Enhanced inhibition of bladder cancer cell growth by simultaneous knockdown of antiapoptotic Bcl-xL and survivin in combination with chemotherapy.

The overexpression of antiapoptotic genes, such as Bcl-xL and survivin, contributes to the increased survival of tumor cells and to the development of treatment resistances. In the bladder cancer cell lines EJ28 and J82, the siRNA-mediated knockdown of survivin reduces cell proliferation and the inhibition of Bcl-xL sensitizes these cells towards subsequent chemotherapy with mitomycin C and cisplatin. Therefore, the aim of this study was to analyze if the simultaneous knockdown of Bcl-xL and survivin might represent a more powerful treatment option for bladder cancer than the single inhibition of one of these target genes. At 96 h after transfection, reduction in cell viability was stronger after simultaneous inhibition of Bcl-xL and survivin (decrease of 40%-48%) in comparison to the single target treatments (decrease of 29% at best). Furthermore, simultaneous knockdown of Bcl-xL and survivin considerably increased the efficacy of subsequent chemotherapy. For example, cellular viability of EJ28 cells decreased to 6% in consequence of Bcl-xL and survivin inhibition plus cisplatin treatment whereas single target siRNA plus chemotherapy treatments mediated reductions down to 15%-36% only. In conclusion, the combination of simultaneous siRNA-mediated knockdown of antiapoptotic Bcl-xL and survivin-a multitarget molecular-based therapy-and conventional chemotherapy shows great potential for improving bladder cancer treatment.

Antisense-mediated inhibition of survivin, hTERT and VEGF in bladder cancer cells in vitro and in vivo.

Since cancer cells are characterised by multiple genetic alterations the single inhibition of one tumour- associated gene might not be sufficient as a therapeutic strategy. We examined the effects of a combined inhibition of survivin, human telomerase reverse transcriptase (hTERT) and vascular endothelial growth factor (VEGF) with antisense oligodeoxynucleotides (AS-ODNs) and small interfering RNAs (siRNAs) in EJ28 and 5637 bladder cancer (BCa) cells. Following verification of the uptake of intraperitoneally applied fluorescence-labelled AS-ODNs and siRNAs in subcutaneous BCa xenografts, the target-directed constructs were tested as single agents in SCID mice bearing subcutaneous EJ28. Simultaneous inhibition of two of the selected transcripts significantly enhanced cell viability reduction compared to the controls consisting of a target directed construct and an appropriate control construct without any homology to the human genome. The uptake of both antisense inhibitor types in the subcutaneous BCa was achieved even without a carrier. In vivo studies with 9 consecutive intraperitoneal injections with 20 mg/kg AS-ODNs or 4.6 mg/kg siRNAs revealed the biocompatibility of both antisense inhibitor types and showed anti-tumoural activity of the AS-ODNs used.

Multitarget siRNA inhibition of antiapoptotic genes (XIAP, BCL2, BCL-X(L)) in bladder cancer cells.

BACKGROUND: The knockdown of XIAP, BCL2 and BCL-X(L) by siRNAs represents a promising treatment option for bladder cancer (BCa) since the overexpression of antiapoptotic genes is often associated with tumor progression and treatment resistance. MATERIALS AND METHODS: EJ28 BCa cells were transfected with siRNAs--separately and combined--followed by analysis of target expression, viability, clonogenic survival, apoptosis and cell cycle. Furthermore, a possible chemosensitization by siRNA pretreatment was investigated. RESULTS: The siRNA-mediated inhibition of these targets--either separately or combined--reduced the targets' expression, reduced cell growth and sensitized cells to a subsequent chemotherapy. CONCLUSION: Since tumor cells may bypass the inhibition of a single gene by changing their expression profile, e.g. switch from BCL2 to BCL-X(L), the combined knockdown of multiple genes of the same pathway might be more effective in killing cancer cells. The siRNAs used represent appropriate tools for this aim since they reduced their targets' expression significantly and long-lastingly.

Functional analyses of C13orf19/P38IP in prostate cell lines.

Human C13orf19 was previously identified to be downregulated in prostate cancer (PCa) but its function is unknown to date. In the present study, C13orf19 mRNA expression was inhibited by siRNA transfection. Furthermore, a possible regulation by androgens and the previously postulated interaction with p38 MAP kinase (p38MAPK) was investigated. The siRNA-mediated downregulation of the C13orf19 mRNA expression in the prostate cell lines PC-3 and BPH-1 was examined by quantitative PCR. Cellular viability, apoptosis, cell cycle distribution and clonogenic survival were investigated. In addition, the effects of C13orf19 downregulation in combination with chemotherapy on overall cell survival were studied. The inhibition of C13orf19 mRNA expression to 12% (after 12 h) and 55% (after 96 h) in PC-3 cells attested to a strong and persistent molecular effect provoked by the siRNA-D5 construct. However, no obvious effects on doubling time and cellular morphology were observed. Cell cycle distribution, clonogenic survival, apoptosis and cell viability showed no alterations, even after combining siRNA transfection with chemotherapy. Therefore, it can be concluded that the reduced expression of C13orf19 in PCa is not involved in the malignant transformation of the cells. A possible androgen dependence of C13orf19 mRNA expression was investigated by treating LNCaP cells with the androgen R1881 and in combination with the antiandrogen, bicalutamide. C13orf19 is expressed independently of the androgen. To analyze the putative interaction between C13orf19 and p38MAPK, PC-3 and BPH-1 cells were treated with the p38MAPK inhibitor, SB203580, and C13orf19 mRNA expression was examined. Additionally, the expression and phosphorylation status of p38MAPK after the inhibition of C13orf19 was investigated by Western blotting. No interaction between C13orf19 and p38MAPK was identified. Therefore, the gene should forthwith be named C13orf19 or Fam48A and not P38IP.

Carbon nanofibers and carbon nanotubes sensitize prostate and bladder cancer cells to platinum-based chemotherapeutics.

Recent data suggest that carbon nanomaterials can act as antitumor agents themselves by increasing the efficiency of cytotoxic agents when applied in combination. Here, carbon nanofibers (CNFs) and multi-walled carbon nanotubes (CNTs) were investigated regarding their impact on cellular function, cellular uptake and ability to sensitize cancer cells of urological origin to the conventional chemotherapeutics cisplatin and carboplatin. CNFs and CNTs (1-200 microg/ml) showed a low to moderate impairment of cellular function with CNFs being more deleterious than CNTs. Inhibition of cellular viability by the nanomaterials was about 20% at most. In combinatory treatments, CNFs and CNTs markedly enhanced the effects of cisplatin and carboplatin on cellular viability by 1.2- to 2.8-fold in prostate, bladder and cisplatin-resistant prostate cancer cells in comparison to the individual effects of the chemotherapeutics. Particularly the cell viability-diminishing effect of CNFs alone and in combination with the chemotherapeutics was more pronounced with dispersions prepared with human serum albumin than with phospholipid-polyethylene glycol. Albumin might mediate the cellular uptake of carbon nanomaterials which was underlined by the co-localization of albumin and carbon nanomaterials along the cellular surface as evidenced by fluorescence microscopy. Transmission electron microscopy revealed that both carbon nanomaterials were internalized by cancer cells, thereby possibly leading to an enhanced accumulation of the chemotherapeutic drugs. In fact, CNFs enhanced the cellular accumulation of carboplatin by 28% as compared to the single treatment with carboplatin. In conclusion, carbon nanomaterial-based applications could present a new strategy to overcome chemoresistance by sensitizing cancer cells to conventional chemotherapeutics.

siRNA-mediated inhibition of antiapoptotic genes enhances chemotherapy efficacy in bladder cancer cells.

BACKGROUND: The up-regulation of antiapoptotic B-cell CLL/lymphoma 2 (BCL2), BCL2-like 1 (BCLXL), X-linked inhibitor of apoptosis (XIAP) and survivin is one mechanism by which cancer cells develop resistance towards chemotherapeutics. Therefore, the knockdown of these four genes could sensitise bladder cancer (BCa) cells towards chemotherapy. MATERIALS AND METHODS: BCL2, BCLXL, XIAP and survivin were inhibited using siRNAs--either one target-alone or all four targets simultaneously--in EJ28 and J82 BCa cells. After 24 h, cells were treated with mitomycin C or cisplatin. Treatment effects were analysed regarding cell viability, cell count and apoptosis induction. RESULTS: Knockdown of BCLXL and survivin, as well as the simultaneous inhibition of all four antiapoptotic genes, sensitised EJ28 and J82 cells towards mitomycin C and cisplatin. CONCLUSION: Since the contribution of one antiapoptotic gene to chemotherapy response can vary between BCa cell lines, the simultaneous knockdown of multiple inhibitors of apoptosis might represent a more promising option for enhancing chemotherapy efficacy in BCa treatment.

Simultaneous siRNA-mediated knockdown of antiapoptotic BCL2, Bcl-xL, XIAP and survivin in bladder cancer cells.

Bladder cancer (BCa) represents the ninth most common malignancy worldwide. Despite intensive treatment with surgery and chemotherapy the prognosis for BCa patients particularly at advanced stages is poor. The ability to evade apoptosis is a hallmark of cancer cells. Since the antiapoptotic genes BCL2, Bcl-xL, XIAP and survivin are frequently upregulated in BCa tissues, their combined siRNA-mediated knockdown might be more potent in decreasing BCa growth than the single inhibition of one target. Against each target two siRNAs were selected that specifically reduced the mRNA and protein levels of their appropriate target in EJ28 and J82 BCa cells. Inhibition of survivin provoked the strongest antiproliferative effect of all single target treatments, for example cell counts decreased by 50%. Simultaneous targeting of all four antiapoptotic genes downregulated expression levels of all targets and mediated significant reductions in cell viability and cell counts as well as induction of apoptosis. In EJ28 cells, combined knockdown of BCL2, Bcl-xL, XIAP and survivin caused a 2.5-fold enhancement in apoptosis rate and reduced cellular viability by 40%. Therefore, simultaneous knockdown of antiapoptotic BCL2, Bcl­xL, XIAP and survivin may represent a promising treatment option for bladder cancer.

Carbon nanotubes filled with a chemotherapeutic agent: a nanocarrier mediates inhibition of tumor cell growth.

AIM: In this paper, carbon nanotubes (CNTs) are presented as feasible carriers for carboplatin, a therapeutic agent for cancer treatment. The drug was introduced into CNTs to demonstrate that they are suited as nanocontainers and nanocarriers and can release the drug to initialize its medical virtue. METHOD: The filling was accomplished by a wet-chemical approach after the CNTs were opened. The effect on cell proliferation and cytotoxicity of the carboplatin-filled CNT was investigated by using a viability assays. RESULTS: Using different analysis methods such as electron energy loss spectroscopy and x-ray photoelectron spectroscopy the structure of carboplatin incorporated into the CNTs was found to be retained. In vitro studies showed that carboplatin-filled CNTs inhibited growth of bladder cancer cells whereas unfilled, opened CNTs barely affected cancer cell growth. CONCLUSION: A reversible filling-emptying process could be performed successfully within this work. This highlights the potential of CNTs for applications in the field of drug delivery.

Synthetic nucleic acids as potential therapeutic tools for treatment of bladder carcinoma.

OBJECTIVES: Abnormal gene activation in human tumours including bladder cancers (bCAs) may cause altered proliferation, maturation, and apoptosis as well as development of resistance to therapeutic interventions. Therefore, silencing of abnormally activated genes appears to be a rational approach for specific target-directed and sensitising therapies. METHODS: Of the available strategies for gene silencing, antisense-based techniques have attracted much attention and are the focus of this review. Putative target genes should be involved in essential tumour-promoting pathways, such as growth signalling, immortalisation, cell cycle regulation, apoptosis, angiogenesis, and development of therapy resistances. This review gives an overview of selected studies performed on bCA-derived cell lines and xenografts reporting down-regulation of potential target genes by antisense-based synthetic nucleic acids such as antisense oligodeoxynucleotides (AS-ODNs) and small interfering RNAs (siRNAs). Effects on proliferation of bCA cells and enhancement of the cytotoxic action of different chemotherapeutics were evaluated. RESULTS: Knock-down of the selected target genes frequently caused an impairment of growth of different bCA cell lines originating from cell cycle arrest or increased apoptosis. In numerous studies, the pretreatment with AS-ODNs or siRNAs provoked strong enhancement of subsequent chemotherapies, emphasising the effectiveness of these inhibition approaches. CONCLUSIONS: The application of antisense-based inhibitors in combination with chemotherapeutics might represent an alternative strategy for the adjuvant treatment of superficial bCA. Nevertheless, translation of this technology to the clinic might be hampered by inestimable off-target effects caused by AS-ODNs and their behaviour after intravesical instillation has to be evaluated in preclinical and clinical trials.