Ruthenium(II) complexes with 6-methyl-2-thiouracil selectively reduce cell proliferation, cause DNA double-strand break and trigger caspase-mediated apoptosis through JNK/p38 pathways in human acute promyelocytic leukemia cells

Ruthenium(II) complexes with 6-methyl-2-thiouracil cis-[Ru(6m2tu)2(PPh3)2] (1) and [Ru(6m2tu)2(dppb)] (2) (where PPh3 = triphenylphosphine; dppb = 1,4-bis(diphenylphosphino)butane; and 6m2tu = 6-methyl-2-thiouracil) are potent cytotoxic agents and able to bind DNA. The aim of this study was to evaluate in vitro cellular underlying mechanism and in vivo effectiveness of these ruthenium(II) complexes in human acute promyelocytic leukemia HL-60 cells. Both complexes displayed potent and selective cytotoxicity in myeloid leukemia cell lines, and were detected into HL-60 cells. Reduction of the cell proliferation and augmented phosphatidylserine externalization, caspase-3, -8 and -9 activation and loss of mitochondrial transmembrane potential were observed in HL-60 cells treated with both complexes. Cotreatment with Z-VAD(OMe)-FMK, a pan-caspase inhibitor, reduced Ru(II) complexes-induced apoptosis. In addition, both metal complexes induced phosphorylation of histone H2AX (S139), JNK2 (T183/Y185) and p38α (T180/Y182), and cotreatment with JNK/SAPK and p38 MAPK inhibitors reduced complexes-induced apoptosis, indicating DNA double-strand break and activation of caspase-mediated apoptosis through JNK/p38 pathways. Complex 1 also reduced HL-60 cell growth in xenograft model. Overall, the outcome indicated the ruthenium(II) complexes with 6-methyl-2-thiouracil as a novel promising antileukemic drug candidates.

In vitro assays. Cells. HL-60 (human acute promyelocytic leukemia), K-562 (human chronic myelogenous leukemia), HCT116 (human colon carcinoma), HepG2 (human hepatocellular carcinoma), HSC-3 (human oral squamous cell carcinoma), SCC-9 (human oral squamous cell carcinoma), B16-F10 (mouse melanoma), MRC-5 (human lung fibroblast), WT SV40 MEF (wild-type immortalized mouse embryonic fibroblast) and BAD KO SV40 MEF (BAD gene knockout immortalized mouse embryonic fibroblast) cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Human peripheral blood mononuclear cells (PBMC) were isolated using standard Ficoll density gradient from heparinized blood collected from 20-to 35-year-old, non-smoker healthy donors with informed consent (number 031019/2013) approved by Human Ethics Committee of Gonçalo Moniz Institute from Oswaldo Cruz Foundation (IGM-FIOCRUZ/BA), and all experiments were performed in accordance with relevant guidelines and regulations. Cells were cultured as recommended by ATCC guidelines and a mycoplasma stain kit (Sigma-Aldrich) was used to validate the use of cells free from contamination. Cell viability in all experiments was examined using the trypan blue exclusion (TBE) assay. Over 90% of the cells were viable at the beginning of the culture. Apoptosis quantification assay. FITC Annexin V Apoptosis Detection Kit I (ID 556547) (BD Biosciences) was used for apoptosis quantification and the analysis was performed according to the manufacturer's instructions. Shortly, cells were washed twice with saline solution and resuspended in 100 μL of binding buffer plus 5 μL of propidium iodide (PI) and 5 μL of FITC Annexin V. Then, cells were gently mixed by vortexing and incubated for 15 min at room temperature in the dark. Finally, 400 μL of binding buffer was added to each tube, and the cell fluorescence was determined by flow cytometry, as described above. Percentage of viable, early apoptotic, late apoptotic and necrotic cells were measured. Protection assays using a pan-caspase inhibitor (Z-VAD(Ome)-FMK, Cayman Chemical; Ann Arbor, MI, USA), JNK/SAPK inhibitor (SP 600125; Cayman Chemical), p38 MAPK inhibitor (PD 169316; Cayman Chemical) and MEK inhibitor (U-0126; Cayman Chemical), were also evaluated. In these assays, cells were preincubated for 2 h with 50 µM Z-VAD(Ome)-FMK, 5 µM U-0126, 5 µM SP 600125 or 5 µM PD 169316, followed by incubation with 4 µM of complexes 1 and 2 for 24 h. Cells were then analyzed by FITC Annexin V Apoptosis Detection assay as described above.
Measurement of mitochondrial transmembrane potential. Mitochondrial transmembrane potential was determined by retention of dye rhodamine 123 as described previously 24 . Briefly, cells were incubated with rhodamine 123 (5 μg/mL, Sigma-Aldrich Co.) at room temperature for 15 min in dark and washed with saline solution. Cells were incubated again in saline solution for more 30 min in dark and cell fluorescence was determined by flow cytometry as described above.
Caspase-3, -8 and -9 activation assays. To investigate the activation of caspase-3, -8 and -9, we used caspase-3 colorimetric assay kit (ID K106-100), caspase-8 colorimetric assay kit (ID K113-100) and caspase-9 colorimetric assay kit (ID K119-100) (all from BioVision Inc.; Milpitas, CA, USA), and the analysis were performed according to the manufacturer's instructions. Enzyme reactions were performed in a 96-well microplate, and to each reaction mixture, 5 μL of cell lysate was added. Total protein quantification was performed in each sample by Bradford assay using bovine serum albumin (BSA) as standard. Absorbance at 405 nm was measured using a SpectraMax 190 Microplate Reader (Molecular Devices).
Measurement of cellular reactive oxygen species levels. The levels of intracellular reactive oxygen species (ROS) were measured according to previously described 25 using 2′,7′-dichlorofluorescin diacetate (DCF-DA, Sigma-Aldrich Co.). Shortly, cells were washed with saline solution and resuspended in saline solution containing 5 μM of DCF-DA for 30 min in dark at room temperature. Finally, cells were washed with saline solution and cell fluorescence was measured by flow cytometry as described above. Protection assay using the antioxidant N-acetyl-L-cysteine (NAC, Sigma-Aldrich Co.) was also evaluated. In brief, cells were preincubated for 1 h with 5 mM of NAC, followed by incubation with 4 µM of complexes 1 and 2 for 24 h. Cells were then analyzed by FITC Annexin V Apoptosis Detection assay as described above.
Human myeloid leukemia xenograft model. Human myeloid leukemia xenograft model was carry out as described previously by Rodrigues et al. 26 with minor modifications. HL-60 cells (2.5 × 10 7 cells/500 µL) were implanted subcutaneously into the left front armpit of the mice. At the beginning of the experiment, mice were randomly divided into four groups: group 1 animals treated with the vehicle 5% DMSO solution (negative control, n = 14); group 2 animals treated with doxorubicin (positive control, 0.1 mg/kg, n = 14); group 3 animals treated with complex 1 at 20 mg/kg (n = 14); and group 4 animals treated with complex 1 at 40 mg/kg (n = 14). When the tumors reached 100 to 200 mm 3 (22 days after HL-60 cells injection), the animals were treated through the intraperitoneal route (200 µL per animal) once a day for 13 consecutive days. One day after the end of the treatment, the animals were anesthetized, and peripheral blood samples were collected from brachial artery. Animals were euthanized by anesthetic overdose, and tumors were excised and weighed.
Toxicological evolution. To assess toxicological aspects, mice were weighed at the beginning and at the end of the experiment as described previously by Rodrigues et al. 26 . Animals were observed for signs of abnormalities throughout the study. Hematological analysis was performed using the Advia 60 hematology system (Bayer, Leverkusen, Germany). Livers, kidneys, lungs and hearts were removed, weighed and examined for any signs of macroscopic lesions, color changes and/or hemorrhages. After macroscopic examination, tumors, livers, kidneys, lungs and hearts were fixed in 4% formalin buffer and embedded in paraffin. Tissue sections were stained with hematoxylin/eosin staining, and a pathologist performed the histological analyses under optical microscopy.
Statistical analysis. Data are presented as mean ± S.E.M. or inhibitory concentration of 50% (IC 50 ) values with their respective 95% confidence intervals obtained by nonlinear regression. Analysis of variance (ANOVA) followed by Student-Newman-Keuls test was used to check differences between experimental groups (p < 0.05). Statistical analysis was carry out using GraphPad Prism software (Intuitive Software for Science, San Diego, CA, USA).
We confirmed the effect of ruthenium(II) complexes with 6-methyl-2-thiouracil on cell viability and proliferation by trypan blue exclusion assay in HL-60 cells, after 12 and 24 h of treatment (Fig. 2). After 12 h of treatment, complex 1 reduced the number of viable cells by 36.9%, and complex 2 reduced 44.1% at concentration of 2 μM, respectively (no statistically significant reduction was observed with treatment of 1 μM). www.nature.com/scientificreports www.nature.com/scientificreports/ Intracellular quantification of ruthenium was assessed with an energy dispersive X-ray spectrometer in HL-60 cells treated with ruthenium(II) complexes with 6-methyl-2-thiouracil after 3 h of incubation (Fig. 3). Cisplatin and oxaliplatin were used as positive controls, and intracellular quantification of platinum was also measured. We were able to detect ruthenium in HL-60 cells treated with both complexes, as well as, we were able to quantify platinum in HL-60 cells treated with cisplatin and oxaliplatin.
Ruthenium(II) complexes with 6-methyl-2-thiouracil trigger caspase-mediated apoptosis in HL-60 cells. Using a light microscope, we analyzed the effect of ruthenium(II) complexes with 6-methyl-2-thiouracil in the cell morphology of HL-60 cells stained with May-Grunwald-Giemsa. Both complexes caused reduction in the cell volume, vacuolization, chromatin condensation and DNA fragmentation were observed after treatment with both complexes (Fig. 4). Moreover, we also found cell shrinkage, as observed by the decrease in forward light scattering (FSC), and nuclear condensation, as observed by an increase in lateral dispersion (SSC), in HL-60 cells treated with both complexes (Fig. 5). These alterations are consistent with apoptotic cell death. The treatment with doxorubicin also presented changes associated with apoptosis.  www.nature.com/scientificreports www.nature.com/scientificreports/ To confirm the apoptosis induction by ruthenium(II) complexes with 6-methyl-2-thiouracil in HL-60 cells, annexin V/propidium iodide double staining was performed to measure phosphatidylserine exposure and loss of membrane integrity, respectively, and the numbers of viable, early apoptotic, late apoptotic and necrotic cells were quantified. Both complexes strongly increased apoptotic cells after 12 and 24 h of treatment (Fig. 6). After 12 h of treatment, complex 1 led 37.2% of apoptosis, while complex 2 caused 31.1% at concentration of 2 μM, respectively (no statistically significant apoptosis induction was observed with the treatment of 1 μM). After 24 h of treatment, complex 1 led 38.5 and 66.4% of apoptosis at concentrations of 1 and 2 μM, respectively, and complex 2 caused 32.1 and 55.9%. Doxorubicin also induced apoptosis in HL-60 cells.  www.nature.com/scientificreports www.nature.com/scientificreports/ Mitochondrial transmembrane potential was also examined in HL-60 cells treated with ruthenium(II) complexes with 6-methyl-2-thiouracil using the retention of dye rhodamine 123 assay by flow cytometry. Both complexes caused loss of the mitochondrial transmembrane potential (Fig. 7). Next, activation of the effector (caspase-3) and initiator (caspases-8 and -9) caspases was also studied. Both complexes induced the activation of all caspases analyzed (Fig. 8). In addition, cotreatment with a pan-caspase inhibitor (Z-VAD(OMe)-FMK) reduced the apoptosis caused by both complexes, indicating a caspase-mediated apoptotic cell death. (Fig. 9). Doxorubicin also induced depolarization of mitochondrial transmembrane potential and led to apoptosis through caspases pathways in HL-60 cells.
Finally, viability of BAD (Bcl-2-associated death promoter) mutant cell line BAD KO SV40 MEF (immortalized mouse embryonic fibroblast with the BAD gene knocked out) and its parental cell line WT SV40 MEF (wild-type immortalized mouse embryonic fibroblasts) were examined after 72 h of treatment with ruthenium(II) complexes with 6-methyl-2-thiouracil by alamar blue assay to assess the role of BAD protein in cytotoxicity caused by these complexes. BAD is an important pro-apoptotic protein belong to Bcl-2 family that is involved in early stages of apoptosis. The effect of ruthenium(II) complexes with 6-methyl-2-thiouracil on ROS levels was also evaluated in HL-60 cells. However, the complexes did not increase significantly ROS levels after 1 or 3 h of incubation (data not shown). In addition, cotreatment with the antioxidant NAC did not reduce the apoptosis induced by complexes (data not shown).

Ruthenium(II) complex with 6-methyl-2-thiouracil reduces HL-60 cell growth in xenograft model.
In vivo anti-leukemia activity of ruthenium(II) complex with 6-methyl-2-thiouracil was studied in C.B-17 SCID mice engrafted with HL-60 cells. Since complex 1 was more potent than complex 2, only complex 1 was used in the in vivo anti-leukemia model. When the tumors reached 100 to 200 mm 3 (22 days after HL-60 cells injection), the animals were treated with complex 1 at doses of 20 and 40 mg/kg by intraperitoneal injections once a day for 13 consecutive days. Both doses were able to inhibit HL-60 cell development in mice (Fig. 13A,B). In the end of the treatment, the mean of tumor mass weight of the negative control animals was 1.4 ± 0.4 g. In the group of the animals treated with lower and higher doses of complex 1, the mean of tumor mass weights was 0.4 ± 0.1 and 0.3 ± 0.1 g, respectively. Tumor mass inhibition rate were 73.1 and 79.9%, respectively. Doxorubicin, at dose www.nature.com/scientificreports www.nature.com/scientificreports/ of 0.1 mg/kg, reduced tumor weight by 62.0%. In the histomorphological analyses, tumors exhibited malignant cells of abundant and granular cytoplasm, with 2-or more distinct nucleoli with a predominant myeloid morphology. These cells were often arranged in agglomerates with sparse/or without extracellular matrix, mostly in negative control and doxorubicin groups. For the groups treated with complex 1 (20 and 40 mg), areas with nodular/ encapsulated growth and extracellular matrix formation were more evident, but multifocal areas of necrosis was more frequent in the negative control and doxorubicin groups (Fig. 13B).
Body weight of the animals at the beginning and at the end of the experiment, hematological analysis of peripheral blood, wet weight of liver, kidney, lung and heart, and their histological analysis, were performed to evaluate toxicology characteristics of ruthenium(II) complex with 6-methyl-2-thiouracil treatment in mice. Only doxorubicin treatment decreased body weight of C.B-17 SCID mice bearing HL-60 cells (P < 0.05). No significant chances were found in liver, kidney, lung or heart weight of none group (P > 0.05) (data not shown). Moreover, no alterations were found in hematological parameters of peripheral blood of any group (P > 0.05) (data not shown).
Morphological analyses of liver, kidneys, lungs and hearts in all groups were performed. In the livers, acinar architecture and centrilobular vein were also preserved in all groups. Focal areas of inflammation and coagulation necrosis were observed in all experimental groups. Additionally, focal areas of steatosis were observed in the negative control and complex 1 (20 and 40 mg/kg) groups. Other findings, such as congestion and hydropic www.nature.com/scientificreports www.nature.com/scientificreports/ degeneration were found in all groups, ranging from mild to moderate. In the lungs, architecture of the parenchyma was partially maintained in all groups, observing a thickening of the alveolar septum with decreased airspace, ranging from mild to moderate. Histopathological analyses of lungs revealed significant inflammation predominantly of mononuclear cells, edema, congestion and hemorrhage, ranging mild to severe. It is important to note that the inflammation was more evident in animals treated with complex 1 (20 and 40 mg/kg). In addition, tumor nodules were observed only in one animal treated with doxorubicin. In the kidneys, tissue architecture was preserved in all experimental groups. Histopathological changes included vascular congestion and thickening of basal membrane of renal glomerulus with decreased urinary space were observed in all kidneys, ranging from mild to moderate. Coagulation necrosis was observed in renal tubules in groups treated with doxorubicin and complex 1 (20 and 40 mg/kg). Histopathological analysis of animal hearts did not show alterations in any group.
JNK/SAPK (isoforms JNK-1, JNK-2 and JNK-3), p38 MAPK (isoforms p38α, p38β, p38γ and p38δ) and ERK1/2 pathways belong to the MAPK family and are involved in different cellular responses, including both cancer cell proliferation and cell death. Interestingly, DNA damage-induced cell death have been involved with activation of JNK and p38 MAPK by expression of pro-apoptotic factors 28 . In fact, we demonstrated that ruthenium(II) complexes with 6-methyl-2-thiouracil induced DNA double-strand break and trigger caspase-mediated apoptosis through JNK/p38 pathways in HL-60 cells. On the other hand, ERK1/2 pathway may active both pro-survival and pro-apoptotic factors. During DNA damage stimuli, e.g. exposition to platinum complexes and ionizing radiation, activation of ERK1/2 pathway causes apoptosis [29][30][31] . Herein, we demonstrated that ERK1/2 pathway is not essential to the apoptosis induced by ruthenium(II) complexes with 6-methyl-2-thiouracil in HL-60 cells.
In recent studies, ruthenium(II) complex with methylimidazole induced cell cycle arrest at G 0 /G 1 phase and caused apoptosis through ROS, MAPK and AKT signaling pathways in human lung carcinoma A549 cells 32 , meanwhile ruthenium(II) complex with xanthoxylin caused S-phase arrest and ERK1/2-mediated apoptosis in HepG2 cells by a p53-independent pathway 10 . Similar results were found by Neves and collaborators 16 with ruthenium complexes containing heterocyclic thioamidates that caused caspase-mediated apoptosis through MAPK signaling in HepG2 cells. Ruthenium(II) complexes with piplartine induced MAPK-and p53-dependent apoptosis in HCT116 cells by a ROS-mediated pathway 7,15 . Apoptosis in A549 cells by mitochondrial homeostasis . Cells were pre-treated for 2 h with 50 µM Z-VAD(OMe)-FMK, then incubated with the complexes at 2 μM for 24 h. Negative control (CTL) was treated with vehicle (0.2% DMSO) used for diluting the complexes, and doxorubicin (DOX, 1 µM) was used as positive control. Data are presented as mean ± S.E.M. of at least three independent experiments performed in duplicate. Ten thousand events were evaluated per experiment, and cellular debris was omitted from analysis. *p < 0.05 compared with negative control by ANOVA followed by Student Newman-Keuls test. # p < 0.05 compared with respective treatment without inhibitor by ANOVA followed by Student Newman-Keuls test. Figure 10. Effect of ruthenium(II) complexes with 6-methyl-2-thiouracil in phospho-histone H2AX (S139) expression, as determined by phospho-specific ELISA in HL-60 cells treated with the complexes at 2 µM for 24 h incubation. Negative control (CTL) was treated with vehicle (0.2% DMSO) used for diluting the complexes, and doxorubicin (DOX, 1 µM) was used as positive control. Data are presented as mean ± S.E.M. of at least three independent experiments performed in duplicate. *p < 0.05 compared with negative control by ANOVA followed by Student Newman-Keuls test.  . For protection assays, cells were pretreated for 2 h with 5 µM U-0126, 5 µM SP 600125 or 5 µM PD 169316 and then incubated with the complexes at 2 µM for 24 h. Negative control (CTL) was treated with vehicle (0.2% DMSO) used for diluting the complexes, and doxorubicin (DOX, 1 µM) was used as positive control. Data are presented as mean ± S.E.M. of at least three independent experiments performed in duplicate. Ten thousand events were evaluated per experiment, and cellular debris was omitted from analysis. *P < 0.05 compared with negative control by ANOVA, followed by Student-Newman-Keuls test. # P < 0.05 compared with respective treatment without inhibitor by ANOVA, followed by Student-Newman-Keuls test. (2019) 9:11483 | https://doi.org/10.1038/s41598-019-47914-x www.nature.com/scientificreports www.nature.com/scientificreports/ destruction and death receptor signaling pathways can be also induced by ruthenium(II) polypyridyl complex 33 . Mazuryk and collaborators 34 revealed that ruthenium(II) complexes with nitroimidazole derivatives of polypyridyl caused caspase-independent cell death by ROS formation, including hydrogen peroxide and peroxyl radicals, and intracellular Ca 2+ homeostasis disruption in human pancreas carcinoma PANC-1 cells. Ruthenium(II) complexes containing 5-fluorouracil 11 and thymine 12 also led caspase-mediated apoptosis in HCT116 and HL-60 cells, respectively.
Complex 1 also inhibited HL-60 cell growth in xenograft model. In mice bearing A549 xenografts, ruthenium(II) imidazole complex also reduced the cancer cell growth 35 . Combination of ruthenium(II)-arene complex and erlotinib inhibited in vivo A2780 cell (human ovarian carcinoma) development in a xenograft tumor model 36 . Ruthenium complex with phenylterpyridine derivative inhibited in vivo A375 (human skin melanoma) cell development in a xenograft tumor model 37 . Moreover, ruthenium(II) complex with xanthoxylin also presented in vivo antitumor effect in C.B-17 SCID mice engrafted with HepG2 cells 10 . When the tumors reached 100 to 200 mm 3 , the animals were treated through the intraperitoneal route for 13 consecutive days. Negative control (CTL) was treated with vehicle (5% DMSO) used for diluting the complexes, and doxorubicin (DOX, 0.1 mg/kg) was used as positive control.