Biological, biochemical and radiopharmacological properties of two Cdk4 inhibitors.

Ziel/Aim:

The aim of our study was the biological, biochemical and radiopharmacological characterization of a known Cdk4 inhibitor CKIA, a derivative compound CKIB and their corresponding iodine-124 radiolabeled compounds in different tumor cell lines. Further we wanted to prove the hypothesis that potent Cdk4 inhibitors, which are interesting for cancer therapy are also suitable as radiotracers for imaging of cell proliferation processes in vivo and tumor visualization by positron emission tomography (PET).

Methodik/Methods:

Cell growth and cell cycle studies were performed in human tumor cell lines HT-29 (colon carcinoma), FaDu (head- and neck squamous cell carcinoma), and THP-1 (acute monocytic leukemia). Cdk4 specific pRb phosphorylation at Ser780 was analyzed in cell lysates by western blotting. mRNA expression of the pRb affected genes E2F-1 and PCNA was measured with quantitative real-time RT-PCR. Radiotracer uptake studies were performed using iodine-124 radiolabeled CKIA ([¹²⁴I]CKIA) or CKIB ([¹²⁴I]CKIB).

Ergebnisse/Results:

Tumor cell growth studies indicate a significantly reduced cell proliferation in all cell lines after 48 h of treatment with 0.1 (< 50%) and 1 µM (< 25%) CKIA. This effect is confirmed by cell cycle studies. Already 24 h after incubation with CKIA the percentage of tumor cells in G1 phase showed a concentration dependent increment up to 90%. pRb phosphorylation is decreased three- to tenfold after 24 h of treatment with 0.1 and 1 µM CKIA in all cell lines. Furthermore, a substantial downregulation of E2F-1 and PCNA mRNA expression could be demonstrated. In vitro radiotracer uptake studies in adherent tumor cells using [¹²⁴I]CKIA (A_s = 20 GBq/µmol) showed a substantial uptake in HT-29 (1,649±117 %ID/mg protein) and FaDu cells (1,033±84 %ID/mg protein) after 2 h at 37°C. At 4°C an obvious lower uptake was detectable in both cell lines (258±30 %ID/mg protein in HT-29, 169±14 %ID/mg protein in FaDu).
Further, a new compound derived from CKIA was studied. CKIB shows similar effects on cell growth and cell cycle distribution. After 48 h of treatment with CKIB cell proliferation was reduced by 36% (0.1 µM) or 62% (1 µM) in HT-29 cells. Over 90% of HT-29 cells in G1 phase were obtained after 24 h of incubation with 1 µM CKIB. Results of pRb phosphorylation and both E2F-1 and PCNA mRNA expression analyses suggest a downregulation after 24 h of incubation with 1 µM CKIB in the tumor cells. In vitro studies with [¹²⁴I]CKIB (A_s = 3.5 GBq/µmol) demonstrate a radiotracer uptake of 904±43 %ID/mg protein in HT-29 and 856±45 %ID/mg protein in FaDu cells after 2 h at 37°C. Radiotracer uptake at 4°C is substantially lower.

Schlussfolgerungen/Conclusions:

In conclusion, our data demonstrate a definite and specific inhibition of tumor cell proliferation after incubation with CKIA and CKIB due to an arrest of tumor cells in G1 phase as a result of Cdk4-cyclin D/ pRb/ E2F pathway inhibition. The radiotracer uptake of [¹²⁴I]CKIA and [¹²⁴I]CKIB observed in human tumor cells are a promising prerequisite for in vivo biodistribution and imaging studies by PET to further support our hypothesis that radiolabeled CKIA and corresponding compounds are suitable tracers for visualization of tumors using PET.