Cancer-associated Endothelial Cells Research Articles

Targeting RhoA expression with formononetin and salvianolic acid B to mitigate pancreatic cancer-associated endothelial cells changes

Ethnopharmacological relevanceAccording to the theory of Qi and blood in Traditional Chinese Medicine (TCM), the combination of Qi-reinforcing herbs and blood-activating herbs has a synergistic effect in improving blood stasis syndrome, especially in tumor treatment. The classic “Radix Astragali - Salvia miltiorrhiza” duo exemplifies this principle, renowned for invigorating Qi and activating blood flow, employed widely in tumor therapies. Our prior research underscores the potent inhibition of pancreatic tumor xenografts by the combination of Formononetin (from Radix Astragali) and Salvianolic acid B (from Salvia miltiorrhiza) in vitro. However, it remains unclear whether this combination can inhibit the abnormal vascularization of pancreatic tumors to achieve its anti-cancer effect. Aim of the studyAbnormal vasculature, known to facilitate tumor growth and metastasis. Strategies to normalize tumor-associated blood vessels provide a promising avenue for anti-tumor therapy. This study aimed to unravel the therapeutic potential of Formononetin combined with Salvianolic acid B (FcS) in modulating pancreatic cancer's impact on endothelial cells, illuminate the underlying mechanisms that govern this therapeutic interaction, thereby advancing strategies to normalize tumor vasculature and combat cancer progression. Materials and methodsA co-culture system involving Human Umbilical Vein Endothelial Cells (HUVECs) and PANC-1 cells was established to investigate the potential of targeting abnormal vasculature as a novel anti-tumor therapeutic strategy. We systematically compared HUVEC proliferation, migration, invasion, and lumenogenesis in both mono- and co-culture conditions with PANC-1 (H-P). Subsequently, FcS treatment of the H-P system was evaluated for its anti-angiogenic properties. Molecular docking was utilized to predict the interactions between Formononetin and Salvianolic acid B with RhoA, and the post-treatment expression of RhoA in HUVECs was assessed. Furthermore, we utilized shRhoA lentivirus to elucidate the role of RhoA in FcS-mediated effects on HUVECs. In vivo, a zebrafish xenograft tumor model was employed to assess FcS's anti-tumor potential, focusing on cancer cell proliferation, migration, apoptosis, and vascular development. ResultsFcS treatment demonstrated a significant, dose-dependent inhibition of PANC-1-induced alterations in HUVECs, including proliferation, migration, invasion, and tube formation capabilities. Molecular docking analyses indicated potential interactions between FcS and RhoA. Further, FcS treatment was found to downregulate RhoA expression and modulated the PI3K/AKT signaling pathway in PANC-1-induced HUVECs. Notably, the phenotypic inhibitory effects of FcS on HUVECs were attenuated by RhoA knockdown. In vivo zebrafish studies validated FcS's anti-tumor activity, inhibiting cancer cell proliferation, metastasis, and vascular sprouting, while promoting tumor cell apoptosis. ConclusionsThis study underscores the promising potential of FcS in countering pancreatic cancer-induced endothelial alterations. FcS exhibits pronounced anti-abnormal vasculature effects, potentially achieved through downregulation of RhoA and inhibition of the PI3K/Akt signaling pathway, thereby presenting a novel therapeutic avenue for pancreatic cancer management.

Primary Human Breast Cancer-Associated Endothelial Cells Favor Interactions with Nanomedicines.

Cancer nanomedicines predominately rely on transport processes controlled by tumor-associated endothelial cells to deliver therapeutic and diagnostic payloads into solid tumors. While the dominant role of this class of endothelial cells for nanoparticle transport and tumor delivery is established in animal models, the translational potential in human cells needs exploration. Using primary human breast cancer as a model, the differential interactions of normal and tumor-associated endothelial cells with clinically relevant nanomedicine formulations are explored and quantified. Primary human breast cancer-associated endothelial cells exhibit up to ≈2times higher nanoparticle uptake than normal human mammary microvascular endothelial cells. Super-resolution imaging studies reveal a significantly higher intracellular vesicle number for tumor-associated endothelial cells, indicating a substantial increase in cellular transport activities. RNA sequencing and gene expression analysis indicate the upregulation of transport-related genes, especially motor protein genes, in tumor-associated endothelial cells. Collectively, the results demonstrate that primary human breast cancer-associated endothelial cells exhibit enhanced interactions with nanomedicines, suggesting a potentially significant role for these cellsin nanoparticle tumor delivery in human patients. Engineering nanoparticles that leverage the translational potential of tumor-associated endothelial cell-mediated transport into human solid tumors may lead to the development of safer and more effective clinical cancer nanomedicines.

Detection of Disease-Specific Parent Cells Via Distinct Population of Nano-Vesicles by Machine Learning.

The diagnosis and prognosis of pathological conditions, such as age-related macular degeneration (AMD) and cancer still need improvement. AMD is primarily caused due to the dysfunction of retinal pigment epithelium (RPE), whereas endothelial cells (ECs) play one of the major roles in angiogenesis; an important process which occurs in malignant progression of cancer. Several reports suggested the augmented release of nano-vesicles under pathological conditions, including from RPE as well as cancer-associated ECs, which take part in various biological processes, including intercellular communication in disease progression. Importantly, these nano-vesicles are around 30-1000 nm and carry the fingerprint of their initiating parent cells (IPCs). Therefore, these nano-vesicles could be utilized as the diagnostic tool for AMD and cancer, respectively. However, the analysis of nano-vesicles for biomarker study is confounded by their extensive heterogeneous nature. To confront this challenge, we utilized artificial intelligence (AI) based machine learning (ML) algorithms such as support vector machine (SVM) and decision tree model on the dataset of nano-vesicles from RPE and ECs cell lines with low dimensionality. Overall, Gaussian SVM demonstrated the highest prediction accuracy of the IPCs of nano-vesicles, among all the chosen SVM classifiers. Additionally, the bagged tree showed the highest prediction among the chosen decision tree-based classifiers. Therefore, the overall bagged tree showed the best performance for the prediction of IPCs of nanovesicles, suggesting the applicability of AI-based prediction approach in diagnosis and prognosis of pathological conditions, including non-invasive liquid biopsy via various biofluids-derived nano-vesicles.

MicroRNAs in the Tumor Microenvironment.

The tumor microenvironment (TME) is decisive for the eradication or survival of any tumor mass. Moreover, it plays a pivotal role for metastasis and for providing the metastatic niche. The TME offers special physiological conditions and is composed of, for example, surrounding blood vessels, the extracellular matrix (ECM), diverse signaling molecules, exosomes and several cell types including, but not being limited to, infiltrated immune cells, cancer-associated endothelial cells (CAEs), and cancer-associated fibroblasts (CAFs). These cells can additionally and significantly contribute to tumor and metastasis progression, especially also by acting via their own deregulated micro (mi) RNA expression or activity. Thus, miRNAs are essential players in the crosstalk between cancer cells and the TME. MiRNAs are small non-coding (nc) RNAs that typically inhibit translation and stability of messenger (m) RNAs, thus being able to regulate several cell functions including proliferation, migration, differentiation, survival, invasion, and several steps of the metastatic cascade. The dynamic interplay between miRNAs in different cell types or organelles such as exosomes, ECM macromolecules, and the TME plays critical roles in many aspects of cancer development. This chapter aims to give an overview on the multiple contributions of miRNAs as players within the TME, to summarize the role of miRNAs in the crosstalk between different cell populations found within the TME, and to illustrate how they act on tumorigenesis and the behavior of cells in the TME context. Lastly, the potential clinical utility of miRNAs for cancer therapy is discussed.

Emerging Roles of Long non-coding RNAs in The Tumor Microenvironment.

Long non-coding RNAs (lncRNAs) are a diverse class of longer than 200 nucleotides RNA transcripts that have limited protein coding capacity. LncRNAs display diverse cellular functions and widely participate in both physiological and pathophysiological processes. Aberrant expressions of lncRNAs are correlated with tumor progression, providing sound rationale for their targeting as attractive anti-tumor therapeutic strategies. Emerging evidences support that lncRNAs participate in tumor-stroma crosstalk and stimulate a distinctive and suitable tumor microenvironment (TME). The TME comprises several stromal cells such as cancer stem cells (CSCs), cancer-associated endothelial cells (CAEs), cancer-associated fibroblasts (CAFs) and infiltrated immune cells, all of which are involved in the complicated crosstalk with tumor cells to affect tumor progression. In this review, we summarize the essential properties and functional roles of known lncRNAs in related to the TME to validate lncRNAs as potential biomarkers and promising anti-cancer targets.

Abstract 999: In vivo breast tumor stiffness and vascular drug delivery recapitulated in a microfluidic tumor-on-a-chip

Abstract Biomimetic tissue engineered microfluidic cancer models offer a higher degree of spatial, temporal and structural precision in controlling the physical parameters and component characteristics of the native tumor microenvironment (TME). Current models working to establish a biomimetic in vitro breast TME are limited by their ability to recapitulate various degrees of in vivo complexities and poor correlation of the diffusional gradients of oxygen, nutrients and anti-cancer drugs. To establish a model and address these challenges, we have used poly(ethylene glycol)-fibrinogen (PEG-Fb) as our biomimetic material to engineer 3D breast tumor tissues and recapitulate the mechanical stiffness of core, midpoint and peripherial zones of the native tumor in a vascularized microfluidic chip. To assess the mechanical stiffness, the in vivo breast tumor (MDA-MB-231 flank xenograft in Athymic nude mice) and engineered tumor constructs were subjected to parallel plate compression test using Cell Scale Microsquisher and the resulting force versus displacement data was acquired to calculate Young’s modulus. Tumor mimetic (“high perfusion chip” (HPC) and “low perfusion chip” (LPC), differ with respect to the vascular network surrounding their respective primary and secondary tumor compartments were used in this study. Breast cancer-associated endothelial cells (hBTEC) were seeded in the vascular network and allowed to form a lumen. Metastatic breast cancer cells MDA-MB-231/ human foreskin fibroblast BJ5ta (ATCC) cells were mixed with polymer precursor solution containing PEG-Fb and Eosin Y. The precursor was loaded into the primary tumor compartment and cross-linked for 2 minutes under visible light. Stiffness was modulated by adding poly(ethylene glycol) diacrylate (PEGDA) to the polymer precursor for recapitulating the different zones of the in vivo tumor. hBTEC media was perfused through the endothelial cell networks were continuously monitored for cell behavior and metastasis. In vivo breast tumor stiffness at core, midpoint and periphery was found to be within the range of the 3D engineered breast tumor tissues with time in culture through day 29. In the vascularized microfluidic chip, cell laden biomaterial was incorporated, the cancer cells were observed to undergo key events of the TME such as intravasation, circulating tumor cells in the endothelial vascular channel, adherence and migration to the secondary chamber resulting in metastasis. In the native TME there are regional differences in drug diffusion; TRITC dextran (4.4 kDa) was administered at a constant flow rate through the chips’ vascularized networks and found to have vascular network geometry and engineered tumor construct stiffness dependent differences in diffusion into the primary tumor chamber, mimicking the in vivo phenomena. Citation Format: Benjamin Anbiah, Iman Hassani, Nicole Habbit, Lani Jasper, Deborah Ramsay, Balabhaskar Prabhakarpandian, Robert Arnold, Elizabeth Lipke. In vivo breast tumor stiffness and vascular drug delivery recapitulated in a microfluidic tumor-on-a-chip [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 999.

MicroRNAs: Pleiotropic Regulators in the Tumor Microenvironment.

MicroRNAs (miRNAs) are small non-coding RNAs that typically inhibit the translation and stability of messenger RNAs (mRNAs). They are ~22 nucleotides long and control both physiological and pathological processes. Altered expression of miRNAs is often associated with human diseases. Thus, miRNAs have become important therapeutic targets, and some clinical trials investigating the effect of miRNA-based therapeutics in different types of diseases have already been conducted. The tumor microenvironment (TME) comprises cells such as infiltrated immune cells, cancer-associated endothelial cells (CAEs) and cancer-associated fibroblasts (CAFs), and all the components participate in the complicated crosstalk with tumor cells to affect tumor progression. Altered miRNAs expression in both these stromal and tumor cells could drive tumorigenesis. Thus, in this review, we discuss how aberrantly expressed miRNAs influence tumor progression; summarize the crosstalk between infiltrated immune cells, CAEs, CAFs, and tumor cells through miRNAs, and clarify the important roles of miRNAs in the tumor microenvironment, which may facilitate the clinical application of miRNA-based therapies.

Improving vascular maturation using noncoding RNAs increases antitumor effect of chemotherapy.

Current antiangiogenesis therapy relies on inhibiting newly developed immature tumor blood vessels and starving tumor cells. This strategy has shown transient and modest efficacy. Here, we report a better approach to target cancer-associated endothelial cells (ECs), reverse permeability and leakiness of tumor blood vessels, and improve delivery of chemotherapeutic agents to the tumor. First, we identified deregulated microRNAs (miRs) from patient-derived cancer-associated ECs. Silencing these miRs led to decreased vascular permeability and increased maturation of blood vessels. Next, we screened a thioaptamer (TA) library to identify TAs selective for tumor-associated ECs. An annexin A2-targeted TA was identified and used for delivery of miR106b-5p and miR30c-5p inhibitors, resulting in vascular maturation and antitumor effects without inducing hypoxia. These findings could have implications for improving vascular-targeted therapy.

Abstract P5-09-02: Tβ4 expression in cancer-associated endothelial cells enhances progression of invasive breast cancer

Abstract Background: Invasive breast cancer is a highly aggressive primary breast tumour with poor prognosis. As tumour angiogenesis is exhibited by invasive breast cancer, anti-angiogenic therapy has been intensively evaluated over the past decade. However, clinical studies were disappointing. Thymosin β4 (Tβ4), a multi-functional peptide, is associated with induction of angiogenesis, but the role of Tβ4 in tumorigenesis of invasive breast cancer is unknown. We tested whether Tβ4 inhibition could be a new target involved in tumour development and tumour angiogenesis for treatment of invasive breast cancer. Methods: We have adapted a 3D co-culture system to acquire invasive breast cancer cells-associated endothelial cells (ECs), which is followed by characterising the differential gene expressions by PCR-based subtraction analysis. We performed Western blot analysis on the protein samples obtained from ECs-stimulated by invasive breast cancer cells. Expression of Tβ4 in invasive breast cancer was assessed by qPCR in the breast tissues (tumour, n=119; background, n=55), and protein expression confirmed by immunohistochemical examination in an invasive breast cancer tissue microarray. Finally, mice breast cancer xenografts plus non-invasively photoacoustic microscopy were used to analyse the effect of Tβ4 knockdown on the tumorigenesis of invasive breast cancer. Results: We found that invasive breast cancer cells could stimulate an increase in Tβ4 expression in microvascular ECs. High Tβ4 levels were strongly associated with high malignant invasive breast cancer and poor clinical outcome. In vivo study showed that siRNA targeting Tβ4 blocked growth of invasive breast cancer in mice. Photoacoustic imaging revealed that knockdown of Tβ4 elicited anti-breast cancer growth, in part, through disruption of tumour vasculature. Our results demonstrate that endothelial cell-derived Tβ4 enhances the progression of breast cancer by up-regulating tumour angiogenesis and it indicates that a high level of tumour stromal Tβ4 is an independent predictor of poor outcome. Conclusions: Inhibiting endothelial Tβ4 could be a new therapeutic target in anti-angiogenic strategy for treatment of invasive breast cancer. Citation Format: Cai J, Jiang W. Tβ4 expression in cancer-associated endothelial cells enhances progression of invasive breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-09-02.

Single-cell RNA sequencing reveals gene expression signatures of breast cancer-associated endothelial cells.

Tumor endothelial cells (TEC) play an indispensible role in tumor growth and metastasis although much of the detailed mechanism still remains elusive. In this study we characterized and compared the global gene expression profiles of TECs and control ECs isolated from human breast cancerous tissues and reduction mammoplasty tissues respectively by single cell RNA sequencing (scRNA-seq). Based on the qualified scRNA-seq libraries that we made, we found that 1302 genes were differentially expressed between these two EC phenotypes. Both principal component analysis (PCA) and heat map-based hierarchical clustering separated the cancerous versus control ECs as two distinctive clusters, and MetaCore disease biomarker analysis indicated that these differentially expressed genes are highly correlated with breast neoplasm diseases. Gene Set Enrichment Analysis software (GSEA) enriched these genes to extracellular matrix (ECM) signal pathways and highlighted 127 ECM-associated genes. External validation verified some of these ECM-associated genes are not only generally overexpressed in various cancer tissues but also specifically overexpressed in colorectal cancer ECs and lymphoma ECs. In conclusion, our data demonstrated that ECM-associated genes play pivotal roles in breast cancer EC biology and some of them could serve as potential TEC biomarkers for various cancers.

Improving vascular maturation using noncoding RNAs increases antitumor effect of chemotherapy.

Current antiangiogenesis therapy relies on inhibiting newly developed immature tumor blood vessels and starving tumor cells. This strategy has shown transient and modest efficacy. Here, we report a better approach to target cancer-associated endothelial cells (ECs), reverse permeability and leakiness of tumor blood vessels, and improve delivery of chemotherapeutic agents to the tumor. First, we identified deregulated microRNAs (miRs) from patient-derived cancer-associated ECs. Silencing these miRs led to decreased vascular permeability and increased maturation of blood vessels. Next, we screened a thioaptamer (TA) library to identify TAs selective for tumor-associated ECs. An annexin A2-targeted TA was identified and used for delivery of miR106b-5p and miR30c-5p inhibitors, resulting in vascular maturation and antitumor effects without inducing hypoxia. These findings could have implications for improving vascular-targeted therapy.

Abstract C171: Human anti-Nucleolin recombinant immunoagents as new potential tools for melanoma treatment

Abstract Immunotherapy and immune-based anti-cancer molecules represent a valid strategy to fight cancer. However, the choice of tumor-specific surface molecules for the selective targeting of cancer cells still represents a critical step in the study design for the development of new therapeutic approaches. Notably, the development of phage-display technology for the selection of fully human single chain antibody fragments (scFvs) and complete antibodies directed toward tumor-associated antigens has represented a significant advancement for immunotherapy. Nucleolin (NCL) is one of the most abundant non-ribosomal proteins in the nucleolus. NCL is frequently up-regulated in cancer and in cancer-associated endothelial cells compared to normal tissues, where it is also present on the cell surface. Altered NCL expression and localization results in oncogenic effects such as stabilization of oncogenic mRNAs and microRNAs (miRNAs). Particularly, we demonstrated that NCL enhances the maturation of specific miRNAs (including miR-21, miR-221 and miR-222) causally involved in cancer pathogenesis, aggressiveness, metastatic potential and resistance to several anti-neoplastic treatments. Because of its oncogenic role and specific expression on cancer cell surface, NCL represents an attractive target for anti-neoplastic therapies. To produce a new anti-NCL molecule with significant potential for clinical applications, we took advantage of phage-display technology to isolate a fully human single chain Fragment variable (scFv), named 4LB5, which binds with high affinity to the RNA binding domain (RBD) of NCL. In our previous study we demonstrated that 4LB5 binds NCL on the surface of aggressive breast cancer cells and inhibits their proliferation both in vitro and in vivo, representing the prototype of a new class of immune-based anti-NCL compounds. Since NCL expression has been previously reported on the cell surface of skin cancer cell lines and up-regulation of NCL-dependent microRNAs was described in human melanomas, the objective of this project was the assessment of 4LB5 as a potential tool for melanoma therapy. To this aim, the recombinant scFv was expressed as His6-fusion protein in E.Coli and purified by affinity chromatography, as previously described. By using Enzyme-Linked Immunosorbent Assays (ELISA), we demonstrated a significant binding of 4LB5 to the cell surface of different melanoma cell lines of both human and mouse origins. Notably, inhibition of NCL expression by siRNA transfection reduced the binding of 4LB5 to the cell surface of these cell lines, further supporting its specificity against NCL. Then, we assessed the potential effects of 4LB5 treatment on cell proliferation. Colony formation assays demonstrated that 4LB5 significantly affected cell proliferation of both human and mouse melanoma cell lines. Our results, in agreement with previously reported data, further support the potential activity of 4LB5 as a tool for cancer therapy, paving the way for additional investigations aimed to fully elucidate the molecular mechanisms affected by this scFv and resulting in its anti-neoplastic therapy in human melanomas. Furthermore, this study supports the idea that anti-NCL immunoagents might represent a class of new anti-cancer compounds with a strong clinical relevance for a wide range of human tumors. Citation Format: Ashley Braddom, Timothy Richmond, Tyler Sheetz, Erika Reese, Anna Tessari, Kathleen Tober, Christin E. Burd, Claudia De Lorenzo, Edward W. Martin, Jr., Vincenzo Coppola, Michael F. Tweedle, Tatiana Oberyszyn, Carlo M. Croce, Dario Palmieri. Human anti-Nucleolin recombinant immunoagents as new potential tools for melanoma treatment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C171.

Gene expression changes after ionizing radiation in endothelial cells derived from human endometrial cancer-preliminary outcomes

Accumulating evidence has demonstrated that death of microvascular endothelial cells plays a decisive role in the tumor response against radiotherapy. Nevertheless, radiation-induced gene alterations on cancer-associated endothelial cells of human endometrial carcinoma remain poorly understood. The purpose of this study was to elucidate the gene expression changes after X-ray radiation in human endometrial carcinoma vascular endothelial cells and to provide new targets for combined treatment of radiation and anti-angiogenesis in human endometrial carcinoma. Endometrial cancer-derived endothelial cells, which obtained before and 4 h after 400 cGy X-ray radiation from four endometrial carcinomas, were analyzed by gene expression profile. The selected meaningful genes from gene microarray experiments were validated by real-time quantitative PCR. Microarray analyses showed 49 significantly changed genes which were common to all the microarray experiments. There into, 14 genes were found to be in persistent up-regulation and 14 in persistent down-regulation 4 h after X-ray radiation when compared with the control group. These genes were involved in cell cycle and growth regulation, cell-apoptosis, chemokine, cell signaling, cellular stress response, angiogenesis, DNA synthesis and repair and cell adhesion. Eight randomly selected genes were validated by real-time PCR. The genes of cancer-derived endothelial cells regulated by X-ray radiation as well as their related signal pathways, which obtained from gene expression profiling data, were relevant to radiosensitivity of endometrial cancer. This study shows that the identified genes and their related signaling pathways are candidated biomarkers for radiation and anti-angiogenesis of human endometrial carcinoma.

E3330 Enhanced Antitumor Effect of Adrinmycin on Human Extranodal Nasal-Type NK/T Cell Lymphoma Xenografted–Nude Mice

Abstract 2723 Abstract:Extranodal Nasal-Type NK/T Cell Lymphoma (EN-NK/T-L) is a distinct clinicopathological entity of non-Hodgkin’s lymphoma, which is characterized by a highly aggressive clinical process and poor prognosis especially when disseminated or recurrent after radiotherapy. Human purinic/apyrimidinic endonuclease/redox factor-1 (hAPE/Ref-1) is a multifunctional protein involved in the repair of DNA damaged by oxidative. Because of its involvement in DNA repair and apoptosis-related signaling mechanisms, APE/Ref-1 is also being discussed as a novel target for tumor-therapeutic approaches. In studies, high level of APE1 expression is associated with an resistance to chemotherapeutic agents and adverse prognosis. And recent studies show that E3330, a small molecule inhibitor of the Ape-1/Ref-1 redox domain, induce inhibition of growth in cancer cell in vitro by specific inhibiting the function of APE/REF-1. E3330 blocks the in vitro growth of pancreatic cancer-associated endothelial cells(J Cell Physiol. 2009 Apr;219(1):209–18.) and of the NK/T-cell lineage in our sdudy already and provides a potential therapeutic strategy in tumor. Thus far, no study has reported the experimental research on E3330 combined with chemotherapy in treating NK/T cell lymphoma. Therefore, we try to establish xenografted-nude mice model of human extranodal nasal type NK/T cell lymphoma and to investigate the effect of E3330 combined with adrinmycin on the human EN-NK/T-L xenografted-nude mice and its mechanism. Methods: EN-NK/T-L Xenografted-nude mice model was established by intraperitoneal injection of NK/T cell lymohoma cells SNK-6. IC50 of E3330 for SNK-6 was calculated according to the result of MTT assay. Xenografted- nude mice were divided into four groups: Control group were given equal volume sodium,po,d1-7□ G E3330 group were given E3330, 6mg/kg, po, d1-7□ GADM group were given ADM, 0.5mg/kg, ip,d1,4,7□ GE3330 + ADM group were given both E3330 and ADM, E3330, 6mg/kg, po, d1-7, ADM 0.5mg/kg, ip, D1,4,7. The tumor weight and the survival rate of tumor–bearing mice were assayed, the apoptosis cells were determined by TdT-mediated Dutp Nick-end-Labeling(Tunel). Immunohistochemistry were used to study the expression of ki-67. Immunofluorescence were used to study the expression of APE/REF-1. Results: Nude mice model of human extranodal nasal type NK/T cell lymphoma was established successfully. The results showed that the expression level of APE/REF-1 was higher in tumor tissues compared with that in normal tissues (Fig 1 A 1B). The transplanted tumor volumes of mice were significantly smaller in E3330 + ADM group than those in other three groups(p<0.05)(Table 1). The expression of ki-67 in E3330 + ADM group was lower than other groups(p<0.05) (Fig 2 A 2B 2C 2D) and the apoptosis cells were increased in the same group (Table 2). Conclusion: Our study showed that E3330 + adrinmycin can significantly inhibit the growth of transplanted tumor of extranodal nasal type NK/T cell lymphoma Xenografted-nude mice. Disclosures:No relevant conflicts of interest to declare.

Impact of APE1/Ref-1 Redox Inhibition on Pancreatic Tumor Growth

Pancreatic cancer is especially a deadly form of cancer with a survival rate less than 2%. Pancreatic cancers respond poorly to existing chemotherapeutic agents and radiation, and progress for the treatment of pancreatic cancer remains elusive. To address this unmet medical need, a better understanding of critical pathways and molecular mechanisms involved in pancreatic tumor development, progression, and resistance to traditional therapy is therefore critical. Reduction-oxidation (redox) signaling systems are emerging as important targets in pancreatic cancer. AP endonuclease1/Redox effector factor 1 (APE1/Ref-1) is upregulated in human pancreatic cancer cells and modulation of its redox activity blocks the proliferation and migration of pancreatic cancer cells and pancreatic cancer-associated endothelial cells in vitro. Modulation of APE1/Ref-1 using a specific inhibitor of APE1/Ref-1's redox function, E3330, leads to a decrease in transcription factor activity for NFκB, AP-1, and HIF1α in vitro. This study aims to further establish the redox signaling protein APE1/Ref-1 as a molecular target in pancreatic cancer. Here, we show that inhibition of APE1/Ref-1 via E3330 results in tumor growth inhibition in cell lines and pancreatic cancer xenograft models in mice. Pharmacokinetic studies also show that E3330 attains more than10 μmol/L blood concentrations and is detectable in tumor xenografts. Through inhibition of APE1/Ref-1, the activity of NFκB, AP-1, and HIF1α that are key transcriptional regulators involved in survival, invasion, and metastasis is blocked. These data indicate that E3330, inhibitor of APE1/Ref-1, has potential in pancreatic cancer and clinical investigation of APE1/Ref-1 molecular target is warranted.

Angiogenin as a Molecular Target for the Treatment of Prostate Cancer

Angiogenin (ANG), a 14 kDa angiogenic ribonuclease, is upregulated in human prostate cancers, especially in hormone refractory diseases, and is the highest upregulated gene in Akt-driven prostate intraepithelial neoplasia (PIN) in mice. ANG has been shown to undergo nuclear translocation in both prostate cancer cells and cancer-associated endothelial cells where it binds to the promoter region of ribosomal DNA (rDNA) and stimulates ribosomal RNA (rRNA) transcription. ANG thus plays an essential role in prostate cancer progression by stimulating both cancer cell proliferation and tumor angiogenesis. A variety of ANG antagonists, including its antisense oligonucleotide, siRNA, soluble binding proteins, monoclonal antibody, enzymatic inhibitors, and nuclear translocation blockers, have all been shown to inhibit prostate cancer in various animal models. Accumulating evidence indicates that ANG is a molecular target for prostate cancer drug development.

The Ape‐1/Ref‐1 redox antagonist E3330 inhibits the growth of tumor endothelium and endothelial progenitor cells: Therapeutic implications in tumor angiogenesis

The apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ape-1/Ref-1) is a multi-functional protein, involved in DNA repair and the activation of redox-sensitive transcription factors. The Ape-1/Ref-1 redox domain acts as a cytoprotective element in normal endothelial cells, mitigating the deleterious effects of apoptotic stimuli through induction of survival signals. We explored the role of the Ape-1/Ref-1 redox domain in the maintenance of tumor-associated endothelium, and of endothelial progenitor cells (EPCs), which contribute to tumor angiogenesis. We demonstrate that E3330, a small molecule inhibitor of the Ape-1/Ref-1 redox domain, blocks the in vitro growth of pancreatic cancer-associated endothelial cells (PCECs) and EPCs, which is recapitulated by stable expression of a dominant-negative redox domain mutant. Further, E3330 blocks the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into CD31(+) endothelial progeny. Exposure of PCECs to E3330 results in a reduction of H-ras expression and intracellular nitric oxide (NO) levels, as well as decreased DNA-binding activity of the hypoxia-inducible transcription factor, HIF-1alpha. E3330 also reduces secreted and intracellular vascular endothelial growth factor expression by pancreatic cancer cells, while concomitantly downregulating the cognate receptor Flk-1/KDR on PCECs. Inhibition of the Ape-1/Ref-1 redox domain with E3330 or comparable angiogenesis inhibitors might be a potent therapeutic strategy in solid tumors.