Inhibited Cell Motility Research Articles

Influence of the ectopic location on the antigen expression and functional characteristics of endometrioma stromal cells

Are the alterations observed in the endometriotic cells, such as progesterone resistance, already present in the eutopic endometrium or acquired in the ectopic location? The response to decidualization with progesterone and cyclic AMP for up to 28 days was compared in different endometrial stromal cell (EnSC) lines established from samples of endometriomas (eEnSC), eutopic endometrium from women with endometriosis (eBEnSC), endometrial tissue from healthy women (BEnSC) and menstrual blood from healthy donors (mEnSC). Usual features of decidualized cells, such as changes in cell morphology and expression of prolactin, were similarly observed in the three types of eutopic EnSC studied, but not in the ectopic cells upon decidualization. Among the phenotypic markers analysed, CD105 was down-regulated under decidualization in all cell types (mEnSC, P = 0.005; BEnSC, P = 0.029; eBEnSC, P = 0.022) except eEnSC. mEnSC and BEnSC underwent apoptosis during decidualization, whereas eBEnSC and eEnSC were resistant to the induction of cell death. Lastly, migration studies revealed that mEnSC secreted undetermined factors during decidualization that inhibited cell motility, whereas eEnSC showed a significantly lower ability to produce those migration-regulating factors (P < 0.0001, P < 0.001 and P = 0.0013 for the migration of mEnSC at 24, 48 and 72 h, respectively; P < 0.0001 for the migration of eEnSC at all times studied). This study provides novel insights into the differences between endometriotic and eutopic endometrial cells and reinforces the idea that the microenvironment in the ectopic location plays additional roles in the acquisition of the alterations that characterize the cells of the endometriotic foci.

CXC- receptor 2 promotes extracellular matrix production and attenuates migration in peripapillary human scleral fibroblasts under mechanical strain.

As the main loading-bearing tissue of eye, sclera exerts an important role in the pathophysiology of glaucoma. Intraocular pressure (IOP) generates mechanical strain on sclera. Recent studies have demonstrated that sclera, especially the peripapillary sclera, undergoes complicated remodelling under the mechanical strain. However, the mechanisms of the hypertensive scleral remodelling in human eyes remained uncertain. In this study, peripapillary human scleral fibroblasts (ppHSFs) were applied cyclic mechanical strain by Flexcell-5000™ tension system. We found that CXC- ligands and CXCR2 were differentially expressed after strain. Increased cell proliferation and inhibited cell motility were observed when CXCR2 was upregulated under the strain, whereas cell proliferation and motility did not have a significant change when CXCR2 was knocked down. CXCR2 could facilitate cell proliferation ability, modulate the mRNA and protein expressions of type I collagen and matrix metalloproteinase 2 via JAK1/2-STAT3 signalling pathway. In addition, CXCR2 might inhibit cell migration via FAK/MLC2 pathway. Taken together, CXCR2 regulated protein production and affected cell behaviours of ppHSFs. It might be a potential therapeutic target for the hypertensive scleral remodelling.

Calotropis Procera Induced Caspase-Dependent Apoptosis and Impaired Akt/mTOR Signaling in 4T1 Breast Cancer Cells.

Calotropis procera (Aiton) Dryand (Apocynaceae) is an herb that has been commonly used in folk medicine to treat various diseases for more than 1500 years. Our goal was to investigate the anti-metastatic effects of phenolics extracted from C. procera (CphE) against 4T1 breast cancer cells and in BALB/c mice. 4T1 cells were treated with CphE and quercetin (positive control) at concentrations that inhibited cell viability by 50% (IC50). Levels of reactive oxygen species (ROS), wound healing, and protein expressions were determined following standard protocols. For the in vivo pilot study, the syngeneic BALB/c mouse model was used. 4T1 cells were injected into mammary fat pads. Tumors were allowed to grow for 9 days before gavage treatment with CphE (150 mg GAE/kg/day) or PBS (controls) for one week. Excised tumors, liver, and lungs were analyzed for gene and protein expression and histology. In vitro results showed that CphE suppressed cell viability through apoptosis induction, via caspase-3 cleavage and total PARP reduction. CphE also scavenged ROS and suppressed Akt, mTOR, ERK1/2, CREB, and Src activation contributing to cell motility inhibition. CphE reduced IR, PTEN, TSC2, p70S6, and RPS6, protein levels, which are proteins involved in the PI3K/Akt/mTOR pathway, suggesting this pathway as CphE primary target. In vivo results showed downregulation of ERK1/2 activation by phosphorylation in tumor tissues, accompanied by angiogenesis reduction in tumor and lung tissues. A reduction of Cenpf mRNA levels in liver and lung tissues strongly suggested anti-invasive cancer activity of CphE. CphE inhibited 4T1 cell signal pathways that play a key role in cell growth and invasion. The potential for in vitro results to be translated in vivo was confirmed. A complete animal study is a guarantee to confirm the CphE anticancer and antimetastatic activity in vivo.

Bisdemethoxycurcumin suppresses human osteosarcoma U‑2 OS cell migration and invasion via affecting the PI3K/Akt/NF‑κB, PI3K/Akt/GSK3β and MAPK signaling pathways invitro.

The metastasis of human osteosarcoma (OS) shows a difficult‑to‑treat clinical scenario and results in decreased quality of life and diminished survival rates. Finding or developing novel treatments to improve the life quality of patients is urgent. Bisdemethoxycurcumin (BDMC), a natural product, was obtained from the rhizome of turmeric (Curcumalonga) and exerts antitumor activities in numerous human cancer cell lines. At present, there is no study showing BDMC effects on OS cell migration and invasion. In the present study, the effects of BDMC on cell migration and invasion of OS U‑2 OScells were investigated invitro. Cell viability and proliferation were measured by flow cytometric and MTT assays, respectively. Cell motility, MMP‑2 and ‑9 activity, and cell migration and invasion were assayed by scratch wound healing, gelatin zymography, and Transwell chamber assays, respectively. The protein expression levels were measured by western blotting. BDMC at 20 and 40µM significantly reduced total cell viability, and BDMC at 5 and 10µM significantly inhibited cell motility in U‑2 OS cells. BDMC significantly suppressed the activities of MMP‑2 and MMP‑9 in U‑2 OS cells. BDMC suppressed cell invasion and migration after 24 h treatment in U‑2 OS cells, and these effects were in a dose‑dependently manner. Results from western blotting indicated that BDMC significantly decreased the protein expression levels of PI3K/Akt/NF‑κB, PI3K/Akt/GSK3β, and MAPK pathway in U‑2 OS cells. Furthermore, BDMC inhibited uPA, MMP‑2, MMP‑9, MMP‑13, N‑cadherin, VE‑cadherin, and vimentin but increased E‑cadherin in U‑2 OS cells. Based on these observations, it was suggested that BDMC may be a potential candidate against migration and invasion of human OS cells in the future.

Oxygen-Deficient Tungsten Oxide (WOx) Nanobelts with pH-Sensitive Degradation for Enhanced Sonodynamic Therapy of Cancer.

The further bioapplications of sonodynamic therapy (SDT) were hindered by the inadequate efficiency and poor degradability of sonosensitizers and the hypoxic tumor microenvironment (TME). Therefore, it is ideal to develop pH-sensitive sonosensitizers that generate abundant reactive oxygen species (ROS) and rapidly degrade in a neutral environment while slowly degrading in an acidic environment to reduce their long-term toxicity. Herein, the defective tungsten oxide nanobelts (WOx NBs) were developed as a type of pH-sensitive and biodegradable sonosensitizers with a high SDT efficiency and low toxicity for enhanced SDT. The defective oxygen sites of WOx NBs could inhibit the recombination of electrons and holes, making WOx NBs promising sonosensitizers that could generate abundant ROS under ultrasound (US) irradiation. Enhanced by the catalase (CAT) that reacted with H2O2 to generate O2, the WOx NBs exhibited better SDT performance against 4T1 cells in both normoxic and hypoxic environments. In addition, the WOx NBs could degrade by releasing protons (H+), resulting in intracellular acidification and inhibited cell motility that further enhanced the therapeutic effects of SDT. Assisted with CAT and ALG for hypoxia refinement and better retention, the WOx NBs enabled effective SDT and antimetastasis against 4T1 tumors in vivo. Most importantly, the WOx NBs could degrade rapidly in normal tissues but slowly in an acidic TME, which was favorable for their fast clearance, without any obvious long-term toxicity. Our work developed defective WOx NBs with a high SDT efficiency and pH-sensitive degradation for enhanced SDT, which extended the biomedical application of tungsten-based nanomaterials and the further development of SDT.

A novel O2- (2,4-dinitrophenyl) diazeniumdiolate inhibits hepatocellular carcinoma migration, invasion, and EMT through the Wnt/β-catenin pathway

Targeted Wnt/β-catenin pathway is considered to be a promising therapy for cancer metastasis. The novel O2 -(2,4-dinitrophenyl) diazeniumdiolate (JS-K) plays a potent inhibitory role in the proliferation of cancers. In this study, HepG2 and SMMC7721 were used to clarify the efficacy of JS-K inhibition of HCC metastasis. JS-K significantly inhibited cell motility through a wound-healing assay and restrained cell migration and invasion at noncytotoxic concentrations. However, the inhibitory effects of migration and invasion were abolished after the addition of NO scavenger, Carboxy-PTIO. In addition, JS-K inhibited the Wnt/β-catenin pathway by a decrease of p-GSK-3β at Ser9, cytosolic β-catenin, and nuclear β-catenin accumulation whereas an increase of p-β-catenin. Furthermore, the transcription regulators c-Myc, survivin, and Cyclin D1 were down-regulated after treating with JS-K. The inhibitory of the Wnt/β-catenin pathway was reversed after the addition of Carboxy-PTIO or LiCl. Meanwhile, JS-K also inhibited the epithelial-mesenchymal transition (EMT)-mediated cell migration and invasion. The characteristics of the inhibition were reflected by the upregulation of E-cadherin whereas the downregulation of Vimentin, Snail, and Slug. Taking together, these results demonstrated that JS-K inhibited HepG2 and SMMC7721 cells migration and invasion by reversing EMT via the Wnt/β-catenin pathway.

Integrated Stress Response Regulation of Corneal Epithelial Cell Motility and Cytokine Production.

PurposeTo investigate the effect of an active integrated stress response (ISR) on human corneal epithelial cell motility and cytokine production.MethodsISR agonists tunicamycin (TUN) and SAL003 (SAL) were used to stimulate the ISR in immortalized corneal epithelial cell lines, primary human limbal epithelial stem cells, and ex vivo human corneas. Reporter lines for ISR-associated transcription factors activating transcription factor 4 (ATF4) and XBP1 activity were generated to visualize pathway activity in response to kinase-specific agonists. Scratch assays and multiplex magnetic bead arrays were used to investigate the effects of an active ISR on scratch wounds and cytokine production. A C/EBP homologous protein (CHOP) knockout cell line was generated to investigate the effects of ISR ablation. Finally, an ISR antagonist was assayed for its ability to rescue negative phenotypic changes associated with an active ISR.ResultsISR stimulation, mediated through CHOP, inhibited cell motility in both immortalized and primary human limbal epithelial cells. Scratch wounding of ex vivo corneas elicited an increase in the ISR mediators phosphorylated-eIF2α and ATF4. ISR stimulation also increased the production of vascular endothelial growth factor (VEGF) and proinflammatory cytokines. ISR ablation, through CHOP knockout or inhibition with integrated stress response inhibitor (ISRIB) rescued epithelia migration ability and reduced VEGF secretion.ConclusionsWe demonstrate that the ISR has dramatic effects on the ability of corneal epithelial cells to respond to wounding models and increases the production of proinflammatory and angiogenic factors. Inhibition of the ISR may provide a new therapeutic option for corneal diseases in which the ISR is implicated.

Scorpion Peptide Smp24 Exhibits a Potent Antitumor Effect on Human Lung Cancer Cells by Damaging the Membrane and Cytoskeleton In Vivo and In Vitro.

Smp24, a cationic antimicrobial peptide identified from the venom gland of the Egyptian scorpion Scorpio maurus palmatus, shows variable cytotoxicity on various tumor (KG1a, CCRF-CEM and HepG2) and non-tumor (CD34+, HRECs, HACAT) cell lines. However, the effects of Smp24 and its mode of action on lung cancer cell lines remain unknown. Herein, the effect of Smp24 on the viability, membrane disruption, cytoskeleton, migration and invasion, and MMP-2/-9 and TIMP-1/-2 expression of human lung cancer cells have been evaluated. In addition, its in vivo antitumor role and acute toxicity were also assessed. In our study, Smp24 was found to suppress the growth of A549, H3122, PC-9, and H460 with IC50 values from about 4.06 to 7.07 µM and show low toxicity to normal cells (MRC-5) with 14.68 µM of IC50. Furthermore, Smp24 could induce necrosis of A549 cells via destroying the integrity of the cell membrane and mitochondrial and nuclear membranes. Additionally, Smp24 suppressed cell motility by damaging the cytoskeleton and altering MMP-2/-9 and TIMP-1/-2 expression. Finally, Smp24 showed effective anticancer protection in a A549 xenograft mice model and low acute toxicity. Overall, these findings indicate that Smp24 significantly exerts an antitumor effect due to its induction of membrane defects and cytoskeleton disruption. Accordingly, our findings will open an avenue for developing scorpion venom peptides into chemotherapeutic agents targeting lung cancer cells.

Bisdemethoxycurcumin suppresses human brain glioblastoma multiforme GBM 8401 cell migration and invasion via affecting NF‐κB and MMP‐2 and MMP‐9 signaling pathway in vitro

Human glioblastoma (GBM) is one of the common cancer death in adults worldwide, and its metastasis will lead to difficult treatment. Finding compounds for future to develop treatment is urgent. Bisdemethoxycurcumin (BDMC), a natural product, was isolated from the rhizome of turmeric (Curcuma longa), which has been shown to against many human cancer cells. In the present study, we evaluated the antimetastasis activity of BDMC in human GBM cells. Cell proliferation, cell viability, cellular uptake, wound healing, migration and invasion, and western blotting were analyzed. Results indicated that BDMC at 1.5-3μM significantly decreased the cell proliferation by MTT assay. BDMC showed the highest uptake by cells at 3h. After treatment of BDMC at 12-48 h significantly inhibited cell motility in GBM 8401 cells by wound healing assay. BDMC suppressed cell migration and invasion at 24 and 48 h treatment by transwell chamber assay. BDMC significantly decreased the levels of proteins associated with PI3K/Akt, Ras/MEK/ERK pathways and resulted in the decrease in the expressions of NF-κB, MMP-2, MMP-9, and N-cadherin, leading to the inhibition of cell migration and invasion. These findings suggest that BDMC may be a potential candidate for the antimetastasis of human GBM cells in the future.

Abstract 5127: Significance of Trop-2 in HER2-positive breast cancer

Abstract Purpose: The glycoprotein Trop-2 is frequently overexpressed in human malignancies and linked to worse outcome. A Trop2-targeting antibody-drug conjugate, sacituzumab govitecan, has been approved by the FDA for patients with unresectable locally advanced or metastatic triple-negative breast cancer. Trop-2 enhances Akt phosphorylation in numerous cancer cell lines, while the opposite findings are also reported suggesting the cell context-dependent effect of Trop-2. Blockade of PI3K/Akt pathway mediates anti-tumor mechanism of anti-HER2 agents. In this study, we aim to investigate the role of Trop-2 in HER2-positive breast cancer. Experimental design: The archival samples comprising different breast cancer subtypes were analyzed using immunohistochemical staining. The public databases, including, Metabric, KM plotter, ROC plotter, were used to verify the prognostic role and clinical significance of Trop-2 in HER2-positive breast cancer. In vitro tumor models, the transwell migration, invasion and sphere assays, were applied for evaluation of cancer progression. The apoptotic cells were determined by flow cytometry using Annexin V/propidium iodide double staining. Results: We analyzed a cohort of 986 breast cancer patients and found that HER2-positive breast cancer harbored higher Trop-2 protein expression than other breast cancer subtypes. Higher levels of Trop-2 and phospho-Akt in HER2-positive breast cancer tissues compared with normal tissues. Trop-2 expression was correlated with tumor stage and phospho-Akt level. HER2-positive breast cancer patients with high level of Trop-2 protein were linked to worse recurrence-free survival. Consistently, data from the public databases exhibited patients with higher level of Trop-2 transcript had shorter recurrence-free survival and overall survival of HER2-positive breast cancer. Overexpression of Trop-2 increased cell migration, invasion and sphere number in SK-BR-3 and BT-474 cells. In contrast, knockdown of Trop-2 suppressed cell motility and sphere formation. Trop-2-expressing cells had better cell viability compared to control cells in the presence of lapatinib or neratinib treatment. Lapatinib- and neratinib-induced cell apoptosis was rescued by Trop-2 overexpression. Notably, patients responded to anti-HER2 therapy had lower Trop-2 transcript expression than non-responders. Conclusion: These results suggested that the oncogenic role of Trop-2 in HER2-positive breast cancer and Trop-2 expression might confer resistance to anti-HER2 therapy. Citation Format: Chun-Yu Liu, Ji-Lin Chen, Pei-Yi Chu, Chun-Teng Huang, Wan-Lun Wang, Chi-Cheng Huang, Po-Han Lin, Ling-Ming Tseng. Significance of Trop-2 in HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5127.

Circular RNA circ-ABCB10 Promotes Proliferation and Inhibits Apoptosis of Laryngeal Carcinoma by Inhibiting KLF6.

Objective To explore the effect of circular RNA circ-ABCB10 on the proliferation and apoptosis of laryngeal carcinoma via inhibiting KLF6. Methods RT-qPCR assay was adopted to detect the expression of circ-ABCB10 and KFL6 in laryngeal carcinoma tissues and cell lines. Cell counting kit-8 (CCK-8) and clone formation assay were employed to detect laryngeal cancer cell viability and proliferation when circ-ABCB10 was silenced or upregulated. In this study, the apoptosis rate was detected by flow cytometry and the protein expression was detected by Western blotting. Wound healing and cross-hole invasion were used to study the migration and invasion of laryngeal cancer cells when circ-ABCB10 was silenced or upregulated. Results The results of RT-qPCR detection indicated that the expression of circ-ABCB10 in all three laryngeal carcinoma cells was downregulated by 3.2 times compared with that of HaCat cells. There is low expression of circ-ABCB10 in most laryngeal carcinoma tissues, the diagnostic cutoff value of circ-ABCB10 is 0.0008, the area under the curve is 0.718, the sensitivity is 0.981, and the specificity is 0.556. The expression level of KLF6 in laryngeal carcinoma is on the rise, which is significantly higher compared to healthy tissues (P < 0.05); 48 hours after transfection, RT-qPCR analysis confirmed the transfection efficiency, and upregulation of circ-ABCB10 could significantly promote cell proliferation. Compared with the control group, silencing circ-MTCL1 could inhibit cell proliferation, overexpression of circ-ABCB10 promoted cell migration, and downregulation of circ-ABCB10 significantly inhibited cell movement (P < 0.001). Upregulation of circ-ABCB10 significantly enhanced the invasiveness and motility of laryngeal cancer cells, while downregulation of circ-ABCB10 was the opposite. Compared with the KLF6 NC group, KLF6 level increased significantly in the KLF6 group, while cell viability, colony formation, scratch healing rate, invasive cell number, and Bcl-2 expression level decreased significantly in the KLF6 group, while apoptosis rate and Bax expression level increased significantly (P < 0.05). KLF6 level in the si-circ-ABCB10+anti-KLF6 group was significantly lower than that in the si-circ-ABCB10+anti-KLF6-NC group (P < 0.05). Meanwhile, the cell activity, colony formation number, cell scratch healing rate, number of invaded cells, and Bcl-2 all indicated an upward trend, while the cell apoptosis rate and Bax expression indicated a downward trend (P < 0.05). Conclusion The expression of circ-ABCB10 in laryngeal carcinoma was significantly higher compared to that in paracancerous tissues. Silencing circ-ABCB10 could significantly inhibit the growth and proliferation of laryngeal adenocarcinoma cells, while overexpression of circ-ABCB10 could significantly promote the growth of laryngeal adenocarcinoma cells, probably by inhibiting KLF6 to enhance the proliferation of laryngeal carcinoma and inhibit apoptosis.

Potential therapeutic targets discovery by transcriptome analysis of an in vitro human gastric signet ring carcinoma model.

Loss of E-cadherin expression is frequently observed in signet ring carcinoma (SRCC). People with germline mutations in CDH1, which encodes E-cadherin, develop diffuse gastric cancer at a higher rate. Loss of E-cadherin expression is thus assumed to trigger oncogenic development. To investigate novel therapeutic targets for gastric SRCC, we engineered an E-cadherin-deficient SRCC model in vitro using a human gastric organoid (hGO) with CDH1 knockout (KO). CDH1 KO hGO cells demonstrated distinctive morphological changes similar to SRCC and high cell motility. RNA-sequencing revealed up-regulation of matrix metalloproteinase (MMP) genes in CDH1 KO hGO cells compared to wild type. MMP inhibitors suppressed cell motility of CDH1 KO hGO cells and SRCC cell lines in vitro. Immunofluorescent analysis with 95 clinical gastric cancer tissues revealed that MMP-3 was specifically abundant in E-cadherin-aberrant SRCC. In addition, CXCR4 molecules translocated onto the cell membrane after CDH1 KO. Addition of CXCL12, a ligand of CXCR4, to the culture medium prolonged cell survival of CDH1 KO hGO cells and was abolished by the inhibitor, AMD3100. In clinical SRCC samples, CXCL12-secreting fibroblasts showed marked infiltration into the cancer area. E-cadherin deficient SRCCs might gain cell motility through upregulation of MMPs. CXCL12-positive cancer-associated fibroblasts could serve to maintain cancer-cell survival as a niche. MMPs and the CXCL12/CXCR4 axis represent promising candidates as novel therapeutic targets for E-cadherin-deficient SRCC.

HGG-43. Abrogation of exosome biogenesis significantly affects cell motility in heterogenous sub-populations of paediatric-type diffuse high-grade glioma

Paediatric-type diffuse High-Grade Gliomas (PDHGG) are highly heterogenous tumours comprised of distinct cell sub-populations co-existing within the same tumour mass. We have shown that primary patient-derived sub-clones, as well as optical-barcoded sub-clones, function as an interconnected network conferring an aggressive phenotype. Here, we explored the role of exosomes in mediating PDHGG inter-clonal communication. A comprehensive characterization of 7 optical-barcoded single cell-derived clones obtained from two patient-derived cell lines (one DMGH3K27-altered and one diffuse high-grade paediatric-type glioma H3WT), confirmed extensive genomic and phenotypic heterogeneity. Live single-cell tracking in 3D migration and invasion assays demonstrated the key role of the inter-clonal crosstalk in driving a more aggressive phenotype. To determine the exosome role in this crosstalk, we first characterised them in terms of size, marker expression and cargo. Moreover, we demonstrated that exosomes were actively internalized by the sub-clones. Exosomal proteomic analysis showed differential protein contents implicated in the regulation of biological processes such as focal adhesion and extracellular matrix organization. The analysis of exosomal miRNome did not show differentially expressed miRNAs between sub-clones, however, specific and distinct exosomal miRNAs were found uniquely expressed by each sub-clone. The abrogation of the exosome biogenesis by GW4869 phospholipase inhibitor did not affect sub-clones viability, but significantly inhibited their motility, when cultured individually and more prominently in co-culture condition. Analysis of the exo-miRNAs uniquely expressed by the sub-clones highlighted a set of target genes regulating cell motility/invasion/migration. These target genes were differentially expressed when sub-clones were co-cultured compared to mono-culture. Moreover, the expression levels of these genes (e.g. CD44, PTRZ, GLI3, NTRK2) were significantly modulated upon GW4869 treatments. In conclusion, our study highlights the importance of the exosomes in the inter-clonal communication and suggests that interfering with the exosome biogenesis may be a valuable strategy to inhibit cell motility in PDHGG.

Effect of berberine on cancer cell motility in glioma, lung cancer, and prostate cancer cell cultures.

e15079 Background: Initially, berberine was used as an antimicrobial agent in traditional medicine, but its anticancer properties were later discovered. In addition to its cytotoxic effect, berberine also has the ability to inhibit cell motility, which has been demonstrated in some permanent cancer cell lines. The objective of this study was to assess berberine anti-migratory activity in permanent cell cultures compared to primary cell cultures which are generally thought to better reflect tumor characteristics. Methods: H1299 lung cancer, PC3 prostate cancer, and T98G glioma cells, as well as primary cell cultures of the corresponding cancer obtained in our laboratory, were planted in an amount of 15*104 cells per well of a 24-well plate (Biofil, China) in DMEM medium (Gibco, USA) supplemented with 10% FBS (HyClone, USA). After cell adhesion berberine was added in concentration 5 µM, and a wound healing assay was performed according to the standard procedure. Cell plates were continuously incubated and photographed in Lionheart FX imager (BioTek, USA) at 37°C and 5.0% CO2. The extent of cell migration was measured as the percentage reduction in wound area after 48 hours of incubation relative to baseline. Data are presented as Mean ± 95% confidence interval (n = 12). Results: The use of berberine at a concentration of 5 µM led to a significant decrease in cell motility in permanent cultures of lung cancer, glioma and prostate cancer. Namely, the reduction in the wound area after 48 hours of incubation with bereberine was 74.52±12.3% (compared with 94.56±6.2% in the control) in H1299 cell culture, 38.22±10.6% (compared with 83.89±15.5% in the control) in T98G cell culture, and 48.6±7.5% (compared with 69.56±8.1% in the control) in PC3 cell culture. The resulting difference between the control and experimental groups in permanent cell cultures was statistically significant at a significance level of 5% (df = 22). At the same time, the values of wound area reduction for primary cultures of the same cancers did not differ significantly in the control and under the influence of 5 μM berberine at the accepted level of significance. Namely, the reduction in the wound area after 48 hours of incubation with bereberine was 84.79±11.2% (compared with 81.47±15.3% in the control) in lung cancer primary cell culture, 94.64±5.1% (compared with 91.73±6.8% in the control) in glioma primary culture, and 62.63±5.8% (compared with 61.1±8.9% in the control) in prostate cancer primary cell culture. Conclusions: Permanent cell lines are more sensitive to berberine anti-migratory activity than primary cancer cell lines of the same localization.

Platinum(IV) Prodrugs with Cancer Stem Cell Inhibitory Effects on Lung Cancer for Overcoming Drug Resistance.

Acquired resistance remains a major barrier for the treatment of non-small cell lung cancer, while cancer stem cells (CSCs) usually lead to the occurrence of drug resistance. We herein report a series of platinum(IV) prodrugs containing CSCs-inhibitory species derived from a known CSCs inhibitor BBI608 in the axial position. Among them, complex 15 exerted the most potent cytotoxicity against A549 and A549/CDDP cancer cells. Besides, 15 could suppress cancer cell stemness, superior to BBI608, and overcome cisplatin resistance. Following assays indicated that an enhanced intracellular accumulation of 15 effectively triggered DNA damage, induced ROS generation, activated mitochondrial apoptosis pathway, and suppressed cell motility via p53 pathway in A549/CDDP cells. In vivo tests indicated that 15 exhibited potent antitumor effects without causing loss in the body weight. Our study provides a novel and efficient approach to promote the antitumor activity of platinum-based prodrugs and overcome cisplatin resistance via inhibiting cancer cell stemness.

TLR9 agonist suppresses choroidal neovascularization by restricting endothelial cell motility via ERK/c-Jun pathway.

Choroidal neovascularization (CNV) is the feature of neovascular age-related macular degeneration (AMD). It has been demonstrated that inflammation plays a key role in the development of CNV. Here we aim to investigate how TLR9 agonist (CpG-ODN), one of the key regulators of inflammatory responses, suppresses CNV in vivo. The cell viability was assessed by MTT and EdU test after CpG-ODN treatment. Endothelial cells gap assay, tube formation assay and transwell assay were practiced to observe how CpG-ODN affected the endothelial cells functions. The choroidal explants and laser-induced CNV model were built to investigate how CpG-ODN suppressed angiogenesis. The ERK and c-Jun expression were evaluated to assess if CpG-ODN affected cell proliferation. Flow cytometry and qPCR was practiced to observe how CpG-ODN regulated cell proliferation. Our data showed that CpG-ODN not only reduced CNV area in vivo, but also decreased the RPE damage. CpG-ODN inhibited endothelial cells from migration and forming tubes, while the effect was not toxic. EdU test and MTT test suggested that CpG-ODN inhibited endothelial cells proliferation. CpG-ODN significantly increased protein expression of phosphorylated c-Jun but reduced phosphorylated ERK in HUVECs, which was confirmed in ERK transfected 293T cells. JNK inhibitor abolished the suppression of endothelial cells migration and tube formation by CpG-ODN. The findings were also in agreement with the observation in CpG-ODN treated CNV eyes in vivo. The flow cytometry and qPCR data revealed that the suppression of cell motility by CpG-ODN was achieved by arresting endothelial cells cell cycle at G0/G1 phase. Our study demonstrated that CpG-ODN suppressed endothelial cell motility by restricting the cell cycle progression at G0/G1 phase, the effect of which was achieved by interacting with ERK/c-Jun pathways.

New Approach against Chondrosoma Cells-Cold Plasma Treatment Inhibits Cell Motility and Metabolism, and Leads to Apoptosis.

(1) Background: Chondrosarcoma (CS) is a malignant primary bone tumor with a cartilaginous origin. Its slow cell division and severely restricted vascularization are responsible for its poor responsiveness to chemotherapy and radiotherapy. The decisive factor for the prognosis of CS patients is the only adequate therapy—surgical resection. Cold atmospheric pressure plasma (CAP) is emerging as a new option in anti-cancer therapy. Its effect on chondrosarcomas has been poorly investigated. (2) Methods: Two CS cell lines—SW 1353 and CAL 78—were used. Various assays, such as cell growth kinetics, glucose uptake, and metabolic activity assay, along with two different apoptosis assays were performed after CAP treatment. A radius cell migration assay was used to examine cell motility. (3) Results: Both cell lines showed different growth behavior, which was taken into account when using the assays. After CAP treatment, a reduction in metabolic activity was observed in both cell lines. The immediate effect of CAP showed a reduction in cell numbers and in influence on this cell line’s growth rate. The measurement of the glucose concentration in the cell culture medium showed an increase after CAP treatment. Live-dead cell imaging shows an increase in the proportion of dead cells over the incubation time for both cell lines. There was a significant increase in apoptotic signals after 48 h and 72 h for both cell lines in both assays. The migration assay showed that CAP treatment inhibited the motility of chondrosarcoma cells. The effects in all experiments were related to the duration of CAP exposure. (4) Conclusions: The CAP treatment of CS cells inhibits their growth, motility, and metabolism by initiating apoptotic processes.

In Silico Optimized Stapled Peptides Targeting WASF3 in Breast Cancer.

Wiskott-Aldrich Syndrome Protein Family (WASF) members regulate actin cytoskeletal dynamics, and WASF3 is directly associated with breast cancer metastasis and invasion. WASF3 forms a heteropentameric complex with CYFIP, NCKAP, ABI, and BRK1, called the WASF Regulatory Complex (WRC), which cooperatively regulates actin nucleation by WASF3. Since aberrant deployment of the WRC is observed in cancer metastasis and invasion, its disruption provides a novel avenue for targeting motility in breast cancer cells. Here, we report the development of a second generation WASF3 mimetic peptide, WAHMIS-2, which was designed using a combination of structure-guided design, homology modeling, and in silico optimization to disrupt binding of WASF3 to the WRC. WAHMIS-2 was found to permeate cells and inhibit cell motility, invasion, and MMP9 expression with greater potency than its predecessor, WAHM1. Targeted disruption of WASF3 from the WRC may serve as a useful strategy for suppression of breast cancer metastasis.

Anti-rotavirus Properties and Mechanisms of Selected Gram-Positive and Gram-Negative Probiotics on Polarized Human Colonic (HT-29) Cells.

Probiotics have been investigated to improve the universal rotavirus (RV) vaccination as well as to ameliorate the RV infection. However, underlying mechanisms how probiotics mediate beneficial effects needs more investigation. Thus, in the present study, we used polarized HT-29 cells to assess the anti-RV properties of Gram-positive, (Lactobacillus acidophilus, Lacticaseibacillus rhamnosus GG, and Bifidobacterium subsp. Lactis Bb12) and Gram negative, (Escherichia coli Nissle 1917) probiotics and study their underlying mechanisms. Our results showed that pre-treatment of HT-29 cells for 4h with probiotics, significantly reduced (p < 0.05) human RV replication and this effect was most pronounced for E. coli Nissle followed by L. acidophilus and L. rhamnosus GG. Strikingly, only pre-treatment with live bacteria or their supernatants demonstrated anti-RV properties. Except Gram negative E. coli Nissle, the Gram-positive probiotics tested did not bind to RV. Ingenuity pathway analysis of tight junction (TJ)- and innate immune-associated genes indicated that E. coli Nissle or E. coli Nissle + RV treatments improved cell-cell adhesion and cell contact, while L. acidophilus or L. acidophilus + RV treatments also activated cell-cell contact but inhibited cell movement functions. RV alone inhibited migration of cells event. Additionally, E. coli Nissle activated pathways such as the innate immune and inflammatory responses via production of TNF, while RV infection activated NK cells and inflammatory responses. In conclusion, E. coli Nissle's ability to bind RV, modulate expression of TJ events, innate immune and inflammatory responses, via specific upstream regulators may explain superior anti-RV properties of E. coli Nissle. Therefore, prophylactic use of E. coli Nissle might help to reduce the RV disease burden in infants in endemic areas.

RNA m6A methylation regulates dissemination of cancer cells by modulating expression and membrane localization of β-catenin

N6-methyladenosine (m6A) methylation, which is modified by the METTL3/METTL14 complex, is a dominant internal modification in mammalian RNA and tightly linked to cancer progression. Here we reveal that METTL3-promoted cell migration, invasion, and epithelial-to-mesenchymal transition (EMT) are associated with expression and membrane localization of β-catenin (encoded by CTNNB1), as opposed to Wnt signaling activation in various types of cancer cells, including cervical, lung, and liver cancer. Specifically, METTL3 regulates the transcription, mRNA decay, translation, and subcellular localization of β-catenin. For CTNNB1 expression, METTL3 indirectly suppresses CTNNB1 transcription by stabilizing its transcription suppressor E2F1 mRNA； deposition of 5'UTR m6A in CTNNB1 promotes its decay in a content-dependent manner via YTHDF2 recognition; 5'UTR m6A in CTNNB1 suppresses its translation efficiency, whereas the global METTL3 level controls the canonical and non-canonical translation of CTNNB1, which is probably associated with the interaction between YTHDF1 and eIF4E1/eIF4E3. For β-catenin translocation, METTL3 represses membrane localization of β-catenin and its interaction with E-cadherin by downregulating c-Met kinase, leading to inhibition of cell motility. Invitro, invivo, and clinical analyses confirm the essential role of β-catenin and its expression regulators in cancer cell dissemination. The findings not only expand our understanding of m6A modification and its roles in gene expression and subcellular localization of targets but also suggest that the METTL3/β-catenin axis might be a potential target to inhibit cancer metastasis.