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1.  Upregulation of Survivin in G2/M Cells and Inhibition of Caspase 9 Activity Enhances Resistance in Staurosporine-Induced Apoptosis 
Neoplasia (New York, N.Y.)  2004;6(1):29-40.
Survivin, a member of the inhibitor of apoptosis (IAP) gene family, plays an important role in both the regulation of cell cycle and the inhibition of apoptosis, and is frequently overexpressed in many tumor types. In neuroblastomas, the expression of survivin correlates with a more aggressive and histologically unfavorable disease. Survivin is predominantly a cytoplasmic protein that is expressed in a cell cycle-dependent manner, increasing in the G2/M phase of the cell cycle followed by a rapid decline in the G1 phase. Recently, the role of survivin in resistance to chemotherapy has become an area of intensive investigation. In this study, we demonstrate a phase-specific resistance due to survivin in staurosporine (STS)-induced apoptosis in the human neuroblastoma cell line SK-N-MC. G2/M-arrested cultures show an upregulation of survivin expression and are more resistant, whereas G1-phase cells that show decreased levels of survivin are more sensitive to apoptosis. Localization studies revealed differences in the distribution of survivin in two synchronized populations, with G1 cells having weakly positive staining confined to the nucleus, in contrast to G2/M cells that depicted a more uniform and intense expression of survivin throughout the cell. In our experimental system, STS induced apoptosis through the mitochondrial-caspase 9-mediated pathway. Retention of survivin in G1 cells by inhibition of the ubiquitin-proteosome pathway or inhibition of caspase 9 protected the cells against apoptosis. Our data suggest that survivin exerts its antiapoptotic effect by inhibiting caspase 9 activity, an important event in STS-mediated apoptosis. In context with cell cycle-dependent responses to chemotherapy, the data from this study suggest the possibility of exploiting the survivin pathway for inducing apoptosis in tumor cells.
PMCID: PMC1679816  PMID: 15068669
Survivin; caspase 9; G2/M; staurosporine; neuroblastoma
2.  The RNA-Binding Protein CUG-BP1 Increases Survivin Expression in Esophageal Cancer Cells through Enhanced mRNA Stability 
The Biochemical journal  2012;446(1):113-123.
Survivin, a member of the Inhibitor of Apoptosis Protein (IAP) family, plays important roles in maintaining cellular homeostasis and regulating cell cycle progression. This IAP is overexpressed in esophageal cancer cells leading to uncontrolled cell growth and resistance to apoptosis. CUG-binding protein 1 (CUG-BP1) is an RNA-binding protein that regulates the stability and translational efficiency of target mRNAs. In this paper, we report that CUG-BP1 is overexpressed in esophageal cancer cell lines and human esophageal cancer specimens. CUG-BP1 associates with the 3′ untranslated region of survivin mRNA, thereby stabilizing the transcript and elevating its expression in esophageal cancer cells. Our results show that overexpression of CUG-BP1 in esophageal epithelial cells results in increased survivin mRNA stability and consequently survivin protein expression. Conversely, silencing CUG-BP1 in esophageal cancer cells destabilizes survivin mRNA, lowering survivin protein levels. In addition, we have found that altering CUG-BP1 expression modulates susceptibility to chemotherapy-induced apoptosis. Overexpression of CUG-BP1 in esophageal epithelial cells increases resistance to apoptosis, while silencing CUG-BP1 makes esophageal cancer cells more susceptible to chemotherapy-induced apoptosis. Cotransfection experiments with siRNA directed against survivin suggest that the anti-apoptotic role of CUG-BP1 is not entirely dependent on its effect on survivin expression.
PMCID: PMC4118298  PMID: 22646166
CUG-BP1; survivin; mRNA stability; apoptosis; 3′-untranslated region; post-transcriptional regulation
3.  Survivin family proteins as novel molecular determinants of doxorubicin resistance in organotypic human breast tumors 
The molecular determinants of breast cancer resistance to first-line anthracycline-containing chemotherapy are unknown.
We examined the response to doxorubicin of organotypic cultures of primary human breast tumors ex vivo with respect to cell proliferation, DNA damage and modulation of apoptosis. Samples were analyzed for genome-wide modulation of cell death pathways, differential activation of p53, and the role of survivin family molecules in drug resistance. Rational drug combination regimens were explored by high-throughput screening, and validated in model breast cancer cell types.
Doxorubicin treatment segregated organotypic human breast tumors into distinct Responder or Non Responder groups, characterized by differential proliferative index, stabilization of p53, and induction of apoptosis. Conversely, tumor histotype, hormone receptor or human epidermal growth factor receptor-2 (HER2) status did not influence chemotherapy sensitivity. Global analysis of cell death pathways identified survivin and its alternatively spliced form, survivin-ΔEx3 as uniquely overexpressed in Non Responder breast tumors. Forced expression of survivin-ΔEx3 preserved cell viability and prevented doxorubicin-induced apoptosis in breast cancer cell types. High-throughput pharmacologic targeting of survivin family proteins with a small-molecule survivin suppressant currently in the clinic (YM155) selectively potentiated the effect of doxorubicin, but not other chemotherapeutics in breast cancer cell types, and induced tumor cell apoptosis.
Survivin family proteins are novel effectors of doxorubicin resistance in chemotherapy-naive breast cancer. The incorporation of survivin antagonist(s) in anthracycline-containing regimens may have improved clinical activity in these patients.
PMCID: PMC4076638  PMID: 24886669
4.  Liposome-Formulated siRNA Against Msi1 and Docetaxel Treatment in an Animal Model of Advanced Gastric Adenocarcinoma Eradicates Cancer Stem Cells and Metastasis 
In advanced gastric adenocarcinoma, potent chemoresistant cancer stem cells (CSCs) differentiate into progenitor cells that form all of the cell types in the patient's tumor. Cancer stem cells have genetic mutations that render them resistant to chemotherapy- or radiation-induced injury due to rapid repair of DNA damage. The goal of this study was to use antisense molecular targeting to eradicate cancer stem cells, as well as docetaxel, thus eliminating tumor cells and potential recurrence and metastasis in a gastric cancer model.
Materials and Methods:
Tumor cells that overexpressed bcl-2 and CSCs were obtained from chemoresistant patients with metastatic advanced gastric adenocarcinoma. The CSCs overexpressed Msi1, which activates Notch and Wnt pathways. The tumor cells were orthotopically transplanted into genetically engineered immunodeficient mice, which developed a tumor with the same cell types as in the human tumor. The animals were treated with pegylated liposomes composed of phospholipids with high transition temperature (Tc). We entrapped docetaxel in the acyl chains, and encapsulated a 21-base pair of small interfering RNA (siRNA) strand targeted to Msi1 in the liposomes. This colloidal formulation was termed LP/AS-Msi1/TXT (under patent).
Post-treatment, the endocytosed siRNA unwound and was incorporated into RNA-induced silencing complex (RISC), which is a stable protein-RNA complex. siRNA was then directed to the targeted Msi1 messenger RNA (mRNA), which is involved in the cancer stem cell pathway. The Msi1 mRNA undergoes cleavage and degradation, interrupting the protein synthesis of the targeted Msi1 gene. This causes downregulation of Wnt-suppressing cell proliferation, migration of cancer stem cells by inhibiting angiogenesis after VEGF downregulation, and induction of apoptosis after downregulation of antiapoptotic caspase inhibitor survivin. Furthermore, siRNA against Msi1 inhibits degrading proteases of extracellular matrix, such as MMP26 and matrilysin, and cell adhesion molecules, such as neuronal cell adhesion molecule (NRCAM) and CD44, inhibiting invasion and metastasis. Also, blockage of the Wnt signaling cascade led to inhibition of cancer progenitor cells by downregulation of NRSF/REST and ENC1 with BTB-like domain genes. It also blocked tumorigenesis by downregulating claudin1, which leads to inhibition of the Ctnn-Beta-TCF/LEF signaling pathway. Downregulation of Msi1 inhibited the Notch signaling pathway, blocking nuclear transcription factors, downregulating genes, and inhibiting proteins involved in the self-renewal and regeneration of cancer stem cells (which might be considered the roots of the gastric adenocarcinoma tree), leading to their eradication by inhibiting mitotic divisions. Docetaxel treatment, via cell signaling mechanisms, eradicated tumor cells (the leaves of the gastric adenocarcinoma tree) by phosphorylating antiapoptotic oncogene bcl-2, leading to induction of apoptosis, or type I programmed cell death (PCD). Downregulation of bcl-2 led to upregulation of tumor suppressor gene Beclin-1, inducing autophagy, or type II PCD. Polymerization of microtubules led to cell cycle blockage, inhibiting mitosis. BrdU and MTT assays exhibited inhibition of DNA synthesis and metabolic activity, respectively. Polymerization of microtubules led to cell cycle blockage, inhibiting mitosis. Transmission electron microscopy demonstrated a phagocytic bystander killing effect mediated by APCs, and adjacent tumor cells. Finally, we observed morphologic and metabolic evidence of inhibition of tumor recurrence and metastasis on computed tomography and positron emission tomography scans, respectively.
The novel therapy, LP/AS-Msi1/TXT, is designed to target cancer stem cells by inhibiting vital pathways, thus eradicating the “roots” of advanced gastric adenocarcinoma recurrence and metastasis, while the co-administered, conventional chemotherapeutic agent docetaxel eradicates the tumor cells (or the “leaves” of advanced gastric adenocarcinoma). LP/AS-Msi1/TXT represents a potential tailored approach to target cancer stem cells with less toxicity than observed with conventional chemotherapy.
PMCID: PMC3056302
5.  Modified vaccinia Ankara expressing survivin combined with gemcitabine generates specific antitumor effects in a murine pancreatic carcinoma model 
Cancer Immunology, Immunotherapy  2010;60(1):99-109.
Survivin is overexpressed by 70–80% of pancreatic cancers, and is associated with resistance to chemotherapy and a poor prognosis. Gemcitabine has been a standard treatment for patients with advanced pancreatic cancer for a decade. Recent reports have demonstrated that gemcitabine treatment attenuates the tumor-suppressive environment by eliminating CD11b+/Gr-1+ myeloid-derived suppressor cells (MDSCs). We hypothesize that a cancer vaccine targeting survivin can achieve enhanced efficacy when combined with gemcitabine. In this study, we tested this hypothesis using modified vaccinia Ankara (MVA) expressing full-length murine survivin. The poorly immunogenic mouse pancreas adenocarcinoma cell line, Pan02, which expresses murine survivin and is syngeneic to C57BL/6, was used for this study. Immunization with MVA-survivin resulted in a modest therapeutic antitumor effect on established Pan02 tumors. When administered with gemcitabine, MVA-survivin immunization resulted in significant tumor regression and prolonged survival. The enhanced vaccine efficacy was associated with decreased CD11b+/Gr-1+ MDSCs. To analyze the survivin-specific immune response to MVA-survivin immunization, we utilized a peptide library of 15mers with 11 residues overlapping from full-length murine survivin. Splenocytes from mice immunized with MVA-survivin produced intracellular γ-interferon in response to in vitro stimulation with the overlapping peptide library. Increased survivin-specific CD8+ T cells that specifically recognized the Pan02 tumor line were seen in mice treated with MVA-survivin and gemcitabine. These data suggest that vaccination with MVA-survivin in combination with gemcitabine represents an attractive strategy to overcome tumor-induced peripheral immune tolerance, and this effect has potential for clinical benefit in pancreatic cancer.
PMCID: PMC3289969  PMID: 20960189
Modified vaccinia Ankara (MVA); Survivin Gemcitabine; Pancreatic cancer; Cancer vaccine
6.  Silencing Survivin Splice Variant 2B Leads to Antitumor Activity in Taxane-Resistant Ovarian Cancer 
To study the role of survivin and its splice variants in taxane-resistant ovarian cancer.
Experimental Design
We assessed the messenger RNA levels of survivin splice variants in ovarian cancer cell lines and ovarian tumor samples. Small-interference RNAs (siRNAs) targeting survivin were designed to silence all survivin splice variants (T-siRNA) or survivin 2B (2B-siRNA) in vitro and orthotopic murine models of ovarian cancer. The mechanism of cell death was studied taxane-resistant ovarian cancer cells and in tumor sections obtained from different mouse tumors.
Taxane-resistant ovarian cancer cells express higher survivin mRNA levels than their taxane-sensitive counterparts. Survivin 2B expression was significantly higher in taxane-resistant compared to sensitive cells. Silencing survivin 2B induced growth inhibitory effects similar to silencing total survivin in vitro. In addition, survivin 2B siRNA incorporated into DOPC nanoliposomes resulted in significant reduction in tumor growth (p<0.05) in orthotopic murine models of ovarian cancer, and these effects were similar to T-siRNA-DOPC. The anti-tumor effects were further enhanced in combination with docetaxel chemotherapy (p<0.01). Finally, we found a significant association between survivin 2B expression and progression free survival in 117 epithelial ovarian cancers obtained at primary debulking surgery.
These data identify survivin 2B as an important target in ovarian cancer, and provide a translational path forward for developing new therapies against this target.
PMCID: PMC3108184  PMID: 21512144
survivin; siRNA; nanoliposomes; ovarian cancer; taxanes
7.  BRCA1-IRIS inactivation overcomes paclitaxel resistance in triple negative breast cancers 
Intrinsic or acquired chemoresistance is a major problem in oncology. Although highly responsive to chemotherapies such as paclitaxel, most triple negative breast cancer (TNBC) patients develop chemoresistance. Here we investigate the role of BRCA1-IRIS as a novel treatment target for TNBCs and their paclitaxel-resistant recurrences.
We analyzed the response of BRCA1-IRIS overexpressing normal mammary cells or established TNBC cells silenced from BRCA1-IRIS to paclitaxel in vitro and in vivo. We analyzed BRCA1-IRIS downstream signaling pathways in relation to paclitaxel treatment. We also analyzed a large cohort of breast tumor samples for BRCA1-IRIS, Forkhead box class O3a (FOXO3a) and survivin expression. Finally, we analyzed the effect of BRCA1-IRIS silencing or inactivation on TNBCs formation, maintenance and response to paclitaxel in an orthotopic model.
We show that low concentrations of paclitaxel triggers BRCA1-IRIS expression in vitro and in vivo, and that BRCA1-IRIS activates two autocrine signaling loops (epidermal growth factor (EGF)/EGF receptor 1 (EGFR)-EGF receptor 2 (ErbB2) and neurogulin 1 (NRG1)/ErbB2-EGF receptor 3 (ErbB3), which enhances protein kinase B (AKT) and thus survivin expression/activation through promoting FOXO3a degradation. This signaling pathway is intact in TNBCs endogenously overexpressing BRCA1-IRIS. These events trigger the intrinsic and acquired paclitaxel resistance phenotype known for BRCA1-IRIS-overexpressing TNBCs. Inactivating BRCA1-IRIS signaling using a novel inhibitory mimetic peptide inactivates these autocrine loops, AKT and survivin activity/expression, in part by restoring FOXO3a expression, and sensitizes TNBC cells to low paclitaxel concentrations in vitro and in vivo. Finally, we show BRCA1-IRIS and survivin overexpression is correlated with lack of FOXO3a expression in a large cohort of primary tumor samples, and that BRCA1-IRIS overexpression-induced signature is associated with decreased disease free survival in heavily treated estrogen receptor alpha-negative patients.
In addition to driving TNBC tumor formation, BRCA1-IRIS overexpression drives their intrinsic and acquired paclitaxel resistance, partly by activating autocrine signaling loops EGF/EGFR-ErbB2 and NRG1/ErbB2-ErbB3. These loops activate AKT, causing FOXO3a degradation and survivin overexpression. Taken together, this underscores the need for BRCA1-IRIS-specific therapy and strongly suggests that BRCA1-IRIS and/or signaling loops activated by it could be rational therapeutic targets for advanced TNBCs.
PMCID: PMC4322455  PMID: 25583261
8.  Survivin-responsive conditionally replicating adenovirus kills rhabdomyosarcoma stem cells more efficiently than their progeny 
Effective methods for eradicating cancer stem cells (CSCs), which are highly tumorigenic and resistant to conventional therapies, are urgently needed. Our previous studies demonstrated that survivin-responsive conditionally replicating adenoviruses regulated with multiple factors (Surv.m-CRAs), which selectively replicate in and kill a broad range of cancer-cell types, are promising anticancer agents. Here we examined the therapeutic potentials of a Surv.m-CRA against rhabdomyosarcoma stem cells (RSCs), in order to assess its clinical effectiveness and usefulness.
Our previous study demonstrated that fibroblast growth factor receptor 3 (FGFR3) is a marker of RSCs. We examined survivin mRNA levels, survivin promoter activities, relative cytotoxicities of Surv.m-CRA in RSC-enriched (serum-minus) vs. RSC-exiguous (serum-plus) and FGFR3-positive vs. FGFR3-negative sorted rhabdomyosarcoma cells, and the in vivo therapeutic effects of Surv.m-CRAs on subcutaneous tumors in mice.
Both survivin mRNA levels and survivin promoter activities were significantly elevated under RSC-enriched relative to RSC-exiguous culture conditions, and the elevation was more prominent in FGFR3-positive vs. FGFR3-negative sorted cells than in RSC-enriched vs. RSC-exiguous conditions. Although Surv.m-CRA efficiently replicated and potently induced cell death in all populations of rhabdomyosarcoma cells, the cytotoxic effects were more pronounced in RSC-enriched or RSC-purified cells than in RSC-exiguous or progeny-purified cells. Injections of Surv.m-CRAs into tumor nodules generated by transplanting RSC-enriched cells induced significant death of rhabdomyosarcoma cells and regression of tumor nodules.
The unique therapeutic features of Surv.m-CRA, i.e., not only its therapeutic effectiveness against all cell populations but also its increased effectiveness against CSCs, suggest that Surv.m-CRA is promising anticancer agent.
PMCID: PMC3925355  PMID: 24467821
Cancer stem cells; Conditionally replicating adenovirus; Fibroblast growth factor receptor 3; Gene therapy; Oncolytic adenovirus; Promoter; Rhabdomyosarcoma; Survivin; Tumor-initiating cell; Virotherapy
9.  Therapeutic targeting of erbB3 with MM-121/SAR256212 enhances antitumor activity of paclitaxel against erbB2-overexpressing breast cancer 
Breast Cancer Research : BCR  2013;15(5):R101.
Elevated expression of erbB3 rendered erbB2-overexpressing breast cancer cells resistant to paclitaxel via PI-3 K/Akt-dependent upregulation of Survivin. It is unclear whether an erbB3-targeted therapy may abrogate erbB2-mediated paclitaxel resistance in breast cancer. Here, we study the antitumor activity of an anti-erbB3 antibody MM-121/SAR256212 in combination with paclitaxel against erbB2-overexpressing breast cancer.
Cell growth assays were used to determine cell viability. Cells undergoing apoptosis were quantified by a specific apoptotic ELISA. Western blot analyses were performed to assess the protein expression and activation. Lentiviral vector containing shRNA was used to specifically knockdown Survivin. Tumor xenografts were established by inoculation of BT474-HR20 cells into nude mice. The tumor-bearing mice were treated with paclitaxel and/or MM-121/SAR256212 to determine whether the antibody (Ab) enhances paclitaxel’s antitumor activity. Immunohistochemistry was carried out to study the combinatorial effects on tumor cell proliferation and induction of apoptosis in vivo.
MM-121 significantly facilitated paclitaxel-mediated anti-proliferative/anti-survival effects on SKBR3 cells transfected with a control vector or erbB3 cDNA. It specifically downregulated Survivin associated with inactivation of erbB2, erbB3, and Akt. MM-121 enhances paclitaxel-induced poly(ADP-ribose) polymerase (PARP) cleavage, activation of caspase-8 and -3, and apoptosis in both paclitaxel-sensitive and -resistant cells. Specific knockdown of Survivin in the trastuzumab-resistant BT474-HR20 cells dramatically enhanced paclitaxel-induced apoptosis, suggesting that increased Survivin caused a cross-resistance to paclitaxel. Furthermore, the studies using a tumor xenograft model-established from BT474-HR20 cells revealed that either MM-121 (10 mg/kg) or low-dose (7.5 mg/kg) paclitaxel had no effect on tumor growth, their combinations significantly inhibited tumor growth in vivo. Immunohistochemical analysis showed that the combinations of MM-121 and paclitaxel significantly reduced the cells with positive staining for Ki-67 and Survivin, and increased the cells with cleaved caspase-3.
The combinations of MM-121 and paclitaxel not only inhibit tumor cell proliferation, but also promote erbB2-overexpressing breast cancer cells to undergo apoptosis via downregulation of Survivin in vitro and in vivo, suggesting that inactivation of erbB3 with MM-121 enhances paclitaxel-mediated antitumor activity against erbB2-overexpressing breast cancers. Our data supports further exploration of the combinatorial regimens consisting of MM-121 and paclitaxel in breast cancer patients with erbB2-overexpressing tumors, particularly those resistant to paclitaxel.
PMCID: PMC3978722  PMID: 24168763
10.  Cross-talk between Epidermal Growth Factor Receptor and Hypoxia-inducible Factor-1α Signal Pathways Increases Resistance to Apoptosis by Up-regulating Survivin Gene Expression* 
The Journal of biological chemistry  2006;281(36):25903-25914.
Although increasing evidence supports a link between epidermal growth factor receptor (EGFR) signaling and resistance to apoptosis, the mechanism by which the EGFR signaling pathway inhibits apoptosis is not well understood. In this study, we found that epidermal growth factor (EGF) stimulation increased the level of expression of the inhibitor of apoptosis protein survivin in breast cancer cells but not in normal mammary epithelial cells. We further demonstrated that activation of survivin gene expression is mediated by oxygen-independent hypoxia-inducible factor (HIF)-1α up-regulation in EGF-treated cancer cells. EGFR signaling activated the phosphoinositide 3-kinase/AKT pathway, subsequently increasing the level of HIF-1α under normoxic conditions. HIF-1α then activated survivin gene transcription through direct binding to the survivin promoter. Furthermore, we found that overexpression of HIF-1α small interfering RNA blocks EGF-induced survivin gene up-regulation and increases apoptosis induced by the chemotherapy drug docetaxel. However, transfection of a plasmid expressing HIF-1α gene activates survivin gene expression and reduces the apoptotic response. Our results demonstrate a novel pathway for EGFR signaling-mediated apoptosis resistance in human cancer cells. Although the role of HIF-1α in regulating cell survival under hypoxic conditions has been studied extensively, our results show that normoxic breast cancer cells utilize cross-talk between EGFR signals and HIF-1α to up-regulate the anti-apoptotic survivin gene, providing a strong rationale for the targeting of HIF-1α as a therapeutic approach for both hypoxic and normoxic tumor cells. Understanding key molecular events in EGFR signaling-induced apoptosis resistance should provide new information for the development of novel therapeutic agents targeting EGFR, HIF-1α, and/or survivin.
PMCID: PMC3132567  PMID: 16847054
11.  A cell-permeable dominant-negative survivin protein induces apoptosis and sensitizes prostate cancer cells to TNF-α therapy 
Survivin is a member of the inhibitor-of-apoptosis (IAP) family which is widely expressed by many different cancers. Overexpression of survivin is associated with drug resistance in cancer cells, and reduced patient survival after chemotherapy and radiotherapy. Agents that antagonize the function of survivin hold promise for treating many forms of cancer. The purpose of this study was to investigate whether a cell-permeable dominant-negative survivin protein would demonstrate bioactivity against prostate and cervical cancer cells grown in three dimensional culture.
A dominant-negative survivin (C84A) protein fused to the cell penetrating peptide poly-arginine (R9) was expressed in E. coli and purified by affinity chromatography. Western blot analysis revealed that dNSurR9-C84A penetrated into 3D-cultured HeLa and DU145 cancer cells, and a cell viability assay revealed it induced cancer cell death. It increased the activities of caspase-9 and caspase-3, and rendered DU145 cells sensitive to TNF-α via by a mechanism involving activation of caspase-8.
The results demonstrate that antagonism of survivin function triggers the apoptosis of prostate and cervical cancer cells grown in 3D culture. It renders cancer cells sensitive to the proapoptotic affects of TNF-α, suggesting that survivin blocks the extrinsic pathway of apoptosis. Combination of the biologically active dNSurR9-C84A protein or other survivin antagonists with TNF-α therapy warrants consideration as an approach to cancer therapy.
PMCID: PMC2958862  PMID: 20920299
12.  Survivin enhances motility of melanoma cells by supporting Akt activation and α5 integrin upregulation 
Cancer research  2010;70(20):7927-7937.
Survivin expression in melanoma is inversely correlated with patient survival. Transgenic mice harboring melanocyte-specific overexpression of survivin exhibit increased susceptibility to UV-induced melanoma and metastatic progression. To understand the mechanistic basis for metastatic progression, we investigated the effects of Survivin on the motility of human melanocytes and melanoma cells. We found that Survivin overexpression enhanced migration on fibronectin and invasion through Matrigel, whereas Survivin knockdown under sub-apoptotic conditions blocked migration and invasion. In melanocytes, Survivin overexpression activated the Akt and MAPK pathways. Akt phosphorylation was required for Survivin-enhanced migration and invasion, whereas Erk phosphorylation was required only for enhanced invasion. In both melanocytes and melanoma cells, Survivin overexpression was associated with upregulation of α5 integrin (fibronectin receptor component), the antibody-mediated blockade or siRNA targeting of which blocked Survivin-enhanced migration. Knockdown of α5 integrin did not affect Akt activation, but inhibition of Akt phosphorylation prevented α5 integrin upregulation elicited by Survivin overexpression. Together, our results showed that Survivin enhanced the migration and invasion of melanocytic cells and suggested that Survivin may promote melanoma metastasis by supporting Akt-dependent upregulation of α5 integrin.
PMCID: PMC2955769  PMID: 20807805
Survivin; migration; invasion; melanoma; α5 integrin
13.  Inhibition of Epithelial Mesenchymal Transition (EMT) With Immunochemogene Treatment in Metastatic Colorectal Cancer 
Epithelial to mesenchymal transition (EMT) causes resistance to epidermal growth factor receptor (EGFR) inhibitors. We used immunochemogene treatment composed of a stealth nanoparticle formulation, consisting of clamp PNA against mRNA of FOXC2, anti-CD44 chimeric MAb, and vinorelbine, in an attempt to eradicate metastatic colorectal cancer (mCRC) cells and inhibit metastasis by blocking EMT.
Tumor cells from patients with stage IV chemoresistant CRC characterized by upregulation of FOXC2, CD44, and bcl-2 were obtained surgically. We synthesized antisense clamp peptide nucleic acid (PNA) oligomers (DNA analogs), in which the 6 mer homopyrimidine triplex [(PNA)2/RNA)] hybridized to the 5-end (Leader), and the 10 mer purine/pyrimidine duplex (PNA/RNA) hybridized to the 3-end (Trailer) of the AUG start codon region on the mRNA of FOXC2. The uncharged and hydrophilic antisense clamp PNA anti-FOXC2 was incorporated in the polar phase, and the vinorelbine molecules were entrapped in the acyl-chains of the lipid phase. This was surrounded by the stealth/biocompatibility polymer layer and biological recognition layer with linked chimeric MAbs against CD44 of the nanoparticle formulation. This was used to treat xenograft animal models developed from CRC cells obtained from the stage IV patients. Tumor cells were analyzed with microarray, single-nucleotide polymorphism (SNP) assay, polymerase chain reaction (PCR), western blot (WB), Southern blot (SB), immunoblotting (LC-MS/MS), immunofluorescence staining, immunohistochemistry (IHC), fluorescent activated cell sorter (FACS), confocal microscopy, transmission electron microscopy (TEM), bromodeoxyuridine (BrdU), MTT, and flow cytometry.
Post-treatment, we observed downregulation of CD44 and Fra-2, and induction of antibody-dependent cellular cytotoxicity (ADCC). The clamp PNA inhibited translation of FOXC2, resulting in activation of Jak2/Stat5a genes, which led to suppression of EMT of cancer cells. This blocked CRC metastatic invasion by reversing the mesenchymal phenotype; reconstituted homotypic adhesion; and promoted differentiation in CRC cells. Undifferentiated epithelial cells undergoing EMT exhibited overexpression of FOXC2, and this expression was lost when these cells returned to their initial differentiated epithelial state, blocking invasion and metastasis. Inhibition of EMT downregulated EGFR and inactivated NF-kB, inhibiting its downstream signaling pathway. Epithelial cell junction proteins claudin 4, claudin 7, and E-cadherin were overexpressed, upregulating beta-catenin; while mesenchymal markers vimentin and fibronectin were downregulated. Downregulation of Twist, Snail, and transcription 3 and 5 blocked the migratory potential of tumor cells, inhibiting metastasis. Calcium-independent cell-cell adhesion molecules EpCAM and TROP2 were upregulated. Vinorelbine blocked tumor cells at G2/M cell cycle, and phosphorylated bcl-2. This circumvented resistance to anoikis, inducing apoptosis in tumor cells due to lack of adhesion, inhibiting invasion and metastasis. In addition to the induction of caspase-dependent apoptosis or programmed cell death (PCD) type I in tumor cells, bcl-2 downregulation caused release of beclin-1 and upregulation of bcl-2–interacting mediator of cell death (BIM), inducing type II PCD or autophagy. TEM exhibited bystander killing effect of tumor cells by adjacent cells, and activated phagocytic cells such as macrophages. DNA synthesis and metabolic activity of tumor cells were inhibited according to BrdU and MTT tests, respectively.
This immunochemogene treatment induced epithelial differentiation by reversing the mesenchymal phenotype, promoted homotypic adhesion, inhibited the multigene signature indicative of EMT, blocking metastatic cell motility/invasiveness, and eradicated mCRC cells resistant to EGFR inhibitors by induction of PCD type-I and type-II, apoptosis and autophagy, leading to a bystander killing effect.
PMCID: PMC3056306
14.  Repression of BIRC5/Survivin by FOXO3/FKHRL1 Sensitizes Human Neuroblastoma Cells to DNA Damage-induced Apoptosis 
Molecular Biology of the Cell  2009;20(7):2041-2048.
The phosphatidylinositol 3-kinase (PI3K)–protein kinase B (PKB) pathway regulates survival and chemotherapy resistance of neuronal cells, and its deregulation in neuroblastoma (NB) tumors predicts an adverse clinical outcome. Here, we show that inhibition of PI3K-PKB signaling in human NB cells induces nuclear translocation of FOXO3/FKHRL1, represses the prosurvival protein BIRC5/Survivin, and sensitizes to DNA-damaging agents. To specifically address whether FKHRL1 contributes to Survivin regulation, we introduced a 4-hydroxy-tamoxifen-regulated FKHRL1(A3)ERtm allele into NB cells. Conditional FKHRL1 activation repressed Survivin transcription and protein expression. Transgenic Survivin exerted a significant antiapoptotic effect and prevented the accumulation of Bim and Bax at mitochondria, the loss of mitochondrial membrane potential as well as the release of cytochrome c during FKHRL1-induced apoptosis. In concordance, Survivin knockdown by retroviral short hairpin RNA technology accelerated FKHRL1-induced apoptosis. Low-dose activation of FKHRL1 sensitized to the DNA-damaging agents doxorubicin and etoposide, whereas the overexpression of Survivin diminished FKHRL1 sensitization to these drugs. These results suggest that repression of Survivin by FKHRL1 facilitates FKHRL1-induced apoptosis and sensitizes to cell death induced by DNA-damaging agents, which supports the central role of PI3K-PKB-FKHRL1 signaling in drug resistance of human NB.
PMCID: PMC2663932  PMID: 19211844
15.  Enhancing effectiveness of the MDR-sensitive compound T138067 using advanced treatment with negative modulators of the drug-resistant protein survivin 
Growing evidence indicates that the antiapoptotic protein survivin is a major factor of drug and radiation resistance in cancer cells. However, application of this finding to therapeutic drug combination is largely unexplored. In this study, breast cancer cells were used for treatment with anticancer compounds alone or in combination. We report that T138067, a better drug against multiple drug resistance (MDR) tumor cells than taxol (Shan et al., PNAS 96:5686–91,1999), induces survivin expression and consequently decreases its effectiveness on the induction of cancer cell death. Treatment of breast cancer cells with T138067 induced survivin expression in these cells while showing no effect on Bcl-2, indicating its specificity. Upregulation of survivin by T138067 was concomitant with an increased drug resistance and associated with an increased phosphorylation of Akt and Erk1/2 MAPK, and a decreased phosphorylation of p38 MAPK without affecting the phosphorylation of ErbB2. Therefore, it is possible that inhibition of T138067-induced survivin expression by alternative approaches may sensitize cells to T138067-induced cell death. We found that treatment of breast cancer cells with SN38, the active metabolite of irinotecan, inhibits survivin expression. Intriguingly, inhibition of survivin expression by SN38 was more effective at a low concentration than at the high concentration, which makes SN38 a good survivin modulator. Furthermore, in contrast with the decreased phosphorylation of p38 MAPK after T138067 treatment, inhibition of survivin expression by SN38 was associated with an increased phosphorylation of the p38 MAPK, suggesting opposing signals converging to survivin. Consistent with these observations, T138067 in combination with SN38 strongly induced cell death in comparison with each drug alone. Similarly, sequential combination of resveratrol, a component of red grapes that inhibits survivin expression, with T138067 also provoked massive breast cancer cell death compared with T138067 alone. Together, these results highlight a new concept that unique signaling cross talk converged to survivin may be considered for rational drug combination in the clinic.
PMCID: PMC2780039  PMID: 19956451
Survivin; T138067; SN38; irinotecan; resveratrol; breast cancer cells
16.  Survivin is a Key Factor in the Differential Susceptibility of Gastric Endothelial and Epithelial Cells to Alcohol-Induced Injury 
We previously demonstrated that the anti-apoptosis protein, survivin, plays a protective role against alcohol-induced gastric injury. Since the endothelium is a primary target of alcohol-induced gastric damage, we investigated whether survivin expression is a key factor in the greater susceptibility of gastric endothelial vs. epithelial cells to alcohol-induced injury. Here, we demonstrate that rat gastric epithelial cells (RGM1 cells, an epithelial cell line derived from normal rat gastric mucosa) expressed 7.5-fold greater survivin protein levels vs. rat gastric endothelial cells. Survivin expression correlated with resistance of gastric epithelial vs. endothelial cells to both alcohol-induced cell damage and alcohol-induced apoptosis. Suppression of survivin protein expression levels using siRNA rendered the gastric epithelial cells as susceptible to both alcohol-induced cell damage and apoptosis as the gastric endothelial cells. Conversely, forced overexpression of survivin by transient transfection rendered gastric endothelial cells as resistant to both alcohol-induced cell damage and apoptosis as mock-transfected gastric epithelial cells. Moreover, overexpression of a threonine-34 to glutamate phosphorylation mimic mutant survivin construct rendered gastric endothelial cells significantly more resistant to alcohol-induced damage and apoptosis vs. mock-transfected gastric epithelial cells. These findings indicate that disparate survivin expression levels can explain the discrepancy between gastric epithelial and endothelial cell susceptibility to alcohol-induced injury; and, that a negative charge at amino acid residue 34 on survivin, such as that which naturally occurs by phosphorylation of threonine-34, enhances its property in conferring gastric mucosal protection.
PMCID: PMC2953945  PMID: 20610854
Apoptosis; Cell death; In vitro Mutagenesis; Overexpression; Phosphorylation mimic; si RNA Knockdown; Transfection
17.  Differential regulation of survivin expression and apoptosis by vitamin D3 compounds in two isogenic MCF-7 breast cancer cell sublines 
Oncogene  2005;24(8):1385.
Although both the antiapoptotic function of survivin and vitamin D3 (VD3)-mediated cell growth inhibition and apoptosis have been extensively studied, it is not known whether survivin plays a role in VD3 compound-mediated cell growth inhibition and apoptosis induction. Using an isogenic model of MCF-7 breast adenocarcinoma cells (MCF-7E and MCF-7L sublines that are sensitive and resistant to VD3 compounds), we found that VD3 compounds effectively downregulated survivin in VD3-sensitive MCF-7E cells, which was associated with VD3-induced apoptosis. In contrast, VD3 compounds failed to downregulate survivin in VD3-resistant MCF-7L cells, which showed resistant to VD3-induced apoptosis. However, inhibition of survivin expression by small interfering RNA (siRNA) induced cell death per se and further sensitized VD3-induced apoptosis in MCF-7L cells, indicating that the inability of these cells to respond to VD3 is due to the failure to downregulate survivin. Forced expression of survivin not only blocked VD3-mediated G1 cell accumulation but also increased S and G2/M cell populations. VD3 treatment rapidly triggered the activation of p38 MAPK signaling in MCF-7E cells but not in MCF- 7L cells. Moreover, inhibition of p38 activation diminished VD3-mediated survivin inhibition and partially rescued VD3-induced cell death. We further showed that VD3 increased the expression of TGFβ1 and TGFβ receptor 2, and that blocking the function of TGFβ receptor 2 diminished VD3 compound-mediated survivin downregulation. Thus, we propose that the VD3 compound-induced growth inhibition and apoptosis induction are at least partially dependent on survivin downregulation via VD3-induced TGFβ signaling and the activation of p38 MAPK pathway. Targeting survivin through these pathways may lead to novel applications for cancer therapeutics.
PMCID: PMC2820410  PMID: 15608672
vitamin D3; survivin; apoptosis; p38 MAPK; MCF-7 breast cancer cell
18.  Induction of HLA-DP4-restricted anti-Survivin Th1 and Th2 responses using an artificial antigen-presenting cell 
In previous cancer vaccine clinical trials targeting Survivin, induction of specific CD8+ T cell responses did not consistently lead to clinical responses. Considering the critical role of CD4+ T cell help in generating anti-tumor immunity, integration of anti-Survivin CD4+ T cell responses may enhance the efficacy of anti-Survivin cancer immunotherapy. HLA-DP4 is emerging as an attractive MHC target allele of CD4+ T cell-mediated immunotherapy, since it is one of the most frequent HLA alleles in many ethnic groups. In this paper, we aimed to elucidate DP4-restricted CD4+ T cell responses against Survivin in cancer patients.
Experimental Design
We generated a human cell-based artificial antigen-presenting cell (aAPC) expressing HLA-DP4, CD80, and CD83 and induced DP4-restricted antigen-specific CD4+ T cells. The number, phenotype, effector function, and in vitro longevity of generated CD4+ T cells were determined.
We first determined previously unknown DP4-restricted CD4+ T cell epitopes derived from CMV pp65, to which sustained Th1-biased recall responses were induced in vitro using DP4-aAPC. In contrast, DP4-aAPC induced in vitro both Th1 and Th2 long-lived anti-Survivin CD4+ T cells from cancer patients. Both Survivin-specific Th1 and Th2 cells were able to recognize Survivin-expressing tumors in a DP4-restricted manner. Neither Survivin-specific IL-10 secreting Tr1 cells nor Th17 cells were induced by DP4-aAPC.
DP4-restricted anti-Survivin Th1 and Th2 immunity with sufficient functional avidity can be induced from cancer patients. The development of strategies to concurrently induce both CD4+ and CD8+ T cell responses against Survivin is warranted for optimal anti-Survivin cancer immunotherapy.
PMCID: PMC3156899  PMID: 21705450
HLA-DP4; Survivin; CD4+ T cells; artificial APC; K562
19.  Localization, Dynamics, and Function of Survivin Revealed by Expression of Functional SurvivinDsRed Fusion Proteins in the Living Cell 
Molecular Biology of the Cell  2003;14(1):78-92.
Survivin, a member of the inhibitor of apoptosis protein family, has attracted growing attention due to its expression in various tumors and its potential application in tumor therapy. However, its subcellular localization and function have remained controversial: Recent studies revealed that survivin is localized at the mitotic spindle, binds caspases, and could thus protect cells from apoptosis. The cell cycle-dependent expression of survivin and its antiapoptotic function led to the hypothesis that survivin connects the cell cycle with apoptosis, thus providing a death switch for the termination of defective mitosis. In other studies, survivin was detected at kinetochores, cleavage furrow, and midbody, localizations being characteristic for chromosomal passenger proteins. These proteins are involved in cytokinesis as inferred from the observation that RNA interference and expression of mutant proteins led to cytokinesis defects without an increase in apoptosis. To remedy these discrepancies, we analyzed the localizations of a survivinDsRed fusion protein in HeLa cells by using confocal laser scanning microscopy and time-lapse video imaging. SurvivinDsRed was excluded from the interphase nucleus and was detected in centrosomes and at kinetochores. It dissociated from chromosomes at the anaphase/telophase transition and accumulated at the ends of polar microtubuli where it was immediately condensed to the midbody. Overexpression of both survivinDsRed and of a phosphorylation-defective mutant conferred resistance against apoptosis-inducing reagents, but only the overexpressed mutant protein caused an aberrant cytokinesis. These data characterize in detail the dynamics of survivin in vertebrate cells and confirm that survivin represents a chromosomal passenger protein.
PMCID: PMC140229  PMID: 12529428
20.  Ubiquitin-proteasomal degradation of antiapoptotic survivin facilitates induction of apoptosis in prostate cancer cells by pristimerin 
International Journal of Oncology  2014;45(4):1735-1741.
Pristimerin (PM), a quinonemethide triterpenoid, is a promising anticancer agent with potent antiproliferative and apoptosis-inducing activities against cancer cell lines. However, the anticancer activity and mechanisms of PM in prostate cancer cells have not been adequately investigated. Here we report that the degradation of survivin plays an important role in the antiproliferative and proapoptotic effects of PM in carcinoma of the prostate (CaP) cell lines. Treatment with PM inhibited proliferation and induced apoptosis in LNCaP and PC-3 cells as characterized by the loss of cell viability and an increase in Annexin V-binding and cleavage of PARP-1, respectively. The antiproliferative and apoptosis-inducing effects of PM were associated with the inhibition of cell cycle regulatory proteins, antiapoptotic survivin and members of the Bcl-2 family. Data showed that response to PM is regulated by survivin since overexpression of survivin rendered CaP cells resistant to PM. Furthermore, downregulation of survivin by PM was mediated through the ubiquitin-proteasomal degradation. Together, these data demonstrate that pristimerin inhibits proliferation and induces apoptosis in CaP cells by abolishing survivin through the ubiquitin-proteasome pathway.
PMCID: PMC4151800  PMID: 25175770
pristimerin; prostate cancer; apoptosis; survivin; ubiquitin; proteasome
21.  Treatment of lymphoma with adoptively transferred T cells 
Expert opinion on biological therapy  2009;9(11):1407-1425.
Chemotherapy-resistant lymphomas can be cured with allogeneic hematopoietic cell transplantation, demonstrating the susceptibility of these tumors to T cell-mediated immune responses. However, high rates of transplant-related morbidity and mortality limit this approach. Efforts have therefore been made to develop alternative T cell-based therapies, and there is growing evidence that adoptive therapy with T cells targeted to lymphoma-associated antigens may be a safe and effective new method for treating this group of diseases.
We review publications on adoptive therapy with ex-vivo-expanded T cells targeting viral antigens, as well as genetically modified autologous T cells, as strategies for the treatment of lymphoma, with the goal of providing an overview of these approaches.
Epstein-Barr virus (EBV)-specific T cell therapy is an effective and safe method of treating EBV-associated lymphomas; however, most lymphoma subtypes do not express EBV antigens. For these diseases, adoptive immunotherapy with genetically modified T cells expressing chimeric T cell receptors targeting lymphoma-associated antigens such as CD19 and CD20 appears to be a promising alternative. Recent innovations including enhanced co-stimulation, exogenous cytokine administration, and use of memory T cells promise to overcome many of the limitations and pitfalls initially encountered with this approach.
PMCID: PMC2776697  PMID: 19723016
Adoptive T cell therapy; Chimeric antigen receptors; Cellular therapy; Chimeric T cell receptors; Epstein-Barr virus; Hematologic malignancies; Hodgkin lymphoma; Immunotherapy; Lymphoma; Non-Hodgkin lymphoma; Post-transplant lymphoproliferative disease; T bodies
22.  Synergistic Effect of Sulindac and Simvastatin on Apoptosis in Lung Cancer A549 Cells through AKT-Dependent Downregulation of Survivin 
Non-steroidal anti-inflammatory drugs (NSAIDs) and statins are potential chemopreventive or chemotherapeutic agents. The mechanism underlying the deregulation of survivin by NSAIDs and statins in human non-small cell lung cancer cells has not been elucidated. In this study, we investigated the synergistic interaction of sulindac and simvastatin in lung cancer A549 cells.
Materials and Methods
Cell viability was measured by an MTT assay, while the expression of apoptotic markers, AKT, and survivin in response to sulindac and simvastatin was examined by Western blotting. DNA fragmentation by apoptosis was analyzed by flow cytometry in A549 cells. Reactive oxygen species (ROS) generation was measured by flow cytometry using H2DCFDA and MitoSOX Red, and the effects of pretreatment with N-acetylcysteine were tested. The effects of AKT on survivin expression in sulindac- and simvastatin-treated cells were assessed. Survivin was knocked down or overexpressed to determine its role in apoptosis induced by sulindac and simvastatin.
Sulindac and simvastatin synergistically augmented apoptotic activity and intracellular ROS production in A549 cells. Inhibition of AKT by siRNA or LY294002 inhibited survivin, while AKT overexpression markedly increased survivin expression, even in the presence of sulindac and simvastatin. Moreover, survivin siRNA enhanced sulindac- and simvastatininduced apoptosis. In contrast, survivin upregulation protected against sulindac- and simvastatin-induced apoptosis.
Combined treatment with sulindac and simvastatin augmented their apoptotic potential in lung cancer cells through AKT signaling-dependent downregulation of survivin. These results indicate that sulindac and simvastatin may be clinically promising therapies for the prevention of lung cancer.
PMCID: PMC4296852  PMID: 25520153
Sulindac; Simvastatin; Apoptosis; Lung neoplasms; Oncogene protein AKT; Survivin
23.  Survivin counteracts the therapeutic effect of microtubule de-stabilizers by stabilizing tubulin polymers 
Molecular Cancer  2009;8:43.
Survivin is a dual function protein. It inhibits the apoptosis of cells by inhibiting caspases, and also promotes cell growth by stabilizing microtubules during mitosis. Over-expression of survivin has been demonstrated to induce drug-resistance to various chemo-therapeutic agents such as cisplatin (DNA damaging agent) and paclitaxel (microtubule stabilizer) in cancers. However, survivin-induced resistance to microtubule de-stabilizers such as Vinca alkaloids and Combretastatin A-4 (CA-4)-related compounds were seldom demonstrated in the past. Furthermore, the question remains as to whether survivin plays a dominant role in processing cytokinesis or inhibiting caspases activity in cells treated with anti-mitotic compounds. The purpose of this study is to evaluate the effect of survivin on the resistance and susceptibility of human cancer cells to microtubule de-stabilizer-induced cell death.
BPR0L075 is a CA-4 analog that induces microtubule de-polymerization and subsequent caspase-dependent apoptosis. To study the relationship between the expression of survivin and the resistance to microtubule de-stabilizers, a KB-derived BPR0L075-resistant cancer cell line, KB-L30, was generated for this study. Here, we found that survivin was over-expressed in the KB-L30 cells. Down-regulation of survivin by siRNA induced hyper-sensitivity to BPR0L075 in KB cells and partially re-stored sensitivity to BPR0L075 in KB-L30 cells. Western blot analysis revealed that down-regulation of survivin induced microtubule de-stabilization in both KB and KB-L30 cells. However, the same treatment did not enhance the down-stream caspase-3/-7 activities in BPR0L075-treated KB cells. Translocation of a caspase-independent apoptosis-related molecule, apoptosis-inducing factor (AIF), from cytoplasm to the nucleus was observed in survivin-targeted KB cells under BPR0L075 treatment.
In this study, survivin plays an important role in the stability of microtubules, but not with caspases inhibition. Over-expression of survivin counteracts the therapeutic effect of microtubule de-stabilizer BPR0L075 probably by stabilizing tubulin polymers, instead of the inhibition of caspase activity in cancer cells. Besides microtubule-related caspase-dependent cell death, caspase-independent mitotic cell death could be initiated in survivin/BPR0L075 combination treatments. We suggest that combining microtubule de-stabilizers with a survivin inhibitor may attribute to a better clinical outcome than the use of anti-mitotic monotherapy in clinical situations.
PMCID: PMC2714493  PMID: 19575780
24.  The Anti-Apoptosis Protein, Survivin, Mediates Gastric Epithelial Cell Cytoprotection Against Ethanol-Induced Injury via Activation of the p34cdc2 Cyclin Dependent Kinase 
Journal of cellular physiology  2008;215(3):750-764.
The anti-apoptosis protein, survivin, promotes cell survival and mitosis. Recent studies have demonstrated that survivin is expressed in normal gastric mucosa. Using an in vitro model, we examined whether survivin plays a role in the cytoprotection produced in gastric mucosa by mild irritant ethanol (ETOH) against subsequent exposure to concentrated ETOH. Pretreatment of rat gastric epithelial cells with 1% ETOH reduced cell death, in response to subsequent incubation with 5% ETOH, by 94% (P<0.005). This pretreatment also resulted in increased total and phosphorylated survivin protein levels by 180% (P<0.0001) and 540% (P<0.0002), respectively, which required the p34cdc2 cell cycle-dependent kinase. The cytoprotective effect was abrogated upon siRNA knockdown of survivin protein levels. Further, overexpression of exogenous survivin resulted in significant cytoprotection by 62% (P<0.02) in the absence of any pretreatment. We further examined the in vivo relevance of these findings. In fasted rats, administration of 20% ETOH, which we found to be 93% (P<0.0001) cytoprotective against 50% ETOH challenge, resulted in increased total and phosphorylated survivin protein levels by 234% (P<0.001) and 214% (P<0.02), respectively. Administration of 20% ETOH resulted in increased gastric p34cdc2 activity by 146% (P<0.01). Inhibition of p34cdc2 by the potent inhibitor, roscovitine, abolished the increased survivin levels in response to pre-administration of 20% ETOH and reduced the cytoprotection against 50% ETOH challenge by 59% (P<0.01). These results indicate that survivin is a key mediator of cytoprotection against ETOH-induced gastric injury, acting at the epithelial cell level, by a mechanism that is dependent, in part, on p34cdc2.
PMCID: PMC2288653  PMID: 18181150
• Adaptation; • Apoptosis; • Cell death; • Inhibitor; • Phosphorylation
25.  Selenium inhibition of survivin expression by preventing Sp1 binding to its promoter 
Survivin, an antiapoptotic protein highly expressed in cancer, regulates multiple cellular network associated with cancer cell viability and drug resistance. Inhibition of survivin expression has been pursued as a valid cancer therapeutic target. In this study, we showed that selenium, an effective chemopreventive agent for many types of cancers, down-regulated survivin expression. Selenium inhibited survivin expression in both mRNA and protein levels in a dose- and time-dependent manner. Using a series of survivin promoter–luciferase constructs, a 37-bp DNA element in the survivin core promoter region that mediates the ability of selenium to inhibit survivin transcription was identified. Gel mobility shift assays and chromatin immunoprecipitation analyses revealed that selenium prevents the binding of Sp1 or Sp1-like proteins to the 37-bp cis-acting DNA element in the survivin promoter. Furthermore, inhibition of survivin expression by small interfering RNA enhanced selenium’s inhibitory effects on cell growth, whereas overexpression of survivin in LNCaP human prostate cancer cells desensitized cancer cells to selenium effect, suggesting that the expression of survivin plays an important role in determining the response of cancer cells to selenium. Taken together, these results suggest that selenium down-regulated survivin expression by preventing the binding of Sp1 or Sp1-like proteins to the promoter of survivin, which contributes at least in part to the inhibitory effect of selenium on survivin gene transcription. In addition, down-regulation of survivin expression may account for one of the molecular mechanisms of the anticancer effects of selenium.
PMCID: PMC2821810  PMID: 17876054

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