Saracatinib inhibits necroptosis and ameliorates psoriatic inflammation by targeting MLKL.

Necroptosis is a kind of programmed cell death that causes the release of damage-associated molecular patterns and inflammatory disease including skin inflammation. Activation of receptor-interacting serine/threonine kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL) is the hallmark of tumour necrosis factor α (TNF)-induced necroptosis. Here, we screened a small-molecule compound library and found that saracatinib inhibited TNF-induced necroptosis. By targeting MLKL, Saracatinib interfered with the phosphorylation, translocation, and oligomerization of MLKL induced by TNF. Consistently, mutation of the saracatinib-binding site of MLKL reduced the inhibitory effect of saracatinib on TNF-induced necroptosis. In an imiquimod (IMQ)-induced psoriasis mouse model, saracatinib effectively blocked MLKL phosphorylation and inflammatory responses in vivo. Taken together, these findings indicate that saracatinib inhibits necroptosis by targeting MLKL, providing a potential therapeutic approach for skin inflammation-related diseases such as psoriasis.

Guanine nucleotide exchange factor RABGEF1 facilitates TNF-induced necroptosis by targeting cIAP1.

Necroptosis is a form of regulated cell death that depends on the receptor-interacting serine-threonine kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL). The molecular mechanisms underlying distinct instances of necroptosis have only recently begun to emerge. In the present study, we characterized RABGEF1 as a positive regulator of RIPK1/RIPK3 activation in vitro. Based on the overexpression and knockdown experiments, we determined that RABGEF1 accelerated the phosphorylation of RIPK1 and promoted necrosome formation in L929 cells. The pro-necrotic effect of RABGEF1 is associated with its E3 ubiquitin ligase activity and guanine nucleotide exchange factor (GEF) activity. We further confirmed that RABGEF1 interacts with cIAP1 protein by inhibiting its function and plays a regulatory role in necroptosis, which can be abolished by treatment with the antagonist Smac mimetic (SM)-164. In conclusion, our study highlights a potential and novel role of RABGEF1 in promoting TNF-induced cell necrosis.

Repurposing of Ibrutinib and Quizartinib as potent inhibitors of necroptosis.

Necroptosis is a form of regulated cell death that has been implicated in multiple diseases. TNF-induced necroptosis is regulated by necrosomes, complexes consisting of RIPK1, RIPK3 and MLKL. In this study, by screening of a small-compound library, we identified dozens of compounds that inhibited TNF-induced necroptosis. According to the mechanisms by which they inhibited necroptosis, these compounds were classified into different groups. We then identified Ibrutinib as an inhibitor of RIPK3 and found that Quizartinib protected against the TNF-induced systemic inflammatory response syndrome in mice by inhibiting the activation of RIPK1. Altogether, our work revealed dozens of necroptosis inhibitors, suggesting new potential approaches for treating necroptosis-related diseases.

Design, synthesis of novel triptolide-glucose conjugates targeting glucose Transporter-1 and their selective antitumor effect.

Six positional isomers of triptolide-glucose conjugates (TG1α, TG1ß, TG2, TG3, TG4 and TG6) were designed and synthesized. These conjugates exhibited better water solubility, and had selective cytotoxicity between tumor cells with high expression of glucose transport-1 (Glut-1) and non-tumor cells with low expression of Glut-1, in which TG2 formed by triptolide (TPL) and d-glucose C2-OH had the strongest cytotoxicity to tumor cells and lowest toxicity in non-tumor cells, therefore the highest relative therapeutic index, which was 5.7 times that of triptolide and consequent the most powerful selective antitumor activity in vitro. The cytotoxicity of TG2 was highly correlated with Glut-1 function. As a prodrug of triptolide, TG2 could promote RNA Pol II degradation and induce apoptosis as TPL does. TG2 had a stronger dose-dependent antitumor effect in vivo than TPL and no adverse reaction occurred when its tumor inhibition was higher than 90%, which was associated with its selective distribution in tumor tissues. TG2 could be used as a promising drug candidate for the treatment of solid tumors with high expression of Glut-1, which is worthy of further study.

Sterols and triterpenoids from Ganoderma lucidum and their reversal activities of tumor multidrug resistance.

Two sterols and seven triterpenoids were isolated and identified from Ganoderma lucidum by silica gel column chromatography, preparative high-performance liquid chromatography and spectra analysis. Then, the multidrug resistance reversal activities of these compounds were assessed using MTT assay. Among these compounds, ganoderol B (3), ganoderone A (4), ganodermanondiol (6) and ganoderiol F (8) were shown to reverse the resistance of human oral epidermoid carcinoma cell line KBv200 to doxorubicin, and the reversal folds were 6.59, 4.70, 4.01 and 7.09, respectively. Ganoderiol F could increase the intracellular accumulation of doxorubicin in KBv200 cells through inhibiting P-glycoprotein transport function. Further mechanistic investigation found that ganoderiol F did not alter P-glycoprotein expression. In conclusion, ganoderiol F has potent effect in reversing P-glycoprotein mediated tumor multidrug resistance. Potential reversal agents against multidrug resistance in tumor may be found in triterpenoids from Ganoderma lucidum.[Formula: see text].

Identification of Methicillin-Resistant Staphylococcus Aureus From Methicillin-Sensitive Staphylococcus Aureus and Molecular Characterization in Quanzhou, China.

To distinguish Methicillin-Resistant Staphylococcus aureus (MRSA) from Methicillin-Sensitive Staphylococcus aureus (MSSA) in the protein sequences level, test the susceptibility to antibiotic of all Staphylococcus aureus isolates from Quanzhou hospitals, define the virulence factor and molecular characteristics of the MRSA isolates. MRSA and MSSA Pfam protein sequences were used to extract feature vectors of 188D, n-gram and 400D. Weka software was applied to classify the two Staphylococcus aureus and performance effect was evaluated. Antibiotic susceptibility testing of the 81 Staphylococcus aureus was performed by the Mérieux Microbial Analysis Instrument. The 65 MRSA isolates were characterized by Panton-Valentine leukocidin (PVL), X polymorphic region of Protein A (spa), multilocus sequence typing test (MLST), staphylococcus chromosomal cassette mec (SCCmec) typing. After comparing the results of Weka six classifiers, the highest correctly classified rates were 91.94, 70.16, and 62.90% from 188D, n-gram and 400D, respectively. Antimicrobial susceptibility test of the 81 Staphylococcus aureus: Penicillin-resistant rate was 100%. No resistance to teicoplanin, linezolid, and vancomycin. The resistance rate of the MRSA isolates to clindamycin, erythromycin and tetracycline was higher than that of the MSSAs. Among the 65 MRSA isolates, the positive rate of PVL gene was 47.7% (31/65). Seventeen sequence types (STs) were identified among the 65 isolates, and ST59 was the most prevalent. SCCmec type III and IV were observed at 24.6 and 72.3%, respectively. Two isolates did not be typed. Twenty-one spa types were identified, spa t437 (34/65, 52.3%) was the most predominant type. MRSA major clone type of molecular typing was CC59-ST59-spa t437-IV (28/65, 43.1%). Overall, 188D feature vectors can be applied to successfully distinguish MRSA from MSSA. In Quanzhou, the detection rate of PVL virulence factor was high, suggesting a high pathogenic risk of MRSA infection. The cross-infection of CA-MRSA and HA-MRSA was presented, the molecular characteristics were increasingly blurred, HA-MRSA with typical CA-MRSA molecular characteristics has become an important cause of healthcare-related infections. CC59-ST59-spa t437-IV was the main clone type in Quanzhou, which was rare in other parts of mainland China.

The deubiquitinating enzyme OTUD1 antagonizes BH3-mimetic inhibitor induced cell death through regulating the stability of the MCL1 protein.

BACKGROUND: Myeloid cell leukaemia 1 (MCL1) is a pro-survival Bcl-2 family protein that plays important roles in cell survival, proliferation, differentiation and tumourigenesis. MCL1 is a fast-turnover protein that is degraded via an ubiquitination/proteasome-dependent mechanism. Although several E3 ligases have been discovered to promote the ubiquitination of MCL1, the deubiquitinating enzyme (DUB) that regulates its stability requires further investigation. METHODS: The immunoprecipitation was used to determine the interaction between OTUD1 and MCL1. The ubiquitination assays was performed to determine the regulation of MCL1 by OTUD1. The cell viability was used to determine the regulation of BH3-mimetic inhibitor induced cell death by OTUD1. The survival analysis was used to determine the relationship between OTUD1 expression levels and the survival rate of cancer patients. RESULTS: By screening a DUB expression library, we determined that the deubiquitinating enzyme OTUD1 regulates MCL1 protein stability in an enzymatic-activity dependent manner. OTUD1 interacts with MCL1 and promotes its deubiquitination. Knockdown of OTUD1 increases the sensitivity of tumour cells to the BH3-mimetic inhibitor ABT-263, while overexpression of OTUD1 increases tumour cell tolerance of ABT-263. Furthermore, bioinformatics analysis data reveal that OTUD1 is a negative prognostic factor for liver cancer, ovarian cancer and specific subtypes of breast and cervical cancer. CONCLUSIONS: The deubiquitinating enzyme OTUD1 antagonizes BH3-mimetic inhibitor induced cell death through regulating the stability of the MCL1 protein. Thus, OTUD1 could be considered as a therapeutic target for curing these cancers.

Chemical and genetic inhibition of STAT3 sensitizes hepatocellular carcinoma cells to sorafenib induced cell death.

Hepatocellular carcinoma (HCC) has become the second leading cause of cancer related death, with an increasing death rate in recent years. For advanced HCC, sorafenib is the first-line FDA approved drug, with no more than 3 months' overall survival advantage. Recently, a novel strategy has been proposed to improve sorafenib efficacy through enhancing the ability of sorafenib to induce cell death. STAT3 plays a key role in cancer development and recurrence by promoting cell proliferation, survival and immune evasion through its well-established function as a transcription factor in cancer. Notably, STAT3 transcription activity, indicated by its phosphorylation on Y705 is heterogeneous in different liver cancer cell lines. And sorafenib attenuates STAT3 phosphorylation on Y705. However, the role of STAT3 in sorafenib induced cell death is still largely unknown. Here, we show that liver cancer cells also exhibit heterogeneous sensitivities to sorafenib induced cell death, which co-relates with the STAT3-Y705 phosphorylation levels and JAK1/2 expression levels in Hep3B, Huh7 and HepG2 cells. Furthermore, overexpression or knockdown of STAT3 could switch HCC cells between resistant and sensitive to sorafenib induced cell death, which could be partially due to its regulation on Mcl-1, an anti-apoptotic protein. Finally, both inhibitors of STAT3 SH2 domain (S3i-201) or STAT3 upstream kinases JAKs (JAK inhibitor I) could synergistically enhance sorafenib induced cell death. Taken together, these data strongly suggest that STAT3 is not only a downstream effector of sorafenib, but also a key regulator of cellular sensitivity to sorafenib induced cell death, which provide support for the notion to develop STAT3-targeting drugs to improve clinical efficacy of sorafenib in liver cancer.

C1206, a novel curcumin derivative, potently inhibits Hsp90 and human chronic myeloid leukemia cells in vitro.

4-(4-Pyridinyl methylene) curcumin (C1206) is a new derivative of curcumin that is more active than curcumin in inhibition of heat shock protein 90 (Hsp90) and antitumor action. In this study we investigated the relationship between C1206-induced inhibition of Hsp90 and its anti-leukemic effects. The fluorescence quenching experiments showed that C1206 seemed to bind the middle dimerization domain of Hsp90. The interaction between C1206 and Hsp90 was driven mainly by electrostatic interaction. In in vitro enzyme activity assay, C1206 dose-dependently inhibited Hsp90 ATPase activity with an IC50 value of 4.17 µmol/L. In both imatinib-sensitive K562 chronic myeloid leukemia cells and imatinib-resistant K562/G01 chronic myeloid leukemia cells, C1206 (0.4-3.2 µmol/L) dose-dependently caused the degradation of Hsp90 client proteins and downstream proteins (AKT, MEK, ERK, C-RAF, P-AKT, P-MEK and P-ERK). Furthermore, C1206 (0.4-3.2 µmol/L) dose-dependently induced apoptosis of K562 and K562/G01 cells through triggering mitochondrial pathway. Consistent with this result, C1206 inhibited the proliferation of K562 and K562/G01 cells with IC50 values of 1.10 and 0.60 µmol/L, respectively. These results suggest that C1206 is a novel Hsp90 inhibitor and a promising therapeutic agent for chronic myeloid leukemia.

Ubiquitin-Specific Protease USP6 Regulates the Stability of the c-Jun Protein.

The c-Jun gene encodes a transcription factor that has been implicated in many physiological and pathological processes. c-Jun is a highly unstable protein that is degraded through a ubiquitination/proteasome-dependent mechanism. However, the deubiquitinating enzyme (DUB) that regulates the stability of the c-Jun protein requires further investigation. Here, by screening a DUB expression library, we identified ubiquitin-specific protease 6 (USP6) and showed that it regulates the stability of the c-Jun protein in a manner depending on its enzyme activity. USP6 interacts with c-Jun and antagonizes its ubiquitination. USP6 overexpression upregulates the activity of the downstream signaling pathway mediated by c-Jun/AP-1 and promotes cell invasion. Moreover, many aberrant genes that are upregulated in USP6 translocated nodular fasciitis are great potential targets regulated by c-Jun. Based on our data, USP6 is an enzyme that deubiquitinates c-Jun and regulates its downstream cellular functions.