ABSTRACT
BACKGROUND: Soothing the liver (called Shu Gan Jie Yu in Chinese, SGJY) is a significant therapeutic method for breast cancer in TCM. In this study, 3 liver-soothing herbs, including Cyperus rotundus L., Citrus medica L. var. sarcodactylis Swingle and Rosa rugosa Thunb. were selected and combined to form a SGJY herbal combinatory. THE AIM OF THE STUDY: To investigate the inhibiting effect of SGJY on breast cancer in vivo and vitro, and to explore the potential mechanisms. MATERIALS AND METHODS: SGJY herbal combination was extracted using water. A breast cancer rat model was developed by chemical DMBA by gavage, then treated with SGJY for 11 weeks. The tumor tissue was preserved for RNA sequencing and analyzed by IPA software. The inhibition effects of SGJY on MCF-7 and T47D breast cancer cells were investigated by SRB assay and cell apoptosis analysis, and the protein expression levels of SNCG, ER-α, p-AKT and p-ERK were measured by western blotting. RESULTS: SGJY significantly reduced the tumor weight and volume, and the level of estradiol in serum. The results of IPA analysis reveal SGJY upregulated 7 canonical pathways and downregulated 16 canonical pathways. Estrogen receptor signaling was the key canonical pathway with 9 genes downregulated. The results of upstream regulator analysis reveal beta-estradiol was the central target; the upstream regulator network scheme showed that 86 genes could affect the expression of the beta-estradiol, including SNCG, CCL21 and MB. Additionally, SGJY was verified to significantly alter the expression of SNCG mRNA, CCL21 mRNA and MB mRNA which was consistent with the data of RNA-Seq. The inhibition effects of SGJY exhibited a dose-dependent response. The apoptosis rates of MCF7 and T47D cells were upregulated. The protein expression of SNCG, ER-α, p-AKT and p-ERK were all significantly decreased by SGJY on MCF-7 and T47D cells. CONCLUSION: The results demonstrate that SGJY may inhibit the growth of breast cancer. The mechanism might involve downregulating the level of serum estradiol, and suppressing the protein expression in the SNCG/ER-α/AKT-ERK pathway.
Subject(s)
Breast Neoplasms , MAP Kinase Signaling System , Animals , Female , Humans , Rats , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation , Estradiol , gamma-Synuclein/genetics , gamma-Synuclein/metabolism , MCF-7 Cells , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Estrogen/metabolism , RNA, Messenger/metabolism , RNA-SeqABSTRACT
Objective: This study investigated the expression and clinical significance of Melanoma Associated Antigen (MAGE)-A proteins and mRNA in patients with non-small cell lung cancer (NSCLC). Methods: A retrospective study was conducted, and we selected a cohort of 88 NSCLC patients treated at our hospital from January 2015 to January 2020. Adjacent tissues were chosen as controls. The expression of MAGE-A proteins in lung cancer and adjacent tissues was assessed via Western blot, while MAGE-As mRNA expression was measured using RT-PCR. Results: The relative expression levels of MAGE-A proteins and mRNA in NSCLC tissues were significantly higher than those in adjacent tissues (P < .05), with values of (0.343 ± 0.101) and (0.728 ± 0.112), respectively. Furthermore, MAGE-As protein expression was significantly higher in stage III - IV lung cancer compared to stage I - II (P < .05). No significant differences were observed in MAGE-A protein expression concerning gender, age, tumor diameter, pathological type, and differentiation degree (P > .05). The relative expression of MAGE-As mRNA was significantly higher in clinical stage III - IV and moderately differentiated lung cancer tissues compared to stage I - II and well-differentiated tissues (P < .05). No significant differences were found in MAGE-As mRNA expression concerning gender, age, tumor diameter, and pathological type (P > .05). Patients with high MAGE-As mRNA expression had a significantly shorter median overall survival of 33 months (95% CI: 31.64-34.36) compared to those with low MAGE-As mRNA expression (P < .05). However, no significant difference was observed in median overall survival between patients with high and low MAGE-As protein expression (P > .05). Conclusions: In NSCLC, the up-regulation of MAGE-A proteins and mRNA is associated with clinical stage and differentiation degree, warranting further investigation.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , Lung Neoplasms/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Retrospective Studies , RNA, Messenger , Clinical Relevance , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolismABSTRACT
Hirudo nipponia, a blood-sucking leech native to East Asia, possesses a rich repertoire of active ingredients in its saliva, showcasing significant medical potential due to its anticoagulant, anti-inflammatory, and antibacterial effects against human diseases. Despite previous studies on the transcriptomic and proteomic characteristics of leech saliva, which have identified medicinal compounds, our knowledge of tissue-specific transcriptomes and their spatial expression patterns remains incomplete. In this study, we conducted an extensive transcriptomic profiling of the salivary gland tissue in H. nipponia based on de novo assemblies of tissue-specific transcriptomes from the salivary gland, teeth, and general head region. Through gene ontology (GO) analysis and hierarchical clustering, we discovered a novel set of anti-coagulant factors-i.e., Hni-Antistasin, Hni-Ghilanten, Hni-Bdellin, Hni-Hirudin-as well as a previously unrecognized immune-related gene, Hni-GLIPR1 and uncharacterized salivary gland specific transcripts. By employing in situ hybridization, we provided the first visualization of gene expression sites within the salivary gland of H. nipponia. Our findings expand on our understanding of transcripts specifically expressed in the salivary gland of blood-sucking leeches, offering valuable resources for the exploration of previously unidentified substances with medicinal applications.
Subject(s)
Hirudo medicinalis , Leeches , Animals , Gene Expression Profiling , Hirudo medicinalis/genetics , Hirudo medicinalis/metabolism , Leeches/genetics , Leeches/metabolism , Membrane Proteins/genetics , Neoplasm Proteins/genetics , Nerve Tissue Proteins/genetics , Proteomics , Salivary Glands/metabolismABSTRACT
Endosulfan belongs to persistent organic pollutants (POPs), closely related to an increased risk of prostate cancer (PCa). The existing evidence shows that lncRNAs compete with miRNAs for binding sites and contribute to the onset and progression of human malignancies. In this study we investigate how endosulfan promotes cell migration and invasion in DU145 and PC3 prostate cancer cells through epigenetic mechanism of lncRNA-miRNA regulation. Based on our past research we focused on PTP4A3 and constructed wild-type (WT) and mutant PTP4A3 plasmids for further analysis. Our results revealed that transfection of PTP4A3-WT can lead to changes in the expression of epithelial-mesenchymal transition (EMT) biomarkers and critical proteins in the TGF-ß signaling pathway, and promote cell migration and invasion in PCa cells. Bioinformatics analysis shows that there were complementary sequences in PTP4A3 3'-UTR and KCNQ1OT1 3'-UTR to the seed sequence of hsa-miR-137-3p, and dual luciferase reporter assay indicates the potential binding capacity of miR-137-3p to 3'-UTR of PTP4A3 and KCNQ1OT1. We found that miR-137-3p mimic inhibited cell migration and invasion, as well as repressed alterations of EMT biomarkers and critical proteins in the TGF-ß signaling pathway. Rescue experiment results revealed that co-transfection of miR-137-3p mimic and PTP4A3-WT plasmid reversed these changes following transfection with miR-137-3p mimic alone. We found that KCNQ1OT1 was predominantly distributed in the cytoplasm from a subcellular fractionation assay. Functionally, silencing of KCNQ1OT1 repressed cell migration and invasion, and caused alterations of EMT biomarkers and critical proteins in the TGF-ß signaling pathway, which were all restored by co-transfection with anti-miR-137-3p or PTP4A3-WT plasmid. Furthermore, overexpression of miR-137-3p or silencing of KCNQ1OT1 dramatically rescued the effects of endosulfan on promoting cell migration and invasion. These findings suggest that endosulfan can indeed promote cell migration and invasion via the KCNQ1OT1/miR-137-3p/PTP4A3 axis in PCa cells.
Subject(s)
MicroRNAs , Prostatic Neoplasms , Protein Tyrosine Phosphatases , RNA, Long Noncoding , Cell Line, Tumor , Cell Movement , Cell Proliferation , Endosulfan/toxicity , Humans , Male , MicroRNAs/genetics , Neoplasm Proteins/genetics , Potassium Channels, Voltage-Gated , Protein Tyrosine Phosphatases/genetics , RNA, Long Noncoding/genetics , Transforming Growth Factor beta/metabolismABSTRACT
Hexavalent chromium [Cr(VI)], one common environmental contaminant, has long been recognized as a carcinogen associated with several malignancies, such as lung cancer, but little information was available about the effects of its low-dose environmental exposure in prostate cancer. Our previous study has shown that low-dose Cr(VI) exposure could promote prostate cancer(PCa) cell growth in vitro and in vivo. In the present study, we furthermore found that low-dose Cr(VI) exposure could induce DNA demethylation in PCa cells. Based on our transcriptome sequencing data and DNA methylation database, we further identified MAGEB2 as a potential effector target that contributed to tumor-promoting effect of low-dose Cr(VI) exposure in PCa. In addition, we demonstrated that MAGEB2 was upregulated in PCa and its knockdown restrained PCa cell proliferation and tumor growth in vitro and in vivo. Moreover, Co-IP and point mutation experiments confirmed that MAGEB2 could bind to the NH2-terminal NTD domain of AR through the F-box in the MAGE homology domain, and then activated AR through up-regulating its downstream targets PSA and NX3.1. Together, low-dose Cr(VI) exposure can induce DNA demethylation in prostate cancer cells, and promote cell proliferation via activating MAGEB2-AR signaling pathway. Thus, inhibition of MAGEB2-AR signaling is a novel and promising strategy to reverse low-dose Cr(VI) exposure-induced prostate tumor progression, also as effective adjuvant therapy for AR signaling-dependent PCa.
Subject(s)
Antigens, Neoplasm , Carcinogens, Environmental , Neoplasm Proteins , Prostatic Neoplasms , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Carcinogens, Environmental/toxicity , Cell Proliferation/drug effects , Chromium/toxicity , Humans , Male , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Receptors, Androgen/metabolism , Signal Transduction/drug effectsABSTRACT
Honey-processed Astragalus is a dosage form of Radix Astragalus mixed with honey by a traditional Chinese medicine processing method which improves immune activity. This pharmacological activity of honey-processed Astragalus polysaccharide (HP-APS) might be due to structural changes during the honey roasting process. Previously, we have prepared and characterized HP-APS and preliminarily found its anti-inflammatory effects. However, whether the pharmacodynamic activity of HP-APS induces tumor cell apoptosis and the mechanisms responsible for the immunogenic death (ICD) have not been elucidated. Here, A549, MC38 and B16 cells were used to evaluate the cells viability, apoptosis and cell cycles, respectively. Cellular immunogenic cell death-related molecules calreticulin (CRT), Heat Shock Proteins (HSP)70, major histocompatibility complex I (MHC-I), and co-stimulator molecules CD80/CD86 were determined by flow cytometry. The extracellular ATP release was also detected. B16-OVA and MC38-OVA cells were treated with HP-APS and co-cultivated with OT1 mouse of CD3+T cells for assessment of proliferation, in mice model, and the establishment of C57BL/B6 mouse model bearing B16 cells for assessment of HP-APS the regulation of immune activity in vivo. Our results showed that HP-APS has an inhibitory effect on tumor cell proliferation, which induces tumor cell apoptosis, preventing cells-transforming from G1 phase to S phase in cell cycles. Furthermore, HP-APS could effectively increase the expression of HSP70, CRT, MHC-I, CD86, CD80 and ATP release. T cell proliferation index is significantly improved. CD3 cell proliferation in OT1 mice was significantly increased from the 4th generation to the 5th generation. Moreover, the results have also shown that HP-APS could inhibit tumor growth by increasing immune cell infiltration in the tumor tissues. In the mouse melanoma model with HP-APS treatment, the tumor weight and volume were significantly reduced, and the growth of melanoma was inhibited. CD8+ T is significantly increased. The ratio of CD4+ T and CD8+ T cells numbers are also significantly increased in mouse spleen, but it is less than PD-1 alone treatment separately. Altogether, these findings suggest that HP-APS exerts anti-tumor effects, and that its underlying mechanisms might be associated with the expression of immunogenicity cell death related molecules and the immunomodulatory effects of immune cells.
Subject(s)
Astragalus Plant/chemistry , Drugs, Chinese Herbal/pharmacology , Immunogenic Cell Death/drug effects , Neoplasms/drug therapy , A549 Cells , Animals , Apoptosis/drug effects , Astragalus propinquus/chemistry , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Cell Proliferation/drug effects , Cell Survival/drug effects , Drugs, Chinese Herbal/chemistry , Gene Expression Regulation, Neoplastic/drug effects , Honey/analysis , Humans , Immunogenic Cell Death/immunology , Immunomodulation/drug effects , Immunomodulation/immunology , Lymphocyte Activation/drug effects , Melanoma, Experimental , Mice , Neoplasm Proteins/genetics , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/pathology , Polysaccharides/chemistry , Polysaccharides/pharmacologyABSTRACT
Vitamin D3 (VD3) deficiency has been associated with increased risk for cirrhosis and hepatocellular carcinoma, a highly incident malignant neoplasia worldwide. On the other hand, VD3 supplementation has shown some beneficial effects in clinical studies and rodent models of chronic liver disease. However, preventive effects of dietary VD3 supplementation in cirrhosis-associated hepatocarcinogenesis is still unknow. To investigate this purpose, male Wistar rats submitted to a combined diethylnitrosamine- and thioacetamide-induced model were concomitantly supplemented with VD3 (5,000 and 10,000 IU/kg diet) for 25 weeks. Liver samples were collected for histological, biochemical and molecular analysis. Serum samples were used to measure 25-hydroxyvitamin D [25(OH)D] and alanine aminotransferase levels. Both VD3 interventions decreased hepatic collagen deposition and pro-inflammatory p65 protein levels, while increased hepatic antioxidant catalase and glutathione peroxidase activities and serum 25(OH)D, without a clear dose-response effect. Nonetheless, only the highest concentration of VD3 increased hepatic protein levels of VD receptor, while decreased the number of large preneoplastic glutathione-S-transferase- (>0.5 mm²) and keratin 8/18-positive lesions, as well the multiplicity of hepatocellular adenomas. Moreover, this intervention increased hepatic antioxidant Nrf2 protein levels and glutathione-S-transferase activity. In summary, dietary VD3 supplementation - in special the highest intervention - showed antifibrotic and antineoplastic properties in chemically-induced cirrhosis-associated hepatocarcinogenesis. The positive modulation of Nrf2 antioxidant axis may be mechanistically involved with these beneficial effects, and may guide future clinical studies.
Subject(s)
Adenoma, Liver Cell/prevention & control , Carcinoma, Hepatocellular/prevention & control , Dietary Supplements , Liver Cirrhosis/drug therapy , Liver Neoplasms/prevention & control , Vitamin D/administration & dosage , Adenoma, Liver Cell/chemically induced , Adenoma, Liver Cell/metabolism , Adenoma, Liver Cell/pathology , Alanine Transaminase/blood , Alanine Transaminase/genetics , Animals , Carcinoma, Hepatocellular/chemically induced , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Catalase/blood , Catalase/genetics , Chemoprevention/methods , Collagen/genetics , Collagen/metabolism , Diethylnitrosamine/toxicity , Gene Expression Regulation/drug effects , Glutathione Peroxidase/blood , Glutathione Peroxidase/genetics , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Keratins/genetics , Keratins/metabolism , Liver/drug effects , Liver/metabolism , Liver/pathology , Liver Cirrhosis/chemically induced , Liver Cirrhosis/metabolism , Liver Cirrhosis/pathology , Liver Neoplasms/chemically induced , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Male , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Nucleocytoplasmic Transport Proteins/genetics , Nucleocytoplasmic Transport Proteins/metabolism , Rats , Rats, Wistar , Receptors, Calcitriol/genetics , Receptors, Calcitriol/metabolism , Thioacetamide/toxicity , Vitamin D/analogs & derivatives , Vitamin D/bloodABSTRACT
BACKGROUND: Studies have shown that long non-coding RNAs (lncRNAs) play essential roles in tumor progression and can affect the response to radiotherapy, including in clear cell renal cell carcinoma (ccRCC). LINC02532 has been found to be upregulated in ccRCC. However, not much is known about this lncRNA. Hence, this study aimed to investigate the role of LINC02532 in ccRCC, especially in terms of radioresistance. METHODS: Quantitative real-time PCR was used to detect the expression of LINC02532, miR-654-5p, and YY1 in ccRCC cells. Protein levels of YY1, cleaved PARP, and cleaved-Caspase-3 were detected by Western blotting. Cell survival fractions, viability, and apoptosis were determined by clonogenic survival assays, CCK-8 assays, and flow cytometry, respectively. The interplay among LINC02532, miR-654-5p, and YY1 was detected by chromatin immunoprecipitation and dual-luciferase reporter assays. In addition, in vivo xenograft models were established to investigate the effect of LINC02532 on ccRCC radioresistance in 10 nude mice. RESULTS: LINC02532 was highly expressed in ccRCC cells and was upregulated in the cells after irradiation. Moreover, LINC02532 knockdown enhanced cell radiosensitivity both in vitro and in vivo. Furthermore, YY1 activated LINC02532 in ccRCC cells, and LINC02532 acted as a competing endogenous RNA that sponged miR-654-5p to regulate YY1 expression. Rescue experiments indicated that miR-654-5p overexpression or YY1 inhibition recovered ccRCC cell functions that had been previously impaired by LINC02532 overexpression. CONCLUSIONS: Our results revealed a positive feedback loop of LINC02532/miR-654-5p/YY1 in regulating the radiosensitivity of ccRCC, suggesting that LINC02532 might be a potential target for ccRCC radiotherapy. This study could serve as a foundation for further research on the role of LINC02532 in ccRCC and other cancers.
Subject(s)
Carcinoma, Renal Cell/metabolism , Kidney Neoplasms/metabolism , MicroRNAs/metabolism , Neoplasm Proteins/metabolism , RNA, Long Noncoding/metabolism , RNA, Neoplasm/metabolism , Radiation Tolerance , YY1 Transcription Factor/metabolism , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/radiotherapy , Cell Line, Tumor , Humans , Kidney Neoplasms/genetics , Kidney Neoplasms/radiotherapy , MicroRNAs/genetics , Neoplasm Proteins/genetics , RNA, Long Noncoding/genetics , RNA, Neoplasm/genetics , YY1 Transcription Factor/geneticsABSTRACT
Cancer cells are able to proliferate in an unregulated manner. There are several mechanisms involved that propel such neoplastic transformations. One of these processes involves bypassing cell death through changes in gene expression and, consequently, cell growth. This involves a complex epigenetic interaction within the cell, which drives it towards oncogenic transformations. These epigenetic events augment cellular growth by potentially altering chromatin structures and influencing key gene expressions. Therapeutic mechanisms have been developed to combat this by taking advantage of the underlying oncogenic mechanisms through chemical modulation. Camptothecin (CPT) is an example of this type of drug. It is a selective topoisomerase I inhibitor that is effective against many cancers, such as colorectal cancer. Previously, we successfully formulated a magnetic nanocarrier-conjugated CPT with ß-cyclodextrin and iron NPs (Fe3O4) cross-linked using EDTA (CPT-CEF). Compared to CPT alone, it boasts higher efficacy due to its selective targeting and increased solubility. In this study, we treated HT29 colon cancer cells with CPT-CEF and attempted to investigate the cytotoxic effects of the formulation through an epigenetic perspective. By using RNA-Seq, several differentially expressed genes were obtained (p < 0.05). Enrichr was then used for the over-representation analysis, and the genes were compared to the epigenetic roadmap and histone modification database. The results showed that the DEGs had a high correlation with epigenetic modifications involving histone H3 acetylation. Furthermore, a subset of these genes was shown to be associated with the Wnt/ß-catenin signaling pathway, which is highly upregulated in a large number of cancer cells. These genes could be investigated as downstream therapeutic targets against the uncontrolled proliferation of cancer cells. Further interaction analysis of the identified genes with the key genes of the Wnt/ß-catenin signaling pathway in colorectal cancer identified the direct interactors and a few transcription regulators. Further analysis in cBioPortal confirmed their genetic alterations and their distribution across patient samples. Thus, the findings of this study reveal that colorectal cancer could be reversed by treatment with the CPT-CEF nanoparticle-conjugated nanocarrier through an epigenetic mechanism.
Subject(s)
Camptothecin , Colorectal Neoplasms , Genes, Neoplasm , Histones , Nanocapsules , Neoplasm Proteins , Wnt Signaling Pathway/drug effects , Camptothecin/chemistry , Camptothecin/pharmacology , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , HT29 Cells , Histones/genetics , Histones/metabolism , Humans , Nanocapsules/chemistry , Nanocapsules/therapeutic use , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolismABSTRACT
Cranberry is a dietary supplement popularly used for the prophylaxis of urinary tract infection. Interestingly, cranberry-warfarin interactions in clinical reports have shown bidirectional outcomes. (±) Warfarin, a widely prescribed anticoagulant, but with a narrow therapeutic index, contains equal amounts of S- and R-warfarin, of which S-warfarin is more active. The aim of this study was to investigate the effects of different ingestion times of cranberry on the pharmacokinetics and pharmacodynamics of warfarin. Rats were orally administered (±) warfarin (0.2 mg/kg) with and without cranberry (5.0 g/kg) at 0.5 h prior to the warfarin, and at 10 h after the warfarin. The plasma concentrations of S- and R-warfarin were determined by LC/MS. The results indicate that cranberry ingested at 0.5 h before (±) warfarin significantly decreased the systemic exposures of S-warfarin and R-warfarin. Conversely, when cranberry was ingested at 10 h after (±) warfarin, the elimination of S-warfarin was significantly inhibited, and the anticoagulation effect of (±) warfarin was significantly enhanced. The results of the mechanism studies indicate that cranberry activated the breast cancer resistance protein (BCRP), which mediated the efflux transports of S-warfarin and R-warfarin. Moreover, the metabolites of cranberry inhibited cytochrome P450 (CYP) 2C9, the main metabolizing enzyme for S-warfarin. In conclusion, cranberry affected the pharmacokinetics of (±) warfarin in a bidirectional manner by activating the BCRP by CJ during absorption and inhibiting the BCRP and CYP2C9 by CMs during elimination, depending on the ingestion time of CJ. The combined use of cranberry with warfarin should be avoided.
Subject(s)
ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism , Cytochrome P-450 Enzyme System/metabolism , Fruit and Vegetable Juices , Gene Expression Regulation/drug effects , Neoplasm Proteins/metabolism , Vaccinium macrocarpon , Warfarin/pharmacokinetics , ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics , Administration, Oral , Animals , Cytochrome P-450 Enzyme System/genetics , Dogs , Food-Drug Interactions , Humans , Madin Darby Canine Kidney Cells , Male , Neoplasm Proteins/genetics , Rats , Rats, Sprague-Dawley , Warfarin/bloodABSTRACT
The essential microelement zinc plays immunoregulatory roles via its ability to influence signaling pathways. Zinc deficiency impairs overall immune function and resultantly increases susceptibility to infection. Thus, zinc is considered as an immune-boosting supplement for populations with hypozincemia at high-risk for infection. Besides its role as a structural cofactor of many proteins, zinc also acts as an intracellular messenger in immune cell signaling. T-cell activation instructs zinc influx from extracellular and subcellular sources through the Zip6 and Zip8 zinc transporters, respectively. Increased cytoplasmic zinc participates in the regulation of T-cell responses by modifying activation signaling. However, the mechanism underlying the activation-dependent movement of zinc ions by Zip transporters in T cells remains elusive. Here, we demonstrate that Zip6, one of the most abundantly expressed Zip transporters in T cells, is mainly localized to lipid rafts in human T cells and is recruited into the immunological synapse in response to TCR stimulation. This was demonstrated through confocal imaging of the interaction between CD4+ T cells and antigen-presenting cells. Further, immunoprecipitation assays show that TCR triggering induces tyrosine phosphorylation of Zip6, which has at least three putative tyrosine motifs in its long cytoplasmic region, and this phosphorylation is coupled with its physical interaction with Zap70. Silencing Zip6 reduces zinc influx from extracellular sources and suppresses T-cell responses, suggesting an interaction between Zip6-mediated zinc influx and TCR activation. These results provide new insights into the mechanism through which Zip6-mediated zinc influx occurs in a TCR activation-dependent manner in human CD4+ T cells.
Subject(s)
Antigen-Presenting Cells/metabolism , CD4-Positive T-Lymphocytes/metabolism , Cation Transport Proteins/metabolism , Immunological Synapses/metabolism , Membrane Microdomains/metabolism , Neoplasm Proteins/metabolism , Receptors, Antigen, T-Cell/metabolism , ZAP-70 Protein-Tyrosine Kinase/metabolism , Antigen-Presenting Cells/immunology , CD4-Positive T-Lymphocytes/immunology , Cation Transport Proteins/genetics , Humans , Immunological Synapses/immunology , Jurkat Cells , Lymphocyte Activation , Membrane Microdomains/immunology , Neoplasm Proteins/genetics , Phosphorylation , Signal Transduction , TyrosineABSTRACT
We have previously reported that F1012-2, a sesquiterpene lactone isolated from the Chinese herbal medicine Eupatorium lindleyanum DC., exhibits strong effects against Triple Negative Breast Cancer (TNBC). In this study, we found F1012-2 effectively inhibited cell migration and invasion detected by wound healing and transwell assays. In order to elucidate the potential mechanisms of F1012-2, we further studied its effect on DNA damage in TNBC cell lines. Using single cell gel electrophoresis (comet assay), immunofluorescence, and western blotting assays, we found that F1012-2 treatment induced significant DNA strand breaks and γ-H2AX activation. Moreover, exposure to F1012-2 led to overproduction of reactive oxygen species (ROS). NAC treatment completely eliminated ROS, which may be due to the interaction between NAC and F1012-2. A further study of the molecular mechanisms demonstrated that the MAPK signaling pathway participated in the anti-TNBC effect of F1012-2. Pretreatment with specific inhibitors targeting JNK (SP600125) and ERK (PD98059) could rescue the decrease in cell viability and inhibit expressions of JNK and ERK phosphorylation, but SB203580 had no effects. Finally, in the acute toxicity experiment, there were no obvious symptoms of poisoning in the F1012-2 treatment group. An in vivo study demonstrated that F1012-2 significantly suppressed the tumor growth and induced DNA damage. In conclusion, the activity of F1012-2-induced DNA damage in TNBC was found in vivo and in vitro, which might trigger the MAPK pathway through ROS accumulation. These results indicate that F1012-2 may be an effective anti-TNBC therapeutic agent.
Subject(s)
DNA Damage , DNA, Neoplasm/metabolism , Lactones/pharmacology , MAP Kinase Signaling System/drug effects , Reactive Oxygen Species/metabolism , Sesquiterpenes/pharmacology , Triple Negative Breast Neoplasms/metabolism , Cell Line, Tumor , DNA, Neoplasm/genetics , Female , Humans , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/pathologyABSTRACT
Rationale: Tanshinone, a type of diterpenes derived from salvia miltiorrhiza, is a particularly promising herbal medicine compound for the treatment of cancers including acute myeloid leukemia (AML). However, the therapeutic function and the underlying mechanism of Tanshinone in AML are not clear, and the toxic effect of Tanshinone limits its clinical application. Methods: Our work utilizes human leukemia cell lines, zebrafish transgenics and xenograft models to study the cellular and molecular mechanisms of how Tanshinone affects normal and abnormal hematopoiesis. WISH, Sudan Black and O-Dianisidine Staining were used to determine the expression of hematopoietic genes on zebrafish embryos. RNA-seq analysis showed that differential expression genes and enrichment gene signature with Tan I treatment. The surface plasmon resonance (SPR) method was used with a BIAcore T200 (GE Healthcare) to measure the binding affinities of Tan I. In vitro methyltransferase assay was performed to verify Tan I inhibits the histone enzymatic activity of the PRC2 complex. ChIP-qPCR assay was used to determine the H3K27me3 level of EZH2 target genes. Results: We found that Tanshinone I (Tan I), one of the Tanshinones, can inhibit the proliferation of human leukemia cells in vitro and in the xenograft zebrafish model, as well as the normal and malignant definitive hematopoiesis in zebrafish. Mechanistic studies illustrate that Tan I regulates normal and malignant hematopoiesis through direct binding to EZH2, a well-known histone H3K27 methyltransferase, and inhibiting PRC2 enzymatic activity. Furthermore, we identified MMP9 and ABCG2 as two possible downstream genes of Tan I's effects on EZH2. Conclusions: Together, this study confirmed that Tan I is a novel EZH2 inhibitor and suggested MMP9 and ABCG2 as two potential therapeutic targets for myeloid malignant diseases.
Subject(s)
ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism , Abietanes/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Enhancer of Zeste Homolog 2 Protein/antagonists & inhibitors , Hematopoiesis/drug effects , Leukemia/drug therapy , Leukemia/metabolism , Matrix Metalloproteinase 9/metabolism , Neoplasm Proteins/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics , Animals , Animals, Genetically Modified , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Chromatin Immunoprecipitation , Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/metabolism , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Hematopoiesis/genetics , Histones/metabolism , Humans , Leukemia/enzymology , Leukemia/genetics , Matrix Metalloproteinase 9/genetics , Neoplasm Proteins/genetics , Polycomb Repressive Complex 2/metabolism , Protein Binding , RNA-Seq , Salvia miltiorrhiza/chemistry , Surface Plasmon Resonance , Transcriptome/genetics , Xenograft Model Antitumor Assays , ZebrafishABSTRACT
Maytenus roylanus (MEM) is a plant with anti-proliferative effects against prostate cancer. We aimed to explore the mechanism of action of MEM in prostate cancer (PCa) by employing an in vitro global proteome approach to get useful information of various signaling pathways and effected genes to define the mechanism of MEM action in prostate cancer. We conducted a global proteome analysis of CWR22Rv1after treatment with methanolic extract of MEM. The result of the proteomic profiling of in vitro PCa cells demonstrated the reduction in tumor protein D52 (TPD52) expression after treatment with methanolic extract of MEM. Down-regulation of TPD52 expression at mRNA level was observed by MEM treatment in CWR22Rν1 and C4-2 cells in a dose-dependent fashion probably by cleavage of Caspase 3 and PARP, or by modulation of cyclin-dependent kinases in CWR22Rν1 and C4-2 cells. The progressive character of the TRAMP model demonstrates a chance to evaluate the potential of chemo-preventive agents for both initial and late stages of prostate cancer development, and induction in TPD52 protein expression with development as well as the progression of prostate cancer was observed in the TRAMP model. Analyses of the tissue microarray collection of 25 specimens confirmed the clinical significance of our findings identifying TPD52 as a potential marker for PCa progression. We determined that knockdown of TPD52 (CWR22Rν1 cells), a considerable downregulation was seen at the protein level. Downregulation of TPD52 inhibited the migration and invasive behavior of prostate cancer cells as observed. Moreover, we observed that the siRNA-TPD52 transfection of CWR22Rν1 cells resulted in tumor growth inhibition with a marked reduction in the secretion of prostate-specific antigen (PSA) in the serum. Intraperitoneal injection of MEM considerably slowed tumor growth in athymic mice, inhibited TPD52 expression, and caused a marked reduction in PSA levels of serum as demonstrated by immunoblot screening and immune-histochemical staining. This report illustrates a molecular overview of pathological processes in PCa, indicating possible new disease biomarkers and therapeutic targets.
Subject(s)
Maytenus/chemistry , Neoplasm Proteins/metabolism , Plant Extracts/pharmacology , Prostatic Neoplasms/metabolism , Proteomics/methods , Tissue Array Analysis/methods , Animals , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Male , Mice, Nude , Neoplasm Proteins/genetics , PC-3 Cells , Plant Extracts/administration & dosage , Plant Leaves/chemistry , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/genetics , RNA Interference , Xenograft Model Antitumor Assays/methodsABSTRACT
In this study, we investigated the effects of treatment with Gingko biloba leaf extract (GLE) on intestinal transporter expression and gut microbiota composition in mice and the correlation between intestinal transporter expression and gut microbiota composition in mice. When GLE was orally administered to mice, intestinal BCRP expression was significantly suppressed. Pharmacokinetic studies showed that the maximum plasma concentration and area under the curve values of sulfasalazine were increased more than twice by treatment with GLE compared with those in the control group. GLE treatment significantly decreased the populations of Proteobacteria and Deferribacteres at the phylum level. Correlation analysis showed that BCRP expression was positively or negatively correlated with the composition of gut bacteria. In Caco-2 cells, GLE treatment did not affect BCRP expression, but treatment with the lysates of GLE-treated mouse feces significantly suppressed BCRP expression. These findings demonstrate that the suppression of intestinal BCRP expression following GLE treatment may occur through modulation of the gut microbiota composition. Thus, the present study suggests that modulation of gut microbiota composition may cause drug transporter-mediated herb-drug interactions.
Subject(s)
ATP Binding Cassette Transporter, Subfamily G, Member 2/metabolism , Gastrointestinal Microbiome/drug effects , Herb-Drug Interactions , Neoplasm Proteins/metabolism , Plant Extracts/pharmacology , Sulfasalazine/pharmacokinetics , ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics , Animals , Caco-2 Cells , Feces/chemistry , Feces/microbiology , Ginkgo biloba , Humans , Male , Metabolome , Mice, Inbred C57BL , Neoplasm Proteins/genetics , Sulfasalazine/bloodABSTRACT
Hemistepta lyrata (Bunge) Bunge is a biennial medicinal plant possessing beneficial effects including anti-inflammation, and hemistepsin A (HsA) isolated from H. lyrata has been known as a hepatoprotective sesquiterpene lactone. In this report, we explored the cytotoxic effects of H. lyrata on hepatocellular carcinoma (HCC) cells and investigated the associated bioactive compounds and their relevant mechanisms. From the viability results of HCC cells treated with various H. lyrata extracts, HsA was identified as the major compound contributing to the H. lyrata-mediated cytotoxicity. HsA increased expression of cleaved PARP and cells with Sub-G1 phase, Annexin V binding, and TUNEL staining, which imply HsA induces apoptosis. In addition, HsA provoked oxidative stress by decreasing the reduced glutathione/oxidized glutathione ratio and accumulating reactive oxygen species and glutathione-protein adducts. Moreover, HsA inhibited the transactivation of signal transducer and activator of transcription 3 (STAT3) by its dephosphorylation at Y705 and glutathione conjugation. Stable expression of a constitutive active mutant of STAT3 prevented the reduction of cell viability by HsA. Finally, HsA enhanced the sensitivity of sorafenib-mediated cytotoxicity by exaggerating oxidative stress and Y705 dephosphorylation of STAT3. Therefore, HsA will be a promising candidate to induce apoptosis of HCC cells via downregulating STAT3 and sensitizing conventional chemotherapeutic agents.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Carcinoma, Hepatocellular/pathology , Gene Expression Regulation, Neoplastic/drug effects , Lactones/pharmacology , Liver Neoplasms/pathology , Neoplasm Proteins/biosynthesis , STAT3 Transcription Factor/biosynthesis , Sesquiterpenes/pharmacology , Transcriptional Activation/drug effects , Cell Line, Tumor , Down-Regulation/drug effects , Drug Screening Assays, Antitumor , Genes, Reporter , Humans , Neoplasm Proteins/genetics , Oxidative Stress , Protein Kinase Inhibitors/pharmacology , Recombinant Proteins/metabolism , STAT3 Transcription Factor/genetics , Sorafenib/pharmacologyABSTRACT
BACKGROUND: Peripheral blood leukocyte (PBL) DNA methylation may serve as a surrogate marker to evaluate the susceptibility to and prognosis of gastric cancer (GC). In this study, blood-derived DNA methylation levels of two tumour-related genes, namely, ZNF331 and WIF1, and their impacts on the risk and prognosis of GC were evaluated. METHODS: In total, 398 GC cases and 397 controls were recruited for the study. Then, all cases were followed up for 5 years. ZNF331 and WIF1 promoter methylation status in PBLs was measured using a methylation-sensitive high-resolution melting method. Logistic and Cox regression models were used to analyse the correlation between gene methylation and the risk and prognosis of GC. Confounders were balanced through propensity score (PS) matching. RESULTS: High ZNF331 methylation significantly decreased GC risk after PS adjustment (OR = 0.580, 95% CI: 0.375-0.898, P = 0.015), which also presented in males (OR = 0.577, 95% CI: 0.343-0.970, P = 0.038). However, WIF1 methylation was not associated with GC risk. Additionally, significant combined effects between ZNF331 methylation and the intake of green vegetables and garlic were observed (OR = 0.073, 95% CI: 0.027-0.196, P < 0.001 and OR = 0.138, 95% CI: 0.080-0.238, P < 0.001, respectively). Furthermore, ZNF331 and WIF1 methylation had no impact on the prognosis of GC. CONCLUSION: ZNF331 methylation in PBLs may affect GC risk in combination with the consumption of green vegetables and garlic and may act as a potential biomarker of GC.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Biomarkers, Tumor/genetics , DNA Methylation , DNA-Binding Proteins/genetics , Neoplasm Proteins/genetics , Stomach Neoplasms/epidemiology , Adaptor Proteins, Signal Transducing/blood , Adaptor Proteins, Signal Transducing/metabolism , Aged , Biomarkers, Tumor/blood , Biomarkers, Tumor/metabolism , Case-Control Studies , DNA-Binding Proteins/blood , DNA-Binding Proteins/metabolism , Diet Surveys/statistics & numerical data , Epigenesis, Genetic , Female , Garlic , Gastric Mucosa/pathology , Gene Expression Regulation, Neoplastic , Humans , Leukocytes/metabolism , Male , Middle Aged , Neoplasm Proteins/blood , Neoplasm Proteins/metabolism , Prognosis , Promoter Regions, Genetic/genetics , Propensity Score , Protective Factors , Risk Assessment/methods , Risk Assessment/statistics & numerical data , Stomach Neoplasms/blood , Stomach Neoplasms/diagnosis , Stomach Neoplasms/prevention & control , VegetablesABSTRACT
Weight loss and cachexia are common problems in colorectal cancer patients; thus, parenteral and enteral nutrition support play important roles in cancer care. However, the impact of nonessential amino acid components of nutritional intake on cancer progression has not been fully studied. In this study, we discovered that gastrointestinal cancer patients who received cysteine as part of the parenteral nutrition had shorter overall survival (P < 0.001) than those who did not. Cystine indeed robustly promotes colon cancer cell growth in vitro and in immunodeficient mice, predominately by inhibiting SESN2 transcription via the GCN2-ATF4 axis, resulting in mTORC1 activation. mTORC1 inhibitors Rapamycin and Everolimus block cystine-induced cancer cell proliferation. In addition, cystine confers resistance to oxaliplatin and irinotecan chemotherapy by quenching chemotherapy-induced reactive oxygen species via synthesizing glutathione. We demonstrated that dietary deprivation of cystine suppressed colon cancer xenograft growth without weight loss in mice and boosted the antitumor effect of oxaliplatin. These findings indicate that cyst(e)ine, as part of supplemental nutrition, plays an important role in colorectal cancer and manipulation of cyst(e)ine content in nutritional formulations may optimize colorectal cancer patient survival.
Subject(s)
Colonic Neoplasms/metabolism , Cystine/adverse effects , Drug Resistance, Neoplasm/drug effects , Mechanistic Target of Rapamycin Complex 1/metabolism , Neoplasm Proteins/metabolism , Reactive Oxygen Species/metabolism , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Cystine/pharmacology , Drug Resistance, Neoplasm/genetics , HCT116 Cells , HT29 Cells , Humans , Mechanistic Target of Rapamycin Complex 1/genetics , Neoplasm Proteins/geneticsABSTRACT
The bioassay-guided fractionation of a CHCl3-MeOH extract from the stems of Cissus trifoliata identified an active fraction against PC3 prostate cancer cells. The treatment for 24 h showed an 80% reduction in cell viability (p ≤ 0.05) by a WST-1 assay at a concentration of 100 µg/mL. The HPLC-QTOF-MS analysis of the fraction showed the presence of coumaric and isoferulic acids, apigenin, kaempferol, chrysoeriol, naringenin, ursolic and betulinic acids, hexadecadienoic and octadecadienoic fatty acids, and the stilbene resveratrol. The exposure of PC3 cells to resveratrol (IC25 = 23 µg/mL) for 24 h induced significant changes in 847 genes (Z-score ≥ ±2). The functional classification tool of the DAVID v6.8 platform indicates that the underlying molecular mechanisms against the proliferation of PC3 cells were associated (p ≤ 0.05) with the process of differentiation and metabolism. These findings provide experimental evidence suggesting the potential of C. trifoliata as a promising natural source of anticancer compounds.
Subject(s)
Antineoplastic Agents, Phytogenic/chemistry , Cell Proliferation/drug effects , Cissus/chemistry , Neoplasm Proteins/genetics , Transcriptome , Antineoplastic Agents, Phytogenic/isolation & purification , Antineoplastic Agents, Phytogenic/pharmacology , Apigenin/chemistry , Apigenin/isolation & purification , Apigenin/pharmacology , Biological Assay , Cell Survival/drug effects , Flavanones/chemistry , Flavanones/isolation & purification , Flavanones/pharmacology , Flavones/chemistry , Flavones/isolation & purification , Flavones/pharmacology , Gene Expression Profiling , Humans , Kaempferols/chemistry , Kaempferols/isolation & purification , Kaempferols/pharmacology , Male , Microarray Analysis , Neoplasm Proteins/classification , Neoplasm Proteins/metabolism , PC-3 Cells , Pentacyclic Triterpenes/chemistry , Pentacyclic Triterpenes/isolation & purification , Pentacyclic Triterpenes/pharmacology , Plant Extracts/chemistry , Resveratrol/chemistry , Resveratrol/isolation & purification , Resveratrol/pharmacology , Betulinic AcidABSTRACT
One of the primary mechanisms of tumor cell immune evasion is the loss of antigenicity, which arises due to lack of immunogenic tumor antigens as well as dysregulation of the antigen processing machinery. In a screen for small-molecule compounds from herbal medicine that potentiate T cell-mediated cytotoxicity, we identified atractylenolide I (ATT-I), which substantially promotes tumor antigen presentation of both human and mouse colorectal cancer (CRC) cells and thereby enhances the cytotoxic response of CD8+ T cells. Cellular thermal shift assay (CETSA) with multiplexed quantitative mass spectrometry identified the proteasome 26S subunit non-ATPase 4 (PSMD4), an essential component of the immunoproteasome complex, as a primary target protein of ATT-I. Binding of ATT-I with PSMD4 augments the antigen-processing activity of immunoproteasome, leading to enhanced MHC-I-mediated antigen presentation on cancer cells. In syngeneic mouse CRC models and human patient-derived CRC organoid models, ATT-I treatment promotes the cytotoxicity of CD8+ T cells and thus profoundly enhances the efficacy of immune checkpoint blockade therapy. Collectively, we show here that targeting the function of immunoproteasome with ATT-I promotes tumor antigen presentation and empowers T cell cytotoxicity, thus elevating the tumor response to immunotherapy.