F. Marampon

Testosterone insulin-like effects: an in vitro study on the short-term metabolic effects of testosterone in human skeletal muscle cells

PurposeTestosterone by promoting different metabolic pathways contributes to short-term homeostasis of skeletal muscle, the largest insulin-sensitive tissue and the primary site for insulin-stimulated glucose utilization. Despite evidences indicate a close relationship between testosterone and glucose metabolism, the molecular mechanisms responsible for a possible testosterone-mediated insulin-like effects on skeletal muscle are still unknown.MethodsHere we used undifferentiated proliferating or differentiated human fetal skeletal muscle cells (Hfsmc) to investigate the short-term effects of testosterone on the insulin-mediated biomolecular metabolic machinery. GLUT4 cell expression, localization and the phosphorylation/activation of AKT, ERK, mTOR and GSK3β insulin-related pathways at different time points after treatment with testosterone were analyzed.ResultsIndependently from cells differentiation status, testosterone, with an insulin-like effect, induced Glut4-mRNA expression, GLUT4 protein translocation to the cytoplasmic membrane, while no effect was observed on GLUT4 protein expression levels. Furthermore, testosterone treatment modulated the insulin-dependent signal transduction pathways inducing a rapid and persistent activation of AKT, ERK and mTOR, and a transient inhibition of GSK3β. T-related effects were shown to be androgen receptor dependent.ConclusionAll together our data indicate that testosterone through the activation of non-genomic pathways, participates in skeletal muscle glucose metabolism by inducing insulin-related effects.

The phosphodiesterase 5 inhibitor tadalafil regulates lipidic homeostasis in human skeletal muscle cell metabolism

PurposeTadalafil seems to ameliorate insulin resistance and glucose homeostasis in humans. We have previously reported that tadalafil targets human skeletal muscle cells with an insulin (I)-like effect. We aim to evaluate in human fetal skeletal muscle cells after tadalafil or I: (i) expression profile of I-regulated genes dedicated to cellular energy control, glycolitic activity or microtubule formation/vesicle transport, as GLUT4, PPARγ, HK2, IRS-1, KIF1C, and KIFAP3; (ii) GLUT4, Flotillin-1, and Caveolin-1 localization, all proteins involved in energy-dependent cell trafficking; (iii) activation of I-targeted paths, as IRS-1, PKB/AKT, mTOR, P70/S6K. Free fatty acids intracellular level was measured. Sildenafil or a cGMP synthetic analog were used for comparison; PDE5 and PDE11 gene expression was evaluated in human fetal skeletal muscle cells.MethodsRTq-PCR, PCR, western blot, free fatty acid assay commercial kit, and lipid stain non-fluorescent assay were used.ResultsTadalafil upregulated I-targeted investigated genes with the same temporal pattern as I (GLUT4, PPARγ, and IRS-1 at 3 h; HK2, KIF1C, KIFAP3 at 12 h), re-localized GLUT4 in cell sites positively immune-decorated for Caveolin-1 and Flotillin-1, suggesting the involvement of lipid rafts, induced specific residue phosphorylation of IRS-1/AKT/mTOR complex in association with free fatty acid de novo synthesis. Sildenafil or GMP analog did not affect GLUT4 trafficking or free fatty acid levels.ConclusionIn human fetal skeletal muscle cells tadalafil likely favors energy storage by modulating lipid homeostasis via IRS-1-mediated mechanisms, involving activation of I-targeted genes and intracellular cascade related to metabolic control. Those data provide some biomolecular evidences explaining, in part, tadalafil-induced favorable control of human metabolism shown by clinical studies.

Testosterone-mediated activation of androgenic signalling sustains in vitro the transformed and radioresistant phenotype of rhabdomyosarcoma cell lines

PurposeRhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in childhood, rarely affects adults, preferring male. RMS expresses the receptor for androgen (AR) and responds to androgen; however, the molecular action of androgens on RMS is unknown.MethodsHerein, testosterone (T) effects were tested in embryonal (ERMS) and alveolar (ARMS) RMS cell lines, by performing luciferase reporter assay, RT-PCR, and western blotting experiments. RNA interference experiments or bicalutamide treatment was performed to assess the specific role of AR. Radiation treatment was delivered to characterise the effects of T treatment on RMS intrinsic radioresistance.ResultsOur study showed that RMS cells respond to sub-physiological levels of T stimulation, finally promoting AR-dependent genomic and non-genomic effects, such as the transcriptional regulation of several oncogenes, the phosphorylation-mediated post-transductional modifications of AR and the activation of ERK, p38 and AKT signal transduction pathway mediators that, by physically complexing or not with AR, participate in regulating its transcriptional activity and the expression of T-targeted genes. T chronic daily treatment, performed as for the hormone circadian rhythm, did not significantly affect RMS cell growth, but improved RMS clonogenic and radioresistant potential and increased AR mRNA both in ERMS and ARMS. AR protein accumulation was evident in ERMS, this further developing an intrinsic T-independent AR activity.ConclusionsOur results suggest that androgens sustain and improve RMS transformed and radioresistant phenotype, and therefore, their therapeutic application should be avoided in RMS post puberal patients.

Disruption of MEK/ERK/c-Myc signaling radiosensitizes prostate cancer cells in vitro and in vivo

PurposeProstate cancer (PCa) cell radioresistance causes the failure of radiation therapy (RT) in localized or locally advanced disease. The aberrant accumulation of c-Myc oncoprotein, known to promote PCa onset and progression, may be due to the control of gene transcription and/or MEK/ERK-regulated protein stabilization. Here, we investigated the role of MEK/ERK signaling in PCa.MethodsLnCAP, 22Rv1, DU145, and PC3 PCa cell lines were used in in vitro and in vivo experiments. U0126, trametinib MEK/ERK inhibitors, and c-Myc shRNAs were used. Radiation was delivered using an x-6 MV photon linear accelerator. U0126 in vivo activity alone or in combination with irradiation was determined in murine xenografts.ResultsInhibition of MEK/ERK signaling down-regulated c-Myc protein in PCa cell lines to varying extents by affecting expression of RNA and protein, which in turn determined radiosensitization in in vitro and in vivo xenograft models of PCa cells. The crucial role played by c-Myc in the MEK/ERK pathways was demonstrated in 22Rv1 cells by the silencing of c-Myc by means of short hairpin mRNA, which yielded effects resembling the targeting of MEK/ERK signaling. The clinically approved compound trametinib used in vitro yielded the same effects as U0126 on growth and C-Myc expression. Notably, U0126 and trametinib induced a drastic down-regulation of BMX, which is known to prevent apoptosis in cancer cells.ConclusionsThe results of our study suggest that signal transduction-based therapy can, by disrupting the MEK/ERK/c-Myc axis, reduce human PCa radioresistance caused by increased c-Myc expression in vivo and in vitro and restores apoptosis signals.

EP-2062: c-Myc silencing impairs oncophenotype and radioresistance of Embrional Rhabdomyosarcoma Cell Lines

S973 ________________________________________________________________________________ Material and Methods: 118 patients with FIGO IB2-IVA st. were treated with RT-CT radical (Tab A). Anti-EGFR monoclonal Ac.(clone H11 Ref. M-3363 -Dako). The Immunoreactivity was based on semiquantitative analysis scored as the % of stained cells. Moderate/high EGFR staining (>31 to ≥70%, respectively) were considered (+). Anti -COX-2 monoclonal Ac. (clone CX-2949 Ref. M361 Dako). Moderate/high COX-2 staining (>31 to ≥70%, respectively) were considered (+). Pelvic radiotherapy in 21 patients with RT-3D, dose 46 Gy. In 97 cases (82%) was extended to the para-aortics, dose 45 Gy. A single application of LDR BCT was delivered on 51 pts (43.5%). Isotope: Cs-137. Dose to point A: 30 Gy. HDR BCT to 64 pts (54%) in 3 or 4 applications. Isotope: Ir-192; Microselectron®. Dose 30 Gy to point A (33 pts) and a dose of 7 Gy to CTV/application (31 pts). CT: CDDP: 40 mg/m2/ iv weekly.