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Nucleus-enriched Ruthenium Polypyridine Complex Acts as a Potent Inhibitor to Suppress Triple-negative Breast Cancer Metastasis In vivo.

Polypyridine Ru(II) complexes have long been deemed to excellent antitumor agents that inhibit the proliferation of breast cancer cells. Nevertheless, their effects on the metastatic potency of breast cancer cells need further research. Herein, a class of polypyridine Ru(II) complexes coordinated with phenazine derivates (DPPZ) ([Ru(bpy)2(DPPZ-R)](ClO4)2, Ru(bpy) 2 DPPZ: R = -H, Ru(bpy) 2 BrDPPZ: R = -Br, Ru(bpy) 2 MDPPZ: R = -CH3, Ru(bpy)2BnDPPZ: R = -acene, Ru(bpy) 2 BEDPPZ: R = -C ≡ C(C6H5)) was synthesized by introducing different substituent groups to regulate the electron cloud density and planarity of the main ligands. Results indicated that this class of DPPZ-based Ru(II) complexes exhibited promising inhibitory effect against MDA-MB-231 triple-negative breast cancer cells, especially for Ru(bpy) 2 BEDPPZ, which is comparable with that of cisplatin. In addition, Ru(bpy) 2 BEDPPZ effectively inhibited the migration and invasion of MDA-MB-231 cells in vitro and suppressed focal adhesion and stress fiber formation. Moreover, it effectively blocked MDA-MB-231 cell metastasis in blood vessels and restrained angiogenesis formation in a zebrafish xenograft breast cancer model. Further studies showed that the mechanisms may involve DNA damage-mediated apoptosis probably due to Ru(bpy) 2 BEDPPZ, which was enriched in the cell nucleus and induced DNA damage. All these results suggested that the DPPZ-based Ru(II) complexes can act as potent anti-metastasis agents.


Introduction
Breast cancer is a disease that significantly threatens women's life. The triple-negative breast cancer (TNBC) is considered to be the most dangerous in the different types of breast cancer [1]. It is a common malignant tumor in China and has high degree of malignancy, incidence of recurrence, and metastasis [2]. The site of distant metastasis occurs mainly in the bone, lungs, liver, and brain [3]. Although methods such as endocrine and molecular targeted therapy are used in the treatment of TNBC, they are not effective. Currently, the commonly used treatment for TNBC is chemotherapy [4,5]. Anthracycline-based drugs, paclitaxel, and platinum antitumor drugs mainly target the DNA [6][7][8]. At the gene level, they affect the function of TNBC cells. However, they have no selectivity against normal cells, causing serious side effects to patients [9]. In TNBC, once metastasis develops in the surrounding tissue, the patient's life is in danger [10]. Thus, developing drugs that can restrain TNBC metastasis and clarifying their mechanism are important [11].
Recently, Ru-based complexes have attracted considerable attention because of their high antitumor activity, low toxicity, and powerful DNA-binding affinity. Two Ru(II) complexes (NAMI-A and KP1019) have entered phase II clinical trials due to their potential antitumor behavior [12]. A number of evidence suggests that Ru(II) complexes have wonderful DNA binding properties [13,14]. DNA is considered as an accepted target for ruthenium complexes. Ru(II) complexes can enter into the cell nucleus and play a key role in biological activity [15]. Barton et al. reported that DPPZ-based Ru(II) complex ([Ru(DIP) 2 (dppz)] 2+ ) with lipophilic DIP ligand facilitates complex cellular uptake into the cell cytoplasm but has low inhibition against different tumor cells as it cannot enter into the cell nucleus [16]. Moreover, Chao et al. found that a type of Ru(II) complex ([Ru(bpy)(phpy)DPPZ] 2+ ) exhibits high antitumor activity against a series of tumor cell lines through accumulating in the nucleic region to bind DNA [17].
In this study, we designed a class of polypyridine Ru(II)-DPPZ complexes by introducing different substituent groups to regulate the electron cloud density of phenazine ligand. This process may improve molecular planarity and modify the terminal benzene ring to increase lipophilicity, thereby helping complexes enriched into the nucleus to enhance antitumor activity. We described the synthesis and the enhanced potency of this new series of polypyridine Ru(II)-DPPZ complexes to block the metastasis of breast cancer in vitro and in vivo. Further studies showed that this class of Ru(II) complexes may enter into breast cancer cells and localize at the nucleus, which ultimately may induce DNA damage to cause cell apoptosis [18,19]. Therefore, the Ru(II) complexes with low-toxicity can act as potential inhibitors against the metastasis of breast cancer for chemotherapy (Scheme 1).

In vitro Tumor Growth Inhibition of the Synthetic Ru(II) Complexes
To better assess the antitumor properties of Ru(II) complexes involving bidentate N-donor ligands with planar aromatic rings, we first evaluated the antiproliferative effects of these complexes on normal and some cancer cell lines (lung A549, breast MCF-7, liver HepG2, triple-negative breast MDA-MB-231, and HaCaT keratinocytes) and compared them with cisplatin. As shown in Table 1, the synthetic Ru (II) complexes emerged acceptable inhibition to various tumor cells after 72 h treatment, especially Ru(bpy) 2 BEDPPZ, which exhibited better antitumor activity than other complexes. The inhibitory effect (IC 50 ) of Ru(bpy) 2 BEDPPZ against triple-negative breast MDA-MB-231 cell lines was approximately 17.2 μM, which was better than that of cisplatin (20.9 μM). Its construction is divalent platinum binds to two chlorine atoms and two ammonia molecules and it can bind to DNA and cause cross connection, thus damaging the function of DNA and inhibiting cell mitosis. It is a cellular non-specific drug. However, the ruthenium compound based on a phenazine ligand containing a large aromatic planar structure can better bind to DNA than cisplatin.
The electric effect, planarity, and steric hindrance of substituted groups in ligands play key roles in antitumor activity of Ru(II) complexes [20]. Although for the complexes of the electron-donating group, with Ru(bpy) 2 BnDPPZ modified complexes (with IC 50 of 32.5 μM) were better than Ru(bpy) 2 MDPPZ (with IC 50 of 93.3 μM) and Ru (bpy) 2 BrDPPZ (with IC 50 of 94.1 μM) modified one against A549 cells, the best one was Ru(bpy) 2 BEDPPZ substituted complexes (with IC 50 of 20.0 μM). Unlike the IC 50 value for different sized planar ligands of complexes to MDA-MB-231 cells, the complexes with high quantity of terminal benzene rings in the ligands had enhanced growth suppression to tumor cells [21][22][23]. Comprehensive analysis of these results showed that the complexes modified by the electron-donating group can enhance antitumor activities, suggesting that the introduction of a phenylethynyl group in the phenazine ligand can availably improve the growth inhibition of tumor cells by exhibiting certain sensitivity to MDA-MB-231 cells. On the basis of its promising in vitro activity, Ru (bpy) 2 BEDPPZ was selected for preliminary evaluation in further studies.

Invasion and Migration of triple-negative breast cells inhibited by Ru (bpy) 2 BEDPPZ
Considering that metastasis is a severe risk in TNBC treatment, it is important to search for effective drugs that can inhibit it [24]. To investigate the anti-metastatic activity of the complexes, wound healing assay was performed to estimate cell migration and repair ability because the healing degree of scratches can reflect the inhibition effect of drugs on the migration ability of tumor cells. Fig. 1A shows an distinct reduction in the distance of wound closure without drug treatment for 72 h, but wound closure was suppressed with the addition of Ru(bpy) 2 BEDPPZ. When 2 μM of Ru(bpy) 2 BEDPPZ was used for treatment, the wound-healing rate was less than half of the control group. When the dose of Ru(bpy) 2 BEDPPZ was increased to 4 μM, the woundhealing rate further decreased, which suggested that Ru(bpy) 2 BEDPPZ inhibited migration in a dose-dependent manner.
Moreover, the invasion of MDA-MB-231 cells blocked by Ru (bpy) 2 BEDPPZ was studied using FITC-gelatin assay. The quantity of black area (degraded area) in the FITC-gelatin showed the invasion capability of tumor cells, because highly invasive MDA-MB-231cells can release matrix metalloproteinases to degrade the FITC-gelatin; that is, high numbers of black area indicated high invasiveness of MDA-MB-231 cells [25]. Fig. 1C shows a high number of black holes in the FITC-gelatin without drug treatment, which suggested that MDA-MB-231 exerted high invasive capability. With 5 μM of Ru(bpy) 2 BEDPPZ treatment, the invasive capability of MDA-MB-231 cells was markedly suppressed, and the quantity of black holes in FITC-gelatin was decreased. These results suggested that the migration and invasion of MDA-MB-231 cells were effectively inhibited by this class of Ru(II) complexes [26].
The possible mechanism of anti-metastasis by Ru(bpy) 2 BEDPPZin vitro was studied (Fig. 2). Here, filamentous actin bundles and FA protein points were observed using paxillin [27], a focal adhesion-associated adaptor protein, suggesting cell adhesion ability on the surface [28]. Without treatment with Ru(bpy) 2 BEDPPZ, a good deal of focal adhesions around the edge of MDA-MB-231 cells were observed. Nevertheless, after Ru(bpy) 2 BEDPPZ treatment, it is found that a decreased quantity of paxillins in MDA-MB-231 cells with increasing concentration of Ru(bpy) 2 BEDPPZ, and the cytoskeletal structure became loose. The stress fibers were also inhibited, after Ru(bpy) 2 BEDPPZ treatment, as represented by F-actin with red fluorescence. The results indicated that Ru(bpy) 2 BEDPPZ might suppress focal adhesions and stress fibers to inhibit the migration and invasion of MDA-MB-231 cells [29].

Suppression of Breast Cancer Growth and Metastasis in vivo
To detect the anticancer activity of the complexes in vivo, we treated the zebrafish breast cancer model with Ru(bpy) 2 BEDPPZ. The model was constructed by using transgenic zebrafish (fli1:EGFP), and quantificational breast cancer cells were microinjected into the zebrafish near the subintestinal vessel (SIV) area, in which blood vessels were labeled with green fluorescence and MDA-MB-231 cells with blue fluorescence [30,31]. As shown in Fig. 3, without drug treatment, a spot of blue breast into the blood vessels of zebrafish tail, which suggested that MDA-MB-231 cells possessed the ability for infinite proliferation and blood vessel invasion [32]. However, with Ru(bpy) 2 BEDPPZ (5 μM) treatment  for 72 h, the number of MDA-MB-231 cells was notably reduced compared with the control group, which implied that Ru(bpy) 2 BEDPPZ can effectively suppress the proliferation of TNBC cells in zebrafish. Moreover, scarce MDA-MB-231 cells were found in the blood vessel of zebrafish, indicating that Ru(bpy) 2 BEDPPZ can suppressed the metastasis of MDA-MB-231 cells in zebrafish xenografts. Moreover, the total area of SIV in the Ru(bpy) 2 BEDPPZ treatment group zebrafish was obviously less than that in the control group, which indicated that Ru(bpy) 2 BEDPPZ exhibited a certain inhibitory effect on tumor angiogenesis. The above results suggested that Ru(bpy) 2 BEDPPZ effectively suppressed the metastasis and proliferation of MDA-MB-231 cells in vivo and can be developed to become a potential candidate to block TNBC metastasis.

Angiogenesis Inhibited by Ru(bpy) 2 BEDPPZ in vitro and in vivo
In general, angiogenesis formation act a pivotal part in tumor proliferation and metastasis, some researchers have been studied that the application of Nanoparticles to the inhibition or synergistic treatment tumor through inhibite endothelial tubes formation [33,34], such as nanodiamond, mesoporous silica nanoparticles, etc. [35,36]. Besides, the vascular endothelial growth factor (VEGF) is one of the most critical factor inducing tumor vessel formation [37,38]. As tumor growth requires angiogenesis to provide adequate oxygen and nutrients, inhibiting angiogenesis formation is considered as an important way to overcome breast cancer. Angiogenesis inhibitors are widely used because of their safety and ability to block new blood vessel formation in tumors [39].
Given that tube formation indicates angiogenesis progression, we also demonstrated whether Ru(bpy) 2 BEDPPZ affects HUVECs' angiogenic effect in vitro by using tube formation assay. As shown in Fig. 4A, without drug interference, HUVECs incubated with VEGF (200 ng/mL) established tubular structures on Matrigel. Then, we treated HUVECs with Ru(bpy) 2 BEDPPZ in the presence of VEGF and found that the tube formation of these cells was inhibited notably compared with control cells. With increasing Ru(bpy) 2 BEDPPZ treatment dose, the matrix-induced tube formation of HUVECs was suppressed, indicating its dose-dependent effects [40,41]. Moreover, comprehensive analysis of the tube number, mean node number, and total length of tube suggested that Ru(bpy) 2 BEDPPZ is responsible for the inhibitory effect of VEGF on the matrix-induced tube formation of HUVECs. These observations indicated that Ru(bpy) 2 BEDPPZ can suppress the angiogenesis in vitro.
Furthermore, the inhibitory effect of Ru(bpy) 2 BEDPPZ against angiogenesis formation was evaluated in zebrafish model. It is found that Ru(bpy) 2 BEDPPZ equally induces distinct defects in ISV formation.

Ru(bpy) 2 BEDPPZ Cellular Localization
To investigate the potential antitumor mechanism of the Ru(II) complex, cellular localization of Ru(bpy) 2 BEDPPZ in MDA-MB-231 cells at different times was investigated, and the results are shown in Fig. 5. The intracellular localization of Ru(bpy) 2 BEDPPZ with red phosphorescence was slightly distributed in the cell nucleus and primarily enriched in the cell cytoplasm, after treatment for 6 h, which showed a mismatch with DAPI. However, with the increase of time, Ru(bpy) 2 BEDPPZ entered into the cell nucleus. At 24 h, a small quantity of Ru(bpy) 2 BEDPPZ was discovered in the cell cytoplasm surrounding the cell nucleus (blue phosphorescence) of the MDA-MB-231 cells. At 72 h, however, Ru(bpy) 2 BEDPPZ was only gathered in the cell nucleus, but perfectly matched with DAPI. The common target for Ru(II) complex to inhibit tumor cells is DNA. Thus, the localization of Ru(bpy) 2 BEDPPZ in the cell nucleus may play a key role in inhibiting breast cancer metastasis.

Growth Inhibition of Ru(bpy) 2 BEDPPZ by DNA Damage-mediated Apoptosis Induction
As Ru(bpy) 2 BEDPPZ emitted strong red fluorescence, which would interfere the PI signal, we used TUNEL assay to detect the apoptosis induction properties of 5 in MDA-MB-231 cells. Fig. 6 shows that the number of apoptotic cells was obviously decreased by upon the addition of Ru(bpy) 2 BEDPPZ. Without drug treatment, a small number of apoptotic cells were observed. However, with 20 μM of Ru(bpy) 2 BEDPPZ treatment, many apoptotic cells were observed. The apoptotic cells with fragmented DNA can be detected by TUNEL. We posited that Ru (bpy) 2 BEDPPZ may induce apoptosis of MDA-MB-231 by inducing DNA damage .  In order to further confirm the DNA damage induced by Ru(bpy) 2 BEDPPZ, we performed comet assay. Moreover, we also detected the induction of DSBs in MDA-MB-231 cells stained with DSB biomarker γH2AX by neutral comet assays and confocal immunofluorescence assays. As indicated by the statistical increase in olive tail moment, Ru (bpy) 2 BEDPPZ extremely increased the generation of endogenous DSBs in MDA-MB-231 cells (Fig. 7A). The percentages of γH2AX focal positive cells were obviously increased in MDA-MB-231 cells treated with different concentrations of Ru(bpy) 2 BEDPPZ (0, 5 and 10 μM). These results indicated that Ru(bpy) 2 BEDPPZ mainly induced apoptosis of MDA-MB-231 cells through DNA damage, which led to endogenous DSBs [43].

Conclusions
In this work, we designed and synthesized a class of polypyridine Ru-DPPZ derivatives by microwave-assisted synthesis technology [44,45]. This class of Ru(II) complexes exerted certain inhibition effects against TNBC cells, especially Ru(bpy) 2 BEDPPZ, which displayed commendable inhibitory activity against the proliferation, migration, and invasion of triplenegative breast cancer MDA-MB-231 cells. The structure-activity relationship analysis of these results suggested that the increased number of aromatic planar rings in ligands can effectively enhance antitumor activity and provided a guiding significance for further optimization of Ru(II) complexes [46,47]. The results of our mechanistic study showed that Ru(bpy) 2 BEDPPZ was enriched in the cell nucleus to induce DNA damage, leading to tumor cell apoptosis. Further evaluation of in vivo activity indicated that low concentrations of Ru(bpy) 2 BEDPPZ effectively inhibited the proliferation and metastasis of MDA-MB-231 cells in zebrafish xenografts. In summary, our study showed that the introduction of a phenylethynyl group in Ru(II) complexes can effectively enhance antitumor activity, which provided a guiding significance for optimizing antitumor drugs.

Synthesis of [Ru(bpy) 2 DPPZ](ClO 4 ) 2 (Ru(bpy) 2 DPPZ)
According to the literature, Ru(bpy) 2 DPPZ was synthesized by using similar metheod but with a few modifications. A mixture of cis-[Ru(bpy) 2 Cl 2 ]•2H 2 O (103.9 mg, 0.2 mmol) and DPPZ (56.4 mg, 0.2 mmol) were heated at reflux for 6 h under N 2 atmosphere in 30 mL of 9:1 glycol/ water mixture. The solution was cooled to room temperature before the reaction mixture was diluted with 80 mL of water. Then the mixture was filter, and excess sodium perchlorate was added to give a orange solid. Using cooled water and ether to filter and wash the crude products, and purified by flash silica gel column chromatography to afford a red powder, yields 67%. ESI-MS (in CH 3

Ru(bpy) 2 BEDPPZ Wound-Healing Assay
MDA-MB-231 cells (1 × 10 5 cells/well) were seeded in a 6-well plates with two marked lines on the back. When the cells covered N85% of the culture plate, using a tip (200 μL pipet) to scratch a line on the plate and orthogonal to the mark. Then, treatment with Ru(bpy) 2 BEDPPZ in different concentrations (0, 2 and 4 μM) in DMEM without FBS and observe migrating cells in the same visual field every 24 h. Based on 10 fields of view for each cell type, the average migration rate and the cell trajectory were computed through Slidebook and Excel software.

Evaluation of the Angiogenic Effect In vivo
24 hpf transgenic Tg(fli1a:EGFP) zebrafish embryos were dechorionated ata petri dishusing protease, then randomly distributed into 6-well microplate, treated with Ru(bpy) 2 BEDPPZ (0, 1.25, 2.5 and 5 μM). After 48 h treatment at 28.5°C, using fresh water to wash zebrafish larvae for two times and then transferredinto the 96-well plate, the subintestinal vessel plexus (SIVs) of zebrafish larvae were observed andimaged under the fluorescence microscope. The number of SIVs of each zebrafish was measured via manual counting, average number of SIVs of zebrafish larvae was calculated in each group [50].

Single Cell Gel Electrophoresis
MDA-MB-231 cells (5 × 10 4 cells/mL) were incubated with Ru(bpy) 2 BEDPPZ (0, 5 and 10 μM) for 72 h. According to 1:7 percentage mix with cell suspension and melted LM agarose. Pipetting the mixture (80 μL) to the slide (Comet Slide TM). Using prechilled lysis solution (4°C 60 min) immerse the slide before refrigeration for 10 min, and it was wash PBS twice times. Next, using freshly prepared alkaline solution (6 g NaOH, 0.25 mM EDTA (2.5 mL), pH = 13) immerse for 20 min, and it was suffered alkaline solution for electrophoresis (25 V 30 min),when the DNA unwinding. After, using PBS wash the slide twice, DNA was stained with EB after observed under a fluorescence microscope.

Growth and Metastasis Inhibition of Ru(bpy) 2 BEDPPZ in vivo
MDA-MB-231 cells were labeled fluorescence by DiI and gathered at a density of 10 7 cells/mL. Then, cells (50-500 cells) were injected into the perivitelline space near the subintestinal vessels (SIVs) of the transgenic zebrafish (fil1:EGFP) embryos at 48 hpf to establish this zebrafish embryo breast cancer model. Juvenile zebrafish of breast cancer model (48 h old) were incubated in 6well plates (10 fishes in every well) with 2 mL solutions without or with Ru(bpy) 2 BEDPPZ (0 and 5 μM) in aquaculture water. The inhibitory effect of Ru(bpy) 2 BEDPPZ in breast cancer zebra fish were captured pictures every 24 h with a fluorescence microscopy [32,51].