Water-Soluble Ruthenium(II) Complexes with Chiral 4-(2,3

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Water-Soluble Ruthenium(II) Complexes with Chiral 4-(2,3dihydroxypropyl)-formamide oxoaporphine (FOA): in Vitro and in Vivo Anticancer Activity by Stabilization of G-Quadruplex DNA, Inhibition of Telomerase Activity and Induction of Tumor Cell Apoptosis Zhen-Feng Chen, Qi-Pin Qin, Jiao-lan Qin, Jie Zhou, Yu-Lan Li, Nan Li, Yan-Cheng Liu, and Hong Liang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00444 • Publication Date (Web): 19 May 2015 Downloaded from http://pubs.acs.org on May 22, 2015

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Journal of Medicinal Chemistry

Water-Soluble

Ruthenium(II)

Complexes

with

Chiral

4-(2,3-dihydroxypropyl)-formamide oxoaporphine (FOA): in Vitro and in Vivo Anticancer Activity by Stabilization of G-Quadruplex DNA, Inhibition of Telomerase Activity and Induction of Tumor Cell Apoptosis Zhen-Feng Chen,* Qi-Pin Qin, Jiao-Lan Qin, Jie Zhou, Yu-Lan Li, Nan Li, Yan-Cheng Liu, Hong Liang*

State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmacy, Guangxi Normal University, 15 Yucai Road, Guilin 541004, P. R. China. TITLE RUNNING HEAD: Chiral oxoaporphine-ruthenium (II) as anticancer agents KEYWORDS: ruthenium(II) complex, chiral oxoaporphine, G-quadruplex DNA, telomerase, anticancer activity, apoptosis

Correspondence to: Professor Zhen-Feng Chen and Hong Liang State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmacy, Guangxi Normal University, 15 Yucai Road, Guilin 541004, P. R. China. Fax, 086-773-2120958 E-mail: [email protected] (Z.-F. Chen) ; [email protected] (H. Liang)

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ABSTRACT:

Three

water-soluble

ruthenium(II)

complexes

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with

chiral

4-(2,3-dihydroxypropyl)-formamide oxoaporphine (FOA), were synthesized and characterized. It was found that these ruthenium(II) complexes exhibited considerable in vitro anticancer activities and that they were the effective stabilizers of telomeric, and G-quadruplex-DNA (G4-DNA) in promoter of c-myc, which acted as a telomerase inhibitor targeting G4-DNA and induced cell senescence and apoptosis. Interestingly, the in vitro anticancer activity of 6 (LC-003) was higher than those of 4 (LC-001) and 5 (LC-002), more selective for BEL-7404 cells than for normal HL-7702 cells, and preferred to activate caspases-3/9. The different biological behaviors of the ruthenium complexes could be correlated with the chiral nature of 4-(2,3-dihydroxypropyl)-formamide oxoaporphine. More significantly, 6 exhibited effective inhibitory on tumor growth in BEL-7402 xenograft mouse model, and higher in vivo safety than cisplatin. These mechanistic insights indicate that 6 displays low toxicity and can be a novel anticancer drug candidate.

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INTRODUCTION

G-quadruplexes (G4s), the nucleic acid sequences rich in guanine and capable of forming a four-stranded structure, are highly associated with important human diseases such as cancer1, HIV2, and diabetes3. G4-forming sequences are found at the ends of eukaryotic chromosome−the telomeres−as well as in other important regions of the genome, as oncogene promoters (proto-oncogenes such as c-myc, bcl-2, c-kit, and VEGF), 5'-UTR regions, and introns.4 Targeting G-quadruplexes (G4s) is now considered an attractive approach toward drug intervention in anticancer therapy.5 Because of its essential role in telomere maintenance and cancer biology,6 hTERT has also become a main target toward developing anticancer drugs.7 A recent study indicated that some G4-DNA ligands efficiently stabilized the G4 forms of nucleic acids, frequently leading to inhibition of telomerase.8 Subtle changes (such as enantiomer) in the structures of metal complexes can have a profound effect on biological activity. It has been well established that different optical isomers, or enantiomers, of a compound, such as thalidomide, can have greatly different biological activities.9 Furthermore, the different enantiomers may also have distinguishing pharmacokinetics and affinities toward their targeting molecules, as is the case for the opioid substitute methadone.10 Such observation is not only true for organic drugs, such as the clinically approved Pt(II) drug oxaliplatin, which contains the chiral 1R,2R-cyclohexadiamine ligand, is not only more active than the corresponding enantiomer (with 1S,2S-cyclohexadiamide), but also shows increased cellular uptake and DNA binding properties.11 More recently, Lippard reported the chiral potential of phenanthriplatin cis-[Pt(NH3)2Cl(Am)]+ (where Am is the N-heterocyclic base phenanthridine) and its influence on guanine binding.12 Over the last decade, many researches indicated that chiral complexes enhanced their binding

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abilities to G4-DNA and exhibited different cytotoxicities. For example, in [PtCl2L2](L2), which are aromatic bisphosphanes and aromatic diamines, the S-(−) isomer was more cytotoxically active and had higher affinity to G4-DNA than its R-(+) counterpart.13 Qu et al. reported that chiral metallo-supramolecular complexes selectively induced human telomeric G4 formation under salt-deficient conditions14 and the chiral metallo-supramolecular complex preferred binding to a higher-order dimeric G4 over monomeric G-quadruplex unit.15 Ma et al. developed a number of kinetically-inert, organometallic iridium(III) complexes bearing various C∧N and N∧N ligands selective for G4-DNA.16 On the other hand, although the platinum(II)-containing drugs (e.g. cisplatin, carboplatin and oxaliplatin) have become the widely used anticancer agents, their drawbacks, such as high levels of in vivo toxicity, drug resistance and low aqueous solubility,17 underline the need for alternative transition metal-containing anticancer drugs. To overcome these drawbacks, ruthenium-based complexes have been developed and shown considerable promise,18 because some of them have exhibited favorable in vitro and in vivo pharmacological profiles in different models including platinum-resistant cells.19 For instance, the ligand exchange kinetics of Pt(II) and Ru(II) complexes in aqueous solution, which is crucial for anticancer activity, are very similar;20 Ruthenium compounds are not very toxic and some of them are quite selective for cancer cells, probably because of the ability of ruthenium to mimic iron in binding to biomolecules.21 Currently, two Ru(III) complexes are in clinical studies.22 According to Contel's classification,23 there are four main groups of ruthenium-based potential anticancer agents that have been studied in more detail and display important antitumor and/or antimetastic activities with lower toxicity.24 They are: 1) NAMI-A type ruthenium(III) coordination complexes based on N, Cl donor

4


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ligands,25 such as NAMI-A,26 KP1019 and KP133927 (Figure 1); 2) organometallic ruthenium(II) compounds with arene ligands (piano-stool structure),28 such as RM17529 and RAPTA-T30 (Figure 1) which are undergoing preclinical studies; 3) cyclometalated complexes with pincer C∧N ligands (RDC family);31 and 4) ruthenium(II/III) complexes with bioactive ligands (such as the well-known therapeutic drug, anticancer active natural ligand),32 such as DW1/233 (Figure 1).

Figure 1. Examples of anticancer ruthenium compounds. With increasing interest in the new antitumor targets G4 and telomerase, a number of ruthenium(II/III)-based anticancer agents targeting G4 and telomerase such as [Ru(bpy)2(tip)]2+

[Ru(phen)2(tip)]2+,34

and

[(C6H6)Ru(o-ClPIP)Cl]Cl,35

[Ru(bpy)(biim)], [Ru(phen)(biim)] and [Ru(p-mopip)(biim)],36 [Ru(bpy)(pedppz)] and

[Ru(bpy)(pemitatp)],37

[(η6-arene)Ru(en)Cl]+,39

[Ru(bpy)2(ptpn)]2+ [Ru(ip)3](ClO4)2

and and

[Ru(pehn)2(ptpn)]2+,38 [Ru(pip)3](ClO4)2,40

[(dmb)2Ru(obip)Ru(dmb)2]4+,41 have been reported. However, only a few chiral anticancer ruthenium complexes, such as ruthenium(II) arene PTS (RAPTA) 5



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complexes,42

Λ-[Ru(phen)2(p-MOPIP)]2+

and

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∆-[Ru(phen)2(p-MOPIP)]2+,43

[{Ru(phen)2}2tpphz]4+,44 Λ-[Ru(phen)2(p-HPIP)]2+ and ∆-[Ru(phen)2(p-HPIP)]2+,45 which display inhibitory effects on telomerase activity by stabilization of G4-DNA, have been developed. It is clear that there is still a need to study the targeting of these ruthenium complexes and gain better understanding of the molecular mechanisms of their action in order to develop more active and selective chemotherapeutics. In addition, more in vivo data are needed to make more reliable predictions of

their

structure-biological activity relationships. In

this

study,

we

designed

and

synthesized

R/S-(±)-4-(2,3-dihydroxypropyl)-formamide R-(+)-4-(2,3-dihydroxypropyl)-formamide

three

oxoaporphine oxoaporphine

chiral

ligands:

(R/S-(±)-FOA),

(R-(+)-FOA)

and

S-(–)-4-(2,3-dihydroxypropyl)-formamide oxoaporphine (S-(–)-FOA), and their corresponding

water-soluble

ruthenium(II)

complexes.

i.e.

4

(LC-001)

cis-[RuCl2(R/S-(±)-FOA)(dmso)2], 5 (LC-002) cis-[RuCl2(R-(+)-FOA)(dmso)2] and 6 (LC-003) cis-[RuCl2(S-(−)-FOA)(dmso)2] (Figure 2). These ruthenium(II) complexes exhibited remarkable and different in vitro cytotoxicities, demonstrated strong affinity to G4-DNA and caused inhibitory effects on the telomerase activity, which are significantly correlated with the chiral nature of the ligands. In addition, we demonstrated for the first time that the high in vitro cytotoxicity, in vivo inhibitory activity on tumor growth and low in vivo toxicity of the water soluble ruthenium(II) complex 6 were mediated via multiple mechanisms, thus providing a potentially promising candidate for the development of more effective anticancer agent.

RESULTS AND DISCUSSION Synthesis and Characterization. Three chiral ligands including 1 (R/S-(±)-FOA),

2 (R-(+)-FOA) and 3 (S-(–)-FOA) were firstly synthesized through the synthetic

6


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routes shown in Figure 2, starting from 4-carboxyl-7-oxo-7H-dibenzo[de,g]quinoline. Their structures were characterized by elemental analyses, circular dichroism (CD) spectra, infrared (IR) spectroscopy, electrospray ionization-mass spectroscopy (ESI-MS), 1H and

13

C nuclear magnetic resonance (NMR) spectroscopy (Supporting

Information, Figures S1−S14). In addition, the crystal structure of racemic 1 was determined by single-crystal X-ray diffraction analysis. As shown in Figure 3, 1 is a planar structure. Selected bond lengths (Å) and bond angles (°) for 1 are reported in Table S1 (Supporting Information), and crystal data and structure refinement parameters for 1 are listed Table S2 (Supporting Information). Except for the chiral moiety, the structure of 1 resembles those of oxoisoaporphine and oxoaporphine derivatives.46

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Figure 2. Synthetic routes chiral 4-(2,3-dihydroxypropyl)-formamide oxoaporphine, 1 (R/S-(±)-FOA), 2 (R-(+)-FOA) ruthenium(II)

complexes.

and 3 (S-(–)-FOA), and their water-soluble i.e.

4,

R/S-(±)-2,3-dihydroxypropyl/PyBOP/DMF

5

and

(65°C);

6. (b)

Reagents:

(a)

R-(+)-2,3-dihydroxy

propyl/PyBOP/DMF (65°C); (c) S-(–)-2,3-dihydroxypropyl/PyBOP/DMF (65 °C); and (d−f) ethanol/chloroform (1:1) (reflux). Structural Characterization of Ruthenium(II) Complexes. Compounds 4, 5 and 6 were prepared by the reactions of cis-RuCl2(dmso)4 with 1, 2, 3 in the presence of ethanol and chloroform under solvothermal conditions (Figure 2), respectively. Their

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structures were characterized by elemental analysis, IR, 1H and 13C NMR, CD spectra and ESI-MS spectroscopy (Supporting Information, Figures S5 and S15−S26). The single-crystal X-ray diffraction analysis for racemic 4 revealed that the ruthenium(II) center was six-coordinated by one bidentate chelating ligand 1 (O^N) via the heterocyclic N and carbonyl O atom, two dmso ligands via S atom, and two chloride anions, to form a distorted octahedral geometry (Figure 3), which is comparable to the liriodenine-ruthenium(II) complex cis-[RuCl2(L)(dmso)2].47 Selected bond lengths (Å) and bond angles (°) are reported in Table S3 (Supporting Information), and crystal data and structure refinement parameters are listed Table S4 (Supporting Information).

Figure 3. Crystal structures of 1 (left) and 4 (right) showing atom labeling. The hydrogen atoms, the perchlorate anion in 1, and hydrate molecules in 4 were omitted for clarity. Solubility of 1− −6 in Water. In addition, the solubility of 1− −6 was investigated gravimetrically in distilled water by UV-vis spectroscopy (Figure S27, Supporting Information) according to the reported method.48 The solubility of 1− −6 was over 1.75, 1.60, 1.25, 1.05, 3.50 and 7.5 mg/mL in distilled water at room temperature, respectively. Stability of 4− −6 in Solution. The stabilities of 4− −6 were investigated under physiological conditions (Tris-KCl-HCl buffer solution with pH value of 7.35, 9



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containing 1% dmso) by the mean of UV-vis spectroscopy. As shown in Figure S28 (Supporting Information), the time-dependent (at 0, 4, 12, and 24 h) UV-vis spectra of each complex indicated that 4− −6 were stable in TBS (Tris-KCl-HCl buffer solution) for 24 h at room temperature. Their stabilities were further confirmed by high performance liquid chromatography (HPLC) experiments. As shown in Figure S29 (Supporting Information), all three complexes were stable in dmso stock solution for 24 h. In Vitro Cytotoxicity. The in vitro cytotoxicities of 1− −3 and their ruthenium complexes 4− −6 against six human tumor cell lines including BEL-7404, A549, MGC80-3, HeLa, Hep-G2, BEL-7402 and one normal liver cell line, HL-7702, were assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Their inhibitory rates were listed in Table S5 (supporting information). For comparison, the cytotoxicities of cisplatin and cis-RuCl2(dmso)4 were also examined and used as references. After incubation of these cells with the indicated compounds at the concentration of 20 µM for 48 h under the identical conditions, they exhibited different cytotoxicities and certain extent of selectivities. As shown in Table S5, except for 5 against Hep-G2 cells, toward the tested tumor cells, the inhibitory rates of 4, 5, and 6 were higher than those of 1, 2, 3, and cis-RuCl2(dmso)4, respectively. More interestingly, ligands 1− −3 and ruthenium(II) complexes 4− −6 displayed lower inhibitory rates against the normal liver cells, HL-7702. The in vitro cytotoxicities of 4, 5, and 6 were further quantified by determining the corresponding IC50 values. As shown in Table 1, except for 5 against A359 and Hep-G2 cells, on the tested tumor cells, 4, 5, and 6 exhibited significantly enhanced cytotoxicities as compared with those of free ligands 1, 2, and 3, suggesting a synergistic effect upon the combination of ruthenium(II) with the corresponding ligands 1, 2, and 3, especially 6. For instance, 10


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the ruthenium(II) complexes showed lower IC50 values (7.4−11.2 µM) on BEL-7404 cells, 1.8−2.1 times more cytotoxic than those of the corresponding free ligands 1, 2, and 3, and even 1.9−2.9 times more cytotoxic than cisplatin alone. In general, the in vitro cytotoxicities of 4, 5, and 6 follows the order of 6>4>5. In contrast to the similar natural oxoaporphine liriodenine-ruthenium(II) complex, cis-[RuCl2(L)(dmso)2], on BEL-7404 and HeLa, 4, 5, and 6 exhibited higher cytotoxicities.47 Moreover, the tested cells incubated with 4, 5, 6 and cisplatin were observed by morphological analyses using fluorescence microscopy. As shown in Figure S30, the observations on the cellular morphology (such as shrinkage, floatage and cytolysis) well agreed with the results with the MTT assay. Of course, 6 was nearly comparable to cisplatin, which was more cytotoxic than 4 and 5 toward BEL-7402 cells. Table 1. IC50a (µM) values of 1− −6 on the selected cells for 48 h.

a

Compds

BEL-7404

A549

MGC80-3

HeLa

Hep-G2

BEL-7402

HL-7702

1

17.2 ± 0.1

20.2± 0.5

32.4 ± 0.8

25.7± 2.2

32.9 ± 0.5

30.4 ± 1.5

>100

2

20.5 ± 0.1

24.1± 0.4

45.8 ± 2.8

28.5 ± 0.3

38.9 ± 1.3

68.6 ± 0.8

>100

3

15.4 ± 0.2

20.4± 0.3

35.7 ± 2.9

22.9 ± 0.7

24.9 ± 0.4

28.5 ± 2.4

81.4 ± 2.7

4

9.6 ± 0.4

11.5± 0.4

12.7 ± 0.4

10.2 ± 0.3

16.5 ± 0.1

19.1 ± 0.6

43.9 ± 0.6

5

11.2 ± 0.4

24.6± 0.4

20.5 ± 1.6

21.2 ± 1.5

39.1 ± 0.2

40.3 ± 2.1

59.7 ± 0.3

6

7.4 ± 0.4

10.8± 0.2

10.4 ± 0.1

9.0 ± 0.6

16.2 ± 0.7

14.7 ± 0.3

69.2 ± 0.3

Cisplatinb

21.4 ± 0.4

17.0± 2.3

5.4 ± 0.5

5.8 ± 0.9

9.5 ± 0.4

13.5 ± 0.9

15.6 ± 0.3

IC50 values are presented as the mean ± SE (standard error of the mean) from five

independent experiments. bCisplatin was dissolved at a concentration of 1 mM in 0.154 M NaCl.49 Cellular Uptake and Distribution of Metals in BEL-7404 Cells. Cell uptake can affect bioactivity,50 moreover, it was observed that cell membranes is a natural phospholipid chiral molecule showing very high preference to L-enantiomer.51 The three ruthenium(II) complexes 4− −6 exhibited the highest in vitro cytotoxicity on

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BEL-7404 cells comparing to the other tested cells, thus we selected BEL-7404 cells to investigate the cellular uptake and distribution of Ru or Pt in these cells treated with the three ruthenium(II) complexes or cisplatin using the inductively coupled plasma mass spectrometry (ICP-MS). Compared with the control group cells, treatment of BEL-7404 cells with 4− −6 (10.0 µM) or cisplatin (10.0 µM) for 8 h resulted in a substantial increase in the cellular concentrations of metals Ru or Pt, respectively, suggesting that a facile internalization of 4− −6 or cisplatin into BEL-7404 cells within 8 h. As shown in Figure 4A, 6 ((3.81 ± 0.14 nmol ruthenium)/106 cells) was taken up by the cells by approximately 1.8-times, 1.7-times and 2.0-times more efficiently than cisplatin ((2.08 ± 0.11 nmol platinum)/106 cells), 4 ((2.26 ± 0.08 nmol ruthenium)/106 cells) and 5 ((1.94 ± 0.16 nmol ruthenium)/106 cells), respectively. These results indicated that chirality will cause a certain influence on cellular uptake. In order to verify the cellular distributions of 4− −6 or cisplatin, the concentrations of 4− −6 and cisplatin in the nuclear fraction, cytoplasmic proteins, membrane proteins, nuclear proteins, and other extracts fractionated from BEL-7404 cells after the exposure of the cells to 4− −6 or cisplatin for 8 h, respectively, were determined according to the method reported by Schreiber et al.52 As shown in Figure 4B, the percentage of metal (ruthenium for 4− −6 or platinum for cisplatin) from 4− −6 in the nuclear fraction were slightly higher than that from cisplatin, especially in 6 treated cells. In addition, 4− −6 accumulated to a higher extent in both nuclear fraction and cytoplasmic proteins, while cisplatin accumulated only in the cytoplasmic proteins. It was also notable that certain amount of ruthenium from the 5 and 6 were also accumulated in the other fractions, including the total soluble proteins. The differences in subcellular distribution of Ru can be attributed to the different cellular pathways involved in both the uptake and efflux of the 4− −6, which, in turn, may be 12


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related to its ability to activate apoptotic pathways.

Figure 4. Cellular uptake and distribution of Ru and Pt in BEL-7404 cells. BEL-7404 cells were treated with 4− −6 (10.0 µM) and cisplatin (10.0 µM) for 8 h at 37°C, respectively. Ru or Pt contents in the whole cell (A) and different fractions (B) were measured by ICP-MS. Control BEL-7404 cells were treated with vehicle (1% dmso). The data shown are mean values ± SD (standard deviations) of three independent measurements for each experiment. Morphological Characterization of Apoptotic BEL-7404 Cells by Hoechst 33258. Apoptosis of BEL-7404 cells treated with 4, 5, and 6 were confirmed by analyzing the nuclear morphology. Nuclei of BEL-7404 cells treated with 4, 5, and 6 for 24 h, respectively, were stained by membrane-permeable blue Hoechst 33258 to detect apoptotic cells morphologically. Figure 5 showed the ﬂuorescence photomicrographs stained by Hoechst 33258 for the cultured BEL-7404 cells treated with 0, 5, 10 and 20 µM of 4, 5, and 6 for 24 h, respectively. In the control cultures, nuclei of the BEL-7404 cancer cells appeared as regular round contours, and the BEL-7404 cells with smaller nuclei and condensed chromatin were rarely observed. After being treated with 5 µM of 4, nuclei morphology of the cells were slightly changed, as shown in Figure 5. However, when the concentration of 4 was increased to 10 µM, more nuclei of the BEL-7404 cells appeared hyper condensed (brightly stained) (Figure 5). Remarkably, the number of apoptotic nuclei containing condensed chromatin was increased significantly in the BEL-7404 cells treated with 20 µM of 4. 13



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Similar results were observed in the cells treated with 5, and 6. With increasing the concentrations of 6, the number of apoptotic nuclei was increased more significantly than those of the cells treated with 4 and 5. Thus, the apoptosis of BEL-7404 cells was induced by 4, 5, and 6 in a concentration-dependent manner. In general, 6 exhibited the most effective one in inducing apoptosis of these cells.

Figure 5. Morphological changes in the nuclei (typical of apoptosis) of cultured BEL-7404 cells induced by 4, 5, and 6. BEL-7404 cells treated with 5, 10 and 20 µM of 4, 5, and 6 for 24 h, respectively, and stained with Hoechst 33258. Selected fields illustrated occurrence of apoptotic cells. Cells with condensed chromatin (brightly stained) were defined as apoptotic BEL-7404 cancer cells. Images were acquired using a Nikon Te2000 deconvolution microscope (magnification 200×). Induction of Senescence. To evaluate the long-term effects of 4, 5, and 6 on BEL-7404 cells, subcytotoxic concentration (2.0 µM) of three complexes was employed to avoid acute cytotoxicity and other nonspecific events that could lead to difficulty in interpretation of the results. On day 7 after being treated with 2.0 µM of 4, 5, and 6, BEL-7404 cells displayed an increased proportion of flat and giant cells with

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phenotypic

characteristics

of

cellular

senescence

as

revealed

by

the

senescence-associated β-galactosidase (SA-β-Gal) assay.53 The results shown in Figure 6 indicated that 6 might induce senescence mainly due to the shortening of telomere length, whereas the effects of 4 and 5 on BEL-7404 cells were not so obvious, implying that dysfunctional telomeres could initiate cellular senescence or apoptosis to suppress tumorigenesis.

Figure 6. Expression of SA-β-Gal in BEL-7404 cells after a long-term treatment with 4, 5, and 6, respectively. BEL-7404 cells were treated continuously with 2.0 µM of 4, 5, 6 or 0.1% dmso (control) for 7 days. These cells were then fixed, stained with SA-β-Gal staining kit, and photographed. Images were acquired using a Nikon Te2000 deconvolution microscope (magnification 200×). Effects on Cell Cycle Progression and Apoptosis. To verify the results of cell morphological analysis, we further investigated the changes in cell cycle and apoptosis of BEL-7404 cells after being treated with three ruthenium(II) complexes. The cell cycle analysis of the control group cells and BEL-7404 cells treated with 20 µM of 4, 5, and 6 for 48 h showed that all the compounds induced evident perturbation of the cell cycle and arrested the cells in S phases, respectively, as shown

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in Figures 7 and S31 (supporting information). The S phase populations observed in 4, 5, and 6 groups were 41.61, 33.17, and 45.13%, respectively, while the corresponding S population observed in the cells of the control group was 24.76%.

Figure 7. Populations for cell cycle of BEL-7404 cells treated with 4, 5, and 6 for 48 h comparing to those of the controlled cells, respectively. Since cell cycle arrest plays an important role in apoptosis of tumor cells,54 we further investigated the abilities of 4, 5, and 6 to induce cell apoptosis or cell death in BEL-7404 cells by flow cytometry. The results are shown in Figures 8 and S32 (Supporting Information), which indicated that 6 induced apoptotic cell death in a dose-dependent manner, as demonstrated by the combined staining with both propidium iodide (PI) and Annexin V-FITC. Compared with those of the control group, in BEL-7404 cells treated with 20 µM of each complex for 24 h, the populations of Annexin V+/PI– cells from early-stage and late-stage were 15.4% and 39.3% for 6, 6.3% and 13.9% for 5, and 6.4% and 25.0% for 4, respectively, which showed significant increase in the percentage of apoptotic cells. However, 4 and 5 failed to significantly induce the early-stage apoptosis under the same conditions.

16


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Figure 8. Populations for early and late-stages of apoptosis of BEL-7404 cells treated with 4, 5, and 6 for 24 h comparing to those the control cells, respectively. Detection of Changes in Protein Levels of 53BP1, TRF1 and TRF2 by Western Blot. Telomeres are specialized structures located at the ends of eukaryotic chromosomes that are essential for chromosome stability and for protection from degradation and DNA repair activities.55 It consists of tandem TTAGGG repeats, and in double strands, they are specifically recognized by the telomeric repeat binding factor 1 (TRF1) and telomeric repeat binding factor 2 (TRF2) in humans cells, which is directly bound to duplex telomeric DNA and anchors the shelterin along the telomere repeats.55(b) TRF1 is a homodimer and a flexible linker between the DNA binding and dimerisation domains, imbuing it with remarkable spatial flexibility. TRF1 also acts a negative regulator of telomere length by recruiting other shelterin components to the DNA.55(c),56 Specifically, telomeres that are severely or completely stripped of the protective telomere repeat binding factors, such as TRF2, evoke a DNA damage response and/or become the target of recombination repair.57 Furthermore, a number of proteins have been identified to associate with the telomere repeat binding factors, i.e. DNA repair/damage checkpoint proteins including tumor

17



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Page 18 of 65

suppressor p53-binding protein 1(53BP1), Rif1, Mre11, γ-H2AX, and ataxia telangiectasia mutated (ATM) complex. Moreover, 53BP1 plays a potential role in DNA damage responses and binds to the tumor suppressor protein p53.58 Therefore, to demonstrate the effects of 4, 5, and 6 on telomere function, we carried out Western blot analyses on TRF1, TRF2 and 53BP1 in BEL-7404 cells and showed the results in the Figure 9. Compared with those of the control, treatment of cells with 6 led to the increased protein levels by 2.9355 ± 0.1056, 2.3899 ± 0.1062, and 3.1835 ± 0.2121 folds for TRF1, TRF2, and 53BP1, respectively, suggesting that 6 significantly induced DNA damages including the damage to telomeres and other chromosomal regions in BEL-7404 cells. Recently, it was reported that TRF1 and TRF2 were the negative regulators of telomere length.55(a),57(b) In the present work, we found that the percentage of TRF1- and TRF2-induced damage to telomeres by 6 was 75.54%. In contrast, treatment of BEL-7404 cells with 10 µM 4, and 5 induced slight increases in protein levels of TRF1, TRF2, and 53BP1, respectively. The accumulation of TRF1, TRF2, and 53BP1 were 2.5932 ± 0.1401, 2.0982 ± 0.2065, and 1.9150 ± 0.1723 for 4, 1.8289 ± 0.1831, 1.7406 ± 0.3549, and 1.7982 ± 0.1124 for 5, respectively, showing that the percentages of TRF1- and TRF2-induced telomeres damage by 4, and 5 are 54.45% and 41.36%, respectively, which also can induce moderate telomere dysfunction. Taken together, these results suggested that the DNA damage response induced by 6 focused on telomeres rather than on other chromosomal regions. The selectivity of 6 on telomeric regions is higher than that of 4, 5, and complies with the order of 6> 4>5.

18


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Figure 9. Changes in protein levels of 53BP1, TRF1 and TRF2 in BEL-7404 cells treated with 4, 5, and 6 (10 µM) for 24 h, respectively. A: Western blot was used to determine the protein levels of 53BP1, TRF1 and TRF2 in BEL-7404 cells treated with 4, 5, and 6 (10 µM) for 24 h, respectively. B: Densitometric analyses of TRF1, TRF2 and 53BP1: the density of each of these protein bands was normalized with that of the β-actin band shown in part A. The relative expression level of each band = (density of each band/density of β-actin band). Mean ± SD was calculated from three independent measurements. Inhibition of p53 Expression. Disruption of the p53 pathway is strongly correlated with tumorigenesis, as it is considered as the key factor in maintaining genomic stability. As an important tumour suppressor, p53 plays a protective role in many cellular processes that are capable of activating multiple target genes, leading to cell cycle arrest. Inactivation of p53, which accounts for at least 50% of all cases,59 is the most common event in human cancers which impairs the DNA-damage response, making the tumour cells more resistant to drug-induced apoptosis. In addition, interference with p53 expression may be one of the action mechanisms for cell cycle arrest. Therefore, in order to further investigate whether the expression of mutant p53 protein is affected by 4, 5, and 6, the BEL-7404 cells containing a mutant form of p53 protein were used for our detailed investigation. 19



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BEL-7404 cells treatment with 10 µM 4, 5, or 6 caused a significant reduction of mutant p53 protein after 24 h, as shown in Figure 10. These results clearly demonstrated that the effects of 4, 5, and 6 on the expression of mutant p53 may be another pathway for their growth inhibition on BEL-7404 cells, especially 6.

Figure 10. Significant reduction of mutant p53 protein in BEL-7404 cells treated with 10 µM 4, 5, 6 or cisplatin. A: Western blot was used to determine the protein level of mutant p53 in BEL-7404 cells treated with 10 µM of 4, 5, 6 and cisplatin for 24 h, respectively. B: Quantitative analysis of 53-kDa mutant p53 from part A, the density of p53 fragment was normalized to that of 42-kDa β-actin band. The relative expression of each protein = (density of each band/density of actin band). Mean ± SD was calculated from three independent measurements. Caspase 3/9 Activation Assay. Caspases, in general, are a family of cysteine proteases that play essential roles in necrosis, inflammation, and apoptosis.60 Caspase-3/9, the executioner caspase, is able to directly degrade multiple substrates including the structural and regulatory proteins.61 Therefore, therapeutic strategies designed and developed to stimulate apoptosis by activating caspase-3/9 may help in combating cancer caused by deficient apoptosis. For this aim, we investigated whether the effector caspase-3/9 is activated when BEL-7404 cells are exposed to 4, 5, and 6, respectively. As shown in Figures 11 and 12, BEL-7404 cells treated with 4, 5, and 6

20


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exhibited a significant increase in caspase-3/9 activity, as indicated by the increased caspase-3 activation in the cells treated with 4, 5, and 6 by 10.5, 6.4 and 90.8%, respectively, and by the increased caspase-9 activation in the 4, 5, and 6 groups by 10.9, 2.4 and 37.7%, respectively, compared with those of the control group cells. The results suggested that 4 and 6 are efficient activators of caspases-3/9, and that 6 is more effective than 4 and 5, abiding by the order of 6>4>5. The up-regulation of the activation of caspase-3/9 is an important pathway in 4- and 6-induced apoptosis in BEL-7404 cells, especially that of 6.

Figure 11. The activation levels of caspase-3 protein in BEL-7404 cells treated with 10 µM of 4, 5, and 6 for 24 h, respectively. Activation of caspase-3 was assessed by the CasPGLOW fluorescein activate caspase-3 staining kit.

21



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Figure 12. The activation levels of caspase-9 protein in BEL-7404 cells treated with 10 µM of 4, 5, and 6 for 24 h, respectively. Activation of caspase-9 was assessed by the CasPGLOW fluorescein activate caspase-9 staining kit.

Selective Binding for G4 and Duplex DNA by 1− −3 and Their Complexes 4− −6. To verify the binding abilities of 1− −3 and their ruthenium(II) complexes 4, 5, and 6 to G4 (Pu27, HTG21, c-kit-1, c-kit-2 and Pu22) and duplex DNA (ds26, ctDNA or F32T+H20M), FID assay, CD, spectral analysis, fluorescence titration analysis, and FRET assay were carried out, and the detailed results are shown in Supporting Information. As shown in Table 2 and Figure S33, the FID assay indicated that 1− −3 and their complexes 4, 5, and 6 exhibited higher selectivities for Pu27 and HTG21 G4- DNAs than for other DNAs, which are the most efficient TO-displacers. HTG21

DC50 was reduced to 2.48, 2.25, 1.85, 0.73, 0.81, and 0.71 µM with 1, 2, 3, 4, 5,

and 6, respectively. Pu27DC50 was reduced to 1.63, 1.77, 1.43, 0.61, 0.65, and 0.52 µM with 1, 2, 3, 4, 5, and 6, respectively. These results illustrated that the selectivities of 1, 2, 3, 4, 5, and 6 for Pu27 and HTG21 G4-DNA over duplex DNA (ctDNA) were moderate. The ratios of

ctDNA

DC50/G4DC50 for HTG21 and Pu27 G-quadruplex DNA

(or Est. Sel.)62 after being treated with 1, 2, 3, 4, 5, and 6 were in the ranges of 17.78 and 136.11 fold and 22.59 and 185.85 fold, respectively. As expected, 4, 5, and 6 had higher selectivities for quadruplex-DNA than their corresponding ligands 1, 2, 3, especially 6. The data from fluorescence titration analysis (FTA) and CD spectra showed that the binding of 1, 2, 3, 4, 5, and 6 with G4-DNA might increase the structural stability of G4-DNA and hence interferes with the DNA function. As shown in Figures S34−S39 and Tables S6 and S7, in the absence and presence of K+, FTA and CD spectra indicated that the Pu27 DNA preferred to form a parallel 22


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conformation,63 whereas human telomeric G4-DNA (HTG21 DNA) preferred to fold into an intramolecularly mixed G4.64 As shown in Table 3, Figures S40 and S41, the FRET results clearly demonstrated that 2.0 µM of 1, 2, 3, 4, 5, and 6 produced the ∆Tm values of 11.68, 10.36, 10.58, 20.86, 16.81, and 23.05 °C for F21T, 14.43, 10.83, 16.55, 19.92, 17.84, and 23.76 °C for FPu18T, respectively. However, under the same conditions, the ∆Tm values for duplex DNA (F32T+H20M) generated by 1, 2, 3, 4, 5, and 6 were 4.16, 4.25, 4.04, 1.90, 3.24, and 1.62 °C, respectively. Therefore, 1, 2, 3, 4, 5, and 6 exerted a stronger stabilizing effect of F21T and FPu18T G-quadruplex DNA than the duplex DNA (F32T+H20M) (Table 3, Figures S40−S43). These results also illustrated that 4, 5, and 6 had higher selectivities for quadruplex-DNA than for their corresponding ligands, 1, 2, and 3. Moreover, 6 had higher selectivity for the Pu27 and HTG21 G4-DNAs over duplex-DNA than that of 4 and 5. Taken together, the favorable binding of 6 with G4 in human telomeric and in promoter region of c-myc gene resulted in relatively high thermodynamic stability, high binding constants and strong ability to induce conformational changes in Pu27 and HTG21 G4 DNAs. Table 2. FID assay for 1− −3 and their complexes 4− −6 on DNAs.

Pu27

DC50

(ctDNADC50/Pu27DC50#) HTG21

DC50

(ctDNADC50/HTG21DC50) c-kit-2

DC50

(ctDNADC50/c-kit-2DC50) c-kit-1

DC50

(ctDNADC50/c-kit-1DC50)

1

2

3

4

5

6

1.63

1.77

1.43

0.61

0.65

0.52

(39.24)

(22.59)

(62.81)

(150.13)

(66.48)

(185.85)

2.48

2.25

1.85

0.73

0.81

0.71

(25.79)

(17.78)

(48.55)

(125.45)

(53.35)

(136.11)

>100

>100

22.94

14.01

32.26

1.98

(3.92)

(6.54)

(1.34)

(48.81)

(＜0.64)

(＜0.40)

66.35

>100

2.45

1.98

1.69

1.54

(0.96)

(＜0.40)

(36.66)

(46.25)

(25.57)

(62.75)

23



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Pu22

DC50

(ctDNADC50/Pu22DC50) ds26

DC50

(ctDNADC50/ds26DC50) ctDNA

DC50

(ctDNADC50/ctDNADC50) #

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33.97

57.62

25.75

1.03

0.99

0.88

(1.88)

(0.69)

(3.49)

(88.91)

(43.65)

(109.82)

12.05

14.41

7.88

1.31

1.27

1.15

(5.31)

(2.77)

(11.40)

(69.91)

(34.02)

(84.03)

63.96

39.98

89.82

91.58

43.21

96.64

(1.00)

(1.00)

(1.00)

(1.00)

(1.00)

(1.00)

Sel. stands for G4-FID selectivity, defined as the ratio of ctDNADC50/G4DC50; DC50 was obtained in

µM.

Table 3. ∆Tm data (°C) of G4-DNA and the duplex DNA (F32T+H20M) obtained by real time-qPCR. Compds 1

2

3

4

5

DNA (1.0 µM) Conc. of Compd ∆Tm (°C) F21T

2.0 µM

11.68

FPu18T

2.0 µM

14.43

F32T+H20M

2.0 µM

4.16

F21T

2.0 µM

10.36

FPu18T

2.0 µM

10.83

F32T+H20M

2.0 µM

4.25

F21T

2.0 µM

10.58

FPu18T

2.0 µM

16.55

F32T+H20M

2.0 µM

4.04

F21T

2.0 µM

20.86

FPu18T

2.0 µM

19.92

F32T+H20M

2.0 µM

1.90

F21T

2.0 µM

16.81

FPu18T

2.0 µM

17.84

F32T+H20M

2.0 µM

3.24

24


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6

F21T

2.0 µM

23.05

FPu18T

2.0 µM

23.76

F32T+H20M

2.0 µM

1.62

Transcriptional and Translational Levels of hTERT and c-myc Genes. G4-ligands may affect the telomerase activity mainly via two pathways, i.e. c-myc-regulated transcriptional activation of hTERT and/or stabilization of the telomeric G-rich end to block the association with the catalytic enzyme.65 Furthermore, hTERT is a key catalytic domain of the telomerase enzyme and is closely related to telomerase function, due to the regulation by c-myc-induced hTERT promoter activity.66 Because the inhibitory activities of 4, 5, and 6 are stronger than those of their corresponding ligands 1, 2, and 3 in telomerase activity assay, only 4, 5, and 6 were selected for RT-qPCR and Western blot assay in BEL-7404 cells. As shown in Figure 13, 4, 5, and 6 down-regulated the transcriptional levels of c-myc and hTERT genes in BEL-7404 cells, indicating that 6 causes stronger effects on down-regulation of these two genes than 4 and 5. In order to further confirm whether the inhibitory effects of 4, 5, and 6 on the expression of these two genes, we performed Western blot analysis and the results are shown in Figures 14 and 15, which demonstrated that 6 also caused stronger inhibition on the protein expression levels of c-myc and hTERT in BEL-7404 cells than 4 and 5, consistent with the results of RT-qPCR.

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Figure 13. Inhibitory effects of 4, 5, and 6 on the transcriptional levels of hTERT and c-myc in the BEL-7404 cells. RT-qPCR was used to determine the transcriptional expression levels of hTERT and c-myc in the BEL-7404 cells treated with 4, 5, and 6, respectively. 5×105 BEL-7404 cells were cultured in a 25-cm2 flask with medium (no drug) and then treated with 10 µM of 4, 5, or 6 for 24 h, respectively. Total RNA was extracted, subjected to reverse transcription and analyzed by RT-qPCR for mRNA levels of hTERT (A), c-myc (B) and GAPDH (as the internal control).

Figure 14. Inhibitory effects of 4, 5, 6 and cisplatin on the protein levels of hTERT in the BEL-7404 cells. A: Western blot was used to determine the protein level of hTERT on BEL-7404 cells treated with 10 µM of 4, 5, 6 and cisplatin for 24 h, respectively. B: Densitometric analysis of 122-kDa hTERT and its cleaved fragment (80 kDa) normalized to the density of 42-kDa β-actin band from part A. The relative expression of each band = (density of each band/density of β-actin band). Mean ± SD 26


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was derived from three independent measurements.

Figure 15. Inhibitory effects of 4, 5, 6 and cisplatin on the protein levels of c-myc in the BEL-7404 cells. A: Western blot was used to determine the translation of c-myc in BEL-7404 cells treated with 10 µM of 4, 5, 6 and cisplatin for 24 h, respectively. B: Densitometric analysis of 60-kDa c-myc protein normalized to the density of 42-kDa β-actin band from part A. The relative expression of each band = (density of each band/density of β-actin band). Mean ± SD was derived from three independent measurements. Transfection of BEL-7404 Cells with Enhanced Green Fluorescent Protein and c-myc Gene Vectors. In order to verify whether 4, 5, and 6 can directly regulate the activity of c-myc promoter (Pu27), and affect the expression of hTRET in BEL-7404 cells, and consequently inhibit the telomerase function, we constructed enhanced green fluorescent protein (EGFP) and c-myc gene vectors according to the methods reported previously.67 After being successfully transfected with EGFP vector or c-myc gene vector, BEL-7404 cells were treated with 10 µM of 4, 5, and 6, respectively, for 24 h, and then assayed using fluorescence microcopy or luciferase reporter gene assay kit. As shown in Figure 16(A), BEL-7404 cells displayed green fluorescence after transfection with EGFP plasmid, indicating the successful transfection. Figure 16(B) further demonstrated that compare with those of the control group cells, treatments 27



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with 6 down-regulated the expression of hTRET in BEL-7404 cells, resulting in inhibition of telomerase via down-regulating the c-myc promoter, which was different from 4 and 5. The significantly improved biological behavior by 6 may be correlated with 3, a S-enantiomer ligand similar to S-ibuprofen, which is over 100 times more potent than R-ibuprofen is.10

Figure 16. Successful transfections of EGFP (A) and c-myc (B) plasmid vectors into BEL-7404 cells, respectively. 2.0 µg of EGFP-carrying plasmid vector or 2.0 µg of c-myc-carrying plasmid vector was co-tranfected into BEL-7404 cells using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA), respectively. 4, 5, and 6 were then added, respectively, into medium at 6 h after transfection of c-myc plasmid. At another 24 h after treatment with each complex, these cells were imaged using Nikon TE2000 (Japan) scanning fluorescent microscope or examined with luciferase reporter gene assay kit. Telomerase Inhibition with TRAP Assay. Inhibition of telomerase activity may be mediated via stabilization of G4s by ligands or ligand-induced quadruplex formation

by telomeric G-rich strand.68 At the same time, telomerase is

overexpressed in 85−90% of the human tumor cells but has undetectable activity in

28


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most of the human normal somatic cells.69 Therefore, we conducted telomeric repeat amplification protocol (TRAP) assay to further characterize telomerase activity. 1, 2, 3, 4, 5, and 6 were added to a telomerase reaction mixture containing the extract prepared from Hep-G2 cells,70 respectively, which express high levels of telomerase. The inhibition ratios of 1, 2, 3, 4, 5, and 6 on telomerase were summarized in Figure 17 and Table S8 (Support Information). As indicated in Table S8 and Figure 17, the inhibitory properties of 4, 5, and 6 on telomerase activity were significantly improved upon an enantioselective ligand 4-(2,3-dihydroxypropyl)-formamide oxoaporphine ligating Ru(II).

Figure 17. Inhibitory rates (%) and

Tel

IC50 (µM) of 1, 2, 3, 4, 5, and 6 on the

telomerase activity, respectively. In order to further determine the abilities of 1, 2, 3, 4, 5, and 6 to inhibit the telomerase in BEL-7404 cells, we treated BEL-7404 cells with 10 µM of the each compound, respectively, for 24 h. The telomerase activity was assayed using a PCR-TRAP ELISA kit (Roche, USA) according to the manufacturer’s guide. As shown in Figure 18 and Table S9, the cellular inhibitory effects of 4, 5, and 6 on telomerase were obviously higher than those of the corresponding ligands 1, 2, and 3, 29



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Page 30 of 65

respectively. Moreover, the inhibitory property of 6 on telomerase was significantly better than those of 4 and 5, and followed the order of 6>4>5, agreeing well with the results of all of experiments mentioned above.

Figure 18. Inhibitory rates (%) of 1, 2, 3, 4, 5, and 6 on telomerase in BEL-7404 cells, respectively. Effects

on

the

Expression

Profiles

of

Telomeres-associated

and

Telomerase-related Genes Using a RT-qPCR Array. Recently, it was indicated that it is important to discover and validate cancer biomarkers and therapeutic targets by gene expression profiling.71 Therefore, we examined the gene expression profiles and compared the relative expression levels of a panel of telomeres-associated and telomerase-related genes in BEL-7404 cells using a RT-qPCR array. In order to determine the effects of 6 (10.0 µM) on the expression of genes associated with telomeres and the telomerase-related genes, BEL-7404 cells were treated with 10.0 µM 6 and compared with those in the control group. We studied the mRNA expression profiles using a Telomeres & Telomerase PCR Array (RT² Profiler™ PCR Array Human Telomeres & Telomerase) which contains 84 well-known telomeres/telomerase-related genes. As shown in Table S10 and Figure 19, of the 84 genes examined, 19 genes were differentially expressed in mRNA levels by

30


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1.4-fold or higher in BEL-7404 cells after being treated with 6 (10.0 µM) for 24 h. The genes whose expression was down-regulated included IGF1 (up to 83.85 fold), PPP2R1B, RB1, RTEL1, SMAD3 and TNKS2 whereas the genes whose expression was up-regulated included TGFB1, TEP1, SIRT6, SART1, RIF1, RFC1, RAD50, PAX8 (up to 4.55 fold), HSPA1L, HAT1, EGF, DCLRE1C and ATM. Subsequently, the up-regulated TERF1 (TRF1), TERF2 (TRF2), TP53BP1 (53BP1) and the down-regulated MYC (c-myc) and TP53 (mutant p53 in BEL-7404 cells) were further validated in their protein levels using Western blot in BEL-7404 cells under the same conditions, respectively. The results of Western blot analysis agreed with the those of a panel of genes for RT-qPCR array in BEL-7404 cells. These results suggest that one of the inhibitory effects of 6 (10.0 µM) on telomeres/telomerase-related and telomerase might be mediated via its interactions with TERF1 (TRF1), TERF2 (TRF2), TP53BP1 (53BP1) and MYC (c-myc) in BEL-7404 cells.

31



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Figure 19. Relative expression profiles of 84 telomeres/telomerase-related genes in 32


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BEL-7404 cells after being treated with 6 (10.0 µM) for 24 h. The figure depicts a log △Ct

transformation plot of the relative expression level of each gene (2-

) in BEL-7404

cells. In summary, 6 was the effective stabilizer of telomeric, and G-quadruplex-DNA (G4-DNA) in promoter of c-myc, which acted as a telomerase inhibitor targeting G4-DNA and induced cell senescence and apoptosis. However, such antitumor mechanism is distinguished from those polypyridyl ruthenium complexes, for example, anticancer ruthenium−arene Schiff-base complexes may act independently of the p53 tumor suppressor gene frequently mutated in cancer;28(f) The iodido and chlorido ruthenium arene azo- and imino-pyridine anticacner complexes are not dependent on p53 for activity, induce a high incidence of late-stage apoptosis, and possess potency which is greatly increased by a low dose of the redox modulator L-BSO;30 Ruthenium polypyridyl complex (RuPOP) that is able to induce mitochondria-mediated and caspase-dependent apoptosis in human cancer cells via regulation of Bcl-2 family members and activation of caspases.31(c) Similar to RuPOP's activation of caspases, 6 could activate caspases-3/9 to initiate cell apoptosis. Preliminary Safety Evaluation. Investigation of toxicity at the maximal administration dose of 6 was illustrated in Figure 19. The highest soluble 6 in solvent (5% v/v dmso/saline) was used as the solution to conduct preliminary study on its safety, and the ICR mice received a possible maximal administration values72 (1.0 mL/20 g) by intraperitoneal injection. One group of ICR mice (n=6) were treated with 6 at a dose of 25 mg/kg once a day (qd). Another group of ICR mice (n=6) received the same dose once every two days (q2d) for consecutive 10 days and the third group of the ICR mice (the control group) received the same volume of solvent. The ICR

33



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mice in all groups were monitored for 4 days, respectively. The animals were observed every day for the signs of toxicity and mortality. The body weight was measured daily and was taken as a parameter of systemic toxicity. During the 10-day period of treatment, no other adverse effects were observed and the body weight of mice displayed a rising trend. As shown in Figure 20, the average body weight of mice treated with 6 at dose of 25 mg/kg per day or per two days had similar growth rates similar to that of the control group. The mice were killed on the day 14 and no signs of peritonitis or damage to organs were observed. These results indicate that no significant toxicity is caused to the mice by the treatment with 6 at these doses daily or one time per two days.

Figure 20. Changes in body weight of ICR mice administrated with maximum administration dose of 6 daily or once every two days. A groups of ICR mice (n = 6) received either vehicle; the second group of mice received 6 at doses of 25 mg/kg daily and the third group of mice received 6 at dose of 25 mg/kg once per two days for 10 days and then monitored for 4 days. The animals were weighed daily. Inhibition of Human Hepatocarcinoma by 6 in Nude Mice. 6 was selected for in vivo antitumor evaluation in mice and BEL-7402 tumor xenograft model was used. In the BEL-7402 tumor model, the mice were randomly divided into the vehicle control, 34


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the 6-treated group and cisplatin-treated group. The mice in 6-treated group or cisplatin-treated group were treated via ip injection daily with either 6 or cisplatin, respectively, once the tumor had grown to a volume of 40−90 mm3 (Figure 21 and Tables S11−S13). As shown in Figure 21A, treatment of mice with 6 resulted in a significant reduction in tumor volume as compared to that observed in mice of the control group. A dose-dependent inhibition of tumor growth by 6 at 12.5 and 25 mg/kg showed relative tumor increment rates (T/C) of 60.3% and 47.6% (P5. Moreover, 6 displayed a stronger ability to inhibit telomerase activity and a higher selectivity for G4-DNA and telomeric region, and led to more severe damage to telomeres in BEL-7404 cells than that of 4 and 5, following the order of 6>4>5. In addition,

6

caused

more

significant

changes

in

the

expression

of

telomeres/telomerase-related genes than those caused by 4 and 5 and induced stronger down-regulation of the expression of c-myc and hTERT in BEL-7404 cells. In summary, the different biological behaviors of 4, 5 and 6 are correlated with the chiral nature of their ligands, exhibiting certain enantiomeric selectivity.

CONCLUSION

To develop effective G4-DNA binders exerting inhibitory effect on telomerase activity, we prepared three ruthenium(II) complexes, 4 (racemate), 5 (R-enantiomer) and 6 (S-enantiomer), with chiral 4-(2,3-dihydroxypropyl)-formamide oxoaporphine ligands, 1, 2 and 3, and then evaluated their in vitro and in vivo antitumor activities. 4, 5 and 6 displayed a synergistic effect upon the combination of ruthenium(II) with the corresponding free chiral oxoaporphine ligands 1, 2 and 3. The results of binding experiments, cellular and molecular assays from various biological assays, including FID assay, FRET assay, fluorescence titration, CD, uptake assay, flow cytometry, RT-qPCR, Western blot, transfection, TRAP assay, and gene expression profiling revealed that 6 had relatively stronger binding affinity to G4-DNA in telomere and promoters of c-myc gene than 4 and 5 did. 4, 5 and 6, especially 6, served as

37



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stabilizers of telomere and G4-DNA present in c-myc promoter and induced DNA damage, leading to cell senescence and apoptosis. Both 4 and 6 could activate caspases-3/9 to initiate cell apoptosis, and the effect of 6 appeared to be stronger, whereas 5 did not. In addition, 4, 5 and 6 caused the arrest of cell growth at S phase. Taken together, 4, 5 and 6 exhibited cytotoxic effects in BEL-7404 cells via both senescence- and apoptosis-mediated mechanisms. The different biological activities of 4,

5

and

6

were

correlated

with

the

chiral

nature

of

4-(2,3-dihydroxypropyl)-formamide oxoaporphine ligands, 1, 2 and 3. Remarkably, 6 exhibited higher in vivo safety than cisplatin did, and high in vivo inhibition efficacy on tumor growth in the BEL-7402 xenograft mouse model. Therefore, 6 may have the potential to be further developed as a safe and effective anticancer agent.

EXPERIMENTAL MEHTODS

The purity of all target compounds used in the biophysical and biological studies was ≥95%. 4− −6 used were routinely checked by HPLC. Synthesis

of

4-Carboxyl-7-oxo-7H-dibenzo[de,g]quinoline.

Synthesis

and

characterization of 4-carboxyl-7-oxo-7H-dibenzo[de,g]quinoline were performed as reported previously by Tang et al.46 Synthesis of R/S-(±)-4-(2,3-Dihydroxypropyl)-formamide Oxoaporphine 1. General acieration procedure: Pylon (7.5 g, 0.015 mol) dissolved in 100 mL of dimethylacetamide (DMA) was added over a 30-min period to a solution of 4-carboxyl-7-oxo-7H-dibenzo[de,g]quinoline

(2.75

g,

0.01

mol)

and

R/S-(±)-3-amino-1,2-propanediol (1.37g, 0.015 mol) in DMA (10 mL) at 65 °C. The mixture was stirred at 65 °C for 1 h and concentrated under vacuum. The residue was taken up with dichloromethane and the solution was washed with a solution of sodium

38


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carbonate, dried (Na2SO4), and concentrated under vacuum. Column chromatography of the residue, after being eluted with a 5% methanol in dichloromethane, gave a yellow 1 (2.53 g, 72.9%). 1H NMR (500 MHz, demos-d6) δ 9.05 (s, 1H), 8.94−8.91 (m, 1H), 8.78 (d, J = 7.1 Hz, 1H), 8.57 (d, J = 8.0 Hz, 1H), 8.36 (did, J = 8.5, 0.7 Hz, 1H), 8.30 (did, J = 7.9, 1.4 Hz, 1H), 8.02 (did, J = 8.5, 7.5 Hz, 1H), 7.88−7.86 (m, 1H), 7.69−7.64 (m, 1H), 4.96 (d, J = 5.2 Hz, 1H), 4.68 (t, J = 5.8 Hz, 1H), 3.78−3.74 (m, 1H), 3.57−3.54 (m, 1H), 3.46−3.43 (m, 1H). 13C NMR (125 MHz, demos-d6) δ 181.7, 166.5, 146.9, 143.6, 135.2, 135.0, 134.1, 132.4, 131.3, 129.9, 128.4, 128.3, 127.3, 126.4, 124.7, 124.1, 70.7, 64.5, 43.6. ESI-MS m/z: 371.0 [M+Na]+. IR (KBr): 3284, 2923, 2879, 1639, 1599, 1550, 1484, 1465, 1432, 1385, 1320, 1273, 1226, 1204, 1163, 1103, 1068, 1043, 934, 905, 804, 772, 749, 697, 604, 584, 565 cm−1. Elemental analysis calcd (%) for C20H16N2O4: C 68.96, H 4.63, N 8.04; found: C 68.98, H 4.68, N 7.99. Synthesis of R-(+)-4-(2,3-Dihydroxypropyl)-formamide Oxoaporphine 2. General acylation procedure: PyBOP (7.5 g, 0.015 mol) dissolved in 100 mL of DMA was

added

over

a

30-min

4-carboxyl-7-oxo-7H-dibenzo[de,g]quinoline

period (2.75

to

a

g,

0.01

solution mol)

of and

R-(+)-3-amino-1,2-propanediol (1.37 g, 0.015 mol) in DMA (10 mL) at 65 °C. The mixture was stirred at 65 °C for 1 h and concentrated under vacuum. The residue was taken up with dichloromethane and the solution was washed with a solution of sodium carbonate, dried (Na2SO4), and concentrated under vacuum. Column chromatography of the residue, after being eluted with a 5% methanol in dichloromethane, gave a yellow 2 (2.25 g, 61.8%). 1H NMR (500 MHz, dmso-d6) δ 9.06 (s, 1H), 8.99−8.97 (m, 1H), 8.80 (d, J = 7.5 Hz, 1H), 8.60 (d, J = 8.1 Hz, 1H), 8.37 (d, J = 8.5 Hz, 1H), 8.31 (d, J = 7.6 Hz, 1H), 8.04−8.03 (m, 1H), 8.01−7.89 (m, 1H), 7.69−7.66 (m, 1H), 5.01 (s, 39



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1H), 4.72 (s, 1H), 3.76–3.74 (m, 1H), 3.58–3.53 (m, 1H), 3.45–3.43 (m, 2H).

Page 40 of 65

13

C

NMR (125 MHz, dmso-d6) δ 181.7, 166.5, 147.0, 143.7, 135.3, 135.1, 134.1, 132.5, 131.4, 130.0, 128.5, 128.4, 127.3, 126.5, 124.8, 124.2, 70.8, 64.5, 43.7. ESI-MS m/z: 371.0 [M+Na]+. IR (KBr): 3456, 3300, 3077, 2945, 2374, 2225, 1657, 1597, 1558, 1462, 1435, 1383, 1320, 1276, 1263, 1226, 1103, 1037, 930, 845, 804, 771, 606, 587 cm−1. Elemental analysis calculated (%) for C20H16N2O4: C 68.96, H 4.63, N 8.04; found: C 68.94, H 4.70, N 7.98. Synthesis of S-(− −)-4-(2,3-Dihydroxypropyl)-formamide Oxoaporphine 3. General acylation procedure: PyBOP (7.5 g, 0.015 mol) in DMA (100 mL) was added over a 30-min period to a solution of 4-carboxyl-7-oxo-7H-dibenzo[de,g]quinoline (2.75 g, 0.01 mol) and S-(−)-3-amino-1,2-propanediol (1.37g, 0.015 mol) in DMA (10 mL) at 65 °C. The mixture was stirred at 65 °C for 1 h and concentrated under vacuum. The residue was taken up with dichloromethane and the solution washed with a solution of sodium carbonate, dried (Na2SO4), and concentrated under vacuum. Column chromatography of the residue, after being eluted with a 5% methanol in dichloromethane, gave yellow 3 (2.66 g, 78.6%). 1H NMR (500 MHz, dmso-d6) δ 9.05 (s, 1H), 8.96−8.94 (m, 1H), 8.79 (d, J = 7.2 Hz, 1H), 8.58 (d, J = 7.9 Hz, 1H), 8.37 (d, J = 8.5 Hz, 1H), 8.31 (d, J = 7.8 Hz, 1H), 8.02 (t, J = 7.9 Hz, 1H), 7.90−7.87 (m, 1H), 7.69−7.66 (m, 1H), 4.99 (s, 1H), 4.70 (s, 1H), 4.38 (t, J = 4.9 Hz, 1H), 3.76 (s, 1H), 3.58−3.54 (m, 1H).

13

C NMR (125 MHz, dmso-d6) δ 181.7, 166.5, 147.0,

143.6, 135.2, 135.1, 134.1, 132.5, 131.3, 129.9, 128.4, 128.3, 127.3, 126.4, 124.7, 124.1, 70.7, 64.5, 43.6. ESI-MS m/z: 371.0 [M+Na]+. IR (KBr): 3299, 3220, 2940, 2874, 2670, 1964, 1841, 1644, 1595, 1561, 1484, 1465, 1436, 1383, 1320, 1273, 1263, 1226, 1204, 1163, 1106, 1078, 1039, 996, 933, 842, 804, 771, 755, 700, 678, 606, 562, 488, 458 cm−1. Elemental analysis calcd (%) for C20H16N2O4: C 68.96, H 4.63, N 8.04; 40


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found: C 68.95, H 4.64, N 8.02. Synthesis of cis-[RuCl2(R/S-(±)-FOA)(dmso)2] 4. The Ru(II) complex 4 was prepared by treating 1 (0.017 g, 0.05 mmol) with cis-RuCl2(dmso)4 (0.025 g, 0.05 mmol) in ethanol/chloroform (1:1) under solvothermal conditions. Dark green block crystals of 4 suitable for X-ray diffraction analysis were harvested. Yield (0.029 g, 85.8%). 1H NMR (500 MHz, dmso-d6) δ 10.53 (s, 1H), 9.22 (s, 1H), 9.02 (s, 1H), 8.71 (s, 2H), 8.40 (s, 1H), 8.14 (s, 1H), 8.03 (s, 1H), 7.78 (s, 1H), 4.96 (s, 2H), 4.69 (s, 2H). ESI-MS m/z: 712.9 [M+Cl]−. IR (KBr): 3400, 3011, 2918, 1646, 1602, 1550, 1492, 1407, 1385, 1350, 1320, 1292, 1259, 1202, 1139, 1099, 1015, 971, 938, 798, 768, 719, 680, 639, 617, 576, 505 cm−1. Elemental analysis calcd (%) for C24H28Cl2N2O6RuS2: C 42.60, H 4.17, N 4.14; found: C 42.63, H 4.19, N 4.13. Synthesis of cis-[RuCl2(R-(+)-FOA)(dmso)2] 5. The Ru(II) complex 5 was prepared by treating 2 (0.017 g, 0.05 mmol) with cis-RuCl2(dmso)4 (0.025 g, 0.05 mmol) in ethanol/chloroform (1:1) under solvothermal conditions. Dark green products of 5 for analysis were harvested. Yield (0.026 g, 76.3%). 1H NMR (500 MHz, dmso-d6) δ 10.58 (s, 1H), 9.27 (t, J = 5.7 Hz, 1H), 8.95 (d, J = 7.6 Hz, 1H), 8.69−8.61 (m, 2H), 8.40 (d, J = 8.5 Hz, 1H), 8.11 (dd, J = 8.4, 7.5 Hz, 1H), 8.02–7.96 (m, 1H), 7.79−7.71 (m, 1H), 5.00 (d, J = 5.2 Hz, 1H), 4.72 (s, 1H), 3.80 (dt, J = 6.9, 5.2 Hz, 1H). 13C NMR (125 MHz, dmso-d6) δ 191.0, 164.9, 147.7, 146.7, 136.8, 136.1, 134.1, 133.3, 132.5, 130.1, 129.2, 128.6, 128.0, 127.6, 126.5, 124.7, 123.8, 70.3, 63.9, 45.2. ESI-MS m/z: 705.0 [M–Cl+2CH3OH]+. IR (KBr): 3442, 3060, 2918, 2374, 1651, 1602, 1568, 1553, 1473, 1449, 1416, 1388, 1353, 1317, 1281, 1224, 1202, 1166, 1147, 1086, 1020, 968, 938, 834, 802, 722, 680, 609, 576, 532 cm−1. Elemental analysis calcd. (%) for C24H28Cl2N2O6RuS2: C 42.60, H 4.17, N 4.14; found: C 42.65, H 4.20, N 4.12.

41



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Page 42 of 65

Synthesis of cis-[RuCl2(S-(− −)-FOA)(dmso)2] 6. The Ru(II) complex 6 was prepared by treating 3 (0.017 g, 0.05 mmol) with cis-RuCl2(dmso)4 (0.025 g, 0.05 mmol) in ethanol/chloroform (1:1) under solvothermal conditions. Dark green products of 6 for analysis were harvested. Yield (0.031 g, 91.2%). 1H NMR (500 MHz, dmso-d6) δ 10.57 (s, 1H), 9.24−9.22 (m, 1H), 9.09 (d, J = 7.2 Hz, 1H), 8.81−8.77 (m, 2H), 8.47 (d, J = 8.5 Hz, 1H), 8.23−8.20 (m, 1H), 8.11−8.08 (m, 1H), 7.88−7.84 (m, 1H), 4.96 (d, J = 5.2 Hz, 1H), 4.69 (t, J = 5.7 Hz, 1H), 4.36 (t, J = 5.0 Hz, 1H), 3.80−3.77 (m, 1H). 13C NMR (125 MHz, dmso-d6) δ 191.5, 165.3, 148.2, 147.4, 137.3, 136.6, 134.8, 133.8, 132.9, 130.7, 129.8, 129.1, 128.5, 128.2, 127.1, 125.3, 124.3, 70.7, 64.4, 45.6. ESI-MS m/z 705.1: [M–Cl+2CH3OH]+. IR (KBr): 3369, 3066, 2962, 2923, 2368, 1651, 1602, 1566, 1556, 1473, 1440, 1416, 1388, 1353, 1320, 1281, 1259, 1224, 1202, 1166, 1141, 1094, 1048, 1018, 968, 938, 834, 801, 771, 680, 661, 609, 532, 499 cm−1. Elemental analysis calcd (%) for C24H28Cl2N2O6RuS2: C 42.60, H 4.17, N 4.14; found: C 42.62, H 4.18, N 4.15. X-Ray Crystallography. The data collections of single crystals of 1 and 4 were performed

on

a

SuperNova

CCD

diffractometer

equipped

with

graphite

monochromated Mo-Kα radiation (λ = 0.71073 Å) at room temperature. The structures were resolved with direct methods and refined using SHELX-97 programs.73 The non-hydrogen atoms were located in successive difference Fourier synthesis. The final refinement was performed by full-matrix least-squares methods with anisotropic thermal parameters for no-hydrogen atoms on F2. The hydrogen atoms were added theoretically and riding on the concerned atoms. The parameters used in intensity collection and refinements were summarized in Tables S1−S4 together with the crystal data. Materials, Instrumentation and Other Experimental Methods. The materials, 42


Page 43 of 65

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instrumentation, cytotoxicity assay and uptake of Ru complexes in BEL-7404 cells, morphological examination, fluorescence morphological examination, SA-β-Gal assay, cell cycle analysis, apoptosis analysis, determination of caspase-3/9 activity by flow cytometric analysis, RNA extraction, reverse transcriptase–polymerase chain reaction (RT-PCR), Western blot, and transfection assay of 4, 5 and 6, and selective binding for G-quadruplex and duplex DNA by 1, 2, 3 and their complexes 4, 5 and 6 were similar to those illustrated in our previous work.74 Gene Expression by a Panel of Genes for RT-qPCR Array. Differential expression profiles of telomeres/telomerase-related genes were analyzed using the Telomeres & Telomerase PCR Array (RT² Profiler™ PCR Array Human Telomeres & Telomerase, PAHS-010Z, SABioscience, USA) obtained from Kangcheng Bio-tech (Shanghai, China). RNA was extracted according to standard protocols and converted to first strand cDNA using the RT2 First Strand Kit. The template was added to an instrument specific, ready-to-use RT2 SYBR Green qPCR Master Mix. The resulting mixture was added to the wells (25 µL/well) of the PCR Array plate containing the telomere maintenance, telomere-associated complexes, telomere regulation and primer sets specific for the telomeres-associated and telomerase-related genes (25 µL for 96-well plates) and RT-qPCR was performed. The threshold cycle (Ct) values for all the genes on each PCR Array were calculated using the instrument specific software and the fold-changes in gene expression for pair-wise comparison were calculated using the 2-△Ct method. Animal Used. Kunming (KM) mice (both male and female, 20−22 g, 5−6 week old) and BALB/c nude mice (male, 20−22 g, 6−7 weeks old) were supplied by Beijing HFK Bioscience Co., LTD (Beijing, China), and used for the human hepatocarcinoma (BEL-7402) xenograft. The in vivo antitumor studies were carried out at the Institute 43



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Page 44 of 65

of Radiation Medicine Chinese Academy of Medical Sciences (Tian Jin, China). The handling of animals and the experimental design were approved by the Ethnics Committee and Animal Care Committee of the Institute. Nude mice were housed at individual ventilated caging system (IVC Rack) with sterile environment with conditions at a constant photoperiod (12 h light/12 h dark at 25-28 °C and 45-65% relative humidity). Acute Toxicity Studies. Six-week old male and female KM mice (weight 20−22 g) were randomly divided into 3 groups (n = 6) and used to study the in vivo safety of 6. The highest solubility of 6 in solvent (5% v/v dmso/saline) was used as the solution, and a good practice volume (0.6 mL/20 g) by intraperitoneal injection was used. Two groups of KM mice were treated with 6 at a dose of 16 mg/kg twice a day (bid) or once a day (qd) for 10 day, respectively, and one group received the same volume of solvent and used as the control. The signs of toxicity were observed and body weight was recorded daily. The In Vivo Antitumor Activity toward BEL-7402 by 6. Nude mice received subcutaneous injection of 5×107 BEL-7402 cells in right flank. When the xenograft tumor growth to the volume about 1000 mm3, the mice were killed and the tumor tissue were cut into small pieces at about 1.5 mm3, and then transplanted into the right flank of male nude mice. When the average tumors reached the volumes of 100-150 mm3, the mice were randomly divided into solvent control and treatment groups (n=7/group). 6 at doses of 16 and 8 mg/kg (5% v/v dmso/saline) were given twice a day for 17 days (ip). Cisplatin was given to mice by ip administration at a dosage of 2 mg/kg/per two days and used as a positive reference for comparison, and control mice received the solvent (5% v/v dmso/saline). Tumor size and body weight were monitored every three days. On day 18, the animals were sacrificed for humane 44


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reasons, and the tumors were weighted and recorded. The tumor volumes were determined every three days by measuring length (l) and width (w) and calculating with the formula of V = lw2/2 as described elsewhere.75 Meanwhile, body weight of mice was measured and taken as a parameter of systemic toxicity. All mice were sacrificed on day 18 after treatment (grouping), and tumor weight was recorded. The rate of tumor growth was calculated using a formula of (1−TWt / TWc)×100, where TWt was the tumor weight of complex-treated mice and the TWc was the tumor weight of vehicle-treated animals.76 Statistical Analysis. The experiments have been repeated three to five times, and the results obtained were presented as means ± standard deviation (SD). Significant changes were assessed by using Student’s t test for unpaired data, and the differences between groups with P values of

Water-Soluble Ruthenium(II) Complexes with Chiral 4-(2,3

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