Extracellular vesicles (EVs) are considered as cornerstones of cell-cell communication. EVs are bio-membrane naturally occurring vesicles generated by both eukaryotic and prokaryotic cells to transfer myriad cargo between close and distant cells to modulate them genetically or at functional levels. Recently, attention has been attracted towards the use of EVs derived from different sources like mammalian, bacterial and plant cells for drug delivery and diagnostic applications. Although all types of EVs vary in their compositions and biogenesis, they have similar features which made them desirable and novel theranostics. Today, breast cancer is a challenging disease to humans around the world, hence, accurate and timely diagnosis and selecting the most appropriate therapeutic plan are pivotal steps forward reducing mortality. In this chapter, we have focused on unique features of EVs to be employed in the diagnosis of breast cancer because of the presence of the data inside the EVs which are related to their origin cells. Also, imaging agents can be enclosed by EVs, providing a high sensitive and reproducible imaging technique. Moreover, EVs are recruited as novel strategies to reach chemotherapeutic molecules to breast cancer in targeted drug delivery. Lastly, by discussing the pros and cons of various types of EVs as theranostics, we shed a light on the role of EVs in breast cancer and underlined the challenges that have to be overcome.

The poor prognosis and inefficiency of the therapeutic agents in treating triple negative breast cancer (TNBC) have raised significant concerns, driving the quest for designing novel and potent chemotherapeutic compounds. In this regard, inducing programmed cell death (PCD) has emerged as a promising approach for breast cancer therapy. Accordingly, a series of hybrid molecules comprising hydrazone and oxamide moieties (5a-5q) were designed, synthesized, and assessed for their anticancer activity against various cancer cells. Among these synthesized hybrids, compound 5q was selected as the lead compound with remarkable ability to disrupt MDA-MB-231 cell growth, achieving an IC50-72h of 9.79 μM, while exhibiting lower toxicity in normal human cells. The in vitro experiments revealed that this compound triggers neither apoptosis nor autophagy in TNBC cells. Furthermore, the in vivo outcomes corroborated the in vitro results, showing a significant delay in tumor growth at a dose of 1 mg/kg/day following three weeks of treatment in the 4T1 mouse model of TNBC. The findings of this study suggested that compound 5q acts through necroptosis by overexpression of P-RIPK3 and phosphorylation of its downstream effector, MLKL. Compound 5q holds promise as a potential candidate for the development of anti-TNBC drugs.

In order to investigate the effects of foliar spraying of salicylic acid and beta-aminobutyric acid on yield, physiological and biochemical characteristics of Dracocephalum moldavica (L.), during cropping year of 2017-2018, in a experiment based on a randomized complete block design with 4 replications was carried out in Kerman. Foliar spraying was done one month before harvesting with three concentrations (0, 0.5 and 1 mM). Plant height, stem diameter, number of leaves, fresh matter yield, dry matter yield, number of lateral branches, yield of total extract and essential oil were measured. Identification of essential oil components was done by using a gas chromatography (GC) which was connected to a mass spectrometer (GC/MS) by comparing the inhibition indices and mass spectrometry study. According to the results, the effect of both elicitors of salicylic acid and beta aminobutyric acid on plant height, number of leaves, fresh matter yield and dry matter yield were significant at 1% level of probability; But, there was no significant difference in stem diameter and number of lateral branches. The interaction effect of both elicitors on plant height and dry matter yield was significant at 5% and 1% probability levels, respectively. While there was no significant difference on other traits under study. Also, the use of both elicitors increased the total extract and essential oil yields. The highest percentage of main compounds in essential oil was affected under the influence of the amount of 1 mM salicylic acid in absence of beta aminobutyric acid, including 36.8% geranyl acetate, 20.5% geraniol, 19.7% geranial, 14.9% neral and neral acetate 2.9%. In general, the use of salicylic acid was found and beta-aminobutyric acid to increase Dracocephalum moldavica (L.) yield.

Breast cancer remains a significant global health concern, emphasizing the critical need for effective treatment strategies, especially targeted therapies. This systematic review aims to summarize the findings from in vitro and in vivo studies regarding the therapeutic potential of exosomes as drug delivery platforms in the field of breast cancer treatment. A comprehensive search was conducted across bibliographic datasets, including Web of Science, PubMed, and Scopus, using relevant queries from several related published articles and the Medical Subject Headings Database. Then, all morphological, biomechanical, histopathological, and cellular-molecular outcomes were systematically collected. A total of 30 studies were identified based on the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. These studies underwent assessment using the Systematic Review Centre for Laboratory Animal Experimentation risk of bias assessment tool. The results indicate that exosomes exhibit promise as effective drug delivery platforms, capable of hindering cancer cell viability, proliferation, migration, and angiogenesis. However, a comprehensive assessment is challenging due to some studies deviating from guidelines and having incomplete methodology. Addressing these, future studies should detail methodologies, optimize dosing, and enhance exosome production. Standardization in reporting, consistent protocols, and exploration of alternative sources are crucial.

Triple-negative breast cancer is a subtype of breast cancer with poor clinical outcome, and currently, no effective targeted therapies are available. Since cancer develops owing to deregulation of apoptosis, employing therapeutic strategies with the ability to target the molecules involved in apoptosis induction would provide a valid approach to hinder tumor progression. Hydrazide-hydrazones and oxamide molecules are the subject of intense studies due to their anticancer effects via apoptosis induction. In the present study, we attempted to elucidate the mechanism of action of a synthesized compound (compound A) in inducing cell death. Annexin/PI and Western blotting analyses, DAPI staining, mitochondrial membrane potential probe, and flow cytometry were applied for the in vitro evaluations. 4T1 syngeneic mouse model and immunohistochemistry were used for the in vivo assessments. Compound A caused cell death by inducing apoptosis in MDA-MB-231 cells in a mitochondrial-dependent manner at high concentrations after 72 h of incubation. Compound A also impeded tumor growth in a 4T1 syngeneic mouse model as evidenced by hematoxylin and eosin staining of the tumors. Furthermore, it significantly diminished the expression of pro-caspase-3, Ki67, and CD31 markers in the tumor sections. Conclusively, this study for the first time reports the anti-cancer efficacy of compound A in both in vitro and in vivo models and its potential in the treatment of triple-negative breast cancer.

Pentavalent antimonials are the mainstay treatment against different clinical forms of leishmaniasis. The emergence of resistant isolates in endemic areas has led to treatment failure. Unraveling the underlying resistance mechanism would assist in improving the treatment strategies against resistant isolates. This study aimed to investigate the RNA expression level of glutathione synthetase (GS), Spermidine synthetase (SpS), trypanothione synthetase (TryS) genes involved in trypanothione synthesis, and thiol-dependent reductase (TDR) implicated in drug reduction, in antimony-sensitive and -resistant Leishmania tropica isolates. We investigated 11 antimony-resistant and 11 antimony-sensitive L. tropica clinical isolates from ACL patients. Drug sensitivity of amastigotes was determined in mouse macrophage cell line J774A.1. The RNA expression level in the promastigote forms was analyzed by quantitative real-time PCR. The results revealed a significant increase in the average expression of GS, SpS, and TrpS genes by 2.19, 1.56, and 2.33-fold in resistant isolates compared to sensitive ones. The average expression of TDR was 1.24-fold higher in resistant isolates, which was insignificant. The highest correlation coefficient between inhibitory concentration (IC50) values and gene expression belonged to the TryS, GS, SpS, and TDR genes. Moreover, the intracellular thiol content was increased 2.17-fold in resistant isolates compared to sensitive ones and positively correlated with IC50 values. Our findings suggest that overexpression of trypanothione biosynthesis genes and increased thiol content might play a key role in the antimony resistance of L. tropica clinical isolates. In addition, the diversity of gene expression in the trypanothione system and thiol content among L. tropica clinical isolates highlighted the phenotypic heterogeneity of antimony resistance among the parasite population.

Background:: Exosomal RNAs (ExoRNAs) offer valuable insights into their cellular origin. ExoRNA studies were faced with challenges in obtaining sufficient amounts of high-quality RNA. Herein, we aimed to compare three traditional exosome isolation methods to introduce an appropriate strategy to extract RNA from cancer-derived exosomes for further RNA analysis. Methods:Exosomes were isolated through ultracentrifugation, precipitation kit, and size exclusion column chromatography, and then characterized by dynamic light scattering and transmission electron microscopy, followed by extracting total RNA. The quality and quantity of the extracted RNAs were assessed by a NanoDrop and 2.5% agarose gel electrophoresis. Results:Extracted exosomes displayed a similar range of size and morphology. We found that polyethylene glycol-precipitation method resulted in a higher RNA yield with a 260/280 ratio of 1.9. The obtained exoRNA appeared as a smear in the agarose gel, indicative of small exoRNAs. Conclusion:We provide researchers a suitable approach to isolate exosomes based on yield and purity of exoRNA.

Background:Neuroinflammation and oxidative stress are critical factors involved in the pathogenesis of Parkinson's disease (PD), the second most common progressive neurodegenerative disease. Additionally, lipid peroxidation end products contribute to inflammatory responses by activating pro-inflammatory genes. Lipid peroxidation occurs as a result of either the overproduction of intracellular reactive oxygen species (ROS) or the reaction of cyclooxygenases (COXs).Objectives:In this study, we examined the role of 1,5-diaryl pyrrole derivatives against the neurotoxic effects of 6-hydroxydopamine (6-OHDA) in a cellular model of PD.Methods:PC12 cells were pre-treated with compounds 2-(4-chlorophenyl)-5-methyl-1-(4-(trifluoromethoxy)phenyl)-1H-pyrrole (A), 2-(4-chlorophenyl)-1-(4-methoxyphenyl)-5-methyl-1H-pyrrole (B), and 1-(2-chlorophenyl)-2-(4-chlorophenyl)-5-methyl-1H-pyrrole (C), respectively, 24 h before exposure to 6-OHDA. We conducted various assays, including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT), ROS, and lipid peroxidation assays, Hoechst staining, Annexin V/PI, Western blotting analysis and ELISA method, to assess the neuroprotective effects of pyrrole derivatives on 6-OHDA-induced neurotoxicity.Results:Our results demonstrated that apoptosis induction was inhibited by controlling the lipid peroxidation process in the in vitro model following pre-treatment with compounds A, B, and, somehow, C. Furthermore, compounds A and C likely act by suppressing the COX-2/PGE2 pathway, a mechanism not attributed to compound B.Conclusions:These findings suggest that the novel synthetic pyrrolic derivatives may be considered promising neuroprotective agents that can potentially prevent the progression of PD.

AbstractTriple-negative breast cancer (TNBC) is a subtype of breast cancer with poor clinical outcome, and currently no effective targeted therapies are available. Since cancer develops owing to deregulation of apoptosis, employing therapeutic strategies with ability to target the molecules involved in apoptosis induction, would provide a valid approach to hinder tumor progression. Hydrazide-hydrazones and oxamide molecules are the subject of intense studies due to their anticancer effects via apoptosis induction. In the present study, we attempted to elucidate the mechanism of action of a synthesized compound (compound A) and understand if the cell death occurs through inducing the apoptosis. These properties were investigated using the, annexin/PI and Western blotting analyses, DAPI staining, and mitochondrial membrane potential probe. Compound A also impeded the tumor growth in a 4T1 syngeneic mouse model as evidenced by hematoxylin and eosin staining of the tumors. Apart from that, it significantly diminished the expression of pro-caspase-3, Ki67 and CD31 markers in the tumor sections. Conclusively, this study for the first time reports the anti-cancer efficacy of compound A in both in vitro and in vivo models, which may serve as a potent candidate in triple negative breast cancer (TNBC) treatment.

Consumption of Medicago sativa (alfalfa) has been linked to infection control in livestock. Therefore, studying the mechanism of this action may lead to the development of enriched food supplements. The purpose of this study was to investigate how M. sativa extract affects the growth of Lacticaseibacillus casei, Lactiplantibacillus plantarum, Staphylococcus aureus, and Escherichia coli. The effects of M. sativa extract on the bacterial cell adherence to Caco-2 cells (Human colon epithelial adenocarcinoma cell lines) and expression of genes involved in the formation of E. coli biofilm were also studied. Generally, in concentrations lower than 100 mg/ml, alfalfa extract had no antimicrobial effect on the probiotic and pathogenic bacteria. However, it not only acted as a growth promoter for two probiotic strains at 2.5% concentration but also increased the antibacterial effect of probiotics supernatants. Furthermore, alfalfa extract reduced the adhesion of pathogenic bacteria to Caco-2 cells. M. sativa extract was identified to inhibit the biofilm formation by E. coli through decreasing the genes expression, papC, and rcsA. As a result, it looks that alfalfa can improve the growth and function of beneficial bacteria, but further studies are needed to elucidate its mechanism of action.

Aims: Doxorubicin is a potent and broad-spectrum antineoplastic medication prescribed for both solid and hematological malignancies. Despite its value, the clinical use of doxorubicin is limited due to cardio-oncologic complication and cardiotoxic adverse effect. Among the mechanisms proposed for its toxicity, mitochondrial dysfunction has gained more attention. Therefore, if damaged mitochondria are replaced by normal efficient mitochondria, cardiac toxicity is expected to be reduced or improved. In this way, we have studied the efficiency of transplantation of freshly isolated rat liver mitochondria in neonatal rat cardiomyocytes that have been damaged by doxorubicin. Materials and methods: For this purpose, isolated mitochondria were characterized using mitochondrial complex II, membrane potential and swelling evaluations, and also fluorescence and electron microscopy. Afterward, the effect of mitotherapy on the damaged cardiomyocytes was investigated by using annexin V/PI staining, MTT, ROS, MMP, lipid peroxidation, GSH and ATP evaluations. Key findings and significance: Transplanted mitochondria could remarkably enter the neonatal rat cardiomyocytes. Addition of mitochondria to the damaged cardiomyocytes, significantly increased cell viability by reducing the level of reactive oxygen species and lipid peroxidation, increasing of Delta psi, ATP and GSH contents and decreasing of apoptotic and necrotic cell death. Our results showed that mitotherapy has a significant restorative effect on cardiotoxicity induced by doxorubicin, which promises a better future to reduce the complications of cancer treatment.

In this research, the involvement of CB1 and TRPV1 receptors in the possible protective effects of anandamide were investigated in the kindling model of epilepsy. The basolateral amygdala of the rat brain was chosen to put stimulating electrodes. Semi-rapid kindling was induced by a repetitive sub-threshold stimulation for 5–9 consecutive days. There were seven groups, six of which were kindled and used for drug testing by intracerebroventricular (i.c.v.) microinjection. (i) Sham, (ii) control group received vehicles, (iii) anandamide (AEA; 100 ng/rat), (iv) capsazepine (TRPV1 antagonist; 100 ng/rat), (v) AM251 (CB1 antagonist; 100 ng/rat), (vi) AM251 + anandamide, and (vii) capsazepine + anandamide. The after-discharge duration, seizure duration, and stage five duration were measured in rats. Moreover, the expressions of the extracellular signal-regulated kinase (ERK) and the cAMP responsive element binding (CREB) proteins in the hippocampus were also studied. The anandamide-treated group showed a significant decrease in seizure scores, while no change was shown in seizure scores in the capsazepine- and AM251-treated groups compared with the control group. Co-administrations of either capsazepine + AEA or AM251 + AEA attenuated the protective effect of AEA against seizure. Furthermore, the group received AEA showed a decrease in the expressions of CREB and p-CREB possibly through the activation of the CB1 and TRPV1 receptors. Activation of CB1 and TRPV1 receptors might be involved in AEA anticonvulsant effect in kindling model of epilepsy. This effect could be due to suppression of CREB phosphorylation in hippocampal neurons.

Low-density lipoprotein receptor-related protein 6 (LRP6) is an important therapeutic target for diseases such as osteoporosis, Alzheimer, cancer, and neurodegenerative disease. Computational methods such as ligand-based and structure-based virtual screening have been introduced as an extremely efficient and accurate tool for finding new drug targets and candidates. In this study, we aimed to screen the National Cancer Institute (NCI) Diversity Set II and parts of the ZINC database by virtual screening to identify potential and safe compounds that can inhibit the LRP6 protein. By utilizing various screening methods such as rigid and flexible molecular docking and Lipinski's rule of five, we identified 10 potential compounds. Then, their validity was further tested by molecular dynamics simulation and MMPBSA binding free energy calculations. Eventually, it was concluded that ZINC03954520, ZINC01729523, ZINC03898665, ZINC13152226, ZINC26730911 and ZINC01069082 compounds can be potential drug compounds for inhibiting LRP6 protein. These compounds in complex with β-propeller domains of LRP6 showed that they are capable of altering the backbone of these domains and interfere with their structural dynamics which may lead to the inhibition of the signal transmission. Communicated by Ramaswamy H. Sarma.

Background and purpose: Considering various studies implying anticancer activity of the hydrazone and oxamide derivatives through different mechanisms such as kinases and calpain inhibition, herein, we report the synthesis, characterization, and evaluation of the antiproliferative effect of a series of hydrazones bearing oxamide moiety compounds (7a-7n) against a panel of cancer cell lines to explore a novel and promising anticancer agent (7k). Experimental approach: Chemical structures of the synthesized compounds were confirmed by FTIR, 1H-NMR, 13C-NMR, and mass spectra. The antiproliferative activity and cell cycle progression of the target compound were investigated using the MTT assay and flow cytometry. Findings/Results: Compound 7k with 2-hydroxybenzylidene structure was found to have a significant in vitro anti-proliferative influence on MDA-MB-231 (human adenocarcinoma breast cancer) and 4T1 (mouse mammary tumor) cells as the model of triple-negative breast cancer, with the IC50-72h values of 7.73 ± 1.05 and 1.82 ± 1.14 μM, respectively. Following 72-h incubation with compound 7k, it caused MDA-MB-231 cell death through G1/S cell cycle arrest at high concentrations (12 and 16 μM). Conclusion and implications: Conclusively, this study for the first time reports the anti-proliferative efficacy of compound 7k possessing 2-hydroxyphenyl moiety, which may serve as a potent candidate in triple-negative breast cancer treatment.

Abstract Background Compounds possessing urea/thiourea moiety have a wide range of biological properties including anticancer activity. On the other hand, taking advantage of the low toxicity and structural diversity of hydrazone derivatives, they are presently being considered for designing chemical compounds with hydrazone moiety in the field of cancer treatment. With this in mind, a series of novel ureido/thioureido derivatives possessing a hydrazone moiety bearing nitro and chloro substituents (4a–4i) have been designed, synthesized, characterized and evaluated for their in vitro cytotoxic effect on HT-29 human colon carcinoma and HepG2 hepatocarcinoma cell lines. Results Two compounds (4c and 4e) having the chloro phenylurea group hybridized with phenyl hydrazone bearing nitro or chloro moieties demonstrated potent anticancer effect with the IC50 values between 2.2 and 4.8 µM at 72 h. The mechanism of action of compound 4c was revealed in hepatocellular carcinoma cells as an inducer of apoptosis in a caspase-independent pathway. Conclusion Taken together, the current work presented compound 4c as a potential lead compound in developing future hepatocellular carcinoma chemotherapy drugs. Methods The compounds were synthesized and then characterized by physical and spectral data (FT-IR, 1H-NMR, 13C-NMR, Mass). The anticancer activity was assessed using MTT assay, flowcytometry, annexin-V, DAPI staining and Western blot analysis. Graphical Abstract

There is a growing interest in designing a nanocarrier containing an EGFR targeting affibody to direct toward cancer cells. Here, cytolysin A was cloned at the N-terminus of ZEGFR:1907 affibody to guarantee its surface presentation on the OMVs while targeting the epidermal growth factor receptors (EGFRs). A separate construct including a fusogenic peptide (GALA) was also designed for the endosomal escape of the nanocarrier. Binding of the two constructs ClyA-affiEGFR and ClyA-affiEGFR-GALA to domain III of EGFR was investigated using molecular docking and molecular dynamic simulations. The higher stability of the ClyA-affiEGFR-GALA/EGFR as compared to the ClyA-affiEGFR/EGFR complex was evident. The ClyA-affiEGFR-GALA structure showed a higher RMSD during the first half of the simulation time implying a much less stable behavior. Plateau state of the radius of gyration plot of ClyA-affiEGFR-GALA confirmed a well-folded structure in the presence of the GALA sequence. Solvent accessible surface area for both proteins was in the same range. The data obtained from hydrogen bond analysis revealed a more equilibrated and stable form of the ClyA-affiEGFR-GALA structure upon interaction with EGFR. The data provided here was a requisite for our biological evaluation of the synthesized constructs as a component of a novel drug delivery system.

Introduction: We have reported that thymol and carvacrol can improve cognitive abilities in Alzheimer Disease (AD) rat models. However, the mechanism of their action is not yet fully understood. Recently, our in vitro results suggested that PC12 cell death induced by Aβ25–35 can be protected by thymol and carvacrol via Protein Kinase C (PKC) and Reactive Oxygen Species (ROS) pathways. So, we hypothesize that the mechanisms of thymol and carvacrol in improving the learning impairment in the AD rat model may be related to their effects on PKC. So, the activity of PKC and protein expression levels of PKCα were examined in the hippocampal cells of the AD rat model. Methods: To examine the thymol and carvacrol effects, we performed a behavioral test in AD rat models induced by Aβ25–35 neurotoxicity. To access the underlying mechanism of the protective effects, western blotting was performed with antibodies against PKCα. We also measured the PKC activity assay by Elisa. Histopathological studies were carried out in the hippocampus with Hematoxylin and Eosin (H&E) staining. Results: The escape latency increased in Aβ-received rats compared to the control group, and thymol and carvacrol reversed this deficit. Furthermore, these compounds could enhance the PKC activity and increase the PKCα expression ratio. Moreover, H&E staining showed that Aβ caused shrinkage of the CA1 pyramidal neurons. However, thymol and carvacrol treatments could prevent this effect of Aβ peptides. Conclusion: This study suggests that Amyloid-Beta (Aβ) results in memory decline and histochemical disturbances in the hippocampus. Moreover, these results revealed that thymol and carvacrol could have protective effects on cognition in AD-like models via PKC activation. Highlights Rat’s ability to find the invisible platform in the Morris Water Maze (MWM) was impaired by Amyloid-Beta (Aβ) infusion in the hippocampus, while this effect was reversed by thymol or carvacrol administration. Aβ significantly downregulated the Protein Kinase C (PKC) activity in rats’ hippocampus. Western blot analysis demonstrated that Aβ significantly reduced PKCα protein expression in AD rat model hippocampal cells. The expression ratio of PKCα was upregulated following the injection of thymol and carvacrol in rats. Injection of Aβ in the hippocampus resulted in histochemical disturbances in CA1 pyramidal neurons. Carvacrol and thymol can prevent several histological changes induced by Aβ. Plain Language Summary Alzheimer's disease is one of the most important brain diseases in which the learning and memory are impaired. One of the main causes of Alzheimer’s disease is the presence of amyloid beta plaques in the neurons. Protein kinase C enzyme reduces amyloid production and accumulation in the brain. In the present study, we tested the possible effects of carvacrol and thymol in a rat model of Alzheimer’s disease. Memory impairment was induced in adult rats by intra-cerebral infusion of amyloid β. One week later, the memory-impaired animals were treated with carvacrol and thymol. Finally, we tested their memory in a Morris water maze apparatus. Furthermore, their hippocampus was dissected and PKC activity and the neuronal injury was evaluated. Our findings exhibited that thymol and carvacrol improved rats' memory performance. In addition, thymol and carvacrol significantly increased PKC activity and prevented neuronal cell loss in the rat hippocampus. This study shows that thymol and carvacrol have beneficial effects on memory and cognitive function via PKC activation.