Matrix metalloproteinase-9 expression correlates with prognosis and involved in ovarian cancer cell invasion.

PURPOSE: One of the most important characteristics of ovarian cancer is invasion and metastasis. Matrix metalloproteinases (MMPs) are known to play an important role in cancer cell invasion by mediating the degradation of extracellular matrix (ECM). The activities of MMPs are regulated by tissue inhibitors of metalloproteinases (TIMPs). In this study, we investigated the clinical significance of MMP-2, -7 and -9 and TIMP-1, -2 and -3 expression and MMP-9 functional role in cell invasion and adhesion in ovarian cancer. METHODS: RT-PCR was used to determine mRNA expression of MMP-2, -7 and -9 and TIMP-1, -2 and -3 in ovarian tissues; ELISA was used to detect the serum level of MMP-9; RNA interference (RNAi) was performed to determine the function of MMP-9 in cell invasion and adhesion in ovarian cancer cells. RESULTS: mRNA expression of MMP-2, MMP-7, MMP-9, TIMP-2 and TIMP-3 and serum level of MMP-9 were significantly high in patients with ovarian cancer. MMP-9 expression was significantly high in patients with advanced ovarian cancer and correlated with poor prognosis. The ability of cells for invasion and adhesion was significantly reduced by treatment of cells with MMP-9 siRNA. CONCLUSIONS: Our results suggest that MMP-9 is a potential prognostic factor for ovarian cancer and could be a novel treatment target in ovarian cancer patients.

Impact of post-operative hormone replacement therapy on life quality and prognosis in patients with ovarian malignancy.

The present study aimed to assess the impact of post-surgical hormone replacement therapy (HRT) on life quality and prognosis in women with ovarian malignancy. HRT (Premarin, Nilestriol and medroxyprogesterone) was administered following surgery in 31 patients with ovarian cancer. A total of 44 ovarian cancer patients of similar age, clinical stage and pathological features did not receive HRT following surgery. The expression of estrogen receptor (ER)-α, ERß and progesterone receptor (PR) in cancer tissues was detected by immunohistochemical staining. Serum levels of calcitonin (CT) and transforming growth factor (TGF)-α were determined by radioimmunoassay and enzyme-linked immunosorbent assay, respectively. Data were analyzed using Kaplan-Meier survival curves, a log-rank test and a Cox scale risk model. Quality of life was assessed in the patient groups and in healthy post-menopausal women (control) based on a questionnaire developed by the European Organization of Research and Treatment of Cancer (EORTC-C30), as well as our own specific questionnaire. A log-rank test revealed no difference in survival between the patients with and without HRT (p>0.05), and a Cox model showed that HRT was not an independent prognostic factor. The accumulated survival rate did not differ significantly based on the expression of ERα, ERß or PR in patients with or without HRT (p>0.05). The serum TGFα levels prior to and following surgery were not significantly different in either of the two patient groups (p>0.05). Serum CT levels were higher in patients without HRT at 1.5 years following surgery (p<0.05), but no significant difference was found in the serum CT levels of patients receiving HRT. The HRT and non-HRT groups differed significantly with regard to the body and emotional functional sub-scales of the EORTC-C30 (p<0.05) and the sex quality and autonomic nerve maladjustment categories of our specific questionnaire (p<0.05). Findings of this study showed that HRT administered following surgery exhibited no apparent negative effect on prognosis in patients with ovarian cancer, regardless of ERα, ERß or PR expression in cancer tissues, and had no effect on serum transforming growth factor (TGF)-α levels. Post-surgical HRT aided in the stabilization of serum CT levels and improved the quality of life in these patients.

Clinical value of combined detection of serum matrix metalloproteinase-9, heparanase, and cathepsin for determining ovarian cancer invasion and metastasis.

AIM: This study evaluated the clinical value of the combined detection of serum cathepsin L (CL), heparanase (Hpa), and matrix metalloproteinase-9 (MMP-9) for determining the degree of ovarian cancer invasion and metastasis before surgery. PATIENTS AND METHODS: Enzyme-linked immunosorbent assays were used to measure the serum content of CL, Hpa, and MMP-9 in 217 patients with untreated ovarian cancer before surgery, 100 patients with benign ovarian tumors, and 101 healthy women as controls. In addition, the degrees of invasion and metastasis were assessed by the 'gold standard' of clinicopathological diagnosis. The associations of the preoperative serum CL, Hpa, and MMP-9 levels with the clinicopathological factors and metastatic status were analyzed. Receiver operating characteristic (ROC) curve analysis was used to evaluate the usefulness of these markers for determining the degree of ovarian cancer invasion before surgery. RESULTS: The serum CL, Hpa, and MMP-9 levels were significantly higher (p=0.001) in patients with malignant ovarian cancer compared with patients with benign ovarian tumors and healthy controls. The serum CL level was significantly higher in patients with epithelial ovarian carcinoma compared with non-epithelial ovarian carcinoma (p=0.048), whereas the serum levels of Hpa (p=0.109) and MMP-9 (p=0.544) did not differ significantly between these two groups. The serum CL, Hpa, and MMP-9 levels correlated with the degree of differentiation and the FIGO staging (p>0.05). The serum CL (p=0.030) and MMP-9 (p=0.010) levels were significantly associated with peritoneal metastasis, and the serum Hpa level (p=0.042) was associated with distant metastasis. A ROC curve analysis revealed sensitivity of 60.9%, 69.6%, and 72.2%, and specificity of 57.4%, 67.2%, and 68.9% for the preoperative serum levels of CL, Hpa, and MMP-9, respectively, as tumor markers for the degree of extra-pelvic metastasis. CONCLUSION: Elevated serum CL, Hpa, and MMP-9 levels are correlated with malignant invasion and progression in ovarian cancer. The combined detection of serum CL, Hpa, and MMP-9 may be useful for determining the extent of ovarian cancer metastasis before surgery.

Evaluation of proteomics-identified CCL18 and CXCL1 as circulating tumor markers for differential diagnosis between ovarian carcinomas and benign pelvic masses.

A lack of sensitive and specific tumor markers for early diagnosis and treatment is a major cause for the high mortality rate of ovarian cancer. The purpose of this study was to identify potential proteomics-based biomarkers useful for the differential diagnosis between ovarian cancer and benign pelvic masses. Serum samples from 41 patients with ovarian cancer, 32 patients with benign pelvic masses, and 41 healthy female blood donors were examined, and proteomic profiling of the samples was assessed by surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectroscopy (MS). A confirmatory study was also conducted with serum specimens from 58 patients with ovarian carcinoma, 37 patients with benign pelvic masses, and 48 healthy women. A classification tree was established using Biomarker Pattern Software. Six differentially expressed proteins (APP, CA 125, CCL18, CXCL1, IL-8, and ITIH4) were separated by high-performance liquid chromatography and identified by matrix-assisted laser desorption/ionization (MALDI)-MS/MS and database searches. Two of the proteins overexpressed in ovarian cancer patients, chemokine CC2 motif ligand 18 (CCL18) and chemokine CXC motif ligand 1 (CXCL1), were automatically selected in a multivariate predictive model. These two protein biomarkers were then validated and evaluated by enzyme-linked immunosorbent assay (ELISA) in 535 serum specimens (130 ovarian cancer, 64 benign ovarian masses, 36 lung cancer, 60 gastric cancer, 55 nasopharyngeal carcinoma, 48 hepatocellular carcinoma, and 142 healthy women). The combined use of CCL18 and CXCL1 as biomarkers for ovarian cancer had a sensitivity of 92% and a specificity of 97%. The multivariate ELISA analysis of the two putative markers in combination with CA 125 resulted in a sensitivity of 99% for healthy women and 94% for benign pelvic masses, and a specificity of 92% for both groups; these values were significantly higher than those obtained with CA 125 alone (p and lt;0.05). We conclude that serum CCL18 and CXCL1 are potentially useful as novel circulating tumor markers for the differential diagnosis between ovarian cancer and benign ovarian masses.

[Relationship between cathepsin L and invasion and metastasis of ovarian carcinoma cells].

OBJECTIVE: To clone cathepsin L (CTSL) gene and construct the eukaryotic expression plasmid pcDNA3.1-CTSL and study the relationship between CTSL and invasion and metastasis in ovarian cancer cells in vitro. METHODS: The total RNA was extracted from the ovarian cancer tissue and the intact cDNA of CTSL was applied by reverse transcription (RT)-PCR. The product of RT-PCR was cloned to pMD18-T vector, and subcloned to pcDNA3.1 vector. It was tested by the enzymation and DNA sequencing. The eukaryotic expression plasmid of CTSL was introduced into HO8910 cells by liposome transfection reagent. RT-PCR was used to confirm the recombinant plasmid DNA integrated with the genomic DNA of HO8910 cells. Western blot was used to confirm the CTSL protein expression in positive clones cells. The cell growth curves, clonogenicity efficiency were observed. The cell cycles were measured by flow cytometer. The ability of invasion, metastasis and adhesion of ovarian cancer cells were detected by the matrigel invasion assay, transwell migration assay and adhesion assay, respectively. RESULTS: The results from restrictive enzyme analysis and sequencing showed that the CTSL gene was successfully inserted into pcDNA3.1. Result from RT-PCR and western blot showed that the ovarian cancer cells which transfected by recombinant plasmid could express CTSL gene and protein. There was no difference between HO8910-CTSL and HO8910-pcDNA3.1 cells in proliferation and adhesion ability (0.16 ± 0.04 versus 0.19 ± 0.04) of the cells (P > 0.05). There was difference between HO8910-CTSL and HO8910-pcDNA3.1 cells in matrigel invasion ability (0.34 ± 0.18 versus 0.17 ± 0.04) and metastasis ability (1.252 ± 0.114 versus 0.486 ± 0.027) of cancer (all P < 0.05). CONCLUSION: CTSL maybe increase the ability of invasion and metastasis of ovarian cancer cells in vitro, which may be a molecular target of blocking invasion and metastasis of ovarian cancer.

[Establishment and biological characteristics of a human ovarian carcinoma cell line originated from peritoneal effusion].

BACKGROUND AND OBJECTIVE: Establishing ovarian cancer cell lines will benefit researches on biological characteristics of ovarian cancer stem cells and antitumor drugs. This study was to establish a human ovarian carcinoma cell line from peritoneal effusion, and explore its biological characteristics. METHODS: Cells were isolated from peritoneal effusion of an ovarian carcinoma patient, purified and cultured in vitro. The morphology of the cells was observed under electromicroscope. The growth curve of cells was drawn to calculate cell doubling time (TD). Cell karyotype was analyzed. The levels of sex hormone, and the expression of CA125 and CA19-9 in cell culture supernate were detected by radioimmunoassay. The cells were transplanted into nude mice to observe tumor formation. RESULTS: A new ovarian carcinoma cell line was established, which had been maintained in vitro for over 100 passages in two years. Abnormal nuclei were observed under electromicroscope. TD was 40.8 h. The karyotype of the cells was hyperdiploid with 63-120 chromosomes. The level of estradiol in cell culture supernate was 45.0 pg/mL; the level of testosterone was 0.03 ng/mL; pregnendione was undetected; the level of CA125 was 4.49 U/mL; the level of CA19-9 was 4.09 U/mL. Poorly differentiated ovarian adenocarcinoma was formed subcutaneously in nude mice after transplantation. CONCLUSION: The new ovarian carcinoma cell line is proved to be an immortalized and malignant cell line.

[Inhibition of Topo II alpha gene expression and reversing of drug resistance in multi-drug resistant epithelial ovarian cancer cells induced by RNA interference in vitro].

OBJECTIVE: To explore whether or not multi-drug resistance could be reversed by RNA interference the expression of Topo II alpha gene in epithelial ovarian cancer cell lines in vitro. METHODS: (1) The best silent small interference RNA (siRNA) of Topo II alpha gene was designed and chose and cloned into psilencer4.1-CMV-neo vector. The psilencer4.1-CMV-neo-Topo II alpha was transfected into SKOV3/DDP cell, then Topo II alpha siRNA(+)SKOV3/DDP cells was incubated. (2) The Topo II alpha mRNA and protein expression of the stability-transfecting cell lines were detected by RT-PCR and western blot method, respectively. The resistance index, the cell cycle and the cellular content of cisplatin were detected by methyl thiazolyl tetrazolium assay, the flow cytometry and high performance liquid chromatography method before and after Topo II alpha RNA interference in cells. RESULTS: (1) The Topo II alpha gene expression level in SKOV3/DDP cells could be inhibited after the plasmid DNA psilencer4.1-CMV-neo-Topo II alpha transfeced. The expression level of Topo II alpha mRNA in Topo II alpha siRNA(+)SKOV3/DDP and SKOV3/DDP cells were 0 and 0.92 +/- 0.08; the expression level of Topo II alpha protein in Topo II alpha siRNA(+)SKOV3/DDP and SKOV3/DDP cells were 0.51 +/- 0.04 and 1.95 +/- 0.09 (P < 0.01). (2) The multi-drug resistance index of Topo II alpha siRNA(+)SKOV3/DDP cell was significantly lower compared with that in SKOV3/DDP cell (3.46 vs 5.05, P < 0.05). (3) The percentage of G(0)/G(1) and G(2)/M phase cell in Topo II alpha siRNA(+)SKOV3/DDP cells were higher than that in SKOV3/DDP cells (P < 0.05). (4) The content of cisplatin in Topo II alpha siRNA(+)SKOV3/DDP cells treated with cisplatin for 24 hours was significantly higher than that in SKOV3/DDP cell (157.20 vs 63.99 ng, P < 0.05). CONCLUSION: The results showed that the tolerance of cisplatin would be reversed by blocking the Topo II alpha gene expression in cisplatin-resistant epithelial ovarian cancer cells.

[Relationship between combined multigene detection and response to adjuvant chemotherapy in early-stage non-small cell lung cancer].

OBJECTIVE: To evaluate the relationship between combined multigene detection and response to adjuvant chemotherapy and prognosis in early-stage non-small cell lung cancer (NSCLC). METHODS: Tissue microarray was prepared from samples of 86 cases of early-stage NSCLC who received adjuvant chemotherapy after radical surgery. The expressions of caspase-3, Fas, bax, bcl-2, survivin, PCNA, Ki67, MGMT, p53, p63, p73, p16, p27, VEGF, nm23, P-gp, MRP, LRP, GST-pi, Topo II, c-myc, cyclin-D1, Her-2, Cox-2, Ku70, Ku80, DNA-PKcs, ERCC1, MSH2, BCRP proteins were detected using immunohistochemical two-step method. RESULTS: The positive rate of the 30 genes in lung cancer tissue were 27.9% - 91.9%, respectively. By univariate analysis, the expression of 8 genes was shown to be related with SCLC adjuvant chemotherapy. The cases with higher expression of survivin, P-gp, LRP, Ki67, p53, ERCC1 and lower expression of bax,VEGF had worse prognosis. By logistic regression analysis, the ERCC1, survivin, bax and VEGF were a marker group. Multivariate analysis showed the predict value of the response to adjuvant chemotherapy in early-stage NSCLC was 96.5%. CONCLUSION: Survivin, ERCC1, bax and VEGF are an ideal marker group to predict the effect of adjuvant chemotherapy in early-stage NSCLC.

[Constitutive characteristics and change trend of gynecological malignant tumors in 8009 hospitalized patients in Guangxi Zhuang Autonomous Region].

OBJECTIVE: To investigate the constitutive characteristics and the change trend of gynecologic malignant tumors in hospitalized patients in Guangxi Zhuang Autonomous Region over the recent 20 years. METHODS: Clinical data of 8009 in-patients who suffered from gynecologic malignant tumors in 23 hospitals from 1985 to 2004 in Guangxi Zhuang Autonomous Region were analyzed, with respect to the tumor types and change trend. RESULTS: (1) The leading 4 types of malignant tumors were cervical cancers, ovarian cancers, endometrial cancers, and malignant trophoblastic tumors according to the constitutive ratios of the tumors. The constitutive ratio of cervical cancer patients rose year by year, from 17.48% during the 1985-1989 period to 49.25% during the 2000-2004 period (P < 0.01). While the constitutive ratio of malignant trophoblastic tumors dropped year by year. The changes of ovarian cancer, endometrial cancer, vulvar and vaginal carcinomas, and sarcoma of the uterus were not obvious (P > 0.05). (2) The occurring age of cervical cancers became younger obviously, from > or = 60 years old dropped to < 40 years old. (3) Cervical cancers were found mainly in urban residents in the former 10 years, the constitutive ratio being 67.1%; while in the latter 10 years it gradually shifted to rural residents, accounting for 52.6% of the total gynecological tumors. (4) Patients were usually at stages II, III, IV when they visited a doctor for their diseases. Especially for ovarian cancer, malignant trophoblastic tumors, sarcoma of the uterus, these patients were in the intermediate or advanced stage when they were diagnosed, mainly because of lack of obvious symptoms. The constitutive ratio of these advanced patients was over 60%. CONCLUSIONS: We should strengthen the screening program of cervical cancer, and pay more attention to prevention and control of other gynecological reproductive organ tumors at the same time. On the other hand, we should explore better tumor markers, new methods of diagnosis and treatment to improve early diagnosis and treatment of gynecologic malignant tumors.

[Expression and clinical significance of Ku70, Ku80 and DNA-PKcs proteins in patients with stageI-II non-small cell lung cancer by tissue microarray].

BACKGROUND: Ku70, Ku80 and DNA-PKcs proteins take part in the repairment of DNA double-strand breaks as regulatory subunits of DNA-dependent protein kinase. The aim of this study is to investigate the expression and clinical significance of Ku70, Ku80 and DNA-PKcs proteins in patients with stage I-II non-small cell lung cancer (NSCLC). METHODS: A total of 86 patients with stage I-II NSCLC who received adjuvant chemotherapy after radical surgery were retrospectively analyzed. The expression of Ku70, Ku80 and DNA-PKcs proteins was detected by tissue microarray technique and immunohistochemical two-step method. RESULTS: The positive rate of Ku70, Ku80 and DNA-PKcs in lung cancer tissues was 68.6%, 72.1% and 87.2%, respectively. Their expression was not related to histological classification (P > 0.05). The patients with worse prognosis seemed to have higher expression of Ku70, Ku80 and DNA-PKcs proteins, however there was no statistical significance (P > 0.05). CONCLUSIONS: Ku70, Ku80 and DNA-PKcs are overexpressed in stage I-II lung cancer without prior chemotherapy. They may be not good for guidance of postoperative chemotherapy in completely resected stageI-II NSCLC.

[Inhibitory effects of RNA interference on expression of matrix metalloproteinase-9 gene and invasiveness and adhesion in ovarian cancer cells].

OBJECTIVE: To investigate the inhibitory effects of RNA interference (RNAi) on the expression of matrix metalloproteinase-9 (MMP-9) gene and invasiveness and adhesion of ovarian cancer cells. METHODS: Four groups of different specific target sequence in coding region of MMP-9 and one non-specific sequence were chosen, which were Site1, Site2, Site3, Site4 and Site5. Small interference RNA (siRNA) expression cassettes (SEC) were constructed by PCR and transfected into ovarian cancer HO-8910PM cells. RT-PCR and western blot were used to detect mRNA and protein expression of MMP-9 gene; the abilities of invasion and adhesion were detected by Matrigel invasion assay and cell adhesion assay. RESULTS: The expression of MMP-9 was inhibited and the inhibitory effects of different sequence were varied. The mRNA expression was 0.64 +/- 0.06, 0.47 +/- 0.07, 0.55 +/- 0.10 in Site1, Site2, Site3 group, and protein expression was 0.30 +/- 0.09, 0.27 +/- 0.08, 0.37 +/- 0.12, respectively. Site2 group had the most efficient inhibitory effect, followed by Site1 and Site3 groups. Cell growth curve revealed that cell growth was significantly inhibited in Site2 group. Invasiveness and adhesion were significantly reduced, the inhibitory rate on invasion in Site1, Site2, Site3 groups were 50.0%, 50.0% and 37.5%, respectively; the inhibitory rate on adhesion in Site1, Site2, Site3, Site4 groups were 43.8%, 48.8%, 33.9%, 24.2% at 60 min and 41.6%, 40.2%, 35.1%, 16.0% at 90 min, respectively. CONCLUSIONS: RNAi exists in ovarian HO-8910PM cells. MMP-9 siRNA can specifically down-regulate MMP-9 expression and lead to the inhibition of invasiveness and adhesion in ovarian cancer cells.

[Inhibitory effects of antisense MMP-9 oligodeoxynucleotides on invasiveness and adherence of ovarian cancer cells].

OBJECTIVE: To observe the inhibitory effects of antisense MMP-9 oligodeoxynucleotides on invasiveness and adhesion ability in vitro of ovarian cancer cells, and to investigate the mechanisms of action. METHODS: MMP-9 antisense oligonucleotides were transfected by lipofectinmin into ovarian cancer cell line HO-8910PM cells expressing MMP-9 induced with fibronectin. RT-PCR, Western blot and gelatin zymography were used to detected MMP-9 expression of mRNA and protein and enzymatic activity. The ability of invasion and migration of ovarian cancer cells was assayed in Transwell cell culture chamber. Cell adhersion assay was carried out in a microculture well pre-coated with fibronectin. RESULTS: MMP-9 expressions of mRNA and protein were significantly decreased in the antisense-transfected cells. Comparing with the control group, the inhibition rate was 34. 8% and 42. 5% , respectively (P <0. 05). Its gelatin enzymatic activity was inhibited. Matrigel invasion assay and Transwell migration assay revealed markedly reduction in invasion and migration for the antisense group. The inhibition rates were 22. 4% and 24. 8% , respectively. The adhesion ability was also reduced. The inhibition rates were 49. 8% and 38. 3% at 60 min and 90 min, respectively. CONCLUSION: MMP-9 down-regulation can significantly inhibit the ability of invasion and attachment of ovarian cells in vitro. MMP-9 may play an important role in invasion and metastasis of ovarian cells and potentially be a molecular target of blocking invasion and metastasis of ovarian cancer.

Demonstration of differential gene expression between sensitive and resistant ovarian tumor cells by fluorescence differential display-PCR analysis.

Chemotherapy plays a major role in cancer management; however, acquired drug resistance remains a significant problem for ovarian cancer treatment. Chemoresistance is regulated by the coordinated expression of a set of genes. Thus, the identification of genes specifically modulated in the process provides an important step toward the discovery of underlying molecular mechanisms in drug resistance events. We recently developed five drug-resistant human ovarian carcinoma cell lines, including two cisplatin (cis) resistant cell lines, two carboplatin (car) resistant cell lines and one taxol (tax) resistant cell line. In this study, we investigated differential gene expression between these resistant cell lines and their parental cell lines by the fluorescence differential display-polymerase chain reaction (FDD-PCR) technique. We first screened and identified differentially expressed genes in the resistant ovarian cancer cell lines, and we then sequenced and analyzed these genes by bioinformatics software. A total of 33 fragments were displayed in the two resistant cell lines (S-cis and S-car) derived from the sensitive SKOV-3 cell line, and 36 fragments were displayed in the three resistant cell lines (A-cis, A-car and A-tax) derived from the sensitive A2780 cell line. After purification, cloning, sequencing, and homology analysis on the NCBI BLAST GenBank, 12 gene fragments were identified from the resistant S-cis and S-car cells, and 23 gene fragments were identified from the resistant A-cis, A-car and A-tax cells. Although a homolog search of the NIH GenBank revealed that most of the gene fragments were not significantly associated with the known drug resistance-related genes, our study conclusively demonstrates that FDD-PCR is a useful tool for analyzing the differential gene expression between resistant and sensitive tumor cells and for identifying novel chemoresistance-associated genes and potential biological markers or genetic markers of drug resistance.

Characterization of the mechanisms of electrochemotherapy in an in vitro model for human cervical cancer.

Electrochemical treatment is among the most effective therapies in the management of cervical malignancy. However, the mechanism of action of this treatment remains largely unknown. Therefore, the purpose of the current project was to establish a suitable eletrochemotherapy regimen for cervical cancer and to investigate the mechanism of the therapy in an in vitro model for human cervical carcinoma. HeLa cells were used as a model for cervical cancer in this study, and the effect of electrochemical treatment on these cells was examined in four different dosage groups (5 V + 5 C, 10 V + 5 C, 5 V + 10 C and 10 V + 10 C). Our results showed that the combinations of lower voltage and higher current (5 V/10 V + 10 C) had a greater anticancer effect in this model as compared to other groups. In addition, we compared the cytotoxic effect between electrochemical treatment and different pH condition treatments in this system, and found that the efficacy of electrochemical treatment in cell killing was better than that of acidic or basic medium treatment. Moreover, we demonstrated that the efficacy of electrochemical treatment was correlated with the degree of ionization and alteration in pH scale. The electrodes were basic on the cathode side which elevated the cations K+, Ca2+ and Mg2+, while the electrodes were acidic on the anode side which reduced the anion Cl-. We also assessed the effect of electrochemical treatment on cell cycle distribution in HeLa cells and showed that the percentage of cells in the G1 phase of the cell cycle was increased (G1 arrest), while the cell population in the S phase was decreased. Furthermore, we demonstrated that the levels of the cell cycle regulator cyclin D1 expression were dramatically reduced when 5 V/10 V + 10 C treatments were applied to these cells, as determined by RT-PCR analysis. By contrast, no significant changes in the levels of cyclin B1, CDK1 or CDK4 were detected. Based on these observations, we conclude that the combination of lower voltage and higher current may be a potentially effective eletrochemotherapy regimen for cervical cancer in the clinic, and that the antitumor effect of electrochemical treatment on cervical carcinoma cells is mediated partly via regulating ionization degree, pH state and cell cycle control.

[Expression of matrix metalloproteinases-9,2,7,and tissue inhibitor of metalloproteinases-1,2,3 mRNA in ovarian tumors and their clinical significance].

BACKGROUND & OBJECTIVE: Matrix metalloproteinases (MMPs)play an important role in cancer cell invasion,and metastasis by degrading the extracellular matrix (ECM). The activities of MMPs are regulated by tissue inhibitor of metalloproteinases (TIMPs). This study was to detect mRNA expression of MMP-9, 2, 7, and TIMP-1, 2, 3 in ovarian tumor tissues,and explore their correlations with clinicopathologic characteristics,and prognosis of patients with ovarian cancer. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR)was used to assay the positive rates,and half-quantity values of MMP-9, 2, 7, and TIMP-1, 2, 3 mRNA in 48 cases of malignant ovarian tumor tissues, 21 cases of benign ovarian tumor tissues, and 22 cases of normal ovarian tissues. RESULTS: The positive rate of MMP-9 mRNA, and the half-quantity values of MMP-9,MMP-2 were significantly higher in malignant tumor tissues than those in normal ovarian tissues (P< 0.05). The positive rates and half-quantity values of TIMP-2,MMP-7,TIMP-3 mRNA were significantly higher in malignant and benign ovarian tumor tissues than those in normal ovarian tissues (P< 0.05). The ratio of MMP-9/TIMP-1 was higher in malignant ovarian tumor tissues(0.91+/-0.67) than that in normal ovarian tissues (0.14+/-0.33)(P< 0.05). The expression level of MMP-9 mRNA was higher in ovarian cancer tissues of stage III-IV(1.13+/-0.66) than those of stage I-II(0.60+/-0.54)(P< 0.05). The mean survival time of MMP-9 positive patients was (43.00+/-17.12) months,and cumulate survival rate was 47.37%,significantly lower than that of MMP-9 negative patients (100%). CONCLUSIONS: MMP-9, MMP-2, MMP-7, TIMP-2, TIMP-3 are over-expressed in ovarian malignant tissues. The over-expression of MMP-9, and the imbalance between MMP-9 and TIMP-1 play important roles in the progress of advanced ovarian cancer. MMP-9 may be a useful prognostic marker for patients with advanced ovarian cancer.

[Establishment of 5 resistant ovarian cancer cell strains and expression of resistance-related genes].

OBJECTIVE: To investigate expression difference of several drug resistance related genes between sensitive and resistant ovarian carcinoma cells. METHODS: Cell lines resistant to cisplatin, carboplatin and taxol were established from ovarian carcinoma cell lines of SKOV3 and A2780, and their biological features were detected. The expressions of several genes related to drug resistance were measured by RT-PCR method. RESULTS: (1) The values of resistance index (RI) of resistant cells to relevant drugs were elevated 3 times or more, with different degrees of cross-resistance to several other drugs (RI 2 approximately 20). They grew more slowly than primary cells (Td elongated 1.4 approximately 2.4 times, P < 0.01) without obvious changes in G(1), G(2), and S ratios (P > 0.05). Intracellular concentrations of relevant drugs were reduced 2.0 approximately 8.5 times in resistant cells (P < 0.05). (2) p53, lung resistance protein-1 (LRP-1), multiple drug resistance related protein-1 (MRP-1) genes were expressed at lower levels in resistant cells than in sensitive cells; while protein kinase C (PKC), topoisomerase (topo) I, and topo II beta were expressed higher, no obvious alterations were found concerning glutathione S transferase-pi (GST-pi), and topo II alpha. Expression of multiple drug resistance-1 (MDR-1) gene was either elevated or reduced in different cells. CONCLUSIONS: The expressions of resistance related genes were widely different in different kinds of resistant cells, suggesting more than one pathway leading to resistance transformation. This adds more difficulties for clinical management.

Correlation of serum VEGF levels with clinical stage, therapy efficacy, tumor metastasis and patient survival in ovarian cancer.

Vascular endothelial growth factor (VEGF) has been shown to play an important role in tumor growth and progression. However, the clinical implications of VEGF expression in ovarian tumors are not fully understood. We therefore investigated the serum level of VEGF in patients with ovarian tumors and explored the potential use of VEGF as a tumor marker for diagnosis, treatment and prognosis of human ovarian cancer. The serum VEGF (sVEGF) levels in 120 patients with ovarian carcinoma, 25 patients with benign ovarian tumor and 90 healthy female blood donors were measured by an enzyme-linked immunosorbent assay in this study. We also determined the levels of sVEGF in patients with epithelial ovarian cancer before and after surgery. Our results showed that: (i) ovarian cancer patients had significantly higher levels of sVEGF compared to those of patients with benign ovarian tumor or those of healthy individuals. As a cut-off at 100 pg/ml, the sensitivity and specificity of sVEGF levels for diagnosing ovarian carcinoma were 77.1% and 87%, respectively. (ii) sVEGF levels were markedly elevated in patients with advanced stage or poorly-differentiated ovarian cancer, as well as in those with more ascites (>500 ml), as compared to patients with early stage and well-differentiated ovarian cancer, or those with less ascites (<500 ml). However, there was no significant difference in sVEGF levels among different pathological subtypes of ovarian carcinoma. (iii) The post-operative sVEGF levels were significantly lower than the pre-operative sVEGF levels. (iv) We measured significantly higher levels of sVEGF in patients with metastasis as compared to patients lacking metastasis. Lastly (v) the average survival-time in patients with higher levels of sVEGF (>100 pg/ml) was 28 months, while the average survival-time in patients with lower levels of sVEGF (<100 pg/ml) was 35 months, indicating that the elevations in sVEGF level are correlated with patient survival and tumor metastasis in ovarian carcinoma. These data suggest that VEGF may be a useful serological biomarker for clinical diagnosis and prognosis of ovarian cancer, for follow-up of ovarian tumor metastasis and for monitoring the efficacy of therapy in patients with ovarian carcinomas.

Development and characterization of five cell models for chemoresistance studies of human ovarian carcinoma.

Platinum agents and paclitaxel (taxol) are among the most effective drugs currently available for treatment of ovarian cancer. One of the hurdles with taxol and platinum- based therapy is the clinical development of resistance to these agents. To investigate the mechanism of drug resistance in human ovarian cancer, we developed and characterized 5 cell models for chemoresistance studies of cisplatin, carboplatin and taxol. We report in this study that these human ovarian carcinoma cell model systems include 2 models for cisplatin resistance, 2 models for carboplatin resistance, and 1 model for taxol resistance. The biological and biochemical characteristics of the models showed that (i), the IC50 values of the drugs for all these resistant cell models were 3 times (or more) higher than those for the parental tumor cells. There also exist varying degrees of cross-resistance to several other chemotherapeutic agents in these systems. Moreover, the intracellular drug accumulations in these cells were significantly reduced as compared to those in the parental cells. (ii), The proliferation rates of these resistant cells were markedly decreased. However, there were no obvious changes in cell cycle distribution in these model systems. (iii), Our results for the expression of a few major drug resistance-related genes revealed that the expression of p53, lrp-1 and mrp-1 was decreased, while the expression of pkc, topo I and topo II beta was increased in the resistant tumor cells as compared with the parental cells. In contrast, no significant alterations in gst-pi and topo II alpha expression were found. Interestingly, the levels of mdr-1 expression were elevated in some models, but were reduced in others, thus suggesting that different pathways are involved in the formation of drug resistance in different cell model systems, and that different mechanisms are responsible for the development of different drug resistances in tumor cells. Taken together, our findings indicate that these models may be potentially used to assess the biochemical and genetic mechanisms of drug resistance in human ovarian cancer and to identify new drug resistance-related genes.