Differential SELEX in Human Glioma Cell Lines

The hope of success of therapeutic interventions largely relies on the possibility to distinguish between even close tumor types with high accuracy. Indeed, in the last ten years a major challenge to predict the responsiveness to a given therapeutic plan has been the identification of tumor specific signatures, with the aim to reduce the frequency of unwanted side effects on oncologic patients not responding to therapy. Here, we developed an in vitro evolution-based approach, named differential whole cell SELEX, to generate a panel of high affinity nucleic acid ligands for cell surface epitopes. The ligands, named aptamers, were obtained through the iterative evolution of a random pool of sequences using as target human U87MG glioma cells. The selection was designed so as to distinguish U87MG from the less malignant cell line T98G. We isolated molecules that generate unique binding patterns sufficient to unequivocally identify any of the tested human glioma cell lines analyzed and to distinguish high from low or non-tumorigenic cell lines. Five of such aptamers act as inhibitors of specific intracellular pathways thus indicating that the putative target might be important surface signaling molecules. Differential whole cell SELEX reveals an exciting strategy widely applicable to cancer cells that permits generation of highly specific ligands for cancer biomarkers.     

Identification of Proteins Sensitive to Thermal Stress in Human Neuroblastoma and Glioma Cell Lines

Heat-shock is an acute insult to the mammalian proteome. The sudden elevation in temperature has far-reaching effects on protein metabolism, leads to a rapid inhibition of most protein synthesis, and the induction of protein chaperones. Using heat-shock in cells of neuronal (SH-SY5Y) and glial (CCF-STTG1) lineage, in conjunction with detergent extraction and sedimentation followed by LC-MS/MS proteomic approaches, we sought to identify human proteins that lose solubility upon heat-shock. The two cell lines showed largely overlapping profiles of proteins detected by LC-MS/MS. We identified 58 proteins in detergent insoluble fractions as losing solubility in after heat shock; 10 were common between the 2 cell lines. A subset of the proteins identified by LC-MS/MS was validated by immunoblotting of similarly prepared fractions. Ultimately, we were able to definitively identify 3 proteins as putatively metastable neural proteins; FEN1, CDK1, and TDP-43. We also determined that after heat-shock these cells accumulate insoluble polyubiquitin chains largely linked via lysine 48 (K-48) residues. Collectively, this study identifies human neural proteins that lose solubility upon heat-shock. These proteins may represent components of the human proteome that are vulnerable to misfolding in settings of proteostasis stress.     

Ceramide Induces Non-Apoptotic Cell Death in Human Glioma Cells

Ceramide causes either apoptosis or non-apoptotic cell death depending on model system and experimental conditions. The present study was undertaken to examine the effect of ceramide on cell viability and its molecular events leading to cell death in A172 human glioma cells. Ceramide induced cell death in a dose-dependent manner and the cell death was dependent on generation of reactive oxygen species and lipid peroxidation. TUNEL assay, Hoechst 33258 staining, and flow cytometric analysis did not show typical apoptotic morphological features. Ceramide caused phosphorylation of extracellular signal-regulated kinase (ERK) and p38, but the cell death was not affected by inhibitors of MAPK subfamilies. Ceramide caused ATP depletion without loss of mitochondrial membrane potential. Ceramide did not induce caspase activation and ceramide-induced cell death was also not altered by inhibitors of caspase activation. Transfection of dominant inhibitory mutant of IκBα (S32A/36A) and pretreatment of pyrrolidinedithiocarbamate, an inhibitor of NF-κB, enhanced ceramide-induced cell death. These results indicate that ceramide causes non-apoptotic, caspase-independent cell death by inducing reactive oxygen species generation in A172 human glioma cells. NF-κB is involved in the regulation of ceramide-induced cell death in human glioma cells.     

Bufalin Induces the Interplay between Apoptosis and Autophagy in Glioma Cells through Endoplasmic Reticulum Stress

Malignant gliomas are common primary tumors of the central nervous system. The prognosis of patients with malignant glioma is poor in spite of current intensive therapy and thus novel therapeutic modalities are necessary. Bufalin is the major component of Chan-Su (a traditional Chinese medicine) extracts from the venom of Bufo gargarizan. In this study, we evaluated the growth inhibitory effect of bufalin on glioma cells and explored the underlying molecular mechanisms. Our results showed that bufalin inhibited the growth of glioma cells significantly. Mechanistic studies demonstrated that bufalin induced apoptosis through mitochondrial apoptotic pathway. In addition, bufalin was also found to induce ER stress-mediated apoptosis, which was supported by the up- regulation of ER stress markers, CHOP and GRP78, and augmented phosphorylation of PERK and eIF2α as well as cleavage of caspase-4. Downregulation of CHOP using siCHOP RNA attenuated bufalin-induced apoptosis, further confirming the role of ER stress response in mediating bufalin-induced apoptosis. Evidence of bufalin-induced autophagy included formation of the acidic vesicular organelles, increase of autophagolysosomes and LC3-II accumulation. Further experiments showed that the mechanism of bufalin-induced autophagy associated with ATP deleption involved an increase in the active form of AMPK, decreased phosphorylation levels of mTOR and its downstream targets 4EBP1 and p70S6K1. Furthermore, TUDC and silencing of eIF2α or CHOP partially blocked bufalin-induced accumulation of LC3-II, which indicated that ER stress preceded bufalin-induced autophagy and PERK/eIF2α/CHOP signaling pathway played a major part in the process. Blockage of autophagy increased expression of ER stress associated proteins and the ratio of apoptosis, indicating that autophagy played a cytoprotective role in bufalin induced ER stress and cell death. In conclusion, bufalin inhibits glioma cell growth and induces interplay between apoptosis and autophagy through endoplasmic reticulum stress. It will provide molecular bases for developing bufalin into a drug candidate for the treatment of maglinant glioma.     

Autophagy Protects against Oxaliplatin-Induced Cell Death via ER Stress and ROS in Caco-2 Cells

Oxaliplatin is included in a number of effective combination regimens used as first and subsequent lines of therapy for metastatic colorectal cancer. Accumulating evidence indicates that autophagy plays a significant role in response to cancer therapy. However, the role of autophagy in oxaliplatin-induced cell death remains to be clarified. In this study, we showed that oxaliplatin induced cell death and autophagy in Caco-2 colorectal cancer cells. The suppression of autophagy using either pharmacologic inhibitors (3-methyladenine, bafilomycin A1) or RNA interference in essential autophagy genes (ATG5 or Beclin1) enhanced the cell death and reactive oxygen species (ROS) production induced by oxaliplatin in Caco-2 cells. Blocking oxaliplatin-induced ROS production by using ROS scavengers (NAC or Tiron) decreased autophagy. Furthermore, numerous dilated endoplasmic reticula (ER) were present in oxaliplatin-treated Caco-2 cells, and blocking ER stress by RNA interference against candidate of metastasis-1 (P8) and C/EBP-homologous protein (CHOP) decreased autophagy and ROS production. Taken together, these data indicate that oxaliplatin activates autophagy as a cytoprotective response via ER stress and ROS in human colorectal cancer cells.     

Coibamide A Induces mTOR-Independent Autophagy and Cell Death in Human Glioblastoma Cells

Coibamide A is an N-methyl-stabilized depsipeptide that was isolated from a marine cyanobacterium as part of an International Cooperative Biodiversity Groups (ICBG) program based in Panama. Previous testing of coibamide A in the NCI in vitro 60 cancer cell line panel revealed a potent anti-proliferative response and “COMPARE-negative” profile indicative of a unique mechanism of action. We report that coibamide A is a more potent and efficacious cytotoxin than was previously appreciated, inducing concentration- and time-dependent cytotoxicity (EC₅₀<100 nM) in human U87-MG and SF-295 glioblastoma cells and mouse embryonic fibroblasts (MEFs). This activity was lost upon linearization of the molecule, highlighting the importance of the cyclized structure for both anti-proliferative and cytotoxic responses. We show that coibamide A induces autophagosome accumulation in human glioblastoma cell types and MEFs via an mTOR-independent mechanism; no change was observed in the phosphorylation state of ULK1 (Ser-757), p70 S6K1 (Thr-389), S6 ribosomal protein (Ser-235/236) and 4EBP-1 (Thr-37/46). Coibamide A also induces morphologically and biochemically distinct forms of cell death according to cell type. SF-295 glioblastoma cells showed caspase-3 activation and evidence of apoptotic cell death in a pattern that was also seen in wild-type and autophagy-deficient (ATG5-null) MEFs. In contrast, cell death in U87-MG glioblastoma cells was characterized by extensive cytoplasmic vacuolization and lacked clear apoptotic features. Cell death was attenuated, but still triggered, in Apaf-1-null MEFs lacking a functional mitochondria-mediated apoptotic pathway. From the study of ATG5-null MEFs we conclude that a conventional autophagy response is not required for coibamide A-induced cell death, but likely occurs in dying cells in response to treatment. Coibamide A represents a natural product scaffold with potential for the study of mTOR-independent signaling and cell death mechanisms in apoptotic-resistant cancer cells.     

hSef基因在不同人组织和细胞系的表达分布

将人源Sef蛋白胞内区编码序列与GST融合构建原核表达质粒进行重组蛋白的表达与纯化并制备多抗 .在COS 7细胞中转染表达hSef显示 ,其分子量分别为 80kD ,10 0kD ,比体外翻译的分子量偏大 ,提示可能有糖基化存在 .Northern印迹的结果表明 ,hSefmRNA主要分布在人肾和睾丸组织 .RT PCR检测到hSefmRNA在众多细胞系有广泛存在 .免疫组化的结果显示 ,hSef蛋白在人肾和睾丸及相应癌组织表达水平较高 .     

Calmodulin-Binding Proteins in Human Y-79 Retinoblastoma and HTB-14 Glioma Cell Lines

The Y-79 human retinoblastoma cell line has been used as a model system for studying differentiation of primitive neuroectodermal cells into either glial-like (glial fibrillary acidic protein positive) or neuron-like (neuron-specific enolase-positive) cells. To determine whether Y-79 retinoblastoma cells express neuronotypic calmodulin-binding proteins, Y-79 cells were either treated with butyrate or dibutyryl cyclic AMP (dbcAMP) in serum-containing medium or were maintained in serum-free media. Using a biotinylated calmodulin blot overlay technique, we found that Y-79 cells treated with dbcAMP or butyrate expressed low levels of membrane-bound calmodulin-binding proteins of 150, 147, 127, and 126 kilodaltons (kDa); butyrate-treated cells also expressed a calmodulin-binding peptide of 135 kDa. Since butyrate treatment of Y-79 cells induces the expression and the secretion of interphotoreceptor retinoid-binding protein (IRBP, 140 kDa), we tested the hypothesis that the calmodulin-binding protein of 135 kDa induced by butyrate treatment was IRBP. Purified bovine IRBP did not bind calmodulin; further, the 135-kDa calmodulin binding protein was not immunoreactive with antisera directed against IRBP. Since dbcAMP and butyrate induce some glial-like characteristics in Y-79 cells, we compared the calmodulin-binding protein pattern in these cells with that seen in human HTB-14 glioma cells. The HTB-14 line did not express calmodulin-binding proteins, even after treatments with agents that induce morphologic change in these cells. Thus, we conclude that Y-79 cells express membrane-bound calmodulin-binding proteins, but in a pattern different from that seen with adult, differentiated neurons or from human HTB-14 glioma cells.     

Epizootic Hemorrhagic Disease Virus Induces and Benefits from Cell Stress,Autophagy, and Apoptosis

The mode and timing of virally induced cell death hold the potential of regulating viral yield, viral transmission, and the severity of virally induced disease. Orbiviruses such as the epizootic hemorrhagic disease virus (EHDV) are nonenveloped and cytolytic. To date, the death of cells infected with EHDV, the signal transduction pathways involved in this process, and the consequence of their inhibition have yet to be characterized. Here, we report that the Ibaraki strain of EHDV2 (EHDV2-IBA) induces apoptosis, autophagy, a decrease in cellular protein synthesis, the activation of c-Jun N-terminal kinase (JNK), and the phosphorylation of the JNK substrate c-Jun. The production of infectious virions decreased upon inhibition of apoptosis with the pan-caspase inhibitor Q-VD-OPH (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone), upon inhibition of autophagy with 3-methyladenine or via the knockout of the autophagy regulator Atg5, or upon treatment of infected cells with the JNK inhibitor SP600125 or the cyclin-dependent kinase (CDK) inhibitor roscovitine, which also inhibited c-Jun phosphorylation. Moreover, Q-VD-OPH, SP600125, and roscovitine partially reduced EHDV2-IBA-induced cell death, and roscovitine diminished the induction of autophagy by EHDV2-IBA. Taken together, our results imply that EHDV induces and benefits from the activation of signaling pathways involved in cell stress and death.     

多种胶质瘤细胞系中获取胶质瘤干细胞方法的研究

目的:进一步证明胶质瘤干细胞是广泛存在的,并寻找一种简洁的方法从不同胶质瘤细胞系中提取肿瘤干细胞。方法:将胶质瘤细胞以合适的密度接种于96孔板中,获取胶质瘤干细胞,并通过检测其自我更新能力、多向分化能力、成瘤能力及胶质瘤干细胞标记物的表达情况对其进行鉴定。结果:多种细胞系中均成功获取了胶质瘤干细胞。并且这细胞球表达神经干细胞的标志物,不表达神经细胞分化标志物,同时又有多向分化的能力,仅5000个细胞就可以在裸鼠颅内成瘤。结论:我们的研究结果表明胶质瘤干细胞是广泛存在的,并为以后进一步研究胶质瘤干细胞的特性及靶向胶质瘤干细胞的药物做铺垫。     

Lithium Induces ER Stress and N-Glycan Modification in Galactose-Grown Jurkat Cells

We previously reported that lithium had a significant impact on Ca²⁺ regulation and induced unfolded protein response (UPR) in yeast cells grown on galactose due to inhibition of phosphoglucomutase (PGM), however the exact mechanism has not been established yet. In this study, we analysed lithium''s effect in galactose-fed cells to clarify whether these ER-related changes are the result of a relative hypoglycemic state. Furthermore, we investigated whether the alterations in galactose metabolism impact protein post-translational modifications. Thus, Jurkat cells were incubated in glucose or galactose containing media with or without lithium treatment. We found that galactose-fed and lithium treated cells showed better survivability than fasting cells. We also found higher UDP-Hexose and glycogen levels in these cells compared to fasting cells. On the other hand, the UPR (X-box binding protein 1 mRNA levels) of galactose-fed and lithium treated cells was even greater than in fasting cells. We also found increased amount of proteins that contained N-linked N-acetyl-glucosamine, similar to what was reported in fasting cells by a recent study. Our results demonstrate that lithium treatment of galactose-fed cells can induce stress responses similar to hypoglycemia, however cell survival is still secured by alternative pathways. We propose that clarifying this process might be an important addition toward the better understanding of the molecular mechanisms that regulate ER-associated stress response.     

Metabolic Stress in Autophagy and Cell Death Pathways

Growth factors and oncogenic kinases play important roles in stimulating cell growth during development and transformation. These processes have significant energetic and synthetic requirements and it is apparent that a central function of growth signals is to promote glucose metabolism to support these demands. Because metabolic pathways represent a fundamental aspect of cell proliferation and survival, there is considerable interest in targeting metabolism as a means to eliminate cancer. A challenge, however, is that molecular links between metabolic stress and cell death are poorly understood. Here we review current literature on how cells cope with metabolic stress and how autophagy, apoptosis, and necrosis are tightly linked to cell metabolism. Ultimately, understanding of the interplay between nutrients, autophagy, and cell death will be a key component in development of new treatment strategies to exploit the altered metabolism of cancer cells.Although single-celled organisms grow and proliferate based on nutrient availability, metazoan cells rely on growth factor input to promote nutrient uptake, regulate growth and proliferation, and survive (Raff 1992; Rathmell et al. 2000). Access and competition for these signals are critical in developmental patterning and to maintain homeostasis of mature tissues. Cells that do not receive proper growth factor signals typically atrophy, lose the ability to uptake and use extracellular nutrients, and instead induce the self-digestive process of autophagy as an intracellular energy source before ultimately undergoing programmed cell death. Cancer cells, in contrast, often become independent of extracellular growth signals by gaining mutations or expressing oncogenic kinases to drive intrinsic growth signals that mimic growth factor input, which can be the source of oncogene addiction. Growth factor input or oncogenic signals often drive highly elevated glucose uptake and metabolism (Rathmell et al. 2000; DeBerardinis et al. 2008; Michalek and Rathmell 2010). First described in cancer by Warburg in the 1920s, this highly glycolytic metabolic program is termed aerobic glycolysis and is a general feature of many nontransformed proliferative cells (Warburg 1956; DeBerardinis et al. 2008).Nutrient uptake and aerobic glycolysis induced by growth signals play key roles in cell survival (Vander Heiden et al. 2001). Manipulating cell metabolism as a means to promote the death of inappropriately dividing cells, therefore, is a promising new avenue to treat disease. Targeting the altered metabolism of cancer cells in particular is of great interest. It is still unclear at the molecular level, however, how inhibiting or modulating cell metabolism leads to apoptosis, and how these pathways may best be exploited (Dang et al. 2009; Wise and Thompson 2010).Growth factor or oncogenic kinases promote multiple metabolic pathways that are essential to prevent metabolic stress and may be targets in efforts to link metabolism and cell death (Vander Heiden et al. 2001). Decreased glucose metabolism on loss of growth signals leads to decreased ATP generation as well as loss in generation of many biosynthetic precursor molecules, including nucleic acids, fatty acids, and acetyl-CoA for acetylation (Zhao et al. 2007; Wellen et al. 2009; Coloff et al. 2011). Glucose is also important as a precursor for the hexosamine pathway, to allow proper glycosylation and protein folding in the endoplasmic reticulum (Dennis et al. 2009; Kaufman et al. 2010). If glucose metabolism remains insufficient or disrupted, the cells can switch to rely on mitochondrial oxidation of fatty acids and amino acids, which are energy rich but do not readily support cell growth and can lead to potentially dangerous levels of reactive oxygen species (Wellen and Thompson 2010). Amino acid deficiency can directly inhibit components of the signaling pathways downstream from growth factors and activate autophagy (Lynch 2001; Beugnet et al. 2003; Byfield et al. 2005; Nobukuni et al. 2005). Finally, hypoxia induces a specific pathway to increase nutrient uptake and metabolism via the hypoxia-inducible factor (HIF1/2α) that promotes adaptation to anaerobic conditions, but may lead to apoptosis if hypoxia is severe (Saikumar et al. 1998; Suzuki et al. 2001; Fulda and Debatin 2007).Typically a combination of metabolic stresses rather than loss of a single nutrient input occur at a given time (Degenhardt et al. 2006) and autophagy is activated to mitigate damage and provide nutrients for short-term survival (Bernales et al. 2006; Tracy et al. 2007; Altman et al. 2011; Guo et al. 2011). Autophagy is a cellular process of bulk cytoplasmic and organelle degradation common to nearly all eukaryotes. Unique double-membraned vesicles known as autophagosomes engulf cellular material and fuse with lysosomes to promote degradation of the contents (Kelekar 2005). Described in greater detail below, autophagy can reduce sources of stress, such as protein aggregates and damaged or dysfunctional intracellular organelles, and provide nutrients during times of transient and acute nutrient withdrawal.Despite the protective effects of autophagy, cells deprived of growth signals, nutrients, or oxygen for prolonged times will eventually succumb to cell death. Apoptosis is the initial death response on metabolic stress and is regulated by Bcl-2 family proteins. In healthy cells, antiapoptotic Bcl-2 family proteins, such as Bcl-2, Bcl-xl, and Mcl-1, bind and inhibit the multidomain proapoptotic proteins Bax and Bak (van Delft and Huang 2006; Walensky 2006; Chipuk et al. 2010). In metabolic stress, proapoptotic “BH3-only” proteins of the Bcl-2 family are induced or activated and bind to and inhibit the antiapoptotic Bcl-2 family proteins to allow activation of the proapoptotic Bax and Bak (Galonek and Hardwick 2006). The BH3-only proteins Bim, Bid, and Puma can also directly bind and activate Bax and Bak (Letai et al. 2002; Ren et al. 2010). Active Bax and Bak disrupt the outer mitochondrial membrane (termed mitochondrial outer-membrane permeabilization, or MOMP) and release several proapoptotic factors including cytochrome-C that activate the apoptosome that in turn activates effector caspases to cleave a variety of cellular proteins and drive apoptosis (Schafer and Kornbluth 2006). In cases in which these apoptotic pathways are suppressed, metabolic stress can instead lead to necrotic cell death (Jin et al. 2007).     

PERK-mediated Autophagy in Osteosarcoma Cells Resists ER Stress-induced Cell Apoptosis

Osteosarcoma is a bone cancer that develops commonly in children and adolescents. However, osteosarcoma treatments often fail by the development of chemoresistance to apoptosis, and the molecular mechanisms remain unclear. In this study, we propose that autophagy is responsible for osteosarcomatous resistance to apoptosis. We implicate PERK-mediated autophagy as a significant contributor to apoptosis resistance due to ER stress in osteosarcoma cells. By immunostainings and western blots, we identified that PERK activated osteosarcomatous autophagy via inhibiting mTORC1 pathway, thereby preventing cell apoptosis. While using RNAi, we knocked down PERK and found that autophagy was suppressed, result in osteosarcomatous apoptosis. Our results identify a novel role of PERK-mediated autophagy as a significant mechanism for osteosarcoma cell survival. These results will help to understand the mechanism of chemoresistance in osteosarcoma cells, and indicate a novel target for improving osteosarcoma therapy.     

Salvianolic Acid B Induces Apoptosis in Human Glioma U87 Cells Through p38-Mediated ROS Generation

Salvianolic acid B (SalB), the main water-soluble bioactive compounds isolated from the traditional Chinese medical herb Danshen, has been shown to exert anti-cancer effect in several cancer cell lines. The aim of our study was to investigate the potential anti-cancer effect of SalB in human glioma U87 cells. We found that treatment with SalB significantly decreased cell viability of U87 cells in a dose- and time-dependent manner. SalB also enhanced the intracellular ROS generation and induced apoptotic cell death in U87 cells. Western blot analysis suggested that SalB increased the phosphorylation of p38 MAPK and p53 in a dose-dependent manner. Moreover, blocking p38 activation by specific inhibitor SB203580 or p38 specific siRNA partly reversed the anti-proliferative and pro-apoptotic effects, and ROS production induced by SalB treatment. The anti-tumor activity of SalB in vivo was also demonstrated in U87 xenograft glioma model. All of these findings extended the anti-cancer effect of SalB in human glioma cell lines, and suggested that these inhibitory effects of SalB on U87 glioma cell growth might be associated with p38 activation mediated ROS generation. Thus, SalB might be concerned as an effective and safe natural anticancer agent for glioma prevention and treatment.     

ER Stress and Autophagy Dysfunction Contribute to Fatty Liver in Diabetic Mice

Diabetes mellitus and nonalcoholic fatty liver disease (NAFLD) are often identified in patients simultaneously. Recent evidence suggests that endoplasmic reticulum (ER) stress and autophagy dysfunction play an important role in hepatocytes injury and hepatic lipid metabolism, however the mechanistic interaction between diabetes and NAFLD is largely unknown. In this study, we used a diabetic mouse model to study the interplay between ER stress and autophagy during the pathogenic transformation of NAFLD. The coexist of inflammatory hepatic injury and hepatic accumulation of triglycerides (TGs) stored in lipid droplets indicated development of steatohepatitis in the diabetic mice. The alterations of components for ER stress signaling including ATF6, GRP78, CHOP and caspase12 indicated increased ER stress in liver tissues in early stage but blunted in the later stage during the development of diabetes. Likewise, autophagy functioned well in the early stage but suppressed in the later stage. The inactivation of unfolded protein response and suppression of autophagy were positively related to the development of steatohepatitis, which linked to metabolic abnormalities in the compromised hepatic tissues in diabetic condition. We conclude that the adaption of ER stress and impairment of autophagy play an important role to exacerbate lipid metabolic disorder contributing to steatohepatitis in diabetes.     

土贝母苷甲经miR-21/ PDCD4 途径诱导胶质瘤U251细胞凋亡

目的:研究土贝母苷甲(TBMS I)对胶质瘤U251细胞的抗肿瘤作用以及探究土贝母苷甲对miR-21及其靶基因PDCD4基因表达的影响。方法:U251细胞在体外进行培养,四甲基偶氮唑盐(MTT)法检测不同浓度的土贝母苷甲对细胞增殖的影响;Hoechst33258染色观察细胞核形态的变化;实时荧光定量PCR检测miR-21和PDCD4基因的表达情况;Western blot检测PDCD4蛋白的表达情况。结果:土贝母苷甲能够显著抑制U251细胞的增殖,其抑制作用呈剂量和时间依赖性。Hoechst33258染色观察到土贝母苷甲处理组细胞的细胞核形态表现出典型的凋亡特征。PCR结果显示:随着土贝母苷甲浓度增加,miR-21的表达逐渐降低(P0.05),PDCD4基因表达显著增加(P0.05)。Western blot结果提示:与阴性对照组相比,15、30μg/mL土贝母苷甲显著上调了PDCD4蛋白的表达(P0.05)。结论:土贝母苷甲能够显著抑制人胶质瘤U251细胞的增殖并诱导细胞发生凋亡,其机制可能与下调miR-21的表达和上调PDCD4的表达有关。