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.     

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。     

Dominant negative pleiotrophin induces tetraploidy and aneuploidy in U87MG human glioblastoma cells

Pleiotrophin (PTN, Ptn) is an 18kDa secretory cytokine that is expressed in many human cancers, including glioblastoma. In previous experiments, interruption of the constitutive PTN signaling in human U87MG glioblastoma cells that inappropriately express endogenous Ptn reversed their rapid growth in vitro and their malignant phenotype in vivo. To seek a mechanism for the effect of the dominant-negative PTN, flow cytometry was used to compare the profiles of U87MG cells and four clones of U87MG cells that express the dominant-negative PTN (U87MG/PTN1-40 cells); here, we report that the dominant-negative PTN in U87MG cells induces tetraploidy and aneuploidy and arrests the tetraploid and aneuploid cells in the G1 phase of the cell cycle. The data suggest that PTN signaling may have a critical role in chromosomal segregation and cell cycle progression; the data suggest induction of tetraploidy and aneuploidy in U87MG glioblastoma cells may be an important mechanism that contributes to the loss of the malignant phenotype of U87MG cells.     

Quantitative proteomic analysis and functional studies reveal that nucleophosmin is involved in cell death in glioblastoma cell line transfected with siRNA

Previously, we reported that nucleophosmin (NPM) was increased in glioblastoma multiforme (GBM). NPM is a phosphoprotein related to apoptosis, ribosome biogenesis, mitosis, and DNA repair, but details about its function remain unclear. We treated U87MG and A172 cells with small interference RNA (siRNA) and obtained a reduction of 80% in NPM1 expression. Knockdown at the protein level was evident after the 4th day and was maintained until the 7th day of transfection that was investigated by quantitative proteomic analysis using isobaric tags. The comparison of proteomic analysis of NPM1-siRNA against controls allowed the identification of 14 proteins, two proteins showed increase and 12 presented a reduction of expression levels. Gene ontology assigned most of the hypoexpressed proteins to apoptosis regulation, including GRP78. NPM1 silencing did not impair cell proliferation until the 7th day after transfection, but sensitized U87MG cells to temozolomide (TMZ), culminating with an increase in cell death and provoking at a later period a reduction of colony formation. In a large data set of GBM patients, both GRP78 and NPM1 genes were upregulated and presented a tendency to shorter overall survival time. In conclusion, NPM proved to participate in the apoptotic process, sensitizing TMZ-treated U87MG and A172 cells to cell death, and in association with upregulation of GRP78 may be helpful as a predictive factor of poor prognosis in GBM patients.     

EMAP-II sensitize U87MG and glioma stem-like cells to temozolomide via induction of autophagy-mediated cell death and G2/M arrest

Despite the fact that temozolomide (TMZ) has been widely accepted as the key chemotherapeutic agent to prolong the survival of patients with glioblastoma, failure and recurrence cases can still be observed in clinics. Glioma stem-like cells (GSCs) are thought to be responsible for the drug resistance. In this study, we investigate whether endothelial monocyte-activating polypeptide-II (EMAP-II), a pro-inflammatory cytokine, can enhance TMZ cytotoxicity on U87MG and GSCs or not. As described in prior research, GSCs have been isolated from U87MG and maintained in the serum-free DMEM/F12 medium containing EGF, b-FGF, and B27. TMZ and/or EMAP-II administration were performed for 72 h, respectively. The results showed that TMZ combined with EMAP-II inhibit the proliferation of U87MG and GSCs by a larger measure than TMZ single treatment by decreasing the IC₅₀. EMAP-II also enhanced TMZ-induced autophagy-mediated cell death and G2/M arrest. Moreover, we found that EMAP-II functioned a targeted suppression on mTOR, which may involve in the anti-neoplasm mechanism. The results suggest that EMAP-II could be considered as a combined chemotherapeutic agent against glioblastoma by sensitizing U87MG and GSCs to TMZ.     

Angiogenin Promotes U87MG Cell Proliferation by Activating NF-κB Signaling Pathway and Downregulating Its Binding Partner FHL3

Angiogenin (Ang) is known to induce cell proliferation and inhibit apoptosis by cellular signaling pathways and its direct nuclear functions, but the mechanism of action for Ang in astrocytoma is not yet clear. Astrocytoma is the most frequent one among various neurogliomas, of which a subtype known as glioblastoma multiforme (GBM) is the most malignant brain glioma and seriously influences the life quality of the patients. The expression of Ang and Bcl-xL were detected in 28 cases of various grades of astrocytoma and 6 cases of normal human tissues by quantitative real-time PCR. The results showed that the expression of Ang and Bcl-xL positively correlated with the malignant grades. Cytological experiments indicated that Ang facilitated human glioblastoma U87MG cell proliferation and knock-down of endogenous Ang promoted cell apoptosis. Furthermore, Ang activated NF-κB pathway and entered the U87MG cell nuclei, and blocking NF-κB pathway or inhibiting Ang nuclear translocation partially suppressed Ang-induced cell proliferation. The results suggested that Ang participated in the regulation of evolution process of astrocytoma by interfering NF-κB pathway and its nucleus function. In addition, four and a half LIM domains 3 (FHL3), a novel Ang binding partner, was required for Ang-mediated HeLa cell proliferation in our previous study. We also found that knockdown of FHL3 enhanced IκBα phosphorylation and overexpression of Ang inhibited FHL3 expression in U87MG cells. Together our findings suggested that Ang could activate NF-κB pathway by regulating the expression of FHL3. In conclusion, the present study established a link between Ang and FHL3 proteins and identifies a new pathway for regulating astrocytoma progression.     

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.     

Curcumin Suppressed Anti-apoptotic Signals and Activated Cysteine Proteases for Apoptosis in Human Malignant Glioblastoma U87MG Cells

Glioblastoma is the most malignant human brain tumor that shows poor response to existing therapeutic agents. Search continues for an effective therapy for controlling this deadliest brain tumor. Curcumin (CCM), a polyphenolic compound from Curcuma longa, possesses anti-cancer properties in both in vitro and in vivo. In the present investigation, we evaluated the therapeutic efficacy of CCM against human malignant glioblastoma U87MG cells. Trypan blue dye exclusion test showed decreased viability of U87MG cells with increasing dose of CCM. Wright staining and ApopTag assay, respectively, showed the morphological and biochemical features of apoptosis in U87MG cells treated with 25 μM and 50 μM of CCM for 24 h. Western blotting showed activation of caspase-8, cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, and release of cytochrome c from mitochondria followed by activation of caspase-9 and caspase-3 for apoptosis. Also, CCM treatments increased cytosolic level of Smac/Diablo to suppress the inhibitor-of-apoptosis proteins and down regulated anti-apoptotic nuclear factor kappa B (NFκB), favoring the apoptosis. Increased activities of calpain and caspase-3 cleaved 270 kDa α-spectrin at specific sites generating 145 kDa spectrin break down product (SBDP) and 120 kDa SBDP, respectively, leading to apoptosis in U87MG cells. Results show that CCM is an effective therapeutic agent for suppression of anti-apoptotic factors and activation of calpain and caspase proteolytic cascades for apoptosis in human malignant glioblastoma cells. Special issue in honor of Naren Banik.     

M2 receptor activation inhibits cell cycle progression and survival in human glioblastoma cells

Muscarinic receptors, expressed in several primary and metastatic tumours, appear to be implicated in their growth and propagation. In this work we have demonstrated that M2 muscarinic receptors are expressed in glioblastoma human specimens and in glioblastoma cell lines. Moreover, we have characterized the effects of the M2 agonist arecaidine on cell growth and survival both in two different glioblastoma cell lines (U251MG and U87MG) and in primary cultures obtained from different human biopsies. Cell growth analysis has demonstrated that the M2 agonist arecaidine strongly decreased cell proliferation in both glioma cell lines and primary cultures. This effect was dose and time dependent. FACS analysis has confirmed cell cycle arrest at G1/S and at G2/M phase in U87 cells and U251 respectively. Cell viability analysis has also shown that arecaidine induced severe apoptosis, especially in U251 cells. Chemosensitivity assays have, moreover, shown arecaidine and temozolomide similar effects on glioma cell lines, although IC50 value for arecaidine was significantly lower than temozolomide. In conclusion, we report for the first time that M2 receptor activation has a relevant role in the inhibition of glioma cell growth and survival, suggesting that M2 may be a new interesting therapeutic target to investigate for glioblastoma therapy.     

The curry spice curcumin selectively inhibits cancer cells growth in vitro and in preclinical model of glioblastoma

Previous studies suggested that curcumin is a potential agent against glioblastomas (GBMs). However, the in vivo efficacy of curcumin in gliomas remains not established. In this work, we examined the mechanisms underlying apoptosis, selectivity, efficacy and safety of curcumin from in vitro (U138MG, U87, U373 and C6 cell lines) and in vivo (C6 implants) models of GBM. In vitro, curcumin markedly inhibited proliferation and migration and induced cell death in liquid and soft agar models of GBM growth. Curcumin effects occurred irrespective of the p53 and PTEN mutational status of the cells. Interestingly, curcumin did not affect viability of primary astrocytes, suggesting that curcumin selectivity targeted transformed cells. In U138MG and C6 cells, curcumin decreased the constitutive activation of PI3K/Akt and NFkappaB survival pathways, down-regulated the antiapoptotic NFkappaB-regulated protein bcl-xl and induced mitochondrial dysfunction as a prelude to apoptosis. Cells developed an early G2/M cell cycle arrest followed by sub-G1 apoptosis and apoptotic bodies formation. Caspase-3 activation occurred in the p53-normal cell type C6, but not in the p53-mutant U138MG. Besides its apoptotic effect, curcumin also synergized with the chemotherapeutics cisplatin and doxorubicin to enhance GBM cells death. In C6-implanted rats, intraperitoneal curcumin (50 mg kg(-1) d(-1)) decreased brain tumors in 9/11 (81.8%) animals against 0/11 (0%) in the vehicle-treated group. Importantly, no evidence of tissue (transaminases, creatinine and alkaline phosphatase), metabolic (cholesterol and glucose), oxidative or hematological toxicity was observed. In summary, data presented here suggest curcumin as a potential agent for therapy of GBMs.     

A non-crosslinking platinum-acridine hybrid agent shows enhanced cytotoxicity compared to clinical BCNU and cisplatin in glioblastoma cells

Using clonogenic survival assays, we demonstrated that a new platinum-acridine hybrid agent, PT-ACRAMTU, is cytotoxic in SNB19 and U87MG glioblastoma cells at low-micromolar concentrations. PT-ACRAMTU is more cytotoxic than ACRAMTU (the platinum-free acridine), acts in a time and dose dependent manner, and appears to generate an apoptotic response in both cell lines on the basis of increased caspase-3 activity.     

Photofrin Based Photodynamic Therapy and miR-99a Transfection Inhibited FGFR3 and PI3K/Akt Signaling Mechanisms to Control Growth of Human Glioblastoma In Vitro and In Vivo

Glioblastoma is the most common malignant brain tumor in humans. We explored the molecular mechanisms how the efficacy of photofrin based photodynamic therapy (PDT) was enhanced by miR-99a transfection in human glioblastoma cells. Our results showed almost similar uptake of photofrin after 24 h in different glioblastoma cells, but p53 wild-type cells were more sensitive to radiation and photofrin doses than p53 mutant cells. Photofrin based PDT induced apoptosis, inhibited cell invasion, prevented angiogenic network formation, and promoted DNA fragmentation and laddering in U87MG and U118MG cells harvoring p53 wild-type. Western blotting showed that photofrin based PDT was efficient to block the angiogenesis and cell survival pathways. Further, photofrin based PDT followed by miR-99a transfection dramatically increased miR-99a expression and also increased apoptosis in glioblastoma cell cultures and drastically reduced tumor growth in athymic nude mice, due to down regulation of fibroblast growth factor receptor 3 (FGFR3) and PI3K/Akt signaling mechanisms leading to inhibition of cell proliferation and induction of molecular mechanisms of apoptosis. Therefore, our results indicated that the anti-tumor effects of photofrin based PDT was strongly augmented by miR-99a overexpression and this novel combination therapeutic strategy could be used for controlling growth of human p53 wild-type glioblastomas both in vitro and in vivo.     

The short chain cell-permeable ceramide (C6) restores cell apoptosis and perifosine sensitivity in cultured glioblastoma cells

Primary glioblastoma multiforme is the most malignant form of astrocytic tumor with an average survival of approximately 12–14 months. The combination of novel Akt inhibitors with anti-cancer therapeutics has achieved improved anti-tumor efficiency. In the current study, we examined the synergistic anti-cancer ability of Akt inhibitor perifosine in combination with short-chain ceramide (C6) against glioblastoma cells (U87MG and U251MG), and studied the underlying mechanisms. We found that perifosine, which blocked Akt/mammalian target of rapamycin activation, only induced moderate cell death and few cell apoptosis in cultured glioblastoma cells. On the other hand, perifosine administration induced significant protective autophagy, which inhibited cell apoptosis induction. Inhibition of autophagy by 3-methyaldenine or by autophagy-related gene-5 RNA interference significantly enhanced perifosine-induced apoptosis and cytotoxicity. We found that the short chain cell-permeable ceramide (C6) significantly enhanced cytotoxic effects of perifosine in cultured glioblastoma cells. For mechanism study, we observed that ceramide (C6) inhibited autophagy induction to restore cell apoptosis and perifosine sensitivity. In conclusion, our study suggests that autophagy inhibition by ceramide (C6) restores perifosine-induced apoptosis and cytotoxicity in glioblastoma cells.     

MicroRNA-330 Is an Oncogenic Factor in Glioblastoma Cells by Regulating SH3GL2 Gene

MicroRNAs have recently emerged as key regulators of cancers. This study was therefore conducted to investigate the role of miR-330 in biological behaviors of human glioblastoma U87 and U251 cell lines and its molecular mechanism. SH3GL2 gene was identified as the target of miR-330. MiR-330 overexpression was established by transfecting miR-330 precursor into U87 and U251 cells, and its effects on proliferation, migration, invasion, cell cycle and apoptosis were studied. Overexpression of miR-330 can enhance cellular proliferation, promote migration and invasion, activate cell cycle and also inhibit apoptosis in U87 and U251 cells. Collectively, these above-mentioned results suggest that miRNA-330 plays an oncogenic role in human glioblastoma by regulating SH3GL2 gene and might be a new therapeutic target of human glioblastoma.     

Opposing Effects of PI3K/Akt and Smad-Dependent Signaling Pathways in NAG-1-Induced Glioblastoma Cell Apoptosis

Nonsteroidal anti-inflammatory drug (NSAID) activated gene-1 (NAG-1) is a divergent member of the transforming growth factor-beta (TGF-β) superfamily. NAG-1 plays remarkable multifunctional roles in controlling diverse physiological and pathological processes including cancer. Like other TGF-β family members, NAG-1 can play dual roles during cancer development and progression by negatively or positively modulating cancer cell behaviors. In glioblastoma brain tumors, NAG-1 appears to act as a tumor suppressor gene; however, the precise underlying mechanisms have not been well elucidated. In the present study, we discovered that overexpression of NAG-1 induced apoptosis in U87 MG, U118 MG, U251 MG, and T98G cell lines via the intrinsic mitochondrial pathway, but not in A172 and LN-229 cell lines. NAG-1 could induce the phosphorylation of PI3K/Akt and Smad2/3 in all six tested glioblastoma cell lines, except Smad3 phosphorylation in A172 and LN-229 cell lines. In fact, Smad3 expression and its phosphorylation were almost undetectable in A172 and LN-229 cells. The PI3K inhibitors promoted NAG-1-induced glioblastoma cell apoptosis, while siRNAs to Smad2 and Smad3 decreased the apoptosis rate. NAG-1 also stimulated the direct interaction between Akt and Smad3 in glioblastoma cells. Elevating the level of Smad3 restored the sensitivity to NAG-1-induced apoptosis in A172 and LN-229 cells. In conclusion, our results suggest that PI3K/Akt and Smad-dependent signaling pathways display opposing effects in NAG-1-induced glioblastoma cell apoptosis.     

UMSCs对U87MG细胞增殖的影响

目的：通过对研究脐带间充质干细胞（Umbilical cord mesenchymalstellcells,UCMSCs）与人恶性胶质母细胞瘤细胞U87MG细胞（U87 Malignant glioma cells）体外共培养,模拟肿瘤生长的内环境,以及其对U87MG细胞增值作用的影响及肿瘤细胞与间充质干细胞的共培养方法。方法：提取人脐带间充质干细胞进行体外培养、扩增,用MTT法测定uMSCS上清液对U87MG的影响,用瑞士染色法检测U87MG形态学变化。结果：MTT比色法结果显示UMSCS对U87MG有抑制作用。96小时培养后1：8、1：4、1：2及未稀释的UMSCs上清液对u87MG的抑制率分别为17％,24％,37．2％及46．4％,u87MG细胞形态亦随着培养时间的延长由多角形变为梭形,突起消失,细胞间骨架结构断裂。结论：通过对共培养前后U87MG与UMSCs共培养后形态学变化、生长曲线变化及对生长周期的影响作用的观察分析,得出UMSCs及其上清液对U87MG有抑制作用,而且呈时间及浓度依赖性。     

RhoE interferes with Rb inactivation and regulates the proliferation and survival of the U87 human glioblastoma cell line

Rho GTPases are important regulators of actin cytoskeleton, but they are also involved in cell proliferation, transformation and oncogenesis. One of this proteins, RhoE, inhibits cell proliferation, however the mechanism that regulates this effect remains poorly understood. Therefore, we undertook the present study to determine the role of RhoE in the regulation of cell proliferation. For this purpose we generated an adenovirus system to overexpress RhoE in U87 glioblastoma cells. Our results show that RhoE disrupts actin cytoskeleton organization and inhibits U87 glioblastoma cell proliferation. Importantly, RhoE expressing cells show a reduction in Rb phosphorylation and in cyclin D1 expression. Furthermore, RhoE inhibits ERK activation following serum stimulation of quiescent cells. Based in these findings, we propose that RhoE inhibits ERK activation, thereby decreasing cyclin D1 expression and leading to a reduction in Rb inactivation, and that this mechanism is involved in the RhoE-induced cell growth inhibition. Moreover, we also demonstrate that RhoE induces apoptosis in U87 cells and also in colon carcinoma and melanoma cells. These results indicate that RhoE plays an important role in the regulation of cell proliferation and survival, and suggest that this protein may be considered as an oncosupressor since it is capable to induce apoptosis in several tumor cell lines.