Arene Ruthenium(II) Complexes as Low-Toxicity Inhibitor against the Proliferation, Migration, and Invasion of MDA-MB-231 Cells through Binding and Stabilizing <i>c‑myc</i> G‑Quadruplex DNA

Arene Ru­(II) complexes have long been extensively studied as potential inhibitors against the proliferation of tumor cells, but their behavior against the migration and invasion of tumor cells needs further research. In this work, a series of arene Ru­(II) complexes, (η⁶-C₆H₆)­Ru­(<i>p</i>-XPIP)­Cl]Cl (X = H, <b>1</b>; F, <b>2</b>; Cl, <b>3</b>; Br, <b>4</b>; and I, <b>5</b>), have been synthesized, and their inhibitory activity against the migration and invasion of MDA-MB-231 breast cancer cells have been investigated. It is found that all of these complexes exhibit excellent inhibitory activity (IC₅₀) against the growth of MDA-MB-231 breast cancer cells, and the value of IC₅₀ for <b>1</b>, <b>2</b>, <b>3</b>, <b>4</b>, and <b>5</b> is about >300, 52.6, 11.4, 45.5, and 59.1 μM, respectively. Further studies by wound-healing assay, FITC-geltain assay, and flow cytometry assay showed that <b>3</b> can apparently suppress the migration and invasion of MDA-MB-231 cells via the joint action of S-phase arrest and apoptosis. Moreover, the binding behavior of these arene Ru­(II) complexes with <i>c-myc</i> G-quadruplex DNA has also been studied, and the results showed that these complexes can bind and stabilize <i>c-myc</i> G-quadruplex DNA in groove binding mode. Also, the low toxicity of <b>3</b> was confirmed by its low inhibitory activity against the growth of normal MCF-10A breast cells <i>in vitro</i> and the development of zebrafish embryos <i>in vivo</i>. In other words, these results indicated that synthetic arene Ru­(II) complexes can be developed as low-toxicity agents against the proliferation, migration, and invasion of breast cancer cells

LC treatment inhibits cancer cell proliferation <i>in vivo</i> and <i>in vitro</i>.

<p>(<b>a</b>) LC selectively inhibits HepG2 tumor growth compared with normal tissues. BALB/c nude mice were <i>s.c</i>. inoculated in the left armpit of each mouse with HepG2 cells (1×10⁶ cells/mouse). When the tumor size reaches 50–75 mm³, nude mice were <i>i.p</i>. injected with 400 mg/kg for consecutive 15 days. Normal Balb/c mice were treated as the nude mice. Body and organ weight were detected. B. W: Body weight. Mean+SD (n = 8). *<i>P</i><0.01, **<i>P</i><0.05, <i>versus</i> each control respectively. % inhibition = body weight or organ weight in the LC-treated group/ average body weight or organ weight in the control group×100. (<b>b</b>) LC inhibits HepG2 cell proliferation in a dose-dependent manner. HepG2 cells were treated with various doses of LC (1.25, 2.5, 5, 10 mM) for 24 h or 48 h, cell proliferation was detected by MTS assay. Mean+SD (n = 3). *<i>P</i><0.01, **<i>P</i><0.05, compared with the control. (<b>c</b>) LC induces cell cycle arrest at Go/G1 phase. HepG2 cells were treated with different doses of LC for 24 h, cell cycle were detected by flow cytometry. Representative results were shown. (<b>d, e</b>) LC slightly induces cell death. HepG2 cells were exposed to various doses of LC for 24 h and cell apoptosis was detected by flow cytometry. Summary of the data were shown in (<b>d</b>) and representive flow images were shown in (<b>e</b>). **P<0.05, versus control. (<b>f</b>) LC does not dramatically affect thymocyte cell viability. Mouse thymocytes were treated with various doses of LC (2.5, 5, 10 mM) for 24 h, cell viability was detected by MTS assay and cell number was count.</p

Computional molecular docking of LC with HDAC.

<p>(<b>a</b>) The chemical structure of LC was shown. (<b>b</b>) The docking model of L-carnitine in active site of HDAC.</p

LC treatment selectively induces expression of p21^cip1 gene, mRNA and protein in cancer cells.

<p>(<b>a</b>) LC induces p21^cip1 gene expression but not p27 and GAPDH. HepG2 cells were treated with LC (2.5, 5.0, 10 mM) for 24 h; cells were collected for gene expression profile analysis. In the gene chip, there are 2 probes for p21^cip1 and 1 probe for p27^kip1. All the fold increases of p21^cip1 and p27^kip1 gene expression <i>versus</i> control were shown. (<b>b</b>) LC dose-dependently induces p21^cip1 mRNA expression but not p27^kip1 in HepG2 cells. HepG2 cells were incubated with different concentrations of LC (2.5, 5, 10 mM) for either 12 h or 24 h; the cells were collected for mRNA assay of p21^cip1 and p27^kip1 by real-time PCR. Fold increase of the LC-treated <i>versus</i> control was shown. Mean+SD (n = 3). *<i>P</i><0.01, **<i>P</i><0.05, compared with control. (<b>c</b>) LC dose-dependently and time-dependently induces p21^cip1 protein accumulation in HepG2 cancer cells. HepG2 and SMMC7721 cells were treated with various doses of LC for 48 h or HepG2 cells were exposed to 5 mM of LC for 12, 24, 36, 48 h; p21 and p27 proteins were detected by Western blot. (<b>d</b>) LC dose-dependently decreases Rb phosphorylation. HepG2 cells were treated with LC for 48 h; Rb and phosphorylated Rb were dectected by Western blot. Typical Western images were shown (left) and band intensity was quantified (right).</p

L-carnitine treatment fails to increase ATP concentration in cancer cells.

<p>(<b>a</b>) Cancer cells are resistant to oligomycin in the presence of D-glucose (2 g/L) but not L-glucose (2 g/L). Human HepG2 cancer cells were cultured in the presence or absence of either D-glucose or L-glucose in the culture medium and treated with various doses of oligomycin (0.1, 0.25, 0.5. 1.0 µg/ml) for 6 h and ATP content was assessed. Mean+SD (n = 3). *<i>P</i><0.01, <i>versus</i> control. DM: DMSO. (<b>b</b>) LC does not increase intracellular ATP content in cancer cells. Human hepatic HepG2 and SMMC-7721 cells were cultured in the normal culture medium respectively and treated with different doses of LC for 6 h, ATP content was detected. LC: L-carnitine. (<b>c</b>) Thymotytes are sensitive to oligomycin in the presence of D-glucose (2 g/L). Mouse thymocytes were treated with oligomycin (1 mg/ml) for different time points (1, 3, 6, 9 h), total ATP content was detected. (<b>d</b>) LC efficiently increases cellular ATP content. Mouse thymocytes were treated with LC (1 mM) for various times, cellular ATP content was assassed. Veh: vehicle.</p

LC induces Histone acetylation in cultured cells.

<p>(<b>a</b>) LC dose-dependently induces accumulation of acetylated histones. HepG2 cells were exposed to LC (1.25, 2.5, 5.0 mM) for 48 h, acetylated histones H3 and H4 were detected by Western blot. GAPDH was used as a loading control. (<b>b</b>) LC time-dependently induces accumulation of acetylated histones. HepG2 cells were treated with LC (5 mM) for different time points (12 h, 24 h, 36 h, 48 h) and acetylated histones were detected. (<b>c</b>) LC treatment increases lysine-acetylated protein accumulation in human HepG2 and SMMC-7721 cancer cells. Human HepG2 and SMMC-7721 cells were treated with LC (10 mM) for 12 h, and then cells were collected for Western blot to detect acetylated proteins with lysine acetylated antibody. (<b>d</b>) LC dose-depentently increases accumulation of acetylated histones in mouse thymocytes. Mouse thymocytes were treated with LC for 24 h, histone acetylation was detected by Western Blot. Buty (1 mM) was used as a positive control.</p

LC treatment induces accumulation of acetylated histones in chromatin associated with p21^cip1 gene but not p27^kip1 gene.

<p>(<b>a, b</b>) HepG2 cells were treated with LC (10 mM) and Buty (1 mM) for 12 h; cells were collected for CHIP assay as described in the Materials and Methods part. The PCR data and fold enrichment of p21^cip1 and p27^kip1 promoter gene in LC- or Buty-treated <i>versus</i> vehicle control were shown in (<b>a</b>) and (<b>b</b>) respectively. IP: immunoprecipitation.; Neg: negative.</p

LC and Vel synergistically inhibit cancer growth and increase histone acetylation and p21^cip1 expression <i>in vivo</i>.

<p>Nude mice bearing HepG2 tumor were <i>i.p</i> injected with vehicle or LC (400 mg/kg, <i>i.p.</i> once/day except day 8), Vel (0.75 mg/kg, <i>i.v.</i> once/3 days) alone and the combination respectively for 15 days. A, Tumor images, tumor weight, body weight, tumor growth curve including Box-plot image and the relative tumor volume in all the four groups (Vehicle-, LC-, Vel- and LC+Vel-) were shown. *<i>P</i><0.05, compared with the Veh control; #<i>P</i><0.05, compared with the treatment alone, ##<i>P</i>>0.05, compared with the treatment alone. B, p21^cip1 and acetylated H3 proteins in tumor tissues of various groups were detected by immunochemistry and the results were shown respectively. All the immunostaining were repeated in three mouse tumor tissues and the typical images were shown.</p

Vel and LC synergistically induces Bax accumulation and silencing Bax by siRNA reversed PARP cleavage.

<p>(<b>A</b>) As treated and analyzed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052576#pone-0052576-g002" target="_blank">Figure 2A</a>, Bax and Bcl-2 gene expression was shown. (<b>B</b>) As treated and analyzed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052576#pone-0052576-g002" target="_blank">Figure 2B</a>, Bax mRNA was detected by real-time PCR and fold increase of Bax mRNA was shown. (<b>C</b>) As treated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052576#pone-0052576-g004" target="_blank">Figure 4A</a>, Bax and Bcl-2 protein levels were detected. Representative images were shown. (<b>D</b>) HepG2 cells were transfected with Bax-siRNA (#1) for 48 h and then treated with the combination of LC (5 mM) and Vel (50 nM) for 24 h, Western blot was performed to detect Bax and PARP cleavage. GAPDH was used as a loading control.</p

A proposed mechanism of the synergistic effect of LC and Vel on cytotoxicity.

<p>Proteasome inhibitor Vel induces proteasome inhibition and histone acetylation which is enhanced by the HDAC inhibitor LC. Proteasome inhibition enhances accumulation of Bax protein and cell cycle inhibitors p21 and p27 proteins, and histone acetylation also further induces p21 expression in cancer cells, both of which contributes to cytotoxicity mediated by the combination of LC and Vel.</p