Cell Scratch Research Articles

Pirfenidone Attenuates the EMT Process and the Secretion of VEGF in TGF-β2-Induced ARPE-19 Cells via Inhibiting the Activation of the NF-κB/Snail Signaling Pathway.

Pirfenidone (PFD), an antifibrotic drug, has various beneficial functions such as antioxidant, antifibrotic, and anti-inflammatory effects. This study aimed to explore the molecular mechanisms underlying how PFD modulates retinal pigment epithelial (RPE) cells involved in neovascularization and subretinal fibrosis. ARPE-19 cell lines were treated with transforming growth factor-beta 2 (TGF-β2) alone or in combination with PFD. RPE cell viability, as a consequence of PFD use, was determined by the CCK-8 assay. Cell migration was assessed by the wound closure assay and quantified by the Image J software. Protein expression of the following markers was measured by the western blot analysis: an epithelial cell marker and E-cadherin; mesenchymal cell markers, fibronectin, matrix metalloprotein-9 (MMP-9), and alpha-smooth muscle actin (α-SMA); a fibrotic marker and connective tissue growth factor (CTGF); an angiogenesis marker and vascular endothelial growth factor (VEGF); NF-κB/Snail. The mRNA levels of fibronectin and α-SMA were determined by quantitative real-time PCR. VEGF was quantitatively measured by the enzyme-linked immunosorbent assay. The cell viability assay revealed that PFD had no significant cytotoxic effect on RPE cells at concentrations of less than 1 mg/mL. The cell scratch assay showed that TGF-β2 stimulation significantly improved the migration of RPE cells and that PFD attenuated this effect. PFD significantly inhibited the TGF-β2-induced protein expression of E-cadherin and increased the TGF-β2-induced protein expression of fibronectin, MMP-9, α-SMA, CTGF, and VEGF in ARPE-19 cells. The mRNA expression of fibronectin and α-SMA was inhibited by PFD in TGF-β2-inducedARPE-19 cells. Additionally, the increased intracellular and supernatant expression of VEGF protein was suppressed by PFD. Mechanistically, RPE cells treated with PFD + TGF-β2 exhibited a decrease in phosphorylation of the NF-κB P65 subunit and activation of Snail, compared with the RPE cells treated with TGF-β2 alone. PFD ameliorated TGF-β2-induced neovascularization and fibrosis by suppressing the NF-κB/Snail signaling pathway. Therefore, PFD may be a potential drug in the treatment of age-related macular degeneration.

The Expression of LINC00673 in Colon Cancer and the Melatonin's Effect on Its Expression

Objective: In this study, the cell lines SW620 and DLD-1 which belongs to colon cancer were employed with the aim of observing melatonin's effect on the growth of colon cancer cells and exploring the influence of melatonin about the autophagy of colon cancer. Besides. It uses qRT-PCR to identify LINCRNA00673 (LINC00673) expression in colon cancer and the influence of melatonin on LINC00673 expression.For the purpose of providing a fundamental theory for melatonin in anti-cancer therapy and also presenting as a biomarker for the early detection of colon cancer. Method: 1) Cultivating the colon cancer cell lines SW620 and DLD-1, and subdividing them in accordance with distinct concentrations of melatonin: 0 mM, 1mM, and 2mM, to detect the impact of melatonin for migration and growth of colon cancer cells by Cell Scratch Test and Colony Formation Assay.2) Detecting the expression level of autophagy-related molecules in colon cancer cells in response to melatonin employing Western Blot Assay. 3) Utilizing QT-PCR, extract RNA from two groups of cells and detect the expression of LINC00673 and LINC00673 following melatonin treatment. Result: 1) The Cell Scratch test and Colony Formation Assay indicated that compared with the normal group, the scratch healing of SW620 cells and DLD-1 cells decreased. Furthermore, following melatonin administration, the number of cell colonies decreased. And the results are statistically significant. 2) Western blotting results illustrated that after the interferon of melatonin, the expression level of the autophagy-related molecule Beclin-1 increased significantly, whereas the expression of P62 decreased. 3) QT-PCR results demonstrated that the expression of LINC00673 in colon cancer cells SW620 and DLD-1 was significantly increased. Moreover, melatonin had an inhibiting effect on it. Conclusion: These in vitro experiments managed to prove that melatonin possesses a powerful anti-colon cancer effect and can inhibit the growth and spread of colon cancer. Western blotting indicated that melatonin can inhibit colon cancer through autophagy. Besides which, it is discovered that melatonin may exert its anticancer effect by influencing LINC00673.

Lipoic Acid Conjugated Boron Hybrids Enhance Wound Healing and Antimicrobial Processes.

Complications of chronic non-healing wounds led to the emergence of nanotechnology-based therapies to enhance healing, facilitate tissue repair, and prevent wound-related complications like infections. Here, we design alpha lipoic acid (ALA) conjugated hexagonal boron nitride (hBN) and boron carbide (B4C) nanoparticles (NPs) to enhance wound healing in human dermal fibroblast (HDFa) cell culture and characterize its antimicrobial properties against Staphylococcus aureus (S. aureus, gram positive) and Escherichia coli (E. coli, gram negative) bacterial strains. ALA molecules are integrated onto hBN and C4B NPs through esterification procedure, and molecular characterizations are performed by using transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and UV-vis spectroscopy. Wound healing and antimicrobial properties are investigated via the use of cell viability assays, scratch test, oxidative stress, and antimicrobial activity assays. Based on our analysis, we observe that ALA-conjugated hBN NPs have the highest wound-healing feature and antimicrobial activity compared to ALA-B4C. On the other hand, hBN, ALA-B4C, and ALA compounds showed promising regenerative and antimicrobial properties. Also, we find that ALA conjugation enhances wound healing and antimicrobial potency of hBN and B4C NPs. We conclude that the ALA-hBN conjugate is a potential candidate to stimulate regeneration process for injuries.

YAP regulates intestinal epithelial cell proliferation through activation of STAT3 in DSS-induced colitis and associated cancer.

Ulcerative colitis (UC) is a chronic, relapsing inflammation of the colon. Impaired epithelial repair is an important biological features of UC. Accelerating intestinal epithelial repair to achieve endoscopic mucosal healing has become a key goal in UC. Yes-associated protein (YAP) is a key transcriptional coactivator that regulates organ size, tissue growth and tumorigenesis. Growing studies have focused on the role of YAP in intestinal epithelial regeneration. This study explore the molecular mechanism for the role YAP in modulating colonic epithelial proliferation, repair, and the development of colitis associated cancer. We constructed the acute colitis mouse model through successive 5 days of 3% dextran sulfate sodium salt (DSS) induction. Then YAP-overexpressed mouse model was constructed by intraperitoneal injection the YAP overexpressed and negative control lentivirus into DSS mice. On the 5th day of DSS induction and the 5th day of normal drinking water after removing DSS (5+5 d), the mice were killed by spinal dislocation. The colon was taken to measure the length, and the bowel 1-2 cm near the anal canal was selected for immunohistochemical and Western blotting. We used YAP over-expressed colonic epithelial cells and small interfering signal transducer and activator of transcription 3 (STAT3) RNA to probe the regulation of YAP on STAT3, using cell counting kit-8 and scratch assays to explore the role of YAP on colonic epithelial cell proliferation. Finally, we conducted co-immunoprecipitation to test the relationship between YAP and STAT3. After DSS treatment, the expression of YAP was dramatically diminished in crypts. Compared with the empty control mice, overexpression of YAP drastically accelerated epithelial regeneration after DSS induced colitis, presenting with more intact of structural integrity in intestinal epithelium and a reduction in the number of inflammatory cells in the mucosa. Further Western blotting, functional experiment and co-immunoprecipitation analyses showed that the expression of YAP in nucleus was significantly increased by 2 h post DSS cessation, accompanied with up-regulated total protein levels of STAT3 and phosphorylated-STAT3 (p-STAT3). Overexpression of YAP enhanced the expression of STAT3, p-STAT3, and their transcriptional targets including c-Myc and Cyclin D1. In addition, it promoted the proliferation and the "wound healing" of colonic cells. However, these effects were reversed when silencing STAT3 on YAP-overexpressed FHC cells. Moreover, protein immunoprecipitation indicated that YAP could directly interact with STAT3 in the nucleus, up-regulatvng the expressvon of STAT3. Finally, during the process of CAC, overexpression of YAP mutant caused the down-regulated expression of STAT3 and inhibited the development and progress of CAC. YAP activates STAT3 signaling in regulation of epithelial cell proliferation and promotes mucosal regeneration after DSS induced colitis, which may serve as a potential therapeutic target in UC. However, persistent and excessive YAP activation may promote CAC development.

Corylin inhibits the progression of Non-small cell lung cancer cells by regulating NF-κB signaling pathway via targeting p65

Lung cancer is characterized by high-risk and high mortality, among which non-small cell lung cancer (NSCLC) conquers a dominant position. Previous studies have reported that corylin has anti-inflammatory, anti-oxidant, and anti-tumor effects; however, its role in NSCLC cells remains unclear. Corylin inhibits the progression of NSCLC cells. A lentivector NF-κB luciferase reporter was constructed by molecular cloning. Corylin was screened and identified as an NF-κB pathway inhibitor by luciferase reporter assay. Corylin inhibited the expression of NF-κB downstream genes, which was detected by qRT-PCR. The effect of corylin on NSCLC cells was detected by colony formation assay, cell apoptosis, cell proliferation, in vitro invasion, and cell scratch assay. Corylin inhibited p65 nuclear translocation and was detected by molecular docking, immunofluorescence assay, and Western blot analysis. We constructed a lentiviral expression vector, containing an NF-κB luciferase reporter and established a stable A549 cell line for its expression. Using this cell line, corylin was screened and identified as an NF-κB pathway inhibitor. It was found that corylin inhibited the expression of NF-κB downstream genes and inhibited the proliferation and migration of NSCLC cells. Meanwhile, it was also found that corylin significantly reversed the increased proliferation of NSCLC cell lines induced by p65 overexpression. Molecular docking analysis showed that corylin could bind to p65 by hydrogen bonding. Further study showed that corylin inhibited the NF-κB signaling pathway by blocking p65 nuclear translocation. Our study screened and identified corylin as an NF-κB inhibitor and elucidated the molecular mechanism by which corylin inhibits the growth of NSCLC cells. The present study provides a novel strategy for improving the prognosis and treatment of NSCLC patients.

Comprehensive analysis of a glycolysis and cholesterol synthesis-related genes signature for predicting prognosis and immune landscape in osteosarcoma.

Glycolysis and cholesterol synthesis are crucial in cancer metabolic reprogramming. The aim of this study was to identify a glycolysis and cholesterol synthesis-related genes (GCSRGs) signature for effective prognostic assessments of osteosarcoma patients. Gene expression data and clinical information were obtained from GSE21257 and TARGET-OS datasets. Consistent clustering method was used to identify the GCSRGs-related subtypes. Univariate Cox regression and LASSO Cox regression analyses were used to construct the GCSRGs signature. The ssGSEA method was used to analyze the differences in immune cells infiltration. The pRRophetic R package was utilized to assess the drug sensitivity of different groups. Western blotting, cell viability assay, scratch assay and Transwell assay were used to perform cytological validation. Through bioinformatics analysis, patients diagnosed with osteosarcoma were classified into one of 4 subtypes (quiescent, glycolysis, cholesterol, and mixed subtypes), which differed significantly in terms of prognosis and tumor microenvironment. Weighted gene co-expression network analysis revealed that the modules strongly correlated with glycolysis and cholesterol synthesis were the midnight blue and the yellow modules, respectively. Both univariate and LASSO Cox regression analyses were conducted on screened module genes to identify 5 GCSRGs (RPS28, MCAM, EN1, TRAM2, and VEGFA) constituting a prognostic signature for osteosarcoma patients. The signature was an effective prognostic predictor, independent of clinical characteristics, as verified further via Kaplan-Meier analysis, ROC curve analysis, univariate and multivariate Cox regression analysis. Additionally, GCSRGs signature had strong correlation with drug sensitivity, immune checkpoints and immune cells infiltration. In cytological experiments, we selected TRAM2 as a representative gene to validate the validity of GCSRGs signature, which found that TRAM2 promoted the progression of osteosarcoma cells. Finally, at the pan-cancer level, TRAM2 had been correlated with overall survival, progression free survival, disease specific survival, tumor mutational burden, microsatellite instability, immune checkpoints and immune cells infiltration. Therefore, we constructed a GCSRGs signature that efficiently predicted osteosarcoma patient prognosis and guided therapy.

Tumor-promoting roles of HMMR in lung adenocarcinoma

Searching for differential genes in lung adenocarcinoma (LUAD) is vital for research. Hyaluronan mediated motility receptor (HMMR) promotes malignant progression of cancer patients. However, the molecular regulators of HMMR-mediated LUAD onset are unknown. This work aimed to study the relevance of HMMR to proliferation, migration and invasion of LUAD cells. Let-7c-5p and HMMR levels in LUAD cells and HLF-a cells were assessed, and their correlation was also detected. Their interaction was determined by dual-luciferase experiments and qRT-PCR. Cell proliferation, migration and invasion potentials in vitro were validated through cell counting kit-8 (CCK-8), colony formation, scratch healing, and transwell assays. The expression of HMMR was examined by qRT-PCR and western blot and the expression of let-7c-5p was assayed by qRT-PCR. It was found that HMMR level was increased in LUAD and negatively correlated with let-7c-5p level. Let-7c-5p directly targeted HMMR to repress LUAD cell proliferation, migration and invasion. The above data illustrated that the let-7c-5p/HMMR axis may provide certain therapeutic value for LUAD patients.

Effect of CTP-mediated PTEN on 5637 bladder cancer cells and the underlying molecular mechanism.

The aim of the present study was to explore the effect of cytoplasmic transduction peptide (CTP)-phosphatase and tensin homolog (PTEN) on the proliferation, cell cycle, apoptosis, migration and invasion of bladder cancer cells and the underlying molecular mechanism. A eukaryotic expression vector, pTT5-CTP-PTEN, was constructed. The constructed vector was transfected into HEK 293-6E cells to express a fusion protein, CTP-PTEN. The fusion protein was purified. 5637 bladder cancer cells were cocultured with purified CTP-PTEN fusion protein. Target gene expression, protein expression, cell proliferation, cell cycle, apoptosis, cell invasion and cell migration were examined by reverse transcription polymerase chain reaction (RT-PCR), western blot, MTT assay, flow cytometry, Transwell assay, and cell scratch assay, respectively. Both PTEN and CTP-PTEN fusion protein inhibited the proliferation, cell cycle, invasion and migration of bladder cancer cells and promoted the apoptosis of bladder cancer cells. The effect of CTP-PTEN was more significant. The fused expression of CTP and PTEN significantly increased the penetrability of the tumor suppressor gene PTEN into cancer cells. The CTP-PTEN fusion protein exhibited a significant carcinostatic effect on 5637 bladder cancer cells.

Integrated miRNA and mRNA omics reveal dioscin suppresses migration and invasion via MEK/ERK and JNK signaling pathways in human endometrial carcinoma in vivo and in vitro

Ethnopharmacological relevancePolygonatum sibiricum Redouté (PS, also called Huangjing in traditional Chinese medicine), is a perennial herb as homology of medicine and food. According to the traditional Chinese medicine theory “Special Records of Famous Doctors”, its functions include invigorating qi and nourishing yin, tonifying spleen and kidney. Traditionally, qi and blood therapy has been believed as most applicable to the treatment of uterine disease. The current research has focused on the effect and mechanism of dioscin, the main active component of PS, on Endometrial carcinoma (EC). Aim of the studyTo study the efficacy of dioscin on proliferation and migration of Endometrial carcinoma cell line, we conducted experiments by using xenograft model and Ishikawa cells, and explored the potential molecular mechanism. Materials and methodsmRNA and miRNA omics techniques were employed to investigate the regulatory mechanism of dioscin on EC Ishikawa cells. Based on in vivo and in vitro experiments, cell clone formation, cell scratching, Transwell, H&E staining, immunohistochemistry, q-PCR, and Western blot techniques were used to determine the molecular effects and mechanisms of dioscin on cell migration. ResultsIntegrated miRNA and mRNA omics data showed that 513 significantly different genes marked enrichment in MAPK signaling pathway. The in vivo data showed that dioscin (24 mg/kg) significantly inhibited tumor growth. The in vitro proliferation and invasiveness of dioscin on Ishikawa cells showed that dioscin could significantly decrease the colony numbers, and suppress the Ishikawa cell wound healing, migration and invasion. Molecular data revealed that dioscin decreased the MMP2 and MMP9 expression in vitro and in vivo. The p-MEK, p-ERK, and p-JNK expression levels were also confirmed to be significantly reduced. Key regulators in the MAPK signaling pathway were further validated in xenograft tumors. ConclusionOur data indicated that dioscin inhibited Ishikawa cell migration and invasion mediated through MEK/ERK and JNK signaling. More importantly, screened hub miRNAs and genes can be regarded as potential molecular targets for future EC treatment.

Heteronuclear Ru(II)-Re(I) complexes as potential photodynamic anticancer agents with anti-metastatic and anti-angiogenic activities

Herein, three heterometallic Ru(II)-Re(I) complexes, [Ru(NN)2(tpphz)Re(CO)3Cl](PF6)2 (N-N = 2,2′-bipyridine (bpy, in RuRe1), 1,10-phenanthroline (phen, in RuRe2), 4,7-diphenyl-1,10-phenanthroline (DIP, in RuRe3), tpphz = tetrapyrido[3,2-a:2′,3′-c:3″,2″-h:2″‘,3″‘-j]phenazine), using tpphz as a bridging ligand to connect Ru(II) polypyridyl moiety and Re(I) tricarbonyl moiety were designed and synthesized. Cytotoxicity tests revealed that RuRe1–3 exhibited high phototoxicities against several cancer cell lines tested, with IC50 values ranging from 0.8 to 6.8 μM. Notably, RuRe2 exhibited the most significant increase in cytotoxicity against human prostate cancer (PC3) cells under light (450 nm) irradiation, with phototoxicity index (PI) value increasing by >112.3-fold. Further mechanistic studies of RuRe2 revealed that RuRe2-mediated PDT could induce tumor cell apoptosis through the mitochondrial pathway. Moreover, RuRe2-mediated PDT could inhibit PC3 cell scratch healing and reduce the expression levels of matrix metalloproteinases 2 (MMP-2), matrix metalloproteinases 9 (MMP-9) and vascular endothelial growth factor receptor VEGFR2. Finally, angiogenic activity assays performed in human umbilical vein endothelial cells (HUVECs) showed that RuRe2 exerted an anti-angiogenesis effect. Our study demonstrated that RuRe1–3 were promising PDT antitumor agents with potential anti-metastatic and anti-angiogenic activities.

Inhibition of Wnt7b reduces the proliferation, invasion, and migration of colorectal cancer cells.

Colorectal cancer is one of the most common gastrointestinal tumors. The role of Wnt7b as a ligand of the Wnt signaling pathway in colorectal cancer remains to be studied. Through bioinformatics online analysis, we found that Wnt7b is abnormally highly expressed in a variety of gastrointestinal tumors. This study mainly explored the effects of Wnt7b regulating the Wnt/β-catenin signaling pathway on the proliferation, migration, and invasion of SW480 cells in colorectal cancer. Applying the TCGA data set, Wnt7b was found to be highly expressed in most gastrointestinal tumor samples. Real-time quantitative PCR(q-PCR), Western blotting(WB) results showed that Wnt7b was significantly higher expressed in colorectal cancer cell lines compared with normal intestinal epithelial cells. SW480 cells transfected with the sh-Wnt7b showed successful knockdown of Wnt7b. MTT colorimetry showed the proliferation ability of sh-Wnt7b group decreased significantly compared with the non-transfected group. The results of double staining flow cytometry showed that the sh-Wnt7b group had more apoptosis. Cell scratch test showed that the cell migration rate of sh-wnt7b group considerably reduced. The Transwell invasion experiment demonstrated that the number of cell invasions in the sh-Wnt7b group decreased significantly. After SW480cells was transfected with sh-Wnt7b, the protein levels of β-catenin, CCND1, and CD44 in this group of cells were detected to be reduced by WB, and the same results were obtained by q-PCR detection of mRNA. Wnt7b is highly expressed in colorectal cancer cells, which may affect the proliferation, migration, and invasion of colorectal cancer cells by activating the Wnt/β-catenin signaling pathway.

Paclitaxel-resistant related lncRNA DBH-AS1 promotes the proliferation and invasion of esophageal cancer.

Chemoresistance is one of the main obstacles in the clinical treatment of cancer. However, secondary resistance to paclitaxel poses new challenges for cancer treatment. Long noncoding RNAs regulate cellular functions at different levels and mechanisms and play an important role in the biological behavior of tumors. LncRNA microarrays were used to detect lncRNAs in Paclitaxel-resistant cells and corresponding parental cells. Cell counting kit 8 and Transwell analysis were used to test the effect of lncRNA on function. The expression of lncRNA DBH-AS1 in TE-4 TAX-R cells was significantly higher than that in TE-4 cells. Transwell analysis showed that the overexpression of lncRNA DBH-AS1 increased the invasion of Eca cells. Cell scratches and Transwell analysis showed that the overexpression of lncRNA DBH-AS1 in Eca cell culture supernatants promoted the migration and invasion of HUVEC. In addition, lncRNA DBH-AS1 relies on miR-21 to regulate the expression of YOD1. Paclitaxel-resistant lncRNA DBH-AS1 appears to promote ECa cell proliferation and invasion by acting as a ceRNA and regulating miR-21-5p /YOD1 signaling pathway.

The Abnormal Proliferation of Hepatocytes is Associated with MC-LR and C-Terminal Truncated HBX Synergistic Disturbance of the Redox Balance.

Microcystin-LR (MC-LR) and hepatitis B virus (HBV) are associated with hepatocellular carcinoma (HCC). However, the concentrations of MC-LR in drinking water and the synergistic effect of MC-LR and HBV on hepatocellular carcinogenesis through their disturbance of redox balance have not been fully elucidated. We measured the MC-LR concentrations in 168 drinking water samples of areas with a high incidence of HCC. The relationships between MC-LR and both redox status and liver diseases in 177 local residents were analyzed. The hepatoma cell line HepG2 transfected with C-terminal truncated hepatitis B virus X gene (Ct-HBX) were treated with MC-LR. Reactive oxygen species (ROS), superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA) were measured. Cell proliferation, migration, invasion, and apoptosis were assessed with cell activity assays, scratch and transwell assays, and flow cytometry, respectively. The mRNA and protein expression-related redox status genes were analyzed with qPCR and Western blotting. The average concentration of MC-LR in well water, river water and reservoir water were 57.55 ng/L, 76.74 ng/L and 132.86 ng/L respectively, and the differences were statistically significant (P < 0.05). The MC-LR levels in drinking water were correlated with liver health status, including hepatitis, clonorchiasis, glutamic pyruvic transaminase abnormalities and hepatitis B surface antigen carriage (all P values < 0.05). The serum MDA increased in subjects who drank reservoir water and were infected with HBV (P < 0.05). In the cell experiment, ROS increased when Ct-HBX-transfected HepG2 cells were treated with MC-LR, followed by a decrease in SOD and GSH and an increase in MDA. MC-LR combined with Ct-HBX promoted the proliferation, migration and invasion of HepG2 cells, upregulated the mRNA and protein expression of MAOA gene, and downregulated UCP2 and GPX1 genes. MC-LR and HBV may synergistically affect redox status and play an important role in hepatocarcinoma genesis.

LncRNA surfactant associated 1 activates large tumor suppressor kinase 1/Yes-associated protein pathway via modulating hypoxic exosome-delivered miR-4766-5p to inhibit lung adenocarcinoma metastasis.

LncRNA surfactant associated 1 (SFTA1P) exhibits low expression in non-small cell lung cancer (NSCLC) tissues as compared with that in adjacent tissues, and may play a suppressing role in NSCLC. However, the effect and mechanism of SFTA1P on the metastasis of lung adenocarcinoma (LUAD) remain undefined, which are thus investigated in this research. Herein, potential impacts of SFTA1P on LUAD were determined through the Cancer Genome Atlas (TCGA) database and Gene Expression Profiling Interactive Analysis (GEPIA). After knockdown/overexpression of SFTA1P, the metastatic ability of LUAD cells was evaluated by molecular biology experiments (cell counting kit-8 assay, scratch test, Transwell assay and Western blot). The effect of SFTA1P on Yes-associated protein (YAP) nuclear translocation was assessed by Western blot. Hypoxia-induced exosomes were extracted for LUAD metastasis analysis. The targeting relationship of SFTA1P/miR-4766-5p/large tumor suppressor kinase 1 (LATS1) was verified by dual-luciferase reporter assay and molecular biology experiments. Xenograft and lung metastasis models were constructed for in vivo validation. SFTA1P was lowly expressed in LUAD, which was associated with the poor prognosis of patients with LUAD. Up-regulated SFTA1P prevented the metastasis of LUAD cells and the nuclear translocation of YAP. Hypoxia-induced exosomes stimulated LUAD cell metastasis, but inhibited the SFTA1P and LATS1/YAP axes. MiR-4766-5p acted as an intermediate "bridge" for SFTA1P to regulate LATS1. SFTA1P repressed xenograft growth and LUAD cell metastasis. To sum up, SFTA1P activates hypoxic exosome-delivered miR-4766-5p through modulating LATS1/YAP pathway, thereby suppressing LUAD cell metastasis, which may serve as a suitable target for the LUAD therapy.

Anti-tumor effect of Huaier extract supernatant on human gastric cancer HGC-27 and MGC-803 cells

The purpose of this study was to investigate the effect of Huaier extract supernatant(HES) on the proliferation, apoptosis, autophagy, and migration of human gastric cancer HGC-27 and MGC-803 cells and its molecular mechanisms. The main components in HES were preliminarily analyzed by high-performance liquid chromatography-mass spectrometry(HPLC-MS). Methyl thiazolyl tetrazolium(MTT) assay, colony formation assay, and 5-ethynyl-2'-deoxyuridine(EdU) staining assay were used to explore the effect of HES on the proliferation of human gastric cancer HGC-27 and MGC-803 cells. Hoechst staining and flow cytometry assay were used to determine the effect of HES on apoptosis of human gastric cancer HGC-27 and MGC-803 cells. Acridine orange staining and cell scratch assay were used to determine the effect of HES on autophagy and migration of human gastric cancer HGC-27 and MGC-803 cells, respectively. Western blot was used to investigate the regulatory effect of HES on the expression levels of proteins related to apoptosis, epithelial-mesenchymal transition(EMT), and signaling pathways in human gastric cancer HGC-27 and MGC-803 cells. The results showed that HES mainly contained some components with high polarities. HES significantly reduced the cell viability of human gastric cancer cells in a dose-and time-dependent manner. The IC_(50 )values after 48 h of HES treatment in human gastric cancer HGC-27 and MGC-803 cells were 7.56 and 10.77 g·L~(-1), respectively. Meanwhile, HES inhibited the colony-forming ability and short-term proliferation of human gastric cancer cells. The apoptosis rates of HGC-27 and MGC-803 cells treated with 8 g·L~(-1) HES for 72 h were 62.13%±8.92% and 54.50%±3.26%, respectively. HES also promoted autophagy in human gastric cancer cells and impaired their migration ability in vitro. Moreover, HES up-regulated the cleavage of the apoptosis marker poly ADP-ribose polymerase(PARP) and the protein expression level of the epithelial cell marker E-cadherin, and down-regulated the protein levels of phosphorylated-mammalian target of rapamycin(p-mTOR), phosphorylated-S6(p-S6), and phosphorylated-extracellular signal-regulated kinase(p-ERK) in human gastric cancer cells. Therefore, HES is one of the effective anti-tumor components of Huaier, which inhibits the proliferation and migration of human gastric cancer cells, and induces apoptosis and autophagy. Moreover, the mTOR signal and ERK signal may be involved in the anti-gastric cancer effect of HES. This study provides novel references for the in-depth research and clinical application of Huaier. It is also of great significance to promote the scientific development and utilization of Huaier.

UCHL1 promotes cancer stemness in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a special kind of breast cancer with strong ability of invasion and metastasis. UCHL1 belongs to the ubiquitin carboxy-terminal hydrolase family and is found to be increased in a variety of malignancies, but its expression in TNBC is unknown. First, we analyzed the expression of UCHL1 in 128 TNBC specimens and paired adjacent normal tissues from 17 TNBC patients undergoing curative resection by immunohistochemistry. Then, the relationship between UCHL1 and cancer stemness was investigated by cell flow cytometry, spheroid formation assays and western blot. Moreover, cell scratch assay and Transwell assays were performed to explore whether UCHL1 promotes the migration and invasion of TNBC cells. Finally, we constructed a xenografts model of TNBC cell lines to observe the effect of UCHL1 on tumorigenesis in vivo. UCHL1 was overexpressed in TNBC tissues and associated with poor prognosis. UCHL1 promoted stem cancer cells properties, including the percentage of CD44+/CD24- cells, sphere-forming ability and CSCs related markers. Furthermore, Scratch assay and Transwell assay proved that UCHL1 enhanced the migration and invasion of TNBC cells. The experimental results of xenografts model in nude mice showed that UCHL1 promoted tumorigenesis of TNBC in vivo. UCHL1 may play a role in the malignant progression of TNBC by maintaining the stemness and promoting cell invasion and is expected to become a potential therapeutic target for TNBC patients.

Liraglutide Improves the Angiogenic Capability of EPC and Promotes Ischemic Angiogenesis in Mice under Diabetic Conditions through an Nrf2-Dependent Mechanism.

The impairment in endothelial progenitor cell (EPC) functions results in dysregulation of vascular homeostasis and dysfunction of the endothelium under diabetic conditions. Improving EPC function has been considered as a promising strategy for ameliorating diabetic vascular complications. Liraglutide has been widely used as a therapeutic agent for diabetes. However, the effects and mechanisms of liraglutide on EPC dysfunction remain unclear. The capability of liraglutide in promoting blood perfusion and angiogenesis under diabetic conditions was evaluated in the hind limb ischemia model of diabetic mice. The effect of liraglutide on the angiogenic function of EPC was evaluated by cell scratch recovery assay, tube formation assay, and nitric oxide production. RNA sequencing was performed to assess the underlying mechanisms. Liraglutide enhanced blood perfusion and angiogenesis in the ischemic hindlimb of db/db mice and streptozotocin-induced type 1 diabetic mice. Additionally, liraglutide improved tube formation, cell migration, and nitric oxide production of high glucose (HG)-treated EPC. Assessment of liraglutide target pathways revealed a network of genes involved in antioxidant activity. Further mechanism study showed that liraglutide decreased the production of reactive oxygen species and increased the activity of nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 deficiency attenuated the beneficial effects of liraglutide on improving EPC function and promoting ischemic angiogenesis under diabetic conditions. Moreover, liraglutide activates Nrf2 through an AKT/GSK3β/Fyn pathway, and inhibiting this pathway abolished liraglutide-induced Nrf2 activation and EPC function improvement. Overall, these results suggest that Liraglutide represents therapeutic potential in promoting EPC function and ameliorating ischemic angiogenesis under diabetic conditions, and these beneficial effects relied on Nrf2 activation.

Discovery of Mcl-1 inhibitors through virtual screening, molecular dynamics simulations and in vitro experiments

As a member of the B-cell lymphoma 2 (Bcl-2) protein family, the myeloid leukemia cell differentiation protein (Mcl-1) can inhibit apoptosis and plays an active role in the process of tumor escape from apoptosis. Therefore, inhibition of Mcl-1 protein can effectively promote the apoptosis of tumor cells and may also reduce tumor cell resistance to drugs targeting other anti-apoptotic proteins. This research is dedicated to the development of Mcl-1 inhibitors, aiming to provide more references for lead compounds with different scaffolds for the development of targeted anticancer drugs. We obtained a series of small molecules with a common core skeleton through molecular docking from Specs database and searched the core structure in ZINC database for more similar small molecules. Collecting these small molecules for preliminary experimental screening, we found a batch of active compounds, and selected two small molecules with the strongest inhibitory activity on B16F10 cells: compound 7 and compound 1. Their IC50s are 7.86 ± 1.25 and 24.72 ± 1.94 μM, respectively. These two compounds were also put into cell scratch test for B16F10 cells and cell viability assay of other cell lines. Furthermore, through molecular dynamics (MD) simulation analysis, we found that compound 7 formed strong binding with the key P2, P3 pocket and ARG 263 of Mcl-1. Finally, ADME results showed that compound 7 performs well in terms of drug similarity. In conclusion, this study provides hits with co-scaffolds that may aid in the design of effective clinical drugs targeting Mcl-1 and the future drug development.

Discovery of pyrazole-carbohydrazide with indole moiety as tubulin polymerization inhibitors and anti-tumor candidates.

In this work, a series of indole-containing pyrazole-carbohydrazide derivatives A1-A25 were synthesized, and their biological activity on tubulin polymerization inhibition and mitotic catastrophe was evaluated. For introducing indole group to CA-4 pattern, the carbohydrazide linker was used for the first time. As the top hit, A18 suggested notable antiproliferation efficacy and tubulin polymerization inhibitory activity. Inferring comparable antitubulin effect with the positive control Colchicine, A18 indicated obviously lower cyto-toxicity. The cell scratch test showed that A18 could block the cell migration, while the confocal imaging depicted that A18 could induce the mitotic catastrophe via a Colchicine-like approach. The docking simulation visualized the probable binding pattern of A18. With the information in this work, some new hints on modification might be involved in further tubulin-related investigations.

CBL inhibits proliferation and invasion of breast cancer cells by ubiquitylation-mediated degradation of NCK2

To observe the effects of Casitas B lymphoma (CBL) protein on proliferation, migration and invasion of breast cancer cells and explore its mechanism of action. Cultured breast cancer cell lines MDA-MB-231 and MCF7A were transfected with a CBL-overexpressing plasmid and a specific siRNA targeting CBL (siRNA-CBL), respectively, and the changes in cell proliferation, migration and invasion were examined using colony-forming assay, cell counting kit-8 (CCK-8), scratch test and Transwell assay. Flow cytometry and Western blotting were performed to examine the effects of CBL overexpression on cell cycle and epithelial-mesenchymal transition (EMT) of MDA-MB-231 cells, and the changes in the number of filamentous pseudopodia were observed by rhodamine- labeled phalloidin staining of the cytoskeleton. IP-mass spectrometry identified NCK2 as the interacting proteins of CBL, and their interaction was verified by immunoprecipitation and immunofluorescence co-localization experiments in HEK-293T cells transfected with the plasmids for overexpression of CBL, NCK2, or both. Cycloheximide tracking and ubiquitination assays were used for assessing the effects of CBL on stability and ubiquitination of NCK2 protein in MDA-MB-231 cells; CCK-8 and Transwell assays were used to determine the effect of NCK2 overexpression on CBL-mediated proliferation and migration of the cells. The proliferation, migration and invasion were significantly suppressed in MDA-MB-231 cells overexpressing CBL (P < 0.05) and significantly enhanced in MCF7 cells with CBL silencing (P < 0.01). Silencing of CBL promoted G1/S transition in MCF7 cells (P < 0.05). Overexpression of CBL significantly decreased the expressions of CDK2/4 (P < 0.01), cyclinA2/B1/D1/D3/E2 (P < 0.05), Snail, N-cadherin, claudin-1 (P < 0.05), and upregulated the expression of E-cadherin (P < 0.05). CBL silencing upregulated the expressions of CDK2/4/6 (P < 0.05), cyclin A2/B1/D1/D3/E2 (P < 0.05), Snail, vimentin, and claudin-1 (P < 0.05) and down-regulated E-cadherin expression (P < 0.05). CBL overexpression obviously reduced the number of filamentous pseudopodia in MDA-MB-231 cells, and the reverse changes were observed in MCF7 cells with CBL silencing. In MDA-MB-231 cells, CBL overexpression lowered NCK2 protein stability (P < 0.05) and promoted its ubiquitin-mediated degradation (P < 0.01). Overexpression of NCK2 obviously reversed CBL-mediated inhibition of cell proliferation and migration (P < 0.01). CBL can inhibit the proliferation, migration and invasion of breast cancer cells through ubiquitination-mediated degradation of NCK2.