Teleost-specific TLR23 in Takifugu rubripes recruits MyD88 to trigger ERK pathway and promotes antibacterial defense.

Takifugu rubripes is a highly valued cultured fish in Asia, while pathogen infections can result in severe diseases and lead to substantial economic losses. Toll-like receptors (TLRs), as pattern recognition receptors, play a crucial role on recognition pathogens and initiation innate immune response. However, the immunological properties of teleost-specific TLR23 remain largely unknown. In this study, we investigated the biological functions of TLR23 (TrTLR23) from T. rubripes, found that TrTLR23 existed in various organs. Following bacterial pathogen challenge, the expression levels of TrTLR23 were significantly increased in immune related organs. TrTLR23 located on the cellular membrane and specifically recognized pathogenic microorganism. Co-immunoprecipitation and antibody blocking analysis revealed that TrTLR23 recruited myeloid differentiation primary response protein (MyD88), thereby mediating the activation of the ERK signaling pathway. Furthermore, in vivo showed that, when TrTLR23 is overexpressed in T. rubripes, bacterial replication in fish tissues is significantly inhibited. Consistently, when TrTLR23 expression in T. rubripes is knocked down, bacterial replication is significantly enhanced. In conclusion, these findings suggested that TrTLR23 played a critical role on mediation TLR23-MyD88-ERK axis against bacterial infection. This study revealed that TLR23 involved in the innate immune mechanism, and provided the foundation for development disease control strategies in teleost.

Inhibition of Delta-like Ligand 4 enhances the radiosensitivity and inhibits migration in cervical cancer via the reversion of epithelial-mesenchymal transition.

BACKGROUND: Concurrent chemoradiotherapy is the common first-line treatment for patients with advanced cervical cancer. However, radioresistance remains a major clinical challenge, which results in recurrence and poor survival. Many studies have shown the potential of Delta-like Ligand 4 (DLL4) as a novel prognostic biomarker and therapeutic target in many solid tumors. Previously, we have found that high DLL4 expression in tumor cells may predict the pelvic lymph node metastasis and poor prognosis in patients with cervical cancer. In our present study, we further studied the effects of DLL4 on the biological behavior and radiosensitivity of cervical cancer cells. METHODS: The expression of DLL4 and epithelial-mesenchymal transition (EMT) phenotype markers in cervical cancer cell lines or tissues were detected using Western blotting, and the expression of DLL4 mRNA in cervical cancer cell lines or tissues was detected using Quantitative real-time PCR. The effect of DLL4 on cell proliferation, migration, and radiosensitivity was evaluated using the CCK8 assay, flow cytometry, Transwell assays for cell invasion and migration, and Immunofluorescence staining in vitro. RESULTS: The expression of DLL4 in radiotherapy-resistant SiHa cells was significantly higher than that in radiotherapy-sensitive Me-180 cells. Furthermore, downregulation of DLL4 enhanced the radiosensitivity of SiHa and Caski cells via the inhibition of cell proliferation, promotion of radiation-induced apoptosis, and inhibition of the DNA damage repair. Moreover, downregulation of DLL4 inhibited the EMT and reduced the proliferation, invasion, and migration ability in SiHa and Caski cells. Consistent with the DLL4 expression in the cell lines, the expression of DLL4 in the tissues of the radioresistant group was also higher than that of the radiosensitive group. CONCLUSIONS: Downregulation of DLL4 inhibited the progression and increased the radiosensitivity in cervical cancer cells by reversing EMT. These results indicated the promising prospect of DLL4 against the radioresistance and metastasis of cervical cancer and its potential as a predictive biomarker for radiosensitivity and prognosis in patients with cervical cancer patients receiving concurrent chemoradiotherapy (cCRT).

Tailoring 3,3'-dihydroxyisorenieratene to hydroxystilbene: finding a resveratrol analogue with increased antiproliferation activity and cell selectivity.

Four novel compounds were designed by "tailoring" 3,3'-dihydroxyisorenieratene (a natural carotenoid) based on an isoprene unit retention truncation strategy. Among them, the smallest molecule 1 (2,3,6,2',3',6'-hexamethyl-4,4'-dihydroxy-trans-stilbene) was concisely synthesized in a one-pot Stille-Heck tandem sequence, and surfaced as a promising lead molecule in terms of its selective antiproliferative activity mediated by blocking the NCI-H460 cell cycle in G1 phase. Additionally, theoretical calculations and cell uptake experiments indicate that the unique polymethylation pattern of compound 1 significantly induces a conformational change shift out of planarity and increases its cell uptake and metabolic stability. The observation should be helpful to rationally design resveratrol-inspired antiproliferative agents.

Prooxidative inhibition against NF-κB-mediated inflammation by pharmacological vitamin C.

Vitamin C (VC), widely found in vegetables and fruits, operates as an electron donor to perform various biological functions including anti-inflammatory activity. However, the mechanisms by which VC inhibits inflammation remain insufficiently understood. Accordingly, we performed a detail mechanistic study on anti-inflammatory activity of VC at millimolar (pharmacological) concentrations in lipopolysaccharides-stimulated RAW264.7 cells. It was found that VC and its two-electron oxidative product, dehydroascorbate (DHA) constructs an efficient redox cycle with the aid of intracellular glutathione and copper ions, thereby facilitating the generation of reactive oxygen species (ROS) and the ROS-dependent inhibition against the NF-κB-mediated inflammation.

Inhibiting NF-κB-Mediated Inflammation by Catechol-Type Diphenylbutadiene via an Intracellular Copper- and Iron-Dependent Pro-Oxidative Role.

Chronic inflammation mediated by nuclear factor-κB (NF-κB) plays a crucial role in the development of cancer. As part of our continuous efforts placed on investigating anticancer mechanisms of dietary catechols, we further applied catechol-type diphenylbutadiene (3,4-DHB) as a model molecule to probe whether it inhibits inflammation by its pro-oxidative role. Employing lipopolysaccharide-stimulated RAW264.7 cells as a model of inflammation, we validated that benefiting from its catechol moiety, 3,4-DHB inhibited significantly the LPS-induced formation of NO (11.48 ± 0.39 µM) compared with the only LPS-stimulated group (31.8 ± 1.78 µM) with an inhibitory rate of 64% at 5 µM, expression of iNOS and COX-2 proteins, phosphorylation of IkB kinase and IkBα, and nuclear translocation of NF-κB. Noticeably, its inhibitory activity against the NF-κB-mediated inflammation can be obviously revised by pretreatment of the cells with dithiothreitol (a quencher of both electrophilic o-quinone and ROS), neocuproine (a specific chelating agent for copper ions), and deferoxamine (a specific chelating agent for iron ions). The above results support that depending on intracellular copper and iron ions, 3,4-DHB, a pro-electrophile, can be converted into its corresponding o-quinone electrophile together with the generation of ROS, a pro-oxidative event that mediates its inhibitory activity against NF-κB signaling and inflammation. The copper- and iron-dependent inhibition against inflammation supports that dietary catechols are probably pro-oxidative anti-inflammatory agents.

A promising redox cycle-based strategy for designing a catechol-type diphenylbutadiene as a potent prooxidative anti-melanoma agent.

Developing anti-melanoma agents with increased activity and specificity is highly desirable due to the increasing incidence, highly metastatic malignancy, and high mortality rate of melanoma. Abnormal redox characteristics such as higher levels of tyrosinase, NAD(P)H: quinone oxidoreductase-1 (NQO1) and reactive oxygen species (ROS) observed in melanoma cells than in other cancer cells and normal cells illustrate their redox vulnerability and have opened a window for developing prooxidative anti-melanoma agents (PAAs) to target the vulnerability. However, how to design PAAs which promote selectively the ROS accumulation in melanoma cells remains a challenge. This work describes a promising redox cycle-based strategy for designing a catechol-type diphenylbutadiene as such type of PAA. This molecule is capable of constructing an efficient catalytic redox cycle with tyrosinase and NQO1 in melanoma B16F1 cells to induce selectively the ROS (mainly including hydrogen peroxide, H2O2) accumulation in the cells, resulting in highly selective suppression of melanoma B16F1 cells over tyrosinase-deficient HeLa and normal L-02 cells.

Maternal control of suspensor programmed cell death via gibberellin signaling.

Plant embryos are generated and develop in a stable and well-protected microenvironment surrounded by maternal tissue, which is vital for embryogenesis. However, the signaling mechanisms responsible for maternal tissue-to-proembryo communication are not well understood. Here, we report a pathway for maternal tissue-to-proembryo communication. We identify a DELLA protein, NtCRF1 (NtCYS regulative factor 1), which regulates suspensor programmed cell death (PCD). NtCRF1 can bind to the promoter of NtCYS and regulate the suspensor PCD-switch module NtCYS-NtCP14 in response to gibberellin (GA). We confirm that GA4, as a primary signal triggering suspensor PCD, is generated in the micropylar endothelium by the transient activation of NtGA3oxs in the maternal tissue. Thus, we propose that GA is a maternal-to-proembryo communication signal that is decoded in the proembryo by a GID1-CRF1-CYS-CP14 signaling cascade. Using this mode of communication, maternal tissue precisely controls the embryonic suspensor PCD and is able to nurse the proembryo in a stage-dependent manner.

ROS-driven and preferential killing of HepG2 over L-02 cells by a short-term cooperation of Cu(II) and a catechol-type resveratrol analog.

This study was aimed at understanding why dietary polyphenols with a catechol skeleton tend to exhibit cancer chemopreventive activity by using a catechol-type stilbene (3,4-DHS) as a model molecule. Only a short-term cooperation of 3,4-DHS and exogenous Cu(II) exhibited a strong preferential ability to kill HepG2 cells over normal L02 cells. Mechanism studies reveal that this 3,4-DHS/Cu(II) system could produce extracellularly reactive oxygen species (ROS) and o-quinone through two sequential proton loss electron transfer followed by diffusion of ROS into cells, leading to higher intracellular accumulation of ROS, preferential disruption of redox homeostasis and more effective mitochondria-dependent apoptosis as well as necrosis of HepG2 cells than L-02 cells. This work provides further evidence that dietary catechol-type molecules show chemopreventive activity by virtue of their copper-dependent prooxidant action.

Applying an Electrophilicity-Based Strategy to Develop a Novel Nrf2 Activator Inspired from Dietary [6]-Shogaol.

Activation of nuclear factor erythroid-2-related factor 2 (Nrf2) is a crucial cellular defense mechanisms against oxidative stress and also an effective means to decrease the risk of oxidative stress-related diseases including cancer. Thus, identifying novel Nrf2 activators is highly anticipated. Inspired from [6]-shogaol (6S), an active component of ginger, herein we developed a novel potent Nrf2 activator, (1E,4E)-1-(4-hydroxy-3-methoxyphenyl)-7-methylocta-1,4,6-trien-3-one (SA) by an electrophilicity-based strategy. Compared with the parent 6S, SA bearing a short but entirely conjugated unsaturated ketone chain manifested the improved electrophilicity and cytoprotection (cell viability for the 10 µM 6S- and SA-treated group being 48.9 ± 5.3% and 76.1 ± 3.2%, respectively) against tert-butylhydroperoxide ( t-BHP)-induced cell death (cell viability for the t-BHP-stimulated group being 42.4 ± 0.4%) of HepG2. Mechanistic study uncovers that SA works as a potent Nrf2 activator by inducing Keap1 modification, inhibiting Nrf2 ubiquitylation and phosphorylating ERK in a Michael acceptor-dependent fashion. Taking 6S as an example, this works illustrates the feasibility and importance of applying an electrophilicity-based strategy to develop Nrf2 activators with dietary molecules as an inspiration due to their low toxicity and extraordinarily diverse chemical scaffolds.

Designing dichlorobinaphthoquinone as a prooxidative anticancer agent based on hydrogen peroxide-responsive in situ production of hydroxyl radicals.

Compared with normal cells, cancer cells harbor increased levels of reactive oxygen species (ROS) including hydrogen peroxide (H2O2), and therefore are more vulnerable to further ROS production. This biochemical difference favors the idea of developing new powerful selective prooxidative anticancer agents. However, it still remains a challenge to design them by targeting this difference. Herein, we report the designed dichlorobinaphthoquinone as a prooxidative anticancer agent which is capable of exploiting increased levels of H2O2 of cancer cells to produce in situ lethal hydroxyl radicals (HO•) and thereby kill them selectively, a design strategy inspired from Zhu et al.'s work on the molecular mechanism for metal-independent production of HO•.

Keto-enol-based modification on piperlongumine to generate a potent Cu(II) ionophore that triggers redox imbalance and death of HepG2 cells.

Altered redox status including higher levels of copper in cancer cells than in normal cells inspired many researchers to develop copper ionophores targeting this status. We have recently found that flavon-3-ol (3-HF) works as a potent Cu(II) ionophore by virtue of its keto-enol moiety. To further emphasize the significance of this moiety for developing Cu(II) ionophores, we herein designed a ß-diketo analog of piperlongumine, PL-I, characterized by the presence of high proportion of the keto-enol form in dimethylsulfoxide and chloroform, and identified its keto-enol structure by NMR and theoretical calculations. Benefiting from deprotonation of its enolic hydroxyl group, this molecule is capable of facilitating the transport of Cu(II) through cellular membranes to disrupt redox homeostasis of human hepatoma HepG2 cells and trigger their death.

Structural basis, chemical driving forces and biological implications of flavones as Cu(II) ionophores.

A main biochemical property of cancer cells, compared with normal cells, is altered redox status including increased levels of copper to maintain their malignant phenotypes. Thus, increasing copper accumulation, by using ionophores, to disrupt abnormal redox homeostasis of cancer cells may be an important anticancer strategy. Naturally occurring molecules with extraordinarily diverse chemical scaffolds are an important source of inspiration for developing copper ionophores. Dietary flavonoids are well-characterized copper chelators and show cancer chemopreventive potential, but their ionophoric role for redox-active copper and the related biological implications have remained unknown. This study reports, for the first time, the structural basis, chemical driving forces and biological implications of flavones (a widely distributed subgroup of flavonoids) as Cu(II) ionophores, and also provides new insights into cancer chemopreventive mechanism of flavones bearing 3(or 5)-hydroxy-4-keto group. 3-Hydroxyflavone surfaced as a potent Cu(II) ionophore to induce the mitochondria-dependent apoptosis of cancer cells in a redox intervention fashion via sequential proton-loss Cu(II) chelation, GSH-driving releasing of copper and protonation-dependent efflux of the neutral ligand.