Interestingly, knockdown of p21 using siRNA reduced the growth inhibitory efficiency of both inhibitors only in cell line with wt-p53 confirming the involvement of p21 in the regulation of p53 (Fig. Immunoprecipitation-western blot analysis revealed reduced association of MDM2-p53 conversation in drug uncovered 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 (KD 170 nM). Western blot Hyal2 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 conversation. 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. as well as in xenograft models [21,24,25]. However Nutlin 3 remained the only inhibitor in its class specific for MDM2 and thus newer inhibitors with higher specificity and minimum toxicity are required. In this paper we show that specific and orally active MDM2 inhibitors MI-219 and MI-319 transiently reactivates p53, resulting in growth inhibition and apoptosis in wild wt-p53 PC cells as well as tumor growth retardation in a Capan-2 [wt-p-53] and not in BxPC-3 DRI-C21045 [mut-p53] PC xenograft model. Further, we also identify two new targets of MDM2 (SIRT1 and Ku70) that play crucial role in the biology of p53. MATERIALS AND METHODS Cell Culture and Experimental Reagents Five human PC cell lines Capan-1, Capan-2, Colo-357, HPAC and BxPC-3 were used in this study. All cells except Colo-357 were purchased from [American Type Culture Collection [ATCC]]. Colo-357 was a gift from Dr. Paul Chiao [M.D. Anderson Cancer Center, Houston, TX]. Capan-1, HPAC, BxPC-3 and Colo-357 were cultured in DMEM [In-vitrogen] supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. Capan-2 was produced in McCoy 5 A media [Invitrogen]. All cells were cultured in a 5% CO2-humidified atmosphere at 37C. Primary antibodies for p53, acetyl-p53, p21WAF1 p21], Puma, MDM2, Bax and SIRT1 were purchased from Cell Signaling [Danvers, MA]. Ku70 antibody was purchased from Abcam [Cambridge, MA]. Anti–actin and all secondary antibodies were obtained from Sigma [Saint Louis]. LipofectAMINE 2000 was purchased from Cell Signaling [Danvers, MA]. Vectastain ABC Kit coupled with avidin-biotin linked system was purchased from Vectastain [Burlingame, CA]. All chips buffers and reagents used for Surface plasmon resonance Biacore experiments were from GE Healthcare (Biacore Life Sciences), unless noted otherwise. The instrument used was Biacore 3000 (instrument ID 3442), which is usually maintained by Dr. Stanley R. Terlecky, in the Department of Pharmacology at Wayne State University School of Medicine and Calibrated by Jose A. Gutierrez from GE Healthcare every six months. Instrument configuration is usually IFC6 carrying Biacore 3000 Control software version 3.2. All analysis was done in DRI-C21045 BI-Aevaluation version 4.1 and all experiments were performed at 25C. Chemical Synthesis of MI-219 and MI-319 MI-219 and MI-319 were synthesized by using our previously published methods . Nutlin-3 (()-4-[4,5-Bis(4-chlorophenyl)-2-(2-isopropoxy-4-methoxy-phenyl)-4,5-dihydro-imidazole-1-carbonyl]-piperazin-2-one) was purchased from Sigma [Sigma St Louis USA]. The binding of small molecules to human MDM2 was predicted by using the GOLD program . SiRNA and Transfections The p21WAF1 siRNA and siRNA control were obtained from Cell Signaling. Human PC cells were transfected with p2WAF1 siNA and control siNA respectively, using LipofectAMINE 2000 as described in the manufacturers protocol [Cell Signaling]. Cell Growth Inhibition Studies by MTT Assay The cells [3 103] were seeded in a 96-well culture plate and treated with MDM2 inhibitors or gemcitabine or combination of both for indicated time and subsequently incubated with MTT reagent [0.5 mg/mL] at 37C for 2 h, and DRI-C21045 MTT assay was done as described earlier . In another set of experiments seeded cells were transfected with p21 siRNA or control siRNA for 5 hours followed by MDM2 inhibitor treatment for 72 hrs and MTT assay was performed. The results were plotted as means SD of three individual experiments having six determinations per experiment for each experimental condition. Flow Cytometry and Cell Cycle Analysis Cell cycle analysis on MI-219 and MI-319 treated cells were performed using DRI-C21045 PI staining. The percent of cells in different phases of the cell cycle was analyzed with CELLQUest software [BDIS] using a Power Macintosh 7500/100 computer [Apple Computer]. Quantification of Apoptosis by ELISA The cell apoptosis ELISA detection kit [Roche, Palo Alto, CA] was used to detect apoptosis in PC cells treated with.
Interestingly, knockdown of p21 using siRNA reduced the growth inhibitory efficiency of both inhibitors only in cell line with wt-p53 confirming the involvement of p21 in the regulation of p53 (Fig