ABSTRACT
Ferroptosis, a form of regulated cell death that is driven by iron-dependent phospholipid peroxidation, has been implicated in multiple diseases, including cancer1-3, degenerative disorders4 and organ ischaemia-reperfusion injury (IRI)5,6. Here, using genome-wide CRISPR-Cas9 screening, we identified that the enzymes involved in distal cholesterol biosynthesis have pivotal yet opposing roles in regulating ferroptosis through dictating the level of 7-dehydrocholesterol (7-DHC)-an intermediate metabolite of distal cholesterol biosynthesis that is synthesized by sterol C5-desaturase (SC5D) and metabolized by 7-DHC reductase (DHCR7) for cholesterol synthesis. We found that the pathway components, including MSMO1, CYP51A1, EBP and SC5D, function as potential suppressors of ferroptosis, whereas DHCR7 functions as a pro-ferroptotic gene. Mechanistically, 7-DHC dictates ferroptosis surveillance by using the conjugated diene to exert its anti-phospholipid autoxidation function and shields plasma and mitochondria membranes from phospholipid autoxidation. Importantly, blocking the biosynthesis of endogenous 7-DHC by pharmacological targeting of EBP induces ferroptosis and inhibits tumour growth, whereas increasing the 7-DHC level by inhibiting DHCR7 effectively promotes cancer metastasis and attenuates the progression of kidney IRI, supporting a critical function of this axis in vivo. In conclusion, our data reveal a role of 7-DHC as a natural anti-ferroptotic metabolite and suggest that pharmacological manipulation of 7-DHC levels is a promising therapeutic strategy for cancer and IRI.
Subject(s)
Dehydrocholesterols , Ferroptosis , Humans , Cell Membrane/metabolism , Cholesterol/biosynthesis , Cholesterol/metabolism , CRISPR-Cas Systems/genetics , Dehydrocholesterols/metabolism , Genome, Human , Kidney Diseases/metabolism , Mitochondrial Membranes/metabolism , Neoplasm Metastasis , Neoplasms/metabolism , Neoplasms/pathology , Phospholipids/metabolism , Reperfusion Injury/metabolismABSTRACT
Growth differentiation factor 15 (GDF15) is a pleiotropic cytokine, which is involved in the cellular stress response following acute damage. However, the functional role of GDF15 in triple-negative breast cancer (TNBC) has not been fully elucidated. ELISA, Western blot, and PCR assays as well as bioinformatics analyses were conducted to observe the expression of GDF15. Cell Counting Kit-8, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet staining assays were conducted to evaluate paclitaxel resistance and cell viability. Cell apoptosis was analyzed by Western blotting. Murine xenograft model assay was employed to evaluate tumor growth in vivo . Our data indicate that GDF15 is markedly elevated in paclitaxel-resistant TNBC cells, which is significantly associated with unfavorable prognosis. Silencing of GDF15 robustly inhibits the proliferation of tumor cells and increases their sensitivity to paclitaxel in vitro and in vivo , whereas the treatment of purified GDF15 protein confers breast cancer cells with chemoresistance ability. Moreover, GDF15 activates protein kinase B (AKT) /mammalian target of rapamycin (mTOR) signaling, inhibition of AKT or mTOR reverses the prosurvival effect of GDF15 and enhances the antitumor efficacy of paclitaxel in TNBC cells. Altogether, our study uncovers the role of GDF15 in tumor growth and paclitaxel resistance, implicating a potential therapeutic target for TNBC.
Subject(s)
Proto-Oncogene Proteins c-akt , Triple Negative Breast Neoplasms , Animals , Humans , Mice , Cell Line, Tumor , Cell Proliferation , Drug Resistance, Neoplasm , Growth Differentiation Factor 15/metabolism , Growth Differentiation Factor 15/pharmacology , Growth Differentiation Factor 15/therapeutic use , Mammals/metabolism , Paclitaxel/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , TOR Serine-Threonine Kinases , Triple Negative Breast Neoplasms/pathologyABSTRACT
A series of N-sulfonaminoethyloxime derivatives of dehydroabietic acid were synthesized and investigated for their antibacterial activity against Staphylococcus aureus Newman strain and multidrug-resistant strains (NRS-1, NRS-70, NRS-100, NRS-108 and NRS-271). Most of the target compounds having chloro, bromo, trifluoromethyl phenyl moiety exhibited potent in vitro antistaphylococcal activity. The meta-CF3 phenyl derivative T23 showed the highest activity with MIC of 0.39-0.78⯵g/mL against S. aureus Newman, while several analogues showed similar potent antibacterial activity with MIC values between 0.78 and 1.56⯵g/mL against five multidrug-resistant S. aureus. The stability of T35 in plasma of SD rat and the cellular cytotoxicity were also evaluated.
Subject(s)
Abietanes/chemistry , Anti-Bacterial Agents/chemical synthesis , Oximes/chemistry , Animals , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Drug Resistance, Multiple, Bacterial/drug effects , Drug Stability , Microbial Sensitivity Tests , Oximes/metabolism , Oximes/pharmacology , Rats , Rats, Sprague-Dawley , Staphylococcus aureus/drug effects , Structure-Activity RelationshipABSTRACT
Detergents and salts are widely used in lysis buffers to enhance protein extraction from biological samples, facilitating in-depth proteomic analysis. However, these detergents and salt additives must be efficiently removed from the digested samples prior to LC-MS/MS analysis to obtain high-quality mass spectra. Although filter-aided sample preparation (FASP), acetone precipitation (AP), followed by in-solution digestion, and strong cation exchange-based centrifugal proteomic reactors (CPRs) are commonly used for proteomic sample processing, little is known about their efficiencies at removing detergents and salt additives. In this study, we (i) developed an integrative workflow for the quantification of small molecular additives in proteomic samples, developing a multiple reaction monitoring (MRM)-based LC-MS approach for the quantification of six additives (i.e., Tris, urea, CHAPS, SDS, SDC, and Triton X-100) and (ii) systematically evaluated the relationships between the level of additive remaining in samples following sample processing and the number of peptides/proteins identified by mass spectrometry. Although FASP outperformed the other two methods, the results were complementary in terms of peptide/protein identification, as well as the GRAVY index and amino acid distributions. This is the first systematic and quantitative study of the effect of detergents and salt additives on protein identification. This MRM-based approach can be used for an unbiased evaluation of the performance of new sample preparation methods. Data are available via ProteomeXchange under identifier PXD005405.
Subject(s)
Proteomics/methods , Specimen Handling/methods , Buffers , Chromatography, Liquid , Detergents/isolation & purification , Peptides/analysis , Proteins/analysis , Salts/isolation & purification , Tandem Mass SpectrometryABSTRACT
Although Janus kinase 2 (JAK2) plays a critical role in the progression of triple-negative breast cancer (TNBC), its inhibitors are incapable of eradicating these tumor cells, implicating drug resistance mechanisms exist. Our evidences show that TNBC cells express high level of Serine/Threonine Kinase 16 (STK16) when JAK2 signaling is blocked. Pharmacological inhibition or silencing of STK16 significantly enhances the sensitivity of TNBC cells to JAK2 inhibition, while over-expression of STK16 alleviates the anti-tumor effect of JAK2-inhibitor. Mechanistically, elevated STK16 expression rescues the phosphorylation status and transcriptional activity of STAT3, as STK16 is able to directly catalyze the phosphorylation of STAT3 at ser-727 residue. Our data indicate that upon JAK2 inhibition, TNBC cells express STK16 to maintain STAT3 transcriptional activity, dual-inhibition of JAK2/STK16 offers a potential way to treat TNBC patients.
Subject(s)
Drug Resistance, Neoplasm , Janus Kinase 2 , Protein Serine-Threonine Kinases , STAT3 Transcription Factor , Triple Negative Breast Neoplasms , STAT3 Transcription Factor/metabolism , STAT3 Transcription Factor/genetics , Janus Kinase 2/metabolism , Janus Kinase 2/antagonists & inhibitors , Humans , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/genetics , Phosphorylation , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/physiology , Cell Line, Tumor , Female , Animals , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/antagonists & inhibitors , Mice, Nude , Mice , Phenotype , Protein Kinase Inhibitors/pharmacologyABSTRACT
Basophils are the rarest circulating white blood cells (WBCs), but they play important roles in allergic disorders and other diseases. To enhance diagnostic capabilities, it would be desirable to isolate and analyze basophils efficiently from small blood samples. In 100 µL of whole blood, there are typically ~103 basophils, outnumbered by ~105 WBCs and ~108 red blood cells (RBCs). Basophils' low abundance has therefore presented a significant challenge in their isolation from whole blood. Conventional in-bulk basophil isolation methods require lengthy processing steps and cannot work with small volumes of blood. Here we report a parallelized integrated basophil isolation device (pi-BID) for the negative immunomagnetic selection of basophils directly from 4 samples of 100 µL of whole blood, in parallel, within 14 minutes including sample preparation time. The pi-BID interfaces directly with standard sample tubes, and uses a single pressure source to drive the flow in parallel microfluidic channels. Compared with conventional in-bulk basophil isolation, the pi-BID is >3× faster, and has higher purity (~93%) and similar recovery (~67%). Compared with other microfluidic devices for the immunomagnetic isolation of WBC sub-types, our pi-BID achieves 10× higher enrichment of target cells from whole blood, with no prior removal of RBCs necessary.
ABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: According to traditional Chinese medicine (TCM) theory, cholestasis belongs to category of jaundice. Artemisia capillaris Thunb. has been widely used for the treatment of jaundice in TCM. The polysaccharides are the one of main active components of the herb, but its effects on cholestasis remain unclear. AIM OF THE STUDY: To investigate the protective effect and mechanism of Artemisia capillaris Thunb. polysaccharide (APS) on cholestasis and liver injury. MATERIALS AND METHODS: The amelioration of APS on cholestasis was evaluated in an alpha-naphthyl isothiocyanate (ANIT)-induced mice model. Then nuclear Nrf2 knockout mice, mass spectrometry, 16s rDNA sequencing, metabolomics, and molecular biotechnology methods were used to elucidate the associated mechanisms of APS against cholestatic liver injury. RESULTS: Treatment with low and high doses of APS markedly decreased cholestatic liver injury of mice. Mechanistically, APS promoted nuclear translocation of hepatic nuclear factor erythroid 2-related factor (Nrf2), upregulated downstream bile acid (BA) efflux transporters and detoxifying enzymes expression, improved BA homeostasis, and attenuated oxidative liver injury; however, these effects were annulled in Nrf2 knock-out mice. Furthermore, APS ameliorated the microbiota dysbiosis of cholestatic mice and selectively increased short-chain fatty acid (SCFA)-producing bacteria growth. Fecal microbiota transplantation of APS also promoted hepatic Nrf2 activation, increased BA efflux transporters and detoxifying enzymes expression, ameliorated intrahepatic BA accumulation and cholestatic liver injury. Non-targeted metabolomics and in vitro microbiota culture confirmed that APS significantly increased the production of a microbiota-derived SCFA (butyric acid), which is also able to upregulate Nrf2 expression. CONCLUSIONS: These findings indicate that APS can ameliorate cholestasis by modulating gut microbiota and activating the Nrf2 pathway, representing a novel therapeutic approach for cholestatic liver disease.
Subject(s)
Artemisia , Cholestasis , Gastrointestinal Microbiome , Jaundice , Mice , Animals , NF-E2-Related Factor 2/metabolism , Liver , Cholestasis/chemically induced , Signal Transduction , Jaundice/metabolism , Bile Acids and Salts/metabolismABSTRACT
AIMS: Janus kinase 2 (JAK2)/signal transducer and activator of transcription (STAT) signaling plays a critical role in the progression of breast cancer. However, a small part of tumor cells survived from the killing effect of JAK2 inhibitor. We aimed to find out the mechanism of drug resistance in breast cancer cells and develop new therapeutic strategies. MATERIALS AND METHODS: The anti-tumor effect of TG101209 in breast cancer cells was confirmed by cell counting kit 8 and flow cytometry. Western blotting was used to determine the up-regulation of zinc finger SWIM-type containing 4 (ZSWIM4) induced by TG101209. In vitro and in vivo experiments were performed to evaluate the role of ZSWIM4 in the resistance of breast cancer cells to TG101209. Through the determination and analysis of 50% inhibiting concentration (IC50) curves, the effect of combination therapy was confirmed. KEY FINDINGS: Our data indicate that the elevated expression of ZSWIM4 contributes to JAK2 inhibition resistance, as knockdown of ZSWIM4 significantly enhances the sensitivity of breast cancer cells to TG101209 and over-expression of this gene mitigates the killing effect. Furthermore, the expression of vitamin D receptor (VDR) and utilization of 1α,25-(OH)2VD3 is decreased in ZSWIM4-knockdown breast cancer cells. VDR-silencing or GW0742-mediated blockade of VDR activity can partially reverse the JAK2 inhibition resistance. SIGNIFICANCE: Our data implicated that ZSWIM4 might be an inducible resistance gene of JAK2 inhibition in breast cancer cells. The combination of JAK2 inhibitor and VDR inhibitor may achieve better coordinated therapeutic effect in breast cancer.
Subject(s)
Breast Neoplasms/drug therapy , DNA-Binding Proteins/metabolism , Drug Resistance, Neoplasm , Gene Expression Regulation, Neoplastic , Janus Kinase 2/antagonists & inhibitors , Pyrimidines/pharmacology , Sulfonamides/pharmacology , Animals , Apoptosis , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Proliferation , DNA-Binding Proteins/genetics , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Hepatic stellate cell (HSC) activation induced by transforming growth factor ß1 (TGF-ß1) plays a pivotal role in fibrogenesis, while the complex downstream mediators of TGF-ß1 in such process are largely unknown. METHODS: We performed pharmacoproteomic profiling of the mice liver tissues from control, carbon tetrachloride (CCl4)-induced fibrosis and NPLC0393 administrated groups. The target gene MAT2A was overexpressed or knocked down in vivo by tail vein injection of AAV vectors. We examined NF-κB transcriptional activity on MAT2A promoter via luciferase assay. Intracellular SAM contents were analyzed by LC-MS method. FINDINGS: We found that methionine adenosyltransferase 2A (MAT2A) is significantly upregulated in the CCl4-induced fibrosis mice, and application of NPLC0393, a known small molecule inhibitor of TGF-ß1 signaling pathway, inhibits the upregulation of MAT2A. Mechanistically, TGF-ß1 induces phosphorylation of p65, i.e., activation of NF-κB, thereby promoting mRNA transcription and protein expression of MAT2A and reduces S-adenosylmethionine (SAM) concentration in HSCs. Consistently, in vivo and in vitro knockdown of MAT2A alleviates CCl4- and TGF-ß1-induced HSC activation, whereas in vivo overexpression of MAT2A facilitates hepatic fibrosis and abolishes therapeutic effect of NPLC0393. INTERPRETATION: This study identifies TGF-ß1/p65/MAT2A pathway that is involved in the regulation of intracellular SAM concentration and liver fibrogenesis, suggesting that this pathway is a potential therapeutic target for hepatic fibrosis. FUND: This work was supported by National Natural Science Foundation of China (No. 81500469, 81573873, 81774196 and 31800693), Zhejiang Provincial Natural Science Foundation of China (No. Y15H030004), the National Key Research and Development Program from the Ministry of Science and Technology of China (No. 2017YFC1700200) and the Key Program of National Natural Science Foundation of China (No. 8153000502).
Subject(s)
Liver Cirrhosis/etiology , Liver Cirrhosis/metabolism , Methionine Adenosyltransferase/metabolism , S-Adenosylmethionine/metabolism , Transcription Factor RelA/metabolism , Transforming Growth Factor beta1/metabolism , Biomarkers , Cell Line , Gene Expression Regulation , Humans , Liver Cirrhosis/pathology , Male , Models, Biological , Phosphorylation , Protein Interaction Maps , Proteome , Proteomics/methods , Signal TransductionABSTRACT
FTO, an mRNA N6-methyladenosine (m6A) demethylase, was reported to promote leukemogenesis. Using structure-based rational design, we have developed two promising FTO inhibitors, namely FB23 and FB23-2, which directly bind to FTO and selectively inhibit FTO's m6A demethylase activity. Mimicking FTO depletion, FB23-2 dramatically suppresses proliferation and promotes the differentiation/apoptosis of human acute myeloid leukemia (AML) cell line cells and primary blast AML cells in vitro. Moreover, FB23-2 significantly inhibits the progression of human AML cell lines and primary cells in xeno-transplanted mice. Collectively, our data suggest that FTO is a druggable target and that targeting FTO by small-molecule inhibitors holds potential to treat AML.
Subject(s)
Alpha-Ketoglutarate-Dependent Dioxygenase FTO/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/chemistry , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/metabolism , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Apoptosis/drug effects , Cell Cycle Checkpoints/drug effects , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Humans , Leukemia, Myeloid, Acute/enzymology , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Methylation , Mice, Inbred BALB C , Mice, Inbred NOD , Mice, Transgenic , Molecular Targeted Therapy , Protein Conformation , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats, Sprague-Dawley , Signal Transduction , Structure-Activity Relationship , U937 Cells , Xenograft Model Antitumor AssaysABSTRACT
OBJECTIVE: To compare the clinical efficacy differences between acupoint catgut embedding and Kuntai capsule for perimenopausal syndrome, so as to provide an effective treatment method for perimenopausal syndrome. METHODS: Thirty-three cases in the embedding group were treated with acupoint catgut embedding at back-shu points and front-mu points of liver, spleen and kidney combined with syndrome differentiation and disease differentiation, ten days per times; the Kuntai group was treated with oral administration of Kuntai capsule, 4 capsules each time, three times per day. The Kupperman index (KI) was observed in the two groups before treatment after 10 days, 30 days and 60 days of treatment, respectively; the efficacy was evaluated according to the ratio of KI. RESULTS: After the treatment, as treatment proceeded, the score of KI and ratio of KI were gradually reduced in two groups; the score of KI and ratio of KI in the embedding group after 10 days of treatment was lower than those in the Kuntai group (both P<0.05); after 10 days of treatment, the total effective rate was 36.4% (12/33) in the embedding group, which was superior to 3.0% (1/33) in the Kuntai group (P<0.05); however, after 30 days and 60 days of treatment, the differences of each index between two groups were not statistically significant (all P>0.05). CONCLUSION: Both the acupoint catgut embedding and Kuntai capsule could reduce the score of KI and improve clinical symptoms, and the acupoint catgut embedding has certain advantage on the early stage of treatment.