A cellular and proteomic approach to assess proteins extracted from cryopreserved human amnion in the cultivation of corneal stromal keratocytes for stromal cell therapy

Abstract Background Human corneal stromal keratocytes propagated in culture media supplemented with human amnion extract (AME) can correct early corneal haze in an animal model. Clinical application of cultivated keratocytes is limited by infectious disease screening before amnion products can be used in humans. It remains unclear if AME from cryopreserved versus fresh human amnion can support human keratocyte propagation, and which components of the extract promote keratocyte growth. Methods Three placentas were collected for the preparation of fresh and cryopreserved amnion tissues followed by homogenization and protein extraction. AME protein profiles were studied using isobaric tagging for relative and absolute quantitation (iTRAQ) proteomics. Enriched gene ontology (GO) terms and functional classes were identified. Primary human keratocytes from 4 donor corneas were cultured in media supplemented with fresh AME (F-AME) or cryopreserved AME (C-AME). Cell viability, proliferation and keratocyte marker expression were examined by confocal immunofluorescence and flow cytometry. Results AME proteomics revealed 1385 proteins with similar expression levels (between 0.5- and 2-fold) between F- and C-AME, while 286 proteins were reduced (less than 0.5-fold) in C-AME. Enriched GO term and biological pathway analysis showed that those proteins with comparable expression between F-AME and C-AME were involved in cell metabolism, epithelial-mesenchymal transition, focal adhesion, cell-extracellular matrix interaction, cell stress regulation and complement cascades. Human corneal stromal keratocytes cultured with F-AME or C-AME showed similar morphology and viability, while cell proliferation was mildly suppressed with C-AME (P > 0.05). Expression of aldehyde dehydrogenase 3A1 (ALDH3A1) and CD34 was similar in both cultures. Conclusion AME from cryopreserved amnion had limited influence on keratocyte culture. It is feasible to use protein extract from cryopreserved amnion to propagate human keratocytes for potential translational applications.

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Biological and medical applications of plasma-activated media, water and solutions

Biological Chemistry ◽

10.1515/hsz-2018-0226 ◽

2018 ◽

Vol 400 (1) ◽

pp. 39-62 ◽

Cited By ~ 64

Author(s):

Nagendra Kumar Kaushik ◽

Bhagirath Ghimire ◽

Ying Li ◽

Manish Adhikari ◽

Mayura Veerana ◽

...

Keyword(s):

Biomedical Applications ◽

Storage Temperature ◽

Epithelial Mesenchymal Transition ◽

Dental Treatment ◽

Culture Media ◽

Atmospheric Pressure Plasma ◽

Medical Applications ◽

Mesenchymal Transition ◽

Safety Issues ◽

Biological And Medical Applications

Abstract Non-thermal atmospheric pressure plasma has been proposed as a new tool for various biological and medical applications. Plasma in close proximity to cell culture media or water creates reactive oxygen and nitrogen species containing solutions known as plasma-activated media (PAM) or plasma-activated water (PAW) – the latter even displays acidification. These plasma-treated solutions remain stable for several days with respect to the storage temperature. Recently, PAM and PAW have been widely studied for many biomedical applications. Here, we reviewed promising reports demonstrating plasma-liquid interaction chemistry and the application of PAM or PAW as an anti-cancer, anti-metastatic, antimicrobial, regenerative medicine for blood coagulation and even as a dental treatment agent. We also discuss the role of PAM on cancer initiation cells (spheroids or cancer stem cells), on the epithelial mesenchymal transition (EMT), and when used for metastasis inhibition considering its anticancer effects. The roles of PAW in controlling plant disease, seed decontamination, seed germination and plant growth are also considered in this review. Finally, we emphasize the future prospects of PAM, PAW or plasma-activated solutions in biomedical applications with a discussion of the mechanisms and the stability and safety issues in relation to humans.

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Microenvironmental Hypoxia Induces Dynamic Changes in Lung Cancer Synthesis and Secretion of Extracellular Vesicles

Cancers ◽

10.3390/cancers12102917 ◽

2020 ◽

Vol 12 (10) ◽

pp. 2917

Author(s):

Shun Wilford Tse ◽

Chee Fan Tan ◽

Jung Eun Park ◽

JebaMercy Gnanasekaran ◽

Nikhil Gupta ◽

...

Keyword(s):

Lung Cancer ◽

Extracellular Vesicles ◽

Cancer Progression ◽

Membrane Trafficking ◽

Epithelial Mesenchymal Transition ◽

Migration And Invasion ◽

Mesenchymal Transition ◽

Functional Clustering ◽

Signalling Molecules ◽

Proteomic Approach

Extracellular vesicles (EVs) mediate critical intercellular communication within healthy tissues, but are also exploited by tumour cells to promote angiogenesis, metastasis, and host immunosuppression under hypoxic stress. We hypothesize that hypoxic tumours synthesize hypoxia-sensitive proteins for packing into EVs to modulate their microenvironment for cancer progression. In the current report, we employed a heavy isotope pulse/trace quantitative proteomic approach to study hypoxia sensitive proteins in tumour-derived EVs protein. The results revealed that hypoxia stimulated cells to synthesize EVs proteins involved in enhancing tumour cell proliferation (NRSN2, WISP2, SPRX1, LCK), metastasis (GOLM1, STC1, MGAT5B), stemness (STC1, TMEM59), angiogenesis (ANGPTL4), and suppressing host immunity (CD70). In addition, functional clustering analyses revealed that tumour hypoxia was strongly associated with rapid synthesis and EV loading of lysosome-related hydrolases and membrane-trafficking proteins to enhance EVs secretion. Moreover, lung cancer-derived EVs were also enriched in signalling molecules capable of inducing epithelial-mesenchymal transition in recipient cancer cells to promote their migration and invasion. Together, these data indicate that lung-cancer-derived EVs can act as paracrine/autocrine mediators of tumorigenesis and metastasis in hypoxic microenvironments. Tumour EVs may, therefore, offer novel opportunities for useful biomarkers discovery and therapeutic targeting of different cancer types and at different stages according to microenvironmental conditions.

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Serum Proteome Alterations in Patients with Cognitive Impairment after Traumatic Brain Injury Revealed by iTRAQ-Based Quantitative Proteomics

BioMed Research International ◽

10.1155/2017/8572509 ◽

2017 ◽

Vol 2017 ◽

pp. 1-13 ◽

Cited By ~ 3

Author(s):

Xin-gui Xiong ◽

Qinghua Liang ◽

Chunhu Zhang ◽

Yang Wang ◽

Wei Huang ◽

...

Keyword(s):

Traumatic Brain Injury ◽

Cognitive Impairment ◽

Brain Injury ◽

Bioinformatic Analysis ◽

Serum Samples ◽

Absolute Quantitation ◽

Proteomic Approach ◽

Healthy Control ◽

Isobaric Tagging ◽

Experimental Findings

Background. Cognitive impairment is the leading cause of traumatic brain injury- (TBI-) related disability; however, the underlying pathogenesis of this dysfunction is not completely understood. Methods. Using an isobaric tagging for relative and absolute quantitation- (iTRAQ-) based quantitative proteomic approach, serum samples from healthy control subjects, TBI patients with cognitive impairment, and TBI patients without cognitive impairment were analysed to identify differentially expressed proteins (DEPs) related to post-TBI cognitive impairment. In addition, DEPs were further analysed using bioinformatic platforms and validated using enzyme-linked immunosorbent assays (ELISA). Results. A total of 56 DEPs were identified that were specifically related to TBI-induced cognitive impairment. Bioinformatic analysis revealed that a wide variety of cellular and metabolic processes and some signaling pathways were involved in the pathophysiology of cognitive deficits following TBI. Five randomly selected DEPs were validated using ELISA in an additional 105 cases, and the results also supported the experimental findings. Conclusions. Despite limitations, our findings will facilitate further studies of the pathological mechanisms underlying TBI-induced cognitive impairment and provide new methods for the research and development of neuroprotective agents. However, further investigation on a large cohort is warranted.

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Curcumin Affects HSP60 Folding Activity and Levels in Neuroblastoma Cells

International Journal of Molecular Sciences ◽

10.3390/ijms21020661 ◽

2020 ◽

Vol 21 (2) ◽

pp. 661 ◽

Cited By ~ 5

Author(s):

Celeste Caruso Bavisotto ◽

Antonella Marino Gammazza ◽

Filippa Lo Cascio ◽

Emanuele Mocciaro ◽

Alessandra Maria Vitale ◽

...

Keyword(s):

Cancer Progression ◽

Epithelial Mesenchymal Transition ◽

Culture Media ◽

Neuroblastoma Cell ◽

Cancer Development ◽

Protective Mechanism ◽

Protein Homeostasis ◽

Matrix Remodeling ◽

Dependent Manner ◽

Mesenchymal Transition

The fundamental challenge in fighting cancer is the development of protective agents able to interfere with the classical pathways of malignant transformation, such as extracellular matrix remodeling, epithelial–mesenchymal transition and, alteration of protein homeostasis. In the tumors of the brain, proteotoxic stress represents one of the main triggering agents for cell transformation. Curcumin is a natural compound with anti-inflammatory and anti-cancer properties with promising potential for the development of therapeutic drugs for the treatment of cancer as well as neurodegenerative diseases. Among the mediators of cancer development, HSP60 is a key factor for the maintenance of protein homeostasis and cell survival. High HSP60 levels were correlated, in particular, with cancer development and progression, and for this reason, we investigated the ability of curcumin to affect HSP60 expression, localization, and post-translational modifications using a neuroblastoma cell line. We have also looked at the ability of curcumin to interfere with the HSP60/HSP10 folding machinery. The cells were treated with 6, 12.5, and 25 µM of curcumin for 24 h, and the flow cytometry analysis showed that the compound induced apoptosis in a dose-dependent manner with a higher percentage of apoptotic cells at 25 µM. This dose of curcumin-induced a decrease in HSP60 protein levels and an upregulation of HSP60 mRNA expression. Moreover, 25 µM of curcumin reduced HSP60 ubiquitination and nitration, and the chaperonin levels were higher in the culture media compared with the untreated cells. Furthermore, curcumin at the same dose was able to favor HSP60 folding activity. The reduction of HSP60 levels, together with the increase in its folding activity and the secretion in the media led to the supposition that curcumin might interfere with cancer progression with a protective mechanism involving the chaperonin.

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Epithelial to Mesenchymal Transition and Cell Biology of Molecular Regulation in Endometrial Carcinogenesis

Journal of Clinical Medicine ◽

10.3390/jcm8040439 ◽

2019 ◽

Vol 8 (4) ◽

pp. 439 ◽

Cited By ~ 12

Author(s):

Hsiao-Chen Chiu ◽

Chia-Jung Li ◽

Giou-Teng Yiang ◽

Andy Tsai ◽

Meng-Yu Wu

Keyword(s):

Growth Factor ◽

Signaling Pathways ◽

Cell Biology ◽

Epithelial To Mesenchymal Transition ◽

Epithelial Mesenchymal Transition ◽

Cell Metabolism ◽

Basic Research ◽

Genetic Mutation ◽

Molecular Regulation ◽

Mesenchymal Transition

Endometrial carcinogenesis is involved in several signaling pathways and it comprises multiple steps. The four major signaling pathways—PI3K/AKT, Ras/Raf/MEK/ERK, WNT/β-catenin, and vascular endothelial growth factor (VEGF)—are involved in tumor cell metabolism, growth, proliferation, survival, and angiogenesis. The genetic mutation and germline mitochondrial DNA mutations also impair cell proliferation, anti-apoptosis signaling, and epithelial–mesenchymal transition by several transcription factors, leading to endometrial carcinogenesis and distant metastasis. The PI3K/AKT pathway activates the ransforming growth factor beta (TGF-β)-mediated endothelial-to-mesenchymal transition (EMT) and it interacts with downstream signals to upregulate EMT-associated factors. Estrogen and progesterone signaling in EMT also play key roles in the prognosis of endometrial carcinogenesis. In this review article, we summarize the current clinical and basic research efforts regarding the detailed molecular regulation in endometrial carcinogenesis, especially in EMT, to provide novel targets for further anti-carcinogenesis treatment.

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Abstract 210: Stem Cell Conditioned Culture Media Attenuated Albumin-induced Epithelial-mesenchymal Transition in Renal Tubular Cells

Hypertension ◽

10.1161/hyp.62.suppl_1.a210 ◽

2013 ◽

Vol 62 (suppl_1) ◽

Author(s):

Junping Hu ◽

Weiqing Han ◽

Qing Zhu ◽

Pin-Lan Li ◽

Ningjun Li

Keyword(s):

Epithelial Mesenchymal Transition ◽

Culture Media ◽

Mesenchymal Transition ◽

Tubular Cells ◽

Protein Levels ◽

Renal Tubular Cells ◽

Proximal Tubular ◽

Renal Proximal Tubular Cells ◽

Renal Proximal Tubular ◽

Renal Tubular

Mesenchymal stem cells (MSCs) have been shown to be a promising therapy for many different diseases. Stem cell conditioned culture media (SCM) exhibit similar beneficial effects as MSCs. Albuminuria-induced epithelial-mesenchymal transition (EMT) plays an important role in progressive renal tubulointerstitial fibrosis in chronic renal disease. The present study tested the hypothesis that SCM inhibit albumin-induced EMT in cultured renal tubular cells. SCM were obtained by culturing rat adult MSCs for 3 days. Cultured renal proximal tubular cells were incubated with rat albumin (20μg/ml) and treated with SCM or control culture media. Our results showed that 48 h albumin incubation stimulated EMT in renal proximal tubular cells as shown by significant decrease in the protein levels of epithelial marker E-cadherin from 2.30 ± 0.27 to 0.87 ± 0.11 ( P < 0.05) and increase in the protein levels of mesenchymal marker fibroblast-specific protein 1 (FSP-1) (2.18±0.33 folds, P < 0.05). SCM treatment significantly inhibited these albumin-induced changes in E-cadherin and FSP-1 by 2.33±0.17 and 1.95±0.23 folds ( P < 0.05), respectively. Meanwhile, albumin increased the mRNA levels of pro-inflammatory factor monocyte chemoattractant protein-1 (MCP)-1 by nearly 30 folds compared with control. SCM almost abolished the increase of MCP-1 induced by albumin. Furthermore, Western blot results displayed that albumin rapidly decreased the cytosolic levels and increased the nuclear levels of NF-κB, indicating a translocation of NF-κB; immunofluorescence microscopy also demonstrated that albumin induced NF-κB translocation from the cytosol into nucleus. SCM blocked the translocation of NF-κB into nucleus. These results suggest that SCM attenuated albumin-induced EMT in renal tubular cells via inhibiting NF-κB activation and inflammation, which may serve as a new therapeutic approach for chronic kidney diseases. (Supported by NIH grant HL89563 and HL106042)

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Abstract PD02-04: Molecular Profiling of Circulating Tumor Cells in Blood of Metastatic Breast Cancer Patients Indicates Therapy Response and Provides Information on Epithelial Mesenchymal Transition and Tumor Stem Cell Metabolism

10.1158/0008-5472.sabcs10-pd02-04 ◽

2010 ◽

Author(s):

B Aktas ◽

M Tewes ◽

R Kimmig ◽

S. Kasimir-Bauer

Keyword(s):

Breast Cancer ◽

Epithelial Mesenchymal Transition ◽

Cell Metabolism ◽

Metastatic Breast ◽

Therapy Response ◽

Molecular Profiling ◽

Tumor Stem Cell ◽

Breast Cancer Patients ◽

Mesenchymal Transition ◽

Stem Cell Metabolism

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The Mitochondrial Protein VDAC1 at the Crossroads of Cancer Cell Metabolism: The Epigenetic Link

Cancers ◽

10.3390/cancers12041031 ◽

2020 ◽

Vol 12 (4) ◽

pp. 1031 ◽

Cited By ~ 4

Author(s):

Zohar Amsalem ◽

Tasleem Arif ◽

Anna Shteinfer-Kuzmine ◽

Vered Chalifa-Caspi ◽

Varda Shoshan-Barmatz

Keyword(s):

Cancer Cell ◽

Dna Microarrays ◽

Epithelial Mesenchymal Transition ◽

Cell Metabolism ◽

Mitochondrial Protein ◽

Residual Tumor ◽

Mesenchymal Transition ◽

Cancer Hallmarks ◽

Acetylation State ◽

Cancer Cell Metabolism

Carcinogenesis is a complicated process that involves the deregulation of epigenetics, resulting in cellular transformational events, such as proliferation, differentiation, and metastasis. Most chromatin-modifying enzymes utilize metabolites as co-factors or substrates and thus are directly dependent on such metabolites as acetyl-coenzyme A, S-adenosylmethionine, and NAD+. Here, we show that using specific siRNA to deplete a tumor of VDAC1 not only led to reprograming of the cancer cell metabolism but also altered several epigenetic-related enzymes and factors. VDAC1, in the outer mitochondrial membrane, controls metabolic cross-talk between the mitochondria and the rest of the cell, thus regulating the metabolic and energetic functions of mitochondria, and has been implicated in apoptotic-relevant events. We previously demonstrated that silencing VDAC1 expression in glioblastoma (GBM) U-87MG cell-derived tumors, resulted in reprogramed metabolism leading to inhibited tumor growth, angiogenesis, epithelial–mesenchymal transition and invasiveness, and elimination of cancer stem cells, while promoting the differentiation of residual tumor cells into neuronal-like cells. These VDAC1 depletion-mediated effects involved alterations in transcription factors regulating signaling pathways associated with cancer hallmarks. As the epigenome is sensitive to cellular metabolism, this study was designed to assess whether depleting VDAC1 affects the metabolism–epigenetics axis. Using DNA microarrays, q-PCR, and specific antibodies, we analyzed the effects of si-VDAC1 treatment of U-87MG-derived tumors on histone modifications and epigenetic-related enzyme expression levels, as well as the methylation and acetylation state, to uncover any alterations in epigenetic properties. Our results demonstrate that metabolic rewiring of GBM via VDAC1 depletion affects epigenetic modifications, and strongly support the presence of an interplay between metabolism and epigenetics.

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Protein Expression Profile in Rat Silicosis Model Reveals Upregulation of PTPN2 and Its Inhibitory Effect on Epithelial-Mesenchymal Transition by Dephosphorylation of STAT3

International Journal of Molecular Sciences ◽

10.3390/ijms21041189 ◽

2020 ◽

Vol 21 (4) ◽

pp. 1189

Author(s):

Ying Zhu ◽

Jingxin Yao ◽

Yuxia Duan ◽

Hong Xu ◽

Qiyun Cheng ◽

...

Keyword(s):

Epithelial Mesenchymal Transition ◽

Tyrosine Phosphatase ◽

Inhibitory Effect ◽

Differentially Expressed ◽

Absolute Quantitation ◽

Opposite Pattern ◽

Mesenchymal Transition ◽

Enhanced Expression ◽

Isobaric Tags

Silicosis is a chronic occupational lung disease caused by long-term inhalation of crystalline silica particulates. We created a rat model that closely approximates the exposure and development of silicosis in humans. Isobaric tags for relative and absolute quantitation (iTRAQ) technologies we used to identify proteins differentially expressed in activated rat lung tissue. We constructed three lentiviral knockdown vectors and an overexpression vector for the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene to achieve stable long-term expression. A total of 471 proteins were differentially expressed in the silicosis group compared with controls. Twenty upregulated, and eight downregulated proteins exhibited a ≥1.5-fold change relative to controls. We next found that the PTPN2, Factor B, and VRK1 concentrations in silicotic rats silicosis and SiO2-stimulated MLE-12 cells were significantly higher than control groups. More importantly, we found that overexpression of PTPN2 simultaneously decreased the expression of phospho–signal transducer and activator of transcription 3 (p-STAT3) and Vimentin, while increasing E-cadherin expression. The opposite pattern was observed for PTPN2-gene silencing. We identified three proteins with substantially enhanced expression in silicosis. Our study also showed that PTPN2 can inhibit epithelial-mesenchymal transition by dephosphorylating STAT3 in silicosis fibrosis.

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