Potentiation of anticancer effect of valproic acid, an antiepileptic agent with histone deacetylase inhibitory activity, by the cyclin-dependent kinase inhibitor P276-00 in human non-small-cell lung cancer cell lines.
BACKGROUND
P276-00 is a novel cyclin-dependent kinase (CDK) inhibitor is in Phase II clinical trials. Valproic acid (VPA), an antiepileptic agent has been associated with anticancer activity, through the inhibition of histone deacetylase I. Here we investigate the effect of the combination of VPA and P276-00, in non-small-cell lung cancer (NSCLC) cell lines.


METHODS
Cell growth inhibition was studied using the Propidium iodide (PI) assay. Cell cycle analysis and recovery were detected by flow cytometry. The expression levels of various proteins were detected by western blot. Inhibition of colony formation in H460 was checked in vitro. In vivo efficacy was studied in H460 xenograft model.


RESULTS
The combination of P276-00 and VPA showed synergistic effect on p53+ and p53- NSCLC cell lines in antiproliferative assay at both constant and non-constant ratio with marked decrease in colony forming potential. Flow cytometric analysis confirmed a significant time dependent increase in apoptosis with 64% apoptotic population at 96 h compared to VPA (1%) and P276-00 (28%) alone (p < 0.0001). Incubation of the cells after treatment, in fresh medium without drugs, led to the recovery of cells treated with P276-00 alone but not the cells treated with the combination of both the drugs. The combination treatment up-regulated tumor suppressor proteins like p53, p21 and p27 along with down-regulation of proliferation and survival proteins viz. cyclin D1 and Bcl-2. This was also associated with the upregulation of the pro-apoptotic protein Bax and significant accumulation of hyperacetylated histones in the combination treatment. Interestingly, VPA in combination with P276-00 was much more effective as an antitumor agent than alone, in the H460 xenograft tumor model in SCID mice.


CONCLUSIONS
This study indicates that the combination of HDAC inhibitor VPA with CDK inhibitor P276-00 is promising novel molecularly targeted therapeutic approach for NSCLC treatment.
Department of Pharmacology, Piramal Life Sciences, Piramal Enterprises Limited, 1 Nirlon Complex, Goregaon (East), Mumbai, Maharashtra 400 063, India.
Nitesh
N
Shirsath
Maggie
M
Rathos
Umesh
U
Chaudhari
H
H
Sivaramakrishnan
Kalpana
K
Joshi
D000107:Acetylation; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000633:toxicity; D017209:Apoptosis / Q000187:drug effects; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D047309:Flavones / Q000494:pharmacology*; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology* / Q000633:toxicity; D006657:Histones / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D051379:Mice; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D014410:Tumor Stem Cell Assay; D014635:Valproic Acid / Q000494:pharmacology* / Q000633:toxicity; D023041:Xenograft Model Antitumor Assays
D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D047309:Flavones; D056572:Histone Deacetylase Inhibitors; D006657:Histones; C520467:P276-00; D047428:Protein Kinase Inhibitors; D014635:Valproic Acid; D018844:Cyclin-Dependent Kinases
CDK inhibitor; Combination studies; HDAC; Non-small-cell lung cancinoma; P276-00; Valproic acid