Reduced Cell Adhesion Research Articles

Anodic Aluminum Oxide Membrane Assisted Extrusion of Polymer Solutions for Hydrogel Formation

AbstractThe potential of polymeric nanostructures obtained by extruding polymer solutions through reusable anodic aluminum oxide (AAO) nanopores for the fabrication of nanostructures is investigated systematically to be able to unveil the potential impact of structure on cell culturing. Thus, nanostructured Na‐alginate and poly(vinyl alcohol) (PVA) polymers as well as blended alginate/PVA hydrogels are fabricated. Na‐alginate and PVA fibers with different dimensions can be obtained. By extruding Na‐alginate solutions into a non‐solvent also networks of uniform nanofibers (diameter <20 nm) are generated. PVA forms agglomerations of nanofibers and nanoparticles with diameter >100 nm. In addition, cell line‐dependent behavior of the cross‐linked PVA/alginate nanofibers is observed, which is characterized by reduced cell adhesion and cluster formation of PaTu 8988t cells on these blend nanofibers compared to normal cell adhesive behavior of NIH 3T3 cells.

Ipatasertib, an oral AKT inhibitor, in combination with carboplatin exhibits anti-proliferative effects in uterine serous carcinoma

Purpose Uterine serous carcinoma (USC) exhibits worse survival rates compared to the endometrioid subtype, and there is currently no effective treatment options for recurrence of this disease after platinum-based chemotherapy. Activation of PIK3CA/AKT/mTOR signaling pathway is a common biological feature in USC. Materials and Methods Ipatasertib (IPAT) is an investigational, orally administered, ATP-competitive, highly selective inhibitor of pan AKT that has demonstrated anti-proliferative activity in a variety of tumor cells and tumor models. In this study, we used IPAT, carboplatin and their combination to investigate the anti-tumor activity in SPEC-2 and ARK-1 cells. Results Our results indicate that IPAT combined with carboplatin at low doses was more effective at reducing proliferation, inducing apoptosis and causing cellular stress than IPAT or carboplatin alone. In particular, inhibition of the PIK3CA/AKT/mTOR pathway and induction of DNA damage were involved in the synergistic inhibition by combination treatment of cell viability in USC cells treated with the combination. Furthermore, IPAT in combination with carboplatin significantly reduced cell adhesion and inhibited cell invasion. Conclusions These findings suggest that the combination of IPAT and carboplatin has potential clinical implications for developing new USC treatment strategies.

The role of activated androgen receptor in cofilin phospho-regulation depends on the molecular subtype of TNBC cell line and actin assembly dynamics.

Triple negative breast cancer (TNBC) is highly metastatic and of poor prognosis. Metastasis involves coordinated actin filament dynamics mediated by cofilin and associated proteins. Activated androgen receptor (AR) is believed to contribute to TNBC tumorigenesis. Our current work studied roles of activated AR and cofilin phospho-regulation during migration of three AR+ TNBC cell lines to determine if altered cofilin regulation can explain their migratory differences. Untreated or AR agonist-treated BT549, MDA-MB-453, and SUM159PT cells were compared to cells silenced for cofilin (KD) or AR expression/function (bicalutamide). Cofilin-1 was found to be the only ADF/cofilin isoform expressed in each TNBC line. Despite a significant increase in cofilin kinase caused by androgens, the ratio of cofilin:p-cofilin (1:1) did not change in SUM159PT cells. BT549 and MDA-MB-453 cells contain high p-cofilin levels which underwent androgen-induced dephosphorylation through increased cofilin phosphatase expression, but surprisingly maintain a leading-edge with high p-cofilin/total cofilin not found in SUM159PT cells. Androgens enhanced cell polarization in all lines, stimulated wound healing and transwell migration rates and increased N/E-cadherin mRNA ratios while reducing cell adhesion in BT549 and MDA-MB-453 cells. Cofilin KD negated androgen effects in MDA-MB-453 except for cell adhesion, while in BT549 cells it abrogated androgen-reduced cell adhesion. In SUM159PT cells, cofilin KD with and without androgens had similar effects in almost all processes studied. AR dependency of the processes were confirmed. In conclusion, cofilin regulation downstream of active AR is dependent on which actin-mediated process is being examined in addition to being cell line-specific. Although MDA-MB-453 cells demonstrated some control of cofilin through an AR-dependent mechanism, other AR-dependent pathways need to be further studied. Non-cofilin-dependent mechanisms that modulate migration of SUM159PT cells need to be investigated. Categorizing TNBC behavior as AR responsive and/or cofilin dependent can inform on decisions for therapeutic treatment.

Breast tumor IGF1R regulates cell adhesion and metastasis: alignment of mouse single cell and human breast cancer transcriptomics.

The acquisition of a metastatic phenotype is the critical event that determines patient survival from breast cancer. Several receptor tyrosine kinases have functions both in promoting and inhibiting metastasis in breast tumors. Although the insulin-like growth factor 1 receptor (IGF1R) has been considered a target for inhibition in breast cancer, low levels of IGF1R expression are associated with worse overall patient survival. To determine how reduced IGF1R impacts tumor phenotype in human breast cancers, we used weighted gene co-expression network analysis (WGCNA) of Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) patient data to identify gene modules associated with low IGF1R expression. We then compared these modules to single cell gene expression analyses and phenotypes of mouse mammary tumors with reduced IGF1R signaling or expression in a tumor model of triple negative breast cancer. WGCNA from METABRIC data revealed gene modules specific to cell cycle, adhesion, and immune cell signaling that were inversely correlated with IGF1R expression in human breast cancers. Integration of human patient data with single cell sequencing data from mouse tumors revealed similar pathways necessary for promoting metastasis in basal-like mammary tumors with reduced signaling or expression of IGF1R. Functional analyses revealed the basis for the enhanced metastatic phenotype including alterations in E- and P-cadherins. Human breast and mouse mammary tumors with reduced IGF1R are associated with upregulation of several pathways necessary for promoting metastasis supporting the conclusion that IGF1R normally helps maintain a metastasis suppressive tumor microenvironment. We further found that reduced IGF1R signaling in tumor epithelial cells dysregulates cadherin expression resulting in reduced cell adhesion.

Different Extracellular β-Amyloid (1-42) Aggregates Differentially Impair Neural Cell Adhesion and Neurite Outgrowth through Differential Induction of Scaffold Palladin.

Extracellular amyloid β-protein (1-42) (Aβ42) aggregates have been recognized as toxic agents for neural cells in vivo and in vitro. The aim of this study was to investigate the cytotoxic effects of extracellular Aβ42 aggregates in soluble (or suspended, SAβ42) and deposited (or attached, DAβ42) forms on cell adhesion/re-adhesion, neurite outgrowth, and intracellular scaffold palladin using the neural cell lines SH-SY5Y and HT22, and to elucidate the potential relevance of these effects. The effect of extracellular Aβ42 on neural cell adhesion was directly associated with their neurotrophic or neurotoxic activity, with SAβ42 aggregates reducing cell adhesion and associated live cell de-adherence more than DAβ42 aggregates, while causing higher mortality. The reduction in cell adhesion due to extracellular Aβ42 aggregates was accompanied by the impairment of neurite outgrowth, both in length and number, and similarly, SAβ42 aggregates impaired the extension of neurites more severely than DAβ42 aggregates. Further, the disparate changes of intracellular palladin induced by SAβ42 and DAβ42 aggregates, respectively, might underlie their aforementioned effects on target cells. Further, the use of anti-oligomeric Aβ42 scFv antibodies revealed that extracellular Aβ42 aggregates, especially large DAβ42 aggregates, had some independent detrimental effects, including physical barrier effects on neural cell adhesion and neuritogenesis in addition to their neurotoxicity, which might be caused by the rigid C-terminal clusters formed between adjacent Aβ42 chains in Aβ42 aggregates. Our findings, concerning how scaffold palladin responds to extracellular Aβ42 aggregates, and is closely connected with declines in cell adhesion and neurite outgrowth, provide new insights into the cytotoxicity of extracellular Aβ42 aggregates in Alzheimer disease.

TRIM67 drives tumorigenesis in oligodendrogliomas through Rho GTPase-dependent membrane blebbing.

IDH mutant gliomas are grouped into astrocytomas or oligodendrogliomas depending on the codeletion of chromosome arms 1p and 19q. Although the genomic alterations of IDH mutant gliomas have been well described, transcriptional changes unique to either tumor type have not been fully understood. Here, we identify Tripartite Motif Containing 67 (TRIM67), an E3 ubiquitin ligase with essential roles during neuronal development, as an oncogene distinctly upregulated in oligodendrogliomas. We used several cell lines, including patient-derived oligodendroglioma tumorspheres, to knock down or overexpress TRIM67. We coupled high-throughput assays, including RNA sequencing, total lysate-mass spectrometry (MS), and coimmunoprecipitation (co-IP)-MS with functional assays including immunofluorescence (IF) staining, co-IP, and western blotting (WB) to assess the in vitro phenotype associated with TRIM67. Patient-derived oligodendroglioma tumorspheres were orthotopically implanted in mice to determine the effect of TRIM67 on tumor growth and survival. TRIM67 overexpression alters the abundance of cytoskeletal proteins and induces membrane bleb formation. TRIM67-associated blebbing was reverted with the nonmuscle class II myosin inhibitor blebbistatin and selective ROCK inhibitor fasudil. NOGO-A/Rho GTPase/ROCK2 signaling is altered upon TRIM67 ectopic expression, pointing to the underlying mechanism for TRIM67-induced blebbing. Phenotypically, TRIM67 expression resulted in higher cell motility and reduced cell adherence. In orthotopic implantation models of patient-derived oligodendrogliomas, TRIM67 accelerated tumor growth, reduced overall survival, and led to increased vimentin expression at the tumor margin. Taken together, our results demonstrate that upregulated TRIM67 induces blebbing-based rounded cell morphology through Rho GTPase/ROCK-mediated signaling thereby contributing to glioma pathogenesis.

Downregulation of odontogenic ameloblast-associated protein in the progression of periodontal disease affects cell adhesion, proliferation, and migration

ObjectiveThis work aimed to examine changes in odontogenic ameloblast-associated protein (ODAM) expression during the progression of periodontal disease and to investigate the effect of ODAM deficiency in vitro by RNA sequencing. DesignGingival tissue samples were collected from three groups, including healthy control, gingivitis and periodontitis patients, and ODAM expression was assessed by immunohistochemistry and quantitative reverse transcription PCR (qRT-PCR). Then, an Odam-knockdown cell line was established by lentiviral infection of small guide RNAs (sgRNAs) into an immortalized ameloblast-lineage cell line. RNA sequencing was carried out in Odam-knockdown and empty lentiviral vector-transfected cells. Differentially expressed genes were compared between these two cell groups to analyze functional enrichment, and a protein-protein interaction network was built. ResultsODAM expression levels in gingival tissue samples were significantly lower in patients with periodontitis than in healthy controls as determined by immunohistochemistry and qRT-PCR. Transcriptomic analysis of Odam-knockdown cells identified 2801 differentially expressed genes, which were enriched in cell-substrate adhesion, proliferation, and migration pathways. The expression of a core of differentially expressed genes was confirmed by qRT-PCR in Odam-knockdown cells and by immunohistochemistry in clinical samples. Knocking down Odam significantly reduced cell adhesion but increased cell proliferation and migration capacity in vitro. ConclusionsODAM is important in cell adhesion, proliferation, and migration, and its downregulation may contribute to periodontitis progression in humans.

Azacitidine and Panobinostat Overcome Bone Marrow Microenvironment-Induced Chemoprotection in KMT2A-rearranged Pediatric AML

KMT2A is the most common gene rearrangement in pediatric acute leukemias and is difficult to treat with a five-year overall survival rate of less than 50%, owing to frequent relapse and poor response to chemotherapy. Recent studies have investigated how interactions within the bone marrow microenvironment help AML cells evade chemotherapy and contribute to relapse by promoting the survival of leukemic blasts. Our group has previously identified that epigenetic drug combination of DNA hypomethylating agent azacitidine and histone deacetylase inhibitor panobinostat reduces cell adhesion and promotes mobilization of leukemic cells to peripheral blood (Weber et al., 132:2637, Blood, 2018). In this study, we used a multi cell coculture of pediatric AML cells with bone marrow stromal cells, endothelial cells and pediatric mesenchymal stem cells to better mimic the bone marrow microenvironment. We observed that the chemoprotection index in multi-cell coculture was much higher than individual cell type (Figure 1A). While 1.5 uM cytarabine reduced MV4;11 cell viability by 50%, there was less than 1% reduction in cell viability in multi-cell coculture. We further investigated if the bone marrow microenvironment induced chemoprotection was mediated by soluble factors, or via adhesive interactions between adherent cell layer and/or the extracellular matrix (ECM). Conditioned media failed to afford chemoprotection, while separation of AML cells from multi-cell layer via Transwells or culturing AML cells in Matrigel-coated dishes showed partial chemoprotection. These results indicated that direct cell-cell or cell-ECM contact was necessary for the chemoprotection. Pre-treatment with azacitidine and panobinostat sensitized five distinct patient-derived xenograft (PDX) lines of KMT2A-rearranged AML to both cytarabine and daunorubicin in multi-cell coculture. These data indicate that azacitidine and panobinostat combination can synergistically overcome chemoprotection mediated by stromal, endothelial and mesenchymal stem cells. We also observed improved survival with pre-treatment of azacitidine panobinostat (2.5 mg/Kg each, Qd5) followed by chemotherapy consisting of cytarabine (50 mg/Kg, Qd5) and daunorubcin (1.5 mg/Kg, Qd3) in a PDX model of KMT2A-rearranged AML (Figure 1B, **P<0.05). To determine if these epigenetic modifers alter the expression of cell adhesion molecules, cells treated with azacitidine panobinostat combination were subjected to a high throughput flow cytometry assay (Lyoplate, Human cell surface marker screening panel). This analysis showed reduced cell surface expression of integrins, ICAMs and chemokine receptors, indicating that epigenetic drug combination likely causes mobilization and chemosensitization via downregulation of cell adhesion molecules which not only mediate adhesive interactions but also promote cell survival. In summary, our data show azacitidine and panobinostat can overcome bone marrow microenvironment induced chemoprotection in vitro and in vivo in KMT2A rearranged AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

20S-ginsenoside Rg3 inhibits the biofilm formation and haemolytic activity of Staphylococcus aureus by inhibiting the SaeR/SaeS two-component system.

Introduction. Staphylococcus aureus is a major cause of chronic diseases and biofilm formation is a contributing factor. 20S-ginsenoside Rg3 (Rg3) is a natural product extracted from the traditional Chinese medicine red ginseng.Gap statement. The effects of Rg3 on biofilm formation and haemolytic activity as well as its antibacterial mechanism against S. aureus have not been reported.Aim. This study aimed to investigate the effects of Rg3 on biofilm formation and haemolytic activity as well as its antibacterial action against clinical S. aureus isolates.Methodology. The effect of Rg3 on biofilm formation of clinical S. aureus isolates was studied by crystal violet staining. Haemolytic activity analysis was carried out. Furthermore, the influence of Rg3 on the proteome profile of S. aureus was studied by quantitative proteomics to clarify the mechanism underlying its antibacterial action and further verified by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR).Results. Rg3 significantly inhibited biofilm formation and haemolytic activity in clinical S. aureus isolates. A total of 63 with >1.5-fold changes in expression were identified, including 34 upregulated proteins and 29 downregulated proteins. Based on bioinformatics analysis, the expression of several virulence factors and biofilm-related proteins, containing CopZ, CspA, SasG, SaeR/SaeS two-component system and SaeR/SaeS-regulated proteins, including leukocidin-like protein 2, immunoglobulin-binding protein G (Sbi) and fibrinogen-binding protein, in the S. aureus of the Rg3-treated group was downregulated. RT-qPCR confirmed that Rg3 inhibited the regulation of SaeR/SaeS and decreased the transcriptional levels of the biofilm-related genes CopZ, CspA and SasG.Conclusions. Rg3 reduces the formation of biofilm by reducing cell adhesion and aggregation. Further, Rg3 can inhibit the SaeR/SaeS two-component system, which acts as a crucial signal transduction system for the anti-virulence activity of Rg3 against clinical S. aureus isolates.

Matrix metalloproteinase 2 is a target of the RAN-GTP pathway and mediates migration, invasion and metastasis in human breast cancer

RAS-related nuclear protein(RAN) is a nuclear shuttle and normally regulates events in the cell cycle. When overexpressed in cultured cells, it causes increases in cell migration/invasion in vitro and its overexpression is associated with early breast cancer patient deaths in vivo. However, the underlying mechanism is unknown. The effect of RAN overexpression on potential targets MMP2, ATF3, CXCR3 was investigated by Real-Time PCR/Western blots in the triple receptor negative breast cancer(TRNBC) cell line MDA-MB231 and consequent biological effects were measured by cell adhesion, cell migration and cell invasion assays. Results showed that knockdown of RAN lead to a reduction of MMP2 and its potential regulators ATF3 and CXCR3. Moreover, knockdown of ATF3 or CXCR3 downregulated MMP2 without affecting RAN, indicating that RAN regulates MMP2 through ATF3 and CXCR3. Knockdown of RAN and MMP2 reduced cell adhesion, cell migration and cell growth in agar, whilst overexpression of MMP2 reversed the knockdown of RAN. Furthermore, immunohistochemical staining for RAN and MMP2 are positively associated with each other in the same tumour and separately with patient survival times in breast cancer specimens, suggesting that a high level of RAN may be a pre-requisite for MMP2 overexpression and metastasis. Moreover, positive immunohistochemical staining for both RAN and MMP-2 reduces further patient survival times over that for either protein separately. Our results suggest that MMP2 expression can stratify progression of breast cancers with a high and low incidence of RAN, both RAN and MMP2 in combination can be used for a more accurate patient prognosis. Simple summaryRan is an important regulator of normal cell growth and behaviour. We have established in cell line models of breast cancer (BC) a molecular pathway between RAN and its protein-degrading effector MMP-2 and properties related to metastasis in culture. Using immunohistochemistry (IHC) staining of primary BCs, we have shown that RAN and MMP-2 are on their own significantly associated with patient demise from metastatic BC. Moreover, when staining for MMP-2 is added to that for RAN in the primary tumours, there is a significant decrease in patient survival time over that for either protein alone. Thus a combination of staining for RAN and MMP2 is an excellent marker for poor prognosis in breast cancer.

Green synthesized nickel doped cobalt ferrite nanoparticles exhibit antibacterial activity and induce reactive oxygen species mediated apoptosis in MCF-7 breast cancer cells through inhibition of PI3K/Akt/mTOR pathway.

This study aimed to synthesize the nickel (Ni) doped cobalt ferrite (CFO) nanoparticles and to determine its anticancer effect on breast cancer MCF-7 cells. The various concentrations of Ni (0.2%, 0.4%, 0.6%, 0.8%, and 1%) doped CFO nanoparticles were synthesized using Coriandrum sativum extracts by precipitation technique. The synthesized Ni-CFO was characterized by X-ray diffraction, Fourier transform infrared spectroscopic, transmission electron microscopy, and vibrating sample magnetometer analyses. The results show that 0.8% Ni-CFO nanoparticles showed good magnetic properties and antioxidant activity than other concentrations of Ni-CFO. The results showed that the administration of 0.8% Ni-CFO nanoparticles promoted apoptosis, and reduced cell adhesion and migration of MCF-7 cells, as demonstrated by increased lipid peroxidation, decreased antioxidant levels such as superoxide dismutase, catalase, and glutathione peroxidase, and increased formation of reactive oxygen species. Moreover, administration of 0.8% Ni-CFO nanoparticles decreased the Bcl-2 expression while activating the expression of p53, Bax, and cleaved caspase 3, 8 and 9 protein expression. Notably, 0.8% Ni-CFO treatment reduced phospho-PI3K, phospho-Akt, and phospho-mTOR expression levels. As a result, via altering apoptotic related proteins, 0.8% Ni-CFO induced cell death. Therefore, the 0.8% Ni-CFO could be utilized to treat breast cancer.

A simple integrated microfluidic platform for the research of hydrogels containing gradients in cell density induced breast cancer electrochemotherapy

Cell density is important for tumour metastasis, treatment and prognosis. Characterizing changes in cell density for electrochemotherapy (ECT) can reveal sub-populations in pathological states, and adjust treatment program. In this work, a simple and convenient microfluidic platform was developed to study the effect cell density on ECT by integrating the improved cell gradient generator, cell culture chamber and indium tin oxide interdigital electrodes. Agarose, as extracellular matrix (ECM), was used to 3D cell culture to imitate in vivo microenvironment. The precision and reproducibility of cell density gradient with agarose solution were achieved because the hydrophobic modification of microchannels surface resulted in reducing cell adhesion and residue. ECT cytotoxicity assay with difference in cell densities was studied. The results showed that tumour cell density is one of the most factors for ECT treatment and ECT cytotoxicity has a certain of cell density-depended. But only electroporation on low cell density level, ECM would be one of the most key factors for ECT cytotoxicity, which would provide a new idea for chip-based cell assay in clinical diagnosis and drug screening in ordinary laboratories.

β1 Integrin induces adhesion and migration of human Th17 cells via Pyk2-dependent activation of P2X4 receptor.

Integrin-mediated T-cell adhesion and migration is a crucial step in immune response and autoimmune diseases. However, the underlying signalling mechanisms are not fully elucidated. In this study, we examined the implication of purinergic signalling, which has been associated with T-cell activation, in the adhesion and migration of human Th17 cells across fibronectin, a major matrix protein associated with inflammatory diseases. We showed that the adhesion of human Th17 cells to fibronectin induces, via β1 integrin, a sustained release of adenosine triphosphate (ATP) from the mitochondria through the pannexin-1 hemichannels. Inhibition of ATP release or its degradation with apyrase impaired the capacity of the cells to attach and migrate across fibronectin. Inhibition studies identified a major role for the purinergic receptor P2X4 in T-cell adhesion and migration but not for P2X7 or P2Y11 receptors. Blockade of P2X4 but not P2X7 or P2Y11 receptors reduced cell adhesion and migration by inhibiting activation of β1 integrins, which is essential for ligand binding. Furthermore, we found that β1 integrin-induced ATP release, P2X4 receptor transactivation, cell adhesion and migration were dependent on the focal adhesion kinase Pyk2 but not FAK. Finally, P2X4 receptor inhibition also blocked fibronectin-induced Pyk2 activation suggesting the existence of a positive feedback loop of activation between β1 integrin/Pyk2 and P2X4 purinergic signalling pathways. Our findings uncovered an unrecognized link between β1 integrin and P2X4 receptor signalling pathways for promoting T-cell adhesion and migration across the extracellular matrix.

Development of Enzymatic-Resistant and Compliant Decellularized Extracellular Matrixes via Aliphatic Chain Modification for Bladder Tissue Engineering.

Here, we report the design and development of highly stretchable, compliant, and enzymatic-resistant transiently cross-linked decellularized extracellular matrixes (dECMs) (e.g., porcine small intestine submucosa/dSIS, urinary bladder matrix/dUBM, bovine pericardium/dBP, bovine dermis/dBD, and human dermis/dHD). Specifically, these dECMs were modified with long aliphatic chains (C9, C14, and C18). Upon modification, dECMs became significantly resistant to enzymatic degradation for extended periods, showed increased water contact angle (>20%-90%), and stretched >200% than their control counterparts. Modified dECMs are compliant, undergoing 100% elongation at only 0.3-0.5 MPa of applied tensile stress (∼10%-25% of their control counterparts), similar to the control bladder tissue. Furthermore, modified dECMs remain structurally stable at the physiological temperature with increased storage and loss modulus values but decreased tan δ values compared to their control counterparts. Although modification reduces cell adhesion, the gene expressions in polarized macrophages remain unchanged (e.g., TGFβ, CD163, and CD86), except for the modified bovine pericardium (dBP) where a significant decrease in TNFα gene expression is observed. When implanted in the rat subcutaneous model, modified dECMs degraded relatively slowly and did not cause significant fibrotic tissue formation. The numbers of pro-regenerative macrophages increased to several folds in a later time point of evaluation. Modified dECM also supported the bladder wall regeneration with formations of the urothelium, lamina propria, blood vessels, and muscle bundles and reduced the occurrence of calculi formation by 50% in a rat bladder augmentation model. We anticipate that the enhanced stretchability, compliance, and physiological stability of dECMs indicate their suitability for urologic tissue regeneration.

The CBP/β-Catenin Antagonist, ICG-001, Inhibits Tumor Metastasis via Blocking of the miR-134/ITGB1 Axis-Mediated Cell Adhesion in Nasopharyngeal Carcinoma.

Simple SummaryMetastatic nasopharyngeal carcinoma (NPC) is incurable and remains the main cause of NPC death. Our previous studies found that the CBP/β-catenin Wnt antagonist, IGC-001, could inhibit the primary tumor formation of NPC tumor cells. Here, we further explored the anti-metastatic activity of ICG-001. We started by screening a panel of microRNAs that are related to epithelial–mesenchymal transition and cancer stem cell phenotypes; both properties can contribute to tumor metastasis. MicroRNA-134 was found to be consistently upregulated by ICG-001. The role of miR-134 in NPC is largely unknown but some studies found an association between low expression of miR-134 and poor prognosis. We examined the role of miR-134 in NPC with both in vitro and in vivo models and found that miR-134 could inhibit cancer cell adhesion, migration, and invasion. Our study provided a functional explanation for the poor prognosis observed in NPC patients with low or loss of miR-134 expression in their tumors and showed that modulation of the Wnt signaling by ICG-001 could effectively inhibit NPC metastasis via the miR-134/ITGB1 axis.Nasopharyngeal carcinoma (NPC) is an Epstein–Barr virus (EBV)-associated malignancy ranking as the 23rd most common cancer globally, while its incidence rate ranked the 9th in southeast Asia. Tumor metastasis is the dominant cause for treatment failure in NPC and metastatic NPC is yet incurable. The Wnt/β-catenin signaling pathway plays an important role in many processes such as cell proliferation, differentiation, epithelial–mesenchymal transition (EMT), and self-renewal of stem cells and cancer stem cells (CSCs). Both the EMT process and CSCs are believed to play a critical role in cancer metastasis. We here investigated whether the specific CBP/β-catenin Wnt antagonist, IGC-001, affects the metastasis of NPC cells. We found that ICG-001 treatment could reduce the adhesion capability of NPC cells to extracellular matrix and to capillary endothelial cells and reduce the tumor cell migration and invasion, events which are closely associated with distant metastasis. Through a screening of EMT and CSC-related microRNAs, it was found that miR-134 was consistently upregulated by ICG-001 treatment in NPC cells. Very few reports have mentioned the functional role of miR-134 in NPC, except that the expression was found to be downregulated in NPC. Transient transfection of miR-134 into NPC cells reduced their cell adhesion, migration, and invasion capability, but did not affect the growth of CSC-enriched tumor spheres. Subsequently, we found that the ICG-001-induced miR-134 expression resulting in downregulation of integrin β1 (ITGB1). Such downregulation reduced cell adhesion and migration capability, as demonstrated by siRNA-mediated knockdown of ITGB1. Direct targeting of ITGB1 by miR-134 was confirmed by the 3′-UTR luciferase assay. Lastly, using an in vivo lung metastasis assay, we showed that ICG-001 transient overexpression of miR-134 or stable overexpression of miR-134 could significantly reduce the lung metastasis of NPC cells. Taken together, we present here evidence that modulation of Wnt/β-catenin signaling pathway could inhibit the metastasis of NPC through the miR-134/ITGB1 axis.

Genome‑wide expression and methylation analyses reveal aberrant cell adhesion signaling in tyrosine kinase inhibitor‑resistant CML cells

Although chronic myeloid leukemia (CML) can be effectively treated using BCR-ABL1 kinase inhibitors, resistance due to kinase alterations or to BCR-ABL1 independent mechanisms remain a therapeutic challenge. For the latter, the underlying mechanisms are widely discussed; for instance, gene expression changes, epigenetic factors and alternative signaling pathway activation. In the present study, in vitro-CML cell models of resistance against the tyrosine kinase inhibitors (TKIs) imatinib (0.5 and 2 µM) and nilotinib (0.1 µM) with biological replicates were generated to identify novel mechanisms of resistance. Subsequently, genome-wide mRNA expression and DNA methylation were analyzed. While mRNA expression patterns differed largely between biological replicates, there was an overlap of 71 genes differentially expressed between cells resistant against imatinib or nilotinib. Moreover, all TKI resistant cell lines demonstrated a slight hypermethylation compared with native cells. In a combined analysis of 151 genes differentially expressed in the biological replicates of imatinib resistance, cell adhesion signaling, in particular the cellular matrix protein fibronectin 1 (FN1), was significantly dysregulated. This gene was also downregulated in nilotinib resistance. Further analyses showed significant FN1-downregulation in imatinib resistance on mRNA (P<0.001) and protein level (P<0.001). SiRNA-mediated FN1-knockdown in native cells reduced cell adhesion (P=0.02), decreased imatinib susceptibility visible by higher Ki-67 expression (1.5-fold, P=0.04) and increased cell number (1.5-fold, P=0.03). Vice versa, recovery of FN1-expression in imatinib resistant cells was sufficient to partially restore the response to imatinib. Overall, these results suggested a role of cell adhesion signaling and fibronectin 1 in TKI resistant CML and a potential target for novel strategies in treatment of resistant CML.

Comprehensively characterizing cellular changes and the expression of THSD7A and PLA2R1 under multiple in vitro models of podocyte injury.

The unique morphology and gene expression of podocytes are critical for kidney function, and their abnormalities lead to nephropathies such as diabetic nephropathy and membranous nephropathy. Podocytes cultured in vitro are valuable tools to dissect the molecular mechanism of podocyte injury relative to nephropathy, however, these models have never been comprehensively compared. Here, we comprehensively compared the morphology, cytoskeleton, cell adhesion, cell spreading, cell migration, and lipid metabolism under five commonly used in vitro models including lipopolysaccharide (LPS), puromycin aminonucleoside (PAN), doxorubicin (Dox), high glucose, and glucose deprivation. Our results indicate that all stimulations significantly downregulate the expression of synaptopodin both in human and mouse podocytes. All stimulations affect podocyte morphology but show different intensity and phenotypes. In general, thefive stimulations reduce cell adhesion, cell spreading, and cell migration, but the effect in human and mouse podocytes is slightly different. Human podocytes show high expression of genes enriched in the pentose phosphate pathway. Dox and PAN treatment show a strong effect on gene expression in lipid metabolism, while the other three stimulations show minimal effect. The expression of phospholipase A2 receptor (PLA2R1) and type-1 domain-containing protein 7 A (THSD7A) show opposite trends in given cells. Stimulations can dramatically affect the expression of PLA2R1 and THSD7A. Inhibition of super-enhancers reduces PLA2R1 and THSD7A expression, but ERK inhibition enhances their expression. Our results demonstrate distinctive responses in five commonly used in vitro podocyte injury models and the dynamic expression of PLA2R1 and THSD7A, which supply novel information to select suitable podocyte injury models.

Responses of promyelocytic leukemia HL60 cells as an inflammatory cell lineage model to silica microparticles used to coat blood collection tubes

BackgroundThe preparation of platelet-rich fibrin (PRF) requires glass blood collection tubes, and thus, the shortage or unavailability of such tubes has driven clinicians to search for suitable substitutes, such as silica-coated plastic tubes. However, we have previously demonstrated the cytotoxicity of silica microparticles (MPs) used in plastic tubes to cultured human periosteal cells. To further establish the effects of silica MPs on inflammation, we examined silica MP-induced changes in a human promyelocytic cell model in vitro.MethodsHuman promyelocytic HL60 cells were used either without chemical induction or after differentiation induced using phorbol myristate acetate (PMA) or dimethyl sulfoxide. HL60 cells, osteoblastic MG63, and Balb/c mouse cells were treated with silica MPs, and their surface ultrastructure and numbers were examined using a scanning electron microscope and an automated cell counter, respectively. Differentiation markers, such as acid phosphatase, non-specific esterase, and CD11b, were visualized by cytochemical and immunofluorescent staining, and superoxide dismutase (SOD) activity was quantified.ResultsRegardless of SOD activity, silica cytotoxicity was observed in MG63 and Balb/c cells. At sub-toxic doses, silica MPs slightly or moderately upregulated the differentiation markers of the control, PMA-induced monocytic, and dimethyl sulfoxide-induced granulocytic HL60 cells. Although SOD activity was the highest (P < 0.05) in PMA-induced cells, a silica-induced reduction in cell adhesion was observed only in those cells (P < 0.05).ConclusionsSilica MP contamination of PRF preparations can potentially exacerbate inflammation at implantation sites. Consequently, unless biomedical advantages can be identified, silica-coated plastic blood collection tubes should not be routinely used for PRF preparations.

Nascent Proteome and Glycoproteome Reveal the Inhibition Role of ALG1 in Hepatocellular Carcinoma Cell Migration.

The online version contains supplementary material available at 10.1007/s43657-022-00050-5.

Kinase Responsive to Stress B negatively regulates Rap1 in Dictyostelium discoideum

Dynamic changes in cell adhesion to the substrate are essential for migration, although details of the pathways regulating adhesion, especially for amoeboid‐type migration are lacking. Social amoeba Dictyostelium discoideum is a commonly used model organism for the study of directed migration since its movement is very similar to that of other amoeboid cells, such as neutrophils and metastatic cancer cells. Rap1 is a small GTPase that regulates adhesion in Dictyostelium cells in part via its effects on myosin II and talin. Kinase responsive to stress B (KrsB), a homolog of mammalian tumor suppressor MST1/2 and DrosophilaHippo, also regulates cell adhesion and migration, although the molecular mechanism of KrsB action is not understood. Since KrsB has been shown to interact with active Rap1 by mass spectroscopy, we investigated the genetic interaction between Rap1 and KrsB. Cells lacking KrsB have increased contact with the substrate and are difficult to detach from the surface, which leads to reduced movement. Expression of constitutively active Rap1G12V increased cell adhesion, and inactive Rap1S17N reduced cell adhesion even in the absence of KrsB, suggesting that Rap1 does not require KrsB to mediate cell adhesion. However, Rap1G12V did not increase cell spreading when KrsB was overexpressed and Rap1‐mediated increase in spreading was dramatically enhanced in the absence of KrsB, suggesting that KrsB might negatively regulate Rap1. In addition, chemoattractant‐induced activation of Rap1, as assessed by transient cortical localization of the biosensor RalGDS, was impaired in krsB¯cells, possibly due to increased basal activity or membrane localization of Rap1. Since mammalian Rap1 localization can be regulated by phosphorylation, KrsB might phosphorylate Rap1, thereby disrupting its activation on the membrane. Overall, our study demonstrates that KrsB negatively regulates Rap1 signaling, and we are currently investigating the molecular mechanism of this regulation.