[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.

[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.

[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.

[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.

[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.

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.

[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.

[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.