tretinoin and entinostat

Histone deacetylase (HDAC) inhibitors affect cell homeostasis, gene expression, and cell cycle progression and promote cell terminal differentiation or apoptosis. However, the effect of HDAC inhibition on SH-SY5Y cells, which are neuroblastoma cells capable of differentiating into neurons under specific conditions, such as in the presence of retinoic acid (RA), is unknown. In this study, we hypothesized that HDAC inhibitors induced the neuronal differentiation of SH-SY5Y cells. To test this hypothesis, we used phase contrast microscopy, immunocytochemistry (ICC), qPCR, and western blotting analysis. MS-275 and valproic acid (VPA), two HDAC inhibitors, were selected to evaluate neuronal differentiation. It was confirmed that cells treated with MS-275 or VPA differentiated into mature neurons, which were distinguished by bipolar or multipolar morphologies with elongated branches. In addition, the mRNA expression of neuronal markers (Tuj1 and NEFH) and the oligodendrocyte marker (CNP) was significantly increased with MS-275 or VPA treatment compared to that with RA treatment. In addition, the protein expression of the other neuronal markers, Tuj1 and NeuN, was highly increased with HDAC inhibitor treatments compared to that with RA treatment. Furthermore, we confirmed that noncanonical Wnt signaling was upregulated by HDAC inhibitors via MAPK signaling and the Wnt/JNK pathway. Therefore, both MS-275 and VPA promoted the differentiation of SH-SY5Y cells into mature neurons via the Wnt signaling pathway.

Topics: Cell Differentiation; Cell Line, Tumor; Histone Deacetylase Inhibitors; Humans; Neuroblastoma; Neurons; Tretinoin; Valproic Acid; Wnt Signaling Pathway

A combination of entinostat, all-trans retinoic acid, and doxorubicin (EAD) induces cell death and differentiation and causes significant regression of xenografts of triple-negative breast cancer (TNBC).. We investigated the mechanisms underlying the antitumor effects of each component of the EAD combination therapy by high-throughput gene expression profiling of drug-treated cells.. Microarray analysis showed that entinostat and doxorubicin (ED) altered expression of genes related to growth arrest, inflammation, and differentiation. ED downregulated MYC, E2F, and G2M cell cycle genes. Accordingly, entinostat sensitized the cells to doxorubicin-induced growth arrest at G2. ED induced interferon genes, which correlated with breast tumors containing a higher proportion of tumor-infiltrating lymphocytes. ED also increased the expression of immune checkpoint agonists and cancer testis antigens. Analysis of TNBC xenografts showed that EAD enhanced the inflammation score in nude mice. Among the genes differentially regulated between the EAD and ED groups, an all-trans retinoic acid (ATRA)-regulated gene, DHRS3, was induced in EAD-treated xenografts. DHRS3 was expressed at lower levels in human TNBC metastases compared to normal breast or primary tumors. High expression of ED-induced growth arrest and inflammatory genes was associated with better prognosis in TNBC patients.. Entinostat potentiated doxorubicin-mediated cell death and the combination induced inflammatory signatures. The ED-induced immunomodulation may improve immunotherapy. Addition of ATRA to ED may potentiate inflammation and contribute to TNBC regression.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzamides; Breast; Cell Cycle Checkpoints; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Datasets as Topic; Doxorubicin; Drug Synergism; Epigenesis, Genetic; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Oligonucleotide Array Sequence Analysis; Pyridines; Survival Analysis; Tretinoin; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays

The differentiation of hematopoietic stem cells into mature blood cells is a highly ordered process and dysregulation of this process can lead to leukemogenesis. Agents that are used to cure acute promyelocytic leukemia (APL) can induce differentiation and/or apoptosis. Here, we describe how effects of all-trans retinoic acid (ATRA) and histone deacetylase inhibitors (HDACi) on APL cell differentiation can be evaluated by immunoblotting and by flow cytometry. We show how the levels of differentiation-associated transcription factors of the CCAAT enhancer binding protein (C/EBP) family can be determined by Western blot and we explain how the cell surface expression of the leukocyte surface antigen CD11b can be measured by flow cytometry.

Topics: Apoptosis; Benzamides; CCAAT-Enhancer-Binding Protein-beta; CCAAT-Enhancer-Binding Proteins; CD11b Antigen; Cell Differentiation; Flow Cytometry; Gene Expression Regulation, Leukemic; Hematopoietic Stem Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunoblotting; Indoles; Panobinostat; Pyridines; Tretinoin

The treatment of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA) induces granulocytic differentiation. This process renders APL cells resistant to cytotoxic chemotherapies. Epigenetic regulators of the histone deacetylases (HDACs) family, which comprise four classes (I-IV), critically control the development and progression of APL. We set out to clarify the parameters that determine the interaction between ATRA and histone deacetylase inhibitors (HDACi). Our assays included drugs against class I HDACs (MS-275, VPA, and FK228), pan-HDACi (LBH589, SAHA), and the novel HDAC6-selective compound Marbostat-100. We demonstrate that ATRA protects APL cells from cytotoxic effects of SAHA, MS-275, and Marbostat-100. However, LBH589 and FK228, which have a superior substrate-inhibitor dissociation constant (Ki) for the class I deacetylases HDAC1, 2, 3, are resistant against ATRA-dependent cytoprotective effects. We further show that HDACi evoke DNA damage, measured as induction of phosphorylated histone H2AX and by the comet assay. The ability of ATRA to protect APL cells from the induction of p-H2AX by HDACi is a readout for the cytoprotective effects of ATRA. Moreover, ATRA increases the fraction of cells in the G1 phase, together with an accumulation of the cyclin-dependent kinase inhibitor p21 and a reduced expression of thymidylate synthase (TdS). In contrast, the ATRA-dependent activation of the transcription factors STAT1, NF-κB, and C/EBP hardly influences the responses of APL cells to HDACi. We conclude that the affinity of HDACi for class I HDACs determines whether such drugs can kill naïve and maturated APL cells.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzamides; CCAAT-Enhancer-Binding Proteins; Cell Cycle; Cell Death; Cell Line, Tumor; DNA Damage; Drug Resistance, Neoplasm; Histone Deacetylase Inhibitors; Humans; Leukemia; NF-kappa B; Pyridines; STAT1 Transcription Factor; Tretinoin

Resistance to aromatase inhibitors (AIs) involves increased HER2. One mechanism by which HER2 may mediate resistance is through expansion of the tumor initiating cell (TIC) population. This study investigates whether combining all-trans retinoic acid (ATRA) and histone deacetylase inhibitor entinostat (ENT) can inhibit TICs and HER2 in AI-resistant cells and tumors. Modulation of cell viability and HER2 expression were assessed in AI-resistant cells treated with ATRA + ENT. Letrozole-resistant LTLT-Ca cells treated with ATRA + ENT were assayed for changes in TIC characteristics, such as TIC markers (BCRP, ALDH, and BMI-1), side population (SP), and mammosphere formation. Xenograft tumors of MCF-7Ca cells made resistant to letrozole were treated with ATRA, ATRA + letrozole, ATRA + ENT, or ATRA + ENT + letrozole. Resulting tumors were assayed for changes in TIC characteristics. Patient samples taken pre- and post-AI treatment were analyzed for changes in ERα and HER2 protein expression. Treatment with ATRA + ENT reduced HER2 expression and viability (P < 0.001) in AI-resistant cells, as well as decreased SP (P < 0.0001), mammosphere formation (P < 0.01), and expression of TIC molecular markers (P < 0.01) in LTLT-Ca. A reduction in tumor growth rate was observed in mice treated with ENT + ATRA + letrozole when compared to mice treated with single agents (P < 0.0001) or ENT + ATRA (P = 0.02). Decreased TIC characteristics, including mammosphere formation (P < 0.05), were observed in tumors from the triple combination. An increase in HER2 and downregulation in ERα protein expression was observed in patients upon resistance to AI (P < 0.005). These studies indicate that the combination of ATRA and ENT inhibits the TIC population of AI-resistant cells and may be effective in reducing tumor recurrence.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Benzamides; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Histone Deacetylase Inhibitors; Humans; Letrozole; Mice, Nude; Nitriles; Pyridines; Receptor, ErbB-2; Tretinoin; Triazoles; Xenograft Model Antitumor Assays

All-trans retinoic acid (ATRA) has only limited single agent activity in AML without the PML-RARα fusion (non-M3 AML). In search of a sensitizing strategy to overcome this relative ATRA resistance, we investigated the potency of the HDAC class-I selective inhibitor entinostat in AML cell lines Kasumi-1 and HL-60 and primary AML blasts. Entinostat alone induced robust differentiation of both cell lines, which was enhanced by the combination with ATRA. This "priming" effect on ATRA-induced differentiation was at least equivalent to that achieved with the DNA hypomethylating agent decitabine, and could overall be recapitulated in primary AML blasts treated ex vivo. Moreover, entinostat treatment established the activating chromatin marks acH3, acH3K9, acH4 and H3K4me3 at the promoter of the RARβ2 gene, an essential mediator of retinoic acid (RA) signaling in different solid tumor models. Similarly, RARβ2 promoter hypermethylation (which in primary blasts from 90 AML/MDS patients was surprisingly infrequent) could be partially reversed by decitabine in the two cell lines. Re-induction of the epigenetically silenced RARβ2 gene was achieved only when entinostat or decitabine were given prior to ATRA treatment. Thus in this model, reactivation of RARβ2 was not necessarily required for the differentiation effect, and pharmacological RARβ2 promoter demethylation may be a bystander phenomenon rather than an essential prerequisite for the cellular effects of decitabine when combined with ATRA. In conclusion, as a "priming" agent for non-M3 AML blasts to the differentiation-inducing effects of ATRA, entinostat is at least as active as decitabine, and both act in part independently from RARβ2. Further investigation of this treatment combination in non-M3 AML patients is therefore warranted, independently of RARβ2 gene silencing by DNA methylation.

Topics: Adult; Aged; Antineoplastic Agents; Benzamides; Cell Differentiation; Cell Line; DNA Methylation; Drug Interactions; Epigenesis, Genetic; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Promoter Regions, Genetic; Pyridines; Tretinoin

Homeobox genes murine Rhox5 and human RHOXF1 are expressed in early embryonic stages and then mostly restricted to germline tissues in normal adult, yet they are aberrantly expressed in cancer cells in vitro and in vivo . Here we study the epigenetic regulation and potential functions of Rhox5 gene.. In Rhox5-silenced or extremely low expresser cells, we observed low levels of active histone epigenetic marks (H3ac, H4ac and H3K4me2) and high levels of repressive mark H3K9me2 along with DNA hypermethylation in the promoter. In Rhox5 low expresser cells, we typically observed modest levels of both active and repressive histone marks along with moderate DNA methylation. In Rhox5 highly expressed CT26 cancer cells, we observed DNA hypomethylation along with high levels of both active and repressive histone marks. Epigenetic drugs (retinoic acid and MS-275) induced F9 cell differentiation with enhanced Rhox5 expression and dynamic changes of epigenetic marks. Finally, Rhox5 knockdown by small hairpin RNA (shRNA) in CT26 colon cancer decreased cell proliferation and migration in vitro and tumor growth in vivo .. Both DNA methylation and histone methylation/acetylation play key roles in modulating Rhox5 expression in various cell types. The stem cell-like "bivalent domain", an epigenetic feature originally identified in key differentiation genes within stem cells, exists in the Rhox5 gene promoter in not only embryonic stem cells but also cancer cells, cancer stem cells, and differentiated Sertoli cells. As Ras signaling-dependent Rhox5 expression promotes tumor growth, Rhox5 may be an ideal target for therapeutic intervention in cancer.

Topics: Animals; Benzamides; Cell Differentiation; Cell Line, Tumor; Cell Movement; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Homeodomain Proteins; Humans; Methylation; Mice; Mice, Nude; Neoplasms; Promoter Regions, Genetic; Pyridines; RNA, Messenger; Stem Cells; Transcription Factors; Tretinoin; Tumor Burden; Xenograft Model Antitumor Assays

To evaluate the antitumoral effect of combined inhibitors of angiogenesis and histone deacetylases in an experimental rat hepatoma model.. MH7777A hepatoma cells were injected into the liver of male Buffalo rats. After 7 d treatment with the vascular endothelial growth factor receptor antagonist PTK787/ZK222584 (PTK/ZK), the histone deacetylase inhibitor MS-275, tamoxifen (TAM) and/or retinoic acid was initiated (n ≥ 8 animals/group). Natural tumor development was shown in untreated control groups (control 1 with n = 12, control 2 with n = 8). The control groups were initiated at different time points to demonstrate the stability of the hepatoma model. For documentation of possible side effects, we documented any change in body weight, loss of fur and diarrhea. After 21 d treatment, the rats were euthanized. Main target parameters were tumor size and metastasis rate. Additionally, immunohistochemistry for the proliferating cell nuclear antigen (PCNA) and TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay were performed.. The control groups developed large tumor nodules with extrahepatic tumor burden in the lung and abdominal organs (control 1: 6.18 cm(3) ± 4.14 cm(3) and control 2: 8.0 cm(3) ± 4.44 cm(3) 28 d after tumor cell injection). The tumor volume did not differ significantly in the control groups (P = 0.13). As single agents MS-275 and PTK/ZK reduced tumor volume by 58.6% ± 2.6% and 48.7% ± 3.2% vs control group 1, which was significant only for MS-275 (P = 0.025). The combination of MS-275 and PTK/ZK induced a nearly complete and highly significant tumor shrinkage by 90.3% ± 1% (P = 0.005). Addition of TAM showed no further efficacy, while quadruple therapy with retinoic acid increased antitumoral efficacy (tumor reduction by 93 ± 1%) and side effects. PCNA positive cells were not significantly reduced by the single agents, while dual therapy (MS-275 and PTK/ZK) and quadruple therapy reduced the PCNA-positive cell fraction significantly by 9.1 and 20.6% vs control 1 (P < 0.05). The number of TUNEL-positive cells, markers for ongoing apoptosis, was increased significantly by the single agents (control 1: 6.9%, PTK/ZK: 11.4%, MS-275: 12.2% with P < 0.05 vs control 1). The fraction of TUNEL-positive cells was upregulated highly significantly by dual therapy (18.4%) and quadruple therapy (24.8%, P < 0.01 vs control 1). For the proliferating (PCNA positive) and apoptotic cell fraction, quadruple therapy was significantly superior to dual therapy (P = 0.01).. Combined PTK/ZK and MS-275 were highly effective in this hepatoma model. Quadruple therapy enhanced the effects microscopically, but not macroscopically. These results should be investigated further.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Benzamides; Drug Therapy, Combination; Histone Deacetylase Inhibitors; Liver Neoplasms; Liver Neoplasms, Experimental; Male; Phthalazines; Protein Kinase Inhibitors; Pyridines; Rats; Receptors, Vascular Endothelial Growth Factor; Tamoxifen; Tretinoin

We have developed new, simple, and efficient procedures for the synthesis of two promising histone deacetylase inhibitors (HDIs), CI-994, (N-(2-aminophenyl)-4-acetylaminobenzamide), and MS-275 (N-(2-aminophenyl)4-[N-(pyridine-3-yl-methoxycarbonyl)aminomethyl]benzamide) from commercially available acetamidobenzoic acid and 3-(hydroxymethyl)pyridine, respectively. The procedures provide CI-994 and MS-275 in 80% and 72% overall yields, respectively. We found that the combination of four HDIs (CI-994, MS-275, SAHA, and TSA) with retinoids all-trans-retinoic acid (ATRA) or 13-cis-retinoic acid (13-CRA) or our atypical retinoic acid metabolism blocking agents (RAMBAs) 1 (VN/14-1) or 2 (VN/66-1) produced synergistic anti-neoplastic activity on human LNCaP prostate cancer cells. The combination of 2 and SAHA induced G1 and G2/M cell cycle arrest and a decrease in the S phase in LNCaP cells. 2+SAHA treatment effectively down-regulated cyclin D1 and cdk4, and up-regulated pro-differentiation markers cytokeratins 8/18 and pro-apoptotic Bad and Bax. Following subcutaneous administration, 2, SAHA or 2+SAHA were well tolerated and caused significant suppression/regression of tumor growth compared with control. These results demonstrate that compound 2 and its combination with SAHA are potentially useful agents that warrant further preclinical development for treatment of prostate cancer.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Benzamides; Cell Cycle; Cell Differentiation; Cell Line, Tumor; Drug Synergism; Histone Deacetylase Inhibitors; Humans; Imidazoles; Male; Neoplasms, Experimental; Phenylenediamines; Prostatic Neoplasms; Pyridines; Transplantation, Heterologous; Tretinoin

Histone deacetylase (HDAC) inhibitors have been shown to reverse epigenetic repression of certain genes, including retinoic acid receptor beta2 (RARbeta2). In this study, we examined whether RARbeta2 expression is repressed in human renal cell carcinoma (RCC) and whether the HDAC inhibitor MS-275 may revert its epigenetic repression.. Six human tumor RCC cell lines were analyzed for RARbeta2 gene expression and for methylation and acetylation status at the promoter level. Modulation of RARbeta2 expression and correlation with antitumor activity by combination of MS-275 with 13-cis-retinoic acid (CRA) was assessed in a RARbeta2-negative RCC cell line.. RARbeta2 expression was either strongly present, weakly expressed, or absent in the RCC cell lines analyzed. Methylation-specific PCR indicated that the RARbeta2 promoter was partially methylated in three of the cell lines. CRA treatment did not inhibit clonogenic growth in the RARbeta2-negative cell line RCC1.18, whereas MS-275 induced a dose-dependent inhibitory effect. A greater inhibitory effect was observed with combination treatment (MS-275 + CRA). Treatment with MS-275 was associated with histone acetylation at the promoter level and synergistic gene reexpression of RARbeta2 in combination with CRA. RARbeta2 reexpression was associated with synergistic induction of the retinoid-responsive gene HOXA5. In vivo, single-agent CRA treatment showed no significant effect, whereas MS-275 and the combination induced a regression of RCC1.18 tumor xenografts. Discontinuation of treatment produced tumor recurrence in MS-275-treated mice, whereas animals treated with the combination remained tumor free.. The HDAC inhibitor MS-275 seems to revert retinoid resistance due to epigenetic silencing of RARbeta2 in a human RCC model and has greater antitumor activity in combination with CRA compared with single agents. Thus, the combination of HDAC inhibitors and retinoids may represent a novel therapeutic approach in patients with RCC.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Benzamides; Blotting, Western; Carcinoma, Renal Cell; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Homeodomain Proteins; Humans; Isotretinoin; Kidney Neoplasms; Male; Mice; Mice, Nude; Mice, SCID; Phosphoproteins; Pyridines; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Tretinoin; Xenograft Model Antitumor Assays