[Impact of the biological function on epithelial ovarian cancer with ITIH4 gene expression down-regulating in vitro].

OBJECTIVE: To investigate the efficiency of biology function of ITIH4 gene silenced by small interfering RNA (siRNA) on ovarian cancer. METHODS: The four pairs ITIH4 gene siRNA interference fragments (ITIH4-546, ITIH4-795, ITIH4-917 and ITIH4-1568) were designed respectively, and transfected into HO8910pm cells with ITIH4 mRNA high expression by liposomal method transiently. Quantitative PCR method was used to detect the ITIH4 mRNA expression in HO8910pm cells transfected with interference fragment. The ITIH4 917 was selected as the best silencing effect of siRNA interference fragment and then the recombinant plasmid expression vector pGPU6/GFP/Neo-shRNA-ITIH4-917 was constructed and transfected into HO8910pm cells. The stably transfected cells- pGPU6/GFP/Neo-shRNA-ITIH4-917-HO8910pm cells was obtained by screening of aminoglycoside antibiotics (G418). The experiment was divided into three groups, namely ITIH4-917 transfection group, the HO8910pm cell group transfected with pGPU6/GFP/Neo-shRNA plasmid (empty vector group), and the HO8910pm cell group transfected with pGPU6/GFP/Neo-shRNA-ITIH4-NC the plasmid (negative control group). Fluorescence quantitative reverse transcription (RT) PCR and western blot were used to detect the ITIH4 mRNA and protein expression. The cell proliferation, the cell cycle, colony formation of cells, cells migration and invasion in vitro were determined by using methyl thiazolyl tetrazolium (MTT), flow cytometry, colony formation assay and transmembrane (transwell) small chamber method [value represented by absorbance (A)], respectively. RESULTS: The fluorescent quantitative PCR results showed that the ITIH4 mRNA expression levels in ITIH4-917 HO8910pm cells was significantly lower than that in the control cells, the relative copy number was only 0.26 ± 0.15. Also the relative copy number of ITIH4 mRNA in ITIH4-917 transfection group cells was 0.34 ± 0.10, it significantly lower than that in empty vector group (1.87 ± 0.12, P = 0.008) and negative control group (1.58 ± 0.21, P = 0.032); Western blot results showed that the ITIH4 relative expression levels of the protein in ITIH4-917 HO8910pm group cells, empty vector group and negative control group were 0.51, 1.64 and 1.74, respectively, there were statistically significant differences (0.51 vs. 1.64, P = 0.012; 0.51 vs. 1.74, P = 0.014). MTT colorimetric assay showed that the proliferation of ITIH4-917 HO8910pm group cells was significantly faster than that in the empty vector group and negative control group, and there were statistically significant differences among them (P = 0.001). The S + G2/M phase cell ratio in ITIH4-917 HO8910pm group cells was 54.2%, which was significantly higher than that in the empty vector group or negative control group (26.3% and 31.3%, respectively, all P < 0.05). The colony formation rate (55.7 ± 0.7)% in ITIH4-917 HO8910pm group cells was also significantly higher than that in empty vector group (29.7 ± 0.9)% (P = 0.037) and negative control group (31.4 ± 0.3)% (P = 0.043). Migration and invasion experiments showed that cell migration in ITIH4-917 HO8910pm group cells was 0.40 ± 0.18, whicht was significantly higher than that in the negative control group or empty vector (0.30 ± 0.03, P = 0.031;0.25 ± 0.03, P = 0.028, respectively). Although the invasive ability of ITIH4-917 HO8910pm group cells (1.31 ± 0.34) was higher than that in the control cells (1.05 ± 0.68) and empty vector group (1.14 ± 0.08), while there were not significant difference (P > 0.05). CONCLUSION: It would be to promote the cell doubling time and increase the migration capability in HO8910pm cells that ITIH4 expression was down-regulating by ITIH4 mRNA interference.

[Isolation and identification of side population cells in human lung adenocarcinoma cell line A549].

OBJECTIVE: To isolate and characterize the side population cells (SP cells) in the lung adenocarcinomas cell line A549. METHODS: The protein expression of ABCG2 in human lung adenocarcinoma cell line A549 was detected by immunohistochemistry. SP and NSP cells in the cell line A549 were isolated by FACS, and their differentiation was analysed. ABCG2 expression in the two cell subsets was detected by RT-PCR. The cell growth curves, cell division indexes, cell cycles, plate clone formation tests, migration and invasion assays, chemotherapeutic susceptibility tests, tests of the intracellular drug levels, and the tumor cell implantation experiments on nude mice were applied to study the biological properties of the two cell subsets. The expression of ABCG2 in the transplanted tumor in nude mice was detected by immunohistochemistry and RT-PCR. RESULTS: The positive rate of ABCG2 expression in the A549 cells by immunohistochemistry was 2.13%. SP and NSP cells were isolated by FACS. The SP cells could produce both SP and NSP cells, while NSP cells only produced NSP cells. SP cells expressed ABCG2, but NSP cells did not. The proliferation and migration abilities of the two cell subsets were similar, but the invasion and tumorigenic ability of SP cells was significantly higher than that of NSP cells. The susceptibilities to DDP and its intracellular levels of the two cell subsets were similar, but the susceptibilities to 5-FU, VP16, NVB and GEM and their intracellular levels of NSP cells were significantly higher than those of the SP cells. CONCLUSIONS: SP cells in the human lung adenocarcinomas cell line A549 is enriched with tumor stem cells. An effective way to get lung adenocarcinomas stem cells is to isolate SP cells by FACS.

[Expression and significance of differentially expressed protein endoplasmic reticulum aminopeptidase 1 in ovarian carcinoma with lymph node metastasis].

OBJECTIVE: To detect the genetic transcription and protein expression level of endoplasmic reticulum aminopeptidase 1 (ERAP1) in human ovarian cancer cells and tissue and study their relationship with directional lymphatic metastasis. METHODS: Real-time fluorescent quantitative PCR and western blot were used to determine the expressions of ERAP1 gene and protein in the human ovarian cancer cell lines between non-directional (SKOV3) and directional highly lymphatic metastasis (SKOV3-pm2, SKOV3-pm3, SKOV3-pm4). Immunohistochemistry method was used to further validate the ERAP1 expressions of the transplanted ovarian tumor primarily focus and the lesions of lymph node metastasis from nude mice and the human ovarian cancer primarily focus and the lesions of lymph node metastasis. RESULTS: The expression level of ERAP1 gene and protein were down-regulated in SKOV3, SKOV3-pm2, SKOV3-pm3, SKOV3-pm4 cell sublines (0.118 ± 0.012, 0.031 ± 0.003, 0.028 ± 0.003, 0.016 ± 0.005; 0.91 ± 0.33, 0.09 ± 0.03, 0.10 ± 0.04, 0.05 ± 0.04; respectively), and the level of ERAP1 in SKOV3 cell was higher than those in the other three kinds of cell lines (P < 0.05). The results showed that there were significant declining trend of expression of ERAP1 in the human ovarian cancer cell lines between non-directional and directional highly lymphatic metastasis; the transplanted ovarian tumor primarily focus and the metastasis lesions of lymph node from nude mice (143 ± 22 vs. 97 ± 12, P < 0.05), the primarily focus (184 ± 14) and the lesions of lymph node metastasis from human ovarian tumors (P < 0.05). CONCLUSION: The absence or down-regulated expression of ERAP1 is closely related to the metastasis and invasion of lymph node in ovarian carcinoma.

[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.

Confirming whether novel rhein derivative 4a induces paraptosis-like cell death by endoplasmic reticulum stress in ovarian cancer cells.

Ovarian cancer is the leading cause of death among gynecologic cancer patients. Although platinum-based chemotherapy as a frontline treatment for ovarian cancer has been widely used in clinical settings, its clinical efficacy is not satisfactory due to the resistance of ovarian cancer cells to apoptosis. Therefore, it is of great significance to induce non-apoptotic programed cell death patterns, such as paraptosis, in ovarian cancer. In this study, we aimed to explore the potential anticancer mechanisms of novel rhein derivative 4a, which was modified with rhein as a lead compound. The results showed that a wide range of vacuoles from the endoplasmic reticulum and mitochondria appeared in ovarian SKOV3, SKOV3-PM4, and A2780 cells treated with derivative 4a, and the cell death caused by derivative 4a is a type of non-apoptotic and non-autophagic death, which is caused by expansion and damage of the endoplasmic reticulum or mitochondria, showing the characteristics of para-apoptotic death. Furthermore, derivative 4a stimulated the unfolded protein reaction of ovarian cancer cells by upregulating the expression of Bip78 and activating the PERK-eIF2α-ATF4 pathways. Notably, rhein derivative 4a-induced cell death was positively correlated with activation of p38, ERK, and JNK, and negatively correlated with Alix, a known protein that inhibits paraptosis. In addition, derivative 4a treatment also induced G2/M phase arrest in ovarian cancer cells. Taken together, our study reveals that derivative 4a induces paraptosis, and this finding can serve as a basis in developing a new strategy for the treatment of antiapoptotic 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.

[Identification of two potential serum biomarkers for ovarian cancer and clinical validation thereof].

OBJECTIVE: To identify practical biomarkers used in diagnosis of ovarian cancer. METHODS: Peripheral blood samples were collected from 59 ovarian cancer patients, 64 ovarian benign tumors patients, and 142 healthy women and underwent column chromatography to purify the target proteins. The proteins thus obtained were identified with matrix-assisted desorption ionization tissue-of-flight mass spectrometry (MALDI-TOF MS). Immunohistochemistry was used to detect the expression of CCL18 and CXCL1 in the cancer tissues. ELISA was used to detect the serum CC118 and CXCL1 contents. Receiver operating characteristic curve was drawn to examine the sensitivity and specificity of the potential biomarker to ovarian cancer. RESULTS: The m/z 7,784 and 7,837 proteins were identified as chemokine CC2 motif ligand 18 (CCL18) and CXC Chemokines ligand 1 (CXCL1) respectively. The serum levels of CCL18 and CXCL1 of the patients with ovarian cancer were 150 +/- 62 ng/ml and 1 +/- 0.4 ng/ml respectively, both were significantly higher than those of the healthy control women and the patients with ovarian benign diseases (all P < 0.05). The diagnostic sensitivity and specificity of CXCL1 to ovarian cancer were 100% and 97.8% respectively. The diagnostic sensitivity of CCL18 was 100% to ovarian cancer of stages I-II and 86.1% to ovarian cancer of stages III-IV. The CCL18 positive rate in the cancer tissue was 84.6% and the CXCL1 expression rate was 96.2%. The model established based the combination of both biomarkers had the sensitivity and specificity to ovarian cancer of both 100%. CONCLUSION: CCL18 and CXCL1 may be used in early screening of ovarian cancer.

[Suppression of WWOX gene by RNA interference reverses platinum resistance acquired in SKOV3/SB cells].

OBJECTIVE: To assess the suppression effect on WWOX gene in SKOV3/SB cell line by small interference RNA (siRNA). METHODS: Transfection of siRNA using lipofectamine 2000 was conducted to silence WWOX gene expression, the expression levels of WWOX mRNA and protein were evaluated, and the effects on the cell cycles at 48 hours of transfection were assessed by RT-PCR, western blot and flow cytometry (FCM) respectively. The cisplatin resistance index was assayed after transfection of SKOV3/SB with siRNA by methyl thiazolyl tetrazolium (MTT) and the cisplatin concentration of SKOV3/SB cells transfected with siRNA of WWOX was measured by high performance liquid chromatography. RESULTS: (1) In SKOV3/SB cells transfected with WWOX interference fragment, whether at the mRNA or protein level, the expression of both of WWOX decreased. There was a significant difference (P < 0.05) compared with SKOV3 cells and non-transfected cells. (2) After transfection of the WWOX interference fragment, the index of platinum resistance of SKOV3/SB decreased from 5.04 to 3.89. (3) The number of cell transfected with the WWOX interference fragment in G(1) phase was increased, while that in S-phase was decreased. (4) The cisplatin concentration of SKOV3/SB cells transfected with the WWOX interference fragment was increased from 9.43 ng/L to 23.45 ng/L compared with SKOV3/SB cells non-transfected with a significant difference (P < 0.05). CONCLUSION: WWOX gene may be involved in cisplatin resistance phenomenon in epithelial ovarian cancer.

[Establishment of human ovarian carcinoma cell lines with directional highly lymphatic metastasis and study of their biological characteristics].

OBJECTIVE: To establish a human ovarian carcinoma cell line with directional highly lymphatic metastasis and to study their biological characteristics. METHODS: The clone cells of ovarian carcinoma, SKOV3, were inoculated into the hind foot pad of nude mice. The cancer cells of lymph node metastatic foci were transplanted into nude mice again when the metastatic nude of mice were observed. After repetition of this procedure for 3 cycles, the metastatic rate and the metastatic paths were observed in nude mice of every passage. We used limited dilution method to separate and select colonial cells with directional highly lymphatic metastatic potentials from the lymphatic metastasis of human ovarian carcinoma cell line SKOV3. The cells with biological characteristics were assayed by growth curve, HE staining, karyotype analysis, nude mice transplantation and immunohistochemistry, respectively. RESULTS: We established a series of cell lines from lymph node metastasis and designated them as SKOV3-PM1, SKOV3-PM2 and SKOV3-PM3 cell strain. When the cells of SKOV3-PM3 were injected into the hind foot pad of nude mice, they produced 100% (10/10) spontaneous lymphatic metastasis. The lymphatic metastatic rates (26/10) were stable and higher than the mother cell line (1/10, P < 0.01). The metastatic paths were single, mostly to lymphatic nodes. The proliferation ability was increased in cancer cells of every passage in vivo and in vitro after passage of cancer cells of lymphatic metastatic foci for 3 cycles in nude mice. Cytogenetics study showed the karyotypes of SKOV3-PM3 had modes from 83 to 89, and the SKOV3 from 91 to 105. In comparison of SKOV3 with SKOV3-PM3, the number of chromosomes was significantly different (P < 0.05). Immunocytochemical study demonstrated all cell lines were still polygonal epithelial cells. The expression of epithelial membranes antigen (EMA) was positive, exhibiting features of human carcinoma. The cell of SKOV3-PM3 grew more quickly than SKOV3, their cell population doubling time being 22.7 hours and 49.6 hours, respectively (P < 0.05). Flow cytometry revealed the proportion of cells in DNA synthesis and mitosis was higher in SKOV3-PM1 (24.2%), SKOV3-PM2 (29.4%), and SKOV3-PM3 (36.7%) than in SKOV3 (21.5%; P < 0.05). CONCLUSIONS: The ovarian carcinoma sublines with directional highly lymphatic metastasis potential have been established successfully. The cells could provide a good experimental material for further investigation of the mechanism of metastasis and invasion of ovarian carcinoma.

[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.

[Identification of serum biomarkers for ovarian cancer using protein chips and time of flight mass spectrometry technology].

OBJECTIVE: To screen relatively specific biomarkers in serum of ovarian cancer patients using surface-enhanced laser desorption and ionization time of flight mass spectrometry and protein chip technology. METHODS: Serum samples from 99 ovarian cancer patients, 87 healthy volunteers and 21 patients with other ovarian diseases were analyzed using metal affinity chromatography protein chip IMAC3 and cation exchange protein chip WCX2, which can specifically bind the metal-combining-proteins. Proteomic spectra were generated by mass spectrometry. The peaks were detected and filtrated by Ciphergen proteinchip software 3.2.0. Using the Biomarker Pattern 5.0 software, a diagnostic system was developed for separating ovarian cancer from the healthy group. RESULTS: Thirty-one protein peaks were significantly different between ovarian cancer patients and healthy controls (P < 0.05). A diagnostic model consisting of protein peaks from IMAC3 and WCX2 was established. Large scale blind test generated a sensitivity of 100% and specificity of 98% respectively. A differentially expressed protein with a mass-to-charge rate of 7769 was identified as potential biomarkers to distinguish ovarian cancer from other ovarian diseases. CONCLUSIONS: Time of flight mass spectrometry is a useful tool for the detection and identification of new protein markers in serum. It will provide a highly accurate and innovative approach for the diagnosis 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.

Protein expression of nucleophosmin, annexin A3 and nm23-H1 correlates with human nasopharyngeal carcinoma radioresistance in vivo.

Radioresistance is a significant obstacle in the treatment of endemic nasopharyngeal carcinoma (NPC). The present study aimed to identify proteins associated with radioresistance in NPC in vitro and in vivo. Proteomics analyses were conducted to screen for differentially-expressed proteins (DEPs) in parental CNE-2 cells and CNE-2R cells. Using proteomics approaches, 16 DEPs were identified. Of these DEPs, nucleophosmin (NPM1), annexin A3 and nm23-H1, were verified using western blot analyses. The tumorigenicity was investigated using mouse xenograft tumorigenicity assays, and tumor growth curves were generated. The protein expression of NPM1, annexin A3 and nm23-H1 was examined by immunohistochemically staining tumor tissues. NPM1 and annexin A3 protein levels were downregulated in the CNE-2R cells, whereas nm23-H1 expression was upregulated. In vivo tests showed that compared with the CNE-2 tumors, CNE-2R tumor growth was significantly retarded (P<0.05). CNE-2 tumor progression was inhibited by irradiation, but CNE-2R tumor progression was not, indicating that the CNE-2R cells were also radioresistant in vivo. NPM1 and annexin A3 expression was significantly lower in non-irradiated (NIR)-CNE-2R tumors compared with NIR-CNE-2 tumors (P<0.01). However, Nm23-H1 protein levels were significantly higher (P<0.05). Overall, the present study established comparable radioresistant and radiosensitive tumor models of human NPC, and identified candidate biomarkers that may correlate with radioresistance. The data showed that dysregulation of NPM1, annexin A3 and nm23-H1 expression correlated with the cellular and tumor radioresponse. These proteins are involved in the regulation of intracellular functions, including stress responses, cell proliferation and DNA repair. However, further clinical evaluations are required.

[Study on the expression and mutation of cadherin 6 gene in human ovarian carcinoma].

OBJECTIVE: To study expression and mutation of cadherin 6 gene in human ovarian tumor tissues and its clinical significance and to explore new molecular biomarker of ovarian tumors. METHODS: The expression and mutation of cadherin 6 in 41 cases of malignant ovarian tumors, 15 cases of benign ovarian tumors and borderline ovarian tumors and 17 cases of normal tissues were detected by RT-PCR and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique respectively. RESULTS: The positive expression rates of cadherin 6 were 71% in normal group, 53% in benign ovarian tumors, 24% in malignant ovarian tumors, being significantly higher in the former two groups than in malignant tumor group (P < 0.05). Statistical analysis revealed a significant correlation between cadherin 6 gene expression and clinical stages and histological subtype of malignant ovarian tumors. Lower expression rate of cadherin 6 gene was found in cancer tissues at stages III approximately IV than at stages I approximately II (P < 0.01). No mutation was found in normal group and benign tumor tissues and it was observed in 2 out of 41 malignant ovarian tumor tissues. CONCLUSIONS: These results suggest that expression of cadherin 6 gene tends to lack in advanced ovarian carcinoma with lymph node metastases, as well as in poorly differentiated carcinomas. It may be a prognostic marker of ovarian carcinoma.

Effects of HLEC on the secreted proteins of epithelial ovarian cancer cells prone to metastasize to lymph nodes.

OBJECTIVE: To study explores the effect of HLEC on the secreted proteins of epithelial ovarian cancer (EOC) cells (SKOV3-PM4) with directional highly lymphatic metastasis. METHODS: Supernatants of four groups of cultured cells, namely, SKOV3 (A), SKOV3+HLEC (B), SKOV3-PM4 (C), SKOV3-PM4+HLEC (D), were collected, and their proteins were detected by antibody arrays and iTRAQ-2D-LC-MALDI-TOF/TOF/MS. Significantly differential proteins were further analyzed via bioinformatics and validated in human serums and cell media via ELISA. RESULTS: Results of antibody arrays and mass spectrometry demonstrated that GRN and VEGFA were upregulated in group C (compared with group A), whereas IGFBP7 and SPARC were downregulated in group D (compared with group C). Comprehensive bioinformatics analysis results showed that IGFBP7 and VEGFA were closely linked to each other. Further validation with serums showed statistical significance in VEGFA and IGFBP7 levels among groups of patients with ovarian cancers, benign tumors, and control groups. Two proteins were upegulated in the first group. VEGFA in the control group was downregulated. For IGFBP, upregulation in the control group and down-regulation in the first group were also observed. CONCLUSION: The HLEC microenvironment is closely associated with directional metastasis to lymph nodes and with differential proteins including cell stromal proteins and adhesion factors. The upregulation of VEGFA and GRN and the downregulation of SPARC and IGFBP7 are closely associated with directional metastasis to lymph nodes in EOC cells.

Identification of genes involved in radioresistance of nasopharyngeal carcinoma by integrating gene ontology and protein-protein interaction networks.

Radioresistance remains one of the important factors in relapse and metastasis of nasopharyngeal carcinoma. Thus, it is imperative to identify genes involved in radioresistance and explore the underlying biological processes in the development of radioresistance. In this study, we used cDNA microarrays to select differential genes between radioresistant CNE-2R and parental CNE-2 cell lines. One hundred and eighty-three significantly differentially expressed genes (p<0.05) were identified, of which 138 genes were upregulated and 45 genes were downregulated in CNE-2R. We further employed publicly available bioinformatics related software, such as GOEAST and STRING to examine the relationship among differentially expressed genes. The results show that these genes were involved in type I interferon-mediated signaling pathway biological processes; the nodes tended to have high connectivity with the EGFR pathway, IFN-related pathways, NF-κB. The node STAT1 has high connectivity with other nodes in the protein-protein interaction (PPI) networks. Finally, the reliability of microarray data was validated for selected genes by semi-quantitative RT-PCR and Western blotting. The results were consistent with the microarray data. Our study suggests that microarrays combined with gene ontology and protein interaction networks have great value in the identification of genes of radioresistance in nasopharyngeal carcinoma; genes involved in several biological processes and protein interaction networks may be relevant to NPC radioresistance; in particular, the verified genes CCL5, STAT1-α, STAT2 and GSTP1 may become potential biomarkers for predicting NPC response to radiotherapy.

[Screening of differentially expressed proteins associated with directional highly lymphatic metastasis in ovarian carcinoma cell lines using SELDI-TOF-MS technology].

BACKGROUND & OBJECTIVE: The ovarian serous papillary adenocarcinoma cell line SKOV3 and its subclones SKOV3-pm2 and SKOV3-pm3 are cell models to investigate the molecular mechanism of invasion and metastasis of ovarian cancers. This study was to screen differentially expressed proteins between ovarian carcinoma cell lines with directional (SKOV3-pm2 and SKOV3-pm3) and non-directional (SKOV3) highly lymphatic metastasis potentials using time-of-flight mass spectrometry technology and protein chips. METHODS: The lymphatic metastasis rates of the three cell lines were detected in animal models. Proteins in endochylema and supernatants of the three cell lines were screened using surface-enhanced laser desorption and ionization-time of flight-mass spectrometry (SELDI-TOF-MS). Each sample was examined using weak cation exchange (CM-10) protein chip assay and immobilized metal affinity capture (IMAC-3) SELDI ProteinChip array. Detected protein peaks were filtrated and analyzed using Ciphergen proteinchip software 3.2.0 and Biomarker Wizard software. Differentially expressed proteins were defined as those whose absolute ratio values were greater than 0.5. RESULTS: Lymphatic metastasis rates in SKOV3, SKOV3-pm2 and SKOV3-pm3 cell xenografts in nude mice were 20%, 90% and 100%, respectively (P<0.05). Proteins in endochylema whose mass-to-charge ratio (m/z) were 6971, 7475, 9089, 9453, 10103, 11655, and the protein in supernatants whose m/z was 4746 were differentially expressed in SKOV3, SKOV3-pm2 and SKOV3-pm3 cells. CONCLUSION: Combined with weak cation exchange protein chip assay and immobilized metal affinity capture SELDI ProteinChip array, SELDI-TOF-MS technology can be used to screen and identify differentially expressed proteins associated with directional highly lymphatic metastasis in ovarian carcinoma cell lines.

[Effects of wogonin on inducing apoptosis of human ovarian cancer A2780 cells and telomerase activity].

BACKGROUND & OBJECTIVE: Inducing apoptosis and inhibiting the telomerase activity of tumor cells became a new therapeutic means for tumor. In vivo and in vitro experiments showed that wogonin possesses antioxidant activities and inhibitory effect on tumor cells growth. This study was designed to evaluate the effect of wogonin on telomerase activity and apoptosis of human ovarian carcinoma cell line A2780. METHODS: MTT assay,fluorescent microscopy,and DNA agarose gel electrophoresis were used to determine the role of wogonin on apoptosis of A2780 cells. The telomerase activity of A2780 cells were observed by using TRAP-ELASA method. RESULTS: A2780 cell growth was significantly inhibited by wogonin. The inhibiting effect showed concentration-dependent and time-dependent manners with IC(50) of 85 microg/ml. After treatment with 50 microg/ml and 100 microg/ml wogonin for 48 hours, A2780 cells showed morphological changes associated with the characters of apoptosis under fluorescent microscope. Typical DNA ladder was found using agarose gel electrophoresis. Telomerase activity of A2780 cells was gradually decreased with the increasing of wogonin concentration. When the concentration of wogonin was higher than 200 microg/ml, telomerase activity of A2780 cells was inhibited markedly. CONCLUSION: Wogonin can inhibit proliferation and induce apoptosis of A2780 cells within a certain concentration range(50-250 microg/ml). Anticancer effects of wogonin were associated with the induction of apoptosis and partly with the suppression of telomerase activity.

[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.