Molecular mechanism of triptolide in myocardial fibrosis through the Wnt/ß-catenin signaling pathway.

Objective. Myocardial fibrosis (MF) is a common manifestation of end-stage cardiovascular diseases. Triptolide (TP) provides protection against cardiovascular diseases. This study was to explore the functional mechanism of TP in MF rats via the Wnt/ß-catenin pathway. Methods. The MF rat model was established via subcutaneous injection of isoproterenol (ISO) and treated with low/medium/high doses of TP (L-TP/M-TP/H-TP) or Wnt agonist BML-284. Cardiac function was examined by echocardiography. Pathological changes of myocardial tissues were observed by HE and Masson staining. Col-I/Col-III/Vimentin/α-SMA levels were detected by immunohistochemistry, RT-qPCR, and Western blot. Collagen volume fraction content was measured. Expression levels of the Wnt/ß-catenin pathway-related proteins (ß-catenin/c-myc/Cyclin D1) were detected by Western blot. Rat cardiac fibroblasts were utilized for in vitro validation experiments. Results. MF rats had enlarged left ventricle, decreased systolic and diastolic function and cardiac dysfunction, elevated collagen fiber distribution, collagen volume fraction and hydroxyproline content. Levels of Col-I/Col-III/Vimentin/α-SMA, and protein levels of ß-catenin/c-myc/Cyclin D1 were increased in MF rats. The Wnt/ß-catenin pathway was activated in the myocardial tissues of MF rats. TP treatment alleviated impairments of cardiac function and myocardial tissuepathological injury, decreased collagen fibers, collagen volume fraction, Col-I, Col-III, α-SMA and Vimentin levels, HYP content, inhibited Wnt/ß-catenin pathway, with H-TP showing the most significant effects. Wnt agonist BML-284 antagonized the inhibitive effect of TP on MF. TP inhibited the Wnt/ß-catenin pathway to repress the proliferation and differentiation of mouse cardiac fibroblasts in vitro. Conclusions. TP was found to ameliorate ISO-induced MF in rats by inhibiting the Wnt/ß-catenin pathway.

Carbonate apatite increases gene expression of osterix and bone morphogenetic protein 2 in the alveolar ridge after socket grafting.

PURPOSE: After tooth extraction, preservation of the alveolar ridge by socket grafting attenuates bone resorption. Runt-related transcription factor 2 (RUNX2) and SP7/Osterix (OSX) are transcription factors playing an important role in osteoblast differentiation. The purpose of this study was to evaluate the effects of carbonate apatite (CO3Ap) on osteoblast-related gene and protein expression after socket grafting. METHODS: Alveolar bone and new bone after CO3Ap grafting were collected at the time of implant placement. Levels of mRNA for RUNX2, SP7/OSX, bone morphogenetic protein 2 (BMP2), BMP7 and platelet derived growth factor B were determined by real-time PCR. Immunostaining was performed using antibodies against RUNX2, SP7/OSX, vimentin and cytokeratin. To evaluate bone resorption rates, cone-beam CT (CBCT) imaging was performed after socket grafting and before implant placement. RESULTS: CBCT imaging showed that the average degree of bone resorption at the CO3Ap graft site was 7.15 ± 3.79%. At the graft sites, levels of SP7/OSX and BMP2 mRNA were significantly increased. Replacement of CO3Ap with osteoid was evident histologically, and in the osteoid osteoblast-like cells were stained for SP7/OSX and vimentin. CONCLUSION: These results show that gene expression of both SP7/OSX and BMP2 can be induced by CO3Ap, suggesting that increased expression of SP7/OSX and vimentin may be involved in the BMP pathway.

HBx Modulates Drug Resistance of Sorafenib-Resistant Hepatocellular Carcinoma Cells.

BACKGROUND: Approximately 50% of hepatocellular carcinoma (HCC) arises due to the infection by hepatitis B virus X protein (HBx). Sorafenib, a unique targeted oral kinase inhibitor, is the therapeutic agent of choice for advanced HCC. The mechanism of HBx in drug resistance of sorafenib-resistant HCC cells was evaluated in this study. METHODS: Employing a stepwise increase of the sorafenib content, Hep3B and HepG2 cells were iteratively induced to establish drug-resistant cell lines (Hep3B/R and HepG2/R). The survival rate of Hep3B, Hep3B/R, HepG2, and HepG2/R cells was estimated using the cell counting kit-8 (CCK-8) assay. The IC50 values of sorafenib were calculated, exploring its effects under varying concentrations. The HBx content was quantified via quantitative reverse transcription PCR (RT-qPCR) and Western Blot. HBx overexpression and interfering virus vectors were constructed and transfected into Hep3B/R and HepG2/R cells. Cell viability and metastasis were assessed by colony formation, wound healing, and transwell assays. E-cadherin, N-cadherin, Vimentin, Slug, and Snail content was evaluated via Western Blot. RESULTS: HBx content was significantly elevated in Hep3B/R and HepG2/R subgroups compared to Hep3B and HepG2 subgroups. The proliferation, clonogenicity, invasiveness, and migratory abilities of Hep3B/R and HepG2/R cells in the HBx subgroup were markedly enhanced; E-cadherin content was significantly reduced, whereas the content of N-cadherin, Vimentin, Slug, and Snail was significantly elevated in the HBx subgroup. Conversely, in the sh-HBx subgroup, the proliferation, clonogenicity, invasion, and migration of Hep3B/R and HepG2/R cells were significantly reduced, E-cadherin content was markedly increased, and N-cadherin, Vimentin, Slug, and Snail content was significantly reduced, compared to the sh-negative control (NC) subgroup. CONCLUSIONS: HBx knockout may affect the development of HCC by reducing the proliferation, invasion, and migration of Hep3B/R and HepG2/R cells through the inhibition of Epithelial-Mesenchymal Transition (EMT).

[Aumolertinib inhibits proliferation, invasion and migration and promotes apoptosis of neuroblastoma cells by downregulating MMP2 and MMP9 expression].

OBJECTIVE: To investigate the effects of aumolertinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), on biological behaviors of neuroblastoma SH-SY5Y cells. METHODS: CCK-8 assay, colony-forming assay, Transwell assay and flow cytometry were used to assess the effects of 2, 4 and 8 µmol/L aumolertinib on proliferation, survival, migration, invasion and apoptosis of SH-SY5Y cells, and the changes in ultrastructure of the cells were observed using transmission electron microscopy. The protein expressions of Bax, Bcl-2, E-cadherin, vimentin, MMP2, and MMP9 in the treated cells were detected using Western blotting. A nude mouse model bearing subcutaneous SH-SY5Y cell xenograft were treated with aumolertinib (15 mg/kg) or cyclophosphamide (20 mg/kg), and the tumor volume and body mass changes was measured. HE staining was used to observe adverse effects of the treatment on the heart, liver, spleen, lungs and kidneys. RESULTS: Aumolertinib significantly inhibited the proliferation and viability of SH-SY5Y cells (P<0.05) with IC50 of 5.004, 3.728 and 3.228 µmol/L at 24, 48 and 72 h, respectively. Aumolertinib treatment induced obvious apoptosis of the cells, which showed characteristic morphological changes of apoptosis under transmission electron microscope. The treatment also inhibited the invasion and migration abilities of SH-SY5Y cells (P<0.01), up-regulated the expression levels of E-cadherin and Bax and lowered the expression levels of Bcl-2, vimentin, MMP2 and MMP9 (P<0.05). In the nude mouse models, treatment with aumolertinib effectively inhibited the growth of neuroblastoma without causing significant toxicity to the vital organs. CONCLUSION: Aumolertinib inhibits proliferation, survival, invasion and migration and induces apoptosis in SH-SY5Y cells by downregulating MMP2 and MMP9 expression.

Effect of emodin combined with cisplatin on the invasion and migration of HepG2 hepatoma cells.

Cisplatin is the leading chemotherapy agent for advanced liver cancer. However, the resistance to cisplatin in liver cancer reduces its efficacy. A potential strategy to increase its effectiveness and reduce toxicity is to combine cisplatin with 1,3,8-trihydroxy-6-methylanthraquinone (emodin). In this study, we examined the effects of emodin combined with cisplatin on the invasion and migration of HepG2 cells and analyzed the role of emodin. The effects of cisplatin, emodin and their combination were assessed in HepG2 cells. Proliferation, invasion and migration of HepG2 cells were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), scar and Transwell assays. The gelatinase spectrum and an ELISA detected the expression of matrix metallopeptidase 2 (MMP-2) and matrix metallopeptidase 9 (MMP-9). The expression of E-cadherin and vimentin was detected by immunofluorescence and Western blots. Emodin inhibited cell invasion and migration in HepG2 hepatoma cells, increased E-cadherin expression, decreased vimentin, MMP-2, and MMP-9 expression. The combination of emodin and cisplatin-induced a more significant effect in a dose-dependent manner. In this study, we found that emodin inhibited hepatocellular carcinoma (HCC) metastasis. Compared with either cisplatin or emodin alone, the combination of both showed a more significant synergistic effect. Emodin can enhance the sensitivity of HepG2 HCC cells to cisplatin by inhibiting epithelial-mesenchymal transition, and thus, play a role in preventing recurrence and metastasis in HCC.

Circular RNA circARPC1B functions as a stabilisation enhancer of Vimentin to prevent high cholesterol-induced articular cartilage degeneration.

BACKGROUND: Osteoarthritis (OA) is a prevalent and debilitating condition, that is, directly associated with cholesterol metabolism. Nevertheless, the molecular mechanisms of OA remain largely unknown, and the role of cholesterol in this process has not been thoroughly investigated. This study aimed to investigate the role of a novel circular RNA, circARPC1B in the relationship between cholesterol and OA progression. METHODS: We measured total cholesterol (TC) levels in the synovial fluid of patients with or without OA to determine the diagnostic role of cholesterol in OA. The effects of cholesterol were explored in human and mouse chondrocytes in vitro. An in vivo OA model was also established in mice fed a high-cholesterol diet (HCD) to explore the role of cholesterol in OA. RNAseq analysis was used to study the influence of cholesterol on circRNAs in chondrocytes. The role of circARPC1B in the OA development was verified through circARPC1B overexpression and knockdown. Additionally, RNA pulldown assays and RNA binding protein immunoprecipitation were used to determine the interaction between circARPC1B and Vimentin. CircARPC1B adeno-associated virus (AAV) was used to determine the role of circARPC1B in cholesterol-induced OA. RESULTS: TC levels in synovial fluid of OA patients were found to be elevated and exhibited high sensitivity and specificity as predictors of OA diagnosis. Moreover, elevated cholesterol accelerated OA progression. CircARPC1B was downregulated in chondrocytes treated with cholesterol and played a crucial role in preserving the extracellular matrix (ECM). Mechanistically, circARPC1B is competitively bound to the E3 ligase synoviolin 1 (SYVN1) binding site on Vimentin, inhibiting the proteasomal degradation of Vimentin. Furthermore, circARPC1B AAV infection alleviates Vimentin degradation and OA progression caused by high cholesterol. CONCLUSIONS: These findings indicate that the cholesterol-circARPC1B-Vimentin axis plays a crucial role in OA progression, and circARPC1B gene therapy has the opportunity to provide a potential therapeutic approach for OA.

Kinesin family member 23 knockdown inhibits cell proliferation and epithelial-mesenchymal transition in esophageal carcinoma by inactivating the Wnt/ß-catenin pathway.

Kinesin family member 23 (KIF23) serves as a tumor-promoting gene with prognostic values in various tumors. However, the role of KIF23 in esophageal carcinoma (ESCA) progression is largely unknown. The overlapping differentially expressed genes (DEGs) in GSE12452, GSE17351, and GSE20347 datasets were identified via GEO2R tool and Venn diagram software. KIF23 expression was analyzed using GSE12452, GSE17351, and GSE20347 datasets, GEPIA database, and qRT-PCR. Cell proliferation was assessed by CCK-8 and EdU incorporation assays. Gene set enrichment analysis (GSEA) analysis was performed to investigate the pathways associated with the regulatory mechanisms of KIF23 in ESCA. The expression of E-cadherin, vimentin, N-cadherin, and matrix metalloproteinase-9 (MMP-9) and alternation of Wnt/ß-catenin pathway were detected by western blot analysis. We identified two overlapping upregulated DEGs, among which KIF23 was selected for subsequent experiments. KIF23 was overexpressed in ESCA samples and cells, and knockdown of KIF23 retarded cell proliferation in ESCA cells. Besides, KIF23 knockdown suppressed epithelial-mesenchymal transition (EMT) process in ESCA cells, as evidenced by the increase of E-cadherin expression and the reduction of vimentin, N-cadherin, and MMP-9 expression. GSEA analysis suggested that Wnt signaling pathway was the significant pathway related to KIF23. Moreover, we demonstrated that KIF23 silencing inhibited the Wnt/ß-catenin pathway in ESCA cells. Activation of Wnt/ß-catenin pathway by SKL2001 reversed the effects of KIF23 silencing on cell proliferation and EMT in ESCA cells. In conclusion, KIF23 knockdown inhibited the proliferation and EMT in ESCA cells through blockage of Wnt/ß-catenin pathway.

Efficacy of Danggui Buxue decoction on diabetic nephropathy-induced renal fibrosis in rats and possible mechanism.

OBJECTIVE: To observe the efficacy of Danggui Buxue decoction (, DBD) on diabetic nephropathy-induced renal fibrosis in rats, and to study the possible mechanism. METHODS: Sixty male Goto Kakizaki (GK) rats were randomly assigned to the model group, gliquidone group, astragaloside IV group, and high-, medium- and low-doses DBD groups. After 8 weeks, changes in body weight, blood glucose, serum creatinine, serum urea nitrogen, and total cholesterol were observed. Changes in transforming growth factor-ß1 (TGF-ß1), Smad3, and Smad5 pathways and the expression of the fibrosis-related proteins collagen IV (col IV), α-smooth muscle actin (α-SMA), and vimentin were assessed. The degree of renal fibrosis was observed by immunohistochemistry and Mason staining. The expression of interleukin 6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor (TNF-α), and C-reactive protein (CRP) in the kidneys was assessed using enzyme linked immunosorbent assay. RESULTS: Our experiments showed that DBD effectively reduced blood glucose, blood urea nitrogen, and creatinine levels after 8 weeks of administration, improved renal function in diabetic rats, alleviated renal fibrosis, and reduced the renal tissue levels of IL-6, IL-10, TNF-α, and CRP. Furthermore, DBD decreased the expression of TGF-ß1, Smad3, col IV, α-SMA, and vimentin in renal tissues and increased the expression of Smad5. CONCLUSIONS: DBD ameliorates diabetic renal interstitial fibrosis by modulating the TGF-ß1/Smads pathway.

ß-Elemene regulates epithelial-mesenchymal transformation and inhibits invasion and metastasis of colorectal cancer cells.

OBJECTIVES: To study the inhibitory effect of ß-elemene on invasion and metastasis of colorectal cancer cells and its possible mechanism. METHODS: Human colon cancer HCT116 cells were treated with different concentrations of ß-elemene. The proliferation inhibition rate of the cells was detected by MTT assay, cell migration rate was detected by scratched assay, and cell invasion rate was evaluated by Transwell cell invasion assay. The expressions of Vimentin, E-cadherin, N-cadherin, and ß-catenin were detected by Western blotting. The mRNA expressions of Vimentin, E-cadherin, N-cadherin, and ß-catenin were detected by real-time PCR. RESULTS: Compared with the control group, the expressions of migration rate, invasion rate, scratch healing rate, N-cadherin, and Vimentin protein of HCT116 cells were decreased after ß-elemene treatment, while the expression of E-cadherin protein was increased, and the inhibition rate of cell proliferation was increased (p<0.05). CONCLUSIONS: ß-Elemene may inhibit cell proliferation and invasion and metastasis by inhibiting EMT signaling pathway in human colon cancer cell line HCT116.

Chlorogenic Acid Inhibits Epithelial-Mesenchymal Transition and Invasion of Breast Cancer by Down-Regulating LRP6.

Epithelial-mesenchymal transition (EMT) is a crucial biologic process for breast cancer metastasis, and inhibition of EMT could be an effective approach to suppress metastatic potential of mammary cancer. High expression of low-density lipoprotein receptor-related protein 6 (LRP6) is usually observed in breast carcinoma and predicts poor prognosis. In the present study, we investigated whether chlorogenic acid (CA) can inhibit the EMT of breast cancer cells and underlying molecular mechanism. We found that CA treatment transformed MCF-7 cell morphology from spindle shape (mesenchymal phenotype) to spherical shape (epithelial phenotype). CA clearly increased epithelial biomarkers' expression (E-cadherin and ZO-1) but decreased mesenchymal proteins' expression (ZEB1, N-cadherin, vimentin, snail, and slug). In addition, CA attenuated MMP-2 and MMP-9 activities and inhibited cell migration and invasion. CA downregulated the expression of LRP6 in MCF-7 cells. Knockdown LRP6 with siRNA repressed cell mobility and invasion, wheras overexpression of LRP6 promoted EMT and antagonized the EMT inhibitory effect of CA on MCF-7 cells. Furthermore, CA directly interacted with Wnt/ß-catenin signaling coreceptor LRP6 and reduced LRP6, p-LRP6, and ß-catenin expression levels in MCF-7 cells. In vivo study revealed that CA notably reduced tumor volume and tumor weight. CA decreased the expression of LRP6, N-cadherin, ZEB1, vimentin, MMP2, MMP9, and increased the expression of E-cadherin and ZO-1. In conclusion, CA inhibited EMT and invasion of breast cancer by targeting LRP6. SIGNIFICANCE STATEMENT: CA, the familiar polyphenol compound in traditional Chinese medicine, repressed EMT and weakened cellular mobility and invasion in MCF-7 cells. The mechanism studies demonstrated that CA could inhibit EMT and invasion of MCF-7 cells via targeting LRP6. Additionally, CA restrained tumor growth and xenograft tumor EMT in vivo. The EMT inhibitory property of CA warrants further studies of CA as a drug candidate for the therapy of metastatic breast carcinoma.

The role of PI3K/AKT-related proteins in di(2-ethyl)hexylphthalate-induced BG-1 and MCF-7 cell proliferation, and inhibition by metformin.

OBJECTIVE: To investigate the molecular mechanism of the proliferation and migration of BG-1 and MCF-7cells induced by DEHP, and the antagonistic effect of metformin. METHODS: The proliferation, cell cycle progression, migration, and invasion abilities of BG-1 and MCF-7 cancer cells were examined via Cell Counting Kit-8, flow cytometry, Transwell, and scratch assays. E2F1, SKP2, cyclin D1, vimentin, E-cadherin, and GSK-3ß, all of which play key roles in cancer development via the PI3K/AKT signaling pathway, were examined by immunofluorescence and immunocytochemistry. RESULTS: Cell proliferation was significantly increased, and the wound closure and number of trans-membrane migrating cells were significantly increased, by DHEP treatment. The numbers of BG-1 and MCF-7 cells in the G0/G1 phase were significantly decreased, while those in the S phase were significantly increased. Increased E2F1, SKP2, cyclin D1, and vimentin levels and decreased E-cadherin and GSK-3ß levels were detected in BG-1 and MCF-7 cells treated with DEHP compared to that in control cells. Furthermore, DEHP-induced effects were markedly inhibited by LY294002 (a PI3K/AKT inhibitor) or metformin. CONCLUSION: We demonstrated that DEHP-induced cell proliferation and migration involves the PI3K/AKT signaling pathway and that metformin can be used to inhibit this proliferation and migration.

GPR55 activation prevents amphetamine-induced conditioned place preference and decrease the amphetamine-stimulated inflammatory response in the ventral hippocampus in male rats.

Inflammatory response in the Central Nervous System (CNS) induced by psychostimulants seems to be a crucial factor in the development and maintenance of drug addiction. The ventral hippocampus (vHp) is part of the reward system involved in substance addiction and expresses abundant G protein-coupled receptor 55 (GPR55). This receptor modulates the inflammatory response in vitro and in vivo, but there is no information regarding its anti-inflammatory effects and its impact on psychostimulant consumption. The aim of the present study was to investigate whether vHp GPR55 activation prevents both the inflammatory response induced by amphetamine (AMPH) in the vHp and the AMPH-induced conditioned place preference (A-CPP). Wistar adult male rats with a bilateral cannula into the vHp or intact males were subjected to A-CPP (5 mg/kg). Upon the completion of A-CPP, the vHp was dissected to evaluate IL-1ß and IL-6 expression through RT-PCR, Western blot and immunofluorescence. Our results reveal that AMPH induces both A-CPP and an increase of IL-1ß and IL-6 in the vHp. The GPR55 agonist lysophosphatidylinositol (LPI, 10 µM) infused into the vHp prevented A-CPP and the AMPH-induced IL-1ß increase. CID 16020046 (CID, 10 µM), a selective GPR55 antagonist, abolished LPI effects. To evaluate the effect of the inflammatory response, lipopolysaccharide (LPS, 5 µg/µl) was infused bilaterally into the vHp during A-CPP acquisition. LPS strengthened A-CPP and increased IL-1ß/IL-6 mRNA and protein levels in the vHp. LPS also increased CD68, Iba1, GFAP and vimentin expression. All LPS-induced effects were blocked by LPI. Our results suggest that GPR55 activation in the vHp prevents A-CPP while decreasing the local neuro-inflammatory response. These findings indicate that vHp GPR55 is a crucial factor in preventing the rewarding effects of AMPH due to its capacity to interfere with proinflammatory responses in the vHp.

Nickel nanoparticles induce epithelial-mesenchymal transition in human bronchial epithelial cells via the HIF-1α/HDAC3 pathway.

We and others have previously demonstrated that exposure to nickel nanoparticles (Nano-Ni) caused fibrogenic and carcinogenic effects; however, the underlying mechanisms are still not fully understood. This study aimed to investigate the effects of Nano-Ni on epithelial-mesenchymal transition (EMT) in human bronchial epithelial cells (BEAS-2B) and its underlying mechanisms since EMT is involved in both cancer pathogenesis and tissue fibrosis. Our results showed that exposure to Nano-Ni, compared to the control Nano-TiO2, caused a remarkable decrease in the expression of E-cadherin and an increase in the expression of vimentin and α-SMA, indicating an inducible role of Nano-Ni in EMT development in human bronchial epithelial cells. HIF-1α nuclear accumulation, HDAC3 upregulation, and decreased histone acetylation were also observed in the cells exposed to Nano-Ni, but not in those exposed to Nano-TiO2. Pretreatment of the cells with a specific HIF-1α inhibitor, CAY10585, or HIF-1α-specific siRNA transfection prior to Nano-Ni exposure resulted in the restoration of E-cadherin and abolished Nano-Ni-induced upregulation of vimentin and α-SMA, suggesting a crucial role of HIF-1α in Nano-Ni-induced EMT development. CAY10585 pretreatment also attenuated the HDAC3 upregulation and increased histone acetylation. Inhibition of HDAC3 with specific siRNA significantly restrained Nano-Ni-induced reduction in histone acetylation and restored EMT-related protein expression to near control levels. In summary, our findings suggest that exposure to Nano-Ni promotes the development of EMT in human bronchial epithelial cells by decreasing histone acetylation through HIF-1α-mediated HDAC3 upregulation. Our findings may provide information for further understanding of the molecular mechanisms of Nano-Ni-induced fibrosis and carcinogenesis.

Cobalt Chloride-induced Hypoxia Can Lead SKBR3 and HEK293T Cell Lines toward Epithelial-mesenchymal Transition.

Hypoxia is a common characteristic of the tumor microenvironment. In response to hypoxia, expression of the hypoxia-inducible factor (HIF) can lead to activation of downstream molecular events such as epithelial-mesenchymal transition (EMT), invasion, and angiogenesis. In this study, CoCl2 was used to simulate hypoxia in SKBR3 and HEK293T cell lines to investigate whether this treatment can induce hypoxia-associated EMT and invasion in the studied cells. SKBR3 and HEK293T cells were treated with different concentrations of CoCl2 at different exposure times and their viability was analyzed. To confirm successful hypoxia induction, the expression levels of HIF1α and vascular endothelial growth factor A (VEGFA) mRNA were assessed. Additionally, the expression of EMT-associated markers including snail, E-cadherin, N-cadherin, and vimentin, as well as invasion-related genes including matrix metalloproteinase-2 (MMP2) and MMP9 was measured. We found that cell viability in CoCl2-treated cells was concentration-dependent and was not affected at low doses. While the expression of HIF and VEGFA genes was upregulated following hypoxia induction. E-cadherin expression was significantly downregulated in HEK293T cells; while, N-cadherin and snail were upregulated in both cell lines. Moreover, an increment of MMP expression was only observed in SKBR3 cells. Taken together, the findings indicated that CoCl2 can mimic hypoxia in both cell lines, but EMT was triggered in SKBR3 cells more effectively than in HEK293T cells, and invasion was only stimulated in SKBR3 cells. In conclusion, SKBR3 cancer cells can be used as an EMT model to better understand its control and manipulation mechanisms and to investigate new therapeutic targets for the suppression of tumor metastasis.

Interferon-beta induces major histocompatibility complex of class I (MHC-I) expression and a proinflammatory phenotype in cultivated human astrocytes.

Major histocompatibility complex class I (MHC-I) has been implicated in several types of neuroplasticity phenomena. Interferon beta-1b (IFN-ß) increases MHC-I expression by motoneurons after sciatic nerve crush in mice, improving axonal growth and functional recovery. Additionally, IFN-ß induces glial hypertrophy associated with upregulation of glial fibrillary acidic protein (GFAP) and MHC-I in murine astrocytes in vitro. As knowledge about MHC-I and its role in synaptic plasticity in human astrocytes (HAs) is scarce, we investigated these aspects in mature HAs obtained from the neocortex of patients undergoing surgery due to hippocampal sclerosis. Cells were exposed to media in the absence (0 IU/ml) or presence of IFN-ß for 5 days (500 IU/ml). Beta-2 microglobulin (ß2m), a component of the MHC-I, GFAP and vimentin proteins, was quantified by flow cytometry (FC) and increased by 100%, 60% and 46%, respectively, after IFN-ß exposure. We also performed qRT-PCR gene expression analyses for ß2m, GFAP, vimentin, and pro- and anti-inflammatory cytokines. Our data showed that IFN-ß-treated astrocytes displayed ß2m and GFAP gene upregulation. Additionally, they presented a proinflammatory profile with increase in the IL-6 and IL-1ß genes and a tendency to upregulate TNF-α. Moreover, we evaluated the effect of HAs conditioned medium (CM) on the formation/maintenance of neurites/synapses by the PC12 lineage. Synaptophysin protein expression was quantified by FC. The CM of IFN-ß-activated astrocytes was not harmful to PC12 neurites, and there was no change in synaptophysin protein expression. Therefore, IFN-ß activated HAs by increasing GFAP, vimentin and MHC-I protein expression. Like MHC-I modulation and astrocyte activation may be protective after peripheral nerve damage and in some neurodegenerative conditions, this study opens perspectives on the pathophysiological roles of astroglial MHC-I in the human CNS.

The effect of neuropilin-1 silencing on the transforming growth factor-ß1-mediated epithelial-mesenchymal transition of colon cancer SW480 cells and its effect on the proliferation and migration of colon cancer cells.

This study aimed to investigate the effect of neuropilin-1 (NRP-1) silencing on epithelial-mesenchymal transformation (EMT) mediated by transforming growth factor-ß1 (TGF-ß1) and on the proliferation and migration of colon cancer SW480 cells. After transfection of small interfering ribonucleic acid (siRNA)-NRP-1 into colon cancer SW480 cells, the messenger RNA (mRNA) and protein expression levels of NRP-1 were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Four EMT models were induced using 0, 2, 5, and 10 ng/mL TGF-ß1, respectively. Cell proliferation was detected using Cell Counting Kit-8, and the protein levels of EMT markers E-cadherin and vimentin were detected using Western blot. EMT was induced in the transfected SW480 cells using TGF-ß1, after which four groups were created: a negative control group (siRNA-Ncontrol), a transfection group (siRNA-NRP-1), an induction group (TGF-ß1), and a transfection + induction group (siRNA-NRP-1+TGF-ß1). Western blot was then used to detect the expression of E-cadherin and vimentin, and cell proliferation and migration were detected using cell counting kit-8 (CCK-8) and scratch assay. After transfection with siRNA-NRP-1, the mRNA and protein expression levels of SW480 cells were significantly decreased (P<0.05). After 48 hours of induction with 10 ng/mL TGF-ß1, cell proliferation was obvious, E-cadherin expression decreased, and vimentin expression significantly increased (P<0.05), indicating that EMT had been successfully induced compared with the induction group, the transfection + induction group had significantly increased E-cadherin expression after corresponding treatments (including transfection and induction alone) (P<0.05), and the proliferation and migration of colon cancer cells decreased (P<0.05). In conclusion: silencing, NRP-1 in colon cancer SW480 cells can partially reverse TGF-ß1-mediated EMT, reduce the proliferation activity of colon cancer cells, and slow their migration ability. Therefore, NRP-1 may become a new target for the treatment of colon cancer.

Effects of plant-based medicinal food on postoperative recurrence and lung metastasis of gastric cancer regulated by Wnt/ß-catenin-EMT signaling pathway and VEGF-C/D-VEGFR-3 cascade in a mouse model.

BACKGROUND: The plant-based medicinal food (PBMF) is a functional compound extracted from 6 medicinal and edible plants: Coix seed, L. edodes, A. officinalis L., H. cordata, Dandelion, and G. frondosa. Our previous studies have confirmed that the PBMF possesses anti-tumor properties in a subcutaneous xenograft model of nude mice. This study aims to further investigate the effects and potential molecular mechanisms of the PBMF on the recurrence and metastasis of gastric cancer (GC). METHODS: Postoperative recurrence and metastasis model of GC was successfully established in inbred 615 mice inoculated with mouse forestomach carcinoma (MFC) cells. After tumorectomy, 63 GC mice were randomly divided into five groups and respectively subject to different treatments for 15 days as below: model control group, 5-Fu group, and three doses of PBMF (43.22, 86.44, 172.88 g/kg PBMF in diet respectively). The inhibition rate (IR) of recurrence tumor weights and organ coefficients were calculated. Meanwhile, histopathological changes were examined and the metastasis IR in lungs and lymph node tissues was computed. The mRNA expressions related to the canonical Wnt/ß-catenin signaling pathway, epithelial-mesenchymal transition (EMT) and lymphangiogenesis were detected by RT-qPCR in recurrence tumors and/or lung tissues. Protein expressions of ß-catenin, p-ß-catenin (Ser33/37/Thr41), GSK-3ß, p-GSK-3ß (Ser9), E-cadherin, and Vimentin in recurrence tumors were determined by Western Blot. LYVE-1, VEGF-C/D, and VEGFR-3 levels in recurrence tumors and/or lung tissues were determined by immunohistochemistry staining. RESULTS: The mRNA, as well as protein expression of GSK-3ß were up-regulated and the mRNA expression of ß-catenin was down-regulated after PBMF treatment. Meanwhile, the ratio of p-ß-catenin (Ser33/37/Thr41) to ß-catenin protein was increased significantly and the p-GSK-3ß (Ser9) protein level was decreased. And PMBF could effectively decrease the mRNA and protein levels of Vimentin while increasing those of E-cadherin. Furthermore, PBMF markedly reduced lymphatic vessel density (LVD) (labeled by LYVE-1) in recurrence tumor tissues, and mRNA levels of VEGF-C/D, VEGFR-2/3 of recurrence tumors were all significantly lower in the high-dose group. CONCLUSIONS: PBMF had a significant inhibitory effect on recurrence and lung metastasis of GC. The potential mechanism may involve reversing EMT by inhabiting the Wnt/ß-catenin signaling pathway. Lymphatic metastasis was also inhibited by PBMF via down-regulating the activation of the VEGF-C/D-VEGFR-2/3 signaling cascade.

MSC-Derived exosomes suppress colorectal cancer cell proliferation and metastasis via miR-100/mTOR/miR-143 pathway.

Exosomes derived from mesenchymal stem cells (MSCs) are mostly responsible for the therapeutic effects of MSCs. To show the therapeutic effects of the human bone marrow MSC-derived exosomes (MSC-Exos) on colorectal cancer (CRC) and explore the molecular cross-talks between them, CRC cells were treated with the MSC-Exos. We found that MSC-Exos were enriched with miR-100 and miR-143, which effectively downregulated mTOR, Cyclin D1, K-RAS, HK2 while upregulated p-27 expression. All these effects were reversed by concurrent treatment with MSC-Exos and antagomiR-100, confirming that they were caused by exosomal transfer of miR-100 into recipient CRC cells. Moreover, exosomal miR-100 promoted endogenous miR-143 expression. The flow cytometry, MTT and trypan blue assays revealed that MSC-Exos could efficiently suppress proliferation and induce apoptosis of the CRC cells. Furthermore, wound healing, transwell migration and invasion assays confirmed their inhibitory effects on the migration and invasiveness of SW480 cells. We further confirmed these effects by analyzing the expression levels of epithelial to mesenchymal transition (EMT) factors and metastasis-related genes. Results showed that MSC-Exos significantly suppressed the expression of MMP2 and MMP9 (metastasis-related genes), SNAIL and TWIST (EMT-inducing transcription factors), Vimentin and N-cadherin (mesenchymal cell markers), whereas E-cadherin (epithelial cell marker) was remarkably up-regulated. Collectively, our data indicated that MSC-Exos could suppress proliferation, migration, invasion and metastasis while inducing the apoptosis of the CRC cells via miR-100/mTOR/miR-143 axis. Our findings highlight that MSC-Exo treatment as well as miR-100 restoration might be considered as potential therapeutic strategies for CRC.

BDE-209 and TCDD enhance metastatic characteristics of melanoma cells after chronic exposure.

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) and BDE-209 (decabromodiphenyl ether) are persistent organic pollutants (POPs) produced by industrial activities and associated with several diseases. TCDD is a known human carcinogen, but few studies investigated about the effects of exposure to both compounds, i.e., whether BDE-209 and TCDD can render tumor cells more aggressive and metastatic. In the current study we investigated if the exposure of B16-F1 and B16-F10 melanoma murine cells to environmental relevant concentrations of TCDD and BDE-209 at 24 h and 15-day exposure modulates the expression of genes related to metastasis, making the cells more aggressive. Both pollutants did not affect cell viability but lead to increase of cell proliferation, including the upregulation of vimentin, MMP2, MMP9, MMP14 and PGK1 gene expression and downregulation of E-cadherin, TIMP2, TIMP3 and RECK, strongly suggesting changes in cell phenotypes defined as epithelial to mesenchymal transition (EMT) in BDE-209 and TCDD-exposed cells. Foremost, increased expression of metalloproteinases and decreased expression of their inhibitors made B16-F1 cells similar the more aggressive B16-F10 cell line. Also, the higher secretion of extracellular vesicles by cells after acute exposure to BDE-209 could be related with the phenotype changes. These results are a strong indication of the potential of BDE-209 and TCDD to modulate cell phenotype, leading to a more aggressive profile.

KMT5A Knockdown Suppresses Osteosarcoma Cell Proliferation and Metastasis Through Ꞵ-Catenin Signalling.

PURPOSE: Osteosarcoma (OS) is the most common malignant solid bone tumor in children and young adults. We aimed to investigate the effects and cellular mechanisms of KMT5A on OS cell activity. METHODS: The protein expression was evaluated in the clinical normal, adjacent and OS osteogenic tissues. Knockdown of KMT5A was achieved by KMT5A siRNAs in a human OS cell line, MG63, to detect cell proliferation and metastasis. RESULTS: KMT5A expression was upregulated in clinical OS tissues. Knockdown of KMT5A inhibited cell proliferation but enhanced cell death, with significantly reduced cyclinD1 and Bcl2 and increased cleaved-caspase9 levels. KMT5A knockdown also suppressed OS cell migration and invasion capacity and deceased MMP3 and vimentin expression. ß-catenin levels were upregulated in OS tissues and blocking KMT5A resulted in a significant decline in ß-catenin expression in the OS cells. Further administration of ß-catenin activator remarkably increased protein levels of KMT5A, cyclinD1, Bcl2, MMP3, and vimentin, which showed reversed effects of KMT5A knockdown on OS cell activity. CONCLUSION: KMT5A knockdown plays an inhibitory role in OS cell proliferation and metastasis through ß-catenin signalling, which provides basic evidence and suggests potential targets for OS therapeutic research.