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Acute myeloid leukemia (AML) is a lethal hematologic malignancy caused by leukemic blasts that fail to mature normally. AML has a high relapse rate, primarily due to a small subset known as leukemic stem cells (LSCs). In this work, we investigated the ability of a Ru(II)-thymine complex (RTC) with the formula [Ru(PPh₃)₂(Thy)(bipy)]PF₆ (where PPh₃ = triphenylphosphine, Thy = thymine, and bipy = 2,2'-bipyridine) to suppress AML LSCs. RTC exhibited potent cytotoxicity toward both solid and hematologic malignancies and suppressed primary AML LSCs, as observed by the reduction in the CD34 +CD38- cell population. In the AML cell line KG-1a, which has an LSC-like population, RTC reduced the number of CD34 + and CD123 + cells. A reduction in leukemic blasts was detected in the bone marrow of RTC-treated NSG mice bearing KG-1a xenografts. Increased DNA fragmentation, YO-PRO-1 staining, active caspase-3 and cleaved PARP (Asp 214) levels, and mitochondrial superoxide levels were detected in RTC-treated KG-1a cells. The pancaspase inhibitor Z-VAD-(OMe)-FMK, but not the antioxidant N-acetylcysteine, partially prevented RTC-induced cell death in KG-1a cells, indicating that RTC induces caspase-mediated apoptosis in KG-1a cells via an oxidative stress-independent pathway. In molecular mechanism studies, transcripts of the NF-κB inhibitor NFKBIA were upregulated, and the level of NF-κB p65 phosphorylated at the Ser529 residue was reduced in RTC-treated KG-1a cells, indicating that RTC may inhibit NF-κB signaling. Overall, these results indicate the anti-AML potential of RTC in AML LSCs via the suppression of NF-κB signaling. Show less

Hepatic cancer is one of the main causes of cancer-related death worldwide. Cancer stem cells (CSCs) are a unique subset of cancer cells that promote tumour growth, maintenance, and therapeutic resistance, leading to recurrence. In the present work, the ability of a ruthenium complex containing 1,3-thiazolidine-2-thione (RCT), with the chemical formula [Ru(tzdt)(bipy)(dppb)]PF₆, to inhibit hepatic CSCs was explored in human hepatocellular carcinoma HepG2 cells. RCT exhibited potent cytotoxicity to solid and haematological cancer cell lines and reduced the clonogenic potential, CD133⁺ and CD44^high cell percentages and tumour spheroid growth of HepG2 cells. RCT also inhibited cell motility, as observed in the wound healing assay and transwell cell migration assay. RCT reduced the levels of Akt1, phospho-Akt (Ser473), phospho-Akt (Thr308), phospho-mTOR (Ser2448), and phospho-S6 (Ser235/Ser236) in HepG2 cells, indicating that interfering with Akt/mTOR signalling is a mechanism of action of RCT. The levels of active caspase-3 and cleaved PARP (Asp214) were increased in RCT-treated HepG2 cells, indicating the induction of apoptotic cell death. In addition, RCT modulated the autophagy markers LC3B and p62/SQSTM1 in HepG2 cells and increased mitophagy in a mt-Keima-transfected mouse embryonic fibroblast (MEF) cell model, and RCT-induced cytotoxicity was partially prevented by autophagy inhibitors. Furthermore, mutant Atg5^-/- MEFs and PentaKO HeLa cells (human cervical adenocarcinoma with five autophagy receptor knockouts) were less sensitive to RCT cytotoxicity than their parental cell lines, indicating that RCT induces autophagy-mediated cell death. Taken together, these data indicate that RCT is a novel potential anti-liver cancer drug with a suppressive effect on CSCs. Show less

Cancer stem cells (CSCs) are defined as a rare population of cancer cells related to tumor initiation and maintenance. These cells are primarily responsible for tumor growth, invasion, metastasis, recurrence, and resistance to chemotherapy. In this paper, we demonstrated the ability of Ru(II)-based complexes containing 2-thiouracil derivatives with the chemical formulas trans-[Ru(2TU)(PPh₃)₂(bipy)]PF₆ (1) and trans-[Ru(6m2TU)(PPh₃)₂(bipy)]PF₆ (2) (where 2TU = 2-thiouracil and 6m2TU = 6-methyl-2-thiouracil) to suppress liver CSCs by targeting NF-κB and Akt/mTOR signaling. Complexes 1 and 2 displayed potent cytotoxic effects on cancer cell lines and suppressed liver CSCs from HepG2 cells. Increased phosphatidylserine exposure, loss of mitochondrial transmembrane potential, increased PARP (Asp214) cleavage, DNA fragmentation, chromatin condensation and cytoplasmic shrinkage were detected in HepG2 cells treated with these complexes. Mechanistically, complexes 1 and 2 target NF-κB and Akt/mTOR signaling in HepG2 cells. Cell motility inhibition was also detected in HepG2 cells treated with these complexes. Complexes 1 and 2 also inhibited tumor progression in mice with HepG2 cell xenografts and exhibited tolerable systemic toxicity. Taken together, these results indicate that these complexes are new anti-HCC drug candidates that can suppress liver CSCs. Show less

In this work, we describe a novel ruthenium-xanthoxylin complex, [Ru(phen)₂(xant)](PF₆) (RXC), that can eliminate colorectal cancer (CRC) stem cells by targeting the chaperone Hsp90. RXC exhibits potent cytotoxicity in cancer cell lines and primary cancer cells, causing apoptosis in HCT116 CRC cells, as observed by cell morphology, YO-PRO-1/PI staining, internucleosomal DNA fragmentation, mitochondrial depolarization, and PARP cleavage (Asp214). Additionally, RXC can downregulate the HSP90AA1 and HSP90B1 genes and the expression of HSP90 protein, as well as the expression levels of its downstream/client elements Akt1, Akt (pS473), mTOR (pS2448), 4EBP1 (pT36/pT45), GSK-3β (pS9), and NF-κB p65 (pS529), implying that these molecular chaperones can be molecular targets for RXC. Moreover, this compound inhibited clonogenic survival, the percentage of the CRC stem cell subpopulation, and colonosphere formation, indicating that RXC can eliminate CRC stem cells. RXC reduced cell migration and invasion, decreased vimentin and increased E-cadherin expression, and induced an autophagic process that appeared to be cytoprotective, as autophagy inhibitors enhanced RXC-induced cell death. In vivo studies showed that RXC inhibits tumor progression and experimental metastasis in mice with CRC HCT116 cell xenografts. Taken together, these results highlight the potential of the ruthenium complex RXC in CRC therapy with the ability to eliminate CRC stem cells by targeting the chaperone Hsp90. Show less

[Ru(5-FU)(PPh₃)₂(bipy)]PF₆ (Ru/5-FU) is a novel ruthenium complex with 5-fluorouracil with promising potential against colorectal cancer (CRC). In the present study, we investigated the molecular mechanism of Ru/5-FU action in HCT116 CRC cells. Ru/5-FU exhibited potent cytotoxicity on a panel of cancer cell lines and on primary cancer cells and induced apoptosis in HCT116 CRC cells. Ru/5-FU reduced AKT1 gene transcripts, as well as the expression of Akt1 and Akt (pS473) and downstream Akt proteins mTOR (pS2448), S6 (pS235/pS236), 4EBP1 (pT36/pT45), GSK-3β (pS9) and NF-κB p65 (pS529), but not Akt upstream proteins Hsp90 and PI3K p85/p55 (pT458/pT199), indicating an inhibitory action of Akt/mTOR signaling. Ru/5-FU increased LC3B expression and reduced p62/SQSTM1 levels, indicating autophagy induction. Curiously, the autophagy inhibitors 3-methyladenine and chloroquine increased Ru/5-FU-induced cell death, indicating an induction of cytoprotective autophagy by this compound. Ru/5-FU also reduced clonogenic survival, as well as the percentage of CD133+ cells and colonosphere formation, indicating that Ru/5-FU can suppress stem cells in HCT116 cells. Ru/5-FU inhibited cell migration and invasion in wound healing assays and Transwell cell invasion assays, along with a reduction in vimentin expression and an increase in E-cadherin levels, indicating that Ru/5-FU can interfere with epithelial-mesenchymal transition. Ru/5-FU also inhibited in vivo HCT116 cell development and experimental lung metastases in mouse xenograft models. Altogether, these results indicate that Ru/5-FU is an anti-CRC chemotherapy drug candidate with the ability to suppress stemness in CRC cells by inhibiting Akt/mTOR signaling. Show less

Two new Ru(II)-based complexes containing 2-thiouracil derivatives, known as 2-thiouracil (2TU) and 6-methyl-2-thiouracil (6m2TU), were synthesized using cis,trans-[RuCl₂(PPh₃)₂(bipy)] as a precursor. The obtained compounds with a general formula trans-[Ru(2TU)(PPh₃)₂(bipy)]PF₆ (1) and trans-[Ru(6m2TU)(PPh₃)₂(bipy)]PF₆ (2) were characterized by analytical techniques such as NMR, UV-vis, and IR spectroscopies, elementary analysis, mass spectrometry, and single-crystal X-ray diffraction. Moreover, the investigation of the complexes-DNA interaction were carried out using spectrophotometric titrations and showed that the complexes present a weak interaction with this biomolecule. The compounds were evaluated against HL-60, K-562, HepG2, and B16-F10 cancer cells and against noncancer cells (PBMCs). The results of the biological assay revealed that complex 2 is more promising than complex 1. Finally, the present study suggests that complexes 1 and 2 causes cell death by apoptosis, significantly increasing the percentage of apoptotic HL-60 cells, in which the compounds altered the cell cycle, reducing the cells in G₁/G₀, G₂/M, and S phases. Show less

We report on chemistry and cytotoxic studies of four new ruthenium (II) complexes containing uracil derivatives. All compounds are neutral, presenting the formula [Ru(PPh₃)₂(2TU)₂] (1), [Ru(PPh₃)₂(6m2TU)₂] (2), [Ru(dppb)(2TU)₂] (3) and [Ru(dppb)(6m2TU)₂] (4), where PPh₃ = triphenylphosphine; dppb = 1,4-bis(diphenylphosphino)butane, 2TU = 2-thiouracil and 6m2TU = 6-methyl-2-thiouracil. They were characterized using NMR, UV-vis and IR spectroscopies, microanalytical analysis and mass spectrometry. Furthermore, the crystal structures of 1-4 were determined by single-crystal X-ray diffraction. The coordination of 2-thiouracil derivatives with ruthenium increases regions able to carry out hydrogen bonds with the biological targets, such as DNA. We evaluated the interaction of the complexes with DNA by UV/Vis spectrophotometric titration, and as a result, the values of DNA-binding constants are in the range of 0.8-1.8 × 10⁴ M^-1. Moreover, the interaction of the complexes with BSA was investigated. In vitro, activities against B16-F10 (mouse melanoma), HepG2 (human hepatocellular carcinoma), HL-60 (human promyelocytic leukemia) and K562 (human chronic myelocytic leukemia) and non-tumor cells: PBMC (human peripheral blood mononuclear cells activated with concanavalin A - human lymphoblast) were carried out. Cytotoxicity assays revealed that complexes (2) and (4) present biological activity against tumor cells comparable with oxaliplatin, the reference platinum drug, revealing that they are promising molecules for developing new antitumor compounds. Show less

Herein, ruthenium complexes containing heterocyclic thioamidates [Ru(mmi)(bipy)(dppb)]PF₆ (1), [Ru(tzdt)(bipy)(dppb)]PF₆ (2), [Ru(dmp)(bipy)(dppb)]PF₆ (3) and [Ru(mpca)(bipy)(dppb)]PF₆ (4) were investigated for their cellular and molecular effects in cancer cell lines. Complexes 1 and 2 were the most potent of the four compounds against a panel of different cancer cell lines in monolayer cultures and showed potent cytotoxicity in a 3D model of multicellular spheroids that formed from human hepatocellular carcinoma HepG2 cells. In addition, both complexes were able to bind to DNA in a calf thymus DNA model. Compared to the controls, a reduction in cell proliferation, phosphatidylserine externalization, internucleosomal DNA fragmentation, and the loss of the mitochondrial transmembrane potential were observed in HepG2 cells that were treated with these complexes. Additionally, coincubation with a pan-caspase inhibitor (Z-VAD(OMe)-FMK) reduced the levels of apoptosis that were induced by these compounds compared to those in the negative controls, indicating that cell death through apoptosis occurred via a caspase-dependent pathway. Moreover, these complexes also induced the phosphorylation of ERK1/2, and coincubation with an MEK inhibitor (U0126), which is known to inhibit the activation of ERK1/2, but not JNK/SAPK and p38 MAPK inhibitors, reduced the complexes-induced apoptosis compared to that in the negative controls, indicating that the induction of apoptotic cell death occurred through ERK1/2 signaling in HepG2 cells. On the other hand, no increase in oxidative stress was observed in HepG2 cells treated with the complexes, and the complexes-induced apoptosis was not reduced with coincubation with the antioxidant N-acetylcysteine or a p53 inhibitor compared to that in the negative controls, indicating that apoptosis occurred via oxidative stress- and p53-independent pathways. Finally, these complexes also reduced the growth of HepG2 cells that were engrafted in C.B-17 SCID mice compared to that in the negative controls. These results indicated that these complexes are novel anticancer drug candidates for liver cancer treatment. Show less

Ruthenium(II) complexes with 6-methyl-2-thiouracil cis-[Ru(6m2tu)₂(PPh₃)₂] (1) and [Ru(6m2tu)₂(dppb)] (2) (where PPh_3 =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. Show less

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. Show less

Ruthenium-based compounds represent a class of potential antineoplastic drugs. Recently, we designed, synthesized, and identified the Ru(II)-thymine complex [Ru(PPh₃)₂(Thy)(bipy)]PF₆ (where PPh = triphenylphosphine, Thy = thymine and bipy = 2,2'-bipyridine) as a potent cytotoxic agent with the ability to bind to DNA and human and bovine serum albumins. In this study, the underlying cytotoxic mechanism of the [Ru(PPh₃)₂(Thy)(bipy)]PF₆ complex was assessed. This complex displayed potent cytotoxicity in different cancer cell lines; the morphology that is associated with 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 human promyelocytic leukemia HL-60 cells that were treated with the complex. Moreover, pretreatment of HL-60 cells with Z-VAD(OMe)-FMK, a pan-caspase inhibitor, partially reduced the apoptosis that was induced by the complex, indicating that the apoptotic cell death occurred through a caspase-mediated pathway. In conclusion, the [Ru(PPh₃)₂(Thy)(bipy)]PF₆ complex displays potent cytotoxicity to different cancer cells and induces caspase-mediated apoptosis in HL-60 cells. Show less

Ruthenium-based compounds have gained great interest due to their potent cytotoxicity in cancer cells; however, much of their potential applications remain unexplored. In this paper, we report the synthesis of a novel ruthenium complex with xanthoxylin (RCX) and the investigation of its cellular and molecular action in human hepatocellular carcinoma HepG2 cells. We found that RCX exhibited a potent cytotoxic effect in a panel of cancer cell lines in monolayer cultures and in a 3D model of multicellular cancer spheroids formed from HepG2 cells. This compound is detected at a high concentration in the cell nuclei, induces DNA intercalation and inhibits DNA synthesis, arresting the cell cycle in the S-phase, which is followed by the activation of the caspase-mediated apoptosis pathway in HepG2 cells. Gene expression analysis revealed changes in the expression of genes related to cell cycle control, apoptosis and the MAPK pathway. In addition, RCX induced the phosphorylation of ERK1/2, and pretreatment with U-0126, an MEK inhibitor known to inhibit the activation of ERK1/2, prevented RCX-induced apoptosis. In contrast, pretreatment with a p53 inhibitor (cyclic pifithrin-α) did not prevent RCX-induced apoptosis, indicating the activation of a p53-independent apoptosis pathway. RCX also presented a potent in vivo antitumor effect in C.B-17 SCID mice engrafted with HepG2 cells. Altogether, these results indicate that RCX is a novel anticancer drug candidate. Show less

Piplartine (piperlongumine) is a plant-derived molecule that has been receiving intense interest due to its anticancer characteristics that target the oxidative stress. In the present paper, two novel piplartine-containing ruthenium complexes [Ru(piplartine)(dppf)(bipy)](PF₆)₂ (1) and [Ru(piplartine)(dppb)(bipy)](PF₆)₂ (2) were synthesized and investigated for their cellular and molecular responses on cancer cell lines. We found that both complexes are more potent than metal-free piplartine in a panel of cancer cell lines on monolayer cultures, as well in 3D model of cancer multicellular spheroids formed from human colon carcinoma HCT116 cells. Mechanistic studies uncovered that the complexes reduced the cell growth and caused phosphatidylserine externalization, internucleosomal DNA fragmentation, caspase-3 activation and loss of the mitochondrial transmembrane potential on HCT116 cells. Moreover, the pre-treatment with Z-VAD(OMe)-FMK, a pan-caspase inhibitor, reduced the complexes-induced apoptosis, indicating cell death by apoptosis through caspase-dependent and mitochondrial intrinsic pathways. Treatment with the complexes also caused a marked increase in the production of reactive oxygen species (ROS), including hydrogen peroxide, superoxide anion and nitric oxide, and decreased reduced glutathione levels. Application of N-acetyl-cysteine, an antioxidant, reduced the ROS levels and apoptosis induced by the complexes, indicating activation of ROS-mediated apoptosis pathway. RNA transcripts of several genes, including gene related to the cell cycle, apoptosis and oxidative stress, were regulated under treatment. However, the complexes failed to induce DNA intercalation. In conclusion, the complexes are more potent than piplartine against different cancer cell lines and are able to induce caspase-dependent and mitochondrial intrinsic apoptosis on HCT116 cells by ROS-mediated pathway. Show less