Stem cell-derived exosomes for chronic wound repair.

Stem cells possess the capability of self-renewal and multipotency, which endows them with great application potential in wound repair fields. Yet, several problems including immune concerns, ethical debates, and oncogenicity impede the broad and deep advance of stem cell-based products. Recently, owing to their abundant resources, excellent biocompatibility, and ease of being engineered, stem cell-derived exosomes were proved to be promising nanomedicine for curing chronic wounds. What is more, stem cell-derived exosomes are almost the mini record of their maternal cells, which even equipped them with the unique characteristics of stem cells. Chronic wound healing efficacy is dominated by several complicated factors, especially the excessive inflammation conditions and impaired vessels. Therefore, this review tries to concentrate on the current advances of stem cell-derived exosomes for reducing inflammation and promoting angiogenesis in chronic wound healing processes. Last but not least, the existing limitations and future perspectives of stem cell-derived exosomes for chronic wound treatment are also outlined.

A Feed-Forward Regulatory Loop between HuR and the Long Noncoding RNA HOTAIR Promotes Head and Neck Squamous Cell Carcinoma Progression and Metastasis.

BACKGROUND/AIMS: The lncRNA Homeobox (HOX) transcript antisense RNA (HOTAIR) is overexpressed in numerous cancers. HuR is also overexpressed during tumourigenesis and is abnormally present within the cytoplasm, where it binds to AU-rich elements in the 3'UTRs of target mRNA and post-transcriptionally regulates the expression of its target genes. However, whether HOTAIR is regulated and the mechanisms by which it affects head and neck squamous cell carcinoma (HNSCC) are not well understood. METHODS: MTT, cell cycle arrest and apoptotic assays were used to examine the effects of HOTAIR and HuR on cell viability in SCC25 and FaDu cells. Wound healing and transwell invasion analysis were performed to detect the effects of HOTAIR and HuR on cell migration and invasion. The interaction between HuR and HOTAIR was confirmed via qRT-PCR, western blots, luciferase reporter and RIP assays. Finally, qRT-PCR analysis was used to detect the levels of HuR and HOTAIR in HNSCC tumours and adjacent normal tissues. RESULTS: Knockdown of HOTAIR and HuR decreased cell viability, cellular migration and invasion. Moreover, HuR interacted and stabilized HOTAIR stability and thus promoted HOTAIR expression. Notably, HOTAIR acted as a miRNA sponge for HuR. HuR also reinforced HOTAIR sponge activity through miRNA recruitment, thus enhancing HuR expression in turn. Finally, HuR and HOTAIR levels were positively correlated and significantly up-regulated in tumours samples. CONCLUSION: We demonstrated the existence of a regulatory loop in which the expression of HOTAIR and HuR is reciprocally and temporally regulated during the metastasis and progression of HNSCC.

Expression of CXCR4 is associated with progression and invasion in patients with nasal-surface basal cell carcinoma.

BACKGROUND: Basal cell carcinoma (BCC) is the most common type of skin cancer with an increasing incidence worldwide that imposes a considerable burden on public health. C-X-C chemokine receptor (CXCR4) plays a vital role in initiation, progression and metastasis of several types of cancers. The aim of the present study was to investigate the expression and clinical significance of CXCR4 in BCC. METHODS: In this study, 80 samples of primary BCC were assessed for CXCR4 expression using immunohistochemistry. The mRNA and protein expression levels of CXCR4 were evaluated by real-time reverse transcription polymerase chain reaction and Western blot analysis, respectively. RESULTS: CXCR4-positive staining was detected in 70% of BCC samples. Overexpression of CXCR4 was significantly associated with tumor size (>2 vs. 2 cm, p = 0.002) and pathological type (invasive vs. noninvasive, p = 0.007). CXCR4 was also upregulated at transcriptional and translational levels. CONCLUSION: Our study revealed that the expression of CXCR4 was associated with progression and invasion in patients with BCC. It may be a considerable biomarker to assess invasiveness of nasal-surface BCC and to guide clinical management of such tumors.

Mesenchymal Stem Cell Derived Extracellular Vesicles: Promising Nanomedicine for Cutaneous Wound Treatment.

A skin wound represents a rupture caused by external damage or the existence of underlying pathological conditions. Sometimes, skin wound healing processes may place a heavy burden on patients, families, and society. Wound healing processes mainly consist of several continuous, dynamic, but overlapping stages, namely, the coagulation stage, inflammation stage, proliferation stage, and remodeling stage. Bacterial infection, excessive inflammation, impaired angiogenesis, and scar formation constitute the four significant factors impeding the recovery efficacy of skin wounds. This encourages scientists to develop multifunctional nanomedicines to meet challenging needs. As we know, mesenchymal stem cells (MSCs) have been widely explored for wound repair owing to their unique capability for self-renewal and multipotency. However, problems including immune concerns and legal restrictions should be properly resolved before MSC-based therapeutics are safely and widely used in clinics. Besides, maintaining the high viability/proliferation capability of MSCs during administration processes and therapy procedures is also one of the biggest technical bottlenecks. Extracellular vesicles (EVs) are cell-derived nanovesicles, that not only possess the basic characteristics and functions of their corresponding maternal cells but also contain several outstanding advantages including abundant sources, excellent biocompatibility, and convenient administration routes. Furthermore, the membrane surface and cavity are easy to flexibly modify to meet versatile application needs. Recently, MSC-derived EVs have emerged as promising therapeutics for skin wound repair. However, current reviews are too broad and rarely focused on the specific roles of EVs in the different stages of wound recovery. Therefore, it is quite necessary to demonstrate the significance of stem cell-derived EVs in promoting wound healing from several specific aspects. Here, this review primarily tries to provide critical comments on current advances in EVs derived from MSCs for wound repair, particularly elaborating on their impressive roles in effectively eliminating infections, inhibiting inflammation, promoting angiogenesis, and reducing scar formation. Last but not least, current limitations and future prospects of EVs derived from MSCs in the areas of wound repair are also objectively analyzed.

Eukaryotic translation initiation factor 3B accelerates the progression of esophageal squamous cell carcinoma by activating ß-catenin signaling pathway.

INTRODUCTION: Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive malignant tumors. Eukaryotic translation initiation factors 3B (EIF3B) is considered to influence tumor proliferation, invasion, apoptosis and cell cycle, which act together to promote the progression of tumors. However, the role of EIF3B in ESCC is unknown. This study aims to explore the clinical and biological role of EIF3B in ESCC. RESULTS: EIF3B expressions were up-regulated in both ESCC tissues and cell lines. Overexpression of EIF3B was associated with tumor depth, lymph node metastasis and advanced TNM stage. Importantly, patients with high EIF3B expression suffered shorter overall and disease-free survival. Knockdown of EIF3B could inhibit cell proliferation and invasion, promote cell apoptosis, and interfere the cell cycle in vitro. EIF3B-knockdown cells could form smaller subcutaneous tumors in vivo. Finally, we demonstrated EIF3B could activate ß-catenin signaling pathway. METHODS: Immunohistochemical staining and Western blot were performed to detect the EIF3B expression in ESCC patient tissues and cell lines. The association between EIF3B expression and patients' prognosis was analyzed by Kaplan-Meier and Cox regression. Then, CCK-8, colony-formation, Transwell and wound-healing assay were performed to compare the bio-functional change after knockdown of EIF3B. Flow cytometry was applied to analyze the change of cell apoptosis and cycle induced by EIF3B knockdown. Tumor xenograft assay was done to verify the in-vitro results. CONCLUSIONS: EIF3B might serve as a novel marker for predicting prognosis of ESCC patients and as a potential therapeutic target, individually or together with other subunits of EIF3 complex.

Hypoxia induced multidrug resistance of laryngeal cancer cells via hypoxia-inducible factor-1α.

OBJECTIVES: To investigate whether hypoxia has an effect on regulation of multidrug resistance (MDR) to chemotherapeutic drugs in laryngeal carcinoma cells and explore the role of hypoxia-inducible factor-1α (HIF- 1α). METHODS: Laryngeal cancer cells were cultured under normoxic and hypoxic conditions. The sensitivity of the cells to multiple drugs and levels of apoptosis induced by paclitaxel were determined by MTT assay and annexin-V/propidium iodide staining analysis, respectively. HIF-1α expression was blocked by RNA interference. The expression of HIF-1α gene was detected by real-time quantitative RT-PCR and Western blotting. The value of fluorescence intensity of intracellular adriamycin accumulation and retention in cells was evaluated by flow cytometry. RESULTS: The sensitivity to multiple chemotherapy agents and induction of apoptosis by paclitaxel could be reduced by hypoxia (P<0.05). A the same time, the adriamycin releasing index of cells was increased (P<0.05). However, resistance acquisition subject to hypoxia in vitro was suppressed by down-regulating HIF-1α expression. CONCLUSION: HIF-1α could be considered as a key regulator for mediating hypoxia-induced MDR in laryngeal cancer cells via inhibition of drug-induced apoptosis and decrease in intracellular drug accumulation.

Potential biomarkers for paclitaxel sensitivity in hypopharynx cancer cell.

Paclitaxel has been proved to be active in treatment and larynx preservation of HNSCC, however, the fact that about 20-40% patients do not respond to paclitaxel makes it urgent to figure out the biomarkers for paclitaxel-based treatment in Hypopharynx cancer (HPC) patients to improve the therapy effect. In this work, Fadu cells, treated or untreated with low dose of paclitaxel for 24 h, were applied to DNA microarray chips. The differential expression in mRNAs and miRs was analyzed and the network between expression-altered mRNAs and miRs was constructed. Differentially expressed genes were mainly enriched in superpathway of cholesterol biosynthesis (ACAT2, MSMO1, LSS, FDFT1 and FDPS etc.), complement system (C3, C1R, C1S, CFR and CFB etc.), interferon signaling (IFIT1, IFIT3, IFITM1 and MX1 etc.), mTOR signaling (MRAS, PRKAA2, PLD1, RND3 and EIF4A1 etc.) and IGF1 signaling (MRAS, IGFBP7, JUN and FOS etc.), most of these pathways are implicated in tumorigenesis or chemotherapy resistance. The first three pathways were predicted to be suppressed, while the last two pathways were predicted to be induced by paclitaxel, suggesting the combination therapy with mTOR inhibition and paclitaxel might be better than single one. The dramatically expression-altered miRs were miR-112, miR-7, miR-1304, miR-222*, miR-29b-1* (these five miRs were upregulated) and miR-210 (downregulated). The 26 putative target genes mediated by the 6 miRs were figured out and the miR-gene network was constructed. Furthermore, immunoblotting assay showed that ERK signaling in Fadu cells was active by low dose of paclitaxel but repressed by high dose of paclitaxel. Collectively, our data would provide potential biomarkers and therapeutic targets for paclitaxel-based therapy in HPC patients.

Gene and microRNA expression reveals sensitivity to paclitaxel in laryngeal cancer cell line.

Paclitaxel is a widely used chemotherapy drug for advanced laryngeal cancer patients. However, the fact that there are 20-40% of advanced laryngeal cancer patients do not response to paclitaxel makes it necessary to figure out potential biomarkers for paclitaxel sensitivity prediction. In this work, Hep2, a laryngeal cancer cell line, untreated or treated with lower dose of paclitaxel for 24 h, was applied to DNA microarray chips for gene and miR expression profile analysis. Expression of eight genes altered significantly following paclitaxel treatment, which was further validated by quantitative real-time PCR. Four up-regulated genes were ID2, BMP4, CCL4 and ACTG2, in which ID2 and BMP4 were implicated to be involved in several drugs sensitivity. While the down-regulated four genes, MAPK4, FASN, INSIG1 and SCD, were mainly linked to the endoplasmic reticulum and fatty acid biosynthesis, these two cell processes that are associated with drug sensitivity by increasing evidences. After paclitaxel treatment, expression of 49 miRs was significantly altered. Within these miRs, the most markedly expression-changed were miR-31-star, miR-1264, miR-3150b-5p and miR-210. While the miRs putatively modulated the mRNA expression of the most significantly expression-altered genes were miR-1264, miR-130a, miR-27b, miR-195, miR-1291, miR-214, miR-1277 and miR-1265, which were obtained by miR target prediction and miRNA target correlation. Collectively, our study might provide potential biomarkers for paclitaxel sensitivity prediction and drug resistance targets in laryngeal cancer patients.

Lack of association between glutathione S-transferase T1 gene polymorphism and laryngeal cancer susceptibility: a meta-analysis based on 2,124 cases and 2,059 controls.

Studies investigating the association between glutathione S-transferase T1 (GSTT1) gene polymorphism and laryngeal cancer susceptibility have reported conflicting results. The aim of the present study was to conduct a meta-analysis assessing the possible association of GSTT1 gene polymorphism with laryngeal cancer risk. The relevant studies were identified through a search of PubMed, Embase, ISI Web of Knowledge and Chinese National Knowledge Infrastructure until May 2011. Twelve studies were included in the present meta-analysis, which described a total of 2124 laryngeal cancer cases and 2059 controls. The overall odds ratio (OR) for GSTT1 null genotype was 1.40 (95% CI=0.90-2.16). When stratifying for race, the pooled ORs for GSTT1 null genotype were 1.07 (95% CI=0.81-1.41) in Caucasians and 5.63 (95% CI=1.00-31.83) in Asians. The pooled ORs for GSTT1 null genotype were 1.03 (95% CI=0.71-1.49) in population-based studies and 2.39 (95% CI=0.73-7.86) in hospital-based studies, stratifying for study design. This meta-analysis suggested that there was lack of association between GSTT1 gene polymorphism and laryngeal cancer risk. However, larger scale primary studies are still required to further evaluate the interaction of GSTT1 gene polymorphism with laryngeal cancer risk.

Glutathione S-transferase M1 gene polymorphism and laryngeal cancer risk: a meta-analysis.

BACKGROUND AND OBJECTIVES: Studies investigating the association between glutathione S-transferase M1 (GSTM1) gene polymorphism and laryngeal cancer risk have reported conflicting results. The aim of the present study was to conduct a meta-analysis assessing the possible associations of GSTM1 gene polymorphism with laryngeal cancer risk. METHODS: The relevant studies were identified through a search of PubMed, Embase, ISI Web of Knowledge and Chinese National Knowledge Infrastructure until May 2011 and selected on the basis of the established inclusion criteria for publications, then a meta-analysis was performed to quantitatively summarize association of GSTM1 polymorphism with laryngeal cancer susceptibility. RESULTS: Seventeen studies were included in the present meta-analysis (2,180 cases and 2,868 controls). The combined results based on all studies showed that GSTM1 null genotype was associated with increased laryngeal cancer risk (OR = 1.17, 95% CI = 1.04∼1.31). When stratifying for race, GSTM1 null genotype exhibited increased laryngeal cancer risk in Caucasians (OR = 1.15, 95% CI = 1.01∼1.31), while no significant association was detected in Asians (OR = 1.25, 95% CI = 0.80∼1.96). In the subgroup analysis based on source of controls, significant associations were observed in the population-based studies (OR = 1.15, 95% CI = 1.01∼1.31) yet not in the hospital-based studies (OR = 1.25, 95% CI = 0.93∼1.67). Furthermore, in the subgroup analysis based on sample size, significant associations were also found in studies with at least 50 cases and 50 controls (OR = 1.15, 95% CI = 1.02∼1.30) but not in studies with fewer than 50 cases or 50 controls (OR = 1.46, 95% CI = 0.87∼2.46). CONCLUSIONS: This meta-analysis supported that the GSTM1 gene polymorphism was associated with laryngeal cancer, particularly in Caucasians, and these associations varied in different subgroup, which indicated that population-based study with larger sample size was more appropriate in design of future study.

[Expression and clinical significance of hypoxia-inducible factor-1alpha and cyclooxygenase-2 in laryngeal squamous cell carcinoma].

OBJECTIVE: To investigate the expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and cyclooxygenase-2 (COX-2) in laryngeal squamous cell carcinoma (LSCC), while determine their relationship with clinicopathologic characteristics and prognosis. METHODS: Tumor tissues were obtained from 60 patients who underwent resection of laryngeal carcinoma in Affiliated First People's Hospital of Shanghai Jiaotong University. Immunohistochemistry (Envision DAKO) was used to detect the expressions of HIF-1alpha and COX-2 in the tumor tissues. As control group, 15 cases of atypical hyperplasia, 10 cases of leukoplakia of vocal cord and 10 cases of polyp of vocal cord were studied. All patients were regularly followed up and the clinical data were collected systematically. RESULTS: Positive staining rates of HIF-1alpha and COX-2 were 95.0% (57/60) and 98.3% (59/60), respectively in all 60 specimen of LSCC. The positive expressions in LSCC were significantly higher than those in atypical hyperplasia, leukoplakia and polyp of vocal cord ( Fisher's exact test, P < 0.01). The expression of HIF-1alpha was correlated with COX-2 in LSCC (r = 0.526, P < 0.01). High level expressions of HIF-1alpha and COX-2 were 35.0% (21/60) and 38.3% (23/60) respectively. High level expression of HIF-1alpha was significantly correlated with clinical stages (chi2 = 4.331, P < 0.05) and lymph nodes metastases (Fisher's exact test, P < 0.05). High level expression of COX-2 was significantly correlated with clinical stage (chi2 = 8.539, P < 0.01) and T stages (chi2 = 6.792, P < 0.01). With univariate analysis, high level expressions of HIF-1alpha and COX-2 were significantly associated with a worse overall survival (chi2 = 6.003, P < 0.05 and chi2 = 9.489, P < 0.01, respectively) and disease-free survival (chi2 = 5.010, P < 0.05 and chi2 = 6.102, P < 0.05, respectively). With multivariate analysis, recurrence and high level expression of COX-2 were two unfavorable prognostic factors (RR = 7.104, P = 0.003; RR = 5.714, P = 0.008). CONCLUSIONS: The expressions of HIF-1alpha and COX-2 played an important role in the process of tumorigenesis and development of LSCC, The expression of HIF-1alpha was correlated with COX-2 in LSCC. COX-2 and recurrence were probably significant risk factors for prognosis of LSCC.

Large-scale purification of human granulocyte-macrophage colony-stimulating factor expressed in Bombyx mori pupae.

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) acts on many different kinds of cells, including monocytes, macrophages, granulocytes, eosinophils, and multipotential stem cells. To explore further explore pharmaceutical action, we expressed hGM-CSF by the Bombyx mori nucleopolyhedrovirus expression system in silkworm pupae. However, purifying recombinant proteins from silkworm pupae on a large scale has been a big challenge. To establish purification methods suitable for mass production, we tried two crude preparation methods: (NH4)2SO4 fractional precipitation and isoelectric precipitation with a combination of gel filtration and ion-exchange chromatography. The isoelectric precipitation method was found to be more efficient. With this method, we eventually obtained approx 11.7 mg of 95% pure product from 1000 g of infected silkworm pupae. The recovery of purified protein was greatly increased, by approx 40%, compared with the other method. The biologic activity of this protein was determined up to 9.0 x 106 colony-forming units/mg in the final purified product.