Cell Migration Capacity Research Articles

Expression of p120 - catenin in oral squamous cell carcinoma and apparently normal mucosa adjacent to oral squamous cell carcinoma.

An essential molecular occurrence in carcinogenesis that can lead to invasion and migration, predisposing cells to malignant transformation, involves alterations in cell adhesion molecules, such as p120 catenin. The destabilization of E-cadherin, caused by the loss or phosphorylation of p120 catenin (p120), regulates cadherin stability and turnover, impacting cell adhesiveness and migratory capacity. Consequently, p120 is associated with the invasiveness and progression of various human epithelial tumor types, including Oral Squamous Cell Carcinomas (OSCC). The present study aimed to assess and establish a correlation between the expression of p120 antibody in OSCC and Apparently Normal Mucosa Adjacent to OSCC (ANMAOSCC). The immunoexpression of p120 in 300 selected cases was categorized into two groups: OSCC (n = 150) and ANMAOSCC (n = 150). Two 4µm-thick tissue sections from the selected blocks were prepared. One section was stained with Hematoxylin and Eosin, while the other underwent immunohistochemical (IHC) staining using anti-p120 catenin antibody (clone No. EP66; Catalog No. PR062; PathnSitu, Wayne, PA, USA). The analysis of p120 immunoexpression included parameters such as intensity, percentage, and the location of staining. In OSCC, over 80% of cases expressed p120, with only 16% exhibiting loss of expression. In ANMAOSCC, all cells maintained p120 expression. In OSCC, p120 was predominantly localized to the membrane and cytoplasm in 76%, while in ANMAOSCC, over 90% showed membrane localization. Regarding positivity, only 19% of OSCC cases reported positivity in >50% of cells, compared to 64.7% in ANMAOSCC. The extent of staining in ANMAOSCC was observed up to the granular layer (45%) and corneal layer (19%). The atypical staining pattern of p120 may indicate a loss of adhesion and could serve as a marker for identifying the malignant potential of ANMAOSCC and the aggressiveness of OSCC. Key words:Oral Squamous Cell Carcinoma, P120 Catenin, Cell Adhesion Molecules, Gene Expression Regulation.

The Role of Torkinib in Inhibiting the Proliferation and Migration of Rheumatoid Arthritis Fibroblast-Like Synoviocytes

This study aims to investigate the potential of an inhibitor of the mammalian target of rapamycin (Torkinib) as a therapeutic drug targeting fibroblast-like synoviocytes (FLS) presenting it as a promising alternative for the treatment of rheumatoid arthritis (RA). FLS were isolated and cultured from RA patients for cellular experiments. The collagen-induced arthritis model in mice was constructed using type II collagen emulsion for animal experiments. Cell viability was assessed using cell counting kit-8 and 5-ethynyl-2′-deoxyuridine staining assays, and flow cytometry was utilized to detect cell apoptosis. The scratch assay assessed cell migratory capacity, while the transwell assay evaluated cell invasive potential. Electron microscopy and monodansylcadaverine staining were employed for autophagy detection, and western blot analysis assisted by nanomagnetic beads examined the expression of relevant proteins. In animal experiments, arthritis development was evaluated using clinical scores and hematoxylin and eosin staining. As a result, Torkinib effectively inhibits the proliferation of RA-FLS with a 50% inhibitory concentration of 8.53 nM. Furthermore, Torkinib induces apoptosis in FLS and activates cellular autophagy while suppressing cell migration and invasion by inhibiting the mammalian target of the rapamycin (mTOR) signaling pathway. Additionally, we observed an antagonistic effect between Torkinib and 3-methyladenine. In summary, Torkinib demonstrates its potential in alleviating RA symptoms in CIA mice and enhancing disease progression.

Identification of diagnostic genes and drug prediction in metabolic syndrome-associated rheumatoid arthritis by integrated bioinformatics analysis, machine learning, and molecular docking.

Interactions between the immune and metabolic systems may play a crucial role in the pathogenesis of metabolic syndrome-associated rheumatoid arthritis (MetS-RA). The purpose of this study was to discover candidate biomarkers for the diagnosis of RA patients who also had MetS. Three RA datasets and one MetS dataset were obtained from the Gene Expression Omnibus (GEO) database. Differential expression analysis, weighted gene co-expression network analysis (WGCNA), and machine learning algorithms including Least Absolute Shrinkage and Selection Operator (LASSO) regression and Random Forest (RF) were employed to identify hub genes in MetS-RA. Enrichment analysis was used to explore underlying common pathways between MetS and RA. Receiver operating characteristic curves were applied to assess the diagnostic performance of nomogram constructed based on hub genes. Protein-protein interaction, Connectivity Map (CMap) analyses, and molecular docking were utilized to predict the potential small molecule compounds for MetS-RA treatment. qRT-PCR was used to verify the expression of hub genes in fibroblast-like synoviocytes (FLS) of MetS-RA. The effects of small molecule compounds on the function of RA-FLS were evaluated by wound-healing assays and angiogenesis experiments. The CIBERSORT algorithm was used to explore immune cell infiltration in MetS and RA. MetS-RA key genes were mainly enriched in immune cell-related signaling pathways and immune-related processes. Two hub genes (TYK2 and TRAF2) were selected as candidate biomarkers for developing nomogram with ideal diagnostic performance through machine learning and proved to have a high diagnostic value (area under the curve, TYK2, 0.92; TRAF2, 0.90). qRT-PCR results showed that the expression of TYK2 and TRAF2 in MetS-RA-FLS was significantly higher than that in non-MetS-RA-FLS (nMetS-RA-FLS). The combination of CMap analysis and molecular docking predicted camptothecin (CPT) as a potential drug for MetS-RA treatment. In vitro validation, CPT was observed to suppress the cell migration capacity and angiogenesis capacity of MetS-RA-FLS. Immune cell infiltration results revealed immune dysregulation in MetS and RA. Two hub genes were identified in MetS-RA, a nomogram for the diagnosis of RA and MetS was established based on them, and a potential therapeutic small molecule compound for MetS-RA was predicted, which offered a novel research perspective for future serum-based diagnosis and therapeutic intervention of MetS-RA.

Analysis of m6A-related lncRNAs for prognostic and immunotherapeutic response in hepatocellular carcinoma.

Background: RNA methylation modifications are important post-translational modifications that are regulated in an epigenetic manner. Recently, N6-methyladenosine (m6A) RNA modifications have emerged as potential epigenetic markers in tumor biology. Methods: Gene expression and clinicopathological data of LIHC were obtained from the cancer genome atlas (TCGA) database. The relationship between long non-coding RNAs (lncRNAs) and m6A-related genes was determined by gene expression analysis using Perl and R software. Co-expression network of m6A-lncRNA was constructed, and the relevant lncRNAs associated with prognosis were identified using univariate Cox regression analysis. These lncRNAs were then divided into two clusters (cluster 1 and cluster 2) to determine the differences in survival, pathoclinical parameters, and immune cell infiltration between the different lncRNA subtypes. The least absolute shrinkage and selection operator (LASSO) was carried out for regression analysis and prognostic model. The HCC patients were randomly divided into a train group and a test group. According to the median risk score of the model, HCC patients were divided into high-risk and low-risk groups. We built models using the train group and confirmed them through the test group. The m6A-lncRNAs derived from the models were analyzed for the tumor mutational burden (TMB), immune evasion and immune function using R software. AL355574.1 was identified as an important m6A-associated lncRNA and selected for further investigation. Finally, in vitro experiments were conducted to confirm the effect of AL355574.1 on the biological function of HCC and the possible biological mechanisms. Huh7 and HepG2 cells were transfected with AL355574.1 siRNA and cell proliferation ability was measured by CCK-8, EdU and colony formation assays. Wound healing and transwell assays were used to determine the cell migration capacity. The expression levels of MMP-2, MMP-9, E-cadherin, N-cadherin and Akt/mTOR phosphorylation were all determined by Western blotting. Results: The lncRNAs with significant prognostic value were classified into two subtypes by a consistent clustering analysis. We found that the clinical features, immune cell infiltration and tumor microenvironment (TME) were significantly different between the lncRNA subtypes. Our analysis revealed significant correlations between these different lncRNA subtypes and immune infiltrating and stromal cells. We created the final risk profile using LASSO regression, which notably included three lncRNAs (AL355574.1, AL158166.1, TMCC1-AS1). A prognostic signature consisting of the three lncRNAs was constructed, and the model showed excellent prognostic predictive ability. The overall survival (OS) of the low-risk cohort was significantly higher than that of the high-risk cohort in both the train and test group. Both risk score [hazard ratio (HR)=1.062; P<0.001] and stage (HR=1.647; P< 0.001) were considered independent indicators of HCC prognosis by univariate and multivariate Cox regression analysis. In Huh7 and HepG2 cells, AL355574.1 knockdown inhibited cell proliferation and migration, suppressed the protein expression levels of MMP-2, MMP-9, N-cadherin and Akt/mTOR phosphorylation, but promoted the protein expression levels of E-cadherin. Conclusions: This study established a predictive model for the OS of HCC patients, and these OS-related m6A-lncRNAs, especially AL355574.1 may play a potential role in the progression of HCC. In vitro experiments also showed that AL355574.1 could enhance the expression of MMPs and EMT through the Akt/mTOR signaling pathway, thereby affected the proliferation and migration of HCC. This provides a new perspective on the anticancer molecular mechanism of AL355574.1 in HCC.

M-CSF secreted by gastric cancer cells exacerbates the progression of gastric cancer by increasing the expression of SHP2 in tumor-associated macrophages.

To investigate the effect of Src homology 2 domain-containing tyrosine phosphatase-2 (SHP2) in tumor-associated macrophages (TAMs), which is mediated by macrophage colony-stimulating factor (M-CSF) secreted by gastric cancer cells, on the development of gastric cancer and its molecular mechanism. The progression of gastric cancer was detected by nude mouse tumor-bearing experiments. Colony formation assay and cell counting kit-8 (CCK8) assay were used to detect the proliferation capacity of gastric cancer cells. The migration capacity of gastric cancer cells was examined by wound healing assay. Transwell migration and invasion assays were performed on gastric cancer cells. Detection of relevant protein expression using western blotting. Overexpression of SHP2 could promote the progression of gastric cancer in nude mice. The results of colony formation assay and CCK8 assay showed that overexpression of SHP2 could enhance the proliferation of gastric cancer cells. It was found by wound healing assay and Transwell assay that overexpression of SHP2 could facilitate the migration and invasion of gastric cancer cells. The results of Western blotting revealed that overexpression of SHP2 could increase the expressions of p-STAT3, s-PD-1, p-Src, p-Lyn, p-PI3K, p-AKT, Arginase-1, MMP1 and MMP3 but decrease the expressions of TBK1 and SOCS1 in TAMs, and also increase the expressions of CD9, TSG101 and s-PD-1 in exosomes. M-CSF secreted by gastric cancer cells can promote the proliferation, invasion and migration of gastric cancer cells by increasing the expression of SHP2 in TAMs.

GLP-1 receptor agonist liraglutide inhibits the proliferation and migration of thyroid cancer cells.

This research was done to find out how liraglutide affected the growth and movement of two human thyroid cancer cell lines overexpressing GLP-1 receptor (migration of medullary thyroid cancer TT/GLP-1R and papillary thyroid carcinoma TCP-1/GLP-1R). Flow cytometer and cAMP assays were used to identify the expression and activation of GLP-1R in two stable cell lines. Counting Kit-8 for Cells was applied to examine the proliferative ability of cells in two stable cell lines after treatment with different concentrations of liraglutide at indicated time points. To track the capacity for cell migration, the Transwell test was utilized. We found that liralutide-activated GLP-1R could significantly reduce the growth and metastasis of two kinds of thyroid tumor cells, and the inhibitory effect was dose- and time- dependent. The phosphorylatios of Akt, S6K1, and 70SK declined after receiving liraglutide therapy In our previous studies, we found that the GLP-1 receptor agonist Liraglutide inhibited the proliferation and migration of thyroid cancer cells through the PI3K/Akt/mTOR pathway. This finding provides a theoretical basis for the treatment of diabetes mellitus complicated with medullary thyroid cancer, and is relatively safe.

Effect of Punicalagin and Ellagic Acid on Human Fibroblasts In Vitro: A Preliminary Evaluation of Their Therapeutic Potential.

Pomegranate is a fruit that contains various phenolic compounds, including punicalagin and ellagic acid, which have been attributed to anti-inflammatory, antioxidant, and anticarcinogenic properties, among others. To evaluate the effect of punicalagin and ellagic acid on the viability, migration, cell cycle, and antigenic profile of cultured human fibroblasts (CCD-1064Sk). MTT spectrophotometry was carried out to determine cell viability, cell culture inserts were used for migration trials, and flow cytometry was performed for antigenic profile and cell cycle analyses. Cells were treated with each phenolic compound for 24 h at doses of 10-5 to 10-9 M. Cell viability was always significantly higher in treated versus control cells except for punicalagin at 10-9 M. Doses of punicalagin and ellagic acid in subsequent assays were 10-6 M or 10-7 M, which increased the cell migration capacity and upregulated fibronectin and α-actin expression without altering the cell cycle. These in vitro findings indicate that punicalagin and ellagic acid promote fibroblast functions that are involved in epithelial tissue healing.

EHMT2 promotes tumorigenesis in GNAQ/11-mutant uveal melanoma via ARHGAP29-mediated RhoA pathway

Constitutive activation of GNAQ/11 is the initiative oncogenic event in uveal melanoma (UM). Direct targeting GNAQ/11 has yet to be proven feasible as they are vital for a plethora of cellular functions. In search of genetic vulnerability for UM, we found that inhibition of euchromatic histone lysine methyltransferase 2 (EHMT2) expression or activity significantly reduced the proliferation and migration capacity of cancer cells. Notably, elevated expression of EHMT2 had been validated in UM samples. Furthermore, Kaplan–Meier survival analysis indicated high EHMT2 protein level was related to poor recurrence-free survival and a more advanced T stage. Chromatin immunoprecipitation sequencing analysis and the following mechanistic investigation showed that ARHGAP29 was a downstream target of EHMT2. Its transcription was suppressed by EHMT2 in a methyltransferase-dependent pattern in GNAQ/11-mutant UM cells, leading to elevated RhoA activity. Rescuing constitutively active RhoA in UM cells lacking EHMT2 restored oncogenic phenotypes. Simultaneously blocking EHMT2 and GNAQ/11 signaling in vitro and in vivo showed a synergistic effect on UM growth, suggesting the driver role of these two key molecules. In summary, our study shows evidence for an epigenetic program of EHMT2 regulation that influences UM progression and indicates inhibiting EHMT2 and MEK/ERK simultaneously as a therapeutic strategy in GNAQ/11-mutant UM.

Chemokine- and chemokine receptor-based signature predicts immunotherapy response in female colorectal adenocarcinoma patients

The clinical significance and comprehensive characteristics of chemokines and chemokine receptors in female patients with advanced colorectal adenocarcinoma have not ever been reported. Our study explored the expression profiles of chemokines and chemokine receptors and constructed a chemokine- and chemokine receptor-based signature in female patients with advanced colorectal adenocarcinoma. Four independent cohorts containing 1335 patients were enrolled in our study. Univariate Cox regression and least absolute shrinkage and selection operator (LASSO) analyses were performed to construct the signature. CIBERSORT was used to evaluate the landscape of immune cell infiltration. Thirty-two pairs of tissue specimens of female advanced colorectal cancer (CRC) patients and two CRC cell lines were used to validate the signature in vitro. Quantitative real-time PCR and western blotting were performed to validate the mRNA and protein expression levels of signature genes. EdU and colony formation assays were performed to examine proliferative ability. Transwell and wound healing assays were used to evaluate cell invasion and migration capacity. During the signature construction and validation process, we found that the signature was more applicable to female patients with advanced colorectal adenocarcinoma. Hence, the subsequent study mainly focused on the particular subgroup. Enrichment analyses revealed that the signature was closely related to immunity. The landscape of immune cell infiltration presented that the signature was significantly associated with T cells CD8 and neutrophils. Gene set enrichment analysis (GSEA) confirmed that the high-risk group was chiefly enriched in the tumor-promoting related pathways and biological processes, whereas the low-risk group was mainly enriched in anti-tumor immune response pathways and biological processes. The signature was closely correlated with CTLA4, PDL1, PDL2, TMB, MSI, and TIDE, indicating that our signature could serve as a robust biomarker for immunotherapy and chemotherapy response. ROC curves verified that our signature had more robust prognostic power than all immune checkpoints and immunotherapy-related biomarkers. Finally, we used 32 pairs of tissue specimens and 2 CRC cell lines to validate our signature in vitro. We first provided a robust prognostic chemokine- and chemokine receptor-based signature, which could serve as a novel biomarker for immunotherapy and chemotherapy response to guide individualized treatment for female patients with advanced colorectal adenocarcinoma.

Research of restricted migration evaluation of MDA-MB-231 cells in 2D and 3D co-culture models.

The restricted migration evaluation is conducive to more complex tumor migration research because of the conformity with in vivo tumors. However, the differences between restricted and unrestricted cell migration and the distinction between different evaluation methods have not been systematically studied, hindering related research. In this study, by constructing the restricted environments on chips, the influence of co-culture conditions on the cancer cell migration capacity was studied. The results showed that the restricted channels can discriminate the influence of weak tumor environmental factors on complex tumor migration behaviors by limiting the free growth instinct of tumor cells. Through the comparison of 2D and 3D restricted migration methods, the extracellular matrix (ECM) restriction was also helpful in distinguishing the influence of the weak tumor environmental factor. However, the 3D ECM can better reflect the tortuosity of the cell migration process and the cooperative behavior among cancer cells. In the anticancer drug evaluation, 3D ECM can more accurately reflect the cytotoxicity of drugs and is more consistent with the drug resistance in the human body. In conclusion, the research will help to distinguish different evaluation methods of cancer cell migration, help researchers select appropriate evaluation models, and promote the research of tumor metastasis.

Aligned electrospun fibers of different diameters for improving cell migration capacity

Electrospun fibers have gained significant attention as scaffolds in skin tissue engineering due to their biomimetic properties, which resemble the fibrous extracellular matrix. The morphological characteristics of electrospun fibers play a crucial role in determining cell behavior. However, the effects of electrospun fibers’ arrangement and diameters on human skin fibroblasts (HSFs) remain elusive. Here, we revealed the impact of electrospun fiber diameters (700 nm, 2000 nm, and 3000 nm) on HSFs' proliferation, migration, and functional expression. The results demonstrated that all fibers exhibited good cytocompatibility. HSFs cultured on nanofibers (700 nm diameter) displayed a more dispersed and elongated morphology. Conversely, fibers with a diameter of 3000 nm exhibited a reduced specific surface area and lower adsorption of adhesion proteins, resulting in enhanced cell migration speed and effective migration rate. Meanwhile, the expression levels of migration-related genes and proteins were upregulated at 48 h for the 3000 nm fibers. This study demonstrated the unique role of fiber diameters in controlling the physiological functions of cells, especially decision-making and navigating migration in complex microenvironments of aligned electrospun fibers, and highlights the utility of these bioactive substitutes in skin tissue engineering applications.

Intracellular Fate of Sub-Toxic Concentration of Functionalized Selenium Nanoparticles in Aggressive Prostate Cancer Cells

Selenium 0 (Se0) is a powerful anti-proliferative agent in cancer research. We investigated the impact of sub-toxic concentrations of Se0 functionalized nanoparticles (SeNPs) on prostate cancer PC-3 cells and determined their intracellular localization and fate. An in-depth characterization of functionalized selenium nanoparticles composition is proposed to certify that no chemical bias relative to synthesis issues might have impacted the study. Selenium is an extremely diluted element in the biological environment and therefore requires high-performance techniques with a very low detection limit and high spatial resolution for intracellular imaging. This was explored with state-of-the-art techniques, but also with cryopreparation to preserve the chemical and structural integrity of the cells for spatially resolved and speciation techniques. Monodisperse solutions of SeNPs capped with bovine serum albumin (BSA) were shown to slow down the migration capacity of aggressive prostate cancer cells compared to polydisperse solutions of SeNPs capped with chitosan. BSA coating could prevent interactions between the reactive surface of the nanoparticles and the plasma membrane, mitigating the generation of reactive oxygen species. The intracellular localization showed interaction with mitochondria and also a localization in the lysosome-related organelle. The SeNPs-BSA localization in mitochondria constitute a possible explanation for our result showing a very significant dampening of the PC-3 cell proliferation capabilities. The purpose of the use of sublethal compound concentrations was to limit adverse effects resulting from high cell death to best evaluate some cellular changes and the fate of these SeNPs on PC-3. Our findings provide new insight to further study the various mechanisms of cytotoxicity of SeNPs.

Iron inhibits glioblastoma cell migration and polarization.

Glioblastoma is one of the deadliest malignancies facing modern oncology today. The ability of glioblastoma cells to diffusely spread into neighboring healthy brain makes complete surgical resection nearly impossible and contributes to the recurrent disease faced by most patients. Although research into the impact of iron on glioblastoma has addressed proliferation, there has been little investigation into how cellular iron impacts the ability of glioblastoma cells to migrate-a key question, especially in the context of the diffuse spread observed in these tumors. Herein, we show that increasing cellular iron content results in decreased migratory capacity of human glioblastoma cells. The decrease in migratory capacity was accompanied by a decrease in cellular polarization in the direction of movement. Expression of CDC42, a Rho GTPase that is essential for both cellular migration and establishment of polarity in the direction of cell movement, was reduced upon iron treatment. We then analyzed a single-cell RNA-seq dataset of human glioblastoma samples and found that cells at the tumor periphery had a gene signature that is consistent with having lower levels of cellular iron. Altogether, our results suggest that cellular iron content is impacting glioblastoma cell migratory capacity and that cells with higher iron levels exhibit reduced motility.

Carbon dots efficiently promote vascularization for enhanced repairing of orthopedic diseases with diabetic mellitus based on nanocatalytic medicine

Insufficient angiogenesis, a prominent characteristic of diabetic mellitus, significantly affected the reparative capacity of orthopedic diseases. Imbalanced redox caused by excessive reactive oxygen species is one of the crucial factors in weakening vascularization. Conditions such as implant osseointegration failure and delayed wound healing in orthopedic diseases are significantly influenced by the vascularization deficits in diabetic mellitus. Promoting angiogenesis by modulating endothelial function is considered as an effective therapy for diabetes, yet current efforts remain insufficient. Nanocatalytic medicine has emerged as promising candidates for therapy. Herein, carbon dots (CDs) with preferable catalytic effect by mimicking both superoxide dismutase (SOD) and catalase (CAT)-like activity were designed and synthesized using anhydrous citric acid and ethane diamine. CDs effectively scavenged superoxide anion (O2•−) and hydrogen peroxide (H2O2) with excellent biocompatibility. Meanwhile, CDs promoted the proliferation, migration, and angiogenesis capacity of human umbilical vein endothelial cells with enhanced SOD and CAT activity in simulated oxidative stress of diabetes. Notably, CDs promoted vascularization by activate PI3K-AKT signaling pathway in impaired endothelial cells, which was demonstrated through transcriptome sequencing, western blot, and reverse transcription-polymerase chain reaction. The therapeutic effect of CDs based on promoting vascularization was found in orthopedic disease models of subcutaneous implant and wound healing of diabetic rat. These findings highlight a promising clinical application for CDs as a nanocatalytic medicine in enhancing vascularization for diabetic mellitus.

Downregulation of RNA binding protein 47 predicts low survival in patients and promotes the development of renal cell malignancies through RNA stability modification

RNA binding proteins (RBPs) are crucial for cell function, tissue growth, and disease development in disease or normal physiological processes. RNA binding motif protein 47 (RBM47) has been proven to have anti-tumor effects on many cancers, but its effect is not yet clear in renal cancer. Here, we demonstrated the expression and the prognostic role of RBM47 in public databases and clinical samples of clear cell renal carcinoma (ccRCC) with bioinformatics analysis. The possible mechanism of RBM47 in renal cancer was verified by gene function prediction and in vitro experiments. The results showed that RBM47 was downregulated in renal cancers when compared with control groups. Low RBM47 expression indicated poor prognosis in ccRCC. RBM47 expression in renal cancer cell lines was reduced significantly when compared to normal renal tubular epithelial cells. Epithelial-mesenchymal transition (EMT) and transforming growth factor-β signaling pathway was associated with RBM47 in ccRCC by Gene set enrichment analysis. RBM47 expression had a positive correlation with e-cadherin, but a negative correlation with snail and vimentin. RBM47 overexpression could repress the migration, invasion activity, and proliferation capacity of renal cancer cells, while RBM47 inhibition could promote the development of the malignant features through EMT signaling by RNA stability modification. Therefore, our results suggest that RBM47, as a new molecular biomarker, may play a key role in the cancer development of ccRCC.

DUSP22 suppresses tumor progression by directly dephosphorylating AKT in non-small cell lung cancer.

Protein kinase B (AKT) plays a pivotalin regulating cell migration, proliferation, apoptosis, and survival, making it a prominent target for anticancer therapy. While the kinase activity of AKT has been extensively explored, its dephosphorylation have largely remained uncharted. Herein, we aimed to unravel the molecular mechanisms governing AKT dephosphorylation, with a specific emphasis on dual-specificity phosphatases DUSP22. Our investigation sought to shed light on the potential of DUSP22 as a potential therapeutic target for non-small cell lung cancer (NSCLC). To determine the expression level of DUSP22 in NSCLC cell lines, the gene expression profiling interactive analysis (GEPIA) and Oncomine database were searched. Additionally, the effect of DUSP22 on patient survival was analyzed with Kaplan-Meier database. Antitumor effects of DUSP22 were tested in A549 and H1299 cell lines. Experiments are based on: (1) cell viability determined by the cell counting kit-8 assay and colony-formation assay; (2) cell migratory ability assessed throughthe scratch assay and the transwell migration assay; (3) the mechanism behind the antitumor effects of DUSP22 dissected with co-immunoprecipitation (Co-IP) and in vitro kinase assays. Our study revealed a significant downregulation ofDUSP22 in both NSCLC cell lines and tissues. Meanwhile, survival rate analysis results demonstratedthat reduced DUSP22 expression was correlated with pooreroverall survival in lung cancer patients. Moreover, DUSP22 exhibited an inhibitory effect on the cell viability and migratory capacity of A549 and H1299 cells. This inhibition was accompanied by the decrease in the phosphorylation of AKT and p38. Mechanistically, the phosphatase domain of DUSP22 interacted with AKT, resulting in theinhibitionof AKT phosphorylation. This inhibitory effect was contingent upon the phosphatase activity of DUSP22. These findings provide compelling evidence that DUSP22 directly interacted with AKT, leading to the dephosphorylation ofAKT at S473 and T308 residues, ultimately curbing the proliferation and migration of lung cancer cells. Additionally, our results also highlight a preclinical rationale for utilizing DUSP22 as a prognostic marker in NSCLC.

The Role of Cyanidin-3-O-glucoside in Modulating Oxaliplatin Resistance by Reversing Mesenchymal Phenotype in Colorectal Cancer.

Epithelial-mesenchymal transition (EMT) plays an important role in the biological and biochemical processes of cells, and it is a critical process in the malignant transformation, and mobility of cancer. Additionally, EMT is one of the main mechanisms contributing to chemoresistance. Resistance to oxaliplatin (OXA) poses a momentous challenge in the chemotherapy of advanced colorectal cancer (CRC) patients, highlighting the need to reverse drug resistance and improve patient survival. In this study, we explored the response of cyanidin-3-O-glucoside (C3G), the most abundant anthocyanin in plants, on the mechanisms of drug resistance in cancer, with the purpose of overcoming acquired OXA resistance in CRC cell lines. We generated an acquired OXA-resistant cell line, named HCT-116-ROx, by gradually exposing parental HCT-116 cells to increasing concentrations of OXA. To characterize the resistance, we performed cytotoxicity assays and shape factor analyses. The apoptotic rate of both resistant and parental cells was determined using Hoechst 33342/Propidium Iodide (PI) fluorescence staining. Migration capacity was evaluated using a wound-healing assay. The mesenchymal phenotype was assessed through qRT-PCR and immunofluorescence staining, employing E-cadherin, N-cadherin, and Vimentin markers. Resistance characterization announced decreased OXA sensitivity in resistant cells compared to parental cells. Moreover, the resistant cells exhibited a spindle cell morphology, indicative of the mesenchymal phenotype. Combined treatment of C3G and OXA resulted in an augmented apoptotic rate in the resistant cells. The migration capacity of resistant cells was higher than parental cells, while treatment with C3G decreased the migration rate of HCT-116-ROx cells. Analysis of EMT markers showed that HCT-116-ROx cells exhibited loss of the epithelial phenotype (E-cadherin) and gain of the mesenchymal phenotype (N-cadherin and Vimentin) compared to HCT-116 cells. However, treatment of resistant cells with C3G reversed the mesenchymal phenotype. The morphological observations of cells acquiring oxaliplatin resistance indicated the loss of the epithelial phenotype and the acquisition of the mesenchymal phenotype. These findings suggest that EMT may contribute to acquired OXA resistance in CRC. Furthermore, C3G decreased the mobility of resistant cells, and reversed the EMT process, indicating its potential to overcome acquired OXA resistance.

Synergistic Effects of 2-Deoxy-D-Glucose and Cinnamic Acid with Erlotinib on NSCLC Cell Line

Background: In spite of all efforts, Non-small cell lung cancer (NSCLC) is a fatal solid tumor with a poor prognosis as of its high metastasis and resistance to present treatments. Tyrosine kinase inhibitors (TKI) such as erlotinib are efficient in treating NSCLC but the emergence of chemoresistance and adverse effects substantially limits their single use. Objective: in this study, the combination treatments of either 2-deoxy-D-glucose (2DG) or cinnamic acid (CINN) with erlotinib (ERL) were tested for their possible synergistic effect on the proliferation and migration capacity of NSCLC cells. Methods: In this study, NSCLC model cell line A549 was used to investigate the effects of single compounds and their combination on cell growth inhibition, clonogenic potential, and migration capacity. Next, the Combination index (CI) and the Dose-Reduction Index (DRI) were determined to determine the nature of the drug’s combination and to measure how many folds the dose could be lowered for each drug in a synergistic combination. Results: the combination treatment demonstrated more significant inhibition of viability of A549 cells compared to individual therapy. Our data show that CINN augments the sensitivity to erlotinib in all doses tested. The combination of 2DG or CINN with erlotinib also reduced the clonogenicity of NSCLC cells up to 67% and 85%, respectively, as compared to the erlotinib single treatment. Furthermore, CINN +ERL decreased the migratory ability of A549 cells by 3-fold and further induced much more apoptotic cell death phenotypes. Conclusion: in summary, co-administration of 2DG or cinnamic acid with erlotinib increases the inhibitory effects of erlotinib on NSCLC cell tumorigenicity and migration.

Targeting Covalent and Non-Covalent Btki-Resistant CLL Using the Dual Irreversible/Reversible 4 th Generation BTK Inhibitor LP-168

Background: Treatment of chronic lymphocytic leukemia (CLL) has been transformed with targeted therapies including inhibitors of Bruton's tyrosine kinase (BTKi). Currently three covalent BTK inhibitors (cBTKi) are approved for CLL, but most patients eventually relapse, commonly through acquisition of the C481S BTK mutation (Woyach et al. 2014). Pirtobrutinib and nemtabrutinib are non-covalent BTK inhibitors (ncBTKi) developed to target and inhibit C481S mutant BTK. However novel secondary site mutations in BTK, namely T474I and L528W, have been found to confer resistance to both cBTKi and ncBTKi (Wang et al. 2022). Regardless of these mutations, BCR signaling remains intact, suggesting that inhibition of BTK maintains its therapeutic importance. LP-168 is a novel ultra-selective 4 th generation BTKi with an active warhead capable of covalent interaction with WT BTK or non-covalent binding when a BTK C481 mutation is present. Methods: To determine the target selectivity of LP-168 we screened the compound against 468 kinases in the scanMAX Kinase Assay. For validation of covalent binding, dialysis against WT BTK was performed. Primary CLL B cells were isolated by negative selection and treated with LP-168 for all experiments. BCR signaling alterations were assessed via immunoblot to observe changes in target protein phosphorylation. Changes in CLL migration towards CXCL12 or CXCL13 were interrogated by transwell migration. Cytotoxicity towards primary CLL cells was measured via Annexin V/PI flow cytometry with and without HS-5 stromal cell support. Experiments using TMD8 cells were performed following CRISPR modification to insert WT, C481S, or T474I BTK. In vivo survival studies were performed using both the Eμ-TCL1 and Eμ-MTCP1 mouse engraftment models. Results: LP-168 demonstrated selectivity towards BTK with roughly 700-fold selectivity for BTK vs its next off-target. In an enzymatic assay, LP-168 displayed nanomolar potency towards both WT BTK (IC 50=0.11 nM) and C481S BTK (IC 50=1.0 nM). Following 2-hour drugging, LP-168 inhibited BCR signaling in primary CLL B cells at increasing concentrations, demonstrated by reduced phosphorylation of BTK (92%, p&amp;lt;0.0001, n=8) and its immediate downstream target PLCγ2 (41%, p=0.0013, n=8). Migration capacity of primary CLL cells towards CXCL12 and CXCL14 was also found to decrease after 2-hours of treatment with LP-168 (52%, p&amp;lt;0.001, n=10 and 51%, p&amp;lt;0.001, n=10, respectively). After 48 hours of exposure, LP-168 was able to induce cytotoxicity of primary CLL cells alone and in co-culture with HS-5 stromal cells in a dose-dependent manner, with similar or improved efficacy to other BTKi. LP-168 decreased CLL cell production of CCL3 and CCL4 chemokines (CCL3: 93%, p&amp;lt;0.001, n=8; CCL4: 93%, p&amp;lt;0.001, n=8). LP-168 also demonstrated modest cytotoxicity towards TMD8 cells harboring either C481S BTK (24%, p=0.0008 , n=3) or T474I BTK (13%, p=0.0690, n=3), as well as decreased production of CCL3 (C481S: 95%, p&amp;lt;0.0001, n=3; T474I: 99%, p&amp;lt;0.0001, n=3) and CCL4 (C481S: 97%, p&amp;lt;0.0001, n=3; T474I: 98%, p&amp;lt;0.0001, n=3) (Figure 1A). Further, we observed inhibition of downstream BCR signaling in primary CLL B cells harboring C481S via immunoblotting and cytokine production. Finally, to evaluate the in vivo efficacy of LP-168 we tested this compound in both the Eµ-TCL1 and Eµ-MTCP1 models. We treated mice daily via oral gavage and found that 50 mg/kg of LP-168 significantly improved survival in the Eµ-TCL1 model when compared to vehicle (median 51 vs 44 days; p=0.0018) or ibrutinib at 50 mg/kg (median 51 vs 45 days; p=0.0098) and the Eµ-MTCP1 model when compared with vehicle (median 122 vs 62 days; p&amp;lt;0.0001) or ibrutinib at 50 mg/kg (median 122 vs 96 days; p=0.0162) (Figure 1B). Conclusions: Collectively, our data demonstrate that LP-168 is a potent and selective inhibitor of BTK with activity against CLL, even in the presence of mutations that mediate resistance to cBTKi and ncBTKi. These data support the continued preclinical and clinical investigation of LP-168, which is currently being studied in the phase 1 setting of CLL (NCT04775745) and NHL (NCT04993690).

Periprostatic adipose tissue thromboinflammation triggers prostatic neoplasia in early metabolic impairment: Interruption by rivaroxaban

AimsProstate cancer is among the highest incidence malignancies in men with a prevalence rate increasing in parallel to the rising global trends in metabolic disorders. Whereas a sizeable body of evidence links metabolic impairment to negative prognosis of prostate cancer, the molecular mechanism underlying this connection has not been thoroughly examined. Our previous work showed that localized adipose tissue inflammation occurring in select adipose depots in early metabolic derangement instigated significant molecular, structural, and functional alterations in neighboring tissues underlying the complications observed at this stage. In this context, the periprostatic adipose tissue (PPAT) constitutes an understudied microenvironment with potential influence on the prostatic milieu. Main methods and resultsWe show that PPAT inflammation occurs in early prediabetes with signs of increased thrombogenic activity including enhanced expression and function of Factor X. This was mirrored by early neoplastic alterations in the prostate with fibrosis, increased epithelial thickness with marked luminal cellular proliferation and enhanced formation of intraepithelial neoplasia. Significantly, interruption of the procoagulant state in PPAT by a 10-day anticoagulant rivaroxaban treatment not only mitigated PPAT inflammation, but also reduced signs of prostatic neoplastic changes. Moreover, rivaroxaban decreased the murine PLum-AD epithelial prostatic cell viability, proliferation, migration, and colony forming capacity, while increasing oxidative stress. A protease-activated receptor-2 agonist reversed some of these effects. SignificanceWe provide some evidence of a molecular framework for the crosstalk between PPAT and prostatic tissue leading to early neoplastic changes in metabolic impairment mediated by upregulation of PPAT thromboinflammation.