Arsenic trioxide induces different gene expression profiles of genes related to growth and apoptosis in glioma cells dependent on the p53 status

We have previously reported that As(2)O(3) affected cell cycle progression and cyclins D1 and B1 expression in two glioma cell lines differing in p53 status (U87MG-wt; T98G-mutated). In the present study, we further demonstrated that As(2)O(3) affected proliferation, viability and apoptosis of the two cell lines in a dose- and time-dependent manner, and T98G cells were more sensitive than U87MG cells to As(2)O(3) -induced apoptosis and inhibition of proliferation and viability. We further investigated the expression profiles of genes related with apoptosis and cell cycle in the two cell lines with a human cDNA-microarray (SuperArray) spotted with 267 genes of apoptosis and cell cycle. Thirty five genes were upregulated and 15 genes downregulated at least 2-fold by As(2)O(3) in U87-MG cells; whereas, 38 genes were upregulated and 21 genes downregulated at least 2-fold in T98G cells by As(2)O(3). After As(2)O(3) treatment, p53 expression was upregulated 56.5-fold in T98G cells, but only 6.0-fold in U87MG cells. The results indicate that As(2)O(3) suppresses the growth of U87MG cells mainly by regulating expression of genes of cell cycle arrest, stress and toxicity; whereas As(2)O(3) affects T98G cells mainly by regulating expression of genes belonging to Bcl-2, tumor necrotic factor receptor and ligand families. The data may be helpful for optimizing As(2)O(3) as an anti-cancer drug in the treatment of gliomas.     

Synthesis and biological assay of new 2’-deoxyuridine dimers containing a 1,2,3-triazole linker. Part I

We describe a simple method for the synthesis of modified dinucleosides containing pyrimidine nucleoside analogues (2’-deoxyuridine, thymidine and 5-fluoro-2’-deoxyuridine). Six different dimers with a 1,2,3-triazole linkage were obtained by azide–alkyne 1,3-dipolar cycloaddition (click reaction), starting from propargylated 2’-deoxyuridine and 5’-azido-nucleoside derivatives. Their cytotoxic activity was tested in five human cancer cell lines: cervical (HeLa), high grade gliomas (U-118?MG, U-87?MG, T98G), liver (HepG2), and normal human fibroblast cell line (MRC-5) using the sulforhodamine B (SRB) assay. The experiment showed that the obtained dimers with a 1,2,3-triazole moiety were very stable compounds, also in the physiological-like media, and had no anticancer activity.     

Changes in the expression of proteins associated with aerobic glycolysis and cell migration are involved in tumorigenic ability of two glioma cell lines

ABSTRACT: BACKGROUND: The most frequent and malignant brain cancer is glioblastoma multiforme (GBM). In gliomas, tumor progression and poor prognosis are associated with the tumorigenic ability of the cells. U87MG cells (wild-type p53) are known to be tumorigenic in nude mice, but T98G cells (mutant p53) are not tumorigenic. We investigated the proteomic profiling of these two cell lines in order to gain new insights into the mechanisms that may be involved in tumorigenesis. RESULTS: We found 24 differentially expressed proteins between T98G and U87MG cells. Gene Ontology supports the notion that over-representation of differentially expressed proteins is involved in glycolysis, cell migration and stress oxidative response. Among those associated with the glycolysis pathway, TPIS and LDHB are up-regulated in U87MG cells. Measurement of glucose consumption and lactate production suggests that glycolysis is more effective in U87MG cells. On the other hand, G6PD expression was 3-fold higher in T98G cells and this may indicate a shift to the pentose-phosphate pathway. Moreover, GRP78 expression was also three-fold higher in T98G than in U87MG cells. Under thapsigargin treatment both cell lines showed increased GRP78 expression and the effect of this agent was inversely correlated to cell migration. Quantitative RT-PCR and immunohistochemistry of GRP78 in patient samples indicated a higher level of expression of GRP78 in grade IV tumors compared to grade I and non-neoplastic tissues, respectively. CONCLUSIONS: Taken together, these results suggest an important role of proteins involved in key functions such as glycolysis and cell migration that may explain the difference in tumorigenic ability between these two glioma cell lines and that may be extrapolated to the differential aggressiveness of glioma tumors.     

Increase in secretion of glial cell line-derived neurotrophic factor from glial cell lines by inhibitors of vacuolar ATPase

Glial cell line-derived neurotrophic factor (GDNF) was reported to be effective for treating subjects with neurodegenerative diseases such as Parkinson's disease. In search of finding a compound which promotes GDNF secretion, we found that concanamycin A (ConA), a vacuolar ATPase (V-type ATPase) inhibitor purified from Streptomyces diastatochromogens, enhanced GDNF secretion from glioma cells. The rat glioma cell line, C6, and the human glioma cell lines, U87MG and T98G, abundantly expressed GDNF mRNA, and secreted GDNF into culture media, and this event was potently enhanced by a Ca(2+) ionophore and by phorbol ester, as noted in other cells. ConA concentration dependently and potently increased GDNF release from C6, U87MG and T98G cells into culture media. In addition, ConA enhanced GDNF secretion from astrocyte primary cultures prepared from the human fetus with the same potency seen in glioma cell lines. Likewise, another V-type ATPase inhibitor, bafilomycinA1 facilitated GDNF release from C6, U87MG and T98G glioma cells, in a concentration-dependent manner. The potencies of these V-type ATPase inhibitors in enhancing GDNF secretion were consistent with those which inhibited V-type ATPase activity. These results suggest that blockade of V-type ATPase potently stimulates the secretion of GDNF from glial cells. The V-type ATPase inhibitors may be beneficial to use for the treatment of diseases in which increase in GDNF could be effective.     

太白银莲花皂苷B对胶质瘤细胞生长抑制的研究

目的:太白银莲花皂苷B(Anemone taipaiensis saponin B)是第一次从太白银莲花中经过系统化学分析和分离鉴定的皂苷之一,所以它的生物学效应目前仍然不清楚。在本研究中,我们首次体外研究太白银莲花皂苷B对胶质瘤细胞系的生物学效应,观察它对胶质瘤细胞增殖的的抑制作用。方法:采用四甲基偶氮唑蓝(MTT)法测定太白银莲花皂苷B对胶质瘤细胞生长曲线的影响,Hoechst 33342细胞核染色后荧光显微镜观察,采用光学显微镜观察细胞的形态学变化。结果:MTT实验结果显示太白银莲花皂苷B对胶质瘤细胞U87MG和U251MG有强烈的生长抑制作用,且具有剂量依赖性,应用SPSS18.0统计软件得出太白银莲花皂苷B对U87MG细胞72 h的抑制浓度为IC25=5.2μmol/L,IC50=6.7μmol/L and IC75=8.7μmol/L,U251细胞的抑制浓度为IC25=6.2μmol/L,IC50=7.9μmol/L and IC75=10.5μmol/L。Hoechst 33342细胞核染色荧光显微镜观察以及光学显微镜下细胞形态观察显示出典型的凋亡细胞形态学特征,经过皂苷B处理后,细胞皱缩成圆球形,细胞核碎裂或者致密浓染,向核膜边缘聚集,染色质浓缩为半月状、车轮状或者马蹄状,凋亡小体出现。这些特征在24 h时更明显。结论:体外实验初步显示,太白银莲花皂苷B对U87MG和U251MG细胞具有明显的增殖抑制作用,并具有促凋亡作用。     

Identification of imidazo[4,5-c]pyridin-2-one derivatives as novel Src family kinase inhibitors against glioblastoma

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumour in the central nervous system (CNS). As the ideal targets for GBM treatment, Src family kinases (SFKs) have attracted much attention. Herein, a new series of imidazo[4,5-c]pyridin-2-one derivatives were designed and synthesised as SFK inhibitors. Compounds 1d, 1e, 1q, 1s exhibited potential Src and Fyn kinase inhibition in the submicromolar range, of which were next tested for their antiproliferative potency on four GBM cell lines. Compound 1s showed effective activity against U87, U251, T98G, and U87-EGFRvIII GBM cell lines, comparable to that of lead compound PP2. Molecular dynamics (MDs) simulation revealed the possible binding patterns of the most active compound 1s in ATP binding site of SFKs. ADME prediction suggested that 1s accord with the criteria of CNS drugs. These results led us to identify a novel SFK inhibitor as candidate for GBM treatment.     

Fever-Range Hyperthermia vs. Hypothermia Effect on Cancer Cell Viability,Proliferation and HSP90 Expression

Purpose

The current study examines the effect of fever-range hyperthermia and mild hypothermia on human cancer cells focusing on cell viability, proliferation and HSP90 expression.

Materials and Methods

A549 and H1299 lung carcinoma, MCF7 breast adenocarcinoma, U87MG and T98G glioblastoma, DU145 and PC3 prostate carcinoma and MRC5 normal fetal lung fibroblasts cell lines were studied. After 3-day exposure to 34°C, 37°C and 40°C, cell viability was determined. Cell proliferation (ki67 index), apoptosis (Caspase 9) and HSP90 expression was studied by confocal microscopy.

Results

Viability/proliferation experiments demonstrated that MRC5 fibroblasts were extremely sensitive to hyperthermia, while they were the most resistant to hypothermia. T98G and A549 were thermo-tolerant, the remaining being thermo-sensitive to a varying degree. Nonetheless, as a universal effect, hypothermia reduced viability/proliferation in all cell lines. Hyperthermia sharply induced Caspase 9 in the U87MG most thermo-sensitive cell line. In T98G and A549 thermo-tolerant cell lines, the levels of Caspase 9 declined. Moreover, hyperthermia strongly induced the HSP90 levels in T98G, whilst a sharp decrease was recorded in the thermo-sensitive PC3 and U87MG cell lines. Hyperthermia sensitized thermo-sensitive cancer cell lines to cisplatin and temozolomide, whilst its sensitizing effect was diminished in thermo-tolerant cell lines.

Conclusions

The existence of thermo-tolerant and thermo-sensitive cancer cell lines was confirmed, which further encourages research to classify human tumor thermic predilection for patient stratification in clinical trials. Of interest, mild hypothermia had a universal suppressing effect on cancer cell proliferation, further supporting the radio-sensitization hypothesis through reduction of oxygen and metabolic demands.     

The role of protein arginine-methyltransferase 1 in gliomagenesis

Protein arginine methyltransferase 1 (PRMT1), a type-I arginine methyltransferase, has been implicated in diverse cellular events. We have focused on the role of PRMT1 in gliomagenesis. In this study, we showed that PRMT1 expression was up-regulated in glioma tissues and cell lines compared with normal brain tissues. The knock-down of PRMT1 resulted in an arrest in the G1-S phase of the cell cycle, proliferation inhibition and apoptosis induction in four glioma cell lines (T98G, U87MG, U251, and A172). Moreover, an in vivo study confirmed that the tumor growth in nude mouse xenografts was significantly decreased in the RNAi-PRMT1 group. Additionally, we found that the level of the asymmetric dimethylated modification of H4R3, a substrate of PRMT1, was higher in glioma cells than in normal brain tissues and decreased after PRMT1 knock-down. Our data suggest a potential role for PRMT1 as a novel biomarker of and therapeutic target in gliomas. [BMB Reports 2012; 45(8): 470-475].     

Inactivation of PI3K/Akt signaling mediates proliferation inhibition and G2/M phase arrest induced by andrographolide in human glioblastoma cells

AimsAndrographolide, a principal diterpenoid lactone isolated from the traditional herbal medicine Andrographis paniculata, has been reported to show anti-tumor activity. Since the high lipid solubility of andrographolide permits it to penetrate the blood–brain barrier and concentrate in the brain, we hypothesized that andrographolide may be a potential chemotherapeutic agent for the treatment of glioblastomas. To clarify this point, we investigated the growth inhibitory effect and mechanisms of actions of andrographolide on human glioblastoma U251 and U87 cells.Main methodsMTT assay and trypan blue exclusion assay were used to investigate the proliferation inhibitory and cytotoxic effects of andrographolide, respectively. Cell cycle distribution was analyzed using flow cytometry. Apoptosis analysis proceeded by detecting the cleavage of caspase-3. The levels of proteins were probed by Western blotting.Key findingsThe results showed that non-toxic concentrations of andrographolide inhibited the proliferation of human glioblastoma cells through induction of G2/M arrest, which was accompanied by down-regulating Cdk1 and Cdc25C proteins. Additionally, andrographolide decreased the activity of PI3K/Akt signaling, as demonstrated by down-regulation of the expression of phos-PI3K, phos-Akt, phos-mTOR and phos-p70s6k in U251 and U87 cells. Furthermore, additive effects on the proliferation inhibition, G2/M arrest and down-regulation of G2/M phase-related proteins were observed, when a combined treatment of andrographolide with PI3K inhibitor LY294002 was used in U251 and U87 cells.SignificanceWe prove that andrographolide inhibits the proliferation of human glioblastoma cells via inducing G2/M arrest, which is mediated by inhibiting the activity of PI3K/Akt signaling.     

Cloning, expression, and characterization of TNFSF14 (LIGHT) gene in mefugu, Takifugu obscurus

Tumor necrosis factor receptor-associated factor 6 (TRAF6), which plays an important role in inflammation and immune response, is an essential adaptor protein for the NF-κB (nuclear factor κB) signaling pathway. Recent studies have shown that TRAF6 played an important role in tumorigenesis and invasion by suppressing NF-κB activation. However, up to now, the biologic role of TRAF6 in glioma has still remained unknown. To address the expression of TRAF6 in glioma cells, four glioma cell lines (U251, U-87MG, LN-18, and U373) and a non-cancerous human glial cell line SVG p12 were used to explore the protein expression of TRAF6 by Western blot. Our results indicated that TRAF6 expression was upregulated in human glioma cell lines, especially in metastatic cell lines. To investigate the role of TRAF6 in cell proliferation, apoptosis, invasion, and migration of glioma, we generated human glioma U-87MG cell lines in which TRAF6 was either overexpressed or depleted. Subsequently, the effects of TRAF6 on cell viability, cell cycle distribution, apoptosis, invasion, and migration in U-87MG cells were determined with 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry analysis, transwell invasion assay, and wound-healing assay. The results showed that knockdown of TRAF6 could decrease cell viability, suppress cell proliferation, invasion and migration, and promote cell apoptosis, whereas overexpression of TRAF6 displayed the opposite effects. In addition, the effects of TRAF6 on the expression of phosphor-NF-κB (p-p65), cyclin D1, caspase 3, and MMP-9 were also probed. Knockdown of TRAF6 could lower the expression of p-p65, cyclin D1, and MMP-9, and raise the expression of caspase 3. All these results suggested that TRAF6 might be involved in the potentiation of growth, proliferation, invasion, and migration of U-87MG cell, as well as inhibition of apoptosis of U-87MG cell by abrogating activation of NF-κB.     

Synthesis and evaluation of diterpenic Mannich bases as antiviral agents against influenza A and SARS-CoV-2

A chemical library was constructed based on the resin acids (abietic, dehydroabietic, and 12-formylabietic) and its diene adducts (maleopimaric and quinopimaric acid derivatives). The one-pot three-component CuCl-catalyzed aminomethylation of the abietane diterpenoid propargyl derivatives was carried out by formaldehyde and secondary amines (diethylamine, pyrrolidine, morpholine, and homopiperazine). All compounds were tested for cytotoxicity and antiviral activity against influenza virus A/Puerto Rico/8/34 (H1N1) in MDCK cells and SARS-CoV-2 pseudovirus in BHK-21-hACE2 cells. Among 21 tested compounds, six derivatives demonstrated a selectivity index (SI) higher than 10, and their IC₅₀ values ranged from 0.19 to 5.0 μM. Moreover, two derivatives exhibited potent anti-SARS-CoV-2 infection activity. The antiviral activity and toxicity strongly depended on the nature of the diterpene core and heterocyclic substituent. Compounds 12 and 21 bearing pyrrolidine moieties demonstrated the highest virus-inhibiting activity with SIs of 128.6 and 146.8, respectively, and appeared to be most effective when added at the time points 0–10 and 1–10 h of the viral life cycle. Molecular docking and dynamics modeling were adopted to investigate the binding mode of compound 12 into the binding pocket of influenza A virus M2 protein. Compound 9 with a pyrrolidine group at C20 of 17-formylabietic acid was a promising anti-SARS-CoV-2 agent with an EC₅₀ of 10.97 µM and a good SI value > 18.2. Collectively, our data suggested the potency of diterpenic Mannich bases as effective anti-influenza and anti-COVID-19 compounds.     

Susceptibility to exogenously added interferon-beta protein depends on intracellular interferon-beta mRNA level in human glioma cells

Exogenously added human interferon-beta (HuIFN-beta) protein possesses a remarkable antiproliferative activity in human glioma and melanoma. Endogenous HuIFN-beta protein, which is produced by its gene transfer using cationic liposomes, has much more effective antiproliferative activity against these tumors, even in cells resistant to exogenously added HuIFN-beta protein. As the first step to elucidate the possible difference in antiproliferative mechanisms between exogenous and endogenous HuIFN-beta protein, we here investigated the relationship between the intracellular level of its mRNA and susceptibility to exogenously added HuIFN-beta protein. In this study, we used seven human glioma cell lines (SK-MG-1, SK-MG-4, SK-AO2, U87MG, U251SP, U251MG and T98) and one human melanoma cell line (MMAN). At first, we examined the relationship between spontaneous expression of HuIFN-beta mRNA and susceptibility to exogenously added HuIFN-beta protein (50 IU/ml) in human glioma cells and then confirmed a significant correlation between them. Next, we confirmed that administration of 0-100 IU/ml exogenously added HuIFN-beta protein upregulated the HuIFN-beta mRNA in a dose-dependent manner using the RT-PCR technique and that the HuIFN-beta mRNA was suppressed by siRNA for HuIFN-beta in SK-MG-1 and MMAN cells. Furthermore, we confirmed that the siRNA for HuIFN-beta significantly suppressed the antiproliferative effect of SK-MG-1 cells treated with 10-100 IU/ml HuIFN-beta protein and MMAN cells with 25 and 50 IU/ml HuIFN-beta protein. We found this phenomenon in another human glioma cell line, U87MG cells, as well. This finding would suggest that susceptibility to exogenously added HuIFN-beta protein is related to the amount of intracellular HuIFN-beta mRNA in human glioma and melanoma cells.     

Effects of 1α,25-dihydroxyvitamin D3 and tacalcitol on cell signaling and anchorage-independent growth in T98G and U251 glioblastoma cells

The active hormonal form of vitamin D, 1α,25-dihydroxyvitamin D₃, is reported to have 1000s of biological targets. The growth-suppressive properties of 1α,25-dihydroxyvitamin D₃ and its synthetic analogs have attracted interest for the development of treatment and/or prevention of cancer. We examined effects of 1α,25-dihydroxyvitamin D₃ and the vitamin D analog tacalcitol on signaling pathways and anchorage-independent growth in T98G and U251 glioblastoma cells. Assay of signaling proteins important for cellular growth indicated suppression of p70-S6 kinase levels by 1α,25-dihydroxyvitamin D₃ and tacalcitol in T98G cells, whereas the levels of PLCγ, a target for phospholipid signaling, was slightly increased.Activation of STAT3, an important regulator of malignancy, was suppressed by 1α,25-dihydroxyvitamin D₃ and tacalcitol in T98G and U251 cells. However, despite the close structural similarity of these compounds, suppression was stronger by tacalcitol (1α,24-dihydroxyvitamin D₃), indicating that even minor modifications of a vitamin D analog can impact its effects on signaling. Experiments using soft agar colony formation assay in T98G and U251 cells revealed significant suppression by 1α,25-dihydroxyvitamin D₃ and tacalcitol on anchorage-independent growth, a property for cancer invasion and metastasis known to correlate with tumorigenicity. These findings indicate that vitamin D and its analogs may be able to counteract the oncogenic transformation, invasion and metastatic potential of glioblastoma and prompt further study of these compounds in the development of improved therapy for brain cancer.     

Saponin 1 Induces Apoptosis and Suppresses NF-κB-Mediated Survival Signaling in Glioblastoma Multiforme (GBM)

Saponin 1 is a triterpeniod saponin extracted from Anemone taipaiensis, a traditional Chinese medicine against rheumatism and phlebitis. It has also been shown to exhibit significant anti-tumor activity against human leukemia (HL-60 cells) and human hepatocellular carcinoma (Hep-G2 cells). Herein we investigated the effect of saponin 1 in human glioblastoma multiforme (GBM) U251MG and U87MG cells. Saponin 1 induced significant growth inhibition in both glioblastoma cell lines, with a 50% inhibitory concentration at 24 h of 7.4 µg/ml in U251MG cells and 8.6 µg/ml in U87MG cells, respectively. Nuclear fluorescent staining and electron microscopy showed that saponin 1 caused characteristic apoptotic morphological changes in the GBM cell lines. Saponin 1-induced apoptosis was also verified by DNA ladder electrophoresis and flow cytometry. Additionally, immunocytochemistry and western blotting analyses revealed a time-dependent decrease in the expression and nuclear location of NF-κB following saponin 1 treatment. Western blotting data indicated a significant decreased expression of inhibitors of apoptosis (IAP) family members,(e.g., survivin and XIAP) by saponin 1. Moreover, saponin 1 caused a decrease in the Bcl-2/Bax ratio and initiated apoptosis by activating caspase-9 and caspase-3 in the GBM cell lines. These findings indicate that saponin 1 inhibits cell growth of GBM cells at least partially by inducing apoptosis and inhibiting survival signaling mediated by NF-κB. In addition, in vivo study also demonstrated an obvious inhibition of saponin 1 treatment on the tumor growth of U251MG and U87MG cells-produced xenograft tumors in nude mice. Given the minimal toxicities of saponin 1 in non-neoplastic astrocytes, our results suggest that saponin 1 exhibits significant in vitro and in vivo anti-tumor efficacy and merits further investigation as a potential therapeutic agent for GBM.     

Anti-tumor activities of luteolin and silibinin in glioblastoma cells: overexpression of miR-7-1-3p augmented luteolin and silibinin to inhibit autophagy and induce apoptosis in glioblastoma in vivo

Glioblastoma is the deadliest brain tumor in humans. High systemic toxicity of conventional chemotherapies prompted the search for natural compounds for controlling glioblastoma. The natural flavonoids luteolin (LUT) and silibinin (SIL) have anti-tumor activities. LUT inhibits autophagy, cell proliferation, metastasis, and angiogenesis and induces apoptosis; while SIL activates caspase-8 cascades to induce apoptosis. However, synergistic anti-tumor effects of LUT and SIL in glioblastoma remain unknown. Overexpression of tumor suppressor microRNA (miR) could enhance the anti-tumor effects of LUT and SIL. Here, we showed that 20 µM LUT and 50 µM SIL worked synergistically for inhibiting growth of two different human glioblastoma U87MG (wild-type p53) and T98G (mutant p53) cell lines and natural combination therapy was more effective than conventional chemotherapy (10 µM BCNU or 100 µM TMZ). Combination of LUT and SIL caused inhibition of growth of glioblastoma cells due to induction of significant amounts of apoptosis and complete inhibition of invasion and migration. Further, combination of LUT and SIL inhibited rapamycin (RAPA)-induced autophagy, a survival mechanism, with suppression of PKCα and promotion of apoptosis through down regulation of iNOS and significant increase in expression of the tumor suppressor miR-7-1-3p in glioblastoma cells. Our in vivo studies confirmed that overexpression of miR-7-1-3p augmented anti-tumor activities of LUT and SIL in RAPA pre-treated both U87MG and T98G tumors. In conclusion, our results clearly demonstrated that overexpression of miR-7-1-3p augmented the anti-tumor activities of LUT and SIL to inhibit autophagy and induce apoptosis for controlling growth of different human glioblastomas in vivo.