Angiogenesis In Xenograft Model Research Articles

Ultrasound-stimulated microbubbles enhances radiosensitivity in cervical cancer

Background Ultrasound-stimulated microbubble (USMB) therapy has proven efficacy of targeting tumor vasculature and enhancing the effect of radiation in tumor xenografts. In this investigation, we studied whether this treatment enhances the sensitivity of cervical cancer to radiation. Methods Human cervical cancer (ME-180 and SiHa) cells were treated with USMB or exposed to radiation (0, 2, 4, 6 and 8 Gy) or radiation (8 Gy) in combination with USMB. Clone formation assay and CCK-8 assay were used to analyze the proliferation capacity of cells. Apoptosis and DNA double-strand breaks were detected using flow cytometry and immunofluorescence staining of gamma-H2AX (γ-H2AX), respectively. Matrigel tubule formation was performed to evaluate the angiogenesis of human umbilical vein endothelial cells. In xenograft model of SiHa cells, tumor tissue expression of CD31 was detected by immunohistochemistry. Results USMB and radiation synergistically restrained the growth of ME-180 and SiHa cells. USMB promoted radiation-induced apoptosis by enhancing the levels of proapoptotic proteins. Furthermore, USMB enhanced radiation-induced γ-H2AX foci to induce DNA double-strand breaks in cervical cancer cells. USMB in combination with radiation reduced the angiogenic capacity of endothelial cells in vitro. Moreover, USMB strengthened the inhibitory effect of radiation on tumor growth and angiogenesis in xenograft models. Conclusion In conclusion, USMB exposure effectively enhanced the destructive effect of radiation on cervical cancer, suggesting that USMB might be a promising sensitizer of radiotherapy to treat cervical cancer.

Hypoxia inducible factor‐1 activator munc‐18‐interacting protein 3 promotes tumour progression in urothelial carcinoma

AbstractBackgroundAlthough the introduction of multiple agents targeting immune and oncogenic signaling pathways has substantially changed the treatment strategies for urothelial carcinoma (UC), these drugs still have some limitations, such as adverse effects and limited responses depending on the molecular subtype of UC. Therefore, the development of novel molecular‐targeted therapies is required. Munc18‐1‐interacting protein 3 (Mint3), which specifically activates hypoxia inducible factor‐1 (HIF‐1) during normoxia in cancer and some stromal cells, might be a good target for UC treatment without severe adverse effects. However, the expression and function of Mint3 in UC remain unclear.MethodsWe prepared bladder cancer (n = 117) and upper tract UC (n = 207) cohorts and analyzed the correlation between Mint3 expression in the tissue microarray and the clinicopathological factors/prognosis. To clarify the function of Mint3 in UC cells, control and Mint3‐depleted UC cells were analyzed for HIF‐1 activity, HIF‐1 target gene expression, cell proliferation, glycolysis, motility and invasion in vitro. Tumorigenicity and angiogenesis in control and Mint3‐depleted UC cells were analyzed in mouse xenograft models. A Mint3 inhibitor, naphthofluorescein and gemcitabine were administrated into UC xenograft‐bearing mice.ResultsMint3 was expressed at various levels in UC cells; high expression was correlated with a poor prognosis but not with the molecular subtype of UC in two independent UC cohorts. Mint3 depletion did not affect cell proliferation but attenuated HIF‐1 activity, HIF‐1 target gene expression, glycolysis and motility/invasion in both luminal‐ and basal‐molecular‐subtype UC cells in vitro. Mint3 depletion in UC cells also attenuated tumour growth and angiogenesis in xenograft models. In addition, pharmacological inhibition of Mint3 sensitized chemo‐resistant UC xenografts to gemcitabine.ConclusionsOur findings indicate that Mint3 is a potential target for UC therapy with or without chemotherapy, independent of the molecular subtype of UC.

Effect of Auraptene on angiogenesis in Xenograft model of breast cancer.

Angiogenesis is the most important challenge in breast cancer treatment. Recently, scientists become interesting in rare natural products and intensive researches was performed to identify their pharmacological profile. Auraptene shows helpful effects such as cancer chemo-preventive, anti-inflammatory, anti-oxidant, immuno-modulatory. In this regard, we investigated the anti-angiogenesis effect of Auraptene in in-vitro and in-vivo model of breast cancer. In this study, 4T, MDA-MB-231 and HUVEC cell lines were used. The proliferation study was done by MTT assay. For tube formation assay, 250 matrigel, 1×104 HUVEC treated with Auraptene, 20ng/mL EGF, 20ng/mL bFGF and 20ng/mL VEGF were used. Gene expression of important gene related to angiogenesis in animal model of breast cancer was investigated by Real-time PCR. Protein expression of VCAM-1 and TNFR-1 gene related to angiogenesis in animal model of breast cancer was investigated by western-blot. Auraptene treatment led to reduction in cell viability of MDA-MB-231 in a concentration-dependent manner. Also, we observed change in the number of tubes or branches formed by cells incubated with 40 and 80μM Auraptene. Auraptene effect the gene expression of important gene related to angiogenesis (VEGF, VEGFR2, COX2, IFNɣ). Moreover, the western blot data exhibited that Auraptene effect the protein expression of VCAM-1 and TNFR-1. Overall, this study shows that Auraptene significantly suppressed angiogenesis via down-regulation of VEGF, VEGFR2, VCAM-1, TNFR-1, COX-2 and up-regulation of IFNγ.

Apatinib exhibits cytotoxicity toward leukemia cells by targeting VEGFR2-mediated prosurvival signaling and angiogenesis

ObjectiveVascular permeability contributes to disease progression and drug resistance in hematological malignancies, including AML. Thus, targeting angiogenic signaling is a promising treatment strategy, especially for relapsed and resistant AML. The aim of this study was to evaluate the efficacy of apatinib, a novel receptor tyrosine kinase inhibitor that selectively targets VEGFR2. MethodsSeveral AML cell lines were exposed to various concentrations of apatinib, and then CCK8 and Annexin V/PI assays were performed to determine IC50 values and apoptosis, respectively. The effect of apatinib against primary AML cells from 57 adult patients and 11 normal controls was also analyzed utilizing an apoptosis assay. Next, we tested the underlying mechanism of apatinib in AML using western blotting and mass cytometry (CyTOF). Finally, the activity of apatinib against tumor growth and angiogenesis was further evaluated in vivo in xenograft models. ResultsWe found apatinib significantly inhibited growth and promoted apoptosis in AML cell lines in vitro. Similarly, apatinib showed cytotoxicity against primary AML cells but didn't affect normal BMMCs. Its effect was highly correlated with several clinical features, such as NPM1 mutation, extramedullary infiltration, relapsed/refractory disease, and M2 and M5 FAB subtypes. In addition, apatinib suppressed AML growth and attenuated angiogenesis in xenograft models. Mechanistically, apatinib-induced cytotoxicity was closely associated with inhibition of the VEGFR2-mediated Src/STAT3 and AKT/mTOR pathways and induction of mitochondria-mediated apoptosis. ConclusionApatinib exerts antileukemia effects by targeting VEGFR2-induced prosurvival signaling and angiogenesis, thus providing a rationale for the application of apatinib in AML.

Apatinib Exhibits Cytotoxicity to Acute Myeloid Leukemia Cell Via Targeting VEGFR2-Mediated Pro-Survival Signaling and Angiogenesis

Objective:Vascular permeability contributes to disease progression and drug resistance in acute myeloid leukemia (AML). Thus, targeting angiogenetic signaling is a promising strategy for the treatment of AML, especially for relapsed and resistant diseases. The aim of the present study was to evaluate the potential efficacy of Apatinib, a novel receptor tyrosine kinase inhibitor selectively targeting VEGFR-2, in AML treatment. Method: CCK8 and Annexin V/PI assay were separately utilized to determine the IC50 value and apoptosis of AML cell lines. The cytotoxic efficacy of Apatinib against primary AML cells from 57 adult patients and 11 normal counterparts were then evaluated by analysis of apoptosis. Using JC-1 kit to detect the mitochondrial membrane potential, and western bolting and mass cytometry assay as well as immunohistochemistry to explore the underlying mechanism. Finally, the anti-leukemia activity was further evaluated in in vivo xenograft models. Results: Our findings demonstrated apatinib markedly inhibited proliferation and promoted apoptosis in AML cell lines. Similarly, apatinib showed cytotoxicity against primary AML cells while harmless to normal hematopoiesis. Its effect was correlated with several clinical features such as NPM1 mutation, extramedullary infiltration, relapse/refractory disease and FAB subtypes. Additionally, apatinib suppressed AML cells growth and attenuated angiogenesis in xenograft model. Mechanistically, apatinib-induced cytotoxicity was closely associated with the inhibition of VEGFR2-meidated Src/STAT3 and AKT/mTOR pathway and the induction of mitochondrial-mediated apoptosis. Conclusion: In summary, apatinib exerts anti-leukemia function via targeting VEGFR2-triggered pro-survival signaling and angiogenesis, providing a rationale for clinical trials of apatinib in the treatment of AML. Disclosures No relevant conflicts of interest to declare.

Highlights of This Issue

Treatment of adult glioma has not changed for over a decade, and it remains unclear how to best translate insights from genomic studies to bedside. Zheng and colleagues tested the utility of panel sequencing for assisting in glioma diagnosis, prognosis prediction, and informing targeted therapy using a prospective cohort of 237 gliomas. The study showed that the platform can identify IDH mutations that align with disease histopathology and outcome, and had high specificity and modest sensitivity in predicting 1p/19q codeletion. It also enabled stratification of hypermutators. Three out of ten patients whose treatments were tailored based on this panel achieved exceptional responses.Treatment of aggressive breast cancer remains challenging. Finding novel anti-cancer targets is critical for progress. In this study, Karginova and colleagues evaluate the efficacy and biological activity of the small molecule DCAC50, designed to inhibit intracellular copper transport through chaperones ATOX1 and CCS. DCAC50 triggers apoptosis in breast cancer cells and suppresses tumor angiogenesis in xenograft models. Co-treatment with paclitaxel enhances cytotoxicity in breast cancer cells. Mechanistically, DCAC50 disrupts copper homeostasis and generates oxidative stress. The ability of DCAC50 to induce apoptosis in cancer cells and suppress angiogenesis supports intracellular copper transport as a promising target for breast cancer therapy.This study by Shatsky and colleagues compares and contrasts solid tumor tissue NGS and ctDNA NGS technology to assess for potentially actionable genomic alterations in advanced breast malignancies. Both technologies were found to be useful and potentially complementary in the discovery of actionable alterations. In addition, ctDNA was found to have prognostic significance, as patients with greater than or equal to 5% ctDNA had a poorer overall survival. Both types of NGS have potential value that may add to targeted treatment options and further our understanding of advanced breast cancer.Peptide receptor radiotherapies with β-emitters are currently used to treat neuroendocrine tumors in patients. However, not all patients respond to treatment and most patients experience hematological toxicity. α-emitters, however, have a shorter pathlength and higher linear energy transfer allowing them to be more potent agents. This study by Stallons and colleagues examined 212Pb-DOTAMTATE in a preclinical neuroendocrine model and was more effective at controlling tumor growth in a single dose efficacy than previously published data with a beta-emitting somatostatin analog with little hematological toxicity. These studies support the therapeutic potential and possible advantage of 212Pb-DOTAMTATE and have led to the initiation of a phase I clinical trial.

Notch3 Targeting: A Novel Weapon against Ovarian Cancer Stem Cells.

Notch signaling is frequently activated in ovarian cancer (OC) and contributes to the proliferation and survival of cultured OC cells as well as to tumor formation and angiogenesis in xenograft models. Several studies demonstrate that Notch3 expression renders cancer cells more resistant to carboplatin, contributing to chemoresistance and poor survival of OC-bearing patients. This suggests that Notch3 can represent both a biomarker and a target for therapeutic interventions in OC patients. Although it is still unclear how chemoresistance arises, different lines of evidence support a critical role of cancer stem cells (CSCs), suggesting that CSC targeting by innovative therapeutic approaches might represent a promising tool to efficiently reduce OC recurrence. To date, CSC-directed therapies in OC tumors are mainly targeted to the inhibition of CSC-related signaling pathways, including Notch. As it is increasingly evident the involvement of Notch signaling, and in particular of Notch3, in regulating stem-like cell maintenance and expansion in several tumors, here we provide an overview of the current knowledge of Notch3 role in CSC-mediated OC chemoresistance, finally exploring the potential design of innovative Notch3 inhibition-based therapies for OC treatment, aimed at eradicating tumor through the suppression of CSCs.

A prodrug of green tea polyphenol (–)-epigallocatechin-3-gallate (Pro-EGCG) serves as a novel angiogenesis inhibitor in endometrial cancer

Anti-angiogenesis effect of a prodrug of green tea polyphenol (–)-epigallocatechin-3-gallate (Pro-EGCG) in malignant tumors is not well studied. Here, we investigated how the treatment with Pro-EGCG inhibited tumor angiogenesis in endometrial cancer. Tumor xenografts of human endometrial cancer were established and subjected to microarray analysis after Pro-EGCG treatment. First, we showed Pro-EGCG inhibited tumor angiogenesis in xenograft models through down-regulation of vascular endothelial growth factor A (VEGFA) and hypoxia inducible factor 1 alpha (HIF1α) in tumor cells and chemokine (C-X-C motif) ligand 12 (CXCL12) in host stroma by immunohistochemical staining. Next, we investigated how HIF1α/VEGFA was down-regulated and how the reduction of CXCL12 inhibited tumor angiogenesis. We found that VEGFA secretion from endometrial cancer cells was decreased by Pro-EGCG treatment through inhibiting PI3K/AKT/mTOR/HIF1α pathway. Furthermore, the down-regulation of CXCL12 in stromal cells by Pro-EGCG treatment restricted migration and differentiation of macrophages thereby inhibited infiltration of VEGFA-expressing tumor-associated macrophages (TAMs). Taken together, we demonstrated that treatment with Pro-EGCG not only decreases cancer cell-secreted VEGFA but also inhibits TAM-secreted VEGFA in endometrial cancer. These findings demonstrate that Pro-EGCG is a novel angiogenesis inhibitor for endometrial cancer.

The selective MEK1 inhibitor Selumetinib enhances the antitumor activity of everolimus against renal cell carcinoma in vitro and in vivo.

Renal cell carcinoma (RCC) is a urologic malignant cancer and often diagnosed at an advanced stage, which results in high mortality. Targeted therapy may improve the quality of life and survival of patients who are not suitable for nephrectomy. Everolimus, an mTOR inhibitor, is currently used as sequential or second-line therapy for RCC refractory to Sunitinib or sorafenib. However, its efficiency is palliative. In this study, we evaluated whether the antitumor activity of everolimus against RCC is enhanced by selumetinib, a selective MEK1 inhibitor. We discovered that everolimus in combination with selumetinib synergistically inhibited the proliferation of Caki-1, 786-O and 769-P cells in vitro. Mechanistically, this combination decreased p-RPS6 and p-4E-BP1 dramatically, which causes G1 cell cycle arrest and prevents reactivation of AKT and ERK. In vivo, the antitumor efficacy and pharmacodynamic biomarkers of the combination therapy were recapitulated in Caki-1 xenograft model. In addition, this combination treatment potently inhibited angiogenesis in xenograft models by impairing VEGF secretion from tumor cells. Our findings provide a sound evidence that combination of everolimus and selumetinib is a potential dual-targeted strategy for renal cell carcinoma.

Abstract 1365: Visualizing the effect of BIBF1120 in lung cancer cells by imaging-massspectrometry (MS)

Abstract Background: Angiogenesis plays an important role for tumor maintenance, progression, and metastasis. BIBF1120 is a multiple tyrosine kinase inhibitor, targeting VEGFR, FGFR and PDGFR, and is known as an anti-angiogenic agent. The mechanism by which anti-angiogenic agents suppress the growth of tumors is supposed to be induction of under-nutrition. However, no study has demonstrated the regional differences in the nutrition status of tumors treated by anti-angiogenic agents. Imaging MS is a novel technology that quantitatively visualizes the distribution of hundreds of metabolites in the selected area of tissues. Aim: The aim of this study is to evaluate the efficacy of BIBF1120 in lung cancer cells, and to examine whether BIBF1120 induces under-nutrition status in association with suppressed angiogenesis in xenograft models of lung cancer using Imaging MS. Method: We used lung cancer cell lines driven by several types of histology, PC9, H1975, H3122, A549, H69, and H520. The efficacy of BIBF1120 was evaluated by MTS assay and mouse xenograft model. We treated the xenografted mice with BIBF1120 or vehicle for 4 weeks and harvested xenograft tumors. Micro vessel density and percentage of apoptosis cells in the tumors was evaluated by immunohistochemistry using anti-Ki-67 antibody and anti-cleaved caspase 3 antibody, respectively. The effect of BIBF1120 on metabolic status in xenograft tumors was evaluated by imaging MS. Result: BIBF1120 inhibited the growth of all of the xenograft tumors tested, although it did not directly affect the proliferation rate of those cells in vitro. BIBF1120-treated tumors exhibited significantly lower micro vessel density compared to the vehicle-treated tumors (p<0.05), but BIBF1120 didn't increased the percentage of apoptosis cells. Imaging MS revealed that BIBF1120 treatment reduced 2-3 DPG (a maker of blood flow), glucose intake, and amount of ATP, whereas activated glycolysis via anaerobic metabolic pathway rather than oxidative phosphorylation in peripheral area of tumors. Conclusion: To our knowledge, this is the first report to demonstrate the effect of BIBF1120 on the nutrition status of tumor cells in a site-specific manner. Citation Format: Daisuke Arai, Kenzo Soejima, Hiroyuki Yasuda, Kota Ishioka, Shizuko Kagawa, Junko Hamamoto, Katsuhiko Naoki, Katsura Emoto, Yuki Sugiura, Makoto Suematsu, Tomoko Betsuyaku. Visualizing the effect of BIBF1120 in lung cancer cells by imaging-massspectrometry (MS). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1365. doi:10.1158/1538-7445.AM2015-1365

Effect of Melatonin on Tumor Growth and Angiogenesis in Xenograft Model of Breast Cancer

As neovascularization is essential for tumor growth and metastasis, controlling angiogenesis is a promising tactic in limiting cancer progression. Melatonin has been studied for their inhibitory properties on angiogenesis in cancer. We performed an in vivo study to evaluate the effects of melatonin treatment on angiogenesis in breast cancer. Cell viability was measured by MTT assay after melatonin treatment in triple-negative breast cancer cells (MDA-MB-231). After, cells were implanted in athymic nude mice and treated with melatonin or vehicle daily, administered intraperitoneally 1 hour before turning the room light off. Volume of the tumors was measured weekly with a digital caliper and at the end of treatments animals underwent single photon emission computed tomography (SPECT) with Technetium-99m tagged vascular endothelial growth factor (VEGF) C to detect in vivo angiogenesis. In addition, expression of pro-angiogenic/growth factors in the tumor extracts was evaluated by membrane antibody array and collected tumor tissues were analyzed with histochemical staining. Melatonin in vitro treatment (1 mM) decreased cell viability (p<0.05). The breast cancer xenografts nude mice treated with melatonin showed reduced tumor size and cell proliferation (Ki-67) compared to control animals after 21 days of treatment (p<0.05). Expression of VEGF receptor 2 decreased significantly in the treated animals compared to that of control when determined by immunohistochemistry (p<0.05) but the changes were not significant on SPECT (p>0.05) images. In addition, there was a decrease of micro-vessel density (Von Willebrand Factor) in melatonin treated mice (p<0.05). However, semiquantitative densitometry analysis of membrane array indicated increased expression of epidermal growth factor receptor and insulin-like growth factor 1 in treated tumors compared to vehicle treated tumors (p<0.05). In conclusion, melatonin treatment showed effectiveness in reducing tumor growth and cell proliferation, as well as in the inhibition of angiogenesis.

E7050: A dual c‐Met and VEGFR‐2 tyrosine kinase inhibitor promotes tumor regression and prolongs survival in mouse xenograft models

c-Met is the cellular receptor for hepatocyte growth factor (HGF) and is known to be dysregulated in various types of human cancers. Activation of the HGF/c-Met pathway causes tumor progression, invasion, and metastasis. Vascular endothelial growth factor (VEGF) is also known as a key molecule in tumor progression through the induction of tumor angiogenesis. Because of their key roles in tumor progression, these pathways provide attractive targets for therapeutic intervention. We have generated a novel, orally active, small molecule compound, E7050, which inhibits both c-Met and vascular endothelial growth factor receptor (VEGFR)-2. In vitro studies indicate that E7050 potently inhibits phosphorylation of both c-Met and VEGFR-2. E7050 also potently represses the growth of both c-met amplified tumor cells and endothelial cells stimulated with either HGF or VEGF. In vivo studies using E7050 showed inhibition of the phosphorylation of c-Met and VEGFR-2 in tumors, and strong inhibition of tumor growth and tumor angiogenesis in xenograft models. Treatment of some tumor lines containing c-met amplifications with high doses of E7050 (50-200 mg/kg) induced tumor regression and disappearance. In a peritoneal dissemination model, E7050 showed an antitumor effect against peritoneal tumors as well as a significant prolongation of lifespan in treated mice. Our results indicate that E7050 is a potent inhibitor of c-Met and VEGFR-2 and has therapeutic potential for the treatment of cancer.

Estrogenic or antiestrogenic therapies for multiple myeloma?

Multiple myeloma (MM) is a common hematological malignancy which remains incurable due to both intrinsic and acquired resistance to conventional or more novel drugs. Estrogenic and antiestrogenic compounds are very promising drugs for the treatment of MM. Indeed, they inhibit cell proliferation in vitro. They block cell cycle and/or induce apoptosis even in drug-resistant MM cells but not normal B cells. They interfere with survival pathways often deregulated in myelomas. They co-operate with conventional drugs to enhance apoptosis or to overcome resistance. In vivo, they act also on tumoral angiogenesis in xenograft models. As a whole, they possess all the criteria which render them attractive for a new therapeutic strategy. Importantly, they are well-tolerated at the doses tested in vitro or in vivo, encouraging the rapid onset of critical trials.

Recombinant non‐collagenous domain of α2(IV) collagen causes involution of choroidal neovascularization by inducing apoptosis

Vascular endothelial cells receive proangiogenic or antiangiogenic signals from components of extracellular matrix (ECM) depending upon the situation and many molecular signals can have opposite effects in different vascular beds. Tissue inhibitor of metalloproteinase 1 is antiangiogenic in several tissues, but promotes retinal neovascularization. When cleaved from native collagens, several of the non-collagenous domains (NC1) of basement membrane collagens have antiangiogenic effects in some tissues, but this is context dependent for the NC1 of the alpha 1 chain of collagen IV. It is critical to examine effects in several well-defined model systems before assuming that an ECM component is universally antiangiogenic. In this study, we examined the effects of a recombinant fragment of NC1 of the alpha 2 chain of type IV collagen (alpha2(IV)NC1) in a well-characterized model of ocular neovascularization. Intravitreous or periocular injections of alpha2(IV)NC1 caused selective apoptosis of endothelial cells participating in neovascularization resulting in suppression of neovascularization when the peptide was given prior to onset of new vessel sprouting. Importantly, when the peptide was given after neovascularization had already developed, it caused the new vessels to regress. This suggests that alpha2(IV)NC1, which has previously been shown to suppress tumor angiogenesis in xenograft models, is also a strong antiangiogenic agent in the choroid and is a therapeutic candidate for treatment of neovascular age-related macular degeneration.