Reactivation of p53 by novel MDM2 inhibitors: implications for pancreatic cancer therapy.
The present study is the first to show in pancreatic cancer (PC) the growth inhibition and apoptosis by novel MDM2 inhibitors (MI-319 & 219) through reactivation of p53 pathway. Our results highlight two new secondary targets of MDM2 inhibitor 'SIRT1' and Ku70. SIRT1 which has a role in ageing and cancer and is known to regulate p53 signaling through acetylation. Ku70 is a key component of non-homologous end joining machinery in the DNA damage pathway and is known to regulate apoptosis by blocking Bax entry into mitochondria. Growth inhibition and apoptosis by MI-219, MI-319 was accompanied by increase in levels of p53 along with p21(WAF1) and the proapoptotic Puma. SiRNA against p21(WAF1) abrogated the growth inhibition of PC cells confirming p21(WAF1) as a key player downstream of activated p53. Immunoprecipitation-western blot analysis revealed reduced association of MDM2-p53 interaction in drug exposed PC cells. In combination studies, the inhibitors synergistically augmented anti-tumor effects of therapeutic drug gemcitabine both in terms of cell growth inhibition as well as apoptosis. Surface plasmon resonance studies confirmed strong binding between MI-319 and Ku70 (K(D) 170 nM). Western blot revealed suppression of SIRT1 and Ku70 with simultaneous upregulation of acetyl-p53 (Lys379) and Bax. Co-Immunoprecipitation studies confirmed that MI-319 could disrupt Ku70-Bax and SIRT1-Bax interaction. Further, using wt-p53 xenograft of Capan-2, we found that oral administration of MI-319 at 300 mg/kg for 14 days resulted in significant tumor growth inhibition without any observed toxicity to the animals. No tumor inhibition was found in mut-p53 BxPC-3 xenografts. In light of our results, the inhibitors of MDM2 warrant clinical investigation as new agents for PC treatment.
Azmi
Asfar S
AS
Department of Pathology, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Philip
Philip A
PA
Aboukameel
A
A
Wang
Zhiwei
Z
Banerjee
Sanjeev
S
Zafar
Syed F
SF
Goustin
Anton-Scott
AS
Almhanna
K
K
Yang
Dajun
D
Sarkar
Fazlul H
FH
Mohammad
Ramzi M
RM
D000818:Animals; D034961:Antigens, Nuclear / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D006801:Humans; D047468:Immunoprecipitation; D007211:Indoles / Q000494:pharmacology*; D000072200:Ku Autoantigen; D051379:Mice; D008813:Mice, Inbred ICR; D016513:Mice, SCID; D010190:Pancreatic Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics / Q000473:pathology; D011485:Protein Binding; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D034622:RNA Interference; D056564:Sirtuin 1 / Q000378:metabolism; D013141:Spiro Compounds / Q000494:pharmacology*; D020349:Surface Plasmon Resonance; D013997:Time Factors; D014162:Transfection; D047368:Tumor Burden; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D023041:Xenograft Model Antitumor Assays; D051028:bcl-2-Associated X Protein / Q000378:metabolism; D000093542:Gemcitabine
D034961:Antigens, Nuclear; D000970:Antineoplastic Agents; C494826:BAX protein, human; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D007211:Indoles; C548961:MI 319; C551433:MI-291; D013141:Spiro Compounds; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D003841:Deoxycytidine; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; C447939:SIRT1 protein, human; D056564:Sirtuin 1; C000606509:Xrcc6 protein, human; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen; D000093542:Gemcitabine