Vimentin Levels Research Articles

Nanoapproach targeting TGFβ1-Smad pathway and modulating lung microenvironment

Lung microenvironment is altered by injurious stimuli resulting in alveolar epithelial cell (AEC) injury, epithelial mesenchymal transition (EMT) and aberrant remodeling. This pathogenetic process reduces the drug amount delivered to the target site, both neoplastic and non-neoplastic and necessitates the use of nanocarriers based targeted drug delivery. Pirfenidone, a partially water-soluble drug was encapsulated; poly(ε-caprolactone) nanoparticles (PCL-NPs) and to MePEG-PCL diblock by nanoprecipitation. Their physicochemical properties were characterized by Nanosight, DLS, Zeta potential, TEM, FTIR and 1H NMR. Drug encapsulation and release efficiency was evaluated by HPLC. Their efficacy in modulating TGF-β1, SMAD-2,3,5 signaling and regulating EMT was assessed in-vitro on A-549 cells by measuring Vimentin and E-cadherin mRNA levels. The pirfenidone loaded PCL and MePEG-PCL-NPs were spherical with uniform particle size (~150 nm), polydispersity index (0.095–0.224), high encapsulation efficacy (50–75 %), drug loading capacity (20 %) and good colloidal stability. Bleomycin induced EMT of AECs with an upregulation of mesenchymal protein, vimentin and loss of epithelial marker, E-cadherin. Bimodal increase in TGFβ1, SMAD-2,3.5 genes at 4 h and then at 24 & 48 h was seen. The pirfenidone nanoformulations efficiently reversed EMT by downregulation of TGF-β1, SMAD-2,3,5, Vimentin and upregulation of E-cadherin gene. The effect of nanoformulations was similar to standard dose pirfenidone.

Application of circulating tumour cells to predict response to treatment in head and neck cancer

BackgroundLocal recurrence and metastasis remain the major causes of death in head and neck cancer (HNC) patients. Circulating tumour cells (CTCs) are shed from primary and metastatic sites into the circulation system and have been reported to play critical roles in the metastasis and recurrence of HNC. Here, we explored the use of CTCs to predict the response to treatment and disease progression in HNC patients.MethodsBlood samples were collected at diagnosis from HNC patients (n = 119). CTCs were isolated using a spiral microfluidic device and were identified using immunofluorescence staining. Correlation of baseline CTC numbers to 13-week PET-CT data and multidisciplinary team consensus data were conducted.ResultsCTCs were detected in 60/119 (50.4%) of treatment naïve HNC patients at diagnosis. Baseline CTC numbers were higher in stage III vs. stage I-II p16-positive oropharyngeal cancers (OPCs) and other HNCs (p = 0.0143 and 0.032, respectively). In addition, we found that baseline CTC numbers may serve as independent predictors of treatment response, even after adjusting for other conventional prognostic factors. CTCs were detected in 10 out of 11 patients exhibiting incomplete treatment responses.ConclusionsWe found that baseline CTC numbers are correlated with treatment response in patients with HNC. The expression level of cell-surface vimentin (CSV) on CTCs was significantly higher in patients with persistent or progressive disease, thus providing additional prognostic information for stratifying the risk at diagnosis in HNC patients. The ability to detect CTCs at diagnosis allows more accurate risk stratification, which in the future may be translated into better patient selection for treatment intensification and/or de-intensification strategies.

Cancer stem cell-like cells-derived exosomal CDKN2B-AS1 stabilizes CDKN2B to promote the growth and metastasis of thyroid cancer via TGF-β1/Smad2/3 signaling

As CDKN2B-AS1 is demonstrated to exert promotive effects on thyroid cancer (TC), this research aims to investigate the role of cancer stem cell-like cells (CSCs)-derived exosomal CDKN2B-AS1 in TC and the underlying regulatory mechanism. Specifically, CDKN2B expression and the correlation of CDKN2B with CDKN2B-AS1 in TC were determined via bioinformatics analysis and further verified by qRT-PCR. After transfection or co-culture with CSCs-derived exosomes, viability, migration, and invasion of TPC-1 and SW579 cells were evaluated by CCK-8, wound healing, and transwell assays, respectively. The uptake of exosomes by TC cells was detected by PKH67 labeling. In vivo tumor formation and metastasis models were established. Tumor volume and weight were calculated. Metastasis loci in lung tissues were observed by hematoxylin-eosin staining. The expression levels of CDKN2B-AS1, CDKN2B, and epithelial-mesenchymal transition- and TGF-β1/Smad2/3 signaling-related factors were detected by qRT-PCR or Western blot. Concretely, CDKN2B and CDKN2B-AS1 were highly expressed in TC, and there was a positive correlation between the two. In addition, CDKN2B-AS1 promoted the translation and stability of CDKN2B. Furthermore, CDKN2B-AS1 was highly expressed in CSCs and CSCs-derived exosomes which could be absorbed by TC cells. CDKN2B silencing inhibited viability, migration, invasion, protein levels of CDKN2B, N-cadherin and Vimentin, and TGF-β1/Smad2/3 signaling, while promoting E-cadherin expression in TC cells. CSCs-derived exosomal CDKN2B-AS1 did oppositely and reversed the effects of CDKN2B silencing on TC cells. CDKN2B silencing impeded tumor growth and metastasis in TC mice, while TGF-β1 performed inversely and impaired the effects of CDKN2B silencing. Collectively, CSCs-derived exosomal CDKN2B-AS1 stabilizes CDKN2B to promote growth and metastasis of TC via TGF-β1/Smad2/3 signaling.

NEK2 promotes the migration and proliferation of ESCC via stabilization of YAP1 by phosphorylation at Thr-143

BackgroundEsophageal Squamous Cell Carcinoma (ESCC) was characterized as a regional-prevalent and aggressive tumor with high morbidity and mortality. NIMA-related kinase 2 (NEK2) is an interesting oncogene, the alteration of which leads to patients-beneficial outcomes. We aimed to explore the role of NEK2 in ESCC and excavate its mechanism.MethodsRNA-seq data were downloaded from TCGA and GEO and analyzed by R software. The protein levels were detected by immunohistochemistry (IHC) or western blot (WB), and mRNA expression was detected by qRT-PCR. The in vitro role of proliferation and migration was detected by Transwell migration assay and by colony formation assay, respectively. The in vivo roles were explored using a subcutaneous xenograft tumor model, where immunofluorescence (IF) and IHC were employed to investigate expression and localization. The interaction between proteins was detected by immunoprecipitation. The stability of proteins was measured by WB in the presence of cycloheximide.ResultsA higher level of NEK2 was found in ESCC than normal esophageal epithelia in GEO, TCGA, and tissue microarray, which was associated with worse prognoses. The NEK2 knockdown impaired the proliferation and migration of ESCC, which also downregulated YAP1 and EMT markers like N-cadherin and Vimentin in vitro. On the contrary, NEK2 overexpression enhanced the migration of ESCC and elevated the levels of YAP1, N-cadherin, and Vimentin. Additionally, the overexpression of YAP1 in NEK2 knocked down ESCCs partly rescued the corresponding decrease in migration. The knockdown of NEK2 played an anti-tumor role in vivo and was accompanied by a lower level and nucleus shuffling of YAP1. In mechanism, NEK2 interacted with YAP1 and increased the stability of both endogenous and exogenous YAP1 by preventing ubiquitination. Moreover, the computer-predicted phosphorylation site of YAP1, Thr-143, reduced the ubiquitination of HA-YAP1, strengthened its stability, and thus influenced the migration in vitro.ConclusionsNEK2 is a prognostic oncogene highly expressed in ESCC and promotes the progression of ESCC in vitro and in vivo. Mechanistically, NEK2-mediated phosphorylation of YAP1 at Thr-143 protects it from proteasome degradation and might serve as a promising therapeutic target in ESCC.3BX3sTS59bhAxgWmCYQN5gVideo

Ferrostatin‑1 alleviates oxalate‑induced renal tubular epithelial cell injury, fibrosis and calcium oxalate stone formation by inhibiting ferroptosis.

The present study aimed to evaluate the role and mechanism of ferrostatin-1 (Fer-1) in oxalate (Ox)-induced renal tubular epithelial cell injury, fibrosis, and calcium oxalate (CaOx) stone formation. A CaOx model in mice kidneys was established via intraperitoneal injection of 80 mg/kg glyoxylic acid for 14 days. The mice were randomly divided into three groups (n=6), namely, the control (Con), the CaOx group, and the CaOx + Fer-1 group. Cultured human renal tubular epithelial cells (HK-2 cells) were randomly divided into three groups (n=3), namely, the control (Con), the Ox group, and the Ox + Fer-1 group. The levels of heme oxygenase 1 (HO-1), superoxide dismutase 2 (SOD2), glutathione peroxidase 4 (GPX4), and solute carrier family 7 member 11 (SLC7A11) were assessed by immunofluorescence and western blot analysis. Renal tubular injury and apoptosis were evaluated by H&E and TUNEL staining. Kidney interstitial fibrosis was evaluated by Masson and Sirius red staining, and the levels of E-cadherin, vimentin and α-SMA were detected by immunofluorescence or western blot analysis. Mitochondrial structure was observed using a transmission electron microscope. The levels of reactive oxygen species (ROS) were determined by flow cytometry and CaOx stone formation was evaluated by von Kossa staining. The results revealed that in comparison with the Con group, mitochondrial injury under glyoxylic acid treatment was observed by TEM. The expression of GPX4 and SLC7A11 in the CaOx and Ox groups was downregulated (P<0.05), whereas the expression of HO-1 and SOD2 was upregulated (P<0.05). Renal tissue damage, apoptosis of renal tubular epithelial cells, and interstitial fibrosis were increased in the CaOx and Ox groups (P<0.05). In comparison with the CaOx or Ox group, the expression of GPX4 and SLC7A11 in the CaOx + Fer-1 or Ox + Fer-1 group was upregulated (P<0.05), whereas that of HO-1 and SOD2 was downregulated (P<0.05). Renal tissue damage, apoptosis of renal tubular epithelial cells and interstitial fibrosis were decreased following Fer-1 treatment (P<0.05). The ROS level was also decreased following Fer-1 treatment. Moreover, CaOx stone formation was decreased in the CaOx + Fer-1 group (P<0.05). In conclusion, Fer-1 alleviated Ox-induced renal tubular epithelial cell injury, fibrosis, and CaOx stone formation by inhibiting ferroptosis.

Abstract 6284: Exosomal miR-29b derived from mesenchymal stem cell (MSC) may suppress peritoneal metastasis via the effects on peritoneal mesothelial cells

Abstract Background: Mesothelial cells play tumor-promoting roles in the development of peritoneal metastasis (PM) through mesothelial-mesenchymal transition (MMT). We reported miR-29b suppressed MMT of human peritoneal mesothelial cell (HPMC). Methods: MiR-29b mimics and negative control miRNA (NC) were transfected to human bone marrow derived MSC by lipofection method. Exosomes were purified from the culture supernatants of the MSC with ultracentrifugation, and added to HPMC. Proliferation and migration of HPMC were examined with MTT assay and transwell assay, respectively. MMT of HPMC induced by 10 ng/ml TGF-β, and evaluated with immunofluorescence staining. Results: MSC transfected with miR-29b mimics produced significantly increased amounts of miR-29b-containing exosomes (p&amp;lt;0.0001, n=3) as compared with negative control. After 24h-culture with PKH-26 stained exosomes, HPMC actively took the exosomes. After 48 hr-culture with TGF-β, HPMC apparently changed the morphology from round to spindle shape. The expression level of E-cadherin and Calretinin in HPMC was decreased while vimentin and Fibronectin tended to be upregulated by TGF-β. However, exosomes from miR-29b transfected MSC significantly suppressed the morphological changes and reduced the expression levels of vimentin and Fibronectin with restored expression of E-cadherin and Calretinin, suggesting the inhibition of MMT in HPMC. The exosome inhibited the proliferation and migration of HPMC by 31±14% (p&amp;lt;0.01, n=5) and 67±9% (p&amp;lt;0.01, n=3), and inhibited the adhesion of NUGC by 90±3% (p&amp;lt;0.001, n=5) as compared with NC exosome. Conclusion: Exosomes from MSC transfected with miR-29b mimic efficiently suppresses MMT. Replacement of the miR-29b in peritoneal cavity might be used for the treatment of PM partially via the effects on mesothelial cell. Citation Format: Yuki Kimura, Hideyuki Ohzawa, Yuki Kaneko, Kohei Tamura, Yurie Futoh, Kazuya Takahashi, Akira Saito, Mineyuki Tojo, Hideyo Miyato, Joji Kitayama. Exosomal miR-29b derived from mesenchymal stem cell (MSC) may suppress peritoneal metastasis via the effects on peritoneal mesothelial cells [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 6284.

Abstract 5540: Tumor phenotypic plasticity and resistance to immune-mediated cytotoxicity in models of castration resistant prostate cancer

Abstract Androgen receptor (AR)-targeted therapies are routinely used to treat prostate cancer, including the use of next-generation hormonal therapies such as abiraterone or enzalutamide. Although these therapies are initially effective, a significant proportion of patients exhibit resistance to treatment, therefore developing castration-resistant prostate cancer (CRPC). While immune checkpoint blockade therapies have led to remarkable clinical responses in patients with several tumor types, CRPC remains resistant to immunotherapy. The characterization of tumor resistance mechanisms has led to the identification of various tumor variants that could emerge along the progression of prostate cancer, including tumors undergoing phenotypic plasticity. Our laboratory has previously shown that phenotypic plasticity is a driver of resistance to immunotherapy. Here we investigated whether changes in tumor phenotype could affect the response of CRPC to immune-based therapies, and ways these mechanisms can be mitigated. Models of resistance to enzalutamide (LNCAP-EnzaR) or abiraterone (LNCAP-AbiR) were derived from the androgen sensitive LNCAP prostate cancer cell line. Resistant and parental LNCAP cells were comparatively evaluated for phenotypic features via RT-PCR, ELISA, western blot, immunofluorescence, and RNAseq analysis. Changes in the susceptibility to NK-cell mediated cytotoxicity were evaluated with NK cells isolated from peripheral blood from healthy donors. LNCAP-EnzaR and LNCAP parental cells were also grown in vivo in NSG MHCI/II-deficient mice, and tumors were characterized for phenotypic markers and potential therapeutic targets. Our results demonstrated that acquisition of resistance to both enzalutamide and abiraterone was associated with a significant increase in mesenchymal tumor features, including increased levels of vimentin and fibronectin, and the loss of epithelial E-cadherin. The susceptibility of LNCAP-Enza-R and LNCAP-AbiR cells to NK-cell mediated cytotoxicity and antibody-dependent cell cytotoxicity (ADCC) were significantly reduced (up to 90%), compared with parental cells. RNAseq analysis identified several upregulated gene and gene pathways, including estrogen receptor 1 (ESR1), which was particularly overexpressed in LNCAP-EnzaR cells. In a xenograft model of LNCAP-EnzaR cells, we corroborated the maintenance of tumor phenotypic plasticity and the expression of actionable targets. Our data indicates that acquisition of resistance to AR inhibition is associated with significant reduction of susceptibility to immune attack, and the acquisition of tumor phenotypic plasticity features. Future studies plan to investigate approaches that revert tumor plasticity, including blockade of increased markers such as ESR1, TGF-beta or IL-8, for potential improvement of tumor susceptibility to immune attack in CRPC. Citation Format: Madeline Dahut, Kristen Fousek, Lucas Horn, Haiyan Qin, Jeffrey Schlom, Claudia Palena. Tumor phenotypic plasticity and resistance to immune-mediated cytotoxicity in models of castration resistant prostate 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 5540.

Dual STAT‑3 and IL‑6R inhibition with stattic and tocilizumab decreases migration, invasion and proliferation of prostate cancer cells by targeting the IL‑6/IL‑6R/STAT‑3 axis

Prostate cancer (PCa) is a key public health problem worldwide; at diagnosis, a high percentage of patients exhibit tumor cell invasion of adjacent tissue. STAT-3, IL-6 receptor (R) and IL-6 serum levels are associated with enhanced PCa migratory, invasive, clonogenic and metastatic ability. Inhibiting the STAT-3 pathway at different levels (cytokines, receptors, and kinases) exhibits relative success in cancer. The present study investigated the effect of Stattic (Stt) + Tocilizumab (Tcz) on proliferative, clonogenic, migratory and invasive ability of human metastatic PCa (assessed by colony formation, wound healing and migration assay). RWPE-1 (epithelial prostate immortalized cells), 22Rv1 (Tumor cells), LNCaP (Metastatic cells) and DU-145 (metastatic, castration-resistant prostate cells) cells were used in vitro to evaluate levels of cytokines, chemokines, growth factors (Cytometric Bead Array), STAT-3, phosphorylated STAT-3 (In-Cell Western), IL-6R, vimentin and epithelial (E-) cadherin (Western Blot). The effect of inhibition of STAT-3 (expressed constitutively in DU-145 cells) with Stt and/or Tcz on expression levels of vimentin, VEGF, and E-cadherin, as well as proliferative, clonogenic, migratory and invasive capacity of metastatic PCa cells was assessed. The expression levels of IL-6, C-X-C chemokine ligand 8, VEGF and vimentin, as well as proliferation and migration, were increased in metastatic PCa cells. Treatment with Stt or Tcz decreased vimentin and VEGF and increased E-cadherin expression levels and inhibited proliferative, clonogenic, migratory and invasive capacity of DU-145 cells; addition of IL-6 decreased this inhibitory effect. However, Stt + Tcz maintained inhibition even in the present of high concentrations of IL-6. Stt + Tcz decreased expression of vimentin and VEGF and inhibited the proliferative, clonogenic, migratory and invasive capacity of metastatic PCa cells. To the best of our knowledge, the present study is the first to combine Stt, a STAT-3 inhibitor, with Tcz, an antibody against IL-6R, to target tumor cells.

Coptidis Rhizoma Suppresses Metastatic Behavior by Inhibiting TGF-β-Mediated Epithelial-Mesenchymal Transition in 5-FU-Resistant HCT116 Cells.

Colorectal cancer (CRC) is the second most lethal malignancy worldwide. The high mortality rate of CRC is largely due to cancer metastasis. Recently, suppressing epithelial-to-mesenchymal transition (EMT) has been considered a promising strategy for treating metastatic cancer, especially drug-resistant metastatic cancer. The present study aimed to evaluate the antimetastatic effect of Coptidis Rhizoma, as well as the potential underlying mechanisms, using a 5-fluorouracil-resistant colon tumor cell model (HCT116/R). Coptidis Rhizoma 30% ethanol extract (CRE) significantly inhibited HCT116/R cells migration and invasion. CRE effectively inhibited EMT in HCT116/R cells by upregulating the expression of an epithelial marker (E-cadherin) and downregulating the expression of mesenchymal markers (vimentin, Snail, and ZEB2) at both the protein and gene levels. Immunofluorescence assays also confirmed consistent patterns in the levels of E-cadherin and vimentin. In addition, the anti-EMT activity of CRE and its related effects were associated with the CRE-mediated suppression of the TGF-β pathway, as shown by changes in the levels of downstream molecules (phosphorylated Akt and p38), and inhibition of migration, invasion, and protein expression of TGF-β after treatment/cotreatment with a TGF-β inhibitor (SB431542). In conclusion, Coptidis Rhizoma exerts an antimetastatic effect, especially in the treatment of drug-resistant cancer, and the possible mechanisms are associated with inhibiting EMT via TGF-β signaling. Thus, Coptidis Rhizoma will likely become a potential therapeutic candidate for simultaneously mitigating drug resistance and metastasis in CRC.

Environmental microcystin exposure triggers the poor prognosis of prostate cancer: Evidence from case-control, animal, and in vitro studies

Microcystin-leucine-arginine (MC-LR) is positively linked with multiple cancers in humans. However, the association between MC-LR and the risk and prognosis of prostate cancer has not been conducted in epidemiological studies. No reported studies have linked MC-LR exposure to the poor prognosis of prostate cancer by conducting experimental studies. The content of MC-LR was detected in most of the aquatic food in wet markets and supermarkets in Nanjing and posed a health risk for consumers. MC-LR levels in both prostate cancer tissues and serum were significantly higher than controls. The adjusted odds ratio (OR) for prostate cancer risk by serum MC-LR was 1.75 (95%CI: 1.21-2.52) in the whole subjects, and a positive correlation between MC-LR and advanced tumor stage was observed. Survival curve analysis indicated patients with higher MC-LR levels in tissues exhibited poorer overall survival. Human, animal, and cell studies confirmed that MC-LR exposure increases the expression of estrogen receptor-α (ERα) and promotes epithelial-mesenchymal transition (EMT) in prostate cancer. Moreover, MC-LR-induced decreased E-cadherin levels, increased vimentin levels, and increased migratory and invasive capacities of prostate cancer cells were markedly suppressed upon ERα knockdown. MC-LR-induced xenograft tumor growth and lung metastasis in BALB/c nude mice can be effectively alleviated with ERα knockdown. Our data demonstrated that MC-LR upregulated vimentin and downregulated E-cadherin through activating ERα, promoting migration and invasion of prostate cancer cells. Our findings highlight the role of MC-LR in prostate cancer, providing new perspectives to understand MC-LR-induced prostatic toxicity.

Pirfenidone inhibits cell fibrosis in connective tissue disease-associated interstitial lung disease by targeting the TNF-α/STAT3/KL6 pathway.

BackgroundTo explore the effect and mechanism of pirfenidone in inhibiting pulmonary fibrosis in connective tissue disease-associated interstitial lung disease (CTD-ILD).MethodsFrom 2018 to 2020, 50 CTD-ILD patients were enrolled in the clinical study. Based on whether pirfenidone was used during treatment, patients were enrolled into the pirfenidone group and the control group. Pulmonary function tests were compared before and after treatment. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the expression of tumor necrosis factor-α (TNF-α), signal transducer and activator of transcription 3 (STAT3), and Krebs Von den Lungen-6 (KL-6) in venous blood before and after treatment. Rat type II (RLE-6TN) lung epithelial cells were cultivated for in vitro experiments, and they were sorted into the control group, bleomycin group, pirfenidone group, TNF-α group, Stattic group, and TNF-α/Stattic combined treatment group. For the in vitro experiments, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tests were used to evaluate cell proliferation, Reverse Transcription-Polymerase Chain Reaction(RT–PCR) was performed to detect STAT3 and KL-6 mRNA expression levels, ELISA was utilized to detect TNF-α and E-cadherin expression levels, and Western blotting (WB) was performed to determine α-smooth muscle actin (α-SMA), vimentin, TNF-α, STAT3, phosphorylated signal transducer and activator of transcription 3 (PSTAT3) and KL-6 expression.ResultsIn the clinical study, the pulmonary function indices including forced expiratory volume in one second (FEV1), forced vital capacity (FVC), FEV1/FVC, peak expiratory flow (PEF) and partial pressure (PaO2) of the patients in the study group were superior to those in the control group (P<0.05). The serum TNF-α, STAT3 and KL-6 levels in the study group were significantly lower than those in the control group (P<0.05). In the in vitro experiments, the α-SMA, vimentin, STAT3 and KL-6 levels in the treatment group were significantly lower than those in the bleomycin group (P<0.05). Compared with those in the pirfenidone group, the α-SMA, vimentin, STAT3 and KL-6 levels in the TNF-α-treated group were significantly upregulated (P<0.05). Meanwhile, cell viability was further upregulated (P<0.05), and the expression of STAT3 and KL-6 was further decreased in the Stattic-treated group (P<0.05). In the group treated with infliximab combined with Stattic, TNF-α expression was significantly increased (P<0.05), cell activity was significantly restored (P<0.05), and the STAT3, KL-6 and E-cadherin expression levels were inhibited (P<0.05).ConclusionsPirfenidone improved pulmonary function 1 and decreased TNF-α, STAT3, and KL-6 expression in CTD-ILD patients. Moreover, pirfenidone inhibits cell fibrosis through the TNF-α/STAT3/Mucin 1(MUC1) pathway.

Co-Culture of Bone Marrow Mesenchymal Stem Cells Regulates the Fibrotic Response of Endometrial Stromal Cells

This study explored the protective effects and mechanisms of bone marrow mesenchymal stem cells (BMSCs) on the fibrotic response of endometrial stromal cells. The endometrial cells were isolated from intrauterine adhesions (IUA) patients and assigned into control group, TGF-β1 group, BMSC group which was co-cultured with BMSCs in presence of TGF-β1; Exo group (treated with BMSCs-originated exosomes); Exo-inhibitor group (treated with exosome-specific inhibitors) followed by analysis of α-SMA and Vimentin level, cell viability and expression of TGFBR2, TGFβ-1, Fibronectin (FN), α-SMA, Collagen 1α1 (Col1a1), Smad2/3 and p-Smad2/3. After TGF-β1 treatment, cells exhibited higher expression of p-Smad2/3, TGFBR2, FN, TGF-SMA and COL1A1 along with reduced cell proliferation. However, BMSCs-originated exosomes or co-culture with BMSCs reversed these changes which could be inverted by exosome-specific inhibitors. In conclusion, BMSCs-originated exosomes and BMSCs exerted an anti-fibrosis effect, which was possibly through regulation of TGFβ1/Smad2/Smad3 signalling pathway in endometrial stromal cells.

ZIC5 promotes human hepatocellular carcinoma cell proliferation through upregulating COL1A1.

Zinc finger of the cerebellum 5 (ZIC5) has been found to be abnormally expressed in a variety of tumors. This study aimed to reveal the expression and functional mechanism of ZIC5 in hepatocellular carcinoma (HCC). Analysis of ZIC5 expression in various tumors and its relationship with survival were derived from The Cancer Genome Atlas (TCGA) database. Cell counting kit-8 (CCK-8) and colony formation assays were performed for the detection of HCC cell proliferation. Differentially expressed genes (DEGs) after ZIC5 overexpression in Huh1 cells were determined by RNA sequencing. Western blot assays were conducted to detect the protein levels of c-Myc, Bcl2, p21, E-cadherin, N-cadherin, vimentin, and collagen type I alpha 1 (COL1A1). Dramatically increased expression of ZIC5 was observed in various tumor tissues, including HCC. Pearson's correlation analysis revealed that the mRNA levels of ZIC5 had a positive correlation with the mRNA levels of MKI67 in HCC tissues. Patients with high ZIC5 expression had a shorter overall survival time. Moreover, ZIC5 overexpression promoted HCC cell proliferation. Then, we found that COL1A1 was significantly upregulated by ZIC5 overexpression to promote the proliferation, migration, and invasion of HCC cells. ZIC5 could accelerate the proliferation, migration, and invasion of HCC cells through upregulating COL1A1. This is the first report that ZIC5 and COL1A1 are intrinsically linked, expanding new research ideas for subsequent HCC research.

The Role of GLI in the Regulation of Hepatic Epithelial-Mesenchymal Transition in Biliary Atresia.

ObjectiveTo study the regulatory role of GLI1/GLI2, a nuclear transcription factor of the Sonic hedgehog (Shh) signaling pathway, in epithelial–mesenchymal transition (EMT) related to hepatic fibrosis in patients with biliary atresia (BA).MethodsThe messenger RNA (mRNA) and protein expression levels of GLI1/GLI2, Snail/Slug, and other Shh- and EMT-related cytokines were tested in the liver tissues of BA patients and animals. Then, GLI1/GLI2 was silenced and overexpressed in mouse intrahepatic bile duct epithelial cells (mIBECs) and BA animals to investigate changes in the mRNA and protein expression of EMT key factors and liver fibrosis indicators. After silencing and overexpression of GLI1/GLI2, immunofluorescence was used to detect the expression of cytokeratin-19 (CK19) and α-smooth muscle actin (α-SMA) in mIBECs, and hematoxylin and eosin (HE) staining and Masson staining were used to observe the degree of liver fibrosis in the BA animals.ResultsCompared with the control, the mRNA and protein expression levels of GLI2, Snail, vimentin, and α-SMA were significantly increased and those of E-cadherin were significantly decreased in liver tissue from BA patients and animals. Overexpression of GLI2 increased the mRNA and protein expression levels of Snail, vimentin, and α-SMA and that of E-cadherin was significantly decreased in mIBECs and BA animals. After GLI2 silencing, the opposite pattern was observed. Immunofluorescence detection showed enhanced expression of the bile duct epithelial cell marker CK19 in mIBECs after GLI2 silencing and enhanced expression of the mesenchymal cell marker α-SMA after GLI2 overexpression. HE and Masson staining suggested that the GLI2-overexpressing group had a significantly higher degree of fibrosis.ConclusionThe Shh signaling pathway plays an important role in fibrogenesis in BA. GLI2 can significantly regulate EMT in mIBECs and livers of BA mice.

Pentagalloylglucose suppresses the growth and migration of human nasopharyngeal cancer cells via the GSK3β/β-catenin pathway in vitro and in vivo

BackgroundNasopharyngeal carcinoma (NPC) is a type of malignant squamous cell tumour originating from the nasopharynx epithelium. Pentagalloylglucose (PGG) is a natural polyphenolic compound that exerts anticancer effects in many types of tumours. However, the role and underlying mechanism of PGG in NPC cells have not been fully defined. PurposeThis study aimed to investigate the anticancer activity of PGG as well as the potential mechanism in NPC cells. MethodsThe effects of PGG on the proliferation, apoptosis and cell cycle distribution of CNE1 and CNE2 cells were assessed by MTT and flow cytometry assays. Cell migration was evaluated using wound healing and transwell assays. The expression of microtubule-associated protein 1 light chain 3 beta (LC3B) was observed by immunofluorescence staining. Western blotting was used to explore the levels of related proteins and signalling pathway components. Furthermore, the effects of PGG on NPC cell growth were analysed in a xenograft mouse model in vivo using cisplatin as a positive control. ResultsPGG dose-dependently inhibited the proliferation of CNE1 and CNE2 cells. PGG regulated the cell cycle by altering p53, cyclin D1, CDK2, and cyclin E1 protein levels. PGG induced apoptosis and autophagy in NPC cells and elevated the Bax/Bcl-2 ratio and the protein levels of LC3B. Moreover, PGG decreased NPC cell migration by increasing E-cadherin and decreasing N-cadherin, vimentin and CD44 protein levels. Mechanistically, PGG treatment downregulated p-mTOR and β-catenin expression but upregulated p-p38 MAPK and p-GSK3β expression. In addition, PGG significantly inhibited NPC cell tumour growth and lung metastasis in vivo. ConclusionPGG may suppress cell proliferation, induce apoptosis and autophagy, and decrease the metastatic capacity of NPC cells through the p38 MAPK/mTOR and Wnt/β-catenin pathways. The present study provides evidence for PGG as a potential therapy for NPC.

Danshensu Attenuated Epithelial-Mesenchymal Transformation and Chemoresistance of Colon Cancer Cells Induced by Platelets.

The interactions between platelets and tumor cells are well-known to play important roles in the progression of malignant tumors. Danshensu, a main water-soluble component of Salvia miltiorrhiza, can resist platelet aggregation and exert significant anti-tumor effects on various types of tumors. However, whether Danshensu could inhibit the progression of malignant tumors by suppressing the activities of platelets had not been reported. The effects of Danshensu on the platelet activity and epithelial-mesenchymal transformation (EMT)-like invasive phenotype of SW620 colon cancer cells were assessed by stimulating with the supernatants from co-cultured platelets and SW620 cells with direct contact (SCP). The expression and secretion of proteins were determined by western blot and enzyme-linked immunosorbent assay (ELISA), respectively. Hematoxylin and eosin (H&E) staining was performed to analyzed the histopathology of tumor tissues and immunohistochemical staining was conducted to examine the protein expression in tumors. Co-incubation of SW620 cells with platelets directly or SCP both generated long spindle-shaped invasive phenotype. Pretreatment of platelets with Danshensu (25 μM) inhibited the morphological changes of SW620 cells induced by SCP, which was associated with the inhibitory effects of Danshensu on platelet secretion. Danshensu diminished the secretion of a list of biological factors in SCP, including interleukin (IL)-6, tumor necrosis factor alpha (TNF-α), IL-1β and vascular endothelial growth factor (VEGF) that are all involved in tumor cell EMT and chemoresistance. Moreover, Danshensu up-regulated the expression of E-cadherin but down-regulated the levels of N-cadherin and Vimentin, resulting in the repression of SW620 cell migration. It was also shown that Danshensu enhanced the sensitivity of SW620 cells to oxaliplatin by suppressing the expression of MDR1. Furthermore, Danshensu could not only reduced the growth of subcutaneous tumors and liver metastasis that induced by SCP, but also down-regulated the expression of MDR1 in vivo. Mechanistic studies revealed that Danshensu suppressed the activation of the TGF-β/Smad signaling pathway. Danshensu attenuated EMT-like characteristics and chemoresistance by inhibiting secretion capability of platelets and activation of the TGF-β/Smad signaling pathway, suggesting that it may be optimized to be a therapeutic agent for fighting against colon cancer.

BHLHE41 Overexpression Alleviates the Malignant Behavior of Colon Cancer Cells Induced by Hypoxia via Modulating HIF-1α/EMT Pathway

Objective BHLHE41 has been shown to be a marker of tumorigenesis. Colon cancer (CC) is a common malignant tumor of colonic mucosa. This study mainly explored the mechanism of BHLHE41 in alleviating malignant behavior of hypoxia-induced CC cells. Methods The levels of BHLHE41 in CC and normal cell lines were tested by Western blot and qRT-PCR. After, CC cells were subjected to hypoxia treatment and BHLHE41 overexpression transfection, and the BHLHE41 expression, the effect of BHLHE41 on CC cell viability, apoptosis, migration, and invasion and cell cycle were tested by qRT-PCR and relevant cell functional experiments. HIF-1α and epithelial-mesenchymal transition- (EMT-) related proteins were tested by Western blot. Moreover, CC tumor-bearing model was established in nude mice, and the effect of BHLHE41 on the tumor was evaluated by measuring the tumor volume and weight. Then, the expressions of BHLHE41 and EMT-related proteins were detected by immunohistochemistry and Western blot. Results Western blot and qRT-PCR showed that BHLHE41 was lowly expressed in CC cells. BHLHE41 overexpression could inhibit the hypoxia-induced CC cell viability, migration, and invasion, induce apoptosis, and alter cell cycle. Besides, BHLHE41 overexpression could enhance the levels of E-cadherin but reduce the levels of HIF-1α, N-cadherin, vimentin, and MMP9 in hypoxia-induced CC cells. Moreover, BHLHE41 overexpression reduced tumor volume, weight, and EMT-related proteins levels in tumor tissues. Conclusions BHLHE41 overexpression could mitigate the malignant behavior of hypoxia-induced CC via modulating the HIF-1α/EMT pathway.

NF-κB as a regulator of cancer metastasis and therapy response: A focus on epithelial-mesenchymal transition.

Metastasis of tumor cells is a complex challenge and significantly diminishes the overall survival and prognosis of cancer patients. The epithelial-to-mesenchymal transition (EMT) is a well-known mechanism responsible for the invasiveness of tumor cells. A number of molecular pathways can regulate theEMT mechanism in cancer cells and nuclear factor-kappaB (NF-κB) is one of them. The nuclear translocation of NF-κB p65 can induce the transcription of several genes involved in EMT induction. The present review describes NF-κB and EMT interaction in cancer cells and their association in cancer progression. Due to the oncogenic role NF-κB signaling, its activation enhances metastasis of tumor cells via EMT induction. This has been confirmed in various cancers including brain, breast, lung and gastric cancers, among others. The ZEB1/2, transforming growth factor-β, and Slug as inducers of EMT undergo upregulation by NF-κB to promote metastasis of tumor cells. After EMT induction driven by NF-κB, a significant decrease occurs in E-cadherin levels, while N-cadherin and vimentin levels undergo an increase. The noncoding RNAs can potentially also function as upstream mediators and modulate NF-κB/EMT axis in cancers. Moreover, NF-κB/EMT axis is involved in mediating drug resistance in tumor cells. Thus, suppressing NF-κB/EMT axis can also promote the sensitivity of cancer cells to chemotherapeutic agents.

Serum vimentin predicts mortality in pediatric severe sepsis: A prospective observational study

ObjectivesVascular hyperpermeability by loss of endothelial barrier integrity is a hallmark of sepsis. Vimentin is involved in the regulation of the endothelial function and inflammatory response. However, the serum level of vimentin and its clinical relevance in pediatric severe sepsis (PSS) remain unknown. MethodsWe conducted a prospective study of PSS cases who were admitted to the pediatric intensive care unit (PICU) from January 2018 to December 2020. ResultsA total of 108 patients with PSS with a median age of 19.5 month were enrolled. The hospital mortality rate was 19.44% (21/108). Comparing with healthy controls, serum vimentin levels on PICU admission were significantly higher in patients with PSS (P < 0.001). The area under the ROC curve for vimentin to predict the hospital mortality was 0.712 (95% CI: 0.578–846) with a sensitivity of 71.43% and a specificity of 70.11%. Moreover, hospital mortality was significantly higher in patients with vimentin level over the cutoff value of 24.53 ng/ml than in patients with vimentin level below 24.53 ng/ml (P < 0.001). ConclusionsSerum vimentin level as an indicator of endothelial injury is associated with the prognosis of PSS, and serum vimentin level ≥24.53 ng/ml on PICU admission predicts high risk for hospital mortality in PSS.

Jieduquyuzishen Prescription Attenuates Renal Fibrosis in MRL/lpr Mice via Inhibiting EMT and TGF-β1/Smad2/3 Pathway.

Jieduquyuziyin prescription (JP) has been used to treat lupus nephritis (LN) and its effectiveness in the treatment of LN has been clinically proven, but the underlying mechanisms have yet to be completely understood. This aim of this study was to clarify the efficacy of JP on the epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells and the molecular mechanisms of JP in MRL/lpr mice. In vivo, we observed the therapeutic actions of JP in MRL/lpr mice as well as its antifibrosis effect and potential mechanism. In vitro, we evaluated the role of JP in EMT and its possible mechanism through the EMT of human renal proximal tubular epithelial cells (HK-2) induced by transforming growth factor-beta 1 (TGF-β1) and M2c macrophages. HK-2 cells were treated with JP-treated serum, and MRL/lpr mice were treated by JP for 8 weeks. The results showed that JP alleviated disease activity, improved renal function, decreased proteinuria, and improved renal injury and fibrosis in MRL/lpr mice. Furthermore, JP suppressed the activation of the TGF-β1/Smad2/3 signaling pathway, upregulated the E-cadherin levels, and downregulated the Vimentin and mesenchymal α-smooth muscle actin (α-SMA) levels in the kidney of MRL/lpr mice. JP was further found to prevent the TGF-β1 and M2c macrophages-induced EMT of HK-2 cells. Collectively, JP could alleviate the disease activity of MRL/lpr mice, improve renal function, and attenuate renal fibrosis, and its underlying mechanisms may be related to the inhibition of EMT and TGF-β1/Smad2/3 signaling pathway.