Cell Cycle Regulating Kinase Cdk4 as a Potential Target for Tumor Cell Treatment and Tumor Imaging

Abstract

The cyclin-dependent kinase (Cdk)-cyclin D/retinoblastoma (pRb)/E2F cascade, which controls the G1/S transition of cell cycle, has been found to be altered in many neoplasias. Inhibition of this pathway by using, for example, selective Cdk4 inhibitors has been suggested to be a promising approach for cancer therapy. We hypothesized that appropriately radiolabeled Cdk4 inhibitors are suitable probes for tumor imaging and may be helpful studying cell proliferation processes in vivo by positron emission tomography. Herein, we report the synthesis and biological, biochemical, and radiopharmacological characterizations of two (124)I-labeled small molecule Cdk4 inhibitors (8-cyclopentyl-6-iodo-5-methyl-2-(4-piperazin-1-yl-phenylamino)-8H-pyrido[2,3-d]-pyrimidin-7-one (CKIA) and 8-cyclopentyl-6-iodo-5-methyl-2-(5-(piperazin-1-yl)-pyridin-2-yl-amino)-8H-pyrido[2,3-d]pyrimidin-7-one (CKIB)). Our data demonstrate a defined and specific inhibition of tumor cell proliferation through CKIA and CKIB by inhibition of the Cdk4/pRb/E2F pathway emphasizing potential therapeutic benefit of CKIA and CKIB. Furthermore, radiopharmacological properties of [(124)I]CKIA and [(124)I]CKIB observed in human tumor cells are promising prerequisites for in vivo biodistribution and imaging studies.

Scheme 1

Radiolabeling of CKIAP and CKIBP. a: [¹²⁴I]NaI, Iodogen, rt, 10 minutes; b: TFA, 50°C, 30 minutes.

Figure 1

Cell proliferation rate of HT-29, FaDu, THP-1, and THP-1 macrophage after treatment with (a) CKIA or (b) CKIB (every 24 hours) in percentage of control without treatment. (c) Comparison of daily treatment and single treatment after 72 hours of incubation with 1 μM CKIA or CKIB, respectively. (means ± standard deviations, n ≥ 6, ANOVA, *P < .05, compared to control (100%)).

Figure 2

Cell cycle distribution of HT-29 cells at 24 hours after treatment with CKIA or CKIB (representative histograms).

Figure 3

(a) Representative Western blots of HT-29 and FaDu cell lysates. (b) pRb phosphorylation at Ser780 relative to total pRb (means ± standard deviations, n = 2–5).

Figure 4

mRNA expression level of E2F-1 and PCNA after 24-hour-incubation with (a) CKIA or (b) CKIB and in G1 arrested adherent tumor cells after serum-deprivation relative to control cells without treatment (means ± standard deviations, n ≥ 6).

Figure 5

Cellular radiotracer uptake of (a) [¹²⁴I]CKIA and (b) [¹²⁴I]CKIB over a period of 5 hours at 37°C and 4°C (means ± standard deviations, n = 4, ANOVA, P < .05, 4°C compared to 37°C, resp.). Symbols represent observed data, lines represent computer-derived fits.