A ruthenium-based 5-fluorouracil complex with enhanced cytotoxicity and apoptosis induction action in HCT116 cells

Combination of multifunctionalities into one compound is a rational strategy in medicinal chemical design, and have often been used with metallodrug-based compounds. In the present study, we synthesized a novel ruthenium-based 5-fluorouracil complex [Ru(5-FU)(PPh3)2(bipy)]PF6 (PPh3 = triphenylphosphine; and bipy = 2,2′-bipyridine) with enhanced cytotoxicity in different cancer cells, and assessed its apoptosis induction action in human colon carcinoma HCT116 cells. The complex was characterized by infrared, cyclic voltammetry, molar conductance measurements, elemental analysis, NMR experiments and X-ray crystallographic analysis. In both 2D and 3D cell culture models, the complex presented cytotoxicity to cancer cells more potent than 5-FU. A typical morphology of apoptotic cell death, increased internucleosomal DNA fragmentation, without cell membrane permeability, loss of the mitochondrial transmembrane potential, increased phosphatidylserine externalization and caspase-3 activation were observed in complex-treated HCT116 cells. Moreover, the pre-treatment with Z-DEVD-FMK, a caspase-3 inhibitor, reduced the apoptosis induced by the complex, indicating cell death by apoptosis through caspase-dependent and mitochondrial intrinsic pathways. The complex failed to induce reactive oxygen species production and DNA intercalation. In conclusion, the novel complex displays enhanced cytotoxicity to different cancer cells, and is able to induce caspase-mediated apoptosis in HCT116 cells.

bonds present values of 1.272 and 1.350 Å, respectively, while in the metal-free 5-FU the values found to these bonds are 1.24 and 1.39 Å. When 5-FU is coordinated to Ru(II) the length of these bonds changes significantly in which the C4-O4 is longer, whereas C4-N3 is shorter. This suggests that the molecule presents an electron delocalization on the [O4-C4-N3-Ru1] moiety, giving stabilization to the chelating system. The metal-free 5-FU and coordinated to Ru presents a planar conformation. In the complex, six-membered rings of 5-FU, bipy and PPh 3 are stacked to form intramolecular π-π interactions with the adjacent ligands, stabilizing the molecular structure of the complex (Supplementary Figure 5).

The complex [Ru(5-FU)(PPh 3 ) 2 (bipy)]PF
The IC 50 value for non-cancer cells was 9.5 and 5.7 μM for the complex and 57.9 and 78.0 μM for 5-FU in MRC5 and PBMC cells, respectively. In addition, the IC 50 value in non-cancer cells was 1.6 and 5.1 μM for doxorubicin, 1.4 and 12.4 μM for oxaliplatin in MRC5 and PBMC cells, respectively. Table 2 shows the calculated selectivity index (SI). The complex exhibited SI similar to 5-FU, doxorubicin and oxaliplatin to most of the cell lines tested.
HCT116 cell line was the most sensitive cell line to the complex [Ru(5-FU)(PPh 3 ) 2 (bipy)]PF 6 and was used as a cellular model to the next experiments. Therefore, the cytotoxicity of the complex was confirmed by trypan blue exclusion (TBE) assay in HCT116 cells, after 24 and 48 h incubation. The complex significantly reduced (p < 0.05) the number of viable cells (Fig. 3 The cytotoxicity of the complex [Ru(5-FU)(PPh 3 ) 2 (bipy)]PF 6 was also performed in an in vitro three-dimensional (3D) model of cancer multicellular spheroids formed from HCT116 cells. The treatment with the complex disrupted these cell aggregations, resulting in the presence of cell debris (Fig. 4A). The IC 50 of the complex was 1.7 μM (Fig. 4B), while 5-FU presented IC 50 > 192.2 μM. The complex was more potent than 5-FU at the least 113-fold. Doxorubicin and oxaliplatin showed IC 50 of 3.5 and 4.6 μM, respectively.     Table 3 shows the obtained cell cycle distribution. All DNA that was sub-diploid in size (sub-G 0 /G 1 ) was considered fragmented. The treatment with the complex caused a significant increase in the internucleosomal DNA fragmentation (p < 0.05) that became more pronounced at highest concentration and at longest time of incubation. No accumulation of cells in any phase of the cell cycle was observed. On the other hand, 5-FU treatment resulted in a significantly increase in the number of cells in S phase compared to the negative control (30.7% at control against 54.0% at 5-FU after 24 h incubation; and 23.8% at control against 40.8% at 5-FU after 48 h incubation, respectively). Doxorubicin and oxaliplatin caused cell cycle arrest at the phase G 2 /M in HCT116 cells.

The complex [Ru(5-FU)(PPh 3 ) 2 (bipy)]PF 6 induces apoptosis through caspase-dependent and mitochondrial intrinsic pathway in HCT116 cells.
As observed by May-Grunwald-Giemsa staining, the treatment with the complex induced cell shrinkage and fragmentation of the nuclei of HCT116 cells (Fig. 5). Doxorubicin, oxaliplatin and 5-FU also induced cell shrinkage and nuclear fragmentation. Annexin V-FITC and propidium iodide (PI) double staining was performed by flow cytometry to measure the percentage of cells in viable, early apoptotic, late apoptotic and necrotic stages. The treatment with the complex [Ru(5-FU)(PPh 3 ) 2 (bipy)]PF 6 resulted in increasing in early and late apoptotic cells in a time-and concentration-dependent manners ( Fig. 6A and B). No significant increase in necrotic cells was observed in HCT116 cells treated with the complex. In addition, pre-incubation with a caspase-3 inhibitor, Z-DEVD-FMK, prevented the increase of apoptotic cells caused by the complex (Fig. 7A and B). A significant increase in caspase-3 activation was also observed in HCT116 cells treated with the complex, as measured by colorimetric assay using DEVD-pNA as the substrate (Fig. 8A). Moreover, the mitochondrial membrane potential was measured by the incorporation of rhodamine 123 using flow cytometry, and the treatment with the complex also induced mitochondrial depolarization in HCT116 cells (Fig. 8B). At the concentration tested (4 μM, based on its IC 50 value), 5-FU did not induce increasing in the early and late apoptosis, suggesting that 5-FU has only anti-proliferative action. Mitochondrial depolarization and activation of caspase-3 were also not affected in 5-FU-treated HCT116 cells, at the concentration tested.
The cytotoxicity of the complex [Ru(5-FU)(PPh 3 ) 2 (bipy)]PF 6 in BAD KO SV40 MEF (BAD gene knockout immortalized mouse embryonic fibroblast) and its parental cell line WT SV40 MEF (wild-type immortalized mouse embryonic fibroblast) was also evaluated by alamar blue assay after 72 h incubation. The IC 50 values for the complex was 1.8 and 1.9 μM for BAD KO SV40 MEF and WT SV40 MEF cell lines, respectively, indicating that BAD gene is not essential for its cytotoxic activity

Discussion
5-FU is an important chemotherapeutic drug widely used in cancer treatment, and ruthenium-based complexes have been shown as potent cytotoxic agents to cancer cells, possibly becoming as a new class of chemotherapeutic drugs. Herein, we combined these two components into one compounds and found a novel ruthenium-based 5-fluorouracil complex with enhanced cytotoxicity. As mentioned above, some ruthenium complexes containing 5-FU as ligand were previously synthesized, but with weak cytotoxicity 13,14 . These ruthenium-based compounds were synthesized using precursor of type [Ru(η 6 -arene)]. In this present paper, we used precursor of type [RuCl 2 (N-N)(P-P)] and obtained the complex [Ru(5-FU)(PPh 3 ) 2 (bipy)]PF 6 , a compound with cytotoxicity to cancer cells more potent than 5-FU in both 2D and 3D cell culture models. The SI of the complex was similar to 5-FU, doxorubicin and oxaliplatin to most of the cell lines tested. In fact, the precursor of type [RuCl 2 (N-N)(P-P)] has been previously used by us to synthesize potent cytotoxic agents 12,18 . 5-FU is an antimetabolite drug that is incorporated into the DNA and RNA, and inhibits the enzyme thymidylate synthase. The incorporation of 5-FU into DNA and RNA occurs mainly during the S phase of cell cycle. Moreover, the inhibition of thymidylate synthase can cause deoxynucleotide imbalance during DNA synthesis. Therefore, 5-FU acts targeting S phase of the cell cycle 6,19,20 . In fact, we observed that 5-FU block S phase of the cell cycle in HCT116 cells. However, although the complex [Ru(5-FU)(PPh 3 ) 2 (bipy)]PF 6 increased significantly the internucleosomal DNA fragmentation, no accumulation of the cells in any phase of the cell cycle was observed, indicating different mechanism of action between 5-FU and its ruthenium-based complex. Many ruthenium complexes have been reported to cause block at different phases of the cell cycle. The ruthenium methylimidazole complex caused cell cycle arrest at G 0 /G 1 phase and induced apoptosis via the mitochondrial pathway, which involved ROS accumulation, mitochondrial dysfunction and Bcl-2 and caspase activation in lung cancer A549 cells 21 . Three ruthenium(II)-arene complexes, namely [(η 6 -p-cymene)Ru(Me 2 dppz)Cl]PF 6 , caused arrest of the cell cycle in G 2 /M and S phases in cervical carcinoma HeLa cells 22 . The ruthenium(II) polypyridyl complexes induce cell cycle arrest at G 2 /M phase in hepatocellular carcinoma BEL-7402 cells 11 .
In conclusion, the complex [Ru(5-FU)(PPh 3 ) 2 (bipy)]PF 6 was synthesized for the first time at this communication and tested against cancer cells with different histological type. In both 2D and 3D cell culture models, the complex presented cytotoxicity to cancer cells more potent than 5-FU. Unlike 5-FU, the complex does not induce S phase arrest, indicating different mechanism of action between 5-FU and its ruthenium-based complex. In addition, the complex was able to induce apoptosis through caspase-dependent and mitochondrial intrinsic pathway in HCT116 cells.

Material and Methods
Chemistry. General. Elemental analysis (C, H and N) was performed using an FISONS instrument, CHNS EA-1108. The FT-IR spectra of complex and metal-free 5-FU was recorded on a FT-IR Bomem-Michelson 102 spectrometer as CsI pellets in the range of 4000-200/cm. The electronic spectra on a Hewlett Packard diode array-8452A scanning spectrophotometer and conductivity value were obtained using a Meter Lab CDM2300 instrument at room temperature, using 10 −3 M solutions of the complex, both experiments were carried out in CH 2 Cl 2 . Cyclic voltammetry measurements were carried out at room temperature in an electrochemical analyzer BAS, model 100B. These experiments were also performed using CH 2 Cl 2 containing Bu 4 NClO 4 (TBAP) (Fluka Purum) at concentration of 0.10 M, as a supporting electrolyte and a one-compartment electrochemical cell based on three electrodes: Pt foils as the working and auxiliary electrodes and an Ag/AgCl (0.10 M TBAP in CH 2 Cl 2 ) as the reference electrode. Under these conditions, the ferrocene (Fc) is oxidized at +0.43 V (Fc+/Fc). Highresolution electrospray ionization mass spectrometry (HRESIMS) spectrum was measured by direct infusion in a MicroTof-Q II Bruker Daltonics Mass Spectrometer (Le) in the positive ion mode, employing methanol as solvent (LC/MS grade from Honeywell/B&J Brand). 31 P{1 H} and 1 H NMR spectra were recorded in acetone-d 6 , on a Bruker DRX, with the 31 P{ 1 H} and 1 H chemical shifts reported in relation to H 3 PO 4 (85%) and tetramethylsilane, respectively. Single-crystals of the complex were obtained from ethyl ether/dichloromethane solvent diffusion. X-ray diffraction experiment was carried out at room temperature on an Enraf-Nonius Kappa-CCD diffractometer with graphite monochromated MoKα radiation (λ = 0.71073 Å). The structure was solved by direct methods and refined by full-matrix least-squares method on F 2 with anisotropic thermal parameters for all non-hydrogen atoms using SHELXS-97 26 . All hydrogen atoms were placed in calculated positions and refined isotropically. Representation of the structure was drawn with the program Mercury 3.8 27 .  3173, 3078, 3055, 3022, 2960, 1659, 1606, 1600,  1537, 1481, 1435, 1329, 1300, 1270, 1236, 1188, 1161, 1092, 1028, 999, 845, 766, 746, 698, 617, 557, 519, 496,  Cytotoxic activity assay. Cell viability was quantified using the alamar blue assay, according to Ahmed et al. 28 . Cells were inserted in 96-well plates for all experiments (7 × 10 4 cells/mL for adherent cells or 3 × 10 5 cells/mL for suspended cells in 100 μL of medium). After 24 h, the complex was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich co.) and added to each well and incubated for 72 h. Doxorubicin (purity ≥ 95%, doxorubicin hydrochloride, Laboratory IMA S.A.I.C., Buenos Aires, Argentina), oxaliplatin (Sigma-Aldrich Co.) and 5-FU (Sigma-Aldrich Co.) were used as the positive controls. Four (for cell lines) or 24 h (for PBMCs) before the end of incubation, 20 μL of a stock solution (0.312 mg/mL) of the alamar blue (resazurin, Sigma-Aldrich Co.) were added to each well. Absorbance at 570 nm and 600 nm was measured using the SpectraMax 190 Microplate Reader (Molecular Devices, Sunnyvale, CA, EUA), and the drug effect was quantified as the percentage of control absorbance.
3D multicellular spheroids culture. HCT116 cells were cultivated in 3D multicellular spheroids. Briefly, 100 μL of a solution of cells (0.5 × 10 6 cells/mL) were inserted in 96-well plate with a cell-repellent surface (Greiner Bio-One; Kremsmünster, Austria) and cultured in complete medium plus 3% matrigel (BD Biosciences; San Jose, CA, EUA). Spheroids with stable structures had formed after three days. Then, the spheroids were exposed to a range of drug concentrations for 72 h, after which the cell viability was quantified by alamar blue assay as described above.
Trypan blue exclusion assay. TBE assay was used to confime the cytotoxic effect of the complex tested. The number of viable cells and non-viable cells (take up trypan blue) were counted. Briefly, 90 μL was removed from the cell suspension and 10 μL of trypan blue (0.4%) was added. Cell counting was performed using a light microscope with a hemocytometer filled with an aliquot of the homogenized cell suspension.
Internucleosomal DNA fragmentation and cell cycle distribution. Cells were harvested in a permeabilization solution containing 0.1% triton X-100, 2 µg/mL PI, 0.1% sodium citrate and 100 µg/mL RNAse (all from Sigma Chemical Co.) and incubated in the dark for 15 min at room temperature 29 . Finally, cell fluorescence was measured by flow cytometry on a BD LSRFortessa cytometer using the BD FACSDiva Software (BD Biosciences, San Jose, CA, EUA) and Flowjo Software 10 (Flowjo LCC, Ashland, OR, EUA). Ten thousand events were evaluated per experiment and cellular debris was omitted from the analysis.
Morphological analysis. To evaluate alterations in morphology, cells were cultured under coverslip and stained with May-Grunwald-Giemsa. Morphological changes were examined by light microscopy using Image-Pro software.
Annexin-V/PI staining assay. For apoptosis detection, we used the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences), and the analysis was performed according to the manufacturer's instructions. The cell fluorescence was determined by flow cytometry as described above. The protection assays using the caspase-3 inhibitor (Z-DEVD-FMK, BD Biosciences) and the antioxidant NAC (Sigma-Aldrich Co.) were performed. In brief, the cells were pre-treated for 2 h with 50 µM Z-DEVD-FMK and for 1 h with 5 mM NAC, followed by incubation with 4 µM of the complex for 48 h. The cells were then trypsinized and the FITC Annexin V Apoptosis Detection assay was conducted as described above.
Caspase-3 activation assay. A caspase-3 colorimetric assay kit (Sigma-Aldrich Co.) was used to investigate caspase-3 activation on complex-treated HCT116 cells based on the cleavage of DEVD-pNA and the analysis was performed according to the manufacturer's instructions. Briefly, cells were lysed by incubation with cell lysis buffer on ice for 10 min and then centrifuged. Enzyme reactions were carried out in a 96-well flat-bottom microplate. To each reaction mixture, 5 μL cell lysate was added. Absorbance at 405 nm was measured using the SpectraMax 190 Microplate Reader (Molecular Devices). The results were expressed as specific activity (IU/mg protein) of caspase-3.
Measurement of the mitochondrial transmembrane potential. Mitochondrial transmembrane potential was determined by the retention of the dye rhodamine 123 30 . Cells were incubated with rhodamine 123 (5 μg/mL, Sigma-Aldrich Co,) at room temperature for 15 min in the dark and washed with saline. The cells were then incubated again in saline at room temperature for 30 min in the dark and cell fluorescence was determined by flow cytometry, as described above.
Measurement of cellular reactive oxygen species levels. The levels of ROS were measured according to previously described 31 using DCF-DA (Sigma-Aldrich Co.). In brief, cells were treated with the complex for 1 and 3 h. Then, the cells were collected, washed with saline and re-suspended in FACS tubes with saline containing 5 μM DCF-DA for 30 min. Finally, the cells were washed with saline and the cell fluorescence was determined by flow cytometry, as described above.
DNA intercalation assay. DNA intercalation was assessed by examining the ability of the complex to displace ethidium bromide from ctDNA (Sigma-Aldrich Co.) 32 . The DNA intercalation assay was conducted in 96-well plate (100 µL) and contained 15 µg/mL ctDNA, 1.5 µM ethidium bromide and 5, 10 and 20 µM of complex in saline solution. The vehicle (0.1% DMSO) used for diluting the compound tested was used as the negative control. Doxorubicin (10 µM) was used as the positive control. Fluorescence was measured using excitation and emission wavelengths of 320 nm and 600 nm, respectively using the spectraMax Microplate Reader (Molecular Devices).
Statistical analysis. Data are presented as mean ± S.E.M. or IC 50 values and their 95% confidence intervals obtained by nonlinear regression. Differences among experimental groups were compared using analysis of variance (ANOVA) followed by the Student-Newman-Keuls test (p < 0.05). All statistical analyses were performed using GraphPad Prism (Intuitive Software for Science, San Diego, CA, USA).