Scaffold-based spheroid models of glioblastoma multiforme and its use in drug screening

Among several types of brain cancers, glioblastoma multiforme (GBM) is a terminal and aggressive disease with a median survival of 15 months despite the most intensive surgery and chemotherapy. Preclinical models that accurately reproduce the tumor microenvironment are vital for developing new therapeutic alternatives. Understanding the complicated interactions between cells and their surroundings is essential to comprehend the tumor's microenvironment, however the monolayer cell culture approach falls short. Numerous approaches are used to develop GBM cells into tumor spheroids, while scaffold-based spheroids provides the opportunity to investigate the synergies between cells as well as cells and the matrix. This review summarizes the development of various scaffold-based GBM spheroid models and the prospective for their use as drug testing systems.     

A curcumin derivative hydrazinobenzoylcurcumin suppresses stem-like features of glioblastoma cells by targeting Ca2+/calmodulin-dependent protein kinase II

Glioblastoma multiforme (GBM) is the most aggressive and common type of human primary brain tumor. Glioblastoma stem-like cells (GSCs) have been proposed to contribute to tumor initiation, progression, recurrence, and therapeutic resistance of GBM. Therefore, targeting GSCs could be a promising strategy to treat this refractory cancer. Calmodulin (CaM), a major regulator of Ca²⁺-dependent signaling, controls various cellular functions via interaction with multiple target proteins. Here, we investigated the anticancer effect of hydrazinobenzoylcurcumin (HBC), a Ca ²⁺/CaM antagonist, against GSCs derived from U87MG and U373MG cells. HBC significantly inhibited not only the self-renewal capacity, such as cell growth and neurosphere formation but also the metastasis-promoting ability, such as migration and invasion of GSCs. HBC induced apoptosis of GSCs in a caspase-dependent manner. Notably, HBC repressed the phosphorylation of Ca ²⁺/CaM-dependent protein kinase II (CaMKII), c-Met, and its downstream signal transduction mediators, thereby reducing the expression levels of GSC markers, such as CD133, Nanog, Sox2, and Oct4. In addition, the knockdown of CaMKIIγ remarkably decreased the cancer stem cell-like phenotypes as well as the expression of stemness markers by blocking c-Met signaling pathway in U87MG GSCs. These results suggest that HBC suppresses the stem-like features of GBM cells via downregulation of CaM/CaMKII/c-Met axis and therefore CaMKII may be a novel therapeutic target to eliminate GSCs.     

Inhibition of anthrax lethal factor by curcumin and chemically modified curcumin derivatives

Abstract

Curcumin (diferuloylmethane), the active ingredient in the eastern spice turmeric (Curcuma longa), has been shown to inhibit the activities of numerous enzymes and signaling molecules involved in cancer, bacterial and viral infections and inflammatory diseases. We have investigated the inhibitory activities of curcumin and chemically modified curcumin (CMC) derivatives toward lethal factor (LF), the proteolytic component of anthrax toxin produced by the bacterium Bacillus anthracis. Curcumin (Compound 1) appears to inhibit the catalytic activity of LF through a mixture of inhibitory mechanisms, without significant compromise to the binding of oligopeptide substrates, and one CMC derivative in particular, Compound 3 (4-phenylaminocarbonylbis-demethoxycurcumin), is capable of inhibiting LF with potency comparable with the parent compound, while also showing improved solubility and stability. The quantitative reduction in catalytic activity achieved by the different CMC derivatives appears to be a function of the proportion of the multiple mechanisms through which they inhibit the enzyme.     

Anti-apoptotic effects of curcumin on photosensitized human epidermal carcinoma A431 cells

Photodynamic treatment (PDT) can elicit a diverse range of cellular responses, including apoptotic cell death. Previously, we showed that PDT stimulates caspase-3 activation and subsequent cleavage and activation of p21-activated kinase 2 (PAK2) in human epidermal carcinoma A431 cells. Curcumin, the yellow pigment of Curcuma longa, is known to have anti-oxidant and anti-inflammatory properties. In the present study, using Rose Bengal (RB) as the photosensitizer, we investigated the effect of curcumin on PDT-induced apoptotic events in human epidermal carcinoma A431 cells. We report that curcumin prevented PDT-induced JNK activation, mitochondrial release of cytochrome c, caspase-3 activation, and cleavage of PAK2. Using the cell permeable dye DCF-DA as an indicator of reactive oxygen species (ROS) generation, we found that both curcumin and ROS scavengers (i.e., l-histidine, a-tocopherol, mannitol) abolished PDT-stimulated intracellular oxidative stress. Moreover, all these PDT-induced apoptotic changes in cells could be blocked by singlet oxygen scavengers (i.e., l-histidine, a-tocopherol), but were not affected by the hydroxyl radical scavenger mannitol. In addition, we found that SP600125, a JNK-specific inhibitor, reduced PDT-induced JNK activation as well as caspase-3 activation, indicating that JNK activity is required for PDT-induced caspase activation. Collectively, these results demonstrate that singlet oxygen triggers JNK activation, cytochrome c release, caspase activation and subsequent apoptotic biochemical changes during PDT and show that curcumin is a potent inhibitor for this process.     

Fenofibrate triggers apoptosis of glioblastoma cells in vitro

Comment on: Wilk A, et al. Cell Cycle 2012; 11:2660-71.     

Impact of curcumin on toll-like receptors

Toll-like receptors (TLRs) have a pivotal role in the activation of innate immune response and inflammation. TLRs can be divided into two subgroups including extracellular TLRs that recognize microbial membrane components (TLR1, 2, 4, 5, 6, and 10), and intracellular TLRs that recognize microbial nucleic acids (TLR3, 7, 8, and 9). Curcumin is a dietary polyphenol from Curcuma longa L. that is reputed to have diverse biological and pharmacological effects. Extensive research has defined the molecular mechanisms through which curcumin mediates its therapeutic effects. One newly defined and important target of curcumin is the TLR, where it exerts an inhibitory effect. In the current study, we focus upon the TLR antagonistic effect of curcumin and curcumin's therapeutic effect as mediated via TLR inhibition. The available evidence indicates that curcumin inhibits the extracellular TLR 2 and 4 and intracellular TLR9 and thereby exerts a therapeutic effect in diseases such as cancer, inflammation, infection, autoimmune, and ischemic disease. Curcumin effectively modulates the TLR response and thereby exerts its potent therapeutic effects.     

A BMP7 variant inhibits the tumorigenic potential of glioblastoma stem-like cells

Glioblastoma multiforme (GBM) is among the most aggressive tumor types and is essentially an incurable malignancy characterized by resistance to chemo-, radio-, and immunotherapy. GBM is maintained by a hierarchical cell organization that includes stem-like, precursor, and differentiated cells. Recurrence and maintenance of the tumor is attributed to a small population of undifferentiated tumor-initiating cells, defined as glioblastoma stem-like cells (GSLCs). This cellular hierarchy offers a potential treatment to induce differentiation of GSLCs away from tumor initiation to a more benign phenotype or to a cell type more amenable to standard therapies. Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, have numerous biological activities including control of growth and differentiation. In vitro, a BMP7 variant (BMP7v) decreased primary human GSLC proliferation, endothelial cord formation, and stem cell marker expression while enhancing neuronal and astrocyte differentiation marker expression. In subcutaneous and orthotopic GSLC xenografts, which closely reproduce the human disease, BMP7v decreased tumor growth and stem cell marker expression, while enhancing astrocyte and neuronal differentiation compared with control mice. In addition, BMP7v reduced brain invasion, angiogenesis, and associated mortality in the orthotopic model. Inducing differentiation of GSLCs and inhibiting angiogenesis with BMP7v provides a potentially powerful and novel approach to the treatment of GBM.     

Interaction of carbon nanotubes with curcumin: Effect of temperature and pH on simultaneous static and dynamic fluorescence quenching of curcumin using carbon nanotubes

Curcumin (Cur) has medicinal properties, undergoes hydrolysis, and has low water solubility that limits its bioavailability and industrial usage. Different host molecules such as carbon nanotubes (CNT) can be useful in improving solubility and stabilizing Cur, therefore understanding the interaction of Cur with host molecules such as CNT is crucial. In this study, UV–visible light absorption and fluorescence spectroscopic techniques have been applied to reveal the interaction of Cur with CNT. Visible light absorption of Cur increases with CNT concentration, whereas fluorescence intensity of Cur is quenched in the presence of CNT. The obtained results confirm that fluorescence reduction is due to both static and dynamic quenching and is a result of the ground state and excited‐state complex formation. The pH environment influences the quenching rate due to deprotonation of Cur at higher pH; excess OH‐ ion concentration in the solution further discourages electrostatic interaction between the deprotonated form of Cur with CNT. It is found that at lower temperatures (<35°C) dynamic quenching is much more dominant and at higher temperatures (45°C) static quenching is more dominant. The interaction is further supported using X‐ray diffraction patterns and Fourier transform infrared spectra in the solid state, and suggests encapsulation of curcumin within the CNT. It is further evident that fluorescence quenching of Cur using CNT is further enhanced in the presence of several salts, as increase in ionic strength of the solution pushes the hydrophobic Cur molecule towards the CNT core by increasing the proximity between them.     

Inhibitory effect of curcumin on the invasion of rat ascites hepatoma cells in vitro and ex vivo

Curcumin, a yellow pigment in turmeric, is a food factor withantioxidative activity. The effect of curcumin on the proliferation and invasion of the rat ascites hepatoma AH109Acells was studied in vitro and ex vivo assay systems. Especially, a co-culture system of the hepatoma cellswith mesothelial cells derived from rat mesentery was employed to investigate the invasive motility. Curcumin suppressed thehepatoma slipping motility in a dose-dependent manner up to 5 M and thereafter maintained the effect up to 20 M, whereas this substance exerted little influence on the proliferation of the hepatoma cells at the same concentrations. Sera obtained from rats orally given curcumin also inhibited the AH109A cellular invasive movement when added to the culturemedium. Hepatoma cells previously cultured with hypoxanthineand xanthine oxidase showed a highly invasive activity. Curcumin and curcumin-loaded rat sera suppressed this reactive oxygen species-potentiated invasive capacity by simultaneously treating AH109A cells with hypoxanthine, xanthine oxidase and either of curcumin samples. These resultssuggest that the antioxidative property of curcumin may beinvolved in its anti-invasive action.     

Inhibition of autophagic flux by the curcumin analog EF-24 and its antiproliferative effect on MCF-7 cancer cells

5-Bis[(2-fluorophenyl)methylene]-4-piperidinone (EF-24) is a curcumin analog, which was identified for its physiochemical stability and diverse pharmacological functions. In the present study, EF-24 was added to the breast cancer cell line MCF-7 and its cellular effects were characterized. The results indicated that EF-24 possessed antiproliferative and antimigratory activities on MCF-7 cells as determined by MTT assay, wound healing, and transwell assay, respectively. In addition, the autophagosomal vesicles could be detected by acridine orange staining and electron microscope analysis in EF-24-treated cells. Conversion of LC3-I to LC3-II was also investigated following EF-24 treatment of the cells. However, the expression analysis of p62 and LC3 revealed that EF-24 could inhibit autophagic flux in MCF-7 cells. Confocal microscopy suggested that EF-24 could inhibit the degradation of autophagic vesicles by blocking the fusion of autophagosomes with lysosomes. EF-24 could also induce apoptosis of MCF-7 cells as determined by Hoechst 33342 staining, flow cytometry analysis, and western blot analysis. Moreover, treatment of the cells with the autophagy inhibitor 3-MA enhanced the PARP1 cleavage of EF-24-treated MCF-7 cells, which indicated the crosstalk between autophagy and apoptosis in breast cancer cells. Additional investigation of EF-24 should be performed in future studies to assess its antiproliferation and antimigratory effects on MCF-7 cells. However, the current results provide a solid foundation for the potential in vivo anticancer activity of this compound.     

姜黄素对慢性低氧高二氧化碳大鼠肺动脉高压及肺动脉管壁胶原的影响

目的：研究姜黄素对慢性低氧高二氧化碳大鼠肺动脉压力及肺动脉管壁Ⅰ型胶原的影响。方法：36只SD大鼠随机分为正常对照组（NC组）,低O2高CO2 4周组（HH组）,低O2高CO2 4周＋姜黄素组（HC组）,采用免疫组化、图像分析等方法观察姜黄素对慢性低O2高CO2大鼠肺动脉压力、肺细小动脉显微和超微结构及肺动脉管壁Ⅰ型胶原的影响。结果：①血流动力学检测显示HH组mPAP明显高于NC组（P〈0．01）,HC组mPAP明显低于HH组（P〈0．01）,三组间mCAP无明显差异（P〉0．05）;②光镜下,肺细小动脉管壁面积／管总面积比值（WA／TA）、肺细小动脉中膜平滑肌细胞核密度（SMC）、肺细小动脉中膜厚度（PAMT）HH组较NC组明显增高（均P〈0．01）,HC组WA／TA、SMC和PAMT较HH组明显降低（均P〈0．01）;③电镜下,HH组肺细小动脉中膜平滑肌细胞增生,面积增大,染色质增多,外膜胶原纤维密集,HC组大鼠肺细小动脉内皮细胞结构基本正常,胶原少见,中膜平滑肌细胞和外膜胶原纤维增生较HH组明显为轻;④免疫组化法发现肺细小动脉Ⅰ型胶原平均吸光度值HH组明显高于Nc组（P〈0．01）,HC组明显低于HH组（P〈0．01）。结论：姜黄素具有降低慢性低O2高CO2性肺动脉高压、改善肺血管重建及抑制肺动脉管壁Ⅰ型胶原沉积的作用。     

Sorafenib selectively depletes human glioblastoma tumor-initiating cells from primary cultures

Glioblastomas are grade IV brain tumors characterized by high aggressiveness and invasiveness, giving patients a poor prognosis. We investigated the effects of the multi-kinase inhibitor sorafenib on six cultures isolated from human glioblastomas and maintained in tumor initiating cells-enriching conditions. These cell subpopulations are thought to be responsible for tumor recurrence and radio- and chemo-resistance, representing the perfect target for glioblastoma therapy. Sorafenib reduces proliferation of glioblastoma cultures, and this effect depends, at least in part, on the inhibition of PI3K/Akt and MAPK pathways, both involved in gliomagenesis. Sorafenib significantly induces apoptosis/cell death via downregulation of the survival factor Mcl-1. We provide evidence that sorafenib has a selective action on glioblastoma stem cells, causing enrichment of cultures in differentiated cells, downregulation of the expression of stemness markers required to maintain malignancy (nestin, Olig2 and Sox2) and reducing cell clonogenic ability in vitro and tumorigenic potential in vivo. The selectivity of sorafenib effects on glioblastoma stem cells is confirmed by the lower sensitivity of glioblastoma cultures after differentiation as compared with the undifferentiated counterpart. Since current GBM therapy enriches the tumor in cancer stem cells, the evidence of a selective action of sorafenib on these cells is therapeutically relevant, even if, so far, results from first phase II clinical trials did not demonstrate its efficacy.     

PDGF signaling inhibits mitophagy in glioblastoma stem cells through N6-methyladenosine

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Heterogeneous phenotype of human glioblastoma: In vitro study

Glioblastomas (GBMs) are the most lethal primary brain tumours. Increasing evidence shows that brain tumours contain the population of stem cells, so‐called cancer stem cells (CSCs). Stem cell marker CD133 was reported to identify CSC population in GBM. Further studies have indicated that CD133 negative cells exhibiting similar properties and are able to initiate the tumour, self‐renew and undergo multilineage differentiation. GBM is a highly heterogeneous tumour and may contain different stem cell populations with different functional properties. We characterized five GBM cell lines, established from surgical samples, according to the marker expression, proliferation and differentiation potential. CD133 positive cell lines showed increased proliferation rate in neurosphere condition and marked differentiation potential towards neuronal lineages. Whereas two cell lines low‐expressing CD133 marker showed mesenchymal properties in vitro, that is high proliferation rate in serum condition and differentiation in mesenchymal cell types. Further, we compared therapy resistance capacity of GBM cell lines treated with hydroxyurea. Our results suggest that CSC concept is more complex than it was believed before, and CD133 could not define entire stem cell population within GBM. At least two different subtypes of GBM CSCs exist, which may have different biological characteristics and imply different therapeutic strategies. Copyright © 2013 John Wiley & Sons, Ltd.     

Evidence of curcumin and curcumin analogue effects in skin diseases: A narrative review

Curcumin, a natural polyphenolic and yellow pigment obtained from the spice turmeric, has strong antioxidative, anti-inflammatory, and antibacterial properties. Due to these properties, curcumin has been used as a remedy for the prevention and treatment of skin aging and disorders such as psoriasis, infection, acne, skin inflammation, and skin cancer. Curcumin has protective effects against skin damage caused by chronic ultraviolet B radiation. One of the challenges in maximizing the therapeutic potential of curcumin is its low bioavailability, limited aqueous solubility, and chemical instability. In this regard, the present review is focused on recent studies concerning the use of curcumin for the treatment of skin diseases, as well as offering new and efficient strategies to optimize its pharmacokinetic profile and increase its bioavailability.     

Ophiobolin A induces paraptosis-like cell death in human glioblastoma cells by decreasing BKCa channel activity

Glioblastoma multiforme (GBM) is the most lethal and common malignant human brain tumor. The intrinsic resistance of highly invasive GBM cells to radiation- and chemotherapy-induced apoptosis accounts for the generally dismal treatment outcomes. This study investigated ophiobolin A (OP-A), a fungal metabolite from Bipolaris species, for its promising anticancer activity against human GBM cells exhibiting varying degrees of resistance to proapoptotic stimuli. We found that OP-A induced marked changes in the dynamic organization of the F-actin cytoskeleton, and inhibited the proliferation and migration of GBM cells, likely by inhibiting big conductance Ca²⁺-activated K⁺ channel (BKCa) channel activity. Moreover, our results indicated that OP-A induced paraptosis-like cell death in GBM cells, which correlated with the vacuolization, possibly brought about by the swelling and fusion of mitochondria and/or the endoplasmic reticulum (ER). In addition, the OP-A-induced cell death did not involve the activation of caspases. We also showed that the expression of BKCa channels colocalized with these two organelles (mitochondria and ER) was affected in this programmed cell death pathway. Thus, this study reveals a novel mechanism of action associated with the anticancer effects of OP-A, which involves the induction of paraptosis through the disruption of internal potassium ion homeostasis. Our findings offer a promising therapeutic strategy to overcome the intrinsic resistance of GBM cells to proapoptotic stimuli.