Towards an Improvement of Anticancer Activity of Benzyl Adenosine Analogs

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.


Introduction
N6-Isopentenyladenosine (i6A) is a modified nucleoside formed by adenosine, harboring an isopentenyl chain derived from dimethylallyl pyrophosphate in the N6 position ( Figure 1). It belongs to the cytokinin family, initially discovered as inducers of plant cell division in culture [1]. The cytokinin phytohormones are now recognized as involved in a plethora of regulating plants processes [2,3], such as seed germination, root growth and leaf senescence [4][5][6]. In humans, many biological actions, both in vitro and in vivo, including antitumor effects, can be attributed to i6A [7][8][9][10][11][12][13]. For example, we demonstrated that i6A exerts antiproliferative effects in thyroid K-RAS (KiMol) transformed cells and untransformed FRTL-5 wild-type cells; in vivo, it shows antiproliferative action by inhibiting the growth of murine xenograft (where cancer KiMol cells were implanted subcutaneously) [14]. N6-benzyladenosine (2, Figure 1) and its substituted-benzyl derivatives have been studied as anticancer agents in different cell lines also as molecules capable of complexing transition metals [15][16][17]. N6-benzyladenine, the cytokinin free base form, has been shown to induce cell differentiation -inducing effects at concentrations between 25-100 µmol/L in acute myeloid leukemia cell lines. The growth inhibition and differentiation of cells induced by N6-benzyladenine may require its conversion into nucleotide [18], whereas riboside forms cause apoptosis of leukemia cell lines at much lower µmol/L concentrations [19,20]. It was shown that the N6-cytokinins induced apoptosis after their intracellular phosphorylation and activation of caspases [21][22][23][24]. Moreover, the cytokinins interconversion into their various structural derivatives was tracked in mammalian cells (HeLa), and considering the cytotoxic activity of the riboside 2, it was expected to induce cytotoxicity at 1 µmol/L concentration. In these cell line however, this effect was not observed and the nucleotide derivative of 2 was barely detected. More importantly, a conversion of 2 into the N6-benzyladenine was observed, hypothesizing that the HeLa cells can circumvent the cytotoxicity of 2 through its modification into a nitrogenous base instead of the nucleotide form [25]. The FPPS enzyme catalyzes the synthesis of several essential metabolites and is critical in protein prenylation and cell membrane synthesis [26][27][28]. In searching for the molecular mechanism explaining the cytotoxic and antiproliferative activity of i6A, we found that it modulated FPPS expression and activity in several experiments. In particular, on human natural killer (NK) cells, i6A directly stimulated the proliferation, the chemokine secretion, and the cytotoxic activity vs. conventional cancer target cells by the induction of the expression and the activity of FPPS [29,30]. The mechanisms relating the antitumor activity of i6A to FPPS are not fully understood; however, several pieces of evidence have shown an i6A-FPPS structural interaction. Specifically, the interaction of i6A with FPPS was first predicted by us in inverse virtual screening [31] and then proved by NMR experiments [32][33][34][35]. Based on these data, we designed new i6A analogs endowed with improved biological activity. Among these, compound 2 ( Figure 1) was confirmed as the most promising i6A derivative, characterized by increased cytostatic and antiproliferative activities compared to i6A. The N6-benzyladenosine (2) selectively targeted glioma cells -with no cytotoxicity on healthy brain cells-inducing intrinsic pathways of apoptosis and inhibition of proliferation. Moreover, it counteracted the oncogenic signaling mediated by the epidermal growth factor receptor (EGFR) [32].
Our molecular docking calculations showed that the isopentenyl moiety and the benzyl ring laid in the allylic sub-pocket of FPPS could contain additional bulky substituents. Intending to probe the FPPS binding site, we designed, synthesized and screened a panel of N6-benzyladenosine derivatives bearing different chemical moieties on the N6-benzyl ring of 2 (2a-m, Figure 2) [33]. NMR data and the 3D superposition of the best binding poses derived from the docking calculations indicated that all the designed molecules had a similar orientation and the para-substituted benzyl ring (mostly p-tertbutyl benzyl, 2f) engaged in cation-π interactions with F98, F99, Y204, and L100 of the hydrophobic sub-pocket. Indeed, cytostatic activity in the micromolar range of 2a-m was observed on glioma cells, whereas 2 and 2f showed a millimolar FPPS inhibition, indicating that the structural requirements may affect the FPPS enzyme inhibition differently from cellular toxicity and proliferation [12,33,[36][37][38].
In the attempt to elucidate the putative molecular mechanism subtending these apparently odd activities of N6-benzyladenosine derivatives, in the present work, we investigate their biomolecular behavior on HCT116, DLD-1 and MC38 colorectal cancer (CRC) cells.

Results and Discussion
To evaluate drug effects on cell viability and proliferation, HCT116, DLD-1 (human colorectal adenocarcinoma), and MC38 (murine colon adenocarcinoma) cell lines characterized by different genetic profiles and sensitivity to chemotherapies were treated for 24 and 48 h with N6-benzyladenosine (2) and its analogs (concentrations ranging from 1 to 20 µM). Assessment of cell viability and proliferation was performed by the MTT assay and IncuCyte live-cell imaging and analysis system. Results showed that the lead compound 2 and the analogs 2a and 2c exerted a significant and persistent cytotoxic effect, starting from 24 h and more evident at 48 h, on both human colon cancer cell lines (Figures 3 and 4 and Table 1). On the other hand, these analogs showed no significant effect on murine colon cell line MC38 ( Figure S1 in Supplementary Materials).
For other analogs, although some of them (2g, 2l) achieved statistical significance, at least at 10 µM after 48 h treatment, no precise dose or time-dependent cytotoxic effect can be evidenced in all CRC cell lines. In particular, the analogue 2g showed a significant inhibitory effect of viability in DLD-1 cells and a detectable antiproliferative effect in MC38 at a dose of 10 µM ( Figure S2 in Supplementary Materials); the compound 2l was found to be effective in HCT116 but not both in DLD-1 and MC38, while the analogs 2d, 2f and 2g exerted a slight proliferative effect in HCT116 ( Figure 3 and Table 1) and in MC38 for 2f after 48 h treatment ( Figure S2 in Supplementary Materials).  Quite the opposite, the analog 2d showed a good antiproliferative effect on murine CRC cells at a dose of 10 µM and 20 µM regardless, with no dose-dependent effect. Indeed, at both 24 and 48 h post-treatment with the highest concentrations of 2d compound, we observed a significant reduction of cell proliferation ( Figure 5).
To elucidate the mechanisms responsible for the observed cytotoxicity, we analyzed the apoptotic pathway induction through Western blots performed in human CRC cells treated with selected compounds. For this purpose, we focused further analyses on the most promising compounds, such as (i) the lead compound (2) and the analogues 2a and 2c, (ii) the analogues 2b and 2l, able to induce cytotoxicity in DLD-1 or HCT116, respectively, and (iii) the compound 2f that seemed to be the most promising from NMR and docking analysis, but substantially ineffective in the control of cell viability and cell proliferation. Apoptotic cell death involves activating of the caspase family, and is accomplished through the cleavage of some proteins essential for normal cell maintenance and survival, such as caspase-3 and PARP cleaved forms [16]. According to the previous assay, the lead compound 2, 2a and 2c induced apoptosis highlighted by the increase of caspase-3 and PARP cleaved forms revealed in DLD-1 and HCT116 treated for 24 h and 48 h ( Figure 6A,B panels respectively). Conversely, the apoptotic process was not triggered by treatment with 2b, 2f or 2l ( Figures S3 and S4 in Supplementary Materials). Results are expressed as percentage (mean ± SD) versus control (* p < 0.05; ** p < 0.01; *** p < 0.001; unpaired Student's t-test).  To prove that the antitumor activity of i6A analogs was related to the ability to impair FPPS activity, we measured their effect on protein prenylation in HCT116 cells. Protein prenylation is necessary for membrane localization and the proper function of otherwise cytosolic proteins; therefore, an effect on protein prenylation may have significant consequences on the survival of the cells. However, reliable measurement of protein prenylation in cell lines or tumor specimens is significantly interfered by the multiple posttranslational lipid modifications, such as farnesylation and geranylgeranylation, which are specific to each protein, and rapidly occur throughout the cell cycle and in response to selected treatments [39]. Moreover, the lack of commercial antibodies to discriminate prenylated proteins made it impossible to evaluate post-translational modifications by simple biological tests. Thus, protein prenylation was measured by observing through sucrose density centrifugation the localization of Rat Sarcoma Viral Oncogene Homolog (RAS) and RAS-Related Protein Rap-1A (Rap-1A), generally prenylated proteins with membrane localization. If the prenylation is impaired, they are found as soluble proteins in the cytosolic fraction. Quantification of the unprenylated fraction of RAS and Rap-1A through Western blot analysis (Figure 7) relates to the inhibition of protein prenylation. Sucrose density centrifugation of HCT116 cells treated with compound 2 and 2a-m (10 µM for 24 h), (Figure 7), indicated the ability of 2f to interfere with protein prenylation and therefore to induce an accumulation of both RAS and Rap1A proteins into the top-heavy fractions of the gradient (6-10), enriched in soluble proteins. Sucrose density treatment performed on CRC cells confirmed the ability of compound 2f to inhibit RAS, and Rap-1A prenylation, which was evident in the detachment of the two proteins from the membrane compartments. A less important action was observable for 2c, which induced the disappearance of the proteins located in fraction 5 of the gradient (Figure 7). In brief, the results confirmed a potent inhibitory effect of the lead compound and some synthetic analogues in CRC cell lines. However, the observed cytotoxic effects do not seem exclusively ascribable to direct inhibition of FPPS, at least in human CRC.

Cell Cultures, Treatments and Cell Viability Assay
Human colorectal cancer cell lines HCT116 and DLD-1 were obtained from the Interlab Cell Line Collection (IST, Genoa, Italy) and grown in McCoy's 5A and RPMI-1640 medium respectively, at 37 • C in a 5% CO 2 atmosphere. To evaluate cell viability, the colorimetric MTT (3-(4,5 di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used as previously described [40]. Briefly, HCT116 and DLD-1 cells were seeded at a density of 1 × 10 5 cells/well and exposed to increasing concentrations of compounds for 24 h or 48 h. MTT stock solution (5 mg/mL in PBS, Sigma) was added to each well and incubated for 4 h at 37 • C in humidified CO 2 . The reduction of tetrazolium to colored formazan only occurred in metabolically active cells and was monitored by spectrophotometer at an optical density of 595 nm after solubilization of the formazan crystals with acidic isopropanol (0.1 N HCl in absolute isopropanol). Each data point represents the average of three separate experiments in triplicate.

Cell Cultures, Treatments and Proliferation Assay
Mouse colon adenocarcinoma cell line MC38 was obtained from American Type Culture Collection (ATCC Catalog #PTA-2920, Manassas, VA, USA) and grown in Dulbecco's modified MEM (DMEM; Gibco TM ) supplemented with 10% fetal bovine serum (FBS; Gibco), 2 mM penicillin-streptomycin, 2 mM L-glutamine (Lonza TM BioWhittaker TM ), 2 mM sodium pyruvate (Gibco TM ) and 1 mM non-essential amino acids (Gibco ® MEM Non-Essential Amino Acids, 100X), at 37 • C in a 5% CO 2 atmosphere. The proliferation assay was performed according to the IncuCyte ® Label-Free Cell Proliferation Assay methodology. Briefly, 5 × 10 3 cells/well were seeded into 96-well flat bottom plate and incubated for 24 h at 37 • C in 5% humidified CO 2 . By day 1, when the cell confluence was approximately 30-40%, the cells were triggered for 48 h with the vehicle (DMSO) (Gibco TM ) and different concentrations of N6-benzyladenosine derivatives (2a-m) (concentrations range 1,2-20 µM). The 96-well flat bottom plates were placed into the IncuCyte ® ZOOM System (Sartorius, Göttingen, Germany) and cell growth was assessed for 48 h, with scan interval every 2 h.

Sucrose Density Gradient
To perform cells fractionation by sucrose density gradient, HCT116 cells were seeded at a concentration of 3.5 × 10 6 in 100 mm dishes and, when they reached 60-70% confluency, they were treated with 10 µM of the compounds for 24 h. Cells were harvested by Trypsin-EDTA, suspended in Mc Coy's 5A supplemented with 10% FBS and incubated at 37 • C with 5% CO 2 for 1 h. About 7.5 × 10 6 cells were washed three times with ice-cold PBS. Lysis buffer, containing 1% Triton X-100 (AppliChem GmbH, Darmstadt, Germany) and protease/phosphatase inhibitors in MOPS-buffered saline (25 mM 3-(N-Morpholino) propanesulfonic acid, 0.15 M NaCl, pH 6.5), was added to the cell pellet and left 30 min on ice. The lysate was adjusted to 40% sucrose (Sigma-Aldrich, Inc.) by mixing with equal volume of 80% sucrose prepared in MOPS-buffered saline and placed at the bottom of a Beckman ultracentrifuge tube. A discontinuous density gradient was prepared above by layering progressively 30% sucrose solution upon the first one and 5% sucrose solution at the top. Tubes were centrifuged at 32,500 rpm for 20-24 h at 4 • C in an SW-50.1 rotor (Beckman Instruments, Palo Alto, CA, USA). Ten fractions were collected from the top to the bottom of the tube and analyzed by Western blot to determine proteins distribution.

Western Blot Analysis
After treatment, HCT116 and DLD1 cells were washed with PBS, detached using trypsin and collected in ice-cold RIPA Buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% Triton X-100, 0.5% deoxycholic acid, 10 mg/mL leupeptin, 2 mM phenylmethylsulfonyl fluoride and 10 mg/mL aprotinin). Equal amount of whole cell extracts (15-30 µg of proteins), or equal volume of each sucrose density fraction was loaded on a 10% SDS-PAGE gel and separated by electrophoresis as described previously [19]. Proteins were transferred to nitrocellulose membranes that were blocked with 5% milk (Bio-Rad Laboratories, Inc., Hercules, CA 94547 USA) and probed with specific antibodies, anti-Caveolin-1 as marker of lipid rafts, or anti-GAPDH used as loading controls. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies and proteins detected by a chemiluminescence system (Amersham™ ECL™, Sigma-Aldrich S.r.l.).

Chemistry
Lead compound 2 and the analogs 2a-m were prepared and purified as described previously [15,33,[41][42][43][44][45][46]. NMR spectra were recorded at room temperature on Bruker Avance 600 MHz spectrometer. Chemical shifts (δ) are reported in ppm relative to the residual solvent peak (MeOD) and the multiplicity of each signal is designated by the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad; app, apparent. Coupling constants (J) are quoted in Hz. High resolution mass spectra (HRMS), recorded on a high-resolution mass spectrometer equipped with electrospray (ESI) and nanospray sources, a quadrupole-time of flighthy brid analyser coupled with capillary UPLC system (Q-TOF Premier/nano Aquity, Waters) in positive mode, and protonated molecular ions [M + H] + , were used for empirical formula confirmation. Liquid chromatography was performed on a Waters system (Milford, MA, USA) consisting of a Waters 486 tunable absorbance detector and a Varian 9012 pump. Samples were prepared by dissolving compounds 2a-m in methanol (0.5 mg/mL) at 0.2 mL/min. The injection volume was 10 µL. Compounds 2a-m were analyzed on a Symmetry ® C18 column (4.6 × 250 mm, 5 µm) under gradient elution at a flow rate of 0.9 mL/min. The mobile phases consisted of 2% (v/v) acetic acid in water (solvent A) and 2% (v/v) acetic acid in acetonitrile (solvent B). The following gradient was used: 0-20 min 10-90% B, 20-24 min 90-10% B, and return to the initial conditions over 3 min. UV detection was obtained at λ = 254 nm. Purity of all compounds (≥99%) was verified by HPLC, NMR and mass spectrometry measurements. NMR, HRMAS and HPLC details are shown in Supplementary Materials for each compound, reported using the IUPAC nomenclature ( Figures S5-S18). Structures of these compounds are presented in Figures 1 and 2.

Statistical Analysis
Significance between two groups was determined by unpaired two-tailed Student's t-test, while two-way analysis of variance (two-way ANOVA) followed by Dunnett's Multiple comparison test was performed to compare multiple mean groups. Graph Pad Prism 9.2.0 software (GraphPad Software, Inc., San Diego, CA 92108, USA) [47] was used to generate graphs and perform statistical analysis.

Conclusions
Taken together, our results showed that the lead compound 2 and the analogs 2a and 2c exerted a significant and persistent antiproliferative effect on the HCT116, DLD-1 human colorectal adenocarcinoma cell lines, while there was no significant effect on murine colon cell line MC38. By contrast, the analog 2d showed a good antiproliferative effect on murine CRC cells at a dose of 10 µM and 20 µM. The time and concentration-dependent effect of some analogs, such as 2g and 2l, on the cell viability was not observed in all CRC cell lines, although statistical significance was achieved, at least at 10 µM after 48 h treatment. To investigate the molecular mechanism related to the observed cytotoxicity, we analyzed the induction of apoptotic pathways through Western blots performed in human CRC cells. The increased expression of caspase-3 and PARP cleaved forms confirmed that compound 2 and the analogs 2a and 2c induced the apoptotic pathway. Finally, to investigate the possibility that the antitumor activity of 2 and 2a-m compounds would be related to the modulation of the proteins prenylation, RAS and Rap-1A prenylation was measured in HCT116 cell line. Sucrose density centrifugation coupled with Western blot analyses proved that compound 2f had a significant effect in impairing the prenylation of RAS and Rap-1A proteins, proving that the antitumor activity of 2f was related to the ability to inhibit FPPS activity. These new results confirmed our previously reported data, highlighting the significant capabilities of benzyl adenosine analogs to exert anticancer activity through FPPS enzyme inhibition. Nevertheless, our apparently contradictory results, with respect to cell viabilities and cytotoxic activity, might be caused by several phenomena corroborating the hypothesis that cytokinins in situ interconversion into their various structural derivatives may occur in a time-dependent manner, mimicking the interconversion hormetic effect. Cellular metabolism or a temporary cell cycle arrest due to the ability of cancer cells to evade the cell-cycle exit somehow might explain our findings. Additionally, unspecific inhibition of regulatory or feedback loops in the mevalonate pathway might contribute to sustaining this process. However, whether and how our compounds exhibit a hormetic effect through the mevalonate pathway need to be further investigated.
Supplementary Materials: The following are available online, Figure S1. Assessment of cell proliferation was performed by IncuCyte live-cell imaging and analysis system. Results showed that compounds 2a and 2c showed no significant effect on murine colon cell line MC38. Data are expressed as mean values ± SEM. Data sets were compared with two-way analysis of variance (ANOVA) test followed by Dunnett's correction; Figure S2. Assessment of cell proliferation was performed by IncuCyte live-cell imaging and analysis system for 2f and 2g compounds. Results showed that compound 2g displayed detectable antiproliferative effect in MC38 at the dose of 10 µM (* p < 0.05) and 20 µM (*** p < 0.001) at early time point (24 h). On the contrary, the compound 2f exerted a slight proliferative effect after 48 h of treatment. Data are expressed as mean values ± SEM. Data sets were compared with two-way analysis of variance (ANOVA) test followed by Dunnett's correction; Figure S3. Western blots analysis of caspase-3 and PARP expression performed in DLD-1 cell line. The apoptotic process was not triggered by treatment with 2b, 2f or 2l; Figure S4. Western blots analysis of caspase-3 and PARP expression performed in HCT116 cell line. The apoptotic process was not triggered by treatment with 2b, 2f or 2l; Figures S5-S18. 2D HSQC and 2D HMBC NMR experiments in MeOD solvent allowed the 1 H and 13 C chemical shift assignment of 2 and 2a-m compounds proton and carbon signals.