trichostatin-a and Bone-Neoplasms

Epigenetic modulation participates in the mechanism of multiple types of pathological pain, so targeting the involved regulators may be a promising strategy for pain treatment. Our previous research identified the analgesic effect of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) on mechanical hyperalgesia in a rat model of bone cancer pain (BCP) via restoration of μ-opioid receptor (MOR) expression. However, the specific types of HDACs contributing to BCP have not been explored. The present study investigated the expression pattern of some common HDACs and found that HDAC2 was up-regulated in a time-dependent manner in the lumbar spinal cord of BCP rats. TSA application suppressed HDAC2 expression in cultured PC12 cells and reversed the augmented HDAC2 in BCP rats. An RNA-interfering strategy confirmed the essential role of HDAC2 in the modulation of mechanical hyperalgesia following tumor cell inoculation, and we further examined its possible downstream targets. Notably, HDAC2 knock-down did not restore MOR expression, but it robustly reversed the down-regulation of potassium-chloride cotransporter 2 (KCC2). The impaired KCC2 expression is a vital mechanism of many types of pathological pain. Therefore, our results demonstrated that HDAC2 in spinal cord contributed to the mechanical hyperalgesia in BCP rats, and this effect may be associated with KCC2 modulation.

Topics: Analgesics, Non-Narcotic; Animals; Bone Neoplasms; Cancer Pain; Cell Line, Tumor; Female; Gene Expression Regulation; Gene Knockdown Techniques; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Hydroxamic Acids; Hyperalgesia; K Cl- Cotransporters; Musculoskeletal Pain; Neoplasm Transplantation; Rats; Rats, Sprague-Dawley; Rats, Wistar; RNA Interference; Spinal Cord; Symporters; Time Factors

Bone cancer pain (BCP) is a common complication with inadequate management in patients suffering from advanced cancer. Histone deacetylase inhibitors showed significant analgesic effect in multiple inflammatory and neuropathic pain models, but their effect in bone cancer pain has never been explored. In this study, we utilized a BCP rat model with intra-tibial inoculation of Walker 256 mammary gland carcinoma cells, which developed progressive mechanical hypersensitivity but not thermal hypersensitivity. Intrathecal application of trichostatin A (TSA), a classic pan-HDAC inhibitor, ameliorated tactile hypersensitivity and enhanced the analgesic effect of morphine in BCP rats. The analgesic effect of TSA was blocked by co-administration of CTAP, a specific MOR antagonist, confirming the involvement of mu-opioid receptor (MOR). A reduction of MOR expression was observed in the lumbar spinal cord of BCP rats and TSA treatment was able to partially reverse it. In vitro study in PC12 cells also demonstrated the dose-dependent enhancement of MOR expression by TSA treatment. Taking all into consideration, we could draw the conclusion that HDAC inhibitor TSA ameliorates mechanical hypersensitivity and potentiates analgesic effect of morphine in BCP rats, probably by restoring MOR expression in spinal cord.

Topics: Analgesics, Opioid; Animals; Bone Neoplasms; Cancer Pain; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Synergism; Drug Therapy, Combination; Female; Histone Deacetylase Inhibitors; Hot Temperature; Hydroxamic Acids; Hyperalgesia; Lumbar Vertebrae; Morphine; PC12 Cells; Rats; Receptors, Opioid, mu; RNA, Messenger; Spinal Cord; Touch

Oral hypoglycemic agent metformin is commonly used for treating type II diabetes; however, initial reports demonstrated that it could be used for suppressing tumor growth in vitro and in vivo. Moreover, novel potential anticancer drug histone deacetylase (HDAC) and inhibitor trichostatin A (TSA) have been extensively studied for inducing various malignancies growth inhibition, cell cycle arrest, and apoptosis. The object of the present study was to investigate the anti-proliferation and apoptosis induction effects of metformin and TSA in osteosarcoma cell line, and to explore the mechanism of metformin and TSA in combination to inhibit the proliferation of osteosarcoma cells. After treating with metformin and TSA, the viability of osteosarcoma cell lines (MG-63 and LM8) was analyzed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) at various concentrations, cell cycle analysis of MG-63 and LM8 cell was performed by flow cytometry. Real-time polymerase chain reaction and Western Blotting were performed to determine the expression of apoptosis-related genes and proteins such as Caspase-3, Bcl-2/Bax, Cyclin D1, and p21. Protein expression of the molecules involved in 5'-adenosine monophosphate-activated protein kinase (AMPK) signaling pathway after treatment with combination was determined by Western blotting. Moreover, orthotopic xenograft tumors were challenged in nude mice to establish the murine model; tumor weight and tumor volume were monitored after drug administration separately or combined via the intraperitoneal (i.p.) route. MTT assays showed that the viability of osteosarcoma cell lines in the combination group (10 mM metformin, 0.3 μM TSA) decreased in a concentration- and time-dependent manner; moreover, the cell cycle of MG-63 and LM8 in the combination group could be arrested in G1/G2 phase higher number compared with drug use separately. Furthermore, a combination of these drugs does not act via the AMPK signaling pathway to induce MG-63 osteosarcoma cell line growth inhibition and apoptosis. As data have showed here, metformin cotreatment increased TSA antitumor effects and have a synergistic effect on osteosarcoma cell line proliferation and apoptosis.

Topics: AMP-Activated Protein Kinase Kinases; Animals; Apoptosis; Blotting, Western; Bone Neoplasms; Cell Cycle; Cell Proliferation; Drug Synergism; Female; Flow Cytometry; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Hypoglycemic Agents; Metformin; Mice; Mice, Inbred BALB C; Osteosarcoma; Protein Kinases; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

Here, we report a complex regulation of endothelin-1 (ET-1) axis driven by epigenetic reactions in 1833-bone metastatic cells, emphasizing the importance in skeletal metastasis from breast carcinoma. Inhibitors of histone deacetylases, trichostatin A (TSA), and of DNA methylases, 5'-Azacytidine (Aza), caused, respectively, reduction and increase in 1833 cell invasiveness, without affecting the basal migration of parental MDA-MB231 cells. Of note, in the two cell lines exposed to Aza the blockade of the ET-1 receptor ETAR with BQ-123 oppositely changed invasive properties. Even if in MDA-MB231 cells the ET-1 axis was scarcely influenced by epigenetic reactions, ETAR remarkably decreased after Aza. In contrast, in 1833 cells Aza exposure enhanced ET-1 coupled to ETAR wild type, being also ETAR truncated form increased, and invasiveness was stimulated. Under demethylation, the increase in ET-1 steady state protein level in 1833 clone seemed regulated at transcriptional level principally via Ets1 transcription factor. In fact, actinomycin D almost completely prevented ET-1 mRNA induction due to Aza. Only in 1833 cells, TSA exposure inactivated ET-1 axis, with reduction of the expression of ET-1 and ETAR mutated form, in agreement with Matrigel invasion decrease. This treatment favoured the ET-1 repressional control, taking place at the level of mRNA stability due to the 3'-untranslated region in the ET-1 gene, and also decreased transcription via NF-kB. Environmental conditions that alter the balance between epigenetic reactions might, therefore, affect metastasis migratory mode influencing ET-1 axis.

Topics: Azacitidine; Bone Neoplasms; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Movement; Collagen; Dactinomycin; DNA Methylation; Drug Combinations; Endothelin A Receptor Antagonists; Endothelin-1; Enzyme Inhibitors; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Laminin; Mutation; Neoplasm Invasiveness; Peptides, Cyclic; Proteoglycans; Proto-Oncogene Protein c-ets-1; Receptor, Endothelin A; RNA Stability; RNA, Messenger; Transcription, Genetic

The chromosomal translocation t(11;22)(q24;q12) yields the EWS-Fli1 fusion gene, which contributes to the development of Ewing Family Tumors (EFTs). Previous studies have shown the ability of EWS-Fli1 chimeric protein to silence p53 activity. Here we demonstrate that the introduction of EWS-Fli1 significantly inhibited p300-mediated acetylation of p53 at Lys-382 and depletion of EWS-Fli1 protein by small interfering RNAs (siRNA) in EFTs cells facilitated it in response to DNA damage. Furthermore, the deacetylation of p53 by EWS-Fli1 suppressed its transcriptional activity and enhanced mdm2-mediated p53 degradation. On the other hand, immunoprecipitation study shows that N-terminal region of EWS-Fli1 associated with histone deacetylase 1 (HDAC1) to forms a complex with p53. Knockdown of HDAC1, but not HDAC2 or HDAC3 protein restored the expression of p53 Lys-382 in EFTs cells. Overexpression of HDAC1 also significantly inhibited p53 transcriptional activity. Pharmacologic inhibitor of HDAC, trichostatin A (TSA) promoted p53-p300 interaction and recruitment of p53 Lys-382 to promoter regions of its target genes p21 and Puma, consequently inducing apoptosis and stabilizing the acetylation of p53 at Lys-382 together with the upregulation of p21 and Puma, which were impaired in EFTs cells after the knockdown of p53 expression. Our data indicate EWS-Fli1 might deacetylate p53 to inhibit its transcriptional function and protein stability via the recruitment of HDAC1. These results might elucidate a novel molecular mechanism about the abrogation of p53 pathway by EWS-Fli1 in EFTs pathogenesis.

Topics: Acetylation; Animals; Apoptosis; Bone Neoplasms; Cell Line, Tumor; E1A-Associated p300 Protein; Histone Deacetylase 1; Humans; Hydroxamic Acids; Mice; NIH 3T3 Cells; Oncogene Proteins, Fusion; Proto-Oncogene Protein c-fli-1; Proto-Oncogene Proteins c-mdm2; RNA-Binding Protein EWS; Sarcoma, Ewing; Transcriptional Activation; Tumor Suppressor Protein p53

Histone deacetylases are enzymes involved in the remodeling of chromatin structure, in the regulation of transcriptional activity, and in epigenetic integrity. Histone deacetylase inhibitors such as trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) have emerged as potent anticancer drugs that have proved useful in preclinical and early clinical trials. The role of histone deacetylase inhibitors in regulating osteoclast differentiation, however, is not well established. In this study, we analyzed the effects of TSA on osteoclast differentiation induced by the differentiation factor RANKL (receptor activator of NF-kappaB ligand). TSA strongly inhibited osteoclast formation in coculture of bone marrow cells and osteoblasts without reducing RANKL expression in osteoblasts. Furthermore, TSA suppressed RANKL-induced osteoclast formation from primary bone marrow-derived macrophages. TSA was only effective when present during the early stage of osteoclast differentiation. This effect was accompanied by a significant decrease in the RANKL-stimulated induction of c-Fos and NFATc1, which are key transcription factors during early osteoclastogenesis. The ectopic introduction of c-Fos and a constitutively active form of NFATc1 reversed the TSA-induced antiosteoclastogenic effect. Consistent with the in vitro results, TSA inhibited lipopolysaccharide- and interleukin-1-induced bone resorption and osteoclast formation in an in vivo model. Taken together, our findings suggest a novel action of TSA: inhibiting RANKL-induced osteoclast formation by suppressing the induction of the osteoclastogenic transcription factor c-Fos. Also, the inhibitory effect of TSA on bone destruction in vivo suggests that histone deacetylase inhibitors may be novel therapeutics for treating typical bone diseases.

Topics: Animals; Bone Marrow Cells; Bone Neoplasms; Bone Resorption; Cell Differentiation; Cells, Cultured; Coculture Techniques; Histone Deacetylase Inhibitors; Hydroxamic Acids; Interleukin-1; Lipopolysaccharides; Macrophages; Mice; Mice, Inbred ICR; NFATC Transcription Factors; Osteitis; Osteoblasts; Osteoclasts; Osteoprotegerin; Proto-Oncogene Proteins c-fos; RANK Ligand; Skull

ARHI is a maternally imprinted tumor suppressor gene whose expression is markedly downregulated in breast cancer. Reactivation of ARHI expression in breast cancer cells is associated with increased histone H3 acetylation and decreased lysine 9 methylation of histone H3. An ARHI promoter segment that spanned bases -420 to +58 (designated the P2 region) exhibits significantly higher promoter activity in normal cells than in cancer cells. To better understand the molecular mechanisms contributing to this differential transcriptional activity, we sought to identify transcription factors that bind to the P2 region of the ARHI promoter and regulate its activity. Sequence analysis and oligonucleotide competition in electrophoretic mobility shift assays identified an A2 fragment containing an E2F-binding site. Using specific antibodies in supershift assays, we have shown that anti-E2F1 and 4 antibodies can supershift the A2-protein complexes, whereas anti-E2F2 and 6 antibodies cannot, demonstrating that the A2 fragment interacts with specific members of the E2F family proteins. When compared with normal breast epithelial cells, breast cancer cells have significantly elevated expression of E2F1, 4 and increased E2F DNA-binding activity. Moreover, chromatin immunoprecipitation experiments revealed that both E2F1 and 4 bind to the ARHI promoter in breast cancer cells in vivo. This binding was reduced when the cells were treated with the histone deacetylase (HDAC) inhibitor--trichostatin A (TSA). When SKBr3 cells were cotransfected with an ARHI/luciferase reporter and E2F-expression vectors, E2F1 and 4 reduced ARHI promoter activity 2-3-fold, and this reduction could be reversed by TSA treatment. The negative regulation by E2F-HDAC complexes could also be reduced by small interfering RNA of E2F1 and 4. While the retinoblastoma protein, pRB, alone had no effect on ARHI promoter activity, repression by E2F1, but not E2F4, was enhanced by the coexpression of pRB. Taken together, our results suggest that E2F1, 4 and their complexes with HDAC play an important role in downregulating the expression of the tumor suppressor gene ARHI in breast cancer cells.

Topics: Acetylation; Binding Sites; Bone Neoplasms; Breast Neoplasms; Cell Nucleus; Cells, Cultured; Chromatin Immunoprecipitation; E2F1 Transcription Factor; E2F2 Transcription Factor; E2F4 Transcription Factor; E2F6 Transcription Factor; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Epithelial Cells; Female; Gene Expression Regulation; Genes, Tumor Suppressor; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Luciferases; Mammary Glands, Human; Osteosarcoma; Promoter Regions, Genetic; Response Elements; Retinoblastoma Protein; rho GTP-Binding Proteins; RNA, Small Interfering

The purpose of this study is to determine whether trichostatin A (TSA), a HDAC specific inhibitor, inhibited the induction and functional activity of hypoxia-inducible factor-1 a(HIF-1a) and hypoxia-induced angiogenesis in vitro in human osteosarcoma. The relationship between expression of HIF-1a proteion and angiogenesis in tumor specimens was also studied. Hypoxic regulation of VEGF was studied by RT-PCR, western blotting analysis and enzyme linked immunosorbent assay. The expression of HIF-la and VEGF in human osteosarcoma specimens was studied by immunohistochemical analysis. Under hypoxia, no regulation of HIF-1a mRNA expression was found. However, HIF-1a protein levels increased dramatically in response to hypoxia. Hypoxia increased VEGF mRNA level, but it was significantly inhibited by trichostatin A in a time- and dose-dependent manner (p < 0.05). Strongly positive immunostaining for HIF-1a and VEGF were detectable in the nuclear and cytoplasm of osteosarcoma cells. HIF-1a expressing cells were prominent in areas with high MVD. Significant correlation were found between HIF-1a expression and MVD (p = 0.005, r = 0.767), as well as between VEGF and MVD (p < 0.002, r = 0.701) by Spearman's rank coefficient analysis. These results indicated that HIF-1a is a key factor responsible for angiogenesis by the induction of VEGF. TSA downregulates hypoxia-response genes and hypoxia-induced angiogenesis by the suppression of HIF-1a activity.

Topics: Bone Neoplasms; Cell Hypoxia; Cell Line, Tumor; Enzyme Inhibitors; Humans; Hydroxamic Acids; Hypoxia-Inducible Factor 1, alpha Subunit; Neovascularization, Pathologic; Osteosarcoma; Reverse Transcriptase Polymerase Chain Reaction; Vascular Endothelial Growth Factor A

Although histone deacetylase (HDAC) inhibitors are emerging as a promising new treatment strategy in malignancy, how they exert their effect on osteosarcoama cells is as yet unclear. This study was undertaken to investigate the underlying mechanism of a HDAC inhibitor Trichostatin A (TSA)-induced apoptosis in a osteosarcoma cell line HOS. We observed that TSA treatment decreased the viability of the cells and prominently increased acetylation of histone H3. Evidence was obtained indicating that TSA induced apoptosis of HOS cells as follows: (1) Generation of DNA fragmentation; (2) activation of procaspase-3; (3) cleavage of PARP; and (4) increase of DNA hypoploidy. The reduction of MMP and the release of cytochrome c to cytosol were also shown, indicating that TSA induces apoptosis in HOS cells in a histone acetylation- and mitochondria-dependent fashions. We also examined whether TSA can sensitize HOS cells to the action of an antitumor agent genistein. The combination therapy of TSA and genistein showed synergistic anticancer effect indicating that TSA can be considered as a novel therapeutic strategy for osteosarcoma not only from its direct apoptosis-inducing activity but also from the possibility of sensitization to other antitumor agents.

Topics: Apoptosis; Bone Neoplasms; Caspase Inhibitors; Cell Line, Tumor; Cell Survival; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Membrane Potentials; Mitochondria; Osteosarcoma