[10]-gingerol induces apoptosis and inhibits metastatic dissemination of triple negative breast cancer in vivo

There is increasing interest in the use of non-toxic natural products for the treatment of various pathologies, including cancer. In particular, biologically active constituents of the ginger oleoresin (Zingiber officinale Roscoe) have been shown to mediate anti-tumour activity and to contribute to the anti-inflammatory, antioxidant, antimicrobial, and antiemetic properties of ginger. Here we report on the inhibitory properties of [10]-gingerol against metastatic triple negative breast cancer (TNBC) in vitro and in vivo. We show that [10]-gingerol concentration-dependently induces apoptotic death in mouse and human TNBC cell lines in vitro. In addition, [10]-gingerol is well tolerated in vivo, induces a marked increase in caspase-3 activation and inhibits orthotopic tumour growth in a syngeneic mouse model of spontaneous breast cancer metastasis. Importantly, using both spontaneous and experimental metastasis assays, we show for the first time that [10]-gingerol significantly inhibits metastasis to multiple organs including lung, bone and brain. Remarkably, inhibition of brain metastasis was observed even when treatment was initiated after surgical removal of the primary tumour. Taken together, these results indicate that [10]-gingerol may be a safe and useful complementary therapy for the treatment of metastatic breast cancer and warrant further investigation of its efficacy, either alone or in combination with standard systemic therapies, in pre-clinical models of metastatic breast cancer and in patients

Cytotoxicity and anti-tumor effects of new ruthenium complexes on triple negative breast cancer cells

<div><p>Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype. The high rate of metastasis associated to the fact that these cells frequently display multidrug resistance, make the treatment of metastatic disease difficult. Development of antitumor metal-based drugs was started with the discovery of cisplatin, however, the severe side effects represent a limitation for its clinical use. Ruthenium (Ru) complexes with different ligands have been successfully studied as prospective antitumor drugs. In this work, we demonstrated the activity of a series of biphosphine bipyridine Ru complexes <b>(1)</b> [Ru(SO₄)(dppb)(bipy)], <b>(2)</b> [Ru(CO₃)(dppb)(bipy)], <b>(3)</b> [Ru(C₂O₄)(dppb)(bipy)] and <b>(4)</b> [Ru(CH₃CO₂)(dppb)(bipy)]PF₆ [where dppb = 1,4-bis(diphenylphosphino)butane and bipy = 2,2’-bipyridine], on proliferation of TNBC (MDA-MB-231), estrogen-dependent breast tumor cells (MCF-7) and a non-tumor breast cell line (MCF-10A). Complex <b>(4)</b> was most effective among the complexes and was selected to be further investigated on effects on tumor cell adhesion, migration, invasion and in apoptosis. Moreover, DNA and HSA binding properties of this complex were also investigated. Results show that complex <b>(4)</b> was more efficient inhibiting proliferation of MDA-MB-231 cells over non-tumor cells. In addition, complex <b>(4)</b> was able to inhibit MDA-MB231 cells adhesion, migration and invasion and to induce apoptosis and inhibit MMP-9 secretion in TNBC cells. Complex <b>(4)</b> should be further investigated <i>in vivo</i> in order to stablish its potential to improve breast cancer treatment.</p></div

Effects of complex (4) on MDA-MB-231 cell adhesion and cytoskeleton structure.

<p><b>(A)</b> Type I collagen, fibronectin, laminin and vitronectin were coated on the wells of a 96-well plate. Complex <b>(4)</b> was incubated with MDA-MB-231 cells, which were subsequently plated on the ECM coatings. After washes, remaining cells were quantified by colorimetry. <b>(B)</b> MDA-MB-231 cells were incubated with complex <b>(4)</b> and then with Phalloidin and DAPI. Significant at the *<i>p</i><0.005, **<i>p</i><0.001 levels using ANOVA and Bonferroni tests.</p

Interactions with DNA and HSA.

<p>Binding constants for the interaction between Ru(II) complexes <b>(1–4)</b> and calf thymus <i>ct</i>-DNA and stern-Volmer quenching constant (K_sv, M^-1), biomolecular quenching rate constant (Kq, M^-1s^-1), binding constant (Kb, M^-1), the number of binding sites (n), ΔG(KJ.mol^-1), ΔH (KJ.mol^-1) and ΔS (J.mol^-1K) values for the complex-HSA system at different temperatures.</p

Crystal structures of the complexes.

<p><b>(A) (1)</b> [Ru(SO₄)(dppb)(bipy)], <b>(B) (2)</b> [Ru(CO₃)(dppb)(bipy)], <b>(C) (3)</b> [Ru(C₂O₄)(dppb)(bipy)] and <b>(D) (4)</b> [Ru(CH₃CO₂)(dppb)(bipy)]PF₆, showing the atoms labelling and the 30% probability ellipsoids. For 4, the PF₆^- anion was omitted, for sake of clarity.</p

Absorption spectral titration.

<p><b>(A)</b> Absorption spectral titration of complex <b>(4)</b> in the presence of increasing concentrations of <i>ct</i>-DNA at 298 K and fluorescence emission spectra of HSA at 37°C. Inset graph represents the plots of (ε_a-ε_f)/(ε_b-ε_f) versus [DNA] for the titration of DNA with Ru(II) complexes. <b>(B)</b> CD spectrum of <i>ct</i>-DNA (100 μM) in the presence of complexes <b>(1–4)</b> in Tris-HCl buffer after incubating by 18 h at 37°C. <b>(C)</b> Electrophoretic mobility pattern of pBR322 plasmid DNA incubated with metal complexes <b>(1–4)</b> by 18 h at 37°C, at indicated concentrations (μM). Ri = ratio complex/DNA; MM = molecular marker. <b>(D)</b> Fluorescence quenching spectra of HSA with different concentrations of complex (4) with the excitation wavelength at 270 nm at 37°C in a Trizma buffer, pH 7.4. The arrow shows the intensity changes upon increasing the concentration of the quencher (0 to 50 μM, orange line to light blue line, respectively).</p

Effect of complex (4) on apoptosis in MDA-MB-231 breast tumor cells and MCF-10A non-tumor breast cells.

<p><b>(A)</b> After treatment with the indicated concentrations of complex <b>(4)</b>, cells were incubated with PE-Annexin-V and 7AAD for 15min, harvested and then analyzed by cytometry. <b>(B)</b> The percentage of apoptotic and necrotic cells was plotted in a graph for MDA-MB-231 and MCF-10A cells. The fluorescence of 7AAD is detected in the FL3-A channel and the fluorescence of PE-Annexin-V is detected in the FL2-A channel. Camptothecin (campto) was used as a positive control for apoptosis. <b>(C)</b> Nuclear fragmentation promoted by complex <b>(4)</b> in MDA-MB-231 cells was investigated using DAPI staining. Staurosporine was used as a positive control for nuclear fragmentation. White arrows show fragmented nuclei. The expression of apoptotic and anti-apoptotic molecules was investigated by <b>(D)</b> qRT-PCR and <b>(E)</b> Western blotting analysis. Significant at the *<i>p</i><0.005, **<i>p</i><0.001, ***<i>p</i><0.0001 levels using ANOVA and Bonferroni tests.</p

Effect of complex 4 on MDA-MB-231, MCF-7 and MCF-10A cells.

<p><b>(A)</b> Cell morphology was examined after 2 h and 24 h of treatment. <b>(B)</b> Clonogenic assay of untreated MDA-MB-231 cells (control) or cells treated with complex <b>(4)</b>. A photograph of a representative experiment is shown along with graph quantifications of colony number and size. Significant at the *<i>p</i><0.005, **<i>p</i><0.001 levels using ANOVA and Bonferroni tests.</p

Effects of complex (4) on MDA-MB-231 cell migration and invasion.

<p><b>(A)</b> Complex was incubated with MDA-MB-231 and cells were allowed to migrate in Boyden chambers. Migrated cells were fixed and quantified by manual counting. Representative images of the inserts are represented above each condition. The wells were photographed under a light microscope under 40× magnifications. Positive control (C+) represents migrating cells without any treatment and negative control (C-) was cells migrating toward an FBS-free medium. <b>(B)</b> Wound healing assay at 0, 24 and 48 h of treatment with the complex. <b>(C)</b> Effect of complex <b>(4)</b> on MDA-MB-231 cell invasion through matrigel. <b>(D)</b> Zymography in 1% gelatin-SDS-PAGE. A photograph of a representative zymography gel is shown. Gels were analyzed by densitometry, and data were normalized in percentage compared to untreated control cell lysate. Significant at the * <i>p</i><0.005, **<i>p</i><0.001 levels using ANOVA and Bonferroni tests.</p