An arresten-derived anti-angiogenic peptide triggers apoptotic cell death in endothelial cells.

BACKGROUND: In recent years, anti-angiogenic peptides have received considerable attention as candidates for cancer treatment. Arresten is an angiogenesis inhibitor that cleaves from the α1 chain of type IV collagen and stimulates apoptosis in endothelial cells. We have recently indicated that a peptide corresponding to the amino acid 78 to 86 of arresten, so-called Ars, prevented the migration and tube formation of HUVECs and the colon carcinoma growth in mice significantly. The current study aimed to determine whether induction of apoptotic cell death in endothelial cells is one of the biochemical mechanisms of this anti-angiogenic peptide. METHODS AND RESULTS: This hypothesis was assessed using the MTT assay, cell cycle analysis, Annexin V-FITC/PI staining, BCL2, CASP8, CASP9, p53, and CDKN2A gene expression studies as well as evaluating apoptosis in tumor tissues by TUNEL assay. Results demonstrated that 40 µM of Ars significantly stimulated 46.2% of early and late apoptosis in HUVECs compared to 13.6% in the untreated cells and did not significantly alter the cell cycle distribution. Moreover, BCL2 and CASP8 were down-regulated, while CASP9 and p53 were up-regulated in endothelial cells. CDKN2A gene expression, the regulator of G1 cell cycle arrest, was not significantly altered. CONCLUSIONS: It might be suggested that Ars induced apoptosis in endothelial cells through the mitochondrial pathway and had no effect on the cell cycle. Besides, Ars induced apoptosis significantly in vivo. However, further studies are required to confirm the detailed molecular mechanism of Ars, this peptide has the potential to be optimized for clinical translations.

Combination Therapy in Cancer: Doxorubicin in Combination with an N-terminal Peptide of Endostatin Suppresses Angiogenesis and Stimulates Apoptosis in the Breast Cancer.

The combination of chemotherapy drugs with angiogenesis inhibitors improves response and survival and reduces the cytotoxic side effects and drug resistance in patients compared to chemotherapy alone. Here, we investigated the efficacy of the concomitant administration of doxorubicin and a peptide derived from the N-terminal domain of Endostatin (called ES-SS) in the 4T1 mammary carcinoma tumor model. Tumor-bearing mice were divided into the control and three treatment groups, including ES-SS, doxorubicin, and the combination. Injections were performed daily for two weeks and tumor volumes were measured during the treatment. Immunohistochemical analysis of Ki-67, CD31, CD34, Bcl-2, p53 expression, and TUNEL assay were performed on tumor tissues at the end of treatment. Besides, molecular dynamics and docking simulations were performed. It was demonstrated that tumor growth was inhibited in mice treated with peptide plus doxorubicin more significantly than in each treatment alone (P<0.05). No weight loss or adverse effects were observed. Moreover, combination therapy was more effective in tumor angiogenesis suppression and apoptosis stimulation (P<0.05). Docking simulations by ClusPro server demonstrated that ES-SS binds to integrin α5ß1, Transglu-taminase 2, and Matrix metalloproteinase 2 with more negative binding energy and hydrogen bonds compared to the native peptide. Generally, we proposed that ES-SS can augment the therapeutic efficacy of doxorubicin through angiogenesis prevention and apoptosis induction in breast tumor. Owing to the advantages of peptides to recombinant proteins or monoclonal antibodies, further preclinical and clinical evaluations of this combination strategy are worth taking into consideration.

Mutational and bioinformatics analysis of the NKX2.1 gene in a cohort of Iranian pediatric patients with congenital hypothyroidism (CH).

Congenital hypothyroidism (CH) occurs with a relatively alarming prevalence in infants, and if not diagnosed and treated in time, it can have devastating consequences for the development of the nervous system. CH is associated with genetic changes in several genes that encode transcription factors responsible for thyroid development, including mutations in the NK2 homeobox 1 (NKX2.1) gene, which encodes the thyroid transcription factor-1 (TTF-1). Although CH is frequently observed in pediatric populations, there is still a limited understanding of the genetic factors and molecular mechanisms contributing to this disease. The sequence of the NKX2.1 gene was investigated in 75 pediatric patients with CH by polymerase chain reaction (PCR), single-stranded conformation polymorphism (SSCP), and direct DNA sequencing. Four missense heterozygous variations were identified in exon 3 of the NKX2.1 gene, including three novel missense variations, namely c.708A>G, p.Gln202Arg; c.713T>G, p.Tyr204Asp; c.833T>G, p.Tyr244Asp, and a previously reported variant rs781133468 (c.772C>G, p.His223Gln). Importantly, these variations occur in highly conserved residues of the TTF-1 DNA-binding domain and were predicted by bioinformatics analysis to alter the protein structure, with a probable alteration in the protein function. These results indicate that nucleotide changes in the NKX2.1 gene may contribute to CH pathogenesis.

Mutational Analysis of the VPREB1 Gene of Pre-BCR Complex in a Cohort of Sporadic Pediatric Patients With B-Cell Acute Lymphoblastic Leukemia.

During bone marrow B-cell development, the pre-B-cell receptor is formed by the association of the immunoglobulin heavy chain with a surrogate light chain, which is encoded by the VPREB1, and λ5 genes. It is known that pre-BCR signaling signifies a critical checkpoint at the pre-B-cell stage. Thus, failure pre-BCR signaling is proposed as a critical factor for the development of B-cell acute lymphoblastic leukemia (B-ALL). B­ALL is the most common pediatric cancer and is one of the leading causes of death in children. Until now, several molecular analyses were performed for genomic alterations in B-ALL, but for genomic analysis of the VPREB1 gene and its rare variations, limited studies have been conducted. In this study, using polymerase chain reaction and direct sequencing of 88 pediatric patients with B-ALL, we investigated the genomic region of the VPREB1 gene to find sequence variations of this gene. Our study presented ten homozygous and heterozygous point mutations and heterozygous nucleotide deletions, in the VPREB1 gene in 36 boys and 32 girls' patients. Our Bioinformatics assay results presented that these variations may alter the RNA folding, protein structure, and therefore probable effect on the protein function. These results propose that nucleotide changes probably contribute to B-ALL pathogenesis.

New insights into short peptides derived from the collagen NC1 α1, α2, and α3 (IV) domains: An experimental and MD simulations study.

Proteolytically cleavage of the collagen NC1 α1 to α3 (IV) domains leads to antiangiogenic proteins called Arresten, Canstatin, and Tumstatin, respectively. The research identified that the two overlapping peptides derived from Tumstatin are more effective than other fragments and amino acids L78, V82, and D84 are essential for their activity. In the present study, the efficacy of a nine amino acid peptide derived from Tumstatin (Tum), containing amino acids L78, V82, and D84 was compared to the corresponding sequence in Arresten (Ars) and Canstatin (Can) in vitro and in vivo. Moreover, CD spectroscopy, MD, and docking simulations were performed to evaluate the structure and the interaction of peptides to integrin αvß3, respectively. Results demonstrated that peptides inhibit viability, migration, and tube formation in vitro, as well as the growth of tumor in vivo and Canstatin-derived peptide was more potent than others. CD measurement and DSSP calculation revealed that Can had more coil conformation. According to MD simulations, Can had more fluctuation, less intramolecular interactions, and less structural compactness compared to Tum and Ars. It can be assumed that amino acid variations lead to a more flexible and loose structure compared to the other peptides. The Canstatin-derived peptide interacts with the integrin αvß3 extremely close to RGD binding site by the most negative binding energy and more interactions. In conclusion, we for the first time identified an active peptide derived from Canstatin and showed that the sequence affected structure and thereby interaction of peptide to its receptor.

Application of E75 peptide vaccine in breast cancer patients: A systematic review and meta-analysis.

The E75 peptide vaccine, derived from tumor-associated antigen HER2, is the most frequently studied anti-HER2 vaccination strategy for the treatment of breast cancer patients. It has been investigated in the several phases Ι/Π of the clinical trials and is currently being evaluated in a randomized multicenter phase III clinical trial. We conducted a systematic review and meta-analysis to clarify the outcomes of the E75 peptide vaccine including the therapeutic efficacy, the disease recurrence, the survival rate, and the side effects. Three peer-reviewed literature databases including the PubMed, Web of Science, and Scopus were sought. Of 29 trials assessed for eligibility, 16 were considered based on our inclusion criteria. Statistical analyses were performed by The Excel and STATA v.11.0. Meta-analysis of delayed-type hypersensitivity)DTH( reactions and CD8+-T cell levels, as immune responses, displayed the significant differences in the vaccinated groups compared to their non-vaccinated counterparts. In addition, the recurrence, and the overall and the disease-free survival were significantly different in the vaccinated subjects versus the control. Evaluation of the local and systemic toxicity of the E75 peptide vaccine demonstrated the minimal side effects. It seems that the E75 peptide vaccine is safe and effective, and can be used for further randomized clinical trials.

The antiangiogenic and antitumor activities of the N-terminal fragment of endostatin augmented by Ile/Arg substitution: The overall structure implicated the biological activity.

The antiangiogenic and antitumor activities of the 27-amino acid fragment corresponding to the N-terminal domain of endostatin were shown to be dependent on a Zn-binding loop in the N-terminus. To investigate whether the regions outside of the N-terminal loop play a role in the peptide function, the structure and function of a variant containing Ile26Arg mutation (ES-R) were compared with those of the native peptide (ES-Zn). Structural analysis using far-UV CD, intrinsic fluorescence and molecular dynamics simulation provided information regarding the overall changes upon the mutation. In addition, the docking simulations predicted a higher affinity of ES-R to integrins αvß3 and α5ß1 than ES-Zn and a profound reorganization of the binding residues throughout the sequence. In Human Umbilical Vein Endothelial Cells (HUVECs), ES-R inhibited the tube formation and activated caspase-3 more strongly than do ES-Zn. Based on in vivo studies, the growth of breast tumor and expression of CD31, Bcl-2 and nonfunctional p53 were inhibited more effectively by ES-R than by ES-Zn. We conclude that the C-terminal region is involved in the peptide function through some global structural effects.

Engineering of a disulfide loop instead of a Zn binding loop restores the anti-proliferative, anti-angiogenic and anti-tumor activities of the N-terminal fragment of endostatin: Mechanistic and therapeutic insights.

Although considerable effort has been devoted to understanding the molecular mechanism of endostatin's anti-cancer activity, the role of its Zn bound N-terminal loop has not been completely clarified. To investigate whether Zn binding or the N-terminal loop is involved in the anti-cancer properties of endostatin, we compared the structure and biological activity of a native Zn binding endostatin peptide (ES-Zn) with three variants: a Zn free variant (ES), a variant containing both a Zn binding site and a disulfide bond (ES-SSZn), and a variant including a disulfide loop but incapable of Zn binding (ES-SS). Spectroscopic studies indicated that ES-Zn and ES-SS consist of random coil and ß structures, whereas ES-SSZn and ES fold into random coils. Theoretical analysis proposed that ES-Zn and ES-SS have a similar binding site to αVß3 integrin. The anti-proliferative activity of endostatin was retained by all peptides except ES, and the in vitro anti-angiogenic property was preserved in ES-Zn and ES-SS. Remarkably, breast tumor growth and CD31 activity were inhibited more effectively by ES-SS than by ES-Zn. Therefore, a correlation exists between the N-terminal loop and anti-cancer properties of endostatin fragment and a disulfide loop may be more promising than a Zn binding loop for inhibiting tumor growth.