Asparagus racemosus mediated silver chloride nanoparticles induce apoptosis in glioblastoma stem cells in vitro and inhibit Ehrlich ascites carcinoma cells growth in vivo

Glioblastoma multiforme (GBM) is a type of brain tumor that is most aggressive, proliferates rapidly and intensive invasion is governed by cell migration and destruction of the extracellular matrix. In the present study, we evaluated the antiproliferative efficacy of the synthesized silver chloride nanoparticles (AgCl-NPs) from Asparagus racemosus root extract against human glioblastoma stem cells (GSCs) and Ehrlich ascites carcinoma (EAC) cells. Biosynthesis of A. racemosus-AgCl-NPs was confirmed by color change, UV–visible spectroscopy and characterized by transmitted electron microscope, energy dispersive spectroscopy, x-ray powder diffraction and Fourier-transform infrared spectroscopy. The A. racemosus-AgCl-NPs inhibited GSCs and EAC cells growth with the IC₅₀ values of 4.8 and 4.69 µg/ml, respectively. A. racemosus-AgCl-NPs induced apoptosis in GSCs which was confirmed by annexin V/PI assay, various genes expression, and caspase-3 protein expression as detected by the immunofluorometric assay. The expression level of the TLR9, NFκB, TNFα, p21 and IKK genes were increased consequently with the decrease of PARP, EGFR, NOTCH2, mTOR and STAT3 genes in GSCs as examined by real-time PCR. The cell cycle arrest at G₀/G₁ phase was detected by flow cytometry. In addition, A. racemosus-AgCl-NPs caused significant inhibition of EAC cells growth, reduced tumor burden, increased the survival of EAC-bearing mice and restored the hematological parameters when compared with the control mice. The synthesized AgCl-NPs inhibited the proliferation of GSCs in vitro with the induction of apoptosis and inhibited the growth of EAC cells in vivo in mice by reducing the tumor burden and increasing the survival periods.     

Co-Administration of Fendiline Hydrochloride Enhances Chemotherapeutic Efficacy of Cisplatin in Neuroblastoma Treatment

Despite significant improvement of neuroblastoma (NB) patients’ survival due to recent treatment advancements in recent years, NB is still associated with high mortality rate. In search of novel strategies to increase NB’s susceptibility to pharmacological treatments, we investigated the in vitro and in vivo effects of fendiline hydrochloride as an enhancer of cisplatin antitumor activity. To assess the modulation of fendiline treatment on cisplatin responses, we used in vitro (evaluating NB cell proliferation by XCELLigence technology and colony formation, and gene expression by RT-PCR) and in vivo (NB cell grafts in NOD-SCID mice) models of NB. NB cell treatment with fendiline induced the expression of the ncRNA NDM29, leading to cell differentiation and to the reduction of the expression of MDRs/ABC transporters linked to multidrug resistance. These events were correlated to higher NB cell susceptibility to cisplatin and, consequently, increased its cytotoxic potency. In vivo, this drug interaction causes an enhanced ability of cisplatin to induce apoptosis in NB masses, resulting in tumor growth reduction and prolonged animal survival rate. Thus, the administration of fendiline might be a possible novel therapeutic approach to increase cisplatin efficacy in aggressive and poorly responsive NB cases.     

Curcumin Loaded Dendrimers Specifically Reduce Viability of Glioblastoma Cell Lines

Glioblastoma (GB) is a deadly and aggressive cancer of the CNS. Even with extensive resection and chemoradiotherapy, patient survival is still only 15 months. To maintain growth and proliferation, cancer cells require a high oxidative state. Curcumin, a well-known anti-inflammatory antioxidant, is a potential candidate for treatment of GB. To facilitate efficient delivery of therapeutic doses of curcumin into cells, we encapsulated the drug in surface-modified polyamidoamine (PAMAM) dendrimers. We studied the in vitro effectiveness of a traditional PAMAM dendrimer (100% amine surface, G4 NH₂), surface-modified dendrimer (10% amine and 90% hydroxyl-G4 90/10-Cys), and curcumin (Cur)-encapsulated dendrimer (G4 90/10-Cys-Cur) on three species of glioblastoma cell lines: mouse-GL261, rat-F98, and human-U87. Using an MTT assay for cell viability, we found that G4 90/10-Cys-Cur reduced viability of all three glioblastoma cell lines compared to non-cancerous control cells. Under similar conditions, unencapsulated curcumin was not effective, while the non-modified dendrimer (G4 NH₂) caused significant death of both cancerous and normal cells. By harnessing and optimizing the components of PAMAM dendrimers, we are providing a promising new route for delivering cancer therapeutics. Our results with curcumin suggest that antioxidants are good candidates for treating glioblastoma.     

The feasibility of using dielectrophoresis for isolation of glioblastoma subpopulations with increased stemness

Cancer stem cells (CSCs) are aggressive subpopulations with increased stem‐like properties. CSCs are usually resistant to most standard therapies and are responsible for tumor repropagation. Similar to normal stem cells, isolation of CSCs is challenging due to the lack of reliable markers. Antigen‐based sorting of CSCs usually requires staining with multiple markers, making the experiments complicated, expensive, and sometimes unreliable. Here, we study the feasibility of using dielectrophoresis (DEP) for isolation of glioblastoma cells with increased stemness. We culture a glioblastoma cell line in the form of neurospheres as an in vitro model for glioblastoma stem cells. We demonstrate that spheroid forming cells have higher expression of stem cell marker, nestin. Next, we show that dielectric properties of neurospheres change as a result of changing culture conditions. Our results indicate that spheroid forming cells need higher voltages to experience the same DEP force magnitude compared to normal monolayer cultures of glioblastoma cell line. This study confirms the possibility of using DEP to isolate glioblastoma stem cells.     

Insights into Multifunctional Nanoparticle-Based Drug Delivery Systems for Glioblastoma Treatment

Glioblastoma (GB) is an aggressive cancer with high microvascular proliferation, resulting in accelerated invasion and diffused infiltration into the surrounding brain tissues with very low survival rates. Treatment options are often multimodal, such as surgical resection with concurrent radiotherapy and chemotherapy. The development of resistance of tumor cells to radiation in the areas of hypoxia decreases the efficiency of such treatments. Additionally, the difficulty of ensuring drugs effectively cross the natural blood–brain barrier (BBB) substantially reduces treatment efficiency. These conditions concomitantly limit the efficacy of standard chemotherapeutic agents available for GB. Indeed, there is an urgent need of a multifunctional drug vehicle system that has potential to transport anticancer drugs efficiently to the target and can successfully cross the BBB. In this review, we summarize some nanoparticle (NP)-based therapeutics attached to GB cells with antigens and membrane receptors for site-directed drug targeting. Such multicore drug delivery systems are potentially biodegradable, site-directed, nontoxic to normal cells and offer long-lasting therapeutic effects against brain cancer. These models could have better therapeutic potential for GB as well as efficient drug delivery reaching the tumor milieu. The goal of this article is to provide key considerations and a better understanding of the development of nanotherapeutics with good targetability and better tolerability in the fight against GB.     

MicroRNA Delivery by Graphene-Based Complexes into Glioblastoma Cells

Glioblastoma (GBM) is the most common primary and aggressive tumour in brain cancer. Novel therapies, despite achievements in chemotherapy, radiation and surgical techniques, are needed to improve the treatment of GBM tumours and extend patients’ survival. Gene delivery therapy mostly uses the viral vector, which causes serious adverse events in gene therapy. Graphene-based complexes can reduce the potential side effect of viral carries, with high efficiency of microRNA (miRNA) or antisense miRNA delivery to GBM cells. The objective of this study was to use graphene-based complexes to induce deregulation of miRNA level in GBM cancer cells and to regulate the selected gene expression involved in apoptosis. The complexes were characterised by Fourier transform infrared spectroscopy (FTIR), scanning transmission electron microscopy and zeta potential. The efficiency of miRNA delivery to the cancer cells was analysed by flow cytometry. The effect of the anticancer activity of graphene-based complexes functionalised by the miRNA sequence was analysed using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide salt (XTT) assays at the gene expression level. The results partly explain the mechanisms of miRNA deregulation stress, which is affected by graphene-based complexes together with the forced transport of mimic miR-124, miR-137 and antisense miR-21, -221 and -222 as an anticancer supportive therapy.     

A New Chalcone Derivative with Promising Antiproliferative and Anti-Invasion Activities in Glioblastoma Cells

Glioblastoma (GBM) is the most common and most deadly primary malignant brain tumor. Current therapies are not effective, the average survival of GBM patients after diagnosis being limited to few months. Therefore, the discovery of new treatments for this highly aggressive brain cancer is urgently needed. Chalcones are synthetic and naturally occurring compounds that have been widely investigated as anticancer agents. In this work, three chalcone derivatives were tested regarding their inhibitory activity and selectivity towards GBM cell lines (human and mouse) and a non-cancerous mouse brain cell line. The chalcone 1 showed the most potent and selective cytotoxic effects in the GBM cell lines, being further investigated regarding its ability to reduce critical hallmark features of GBM and to induce apoptosis and cell cycle arrest. This derivative showed to successfully reduce the invasion and proliferation capacity of tumor cells, both key targets for cancer treatment. Moreover, to overcome potential systemic side effects and its poor water solubility, this compound was encapsulated into liposomes. Therapeutic concentrations were incorporated retaining the potent in vitro growth inhibitory effect of the selected compound. In conclusion, our results demonstrated that this new formulation can be a promising starting point for the discovery of new and more effective drug treatments for GBM.     

Bimodal Therapeutic Agents Against Glioblastoma,One of the Most Lethal Forms of Cancer

About 95 % of people diagnosed with glioblastoma die within five years. Glioblastoma is the most aggressive central nervous system tumour. It is necessary to make progress in the glioblastoma treatment so that advanced chemotherapy drugs or radiation therapy or, ideally, two-in-one hybrid systems should be implemented. Tyrosine kinase receptors–inhibitors and boron neutron capture therapy (BNCT), together, could provide a therapeutic strategy. In this work, sunitinib decorated-carborane hybrids were prepared and biologically evaluated identifying excellent antitumoral- and BNCT-agents. One of the selected hybrids was studied against glioma-cells and found to be 4 times more cytotoxic than sunitinib and 1.7 times more effective than ¹⁰B-boronophenylalanine fructose complex when the cells were irradiated with neutrons.     

Resveratrol augments ER stress and the cytotoxic effects of glycolytic inhibition in neuroblastoma by downregulating Akt in a mechanism independent of SIRT1

Cancer cells typically display increased rates of aerobic glycolysis that are correlated with tumor aggressiveness and a poor prognosis. Targeting the glycolytic pathway has emerged as an attractive therapeutic route mainly because it should spare normal cells. Here, we evaluate the effects of combining the inhibition of glycolysis with application of the polyphenolic compound resveratrol (RSV) in neuroblastoma (NB) cancer cell lines. Inhibiting glycolysis with 2-deoxy-D-glucose (2-DG) significantly reduced NB cell viability and was associated with increased endoplasmic reticulum (ER) stress and Akt activity. Administration of 2-DG increased the expression of the ER molecular chaperones GRP78 and GRP94, the prodeath protein C/EBP homology protein (CHOP) and the phosphorylation of Akt at S473, T450 and T308. Combined treatment with both RSV and 2-DG reduced GRP78, GRP94 and Akt phosphorylation but increased CHOP and NB cell death when compared with the administration of 2-DG alone. The selective inhibition of Akt activity also decreased 2-DG-induced GRP78 and GRP94 expression and increased CHOP expression, suggesting that Akt can modulate ER stress. Protein phosphatase 1α (PP1α) was activated by RSV, as indicated by a reduction in PP1α phosphorylation at T320. Pretreatment of cells with tautomycin, a selective PP1α inhibitor, prevented the RSV-mediated decrease in Akt phosphorylation, suggesting that RSV enhances 2-DG-induced cell death by activating PP1 and downregulating Akt. The RSV-mediated inhibition of Akt in the presence of 2-DG was not prevented by the selective inhibition of SIRT1, a known target of RSV, indicating that the effects of RSV on this pathway are independent of SIRT1. We propose that RSV inhibits Akt activity by increasing PP1α activity, thereby potentiating 2-DG-induced ER stress and NB cell death.     

Deoxynivalenol-mimic nanobody isolated from a naïve phage display nanobody library and its application in immunoassay

In this study, using mycotoxin deoxynivalenol (DON) as a model hapten, we developed a nanobody-based environmental friendly immunoassay for sensitive detection of DON. Two nanobodies (N-28 and N-31) which bind to anti-DON monoclonal antibody (MAb) were isolated from a naive phage display library. These nanobodies are clonable, thermally stable and mycotoxin-free products and can be served as coating antigen mimetics in heterologous immunoassay. The half inhibition concentration (IC₅₀) of the immunoassay developed with N-28 and N-31 was 8.77 ± 0.41 ng mL⁻¹ and 19.97 ± 0.84 ng mL⁻¹, respectively, which were 18- and 8-fold more sensitive than the conventional coating antigen (DON-BSA) based immunoassay. In order to better understand the molecular mechanism of antigen mimicry by nanobody, the 3D structure of “nanobody (N-28) - anti-DON MAb” complex was presented and verified by molecular modeling and alanine-scanning mutagenesis. The results showed that hydrogen bond and hydrophobic interaction formed between Thr 102 – Ser 106 of N-28 and CDR H3 residues of anti-DON antibody may contribute to their binding. This novel concept of enhancing sensitivity of immunoassay for DON based on nanobody may provide potential applications in a general method for immunoassay of various food chemical contaminants.     

Garcinia Biflavonoid 1 Improves Lipid Metabolism in HepG2 Cells via Regulating PPARα

Garcinia biflavonoid 1 (GB1) is one of the active chemical components of Garcinia kola and is reported to be capable of reducing the intracellular lipid deposition, which is the most significant characteristic of non-alcoholic fatty liver disease. However, its bioactive mechanism remains elusive. In the current study, the lipid deposition was induced in HepG2 cells by exposure to oleic acid and palmitic acid (OA&PA), then the effect of GB1 on lipid metabolism and oxidative stress and the role of regulating PPARα in these cells was investigated. We found that GB1 could ameliorate the lipid deposition by reducing triglycerides (TGs) and upregulate the expression of PPARα and SIRT6, suppressing the cell apoptosis by reducing the oxidative stress and the inflammatory factors of ROS, IL10, and TNFα. The mechanism study showed that GB1 had bioactivity in a PPARα-dependent manner based on its failing to improve the lipid deposition and oxidative stress in PPARα-deficient cells. The result revealed that GB1 had significant bioactivity on improving the lipid metabolism, and its potential primary action mechanism suggested that GB1 could be a potential candidate for management of non-alcoholic fatty liver disease.     

Strengthening Anti-Glioblastoma Effect by Multi-Branched Dendrimers Design of a Scorpion Venom Tetrapeptide

Glioblastoma is the most aggressive and invasive form of central nervous system tumors due to the complexity of the intracellular mechanisms and molecular alterations involved in its progression. Unfortunately, current therapies are unable to stop its neoplastic development. In this context, we previously identified and characterized AaTs-1, a tetrapeptide (IWKS) from Androctonus autralis scorpion venom, which displayed an anti-proliferative effect against U87 cells with an IC₅₀ value of 0.57 mM. This peptide affects the MAPK pathway, enhancing the expression of p53 and altering the cytosolic calcium concentration balance, likely via FPRL-1 receptor modulation. In this work, we designed and synthesized new dendrimers multi-branched molecules based on the sequence of AaTs-1 and showed that the di-branched (AaTs-1-2B), tetra-branched (AaTs-1-4B) and octo-branched (AaTs-1-8B) dendrimers displayed 10- to 25-fold higher effects on the proliferation of U87 cells than AaTs-1. We also found that the effects of the newly designed molecules are mediated by the enhancement of the ERK1/2 and AKT phosphorylated forms and by the increase in p53 expression. Unlike AaTs-1, AaTs-1-8B and especially AaTs-1-4B affected the migration of the U87 cells. Thus, the multi-branched peptide synthesis strategy allowed us to make molecules more active than the linear peptide against the proliferation of U87 glioblastoma cells.     

Mass spectrometry based proteomic analysis of human stem cells: a brief review

Stem cells can give rise to various cell types and are capable of regenerating themselves over multiple cell divisions. Pluripotency and self-renewal potential of stem cells have drawn vast interest from different disciplines, with studies on the molecular properties of stem cells being one example. Current investigations on the molecular basis of stem cells pluripotency and self-renewal entail traditional techniques from chemistry and molecular biology. In this mini review, we discuss progress in stem cell research that employs proteomics approaches. Specifically, we focus on studies on human stem cells from proteomics perspective. To our best knowledge, only the following types of human stem cells have been examined via proteomics analysis: human neuronal stem cells, human mesenchymal stem cells, and human embryonic stem cells. Protein expression serves as biomarkers of stem cells and identification and expression level of such biomarkers are usually determined using two-dimensional electrophoresis coupled mass spectrometry or non-gel based mass spectrometry.     

High Content Screening for Compounds that Induce Early Stages of Human Embryonic Stem Cell Differentiation

Embryonic stem cells, due to their self-renewal and pluripotency properties, can be used to repair damaged tissues and as an unlimited source of differentiated cells. Although stem cells represent an important opportunity for cell based therapy and small molecules screening (in the context of drug or target discovery) many drawbacks are still preventing their widespread use. One of the most significant limitations is related to the complexity, as well as the reliability, of current protocols driving stem cells into any homogeneously differentiated cellular population. In this respect there is a strong demand for molecular agents promoting differentiation and thereby enabling robust, efficient and safe production of differentiated cells. In order to identify novel molecules that enhance early stages of differentiation, we developed an image based high content screening (HCS) approach using human embryonic stem cells (hESC). In our approach, we took advantage of custom image mining software specifically adapted for the selection of stem cell differentiation agents and the rejection of false positive hits. As a proof of concept ～3500 small molecules originating from commercial libraries were screened and a number of molecules of interests were identified. These molecules show stem cell differentiation properties comparable to the phenotypic signature obtained with the reference compound retinoic acid.     

5-Aminolaevulinic acid-mediated photodynamic therapy in multidrug resistant leukemia cells

To verify if photodynamic therapy (PDT) could overcome multidrug resistance (MDR) when it it applied to eradicate minimal residual disease in patients with leukemia, we investigated the fluorescence kinetics of 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) and the effect of subsequent photodynamic therapy on MDR leukemia cells, which express P-glycoprotein (P-gp), as well as on their parent cells. Evaluation of PpIX accumulation by flow cytometry showed that PpIX accumulated at higher levels in mdr-1 gene-transduced MDR cells (NB4/MDR) and at lower levels in doxorubicin-induced MDR cells (NOMO-1/ADR) than in their parent cells. A P-gp inhibitor could not increase PpIX accumulation. Measurement of extracellular PpIX concentration by fluorescence spectrometry showed that P-gp did not mediate the fluorescence kinetics of ALA-induced PpIX production. Assessment of ferrochelatase activity using high-performance liquid chromatography indicated that PpIX accumulation in drug-induced MDR cells was probably regulated by this enzyme. Assessment of phototoxicity of PDT using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that PDT was effective in NB4, NB4/MDR, NOMO-1 and NOMO-1/ADR cells, which accumulated high levels of PpIX, but not effective in K562 and K562/ADR cell lines, which accumulated relatively low levels of PpIX. These findings demonstrate that P-gp does not mediate the ALA-fluorescence kinetics, and multidrug resistant leukemia cells do not have cross-resistance to ALA-PDT.     

AaTs-1: A Tetrapeptide from Androctonus australis Scorpion Venom,Inhibiting U87 Glioblastoma Cells Proliferation by p53 and FPRL-1 Up-Regulations

Glioblastoma is an aggressive cancer, against which medical professionals are still quite helpless, due to its resistance to current treatments. Scorpion toxins have been proposed as a promising alternative for the development of effective targeted glioblastoma therapy and diagnostic. However, the exploitation of the long peptides could present disadvantages. In this work, we identified and synthetized AaTs-1, the first tetrapeptide from Androctonus australis scorpion venom (Aa), which exhibited an antiproliferative effect specifically against human glioblastoma cells. Both the native and synthetic AaTs-1 were endowed with the same inhibiting effect on the proliferation of U87 cells with an IC₅₀ of 0.56 mM. Interestingly, AaTs-1 was about two times more active than the anti-glioblastoma conventional chemotherapeutic drug, temozolomide (TMZ), and enhanced its efficacy on U87 cells. AaTs-1 showed a significant similarity with the synthetic peptide WKYMVm, an agonist of a G-coupled formyl-peptide receptor, FPRL-1, known to be involved in the proliferation of glioma cells. Interestingly, the tetrapeptide triggered the dephosphorylation of ERK, p38, and JNK kinases. It also enhanced the expression of p53 and FPRL-1, likely leading to the inhibition of the store operated calcium entry. Overall, our work uncovered AaTs-1 as a first natural potential FPRL-1 antagonist, which could be proposed as a promising target to develop new generation of innovative molecules used alone or in combination with TMZ to improve glioblastoma treatment response. Its chemical synthesis in non-limiting quantity represents a valuable advantage to design and develop low-cost active analogues to treat glioblastoma cancer.