Genetically Engineered Lung Cancer Cells for Analyzing Epithelial–Mesenchymal Transition

Kiełbus, Michał; Czapiński, Jakub; Kałafut, Joanna; Woś, Justyna; Stepulak, Andrzej; Rivero-Müller, Adolfo

doi:10.3390/cells8121644

Cited by 7 publications

(6 citation statements)

References 57 publications

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“…The expression of vimentin may play a key role in cell migratory ability, since migratory ability can be inhibited when vimentin expression is suppressed (Wawruszak et al 2019). The expression level of vimentin is positively associated with the invasive and migratory ability of tumor cells, such as ovarian cancer, cervical cancer, endometrial cancer (Zhou et al 2014), breast cancer (Xu et al 2020), tongue squamous cell carcinoma (Hussein et al 2018), lung cancer (Kielbus et al 2019), and prostate cancer (Kaufhold and Bonavida 2014). In the present study, overexpression of HSP70 can downregulate the expression of vimentin and abate the process of EMT; however, the specific molecular mechanism and signaling pathways involved remain unclear; thus, further studies are required.…”

Section: Discussionmentioning

confidence: 99%

Overexpression of heat shock protein 70 inhibits epithelial-mesenchymal transition and cell migration induced by transforming growth factor-β in A549 cells

Shi

Zhai

et al. 2021

Cell Stress and Chaperones

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Heat shock protein 70 (HSP70) is a key member of the HSP family that contributes to a pre-cancerous environment; however, its role in lung cancer remains poorly understood. The present study used geranylgeranylacetone (GGA) to induce HSP70 expression, and transforming growth factor-β (TGF-β) was used to construct an epithelial-mesenchymal transition (EMT) model by stimulating A549 cells in vitro. Western Blot was performed to detect protein levels of NADPH oxidase 4 (NOX4) and the EMTassociated proteins E-cadherin and vimentin both before and after HSP70 expression. Cell morphological changes were observed, and the effect of HSP70 on cell migration ability was detected via the wound healing. The results demonstrated that GGA at 50 and 200 μmol/L could significantly induce HSP70 expression in A549 cells (P < 0.05). Furthermore, HSP70 induced by 200 μmol/L GGA significantly inhibited the changes of E-cadherin, vimentin, and cell morphology induced by TGF-β (P < 0.05), while HSP70 induced by 50 μmol/L GGA did not. The results of the wound healing assay indicated that 200 μmol/L GGA significantly inhibited A549 cell migration induced by TGF-β. Taken together, the results of the present study demonstrated that overexpression of HSP70 inhibited the TGF-β induced EMT process and changed the cell morphology and migratory ability induced by TGF-β in A549 cells.

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Section: Discussionmentioning

confidence: 99%

Overexpression of heat shock protein 70 inhibits epithelial-mesenchymal transition and cell migration induced by transforming growth factor-β in A549 cells

Shi

Zhai

et al. 2021

Cell Stress and Chaperones

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“…TGFβ was produced in HEK293 cells, as previously reported [44], where the medium of cells transfected with a plasmid coding TGFβ was collected and transferred to 96 well plate containing cells expressing the TWIST1-GLuc reporter construct. As a control, we used media from mock-transfected HEK-293T cells.…”

Section: Methodsmentioning

confidence: 99%

Development of a Bioassay for Continuous Monitoring of TWIST1 Activity

Czapiński¹,

Kałafut²,

Przybyszewska-Podstawka³

et al. 2021

Preprint

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TWIST1 is a transcription factor that affects cell behavior during development and cell differentiation. Yet, it is better known for its roles in neoplasia through regulation of cell plasticity. The pathological contributions of TWIST1 in tumor initiation, angiogenesis, invasion, metastasis, and chemo-resistance have been the focus of much research. To-date, the only way to quantitatively measure the abundance of TWIST is by immunoblots. Yet, no bioassay exists that can detect TWIST1 activity. Thus, we present here a TWIST1 cell-based assay that allows measuring the amount of active TWIST1 non-invasively in living cells. The bioassay was characterized against previously described TWIST1 “inhibitors”, as well as by epigenetic modulators of TWIST1 gene expression. Moreover, we tested multiple cell lines, showing that the level of TWIST1 mRNA resembles that of the bioassay. We show that prostate cancer cells (PC3) undergoing EMT, migrate out of 3D-spheroids and have increased TWIST1 activity. This fast and reliable system to detect active TWIST1 in different biological conditions allows a detailed analysis of this factor, as well as it can be used for drug discovery, since TWIST1 is a potential target for cancer chemotherapeutics.

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“…Wound-healing assay. For the Wound-healing assay cell lines of H1299 and H2170 VRCs (vimentin reporter cells) 38 were seeded into 12-well plates at a density 5 × 10 4 cells/mL. One day later were co-transfected with one half of Cas9 protein (pCMV-Cas4.5C) driven by general promoter, construct with EGxxFP reporter plasmid, Cetn1 gRNA and second half of Cas9 TBD-Cas4.5N under the specific promoter activated during EMT -under the control of TWIST1, resulted in EGFP expression.…”

Section: Molecular Cloningmentioning

confidence: 99%

Synthetic Circuits Based on Split Cas9 to Detect Cellular Events

Przybyszewska-Podstawka

Czapiński

Kałafut

et al. 2023

Preprint

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Synthetic biology involves the generation of logic circuits to create or control biological functions and behaviours by engineering interconnected genetic elements such as promoters, repressors, and transcriptional activators. CRISPR discovery, and its adaptations to mammalian cells, has made it the tool of choice in molecular biology and revolutionized genome engineering in biomedical sciences. Here, we describe an adaptation of a split Cas9 to generate synthetic logic gates to sense biological events. As proof-of-concept, the complementing halves of split Cas9 were placed under different promoters, one unique to cancer cells of epithelial origin (phCEA) and one universal promoter (pCMV). We used self-assembling inteins to reunite the halves when co-expressed. Only cancer cells with epithelial origin expressed both halves and activated a reporter becoming green fluorescent. We then investigated whether we could apply this system to the detection of biological processes such as epithelial to mesenchymal transition (EMT). We designed another logic gate where one halve is expressed only by cancer cells of epithelial origin, while the other is activated during EMT - under the control of TWIST1. Indeed, cells undergoing EMT were detected by the activation of the reporter. Finally, the split-Cas9 logic gate was applied as a sensor to detect cell-cell fusion events in multiple cell lines. Each cell type expressed only one halve of split Cas9, and only induction of fusion resulted in the appearance of multinucleated syncytia and the expression of the reporter system. The simplicity and flexibility of the split Cas9 system reported here can be integrated to many other cellular processes, not only as a sensor but as an actuator.

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Genetically Engineered Lung Cancer Cells for Analyzing Epithelial–Mesenchymal Transition

Cited by 7 publications

References 57 publications

Overexpression of heat shock protein 70 inhibits epithelial-mesenchymal transition and cell migration induced by transforming growth factor-β in A549 cells

Overexpression of heat shock protein 70 inhibits epithelial-mesenchymal transition and cell migration induced by transforming growth factor-β in A549 cells

Development of a Bioassay for Continuous Monitoring of TWIST1 Activity

Synthetic Circuits Based on Split Cas9 to Detect Cellular Events

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