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Chemoproteomics reveals berberine directly binds to PKM2 to inhibit the progression of colorectal cancer.

ABSTRACT: Colorectal cancer is one of the most serious tumors and berberine can inhibit the recurrence and transformation of colorectal adenoma into colorectal cancer. However, the direct binding target proteins of berberine in inhibiting colorectal cancer remain unclear. In this study, the chemical proteomics method was used and demonstrated that berberine is directly bound to pyruvate kinase isozyme type M2 (PKM2) in colorectal cancer cells. The triangular N-O-O triangular structure of berberine contributed to hydrophobic interaction with I119 amino acid residues and π-π interaction with F244 amino acid residues of PKM2 protein. Moreover, berberine was shown to inhibit the reprogramming of glucose metabolism and the phosphorylation of STAT3, down regulate the expression of Bcl-2 and Cyclin D1 genes, ultimately inhibiting the progression of colorectal cancer. This study uncovered the direct binding target protein and mechanism of berberine to improve metabolic reprogramming in colorectal cancer, which is helpful to guide the optimization of berberine.

SUBMITTER: Yan SH

PROVIDER: S-EPMC9386086 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Chemoproteomics reveals berberine directly binds to PKM2 to inhibit the progression of colorectal cancer.

Yan Shi-Hai SH Hu Li-Mu LM Hao Xue-Hui XH Liu Jiang J Tan Xi-Ying XY Geng Zhi-Rong ZR Ma Jing J Wang Zhi-Lin ZL

iScience 20220720 8

Colorectal cancer is one of the most serious tumors and berberine can inhibit the recurrence and transformation of colorectal adenoma into colorectal cancer. However, the direct binding target proteins of berberine in inhibiting colorectal cancer remain unclear. In this study, the chemical proteomics method was used and demonstrated that berberine is directly bound to pyruvate kinase isozyme type M2 (PKM2) in colorectal cancer cells. The triangular N-<i>O</i>-O triangular structure of berberine ...[more]

PMID: 35992091

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Project description:Background: Colorectal cancer (CRC) is a common malignant tumor with a poor prognosis. Long noncoding RNAs (lncRNAs) have recently gained attention for their pivotal role in regulating cancer progression, including CRC. This study aimed to investigate the biological mechanisms underlying the participation of long intergenic non-protein coding RNA 1134 (LINC01134) in the progression of CRC. Material and Methods: Quantitative Real-time-PCR (RT-qPCR) and western blot were applied to assess the expression levels of mRNA and protein. Functional experiments (CCK8 assay, colon formation assay, EdU assay and flow cytometry) were applied to assess cell viability and apoptosis. RNA-RNA interaction assays, subcellular fractionation analysis and dual luciferase reporter assays were employed to explore molecular interactions between LINC01134 and solute carrier family 1 member 5 (SLC1A5). The mRNA stability was analyzed using actinomycin D (ActD). Results: We found that LINC01134 expression was highly expressed in CRC tissues and positively correlated with advanced clinical stages and unfavorable prognosis, which is consistent with findings from CRC cell lines. Functional experiments showed that suppressing LINC01134 restrained the proliferation of CRC both in vitro and in vivo and induced apoptosis of CRC cells. Gene co-expression analysis revealed a positive relationship between LINC01134 and SLC1A5, which was also upregulated and associated with unfavorable prognosis in CRC. Further analysis of RNA interactions and mRNA stability revealed that LINC01134 directly binds to SLC1A5 mRNA, enhancing its stability. Remarkably, silencing SLC1A5 expression partially counteracted the promotion of CRC cell proliferation by LINC01134 overexpression and alleviated its inhibition of apoptosis. Conclusions: Our findings indicated that LINC01134 functioned as an oncogene in CRC by binding directly to SLC1A5 mRNA and increasing its stability. Therefore, targeting LINC01134 could be a potential therapeutic target for treating CRC.

Project description:BackgroundAerobic glycolysis is a tumor cell phenotype and a hallmark in cancer research. The alternative splicing of the pyruvate kinase M (PKM) gene regulates the expressions of PKM1/2 isoforms and the aerobic glycolysis of tumors. Polypyrimidine tract binding protein (PTBP1) is critical in this process; however, its impact and underlying mechanisms in colorectal cancer (CRC) remain unclear. This study aimed to investigate the role of PTBP1 crotonylation in CRC progression.MethodsThe crotonylation levels of PTBP1 in human CRC tissues and cell lines were analyzed using crotonylation proteomics and immunoprecipitation. The main crotonylation sites were identified by immunoprecipitation and immunofluorescent staining. The glycolytic capacities of CRC cells were evaluated by measuring the glucose uptake, lactate production, extracellular acidification rate, and glycolytic proton efflux rate. The role and mechanism of PTBP1 crotonylation in PKM alternative splicing were determined by Western blot, quantitative real-time PCR (RT-qPCR), RNA immunoprecipitation, and immunoprecipitation. The effects of PTBP1 crotonylation on the behaviors of CRC cells and CRC progression were assessed using CCK-8, colony formation, cell invasion, wound healing assays, xenograft model construction, and immunohistochemistry.ResultsThe crotonylation level of PTBP1 was elevated in human CRC tissues compared to peritumor tissues. In CRC tissues and cells, PTBP1 was mainly crotonylated at K266 (PTBP1 K266-Cr), and lysine acetyltransferase 2B (KAT2B) acted as the crotonyltranferase. PTBP1 K266-Cr promoted glycolysis and lactic acid production, increasing the PKM2/PKM1 ratio in CRC tissues and cells. Mechanistically, PTBP1 K266-Cr enhanced the interaction of PTBP1 with heterogeneous nuclear ribonucleoprotein A1 and A2 (hnRNPA1/2), thus affecting the PKM alternative splicing. PTBP1 K266-Cr facilitated CRC cell proliferation, migration, and metastasis in vitro and in vivo. Pathologically, a high level of PTBP1 K266-Cr was associated with poor prognosis in CRC patients.ConclusionsCrotonylation of PTBP1 coordinates tumor cell glycolysis and promotes CRC progression by regulating PKM alternative splicing and increasing PKM2 expression.

Project description:BackgroundGastric cancer (GC) is marked by high incidence, malignancy, and poor prognosis. Understanding its development mechanisms and discovering effective drugs are urgent needs. Elevated oxidative stress levels in GC patients have been linked to disease progression. Berberine, an isoquinoline alkaloid from Coptis chinensis, exhibits strong anti-GC properties without notable side effects. However, its impact and mechanisms regarding oxidative stress in GC remain unclear. This study aims to explore berberine's anti-GC mechanisms through network pharmacology and validate findings via in vitro experiments.MethodsBerberine's target genes were sourced from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) and Comparative Toxicogenomics Database (CTD). GC-related targets were gathered from GeneCards, Online Mendelian Inheritance in Man (OMIM), PharmGkb, the Therapeutic Target Database (TTD), and DrugBank. The intersection of these targets facilitated the construction of a "drug-disease-target" network using Cytoscape 3.9.1. A protein-protein interaction (PPI) network was developed via the STRING database, and core targets were identified through visualization and topological analysis. Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using R. Subsequently, in vitro experiments validated the pharmacology predictions, evaluating berberine's effects on AGS and MKN45 GC cell viability and migration through Cell Counting Kit-8 (CCK-8) and cell scratch assays. The impact of berberine on reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) levels was assessed using specific detection kits. Additionally, the influence of berberine on oxidative stress-related signaling pathways nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1), hypoxia-inducible factor-1α (HIF-1α), and epithelial-mesenchymal transition (EMT) was assessed through Western blot analysis.ResultsNetwork pharmacology analysis identified 281 targets for berberine and 8,953 targets related to GC, revealing 224 common targets. GO enrichment analysis encompassed 3,001 biological processes, with the top 10 including responses to external biotic stimuli, oxidative stress, nutrient levels, chemical stress, oxygen levels, and hypoxia. Additionally, 122 cellular components and 213 molecular functions were identified. KEGG pathway enrichment analysis indicated 176 related signaling pathways, with key pathways for berberine's anti-GC effects potentially including phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT), forkhead box O (FOXO), and HIF-1. In vitro experiments demonstrated that berberine significantly inhibited GC cell activity and migration, increased intracellular levels of ROS and MDA, reduced levels of SOD, and suppressed the expression of Nrf2/HO-1, HIF-1α, and EMT pathway proteins.ConclusionsRegulation of oxidative stress may be one of the key mechanisms by which berberine inhibits the progression of gastric cancer.

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Chemoproteomics reveals berberine directly binds to PKM2 to inhibit the progression of colorectal cancer.

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Chemoproteomics reveals berberine directly binds to PKM2 to inhibit the progression of colorectal cancer.

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