Target Genes Cyclin D1 Research Articles

FLCN Regulates HIF2α Nuclear Import and Proliferation of Clear Cell Renal Cell Carcinoma.

Aims and Hypothesis: This study aims to explore the specific molecular mechanism of folliculin (FLCN)-induced proliferation, migration, and invasion in clear cell renal cell carcinoma (ccRCC) and to investigate the relationship of FLCN and HIF2α. Folliculin was identified as a tumor suppressor gene. Its deletions and mutations are associated with a potential risk of renal cancer. At present, the specific molecular mechanism of FLCN-induced proliferation, invasion, and migration in ccRCC remains elusive.Methods: Cell proliferation was measured by flow cytometry analysis, while cell migration and invasion were measured by wound healing assay and Matrigel invasion assay. The expression of FLCN, HIF2α, MMP9, and p-AKT was examined by Western blotting. The cells were transfected with plasmids or siRNA to upregulate or downregulate the expression of FLCN. Immunofluorescence microscopy was carried out to display the HIF2α location. We also determined the correlation of FLCN and HIF2α in human renal cancer samples.Results: FLCN was combined with HIF2α in renal tubular epithelial and cancer cells, and it effectively alleviates the deterioration of renal cancer cells by degrading HIF2α. The silencing of FLCN showed a promotion of HIF2α protein expression via PI3K/mTORC2 pathway, which in turn led to an increase in downstream target genes Cyclin D1 and MMP9. Moreover, interfering with siFLCN advanced the time of HIF2α entry into the nucleus.Conclusions: Our study illustrated that FLCN could be a new therapeutic target in ccRCC. FLCN combined with HIF2α and identified a novel PI3K/mTORC2/HIF2α signaling in ccRCC cells.

Wnt-C59 Attenuates Pressure Overload-Induced Cardiac Hypertrophy via Interruption of Wnt Pathway.

BackgroundCardiac hypertrophy usually results in heart failure and is an important cause of mortality worldwide. Wnt/β-catenin signaling pathway hyper-activation is involved in the pathogenesis and progression of cardiac hypertrophy. Wnt-C59 is a small molecular compound, which strongly and specifically targets at Porcupine to pharmacologically inhibit Wnt palmitoylation, secretion, and other biological activities. However, the role of Wnt-C59 in cardiac hypertrophy remains unknown.Material/MethodsWe performed transverse aortic constriction (TAC) in adult male mice to induce pressure overload and establish an in vivo model of cardiac hypertrophy. Angiotensin II (Ang-II) was utilized to culture cardiomyocyte to establish a model of in vitro cardiomyocyte hypertrophy. Daily administration of Porcupine inhibitor Wnt-C59 was performed for 4 weeks after TAC surgery.ResultsWnt-C59 significantly improved cardiac function and enhanced survival of mice subjected to TAC surgery. Histologically, Wnt-C59 attenuated TAC-induced increase in heart mass, cross-section area of cardiomyocyte, cardiac fibrosis, cardiomyocyte apoptosis, and expression of the hypertrophic biomarkers β-MHC, ANP, and BNP. TAC-induced oxidative stress was also ameliorated by Wnt-C59. Wnt-C59 attenuated Ang-II-induced in vitro cardiomyocyte hypertrophy, as indicated by decreased cell size and lower expression of ANP, BNP, and β-MHC. Moreover, Wnt/β-catenin activation was blocked by Wnt-C59 in cardiac hypertrophy, as indicated by decreased protein expression of Wnt3a and β-catenin and the Wnt target genes cyclin D1 and c-Myc.ConclusionsCollectively, Porcupine inhibitor Wnt-C59 attenuates pressure overload-induced cardiac hypertrophic via interruption of the Wnt/β-catenin signaling pathway, and it might be a promising drug for patients with cardiac hypertrophy.

Polyphyllin I attenuates pressure over-load induced cardiac hypertrophy via inhibition of Wnt/β-catenin signaling pathway

AimsCardiac hypertrophy is one of most important risk factors for cardiovascular mortality. Activation of Wnt/β-catenin signaling pathway is acknowledged to be an important mechanism for pathogenesis of cardiac hypertrophy. Polyphyllin I (PPI), a component in the traditional Chinese medicinal herb, has shown anticancer effect partially via interruption of Wnt/β-catenin signaling pathway. Our aim was to test whether PPI attenuates cardiac hypertrophy. Materials and methodsAdult male C57BL/6J mice were subjected to either pressure overload generated by transverse aortic constriction (TAC) or sham surgery (control group). Angiotensin-II (Ang-II) was used to induce cardiomyocyte hypertrophy in vitro. PPI was intraperitoneally administrated daily for 4 weeks after TAC surgery and then cardiac function was determined by echocardiography and histological analysis was performed. Key findingsPPI significantly ameliorated cardiac dysfunction of mice subjected to TAC. Meanwhile, PPI attenuated TAC induced cardiac hypertrophy indicated by blunted increase in heart mass, cross section area of cardiomyocyte, cardiac fibrosis and expression of hypertrophic biomarkers ANP, BNP and β-MHC. In addition, PPI also ameliorated Ang-II induced cardiomyocyte hypertrophy in vitro. Importantly, PPI decreased protein expression of active β-catenin/total β-catenin, phosphorylation of GSK3β and Wnt target genes c-myc, c-jun, c-fos and cyclin D1 and its anti-hypertrophic effect was blunted by supplementation of Wnt 3a. SignificanceOur results suggest that PPI attenuates cardiac dysfunction and attenuate development of pressure over-load induced cardiac hypertrophic via suppressing Wnt/β-catenin signaling pathway. PPI might be a candidate drug for treatment of cardiac hypertrophy.

The Canonical Wnt Pathway in the Pathogenesis of Cardiac Allograft Vasculopathy

The canonical wnt pathway with its central signaling molecule β-catenin is important for tissue development and homeostasis and its dysregulation leads to many diseases including pathologies of the cardiovascular system. Inhibition of the wnt pathway has the potential to protect against cardiac allograft vasculopathy (CAV), because it reduces the proliferation of smooth muscle cells (SMCs), which is a main pathomechanism of CAV and causes neointima formation and vessel occlusion. For in vitro experiments human (hu) and murine (mu) aortic SMCs were grown in the presence of increasing concentrations of wnt inhibitors, such as XAV-939, ICG-001 or PKF118-310. Cell proliferation was measured as the extent of PrestoBlue Cell Viability Reagent metabolism and expression of representative wnt target genes was assessed by Taqman PCR. Additionally, the wnt inhibitors were used in the mouse model of aortic transplantation (n=8 mice/group) and the extent of neointima formation as an indicator for CAV was analyzed. We also measured the expression of β-catenin (immunofluorescence staining), wnt target genes (Taqman PCR) and several inflammatory cytokines (Taqman PCR) in the transplant. In vitro experiments revealed dose-dependent proliferation inhibition in huSMCs and muSMCs after treatment with each wnt inhibitor. PKF118-310 induced cell death in the highest concentrations tested. While XAV-939 had only minor influence on the expression of the wnt target genes cyclin D1, axin2 and p21, ICG-001 and PKF118-310 induced the expression of p21 and cyclin D1 and reduced the expression of axin2. Interestingly, in vivo experiments showed no effect of XAV-939 and ICG-001 on neointima formation, whereas neointimal amounts of β-catenin were significantly reduced. ICG-001 also reduced the expression of inflammatory cytokines (INFγ, TNFα and IL-6) and of p21, but did not influence cyclin D1 expression. XAV-939 had no relevant influence on cytokine expression. Results for PKF118-310 and wnt target gene expression in transplants after XAV-939 treatment are currently assessed. Strong anti-proliferative effects were found in vitro for all three wnt inhibitors, but this did not lead to reduced CAV development in a mouse model of vessel transplantation. Effects on SMCs themselves after ICG-001 and PKF118-310 seem indeed mediated via regulation of wnt target genes.

Porcupine Inhibitor LGK974 Downregulates the Wnt Signaling Pathway and Inhibits Clear Cell Renal Cell Carcinoma.

Targeted therapy for kidney cancer has achieved significant clinical results. However, because most patients who use targeted therapy will develop drug resistance, we still need to constantly explore new therapeutic targets. Although porcupine (PORCN) as a palmitoyltransferase plays a crucial role in the activation and secretion of Wnt proteins and affects the activity of the Wnt signaling pathway, little is known about the role of PORCN in clear cell renal cell carcinoma (ccRCC). We found that PORCN is highly expressed in renal cancer cell lines and patients with renal cell carcinoma with high expression of PORCN have a poor prognosis. Pathway analysis of PORCN and its related proteins showed that PORCN played a role through the Wnt signaling pathway, and there was a strong coexpression relationship between PORCN and Wnt proteins. Therefore, PORCN may be a potential and effective target for ccRCC. In the present study, we found that LGK974 could inhibit proliferation and colony formation and induce apoptosis in ccRCC cells. We also found that LGK974 could inhibit the migration and invasion of renal cell carcinoma and reduce the expression of mesenchymal markers. After treatment with LGK974, the expression level of β-catenin, a key protein in the classical Wnt pathway, was significantly decreased, and the expression levels of the target genes cyclin D1, c-Myc, MMP9, and MMP2 in the Wnt signaling pathway were also significantly decreased, which represented a significant decrease in the activity of the Wnt signaling pathway. At the same time, the cycle of renal cancer cells was significantly blocked. In conclusion, our results indicate that LGK974 could significantly inhibit the progression of renal cancer cells in a safe concentration range, so PORCN may be a safe and effective target for patients with renal cancer.

β-Elemonic acid inhibits the growth of human Osteosarcoma through endoplasmic reticulum (ER) stress-mediated PERK/eIF2α/ATF4/CHOP activation and Wnt/β-catenin signal suppression

BackgroundOsteosarcoma (OS) is a significant threat to the lives of children and young adults. Although neoadjuvant chemotherapy is the first choice of treatment for OS, it is limited by serious side-effects and cancer metastasis. β-Elemonic acid (β-EA), an active component extracted from Boswellia carterii Birdw., has been reported to exhibit potential anti-inflammatory and anticancer activities. However, the anti-tumor effects and underlying mechanisms on OS as well as pharmacokinetic characteristics of β-EA remain unknown. PurposeThis study was aimed to investigating the anti-tumor effects of β-EA on human OS, the underlying mechanisms, and the pharmacokinetic and tissue distribution characteristics. Study design and methodsCell viability and colony formation assays were performed to determine the effect of β-EA cell on cell proliferation. Apoptosis rates, mitochondrial membrane potential and cell cycle features were analyzed by flow cytometry. qRT-PCR, Western blot, immunofluorescence and immunohistochemical assays were conducted to evaluate the expression levels of genes or proteins related to the pathways affected by β-EA in vitro and in vivo. Cell migration and invasion were evaluated in wound healing and Transwell chamber assays. The effects and pharmacokinetic characteristics of β-EA in vivo were evaluated by analyzing tumor suppression, pharmacokinetics and tissue distribution. ResultsExplorations indicated that endoplasmic reticulum (ER) stress conditions provoked by β-EA activated the PERK/eIF2α/ATF4 branch of the unfolded protein reaction (UPR), stimulating C/EBP homologous protein (CHOP)-regulated apoptosis and inducing Ca2+ leakage leading to caspase-dependent apoptosis. Furthermore, β-EA induced G0/G1 cell cycle arrest and inhibited metastasis of HOS and 143B cells by attenuating Wnt/β-catenin signaling effects, which included decreased levels of p-Akt(Ser473), p-Gsk3β (Ser9), Wnt/β-catenin target genes (c-Myc and CyclinD1) along with a decline in nuclear β-catenin accumulation. The fast absorption, short elimination half-life, and linear pharmacokinetic characteristics of β-EA were also revealed. The distribution of β-EA was detected in the tumor and bone tissues. ConclusionsOverall, both in vitro and in vivo investigations showed the potential of β-EA for the treatment of human OS. The pharmacokinetic profile and considerable distribution in the tumor and bone tissues warrant further preclinical or even clinical studies.

Down-regulation of SLC35C1 induces colon cancer through over-activating Wnt pathway.

The canonical Wnt signalling pathway is a critical pathway involved in the proliferation of cells. It has been well‐established that it plays the central role during colorectal carcinogenesis and development. Yet the exact molecular mechanism of how the canonical Wnt pathway is fine‐tuned remains elusive. We found that SLC35C1, a GDP‐fucose transporter, negatively regulates the Wnt signalling pathway. We show here that SLC35C1 is reduced in all colon cancer by both immunohistochemistry images and TCGA data, whereas β‐catenin is increased. Down‐regulation of SLC35C1 is also detected by real‐time PCR in stage 3 and stage 4 colorectal cancer tissues. Moreover, analysing the TCGA database with cBioPortal reveals the negative correlation of SLC35C1 mRNA level to the expression of β‐catenin. Reduced SLC35C1 significantly promotes cell proliferation and colony formation of HEK293 cells. Meanwhile, in HEK293 cells silencing SLC35C1 activates canonical Wnt pathway, whereas overexpressing SLC35C1 inhibits it. Consistently, the reduction of SLC35C1 in HEK293 cells also elevated the mRNA level of Wnt target genes C‐myc, Axin2 and Cyclin D1, as well as the secretion of Wnt3a. In conclusion, we identified SLC35C1 as a negative regulator of the Wnt signalling pathway in colon cancer. Decreased SLC35C1 may cause over‐activation of Wnt signalling in colorectal cancer.

NFE2L3 promotes malignant behavior and EMT of human hepatocellular carcinoma (HepG2) cells via Wnt/β‑catenin pathway.

Objective: NFE2L3 is a member of the cap 'n' collar basic-region leucine zipper family. NFE2L3 has turned out to be associated with oxidative stress, but the relevance of NFE2L3 in hepatocellular carcinoma (HCC) has remained elusive. This study aimed to investigate the role of NFE2L3 in HCC and explore underlying mechanisms.Methods: Quantitative real-time PCR, western blot and immunohistochemistry were used to detect the mRNA and protein expression of NFE2L3, the expression of epithelial-mesenchymal transition (EMT) markers and Wnt/β-catenin signaling pathway-related proteins. In loss-function experiments, HepG2 cells were transfected with lentiviral vector containing NFE2L3 short hairpin RNA or scramble control. Cell proliferation and migration were measured by Cell Counting Kit-8, Colony formation, EdU incorporation and Transwell assays respectively. Flow cytometry was used to analyze cell cycle and apoptosis. HepG2 cells were subcutaneously injected into nude mice and tumor size was measured once every other day.Results: The results revealed that high expression of NFE2L3 was positively associated with malignant behavior and EMT in HCC. Knockdown of NFE2L3 inhibited cell proliferation and migration, led to cell cycle G0/G1 arrest and induction of cell apoptosis, increased expression of E-cadherin and decreased expression of N‑cadherin, Vimentin, MMP2, CDK2 and PCNA. In addition, tumor growth was inhibited by silencing of NFE2L3 in vivo. Expression of β-catenin and Wnt target genes cyclin D1 and TCF4 was reduced in HepG2-shNFE2L3 cells.Conclusions: NFE2L3 promotes cell proliferation, metastasis, and induces EMT of hepatocellular carcinoma (HepG2) cells via activation of Wnt/β-catenin pathway.

ZNF671 Inhibits the Proliferation and Metastasis of NSCLC via the Wnt/β-Catenin Pathway.

BackgroundLung cancer is the most common cancer in the world and is the main cause of cancer-related death. Revealing the potential mechanism of malignant characteristics of lung cancer is urgent for treating this disease effectively. Zinc finger protein 671 (ZNF671) is a member of the largest transcription factor family in the human genome. The role of ZNF671 in non-small-cell lung cancer (NSCLC) remains unknown. The purpose of this study was to investigate the function and mechanism of ZNF671 in NSCLC.MethodsZNF671 expression in NSCLC cells and tissues were detected by Real-Time PCR, Western blot and TCGA databases. Then, we evaluated the prognostic value of ZNF671 expression in NSCLC using the Kaplan–Meier plotter (KM plotter) and TCGA databases. Moreover, the function of ZNF671 in the proliferation and metastasis of lung cancer was investigated by MTT assay, colony formation assay, in vivo experiment, EdU assay, wound healing assay, transwell assay, and 3D culture assay. Luciferase reporter and subcellular fractionation assays were performed to determine the underlying mechanism of ZNF671-mediated proliferation and metastasis of NSCLC.ResultsZNF671 expression was significantly reduced in both NSCLC cell lines and clinical specimens compared to that in normal controls. The survival analysis results indicated that the downregulation of ZNF671 significantly correlates with poor prognosis and predicts a shorter overall survival and post-progression survival among NSCLC patients. Ectopic overexpression of ZNF671 dramatically restrains, whereas silencing ZNF671 enhanced, cell proliferation and metastasis of NSCLC. Mechanically, gene set enrichment analysis (GSEA) showed that the expression of ZNF671 was significantly correlated with Wnt/β-catenin signaling. Simultaneously, our results confirm that the overexpression of ZNF671 inhibits cell cycle progression and metastasis by weakening the Wnt/β-catenin pathway, and then downregulating the expression of downstream target genes CyclinD1 and MMP9.ConclusionThis study found that the overexpression of ZNF671 restrains the proliferation and metastasis of lung cancer through inhibiting Wnt/β-catenin signaling pathway. Furthermore, our current results provide important insights into ZNF671 as an excellent predictive biomarker for NSCLC, thus providing a novel perspective for the treatment of NSCLC.

The Wnt/β-catenin/VASP positive feedback loop drives cell proliferation and migration in breast cancer.

Previous studies have shown that the main function of VASP is to regulate the cytoskeleton and play an important role in promoting tumor cell metastasis. In this study, we first reveal that VASP is located in the nucleus of breast cancer cells and elucidate a Wnt/β-catenin/VASP positive feedback loop. We identify that VASP is a target gene of Wnt/β-catenin signaling pathway, and activation of Wnt/β-catenin signaling pathway can significantly upregulate VASP protein expression, while upregulated VASP protein can in turn promote translocation of β-catenin and DVL3 proteins into the nucleus. In the nucleus, VASP, DVL3, β-catenin, and TCF4 can form VASP/DVL3/β-catenin/TCF4 protein complex, activating Wnt/β-catenin signaling pathway, and promoting the expression of target genes VASP, c-myc, and cyclin D1. Thus, our study reveals that there is a Wnt/β-catenin/VASP malignant positive feedback loop in breast cancer, which promotes the proliferation and migration of breast cancer cells, and breaking this positive feedback loop may provide new strategy for breast cancer treatment.

Capsaicin suppressed activity of prostate cancer stem cells by inhibition of Wnt/β-catenin pathway.

Cancer stem cells (CSCs) are considered as the origin and driving cells of cancer, and play a key role in the progress of cancer. Studies have shown that capsaicin exerted inhibitory effect on prostate cancer cells, however, the effects of capsaicin on prostate CSCs remain undefined. In the present study, we showed that capsaicin could downregulate prostate CSCs markers and inhibit the growth of PC-3 and DU145 prostate cancer stem cells. Further, we found capsaicin suppressed the expression of Wnt-2, p-GSK3β and β-catenin, along with downregulation of Wnt/β-catenin pathway target genes c-myc and cyclinD1. Using LiCl, a activator of Wnt/β-catenin pathway, we found activation of Wnt/β-catenin pathway could ameliorate the downregulation of prostate CSCs markers and the growth inhibition induced by capsaicin in prostate cancer stem cells. Those data suggested that the inhibition effect of capsaicin on prostate cancer stem cells and the anti-cancer effect of capsaicin on prostate cancer stem cell may be mediated by Wnt/β-catenin pathway. Findings from this study reveals for the first time the potential role and mechanisms of capsaicin on prostate cancer stem cells.

PTPRT epigenetic silencing defines lung cancer with STAT3 activation and can direct STAT3 targeted therapies

ABSTRACT Signal Transducers and Activators of Transcription-3 (STAT3), a potent oncogenic transcription factor, is constitutively activated in lung cancer, but mutations in pathway genes are infrequent. Protein Tyrosine Phosphatase Receptor-T (PTPRT) is an endogenous inhibitor of STAT3 and PTPRT loss-of-function represents one potential mechanism of STAT3 hyperactivation as observed in other malignancies. We determined the role of PTPRT promoter methylation and sensitivity to STAT3 pathway inhibitors in non-small cell lung cancer (NSCLC). TCGA and Pittsburgh lung cancer cohort methylation data revealed hypermethylation of PTPRT associated with diminished mRNA expression in a subset of NSCLC patients. We report frequent hypermethylation of the PTPRT promoter which correlates with transcriptional silencing of PTPRT and increased STAT3 phosphorylation (Y705) as determined by methylation-specific PCR (MSP) and real time quantitative reverse transcription (RT)-PCR in NSCLC cell lines. Silencing of PTPRT using siRNA in H520 lung cancer cell line resulted in increased pSTAT3Tyr705 and upregulation of STAT3 target genes such as Cyclin D1 and Bcl-XL expression. We show this association of PRPRT methylation with upregulation of the STAT3 target genes Cyclin D1 and Bcl-XL in patient derived lung tumour samples. We further demonstrate that PTPRT promoter methylation associated with different levels of pSTAT3Ty705 in lung cancer cell lines had selective sensitivity to STAT3 pathway small molecule inhibitors (SID 864,669 and SID 4,248,543). Our data strongly suggest that silencing of PTPRT by promoter hypermethylation is an important mechanism of STAT3 hyperactivation and targeting STAT3 may be an effective approach for the development of new lung cancer therapeutics.

Growth inhibitory efficacy and anti-aromatase activity of Tabebuia avellanedae in a model for post-menopausal Luminal A breast cancer.

Aromatase inhibitors (AIs) represent a treatment option for post-menopausal estrogen receptor-positive (ER+) breast cancer as monotherapy, or in combination with cyclin-dependent kinase 4/6 or mTOR inhibitors. Long-term treatment with these agents leads to dose-limiting toxicity and drug resistance. Natural substances provide testable alternatives to current therapy. Tabebuia avellanedae (TA) tree is indigenous to the Amazon rainforest. The inner bark of TA represents a medicinal dietary supplement known as Taheebo. Non-fractionated aqueous extract from TA is an effective growth inhibitor in the Luminal A and triple negative breast cancer models. The quinone derivative naphthofurandione (NFD) is a major bioactive agent in TA. The present study examined the efficacy of finely ground powder from the inner bark of TA, available under the name of Taheebo-NFD-Marugoto (TNM). The ER+ MCF-7 cells stably transfected with the aromatase gene MCF-7AROM represented a model for aromatase-expressing post-menopausal breast cancer. Anchorage-independent colony formation, cell cycle progression, pro-apoptotic caspase 3/7 activity, apoptosis-specific gene expression, aromatase activity and select estradiol (E2) target gene expression represented the mechanistic end points. Treatment of MCF-7AROM cells with TNM induced a dose-dependent reduction in E2-promoted anchorage-independent colony number. Mechanistic assays on TNM-treated MCF-7AROM cells demonstrated that TNM at a concentration of 10 µg (NFD content: 2 ng), induced S-phase arrest, increased pro-apoptotic caspase 3/7 activity, increased pro-apoptotic BAX and decreased anti-apoptotic BCL-2 gene expression, and inhibited aromatase activity. Additionally, TNM treatment downregulated ESR-1 (gene for ER-α), aromatase and progesterone gene expression and reduced mRNA levels of E2 target genes pS2, GRB2 and cyclin D1. Inhibition of aromatase activity, based on the NFD content of TNM was superior to the clinical AIs Letrozole and Exemestane. These data demonstrated the potential efficacy of TNM as a nutritional alternative for current therapy of aromatase positive, post-menopausal breast cancer.

Thymoquinone induces apoptosis of human epidermoid carcinoma A431 cells through ROS-mediated suppression of STAT3

Black seed (Nigella sativa) oil has been used in various dermatological applications, and its major constituent, thymoquinone (TQ) has been shown to exhibit antiproliferative activity against various cancer cells. In this study, we tried to provide a mechanistic basis of apoptosis induced by TQ. Skin squamous carcinoma A431 cells were treated with TQ to monitor the apoptosis induced by TQ. Western blot analysis was performed to detect expression of apoptotic or anti-apoptotic proteins. Cell viability and apoptosis were measured by using the MTT test and FACS analysis, respectively. The induction of intracellular reactive oxygen species (ROS) by TQ was evaluated by 2′,7′-dichlorofluorescein diacetate staining. In vivo xenograft study was followed to confirm the antiproliferative effect of TQ. Treatment of A431 cells with TQ-induced apoptosis, which was associated with the induction of p53 and Bax, inhibition of Mdm2, Bcl-2, and Bcl-xl expression, and activation of caspase-9, -7, and -3. TQ inhibited the constitutive phosphorylation and DNA binding activity of signal transducer and activator of transcription-3 (STAT3) in A431 cells by blocking the phosphorylation of the upstream kinase, Src. Moreover, the expression of STAT3 target gene products, cyclin D1 and survivin, was attenuated by TQ treatment. The generation of ROS was increased during TQ-induced apoptosis, and the pretreatment of N-acetyl cysteine, a ROS scavenger, reversed the apoptotic effect of TQ. In vivo study with NOD scid gamma (NSG) mice confirmed the inhibitory effect of TQ on the growth of A431 cells. Our results provide the first demonstration that TQ induces the apoptosis of A431 cells through generation of ROS and inhibition of STAT3 signaling.

Baicalein Inhibits Dextran Sulfate Sodium-induced Mouse Colitis.

BackgroundBaicalein is a bioactive flavone that is originally extracted from the root of Scutellaria baicalensis Georgi. This plant has long served as Chinese herbal medicine in the management of multiple diseases including inflammatory bowel diseases. Although it has been revealed that baicalein inhibits experimental colitis in mice, the molecular mechanisms still remain largely unrecognized.MethodsThe experimental colitis was induced in mice by 3% dextran sulfate sodium (DSS) in drinking water. The mice were given baicalein (10 or 25 mg/kg) by gavage for 7 days before and after DSS administration. Expression of COX-2 and inducible nitric oxide synthase (iNOS) and molecules involved in NF-κB signaling, such as inhibitor of κBα (IκBα), pIκBα, p65, and phospho-p65 was examined by Western blot analysis in the tissue of the mouse colon. Activity of IκB kinase β (IKKβ) was assessed by measuring the relative amount of radioactive γ-phosphate of ATP transferred to the IκBα substrate protein. The expression and phosphorylation of STAT3 and its target gene cyclin D1 were also measured.ResultsBaicalein prominently mitigated the severity of DSS-induced colitis in mice. It inhibited the expression of COX-2 and iNOS. Moreover, baicalein attenuated activity and phosphorylation of IKKβ and subsequent degradation of IκBα. Baicalein suppressed the phosphorylation and nuclear translocation of p65, resulting in a reduced DNA binding activity of NF-κB. Baicalein also suppressed the phosphorylation of STAT3 and expression of cyclin D1. Baicalein exhibited the synergistic effect on inhibition of COX-2 induced by DSS with curcumin, an ingredient of turmeric.ConclusionsProtective effects of baicalein on DSS-induced colitis are associated with suppression of NF-κB and STAT3 signaling pathways, which may contribute to its cancer preventive effects on colon carcinogenesis.

Porphyromonas gingivalis Infection Promoted the Proliferation of Oral Squamous Cell Carcinoma Cells through the miR-21/PDCD4/AP-1 Negative Signaling Pathway.

Recent epidemiological studies have revealed that Porphyromonas gingivalis, a major pathogen in periodontal disease, is associated with the development of oral squamous cell carcinoma (OSCC). However, the underlying mechanisms induced by P.gingivalis have not been well-defined. We aimed to determine the role of P.gingivalis in OSCC proliferation and the relevant molecular mechanisms. A cellular proliferation model of OSCC Tca8113 cells infected by P.gingivalis at a multiplicity of infection (MOI) of 50 was established. Cell proliferation was drastically increased in the infected cells compared with the control cells, while the proportion of cells in S phase was increased and the proportion of cells in G1 phase was decreased in the infected cells compared with the control cells. Additionally, the levels of activator protein 1 (AP-1; c-Jun and c-Fos) and its target gene cyclin D1 were increased in P.gingivalis-infected Tca8113 cells compared with control cells. miR-21 expression was elevated when programmed cell death 4 (PDCD4) expression was downregulated. Cyclin D1 expression was regulated by miR-21, PDCD4, and AP-1. The disruption of the pathway by silencing c-Jun, blocking miR-21 expression, or overexpressing PDCD4 led to decreased cyclin D1 expression and inhibited cell proliferation. P.gingivalis DNA levels were positively correlated with miR-21 and c-Jun expression and negatively correlated with PDCD4 expression in clinical OSCC samples. Our findings indicated that P.gingivalis might promote OSCC proliferation by regulating cyclin D1 expression via the miR-21/PDCD4/AP-1 negative feedback signaling pathway.

Discovery of fluorescent coumarin-benzo[b]thiophene 1, 1-dioxide conjugates as mitochondria-targeting antitumor STAT3 inhibitors

STAT3 has been extensively studied as a potential antitumor target. Though studies on regulating STAT3 mainly focus on the inhibition of STAT3 phosphorylation at Tyr705 residue, the phosphorylation at Ser727 residue of STAT3 protein is also closely associated with the mitochondrial import of STAT3 protein. N, N-diethyl-7-aminocoumarin is a fluorescent mitochondria-targeting probe. In this study, a series of STAT3 inhibitors were developed by connecting N, N-diethyl-7-aminocoumarin fluorophore with benzo [b]thiophene 1, 1-dioxide moiety. All designed compounds displayed potent anti-proliferative activity against cancer cells. The representative compound 7a was mainly accumulated in mitochondria visualized by its fluorescence. STAT3 phosphorylation was inhibited by compound 7a at both Tyr705 and Ser727 residues. Compound 7a inhibited STAT3 phosphorylation whereas had no influence on the phosphorylation levels of STAT1, JAK2, Src and Erk1/2, indicating good selectivity of compound 7a. Moreover, compound 7a down-regulated the expression of STAT3 target genes Bcl-2 and Cyclin D1, increased ROS production and remarkably reduced the mitochondrial membrane potential to induce mitochondrial apoptotic pathway. Furthermore, compound 7ain vivo suppressed breast cancer 4T1 implanted tumor growth. Taken together, these results highlighted that compound 7a might be a promising mitochondria-targeting STAT3 inhibitor for cancer therapy.

MiRNA-451a regulates RPE function through promoting mitochondrial function in proliferative diabetic retinopathy.

The purpose of this study was to explore the role of microRNA-451a (miR-451a) in diabetic retinopathy through activating transcription factor 2 (ATF2). The epiretinal membrane samples from patients with proliferative diabetic retinopathy (PDR) were immunolabeled with an antibody for Ki-67 to identify the proliferative cells. The expression of miR-451a was measured by qRT-PCR in the retina of Akita mice and in RPE cells under diabetic conditions. The potential downstream targets of miR-451a were predicted by bioinformatics and confirmed by dual luciferase assay, qRT-PCR, and Western blotting. Mitochondrial function, cell proliferation, and migration assays were used to detect the functional change after transfection of miR-451a mimic and inhibitor. Proliferative RPE cells were identified in the epiretinal membrane from PDR patients. The expression of miR-451a was downregulated both in the retina of Akita mice and 4-hydroxynonenal (4-HNE)-treated RPE cells. Bioinformatic analysis and luciferase assay identified ATF2 as a potential target of miR-451a. miR-451a inhibited proliferation and migration of RPE cells. The mitochondrial function was enhanced by miR-451a mimic, but suppressed by miR-451a inhibitor. In diabetic conditions, miR-451a showed a protective effect on mitochondrial function. The results of qRT-PCR and Western blotting revealed that overexpression of miR-451a downregulated the expression of ATF2 and its downstream target genes CyclinA1, CyclinD1, and MMP2. In conclusion, miR-451a/ATF2 plays a vital role in the regulation of proliferation and migration in RPE cells through regulation of mitochondrial function, which may provide new perspectives for developing effective therapies for PDR.

Long noncoding RNA H19 promotes vascular remodeling by sponging let-7a to upregulate the expression of cyclin D1

Vascular remodeling is mainly caused by excessive proliferation of vascular smooth muscle cells (VSMCs). Noncoding RNAs (ncRNAs) have emerged as important regulators in diverse pathological processes. Previous work has shown the functions and mechanisms of long noncoding RNA H19 (LncRNA H19) on VSMCs. As long noncoding RNAs (lncRNAs) are complex in their mechanisms of action, the aim of the study is to identify if there are any other molecular mechanisms of LncRNA H19 on VSMCs. In vivo studies demonstrated that cyclin D1 was overexpressed in neointima of balloon-injured artery. In vitro studies identified that the overexpression of LncRNA H19 promoted VSMCs proliferation and cyclin D1 upregulation. On the contrary, cellular proliferation and expression of cyclin D1 were inhibited in VSMCs after infection with let-7a. Furthermore, luciferase reporter assays and RNA pull-down assays were used to explore the regulatory mechanism, we found that LncRNA H19 functioned as a competing endogenous RNA (ceRNA) by sponging let-7a to promote the expression of the target gene cyclin D1. In conclusion, LncRNA H19 positively regulated cyclin D1 expression through directly binding to let-7a in VSMCs. Our findings provide new insight into the mechanism of LncRNA H19 in VSMCs proliferation and vascular remodeling, and further indicate the implications of LncRNA H19 in the diagnosis and treatment of vascular proliferative diseases.

Paris Saponin II inhibits colorectal carcinogenesis by regulating mitochondrial fission and NF-κB pathway.

Colorectal cancer (CRC) is one of the leading causes of cancer-related morbidity and mortality worldwide. Accumulating evidence suggests that mitochondrial dynamics are closely implicated in carcinogenesis including CRC. Paris Saponin II (PSII), a major steroidal saponin extracted from Rhizoma Paris polyphylla, has emerged as a potential anticancer agent. However, the effects of PSII on CRC and its underlying mechanisms remain unknown. In the present study, we found PSII induced apoptosis and inhibited colony formation in HT 29 and HCT 116 cells, and cell cycle arrest in G1 phase. PSII inhibited the phosphorylation of ERK1/2 and mitochondrial translocation of dynamin-related protein 1 (Drp1) by dephosphorylating Drp1 at Ser616, leading to the suppression of mitochondrial fission. PSII also suppressed NF-κB activation as a result of the inhibition of IKKβ and p65 translocation. Drp1 knockdown remarkably downregulated the nuclear expression of p65 and its target genes cyclin D1 and c-Myc in HCT 116 cell, confirming the link between mitochondrial fission and NF-κB pathway. Silencing of Drp 1 enhanced the inhibitory effects of PSII on p65 phosphorylation and the expressions of cyclin D1 and c-Myc, revealing that the inhibitory effects of PSII on cyclin D1 and c-Myc were relevant in the suppression of Drp1 and NF-κB activation. An in vivo study demonstrated PSII remarkably decreased the xenograft tumor size and suppressed the phosphorylation of ERK1/2 and Drp1 at Ser616. Taken together, our results suggested that PSII could inhibit colorectal carcinogenesis, at least in part, by regulating mitochondrial fission and NF-κB pathway.