Towards an Improvement of Anticancer Activity of Benzyl Adenosine Analogs

Abstract

N6-Isopentenyladenosine (i6A) is a naturally occurring modified nucleoside displaying in vitro and in vivo antiproliferative and pro-apoptotic properties. In our previous studies, including an in silico inverse virtual screening, NMR experiments and in vitro enzymatic assays, we demonstrated that i6A targeted farnesyl pyrophosphate synthase (FPPS), a key enzyme involved in the mevalonate (MVA) pathway and prenylation of downstream proteins, which are aberrant in several cancers. Following our interest in the anticancer effects of FPPS inhibition, we developed a panel of i6A derivatives bearing bulky aromatic moieties in the N6 position of adenosine. With the aim of clarifying molecular action of N6-benzyladenosine analogs on the FPPS enzyme inhibition and cellular toxicity and proliferation, herein we report the evaluation of the N6-benzyladenosine derivatives' (compounds 2a-m) effects on cell viability and proliferation on HCT116, DLD-1 (human) and MC38 (murine) colorectal cancer cells (CRC). We found that compounds 2, 2a and 2c showed a persistent antiproliferative effect on human CRC lines and compound 2f exerted a significant effect in impairing the prenylation of RAS and Rap-1A proteins, confirming that the antitumor activity of 2f was related to the ability to inhibit FPPS activity.

Keywords: FPPS; N6-benzyladenosine derivatives; colorectal cancer.

Figure 1

Chemical structures of i6A (1) and N6-benzyladenosine (2).

Figure 2

Chemical structures of N6-benzyladenosine derivatives (2a–m).

Figure 3

Effect of 2 and its analogues on HCT116 cell viability. Values are means ± SD from three individual experiments (* p < 0.05; *** p < 0.001; unpaired Student’s t-test).

Figure 4

Effect of 2 and its analogues on DLD-1 cell viability. Values are means ± SD from three individual experiments (* p < 0.05; ** p < 0.01; *** p < 0.001; unpaired Student’s t-test).

Figure 5

Antiproliferative effect of the analog compound 2d on MC38 cell line treated for 48 h (1.2–20 μM). Data are expressed as mean values ± SEM. Data sets were compared with two-way analysis of variance (ANOVA) test followed by Dunnett’s correction (* p < 0.05, *** p < 0.001).

Figure 6

Western blots analysis of caspase-3 and PARP expression performed in DLD-1 cells (A) and HCT116 (B) treated with compounds 2, 2a and 2c. The apoptotic process was activated by treatment with these three compounds.

Figure 7

Western blot analysis of sucrose density gradient fractions of HCT116 cells treated with compound 2 and 2a–m (10 μM for 24 h). 2f induced an accumulation of both RAS and Rap-1A proteins into the top-heavy fractions of the gradient (6–10).