Antisense MDM2 sensitizes prostate cancer cells to androgen deprivation, radiation, and the combination.
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. Because AD + radiation (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.


METHODS
LNCaP cells were cultured in vitro in either complete, androgen deprived (AD), or AD+R1881 (synthetic androgen) medium for 2-3 days before AS was administered. Radiation at 5 Gy was given 18-24 h later. Processing of the cells after RT was done at 3 h for Western blots, 24 and 48 h for trypan blue dye exclusion, 18 h for Annexin V staining by flow cytometric analysis, 18 h for Caspase 3+7 quantification by fluorometric assay, and immediately for clonogenic survival measured 12-14 days later. There were 18 treatment groups that were studied: lipofectin control, AS, antisense mismatch (ASM), AD, AD+R1881, and RT in all possible combinations. 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 lipofectin control, AD, and RT controls. Apoptosis was further increased significantly by the addition of AD or RT to AS. When AS, AD, and 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 measured by clonogenic assay revealed synergistic cell killing of AS+RT beyond that of ASM+RT and RT alone, and AS+RT+AD beyond that of AS+RT, AS+RT+AD+R1881, ASM+RT+AD, and ASM+RT+AD+R1881.


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 AD+RT and metastasis to AD.
Mu
Zhaomei
Z
Department of Radiation Oncology, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111, USA.
Hachem
Paul
P
Agrawal
Sudhir
S
Pollack
Alan
A
D000726:Androgen Antagonists / Q000627:therapeutic use*; D017209:Apoptosis; D015153:Blotting, Western; D045744:Cell Line, Tumor; D003131:Combined Modality Therapy; D006801:Humans; D008297:Male; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense / Q000627:therapeutic use*; D011471:Prostatic Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000532:radiotherapy*; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2; D011836:Radiation Tolerance; D014410:Tumor Stem Cell Assay
D000726:Androgen Antagonists; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense; D011518:Proto-Oncogene Proteins; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2