Designing a novel fusion protein from Streptococcus agalactiae with apoptosis induction effects on cervical cancer cells.

Cervical cancer remains life-threatening cancer in women around the world. Due to the limitations of conventional treatment approaches, there is an urgent need to develop novel and more efficient strategies against cervical cancer. Therefore, the researchers attend to the alternative anti-cancer compounds like bacterial products. Rib and α are known as surface proteins of Streptococcus agalactiae with immunologic effects. In the present study, we designed a new anti-cancer fusion protein (Rib-α) originating from S. agalactiae with in silico methods, and then, the recombinant gene was cloned in the pET-22 (+) expression vector. The recombinant protein was expressed in E. coli BL21. To purify the expressed protein, we applied the Ni-NTA column. The molecular mechanism by which Rib-α is cytotoxic to cancer cells has been discussed based on MTT, flow cytometry, and real-time PCR methods. The engineered fusion protein suppressed the proliferation of the cancer cells at 180 µg/ml. Cytotoxic assessment and morphological changes, augmentation of apoptotic-related genes, upregulation of caspase-3 mRNA, and flow cytometric analysis confirmed that apoptosis might be the principal mechanism of cell death. According to our findings, Rib-α fusion protein motivated the intrinsic apoptosis pathway. Therefore, it can be an exciting candidate to discover a new class of antineoplastic agents.

Anti-angiogenic peptides application in cancer therapy; a review.

Cancer is a disease advanced via surplus angiogenesis. The development of new anti-angiogenic therapeutic agents with more efficacy and fewer side effects is still quite necessary. Conventional therapies saving the life of many cancer patients but due to drug resistance and lack of specificity utilizing these methods is faced with limits. Recently, new therapeutic agents have been developed and used to treat cancers such as scaffold proteins, monoclonal antibodies, tyrosine kinase inhibitors, and peptides. In antiangiogenic drug development, anti-angiogenic peptides design is a significant aim. Peptides have developed as substantial therapeutics that are being carefully investigated in angiogenesis-dependent diseases because of their high penetrating rate into the cancer cells, high specificity, and low toxicity. In this review, we focus on anti-angiogenic peptides in the field of cancer therapy that are designed, screened, or derived from nanobodies, mimotopes, phage displays, and natural resources.

Designing and Development of a Tandem Bivalent Nanobody against VEGF165.

BACKGROUND: Inhibition of angiogenesis using monoclonal antibodies is an effective strategy in cancer therapy. However, they could not penetrate sufficiently into solid tumors. Antibody fragments have solved this issue. However, they suffer from short in vivo half-life. In the current study, a tandem bivalent strategy was used to enhance the pharmacokinetic parameters of an anti-VEGF165 nanobody. METHODS: Homology modeling and MD simulation were used to check the stability of protein. The cDNA was cloned into pHEN6C vector and the expression was investigated in WK6 Escherichia coli (E. coli) cells by SDS-PAGE and western blot. After purification, the size distribution of tandem bivalent nanobody was investigated by dynamic light scattering. Moreover, in vitro antiproliferative activity and pharmacokinetic study were studied in HUVECs and Balb/c mice, respectively. RESULTS: RMSD analysis revealed the tandem bivalent nanobody had good structural stability after 50 ns of simulation. A hinge region of llama IgG2 was used to fuse the domains. The expression was induced by 1 mM IPTG at 25°C for overnight. A 30 kDa band in 12% polyacrylamide gel and nitrocellulose paper has confirmed the expression. The protein was successfully purified using metal affinity chromatography. MTT assay revealed there is no significant difference between the antiproliferative activity of tandem bivalent nanobody and the native protein. The hydrodynamic radius and terminal half-life of tandem bivalent nanobody increased approximately 2-fold by multivalency compared to the native protein. CONCLUSION: Our data revealed that the physicochemical as well as in vivo pharmacokinetic parameters of tandem bivalent nanobody was significantly improved.

Challenges and advancements in the pharmacokinetic enhancement of therapeutic proteins.

Nowadays, proteins are frequently administered as therapeutic agents in human diseases. However, the main challenge regarding the clinical application of therapeutic proteins is short circulating plasma half-life that leads to more frequent injections for maintaining therapeutic plasma levels, increased therapy costs, immunogenic reactions, and low patient compliance. So, the development of novel strategies to enhance the pharmacokinetic profile of therapeutic proteins has attracted great attention in pharmaceuticals. So far, several techniques, each with their pros and cons, have been developed including chemical bonding to polymers, hyper glycosylation, Fc fusion, human serum albumin fusion, and recombinant PEG mimetics. These techniques mainly classify into three strategies; (i) the endosomal recycling of neonatal Fc receptor which is observed for immunoglobulins and albumin, (ii) decrease in receptor-mediated clearance, and (iii) increase in hydrodynamic radius through chemical and genetic modifications. Recently, novel PEG mimetic peptides like proline/alanine/serine repeat sequences are designed to overcome pitfalls associated with the previous technologies. Biodegradability, lack of or low immunogenicity, product homogeneity, and a simple production process, currently make these polypeptides as the preferred technology for plasma half-life extension of therapeutic proteins. In this review, challenges and pitfalls in the pharmacokinetic enhancement of therapeutic proteins using PEG-mimetic peptides will be discussed in detail.

New Proline, Alanine, Serine Repeat Sequence for Pharmacokinetic Enhancement of Anti-VEGF Single-Domain Antibody.

Therapeutic fragmented antibodies show a poor pharmacokinetic profile that leads to frequent high-dose administration. In the current study, for the first time, a novel proline, alanine, serine (PAS) repeat sequence called PAS#208 was designed to extend the plasma half-life of a nanosized anti-vascular endothelial growth factor-A single-domain antibody. Polyacrylamide gel electrophoresis, circular dichroism, dynamic light scattering, and electrophoretic light scattering were used to assess the physicochemical properties of the newly designed PAS sequence. The effect of PAS#208 on the biologic activity of a single-domain antibody was studied using an in vitro proliferation assay. The pharmacokinetic parameters, including terminal half-life, the volume of distribution, elimination rate constant, and clearance, were determined in mice model and compared with the native protein and PAS#1(200) sequence. The novel PAS repeat sequence showed comparable physicochemical, biologic, and pharmacokinetic features to the previously reported PAS#1(200) sequence. The PAS#208 increased the hydrodynamic radius and decreased significantly the electrophoretic mobility of the native protein without any change in zeta potential. Surprisingly, the fusion of PAS#208 to the single-domain antibody increased the binding potency. In addition, it did not alter the biologic activity and did not show any cytotoxicity on the normal cells. The PAS#208 sequence improved the terminal half-life (14-fold) as well as other pharmacokinetic parameters significantly. The simplicity as well as superior effects on half-life extension make PAS#208 sequence a novel sequence for in vivo pharmacokinetic enhancement of therapeutic fragmented antibodies. SIGNIFICANCE STATEMENT: In the current study, a new proline, alanine, serine (PAS) sequence was developed that showed comparable physicochemical, biological, and pharmacokinetic features to the previously reported PAS#1(200) sequence. The simplicity as well as superior effects on half-life extension make PAS#208 sequence a novel sequence for in vivo pharmacokinetic enhancement of recombinant small proteins.

The effect of mild agonist stimulation on the platelet reactivity in patients with type 2 diabetes mellitus.

BACKGROUND: Patients with type 2 diabetes mellitus (T2DM) have accelerated atherosclerosis as a pro thrombotic state that is associated with the platelet activation priming. Platelets, which undergo the continuous mild stimulation, may lose their sensitivity to react to a strong stimulation. The present study aimed to investigate activation responses of platelets to mild and subsequent strong stimulations in patients with T2DM and healthy individuals. METHODS: Blood samples, which were taken from 40 patients with T2DM and 35 healthy individuals, were collected into the citrate containing tubes. The samples were subjected to the soft centrifugation to prepare the platelet rich plasma (PRP). Platelets in PRP samples were treated at a low (1 µM) concentration and then at a high (10 µM) concentration of ADP. Before and after stimulation with different doses of ADP, levels of CD62P expression and formation of platelet micro particles (PMPs) were measured using a flow cytometry method. RESULTS: The platelets from patients with T2DM had higher levels of CD62P expression before any stimulation (P = 0.003) than control samples. Platelets, which underwent the mild stimulation, indicated lower responses to CD62P expression, but higher PMPs formation after stimulation with high dose of ADP. Patients with T2DM had higher platelet micro particles in all states with the ADP stimulation. (P = 0.004, SD: ±74.52). CONCLUSIONS: The flow cytometry data indicated that platelets were pre-active and associated with metabolic conditions in patients with type 2 diabetes mellitus. The induction of desensitization state helped platelets to reduce the platelet activation and sensitivity to ADP in a diabetic environment. Furthermore, the production of platelets micro-particles was high in the patients; and desensitized platelets were more susceptible to shedding of micro-particles.

Differential Expression of HSP90ß in MDA-MB-231 and MCF-7 Cell Lines after Treatment with Doxorubicin.

BACKGROUND: Breast cancer is a complex, heterogeneous disease and one of the most common malignancies in women worldwide. The efficacy of chemotherapy as an important breast cancer treatment option has been severely limited because of the inherent or acquired resistance of cancer cells. The molecular chaperone heat shock protein 90 (HSP90) upregulated in response to cellular stress is required for functions such as conformational maturation, activation and stability in more than 200 client proteins, mostly of the signaling type. In this study, the expression of HSP90 isoforms including HSP90α and HSP90ß in breast cancer cell lines before and after treatment with doxorubicin (DOX) was assessed. MATERIAL AND METHODS: The cell cytotoxicity of DOX in MDA-MB-231 and MCF-7 cell lines was determined using the MTT assay. Immunofluorescence and western blotting techniques were used to determine the expression of HSP90ß in the cell lines before and after DOX treatment. Immunofluorescence was also conducted to ascertain the expression of HSP90α. RESULTS: The MTT assay results showed that the MDA-MB- 231 cells (IC50=14.521 µM) were more sensitive than the MCF-7 cells (IC50=16.3315 µM) to DOX. The immunofluorescence results indicated that the expression of HSP90α in both cell lines decreased after exposure to DOX. The western blot and immunofluorescence analyses showed that HSP90ß expression decreased in the MCF-7 cells but increased in the MDA-MB- 231 cells after DOX treatment. Conclusion: The obtained results suggested that HSP90α and HSP90ß expression levels were reduced in the MCF-7 cells after exposure to DOX. In the MDA-MB-231 cells, HSP90α expression was reduced while HSP90ß was found to be overexpressed following DOX treatment.

The Radioprotective Effects of Curcumin and Trehalose Against Genetic Damage Caused By I-131.

Background: Thyroid cancer has been growing rapidly during the last decades. Radioiodine-131 (I-131) as an appropriate therapy modality is currently using in the treatment of cancer and hyperthyroidism diseases. This radiotracer is considered as a cause of oxidative DNA damage in nontarget cells and tissues. The aim of this study was to investigate the effects of curcumin and trehalose on the level of DNA double-strand breaks (DSBs) caused by I-131 in human lymphocytes. Materials and Methods: First, 6-mL blood samples were taken from each of the five volunteers. After 1 h of preincubation with the antioxidants, a total of 20 µCi I-131/2 mL (blood + NaCl) was added to each sample, and then, the samples were reincubated for 1 h. Lymphocytes were separated and the mean DSB levels were measured for each sample through γ-H2AX assay to evaluate the effects of antioxidants. Results: After 1-h incubation with I-131, the DSBs increased by 102.9% compared to the control group (0.343 vs. 0.169 DSB/cell; P = 0.00). Furthermore, compared to the control + I-131 group, curcumin and trehalose reduced the DSBs by 42% and 38%, respectively. There was a significant decrement (P = 0.00) in the levels of DSBs of the curcumin + I-131 and trehalose + I-131 subgroups compared to the control + I-131 subgroup. Furthermore, there was no significant relationship between the radioprotective effect of curcumin and trehalose (P = 0.95). Conclusion: The use of curcumin and trehalose as antioxidant can reduce the numbers of DSBs caused by I-131. Meanwhile, the radioprotective effect of curcumin was more than trehalose.

Vitamins E and C Prevent DNA Double-strand Breaks in Peripheral Lymphocytes Exposed to Radiations from Iodine-131.

PURPOSE: Iodine-131 is used as a radiopharmaceutical to treat thyroid cancer. The current study aimed to evaluate the effects of vitamins E and C on the level of DNA double-strand breaks (DSBs) caused by Radioiodine-131 (I-131) in human lymphocytes. MATERIALS AND METHODS: Whole blood samples from human volunteers were incubated with a certain concentration of vitamins. After 1-h incubation, the samples were incubated with 20 µCi I-131/2 mL (blood + NaCl) for 1 h. To evaluate the effects of antioxidants, lymphocytes were separated, and the mean DSBs/cell was measured for each sample through γ-H2AX assay. RESULTS: After 1-h incubation with 20 µCi I-131/2 mL (blood + NaCl), iodine-131 increased the level of DSBs by 102.9%, compared with the background group. Vitamins E and C reduced the level of DSBs by 21.5% and 36.4%, respectively. CONCLUSION: Using vitamins E and C as antioxidants can reduce the toxicity of I-131. Furthermore, vitamin C provided the more protection for DNA, compared with vitamin E.

Platelet activation and function in response to high intensity interval exercise and moderate continuous exercise in CABG and PCI patients.

BACKGROUND: The effects of high intensity interval training (HIIT) on inflammatory markers and endothelial function have been extensively shown. However, the acute effect of HIIT on platelet activation and function in patients with recent revascularization is unclear. OBJECTIVE: The purpose of present study was to compare the responses of platelet activation (CD62P) and function (platelet aggregation) to high intensity interval exercise (HIIE) and moderate continuous exercise (MCE) in coronary artery bypass grafting (CABG) and percutaneous coronary interventions (PCI) patients. METHODS: Thirty patients who had CABG or PCI were randomly divided into HIIE, MCE and control groups. After determining the VO2peak, subjects in the MCE group carried out 30 min of continuous exercise at 60% of VO2peak, whereas, the subjects in HIIE group performed an interval protocol consisted of 8 repetitions of 2 min activity (running on treadmill) at 90% of VO2peak interspersed by 2 min of active recovery between repetitions at 30% of VO2peak .  Subjects in control group were seated and had no activity for the same period of time. Two blood samples were collected before and immediately after exercise and were analyzed for markers of platelet activation and function. RESULTS: Data analyzes revealed that increases in platelet aggregation induced by ADP and corrected for increases in platelet count in response to MCE trial was significantly lower than HIIE group (P < 0.05). In addition, responses of CD62P to MCE trial was significantly lower compared to HIIE group (P < 0.05). Changes in plateletcrit and platelet distribution width were significantly different among the three trials where the PCT and PDW following the HIIE were higher than MCE. Platelet count increased significantly (P < 0.05) by 13% following HIIE trial. CONCLUSIONS: Based on the findings of the present study it could be concluded that the risk of exercise-induced thrombosis is higher during HIIE than MCE in patients with recent revascularization.

Variations in Intraplatelet Phospho-VASP Expression Due to Pre-analytical Sample Preparations, Illustration of a Quality Control Issue in Platelet Pharmacology.

Intraplatelet vasodilator-stimulated phosphoprotein (VASP) analysis is a commonly used laboratory approach for monitoring of the anti-platelet therapy with adenosine diphosphate (ADP) receptor blocking agents; however, it's testing in clinical laboratory needs a high level of experience and proficiency. The ability to recognize how the pre-analytical variations can change the results would be helpful for the interpretation of data from intraplatelet VASP analysis. The aim of this study was to describe the possible differences of intraplatelet phospho-VASP expression between washed and platelet rich plasma (PRP) samples, both at baseline levels and following experimentally induction of VASP phosphorylation. PRP and washed platelet samples were treated with different inducers of VASP phosphorylation, including forskolin (10 µM), prostaglandin E1 (PGE1) (50 nM) and sodium nitro-prusside (SNP) (100 µM). Untreated PRP and washed platelet samples were also included in study as baseline controls. After labeling of platelets with either anti P-Serine(157)-VASP or anti P-Serine(239)-VASP, the samples were subjected to flow cytometric analysis to monitor the levels of intraplatelet phospho-VASP expression. Washed platelet samples tend to show increased expression of intraplatelet P-Serine(157)-VASP at baseline state and also more expression of P-Serine(157)-VASP and P-Serine(239)-VASP in response to forskolin and SNP, compared with PRP samples. Though, reduced levels of PGE1-induced VASP phosphorylation at both residues were detected for washed platelets. In this study we have provided some background information required for performing of intraplatelet VASP analysis on differently handled platelet samples and interpretation of the obtained results.

Induction of multipotency in umbilical cord-derived mesenchymal stem cells cultivated under suspension conditions.

Due to the limitations in the clinical application of embryonic stem cells (ESC) and induced pluripotent stem cells, mesenchymal stem cells (MSCs) are now much more interesting for cell-based therapy. Although MSCs have several advantages, they are not capable of differentiating to all three embryonic layers (three germ layers) without cultivation under specific induction media. Hence, improvement of MSCs for cell therapy purposes is under intensive study now. In this study, we isolated MSCs from umbilical cord tissue at the single-cell level, by treatment with trypsin, followed by cultivation under suspension conditions to form a colony. These colonies were trypsin resistant, capable of self-renewal differentiation to the three germ layers without any induction, and they were somewhat similar to ESC colonies. The cells were able to grow in both adherent and suspension culture conditions, expressed both the MSCs markers, especially CD105, and the multipotency markers, i.e., SSEA-3, and had a limited lifespan. The cells were expanded under simple culture conditions at the single-cell level and were homogenous. Further and complementary studies are required to understand how trypsin-tolerant mesenchymal stem cells are established. However, our study suggested non-embryonic resources for future cell-based therapy.

Modulation of hyperthermia-induced platelet aggregation inhibition in the presence of urea.

PURPOSE: This study has been conducted to evaluate the effect of urea on aggregation responses of heat-treated platelets. MATERIALS AND METHODS: The urea was added to platelet-rich plasma (PRP) samples in final concentrations of 50 and 100 mM. PRP samples, with or without exogenous urea, were incubated at 37 °C, 39 °C and 41 °C for 90 min and then were stimulated with adenosine diphosphate (ADP) or epinephrine for measuring of platelet aggregation responses. The average reduction in aggregability of heat-treated samples with reference to mean value obtained for control samples treated at 37 °C was expressed as inhibition percentage. RESULTS: Aggregation responses of the samples treated in the presence of 50 mM and 100 mM urea were significantly less inhibited by hyperthermia treatments compared with those treated without exogenous urea. CONCLUSION: The results indicate that the inhibitory effect of hyperthermia on platelet aggregation responses could be significantly modulated by urea.