PKN2 is involved in aggregation and spheroid formation of fibroblasts in suspension culture by regulating cell motility and N-cadherin expression

Kubouchi, Koji; Mukai, Hideyuki

doi:10.1016/j.bbrep.2020.100895

Cited by 5 publications

(4 citation statements)

References 34 publications

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“…The role of Ncadherin was demonstrated to be similar in spheroid aggregation and compaction. 45 Although each cell line posed a unique set of challenges in generating large and compact spheroids, we succeed in developing a robust protocol to generate tight spheroids for each cell line using 40 μg mL −1 type-I collagen. To better describe the mechanism of spheroid compaction using collagen, we can compare the data related to collagen-based U87MG spheroids with collagen-free U251 spheroids.…”

Section: Resultsmentioning

confidence: 99%

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Type-I Collagen/Collagenase Modulates the 3D Structure and Behavior of Glioblastoma Spheroid Models

Calori

Alves

Hong

et al. 2022

ACS Appl. Bio Mater.

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Multicellular tumor spheroids have emerged as well-structured, three-dimensional culture models that resemble and mimic the complexity of the dense and hypoxic cancer microenvironment. However, in brain tumor studies, a variety of glioblastoma multiforme (GBM) cell lines only self-assemble into loose cellular aggregates, lacking the properties of actual glioma tumors in humans. In this study, we used type-I collagen as an extracellular matrix component to promote the compaction of GBM aggregates forming tight spheroids to understand how collagen influences the properties of tumors, such as their growth, proliferation, and invasion, and collagenase to promote collagen degradation. The GBM cell lines U87MG, T98G, and A172, as well as the medulloblastoma cell line UW473, were used as standard cell lines that do not spontaneously self-assemble into spheroids, and GBM U251 was used as a self-assembling cell line. According to the findings, all cell lines formed tight spheroids at collagen concentrations higher than 15.0 μg mL–1. Collagen was distributed along the spheroid, similarly to that observed in invasive GBM tumors, and decreased cell migration with no effect on the cellular uptake of small active molecules, as demonstrated by uptake studies using the photosensitizer verteporfin. The enzymatic cleavage of collagen affected spheroid morphology and increased cell migration while maintaining cell viability. Such behaviors are relevant to the physiological models of GBM tumors and are useful for better understanding cell migration and the in vivo infiltration path, drug screening, and kinetics of progression of GBM tumors.

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Section: Resultsmentioning

confidence: 99%

“…Type-I collagen might be a “linker” between collagen-binding integrins at downregulated E-cadherin expression cells, keeping cells in tight compaction. The role of N-cadherin was demonstrated to be similar in spheroid aggregation and compaction …”

Section: Results and Discussionmentioning

confidence: 99%

Type-I Collagen/Collagenase Modulates the 3D Structure and Behavior of Glioblastoma Spheroid Models

Calori

Alves

Hong

et al. 2022

ACS Appl. Bio Mater.

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“…TcnA treatment led to significantly (−log 10 p -value: 3) increased phosphorylation at this site with a log 2 regulation factor of 2.7 ( Figure 5 ). Pkn2 is a Rho/Rac effector protein which takes part in various cellular processes such as signal transduction, cell cycle progression, actin cytoskeleton assembly, cell adhesion and others [ 60 , 61 , 62 , 63 ]. Pkn2 is known for binding RhoA, subsequently promoting a variety of RhoA-regulated processes.…”

Section: Discussionmentioning

confidence: 99%

Clostridium novyi’s Alpha-Toxin Changes Proteome and Phosphoproteome of HEp-2 Cells

Schweitzer

Genth

Pich

2022

IJMS

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C. novyi type A produces the alpha-toxin (TcnA) that belongs to the large clostridial glucosylating toxins (LCGTs) and is able to modify small GTPases by N-acetylglucosamination on conserved threonine residues. In contrast, other LCGTs including Clostridioides difficile toxin A and toxin B (TcdA; TcdB) modify small GTPases by mono-o-glucosylation. Both modifications inactivate the GTPases and cause strong effects on GTPase-dependent signal transduction pathways and the consequent reorganization of the actin cytoskeleton leading to cell rounding and finally cell death. However, the effect of TcnA on target cells is largely unexplored. Therefore, we performed a comprehensive screening approach of TcnA treated HEp-2 cells and analyzed their proteome and their phosphoproteome using LC-MS-based methods. With this data-dependent acquisition (DDA) approach, 5086 proteins and 9427 phosphosites could be identified and quantified. Of these, 35 proteins were found to be significantly altered after toxin treatment, and 1832 phosphosites were responsive to TcnA treatment. By analyzing the TcnA-induced proteomic effects of HEp-2 cells, 23 common signaling pathways were identified to be altered, including Actin Cytoskeleton Signaling, Epithelial Adherens Junction Signaling, and Signaling by Rho Family GTPases. All these pathways are also regulated after application of TcdA or TcdB of C. difficile. After TcnA treatment the regulation on phosphorylation level was much stronger compared to the proteome level, in terms of both strength of regulation and the number of regulated phosphosites. Interestingly, various signaling pathways such as Signaling by Rho Family GTPases or Integrin Signaling were activated on proteome level while being inhibited on phosphorylation level or vice versa as observed for the Role of BRCA1 in DNA Damage Response. ZIP kinase, as well as Calmodulin-dependent protein kinases IV & II, were observed as activated while Aurora-A kinase and CDK kinases tended to be inhibited in cells treated with TcnA based on their substrate regulation pattern.

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“…Human iPSCs were seeded in 96-well ultra-low attachment plates to create a model of multicellular human liver organoids [45]. The liquid overlay technique was successfully employed to obtain other 3D cell cultures, such as neural organoids [46], fibroblast spheroids [47], cancer spheroids [48], and MSC spheroids [49].…”

Section: Liquid Overlaymentioning

confidence: 99%

Three-Dimensional Cell Cultures: The Bridge between In Vitro and In Vivo Models

Urzì,

Gasparro,

Costanzo

et al. 2023

IJMS

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Although historically, the traditional bidimensional in vitro cell system has been widely used in research, providing much fundamental information regarding cellular functions and signaling pathways as well as nuclear activities, the simplicity of this system does not fully reflect the heterogeneity and complexity of the in vivo systems. From this arises the need to use animals for experimental research and in vivo testing. Nevertheless, animal use in experimentation presents various aspects of complexity, such as ethical issues, which led Russell and Burch in 1959 to formulate the 3R (Replacement, Reduction, and Refinement) principle, underlying the urgent need to introduce non-animal-based methods in research. Considering this, three-dimensional (3D) models emerged in the scientific community as a bridge between in vitro and in vivo models, allowing for the achievement of cell differentiation and complexity while avoiding the use of animals in experimental research. The purpose of this review is to provide a general overview of the most common methods to establish 3D cell culture and to discuss their promising applications. Three-dimensional cell cultures have been employed as models to study both organ physiology and diseases; moreover, they represent a valuable tool for studying many aspects of cancer. Finally, the possibility of using 3D models for drug screening and regenerative medicine paves the way for the development of new therapeutic opportunities for many diseases.

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PKN2 is involved in aggregation and spheroid formation of fibroblasts in suspension culture by regulating cell motility and N-cadherin expression

Cited by 5 publications

References 34 publications

Type-I Collagen/Collagenase Modulates the 3D Structure and Behavior of Glioblastoma Spheroid Models

Type-I Collagen/Collagenase Modulates the 3D Structure and Behavior of Glioblastoma Spheroid Models

Clostridium novyi’s Alpha-Toxin Changes Proteome and Phosphoproteome of HEp-2 Cells

Three-Dimensional Cell Cultures: The Bridge between In Vitro and In Vivo Models

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