Objective To construct the autocatalytic caspase-3 and investigate its apoptosis- inducing effect in ovarian cancer in vitro and in vivo.Methods PCR recombination technique was used to construct autocatalytic caspase-3 which is named as rev-caspase-3,and Ad-Max system was used to prepare recombinant adenovirus containing rev-caspase-3,which is named as Ad-rev-easp3.Immunohistochemistry was used to detect active caspase-3 expression.Cell counting kit,flow cytometry and western blot were used to measure cell survival rate,apoptotic rate,cell cycle distribution and the expressions of plT,active subunit of caspase-3,and p85,the poly(adenosine diphosphate-ribose)polymerase(PARP)cleavage segment,respectively.Transmission electron microscope was used to detect cell ultrastrueture,and real time PCR was used to detect apoptosis-related gene expression.Subcutaneous tumor models and abdominally spread tumor models of human ovarian carcinoma were established using AO cells in BALB/c nude mice. The mouse survival rates were measured for abdominally spread tumor models,and the volume of tumor nodules were determined for subcutaneous tumor models following the treatments of rev-caspase-3.Results Active caspase-3 protein was significantly expressed,and the expression levels of active subunit of caspase- 3,p17,and the PARP cleavage segment,p85,were significantly elevated in cells treated with rev-caspase- 3.The decrease of cell survival rate and the increase of cell apoptotic rate were detected following Ad-rev- casp3 treatment.Treatments with Ad-rev-casp3 [ multiplicity of infection(MOI)was 70 ] resulted in survival rate of 30.3% and apoptotic rate of 40.2%.There was a significant increase in cell number of S- phase(56.5%),while there was no significant apoptosis(3.4%)following treatments with Ad-rev-casp3 at a low dosage of MOI=10.Cells treated with rev-caspase-3 displayed significant apoptotic morphology. The levels of active caspase-3 gene expressions(9.44)significantly increased.Rev-caspase-3 treatment significantly prolonged survival,the mean survival duration was(213?16)days,and suppressed tumor growth(tumor growth suppression rate was 70%),when compared with treatment with phosphate buffered saline(PBS).Conclusion Recombinant adenovirus containing rev-caspase-3 can significantly induce apoptosis of ovarian carcinoma cells,suppress tumor growth and prolong the mouse survival duration.

Objective　To evaluate the ability of a risk of malignancy index (RMI), based on a serum CA125 level, ultrasound findings and menopausal status,to discriminate a benign from a malignant pelvic mass. Methods　One hundred and forty women with a pelvic mass, 30 years or older,admitted between January 1998 and June 1999, were studied. The sensitivity,specificity and positive predictive value of serum CA125 level, ultrasound findings and the menopausal status separately and combined into the RMI,to diagnose ovarian cancer. Results　RMI was more accurate than any individual criterion in diagnosing cancer. Using a RMI cut-off level of 200 to indicate malignancy, the RMI derived from this data set gave a sensitivity of 87.3%, specificity of 84.4%, and positive predictive value of 82.1%. Conclusions　RMI is able to correctly discriminate between malignant and benign pelvic mass. It can be introduced easily into clinical practice to facilitate the selection of patients for primary surgery.

Objective To isolate and identify the aseites-derived exosomes from patients with ovarian epithelial carcinoma,and explore the relationship between exosomes and the prognosis of ovarian epithelial carcinoma.Methods Ascites-derived egosomes were isolated by ultraeentrifugation on sucrose and D2O gradients from 41 ovarian epithelial carcinoma patients,and identified by transmission electron microscope and western blot analyses.Ascites-derived exosomes were evaluated for effect on prognosis of ovarian epithelial carcinoma.Resulls Exosomes were isolated and purified from the ascites in 85% (35/41)of ovarian epithelial carcinoma patients;major histocompability complex-I(MHC-I)could be detected in 100%(35/35)of the aseites-derived exosomes samples,heat shock protein-70(Hsp70)in 91%(32/35),and CD81 in 86%(30/35).The patients with positive ascites-derived exosomes had no significant difference in age,pathological type and the degree of differentiation of tumor,surgical-pathological staging,the optimal operation and the responsibility to chemotherapy(P>0.05).The patients with positive ascites-derived exosomes,the reduction of CA125 level after cytoreduetive surgery was(66±27)%,which was more than that of the patients without ascites-derived exosomes(37±86)%and all the whole patients not considering the condition of exosomes(61±44)%(P<0.01),and there were also no difference whether or no optimal operation ration(P<0.01).Conclusions There are ascites-derived exosomes in ascites from most patients with ovarian epithelial carcinoma.While the relationship between exosomes and prognosis of ovarian epithelial carcinoma needs further research.

Objective To investigate the antitumor effects on ovarian cancer using recombinant adenoviruses expressing autocatalytic caspase-3 driven by amplified human telomerase reverse transcriptase promoter (AdHTVP2G5-rev-casp3) combined with flavopiridol. Methods Following the treatment with AdHTVP2G5-rev-casp3 combined with flavopiridol, cell survival rate was measured by cell counting kit 8;cell apoptotic rate and cell cycle distribution were detected by flow cytometry. Western blot was performed to observe the expression of p17, the active subunit of caspase-3, and p85, the cleavage segment of substrate of caspase-3, in AO cells. The mice survival rates were measured for abdominally metastatic tumor models and the volume of tumor nodules were determined for subcutaneous tumor models following the treatments of AdHTVP2G5-rev-casp3 combined with flavopiridol. HE staining was used to detect the histopathological changes of various organs, and the serum level of alanine transaminase (ALT) and aspartate aminotransferase (AST) were measured to monitor liver damages following the intraperitoneal administration of AdHTVP2G5-rev-casp3 and flavopiridol. Results There was no significant cell-killing effects or apoptosis in AO cells following treatments with AdHTVP2G5-rev-casp3 or flavopiridol at low dosage alone (apoptotic rate all ＜ 11% ), whereas significant synergism of their sequential combination was observed in AO cells. This sequential treatment of AdHTVP2G5-rev-casp3 [multiplicity of infection (MOI) was 20]infection for 72 hours, followed by flavopiridol ( 300 nmol/L) for 48 hours, could result in the most substantial cell death, and AO cells survival rate and apoptotic rate were 73. 5% and 11.6%, respectively.Following treatments with AdHTVP2G5-rev-casp3 at low doses ( MOI = 10), there was a significant increase in cell number with S-phase content ( 62. 5% ), which resulted in the most marked apoptosis induced by sequential treatments with flavopiridol. The sequential combination could induce significantly higher levels of p17 and p85 expression than that when their applications alone. Combined AdHTVP2G5-rev-casp3 and flavopiridol treatment prolonged mouse survival [ mean survival time of ( 286 ± 6) days ] and suppressed tumor growth significantly (tumor growth suppression rate of 81% ), when compared with treatment using either alone. The levels of serum ALT and AST were not significantly elevated and no obvious lesions were found in any organs in treatments with AdHTVP2G5-rev-casp3 of low doses combined with flavopiridol.Conclusions AdHTVP2G5-rev-casp3 at low doses results in a significant increase in cell number with Sphase content, which significantly enhanced the sensitivity of cells to flavopiridol. Treatments of autocatalytic caspase-3 combined at low doses with flavopiridol result in significant synergistic antitumor effects,significant tumor growth suppression and prolonged survival of mice. When compared with normal dose flavopiridol alone, the combination could resulted in minimal liver toxicity.

ObjectiveThe apoptosis and anti-proliferation effects of photodynamic methods on cervical cancer and ovarian cancer cell lines were studied to explore the advantage of photodynamic therapy in gynecological cancer.MethodsCervical cancer cell line (Hela) and ovarian cancer cell line (SK()V3) were treated with photosensitizer (5-ALA) at different doses and then radiated by laser with energy scopes. The morphological characteristics were observed by light microscopy and electronic microscopy. Inhibition effects and cell apoptosis were studied by MTT assay and flow cytometry (FCM) ResultsThe cytomorphosis and shrinkage, nucleus condensation and anachromasia, dense chromatin lining along cell membrane were observed in both cell line receiving photodynamic therapy.Through transmission electron microscope (TEM), we found nucleus chromatin gathering along membrane of cell nuclear and apoptotic body formed. The cell inhibition rates were 12.9％-90.7％ and 3.9 ％-70.0％, respectively in Skov3 cell and HeLa cell treated with 5-ALA of different concentrations (0.1, 0.5, 1.0, 2.5 mol/L) and receiving laser of different doses (0.1,0.5, 2.5, 12.5 J/cm2). The concentration of the photosensitizer and the dose of laser influenced the optical density(A) of cell lines (P＜0.01). The photo sensitizer was related strongly to laser (P＜0.01). The optical density of the two cell lines decreased with the increases of photo sensitizer concentration and laser dose ( P＜0.01 ),showing concentration- and time-dependent manner.Conclusions Photodynamic therapy inhibits the growth of ovarian cancer and cervical cancer cell line.

Objective To construct recombinant adenoviral vector expressing autocatalysis caspase- 3 driven by human telomerase reverse transcriptase promoter amplified by two-step transcription amplification (hTERTp-TSTA),and investigate its antitumor effect in ovarian cancer iri vitro and in vivo.Methods Recombinant adenoviruses expressing autocatalytic caspase-3(rev-caspase-3)driven by hTERTp-TSTA were prepared,which were named as AdHTVP2G5-rev-casp3.AdHT-rev-casp3,Ad-rev-casp3 and AdHTVP2G5- EGEP,which express rev-easpase-3 driven by hTERTp,cytomegalovirus promoter(CMVp)and enhanced green fluorescent protein(EGFP),respectively,were used as controls.Western blot,cell counting kit (CCK-8),flow cytometry(FCM)and TdT-mediated dUTP-biotin nick end labeling(TUNEL)were used to detect the expression of p17,active subunit of caspase-3,and p85,and to measure cell survival rates, apoptotic rates and cell cycle distribution in ovarian cell line AO and normal human umbilical vein endothelial cell line HUVEC,following treatments of AdHTVP2G5-rev-casp3.subcutaneous tumor models and abdominally spread tumor models of human ovarian carcinoma using AO cells in BALB/e nude mice were established.Following treatments of AdHTVP2G5-rev-easp3,western blot was used to detect the expression of active caspase-3 in abdominally spread tumors and liver tissues,respectively,and the mouse survival rates and the volume of tumor nodules were measured,and the serum level of alanine transaminase (ALT)and aspartate transaminase(AST)were analyzed to monitor liver damages and HE staining was used to detect the histopathological changes of various organs.Results The levels of p17 expression in AdHTVP2G5-rev-casp3-treated AO cells were significantly higher than that in Ad-rev-casp3 or AdHT-rev- casp3 treated AO cells,while no expression was observed in AdHTVP2G5-rev-casp3-treated HUVEC.There was strong cell killing of AdHTVP2G5-rev-casp3 of hTERT positive AO cells,but not of the hTERT-negative HUVEC cells.Cell survival rate and apoptotic rate of AO cells treated with AdHTVP2G5-rev-casp3 were 17.8% and 40.2%,respectively,significantly different from that treated with AdHT-rev-casp3(75.2% and 16.1%)at the multiplicity of infection(MOI)of 70(P0.05) .Significant expressions of active caspase-3 were shown in AdHTVP2G5-rev-casp3-treated tumors,whereas no expression was shown in liver.In contrast,both tumors and liver tissues showed active caspase-3 expression following treatments of Ad-rev-casp3.AdHTVP2G5-rev-casp3 and Ad-rev-casp3 prolonged mouse survival[mean survival time of(259?14)d and(213?16)d],when compared with treatment with AdHT- rev-casp3[(177?12)d]and AdHTVP2G5-EGFP[(109?7)d;P

Objective To investigate the antitumor effect of flavopiridol in ovarian cancer. Methods After the treatment with flavopiridol of AO cells,cell apoptotic rate and cell cycle distribution were detected by flow eytometer and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling(TUNEL).Real time PCR was used to detect the expression of cyclin D and active caspase-3 in AO cells.Subcutaneous tumor models and abdominally spread tumor models of human ovarian carcinoma using AO ceils in BALB/c nude mice were established.The mouse survival rates were measured for abdominally spread tumor models and the volume of tumor nodules was determined for subcutaneous tumor models following the treatments of flavopiridol.TUNEL was used to detect cell apoptosis,and immunohistochemistry was used to measure microvessel density(MVD)in tumor tissues. Results AO cells showed apoptotic rates of 4.1%,10.7% and 7.6% following the treatments with flavopiridol at 150,300 and 500 nmol/L respectively,accompanied by an increase in G_1 progression and a decrease in S phase progression.The level of active caspase-3 increased(2.55 vs 2.49)and the level of cyclin D expression decreased significantly(0.25 vs 0.69,P

Objective To compare the clinical and histological features and prognosis of patients with ovarian cancer from different genetic background, and to make further understanding of the genetic model of BRCA genes used pedigree analysis. Methods There were 71 patients from 67 independent families enrolled in our study from Apr. 2000 to Jun. 2009 in Peking Union Medical College Hospital. All exons of BRCA1/2 genes were analyzed using denaturing high-performance liquid chromatography(DHPLC) followed by direct sequencing, and clinical features of patients were compared by statistical analysis. Pedigree analysis of two families with BRCA genes mutation were performed. Results The mutation rate of BRCA genes was 28%(20/71). The frequency of BRCA1 and BRCA2 gene mutation was 23%(16/71) and 6%(4/71), respectively (P=0.004). Histology types of patients with and without BRCA genes mutation were different. The onset age between patients with and without BRCA genes mutation was similar (52.6 versus 54.6 years old, P=0.393), and tend to be early-onset breast or ovarian cancer in high-risk group. There was no significant difference of platinum-resistant rate, disease free survival and overall survival rate between patients with and without BRCA genes mutation (all P>0.05). According to the pedigree analysis, up to 100% of female offspring inherited pathogenic mutations, and male offspring could be a mutation carrier. Conclusions The genetic screening and clinical intervention should be performed as early as possible for the members from families at risk of hereditary ovarian cancer. Genetic consulting is important for patients with high-grade papillary serous adenocarcinoma of ovary. It is still unknown that whether the patients with BRCA gene mutations have better prognosis than sporadic ones, and further perspective, randomized controlled trial is still needed.

Objective To determine the effect of dihydroartiminisin on the proliferation and phosphorylation of mitogen-activated protein kinase (MAPK) in SKOV3 and OVCAR3 ovarian cancer cell lines.Methods Methyl thiazolyl tetrazolium assay was performed to evaluate the anti-proliferative effect of dihydroartiminisin in SKOV3 and OVCAR3 cells,and Western blot was used to determine its effect on phosphorylation level of MAPK,including extra-cell regulated kinase (ERK)1/2 and p38 protein kinase,in the two cell lines.Results Dihydroartiminisin inhibited the proliferation of ovarian cancer cells in vitro,with a mean of 50% inhibition concentration (IC50) at 72 h of (9.0 ±1.4) μmol/L for SKOV3 and (5.5 ±1.2)μmol/L for OVCAR3 respectively. Compared to cells without dihydroartiminisin treatment,phosphorylation level of ERK 1/2 in SKOV3 and OVCAR3 cells treated with dihydroartiminisin decreased by 64.2% and 75.3% respectively (P＜0.05),while phosphorylation of p38 protein kinase in SKOV3 and OVCAR3 only decreased by 8.5% and 6.4% respectively (P ＞0.05).Conclusion Dihydroartiminisin can inhibit the proliferation of ovarian cancer cell in vitro, probably through down-regulation of the phosphorylation of ERK 1/2 in ovarian cancer cells.

Objective To study the antitumor effects of photodynamic therapy (PDT)treated by ovarian cancer cell lysates in rat epithelial ovarian cancer in vivo. Methods Female Fischer344 rats of 6 -8 weeks were allocated to four groups ( n = 8 each) : PDT group ( inoculated intraperitoneal with PDT tumor cell lysates), freeze/thaw group ( inoculated intraperitonealy with freeze-thaw tumor cell lysates), normal saline group (inoculated intraperitoneal with normal saline) and control group. Rat epithelial ovarian cancer NuTu19 cells were injected into all rats by intraperitoneal at day 7,while injected with normal saline in control group. The number of tumor specific interferon-γ (IFN-γ) secreting splenocytes was quantified by enzyme linked immunospot(ELISPOT) assay, the cytotoxic T lymphocyte(CTL) activity of splenocytes was measured by lactate dehydrogenase(LDH) analysis and tumor growth and the survival time of rats were also observe& Results Stimulated by PDT tumor cell lysates, the number of tumor specific IFN-γ secreting splenocytes in PDT group, freeze/thaw group, normal saline group and control group were 448. 8±34. 2, 211.2±47.9,43.3 ± 11.1,16.1 ± 2.4 respectively, which were significant differences among of them ( P < 0.05). Stimulated by freeze/thaw tumor cell lysates, the number of tumor specific IFN-γ, secreting splenocytes in four groups were 151.7 ± 22.6,188.7 ± 53.0, 18.2 ± 12.2,8.8 ± 7.7 respectively, which were not significant differences among of them ( P>0.05 ). Cytotoxicity of splenocytes of PDT group increased significantly than that in other three groups(P <0.05). Except rats in control group were all alive until the experiment ended, the mean survival time of other rats were 234 d in PDT group, 171 d in freeze/ thaw group and 168 d in normal saline group, which in PDT group was significantly higher than those in freeze/thaw group and normal saline group ( P<0.05 ). Conclusions Rats treated by PDT tumor cell lysates could produce antitumor effects in vivo, which shown that induce tumor-specific immune response and prolong the life span.