Cytotoxic effects of the compound cis-tetraammine(oxalato)ruthenium(III) dithionate on K-562 human chronic myelogenous leukemia cells

Leukemia is a major type of cancer affecting a significant segment of the population, and especially children. In fact, leukemia is the most frequent childhood cancer, with 26% of all cases, and 20% mortality (Canadian Cancer Society/National Cancer Institute of Canada2005). The American Cancer Society (ACS) estimated that 47,150 new cases of leukemia would be diagnosed in the United States in 2012, whereas about 23,540 adults and children would die of leukemia during 2012 (American Cancer Society2009).

Although the incidence rate for this disease remains relatively unchanged, some success has fortunately been attained in its treatment. But even if the success of clinical trials in identifying new agents and treatment modalities has been significant, current treatments have many limitations related to their side effects and the development of acquired drug resistance (Robert and Jarry2003) The new therapeutic agents thus needed should be more active and produce less side effects and they also should act through a mechanism different from that of cytotoxic agents already used (Menezes et al.2007; Silveira-Lacerda et al.2009).

Chemotherapy is a common treatment for leukemia (Ge et al.2009). In general the therapy uses a number of different anticancer drugs, which destroy cancer cells by preventing them from growing and dividing rapidly. Unfortunately, a number of the body’s normal, non-cancerous cells (e.g., hair cells, red and white blood cells, blood-clotting platelets, and cells of the gastrointestinal mucosa) also divide rapidly and are harmed by chemotherapy (Ge et al.2009; Kim et al.2002). The side effects of chemotherapy hamper many normal activities of patients undergoing treatment (Kim et al.2002).

The preparations of metallo complexes with potential antitumor activity has been one of the main targets of transition metal chemistry since Rosenberg’s discovery of cisplatin {cis-diamminedichloridoplatinum(II), cis-[Pt(NH₃)₂Cl₂]} cytotoxic activity in the 1960s (Rosenberg et al.1965). In 1978, cisplatin was approved as the first platinum based drug for the oncology treatment, although several negative side-effects (nephrotoxicity, neurotoxicity, nausea, etc.) had been induced on treated patients (Kelland and Farrell2000). Nevertheless, cisplatin was followed by carboplatin {cis-diammine-1,1′ -cyclobutanedicarboxylateplatinum(II), [Pt(NH₃)₂(cbdc)], approved in 1985} and oxaliplatin {1R,2R-diamminocyclohexaneoxalatoplatinum(II), [Pt(dach)(ox)], approved in 1996}, which met requirements of improving antitumor activity and reducing disadvantages of cisplatin, carboplatin and oxaliplatin represent the second, and third platinum-based drug generations, respectively ((Kelland and Farrell2000; Štarha et al.2009). Nowadays, not only platinum-bearing complexes are extensively studied with the aim to broaden a spectrum of transition metal-based complexes which could be used in the treatment of cancer (Štarha et al.2009).

In addition to the other metal complexes cisplatin have been developed using heavy metals. For example, gold complexes have been developed for the treatment of rheumatoid arthritis, silver complexes as anti-microbial agents, antimony complexes for the treatment of leishmaniasis, vanadium(IV) complexes as antiviral and antidiabetic agents, arsenic trioxide (Trisenox) for the treatment of acute promyelocytic leukaemia, and metal-activated bleomycin for the treatment of Hodgkin’s lymphoma and testicular cancer. Transition-metal-based therapeutic agents currently under clinical trials include third generation antitumor platinum complexes such as liposomal cisplatin (Lipoplatin), satraplatin, and picoplatin, the antimalarial ferrocene–quinoline conjugate ferroquine (Lung et al.2013).

Rhodium and Iridium has also been used for the synthesis of these metal complexes, as they allow greater selectivity and affinity molecules. The iridium (III) already shows results inhibiting TNF-α in HepG2 line through a hydrophobic binding (Meggers2009; Leung et al.2012). These metal complexes possess many advantages that make them suitable for the development of new therapeutic agents and compounds of ruthenium has been highlighted because ruthenium complexes have shown potential utility in chemotherapy and photodynamic therapy (Clarke2003; Ronconi and Sadler2007). Ruthenium complexes generally have lower toxicities compared to cisplatin attributed to their specific accumulation in cancer tissues (Sava et al.1998). In vitro and in vivo studies show high anticancer activity of ruthenium complexes and some of them are currently undergoing clinical trials (Jakupec et al.2005; Hartinger et al.2006).

The identification of new chemotherapeutics agents is critical for further progress in the treatment of leukemia. In comparison to the platinum(II) antitumor complexes currently used in the clinic, ruthenium compounds offer potentially reduced toxicity, a novel mechanism of action, the prospect of non-cross-resistance, and a different spectrum of activity (Clarke2003). The reduced toxicity is in part due to the ability of ruthenium complexes to mimic the binding of iron to molecules of biological significance, exploiting the mechanisms that the body has evolved for non-toxic transport of iron (Frasca et al.2001). This reduced toxicity, together with non-cross-resistance in cisplatin-resistant cancer cells, is particularly attractive attributes of these complexes (Allardyce et al.2003).

Based on these evidences, in the present work we studied the cytotoxic activity of the ruthenium(III) compound cis-tetraammine(oxalato)ruthenium(III) dithionate {cis-[Ru(C₂O₄)(NH₃)₄]₂(S₂O₆)} against human chronic myelogenous leukemia (K-562) tumor cell line.

Apoptosis is an active physiological process resulting in cellular self-destruction that involves specific morphological and biochemical changes in the nucleus and cytoplasm (Kostova2006). Agents that suppress the proliferation of malignant cells by inducing apoptosis may represent a useful mechanistic approach to both cancer chemoprevention and chemotherapy. While many anticancer agents have been developed, unfavourable side effects and resistance are serious problems (Panchal1998). Since the introduction of cisplatin in cancer therapy, metal complexes and organometallic compounds have been gaining importance in oncology (Panchal1998; Sanna et al.2002). Metal-based compounds increase the possibility of developing molecules better-suited for binding to specific biological targets (Sanna et al.2002; Hartinger et al.2006).

Ruthenium complexes appear particularly promising; although they exhibit lower cytotoxicity as compared to cisplatin, they are better tolerated in vivo (Sanna et al.2002; Hartinger et al.2006; Menezes et al.2007). Research on bioactive ruthenium(II) complexes is very active (Chen and Wong2008). These studies have led to the development of ruthenium based anticancer agents (Yan et al.2005; Ang and Dyson2006; Hartinger et al.2006). Research groups of Sadler, Dyson, Keppler and Reedijk have synthesized a remarkably large number of Ru(II)/Ru(III) organometallic complexes that are being tested for anticancer activity. Ruthenium complexes are slitly cytotoxic but do not affect normal cells significantly. Ruthenium drugs are very promising candidates for novel cancer therapy, with two drugs already in clinical trials, NAMI-A and KP1019 (Cocchietto and Sava2000; Zorzet et al.2001; Hartinger et al.2006). Thus, there is growing interest in the use of new organometallics for the treatment of various cancers and the development of safer and more effective therapeutic agents (Panchal1998; Silveira-Lacerda et al.2009). In the present work, we studied the cytotoxic activity of cis-tetraammine(oxalato)ruthenium(III) dithionate cytotoxicity towards human chronic myelogenous leukemia (K-562) tumor cell line.

The antiproliferative activity of cis-tetraammine(oxalato)ruthenium(III) dithionate, a ruthenium-based complex, have been evaluated in vitro against human leukemia (K-562) cells using trypan blue and MTT assay. Inhibition of cell proliferation is an important potency indicator for chemotherapeutic drugs. As shown in Figures 1 and2a and b, the tested compound induces cell death in a dose and time dependent manner on K-562 cells. It is found that the effect was improved linearly while prolonging the incubation time. The determined IC₅₀ values of this complex, 18.28 μM (Figure 2a), is considerably the same of those of the commercially used antineoplastic drugs cisplatin (IC₅₀ = 11 μM) and oxaliplatin (IC₅₀ = 18 μM) on the same tumor cell line (Štarha et al.2009). These results corroborate previous observations that r(III) complexes induces cytotoxicity towards tumor cells such as human Jurkat, HeLa and SK-BR-3, and murine S-180 and A-20 tumor cell lines (Frasca et al.2001; Silveira-Lacerda et al.2009).

For ruthenium(II) complexes as methylimidazole (RMC1) he also found having cytotoxicity of 17.34 mg mL^-1 for A549, 18.89 mg mL^-1 for A375 and 20.25 mg mL^-1 for Hep G2, respectively. The same compound exhibits cytotoxicity of 51.59 mg mL^-1 for HBE (basal lineage), as well as demonstrating that the compound RMC1 ruthenium II (Yang et al.2012). The complex [Ru(phen)₂(ƥ-MOPIP)]²⁺ can effectively inhibit proliferation of the A375 cell line with a low IC₅₀ (5.9 ± 1.1 mM). [Ru(bpy)₂(dppn)]²⁺ exhibits high cytotoxicity against human HT-29 and MCF-7 cancer cell lines comparable to that of cisplatin induces cell death in a dose and time dependent manner (Schatzschneider et al.2008), and [Ru(dmp)₂(DBHIP)]²⁺ can effectively induce apoptosis of the BEL-7402 cell line (Liu et al.2010).

The lower general toxicity of ruthenium compounds compared to platinum drugs has been attributed to the ability of ruthenium compounds to specifically accumulate in cancer tissues. The higher specificity of these compounds for their targets may also be linked to their selective uptake by the tumor compared with healthy tissue and to selective activation by reduction to cytotoxic species within the tumor (Bergamo et al.1999; Allardyce et al.2003; Clarke2003).

Ruthenium-chloro complexes tend to undergo hydrolysis in aqueous media leading to the generation of cationic Ru–OH₂ complexes capable of reacting with DNA with greater ease than the corresponding chloro complexes (Melchart et al.2007; Bacac et al.2004; Hotze et al.2004). The hydrolyzed complexes interact with the N7 of guanine in DNA duplexes leading to disruption of the structure of genetic material (Chen et al.2003).

To explore the mechanisms of the cytotoxic effects produced by cis-tetraammine(oxalato)Ruthenium(III) dithionate, drug-induced changes in cell cycle distribution were examined. Compared to the cell cycle profiles of untreated cells, flow cytometric analysis indicated the sub-G1 arresting effect of ruthenium compound on K-562 cells, as ruthenium(III) dithionate already induced a 48.2% increase in the number of sub-G0/G1 cells after a 48 h incubation. The ruthenium(III) compound caused a minor S phase accumulation after incubation at concentrations of 40 and 150 μM for all periods of exposition Figure 4. Treatment with 40 μM of cis-[Ru(C₂O₄)(NH₃)₄]₂(S₂O₆) induced respectively a 1.7-fold, 2.2-fold and 2.4-fold increase in the number of sub-G0/G1 cells when compared to control for 24, 48 and 72 h, respectively Table 1.

Furthermore, another important biochemical sign of apoptosis was studied: DNA laddering and fragmentation (data not shown), possibly representing DNA cleavage into oligonucleosomal fragments through activation of the cysteine protease caspase-3, which is involved in the proteolytic cleavage of key downstream proteins such as poly (ADP-ribose) polymerase (PARP) (Bortner et al.1995; Clarke and Stubbs1996). This process ultimately results in DNA fragmentation and apoptotic death (Bortner et al.1995). Although this type of binding has been suggested to mimic, to some extent, cisplatin-induced DNA crosslinking, other types of damage may exist that dominate or contribute to the biological activity of this drug prototype.

Several studies have been using this test to corroborate the results obtained in other assays. In the present study, results derived from DNA laddering assay did not show K-562 DNA fragmentation, instead the results shows DNA integrity and even differences on DNA content. Is important note that this data does not corroborate with more sensitive techniques such as flow cytometry and comet assay, used in this work. These techniques confirmed that the ruthenium(III) compound interfere on cell cycle and induces cells to enter apoptosis. Thus, we can infer that the technique of DNA gel electrophoresis is not best assay to assess the degradation of genome DNA.

The present study also evaluated the DNA-damaging effects detected by the alkaline version of the comet assay in K-562 cancer cells. This assay has been used as a test to predict the risk to develop certain diseases (renal cell carcinoma, cancers of the bladder, esophagus, and lung) due to susceptibility of the individual to DNA damage (Ribeiro et al.2009). The in vitro comet assay is proposed as an alternative to cytogenetic assays in early genotoxicity/photogenotoxicity screening of drug candidates as well as for neurotoxicity (Witte et al.2007).

The alkaline comet assay has been used to assess the genotoxicity of chemicals, environmental exposures to carcinogens, toxins, and physical agents both in vitro and in vivo (Trzeciak et al.2000; Sekihashi et al.2002). This method was also used to measure DNA repair capacity in live cells (Banath et al.1998) and acellular systems (Dusinská et al.2004).

In our study, cis-[Ru(C₂O₄)(NH₃)₄]₂(S₂O₆) shows a significant increase in tailed cells in any of the concentrations tested compared with negative control (Figure 3). Consequently, the concentration of cis-[Ru(C₂O₄)(NH₃)₄]₂(S₂O₆) might be associated cytotoxicity with direct effect on K-562 cells DNA. Thus, it can be deducted that ruthenium-based compounds present selectivity to enter both tumor and normal cells. It is known that all the body cells present transferrin receptors, particularly tumor cells, in which these receptors are found in higher numbers. Due to these features, higher quantities of ruthenium complexes penetrate tumor tissue, reduce ruthenium(III) to ruthenium(II) and binding to DNA (guanine), and promote strand breaks (Clarke and Stubbs1996; Lebwohl and Canetta1998; Sava and Bergamo2000; Katsaros and Anagnostopoulou2002; Galanski et al.2003; Desoize2004). Another hypothesis is that even in small quantities this compound might enter normal cells, because ruthenium(III) complexes are activated only by its reduction when it finds an environment with high concentration of glutathione, low pH, and occurrence of hypoxia. It is very common to find tumor cells presenting these conditions since it is known that these cells have high levels of glutathione and oxygen consumption when the nutrients are quickly used due to their accelerated development promoting hypoxia (Sava and Bergamo2000). Consequently, these cells need more glycolitic energy, which increases the level of lactic acid in the tissues and causes a decrease in the media pH. It is worth emphasizing that ruthenium(III) compounds can be used as pro-drugs that are activated by in vivo reduction to ruthenium(II) (Desoize2004; Sava and Bergamo2000).

Broadening the chemotherapeutic arsenal depends on understanding existing agents with a view toward developing new modes of attack. Indeed, few of the organometallics compounds may function in a manner analogous to cisplatin, which appears to bend DNA by cross-linking adjacent guanines, thereby causing a class of DNA binding proteins to adhere to the site. Overall, the broad class of ruthenium(III) antitumor agents appears to differ from cisplatin by favoring interstrand rather than intrastrand cross-links (Clarke et al.1999). In agreement with this hypothesis, it was demonstrated that NAMI-A induced apoptosis in the ECV304 transformed human endothelial and KP1019 in colorectal carcinoma cell lines via activation of caspase-3 and DNA fragmentation (Bortner et al.1995; Nunez et al.1998; Kapitza et al.2005).

Despite the resounding success of cisplatin and closely related platinum antitumor agents, the movement of other transition-metal antitumor agents toward the clinic has been exceptionally slow (Clarke et al.1999). Non-Platinum chemotherapeutic metallopharmaceuticals hold much promise for the future, and needs to be actively explored in a large variety of tumor types in combination therapies. Besides the already well established NAMI-A and KP-1019 activities on gastrointestinal, breast, prostate, and ovarian cancers (Pluim et al.2004; Bergamo and Sava2007), development as a potential anti-tumor drug.

The present results indicate that cis-[Ru(C₂O₄)(NH₃)₄]₂(S₂O₆) is worthy of further development as a potential anti-tumor drug that could be used in the treatment of leukemia. Thus, additional studies are needed to determine the molecular mechanisms of the active components and to evaluate the potential in vivo anticancer activity of the cis-tetraammine(oxalato)ruthenium(III) dithionate.