Targeted inhibition of ubiquitin signaling reverses metabolic reprogramming and suppresses glioblastoma growth.

Glioblastoma multiforme (GBM) is the most frequent and aggressive form of primary brain tumor in the adult population; its high recurrence rate and resistance to current therapeutics urgently demand a better therapy. Regulation of protein stability by the ubiquitin proteasome system (UPS) represents an important control mechanism of cell growth. UPS deregulation is mechanistically linked to the development and progression of a variety of human cancers, including GBM. Thus, the UPS represents a potentially valuable target for GBM treatment. Using an integrated approach that includes proteomics, transcriptomics and metabolic profiling, we identify praja2, a RING E3 ubiquitin ligase, as the key component of a signaling network that regulates GBM cell growth and metabolism. Praja2 is preferentially expressed in primary GBM lesions expressing the wild-type isocitrate dehydrogenase 1 gene (IDH1). Mechanistically, we found that praja2 ubiquitylates and degrades the kinase suppressor of Ras 2 (KSR2). As a consequence, praja2 restrains the activity of downstream AMP-dependent protein kinase in GBM cells and attenuates the oxidative metabolism. Delivery in the brain of siRNA targeting praja2 by transferrin-targeted self-assembling nanoparticles (SANPs) prevented KSR2 degradation and inhibited GBM growth, reducing the size of the tumor and prolonging the survival rate of treated mice. These data identify praja2 as an essential regulator of cancer cell metabolism, and as a potential therapeutic target to suppress GBM growth.

Ageritin-The Ribotoxin-like Protein from Poplar Mushroom (Cyclocybe aegerita) Sensitizes Primary Glioblastoma Cells to Conventional Temozolomide Chemotherapy.

Here, we propose Ageritin, the prototype of the ribotoxin-like protein family, as an adjuvant treatment to control the growth of NULU and ZAR, two primary human glioblastoma cell lines, which exhibit a pharmacoresistance phenotype. Ageritin is able to inhibit NULU and ZAR growth with an IC50 of 0.53 ± 0.29 µM and 0.42 ± 0.49 µM, respectively. In this study, Ageritin treatment highlighted a macroscopic genotoxic response through the formation of micronuclei, which represents the morphological manifestation of genomic chaos induced by this toxin. DNA damage was not associated with either the deregulation of DNA repair enzymes (i.e., ATM and DNA-PK), as demonstrated by quantitative PCR, or reactive oxygen species. Indeed, the pretreatment of the most responsive cell line ZAR with the ROS scavenger N-acetylcysteine (NAC) did not follow the reverse cytotoxic effect of Ageritin, suggesting that this protein is not involved in cellular oxidative stress. Vice versa, Ageritin pretreatment strongly enhanced the sensitivity to temozolomide (TMZ) and inhibited MGMT protein expression, restoring the sensitivity to temozolomide. Overall, Ageritin could be considered as a possible innovative glioblastoma treatment, directly damaging DNA and downregulating the MGMT DNA repair protein. Finally, we verified the proteolysis susceptibility of Ageritin using an in vitro digestion system, and considered the future perspective use of this toxin as a bioconjugate in biomedicine.

Inhibiting effect of p-Coumaric acid on U87MG human glioblastoma cell growth.

p-Coumaric acid (pCA) is a hydroxycinnamic acid derivative commonly found in many natural products that has been extensively studied for its anticancer activity in multiple cell lines. In this report we investigated the effects of this phytochemical as adjuvant therapy to treat glioblastoma, an infaust brain tumour characterized by the acquired or innate resistance to the conventional chemotherapy temozolomide (TMZ). U87Mg glioblastoma cell growth and viability was assessed by growth rate curves and MTT assay incubating cells with 0.5 and 1 mM pCA for 24 h, 48 h and 72 h. Cell cycle analysis, performed by flow cytometry, showed that pCA led the accumulation of GBM cells in G2/M phase. Western blot analysis shows that pCA induced CDK4 cyclin-dependent kinase reduction and p53 increase, followed by induction of the CDK inhibitor p21. Furthermore, pCA treatment mediated the activation of apoptosis and the inhibition of migration of U87Mg cells. Finally, the treatment of glioblastoma cells in vitro with pCA concomitantly with the TMZ revealed a synergistic effect between the natural substance and the chemotherapy. In conclusion, our results demonstrated that pCA acts influencing the cell viability and cell cycle of U87Mg cells by promoting cell cycle arrest in G2/M phase and apoptosis.

Cytotoxicity Effect of Quinoin, Type 1 Ribosome-Inactivating Protein from Quinoa Seeds, on Glioblastoma Cells.

Ribosome-inactivating proteins (RIPs) are found in several edible plants and are well characterized. Many studies highlight their use in cancer therapy, alone or as immunoconjugates, linked to monoclonal antibodies directed against target cancer cells. In this context, we investigate the cytotoxicity of quinoin, a novel type 1 RIP from quinoa seeds, on human continuous and primary glioblastoma cell lines. The cytotoxic effect of quinoin was assayed on human continuous glioblastoma U87Mg cells. Moreover, considering that common conventional glioblastoma multiforme (GBM) cell lines are genetically different from the tumors from which they derive, the cytotoxicity of quinoin was subsequently tested towards primary cells NULU and ZAR (two cell lines established from patients' gliomas), also in combination with the chemotherapeutic agent temozolomide (TMZ), currently used in glioblastoma treatment. The present study demonstrated that quinoin (2.5 and 5.0 nM) strongly reduced glioblastoma cells' growth. The mechanisms responsible for the inhibitory action of quinoin are different in the tested primary cell lines, reproducing the heterogeneous response of glioblastoma cells. Interestingly, primary cells treated with quinoin in combination with TMZ were more sensitive to the treatment. Overall, our data highlight that quinoin could represent a novel tool for glioblastoma therapy and a possible adjuvant for the treatment of the disease in combination with TMZ, alone or as possible immunoconjugates/nanoconstructs.

Gymnema sylvestre Extract Restores the Autophagic Pathway in Human Glioblastoma Cells U87Mg.

Glioblastoma is a brain tumour, characterised by recurrent or innate resistance to conventional chemoradiotherapy. Novel natural molecules and phyto-extracts have been proposed as adjuvants to sensitise the response to Temozolomide (TMZ). In this study, we investigated the effect of GS extract on human glioblastoma cells U87Mg. According to the IC50-values, GS extract displayed a significant cytotoxicity. This was confirmed by cell growth inhibition and alteration in metabolic activity evaluated by cell count and MTT assay. GS induced reduction in Pro-caspase 9, 3, but not PARP cleavage nor DNA fragmentation. Thus, in GS-induced cytotoxicity, cell death is not associated with apoptosis. In this context, short-term treatment of U87Mg cells with GS extract (1 mg/mL) reduced the phosphorylation levels of mTOR and of its downstream target P70 S6 kinase, highlighting the role of GS extract into autophagy induction. The activation of autophagic flux by GS extract was confirmed by Western blot analysis, which revealed the reduction in p62 and the concomitant increase in LC3B II/I ratio. Immunofluorescence evidenced the accumulation of LC3B puncta in U87Mg cells pretreated with autophagy inhibitor Bafilomycin A1. Furthermore, as main key regulators of type II programmed cell death, p53, p21 and CDK4 were also investigated and were inhibited by GS treatment. In conclusion, GS extract could be considered as an autophagy inducer in glioblastoma cells U87Mg.

Characterization of primary glioma cell lines derived from the patients according to 2016 CNS tumour WHO classification and comparison with their parental tumours.

BACKGROUND: Gliomas represent about 80% of primary brain tumours and about 30% of malignant ones, which today don't have a resolution therapy because of their variability. A valid model for the study of new personalized therapies can be represented by primary cultures from patient's tumour biopsies. METHODS: In this study we consider 12 novel cell lines established from patients' gliomas and immunohistochemically and molecularly characterized according to the newly updated 2016 CNS Tumour WHO classification. RESULTS: Eight of these lines were glioblastoma cells, two grade III glioma cells (anaplastic astrocytoma and oligo astrocytoma) and two low grade glioma cells (grade II astrocytoma and oligodendroglioma). All cell lines were analysed by immunohistochemistry for specific glioma markers, respectively VIMENTIN, GFAP, IDH1R132, and ATRX. The methylation status of the MGMT gene promoter was also determined in all lines. The comparison of the immunohistochemical characteristics and of the MGMT methylation status of the lines with the tissues of origin shows that the cells in culture maintain the same characteristics. CONCLUSIONS: Human cancer cell lines represent a support in the knowledge of tumour biology and in drug discovery through its facile experimental manipulation. TRIAL REGISTRATION: NCT04180046.

Implication of Lactucopicrin in Autophagy, Cell Cycle Arrest and Oxidative Stress to Inhibit U87Mg Glioblastoma Cell Growth.

In this study, we propose lactucopicrin (LCTP), a natural sesquiterpene lactone from Lactucavirosa, as a molecule able to control the growth of glioblastoma continuous cell line U87Mg. The IC50 of U87Mg against LCTP revealed a strong cytotoxic effect. Daily administration of LCTP showed a dose and time-dependent reduction of GBM cell growth and viability, also confirmed by inhibition of clonogenic potential and mobility of U87Mg cells. LCTP activated autophagy in U87Mg cells and decreased the phosphorylation of proliferative signals pAKT and pERK. LCTP also induced the cell cycle arrest in G2/M phase, confirmed by decrease of CDK2 protein and increase of p53 and p21. LCTP stimulated apoptosis as evidenced by reduction of procaspase 6 and the increase of the cleaved/full-length PARP ratio. The pre-treatment of U87Mg cells with ROS scavenger N-acetylcysteine (NAC), which reversed its cytotoxic effect, showed the involvement of LCTP in oxidative stress. Finally, LCTP strongly enhanced the sensitivity of U87Mg cells to canonical therapy Temozolomide (TMZ) and synergized with this drug. Altogether, the growth inhibition of U87Mg GBM cells induced by LCTP is the result of several synergic mechanisms, which makes LCTP a promising adjuvant therapy for this complex pathology.

Stimulation of S1PR5 with A-971432, a selective agonist, preserves blood-brain barrier integrity and exerts therapeutic effect in an animal model of Huntington's disease.

Huntington's disease (HD) is the most common neurodegenerative disorder for which no effective cure is yet available. Although several agents have been identified to provide benefits so far, the number of therapeutic options remains limited with only symptomatic treatment available. Over the past few years, we have demonstrated that sphingolipid-based approaches may open the door to new and more targeted treatments for the disease. In this study, we investigated the therapeutic potential of stimulating sphingosine-1-phosphate (S1P) receptor 5 by the new selective agonist A-971432 (provided by AbbVie) in R6/2 mice, a widely used HD animal model. Chronic administration of low-dose (0.1 mg/kg) A-971432 slowed down the progression of the disease and significantly prolonged lifespan in symptomatic R6/2 mice. Such beneficial effects were associated with activation of pro-survival pathways (BDNF, AKT and ERK) and with reduction of mutant huntingtin aggregation. A-971432 also protected blood-brain barrier (BBB) homeostasis in the same mice. Interestingly, when administered early in the disease, before any overt symptoms, A-971432 completely protected HD mice from the classic progressive motor deficit and preserved BBB integrity. Beside representing a promising strategy to take into consideration for the development of alternative therapeutic options for HD, selective stimulation of S1P receptor 5 may be also seen as an effective approach to target brain vasculature defects in the disease.