Mitomycin C modulates intracellular matrix metalloproteinase-9 expression and affects corneal fibroblast migration

Chen, Tsan‐Chi; Ho, Wei‐Ting; Lai, Chien-Hsueh; Chang, Shu‐Wen

doi:10.1016/j.ejphar.2019.172752

Cited by 6 publications

(8 citation statements)

References 28 publications

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“…Using this lentiviral transduction system, we previously showed that several transgenes could be expressed in primary HCFs, e . g ., EGFP alone and EGFP-fused paxillin [ 14 ], EGFP-fused ZO-1 [ 4 ], and matrix metalloproteinase-9 fused with EGFP driven by an internal ribosome entry site [ 3 ]. Our current results indicate that lentiviral EGFP expression in ocular cells was most difficult in HCFs, followed by HCnEs and HCjEs ( Fig 1 ).…”

Section: Discussionmentioning

confidence: 99%

“…To express the lentiviral EGFP, pseudovirions encoding EGFP were produced according to a published procedure with appropriate modifications [ 3 ]. Briefly, 1.0 × 10 6 HEK-293T cells were seeded in a 10-cm culture dish with antibiotic-free DMEM containing 10% FBS at 37°C under 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

“…Various types of genetic elements, such as small interfering RNAs and plasmids that may encode diverse molecules, can be transferred into mammalian cells by various techniques including electroporation ( in vitro or in vivo ) [ 7 ], lipid-based reagents [ 8 ], and virus-based vectors [ 3 , 9 , 10 ]. Regardless of the delivery process, mammalian cells often suffer post-electroporation damage [ 11 ], chemical insults [ 12 ], or off-target integration of the transgene (in the case of lentiviral transduction) [ 13 ].…”

Section: Introductionmentioning

confidence: 99%

“…To track transgene expression in mammalian cells that have been transfected with a vector, various fluorescent proteins, including enhanced green fluorescent protein (EGFP), yellow fluorescent protein, and red fluorescent protein, are frequently encoded by the transgene open-reading frame by gene fusion [ 14 , 15 ] or an internal ribosome entry site–driven bicistronic expression system [ 3 , 16 , 17 ]. The expression and intracellular dynamics of the fusion protein can be monitored by fluorescence microscopy and/or flow cytometry.…”

Section: Introductionmentioning

confidence: 99%

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Repeated cell sorting ensures the homogeneity of ocular cell populations expressing a transgenic protein

Chen

Chang

2022

PLoS ONE

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Transgenic proteins can be routinely expressed in various mammalian cell types via different transgenic systems, but the efficiency of transgene expression is constrained by the complex interplay among factors such as the temporal consistency of expression and compatibility with specific cell types, including ocular cells. Here, we report a more efficient way to express an enhanced green fluorescent protein (EGFP) in human corneal fibroblasts, corneal epithelial cells, and conjunctival epithelial cells through a lentiviral expression system. The relative transducing unit criterion for EGFP-expressing pseudovirions was first determined in HEK-293T cells. Homogeneous populations of EGFP-positive and EGFP-negative cells could be isolated by cell sorting. The half-maximal inhibitory concentration (IC50) value for puromycin was calculated according to viability curves for each cell type. The results revealed that cell types differed with respect to EGFP expression efficiency after transduction with the same amount of EGFP-encoding pseudovirions. Using a cell sorter, the homogeneity of EGFP-positive cells reached >95%. In the initial sorting stage, however, the efficiency of EGFP expression in the sorted cells was noticeably reduced after two rounds of sequential culture, but repeated sorting for up to four rounds yielded homogeneous EGFP-positive human corneal fibroblasts that could be maintained in continuous culture in vitro. The sorted EGFP-positive cells retained their proper morphology and cell type-specific protein expression patterns. Puromycin resistance was found to depend on cell type, indicating that the IC50 for puromycin must be determined for each cell type to ensure the isolation of homogeneous EGFP-positive cells. Taken together, repeated cell sorting is an efficient means of obtaining homogeneous populations of ocular cells expressing a transgenic protein during continuous culture without the potential confounding effects of antibiotics.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Repeated cell sorting ensures the homogeneity of ocular cell populations expressing a transgenic protein

Chen

Chang

2022

PLoS ONE

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“…The concentration of MMC to induce cytotoxicity could be much lower in other organs. A number of studies using MMC in clinic showed its safety and effectiveness (Sui et al, 2017;Chen et al, 2019). However, in an animal study by Wu et al, injection of MMC at the concentrations of 0.5 mg/ml or 0.05 mg/ml was used in porcine models after endoscopic mucosectomy.…”

Section: Discussionmentioning

confidence: 99%

Mitomycin C Inhibits Esophageal Fibrosis by Regulating Cell Apoptosis and Autophagy via lncRNA-ATB and miR-200b

Zhang

Wang

et al. 2021

Front. Mol. Biosci.

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Benign esophageal strictures (BESs) frequently results from esophageal fibrosis. The transformation of fibroblasts into fibrocyte is an important cause of fibrosis. The treatment of fibrosis is challenging. Some previous studies have indicated the antifibrotic effect of mitomycin C (MMC). However, the mechanism of action of MMC and its optimal dose for treatment remains unclear. In the present study, the role of MMC in fighting fibrosis and its mechanism was investigated. Human esophageal fibroblast cells (HEFs)were treated without or with MMC, at 2, 5, 10 μg/ml, combining with mimic lncRNA-ATB, miR-200b inhibitor, rapamycin (RAPA), and 3-Methyladenine (3-MA). The cell viability, and cell apoptosis were evaluated. In addition, expression of apoptosis related proteins (caspase8 and caspase3), autophagy related proteins (LC3II and ATG5) and fibrosis related proteins (α-SMA collagen-1 and TGF-β) were also evaluated. Furthermore, autophagosome was observed by transmission electron microscope. Results showed that the expression of lncRNA-ATB was down-regulated and miR-200b was up-regulated after treated with MMC. And MMC induced cell apoptosis and inhibited cell autophagy. On the other hand, RAPA, mimic lncRNA-ATB and miR-200b inhibitor reduced fibrogenic effect of MMC on HEFs. Collectively, this study suggests that MMC inhibited esophageal fibrosis by regulating cell apoptosis and autophagy via downregulating lncRNA-ATB and upregulating miR-200b.

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Moxifloxacin induces random migration in human corneal fibroblasts via the protein kinase C epsilon/zonula occludens-1 signaling pathway

Chen

Chang

2021

European Journal of Pharmacology

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Mitomycin C modulates intracellular matrix metalloproteinase-9 expression and affects corneal fibroblast migration

Cited by 6 publications

References 28 publications

Repeated cell sorting ensures the homogeneity of ocular cell populations expressing a transgenic protein

Repeated cell sorting ensures the homogeneity of ocular cell populations expressing a transgenic protein

Mitomycin C Inhibits Esophageal Fibrosis by Regulating Cell Apoptosis and Autophagy via lncRNA-ATB and miR-200b

Moxifloxacin induces random migration in human corneal fibroblasts via the protein kinase C epsilon/zonula occludens-1 signaling pathway

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