Lewis Lung Carcinoma Mouse Model Research Articles

LBA3 - Novel pyrazolopyrimidine derivatives inhibit angiogenesis and tumor growth in a mouse Lewis lung carcinoma model

ABSTRACT Background: Vascular endothelial growth factor receptors (VEGFR) are tyrosine kinases (TK) known to promote angiogenesis and cancer. Since quinazoline and pyridopyrimidine derivatives have been proved to be potent and highly selective TK inhibitors, in this work we aimed to synthesize novel substituted pyrazolopyridines and explore their capability to modulate angiogenesis and tumor growth. Materials and methods: Pyrazolopyridine derivatives were synthesized using 2-amino-5-nitro-4-picoline as starting material, which upon suitable transformations was converted to 5-chloropyrazolo[3,4-c]pyridine. A 4-methoxybenzyl group was inserted at N1 of the heterocyclic ring system followed by a subsequent addition of a 3-phenyl group in certain derivatives. A second chlorine atom was introduced at position 7 via N-oxide metathesis and then 5- and 7-chlorine atoms were successively displaced by suitable amines. Twelve derivatives were isolated pure and their structure identified using 1D and 2D NMR and mass spectrometry. Compounds were assessed for their ability to modulate basal and/or VEGF-induced endothelial cell proliferation, migration and differentiation using MTT, transwell and matrigel tube-formation assays [ 1 ], respectively. The effect of active compounds on VEGFR2 signaling was determined by immunoblotting. In vivo activity was evaluated on a syngeneic Lewis lung carcinoma (LLC) transplantation mouse model [ 2 ] Results: Amongst test compounds, four (SC1117, SC1119, SC1123, SC1124) significantly inhibited at a concentration-dependent manner (0.2–2 µM), crucial pro-angiogenic attributes of endothelial cells related to proliferation, migration and differentiation capabilities, in part by targeting pathways downstream of VEGFR2. Moreover i.p. administration of SC1117, SC1119 and SC1124 (0.05-0.2 mg/kg body weight thrice/week, for 2 weeks) significantly reduced LLC tumor volume in mice without toxicity. Conclusions: We described three novel pyrazolopyrimidine derivatives targeting VEGFR2 as potential inhibitors of angiogenesis and tumor growth.

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PC-FACS

Abstract 3995: Quantitative protein profiling of tumor angiogenesis and metastasis biomarkers in mouse and human models

Abstract Tumor and stromal cells secrete a variety of proteins acting as extracellular signals and creating a supportive microenvironment for tumor development, angiogenesis, and metastasis. We used the MILLIPLEX MAP cytokine/chemokine, cancer, angiogenesis, metastasis, MMP, bone metabolism, adipocyte, and cell signaling multi-pathway panels, based on the Luminex xMAP technology, to quantitatively evaluate the protein biomarker expression profiles on cultured mouse and human tumor cells and stromal cells as well as in vivo mouse and human tumor tissues. In the mouse system, biomarker profiling was performed to characterize secreted protein levels in Lewis Lung carcinoma (LLC) cells, B16.10 melanoma cells, b.End3 endothelial cells, and LLC tumors. The in vivo characterization, using primary tumors from mice bearing metastatic LLC, has identified differential expression of a panel of biomarkers, including amphiregulin (AREG), EGF, PECAM-1, KC, MCP-1, MIP-2, MMP-12, and resistin, between the high-fat (45% fat) and AIN-93G (15% fat) fed mice (p<0.05, 8 mice per group). These putative biomarkers may be involved in molecular mechanisms of the high-fat enhanced metastasis. Cell signaling pathway analysis revealed the activation of multiple pathways (JNK, p38, ERK, and STAT) in this mouse LLC model. In the human system, a heat map was generated to summarize the secreted protein signatures and the soluble receptor tyrosine kinases (RTK) profile on a series of the cultured human tumor cells (lung, brain, breast, prostate, liver, and colon) and 3 other human cell lines, HUVEC (endothelial), THP-1 (monocyte), and HFF-1 (fibroblast). Lastly, expression profiling of tumor and adjacent normal tissues from 2 patients with metastatic breast cancer was analyzed. Elevated levels of protein biomarkers, including MDC, IL-1RA, IL-8, IP-10, MCP-1, MIP-1α, OPG, OPN, SPARC, periostin, endoglin, PLGF, VEGF-A, MMP-1, MMP-7, MMP-9, and MMP-12, were detected in the breast tumors analyzed compared to their matched adjacent normal breast tissue. In summary, our results demonstrated that multiplexing using the Luminex xMAP bead-based platform for simultaneous quantitative expression profiling is a useful proteomic research tool, providing insights into biomarkers related to tumor progression and metastasis. Citation Format: Wen-Rong Lie, Jonathan Lipsey, Tim Warmke, Lin Yan, Jehangir Mistry. Quantitative protein profiling of tumor angiogenesis and metastasis biomarkers in mouse and human models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3995. doi:10.1158/1538-7445.AM2014-3995

Treatment with Huisheng oral solution inhibits the development of pulmonary thromboembolism and metastasis in mice with Lewis lung carcinoma

The aim of this study was to investigate whether Huisheng oral solution (HSOS) has an inhibitory effect on the development of pulmonary thrombosis and metastasis in mice with Lewis lung carcinoma (LLC), and to explore the possible mechanisms involved. A mouse model of LLC was developed, and model mice were divided into either a treatment group or a control group to undergo treatment with HSOS or normal saline. Normal mice treated with saline were used as normal controls. On day 25 after treatment, blood samples were drawn from the eyes of half the mice in each group to determine blood cell counts and plasma levels of D-Dimer and vascular endothelial growth factor (VEGF), while heart blood samples were collected from the remaining mice to measure the rate of thrombin-induced platelet aggregation. For all mice, pathological analyses of the cerebrum, lung, mesentery, femoral vein, external iliac vein and spleen were performed. Tumors were weighed to assess the impact of HSOS treatment on tumor growth, and the number of thrombi, metastatic nodules and neovessels in the tumor tissue were counted. In addition, 24 normal New Zealand rabbits were divided into two groups and treated with either HSOS or normal saline to determine the rates of ADP-, collagen- or thrombin-induced platelet aggregation. Compared with the model group, HSOS treatment decreased the incidence of pulmonary thromboembolism and metastasis, the number of metastatic nodules, the plasma levels of D-dimer and VEGF, the rate of collagen-induced platelet aggregation in rabbits and the numbers of leukocytes and tumor neovessels (P<0.05 for all). It increased the thymus and spleen coefficients and the number of platelets (P<0.05 for all), but had no significant effect on thrombin-induced platelet aggregation in mice and rabbits, ADP-induced platelet aggregation in rabbits, or the number of red blood cells. The reduced rate of tumor growth was 9.7% in mice treated with HSOS. HSOS treatment effectively reduced the development of pulmonary thromboembolism and metastasis in mice bearing LLC via mechanisms possibly associated with ameliorating a blood hypercoagulable state, decreasing tumor angiogenesis and enhancing immunity.

Development of a New Positron Emission Tomography Tracer for Targeting Tumor Angiogenesis: Synthesis, Small Animal Imaging, and Radiation Dosimetry

Angiogenesis plays a key role in cancer progression and correlates with disease aggressiveness and poor clinical outcomes. Affinity ligands discovered by screening phage display random peptide libraries can be engineered to molecularly target tumor blood vessels for noninvasive imaging and early detection of tumor aggressiveness. In this study, we tested the ability of a phage-display-selected peptide sequence recognizing specifically bone marrow- derived pro-angiogenic tumor-homing cells, the QFP-peptide, radiolabeled with 64Cu radioisotope to selectively image tumor vasculature in vivo by positron emission tomography (PET). To prepare the targeted PET tracer we modified QFP-phage with the DOTA chelator and radiolabeled the purified QFP-phage-DOTA intermediate with 64Cu to obtain QFP-targeted radioconjugate with high radiopharmaceutical yield and specific activity. We evaluated the new PET tracer in vivo in a subcutaneous (s.c.) Lewis lung carcinoma (LLC) mouse model and conducted tissue distribution, small animal PET/CT imaging study, autoradiography, histology, fluorescence imaging, and dosimetry assessments. The results from this study show that, in the context of the s.c. LLC immunocompetent mouse model, the QFP-tracer can target tumor blood vessels selectively. However, further optimization of the biodistribution and dosimetry profile of the tracer is necessary to ensure efficient radiopharmaceutical applications enabled by the biological specificity of the QFP-peptide.

Abstract LB-338: A novel LPA-PKD1-FoxO1 pathway in endothelial cells provides an angiogenic switch via down-regulation of CD36 transcription and induction of arteriogenic responses .

Abstract CD36 is an endothelial cell (EC) receptor that mediates angiostatic activity and oxidative stress, which are important in ischemic disease and tumor progression. CD36 interacts with TSR domain proteins including thrombospondin-1. This results in a signal that is likely to initiate an antiangiogenic “switch," subsequently converting a growth factor-mediated proangiogenic response to an antiangiogenic response. However, in pathologic settings including malignant tumors, robust angiogenesis occurs despite the abundance of TSR-containing proteins in the microenvironment. This suggests that there is a mechanism by which TSR-mediated antiangiogenesis could be blunted via the localized downregulation of EC CD36 transcription. We previously reported that lysophosphatidic acid (LPA), a bioactive signaling phospholipid mediator, down-regulates microvascular EC (MVEC) CD36 transcription by protein kinase PKD-1 signaling via G protein coupled receptor LPA1,3. However, further elucidating the CD36 transcriptional mechanisms is critical to understanding the antiangiogenic switch. To this end, we recently discovered that obesity-derived LPA inhibited CD36 expression in tumor-associated ECs (TAECs). Similarly, inducing the constitutively active PKD-1 or PKD-CA expression also resulted in CD36 downregulation. Mechanistically, LPA treatment led to nuclear accumulation of PKD-1, HDAC7 and FoxO1. Furthermore, LPA-mediated PKD-1 signaling enhanced FoxO1-HDAC7 interaction in the nucleus. This interaction was attenuated with PKD-1 silencing. Intriguingly, doxycycline induced PKD-CA expression increased ephrin B2 expression and ERK activation, which are two critical “molecular signatures” involved in arteriogenesis. Functionally, FGF-2 induced a potent proangiogenic response even in the presence of TSP-1 in ECs pre-treated with LPA. Doxycycline induced PKD-CA expression stimulated robust arterial EC branching in a 3D spheroid assay. This angiogenic response was inhibited by a novel PKD-1 specific inhibitor. Moreover, in a mouse Lewis lung carcinoma model, we detected a minimal level of endothelial CD36 expression with robust tumor angiogenesis. The role of PKD-1 signaling in CD36 transcriptional repression and tumor arteriogenesis is a critical yet underexplored and underappreciated field. We propose that the LPA-PKD-FoxO1 signaling axis is essential for CD36 transcriptional repression and arteriogenic signaling and responses. The PKD-1 stimulated nuclear signaling network is critical to epigenetic suppression of CD36 transcription and silencing of CD36 antiangiogenic switch, subsequently turning on the angiogenic switch and stimulating the arteriogenic responses. Targeting the LPA-PKD1-FoxO1 signaling axis could have therapeutic potential in the ischemic disease and malignant tumors. Citation Format: Jacob D. Kohlenberg, Yiliang Chen, Brad Best, Peter Storz, Randall T. Peterson, Roy Silverstein, Bin Ren. A novel LPA-PKD1-FoxO1 pathway in endothelial cells provides an angiogenic switch via down-regulation of CD36 transcription and induction of arteriogenic responses . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-338. doi:10.1158/1538-7445.AM2013-LB-338

Development of a metastatic fluorescent Lewis Lung carcinoma mouse model: Identification of mRNAs and microRNAs involved in tumor invasion

Cancer metastasis is the foremost cause of death in cancer patients. A series of observable pathological changes takes place during progression and metastasis of cancer, but the underlying genetic changes remain unclear. Therefore, new approaches are required, including insights from cancer mouse models. To examine the mechanisms involved in tumor metastasis, we first generated a stably transfected Lewis Lung carcinoma cell line expressing a far-red fluorescent protein, called Katushka. After in vivo growth in syngeneic mice, two fluorescent Lewis Lung cancer subpopulations were isolated from primary tumors and lung metastases. The metastasis-derived cells exhibited a significant improvement in in vitro invasive activity compared to the primary tumor-derived cells, using a quantitative invasion chamber assay. Moreover, expression levels of 84 tumor metastasis-related mRNAs, 88 cancer-related microRNAs as well as Dicer and Drosha were determined using RT-qPCR. Compared to the primary Lewis Lung carcinoma subculture, the metastasis-derived cells exhibited statistically significantly increased mRNA levels for several matrix metalloproteinases as well as hepatocyte growth factor (HGF) and spleen tyrosine kinase (SYK). A modest decrease in Drosha and Dicer mRNA levels was accompanied by significant downregulation of ten microRNAs, including miR-9 and miR-203, in the lung metastatic Lewis Lung carcinoma cell culture. Thus, a tool for cancer metastasis studies has been established and the model is well suited for the identification of novel microRNAs and mRNAs involved in malignant progression. Our results suggest that increases in metalloproteinase expression and impairment of microRNA processing are involved in the acquirement of metastatic ability.

Enhanced radioresponse with a novel recombinant human endostatin protein via tumor vasculature remodeling: Experimental and clinical evidence

PurposeThis study aimed to examine the effect of the novel recombinant human endostatin (rh-Endo) protein on tumor vasculature, and to explore and evaluate the optimal scheduling of rh-Endo and radiotherapy (RT). MethodsTumor-perfusion parameters and hypoxia were monitored after rh-Endo treatment in 10 non-small cell lung-cancer (NSCLC) patients. Eight-week female C57BL/6J mice were randomized to receive rh-Endo or control (saline) once daily for 12days when Lewis lung carcinoma (LLC) reached approximately 100–150mm3. On planned days, tumors were measured for cell apoptosis, microvessel density, pericytes, blood-vessel morphology, and tumor hypoxia. The tumor response under different combinations of rh-Endo and RT schedules was evaluated. ResultsTumor hypoxia was significantly reduced 5days after rh-Endo in NSCLC patients, and a similar result was found in the LLC mouse model. The anti-tumor effect was markedly enhanced when RT was administered within the remodeling period compared to any other treatment schedule. rh-Endo treatment remodeled the tumor vasculature after 5days by reducing microvessel density and increasing pericytic coverage of the vessel endothelium. ConclusionThis study demonstrated decreased hypoxia in animals and patients upon rh-Endo treatment, which also enhanced the radioresponse within the vasculature-remodeling period. The optimal clinical combination of rh-Endo and RT warrants further investigation.

Effect of Heparin and Its Derivatives On the Progression of Tumor Growth in Mouse Lewis Lung Carcinoma Model.

Abstract 2274 Background:Unfractionated heparin and it derivatives are known to produce pleotropic effects including the anticoagulant, anti-inflammatory and antitumor actions. Preclinical evidence suggests that heparins have an effect on tumor progression independent of their anticoagulant activity. Heparin and low molecular weight heparin have also been shown to exhibit interactions with growth factors and other cellular receptors. Recently an ultra low molecular weight heparin namely, semuloparin has also been found to be effective in the management of cancer associated thrombosis (Save ONCO trial). This study was designed to investigate whether heparin and its derivatives are able to inhibit tumor growth in a Lewis Lung carcinoma model. Methods:Female C57BL/6 mice were obtained at 6–8 weeks of age and were implanted with 5×105 LN7 tumor cells by dorsal subcutaneous injection in the upper back. When tumors were initially palpable, at 7–10 days of growth, mice were treated with subcutaneous injections of heparin, a low molecular weight heparin (LMWH), namely enoxaparin, an ultra LMWH, semuloparin or saline, daily for two weeks at approximately 1 cm away from the tumor growth, in a dose range of 1.0 – 0.25 mg/kg. After the treatment period, animals were sacrificed and the spleens and tumors were removed and tumor weight, tumor volume, spleen weight and spleen size were measured. Blood samples were drawn through heart puncture for ex vivo analysis. VEGF levels were measured using a commercially available ELISA method. Results:At the 1.0 and 0.5 mg/kg dosages, both enoxaparin (p<0.01) and semuloparin (p<0.01) showed a decrease in tumor volume compared to the saline control animals. At the 1.0 mg/kg dosage although heparin was effective in reducing the tumor size, the mortality was higher in this group due to bleeding. At 0.5 mg/kg heparin was not different from the saline control in terms of reducing tumor growth. In addition, at a dosage of 0.25 mg/kg, only semuloparin showed a significant difference in reducing the tumor size in comparison to saline control (p<0.01). Similar results were observed for the tumor weight. There were no significant differences noted in spleen weight or spleen size among these agents. The mortality rates in the mice treated with enoxaparin and semuloparin were relatively lower (<10%) and were comparable between the two groups. Ex vivo analysis of blood showed a significant difference in the VEGF levels in the semuloparin group in comparison to enoxaparin and heparin. Conclusions:These studies suggest that heparin and its derivatives are capable of inhibiting tumor growth in a dose dependent manner. Enoxaparin and semuloparin are more effective at reducing tumor growth compared to heparin. In addition, both the enoxaparin and semuloparin were safer than heparin at the 1.0 and 0.5 mg dosages as bleeding was observed in the heparin treated animals. Semuloparin downregulated the VEGF levels in comparison to the other groups suggesting that this mechanism potentially plays a role in the control of the tumor growth. Reported clinical studies have shown that semuloparin is safer and effective for the prevention of venous thromboembolism in cancer patients and compares favorably to enoxaparin in terms of antithrombotic effect and safety profile. Therefore, semuloparin may be a better alternate for the management of cancer associated thrombosis. Disclosures:No relevant conflicts of interest to declare.

Effect of heparin and its derivatives on the progression of tumor growth in mouse Lewis lung carcinoma model.

e13115 Background: Preclinical evidence suggests that heparins have an effect on tumor progression independent of their anticoagulant activity. Heparins have also been shown to exhibit interactions with growth factors and other cellular receptors. This study was designed to investigate whether heparin and its derivatives are able to inhibit tumor growth. Methods: Female C57BL/6 mice were obtained at 6-8 weeks of age and were implanted with 5X105 LN7 tumor cells by dorsal subcutaneous injection of in the upper back. When tumors were first palpable, after 7-10 days of growth, mice were treated with subcutaneous injections of heparin, a low molecular weight heparin (LMWH), namely enoxaparin, an ultra LMWH, semuloparin or saline, daily for two weeks in a dose range of 1.0 – 0.25 mg/kg. After the treatment period, animals were sacrificed and the spleens and tumors were removed and their weight, volume, spleen weight and size were measured. Results: At the 1.0 and 0.5 mg/kg dosages, both enoxaparin (p&lt;0.01) and semuloparin (p&lt;0.01) showed a decrease in tumor volume compared to the saline control animals. At the 1.0 mg/kg dosage, the mortality was high in the heparin group due to bleeding. At 0.5 mg/kg heparin was not different from the saline control. In addition, at a dosage of 0.25 mg/kg, only semuloparin showed a difference compared to the saline control (p&lt;0.01). Similar results were observed for the tumor weight. There were no significant differences noted in spleen weight or spleen size among these agents. The mortality rates between the mice treated with enoxaparin and semuloparin were comparable. Conclusions: These studies suggest that heparin and its derivatives are capable of inhibiting tumor growth in a dose dependent manner. Enoxaparin and semuloparin are more effective at reducing tumor growth compared to heparin. Clinical studies have shown that semuloparin is safe and effective for the prevention of venous thromboembolism in cancer patients and compares favorably to enoxaparin in terms of antithrombotic effect and safety profile. Therefore, semuloparin may be a better alternate for the prevention of cancer associated thrombosis.

The impact of intraoperative vaccination with IL-12 modified autologous tumor cells in the Lewis lung carcinoma mouse model

To improve immunological defense of tumors, we investigated the effect of intraoperative vaccination with IL-12 cDNA transfected cells in an autologous mouse tumor model. Tumors derived from autologous Lewis lung carcinoma cells were established in C57/BL6 mice. At day seven, the tumors were surgically removed. Simultaneously, the mice were vaccinated intraoperatively with Lewis lung carcinoma cells transfected with an IL-12-encoding pRSC construct or with the empty plasmid, or with dead cells either intrasplenically (i.s.) or subcutaneously (s.c.). Control mice did not obtain vaccination. Tumor re-growth, survival, and metastasis were monitored. Mice with no tumor re-growth underwent a second tumor implantation. The same parameters were examined. After tumor resection and vaccination tumors reappeared in 60.0% of the animals of the control group. Lowest tumor reoccurrence rates of 41.4 and 43.5% were seen in animals receiving IL-12 pRSC cells either i.s. or s.c. Survival tended to be enhanced in all vaccinated animals compared with the control group. Following R0 tumor resection, the rate of tumor-free survivors was highest when IL-12 pRSC cells were given i.s. (73%, control 45%). 37-59% of the mice of the therapy groups did not develop a tumor, that is, they were tumor-free survivors. These mice underwent a second tumor implantation 120 days after tumor resection and vaccination. Tumor growth was significantly delayed in mice receiving IL-12 pRSC cells. Intraoperative autologous whole-cell vaccination is practicable and proved to have anti-tumor potential, and therefore, it could be an additional option in adjuvant cancer therapy.

Adenosinergic Regulation of the Expansion and Immunosuppressive Activity of CD11b+Gr1+ Cells

Extracellular adenosine and purine nucleotides are elevated in many pathological situations associated with the expansion of CD11b(+)Gr1(+) myeloid-derived suppressor cells (MDSCs). Therefore, we tested whether adenosinergic pathways play a role in MDSC expansion and functions. We found that A(2B) adenosine receptors on hematopoietic cells play an important role in accumulation of intratumoral CD11b(+)Gr1(high) cells in a mouse Lewis lung carcinoma model in vivo and demonstrated that these receptors promote preferential expansion of the granulocytic CD11b(+)Gr1(high) subset of MDSCs in vitro. Flow cytometry analysis of MDSCs generated from mouse hematopoietic progenitor cells revealed that the CD11b(+)Gr-1(high) subset had the highest levels of CD73 (ecto-5'-nucleotidase) expression (Δmean fluorescence intensity [MFI] of 118.5 ± 16.8), followed by CD11b(+)Gr-1(int) (ΔMFI of 57.9 ± 6.8) and CD11b(+)Gr-1(-/low) (ΔMFI of 12.4 ± 1.0) subsets. Even lower levels of CD73 expression were found on Lewis lung carcinoma tumor cells (ΔMFI of 3.2 ± 0.2). The high levels of CD73 expression in granulocytic CD11b(+)Gr-1(high) cells correlated with high levels of ecto-5'-nucleotidase enzymatic activity. We further demonstrated that the ability of granulocytic MDSCs to suppress CD3/CD28-induced T cell proliferation was significantly facilitated in the presence of the ecto-5'-nucleotidase substrate 5'-AMP. We propose that generation of adenosine by CD73 expressed at high levels on granulocytic MDSCs may promote their expansion and facilitate their immunosuppressive activity.

Mast cells and histamine enhance the proliferation of non-small cell lung cancer cells

Non-small cell lung cancer (NSCLC) is the most common form of lung cancer with an extremely low survival rate. It is characterized by a chronic inflammatory process with intense mast cell infiltrate that is associated with reduced survival. The aim of this study was to test the hypothesis that mast cells have an enhancing effect on NSCLC proliferation. To assess the tumor-promoting potential of mast cells, we used the human alveolar basal adenocarcinoma (A549) and the mouse Lewis lung carcinoma (LLC) cell lines, umbilical cord blood-derived mast cells (CBMC) and the mast cell-deficient mouse Sash model. The proliferation rate of A549/LLC cells was markedly increased by mast cells and histamine. Histamine proliferating activity was mediated via H1, H2 and H4 receptors and caused ERK phosphorylation. LLC induced in Sash mice or in wild-type mice treated with the mast cell stabilizer nedocromil sodium displayed an accelerated growth (number of metastic colonies in the lungs, total lung area and lung/total mice weight ratio). In summary, we have shown a significant effect of mast cells and histamine in enhancing NSCLC/LLCX growth in vitro, while in a mouse LLC model in vivo we have found that mast cells are important negative regulators of cancer development. Therefore our results would indicate a pro-tumorogenic effect of the mast cells in vitro on established lung tumor cell lines, and anti-tumorogenic effect in mice at lung cancer induction. In conclusion, mast cell/anti-histamine targeted therapies should carefully consider this dual effect.

Ethyl pyruvate, an anti-inflammatory agent, inhibits tumor angiogenesis through inhibition of the NF-κB signaling pathway

Ethyl pyruvate (EP), simple derivative of pyruvate, has been shown to have anti-inflammatory properties. Here, we demonstrate EP’s strong anti-angiogenic activity. EP inhibited in vivo angiogenesis in the mouse Matrigel-plug assay and tumor growth in the mouse Lewis lung carcinoma model. EP also interfered with the angiogenic cascade, including growth, invasion, migration, and tube formation. Activation of NF-κB by vascular endothelial cell growth factor was reduced by EP. Taken together, we suggest that EP may have potential as a new multi-functional drug candidate for anti-angiogenesis and cancer therapy.

Effect of Sunitinib Combined with Ionizing Radiation on Endothelial Cells

The aims of present study were to evaluate the efficacy of combining sunitinib with ionizing radiation (IR) on endothelial cells in vitro and in vivo. Human umbilical vein endothelial cells (HUVECs) were exposed to IR with or without sunitinib pretreatment. Apoptosis assay and cell cycle distribution were analyzed by flow cytometry. Clonogenic survival assay at 3 Gy dose with or without sunitinib was performed. The activity of phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway was detected by Western immunoblot. Lewis lung carcinoma mouse model was built to examine the effect of combination therapy on endothelial cells in vivo. Microvasculature changes were detected by immunohistochemistry using anti-CD31 antibody. Our results showed combination therapy of sunitinib and IR significantly increased apoptosis of endothelial cells and inhibited colony formation compared to sunitinib or radiotherapy alone. It also resulted in cell cycle redistribution (decreasing cells in S phase and increasing cells in G2/M phase). The activity of PI3K/Akt signal pathway was inhibited, which could be the potential mechanisms that account for the enhanced radiation response induced by sunitinib. In vivo analysis showed that combination therapy significantly decreased microvasculature formation. The results demonstrated that combination therapy of sunitinib and IR has the potential to increase the cytotoxic effects on endothelial cells.

Immunopharmacology of Thymosin α1 and Cytokine Synergy

Thymosin alpha1 (Talpha1) is a 28 amino acid biologically active protein cleaved from positions 2-29 of a precursor protein, prothymosin alpha. Since its discovery, Talpha1 has been administered to animals and humans in a wide variety of settings and its pharmacologic effects are to enhance cellular immunity. Talpha1 administration is highly effective in settings where irradiation, chemotherapy, tumor burden, or immune senescence have caused a reduction of T cell number and/or function. Recent in vitro studies, including the one reported here, suggest that Talpha1 may act via pathways commonly used by various cytokines. This raises the possibility that Talpha1 and cytokines may have synergistic activity through potentiation of cytokine activity by Talpha1. Improved control of tumor growth when tumor-bearing mice were treated with Talpha1 and high doses of IL-2 has been previously reported. We extended those studies with the Lewis lung carcinoma mouse model using IRX-2, a natural well-defined biologic containing multiple cytokines, in combination with Talpha1 (IRX-3). Although IRX-2 was effective alone (using doses that contain significantly less IL-2 than in most typical studies), adding Talpha1 led to significant improvement in survival of the tumor-bearing mice. Based on these observations, the immunopharmacology of Talpha1 predicts an important clinical role for Talpha1 in the restoration of cellular immune activity when used in combination with cytokines. Patients who experience immune suppression due to the presence of tumor, irradiation, and/or chemotherapy or aging of the host would most benefit from this treatment combination.

Inhibition of Lewis Lung Carcinoma Growth by Toxoplasma gondii through Induction of Th1 Immune Responses and Inhibition of Angiogenesis

Toxoplasma gondii is an obligate intracellular protozoan parasite that induces antitumor activity against certain types of cancers. However, little information is available regarding the immunologic mechanisms that regulate these effects. For this purpose, C57BL/6 mice were administered either the T. gondii Me49 strain orally or Lewis lung carcinoma (LLC) cells intramuscularly. Survival rates, tumor size, histopathology, and immune responses were determined for each group, and angiogenesis was evaluated by in vivo Matrigel plug assay. Toxoplasma-infected (TG-injected) mice survived the entire experimental period, whereas cancer cell-bearing (LLC-injected) mice died within six weeks. Mice injected with both T. gondii and cancer cells (TG/LLC-injected group) showed significantly increased survival rates, CD8+ T-cell percentages, IFN-γ mRNA expression levels, serum IgG2a titers, and CTL responses as compared to the LLC-injected mice. In addition, angiogenesis in the TG/LLC-injected mice was notably inhibited. These effects in TG/LCC-injected mice were similar or were increased by the addition of an adjuvant, Quil-A. However, TG/LLC-injected mice showed decreased percentages of CD4+ and CD8+ T-cells, IFN-γ mRNA expression levels, and serum IgG1 and IgG2a titers as compared to TG-injected mice. Taken together, our results demonstrate that T. gondii infection inhibits tumor growth in the Lewis lung carcinoma mouse model through the induction of Th1 immune responses and antiangiogenic activity.

Antibody-directed targeting of angiostatin's receptor annexin II inhibits Lewis Lung Carcinoma tumor growth via blocking of plasminogen activation: Possible biochemical mechanism of angiostatin's action

Angiostatin, the N-terminal four kringles (K1–4) of parent molecule plasminogen, is reported to block Lewis Lung Carcinoma (LLC) tumor growth and metastasis. However, angiostatin's mechanism of action is unclear. We earlier reported that angiostatin binds to cell surface annexin II through the lysine-binding domain (kringles 1–4) [Tuszynski, G.P., Sharma, M., Rothman, V.L., Sharma, M.C., 2002. Angiostatin binds to tyrosine kinase substrate annexin II through the lysine-binding domain in endothelial cells. Microvasc. Res. 64:448–462.]). We now show that annexin II on the cell surface of LLC cells regulates conversion of plasminogen to plasmin. Activation of plasminogen to plasmin is time-dependent, with the linear activation lasting up to 120 min. Monoclonal antibodies to annexin II reduced plasminogen activation by 92.6%, suggesting a specific role of annexin II in plasmin generation. Angiostatin also reduced plasmin generation by 81.6%, suggesting that angiostatin may be competing with plasminogen through lysine-binding domain. ε-Aminocaproic acid, a lysine analogue, effectively blocked plasminogen activation indicating that, indeed, the lysine-binding site of the kringles domain is required for activation. These data suggest that annexin II may be a receptor target for angiostatin's action. Therefore, we tested the effect of high affinity monoclonal antibody to annexin II in mouse model of LLC. A single dose of antibody treatment inhibited LLC tumor growth almost 70% with concomitant inhibition of circulating plasmin generation and its proteolytic activity. Taken together, it is possible that inhibition of LLC tumor growth and metastasis reported by angiostatin therapy may be due to blocking of annexin-II-dependent plasmin generation. Plasmin is known to influence angiogenic, invasive and metastatic capability of tumors.

Thrombospondin-1 Mimetic Peptide Inhibitors of Angiogenesis and Tumor Growth: Design, Synthesis, and Optimization of Pharmacokinetics and Biological Activities

The heptapeptide 1, NAc-Gly-Val-DIle-Thr-Arg-Ile-ArgNHEt, a structurally modified fragment derived from the second type-1 repeat of thrombospondin-1 (TSP-1), is known to possess antiangiogenic activity. However, therapeutic utility could not be demonstrated because this peptide has a very short half-life in rodents. To optimize the PD/PK profile of 1, we initiated a systematic SAR study. The initial structural modifications were performed at positions 5 and 7 of peptide 1 and at the N- and C-termini. Out of several hundred peptides synthesized, the nonapeptide 5 (ABT-526) emerged as a promising lead. ABT-526 inhibited VEGF-induced HMVEC cell migration and tube formation in the nanomolar range and increased apoptosis of HUAEC cells. ABT-526 showed acceptable PK in rodents, dog, and monkey. ABT-526, when incorporated in an angiogenic pellet implanted in the rat cornea at 10 microM, reduced neovascularization by 92%. Substitution of DalloIle in place of DIle in ABT-526 provided nonapeptide 6 (ABT-510), which was 30-fold less active than ABT-526 in the EC migration but 20-fold more active in the tube formation assay. In comparison to ABT-526, ABT-510 has increased water solubility and slower clearance in dog and monkey. Radiolabeled ABT-510 demonstrated saturable binding to HMVEC cells at 0.02-20 nM concentrations and was displaceable by TSP-1. ABT-510 and ABT-526 were shown to significantly increase apoptosis of HUAEC cells. ABT-510 was effective in blocking neovascularization in the mouse Matrigel plug model and inhibited tumor growth in the mouse Lewis lung carcinoma model. Previous studies had shown that ABT-510 was effective in inhibiting the outgrowth of murine melanoma metastases in syngeneic mice and in blocking the growth of human bladder carcinoma implanted in nude mice. It had been also shown that ABT-510 could regress tumor lesions in pet dogs or cause unexpected stabilization of the disease in advanced canine cancer. ABT-526 and ABT-510 are the first compounds in the class of potent inhibitors of angiogenesis that mimic the antiangiogenic function of TSP-1. ABT-510 is currently in phase II clinical studies.

Inhibition of tumor cell-induced platelet aggregation and lung metastasis by the oral GpIIb/IIIa antagonist XV454.

Platelets are known to play a role in blood borne metastasis. Previous experimental studies have suggested that platelet GpIIb/IIIa may be a therapeutic target. However, the need for intravenous administration limits the potential application of current GpIIb/IIIa inhibitors to cancer therapy. The aim of the present study was to assess the efficacy of a novel, non-peptide oral GpIIb/IIIa antagonist (XV454) on tumor cell-induced platelet aggregation in vivo and on experimental metastasis. A Lewis lung carcinoma (LL2) mouse model of experimental metastasis was used in this study. XV454 (100 micro g) was administered intravenously (via tail vein) or orally (gavages) to 20 g mice. To determine the effect of XV454 on platelet aggregation, blood samples were collected by cardiac puncture 10 minutes after intravenous and 1-24 hrs after oral XV454, and platelet function was assessed by aggregometry, thrombelastography and the Platelet Function Analyzer (PFA100). The effect of XV454 on tumor cell-induced thrombocytopenia was determined 10 minutes after intravenous and 3 hrs after oral XV454 administration. Tumor cells (2 x 10(6)) were injected intravenously and 15 minutes after cell injection, platelet count was measured and compared to baseline (pre-injection) counts. To assess the effect on metastasis, XV454 was administered intravenous or orally 10 minutes and 3 hrs before tumor cell injection, respectively. Eighteen days later, surface lung tumor nodules were counted and the total lung tumor burden assessed. In a fourth group, in addition to the initial oral dose (before tumor cell injection), oral XV454 was given daily for the first week and three times in the second week. Administration of XV454 (5 mg/kg) completely inhibited platelet aggregation and this effect persisted for at least 24 hrs after oral delivery. Both intravenous and oral XV454 significantly inhibited tumor cell-induced thrombocytopenia (P < 0.01), the number of surface lung tumor nodules (80-85%; P < 0.001) and total tumor burden (83% for intravenous group; 50% oral [single treatment] group; 91% oral [multiple treatment] group, P < 0.001). Overall, these data provide further evidence for the effect of oral and intravenous GpIIb/IIIa antagonism on tumor cell-platelet interaction and metastasis.