Survivin Protein Levels Research Articles

Oxymatrine inhibits proliferation of human bladder cancer T24 cells by inducing apoptosis and cell cycle arrest

Oxymatrine has been shown to exert an antitumor effect on several types of cancer cells. However, the role of oxymatrine in bladder cancer has not yet been evaluated. The present study was designed to investigate the potential anti-proliferative effect of oxymatrine on bladder cancer T24 cells and the possible mechanisms involved. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine cell growth, and the cell morphology was examined using hematoxylin and eosin staining, wrights' staining and electron microscopy. The caspase-3 and survivin mRNA and protein levels were assessed using reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. The expression of tumor protein p53 (p53), Bcl-2-associated X protein (Bax) and B-cell lymphoma 2 (Bcl-2) were analyzed using immunohistochemistry. Oxymatrine inhibited the proliferation of the T24 cells in a dose- and time-dependent manner. Oxymatrine also induced apoptosis and cell cycle arrest in the cells, in association with the upregulation of caspase-3 and Bax, and the downregulation of survivin, Bcl-2 and p53 expression. Overall, oxymatrine inhibits the proliferation of human bladder cancer cells by inducing apoptosis and cell cycle arrest via mechanisms that involve p53-Bax signaling and the downregulation of survivin expression.

Methyl Protodioscin Induces Apoptosis in Human Osteosarcoma Cells by Caspase-Dependent and MAPK Signaling Pathways.

Methyl protodioscin (MPD), a furostanol saponin derived from the rhizomes of Dioscorea collettii var. hypoglauca (Dioscoreaceae), has been shown to exhibit broad bioactivities such as anti-inflammation and antitumor activities. Here, we explored the molecular mechanisms by which MPD induced apoptosis in MG-63 cells. The data showed that MPD significantly suppressed cell growth (cell viabilities: 22.5 ± 1.9% for 8 μM MPD versus 100 ± 1.4% for control, P < 0.01) and enhanced cell apoptosis. The exposure to MPD resulted in a significant induction of reactive oxygen species, loss of mitochondrial membrane potential, and activation of caspase-9 and caspase-3 (P < 0.01, all cases). Furthermore, treatment with MPD increased the levels of phosphorylated JNK and p38 MAPK and markedly decreased the levels of phosphorylated ERK in MG-63 cells. Co-administration of the JNK-specific antagonist, the p38-specific antagonist, or the caspase antagonist (P < 0.05, all cases) has reversed the apoptotic effects in MPD treatment. We also found that exposure to MPD resulted in a significant reduction in the protein level of anti-apoptotic proteins Bcl-2, survivin, and XIAP (P < 0.05, all cases). In conclusion, our results indicate that MPD induces apoptosis of human osteosarcoma MG-63 cells, at least in part, by caspase-dependent and MAPK signaling pathways.

Survivin and NAIP in Human Benign Prostatic Hyperplasia: Protective Role of the Association of Serenoa repens, Lycopene and Selenium from the Randomized Clinical Study.

Benign prostatic hyperplasia (BPH) treatment includes the apoptosis machinery modulation through the direct inhibition of caspase cascade. We previously demonstrated that Serenoa repens (Ser) with lycopene (Ly) and selenium (Se) reawakened apoptosis by reducing survivin and neuronal apoptosis inhibitory protein (NAIP) levels in rats. The aim of this study was to evaluate the effectiveness of Ser-Se-Ly association on survivin and NAIP expression in BPH patients. Ninety patients with lower urinary tract symptoms (LUTS) due to clinical BPH were included in this randomized, double-blind, placebo-controlled trial. Participants were randomly assigned to receive placebo (Group BPH + placebo, n = 45) or Ser-Se-Ly association (Group BPH + Ser-Se-Ly; n = 45) for 3 months. At time 0, all patients underwent prostatic biopsies. After 3 months of treatment, they underwent prostatic re-biopsy and specimens were collected for molecular, morphological, and immunohistochemical analysis. After 3 months, survivin and NAIP were significantly decreased, while caspase-3 was significantly increased in BPH patients treated with Ser-Se-Ly when compared with the other group. In BPH patients treated with Ser-Se-Ly for 3 months, the glandular epithelium was formed by a single layer of cuboidal cells. PSA showed high immunoexpression in all BPH patients and a focal positivity in Ser-Se-Ly treated patients after 3 months. Evident prostate specific membrane antigen (PSMA) immunoexpression was shown in all BPH patients, while no positivity was present after Ser-Se-Ly administration. Ser-Se-Ly proved to be effective in promoting apoptosis in BPH patients.

TGF-β1 signaling pathway serves a role in HepG2 cell regulation by affecting the protein expression of PCNA, gankyrin, p115, XIAP and survivin.

The transforming growth factor-β (TGF-β) signaling pathway serves a key role in the pathogenesis of liver cancer. To investigate the association between TGF-β1 and the following proteins: Proliferating cell nuclear antigen (PCNA), gankyrin, general vesicular transport factor p115 (p115), X-linked inhibitor of apoptosis protein (XIAP) and survivin, HepG2 liver cancer cells were transfected with small interfering RNA (siRNA) directed against TGF-β1, or were treated with exogenous TGF-β1. TGF-β1 protein expression levels were assessed at 72 and 96 h using western blotting, cell growth was evaluated using a Cell Counting kit-8 assay, and flow cytometry was used to examine cell cycle distribution and apoptosis. In addition, PCNA, gankyrin, p115, XIAP and survivin protein levels were evaluated using western blotting. TGF-β1 protein expression levels were decreased at 72 and 96 h following siRNA transfection, indicating that the siRNA against TGF-β1 was effective. In the TGF-β1-knockdown group, the HepG2 cells exhibited G1 or S-phase cell cycle arrest; therefore, the number of G2-phase cells was decreased, cell growth was inhibited and apoptotic peaks were observed. By contrast, no significant alteration in cell cycle distribution or apoptosis was observed in the cells treated with exogenous TGF-β1. In the exogenous TGF-β1 group, PCNA and XIAP protein expression levels were increased, whereas gankyrin, p115 and survivin protein expression was observed to be dependent on the duration of treatment. By contrast, PCNA, gankyrin, XIAP and survivin protein expression decreased following TGF-β1 knockdown; however, p115 protein expression increased. In conclusion, the TGF-β1 signaling pathway may affect cell growth, cell cycle distribution and apoptosis through the regulation of PCNA, gankyrin, p115, XIAP and survivin protein expression in liver cancer. The results of the present study may improve the current understanding of the role of the TGF-β signaling pathway during the pathogenesis of liver cancer.

Aurora kinase A regulates Survivin stability through targeting FBXL7 in gastric cancer drug resistance and prognosis

Aurora kinase A (AURKA) has been implicated in the regulation of cell cycle progression, mitosis and a key number of oncogenic signaling pathways in various malignancies. However, little is known about its role in gastric cancer prognosis and genotoxic resistance. Here we found that AURKA was highly overexpressed in gastric cancer and inversely correlated with disease prognosis. Overexpression of AURKA exacerbated gastric cancer drug resistance through upregulating the expression of the anti-apoptotic protein Survivin. Conversely, we demonstrated that AURKA depletion caused a decrease in Survivin protein levels by increasing its ubiquitylation and degradation. Mass spectrometric analysis revealed that upon AURKA depletion, Survivin bound to the FBXL7 E3 ubiquitin ligase, which induced ubiquitin-proteasome degradation of Survivin. In addition, we showed that AURKA regulated FBXL7 both at the levels of transcription and translation. Moreover, proteomic analysis of nuclear AURKA-interacting proteins identified Forkhead box protein P1 (FOXP1). We next showed that AURKA was required for FBXL7 transcription and that AURKA negatively regulated FOXP1-mediated FBXL7 expression. The physiological relevance of the regulation of Survivin by AURKA through the FOXP1–FBXL7 axis was further underscored by the significant positive correlations between AURKA and Survivin expression in gastric cancer patient samples. Moreover, the AURKA depletion or kinase inhibition-induced apoptotic cell death could be reversed by Survivin ectopic overexpression, further supporting that AURKA regulated Survivin to enhance drug resistance. In agreement, inhibition of AURKA synergistically enhanced the cytotoxic effect of DNA-damaging agents in cancer cells by suppressing Survivin expression. Taken together, our data suggest that AURKA restricts Survivin ubiquitylation and degradation in gastric cancer to promote drug resistance and hence the AURKA–Survivin axis can be targeted to promote the efficacy of DNA-damaging agents in gastric cancer.

Molecular-Based Recursive Partitioning Analysis Model for Glioblastoma in the Temozolomide Era

There is a need for a more refined, molecularly based classification model for glioblastoma (GBM) in the temozolomide era. To refine the existing clinically based recursive partitioning analysis (RPA) model by incorporating molecular variables. NRG Oncology RTOG 0525 specimens (n = 452) were analyzed for protein biomarkers representing key pathways in GBM by a quantitative molecular microscopy-based approach with semiquantitative immunohistochemical validation. Prognostic significance of each protein was examined by single-marker and multimarker Cox regression analyses. To reclassify the prognostic risk groups, significant protein biomarkers on single-marker analysis were incorporated into an RPA model consisting of the same clinical variables (age, Karnofsky Performance Status, extent of resection, and neurologic function) as the existing RTOG RPA. The new RPA model (NRG-GBM-RPA) was confirmed using traditional immunohistochemistry in an independent data set (n = 176). Overall survival (OS). In 452 specimens, MGMT (hazard ratio [HR], 1.81; 95% CI, 1.37-2.39; P < .001), survivin (HR, 1.36; 95% CI, 1.04-1.76; P = .02), c-Met (HR, 1.53; 95% CI, 1.06-2.23; P = .02), pmTOR (HR, 0.76; 95% CI, 0.60-0.97; P = .03), and Ki-67 (HR, 1.40; 95% CI, 1.10-1.78; P = .007) protein levels were found to be significant on single-marker multivariate analysis of OS. To refine the existing RPA, significant protein biomarkers together with clinical variables (age, Karnofsky Performance Status, extent of resection, and neurological function) were incorporated into a new model. Of 166 patients used for the new NRG-GBM-RPA model, 97 (58.4%) were male (mean [SD] age, 55.7 [12.0] years). Higher MGMT protein level was significantly associated with decreased MGMT promoter methylation and vice versa (1425.1 for methylated vs 1828.0 for unmethylated; P < .001). Furthermore, MGMT protein expression (HR, 1.84; 95% CI, 1.38-2.43; P < .001) had greater prognostic value for OS compared with MGMT promoter methylation (HR, 1.77; 95% CI, 1.28-2.44; P < .001). The refined NRG-GBM-RPA consisting of MGMT protein, c-Met protein, and age revealed greater separation of OS prognostic classes compared with the existing clinically based RPA model and MGMT promoter methylation in NRG Oncology RTOG 0525. The prognostic significance of the NRG-GBM-RPA was subsequently confirmed in an independent data set (n = 176). This new NRG-GBM-RPA model improves outcome stratification over both the current RTOG RPA model and MGMT promoter methylation, respectively, for patients with GBM treated with radiation and temozolomide and was biologically validated in an independent data set. The revised RPA has the potential to contribute to improving the accurate assessment of prognostic groups in patients with GBM treated with radiation and temozolomide and to influence clinical decision making. clinicaltrials.gov Identifier: NCT00304031.

Gambogic acid potentiates the chemosensitivity of colorectal cancer cells to 5-fluorouracil by inhibiting proliferation and inducing apoptosis.

Chemotherapy using 5-fluorouracil (5-FU) for colorectal cancer (CRC) has low specificity and response rates, leading to severe side effects. Gambogic acid (GA), a traditional Chinese medicine, has multi-targeted anticancer effects, including growth inhibition and apoptosis induction. However, it is unclear whether a combination of 5-FU and GA has synergistic anticancer effects in CRC cells. In this study, SW480 and HCT116 human CRC cells and human intestinal epithelial cells (IECs) were treated with different concentrations of 5-FU, GA or 5-FU+GA. A Cell Counting kit-8 assay was conducted to quantify cell proliferation. The combination index (CI) was calculated and the median-effect principle was applied to analyze the interaction between 5-FU and GA. Flow cytometry was used to determine the percentage of cells undergoing apoptosis. Reverse transcription-quantitative polymerase chain reaction and western blotting were applied to measure P53, survivin and thymidylate synthase (TS) mRNA and protein levels. It was found that 5-FU+GA more pronouncedly inhibited cell growth and induced apoptosis, compared with either monotherapy. CI values <1 indicated the synergistic effects of the drugs. 5-FU+GA further decreased P53, survivin and TS mRNA and protein levels in the two CRC cell lines compared with single drugs, whereas increased P53 protein levels were observed in HCT116 cells. Moreover, 5-FU+GA did not increase cytotoxicity to IECs. These results demonstrate that GA enhances the anticancer effects of 5-FU on CRC cells. Combined treatment with 5-FU and GA is effective and safe for CRC cells, and may become a promising chemotherapy treatment.

Mechanism of reversal of multidrug resistance by curcumin in human colorectal cancer cell line HCT-8/5-FU.

Since there are no studies on the reversal of multidrug resistance by curcumin in the human colorectal cancer cell line HCT-8/5-FU, our aim was to search for highly efficient reversal agents and investigate the underlying mechanisms of this reversal. The cytotoxic effects of curcumin and 5-FU on HCT-8 and HCT-8/5-FU cells and the reversal effects of 5-FU in combination with curcumin on HCT-8/5-FU cells were measured using cell counting kit-8. Apoptosis and the cell cycle were analyzed by flow cytometry. Protein and mRNA expression levels of BCL-2, survivin, P-gp, and HSP-27 were detected by western blotting and quantitative real-time reverse transcription polymerase chain reaction, respectively. Curcumin inhibited the growth of HCT-8 and HCT-8/5-FU cells. It significantly reduced the IC50 of 5-FU for HCT-8/5-FU cells (P < 0.01) and the expression of BCL-2, survivin, P-gp, and HSP-27 in the cells. Curcumin can effectively reverse multidrug resistance in human colorectal cancer drug-resistant HCT-8/5-FU cells. The mechanism through which this occurs may be associated with decreased expression of BCL-2, survivin, P-gp, and HSP-27. Curcumin may therefore have clinical implications as a new agent for colorectal cancer.

Targeting specificity protein 1 transcription factor and survivin using tolfenamic acid for inhibiting Ewing sarcoma cell growth.

Transcription factor Specificity protein1 (Sp1) and its downstream target survivin (inhibitor of apoptosisprotein), play major roles in the pathogenesis of various cancers. Ewing Sarcoma (ES) is a common soft tissue/bone tumor in adolescent and young adults. Overexpression of survivin is also linked to the aggressiveness and poor prognosis of ES. Small molecule Tolfenamic acid (TA) inhibits Sp1 and survivin in cancer cells. In this investigation, we demonstrate a strategy to target Sp1 and survivin using TA and positive control Mithramycin A (Mit) to inhibit ES cell growth. Knock down of Sp1 using small interfering RNA (siRNA) resulted in significant (p<0.05) inhibition of CHLA-9 and TC-32 cell growth as assessed by CellTiter-Glo assay kit. TA or Mit treatment caused dose/time-dependent inhibition of cell viability, and this inhibition was correlated with a decrease in Sp1 and survivin protein levels in ES cells. Quantitative PCR results showed that Mit treatment decreased the mRNA expression of both survivin and Sp1, whereas TA diminished only survivin but not Sp1. Proteasome inhibitor restored TA-induced inhibition of Sp1 protein expression suggesting that TA might cause proteasome-dependent degradation. Gel shift assay using ES cell nuclear extract and biotinylated Sp1 consensus oligonucleotides confirmed that both TA and Mit decreased DNA-binding activity of Sp1. These results demonstrate that both Mit and TA reduce expression of Sp1 and survivin, disrupt Sp1 DNA-binding and inhibit ES cell proliferation. This investigation suggests that targeting Sp1 and survivin could be an effective strategy for inhibiting ES cell growth.

Inhibition of cell survival and proliferation by nimbolide in human androgen-independent prostate cancer (PC-3) cells: involvement of the PI3K/Akt pathway.

Prostate cancer is most common malignancy among men in the world. PI3K-Akt signaling appears to be critical to prostate cancer cell proliferation and survival. Our earlier study reveals that nimbolide (2µM) prevents cell survival via IGF signaling pathway through PI3K/Akt and induces apoptosis in PC-3 cell line. Akt mediates the phosphorylation and activation of mTOR that plays a critical role in the regulation of protein translation and synthesis, angiogenesis, and cell cycle progression. The present study was aimed to investigate the effect of nimbolide on tPI3K, tAkt, pAkt, tmTOR, GSK3β, pGSK3β, PCNA, c-Myc, Cyclin D1, and Survivin protein levels by western blot analysis. Apoptosis was visualized by Ao/EtBr dual staining (20×), and protein expression of PCNA by immunocytochemistry was performed. Molecular docking was performed to understand the possible interaction between nimbolide and Akt, PCNA, and Cyclin D1. Nimbolide altered the PI3K-Akt-mediated cell survival and proliferative molecules. Thus, nimbolide exerted anticancer effects in vitro by representing the PI3K-Akt-mTOR pathway in PC-3 cells. Thereby, it acts as a potent anticancer drug for prostate cancer.

MiR-138-5p contributes to cell proliferation and invasion by targeting Survivin in bladder cancer cells.

BackgroundSurvivin (encoded by the gene BIRC5) plays an important role in the carcinogenesis of bladder cancer. Identifying miRNAs that target Survivin in the setting of bladder cancer will help to develop Survivin-based therapies for bladder cancer.MethodsThe expression levels of miR-138-5p and Survivin protein were measured in 12 resected bladder cancer specimens. The correlation between miR-138-5p and Survivin was further examined by evaluating Survivin expression in human bladder cancer cell lines that either overexpressed or knocked down miR-138-5p. A luciferase reporter assay was performed to test the direct binding of miR-138-5p to the target gene BIRC5. We also investigated the biological role of miR-138-5p targeting to Survivin in bladder cancer cell lines both in vivo and in vitro.ResultsIn this study, we found that the Survivin protein was either absent or weakly expressed in normal adjacent tissues and consistently up-regulated in bladder cancer tissues; however, the mRNA levels did not vary as much, suggesting that a post-transcriptional mechanism was involved. Because microRNAs are powerful post-transcriptional regulators of gene expression, we used bioinformatic analyses to search for microRNAs that could potentially target BIRC5 in the setting of bladder cancer. We identified 2 specific targeting sites for miR-138-5p in the 3′ untranslated region (3′-UTR) of BIRC5. We further identified an inverse correlation between miR-138-5p and Survivin protein levels in bladder cancer tissue samples. By overexpressing or knocking down miR-138-5p in bladder cancer cells, we experimentally confirmed that miR-138-5p directly recognizes the 3′-UTR of the BIRC5 transcript and regulates Survivin expression. Furthermore, the biological consequences of the targeting of BIRC5 by miR-138-5p were examined in vitro via cell proliferation and invasion assays and in vivo using a mouse xenograft tumor model. We demonstrated that BIRC5 repression by miR-138-5p suppressed the proliferative and invasive characteristics of bladder cancer cells and that miR-138-5p exerted an anti-tumor effect by negatively regulating BIRC5 in a xenograft mouse model.ConclusionsTaken together, our findings provide the first clues regarding the role of miR-138-5p as a tumor suppressor in bladder cancer by inhibiting BIRC5 translation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0569-4) contains supplementary material, which is available to authorized users.

Fish-Oil-Derived DHA-mediated Enhancement of Apoptosis in Acute Lymphoblastic Leukemia Cells is Associated with Accumulation of p53, Downregulation of Survivin, and Caspase-3 Activation

ABSTRACTIn acute lymphoblastic leukemia (ALL), resistance to chemotherapy is associated with inactivation of p53 and upregulation of survivin. Thus, targeting the p53 and survivin expression may provide an attractive strategy for ALL treatment. It has been shown that fish-oil-derived docosahexaenoic acid (DHA) activates several antitumorigenic mechanisms in tumor cells, but little is known regarding the role of DHA on modulating p53 and survivin expression in ALL cells. In this study, we investigated the alterations of the p53 and survivin expression and induction of apoptosis in DHA-treated Molt-4 cells that serve as a model for ALL cells. Molt-4 cells were treated with 50, 100, 150, and 200 μM DHA after which cell proliferation, survivin mRNA and protein levels, p53 protein level, caspase-3 activation, and apoptotic rates were evaluated by different cellular and molecular techniques. After 48- and 72-h treatments with DHA at concentrations ranging from 50 to 200 μM, cell proliferation rates were measured to be 80.5–44.4%, and 73.4–14.4%, respectively, compared to untreated cells. We also found that treatment for 48 h with 200 μM DHA resulted in 10.8- and 3.6-fold increase in p53 protein level and caspase-3 activation followed by 4.7-and 1.6-fold decrease in survivin mRNA and protein levels, respectively, compared to untreated cells. Treatment of cells with different concentrations of DHA dramatically increased the p53/survivin and caspase-3/survivin ratios by 2.8- to 16.9-fold and 3.3 to 5.6-fold increases, respectively, compared to untreated cells. A decrease in the number of cells ranging from 16% to 70% and an increase in the number of apoptotic cells ranging from 9.3% to 93% was also observed with increasing DHA concentrations. In conclusion, p53 and survivin may provide promising targets of DHA in ALL cells and this compound with high proapoptotic capacity represents the possibility of its therapeutic application for ALL treatment.

Ubiquitin-like protein FAT10 promotes bladder cancer progression by stabilizing survivin.

Human HLA-F adjacent transcript 10 (FAT10) is a member of the ubiquitin-like-modifier family of proteins, which have been implicated in cancer development. In addition, the Survivin protein promotes proliferation in bladder cancer (BC). In this study, we explored the link between FAT10 and Survivin. FAT10 expression was dramatically up-regulated in BC tissue samples, and Kaplan-Meier survival analysis revealed that BC patients with high FAT10 expression had shorter overall survival than those with low FAT10 expression. Moreover, RNAi-mediated FAT10 knockdown decreased Survivin protein levels and inhibited BC proliferation both in vitro and in vivo. FAT10 directly bound to and stabilized Survivin protein, thereby promoting cancer cell proliferation by inhibiting ubiquitin-mediated degradation. These results reveal a novel mechanism by which FAT10 promotes tumor proliferation by directly stabilizing Survivin protein in BC.

Cullin9 protects mouse eggs from aneuploidy by controlling microtubule dynamics via Survivin

The Cullin9 gene encodes a putative E3 ligase that serves a wide variety of biological functions in mitosis, whereas its roles in meiosis have not yet clearly defined. Here, we report that Cullin9 accumulates on the spindle apparatus and colocalizes with the microtubule fibers during mouse oocyte meiotic maturation. Depletion of Cullin9 by morpholino microinjection results in a remarkably higher rate of disorganized spindles and misaligned chromosomes in oocytes, which is coupled with the impaired kinetochore-microtubule attachments. Resultantly, the incidence of aneuploid eggs significantly increases in Cullin9-depleted oocytes. Moreover, we show that Cullin9 controls Survivin's protein level during meiotic maturation, and thus regulates microtubule stability in oocytes. Thus, our study assigns a new meiotic function to Cullin9 and reveals that it prevents mouse eggs from aneuploidy by regulating microtubule dynamics via Survivin.

Immunohistochemical expression and serum level of survivin protein in colorectal cancer patients.

Survivin is one of the apoptosis-related inhibitors that is associated with a more aggressive behavior and a poor prognosis in numerous types of malignancies, including colorectal cancer (CRC). The objective of the present study was to perform immunohistochemical tissue analysis of survivin expression and serum analysis of survivin levels in CRC patients. The study group consisted of 55 CRC patients. Survivin expression was assessed by immunohistochemistry in 38 patients using monoclonal antibodies. Color reactions were observed in the nucleus and cytoplasm of the cancer cells. The expression was defined based on the H-score method. The level of survivin was determined using the enzyme-linked immunosorbent assay method. A positive immunoreaction was observed in the tumor tissues of 84.2% (32/38) of patients with CRC, consisting of nuclear (63.2%; 24/38) and cytoplasmic (81.6%; 31/38) expression. The survivin nuclear expression was associated with tumor mass location and the presence of distant metastases (P=0.048 and P=0.026, respectively). Survivin was detected in the sera of 38.2% (21/55) of CRC patients and in 81.8% (18/22) of healthy individuals. Serum protein levels were found to correlate with hematocrit (P=0.035), hemoglobin (P=0.008) and albumin (P=0.045), but not with any of the investigated clinicopathological parameters. The immunohistochemical positive reaction of survivin in the nuclei of cancer cells may condition their proliferative capacity, which is associated with higher risk of developing metastatic foci. Thus, the present study suggests that the expression of survivin may have diagnostic implications in cancer of the colon and thus requires further research. By contrast, the survivin serum level in CRC patients appears to be diagnostically ineffectual for clinical use.

Inhibition of cyclin dependent kinase 9 by dinaciclib suppresses cyclin B1 expression and tumor growth in triple negative breast cancer.

Cyclin-dependent kinases (CDKs) are potential cancer therapeutic targets because of their critical role in promoting cell growth. Dinaciclib is a novel CDK inhibitor currently under clinical evaluation for the treatment of advanced malignancies. In this study, we demonstrated the anti-tumor activity of dinaciclib in triple negative breast cancer (TNBC) patient derived xenograft (PDX) and cell lines in vitro and in vivo. Treatment with dinaciclib induced cell cycle arrest at G2/M phase and marked apoptosis. These changes were accompanied by reduced phosphorylation of CDK1 and retinoblastoma (Rb) protein and decreased protein levels of cyclin B1, cMYC and survivin. We further demonstrated that siRNA knockdown of CDK9, the kinase subunit of positive transcription elongation factor b (P-TEFb), instead of CDK1 or CDK2, reduced the levels of cyclin B1 and MYC in TNBC cell lines. These data support the importance of CDK9, in addition to CDK1, in mediating the growth inhibitory effect of dinaciclib in TNBC. Further investigation of CDK9 as a therapeutic target in TNBC is needed.

Inhibition of disheveled-2 resensitizes cisplatin-resistant lung cancer cells through down-regulating Wnt/β-catenin signaling

Cisplatin (CDDP) is currently recommended as the front-line chemotherapeutic agent for lung cancer. However, the resistance to cisplatin is widespread in patients with advanced lung cancer, and the molecular mechanism of such resistance remains incompletely understood. Disheveled (DVL), a key mediator of Wnt/β-catenin, has been linked to cancer progression, while the role of DVL in cancer drug resistance is not clear. Here, we found that DVL2 was over-expressed in cisplatin-resistant human lung cancer cells A549/CDDP compared to the parental A549 cells. Inhibition of DVL2 by its inhibitor (3289-8625) or shDVL2 resensitized A549/CDDP cells to cisplatin. In addition, over-expression of DVL2 in A549 cells increased the protein levels of BCRP, MRP4, and Survivin, which are known to be associated with chemoresistance, while inhibition of DVL2 in A549/CDDP cells decreased these protein levels, and reduced the accumulation and nuclear translocation of β-catenin. In addition, shβ-catenin abolished the DVL2-induced the expression of BCRP, MRP4, and Survivin. Furthermore, our data showed that GSK3β/β-catenin signals were aberrantly activated by DVL2, and inactivation of GSK3β reversed the shDVL2-induced down-regulation of β-catenin. Taken together, these results suggested that inhibition of DVL2 can sensitize cisplatin-resistant lung cancer cells through down-regulating Wnt/β-catenin signaling and inhibiting BCRP, MRP4, and Survivin expression. It promises a new strategy to chemosensitize cisplatin-induced cytotoxicity in lung cancer.

Abstract 515: Survivin contributes to radiation resistance in head and neck cancer through regulation of DNA damage repair

Abstract Background: A major challenge affecting outcomes of patients with squamous cell carcinomas of the head and neck (SCCHN) is radiation resistance. Overexpression of survivin is associated with radiation resistance and poor survival. Our study objectives were to: 1) evaluate the effects of survivin expression level on radiosensitivity in SCCHN cell lines; 2) identify the effect of survivin on DNA damage repair following radiation exposure in SCCHN cells; 3) evaluate the feasibility and efficacy of the combination therapy of survivin inhibitor YM155 and radiation in SCCHN. Methods: SCCHN cell lines, including JHU-022, PCI-15A and Tu686 were used to evaluate the role of survivin in DNA double strand break (DSB) repair following radiation exposure. To determine whether survivin specifically targets the sites of DNA damage, we performed ChIP assays using PCI-15A cells carrying the DR-GFP homologous recombination reporter. The in vitro effects of YM155 on sensitizing resistant cells to radiation were also evaluated using these cell lines. Results: The fraction surviving radiation was about 2.5-fold greater in PCI-15A and Tu686 cells compared with that in JHU-022 cells. As expected, survivin protein levels were elevated in PCI-15A and Tu686 cells compared with JHU-022 cells, suggesting an inverse correlation between survivin expression and radiosensitivity. Clonogenic survival following radiation exposure was inhibited by depletion of survivin in PCI-15A cells by siRNA, and increased by overexpression of survivin in JHU-022 cells. Inhibition of survivin by YM155 enhances the efficacy of radiation in PCI-15A cells. Immunoblotting revealed that radiation induced nuclear accumulation of survivin and its complex with γ-H2X, and DNA-PKCs in PCI-15A cells, suggesting survivin was involved in DNA damage response and repair induced by radiation. ChIP assay further confirmed that survivin specifically targeted and bound to the DNA DSB site, which is consistent with results obtained from immunofluorescence analysis. Conclusions: Survivin is involved in DNA damage response and repair induced by radiation exposure in SCCHN cells. Inhibition of survivin by YM155 enhances the efficacy of radiation, and may provide a novel therapeutic approach to improve the efficacy of radiotherapy in SCCHN. (This research was supported by the National Cancer Institute award P50 CA128613, and GCC Distinguished Cancer Scholar to Dong M. Shin, Zhuo (Georgia) Chen, and Jonathan J Beitler) Citation Format: Xu Wang, Jonathan J. Beitler, Wen Huang, Guo Chen, Sreenivas Nannapaneni, Nabil F. Saba, Xingming Deng, Zhuo G. Chen, Dong M. Shin. Survivin contributes to radiation resistance in head and neck cancer through regulation of DNA damage repair. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 515.

Abstract 4608: Survivin downregulation by α-santalol is not mediated through PI3K-Akt pathway in human breast cancer cells

Abstract α-Santalol, a terpenoid found in sandalwood oil, has been shown to inhibit cancer cell growth in vitro by inducing apoptosis. This study was performed to investigate the anticancer properties of α-santalol associated with the induction of apoptosis in cultured MCF-7 (estrogen receptor (ER) positive, and wild type p53) and MDA-MB-231 (ER-negative and mutant p53) breast cancer cells. Expression of major proteins examined in the study were determined using standard Western blot protocol and analyzed by LICOR-Odyssey infra-red scanner. Total protein levels of survivin were confirmed by survivin ELISA kit. Cell viability was assessed by trypan blue dye exclusion assay, and caspase-3 activity was determined by caspase-3 (active) ELISA kit. Treatment of breast cancer cells for 6 and 9 hour time intervals with α-santalol (20, 40 μM) resulted in statistically significant concentration-dependent downregulation of survivin. pAkt levels were found to be slightly upregulated despite the downregulation of survivin. Pharmacological inhibition of the PI3K-Akt pathway did not result in a synergistic/additive increase in cell death or caspase-3 activity caused by α-santalol. The study reveals that survivin downregulation by α-santalol in breast cancer cells is not mediated through the PI3K-Akt pathway. Citation Format: AJAY BOMMAREDDY, KARRYN CRISAMORE, SARAH FILLMAN, SARAH BROZENA, JAMES STEIGERWALT, TERRA LANDIS, ADAM L. VANWERT, CHANDRADHAR DWIVEDI. Survivin downregulation by α-santalol is not mediated through PI3K-Akt pathway in human breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4608.

TanshinoneI induces human colorectal cancer cell apoptosis: The potential roles of AuroraA-p53 and survivin-mediated signaling pathways.

Colorectal cancer (CRC) is one of the most common malignancies worldwide and a leading cause of cancer death. Despite decades of intensive investigations, effective interventional options are still limited and patient prognosis remains poor. TanshinoneI, an active compound from traditional Chinese herbal medicine Salvia miltiorrhiza Bunge, has been shown to inhibit cell growth of leukemia, lung, and breast cancers. However, whether and how TanshinoneI exerts similar effects on CRC needs to be elucidated. TanshinoneI induced CRC cell apoptosis was characterized and the roles of AuroraA-p53 and survivin-mediated pathways were analyzed in different CRC cell lines. TanshinoneI markedly inhibited CRC cell growth and induced apoptosis in CRC cells with functional p53 protein. Interestingly, TanshinoneI did not exert as much inhibitory effect on normal colon epithelial cells or CRC cells with mutant p53, indicating relative selectivity toward colorectal cancer cells with full presence of p53. In tse cells with wild-type p53, data showed that TanshinoneI mediated AuroraA inhibition results in p53 upregulation, which is required for cell apoptosis. In CRC cells with mutant p53 protein (not able to localize to the nucleus), however, AuroraA knockdown failed to induce CRC cell apoptosis. Instead, data showed that protein level of survivin decreased following TanshinoneI treatment. These observations were further substantiated by the pivotal role of survivin in TanshinoneI mediated apoptosis in CRC cells with p53 mutant. TanshinoneI, a novel natural compound, exerts significant inhibitory effect on CRC cell growth via a mechanism involving either AuroraA-p53 axis or survivin-involving mechanism depending on different intrinsic characteristics of tumor cells.