Survivin mRNA Levels Research Articles

Overexpression of the Survivin gene in SGC7901 cell resistance to cisplatin.

The aim of the present study was to investigate the mechanism of SGC7901 cell resistance to cisplatin (CDDP). SGC7901/CDDP cells were established by the long-term continuous exposure of SGC7901 cells to CDDP in stepwise concentration increments. The morphologies of the SGC7901/CDDP and SGC7901 cells were observed by microscopy. The expression levels of Survivin mRNA and protein in the SGC7901/CDDP and SGC7901 cells were examined by reverse transcription polymerase chain reaction and western blotting respectively. The results revealed morphological differences between the SGC7901 and SGC7901/CDDP cells. The expression levels of Survivin mRNA and protein were significantly higher in the SGC7901/CDDP cells than in the SGC7901 cells. Therefore, high expression levels of the Survivin gene may explain SGC7901 cell resistance to CDDP.

Survivin mRNA Level in Blood Predict the Efficacy of Neoadjuvant Chemotherapy in Patients with Stage IIIA-N2 Non-Small Cell Lung Cancer.

In a previous study, survivin mRNA expression in non-small cell lung cancer (NSCLC) tissue had been demonstrated to be associated with unfavorable prognosis of patients treated with chemotherapy. In this study, we investigated the survivin mRNA levels in blood of patients with stage IIIA-N2 NSCLC and their association with the efficacy of neoadjuvant chemotherapy (NCT) and disease-free survival (DFS) and overall survival (OS). Blood specimens were collected from 56 patients with stage IIIA-N2 NSCLC before (N0) and after the complete of NCT (N1). Survivin mRNA was measured by real-time quantitative-PCR assay. Receiver operating characteristics curve analysis was undertaken to determine the best cutoff value for survivin mRNA. Results showed that high blood survivin mRNA levels at N0 and N1 were significantly associated with clinical (P = 0.01 and P = 0.008, respectively) and pathologic response (both P = 0.004, respectively). Moreover, the change of blood survivin mRNA levels in these NSCLC patients is associated with the clinical and pathologic response to NCT. Patients with high survivin mRNA levels at N0 and N1 had significantly shorter DFS and OS than those with low survivin mRNA levels (P = 0.021 and P = 0.014, respectively for DFS; P = 0.009 and P = 0.005, respectively for OS). Multivariate analysis demonstrated that high blood survivin mRNA level was an independent predictor for worse DFS and OS in the NSCLC patients receiving NCT. In conclusion, survivin mRNA level in blood from stage IIIA-N2 NSCLC patients receiving NCT is predictive of cancer outcome.

RRR-α-tocopheryl succinate induces apoptosis in human gastric cancer cells via the NF-κB signaling pathway.

To investigate the effects of the nuclear factor (NF)-κB signaling pathway on the induction of apoptosis by vitaminE succinate (RRR-α-tocopheryl succinate; VES) in human gastric carcinoma cells. Human gastric carcinoma SGC-7901 cells were treated with temperate concentrations of VES and pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB. Cell viability and apoptosis were respectively estimated by methylthiazol tetrazolium (MTT) assay and the AnnexinV‑FITC method. Western blot analysis was used to evaluate the protein expressions of NF-κBp65 and Bcl-2 family members Bcl-2, Bax and cleavage of caspase-3, caspase-9, and poly (ADP-ribose) polymerase (PARP). The DNA-binding activity of NF-κBp65 was measured by electrophoretic mobility shift assay(EMSA). Reverse transcription and polymerase chain reaction(RT-PCR) was implemented to evaluate the transcription of inhibitor of apoptosis (IAP) genes. Apoptosis assessment showed that VES induces apoptotic cell death in human gastric carcinoma cells. In the following experiments, PDTC (100µM) was used in cell treatment 2h before VES. The decreased ratio of the nuclear and cytosolic NF-κBp65 protein level was induced by VES and PDTC reinforced this trend. PDTC treatment significantly enhanced the decrease of NF-κB-DNA binding activity induced by VES in human gastric SGC-7901. The decrease in protein expression of Bcl-2 as well as the increase in the protein expression of Bax were induced by VES treatment. The cleavage of caspase-9, caspase-3 and PARP was induced. There was no effect on the gene transcription of c-IAP-1, c-IAP-2, and x-linked IAP (XIAP) compared with the control group, whereas mRNA levels of survivin and the neuronal apoptosis inhibitory protein (NAIP) markedly decreased. Notably, pretreatment with PDTC reinforced all the above VES-induced effects. In conclusion, VES-induced apoptosis in SGC-7901 cells is accompanied by the inhibition of the NF-κB signaling pathway, including changes in Bcl-2 family members, cleavage of caspases and gene transcription of survivin and NAIP.

AB0033 Intracellular Expression of Survivin and Bcl-6 is Decreased in CD4+ T-Cells of Rheumatoid Arthritis Patients with High Serum Survivin

BackgroundSurvivin is recognized as a marker of persistently active and erosive RA. The findings in experimental arthritis suggested that survivin co-ordinates the antigen-dependent leukocyte maturation and function by regulating transcriptional...

Effects of everolimus on macrophage-derived foam cell behavior.

The purpose of this study was to investigate the effects of everolimus on foam cell (FC) viability, mRNA levels, and inflammatory cytokine production to better understand its potential inhibitory effects on atheroma progression. Human THP1 macrophage-derived FC were formed using acetylated LDL (acLDL, 100 μg/mL) for 72 hours, followed by everolimus treatment (10(-5)-10(-11) M) for 24 hours. FC viability was quantified using fluorescent calcein AM/DAPI staining. FC lysates and media supernatants were analyzed for apoptosis and necrosis using a Cell Death ELISA(PLUS) assay. FC lysates and media supernatants were also analyzed for inflammatory cytokine (IL1β, IL8, MCP1, TNFα) mRNA levels and protein expression using quantitative reverse transcription real-time polymerase chain reaction (QPCR) and a Procarta® immunoassay, respectively. mRNA levels of autophagy (MAP1LC3), apoptosis (survivin, clusterin), and matrix degradation (MMP1, MMP9) markers were evaluated by Quantigene® Plex assay and verified with QPCR. Additionally, hypercholesterolemic rabbits received everolimus-eluting stents (EES) for 28 or 60 days. RAM-11 immunohistochemical staining was performed to compare %RAM-11 positive area between stented sections and unstented proximal sections. Statistical significance was calculated using one-way ANOVA (p≤0.05). Calcein AM/DAPI staining showed that FC exposed to everolimus (10(-5) M) had significantly decreased viability compared to control. FC apoptosis was significantly increased at a high dose of everolimus (10(-5)M), with no necrotic effects at any dose tested. Everolimus did not affect endothelial (HUVEC) and smooth muscle (HCASMC) cell apoptosis or necrosis. Everolimus (10(-5)M) significantly increased MAP1LC3, caused an increased trend in clusterin (p=0.10), and significantly decreased survivin and MMP1 mRNA levels in FC. MCP1 cytokine mRNA levels and secreted protein expression was significantly decreased by everolimus (10(-5) M) in FC. Percentage of RAM-11 positive area exhibited a reduction trend within sections stented with EES compared to unstented proximal sections at 60 days (p=0.09). Everolimus, a potent anti-proliferative agent used in drug-eluting stents and bioresorbable vascular scaffolds, may inhibit atheroma progression and/or promote atheroma stabilization through diminished viability of FC, decreased matrix degradation, and reduced pro-inflammatory cytokine secretion. We explored the effects of everolimus on the behavior of human THP1 macrophage-derived foam cells in culture, including cell viability, mRNA levels, and pro-inflammatory cytokine production. We conclude that everolimus, a potent anti-proliferative agent used in drug-eluting stents/bioresorbable vascular scaffolds, may potentially inhibit atheroma progression and/or promote atheroma stabilization through diminished viability of foam cells, decreased matrix degradation, and reduced pro-inflammatory cytokine secretion.

Inhibitor of DNA-binding protein 1 knockdown arrests the growth of colorectal cancer cells and suppresses hepatic metastasis in vivo.

Inhibitor of DNA-binding protein 1 (ID1) is commonly abnormally overexpressed in colorectal cancer (CRC); yet, the functional significance of ID1 in the growth and invasive properties of CRC cells remains largely unclear. The present study investigated the effects of ID1 downregulation on the cell growth and metastatic features of CRC. Using lentiviral shRNA infection, stable ID1-knockdown (KD) HCT116 and SW620 cells, human metastatic CRC cell lines, were created. In vitro, the migration/invasion capacity of the ID1-KD CRC cells was assessed by a wound healing assay. The activities of MMP2 and MMP-9 were measured by gelatin zymography. The expression of CXC chemokine receptor 4 (CXCR4), PCNA and survivin were determined by immunoblot analysis and qRT-PCR. The effects of ID1 knockdown on tumor growth and hepatic metastasis were demonstrated by a xenograft study in mice. The results showed evident decreases in proliferation, migration and invasion and an increased apoptosis rate in the ID1-KD CRC cells. Similarly, ID1 knockdown significantly decreased mRNA and protein levels of PCNA, survivin, CXCR4, MMP2 and MMP9. Overexpression of CXCR4 antagonized the negative effect on the migration and invasion abilities of the ID1-KD cells. As compared with the control, ID1 knockdown prevented tumor growth and profoundly suppressed hepatic metastasis in vivo. The present study demonstrated the significance of ID1 in colon cancer progression, and its effect on tumor invasiveness and metastatic properties may be partly dependent on CXCR4.

Survivin mRNA expression in blood as a predictor of the response to EGFR-tyrosine kinase inhibitors and prognosis in patients with non-small cell lung cancer

The epidermal growth factor receptor (EGFR) mutations have been proven to be a reliable predictive marker for the response to EGFR-tyrosine kinase inhibitors (TKIs) in patients with non-small cell lung cancer (NSCLC). However, the responses to EGFR-TKIs vary even among patients with EGFR mutation. Recent study showed that survivin overexpression attenuated EGFR-TKI-induced apoptosis and inhibited the antitumor effect of EGFR-TKIs on EGFR mutation NSCLC cells. We investigated the role of survivin mRNA expression in peripheral blood on predicting response and prognosis in NSCLC patients treated with EGFR-TKIs. Survivin mRNA expression levels in blood was detected using quantitative real-time-PCR assay in 62 patients with advanced NSCLC before (D0) and 4 weeks after treatment of EGFR-TKIs (D4w). The associations between survivin mRNA expression in blood and tumor response to treatment, time to progression (TTP), and overall survival (OS) were analyzed. Blood survivin mRNA levels at D0 and D4w were significantly higher in patients with progressive disease than in those with partial response and stable disease. The detections of blood survivin mRNA positivity at D0 and D4w were associated with unfavorable response to EGFR-TKIs treatment and shorter TTP and OS. Multivariate Cox analysis showed that blood survivin mRNA positivity before and 4 weeks after EGFR-TKIs treatment were independent predictor for worse TTP and OS. In conclusion, Blood survivin mRNA positivity was strongly related to a poor treatment outcome of EGFR-TKIs and may be a potential non-invasive biomarker for NSCLC treatment.

Survivin-responsive conditionally replicating adenovirus kills rhabdomyosarcoma stem cells more efficiently than their progeny

BackgroundEffective 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.MethodsOur 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.ResultsBoth 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.ConclusionsThe 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.

Downregulation of the Wnt/β‐catenin signaling pathway is involved in manganese‐induced neurotoxicity in rat striatum and PC12 cells

Manganese (Mn) is an essential trace element. However, exposure to excessive Mn may cause neurodegenerative disorders called manganism. Accumulating evidence indicated that dysregulation of Wnt/β-catenin signaling was tightly associated with the onset of neurodegenerative disorders. However, whether aberrant Wnt/β-catenin signaling contributes to Mn-induced neurotoxicity remains unknown. The present study investigates the involvement of Wnt/β-catenin signaling in Mn-induced neurotoxicity. Western blot and immunohistochemistry analyses showed a remarkable downregulation of p-Ser9-glycogen synthase kinase-3β (GSK-3β) and β-catenin in rat striatum after Mn exposure. TUNEL assay revealed significant neuronal apoptosis following treatment with 25 mg/kg Mn. Immunofluorescent staining showed that β-catenin was expressed predominantly in neurons, and colocalization of β-catenin and active caspase-3 was observed after Mn exposure. Furthermore, Mn exposure resulted in PC12 cells apoptosis, which was accompanied by reduced levels of cellular β-catenin and p-GSK-3β. Accordingly, the mRNA level of the prosurvival factor survivin, a downstream target gene of β-catenin, was synchronously decreased. More importantly, blockage of GSK-3β activity with the GSK-3β inhibitor lithium chloride could attenuate Mn-induced downregulation of β-catenin and survivin as well as neuronal apoptosis. Overall, the present study demonstrates that downregulation of Wnt/β-catenin signaling pathway may be of vital importance in the neuropathological process of Mn-induced neurotoxicity.

Tumor priming enhances siRNA delivery and transfection in intraperitoneal tumors

Cancers originating from the digestive system account for 290,000 or ~20% of all new cancer cases annually in the US. We previously developed paclitaxel-loaded tumor-penetrating microparticles (TPM) for intraperitoneal (IP) treatment of peritoneal tumors (Lu et al., 2008; Tsai et al., 2007; Tsai et al., 2013). TPM is undergoing NIH-supported IND-enabling studies for clinical evaluation. The present study evaluated the hypothesis that TPM, via inducing apoptosis and expanding the interstitial space, promotes the delivery and transfection of lipid vectors containing siRNA. The in vivo model was the metastatic human Hs766T pancreatic tumor that, upon IP injection, produced widely distributed solid tumors and ascites in the peritoneal cavity in 100% of animals. The target gene was survivin, an anti-apoptotic protein induced by chemotherapy and associated with metastases and poor prognosis of patients with gastric and colorectal cancers. The siRNA carrier was pegylated liposomes comprising cationic and neutral lipids plus a fusogenic lipid (PCat). PCat-loaded with survivin siRNA (PCat–siSurvivin) was active in cultured cells (decreased survivin mRNA and protein levels, reduced cell clonogenicity, enhanced paclitaxel activity), but lost its activity in vivo; this difference is consistent with the well-known problem of inadequate delivery and transfection of siRNA in vivo. In comparison, single agent TPM prolonged animal survival and, as expected, induced survivin expression in tumors. Addition of PCat–siSurvivin reversed the TPM-induced survivin expression and enhanced the antitumor activity of TPM. The finding that in vivo survivin knockdown by PCat–siSurvivin was successful only when it was given in combination with TPM provides the proof-of-concept that tumor priming promotes the delivery and transfection of liposomal siRNA. The data further suggest the TPM/PCat–siSurvivin combination as a potentially useful chemo-gene therapy for peritoneal cancer.

Survivin expression in circulating tumor cells of patients with gastric cancer.

65 Background: Detection of circulating tumor cells (CTC) in peripheral blood is thought to be a promising method to evaluate the extent of minimal residual disease that may be useful for predicting recurrence and for monitoring treatment efficacy. However, unlike breast or lung cancer, little information is available in patients with gastric cancer. Among them, evaluation of survivin, a member of the inhibitor of apoptosis (IAP) family, mRNA expression in peripheral blood appears to be a promising procedure with relative good performance for predicting recurrence after curative resection. In order to evaluate the clinical significance of surviving expression in peripheral blood, we performed real-time RT-PCR analysis using whole blood obtained from patients with gastric cancer. Methods: A total of 95 patients with gastric cancer treated at our cancer center between Nov. 2009 and Aug. 2012 were enrolled in this study. Peripheral blood samples were collected using PAX gene and RNA was isolated using Boom method. After cDNA synthesis, quantitative real-time PCR targeting survivin gene was performed. The relative expression level of survivin mRNA was calculated with actin beta as an internal standard. Correlation between survivin expression and clinicopathologic factors was investigated. Results: Survivin mRNA expression level in peripheral blood ranged from 34.8 to 1889.6, and the median level was 370.3. Survivin mRNA level tended to be higher in patients with T4, N3b, CY1, and Stage IV. There was no significant correlation with histological type, lympho-vascular invasion, or macroscopic type. If we set a cut-off value at 540, survivin was positive in 27 patients (28%). Disease-free survival was significantly worse in survivin positive patients than in negative patients (p=0.0216). Conclusions: Survivin expression in CTC was higher in advanced stage and appears to be a good prognostic marker in patients with gastric cancer. Validation with large sample size should be performed to confirm the significance of survivin in CTC.

Small Double-Stranded RNA Mediates the Anti-Cancer Effects of p21WAF1/ClP1Transcriptional Activation in a Human Glioma Cell Line

PurposeThis study was conducted to investigate the small double-stranded RNA (dsRNA) mediated anti-tumor effects of p21WAF1/ClP1 (p21) transcriptional activation in vitro in the human glioma SHG-44 cell line.Materials and MethodsHuman glioma SHG-44 cells were transfected with dsRNA using LipofectAMINE 2000 transfection reagent. Real-time PCR and Western blot analysis were conducted to detect p21 and survivin mRNA and protein levels, respectively. Cell proliferation was examined by MTT assay. Cell cycle distribution and apoptosis were detected by flow-cytometric analysis.ResultsWe found that dsRNA targeting p21 promoter (dsP21) significantly induced the expression of p21 at transcription and protein levels, and reduced the expression of survivin. AS well, dsP21 transcription significantly inhibited human glioma SHG-44 cell proliferation. Analysis of cell cycle distribution revealed that dsP21 transfection increased accumulation of cells in the G0/G1 phase and reduced accumulation of cells in the S phase. Further analysis revealed that dsP21 transcription led to an increase in both early and late stages of apoptosis in human glioma SHG-44 cells.ConclusionIn the present study, P21 activation by RNA-induced gene activation (RNAa) induced anti-tumor activity in vitro in a human glioma SHG-44 cell line. The results suggested that RNAa could be used for human glioma treatment by targeted activation of tumor suppressor genes.

SiRNA-mediated Silencing of Survivin Inhibits Proliferation and Enhances Etoposide Chemosensitivity in Acute Myeloid Leukemia Cells

Overexpression of survivin, a known inhibitor of apoptosis, is associated with tumor progression and drug resistance in numerous malignancies, including leukemias. The aim of this study was to investigate the effect of a specific survivin small interference RNA (siRNA) on proliferation and the sensitivity of HL-60 acute myeloid leukemia (AML) cells to the chemotherapeutic drug etoposide. The cells were transfected with siRNAs using LipofectamineTM2000 transfection reagent. Relative survivin mRNA and protein levels were measured by quantitative real-time PCR and Western blotting, respectively. Trypan blue exclusion assays were performed to monitor tumor cell proliferation after siRNA transfection. The cytotoxic effects of etoposide and survivin siRNA, alone and in combination, on leukemic cells were determined using MTT assay. Apoptosis was assessed by ELISA cell death assay. Survivin siRNA markedly reduced both mRNA and protein expression levels in a time-dependent manner, leading to distinct inhibition of cell proliferation and increased spontaneous apoptosis. Surprisingly, survivin siRNA synergistically increased the cell toxic effects of etoposide. Moreover, survivin down-regulation significantly enhanced its induction of apoptosis. Our study suggests that down-regulation of survivin by siRNA can trigger apoptosis and overcome drug resistance of leukemia cells. Therefore, survivin siRNA may be an effective adjuvant in AML chemotherapy.

Antiproliferative and Apoptotic Effects of Indomethacin on Human Retinoblastoma Cell Line Y79 and the Involvement of ß-Catenin, Nuclear Factor-κB and Akt Signaling Pathways

Background: To determine in vitro if indomethacin inhibits proliferation and induces apoptosis in human retinoblastoma cell line Y79, and to explore possibly involved signaling pathways. Methods: The human retinoblastoma cell line Y79 was cultured with indomethacin at various concentrations (0, 25, 50, 100, 200 and 400 µmol/l). The effect of indomethacin on cell proliferation and apoptosis was examined by the Cell Counting Kit-8 and TUNEL test, respectively. The mRNA level of survivin, ß-catenin and Bcl-2 was detected by RT-PCR. The protein level of survivin was measured by ELISA. Western blot was used to analyze ß-catenin, nuclear factor (NF)-κB/p65, phosphorylated Akt (pAkt) and total Akt (tAkt) expression in cultured cells. Results: Indomethacin treatment inhibits proliferation (at concentrations from 25 to 400 µmol/l) and induces apoptosis (at concentrations from 100 to 400 µmol/l) of human retinoblastoma cell line Y79 in a dose-dependent manner. RT-PCR showed that the mRNA expression of Bcl-2 (F = 20.497; p < 0.001) and of ß-catenin (F = 14.835; p < 0.001) was significantly different among the treated groups. Survivin mRNA levels remained steady, but its protein levels decreased significantly as measured by ELISA (F = 67.633; p < 0.001). Western blot analysis showed a dose-dependent downregulation of ß-catenin (F = 37.411; p < 0.001), NF-κB/p65 (F = 16.302; p < 0.001) and of pAkt (F = 27.700; p < 0.001) after indomethacin treatment, while tAkt protein expression was steady among the groups. Conclusions: Treatment with indomethacin can potently suppress proliferation and induce apoptosis in the retinoblastoma Y79 cell line. Wnt/ß-catenin, NF-κB and Akt/PKB pathways might be implicated in the process.

Eugenol triggers apoptosis in breast cancer cells through E2F1/survivin down-regulation

BackgroundBreast cancer is a major health problem that threatens the lives of millions of women worldwide each year. Most of the chemotherapeutic agents that are currently used to treat this complex disease are highly toxic with long-term side effects. Therefore, novel generation of anti-cancer drugs with higher efficiency and specificity are urgently needed.MethodsBreast cancer cell lines were treated with eugenol and cytotoxicity was measured using the WST-1 reagent, while propidium iodide/annexinV associated with flow cytometry was utilized in order to determine the induced cell death pathway. The effect of eugenol on apoptotic and pro-carcinogenic proteins, both in vitro and in tumor xenografts was assessed by immunoblotting. While RT-PCR was used to determine eugenol effect on the E2F1 and survivin mRNA levels. In addition, we tested the effect of eugenol on cell proliferation using the real-time cell electronic sensing system.ResultsEugenol at low dose (2 μM) has specific toxicity against different breast cancer cells. This killing effect was mediated mainly through inducing the internal apoptotic pathway and strong down-regulation of E2F1 and its downstream antiapoptosis target survivin, independently of the status of p53 and ERα. Eugenol inhibited also several other breast cancer related oncogenes, such as NF-κB and cyclin D1. Moreover, eugenol up-regulated the versatile cyclin-dependent kinase inhibitor p21WAF1 protein, and inhibited the proliferation of breast cancer cells in a p53-independent manner. Importantly, these anti-proliferative and pro-apoptotic effects were also observed in vivo in xenografted human breast tumors.ConclusionEugenol exhibits anti-breast cancer properties both in vitro and in vivo, indicating that it could be used to consolidate the adjuvant treatment of breast cancer through targeting the E2F1/survivin pathway, especially for the less responsive triple-negative subtype of the disease.

Abstract B227: Overexpression of microRNA 214-3p in esophageal cancer cells enhances sensitivity to chemotherapy-induced apoptosis by targeting CUG-binding protein 1 (CUGBP1) and survivin.

Abstract Introduction: Survivin, a key regulator of apoptosis and cell division is overexpressed in esophageal cancer cells and has been correlated with decreased survival. Our previous studies indicate that the RNA-binding protein CUGBP1 is also overexpressed in esophageal cancer cells and contributes to survivin overexpression by stabilizing its mRNA. To date, little information exists on the regulation of CUGBP1 in esophageal cancer cells. Based on sequence analysis, miR 214-3p is predicted to bind both CUGBP1 and survivin mRNA with high binding affinity. The purposes of this study are to determine expression levels of miR 214-3p in esophageal cancer cells and to establish whether miR 214-3p directly interacts with CUGBP1 and survivin mRNA to regulate their expression and modulate sensitivity to chemotherapy-induced apoptosis. Methods: Studies were conducted in human esophageal epithelial (hESO) cells and in TE7 and TE10 human esophageal cancer cells. Global microRNA expression in these cell lines was determined by microarray analysis and confirmed by real-time PCR. MiR 214-3p function was tested by its overexpression and silencing. Levels of protein expression were measured by Western blot. Levels of mRNA were measured by real-time PCR. Binding of miR 214-3p to CUGBP1 and survivin mRNA was examined by biotinylated RNA pull-down assays. mRNA stability was determined by measuring its half-life after addition of Actinomycin D. The apoptotic phenotype was examined by caspase expression using Western blot and flow cytometry. Results: Levels of miR 214-3p in TE7 and TE10 esophageal cancer cells are reduced by approximately 3,000 log-fold in comparison to hESO cells. Biotinylated RNA-pull down assays confirm direct binding of miR 214-3p to CUGBP1 and survivin mRNA. CUGBP1 and survivin mRNA and protein levels are reduced in a time-dependent manner following miR 214-3p overexpression in TE7 and TE10 cells. Conversely, silencing miR 214-3p in hESO cells results in increased CUGBP1 and survivin mRNA and protein levels. The stability of both CUGBP1 and survivin mRNA is decreased following overexpression of miR 214-3p in TE7 cells. Caspase-3 protein levels are increased when TE7 cells are exposed to camptothecin following microRNA 214-3p overexpression. Conclusion: MiR 214-3p expression is markedly reduced in TE7 and TE10 esophageal cancer cells relative to hESO cells. MiR 214-3p binds and destabilizes both CUGBP1 and survivin mRNA. Overexpression of miR 214-3p in esophageal cancer cells results in decreased expression of CUGBP1 and survivin and leads to increased sensitivity to chemotherapy-induced apoptosis. These results strongly support the supposition that the loss of miR 214-3p in esophageal cancer cells contributes to enhanced resistance to chemotherapy-induced apoptosis by leading to increased CUGBP1 and survivin expression. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B227. Citation Format: Pornima Phatak, Daniel Mansour, Kimberly Byrnes, Shan Cao, Lan Liu, Ruiyun Li, Rao Jaladanki, Douglas J. Tuner, Jian Ying Wang, James M. Donahue. Overexpression of microRNA 214-3p in esophageal cancer cells enhances sensitivity to chemotherapy-induced apoptosis by targeting CUG-binding protein 1 (CUGBP1) and survivin. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B227.

Effect of nimesulide on the growth of human laryngeal squamous cell carcinoma

PurposeTo investigate the effect of nimesulide on the growth of human laryngeal squamous cell carcinoma. Materials and methodsThe effect of NIM on Hep-2 cell proliferation was measured by the MTT assay. Flow cytometry was used to evaluate the cell cycle and apoptosis in Hep-2 cells. A Western blot analysis was used to detect changes in the protein expression levels of COX-2, Survivin and proliferating cell nuclear antigen (PCNA) in Hep-2 cells. A Hep-2 tumor xenograft model was established in nude mice to observe tumor growth. The changes in the xenograft tumors were observed after hematoxylin/eosin staining. The expression levels of COX-2, Survivin and PCNA proteins and mRNA were measured by immunohistochemical analysis and RT-PCR, respectively. ResultsNIM had time- and dose-dependent inhibitory effect on the proliferation of Hep-2 cells. NIM could prevent the progression of the cell cycle. After NIM treatment, COX-2, Survivin and PCNA protein levels were reduced in the Hep-2 cells. The volume and weight of the xenograft tumors in the NIM treatment group were significantly reduced. The NIM treatment group also exhibited significantly reduced expression levels of COX-2, Survivin and PCNA at both the protein and mRNA levels. ConclusionsOur results suggested that NIM has significant inhibitory effects on the growth of Hep-2 cells and xenograft tumors in nude mice. Selective COX-2 inhibitors could potentially become part of a comprehensive treatment for laryngeal squamous cell carcinoma. Additional research and development will provide new and broader prospects for the prevention and treatment of laryngeal squamous cell carcinoma.

Survivin Messenger RNA Levels in Epstein-Barr Virus–Positive Patients With Leukemic Low-Grade B-Cell Lymphomas Expressing the Latent Membrane Protein 1: Evidence of Apoptotic Function?

Epstein-Barr virus (EBV) is a ubiquitous pathogen that chronically infects B lymphocytes and is implicated in the pathogenesis of lymphoproliferative diseases. Latent membrane protein 1 (LMP1), the major oncoprotein of the virus, has been shown to inhibit apoptosis and trigger survivin expression in malignant cell lines. LMP1 expression has been detected in patients with chronic lymphocytic leukemia, but its properties have not been studied in patients with low-grade B-cell lymphomas. Recent data show that LMP1 can simultaneously induce and inhibit apoptosis in B cells. We detected LMP1 messenger RNA (mRNA) in patients with leukemic low-grade B-cell lymphoma and correlated the expression of the antiapoptotic molecule survivin to that of LMP1 in this group of patients. Peripheral whole blood from 64 patients with low-grade B-cell lymphoma was tested by quantitative reverse transcriptase-polymerase chain reaction (PCR) for the presence of the BXLF-1 gene of EBV, and positive samples were tested by conventional PCR for LMP1 expression. Accordingly, survivin mRNA levels were measured by quantitative reverse transcriptase PCR in all samples and compared between LMP1-positive (LMP1(+)) and LMP1(-) patients. The BXLF-1 gene was detected in 27 of 64 patients (42%). LMP1 was expressed in 22 of 27 (81%) EBV(+) patients. Survivin expression was found to be 6.36 times higher in LMP1(-) patients than in LMP1(+) patients (P = .008). Our results imply that in patients with non-EBV-related leukemic low-grade B-cell lymphoma, LMP1 expression is possibly correlated to apoptosis, as indicated by the lower survivin mRNA levels in LMP1(+) patients.

Polypurine reverse Hoogsteen hairpins as a gene therapy tool against survivin in human prostate cancer PC3 cells in vitro and in vivo

As a new approach for gene therapy, we recently developed a new type of molecule called polypurine reverse Hoogsteen hairpins (PPRHs). We decided to explore the in vitro and in vivo effect of PPRHs in cancer choosing survivin as a target since it is involved in apoptosis, mitosis and angiogenesis, and overexpressed in different tumors. We designed four PPRHs against the survivin gene, one of them directed against the template strand and three against different regions of the coding strand. These PPRHs were tested in PC3 prostate cancer cells in an in vitro screening of cell viability and apoptosis. PPRHs against the promoter sequence were the most effective and caused a decrease in survivin mRNA and protein levels. We confirmed the binding between the selected PPRHs and their target sequences in the survivin gene. In addition we determined that both the template- and the coding-PPRH targeting the survivin promoter were interfering with the binding of transcription factors Sp1 and GATA-3, respectively. Finally, we conducted two in vivo efficacy assays using the Coding-PPRH against the survivin promoter and performing two routes of administration, namely intratumoral and intravenous, in a subcutaneous xenograft tumor model of PC3 prostate cancer cells. The results showed that the chosen Coding-PPRH proved to be effective in decreasing tumor volume, and reduced the levels of survivin protein and the formation of blood vessels. These findings represent the preclinical proof of principle of PPRHs as a new silencing tool for cancer gene therapy.

Effects of quercetin on the proliferation of breast cancer cells and expression of survivin in vitro

Quercetin is a hydrophobic agent with potential anticancer activity. The aim of the present study was to observe the effects of quercetin on the proliferation of the breast cancer cell line MCF-7 and the gene expression of survivin. The molecular mechanism underlying the antiproliferative effect of quercetin was also investigated. MCF-7 breast cancer cells were treated with various concentrations of quercetin. The inhibitory effect of quercetin on proliferation was detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and the inhibition rate was calculated. Cellular apoptosis was detected by immunocytochemistry and survivin mRNA expression levels were observed using reverse transcription-polymerase chain reaction (RT-PCR). Western blot analysis was used to analyze changes in the expression levels of survivin protein. Quercetin induced the apoptosis of MCF-7 cells and inhibited the proliferation of the MCF-7 breast cancer cells in a time- and concentration-dependent manner. The mRNA and protein expression levels of survivin were reduced as the concentration of quercetin increased. Quercetin inhibited the growth of MCF-7 cells and promoted apoptosis by inducing G0/ G1 phase arrest. It also regulated the expression of survivin mRNA in MCF-7 cells, which may be the mechanism underlying its antitumor effect.