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.

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.

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/ß-catenin complex was assessed by immunofluorescence and Western blot. Transcription activity of ß-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/ß-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.

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/ß 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.

Effect of n-3 fatty acids on patients with advanced lung cancer: a double-blind, placebo-controlled study.

PUFA from fish oil appear to have anti-inflammatory and anti-oxidative effects and improve nutritional status in cancer patients. With this as background, the aim of the present study was to investigate the effect of EPA plus DHA on inflammatory condition, and oxidative and nutritional status in patients with lung cancer. In our multicentre, randomised, double-blind trial, thirty-three patients with a diagnosis of advanced inoperable non-small-cell lung cancer and undergoing chemotherapy were divided into two groups, receiving four capsules/d containing 510 mg of EPA and 340 mg of DHA, or 850 mg of placebo, for 66 d. At the start of chemotherapy (T0), after 8 d (T1), 22 d (T2) and 66 d (T3), biochemical (inflammatory and oxidative status parameters) and anthropometric parameters were measured in both groups. A significant increase of body weight in the n-3 group at T3 v. T0 was observed. Concerning inflammation, C-reactive protein and IL-6 levels differed significantly between the n-3 and placebo groups at T3, and progressively decreased during chemotherapy in the n-3 group, evidencing n-3 PUFA anti-inflammatory action. Concerning oxidative status, plasma reactive oxygen species levels increased in the placebo group v. the n-3 group at the later treatment times. Hydroxynonenal levels increased in the placebo group during the study, while they stabilised in the n-3 group. Our data confirm that the continual assumption of EPA plus DHA determined an anti-inflammatory and anti-oxidative action which could be considered a preliminary goal in anti-cachectic 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.

Effects of the histone deacetylase inhibitor valproic acid on the sensitivity of anaplastic thyroid cancer cell lines to imatinib.

New therapeutic approaches are mandatory for anaplastic thyroid cancer. We investigated the ability of a new combined treatment using valproic acid (VPA), the only clinically available histone deacetylase inhibitor, and the tyrosine-kinase inhibitor imatinib mesylate to control the cell growth of anaplastic thyroid cancer cell lines. We showed that treatment with imatinib alone is unable to affect the cell growth of anaplastic thyroid cancer cells, whereas in ARO cells, the combined treatment resulted in a cytostatic effect, with clinically achievable doses of imatinib and VPA. The effect is mediated by G1 growth arrest, acting through p21 expression and the impairment of AKT phosphorylation.

Pro-inflammatory cytokines and oxidative stress/antioxidant parameters characterize the bio-humoral profile of early cachexia in lung cancer patients.

Cancer-related cachexia, that is present in about 50% of cancer patients and accounts for 20% of all cancer deaths, is clinically characterized by progressive weight loss, anorexia, metabolic alterations, asthenia, depletion of lipid stores and severe loss of skeletal muscle proteins. The main biochemical and molecular alterations that are responsible for the syndrome are prematurely present in the progress of the disease and the identification of the early stages of cachexia can be useful in targetting patients who will benefit from early treatment. The aim of the present study was to delineate the bio-humoral profile of a group of lung cancer patients either non-cachectic or cachectic by evaluating serum pro-inflammatory cytokines and oxidative stress/antioxidant parameters (both recognized to be involved in cachexia pathogenesis) and pro-inflammatory cytokine gene expression in PBMC (Peripheral blood mononuclear cells) of cancer patients. All serum pro-inflammatory cytokines and oxidative stress/antioxidant parameters significantly increased in neoplastic patients, but only TNF-alpha, ROS, GSH and vitamin E showed a significantly greater increase in cachectic patients. Pro-inflammatory cytokine gene expression mirrored serum level behaviour except for IL-6 that was increased in serum but not as gene expression, suggesting its provenience from tumour tissue. Our data support that the simultaneous determination of ROS, GSH, vitamin E, together with TNF-alpha allows the identification of a lung cancer patient developing cancer-related cachexia. This bio-humoral profile should be used for the early diagnosis and follow-up of the syndrome. Moreover, the evaluation of gene expression in patient PBMC was helpful in differentiating tumour vs host factors, therefore being useful in the study of pathogenetic mechanisms in neoplastic cachectic patients.

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.

Valproic acid, a histone deacetylase inhibitor, enhances sensitivity to doxorubicin in anaplastic thyroid cancer cells.

Multimodality treatments (i.e. surgery, chemotherapy, and radiotherapy) are recommended for anaplastic thyroid carcinoma (ATC), an extremely lethal human cancer, but to date there is little evidence that such approaches improve survival rates. It is thus necessary to seek new therapeutic tools. Histone deacetylase (HDAC) inhibitors are a promising class of anti-neoplastic agents that induce differentiation and apoptosis. Moreover, they may enhance the cytotoxicity of drugs targeting DNA through acetylation of histones. Using two ATC cell lines (CAL-62 and ARO), we show here that valproic acid (VPA), a clinically available HDAC inhibitor, enhances the activity of doxorubicin, whose anti-tumor properties involve binding to DNA and inhibiting topoisomerase II. A meager 0.7 mM VPA, which corresponds to serum concentrations in patients treated for epilepsy, is able to increase the cytotoxicity of doxorubicin about threefold in CAL-62 cells and twofold in ARO cells. The sensitizing effect, which is through histone acetylation, involves increased apoptosis, which is also shown by the increased caspase 3 activation and the enhancement of doxorubicin-induced G2 cell cycle arrest. These results might offer a rationale for clinical studies of a new combined therapy in an effort to improve the outcome of patients with anaplastic thyroid cancer.

Valproic acid induces apoptosis and cell cycle arrest in poorly differentiated thyroid cancer cells.

Poorly differentiated thyroid carcinoma is an aggressive human cancer that is resistant to conventional therapy. Histone deacetylase inhibitors are a promising class of drugs, acting as antiproliferative agents by promoting differentiation, as well as inducing apoptosis and cell cycle arrest. Valproic acid (VPA), a class I selective histone deacetylase inhibitor widely used as an anticonvulsant, promotes differentiation in poorly differentiated thyroid cancer cells by inducing Na(+)/I(-) symporter and increasing iodine uptake. Here, we show that it is also highly effective at suppressing growth in poorly differentiated thyroid cancer cell lines (N-PA and BHT-101). Apoptosis induction and cell cycle arrest are the underlying mechanisms of VPA's effect on cell growth. It induces apoptosis by activating the intrinsic pathway; caspases 3 and 9 are activated but not caspase 8. Cell cycle is selectively arrested in G(1) and is associated with the increased expression of p21 and the reduced expression of cyclin A. Both apoptosis and cell cycle arrest are induced by treatment with 1 mm VPA, a dose that promotes cell redifferentiation and that is slightly above the serum concentration reached in patients treated for epilepsy. These multifaceted properties make VPA of clinical interest as a new approach to treating poorly differentiated thyroid cancer.