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1.
J Nat Med ; 76(3): 686-692, 2022 Jun.
Article in English | MEDLINE | ID: mdl-35445961

ABSTRACT

Metastasis is responsible for approximately 90% of cancer-associated mortality and proceeds through multiple steps. Several herbal medicines are reported to inhibit primary tumor growth, but the suppressor effects of the medicines on metastasis progression are still not fully elucidated. Here we report that cinnamon bark extract (CBE) has a suppressor effect on metastatic dissemination of cancer cells. Through a phenotypic screening using zebrafish embryos, CBE was identified to interfere with the gastrulation progression of zebrafish embryos, of which the molecular mechanisms are conserved in metastasis progression. A Boyden chamber assay showed that CBE decreased cell motility and invasion of MDA-MB-231 human breast cancer cells without affecting their cell viability. Furthermore, CBE suppressed metastatic dissemination of the cells in a zebrafish xenotransplantation model. Quantitative metabolome analyses revealed that the productions of glucose-6-phosphate (G6P) and fructose 6-phosphate which are intermediate metabolites of glycolytic metabolism were interrupted in CBE-treated cells. qPCR and western-blotting analyses revealed that CBE-treated cells showed decreased expression of hexokinase 2 (HK2) which yields G6P. Pharmacological inhibition of HK2 with 2-deoxy-D-glucose suppressed cell invasion and migration of the cells without affecting their cell viability. Taken together, CBE suppresses metastatic dissemination of cancer cells through inhibition of glycolysis metabolism.


Subject(s)
Breast Neoplasms , Zebrafish , Animals , Cell Line, Tumor , Cell Proliferation , Cinnamomum zeylanicum , Female , Glycolysis , Humans , Plant Bark , Plant Extracts/pharmacology
2.
Sci Rep ; 11(1): 13474, 2021 06 29.
Article in English | MEDLINE | ID: mdl-34188151

ABSTRACT

Deoxyribonucleotide biosynthesis from ribonucleotides supports the growth of active cancer cells by producing building blocks for DNA. Although ribonucleotide reductase (RNR) is known to catalyze the rate-limiting step of de novo deoxyribonucleotide triphosphate (dNTP) synthesis, the biological function of the RNR large subunit (RRM1) in small-cell lung carcinoma (SCLC) remains unclear. In this study, we established siRNA-transfected SCLC cell lines to investigate the anticancer effect of silencing RRM1 gene expression. We found that RRM1 is required for the full growth of SCLC cells both in vitro and in vivo. In particular, the deletion of RRM1 induced a DNA damage response in SCLC cells and decreased the number of cells with S phase cell cycle arrest. We also elucidated the overall changes in the metabolic profile of SCLC cells caused by RRM1 deletion. Together, our findings reveal a relationship between the deoxyribonucleotide biosynthesis axis and key metabolic changes in SCLC, which may indicate a possible link between tumor growth and the regulation of deoxyribonucleotide metabolism in SCLC.


Subject(s)
Cell Proliferation , Deoxyribonucleotides/biosynthesis , Lung Neoplasms/metabolism , Small Cell Lung Carcinoma/metabolism , Animals , Cell Line, Tumor , DNA Damage , Deoxyribonucleotides/genetics , Female , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mice , Mice, Nude , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Ribonucleoside Diphosphate Reductase/genetics , Ribonucleoside Diphosphate Reductase/metabolism , Small Cell Lung Carcinoma/genetics , Small Cell Lung Carcinoma/pathology
3.
Biochem Biophys Res Commun ; 526(1): 191-198, 2020 05 21.
Article in English | MEDLINE | ID: mdl-32201076

ABSTRACT

Collagen type I (Col I) is one of the major extracellular matrix proteins in the cancer tissue. Previously, we have reported that Col I induces epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance by mTOR activation through Akt and ERK1/2 independent pathway. In this study, we aimed to elucidate the molecular mechanism of Col I induced EGFR-TKI resistance. First, we demonstrated the uptake of fluorescently labeled Col I by EGFR-mutated lung cancer cell line PC-9 cells using confocal microscopy and flow cytometry. Metabolome analysis revealed that the metabolic profiles of PC-9 cells was influenced by Col I treatment. Uptake of Col I into PC-9 cells was not inhibited by MMP inhibitor, GM6001, and endocytosis inhibitors, Pitstop2 and Dyngo4a; however, macropinocytosis inhibitor EIPA prevented its uptake. Moreover, the combination of EIPA and EGFR-TKI abrogated Col I-induced EGFR-TKI resistance in PC-9 cells. Inhibition of Rac1, which is essential for micropinocytosis, also decreased the uptake of Col I in PC-9 cells and restored their sensitivity to EGFR-TKI. Thus, EGFR mutated lung cancer cells could develop EGFR-TKI resistance by Col I uptake by macropinocytosis route.


Subject(s)
Antineoplastic Agents/pharmacology , Collagen Type I/metabolism , Drug Resistance, Neoplasm , Pinocytosis , TOR Serine-Threonine Kinases/metabolism , Amino Acids/metabolism , Cell Line, Tumor , Cytoplasm/drug effects , Cytoplasm/metabolism , Drug Resistance, Neoplasm/drug effects , ErbB Receptors/metabolism , Gefitinib/pharmacology , Humans , Mechanistic Target of Rapamycin Complex 1/metabolism , Metabolomics , Pinocytosis/drug effects , Protein Kinase Inhibitors/pharmacology , rac1 GTP-Binding Protein/metabolism
4.
J Vis Exp ; (148)2019 06 09.
Article in English | MEDLINE | ID: mdl-31233016

ABSTRACT

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.


Subject(s)
Electrophoresis, Capillary/methods , Mass Spectrometry/methods , Metabolomics/methods , Biomarkers/metabolism , Cell Adhesion , Cells, Cultured , Humans , Water/chemistry
5.
Cancer Cell ; 35(2): 177-190.e8, 2019 02 11.
Article in English | MEDLINE | ID: mdl-30686770

ABSTRACT

ARID1A encodes an SWI/SNF chromatin-remodeling factor and is frequently mutated in various cancers. This study demonstrates that ARID1A-deficient cancer cells are specifically vulnerable to inhibition of the antioxidant glutathione (GSH) and the glutamate-cysteine ligase synthetase catalytic subunit (GCLC), a rate-limiting enzyme for GSH synthesis. Inhibition of GCLC markedly decreased GSH in ARID1A-deficient cancer cells, leading to apoptotic cell death triggered by excessive amounts of reactive oxygen species. The vulnerability of ARID1A-deficient cancer cells results from low basal levels of GSH due to impaired expression of SLC7A11. The SLC7A11-encoded cystine transporter supplies cells with cysteine, a key source of GSH, and its expression is enhanced by ARID1A-mediated chromatin remodeling. Thus, ARID1A-deficient cancers are susceptible to synthetic lethal targeting of GCLC.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Enzyme Inhibitors/pharmacology , Glutamate-Cysteine Ligase/antagonists & inhibitors , Glutathione/metabolism , Nuclear Proteins/deficiency , Ovarian Neoplasms/drug therapy , Oxidative Stress/drug effects , Quinuclidines/pharmacology , Transcription Factors/deficiency , Amino Acid Transport System y+/genetics , Amino Acid Transport System y+/metabolism , Animals , DNA-Binding Proteins , Female , Glutamate-Cysteine Ligase/metabolism , HCT116 Cells , Humans , Mice, Inbred BALB C , Mice, Nude , Molecular Targeted Therapy , Nuclear Proteins/genetics , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Transcription Factors/genetics , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
6.
Front Pharmacol ; 9: 1129, 2018.
Article in English | MEDLINE | ID: mdl-30369878

ABSTRACT

Antifolates are a class of drugs effective for treating malignant pleural mesothelioma (MPM). The majority of antifolates inhibit enzymes involved in purine and pyrimidine synthesis such as dihydrofolate reductase (DHFR), thymidylate synthase (TYMS), and glycinamide ribonucleotide formyltransferase (GART). In order to select the most suitable patients for effective therapy with drugs targeting specific metabolic pathways, there is a need for better predictive metabolic biomarkers. Antifolates can alter global metabolic pathways in MPM cells, yet the metabolic profile of treated cells has not yet been clearly elucidated. Here we found that MPM cell lines could be categorized into two groups according to their sensitivity or resistance to pemetrexed treatment. We show that pemetrexed susceptibility could be reversed and DNA synthesis rescued in drug-treated cells by the exogenous addition of the nucleotide precursors hypoxanthine and thymidine (HT). We observed that the expression of pemetrexed-targeted enzymes in resistant MPM cells was quantitatively lower than that seen in pemetrexed-sensitive cells. Metabolomic analysis revealed that glycine and choline, which are involved in one-carbon metabolism, were altered after drug treatment in pemetrexed-sensitive but not resistant MPM cells. The addition of HT upregulated the concentration of inosine monophosphate (IMP) in pemetrexed-sensitive MPM cells, indicating that the nucleic acid biosynthesis pathway is important for predicting the efficacy of pemetrexed in MPM cells. Our data provide evidence that may link therapeutic response to the regulation of metabolism, and points to potential biomarkers for informing clinical decisions regarding the most effective therapies for patients with MPM.

7.
Cancer Res ; 78(9): 2179-2190, 2018 05 01.
Article in English | MEDLINE | ID: mdl-29490947

ABSTRACT

Comprehensive genomic analysis has revealed that the PI3K/AKT/mTOR pathway is a feasible therapeutic target in small-cell lung carcinoma (SCLC). However, biomarkers to identify patients likely to benefit from inhibitors of this pathway have not been identified. Here, we show that metabolic features determine sensitivity to the PI3K/mTOR dual inhibitor gedatolisib in SCLC cells. Substantial phosphatidyl lipid analysis revealed that a specific phosphatidylinositol (3,4,5)-trisphosphate (PIP3) subspecies lipid product PIP3 (38:4) is predictive in assessing sensitivity to PI3K/mTOR dual inhibitor. Notably, we found that higher amounts of purine-related aqueous metabolites such as hypoxanthine, which are characteristic of SCLC biology, lead to resistance to PI3K pathway inhibition. In addition, the levels of the mRNA encoding hypoxanthine phosphoribosyl transferase 1, a key component of the purine salvage pathway, differed significantly between SCLC cells sensitive or resistant to gedatolisib. Moreover, complementation with purine metabolites could reverse the vulnerability to targeting of the PI3K pathway in SCLC cells normally sensitive to gedatolisib. These results indicate that the resistance mechanism of PI3K pathway inhibitors is mediated by the activation of the purine salvage pathway, supplying purine resource to nucleotide biosynthesis. Metabolomics is a powerful approach for finding novel therapeutic biomarkers in SCLC treatment.Significance: These findings identify features that determine sensitivity of SCLC to PI3K pathway inhibition and support metabolomics as a tool for finding novel therapeutic biomarkers. Cancer Res; 78(9); 2179-90. ©2018 AACR.


Subject(s)
Biomarkers, Pharmacological/metabolism , Phosphatidylinositol 3-Kinases/genetics , Protein Kinase Inhibitors/administration & dosage , Small Cell Lung Carcinoma/drug therapy , Animals , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , Female , Humans , Male , Metabolomics , Mice , Morpholines/administration & dosage , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Purines/metabolism , Signal Transduction/drug effects , Small Cell Lung Carcinoma/genetics , Small Cell Lung Carcinoma/metabolism , Small Cell Lung Carcinoma/pathology , TOR Serine-Threonine Kinases/genetics , Triazines/administration & dosage
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