Chemoproteomic mapping of the glycolytic targetome in cancer cells.

Hyperactivated glycolysis is a metabolic hallmark of most cancer cells. Although sporadic information has revealed that glycolytic metabolites possess nonmetabolic functions as signaling molecules, how these metabolites interact with and functionally regulate their binding targets remains largely elusive. Here, we introduce a target-responsive accessibility profiling (TRAP) approach that measures changes in ligand binding-induced accessibility for target identification by globally labeling reactive proteinaceous lysines. With TRAP, we mapped 913 responsive target candidates and 2,487 interactions for 10 major glycolytic metabolites in a model cancer cell line. The wide targetome depicted by TRAP unveils diverse regulatory modalities of glycolytic metabolites, and these modalities involve direct perturbation of enzymes in carbohydrate metabolism, intervention of an orphan transcriptional protein's activity and modulation of targetome-level acetylation. These results further our knowledge of how glycolysis orchestrates signaling pathways in cancer cells to support their survival, and inspire exploitation of the glycolytic targetome for cancer therapy.

Analysis of the active components and metabolites of Taohong Siwu decoction by using ultra high performance liquid chromatography quadrupole time-of-flight mass spectrometry.

Taohong Siwu Decoction is a classic Chinese medicine prescription for treatment of cerebral ischemia and gynecological diseases. However, the active ingredients of Taohong Siwu Decoction have not been identified. In this study, a ultra performance liquid chromatography quadrupole time-of-flight mass system was used to analyze the active components and metabolites of Taohong Siwu Decoction absorbed into the blood and the brain. A total of 39 active compounds and 90 metabolites were identified in the blood and brain by comparing retention times, accurate masses, fragmentation patterns, and literature data. The results showed that flavonoids (Carthamus tinctorius L), aromatic organic acids, and benzoquinones (Angelica sinensis (Oliv.) Diels and Ligusticumchuanxiong hort) were prominent active ingredients in Taohong Siwu Decoction. Furthermore, hydrolysis, glucuronidation, and sulfation were identified as the main metabolic pathways of Taohong Siwu Decoction in vivo. This was the first study to characterize the active components and metabolites of Taohong Siwu Decoction in the blood and brain using ultra performance liquid chromatography quadrupole time-of-flight mass.

Integrative Omics Analysis Revealed that Metabolic Intervention Combined with Metronomic Chemotherapy Selectively Kills Cancer Cells.

Metronomic chemotherapy, a relatively new dosing paradigm for anticancer therapy, is an alternative to traditional chemotherapy that uses maximal tolerated dose (MTD). Although these two dosing regimens both lead to tumor cell death, how cell metabolism is differentially affected during apoptosis remains elusive. Herein, we employed metabolomics to monitor the metabolic profiles of MCF-7 cells in response to the two dosing regimens that mimic MTD and MN treatments using a model chemotherapeutic drug, doxorubicin (Dox), and correlated the changes of metabolic genes examined by PCR array to integratively describe the reprogrammed metabolic patterns. We found glycolysis, amino acid, and nucleotide synthesis-associated metabolic pathways were activated in response to the MN treatment, whereas these pathways were inhibited in a pronounced way in response to the MTD treatment. Direct supplementation of key metabolites and pharmacological modulation of targeted metabolic enzymes can both regulate cell fates. Subsequently, we tested the combined use of MN dosing with targeted metabolic intervention using a normal cell line and found the combined treatment hardly affected its apoptotic rate. Our in vitro findings using MCF-7 and MCF-10A cells thus suggest the promising perspective of combining MN dosing of chemotherapeutic agents with metabolic modulation to selectively kill cancer cells rather than normal cells.

Time-Resolved Acetaldehyde-Based Accessibility Profiling Maps Ligand-Target Interactions.

Elucidating ligand-protein interactions is important in understanding the biochemical machinery for given proteins. Previously, formaldehyde (FH)-based labeling has been employed to obtain such structural knowledge, since reactive residues that participate in ligand-target interactions display reduced accessibility to FH-labeling reagents, and thus can be identified by quantitative proteomics. Although being rapid and efficient for probing proteinaceous lysine accessibility, here, we report an acetaldehyde (AcH)-labeling approach that complements with FH for probing ligand-target interactions. AcH labeling examines lysine accessibility at a more moderate reaction speed and hence delivers a cleaner reaction when compared to that of FH. The subsequent application of AcH to label RNase A without and with ligands has assisted to assign lysines involved in ligand-RNase A binding by detecting the time-dependent changes in accessibility profiles. We further employed multiple reaction monitoring (MRM) to quantify these ligand-binding-responsive sites when a variety of potential ligands were queried. We noted that the time-resolved abundance changes of these peptides can sensitively determine the ligand-binding sites and differentiate binding affinities among these ligands, which was confirmed by native mass spectrometry (MS) and molecular docking. Lastly, we demonstrated that the binding sites can be recognized by monitoring the chemical accessibility of these responsive peptides in cell lysates. Together, we believe that the proposed combined use of AcH-based lysine accessibility profiling, native MS, and MRM screening is a powerful toolbox in characterizing ligand-target interactions, mapping topography, and interrogating affinities and holds promise for future applications in a complex cellular environment.

Cytosolic ME1 integrated with mitochondrial IDH2 supports tumor growth and metastasis.

NADPH is a pivotal cofactor that maintains redox homeostasis and lipogenesis in cancer cells and interference with NADPH production is a promising approach for treating cancer. However, how normal and cancer cells differentially exploit NADPH-producing pathways is unclear, and selective approaches to targeting NADPH are lacking. Here, we show that the assayed cancer cell lines preferentially depend on ME1-mediated NADPH production. ME1 knockdown increases intracellular ROS levels and impairs lipogenesis in cancer cells, leading to retarded proliferation and increased anoikis, while sparing normal cells. Notably, ME1 interference ultimately resulted in adaptive upregulation of mitochondrial IDH2 dependent of AMPK-FoxO1 activation to replenish the NADPH pool and mitigate cytosolic ROS. Combining ME1 ablation and IDH2 inhibition drastically reduces intracellular NADPH and prevents resistance to ME1 interference, resulting in increased apoptosis and impeded tumor growth and metastasis. This study demonstrates that cytosolic ME1 integrated with mitochondrial IDH2 is essential for tumor growth and metastasis, thereby highlighting the blockade of metabolic compensation by disrupting mitochondrial-cytosol NADPH transport as a promising approach to selectively targeting NADPH in cancer cells that rely on NADPH-driven antioxidant systems.

UPLC-Q-TOF-MS Study of the Mechanism of THSWD for Breast Cancer Treatment.

Taohong Siwu decoction (THSWD) is a classic traditional Chinese medicine (TCM) prescription that is widely used in the clinical treatment of gynecological and cerebrovascular diseases. Here we used a method that coupled ultra-performance liquid chromatography to quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) in which both positive and negative ion modes were established to investigate the major constituents in THSWD. A Waters ACQUITY UPLC BEH C18 column (2.1 mm×100 mm, 1.7 µm) was used to separate the aqueous extract of THSWD. The mobile phase consisted of 0.1% aqueous formic acid (A) and acetonitrile (B). Ninety-five components were identified in two different ion modes, including aromatic acids, flavones, polysaccharides, volatile oils monoterpene glycosides, aromatic cyanogenic glycosides, and others. Pathological changes in tumors and serum expression of interleukin-4 in a mouse model of breast cancer were detected after THSWD treatment. The results showed that THSWD had obvious therapeutic effects. This study establishes a material basis for the use of THSWD in the treatment of breast cancer.

Bioinformatics analysis of a long non­coding RNA and mRNA regulation network in rats with middle cerebral artery occlusion based on RNA sequencing.

Long non­coding RNAs (lncRNAs) have been proven to be critical gene regulators of development and disease. The main aim of the present study was to elucidate the lncRNA­mRNA regulation network in ischemic stroke induced by middle cerebral artery occlusion (MCAO) using RNA sequencing (RNA­seq) in rats. lncRNA expression profiles were screened in brain tissues to identify a number of differentially expressed lncRNAs (DELs) and genes (DEGs) by RNA­seq. Reverse transcription­quantitative polymerase chain reaction was performed to further confirm the lncRNA expression data. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used to mine mRNA functions, and a lncRNA­mRNA network was constructed. Additionally, cis­ and trans­regulatory gene analyses of DELs were predicted. A total of 134 DELs (fold change >2, false discovery rate <0.05) and 1,006 DEGs (fold change >2 and P<0.05) were identified. Eighteen lncRNAs were predicted to regulate heme oxygenase 1, mitotic checkpoint serine/threonine kinase B, chemokine ligand 2 and DNA Topoisomerase IIα, amongst other genes. These genes are all associated with a cellular response to inorganic substances, alkaloids, estradiol, reactive oxygen species, metal ions, oxidative stress, and are associated with metabolic pathways, chemokine signaling pathways, malaria, Parkinson's disease, the cell cycle and other GO and KEGG pathway enrichments. The present study identifies novel DELs and an lncRNA­mRNA regulatory network that may allow for an improved understanding of the molecular mechanism of ischemic stroke induced by MCAO.

Identification and functional analysis of microRNAs in rats following focal cerebral ischemia injury.

MicroRNA sequencing (miRNA­seq) was performed in the present study to investigate miRNA expression profiles in infarcted brain areas following focal cerebral ischemia induced by middle cerebral artery occlusion in rats. In total, 20 miRNAs were identified to be upregulated and 17 to be downregulated in the infarct area. The expression levels of six differentially expressed miRNAs (DEmiRs), miR­211­5p, miR­183­5p, miR­10b­3p, miR­182, miR­217­5p and miR­96­5p, were examined by reverse transcription­â€‹quantitative polymerase chain reaction. Subsequently, a miRNA­mRNA network was constructed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to investigate the functions of the mRNAs targeted by these DEmiRs. The present study aimed to investigate the association between miRNAs and cerebral ischemia to provide potential insight into the molecular mechanisms underlying ischemic stroke.

RNA Sequencing for Gene Expression Profiles in a Rat Model of Middle Cerebral Artery Occlusion.

Gene expression regulatory mechanisms in models of middle cerebral artery occlusion (MCAO) have been assessed in some studies, but questions remain. The discovery of differentially expressed genes (DEGs) between MCAO and control rats analyzed by next-generation RNA sequencing is of particular interest. These DEGs may help guide the clinical diagnosis of stroke and facilitate selection of the optimal treatment strategy. Twenty rats were equally divided into control and MCAO groups. Three rats from each group were randomly selected for RNA sequencing analysis. Sequence reads were obtained from an Illumina HiSeq2500 platform and mapped onto the rat reference genome RN6 using Hisat2. We identified 1,007 significantly DEGs with p<0.05, including 785 upregulated (fold change [FC]>2) and 222 downregulated (FC<0.5) DEGs, in brain tissue from MCAO rats compared with that from control rats, and numerous immune response genes were identified. Gene ontology (GO) analysis revealed that the majority of the most enriched and meaningful biological process terms were mainly involved in immune response, inflammatory response, cell activation, leukocyte migration, cell adhesion, response to external stimulus, cell migration, and response to wounding. Also enriched were immune-related pathways and neural-related pathways. Similar to GO molecular function terms, the enriched terms were mainly related to cytokine receptor activity. Six DEGs were verified by quantitative real-time polymerase chain reaction (qRT-PCR). Protein-protein interaction analysis of these hits revealed that MCAO affects complement and coagulation cascades and chemokine signaling pathway. Our study identified novel biomarkers that could help further elucidate MCAO mechanisms and processes.

High Throughput mRNA Sequencing Reveals Potential Therapeutic Targets of Tao-Hong-Si-Wu Decoction in Experimental Middle Cerebral Artery Occlusion.

Background: Experimental and clinical studies have shown that Tao-Hong-Si-Wu decoction (THSWD) improved neurological deficits resulting from Middle Cerebral Artery Occlusion (MCAO). However, the mechanisms of action of THSWD in MCAO have not been characterized. In this study, the mRNA transcriptome was used to study various therapeutic targets of THSWD. Methods: RNA-seq was used to identify differentially expressed genes (DEGs). MCAO-induced up-regulated genes (MCAO vs. control) and THSWD-induced down-regulated genes (compared to MCAO) were identified. Intersection genes were defined as up-regulated differentially expression genes (up-DEGs) identified as MCAO-induced gene expression that were reversed by THSWD. Genes down-regulated by MCAO and up-regulated by THSWD were grouped as another series of intersections. Biological functions and signaling pathways were determined by gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses. In addition, several identified genes were validated by RT-qPCR. Results: A total of 339 DEGs were filtered based on the 2 series (MCAO vs. control and MCAO vs. THSWD), and were represented by genes involved in cell cycle (rno04110), ECM-receptor interaction (rno04512), complement and coagulation cascades (rno04610), focal adhesion (rno04510), hematopoietic cell lineage (rno04640), neuroactive ligand-receptor interaction (rno04080), cocaine addiction (rno05030), amphetamine addiction (rno05031), nicotine addiction (rno05033), fat digestion and absorption (rno04975), glycerophospholipid metabolism (rno00564), and others. The protein-protein interaction (PPI) network consisted of 202 nodes and 1,700 connections, and identified two main modules by MOCDE. Conclusion: Cell cycle (rno04110), ECM-receptor interaction (rno04512), complement and coagulation cascades (rno04610), focal adhesion (rno04510), hematopoietic cell lineage (rno04640), and neuroactive ligand-receptor interactions (rno04080) are potential therapeutic targets of THSWD in MCAO. This study provided a theoretical basis for THSWD prevention of neurological deficits resulting from intracerebral hemorrhage.