The anti-tumor effects of AZD4547 on ovarian cancer cells: differential responses based on c-Met and FGF19/FGFR4 expression.

BACKGROUND: The FGF/FGFR signaling pathway plays a critical role in human cancers. We analyzed the anti-tumor effect of AZD4547, an inhibitor targeting the FGF/FGFR pathway, in epithelial ovarian cancer (EOC) and strategies on overcoming AZD4547 resistance. METHODS: The effect of AZD4547 on cell viability/migration was evaluated and in vivo experiments in intraperitoneal xenografts using EOC cells and a patient-derived xenograft (PDX) model were performed. The effect of the combination of AZD4547 with SU11274, a c-Met-specific inhibitor, FGF19-specific siRNA, or an FGFR4 inhibitor was evaluated by MTT assay. RESULTS: AZD4547 significantly decreased cell survival and migration in drug-sensitive EOC cells but not drug-resistant cells. AZD4547 significantly decreased tumor weight in xenograft models of drug-sensitive A2780 and SKOV3ip1 cells and in a PDX with drug sensitivity but not in models with drug-resistant A2780-CP20 and SKOV3-TR cells. Furthermore, c-Met expression was high in SKOV3-TR and HeyA8-MDR cells, and co-administration of SU11274 and AZD4547 synergistically induced cell death. In addition, expressions of FGF19 and FGFR4 were high in A2780-CP20 cells. Combining AZD4547 with FGF19 siRNA or with a selective FGFR4 inhibitor led to significantly reduced cell proliferation in A2780-CP20 cells. CONCLUSIONS: This study showed that AZD4547 has significant anti-cancer effects in drug-sensitive cells and PDX models but not in drug-resistant EOC cells. In drug-resistant cells, the expression level of c-Met or FGF19/FGFR4 may be a predictive biomarker for AZD4547 treatment response, and a combination strategy of drugs targeting c-Met or FGF19/FGFR4 together with AZD4547 may be an effective therapeutic strategy for EOC.

Ulipristal acetate, a selective progesterone receptor modulator, induces cell death via inhibition of STAT3/CCL2 signaling pathway in uterine sarcoma.

Ulipristal acetate (UPA) is a selective progesterone receptor modulator and is used for the treatment of uterine leiomyoma (a benign tumor). Uterine sarcoma which is highly malignant cancer with a poor prognosis is clinically resembled with uterine leiomyoma. There has been no experimental research on the effect of UPA on uterine sarcoma. In this study, we examined the efficacy of UPA in uterine sarcoma with in vitro and in vivo animal models. Cytotoxicity of UPA was determined in uterine sarcoma cell lines (MES-SA, SK-UT-1, and SK-LMS-1). Apoptotic genes and signaling pathways affected by UPA were analyzed by complementary DNA (cDNA) microarray of uterine sarcoma cell lines and western blot, respectively. An in vivo efficacy of UPA was examined with uterine sarcoma cell line- and patient-derived xenograft (PDX) mice models. UPA inhibited cell growth in uterine sarcoma cell lines and primary culture cells from a PDX mouse (PDX-C). cDNA microarray analysis revealed that CCL2 was highly down-regulated by UPA. Phosphorylation and the total expression of STAT3 were inhibited by UPA. UPA also inhibited CCL2 and STAT3 in PDX-C. The inhibitory effect of UPA had not changed in the overexpression of PR and treatment of progesterone. In vivo efficacy studies with cell line-derived xenografts and a PDX model with leiomyosarcoma, a typical uterine sarcoma, demonstrated that UPA significantly decreased tumor growth. UPA had significant anti-tumor effects in uterine sarcoma through the inhibition of STAT3/CCL2 signaling pathway and might be a potential therapeutic agent to treat this disease.

Anti-cancer effect of fenbendazole-incorporated PLGA nanoparticles in ovarian cancer.

OBJECTIVE: Fenbendazole (FZ) has potential anti-cancer effects, but its poor water solubility limits its use for cancer therapy. In this study, we investigated the anti-cancer effect of FZ with different drug delivery methods on epithelial ovarian cancer (EOC) in both in vitro and in vivo models. METHODS: EOC cell lines were treated with FZ and cell proliferation was assessed. The effect of FZ on tumor growth in cell line xenograft mouse model of EOC was examined according to the delivery route, including oral and intraperitoneal administration. To improve the systemic delivery of FZ by converting fat-soluble drugs to hydrophilic, we prepared FZ-encapsulated poly(D,L-lactide-co-glycolide) acid (PLGA) nanoparticles (FZ-PLGA-NPs). We investigated the preclinical efficacy of FZ-PLGA-NPs by analyzing cell proliferation, apoptosis, and in vivo models including cell lines and patient-derived xenograft (PDX) of EOC. RESULTS: FZ significantly decreased cell proliferation of both chemosensitive and chemoresistant EOC cells. However, in cell line xenograft mouse models, there was no effect of oral FZ treatment on tumor reduction. When administered intraperitoneally, FZ was not absorbed but aggregated in the intraperitoneal space. We synthesized FZ-PLGA-NPs to obtain water solubility and enhance drug absorption. FZ-PLGA-NPs significantly decreased cell proliferation in EOC cell lines. Intravenous injection of FZ-PLGA-NP in xenograft mouse models with HeyA8 and HeyA8-MDR significantly reduced tumor weight compared to the control group. FZ-PLGA-NPs showed anti-cancer effects in PDX model as well. CONCLUSION: FZ-incorporated PLGA nanoparticles exerted significant anti-cancer effects in EOC cells and xenograft models including PDX. These results warrant further investigation in clinical trials.

Hydroxychloroquine reduces hypertension and soluble fms-like kinase-1 in a Nω-nitro-l-arginine methyl ester-induced preeclampsia rat model.

OBJECTIVE: Hydroxychloroquine, a drug used for malaria and autoimmune diseases reportedly has beneficial effects against preeclampsia in pregnant women with lupus. However, its mechanism against preeclampsia remains unclear. We investigated the effect of hydroxychloroquine on an Nω-nitro-l-arginine methyl ester-induced preeclampsia rat model. METHODS: Pregnant Sprague-Dawley rats were divided into four groups based on treatment (administered on gestational days 7-18): control, Nω-nitro-l-arginine methyl ester, hydroxychloroquine, and Nω-nitro-l-arginine methyl ester plus hydroxychloroquine. All animals were sacrificed on gestational day 19. We assayed tube formation and determined reactive oxygen species levels using human umbilical vein endothelial cells. RESULTS: Results showed that hydroxychloroquine significantly lowered mean systolic blood pressure (P  < 0.05) in Nω-nitro-l-arginine methyl ester-treated rats. Hydroxychloroquine did not affect their fetal and placental weights. Hydroxychloroquine mitigated Nω-nitro-l-arginine methyl ester-associated changes in proteinuria (P  < 0.05). It normalized plasma soluble fms-like kinase-1 (P  < 0.05) and endothelin-1 (P  < 0.01) levels. In the tube formation assay, hydroxychloroquine increased the total meshes area (P  < 0.05) and mitigated Nω-nitro-l-arginine methyl ester-induced reactive oxygen species formation (P  < 0.05) in human umbilical vein endothelial cells. CONCLUSION: We conclude that hydroxychloroquine alleviated hypertension, proteinuria, and normalized soluble fms-like kinase-1 and endothelin-1 levels in our preeclampsia model and that these changes may involve the restoration of endothelial dysfunction; thus, hydroxychloroquine could potentially be used for preventing preeclampsia, even in the absence of lupus.

Effect of hydroxychloroquine and chloroquine on syncytial differentiation and autophagy in primary human trophoblasts.

During placentation, cytotrophoblasts differentiate and fuse to form multinucleated cells (syncytiotrophoblasts) in a process that involves autophagy. Appropriate syncytial differentiation is essential for establishing a healthy pregnancy. In this study, we evaluated the effect of two chloroquine compounds, hydroxychloroquine (HCQ) and chloroquine (CQ), on syncytial differentiation and autophagy in cultured primary human trophoblasts (PHTs). PHT cells were isolated from the human term placenta. Bafilomycin, a well-known autophagy inhibitor, was used as a positive control. Biochemical and morphological differentiation was assessed in syncytiotrophoblasts, and autophagy-related proteins and genes were evaluated. Affymetrix Human Gene 2.0 ST Array profiling was used to identify genes affected by HCQ during syncytial differentiation. Chloroquine compounds lowered the production of beta-human chorionic gonadotropin (ß-hCG) and the fusion index in PHTs. Syncytial differentiation in PHT was associated with the increased expression of ATG4C mRNA (autophagy-related gene), and this expression was affected by CQ but not by HCQ. Microarray analysis revealed that HCQ or CQ affected several genes (MMP15, GPC3, CXCL10, TET-1, and S100A7) during syncytial differentiation, which were different from that of the syncytial differentiation suppression (Ham's/Waymouth media) or autophagy inhibition (bafilomycin treatment). Using Kyoto Encyclopedia of Genes and Genomes analysis we identified that HCQ might affect JAK2 signaling in the syncytial differentiation of PHT. In conclusion, chloroquine compounds could mitigate biochemical and morphological syncytial trophoblast differentiation in cultured PHT cells through the JAK signaling pathway rather than the inhibition of autophagic activity.

Anticancer Activity of the Combination of Cabozantinib and Temozolomide in Uterine Sarcoma.

PURPOSE: To evaluate the anticancer effects of cabozantinib, temozolomide, and their combination in uterine sarcoma cell lines and mouse xenograft models. EXPERIMENTAL DESIGN: Human uterine sarcoma cell lines (SK-LMS-1, SK-UT-1, MES-SA, and SKN) were used to evaluate the anticancer activity of cabozantinib, temozolomide, and their combination. The optimal dose of each drug was determined by MTT assay. Cell proliferation and apoptosis were assessed 48 and 72 hours after the drug treatments. The tumor weights were measured in an SK-LMS-1 xenograft mouse model and a patient-derived xenograft (PDX) model of leiomyosarcoma treated with cabozantinib, temozolomide, or both. RESULTS: Given individually, cabozantinib and temozolomide each significantly decreased the growth and viability of cells. This inhibitory effect was more pronounced when cabozantinib (0.50 µmol/L) and temozolomide (0.25 or 0.50 mmol/L) were co-administered (P < 0.05). The combination of the drugs also significantly increased apoptosis in all cells. Moreover, this effect was consistently observed in patient-derived leiomyosarcoma cells. In vivo studies with SK-LMS-1 cell xenografts and the PDX model with leiomyosarcoma demonstrated that combined treatment with cabozantinib (5 mg/kg/d, per os administration) and temozolomide (5 mg/kg/d, per os administration) synergistically decreased tumor growth (both P < 0.05). CONCLUSIONS: The addition of cabozantinib to temozolomide offers synergistic anticancer effects in uterine sarcoma cell lines and xenograft mouse models, including PDX. These results warrant further investigation in a clinical trial.

Combination effect of poly (ADP-ribose) polymerase inhibitor and DNA demethylating agents for treatment of epithelial ovarian cancer.

OBJECTIVE: Poly (ADP)-ribose polymerase inhibitors (PARPi) are effective clinical agents for treatment of epithelial ovarian cancer (EOC) harboring BRCA mutations as well as those without BRCA mutations. In this study, we evaluate the efficacy of combined PARPi and DNA methyltransferase inhibitor (DNMTi) in EOCs. METHODS: Expression levels of DNMT1 and PARP1 proteins in EOC cells were assessed using western blot analysis and immunohistochemistry. To evaluate the effects of co-treatment of PARPi (olaparib) and DNMTi (5-azacitidine, 5-AZA), we performed cell proliferation, apoptosis, and wound-healing assays in EOC cells. In addition, we performed in vivo experiments using both cell-line and patient-derived xenograft (PDX) models of EOC. RESULTS: The combination of olaparib and 5-AZA significantly inhibited cell proliferation and migration and induced apoptosis compared with olaparib or 5-AZA alone in EOC cell lines including A2780, HeyA8, A2780-CP20, and HeyA8-MDR. Moreover, in vivo experiments with this combination showed significantly decreased weight and nodule numbers of tumors in cell-line xenograft models with A2780 cells and a PDX model compared with control, olaparib, and 5-AZA groups. As a potential mechanism, the expression of intracellular reactive oxygen species (ROS) and its related proteins, including p-ERK, NRF2, p-p38, HO-1, and γH2AX, was affected in EOC cells. CONCLUSIONS: Co-treatment with PARPi and DNMTi had a significant anti-tumor effect in EOC cells. This combination might be a potential therapeutic strategy for EOCs.

Establishment and preclinical application of a patient-derived xenograft model for uterine cancer.

BACKGROUND: The patient-derived xenograft (PDX) model is a promising translational platform for duplicating the characteristics of primary tumors. Here, we established and characterized PDX models of uterine cancer to demonstrate their utility for preclinical drug testing. MATERIALS AND METHODS: We generated PDX tumors surgically derived from 58 cases of uterine cancer. Subrenal capsule xenografts and primary tumors were compared using microscopic examination, short tandem repeat analyses, and targeted sequencing analyses. A phosphatidylinositol 3-kinase (PI3K) inhibitor was administered to mice whose PDX tumors harbored a PTEN deletion or PIK3CA mutation. We also generated an orthotopic PDX model using uterine horn implantation. RESULTS: Thirty-three (56.9%) PDXs were successfully generated and passaged to maintain tumors. The histological features of the PDX tumors were stable over subsequent passages. By contrast, the proportions of epithelial and mesenchymal components of carcinosarcoma PDX models varied by generation. Targeted sequencing analyses revealed that all mutated cancer-related genes were stable during establishment and subgrafting. Treatment with a PI3K inhibitor cased a significant decrease in tumor weight in the clear cell carcinoma PDX harboring a frameshift PTEN deletion (p = 0.049) and in the serous carcinoma PDX harboring a missense PI3KCA mutation (p = 0.003) compared with matched controls. We also successfully established orthotopic PDX models (3/3; 100.0%). CONCLUSIONS: The histological and genetic features of PDXs were similar to those of primary tumors. This model is a promising translational platform for preclinical testing of new anticancer drugs and will enable the personalized development of therapeutic options for uterine cancer.

Chloroquine reverses chemoresistance via upregulation of p21WAF1/CIP1 and autophagy inhibition in ovarian cancer.

Overcoming drug-resistance is a big challenge to improve the survival of patients with epithelial ovarian cancer (EOC). In this study, we investigated the effect of chloroquine (CQ) and its combination with cisplatin (CDDP) in drug-resistant EOC cells. We used the three EOC cell lines CDDP-resistant A2780-CP20, RMG-1 cells, and CDDP-sensitive A2780 cells. The CQ-CDDP combination significantly decreased cell proliferation and increased apoptosis in all cell lines. The combination induced expression of γH2AX, a DNA damage marker protein, and induced G2/M cell cycle arrest. Although the CQ-CDDP combination decreased protein expression of ATM and ATR, phosphorylation of ATM was increased and expression of p21WAF1/CIP1 was also increased in CQ-CDDP-treated cells. Knockdown of p21WAF1/CIP1 by shRNA reduced the expression of γH2AX and phosphorylated ATM and inhibited caspase-3 activity but induced ATM protein expression. Knockdown of p21WAF1/CIP1 partly inhibited CQ-CDDP-induced G2/M arrest, demonstrating that knockdown of p21WAF1/CIP1 overcame the cytotoxic effect of the CQ-CDDP combination. Ectopic expression of p21WAF1/CIP1 in CDDP-treated ATG5-shRNA/A2780-CP20 cells increased expression of γH2AX and caspase-3 activity, demonstrating increased DNA damage and cell death. The inhibition of autophagy by ATG5-shRNA demonstrated similar results upon CDDP treatment, except p21WAF1/CIP1 expression. In an in vivo efficacy study, the CQ-CDDP combination significantly decreased tumor weight and increased expression of γH2AX and p21WAF1/CIP1 in A2780-CP20 orthotopic xenografts and a drug-resistant patient-derived xenograft model of EOC compared with controls. These results demonstrated that CQ increases cytotoxicity in combination with CDDP by inducing lethal DNA damage by induction of p21WAF1/CIP1 expression and autophagy inhibition in CDDP-resistant EOC.

Anti-Cancer Activity of As4O6 and its Efficacy in a Series of Patient-Derived Xenografts for Human Cervical Cancer.

PURPOSE: To investigate the anti-cancer effects of tetraarsenic hexoxide (TAO, As4O6) in cervical cancer cell lines and in a series of patient-derived xenograft (PDX) mouse models. METHODS: Human cervical cancer cell lines, including HeLa, SiHa and CaSki, and human umbilical vein endothelial cells (HUVECs), were used to evaluate the anti-cancer activity of TAO. Cellular proliferation, apoptosis, and enzyme-linked immunosorbent assay (ELISA) for matrix metallopeptidase 2 (MMP-2) and 9 (MMP-9) were assessed. The tumor weights of the PDXs that were given TAO were measured. The PDXs included primary squamous cell carcinoma, primary adenocarcinoma, recurrent squamous cell carcinoma, and recurrent adenocarcinoma. RESULTS: TAO significantly decreased cellular proliferation and increased apoptosis in cervical cancer cell lines and HUVEC. The functional studies on the cytotoxicity of TAO revealed that it inhibited the activation of Akt and vascular endothelial growth factor receptor 2 (VEGFR2). It also decreased the concentrations of MMP-2 in both cervical cancer cell lines and HUVECs. Active caspase-3 and p62 were both increased by the treatment of TAO, indicating increased rates of apoptosis and decreased rates of autophagy, respectively. In vivo studies with PDXs revealed that TAO significantly decreased tumor weight for both primary squamous cell carcinoma and adenocarcinoma of the cervix. However, this anti-cancer effect was not seen in PDXs with recurrent cancers. Nevertheless, the combination of TAO with cisplatin significantly decreased tumor weight in PDX models for both primary and recurrent cancers. CONCLUSIONS: TAO exerted inhibitory effects on angiogenesis, cellular migration, and autophagy, and it showed stimulatory effects on apoptosis. Overall, it demonstrated anti-cancer effects in animal models for human cervical cancer.

Autophagy regulates trophoblast invasion by targeting NF-κB activity.

Preeclampsia is one of the most serious complications of pregnancy, affecting 5-10% of parturients worldwide. Recent studies have suggested that autophagy is involved in trophoblast invasion and may be associated with defective placentation underlying preeclampsia. We thus aimed to understand the mechanistic link between autophagy and trophoblast invasion. Using the two most commonly used trophoblast cell lines, JEG-3 and HTR-8/SVneo, we inhibited autophagy by ATG5 and beclin-1 shRNA. Conversion of LC3-II was evaluated in ATG5 and beclin-1 knock-down cells in the presence of the lysosomal protease inhibitors E-64d and pepstatin A, to detect the efficiency of autophagy inhibition. Upon autophagy inhibition, we measured cell invasion, activity of NF-κB and related signaling pathways, MMP-2, MMP-9, sFlt-1, and TNF-α levels. Autophagy inhibition increased the invasiveness of these trophoblastic cell lines and increased Akt and NF-κB activity as well as p65 expression. Of note, an NF-κB inhibitor significantly attenuated the trophoblast invasion induced by autophagy inhibition. Autophagy inhibition was also associated with increased MMP-2 and MMP-9 levels and decreased the production of sFlt-1 and TNF-α. Collectively, our results indicate that autophagy regulates trophoblast invasiveness in which the NF-κB pathway and MMP-2, MMP-9, sFlt-1 and TNF-α levels are affected.

Identification of Candidate Genes Associated with Susceptibility to Ovarian Clear Cell Adenocarcinoma Using cis-eQTL Analysis.

Ovarian clear cell adenocarcinoma (Ov-CCA) has a higher prevalence in the Japanese ancestry than other populations. The ancestral disparities in Ov-CCA prevalence suggests the presence of Ov-CCA-specific genetic alterations and may provide an opportunity to identify the novel genes associated with Ov-CCA tumorigenesis. Using 94 previously reported genes as the phenotypic trait, we conducted multistep expression quantitative trait loci (eQTL) analysis with the HapMap3 project datasets. Four single-nucleotide polymorphisms (SNPs) (rs4873815, rs12976454, rs11136002, and rs13259097) that had different allele frequencies in the Japanese ancestry and seven genes associated in cis (APBA3, C8orf58, KIAA1967, NAPRT1, RHOBTB2, TNFRSF10B, and ZNF707) were identified. In silico functional annotation analysis and in vitro promoter assay validated the regulatory effect of rs4873815-TT on ZNF707 and rs11136002-TT on TNFRSF10B. Furthermore, ZNF707 was highly expressed in Ov-CCA and had a negative prognostic value in disease recurrence in our sample cohort. This prognostic power was consistently observed in The Cancer Genome Atlas (TCGA) clear cell renal cell carcinoma dataset, suggesting that ZNF707 may have prognostic value in clear cell histology regardless of tissue origin. In conclusion, rs4873815-TT/ZNF707 may have clinical significance in the prognosis and tumorigenesis of Ov-CCA, which may be more relevant to clear cell histology. Besides, this study may underpin the evidence that cis-eQTL analysis based on ancestral disparities can facilitate the discovery of causal genetic alterations in complex diseases, such as cancer.

Pharmacogenomic analysis of patient-derived tumor cells in gynecologic cancers.

BACKGROUND: Gynecologic malignancy is one of the leading causes of mortality in female adults worldwide. Comprehensive genomic analysis has revealed a list of molecular aberrations that are essential to tumorigenesis, progression, and metastasis of gynecologic tumors. However, targeting such alterations has frequently led to treatment failures due to underlying genomic complexity and simultaneous activation of various tumor cell survival pathway molecules. A compilation of molecular characterization of tumors with pharmacological drug response is the next step toward clinical application of patient-tailored treatment regimens. RESULTS: Toward this goal, we establish a library of 139 gynecologic tumors including epithelial ovarian cancers (EOCs), cervical, endometrial tumors, and uterine sarcomas that are genomically and/or pharmacologically annotated and explore dynamic pharmacogenomic associations against 37 molecularly targeted drugs. We discover lineage-specific drug sensitivities based on subcategorization of gynecologic tumors and identify TP53 mutation as a molecular determinant that elicits therapeutic response to poly (ADP-Ribose) polymerase (PARP) inhibitor. We further identify transcriptome expression of inhibitor of DNA biding 2 (ID2) as a potential predictive biomarker for treatment response to olaparib. CONCLUSIONS: Together, our results demonstrate the potential utility of rapid drug screening combined with genomic profiling for precision treatment of gynecologic cancers.

Anti-Tumor Effects of Wee1 Kinase Inhibitor with Radiotherapy in Human Cervical Cancer.

Although the concurrent use of a chemotherapeutic agent and radiotherapy improves survival in patients with locally advanced or recurrent cervical cancer, severe side effects related to chemotherapy are frequent and may result in a low quality of life for the patients. In this study, we investigated the effects of a combination of Wee1 inhibitor (AZD1775) and irradiation in cervical cancer. In vitro effects of AZD1775 with irradiation in human cervical cancer cells were assessed by clonogenic survival and apoptosis assays. The effects on DNA damage response signaling and the cell cycle were also explored. Tumor growth delay was evaluated to investigate the in vivo effects of AZD1775 with irradiation in cervical cancer mouse models, including xenografts and patient-derived xenografts (PDXs). The co-treatment of AZD1775 and irradiation significantly decreased clonogenic survival and increased apoptosis in cervical cancer cells. These effects were associated with G2 checkpoint abrogation which resulted in persistent DNA damage. Both in the xenografts and the PDXs, the co-treatment significantly decreased tumor growth compared tothe irradiation alone (p < 0.05). These results demonstrate that the Wee1 inhibitor (AZD1775) can be considered as a potential alternative as a radiosensitizer in cervical cancer instead of a chemotherapeutic agent such as cisplatin.

Changes in calcium channel proteins according to magnesium sulfate administration in placentas from pregnancies with pre-eclampsia or fetal growth restriction.

We aimed to evaluate the changes in plasma membrane Ca2+-ATPase (PMCA) and sarcoendoplasmic reticulum CA2+-ATPase (SERCA-2) according to the antepartal magnesium sulfate (MgSO4) administration in the placentas from pregnancies with pre-eclampsia (PE) or fetal growth restriction (FGR). Pregnant women were classified as follows: (group 1) pregnancies without PE or FGR (n=16), (group 2) pregnancies with PE or FGR but without MgSO4 administration (n=14), and (group 3) pregnancies with PE or FGR and with MgSO4 administration (n=28). We observed the localization of PMCA and SERCA-2 in placentas and compared its expression among 3 groups. And we observed its expression in BeWo cells following treatment with MgSO4 and CoCl2 PMCA staining was more observed in the basal membrane, whereas SERCA-2 staining was observed predominantly under the microvillous membrane. SERCA-2 expression was significantly increased in group 3 compared with that in group 1. Considering the gestational age at delivery, PMCA expression was increased in group 2 and group 3 compared with that in group 1 after 36 weeks of gestation. SERCA-2 was increased in group 3, but not in group 2 compared with that in group 1 after 36 weeks of gestation. In BeWo cells, MgSO4 treatment increased PMCA and SERCA-2 expression. PMCA expression was influenced by gestational age at delivery, and SERCA-2 expression was increased in the presence of PE and antepartal MgSO4 administration. This indicates that antepartal MgSO4 administration has a greater influence on SERCA-2 than PMCA.

The C-terminus of IGFBP-5 suppresses tumor growth by inhibiting angiogenesis.

Insulin-like growth factor-binding protein 5 (IGFBP-5) plays a role in cell growth, differentiation, and apoptosis. In this study, we found that IGFBP5 was markedly downregulated in ovarian cancer tissue. We investigated the functional significance of IGFBP-5 as a tumor suppressor. To determine functional regions of IGFBP-5, truncation mutants were prepared and were studied the effect on tumor growth. Expression of C-terminal region of IGFBP-5 significantly decreased tumor growth in an ovarian cancer xenograft. A peptide derived from the C-terminus of IGFBP-5 (BP5-C) was synthesized to evaluate the minimal amino acid motif that retained anti-tumorigenic activity and its effect on angiogenesis was studied. BP5-C peptide decreased the expression of VEGF-A and MMP-9, phosphorylation of Akt and ERK, and NF-kB activity, and inhibited angiogenesis in in vitro and ex vivo systems. Furthermore, BP5-C peptide significantly decreased tumor weight and angiogenesis in both ovarian cancer orthotopic xenograft and patient-derived xenograft mice. These results suggest that the C-terminus of IGFBP-5 exerts anti-cancer activity by inhibiting angiogenesis via regulation of the Akt/ERK and NF-kB-VEGF/MMP-9 signaling pathway, and might be considered as a novel angiogenesis inhibitor for the treatment of ovarian cancer.

Isolation of basal membrane proteins from BeWo cells and their expression in placentas from fetal growth-restricted pregnancies.

INTRODUCTION: The syncytiotrophoblast, a key barrier between the mother and fetus, is a polarized epithelium composed of a microvillus and basal membrane (BM). We sought to characterize BM proteins of BeWo cells in relation to hypoxia and to investigate their expression in placentas from pregnancies complicated by fetal growth restriction (FGR). METHODS: We isolated the BM fraction of BeWo cells by the cationic colloidal silica method and identified proteins enriched in this fraction by mass spectrometry. We evaluated the effect of hypoxia on the expression and intracellular localization of identified proteins and compared their expression in BM fractions of FGR placentas to those from normal pregnancies. RESULTS: We identified BM proteins from BeWo cells. Among BM proteins, we further characterized heme oxygenase-1 (HO-1), voltage-dependent anion channel-1 (VDAC1), and ribophorin II (RPN2), based on their relevance to placental biology. Hypoxia enhanced the localization of these proteins to the BM of BeWo cells. HO-1, VDAC1, and RPN2 were selectively expressed in the human placental BM fraction. C-terminally truncated HO-1 was identified in placental BM fractions, and its BM expression was significantly reduced in FGR placentas than in normal placentas. Interestingly, a truncated HO-1 construct was predominantly localized in the BM in response to hypoxia and co-localized with VDAC1 in BeWo cells. DISCUSSION: Hypoxia increased the BM localization of HO-1, VDAC1, and RPN2 proteins. FGR significantly reduced the expression of truncated HO-1, which was surmised to co-localize with VDAC1 in hypoxic BeWo cells.

Autophagy induced by tumor necrosis factor α mediates intrinsic apoptosis in trophoblastic cells.

To investigate the interconnection of apoptosis and autophagy in trophoblastic cells, we treated JEG-3 cells with tumor necrosis factor α (TNF-α) after transfecting LC3 or Beclin 1 or calpain small interfering RNA (siRNA), which blocks cleavage of autophagy-related gene 5 (Atg5) into N-terminal truncated Atg5 (tAtg5), a mediator between apoptosis and autophagy, and assessed the changes in LC3-II, caspase 9, caspase 3, and tAtg5. We also assessed the TNF-α-induced changes in LC3-II, caspase 9, and caspase 3 in primary trophoblasts from term placentae after transfecting siRNA for LC3 or Beclin 1. In both types of cells, transfection of LC3 or Beclin 1 siRNA significantly attenuated TNF-α-induced increases in LC3-II and activations of caspase 9 and caspase 3. There was significant abrogation of TNF-α-induced expression of tAtg5 after transfection with LC3 or Beclin 1 siRNA. Moreover, transfection with calpain siRNA significantly decreased TNF-α-induced changes in caspase 3 and caspase 9 in addition to tAtg5 in JEG-3 cells. Our data suggest that TNF-α-induced autophagy mediates intrinsic apoptosis, probably through tAtg5, in trophoblastic cells.

Upregulation of CD9 in ovarian cancer is related to the induction of TNF-α gene expression and constitutive NF-κB activation.

Ovarian cancer is a gynecological cancer with a high death rate. We utilized global gene expression profiles of ovarian carcinomas obtained by complementary DNA (cDNA) microarray to identify ovarian cancer-specific proteins. CD9 was upregulated in ovarian carcinomas, and overexpression of the CD9 protein was detected in ovarian carcinomas by immunohistochemistry. CD9 was also overexpressed in several cancer cell lines, including ovarian cancer cells. In order to elucidate the biological significance of highly expressed CD9 in cancer cells, functional studies of CD9 were performed by ectopic expression, knockdown of CD9 using small interfering RNA (siRNA) and blockage of CD9 activity using the CD9-specific monoclonal antibody ALB6. Ectopic CD9 induced cell survival. In order to identify signaling pathways related to CD9, the gene expressions of CD9/SKOV3 cells were analyzed by cDNA microarray. Among the many upregulated genes, tumor necrosis factor (TNF)-α was induced in CD9/SKOV3 cells. The effect of overexpressed CD9 on the downstream signaling events of TNF-α was further investigated. In CD9/SKOV3 cells, the nuclear factor-kappaB (NF-κB)-signaling pathway was constitutively activated. Knockdown of CD9 by siRNA and blockage of CD9 activity by ALB6 in ovarian cancer cells demonstrated that constitutive activation of NF-κB is CD9 dependent and that CD9 is involved in anti-apoptosis. A CD9 functional study was performed in an ovarian cancer-xenograft mouse by injecting ALB6 into the peritoneum. ALB6 resulted in reduced tumor weight compared with that of control IgG(1). Collectively, these results demonstrate that CD9 functions as an oncogene and represents a target for the development of cancer-specific therapeutics.