Systematic evaluation of the antitumor activity of three ruthenium polypyridyl complexes

https://doi.org/10.1016/j.jinorgbio.2021.111616Get rights and content

Highlights

  • Cinnamaldehyde-derived Ru(II) complexes were designed and synthesized.

  • In vitro antitumor activity screening of the Ru(II) complexes were investigated.

  • The desired Ru(II) complexes localizes in mitochondria.

  • Ru(II) complexes induce apoptosis of HepG-2 cells.

Abstract

Ruthenium-containing complexes have emerged as good alternative to the currently used platinum-containing drugs for malignant tumor therapy. In this work, cytotoxic effects of recently synthesized ruthenium polypyridyl complexes [Ru(bpy)2(CFPIP)](ClO4)2 (bpy = 2,2′-bipyridine, CFPIP = (E)-2-(4-fluorostyryl)-1H-imidazo[4,5-f][1,10]phenanthroline, Ru(II)-1), [Ru(phen)2(CFPIP)](ClO4)2 (phen = 1,10-phenanthroline, Ru(II)-2) and [Ru(dmb)2(CFPIP)](ClO4)2 (dmb = 4,4′-dimethyl-2,2′-bipyridine, Ru(II)-3) toward different tumor cells were investigated in vitro and compared with cisplatin, the most widely used chemotherapeutic drug against hepatocellular carcinoma (HepG-2). The results demonstrate that target complexes show excellent cytotoxicity against HepG-2 cells with low IC50 value of 21.4 ± 1.5, 18.0 ± 2.1 and 22.3 ± 1.7 μM, respectively. It was important noting that target Ru(II) complexes exhibited better antitumor activity than cisplatin (IC50 = 28.5 ± 2.4 μM) against HepG-2 cells, and has no obvious toxicity to normal cell LO2. DNA binding results suggest that Ru(II)-1, Ru(II)-2 and Ru(II)-3 interact with CT DNA (calf thymus DNA) through intercalative mode. Complexes exerted its antitumor activity through increasing anti-migration and inducing cell cycle arrest at the S phase. In addition, the apoptosis was tested by AO (acridine orange)/EB (ethidium bromide) staining and flow cytometry. Mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and colocalization tests were also evaluated by ImageXpress Micro XLS system. Overall, the results show that the ruthenium polypyridyl complexes induce apoptosis in HepG-2 cells through ROS-mediated mitochondria dysfunction pathway.

Graphical abstract

DNA-binding behaviors and antitumor activity of the Ru(II) complexes were investigated. Complexes exerted its antitumor activity through increasing anti-migration and inducing cell cycle arrest at the S phase. In addition, mitochondrial membrane potential, reactive oxygen species, and colocalization tests were also evaluated by ImageXpress Micro XLS system.

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Introduction

Malignant tumor remains one of the most challenging and dangerous health problems for human beings worldwide [[1], [2], [3], [4], [5], [6], [7], [8]]. According to the data from World Health Organization, in 2015, the number of deaths caused by cancer was 8.8 million, and nearly one-sixth of the deaths worldwide were attributed to malignant tumors [9]. Because of the huge therapeutic challenges of malignant tumors, great attention has been devoted to develop efficient protocols for the construction of new chemotherapeutic drugs recently [[10], [11], [12], [13], [14], [15], [16], [17]]. In order to discover more effective anti-tumor drugs, thousands of platinum-containing complexes have been synthesized, but only a few of these complexes have entered clinical trials [[18], [19], [20], [21]]. Moreover, this platinum-based complexes used in clinically has various adverse side effects, such as bone marrow suppression, allergies, tachycardia, renal toxicity, gastrointestinal reactions, etc., which greatly limits its use in the clinic [22,23]. To improve these shortcomings, alternative metal-based agents that lead to antitumor mechanisms different from platinum-based chemotherapeutics have been investigated and proved be a feasible methods. According to the literature, ruthenium metal complexes have attracted a great deal of interest from scientists due to their low cytotoxicity, rich photochemical and photobiological properties and anti-transfer properties [[24], [25], [26], [27], [28], [29], [30]]. In recent years, the application of ruthenium metal complexes in the field of anti-tumor has made significant research progress [[31], [32], [33], [34], [35], [36], [37]]. Two leading types of ruthenium complexes, NAMI-A and KP1019 have entered phase II clinical trials to treat malignant tumors [38]. In 2017, Liu found that [Ru(phen)2(MHPIP)](ClO4)2 (phen = 1,10-phenanthroline; MHPIP = 2-(1-methyl-1H-pyrazol-4-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) induces HepG-2 cells apoptosis. In addition, the target Ru(II) complex can regulate the expression of caspase 3 and Bcl-2 family proteins [21]. Liu et al. reported that [(η6-p-cymene)Ru(N/O^N)Cl]0/+ (N/O^N = schiff base chelating ligands), a class of Half-sandwich Ru compounds with excellent anti-tumor activity, was able to effectively increase the level of reactive oxygen species (ROS) in HepG-2 cells and synergistically decrease the mitochondrial membrane potential (MMP) [39]. Recently, our group reported that ruthenium polypyridyl complexes containing heterocycle and alkynyl in their main ligands could enter the cytoplasm through the cell membrane and accumulate in the mitochondria [40].

Cinnamaldehyde, especially cinnamaldehyde derivatives, are a significant scaffold of various functional molecules [[41], [42], [43], [44]]. They not only serve as useful and important precursors for the synthesis of olefin, carboxylic acids and heterocycles, but also show a broad range of biological and pharmaceutical activities, such as antitumor, antifungal, antibacterial and anti-inflammatory [[45], [46], [47]]. Nonetheless, cinnamaldehyde-derived Ru(II) polypyridyl complexes are rarely studied as antitumor drugs. In continuation of our interest in the field of antitumor [[48], [49], [50]], herein, three Ru(II) polypyridyl complexes, [Ru(Nsingle bondN)2(CFPIP)](ClO4)2 (CFPIP = (E)-2-(4-fluorostyryl)-1H-imidazo[4,5-f][1,10]phenanthroline, N-N = 2′-bipyridine (bpy, Ru(II)-1), 1,10-phenanthroline (phen, Ru(II)-2) and 4,4′-dimethyl-2,2′-bipyridine (dmb, Ru(II)-3)) (Scheme 1), have been synthesized and characterized by IR, HRMS, 1H NMR, and 13C NMR. Complexes Ru(II)-1, Ru(II)-2, and Ru(II)-3 show excellent activity on the cell proliferation in HepG-2 cells with IC50 values of 21.4 ± 1.5, 18.0 ± 2.1, 22.3 ± 1.7 μM, respectively. Subsequently, the antitumor mechanism of the Ru(II) complexes was investigated by DNA binding behaviors, morphological changes, apoptosis, MMP (mitochondrial membrane potentials), ROS levels, cellular localization, cell cycle arrest and cell invasion. To sum up, our study demonstrates that target ruthenium complexes exhibited the excellent cytotoxicity against HepG-2 cells, and further induced apoptosis in HepG-2 cells through ROS-mediated mitochondrial dysfunction pathways.

Section snippets

Materials and methods

All reagents and solvents were purchased commercially and used without further purification unless otherwise noted. Calf thymus DNA (CT DNA) was obtained from the Sino American Biotechnology Company. Ltd. Ultrapure MilliQ water was used in all experiments. DMSO and RPMI 1640 were purchased from Sigma. Cell lines of HeLa (Human cervical cancer cell line), SGC-7901 (human gastric carcinoma cells), HepG-2 (Hepatocellular carcinoma cells), A549 (Human lung carcinoma cells), and normal LO2 cells

Synthesis and characterization

The ligands (E)-2-(4-fluorostyryl)-1H-imidazo[4,5-f][1,10]phenanthroline (CFPIP) and corresponding Ru(II) complexes were prepared using the literature method. The synthetic route of the ligands and complexes are elucidated in Scheme 1. Experiments have shown that the complexes are very stable (Fig. S3). Furthermore, the desired ruthenium polypyridyl complexes (Ru(II)-1, Ru(II)-2 and Ru(II)-3) were characterized by HRMS, IR, 1H NMR and 13C NMR. In the IR spectra, the peaks of 1639 cm−1 for

Conclusions

Three new ruthenium polypyridyl complexes containing CFPIP moiety were designed, synthesized and characterized, and theirs in vitro antitumor activity were investigated. The CFPIP moiety endows the Ru(II) complexes excellent physical and chemical properties, and Ru(II)-1, Ru(II)-2 and Ru(II)-3 exhibit higher antitumor activity than cisplatin against HepG-2 cells. The DNA binding experiments show that desired complexes interact with CT DNA through intercalative mode. Mechanism investigations

Declaration of Competing Interest

We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. All authors have read and agreed to the submission.

Acknowledgements

The authors thank the Guangxi Natural Science Foundation (2018GXNSFBA050024, 2020GXNSFAA297227), Guangxi Science and Technology Base and Special Fund for Talents (AD19245008), Key Laboratory of Electrochemical and Magneto-chemical Function Materials (EMFM20211123) and the Ph. D. Scientific Research Foundation of Guilin University of Technology.

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