The role of A-kinase interacting protein 1 in regulating progression and stemness as well as indicating the prognosis in glioblastoma

BackgroundA-kinase interacting protein 1 (AKIP1) is recently implicated in the pathogenesis of several solid tumors, while its role in glioblastoma multiforme (GBM) is largely unknown. Therefore, the current study aimed to investigate the effect of AKIP1 on GBM cell malignant behaviors, stemness, and its underlying molecular mechanisms.MethodsU-87 MG and A172 cells were transfected with control or AKIP1 overexpression plasmid; control or AKIP1 siRNA plasmid. Then cell proliferation, apoptosis, invasion, CD133⁺ cell proportion, and sphere formation assays were performed. Furthermore, RNA-Seq was performed in U-87 MG cells. Besides, AKIP1 expression was detected in 25 GBM and 25 low-grade glioma (LGG) tumor samples.ResultsAKIP1 was increased in several GBM cell lines compared to the control cell line. After transfections, it was found that AKIP1 overexpression increased cell invasion, CD133⁺ cell proportion, and sphere formation ability while less affecting cell proliferation or cell apoptosis in U-87 MG and A172 cells. Moreover, AKIP1 siRNA achieved the opposite effect in these cells, except that it inhibited cell proliferation but induced cell apoptosis to some extent. Subsequent RNA-Seq assay showed several critical carcinogenetic pathways, such as PI3K/AKT, Notch, EGFR tyrosine kinase inhibitor resistance, Ras, ErbB, mTOR pathways, etc. were potentially related to the function of AKIP1 in U-87 MG cells. Clinically, AKIP1 expression was higher in GBM tissues than in LGG tissues, which was also correlated with the poor prognosis of GBM to some degree.ConclusionsAKIP1 regulates the malignant behaviors and stemness of GBM via regulating multiple carcinogenetic pathways.     

Effects of the PKC inhibitor PD 406976 on cell cycle progression, proliferation, PKC isozymes and apoptosis in glioma and SVG-transformed glial cells

It is well established that protein kinase C (PKC) isozymes are involved in the proliferation of glioma cells. However, reports differ on which PKC isozymes are responsible for glioma proliferation. As a means to further elucidate this, the objectives of our research were to determine how inhibition of PKC-alpha, PKC-beta and PKCmu with PD 406976 regulates the cell cycle, cell proliferation and PKC during glioma growth and development. To establish the cell cycle effects of PD 406976 on brain cells (SVG, U-138MG and U-373MG glioma cells), specimens were treated with either dimethylsulfoxide (DMSO; control) or PD 406976 (2 microm). Results from flow cytometry demonstrated that PD 406976 delayed the entry DNA synthesis phase in SVG cells and delayed the number of cells entering and exiting the DNA synthesis phase in both U-138MG and U-373MG cells, indicating that PD 406976 may inhibit G(1)/S and S phase progression. Assessment of cell viability demonstrated a cytostatic effect of PD 406976 on SVG, U-138MG and U-373MG glioma cell proliferation. The PD 406976-induced decreased proliferation was sustained at 48-96 h. A PKC activity assay was quantified and demonstrated that exposure of SVG and U-373MG glioma cells to PD 406976 suppressed PKC activity. Western blotting demonstrated reduced PKC-beta1, PKC-gamma and PKC-tau protein content in cells treated with PD 406976. We determined that the growth inhibitory effect of PD 406976 was not as a result of apoptosis.     

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.     

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.     

NDV-induced apoptosis in absence of Bax; evidence of involvement of apoptotic proteins upstream of mitochondria

ABSTRACT: BACKGROUND: Recently it was shown that following infection of HeLa cells with Newcastle disease virus (NDV), the matrix (M) protein binds to Bax and subsequently the intrinsic pathway of apoptosis is activated. Moreover, there was very little alteration on mRNA and protein levels of Bax and Bcl-2 after infection with NDV.FindingIn order to further investigate the role of members of the Bcl-2 family, Bax-knockout and wild-type HCT116 cells were infected with NDV strain AF2240. Although both cells underwent apoptosis through the activation of the intrinsic pathway and the release of cytochrome c from mitochondria, the percentage of dead Bax-knockout cells was significantly lower than wt cells (more than 10 % at 48 h post-infection). In a parallel experiment, the effect of NDV on HT29 cells, that are originally Bcl-2-free, was studied. Apoptosis in HT29 cells was associated with Bax redistribution from cytoplasm to mitochondria, similar to that of HeLa and wt HCT116 cells. CONCLUSION: Although the presence of Bax during NDV-induced apoptosis contributes to a faster cell death, it was concluded that other apoptotic protein(s) upstream of mitochondria are also involved since cancer cells die whether in the presence or absence of Bax. Therefore, the classic Bax/Bcl-2 ratio may not be a major determinant in NDV-induced apoptosis.     

Tubeimoside V (1), a new cyclic bisdesmoside from tubers of Bolbostemma paniculatum, functions by inducing apoptosis in human glioblastoma U87MG cells

Malignant glioblastoma is one of the most common malignant tumors in the neurological system. Tubeimoside V (1), a new cyclic bisdesmoside from tubers of Bolbostemma paniculatum, appears to exhibit various biological activities, including antitumor effect, but the function and mechanism of this new agent on glioblastoma cells has not previously been determined. In the present study, we investigated the proliferation change of human glioblastoma U87MG cells exposured to different concentrations (0.9-14.8 microM) of Tubeimoside V (1) for a certain time. The results showed that Tubeimoside V (1) significantly suppressed U87MG cell proliferation in a time- and dose-dependent manner (IC(50) = 3.6 microM). Flow cytometric analysis of DNA in U87MG cells showed that Tubeimoside V (1) induces the prominent appearance of a sub-G1 peak in the cell cycle suggestive of apoptosis. Furthermore, U87MG cells' treatment with Tubeimoside V (1) resulted in nuclear condensation with apoptotic bodies observed by both fluorescence and electron microscopy. The result of annexin V/PI assay showed that phosphatidylserine externalization began after treatment, and then increased in the following 24h. Molecular changes explored through Western-blot staining showed Tubeimoside V (1) decreased the expression levels of Bcl-2 protein and increased the expression levels of Bax protein. The novel findings suggest that the cytotoxic actions of Tubeimoside V (1) toward U87MG cells result from the induction of cell apoptosis. Overall, our data demonstrate that Tubeimoside V (1) is an efficient apoptotic killing agent of glioblastoma cells and suggest that this mechanism may play a critical role in anti-tumor chemotherapy.     

The tricyclic antidepressant imipramine induces autophagic cell death in U-87MG glioma cells

In this study, we investigated the antitumor effects of the tricyclic antidepressant 3-(10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)-N,N-dimethylpropan-1-amine (imipramine) on glioma cells. We found that exposure of U-87MG cells to imipramine resulted in the inhibition of PI3K/Akt/mTOR signaling, reduction of clonogenicity, and induction of cell death. Imipramine stimulated the formation of acidic vesicular organelles, the conversion of LC3-I to LC3-II, and the redistribution of LC3 to autophagosomes, suggesting that it stimulates the progression of autophagy. It did not, however, induce apoptosis. We further showed that knockdown of Beclin-1 using siRNA abrogated imipramine-induced cell death. These results suggest that imipramine exerts antitumor effects on PTEN-null U-87MG human glioma cells by inhibiting PI3K/Akt/mTOR signaling and by inducing autophagic cell death.     

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.     

NDV‐D90 inhibits 17β‐estradiol‐mediated resistance to apoptosis by differentially modulating classic and nonclassic estrogen receptors in breast cancer cells

Newcastle disease virus (NDV) is endowed with the oncolytic ability to kill tumor cells, while rarely causing side effects in normal cells. Both estrogen receptor α (ERα) and the G protein estrogen receptor (GPER) modulate multiple biological activities in response to estrogen, including apoptosis in breast cancer (BC) cells. Here, we investigated whether NDV‐D90, a novel strain isolated from natural sources in China, promoted apoptosis by modulating the expression of ERα or the GPER in BC cells exposed to 17β‐estradiol (E2). We found that NDV‐D90 significantly killed the tumor cell lines MCF‐7 and BT549 in a time‐ and dose‐dependent manner. We also found that NDV‐D90 exerted its effects on the two cell lines mainly by inducing apoptosis but not necrosis. NDV‐D90 induced apoptosis via the intrinsic and extrinsic signaling pathways in MCF‐7 cells (ER‐positive cells) during E2 exposure not only by disrupting the E2/ERα axis and enhancing GPER expression but also by modulating the expression of several apoptosis‐related proteins through ERα‐and GPER‐independent processes. NDV‐D90 promoted apoptosis via the intrinsic signaling pathway in BT549 cells (ER‐negative cells), possibly by impairing E2‐mediated GPER expression. Furthermore, NDV‐D90 exerted its antitumor effects in vivo by inducing apoptosis. Overall, these results demonstrated that NDV‐D90 promotes apoptosis by differentially modulating the expression of ERα and the GPER in ER‐positive and negative BC cells exposed to estrogen, respectively, and can be utilized as an effective approach to treating BC.     

Reduced Newcastle disease virus-induced oncolysis in a subpopulation of cisplatin-resistant MCF7 cells is associated with survivin stabilization

ABSTRACT: BACKGROUND: Cisplatin resistance is a serious problem in cancer treatment. To overcome it, alternative approaches including virotherapy are being pursued. One of the candidates for anticancer virotherapy is the Newcastle disease virus (NDV). Even though NDV's oncolytic properties in various cancer cells have been widely reported, information regarding its effects on cisplatin resistant cancer cells is still limited. Therefore, we tested the oncolytic efficacy of a strain of NDV, designated as AF2240, in a cisplatin-resistant breast cancer cell line. METHODS: Cisplatin-resistant cell line (MCF7-CR) was developed from the MCF7 human breast adenocarcinoma cell line by performing a seven-cyclic exposure to cisplatin. Following NDV infection, fluorescence-activated cell sorting (FACS) analysis and immunoblotting were used to measure cell viability and viral protein expression, respectively. Production of virus progeny was then assessed by using the plaque assay technique. RESULTS: Infection of a mass population of the MCF7-CR with NDV resulted in 50% killing in the first 12 hours post-infection (hpi), comparable to the parental MCF7. From 12 hpi onwards, the remaining MCF7-CR became less susceptible to NDV killing. This reduced susceptibility led to increased viral protein synthesis and virus progeny production. The reduction was also associated with a prolonged cell survival via stabilization of the survivin protein. CONCLUSIONS: Our findings showed for the first time, the involvement of survivin in the reduction of NDV-induced oncolysis in a subpopulation of cisplatin-resistant cells. This information will be important towards improving the efficacy of NDV as an anticancer agent in drug resistant cancers.     

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。     

Oncolytic specificity of Newcastle disease virus is mediated by selectivity for apoptosis-resistant cells

Newcastle disease virus (NDV) is a negative-sense RNA virus that has been shown to possess oncolytic activity. NDV's selective replication in tumor cells has been previously suggested to be due to the lack of a proper antiviral response in these cells. Here we demonstrate that NDV possesses oncolytic activity in tumor cells capable of a robust type I interferon (IFN) response, suggesting that another mechanism underlies NDV's tumor specificity. We show that the oncolytic selectivity of NDV for tumor cells is dependent upon tumor cell resistance to apoptosis. Utilizing the human non-small-cell lung cancer cell line A549 overexpressing the antiapoptotic protein Bcl-xL, we show significant enhancement of oncolytic activity and NDV replication. Interestingly, while the Bcl-xL-overexpressing cells were resistant to apoptotic stimuli induced by chemotherapeutic agents and early viral replication, during the subsequent viral cycles, we observed a paradoxical increase in apoptosis in response to NDV. The increased oncolytic activity seen was secondary to enhanced viral replication and syncytium formation. The induction of a type I IFN response was enhanced in Bcl-xL cells. Overall, these findings propose a new mechanism for cancer cell specificity for NDV, making it an attractive anticancer agent for chemoresistant tumors with enhanced antiapoptotic activity.     

Silencing of the human microsomal glucose-6-phosphate translocase induces glioma cell death: potential new anticancer target for curcumin

G6P translocase (G6PT) is thought to play a crucial role in transducing intracellular signaling events in brain tumor-derived cancer cells. In this report, we investigated the contribution of G6PT to the control of U-87 brain tumor-derived glioma cell survival using small interfering RNA (siRNA)-mediated suppression of G6PT. Three siRNA constructs were generated and found to suppress up to 91% G6PT gene expression. Flow cytometry analysis of propidium iodide/annexin-V-stained cells indicated that silencing the G6PT gene induced necrosis and late apoptosis. The anticancer agent curcumin, also inhibited G6PT gene expression by more than 90% and triggered U-87 glioma cells death. Overexpression of recombinant G6PT rescued the cells from curcumin-induced cell death. Targeting G6PT expression may provide a new mechanistic rationale for the action of chemopreventive drugs and lead to the development of new anti-cancer strategies.     

Caveolin-1 is a negative regulator of tumor growth in glioblastoma and modulates chemosensitivity to temozolomide

Caveolin-1 (Cav-1) is a critical regulator of tumor progression in a variety of cancers where it has been shown to act as either a tumor suppressor or tumor promoter. In glioblastoma multiforme, it has been previously demonstrated to function as a putative tumor suppressor. Our studies here, using the human glioblastoma-derived cell line U-87MG, further support the role of Cav-1 as a negative regulator of tumor growth. Using a lentiviral transduction approach, we were able to stably overexpress Cav-1 in U-87MG cells. Gene expression microarray analyses demonstrated significant enrichment in gene signatures corresponding to downregulation of MAPK, PI3K/AKT and mTOR signaling, as well as activation of apoptotic pathways in Cav-1-overexpressing U-87MG cells. These same gene signatures were later confirmed at the protein level in vitro. To explore the ability of Cav-1 to regulate tumor growth in vivo, we further show that Cav-1-overexpressing U-87MG cells display reduced tumorigenicity in an ectopic xenograft mouse model, with marked hypoactivation of MAPK and PI3K/mTOR pathways. Finally, we demonstrate that Cav-1 overexpression confers sensitivity to the most commonly used chemotherapy for glioblastoma, temozolomide. In conclusion, Cav-1 negatively regulates key cell growth and survival pathways and may be an effective biomarker for predicting response to chemotherapy in glioblastoma.     

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.     

Acute and persistent infection of human neural cell lines by human coronavirus OC43

Human coronaviruses (HuCV) are recognized respiratory pathogens. Data accumulated by different laboratories suggest their neurotropic potential. For example, primary cultures of human astrocytes and microglia were shown to be susceptible to an infection by the OC43 strain of HuCV (A. Bonavia, N. Arbour, V. W. Yong, and P. J. Talbot, J. Virol. 71:800-806, 1997). We speculate that the neurotropism of HuCV will lead to persistence within the central nervous system, as was observed for murine coronaviruses. As a first step in the verification of our hypothesis, we have characterized the susceptibility of various human neural cell lines to infection by HuCV-OC43. Viral antigen, infectious virus progeny, and viral RNA were monitored during both acute and persistent infections. The astrocytoma cell lines U-87 MG, U-373 MG, and GL-15, as well as neuroblastoma SK-N-SH, neuroglioma H4, oligodendrocytic MO3.13, and the CHME-5 immortalized fetal microglial cell lines, were all susceptible to an acute infection by HuCV-OC43. Viral antigen and RNA and release of infectious virions were observed during persistent HuCV-OC43 infections ( approximately 130 days of culture) of U-87 MG, U-373 MG, MO3.13, and H4 cell lines. Nucleotide sequences of RNA encoding the putatively hypervariable viral S1 gene fragment obtained after 130 days of culture were compared to that of initial virus input. Point mutations leading to amino acid changes were observed in all persistently infected cell lines. Moreover, an in-frame deletion was also observed in persistently infected H4 cells. Some point mutations were observed in some molecular clones but not all, suggesting evolution of the viral population and the emergence of viral quasispecies during persistent infection of H4, U-87 MG, and MO3.13 cell lines. These results are consistent with the potential persistence of HuCV-OC43 in cells of the human nervous system, accompanied by the production of infectious virions and molecular variation of viral genomic RNA.