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Cite this: DOI: 10.1039/c4mt00158c
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Arene ruthenium(II) complex, a potent inhibitor against proliferation, migration and invasion of breast cancer cells, reduces stress fibers, focal adhesions and invadopodia† Qiong Wu,a Jiangtu He,b Wenjie Mei,*a Zhao Zhang,a Xiaohui Wuc and Fenyong Sun*b Effective chemotherapy drugs for cancer that would inhibit tumor growth and suppress metastasis are currently lacking. In this study, a series of arene ruthenium complexes, [(Z6-arene)Ru(H2iip)Cl]Cl (arene = p-cymene, RAWQ03; CH3C6H5, RAWQ04; and C6H6, RAWQ11), were synthesized and their inhibitory activity against tumor cells were evaluated. The results showed that the complex RAWQ11 inhibited the growth of MDA-MB-231 breast cancer cells by inducing S-phase arrest, which is closely related to the inhibition of cell mitosis-mediated cell nucleus damage. Further studies showed that RAWQ11 can inhibit the invasion and metastasis of MDA-MB-231 cells. The morphology of MDA-MB-231 cells changed, the number of focal adhesions decreased, and the stress fibers de-polymerized upon dealing with the complex RAWQ11. The FITC-gelatin assay confirmed that the formation of invadopodia in MDA-MB-231 cells was
Received 8th June 2014, Accepted 1st August 2014 DOI: 10.1039/c4mt00158c
significantly blocked by RAWQ11. Furthermore, RAWQ11 can block the AKT signal pathway by upregulating the PTEN expression through binding and downregulating miR-21. These results demonstrated that this type of arene ruthenium(II) complex can block the invadopodia formation by regulating the PTEN/AKT signal pathway mediated by miR-21 to inhibit the invasion and metastasis of breast cancer cells. Therefore, this
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complex can be used as a potential dual functional agent to inhibit the growth and metastasis of tumor cells.
Introduction Breast cancer is one of the leading causes of death in women because of its high metastasis and invasion of the lymph nodes, lungs, and bones, even the brain, during the terminal phase of the cancer.1–3 A series of treatments have been utilized to relieve cancer-related symptoms, delay cancer progression, and prolong and improve the quality of life; however, metastatic breast cancer (MBC) still remains an incurable condition.4,5 Recently, Ru complexes, especially arene Ru(II) complexes, have shown promising potential to cure breast cancer. Accumulating evidence has shown that arene Ru complexes exhibit high
a
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006, P. R. China. E-mail: [email protected] b Department of Clinical Laboratory Medicine, Shanghai Tenth People’s Hospital of Tongji University, Shanghai, 200072, P. R. China. E-mail: [email protected] c College of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou, 510006, P. R. China † Electronic supplementary information (ESI) available: Experimental details of the arene ruthenium(II) complex, a potent inhibitor against proliferation, migration and invasion of breast cancer cells that reduces stress fibers, focal adhesion and invadopodia. CCDC 916819. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4mt00158c
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anti-invasion and anti-metastasis activities in vitro and in vivo, with low toxicity.6–8 For example, NAMI-A (imidazolium trans[tetrachloro(dimethylsulfoxide)(1H-imidazole)ruthenate(III)]) can selectively reduce tumor metastasis and inhibit tumor cell invasion in vitro.9 In addition, KP1019 (trans-[tetrachlorobis(1H-indazole)ruthenate(III)]) can inhibit migration and invasion of MDA-MB-231 breast cancer cells by reducing the release of the extracellular matrix (MMP-2/9).10 Arene Ru complexes reported by Dyson, such as RAPTA-B ([Ru(Z6-C6H6)(pta)Cl2]) and RAPTA-C ([Ru(Z6p-C6H4MeiPr)(pta)Cl2]), can prevent the tumor growth and metastasis in CBA mice bearing the MCa mammary carcinoma by blocking angiogenesis.6 In addition, the arene Ru complex RM175 ([(Z6-biphenyl)Ru(ethylenediamine)Cl]+) also exhibits tumor metastasis inhibition in vivo and reduces the invasion and metastasis by promoting cell–cell re-adhesion and decreasing the release of metalloproteinases (MMPs).7 Recently, invadopodia formation, a key step in epithelial mesenchymal transition (EMT) of tumor cells, has been found to play an important role in the invasion and metastasis of tumors.11 EMT endows tumor cells with a more motile and invasive phenotype largely ascribed to the expression of EMT markers associated with poor prognosis; invadopodia is a direct performer in the extracellular matrix (ECM) degradation in the
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EMT process.12 Maturation of invadopodia allows tumor cells to possess the ability to invade the surrounding tissues and spread to distant organs. Moreover, targeting invadopodia is a possible approach to block breast cancer metastasis.13 Thus, invasion of breast cancer cells in vivo can be markedly inhibited by regulating the key gene or protein, such as Twist1, PDGFR, and Src, but without an effective agent to block the formation of invadopodia.14,15 Our previous study has demonstrated that arene Ru complexes coordinated with phenanthroimidazole derivatives can inhibit the growth of tumor cells by inducing S-phase arrest.16 However, whether or not arene Ru(II) complexes can inhibit the metastasis and invasion of tumor cells by blocking the invadopodia formation still remains unclear. Therefore, understanding the mechanism of these complexes would be helpful in developing novel therapeutic agents and obtaining effective drugs to inhibit metastatic breast cancer (MBC). Moreover, miR-21, a small non-coding RNA molecule that helps tumor cells lose their resistance to undergo EMT, negatively regulates gene expression at the post-transcriptional level and is overexpressed in MBC.17,18 A number of evidence from experimental and clinical studies have suggested that deregulated expression of miR-21 is associated with the early development of the EMT genetic program and promotes the formation of invadopodia.19 Our previous study also demonstrated that arene Ru complexes coordinated with phenanthroimidazole derivatives can downregulate the transcription of miR-21 in tumor cells.20 Thus, developing drugs that can inhibit the metastasis and invasion of tumor cells by blocking the formation of invadopodia through the regulation of the miR-21 transcription is essential. In this study, a series of arene ruthenium complexes, [(Z6-arene)Ru(H2iip)Cl]Cl (arene = p-cymene, RAWQ03; CH3C6H5, RAWQ04; and C6H6, RAWQ11), have been synthesized and demonstrated to inhibit the invasion and metastasis of MDAMB-231 breast cancer cells; the underlying mechanism of RAWQ11 has also been investigated. To the best of our knowledge, this study is the first to demonstrate that arene ruthenium complexes prevent the invasion and metastasis of breast cancer cells by blocking invadopodia formation. Further studies have demonstrated that RAWQ11 can bind and downregulate miR-21 expression to regulate the PTEN/AKT signal pathway and block invadopodia formation.
Experimental Microwave-assisted synthesis of arene Ru(II) complexes The binuclear arene Ru(II) complexes, namely, [(Z6-p-cymene)RuCl2]2, [(Z6-CH3C6H5)RuCl2]2, and [(Z6-C6H6)RuCl2]2, were prepared according to the literature.21 H2iip (2-indole-3-imidazole[4,5-f ][1,10] phenanthroline) was synthesized according to the literature.22 Arene ruthenium(II) complexes were prepared according to the literature.23 The precursor [(Z6-p-cymene)RuCl2]2, [(Z6-CH3C6H5)RuCl2]2, or [(Z6-C6H6)RuCl2]2 (0.1 mmol), and the ligand H2iip (0.22 mmol, 67.1 mg) in dichloromethane (20 mL) were stirred for 30 min in a Pyrex vessel at 90 1C in a microwave reactor. The solvent was removed by rotary evaporation,
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the solids were dissolved in distilled water, and the yellow powder was obtained after recrystallization in distilled water. RAWQ03: ESI-MS (in EtOH): m/z 606.1, ([M Cl]+). 1H NMR (500 MHz, DMSO) d 11.86 (s, 1H), 9.84 (d, J = 4.9 Hz, 2H), 8.67 (m, 2H), 8.19 (dd, J = 8.1, 5.2 Hz, 2H), 7.54 (dt, J = 17.6, 8.8 Hz, 1H), 7.26 (m, 2H), 6.34 (d, J = 6.4 Hz, 2H), 6.12 (d, J = 6.4 Hz, 2H), 2.51 (dt, J = 3.6, 1.7 Hz, 6H), 2.21 (s, 3H). 13C NMR (126 MHz, DMSO) d 153.23 (s), 136.60 (s), 132.52 (s), 130.08– 129.05 (m), 127.28 (s), 125.93 (s), 125.19 (s), 122.48 (s), 121.56 (s), 120.55 (s), 115.37 (s), 112.14 (s), 106.16 (s), 103.75 (s), 102.96 (s), 86.26 (s), 83.96 (s). RAWQ04: ESI-MS (in EtOH): m/z 564.1, ([M Cl]+). 1H NMR (500 MHz, DMSO) d 11.81 (s, 1H), 9.83 (d, J = 4.5 Hz, 2H), 9.39 (d, J = 6.4 Hz, 2H), 8.71 (m, 1H), 8.62 (d, J = 2.0 Hz, 1H), 8.15 (dd, J = 8.2, 5.2 Hz, 2H), 7.53 (m, 1H), 7.24 (m, 2H), 6.44 (t, J = 6.0 Hz, 2H), 6.09 (d, J = 6.2 Hz, 2H), 5.88 (m, 1H). 13C NMR (126 MHz, DMSO) d 153.56 (s), 143.12 (s), 137.00 (s), 132.78 (s), 127.49 (s), 126.13 (s), 125.62 (s), 122.84 (s), 122.04 (s), 120.89 (s), 112.51 (s), 105.85 (s), 90.47 (s), 83.40 (s), 80.27 (s). RAWQ11: ESI-MS (in MeOH): m/z 550.1, ([M Cl]+). 1 H NMR (DMSO-d6, ppm) d 14.61 (s, 1H), 11.84 (s, 1H), 9.94 (dd, J = 25.2, 5.1 Hz, 2H), 9.43 (s, 1H), 9.29 (d, J = 8.1 Hz, 1H), 8.70 (dd, J = 8.1, 6.0 Hz, 1H), 8.51 (d, J = 15.1 Hz, 1H), 8.18 (dd, J = 25.2, 6.1 Hz, 2H), 7.55 (dd, J = 15.2, 6.0 Hz, 1H), 7.28 (dd, J = 6.1, 5.1 Hz, 2H), 6.33 (s, 6H). 13C NMR (DMSO-d6, ppm) 152.52 (s), 150.52 (s), 137.01 (s), 132.92 (s), 128.80 (s), 127.61 (s), 126.23 (s), 125.54 (s), 122.94 (s), 121.95 (s), 121.00 (s), 112.58 (s), 106.53 (s), 88.11 (s), 87.20 (s). Crystal structure The crystal suitable for X-ray diffraction was obtained by slow recrystallization in a distilled water solution. The solution was kept in the dark at room temperature to obtain a yellow brown crystal. The X-ray intensity data for RAWQ11 was collected on an X-ray diffractometer equipped with a graphitemonochromated MoKa radiation (l = 0.71073 Å), using an o scan mode (0.991 o y o 27.121). MTT assay The tested compounds were dissolved in DMSO with 1 mM stock solution. Cells were incubated with different concentrations of the complexes for 24 h. Cell viability was determined by measuring the ability of cells to transform MTT into a purple formazan dye, which was carried out as described previously.9 Cells were seeded in 96-well tissue culture plates for 24 h. After incubation, 20 mL per well of MTT solution (5 mg mL1 phosphate buffered saline) was added and incubated for 5 h. The color intensity of the formazan solution, which reflects the cell growth conditions, was measured at 570 nm using a microplate spectrophotometer (SpectroAmaxt 250). 3D cell culture Growth factor reduced Matrigel (BD Biosciences) was prepared as per the manufacturer’s protocol. Cells were dissociated by 0.05% trypsin–EDTA (invitrogen) and re-suspended at a cell density of 2 106 cells per mL. 15 mL of Hoechst 33258 suspension a cell intravital stain was mixed with an equal volume of Matrigel on ice
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and quickly plated onto a Matrigel-coated glass bottom dish. After 30 minutes of incubation at 37 1C, DMEM media with feeder MDA-MB-231 cells were added to the 3D gel-containing dish.17 The cells were dyed by using Hoechst 33258 in DMEM and dealt with different concentrations of RAWQ11 (0, 1, 5 and 10 mM) in 96-well plates used especially for laser confocal microscopy on ice.
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Flow cytometry analysis Cell cycle distribution was analyzed by flow cytometry as previously reported.24 Treated or untreated cells were trypsinized, washed with PBS, and fixed with 75% ethanol overnight at 20 1C. Fixed cells were washed with PBS and stained with propidium iodide (PI) (1.21 mg mL1 Tris, 700 U mL1 RNase, 50.1 mg mL1 PI, pH = 8.0) for 30 min in the dark. Cell cycle distribution was analyzed using the MultiCycle software (Phoenix Flow Systems, San Diego, CA). Apoptotic cells with hypodiploid DNA content were measured by quantifying the sub-G1 peak in the cell cycle pattern. Western blot analysis Total cellular proteins were extracted by incubating the cells in lysis buffer obtained from Cell Signaling Technology; protein concentrations were determined by BCA assay. Anti-body p21, p-p21 (Thr 145), AKT, p-AKT (Thr308), GSK3b, MMP-9, FAK and PTEN were purchased from Abcam, Cell Signaling Technology and Proteintech. SDS-PAGE was carried out in 10% tricine gels, loading equal amounts of protein per lane, which was carried out as described previously.9 After electrophoresis, separated proteins were transferred to nitrocellulose membranes and blocked with 5% non-fat milk in TBST buffer for 1 h. After that, the membranes were incubated with primary antibodies at 1 : 1000 dilutions in 5% non-fat milk overnight at 4 1C, and then secondary antibodies were conjugated with horseradish peroxidase at 1 : 2000 dilution for 1 h at room temperature. Protein bands were visualized on X-ray films using an enhanced chemiluminescence system (Kodak). Wound healing assay Cells were seeded in a 6-well tissue culture plate marked on the back (1 105 cells per well) until the monolayer cells spread to more than 80% of the bottom of the culture plate. A line was scratched orthogonally using a tip (200 mL) to mark the plate. The cells were then incubated with the tested compounds at different concentrations (0, 1, 2, 5, and 10 mM) for 48 h. Migrating cells were imaged in the same visual field every 12 h for two days, which was carried out as described previously.7 Transwell invasion assay Up to 50 mL of Matrigel was added into the top chamber of the 24-well transwell on ice, and the plate was then incubated at 37 1C and 5% CO2 for 30 min. Afterward, the cells were placed in the top chamber with serum-free media having different concentrations of RAWQ11 (0, 1, 2, 5 10, and 20 mM) while the bottom chamber was added with DMEM containing 10% FBS and incubated for 24 h. Cell invasion was imaged using a fluorescence microscope.8
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Fluorescence real-time observation Transfection reagent LipoFiltert was provided by Shanghai Hanbio. To visualize the actin cytoskeleton reagent pEGFP-actin was purchased from Clontech. Cells transfected with EGFP-actin showing green fluorescence in plates were trypsinized and resuspended in 0.5 mL of DMEM media containing 200 mL of Hoechst 33258 and different concentrations of RAWQ11 (50 mM). Cell morphology was observed real-time using a laser confocal microscope for 2 h. Immunofluorescence MDA-MB-231 cells were treated with different concentrations of RAWQ11 (10 mM). MDA-MB-231 cells in complete growth medium at 5 104 cells per mL were incubated for 24 h at 37 1C, unless otherwise stated. Cells were washed once in PBS, fixed, and permeabilized simultaneously using 4% paraformaldehyde with 1% Triton X-100 in PBS, quenched with 0.1 M glycine in PBS, and blocked overnight at 4 1C with 3% (wt/vol) BSA. Fixed cells were stained with primary antibodies as indicated.4 Cell morphology was observed using a laser confocal microscope. FITC-gelatin invasion assay FITC-gelatin invasion assay was performed according to the procedures of the manufacturer (SAGENE). In brief, coverslips (18 mm diameter) were coated with 50 mg mL1 poly-L-lysine for 20 min at room temperature, washed with PBS, fixed with 0.5% glutaraldehyde for 15 min and washed with PBS 3 times. After washing, the coverslips were inverted on a drop of 0.2% FITC conjugated gelatin in PBS containing 2% sucrose, incubated for 10 min at room temperature, washed with PBS 3 times, quenched with sodium borohydride (5 mg mL1) for 3 min and finally incubated in 2 mL of complete medium for 2 h. Cells (2 105 each well) with different concentrations of RAWQ11 (0 and 1 mM) were plated in FITC gelatin-coated coverslips, incubated at 37 1C for 12 h. The ECM degradation status was evaluated and photographed using a laser confocal microscope. Cells (2 105 each well) with different concentrations of RAWQ11 and NAMI-A (0, 1, 2, and 5 mM) were plated in FITC gelatincoated coverslips and incubated at 37 1C for 12 h.15 The ECM degradation status was evaluated and photographed using a laser confocal microscope. RT-qPCR analysis The primers were purchased from BGI, Trizol was purchased from Invitrogen and an SYBR green PCR kit for quantitative PCR analysis was purchased from Takara. Total RNA was isolated using Trizol according to the recommendations of the manufacturer. Up to 2 mg of the total RNA from each samples were reverse transcribed using oligo (dT) primers at 37 1C for 90 min. The relative mRNA levels were evaluated by quantitative PCR using a SYBR green PCR kit. The signals were normalized to 18 S as internal control. The quantity of miR-21 in each BC, relative to the average expression in 40 NATs, was calculated using the following equation: RQ = 2DDCT, where DDCT = (CT miRNA CT U6 RNA)S (CT miRNA CT U6 RNA)MeanC. The primer sequences are listed in the ESI.†
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[(Z6-CH3C6H5)RuCl2]2, and [(Z6-C6H6)RuCl2]2) and the ligand (H2iip) under microwave irradiation (Fig. 1A). Microwave-assisted heating technology can produce the objective compounds with higher yield and faster than the conventional heating method.
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X-ray structure of the complex RAWQ11 The crystal structure of the complex RAWQ11 was obtained from mixed solutions of DMF and CH2Cl2. The molecular structure of the lattice of RAWQ11 is illustrated in Fig. 1B. The selected crystallographic data, bond distances, and bond angles are listed in Tables S1 and S2 (ESI†). The crystal structure of RAWQ11 has a P4% 21c space group and a typical "three-leg piano-stool" stereochemical structure that is well-documented for the arene ruthenium complex, as shown in Fig. 1B.23 Cytotoxicity of arene Ru(II) complexes Fig. 1 (A) Molecular structure of the arene Ru complexes (R1, RAWQ03; R2, RAWQ04; and R3, RAWQ11). (B) Crystal structure of the arene Ru complex RAWQ11.
miR21-binding properties The interaction between the complexes and miR-21 was examined by UV-vis spectroscopy, CD spectroscopy, and ITC and FRET assays. The UV absorption spectra of the complex RAWQ11 were recorded at 270 nm and 293 nm; titration was terminated when the intensity of the two bands did not change significantly upon the addition of miR-21. The CD spectrum of miR-21 RNA in the presence or absence of RAWQ11 was also recorded. The binding of various small molecules was assessed by isothermal titration calorimetry on a thermostated Micro Cal VP-ITC system. The stability of miR-21 was evaluated by FRET assay.23 The fluorescent labeled oligonucleotide, miR-21 RNA (5 0 -FAM-TGGGGAGGGTGGGGAGGGTGGGGAAGGTAMRA-3 0 , FAM: carboxyfluorescein, and TAMRA: 6-carboxytetramethyl-rhodamine) used as the FRET probes were diluted in Tris-HCl buffer and then annealed by being heated to 92 1C for 5 min, followed by slowly cooling to room temperature. A constant temperature was maintained for 30 s prior to each reading to ensure a stable value. Final analysis of the data was carried out by using Origin7.5 (Origin Lab Corp.).
Results and discussion Microwave-assisted synthesis of arene ruthenium(II) complexes Arene ruthenium complexes RAWQ03, RAWQ04, and RAWQ11 were synthesized using the precursors ([(Z6-p-cymene)RuCl2]2,
The inhibitory activity of synthetic arene ruthenium(II) complexes against various breast cancer cells and normal human cells was evaluated by MTT assay. The synthetic complexes exhibited significant inhibition to MDA-MB-231 and MCF-7 breast cancer cells and low toxicity to human normal MCF-10A, HK-2, and HaCat cells, especially the complex RAWQ11, as shown in Table 1. Compared with complexes 1, 2, and RAWQ11, the antitumor activity against MDA-MB-231 and MCF-7 breast cancer cells decreased with the increase in sterically rigid linking groups in the arene ring.25 However, no significant regularity was found in normal human cells. The complex RAWQ11 can effectively and sensitively inhibit the growth of MDA-MB-231 highly metastatic human breast adenocarcinoma cells (Table 1). After being treated with RAWQ11 for 24 h, the number of MDA-MB-231 cells decreased and a dosage-dependent cell shrinkage was observed (Fig. S11, ESI†). The inhibitory activity (IC50) of RAWQ11 against MDA-MB-231 cells was about 20.8 mM, which is approximately 1.7 times better than that of cisplatin under the same conditions and much better than NAMI-A that exhibits a great anti-metastasis activity in vivo. The cytotoxicity of RAWQ11 against MCF-10A normal human breast cells was approximately IC50 4 300 mM. These data are far lower than those of cisplatin (6.32 mM) and give a wider safety range in chemotherapy.24 Growth inhibition or death of cells is the result of apoptosis, cell cycle arrest, or a combined action of both,19 so, flow cytometry was carried out. After the MDA-MB-231 cells were exposed to different concentrations of RAWQ11 for 24 h, a significant increase in cell proportion in the S-phase of MDA-MB-231 cells was observed (Fig. 2A), but no obvious apoptosis occurred (Fig. 2B).
Table 1 In vitro cytotoxicity [given as IC50 values (mM)] of complexes RAWQ03, RAWQ04, RAWQ11, NAMI-A and cisplatin against MDA-MB-231 and MCF-7 human breast cancer cell lines, MCF-10A human breast epithelial cells, HK-2 human normal kidney 2 cells, and immortalized human keratinocyte HaCat cells
IC50 (mM) Comp.
MDA-MB-231
MCF-7
MCF-10A
HK-2
HaCat
RAWQ03 RAWQ04 RAWQ11 Cisplatin NAMI-A
107.6 93.2 20.8 36.1 4300
4300 101.3 0.17 17.9 0.32 13.2 0.41 4300
4300 234.7 0.24 4300 6.32 0.14 4300
205.4 0.31 4300 110.3 0.61 13.7 0.58 4300
111.6 0.52 4300 139.2 0.26 7.5 0.43 4300
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0.37 0.53 0.40 0.28
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Fig. 2 (A) S-phase arrest of MDA-MB-231 cells induced by RAWQ11. MDA-MB-231 cells were treated with RAWQ11 (0, 1, 5, and 10 mM) for 24 h, almost 40% cycling cells are in the S phase and the sharp peak suggests that some cells are experiencing S-phase delay or arrest; (B) induction apoptosis of the complex RAWQ11 against MDA-MB-231 cells. MDA-MB231 cells were treated with RAWQ11 (0, 1, 5, and 10 mM) for 24 h; little apoptosis of MDA-MB-231 cells was seen with the increase of RAWQ11. (C) Regulation of the expression of p21 and p-p21 by RAWQ11 at the protein level. (D) Regulation of the expression of p21 by RAWQ11 at the RNA level. MDA-MB-231 cells were treated with RAWQ11 (0, 1, 2, 5, and 10 mM) for 24 h.
These results revealed that RAWQ11 can block MDA-MB-231 cell growth by inducing S-phase arrest rather than apoptosis.26 In addition, the up-regulation of p21 and p-p21, which are the key proteins in the S-phase of the cell cycle, at protein and RNA levels, upon the increase in RAWQ11, indicated that RAWQ11 inhibits the growth of breast cancer cells by inducing S-phase arrest through promoting the expression and phosphorylation of p21. Considering that cell migration is an important step in tumor metastasis, the inhibitory effect of RAWQ11 against migration of MDA-MB-231 cells was further evaluated by wound healing assay.27 Compared with the control cells that spontaneously migrated, MDA-MB-231 cell treatment with RAWQ11 showed a small decrease in the time of wound closure at 48 h, and a dosage-dependent effect (Fig. 3A). Less than confluent cultures showed the significant effect of RAWQ11 (1 and 2 mM) on wound closure, indicating that RAWQ11 effectively inhibits MDA-MB-231 migration.28 Invasive cells can release MMPs to degrade the ECM and spread to the surrounding tissues. Thus, transwell invasion assay was conducted to determine the inhibition of the invasion ability of MDA-MB-231 cells when treated with RAWQ11. One layer Matrigel was added into the top chamber of the 24-well transwell to evaluate the invasion of MDA-MB-231 cells in vitro. Invasive cells with invadopodia can degrade Matrigel to migrate to the other side of the membrane (Fig. 3B), which can be dyed by crystal violet. The more the cells dyed with
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Fig. 3 (A) Wound healing assay to evaluate the migration of MDA-MB-231 cells after being treated with RAWQ11 (0, 1, and 2 mM) and DMEM with 10% FBS. Cells were wounded and monitored using a microscope every 12 h. Migration was determined by the rate of cells filling the scratched area. (B) A diagram of transwell and invasion experiment. (C) Transwell assay to observe the invasion of MDA-MB-231 cells after being treated with RAWQ11 (0, 1, 2, 5, 10, and 20 mM) for 24 h. MDA-MB-231 cells were plated on Matrigel-coated membranes in the upper chamber of transwell with SFM (serum free medium); DMEM containing 10% FBS was placed in the bottom chamber of transwell. Cells penetrating the membrane were fixed and stained with 0.1% crystal violet after 24 h. (D) Regulation of the expression of FAK, GSK3b, and MMP9 by RAWQ11 at the protein level. (E) Regulation of the expression of FAK, GSK3b, MMP2, and MMP9 by RAWQ11 at the RNA level.
crystal violet, the stronger the invasion of the cells.29 Transwell invasion assays were carried out with MDA-MB-231 cells in the presence of different concentrations of RAWQ11 for 24 h. Compared with the control, invasion of MDA-MB-231 cells was blocked as the concentration of RAWQ11 increased (Fig. 3C). After being treated with 1 mM of RAWQ11, the number of MDAMB-231 cells that penetrated the Matrigel decreased significantly. When the concentration of RAWQ11 reached 5 and 10 mM, MDA-MB-231 cells exhibited a slight invasion ability because few cells penetrated the Matrigel. In addition, the expression of MMP9 was downregulated and the transcription of MMP2 and MMP9 was significantly decreased by RAWQ11.
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Moreover, the release of MMPs negatively regulated by GSK3b increased with RAWQ11. These studies highlight that invasion and migration of MDA-MB-231 cells could be significantly suppressed by RAWQ11.30 EMT is often characterized by the change in cell morphology, the reconstruction of stress fibers, and the increase of focal adhesion, which are in tight correlation with the formation of invadopodia.12,31 Transitions in cell morphology and reorganization of actin cytoskeleton are important to cell migration and invasion.32 Changes in cell morphology were observed using real-time fluorescence. As shown in Fig. 4A, cell treatment with RAWQ11 at 50 mM exhibited full morphology, from a triangle at the beginning (0 h), which gradually changed to a round shape over time, and then a completely loose morphology at 2 h. In addition, the actin cytoskeleton marked by green GFP gradually depolymerized and the cell nucleus stained by blue Hoechst 33528 shrunk, which then disappeared eventually. The complex RAWQ11 can inhibit cell mitosis and lead to the disappearance of the cell nucleus because of cell nucleus condensation. The nuclear envelope and nucleoli disappeared during the prophase of mitosis as seen in the video (Video S1, ESI†). Furthermore, a 3D cell culture model constructed using a cell development system to simulate the micro-environment of cell growth in vitro33 was developed to observe the influence of RAWQ11 on the nucleus of MDA-MB-231 cells. The nucleus of MDA-MB-231 cells, whose integrity was kept perfect without the treatment of RAWQ11, broke into tiny pieces with an increase in the amount of RAWQ11 (Fig. 4B). The complex RAWQ11 can reduce the polymerization of the actin cytoskeleton, inhibit the maintenance of cell morphology, and induce cell nucleus damage to block the migration of MDA-MB-231 cells.34
Fig. 4 (A) Real-time imaging using a laser scanning co-focus light microscope of MDA-MB-231 cells treated with RAWQ11 (50 mM) for 2 h. The cell cytoskeleton and nucleus were visualized by green (transfected with GFPactin) and blue (dyed with Hoechst 33258) fluorescence, respectively. See also Video S1 (ESI†). (B) Induction of cell nucleus damage of RAWQ11 by 3D cell culture. MDA-MB-231 cells were dyed with Hoechst 33258 at different concentrations of RAWQ11 (0, 1, 5, and 10 mM).
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To further evaluate the effect on the inhibition of focal adhesions and stress fibers in MDA-MB-231 cells by RAWQ11, immuno-fluorescence experiments were carried out using the laser scanning co-focus light microscope. Stress fibers, the leading actors in the cell migration process, are usually composed of bundles of approximately 10–30 actin filaments.35 In addition, focal adhesions, such as integrin and proteoglycan-mediated adhesion links to the actin cytoskeleton, regulate cell migration.36 The focal adhesions were observed by paxillin because characterization of the protein revealed that paxillin was localized at discrete structures of focal adhesions, which are sites of close cellular contact with the underlying extra-cellular matrix.37 Invadopodia are actin-rich protrusions located in the areas of focal adhesions in contact with the ECM.38–40 As shown in Fig. 5, a number of focal adhesions can be observed in MDA-MB-231 cells without RAWQ11 treatment. However, after being treated with RAWQ11, the number of paxillins in MDA-MB-231 cells decreased significantly and the cellular morphology changed. Moreover, the stress fibers treated by RAWQ11 was reduced as represented by the F-actin with red fluorescence. F-actin was down-regulated (Fig. 3A), indicating that RAWQ11 can inhibit the migration of MDAMB-231 cells through suppressed focal adhesions and stress fibers. Further studies indicated that FAK expression, a direct regulator to control the formation of focal adhesions, was blocked significantly (Fig. 3D and E).41 These results showed that RAWQ11 can inhibit the invasion and migration in MDAMB-231 cells by blocking the formation of focal adhesions and the depolymerization of F-actin, which are bound to the formation of invadopodia. FITC-gelatin (fluorescein isothiocyanate-conjugated gelatin) invasion assay was utilized to further clarify whether or not 3 can block the formation of invadopodia in MDA-MB-231 cells. FITC-gelatin, which is usually used to observe cells under green fluorescence, has been used to observe the number of invadopodia formed and test the invasion ability of tumor cells (Fig. 6).42 Cells with high invasion can form invadopodia to release MMPs that act to degrade FITC-gelatin. The greater the number of the dark (non-fluorescent) areas of degradation in the FITC-gelatin, the greater the invasion of tumor cells.43 Five black holes (non-fluorescent areas) can be counted in
Fig. 5 The distribution of focal adhesion proteins (green) studied by indirect immunofluorescence (FITC-coupled second antibody) using the first antibodies against paxillin. The distribution of stress fibers (red) was studied by indirect immunofluorescence (rhodamine-conjugated phalloidin) using the first antibodies against F-actin.
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Fig. 6 (A) View of a single MDA-MB-231 cell, the number of invadopodia treated without and with RAWQ11. (B) The whole view of many MDA-MB-231 cells, the number of invadopodia treated without and with RAWQ11. Cell cytoskeletons were dyed with rhodamine-conjugated phalloidin. The dark area in the FITC-gelatin was identified as the position degraded by invadopodia.
FITC-gelatin for a single cell without the treatment of RAWQ11 (Fig. 6A). After being treated with RAWQ11, a few black holes were observed, indicating that the complex RAWQ11 can inhibit the formation of invadopodia effectively. Moreover, a number of black holes can be observed in the whole view of the FITC-gelatin for MDA-MB-231 cells without RAWQ11 treatment (Fig. 6B), indicating that up to almost a third of the cells can invade the FITC-gelatin and showed strong invasion ability for MDAMB-231 cells. However, after being treated with RAWQ11 (1 mM), a few black holes were found in the FITC-gelatin, indicating that the invasion effect of MDA-MB-231 cells was inhibited markedly. No black hole was observed and the shape of the cells changed with the high concentration of RAWQ11 (2 and 5 mM), as shown in Fig. S13 (ESI†).44 Importantly, NAMI-A, a classic anti-metastasis agent, cannot effectively inhibit the invasion of MDA-MB-231 cells at the concentrations of 1, 2 and 5 mM, which invaded lots of black holes in the FITCgelatin, as shown in Fig. S14 (ESI†). These results proved that arene Ru complexes can significantly inhibit the formation of invadopodia better than NAMI-A. The AKT signal pathway plays an important role in regulating the signaling of multiple biological processes, such as cell proliferation,
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cell cycle, apoptosis, cell invasion, and metastasis.45 When the AKT signal pathway is inactivated, the expression and phosphorylation of p21 are up-regulated, leading to the S-phase arrest of tumor cells. Meanwhile, the upregulation of the expression of GSK3b results in the inhibition of the release of MMPs to suppress the invasion of tumor cells.46 The AKT signaling pathway was regulated by PTEN, a key phosphorylation inhibitor that suppresses the expression of FAK and paxillin, to inhibit cell migration.47 After being treated with RAWQ11, the expression of AKT and p-AKT was downregulated, whereas the expression of PTEN was upregulated at protein and RNA levels, as shown in Fig. 7A and B. These data indicate that RAWQ11 can regulate the AKT signaling pathway by upregulating PTEN to inhibit cell invasion.48 Regulation of miR-21 by RAWQ11 was evaluated by RT-qPCR assay because miR-21 can directly suppress PTEN expression to activate the invasion and metastasis of tumor cells.49 The RT-qPCR results showed that the expression of miR-21 was downregulated with the increase in RAWQ11 (Fig. 8A). miR-21 is processed from transcripts that can form local RNA hairpin structures, which has been considered as potential targets for antitumor drugs.2 Thus, the interaction between RAWQ11 and miR-21 was further investigated in vitro to show that this complex can bind and stabilize the conformation of miR-21. The results confirmed the occurrence of hypochromism characterized by IL and LMCT absorption bands ascribed to RAWQ11 in the electronic spectra with the increase in the amount of miR-21 (Fig. 8B). Moreover, the positive CD signal of miR-21 at 260 nm significantly decreased upon the addition of RAWQ11, and a reduced CD signal was also observed in the range of 290–330 nm (Fig. 8C). The titration plot strongly supported the proposition that RAWQ11 binds to miR-21 in a one-site mode and completely saturates at a 1 : 1 stoichiometry; the binding constant calculated for RAWQ11 [K] is about 1.3 105 M1 (Fig. 8D). Furthermore, the results of FRET showed that RAWQ11 binds to miR-21 to stabilize the structure of miR-21 with the increase in the melting point of miR-21 to about 20 1C in the presence of 6 mM of RAWQ11 (Fig. 8E). These results showed that RAWQ11 can bind to and stabilize the structure of miR-21 to downregulate the expression of miR-21. Invadopodia is a direct actor in the invasion of the surrounding tissues and organs in tumor cells with high invasion and metastasis. However, the paucity of the agent for effective blocking of the formation of invadopodia in vitro has greatly
Fig. 7 Regulation of the key protein and mRNA by RAWQ11. MDA-MB-231 cells were treated with RAWQ11 (0, 1, 2, 5, and 10 mM) for 24 h. (A) Regulation of the expression of PTEN, AKT, and p-AKT by RAWQ11 at the protein level. (B) Regulation of the expression of PTEN and AKT by RAWQ11 at the RNA level.
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Fig. 8 Regulation of miR-21 by RAWQ11 in cells and the interaction of miR-21 with RAWQ11 in vitro. (A) Regulation of miR-21 in MDA-MB-231 cells downregulated by RAWQ11 (0, 1, 2, 5, and 10 mM). (B) Absorption spectra of RAWQ11 recorded upon the addition of increasing concentrations of miR-21 ([miR-21] = 0.5n mM, n = 0, 1, 2, 3, 4, 5, 6, 7. [RAWQ11] = 20 mM). (C) CD spectra of miR-21 recorded upon the addition of RAWQ11. ([miR-21] = 2 mM, [RAWQ11] = 5n mM, n = 0, 1, 2, 3, 4, 5). (D) ITC experimental curves at 25 1C for titration of RAWQ11 with miR-21. The results were converted to molar heat and plotted against the compound/ RNA molar ratio. The line shows the fit to the results and provides the best-fit DH values for binding, [miR-21] = 2 mM. (E) FRET-melting curves obtained with miR-21 (0.2 mM) alone (’) upon the addition of RAWQ11. Increasing trend of miR-21 melting upon the addition of RAWQ11 (1, 3, and 6 mM).
impeded the discovery of novel agents to inhibit the invasion and metastasis of breast cancer.50 In this study, we found that the arene ruthenium complex RAWQ11 can inhibit the growth of MDA-MB-231 cells effectively and suppress the migration and invasion of cells in vitro. Further studies indicated that RAWQ11 inhibits the growth of MDA-MB-231 cells by inducing the S-phase arrest-mediated cell nucleus damage. Notably, cell mitosis can also be inhibited by RAWQ11 because cell nucleus damage can be ascribed to nuclear envelope and nucleoli disappearance during the prophase of mitosis. Moreover, focal adhesions, structural links on the cell membrane where the actin cytoskeleton inside the cell is connected to the ECM on the outside, will be relieved significantly during the formation of invadopodia. The complex RAWQ11 can reduce the number of focal adhesions and promote the depolymerization of stress fibers. Finally, FITC-gelatin assay was carried out to demonstrate that the formation of invadopodia of MDA-MB-231 cells is blocked by RAWQ11. According to the study of the signal pathway, we found that RAWQ11 can bind to miR-21 and downregulate the expression of miR-21, leading to the up-regulation of PTEN, as a result the AKT signal pathway is inactivated, which can
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Fig. 9 Diagram of the S-phase arrest, metastasis, and invasion of MDAMB-231 cells inhibited by RAWQ11 through blocking the formation of invadopodia by regulating the PTEN/AKT signaling pathway-mediated miR-21.
regulate the cell cycle, invasion, and metastasis (Fig. 9). In other words, we developed a potential agent with dual function in inhibiting the proliferation, invasion, and metastasis of breast cancer cells through the PTEN/AKT signal pathway mediated by miR-21.
Acknowledgements We thank Prof. Jiao Guo for her expert secretarial assistance and Prof. Haiyang Liu for his help in the crystal structure analysis. This work was supported by the Science and Technology Item Foundation of Guangzhou (2013J4100072), the Joint Natural Sciences Fund of the Department of Science and Technology and the First Affiliated Hospital of Guangdong Pharmaceutical University (GYFYLH201309), and Excellent Discipline Leader Training Plan of Shanghai Health System.
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Cite this: DOI: 10.1039/c4mt00158c
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Arene ruthenium(II) complex, a potent inhibitor against proliferation, migration and invasion of breast cancer cells, reduces stress fibers, focal adhesions and invadopodia† Qiong Wu,a Jiangtu He,b Wenjie Mei,*a Zhao Zhang,a Xiaohui Wuc and Fenyong Sun*b Effective chemotherapy drugs for cancer that would inhibit tumor growth and suppress metastasis are currently lacking. In this study, a series of arene ruthenium complexes, [(Z6-arene)Ru(H2iip)Cl]Cl (arene = p-cymene, RAWQ03; CH3C6H5, RAWQ04; and C6H6, RAWQ11), were synthesized and their inhibitory activity against tumor cells were evaluated. The results showed that the complex RAWQ11 inhibited the growth of MDA-MB-231 breast cancer cells by inducing S-phase arrest, which is closely related to the inhibition of cell mitosis-mediated cell nucleus damage. Further studies showed that RAWQ11 can inhibit the invasion and metastasis of MDA-MB-231 cells. The morphology of MDA-MB-231 cells changed, the number of focal adhesions decreased, and the stress fibers de-polymerized upon dealing with the complex RAWQ11. The FITC-gelatin assay confirmed that the formation of invadopodia in MDA-MB-231 cells was
Received 8th June 2014, Accepted 1st August 2014 DOI: 10.1039/c4mt00158c
significantly blocked by RAWQ11. Furthermore, RAWQ11 can block the AKT signal pathway by upregulating the PTEN expression through binding and downregulating miR-21. These results demonstrated that this type of arene ruthenium(II) complex can block the invadopodia formation by regulating the PTEN/AKT signal pathway mediated by miR-21 to inhibit the invasion and metastasis of breast cancer cells. Therefore, this
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complex can be used as a potential dual functional agent to inhibit the growth and metastasis of tumor cells.
Introduction Breast cancer is one of the leading causes of death in women because of its high metastasis and invasion of the lymph nodes, lungs, and bones, even the brain, during the terminal phase of the cancer.1–3 A series of treatments have been utilized to relieve cancer-related symptoms, delay cancer progression, and prolong and improve the quality of life; however, metastatic breast cancer (MBC) still remains an incurable condition.4,5 Recently, Ru complexes, especially arene Ru(II) complexes, have shown promising potential to cure breast cancer. Accumulating evidence has shown that arene Ru complexes exhibit high
a
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006, P. R. China. E-mail: [email protected] b Department of Clinical Laboratory Medicine, Shanghai Tenth People’s Hospital of Tongji University, Shanghai, 200072, P. R. China. E-mail: [email protected] c College of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou, 510006, P. R. China † Electronic supplementary information (ESI) available: Experimental details of the arene ruthenium(II) complex, a potent inhibitor against proliferation, migration and invasion of breast cancer cells that reduces stress fibers, focal adhesion and invadopodia. CCDC 916819. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4mt00158c
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anti-invasion and anti-metastasis activities in vitro and in vivo, with low toxicity.6–8 For example, NAMI-A (imidazolium trans[tetrachloro(dimethylsulfoxide)(1H-imidazole)ruthenate(III)]) can selectively reduce tumor metastasis and inhibit tumor cell invasion in vitro.9 In addition, KP1019 (trans-[tetrachlorobis(1H-indazole)ruthenate(III)]) can inhibit migration and invasion of MDA-MB-231 breast cancer cells by reducing the release of the extracellular matrix (MMP-2/9).10 Arene Ru complexes reported by Dyson, such as RAPTA-B ([Ru(Z6-C6H6)(pta)Cl2]) and RAPTA-C ([Ru(Z6p-C6H4MeiPr)(pta)Cl2]), can prevent the tumor growth and metastasis in CBA mice bearing the MCa mammary carcinoma by blocking angiogenesis.6 In addition, the arene Ru complex RM175 ([(Z6-biphenyl)Ru(ethylenediamine)Cl]+) also exhibits tumor metastasis inhibition in vivo and reduces the invasion and metastasis by promoting cell–cell re-adhesion and decreasing the release of metalloproteinases (MMPs).7 Recently, invadopodia formation, a key step in epithelial mesenchymal transition (EMT) of tumor cells, has been found to play an important role in the invasion and metastasis of tumors.11 EMT endows tumor cells with a more motile and invasive phenotype largely ascribed to the expression of EMT markers associated with poor prognosis; invadopodia is a direct performer in the extracellular matrix (ECM) degradation in the
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EMT process.12 Maturation of invadopodia allows tumor cells to possess the ability to invade the surrounding tissues and spread to distant organs. Moreover, targeting invadopodia is a possible approach to block breast cancer metastasis.13 Thus, invasion of breast cancer cells in vivo can be markedly inhibited by regulating the key gene or protein, such as Twist1, PDGFR, and Src, but without an effective agent to block the formation of invadopodia.14,15 Our previous study has demonstrated that arene Ru complexes coordinated with phenanthroimidazole derivatives can inhibit the growth of tumor cells by inducing S-phase arrest.16 However, whether or not arene Ru(II) complexes can inhibit the metastasis and invasion of tumor cells by blocking the invadopodia formation still remains unclear. Therefore, understanding the mechanism of these complexes would be helpful in developing novel therapeutic agents and obtaining effective drugs to inhibit metastatic breast cancer (MBC). Moreover, miR-21, a small non-coding RNA molecule that helps tumor cells lose their resistance to undergo EMT, negatively regulates gene expression at the post-transcriptional level and is overexpressed in MBC.17,18 A number of evidence from experimental and clinical studies have suggested that deregulated expression of miR-21 is associated with the early development of the EMT genetic program and promotes the formation of invadopodia.19 Our previous study also demonstrated that arene Ru complexes coordinated with phenanthroimidazole derivatives can downregulate the transcription of miR-21 in tumor cells.20 Thus, developing drugs that can inhibit the metastasis and invasion of tumor cells by blocking the formation of invadopodia through the regulation of the miR-21 transcription is essential. In this study, a series of arene ruthenium complexes, [(Z6-arene)Ru(H2iip)Cl]Cl (arene = p-cymene, RAWQ03; CH3C6H5, RAWQ04; and C6H6, RAWQ11), have been synthesized and demonstrated to inhibit the invasion and metastasis of MDAMB-231 breast cancer cells; the underlying mechanism of RAWQ11 has also been investigated. To the best of our knowledge, this study is the first to demonstrate that arene ruthenium complexes prevent the invasion and metastasis of breast cancer cells by blocking invadopodia formation. Further studies have demonstrated that RAWQ11 can bind and downregulate miR-21 expression to regulate the PTEN/AKT signal pathway and block invadopodia formation.
Experimental Microwave-assisted synthesis of arene Ru(II) complexes The binuclear arene Ru(II) complexes, namely, [(Z6-p-cymene)RuCl2]2, [(Z6-CH3C6H5)RuCl2]2, and [(Z6-C6H6)RuCl2]2, were prepared according to the literature.21 H2iip (2-indole-3-imidazole[4,5-f ][1,10] phenanthroline) was synthesized according to the literature.22 Arene ruthenium(II) complexes were prepared according to the literature.23 The precursor [(Z6-p-cymene)RuCl2]2, [(Z6-CH3C6H5)RuCl2]2, or [(Z6-C6H6)RuCl2]2 (0.1 mmol), and the ligand H2iip (0.22 mmol, 67.1 mg) in dichloromethane (20 mL) were stirred for 30 min in a Pyrex vessel at 90 1C in a microwave reactor. The solvent was removed by rotary evaporation,
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the solids were dissolved in distilled water, and the yellow powder was obtained after recrystallization in distilled water. RAWQ03: ESI-MS (in EtOH): m/z 606.1, ([M Cl]+). 1H NMR (500 MHz, DMSO) d 11.86 (s, 1H), 9.84 (d, J = 4.9 Hz, 2H), 8.67 (m, 2H), 8.19 (dd, J = 8.1, 5.2 Hz, 2H), 7.54 (dt, J = 17.6, 8.8 Hz, 1H), 7.26 (m, 2H), 6.34 (d, J = 6.4 Hz, 2H), 6.12 (d, J = 6.4 Hz, 2H), 2.51 (dt, J = 3.6, 1.7 Hz, 6H), 2.21 (s, 3H). 13C NMR (126 MHz, DMSO) d 153.23 (s), 136.60 (s), 132.52 (s), 130.08– 129.05 (m), 127.28 (s), 125.93 (s), 125.19 (s), 122.48 (s), 121.56 (s), 120.55 (s), 115.37 (s), 112.14 (s), 106.16 (s), 103.75 (s), 102.96 (s), 86.26 (s), 83.96 (s). RAWQ04: ESI-MS (in EtOH): m/z 564.1, ([M Cl]+). 1H NMR (500 MHz, DMSO) d 11.81 (s, 1H), 9.83 (d, J = 4.5 Hz, 2H), 9.39 (d, J = 6.4 Hz, 2H), 8.71 (m, 1H), 8.62 (d, J = 2.0 Hz, 1H), 8.15 (dd, J = 8.2, 5.2 Hz, 2H), 7.53 (m, 1H), 7.24 (m, 2H), 6.44 (t, J = 6.0 Hz, 2H), 6.09 (d, J = 6.2 Hz, 2H), 5.88 (m, 1H). 13C NMR (126 MHz, DMSO) d 153.56 (s), 143.12 (s), 137.00 (s), 132.78 (s), 127.49 (s), 126.13 (s), 125.62 (s), 122.84 (s), 122.04 (s), 120.89 (s), 112.51 (s), 105.85 (s), 90.47 (s), 83.40 (s), 80.27 (s). RAWQ11: ESI-MS (in MeOH): m/z 550.1, ([M Cl]+). 1 H NMR (DMSO-d6, ppm) d 14.61 (s, 1H), 11.84 (s, 1H), 9.94 (dd, J = 25.2, 5.1 Hz, 2H), 9.43 (s, 1H), 9.29 (d, J = 8.1 Hz, 1H), 8.70 (dd, J = 8.1, 6.0 Hz, 1H), 8.51 (d, J = 15.1 Hz, 1H), 8.18 (dd, J = 25.2, 6.1 Hz, 2H), 7.55 (dd, J = 15.2, 6.0 Hz, 1H), 7.28 (dd, J = 6.1, 5.1 Hz, 2H), 6.33 (s, 6H). 13C NMR (DMSO-d6, ppm) 152.52 (s), 150.52 (s), 137.01 (s), 132.92 (s), 128.80 (s), 127.61 (s), 126.23 (s), 125.54 (s), 122.94 (s), 121.95 (s), 121.00 (s), 112.58 (s), 106.53 (s), 88.11 (s), 87.20 (s). Crystal structure The crystal suitable for X-ray diffraction was obtained by slow recrystallization in a distilled water solution. The solution was kept in the dark at room temperature to obtain a yellow brown crystal. The X-ray intensity data for RAWQ11 was collected on an X-ray diffractometer equipped with a graphitemonochromated MoKa radiation (l = 0.71073 Å), using an o scan mode (0.991 o y o 27.121). MTT assay The tested compounds were dissolved in DMSO with 1 mM stock solution. Cells were incubated with different concentrations of the complexes for 24 h. Cell viability was determined by measuring the ability of cells to transform MTT into a purple formazan dye, which was carried out as described previously.9 Cells were seeded in 96-well tissue culture plates for 24 h. After incubation, 20 mL per well of MTT solution (5 mg mL1 phosphate buffered saline) was added and incubated for 5 h. The color intensity of the formazan solution, which reflects the cell growth conditions, was measured at 570 nm using a microplate spectrophotometer (SpectroAmaxt 250). 3D cell culture Growth factor reduced Matrigel (BD Biosciences) was prepared as per the manufacturer’s protocol. Cells were dissociated by 0.05% trypsin–EDTA (invitrogen) and re-suspended at a cell density of 2 106 cells per mL. 15 mL of Hoechst 33258 suspension a cell intravital stain was mixed with an equal volume of Matrigel on ice
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and quickly plated onto a Matrigel-coated glass bottom dish. After 30 minutes of incubation at 37 1C, DMEM media with feeder MDA-MB-231 cells were added to the 3D gel-containing dish.17 The cells were dyed by using Hoechst 33258 in DMEM and dealt with different concentrations of RAWQ11 (0, 1, 5 and 10 mM) in 96-well plates used especially for laser confocal microscopy on ice.
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Flow cytometry analysis Cell cycle distribution was analyzed by flow cytometry as previously reported.24 Treated or untreated cells were trypsinized, washed with PBS, and fixed with 75% ethanol overnight at 20 1C. Fixed cells were washed with PBS and stained with propidium iodide (PI) (1.21 mg mL1 Tris, 700 U mL1 RNase, 50.1 mg mL1 PI, pH = 8.0) for 30 min in the dark. Cell cycle distribution was analyzed using the MultiCycle software (Phoenix Flow Systems, San Diego, CA). Apoptotic cells with hypodiploid DNA content were measured by quantifying the sub-G1 peak in the cell cycle pattern. Western blot analysis Total cellular proteins were extracted by incubating the cells in lysis buffer obtained from Cell Signaling Technology; protein concentrations were determined by BCA assay. Anti-body p21, p-p21 (Thr 145), AKT, p-AKT (Thr308), GSK3b, MMP-9, FAK and PTEN were purchased from Abcam, Cell Signaling Technology and Proteintech. SDS-PAGE was carried out in 10% tricine gels, loading equal amounts of protein per lane, which was carried out as described previously.9 After electrophoresis, separated proteins were transferred to nitrocellulose membranes and blocked with 5% non-fat milk in TBST buffer for 1 h. After that, the membranes were incubated with primary antibodies at 1 : 1000 dilutions in 5% non-fat milk overnight at 4 1C, and then secondary antibodies were conjugated with horseradish peroxidase at 1 : 2000 dilution for 1 h at room temperature. Protein bands were visualized on X-ray films using an enhanced chemiluminescence system (Kodak). Wound healing assay Cells were seeded in a 6-well tissue culture plate marked on the back (1 105 cells per well) until the monolayer cells spread to more than 80% of the bottom of the culture plate. A line was scratched orthogonally using a tip (200 mL) to mark the plate. The cells were then incubated with the tested compounds at different concentrations (0, 1, 2, 5, and 10 mM) for 48 h. Migrating cells were imaged in the same visual field every 12 h for two days, which was carried out as described previously.7 Transwell invasion assay Up to 50 mL of Matrigel was added into the top chamber of the 24-well transwell on ice, and the plate was then incubated at 37 1C and 5% CO2 for 30 min. Afterward, the cells were placed in the top chamber with serum-free media having different concentrations of RAWQ11 (0, 1, 2, 5 10, and 20 mM) while the bottom chamber was added with DMEM containing 10% FBS and incubated for 24 h. Cell invasion was imaged using a fluorescence microscope.8
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Fluorescence real-time observation Transfection reagent LipoFiltert was provided by Shanghai Hanbio. To visualize the actin cytoskeleton reagent pEGFP-actin was purchased from Clontech. Cells transfected with EGFP-actin showing green fluorescence in plates were trypsinized and resuspended in 0.5 mL of DMEM media containing 200 mL of Hoechst 33258 and different concentrations of RAWQ11 (50 mM). Cell morphology was observed real-time using a laser confocal microscope for 2 h. Immunofluorescence MDA-MB-231 cells were treated with different concentrations of RAWQ11 (10 mM). MDA-MB-231 cells in complete growth medium at 5 104 cells per mL were incubated for 24 h at 37 1C, unless otherwise stated. Cells were washed once in PBS, fixed, and permeabilized simultaneously using 4% paraformaldehyde with 1% Triton X-100 in PBS, quenched with 0.1 M glycine in PBS, and blocked overnight at 4 1C with 3% (wt/vol) BSA. Fixed cells were stained with primary antibodies as indicated.4 Cell morphology was observed using a laser confocal microscope. FITC-gelatin invasion assay FITC-gelatin invasion assay was performed according to the procedures of the manufacturer (SAGENE). In brief, coverslips (18 mm diameter) were coated with 50 mg mL1 poly-L-lysine for 20 min at room temperature, washed with PBS, fixed with 0.5% glutaraldehyde for 15 min and washed with PBS 3 times. After washing, the coverslips were inverted on a drop of 0.2% FITC conjugated gelatin in PBS containing 2% sucrose, incubated for 10 min at room temperature, washed with PBS 3 times, quenched with sodium borohydride (5 mg mL1) for 3 min and finally incubated in 2 mL of complete medium for 2 h. Cells (2 105 each well) with different concentrations of RAWQ11 (0 and 1 mM) were plated in FITC gelatin-coated coverslips, incubated at 37 1C for 12 h. The ECM degradation status was evaluated and photographed using a laser confocal microscope. Cells (2 105 each well) with different concentrations of RAWQ11 and NAMI-A (0, 1, 2, and 5 mM) were plated in FITC gelatincoated coverslips and incubated at 37 1C for 12 h.15 The ECM degradation status was evaluated and photographed using a laser confocal microscope. RT-qPCR analysis The primers were purchased from BGI, Trizol was purchased from Invitrogen and an SYBR green PCR kit for quantitative PCR analysis was purchased from Takara. Total RNA was isolated using Trizol according to the recommendations of the manufacturer. Up to 2 mg of the total RNA from each samples were reverse transcribed using oligo (dT) primers at 37 1C for 90 min. The relative mRNA levels were evaluated by quantitative PCR using a SYBR green PCR kit. The signals were normalized to 18 S as internal control. The quantity of miR-21 in each BC, relative to the average expression in 40 NATs, was calculated using the following equation: RQ = 2DDCT, where DDCT = (CT miRNA CT U6 RNA)S (CT miRNA CT U6 RNA)MeanC. The primer sequences are listed in the ESI.†
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[(Z6-CH3C6H5)RuCl2]2, and [(Z6-C6H6)RuCl2]2) and the ligand (H2iip) under microwave irradiation (Fig. 1A). Microwave-assisted heating technology can produce the objective compounds with higher yield and faster than the conventional heating method.
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X-ray structure of the complex RAWQ11 The crystal structure of the complex RAWQ11 was obtained from mixed solutions of DMF and CH2Cl2. The molecular structure of the lattice of RAWQ11 is illustrated in Fig. 1B. The selected crystallographic data, bond distances, and bond angles are listed in Tables S1 and S2 (ESI†). The crystal structure of RAWQ11 has a P4% 21c space group and a typical "three-leg piano-stool" stereochemical structure that is well-documented for the arene ruthenium complex, as shown in Fig. 1B.23 Cytotoxicity of arene Ru(II) complexes Fig. 1 (A) Molecular structure of the arene Ru complexes (R1, RAWQ03; R2, RAWQ04; and R3, RAWQ11). (B) Crystal structure of the arene Ru complex RAWQ11.
miR21-binding properties The interaction between the complexes and miR-21 was examined by UV-vis spectroscopy, CD spectroscopy, and ITC and FRET assays. The UV absorption spectra of the complex RAWQ11 were recorded at 270 nm and 293 nm; titration was terminated when the intensity of the two bands did not change significantly upon the addition of miR-21. The CD spectrum of miR-21 RNA in the presence or absence of RAWQ11 was also recorded. The binding of various small molecules was assessed by isothermal titration calorimetry on a thermostated Micro Cal VP-ITC system. The stability of miR-21 was evaluated by FRET assay.23 The fluorescent labeled oligonucleotide, miR-21 RNA (5 0 -FAM-TGGGGAGGGTGGGGAGGGTGGGGAAGGTAMRA-3 0 , FAM: carboxyfluorescein, and TAMRA: 6-carboxytetramethyl-rhodamine) used as the FRET probes were diluted in Tris-HCl buffer and then annealed by being heated to 92 1C for 5 min, followed by slowly cooling to room temperature. A constant temperature was maintained for 30 s prior to each reading to ensure a stable value. Final analysis of the data was carried out by using Origin7.5 (Origin Lab Corp.).
Results and discussion Microwave-assisted synthesis of arene ruthenium(II) complexes Arene ruthenium complexes RAWQ03, RAWQ04, and RAWQ11 were synthesized using the precursors ([(Z6-p-cymene)RuCl2]2,
The inhibitory activity of synthetic arene ruthenium(II) complexes against various breast cancer cells and normal human cells was evaluated by MTT assay. The synthetic complexes exhibited significant inhibition to MDA-MB-231 and MCF-7 breast cancer cells and low toxicity to human normal MCF-10A, HK-2, and HaCat cells, especially the complex RAWQ11, as shown in Table 1. Compared with complexes 1, 2, and RAWQ11, the antitumor activity against MDA-MB-231 and MCF-7 breast cancer cells decreased with the increase in sterically rigid linking groups in the arene ring.25 However, no significant regularity was found in normal human cells. The complex RAWQ11 can effectively and sensitively inhibit the growth of MDA-MB-231 highly metastatic human breast adenocarcinoma cells (Table 1). After being treated with RAWQ11 for 24 h, the number of MDA-MB-231 cells decreased and a dosage-dependent cell shrinkage was observed (Fig. S11, ESI†). The inhibitory activity (IC50) of RAWQ11 against MDA-MB-231 cells was about 20.8 mM, which is approximately 1.7 times better than that of cisplatin under the same conditions and much better than NAMI-A that exhibits a great anti-metastasis activity in vivo. The cytotoxicity of RAWQ11 against MCF-10A normal human breast cells was approximately IC50 4 300 mM. These data are far lower than those of cisplatin (6.32 mM) and give a wider safety range in chemotherapy.24 Growth inhibition or death of cells is the result of apoptosis, cell cycle arrest, or a combined action of both,19 so, flow cytometry was carried out. After the MDA-MB-231 cells were exposed to different concentrations of RAWQ11 for 24 h, a significant increase in cell proportion in the S-phase of MDA-MB-231 cells was observed (Fig. 2A), but no obvious apoptosis occurred (Fig. 2B).
Table 1 In vitro cytotoxicity [given as IC50 values (mM)] of complexes RAWQ03, RAWQ04, RAWQ11, NAMI-A and cisplatin against MDA-MB-231 and MCF-7 human breast cancer cell lines, MCF-10A human breast epithelial cells, HK-2 human normal kidney 2 cells, and immortalized human keratinocyte HaCat cells
IC50 (mM) Comp.
MDA-MB-231
MCF-7
MCF-10A
HK-2
HaCat
RAWQ03 RAWQ04 RAWQ11 Cisplatin NAMI-A
107.6 93.2 20.8 36.1 4300
4300 101.3 0.17 17.9 0.32 13.2 0.41 4300
4300 234.7 0.24 4300 6.32 0.14 4300
205.4 0.31 4300 110.3 0.61 13.7 0.58 4300
111.6 0.52 4300 139.2 0.26 7.5 0.43 4300
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0.37 0.53 0.40 0.28
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Fig. 2 (A) S-phase arrest of MDA-MB-231 cells induced by RAWQ11. MDA-MB-231 cells were treated with RAWQ11 (0, 1, 5, and 10 mM) for 24 h, almost 40% cycling cells are in the S phase and the sharp peak suggests that some cells are experiencing S-phase delay or arrest; (B) induction apoptosis of the complex RAWQ11 against MDA-MB-231 cells. MDA-MB231 cells were treated with RAWQ11 (0, 1, 5, and 10 mM) for 24 h; little apoptosis of MDA-MB-231 cells was seen with the increase of RAWQ11. (C) Regulation of the expression of p21 and p-p21 by RAWQ11 at the protein level. (D) Regulation of the expression of p21 by RAWQ11 at the RNA level. MDA-MB-231 cells were treated with RAWQ11 (0, 1, 2, 5, and 10 mM) for 24 h.
These results revealed that RAWQ11 can block MDA-MB-231 cell growth by inducing S-phase arrest rather than apoptosis.26 In addition, the up-regulation of p21 and p-p21, which are the key proteins in the S-phase of the cell cycle, at protein and RNA levels, upon the increase in RAWQ11, indicated that RAWQ11 inhibits the growth of breast cancer cells by inducing S-phase arrest through promoting the expression and phosphorylation of p21. Considering that cell migration is an important step in tumor metastasis, the inhibitory effect of RAWQ11 against migration of MDA-MB-231 cells was further evaluated by wound healing assay.27 Compared with the control cells that spontaneously migrated, MDA-MB-231 cell treatment with RAWQ11 showed a small decrease in the time of wound closure at 48 h, and a dosage-dependent effect (Fig. 3A). Less than confluent cultures showed the significant effect of RAWQ11 (1 and 2 mM) on wound closure, indicating that RAWQ11 effectively inhibits MDA-MB-231 migration.28 Invasive cells can release MMPs to degrade the ECM and spread to the surrounding tissues. Thus, transwell invasion assay was conducted to determine the inhibition of the invasion ability of MDA-MB-231 cells when treated with RAWQ11. One layer Matrigel was added into the top chamber of the 24-well transwell to evaluate the invasion of MDA-MB-231 cells in vitro. Invasive cells with invadopodia can degrade Matrigel to migrate to the other side of the membrane (Fig. 3B), which can be dyed by crystal violet. The more the cells dyed with
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Fig. 3 (A) Wound healing assay to evaluate the migration of MDA-MB-231 cells after being treated with RAWQ11 (0, 1, and 2 mM) and DMEM with 10% FBS. Cells were wounded and monitored using a microscope every 12 h. Migration was determined by the rate of cells filling the scratched area. (B) A diagram of transwell and invasion experiment. (C) Transwell assay to observe the invasion of MDA-MB-231 cells after being treated with RAWQ11 (0, 1, 2, 5, 10, and 20 mM) for 24 h. MDA-MB-231 cells were plated on Matrigel-coated membranes in the upper chamber of transwell with SFM (serum free medium); DMEM containing 10% FBS was placed in the bottom chamber of transwell. Cells penetrating the membrane were fixed and stained with 0.1% crystal violet after 24 h. (D) Regulation of the expression of FAK, GSK3b, and MMP9 by RAWQ11 at the protein level. (E) Regulation of the expression of FAK, GSK3b, MMP2, and MMP9 by RAWQ11 at the RNA level.
crystal violet, the stronger the invasion of the cells.29 Transwell invasion assays were carried out with MDA-MB-231 cells in the presence of different concentrations of RAWQ11 for 24 h. Compared with the control, invasion of MDA-MB-231 cells was blocked as the concentration of RAWQ11 increased (Fig. 3C). After being treated with 1 mM of RAWQ11, the number of MDAMB-231 cells that penetrated the Matrigel decreased significantly. When the concentration of RAWQ11 reached 5 and 10 mM, MDA-MB-231 cells exhibited a slight invasion ability because few cells penetrated the Matrigel. In addition, the expression of MMP9 was downregulated and the transcription of MMP2 and MMP9 was significantly decreased by RAWQ11.
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Moreover, the release of MMPs negatively regulated by GSK3b increased with RAWQ11. These studies highlight that invasion and migration of MDA-MB-231 cells could be significantly suppressed by RAWQ11.30 EMT is often characterized by the change in cell morphology, the reconstruction of stress fibers, and the increase of focal adhesion, which are in tight correlation with the formation of invadopodia.12,31 Transitions in cell morphology and reorganization of actin cytoskeleton are important to cell migration and invasion.32 Changes in cell morphology were observed using real-time fluorescence. As shown in Fig. 4A, cell treatment with RAWQ11 at 50 mM exhibited full morphology, from a triangle at the beginning (0 h), which gradually changed to a round shape over time, and then a completely loose morphology at 2 h. In addition, the actin cytoskeleton marked by green GFP gradually depolymerized and the cell nucleus stained by blue Hoechst 33528 shrunk, which then disappeared eventually. The complex RAWQ11 can inhibit cell mitosis and lead to the disappearance of the cell nucleus because of cell nucleus condensation. The nuclear envelope and nucleoli disappeared during the prophase of mitosis as seen in the video (Video S1, ESI†). Furthermore, a 3D cell culture model constructed using a cell development system to simulate the micro-environment of cell growth in vitro33 was developed to observe the influence of RAWQ11 on the nucleus of MDA-MB-231 cells. The nucleus of MDA-MB-231 cells, whose integrity was kept perfect without the treatment of RAWQ11, broke into tiny pieces with an increase in the amount of RAWQ11 (Fig. 4B). The complex RAWQ11 can reduce the polymerization of the actin cytoskeleton, inhibit the maintenance of cell morphology, and induce cell nucleus damage to block the migration of MDA-MB-231 cells.34
Fig. 4 (A) Real-time imaging using a laser scanning co-focus light microscope of MDA-MB-231 cells treated with RAWQ11 (50 mM) for 2 h. The cell cytoskeleton and nucleus were visualized by green (transfected with GFPactin) and blue (dyed with Hoechst 33258) fluorescence, respectively. See also Video S1 (ESI†). (B) Induction of cell nucleus damage of RAWQ11 by 3D cell culture. MDA-MB-231 cells were dyed with Hoechst 33258 at different concentrations of RAWQ11 (0, 1, 5, and 10 mM).
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To further evaluate the effect on the inhibition of focal adhesions and stress fibers in MDA-MB-231 cells by RAWQ11, immuno-fluorescence experiments were carried out using the laser scanning co-focus light microscope. Stress fibers, the leading actors in the cell migration process, are usually composed of bundles of approximately 10–30 actin filaments.35 In addition, focal adhesions, such as integrin and proteoglycan-mediated adhesion links to the actin cytoskeleton, regulate cell migration.36 The focal adhesions were observed by paxillin because characterization of the protein revealed that paxillin was localized at discrete structures of focal adhesions, which are sites of close cellular contact with the underlying extra-cellular matrix.37 Invadopodia are actin-rich protrusions located in the areas of focal adhesions in contact with the ECM.38–40 As shown in Fig. 5, a number of focal adhesions can be observed in MDA-MB-231 cells without RAWQ11 treatment. However, after being treated with RAWQ11, the number of paxillins in MDA-MB-231 cells decreased significantly and the cellular morphology changed. Moreover, the stress fibers treated by RAWQ11 was reduced as represented by the F-actin with red fluorescence. F-actin was down-regulated (Fig. 3A), indicating that RAWQ11 can inhibit the migration of MDAMB-231 cells through suppressed focal adhesions and stress fibers. Further studies indicated that FAK expression, a direct regulator to control the formation of focal adhesions, was blocked significantly (Fig. 3D and E).41 These results showed that RAWQ11 can inhibit the invasion and migration in MDAMB-231 cells by blocking the formation of focal adhesions and the depolymerization of F-actin, which are bound to the formation of invadopodia. FITC-gelatin (fluorescein isothiocyanate-conjugated gelatin) invasion assay was utilized to further clarify whether or not 3 can block the formation of invadopodia in MDA-MB-231 cells. FITC-gelatin, which is usually used to observe cells under green fluorescence, has been used to observe the number of invadopodia formed and test the invasion ability of tumor cells (Fig. 6).42 Cells with high invasion can form invadopodia to release MMPs that act to degrade FITC-gelatin. The greater the number of the dark (non-fluorescent) areas of degradation in the FITC-gelatin, the greater the invasion of tumor cells.43 Five black holes (non-fluorescent areas) can be counted in
Fig. 5 The distribution of focal adhesion proteins (green) studied by indirect immunofluorescence (FITC-coupled second antibody) using the first antibodies against paxillin. The distribution of stress fibers (red) was studied by indirect immunofluorescence (rhodamine-conjugated phalloidin) using the first antibodies against F-actin.
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Fig. 6 (A) View of a single MDA-MB-231 cell, the number of invadopodia treated without and with RAWQ11. (B) The whole view of many MDA-MB-231 cells, the number of invadopodia treated without and with RAWQ11. Cell cytoskeletons were dyed with rhodamine-conjugated phalloidin. The dark area in the FITC-gelatin was identified as the position degraded by invadopodia.
FITC-gelatin for a single cell without the treatment of RAWQ11 (Fig. 6A). After being treated with RAWQ11, a few black holes were observed, indicating that the complex RAWQ11 can inhibit the formation of invadopodia effectively. Moreover, a number of black holes can be observed in the whole view of the FITC-gelatin for MDA-MB-231 cells without RAWQ11 treatment (Fig. 6B), indicating that up to almost a third of the cells can invade the FITC-gelatin and showed strong invasion ability for MDAMB-231 cells. However, after being treated with RAWQ11 (1 mM), a few black holes were found in the FITC-gelatin, indicating that the invasion effect of MDA-MB-231 cells was inhibited markedly. No black hole was observed and the shape of the cells changed with the high concentration of RAWQ11 (2 and 5 mM), as shown in Fig. S13 (ESI†).44 Importantly, NAMI-A, a classic anti-metastasis agent, cannot effectively inhibit the invasion of MDA-MB-231 cells at the concentrations of 1, 2 and 5 mM, which invaded lots of black holes in the FITCgelatin, as shown in Fig. S14 (ESI†). These results proved that arene Ru complexes can significantly inhibit the formation of invadopodia better than NAMI-A. The AKT signal pathway plays an important role in regulating the signaling of multiple biological processes, such as cell proliferation,
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cell cycle, apoptosis, cell invasion, and metastasis.45 When the AKT signal pathway is inactivated, the expression and phosphorylation of p21 are up-regulated, leading to the S-phase arrest of tumor cells. Meanwhile, the upregulation of the expression of GSK3b results in the inhibition of the release of MMPs to suppress the invasion of tumor cells.46 The AKT signaling pathway was regulated by PTEN, a key phosphorylation inhibitor that suppresses the expression of FAK and paxillin, to inhibit cell migration.47 After being treated with RAWQ11, the expression of AKT and p-AKT was downregulated, whereas the expression of PTEN was upregulated at protein and RNA levels, as shown in Fig. 7A and B. These data indicate that RAWQ11 can regulate the AKT signaling pathway by upregulating PTEN to inhibit cell invasion.48 Regulation of miR-21 by RAWQ11 was evaluated by RT-qPCR assay because miR-21 can directly suppress PTEN expression to activate the invasion and metastasis of tumor cells.49 The RT-qPCR results showed that the expression of miR-21 was downregulated with the increase in RAWQ11 (Fig. 8A). miR-21 is processed from transcripts that can form local RNA hairpin structures, which has been considered as potential targets for antitumor drugs.2 Thus, the interaction between RAWQ11 and miR-21 was further investigated in vitro to show that this complex can bind and stabilize the conformation of miR-21. The results confirmed the occurrence of hypochromism characterized by IL and LMCT absorption bands ascribed to RAWQ11 in the electronic spectra with the increase in the amount of miR-21 (Fig. 8B). Moreover, the positive CD signal of miR-21 at 260 nm significantly decreased upon the addition of RAWQ11, and a reduced CD signal was also observed in the range of 290–330 nm (Fig. 8C). The titration plot strongly supported the proposition that RAWQ11 binds to miR-21 in a one-site mode and completely saturates at a 1 : 1 stoichiometry; the binding constant calculated for RAWQ11 [K] is about 1.3 105 M1 (Fig. 8D). Furthermore, the results of FRET showed that RAWQ11 binds to miR-21 to stabilize the structure of miR-21 with the increase in the melting point of miR-21 to about 20 1C in the presence of 6 mM of RAWQ11 (Fig. 8E). These results showed that RAWQ11 can bind to and stabilize the structure of miR-21 to downregulate the expression of miR-21. Invadopodia is a direct actor in the invasion of the surrounding tissues and organs in tumor cells with high invasion and metastasis. However, the paucity of the agent for effective blocking of the formation of invadopodia in vitro has greatly
Fig. 7 Regulation of the key protein and mRNA by RAWQ11. MDA-MB-231 cells were treated with RAWQ11 (0, 1, 2, 5, and 10 mM) for 24 h. (A) Regulation of the expression of PTEN, AKT, and p-AKT by RAWQ11 at the protein level. (B) Regulation of the expression of PTEN and AKT by RAWQ11 at the RNA level.
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Fig. 8 Regulation of miR-21 by RAWQ11 in cells and the interaction of miR-21 with RAWQ11 in vitro. (A) Regulation of miR-21 in MDA-MB-231 cells downregulated by RAWQ11 (0, 1, 2, 5, and 10 mM). (B) Absorption spectra of RAWQ11 recorded upon the addition of increasing concentrations of miR-21 ([miR-21] = 0.5n mM, n = 0, 1, 2, 3, 4, 5, 6, 7. [RAWQ11] = 20 mM). (C) CD spectra of miR-21 recorded upon the addition of RAWQ11. ([miR-21] = 2 mM, [RAWQ11] = 5n mM, n = 0, 1, 2, 3, 4, 5). (D) ITC experimental curves at 25 1C for titration of RAWQ11 with miR-21. The results were converted to molar heat and plotted against the compound/ RNA molar ratio. The line shows the fit to the results and provides the best-fit DH values for binding, [miR-21] = 2 mM. (E) FRET-melting curves obtained with miR-21 (0.2 mM) alone (’) upon the addition of RAWQ11. Increasing trend of miR-21 melting upon the addition of RAWQ11 (1, 3, and 6 mM).
impeded the discovery of novel agents to inhibit the invasion and metastasis of breast cancer.50 In this study, we found that the arene ruthenium complex RAWQ11 can inhibit the growth of MDA-MB-231 cells effectively and suppress the migration and invasion of cells in vitro. Further studies indicated that RAWQ11 inhibits the growth of MDA-MB-231 cells by inducing the S-phase arrest-mediated cell nucleus damage. Notably, cell mitosis can also be inhibited by RAWQ11 because cell nucleus damage can be ascribed to nuclear envelope and nucleoli disappearance during the prophase of mitosis. Moreover, focal adhesions, structural links on the cell membrane where the actin cytoskeleton inside the cell is connected to the ECM on the outside, will be relieved significantly during the formation of invadopodia. The complex RAWQ11 can reduce the number of focal adhesions and promote the depolymerization of stress fibers. Finally, FITC-gelatin assay was carried out to demonstrate that the formation of invadopodia of MDA-MB-231 cells is blocked by RAWQ11. According to the study of the signal pathway, we found that RAWQ11 can bind to miR-21 and downregulate the expression of miR-21, leading to the up-regulation of PTEN, as a result the AKT signal pathway is inactivated, which can
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Fig. 9 Diagram of the S-phase arrest, metastasis, and invasion of MDAMB-231 cells inhibited by RAWQ11 through blocking the formation of invadopodia by regulating the PTEN/AKT signaling pathway-mediated miR-21.
regulate the cell cycle, invasion, and metastasis (Fig. 9). In other words, we developed a potential agent with dual function in inhibiting the proliferation, invasion, and metastasis of breast cancer cells through the PTEN/AKT signal pathway mediated by miR-21.
Acknowledgements We thank Prof. Jiao Guo for her expert secretarial assistance and Prof. Haiyang Liu for his help in the crystal structure analysis. This work was supported by the Science and Technology Item Foundation of Guangzhou (2013J4100072), the Joint Natural Sciences Fund of the Department of Science and Technology and the First Affiliated Hospital of Guangdong Pharmaceutical University (GYFYLH201309), and Excellent Discipline Leader Training Plan of Shanghai Health System.
Notes and references 1 M. Marmot, D. Altman, D. Cameron, J. Dewar, S. Thompson and M. Wilcox, Br. J. Cancer, 2013, 108, 2205–2240. 2 J. Kevin, E. G. Cheung, W. Zena and J. E. Andrew, Cell, 2013, 155, 1639–1651. 3 N. Niikura, B. C. Odisio, Y. Tokuda, F. W. Symmans, G. N. Hortobagyi and N. T. Ueno, Nat. Rev. Clin. Oncol., 2013, 10, 711–719. 4 V. Harding, M. Afshar, J. Krell, R. Ramaswami, C. Twelves and J. Stebbing, Br. J. Cancer, 2013, 109, 1543–1548. 5 B. L. Eckhardt, P. A. Francis, B. S. Parker and R. L. Anderson, Nat. Rev. Drug Discovery, 2012, 11, 479–497.
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Metallomics
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Metallomics
