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In order to take advantage of both immunotherapeutic and metabolic antitumor agents, novel dual indoleamine 2,3- dioxygenase 1 (IDO1) and thioredoxin reductase 1 (TrxR1) inhibitors were designed. Thioredoxin reductase 1 (TrxR1) is a main ROS modulator within CRC cells. Indoleamine 2,3-dioxygenase (IDO1) is crucial controller for tryptophan (Trp) metabolism that is also important for CRC immunotherapy. Herein, ten compounds 12a-j containing hydroxyamidine scaffold were designed, synthesized and evaluated for inhibitory activities against IDO1/TrxR1 enzyme and CRC cells. Among these compounds, the most active compound 12d (ZC0109) showed excellent and balanced activity against both IDO1 (IC50 = 0.05 µM) and TrxR1 (IC50 = 3.00 ± 0.25 µM) were selected for further evaluation. Compound ZC0109 exhibited good dual inhibition against IDO1 and TrxR1 both in vitro and in vivo. Further mechanistic studies reveal that, through IDO1 and TrxR1 inhibition by ZC0109 treatment, accumulated ROS effectively induced apoptosis and G1/S cell cycle arrest in cancer cells. In vivo evaluation demonstrated excellent anti-tumor effect of ZC0109 with the notable ability of promoting ROS-induced apoptosis, reducing kynurenine level in plasma and restoring anti-tumor immune response. Thus, ZC0109 represents a potential CRC therapy agent for further development.
Assuntos
Neoplasias Colorretais , Inibidores Enzimáticos , Indolamina-Pirrol 2,3,-Dioxigenase , Espécies Reativas de Oxigênio , Tiorredoxina Redutase 1 , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Tiorredoxina Redutase 1/antagonistas & inibidores , Linhagem Celular Tumoral , Humanos , Apoptose/efeitos dos fármacos , Neoplasias Colorretais/enzimologiaRESUMO
Background: Accumulating evidence suggests that the polymerase I and transcript release factor (PTRF), a key component of the caveolae structure on the plasma membrane, plays a pivotal role in suppressing the progression of colorectal cancers. However, the role of PTRF in the development of functional gastrointestinal (GI) disorders remains unclear. Post-infectious irritable bowel syndrome (PI-IBS) is a common functional GI disorder that occurs after an acute GI infection. Here, we focused on the role of PTRF in the occurrence of PI-IBS and investigated the underlying mechanisms. Methods: Lipopolysaccharide (LPS) (5 µg/ml) was used to induce inflammatory injury in human primary colonic epithelial cells (HCoEpiCs). Furthermore, a rat model of PI-IBS was used to study the role of PTRF. Intestinal sensitivity was assessed based on the fecal water content. A two-bottle sucrose intake test was used to evaluate behavioral changes. Furthermore, shRNA-mediated knockdown of PTRF was performed both in vitro and in vivo. We detected the expression of PTRF in colonic mucosal tissues through immunohistochemistry (IHC), western blotting (WB), and immunofluorescence (IF) analysis. Luciferase activity was quantified using a luciferase assay. Co-localization of PTRF and Toll-like receptor 4 (TLR4) was detected using IF analysis. The activation of the signaling pathways downstream of TLR4, including the iNOs, p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) pathways, was detected via WB. The levels of NO, IL-1ß, IL-6, and TNF-α were measured using enzyme-linked immunosorbent assays. Results: LPS significantly induced PTRF expression and signaling downstream of TLR4, including p38, ERK, and JNK pathways, in HCoEpiCs. Moreover, shRNA-mediated knockdown of PTRF in HCoEpiCs significantly decreased the phosphorylation of JNK, ERK, and p38 and iNOS expression. In PI-IBS rats, the lack of PTRF not only reduced fecal water content and suppressed depressive behavior but also increased the body weight. Furthermore, we found a strong co-localization pattern for PTRF and TLR4. Consistently, the lack of PTRF impaired TLR4 signaling, as shown by the decreased levels of p-JNK, p-ERK, and p-p38, which are upstream factors involved in iNOS expression. Conclusion: PTRF promoted PI-IBS and stimulated TLR4 signaling both in vitro and in vivo. The results of this study not only enlighten the pathogenesis of PI-IBS but also help us understand the biological activity of PTRF and provide an important basis for the clinical treatment of PI-IBS by targeting PTRF.
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Colorectal cancer (CRC) is one of the most common cancer types and a leading cause of cancer-associated mortality in China. Increased thioredoxin reductase 1 (TrxR1) levels have been previously identified as possible target for CRC. The present study revealed that the natural product hydroxytyrosol (HT), which exhibits a polyphenol scaffold, is a potent inhibitor of TrxR1. Inhibition of TrxR1 was indicated to result in accumulation of reactive oxygen species, inhibit proliferation and induce apoptosis and G1/S cell cycle arrest of CRC cells. Using a C-terminal mutant TrxR1 enzyme activity assay, TrxR1 RNA interference assay and HT binding model assay, the present study demonstrated the core character of the selenocysteine residue in the interaction between HT and TrxR1. HT can serve as polyphenol scaffold to develop novel TrxR1 inhibitors for CRC treatment in the future.
RESUMO
Network pharmacology and liver fibrosis(LF) model in vitro were used to analyze the underly mechanism of anti-liver fibrosis effect that induced by Piperis Longi Fructus and its major active compounds. TCMSP and TCMIP were used to search for the chemical constituents of Piperis Longi Fructus, as well as the oral bioavailability(OB), drug-likeness(DL), intercellular permeability of intestinal epithelial cells(Caco-2) and Drug-likeness grading were set as limiting conditions. The related target genes of Piperis Longi Fructus were queried by TCMSP database, while related targets of LF were screened by GeneCards databases. Interaction network was constructed using Cytoscape 3.7.1. These above data were imported into STRING database for PPI network analysis. Enrichment of gene ontology(GO) and pathway analysis(KEGG) within Bioconductor database were utilized to note functions of related targets of Piperis Longi Fructus. Finally, the core targets and pathways were preliminarily verified by in vitro experiments. The effects of piperlongumine(PL), the major active component of Piperis Longi Fructus, on proliferation of rat liver stellate cells(HSC-T6) and expression of α smooth muscle actin(α-SMA) and collagen â were investigated. The major factors TNF-α of tumor necrosis factor(TNF) pathway and NF-κB p65, IL-6 protein expressions of LF process were examined. A total of 12 active compounds such as PL were obtained by analyzing the bioavailability and drug-like properties, which inferred to 48 targets. The functional enrichment analysis of GO obtained 1 240 GO items, mainly involving in process of biology and molecular function. A total of 99 signaling pathways were enriched in the KEGG pathway enrichment analysis, including TNF signaling pathway, cGMP-PKG signaling pathway, calcium signaling pathways. CCK-8 assay showed that PL inhibited proliferation of HSC-T6 induced by transforming growth factor-ß1(TGF-ß1). Western blot analysis found that treated with PL suppressed the protein expressions of α-SMA, collagen â , TNF-α and p65 in HSC-T6. Enzyme linked immunosorbent assay(ELISA) showed that PL inhibited the expressions of TNF-α and IL-6 in the cluture supertant of HSC-T6 cells. In conclusion, PL could play an anti-liver fibrosis role by regulating TNF/NF-κB signaling pathway. This study provided the mechanism basis of anti-LF effects induced by Piperis Longi Fructus and its major active compounds, which might help for the further study of the mechanism and key targets of Piperis Longi Fructus.
Assuntos
Células Estreladas do Fígado , Cirrose Hepática , Animais , Células CACO-2 , Células Estreladas do Fígado/metabolismo , Humanos , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/genética , NF-kappa B/metabolismo , Ratos , Transdução de SinaisRESUMO
Targeting the Trp-Kyn pathway is an attractive approach for cancer immunotherapy. Thioredoxin reductase (TrxR) enzymes are reactive oxygen species (ROS) modulators that are involved in the tumor cell growth and survival processes. The 4-phenylimidazole scaffold is well-established as useful for indoleamine 2,3-dioxygenase 1 (IDO1) inhibition, while piperlongumine (PL) and its derivatives have been reported to be inhibitors of TrxR. To take advantage of both immunotherapy and TrxR inhibition, we designed a first-generation dual IDO1 and TrxR inhibitor (ZC0101) using the structural combination of 4-phenylimidazole and PL scaffolds. ZC0101 exhibited better dual inhibition against IDO1 and TrxR in vitro and in cell enzyme assays than the uncombined forms of 4-phenylimidazole and PL. It also showed antiproliferative activity in various cancer cell lines, and a selective killing effect between normal and cancer cells. Furthermore, ZC0101 effectively induced apoptosis and ROS accumulation in cancer cells. Knockdown of TrxR1 and IDO1 expression induced cellular enzyme inhibition and ROS accumulation effects during ZC0101 treatment, but only reduced TrxR1 expression was able to improve ZC0101's antiproliferation effect. This proof-of-concept study provides a novel strategy for cancer treatment. ZC0101 represents a promising lead compound for the development of novel antitumor agents that can also be used as a valuable probe to clarify the relationships and mechanisms of cancer immunotherapy and ROS modulators.
Assuntos
Antineoplásicos/farmacologia , Dioxolanos/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Tiorredoxina Redutase 1/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dioxolanos/síntese química , Dioxolanos/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Estrutura Molecular , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade , Tiorredoxina Redutase 1/metabolismo , Células Tumorais CultivadasRESUMO
Resistance is a major concern when administering chemotherapy to patients with non-small cell lung cancer (NSCLC). Chemosensitizer are agents that can reverse resistance to chemotherapeutic drugs, thereby enhancing the chemosensitivity of tumor cells. Thus, their development will improve therapeutic efficacy in cancer. However, few effective chemosensitizer have been identified to date. Piperlongumine (PL) has been shown to effectively reverse resistance to chemotherapeutic drugs in several types of cancers. However, the mechanisms associated with the chemotherapy resistance reversal effect of PL and its regulation of target factors in chemotherapy resistance cells are still unclear. This study investigated the reversal effect of PL both in vitro and in vivo, and provided evidence that PL inhibited the phosphorylation of Akt via the accumulation of reactive oxygen species in chemotherapy resistance cells. Consequently, various Akt activation-dependent genes caused a reduction of drug efflux and induction of apoptosis in cisplatin-resistant A549 NSCLC cells. Our results indicate that Akt phosphorylation may play a functional role in the reversal effect of PL and contribute, at least in part, to the treatment outcomes of patients with chemotherapy resistance.
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With the aim of discovering novel cyclin-dependent kinase 8 (CDK8) inhibitors, a combined similarity search and molecular docking approach was employed, which led to 32 hits. Biological tests led to the discovery of several novel submicromolar inhibitors. In particular, compound C768-0769 (ZC0201) showed good CDK8 inhibitory activity, and compound ZC0201 effectively suppressed HCT-116 colorectal cancer cell proliferation by inducing G1/S transition arrest. Furthermore, modulation of phosphorylated signal transducer and activator of transcription 1 (Ser 727) (STAT1SER727), a pharmacodynamic biomarker of CDK8 activity, demonstrated that ZC0201 may cause G1/S transition arrest through CDK8 activity inhibition. Due to its good cellular activity, ZC0201 may be an ideal lead compound for further modification as a potential cancer therapeutic agent.
Assuntos
Quinase 8 Dependente de Ciclina/antagonistas & inibidores , Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/patologia , Ensaios de Seleção de Medicamentos Antitumorais , Fase G1/efeitos dos fármacos , Células HCT116 , Humanos , Simulação de Acoplamento Molecular , Fosforilação , Inibidores de Proteínas Quinases/química , Fase S/efeitos dos fármacos , Fator de Transcrição STAT1/metabolismoRESUMO
Multidrug resistance (MDR) is a major concern when using chemotherapy for the treatment of patients with colorectal cancer. MDR modulators are agents that can reverse MDR and, thus, enhance the chemosensitivity of tumor cells. The development of MDR modulators can improve the therapeutic efficacies of MDR in cancer. However, few effective MDR modulators have been identified so far. Curcumin has been reported to be an effective compound in the reversal of MDR in colorectal cancer cells. However, the mechanisms associated with the reversal effect of curcumin on MDR and its regulation of target factors in MDR cells remain to be fully elucidated. 3(4,5dimethyl2thiazol)2,5diphenyltetrazolium bromide assays, flow cytometer apoptosis assays as well as mRNA and protein expression assays were performed in the present study, and the results confirmed the reversal effect of curcumin on HCT8/5Fu cells and provided evidence that activated nuclear factor erythroid 2related factor (Nrf2) deficiency induced by the curcumin altered the Bcell lymphoma 2 (Bcl2) associated X protein/Bcl2 expression ratio, which led to the induction of apoptosis in HCT8/5Fu cells. These results indicated that Nrf2 may have a functional in the reversal effect of curcumin and contribute, at least in part, to the outcomes of chemotherapy in patients with MDR.
Assuntos
Antineoplásicos/farmacologia , Curcumina/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Fluoruracila/farmacologia , Genes bcl-2 , Humanos , Transdução de Sinais/efeitos dos fármacos , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Rice straw is supposed to be an environment-friendly biomaterial for inhibiting the growth of harmful blooms of the cyanobacterium Microcystis aeruginosa. The effects of rice straw extract(RSE) on algal growth, morphologic parameters(cell size), and physiological parameters(in vivo Chl-a fluorescence) were investigated using flow cytometry. We examined the selective inhibitory potential of rice straw on four cyanobacterial strains(toxic and non-toxic Microcystis aeruginosa, toxic Anabaena flos-aquae, and Microcystis ichthyoblabe), in comparison with inhibitory effects on three common freshwater green algae(Selenastrum capricornutum, Chlorella pyrenoidosa, and Scenedesmus obliqnus). Concentrations from 2.0 to 10.0 g·L-1 of RSE were found to efficiently inhibit the growth of cyanobacteria in a dose-dependent manner, simultaneously modifying the in vivo Chl-a fluorescence and cell size. The 50% growth-inhibition concentration(7 d) of A. flos-aquae, M. ichthyoblabe, M. aeruginosa(toxic strain), M. aeruginosa(non-toxic strain) was 1.72, 2.21, 2.92 and 5.72 g·L-1, respectively. Interestingly, the growth and cell size of C. pyrenoidosa and S. obliqnus increased with the addition of RSE and colony formation was observed. In the case of S. capricornutum, the inhibitory effect of RSE on growth and in vivo Chl-a fluorescence occurred at 1.0-4.0 g·L-1, while RSE induced a stimulatory effect on algal growth at 8.0-10.0 g·L-1. Taken together, the sensitivity of cyanobacteria to RSE was significantly higher than that of S. capricornutum, C. pyrenoidosa and S. obliqnus. The higher sensitivity of PSâ ¡ reaction center of cyanobacteria and the ability to form colonies of green algae may have important implications for the species-specific allelopathic antialgal activity of rice straw.