Manhua Cui

Silencing of cyclooxygenase‑2 inhibits the growth, invasion and migration of ovarian cancer cells

The present study aimed to investigate the effect of downregulating cyclooxygenase‑2 (COX‑2) expression on the growth of human ovarian cancer cells. The COX‑2‑specific small interfering RNA (siRNA) plasmid vector was constructed and then transfected into ovarian cancer cells. The expression of COX‑2 mRNA and protein was detected by quantitative polymerase chain reaction and western blot analysis, respectively. Cell proliferation, apoptosis, cell cycle distribution and cell migration were assessed following knockdown of COX‑2 by RNA interference (RNAi). Western blot analysis was used to identify differentially expressed angiogenesis- and cell cycle‑associated proteins in cells with silenced COX‑2. The expression levels of COX‑2 in ovarian cancer cells transfected with siRNA were decreased, leading to a significant inhibition of ovarian cancer cell proliferation, migration and invasion. Western blot analysis revealed that silencing of COX‑2 may inhibit vascular endothelial growth factor, matrix metalloproteinase (MMP)‑2 and MMP‑9 protein expression. In conclusion, the present study demonstrated that RNAi can effectively silence COX‑2 gene expression and inhibit the growth of ovarian cancer cells, which indicates that there is a potential of targeting COX‑2 as a novel gene therapy approach for the treatment of ovarian cancer.

Downregulation of matrix metalloproteinase 9 by small interfering RNA inhibits the tumor growth of ovarian epithelial carcinoma in vitro and in vivo

Matrix metalloproteinase 9 (MMP-9) is upregulated in various types of malignancy, including human ovarian carcinomas. It promotes invasion, metastasis, growth and the survival of malignant cells. However, relatively little is known about the role of MMP9 in epithelial ovarian carcinoma. Therefore, the aim of the present study was to determine the effects of targeting this molecule on ovarian carcinoma progression. A plasmid, psi-MMP-9, carrying a short hairpin RNA against MMP-9 gene expression was constructed and transfected into the human ovarian cancer cell line SKOV3 using a human U6 promoter-driven DNA template approach to determine the effect of MMP-9 gene RNA interference (RNAi) on the proliferation, apoptosis, migration, invasion and tumorigenicity of the human ovarian carcinoma cells. The results demonstrated that siRNA-mediated knockdown of MMP-9 in the human ovarian cancer cell line SKOV3 inhibited cell proliferation, migration and invasion in vitro. The results also demonstrated that downregulation of MMP-9 led to cell apoptosis in SKOV3 cells, inhibited the expression of anti-apoptotic molecules, including B cell lymphoma-2, survivin and X-linked inhibitor of apoptosis protein, and enhanced the activity of capsase-3 and caspase-8. In addition, knockdown of MMP-9 inhibited tumorigenicity in nude mice. Taken together, MMP-9 gene RNAi in ovarian carcinoma cells inhibited proliferation, migration and invasion, induced cell apoptosis in vitro and suppressed tumor growth in nude mice. These results suggest that MMP-9 is an ovarian cancer-associated gene and is a potential target for therapeutic anti-cancer drugs.

Expression and purification of recombinant ATF-mellitin, a new type fusion protein targeting ovarian cancer cells, in P. pastoris.

Melittin is well known to possess cytolytic activity with wide-spectrum lytic properties and its potential use as an agent to treat several types of cancer has been tested. Due to the non-specific toxicity, melittin can impair not only cancer cells but also normal tissue. Thus, tumor-targeted toxins may be helpful for developing novel anticancer therapeutics. The urokinase-type plasminogen activator (uPA) plays a central role in tissue remodelling events occurring in normal physiology and in pathophysiology, including cancer invasion and metastasis. Heartening findings showed that uPA receptor is predominantly expressed on many types of cancer. Therefore, the amino-terminal fragment (ATF) of uPA which was able to identify and bond with cancer cells was used as the cell-targeting domain to make up tumor-targeted toxin in this study. In the present study, pPICZαC-ATF-melittin eukaryotic expression vector was successfully constructed. After transformed into P. pastoris and induced by methanol, rATF-mellitin was detected by SDS-PAGE and western blot analysis. After induction with methanol, the expression level of rATF-mellitin was 312 mg/l in 80-l fermentor. rATF‑mellitin was purified to >95% purity using SP Sepharose ion exchange chromatography and source™ 30 RPC with 67.2% recovery. Cell proliferation assay showed that rATF-melittin inhibited growth of SKOV3 cells and had no cytotoxicity effect on normal cells. For the first time, we established a stable and effective rATF-mellitin P. pastoris expression system to obtain a high level of expression of secreted rATF-mellitin which was purified by a highly efficient purification procedure.

Focus on Mesenchymal Stem Cell-Derived Exosomes: Opportunities and Challenges in Cell-Free Therapy

Mesenchymal stem cells have been at the forefront of regenerative medicine for many years. Exosomes, which are nanovesicles involved in intercellular communication and the transportation of genetic material transportation that can be released by mesenchymal stem cells, have been recently reported to play a role in cell-free therapy of many diseases, including myocardial infarction, drug addiction, and status epilepticus. They are also thought to help ameliorate inflammation-induced preterm brain injury, liver injury, and various types of cancer. This review highlights recent advances in the exploration of mesenchymal stem cell-derived exosomes in therapeutic applications. The natural contents, drug delivery potency, modification methods, and drug loading methods of exosomes are also discussed.

Expression and anticancer activity analysis of recombinant human uPA1‑43-melittin.

The present study is focused on expression of a target fusion protein which can be used in ovarian cancer target therapy. It aimed to construct human urokinase-type plasminogen activator (uPA)(1-43)-melittin eukaryotic expression vector to express recombinant human uPA(1-43)-melittin (rhuPA(1-43)-melittin) in P. pastoris and to detect its anticancer effects on ovarian cancer cells. The DNA sequences that encode uPA1-43 amino acids and melittin were synthesized according to its native amino acid sequences and consequently inserted into pPICZαC vector. Then uPA1-43-melittin -pPICZαC was transformed into P. pastoris X-33, and rhuPA(1-43)-melittin was expressed by methonal inducing. The bioactivities of recombinant fusion protein were detected with inhibition effects on growth of ovarian cancer cells, cell cycle detection and TUNEL assay. The results of DNA sequence analysis of the recombinant vector uPA(1-43)-melittin -pPICZαC demonstrated that the DNA encoding human uPA 1-43 amino acids and 1-26 amino acids of melittin was correctly inserted into the pPICZαC vector. After being induced by methonal, fusion protein with molecular weight 7.6 kDa was observed on the basis of SDS-PAGE and western blot analysis. The recombinant protein was able to suppress growth of SKOV3, induce cell cycle arrest and apoptosis of SKOV3 cells. The fusion protein does not have any obvious toxicity on normal tissues. RhuPA(1-43)-melittin was successfully expressed in P. pastoris. Taking uPA(1-43) amino acids specifically binding to uPAR as targeted part of fusion protein, and making use of antitumor activity of melittin, the recombinant fusion protein it was able to inhibit growth of ovarian tumors and to be applied for effective targeted treatment.

Endometriosis Malignant Transformation: Epigenetics as a Probable Mechanism in Ovarian Tumorigenesis

Endometriosis, defined as the presence of ectopic endometrial glands and stroma outside the uterine cavity, is a chronic, hormone-dependent gynecologic disease affecting millions of women across the world, with symptoms including chronic pelvic pain, dysmenorrhea, dyspareunia, dysuria, and subfertility. In addition, there is well-established evidence that, although endometriosis is considered benign, it is associated with an increased risk of malignant transformation, with the involvement of various mechanisms of development. More and more evidence reveals an important contribution of epigenetic modification not only in endometriosis but also in mechanisms of endometriosis malignant transformation, including DNA methylation and demethylation, histone modifications, and miRNA aberrant expressions. In this present review, we mainly summarize the research progress about the current knowledge regarding the epigenetic modifications of the relations between endometriosis malignant transformation and ovarian cancer in an effort to identify some risk factors probably associated with ectopic endometrium transformation.

The Progress of Methylation Regulation in Gene Expression of Cervical Cancer

Cervical cancer is one of the most common gynecological tumors in females, which is closely related to high-rate HPV infection. Methylation alteration is a type of epigenetic decoration that regulates the expression of genes without changing the DNA sequence, and it is essential for the progression of cervical cancer in pathogenesis while reflecting the prognosis and therapeutic sensitivity in clinical practice. Hydroxymethylation has been discovered in recent years, thus making 5-hmC, the more stable marker, attract more attention in the field of methylation research. As markers of methylation, 5-hmC and 5-mC together with 5-foC and 5-caC draw the outline of the reversible cycle, and 6-mA takes part in the methylation of RNA, especially mRNA. Furthermore, methylation modification participates in ncRNA regulation and histone decoration. In this review, we focus on recent advances in the understanding of methylation regulation in the process of cervical cancer, as well as HPV and CIN, to identify the significant impact on the prospect of overcoming cervical cancer.

Tumor necrosis factor-related apoptosis inducing ligand overexpression and Taxol treatment suppresses the growth of cervical cancer cells in vitro and in vivo

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a member of tumor necrosis factor (TNF) superfamily and functions to promote apoptosis by binding to cell surface death receptor (DR)4 and DR5. Cancer cells are more sensitive than normal cells to TRAIL-induced apoptosis, and TRAIL-based therapeutic strategies have shown promise for the treatment of cancer. The present study investigated whether enforced overexpression of TRAIL in cervical cancer cells promoted cell death in the presence or absence of Taxol, an important first-line cancer chemotherapeutic drug. Hela human cervical cancer cells were transfected with a TRAIL expression plasmid, and the effects of the combination treatment with Taxol on apoptosis was investigated in vitro and in tumor xenografts in vivo. The results indicated that Taxol treatment and TRAIL overexpression enhanced apoptosis compared with either treatment alone. The present data indicate that Taxol may enhance the pro-apoptotic effects of TRAIL overexpression in HeLa cells by increasing cleaved caspase-3 and DR5 expression levels and decreasing Bcl-2 expression levels. Furthermore, the findings suggest a possible novel treatment option for cervical cancer and uncovers a potential mechanism of the enhancing effects of Taxol on TRAIL-induced apoptosis.

A novel uPAg-KPI fusion protein inhibits the growth and invasion of human ovarian cancer cells in vitro

Urokinase-type plasminogen activator (uPA) acts by breaking down the basement membrane and is involved in cell proliferation, migration and invasion. These actions are mediated by binding to the uPA receptor (uPAR) via its growth factor domain (GFD). The present study evaluated the effects of uPAg-KPI, a fusion protein of uPA-GFD and a kunitz protease inhibitor (KPI) domain that is present in the amyloid β-protein precursor. Using SKOV-3 cells, an ovarian cancer cell line, we examined cell viability, migration, invasion and also protein expression. Furthermore, we examined wound healing, and migration and invasion using a Transwell assay. Our data showed that uPAg-KPI treatment reduced the viability of ovarian cancer SKOV-3 cells in both a concentration and time-dependent manner by arresting tumor cells at G1/G0 phase of the cell cycle. The IC50 of uPAg-KPI was 0.5 µg/µl after 48 h treatment. At this concentration, uPAg-KPI also inhibited tumor cell colony formation, wound closure, as well as cell migration and invasion capacity. At the protein level, western blot analysis demonstrated that uPAg-KPI exerted no significant effect on the expression of total extracellular signal-regulated kinase (ERK)1/ERK2 and AKT, whereas it suppressed levels of phosphorylated ERK1/ERK2 and AKT. Thus, we suggest that this novel uPAg-KPI fusion protein reduced cell viability, colony formation, wound healing and the invasive ability of human ovarian cancer SKOV-3 cells in vitro by regulating ERK and AKT signaling. Further studies using other cell lines will confirm these findings.

Cysteine-rich, angiogenic inducer, 61 expression in patients with ovarian epithelial carcinoma.

Objective Cysteine-rich, angiogenic inducer, 61 (CYR61) is a key gene in the transforming growth factor-β signalling pathway, which is involved in the development of many tumour types. This study aimed to clarify the status and clinical significance of CYR61 expression in patients with ovarian epithelial carcinoma. Methods Tissue from patients with ovarian epithelial carcinoma or benign ovarian tumours were investigated retrospectively for CYR61 expression at mRNA and protein levels, using reverse transcription–polymerase chain reaction and immunohistochemistry, respectively. Correlations between immunohistochemical scores and several clinicopathological parameters were investigated. Results In 50 patients with ovarian epithelial carcinoma and 50 patients with benign ovarian tumours, CYR61 expression on mRNA and protein levels was significantly higher in ovarian epithelial carcinoma tissue than in benign ovarian tissue. CYR61 expression was associated with regional lymph node metastases and progression of clinical disease stage. There was no difference in CYR61 expression between patients aged <50 years and ≥50 years. Conclusions CYR61 expression was significantly upregulated in ovarian carcinoma tissue compared with benign ovarian tumour tissue samples. Protein CYR61 levels were associated with lymph node metastases and Union for International Cancer Control stage. Protein CYR61 may be useful in targeted diagnosis and therapy, for patients with ovarian epithelial carcinoma.