Mariateresa Pugliese

Valproic acid enhances tubulin acetylation and apoptotic activity of paclitaxel on anaplastic thyroid cancer cell lines

The introduction of paclitaxel into multimodal therapy for anaplastic thyroid carcinoma has failed to improve overall survival. Toxicity rules out the high doses required, especially in older patients. The search for strategies to enhance paclitaxel antineoplastic activity and reduce its side effects is thus advisable. The study aimed to determine whether the histone deacetylase (HDAC) inhibitor valproic acid (VPA) improves the anticancer action of paclitaxel and elucidate the mechanisms underlying the effects of combined treatment. We examined the effect of VPA on the sensitivity to paclitaxel of two anaplastic thyroid carcinoma cell lines (CAL-62 and ARO), and the ability of the drug to determine tubulin acetylation and enhance paclitaxel-induced acetylation. The addition of as little as 0.7 mM VPA to paclitaxel enhances both cytostatic and cytotoxic effects of paclitaxel alone. Increased apoptosis explains the enhancement of the cytotoxic effect. The mechanism underlying this effect is through inhibition of HDAC6 activity, which leads to tubulin hyperacetylation. The results suggest a mechanistic link between HDAC6 inhibition, tubulin acetylation, and the VPA-induced enhancement of paclitaxel effects, and provide the rationale for designing future combination therapies.

Cytotoxic activity of the histone deacetylase inhibitor panobinostat (LBH589) in anaplastic thyroid cancer in vitro and in vivo.

Anaplastic thyroid carcinoma (ATC) has a rapidly fatal clinical course, being resistant to multimodal treatments. Microtubules, α/β tubulin heterodimers, are crucial in cell signaling, division and mitosis and are among the most successful targets for anticancer therapy. Panobinostat (LBH589) is a potent deacetylase inhibitor acting both on histones and nonhistonic proteins, including α‐tubulin. In vitro LBH589, evaluated in three ATC cell lines (BHT‐101, CAL‐62 and 8305C), resulted in impairment of cell viability, inhibition of colony formation, cell cycle arrest and apoptosis induction. Mechanistically, we showed that LBH589 not only affected the expression of p21 and cyclin D1, but markedly determined microtubule stabilization as evidenced by tubulin acetylation and increased tubulin polymerization. In a SCID xenograft model implanted with CAL‐62 cells, the cytotoxic properties of LBH589 were confirmed. The drug at the dose of 20 mg/kg significantly impaired tumor growth (final tumor volume 2.5‐fold smaller than in untreated animals); at this dose, no relevant side effects were observed. In tumors of treated animals, a significant reduction of Ki67, which was negatively correlated with tubulin acetylation, was observed. Moreover, acetyl‐tubulin levels negatively correlated with tumor volume at sacrifice, reinforcing the opinion that tubulin acetylation has a role in the inhibition of tumor growth. In conclusion, LBH589, acting on both histones and nonhistonic proteins in anaplastic thyroid cancer, appears to be a promising therapeutic agent for the treatment of this kind of cancer which is known not to respond to conventional therapy.

Histone Deacetylase Inhibition Modulates E-Cadherin Expression and Suppresses Migration and Invasion of Anaplastic Thyroid Cancer Cells

CONTEXT Anaplastic thyroid cancer cells are characterized by a mesenchymal phenotype, as revealed by spindle-shaped cells and absent or reduced levels of E-cadherin. Epigenetic silencing is considered one of the leading mechanisms of E-cadherin impairment, which causes the acquisition of the invasive and metastatic phenotype of anaplastic thyroid cancer. OBJECTIVES In this study we investigated the effects of histone deacetylase inhibition on E-cadherin expression, cell motility, and invasion in anaplastic thyroid cancer cell cultures. DESIGN Three stabilized cell lines and primary cultures of anaplastic thyroid cancer were treated with various histone deacetylase inhibitors. After treatment, we evaluated histone acetylation by Western blotting and E-cadherin expression by RT-real time PCR. The proper localization of E-cadherin/β-catenin complex was assessed by immunofluorescence and Western blot. Transcription activity of β-catenin was measured by luciferase reporter gene and cyclin D1 expression. The effect on cell motility and invasion was studied both in vitro using scratch-wound and transwell invasion assays and in anaplastic thyroid carcinomas tumor xenografts in mice in vivo. RESULTS Histone deacetylase inhibition induced the E-cadherin expression and the proper membrane localization of the E-cadherin/β-catenin complex, leading to reduced cancer cell migration and invasion. CONCLUSIONS We here demonstrate an additional molecular mechanism for the anticancer effect of histone deacetylase inhibition. The antiinvasive effect in addition to the cytotoxic activity of histone deacetylase inhibitors opens up therapeutic perspectives for the anaplastic thyroid tumor that does not respond to conventional therapy.

Emerging molecular therapies of advanced thyroid cancer.

Advanced thyroid cancer refers to thyroid tumors which are resistant to conventional therapies and do not respond to radioiodine and comprises metastatic or recurrent differentiated cancers, poorly differentiated and anaplastic tumors. Progress in the knowledge of genetic/epigenetic alterations in thyroid cancer cells is rapidly offering several opportunities to develop new drugs directed to specific targets. Drugs currently proposed for molecular therapy include: (a) monoclonal antibodies; (b) kinase inhibitors; (c) anti-angiogenetic drugs; (d) proteasome inhibitors; (e) retinoic acid and PPAR-gamma ligands; (f) radionuclide therapy; (g) epigenetic drugs (deacetylase inhibitors and demethylating agents). The results of several phase II trials using molecular drugs look promising. None of the treated patients, however, had a complete response, and only a minority of them had a partial response. The review will focus especially on epigenetic therapy, whose goal is to target the chromatin in rapidly dividing tumor cells and potentially restore normal cell functions. Deacetylases inhibitors modulate both epigenetic and multiple non-epigenetic mechanisms; they are, thus, viewed as a promising class of anticancer drugs. Experimental data show that deacetylase inhibitors are effective against advanced thyroid cancer. However, since multiple pathways need to be inhibited in order to substantially affect thyroid cancer growth, it is likely that a significant increase in the response rate to treatment of advanced thyroid cancer will be achieved through combinatorial drug therapies. Actually, many pre-clinical and clinical studies evaluate the combination of either two epigenetic drugs or a non-epigenetic chemotherapeutic and an epigenetic drug, in the effort to increase response rates.

Histone Deacetylase Inhibition Affects Sodium Iodide Symporter Expression and Induces 131I Cytotoxicity in Anaplastic Thyroid Cancer Cells

BACKGROUND Anaplastic thyroid cancers (ATCs) represent only 1%-2% of all thyroid tumors, but they account for up to 50% of the mortality. Treatment of differentiated thyroid carcinomas is well standardized and the use of radioiodine represents an essential step; in contrast, there is no standardized therapeutic approach for anaplastic tumors and their prognosis is poor. The resistance of ATC to radioiodine treatment is principally due to the absence of expression of the sodium iodide symporter (NIS), mainly due to epigenetic silencing. The acetylation status of histones is involved in the epigenetic control of gene expression and is usually disrupted in advanced thyroid cancer. Histone deacetylase inhibitors have been demonstrated as potent anticancer drugs with several different effects on cell viability and differentiation. METHODS Stabilized ATC cell lines (BHT-101 and CAL-62) and primary cultures from patients who underwent thyroidectomy for ATC were treated with the histone deacetylase inhibitor LBH589. After treatment, we evaluated the expression and function of NIS. Gene expression was evaluated by real-time polymerase chain reaction (RT-PCR), NIS promoter activity was determined with a luciferase reporter assay, and protein expression was assessed through immunofluorescence. We tested the protein function by (125)I uptake and efflux experiments; finally the cytotoxic effect of (131)I was determined with a clonogenic assay. RESULTS Our results demonstrate that treatment with LBH589 leads to NIS RNA expression as shown by RT-PCR and luciferase assay, and to protein expression as determined by immunofluorescence in vitro and by immunohistochemistry in xenograft tumors. Moreover, (125)I uptake and efflux experiments show the correct protein function and iodine retention, which translate into cytotoxicity effects, as demonstrated by a clonogenic assay with (131)I. CONCLUSIONS This study supplies a new potential strategy for the treatment of ATC by modifying gene expression with the aim of inducing responsiveness towards radioiodine therapy.

Tumour necrosis factor alpha and oxidative stress as maintaining factors in the evolution of anorexia nervosa

OBJECTIVE: Aim of the study was to evaluate tumour necrosis factor α (TNF-α) axis and oxidative status in patients with anorexia nervosa (AN) seeking a possible correlation with both nutritional status and evolution of the disease. SUBJECTS AND METHODS: Thirty-nine consecutive women with AN and an age-matched healthy control group were studied. Patients were 26±9 yr, with a body mass index (BMI) of 13.9±2 kg/m2. TNF-α, its receptors TNF-R55 and TNF-R75, and oxidative status markers (selenium, ascorbic/dehydroascorbic acid, retinol, α-tocopherol, selenium-dependent gluthatione peroxidase, reduced/oxidated gluthatione) were measured. A correlation with both nutritional indexes (body weight, BMI, albumin, prealbumin, transferrin, lymphocyte count) and disease duration was investigated. Pearson’s correlation and unpaired Student’s t-test were used to compare patients and controls. RESULTS: TNF-α and oxidative status markers were significantly higher in patients than controls and TNF-α was directly related to dehydroascorbic acid (p<0.05). Both TNF-R55 and TNF-R75 were higher in patients with duration of disease longer than one year as compared to controls and patients with shorter duration. Receptors inversely correlated with BMI (p<0.05 and p<0.01) and directly with disease duration (p<0.05). Inverse correlation between disease duration and BMI was present (p<0.01). CONCLUSIONS: The study showed activation of TNF-α axis and oxidative stress in AN patients, as well as correlation between the two systems. Due to the correlation between TNF receptors and both BMI and disease duration, a possible role of pro-inflammatory cytokines in the evolution of the eating disorder is suggested.

The pan-DAC inhibitor LBH589 is a multi-functional agent in breast cancer cells: cytotoxic drug and inducer of sodium-iodide symporter (NIS)

New drugs with anti-tumor activity, also able to modify the expression of selected molecules, are under evaluation in breast cancer which is becoming resistant to conventional treatment, or in metastatic disease. The sodium-iodide symporter (NIS), which mediates iodide uptake into thyroid cells, and is the molecular basis of radioiodine imaging and therapy in thyroid cancer, is also expressed in a large portion of breast tumors. Since NIS expression in breast cancer is not sufficient for a significant iodide uptake, drugs able to induce its expression and correct function are under evaluation. In the present study, we report for the first time that the pan-deacetylase (DAC) inhibitor LBH589 (panobinostat) significantly induced NIS, both as mRNA and as protein, through the increase of NIS promoter activity, with the final consequence of obtaining a significant up-take of iodide in MCF7, T47D, and MDA-MB231 breast cancer cells. Moreover, we observed that LBH589 causes a significant reduction in cell viability of estrogen-sensitive and -insensitive breast cancer cells within nanomolar range. The anti-tumor effect of LBH589 is sustained by apoptosis induction and cell cycle arrest in G2/M. In conclusion, our data suggest that LBH589 might be a powerful tool in the management of breast cancer due to its multiple effects and support a potential application of LBH589 in the diagnosis and treatment of this disease.

Valproic Acid, a Histone Deacetylase Inhibitor, in Combination with Paclitaxel for Anaplastic Thyroid Cancer: Results of a Multicenter Randomized Controlled Phase II/III Trial

Anaplastic thyroid cancer (ATC) has a median survival less than 5 months and, to date, no effective therapy exists. Taxanes have recently been stated as the main drug treatment for ATC, and the histone deacetylase inhibitor valproic acid efficiently potentiates the effects of paclitaxel in vitro. Based on these data, this trial assessed the efficacy and safety of the combination of paclitaxel and valproic acid for the treatment of ATC. This was a randomized, controlled phase II/III trial, performed on 25 ATC patients across 5 centers in northwest Italy. The experimental arm received the combination of paclitaxel (80 mg/m2/weekly) and valproic acid (1,000 mg/day); the control arm received paclitaxel alone. Overall survival and disease progression, evaluated in terms of progression-free survival, were the primary outcomes. The secondary outcome was the pharmacokinetics of paclitaxel. The coadministration of valproic acid did not influence the pharmacokinetics of paclitaxel. Neither median survival nor median time to progression was statistically different in the two arms. Median survival of operated-on patients was significantly better than that of patients who were not operated on. The present trial demonstrates that the addition of valproic acid to paclitaxel has no effect on overall survival and disease progression of ATC patients. This trial is registered with EudraCT 2008-005221-11.