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Cellular and Molecular Life Sciences :... Aug 2023Human erythroleukemic K562 cells represent the prototypical cell culture model of chronic myeloid leukemia (CML). The cells are pseudo-triploid and positive for the...
Human erythroleukemic K562 cells represent the prototypical cell culture model of chronic myeloid leukemia (CML). The cells are pseudo-triploid and positive for the Philadelphia chromosome. Therefore, K562 cells have been widely used for investigating the BCR/ABL1 oncogene and the tyrosine kinase inhibitor, imatinib-mesylate. Further, K562 cells overexpress transferrin receptors (TfR) and have been used as a model for targeting cytotoxic therapies, via receptor-mediated endocytosis. Here, we have characterized K562 cells focusing on the karyotype of cells in prolonged culture, regulation of expression of TfR in wildtype (WT) and doxorubicin-resistant cells, and responses to histone deacetylase inhibition (HDACi). Karyotype analysis indicates novel chromosomes and gene expression analysis suggests a shift of cultured K562 cells away from patient-derived leukemic cells. We confirm the high expression of TfR on K562 cells using immunofluorescence and cell-surface receptor binding radioassays. Importantly, high TfR expression is observed in patient-derived cells, and we highlight the persistent expression of TfR following doxorubicin acquired resistance. Epigenetic analysis indicates that permissive histone acetylation and methylation at the promoter region regulates the transcription of TfR in K562 cells. Finally, we show relatively high expression of HDAC enzymes in K562 cells and demonstrate the chemotoxic effects of HDACi, using the FDA-approved hydroxamic acid, vorinostat. Together with a description of morphology, infrared spectral analysis, and examination of metabolic properties, we provide a comprehensive characterization of K562 cells. Overall, K562 cell culture systems remain widely used for the investigation of novel therapeutics for CML, which is particularly important in cases of imatinib-mesylate resistance.
Topics: Humans; Imatinib Mesylate; K562 Cells; Fusion Proteins, bcr-abl; Transferrin; Pyrimidines; Drug Resistance, Neoplasm; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Histone Deacetylases; Doxorubicin; Receptors, Transferrin; Chromosomes; Mesylates; Apoptosis
PubMed: 37578596
DOI: 10.1007/s00018-023-04905-6 -
Gene Nov 2023The role of histone deacetylases (HDACs) in the tumor immune microenvironment of gynecologic tumors remains unexplored. We integrated data from The Cancer Genome Atlas...
The role of histone deacetylases (HDACs) in the tumor immune microenvironment of gynecologic tumors remains unexplored. We integrated data from The Cancer Genome Atlas and Human Protein Atlas to examine HDAC expression in breast, cervical, ovarian, and endometrial cancers. Elevated HDAC expression correlated with poor prognosis and highly malignant cancer subtypes. Gene Set Enrichment Analysis revealed positive associations between HDAC expression and tumor proliferation signature, while negative associations were found with tumor inflammation signature. Increased HDAC expression was linked to reduced infiltration of natural killer (NK), NKT, and CD8 T cells, along with negative associations with the expression of PSMB10, NKG7, CCL5, CD27, HLA-DQA1, and HLA-DQB1. In a murine 4T1 breast cancer model, treatment with suberoylanilide hydroxamic acid (SAHA; HDAC inhibitor) and PD-1 antibody significantly inhibited tumor growth and infiltration of CD3 and CD8 T cells. Real-time polymerase chain reaction revealed upregulated expressions of Psmb10, Nkg7, Ccl5, Cd8a, Cxcr6, and Cxcl9 genes, while Ctnnb1 and Myc genes were inhibited, indicating tumor suppression and immune microenvironment activation. Our study revealed that HDACs play tumor-promoting and immunosuppressive roles in gynecologic cancers, suggesting HDAC inhibitors as potential therapeutic agents for these cancers.
Topics: Female; Humans; Animals; Mice; Histone Deacetylases; Genital Neoplasms, Female; Hydroxamic Acids; CD8-Positive T-Lymphocytes; Vorinostat; Histone Deacetylase Inhibitors; Tumor Microenvironment; Membrane Proteins; Proteasome Endopeptidase Complex
PubMed: 37572797
DOI: 10.1016/j.gene.2023.147704 -
International Journal of Molecular... Aug 2023Sepsis is a life-threatening medical emergency triggered by excessive inflammation in response to an infection. High mortality rates and limited therapeutic options pose...
Sepsis is a life-threatening medical emergency triggered by excessive inflammation in response to an infection. High mortality rates and limited therapeutic options pose significant challenges in sepsis treatment. Histone deacetylase inhibitors (HDACi), such as suberoylanilide hydroxamic acid (SAHA), have been proposed as potent anti-inflammatory agents for treating inflammatory diseases. However, the underlying mechanisms of sepsis treatment remain poorly understood. In this study, we investigated the effects of SAHA treatment in the lipopolysaccharide (LPS)-induced endotoxemia mouse model as it closely mimics the early stages of the systemic inflammation of sepsis. Our results demonstrate a reduced inflammatory mediator secretion and improved survival rates in mice. Using quantitative acetylomics, we found that SAHA administration increases the acetylation of lactate dehydrogenase (LDHA), and consequently inhibits LDHA activity. Notably, the reduced enzyme activity of LDHA results in a reduced rate of glycolysis. Furthermore, our experiments with bone marrow-derived macrophages (BMDMs) show that SAHA administration reduced oxidative stress and extracellular ATP concentrations, ultimately blunting inflammasome activation. Overall, our study provides insights into the mechanism underlying SAHA's therapeutic effects in sepsis treatment and highlights LDHA as a potential target for developing novel sepsis treatment.
Topics: Animals; Mice; Vorinostat; Histone Deacetylase Inhibitors; Endotoxemia; Hydroxamic Acids; Sepsis
PubMed: 37569823
DOI: 10.3390/ijms241512448 -
Heliyon Aug 2023Oxidative stress plays an important role in the secondary neuronal damage after traumatic brain injury (TBI). Inhibition of histone deacetylases (HDACs) has been shown...
Oxidative stress plays an important role in the secondary neuronal damage after traumatic brain injury (TBI). Inhibition of histone deacetylases (HDACs) has been shown to reduce reactive oxygen species (ROS) production and NADPH oxidases (Nox) transcription. Vorinostat is an HDAC inhibitor. This study investigated the influence of vorinostat on neurological impairments in a rat model of TBI induced by lateral fluid percussion injury (LFPI). Different concentrations of vorinostat (5, 25, and 50 mg/kg) were administered via intraperitoneal injection. Neurological deficits were evaluated by modified neurological severity scoring (mNSS). Evans blue extravasation was performed to assess blood-brain barrier (BBB) permeability. Morris water maze assay was performed to evaluate cognitive impairments. Protein levels were evaluated through ELISA and Western blot. Vorinostat was found to attenuate TBI induced brain edema and BBB permeability in rats. Vorinostat also alleviated TBI-induced neurological impairments and anxiety-like behavior in rats. Vorinostat attenuated TBI induced apoptosis and oxidative stresses in ipsilateral injury cortical tissue. Vorinostat inhibited HDAC1, HDAC3, and Nox4 while activated AMPK signaling in ipsilateral injury cortical tissue. In conclusion, administration of vorinostat alleviates the secondary damage of TBI in rat model. The oxidative stress in the ipsilateral injury cortical tissues is decreased by the inhibition of Nox4 expression and the activation of AMPK.
PubMed: 37560709
DOI: 10.1016/j.heliyon.2023.e18485 -
Current Issues in Molecular Biology Jul 2023Hepatocellular carcinoma (HCC) is a worldwide health issue. Epigenetic alterations play a crucial role in HCC tumorigenesis. Using epigenetic modulators for HCC...
The Synergistic Antitumor Effect of Decitabine and Vorinostat Combination on HepG2 Human Hepatocellular Carcinoma Cell Line via Epigenetic Modulation of Autophagy-Apoptosis Molecular Crosstalk.
Hepatocellular carcinoma (HCC) is a worldwide health issue. Epigenetic alterations play a crucial role in HCC tumorigenesis. Using epigenetic modulators for HCC treatment confers a promising therapeutic effect. The aim of this study was to explore the effect of a decitabine (DAC) and vorinostat (VOR) combination on the crosstalk between apoptosis and autophagy in the HCC HepG2 cell line at 24 h and 72 h. Median inhibitory concentrations (ICs) of VOR and DAC were assessed in the HepG2 cell line. The activity of caspase-3 was evaluated colorimetrically, and Cyclin D1(CCND1), Bcl-2, ATG5, ATG7, and P62 levels were assessed using ELISA at different time intervals (24 h and 72 h), while and gene expression were measured by using qRT-PCR. The synergistic effect of VOR and DAC was confirmed due to the observed combination indices (CIs) and dose reduction indices (DRIs). The combined treatment with both drugs inhibited the proliferation marker (CCND1), and enhanced apoptosis compared with each drug alone at 24 h and 72 h via active caspase-3 upregulation and Bcl-2 downregulation). Moreover, the combination induced autophagy as an early event via upregulation of , , ATG5, and ATG7 gene expression. The initial induction of autophagy started to decrease after 72 h due to downregulation, and there was decreased expression of compared with the value at 24 h. Herein, epigenetic modulation via the VOR/DAC combination showed an antitumor effect through the coordination of an autophagy-apoptosis crosstalk and promotion of autophagy-induced apoptosis, which ultimately led to the cellular death of HCC cancer cells.
PubMed: 37504291
DOI: 10.3390/cimb45070375 -
Haematologica Feb 2024Outcomes after programmed death-1 (PD-1) blockade in B-cell lymphomas are disappointing with few durable responses. Histone deacetylase inhibitors exhibit favorable...
Outcomes after programmed death-1 (PD-1) blockade in B-cell lymphomas are disappointing with few durable responses. Histone deacetylase inhibitors exhibit favorable immunomodulatory effects and demonstrate synergistic anti-tumor immune responses with anti-PD-1 therapy in preclinical models. We, therefore, developed a phase I study to evaluate the safety and preliminary efficacy of pembrolizumab with vorinostat in relapsed/refractory B-cell lymphomas. Patients were treated in a dose-escalation cohort using a Rolling 6 design followed by an expansion cohort at the recommended phase II dose (R2PD). Fifty-two patients were enrolled (32 Hodgkin and 20 non-Hodgkin lymphoma [NHL]). Here, we report safety data from the dose escalation cohort, and the toxicity and efficacy within NHL patients. Vorinostat was administered twice daily on days 1-5 and 8-12 (dose-level [DL]1: 100 mg; DL2: 200 mg) and pembrolizumab (200 mg) was administered on day 1 of each 3-week cycle. Of six patients treated at DL1, one had a dose-limiting toxicity (DLT) (Stevens-Johnson syndrome [SJS]), and one of six had a DLT at DL2 (thromboembolism); therefore, DL2 was the RP2D. The patient developing SJS was treated with corticosteroids, infliximab, and cyclosporine but ultimately died of invasive fungal infection from the extensive immunosuppression used to treat the SJS. The most common adverse events were hypertension, diarrhea, and cytopenias. Of 20 NHL patients, nine had follicular lymphoma (FL) and 11 had diffuse large B-cell lymphoma (DLBCL). Five DLBCL patients had primary mediastinal B-cell lymphoma (PMBL). The complete and overall response rates (CR and ORR) were 11% and 22% for FL and 45% and 55% for all DLBCL. Amongst DLBCL, the CR and ORR was 80% and 80% for PMBL and 17% and 33% for non-PMBL. In conclusion, pembrolizumab with vorinostat was tolerable and produced responses in relapsed/refractory B-cell NHL, with particularly notable efficacy in PMBL (clinicaltrials gov. Identifier: NCT03150329).
Topics: Humans; Vorinostat; Neoplasm Recurrence, Local; Lymphoma, Non-Hodgkin; Lymphoma, Large B-Cell, Diffuse; Lymphoma, Follicular; Antibodies, Monoclonal, Humanized
PubMed: 37470137
DOI: 10.3324/haematol.2023.283002 -
Journal of Advanced Research Jun 2024Posttranslational modification of proteins by reversible acetylation regulates key biological processes. Histone deacetylases (HDACs) catalyze protein deacetylation and...
INTRODUCTION
Posttranslational modification of proteins by reversible acetylation regulates key biological processes. Histone deacetylases (HDACs) catalyze protein deacetylation and are frequently dysregulated in tumors. This has spurred the development of HDAC inhibitors (HDACi). Such epigenetic drugs modulate protein acetylation, eliminate tumor cells, and are approved for the treatment of blood cancers.
OBJECTIVES
We aimed to identify novel, nanomolar HDACi with increased potency over existing agents and selectivity for the cancer-relevant class I HDACs (HDAC1,-2,-3,-8). Moreover, we wanted to define how such drugs control the apoptosis-autophagy interplay. As test systems, we used human leukemic cells and embryonic kidney-derived cells.
METHODS
We synthesized novel pyrimidine-hydroxamic acid HDACi (KH9/KH16/KH29) and performed in vitro activity assays and molecular modeling of their direct binding to HDACs. We analyzed how these HDACi affect leukemic cell fate, acetylation, and protein expression with flow cytometry and immunoblot. The publicly available DepMap database of CRISPR-Cas9 screenings was used to determine sensitivity factors across human leukemic cells.
RESULTS
Novel HDACi show nanomolar activity against class I HDACs. These agents are superior to the clinically used hydroxamic acid HDACi SAHA (vorinostat). Within the KH-series of compounds, KH16 (yanostat) is the most effective inhibitor of HDAC3 (IC = 6 nM) and the most potent inducer of apoptosis (IC = 110 nM; p < 0.0001) in leukemic cells. KH16 though spares embryonic kidney-derived cells. Global data analyses of knockout screenings verify that HDAC3 is a dependency factor in 115 human blood cancer cells of different lineages, independent of mutations in the tumor suppressor p53. KH16 alters pro- and anti-apoptotic protein expression, stalls cell cycle progression, and induces caspase-dependent processing of the autophagy proteins ULK1 and p62.
CONCLUSION
These data reveal that HDACs are required to stabilize autophagy proteins through suppression of apoptosis in leukemic cells. HDAC3 appears as a valid anti-cancer target for pharmacological intervention.
Topics: Humans; Apoptosis; Autophagy; Hydroxamic Acids; Histone Deacetylase Inhibitors; Leukemia; Cell Line, Tumor; Histone Deacetylases; Acetylation; Antineoplastic Agents; HEK293 Cells
PubMed: 37467961
DOI: 10.1016/j.jare.2023.07.005 -
Journal of Inflammation Research 2023Centromere protein I (CENPI) has been shown to affect the tumorigenesis of breast and colorectal cancers. However, its biological role and prognostic value in other...
BACKGROUND
Centromere protein I (CENPI) has been shown to affect the tumorigenesis of breast and colorectal cancers. However, its biological role and prognostic value in other kinds of cancer, especially adrenocortical carcinoma (ACC), remained to be further investigated.
METHODS
Various bioinformatics tools were adopted for exploring the significance of differential expression of CENPI in several malignant tumors from databases such as Depmap portal, GTEx, and TCGA. ACC was selected for further analyzed, and information such as clinicopathological features, the prognostic outcome of diverse subgroups, differentially expressed genes (DEGs), co-expression genes, as well as levels of tumor-infiltrating immune cells (TIIC), was extracted from multiple databases. To verify the possibility of CENPI as a therapeutic target in ACC, drug sensitivity assay and si-RNA mediate knockdown of CENPI were carried out.
RESULTS
The pan-cancer analyses showed that the CENPI mRNA expression levels differed significantly among most cancer types. Additionally, a high precision in cancer prediction and close relation with cancer survival indicated that CENPI could be a potential candidate biomarker to diagnose and predict cancer prognosis. In ACC, CENPI was closely related to multiple clinical characteristics, such as pathological stage and primary therapy outcome. High CENPI levels predicted poor overall survival (OS), progression-free interval (PFI), and disease-specific survival (DSS) of ACC patients, particularly for different clinical subgroups. Moreover, the expression of CENPI showed positive relationship to Th2 cells but negatively related to most of the TIICs. Furthermore, drug sensitivity assay showed that vorinostat inhibit CENPI expression and ACC cell growth. Additionally, si-RNA mediated knockdown of CENPI inhibited ACC cell growth and invasion and showed synergistic anti-proliferation effect with AURKB inhibitor barasertib.
CONCLUSION
Pan-cancer analysis demonstrated that CENPI is a potential diagnostic and prognostic biomarker in various cancers as well as an anti-ACC therapeutic target.
PubMed: 37465344
DOI: 10.2147/JIR.S408358 -
European Journal of Cell Biology Sep 2023ING1 is a chromatin targeting subunit of the Sin3a histone deacetylase (HDAC) complex that alters chromatin structure to subsequently regulate gene expression. We find...
ING1 is a chromatin targeting subunit of the Sin3a histone deacetylase (HDAC) complex that alters chromatin structure to subsequently regulate gene expression. We find that ING1 knockdown increases expression of Twist1, Zeb 1&2, Snai1, Bmi1 and TSHZ1 drivers of EMT, promoting EMT and cell motility. ING1 expression had the opposite effect, promoting epithelial cell morphology and inhibiting basal and TGF-β-induced motility in 3D organoid cultures. ING1 binds the Twist1 promoter and Twist1 was largely responsible for the ability of ING1 to reduce cell migration. Consistent with ING1 inhibiting Twist1 expression in vivo, an inverse relationship between ING1 and Twist1 levels was seen in breast cancer samples from The Cancer Genome Atlas (TCGA). The HDAC inhibitor vorinostat is approved for treatment of multiple myeloma and cutaneous T cell lymphoma and is in clinical trials for solid tumours as adjuvant therapy. One molecular target of vorinostat is INhibitor of Growth 2 (ING2), that together with ING1 serve as targeting subunits of the Sin3a HDAC complex. Treatment with sublethal (LD25-LD50) levels of vorinostat promoted breast cancer cell migration several-fold, which increased further upon ING1 knockout. These observations indicate that correct targeting of the Sin3a HDAC complex, and HDAC activity in general decreases luminal and basal breast cancer cell motility, suggesting that use of HDAC inhibitors as adjuvant therapies in breast cancers that are prone to metastasize may not be optimal and requires further investigation.
Topics: Female; Humans; Breast Neoplasms; Cell Line, Tumor; Chromatin; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Vorinostat
PubMed: 37459799
DOI: 10.1016/j.ejcb.2023.151341 -
PeerJ 2023This research aimed to ascertain the neuroprotective effect of histone deacetylase (HDAC) inhibition on retinal photoreceptors in mice, a model of retinitis pigmentosa...
PURPOSE
This research aimed to ascertain the neuroprotective effect of histone deacetylase (HDAC) inhibition on retinal photoreceptors in mice, a model of retinitis pigmentosa (RP)
METHODS
Single-cell RNA-sequencing (scRNA-seq) explored HDAC and poly (ADP-ribose) polymerase (PARP)-related gene expression in both -mutant and wild-type (WT) mice. The CUT&Tag method was employed to examine the functions of HDAC in mice. Organotypic retinal explant cultures from WT and mice were exposed to the HDAC inhibitor SAHA (suberoylanilide hydroxamic acid) postnatally, from day 5 to day 11. The terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay was applied to quantify the percentage of photoreceptor loss in the outer nuclear layer (ONL). HDAC activity was confirmed to be inhibited by SAHA through an HDAC activity assay. Moreover, the study evaluated PARP activity, a key driver of the initial response to DNA damage during photoreceptor degeneration, following HDAC inhibition.
RESULTS
The scRNA-seq revealed that diverse roles of HDAC and PARP isoforms in photoreceptor cell death. HDAC-related genes appeared to regulate cell death and primary immunodeficiency. Alterations in HDAC activity were consistent with the TUNEL-positive cells in the ONL at different time points. Notably, SAHA significantly postponed photoreceptor loss and decreased HDAC and PARP activity, thereby implicating both in the same degenerative pathway.
CONCLUSIONS
This study highlights that the interaction between HDAC inhibition and PARP can delay photoreceptor cell death, proposing a promising therapeutic approach for RP.
Topics: Mice; Animals; Histone Deacetylases; Poly(ADP-ribose) Polymerase Inhibitors; Single-Cell Gene Expression Analysis; Retinitis Pigmentosa; Photoreceptor Cells; Poly(ADP-ribose) Polymerases; Vorinostat
PubMed: 37456870
DOI: 10.7717/peerj.15659