Smad3 Inhibitor Research Articles

Inhibition of TGF-β/Smad3 Signaling Disrupts Cardiomyocyte Cell Cycle Progression and Epithelial-Mesenchymal Transition-Like Response During Ventricle Regeneration.

Unlike mammals, zebrafish can regenerate injured hearts even in the adult stage. Cardiac regeneration requires the coordination of cardiomyocyte (CM) proliferation and migration. The TGF-β/Smad3 signaling pathway has been implicated in cardiac regeneration, but the molecular mechanisms by which this pathway regulates CM proliferation and migration have not been fully illustrated. Here, we investigated the function of TGF-β/Smad3 signaling in a zebrafish model of ventricular ablation. Multiple components of this pathway were upregulated/activated after injury. Utilizing a specific inhibitor of Smad3, we detected an increased ratio of unrecovered hearts. Transcriptomic analysis suggested that the TGF-β/Smad3 signaling pathway could affect CM proliferation and migration. Further analysis demonstrated that the CM cell cycle was disrupted and the epithelial–mesenchymal transition (EMT)-like response was impaired, which limited cardiac regeneration. Altogether, our study reveals an important function of TGF-β/Smad3 signaling in CM cell cycle progression and EMT process during zebrafish ventricle regeneration.

CRISPR screens identify tumor‐promoting genes conferring melanoma cell plasticity and resistance

Most genetic alterations that drive melanoma development and resistance to targeted therapy have been uncovered. In contrast, and despite their increasingly recognized contribution, little is known about the non‐genetic mechanisms that drive these processes. Here, we performed in vivo gain‐of‐function CRISPR screens and identified SMAD3, BIRC3, and SLC9A5 as key actors of BRAFi resistance. We show that their expression levels increase during acquisition of BRAFi resistance and remain high in persister cells and during relapse. The upregulation of the SMAD3 transcriptional activity (SMAD3‐signature) promotes a mesenchymal‐like phenotype and BRAFi resistance by acting as an upstream transcriptional regulator of potent BRAFi‐resistance genes such as EGFR and AXL. This SMAD3‐signature predicts resistance to both current melanoma therapies in different cohorts. Critically, chemical inhibition of SMAD3 may constitute amenable target for melanoma since it efficiently abrogates persister cells survival. Interestingly, decrease of SMAD3 activity can also be reached by inhibiting the Aryl hydrocarbon Receptor (AhR), another druggable transcription factor governing SMAD3 expression level. Our work highlights novel drug vulnerabilities that can be exploited to develop long‐lasting antimelanoma therapies.

Inhibition of Smad3 in macrophages promotes Aβ efflux from the brain and thereby ameliorates Alzheimer's pathology

Impaired amyloid-β (Aβ) clearance is believed to be a primary cause of Alzheimer's disease (AD), and peripheral abnormalities in Aβ clearance have recently been linked to AD pathogenesis and progression. Data from recent genome-wide association studies have linked genetic risk factors associated with altered functions of more immune cells to AD pathology. Here, we first identified correlations of Smad3 signaling activation in peripheral macrophages with AD progression and phagocytosis of Aβ. Then, manipulating the Smad3 signaling regulated macrophage phagocytosis of Aβ and induced switch of macrophage inflammatory phenotypes in our cell cultures. In our mouse models, flag-tagged or fluorescent-dye conjugated Aβ was injected into the lateral ventricles or tail veins, and traced. Interestingly, blocking Smad3 signaling efficiently increased Aβ clearance by macrophages, reduced Aβ in the periphery and thereby enhanced Aβ efflux from the brain. Moreover, in our APP/PS1 transgenic AD model mice, Smad3 inhibition significantly attenuated Aβ deposition and neuroinflammation, and ameliorated cognitive deficits, probably by enhancing the peripheral clearance of Aβ. In conclusion, enhancing Aβ clearance by peripheral macrophages through Smad3 inhibition attenuated AD-related pathology and cognitive deficits, which may provide a new perspective for understanding AD and finding novel therapeutic approaches.

MiR-21-3p Promotes Hepatocellular Carcinoma Progression via SMAD7/YAP1 Regulation.

BackgroundHepatocellular carcinoma (HCC) remains a major global health burden due to its high prevalence and mortality. Emerging evidence reveals that microRNA (miRNA) plays a vital role in cancer pathogenesis and is widely involved in the regulation of signaling pathways via their targeting of downstream genes. MiR-21-3p, a liver-enriched miRNA, and SMAD7, the negative regulator of the TGF-β signaling pathway, likely exert a vital influence on HCC progression.AimsHere, we explore the role of the miR-21-3p-SMAD7/YAP1 axis on HCC pathogenesis.MethodsMiRNA microarray analysis was performed for miRNA screening. The dual-luciferase assay was adopted for target verification. Expression of miRNA and related genes were quantified via qRT-PCR, western blotting, and immunohistochemical staining. Flow cytometry and the transwell migration assay were used to detail cell apoptosis, invasion and metastases. Rat models were established to explore the role of the miR-21-3p-SMAD7/YAP1 axis in hepatocarcinogenesis. Bioinformatics analysis was conducted for exploring genes of clinical significance.ResultsMiR-21-3p levels were found to be significantly elevated in hepatocellular carcinoma and indicate poor overall survival. High miR-21-3p levels were associated with advanced tumor stages (P = 0.029), in particular T staging (P = 0.026). Low SMAD7/high YAP1 levels were confirmed in both HCC and rat models with advanced liver fibrosis and cirrhosis. Besides, SMAD7 was demonstrated to be the direct target of miR-21-3p. The effect of MiR-21-3p on tumor phenotypes and YAP1 upregulation could be partly reversed via the restoration of SMAD7 expression in HCC cell lines. Overexpression of YAP1 after miR-21-3p upregulation promoted expression of nuclear transcription effector connective tissue growth factor. Co-survival analysis indicated that lower miR-21-3p/higher SMAD7 (P = 0.0494) and lower miR-21-3p/lower YAP1 (P = 0.0379) group patients had better overall survival rates. Gene Set Variation Analysis revealed that gene sets related to miR-21-3p and SMAD7 were significantly associated with the TGF-β signaling pathway in HCC.ConclusionMiR-21-3p promotes migration and invasion of HCC cells and upregulation of YAP1 expression via direct inhibition of SMAD7, underscoring a major epigenetic mechanism in the pathogenesis of HCC.

Treatment with phosphodiester CpG-ODN ameliorates atopic dermatitis by enhancing TGF-β signaling

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG phosphorothioate (PS CpG-ODN) are known to decrease IgE synthesis in Th2 allergy responses. Nonetheless, the therapeutic role of PS CpG-ODN is limited due to cytotoxicity. Therefore, we developed a phosphodiester (PO) form of CpG-ODN (46O) with reduced toxicity but effective against allergies. In this study, we first compared the toxicity of 46O with CpG-ODNs containing a PS backbone (1826S). We also investigated the therapeutic efficacy and mechanism of 46O injected intravenously in a mouse model of ovalbumin (OVA)-induced atopic dermatitis (AD). To elucidate the mechanism of 46O underlying the inhibition of IgE production, IgE- and TGF-β-associated molecules were evaluated in CD40/IL-4- or LPS/IL-4-stimulated B cells. Our data showed that the treatment with 46O was associated with a lower hematological toxicity compared with 1826S. In addition, injection with 46O reduced erythema, epidermal thickness, and suppressed IgE and IL-4 synthesis in mice with OVA-induced AD. Additionally, 46O induced TGF-β production in LPS/IL-4-stimulated B cells via inhibition of Smad7, which suppressed IgE synthesis via interaction between Id2 and E2A. These findings suggest that enhanced TGF-β signaling is an effective treatment for IgE-mediated allergic conditions, and 46O may be safe and effective for treating allergic diseases such as AD and asthma.

N-myc downstream regulated gene 2 ameliorates myocardial remodeling and cardiac function in heart failure rats.

This study aims to explore the effect of NDRG2 (N-myc downstream regulated gene 2)-mediated Transforming growth factor-beta 1 (TGF-β1)/ Sma- and Mad-related protein (Smad) pathway in heart failure (HF) rats. HF rat models were established and treated with AdEGFP (adenovirus encoding enhanced green fluorescent protein) or AdNDRG2 (adenovirus encoding NDRG2). The echocardiography and hemodynamic parameters were detected, and the infarct size was calculated via 2,3,5-triphenyltetrazolium chloride (TTC) staining. Masson staining was performed to observe the collagen volume fraction (CVF), quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) to detect the expression of Collagen I (Col-I) and Collagen III (Col-III), and Transferase (TdT)-mediated D-UTP-biotin nick end labeling (TUNEL) staining to evaluate the apoptosis. Rats in the Model group presented with the decreases in left ventricular ejection fraction (LVEF), left ventricular shortening fraction (LVFS), left ventricular systolic pressure (LVSP) and maximal/minimum rate of left ventricular pressure (±dp/dt max), and significant increases in left ventricular end-diastolic pressure (LVEDP) and CVF. At the meantime, the expression of Col-I and Col-III as well as the apoptotic rate of myocardial cells was also elevated with increased infarct size in the Model group. The Model rats also had the significant reduction in the expression of NDRG2 and up-regulations of TGF-β1, p-Smad2/Smad2, p-Smad3/Smad3 and tissue inhibitor of metalloproteinases-2 (TIMP-2). However, model rats treated with AdNDRG2 had evident amelioration in aforementioned indicators. In conclusion, NDRG2 reduces the apoptosis of myocardial cells and improves the heart function and myocardial remodeling in HF rats via inhibiting the activity of TGF-β1/Smad.

Akt signaling is activated by TGF\u03b22 and impacts tenogenic induction of mesenchymal stem cells

BackgroundTissue engineered and regenerative approaches for treating tendon injuries are challenged by the limited information on the cellular signaling pathways driving tenogenic differentiation of stem cells. Members of the transforming growth factor (TGF) β family, particularly TGFβ2, play a role in tenogenesis, which may proceed via Smad-mediated signaling. However, recent evidence suggests some aspects of tenogenesis may be independent of Smad signaling, and other pathways potentially involved in tenogenesis are understudied. Here, we examined the role of Akt/mTORC1/P70S6K signaling in early TGFβ2-induced tenogenesis of mesenchymal stem cells (MSCs) and evaluated TGFβ2-induced tenogenic differentiation when Smad3 is inhibited.MethodsMouse MSCs were treated with TGFβ2 to induce tenogenesis, and Akt or Smad3 signaling was chemically inhibited using the Akt inhibitor, MK-2206, or the Smad3 inhibitor, SIS3. Effects of TGFβ2 alone and in combination with these inhibitors on the activation of Akt signaling and its downstream targets mTOR and P70S6K were quantified using western blot analysis, and cell morphology was assessed using confocal microscopy. Levels of the tendon marker protein, tenomodulin, were also assessed.ResultsTGFβ2 alone activated Akt signaling during early tenogenic induction. Chemically inhibiting Akt prevented increases in tenomodulin and attenuated tenogenic morphology of the MSCs in response to TGFβ2. Chemically inhibiting Smad3 did not prevent tenogenesis, but appeared to accelerate it. MSCs treated with both TGFβ2 and SIS3 produced significantly higher levels of tenomodulin at 7 days and morphology appeared tenogenic, with localized cell alignment and elongation. Finally, inhibiting Smad3 did not appear to impact Akt signaling, suggesting that Akt may allow TGFβ2-induced tenogenesis to proceed during disruption of Smad3 signaling.ConclusionsThese findings show that Akt signaling plays a role in TGFβ2-induced tenogenesis and that tenogenesis of MSCs can be initiated by TGFβ2 during disruption of Smad3 signaling. These findings provide new insights into the signaling pathways that regulate tenogenic induction in stem cells.

Chrysin induces osteogenic differentiation of human dental pulp stem cells

ObjectivesAs an ideal cell source for tissue engineering and bone defect repair, dental pulp stem cells (DPSCs) have good osteogenic differentiation potential. Chrysin, a flavonoid extracted from oroxylum seeds, has been proven to promote bone formation of bone marrow stem cells. However, the effect of chrysin on osteogenic differentiation of DPSCs remains unclear. This study aimed to investigate the role of Chrysin in promoting osteogenic differentiation of DPSCs and in DPSC-based bone formation. Material and methodsWe investigated the effects of chrysin on DPSCs from patients by CCK-8 assay, Alizarin Red S staining, qPCR and Western blotting. The effects of chrysin on DPSC-based bone formation in a heterotopic osteogenesis model in nude mice and a rat calvarial defect model were also performed. Finally, we investigated the mechanism of chrysin-treated DPSCs by proteomics. ResultsChrysin upregulated the expression of osteogenic proteins and induced osteogenic differentiation of DPSCs. Moreover, chrysin induced abundant β-TCP-induced formation of mineralized bone tissue and promoted DPSC-based bone formation in a heterotopic osteogenesis model in nude mice and a rat calvarial defect model. Proteomics showed that upregulation of the Smad3 was closely related to osteogenic differentiation. Inhibiting of Smad3 activation by a Smad3 inhibitor could reverse the chrysin-mediated increases in the expression levels of osteogenic genes and osteogenic induction of DPSCs. ConclusionsOur study implies the intriguing potential of chrysin-treated DPSCs in bone regeneration and bone defect repair.

Inhibition of tumor invasion and metastasis by targeting TGF-β-Smad-MMP2 pathway with Asiatic acid and Naringenin

Transforming growth factor β (TGF-β) has been shown to promote tumor invasion and metastasis by activating the matrix metalloproteinases (MMPs); however, signaling mechanisms remain controversial and therapies targeting MMPs are still suboptimal. In the present study, we found that combined therapy with Asiatic acid (AA), a Smad7 agonist, and Naringenin (NG), a Smad3 inhibitor, effectively retrieved the balance between Smad3 and Smad7 signaling in the TGF-β-rich tumor microenvironment and thus significantly suppressed tumor invasion and metastasis in mouse models of melanoma and lung carcinoma. Mechanistically, we unraveled that Smad3 acted as a transcriptional activator of MMP2 and as a transcriptional suppressor of tissue inhibitors of metalloproteinase-2 (TIMP2) via binding to 5′ UTR of MMP2 and 3′ UTR of TIMP2, respectively. Treatment with NG inhibited Smad3-mediated MMP2 transcription while increasing TIMP, whereas treatment with AA enhanced Smad7 to suppress TGF-β/Smad3 signaling, as well as the activation of MMP2 by targeting the nuclear factor-κB (NF-κB)-membrane-type-1 MMP (MT1-MMP) axis. Therefore, the combination of AA and NG additively suppressed invasion and metastasis of melanoma and lung carcinoma by targeting TGF-β/Smad-dependent MMP2 transcription, post-translational activation, and function.

Co-delivery of doxorubicin and SIS3 by folate-targeted polymeric micelles for overcoming tumor multidrug resistance.

Multidrug resistance (MDR) is considered as a critical limiting factor for the successful chemotherapy, which is mainly characterized by the overexpression of ATP-binding cassette (ABC) transporter ABCB1 or ABCG2. In this study, folate-targeted polymeric micellar carrier was successfully constructed to co-delivery of doxorubicin (DOX) and SIS3 (FA/DOX/SIS3 micelles), a specific Smad3 inhibitor which sensitizes ABCB1- and ABCG2-overexpressing cancer cells to chemotherapeutic agents. The ratio of DOX to SIS3 in polymeric micelles was determined based on the anti-tumor activity against resistant breast cells. In addition, FA/DOX/SIS3 micelles exhibited a much longer circulation time in blood and were preferentially accumulated in resistant tumor tissue. Pharmacodynamic studies showed that FA/DOX/SIS3 micelles possessed superior anti-tumor activity than other DOX-based treatments. Overall, FA/DOX/SIS3 micelles are a promising formulation for the synergistic treatment of drug-resistant tumor.

Transforming Growth Factor β Inhibits MUC5AC Expression by Smad3/HDAC2 Complex Formation and NF-κB Deacetylation at K310 in NCI-H292 Cells.

Airway mucus secretion is an essential innate immune response for host protection. However, overproduction and hypersecretion of mucus, mainly composed of the gel-forming MUC5AC protein, are significant risk factors for patients with asthma and chronic obstructive pulmonary disease (COPD). The transforming growth factor β (TGFβ) signaling pathway negatively regulates MUC5AC expression; however, the underlying molecular mechanism is not fully understood. Here, we showed that TGFβ significantly reduces the expression of MUC5AC mRNA and its protein in NCI-H292 cells, a human mucoepidermoid carcinoma cell line. This reduced MUC5AC expression was restored by a TGFβ receptor inhibitor (SB431542), but not by the inhibition of NF-κB (BAY11-7082 or Triptolide) or PI3K (LY294002) activities. TGFβ-activated Smad3 dose-dependently bound to MUC5AC promoter. Notably, TGFβ-activated Smad3 recruited HDAC2 and facilitated nuclear translocation of HDAC2, thereby inducing the deacetylation of NF-κB at K310, which is essential for a reduction in NF-κB transcriptional activity. Both TGFβ-induced nuclear translocation of Smad3/HDAC2 and deacetylation of NF-κB at K310 were suppressed by a Smad3 inhibitor (SIS3). These results suggest that the TGFβ-activated Smad3/HDAC2 complex is an essential negative regulator for MUC5AC expression and an epigenetic regulator for NF-κB acetylation. Therefore, these results collectively suggest that modulation of the TGFβ1/Smad3/HDAC2/NF-κB pathway axis can be a promising way to improve lung function as a treatment strategy for asthma and COPD.

Smad3-Targeted Therapy Protects against Cisplatin-Induced AKI by Attenuating Programmed Cell Death and Inflammation via a NOX4-Dependent Mechanism

Background: Transforming growth factor-β (TGF-β)/Smad signaling is the central mediator in renal fibrosis, yet its functional role in acute kidney injury (AKI) is not fully understood. Recent evidence showed that TGF-β/Smad3 may be involved in the pathogenesis of AKI, but its functional role and mechanism of action in cisplatin-induced AKI are unclear. Objectives: Demonstrating that Smad3 may play certain roles in cisplatin nephropathy due to its potential effect on programmed cell death and inflammation. Methods: Here, we established a cisplatin-induced AKI mouse model with Smad3 knockout mice and created stable in vitro models with Smad3 knockdown tubular epithelial cells. In addition, we tested the potential of Smad3-targeted therapy using 2 in vivo protocols – lentivirus-mediated Smad3 silencing in vivo and use of naringenin, a monomer used in traditional Chinese medicine and a natural inhibitor of Smad3. Results: Disruption of Smad3 attenuated cisplatin-induced kidney injury, inflammation, and NADPH oxidase 4-dependent oxidative stress. We found that Smad3-targeted therapy protected against loss of renal function and alleviated apoptosis, RIPK-mediated necroptosis, renal inflammation, and oxidative stress in cisplatin nephropathy. Conclusions: These findings show that Smad3 promotes cisplatin-induced AKI and Smad3-targeted therapy protects against this pathological process. These findings have substantial clinical relevance, as they suggest a therapeutic target for AKI.

Mitomycin C induces pulmonary vascular endothelial-to-mesenchymal transition and pulmonary veno-occlusive disease via Smad3-dependent pathway in rats.

Pulmonary veno-occlusive disease (PVOD) is a rare disease characterized by the obstruction of small pulmonary veins leading to pulmonary hypertension. However, the mechanisms underlying pulmonary vessel occlusion remain largely unclear. A mitomycin C (MMC)-induced PVOD rat model was used as in vivo animal model, and primarily cultured rat pulmonary microvascular endothelial cells (PMVECs) were used as in vitro cell model. Our data suggested an endothelial-to-mesenchymal transition (EndoMT) may be present in the pulmonary microvessels isolated from either PVOD patients or MMC-induced PVOD rats. In comparison to the control vessels, vessels from both PVOD patients and PVOD rats had co-localized staining of specific endothelial marker von Willebrand factor (vWF) and mesenchymal marker α-smooth muscle actin (α-SMA), suggesting the presence of cells that co-express endothelial and mesenchymal markers. In both the lung tissues of MMC-induced PVOD rats and MMC-treated rat PMVECs there were decreased levels of endothelial markers (e.g. VE-cadherin and CD31) and increased mesenchymal markers (e.g. vimentin, fibronectin and α-SMA) were detected indicating EndoMT. Moreover, MMC-induced activation of the TGFβ/Smad3/Snail axis, while blocking this pathway with either selective Smad3 inhibitor (SIS3) or small interfering RNA (siRNA) against Smad3, dramatically abolished the MMC-induced EndoMT. Notably, treatment with SIS3 remarkably prevented the pathogenesis of MMC-induced PVOD in rats. Our data indicated that targeted inhibition of Smad3 leads to a potential, novel strategy for PVOD therapy, likely by inhibiting the EndoMT in pulmonary microvasculature.

Effect of SIS3 on Extracellular Matrix Remodeling and Repair in a Lipopolysaccharide-Induced ARDS Rat Model.

The remodeling of the extracellular matrix (ECM) in the parenchyma plays an important role in the development of acute respiratory distress syndrome (ARDS), a disease characterized by lung injury. Although it is clear that TGF-β1 can modulate the expression of the extracellular matrix (ECM) through intracellular signaling molecules such as Smad3, its role as a therapeutic target against ARDS remains unknown. In this study, a rat model was established to mimic ARDS via intratracheal instillation of lipopolysaccharide (LPS). A selective inhibitor of Smad3 (SIS3) was intraperitoneally injected into the disease model, while phosphate-buffered saline (PBS) was used in the control group. Animal tissues were then evaluated using histological analysis, immunohistochemistry, RT-qPCR, ELISA, and western blotting. LPS was found to stimulate the expression of RAGE, TGF-β1, MMP2, and MMP9 in the rat model. Moreover, treatment with SIS3 was observed to reverse the expression of these molecules. In addition, pretreatment with SIS3 was shown to partially inhibit the phosphorylation of Smad3 and alleviate symptoms including lung injury and pulmonary edema. These findings indicate that SIS3, or the blocking of TGF-β/Smad3 pathways, could influence remodeling of the ECM and this may serve as a therapeutic strategy against ARDS.

Substantia nigra Smad3 signaling deficiency: relevance to aging and Parkinson\u2019s disease and roles of microglia, proinflammatory factors, and MAPK

BackgroundSmad3 signaling is indicated to regulate microglia activity. Parkinson’s disease (PD) neurodegeneration is shown to be associated with aging and neuroinflammation. However, it remains unclear about the relationship among Smad3 signaling, aging, neuroinflammation, and PD.MethodsRats were treated with SIS3 (a specific inhibitor of Smad3, intranigal injection) and/or lipopolysaccharide (intraperitoneal injection). We investigated the effect of SIS3 and lipopolysaccharide and their mechanism of action on motor behavior and nigrostriatal dopaminergic system in the rats. Furthermore, we explored the effect of SIS3 and LPS and their potential signaling mechanism of action on inflammatory response by using primary microglial cultures. Finally, we investigated the relationship among aging, Smad3 signaling, and neuroinflammation using animals of different ages.ResultsBoth SIS3 and lipopolysaccharide induced significant behavior deficits and nigrostriatal dopaminergic neurodegeneration in the rats compared with the vehicle-treated (control) rats. Significantly increased behavior deficits and nigrostriatal dopaminergic neurodegeneration were observed in the rats co-treated with SIS3 and lipopolysaccharide compared with the rats treated with vehicle, SIS3, or lipopolysaccharide. Furthermore, both SIS3 and lipopolysaccharide induced significant microglia activation and proinflammatory factor (IL-1β, IL-6, iNOS, and ROS) level increase in the SN of rats compared with the control rats. Significantly enhanced microglial inflammatory response was observed in the rats co-treated with SIS3 and lipopolysaccharide compared with the other three groups. For our in vitro study, both SIS3 and lipopolysaccharide induced significant proinflammatory factor level increase in primary microglia cultures compared with the control cultures. Significantly increased inflammatory response was observed in the cultures co-treated with SIS3 and lipopolysaccharide compared with the other three groups. MAPK (ERK/p38) contributed to microglial inflammatory response induced by co-treatment with SIS3 and lipopolysaccharide. Interestingly, there was decrease in Smad3 and pSmad3 expression (protein) and enhancement of neuroinflammation in the mouse SN with aging. Proinflammatory factor levels were significantly inversely correlated with Smad3 and pSmad3 expression.ConclusionOur study strongly indicates the involvement of SN Smad3 signaling deficiency in aging and PD neurodegeneration and provides a novel molecular mechanism underlying the participation of aging in PD and helps to elucidate the mechanisms for the combined effect of multiple factors in PD.

Chemical proteomics reveals ligustilide targets SMAD3, inhibiting collagen synthesis in aortic endothelial cells

Atherosclerosis is a persistent inflammatory state, while vascular endothelial fibrosis is one of the primary causes of atherosclerosis development. Although ligustilide (Lig) was shown to exert obvious antiatherogenic effects in previous studies, its precise mechanism has not been deeply discussed. In this paper, we designed a Lig-derived photoaffinity labelling (PAL) probe to identify potential therapeutic targets of Lig via chemical proteomics approach. Mothers against decapentaplegic homologue 3 (SMAD3), a signal transmitter of transforming growth factor-β (TGF-β) which promotes the development of vascular fibrosis, was identified as a potential target of Lig. Lig suppressed the phosphorylation and nuclear translocation of SMAD3 by blocking the interaction between SMAD3 and TGF-β receptor 1, thereby inhibiting the collagen synthesis process. Hence, developing a novel SMAD3 inhibitor may present a promising therapeutic option for preventing vascular fibrosis.

Graphene oxide inhibits cell migration and invasion by destroying actin cytoskeleton in cervical cancer cells.

Objective: To investigate the antitumor effects of Graphene oxide (GO) on tumor invasion and metastasis in human cervical cancer Hela cells.Results: GO significantly inhibited cell viability and the number of clones, promoted cell apoptosis, as well as suppressed cell migration and invasion, and destroyed the structure of actin cytoskeleton of Hela cells in a dose-dependent manner in. Moreover, the expression of metastasis-related proteins, including MMP2 and Cdc42, were significantly suppressed by the treatment of GO. And the expression of MMP3 was remarkably increased by Smad inhibitor and the protein levels of MMP3 and ICAM were elevated by the JNK inhibitor in GO-treated Hela cells.Conclusion: GO exhibited inhibitory effects on cell migration and invasion possibly by destroying actin cytoskeleton in Hela cells, which is a potential component of the Smad and JNK signalling pathways.Methods: GO was prepared and chracterized by UV visible light absorption spectroscopy and atomic force microscopy. Hela cells were treated with Go at different dose levels. Then, in vitro cytotoxicity of GO was evaluated by the MTT assay, colony-forming assay and cell apoptosis assay. The inhibitory effects of GO on tumor cell migration and invasion as well as actin cytoskeleton were explored using Hela cells.

All-trans-retinoic acid inhibits mink hair follicle growth via inhibiting proliferation and inducing apoptosis of dermal papilla cells through TGF-β2/Smad2/3 pathway

Dermal papilla cells (DPCs), an important component of hair follicles, its proliferation and apoptosis directly regulate and maintain the growth of hair follicles. All-trans-retinoic acid (ATRA) plays a critical role in hair growth. In this study, the effects of ATRA on cultured mink hair follicle growth were studied by administration of different concentrations of ATRA for 12 days in vitro. In addition, the proliferation and apoptosis of DPCs were measured after treating with ATRA. The mRNA and protein levels of hair follicle growth associated factor transforming growth factor-β2 (TGF-β2) and the phosphorylation levels of Smad2/3 were determined. Moreover, TGF-β type I and type II receptor inhibitor LY2109761 and specific inhibitor of Smad3 (SIS3) were administered to investigate the underlying molecular mechanism. The results showed that ATRA inhibited hair follicle growth, promoted TGF-β2 expression and activated phosphorylation of Smad2/3. In addition, ATRA inhibited cell proliferation by arresting the cell cycle at G1 phase and induced apoptosis of DPCs by enhancing the ratio of Bax/Bcl-2 and promoted the cleavage of caspase-3. Furthermore, LY2109761 or SIS3 partially reversed the decreased cell viability, increased apoptosis that were induced by ATRA. In conclusion, ATRA could inhibit hair follicle growth via inhibiting proliferation and inducing apoptosis of DPCs partially through the TGF-β2/Smad2/3 pathway.

Abstract 6336: Non-canonical activation of Gli-2 by TGF-β/Smad3 signaling pathway in modulation of chemoresistance of hepatocellular carcinoma

Abstract BACKGROUND: It has been demonstrated in our previous study that poorly-differentiated hepatoma cells with low α-feto-protein (AFP) expression and a CD133-/EpCAM- surface profile exhibited aberrant hedgehog signaling (Hh) activity, epithelial-mesenchymal transition and chemoresistance. However, expression of canonical Hh molecules (Sonic ligand and SMO) was not increased in poorly-differentiated hepatoma cells in contrast to well-differentiated hepatoma cells, indicating that Hh transcription factors Gli-1/2 may be activated through a non-classic pathway. The present study aims to investigate whether the TGF-β/Smad3 pathway is responsible for Gli-2 activation through a non-canonical pathway. METHODS: Expression of Sonic Hh (Shh) and Smad3 was assessed in both hepatoma cells and HCC specimens. Gli-2 and Smad3 expression was suppressed by lentiviral vector-mediated RNA interference in poorly-differentiated hepatoma Huh-7-DN and HLE cells. Chemosensitivity was evaluated in hepatoma cells with a Gli-2 or Smad3 inhibitor. RESULTS: Significantly increased Gli-1 and Gli-2 protein levels were observed in 78% (11 out of 14) and 50% (7 out of 14) HCC specimens. In 7 HCC patients with a normal serum AFP value (<20 μg/L), Gli-1/2 protein levels were obviously higher than their pericancerous tissue. Moreover, increased Gli-2 and ABCC1 protein levels were accompanied with increased TGF-β1 and p-Smad3 protein levels in 5 out 7 HCC specimens; whereas Shh protein levels were similar to paired pericancerous tissue. Consistent with the findings from HCC specimens, significantly higher Gli-2 and p-Smad3 nuclear protein levels were associated with increased ABCC1 protein levels in poorly-differentiated hepatoma cells (Huh-7-trans, Huh-7-DN, HLE and HLF); whereas Shh and SMO protein levels were not significantly different from well-differentiated hepatoma Huh-7 and Hep3B cells. Suppressing Gli-2 expression by specific shRNA caused marked decreased expression of Gli-2 and ABCC1 in Huh-7-DN, Huh-7-trans and HLE cells. In addition, expression of Smad3, Gli-2 and ABCC1 was significantly down-regulated in HLE cells after infected with a lentiviral vector harboring shSmad3, indicating that Gli-2 is a downstream target of Smad3. Sorafenib IC50 was significantly reduced from 35.76 to 17.45 μM or 14.04 μM in HLE cells when treated with a GLI inhibitor (GANT-61) or a Smad3 inhibitor (SIS3) at 5 μM. A potential GLI-binding site in the ABCC1 promoter was identified. ABCC1-Gli-luciferase reporter activity was increased up to 39-fold in HLE cells when transfected with constructs containing the potential GLI-binding site, compared to the construct that lacked a GLI-binding site. CONCLUSION: TGF-β/Smad3 activated Gli-2 and its direct transcriptional target ABCC1 in poorly-differentiated and chemoresistant hepatoma cells. Suppressing TGF-β/Smad3/Gli-2 signaling activity confers a potential molecular target in partially alleviating Sorafenib resistance for the treatment of refractory hepatoma. Citation Format: Jia Ding, Hui-yan Li, Li Zhang, Xiao-tian Zhou, Jian Wu. Non-canonical activation of Gli-2 by TGF-β/Smad3 signaling pathway in modulation of chemoresistance of hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6336.

Treatment of Hypertensive Heart Disease by Targeting Smad3 Signaling in Mice

Transforming growth factor β (TGF-β)/Smad3 signaling plays a central role in chronic heart disease. Here, we report that targeting Smad3 with a Smad3 inhibitor SIS3 in an established mouse model of hypertension significantly improved cardiac dysfunctions by preserving the left ventricle (LV) ejection fraction (LVEF) and LV fractional shortening (LVFS), while reducing the LV mass. In addition, SIS3 treatment also halted the progression of myocardial fibrosis by blocking α-smooth muscle actin-positive (α-SMA+) myofibroblasts and collagen matrix accumulation, and inhibited cardiac inflammation by suppressing interleukin (IL)-1β, tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein 1 (MCP1), intercellular cell adhesion molecule-1 (ICAM1) expression, and infiltration of CD3+ T cells and F4/80+ macrophages. Interestingly, treatment with SIS3 did not alter levels of high blood pressure, revealing a blood pressure-independent cardioprotective effect of SIS3. Mechanistically, treatment with SIS3 not only directly inactivated TGF-β/Smad3 signaling but also protected cardiac Smad7 from Smurf2-mediated proteasomal ubiquitin degradation. Because Smad7 functions as an inhibitor for both TGF-β/Smad and nuclear factor κB (NF-κB) signaling, increased cardiac Smad7 could be another mechanism through which SIS3 treatment blocked Smad3-mediated myocardial fibrosis and NF-κB-driven cardiac inflammation. In conclusion, SIS3 is a therapeutic agent for hypertensive heart disease. Results from this study demonstrate that targeting Smad3 signaling with SIS3 may be a novel and effective therapy for chronic heart disease.