RESUMO
BACKGROUND: c-MET is a receptor tyrosine kinase which is classically activated by HGF to activate its downstream signaling cascades such as MAPK, PI3K/Akt/mTOR, and STAT3. The c-MET modulates cell proliferation, epithelial-mesenchymal transition (EMT), immune response, morphogenesis, apoptosis, and angiogenesis. The c-MET has been shown to serve a prominent role in embryogenesis and early development. The c-MET pathway is deregulated in a broad range of malignancies, due to overexpression of ligands or receptors, genomic amplification, and MET mutations. The link between the deregulation of c-MET signaling and tumor progression has been well-documented. Overexpression or overactivation of c-MET is associated with dismal clinical outcomes and acquired resistance to targeted therapies. Since c-MET activation results in the triggering of oncogenic pathways, abrogating the c-MET pathway is considered to be a pivotal strategy in cancer therapeutics. Herein, an analysis of role of the c-MET pathway in human cancers and its relevance in bone metastasis and therapeutic resistance has been undertaken. Also, an attempt has been made to summarize the inhibitory activity of selected natural compounds towards c-MET signaling in cancers. METHODS: The publications related to c-MET pathway in malignancies and its natural compound modulators were obtained from databases such as PubMed, Scopus, and Google Scholar and summarized based on PRISMA guidelines. Some of the keywords used for extracting relevant literature are c-MET, natural compound inhibitors of c-MET, c-MET in liver cancer, c-MET in breast cancer, c-MET in lung cancer, c-MET in pancreatic cancer, c-MET in head and neck cancer, c-MET in bone metastasis, c-MET in therapeutic resistance, and combination of c-MET inhibitors and chemotherapeutic agents. The chemical structure of natural compounds was verified in PubChem database. RESULTS: The search yielded 3935 publications, of which 195 reference publications were used for our analysis. Clinical trials were referenced using ClinicalTrials.gov identifier. The c-MET pathway has been recognized as a prominent target to combat the growth, metastasis, and chemotherapeutic resistance in cancers. The key role of the c-MET in bone metastasis as well as therapeutic resistance has been elaborated. Also, suppressive effect of selected natural compounds on the c-MET pathway in clinical/preclinical studies has been discussed.
Assuntos
Neoplasias , Proteínas Proto-Oncogênicas c-met , Transdução de Sinais , Humanos , Proteínas Proto-Oncogênicas c-met/metabolismo , Neoplasias/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/secundário , Neoplasias Ósseas/metabolismoRESUMO
BACKGROUND AND AIMS: Aberrant activation of fatty acid synthase (FASN) is a major metabolic event during the development of HCC. We evaluated the therapeutic efficacy of TVB3664, a FASN inhibitor, either alone or in combination, for HCC treatment. APPROACH AND RESULTS: The therapeutic efficacy and the molecular pathways targeted by TVB3664, either alone or with tyrosine kinase inhibitors or the checkpoint inhibitor anti-programmed death ligand 1 antibody, were assessed in human HCC cell lines and multiple oncogene-driven HCC mouse models. RNA sequencing was performed to elucidate the effects of TVB3664 on global gene expression and tumor metabolism. TVB3664 significantly ameliorated the fatty liver phenotype in the aged mice and AKT-induced hepatic steatosis. TVB3664 monotherapy showed moderate efficacy in NASH-related murine HCCs, induced by loss of phosphatase and tensin homolog and MET proto-oncogene, receptor tyrosine kinase (c-MET) overexpression. TVB3664, in combination with cabozantinib, triggered tumor regression in this murine model but did not improve the responsiveness to immunotherapy. Global gene expression revealed that TVB3664 predominantly modulated metabolic processes, whereas TVB3664 synergized with cabozantinib to down-regulate multiple cancer-related pathways, especially the AKT/mammalian target of rapamycin pathway and cell proliferation genes. TVB3664 also improved the therapeutic efficacy of sorafenib and cabozantinib in the FASN-dependent c-MYC-driven HCC model. However, TVB3664 had no efficacy nor synergistic effects in FASN-independent murine HCC models. CONCLUSIONS: This preclinical study suggests the limited efficacy of targeting FASN as monotherapy for HCC treatment. However, FASN inhibitors could be combined with other drugs for improved effectiveness. These combination therapies could be developed based on the driver oncogenes, supporting precision medicine approaches for HCC treatment.
Assuntos
Carcinoma Hepatocelular , Ácido Graxo Sintase Tipo I , Neoplasias Hepáticas , Anilidas , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Ácido Graxo Sintase Tipo I/antagonistas & inibidores , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Ácido Graxo Sintases/antagonistas & inibidores , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Mamíferos/metabolismo , Camundongos , Monoéster Fosfórico Hidrolases/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Piridinas , Sorafenibe/farmacologia , Serina-Treonina Quinases TOR , TensinasRESUMO
Hepatocellular carcinoma (HCC), the most prevalent liver cancer, is considered one of the most lethal malignancies with a dismal outcome. There is an urgent need to find novel therapeutic approaches to treat HCC. At present, natural products have served as a valuable source for drug discovery. Here, we obtained five known biflavones from the root of Stellera chamaejasme and evaluated their activities against HCC Hep3B cells in vitro. Chamaejasmenin E (CE) exhibited the strongest inhibitory effect among these biflavones. Furthermore, we found that CE could suppress the cell proliferation and colony formation, as well as the migration ability of HCC cells, but there was no significant toxicity on normal liver cells. Additionally, CE induced mitochondrial dysfunction and oxidative stress, eventually leading to cellular apoptosis. Mechanistically, the potential target of CE was predicted by database screening, showing that the compound might exert an inhibitory effect by targeting at c-Met. Next, this result was confirmed by molecular docking, cellular thermal shift assay (CETSA), as well as RT-PCR and Western blot analysis. Meanwhile, CE also reduced the downstream proteins of c-Met in HCC cells. In concordance with above results, CE is efficacious and non-toxic in tumor xenograft model. Taken together, our findings revealed an underlying tumor-suppressive mechanism of CE, which provided a foundation for identifying the target of biflavones.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Biflavonoides/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Extratos Vegetais/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Thymelaeaceae/química , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Biflavonoides/química , Biflavonoides/isolamento & purificação , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Estrutura Molecular , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/isolamento & purificação , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Relação Estrutura-AtividadeRESUMO
Berberine is a natural product that has long been used in traditional Chinese medicine due to its antimicrobial, anti-inflammatory and metabolism-regulatory properties. Osimertinib is the first third-generation EGFR-tyrosine kinase inhibitor (TKI) approved for the treatment of non-small cell lung cancer (NSCLC) with activating EGFR mutations and those resistant to earlier generation EGFR-TKIs due to a T790M mutation. However, emergence of acquired resistance to osimertinib limits its long-term efficacy in the clinic. One known mechanism of acquired resistance to osimertinib and other EGFR-TKIs is MET (c-MET) gene amplification. Here, we report that berberine, when combined with osimertinib, synergistically and selectively decreased the survival of several MET-amplified osimertinib-resistant EGFR mutant NSCLC cell lines with enhanced induction of apoptosis likely through Bim elevation and Mcl-1 reduction. Importantly, this combination effectively enhanced suppressive effect on the growth of MET-amplified osimertinib-resistant xenografts in nude mice and was well tolerated. Molecular modeling showed that berberine was able to bind to the kinase domain of non-phosphorylated MET, occupy the front of the binding pocket, and interact with the activation loop, in a similar way as other known MET inhibitors do. MET kinase assay showed clear concentration-dependent inhibitory effects of berberine against MET activity, confirming its kinase inhibitory activity. These findings collectively suggest that berberine can act as a naturally-existing MET inhibitor to synergize with osimertinib in overcoming osimertinib acquired resistance caused by MET amplification.
Assuntos
Acrilamidas/administração & dosagem , Compostos de Anilina/administração & dosagem , Antineoplásicos/administração & dosagem , Berberina/administração & dosagem , Produtos Biológicos/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos Nus , Proteínas Proto-Oncogênicas c-met/metabolismoRESUMO
Hepatocellular carcinoma (HCC) has been identified as one of the most deadly malignancies with limited therapeutic efficacy worldwide. However, understanding the molecular mechanisms of crosstalk between signaling pathways in HCC and predicting cancer cell responses to targeted therapeutic interventions remain to be challenge. Thus, in this study, we aimed to evaluate the anticancerous efficacy of Silybum marianum total extract (STE), silymarin (Sm), and silibinin (Sb) against experimentally-induced HCC in rats. In vitro investigations were also performed and the anticancer effects against HCC cell lines (HepG2 and Huh7) were confirmed. Wistar rats were given diethylnitrosamine (DEN)/2-acetylaminofluorene (AAF)/carbon tetrachloride (CCl4) and were orally treated with STE (200 mg/kg body weight (bw)), Sm (150 mg/kg bw), and Sb (5 mg/kg bw) every other day from the 1st or 16th week to the 25th week of DEN/AAF/CCl4 injection. Treatment with STE, Sm, and Sb inhibited the growth of cancerous lesions in DEN/AAF/CCl4-treated rats. This inhibition was associated with inhibition of Ki-67 expression and repression of HGF/cMet, Wnt/ß-catenin, and PI3K/Akt/mTOR signaling pathways. STE, Sm, and Sb improved liver function biomarkers and tumor markers (AFP, CEA, and CA19.9) and increased total protein and albumin levels in serum. STE, Sm, and Sb treatment was also noted to reduce the hepatic production of lipid peroxides, increase hepatic glutathione content, and induce the activities of hepatic antioxidant enzymes in DEN/AAF/CCl4-treated rats. These results indicate that STE, Sm, and Sb exert anti-HCC effects through multiple pathways, including suppression of Ki-67 expression and HGF/cMet, Wnt/ß-catenin, and PI3K/Akt/mTOR pathways and enhancement of antioxidant defense mechanisms.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas Experimentais/prevenção & controle , Neoplasias Hepáticas/tratamento farmacológico , Extratos Vegetais/farmacologia , Silybum marianum/química , Animais , Antioxidantes/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Células Hep G2 , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Neoplasias Hepáticas/patologia , Masculino , Fosfatidilinositol 3-Quinase/metabolismo , Extratos Vegetais/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Ratos , Ratos Wistar , Silibina/isolamento & purificação , Silibina/farmacologia , Silimarina/isolamento & purificação , Silimarina/farmacologia , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Thrombotic diseases seriously threaten human life. Justicia, as a common Chinese medicine, is usually used for anti-inflammatory treatment, and further studies have found that it has an inhibitory effect on platelet aggregation. Therefore, it can be inferred that Justicia can be used as a therapeutic drug for thrombosis. This work aims to reveal the pharmacological mechanism of the anti-thrombotic effect of Justicia through network pharmacology combined with wet experimental verification. During the analysis, 461 compound targets were predicted from various databases and 881 thrombus-related targets were collected. Then, herb-compound-target network and protein-protein interaction network of disease and prediction targets were constructed and cluster analysis was applied to further explore the connection between the targets. In addition, Gene Ontology (GO) and pathway (KEGG) enrichment were used to further determine the association between target proteins and diseases. Finally, the expression of hub target proteins of the core component and the anti-thrombotic effect of Justicia's core compounds were verified by experiments. In conclusion, the core bioactive components, especially justicidin D, can reduce thrombosis by regulating F2, MMP9, CXCL12, MET, RAC1, PDE5A, and ABCB1. The combination of network pharmacology and the experimental research strategies proposed in this paper provides a comprehensive method for systematically exploring the therapeutic mechanism of multi-component medicine.
Assuntos
Dioxolanos/farmacologia , Fibrinolíticos/farmacologia , Redes Reguladoras de Genes , Lignanas/farmacologia , Mapas de Interação de Proteínas , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Células Cultivadas , Quimiocina CXCL12/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Dioxolanos/química , Descoberta de Drogas/métodos , Fibrinolíticos/química , Humanos , Justicia/química , Lignanas/química , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
MET, the receptor of hepatocyte growth factor (HGF), is a driving factor in renal cell carcinoma (RCC) and also a proven drug target for cancer treatment. To improve the activity and to investigate the mechanisms of action of Apigenin (APG), novel derivatives of APG with improved properties were synthesized and their activities against Caki-1 human renal cancer cell line were evaluated. It was found that compound 15e exhibited excellent potency against the growth of multiple RCC cell lines including Caki-1, Caki-2 and ACHN and is superior to APG and Crizotinib. Subsequent investigations demonstrated that compound 15e can inhibit Caki-1 cell proliferation, migration and invasion. Mechanistically, 15e directly targeted the MET kinase domain, decreased its auto-phosphorylation at Y1234/Y1235 and inhibited its kinase activity and downstream signaling. Importantly, 15e had inhibitory activity against mutant MET V1238I and Y1248H which were resistant to approved MET inhibitors Cabozantinib, Crizotinib or Capmatinib. In vivo tumor graft study confirmed that 15e repressed RCC growth through inhibition of MET activation. These results indicate that compound 15e has the potential to be developed as a treatment for RCC, and especially against drug-resistant MET mutations.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Apigenina/farmacologia , Carcinoma/tratamento farmacológico , Neoplasias Renais/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Apigenina/química , Domínio Catalítico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Simulação de Acoplamento Molecular , Estrutura Molecular , Mutação , Neoplasias Experimentais/tratamento farmacológico , Fitoterapia , Conformação Proteica , Proteínas Proto-Oncogênicas c-met/genéticaRESUMO
BACKGROUND: Lung cancer has the highest incidence and cancer-related mortality of all cancers worldwide. Its treatment is focused on molecular targeted therapy. c-MET plays an important role in the development and metastasis of various human cancers and has been identified as an attractive potential anti-cancer target. Podophyllotoxin (PPT), an aryltetralin lignan isolated from the rhizomes of Podophyllum species, has several pharmacological activities that include anti-viral and anti-cancer effects. However, the mechanism of the anti-cancer effects of PPT on gefitinib-sensitive (HCC827) or -resistant (MET-amplified HCC827GR) non-small cell lung cancer (NSCLC) cells remains unexplored. PURPOSE: In the present study, we investigated the underlying mechanisms of PPT-induced apoptosis in NSCLC cells and found that the inhibition of c-MET kinase activity contributed to PPT-induced cell death. METHODS: The regulation of c-MET by PPT was examined by pull-down assay, ATP-competitive binding assay, kinase activity assay, molecular docking simulation, and Western blot analysis. The cell growth inhibitory effects of PPT on NSCLC cells were assessed using the MTT assay, soft agar assay, and flow cytometry analysis. RESULTS: PPT could directly interact with c-MET and inhibit kinase activity, which further induced the apoptosis of HCC827GR cells. In contrast, PPT did not significantly affect EGFR kinase activity. PPT significantly inhibited the cell viability of HCC827GR cells, whereas the PPT-treated HCC827 cells showed a cell viability of more than 80%. PPT dose-dependently induced G2/M cell cycle arrest, as shown by the downregulation of cyclin B1 and cdc2, and upregulation of p27 expression in HCC827GR cells. Furthermore, PPT treatment induced Bad expression and downregulation of Mcl-1, survivin, and Bcl-xl expression, subsequently activating multi-caspases. PPT thereby induced caspase-dependent apoptosis in HCC827GR cells. CONCLUSION: These results suggest the potential of PPT as a c-MET inhibitor to overcome tyrosine kinase inhibitor resistance in lung cancer.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Podofilotoxina/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Antineoplásicos Fitogênicos/química , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/genética , Gefitinibe/farmacologia , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular , Podofilotoxina/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/química , Proteínas Proto-Oncogênicas c-met/metabolismoRESUMO
Hepatocellular Carcinoma (HCC) is one of the most common malignant tumors, and high recurrence and metastasis are the major obstacles to successful treatment of HCC. Traditional Chinese medicine has little known and unique advantages in the treatment of HCC. Previous studies have confirmed that Chinese herbal formula Qingrejiedu (clears away heat and toxins), Huoxuehuayu (promotes blood flow to remove stasis) and Fuzhengguben (strengthens healthy qi and root) (QHF) has a significant effect on patients with advanced HCC, improves the quality of life and prolongs the survival time of patients significantly. In this study, we investigated the effect of QHF on proliferation, migration and invasion of human high metastatic hepatocellular carcinoma cell line HCCLM3 and its underlying mechanism. The results from our in vitro experiments showed that QHF has the ability to inhibit the proliferation by inducing G2/M phase cell cycle arrest and induce apoptosis. Moreover, QHF can also inhibit migration and invasion of HCCLM3 cells and the expression of the p-c-Met protein in HCCLM3 cells was down-regulated. c-Met is closely related to the metastasis of HCC, then we constructed a stable transfected cell line HepG2-met with high expression of c-Met by transfection. Further study in vivo revealed that c-Met gene will promote the growth of tumors and lung metastases in nude mice, and QHF intervention can reduce tumor lung metastases by inhibiting the HGF/c-Met signaling pathway. In conclusion, our study reveals that QHF can inhibit the proliferation, migration and invasion of HCCLM3, and this effect may be related to inhibiting HGF/c-Met signaling pathway.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Fator de Crescimento de Hepatócito/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Pulmonares/prevenção & controle , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/secundário , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Células Hep G2 , Fator de Crescimento de Hepatócito/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-met/genética , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Persimmon (Diospyros kaki L.f.) trees are widely cultivated for their edible fruits in Asia. D. kaki leaves are abundant in phytochemicals that have numerous medicinal properties. Hepatocyte growth factor (HGF) and its receptor Met lead to poor prognosis via the promotion of metastasis and chemoresistance in hepatocellular carcinoma (HCC). Therefore, inhibitors targeting the HGF/Met pathway are regarded as promising drugs against HCC. Here, we investigated the effects of D. kaki leaves on HGF-induced epithelial-to-mesenchymal transition (EMT) and stemness traits in HCC. The ethanol extract of D. kaki leaves (EEDK) markedly suppressed HGF-mediated cell migration and invasion through upregulation of CDH1 and downregulation of SNAI1, VIM, MMP1, MMP2, and MMP9. Moreover, EEDK increased the cytotoxicity of sorafenib, which was reduced by HGF, and decreased the expression of the stemness markers KRT19 and CD44. Additionally, we found a clear correlation between stemness and EMT markers in HCC patients. Importantly, EEDK reduced Met activity and attenuated HGF-mediated activation of JNK/c-Jun. Our findings provide new evidence that EEDK can ameliorate HCC with poor prognosis and aggressive phenotype by blocking HGF/Met signaling.
Assuntos
Carcinoma Hepatocelular/patologia , Diospyros/química , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fator de Crescimento de Hepatócito/metabolismo , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Extratos Vegetais/farmacologia , Folhas de Planta/química , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias Hepáticas/metabolismoRESUMO
ETHNOPHARMACOLOGY RELEVANCE: Si-Jun-Zi-Tang (SJZT) is a traditional Chinese medicine formula used to treat chronic and debilitating diseases including melanoma. SJZT-based therapies have achieved good clinical outcomes in melanoma management. However, the pharmacological basis of SJZT for its clinical use in melanoma treatment is not fully understood. AIM OF THE STUDY: To investigate the anti-melanoma effects and mechanism of action of an ethanolic extract of SJZT. MATERIALS AND METHODS: SJZT was extracted using 50% ethanol. A murine B16 melanoma-bearing mouse model was employed to investigate the anti-melanoma effects of SJZT. microRNA (miRNA) and mRNA levels were examined by RT-qPCR, and protein levels were measured by Western blotting. RESULTS: SJZT significantly inhibited B16 tumor growth in mice. Mechanistic investigations revealed that SJZT elevated miR-34b (a tumor suppressing miRNA), and lowered c-Met (a miR-34b target gene) and ß-catenin (a downstream molecule of c-Met signaling) expression levels in the B16 tumors. CONCLUSIONS: In this study we found, for the first time, that SJZT exerts anti-melanoma effects and regulates the miR-34b/c-Met/ß-catenin pathway in a melanoma mouse model. Our findings provide pharmacological justifications for the clinical use of SJZT in treating melanoma.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Melanoma Experimental/tratamento farmacológico , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , beta Catenina/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Masculino , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-met/genética , Transdução de Sinais , Carga Tumoral/efeitos dos fármacosRESUMO
Licochalcone D (LCD), a flavonoid isolated from a Chinese medicinal plant Glycyrrhizainflata, has a variety of pharmacological activities. However, the anti-cancer effects of LCD on non-small cell lung cancer (NSCLC) have not been investigated yet. The amplification of MET (hepatocyte growth factor receptor) compensates for the inhibition of epidermal growth factor receptor (EGFR) activity due to tyrosine kinase inhibitor (TKI), leading to TKI resistance. Therefore, EGFR and MET can be attractive targets for lung cancer. We investigated the anti-proliferative and apoptotic effects of LCD in lung cancer cells HCC827 (gefitinib-sensitive) and HCC827GR (gefitinib-resistant) through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, pull-down/kinase assay, cell cycle analysis, Annexin-V/7-ADD staining, reactive oxygen species (ROS) assay, mitochondrial membrane potential (MMP) assay, multi-caspase assay, and Western blot analysis. The results showed that LCD inhibited phosphorylation and the kinase activity of EGFR and MET. In addition, the predicted pose of LCD was competitively located at the ATP binding site. LCD suppressed lung cancer cells growth by blocking cell cycle progression at the G2/M transition and inducing apoptosis. LCD also induced caspases activation and poly (ADP-ribose) polymerase (PARP) cleavage, thus displaying features of apoptotic signals. These results provide evidence that LCD has anti-tumor effects by inhibiting EGFR and MET activities and inducing ROS-dependent apoptosis in NSCLC, suggesting that LCD has the potential to treat lung cancer.
Assuntos
Chalconas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Chalconas/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Receptores ErbB/metabolismo , Gefitinibe/farmacologia , Humanos , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Anomalous changes of the cell mesenchymal-epithelial transition factor (c-Met) receptor tyrosine kinase signaling pathway play an important role in the occurrence and development of human cancers, including gastric cancer. In this study, we designed and synthesized a novel peptide (CM 7) targeting the tyrosine kinase receptor c-Met, that can inhibit c-Met-mediated signaling in MKN-45 and U87 cells. Its affinity to human c-Met protein or c-Met-positive cells was determined, which showed specific binding to c-Met with high affinity. Its biological activities against MKN-45 c-Met-positive cells were evaluated in vitro and in vivo. As a result, peptide CM 7 exhibited moderate regulation of c-Met-mediated cell proliferation, migration, invasion, and scattering. The inhibitory effect of peptide CM 7 on tumor growth in vivo was investigated by establishing a xenograft mouse model using MKN-45 cells, and the growth inhibition rate of tumor masses for peptide CM 7 was 62%. Based on our data, CM 7 could be a promising therapeutic peptide for c-Met-dependent cancer patients.
Assuntos
Antineoplásicos/farmacologia , Terapia de Alvo Molecular , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Camundongos Nus , Modelos Moleculares , Invasividade Neoplásica , Peptídeos/síntese química , Peptídeos/química , Proteínas Proto-Oncogênicas c-met/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Fridericia platyphylla (Cham.) L.G. Lohmann (FP) has cytotoxic, anti-inflammatory, and analgesic properties. We aimed to characterize the cytotoxic and antiproliferative effects of FP extract on normal (GAS) and tumor-derived (ACP02 and HepG2) cell lines. The effective concentrations (EC50s) by tetrazolium bromide assay (MTT) were 56.16, 43.68, and 42.57 µg mL-1 and 69.38, 41.73, and 52.39 µg mL-1 by neutral red assay for GAS, ACP02, and HepG2 cells, respectively. The extract decreased nuclear division indices, which was not reflected in cell proliferation curves. Flow cytometric analyses showed that even 30 µg mL-1 extract (shown to be noncytotoxic by MTT assay) increased the sub-G1 population, indicating cell death due to apoptosis and necrosis. A cytokinesis-block micronucleus cytome assay showed that 30 µg mL-1 of the extract increased the frequency of nuclear buds in tumor cells. Real-time quantitative polymerase chain reaction showed CCND1 upregulation in doxorubicin-treated GAS cells and BCL-XL, BIRC5, and MET downregulation in 5 or 30 µg mL-1 in FP extract-treated ACP02 cells. In conclusion, FP extract modulated apoptosis- and cell cycle-related genes and presented selective cytotoxicity toward tumor cells that deserves further investigation by testing other cell types. Our results demonstrated that even medicinal plants exert adverse effects depending on the extract concentrations used and tissues investigated.
Assuntos
Bignoniaceae/química , Extratos Vegetais/farmacologia , Raízes de Plantas/química , Neoplasias Gástricas/tratamento farmacológico , Survivina/metabolismo , Proteína bcl-X/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Necrose , Extratos Vegetais/química , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Espécies Reativas de Oxigênio , Survivina/genética , Proteína bcl-X/genéticaRESUMO
Tubeimoside-1 (TBMS1) is one of the extracts of rhizoma bolbostemmae, which has remarkable anti-cancer function in the treatment of esophagus and gastric cancer in traditional Chinese medicine. However the mechanisms of its anti-cancer function is remain unclear. In this study, we demonstrate that TBMS1 could inhibit cell growth and metastasis in glioblastoma. MET is a member of the receptor tyrosine kinase family, which amplifies frequently in various human cancers. As an important proto-oncogene, multiple inhibitors have been developed for the therapy of cancers. Here, we found TBMS1 could reduce/decrease the protein level of MET via increasing its Ubiquitination degradation. Therefore, TBMS1 is a promising compound for the treatment of glioblastoma and an inhibitor of MET.
Assuntos
Glioblastoma/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Saponinas/farmacologia , Triterpenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Glioblastoma/patologia , Humanos , Camundongos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Epidermal growth factor receptor (EGFR) gene alterations are associated with sensitization to tyrosine kinase inhibitors such as gefitinib in lung cancer. Some patients suffering from non-small cell lung cancer (NSCLC) have difficulty in treating the cancer due to resistance acquired to gefitinib with MET amplification. Therefore EGFR and MET may be attractive targets for lung cancer therapy. PURPOSE: This study aimed to investigate the anti-cancer activity of Licochalcone (LC)B extracted from Glycyrrhiza inflata, in gefitinib-sensitive or gefitinib-resistant NSCLC cells, and to define its mechanisms. STUDY DESIGN: We investigated the mechanism of action of LCB by targeting EGFR and MET in human NSCLC cells. METHODS: We used the HCC827 and HCC827GR lines as gefitinib-sensitive and -resistant cells respectively, and determined the effects of LCB on both, by performing cell proliferation assay, flow cytometry analysis and Western blotting. Targets of LCB were identified by pull-down/kinase assay and molecular docking simulation. RESULTS: LCB inhibited both EGFR and MET kinase activity by directly binding to their ATP-binding pockets. The ability of this interaction was verified by computational docking and molecular dynamics simulations. LCB suppressed viability and colony formation of both HCC827 and HCC827GR cells while exhibiting no cytotoxicity to normal cells. The induction of G2/M cell-cycle arrest and apoptosis by LCB was confirmed by Annexin V/7-AAD double staining, ER stress and reactive oxygen species induction, mitochondrial membrane potential loss and caspase activation as well as related-proteins regulation. Inhibition of EGFR and MET by LCB decreased ERBB3 and AKT axis activation. CONCLUSION: We provide insights into the LCB-mediated mechanisms involved in reducing cell proliferation and inducing apoptosis in NSCLC cells. This occurs through dual inhibition of EGFR and MET in NSCLC cells regardless of their sensitivity or resistance to gefitinib. LCB may be a promising novel therapeutic medicine for gefitinib-sensitive or resistant NSCLC treatment.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Chalconas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/metabolismo , Antineoplásicos Fitogênicos/química , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Chalconas/química , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/química , Receptores ErbB/metabolismo , Gefitinibe/farmacologia , Glycyrrhiza/química , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular , Espécies Reativas de Oxigênio/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: The Chinese herbal medicine Fuzheng Kang-Ai (FZKA) decoction obtained from Guangdong Kangmei Pharmaceutical Company, which contains 12 components with different types of constituents, has been used as part of the adjuvant treatment of lung cancer for decades. We previously showed that FZKA decoction enhances the growth inhibition of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-resistant non-small cell lung cancer (NSCLC) cells by suppressing glycoprotein mucin 1 (MUC1) expression. However, the molecular mechanism underlying the therapeutic potential, particularly in sensitizing or/and enhancing the anti-lung cancer effect of EGFR-TKIs, remains unclear. MATERIALS AND METHODS: Cell viability was measured using 3-(4, 5-diMEThylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and 5-ethynyl -2'-deoxyuridine (EdU) assays. Western blot analysis was performed to examine the protein expressions of DNA methyltransferase 1 (DNMT1), specificity protein 1 (SP1), and MET, an oncogene encoding for a trans-membrane tyrosine kinase receptor activated by the hepatocyte growth factor (HGF). The expression of MET mRNA was measured by quantitative real-time PCR (qRT-PCR). Exogenous expression of DNMT1 and SP1, and MET were carried out by transient transfection assays. The promoter activity of MET was tested using Dual-luciferase reporter assays. A nude mouse xenografted tumor model further evaluated the effect of the combination of FZKA decoction and erlotinib in vivo. RESULTS: The combination of FZKA and erlotinib produced an even greater inhibition of NSCLC cell growth. FZKA decreased the expressions of DNMT1, SP1, and MET (c-MET) proteins, and the combination of FZKA and erlotinib demonstrated enhanced responses. Interestingly, there was a mutual regulation of DNMT1 and SP1. In addition, exogenously expressed DNMT1 and SP1 blocked the FZKA-inhibited c-MET expression. Moreover, excessive expressed MET neutralized FZKA-inhibited growth of NSCLC cells. FZKA decreased the mRNA and promoter activity of c-MET, which was not observed in cells with ectopic expressed DNMT1 gene. Similar findings were observed in vivo. CONCLUSION: FZKA decreases MET gene expression through the repression and mutual regulation of DNMT1 and SP1 in vitro and in vivo. This leads to inhibit the growth of human lung cancer cells. The combination of FZKA and EGFR-TKI erlotinib exhibits synergy in this process. The regulatory loops among the DNMT1, SP1 and MET converge in the overall effects of FZKA and EGFR-TKI erlotinib. This in vitro and in vivo study clarifies an additional novel molecular mechanism underlying the anti-lung cancer effects in response to the combination of FZKA and erlotinib in gefitinib-resistant NSCLC cells.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Cloridrato de Erlotinib/farmacologia , Neoplasias Pulmonares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Sinergismo Farmacológico , Medicamentos de Ervas Chinesas/uso terapêutico , Cloridrato de Erlotinib/uso terapêutico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos Endogâmicos BALB C , Camundongos Nus , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Fator de Transcrição Sp1/metabolismoRESUMO
BACKGROUND: Recent studies claimed the important role of cold atmospheric plasma (CAP) with nanotechnology in cancer treatments. In this study, silymarin nanoemulsion (SN) was used along with air CAP as therapeutic agent to counter human melanoma. METHODS: In this study, we examined the combined treatment of CAP and SN on G-361 human melanoma cells by evaluating cellular toxicity levels, reactive oxygen and nitrogen species (RONS) levels, DNA damage, melanoma-specific markers, apoptosis, caspases and poly ADP-ribose polymerase-1 (PARP-1) levels using flow cytometer. Dual-treatment effects on the epithelial-mesenchymal transition (EMT), Hepatocyte growth factor (HGF/c-MET) pathway, sphere formation and the reversal of EMT were also assessed using western blotting and microscopy respectively. SN and plasma-activated medium (PAM) were applied on tumor growth and body weight and melanoma-specific markers and the mesenchymal markers in the tumor xenograft nude mice model were checked. RESULTS: Co-treatment of SN and air CAP increased the cellular toxicity in a time-dependent manner and shows maximum toxicity at 200 nM in 24 h. Intracellular RONS showed significant generation of ROS (< 3 times) and RNS (< 2.5 times) in dual-treated samples compared to control. DNA damage studies were assessed by estimating the level of γ-H2AX (1.8 times), PD-1 (> 2 times) and DNMT and showed damage in G-361 cells. Increase in Caspase 8,9,3/7 (> 1.5 times), PARP level (2.5 times) and apoptotic genes level were also observed in dual treated group and hence blocking HGF/c-MET pathway. Decrease in EMT markers (E-cadherin, YKL-40, N-cadherin, SNAI1) were seen with simultaneously decline in melanoma cells (BRAF, NAMPT) and stem cells (CD133, ABCB5) markers. In vivo results showed significant reduction in SN with PAM with reduction in tumor weight and size. CONCLUSIONS: The use of air CAP using µ-DBD and the SN can minimize the malignancy effects of melanoma cells by describing HGF/c-MET molecular mechanism of acting on G-361 human melanoma cells and in mice xenografts, possibly leading to suitable targets for innovative anti-melanoma approaches in the future.
Assuntos
Antioxidantes/uso terapêutico , Carcinogênese/efeitos dos fármacos , Fator de Crescimento de Hepatócito/metabolismo , Melanoma/tratamento farmacológico , Gases em Plasma/uso terapêutico , Proteínas Proto-Oncogênicas c-met/metabolismo , Silimarina/uso terapêutico , Animais , Antioxidantes/farmacologia , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Sinergismo Farmacológico , Transição Epitelial-Mesenquimal , Humanos , Masculino , Melanoma/metabolismo , Camundongos , Camundongos Nus , Gases em Plasma/farmacologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Silimarina/farmacologiaRESUMO
Savolitinib is a novel small-molecule selective cMet inhibitor. This work characterized its pharmacokinetics in preclinical phase, established the preclinical relationships between PK, cMet modulation and anti-tumor efficacy. In vitro and in vivo animal studies were performed for PK characterization. Savolitinib showed good absorption, moderate tissue distribution, low to intermediate clearance, and low accumulation. Hepatic oxidative metabolism followed by urinary and biliary excretions was the major elimination pathway. Based on preclinical PK data, human PK profiles were predicted using empirical methods. Pharmacodynamic studies for evaluating cMet inhibition and anti-tumor efficacy were conducted in nude mice bearing Hs746t xenograft. PK/PD models were built to link the PD measurements to nude mouse PK. The established integrated preclinical PK/PD model contained a two-compartment non-linear PK model, a biomarker link model and a tumor growth transit model. The IC50 of cMet inhibition and the concentration achieving half of the maximal Hs746t tumor reduction by savolitinib were equal to 12.5 and 3.7â¯nM (free drug), respectively. Based on the predicted human PK data, as well as the established PK/PD model in nude mouse, the human PD (cMet inhibition) profiles were also simulated. This research supported clinical development of savolitinib. Understanding the preclinical PK/PD relationship of savolitinib provides translational insights into the cMet-targeted drug development.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/farmacocinética , Pirazinas/farmacologia , Pirazinas/farmacocinética , Triazinas/farmacologia , Triazinas/farmacocinética , Animais , Células CACO-2 , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Camundongos Nus , Modelos Biológicos , Proteínas Proto-Oncogênicas c-met/metabolismo , Ratos , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Despite previous studies suggesting that choline and betaine ameliorate lipid accumulation in rat livers, the relative effectiveness of the two nutrients is unclear. We examined the efficacy of dietary supplementation with choline or betaine in ameliorating lipid accumulation induced by vitamin B6 (B6) deficiency in the rat liver. Male Wistar rats were fed control, B6-deficient, choline-supplemented B6-deficient, betaine-supplemented B6-deficient, or both choline and betaine-supplemented B6-deficient diets (all containing 9 g of l-methionine (Met)/kg) for 35 d. Two experiments were performed, i.e., one using 17 mmol/kg diet choline bitartrate, betaine anhydrous, and the combination and another using 8.5 mmol/kg diet. Rats fed a B6-deficient diet developed lipid accumulation in the liver with a reduction of plasma lipids induced by the disruption of Met metabolism. However, the addition of 17 mmol/kg diet choline or betaine was sufficient to ameliorate the disruptions of lipid and Met metabolism. Additionally, 8.5 mmol/kg diet choline ameliorated liver lipid deposition, while the same amount of betaine had no significant effects on liver or plasma lipid profiles. Supplementation with choline resulted in a higher liver betaine than that found using the same amount of betaine alone, although the overall liver betaine content was reduced in B6-deficient rats. Our findings indicate that choline is more effective than betaine in ameliorating B6 deficiency-related disruptions in Met metabolism and liver lipid accumulation by increasing liver betaine levels.