ABSTRACT
Malignant mesothelioma (MM) is a highly aggressive tumor of the serous membranes for which there is currently no effective curative modality. Recent data suggest that hyperactivation of the tyrosine kinase SRC has a key role in MM development and therefore this kinase represents an important molecular target for MM therapy. We tested new pyrazolo[3,4-d]pyrimidine SRC inhibitors on a panel of MM cell lines expressing the active form of SRC. These SRC inhibitors exerted a significant proapoptotic effect on MM cells without affecting the normal mesothelial cell line MET-5A, supporting a possible use of these SRC inhibitors for a safe treatment of MM. We also showed that SRC inhibitor-induced apoptosis occurred concomitantly with an increase in the nuclear stability of the cyclin-dependent kinase inhibitor p27. This finding is remarkable considering that loss of nuclear p27 expression is a well-established adverse prognostic factor in MM, and p27 nuclear localization is crucial for its tumor-suppressive function. Consistently, SRC inhibition seems to promote the increase in p27 nuclear level also by inactivating the AKT kinase and downregulating cyclin D1, which would otherwise delay p27 nuclear import and provoke its cytoplasmic accumulation. To determine whether p27 stabilization has a direct role in apoptosis induced by SRC inhibition, we stably silenced the CDKN1B gene, encoding p27, in MSTO-211H and REN mesothelioma cells by transduction with lentiviral vectors expressing short hairpin RNAs against the CDKN1B transcript. Strikingly, p27 silencing was able to suppress the apoptosis induced by these SRC inhibitors in both MM cell lines, suggesting that p27 has a crucial proapoptotic role in MM cells treated with SRC inhibitors. Our findings reveal a new mechanism, dependent on p27 nuclear stabilization, by which SRC inhibition can induce apoptosis in MM cells and provide a new rationale for the use of SRC inhibitors in MM therapy.
Subject(s)
Apoptosis/drug effects , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Pyrazoles/pharmacology , Pyrimidines/pharmacology , src-Family Kinases/antagonists & inhibitors , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Blotting, Western , Cell Cycle/drug effects , Cell Line , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Survival/drug effects , Cyclin D1/metabolism , Cyclin-Dependent Kinase Inhibitor p27/genetics , Dose-Response Relationship, Drug , Enzyme Stability/drug effects , Flow Cytometry , Humans , Mesothelioma/genetics , Mesothelioma/metabolism , Mesothelioma/pathology , Molecular Structure , Proto-Oncogene Proteins c-akt/metabolism , Pyrazoles/chemistry , Pyrimidines/chemistry , RNA InterferenceABSTRACT
BACKGROUND: Although influenza vaccination is widely recommended for immunosuppressed people, the same immune dysfunction that can increase the risk of contracting influenza might also compromise vaccine effectiveness, especially during pandemics. Clinical data have highlighted the role of adjuvants in improving vaccine efficacy. As uremic patients are especially vulnerable to infections, it is recommended that they should be vaccinated yearly against influenza. This paper presents the results of a pilot clinical trial, conducted in hemodialyzed patients with an adjuvated pandemic H1N1v influenza vaccine alone and combined with Thymosin-alpha 1. METHODS: Subjects were subdivided into 3 treatment groups receiving: the adjuvated pandemic influenza vaccine (Focetria) only (first treatment group), and the Vaccine+Thymosin alpha 1 (Zadaxin) at a dose of 3.2 and 6.4 mg (second and third treatment groups respectively). The immunoresponse was assessed on days 0, 21, 42, 84 and 168 after vaccine administration by means of Hemagglutination Inhibition (HI), Microneutralization (MN) and Single Radial Hemolysis (SRH) assays. The CHMP regards HI as the gold standard test to evaluate the immune response to influenza vaccines before influenza vaccines are licensed. The CHMP criteria are slightly different in adults (18-60-year-old subjects) and the elderly (>60 years old). Indeed, 40% of seroconversion, 70% of subjects seroprotected 21 days after vaccination, and a 2.5-fold increase in GMR (Geometric Mean Ratio) are required in adults, while in the elderly, the corresponding threshold values are: 30%, 60% and a 2-fold increase. All these criteria must be met for the licensing of a pandemic influenza vaccine. Safety evaluation was performed by means of Adverse Event (AE) recording, laboratory assays (hematology and chemistry), electrocardiogram, and assessment of vital signs. RESULTS: Three populations were considered: Intention-To-Treat (ITT) (94 patients), Per Protocol (PP) (82 patients), and Safety population (99 patients). With regard to the Geometric Mean Titer (GMT) and the Geometric Mean Ratio (GMR) of HI on Day 21 in the ITT population, both "Vaccine+Thymosin alpha 1" groups presented better results than the "Vaccine only" group. A large proportion of ITT patients in the two Vaccine+Thymosin alpha 1 groups achieved seroconversion by Day 21. On Day 42, the decrease in the GMT of HI was greater in the Vaccine+Thymosin alpha 1 groups than in the vaccine only group. Similar results were obtained in the PP population. The CHMP criteria were fully met in the groups treated with Vaccine+Thymosin alpha 1. No AE was found to be related to Thymosin alpha 1 nor to the Focetria vaccine. CONCLUSIONS: Although further studies in larger hemodialyzed populations are necessary, it can be concluded that Thymosin alpha 1 enhanced the immunogenicity of the pandemic influenza vaccine used. Moreover, it proved safe and well tolerated, and did not affect hematology or blood-chemistry values.
