TGFß1 induces stress fiber formation through upregulation of TRPC6 in vascular smooth muscle cells.

Aberrant transforming growth factor ß1 (TGFß1) signaling plays a crucial role in the pathogenesis of vascular fibrosis. On the other hand, deregulated transient receptor potential canonical 6 (TRPC6) channel expression shows impaired vascular physiology and wound healing. However, it has little been known about the functional association between TGFß1 and TRPC6 in vascular smooth muscle cells (VSMCs). In this study, we analyzed the microarray data obtained from TGFß1-treated A7r5 VSMCs. We found that TGFß1 specifically elevates the expression level of TRPC6 mainly through Smad-dependent canonical pathway. The siRNA against TRPC6 abolished TGFß1-induced molecular and cellular phenotype changes, including myosin light chain phosphorylation, actin stress fiber formation, and cell migration. These results demonstrate that TRPC6 is an important component of TGFß1 signaling pathway in VSMCs. Therefore, our findings provide a basis for future investigation aimed at developing novel therapeutic strategies for treatment of vascular fibrosis.

ROS and endothelial nitric oxide synthase (eNOS)-dependent trafficking of angiotensin II type 2 receptor begets neuronal NOS in cardiac myocytes.

Angiotensin II (Ang II), a potent precursor of hypertrophy and heart failure, upregulates neuronal nitric oxide synthase (nNOS or NOS1) in the myocardium. Here, we investigate the involvement of type 1 and 2 angiotensin receptors (AT1R and AT2R) and molecular mechanisms mediating Ang II-upregulation of nNOS. Our results showed that pre-treatment of left ventricular (LV) myocytes with antagonists of AT1R or AT2R (losartan, PD123319) and ROS scavengers (apocynin, tiron or PEG-catalase) blocked Ang II-upregulation of nNOS. Surface biotinylation or immunocytochemistry experiments demonstrated that AT1R expression in plasma membrane was progressively decreased (internalization), whereas AT2R was increased (membrane trafficking) by Ang II. Inhibition of AT1R or ROS scavengers prevented Ang II-induced translocation of AT2R to plasma membrane, suggesting an alignment of AT1R-ROS-AT2R. Furthermore, Ang II increased eNOS-Ser(1177) but decreased eNOS-Thr(495), indicating concomitant activation of eNOS. Intriguingly, ROS scavengers but not AT2R antagonist prevented Ang II-activation of eNOS. NOS inhibitor (L-NG-Nitroarginine Methyl Ester, L-NAME) or eNOS gene deletion (eNOS(-/-)) abolished Ang II-induced membrane trafficking of AT2R, nNOS protein expression and activity. Mechanistically, S-nitrosation of AT2R was increased by sodium nitroprusside (SNP), a NO donor. Site-specific mutagenesis analysis reveals that C-terminal cysteine 349 in AT2R is essential in AT2R translocation to plasma membrane. Taken together, we demonstrate, for the first time, that Ang II upregulates nNOS protein expression and activity via AT1R/ROS/eNOS-dependent S-nitrosation and membrane translocation of AT2R. Our results suggest a novel crosstalk between AT1R and AT2R in regulating nNOS via eNOS in the myocardium under pathogenic stimuli.

A network perspective on unraveling the role of TRP channels in biology and disease.

Transient receptor potential (TRP) channels are a large family of non-selective cation channels that mediate numerous physiological and pathophysiological processes; however, still largely unknown are the underlying molecular mechanisms. With data generated on an unprecedented scale, network-based approaches have been revolutionizing the way in which we understand biology and disease, discover disease genes, and develop therapeutic strategies. These circumstances have created opportunities to encounter TRP channel research to data-intensive science. In this review, we provide an introduction of network-based approaches in biomedical science, describe the current state of TRP channel network biology, and discuss the future direction of TRP channel research. Network perspective will facilitate the discovery of latent roles and underlying mechanisms of TRP channels in biology and disease.

Oncogenic role of FOXM1 in human prostate cancer (Review).

Prostate cancer is the leading cause of cancer­related mortality among men worldwide. In particular, castration­resistant prostate cancer presents a formidable clinical challenge and emphasizes the need to develop novel therapeutic strategies. Forkhead box M1 (FOXM1) is a multifaceted transcription factor that is implicated in the acquisition of the multiple cancer hallmark capabilities in prostate cancer cells, including sustaining proliferative signaling, resisting cell death and the activation of invasion and metastasis. Elevated FOXM1 expression is frequently observed in prostate cancer, and in particular, FOXM1 overexpression is closely associated with poor clinical outcomes in patients with prostate cancer. In the present review, recent advances in the understanding of the oncogenic role of deregulated FOXM1 expression in prostate cancer were highlighted. In addition, the molecular mechanisms by which FOXM1 regulates prostate cancer development and progression were described, thereby providing knowledge and a conceptual framework for FOXM1. The present review also provided valuable insight into the inherent challenges associated with translating biomedical knowledge into effective therapeutic strategies for prostate cancer.

Icilin inhibits E2F1-mediated cell cycle regulatory programs in prostate cancer.

Aberrant expression of cell cycle regulators have been implicated in prostate cancer development and progression. Therefore, understanding transcriptional networks controlling the cell cycle remain a challenge in the development of prostate cancer treatment. In this study, we found that icilin, a super-cooling agent, down-regulated the expression of cell cycle signature genes and caused G1 arrest in PC-3 prostate cancer cells. With reverse-engineering and an unbiased interrogation of a prostate cancer-specific regulatory network, master regulator analysis discovered that icilin affected cell cycle-related transcriptional modules and identified E2F1 transcription factor as a target master regulator of icilin. Experimental analyses confirmed that icilin reduced the activity and expression levels of E2F1. These results demonstrated that icilin inactivates a small regulatory module controlling the cell cycle in prostate cancer cells. Our study might provide insight into the development of cell cycle-targeted cancer therapeutics.

Emerging role of E2F8 in human cancer.

E2F8 is a multifaceted transcription factor that plays a crucial role in mediating the hallmarks of cancer, including sustaining proliferative signaling, resisting cell death, and activating invasion and metastasis. Aberrant E2F8 expression is associated with poor clinical outcomes in most human cancers. However, E2F8 also exhibits tumor-suppressing activity; thus, the role of E2F8 in cell-fate determination is unclear. In this review, we highlight the recent progress in understanding the role of E2F8 in human cancers, which will contribute to building a conceptual framework and broadening our knowledge pertaining to E2F8. This review provides insight into future challenges and perspectives regarding the translation of biological knowledge into therapeutic strategies for the treatment of cancer.

Cyclosporin A inhibits prostate cancer growth through suppression of E2F8 transcription factor in a MELK­dependent manner.

The treatment of advanced prostate cancer remains a formidable challenge due to the limited availability of effective treatment options. Therefore, it is imperative to identify promising druggable targets that provide substantial clinical benefits and to develop effective treatment strategies to overcome therapeutic resistance. Cyclosporin A (CsA) showed an anticancer effect on prostate cancer in cultured cell and xenograft models. E2F8 was identified as a master transcription factor that regulated a clinically significant CsA specific gene signature. The expression of E2F8 increased during prostate cancer progression and high levels of E2F8 expression are associated with a poor prognosis in patients with prostate cancer. MELK was identified as a crucial upstream regulator of E2F8 expression through the transcriptional regulatory network and Bayesian network analyses. Knockdown of E2F8 or MELK inhibited cell growth and colony formation in prostate cancer cells. High expression levels of E2F8 and androgen receptor (AR) are associated with a worse prognosis in patients with prostate cancer compared with low levels of both genes. The inhibition of E2F8 improved the response to AR blockade therapy. These results suggested that CsA has potential as an effective anticancer treatment for prostate cancer, while also revealing the oncogenic role of E2F8 and its association with clinical outcomes in prostate cancer. These results provided valuable insight into the development of therapeutic and diagnostic approaches for prostate cancer.

SK&F 96365 induces apoptosis and autophagy by inhibiting Akt-mTOR signaling in A7r5 cells.

SK&F 96365 has been widely used as an inhibitor of transient receptor potential (TRP) calcium channels in various physiological settings. However, growing evidence suggests that SK&F 96365 affects several cellular and molecular processes via uncharacterized off-target mechanisms. In this study, we showed that SK&F 96365 induces apoptosis and autophagy in A7r5 vascular smooth muscle cells. The combined suppression of apoptosis and autophagy provoked necrosis rather than rescued cell death in the cells treated with SK&F 96365. In addition, we found that SK&F 96365 inhibits Akt-mTOR signaling pathways, which is comparable with the efficacy of other known Akt inhibitors. Our findings suggest that SK&F 96365 can be a useful agent for delineating the molecular mechanisms underlying crosstalk among cell death pathways.

Menthol induces cell-cycle arrest in PC-3 cells by down-regulating G2/M genes, including polo-like kinase 1.

Menthol, a naturally occurring monoterpene, is used in foods, cosmetic products, and topical therapeutic preparations. It also exerts cytotoxic activity against several cancer cell types, including prostate cancer cells. However, little is known about the mechanism of menthol action against prostate cancer cells. In this study, we investigated the effect of menthol on the gene expression profile of PC-3 prostate cancer cells using DNA microarray analyses. Gene set enrichment analysis revealed that menthol primarily affects the expression of cell cycle-related genes. Subsequent experimental analyses validated that menthol induces G2/M arrest. Particularly, menthol markedly down-regulated polo-like kinase 1 (PLK1), a key regulator of G2/M phase progression and inhibited its downstream signaling. Our computational analyses and experimental validation provide a basis for future investigations that are aimed at elucidating the action of menthol on cell cycle control in prostate cancer cells.

Distribution and Function of Platelet-derived Growth Factor Receptor Alpha-positive Cells and Purinergic Neurotransmission in the Human Colon: Is It Different Between the Right and Left Colon?

Background/Aims: Platelet-derived growth factor receptor alpha-positive (PDGFRα+) cells function in the purinergic regulation of gastrointestinal motility, and purines are reportedly inhibitory neurotransmitters in the enteric nervous system. We explore the distribution and function of PDGFRα+ cells related to purinergic inhibitory neurotransmission in human right and left colons. Methods: Human colonic segments were prepared with mucosa and submucosa intact, and the circular muscle tension and longitudinal muscle tension were recorded. Purinergic neurotransmitters were administered after recording the regular contractions. Immunohistochemistry was performed on the circular muscle layers. Intracellular recording was performed on the colonic muscular layer. SK3, P2RY1, and PDGFR-α mRNA expression was tested by quantitative real-time polymerase chain reaction (qPCR). Results: Adenosine triphosphate (ATP) treatment significantly decreased the frequency and area under the curve (AUC) of the segmental contraction in right and left colons. Beta-nicotinamide adenine dinucleotide (ß-NAD) decreased the frequency in the right colon and the amplitude, frequency and AUC in the left colon. Apamin significantly increased frequency and AUC in the left colon, and after apamin pretreatment, ATP and ß-NAD did not change segmental contractility. Through intracellular recordings, a resting membrane potential decrease occurred after ATP administration; however, the degree of decrease between the right and left colon was not different. PDGFRα+ cells were distributed evenly in the circular muscle layers of right and left colons. SK3, P2RY1, and PDGFRα expression was not different between the right and left colon. Conclusion: Purines reduce right and left colon contractility similarly, and purinergic inhibitory neurotransmission can be regulated by PDGFRα+ cells in the human colon.

Crizotinib attenuates cancer metastasis by inhibiting TGFß signaling in non-small cell lung cancer cells.

Crizotinib is a clinically approved tyrosine kinase inhibitor for the treatment of patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) harboring EML4-ALK fusion. Crizotinib was originally developed as an inhibitor of MET (HGF receptor), which is involved in the metastatic cascade. However, little is known about whether crizotinib inhibits tumor metastasis in NSCLC cells. In this study, we found that crizotinib suppressed TGFß signaling by blocking Smad phosphorylation in an ALK/MET/RON/ROS1-independent manner in NSCLC cells. Molecular docking and in vitro enzyme activity assays showed that crizotinib directly inhibited the kinase activity of TGFß receptor I through a competitive inhibition mode. Cell tracking, scratch wound, and transwell migration assays showed that crizotinib simultaneously inhibited TGFß- and HGF-mediated NSCLC cell migration and invasion. In addition, in vivo bioluminescence imaging analysis showed that crizotinib suppressed the metastatic capacity of NSCLC cells. Our results demonstrate that crizotinib attenuates cancer metastasis by inhibiting TGFß signaling in NSCLC cells. Therefore, our findings will help to advance our understanding of the anticancer action of crizotinib and provide insight into future clinical investigations.

Transcriptome Analysis of the Anti-TGFß Effect of Schisandra chinensis Fruit Extract and Schisandrin B in A7r5 Vascular Smooth Muscle Cells.

Schisandra chinensis fruit extract (SCE) has been used as a traditional medicine for treating vascular diseases. However, little is known about how SCE and schisandrin B (SchB) affect transcriptional output-a crucial factor for shaping the fibrotic responses of the transforming growth factor ß (TGFß) signaling pathways in in vascular smooth muscle cells (VSMC). In this study, to assess the pharmacological effect of SCE and SchB on TGFß-induced transcriptional output, we performed DNA microarray experiments in A7r5 VSMCs. We found that TGFß induced distinctive changes in the gene expression profile and that these changes were considerably reversed by SCE and SchB. Gene Set Enrichment Analysis (GSEA) with Hallmark signature suggested that SCE or SchB inhibits a range of fibrosis-associated biological processes, including inflammation, cell proliferation and migration. With our VSMC-specific transcriptional interactome network, master regulator analysis identified crucial transcription factors that regulate the expression of SCE- and SchB-effective genes (i.e., TGFß-reactive genes whose expression are reversed by SCE and SchB). Our results provide novel perspective and insight into understanding the pharmacological action of SCE and SchB at the transcriptome level and will support further investigations to develop multitargeted strategies for the treatment of vascular fibrosis.

The conflicting role of E2F1 in prostate cancer: A matter of cell context or interpretational flexibility?

The transcription factor E2F1 plays a crucial role in mediating multiple cancer hallmark capabilities that regulate cell cycle, survival, apoptosis, metabolism, and metastasis. Aberrant activation of E2F1 is closely associated with a poor clinical outcome in various human cancers. However, E2F1 has conflictingly been reported to exert tumor suppressive activity, raising a question as to the nature of its substantive role in the control of cell fate. In this review, we summarize deregulated E2F1 activity and its role in prostate cancer. We highlight the recent advances in understanding the molecular mechanism by which E2F1 regulates the development and progression of prostate cancer, providing insight into how cell context or data interpretation shapes the role of E2F1 in prostate cancer. This review will aid in translating biomedical knowledge into therapeutic strategies for prostate cancer.

Altered expression of fucosylation pathway genes is associated with poor prognosis and tumor metastasis in non­small cell lung cancer.

Fucosylation is a post­translational modification that attaches fucose residues to protein­ or lipid­bound oligosaccharides. Certain fucosylation pathway genes are aberrantly expressed in several types of cancer, including non­small cell lung cancer (NSCLC), and this aberrant expression is associated with poor prognosis in patients with cancer. However, the molecular mechanism by which these fucosylation pathway genes promote tumor progression has not been well­characterized. The present study analyzed public microarray data obtained from NSCLC samples. Multivariate analysis revealed that altered expression of fucosylation pathway genes, including fucosyltransferase 1 (FUT1), FUT2, FUT3, FUT6, FUT8 and GDP­L­fucose synthase (TSTA3), correlated with poor survival in patients with NSCLC. Inhibition of FUTs by 2F­peracetyl­fucose (2F­PAF) suppressed transforming growth factor ß (TGFß)­mediated Smad3 phosphorylation and nuclear translocation in NSCLC cells. In addition, wound­healing and Transwell migration assays demonstrated that 2F­PAF inhibited TGFß­induced NSCLC cell migration and invasion. Furthermore, in vivo bioluminescence imaging analysis revealed that 2F­PAF attenuated the metastatic capacity of NSCLC cells. These results may help characterize the oncogenic role of fucosylation in NSCLC biology and highlight its potential for developing cancer therapeutics.

A splice variant of the C(2)H(2)-type zinc finger protein, ZNF268s, regulates NF-kappaB activation by TNF-alpha.

IkappaB kinase (IKK), the pivotal kinase in signal-dependent activation of nuclear factor-kappaB (NF-kappaB), is composed of multiple protein components, including IKK alpha/beta/gamma core subunits. To investigate the regulation of the IKK complex, we immunoaffinity purified the IKK complex, and by MALDI-TOF mass spectrometry identified a splice variant of zinc finger protein 268 (ZNF268) as a novel IKK-interacting protein. Both the full-length and the spliced form of the ZNF268 protein were detected in a variety of mammalian tissues and cell lines. The genes were cloned and expressed by in vitro transcription/translation. Several deletion derivatives, such as KRAB domain (KRAB) on its own, the KRAB/spacer/4-zinc fingers (zF4), and the spacer/ 4-zinc fingers (zS4), were ectopically expressed in mammalian cells and exhibited had different subcellular locations. The KRAB-containing mutants were restricted to the nucleus, while zS4 was localized in the cytosol. TNF-alpha-induced NF-kappaB activation was examined using these mutants and only zS4 was found to stimulate activation. Collectively, the results indicate that a spliced form of ZNF268 lacking the KRAB domain is located in the cytosol, where it seems to play a role in TNF-alpha-induced NF-kappaB activation by interacting with the IKK complex.

Schisandrol B and schisandrin B inhibit TGFß1-mediated NF-κB activation via a Smad-independent mechanism.

Aberrant transforming growth factor ß1 (TGFß1) signaling plays a pathogenic role in the development of vascular fibrosis. We have reported that Schisandra chinensis fruit extract (SCE), which has been used as a traditional oriental medicine, suppresses TGFß1-mediated phenotypes in vascular smooth muscle cells (VSMCs). However, it is still largely unknown about the pharmacologic effects of SCE on various TGFß1 signaling components. In this study, we found that SCE attenuated TGFß1-induced NF-κB activation and nuclear translocation in VSMCs. Among the five active ingredients of SCE that were examined, schisandrol B (SolB) and schisandrin B (SchB) most potently suppressed TGFß1-mediated NF-κB activation. In addition, SolB and SchB effectively inhibited IKKα/ß activation and IκBα phosphorylation in TGFß1-treated VSMCs. The pharmacologic effects of SolB and SchB on NF-κB activation were independent of the Smad-mediated canonical pathway. Therefore, our study demonstrates that SCE and its active constituents SolB and SchB suppress TGFß1-mediated NF-κB signaling pathway in a Smad-independent mechanism. Our results may help further investigations to develop novel multi-targeted therapeutic strategies that treat or prevent vascular fibrotic diseases.

Endocytosis of KATP Channels Drives Glucose-Stimulated Excitation of Pancreatic ß Cells.

Insulin secretion from pancreatic ß cells in response to high glucose (HG) critically depends on the inhibition of KATP channel activity in HG. It is generally believed that HG-induced effects are mediated by the increase in intracellular ATP, but here, we showed that, in INS-1 cells, endocytosis of KATP channel plays a major role. Upon HG stimulation, resting membrane potential depolarized by 30.6 mV (from -69.2 to -38.6 mV) and KATP conductance decreased by 91% (from 0.243 to 0.022 nS/pF), whereas intracellular ATP was increased by only 47%. HG stimulation induced internalization of KATP channels, causing a significant decrease in surface channel density, and this decrease was completely abolished by inhibiting endocytosis using dynasore, a dynamin inhibitor, or a PKC inhibitor. These drugs profoundly inhibited HG-induced depolarization. Our results suggest that the control of KATP channel surface density plays a greater role than ATP-dependent gating in regulating ß cell excitability.

Role of NADPH oxidase 4 in lipopolysaccharide-induced proinflammatory responses by human aortic endothelial cells.

OBJECTIVE: We investigated the role of NADPH oxidase 4 (Nox4) on lipopolysaccharide (LPS)-induced proinflammatory responses by human aortic endothelial cells (HAECs). METHODS AND RESULTS: Yeast two-hybrid and glutathione-S-transferase pull-down assays indicated that the cytosolic Toll/IL-1R region of Toll-like receptor 4 (TLR4) (amino acids 739-769) is the responsible domain for interaction with the COOH terminal of Nox4 (amino acids 451-530). Consistently, overexpression of the COOH-terminal region of Nox4 inhibited nuclear factor-kappaB activation in response to LPS. Downregulation of Nox4 by transfection of siRNA specific to Nox4 in HAECs resulted in a failure to induce reactive oxygen species (ROS) generation and subsequent expression of intercellular adhesion molecule-1 (ICAM-1) and chemokines such as IL-8 and monocyte chemoattractant protein-1 (MCP-1) in response to LPS. Furthermore, transient transfection of endothelial cells with Nox4 siRNA led to a decrease in migration and adhesion of monocytes in response to LPS by 36% and 52%, respectively. CONCLUSIONS: Nox4 plays a central role in LPS-induced proinflammatory responses by endothelial cells in an ROS-dependent manner.

The antitumor effects of geraniol: Modulation of cancer hallmark pathways (Review).

Geraniol is a dietary monoterpene alcohol that is found in the essential oils of aromatic plants. To date, experimental evidence supports the therapeutic or preventive effects of geraniol on different types of cancer, such as breast, lung, colon, prostate, pancreatic, and hepatic cancer, and has revealed the mechanistic basis for its pharmacological actions. In addition, geraniol sensitizes tumor cells to commonly used chemotherapy agents. Geraniol controls a variety of signaling molecules and pathways that represent tumor hallmarks; these actions of geraniol constrain the ability of tumor cells to acquire adaptive resistance against anticancer drugs. In the present review, we emphasize that geraniol is a promising compound or chemical moiety for the development of a safe and effective multi-targeted anticancer agent. We summarize the current knowledge of the effects of geraniol on target molecules and pathways in cancer cells. Our review provides novel insight into the challenges and perspectives with regard to geraniol research and to its application in future clinical investigation.

Data-driven Analysis of TRP Channels in Cancer: Linking Variation in Gene Expression to Clinical Significance.

BACKGROUND: Experimental evidence has suggested that transient receptor potential (TRP) channels play a crucial role in tumor biology. However, clinical relevance and significance of TRP channels in cancer remain largely unknown. MATERIALS AND METHODS: We applied a data-driven approach to dissect the expression landscape of 27 TRP channel genes in 14 types of human cancer using International Cancer Genome Consortium data. RESULTS: TRPM2 was found overexpressed in most tumors, whereas TRPM3 was broadly down-regulated. TRPV4 and TRPA1 were found up- and down-regulated respectively in a cancer type-specific manner. TRPC4 was found to be closely associated with incidence of head and neck cancer and poor survival of patients with kidney cancer. TRPM8 was identified as a new molecular marker for lung cancer diagnosis and TRPP1 for kidney cancer prognosis. CONCLUSION: Our data-driven approach demonstrates that the variation in the expression of TRP channel genes is manifested across various human cancer types and genes, for certain TRP channels have strong predictive diagnostic and prognostic potential.