Histone deacetylase inhibitors, N-butyric acid and trichostatin A, induce caspase-8- but not caspase-9-dependent apoptosis in human malignant glioma cells

Histone deacetylase (HDAC) inhibitors have both apoptotic and differentiating effects on various tumor cells. However, the mechanisms underlying the effect of HDAC inhibitors remain unclear. In this study, we investigated the function of anti-proliferative effects of HDAC inhibitors, N-butyric acid and trichostatin A, on human malignant glioma cell lines, U251-MG and D54. MTT assay showed a dose-dependent inhibition of cellular proliferation in both cell lines. Cell cycle analysis revealed increased sub-G1 population in both lines, and G1 arrest only in U251-MG cells. Induction of apoptosis was also supported by the occurrence of DNA fragmentation in tumor cells treated with HDAC inhibitors. Furthermore, caspase inhibition assay indicated that HDAC inhibitor-induced apoptosis was caspase-dependent. Neither mitochondrial membrane potential nor the expression of caspase-9 was changed by treatment with HDAC inhibitors, suggesting the possibility that HDAC inhibitor-induced apoptosis was not mediated by the mitochondrial cell death pathway. On the other hand, immunoblot assay confirmed increased expression of caspase-8 in both lines, and elevation of p21 but not p27 protein in U251-MG cells following HDAC inhibitor treatment. Taken together, the HDAC inhibitors, N-butyric acid and trichostatin A, induce caspase-8- but not caspase-9-dependent apoptosis with or without p21-mediated G1 arrest in human malignant glioma cells.     

Histone deacetylase inhibition induces apoptosis and autophagy in human neuroblastoma cells

Neuroblastoma (NB) is the most common solid extracranial tumor in children. Here we showed that trichostatin A, a histone deacetylase inhibitor (HDACi), decreases cell viability in three NB cell lines of different phenotypes. The treatment leads to G2/M-phase arrest, apoptosis and autophagy. Autophagy induction accompanies apoptosis in the most proliferative, N-Myc overexpressing cells. In contrast, autophagy precedes apoptosis and acts as a protective mechanism in the less proliferative, non-N-Myc overexpressing cells. Therefore, the autophagy induction is a relevant event in the NB response to HDACis, and it should be considered in the design of new treatments for this malignancy.     

Histone deacetylase inhibitors such as sodium butyrate and trichostatin A inhibit vascular endothelial growth factor (VEGF) secretion from human glioblastoma cells

We investigated the effects of histone deacetylase (HDAC) inhibitors such as sodium butyrate (SB) and trichostatin A (TSA) on the expression of vascular endothelial growth factor (VEGF) by human glioblastoma T98G, U251MG, and U87MG cells. The glioblastoma cells secreted three VEGF isoforms, VEGF (189), (165), and (121), although the expression levels of VEGF differed between the cell types. Treatment with either 5mM SB or 100ng/ml TSA reduced VEGF secretion in conditioned media and reduced VEGF mRNA expression. We also studied the expression of VEGF-B,-C, and-D mRNA in human glioblastoma cells and their modulation by HDAC inhibitors. The PCR products of VEGF-B (357 bp), VEGF-C (501 bp), and VEGF-D (484 bp) were amplified in all glioblastoma cells examined. Treatment with SB reduced the expression of VEGF-D mRNA in U251MG cells and the expression of VEGF-B mRNA in U87MG cells. TSA treatment reduced the expression of VEGF-D in U251MG cells. These results suggest that HDAC inhibitors reduce VEGF secretion and modulate the expression of the other VEGF family members, and therefore may inhibit angiogenesis in glioblastoma tissues.     

Histone deacetylase inhibitors such as sodium butyrate and trichostatin A induce apoptosis through an increase of the bcl-2-related protein Bad

The effects of sodium butyrate (SB) and trichostatin A (TSA) on cell proliferation and apoptosis against human glioma T98G, U251MG, and U87MG cells were investigated. Upon exposure to either SB or TSA, cell proliferation was reduced, and apoptosis detected by DNA fragmentation analysis and the cleavage of CPP32 was induced. Previously, we reported that SB increased the expression levels of p21 (WAF-1) and inhibited G1-S transition of the cell cycle. In this study, we showed that TSA also increased p21 expression, suggesting that histone deacetylase (HDAC) inhibitors may up-regulate p21 protein in common and thus arrest proliferation in the G1 phase of the cell cycle. To further determine the underlying molecular mechanisms of apoptosis with either SB or TSA treatment, we studied the expression levels of apoptosisrelated proteins in human glioma cells. SB increased the expression of the Bad protein, although the expression of Bcl-2, Bcl-xL, Bax, and Fas was not changed by the addition of SB. TSA treatment also up-regulated the expression of Bad protein. The results suggest that HDAC inhibitors such as SB and TSA induce apoptosis through an increase in Bad protein in human glioma cells in vitro.     

Histone deacetylase inhibitors VPA and TSA induce apoptosis and autophagy in pancreatic cancer cells

Purpose

Histone deacetylase inhibitors (HDACi) are anti-neoplastic agents that are known to affect the growth of different cancer types, but their underlying mechanisms are still incompletely understood. Here, we compared the effects of two HDACi, i.e., Trichostatin A (TSA) and Valproic Acid (VPA), on the induction of cell death and autophagy in pancreatic cancer-derived cells that exhibit a high metastatic capacity and carry KRAS/p53 double mutations.

Methods

Cell viability and proliferation tests were carried out using Trypan blue dye exclusion, MTT and BrdU assays. FACS analyses were carried out to assess cell cycle progression, apoptosis, reactive oxygen species (ROS) production and mitochondrial depolarization, while Western blot and immunoprecipitation analyses were employed to detect proteins involved in apoptosis and autophagy.

Results

We found that both VPA and TSA can induce apoptosis in Panc1 and PaCa44 pancreatic cancer-derived cells by triggering mitochondrial membrane depolarization, Cytochrome c release and Caspase 3 activation, although VPA was more effective than TSA, especially in Panc1 cells. As underlying molecular events, we found that ERK1/2 was de-phosphorylated and that the c-Myc and mutant p53 protein levels were reduced after VPA and, to a lesser extent, after TSA treatment. Up-regulation of p21 and Puma was also observed, concomitantly with mutant p53 degradation. In addition, we found that in both cell lines VPA increased the pro-apoptotic Bim level, reduced the anti-apoptotic Mcl-1 level and increased ROS production and autophagy, while TSA was able to induce these effects only in PaCA44 cells.

Conclusions

From our results we conclude that both VPA and TSA can induce pancreatic cancer cell apoptosis and autophagy. VPA appears have a stronger and broader cytotoxic effect than TSA and, thus, may represent a better choice for anti-pancreatic cancer therapy.

     

Histone deacetylase inhibitors: inducers of differentiation or apoptosis of transformed cells

Histone deacetylase (HDAC) inhibitors have been shown to be potent inducers of growth arrest, differentiation, and/or apoptotic cell death of transformed cells in vitro and in vivo. One class of HDAC inhibitors, hydroxamic acid-based hybrid polar compounds (HPCs), induce differentiation at micromolar or lower concentrations. Studies (x-ray crystallographic) showed that the catalytic site of HDAC has a tubular structure with a zinc atom at its base and that these HDAC inhibitors, such as suberoylanilide hydroxamic acid and trichostatin A, fit into this structure with the hydroxamic moiety of the inhibitor binding to the zinc. HDAC inhibitors cause acetylated histones to accumulate in both tumor and normal tissues, and this accumulation can be used as a marker of the biologic activity of the HDAC inhibitors. Hydroxamic acid-based HPCs act selectively to inhibit tumor cell growth at levels that have little or no toxicity for normal cells. These compounds also act selectively on gene expression, altering the expression of only about 2% of the genes expressed in cultured tumor cells. In general, chromatin fractions enriched in actively transcribed genes are also enriched in highly acetylated core histones, whereas silent genes are associated with nucleosomes with a low level of acetylation. However, HDACs can also acetylate proteins other than histones in nucleosomes. The role that these other targets play in the induction of cell growth arrest, differentiation, and/or apoptotic cell death has not been determined. Our working hypothesis is that inhibition of HDAC activity leads to the modulation of expression of a specific set of genes that, in turn, result in growth arrest, differentiation, and/or apoptotic cell death. The hydroxamic acid-based HPCs are potentially effective agents for cancer therapy and, possibly, cancer chemoprevention.     

Histone deacetylase inhibitor valproic acid suppresses the growth and increases the androgen responsiveness of prostate cancer cells

We identified the molecular target by histone deacetylase (HDAC) inhibitors for exploring their potential prostate cancer (PCa) therapy. Upon HDAC inhibitors-treatment, LNCaP cell growth was suppressed, correlating with increased cellular prostatic acid phosphatase (cPAcP) expression, an authentic protein tyrosine phosphatase. In those cells, ErbB-2 was dephosphorylated, histone H3/H4 acetylation and methylation increased and cyclin proteins decreased. In PAcP shRNA-transfected C-81 cells, valproic acid (VPA) efficacy of growth suppression was diminished. Further, VPA pre-treatment enhanced androgen responsiveness of C-81, C4-2 and MDA PCa2b-AI cells. Thus, cPAcP expression is involved in growth suppression by HDAC inhibitors in PCa cells, and VPA pre-treatments increase androgen responsiveness.     

Histone deacetylase inhibitors require caspase activity to induce apoptosis in lung and prostate carcinoma cells

Histone deacetylase inhibitors (HDIs) are a promising new class of antineoplastic agents with the capacity to induce growth arrest and/or apoptosis of cancer cells. However, their precise mechanism of action is uncertain; particularly, the role of caspases in the apoptotic response to HDIs is controversial. Here, we show that the HDIs explored, suberoylanilide hydroxamic acid, sodium butyrate and trichostatin A, activated caspase-3 in A549 and PC-3 carcinoma cells. Additionally, the poly-caspase inhibitor z-VAD-fmk prevented HDI-induced apoptosis, as judged by determining mitochondrial membrane potential and by quantifying internucleosomal DNA fragmentation. Importantly, z-VAD-fmk also significantly inhibited HDI-elicited cell death, as assessed by measuring propidium iodide uptake. As an accessory finding, with the inhibition of caspases, a HDI-induced G2-M arrest became evident. Taken together, these results provide evidence that HDIs require activated caspases to induce apoptosis of carcinoma cells.     

Histone deacetylase inhibitors and hydroxyurea modulate the cell cycle and cooperatively induce apoptosis

Therapy resistance represents a major problem for disease management in oncology. Histone deacetylase inhibitors (HDACi) have been shown to modulate the cell cycle, to induce apoptosis and to sensitize cancer cells for other chemotherapeutics. Our study shows that the HDACi valproic acid (VPA) and the ribonucleotide reductase inhibitor hydroxyurea (HU) potentiate the pro-apoptotic effects of each other towards several cancer cell lines. This correlates with the HU-induced degradation of the cyclin-dependent kinase inhibitors (CDKI) p21 and p27, mediated by the proteasome or caspase-3. Moreover, we found that caspase-3 activation is required for VPA-induced apoptosis. Remarkably, p21 and p27 can confer resistance against VPA and HU. Both CDKI interact with caspase-3 and compete with other caspase-3 substrates. Hence, p21 and p27 may contribute to chemotherapy resistance as apoptosis inhibitors. Since the biological effects of VPA and HU could be achieved at concentrations used in current treatment protocols, the combined application of these compounds might be considered as a potential strategy for cancer treatment.     

Histone deacetylase inhibition by valproic acid down-regulates c-FLIP/CASH and sensitizes hepatoma cells towards CD95- and TRAIL receptor-mediated apoptosis and chemotherapy

Hepatocellular carcinoma (HCC) is highly resistant to chemotherapy, leading to a poor prognosis of advanced disease. Inhibitors of histone deacetylase (HDACi) induce re-differentiation in tumor cells and thereby re-establish sensitivity towards apoptotic stimuli. HDACi are entering the clinical stage of tumor treatment, and several substances are currently being tested in clinical trials to prove their efficacy in the treatment of leukemias and solid tumors. In this study, we investigated the impact of the HDACi valproic acid (VA) on TRAIL- and CD95-mediated apoptosis in hepatoma cells, as well as its sensitizing effect on a chemotherapeutic agent. Treatment of HepG2 cells with VA increased sensitivity to CD95-mediated apoptosis (4% apoptosis vs. 42%), and treatment with epirubicin (74% vs. 90% viability). Caspase-3 activity was significantly enhanced in cells treated with VA plus anti-CD95 antibodies compared to cells treated with antibodies alone. In parallel, VA strongly augmented the effect of TNF-related apoptosis-inducing ligand (TRAIL or Apo2 ligand) on HepG2 cells (10% vs. 58% apoptosis). VA induced down-regulation of cellular FLICE-inhibitory protein (c-FLIP/CASH, also known as Casper/iFLICE/FLAME-1/CLARP/MRIT/usurpin), providing a possible molecular mechanism underlying the increased sensitivity towards cell death-mediated apoptosis. HDAC inhibitors are a promising class for the treatment of leukemias. In addition, among other class members, VA deserves further evaluation as a treatment option for patients with advanced HCC.     

The histone deacetylase inhibitors suberoylanilide hydroxamic (Vorinostat) and valproic acid induce irreversible and MDR1-independent resistance in human colon cancer cells

Histone deacetylase (HDAC) inhibitors such as suberoylanilide hydroxamic acid (SAHA, Vorinostat), valproic acid (VPA), and FK228 are members of a relatively novel class of small molecular weight chemicals that have high antineoplastic activity. They cause growth inhibition and apoptosis specifically in tumor cells, and they act also as chemo- and radio-sensitizers. In the present study, the potential of SAHA and VPA to induce resistance was studied. To that aim HDAC inhibitor-resistant sublines were generated by stepwise exposure of colon tumor cells to increasing concentrations of these compounds. Clonogenic data demonstrated that the SAHA- and VPA-induced sublines were 2-fold resistant to these compounds. This resistance was non-reversible, as it was maintained even when the sublines were cultured in the absence of SAHA or VPA. The SAHA- and VPA-induced resistant sublines were also stably cross-resistant to VPA and SAHA, respectively, but retained sensitivity against non-HDAC inhibitor-type anticancer agents. The SAHA-induced resistance correlated with loss of the G2/M checkpoint but it was not accompanied by reduced induction of the endogenous cell cycle inhibitors p21 and p27. Furthermore, SAHA-induced resistance was not due to reduced apoptosis, and it was neither dependent on MDR expression nor was it due to increased expression of HDAC1 and HDAC3. Taken together, these data demonstrate the potential of SAHA and VPA to induce resistance. This resistance was not dependent on MDR expression, did not involve MMR, and seemed to underlie a mechanism that differs from that underlying the previously observed FK228-induced resistance. The finding that SAHA and VPA induce only modest resistance despite continuous treatment and that the resistance is MDR-independent suggests a preference for these two drugs over FK228 for use in combination treatment with classic anticancer agents.     

Histone deacetylase inhibitors radiosensitize human melanoma cells by suppressing DNA repair activity.

PURPOSE: Histone deacetylase (HDAC) inhibitors have emerged recently as promising anticancer agents. They arrest cells in the cell cycle and induce differentiation and cell death. The antitumor activity of HDAC inhibitors has been linked to their ability to induce gene expression through acetylation of histone and nonhistone proteins. However, it has recently been suggested that HDAC inhibitors may also enhance the activity of other cancer therapeutics, including radiotherapy. The purpose of this study was to evaluate the ability of HDAC inhibitors to radiosensitize human melanoma cells in vitro. EXPERIMENTAL DESIGN: A panel of HDAC inhibitors that included sodium butyrate (NaB), phenylbutyrate, tributyrin, and trichostatin A were tested for their ability to radiosensitize two human melanoma cell lines (A375 and MeWo) using clonogenic cell survival assays. Apoptosis and DNA repair were measured by standard assays. RESULTS: NaB induced hyperacetylation of histone H4 in the two melanoma cell lines and the normal human fibroblasts. NaB radiosensitized both the A375 and MeWo melanoma cell lines, substantially reducing the surviving fraction at 2 Gy (SF2), whereas it had no effect on the normal human fibroblasts. The other HDAC inhibitors, phenylbutyrate, tributyrin, and trichostatin A had significant radiosensitizing effects on both melanoma cell lines tested. NaB modestly enhanced radiation-induced apoptosis that did not correlate with survival but did correlate with functional impairment of DNA repair as determined based on the host cell reactivation assay. Moreover, NaB significantly reduced the expression of the repair-related genes Ku70 and Ku86 and DNA-dependent protein kinase catalytic subunit in melanoma cells at the protein and mRNA levels. Normal human fibroblasts showed no change in DNA repair capacity or levels of DNA repair proteins following NaB treatment. We also examined gamma-H2AX phosphorylation as a marker of radiation response to NaB and observed that compared with controls, gamma-H2AX foci persisted long after ionizing exposure in the NaB-treated cells. CONCLUSIONS: HDAC inhibitors radiosensitize human tumor cells by affecting their ability to repair the DNA damage induced by ionizing radiation and that gamma-H2AX phosphorylation can be used as a predictive marker of radioresponse.     

Effects of the histone deacetylase inhibitor valproic acid on Notch signalling in human neuroblastoma cells

Neuroblastoma (NB), a sympathetically derived childhood tumour, shows characteristics of neuronal precursor cells, suggesting a halted differentiation process. We have previously shown that the Notch signalling cascade, a key player during normal neurogenesis, also might be involved in NB differentiation. Valproic acid (VPA), a well-tolerated antiepileptic drug, has been shown to induce differentiation and cell death of NB cells, possibly associated with its recently described HDAC inhibiting activity. Stimulation of NB cells with VPA led to increased cell death and phenotypic changes associated with differentiation, that is, neurite extension and upregulation of neuronal markers. VPA treatment also led to an activated Notch signalling cascade as shown by increased levels of intracellular Notch-1 and Hes-1, mimicking the initial phase of induced differentiation. These results reinforce that VPA potentially could be used in differentiation therapy of NB and that the effects in part could be a consequence of interference with the Notch signalling cascade.