The Use of Antisense Oligonucleotides in Evaluating Survivin as a Therapeutic Target for Radiation Sensitization in Lung Cancer

Elucidating the mechanism of over and under expression of proteins is critical in developing a better understanding of cancer. Multiple techniques are used to examine differential expression of proteins in cells and assess changes in protein expression in response to therapies such as radiation. Reduced expression can be caused by protein inactivation, mRNA instability, or reduced transcription. The following protocol was used to determine the mechanism for the reduced expression of an antiapoptotic factor, survivin, in normal tissues in response to radiation and the defect in cancer cells that prevents this reduction. We also examined ways to overcome survivin over expression in cancer cells in order to sensitize them to radiation. We will focus on the use of antisense oligonucleotides, cell cycle analysis, and luciferase reporter genes

Endothelium Derived Nitric Oxide Synthase Negatively Regulates the PDGF-Survivin Pathway during Flow-Dependent Vascular Remodeling

Chronic alterations in blood flow initiate structural changes in vessel lumen caliber to normalize shear stress. The loss of endothelial derived nitric oxide synthase (eNOS) in mice promotes abnormal flow dependent vascular remodeling, thus uncoupling mechanotransduction from adaptive vascular remodeling. However, the mechanisms of how the loss of eNOS promotes abnormal remodeling are not known. Here we show that abnormal flow-dependent remodeling in eNOS knockout mice (eNOS (−/−)) is associated with activation of the platelet derived growth factor (PDGF) signaling pathway leading to the induction of the inhibitor of apoptosis, survivin. Interfering with PDGF signaling or survivin function corrects the abnormal remodeling seen in eNOS (−/−) mice. Moreover, nitric oxide (NO) negatively regulates PDGF driven survivin expression and cellular proliferation in cultured vascular smooth muscle cells. Collectively, our data suggests that eNOS negatively regulates the PDGF-survivin axis to maintain proportional flow-dependent luminal remodeling and vascular quiescence

Characterisation of the anti-apoptotic function of survivin-ΔEx3 during TNFα−mediated cell death

Survivin is an oncogenic protein involved in cell division and acts as an anti-apoptotic factor. It is highly expressed in most cancers and is associated with chemotherapy resistance, increased tumour recurrence, and shorter patient survival. This makes anti-survivin therapy an attractive cancer treatment strategy. These functions are mediated by several survivin spliced variants, whose expression may correlate with cancer progression. One of the spliced variants, survivin-ΔEx3, is known to inhibit apoptosis, through undefined mechanisms. Here, we characterised these mechanisms upon TNFα−mediated apoptosis, and showed that survivin-ΔEx3 acts as an adaptor, allowing the formation of a complex between Bcl-2 and activated caspase-3. The Bcl-2/survivin-ΔEx3 complex, but not survivin-ΔEx3 itself, inhibits the activity of caspase-3. Bcl-2 is therefore linked to the postmitochondrial apoptotic machinery by survivin-ΔEx3. Thus, survivin-ΔEx3 plays a key role in the inhibition of caspase-3 activity, and in the control of the mitochondrial checkpoint of apoptosis. This study suggests that targeting survivin-ΔEx3, rather than survivin alone, could be relevant for treating human cancers

The antiapoptotic gene survivin is highly expressed in human chondrosarcoma and promotes drug resistance in chondrosarcoma cells in vitro

Background Chondrosarcoma is virtually resistant to chemotherapy and radiation therapy. Survivin, the smallest member of the inhibitor of apoptosis protein family, is a critical factor for tumor progression and resistance to conventional therapeutic approaches in a wide range of malignancies. However, the role of survivin in chondrosarcoma has not been well studied. We examined the importance of survivin gene expression in chondrosarcoma and analysed its influences on proliferation, apoptosis and resistance to chemotherapy in vitro. Methods Resected chondrosarcoma specimens from which paraffin-embedded tissues could be extracted were available from 12 patients. In vitro experiments were performed in human chondrosarcoma cell lines SW1353 and Hs819.T. Immunohistochemistry, immunoblot, quantitative PCR, RNA interference, gene-overexpression and analyses of cell proliferation and apoptosis were performed. Results Expression of survivin protein was detected in all chondrosarcoma specimens analyzed, while undetectable in adult human cartilage. RNA interference targeting survivin resulted in a G2/M-arrest of the cell cycle and led to increased rates of apoptosis in chondrosarcoma cells in vitro. Overexpression of survivin resulted in pronounced resistance to doxorubicin treatment. Conclusions These findings indicate that survivin plays a role in the pathogenesis and pronounced chemoresistance of high grade chondrosarcoma. Survivin antagonizing therapeutic strategies may lead to new treatment options in unresectable and metastasized chondrosarcoma

Association of polymorphisms in survivin gene with the risk of hepatocellular carcinoma in Chinese han population: a case control study

<p>Abstract</p> <p>Background</p> <p>Survivin, one of the strongest apoptosis inhibitors, plays a critical role in the development and progression of hepatocellular carcinoma (HCC). By comparison, relatively little is known about the effect of <it>survivin </it>gene polymorphisms on HCC susceptibility. Our study aimed to investigate the association of <it>survivin </it>gene polymorphisms with the risk of HCC in Chinese han population.</p> <p>Methods</p> <p>A case-control study was conducted in Chinese han population consisting of 178 HCC cases and 196 cancer-free controls. Information on demographic data and related risk factors was collected for all subjects. Polymorphisms of the <it>survivin </it>gene, including three loci of rs8073069, rs9904341 and rs1042489, were selected and genotyped by a polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) technique. Association analysis of genotypes/alleles and haplotypes from these loci with the risk of HCC was conducted under different genetic models.</p> <p>Results</p> <p>Using univariate analysis of rs8073069, rs9904341 and rs1042489 under different genetic models, no statistically significant difference was found in genotype or allele distribution of HCC cases relative to the controls (<it>P </it>> 0.05). Linkage disequilibrium (LD) analysis showed that these loci were in LD. Multivariate logistic regression indicated that with no G-C-T haplotype as reference, the haplotype of G-C-T from these loci was associated with a lower risk for HCC under the recessive model (<it>OR = </it>0.46, 95% confidence interval (<it>CI</it>): 0.24~0.90, <it>P </it>= 0.023). Both HBsAg+ and the medical history of viral hepatitis type B were risk factors for HCC. However, no statistically significant haplotype-environment interaction existed.</p> <p>Conclusions</p> <p>No association between rs8073069, rs9904341 or rs1042489 in <it>survivin </it>gene and the risk of HCC is found in Chinese han population, but rs8073069G-rs9904341C- rs1042489T is perhaps a protective haplotype for HCC.</p

Survivin gene silencing sensitizes prostate cancer cells to selenium growth inhibition

<p>Abstract</p> <p>Background</p> <p>Prostate cancer is a leading cause of cancer-related death in men worldwide. Survivin is a member of the inhibitor of apoptosis (IAP) protein family that is expressed in the majority of human tumors including prostate cancer, but is barely detectable in terminally differentiated normal cells. Downregulation of survivin could sensitize prostate cancer cells to chemotherapeutic agents <it>in vitro </it>and <it>in vivo</it>. Selenium is an essential trace element. Several studies have shown that selenium compounds inhibit the growth of prostate cancer cells. The objective of this study is to investigate whether survivin gene silencing in conjunction with selenium treatment could enhance the therapeutic efficacy for prostate cancer and to elucidate the underlying mechanisms.</p> <p>Methods</p> <p>Expression of survivin was analyzed in a collection of normal and malignant prostatic tissues by immunohistochemical staining. <it>In vitro </it>studies were conducted in PC-3M, C4-2B, and 22Rv1 prostate cancer cells. The effect of selenium on survivin expression was analyzed by Western blotting and semi-quantitative RT-PCR. Survivin gene knockdown was carried out by transfecting cells with a short hairpin RNA (shRNA) designed against survivin. Cell proliferation was quantitated by the 3-(4,5-Dimethylthiazol-2-yl)- 2,5-Diphenyltetrazolium Bromide (MTT) assay and apoptosis by propidium iodide staining followed by flow cytometry analysis. Finally, <it>in vivo </it>tumor growth assay was performed by establishing PC-3M xenograft in nude mice and monitoring tumor growth following transfection and treatment.</p> <p>Results</p> <p>We found that survivin was undetectable in normal prostatic tissues but was highly expressed in prostate cancers. Survivin knockdown or selenium treatment inhibited the growth of prostate cancer cells, but the selenium effect was modest. In contrast to what have been observed in other cell lines, selenium treatment had little or no effect on survivin expression in several androgen-independent prostate cancer cell lines. Survivin knockdown sensitized these cells to selenium growth inhibition and apoptosis induction. In nude mice bearing PC-3M xenografts, survivin knockdown synergizes with selenium in inhibiting tumor growth.</p> <p>Conclusions</p> <p>Selenium could inhibit the growth of hormone-refractory prostate cancer cells both <it>in vitro </it>and <it>in vivo</it>, but the effects were modest. The growth inhibition was not mediated by downregulating survivin expression. Survivin silencing greatly enhanced the growth inhibitory effects of selenium.</p

Macroscopic transport by synthetic molecular machines

Nature uses molecular motors and machines in virtually every significant biological process, but demonstrating that simpler artificial structures operating through the same gross mechanisms can be interfaced with—and perform physical tasks in—the macroscopic world represents a significant hurdle for molecular nanotechnology. Here we describe a wholly synthetic molecular system that converts an external energy source (light) into biased brownian motion to transport a macroscopic cargo and do measurable work. The millimetre-scale directional transport of a liquid on a surface is achieved by using the biased brownian motion of stimuli-responsive rotaxanes (‘molecular shuttles’) to expose or conceal fluoroalkane residues and thereby modify surface tension. The collective operation of a monolayer of the molecular shuttles is sufficient to power the movement of a microlitre droplet of diiodomethane up a twelve-degree incline.

Both Strands of siRNA Have Potential to Guide Posttranscriptional Gene Silencing in Mammalian Cells

Despite the widespread application of RNA interference (RNAi) as a research tool for diverse purposes, the key step of strand selection of siRNAs during the formation of RNA-induced silencing complex (RISC) remains poorly understood. Here, using siRNAs targeted to the complementary region of Survivin and the effector protease receptor 1 (EPR-1), we show that both strands of the siRNA duplex can find their target mRNA and are equally eligible for assembly into Argonaute 2 (Ago2) of RISC in HEK293 cells. Transfection of the synthetic siRNA duplexes with different thermodynamic profiles or short hairpin RNA (shRNA) vectors that generate double-stranded RNAs (dsRNAs), permitting processing specifically from either the 5′ or 3′ end of the incipient siRNA, results in the degradation of the respective target mRNAs of either strand of the siRNA duplex with comparable efficiencies. Thus, while most RNAi reactions may follow the thermodynamic asymmetry rule in strand selection, our study suggests an exceptional mode for certain siRNAs in which both strands of the duplex are competent in sponsoring RNAi, and implies additional factors that might dictate the RNAi targets

Survivin, Survivin-2B, and Survivin-deItaEx3 expression in medulloblastoma: biologic markers of tumour morphology and clinical outcome

Survivin is an apoptotic inhibitor that is expressed at high levels in a variety of malignancies. Survivin has four known alternative splice forms (Survivin, Survivin-2B, Survivin-deltaEx3, and Survivin-3B), and the recent literature suggests that these splice variants have unique functions and subcellular localisation patterns. We evaluated 19 fresh-frozen paediatric medulloblastomas for the expression of three Survivin isoforms by quantitative PCR. Survivin was most highly expressed when compared with normal cerebellar tissue. We also investigated Survivin protein expression in 40 paraffin-embedded paediatric medulloblastoma tumours by immunohistochemistry. We found a statistically significant association between the percentage of Survivin-positive cells and histologic subtype, with the large-cell-anaplastic variant expressing Survivin at higher levels than the classic subtype. We also found a statistically significant relationship between the percent of Survivin-positive cells in the tumours and clinical outcome, with higher levels of Survivin correlating with a worse prognosis. In summary, our study demonstrates a role for Survivin as a marker of tumour morphology and clinical outcome in medulloblastoma. Survivin may be a promising future prognostic tool and potential biologic target in this malignancy