proportion in G2/M period in Survivin ASODN group(1.75±0.17%)significantly decreased compared with control group ( 13.4±0.05 %) , (t=67.717, p <0.01)4.3 The effect of Survivin ASODN on migration and invasion of BGC-823 cellsMigration rate of BGC-823 cells was lower in Survivin ASODN group (54.53±2.13%) than in control group(67.97±2.21%)(,t =4.378,p<0.05);The cells number of migration was lower in Survivin ASODN group (74.33±2.08) than in control group(140.67±1.53), (t =44.498,p<0.01).Invasion rate of BGC-823 cells was lower in Survivin ASODN group (19.2±0.66%) than in control group(27.3±0.57%),(t = 9.332, p<0.01);The cells number of invasion was lower in Survivin ASODN group (59.33±5.69) than in control group(97.33±4.04), (t = 9.435,p<0.01).Conclusions:1 Survivin might be closely related to proliferation,apoptosis,migration,invasion of SKOV3 cells and BGC-823 cells .2 Survivin ASODN could significantly inhibit proliferation,apoptosis,migration,invasion of SKOV3 cells and BGC-823 cells , which could treat advanced,metastatic and recurrent cancer.Part five: The roles of Antisense oligonucleotides aimed to survivin (survivin ASODN) on expression of survivin,VEGF,Smac/DIABLO in SKOV3 cells and BGC-823 cellsObjective: To investigate the effect of Survivin ASODN on expression of survivin and relative proteins in SKOV3 cells and BGC-823 cells. Methods: Survivin ASODN was synthesized by targeting 232-251 region of Survivin mRNA, and transfection was mediated by LipofectamineTM2000. Expression of Survivn,VEGF,Smac/DIABLO mRNA in Survivin ASODN group and in control group were detected by RT-PCR, and expression of Survivn,VEGF,Smac/DIABLO protein in Survivin ASODN group and in control group were detected by FCM.Results:1 The effect of Survivin ASODN on expression of Survivn,VEGF,Smac/DIABLO mRNA in SKOV3 cellsIn control group,the ratio of integral optical density(IA) about Survivn,VEGF,Smac/DIABLO mRNA were 0.81±0.07,0.35±0.04,0.59±0.28; in Survivin ASODN group, the ratio of IA about Survivn,VEGF,Smac/DIABLO mRNA were 0.45±0.07,0.23±0.03,1.66±0.26. Expression of Survivn,VEGF mRNA significantly decreased compared with control group(t = 3.503,p<0.05;t =2.539,p<0.05);but expression of Smac/DIABLO mRNA significantly increased compared with control group(t = -2.801,p<0.05).2 The effect of Survivin ASODN on expression of Survivn,VEGF,Smac/DIABLO protein in SKOV3 cellsIn control group, Fluorescence Index(FI) about Survivn,VEGF,Smac/DIABLO protein were 1.00±0.03,1.00±0.03,0.99±0.07; in Survivin ASODN group, FI about Survivn,VEGF,Smac/DIABLO protein were 0.64±0.04,0.59±0.03,1.91±0.03. Expression of Survivn,VEGF protein significantly decreased compared with control group(t = 7.815,p<0.05;t = 33.837,p<0.01);but expression of Smac/DIABLO protein significantly increased compared with control group(t = 11.392,p<0.01).3 The effect of Survivin ASODN on expression of Survivn,VEGF,Smac/DIABLO mRNA in BGC-823 cellsIn control group,the ratio of IA about Survivn,VEGF,Smac/DIABLO mRNA were 0.86±0.05,0.78±0.06,0.56±0.04; in Survivin ASODN group, the ratio of IA about Survivn,VEGF,Smac/DIABLO mRNA were 0.52±0.09,0.52±0.08,0.90±0.11. Expression of Survivn,VEGF mRNA significantly decreased compared with control group(t = 3.294,p<0.05;t = 2.700,p<0.05);but expression of Smac/DIABLO mRNA significantly increased compared with control group(t = -2.832,p<0.05). 4 The effect of Survivin ASODN on expression of Survivn,VEGF,Smac/DIABLO protein in BGC-823 cellsIn control group, FI about Survivn,VEGF,Smac/DIABLO protein were 0.99±0.02,1.00±0.01,1.00±0.05; in Survivin ASODN group, FI about Survivn,VEGF,Smac/DIABLO protein were 0.73±0.01,0.75±0.01,1.64±0.02. Expression of Survivn,VEGF protein significantly decreased compared with control group(t = 10.557 p<0.01;t = 21.651,p<0.01);but expression of Smac/DIABLO protein significantly increased compared with control group(t =11.667,p<0.01).Conclusions: Survivin ASODN could not only knock down expression of survivin straightly ,but also knock down expression of VEGF and up-regulate expression of Smac/DIABLO straightly or instraightly, which might be the mechanism for it to treat advanced,metastatic and recurrent cancer.