Matrix stiffening and acquired resistance to chemotherapy: concepts and clinical significance.

Extracellular matrix (ECM) refers to the non-cellular components of the tumour microenvironment, fundamentally providing a supportive scaffold for cellular anchorage and transducing signaling cues that orchestrate cellular behaviour and function. The ECM integrity is abrogated in several cases of cancer, ending in aberrant activation of a number of mechanotransduction pathways and induction of multiple tumorigenic events such as extended proliferation, cell death resistance, epithelial-mesenchymal transition and most importantly the development of chemoresistance. In this regard, the present study mainly aims to elucidate how the ECM-stiffening process may contribute to the development of chemoresistance during cancer progression and what pharmacological approaches are required for tackling this issue. Hence, the first section of this review explains the process of ECM stiffening and the ways it may affect biochemical pathways to induce chemoresistance in a clinic. In addition, the second part focuses on describing some of the most important pharmacological agents capable of targeting ECM components and underlying pathways for overcoming ECM-induced chemoresistance. Finally, the third part discusses the obtained results from the application of these agents in the clinic for overcoming chemoresistance.

Extremely low frequency magnetic field enhances expression of a specific recombinant protein in bacterial host.

Expression of proteins in bacterial host cells, particularly E.coli, has gained much attention in recent years. Low expression outcome is the main technical drawback associated with this procedure, further restricting its largescale application in industry. Therefore, application of new amendments or reformations are required before further proceedings. Extremely low frequency magnetic fields (ELF-MFs) have shown to significantly affect biological processes, including gene expression, in E.coli. In current study, we investigated whether application of ELF-MF could result in overexpression of proteins in E.coli or not. Cluster of differentiation-22 (CD22), as a model protein, was expressed in E.Coli Rosetta (DE3) under continuous exposure to ELF-MF after applying various concentrations of Isopropyl ß-d-1-thiogalactopyranoside (IPTG) (0.25-1.25 mM) as inducer. The strength and frequency of electromagnetic fields (EMFs) ranged between 15 and 100 mT and 2.5-20 Hz respectively. Interestingly, application of 55 mT EMFs with frequencies ranging from 2.5 to 2.8 Hz significantly enhanced the yield of expression at all studied IPTG concentrations. Contrarily, EMFs with intensities other than 55 mT meaningfully declined protein expression at IPTG concentrations equal to 1 and 1.25 mM. In conclusion, application of specific range of ELF-MFs may be exploited as a new modification for enhancing heterologous expression of proteins in E.coli.

Development of a recombinant anti-VEGFR2-EPCAM bispecific antibody to improve antiangiogenic efficiency.

Tumor progression and metastasis, especially in invasive cancers (such as triple-negative breast cancer [TNBC]), depend on angiogenesis, in which vascular epithelial growth factor (VEGF)/vascular epithelial growth factor receptor [1] has a decisive role, followed by the metastatic spread of cancer cells. Although some studies have shown that anti-VEGFR2/VEGF monoclonal antibodies demonstrated favorable results in the clinic, this approach is not efficient, and further investigations are needed to improve the quality of cancer treatment. Besides, the increased expression of epithelial cell adhesion molecule (EpCAM) in various cancers, for instance, invasive breast cancer, contributes to angiogenesis, facilitating the migration of tumor cells to other parts of the body. Thus, the main goal of our study was to target either VEGFR2 or EpCAM as pivotal players in the progression of angiogenesis in breast cancer. Regarding cancer therapy, the production of bispecific antibodies is easier and more cost-effective compared to monoclonal antibodies, targeting more than one antigen or receptor; for this reason, we produced a recombinant antibody to target cells expressing EpCAM and VEGFR2 via a bispecific antibody to decrease the proliferation and metastasis of tumor cells. Following the cloning and expression of our desired anti-VEGFR2/EPCAM sequence in E. coli, the accuracy of the expression was confirmed by Western blot analysis, and its binding activities to VEGFR2 and EPCAM on MDA-MB-231 and MCF-7 cell lines were respectively indicated by flow cytometry. Then, its anti-proliferative potential was indicated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and apoptosis assay to evaluate inhibitory effects of the antibody on tumor cells. Subsequently, the data indicated that migration, invasion, and angiogenesis were inhibited in breast cancer cell lines via the bispecific antibody. Furthermore, cytokine analysis indicated that the bispecific antibody could moderate interleukin 8 (IL-8) and IL-6 as key mediators in angiogenesis progression in breast cancer. Thus, our bispecific antibody could be considered as a promising candidate tool to decrease angiogenesis in TNBC.

Dual l-Carnosine/Aloe vera Nanophytosomes with Synergistically Enhanced Protective Effects against Methylglyoxal-Induced Angiogenesis Impairment.

Microvascular complications are among the major outcomes of patients with type II diabetes mellitus, which are the consequences of impaired physiological functioning of small blood vessels and angiogenic responses in these patients. Overproduction and accumulation of methylglyoxal (MGO), a highly reactive dicarbonyl byproduct of glycolysis pathway, has been acclaimed as the main inducer of impaired angiogenic responses and microvascular dysfunction in diabetic patients with uncontrolled hyperglycemia. Hence, an effective approach to overcome diabetes-associated microvascular complications is to neutralize the deleterious activity of enhanced the concentration of MGO in the body. Owing to the glycation inhibitory activity of Aloe vera whole extract, and capability of l-carnosine, an endogenous dipeptide, in attenuating MGO's destructive activity, we examined whether application of a combination of l-carnosine and A. vera could be an effective way of synergistically weakening this reactive dicarbonyl's impaired angiogenic effects. Additionally, overcoming the poor cellular uptake and internalization of l-carnosine and A. vera, a nanophytosomal formulation of the physical mixture of two compounds was also established. Although l-carnosine and A. vera at whole studied combination ratios could synergistically enhance viability of human umbilical vein endothelial cells (HUVECs) treated with MGO, the 25:1 w/w ratio was the most effective one among the others (27 ± 0.5% compared to 12 ± 0.3 to 18 ± 0.4%; F (4, 15) = 183.9, P < 0.0001). Developing dual nanophytosomes of l-carnosine/A. vera (25:1) combination ratio, we demonstrated superiority of the nanophytosomal formulation in protecting HUVECs against MGO-induced toxicity following a 24-72 h incubation period (17.3, 15.8, and 12.4% respectively). Moreover, 500 µg/mL concentration of dual l-carnosine/A. vera nanophytosomes exhibited a superior free radical scavenging potency (63 ± 4 RFU vs 83 ± 5 RFU; F (5, 12) = 54.81, P < 0.0001) and nitric oxide synthesizing capacity (26.11 ± 0.19 vs 5.1 ± 0.33; F (5, 12) = 2537, P < 0.0001) compared to their physical combination counterpart. Similarly, 500 µg/mL dual l-carnosine/A. vera nanophytosome-treated HUVECs demonstrated a superior tube formation capacity (15 ± 3 vs 2 ± 0.3; F (5, 12) = 30.87, P < 0.001), wound scratch healing capability (4.92 ± 0.3 vs 3.07 ± 0.3 mm/h; F (5, 12) = 39.21, P < 0.0001), and transwell migration (586 ± 32 vs 394 ± 18; F (5, 12) = 231.8, P < 0.001) and invasion (172 ± 9 vs 115 ± 5; F (5, 12) = 581.1, P < 0.0001) activities compared to the physical combination treated ones. Further confirming the proangiogenic activity of the dual l-carnosine/A. vera nanophytosomes, a significant shift toward expression of proangiogenic genes including HIF-1α, VEGFA, bFGF, KDR, and Ang II was reported in treated HUVECs. Overall, dual l-carnosine/A. vera nanophytosomes could be a potential candidate for attenuating type II DM-associated microvascular complications with an impaired angiogenesis background.

The role of activated leukocyte cell adhesion molecule (ALCAM) in cancer progression, invasion, metastasis and recurrence: A novel cancer stem cell marker and tumor-specific prognostic marker.

Activated leukocyte cell adhesion molecule (ALCAM) or CD166 is a 100 to 105 KDa transmembrane immunoglobulin which is involved in activation of T-cells, hematopoiesis, neutrophils trans-endothelial migration, angiogenesis, inflammation and tumor propagation and invasiveness through formation of homophilic and heterophilic interactions. Recently, many studies have proposed that the expression pattern of ALCAM is highly associated with the grade, stage and invasiveness of tumors. Although ALCAM is a valuable prognostic marker in different carcinomas, similar expression patterns in different tumor types may be associated with completely different prognostic states, making it to be a tumor-type-dependent prognostic marker. In addition, ALCAM isoforms provide ways for primary detection of tumor cells with metastatic potential. More importantly, this prognostic marker has shown to be considerably dependent on the cytoplasmic and membranous expression, indirect and direct regulation of post-transcriptional molecules, pro-apoptotic proteins functionalities and several other oncogenic proteins or signalling pathways. This review mainly focuses on the pathways involved in expression of ALCAM and its prognostic value of in different types of cancers and the way in which it is regulated.

Dual in vitro invasion/migration suppressing and tamoxifen response modulating effects of a recombinant anti-ALCAM scFv on breast cancer cells.

It has been shown that overexpression of activated leukocyte cell adhesion molecule (ALCAM) is involved in development of resistance to tamoxifen therapy and promotion of cell invasion, migration and metastasis in ER+ breast cancer cells. Thus, we hypothesized that blockade of ALCAM interconnections with antibodies could be an effective approach for reversing mentioned negative events associated with ALCAM overexpression in breast cancer cells. Here, an anti-ALCAM scFv was recombinantly expressed and used throughout study for examination of the putative anticancer effects of ALCAM blockade. The anti-ALCAM scFv coding sequence was obtained from GenBank database and after addition of a 6× His-tag moiety, signal peptide and flanking sequences, the whole construct was expressed in Escherichia coli. Tamoxifen resistant MCF7 cells were then pretreat for 24 hours with purified recombinant anti-ALCAM scFv prior to administration of tamoxifen. In parallel, the cytotoxicity profile of anti-ALCAM scFv and tamoxifen co-treatments against tamoxifen resistant and sensitive MCF7 cell lines was also evaluated using CompuSyn software. The invasion/migration inhibitory effects of anti-ALCAM scFv on MDA-MB-231 cells were also evaluated. Pretreatment with anti-ALCAM scFv could successfully enhance anti-proliferative effects of tamoxifen against resistant MCF-7 cell lines. Furthermore, the combination of 19.2:1 of tamoxifen to anti-ALCAM scFv demonstrated synergistic cell inhibitory effect against tamoxifen resistant MCF7 cell lines. Also, incubating MDA-MB-231 cell lines with anti-ALCAM scFv resulted in a 30% and 25% reduction in number of invaded and migrated cells respectively. Overall, application of anti-ALCAM scFv could significantly suppress cancer cells metastasis in vitro and modulate tamoxifen resistant ER+ MCF7 cell line's sensitivity to tamoxifen. SIGNIFICANCE OF THE STUDY: Acquisition of resistance to tamoxifen therapy is one of the major challenges associated with cancer chemotherapy, gradually turning a responsive tumour into a refractory more invasive one which ultimately ends in disease progression and relapse. Here, we reported expression of an anti-ALCAM scFv, capable of increasing the sensitivity of tamoxifen resistant ER+ MCF-7 cells to tamoxifen therapy following a 24-hour pretreatment period. In addition, we demonstrated that the anti-ALCAM scFv monotherapy was also capable of suppressing invasion and migration of MDA-MB-231 cells in Boyden chamber assays.

Challenges facing antiangiogenesis therapy: The significant role of hypoxia-inducible factor and MET in development of resistance to anti-vascular endothelial growth factor-targeted therapies.

It is now fully recognized that along with multiple physiological functions, angiogenesis is also involved in the fundamental process and pathobiology of several disorders including cancer. Recent studies have fully established the role of angiogenesis in cancer progression as well as invasion and metastasis. Consequently, many therapeutic agents such as monoclonal antibodies targeting angiogenesis pathway have been introduced in clinic with the hope for improving the outcomes of cancer therapy. Bevacizumab (Avastin®) was the first anti-vascular endothelial growth factor (VEGF) targeting monoclonal antibody developed with this purpose and soon received its accelerated US Food and Drug Administration (FDA) approval for treatment of patients with metastatic breast cancer in 2008. However, the failure to meet expecting results in different follow-up studies, forced FDA to remove bevacizumab approval for metastatic breast cancer. Investigations have now revealed that while suppressing VEGF pathway initially decreases tumor progression rate and vasculature density, activation of several interrelated pathways and signaling molecules following VEGF blockade compensate the insufficiency of VEGF and initially blocked angiogenesis, explaining in part the failure observed with bevacizumab single therapy. In present review, we introduce some of the main pathways and signaling molecules involved in angiogenesis and then propose how their interconnection may result in development of resistance to bevacizumab.

Effects of a combination of Nigella sativa and Vitex agnus-castus with citalopram on healthy menopausal women with hot flashes: results from a subpopulation analysis.

The goal of the current study was to evaluate the efficiency of a phytotherapeutic intervention consisting of a combination of Nigella sativa and Vitex agnus-castus with citalopram in the control of hot flashes in healthy menopausal women. An 8 week, double-blind, randomized, placebo-controlled study was performed among 46 women aged between 40 and 60 years experiencing an average of more than four hot flashes per day recruited during July 2016 to June 2017. Data on severity of vasomotor symptoms were collected at the end of the eighth week. Herbal medication or placebo capsules were administered once daily in morning. At the end of the 8-week treatment period, analyses of covariance demonstrated the superiority of herbal combination with citalopram over placebo and citalopram for three MENQOL domain scores including vasomotor (p < .001), physical (p = .036), psychosocial (p = .001) but no significant differences were observed in terms of sexual function (p = .231). Based on the results, the addition of a combination of N. sativa and V. agnus-castus to the citalopram may be a potential clinical application for improving therapeutic outcomes. Larger randomized, controlled trials are also warranted for further investigations of these symptoms.

Pegylated magnetic mesoporous silica nanoparticles decorated with AS1411 Aptamer as a targeting delivery system for cytotoxic agents.

Fulfilling the purpose of developing a NP with theragnostic capabilities, the current study describes the synthesis of an aptamer-functionalized PEG-coated SPION/mesoporous silica core-shell nanoparticle for concurrent cancer targeted therapy and magnetic resonance imaging. SPIONs were synthesized according to a thermal decomposition method and served as cores for SPION/mesoporous silica core/shell nanoparticles (MMSNs). Doxorubicin was then successfully loaded in MMSNs which were then coated with di-carboxylic acid functionalized polyethylene glycol (PEG-MMSNs). AS1411 aptamers were at the end covalently attached to NPs (APT-PEG-MMSNs). The mean diameter of synthesized NPs was about 89 nm and doxorubicin encapsulation efficacy was ≈67.47%. Results of MTT based cell cytotoxicity assay demonstrated a significantly higher toxicity profile for APT-PEG-MMSNs against MCF7 cells compared to non-decorated MMSNs, while no significant differences were spotted against NIH-3T3 cells. Meanwhile, formation of protein corona around APT-PEG-MMSNs in biological medium significantly attenuated observed cytotoxicity against MCF7 cell line. Examining NPs uptake by MCF7 cells using confocal laser scanning microscopy also confirmed superiority of APT-PEG-MMSNs over PEG-MMSNs. Finally, APT decorated NPs induced highest signal intensity reduction in T2-weighted images during in vitro MRI assay. In conclusion, developed NPs may serve as promising multifunctional vehicles for simultaneous cancer targeted therapy and MRI imaging.

NF-κB as the main node of resistance to receptor tyrosine kinase inhibitors in triple-negative breast cancer.

Graphical abstract Although accounting for merely a minute portion of diagnosed breast cancers, disproportionate number of deaths and associated low survival rate of patients have made triple-negative breast cancer to be considered as the most lethal breast cancer subtype. More importantly, intrinsic or developed resistance to chemotherapeutic regimens and disappointing outcomes of trials associated with many newly developed agents are other obstacles in establishment of a durable response in these patients. Interestingly, these happen despite the outstanding preclinical outcomes observed by these agents, most importantly among them, targeted receptor tyrosine kinase inhibitors. Pursuing these disappointing outcomes, especially in the case of targeted receptor tyrosine kinase inhibitors, many researches have focused on identification of the hidden factors involved. Highly inflammatory, rich in reactive oxygen species, and hypoxic microenvironment of triple-negative breast cancer tumors and the involving mediators were the first suggestions for observed resistance and poor clinical outcomes of targeted receptor tyrosine kinase inhibitors. Interestingly, for all aberrantly expressed mediators observed in microenvironment, downstream pathways converge in a common node, nothing but the nuclear factor-κB, the insidious factor proposed to be the cause of many events opposing achievement of a desired outcome. In first section of current review, we describe the signaling pathways underlying activation of receptor tyrosine kinases and their convergence at the nuclear factor-κB node, and in next section, we demonstrate how unique hypoxic, inflammatory, rich in free-radical microenvironment of triple-negative breast cancer exacerbate pathways in which otherwise could become mostly suppressed by receptor tyrosine kinase inhibitors.

A two-week, double-blind, placebo-controlled trial of Viola odorata, Echium amoenum and Physalis alkekengi mixture in symptomatic benign prostate hyperplasia (BPH) men.

CONTEXT: As an alternative approach, administration of phytotherapeutic agents in management of benign prostate hyperplasia (BPH), is rapidly growing each day. Different authors have indicated effectiveness of Viola odorata L. (Violaceae), Echium amoenum Fisch. & C.A.Mey. (Boraginaceae) and Physalis alkekengi L. (Solanaceae) in treatment of BPH. However, none have reported the beneficial outcomes of the mixture yet. OBJECTIVE: This study evaluates the therapeutical effects of V. odorata, E. amoenum and P. alkekengi mixture on symptomatic BPH patients. MATERIALS AND METHODS: Eighty six symptomatic BPH patients with International Prostate Symptom Score (IPSS) of more than 13 and prostate volume of more than 30 cm3 were randomly allocated to receive a two-week course of placebo (control group) or 1 mL of mixed hydro-alcoholic solution of P. alkekengi, E. amoenum and V. odorata extracts (1.5, 1 and 1.5% respectively) (treatment group). RESULTS: IPSS score of incomplete urination (42.3 ± 2.04%), frequency of urination (20.08 ± 1.02%), intermittency (40.78 ± 2.16%), urgency (60.91 ± 3.14%), weak stream (50.58 ± 2.14%), straining (55.67 ± 2.53%) and nocturia (40.14 ± 1.89%) in treatment group were significantly decreased after treatment compare to placebo receiving group. Furthermore, the prostate volume (16.92 ± 0.89%) and extant urine volume (28.12 ± 1.36%) also significantly decreased in treatment group compared to control group. No significant side effects or abnormalities in biochemical tests and urinalysis were observed throughout the study. DISCUSSION AND CONCLUSIONS: Based on results, mentioned mixture is safe and effective in improving life quality of patients suffering from BPH.

Reduction of chlorpyrifos-induced toxicity in human lymphocytes by selected phosphodiesterase inhibitors.

One of the most famous and commonly used compounds from organophosphate (OP) family is chlorpyrifos (CP) which is widespreadly used as a powerful insecticide. Previous studies have shown that OPs induce oxidative stress, inflammation and apoptosis by generating the free radicals. The protective effects of three members of phosphodiesterase inhibitor (PDEI) family, including rolipram (RLP), milrinon (MLR) and pentoxifylline (PTX) were evaluated in the human lymphocytes against CP's toxicity. In this case, the level of oxidative stress biomarkers, the viability of the cells and the rate of apoptosis by flow cytometry were investigated. The results of this study revealed that CP makes a significant increase in the level of inflammatory and oxidative stress markers such as meyloperoxidase (MPO), lipid peroxidation (LPO), total thiol molecules (TTM) and total antioxidant potential (TAP), and also makes an enhancement in the rate of apoptosis process. On the other hand, PDEIs and specifically the combination of them restored the negative effects of CP and significantly prevented the apoptosis and oxidative stress imbalance. It is concluded that these PDEIs have positive effects in attenuation, recovery, and protection of CP-induced toxicity in the human lymphocytes.

Optimization of paclitaxel-loaded poly (d,l-lactide-co-glycolide-N-p-maleimido benzoic hydrazide) nanoparticles size using artificial neural networks.

The aim of this study was to find a model using artificial neural networks (ANNs) to predict PLGA-PMBH nanoparticles (NPs) size in preparation by modified nanoprecipitation. The input variables were polymer content, drug content, power of sonication and ratio of organic/aqueous phase (i.e. acetone/water), while the NPs size of PLGA-PMBH was assumed as the output variable. Forty samples of PLGA-PMBH NPs containing anticancer drug (i.e. paclitaxel) were synthesized by changing the variable factors in the experiments. The data modeling were performed using ANNs. The effects of input variables (namely, polymer content, drug content, power of sonication and ratio of acetone/water) on the output variables were evaluated using the 3D graphs obtained after modeling. Contrasting the 3D graphs from the generated model revealed that the amount of polymer (PLGA-PMBH) and drug content (PTX) have direct relation with the size of polymeric NPs in the process. In addition, it was illustrated that the ratio of acetone/water was the most important factor affecting the particle size of PLGA-PMBH NPs provided by solvent evaporation technique. Also, it was found that increasing the sonication power (up to a certain amount) indirectly affects the polymeric NPs size however it was directly affected in higher values.

Surface modification of PLGA nanoparticles via human serum albumin conjugation for controlled delivery of docetaxel.

BACKGROUND: Poly lactic-co-glycolic acid (PLGA) based nanoparticles are considered to be a promising drug carrier in tumor targeting but suffer from the high level of opsonization by reticuloendothelial system due to their hydrophobic structure. As a result surface modification of these nanoparticles has been widely studied as an essential step in their development. Among various surface modifications, human serum albumin (HSA) possesses advantages including small size, hydrophilic surface and accumulation in leaky vasculature of tumors through passive targeting and a probable active transport into tumor tissues. METHODS: PLGA nanoparticles of docetaxel were prepared by emulsification evaporation method and were surface conjugated with human serum albumin. Fourier transform infrared spectrum was used to confirm the conjugation reaction where nuclear magnetic resonance was utilized for conjugation ratio determination. In addition, transmission electron microscopy showed two different contrast media in conjugated nanoparticles. Furthermore, cytotoxicity of free docetaxel, unconjugated and conjugated PLGA nanoparticles was studied in HepG2 cells. RESULTS: Size, zeta potential and drug loading of PLGA nanoparticles were about 199 nm, -11.07 mV, and 4%, respectively where size, zeta potential and drug loading of conjugated nanoparticles were found to be 204 nm, -5.6 mV and 3.6% respectively. Conjugated nanoparticles represented a three-phasic release pattern with a 20% burst effect for docetaxel on the first day. Cytotoxicity experiment showed that the IC50 of HSA conjugated PLGA nanoparticles (5.4 µg) was significantly lower than both free docetaxel (20.2 µg) and unconjugated PLGA nanoparticles (6.2 µg). CONCLUSION: In conclusion surface modification of PLGA nanoparticles through HSA conjugation results in more cytotoxicity against tumor cell lines compared with free docetaxel and unconjugated PLGA nanoparticles. Albumin conjugated PLGA nanoparticles may represent a promising drug delivery system in cancer therapy.

Enhanced antibacterial activity of roxithromycin loaded pegylated poly lactide-co-glycolide nanoparticles.

BACKGROUND AND THE PURPOSE OF THE STUDY: The purpose of this study was to prepare pegylated poly lactide-co-glycolide (PEG-PLGA) nanoparticles (NPs) loaded with roxithromycin (RXN) with appropriate physicochemical properties and antibacterial activity. Roxithromycin, a semi-synthetic derivative of erythromycin, is more stable than erythromycin under acidic conditions and exhibits improved clinical effects. METHODS: RXN was loaded in pegylated PLGA NPs in different drug;polymer ratios by solvent evaporation technique and characterized for their size and size distribution, surface charge, surface morphology, drug loading, in vitro drug release profile, and in vitro antibacterial effects on S. aureus, B. subtilis, and S. epidermidis. RESULTS AND CONCLUSION: NPs were spherical with a relatively mono-dispersed size distribution. The particle size of nanoparticles ranged from 150 to 200 nm. NPs with entrapment efficiency of up to 80.0±6.5% and drug loading of up to 13.0±1.0% were prepared. In vitro release study showed an early burst release of about 50.03±0.99% at 6.5 h and then a slow and steady release of RXN was observed after the burst release. In vitro antibacterial effects determined that the minimal inhibitory concentration (MIC) of RXN loaded PEG-PLGA NPs were 9 times lower on S. aureus, 4.5 times lower on B. subtilis, and 4.5 times lower on S. epidermidis compared to RXN solution. In conclusion it was shown that polymeric NPs enhanced the antibacterial efficacy of RXN substantially.

Recombinant nanobody against MUC1 tandem repeats inhibits growth, invasion, metastasis, and vascularization of spontaneous mouse mammary tumors.

Alteration in glycosylation pattern of MUC1 mucin tandem repeats during carcinomas has been shown to negatively affect adhesive properties of malignant cells and enhance tumor invasiveness and metastasis. In addition, MUC1 overexpression is closely interrelated with angiogenesis, making it a great target for immunotherapy. Alongside, easier interaction of nanobodies (single-domain antibodies) with their antigens, compared to conventional antibodies, is usually associated with superior desirable results. Herein, we evaluated the preclinical efficacy of a recombinant nanobody against MUC1 tandem repeats in suppressing tumor growth, angiogenesis, invasion, and metastasis. Expressed nanobody demonstrated specificity only toward MUC1-overexpressing cancer cells and could internalize in cancer cell lines. The IC50 values (the concentration at which the nanobody exerted half of its maximal inhibitory effect) of the anti-MUC1 nanobody against MUC1-positive human cancer cell lines ranged from 1.2 to 14.3 nm. Similar concentrations could also effectively induce apoptosis in MUC1-positive cancer cells but not in normal cells or MUC1-negative human cancer cells. Immunohistochemical staining of spontaneously developed mouse breast tumors prior to in vivo studies confirmed cross-reactivity of nanobody with mouse MUC1 despite large structural dissimilarities between mouse and human MUC1 tandem repeats. In vivo, a dose of 3 µg nanobody per gram of body weight in tumor-bearing mice could attenuate tumor progression and suppress excessive circulating levels of IL-1a, IL-2, IL-10, IL-12, and IL-17A pro-inflammatory cytokines. Also, a significant decline in expression of Ki-67, MMP9, and VEGFR2 biomarkers, as well as vasculogenesis, was evident in immunohistochemically stained tumor sections of anti-MUC1 nanobody-treated mice. In conclusion, the anti-MUC1 tandem repeat nanobody of the present study could effectively overcome tumor growth, invasion, and metastasis.

Cellular stress response to extremely low-frequency electromagnetic fields (ELF-EMF): An explanation for controversial effects of ELF-EMF on apoptosis.

Impaired apoptosis is one of the hallmarks of cancer, and almost all of the non-surgical approaches of eradicating tumour cells somehow promote induction of apoptosis. Indeed, numerous studies have stated that non-ionizing non-thermal extremely low-frequency magnetic fields (ELF-MF) can modulate the induction of apoptosis in exposed cells; however, much controversy exists in observations. When cells are exposed to ELF-EMF alone, very low or no statistically significant changes in apoptosis are observed. Contrarily, exposure to ELF-EMF in the presence of a co-stressor, including a chemotherapeutic agent or ionizing radiation, can either potentiate or inhibit apoptotic effects of the co-stressor. In our idea, the main point neglected in interpreting these discrepancies is "the cellular stress responses" of cells following ELF-EMF exposure and its interplay with apoptosis. The main purpose of the current review was to outline the triangle of ELF-EMF, the cellular stress response of cells and apoptosis and to interpret and unify discrepancies in results based on it. Therefore, initially, we will describe studies performed on identifying the effect of ELF-EMF on induction/inhibition of apoptosis and enumerate proposed pathways through which ELF-EMF exposure may affect apoptosis; then, we will explain cellular stress response and cues for its induction in response to ELF-EMF exposure; and finally, we will explain why such controversies have been observed by different investigators.

Necroptosis triggered by ROS accumulation and Ca2+ overload, partly explains the inflammatory responses and anti-cancer effects associated with 1Hz, 100 mT ELF-MF in vivo.

Whereas the anti-neoplastic activity of extremely low frequency magnetic fields (ELF-EMF) is well-documented in literature, little is known about its underlying anti-cancer mechanisms and induced types of cell death. Here, for the first time, we reported induction of necroptosis, a specific type of programed necrotic cell death, in MC4-L2 breast cancer cell lines following a 2 h/day exposure to a 100 Hz, 1 mT ELF-EMF for five days. For in vivo assessment, inbred BALB/c mice bearing established MC-4L2 tumors were exposed to 100 mT, 1 Hz ELF-EMF 2 h daily for a period of 28-day, following which tumors were dissected and fixed for evaluation of tumor biomarkers expression and types of cell death induced using TUNEL assay, Immunohistochemistry and H&E staining. Peripheral blood samples were also collected for assessing pro-inflammatory cytokine profile following exposure. An exaggerated proinflammatory response evident form enhancement of IFN-γ (4.8 ± 0.24 folds) and TNF-α (3.1 ± 0.19 folds) and number of tumors infiltrating lymphocytes (TILs), specially CD8+ Th cells (~20 folds), proposed occurrence of necroptosis in vivo. Meanwhile, exposure could effectively suppress tumor growth and expression of Ki-67, CD31, VEGFR2 and MMP-9. In vitro studies on ELF-EMF exposed MC-4L2 cells demonstrated a meaningful increase in phosphorylation of RIPK1/RIPK3/MLKL proteins and cleavage of caspase-9/caspase-3, confirming occurrence of both necroptosis and apoptosis. Complementary in vitro studies by treating ELF-EMF exposed MC-4L2 cells with verapamil (a calcium channel inhibitor), N-acetyl cysteine (a ROS scavenger) or calcium chloride confirmed the role of elevated intracellular calcium and ROS levels in ELF-EMF induced necroptosis.

Interferons: role in cancer therapy.

Interferons (IFNs) are a group of signaling cytokines, secreted by host cells to induce protection against various disorders. IFNs can directly impact on tumor cells or indirectly induce the immune system to protect host cells. The expression levels of IFNs and its functions of are excellently modulated in a way to protect host cells from probable toxicities caused by extreme responses. The efficacy of anticancer therapies is correlated to IFNs signaling. Although IFN signaling is involved in induction of antitumor responses, chronic stimulation of the IFN signaling pathway can induce resistance to various antineoplasm therapies. Hence, IFNs are expressed by both cancer and immune cells, and modulate their biological function. Understanding this mechanism of action might be a key target of combination therapies.