Wen Di

FTY720 induces necrotic cell death and autophagy in ovarian cancer cells: a protective role of autophagy.

FTY720, a sphingosine analog, is a novel immunosuppressant currently undergoing multiple clinical trials for the prevention of organ transplant rejection and treatment of various autoimmune diseases. Recent studies indicate an additional cytotoxic effect of FTY720 and its preclinical efficacy in a variety of cancer models, yet the underlying mechanisms remain unclear. We demonstrate here for the first time that FTY720 exhibits a potent, dose- and time-dependent cytotoxic effect in human ovarian cancer cells, even in the cells that are resistant to cisplatin, a commonly prescribed chemotherapeutic drug for treatment of ovarian cancer. In contrast to the previously reported cytotoxicity of FTY720 in many other cancer cell types, FTY720 kills ovarian cancer cells independent of caspase 3 activity and induces cellular swelling and cytoplasmic vacuolization with evident features of necrotic cell death. Furthermore, the presence of autophagic hallmarks, including an increased number of autophagosomes and the formation and accumulation of LC3-II, are observed in FTY720-treated cells before cell death. FTY720 treatment enhances autophagic flux as reflected in the increased LC3 turnover and p62 degradation. Notably, blockade of autophagy by either specific chemical inhibitors or siRNAs targeting Beclin 1 or LC3 resulted in aggravated necrotic cell death in response to FTY720, suggesting that FTY720-induced autophagy plays a self-protective role against its own cytotoxic effect. Thus, our findings not only demonstrate a new death pathway underlying the cytotoxic effect of FTY720, but also suggest that targeting autophagy could augment the anticancer potency, providing the framework for further development of FTY720 as a new chemotherapeutic agent for ovarian cancer treatment.

MiR-199a attenuates endometrial stromal cell invasiveness through suppression of the IKKβ/NF-κB pathway and reduced interleukin-8 expression

MicroRNAs have recently been identified as regulators that modulate target gene expression and are suggested to be involved in the development and progression of endometriosis. This study was undertaken to analyze the expression level of microRNA-199a (miR-199a) in paired ovarian endometrioma and eutopic endometrium from women with endometriosis, and to investigate the contribution of miR-199a to the invasive capability of endometrial stromal cells (ESCs). Cell adhesion, migration and Matrigel invasion assays were carried out to measure the invasiveness of ESCs. Bioinformatics prediction, reporter gene assay, PCR, western blotting and ELISA were performed to identify miR-199a targets and related signaling pathways. The results showed that the expression level of miR-199a was lower in the eutopic endometrium from women with endometriosis, and even lower in the paired ovarian endometrioma, compared with the expression in normal controls. Moreover, ectopic expression of miR-199a attenuated ESC adhesion, migration and invasiveness. MiR-199a targeted and inhibited IkappaB kinase beta (IKKβ) in ESCs. Accompanied by IKKβ reduction, miR-199a suppressed nuclear factor-kappa B (NF-κB) pathway activation and interleukin-8 (IL-8) production in ESCs. All these findings suggest that miR-199a, down-regulated in endometriosis, attenuates the invasive capability of ESCs in vitro partly through IKK/NF-κB pathway suppression and reduced IL-8 expression. In conclusion, miR-199a could be involved in the pathogenesis of endometriosis.

MicroRNA-7 Inhibits Tumor Metastasis and Reverses Epithelial-Mesenchymal Transition through AKT/ERK1/2 Inactivation by Targeting EGFR in Epithelial Ovarian Cancer

Epidermal growth factor receptor (EGFR) overexpression and activation result in increased proliferation and migration of solid tumors including ovarian cancer. In recent years, mounting evidence indicates that EGFR is a direct and functional target of miR-7. In this study, we found that miR-7 expression was significantly downregulated in highly metastatic epithelial ovarian cancer (EOC) cell lines and metastatic tissues, whereas the expression of, EGFR correlated positively with metastasis in both EOC patients and cell lines. Overexpression of miR-7 markedly suppressed the capacities of cell invasion and migration and resulted in morphological changes from a mesenchymal phenotype to an epithelial-like phenotype in EOC. In addition, overexpression of miR-7 upregulated CK-18 and β-catenin expression and downregulated Vimentin expression, accompanied with EGFR inhibition and AKT/ERK1/2 inactivation. Similar to miR-7 transfection, silencing of EGFR with this siRNA in EOC cells also upregulated CK-18 and β-catenin expression and downregulated Vimentin expression, and decreased phosphorylation of both Akt and ERK1/2, confirming that EGFR is a target of miR-7 in reversing EMT. The pharmacological inhibition of PI3K-AKT and ERK1/2 both significantly enhanced CK-18 and β-catenin expression and suppressed vimentin expression, indicating that AKT and ERK1/2 pathways are required for miR-7 mediating EMT. Finally, the expression of miR-7 and EGFR in primary EOC with matched metastasis tissues was explored. It was showed that miR-7 is inversely correlated with EGFR. Taken together, our results suggested that miR-7 inhibited tumor metastasis and reversed EMT through AKT and ERK1/2 pathway inactivation by reducing EGFR expression in EOC cell lines. Thus, miR-7 might be a potential prognostic marker and therapeutic target for ovarian cancer metastasis intervention.

Arsenic trioxide and cisplatin synergism increase cytotoxicity in human ovarian cancer cells: therapeutic potential for ovarian cancer.

Drug resistance is a major concern in the successful treatment of ovarian cancer. In the present study we report a combinational drug regime using arsenic trioxide (ATO) and cisplatin (CDDP) to increase therapeutic potentiality in ovarian cancer cells. ATO‐mediated growth inhibition and apoptosis in human suspension ovarian cancer COC1 cells were evaluated by MTT assay and annexin V assay using flow cytometry, respectively. cDNA arrays were performed to screen ATO‐mediated gene expression. Treatment of COC1 cells with ATO alone resulted in growth inhibition and apoptosis with a dose‐and time‐dependent fashion; further cDNA arrays showed that 34 genes (23 up‐regulated genes and 11 down‐regulated genes) may strongly associate with the antiproliferative and pro‐apoptotic effects induced by ATO. Furthermore, Chou–Talalay analysis was used to evaluate the combinational effect of ATO and CDDP as well as dose‐reduction index (DRI) in a panel of ovarian cancer cells including CDDP‐sensitive and ‐resistant cell lines. The combination index (CI) analysis indicated that the interaction effect of ATO/CDDP exhibited a wide range of synergism in all the adherent ovarian cancer cells (A2780, IGROV‐1, SKOV‐3, and R182) as well as 0.93 to 0.69 for IC50 to IC90 in suspension COC1 cells where CI < 1, =1, and >1, define synergism, additive effect, and antagonism, respectively. More intriguingly, the combination of ATO and CDDP yielded favorable DRIs ranging from 1.23‐fold to 13.51‐fold dose reduction. These results suggest that ATO and its combination with CDDP present therapeutic potential for ovarian cancer, and deserve further preclinical and clinical studies. (Cancer Sci 2009; 100: 2459–2464)

TWEAK promotes ovarian cancer cell metastasis via NF-κB pathway activation and VEGF expression

The poor prognosis of human ovarian cancer is partly due to its metastasis and recurrence. It has been demonstrated that tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor inducible-14 (Fn14) signaling system may be a potential regulator of human tumorigenesis. The objective of this study was to understand the effect of TWEAK on ovarian cancer metastasis. We recently showed that activation of Fn14 signaling by TWEAK promoted cell migration and invasion in human HO-8910PM cells. Treating HO-8910PM cells with TWEAK resulted in the activation of nuclear factor-kappa B (NF-kappaB) and subsequently the translocation of NF-kappaB from cytoplasm to nucleus. In addition, TWEAK promoted vascular endothelial growth factor (VEGF) protein expression, and this effect was dependent upon NF-kappaB transcriptional activity. Blocking the NF-kappaB pathway with PDTC suppressed TWEAK-induced up-regulation of VEGF protein expression and cell metastasis. Our results suggest that TWEAK-Fn14 functions, in part, through the NF-kappaB signaling pathway to up-regulate VEGF expression to foster ovarian cancer cell metastasis. Targeted therapy against TWEAK-Fn14 signaling system as an adjuvant to surgery may improve clinical management of invasive ovarian cancer cells and advance the outcome of this devastating cancer.

Hypoxia inducible factor 1α-mediated LOX expression correlates with migration and invasion in epithelial ovarian cancer

This study investigated the role of LOX in promoting invasion and metastasis of epithelial ovarian cancer in a hypoxic environment and its specific signal transduction pathway. Immunohistochemical detection of HIF-1α and LOX protein expression was performed on formalin-fixed paraffin sections of normal ovary, benign ovarian tumors, borderline and malignant epithelial ovarian tumor paraffin sample, using Mann-Whitney U test for independent comparisons and Wilcoxon signed-ranks test for paired comparisons. HIF-1α and LOX were knocked down in epithelial ovarian cancer cells (EOC), and HIF-1α/LOX regulation mechanism and LOX catalytic activity under hypoxia/reoxygenation microenvironment were explored. Cell migration and invasion ability in LOX inhibited HO8910 cells were investigated under hypoxia/reoxygenation conditions, using matrigel cell invasion and migration assays. We found that HIF-1α and LOX are highly expressed in epithelial ovarian cancer tissues, and the expression of both proteins is significantly correlated with the tumor grade, tumor diameter and lymph node metastasis. HIF-1α expression is positively correlated with the expression of LOX. Specifically, the expression of LOX and HIF-1α markedly increases under hypoxic conditions and decreases after reoxygenation. siRNA knockdown of LOX or β-aminoproprionitrile (βAPN), an inhibitor of LOX activity, that attenuates LOX activity, downregulates HIF-1α protein expression and inhibits HO8910 migratory and invasive abilities. LOX catalytic activity is significantly reduced under hypoxic conditions. Moreover, EOC cells display a marked increase in LOX-dependent FAK/AKT activation and cell migration following hypoxia/reoxygenation. Collectively, our study demonstrates that the hypoxia-HIF-1α, LOX-FAK/AKT pathway regulates the migration and invasion of epithelial ovarian cancer cells under hypoxia/reoxygenation conditions, thus, promoting metastasis of ovarian cancer.

Knockdown of CRM1 inhibits the nuclear export of p27Kip1 phosphorylated at serine 10 and plays a role in the pathogenesis of epithelial ovarian cancer

In a previous study, the nuclear export protein chromosomal region maintenance (CRM1) was correlated with p27(Kip1) in glioma. The aims of the present study were to investigate the expression of CRM1 and pSer10p27 and their functional roles in epithelial ovarian cancer (EOC) tissues. Using immunohistochemical analysis, CRM1 and pSer10p27 expression levels were shown to be associated with histologic stage and grade (P<0.05). High CRM1 and pSer10p27 expression levels were prognostic indicators of overall survival (P<0.05). Knockdown of CRM1 and pSer10p27 expression arrested cell cycle progression and inhibited the proliferation of SKOV3 cells both in vitro and in vivo. These data support the idea that pSer10p27 and CRM1 play cooperative roles in EOC.

Specific cell targeting with APRPG conjugated PEG–PLGA nanoparticles for treating ovarian cancer

Good biocompatibility, specific tumor targeting, effective drug loading capacity and persistence in the circulation in vivo are imperative prerequisites for the antitumor efficiency of nanoparticles and their further clinical application. In this study, APRPG (Ala-Pro-Arg-Pro-Gly) peptide-modified poly (ethylene glycol)-poly (lactic acid) (PEG-PLA) nanoparticles (NP-APRPG) encapsulating inhibitors of angiogenesis (TNP-470) (TNP-470-NP-APRPG) were fabricated. TNP-470-NP-APRPG was designed to feature maleimide-PEG-PLA and mPEG-PLA as carrier materials, the APRPG peptide for targeting angiogenesis, PEG for prolonging circulation in vivo and PLA for loading TNP-470. TNP-470-NP-APRPG was confirmed to be approximately 130 nm in size with negative ζ-potential (-14.3 mV), narrow distribution (PDI = 0.27) and spherical morphology according to dynamic light scattering (DLS) and transmission electron microscopy (TEM) analyses. In addition, X-ray photoelectron spectra (XPS) analyses confirmed 7.73% APRPG grafting on the TNP-470-NP. In vitro, TNP-470-NP-APRPG exhibited effective inhibition of proliferation, migration and tube formation in human umbilical vein endothelial cells (HUVECs). Similar findings were observed for the retardation of tumor growth in SKOV3 ovarian cancer-bearing mice, suggesting the significant inhibition of angiogenesis and antitumor efficiency of TNP-470-NP-APRPG. Moreover, no obvious toxic drug responses were observed. Further evidence obtained from the immunohistochemical examination demonstrated that the tumor growth inhibition was closely correlated with the high rate of apoptosis among endothelial cells and the effective blockade of endothelial cell proliferation. These results demonstrate that NP-APRPG is a promising carrier for delivering TNP-470 to treat ovarian cancer and that this approach has the potential to achieve broad tumor coverage in the clinic.

FOXM1 Modulates Cisplatin Sensitivity by Regulating EXO1 in Ovarian Cancer

Cisplatin is commonly used in ovarian cancer chemotherapy, however, chemoresistance to cisplatin remains a great clinical challenge. Oncogenic transcriptional factor FOXM1 has been reported to be overexpressed in ovarian cancer. In this study, we aimed to investigate the potential role of FOXM1 in ovarian cancers with chemoresistance to cisplatin. Our results indicate that FOXM1 is upregulated in chemoresistant ovarian cancer samples, and defends ovarian cancer cells against cytotoxicity of cisplatin. FOXM1 facilitates DNA repair through regulating direct transcriptional target EXO1 to protect ovarian cancer cells from cisplatin-mediated apoptosis. Attenuating FOXM1 and EXO1 expression by small interfering RNA, augments the chemotherapy efficacy against ovarian cancer. Our findings indicate that targeting FOXM1 and its target gene EXO1 could improve cisplatin effect in ovarian cancer, confirming their role in modulating cisplatin sensitivity.

Highly effective antiangiogenesis via magnetic mesoporous silica-based siRNA vehicle targeting the VEGF gene for orthotopic ovarian cancer therapy

Therapeutic antiangiogenesis strategies have demonstrated significant antitumor efficacy in ovarian cancer. Recently, RNA interference (RNAi) has come to be regarded as a promising technology for treatment of disease, especially cancer. In this study, vascular endothelial growth factor (VEGF)-small interfering RNA (siRNA) was encapsulated into a magnetic mesoporous silica nanoparticle (M-MSN)-based, polyethylenimine (PEI)-capped, polyethylene glycol (PEG)-grafted, fusogenic peptide (KALA)-functionalized siRNA delivery system, termed M-MSN_VEGF siRNA@PEI-PEG-KALA, which showed significant effectiveness with regard to VEGF gene silencing in vitro and in vivo. The prepared siRNA delivery system readily exhibited cellular internalization and ease of endosomal escape, resulting in excellent RNAi efficacy without associated cytotoxicity in SKOV3 cells. In in vivo experiments, notable retardation of tumor growth was observed in orthotopic ovarian tumor-bearing mice, which was attributed to significant inhibition of angiogenesis by systemic administration of this nanocarrier. No obvious toxic drug responses were detected in major organs. Further, the magnetic core of M-MSN_VEGF siRNA@PEI-PEG-KALA proved capable of probing the site and size of the ovarian cancer in mice on magnetic resonance imaging. Collectively, the results demonstrate that an M-MSN-based delivery system has potential to serve as a carrier of siRNA therapeutics in ovarian cancer.