Sascha Venturelli

Natural Killer Cell–Mediated Lysis of Hepatoma Cells via Specific Induction of NKG2D Ligands by the Histone Deacetylase Inhibitor Sodium Valproate

Natural killer (NK) cells as components of the innate immunity substantially contribute to antitumor immune responses. However, the tumor-associated ligands engaging activating NK cell receptors are largely unknown. An exception are the MHC class I chain-related molecules MICA and MICB and the UL16-binding proteins (ULBP) which bind to the activating immunoreceptor NKG2D expressed on cytotoxic lymphocytes. A therapeutic induction of NKG2D ligands that primes cancer cells for NK cell lysis has not yet been achieved. By microarray studies, we found evidence that treatment of human hepatocellular carcinoma cells with the histone deacetylase inhibitor (HDAC-I) sodium valproate (VPA) mediates recognition of cancer cells by cytotoxic lymphocytes via NKG2D. VPA induced transcription of MICA and MICB in hepatocellular carcinoma cells, leading to increased cell surface, soluble and total MIC protein expression. No significant changes in the expression of the NKG2D ligands ULBP1-3 were observed. The induction of MIC molecules increased lysis of hepatocellular carcinoma cells by NK cells which was abolished by addition of a blocking NKG2D antibody. Importantly, in primary human hepatocytes, VPA treatment did not induce MIC protein expression. Taken together, our data show that the HDAC-I VPA mediates specific priming of malignant cells for innate immune effector mechanisms. These results suggest the clinical evaluation of HDAC-I in solid tumors such as hepatocellular carcinoma, especially in combination with immunotherapy approaches employing adoptive NK cell transfer.

HDAC inhibitor treatment of hepatoma cells induces both TRAIL‐independent apoptosis and restoration of sensitivity to TRAIL

Hepatocellular carcinoma (HCC) displays a striking resistance to chemotherapeutic drugs or innovative tumor cell apoptosis–inducing agents such as tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL). Recently, we found 2 histone deacetylase inhibitors (HDAC‐I), valproic acid and ITF2357, exhibiting inherent therapeutic activity against HCC. In TRAIL‐sensitive cancer cells, the mechanism of HDAC‐I–induced cell death has been identified to be TRAIL‐dependent by inducing apoptosis in an autocrine fashion. In contrast, in HCC‐derived cells, a prototype of TRAIL‐resistant tumor cells, we found a HDAC‐I‐mediated apoptosis that works independently of TRAIL and upregulation of death receptors or their cognate ligands. Interestingly, TRAIL resistance could be overcome by a combinatorial application of HDAC‐I and TRAIL, increasing the fraction of apoptotic cells two‐ to threefold compared with HDAC‐I treatment alone, whereas any premature HDAC‐I withdrawal rapidly restored TRAIL resistance. Furthermore, a tumor cell–specific downregulation of the FLICE inhibitory protein (FLIP) was observed, constituting a new mechanism of TRAIL sensitivity restoration by HDAC‐I. In contrast, FLIP levels in primary human hepatocytes (PHH) from different donors were upregulated by HDAC‐I. Importantly, combination HDAC‐I/TRAIL treatment did not induce any cytotoxicity in nonmalignant PHH. In conclusion, HDAC‐I compounds, exhibiting a favorable in vivo profile and inherent activity against HCC cells, are able to selectively overcome the resistance of HCC cells toward TRAIL. Specific upregulation of intracellular FLIP protein levels in nonmalignant hepatocytes could enhance the therapeutic window for clinical applications of TRAIL, opening up a highly specific new treatment option for advanced HCC. (HEPATOLOGY 2006;43:425–434.)

Epigenetic combination therapy as a tumor-selective treatment approach for hepatocellular carcinoma.

Innovative epigenetic therapeutics comprise histone deacetylase inhibitors (HDAC‐I) and demethylating agents (DA). It was recently found that HDAC‐I compounds exhibit profound therapeutic activities against hepatocellular carcinoma (HCC). A comprehensive preclinical investigation was performed on the potential of a combined HDAC‐I/DA epigenetic regimen for the highly chemotherapy‐resistant HCC entity.

Resveratrol as a Pan-HDAC Inhibitor Alters the Acetylation Status of Jistone Proteins in Human-Derived Hepatoblastoma Cells

The polyphenolic alcohol resveratrol has demonstrated promising activities for the prevention and treatment of cancer. Different modes of action have been described for resveratrol including the activation of sirtuins, which represent the class III histone deacetylases (HDACs). However, little is known about the activity of resveratrol on the classical HDACs of class I, II and IV, although these classes are involved in cancer development or progression and inhibitors of HDACs (HDACi) are currently under investigation as promising novel anticancer drugs. We could show by in silico docking studies that resveratrol has the chemical structure to inhibit the activity of different human HDAC enzymes. In vitro analyses of overall HDAC inhibition and a detailed HDAC profiling showed that resveratrol inhibited all eleven human HDACs of class I, II and IV in a dose-dependent manner. Transferring this molecular mechanism into cancer therapy strategies, resveratrol treatment was analyzed on solid tumor cell lines. Despite the fact that hepatocellular carcinoma (HCC) is known to be particularly resistant against conventional chemotherapeutics, treatment of HCC with established HDACi already has shown promising results. Testing of resveratrol on hepatoma cell lines HepG2, Hep3B and HuH7 revealed a dose-dependent antiproliferative effect on all cell lines. Interestingly, only for HepG2 cells a specific inhibition of HDACs and in turn a histone hyperacetylation caused by resveratrol was detected. Additional testing of human blood samples demonstrated a HDACi activity by resveratrol ex vivo. Concluding toxicity studies showed that primary human hepatocytes tolerated resveratrol, whereas in vivo chicken embryotoxicity assays demonstrated severe toxicity at high concentrations. Taken together, this novel pan-HDACi activity opens up a new perspective of resveratrol for cancer therapy alone or in combination with other chemotherapeutics. Moreover, resveratrol may serve as a lead structure for chemical optimization of bioavailability, pharmacology or HDAC inhibition.

Epigenetic activities of flavonoids in the prevention and treatment of cancer

Aberrant epigenetic modifications are described in an increasing number of pathological conditions, including neurodegenerative diseases, cardiovascular diseases, diabetes mellitus type 2, obesity and cancer. The general reversibility of epigenetic changes makes them an attractive and promising target e.g. in the treatment of cancer. Thus, a growing number of epigenetically active compounds are currently tested in clinical trials for their therapeutic potential. Interestingly, many phytochemicals present in plant foods, particularly flavonoids, are suggested to be able to alter epigenetic cellular mechanisms. Flavonoids are natural phenol compounds that form a large group of secondary plant metabolites with interesting biological activities. They can be categorized into six major subclasses, which display diverse properties affecting the two best characterized epigenetic mechanisms: modulation of the DNA methylation status and histone acetylation. High dietary flavonoid intake has strongly been suggested to reduce the risk of numerous cancer entities in a large body of epidemiological studies. Established health-promoting effects of diets rich in fruit and vegetables are faced by efforts to use purified flavonoids as supplements or pharmaceuticals, whereupon data on the latter applications remain controversial. The purpose of this review is to give an overview of current research on flavonoids to further elucidate their potential in cancer prevention and therapy, thereby focusing on their distinct epigenetic activities.

The farnesyl transferase inhibitor lonafarnib inhibits mTOR signaling and enforces sorafenib-induced apoptosis in melanoma cells.

Farnesyl transferase inhibitors (FTIs) inhibit the farnesylation of proteins, including RAS and RHEB (Ras homolog enriched in brain). RAS signals to the RAF-MEK-ERK (MAPK) and PI3K-AKT-mTOR (AKT) signaling pathways, which have a major role in melanoma progression. RHEB positively regulates mammalian target of rapamycin (mTOR). We investigated the effects of the FTI lonafarnib alone and in combination with MAPK (mitogen-activated protein kinase) or AKT (acutely transforming retrovirus AKT8 in rodent T-cell lymphoma) pathway inhibitors on proliferation, survival, and invasive tumor growth of melanoma cells. Lonafarnib alone did not sufficiently inhibit melanoma cell growth. Combinations of lonafarnib with AKT pathway inhibitors did not significantly increase melanoma cell growth inhibition. In contrast, combinations of lonafarnib with MAPK pathway inhibitors yielded additional growth-inhibiting effects. In particular, the combination of the FTI lonafarnib with the pan-RAF inhibitor sorafenib synergistically inhibited melanoma cell growth, significantly enhanced sorafenib-induced apoptosis, and completely suppressed invasive tumor growth in monolayer and organotypic cultures, respectively. Apoptosis induction was associated with upregulation of the endoplasmic reticulum stress-related transcription factors p8 and CHOP (CAAT/enhancer binding protein (C/EBP) homologous protein), and downregulation of the antiapoptotic Bcl-2 (B-cell lymphoma-2) family protein Mcl-1(myeloid cell leukemia 1). Lonafarnib did not affect MAPK and AKT but did affect mTOR signaling. Together, these findings suggest that the FTI lonafarnib inhibits mTOR signaling and enforces sorafenib-induced apoptosis in melanoma cells and may therefore represent an effective alternative for melanoma treatment.

Kaempferol, a new nutrition-derived pan-inhibitor of human histone deacetylases

Kaempferol is a natural polyphenol belonging to the group of flavonoids. Different biological functions like inhibition of oxidative stress in plants or animal cells and apoptosis induction have been directly associated with kaempferol. The underlying mechanisms are only partially understood. Here we report for the first time that kaempferol has a distinct epigenetic activity by inhibition of histone deacetylases (HDACs). In silico docking analysis revealed that it fits into the binding pocket of HDAC2, 4, 7 or 8 and thereby binds to the zinc ion of the catalytic center. Further in vitro profiling of all conserved human HDACs of class I, II and IV showed that kaempferol inhibited all tested HDACs. In clinical oncology, HDAC inhibitors are currently under investigation as new anticancer compounds. Therefore, we studied the effect of kaempferol on human-derived hepatoma cell lines HepG2 and Hep3B as well as on HCT-116 colon cancer cells and found that it induces hyperacetylation of histone complex H3. Furthermore, kaempferol mediated a prominent reduction of cell viability and proliferation rate. Interestingly, toxicity assays revealed signs of relevant cellular toxicity in primary human hepatocytes only starting at 50 μM as well as in an in vivo chicken embryotoxicity assay at 200 μM. In conclusion, the identification of a novel broad inhibitory capacity of the natural compound kaempferol for human-derived HDAC enzymes opens up the perspective for clinical application in both tumor prevention and therapy. Moreover, kaempferol may serve as a novel lead structure for chemical optimization of pharmacokinetics, pharmacology or inhibitory activities.

Differential Induction of Apoptosis and Senescence by the DNA Methyltransferase Inhibitors 5-Azacytidine and 5-Aza-2′-Deoxycytidine in Solid Tumor Cells

Epigenetic alterations are a hallmark of cancer that govern the silencing of genes. Up to now, 5-azacytidine (5-aza-CR, Vidaza) and 5-aza-2′-deoxycytidine (5-aza-dC, Dacogen) are the only clinically approved DNA methyltransferase inhibitors (DNMTi). Current effort tries to exploit DNMTi application beyond acute leukemia or myelodysplastic syndrome, especially to solid tumors. Although both drugs only differ by a minimal structural difference, they trigger distinct molecular mechanisms that are highly relevant for a rational choice of new combination therapies. Therefore, we investigated cell death pathways in vitro in human hepatoma, colon, renal, and lung cancer cells and in vivo in chorioallantoic membrane and xenograft models. Real-time cancer cell monitoring and cytokine profiling revealed a profoundly distinct response pattern to both drugs. 5-aza-dC induced p53-dependent tumor cell senescence and a high number of DNA double-strand breaks. In contrast, 5-aza-CR downregulated p53, induced caspase activation and apoptosis. These individual response patterns of tumor cells could be verified in vivo in chorioallantoic membrane assays and in a hepatoma xenograft model. Although 5-aza-CR and 5-aza-dC are viewed as drugs with similar therapeutic activity, they induce a diverse molecular response in tumor cells. These findings together with other reported differences enable and facilitate a rational design of new combination strategies to further exploit the epigenetic mode of action of these two drugs in different areas of clinical oncology. Mol Cancer Ther; 12(10); 2226–36. ©2013 AACR.

Plants Release Precursors of Histone Deacetylase Inhibitors to Suppress Growth of Competitors

Chemical compounds in plant root exudates influence the growth of neighboring plants by interfering with their chromatin configuration and gene expression. To secure their access to water, light, and nutrients, many plant species have developed allelopathic strategies to suppress competitors. To this end, they release into the rhizosphere phytotoxic substances that inhibit the germination and growth of neighbors. Despite the importance of allelopathy in shaping natural plant communities and for agricultural production, the underlying molecular mechanisms are largely unknown. Here, we report that allelochemicals derived from the common class of cyclic hydroxamic acid root exudates directly affect the chromatin-modifying machinery in Arabidopsis thaliana. These allelochemicals inhibit histone deacetylases both in vitro and in vivo and exert their activity through locus-specific alterations of histone acetylation and associated gene expression. Our multilevel analysis collectively shows how plant-plant interactions interfere with a fundamental cellular process, histone acetylation, by targeting an evolutionarily highly conserved class of enzymes.

Epigenetic Impacts of Ascorbate on Human Metastatic Melanoma Cells

In recent years, increasing evidence has emerged demonstrating that high-dose ascorbate bears cytotoxic effects on cancer cells in vitro and in vivo, making ascorbate a pro-oxidative drug that catalyzes hydrogen peroxide production in tissues instead of acting as a radical scavenger. This anticancer effect of ascorbate is hypoxia-inducible factor-1α- and O2-dependent. However, whether the intracellular mechanisms governing this effect are modulated by epigenetic phenomena remains unknown. We treated human melanoma cells with physiological (200 μM) or pharmacological (8 mM) ascorbate for 1 h to record the impact on DNA methyltransferase (DNMT)-activity, histone deacetylases (HDACs), and microRNA (miRNA) expression after 12 h. The results were analyzed with the MIRUMIR online tool that estimates the power of miRNA to serve as potential biomarkers to predict survival of cancer patients. FACS cell-cycle analyses showed that 8 mM ascorbate shifted BLM melanoma cells toward the sub-G1 fraction starting at 12 h after an initial primary G2/M arrest, indicative for secondary apoptosis induction. In pharmacological doses, ascorbate inhibited the DNMT activity in nuclear extracts of MeWo and BLM melanoma cells, but did not inhibit human HDAC enzymes of classes I, II, and IV. The expression of 151 miRNAs was altered 12 h after ascorbate treatment of BLM cells in physiological or pharmacological doses. Pharmacological doses up-regulated 32 miRNAs (≥4-fold) mainly involved in tumor suppression and drug resistance in our preliminary miRNA screening array. The most prominently up-regulated miRNAs correlated with a significantly increased overall survival of breast cancer or nasopharyngeal carcinoma patients of the MIRUMIR database with high expression of the respective miRNA. Our results suggest a possible epigenetic signature of pharmacological doses of ascorbate in human melanoma cells and support further pre-clinical and possibly even clinical evaluation of ascorbate for melanoma therapy.