Rehmannia glutinosa Libosch and Cornus officinalis Sieb herb couple ameliorates renal interstitial fibrosis in CKD rats by inhibiting the TGF-ß1/MAPK signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Herb couple Rehmannia glutinosa Libosch and Cornus officinalis Sieb (RC), originated from "Liuwei Dihuang Pill" which recorded in Key to Therapeutics of Children's Diseases. Traditionally, they have been used widely for their ability to nourish yin and energize the kidneys. Our previous study indicated that the RC could protect against adenine induced Chronic kidney disease (CKD) rats. Nevertheless, there is still no clear explanation of the mechanisms by which RC affects renal interstitial fibrosis in CKD rats. AIM OF THE STUDY: Current Work aims to explore the amelioration and potential mechanism of RC on renal interstitial fibrosis in CKD rats. MATERIALS AND METHODS: CKD rats were induced by adenine. Two weeks after administration, blood, urine, and kidney tissue were collected for biochemical analysis. Observing the physiological state of rats through the changes of rat body weight and renal index. The pro-inflammatory cytokines were measured by enzyme linked immunosorbent assay (ELISA), while renal tissue damage and fibrosis were assessed with Hematoxylin-eosin staining (H&E) and Masson's trichrome staining. In order to determine the levels of indicators and proteins associated with fibrosis signaling pathways, real time PCR (Rt-PCR), Western blot (WB), and immunofluorescence were employed. RESULTS: The renal interstitial fibrosis led to impaired cellular functions with increased the levels of Blood Urea Nitrogen (BUN), Urine protein (UP), Interleukin-1ß (IL-1ß), Interleukin-6 (IL-6), and Tumor Necrosis Factor alpha (TNF-α). and simultaneous up-regulated collagenⅠ(COL-1), fibronection (FN), α-smooth muscle actin (a-SMA), transforming growth factor-ß1 (TGF-ß1), c-Jun N-terminal kinase (JNK), p38 and extracellular regulated protein kinases (ERK), down-regulated the expression of the E-cadherin proteins. RC notably improved renal dysfunction in CKD rats as indicated by decreases in BUN, UP, and renal index. In addition, consistent with the morphological changes of renal tissue, renal interstitial fibrosis in CKD rats after RC intervention was significantly improved, mainly manifested by a decrease in the positive expression of COL-1, FN, and a-SMA, and increased levels of E-cadherin protein. Meanwhile, RC reduced the classical pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α in adenine-induced CKD rats. Additionally, RC administration also down-regulated TGF-ß1, JNK, p38 and ERK. CONCLUSION: In conclusion, RC may reduce inflammation in adenine induced CKD rats, improve extracellular matrix (ECM) components deposition, and diminish epithelial-mesenchymal transition (EMT) marker protein levels. Furthermore, RC intervention significantly reduces the release of inflammatory cytokines and inhibits the TGF-ß1/MAPK signaling pathway. Based on the results, RC might be useful in the treatment of adenine induced renal fibrosis.

Fibrinogen Fragment X Mediates Endothelial Barrier Disruption via Suppression of VE-Cadherin.

INTRODUCTION: Major traumatic injury is associated with early hemorrhage-related and late-stage deaths due to multiple organ failure (MOF). While improvements to hemostatic resuscitation have significantly reduced hemorrhage-related deaths, the incidence of MOF among trauma patients remains high. Dysregulation of vascular endothelial cell (EC) barrier function is a central mechanism in the development of MOF; however, the mechanistic triggers remain unknown. Accelerated fibrinolysis occurs in a majority of trauma patients, resulting in high circulating levels of fibrin(ogen) degradation products, such as fragment X. To date, the relationship between fragment X and EC dysregulation and barrier disruption is unknown. The goal of this study was to determine the effects of fragment X on EC barrier integrity and expression of paracellular junctional proteins that regulate barrier function. METHODS: Human lung microvascular endothelial cells (HLMVECs) were treated with increasing concentrations of fragment X (1, 10, and 100 µg/mL), and barrier function was monitored using the xCELLigence live-cell monitoring system. Quantitative PCR (qPCR) was performed to measure changes in EC expression of 84 genes. Immunofluorescent (IF) cytostaining was performed to validate qPCR findings. RESULTS: Fragment X treatment significantly increased endothelial permeability over time (P < 0.05). There was also a significant reduction in VE-cadherin mRNA expression in fragment X-treated HLMVECs compared to control (P = 0.01), which was confirmed by IF staining. CONCLUSIONS: Fragment X may induce EC hyperpermeability by reducing VE-cadherin expression. This suggests that a targeted approach to disrupting EC-fragment X interactions could mitigate EC barrier disruption, organ edema, and MOF associated with major trauma.

Effect of alpha lipoic acid on epithelial mesenchymal transition in SKOV-3 cells.

BACKGROUND: Ovarian cancer is the fifth leading cause of cancer-related death in women. Patients are usually diagnosed with advanced tumor metastass. Epithelial over cancer cells spread from primary tumor by undergoing epithelial mesenchymal transition (EMT). It has been suggested that alpha lipoic acid (ALA), a natural antioxidant lipophilic compound, reduces the oxidative stress by causing apoptosis and inhibition of proliferation of cell in cancer cells. The aim of our study was to establish a transforming growth factor ß1 (TGF ß1) dependent epithelial mesenchymal transition model in the SKOV-3 ovarian adenocarcinoma cell line which is an epithelial subtype of ovarian cancer and to investigate the effects of alpha lipoic acid on EMT and ovarian cancer migration. METHODS: For establish an EMT model, SKOV-3 cells were treated with different dose of TGF ß1 and XTT cell viability kit was used to find IC 50 dose of ALA. Four different groups that are control, TGF ß1, ALA and ALA + TGF ß1 were created. Changes in the expression of genes related to EMT markers that are E-cadherin, vimentin, Snail, Slug, Twist and Zeb were analyzed with quantitative real-time PCR. These proteins were determined with the immunocytochemistry method. The migration capacity was analyzed with wound healing assay. Matrigel invasion capacity test was used to show invasion and colonization test to show colonization. RESULTS: The dose of TGF ß1 was determined 100 ng/ml at 72 h, the IC50 dose of ALA 219.033 µM at 48 h was determined. EMT markers in the TGF ß1 group were compatible with EMT and it was shown to inhibit EMT in the groups given ALA. According to wound healing, colonization and invasion experiments, proliferation and invasion increased in TGF ß1 group, but decreased in ALA and combined groups (p < 0.05). CONCLUSION: These results indicate that ALA suppresses the metastasis of ovarian cancer cells by regulating EMT, implying that ALA might be a potential therapeutic agent for the treatment of ovarian cancer.

Aluminium bioaccumulation in colon cancer, impinging on epithelial-mesenchymal-transition and cell death.

We investigated the presence of aluminium (Al) in human colon cancer samples and its potential association with biological processes involved in cancer progression, such as epithelial to mesenchymal transition (EMT) and cell death. 25 consecutive colon samples were collected from patients undergoing colonic resection. Both neoplastic and normal mucosa were collected from each patient and subjected to histological, ultrastructural and immunohistochemical analyses. Moreover, colon samples from two Al-positive patients underwent multi-omic analyses, including whole genome sequencing and RNA sequencing (RNAseq). Morin staining, used to identify in situ aluminium bioaccumulation, showed the presence of Al in tumor areas of 24 % of patients. Transmission electron microscopy and energy-dispersive X-ray microanalysis confirmed the presence of Al specifically in intracytoplasmic electrondense nanodeposits adjacent to mitochondria of colon cancer cells. Immunohistochemical analyses for vimentin and nuclear ß-catenin were performed to highlight the occurrence of the EMT phenomenon in association to Al bioaccumulation. Al-positive samples showed a significant increase in both the number of vimentin-positive and nuclear ß-catenin-positive cancer cells compared to Al-negative samples. Moreover, Al-positive samples exhibited a significant decrease in the number of apoptotic cells, as well as the expression of the anti-apoptotic molecule BCL-2. Multi-omic analyses revealed a higher tumor mutational burden (TMB) in Al-positive colon cancers (n = 2) compared to a control cohort (n = 100). Additionally, somatic mutations in genes associated with EMT (GATA3) and apoptosis (TP53) were observed in Al-positive colon cancers. In conclusion, this study provides the first evidence of Al bioaccumulation in colon cancer and its potential role in modulating molecular pathways involved in cancer progression, such as EMT and apoptosis. Understanding the molecular mechanisms underlying Al toxicity might contribute to improve strategies for prevention, early detection, and targeted therapies for the management of colon cancer patients.

Interplay between the plasma membrane and cell-cell adhesion maintains epithelial identity for correct polarised cell divisions.

Polarised epithelial cell divisions represent a fundamental mechanism for tissue maintenance and morphogenesis. Morphological and mechanical changes in the plasma membrane influence the organisation and crosstalk of microtubules and actin at the cell cortex, thereby regulating the mitotic spindle machinery and chromosome segregation. Yet, the precise mechanisms linking plasma membrane remodelling to cell polarity and cortical cytoskeleton dynamics to ensure accurate execution of mitosis in mammalian epithelial cells remain poorly understood. Here, we manipulated the density of mammary epithelial cells in culture, which led to several mitotic defects. Perturbation of cell-cell adhesion formation impairs the dynamics of the plasma membrane, affecting the shape and size of mitotic cells and resulting in defects in mitotic progression and the generation of daughter cells with aberrant architecture. In these conditions, F- actin-astral microtubule crosstalk is impaired, leading to mitotic spindle misassembly and misorientation, which in turn contributes to chromosome mis-segregation. Mechanistically, we identify S100 Ca2+-binding protein A11 (S100A11) as a key membrane-associated regulator that forms a complex with E-cadherin (CDH1) and the leucine-glycine-asparagine repeat protein LGN (also known as GPSM2) to coordinate plasma membrane remodelling with E-cadherin-mediated cell adhesion and LGN-dependent mitotic spindle machinery. Thus, plasma membrane-mediated maintenance of mammalian epithelial cell identity is crucial for correct execution of polarised cell divisions, genome maintenance and safeguarding tissue integrity.

ZWINT promotes the proliferation, migration, and invasion of cervical cancer cells by regulating the p53/p21 signaling pathway.

Cervical cancer leads to 300,000 deaths annually and the mechanism of cervical carcinogenesis remains unclear. Zeste White 10-interacting kinetochore protein (ZWINT) is uniquely elevated in malignancies, promoting proliferation, migration, and colony formation of cancer cells. To investigate the role of ZWINT in proliferation, migration, invasion of cervical cancer, and evaluate the potential ability of ZWINT as a therapeutic target. First, ZWINT expression in cervical cancer was analyzed using the bioinformatic methods and assessed in several cervical cancer cell lines. The cell viability and colony formation assays were used to evaluate cell proliferation. Then, transwell assay was performed to investigate cell migration and invasion. Moreover, western blot was used to measure the expression level of ZWINT, matrix metalloproteinase 9 (MMP-9), N-cadherin, E-cadherin, p53 and p21 in CaSki and HeLa cells with ZWINT overexpression or knockdown. The bioinformatic analysis and western blot assay revealed the expression of ZWINT was significantly increased in cervical cancer. The cell viability and colony formation analysis illustrated that cell proliferation could be promoted by ZWINT overexpression and suppressed by ZWINT knockdown. Moreover, ZWINT promoted migration and invasion of CaSki and HeLa cells, through regulating the expression of MMP-9, N-cadherin, and E-cadherin. Furthermore, ZWINT attenuated the expression of p53 and p21 in cervical cancer cells. In summary, ZWINT functions in promoting cell proliferation, migration, and invasion of cervical cancer cells by suppressing p53/p21 signaling pathway, which indicated ZWINT is a potential therapeutic target for cervical cancer treatment.

INHBA gene silencing inhibits proliferation, migration, and invasion of osteosarcoma cells by repressing TGF-ß signaling pathway activation.

BACKGROUND: Osteosarcoma (OS) is a refractory malignancy. This study aimed to explore the roles and mechanisms of Inhibin subunit beta A (INHBA) in OS. METHODS: INHBA expression levels in OS tissues and cells were assessed using RT-qPCR and western blotting. The impact of INHBA silencing on OS development was then explored by transfecting the OS cell lines U2OS and MG63 with INHBA-small interfering RNA (siRNA). The influence of INHBA silencing on U2OS and MG63 cell proliferation, migration, and invasion was examined using MTT and Transwell assays. Epithelial-mesenchymal transition (EMT) markers (E-cadherin and N-cadherin) were analyzed by RT-qPCR. The expression of genes involved in cell proliferation, migration, invasion, and the TGF-ß signaling pathway was evaluated by western blotting and RT-qPCR. RESULTS: INHBA levels were elevated in the OS tissues and cells. Furthermore, the transforming growth factor-ß (TGF-ß) signaling pathway of OS cells was suppressed in response to INHBA-siRNA, whereas proliferation, migration, and invasion of OS cells were inhibited. Besides, INHBA-siRNA significantly inhibited OS cell EMT, evidenced by enhanced E-cadherin mRNA expression and reduced N-cadherin mRNA expression. Further mechanistic studies revealed that the TGF-ß1 agonist SRI-011381 hydrochloride increased OS cell proliferation, migration, and invasion after INHBA downregulation. CONCLUSION: We found that INHBA silencing could play a vital role in OS via TGF-ß1-regulated proliferation, migration, and invasion.

Mobility gene expression differences among wild-type, Mmp20 null and Mmp20 over-expresser mice plus visualization of 3D mouse ameloblast directional movement.

Enamel forming ameloblasts move away from the dentino-enamel junction and also move relative to each other to establish enamel shape during the secretory stage of enamel development. Matrix metalloproteinase-20 (MMP20) is a tooth specific proteinase essential for proper enamel formation. We previously reported that MMP20 cleaves cadherins and may regulate ameloblast movement. Here, we used an Amelx promoter driven tdTomato reporter to label mouse ameloblasts. With these transgenic mice, we assessed ameloblast mobility group dynamics and gene expression. Three-dimensional imaging of mouse ameloblasts were observed in hemi-mandibles by using a tissue clearing technique. The three-dimensional ameloblast layer in Tg(Amelx-Mmp20) mice that overexpress MMP20 was uneven and the ameloblasts migrated away from this layer. Mouse ameloblast movement toward incisal tips was monitored by ex vivo time-lapse imaging. Gene expression related to cell migration and adhesion was analyzed in ameloblasts from wild-type mice, Mmp20-/- mice with no functional MMP20 and from Tg(Amelx-Mmp20) overexpressing mice. Gene expression was altered in Mmp20-/- and Tg(Amelx-Mmp20) mice compared to wild type. Among the genes assessed, those encoding laminins and a gap junction protein were upregulated in Mmp20-/- mice. New techniques and findings described in this study may lead to an improved understanding of ameloblast movement during enamel formation.

Mechanism of Human Cytomegalovirus-Induced Epithelial-Mesenchymal Transition in Glioma Cells via the Upregulation of RIP2 Expression.

Human cytomegalovirus (HCMV) is associated with epithelial-mesenchymal transition (EMT) in glioma cells; however, its underlying action mechanism remain ambiguous. In this study, we investigated the effects of receptor-interacting protein 2 (RIP2) and nuclear factor (NF)-κB on EMT in HCMV-infected glioma LN-18 cells. Wound healing and invasion assays were used to evaluate the migration and invasion of cells. Western blotting and immunofluorescence microscopy were used to determine the protein expression levels. We found that HCMV induced enhanced migration and invasion of LN-18 cells, activation of the RIP2/NF-κB signaling pathway, downregulation of epithelial cell marker (E-cadherin) expression, and upregulation of mesenchymal cell marker (N-cadherin and vimentin) expression. Moreover, inhibition of RIP2 or NF-κB inhibited the induction of HCMV in LN-18 cells. Therefore, HCMV induces EMT in glioma cells by promoting the activation of NF-κB signaling pathway via the upregulation of RIP2 expression.

Complete loss of E-cadherin expression in a rare case of metastatic malignant meningioma: a case report.

BACKGROUND: Hematogenous tumor spread of malignant meningiomas occurs very rarely but is associated with very poor prognosis. CASE PRESENTATION: We report an unusual case of a patient with a malignant meningioma who developed multiple metastases in bones, lungs and liver after initial complete resection of the primary tumor. After partial hepatic resection, specimens were histologically analyzed, and a complete loss of E-cadherin adhesion molecules was found. No oncogenic target mutations were found. The patient received a combination of conventional radiotherapy and peptide receptor radionuclide therapy (PRRT). Due to aggressive tumor behavior and rapid spread of metastases, the patient deceased after initiation of treatment. CONCLUSIONS: E-cadherin downregulation is associated with a higher probability of tumor invasion and distant metastasis formation in malignant meningioma. Up to now, the efficacy of systemic therapy, including PRRT, is very limited in malignant meningioma patients.

Effects of electroacupuncture on the microvascular structure and related protein expression in the hippocampus of vascular dementia rats. / ç”µé’ˆå¯¹è¡€ç®¡æ€§ç—´å‘†å¤§é¼ æµ·é©¬å¾®è¡€ç®¡ç»“æž„å’Œç›¸å ³è›‹ç™½è¡¨è¾¾çš„å½±å“.

OBJECTIVES: To explore the effect of electroacupuncture (EA) at "Baihui" (GV20) and "Shenting" (GV24) on the microvascular structure and related protein expression in the hippocampus of vascular dementia (VD) rat model, and to investigate the mechanism of EA in the treatment of VD. METHODS: A total of 24 SD rats were randomly divided into sham operation, model, EA, and oxiracetam groups, with 6 rats in each group. Multiple cerebral infarction method was used to establish VD model. In the EA group, EA was applied to GV20 and GV24 for 30 min, once daily for 14 days. Rats in the oxiracetam group were treated with oxiracetam (50 mg/kg) by intraperitoneal injection, and the course of treatment was the same as that in the EA group. Learning and memory ability were evaluated by using Morris water maze test and new object recognition experiment. The cerebral blood flow was detected by laser doppler. The microvascular structure in the hippocampus was observed by transmission electron microscopy. The expression of vascular structure related proteins of platelet-derived growth factor receptor (PDGFR)-ß, platelet endothelial cell adhesion molecule-1(CD31), neural cadherin N-Cadherin, Zonula occludens protein-1(ZO-1) in the hippocampus were measured by Western blot. RESULTS: Compared with the sham operation group, the rats in the model group had a significant increase in time of first crossing the platform, a significant decrease in the number of crossing platform and the new object preference index (P<0.05), a significant decrease in cerebral blood flow (P<0.05), and a significant increase in the brain weight (P<0.05). The structure boundary of pericyte and endothelial cells in the microvessels of the hippocampal CA1 area of model group was blurred, accompanied by obvious edema around the vessels and the reduction of tight junctions. The protein expression levels of PDGFR-ß, CD31, N-Cadherin, ZO-1 were significantly decreased in the model group compared with those in the sham operation group (P<0.05). Compared with the model group, the time of first crossing the platform of rats in the EA and oxiracetam group was shortened, the number of crossing platform were increased (P<0.05), the cerebral blood flow was increased (P<0.05), the brain weight was decreased (P<0.05), the morphology and structure of pericyte and endothelial cells in the microvessels of hippocampal CA1 area were intact, accompanied by the increase of tight junctions. Additionally, Compared with the model group, the EA group had a significant increase in the new object preference index (P<0.05), the protein expression levels of PDGFR-ß, CD31, ZO-1 in the EA group were increased (P<0.05), and the expression of PDGFR-ß, N-Cadherin, ZO-1 in the oxiracetam group were increased (P<0.05). CONCLUSIONS: EA at GV20 and GV24 can improve the learning and memory ability of VD rats, and the mechanism may be related to the repair of microvascular structures and improvement of cerebral blood flow.

Alteration of Cadherin 3 Expression and DNA Methylation in Association with Aggressive Renal Cell Carcinoma.

Cadherins (calcium-dependent adhesion proteins) are important in cellular adhesion and may play a role in the development and progression of renal cell carcinoma (RCC). This study investigated changes in cadherin 3 (CDH3; P-cadherin) mRNA expression, DNA methylation, and protein expression in RCC and compared the results with the histopathological and clinical characteristics of patients. The possible contribution of CDH3 to tumor cell invasiveness was tested in a functional assay using siRNA-based suppression of CDH3 expression and subsequent real-time impedance analysis using a Matrigel invasion model. Our analyses revealed a tumor-specific loss of CDH3 mRNA expression, CDH3 DNA hypermethylation, and loss of distal tubular and collecting duct CDH3 protein expression in RCC. A relatively higher methylation level in tumors was associated with a loss of cell differentiation and higher clinical stage. siRNA-induced suppression of CDH3 expression modulated the invasion characteristics of tumor cells in the impedance-based real-time cellular analysis. Our results indicate that loss of CDH3 expression is common in RCC and may contribute to the pathogenesis of a subset of RCC. Further studies to reveal the mechanisms of loss of expression and its effects on the invasive behavior of renal tumor cells are required.

Polyphyllin I attenuates the invasion and metastasis via downregulating GRP78 in drug-resistant hepatocellular carcinoma cells.

Drug resistance to chemotherapy agents presents a major obstacle to the effective treatment of hepatocellular carcinoma (HCC), a common type of liver cancer. Increasing evidence indicates a link between drug resistance and the recurrence of HCC. Polyphyllin I (PPI), a promising pharmaceutical candidate, has shown potential therapeutic advantages in the treatment of sorafenib-resistant hepatocellular carcinoma (SR-HCC cells). In this study, we sought to investigate the mechanism underlying the inhibitory effect of PPI on the invasion and metastasis of SR-HCC cells. Our in vitro studies included scratch wound-healing migration assays and transwell assays to examine PPI's effect on HCC cell migration and invasion. Flow cytometry was employed to analyze the accumulation or efflux of chemotherapy drugs. The results of these experiments demonstrated that PPI increased the susceptibility of HCC to sorafenib while inhibiting SR-HCC cell growth, migration, and invasion. Molecular docking analysis revealed that PPI exhibited a higher binding affinity with GRP78. Western blot analysis and immunofluorescence experiments showed that PPI reduced the expression of GRP78, E-cadherin, N-cadherin, Vimentin, and ABCG2 in SR-HCC cells. Interference with and overproduction of GRP78 in vitro impacted the proliferation, migration, invasion, and metastasis of HCC cells. Further examination revealed that PPI hindered the expression of GRP78 protein, resulting in a suppressive effect on SR-HCC cell migration and invasion. Histological examination of tumor tissue substantiated that administering PPI via gavage to HepG2/S xenograft nude mice inhibited tumor growth and significantly reduced tumor size, as evidenced by xenograft experiments involving nude mice. Hematoxylin and eosin (HE) staining of tumor tissue specimens, along with immunohistochemistry (IHC), were conducted to evaluate the expression levels of Ki67, GRP78, N-cadherin, Vimentin, and ABCG2. The results indicated that PPI administration decreased the levels of proteins associated with metastasis and markers of drug resistance in tumor tissues, impeding tumor growth and spread. Overall, our findings demonstrated that PPI effectively suppressed the viability, proliferation, invasion, and metastasis of SR-HCC cells both in vitro and in vivo by modulating GRP78 activity. These findings provide new insights into the mechanism of PPI inhibition of SR-HCC cell invasion and metastasis, highlighting PPI as a potential treatment option for sorafenib-resistant HCC.

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

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

Rap1 small GTPase is essential for maintaining pulmonary endothelial barrier function in mice.

Vascular permeability is dynamically but tightly controlled by vascular endothelial (VE)-cadherin-mediated endothelial cell-cell junctions to maintain homeostasis. Thus, impairments of VE-cadherin-mediated cell adhesions lead to hyperpermeability, promoting the development and progression of various disease processes. Notably, the lungs are a highly vulnerable organ wherein pulmonary inflammation and infection result in vascular leakage. Herein, we showed that Rap1, a small GTPase, plays an essential role for maintaining pulmonary endothelial barrier function in mice. Endothelial cell-specific Rap1a/Rap1b double knockout mice exhibited severe pulmonary edema. They also showed vascular leakage in the hearts, but not in the brains. En face analyses of the pulmonary arteries and 3D-immunofluorescence analyses of the lungs revealed that Rap1 potentiates VE-cadherin-mediated endothelial cell-cell junctions through dynamic actin cytoskeleton reorganization. Rap1 inhibits formation of cytoplasmic actin bundles perpendicularly binding VE-cadherin adhesions through inhibition of a Rho-ROCK pathway-induced activation of cytoplasmic nonmuscle myosin II (NM-II). Simultaneously, Rap1 induces junctional NM-II activation to create circumferential actin bundles, which anchor and stabilize VE-cadherin at cell-cell junctions. We also showed that the mice carrying only one allele of either Rap1a or Rap1b out of the two Rap1 genes are more vulnerable to lipopolysaccharide (LPS)-induced pulmonary vascular leakage than wild-type mice, while activation of Rap1 by administration of 007, an activator for Epac, attenuates LPS-induced increase in pulmonary endothelial permeability in wild-type mice. Thus, we demonstrate that Rap1 plays an essential role for maintaining pulmonary endothelial barrier functions under physiological conditions and provides protection against inflammation-induced pulmonary vascular leakage.

[SLC12A8 promotes proliferation, invasiveness, migration and epithelial-mesenchymal transition of bladder cancer cells by activating JAK/STAT singaling].

OBJECTIVE: To investigate the role of solute carrier family 12 member A8 (SLC12A8) in regulation of biological behaviors of bladder cancer and the mechanism mediating its effect. METHODS: The TCGA database was used to analyze SLC12A8 expression in bladder cancer and is correlation with prognosis and clinicopathological characteristics of the patients. In different bladder cancer cell lines, the effects of transient transfection with SLC12A8 siRNA on cell proliferation, invasion and migration ability were examined using CCK-8 assay, Transwell assay and scratch experiment. Gene set enrichment analysis (GSEA) was carried out to analyze pathway enrichment. The correlation of SLC12A8 with the expressions of epithelial-mesenchymal transition (EMT) markers was analyzed using Western blotting. The effect of colivelin on biological behaviors of the cells with SLC12A8 knockdown was assessed using CCK-8 and Transwell assays. RESULTS: SLC12A8 was highly expressed in bladder cancer (P<0.05) and associated with a poor prognosis and advanced pathological stages of the patients (P<0.05), and could serve as an independent prognostic factor. The bladder cancer cell lines with SLC12A8 knockdown showed significantly attenuated proliferation, invasion and migration capacities (P<0.05). GSEA identified significant gene enrichment in the JAK/STAT signaling pathway (P=0.008). Correlation analysis showed that SLC12A8 expression was negatively correlated with E- cadherin expression (r=-0.167, P<0.001) but positively with N-cadherin (r=0.306, P<0.001) and vimentin (r=0.358, P<0.001) expressions. The bladder cancer cells with SLC12A8 knockdown showed significantly decreased expressions of p-Jak2, p-Stat3, N-cadherin and vimentin proteins with an increased expression of E-cadherin. Treatment with colivelin effectively enhanced proliferation, invasion and migration capacities of the bladder cancer cells with SLC12A8 knockdown (P<0.05). CONCLUSION: SLC12A8 promotes bladder cancer progression by activating the JAK/STAT signaling pathway and its high expression is closely associated with a poor prognosis of the patients.

MiR-484 promotes the malignant progression of glioma by inhibiting CDKN2A expression.

INTRODUCTION: Cyclin Dependent Kinase Inhibitor 2A (CDKN2A) is involved in glioma progression, but the specific molecular mechanism of CDKN2A in glioma cell migration and invasion needs to be further explored. MATERIAL AND METHODS: Data related to CDKN2A expression and glioma overall survival were obtained from The Cancer Genome Atlas (TCGA) database. Then, CDKN2A expression in glioma tissues/cells or paracancer tissues/astrocytes was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot. Afterwards, Wound healing, Transwell and tube formation assay were performed to identify the invasion, migration and angiogenesis of glioma cells, respectively. TargetScan database predicted the targeted binding between miR-484 and CDKN2A, which was verified by dual luciferase reporter gene assay. Western blot and qRT-PCR were performed to detect the expression of VEGF, E-cadherin, N-cadherin and Vimentin in glioma cells. RESULTS: CDKN2A was low-expressed in glioma tissue/cells as compared to paracancer tissue/astrocytes, and was strongly associated to the poor prognosis of glioma. Further studies found that down-regulation of CDKN2A could promote migration, invasion and angiogenesis of glioma cells. Besides, miR-484 was high-expressed in glioma cells compared to astrocytes. Up-regulation of miR-484 could enhance migration, invasion and angiogenesis of glioma cells. In addition, up-regulated miR-484 suppressed the expression of E-cadherin, and promoted the expression of N-cadherin, Vimentin and VEGF. However, there was negative regulation of miR-484 and CDKN2A, and CDKN2A could partially offset the effect of miR-484. CONCLUSIONS: MiR-484 promoted cell migration, invasion and angiogenesis by inhibiting CDKN2A expression.

Ephedrine alleviates bleomycin-induced pulmonary fibrosis by inhibiting epithelial-mesenchymal transition and restraining NF-κB signaling.

Pulmonary fibrosis is a lethal and progressive pulmonary disorder in human beings. Ephedrine is a compound isolated from Ephedra and plays a regulatory role in inflammatory response. This study focused on the anti-pulmonary fibrosis effect of ephedrine and its potential molecular mechanism. After a mouse model of pulmonary fibrosis was established through bleomycin (BLM) induction, the survival percentage, body weight, and pulmonary index were measured. Hematoxylin-eosin staining and Masson's trichrome staining for lung tissues were performed to observe the pathological alterations. The viability of lung epithelial BEAS-2B cells, intracellular production of reactive oxygen species, and the levels of pro-inflammatory cytokines were examined by cell counting kit-8 assays, 2',7'-dichlorofluorescein diacetate (DCF-DA) staining, and enzyme-linked immunosorbent assay, respectively. Immunofluorescence staining was performed to determine E-cadherin and vimentin expression after BLM or ephedrine treatment. The mRNA and protein levels of cytokeratin-8, E-cadherin, α-SMA, and vimentin were subjected to quantitative polymerase chain reaction and immunoblotting. Experimental results revealed that ephedrine treatment rescued the repressive impact of BLM on BEAS-2B cell viability, and ephedrine inhibited BLM-induced overproduction of reactive oxygen species and inflammatory response in BEAS-2B cells. Additionally, ephedrine suppressed epithelial-mesenchymal transition (EMT) process stimulated by BLM treatment, as demonstrated by the reduced α-SMA and vimentin levels together with the increased cytokeratin-8 and E-cadherin levels in BLM + Ephedrine group. In addition, ephedrine inhibited NF-κB and activated Nrf-2 signaling in BLM-treated BEAS-2B cells. Moreover, ephedrine ameliorated pulmonary fibrosis in BLM-induced mice and improved the survival of model mice. In conclusion, ephedrine attenuates BLM-evoked pulmonary fibrosis by repressing EMT process via blocking NF-κB signaling and activating Nrf-2 signaling, suggesting that ephedrine might become a potential anti-pulmonary fibrosis agent in the future.

EGF-Enhanced GnRH-II Regulation in Decidual Stromal Cell Motility through Twist and N-Cadherin Signaling.

Crucial roles in embryo implantation and placentation in humans include the invasion of the maternal decidua by extravillous trophoblasts and the motile behavior of decidual endometrial stromal cells. The effects of the epidermal growth factor (EGF) and GnRH-II in the endometrium take part in early pregnancy. In the present study, we demonstrated the coaction of EGF- and GnRH-II-promoted motility of human decidual endometrial stromal cells, indicating the possible roles of EGF and GnRH-II in embryo implantation and early pregnancy. After obtaining informed consent, we obtained human decidual endometrial stromal cells from decidual tissues from normal pregnancies at 6 to 12 weeks of gestation in healthy women undergoing suction dilation and curettage. Cell motility was evaluated with invasion and migration assays. The mechanisms of EGF and GnRH-II were performed using real-time PCR and immunoblot analysis. The results showed that human decidual tissue and stromal cells expressed the EGF and GnRH-I receptors. GnRH-II-mediated cell motility was enhanced by EGF and was suppressed by the knockdown of the endogenous GnRH-I receptor and EGF receptor with siRNA, revealing that GnRH-II promoted the cell motility of human decidual endometrial stromal cells through the GnRH-I receptor and the activation of Twist and N-cadherin signaling. This new concept regarding the coaction of EGF- and GnRH-promoted cell motility suggests that EGF and GnRH-II potentially affect embryo implantation and the decidual programming of human pregnancy.

Exosomal miR-205-5p Improves Endometrial Receptivity by Upregulating E-Cadherin Expression through ZEB1 Inhibition.

Endometrial receptivity is a complex process that prepares the uterine endometrium for embryo implantation; insufficient endometrial receptivity is one of the causes of implantation failure. Here, we analyzed the microRNA expression profiles of exosomes derived from both receptive (RL95-2) and non-receptive (AN3-CA) endometrial epithelial cell (EEC) lines to identify exosomal miRNAs closely linked to endometrial receptivity. Among the 466 differentially expressed miRNAs, miR-205-5p was the most highly expressed in exosomes secreted from receptive RL95-2 cells. miR-205-5p, enriched at the adhesive junction, was closely related to endometrial receptivity. ZEB1, a transcriptional repressor of E-cadherin associated with endometrial receptivity, was identified as a direct target of miR-205-5p. miR-205-5p expression was significantly lower in the endometrial tissues of infertile women than in that of non-infertile women. In vivo, miR-205-5p expression was upregulated in the post-ovulatory phase, and its inhibitor reduced embryo implantation. Furthermore, administration of genetically modified exosomes overexpressing miR-205-5p mimics upregulated E-cadherin expression by targeting ZEB1 and improved spheroid attachment of non-receptive AN3-CA cells. These results suggest that the miR-205-5p/ZEB1/E-cadherin axis plays an important role in regulating endometrial receptivity. Thus, the use of exosomes harboring miR-205-5p mimics can be considered a potential therapeutic approach for improving embryo implantation.