Study on mechanism of Hippo/YAP pathway inhibition by salvianolic acid B in HuH-7 cells / 中国药理学通报

Aim To investigate whether salvianolic acid B ( Sal B) has inhibitory effect on hepatoma HuH- 7 cells and explore whether it works via Hippo/YAP signaling pathway. Methods HuH-7 cells were induced by TGF-β1 (9 pmol · L

Hippo-YAP pathway is involved in the effect of NaAsO / 中国药理学通报

Aim To explore the effects of NaAsO

Lingnan Fire-needle on Inhibiting Melanocyte Apoptosis in Vitiligo Model Mice by Regulating Hippo-YAP Signaling Pathway / 中山大学学报(医学科学版)

ObjectiveTo observe the effects of fire-needle of Lingnan on the vitiligo model after hydroquinone-induced oxidative stress based on the Hippo-YAP signaling pathway. MethodsC57BL/6 mice were randomly divided into normal group （Control）， model group （HQ）， HQ+fire-needle group （FA）， and positive control group （Halometasone）， with 8 mice in each group. The vitiligo model was prepared by hydroquinone （HQ）. The skin pathological changes were observed by depigmentation score， HE staining and Masson-Fontana. Elisa was used to detect the levels of tyrosinase （TYR）， malondialdehyde （MDA） and monoamine oxidase （MAO）.Western-blot and PCR were used to detect the expression of Yap1 and Tp73 among the groups. ResultsCompared with the control group， the epidermis and dermis were significantly thicker. The number of melanocyte hair follicles， basal melanocytes， epidermal cells containing melanin granules were significantly decreased， and the depigmentation score was significantly reduced（P＜0.01）. The level of TYR decreased， and the levels of MDA and MAO increased after modeling（P＜0.01）. The expression of Yap1 and Tp73 were significantly reduced （P<0.01）. The dermis became thinner in the halometasone and FA group after treatment of 4 weeks. The number of melanocyte hair follicles， basal melanocytes， epidermal cells containing melanin granules increased （P<0.05）. Compared with that of the HQ group， the level of TYR in the halometasone group and FA group was significantly increased （P<0.01）. The levels of MDA and MAO in the FA group were decreased （P<0.05）. The expressions of Yap1 and Tp73 in the FA group were significantly increased （P<0.01）， and their effects were better than those in the Halometasone group （P<0.05）. ConclusionsFire-needle of Lingnan protects melanocytes from oxidative stress by activating the Hippo-YAP pathway. It enhances the synthesis and function of melanocytes and promotes repigmentation by reducing the content and activity of oxidative stress products.

Effects of andrographolide on angiogenesis in diabetic foot rats and its mechanism / 中国药房

OBJECTIVE To investigate the effects of andrographolide （Andro） on angiogenesis in rats with diabetic foot and to explore its mechanism of action based on the Hippo-Yes-associated protein （YAP） signaling pathway. METHODS The rat model of type 2 diabetes was established by using low-dose streptozotocin combined with high-fat and high-glucose diet. On the basis of successful modeling， the rat model of diabetes foot was established by scalding. Model rats were randomly divided into 5 groups with 12 rats in each group： model group， Andro low-dose， medium-dose， and high-dose groups （1， 10， and 20 mg/kg）， as well as inhibitor group （20 mg/kg Andro+100 mg/kg of verteporfin， an specific inhibitor of Hippo-YAP signaling pathway）； other 12 healthy rats were included in the Control group. Rats in each group were intragastrically and intraperitoneally injected with solvents or corresponding drugs， once a day， for 2 consecutive weeks. The wound healing， fasting blood glucose （FBG） and fasting insulin （FINS） were detected in rats after medication. HE staining was performed to observe the tissue damage and capillary number of rat trauma； the number of endothelial progenitor cells （EPCs） in peripheral blood of rats was counted by using flow cytometry； the contents of serum total cholesterol （TC）， triglyceride （TG）， high-density lipoprotein cholesterol （HDL-C） and low-density lipoprotein cholesterol （LDL-C） in rats were determined by fully automatic biochemical analyzer； the expressions of hypoxia- inducible factor 1α （HIF-1α）， vascular endothelial growth factor （VEGF） and Hippo-YAP signaling pathway-related proteins in the traumatic tissues of rats in each group were detected by Western blot. RESULTS Compared with Control group， the wound healing rate， capillary number， the proportion of EPCs， HDL-C content， as well as the protein expression levels of HIF-1α and VEGF and the phosphorylation levels of mammalian Ste20-like kinase 1， large tumor suppressor gene 1 and YAP proteins were significantly reduced in the model group， while the FBG， FINS levels and TC， TG and LDL-C contents were significantly increased （P＜0.05）. Compared with model group， the above indexes were significantly reversed in Andro low-dose， medium-dose and high-dose group， in a dose-dependent manner （P＜ 0.05）； verteporfin attenuated the above reversal effect of Andro （P＜0.05）. CONCLUSIONS Andro has the effects of lowering blood glucose and blood lipids， promoting blood vessel formation and wound healing in rats with diabetic foot， and its mechanism of action may be related to the activation of Hippo-YAP signaling pathway.

Hippo-YAP signaling pathway regulates autophagy of human periodontal ligament cells under cyclic tensile stress / 华西口腔医学杂志

OBJECTIVES@#This work aimed to investigate the molecular mechanism of cyclic tensile stress (CTS) stimulating autophagy in human periodontal ligament cells (hPDLCs).@*METHODS@#hPDLCs were isolated and cultured from normal periodontal tissues. hPDLCs were loaded with tensile stress by force four-point bending extender to simulate the autophagy of hPDLCs induced by orthodontic force du-ring orthodontic tooth movement. XMU-MP-1 was used to inhibit the Hippo signaling pathway to explore the role of the Hippo-YAP signaling pathway in activating hPDLC autophagy by tensile stress. The expression levels of autophagy-related genes (Beclin-1, LC3, and p62) in hPDLCs were detected by real-time quantitative polymerase chain reaction. Western blot was used to detect the expression levels of autophagy-related proteins (Beclin-1, LC3-Ⅱ/LC3-Ⅰ, and p62) and Hippo-YAP pathway proteins (active-YAP and p-YAP) in hPDLCs. Immunofluorescence was used to locate autophagy-related proteins (LC3-Ⅱand p62) and Hippo-YAP pathway proteins (active-YAP) of hPDLCs.@*RESULTS@#CTS-activated autophagy in hPDLCs and expression of autophagy-related proteins initially increased and then decreased; it began to increase at 30 min, peaked at 3 h, and decreased (P<0.05). CTS increased the expression of active-YAP protein and decreased the expression of p-YAP protein (P<0.05). When XMU-MP-1 inhibited the Hippo-YAP signaling pathway (P<0.05), active-YAP protein was promoted to enter the nucleus and autophagy expression was enhanced (P<0.05).@*CONCLUSIONS@#The Hippo-YAP signaling pathway is involved in the regulation of autophagy activation in hPDLCs under CTS.

Research progress on the effect of Hippo-YAP/TAZ signal pathway regulating ferroptosis on tumor / 中国肿瘤生物治疗杂志

@#[摘 要] 铁死亡是一种新型的铁依赖的程序性细胞死亡，常伴随脂质过氧化物的异常累积。Hippo通路是一种高度进化保守的蛋白激酶信号通路，通过调节下游效应蛋白YAP/TAZ的亚细胞定位和蛋白稳定性，参与调节细胞的多种生命活动，包括组织生长、干细胞分化、肿瘤的发生发展等。近年来的研究发现，Hippo-YAP/TAZ信号通路通过细胞密度、细胞接触、细胞代谢、机械信号等多种细胞外途径影响肿瘤细胞对铁死亡的敏感性，在不同类型的肿瘤组织中通过特定的刺激条件、铁死亡靶向蛋白及其分子机制，影响泌尿、生殖、消化、呼吸和内分泌系统等肿瘤的发生和发展。Hippo-YAP/TAZ信号通路作为铁死亡新的调节机制，其激活为转移性及耐药性肿瘤的治疗提供了新的思路和方向。

Effects of sodium arsenite on proliferation, apoptosis and Hippo-YAP pathway of PC12 cells / 中国药理学通报

To investigate the effects of sodium arsenite(NaAsO

Metformin inhibits proliferation and promotes apoptosis of HER-2 positive breast cancer cells possibly through the Hippo-YAP pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effect of metformin on the proliferation and apoptosis of HER-2-positive breast cancer cell line SKBR3 and explore the possible mechanism of its action.@*METHODS@#SKBR3 cells were treated with different concentrations (20-120 μmol/L) of metformin, and the changes in cell proliferation and colony formation ability were assessed using CCK-8 assay and crystal violet staining, respectively. Flow cytometry was performed to analyze cell apoptosis and cell cycle changes. Real-time fluorescent quantitative PCR (qRT-PCR) was used to detect mRNA expressions of YAP, TAZ, EGFR, CTGF, CYR61, E-cadherin, N-cadherin, vimentin and fibronectin in the treated cells, and the protein expressions of YAP and TAZ were detected using Western blotting; immunofluorescence assay was used to observe YAP/TAZ nuclear translocation in the cells.@*RESULTS@#Metformin treatment significantly inhibited the proliferation of SKBR3 cells (P < 0.05) in a concentration- and time-dependent manner. The results of flow cytometry showed that metformin significantly promoted apoptosis and caused cell cycle arrest at G1 phase in SKBR3 cells. Metformin treatment significantly down-regulated the mRNA expressions of YAP, TAZ, EGFR, CTGF and CYR61, N-cadherin, vimentin and fibronectin (P < 0.05) and up-regulated the expression of E-cadherin (P < 0.05); Western blotting results showed that YAP and TAZ protein expressions were significantly down-regulated in the cells after metformin treatment (P < 0.05). Immunofluorescence assay revealed that metformin treatment caused the concentration of YAP and TAZ in the cytoplasm, and significantly reduced their amount in the cell nucleus.@*CONCLUSION@#Metformin can inhibit proliferation and promote apoptosis and epithelal-mesenchymal transition of HER-2 positive breast cancer cells possibly by that inhibing YAP and TAZ expression and their nuclear localization.

ACTL6A Induces Cisplatin Resistance in the Lung Adenocarcinoma by Inhibiting the Hippo / YAP Signaling Pathway / 中国生物化学与分子生物学报

Actin-like 6A (ACTL6A), also known as BAF53A, is an SWI / SNF subunit of chromatin-remodeling factors and plays an important role in regulating stem cell function. Recent studies found that ACTL6A was involved in tumor occurrence and development. However, the mechanism of ACTL6A in cisplatin resistance is still unclear. This study investigated the biological function and molecular mechanism of ACTL6A in maintaining cancer stem cell function and cisplatin resistance. First, analysis from TCGA, GEO, and GEPIA databases showed that ACTL6A expression levels in lung adenocarcinoma (LUAD) tissues and cisplatin resistant cells were dramatically higher than that in adjacent normal tissues and cisplatin sensitive cells (P < 0. 05), and ACTL6A high expression was positively associated with a poor prognosis of LUAD. Knockdown of ACTL6A enhanced cisplatin sensitivity (P < 0. 05), reduced tumor sphere (P<0. 05), inhibited cell migration (P<0. 05), and promoted cell apoptosis (P<0. 05) in A549 cells. Western blotting showed that knockdown of ACTL6A increased the protein expression of E-cadherin, and decreased the protein expression of N-cadherin, vimentin, and twist. Moreover, knockdown of ACTL6A inhibited the expression of cancer stem cell markers, including ALDH3A1, ALDH4A1, SOX2, OCT4, and Nanog. Subsequently, Hippo / YAP signaling-related proteins were analyzed by Western blotting. The results showed the expression of beta-TRCP and YAP was decreased in A549 cell with knockdown of ACTL6A. However, phosphorylation levels at S127 and S397 of YAP were increased and inhibited translocation of YAP into the nucleus for regulating related gene expression. In summary, ACTL6A maintained the stemness of lung cancer stem cells and promoted cisplatin resistance in A549 cells by inhibiting activation of the Hippo signaling pathway.

Research progress on the Hippo-YAP signaling pathway mediated osteoimmunology in modulating implant osseointegration / 口腔疾病防治

@#Ideal osseointegration is intimately related to favorable osteoimmune properties around dental implants. An increasing number of in vitro and in vivo studies have indicated that the Hippo-YAP signaling pathway is involved in this biological process. In this article, the implicated roles of Hippo-YAP the signaling axis in peri-implant osteoimmunology were summarized by reviewing relevant evolving literature. The discrepancy concerning the Hippo-YAP signaling regulatory effect on osteogenesis and polarization direction were analyzed as well as propose the potential mechanism, which may be caused by the maturation of osteogenesis-related cells and heterogeneity of macrophages. More attention should be given to the requirements of promoting osteogenesis and patterns of regulating the immune microenvironment by Hippo-YAP in future studies.

Effects of microRNA-195-5p on growth, migration and invasion of breast cancer cells / 肿瘤

Objective: To investigate the effects of up- or down-regulated microRNA (miRNA, miR)-195-5p on the growth, migration and invasion of human triple negative breast cancer MDAMB-231 and BT-549 cells, and to explore the potential mechanism. Methods: The Cancer Genome Atlas (TCGA) database was used to analyze the expression level of miR-195-5p in breast cancer. MiR-195-5p-mimics was transfected into MDA-MB-231 cells to construct the recombinant cells with exogenous miR-195-5p overexpression. MiR-195-5p-inhibitor was transfected into BT-549 cells to construct the recombinant cells with endogenous miR-195-5p gene silence. The expression level of miR-195-5p in the recombinant cells was detected by realtime fluorescent quantitative PCR. The proliferation of MDA-MB-231 and BT-549 cells was detected by MTT assay, the longitudinal migration and invasion abilities were detected by Transwell migration and invasion assays, the lateral migration ability was evaluated by wound healing assay. The mRNA expression levels of migration-associated factor yes-associated protein-1 (YAP-1), and epithelialmesenchymal transition (EMT) markers (including E-cadherin, snail and vimentin) were detected by real-time fluorescent quantitative PCR. Meantime, the expression levels of EMT markers [including zinc finger E-box binding homeobox 1 (ZEB1), E-cadherin, N-cadherin and vimentin), YAP-1, and phosphorylated YAP-1 (p-YAP-1) were detected by Western blotting. Results: The expression of miR-195-5p in breast cancer tissues was low. The expression of miR-195-5p in MDA-MB-231 cells was lower than that in BT-549 cells (P < 0.001). After the transfection of miR-195-5p-mimics into MDA-MB-231 cells, the expression of miR-195-5p was significantly increased (P < 0.001); while the expression of miR-195-5p in BT-549 cells transfected with miR-195-5p-inhibitor was decreased (P < 0.001). After the over-expression of miR-195-5p, the proliferation, migration and invasion abilities of MDA-MB-231 cells were decreased (all P < 0.05); but the opposite phenomenon was observed in BT-549 cells with down-regulation of miR-195-5p (all P < 0.05). The expression level of E-cadherin mRNA increased, but the expression levels of YAP-1, vimentin and snail mRNAs decreased in MDA-MB-231 cells transfected with miR-195-5p-mimics (all P < 0.05). The expression level of E-cadherin protein increased (P < 0.001), but the expression levels of ZEB1 (P < 0.05), N-cadherin (P < 0.01) and vimentin proteins (P < 0.05) decreased in MDAMB-231 cells with miR-195-5p overexpression, suggesting that the EMT process might be inhibited. The expression levels of migration-related markers YAP-1 and p-YAP-1 proteins in MDA-MB-231 cells with miR-195-5p overexpression were significantly down-regulated (P < 0.05 and P < 0.01). Conclusion: The expression of miR-195-5p is down-regulated in breast cancer, and can inhibit the proliferation, migration, invasion and EMT of breast cancer cells, which may be related to reducing the activity of Hippo/YAP signaling pathway.

Stimulation of Yes-associated protein activity attenuates lung injury repair induced by LPS in mice / 中国临床药理学与治疗学

AIM: To observe the effect and possible mechanism of stimulation of Yes-associated protein (YAP) activity on acute lung injury and repair induced by LPS in mice. METHODS: Ninety adult male ICR mice were divided into two groups: acute lung injury model group induced by LPS (7.5 mg/kg, i.p.) and LPS+XMU treatment (1 mg•kg-1•d-1) group. At 0 h, 24 h, 48 h and 72 h after LPS injection, the contents of TNF-α and IL-6 in bronchoalveolar lavage fluid (BALF) were detected by ELISA method. The activity of myeloperoxidase (MPO) and the content of protein in BALF were evaluated by chemical technique. The protein level of nuclear YAP, cytosol phosphorylated YAP (P-YAP), connective tissue growth factor (CTGF) and proliferating cell nuclear antigen (PCNA) in lung were analyzed by Western blot. RESULTS:The protein level of nuclear YAP at 24 h, 48 h and 72 h in lung of LPS+XMU group were up-regulated than those of LPS group by 39.2%, 148.1% and 42.9%, and the protein level of CTGF in lung were up-regulated than those of LPS group by 186.6%, 10.1% and 146.1% (P<0.05), respectively, while the protein level of cytosol P-YAP was down-regulated. The content of IL-6 and TNF-α at 24 h in BALF of LPS+XMU group were lower than those of LPS group by 28.4% and 23.7%, and the activity of MPO and the content of TNF-α at 48 h in BALF were lower by 13.5% and 39.5%, and the content of IL-6 and TNF-α at 72 h in BALF were lower by 42.8% and 16.7% (P<0.05), respectively. The protein level of PCNA at 48 h and 72 h in lung of LPS+XMU group were up-regulated than those of LPS group by 44.2% and 14.9% (P<0.05), respectively, while there was no significant difference at 24 h. The content of protein at 24 h, 48 h and 72 h in BALF of LPS+XMU group were lower than those of LPS group by 32.4%, 46.0% and 26.3% (P<0.05), respectively. The pathological changes showed a significantly attenuated tissue injury and accelerated recovery from lung injury in LPS+XMU group compared with mice injected with LPS alone at each times point. CONCLUSION: Stimulation of YAP activity attenuates lung injury and promotes lung recovery by alleviating lung inflammation and injury at injury phase, but by promoting inflammation resolution and stimulating cell proliferation at repair phase.

Evodiamine induces apoptosis of human hepatoma cell line BEL-7402 by Hippo-YAP pathway / 中草药

Objective: To investigate the effects and the underlying mechanisms of evodiamine (EVO) on apoptosis of hepatocellular carcinoma (HCC) BEL-7402 cells. Methods: Cell counting kit-8 (CCK-8) assay was used to detect the effect of EVO on proliferation activity of BEL-7402 cells. Hoechst 33258 staining was used for observing morphological changes of apoptosis. The cell apoptosis and cycle distribution were analyzed by flow cytometry. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting assay were used to detect the expression levels of key genes from Hippo-YAP pathway in HL-7702 and BEL-7402 cells, including mammalian STE20-like protein kinase 1/2 (MST1/2), large tumor suppressor 1 (LATS1), and Yes-associated protein (YAP), then to examine the effect of EVO on the expression levels of these genes in BEL-7402 cells. The effect of EVO on the expression of YAP in hepatocellular carcinoma BEL-7402 cells was observed by immunofluorescence assay. Results: The proliferation of BEL-7402 cells were significantly inhibited by EVO in a dose- and time-dependent manners. Hoechst 33258 staining showed that EVO induced BEL-7402 cell typical apoptotic morphology, such as nuclear chromatin concentration and edge accumulation. Besides, flow cytometry tests showed that BEL-7402 cell apotosis were increased and cell cycle arrested in G2/M phase after being treated with EVO (P < 0.01). qRT-PCR and Western blotting showed that the expression level of MST2 and LATS1 were lower in BEL-7402 cell (P < 0.05), while the transcription and protein levels of YAP were significantly higher (P < 0.05). EVO could activate Hippo signal pathway, upregulate the expression of MST2 and LATS1 and then inhibit the expression of YAP in BEL-7402 cell (P < 0.05). Immunofluorescence assay also validated that EVO would significantly inhibit the overexpression of YAP in BEL-7402 cell (P < 0.01). Conclusion: EVO can induce the apoptosis of BEL-7402 cells, which may be through activating Hippo signal pathway and then down-regulate the expression of YAP.

Citron Rho-interacting serine/threonine kinase knockdown suppresses prostate cancer cell proliferation and metastasis by blocking Hippo-YAP pathway / 南方医科大学学报

OBJECTIVE@#Citron Rho-interacting serine/threonine kinase (CIT) was identified recently as an oncogene involved in the progression of various malignant tumors, but its role in prostate cancer (PCa) remains unclear. In this study, we aimed to investigate the biological functions of CIT in PCa.@*METHODS@#We analyzed the expression of CIT in PCa tissues and its clinical correlations based on the Cancer Genome Atlas (TCGA) and Memorial Sloan-Kettering Cancer Center (MSKCC) dataset. We then examined the effects of RNA interference-mediated CIT silencing on the proliferation, migration and invasion of PC-3 cells using cell counting kit-8, wound healing assay and Transwell assay. We also investigated the effect of CIT silencing on epithelial-mesenchymal transition (EMT) and Hippo-Yap signaling pathway in the cells using Western blotting.@*RESULTS@#CIT expression was significantly elevated in PCa tissues from TCGA cohort ( < 0.05). MSKCC dataset analysis showed that an elevated expression of CIT was significantly correlated with N stage (=0.001), distant metastasis ( < 0.001), Gleason score (=0.010) and PSA (=0.004). In cultured PC-3 cells, knockdown of CIT significantly inhibited cell proliferation, migration and invasion, reversed the EMT phenotype and decreased the expression and activity of YAP.@*CONCLUSIONS@#CIT might function as an oncogene in PCa by modulating the Hippo-YAP signaling pathway and serve as a candidate therapeutic target for PCa.

Pseudolaric Acid B Inhibits Proliferation, Invasion and Epithelial-to-Mesenchymal Transition in Human Pancreatic Cancer Cell

PURPOSE: This study was aimed to investigate the effect of pseudolaric acid B (PAB) on proliferation, invasion and epithelial-to-mesenchymal transition (EMT) in pancreatic cancer cells and to explore the possible mechanism. MATERIALS AND METHODS: The pancreatic cancer cell line SW1990 was cultured and treated with PAB dose- and time-dependent manners. Cell proliferation and invasion ability were measured by MTT assay and Matrigel/Transwell test, respectively. Semi-quantitative real-time polymerase chain reaction and Western blotting were conducted to detect the expression of EMT markers and the key molecules. Finally, nude mice subcutaneous transplantation tumor model was used to confirm the therapy efficacy of PAB. RESULTS: PAB could inhibit SW1990 cell proliferation and invasion in time- and dose-dependent manners. Vimentin, fibronectin, N-cadherin, Snail, Slug, YAP, TEAD1, and Survivin were down-regulated (p < 0.01), while E-cadherin, caspase-9, MST1, and pYAP were up-regulated (p < 0.05). Combined PAB and gemcitabine treatment markedly restricted the tumor growth compared with gencitabin or PAB alone groups. CONCLUSION: PAB could inhibit the proliferation and invasion ability of pancreatic cancer cells through activating Hippo-YAP pathway and inhibiting the process of EMT.

Research advances in the role of the Hippo-YAP/TAZ signaling pathway in primary liver cancer / 中华肝脏病杂志

The Hippo-YAP/TAZ signaling pathway is an evolutionarily conserved pathway, which has been confirmed to play an important role in organ volume control, stem cell function, tissue regeneration, and tumorigenesis. Recent research findings show that the Hippo-YAP/TAZ signaling pathway is closely associated with the development and progression of primary liver cancer, and inhibition of the activity of this pathway may be a new method for the treatment of liver cancer. This article reviews the research advances in the role of the Hippo-YAP/TAZ signaling pathway in primary liver cancer.