The metastatic phenotype shift toward myofibroblast of adipose-derived mesenchymal stem cells promotes ovarian cancer progression.

Ovarian cancer metastasizes to organs in the abdominal cavity, such as the omentum that is a rich source of adipose-derived mesenchymal stem cells (ADSCs). In present, ADSCs have received more and more attention for their roles in the development of cancer. In this study, we examined α-smooth muscle actin (α-SMA) expression and carcinoma-associated fibroblast (CAF)-like differentiation capabilities in ADSCs from omentum of different patients. The effects of ADSCs on the proliferation and invasion of epithelial ovarian cancer cells (EOCCs) were also assessed in vitro and in vivo. Our results showed that ADSCs from omentum of ovarian cancer patients, no matter whether metastasis or not, expressed higher levels of α-SMA than ADSCs from patients with benign gynecologic disease. Using direct and indirect co-culture system, we found that EOCCs induced ADSCs to express CAF markers, including α-SMA and fibroblast activation protein, via the transforming growth factor beta 1 (TGF-ß1) signaling pathway. Moreover, co-cultured ADSCs exhibited functional properties similar to those of CAFs, including the ability to promote EOCCs proliferation, progression and metastasis both in vitro and in vivo. Furthermore, blocking the TGF-ß1 pathway can counteract the CAF-like differentiation and tumor promotion effect of ADSCs. Our results suggest that ADSCs are a source of CAFs and that they participate in the interaction between EOCCs and the omental microenvironment. EOCCs could induce ADSCs in the omentum to differentiate before ovarian cancer metastasis, which participate in the formation of omental metastatic niches and promote the proliferation and invasion of ovarian cancer.

Tumor cell­fibroblast heterotypic aggregates in malignant ascites of patients with ovarian cancer.

Ascitic multicellular aggregates (MCAs) promote peritoneal metastasis of ovarian cancer. The aim of the present study was to elucidate the role of cancer­associated fibroblasts (CAFs) in MCA formation and metastasis in patients with high­grade serous ovarian cancer (HGSOC). Immunohistochemistry was used to identify the cell phenotypes and the presence of CAFs in ascitic MCAs. The role of CAFs in tumor­cell MCA formation was assessed by co­culture in suspension. Primary ascitic tumor cells and omental CAFs were used to generate ex vivo MCAs in hanging drops, and the invasiveness of MCAs was evaluated by mesothelial clearance and adhesion assays in vitro and in vivo. MCAs containing CAFs and tumor cells were identified in the ascitic fluid. CAFs facilitated tumor cell aggregation and compaction to form MCAs, and enhanced the mesothelial clearance and adhesion abilities of tumor­cell MCAs. These findings suggest that ascitic CAFs promote peritoneal metastasis by forming heterotypic aggregates with tumor cells, and that they may serve as potential targets for the treatment of HGSOC.

Dicer affects cisplatin­mediated apoptosis in epithelial ovarian cancer cells.

Dicer is an essential enzyme that processes micro (mi)-RNA precursors into mature miRNAs, and serves a critical role in cancer development and progression by regulating gene expression. However, the role of Dicer in cisplatin­mediated apoptosis and chemotherapy resistance in epithelial ovarian cancer (EOC) cells is poorly understood. In the present study, Dicer was expressed at low levels in cisplatin­resistant A2780 cells when compared with parental cells. In addition, knocking down Dicer using short hairpin RNA decreased the sensitivity of A2780 and CAOV3 cells to cisplatin. Furthermore, downregulating Dicer significantly inhibited cisplatin­induced apoptosis in ovarian cancer cells, and decreased the levels of proteins involved in apoptosis signaling pathways, including P73, P63, P53, caspase­9 and caspase­3. These findings indicated that Dicer may be a promising target for overcoming drug resistance in ovarian cancer.

Metformin inhibits ovarian cancer via decreasing H3K27 trimethylation.

Metformin has been used for the treatment of type II diabetes mellitus for decades. Recently, used of metformin in the therapy of diverse human cancer types has received widespread attention, while the underlying mechanisms have been not fully elucidated. In the current study, 5-ethynyl-20-deoxyuridine assay to detect cell proliferation, flow cytometry to detect apoptosis, scratch wound healing and Transwell migration assay to detect cell migration capacity. The current study reported that metformin inhibited cell proliferation and migration, and promoted apoptosis in ovarian cancer cells, particularly under normoglycemic conditions in vitro. Metformin treatment significantly promoted the phosphorylation of AMP-activated protein kinase (AMPK), and reduced histone H3 lysine 27 trimethylation (H3K27me3) and polycomb repressor complex 2 (PRC2) levels. Additionally, overexpression of EZH2 to increase H3K27me3 abrogated the effect of metformin on the cell proliferation, migration and apoptosis in SKOV3 and ES2 cells. Similar to metformin, another AMPK agonist, 2-deoxy-D-glucose, reduced the H3K27me3 level and PRC2 expression. In cells pretreated with Compound C, an AMPK inhibitor, metformin was not able to induce AMPK phosphorylation or reduce H3K27me3. Metformin-mediated AMPK activation and H3K27me3 inhibition were more robust in cells exposed to low glucose (5.5 mM) compared with those exposed to high glucose (25 mM). These findings implicate H3K27me3 repression mediated by AMPK phosphorylation in the antitumor effect of metformin in ovarian cancer, indicating that metformin alters epigenetic modifications by targeting PRC2 and supports the use of metformin in treatment of patients with epithelial ovarian cancer without diabetes.

Adipose­derived mesenchymal stem cells attenuate cisplatin­induced apoptosis in epithelial ovarian cancer cells.

Environment-mediated drug resistance (EMDR) serves an important role in tumor chemotherapy resistance. Adipose­derived mesenchymal stem cells (ADSC) are an important component of the tumor microenvironment. However, the role of ADSC in EMDR remains unclear. Therefore, in order to clarify whether ADSCs contribute to the cisplatin­mediated apoptosis in epithelial ovarian cancer (EOC), the present study isolated ADSCs from the omentum of women with benign disease and collected the ADSC culture medium as conditioned medium. Subsequently, it was revealed that ADSCs decreased the sensitivity of EOC cells to cisplatin via an MTT assay. In addition, it was revealed that ADSCs may reduce cisplatin-induced apoptosis in EOC cells, as determined by Hoechst staining and flow cytometric analysis. Additionally, a lower level of cleaved caspase­3 was observed, accompanied with decreased intracellular platinum accumulation in EOC cells indirectly co­cultured with ADSCs. In conclusion, the findings of the present study suggested that ADSCs reduced cisplatin­induced apoptosis and the intracellular level of platinum in EOC cells, which in turn resulted in cisplatin resistance. Therefore, ADSCs may serve as a therapeutic target for recurrent EOC.

Role of EZH2 in cancer stem cells: from biological insight to a therapeutic target.

Epigenetic modifications in cancer stem cells largely result in phenotypic and functional heterogeneity in many solid tumors. Increasing evidence indicates that enhancer of zeste homolog 2 (EZH2), the catalytic subunit of Polycomb repressor complex 2, is highly expressed in cancer stem cells of numerous malignant tumors and has a critical function in cancer stem cell expansion and maintenance. Here, we review up-to-date information regarding EZH2 expression patterns, functions, and molecular mechanisms in cancer stem cells in various malignant tumors and discuss the therapeutic potential of targeting EZH2 in tumors.

SOX2 is required to maintain cancer stem cells in ovarian cancer.

Ovarian cancer cells can form spheroids under serum-free suspension culture conditions. The spheroids, which are enriched in cancer stem cells, can result in tumor dissemination and relapse. To identify new targetable molecules in ovarian cancer spheroids, we investigated the differential expression of genes in spheroids compared with that under monolayer culture conditions by qPCR microarray. We identified that SOX2 is overexpressed in spheroids. We then proved that SOX2 expression was increased in successive spheroid generations. Besides, knockdown of SOX2 expression in SKOV3 or HO8910 ovarian cancer spheroid cells decreased spheroid formation, cell proliferation, cell migration, resistance to Cisplatin treatment, tumorigenicity, and the expression of stemness-related genes and epithelial to mesenchymal transition-related genes, whereas overexpression of SOX2 in SKOV3 or HO8910 ovarian cancer cells showed the opposite effects. In addition, we found that SOX2 expression was closely associated with chemo-resistance and poor prognosis in EOC patients. These results strongly suggest that SOX2 is required to maintain cancer stem cells in ovarian cancer. Targeting SOX2 in ovarian cancer may be therapeutically beneficial.

Comparison of different sample preparation methods for platinum determination in cultured cells by graphite furnace atomic absorption spectrometry.

BACKGROUND: Platinum-based agents are widely used in chemotherapy against solid tumors and insufficient intracellular drug accumulation is one of the leading causes of platinum resistance which is associated with poor survival of tumor patients. Thus, the detection of intracellular platinum is pivotal for studies aiming to overcome platinum resistance. In the present study, we aimed to establish a reliable graphite furnace atomic absorption spectrometry (GFAAS)-based assay to quantify the intracellular platinum content for cultured cells. METHODS: Several most commonly applied cell preparation methods, including 0.2% HNO3, 0.2% Triton X-100, concentrated nitric acid, RIPA combined with concentrated nitric acid and hydroxide, followed by GFAAS for platinum detection were compared in ovarian, cervical and liver cancer cell lines to obtain the optimal one, and parameters regarding linearity, accuracy, precision and sensitivity were evaluated. Influence of other metals on platinum detection and the storage conditions of samples were also determined. RESULTS: The treatment of cells with 0.2% HNO3 was superior to other approaches with fewer platinum loss and better repeatability. The recovery rate and precision of this method were 97.3%-103.0% and 1.4%-3.8%, respectively. The average recoveries in the presence of other metals were 95.1%-103.1%. The detection limit was 13.23 ug/L. The recovery rate of platinum remained acceptable even in cell samples stored in -20 °C or -80 °C for two months. DISCUSSION: After comparison, we found that 0.2% HNO3 was optimal for intracellular platinum quantification based on GFAAS, which presented values compatible with that of inductively-coupled plasma mass-spectrometry (ICP-MS), and this is partially attributed to the simplicity of this method. Moreover, the assay was proved to be accurate, sensitive, cost-effective and suitable for the research of platinum-based antitumor therapy.

Role of HER family members in predicting prognoses in epithelial ovarian cancer: a meta-analysis.

AIMS AND BACKGROUND: Human epidermal receptor (HER) family receptors are commonly overexpressed in various human tumors, and their overexpression is thought to play a critical role in tumor progression. The aim of this meta-analysis was to evaluate the prognostic significance of HER family members in epithelial ovarian cancer (EOC). METHODS: Relevant studies published between January 1, 1980, and April 24, 2013, that evaluated the associations of HER family members with overall survival (OS), progression-free survival (PFS), response to platinum-based chemotherapy, lymph node metastasis, or ascites in EOC were identified via searches of PubMed and EMBASE. RESULTS: We identified 37 eligible articles that met the inclusion criteria. The results of the meta-analysis revealed that significantly poorer OS of patients with EOC was predicted by high Her-2 expression levels (hazard ratio [HR] 1.69, 95% confidence interval [CI] 1.31-2.19). Furthermore, high Her-2 expression was significantly associated with poor PFS (HR 1.88, 95% CI 1.46-2.41) and an increased risk of ascites (risk ratio 1.21, 95% CI 1.02-1.42). CONCLUSIONS: High levels of expression of Her-2 are significantly related to poor survival and an increased risk of ascites in patients with EOC. Future prospective cohorts with larger samples are needed to verify the prognostic value of Her-2 expression in EOC.

Circulating soluble neuropilin-1 in patients with early cervical cancer and cervical intraepithelial neoplasia can be used as a valuable diagnostic biomarker.

OBJECTIVE: To investigate soluble neuropilin-1 (sNRP-1) in circulating and NRP-1 protein in cervical tissues from patients with cervical cancer or cervical intraepithelial neoplasia (CIN). METHODS: sNRP-1 was measured in 64 preoperative patients and 20 controls. NRP-1 protein in cervical tissue was detected in 56 patients and 20 controls. RESULTS: Both sNRP-1 and NRP-1 proteins were correlated with stage. sNRP-1 presented a high diagnostic ability of cervical cancer and CIN, with a sensitivity of 70.97% and a specificity of 73.68%. CONCLUSIONS: sNRP-1 in circulating can serve as a possible valuable diagnostic biomarker for cervical cancer and CIN.

Tumor-associated macrophages induce lymphangiogenesis in cervical cancer via interaction with tumor cells.

Our studies were conducted to investigate the clinical and functional significance of tumor-associated macrophages (TAMs) in cervical tumor lymphatic metastasis. We found that the increase in macrophages in tumor stroma is significantly associated with lymphatic metastasis (p = 0.017), through performing immunohistochemical staining in 111 cervical samples (55 invasive squamous carcinomas of uterine cervix, 27 cervical intraepithelial neoplasms III, and 29 normal cervix). The human lymphatic endothelial cells (HLEC), which were cultured in conditioned medium of cervical cancer cell-macrophage coculture, formed more tube-like structures in vitro, when compared with those in conditioned mediums of LEC, normal cervical epithelium, single macrophage, and single cervical cancer cell (all p < 0.001). The mRNA expressions of IL-1ß and IL-8 in cervical cancer cells cocultured with macrophages were increased, compared with those in cervical cancer cell cultured alone (pIL-1ß  < 0.05 and pIL-8  < 0.01). Meanwhile, the mRNA expression of VEGF-C and VEGF-A was increased in macrophages cocultured with cervical cancer cells, compared with the expression in those macrophages cultured alone (both p < 0.05). Taken together, the results suggest that TAMs promote lymphangiogenesis mainly through interaction with surrounding cervical cancer cells.

Proteomic identification of target proteins following Drosha knockdown in cervical cancer.

The nuclear microRNA (miRNA) processing enzyme Drosha is upregulated in cervical cancer, and its overexpression is related to an invasive tumour phenotype. However, the mechanisms that underlie this effect remain poorly understood. The aim of this study was to identify the potential targets of Drosha in cervical cancer. Here, we demonstrated that Drosha knockdown (Drosha-KD) inhibited proliferation, colony formation and the migration of cervical cancer cells in vitro. A global upregulation of proteins in Drosha-KD cells was revealed by two-dimensional gel electrophoresis (2-DE). Eighteen proteins were identified by liquid chromatography and tandem mass spectrometry technology (LC-MS/MS) from 21 selected protein spots that exhibited significant alterations in Drosha-KD cells. The majority of the identified proteins have been previously associated with tumour formation. The downregulation of tubulin 5ß in Drosha-KD cervical cancer cells was further confirmed by western blotting. Our results suggest that Drosha affects the biological activity of cervical cancer cells by regulating the expression of numerous tumour-associated proteins.

[Disfigurement of p16INK4A gene expression in development of ovarian cancer and the mechanism].

OBJECTIVE: To evaluate the expression of p16INK4A gene in ovarian cancer and analyze the relation between this alteration and the promoter methylation of p16INK4A DNA. METHODS: The expression of p16INK4A mRNA was detected with RT-PCR in ovarian cancer cell lines and tissues, and the protein was detected by western blot. Methylation specific PCR (MSP) was used to check whether it was methylated in the promoter of p16INK4A, and normal ovarian tissues were used as control. The demethylating agent, 5-aza-2'-deoxycytidine, was used to treat methylated ovarian cancer cells and then, ovarian cancer cell growth was measured in vivo and in vitro. RESULTS: Three ovarian cancer cell lines (Anglne, SW626, OVCAR3) and six ovarian cancer specimens were found methylated in p16INK4A DNA; the methylation rate was 3/7 and 33% (6/18) in ovarian cancer cell lines and specimens respectively. All the methylated cell lines and ovarian cancer tissues expressed decreasing p16INK4A mRNA and protein. Compared with the control, both the expression of p16INK4A mRNA and protein were decreased significantly or absolutely deleted in ovarian cancer cells (P < 0.05). The decrease was partly due to the methylation of p16INK4A. 5-aza-2'-deoxycytidine could reactivate the expression of p16INK4A in methylated cells and decrease the cell growth in vivo and in vitro. CONCLUSION: The disfigurement of p16INK4A gene plays an important role in the development of ovarian cancer, and this alteration is partially caused by methylation of p16INK4A DNA.