The effect of antisense Bcl-2 oligonucleotides on Bcl-2 protein expression and apoptosis in human bladder transitional cell carcinoma

Uro-Oncology Research Group, Cancer Research Centre, Queen's University Belfast, Belfast, Northern Ireland, UK.
The Journal of Urology (Impact Factor: 3.75). 10/2001; 166(3):1098-105. DOI: 10.1016/S0022-5347(05)65929-0
Source: PubMed

ABSTRACT Bcl-2 is an important determinant of transitional cell carcinoma of the bladder recurrence and progression as well as a factor in patient response to chemotherapy or radiotherapy. We determined Bcl-2 down-regulation after antisense oligonucleotide therapy and synergism with mitomycin C in transitional cell carcinoma of the bladder.
Bcl-2 protein was quantified using flow cytometry and immunohistochemistry in 4 bladder cancer cell lines, in bladder washings from 6 patients with carcinoma in situ and in 16 patient tumor samples. The synergistic effects of antisense oligonucleotides G3139 and 2009, and mitomycin C were investigated in 4 cell lines, while 2009 down-regulation was examined in 20 tumor explants in an ex vivo model.
Bcl-2 protein expression was found in all 4 cell lines and in 5 of the 6 cell populations derived from patients with carcinoma in situ. Of the 16 tumors 7 were classified positive by frozen section immunohistochemistry and quantitative flow cytometry. G3139 and 2009 down-regulated Bcl-2 protein expression in all 4 cell lines and 2009 down-regulated Bcl-2 protein expression in half of the Bcl-2 positive tumor specimens. There was only evidence in 1 cell line, T24/83, that Bcl-2 protein expression down-regulation enhanced mitomycin C induced apoptotic cell death.
Bcl-2 was expressed in a significant proportion of bladder tumors and in carcinoma in situ. Therefore, antisense oligonucleotides represent a viable strategy for Bcl-2 protein down-regulation. However, it may not always translate into an increased level of mitomycin C induced apoptosis in transitional cell carcinoma of the bladder.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 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. 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. 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. 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.
    Anticancer research 07/2008; 28(4B):2259-63. · 1.87 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Adenoviral gene therapy is an experimental approach to cancer refractory to standard cancer therapies. Adenoviruses can be utilized as vectors to deliver therapeutic transgenes into cancer cells, while gene therapy with oncolytic adenoviruses exploits the lytic potential of viruses to kill tumor cells. Although adenoviruses demonstrate several advantages over other vectors - such as the unparalleled transduction efficacy and natural tropism to a wide range of tissues - the gene transfer efficacy to cancer cells has been limited, consequently restricting the therapeutic effect. There are, however, several approaches to circumvent this problem. We utilized different modified adenoviruses to obtain information on adenovirus tropism towards non-small cell lung cancer (NSCLC) cells. To enhance therapeutic outcome, oncolytic adenoviruses were evaluated. Further, to enhance gene delivery to tumors, we used mesenchymal stem cells (MSCs) as carriers. To improve adenovirus specificity, we investigated whether widely used cyclooxygenase 2 (Cox-2) promoter is induced by adenovirus infection in nontarget cells and whether selectivity can be retained by the 3 untranslated region (UTR) AU-rich elements. In addition, we investigated whether switching adenovirus fiber can retain gene delivery in the presence of neutralizing antibodies. Our results show that adenoviruses, whose capsids were modified with arginine-glycine-aspartatic acid (RGD-4C), the serotype 3 knob, or polylysins displayed enhanced gene transfer into NSCLC cell lines and fresh clinical specimens from patients. The therapeutic efficacy was further improved by using respective oncolytic adenoviruses with isogenic 24bp deletion in the E1A gene. Cox-2 promoter was also shown to be induced in normal and tumor cells following adenovirus infection, but utilization of 3 UTR elements can increase the tumor specificity of the promoter. Further, the results suggested that use of MSCs could enhance the bioavailability and delivery of adenoviruses into human tumors, although cells had no tumor tropism per se. Finally, we demonstrated that changing adenovirus fiber can allow virus to escape from existing neutralizing antibodies when delivered systemically. In conclusion, these results reveal that adenovirus gene transfer and specificity can be increased by using modified adenoviruses and MSCs as carriers, and fiber modifications simultaneously decrease the effect of neutralizing antibodies. This promising data suggest that these approaches could translate into clinical testing in patients with NSCLC refractory to current modalities. Adenovirus geeniterapia on yksi kehitteillä olevista syövän hoitomuodoista. Adenoviruksia voidaan käyttää joko kuljettimina, joiden avulla kohdekudokseen viedään hoitogeenejä tai syöpäsoluja voidaan tuhota hyödyntämällä adenoviruksien jakautumissykliä, minkä seurauksena infektoitu solu tuhoutuu. Vaikka adenoviruksilla on monia etuja verrattuna muihin viruksiin, niiden kyky infektoida syöpäsoluja on rajallinen. Tässä työssä olemme tutkineet eritavoin muokattujen adenoviruksien kykyä infektoida ei pieni soluisen keuhkosyövän solulinjoja ja lisääntyä niissä. Lisäksi olemme tutkineet mahdollisuutta käyttää mesenkymaalisia kantasoluja kuljettimina, jotta virus saataisiin paremmin kohdennettua tuumorikudokseen. Lisätäksemme adenoviruksien spesifisyyttä, olemme selvittäneet aktivoituuko paljon käytetty tuumorispesifinen promoottori, Cox-2 normaalisoluissa adenovirus infektion seurauksena ja voidaanko promoottorin tuumorispesifisyys palauttaa erillisellä 3'UTR säätelytekijällä. Lopuksi tutkimme, voidaanko adenoviruksen geeninsiirtotehokkuus säilyttää myös adenovirukselle spesifisen immuunivasteen vallitessa vaihtamalla toistamiseen injektoivan viruksen fiber erilaiseen kuin ensimmäisellä hoitokerralla annetulla viruksella. Saadut tulokset osoittavat, että muokatut adenovirukset, jotka on kohdennettu esim. integriineihin tai hepariinisulfaattiproteoglykaaneihin viruksen primäärireseptorin asemesta infektoivat tehokkaammin ei pienisoluisen keuhkosyövän soluja ja lisääntyvät niissä. Cox-2 promoottorin todettiin aktivoituvan adenovirus infektiosta, mutta spesifisyys saatiin palautettua 3'UTR säätelytekijän avulla. Lisäksi adenovirukset saatiin mesenkymaalisien kantasolujen avulla kuljetettua tehokkaammin tuumorikudokseen. Osoitimme myös, että adenoviruksen fiberin vaihtaminen injektiokertojen välillä auttaa virusta välttämään ensimmäisellä kerralla nousseen immuunivasteen vaikutuksen. Yhteenvetona voidaan todeta, että adenoviruksien geeninsiirto tehokuutta ja spesifisyyttä voidaan lisätä käyttämällä muokattuja adenoviruksia ja mesenkymaalisia kantasoluja kuljettimina sekä samanaikaisesti vähentää vasta-aineiden vaikutusta adenoviruksien infektointitehokkuuteen.
  • [Show abstract] [Hide abstract]
    ABSTRACT: The components of the apoptotic program are targets for anticancer therapy. Bcl-2 protein inhibits apoptosis and confers resistance to treatment with traditional cytotoxic chemotherapy, radiotherapy, and monoclonal antibodies (mAb). Oblimersen sodium (G3139, Genasense, Genta Inc., Berkeley Heights, NJ) is an antisense oligonucleotide (AS-ON) compound designed to specifically bind to the first 6 codons of the human bcl-2 mRNA sequence, resulting in degradation of bcl-2 mRNA and subsequent decrease in Bcl-2 protein translation. Oblimersen is the first oligonucleotide to demonstrate proof of principle of an antisense effect in human tumors by the documented downregulation of the target Bcl-2 protein. A growing body of preclinical and clinical evidence suggests that oblimersen synergizes with many cytotoxic and biologic/immunotherapeutic agents against a variety of hematologic malignancies and solid tumors. Randomized clinical trials are currently underway to evaluate the efficacy and tolerability of oblimersen in combination with cytotoxic chemotherapy in chronic lymphocytic leukemia, multiple myeloma, malignant melanoma, and non-small cell lung cancer. In addition, nonrandomized trials are under way to evaluate oblimersen in non-Hodgkin's lymphoma, acute myeloid leukemia, and hormone-refractory prostate cancer. Preclinical data also support the clinical evaluation of oblimersen in additional tumor types, including chronic myelogenous leukemia and breast, small cell lung, gastric, colon, bladder, and Merkel cell cancers. Enhancement of the efficacy of anticancer treatments with oblimersen Bcl-2 antisense therapy represents a promising new apoptosis-modulating strategy, and ongoing clinical trials will test this therapeutic approach.
    Antisense and Nucleic Acid Drug Development 07/2002; 12(3):193-213. DOI:10.1089/108729002760220798
Show more