ABSTRACT
Maritime pine bark is a rich source of polyphenolic compounds and is commonly employed as a herbal supplement worldwide. This study was designed to check the potential of maritime pine tannin extract (MPTE) in anticancer therapy and to determine the underlying mechanism of action. Our results showed that MPTE, containing procyanidin oligomers and lanostane type terpenoids, has an inhibitory effect on cancer cell proliferation through cell cycle arrest in the G2/M phase. Treatment with MPTE also induced apoptosis in a concentration-dependent manner in human cancer cell lines (HeLa and U2OS), as evidenced by the enhanced activation of caspase 3 and the cleavage of PARP along with the downregulation of the antiapoptotic protein Bcl-2. Interestingly, human non-cancerous fibroblasts are much less sensitive to MPTE, suggesting that it preferentially targets cancer cells. MPTE played a pro-oxidant role in cancer cells and promoted the expression of the p73 tumor suppressor gene in p53-deficient cells. It also downregulated the protooncogenic proteins UHRF1 and DNMT1, mediators of the DNA methylation machinery, and reduced the global methylation levels in HeLa cells. Overall, our results show that maritime pine tannin extract can play a favorable role in cancer treatment, and can be further explored by the pharmaceutical industry.
Subject(s)
Antineoplastic Agents/pharmacology , CCAAT-Enhancer-Binding Proteins , Epigenesis, Genetic/drug effects , Pinus/chemistry , Tannins/pharmacology , Ubiquitin-Protein Ligases , Apoptosis/drug effects , CCAAT-Enhancer-Binding Proteins/genetics , CCAAT-Enhancer-Binding Proteins/metabolism , DNA (Cytosine-5-)-Methyltransferase 1/genetics , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , Down-Regulation/drug effects , HeLa Cells , Humans , Plant Bark/chemistry , Plant Extracts/pharmacology , Tumor Protein p73/genetics , Tumor Protein p73/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
Liver fibrosis is the consequence of chronic liver injury and is a major challenge to global health. However, successful therapy for liver fibrosis is still lacking. Sennoside A (SA), a commonly used clinical stimulant laxative, is reported to improve hepatic disease, but the underlying mechanisms remain largely elusive. Here, we show for the first time that SA enhanced suppressor of cytokine signaling 1 (SOCS1) expression in a DNA methyltransferase 1 (DNMT1)-dependent manner and thereby attenuated liver fibrosis. Consistently, SA inhibited the expression of the liver fibrogenesis markers α-smooth muscle actin (α-SMA) and type I collagen alpha-1 (Col1α1) and suppressed inflammatory responses in vivo and in vitro. Coculture experiments with macrophages/hepatic stellate cells (HSCs) revealed that SA suppressed HSC proliferation by downregulating proinflammatory cytokines in macrophages. Mechanically, SA promoted the aberrant expression of SOCS1 in liver fibrosis. However, blocking SOCS1 expression weakened the inhibitory effect of SA on HSC proliferation, indicating that SOCS1 may play an important role in mediating the antifibrotic effect of SA. Furthermore, SA inhibited DNMT1-mediated SOCS1 and reduced HSC proliferation by inhibiting inflammatory responses in carbon tetrachloride (CCl4) -induced liver fibrosis.
Subject(s)
Anti-Inflammatory Agents/therapeutic use , Chemical and Drug Induced Liver Injury/drug therapy , Liver Cirrhosis/drug therapy , Sennosides/therapeutic use , Suppressor of Cytokine Signaling 1 Protein/genetics , Animals , Anti-Inflammatory Agents/pharmacology , Carbon Tetrachloride , Cell Line , Cell Proliferation/drug effects , Chemical and Drug Induced Liver Injury/genetics , Chemical and Drug Induced Liver Injury/metabolism , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , DNA Methylation/drug effects , Hepatic Stellate Cells/drug effects , Hepatic Stellate Cells/metabolism , Liver/drug effects , Liver/pathology , Liver Cirrhosis/chemically induced , Liver Cirrhosis/genetics , Liver Cirrhosis/metabolism , Macrophages/drug effects , Macrophages/metabolism , Male , Mice, Inbred C57BL , Rats , Sennosides/pharmacology , Up-Regulation/drug effectsABSTRACT
The pathological characteristics of Parkinson's disease (PD) include dopaminergic neuron damage, specifically disorders caused by dopamine synthesis, in vivo. Plastrum testudinis extract (PTE) and its bioactive ingredient ethyl stearate (PubChem CID: 8122) were reported to be correlated with tyrosine hydroxylase (TH), which is a biomarker of dopaminergic neurons. This suggests that PTE and its small-molecule active ingredient ethyl stearate have potential for development as a therapeutic drug for PD. In this study, we treated 6-hydroxydopamine (6-OHDA)-induced model rats and PC12 cells with PTE. The mechanism of action of PTE and ethyl stearate was investigated by western blotting, bisulfite sequencing PCR (BSP), real-time PCR, immunofluorescence and siRNA transfection. PTE effectively upregulated the TH expression and downregulated the alpha-synuclein expression in both the substantia nigra and the striatum of the midbrain in a PD model rat. The PC12 cell model showed that both PTE and its active monomer ethyl stearate significantly promoted TH expression and blocked alpha-synuclein, agreeing with the in vivo results. BSP showed that PTE and ethyl stearate increased the methylation level of the Snca intron 1 region. These findings suggest that some of the protective effects of PTE on dopaminergic neurons are mediated by ethyl stearate. The mechanism of ethyl stearate may involve disrupting the abnormal aggregation of DNA (cytosine-5)-methyltransferase 1 (DNMT1) with alpha-synuclein by releasing DNMT1, upregulating Snca intron 1 CpG island methylation, and ultimately, reducing the expression of alpha-synuclein.
Subject(s)
Antiparkinson Agents/pharmacology , Antiparkinson Agents/therapeutic use , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , Dopaminergic Neurons/drug effects , Neuroprotective Agents/pharmacology , Neuroprotective Agents/therapeutic use , Parkinson Disease, Secondary/drug therapy , Plant Extracts/pharmacology , Plant Extracts/therapeutic use , Tissue Extracts/chemistry , alpha-Synuclein/metabolism , Animals , DNA (Cytosine-5-)-Methyltransferase 1/drug effects , Hydroxydopamines , Male , Mesencephalon/drug effects , Mesencephalon/metabolism , PC12 Cells , Parkinson Disease, Secondary/chemically induced , Rats , Rats, Sprague-Dawley , Stearates/pharmacology , Substantia Nigra/drug effects , Substantia Nigra/metabolism , alpha-Synuclein/drug effectsABSTRACT
Glioblastoma multiforme (GBM) is a deadly malignant brain tumor that is resistant to most clinical treatments. Novel therapeutic agents that are effective against GBM are required. Antrodia cinnamomea has shown antiproliferative effects in GBM cells. However, the exact mechanisms and bioactive components remain unclear. Thus, the present study aimed to investigate the effect and mechanism of 4-acetylantrocamol LT3 (4AALT3), a new ubiquinone from Antrodia cinnamomeamycelium, in vitro. U87 and U251 cell lines were treated with the indicated concentration of 4AALT3. Cell viability, cell colony-forming ability, migration, and the expression of proteins in well-known signaling pathways involved in the malignant properties of glioblastoma were then analyzed by CCK-8, colony formation, wound healing, and western blotting assays, respectively. We found that 4AALT3 significantly decreased cell viability, colony formation, and cell migration in both in vitro models. The epidermal growth factor receptor (EGFR), phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR), Hippo/yes-associated protein (YAP), and cAMP-response element binding protein (CREB) pathways were suppressed by 4AALT3. Moreover, 4AALT3 decreased the level of DNA repair enzyme O6-methylguanine-DNA methyltransferase and showed a synergistic effect with temozolomide. Our findings provide the basis for exploring the beneficial effect of 4AALT3 on GBM in vivo.
Subject(s)
4-Butyrolactone/analogs & derivatives , Cyclohexanones/pharmacology , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , DNA Repair/drug effects , Glioblastoma/drug therapy , Guanine/analogs & derivatives , 4-Butyrolactone/chemistry , 4-Butyrolactone/pharmacology , Cell Line, Tumor , Cell Movement/drug effects , Cell Survival/drug effects , Cyclohexanones/chemistry , Down-Regulation , Guanine/metabolism , Humans , Ubiquinone/pharmacologyABSTRACT
Ethanol (EtOH) has been linked to neurotoxic effects on the fetus and prenatal alcohol exposure (PAE) has a negative impact on brain neurodevelopment. Therefore, the present study was aimed to focus on the underlying mechanisms of alcohol-induced oxidative stress and apoptotic cell death in addition to shedding the light on the modulatory effect of nanocurcumin in rats' offspring prefrontal cortices. The current study investigated the effects of prenatal maternal exposure to EtOH intragastric (i.g.) administration of 0.015 mL/g of a 10 % v/v ethanol solution throughout gestation and the concomitant use of nanocurcumin, on 21-day-old offspring Wistar rat prefrontal cortex parameters. CYP2E1, DBN1, DNMT1, miRNA-335, miRNA-21, c-Fos and Cox-2 gene expression as well as the accompanying histological and ultrastructural alterations were assessed. The implemented experimental setting has revealed that ethanol exposure caused significant alterations in the above mentioned parameters. Changes observed in nanocurcumin-treated animals were significantly different to the ethanol-treated group when nanocurcumin was concomitantly administered.
Subject(s)
Curcumin/therapeutic use , Ethanol/pharmacology , Gene Expression/drug effects , Neuroprotective Agents/therapeutic use , Prefrontal Cortex/drug effects , Prenatal Exposure Delayed Effects/genetics , Animals , Curcumin/pharmacology , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Cytochrome P-450 CYP2E1/genetics , Cytochrome P-450 CYP2E1/metabolism , DNA (Cytosine-5-)-Methyltransferase 1/genetics , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , Female , MicroRNAs/genetics , MicroRNAs/metabolism , Neuropeptides/genetics , Neuropeptides/metabolism , Neuroprotective Agents/pharmacology , Prefrontal Cortex/metabolism , Pregnancy , Prenatal Exposure Delayed Effects/metabolism , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-fos/metabolism , Rats , Rats, WistarABSTRACT
Triple-negative breast cancer (TNBC) is an aggressive cancer, and rapidly progresses following relapse in advanced stage. This cancer is usually associated with worse overall survival, so the carcinogenesis of TNBC needs to be further explored to find more effective therapies. In this study, we intended to identify the roles of YY1-mediated long non-coding RNA Kcnq1ot1 in TNBC. First, the paired samples of tumor tissues and adjacent tissues were collected to determine YY1, lncRNA Kcnq1ot1, and PTEN expression using RT-qPCR and Western blot analysis followed by analysis of the relationship between them and patient survival. The results revealed that YY1 and lncRNA Kcnq1ot1 were upregulated in TNBC tissues, and high expression of YY1 and lncRNA Kcnq1ot1 was associated with poor patient survival. Then, ChIP and MSP assays were employed to explore interactions between YY1, lncRNA Kcnq1ot1, and PTEN gene. We obtained that YY1 upregulated lncRNA Kcnq1ot1, which mediated PTEN methylation via DNMT1, thus decreasing PTEN expression. Afterward, TNBC cells were examined for their viability using functional assays with the results displaying that overexpression of YY1 facilitated TNBC cell proliferation, invasion, and migration. Mechanistically, upregulated YY1 repressed tumor growth by inhibiting PTEN via upregulation of lncRNA Kcnq1ot1. Mouse models were also constructed, and the above effects of YY1, lncRNA Kcnq1ot1, and PTEN on TNBC were also established in vivo. Taken together, this study demonstrates that the silencing of YY1 exerted tumor-suppressive effects on TNBC by modulating lncRNA Kcnq1ot1/DNMT1/PTEN pathway, in support of further investigation into anti-tumor therapy for TNBC.
Subject(s)
DNA (Cytosine-5-)-Methyltransferase 1/metabolism , PTEN Phosphohydrolase/metabolism , Triple Negative Breast Neoplasms/genetics , YY1 Transcription Factor/metabolism , Adult , Animals , Cell Line, Tumor , Cell Proliferation , Disease Models, Animal , Disease Progression , Female , Humans , Mice , Middle Aged , Potassium Channels, Voltage-Gated/metabolismABSTRACT
Hepatic stellate cell (HSC) activation is an essential event during liver fibrogenesis. Phosphatase and tension homolog deleted on chromosome 10 (PTEN) is a negative regulator of this process. DNA methyltransferase 1 (DNMT1), which catalyzes DNA methylation and subsequently leads to the transcriptional repression of PTEN, is selectively induced in myofibroblasts from diseased livers. Sennoside A (SA), a major purgative constituent of senna and the Chinese herb rhubarb, is widely used in China and other Asian countries as an irritant laxative. SA is reported to improve hepatic steatosis. However, the effect and mechanism of SA on liver fibrosis remain largely unknown. We recently identified a novel strategy for protecting liver fibrosis via epigenetic modification by targeting DNMT1. A Surface Plasmon Resonance (SPR) assay first reported that SA could directly bind DNMT1 and inhibit its activity. Administration of SA significantly prevented liver fibrosis, as evidenced by the dramatic downregulation of α-smooth muscle actin (α-SMA) and type I collagen alpha-1 (Col1α1) protein levels in a CCl4 -induced mouse hepatic fibrosis model and in TGF-ß1-activated HSC-T6 cells, in vivo and in vitro. SA decreased the expression of Cyclin D1, CDK, and C-myc, indicating that SA may inhibit the activation and proliferation of TGF-ß1-induced HSC-T6. Moreover, SA significantly promoted the expression of PTEN and remarkably inhibited the expression of p-AKT and p-ERK in vitro. Blocking PTEN or overexpressing DNMT1 could reduce the effect of SA on liver fibrosis. These data suggest that SA directly binds and inhibits the activity and that attenuated DNMT1-mediated PTEN hypermethylation caused the loss of PTEN expression, followed by the inhibition of the AKT and ERK pathways and prevented the development of liver fibrosis. Hence, SA might be employed as a promising natural supplement for liver fibrosis drug therapy.
Subject(s)
DNA (Cytosine-5-)-Methyltransferase 1/metabolism , DNA Methylation , Hepatic Stellate Cells/drug effects , Liver Cirrhosis/drug therapy , PTEN Phosphohydrolase/genetics , Sennosides/pharmacology , Actins/genetics , Actins/metabolism , Animals , Cell Line , Cell Proliferation , Collagen Type I/genetics , Collagen Type I/metabolism , Collagen Type I, alpha 1 Chain , Cyclin D1/genetics , Cyclin D1/metabolism , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Hepatic Stellate Cells/metabolism , Hepatic Stellate Cells/physiology , Liver Cirrhosis/prevention & control , Male , Mice , Mice, Inbred C57BL , PTEN Phosphohydrolase/metabolism , Protein Binding , Sennosides/therapeutic use , Signal Transduction , Transforming Growth Factor beta/pharmacologyABSTRACT
We evaluated if chronic consumption of quercetin (Q) with green tea extract (GTE) enhances the bioavailability of GT polyphenols (GTPs) and reduces methylation activity as previously observed in mouse xenograft tumors. In this prospective, randomized, parallel design, placebo controlled study, thirty-one men with prostate cancer consumed daily 1 gram of GTE (830 mg of GTP) with 800 mg of Q (GT + Q) or placebo (GT + PL) for four weeks before prostatectomy. First morning voided urine was collected at baseline, 3 weeks and the day of surgery, and prostate tissue on the day of surgery. In week 3, plasma concentration of GTPs and Q was measured in blood collected before and 2 hours after the morning dose. Prostate tissue epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) were detected in 67 and 93% of participants in the GT + Q group and 75 and 94% of participants in the GT + PL group. Q was increased 14-fold, 12-fold and 4.5-fold in plasma, urine, and prostate tissue, respectively, in the GT + Q compared to the GT + PL-group. There was a trend for decreased EGC levels in urine collected prior to prostatectomy in the GT + Q compared to GT + PL-group (p = 0.053). Plasma epigallocatechin (EGC) showed a trend to increase (p = 0.066) two hours after capsule intake in the GT + Q vs. the GT + PL-group. There was no significant difference between the groups in GTP content or methylation activity in prostate tissue or RBCs. No liver toxicity was observed. Although our findings are suggestive, further studies are warranted evaluating if Q alters GTP metabolism.
Subject(s)
Polyphenols/metabolism , Prostate/metabolism , Prostatic Neoplasms/metabolism , Quercetin/metabolism , Tea/chemistry , Aged , Biomarkers , Catechol O-Methyltransferase/genetics , Catechol O-Methyltransferase/metabolism , DNA (Cytosine-5-)-Methyltransferase 1/genetics , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , Dietary Supplements , Drug Therapy, Combination , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Male , Middle Aged , Polyphenols/chemistry , Quercetin/administration & dosage , Quercetin/chemistryABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: The classical and traditional Chinese medicine prescription, Liuwei Dihuang (LWDH), has been commonly used to treat the menopausal syndrome. It has been reported that LWDH could improve estrogen receptor α (ERα) expression to prevent atherosclerosis (AS), while the mechanism of LWDH on regulating ERα expression was still unknown. AIM OF THE STUDY: To reveal the mechanism of LWDH on regulating the ERα expression. MATERIALS AND METHODS: The protective effect of LWDH on Hcy-induced apoptosis of human umbilical vein endothelial cells (HUVECs) was examined. The expression of ERα and DNA methyltransferases 1 (DNMT1) were detected by Western blot and real-time polymerase chain reaction (RT-PCR). The methylation rate of the ERα gene was assayed by the bisulfite sequencing PCR (BSP). High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS) was applied to determine the level of S-Adenosyl methionine (SAM) and S-Adenosyl homocysteine (SAH). In vivo, the ApoE-/- mice were ovariectomized to establish postmenopausal atherosclerosis (AS) model. RESULTS: In vitro study showed that LWDH protects HUVECs from Hcy-induced apoptosis. Treatment with LWDH significantly increased the ERα expression and reduced the methylation rate of the ERα gene by inhibiting the DNMT1 expression. The level of main methyl donor SAM and the ration of SAM/SAH were reduced by LWDH. In vivo, LWDH prevented the formation of plaque and reduced the concentration of Hcy. In addition, LWDH upregulated the ERα expression, as well as inhibiting the expression of DNMT1 in atherosclerotic mice. CONCLUSIONS: LWDH exerted protective effects on postmenopausal AS mice, and HUVECs treated with Hcy. LWDH increased of ERα expression via inhibiting DNMT1-dependent ERα methylation.
Subject(s)
Atherosclerosis/drug therapy , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , Drugs, Chinese Herbal/pharmacology , Drugs, Chinese Herbal/therapeutic use , Estrogen Receptor alpha/metabolism , Human Umbilical Vein Endothelial Cells/drug effects , Animals , Apoptosis/drug effects , Atherosclerosis/metabolism , Cells, Cultured , DNA (Cytosine-5-)-Methyltransferase 1/genetics , DNA Methylation/drug effects , Diet, High-Fat , Estrogen Receptor alpha/genetics , Female , Human Umbilical Vein Endothelial Cells/physiology , Humans , Mice, Inbred C57BL , Mice, Knockout, ApoE , Postmenopause , Rats, Sprague-DawleyABSTRACT
BACKGROUND: Sulforaphane (SFN), a natural compound present in cruciferous vegetable, has been shown to possess anti-cancer activities. Cancer stem cell (CSC) in bulk tumor is generally considered as treatment resistant cell and involved in cancer recurrence. The effects of SFN on nasopharyngeal carcinoma (NPC) CSCs have not yet been explored. PURPOSE: The present study aims to examine the anti-tumor activities of SFN on NPC cells with CSC-like properties and the underlying mechanisms. METHODS: NPC cells growing in monolayer culture, CSCs-enriched NPC tumor spheres, and also the NPC nude mice xenograft were used to study the anti-tumor activities of SFN on NPC. The population of cells expressing CSC-associated markers was evaluated using flow cytometry and aldehyde dehydrogenase (ALDH) activity assay. The effect of DNA methyltransferase 1 (DNMT1) on the growth of NPC cells was analyzed by using small interfering RNA (siRNA)-mediated silencing method. RESULTS: SFN was found to inhibit the formation of CSC-enriched NPC tumor spheres and reduce the population of cells with CSC-associated properties (SRY (Sex determining Region Y)-box 2 (SOX2) and ALDH). In the functional study, SFN was found to restore the expression of Wnt inhibitory factor 1 (WIF1) and the effect was accompanied with the downregulation of DNMT1. The functional activities of WIF1 and DNMT1 were confirmed using exogenously added recombinant WIF1 and siRNA knockdown of DNMT1. Moreover, SFN was found to inhibit the in vivo growth of C666-1 cells and enhance the anti-tumor effects of cisplatin. CONCLUSION: Taken together, we demonstrated that SFN could suppress the growth of NPC cells via the DNMT1/WIF1 axis.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Antineoplastic Agents, Phytogenic/pharmacology , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , Isothiocyanates/pharmacology , Nasopharyngeal Carcinoma/drug therapy , Nasopharyngeal Neoplasms/drug therapy , Adaptor Proteins, Signal Transducing/genetics , Animals , Antineoplastic Agents, Phytogenic/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Brassicaceae/chemistry , Cell Line, Tumor , Cisplatin/administration & dosage , DNA (Cytosine-5-)-Methyltransferase 1/genetics , Humans , Isothiocyanates/administration & dosage , Male , Mice, Inbred BALB C , Mice, Nude , Nasopharyngeal Carcinoma/metabolism , Nasopharyngeal Carcinoma/pathology , Nasopharyngeal Neoplasms/metabolism , Nasopharyngeal Neoplasms/pathology , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/pathology , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism , Sulfoxides , Xenograft Model Antitumor AssaysABSTRACT
Aberrant epigenetic modifications are responsible for tumor development and cancer progression; however, readily reversible. Bioactive molecules from diets are promising to cure cancer by modulating epigenetic marks and changing immune response. These compounds specifically target the activity of DNMTs and HDACs to cure various human cancers. In view of this, we investigated the anticancer and epigenetic regulatory activities of an edible-plant Paederia foetida. The efficacy of methanolic extract of P. foetida leaves (MEPL) was tested for the modulation of epigenetic factors in gene silencing, i.e. DNMT and HDAC and expression pattern of certain tumor-suppressor genes. After treatment of prostate cancer cells (PC-3 and DU-145) with MEPL, lupeol and ß-sitosterol; induction of apoptosis, decrease in cellular-viability and inhibition of cellular-migration were noticed. Simultaneously there was inhibition of DNMT1, HDACs and pro-inflammatory, IL-6, IL1-ß, TNF-α and anti-inflammatory, IL-10 genes in cancer and THP1 cell lines. The DNMT1 protein content, enzyme activity and Bcl2 expression decreased significantly; however, expression of E-cadherin (CDH1) and pro-apoptotic gene Bax increased significantly after the treatment of cells with drugs. We conclude plant-derived compounds can be considered to target epigenetic machineries involved with malignant transformation and can open new avenues for cancer therapeutics provoking immune response.
Subject(s)
Cell Survival/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Inflammation/metabolism , Plant Extracts/pharmacology , Prostatic Neoplasms , Rubiaceae/chemistry , Cell Line, Tumor , DNA (Cytosine-5-)-Methyltransferase 1/genetics , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , Histone Deacetylase 1/genetics , Histone Deacetylase 1/metabolism , Histone Deacetylase 2/genetics , Histone Deacetylase 2/metabolism , Humans , Inflammation/genetics , Male , Pentacyclic Triterpenes , Phytochemicals , Plant Extracts/chemistry , Plant Leaves/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , SitosterolsABSTRACT
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.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Non-Small-Cell Lung/metabolism , Drugs, Chinese Herbal/pharmacology , Erlotinib Hydrochloride/pharmacology , Lung Neoplasms/metabolism , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Animals , Antineoplastic Agents/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , Drug Synergism , Drugs, Chinese Herbal/therapeutic use , Erlotinib Hydrochloride/therapeutic use , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lung Neoplasms/drug therapy , Mice, Inbred BALB C , Mice, Nude , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-met/genetics , Proto-Oncogene Proteins c-met/metabolism , Sp1 Transcription Factor/metabolismABSTRACT
Breast cancer is a complex disease driven by multiple factors including both genetic and epigenetic alterations. Recent studies revealed that abnormal gene expression induced by epigenetic changes including aberrant promoter methylation plays a critical role in human breast carcinogenesis. Cucurbitacin B has antiproliferative activity against various human breast cancer cells, but the molecular mechanism is not completely understood. In this study, we explore the influence of cucurbitacin B from Trichosanthes cucumerina on the methylation status at the promoter of oncogenes c-Myc, cyclin D1, and survivin in breast cancer cell lines. Growth inhibitory effect of cucurbitacin B on breast cancer cells was assessed by MTT assay and colony formation assay. Methylation status of genomic DNA was determined by methylation-specific PCR. Gene and protein expression levels of all genes studied were analyzed by real-time RT-PCR and western blot. The results indicated that cucurbitacin B could inhibit cell growth in breast cancer cells. The oncogene promoters are usually hypomethylated in cancer cells. Upon cucurbitacin B treatment, upregulation of DNMT1 and obvious heavy methylation in the promoters of c-Myc, cyclin D1, and survivin, which consequently downregulated the expression of all these oncogenes, were observed. Hence, cucurbitacin B proved to be a potential cancer therapeutic agent, in part by inducing hypermethylation and silences the oncogenic activation.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Breast Neoplasms/drug therapy , Gene Expression Regulation, Neoplastic , Trichosanthes/chemistry , Triterpenes/pharmacology , Antineoplastic Agents, Phytogenic/chemistry , Breast Neoplasms/genetics , Cell Line, Tumor , Cyclin D1/genetics , DNA (Cytosine-5-)-Methyltransferase 1/genetics , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , DNA Methylation/drug effects , Female , Humans , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins c-myc/genetics , Survivin/genetics , Triterpenes/chemistryABSTRACT
PURPOSE: Ewing sarcoma (ES) is a rare and highly malignant cancer that occurs in the bone and surrounding tissue of children and adolescents. The EWS/ETS fusion transcription factor that drives ES pathobiology was previously demonstrated to modulate cyclin D1 expression. In this study, we evaluated abemaciclib, a small-molecule CDK4 and CDK6 (CDK4 and 6) inhibitor currently under clinical investigation in pediatric solid tumors, in preclinical models of ES. EXPERIMENTAL DESIGN: Using Western blot, high-content imaging, flow cytometry, ELISA, RNA sequencing, and CpG methylation assays, we characterized the in vitro response of ES cell lines to abemaciclib. We then evaluated abemaciclib in vivo in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models of ES as either a monotherapy or in combination with chemotherapy. RESULTS: Abemaciclib induced quiescence in ES cell lines via a G1 cell-cycle block, characterized by decreased proliferation and reduction of Ki-67 and FOXM1 expression and retinoblastoma protein (RB) phosphorylation. In addition, abemaciclib reduced DNMT1 expression and promoted an inflammatory immune response as measured by cytokine secretion, antigen presentation, and interferon pathway upregulation. Single-agent abemaciclib reduced ES tumor volume in preclinical mouse models and, when given in combination with doxorubicin or temozolomide plus irinotecan, durable disease control was observed. CONCLUSIONS: Collectively, our data demonstrate that the antitumor effects of abemaciclib in preclinical ES models are multifaceted and include cell-cycle inhibition, DNA demethylation, and immunogenic changes.
Subject(s)
Aminopyridines/pharmacology , Benzimidazoles/pharmacology , Cell Cycle , DNA Methylation , Interferons/metabolism , Sarcoma, Ewing/genetics , Sarcoma, Ewing/metabolism , Signal Transduction/drug effects , Animals , Cell Cycle/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin D1/genetics , Cyclin D1/metabolism , DNA (Cytosine-5-)-Methyltransferase 1/genetics , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , Disease Models, Animal , Drug Evaluation, Preclinical , Drug Resistance, Neoplasm/genetics , Humans , Mice , Sarcoma, Ewing/drug therapy , Sarcoma, Ewing/pathology , Xenograft Model Antitumor AssaysABSTRACT
Chronic NP-1 administration reduces body weight and hepatic steatosis despite induction of tolerance in adiponectin gene transcription with respect to the acute actions of this drug. This study explored the hypothesis that NP-1 could exert these effects through mechanisms independent of adiponectin. To this aim, we took advantage of the Zucker (fa/fa) rat model, which exhibits obesity, fatty liver and elevated leptin and adiponectin levels. Body weight and food intake were reduced after chronic NP-1 treatment. Plasma TNFα concentrations were elevated but no increase in adiponectin was found. Even so, NP-1 ameliorated fatty liver and corrected dyslipidemia by mechanisms probably associated with reduced feeding, transcription of Cpt1 and down-regulation of Hmgcr-CoA expression. In brown fat tissue NP-1 increased Dnmt1 (inhibitor of Adipoq) while it reduced Ucp1 expression and heat production, which excludes thermogenesis as a mechanism of the NP-1 slimming effect. The anti-obesity action of chronic NP-1 administration might be mediated by TNFα, which is known to have anorectic actions in the hypothalamus and to regulate both Dmnt1 and Ucp1 expression in adipose tissues. This finding opens up the possibility of using NP-1-mediated TNFα-induced weight loss as an innovative treatment of complicated obesity under strict pharmacologic control.
Subject(s)
Adiponectin/metabolism , Dyslipidemias/prevention & control , Gene Expression Regulation/drug effects , Obesity/complications , Thiazoles/pharmacology , Tumor Necrosis Factor-alpha/blood , Adiponectin/genetics , Adipose Tissue, Brown/drug effects , Adipose Tissue, Brown/metabolism , Animals , Body Weight , DNA (Cytosine-5-)-Methyltransferase 1/genetics , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , Dyslipidemias/etiology , Dyslipidemias/metabolism , Dyslipidemias/pathology , Feeding Behavior , Hypothalamus/drug effects , Hypothalamus/metabolism , Liver/drug effects , Liver/metabolism , Male , Promoter Regions, Genetic , Rats , Rats, Zucker , Thinness/complications , Weight LossABSTRACT
BACKGROUND Shikonin is a component of Chinese herbal medicine. The aim of this study was to investigate the effects of shikonin on cell migration of papillary thyroid cancer cells of the TPC-1 cell line in vitro and expression levels of the phosphate and tensin homolog deleted on chromosome 10 (PTEN) and DNA methyltransferase 1 (DNMT1) genes. MATERIAL AND METHODS The Cell Counting Kit-8 (CCK-8) assay was performed to evaluate the proliferation of TPC-1 papillary thyroid cancer cells, and the normal thyroid cells, HTori-3, in vitro. A transwell motility assay was used to analyze the migration of TPC-1 cells. Western blot was performed to determine the expression levels of PTEN and DNMT1 genes. A methylation-specific polymerase chain reaction (PCR) (MSP) assay was used to evaluate the methylation of PTEN. RESULTS Following treatment with shikonin, the cell survival rate of TPC-1 cells decreased in a dose-dependent manner; the inhibitory effects on HTori-3 cells were less marked. Shikonin inhibited TPC-1 cell migration and invasion in a dose-dependent manner. The methylation of PTEN was suppressed by shikonin, which also reduced the expression of DNMT1 in a dose-dependent manner, and increased the expression of PTEN. Overexpression of DNMT1 promoted the migration of TPC-1 cells and the methylation of PTEN. Levels of protein expression of PTEN in TPC-1 cells treated with shikonin decreased, and were increased by DNMT1 knockdown. CONCLUSIONS Shikonin suppressed the expression of DNMT1, reduced PTEN gene methylation, and increased PTEN protein expression, leading to the inhibition of TPC-1 cell migration.
Subject(s)
Cell Movement/drug effects , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , Down-Regulation/drug effects , Naphthoquinones/pharmacology , Thyroid Neoplasms/enzymology , Thyroid Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Gene Amplification/drug effects , Gene Knockdown Techniques , Humans , Methylation , Neoplasm Invasiveness , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , RNA, Small Interfering/metabolismABSTRACT
Our preliminary study showed that the total flavonoids in Isodon amethystoides(TFIA), a local medicinal herb in Suzhou, had a certain therapeutic effect on adjuvant arthritis, and this therapeutic effect may be achieved through the up-regulation of miR-152 expression. In this paper, the molecular mechanism of TFIA on the pathogenesis of adjuvant arthritis(AA) rats was further studied. AA rats were prepared with complete Freund's adjuvant, and then treated with TFIA by intragastric administration. Real-time qPCR was used to detect the effects of TFIA on the negative regulatory loop of miR-152, methylase DNMT1 and methyl-CpG binding protein MeCP2 in fibroblast like synoviocytes(FLS) of AA rats, as well as the effects of TFIA on the classic Wnt signaling pathway and the expression of fibronectin gene in AA rats. Intragastric administration of TFIA significantly inhibited the expression of DNMT1 and reversed the negative regulatory loop composed of miR-152, DNMT1 and MeCP2 in the pathology of AA rats. After transfection of miR-152 inhibitors into the FLS in treatment group, DNMT1 expression was significantly restored. TFIA significantly up-regulated the expression of SFRP4 and inhibited the expression of ß-catenin, C-myc and ccnd1, the key genes of canonical Wnt signaling pathway. TFIA also significantly inhibited the expression of fibronectin, an AA gene. The effect of TFIA on the expression of SFRP4, ß-catenin, C-myc, ccnd1 and fibronectin was reversed after transfection with miR-152 inhibitors in the treatment group FLS. TFIA may inhibit the DNMT1 expression, up-regulate the SFRP4 expression, inhibit the expression of classical Wnt signaling genes ß-catenin, C-myc, and ccnd1 as well as the RA gene fibronectin expression through the up-regulation of miR-152 expression.
Subject(s)
Arthritis, Experimental/drug therapy , Flavonoids/pharmacology , Isodon/chemistry , Animals , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , Methyl-CpG-Binding Protein 2/metabolism , MicroRNAs/metabolism , Phytotherapy , Plants, Medicinal/chemistry , Proto-Oncogene Proteins/metabolism , Rats , Synoviocytes/drug effects , Wnt Signaling PathwayABSTRACT
Grape pomace is a rich source of phenolic compounds commonly employed for elaboration of dietary supplements. The aim of the present study was to investigate the anticancer effect of a purified white grape pomace extract (PWGPE) in acute lymphoblastic leukemia Jurkat cells and to characterize the underlying mechanism. Apoptosis, mitochondrial membrane potential and reactive oxygen species (ROS) levels were assessed by flow cytometry and protein expression levels were analysed by Western blotting. PWGPE induced apoptosis in Jurkat cells in a time- and concentration-dependent manner. The anticancer effect was associated with an increased expression of p73 and down-regulation of pro-survival factors, including p-Akt, Bcl-2, and survivin. PWGPE reduced the expression of several proteins that block the expression of apoptosis-related genes such as DNMT1, HDAC1/2, UHRF1, and the polycomb group protein members: EZH2, SUZ12, and BMI1. In addition, the extract induced the formation of ROS, whereas pre-treatment with PEG-catalase and N-acetylcysteine prevented the ROS formation and markedly decreased the induction of apoptosis. These findings suggest that PWGPE-induced apoptosis in Jurkat human leukemia cells, is mediated by mitochondrial depolarization and caspase-3 cleavage, and depends on ROS generation and regulation of epigenetic gene silencing. Therefore, PWGPE may play an important role in the treatment of acute lymphoblastic leukemia (ALL).
Subject(s)
Apoptosis/drug effects , Epigenesis, Genetic/drug effects , Phenols/pharmacology , Plant Extracts/pharmacology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Vitis/chemistry , Caspase 3/genetics , Caspase 3/metabolism , DNA (Cytosine-5-)-Methyltransferase 1/genetics , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , Humans , Jurkat Cells , Phenols/analysis , Plant Extracts/analysis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/physiopathology , Reactive Oxygen Species/metabolism , Tumor Protein p73/genetics , Tumor Protein p73/metabolismABSTRACT
DNA methylation is an epigenetic change that results in the addition of a methyl group at the carbon-5 position of cytosine residues. DNA methyltransferase (DNMT) inhibitors can suppress tumour growth and have significant therapeutic value. However, the established inhibitors are limited in their application due to their substantial cytotoxicity. Additionally, the standard drugs for DNMT inhibition are non-selective cytosine analogues with considerable cytotoxic side-effects. In the present study, we have designed a workflow by integrating various ligand-based and structure-based approaches to discover new agents active against DNMT1. We have derived a pharmacophore model with the help of available DNMT1 inhibitors. Utilising this model, we performed the virtual screening of Maybridge chemical library and the identified hits were then subsequently filtered based on the Naïve Bayesian classification model. The molecules that have returned from this classification model were subjected to ensemble based docking. We have selected 10 molecules for the biological assay by inspecting the interactions portrayed by these molecules. Three out of the ten tested compounds have shown DNMT1 inhibitory activity. These compounds were also found to demonstrate potential inhibition of cellular proliferation in human breast cancer MDA-MB-231 cells. In the present study, we have utilized a multi-step virtual screening protocol to identify inhibitors of DNMT1, which offers a starting point to develop more potent DNMT1 inhibitors as anti-cancer agents.
Subject(s)
DNA (Cytosine-5-)-Methyltransferase 1/antagonists & inhibitors , Drug Evaluation, Preclinical , Enzyme Inhibitors/analysis , Enzyme Inhibitors/pharmacology , Models, Molecular , Antineoplastic Agents/analysis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Bayes Theorem , Cell Line, Tumor , Cell Proliferation/drug effects , Cell-Free System , DNA (Cytosine-5-)-Methyltransferase 1/chemistry , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , Enzyme Inhibitors/chemistry , HEK293 Cells , Humans , Inhibitory Concentration 50 , Molecular Docking Simulation , Reproducibility of Results , Small Molecule LibrariesABSTRACT
DNA methyltransferase is the key enzyme in the process of DNA methylation, playing an important role in regulation of gene expression in vivo. According to the Ganoderma lucidum transcriptome data, a full-length cDNA sequence of MET1 from G. lucidum was cloned for the first time, the GenBank registration number is KU239998, and we conducted a comprehensive bioinformatics analysis of the genetic characteristics and spatial structure. The prokaryotic expression analysis showed that E.coli[pET28a(+)-GlMET1] in BL21(DE3) could induce objective protein, shaking the culture at 16 â until the host bacterium(A600) was approximately 0.8, and added IPTG to finally concentration of 0.2 mmolâ¢L⻹, and then the optimal expression of GlMET1 recombinant protein was accumulated for the induction time of 20 h. The real-time PCR results showed that the expression levels of GlMET1 had obvious differences among varieties of G. lucidum. During the maturity stage, the expression levels of GlMET1 were lower than that in juvenile stage, the results showed that with the growth of G. lucidum, the expression levels of GlMET1 were on the decline. The research provided an important basis for studying the mechanism of DNA methyltransferase thoroughly.