β-diketone-cobalt complexes inhibit DNA synthesis and induce S-phase arrest in rat C6 glioma cells

β-diketone-cobalt complexes, a family of newly synthesized non-platinum metal compounds, exhibit potential antitumor activity; however, the antitumor mechanism is unclear. The current study investigated the mechanism by which β-diketone-cobalt complexes inhibit rat C6 glioma cell proliferation. It was found that β-diketone-cobalt complexes suppress rat C6 glioma cell viability in a dose-dependent manner (3.125–100 μg/ml). In rat C6 glioma cells, the IC50 value of β-diketone-cobalt complexes was 24.7±3.395 μg/ml and the IC10 value was 4.37±1.53 μg/ml, indicating a strong inhibitory effect. Further investigation suggested that β-diketone-cobalt complexes inhibit rat C6 glioma cell proliferation, which is associated with S-phase arrest and DNA synthesis inhibition. During this process, β-diketone-cobalt complexes decreased cyclin A expression and increased cyclin E and p21 expression. In addition, β-diketone-cobalt complexes exhibit a stronger antitumor capability than the antineoplastic agent, 5-fluorouracil.


Introduction
Inorganic chemistry has its place in medicine, and metals, particularly transition metals, have various clinical applications (1,2). Cisplatin, as an inorganic antineoplastic agent, has been extensively used to treat tumors, but its clear side effects and tolerance limit its clinical applications. In addition, platinum complex with new ligands did not exhibit marked advantages in previous clinical trials. To date, only carboplatin and oxaliplatin have been used clinically (3,4). β-diketone-cobalt complexes are polyoxometalates containing cobalt and traditional methods have been modified for the synthesis (5). β-diketone-cobalt complexes have been shown to suppress SMMC-7721 and SK-OV-3 tumor cell viability and interact with λ-DN (6); however, the molecular mechanisms of β-diketone-cobalt complexes against tumors remain unclear.
Brain glioma is a common central nervous system tumor, with at least five new cases per 100,000 individuals diagnosed worldwide each year (7,8). Malignant brain glioma extensively infiltrates normal brain tissues and is difficult to completely excise surgically. The relapse rate is high and conventional therapies used are radiotherapy and chemotherapy (9). Although present therapeutic methods are markedly advanced, the majority of patients cannot be cured (10). As present chemotherapeutics do not obtain ideal outcomes, the development of highly effective, low toxicity drugs for the treatment of brain glioma is required.
T he cu r rent st udy focused on the effects of β-diketone-cobalt complexes against C6 rat glioma cell cytotoxicity and their potential molecular mechanisms of action against tumor cells.
Cell lines and culture. Rat C6 glioma cells were incubated in Dulbecco's modified Eagle's medium (Gibco Life Sciences, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco Life Sciences), 2 mM L-glutamate, 100 U/ml penicillin and 100 µg/ml streptomycin at 37˚C in a 5% CO 2 incubator.
MTT assay. Rat C6 glioma cells at 10 4 cells/well were seeded onto 96-well plates for 24 h at 37℃ and then treated with β-diketone-cobalt complexes. Next, 20 µl MTT solution β-diketone-cobalt complexes inhibit DNA synthesis and induce S-phase arrest in rat C6 glioma cells ( Western blot analysis. Rat C6 glioma cells were treated with β-diketone-cobalt complexes for 12 h prior to the preparation of cell lysates. Subsequently the cell lysates were separated through a 12% SDS-PAGE gel. Following electrophoresis, proteins were transferred to PVDF membranes, and blocked with 5% non-fat dry milk in TBST buffer (20 mM Tris-HCl pH 7.6, 150 mM NaCl and 0.05% Tween-20) for 1 h at room

A B
was used to calculate the statistical significance of the experimental results. P<0.05 and P<0.01 were considered to indicate statistically significant differences. Data are presented as the mean ± SD, unless stated otherwise.
The abovementioned results revealed that β-diketone-cobalt complexes exhibit a marked inhibitory effect on rat C6 glioma cells.
β-diketone-cobalt complexes inhibit DNA synthesis and induce S-phase arrest in rat C6 glioma cells. To further investigate the mechanisms by which β-diketone-cobalt complexes suppress rat C6 glioma cell proliferation, flow cytometry was utilized to identify the effects of β-diketone-cobalt complexes and 5-Fu on the rat C6 glioma cell cycle. Compared with the control group, the percentage of S-phase cells significantly increased from 9.17 to 27.04 and 26.48% following rat C6 glioma cell exposure to β-diketone-cobalt complexes for 24 and 48 h, respectively. The percentage of cells in S phase increased from 9.17 to 16.43% following exposure to 5-Fu for 48 h (Fig. 3A). Subsequently, [ 3 H]-thymidine assay was employed to measure the effects of various concentrations of β-diketone-cobalt complexes on the DNA synthesis of rat C6 glioma cells for 24 h. Compared with the control group, with increased concentration of β-diketone-cobalt complexes, DNA synthesis in rat C6 glioma cells was evidently inhibited in a dose-dependent manner (Fig. 3B). It was concluded that β-diketone-cobalt complexes suppress rat C6 glioma cell proliferation by inhibiting DNA synthesis and inducing S-phase cell cycle arrest.
Effects of β-diketone-cobalt complexes on cyclin A,cyclin E and p21 expression in rat C6 glioma cells. To identify proteins involved in S-phase arrest induced by β-diketone-cobalt complexes in rat C6 glioma cells, protein expression was detected in rat C6 glioma cells at 48 h following exposure to β-diketone-cobalt complexes. Results showed that β-diketone-cobalt complexes reduced cyclin A expression in rat C6 glioma cells (13), but increased cyclin E and p21 expression (Fig. 4).

Discussion
β -d i ketone-coba lt complexes, newly synt hesi zed non-platinum metal compounds, have been shown to inhibit SMMC-7721 and SK-OV-3 cell viability, but their antitumor mechanisms remain unclear (14). Brain glioma is a common tumor in the central nervous system and is difficult to completely excise surgically. Its relapse rate is high and conventional therapy is based on radiotherapy and chemotherapy; however, the outcomes of current chemotherapy drugs are not ideal. The present study first explored the mechanisms by which β-diketone-cobalt complexes inhibit rat C6 glioma cell proliferation and confirmed that β-diketone-cobalt complexes suppress rat C6 glioma cell viability in a dose-dependent manner (3.125-100 µg/ml). Of note, in rat C6 glioma cells, the IC 50 value of β-diketone-cobalt complexes was 24.7±3.395 µg/ml and IC 10 value was 4.37±1.53 µg/ml, showing a good inhibitory effect against tumors (Fig. 1B). 5-Fu, a common anticancer drug, is used for the treatment of head and neck cancer (15). 5-Fu interacts with nucleic acid metabolism, leading to cytotoxicity and cell death, thus, exerting its antitumor activity (16,17). Following comparison, the current study confirmed that β-diketone-cobalt complexes exhibit marked antitumor activity in vitro compared with 5-Fu ( Fig. 2A). β-diketone-cobalt complexes at low concentrations significantly inhibited DNA synthesis in rat C6 glioma cells (Fig. 3B). Whether β-diketone-cobalt complexes, similar to conventional chemotherapy drugs, are simple cytotoxic drugs is poorly understood. The present study revealed that the inhibitory effect of β-diketone-cobalt complexes on rat C6 glioma cell proliferation correlates with S-phase arrest ( Figs. 2A and  3A). However, 5-Fu did not suppress cell proliferation by cell cycle arrest (Fig. 3A), in contrast to the antitumor mechanisms of β-diketone-cobalt complexes.
Cell cycle regulation depends on two protein families, the cyclins and cyclin-dependent protein kinases (CDKs). During the cell cycle, cyclin expression dynamically alters and during the transition from G1 to S phase, cyclin E activates CDKs and cyclin E expression increases. Cyclin E expression is downregulated after entering S phase (18,19). In the current study, cyclin E expression increased at 24 h following treatment with β-diketone-cobalt complexes and diminished at 48 h (Fig. 4). Cyclin A plays a key role in S phase, but p21 causes cell cycle arrest by inhibiting CDK activity (20)(21)(22). However, β-diketone-cobalt complexes decreased the expression levels of cyclin A and p21 (Fig. 4). In conclusion, β-diketone-cobalt complexes significantly suppress rat C6 glioma cell proliferation, showing a potential ability for the development of novel antitumor drugs.