Endostatin and Cancer Therapy: A Novel Potential Alternative to Anti-VEGF Monoclonal Antibodies.

Angiogenesis is a physiological process that consists of the formation of new blood vessels from preexisting ones. Angiogenesis helps in growth, development, and wound healing through the formation of granulation tissue. However, this physiological process has also been linked to tumor growth and metastasis formation. Indeed, angiogenesis has to be considered as a fundamental step to the evolution of benign tumors into malignant neoplasms. The main mediator of angiogenesis is vascular endothelial growth factor (VEGF), which is overexpressed in certain cancers. Thus, there are anti-VEGF monoclonal antibodies, such as bevacizumab, used as anti-cancer therapies. However, bevacizumab has shown adverse events, such as hypertension and proteinuria, which in the most severe cases can lead to cessation of therapy, thus contributing to worsening patients' prognosis. On the other hand, endostatin is an endogenous protein that strongly inhibits VEGF expression and angiogenesis and shows a better safety profile. Moreover, endostatin has already given promising results on small scale clinical studies. Hence, in this review, we present data supporting the use of endostatin as a replacement for anti-VEGF monoclonal antibodies.

Anti-tumor effect of endostatin in a sleep-apnea mouse model with tumor.

BACKGROUND: Obstructive sleep apnea (OSA) is associated with cancer incidence and mortality. The underlying mechanism is unclear. This study aims to evaluate the influence of intermittent hypoxia (IH), a novel hallmark of OSA, on tumor and to access the anti-tumor effect of endostatin on a mouse model with OSA. METHODS: The C57BL/6 J mice were randomly classified into four groups: control (normoxia) (CTL), control plus endostatin (CTL + ED), IH, and IH plus endostatin (IH + ED). Mice in IH and IH + ED groups were subjected to IH 8 h per day in 5 weeks. Lewis lung cancer cells were injected into the flank of each mouse after 1 week of IH exposure. Endostatin was also intraperitoneally injected after tumor volume reached about 200 mm3. The maximum standard uptake values (SUVmax) were detected by micro-positron emission tomography-computed tomography (micro-PET-CT) imaging prior and post-endostatin administration. Microvessel density (MVD) and vascular endothelial growth factor (VEGF) were determined for evaluating the anti-tumor effect of endostatin among the normoxia and IH conditions. RESULTS: Mice had higher SUVmax in the IH group than the CTL group (p < 0.01). When compared with mice in the CTL group, those in the IH group had significantly greater MVD values (p < 0.001). The SUVmax can be attenuated by endostatin both in the CTL (p < 0.01) and IH conditions (p < 0.001). When compared with CTL group, mice in the IH group had increased MVD values (p < 0.001) and VEGF expression both at mRNA (p < 0.05) and protein levels (p < 0.001 in western blotting results). Treatment with endostatin attenuated serum and tissue VEGF levels, lowering the MVD values. As compared to normoxia condition, the endostatin-therapeutic effects were more significant under the IH condition (p < 0.05 in western blotting results). CONCLUSIONS: Micro-PET-CT imaging is a promising non-invasive technique to evaluate the tumor metabolic characteristics under IH condition in vivo. The anti-tumor effect of endostatin under IH condition is superior to that of the normoxia condition.

An extracellular proteasome releases endostatin from human collagen XVIII.

Endostatin is a potent anti-angiogenic and anti-tumor protein capable of regressing tumors without inducing acquired resistance. Since it is a fragment of the parental molecule, collagen XVIII, its endogenous production depends on the activity of a specific proteolytic enzyme. While such an enzyme has been described in mice, a human counterpart has not been identified so far. Here, we searched for this enzyme by using a fluorescence resonance energy transfer peptide containing the cleavage site of human collagen XVIII. We found that the cleavage activity was present in various murine and human tumor cells but not in untransformed cells. It was ascribed to a large protein complex identified as an extracellular form of proteasome 20S. Since circulating proteasome 20S has recently emerged as an important marker of tumor progression, the possibility of proteasomes controlling the production of angiostatic endostatin may inspire the development of new anticancer therapies.

Structural studies of the protein endostatin in fusion with BAX BH3 death domain, a hybrid that presents enhanced antitumoral activity.

Endostatin (ES) is an antiangiogenic protein that exhibits antitumor activity in animal models. However, the activity observed in animals was not observed in human clinical trials. ES-BAX is a fusion protein composed of two functional domains: ES, which presents specificity and is internalized by activated endothelial cells and the proapoptotic BH3 domain of the protein BAX, a peptide inductor of cellular death when internalized. We have previously shown (Chura-Chambi et al., Cell Death Dis, 5, e1371, 2014) that ES-BAX presents improved antitumor activity in relation to wild-type ES. Secondary and tertiary structures of ES-BAX are similar to ES, as indicated by homology-modeling studies and molecular dynamics simulations. Tryptophan intrinsic fluorescence and circular dichroism spectroscopy corroborate these data. 15 N HSQC NMR indicates that ES-BAX is structured, but some ES residues have suffered chemical shift perturbations, suggesting that the BH3 peptide interacts with some parts of the ES protein. ES and ES-BAX present similar stability to thermal denaturation. The production of stable hybrid proteins can be a new approach to the development of therapeutic agents presenting specificity for tumoral endothelium and improved antitumor effect.

Endostatin exerts radiosensitizing effect in non-small cell lung cancer cells by inhibiting VEGFR2 expression.

BACKGROUND: To determine the effects of endostatin on vascular growth factor receptor 2 (VEGFR2) expression in non-small cell lung cancer (NSCLC) cells and the mechanisms underlying its radiosensitizing effect. METHODS: VEGFR2 mRNA levels were determined in different NSCLC cell lines using qRT-PCR. RT-PCR and Western blot assays were used to assess the expression of mRNA and proteins. The radiosensitivity of the cells was determined by colony-formation assays; and cell apoptosis and cell cycle distribution were determined by flow cytometry. RESULTS: VEGFR2 mRNA levels differed among the five NSCLC cell lines (P < 0.01), with the highest expression in Calu-1 cells and lowest in A549 cells. Endostatin significantly inhibited the growth of Calu-1 cells (P < 0.01) (IC20 = 296.5 µg/ml), and the expression of VEGFR2 and HIF-1α (P < 0.05). Phosphorylation of protein kinase B (Akt), extracellular signal-regulated kinases 1/2 (ERK1/2), and p38 were significantly lower in endostatin-treated cells than control (P < 0.05). Endostatin enhanced the radiosensitivity of Calu-1 cells to SER = 1.38 and induced apoptosis (P < 0.01) and G2/M blockage (P < 0.01). However, endostatin had limited effects on A549 cells. Compared with Calu-1 cells, there was not significantly effects on cell radiosensitivity (SER = 1.09). CONCLUSIONS: Endostatin induces apoptosis and enhances radiosensitivity of the VEGFR2 high-expressing cell line Calu-1, but it has a limited effect on the VEGFR2 low-expressing cell line A549.

Stimulation of PAR-1 or PAR-4 promotes similar pattern of VEGF and endostatin release and pro-angiogenic responses mediated by human platelets.

BACKGROUND: Platelets mediate angiogenesis through the secretion of several factors, including the pro-angiogenic vascular endothelial growth factor (VEGF) and the anti-angiogenic endostatin. Although previous findings indicated that these molecules are packed into different alpha-granules and selectively released by specific stimulation of protease-activated receptor (PAR)-1 or PAR-4, recent evidences are against this hypothesis. OBJECTIVES: To elucidate the controversies about the VEGF and endostatin release and the overall angiogenic effect of PARs-stimulated platelets. METHODS: VEGF and endostatin were quantified by enzyme linked-immunosorbent assay (ELISA). Endothelial proliferation (pNPP assay), wound healing (scratch assay) and tubule formation (matrigel) of human microvascular endothelial cells (HMEC-1) and endothelial progenitor cells (EPC) were determined using supernatants from PAR-1- or PAR-4-stimulated platelets. RESULTS: Activation of washed platelets (WPs) by PAR-1- or PAR-4-activating peptide (AP) promoted the VEGF and endostatin secretion in a concentration-dependent manner, being PAR-1-AP more potent than PAR-4-AP. The release of both molecules was abrogated by pre-incubation of platelets with PAR antagonists. Activation of platelet-rich plasma (PRP) with either PAR-1-AP or PAR-4-AP induced a significant VEGF secretion. Quantification of platelet-endostatin secretion was not possible in PRP due to the high levels of plasmatic endostatin vs. platelet content. Releasates from PAR-1- or PAR-4-activated WPs promoted similar pattern of angiogenic responses of HMEC-1 or EPC. Moreover, proliferation of HMEC-1 mediated by PAR-stimulated PRP releasates was delayed and significantly lower compared with that induced by PAR-stimulated WPs. CONCLUSIONS: Our results are in contrast with the previously described differential release of VEGF and endostatin induced by the selective PAR-1 or PAR-4 stimulation, and support the notion that while circulating endostatin accounts for the maintenance of a systemic anti-angiogenic state, locally, the release of platelet alpha-granule content promotes angiogenesis.

Involvement of the NF-кB/p50/Bcl-3 complex in response to antiangiogenic therapy in a mouse model of metastatic renal cell carcinoma.

Renal cell carcinoma (RCC) represents approximately 2-3% of human malignancies. Nuclear transcription factor кB (NF-кB) is composed of a family of transcription factors that have been associated with the development and progression of RCC. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we evaluated the expression of NF-кB in metastatic tumor cells from animals treated with ES. Balb/c-bearing Renca-EGFP cells were treated with NIH/3T3-LendSN or NIH/3T3-LXSN cells as a control. At the end of the in vivo experiment, plasma Renca-EGFP-sorted cells and tissue lung samples were collected. A real-time PCR array for NF-κB target genes revealed that ES therapy led to down regulation of Bcl-3 (P<0.031), NF-кB1 (P<0.001) and c-Rel (P<0.004) in the ES-treated group. Using an electrophoretic mobility shift assay (EMSA), we observed a reduction in NF-kB binding activity in ES-treated Renca-EGP cells. Furthermore, a supershift assay showed a clear shift of the NF-кB DNA band in samples incubated with a p50 antibody. By immunohistochemistry analysis, ES treatment resulted in a significant reduction in expression of p50. (ES vs. control P<0.05). The immunoprecipitation experiments confirmed the presence of a p50/Bcl-3 complex in nuclear extracts from cells of metastatic lung tissues. Our findings indicate that p50 and Bcl-3 plays a regulatory role in gene transcription in RCC.

Vascular endothelial growth factor as a biomarker for endostatin gene therapy.

Renal cell carcinoma (RCC) is characterized by high vascular endothelial growth factor (VEGF) production and, consequently, excessive angiogenesis. Several strategies have been developed to target angiogenesis as a method for treating metastatic RCC (mRCC). Endostatin (ES) is a C-terminal fragment of collagen XVIII that has antiangiogenic activity. The aim of this study was to investigate the predictive value of circulating VEGF-A in a murine model of mRCC after ES gene therapy. ES therapy did not affect the levels of collagen XVIII/ES or ES in the tissue. The circulating level of ES was increased in the control and ES-treated groups (normal vs. control, P<0.05 and ES-treated vs. control, P<0.001), and the intratumoral vessels were significantly decreased (ES-treated vs. control, P<0.05). ES therapy decreased the VEGF mRNA levels. The tissue and circulating levels of VEGF in the control group were significantly higher than normal (P<0.01 and P<0.05, respectively). Treatment with ES significantly reduced the VEGF concentrations in both compartments (P<0.001 for tissue and P<0.05 for plasma). Our findings indicate that in addition to the directly targeted tumor vessels, ES exerts its antitumor effect by down-regulating VEGF gene expression in renal tumor cells. Additionally, our findings point to the predictive value of VEGF for ES therapy.

Genotype analysis of the human endostatin variant p.D104N in benign and malignant adrenocortical tumors

OBJECTIVE: Endostatin is a potent endogenous inhibitor of angiogenesis. It is derived from the proteolytic cleavage of collagen XVIII, which is encoded by the COL18A1 gene. A polymorphic COL18A1 allele encoding the functional polymorphism p.D104N impairs the activity of endostatin, resulting in a decreased ability to inhibit angiogenesis. This polymorphism has been previously analyzed in many types of cancer and has been considered a phenotype modulator in some benign and malignant tumors. However, these data are controversial, and different results have been reported for the same tumor types, such as prostate and breast cancer. The purpose of this study was to genotype the p.D104N variant in a cohort of pediatric and adult patients with adrenocortical tumors and to determine its possible association with the biological behavior of adrenocortical tumors. METHODS: DNA samples were obtained from 38 pediatric and 56 adult patients (0.6-75 yrs) with adrenocortical tumors. The DNA samples were obtained from peripheral blood, frozen tissue or paraffin-embedded tumor blocks when blood samples or fresh frozen tissue samples were unavailable. Restriction fragment length polymorphism analysis was used to genotype the patients and 150 controls. The potential associations of the p.D104N polymorphism with clinical and histopathological features and oncologic outcome (age of onset, tumor size, malignant tumor behavior, and clinical syndrome) were analyzed. RESULTS: Both the patient group and the control group were in Hardy-Weinberg equilibrium. The frequencies of the p.D104N polymorphism in the patient group were 81.9 percent (DD), 15.9 percent (DN) and 2.2 percent (NN). In the controls, these frequencies were 80.6 percent, 17.3 percent and 2.0 percent, respectively. We did not observe any association of this variant with clinical or histopathological features or oncologic outcome in our cohort of pediatric and adult patients with adrenocortical tumors.

Collagen XVIII/endostatin expression in experimental endotoxemic acute renal failure

Acute renal failure (ARF) is a frequent complication of Gram-negative sepsis, with a high risk of mortality. Lipopolysaccharide (LPS)-induced ARF is associated with hemodynamic changes that are strongly influenced by the overproduction of nitric oxide (NO) through the cytokine-mediated up-regulation of inducible NO synthase. LPS-induced reductions in systemic vascular resistance paradoxically culminate in renal vasoconstriction. Collagen XVIII is an important component of the extracellular matrix expressed in basement membranes. Its degradation by matrix metalloproteases, cathepsins and elastases results in the formation of endostatin, claimed to have antiangiogenic activity and to be a prominent vasorelaxing agent. We evaluated the expression of endostatin/collagen XVIII in an endotoxemic ARF model. ARF was induced in C57BL/6 mice by intraperitoneal injection of LPS (10 mg/kg) followed by sacrifice 4 and 12 h later. Kidney tissue was the source of RNA and protein and the subject of histological analysis. As early as 4 h after LPS administration, blood urea, creatinine and NO levels were significantly increased compared to control. Endostatin/collagen XVIII mRNA levels were 0.71 times lower than sham-inoculated mice 4 h after LPS inoculation, returning to normal levels 12 h after LPS inoculation. Immunohistological examination revealed that acute injury caused by LPS leads to an increase of endostatin basement membrane staining in association with the decrease of CD31 endothelial basement membrane staining. These results indicate that in the early phase of endotoxemic ARF the endostatin levels were not regulated by gene expression, but by the metabolism of collagen XVIII.

COL18A1 is highly expressed during human adipocyte differentiation and the SNP c.1136C > T in its "frizzled" motif is associated with obesity in diabetes type 2 patients

Collagen XVIII can generate two fragments, NC11-728 containing a frizzled motif which possibly acts in Wnt signaling and Endostatin, which is cleaved from the NC1 and is a potent inhibitor of angiogenesis. Collagen XVIII and Wnt signaling have recently been associated with adipogenic differentiation and obesity in some animal models, but not in humans. In the present report, we have shown that COL18A1 expression increases during human adipogenic differentiation. We also tested if polymorphisms in the Frizzled (c.1136C>T; Thr379Met) and Endostatin (c.4349G>A; Asp1437Asn) regions contribute towards susceptibility to obesity in patients with type 2 diabetes (113 obese, BMI =30; 232 non-obese, BMI < 30) of European ancestry. No evidence of association was observed between the allele c.4349G>A and obesity, but we observed a significantly higher frequency of homozygotes c.1136TT in obese (19.5 percent) than in non-obese individuals (10.9 percent) [P = 0.02; OR = 2.0 (95 percentCI: 1.07-3.73)], suggesting that the allele c.1136T is associated to obesity in a recessive model. This genotype, after controlling for cholesterol, LDL cholesterol, and triglycerides, was independently associated with obesity (P = 0.048), and increases the chance of obesity in 2.8 times. Therefore, our data suggest the involvement of collagen XVIII in human adipogenesis and susceptibility to obesity.

Colágeno XVIII pode gerar dois fragmentos, um correspondendo à região NC11-728 contendo o motivo ''frizzled'', o qual possivelmente atua na sinalização Wnt, e outro correspondendo a Endostatina, que é clivada a partir da região NC1 e é uma potente inibidora de angiogênese. Colágeno XVIII e a via de sinalização Wnt foram recentemente associados à diferenciação adipogênica e obesidade em alguns modelosanimais, porém ainda não em humanos. No presente trabalho, mostramos que os níveis de expressão gênica do COL18A1 aumentam durante o processo de diferenciação adipogênica em humanos. Também testamos se polimorfismos localizados no motivo ''Frizzled'' (c.1136C > T; Thr379Met) e na região da Endostatina (c.4349G > A; Asp1437Asn) contribuem na predisposição a obesidade em pacientes com diabetes tipo 2. (113 obesos, BMI > 30; 232 não-obesos, BMI < 30) de ancestralidade Européia. Nenhuma evidência de associação entre o alelo c.4349G > A e obesidade foi observada, contudo, observamos uma freqüência significativamente maior de homozigotos c.1136TT em obesos (19.5 por cento) do que em não-obesos (10.9 por cento)[P = 0.02; OR = 2.0 (95 por centoCI: 1.07-3.73)], sugerindo que o alelo c.1136T está associado com obesidade conforme ummodelo recessivo. Este genótipo manteve-se associado à obesidade (P = 0.048) mesmo após o controle das variáveis colesterol, LDL e triglicérides, e confere um risco 2.8 vezes maior de obesidade. Portanto, nossos dados sugerem o envolvimento do colágeno XVIII em adipogênese humana e predisposição a obesidade.

Cirurgião diante das novas terapias para o tratamento do câncer / The surgeon facing new therapies for cancer treatment

The cancer treatement has been subjected to substantial changes, mainly concerning clinical specialities. The advances on tumours study, especially related to genetics and molecular biology, greatly increased our understanding about many aspects of carcinogenesis, neoplasic growth and metastasizing process, untill now obscure. In this context, surgery seems to be attained its limits in trying to erradicate completely the disease, and although the great resections made, this aim has not been succeeded in many cases. New treatments are emerging each year and between the most promising we can highlight tumour angiogenesis chemical blocking agents, with highly promising experimental results. At the same time of the beginning of clinical researchs about these drugs, the authors present a review work, with the objective of presenting a general survey of the knowledge achieved about these recently discovered drugs in tumours control.