Inhibitory Effects of Euphorbia ebracteolata Hayata Extract ECB on Melanoma-Induced Hyperplasia of Blood Vessels in Zebrafish Embryos

Melanoma is a serious malignant form of skin cancer. Euphorbiaceae compound B (ECB, 2,4-dihydroxy-6-methoxy-3-methylacetophenone) is an acetophenone compound that is isolated from Euphorbia ebracteolata Hayata (EEH), an herbaceous perennial, and has antitumor activity. Here, we transplanted human melanoma cells into zebrafish embryos to establish a zebrafish/melanoma model. We showed that this model can be used to evaluate the therapeutic effect of EEH and ECB and discussed its potential mechanism of action. The results showed that ECB was an active ingredient of EEH in inhibiting melanoma-induced hyperplasia of blood vessels in zebrafish embryos, similar to the angiogenic inhibitor vatalanib. ECB inhibited the number and length of subintestinal veins ( p < 0.05 ), as well as the distribution of melanoma in zebrafish embryos ( p < 0.05 ). More importantly, unlike vatalanib, ECB only inhibited melanoma-induced abnormal and excessive growth of blood vessels in xenografts. In addition, ECB inhibited the mRNA expression of vegfr2 and vegfr3 in zebrafish. Both vegfr2 and vegfr3 are essential genes that regulate blood vessel formation and upregulate the expression of p53 and casp3a genes in zebrafish. Together, the above-mentioned results indicate that ECB has a potential antimelanoma effect in vivo, which may be mediated by inhibiting vascular endothelial growth factor receptors.

Treatment Strategies of Gastric Cancer—Molecular Targets for Anti-angiogenic Therapy: a State-of-the-art Review

Purpose Recent studies have suggested that molecular targets for the anti-angiogenic therapy might constitute a basis for additional therapy in gastric cancer treatment. A vast number of molecules, receptors, pathways, specific interactions, and thus strategies that target gastric cancer angiogenesis specifically have been reported in numerous research articles and clinical trials. Methods We conducted a systematic literature review of molecularly targeted treatment strategies in gastric cancer on the following databases—PubMed, Google Scholar, and Scopus—on September 20, 2020. Multiple articles and evaluations were searched for studies reporting newly found and promising molecular anti-angiogenic therapy pathways. Eventually, 39 articles regarding the anti-angiogenic therapy in gastric cancer were included in the final analysis. Results As a consequence of the release of the pro-angiogenic molecules from the tumour cells, gastric cancer presents high angiogenic capability. Therefore, potential schemes for future treatment strategies include the decrease of the process ligands as well as the expression of their receptors. Moreover, the increase in the angiogenic inhibitor levels and direct aim for the inner walls of the endothelial cells appear as a promising therapeutic strategy. Beyond that, angiogenesis process inhibition seems to indirectly exaggerate the effects of chemotherapy in the considered patients. Conclusions The anti-angiogenic treatment in gastric cancer patients evaluates its significance especially in the early stages of the malignancy. The studies conducted so far show that most of the meaningful angiogenic factors and receptors with the potential molecular pathways should be further evaluated since they could potentially play a substantial role in future therapies.

Newcastle Disease Virus Expressing an Angiogenic Inhibitor Exerts an Enhanced Therapeutic Efficacy in Colon Cancer Model

Numerous studies demonstrate that the NDV-mediated gene therapy is a promising new approach for treatment of cancer. VEGF-Trap plays a vital role in anti-angiogenesis. Therefore, we hypothesize that a recombinant NDV (rNDV) expressing VEGF-Trap would be an ideal agent for the colon cancer therapy. In this study, VEGF-Trap gene was incorporated into the genome of rNDV (named rNDV-VEGF-Trap). rNDV-VEGF-Trap reduced cell growth ratio by 85.37% and migration ratio by 87.9% in EA.hy926 cells. In vivo studies, treatment with rNDV-VEGF-Trap reduced tumor volume of CT26-bearing mice by more than 3 folds and tumor weight by more than 4 folds. Immunohistochemistry analysis of CD34 showed rNDV-VEGF-Trap significantly decreased the number of vascular endothelial cells in the tumor tissues of the tumor-bearing mice. Moreover, Western blot analysis demonstrated that treatment with rNDV-VEGF-Trap significantly decreased the phosphorylation levels of AKT, ERK1/2 and STAT3 and increased the expression levels of P53, BAX and cleaved caspase-3 in the tumor tissue. In addition, to evaluate the toxicity of VEGF-Trap, serum chemistries were analyzed. The results showed that rNDV-VEGF-Trap caused insignificant changes of creatinine levels, alanine aminotransferase (ALT) and aspartate transaminase (AST). Futhermore, administration of rNDV-VEGF-Trap did not cause the diarrhoea，decreased appetite, weight decrease and haemorrhage of the experimental mice. These data suggest that rNDV-VEGF-Trap exhibits an enhanced inhibition of CT26-bearing mice by enhancing anti-angiogenesis and apoptosis. rNDV-VEGF-Trap is a potential candidate for carcinoma therapy especially for colon cancer.

Abstract 14979: Endostatin as a Predictor of Severity and Survival in Pediatric Congenital Heart Disease Associated Pulmonary Hypertension

Introduction: Pediatric pulmonary arterial hypertension (PAH) is a multifactorial pulmonary vascular disease with high mortality. Endostatin (ES), an angiogenic inhibitor, is associated with disease severity and mortality in adults with PAH and poor lung growth in children. Hypothesis: Increased ES is associated with worse disease severity and outcomes in pediatric PAH. Methods: Serum ES levels were measured in two cohorts of pediatric PAH (IPAH, FPAH and APAH) patients; the cross-sectional CCHMC PAH Biobank (PAHB, N=175) and a longitudinal cohort from Children’s Hospital of Colorado (CHC, N=61). Outcomes included medical therapy, functional and hemodynamic measures, and survival (death, transplant, palliative shunt). Adjusted logistic and linear regressions and Kaplan-Meier analysis were used to assess the relationship between ES and clinical outcomes. Results: In both cohorts, ES was significantly higher in PAH associated with congenital heart disease (APAH-CHD, PAHB n=70, p=0.002; CHC n=29, p=0.007) and was highest in those with a ventricular shunt (n=24, p=0.001). In APAH-CHD, ES was associated with a decreased 6MWD (-108m, -187- -30, p=0.01) and increased mean right atrial pressure (mRAP 1.8mmHg, 0.3-3.3, p=0.02). Longitudinally, in the APAH-CHD subgroup when adjusted for age, sex, and multiple time points, higher ES was associated with increased PVRi and mPAP (PVRi 2.5WU*m 2 , 0.7-4.3, p=0.007; mPAP 10mmHg, 4.5-15, p<0.001). Kaplan-Meier analysis (Figure 1) demonstrated that ES was associated with worse outcomes in both cohorts, with an adjusted Cox proportional hazard ratio of 4.3 (1.13-16, p=0.03) in the PAHB. Conclusions: ES was associated with worse functional measures, pulmonary vascular hemodynamics, and survival in pediatric APAH-CHD. These observations suggest that serum ES could act as a pulmonary vascular specific biomarker to identify those who are at increased risk of developing PAH and predict poor outcomes in APAH-CHD.

A Ib/II study of the combination of lenvatinib (L) and eribulin (E) in advanced liposarcoma (LPS) and leiomyosarcoma (LMS) (LEADER).

11507 Background: For advanced LPS and LMS, the two most common histologies in soft tissue sarcoma, there are limited treatment options that readily balance efficacy and toxicity. Patients (pts) treated with E had an improved median overall survival (OS) in a phase III randomized study compared to dacarbazine but with an unsatisfactory 4% objective response rate (ORR). Early studies of L, a multi-targeted anti-angiogenic inhibitor, had suggested efficacy in sarcoma pts. We hypothesized that the combination of anti-angiogenic agent and chemotherapy could potentiate treatment benefit and aimed to explore the safety and efficacy of L + E in advanced LMS and LPS. Methods: LEADER was a single-arm phase Ib/II study for advanced adult LMS and LPS pts who had received no more than 2 lines of systemic chemotherapy. The phase Ib part (starting dose: L 18mg/day, E 1.1mg/m2) had been reported and the recommended phase 2 dose (RP2D) was determined at L 14mg/day and E 1.1mg/m2 D1, D8 every 21 days. The primary endpoint of the phase II part was ORR by RECIST 1.1, secondary endpoints included ORR by Choi criteria, progression-free survival (PFS), 6-month PFS rate, and OS. With α = 0.05 and 80% power, the pts needed for stage I and total of the Simon 2-stage design was 13 and 27 pts, respectively. Results: As of Jan 22, 2020, 20 pts (F/M 13/7) had been treated with at least one cycle of L + E; 14 were LMS (5 uterine, 9 non-uterine) and 6 were LPS (4 dedifferentiated, 2 myxoid round cell). The median age was 51 (range 29-73); the median lines of treatment(s) received before enrollment was 1 (range 0-3). 18 pts were evaluable for primary endpoint: the ORR by RECIST 1.1 was 27 % (5/18) (95% CI 10-53%). The ORR by Choi criteria was 67 % (12/18) (95% CI 41-87%). With 8 PFS events, the median PFS and 6-month PFS rate was 56 weeks (95% CI 25-not reached) and 72%, respectively. There were no OS events. The ORRs by RECIST 1.1 between different L starting doses were not significantly different (18mg 33% (2/6) vs 14mg 25% (3/12), p = 0.7). 15 pts experienced at-least one grade (gr) 3 or 4 adverse event (AE); gr 3 or 4 AEs occurred in > 1 pts included (% of phase Ib, % of phase II pts) hypertension (n = 4) (67%, 0%); hand-foot-syndrome (n = 4) (50%, 7%), proteinuria (n = 3) (0%, 25%), febrile neutropenia (n = 2) (17% vs 5%), neutropenia (n = 6) (50% vs 25%). The RP2D was associated with overall lower gr3/4 AEs except for proteinuria. Conclusions: L + E had shown promising efficacy in advanced LMS and LPS. L at 14mg/day had a better AE profile without compromising activity. The exploratory biomarker study of LEADER is ongoing. Clinical trial information: NCT03526679 .

Mathematical Model Predicts Effective Strategies to Inhibit VEGF-eNOS Signaling

The endothelial nitric oxide synthase (eNOS) signaling pathway in endothelial cells has multiple physiological significances. It produces nitric oxide (NO), an important vasodilator, and enables a long-term proliferative response, contributing to angiogenesis. This signaling pathway is mediated by vascular endothelial growth factor (VEGF), a pro-angiogenic species that is often targeted to inhibit tumor angiogenesis. However, inhibiting VEGF-mediated eNOS signaling can lead to complications such as hypertension. Therefore, it is important to understand the dynamics of eNOS signaling in the context of angiogenesis inhibitors. Thrombospondin-1 (TSP1) is an important angiogenic inhibitor that, through interaction with its receptor CD47, has been shown to redundantly inhibit eNOS signaling. However, the exact mechanisms of TSP1′s inhibitory effects on this pathway remain unclear. To address this knowledge gap, we established a molecular-detailed mechanistic model to describe VEGF-mediated eNOS signaling, and we used the model to identify the potential intracellular targets of TSP1. In addition, we applied the predictive model to investigate the effects of several approaches to selectively target eNOS signaling in cells experiencing high VEGF levels present in the tumor microenvironment. This work generates insights for pharmacologic targets and therapeutic strategies to inhibit tumor angiogenesis signaling while avoiding potential side effects in normal vasoregulation.

3D Bioprinted Vascularized Tumour for Drug Testing

An in vitro screening system for anti-cancer drugs cannot exactly reflect the efficacy of drugs in vivo, without mimicking the tumour microenvironment (TME), which comprises cancer cells interacting with blood vessels and fibroblasts. Additionally, the tumour size should be controlled to obtain reliable and quantitative drug responses. Herein, we report a bioprinting method for recapitulating the TME with a controllable spheroid size. The TME was constructed by printing a blood vessel layer consisting of fibroblasts and endothelial cells in gelatine, alginate, and fibrinogen, followed by seeding multicellular tumour spheroids (MCTSs) of glioblastoma cells (U87 MG) onto the blood vessel layer. Under MCTSs, sprouts of blood vessels were generated and surrounding MCTSs thereby increasing the spheroid size. The combined treatment involving the anti-cancer drug temozolomide (TMZ) and the angiogenic inhibitor sunitinib was more effective than TMZ alone for MCTSs surrounded by blood vessels, which indicates the feasibility of the TME for in vitro testing of drug efficacy. These results suggest that the bioprinted vascularized tumour is highly useful for understanding tumour biology, as well as for in vitro drug testing.

Effect of 8 weeks of Aerobic Training on Genes Expression of Hypoxia Inducible Factor HIF-1α, Vascular Endothelial Growth Factor (VEGF) and Angiostatin in Hippocampus of Male Rats with Wistar Model

Introduction: Many studies have been done about the effects of exercise on angiogenic inhibitor and stimulator factors in muscles, but few studies have examined the role of these factors in the brain especially the hippocampus. Therefore, the purpose of the current study was to investigate the effect of 8 weeks of aerobic training on gene expression of HIF-1α, VEGF and angiostatin in hippocampus of male rats. Methods: 18 adult male Wistar rats (190±10 gr) were randomly divided into 3 groups: control, sham and aerobic training. Ratsin the training group performed 8 weeks of aerobic training (5 sessions per week) on a treadmill. 24 hours after the last session of exercise, rats were decapitated and the hippocampus were carefully removed and rapidly frozen in liquid nitrogen, then stored at -80°C for further analysis. Real-Time-PCR method was used to measure the expression of genes in the hippocampus. The data were analyzed by SPSS 18 software. Comparisons between groups were performed by one-way ANOVA and followed by post-hoc analysis Tukey test. All statistically significant was set at P<0.05. Results: The results showed aerobic training significantly increased mRNA levels of HIF-1α (P=0.001) and VEGF (P=0.001), but there was no significant difference in the mRNA levels of angiostatin (P=0.316). Conclusion: According to the results of this study and changes in the levels of HIF-1α and VEGF, it seems aerobic training has helpful effects on brain especially on the hippocampus and this type of training is recommended for individuals.

The contrary intracellular and extracellular functions of PEDF in HCC development

Pigment epithelium-derived factor (PEDF), a classic angiogenic inhibitor, has been reported to function as a tumor suppression protein and to downregulate in many types of solid tumors. However, the expression level of PEDF and its role in hepatocellular carcinoma (HCC) are contradictory. The present study investigates the expression and different activities of secreted and intracellular PEDF during HCC development, as well as the underlying mechanism of PEDF on HCC lipid disorders. We found that PEDF had no association with patients’ prognosis, although PEDF was highly expressed and inhibited angiogenesis in HCC tumor tissues. The animal experiments indicated that full-length PEDF exhibited equalizing effects on tumor growth activation and tumor angiogenesis inhibition in the late stage of HCC progression. Importantly, the pro-tumor activity was mediated by the intracellular PEDF, which causes accumulation of free fatty acids (FFAs) in vivo and in vitro. Based on the correlation analysis of PEDF and lipid metabolic indexes in human HCC tissues, we demonstrated that the intracellular PEDF led to the accumulation of FFA and eventually promoted HCC cell growth by inhibiting the activation of AMPK via ubiquitin–proteasome-mediated degradation, which causes increased de novo fatty acid synthesis and decreased FFA oxidation. Our findings revealed why elevated PEDF did not improve the patients’ prognosis as the offsetting intracellular and extracellular activities. This study will lead to a comprehensive understanding of the diverse role of PEDF in HCC and provide a new selective strategy by supplement of extracellular PEDF and downregulation of intracellular PEDF for the prevention and treatment of liver cancer.