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• W2005742227 abstract "Growing evidence suggests that survivin, a member of the inhibitor of apoptosis gene family, is responsible for drug resistance in cancer cells, yet little is known about its role in the endothelial cells of the tumor vasculature. We have previously reported that tumor-associated endothelial cells derived from gliomas (TuBECs) are resistant to anticancer chemotherapy whereas normal brain endothelial cells (BECs) are sensitive. The focus of this study is to investigate the mechanism behind this chemoresistance. Here we show that survivin is constitutively overexpressed in the glioma vasculature but not in the blood vessels of normal brain. To determine whether survivin contributes to TuBEC chemoresistance, we used a lentiviral siRNA system or the drug roscovitine to down-regulate survivin expression. Reduced levels of survivin sensitized TuBECs to the chemotherapeutic agents VP-16, paclitaxel, thapsigargin, and temozolomide. This cell death was mediated through caspases 7 and 4. Conversely, forced expression of survivin in BECs was protective against drug cytotoxicity. These data suggest that overexpression of survivin in endothelial cells serves as a protective mechanism that defends the vasculature from drug cytotoxicity. Our studies demonstrate that targeting survivin may be an effective approach to chemosensitization and anti-vascular therapy for brain tumors. Growing evidence suggests that survivin, a member of the inhibitor of apoptosis gene family, is responsible for drug resistance in cancer cells, yet little is known about its role in the endothelial cells of the tumor vasculature. We have previously reported that tumor-associated endothelial cells derived from gliomas (TuBECs) are resistant to anticancer chemotherapy whereas normal brain endothelial cells (BECs) are sensitive. The focus of this study is to investigate the mechanism behind this chemoresistance. Here we show that survivin is constitutively overexpressed in the glioma vasculature but not in the blood vessels of normal brain. To determine whether survivin contributes to TuBEC chemoresistance, we used a lentiviral siRNA system or the drug roscovitine to down-regulate survivin expression. Reduced levels of survivin sensitized TuBECs to the chemotherapeutic agents VP-16, paclitaxel, thapsigargin, and temozolomide. This cell death was mediated through caspases 7 and 4. Conversely, forced expression of survivin in BECs was protective against drug cytotoxicity. These data suggest that overexpression of survivin in endothelial cells serves as a protective mechanism that defends the vasculature from drug cytotoxicity. Our studies demonstrate that targeting survivin may be an effective approach to chemosensitization and anti-vascular therapy for brain tumors. The tumor vasculature is critical for cancer growth.1Bergers G Benjamin LE Tumorigenesis and the angiogenic switch.Nat Rev Cancer. 2003; 3: 401-410Crossref PubMed Scopus (2791) Google Scholar This is particularly relevant in the highly vascular primary brain tumor, glioblastoma multiforme (glioma), a grade IV astrocytoma.2Louis DN Pomeroy SL Cairncross JG Focus on central nervous system neoplasia.Cancer Cell. 2002; 1: 125-128Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar The blood vessels within tumors have been shown to be abnormal in their structure and function as compared to the normal vasculature.3Papetti M Herman IM Mechanisms of normal and tumor-derived angiogenesis.Am J Physiol. 2002; 28: C947-C970Crossref Scopus (607) Google Scholar, 4Bussolati B Deambrosis I Russo S Deregibus MC Camussi G Altered angiogenesis and survival in human tumor-derived endothelial cells.FASEB J. 2003; 17: 1159-1161Crossref PubMed Scopus (231) Google Scholar Studies from this laboratory have shown that the tumor-associated endothelial cells derived from gliomas (TuBECs) have different properties than normal brain endothelial cells (BECs). TuBECs actively secrete pro-angiogenic factors,5Charalambous C Hofman FH Chen TC Functional and phenotypic differences between glioblastoma multiforme-derived and normal human brain endothelial cells.J Neurosurg. 2005; 102: 699-705Crossref PubMed Scopus (36) Google Scholar have increased migration,6Charalambous C Pen LB Su YS Milan J Chen TC Hofman FM Interleukin-8 differentially regulates migration of tumor-associated and normal human brain endothelial cells.Cancer Res. 2005; 65: 10347-10354Crossref PubMed Scopus (59) Google Scholar undergo G0/G1 cell-cycle arrest,7Charalambous C Virrey JJ Kardosh A Jabbour MN Qazi-Abdullah L Pen L Zidovetzki R Schönthal AH Chen TC Hofman FM Glioma-associated endothelial cells show evidence of replicative senescence.Exp Cell Res. 2007; 313: 1192-1202Crossref PubMed Scopus (18) Google Scholar and are resistant to drugs as compared to BECs.7Charalambous C Virrey JJ Kardosh A Jabbour MN Qazi-Abdullah L Pen L Zidovetzki R Schönthal AH Chen TC Hofman FM Glioma-associated endothelial cells show evidence of replicative senescence.Exp Cell Res. 2007; 313: 1192-1202Crossref PubMed Scopus (18) Google Scholar The mechanism of this chemoresistance in TuBECs is not known and is the focus of this study. Studies have shown that chemoresistance in tumor cells can be correlated to an overexpression of the inhibitor of apoptosis survivin. This bifunctional protein inhibits apoptosis and promotes cell division.8Altieri DC The case for survivin as a regulator of microtubule dynamics and cell-death decisions.Curr Opin Cell Biol. 2006; 18: 609-615Crossref PubMed Scopus (257) Google Scholar Survivin is also localized to various areas of the cell; mitochondrial and cytosolic survivin suppress apoptosis through blocking the activity of caspases 9, 3, and 7,9Dohi T Beltrami E Wall NR Plescia J Altieri DC Mitochondrial survivin inhibits apoptosis and promotes tumorigenesis.J Clin Invest. 2004; 114: 1117-1127Crossref PubMed Scopus (339) Google Scholar, 10Dohi T Okada K Xia F Wilford CE Samuel T Welsh K Marusawa H Zou H Armstrong R Matsuzawa S Salvesen GS Reed JC Altieri DC An IAP–IAP complex inhibits apoptosis.J Biol Chem. 2004; 279: 34087-34090Crossref PubMed Scopus (337) Google Scholar, 11Shin S Sung BJ Cho YS Kim HJ Ha NC Hwang JI Chung CW Jung YK Oh BH An anti-apoptotic protein human survivin is a direct inhibitor of caspase-3 and -7.Biochemistry. 2001; 40: 1117-1123Crossref PubMed Scopus (674) Google Scholar whereas nuclear survivin is induced at G2/M of the cell cycle to ensure proper mitosis and cytokinesis.12Vong QP Cao K Li HY Iglesias PA Zheng Y Chromosome alignment and segregation regulated by ubiquitination of survivin.Science. 2005; 310: 1499-1504Crossref PubMed Scopus (198) Google Scholar Survivin is typically found at low levels in normal cells but is elevated in many solid and hematologenous cancers.13Ambrosini G Adida C Altieri DC A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma.Nat Med. 1997; 3: 917-921Crossref PubMed Scopus (2981) Google Scholar In various tumors, high survivin levels are correlated with poor prognosis, decreased apoptosis, increased angiogenesis, and chemoresistance in cancer cell lines.14Altieri DC Validating survivin as a cancer therapeutic agent.Nat Rev Cancer. 2003; 3: 46-54Crossref PubMed Scopus (1094) Google Scholar, 15Zhen HN Zhang X Hu PZ Survivin expression and its relation with proliferation, apoptosis, and angiogenesis in brain gliomas.Cancer. 2005; 104: 2775-2783Crossref PubMed Scopus (80) Google Scholar However, little is known of survivin's function in the endothelial cells of the tumor vasculature. In this study we present the novel findings that survivin is overexpressed in primary cultures of endothelial cells derived from human glioma tissues and that survivin protects these cells from chemotherapeutic agents. We decreased survivin through genetic and pharmacological approaches and found that survivin is responsible for the chemoresistance in TuBECs. Furthermore, forced expression of survivin protected BECs from cytotoxic drugs. Thus survivin is a prosurvival player in tumor-associated endothelial cells, and reducing survivin in the tumor vasculature can be an effective chemosensitizing mechanism and a valuable target for anti-angiogenic therapy. Isolation of BECs and TuBECs from human normal brain and glioma tissues was previously described.5Charalambous C Hofman FH Chen TC Functional and phenotypic differences between glioblastoma multiforme-derived and normal human brain endothelial cells.J Neurosurg. 2005; 102: 699-705Crossref PubMed Scopus (36) Google Scholar Briefly, tissues were obtained and handled in agreement with the Keck School of Medicine, University of Southern California Institutional Review Board guidelines. The tissues were washed three times with RPMI 1640 medium (Life Technologies, Inc., Grand Island, NY) containing 2% fetal calf serum (Omega Scientific, Tarzana, CA), and 1% penicillin/streptomycin (Life Technologies, Inc.). The tissue was then minced into small pieces, and fresh medium was added. The mixture was transferred to a centrifuge tube, and an equal volume of a 30% dextran solution (Sigma-Aldrich, St. Louis, MO) was added, bringing the mixture to a final concentration of 15% dextran. The resulting mixture was then centrifuged for 10 minutes at 10,000 rpm to pull down the brain microvessels. The microvessel pellet was resuspended in 1 mg/ml of collagenase-dispase in RPMI 1640 medium supplemented with 2% fetal calf serum (FCS) (RPMI-2% FCS) and incubated in a shaking 37°C water bath for 1 hour. Subsequently, 10 ml of RPMI-2% FCS was added to the cells and centrifuged at 1200 rpm for 5 minutes. The pellet was resuspended in 20 ml of the RPMI-2% FCS and centrifuged again. The final pellet was resuspended in the endothelial cell culture medium [RPMI 1640 medium supplemented with 100 ng/ml endothelial cell growth supplement (Upstate Biotechnologies, Rochester, NY), 2 mmol/L l-glutamine (Life Technologies, Inc.), 10 mmol/L HEPES (Life Technologies, Inc.), 24 mmol/L sodium bicarbonate (Life Technologies, Inc.), 300 U heparin USP (Sigma-Aldrich), 1% penicillin/streptomycin, and 10% FCS]. Cells were plated on precoated gelatin flasks, and the medium was changed every 3 or 4 days until the cell cultures became 80% confluent. Endothelial cells were then purified from the cellular mixture by selecting cells that bind diacetylated low-density lipoprotein (di-LDL). Subconfluent cells were incubated with 10 ng/ml of fluorescent di-LDL for 4 hours at 37°C and then analyzed using fluorescence-activated cell sorting analysis (see Supplemental Figure S1 at http://ajp.amjpathol.org). After the sorting procedure, the purity of BECs and TuBECs was confirmed by immunostaining for specific endothelial cell markers: CD31/PECAM-1 (Santa Cruz Biotechnology, Santa Cruz, CA), von Willebrand Factor (DAKO, Carpinteria, CA), VE-cadherin (R&D Systems, Minneapolis, MN), and CD105/endoglin (Santa Cruz Biotechnology) and was found to be 100% positive (see Figure 1). BECs and TuBECs were negative for astrocyte cell marker glial fibrillary acidic protein (DAKO), progenitor endothelial cell marker CD34 (DAKO), and macrophage/microglia marker CD11b (DAKO). BEC and TuBEC cultures were grown onto 1% gelatin-coated surfaces and were used up to passage 6. Digital images were taken of the cultured cells using a Sony (Park Ridge, NJ) DSC-P9 camera and Nikon (Tokyo, Japan) TMS microscope (objective lens: × 10 magnification, 0.25 numerical aperture) at room temperature. BECs and TuBECs were propagated in medium containing endothelial cell growth supplement, as described above. In performing experiments, however, endothelial cell growth supplement was removed from the culture medium. Cells in RPMI with 10% FCS were plated onto 96-well plates at 5 × 103 cells/well. Cells were treated with VP-16 (Calbiochem, La Jolla, CA), paclitaxel (Sigma-Aldrich), temozolomide (Schering-Plough, Kenilworth, NJ), or thapsigargin (Calbiochem), alone or in combination with roscovitine (Sigma-Aldrich). After treatment, cells were lysed and immediately analyzed using the Cell Death Detection ELISAPlus kit (Roche Diagnostics, Indianapolis, IN). Absorbance was measured at 405 nm, and percent cell death was calculated based on a 100% positive cell death control. The groups were treated in duplicate and the experiments were repeated at least twice. Cytocentrifuge cell preparations and cells grown onto chambered slides were fixed with acetone, blocked with 5% goat serum, and incubated overnight with anti-survivin polyclonal antibody (1:100) (Santa Cruz Biotechnology). Samples were incubated with biotinylated goat anti-rabbit antibody (1:400) (Vector Laboratories, Burlingame, CA) for 45 minutes, treated with the avidin-biotin-peroxidase complex (Vector Laboratories) for 30 minutes, and then treated with aminoethyl carbazol substrate for 15 minutes (Vector Laboratories). Samples were counterstained with hematoxylin for 1.5 minutes. A red precipitate denotes positive staining. Specificity of the anti-survivin polyclonal antibody was tested and confirmed through the use of monoclonal survivin antibodies and survivin blocking peptides (see Supplemental Figure S2, A and B, at http://ajp.amjpathol.org). Double staining was performed on glioma and normal brain tissues fixed with acetone. Tissues were co-incubated with anti-survivin polyclonal (1:100) and anti-CD105 monoclonal (1:100) antibodies, and subsequently treated with Texas Red anti-rabbit (1:400) and fluorescein anti-mouse secondary antibodies (1:200) (Vector Laboratories). Mounting medium containing the fluorescent blue 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories) was used to identify nuclear staining. Rabbit and mouse IgG isotype-matched controls and the omission of primary antibody were used as negative controls. The staining was analyzed using a Zeiss LSM510 confocal microscope (Zeiss, Thornwood, NY); red color denotes survivin positivity, green color represents CD105 positivity, and yellow color signifies positive staining for both survivin and CD105. Western blots were performed as previously described.7Charalambous C Virrey JJ Kardosh A Jabbour MN Qazi-Abdullah L Pen L Zidovetzki R Schönthal AH Chen TC Hofman FM Glioma-associated endothelial cells show evidence of replicative senescence.Exp Cell Res. 2007; 313: 1192-1202Crossref PubMed Scopus (18) Google Scholar Membranes were incubated overnight with antibodies to survivin (1:250) (Santa Cruz Biotechnology), caspase 7 (1:1000) (BD Pharmingen, Franklin Lakes, NJ), caspase 4 (1:500) (BD Pharmingen), CHOP (1:500) (Santa Cruz Biotechnology), or GAPDH (1:5000) (Santa Cruz Biotechnology) and then incubated with horseradish peroxidase-conjugated (Santa Cruz Biotechnology) or fluorescent-conjugated (Pierce, Rockford, IL) secondary antibodies (1:5000 to 1:15,000) for 45 minutes. Protein bands were detected either by chemiluminescence using the SuperSignal substrates (Pierce) or by Odyssey infrared imaging (LI-COR Biosciences, Lincoln, NE). Specificity of the anti-survivin polyclonal antibody was confirmed in comparison to a monoclonal survivin antibody (see Supplemental Figure S2C at http://ajp.amjpathol.org). The following siRNA sequences (sense) were used: 5′-GGCTGGCTTCATCCACTGC-3′ (survivin) and 5′-GTGACCAGCGAATACCTGT-3′ (LacZ). The siRNA sequences were subcloned into the lentiviral siRNA delivery vector FG-12 at XbaI and XhoI sites, as previously described.16Qin XF An DS Chen ISY Baltimore D Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5.Proc Natl Acad Sci USA. 2003; 100: 183-188Crossref PubMed Scopus (635) Google Scholar The human wild-type survivin gene (pEYFP-N1-survivin) was kindly provided by Dr. Jeroen Pouwels and Dr. Anu Kukkonen (VTT Medical Biotechnology, Turku, Finland). Survivin was amplified using the primers 5′-CTAGTCTAGAGCCACCATGGGTGCCCCGACGTT-3′ and 5′-CGGGAATTCTCAATCCATGGCAGCCAGCTGCT-3′, and subcloned into the lentiviral expression vector pRRLsinCMV at XbaI and EcoR1 sites. Green fluorescent protein (GFP) was amplified from pEGFP with the primers 5′-CTGTCGGATCCGGAACCGTCAGATCCGCTA-3′ and 5′-CTGCAGAATTCGAAGCTTGAGCTCGAG-3′, and subcloned into pRRLsinCMV at BamH1 and EcoR1 sites. Correct orientation and sequence for both FG-12 and pRRLsinCMV cloning was confirmed through restriction enzyme digestion and DNA sequencing. The FG-12 or pRRLsinCMV constructs were used to transfect 293T cells along with packaging vectors pMDG and pCMVΔR8.2.17Guan S Chen M Woodley DT Li W Nckβ adapter controls neuritogenesis by maintaining cellular paxillin level.Mol Cell Biol. 2007; 17: 6001-6011Crossref Scopus (21) Google Scholar The viral supernatant was collected and used to infect primary endothelial cell cultures. For the FG-12 system, infection efficiency was monitored through GFP labeling.18Bandyopadhyay B Fan J Guan S Li Y Chen M Woodley DT Li W A “traffic control” role for TGFbeta3: orchestrating dermal and epidermal cell motility during wound healing.J Cell Biol. 2006; 172: 1093-1105Crossref PubMed Scopus (127) Google Scholar The cells were then evaluated through Western blot analysis. Cells were plated onto gelatin-coated six-well plates at 5 × 104 cells/well. Cells were treated with VP-16, paclitaxel, or thapsigargin for 96 hours. The culture supernatants were collected, centrifuged at 1000 × g for 10 minutes, and the LDH release assay was performed according to manufacturer's protocol (Sigma-Aldrich). Absorbance was read at 490 nm. The fold increase of LDH release was based on the OD readings from untreated, uninfected cells. Groups were treated in triplicate and experiments were repeated at least twice. Cells were seeded in triplicates onto 96-well plates in RPMI/10%FCS at 3 × 103 cells/well. Cells were treated with the designated drugs: VP-16, paclitaxel, thapsigargin, and/or roscovitine. After treatment, cells were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (Sigma-Aldrich) for 4 hours. Medium was removed and dimethyl sulfoxide (DMSO) was added. Absorbance was measured at 490 nm, and percentage cell viability was calculated relative to untreated controls. Experiments were repeated three times. Values are presented as the mean ± SEM. Statistical significance was evaluated using the Student's two-tailed t-test. P < 0.05 was considered statistically significant. Fresh human glioma and normal brain specimens were processed for the isolation and culture of endothelial cells, as extensively described in the Materials and Methods. GBM tissues were received after surgery from glioma patients, whereas normal brain specimens were obtained from trauma or epileptic patients. The characterization of TuBECs and BECs was validated through 100% positive immunostaining for the following endothelial cell markers: CD31, von Willebrand Factor, CD105, and VE-cadherin (Figure 1). TuBECs and BECs, however, stained negative for the astrocyte/glial cell marker GFAP and for the microglia/macrophage marker CD11b (Figure 1). The glioma cell line U87MG was 100% positive for GFAP expression, whereas glioma tissues stained positive for CD11b and CD34. Interestingly, TuBECs and BECs were also negative for CD34, a marker of precursor endothelial cells, which suggests that TuBECs and BECs are at their end stage of differentiation (Figure 1). TuBECs and BECs were treated with drugs that induce cytotoxicity via different mechanisms, particularly VP-16 (10, 50 μmol/L), paclitaxel (3, 10 ng/ml), thapsigargin (10, 30 nmol/L), and temozolomide (100, 300 μmol/L). VP-16 inhibits topoisomerase II, whereas paclitaxel affects microtubule stability.19Meresse P Dechaux E Monneret C Bertounesque E Etoposide: discovery and medicinal chemistry.Curr Med Chem. 2004; 11: 2443-2466Crossref PubMed Scopus (126) Google Scholar, 20Marupudi NI Han JE Li KW Renard VM Tyler BM Brem H Paclitaxel: a review of adverse toxicities and novel delivery strategies.Expert Opin Drug Safety. 2007; 6: 609-621Crossref PubMed Scopus (298) Google Scholar Thapsigargin targets calcium pumps in the endoplasmic reticulum (ER),21Denmeade SR Isaacs JT The SERCA pump as a therapeutic target: making a “smart bomb” for prostate cancer.Cancer Biol Ther. 2005; 4: 14-22Crossref PubMed Scopus (192) Google Scholar and temozolomide is a DNA alkylating agent.22Friedman HS Kirby T Calvert H Temozolomide and treatment of malignant glioma.Clin Cancer Res. 2000; 6: 2585-2597PubMed Google Scholar After drug treatment, cell death was analyzed using the Cell Death Detection ELISA assay. TuBECs were relatively resistant to VP-16, paclitaxel, and thapsigargin, whereas the BECs were sensitive. After 72 hours TuBECs exhibited much lesser cell death than BECs; BECs underwent 72% cell death with 50 μmol/L VP-16 (Figure 2A). TuBECs and BECs were also treated with paclitaxel, and a similar trend was observed. Paclitaxel at both doses had no significant effect on TuBEC viability as compared to untreated cells (P = 0.116 and P = 0.189), but BECs exhibited four times more cell death at the higher concentration (Figure 2B). This chemoresistance in TuBECs was again observed with thapsigargin treatment. TuBECs treated with 30 nmol/L thapsigargin showed 18% cell death whereas treatment of BECs showed 65% cell death, respectively (Figure 2C). Similar findings for VP-16, paclitaxel, and thapsigargin treatments were also detected in the MTT cell viability assay (data not shown). Interestingly, both TuBECs and BECs were not affected by 7-day temozolomide treatment as compared to untreated cells, even at 300 μmol/L (P = 0.136, TuBECs; and P = 0.227, BECs) (Figure 2D). These studies indicate that TuBECs are resistant to different classes of cytotoxic drugs. Because survivin is overexpressed in a variety of tumors including gliomas,15Zhen HN Zhang X Hu PZ Survivin expression and its relation with proliferation, apoptosis, and angiogenesis in brain gliomas.Cancer. 2005; 104: 2775-2783Crossref PubMed Scopus (80) Google Scholar we wanted to explore whether survivin is also overexpressed in the tumor vasculature. To accomplish this, we immunostained cell preparations of TuBECs and BECs with an antibody to survivin. The results showed that TuBECs stained intensely positive for survivin compared to BECs (Figure 3A). Survivin staining was also performed on cells grown on glass chambered slides; again, survivin expression was greater in TuBECs compared to BECs (Figure 3A). To confirm the specificity of survivin staining, a survivin blocking peptide was added along with the antibody and shown to have reduced the survivin staining (see Supplemental Figure S2B at http://ajp.amjpathol.org). Western blot analysis was also performed on TuBECs and BECs (Figure 3B). This is representative of 10 TuBEC and 11 BEC specimens from different patients. The data demonstrate that survivin is highly expressed in TuBECs and minimally expressed in BECs. We then performed double staining on human glioma tissues to determine whether survivin expression can be detected in the glioma vasculature in situ(Figure 3, C and D). Tissues were co-stained for CD105/endoglin, a marker for endothelial cells, and survivin; DAPI blue staining identified nuclei. Normal brain tissues were stained using the same procedure (Figure 3, E and F). The staining of the tissues was analyzed through confocal microscopy. Merging CD105 and survivin staining revealed that the tumor-associated vasculature within the glioma tissue exhibited positive survivin expression, as denoted by the yellow color (Figure 3, C and D; arrows). Normal brain tissues were negative for survivin (Figure 3D). Two representative fields are shown for both the glioma and normal brain tissues, one at a lower magnification (Figure 3, C and E) and another at a higher magnification (Figure 3, D and F). These studies demonstrate that blood vessels of glioma tissues express elevated levels of the survivin protein. To test whether survivin protects TuBECs from chemotherapeutic agents, a lentiviral vector carrying the siRNA targeted against survivin (siSurv) was constructed. A siRNA against the nonmammalian gene LacZ was also used (siLacZ) as a control. Five days after infection, TuBECs with siSurv or siLacZ were evaluated for survivin expression using Western blot analysis (Figure 4A). Survivin protein was reduced in TuBECs infected with siSurv. This down-regulation of survivin was sustained for at least 3 weeks, as indicated by immunostaining of TuBEC siSurv and TuBEC siLacZ performed 21 days after infection (Figure 4B). These data also emphasize the specificity of the reagents used in these studies. We then examined whether reduced survivin levels would affect TuBEC sensitivity to drugs. TuBECs, TuBEC siSurv, and TuBEC siLacZ were treated with VP-16 for 96 hours. In observing the cells in culture, it was apparent that VP-16 was effective in killing the cells infected with siSurv (Figure 4C). These cultures exhibited floating and rounded cells, typical of cell death. The TuBECs infected with siLacZ, however, had little response to VP-16 and exhibited morphology of healthy cells. To quantify the results observed in culture, the Cell Death Detection ELISA was used. Results show that the drugs had relatively little effect on uninfected TuBECs and TuBECs infected with siLacZ; in contrast, TuBECs infected with siSurv demonstrated significantly increased sensitivity to these agents (Figure 4D). TuBECs infected with siSurv and treated with VP-16 or thapsigargin for 96 hours demonstrated nearly a twofold increase in cell death (72%) (P = 0.007) and (55%) (P = 0.005), respectively. TuBECs infected with siSurv and treated with paclitaxel for 96 hours, exhibited a ninefold increase in cell death to 45% (P = 0.009). Incubation of TuBECs for 4 days with temozolomide, the principle drug used for glioma therapy, had no effect on cell death, in the absence or presence of siSurv (data not shown). However, after 7 days of temozolomide treatment, siSurv-infected TuBECs exhibited increased cell death (25%) compared to untreated or control infected TuBECs (P = 0.001) (Figure 4E). Similar results for VP-16, paclitaxel, and thapsigargin treatments were obtained using the LDH release assay (Figure 4F). These data indicate that decreasing survivin protein levels in TuBECs will chemosensitize these vascular cells to anti-tumor agents. To determine whether survivin overexpression will be sufficient to confer chemoresistance on normal BECs, we infected BECs with a lentiviral expression vector containing the human wild-type survivin gene (BEC-Surv); to serve as a control, BECs were infected with a lentivirus containing GFP (BEC-GFP). Western blot analysis detected the increased survivin expression in BEC-Surv (Figure 5A). Once again the specificity of the antibody to survivin was confirmed. Survivin levels of BEC-GFP remained similar to those of uninfected BECs, suggesting the lentivirus had no secondary effects on survivin expression. Uninfected BECs, BEC-Surv, and BEC-GFP were then treated with drugs (VP-16, thapsigargin, and paclitaxel), and cytotoxicity was measured. The results show that overexpression of survivin resulted in reduced cytotoxicity. In Figure 5B, BEC-Surv were more resistant to VP-16 after 72 hours of treatment as compared to BECs (P = 0.019). A similar trend was also seen with thapsigargin, with significantly less death observed with BEC-Surv (43%) than with BECs (82%) (P = 0.010) (Figure 5C). This protective effect of survivin was also exhibited with paclitaxel treatment after 72 and 144 hours (P = 0.002 and P = 0.008, respectively) (Figure 5D). These data demonstrate that the overexpression of survivin in normal endothelial cells is protective and causes these sensitive cells to become resistant to cytotoxic drugs. We next examined the activation of different caspases involved in apoptosis to better understand the mechanism by which reduced expression of survivin enhances cell death in TuBECs. Cells were treated with VP-16 and tested for caspase 7 cleavage, an effector caspase shown to interact with survivin.9Dohi T Beltrami E Wall NR Plescia J Altieri DC Mitochondrial survivin inhibits apoptosis and promotes tumorigenesis.J Clin Invest. 2004; 114: 1117-1127Crossref PubMed Scopus (339) Google Scholar, 11Shin S Sung BJ Cho YS Kim HJ Ha NC Hwang JI Chung CW Jung YK Oh BH An anti-apoptotic protein human survivin is a direct inhibitor of caspase-3 and -7.Biochemistry. 2001; 40: 1117-1123Crossref PubMed Scopus (674) Google Scholar Treatment of BECs with VP-16 caused cleavage of caspase 7 (Figure 6A), whereas VP-16 had no effect on treated TuBECs. However, TuBECs with reduced survivin demonstrated activation of procaspase 7 when treated with the drug. Similar results were obtained with thapsigargin (Figure 6B) and paclitaxel treatments (Figure 6C). We also detected an intermediate product of procaspase 7 (32 kDa); this intermediate product naturally occurs in cells and was not indicative of caspase 7 activity. We then investigated the initiator caspase 4, a marker of ER stress-induced apoptosis (caspase 12 in mice).23Szegezdi E Logue SE Gorman AM Samali A Mediators of endoplasmic reticulum stress-induced apoptosis.EMBO Rep. 2006; 7: 880-885Crossref PubMed Scopus (1759) Google Scholar The results show that TuBEC siSurv treated with thapsigargin exhibited caspase 4 cleavage (Figure 6D), whereas untreated or control-infected TuBECs demonstrated negligible reactivity. We then analyzed thapsigargin-treated TuBECs and BECs for the induction of CHOP, a pro-apoptotic mediator of the ER stress pathway.23Szegezdi E Logue SE Gorman AM Samali A Mediators of endoplasmic reticulum stress-induced apoptosis.EMBO Rep. 2006; 7: 880-885Crossref PubMed Scopus (1759) Google Scholar The results were similar to those observed with caspase 4 cleavage; thapsigargin-treated TuBEC siSurv demonstrated remarkable CHOP induction (Figure" @default.
• W2005742227 created "2016-06-24" @default.
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• W2005742227 date "2008-08-01" @default.
• W2005742227 modified "2023-10-16" @default.
• W2005742227 title "Increased Survivin Expression Confers Chemoresistance to Tumor-Associated Endothelial Cells" @default.
• W2005742227 cites W1558742074 @default.
• W2005742227 cites W1968262266 @default.
• W2005742227 cites W1980350123 @default.
• W2005742227 cites W1980595945 @default.
• W2005742227 cites W1982491440 @default.
• W2005742227 cites W1983312081 @default.
• W2005742227 cites W1985850927 @default.
• W2005742227 cites W1991852988 @default.
• W2005742227 cites W1998796283 @default.
• W2005742227 cites W2015710917 @default.
• W2005742227 cites W2020967120 @default.
• W2005742227 cites W2021942889 @default.
• W2005742227 cites W2029758127 @default.
• W2005742227 cites W2030891330 @default.
• W2005742227 cites W2035523654 @default.
• W2005742227 cites W2039791984 @default.
• W2005742227 cites W2050499474 @default.
• W2005742227 cites W2053219638 @default.
• W2005742227 cites W2055019397 @default.
• W2005742227 cites W2059890691 @default.
• W2005742227 cites W2064580048 @default.
• W2005742227 cites W2065392301 @default.
• W2005742227 cites W2075971408 @default.
• W2005742227 cites W2083540484 @default.
• W2005742227 cites W2101546624 @default.
• W2005742227 cites W2105253056 @default.
• W2005742227 cites W2115719349 @default.
• W2005742227 cites W2133644385 @default.
• W2005742227 cites W2133653109 @default.
• W2005742227 cites W2135135539 @default.
• W2005742227 cites W2149754404 @default.
• W2005742227 cites W2149829152 @default.
• W2005742227 cites W2151559423 @default.
• W2005742227 cites W2154054522 @default.
• W2005742227 cites W2154795389 @default.
• W2005742227 cites W2163453798 @default.
• W2005742227 cites W2169838007 @default.
• W2005742227 cites W2208349676 @default.
• W2005742227 cites W2804842145 @default.
• W2005742227 cites W3149376927 @default.
• W2005742227 cites W4245093707 @default.
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