VEGFR2 Mimicking Peptide Inhibits the Proliferation of Human Umbilical Vein Endothelial Cells (Huvecs) by Blocking VEGF.

INTRODUCTION: A variety of key human physiological processes rely on angiogenesis, ranging from reproduction and fetal growth to wound healing and tissue repair. Furthermore, this process significantly contributes to tumor progression, invasion, and metastasis. As the strongest inducer of angiogenesis, Vascular Endothelial Growth Factor (VEGF) and its receptor (VEGFR) are targets of therapeutic research for blocking pathological angiogenesis. OBJECTIVE: Preventing the interaction between VEGF and VEGFR2 by a peptide is a promising strategy for developing antiangiogenic drug candidates. This study was aimed at designing and evaluating VEGF-targeting peptides using in silico and in vitro techniques. METHODS: The VEGF binding site of VEGFR2 was considered a basis for peptide design. The interaction of VEGF and all three peptides derived from VEGFR2 were analyzed using ClusPro tools. In a complex with VEGF, the peptide with a higher docking score was evaluated to confirm its stability using molecular dynamics (MD) simulation. The gene coding for the selected peptide was cloned and expressed in E. coli BL21. The bacterial cells were cultured on a large scale, and the expressed recombinant peptide was purified using Ni-NTA chromatography. Refolding of the denatured peptide was carried out by the stepwise removal of the denaturant. The reactivity of peptides was confirmed using western blotting and enzyme-linked immunosorbent assay (ELISA) assays. Finally, the inhibition potency of the peptide on human umbilical vein endothelial cells was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- 2H-tetrazolium bromide (MTT) assay. RESULTS: Among three peptides, the peptide with the best docking pose and the highest affinity for VEGF was selected for further studies. Then the stability of the peptide was confirmed over the 100 ns MD simulation. After in silico analyses, the selected peptide was presented for in vitro analysis. Expression of the selected peptide in E. coli BL21 resulted in a pure peptide with a yield of approximately 200 µg/ml. Analysis by ELISA revealed the high reactivity of the peptide with VEGF. Western blot analysis confirmed the specific reactivity of selected peptides with VEGF. The MTT assay revealed the growth inhibitory effect of the peptide on human umbilical vein endothelial cells with an IC50 value of 247.8 µM. CONCLUSION: In summary, the selected peptide demonstrated a promising inhibitory effect on human umbilical vein endothelial cells that could be a valuable anti-angiogenic candidate for further assessment. Additionally, these in silico and in vitro data provide new insights into peptide design and engineering.

Recent advances for cancer detection and treatment by microfluidic technology, review and update.

Numerous cancer-associated deaths are owing to a lack of effective diagnostic and therapeutic approaches. Microfluidic systems for analyzing a low volume of samples offer a precise, quick, and user-friendly technique for cancer diagnosis and treatment. Microfluidic devices can detect many cancer-diagnostic factors from biological fluids and also generate appropriate nanoparticles for drug delivery. Thus, microfluidics may be valuable in the cancer field due to its high sensitivity, high throughput, and low cost. In the present article, we aim to review recent achievements in the application of microfluidic systems for the diagnosis and treatment of various cancers. Although microfluidic platforms are not yet used in the clinic, they are expected to become the main technology for cancer diagnosis and treatment. Microfluidic systems are proving to be more sensitive and accurate for the detection of cancer biomarkers and therapeutic strategies than common assays. Microfluidic lab-on-a-chip platforms have shown remarkable potential in the designing of novel procedures for cancer detection, therapy, and disease follow-up as well as the development of new drug delivery systems for cancer treatment.

Prevalence of K-RAS mutations and CA125 tumor marker in patients with ovarian carcinoma.

BACKGROUND: Ovarian carcinoma is one of the leading causes of cancer-related death among females. K-ras codon 12 mutations are commonly occurring mutations in different types of cancers and leads to resistance against anti-EGFR therapeutics. Hence, determination of mutations in k-ras gene is crucial for predicting response to anti-EGFR therapies. This study aimed to evaluate the prevalence of k-ras gene mutations and CA125 tumor marker in patients with ovarian carcinoma in Tabriz city. METHODS: Genomic DNA was extracted from 67 tissues pertained to women with ovarian carcinoma. Mutations in codon 12 and 13 of k-ras gene were analyzed by Nested PCR and RFLP methods. Titer of CA125 tumor marker was determined by ELISA. RESULTS: Among the 67 patients, 7 patients (10.4%) had mutation in k-ras codon 12 while none of them had mutation in k-ras codon 13. Based on the results, the frequency of various genotypes were 89.55%, 10.44%, and 0%, for GG, GA, and AA alleles, respectively. The p-value for stage I and Grade I tumors were 0.022 and 0.04, respectively, indicating a statistically significant correlation between codon 12 mutation and stage I and Grade I tumors. Furthermore, we found significant correlations among CA125 tumor marker titers and histological grade (p<0.000) and stage (p<0.000). The mean serum CA125 tumor marker levels in malignant carcinomas were 499.84 U/ml. CONCLUSION: The results in this study indicated high prevalence of k-ras codon 12 mutations and high titer of CA125 tumor marker in patients with ovarian carcinoma in the study region.

Cytoplasmic Chaperones Enhance Soluble Expression of Anti-EGFR huscFv in Escherichia coli.

BACKGROUND: Overexpression of EGFR is associated with carcinogenesis in more than 70% of head and neck cancers. Anti-EGFR monoclonal antibodies bind to the extracellular domain of EGFR and block the EGFR downstream signaling pathway, which results in the suppression of the growth of the tumor cells. Escherichia coli is the preferred system for expressing various recombinant proteins, including single chain antibodies, but the formation of inclusion bodies negatively affects the efficacy of this system. Several strategies have been suggested to solve this problem, notably the utilization of molecular chaperones. OBJECTIVES: In this study, we attempted to increase the soluble expression of huscfv antibody via co-expression with the cytoplasmic chaperones. MATERIALS AND METHODS: To achieve this purpose, chaperones plasmids pG-KJE8, pGro7, pKjE7, pTf16 and pG-Tf2 encoding cytoplasmic chaperones were co-expressed with the humanized anti-EGFR scFv construct in E. coli. Different temperatures, incubations times, and concentrations of IPTG were used to produce an active antibody with the highest solubility. Results were analyzed by SDS-PAGE. Soluble huscFv was purified by Ni-NTA column and the biologic activity of the recombinant protein was determined by ELISA. RESULT: The results indicated that the highest concentrations of humanized anti-EGFR scFv were obtained by co-expression of huscFv via chaperone plasmid pG-KJE8 with 0.2 mM concentration of inducer (IPTG), culture temperature of 25 °C, and 4 h incubation time after induction. CONCLUSION: In conclusion, co-expression with chaperones could be used as an efficient strategy to produce soluble active ScFvs in E. coli.

Granulocyte colony-stimulating factor in repeated IVF failure, a randomized trial.

Recent studies have revealed key roles for granulocyte colony-stimulating factor (GCSF) in embryo implantation process and maintenance of pregnancy, and some studies showed promising results by using local intrauterine infusion of GCSF in patients undergoing in vitro fertilization (IVF). This multicenter, randomized, controlled trial included 112 infertile women with repeated IVF failure to evaluate the efficacy of systemic single-dose subcutaneous GCSF administration on IVF success in these women. In this study, the Long Protocol of ovarian stimulation was used for all participants. Sealed, numbered envelopes assigned 56 patients to receive subcutaneous 300 µg GCSF before implantation and 56 in the control group. The implantation (number of gestational sacs on the total number of transferred embryos), chemical pregnancy (positive serum ß-HCG), and clinical pregnancy (gestational sac and fetal heart) rates were compared between the two groups. This trial is registered at www.irct.ir (IRCT201503119568N11). The successful implantation (18% vs 7.2%, P=0.007), chemical pregnancy (44.6% vs 19.6%, P=0.005), and clinical pregnancy (37.5% vs 14.3%, P=0.005) rates were significantly higher in the intervention group than in the control group. After adjustment for participants' age, endometrial thickness, good-quality oocyte counts, number of transferred embryos, and anti-Mullerian hormone levels, GCSF treatment remained significantly associated with successful implantation (OR=2.63, 95% CI=1.09-6.96), having chemical pregnancy (OR= 2.74, 95% CI=1.11-7.38) and clinical pregnancy (OR=2.94, 95% CI=1.23-8.33). In conclusion, administration of single-dose systemic subcutaneous GCSF before implantation significantly increases the IVF success, implantation, and pregnancy rates in infertile women with repeated IVF failure.