Antisense MDM2 sensitizes prostate cancer cells to androgen deprivation, radiation, and the combination

Abstract

Purpose/Objective: Antisense MDM2 (AS) sensitizes a variety of tumor cell types, including prostate cancer, to radiation and chemotherapy. We have previously described that AS enhances the apoptotic response to androgen deprivation (AD) and that this translates into a reduction in overall cell survival, as measured by clonogenic assay. Since AD+RT is a key strategy for the treatment of men with high risk prostate cancer, AS was tested for the ability to sensitize cells to the combination of AD+RT.Materials/Methods: LNCaP cells were cultured in vitro in either complete medium, charcoal stripped androgen deprived (AD) medium, or AD medium supplemented with the synthetic androgen R1881 (10–10 M) for 2 to 3 days. AS was then administered for 18 to 24 hr before RT was given. Processing of the cells after RT was done at 3 hr for Western blots, 24 and 48 hr for trypan blue dye exclusion, 18 hr for Annexin V staining and flow cytometric analysis, 18 hr for caspases 3+7 quantification by flourometric assay and immediately for clonogenic survival measured 12 to 14 days later. There were 18 treatment groups that were studied: Lipofectin control (LC), AS, antisense mismatch (ASM), AD, AD+R1881, AD+AS, AD+ASM, AD+AS+R1881, AD+ASM+R1881, RT, RT+AS, RT+ASM, RT+AD, RT+AD+R1881, RT+AS+AD, RT+ASM+AD, RT+ASM+AD+R1881, RT+AS+AD+R1881. Statistical comparisons between groups were accomplished with one-way analysis of variance using the Bonferroni test, considering all 18 groups.Results: AS caused a reduction in MDM2 expression and an increase in p53 and p21 expression. Early cell death by trypan blue was found to be reflective of the apoptotic results by Annexin V and Caspase 3+7. AS caused a significant increase in apoptosis over the LC, AD and RT controls. Apoptosis was further increased significantly by the addition of AD or RT to AS. When AS+AD+RT were combined there was a consistent increase in early cell death over AS+AD and AS+RT by all of the assay methods, although this increase was not significant. Overall cell death, as measured by clonogenic assay, revealed synergistic cell killing of AS+RT (2, 4, and 6 Gy) beyond that of ASM+RT and LC (no oligonucleotide) +RT, and AS+RT+AD over ASM+RT+AD, RT+AD, ASM+RT+AD+R1881 and AS+RT+AD+R1881 (See Figs 1A/B).Conclusions: AS sensitizes cells to AD, RT, and AD+RT, and shows promise in the treatment of the full range of patients with prostate cancer. AS has the potential to sensitize the primary tumor to RT and metastasis to AD. View Large Image Figure ViewerDownload (PPT) Purpose/Objective: Antisense MDM2 (AS) sensitizes a variety of tumor cell types, including prostate cancer, to radiation and chemotherapy. We have previously described that AS enhances the apoptotic response to androgen deprivation (AD) and that this translates into a reduction in overall cell survival, as measured by clonogenic assay. Since AD+RT is a key strategy for the treatment of men with high risk prostate cancer, AS was tested for the ability to sensitize cells to the combination of AD+RT. Materials/Methods: LNCaP cells were cultured in vitro in either complete medium, charcoal stripped androgen deprived (AD) medium, or AD medium supplemented with the synthetic androgen R1881 (10–10 M) for 2 to 3 days. AS was then administered for 18 to 24 hr before RT was given. Processing of the cells after RT was done at 3 hr for Western blots, 24 and 48 hr for trypan blue dye exclusion, 18 hr for Annexin V staining and flow cytometric analysis, 18 hr for caspases 3+7 quantification by flourometric assay and immediately for clonogenic survival measured 12 to 14 days later. There were 18 treatment groups that were studied: Lipofectin control (LC), AS, antisense mismatch (ASM), AD, AD+R1881, AD+AS, AD+ASM, AD+AS+R1881, AD+ASM+R1881, RT, RT+AS, RT+ASM, RT+AD, RT+AD+R1881, RT+AS+AD, RT+ASM+AD, RT+ASM+AD+R1881, RT+AS+AD+R1881. Statistical comparisons between groups were accomplished with one-way analysis of variance using the Bonferroni test, considering all 18 groups. Results: AS caused a reduction in MDM2 expression and an increase in p53 and p21 expression. Early cell death by trypan blue was found to be reflective of the apoptotic results by Annexin V and Caspase 3+7. AS caused a significant increase in apoptosis over the LC, AD and RT controls. Apoptosis was further increased significantly by the addition of AD or RT to AS. When AS+AD+RT were combined there was a consistent increase in early cell death over AS+AD and AS+RT by all of the assay methods, although this increase was not significant. Overall cell death, as measured by clonogenic assay, revealed synergistic cell killing of AS+RT (2, 4, and 6 Gy) beyond that of ASM+RT and LC (no oligonucleotide) +RT, and AS+RT+AD over ASM+RT+AD, RT+AD, ASM+RT+AD+R1881 and AS+RT+AD+R1881 (See Figs 1A/B). Conclusions: AS sensitizes cells to AD, RT, and AD+RT, and shows promise in the treatment of the full range of patients with prostate cancer. AS has the potential to sensitize the primary tumor to RT and metastasis to AD.