Epstein-Barr virus-encoded BART9 and BART15 miRNAs are elevated in exosomes of cerebrospinal fluid from relapsing-remitting multiple sclerosis patients.

Epstein-Barr virus (EBV) infection is approved as the main environmental trigger of multiple sclerosis (MS). In this path, we quantified ebv-miR-BART9-3p and ebv-miR-BART15 in exosomes of cerebrospinal fluid (CSF) of untreated relapsing-remitting MS (RRMS) patients in comparison with the control group. Interestingly, patients displayed significant upregulation of ebv-miR-BART9-3p (18.4-fold) and ebv-miR-BART15 (3.1-fold) expression in CSF exosomes. Moreover, the expression levels of hsa-miR-21-5p and hsa-miR-146a-5p were found to be significantly elevated in the CSF samples obtained from the patient group compared to those obtained from the HC group. The levels of Interferon-gamma (IFN-γ), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-17 (IL-17), interleukin-23 (IL-23), transforming growth factor beta (TGF-ß), and tumor necrosis factor-alpha (TNF-α) were observed to be significantly elevated in the serum and CSF exosomes of the patients. The highest increase was observed in TGF-ß (8.5-fold), followed by IL-23 (3.9-fold) in CSF exosomes. These findings are in agreement with the association between EBV infection and inflammatory cytokines induction. Furthermore, the ratios of TGF-ß: TNF-α and TGF-ß: IFN-γ attained values of 4 to 16.4 and 1.3 to 3.6, respectively, in the CSF exosomes of the patients, in comparison to those of the control group. These findings show EBV activity in RRMS patients is different from that of healthy ones. Elevation of ebv-miR-BART9-3p, ebv-miR-BART15, and inflammatory cytokines expression in CSF exosomes in RRMS patients provides a substantial link between EBV activity and the onset of the disease, as well as the transition from EBV infection to MS.

Stimulators of immunogenic cell death for cancer therapy: focusing on natural compounds.

A growing body of evidence indicates that the anticancer effect of the immune system can be activated by the immunogenic modulation of dying cancer cells. Cancer cell death, as a result of the activation of an immunomodulatory response, is called immunogenic cell death (ICD). This regulated cell death occurs because of increased immunogenicity of cancer cells undergoing ICD. ICD plays a crucial role in stimulating immune system activity in cancer therapy. ICD can therefore be an innovative route to improve anticancer immune responses associated with releasing damage-associated molecular patterns (DAMPs). Several conventional and chemotherapeutics, as well as preclinically investigated compounds from natural sources, possess immunostimulatory properties by ICD induction. Natural compounds have gained much interest in cancer therapy owing to their low toxicity, low cost, and inhibiting cancer cells by interfering with different mechanisms, which are critical in cancer progression. Therefore, identifying natural compounds with ICD-inducing potency presents agents with promising potential in cancer immunotherapy. Naturally derived compounds are believed to act as immunoadjuvants because they elicit cancer stress responses and DAMPs. Acute exposure to DAMP molecules can activate antigen-presenting cells (APCs), such as dendritic cells (DCs), which leads to downstream events by cytotoxic T lymphocytes (CTLs) and natural killer cells (NKs). Natural compounds as inducers of ICD may be an interesting approach to ICD induction; however, parameters that determine whether a compound can be used as an ICD inducer should be elucidated. Here, we aimed to discuss the impact of multiple ICD inducers, mainly focusing on natural agents, including plant-derived, marine molecules, and bacterial-based compounds, on the release of DAMP molecules and the activation of the corresponding signaling cascades triggering immune responses. In addition, the potential of synthetic agents for triggering ICD is also discussed.

Copper (II) complexes with N, S donor pyrazole-based ligands as anticancer agents.

A group of bidentate nitrogen and sulfur donor pyrazole derivative ligands abbreviated as Na[RNCS(Pz)], Na[RNCS(PzMe2)], Na[RNCS(PzMe3)], Na[RNCS(PzPhMe)], Na[RNCS(PzPh2)], where (R = Et, Ph), and their Cu (II) complexes were synthesized and characterized by spectroscopic and physicochemical methods. The crystal structure of [Cu(PhNCSPzMe3)2] was determined by X-ray crystallography analysis and the results described a distorted square planar coordination geometry for this complex. Also, the cyclic voltammetry investigations indicated that the synthesized copper complex is an electrochemically active species. Moreover, the cytotoxic activity of all of the twenty synthesized compounds was evaluated using MTT assay against the MCF-7 (human breast carcinoma) cell lines, in vitro. Cu (II) complexes indicate significant cytotoxicity against the MCF-7 cell lines as compared with the free ligands. The docking studies showed that the copper complexes have better interactions with EGFR and CDK2 proteins, compared to the free ligands, and most of the studied compounds have a higher value of binding energy relative to the studied controls. The results of QSAR analysis suggest that dipole moment is in direct correlation with the obtained IC50 values, and it strongly impact the anticancer effects generated by the compounds. Our findings suggest that the developed copper complexes can be good candidates for further evaluations as chemotherapeutic agents in the treatment of cancer.

Hsp70 in cancer: A double agent in the battle between survival and death.

The heat shock protein (Hsps) superfamily, also known as molecular chaperones, are highly conserved and present in all living organisms and play vital roles in protein fate. The HspA1A (Hsp70-1), called Hsp70 in this review, is expressed at low or undetectable levels in most unstressed normal cells, but numerous studies have shown that diverse types of tumor cells express Hsp70 at the plasma membrane that leads to resistance to programmed cell death and tumor progression. Hsp70 is released into the extracellular milieu in three forms including free soluble, complexed with cancer antigenic peptides, and exosome forms. Therefore, it seems to be a promising therapeutic target in human malignancies. However, a great number of studies have indicated that both intracellular and extracellular Hsp70 have a dual function. A line of evidence presented that intracellular Hsp70 has a cytoprotective function via suppression of apoptosis and lysosomal cell death (LCD) as well as that extracellular Hsp70 can promote tumorigenesis and angiogenesis. Other evidence showed intracellular Hsp70 can promote apoptosis and membrane-associated/extracellular Hsp70 can elicit antitumor innate and adaptive immune responses. Given the contradictory functions, as a "double agent," could Hsp70 be a promising tool in the future of targeted cancer therapies? To answer this question, in this review, we will discuss the functions of Hsp70 in cancers besides inhibition and stimulation strategies for targeting Hsp70 along with their challenges.

Clinical application of immune checkpoints in targeted immunotherapy of prostate cancer.

Immunotherapy is considered as an effective method for cancer treatment owing to the induction of specific and long-lasting anti-cancer effects. Immunotherapeutic strategies have shown significant success in human malignancies, particularly in prostate cancer (PCa), a major global health issue regarding its high metastatic rates. In fact, the first cancer vaccine approved by FDA was Provenge, which has been successfully used for treatment of PCa. Despite the remarkable success of cancer immunotherapy in PCa, many of the developed immunotherapy methods show poor therapeutic outcomes. Immunosuppression in tumor microenvironment (TME) induced by non-functional T cells (CD4+ and CD8+), tolerogenic dendritic cells (DCs), and regulatory T cells, has been reported to be the main obstacle to the effectiveness of anti-tumor immune responses induced by an immunotherapy method. The present review particularly focuses on the latest findings of the immune checkpoints (ICPs), including CTLA-4, PD-1, PD-L1, LAG-3, OX40, B7-H3, 4-1BB, VISTA, TIM-3, and ICOS; these checkpoints are able to have immune modulatory effects on the TME of PCa. This paper further discusses different approaches in ICPs targeting therapy and summarizes the latest advances in the clinical application of ICP-targeted therapy as monotherapy or in combination with other cancer therapy modalities in PCa.

Isolation and characterization of a novel scFv antibody fragments specific for Hsp70 as a tumor biomarker.

Many studies have shown that more than 50% of tumors express heat shock protein 70 kDa (Hsp70) at the plasma membrane surface while not seen in normal cells, therefore it is a promising therapeutic target in human cancers. Hence, we used phage display technology to produce a single-chain fragment variable (scFv) antibody against human Hsp70. For this, a target peptide from human Hsp70 was designed using bioinformatics studies and was chemically synthesized. Then, the selection was performed using four rounds of biopanning with a stepwise decreased amount of the target peptide. Fourteen positive scFv clones were selected using monoclonal phage enzyme-linked immunosorbent assay screening, which was further characterized by means of the polymerase chain reaction and DNA sequencing. Among them, the G6 clone was selected to express scFv into the Escherichia coli. Expression and purification of the scFv shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by Western blot analysis. In silico analysis confirmed specific binding of the scFv to Hsp70 in CDR regions. The specificity of the scFv measured by surface plasmon resonance and immunofluorescence of the A549 human lung carcinoma cell line confirmed the in vitro function of the scFv. Based upon these findings, we propose a novel anti-human Hsp70 scFv as potential immunotherapy agents that may be translated into preclinical/clinical applications.

Evaluation of the Physicochemical and Biological Stability of Cetuximab under Various Stress Condition.

Cetuximab is a chimeric monoclonal antibody against epidermal growth factor receptor (EGFR) and it is approved for treatment of human colorectal cancer and squamous cell carcinoma of head and neck. The aim of this research was to study the stability of cetuximab finish product (5 mg/mL) under various stress conditions including mechanical, thermal, light stress, and various freeze-thaw cycles. To determine the effects of environmental stresses on the physicochemical properties and bioactivity of cetuximab, a combination of physicochemical and cell-based biological methods including size exclusion chromatography (SEC), cation exchange chromatography (CEX), flow cytometry-based binding assay, and MTS cell viability/proliferation assay was used. The results obtained by the SEC and CEX methods revealed that incubation of cetuximab at 25 and 30 °C, shaking, and various freeze-thaw cycles caused no physicochemical instability. However, functional analysis of the samples exposed to the above-mentioned conditions revealed a significant decrease in the bioactivity of cetuximab indicated by a significant reduction in the cell binding and growth inhibitory effects of cetuximab in EGFR overexpressing cancer cell line (A431). Incubation of cetuximab at 40 and 50 °C led to polymerization and fragmentation of the mAb and resulted in a significant decrease in the bioactivity of the mAb. Our findings show that the light exposure had the most destructive effects on physicochemical and biological characteristics of cetuximab. In conclusion, we found that all mentioned stress conditions significantly affect the bioactivity of cetuximab. Our finding highlights the importance of bioactivity evaluation of biopharmaceuticals in their quality control assessment.

CDK9 Regulates Apoptosis of Myoblast Cells by Modulation of microRNA-1 Expression.

Cdk9 is the catalytic core of the positive transcription elongation factor b (P-TEFb) and regulates transcriptional elongation factors by phosphorylation of RNA pol II. Apart from its role on myogenic gene expression, Cdk9 regulation of muscle-specific microRNAs in the early stage of cardiomyogenesis is poorly understood. Here we demonstrate that Cdk9 not only regulates myogenic transcription factors, but also controls muscle-specific microRNAs. During cardiac differentiation of mouse embryonic stem cells, high Cdk9 expression preceded up-regulation of miR-1. To investigate potential regulatory roles of Cdk9 on cardiac microRNAs and myogenesis genes, we overexpressed Cdk9 in myoblast C2C12 cells, which resulted in significant induction of miR-1 and miR-206, while miR-133 was downregulated. Moreover, expression levels of MyoD and Srf, key regulators of myogenesis, also increased in cells with overexpression of Cdk9. We further observed Cdk9-mediated apoptosis in C2C12 cells corresponding to induction of miR-1 expression levels. Thus, Cdk9 plays a complex role in myocyte progenitor differentiation and apoptosis by regulating myogenic protein and muscle-specific microRNA expression. J. Cell. Biochem. 119: 547-554, 2018. © 2017 Wiley Periodicals, Inc.

Functionalized Caprolactone-Polyethylene Glycol Based Thermo-Responsive Hydrogels of Silibinin for the Treatment of Malignant Melanoma.

PURPOSE: Silibinin, is a natural compound, which has shown anticancer activity in various malignancies. In this study, we evaluated the anticancer effects of silibinin in B16-F10 melanoma cells and developed a novel thermoresponsive hydrogel for local delivery of this compound. METHOD: A thermoresponsive hydrogel loaded with silibinin was prepared using triblock copolymers of poly[(α-benzyl carboxylate-e-caprolactone)-co-(α-carboxyl-e-caprolactone)]ran-b-PEG-b-[(α-benzyl carboxylate-e-caprolactone) -co-(α-carboxyl-e-caprolactone)]ran (PCBCL-b-PEG-b-PCBCL), namely PolyGelTM, and compared with a Pluronic F-127 formulation of silibinin. Sol-gel transition temperature of hydrogels was measured by inverse flow method and modulated differential scanning calorimetry (MDSC). Silibinin loading efficiency was measured by HPLC. The MTT and clonogenic assays were used to assess the cytotoxicity and anti-proliferative effects of silibinin on B16-F10 melanoma cells. Flow cytotmetry was used to quantify the induced level of apoptosis and measure the intracellular level of activated STAT3 (pSTAT3) following silibinin treatment in B16.F10 cells. The effects of silibinin on the activation of oncogenic proteins were also evaluated by western blot. RESULTS: Silibinin inhibited cell proliferation (IC50 = 67 µM), provoked cell cycle arrest, induced apoptosis, suppressed key oncogenic pathways (i.e STAT3 and MEK/ERK), and enhanced the cytotoxic effects of doxorubicin in B16-F10 cells. Both PolyGelTM and Pluronic F-127 hydrogels were effective in loading silibinin. A lower drug release pattern within 24h, fitting first- order release kinetics, was observed for the release of silibinin from both gels compared to free drug.  PolyGelTM demonstrated enhanced percutaneous absorption of silibinin through increasing mouse skin intracellular lipid fluidity as documented by DSC of skin following PolyGelTM use. Silibinin loaded in PolyGel TM inhibited the growth of B16-F10 cells (IC50 = 30 µM) and effectively suppressed pSTAT3 activity in B16-F10 cells at 10 µM. CONCLUSION: Our results imply a great potential for PolyGel TM formulations of silibinin for local treatment of malignant melanoma. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's content page.

STAT1 is phosphorylated and downregulated by the oncogenic tyrosine kinase NPM-ALK in ALK-positive anaplastic large-cell lymphoma.

The tumorigenicity of most cases of ALK-positive anaplastic large-cell lymphoma (ALK+ ALCL) is driven by the oncogenic fusion protein NPM-ALK in a STAT3-dependent manner. Because it has been shown that STAT3 can be inhibited by STAT1 in some experimental models, we hypothesized that the STAT1 signaling pathway is defective in ALK+ ALCL, thereby leaving the STAT3 signaling unchecked. Compared with normal T cells, ALK+ ALCL tumors consistently expressed a low level of STAT1. Inhibition of the ubiquitin-proteasome pathway appreciably increased STAT1 expression in ALK+ ALCL cells. Furthermore, we found evidence that NPM-ALK binds to and phosphorylates STAT1, thereby promoting its proteasomal degradation in a STAT3-dependent manner. If restored, STAT1 is functionally intact in ALK+ ALCL cells, because it effectively upregulated interferon-γ, induced apoptosis/cell-cycle arrest, potentiated the inhibitory effects of doxorubicin, and suppressed tumor growth in vivo. STAT1 interfered with the STAT3 signaling by decreasing STAT3 transcriptional activity/DNA binding and its homodimerization. The importance of the STAT1/STAT3 functional interaction was further highlighted by the observation that short interfering RNA knockdown of STAT1 significantly decreased apoptosis induced by STAT3 inhibition. Thus, STAT1 is a tumor suppressor in ALK+ ALCL. Phosphorylation and downregulation of STAT1 by NPM-ALK represent other mechanisms by which this oncogenic tyrosine kinase promotes tumorigenesis.

Self-Associating Poly(ethylene oxide)-block-poly(α-carboxyl-ε-caprolactone) Drug Conjugates for the Delivery of STAT3 Inhibitor JSI-124: Potential Application in Cancer Immunotherapy.

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) in tumor cells and tumor associated dendritic cells (DCs) plays a major role in the progression of cancer. JSI-124 (cucurbitacin I) is a potent inhibitor of STAT3; however, its poor solubility and nonspecificity limit its effectiveness in cancer immunotherapy. In order to achieve a nanocarrier for solubilization and passive targeting of JSI-124 to tumor cells and tumor associated DCs, the drug was chemically conjugated to pendent COOH groups of self-associating poly(ethylene oxide)-block-poly(α-carboxylate-ε-caprolactone) (PEO-b-PCCL). Developed PEO-b-P(CL-JSI-124) conjugates self-assembled to polymeric micelles of 40 nm size range with negligible drug release under physiological mimicking conditions. The conjugation of JSI-124 to PEO-b-PCCL was confirmed by 1H NMR, thin layer chromatography (TLC), and HPLC with a conjugation of 8.9% w/w of the polymer. As expected, JSI-124 nanoconjugates showed lower potency in p-STAT3 inhibition and direct anticancer activity in B16-F10 melanoma cells. Interestingly, JSI-124 nanoconjugates were more powerful than free drug in reducing the level of p-STAT3 in tumor exposed bone marrow derived dendritic cells (BMDCs). The JSI-124 nanoconjugates were also significantly more active than free drug in reversing the immunosuppressive effect of B16-F10 tumor and led to significantly better phenotypical and functional stimulation of tumor exposed immature BMDCs in the presence of immune adjuvants like LPS and CpG. Our findings points to great promise for PEO-b-P(CL-JSI-124) micelles for modulation of immunosuppressive microenvironment in melanoma tumors, implicating application of this strategy in cancer immunotherapy.

Silibinin sensitizes chemo-resistant breast cancer cells to chemotherapy.

CONTEXT: Multiple drug resistance is the major obstacle to conventional chemotherapy. Silibinin, a nontoxic naturally occurring compound, has anticancer activity and can increase the cytotoxic effects of chemotherapy in various cancer models. OBJECTIVE: To evaluate the effects of silibinin on enhancing the sensitivity of chemo-resistant human breast cell lines to doxorubicin (DOX) and paclitaxel (PAC). MATERIALS AND METHODS: The cells were treated with silibinin (at 50 to 600 µM concentrations) and/or chemo drugs for 24 and 48 h, then cell viability and changes in oncogenic proteins were determined by MTT assay and Western blotting/RT-PCR, respectively. Flow cytometry was used to study apoptosis in the cells receiving different treatments. The antitumorigenic effects of silibinin (at 200 to 400 µM concentration) were evaluated by mammosphere assay. RESULTS: Silibinin exerted significant growth inhibitory effects with IC50 ranging from 200 to 570 µM in different cell lines. Treatment of DOX-resistant MDA-MB-435 cells with silibinin at 200 µM reduced DOX IC50 from 71 to 10 µg/mL and significantly suppressed the key oncogenic pathways including STAT3, AKT, and ERK in these cells. Interestingly treatment of DOX-resistant MDA-MB-435 cells with silibinin at 400 µM concentration for 48 h induced a 50% decrease in the numbers of colonies as compared with DMSO-treated cells. Treatment of PAC-resistant MCF-7 cells with silibinin at 400 µM concentration generated synergistic effects when it was used in combination with PAC at 250 nM concentration (CI = 0.81). CONCLUSION: Silibinin sensitizes chemo-resistant cells to chemotherapeutic agents and can be useful in treating breast cancers.

The clinical and biological significance of STAT1 in esophageal squamous cell carcinoma.

BACKGROUND: Loss of STAT1 (Signal Transducer and Activator of Transcription-1) has been implicated in the pathobiology of a number of cancer types. Nonetheless, the biological and clinical significance of STAT1 in esophageal squamous cell carcinomas (ESCC) has not been comprehensively studied. METHODS: Using immunohistochemistry, we detected the STAT1 expression in a cohort of ESCC patients; In-vitro experiments, we used enforced gene transfection of STAT1C into two STAT1- weak/negative ESCC cell lines and siRNA knockdown of STAT1 in two STAT1-strong ESCC cell lines to detect STAT1 function in ESCC. RESULTS: We found that the expression of STAT1 was heterogeneous in ESCC, with 64 (49.0%) strongly positive cases, 59 (45.0%) weakly positive cases and 8 (6.1%) negative cases. STAT1 expression inversely correlated with the depth of tumor invasion and tumor size (p=0.047 and p=0.029, respectively, Chi square). Furthermore, patients with STAT1-strong/weak tumors had a significantly longer survival compared to those with STAT1-negative tumors (33.6 months versus 13.1 months, p=0.019). In patients carrying tumors of aggressive cytology (n=50), those with STAT1-strong tumors survived significantly longer than those with STAT1-weak/negative tumors (34.6 months versus 20.5 months, p=0.011). Our in-vitro experiments revealed that STAT1 is proapoptotic and inhibitory to cell-cycle progression and colony formation. Lastly, we found evidence that STAT1 signaling in ESCC cells down-regulated the expression and/or activity of NF-κB and STAT3, both of which are known to have oncogenic potential. CONCLUSION: To conclude, our findings suggest that STAT1 is a tumor suppressor in ESCC. Loss of STAT1, which is frequent in ESCC, contributes to the pathogenesis of these tumors.

Mitochondrial delivery of doxorubicin via triphenylphosphine modification for overcoming drug resistance in MDA-MB-435/DOX cells.

In this study, doxorubicin (DOX) was conjugated to a lipophilic triphenylphosphonium (TPP) that is selectively taken up by the mitochondrial membrane of cells. This new derivative of DOX, i.e., TPP-DOX, was characterized by infrared spectroscopy (IR), nuclear magnetic resonance ((1)H NMR, (13)C NMR), and mass spectrometry. The effect of TPP modification on DOX cell uptake, intracellular trafficking, eventual DOX induced cytotoxicity, and the level of cleaved caspase 3 and PARP in wild type MDA-MB-435/WT and DOX resistant MDA-MB-435/DOX cells was then evaluated and compared to that for free DOX. In general, free DOX cellular uptake appeared to be significantly higher in MDA-MB-435/WT than MDA-MB-435/DOX cells. Moreover, free DOX was able to enter the nucleus of MDA-MB-435/WT cells, but in MDA-MB-435/DOX cells, it was confined within the cytoplasm. The TPP-DOX, on the other hand, was localized in the cytoplasm of both cell phenotypes and showed preferential distribution to the mitochondria. Correspondingly, in MDA-MB-435/DOX cells, an enhanced cytotoxicity was observed for TPP-DOX (IC50 of 33.6 and 21.0 µM at 48 and 72 h incubation, respectively) in comparison to free DOX (IC50 of 126.7 and 77.96 µM at 48 and 72 h incubation, respectively). This observation was accompanied by the increased level of cleaved caspase 3 and PARP indicating enhanced apoptosis in both cell lines, particularly that of MDA-MB-435/DOX, for TPP-DOX compared to free DOX following 24 h treatment. The present study highlights promising application of TPP-DOX in reversing drug resistance in tumor cells.

Nano-delivery of Silibinin Potentiate the Induction of Immunogenic Cell Death (ICD) in Melanoma Cells.

BACKGROUND: Induction of immunogenic cell death (ICD) in tumors can enhance antitumor immunity and modulate immunosuppression in the tumor microenvironment (TME). OBJECTIVE: In the current study, we investigated the effect of silibinin, a natural compound with anticancer activity, and its polymer-based nanoformulations on the induction of apoptosis and ICD in cancer cells. METHODS: Free and nanoparticulate silibinin were evaluated for their growth-inhibitory effects using an MTT assay. Annexin V/PI staining was used to analyze apoptosis. Calreticulin (CRT) expression was measured by flow cytometry. Western blotting was conducted to examine the levels of elf2α, which plays a role in the ICD pathway. The HSP90 and ATP levels were determined using specific detection kits. RESULTS: Compared to the free drug, silibinin-loaded nanocarriers significantly increased the induction of apoptosis and ICD in B16F10 cells. ICD induction was characterized by significantly increased levels of ICD biomarkers, including CRT, HSP90, and ATP. We also observed an increased expression of p-elf-2α/ elf-2α in B16F10 cells treated with silibinin-loaded micelles compared to cells that received free silibinin. CONCLUSION: Our findings showed that the encapsulation of silibinin in polymeric nanocarriers can potentiate the effects of this drug on the induction of apoptosis and ICD in B16F10 melanoma cells.

Exosome Content-Mediated Signaling Pathways in Multiple Sclerosis.

Exosomes are small extracellular vesicles with a complex lipid-bilayer surface and 30-150 nm diameter. These vesicles play a critical role in intercellular signaling networks during physiopathological processes through data trafficking and cell reprogramming. It has been demonstrated that exosomes are involved in a variety of central nervous system (CNS) disorders such as multiple sclerosis (MS). Exosome mediators' cell-to-cell communication is possibly by delivering their contents such as proteins, RNAs (coding and non-coding), DNAs (mitochondrial and genomic), and transposable elements to the target cells. Exosomal microRNAs (miRNAs) differ in their expression patterns in MS disease, thereby providing novel diagnostic and prognostic biomarkers and therapeutic options for better treatment of MS disease. Furthermore, these microvesicles are non-immunogenic and non-toxic therapeutic tools for transferring miRNAs across the blood-brain barrier (BBB). Collectively, exosomes could be used as novel drug delivery devices for the treatment of MS patients. This review summarized research regarding the exosomes from serum, plasma, PBMC, and other cells in MS patients and experimental models. We also provide a critical view of exosome content-mediated signaling pathways in MS, including TNF-α, TGF-ß, NF-κB, and Wnt pathways. The use of exosomes as a therapeutic potential in MS has also been discussed.

Gene methylation and silencing of SOCS3 in mantle cell lymphoma.

The significance of loss of SOCS3, a negative regulator of signalling pathways including those of STAT3 and NF-κB, was examined in mantle cell lymphoma (MCL). The protein expression and gene methylation status of SOCS3 were detected using immunohistochemistry/Western blots and methylation-specific polymerase chain reaction, respectively. To evaluate its functional importance, SOCS3 was restored in two SOCS3-negative MCL cell lines using a lentiviral vector. Loss of SOCS3 protein expression was found in 3/4 MCL cell lines and 18/33 (54.5%) tumours. SOCS3 was found consistently methylated in cell lines (3/4) and tumours (7/7) negative for SOCS3, and was unmethylated in all SOCS3-positive cell line (1/1) and tumours (5/5) examined. Treatment of all three SOCS3-negative cell lines with 2'-deoxy-5-azacytidine restored SOCS3 expression. SOCS3 is biologically important in MCL, as lentiviral transfer of SOCS3 in SOCS3-negative cell lines increased their apoptotic activity, downregulated nuclear factor (NF)-κB-p65, cyclin D1 (CCND1), BCL2 and BCL-XL (BCL2L1), and substantially dampened interleukin 10-induced STAT3 activation. In 19 patients aged ≤ 69 years at time of diagnosis, we found that those that carried SOCS3-negative tumours showed a trend toward a worse outcome (P = 0.1, log-rank).

Silibinin induces immunogenic cell death in cancer cells and enhances the induced immunogenicity by chemotherapy.

Introduction: Silibinin is a natural flavonoid compound known to induce apoptosis in cancer cells. Despite silibinin's safety and efficacy as an anticancer drug, its effects on inducing immunogenic cell death (ICD) are largely unknown. Herein, we have evaluated the stimulating effects of silibinin on ICD in cancer cells treated with silibinin alone or in combination with chemotherapy. Methods: The anticancer effect of silibinin, alone or in combination with doxorubicin or oxaliplatin (OXP), was assessed using the MTT assay. Compusyn software was used to analyze the combination therapy data. Western blotting was conducted to examine the level of STAT3 activity. Flow cytometry was used to analyze calreticulin (CRT) and apoptosis. The heat shock protein (HSP70), high mobility group box protein1 (HMGB1), and IL-12 levels were assessed by ELISA. Results: Compared to the negative control groups, silibinin induced ICD in CT26 and B16F10 cells and significantly enhanced the induction of this type of cell death by doxorubicin, and these changes were allied with substantial increases in the level of damage-associated molecular patterns (DAMPs) including CRT, HSP70, and HMGB1. Furthermore, conditioned media from cancer cells exposed to silibinin and doxorubicin was found to stimulate IL-12 secretion in dendritic cells (DCs), suggesting the link of this treatment with the induction of Th1 response. Silibinin did not augment the ICD response induced by OXP. Conclusion: Our findings showed that silibinin can induce ICD and it potentiates the induction of this type of cell death induced by chemotherapy in cancer cells.

Clinical Significance of Carnitine in the Treatment of Cancer: From Traffic to the Regulation.

Metabolic reprogramming is a common hallmark of cancer cells. Cancer cells exhibit metabolic flexibility to maintain high proliferation and survival rates. In other words, adaptation of cellular demand is essential for tumorigenesis, since a diverse supply of nutrients is required to accommodate tumor growth and progression. Diversity of carbon substrates fueling cancer cells indicate metabolic heterogeneity, even in tumors sharing the same clinical diagnosis. In addition to the alteration of glucose and amino acid metabolism in cancer cells, there is evidence that cancer cells can alter lipid metabolism. Some tumors rely on fatty acid oxidation (FAO) as the primary energy source; hence, cancer cells overexpress the enzymes involved in FAO. Carnitine is an essential cofactor in the lipid metabolic pathways. It is crucial in facilitating the transport of long-chain fatty acids into the mitochondria for ß-oxidation. This role and others played by carnitine, especially its antioxidant function in cellular processes, emphasize the fine regulation of carnitine traffic within tissues and subcellular compartments. The biological activity of carnitine is orchestrated by specific membrane transporters that mediate the transfer of carnitine and its derivatives across the cell membrane. The concerted function of carnitine transporters creates a collaborative network that is relevant to metabolic reprogramming in cancer cells. Here, the molecular mechanisms relevant to the role and expression of carnitine transporters are discussed, providing insights into cancer treatment.

Multiplex Analysis of Cerebrospinal Fluid and Serum Exosomes MicroRNAs of Untreated Relapsing Remitting Multiple Sclerosis (RRMS) and Proposing Noninvasive Diagnostic Biomarkers.

Exosomal microRNAs (miRNAs) are emerging diagnostic biomarkers for neurodegenerative diseases. In this study, we aimed to detect relapsing-remitting multiple sclerosis (RRMS)-specific miRNAs in cerebrospinal fluid (CSF) and serum exosomes with diagnostic potential. One ml of CSF and serum sample were collected from each of the 30 untreated RRMS patients and healthy controls (HCs). A panel of 18 miRNAs affecting inflammatory responses was applied, and qRT-PCR was conducted to detect differentially expressed exosomal miRNAs in CSF and serum of RRMS patients. We identified that 17 out of 18 miRNAs displayed different patterns in RRMS patients compared to HCs. Let-7 g-5p, miR-18a-5p, miR-145-5p, and miR-374a-5p with dual pro-inflammatory and anti-inflammatory actions and miR-150-5p and miR-342-3p with anti-inflammatory action were significantly upregulated in both CSF and serum-derived exosomes of RRMS patients compared to corresponding HCs. Additionally, anti-inflammatory miR-132-5p and pro-inflammatory miR-320a-5p were significantly downregulated in both CSF and serum-derived exosomes of RRMS patients compared to HCs. Ten of 18 miRNAs were differentially expressed in CSF and serum exosomes of the patients. Furthermore, miR-15a-5p, miR-19b-3p, and miR-432-5p were upregulated, and miR-17-5p was downregulated only in CSF exosomes. Interestingly, U6 housekeeping gene was differentially expressed in CSF and serum exosomes, in both RRMS and HCs. As the first report describing CSF exosomal miRNAs expression profile compared to that of serum exosomes in untreated RRMS patients, we showed that CSF and serum exosomes are not identical in terms of biological compounds and display different patterns in miRNAs and U6 expression.