Inhibition and eradication of human glioma with tumor-targeting Salmonella typhimurium in an orthotopic nude-mouse model

Malignant glioma tumors are the most common primary central nervous system tumors. Despite the multidisciplinary approach to treatment, prognosis remains poor. In this study, we demonstrated that the Salmonella typhimurium A1-R tumor-targeting strain can inhibit and eradicate human glioma in an orthotopic nude-mouse model. S. typhimurium A1-R was administered by injection through a craniotomy open-window or intravenously in nude mice. To establish the model, 2 x 105 U87-RFP human glioma cells were injected stereotactically into the mouse brain through the craniotomy open window. Two weeks after glioma-cell implantation, mice were treated with S. typhimurium A1-R [2 x 10⁷ CFU/200 μl intravenous injection (i.v.) or 1 x 10⁶ CFU/1 μl intracranial injection (i.c.)] once a week for 3 weeks. Brain tumors were observed by fluorescence imaging through the craniotomy open window over time. S. typhimurium A1-R, administered i.c., inhibited brain tumor growth 7.6-fold compared with untreated mice (p = 0.009) and improved survival 73% (p = 0.001). Two of ten mice appeared to have their tumors eradicated. Intravenous administration of S. typhimurium A1-R was not effective. The craniotomy open window enabled observation of tumor growth in the brain in real time in both treated and untreated mice. The results of the present study demonstrate that bacterial therapy of brain cancer is a novel, effective and safe treatment strategy in a highly treatment-resistance cancer.     

Inhibition and eradication of human glioma with tumor-targeting Salmonella typhimurium in an orthotopic nude-mouse model

Malignant glioma tumors are the most common primary central nervous system tumors. Despite the multidisciplinary approach to treatment, prognosis remains poor. In this study, we demonstrated that the Salmonella typhimurium A1-R tumor-targeting strain can inhibit and eradicate human glioma in an orthotopic nude-mouse model. S. typhimurium A1-R was administered by injection through a craniotomy open-window or intravenously in nude mice. To establish the model, 2 × 10⁵ U87-RFP human glioma cells were injected stereotactically into the mouse brain through the craniotomy open window. Two weeks after glioma-cell implantation, mice were treated with S. typhimurium A1-R [2 × 10⁷ CFU/200 µl intravenous injection (i.v.) or 1 × 10⁶ CFU/1 µl intracranial injection (i.c.)] once a week for 3 weeks. Brain tumors were observed by fluorescence imaging through the craniotomy open window over time. S. typhimurium A1-R, administered i.c., inhibited brain tumor growth 7.6-fold compared with untreated mice (p = 0.009) and improved survival 73% (p = 0.001). Two of ten mice appeared to have their tumors eradicated. Intravenous administration of S. typhimurium A1-R was not effective. The craniotomy open window enabled observation of tumor growth in the brain in real time in both treated and untreated mice. The results of the present study demonstrate that bacterial therapy of brain cancer is a novel, effective and safe treatment strategy in a highly treatment-resistance cancer.Key words: Salmonella typhimurium A1-R, fluorescent proteins, brain cancer, mouse model, in vivo imaging     

UV light killing efficacy of fluorescent protein‐expressing cancer cells in vitro and in vivo

We investigated the cell‐killing efficacy of UV light on cancer cells expressing GFP in the nucleus and RFP in the cytoplasm (dual‐color cells). After exposure to various doses of UVA, UVB, or UVC, apoptotic and viable cells were quantitated under fluorescence microscopy using dual‐color 143B human osteosarcoma cells, HT‐1080 human fibrosarcoma cells, Lewis lung carcinoma (LLC), and XPA‐1 human pancreatic cancer cells in vitro. UV‐induced cancer cell death was wave‐length and dose dependent, as well as cell‐line dependent. After UVA exposure, most cells were viable even when the UV dose was increased up to 200 J/m². With UVB irradiation, cell death was observed with irradiation at 50 J/m². For UVC, as little as 25 J/m² UVC irradiation killed approximately 70% of the 143B dual‐color cells. This dose of UVB or UVA had almost no effect on the cancer cells. UV‐induced cancer cell death varied among the cell lines. Cell death began about 4 h after irradiation and continued until 10 h after irradiation. UVC exposure also suppressed cancer cell growth in nude mice in a model of minimal residual cancer (MRC). No apparent side effects of UVC exposure were observed. This study opens up the possibility of UVC treatment for MRC after surgical resection. J. Cell. Biochem. 110: 1439–1446, 2010. © 2010 Wiley‐Liss, Inc.     

A transgenic red fluorescent protein‐expressing nude mouse for color‐coded imaging of the tumor microenvironment

The tumor microenvironment (TME) is critical for tumor growth and progression. We have previously developed color‐coded imaging of the TME using a green fluorescent protein (GFP) transgenic nude mouse as a host. However, most donor sources of cell types appropriate for study in the TME are from mice expressing GFP. Therefore, a nude mouse expressing red fluorescent protein (RFP) would be an appropriate host for transplantation of GFP‐expressing stromal cells as well as double‐labeled cancer cells expressing GFP in the nucleus and RFP in the cytoplasm, thereby creating a three‐color imaging model of the TME. The RFP nude mouse was obtained by crossing non‐transgenic nude mice with the transgenic C57/B6 mouse in which the β‐actin promoter drives RFP (DsRed2) expression in essentially all tissues. In crosses between nu/nu RFP male mice and nu/+ RFP female mice, the embryos fluoresced red. Approximately 50% of the offspring of these mice were RFP nude mice. In the RFP nude mouse, the organs all brightly expressed RFP, including the heart, lungs, spleen, pancreas, esophagus, stomach, duodenum, the male and female reproductive systems; brain and spinal cord; and the circulatory system, including the heart, and major arteries and veins. The skinned skeleton highly expressed RFP. The bone marrow and spleen cells were also RFP positive. GFP‐expressing human cancer cell lines, including HCT‐116‐GFP colon cancer and MDA‐MB‐435‐GFP breast cancer were orthotopically transplanted to the transgenic RFP nude mice. These human tumors grew extensively in the transgenic RFP nude mouse. Dual‐color fluorescence imaging enabled visualization of human tumor–host interaction. The RFP nude mouse model should greatly expand our knowledge of the TME. J. Cell. Biochem. 106: 279–284, 2009. © 2008 Wiley‐Liss, Inc.     

EphA2对神经胶质瘤细胞系U251的凋亡﹑增殖﹑迁移和侵袭的研究

为了研究EphA2对神经胶质瘤细胞系U251在增殖、凋亡、迁移和侵袭方面所起的作用,用RT-PCR方法检测正常脑组织标本与两种恶性胶质瘤细胞系中EphA2 mRNA表达水平,然后用化学合成的针对EphA2基因的小干扰RNA(siRNA)下调该基因的表达,以检测其在U251中的生物学功能．证实了EphA2基因在正常脑组织标本中的表达水平远低于两种恶性胶质瘤细胞系．把体外化学合成针对EphA2基因的小干扰RNA(siRNA- EphA2)转染入U251细胞后,Western blot, 实时定量 RT-PCR检测到U251细胞中EphA2蛋白及mRNA表达水平都明显降低,并且细胞增殖受到显著抑制,同时出现了明显的细胞凋亡．伤口愈合实验(检测细胞迁移能力),Transwell小室实验(检测细胞侵袭能力)均表明,下调EphA2的表达后,细胞的迁移和侵袭能力较阴性对照组显著减弱．上述结果表明,在神经胶质瘤U251细胞中,EphA2与其恶性增殖及高度侵染性相关,可作为分子治疗的有效靶点．     

Antitumour and pro-apoptotic actions of highly unsaturated fatty acids in glioma

The highly unsaturated fatty acids (HUFA) of the n-6 and n-3 series are involved in cell signalling in normal and transformed cells and have recently been associated with pathways leading to tumour cell death. The antitumour activity of three HUFA (arachidonic acid, gamma linolenic acid and eicosapentaenoic acid) were studied in glioma cells and tissue. Using five glioma models, including primary cell suspensions prepared from 46 human glioma samples and an in vivo rat C6 glioma model, we obtained evidence that, following exposure to HUFA, either administered into the medium surrounding human glioma cells or in 16 preparations of multicellular spheroids derived from human and rodent glioma cell lines (C6, MOG, U87, U373) or administered intra-tumourally by infusion using osmotic mini-pumps in 48 rats, glioma regression and apoptosis were detected. Additionally, synergy between gamma irradiation and HUFA administration was observed in 13 experiments analyzing C6 glioma cell apoptosis in vitro. These pro-apoptotic and antiproliferative activities were observed using both C18 and C20 fatty acids of the n-6 and n-3 series, but not when saturated and monounsaturated C18 and C20 fatty acid preparations were used. In the glioma infusion model, in addition to the apoptosis detected in glioma tissue infused with HUFA for 3-7 days, preservation of normal neural tissue and vasculature in adjacent brain was observed. Also, there was little evidence of acute inflammatory infiltration in regressing tumours. Our findings suggest that intraparenchymal infusion of HUFA may be effective in stimulating glioma regression.     

Simultaneous color‐coded imaging to distinguish cancer “stem‐like” and non‐stem cells in the same tumor

In this study, we demonstrate that the differential behavior, including malignancy and chemosensitivity, of cancer stem‐like and non‐stem cells can be simultaneously distinguished in the same tumor in real time by color‐coded imaging. CD133⁺ Huh‐7 human hepatocellular carcinoma (HCC) cells were considered as cancer stem‐like cells (CSCs), and CD133^? Huh‐7 cells were considered as non‐stem cancer cells (NSCCs). CD133⁺ cells were isolated by magnetic bead sorting after Huh‐7 cells were genetically labeled with green fluorescent protein (GFP) or red fluorescent protein (RFP). In this scheme, CD133⁺ cells were labeled with GFP and CD133^? cells were labeled with RFP. CSCs had higher proliferative potential compared to NSCCs in vitro. The same number of GFP CSCs and the RFP NSCCs were mixed and injected subcutaneously or in the spleen of nude mice. CSCs were highly tumorigenic and metastatic as well as highly resistant to chemotherapy in vivo compared to NSCCs. The ability to specifically distinguish stem‐like cancer cells in vivo in real time provides a visual target for prevention of metastasis and drug resistance. J. Cell. Biochem. 111: 1035–1041, 2010. © 2010 Wiley‐Liss, Inc.     

Color-coded intravital imaging demonstrates a transforming growth factor-β (TGF-β) antagonist selectively targets stromal cells in a human pancreatic-cancer orthotopic mouse model

Pancreatic cancer is a recalcitrant malignancy, partly due to desmoplastic stroma which stimulates tumor growth, invasion, and metastasis, and inhibits chemotherapeutic drug delivery. Transforming growth factor-β (TGF-β) has an important role in the formation of stromal desmoplasia. The present study describes the ability of color-coded intravital imaging to demonstrate the efficacy of a TGF-β inhibitor to target stroma in an orthotopic mouse model of pancreatic cancer. The BxPC-3 human pancreatic adenocarcinoma cell line expressing green fluorescent protein (GFP), which also has a high TGF-β expression level, was used in an orthotopic model in transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). Fourteen mice were randomized into a control group (n = 7, vehicle, i.p., weekly, for 3 weeks) and a treated group (n = 7, SB431542 [TGF-β receptor type I inhibitor] 0.3 mg, i.p., weekly, for 3 weeks). Stromal cells expressing RFP and cancer cells expressing GFP were observed weekly for 3 weeks by real-time color-coded intravital imaging. The RFP fluorescence area from the stromal cells, relative to the GFP fluorescence area of the cancer cells, was significantly decreased in the TGF-β-inhibitor-treatment group compared to the control group. The present study demonstrated color-coded imaging in an orthotopic pancreatic-cancer cell-line mouse model can readily detect the selective anti-stromal-cell targeting of a TGF-β inhibitor.     

Capsaicin-induced apoptosis of glioma cells is mediated by TRPV1 vanilloid receptor and requires p38 MAPK activation

We provide evidence on the expression of the transient receptor potential vanilloid type-1 (TRPV1) by glioma cells, and its involvement in capsaicin (CPS)-induced apoptosis. TRPV1 mRNA was identified by quantitative RT-PCR in U373, U87, FC1 and FLS glioma cells, with U373 cells showing higher, and U87, FC1 and FLS cells lower TRPV1 expression as compared with normal human astrocytes. By flow cytometry we found that a substantial portion of both normal human astrocytes, and U87 and U373 glioma cells express TRPV1 protein. Moreover, we analyzed the expression of TRPV1 at mRNA and protein levels of glioma tissues with different grades. We found that TRPV1 gene and protein expression inversely correlated with glioma grading, with marked loss of TRPV1 expression in the majority of grade IV glioblastoma multiforme. We also described that CPS trigger apoptosis of U373, but not U87 cells. CPS-induced apoptosis involved Ca(2+) influx, p38 but not extracellular signal-regulated mitogen-activated protein kinase activation, phosphatidylserine exposure, mitochondrial permeability transmembrane pore opening and mitochondrial transmembrane potential dissipation, caspase 3 activation and oligonucleosomal DNA fragmentation. TRPV1 was functionally implicated in these events as they were markedly inhibited by the TRPV1 antagonist, capsazepine. Finally, p38 but not extracellular signal-regulated protein kinase activation was required for TRPV1-mediated CPS-induced apoptosis of glioma cells.     

Activation of protease-activated receptor 2 reduces glioblastoma cell apoptosis

Background

The pathogenesis of glioma is unclear. The disturbance of the apoptosis process plays a critical role in glioma growth. Factors regulating the apoptosis process are to be further understood. This study aims to investigate the role of protease activated receptor-2 (PAR2) in regulation the apoptosis process in glioma cells.

Results

The results showed that U87 cells and human glioma tissue expressed PAR2. Exposure to tryptase, or the PAR2 active peptide, increased STAT3 phosphorylation in the radiated U87 cells, reduced U87 cell apoptosis, suppressed the expression of p53 in U87 cells.

Conclusions

Activation of PAR2 can reduce the radiated U87 cell apoptosis via modulating the expression of p53. The results implicate that PAR2 may be a novel therapeutic target in the treatment of glioma.     

A Novel Zebrafish Xenotransplantation Model for Study of Glioma Stem Cell Invasion

Invasion and metastasis of solid tumors are the major causes of death in cancer patients. Cancer stem cells (CSCs) constitute a small fraction of tumor cell population, but play a critical role in tumor invasion and metastasis. The xenograft of tumor cells in immunodeficient mice is one of commonly used in vivo models to study the invasion and metastasis of cancer cells. However, this model is time-consuming and labor intensive. Zebrafish (Danio rerio) and their transparent embryos are emerging as a promising xenograft tumor model system for studies of tumor invasion. In this study, we established a tumor invasion model by using zebrafish embryo xenografted with human glioblastoma cell line U87 and its derived cancer stem cells (CSCs). We found that CSCs-enriched from U87 cells spreaded via the vessels within zebrafish embryos and such cells displayed an extremely high level of invasiveness which was associated with the up-regulated MMP-9 by CSCs. The invasion of glioma CSCs (GSCs) in zebrafish embryos was markedly inhibited by an MMP-9 inhibitor. Thus, our zebrafish embryo model is considered a cost-effective approach tostudies of the mechanisms underlying the invasion of CSCs and suitable for high-throughput screening of novel anti-tumor invasion/metastasis agents.     

A color-coded orthotopic nude-mouse treatment model of brain-metastatic paralyzing spinal cord cancer that induces angiogenesis and neurogenesis

Objective: Cancer of the spinal cord is highly malignant and often leads to paralysis and death. A realistic mouse model would be an important benefit for the better understanding and treatment of spinal cord glioma.
Materials and methods: To develop an imageable, patient-like model of this disease, U87 human glioma tumour fragments (expressing red fluorescent protein), were transplanted by surgical orthotopic implantation into the spinal cord of nontransgenic nude mice or transgenic nude mice expressing nestin-driven green fluorescent protein (ND-GFP). In ND-GFP mice, GFP is expressed in nascent blood vessels and neural stem cells. The animals were treated with temozolomide or vehicle control.
Results: The intramedullary spinal cord tumour grew at the primary site, caused hind-limb paralysis and also metastasized to the brain. Temozolomide inhibited tumour growth ( P  < 0.01) and prevented metastasis, as well as prevented paralysis in four mice and delayed paralysis in two mice of the six tested ( P  = 0.005). In the ND-GFP-expressing host, ND-GFP cells staining positively for neuronal class III-β-tubulin or CD31, surrounded the tumour. These results suggest that the tumour stimulated both neurogenesis and angiogenesis, respectively.
Conclusion: A patient-like model of spinal cord glioma was thus developed, which can be used for the discovery of new agents, including those that inhibit invasion and metastasis of the disease as well as those that prevent paralysis.     

Mesenchymal Stem Cells Modified with a Single-Chain Antibody against EGFRvIII Successfully Inhibit the Growth of Human Xenograft Malignant Glioma

Background

Glioblastoma multiforme is the most lethal brain tumor with limited therapeutic options. Antigens expressed on the surface of malignant cells are potential targets for antibody-mediated gene/drug delivery.

Principal Findings

In this study, we investigated the ability of genetically modified human mesenchymal stem cells (hMSCs) expressing a single-chain antibody (scFv) on their surface against a tumor specific antigen, EGFRvIII, to enhance the therapy of EGFRvIII expressing glioma cells in vivo. The growth of U87-EGFRvIII was specifically delayed in co-culture with hMSC-scFvEGFRvIII. A significant down-regulation was observed in the expression of pAkt in EGFRvIII expressing glioma cells upon culture with hMSC-scFvEGFRvIII vs. controls as well as in EGFRvIII expressing glioma cells from brain tumors co-injected with hMSC-scFvEGFRvIII in vivo. hMSC expressing scFvEGFRvIII also demonstrated several fold enhanced retention in EGFRvIII expressing flank and intracranial glioma xenografts vs. control hMSCs. The growth of U87-EGFRvIII flank xenografts was inhibited by 50% in the presence of hMSC-scFvEGFRvIII (p<0.05). Moreover, animals co-injected with U87-EGFRvIII and hMSC-scFvEGFRvIII intracranially showed significantly improved survival compared to animals injected with U87-EGFRvIII glioma cells alone or with control hMSCs. This survival was further improved when the same animals received an additional dosage of hMSC-scFvEGFRvIII two weeks after initial tumor implantation. Of note, EGFRvIII expressing brain tumors co-injected with hMSCs had a lower density of CD31 expressing blood vessels in comparison with control tumors, suggesting a possible role in tumor angiogenesis.

Conclusions/Significance

The results presented in this study illustrate that genetically modified MSCs may function as a novel therapeutic vehicle for malignant brain tumors.     

PI3K通路抑制剂LY294002对人脑胶质瘤细胞株U87细胞衰老和凋亡的影响

目的:PI3K/Akt信号通路是与胶质瘤发生发展密切相关的核心通路之一,LY294002是该通路的特异性抑制剂。本研究通过探讨PI3K通路抑制剂LY294002对U87胶质瘤细胞系细胞衰老及凋亡的影响,从而为胶质瘤患者治疗的新策略奠定理论基础。方法:将体外培养的人脑胶质瘤U87细胞株分为DMSO处理的对照组和LY294002(100μM)处理的实验组,采用β-半乳糖苷酶染色和流式细胞术的方法,分别检测并比较两组肿瘤细胞衰老和凋亡的情况。结果:LY294002处理组U87胶质瘤细胞的衰老指数(32.20±4.46%)显著高于DMSO对照组(3.40±1.61%,t=6.254,P0.001)。另外,与DMSO对照组相比,凋亡蛋白caspase-3mRNA的表达在LY294002处理组胶质瘤细胞中显著上调(t=8.923,P0.05)。LY294002处理组肿瘤细胞的凋亡指数(80.10±4.832%)明显高于DMSO对照组(4.260±1.073%,t=8.923,P0.05)。结论:LY294002既能够诱导肿瘤细胞衰老,又能够诱导肿瘤细胞凋亡,然而其诱导胶质瘤细胞凋亡的能力占据主导地位,为其发挥抗胶质瘤效应的主要途径。另外,在LY294002的持续作用下,部分衰老的肿瘤细胞或许会发生凋亡。这些结论为为临床增强胶质瘤患者的联合化疗奠定了理论基础。     

Mesenchymal stem cells overexpressing PEDF decrease the angiogenesis of gliomas

The present study is an exploration of a novel strategy to target a therapeutic gene to brain tumour tissues. In the present study, we evaluated the feasibility of using hMSCs (human mesenchymal stem cells) to deliver PEDF (pigment epithelium-derived factor), a potent inhibitor of tumour angiogenesis, in a model of intracranial gliomas. To assess its potential of tracking gliomas, MSCs (mesenchymal stem cells) were injected into the cerebral hemisphere and it showed that MSCs infiltrated into the vessel beds and scattered throughout the tumour. In vitro migration assay showed that the VEGF (vascular endothelial growth factor) enhanced MSC migration. In contrast, the migratory activity of MSCs was significantly inhibited with the presence of PEDF. Systematic delivery of AAV (adeno-associated virus)–PEDF to established glioma xenografts resulted in increased apoptosis of gliomas. In addition, MSC–PEDF treatment prolonged the survival of mice bearing U87 gliomas. Taken together, these data validate that MSCs–PEDF can migrate and deliver PEDF to target glioma cells, which may be a novel and promising therapeutic approach for refractory brain tumour.     

Assessment of alterations in X-ray irradiation-induced DNA damage of glioma cells by using proton nuclear magnetic resonance spectroscopy

Glioma is one of the most common types of brain tumors. DNA damage is closely associated with glioma cell apoptosis induced by X-ray irradiation. Alterations of metabolites in glioma can be detected noninvasively by proton nuclear magnetic resonance (1H NMR) spectroscopy. To noninvasively explore the micro mechanism in X-ray irradiation-induced apoptosis, the relationship between metabolites and DNA damage in glioma cells was investigated. Three glioma cell lines (C6, U87 and U251) were randomly designated as control (0 Gy) and treatment groups (1, 5, 10, 15 Gy). After X-ray exposure, each group was separated into four parts: (i) to detect metabolites by 1H NMR spectroscopy; (ii) to make cell colonies; (iii) to detect cell cycle distribution and apoptosis rate by flow cytometry; and (iv) to measure DNA damage by comet assay. The metabolite ratios of lactate/creatine and succinate/creatine decreased (lactate/creatine: C6, 22.17–66.27%; U87, 15.93–44.56%; U251, 26.27–74.48%. succinate/creatine: C6, 14.41–48.35%; U87, 22.03–70.62%; U251, 17.33–60.06%) and choline/creatine increased (C6, 52.22–389.68%; U87, 56.15–82.36%; U251, 31.87–278.62%) in the treatment groups compared with the control group (each P < 0.05), which linearly depended on DNA damage. An increasing dose of X-ray irradiation increased numbers of apoptotic cells (P < 0.01), and the DNA damage parameters were dose-dependent (P < 0.05). The colony-forming rate declined (P < 0.01) and the percentage of cells at G1 stage increased when exposed to 1 Gy X-ray (three cell lines, P < 0.05). Metabolite alterations detected by 1H NMR spectroscopy can be used to determine DNA damage induced by X-ray irradiation. 1H NMR spectroscopy is a noninvasive method to predict DNA damage of glioma cell at the micro level.     

肿瘤干细胞标记分子CD133在胶质瘤细胞U87中的稳定表达

目的：在体外胶质瘤U87细胞中稳定表达肿瘤干细胞标记分子CD133。方法：通过脂质体介导将表达载体质粒CD133-1／pCR3．1-Uni转染U87细胞,G418筛选稳定表达抗性的细胞株;用细胞免疫荧光染色鉴定表达CD133分子的U87细胞。结果：转染CD133表达载体的U87细胞可以被CD133单抗识别,而转染空载体的U87细胞免疫染色结果为阴性,表明CD133分子在U87细胞中稳定表达。结论：U87细胞稳定表达CD133分子,为体内外分析CD133阳性U87细胞特性奠定了基础：U87CD133阳性细胞可以作为免疫组化或流式细胞术等检测其他肿瘤干细胞CD133表达的阳性对照细胞。     

Pro-apoptotic role of integrin β3 in glioma cells

Malignant gliomas are the most destructive type of brain cancer. In order to gain a better understanding of the molecular mechanisms of glioma cell death and survival, we previously established an alkylating agent 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-resistant variant of C6 rat glioma cells. Proteomic analysis indicated a significant down-regulation of integrin beta 3 (ITGB3) in the BCNU-resistant C6R cells. Re-expression of ITGB3 in C6R cells restored the BCNU sensitivity. In U87MG, U373MG, and T98G human glioma cells, there was a positive correlation between ITGB3 expression and the sensitivity to BCNU and etoposide, suggesting an important role of ITGB3 in glioma cell death. Over-expression of ITGB3 cDNA significantly increased the sensitivity of the human glioma cells to the anticancer drug-induced apoptosis. Nitric oxide showed an additive effect on the anticancer drug-induced glioma cell death by increasing ITGB3 expression. Subsequent dissection of signaling pathways indicated that extracellular signal-regulated kinase and unligated integrin-mediated cell death pathway may be involved in the pro-apoptotic role of ITGB3 in glioma cells. These results implicate ITGB3 in glioma cell death/survival and drug resistance.