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American Journal of Physiology. Cell... Jun 2024NBCn1 (SLC4A7) is one of the two major Na-HCO cotransporters in the human colonic epithelium, expressed predominantly in the highly proliferating colonocytes at the...
NBCn1 (SLC4A7) is one of the two major Na-HCO cotransporters in the human colonic epithelium, expressed predominantly in the highly proliferating colonocytes at the cryptal base. Increased NBCn1 expression levels are reported in tumors, including colorectal cancer. The study explores its importance for maintenance of the intracellular pH (pH), as well as the proliferative, adhesive, and migratory behavior of the self-differentiating Caco2BBe colonic tumor cell line. In the self-differentiating Caco2BBe cells, mRNA was highly expressed from the proliferative stage until full differentiation. The downregulation of NBCn1 expression by RNA interference affected proliferation and differentiation and decreased intracellular pH (pH) of the cells in correlation with the degree of knockdown. In addition, a disturbed cell adhesion and reduced migratory speed were associated with NBCn1 knockdown. Murine colonic enteroids also displayed reduced proliferative activity. In the migrating Caco2BBe cells, NBCn1 was found at the leading edge and in colocalization with the focal adhesion markers vinculin and paxillin, which suggests that NBCn1 is involved in the establishment of cell-matrix adhesion. Our data highlight the physiological significance of NBCn1 in modulating epithelial pH homeostasis and cell-matrix interactions in the proliferative region of the colonic epithelium and unravel the molecular mechanism behind pathological overexpression of this transporter in human colorectal cancers. The transporter NBCn1 plays a central role in maintaining homeostasis within Caco2BBe colonic epithelial cells through its regulation of intracellular pH, matrix adhesion, migration, and proliferation. These observations yield valuable insights into the molecular mechanism of the aberrant upregulation of this transporter in human colorectal cancers.
Topics: Humans; Sodium-Bicarbonate Symporters; Cell Movement; Animals; Cell Proliferation; Hydrogen-Ion Concentration; Caco-2 Cells; Colon; Enterocytes; Cell Adhesion; Mice; Mice, Knockout; Cell Differentiation; Mice, Inbred C57BL
PubMed: 38646790
DOI: 10.1152/ajpcell.00079.2024 -
Bioorganic & Medicinal Chemistry Letters Jun 2024The naturally occurring bile acid lithocholic acid (LCA) has been a crucial core structure for many non-sugar-containing sialyltranferase (ST) inhibitors documented in...
Biological evaluation of sulfonate and sulfate analogues of lithocholic acid: A bioisosterism-guided approach towards the discovery of potential sialyltransferase inhibitors for antimetastatic study.
The naturally occurring bile acid lithocholic acid (LCA) has been a crucial core structure for many non-sugar-containing sialyltranferase (ST) inhibitors documented in literature. With the aim of elucidating the impact of the terminal carboxyl acid substituent of LCA on its ST inhibition, in this present study, we report the (bio)isosteric replacement-based design and synthesis of sulfonate and sulfate analogues of LCA. Among these compounds, the sulfate analogue SPP-002 was found to selectively inhibit N-glycan sialylation by at least an order of magnitude, indicating a substantial improvement in both potency and selectivity when compared to the unmodified parent bile acid. Molecular docking analysis supported the stronger binding of the synthetic analogue in the enzyme active site. Treatment with SPP-002 also hampered the migration, adhesion, and invasion of MDA-MB-231 cells in vitro by suppressing the expression of signaling proteins involved in the cancer metastasis-associated integrin/FAK/paxillin pathway. In totality, these findings offer not only a novel structural scaffold but also valuable insights for the future development of more potent and selective ST inhibitors with potential therapeutic effects against tumor cancer metastasis.
Topics: Lithocholic Acid; Humans; Sialyltransferases; Cell Line, Tumor; Molecular Docking Simulation; Cell Movement; Enzyme Inhibitors; Structure-Activity Relationship; Sulfates; Neoplasm Metastasis; Sulfonic Acids; Antineoplastic Agents; Molecular Structure; Cell Adhesion; Dose-Response Relationship, Drug; Paxillin; Focal Adhesion Kinase 1; Drug Discovery
PubMed: 38641151
DOI: 10.1016/j.bmcl.2024.129760 -
Gastroenterology Apr 2024High expression of phosphatidylinositol 4-kinase III alpha (PI4KIIIα) correlates with poor survival rates in patients with hepatocellular carcinoma. In addition,...
BACKGROUND & AIMS
High expression of phosphatidylinositol 4-kinase III alpha (PI4KIIIα) correlates with poor survival rates in patients with hepatocellular carcinoma. In addition, hepatitis C virus (HCV) infections activate PI4KIIIα and contribute to hepatocellular carcinoma progression. We aimed at mechanistically understanding the impact of PI4KIIIα on the progression of liver cancer and the potential contribution of HCV in this process.
METHODS
Several hepatic cell culture and mouse models were used to study the functional importance of PI4KIIIα on liver pathogenesis. Antibody arrays, gene silencing, and PI4KIIIα-specific inhibitor were applied to identify the involved signaling pathways. The contribution of HCV was examined by using HCV infection or overexpression of its nonstructural protein.
RESULTS
High PI4KIIIα expression and/or activity induced cytoskeletal rearrangements via increased phosphorylation of paxillin and cofilin. This led to morphologic alterations and higher migratory and invasive properties of liver cancer cells. We further identified the liver-specific lipid kinase phosphatidylinositol 3-kinase C2 domain-containing subunit gamma (PIK3C2γ) working downstream of PI4KIIIα in regulation of the cytoskeleton. PIK3C2γ generates plasma membrane phosphatidylinositol 3,4-bisphosphate-enriched, invadopodia-like structures that regulate cytoskeletal reorganization by promoting Akt2 phosphorylation.
CONCLUSIONS
PI4KIIIα regulates cytoskeleton organization via PIK3C2γ/Akt2/paxillin-cofilin to favor migration and invasion of liver cancer cells. These findings provide mechanistic insight into the contribution of PI4KIIIα and HCV to the progression of liver cancer and identify promising targets for therapeutic intervention.
PubMed: 38636680
DOI: 10.1053/j.gastro.2024.04.009 -
BioRxiv : the Preprint Server For... Mar 2024Breast cancers are categorized into subtypes with distinctive therapeutic vulnerabilities and prognoses based on their expression of clinically targetable receptors and...
Breast cancers are categorized into subtypes with distinctive therapeutic vulnerabilities and prognoses based on their expression of clinically targetable receptors and gene expression patterns mimicking different cell types of the normal gland. Here, we tested the role of Mcam in breast cancer cell state control and tumorigenicity in a luminal progenitor-like murine tumor cell line (Py230) that exhibits lineage and tumor subtype plasticity. Mcam knockdown Py230 cells show augmented Stat3 and Pi3K/Akt activation associated with a lineage state switch away from a hormone-sensing/luminal progenitor state toward alveolar and basal cell related phenotypes that were refractory to growth inhibition by the anti-estrogen therapeutic, tamoxifen. Inhibition of Stat3, or the upstream activator Ck2, reversed these cell state changes. Mcam binds Ck2 and acts as a regulator of Ck2 substrate utilization across multiple mammary tumor cell lines. In Py230 cells this activity manifests as increased mesenchymal morphology, migration, and Src/Fak/Mapk/Paxillin adhesion complex signaling , in contrast to Mcam's reported roles in promoting mesenchymal phenotypes. , Mcam knockdown reduced tumor growth and take rate and inhibited cell state transition to Sox10+/neural crest like cells previously been associated with tumor aggressiveness. This contrasts with human luminal breast cancers where MCAM copy number loss is highly coupled to Cyclin D amplification, increased proliferation, and the more aggressive Luminal B subtype. Together these data indicate a critical role for Mcam and its regulation of Ck2 in control of breast cancer cell state plasticity with implications for progression, evasion of targeted therapies and combination therapy design.
PubMed: 38562809
DOI: 10.1101/2023.05.10.540211 -
BioRxiv : the Preprint Server For... Mar 2024Many cells adhere to extracellular matrix for efficient cell migration. This adhesion is mediated by focal adhesions, a protein complex linking the extracellular matrix...
Many cells adhere to extracellular matrix for efficient cell migration. This adhesion is mediated by focal adhesions, a protein complex linking the extracellular matrix to the intracellular cytoskeleton. Focal adhesions have been studied extensively in mesenchymal cells, but recent research in physiological contexts and amoeboid cells suggest focal adhesion regulation differs from the mesenchymal focal adhesion paradigm. We used to uncover new mechanisms of focal adhesion regulation, as are amoeboid cells that form focal adhesion-like structures for migration. We show that PaxillinB, the homologue of Paxillin, localizes to dynamic focal adhesion-like structures during migration. Unexpectedly, reduced PaxillinB recruitment to these structures increases cell migration. Quantitative analysis of focal adhesion size and dynamics show that lack of PaxillinB recruitment to focal adhesions does not alter focal adhesion size, but rather increases focal adhesion turnover. These findings are in direct contrast to Paxillin function at focal adhesions during mesenchymal migration, challenging the established focal adhesion model.
PubMed: 38562712
DOI: 10.1101/2024.03.19.585764 -
Investigational New Drugs Jun 2024Breast cancer is a leading cause of death in women worldwide. Cancer therapy based on stem cells is considered as a novel and promising platform. In the present study,...
Breast cancer is a leading cause of death in women worldwide. Cancer therapy based on stem cells is considered as a novel and promising platform. In the present study, we explore the therapeutic effects of human amniotic mesenchymal stromal cells (hAMSCs) through the reduction of focal adhesion kinase (FAK) activity, SHP-2, and cell adhesion proteins such as Paxillin, Vinculin, Fibronectin, Talin, and integrin αvβ3 expression in MDA-MB-231 breast cancer cells. For this purpose, we employed a co-culture system using 6-well plate transwell. After 72 h, hAMSCs-treated MDA-MB-231 breast cancer cells, the activity of focal adhesion kinase (FAK) and the expression of SHP-2 and cell adhesion proteins such as Paxillin, Vinculin, Fibronectin, Talin, and integrin αvβ3 expression were analyzed using western blot. The shape and migration of cells were also analyzed. Based on our results, a significant reduction in tumor cell motility through downregulation of the tyrosine phosphorylation level of FAK (at Y397 and Y576/577 sites) and cell adhesion expression in MDA-MB-231 breast cancer cells was demonstrated. Our findings indicate that hAMSCS secretome has therapeutic effects on cancer cell migration through downregulation of FAK activity and expression of cell adhesion proteins.
Topics: Humans; Cell Movement; Breast Neoplasms; Female; Cell Line, Tumor; Mesenchymal Stem Cells; Cell Adhesion; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Phosphorylation; Coculture Techniques; Protein Tyrosine Phosphatase, Non-Receptor Type 11
PubMed: 38536544
DOI: 10.1007/s10637-024-01434-2 -
Toxics Feb 2024Various PFAS have been identified as potential endocrine-disrupting chemicals due to estrogen receptor activation, impacts on puberty timing, or impacts on hormonally...
Various PFAS have been identified as potential endocrine-disrupting chemicals due to estrogen receptor activation, impacts on puberty timing, or impacts on hormonally sensitive endpoints in fish. This study screened multiple PFAS in the rat uterotrophic assay to determine potential estrogenic effects on the uterus with PFAS exposure. This study also explored PFAS-dependent uterine signaling with an osmotic stress mRNA gene expression array. Briefly, Sprague-Dawley rats (26-39 days old) were ovariectomized, and uterine tissue was allowed to regress for a 3-week period of recovery. Animals were then exposed daily via oral gavage to PFAS for 4 days, and then uterine weight was determined. In contrast to the positive control estrogens, the PFAS tested (4:2, 6:2, and 8:2FTOH; perfluorooctanesulfonamide (PFOSA), perfluorononanoic acid (PFNA), perfluorohexane sulfonate (PFHxS), perfluorooctane sulfonate (PFOS), nafion byproduct 2 (NBP2), 1H,1H,8H,8H-perfluorooctane-1,8-diol (FC8-diol) and 1H,1H,10H,10H-perfluorodecane-1,10-diol (FC10-diol)) caused no significant changes in the uterine weight. Hormonally active compounds can act as carcinogens, and because earlier rodent work has demonstrated that chronic PFOA exposure is associated with increased risk of uterine cancer, uterine mRNA gene expression was explored with an osmotic stress RT-qPCR array. PFAS exposure significantly upregulated multiple genes across the array, with PFOSA being the compound most similar to the reference estrogens (estradiol benzoate and ethinyl estradiol) in its expression pattern. Also, across all PFAS, pathway analysis revealed that the paxillin pathway, a pathway important in tumor suppressor gene SHP-2 signaling, was significantly upregulated with PFAS exposure. These results demonstrate that in vitro estrogen screens or impacts in fish may show different responses from direct impacts on mammalian uterine weight as assessed with the uterotrophic assay. This study also builds out new mechanisms that may contribute to understanding of carcinogenic changes seen in the uterus after PFAS exposure.
PubMed: 38535903
DOI: 10.3390/toxics12030170 -
OncoTargets and Therapy 2024Progerin, the underlying cause of Hutchinson-Gilford Progeria Syndrome (HGPS), has been extensively studied for its impact on normal cells and premature aging patients....
OBJECTIVE
Progerin, the underlying cause of Hutchinson-Gilford Progeria Syndrome (HGPS), has been extensively studied for its impact on normal cells and premature aging patients. However, there is a lack of research on its specific effects on tumor cells. Melanoma is one of the most common malignant tumors with high morbidity and mortality. This study aimed to elucidate the potential therapeutic role of progerin in melanoma.
MATERIALS AND METHODS
We constructed the melanoma A375 cell line and M14 cell line with stable expression of progerin. The expression of progerin, paxillin, and epithelial-mesenchymal transition (EMT) marker proteins in each cell group was measured using Western blot. The migration, proliferation, and cell cycle of cancer cells were assessed using the transwell assay, wound healing assay, colony formation assay, CCK 8 assay, and flow cytometry. RT-qPCR technology was used to examine the impact of progerin overexpression on microRNA expression. Finally, we transfected paxillin into the progerin overexpression cell group to verify whether progerin regulates the phenotype of tumor cells through paxillin.
RESULTS
Our study demonstrated that overexpression of progerin leads to decreased expression of paxillin and inhibits cancer cell migration, proliferation, EMT process and cell cycle progression. Additionally, rescue experiments revealed that the migration, proliferation ability, and EMT marker protein expression in progerin overexpressing cancer cells could be partially restored by transfecting a plasmid containing the paxillin gene. Mechanistic investigations further revealed that progerin achieves this inhibition of paxillin expression by upregulating miR-212.
CONCLUSION
This study reveals that progerin may inhibit the migration and proliferation of melanoma cells through the miR-212/paxillin axis, which provides a new approach for the future treatment of this disease.
PubMed: 38533131
DOI: 10.2147/OTT.S442504 -
BioRxiv : the Preprint Server For... Mar 2024Zipper-interacting protein kinase (ZIPK) is a Ser/Thr protein kinase with regulatory involvement in vascular smooth muscle cell (VSMC) actin polymerization and focal...
Cooperative involvement of zipper-interacting protein kinase (ZIPK) and the dual-specificity cell-division cycle 14A phosphatase (CDC14A) in vascular smooth muscle cell migration.
Zipper-interacting protein kinase (ZIPK) is a Ser/Thr protein kinase with regulatory involvement in vascular smooth muscle cell (VSMC) actin polymerization and focal adhesion assembly dynamics. ZIPK silencing can induce cytoskeletal remodeling with disassembly of actin stress fiber networks and coincident loss of focal adhesion kinase (FAK)-pY397 phosphorylation. The link between ZIPK inhibition and FAK phosphorylation is unknown, and critical interactor(s) and regulator(s) are not yet defined. In this study, we further analyzed the ZIPK-FAK relationship in VSMCs. The application of HS38, a selective ZIPK inhibitor, to coronary artery vascular smooth muscle cells (CASMCs) suppressed cell migration, myosin light chain phosphorylation (pT18&pS19) and FAK-pY397 phosphorylation as well. This was associated with the translocation of cytoplasmic FAK to the nucleus. ZIPK inhibition with HS38 was consistently found to suppress the activation of FAK and attenuate the phosphorylation of other focal adhesion protein components (i.e., pCas130, paxillin, ERK). In addition, our study showed a decrease in human cell-division cycle 14A phosphatase (CDC14A) levels with ZIPK-siRNA treatment and increased CDC14A with transient transfection of ZIPK. Proximity ligation assays (PLA) revealed CDC14A localized with ZIPK and FAK. Silencing CDC14A showed an increase of FAK-pY397 phosphorylation. Ultimately, the data presented herein strongly support a regulatory mechanism of FAK in CASMCs by a ZIPK-CDC14A partnership; ZIPK may act as a key signal integrator to control CDC14A and FAK during VSMC migration.
PubMed: 38496458
DOI: 10.1101/2024.03.06.583600 -
Phytotherapy Research : PTR Jun 2024Insufficient vessel maintenance adversely impacts patients in terms of tissue reperfusion following stroke or myocardial infarction, as well as during wound healing....
Insufficient vessel maintenance adversely impacts patients in terms of tissue reperfusion following stroke or myocardial infarction, as well as during wound healing. Angiogenesis impairment is a feature typical of metabolic disorders acting at the cardiovascular level, such as diabetes. Therapeutic angiogenesis regulation offers promising clinical implications, and natural compounds as pro-angiogenic nutraceuticals hold valuable applications in regenerative medicine. By using cultured endothelial cells from human umbilical veins (HUVEC) we studied functional and molecular responses following exposure to erucin, a natural isothiocyanate derived from Brassicaceae plants and extracted from the seeds of rocket. Erucin (at nanomolar concentrations) promotes cell migration and tube formation, similar to vascular endothelial growth factor (VEGF), through mobilizing paxillin at endothelial edges. At the molecular level, erucin induces signaling pathways typical of angiogenesis activation, namely Ras, PI3K/AKT, and ERK1/2, leading to VEGF expression and triggering its autocrine production, as pharmacological inhibition of soluble VEGF and VEGFR2 dampens endothelial functions. Furthermore, erucin, alone and together with VEGF, preserves endothelial angiogenic functions under pathological conditions, such as those induced in HUVEC by high glucose (HG) exposure. Erucin emerges as a compelling candidate for therapeutic revascularization applications, showcasing promising prospects for natural compounds in regenerative medicine, particularly in addressing angiogenesis-related disorders.
Topics: Humans; Human Umbilical Vein Endothelial Cells; Glucose; Vascular Endothelial Growth Factor A; Isothiocyanates; Cell Movement; Paxillin; Angiogenesis Inducing Agents; Vascular Endothelial Growth Factor Receptor-2; Signal Transduction; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Brassicaceae; Neovascularization, Physiologic; Sulfides; Thiocyanates
PubMed: 38488442
DOI: 10.1002/ptr.8183