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.     

Combination of Heme Oxygenase-1 Inhibition and Sigma Receptor Modulation for Anticancer Activity

Cancer is a multifactorial disease that may be tackled by targeting different signaling pathways. Heme oxygenase-1 (HO-1) and sigma receptors (σRs) are both overexpressed in different human cancers, including prostate and brain, contributing to the cancer spreading. In the present study, we investigated whether HO-1 inhibitors and σR ligands, as well a combination of the two, may influence DU145 human prostate and U87MG human glioblastoma cancer cells proliferation. In addition, we synthesized, characterized, and tested a small series of novel hybrid compounds (HO-1/σRs) 1–4 containing the chemical features needed for HO-1 inhibition and σR modulation. Herein, we report for the first time that targeting simultaneously HO-1 and σR proteins may be a good strategy to achieve increased antiproliferative activity against DU145 and U87MG cells, with respect to the mono administration of the parent compounds. The obtained outcomes provide an initial proof of concept useful to further optimize the structure of HO-1/σRs hybrids to develop novel potential anticancer agents.     

Monitoring the Glutathione Redox Reaction in Living Human Cells by Combining Metabolic Labeling with Heteronuclear NMR

The glutathione (GSH) redox reaction is critical for defense against cellular reactive oxygen species (ROS). However, direct and real‐time monitoring of this reaction in living mammalian cells has been hindered by the lack of a facile method. Herein, we describe a new approach that exploits the GSH biosynthetic pathway and heteronuclear NMR. [U‐¹³C]‐labeled cysteine was incorporated into GSH in U87 glioblastoma cells, and the oxidation of GSH to GSSG by a ROS‐producing agent could be monitored in living cells. Further application of the approach to cells resistant to temozolomide (TMZ), an anti‐glioblastoma drug, suggested a possible new resistance mechanism involving neutralization of ROS. This result was corroborated by the observation of up‐regulation of glutathione peroxidase 3 (GPx3). This new approach could be easily applied to redox‐dependent signaling pathways and drug resistance involving ROS.     

Cytotoxic constituents of Abutilon indicum leaves against U87MG human glioblastoma cells

The study was aimed to identify cytotoxic leads from Abutilon indicum leaves for treating glioblastoma. The petroleum ether extract, methanol extract (AIM), chloroform and ethyl acetate sub-fractions (AIM-C and AIM-E, respectively) prepared from AIM were tested for cytotoxicity on U87MG human glioblastoma cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. These extracts exhibited considerable activity (IC₅₀ values of 42.6–64.5 μg/mL). The most active AIM-C fraction was repeatedly chromatographed to yield four known compounds, methyl trans-p-coumarate (1), methyl caffeate (2), syringic acid (3) and pinellic acid (4). Cell viability assay of 1–4 against U87MG cells indicated 2 as most active (IC₅₀ value of 8.2 μg/mL), whereas the other three compounds were much less active. Interestingly, compounds 1–4 were non-toxic towards normal human cells (HEK-293). The content of 2 in AIM-C was estimated as 3% by HPLC. Hence, presence of some more active substances besides methyl caffeate (2) in AIM-C is anticipated.     

Cyclic RGD–Polyethylene Glycol–Polyethylenimine for Intracranial Glioblastoma‐Targeted Gene Delivery

Even though the blood–brain barrier (BBB) is compromised for angiogenesis, therapeutic agents for glioblastoma multiforme (GBM) are particularly inefficient due to the existence of a blood–tumor barrier (BTB), which hampers tumor accumulation and uptake. Integrin α_vβ₃ is overexpressed on glioblastoma U87 cells and neovasculture, thus making its ligands such as the RGD motif target glioblastoma in vitro and in vivo. In the present work, we have designed a modified polyethylene glycol–polyethylenimine (PEG–PEI) gene carrier by conjugating it with a cyclic RGD sequence, c(RGDyK) (cyclic arginine‐glycine‐aspartic acid‐D ‐tyrosine‐lysine). When complexed with plasmid DNA, this gene carrier, termed RGD–PEG–PEI, formed homogenous nanoparticles with a mean diameter of 73 nm. These nanoparticles had a high binding affinity with U87 cells and facilitated targeted gene delivery against intracranial glioblastoma in vivo, thereby leading to a higher gene transfer efficiency compared to the PEG–PEI gene carrier without RGD decoration. This intracranial glioblastoma‐targeted gene carrier also enhanced the therapeutic efficacy of pORF‐hTRAIL, as evidenced by a significantly prolonged survival of intracranial glioblastoma‐bearing nude mice. Considering the contribution of glioblastoma neovasculature to the BBB under angiogenic conditions, our results demonstrated the therapeutic feasibility of treating a brain tumor through mediation of integrin α_vβ₃, as well as the potential of using RGD–PEG–PEI as a targeted gene carrier in the treatment of intracranial glioblastoma.     

Synthesis and Biological Evaluation of Novel 2‐Aryl Benzimidazoles as Chemotherapeutic Agents

Here, we describe the synthesis and preliminary biological evaluation of novel N‐unsubstituted and N‐methylated 2‐aryl benzimidazole derivatives that contain fluorinated or hydroxylated alkyl substituents in the 4‐N‐aryl position and different substitution patterns (H vs Br vs I) in the benzimidazole ring. For the selected compounds and for comparison purposes, the congener benzothiazoles were also tested. The cytotoxic effect of 11 benzazole derivatives was evaluated in a panel of human cancer cell lines, such as breast (MCF7), melanoma (A375), cervix (HeLa), and glioblastoma (U87). In general, the compounds exerted a moderate cytotoxic activity against all cells tested. In particular, for the A375 and HeLa cells, the N‐unsubstituted benzimidazoles 2 and 3 displayed a better cytotoxic profile than the respective N‐methylated benzimidazole congeners ( 5 and 7 ). The biodistribution of compound 2 , which has shown the highest cytotoxic activity active in the U87 glioblastoma cells (IC₅₀ = 45.2 ± 13.0), was evaluated in CD1 mice using its ¹⁸F‐labeled counterpart ( [¹⁸F]2 ). These studies showed that compound 2 can cross the blood brain barrier with a reasonable brain uptake (1.24 and 2.81%I.A./g at 5 and 60 min p.i., respectively), which is a crucial issue for systemic chemotherapy of glioblastoma. Altogether, the in vitro antitumoral activity of benzimidazole 2 against the U87 cells and the ability of its ¹⁸F‐congener to cross the blood brain barrier provide a strong rationale to consider the reported fluoroalkylated 2‐aryl benzimidazoles as lead candidates for the generation of chemotherapeutic agents, in particular, against highly aggressive brain tumors such as glioblastoma.     

Hydrogel microfluidic co‐culture device for photothermal therapy and cancer migration

We developed the photo‐crosslinkable hydrogel microfluidic co‐culture device to study photothermal therapy and cancer cell migration. To culture MCF7 human breast carcinoma cells and metastatic U87MG human glioblastoma in the microfluidic device, we used 10 w/v% gelatin methacrylate (GelMA) hydrogels as a semi‐permeable physical barrier. We demonstrated the effect of gold nanorod on photothermal therapy of cancer cells in the microfluidic co‐culture device. Interestingly, we observed that metastatic U87MG human glioblastoma largely migrated toward vascular endothelial growth factor (VEGF)‐treated GelMA hydrogel‐embedding microchannels. The main advantage of this hydrogel microfluidic co‐culture device is to simultaneously analyze the physiological migration behaviors of two cancer cells with different physiochemical motilities and study gold nanorod‐mediated photothermal therapy effect. Therefore, this hydrogel microfluidic co‐culture device could be a potentially powerful tool for photothermal therapy and cancer cell migration applications.     

99mTc-radiolabeled imidazo[2,1-b]benzothiazole derivatives as potential radiotracers for glioblastoma

This study presents [^99mTc]BPTG-1 and [^99mTc]BPTG-2 for glioblastoma imaging. In vitro cellular uptakes of these radiotracers were examined in SKOV-3, MCF-7, U87-MG, HT-29, and A549 cell lines. U87-MG cell line displayed the highest radiotracers uptakes. Biodistribution study in U87-MG tumor bearing mice revealed higher uptake of radiotracers in tumor than muscle and brain. Liver, intestine, and kidneys displayed the highest radioactivity uptakes. The main route of radiotracers elimination was hepatobiliary. Due to the brain uptake of these radiotracers, they are promising radiotracers for future studies in the diagnosis of glioblastoma.

     

Harnessing the Potential Synergistic Interplay Between Photosensitizer Dark Toxicity and Chemotherapy

The combination of photodynamic therapy and taxol- or platinum-based chemotherapy (photochemotherapy) is an effective and promising cancer treatment. While the mechanisms of action of photochemotherapy are actively studied, relatively little is known about the cytotoxicity and molecular alterations induced by the combination of chemotherapy and photosensitizers without light activation in cancer cells. This study investigates the interplay between the photosensitizer benzoporphyrin derivative (BPD) without light activation and cisplatin or paclitaxel in two glioblastoma lines, U87 and U251. The combination effect of BPD and cisplatin in U87 cells is slightly synergistic (combination index, CI = 0.93), showing 1.8- to 2.6-fold lower half-maximal inhibitory concentrations (IC₅₀) compared to those of individual drugs. In contrast, combining BPD and paclitaxel is slightly antagonistic (CI = 1.14) in U87 cells. In U251 cells, the combinations of BPD and cisplatin or paclitaxel are both antagonistic (CI = 1.24 and 1.34, respectively). Western blotting was performed to investigate changes in the expression levels of YAP, TAZ, Bcl-2 and EGFR in U87 and U251 cells treated with BPD, cisplatin and paclitaxel, both as monotherapies and in combination. Our study provides insights into the molecular alterations in two glioma lines caused by each monotherapy and the combinations, in order to inform the design of effective treatments.     

Green synthesis of Zn nanoparticles and in situ hybridized with BSA nanoparticles for Baicalein targeted delivery mediated with glutamate receptors to U87-MG cancer cell lines

Glioblastoma multiforme (GBM) is the most aggressive malignant tumor of the brain. It has different glutamate receptor types. So, these receptors can be a suitable target for GBM treatment. The current study investigated the anticancer effects of bovine serum albumin (BSA)-Baicalein @Zn-Glu nanostructure mediated-GluRs in human glioblastoma U87 cells. BSA-Ba@Zn-Glu hybrid nanoparticles (NPs) were set and considered transporters for Baicalein (Ba) active compound delivery. BSA-Ba@Zn-Glu NPs were synthesized by a single-step reduction process. The successful production was confirmed through transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FT-IR), and hemolysis test. The cytotoxic efficacy and apoptosis rate of the nanostructures on U87 glioblastoma cells were investigated by 3-(4,5-dimethylthialzol-a-yl)-2,5 diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. The synthesized BSA-Ba@Zn-Glu nanostructures with a diameter of 142.40 ± 1.91 to 177.10 ± 1.87 nm and zeta potential of −10.57 ± 0.71 to −35.77 ± 0.60 mV are suitable for extravasation into tumor cells. The drug release from the BSA-Ba@Zn NPs showed controlled and pH-dependent behavior. In vitro results indicated that the BSA-Ba@Zn-Glu NPs significantly reduce cell viability and promote apoptosis of U87 cancer cells. It revealed the cytotoxic effect of the Baicalein and an increase in cellular uptake of nanoparticles by Glu receptors. Zn NPs were synthesized based on a green synthesis method. BSA NPs were used as a nano-platform for Glu conjugation and Ba drug delivery. BSA-Ba@Zn-Glu NPs induce cytotoxicity and apoptosis in human brain cancer cells (U87) in a dose-dependent manner. Finally, this nanostructure could be served in targeted drug delivery in vivo studies and applied along with other strategies such as X-ray irradiation as combinational therapies in future studies.     

Antagonistic Effects of Endogenous Nitric Oxide in a Glioblastoma Photodynamic Therapy Model

Gliomas are aggressive brain tumors that are resistant to conventional chemotherapy and radiotherapy. Much of this resistance is attributed to endogenous nitric oxide (NO). Recent studies revealed that 5‐aminolevulinic acid (ALA)‐based photodynamic therapy (PDT) has advantages over conventional treatments for glioblastoma. In this study, we used an in vitro model to assess whether NO from glioblastoma cells can interfere with ALA‐PDT. Human U87 and U251 cells expressed significant basal levels of neuronal NO synthase (nNOS) and its inducible counterpart (iNOS). After an ALA/light challenge, iNOS level increased three‐ to fourfold over 24 h, whereas nNOS remained unchanged. Elevated iNOS resulted in a large increase in intracellular NO. Extent of ALA/light‐induced apoptosis increased substantially when an iNOS inhibitor or NO scavenger was present, implying that iNOS/NO was acting cytoprotectively. Moreover, cells surviving a photochallenge exhibited a striking increase in proliferation, migration and invasion rates, iNOS/NO again playing a dominant role. Also observed was a large iNOS/NO‐dependent increase in matrix metalloproteinase‐9 activity, decrease in tissue inhibitor of metalloproteinase‐1 expression and increase in survivin and S100A4 expression, each effect being consistent with accelerated migration/invasion as a prelude to metastasis. Our findings suggest introduction of iNOS inhibitors as pharmacologic adjuvants for glioblastoma PDT.     

Entelon® (Vitis vinifera Seed Extract) Prevents Cancer Metastasis via the Downregulation of Interleukin-1 Alpha in Triple-Negative Breast Cancer Cells

Interleukin-1 (IL1) is a proinflammatory cytokine and promotes cancer cell proliferation and invasiveness in a diversity of cancers, such as breast and colon cancer. Here, we focused on the pharmacological effect of Entelon^® (ETL) on the tumorigenesis of triple-negative breast cancer (TNBC) cells by IL1-alpha (IL1A). IL1A enhanced the cell growth and invasiveness of TNBC cells. We observed that abnormal IL1A induction is related with the poor prognosis of TNBC patients. IL1A also increased a variety of chemokines such as CCL2 and IL8. Interestingly, IL1A expression was reduced by the ETL treatment. Here, we found that ETL significantly decreased the MEK/ERK signaling pathway in TNBC cells. IL1A expression was reduced by UO126. Lastly, we studied the effect of ETL on the metastatic potential of TNBC cells. Our results showed that ETL significantly reduced the lung metastasis of TNBC cells. Our results showed that IL1A expression was regulated by the MEK/ERK- and PI3K/AKT-dependent pathway. Taken together, ETL inhibited the MEK/ERK and PI3K/AKT signaling pathway and suppressing the lung metastasis of TNBC cells through downregulation of IL1A. Therefore, we propose the possibility of ETL as an effective adjuvant for treating TNBC.     

Liposomes loaded with lipophilic derivative of closo-carborane as a potential boron delivery system for boron neutron capture therapy of tumors

Liposomes encapsulated with lipophilic derivative of 1,2-dicarba-closo-dodecaborane have been obtained and tested for toxicity to glioblastoma U87 cells and biodistribution on a U87MG xenograft mouse model. The liposomes are able to penetrate the tumor and provide boron concentration up to 1.5 mmmmg g–1 with tumor-to-muscle ratio up to 2.4.     

Combinatorial Therapeutic Effect of Inhibitors of Aldehyde Dehydrogenase and Mitochondrial Complex I,and the Chemotherapeutic Drug,Temozolomide against Glioblastoma Tumorspheres

Resident cancer cells with stem cell-like features induce drug tolerance, facilitating survival of glioblastoma (GBM). We previously showed that strategies targeting tumor bioenergetics present a novel emerging avenue for treatment of GBM. The objective of this study was to enhance the therapeutic effects of dual inhibition of tumor bioenergetics by combination of gossypol, an aldehyde dehydrogenase inhibitor, and phenformin, a biguanide compound that depletes oxidative phosphorylation, with the chemotherapeutic drug, temozolomide (TMZ), to block proliferation, stemness, and invasiveness of GBM tumorspheres (TSs). Combination therapy with gossypol, phenformin, and TMZ induced a significant reduction in ATP levels, cell viability, stemness, and invasiveness compared to TMZ monotherapy and dual therapy with gossypol and phenformin. Analysis of differentially expressed genes revealed up-regulation of genes involved in programmed cell death, autophagy, and protein metabolism and down-regulation of those associated with cell metabolism, cycle, and adhesion. Combination of TMZ with dual inhibitors of tumor bioenergetics may, therefore, present an effective strategy against GBM by enhancing therapeutic effects through multiple mechanisms of action.     

Alkaloid extracts of Ficus species and palm oil-derived tocotrienols synergistically inhibit proliferation of human cancer cells

Tocotrienols have been reported to possess anticancer effects other than anti-inflammatory and antioxidant activities. This study explored the potential synergism of antiproliferative effects induced by individual alkaloid extracts of Ficus fistulosa, Ficus hispida and Ficus schwarzii combined with δ- and γ-tocotrienols against human brain glioblastoma (U87MG), lung adenocarcinoma (A549) and colorectal adenocarcinoma (HT-29) cells. Cell viability and morphological results demonstrated that extracts containing a mixture of alkaloids from the leaves and bark of F. schwarzii inhibited the proliferation of HT-29 cells, whereas the alkaloid extracts of F. fistulosa inhibited the proliferation of both U87MG and HT-29 cells and showed synergism in combined treatments with either δ- or γ-tocotrienol resulting in 2.2–34.7 fold of reduction in IC₅₀ values of tocotrienols. The observed apoptotic cell characteristics in conjunction with the synergistic antiproliferative effects of Ficus species-derived alkaloids and tocotrienols assuredly warrant future investigations towards the development of a value-added chemotherapeutic regimen against cancers.     

A new cyclopamine glucuronide prodrug with improved kinetics of drug release

We prepared a new glucuronide prodrug of cyclopamine designed to target selectively the Hedgehog signalling pathway of cancer cells. This prodrug includes a novel self-immolative linker bearing a hydrophilic side chain that can be easily introduced via"click chemistry". With this design, the prodrug exhibits reduced toxicity compared to the free drug on U87 glioblastoma cells. However, in the presence of β-glucuronidase, the prodrug conducts to the quick release of cyclopamine thereby restoring its antiproliferative activity.     

Phospholipase D activates HIF-1-VEGF pathway via phosphatidic acid

Growth factor-stimulated phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC), generating phosphatidic acid (PA) which may act as a second messenger during cell proliferation and survival. Therefore, PLD is believed to play an important role in tumorigenesis. In this study, a potential mechanism for PLD-mediated tumorigenesis was explored. Ectopic expression of PLD1 or PLD2 in human glioma U87 cells increased the expression of hypoxia-inducible factor-1α (HIF-1α) protein. PLD-induced HIF-1 activation led to the secretion of vascular endothelial growth factor (VEGF), a HIF-1 target gene involved in tumorigenesis. PLD induction of HIF-1α was significantly attenuated by 1-butanol which blocks PA production by PLD, and PA per se was able to elevate HIF-1α protein level. Inhibition of mTOR, a PA-responsive kinase, reduced the levels of HIF-1α and VEGF in PLD-overexpressed cells. Epidermal growth factor activated PLD and increased the levels of HIF-1α and VEGF in U87 cells. A specific PLD inhibitor abolished expression of HIF-1α and secretion of VEGF. PLD may utilize HIF-1-VEGF pathway for PLD-mediated tumor cell proliferation and survival.