Rational Exploration of Novel SDHI Fungicide through an Amide-ß-ketonitrile Bioisosteric Replacement Strategy.

The identification of succinate dehydrogenase inhibitor (SDHI) fungicides bearing a novel scaffold is of great importance to control pathogenic fungi. Difluoromethyl-pyrazole ß-ketonitrile derivatives were rationally designed through an innovative amide-ß-ketonitrile bioisosteric replacement strategy and evaluated for their antifungal activities. In preliminary fungicidal screening, our new ß-ketonitrile compounds showed outstanding in vitro activity. Compounds A7 and A14 exhibited EC50 values of 0.116 and 0.165 µg/mL against Sclerotinia sclerotiorum, respectively, and A14 also displayed an EC50 of 0.0774 µg/mL against Rhizoctonia solani. Furthermore, A14 exhibited moderate in vivo protective activity against rice sheath blight on rice plants. Results from SDH enzymatic assays demonstrated that A14 possesses significant inhibitory effect toward porcine heart SDH, with an IC50 value of 0.183 µM, which was 20-fold more potent than that of fluxapyroxad (IC50 = 3.76 µM). A docking study indicated that H-bonds, cation-π interactions, and edge-to-face π-π interactions play key roles in the binding of A14 with R. solani SDH. The CoMSIA model guided the approach to further structural optimizations and indicated that hydrophobic and steric substituents on the benzene ring have decisive effects on the fungicidal activity against R. solani. The present work describes for the first time the successful bioisosteric replacement of the common SDHI amide moiety by a ß-ketonitrile group and highlights the potential of ß-ketonitriles as an innovative novel SDHI subclass.

Reversible Regulation of Succinate Dehydrogenase by Tools of Photopharmacology.

Succinate dehydrogenase (SDH) is extremely important in metabolic function and biological processes. Modulation of SDH has been reported to be a promising therapeutic target to SDH mutations. Current measures for the regulation of SDH are scarce, and precise and reversible modulation of SDH still remains challenging. Herein, a powerful tool for reversible optical control of SDH was proposed and evaluated utilizing the technology of photopharmacology. We reported photochromic ligands (PCLs), azobenzene-pyrazole amides (APAs), that exert light-dependent inhibition effects on SDH. Physicochemical property tests and biological assays were conducted to demonstrate the feasibility of modulating SDH. In this paper, common agricultural pathogens were used to develop a procedure by which our PCLs could reversibly and precisely control SDH utilizing green light. This research would help us to understand the target-ligand interactions and provide new insights into modulation of SDH.

Azo-incorporating Increases Inhibitory Activity of Succinate Dehydrogenase.

Diversity of pesticide discovery provided a solution to resistance. Here, we presented a strategy of azo-incorporating to promote the diverse developments of fungicide. A series of novel fungicides were synthesized by incorporating azobenzene derivatives into fluxapyroxad. Much better in vitro fungicidal activity increases for compound 9d were observed compared to the positive control, fluxapyroxad against Botrytis cinerea and Rhizoctonia solani. Compound 9d (IC50 = 0.03 µM) also had a great enzyme-inhibiting activity increase toward succinate dehydrogenase in comparison with fluxapyroxad (IC50 = 4.40 µM). A comparatively equivalent biological activity was observed between compounds 8a and 9d. SEM analysis helped us to observe clearly the morphology of the fungi before and after active ingredient delivery. Our results of molecular docking analysis, fluorescence quenching analysis, and enzymatic assays demonstrated that compound 8a and 9d act on SDH. An increase in inhibitory activity could be occurring after incorporation of azobenzene, which provided a new strategy for molecular design in pesticide discovery.

A novel ETV6-miR-429-CRKL regulatory circuitry contributes to aggressiveness of hepatocellular carcinoma.

BACKGROUND: Tumor metastasis is one of the main causes of the high mortality of hepatocellular carcinoma (HCC). E-Twenty Six variant gene 6 (ETV6) is a strong transcriptional repressor, associated with the development and progression of tumors. However, the exact role and underlying mechanism of ETV6 in HCC remain unclear. METHODS: Western blotting, quantitative real-time PCR and immunohistochemistry were used to detect the expression levels of ETV6, CRKL (v-crk sarcoma virus CT10 oncogene homologue (avian)-like) and miR-429 in HCC tissues and cells; Transwell chamber and F-actin cytoskeleton staining assay to examine the effects of ETV6 and CRKL deregulation on the migration, invasion and cytoskeleton of HCC cells; Co-immunoprecipitation assay to determine the interaction between CRKL and ETV6; Chromatin immunoprecipitation assay to investigate the interaction between ETV6 and miR-429. RESULTS: We established a novel ETV6-miR-429-CRKL regulatory circuitry contributes to HCC metastasis. ETV6 and CRKL were frequently increased, while miR-429 was downregulated in both hepatocarcinoma tissues and hepatocarcinoma cells. Moreover, ETV6 upregulation was positively correlated with CRKL upregulation, and two negative correlations were also established for ETV6 and CRKL upregulation with miR-429 downregulation in both hepatocarcinoma patients' tumorous tissues and hepatocarcinoma cells. Functional investigations revealed that overexpression and knockdown of ETV6 was remarkably effective in promoting and suppressing HCC cell migration, invasion, cytoskeleton F-actin expression and arrangement, whereas, CRKL overexpression exhibited similar effects to the overexpression of ETV6. Mechanistically, ETV6 negatively regulates miR-429 expression by directly binding to the promoter region of miR-429; miR-429 negatively regulates CRKL expression by selectively targeting CRKL-3'-UTR; ETV6 directly binds to CRKL and positively regulates its expression, which in turn CRKL positively regulates ETV6 expression. CONCLUSIONS: Our data demonstrated that ETV6 promotes migration and invasion of HCC cells by directly binding to promoter region of miR-429 via modulating CRKL expression. The newly identified ETV6-miR-429-CRKL regulatory circuitry contributes to the aggressiveness of HCC, which provides new clues for fundamental research on diagnosis and treatment parameters for HCC.

Annexin A5 regulates hepatocarcinoma malignancy via CRKI/II-DOCK180-RAC1 integrin and MEK-ERK pathways.

As a calcium-dependent phospholipid binding annexin protein, annexin A5 (Anxa5) links to the progression, metastasis, survival, and prognosis of a variety of cancers. Current work showed ANXA5 overexpression was positively correlated with the upregulations of CRKI/II and RAC1 in hepatocarcinoma (HCC) patients' tissues, which potentially enhanced the clinical progression and lymphatic metastasis of HCC. The role and action mechanism of ANXA5 in hepatocarcinoma was then investigated using a hepatocarcinoma Hca-P cell line, an ideal and well-established murine cell model with 100% inducible tumorigenicity of implanted mice with low (~25%) lymph node metastatic (LNM) rate. In vitro evidences indicated ANXA5 stable knockdown resulted in decreased proliferation, migration, invasion and adhesion to lymph node (LN), and increased intercellular cohesion behaviors of hepatocarcinoma Hca-P cells. Consistently, stable ANXA5 knockdown led to reduced in vivo tumorigenicity and malignancy, LNM rate and level potentials of Hca-P- transplanted mice via inhibiting CD34 and VEGF3. The levels of CRKI/II and RAC1 were reduced in tumor tissues from mice transplanted with Hca-P cells with stable ANXA5 knockdown. Molecular action investigation further showed ANXA5 downregulation apparently suppressed the expressions of molecules CRKI/II, DOCK180, RAC1 in integrin pathway, p-MEK, p-ERK, c-Myc, and MMP-9 in MEK- ERK pathway together with VIMINTIN in Hca-P cells in appropriate to knockdown extent. Collectively, Anxa5 was able to mediate HCC carcinogenesis via integrin and MEK-ERK pathways. It is of potential use in the research and treatment of HCC.

Anxa5 mediates the in vitro malignant behaviours of murine hepatocarcinoma Hca-F cells with high lymph node metastasis potential preferentially via ERK2/p-ERK2/c-Jun/p-c-Jun(Ser73) and E-cadherin.

OBJECTIVE: Annexin A5 (Anxa5) is associated with the progression of some cancers, while its role and regulation mechanism in tumor lymphatic metastasis is rarely reported. This study aims to investigate the influence of Anxa5 knockdown on the malignant behaviours of murine hepatocarcinoma Hca-F cell line with high lymph node metastatic (LNM) potential and the underlying regulation mechanism. METHODS: RNA interfering was performed to silence Anxa5 in Hca-F. Monoclonal shRNA-Anxa5- Hca-F cells were obtained via G418 screening by limited dilution method. Quantitative real-time RT-PCR (qRT-PCR) and Western blotting (WB) were applied to measure Anxa5 expression levels. CCK-8, Boyden transwell-chamber and in situ LN adhesion assays were performed to explore the effects of Anxa5 on the proliferation, migration, invasion and adhesion capacities of Hca-F. WB and qRT-PCR were used to detect the level changes of key molecules in corresponding signal pathways. RESULTS: We obtained two monoclonal shRNA-Anxa5-transfected Hca-F cell lines with stable knockdowns of Anxa5. Anxa5 knockdown resulted in significantly reduced proliferation, migration, invasion and in situ LN adhesion potentials of Hca-F in proportion to its knockdown extent. Anxa5 downregulation enhanced E-cadherin levels in Hca-F. Moreover, Anxa5 affected Hca-F behaviours specifically via ERK2/p-ERK2/c-Jun/p-c-Jun(Ser73) instead of p38MAPK/c-Jun, Jnk/c-Jun and AKT/c-Jun pathways. CONCLUSIONS: Anxa5 mediates the in vitro malignant behaviours of murine hepatocarcinoma Hca-F cells via ERK2/c-Jun/p-c-Jun(Ser73) and ERK2/E-cadherin pathways. It is an important molecule in metastasis (especially LNM) and a potential therapeutic target for hepatocarcinoma.

ANXA5 level is linked to in vitro and in vivo tumor malignancy and lymphatic metastasis of murine hepatocarcinoma cell.

AIM: To investigate ANXA5 overexpression on in vitro and in vivo malignancies of murine Hca-P cells. MATERIALS & METHODS: Hca-P with low lymph node metastasis (LNM) potential was used as cell model. TEM, CCK-8 and Boyden transwell assays were performed for in vitro Hca-P behaviors. Hca-P-transplanted mouse model was established for in vivo experiment. RESULTS: ANXA5-overexpressing monoclonal Anxa5-Hca-P-1, Anxa5-Hca-P-2 and Anxa5-Hca-P-3 cells were obtained. ANXA5 upregulation alters the proliferation, morphology and rough endoplasmic reticulum of Hca-P cells, enhances in vitro migration and invasions of Hca-P, promotes in vivo malignant degree and LNM rate of Anxa5-Hca-P-3-transplanted mice. CONCLUSION: As a potential indicator for malignancy and lymphatic metastasis, ANXA5 overexpression increases in vitro migration and invasion of Hca-P cell, promotes in vivo malignancy, LNM rate and level of Hca-P-transplanted mice.

CRKL knockdown promotes in vitro proliferation, migration and invasion, in vivo tumor malignancy and lymph node metastasis of murine hepatocarcinoma Hca-P cells.

Our previous study (Biomed Pharmacother 2015;69:11) demonstrated that the over-expression of CRKL, a chicken tumor virus number 10 regulator of kinase-like protein, suppresses in vitro proliferation, invasion and migration of murine hepatocarcinoma Hca-P cell, a murine HCC cell with lymph node metastatic (LNM) rate of ∼25%. In current work, we investigated the effects of CRKL knockdown on the in vitro cell proliferation, migration and invasion, and on the in vivo tumor malignancy and LNM rate and level for Hca-P cells. Western blotting assay indicated that CRKL was down-regulated by ∼90% in a monoclonal CrkL-shRNA-transfected Hca-P cells. Compared with Hca-P and unrelated-shRNA-transfected Hca-P cell, the in vitro proliferation, migration and invasion potentials were significantly enhanced following CRKL stable deregulation. CRKL knock-down significantly promoted the tumorigenicity malignancy, LNM rates and level of Hca-P-transplanted mice. Consistent with our previous work, it can be concluded CRKL plays an important role in hepatocarcinoma cell proliferation, invasion and migration as well hepatocarcinoma malignancy and metastasis. It functions as a potential tumor suppressor in hepatocarcinoma.

Experimental observation of classical subwavelength interference with a pseudothermal light source.

We report the experimental observation of classical subwavelength double slit interference with a pseudothermal light source. The experimental results are in good agreement with the theoretical simulation using the second order correlation function for the thermal light.