Background: Excess body adipose tissue accumulation is a common and growing health problem caused by an unbalanced diet and/or junk food. Although the effects of dietary fat and glucose on lipid metabolism regulation are well known, those of essential amino acids (EAAs) have been poorly investigated. Our aim was to study the influence of a special diet containing all EAAs on retroperitoneal white adipose tissue (rpWAT) and interscapular brown adipose tissue (BAT) of mice. Methods: Two groups of male Balb/C mice were used. The first was fed with a standard diet. The second was fed with an EAAs-rich diet (EAARD). After 3 weeks, rpWAT and BAT were removed and prepared for subsequent immunohistochemical analysis. Results: EAARD, although consumed significantly less, moderately reduced body weight and BAT, but caused a massive reduction in rpWAT. Conversely, the triceps muscle increased in mass. In rpWAT, the size of adipocytes was very small, with increases in leptin, adiponectin and IL-6 immunostaining. In BAT, there was a reduction in lipid droplet size and a simultaneous increase in UCP-1 and SIRT-3. Conclusions: A diet containing a balanced mixture of free EAA may modulate body adiposity in mice, promoting increased thermogenesis.

Identifying the molecular mechanisms underlying radioresistance is a priority for the treatment of RMS, a myogenic tumor accounting for approximately 50% of all pediatric soft tissue sarcomas. We found that irradiation (IR) transiently increased phosphorylation of Akt1, Src, and Cav1 in human RD and RH30 lines. Synthetic inhibition of Akt1 and Src phosphorylation increased ROS levels in all RMS lines, promoting cellular radiosensitization. Accordingly, the elevated activation of the Akt1/Src/Cav1 pathway, as detected in two RD lines characterized by overexpression of a myristoylated Akt1 form (myrAkt1) or Cav1 (RDCav1), was correlated with reduced levels of ROS, higher expression of catalase, and increased radioresistance. We found that treatment with cholesterol-lowering drugs such as lovastatin and simvastatin promoted cell apoptosis in all RMS lines by reducing Akt1 and Cav1 levels and increasing intracellular ROS levels. Combining statins with IR significantly increased DNA damage and cell apoptosis as assessed by γ histone 2AX (γH2AX) staining and FACS analysis. Furthermore, in combination with the chemotherapeutic agent actinomycin D, statins were effective in reducing cell survival through increased apoptosis. Taken together, our findings suggest that the molecularly linked signature formed by Akt1, Src, Cav1, and catalase may represent a prognostic determinant for identifying subgroups of RMS patients with higher probability of recurrence after radiotherapy. Furthermore, statin-induced oxidative stress could represent a treatment option to improve the success of radiotherapy.

The aim of the present study was to investigate the effects of targeting the mevalonate pathway (MVP) in rhabdomyosarcoma (RMS), a soft tissue tumor with a prevalence in young people. In silico analyses of RNA datasets were performed to correlate MVP with RMS patient survival. The sensitivity of RMS cell lines to MVP inhibitors was assessed in vitro by analysis of cell growth (crystal violet and clonogenic assays), cell migration (wound healing assay), cell survival (neutral red assay), and oxidative stress (ROS assay). The effects of MVP inhibitors were tested in vivo by analyzing RMS xenografts grown in NOD/SCID mice. Quantification of protein targets was performed using immunoblotting or immunohistochemistry analyses. In silico analysis showed upregulation of sterol regulatory element-binding protein 2 (SREBP2) and MVP genes, including 3-Hydroxy-3-Methylglutaryl-CoA Reductase (HMGCR), farnesyl-diphosphate synthase (FDPS), squalene epoxidase (SQLE), which correlated with worse overall patient survival. Targeting of MVP in human RD and RH30 lines by inhibitors of SREBP2 (fatostatin), HMGCR (lovastatin and simvastatin), and FDPS (zoledronic acid) resulted in impaired cell growth, migration, and viability, and increased oxidative cell death in combination with actinomycin D. Conversely, cholesterol (CHO) supplementation enhanced cell growth and migration. Fatostatin and lovastatin produced rapid attenuation of Erk1/2 and Akt1 signaling in RMS lines, and oral administration of lovastatin reduced tumor mass growth of xenografted RD cells in NOD/SCID mice. Finally, we found that forced Akt1 activation in RD cells was sufficient to drive SREBP2, HMGCR and SQLE protein expression, promoting increased susceptibility to MVP inhibitors. These data suggest that the Akt1, SREBP2 and MVP axis is critical for RMS tumor growth, migration, and oxidative stress protection primarily through maintaining adequate CHO levels that enable proper intracellular signaling. Therefore, stimulating CHO depletion via SREBP2 and MVP inhibition may represent a viable option to improve the combination therapy protocol, especially in pAkt1-positive RMS.

The metabolic hyperactivity of tumor cells demands a substantial amount of energy and molecules to build new cells and expand the tumor, diverting these resources from healthy cells. Amino acids (AAs) are the only totipotent and essential molecules for protein construction. Previous in vitro studies in human and murine cancer cells, along with in vivo studies in mice, have shown that an excess of essential amino acids (EAAs) exerts an inhibitory effect on tumor proliferation by promoting apoptosis and autophagy. In this study, both in vitro and in vivo, we evaluated whether a mixture based on EAA can influence the development of human colon cancer (HCT116). To this end, in vitro, we assessed the proliferation of HCT116 cells treated with a special mix of EAA. In vivo, immunosuppressed athymic nude mice, injected with HCT116 cells subcutaneously (s.c.) or intraperitoneally (i.p.), were given a modified EAAs-rich diet (EAARD) compared to the standard laboratory diet (StD). In vitro data showed that the EAA mix impairs cancer growth by inducing apoptosis and autophagy. In vivo, the results demonstrated that EAARD-fed mice developed s.c. tumors significantly smaller than those of StD-fed mice (total mass 3.24 vs. 6.09 g, respectively). Mice injected i.p. and fed with EAARD showed a smaller and more limited number of intra-peritoneal tumors than StD-fed mice (total mass 0.79 vs. 4.77 g, respectively). EAAs prevents the growth of HCT116 cells by inducing autophagy and apoptosis, increasing endoplasmic reticulum stress, and inhibiting inflammation and neo-vascularization. In addition, the EAARD-fed mice, maintained muscle mass and white and brown adipose tissues. A diet with an excess of EAAs affects the survival and proliferative capacity of human colon cancer cells, maintaining anabolic stimuli in muscular cells.

Rhabdomyosarcoma (RMS), the most common form of sarcoma typical of pediatric age, arises from the malignant transformation of the mesenchymal precursors that fail to differentiate into skeletal muscle cells. Here, we investigated whether the protein phospholipase C δ4 (PLCδ4), a member of the PLC family involved in proliferation and senescence mechanisms of mesenchymal stromal stem cells, may play a role in RMS. Our molecular and morpho-functional data reveal that PLCδ4 is highly expressed in the fusion-negative, p53-positive, SMARCB1 heterozygous mutated embryonal RMS (ERMS) cell line A204, while it is poorly expressed in the ERMS cell lines RD (fusion-negative, MYC amplification, N-RAS (Q61H), homozygous mutated p53) and Hs729 (homozygous mutated p53) and the alveolar rhabdosarcoma (ARMS) cell line SJCRH30 (RH30; fusion positive, heterozygous mutated RARA, polyheterozygous mutated p53). To characterize the role of PLCδ4, the RD cell line was stably transfected with wild-type PLCδ4 (RD/PLCδ4). Overexpressed PLCδ4 mainly localized to the nucleus in RD cells and contributed to the phosphorylation of PRAS40 (T246), Chk2(T68), WNK1(T60), and Akt 1/273 (S473), as revealed by proteome profiler array analysis. Overexpression of PLCδ4 in RD cells enhanced cyclin B1 expression and resulted in G2/M-phase cell cycle arrest. In contrast, siRNA-mediated knockdown of PLCδ4 in A204 cells resulted in reduced cyclin B1 expression. Our study identifies a novel role for nuclear PLCδ4 as a regulator of cyclin B1 via Akt-dependent phosphorylation. The modulation of PLCδ4 expression and its downstream targets could represent a crucial signaling pathway to block embryonal RMS cell proliferation.