Death by histone deacetylase inhibitor quisinostat in tongue squamous cell carcinoma via apoptosis, pyroptosis, and ferroptosis.

Tongue cancer is one of the most common oral malignancies. Quisinostat is a histone deacetylase inhibitor with antitumor activity. The aim of this study was to evaluate the effects of quisinostat on the viability of tongue squamous cell carcinoma (TSCC) cells (CAL-27, TCA-8113) in vitro and in vivo. Cell viability, cell morphological observation, scratch wound-healing assay, transwell migration assay, transmission electron microscope, flow cytometry and cellular reactive oxygen species were assessed in vitro. The results showed that quisinostat can significantly inhibit the viability, growth and migration of TSCC cells. And quisinostat could significantly induce TSCC cells apoptosis, pyroptosis, and ferroptosis. Quisinostat significantly inhibited tumor tissue growth in animal experiments. Up-regulation of the expression of Bax, cleaved-caspase3, caspase-1, p53, phospho-p53 and down-regulated of the expression of caspase-3, Bcl-2, GPX4 in cell lines and tumor tissues of nude mice were observed by Western blotting analysis. Up-regulation of the expression of caspase-1, Bax, cleaved-caspase3, p53 and down-regulated of the expression of ki67, caspase-3, Bcl-2, GPX4 in tumor tissues of nude mice were observed by immunohistochemistry. TUNEL analysis showed that quisinostat could increase the apoptosis rate in the tumor tissues of nude mice. Up-regulation of the expression of p53 and down-regulated expression of GPX4 in cell lines were observed by immunofluorescent staining, and the expression locations of p53 and GPX4 proteins in TSCC cells were observed. Based on these findings, quisinostat may be a potential drug for the treatment of tongue squamous cell carcinoma.

Knockdown of endogenous RNF4 exacerbates ischaemia-induced cardiomyocyte apoptosis in mice.

RNF4, a poly-SUMO-specific E3 ubiquitin ligase, is associated with protein degradation, DNA damage repair and tumour progression. However, the effect of RNF4 in cardiomyocytes remains to be explored. Here, we identified the alteration of RNF4 from ischaemic hearts and oxidative stress-induced apoptotic cardiomyocytes. Upon myocardial infarction (MI) or H2 O2 /ATO treatment, RNF4 increased rapidly and then decreased gradually. PML SUMOylation and PML nuclear body (PML-NB) formation first enhanced and then degraded upon oxidative stress. Reactive oxygen species (ROS) inhibitor was able to attenuate the elevation of RNF4 expression and PML SUMOylation. PML overexpression and RNF4 knockdown by small interfering RNA (siRNA) enhanced PML SUMOylation, promoted p53 recruitment and activation and exacerbated H2 O2 /ATO-induced cardiomyocyte apoptosis which could be partially reversed by knockdown of p53. In vivo, knockdown of endogenous RNF4 via in vivo adeno-associated virus infection deteriorated post-MI structure remodelling including more extensive interstitial fibrosis and severely fractured and disordered structure. Furthermore, knockdown of RNF4 worsened ischaemia-induced cardiac dysfunction of MI models. Our results reveal a novel myocardial apoptosis regulation model that is composed of RNF4, PML and p53. The modulation of these proteins may provide a new approach to tackling cardiac ischaemia.

TGF-ß1-PML SUMOylation-peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) form a positive feedback loop to regulate cardiac fibrosis.

Transforming growth factor-ß (TGF-ß) signaling pathway is involved in fibrosis in most, if not all forms of cardiac diseases. Here, we evaluate a positive feedback signaling the loop of TGF-ß1/promyelocytic leukemia (PML) SUMOylation/Pin1 promoting the cardiac fibrosis. To test this hypothesis, the mice underwent transverse aortic constriction (3 weeks) were developed and the morphological evidence showed obvious interstitial fibrosis with TGF-ß1, Pin1 upregulation, and increase in PML SUMOylation. In neonatal mouse cardiac fibroblasts (NMCFs), we found that exogenous TGF-ß1 induced the upregulation of TGF-ß1 itself in a time- and dose-dependent manner, and also triggered the PML SUMOylation and the formation of PML nuclear bodies (PML-NBs), and consequently recruited Pin1 into nuclear to colocalize with PML. Pharmacological inhibition of TGF-ß signal or Pin1 with LY364947 (3 µM) or Juglone (3 µM), the TGF-ß1-induced PML SUMOylation was reduced significantly with downregulation of the messenger RNA and protein for TGF-ß1 and Pin1. To verify the cellular function of PML by means of gain- or loss-of-function, the positive feedback signaling loop was enhanced or declined, meanwhile, TGF-ß-Smad signaling pathway was activated or weakened, respectively. In summary, we uncovered a novel reciprocal loop of TGF-ß1/PML SUMOylation/Pin1 leading to myocardial fibrosis.

PMLIV overexpression promotes TGF-ß-associated epithelial-mesenchymal transition and migration in MCF-7 cancer cells.

The epithelial-mesenchymal transition (EMT) is a key event associated with metastasis and dissemination in breast tumor pathogenesis. Promyelocytic leukemia (PML) gene produces several isoforms due to alternative splicing; however, the biological function of each specific isoform has yet to be identified. In this study, we report a previously unknown role for PMLIV, the most intensely studied nuclear isoform, in transforming growth factor-ß (TGF-ß) signaling-associated EMT and migration in breast cancer. This study demonstrates that PMLIV overexpression promotes a more aggressive mesenchymal phenotype and increases the migration of MCF-7 cancer cells. This event is associated with activation of the TGF-ß canonical signaling pathway through the induction of Smad2/3 phosphorylation and the translocation of phospho-Smad2/3 to the nucleus. In this study, we report a previously unknown role for PMLIV in TGF-ß signaling-induced regulation of breast cancer-associated EMT and migration. Targeting this pathway may be therapeutically beneficial.

Pharmacological inhibition of SUMO-1 with ginkgolic acid alleviates cardiac fibrosis induced by myocardial infarction in mice.

BACKGROUND AND PURPOSE: Protein modification by small ubiquitin-like modifier (SUMO) plays a critical role in the pathogenesis of heart diseases. The present study was designed to determine whether ginkgolic acid (GA) as a SUMO-1 inhibitor exerts an inhibitory effect on cardiac fibrosis induced by myocardial infarction (MI). EXPERIMENTAL APPROACH: GA was delivered by osmotic pumps in MI mice. Masson staining, electron microscopy (EM) and echocardiography were used to assess cardiac fibrosis, ultrastructure and function. Expression of SUMO-1, PML, TGF-ß1 and Pin1 was measured with Western blot or Real-time PCR. Collagen content, cell viability and myofibroblast transformation were measured in neonatal mouse cardiac fibroblasts (NMCFs). Promyelocytic leukemia (PML) protein was over-expressed by plasmid transfection. KEY RESULTS: GA improved cardiac fibrosis and dysfunction, and decreased SUMO-1 expression in MI mice. GA (>20 µM) inhibited NMCF viability in a dose-dependent manner. Nontoxic GA (10 µM) restrained angiotensin II (Ang II)-induced myofibroblast transformation and collagen production. GA also inhibited expression of TGF-ß1 mRNA and protein in vitro and in vivo. GA suppressed PML SUMOylation and PML nuclear body (PML-NB) organization, and disrupted expression and recruitment of Pin1 (a positive regulator of TGF-ß1 mRNA), whereas over-expression of PML reversed that. CONCLUSIONS AND IMPLICATIONS: Inhibition of SUMO-1 by GA alleviated MI-induced heart dysfunction and fibrosis, and the SUMOylated PML/Pin1/TGF-ß1 pathway is crucial for GA-inhibited cardiac fibrosis.

Manipulating PML SUMOylation via Silencing UBC9 and RNF4 Regulates Cardiac Fibrosis.

The promyelocytic leukemia protein (PML) is essential in the assembly of dynamic subnuclear structures called PML nuclear bodies (PML-NBs), which are involved in regulating diverse cellular functions. However, the possibility of PML being involved in cardiac disease has not been examined. In mice undergoing transverse aortic constriction (TAC) and arsenic trioxide (ATO) injection, transforming growth factor ß1 (TGF-ß1) was upregulated along with dynamic alteration of PML SUMOylation. In cultured neonatal mouse cardiac fibroblasts (NMCFs), ATO, angiotensin II (Ang II), and fetal bovine serum (FBS) significantly triggered PML SUMOylation and the assembly of PML-NBs. Inhibition of SUMOylated PML by silencing UBC9, the unique SUMO E2-conjugating enzyme, reduced the development of cardiac fibrosis and partially improved cardiac function in TAC mice. In contrast, enhancing SUMOylated PML accumulation, by silencing RNF4, a poly-SUMO-specific E3 ubiquitin ligase, accelerated the induction of cardiac fibrosis and promoted cardiac function injury. PML colocalized with Pin1 (a positive regulator for TGF-ß1 mRNA expression in PML-NBs) and increased TGF-ß1 activity. These findings suggest that the UBC9/PML/RNF4 axis plays a critical role as an important SUMO pathway in cardiac fibrosis. Modulating the protein levels of the pathway provides an attractive therapeutic target for the treatment of cardiac fibrosis and heart failure.

Transient Receptor Potential Melastatin 4 (TRPM4) Contributes to High Salt Diet-Mediated Early-Stage Endothelial Injury.

BACKGROUND/AIMS: The present study investigated whether the transient receptor potential melastatin 4 (TRPM4) channel plays a role in high salt diet (HSD)-induced endothelial injuries. METHODS: Western blotting and immunofluorescence were used to examine TRPM4 expression in the mesenteric endothelium of Dahl salt-sensitive (SS) rats fed a HSD. The MTT, TUNEL, and transwell assays were used to evaluate the cell viability, cell apoptosis, and cell migration, respectively, of human umbilical vein endothelial cells (HUVECs). Enzyme-linked immunosorbent assays were used to determine the concentrations of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion protein 1 (VCAM-1), and E-selectin. Carboxy-H2DCFDA, a membrane-permeable reactive oxygen species (ROS)-sensitive fluorescent probe, was used to detect intracellular ROS levels. RESULTS: TRPM4 was mainly expressed near the plasma membrane of mesenteric artery endothelial cells, and its expression level increased in SS hypertensive rats fed a HSD. Its protein expression was significantly upregulated upon treatment with exogenous hydrogen peroxide (H2O2) and aldosterone in cultured HUVECs. Cell viability decreased upon treatment with both agents in a concentration-dependent manner, which could be partially reversed by 9-phenanthrol, a specific TRPM4 inhibitor. Exogenous H2O2 induced apoptosis, enhanced cell migration, and increased the release of adhesion molecules, including ICAM-1, VCAM-1, and E-selectin, all of which were significantly attenuated upon treatment with 9-phenanthrol. Aldosterone and H2O2 induced the accumulation of intracellular ROS, which was significantly inhibited by 9-phenanthrol, suggesting that oxidative stress is one of the mechanisms underlying aldosterone-induced endothelial injury. CONCLUSIONS: Given the fact that oxidative stress and high levels of circulating aldosterone are present in hypertensive patients, we suggest that the upregulation of TRPM4 in the vascular endothelium may be involved in endothelial injuries caused by these stimuli.

MicroRNA-328, a Potential Anti-Fibrotic Target in Cardiac Interstitial Fibrosis.

BACKGROUND/AIMS: Deregulated myocardial fibrosis is associated with a wide spectrum of cardiac conditions, being considered one of the major causes for heart disease. Our study was designed to investigate the role of microRNA-328 (miR-328) in regulating cardiac fibrosis. METHODS: We induced cardiac fibrosis following MI by occlusion of the left coronary artery in C57BL/6 mice. Real-time PCR was employed to evaluate the level of miR-328. Masson's Trichrome stain was used to evaluate the development of fibrosis. Luciferase activity assay was performed to confirm the miRNA's binding site in the TGFßRIII gene. Western blot analysis was used to examine TGFßRIII, p-smad2/3 and TGF-ß1 at protein level. RESULTS: In this study, we found that miR-328 was significantly upregulated in the border zone of infarcted myocardium of wild type (WT) mice; TGFßRIII was downregulated whereas TGF-ß1 was upregulated along with increased cardiac fibrosis. And miR-328 stimulated TGF-ß1 signaling and promoted collagen production in cultured fibroblasts. We further found that the pro-fibrotic effect of miR-328 was mediated by targeting TGFßRIII. Additionally, cardiac fibrosis was significantly reduced in infarcted heart when treated with miR-328 antisense. CONCLUSIONS: These data suggest that miR-328 is a potent pro-fibrotic miRNA and an important determinant of cardiac fibrosis in diseased heart.

let-7e replacement yields potent anti-arrhythmic efficacy via targeting beta 1-adrenergic receptor in rat heart.

Beta-adrenoceptor (ß-AR) exerts critical regulation of cardiac function. MicroRNAs (miRNAs) are potentially involved in a variety of biological and pathological processes. This study aimed to investigate the role of miRNA let-7e in the up-regulation of ß(1) -AR and arrhythmogenesis in acute myocardial infarction (AMI) in rats. ß(1) -AR expression was significantly up-regulated and let-7a, c, d, e and i were markedly down-regulated in the infarcted heart after 6 and 24 hrs myocardial infarction. Forced expression of let-7e suppressed ß(1) -AR expression at the protein level, without affecting ß(1) -AR mRNA level, in neonatal rat ventricular cells (NRVCs). Silencing of let-7e by let-7e antisense inhibitor (AMO-let-7e) enhanced ß(1) -AR expression at the protein level in NRVCs. Administration of the lentivirus vector containing precursor let-7e (len-pre-let-7e) significantly inhibited ß(1) -AR expression in rats, whereas len-AMO-let-7e up-regulated ß(1) -AR relative to the baseline control level, presumably as a result of depression of tonic inhibition of ß(1) -AR by endogenous let-7e. Len-negative control (len-NC) did not produce significant influence on ß(1) -AR expression. Len-pre-let-7e also profoundly reduced the up-regulation of ß(1) -AR induced by AMI and this effect was abolished by len-AMO-let-7e. Importantly, len-pre-let-7e application significantly reduced arrhythmia incidence after AMI in rats and its anti-arrhythmic effect was cancelled by len-AMO-let-7e. Notably, anti-arrhythmic efficacy of len-pre-let-7e was similar to propranolol, a non-selective ß-AR blocker and metoprolol, a selective ß(1) -AR blocker. Down-regulation of let-7e contributes to the adverse increase in ß(1) -AR expression in AMI and let-7e supplement may be a new therapeutic approach for preventing adverse ß(1) -AR up-regulation and treating AMI-induced arrhythmia.

Mild hypoxia-induced cardiomyocyte hypertrophy via up-regulation of HIF-1α-mediated TRPC signalling.

Hypoxia-inducible factor-1 alpha (HIF-1α) is a central transcriptional regulator of hypoxic response. The present study was designed to investigate the role of HIF-1α in mild hypoxia-induced cardiomyocytes hypertrophy and its underlying mechanism. Mild hypoxia (MH, 10% O(2)) caused hypertrophy in cultured neonatal rat cardiac myocytes, which was accompanied with increase of HIF-1α mRNA and accumulation of HIF-1α protein in nuclei. Transient receptor potential canonical (TRPC) channels including TRPC3 and TRPC6, except for TRPC1, were increased, and Ca(2+)-calcineurin signals were also enhanced in a time-dependent manner under MH condition. MH-induced cardiomyocytes hypertrophy, TRPC up-regulation and enhanced Ca(2+)-calcineurin signals were inhibited by an HIF-1α specific blocker, SC205346 (30 µM), whereas promoted by HIF-1α overexpression. Electrophysiological voltage-clamp demonstrated that DAG analogue, OAG (30 µM), induced TRPC current by as much as 170% in neonatal rat cardiomyocytes overexpressing HIF-1α compared to negative control. These results implicate that HIF-1α plays a key role in development of cardiac hypertrophy in responses to hypoxic stress. Its mechanism is associated with up-regulating TRPC3, TRPC6 expression, activating TRPC current and subsequently leading to enhanced Ca(2+)-calcineurin signals.

ß-adrenoceptor regulates miRNA expression in rat heart.

BACKGROUND: MicroRNAs (miRNAs) are noncoding RNAs of 18-25 nucleotides that post-transcriptionally regulate gene expression and are involved in a wide range of physiological and pathological conditions. The ß-adrenergic signaling pathway plays a fundamental role in regulation of heart function. The present study was designed to investigate the expression profile of miRNAs and functional implications under conditions of ß-adrenoceptor activation or inhibition in rat heart. MATERIAL/METHODS: Hemodynamic parameters were measured to assess heart function in Wistar rats treated with isoproterenol (ISO) or propranolol (PRO). miRNA expression was analyzed by miRNA Microarray and confirmed by real-time quantitative reverse transcription PCR (real-time qRT-PCR). RESULTS: Isoproterenol (ISO, a ß-adrenoceptor activator) and propranolol (PRO, a ß-adrenoceptor inhibitor) induced differential miRNA expression profiles. Out of 349 miRNAs measured, 43 were upregulated and nine downregulated in the ISO group, while five miRNAs were upregulated and 28 downregulated in PRO group. Among these altered miRNAs in both PRO and ISO groups, 11 were cardiac abundant and 11 showed opposite profiles between the PRO and ISO groups. The recognized anti-hypertrophic miRNAs miR-1, miR-21 and miR-27b, and the pro-hypertrophic miRNAs miR-22, miR-24, miR-199a, miR-212 and miR-214, were upregulated in the ISO group. In the PRO group, pro-hypertrophic miRNA miR-30c was upregulated, whereas miR-212 was downregulated. CONCLUSIONS: ß-adrenoceptor intervention alters miRNA expression profile, and miRNAs may be involved in the ß-adrenoceptor signaling pathway. Cardiomyocyte hypertrophy is a balanced process between pro-hypertrophic and anti-hypertrophic regulation and involves, at the very least, miRNA participation.

TGFBR3, a potential negative regulator of TGF-ß signaling, protects cardiac fibroblasts from hypoxia-induced apoptosis.

A lot of evidence indicates that cardiac fibroblasts are essential for maintaining the structure and function of heart. The present study examined whether TGFBR3 (transforming growth factor type III receptor, also known as betaglycan) could prevent hypoxia-induced injury in neonatal mice cardiac fibroblasts, if so, its possible molecular targets. MTT, electron microscopy and TUNEL assay were used to identify cell viability and apoptosis in neonatal mice cardiac fibroblasts. Results showed that hypoxia for 24 h markedly reduce cell viability by 49.8 ± 8.9%, largely via apoptosis. However, hypoxia-induced apoptosis in cardiac fibroblasts were almost completely prevented by overexpression of TGFBR3. In the present study, hypoxia also induced TGF-ß1, p-Smad2/3 expression, TGFBR1-TGFBR2 complex formation and collagen production in cardiac fibroblasts, which were attenuated substantially by TGFBR3 overexpression. TGFBR3 also reversed Bax up-regulation, Bcl-2 down-regulation and Caspase-3 activation induced by hypoxia in cardiac fibroblasts. Hypoxia or TGF-ß1 itself triggered an increase of [Ca(2+) ](i) in cardiac fibroblasts, which were both inhibited by TGFBR3 overexpression. Taken together, our results indicate that TGFBR3 may act as a protective factor in apoptotic process of cardiac fibroblasts by negative regulation of TGF-ß signaling and represent a potential therapeutic target for heart remodeling after hypoxia injury.

HYD-PEP06 suppresses hepatocellular carcinoma metastasis, epithelial-mesenchymal transition and cancer stem cell-like properties by inhibiting PI3K/AKT and WNT/ß-catenin signaling activation.

HYD-PEP06, an endostatin-modified polypeptide, has been shown to produce effective anti-colorectal carcinoma effects through inhibiting epithelial-mesenchymal transition (EMT). However, whether HYD-PEP06 has similar suppressive effect on hepatocellular carcinoma (HCC) remained unknown. In this study, HYD-PEP06 inhibited metastasis and EMT but not proliferation in vitro. Cignal finder pathway reporter array and Western blot analysis revealed that HYD-PEP06 suppressed HCCLM3 cell metastasis and EMT by inhibiting the PI3K/AKT pathway. Moreover, HYD-PEP06 exerted anti-metastasis effects in HepG2 cancer stem-like cells (CSCs) via suppressing the WNT/ß-catenin signaling pathway. Finally, in HCCLM3 tumor-bearing BALB/c nu/nu nude mice, HYD-PEP06 substantially suppressed tumor growth, lung metastasis and HCC progress. Our results suggest that HYD-PEP06 inhibits the metastasis and EMT of HCC and CSCs as well, and thus has the potential as an agent for HCC treatment.

Tanshinone IIA inhibits miR-1 expression through p38 MAPK signal pathway in post-infarction rat cardiomyocytes.

Tanshinone IIA is a fat-soluble pharmacologically active ingredient of Danshen, a well-known traditional Chinese medicine used for cardiovascular diseases such as coronary heart disease. Tanshinone IIA has been confirmed to suppress miR-1 and reduce the arrhythmogenesis after myocardial infarction (MI). However, the modulation mechanism is not clear. Tanshinone IIA was administrated daily for 7 days before ligation of the left anterior descending artery (LAD) and lasted for 3 months after LAD. Neonatal cardiomyocytes were exposed to 2% O(2)+95% N(2) condition for 24 h to simulate ischemia in vivo. Protein expression was examined with Western blot and miR-1 level was quantified by Real-time PCR. Our results showed that tanshinone IIA relieved ischemia-induced injury by improving the cardiac function. This beneficial effect may due to the depression of the elevated miR-1 level in ischemic and hypoxic cardiomyocytes, which subsequently restored its target Cx43 protein. Furthermore, tanshinone IIA could inhibit activated p38 MAPK and heart special transcription factors SRF and MEF2, in ischemic and hypoxic cardiomyocytes. Pretreatment with p38 MAPK inhibitor, SB203580 (10 uM), significantly relieved hypoxia-induced miR-1 increment and restored its downstream target Cx43 protein expression. These data suggest that tanshinone IIA play a role in protection cardiomyocytes from ischemic and hypoxic injury. The effect is based on inhibiting miR-1 expression through p38 MAPK signal pathway. This might provide us a new target to explore the novel strategy for ischemic cardioprotection.

Ascorbic Acid Sensitizes Colorectal Carcinoma to the Cytotoxicity of Arsenic Trioxide via Promoting Reactive Oxygen Species-Dependent Apoptosis and Pyroptosis.

Arsenic trioxide (ATO) is an effective therapeutic agent against acute promyelocytic leukemia (APL); however, its anti-tumor effect on solid tumors such as colorectal cancer (CRC) is still in debate. Ascorbic acid (AA) also produces a selective cytotoxic activity against tumor cells. Here, we exploit the potential benefit of ATO/AA combination in generating cytotoxicity to CRC cells, which may lay the groundwork for the potential combinational chemotherapy of CRCs. According to the results, we found that ATO and AA effectively inhibited the viability of human CRC cells in a synergistic manner. AA and ATO corporately activated caspase-3 to trigger apoptosis and upregulated the expression of caspase-1 and promoted formation of inflammasomes to induce pyroptosis. Furthermore, the stimulation of reactive oxygen species (ROS) overproduction was demonstrated as a subcellular mechanism for apoptosis and pyroptosis induced by ATO/AA combination treatment. Our findings suggest that ATO combination with a conventional dosage of AA offers an advantage for killing CRC cells. The synergistic action of ATO/AA combination might be considered a plausible strategy for the treatment of CRC and perhaps other solid tumors as well.

Ginkgolic Acid, a SUMO-1 Inhibitor, Inhibits the Progression of Oral Squamous Cell Carcinoma by Alleviating SUMOylation of SMAD4.

Small ubiquitin-related modifiers (SUMO) represent a class of ubiquitin-like proteins that are conjugated, like ubiquitin, by a set of enzymes to form cellular regulatory proteins, and play key roles in the control of cell proliferation, differentiation, and apoptosis. We found that ginkgolic acid (GA) can significantly reduce cell vitality in a dose- and time-dependent manner and can also accelerate cyto-apoptosis in both Tca8113 and Cal-27 cells. Migration and wound-healing assays were executed to determine the anti-migration effect of GA in oral squamous cell carcinoma (OSCC) cell lines. GA represses transforming growth factor-ß1 (TGF-ß1)-induced epithelial-mesenchymal transition (EMT) markers in OSCC cell lines. This investigation is the first evidence that GA suppresses TGF-ß1-induced SUMOylation of SMAD4. We show that GA affects the phosphorylation of SMAD2/3 protein and the release of SMAD4. In the xenograft mouse model, the OSCC progression was reduced by GA, effectively suppressing the growth of tumors. In addition, siSMAD4 improved cell migration and viability, which was inhibited by GA in Tca8113 cells. GA suppresses tumorigenicity and tumor progression of OSCC through inhibition of TGF-ß1-induced enhancement of SUMOylation of SMAD4. Thus, GA could be a promising therapeutic for OSCC.

Scutellarin-induced endothelium-independent relaxation in rat aorta.

Scutellarin is a flavonoid extracted from the traditional Chinese herb, Erigeron breviscapus Hand Mazz. In the present study, the vasorelaxant effects of scutellarin and the underlying mechanism were investigated in isolated rat aorta. Scutellarin (3, 10, 30, 100 microm) caused a dose-dependent relaxation in both endothelium-intact and endothelium-denuded rat aortic rings precontracted with noradrenaline bitartrate (IC(50) = 7.7 +/- 0.6 microm), but not with potassium chloride. Tetraethylammonium, glibenclamide, atropine, propranolol, indomethacin and N(G)-nitro-l-arginine methyl ester had no influence on the vasorelaxant effect of scutellarin, which further excluded the involvement of potassium channels, muscarinic receptor, nitric oxide pathway and prostaglandin in this effect. Pretreatment with scutellarin decreased the tonic phase, but not the phasic phase of the noradrenaline bitartrate induced tension increment. Scutellarin also alleviated Ca(2+)-induced vasoconstriction in Ca(2+)-depleted/noradrenaline bitartrate pretreated rings in the presence of voltage-dependent calcium channel blocker verapamil. The noradrenaline bitartrate evoked intracellular calcium increase was inhibited by scutellarin. Scutellarin had no effect on phorbol-12,13-diacetate induced contraction in a calcium-free bath solution. These results showed that scutellarin could relax thoracic artery rings in an endothelium-independent manner. The mechanism seems to be the inhibition of extracellular calcium influx independent of the voltage-dependent calcium channel.

PEP06 polypeptide 30 exerts antitumour effect in colorectal carcinoma via inhibiting epithelial-mesenchymal transition.

BACKGROUND AND PURPOSE: PEP06, a polypeptide modified from endostatin, was investigated for its antitumour effects on colorectal cancer (CRC) and the possible mechanisms of this antitumour activity were examined in in vitro and in vivo models. EXPERIMENTAL APPROACH: After PEP06 treatment, cell proliferation and migration assays were performed in CRC cells. Epithelial-mesenchymal transition (EMT) progression was determined by Western blotting, immunofluorescent staining and immunohistochemistry in vitro and in a residual xenograft model. MiRNAs regulated by PEP06 were identified by miRNA microarray and verified by in situ hybridization and quantitative real-time PCR. The interactions between PEP06 and integrin αvß3 were determined with Biacore SA biochips. The cellular function of miR-146b-5p was validated by gain-of-function and loss-of-function approaches. A mouse model of lung metastasis was used to determine the effect of PEP06 on metastatic growth. KEY RESULTS: PEP06 did not affect cell viability but reduced migration and EMT in SW620 and HCT116 cells. PEP06 significantly repressed the expression of miR-146b-5p in these two cell lines through binding to integrin αvß3. MiR-146b-5p was shown to increase EMT by targeting Smad4, and the miR-146b-5p-Smad4 cascade regulated EMT in CRC. PEP06 also suppressed CRC pulmonary metastasis, increased survival of mice and hampered residual tumour growth by inhibiting EMT through down-regulating miR-146b-5p. CONCLUSIONS AND IMPLICATIONS: PEP06 is a polypeptide that inhibits the growth and metastasis of colon cancer through its RGD motif binding to integrin αvß3, thereby down-regulating miR-146b-5p to inhibit EMT in vitro and in vivo. It might have potential as a therapeutic for CRC.

MIAT Is a Pro-fibrotic Long Non-coding RNA Governing Cardiac Fibrosis in Post-infarct Myocardium.

A long non-coding RNA (lncRNA), named myocardial infarction associated transcript (MIAT), has been documented to confer risk of myocardial infarction (MI). The aim of this study is to elucidate the pathophysiological role of MIAT in regulation of cardiac fibrosis. In a mouse model of MI, we found that MIAT was remarkably up-regulated, which was accompanied by cardiac interstitial fibrosis. MIAT up-regulation in MI was accompanied by deregulation of some fibrosis-related regulators: down-regulation of miR-24 and up-regulation of Furin and TGF-ß1. Most notably, knockdown of endogenous MIAT by its siRNA reduced cardiac fibrosis and improved cardiac function and restored the deregulated expression of the fibrosis-related regulators. In cardiac fibroblasts treated with serum or angiotensin II, similar up-regulation of MIAT and down-regulation of miR-24 were consistently observed. These changes promoted fibroblasts proliferation and collagen accumulation, whereas knockdown of MIAT by siRNA or overexpression of miR-24 with its mimic abrogated the fibrogenesis. Our study therefore has identified MIAT as the first pro-fibrotic lncRNA in heart and unraveled the role of MIAT in the pathogenesis of MI. These findings also promise that normalization of MIAT level may prove to be a therapeutic option for the treatment of MI-induced cardiac fibrosis and the associated cardiac dysfunction.

Arsenic trioxide and angiotensin II have inhibitory effects on HERG protein expression: Evidence for the role of PML SUMOylation.

The human ether-a-go-go-related gene (HERG) channel is a novel target for the treatment of drug-induced long QT syndrome, which causes lethal cardiotoxicity. This study is designed to explore the possible role of PML SUMOylation and its associated nuclear bodies (NBs) in the regulation of HERG protein expression. Both arsenic trioxide (ATO) and angiotensin II (Ang II) were able to significantly reduce HERG protein expression, while also increasing PML SUMOylation and accelerating the formation of PML-NBs. Pre-exposure of cardiomyocytes to a SUMOylation chemical inhibitor, ginkgolic acid, or the silencing of UBC9 suppressed PML SUMOylation, subsequently preventing the downregulation of HERG induced by ATO or Ang II. Conversely, knockdown of RNF4 led to a remarkable increase in PML SUMOylation and the function of PML-NBs, further promoting ATO- or Ang II-induced HERG protein downregulation. Mechanistically, an increase in PML SUMOylation by ATO or Ang II dramatically enhanced the formation of PML and Pin1 complexes in PML-NBs, leading to the upregulation of TGF-ß1 protein, eventually inhibiting HERG expression through activation of protein kinase A. The present work uncovered a novel molecular mechanism underlying HERG protein expression and indicated that PML SUMOylation is a critical step in the development of drug-acquired arrhythmia.