Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that are involved in the degradation of various proteins in the extracellular matrix (ECM). Typically, MMPs have a propeptide sequence, a catalytic metalloproteinase domain with catalytic zinc, a hinge region or linker peptide, and a hemopexin domain. MMPs are commonly classified on the basis of their substrates and the organization of their structural domains into collagenases, gelatinases, stromelysins, matrilysins, membrane-type (MT)-MMPs, and other MMPs. MMPs are secreted by many cells including fibroblasts, vascular smooth muscle (VSM), and leukocytes. MMPs are regulated at the level of mRNA expression and by activation of their latent zymogen form. MMPs are often secreted as inactive pro-MMP form which is cleaved to the active form by various proteinases including other MMPs. MMPs cause degradation of ECM proteins such as collagen and elastin, but could influence endothelial cell function as well as VSM cell migration, proliferation, Ca2+ signaling, and contraction. MMPs play a role in tissue remodeling during various physiological processes such as angiogenesis, embryogenesis, morphogenesis, and wound repair, as well as in pathological conditions such as myocardial infarction, fibrotic disorders, osteoarthritis, and cancer. Increases in specific MMPs could play a role in arterial remodeling, aneurysm formation, venous dilation, and lower extremity venous disorders. MMPs also play a major role in leukocyte infiltration and tissue inflammation. MMPs have been detected in cancer, and elevated MMP levels have been associated with tumor progression and invasiveness. MMPs can be regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs), and the MMP/TIMP ratio often determines the extent of ECM protein degradation and tissue remodeling. MMPs have been proposed as biomarkers for numerous pathological conditions and are being examined as potential therapeutic targets in various cardiovascular and musculoskeletal disorders as well as cancer.

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Biochemical and Biological Attributes of Matrix Metalloproteinases.

Exosomes are common membrane-bound nanovesicles that contain diverse biomolecules, such as lipids, proteins, and nucleic acids. Exosomes are derived from cells through exocytosis, are ingested by target cells, and can transfer biological signals between local or distant cells. Exosome secretion is a constitutive phenomenon that is involved in both physiological and pathological processes and determines both the exosomal surface molecules and the contents. Hence, we can exploit exosomes as biomarkers, vaccines and drug carriers and modify them rationally for therapeutic interventions. However, it is still a challenge to identify, isolate and quantify exosomes accurately, efficiently and selectively. Further studies on exosomes will explore their potential in translational medicine and provide new avenues for the creation of effective clinical diagnostics and therapeutic strategies; the use of exosomes in these applications can be called exosome theranostics. This review describes the fundamental processes of exosome formation and uptake. In addition, the physiological and pathological roles of exosomes in biology are also illustrated with a focus on how exosomes can be exploited or engineered as powerful tools in translational medicine.

Exosome Theranostics: Biology and Translational Medicine.

Exosomes have emerged as a novel mode of intercellular communication. Exosomes can shuttle bioactive molecules including proteins, DNA, mRNA, as well as non-coding RNAs from one cell to another, leading to the exchange of genetic information and reprogramming of the recipient cells. Increasing evidence suggests that tumor cells release excessive amount of exosomes, which may influence tumor initiation, growth, progression, metastasis, and drug resistance. In addition, exosomes transfer message from tumor cells to immune cells and stromal cells, contributing to the escape from immune surveillance and the formation of tumor niche. In this review, we highlight the recent advances in the biology of exosomes as cancer communicasomes. We review the multifaceted roles of exosomes, the small secreted particles, in communicating with other cells within tumor microenvironment. Given that exosomes are cell type specific, stable, and accessible from body fluids, exosomes may provide promising biomarkers for cancer diagnosis and represent new targets for cancer therapy.

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Exosomes in cancer: small particle, big player

Hypoxia is a common feature of solid tumors and is associated with aggressiveness and poor patient outcomes. Exosomes, initially considered to be cellular "garbage dumpsters," are now implicated in mediating interactions with the cellular environment. However, the mechanisms underlying the association between exosomes and hypoxia during cancer progression remain poorly understood. In this study, we found that exosomes derived from hypoxic oral squamous cell carcinoma (OSCC) cells increased the migration and invasion of OSCC cells in a HIF-1α and HIF-2α-dependent manner. Given that exosomes have been shown to transport miRNAs to alter cellular functions, we performed miRNA sequencing of normoxic and hypoxic OSCC-derived exosomes. Of the 108 miRNAs that were differentially expressed, miR-21 stood out as one of the most significantly upregulated miRNAs under hypoxic conditions. miR-21 depletion in hypoxic OSCC cells led to decreased miR-21 levels in exosomes and significantly reduced cell migration and invasion. Conversely, restoration of miR-21 expression in HIF-1α and HIF-2α-depleted exosomes rescued OSCC cell migration and invasion. Moreover, exosomal miR-21 markedly enhanced snail and vimentin expression, while significantly decreasing E-cadherin levels in OSCC cells, in vitro and in vivo Finally, circulating exosomal miR-21 levels were closely associated with HIF-1α/HIF-2α expression, T stage, and lymph node metastasis in patients with OSCC. In conclusion, our findings suggest that the hypoxic microenvironment may stimulate tumor cells to generate miR-21-rich exosomes that are delivered to normoxic cells to promote prometastatic behaviors and prompt further investigation into the therapeutic value of exosome inhibition for cancer treatment. Cancer Res; 76(7); 1770-80. ©2016 AACR.

https://cancerres.aacrjournals.org/content/canres/76/7/1770.full.pdf

Exosomes Derived from Hypoxic Oral Squamous Cell Carcinoma Cells Deliver miR-21 to Normoxic Cells to Elicit a Prometastatic Phenotype.

Exosomes, which are nanosized endocytic vesicles that are secreted by most cells, contain an abundant cargo of different RNA species that can modulate the behavior of recipient cells and may be used as circulating biomarkers for diseases. Here, we develop a web-accessible database (http://www.exoRBase.org), exoRBase, which is a repository of circular RNA (circRNA), long non-coding RNA (lncRNA) and messenger RNA (mRNA) derived from RNA-seq data analyses of human blood exosomes. Experimental validations from the published literature are also included. exoRBase features the integration and visualization of RNA expression profiles based on normalized RNA-seq data spanning both normal individuals and patients with different diseases. exoRBase aims to collect and characterize all long RNA species in human blood exosomes. The first release of exoRBase contains 58 330 circRNAs, 15 501 lncRNAs and 18 333 mRNAs. The annotation, expression level and possible original tissues are provided. exoRBase will aid researchers in identifying molecular signatures in blood exosomes and will trigger new exosomal biomarker discovery and functional implication for human diseases.

exoRBase: a database of circRNA, lncRNA and mRNA in human blood exosomes.

Serum exosomes containing noncoding RNA (ncRNA) play an important role in both physiological and pathological conditions. However, biological function of exosomal ncRNA remains unclear. The aim of the study was to investigate the prognostic and diagnostic values of exosomal ncRNA by comparing the amounts of exosomal miR-21 and HOTAIR in serum of laryngeal squamous cell carcinoma (LSCC) patients with those of polyps of vocal cords, and by determinating whether combined detection of the two molecules could provide useful information in the diagnosis of LSCC. Exosomes were isolated from the serum samples of 52 LSCC patients and those of 49 patients with polyps of vocal cords. TEM and Western blot were applied for the confirmation of isolated exosomes by observing the ultra structure and testing CD63 marker protein, respectively. RT-PCR was performed to detect the expression of miR-21 and HOTAIR in the exosomes. The receiver-operating characteristic (ROC) curve was generated to examine the prognostic value of the two molecules. The expression of exosomal miR-21 and HOTAIR was significantly higher in patients with LSCC than those with vocal cord polyps. There were significant differences of serum exosomal miR-21 and HOTAIR expressions between the advanced T classifications (T3/T4) or clinical stages (III/IV) and the early stages. The patients with lymph node metastasis had higher serum exosomal miR-21 and HOTAIR expressions than those without. There were no differences between patient sex, tumor locations and differentiations. The area under the ROC curve of combined examination of exosomal HOTAIR and miR-21 for diagnosing LSCC was 87.6 %, which was significantly higher than 80.1 % of miR-21 (p = 0.0359) or 72.7 % of HOTAIR (p = 0.0012), showing 94.2 and 73.5 % of sensitivity and specificity, respectively, in differentiating the malignant from benign laryngeal disease. Serum exosomal miR-21 and HOTAIR were significantly correlated with clinical parameters of LSCC, and combined evaluation of their serum expressions may be a valuable biomarker to screen LSCC and might be a promising predicting tool for LSCC patient.

Combined detection of serum exosomal miR-21 and HOTAIR as diagnostic and prognostic biomarkers for laryngeal squamous cell carcinoma

Biosynthesis of Zinc oxide nanoparticles (ZnONPs) from natural plants stands as a promising nanodrug delivery system in cancer therapeutics. Marsdenia tenacissima (M.t), a Chinese medicinal plant has been extensively used as clinical remedy for treating several types of cancer. In this present study, ZnONPs were synthesized from Marsdenia tenacissima and its anti cancer potency was assessed against in vitro laryngeal cancer cell line Hep-2. The biosynthesized Marsdenia tenacissima Zinc Oxide Nanoparticles [M.t-ZnONPs] was characterized using UV-visible Spec, SEM, TEM and EDAX analysis. The cytotoxic and apoptotic inducing potential of M.t-ZnONPs was assessed by MTT assay and staining such as DCFH-DA, AO/EtBr, Rhodamine 123, DAPI and comet assay. The anticancer potential of M.t-ZnONPs was analysed by Real time PCR analysis of proapoptotic, antiapoptotic and caspases proteins. Our present findings showed characteristic and morphological representation of synthesized M.t-ZnONPs by UV-visible Spec, SEM, TEM and EDAX analysis. M.t-ZnONPs exhibits its cytotoxicity by inhibiting the viability of Hep-2 cells and IC50 value was obtained by MTT assay. The results of apoptotic staining techniques in M.t-ZnONPs treated Hep-2 cells confirm with excess ROS generation, disruption of mitochondrial membrane potential and nuclear damage. The apoptotic inducing potential of M.t-ZnONPs was also evidenced by upregulation of proapoptotic proteins Bax, Caspase 3 & 9 and downregultion of antiapoptotic protein Bcl-2 by RT-PCR analysis. Finally, these results suggested that biosynthesized M.t-ZnONPs is an effective anticancer agent which induces apoptosis in Hep-2 laryngeal cell line and thus conclude that M.t-ZnONPs, a valid anticancer strategy in treating various cancer.

Synthesis of Zinc oxide nanoparticles from Marsdenia tenacissima inhibits the cell proliferation and induces apoptosis in laryngeal cancer cells (Hep-2).

miRNAs regulate gene expression and are key mediators of tumourigenesis. miR-129 has diverse effects in tumours, but its role in laryngeal squamous cell carcinoma (LSCC) remains unknown. This article focuses on the role of miR-129-5p in LSCC. We show miR-129-5p is upregulated in primary LSCC tumours and correlated with advanced disease. Down-regulating miR-129-5p suppressed cell proliferation and migration, and caused cell cycle arrest in Hep-2 cell lines. Downregulation of miR-129-5p alone is sufficient to induce apoptosis both in vivo and in vitro. Moreover, the growth of LSCC xenograft exposed to miR-129-5p antisense oligonucleotides (ASO) in BALB/c mice was markedly inhibited. In addition, we found that miR-129-5p targeted adenomatous polyposis coli (APC) to release inhibition of Wnt signalling causing cell growth and tumourigenesis. Our results suggest miR-129-5p functions as an oncogene in LSCC by repressing APC and is a potential therapeutic target for LSCC.

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Down-Regulation of miR-129-5p Inhibits Growth and Induces Apoptosis in Laryngeal Squamous Cell Carcinoma by Targeting APC

Is E-cadherin immunoexpression a prognostic factor for head and neck squamous cell carcinoma (HNSCC)? A systematic review and meta-analysis

MicroRNAs were reported to be involved in the modulation of tumor development. The aim of our study was to investigate the effect of miR-205 on proliferation and apoptosis of laryngeal squamous cell carcinoma (LSCC) and seek associations between miR-205 and Bcl-2 using in vitro and in vivo methods. Real-time qPCR was used to analyze the expression of miR-205 in LSCC samples and Hep-2 cell line. Apoptosis, cell cycle, and proliferation (MTT) assays were performed to test the apoptosis and proliferation of LSCC cells after miR-205 transfection. Bcl-2 expression in cells was assessed with Western blotting. The tumorigenicity of LSCC cells was evaluated in nude mice model. MiR-205 was significantly down-regulated in LSCC tissues compared to adjacent normal tissues. Lower expression of miR-205 was indicated to be statistically related with advanced clinical stage and T3–4 grades. We found that restoration of miR-205 down-regulated the proliferative markers of dihydrofolate reductase and proliferating cell nuclear antigen and apoptotic regulator of Bcl-2. The findings in vitro and in vivo showed miR-205 could suppress cell proliferation and induce cell apoptosis. In addition, Bcl-2 was identified as one of the direct targets of miR-205 in LSCC cells. These results suggest that miR-205 may play as a tumor suppressor in LSCC, probably by targeting Bcl-2 and serve as a potential target for therapeutic intervention.

Jianguang Lu

Papers

Combined detection of serum exosomal miR-21 and HOTAIR as diagnostic and prognostic biomarkers for laryngeal squamous cell carcinoma

Synthesis of Zinc oxide nanoparticles from Marsdenia tenacissima inhibits the cell proliferation and induces apoptosis in laryngeal cancer cells (Hep-2).

Down-Regulation of miR-129-5p Inhibits Growth and Induces Apoptosis in Laryngeal Squamous Cell Carcinoma by Targeting APC

Is E-cadherin immunoexpression a prognostic factor for head and neck squamous cell carcinoma (HNSCC)? A systematic review and meta-analysis

MicroRNA-205 suppresses proliferation and promotes apoptosis in laryngeal squamous cell carcinoma.