Rnd3 Is a Crucial Mediator of the Invasive Phenotype of Glioblastoma Cells Downstream of Receptor Tyrosine Kinase Signalling.

Enhanced invasiveness is one of the defining biological traits of glioblastoma cells, which exhibit an infiltrative nature that severely hinders surgical resection. Among the molecular lesions responsible for GBM aggressiveness, aberrant receptor tyrosine kinase (RTK) signalling is well-characterised. Enhanced RTK signalling directly impacts a myriad of cellular pathways and downstream effectors, which include the Rho GTPase family, key regulators of actin cytoskeletal dynamics. Here, we have analysed the functional crosstalk between oncogenic signals emanating from RTKs and Rho GTPases and focused on the specific contribution of Rnd3 to the invasive phenotype of GBM in this context. We found that RTK inhibition with a panel of RTK inhibitors decreased cell motility and cell invasion and promoted dramatic actin cytoskeleton reorganisation through activation of the RhoA/Rho-associated protein kinase 1 (ROCK) axis. RTK inhibition also significantly decreased Rnd3 expression levels. Consistently, shRNA-mediated Rnd3 silencing revealed that Rnd3 depletion promoted substantial changes in the actin cytoskeleton and reduced cell motility and invasion capacity, recapitulating the effects observed upon RTK inhibition. Our results indicate that Rnd3 is a crucial mediator of RTK oncogenic signalling involved in actin cytoskeletal reorganisation, which contributes to determining the invasive phenotype of GBM cells.

Glioblastoma Embryonic-like Stem Cells Exhibit Immune-Evasive Phenotype.

BACKGROUND: Glioma stem cells (GSCs) have self-renewal and tumor-initiating capacities involved in drug resistance and immune evasion mechanisms in glioblastoma (GBM). METHODS: Core-GSCs (c-GSCs) were identified by selecting cells co-expressing high levels of embryonic stem cell (ESC) markers from a single-cell RNA-seq patient-derived GBM dataset (n = 28). Induced c-GSCs (ic-GSCs) were generated by reprogramming GBM-derived cells (GBM-DCs) using induced pluripotent stem cell (iPSC) technology. The characterization of ic-GSCs and GBM-DCs was conducted by immunostaining, transcriptomic, and DNA methylation (DNAm) analysis. RESULTS: We identified a GSC population (4.22% ± 0.59) exhibiting concurrent high expression of ESC markers and downregulation of immune-associated pathways, named c-GSCs. In vitro ic-GSCs presented high expression of ESC markers and downregulation of antigen presentation HLA proteins. Transcriptomic analysis revealed a strong agreement of enriched biological pathways between tumor c-GSCs and in vitro ic-GSCs (κ = 0.71). Integration of our epigenomic profiling with 833 functional ENCODE epigenetic maps identifies increased DNA methylation on HLA genes' regulatory regions associated with polycomb repressive marks in a stem-like phenotype. CONCLUSIONS: This study unravels glioblastoma immune-evasive mechanisms involving a c-GSC population. In addition, it provides a cellular model with paired gene expression, and DNA methylation maps to explore potential therapeutic complements for GBM immunotherapy.

Toward a Rational Design of Polyamine-Based Zinc-Chelating Agents for Cancer Therapies.

In vitro viability assays against a representative panel of human cancer cell lines revealed that polyamines L1a and L5a displayed remarkable activity with IC50 values in the micromolar range. Preliminary research indicated that both compounds promoted G1 cell cycle arrest followed by cellular senescence and apoptosis. The induction of apoptotic cell death involved loss of mitochondrial outer membrane permeability and activation of caspases 3/7. Interestingly, L1a and L5a failed to activate cellular DNA damage response. The high intracellular zinc-chelating capacity of both compounds, deduced from the metal-specific Zinquin assay and ZnL2+ stability constant values in solution, strongly supports their cytotoxicity. These data along with quantum mechanical studies have enabled to establish a precise structure-activity relationship. Moreover, L1a and L5a showed appropriate drug-likeness by in silico methods. Based on these promising results, L1a and L5a should be considered a new class of zinc-chelating anticancer agents that deserves further development.

The tumor suppressor FOXO3a mediates the response to EGFR inhibition in glioblastoma cells.

PURPOSE: Although EGFR activation is a hallmark of glioblastoma (GBM), anti-EGFR therapy has so far not yielded the desired effects. Targeting PI3K/Akt has been proposed as a strategy to increase the cellular sensitivity to EGFR inhibitors. Here we evaluated the contribution of FOXO3a, a key Akt target, in the response of GBM cells to EGFR inhibition. METHODS: FOXO3a activation was assessed by immunofluorescence and gene reporter assays, and by evaluating target gene expression using Western blotting and qRT-PCR. Cellular effects were evaluated using cell viability and apoptosis assays, i.e., Annexin V/PI staining and caspase 3/7 activity measurements. Drug synergism was evaluated by performing isobolographic analyses. Gene silencing experiments were performed using stable shRNA transfections. RESULTS: We found that EGFR inhibition in GBM cells led to FOXO3a activation and to transcriptional modulation of its key targets, including repression of the oncogene FOXM1. In addition, we found that specific FOXO3a activation recapitulated the molecular effects of EGFR inhibition, and that the FOXO3a activator trifluoperazine, a FDA-approved antipsychotic agent, reduced GBM cell growth. Subsequent isobolographic analyses of combination experiments indicated that trifluoperazine and erlotinib cooperated synergistically and that their concomitant treatment induced a robust activation of FOXO3a, leading to apoptosis in GBM cells. Using gene silencing, we found that FOXO3a is essential for the response of GBM cells to EGFR inhibition. CONCLUSIONS: Our data indicate that FOXO3a activation is a crucial event in the response of GBM cells to EGFR inhibition, suggesting that FOXO3a may serve as an actionable therapeutic target that can be modulated using FDA-approved drugs.

N-(2-methyl-indol-1H-5-yl)-1-naphthalenesulfonamide: A novel reversible antimitotic agent inhibiting cancer cell motility.

A series of compounds containing the sulfonamide scaffold were synthesized and screened for their in vitro anticancer activity against a representative panel of human cancer cell lines, leading to the identification of N-(2-methyl-1H-indol-5-yl)-1-naphthalenesulfonamide (8e) as a compound showing a remarkable activity across the panel, with IC50 values in the nanomolar-to-low micromolar range. Cell cycle distribution analysis revealed that 8e promoted a severe G2/M arrest, which was followed by cellular senescence as indicated by the detection of senescence-associated ß-galactosidase (SA-ß-gal) in 8e-treated cells. Prolonged 8e treatment also led to the onset of apoptosis, in correlation with the detection of increased Caspase 3/7 activities. Despite increasing γ-H2A.X levels, a well-established readout for DNA double-strand breaks, in vitro DNA binding studies with 8e did not support interaction with DNA. In agreement with this, 8e failed to activate the cellular DNA damage checkpoint. Importantly, tubulin staining showed that 8e promoted a severe disorganization of microtubules and mitotic spindle formation was not detected in 8e-treated cells. Accordingly, 8e inhibited tubulin polymerization in vitro in a dose-dependent manner and was also able to robustly inhibit cancer cell motility. Docking analysis revealed a compatible interaction with the colchicine-binding site of tubulin. Remarkably, these cellular effects were reversible since disruption of treatment resulted in the reorganization of microtubules, cell cycle re-entry and loss of senescent markers. Collectively, our data suggest that this compound may be a promising new anticancer agent capable of both reducing cancer cell growth and motility.

Efficacy of the GemOx-R regimen leads to the identification of Oxaliplatin as a highly effective drug against Mantle Cell Lymphoma.

Mantle Cell Lymphoma (MCL) is an aggressive lymphoma subtype that accounts for 6-8% of non-Hodgkin lymphomas. The disease is mostly incurable and characterized by a continuous pattern of relapse. Major changes have recently been implemented in the management of MCL, but continuous relapses still mark this disease as a challenge for clinicians. We previously reported the efficacy of GemOx-R (Gemcitabine, Oxaliplatin and Rituximab) in patients with refractory and relapsing MCL. We present results for a larger series with longer follow-up and including high-risk frontline patients, showing an overall response rate of 83%. The efficacy of each component of GemOx-R was evaluated in a panel of MCL cell lines. Also, patient-derived primary cells were used in ex vivo experiments. The results show that oxaliplatin has a profound effect on cellular viability and is the most effective drug within this regimen. We further present synergistic efficacy of oxaliplatin combined with cytarabine in MCL cells. Interestingly, this synergistic effect was not seen when cisplatin and cytarabine were combined, indicating that among the platinum-derived agents oxaliplatin may be the preferred approach. Taken together our findings suggest that oxaliplatin alone or combined with cytarabine could constitute an alternative backbone for MCL regimens.

Pro-Oxidant Activity of Amine-Pyridine-Based Iron Complexes Efficiently Kills Cancer and Cancer Stem-Like Cells.

Differential redox homeostasis in normal and malignant cells suggests that pro-oxidant-induced upregulation of cellular reactive oxygen species (ROS) should selectively target cancer cells without compromising the viability of untransformed cells. Consequently, a pro-oxidant deviation well-tolerated by nonmalignant cells might rapidly reach a cell-death threshold in malignant cells already at a high setpoint of constitutive oxidative stress. To test this hypothesis, we took advantage of a selected number of amine-pyridine-based Fe(II) complexes that operate as efficient and robust oxidation catalysts of organic substrates upon reaction with peroxides. Five of these Fe(II)-complexes and the corresponding aminopyridine ligands were selected to evaluate their anticancer properties. We found that the iron complexes failed to display any relevant activity, while the corresponding ligands exhibited significant antiproliferative activity. Among the ligands, none of which were hemolytic, compounds 1, 2 and 5 were cytotoxic in the low micromolar range against a panel of molecularly diverse human cancer cell lines. Importantly, the cytotoxic activity profile of some compounds remained unaltered in epithelial-to-mesenchymal (EMT)-induced stable populations of cancer stem-like cells, which acquired resistance to the well-known ROS inducer doxorubicin. Compounds 1, 2 and 5 inhibited the clonogenicity of cancer cells and induced apoptotic cell death accompanied by caspase 3/7 activation. Flow cytometry analyses indicated that ligands were strong inducers of oxidative stress, leading to a 7-fold increase in intracellular ROS levels. ROS induction was associated with their ability to bind intracellular iron and generate active coordination complexes inside of cells. In contrast, extracellular complexation of iron inhibited the activity of the ligands. Iron complexes showed a high proficiency to cleave DNA through oxidative-dependent mechanisms, suggesting a likely mechanism of cytotoxicity. In summary, we report that, upon chelation of intracellular iron, the pro-oxidant activity of amine-pyrimidine-based iron complexes efficiently kills cancer and cancer stem-like cells, thus providing functional evidence for an efficient family of redox-directed anti-cancer metallodrugs.

Cell uptake and localization studies of squaramide based fluorescent probes.

Cell internalization is a major issue in drug design. Although squaramide-based compounds are receiving much attention because of their interesting bioactivity, cell uptake and trafficking within cells of this type of compounds are still unknown. In order to monitor the cell internalization process of cyclosquaramide compounds we have prepared two fluorescent probes by covalently linking a fluorescent dye (BODIPY derivative or fluorescein) to a noncytotoxic cyclosquaramide framework. These two probes (C2-BDP and C2-FITC) rapidly internalize across live cell membranes through endocytic receptor-mediated mechanisms. Due to its higher fluorescence and photochemical stability, C2-BDP is a superior dye than C2-FITC. C2-BDP remains sequestered in late endosomes allowing their fast and selective imaging in various live cell types. Cyclosquaramide-cell membrane interactions facilitate cell uptake and have been investigated by binding studies in solution as well as in live cells. Cyclosquaramide 1 (C2-BDP) can be used as a highly fluorescent probe for the rapid and selective imaging of late endosomes in live cells.

Retama monosperma n-hexane extract induces cell cycle arrest and extrinsic pathway-dependent apoptosis in Jurkat cells.

BACKGROUND: Retama monosperma L. (Boiss.) or Genista monosperma L. (Lam.), locally named as "R'tam", is an annual and spontaneous plant belonging to the Fabaceae family. In Morocco, Retama genus is located in desert regions and across the Middle Atlas and it has been widely used in traditional medicine in many countries. In this study, we show that Retama monosperma hexane extract presents significant anti-leukemic effects against human Jurkat cells. METHODS: Human Jurkat cells, together with other cell lines were screened with different concentrations of Retama monosperma hexane extract at different time intervals. Growth inhibition was determined using luminescent-based viability assays. Cell cycle arrest and apoptosis were measured by flow cytometry analysis. Combined caspase 3 and 7 activities were measured using luminometric caspase assays and immunoblots were performed to analyze expression of relevant pro- and anti-apoptotic proteins. GC-MS were used to determine the chemical constituents of the active extract. RESULTS: Retama monosperma hexane extract (Rm-HE) showed significant cytotoxicity against Jurkat cells, whereas it proved to be essentially ineffective against both normal mouse fibroblasts (NIH3T3) and normal lymphocytes (TK-6). Cytometric analysis indicated that Rm-HE promoted cell cycle arrest and apoptosis induction accompanied by DNA damage induction indicated by an increase in p-H2A.X levels. Rm-HE induced apoptosis was partially JNK-dependent and characterized by an increase in Fas-L levels together with activation of caspases 8, 3, 7 and 9, whereas neither the pro-apoptotic nor anti-apoptotic mitochondrial membrane proteins analyzed were significantly altered. Chemical identification analysis indicated that α-linolenic acid, campesterol, stigmasterol and sitosterol were the major bioactive components within the extract. CONCLUSIONS: Our data suggest that bioactive compounds present in Rm-HE show significant anti leukemic activity inducing cell cycle arrest and cell death that operates, at least partially, through the extrinsic apoptosis pathway.