Qian Yang Yu Yin Granule protects against hypertension-induced renal injury by epigenetic mechanism linked to Nicotinamide N-Methyltransferase (NNMT) expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Qian Yang Yu Yin Granule (QYYY) is a Chinese herbal formulation. It is used to treat hypertensive nephropathy for decades in China, but it is unknown that the exact mechanism of QYYY on hypertensive nephropathy. AIMS OF STUDY: The present study was to elucidate its epigenetic mechanism of QYYY on hypertensive nephropathy. MATERIALS AND METHODS: In the current study, HEK293T cells' proliferation induced by Ang II was chosen to observe epigenetic mechanisms of QYYY on renal damage. The cell proliferation was examined by MTT assays and ethynyldeoxyuridine analysis. Cell cycle analysis was performed. After treatment with QYYY, expression of Nicotinamide N-methyltransferase (NNMT), sirtuin1(SIRT1), S-adenosylhomocysteine(SAH), histone H3K4 methylation, and cortactin acetylation(acetyl-cortactin,ac-cortactin) were further investigated by western-blotting and real time PCR. DNA methylation was detected by ELISA. The study also observed the changes of SIRT1, SAH, H3K4 methylation, acetyl-cortactin when NNMT over-expressed by lentivirus transfection. Angiotensin II(Ang II) induced renal damage in spontaneously hypertensive rats(SHR). After eight weeks treatment of QYYY, blood pressure, serum and urine creatinine, and urinary microalbumin(mAlb) were assessed. The concentration of N1 -methylnicotinamide were detected by liquid chromatography with tandem mass spectrometry. The protein of NNMT, ac-cortactin, H3K3me3 were also assessed in vivo. RESULTS: QYYY inhibited HEK293T cells' proliferation, down-regulated the expression of NNMT, SAH, acetyl-cortactin and DNA methylation, up-regulated the expression of SIRT1, histone H3K4 trimethylation(H3K4me3). Over-expression of NNMT increased the expression of SAH and acetyl-cortactin, and reduced the expression of SIRT1 and H3K4me3. The study also demonstrated that QYYY promoted urinary creatinine excretion and reduced serum creatinine and urinary mAlb in SHR. QYYY decreased the concentration of N1 -methylnicotinamide in Ang II group. QYYY decreased the protein of NNMT, ac-cortactin and increased H3K4me3 in vivo. CONCLUSION: The results showed that QYYY alleviated renal impairment of SHR and inhibited HEK293T cells' proliferation induced by Ang II through the pathway of epigenetic mechanism linked to Nicotinamide N-Methyltransferase (NNMT) expression, including histone methylation, DNA methylation and acetyl-cortactin. This study unveiled a novel molecular mechanism by which QYYY controlled the progression of hypertensive nephropathy.

Cortactin is a prognostic marker for oral squamous cell carcinoma and its overexpression is involved in oral carcinogenesis.

EMS1 (chromosome eleven, band q13, mammary tumor and squamous cell carcinoma-associated gene 1) gene amplification and the concomitant cortactin overexpression have been reported to associate with poor prognosis and tumor metastasis. In this study, we examined cortactin expression by immunohistochemistry in human oral tumors and murine tongue tumors which were induced by the carcinogen 4-nitroquinoline 1-oxide (4-NQO). The immunostaining results show over- to moderate expression of cortactin in 85% (104/122) of oral squamous cell carcinoma (OSCC) tissues and in all 15 leukoplakia tissues examined. Further, statistical analysis indicates that cortactin overexpression appears to be a predictor for shorter survival and poorer prognosis in OSCC patients. In an animal model, cortactin is shown to upregulate in infiltrating squamous cell carcinoma, papilloma, and epithelia with squamous hyperplasia, indicating that cortactin induction is an early event during oral carcinogenesis. It is suggested that cortactin expression is mediated in the progression of pre-malignancy to papilloma, based on earlier cortactin induction in pre-malignancy preceding cyclin D1 in papilloma. In conclusion, cortactin overexpression is frequently observed in human OSCC and mouse tongue tumors. Thus, cortactin may have an important role in the development of oral tumors in human and mice. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 799-812, 2017.

Chansu inhibits the expression of cortactin in colon cancer cell lines in vitro and in vivo.

BACKGROUND: Chansu is a transitional Chinese medicine that has been used for centuries as therapy for inflammation, anaesthesia and arrhythmia in China and other Asian countries. Recently, it has also been used for anti-cancer purposes. We have previously shown that Chansu has a huge pro-apoptotic potential on colon cancer cells, but to date the detailed mechanism of this action is not well understood. METHODS: One of the major components of Chansu, Cinobufagin (CBF) was used to treat cancer cells. The expressions of levels of cortactin, an important factor in tumour progression and cancer invasion, were assessed in in vitro and in vivo experiments. Additional analyses were performed in subcellular protein fractions and immune-fluorescent staining was used to define cortactin protein expression and the changes of location in CBF-treated cells. RESULTS: CBF strongly inhibited the expression of cortactin in HCT116 cells. There were reductions of both mRNA transcription and protein synthesis, which were more significant in the absence of oxygen in vitro. In addition, nuclear translocation of cortactin was observed in HCT116 cells post CBF exposure but not in the negative control, indicating that CBF is likely to interrupt co-localisation of cortactin to cytoskeletal proteins. Most importantly, CBF could diminish the expression of cortactin in human HCT116 xenograft tumours in nude mouse in vivo. CONCLUSIONS: CBF inhibits cortactin expression and nuclear translocation in colon cancer cells in vitro and in mouse models bearing human colon tumour in vivo, suggesting it might disrupt actin-regulated cell movement. Thus, CBF or Chansu could be developed as an effective anti-cancer therapy to stop local invasion and metastasis.

Denbinobin, a phenanthrene from Dendrobium nobile, impairs prostate cancer migration by inhibiting Rac1 activity.

Prostate cancer is the most prevalent type of cancer in the United States. The most common site of prostate cancer metastasis is bone. CXCL12 is preferentially expressed in bone and is targeted by prostate cancer cells, which over-express the receptor for CXCL12, CXCR4. In response to CXCL12 stimulation, Rac1, a GTPase, along with its effectors, regulates actin polymerization to form lamellipodia, which is a critical event for cell migration. Cortactin, an actin-binding protein, is recruited to the lamellipodia and is phosphorylated at tyrosine residues. The phosphorylated cortactin is also involved in cell migration. The inhibition of Rac1 activity using a dominant negative Rac1 impairs lamellipodial protrusion as well as cortactin translocation and cortactin phosphorylation. Denbinobin, a substance extracted from Dendrobium nobile, has anticancer effects in many cancer cell lines. Whether denbinobin can inhibit prostate cancer cell migration is not clear. Here, we report that denbinobin inhibited Rac1 activity. The inhibition of Rac1 activity prevented lamellipodial formation. Cortactin phosphorylation and translocation to the lamellipodia were also impaired, and PC3 cells were unable to migrate. These results indicate that denbinobin prevents CXCL12-induced PC3 cell migration by inhibiting Rac1 activity.

Cortactin tyrosine phosphorylation promotes its deacetylation and inhibits cell spreading.

BACKGROUND: Cortactin is a classical Src kinase substrate that participates in actin cytoskeletal dynamics by activating the Arp2/3 complex and interacting with other regulatory proteins, including FAK. Cortactin has various domains that may contribute to the assembly of different protein platforms to achieve process specificity. Though the protein is known to be regulated by post-translational modifications such as phosphorylation and acetylation, how tyrosine phosphorylation regulates cortactin activity is poorly understood. Since the basal level of tyrosine phosphorylation is low, this question must be studied using stimulated cell cultures, which are physiologically relevant but unreliable and difficult to work with. In fact, their unreliability may be the cause of some contradictory findings about the dynamics of tyrosine phosphorylation of cortactin in different processes. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we try to overcome these problems by using a Functional Interaction Trap (FIT) system, which involves cotransfecting cells with a kinase (Src) and a target protein (cortactin), both of which are fused to complementary leucine-zipper domains. The FIT system allowed us to control precisely the tyrosine phosphorylation of cortactin and explore its relationship with cortactin acetylation. CONCLUSIONS/SIGNIFICANCE: Using this system, we provide definitive evidence that a competition exists between acetylation and tyrosine phosphorylation of cortactin and that phosphorylation inhibits cell spreading. We confirmed the results from the FIT system by examining endogenous cortactin in different cell types. Furthermore, we demonstrate that cell spreading promotes the association of cortactin and FAK and that tyrosine phosphorylation of cortactin disrupts this interaction, which may explain how it inhibits cell spreading.

A novel approach to prevent endothelial hyperpermeability: the Crataegus extract WS® 1442 targets the cAMP/Rap1 pathway.

Endothelial hyperpermeability followed by edema formation is a hallmark of many severe disorders. Effective drugs directly targeting endothelial barrier function are widely lacking. We hypothesized that the hawthorn (Crataegus spp.) extract WS® 1442, a proven multi-component drug against moderate forms of heart failure, would prevent vascular leakage by affecting endothelial barrier-regulating systems. In vivo, WS® 1442 inhibited the histamine-evoked extravasation of FITC-dextran from mouse cremaster muscle venules. In cultured human endothelial cells, WS® 1442 blocked the thrombin-induced FITC-dextran permeability. By applying biochemical and microscopic techniques, we revealed that WS® 1442 abrogates detrimental effects of thrombin on adherens junctions (vascular endothelial-cadherin), the F-actin cytoskeleton, and the contractile apparatus (myosin light chain). Mechanistically, WS® 1442 inhibited the thrombin-induced rise of intracellular calcium (ratiometric measurement), followed by an inactivation of PKC and RhoA (pulldown assay). Moreover, WS® 1442 increased endothelial cAMP levels (ELISA), which consequently activated PKA and Rap1 (pulldown assay). Utilizing pharmacological inhibitors or siRNA, we found that PKA is not involved in barrier protection, whereas Epac1, Rap1, and Rac1 play a crucial role in the WS® 1442-induced activation of cortactin, which triggers a strong cortical actin rearrangement. In summary, WS® 1442 effectively protects against endothelial barrier dysfunction in vitro and in vivo. It specifically interacts with endothelial permeability-regulating systems by blocking the Ca(2+)/PKC/RhoA and activating the cAMP/Epac1/Rap1 pathway. As a proven safe herbal drug, WS® 1442 opens a novel pharmacological approach to treat hyperpermeability-associated diseases. This in-depth mechanistic work contributes to a better acceptance of this herbal remedy.