Abrogation of p21 expression by flavopiridol enhances depsipeptide-mediated apoptosis in malignant pleural mesothelioma cells.
OBJECTIVE
Recent insights regarding the pathogenesis of malignant pleural mesothelioma (MPM) provide new opportunities for targeted molecular therapies for this highly lethal disease. The present study was undertaken to examine the effects of the histone deacetylase inhibitor, Depsipeptide (DP) FK228, in conjunction with the cyclin-dependent kinase inhibitor, Flavopiridol (FLA), in cultured MPM cells.


METHODS
Proliferation and apoptosis in drug-treated, virally transduced, or control cells were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Apo-bromodeoxyuridine techniques. Western blot and ELISA techniques were used to examine signal transduction and cell cycle-related protein levels in MPM cells exposed to DP and/or FLA in the presence or absence of calphostin, phorbol-12,13-dibutyrate, 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole, or adenoviral p21 transduction.


RESULTS
DP (1-50 ng/ml x 6 h) or FLA (100-200 nM x 72 h) alone, mediated low-level, dose-dependent growth inhibition in MPM cells. In contrast, sequential DP/FLA treatment mediated marked growth inhibition and apoptosis in these cell lines. The cytotoxic effects of DP/FLA were considerably less pronounced in cultured normal cells. The proapoptotic effects of DP/FLA treatment coincided with inhibition of DP-mediated induction of p21 by FLA. Overexpression of p21 by adenoviral gene transfer techniques rendered MPM cells refractory to the cytotoxic effects of this treatment regimen. In p21 reporter assays, promoter activation by DP was antagonized by FLA. The magnitude of inhibition of DP-mediated p21 induction by FLA exceeded that observed with the pTEFb antagonist 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole. Calphostin C abrogated p21 induction mediated by DP and enhanced DP-mediated apoptosis in a manner comparable with FLA in MPM cells; in contrast, phorbol-12,13-dibutyrate blocked FLA-mediated inhibition of p21 induction by DP and markedly protected these cells from the apoptotic effects of sequential DP/FLA.


CONCLUSIONS
FLA abrogates DP-mediated induction of p21 expression, in part, via inhibition of protein kinase C signaling and markedly potentiates the cytotoxic effects of DP in MPM cells.
Nguyen
Dao M
DM
Thoracic Oncology Section, Surgery Branch, and Medical Oncology Clinical Research Unit, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892-1502, USA.
Schrump
William D
WD
Chen
G Aaron
GA
Tsai
Wilson
W
Nguyen
Phuongmai
P
Trepel
Jane B
JB
Schrump
David S
DS
D017209:Apoptosis / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics*; D004791:Enzyme Inhibitors; D005419:Flavonoids / Q000494:pharmacology*; D006801:Humans; D008654:Mesothelioma; D010880:Piperidines / Q000494:pharmacology*; D010997:Pleural Neoplasms
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D010880:Piperidines; C077990:alvocidib