NAHA, a novel hydroxamic acid-derivative, inhibits growth and angiogenesis of breast cancer in vitro and in vivo

Background

We have recently synthesized novel N-alkylated amino acid-derived hydroxamate, 2-[Benzyl-(2-nitro-benzenesulfonyl)-amino]-N-hydroxy-3-methyl-N-propyl-butyramide (NAHA). Here, we evaluate the anticancer activity of NAHA against highly invasive human breast cancer cells MDA-MB-231 in vitro and in vivo.

Methodology/Principal Findings

Cell growth was evaluated by MTT and soft agar assays. Protein expression was determined by DNA microarray and Western blot analysis. Metastatic potential was evaluated by cell adhesion, migration, invasion, capillary morphogenesis, and ELISA assays. The anticancer activity in vivo was evaluated in mouse xenograft model. NAHA inhibited proliferation and colony formation of MDA-MB-231 cells together with the down-regulation of expression of Cdk2 and CDC20 proteins. NAHA inhibited cell adhesion, migration, and invasion through the suppression of secretion of uPA. NAHA suppressed secretion of VEGF from MDA-MB-231 cells and inhibited capillary morphogenesis of human aortic endothelial cells (HAECs). Finally, NAHA at 50 mg/kg was not toxic and decreased tumor volume and tumor weight in vivo. This suppression of tumor growth was associated with the inhibition of mitotic figures and induction of apoptosis, and the reduction of CD31 and VEGF positive cells in tumors.

Conclusion

NAHA could be a novel promising compound for the development of new drugs for the therapy of invasive breast cancers.     

Fucoidan Induces Cancer Cell Apoptosis by Modulating the Endoplasmic Reticulum Stress Cascades

Background

Cancer metastasis is the main cause leading to disease recurrence and high mortality in cancer patients. Therefore, inhibiting metastasis process or killing metastatic cancer cells by inducing apoptosis is of clinical importance in improving cancer patient survival. Previous studies revealed that fucoidan, a fucose-rich polysaccharide isolated from marine brown alga, is a promising natural product with significant anti-cancer activity. However, little is known about the role of endoplasmic reticulum (ER) stress in fucoidan-induced cell apoptosis.

Principal Findings

We reported that fucoidan treatment inhibits cell growth and induces apoptosis in cancer cells. Fucoidan treatments resulted in down-regulation of the glucose regulated protein 78 (GRP78) in the metastatic MDA-MB-231 breast cancer cells, and of the ER protein 29 (ERp29) in the metastatic HCT116 colon cancer cells. However, fucoidan treatment promoted ER Ca²⁺-dependent calmodulin-dependent kinase II (CaMKII) phosphorylation, Bcl-associated X protein (Bax) and caspase 12 expression in MDA-MB-231 cells, but not in HCT116 cells. In both types of cancer cells, fucoidan activated the phosphorylation of eukaryotic initiation factor 2 alpha (p-eIF2α)\CCAAT/enhancer binding protein homologous protein (CHOP) pro-apoptotic cascade and inhibited the phosphorylation of inositol-requiring kinase 1 (p-IRE-1)\X-box binding proteins 1 splicing (XBP-1s) pro-survival cascade. Furthermore, CHOP knockdown prevented DNA damage and cell death induced by fucoidan.

Conclusion/Significance

Fucoidan exerts its anti-tumor function by modulating ER stress cascades. Contribution of ER stress to the fucoidan-induced cell apoptosis augments our understanding of the molecular mechanisms underlying its anti-tumour activity and provides evidence for the therapeutic application of fucoidan in cancer.     

Combination Therapy of VEGF-Trap and Gemcitabine Results in Improved Anti-Tumor Efficacy in a Mouse Lung Cancer Model

Background

Angiogenesis is essential for the growth and metastasis of cancer. Although anti-angiogenic agents, particularly vascular endothelial growth factor (VEGF) inhibitors, have exhibited single-agent activity, there is considerable interest in combining these novel drugs with conventional chemotherapy reagents to achieve an optimal clinical efficacy. The objective of this study was to evaluate the benefits of the combination therapy of vascular endothelial growth factor trap (VEGF-Trap) with gemcitabine in a lung tumor model.

Methods

A luciferase-expressing Lewis lung carcinoma (LLC) model was established in C57BL/6J mice and tumor-bearing mice were randomized into control, VEGF-Trap, gemcitabine and VEGF-Trap/gemcitabine combination groups. Tumor growth and animal survival were monitored. Tumor microvessel density and cell proliferation were evaluated by CD31 and Ki-67 immunohistochemical analysis. TUNEL assay was performed to detect apoptotic cells. The protein levels of Cyclin D1, Pro-Caspase-3, Bcl-2, MMP2 and MMP9 in tumor extracts were examined by western blot.

Results

VEGF-Trap in combination with gemcitabine showed significantly enhanced inhibition of tumor growth and prolonged mouse survival compared to the VEGF-Trap or gemcitabine monotherapy. The VEGF-Trap/gemcitabine combination therapy not only potently inhibited tumor angiogenesis and cell proliferation, but also increased cellular apoptosis within tumor tissues. In addition, the combination treatment markedly down-regulated the expression of proliferation, anti-apoptosis and invasion related proteins.

Conclusion

Combination therapy using VEGF-Trap and gemcitabine resulted in improved anti-tumor efficacy in a lung cancer model and VEGF-Trap/gemcitabine combination might represent a promising strategy in the treatment of human lung cancer.     

miR-30b,Down-Regulated in Gastric Cancer,Promotes Apoptosis and Suppresses Tumor Growth by Targeting Plasminogen Activator Inhibitor-1

Background

Gastric cancer is one of the most common malignant diseases worldwide. Emerging evidence has shown that microRNAs (miRNAs) are associated with tumor development and progression. Our previous studies have revealed that H. pylori infection was able to induce the altered expression of miR-30b in gastric epithelial cells. However, little is known about the potential role of miR-30b in gastric cancer.

Methods

We analyzed the expression of miR-30b in gastric cancer cell lines and human gastric cancer tissues. We examined the effect of miR-30b mimics on the apoptosis of gastric cancer cells in vitro by flow cytometry (FCM) and caspase-3/7 activity assays. Nude mouse xenograft model was used to determine whether miR-30b is involved in tumorigenesis of gastric cancer. The target of miR-30b was identified by bioinformatics analysis, luciferase assay and Western blot. Finally, we performed the correlation analysis between miR-30b and its target expression in gastric cancer.

Results

miR-30b was significantly down-regulated in gastric cancer cells and human gastric cancer tissues. Enforced expression of miR-30b promoted the apoptosis of gastric cancer cells in vitro, and miR-30b could significantly inhibit tumorigenicity of gastric cancer by increasing the apoptosis proportion of cancer cells in vivo. Moreover, plasminogen activator inhibitor-1 (PAI-1) was identified as the potential target of miR-30b, and miR-30b level was inversely correlated with PAI-1 expression in gastric cancer. In addition, silencing of PAI-1 was able to phenocopy the effect of miR-30b overexpression on apoptosis regulation of cancer cells, and overexpression of PAI-1 could suppressed the effect of promoting cell apoptosis by miR-30b, indicating PAI-1 is potentially involved in miR-30b-induced apoptosis on cancer cells.

Conclusion

miR-30b may function as a novel tumor suppressor gene in gastric cancer by targeting PAI-1 and regulating the apoptosis of cancer cells. miR-30b could serve as a potential biomarker and therapeutic target against gastric cancer.     

Oleanolic acid initiates apoptosis in non-small cell lung cancer cell lines and reduces metastasis of a B16F10 melanoma model in vivo

Background

Drug resistance, a process mediated by multiple mechanisms, is a critical determinant for treating lung cancer. The aim of this study is to determine if oleanolic acid (OA), a pentacyclic triterpene present in several plants, is able to circumvent the mechanisms of drug resistance present in non-small cell lung cancer (NSCLC) cell lines and to induce their death.

Principal Findings

OA decreased the cell viability of the NSCLC cell lines A459 and H460 despite the presence of active, multidrug-resistant (MDR) MRP1/ABCC1 proteins and the anti-apoptotic proteins Bcl-2 and survivin. These effects are due to apoptosis, as evidenced by the capacity of OA to induce fragmentation of DNA and activate caspase 3. Induction of NSCLC cell death by OA cannot be explained by inhibition of the MDR proteins, since treatment with triterpene had little or no effect on the activity or expression of MRP1. Moreover, treatment with OA had no effect on the expression of the anti-apoptotic protein Bcl-2, but increased the expression of the pro-apoptotic protein Bax, altering the Bcl-2/Bax balance towards a pro-apoptotic profile. OA also decreased the expression of the anti-apoptotic protein survivin. Furthermore, OA decreased the expression of the angiogenic vascular endothelial growth factor (VEGF) and decreased the development of melanoma-induced lung metastasis.

Conclusion

Our data provide a significant insight into the antitumoral and antimetastatic activity of OA in NSCLC and suggest that including OA in the NSCLC regimens may help to decrease the number of relapses and reduce the development of metastases.     

Anti-tumor activity of a novel compound-CDF is mediated by regulating miR-21, miR-200, and PTEN in pancreatic cancer

Background

The existence of cancer stem cells (CSCs) or cancer stem-like cells in a tumor mass is believed to be responsible for tumor recurrence because of their intrinsic and extrinsic drug-resistance characteristics. Therefore, targeted killing of CSCs would be a newer strategy for the prevention of tumor recurrence and/or treatment by overcoming drug-resistance. We have developed a novel synthetic compound-CDF, which showed greater bioavailability in animal tissues such as pancreas, and also induced cell growth inhibition and apoptosis, which was mediated by inactivation of NF-κB, COX-2, and VEGF in pancreatic cancer (PC) cells.

Methodology/Principal Findings

In the current study we showed, for the first time, that CDF could significantly inhibit the sphere-forming ability (pancreatospheres) of PC cells consistent with increased disintegration of pancreatospheres, which was associated with attenuation of CSC markers (CD44 and EpCAM), especially in gemcitabine-resistant (MIAPaCa-2) PC cells containing high proportion of CSCs consistent with increased miR-21 and decreased miR-200. In a xenograft mouse model of human PC, CDF treatment significantly inhibited tumor growth, which was associated with decreased NF-κB DNA binding activity, COX-2, and miR-21 expression, and increased PTEN and miR-200 expression in tumor remnants.

Conclusions/Significance

These results strongly suggest that the anti-tumor activity of CDF is associated with inhibition of CSC function via down-regulation of CSC-associated signaling pathways. Therefore, CDF could be useful for the prevention of tumor recurrence and/or treatment of PC with better treatment outcome in the future.     

Epidermal Expression of Neuropilin 1 Protects Murine keratinocytes from UVB-induced apoptosis

Background

Neuropilin 1 (NRP1) is expressed on several cell types including neurons and endothelial cells, where it functions as an important regulator in development and during angiogenesis. As a cell surface receptor, NRP1 is able to bind to members of the VEGF family of growth factors and to secreted class 3 semaphorins. Neuropilin 1 is also highly expressed in keratinocytes, but the function of NRP1 in epidermal physiology and pathology is still unclear.

Methods and Results

To elucidate the role of NRP1 in skin in vivo we generated an epidermis-specific neuropilin 1 knock out mouse model by using the Cre-LoxP-System. Mice were viable and fertile and did not display any obvious skin or hair defects. After challenge with UVB irradiation, we found that deletion of epidermal NRP1 leads to increased rates of apoptosis both in vitro and in vivo. NRP1-deficient primary keratinocytes cultured in vitro showed significantly higher rates of apoptosis 24 hours after UVB. Likewise, there is a significant increase of active caspase 3 positive cells in the epidermis of Keratin 14-Cre-NRP1 (−/−) mice 24 hours after UVB irradiation. By Western Blot analysis we could show that NRP1 influences the cytosolic levels of Bcl-2, a pro-survival member of the Bcl-2 family. After UVB irradiation the amounts of Bcl-2 decrease in both protein extracts from murine epidermis and in NRP1-deficient keratinocytes in vitro, whereas wild type cells retain their Bcl-2 levels. Likewise, levels of phospho-Erk and Rac1 were lower in NRP1-knock out keratinocytes, whereas levels of pro-apoptotic p53 were higher.

Conclusion

NRP1 expression in keratinocytes is dispensable for normal skin development. Upon UVB challenge, NRP1 contributes to the prevention of keratinocyte apoptosis. This pro-survival function of NRP1 is accompanied by the maintenance of high levels of the antiapoptotic regulator Bcl-2 and by lower levels of pro-apoptotic p53.     

CD1d-expressing breast cancer cells modulate NKT cell-mediated antitumor immunity in a murine model of breast cancer metastasis

Background

Tumor tolerance and immune suppression remain formidable obstacles to the efficacy of immunotherapies that harness the immune system to eradicate breast cancer. A novel syngeneic mouse model of breast cancer metastasis was developed in our lab to investigate mechanisms of immune regulation of breast cancer. Comparative analysis of low-metastatic vs. highly metastatic tumor cells isolated from these mice revealed several important genetic alterations related to immune control of cancer, including a significant downregulation of cd1d1 in the highly metastatic tumor cells. The cd1d1 gene in mice encodes the MHC class I-like molecule CD1d, which presents glycolipid antigens to a specialized subset of T cells known as natural killer T (NKT) cells. We hypothesize that breast cancer cells, through downregulation of CD1d and subsequent evasion of NKT-mediated antitumor immunity, gain increased potential for metastatic tumor progression.

Methodology/Principal Findings

In this study, we demonstrate in a mouse model of breast cancer metastasis that tumor downregulation of CD1d inhibits iNKT-mediated antitumor immunity and promotes metastatic breast cancer progression in a CD1d-dependent manner in vitro and in vivo. Using NKT-deficient transgenic mouse models, we demonstrate important differences between type I and type II NKT cells in their ability to regulate antitumor immunity of CD1d-expressing breast tumors.

Conclusions/Significance

The results of this study emphasize the importance of determining the CD1d expression status of the tumor when tailoring NKT-based immunotherapies for the prevention and treatment of metastatic breast cancer.     

Evaluating the Potential Bioactivity of a Novel Compound ER1626

Background

ER1626, a novel compound, is a derivate of indeno-isoquinoline ketone. This study was designed to evaluate the biological activity and potential anti-tumor mechanism of ER1626.

Method

MTT assay, scratch assay and flow cytometry were used to determine cell proliferation, cell migration and cell cycle distribution as well as cell apoptosis on human breast cancer MCF-7 cells and endometrial cancer Ishikawa cells. We also explored the antiangiogenic effect of ER1626 on HUVEC cells and chicken embryos. The expression of estrogen receptor protein was investigated with western-blot analysis.

Results

ER1626 down-regulated the expression of estrogen receptor α protein and up-regulated β protein in MCF-7 and Ishikawa cells. The value of IC₅₀ of ER1626 on MCF-7 and Ishikawa cells were respectively 8.52 and 3.08 µmol/L. Meanwhile, ER1626 decreased VEGF secretion of MCF-7 and Ishikawa cells, disturbed the formation of VEGF-stimulated tubular structure in HUVEC cells, and inhibited the angiogenesis on the chicken chorioallantoic membrane. Scratch assay revealed that ER1626 suppressed the migration of MCF-7, Ishikawa and HUVEC cells. In addition to induction tumor cell apoptosis, ER1626 arrested cell cycle in G1/G0 phase in MCF-7 cells and G2/M phase in Ishikawa cells.

Conclusion

In conclusion, our results demonstrated that ER1626 has favorable bioactivities to be a potential candidate against breast cancer and angiogenesis.     

Antitumor Activity of Emodin against Pancreatic Cancer Depends on Its Dual Role: Promotion of Apoptosis and Suppression of Angiogenesis

Background

Emodin has been showed to induce apoptosis of pancreatic cancer cells and inhibit tumor growth in our previous studies. This study was designed to investigate whether emodin could inhibit the angiogenesis of pancreatic cancer tissues and its mechanism.

Methodology/Principal Finding

In accordance with our previous study, emodin inhibited pancreatic cancer cell growth, induced apoptosis, and enhanced the anti-tumor effect of gemcitabine on pancreatic caner cells in vitro and in vivo by inhibiting the activity of NF-κB. Here, for the first time, we demonstrated that emodin inhibited tumor angiogenesis in vitro and in implanted pancreatic cancer tissues, decreased the expression of angiogenesis-associated factors (NF-κB and its regulated factors VEGF, MMP-2, MMP-9, and eNOS), and reduced eNOS phosphorylation, as evidenced by both immunohistochemistry and western blot analysis of implanted tumors. In addition, we found that emodin had no effect on VEGFR expression in vivo.

Conclusions/Significance

Our results suggested that emodin has potential anti-tumor effect on pancreatic cancer via its dual role in the promotion of apoptosis and suppression of angiogenesis, probably through regulating the expression of NF-κB and NF-κB-regulated angiogenesis-associated factors.     

Enhanced anticancer activity of gemcitabine in combination with noscapine via antiangiogenic and apoptotic pathway against non-small cell lung cancer

Background

The aim of this investigation was to evaluate the anticancer activity of Noscapine (Nos) and Gemcitabine (Gem) combination (NGC) against non-small cell lung cancer (NSCLC) and to elucidate the underlying mechanism of action.

Methods

Isobolographic method was used to calculate combination index values from cytotoxicity data. In vitro antiangiogenic and apoptotic activity of Nos, Gem and NGC was evaluated. For in vivo studies, female athymic Nu/nu mice were xenografted with H460 tumors and the efficacy of Nos, Gem, or NGC was determined. Protein expressions by immunohistochemical staining were evaluated in harvested tumor tissues.

Results

The CI values (<0.59) were suggestive of synergistic behavior between Nos and Gem. NGC treatment showed significantly inhibited tube formation and increased percentage of apoptotic cells. NGC, Gem and Nos treatment reduced tumor volume by 82.9±4.5 percent, 39.4±5.8 percent and 34.2±5.7 percent respectively. Specifically, NGC treatment decreased expression cell survival proteins; VEGF, CD31 staining and microvessel density and enhanced DNA fragmentation and cleaved caspase 3 levels compared to single agent treated and control groups.

Conclusion

Nos potentiated the anticancer activity of Gem in an additive to synergistic manner against lung cancer via antiangiogenic and apoptotic pathways. These findings suggest potential benefit for use of NGC chemotherapy for treatment of lung cancer.     

Forkhead Box L1 Is Frequently Downregulated in Gallbladder Cancer and Inhibits Cell Growth through Apoptosis Induction by Mitochondrial Dysfunction

Background

Forkhead box L1 (FOXL1), considered as a novel candidate tumor suppressor, suppresses proliferation and invasion in certain cancers. However, the regulation and function of FOXL1 in gallbladder cancer (GBC) remains unclear.

Methods

FOXL1 expression at mRNA and protein levels in GBC tissues and cell lines were examined by RT-PCR, immunohistochemistry and western blot assay. FOXL1 expression in GBC cell lines was up-regulated by transfection with pcDNA-FOXL1. The effects of FOXL1 overexpression on cell proliferation, apoptosis, migration and invasion were evaluated in vitro or in vivo. In addition, the status of mediators involved in migration, invasion and apoptosis was examined using western blot after transfection with pcDNA-FOXL1.

Results

FOXL1 was frequently downregulated in GBC tissues and cell lines. Its higher expression is associated with better prognosis, while its lower expression is correlated with advanced TNM stage and poor differentiation. FOXL1 overexpression in NOZ cells significantly suppresses cell proliferation, migration and invasion in vitro and tumorigenicity in nude mice. FOXL1 overexpression disrupted mitochondrial transmembrane potential and triggered mitochondria-mediated apoptosis in NOZ cells. In addition, FOXL1 overexpression suppressed ZEB1 expression and induced E-cadherin expression in NOZ cells.

Conclusion

Our findings suggested that dysregulated FOXL1 is involved in tumorigenesis and progression of GBC and may serve as a predictor of clinical outcome or even a therapeutic target for patients with GBC.     

Identification of LZAP as a new candidate tumor suppressor in hepatocellular carcinoma

Background

LZAP was isolated as a binding protein of the Cdk5 activator p35. LZAP has been highly conserved during evolution and has been shown to function as a tumor suppressor in various cancers. This study aimed to investigate LZAP expression and its prognostic value in hepatocellular carcinoma (HCC). Meanwhile, the function of LZAP in hepatocarcinogenesis was further investigated in cell culture models and mouse models.

Methods

Real-time quantitative PCR, western blot and immunohistochemistry were used to explore LZAP expression in HCC cell lines and primary HCC clinical specimens. The functions of LZAP in the proliferation, colony formation, cell cycle, migration, invasion and apoptosis of HCC cell lines were also analyzed by infecting cells with an adenovirus containing full-length LZAP. The effect of LZAP on tumorigenicity in nude mice was also investigated.

Results

LZAP expression was significantly decreased in the tumor tissues and HCC cell lines. Clinicopathological analysis showed that LZAP expression was significantly correlated with tumor size, histopathological classification and serum α-fetoprotein (AFP). The Kaplan–Meier survival curves revealed that decreasing LZAP expression was associated with poor prognosis in HCC patients. LZAP expression was an independent prognostic marker of overall HCC patient survival in a multivariate analysis. The re-introduction of LZAP expression in the HepG2 and sk-Hep1 HCC cell lines significantly inhibited proliferation and colony formation in the HCC cells and induced G1 phase arrest and apoptosis of the HCC cells in vitro. Restoring LZAP expression in the HCC cell lines also inhibited migration and invasion. In addition, experiments with a mouse model revealed that LZAP overexpression could suppress HCC tumorigenicity in vivo.

Conclusions

Our data suggest that LZAP may play an important role in HCC progression and could be a potential molecular therapy target for HCC.     

Genistein Inhibits Prostate Cancer Cell Growth by Targeting miR-34a and Oncogenic HOTAIR

Objective

Genistein is a soy isoflavone that has antitumor activity both in vitro and in vivo. It has been shown that genistein inhibits many type of cancers including prostate cancer (PCa) by regulating several cell signaling pathways and microRNAs (miRNAs). Recent studies suggest that the long non-coding RNAs (lncRNAs) are also involved in many cellular processes. At present there are no reports about the relationship between gensitein, miRNAs and lncRNAs. In this study, we focused on miRNAs, lncRNA that are regulated by genistein and investigated their functional role in PCa.

Method

Microarray (SurePrint G3 Human GE 8×60K) was used for expression profiling of genistein treated and control PCa cells (PC3 and DU145). Functional assay (cell proliferation, migration, invasion, apoptosis and cell cycle assays) were performed with the PCa cell lines, PC3 and DU145. Both in vitro and in vivo (nude mouse) models were used for growth assays. Luciferase reporter assays were used for binding of miR-34a to HOTAIR.

Results

LncRNA profiling showed that HOTAIR was highly regulated by genistein and its expression was higher in castration-resistant PCa cell lines than in normal prostate cells. Knockdown (siRNA) of HOTAIR decreased PCa cell proliferation, migration and invasion and induced apoptosis and cell cycle arrest. miR-34a was also up-regulated by genistein and may directly target HOTAIR in both PC3 and DU145 PCa cells.

Conclusions

Our results indicated that genistein inhibited PCa cell growth through down-regulation of oncogenic HOTAIR that is also targeted by tumor suppressor miR-34a. These findings enhance understanding of how genistein regulates lncRNA HOTAIR and miR-34a in PCa.     

Simultaneous silencing of VEGF and KSP by siRNA cocktail inhibits proliferation and induces apoptosis of hepatocellular carcinoma Hep3B cells

Background

Vascular endothelial growth factor (VEGF) is involved in the growth of new blood vessels that feed tumors and kinesin spindle protein (KSP) plays a critical role in mitosis involving in cell proliferation. Simultaneous silencing of VEGF and KSP, an attractive and viable approach in cancer, leads on restricting cancer progression. The purpose of this study is to examine the therapeutic potential of dual gene targeted siRNA cocktail on human hepatocellular carcinoma Hep3B cells.

Results

The predesigned siRNAs could inhibit VEGF and KSP at mRNA level. siRNA cocktail showed a further downregulation on KSP mRNA and protein levels compared to KSP-siRNA or VEGF-siRNA, but not on VEGF expression. It also exhibited greater suppression on cell proliferation as well as cell migration or invasion capabilities and induction of apoptosis in Hep3B cells than single siRNA simultaneously. This could be explained by the significant downregulation of Cyclin D1, Bcl-2 and Survivin. However, no sigificant difference in the mRNA and protein levels of ANG2, involving inhibition of angiogenesis was found in HUVECs cultured with supernatant of Hep3B cells treated with siRNA cocktail, compared to that of VEGF-siRNA.

Conclusion

Silencing of VEGF and KSP plays a key role in inhibiting cell proliferation, migration, invasion and inducing apoptosis of Hep3B cells. Simultaneous silencing of VEGF and KSP using siRNA cocktail yields promising results for eradicating hepatocellular carcinoma cells, a new direction for liver cancer treatment.     

Overexpression of EFEMP1 Correlates with Tumor Progression and Poor Prognosis in Human Ovarian Carcinoma

Objective

This study was to explore the role of EFEMP1 in ovarian tumor progression and its relationship with prognosis of ovarian carcinoma.

Methods

EFEMP1 mRNA and protein expressions in normal ovarian tissue, ovarian tumor, high invasive subclones and low invasive subclones were evaluated by immunohistochemistry and real time RT-PCR. Serum EFEMP1 levels in patients with ovarian tumor were measured by ELISA assay. To assess the angiogenic properties of EFEMP1, VEGF and tumor microvessel density were analyzed in ovarian carcinoma by immunohistochemistry.

Results

EFEMP1 expression was up-regulated in ovarian carcinoma, positively correlated with MVD and VEGF, and its overexpression and high serum levels were significantly associated with high stage, low differentiation, lymph node metastasis and poor prognosis of ovarian cancer. EFEMP1 expression was also found to be over-expressed in the highly invasive subclones compared with the low invasive subclones.

Conclusion

EFEMP1 is a newly identified gene over-expressed in ovarian cancer, associated with poor clinicopathologic features and promotes angiogenesis. This study shows that EFEMP1 may serve as a new prognostic factor and a therapeutic target for patients with ovarian cancer in the future.     

Copper deficiency induced emphysema is associated with focal adhesion kinase inactivation

Background

Copper is an important regulator of hypoxia inducible factor 1 alpha (HIF-1α) dependent vascular endothelial growth factor (VEGF) expression, and is also required for the activity of lysyl oxidase (LOX) to effect matrix protein cross-linking. Cell detachment from the extracellular matrix can induce apoptosis (anoikis) via inactivation of focal adhesion kinase (FAK).

Methodology

To examine the molecular mechanisms whereby copper depletion causes the destruction of the normal alveolar architecture via anoikis, Male Sprague-Dawley rats were fed a copper deficient diet for 6 weeks while being treated with the copper chelator, tetrathiomolybdate. Other groups of rats were treated with the inhibitor of auto-phosphorylation of FAK, 1,2,4,5-benzenetetraamine tetrahydrochloride (1,2,4,5-BT) or FAK small interfering RNA (siRNA).

Principal Findings

Copper depletion caused emphysematous changes, decreased HIF-1α activity, and downregulated VEGF expression in the rat lungs. Cleaved caspase-3, caspase-8 and Bcl-2 interacting mediator of cell death (Bim) expression was increased, and the phosphorylation of FAK was decreased in copper depleted rat lungs. Administration of 1,2,4,5-BT and FAK siRNA caused emphysematous lung destruction associated with increased expression of cleaved capase-3, caspase-8 and Bim.

Conclusions

These data indicate that copper-dependent mechanisms contribute to the pathogenesis of emphysema, which may be associated with decreased HIF-1α and FAK activity in the lung.     

Endogenous VEGF is required for visual function: evidence for a survival role on müller cells and photoreceptors

Background

Vascular endothelial growth factor (VEGF) is well known for its role in normal and pathologic neovascularization. However, a growing body of evidence indicates that VEGF also acts on non-vascular cells, both developmentally as well as in the adult. In light of the widespread use of systemic and intraocular anti-VEGF therapies for the treatment of angiogenesis associated with tumor growth and wet macular degeneration, systematic investigation of the role of VEGF in the adult retina is critical.

Methods and Findings

Using immunohistochemistry and Lac-Z reporter mouse lines, we report that VEGF is produced by various cells in the adult mouse retina and that VEGFR2, the primary signaling receptor, is also widely expressed, with strong expression by Müller cells and photoreceptors. Systemic neutralization of VEGF was accomplished in mice by adenoviral expression of sFlt1. After 14 days of VEGF neutralization, there was no effect on the inner and outer retina vasculature, but a significant increase in apoptosis of cells in the inner and outer nuclear layers. By four weeks, the increase in neural cell death was associated with reduced thickness of the inner and outer nuclear layers and a decline in retinal function as measured by electroretinograms. siRNA-based suppression of VEGF expression in a Müller cell line in vitro supports the existence of an autocrine role for VEGF in Müller cell survival. Similarly, the addition of exogenous VEGF to freshly isolated photoreceptor cells and outer-nuclear-layer explants demonstrated VEGF to be highly neuroprotective.

Conclusions

These results indicate an important role for endogenous VEGF in the maintenance and function of adult retina neuronal cells and indicate that anti-VEGF therapies should be administered with caution.