Jürgen Sonnemann

Histone deacetylase inhibitors induce cell death and enhance the apoptosis-inducing activity of TRAIL in Ewing’s sarcoma cells

PurposeThe present in vitro study was conducted to evaluate the effects of the histone deacetylase inhibitors (HDIs) suberoyl anilide hydroxamic acid (SAHA), sodium butyrate (NaB) and MS-275 applied as single agents or in combination with TRAIL in Ewing’s sarcoma.MethodsCytotoxic activities were assessed by cytofluorometric analysis of propidium iodide uptake, DNA fragmentation and mitochondrial depolarisation as well as by measuring caspase-9 and -3 activities. Cell-surface expression of TRAIL receptors was determined by cytofluorometry, and histone H4 acetylation was assessed by western blot.ResultsAll three HDIs potently induced cell death in the two cell lines explored, SK-ES-1 and WE-68. However, they seemed to differ in their modes of action. SAHA and NaB induced mitochondrial depolarisation as well as caspase-9 and -3 activities, and their cytotoxic effects could be significantly reduced by the pan-caspase inhibitor z-VAD-fmk. MS-275 was a much weaker inducer of caspase-9 and -3 activities as well as mitochondrial injury; consistently, z-VAD-fmk had little effect on MS-275-mediated activities. Combined treatment of HDIs and TRAIL led to an additive effect in SK-ES-1 cells and a supra-additive effect in WE-68 cells. Yet, HDIs did not increase cell-surface expression of TRAIL receptor 2, but rather decreased it. Selective inhibition of caspase-8 in WE-68 cells and HDI treatment of CADO-ES-1 cells, a Ewings sarcoma cell line deficient in caspase-8 expression, revealed that caspase-8 was not required for HDI-mediated apoptosis.ConclusionsThese results suggest that HDIs may be considered as a novel treatment strategy for Ewing’s sarcoma either applied as monotherapy or in combination with TRAIL.

Histone deacetylase inhibitors interact synergistically with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to induce apoptosis in carcinoma cell lines

SummaryBoth tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and histone deacetylase inhibitors (HDIs) show promise for the treatment of cancer. However, in a number of reports they have been proven ineffective to induce cell death when applied as single agents. In this study, we show that A549 lung carcinoma cells and PC-3 prostate carcinoma cells underwent substantial apoptosis when coexposed to TRAIL and either suberoylanilide hydroxamic acid, sodium butyrate or trichostatin A. HDIs and TRAIL synergized in activation of capase-3, induction of internucleosomal DNA fragmentation and promoting mitochondrial damage. Significantly, cotreatment with minimally toxic doses of HDIs and TRAIL resulted in a marked apoptotic response in both cell lines. These data provide a rationale for a more in-depth exploration into the potential of combining TRAIL and HDIs as a valuable anticancer strategy.

Histone deacetylase inhibitors sensitize tumour cells for cytotoxic effects of natural killer cells

Histone deacetylase inhibitors (HDIs) are emerging as potent anti-tumour agents which induce cell cycle arrest, differentiation and/or apoptosis in many tumour cells. Furthermore, they render tumour cells more sensitive to other therapeutic regimens like ionizing radiation, chemotherapy and recombinant tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Here, we show that the HDIs suberoylanilide hydroxamic acid (SAHA; vorinostat), sodium butyrate (NaB) and MS-275 sensitized DAOY and PC3 tumour cells for the cytotoxic effects of IL-2-activated PBMCs. In (51)Cr-release assays, blockade of the activating NK receptors DNAM-1, NKG2D and the NCRs completely abrogated tumour cell lysis, revealing that NK cells were the main effector cells involved. HDIs increased the tumour surface expression of ligands for the activating NK receptors NKG2D and DNAM-1 thereby facilitating tumour cell recognition by NK cells. These results suggest that the combination of HDIs and immunotherapy may be an effective strategy for anti-cancer therapy.

The expression of histone deacetylase 4 is associated with prednisone poor-response in childhood acute lymphoblastic leukemia

This study aimed at the identification of histone deacetylase (HDAC) isoforms relevant for childhood acute lymphoblastic leukemia (ALL). Expression of HDAC1-11 was determined in 93 primary ALL and eight healthy donor samples. HDAC1, HDAC2 and HDAC8 showed significantly higher expressions in ALL samples. Correlation analysis of HDAC expression with clinicopathological parameters revealed that high HDAC1, HDAC2, HDAC4 and HDAC11 levels were significantly associated with unfavorable prognostic factors. Particularly, high HDAC4 expression was associated with high initial leukocyte count, T cell ALL and prednisone poor-response. siRNA-mediated inhibition of HDAC4 sensitized a T-ALL cell line to etoposide-induced cell death. In conclusion, our data point to HDAC4 as drug target in childhood ALL, especially in prednisone poor-responders.

Anticancer effects of the p53 activator nutlin-3 in Ewing’s sarcoma cells

Mutation of p53 is rare in Ewings sarcoma (ES), suggesting that targeting and activation of wild-type p53 may be an effective therapeutic strategy for ES. The recently developed small-molecule MDM2 inhibitor nutlin-3 restores wild-type p53 function, resulting in the inhibition of cancer cell growth and the induction of apoptosis. In the present study, we explored the responsiveness of ES cell lines with wild-type or mutated p53 to nutlin-3. We found that treatment with nutlin-3 increased p53 level and induced p53 target gene expression (MDM2, p21, PUMA) in ES cells with wild-type p53, but not in ES cells with mutated p53. Consistently, nutlin-3 elicited apoptosis only in wild-type p53 cells, as assessed by caspase-3 activity assay and flow cytometric analyses of mitochondrial depolarisation and DNA fragmentation. In addition, we found nutlin-3 to evoke cellular senescence, indicating that nutlin-3 induces pleiotropic anticancer effects in ES. Furthermore, combined treatment with nutlin-3 and an inhibitor of NF-κB produced synergistic antineoplastic activity in ES cells. Our findings suggest that the direct activation of p53 by nutlin-3 treatment may be a useful new therapeutic approach for patients with ES.

Peroxiredoxin III protects pancreatic β cells from apoptosis

Type 1 diabetes mellitus is characterized by a progressive autoimmune destruction of insulin-producing β cells. Macrophages and T lymphocytes release cytokines, which induce the synthesis of oxygen and nitrogen radicals in the pancreatic islets. The resulting cellular and mitochondrial damage promotes β cell death. β cells are very sensitive to the autoimmune free radical-dependent attack due to their low content of antioxidant enzymes such as glutathione peroxidase and catalase. A focal point of β cell protection should be the control of the mitochondrial redox status, which will result in the preservation of metabolic stimulus-secretion coupling. For this reason, there is a considerable interest in the mitochondrial peroxiredoxin III (PRX III), a thioredoxin-dependent peroxide reductase, which was shown to be able to protect against both oxidative and nitrosative stress. Using the Tet-On-system, we generated stably transfected rat insulinoma cells over- or under-expressing PRX III in a doxycyclin-dependent manner to analyze the effect of increased or decreased amounts of cellular PRX III, following treatment with several stressors. We provide evidence that PRX III protects pancreatic β cells from cell stress induced by accumulation of hydrogen peroxide, or the induction of inducible nitric oxide synthase or caspase-9 and -3 by pro-inflammatory cytokines or streptozotocin. Basal insulin secretion was markedly decreased in cells expressing lower levels of PRX III. We suggest PRX III may be a suitable target for promoting deceleration or even prevention of stress-associated apoptosis in pancreatic β cells and the manifestation of insulin-dependent diabetes mellitus.

Inhibition of novel protein kinase Cɛ augments TRAIL-induced cell death in A549 lung cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has great potential for cancer treatment since it provokes cell death in most tumor cells while leaving most normal cells unscathed. Some cancers, however, show resistance to TRAIL, indicating that TRAIL alone may be insufficient for cancer therapy. Here we studied whether the apoptotic susceptibility of A549 non-small cell lung cancer cells could be modulated by inhibiting protein kinase C (PKC). We show that an inhibitor with preference for novel PKC isozymes, NPC 15437, significantly augmented TRAIL sensitivity of A549 cells, as judged by assessing cell death and mitochondrial membrane potential. Likewise, NPC 15437 also significantly potentiated the responsiveness of DAOY medulloblastoma cells to TRAIL. In contrast, an inhibitor with preference for conventional PKC isozymes, Gö6976, did not augment TRAIL sensitivity of A549 cells. To further specify the PKC isozyme responsible for TRAIL sensitization, we used a peptide inhibitor with selectivity for the novel PKC isozyme ɛ, myr-PKCɛ V1-2. The inhibition of PKCɛ resulted in a significant amplification of the cytotoxic activity of TRAIL in A549 cells. Altogether, our study provides evidence for a considerable role of PKCɛ in the apoptotic responsiveness of A549 lung cancer cells, and possibly other malignancies, to TRAIL.

Synergistic activity of the histone deacetylase inhibitor suberoylanilide hydroxamic acid and the bisphosphonate zoledronic acid against prostate cancer cells in vitro

Bisphosphonates are widely used agents for the treatment of malignant bone disease. They inhibit osteoclast-mediated bone resorption and can have direct effects on cancer cells. In this study, we investigated whether the anticancer activity of the third-generation bisphosphonate zoledronic acid (ZOL) could be enhanced by combination with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). We found that ZOL and SAHA cooperated to induce cell death in the prostate cancer cell lines LNCaP and PC-3. The effect was synergistic, as evidenced by combination index isobologram analysis. ZOL and SAHA synergized to induce dissipation of the mitochondrial transmembrane potential, to activate caspase-3, and to trigger DNA fragmentation, showing that the combination of ZOL and SAHA resulted in the initiation of apoptosis. Because ZOL acts by inhibiting the mevalonate pathway, thereby preventing protein prenylation, we explored whether the mevalonate pathway was also the target of the cooperative action of ZOL and SAHA. We found that geranylgeraniol, but not farnesol, significantly reduced ZOL/SAHA-induced cell death, indicating that the synergistic action of the agents was due to the inhibition of geranylgeranylation. Consistently, a direct inhibitor of geranylgeranylation, GGTI-298, synergized with SAHA to induce cell death, whereas an inhibitor of farnesylation, FTI-277, had no effect. In addition, SAHA synergized with mevastatin, an inhibitor of the proximal enzyme in the mevalonate pathway. These in vitro findings provide a rationale for an in vivo exploration into the potential of combining SAHA and ZOL, or other inhibitors of the mevalonate pathway, as an effective strategy for anticancer therapy. [Mol Cancer Ther 2007;6(11):2976–84]

Increased activity of histone deacetylases in childhood acute lymphoblastic leukaemia and acute myeloid leukaemia: support for histone deacetylase inhibitors as antileukaemic agents.

Butterfield, Y., Birol, I., Holt, R., Schein, J., Horsman, D.E., Moore, R., Jones, S.J., Connors, J.M., Hirst, M., Gascoyne, R.D. & Marra, M.A. (2011) Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma. Nature, 476, 298–303. Ngo, V.N., Young, R.M., Schmitz, R., Jhavar, S., Xiao, W., Lim, K.H., Kohlhammer, H., Xu, W., Yang, Y., Zhao, H., Shaffer, A.L., Romesser, P., Wright, G., Powell, J., Rosenwald, A., MullerHermelink, H.K., Ott, G., Gascoyne, R.D., Connors, J.M., Rimsza, L.M., Campo, E., Jaffe, E.S., Delabie, J., Smeland, E.B., Fisher, R.I., Braziel, R.M., Tubbs, R.R., Cook, J.R., Weisenburger, D. D., Chan, W.C. & Staudt, L.M. (2011) Oncogenically active MYD88 mutations in human lymphoma. Nature, 470, 115–119. Rinaldi, A., Mian, M., Chigrinova, E., Arcaini, L., Bhagat, G., Novak, U., Rancoita, P.M., De Campos, C.P., Forconi, F., Gascoyne, R.D., Facchetti, F., Ponzoni, M., Govi, S., Ferreri, A.J., Mollejo, M., Piris, M.A., Baldini, L., Soulier, J., Thieblemont, C., Canzonieri, V., Gattei, V., Marasca, R., Franceschetti, S., Gaidano, G., Tucci, A., Uccella, S., Tibiletti, M.G., Dirnhofer, S., Tripodo, C., Doglioni, C., Dalla Favera, R., Cavalli, F., Zucca, E., Kwee, I. & Bertoni, F. (2011) Genome-wide DNA profiling of marginal zone lymphomas identifies subtype-specific lesions with an impact on the clinical outcome. Blood, 117, 1595–1604. Wang, L., Lawrence, M.S., Wan, Y., Stojanov, P., Sougnez, C., Stevenson, K., Werner, L., Sivachenko, A., DeLuca, D.S., Zhang, L., Zhang, W., Vartanov, A.R., Fernandes, S.M., Goldstein, N.R., Folco, E.G., Cibulskis, K., Tesar, B., Sievers, Q.L., Shefler, E., Gabriel, S., Hacohen, N., Reed, R., Meyerson, M., Golub, T.R., Lander, E. S., Neuberg, D., Brown, J.R., Getz, G. & Wu, C. J. (2011) SF3B1 and other novel cancer genes in chronic lymphocytic leukemia. New England Journal of Medicine, 365, 2497–2506. Xu, L., Sohani, A.R., Arcaini, L., Hunter, Z., Yang, G., Zhou, Y., Liu, X., Cao, Y., Manning, R., Patterson, C.J., Ioakimidis, L., Tripsas, C., Pinkus, G.S., Harris, N.L., Rodig, S.J. & Treon, S. (2011) A somatic variant in MYD88 (L265P) revealed by whole genome sequencing differentiates lymphoplasmacytic lymphoma from marginal zone lymphomas. Blood (ASH Annual Meeting Abstracts), 118, 261. Yan, Y., Huang, Y., Watkins, A.J., Kocialkowski, S., Zeng, N., Hamoudi, R.A., Isaacson, P.G., de Leval, L., Wotherspoon, A. & Du, M.Q. (2012) BCR and TLR signaling pathways are recurrently targeted by genetic changes in splenic marginal zone lymphomas. Haematologica, 97, 595–598.

Histone deacetylase inhibitors enhance the anticancer activity of nutlin-3 and induce p53 hyperacetylation and downregulation of MDM2 and MDM4 gene expression

SummaryNutlin-3, a small-molecule MDM2 inhibitor, restores p53 function and is, thus, an appealing candidate for the treatment of cancers retaining wild-type p53. However, nutlin-3 applied as single agent may be insufficient for cancer therapy. Therefore, we explored whether the anticancer activity of nutlin-3 could be enhanced by combination with histone deacetylase inhibitors (HDACi), i.e. vorinostat, sodium butyrate, MS-275 and apicidin. We found that nutlin-3 and HDACi cooperated to induce cell death in the p53 wild-type cell lines A549 and A2780, but not in the p53 null cell line PC-3, as assessed by Alamar Blue assay and flow cytometric analyses of propidium iodide uptake and mitochondrial depolarization. Combination index analysis showed that the effect was synergistic. For comparison, we tested nutlin-3 in combination with paclitaxel, revealing that nutlin-3 antagonized the cytotoxic activity of paclitaxel. To shed light on the underlying mechanism of the synergistic action of nutlin-3 and HDACi, we determined the acetylation status of p53 by immunoblotting and the mRNA levels of MDM2 and MDM4 by real-time RT-PCR. We observed vorinostat to induce p53 hyperacetylation, to reduce the constitutive gene expression of MDM2 and MDM4, and to counteract the nutlin-3-induced upregulation of MDM2 gene expression. In conclusion, our study shows that HDACi amplify the antitumor activity of nutlin-3—possibly by inducing p53 hyperacetylation and/or MDM2 and/or MDM4 downregulation—suggesting that treatment with a combination of nutlin-3 and HDACi may be an effective strategy for treating tumors with wild-type p53.