Bcl-xL antisense oligonucleotide and cisplatin combination therapy extends survival in SCID mice with established mesothelioma xenografts.

Bcl-xL functions as a dominant regulator of apoptotic cell death and is implicated in chemotherapeutic resistance of malignant pleural mesothelioma (MPM). Mesothelioma cell lines demonstrate increasing levels of Bcl-xL as resistant clones are selected in vitro. Moreover, upon introduction of antisense oligonucleotides specific to Bcl-xL mRNA, MPM cells are sensitized to chemotherapeutic agents. Here we describe the therapeutic effects of a novel combination therapy, Bcl-xL antisense oligonucleotide (ASO 15999) and cisplatin, on mesothelioma cell lines in vitro and in vivo; in addition, efficacy of ASO 15999 in decreasing tumor load as well as its effect on survival in an animal model. Finally, we initiated preliminary toxicity studies involved with intraperitoneal (IP) injections of ASO 15999 into mice. This novel combination, with doses of cisplatin four times below established IC(50) levels, significantly decreased viability of MPM cell lines after 48 hr. The growth of established mouse flank human tumor xenografts was reduced with intra-tumor administration of ASO 15999. Local spread and development of IP xenografts was reduced with treatments of ASO alone, and survival of mice afflicted with these xenografts was prolonged after administration of ASO alone and ASO 15999 + cisplatin combination therapy. These findings suggest that ASO 15999 sensitizes MPM cell lines to the toxic effects of cisplatin. ASO 15999 induced reduction of Bcl-xL is effective in slowing the progression of human mesothelioma cell lines both in vitro and in vivo. More notably, the combination of Bcl-xL ASO and cisplatin extends survival in an orthotopic tumor xenograft model.

Histopathological diagnosis of endobronchial endometriosis treated with argon laser.

The authors present a case of endobronchial endometriosis with catamenial haemoptysis. The lesion was diagnosed as endobronchial endometriosis based on histopathological examination of a bronchial biopsy from the right second carina. Fibreoptic bronchoscopic examination revealed a tiny hyperaemic submucosal area with bleeding and a brown-coloured diverticulum at bottom of this lesion encompassing a 2-cm2 area at the right second carina. Multiplanar reconstructions of a spiral CT scan revealed a 0.5-cm lesion that looked like a diverticulum at the right second carina. The patient was treated with argon laser at bronchoscopy. Following treatment, the patient has been asymptomatic with no recurrence of haemoptysis.

Gene therapy for sarcoma utilizing adenoviral transfer of the beta-glucuronidase and bax genes and an anthracyline prodrug.

BACKGROUND: When acted on by beta-glucuronidase (BG), HMR1826 is metabolized to doxorubicin. Use of this prodrug with adenoviral transfer of beta-glucuronidase (AdBG) is limited by the drug's inability to enter cells and intracellular retention of BG after transduction. We evaluated a system combining AdBG, transfer of the proapoptotic gene bax (AdBax) at a low multiplicity of infection, and HMR1826 administration. MATERIALS AND METHODS: Fibrosarcoma cells were treated with AdBG alone, AdBG plus HMR1826, AdBG followed by beta-galactosidase (AdLacZ) plus HMR1826, and AdBG followed by AdBax with no prodrug. In the experimental group, cells were transfected with AdBG, followed by AdBax plus HMR1826. Viability was measured 24 h after transfection and prodrug administration. Western blots for BG were performed on cell lysates and supernatants. RESULTS: Minimal cellular killing was noted in the AdBG alone, AdBG plus HMR 1826, or AdBG:AdLacZ plus HMR 1826 groups, and Western blot did not demonstrate BG in the supernatant even though all AdBG-transfected cell lysates were positive. Cell killing was noted in the AdBG:AdBax group, but less than in the AdBG:AdBax plus HMR 1826 group (without prodrug versus with prodrug: 1:1 to 55.5% versus 75.9%, 5:1 to 10.0% versus 75.9%, 10:1 7.6% versus 49.0%, 20:1 4.6% versus 24.9%, P = 0.037). Western blot demonstrated BG in the supernatant of the AdBG:AdBax groups. CONCLUSIONS: We have devised a novel enzyme prodrug method of killing tumor cells and engendering a bystander effect. AdBax leads to BG release from dying cells after AdBG transduction and conversion of HMR1826 to an active anthracycline.

Antisense oligonucleotides directed at the bcl-xl gene product augment chemotherapy response in mesothelioma.

OBJECTIVE: Malignant pleural mesothelioma (MPM) is resistant to both conventional chemotherapy and apoptosis. The bcl-2 family proteins are major determinants of apoptotic homeostasis. MPM lines and tumors routinely overexpress the anti-apoptotic protein BCL-XL. We have previously shown that antisense inhibition of BCL-XL in MPM cells leads to apoptosis. We sought to determine whether antisense oligonucleotides directed at the bcl-xl gene product would augment response to a conventional chemotherapeutic agent in human mesothelioma cell lines. METHODS: The human MPM cell lines REN and I-45 were exposed to two bcl-xl antisense oligonucleotides (15999, 16009) and one sense oligonucleotide (113529) construct at varying doses, followed by IC(50) cisplatin. Cellular viability was assessed by a calorimetric assay, and apoptosis was evaluated by Hoechst staining, Annexin V staining, and sub-G(1) fluorescence-activated cell sorter analysis. Western blot analysis of BCL-2 family proteins was performed following single agent and combined treatment. Isobologram mathematical analysis was used to determine whether or not combination therapies were additive or synergistic. RESULTS: Cell viability was most affected with the 15999 antisense oligonucleotides plus IC(50) cisplatin combination (70% of I-45 and 90% of REN cells killed), and apoptosis was markedly increased with this combination by all measures. Western blot demonstrated 15999 antisense oligonucleotides construct down-regulation of BCL-XL, but no further effect on expression of BCL-2 proteins with cisplatin. Isobologram analysis demonstrated 15999 + cisplatin synergistic effect. CONCLUSIONS: Exposure of human MPM cells to bcl-xl antisense oligonucleotides sensitizes human mesothelioma cells to the conventional chemotherapeutic agent cisplatin. Similar approaches using a combination of molecular and conventional treatment may have clinical utility for this tumor.

Phosphatase and tensin analog gene overexpression engenders cellular death in human malignant mesothelioma cells via inhibition of AKT phosphorylation.

BACKGROUND: Abnormal phosphatase and tensin analog (PTEN) gene expression has been noted in neoplasms. The PTEN protein cleaves phosphate groups from cellular growth kinases, inhibiting tumor propagation. A downstream target of PTEN is AKT, a serine-threonine kinase that when activated inhibits apoptosis. We sought to determine whether PTEN overexpression in mesothelioma cells would engender hypophosphorylation of AKT and apoptosis. METHODS: Human malignant mesothelioma cell lines REN and I-45 were transfected with adenoviral vectors AdPTEN and AdBgal (marker gene) at various multiplicities of infection (MOI). Cell viability was measured using a colorimetric assay, and apoptosis was assessed by morphology and subG1 fluorescence-activated cell sorter (FACS) analysis. PTEN protein and AKT phophorylation were evaluated by Western blot, and AKT kinase activity was evaluated by functional assay. RESULTS: Increased cellular killing was noted with AdPTEN gene transfer. The ratio of cell killing with AdPTEN to AdBgal widened with increasing MOI and was statistically significant at all MOI. Cells demonstrated apoptosis by morphologic and subG1 FACS analyses. Cells overexpressing PTEN demonstrated decreased phosphorylated (active) AKT and AKT kinase activity compared with controls. CONCLUSIONS: Overexpression of PTEN engenders apoptosis in mesothelioma by AKT hypophosphorylation. The forced overexpression of PTEN may prove useful clinically in this treatment-resistant neoplasm.

Adenoviral transfer of mda-7 leads to BAX up-regulation and apoptosis in mesothelioma cells, and is abrogated by over-expression of BCL-XL.

BACKGROUND: Malignant pleural mesothelioma (MPM) is unresponsive to conventional therapies. Forced expression of the novel tumor suppressor mda-7 gene in other cell types has resulted in decreased growth and apoptosis. We evaluated cell growth, apoptosis and tumor suppressor characteristics following forced expression of this gene in mesothelioma cell lines. METHODS: MDA-7 expression in human MPM cells at baseline, following pharmacologic differentiation and viral mda-7 transduction (Ad-mda7) were evaluated with Western blot. Cell viability was evaluated with a colorimetric (XTT) assay, and apoptosis with subG1 FACS and Hoescht. Caspase-3 expression was evaluated by functional assay. These parameters were also evaluated in a stable bcl-xl hyper-expressing MPM cell line. Bax mRNA levels were evaluated with real-time PCR. RESULTS: No baseline or differentiated MPM MDA7 expression was found, but was noted following Ad-mda7 exposure. More than 50% of MPM cells were killed at 5 days following Ad-mda7 exposure (p < 0.001). Apoptosis was accompanied by caspase-3 cleavage and increased BAX expression at both the protein (translational) and mRNA (transcriptional) level. These findings were reduced in a bcl-xl hyper-expressing cell line (P < 0.01). CONCLUSIONS: Although mda-7 does not appear to be a MPM suppressor gene, adenoviral-mediated expression in cell lines induces apoptotic cellular death related to BAX upregulation and caspase cleavage. This is supported by abrogation of effect in a bcl-xl hyper-expressing cell line.