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Although doxorubicin is widely used to treat cancer, severe side effects limit its clinical use. Combination of standard chemotherapy with natural products can increase the efficacy and attenuate the side effects of current therapies. Here we studied the anticancer effects of a combined regimen comprising doxorubicin and [10]-gingerol against triple-negative breast cancer, which does not respond to hormonal or targeted therapies. Cytotoxicity was evaluated by MTT assay, cell cycle progression and apoptosis were analyzed by flow cytometry and signaling pathways were analyzed by Western blotting in human and murine triple negative breast cancer cell systems. The anticancer/antimetastatic and toxic effects of the combined regimen was evaluated using syngeneic and xenograft orthotopic models. The combination of doxorubicin and [10]-gingerol significantly increased the number of apoptotic cells, compared to each compound alone. In 4T1Br4 cells, the combined regimen was the only condition able to increase the levels of active caspase 3 and γH2AX and to decrease the level of Cdk-6 cyclin. In vivo, doxorubicin (3 mg/Kg, D3) and [10]-gingerol (10 mg/Kg, G10) resulted in a significant reduction in the volume of primary tumors and a decrease in the number of circulating tumor cells (CTCs). Interestingly, only the combined regimen led to decreased tumor burdens to distant organs (i.e., metastasis) and reduced chemotherapy-induced weight loss and hepatotoxicity in tumor-bearing animals. Likewise, in a xenograft model, only the combined regimen was effective in significantly reducing the primary tumor volume and the prevalence of CTCs. Our data indicate that [10]-gingerol has potential to be used as a neoadjuvant or in combined therapy with doxorubicin, to improve its anticancer activity.

Triple-negative breast cancer subtype is the most aggressive type of breast cancer due to the lack of specific therapeutic targets, having limited treatment options, low survival prognosis and high recurrence rates. In this work, we describe the effects of a semisynthetic derivative of [6]-gingerol (6G) called SSi6, produced by the addition of a 2,4-dinitrophenylhydrazine reagent on several aspects of triple-negative breast cancer biology. Human breast cancer cell lines MDA-MB-231 and MCF-10A were used in the experiments. MTT assays were used to detect cell viability. Cell cycle and apoptosis assay were analyzed using flow cytometer Accuri C6 and analysis of proteins as retinoblastoma Rb and kinases Cdk4/6 were analyzed by western blotting. SSi6 induced cytotoxic effects on triple-negative breast cancer cells, with higher selectivity when compared to the non-tumor MCF-10A cells. In addition, SSi6 inhibited migration and invasion of triple-negative breast cancer cells and was able to arrest cell cycle at the G1-phase, mainly by decreasing Cdk4/6-Rb axis levels. Therefore, SSi6 provoked the induction of apoptosis in triple-negative breast cancer cells. SSi6 was more efficient in producing these effects, compared to the original 6G natural product. This study may contribute to a better understanding of the effects of natural and semisynthetic products on the in-vitro metastatic processes in the MDA-MB-231 triple-negative breast cancer cell line. Additional, it can be useful to understand the effects of chemical modifications on already effective natural compounds aiming at the improvement of their bioactive properties, such as in the increase of the cytotoxic selectivity against tumor cells, compared to non-tumor ones.

Miconia genus, belonging to Melastomataceae family, is widely distributed in tropical America. Isolated compounds and semi-synthetic derivatives from Miconia ferruginata leaves were evaluated both for their cytotoxicity on tumor cell lines and against important proteolytic enzymes such as cathepsins B and K. Among the evaluated compounds, the mixture of methyl ursolate ( 2a ) and oleanate ( 3a ) showed considerable cytotoxicity against melanoma (MDA-MB435) and colon cancer (HCT-8) cell lines. In addition, it was also demonstrated that the mixture of ursolic ( 2 ) and oleanolic ( 3 ) acids inhibited of the cathepsin B, with IC 50 value of 13.02 μM. On the other hand, mixture of compounds 2a and 3a showed considerable activity against cathepsin K, with IC 50 value of 1.42 μM. The compound 5,6,7-trihydroxy 4’-methoxyflavone ( 1) showed no activity.

Mesosphaerum pectinatum (L.) Kuntze (Lamiaceae), also known as sambacaitá, is a medicinal plant widely used in northeastern Brazil for the treatment of inflammatory and painful conditions, bacterial infections and cancer. Hence, the medicinal use of this species is quite meaningful to the search for bioactive compounds.To evaluate the antinociceptive and anti-inflammatory activities of the pectinolide-enriched fraction of Mesosphaerum pectinatum (PEF) in animal models.The PEF was analyzed with HPLC-DAD andThe PEF significantly decreased the licking time of the animals treated when compared to the control group (second phase). In the carrageenan-induced peritonitis model, PEF (100 and 200 mg/kg) significantly decreased total and differential leukocyte counts. The PEF (0.3, 1.0 and 3.0 mg/ear) significantly reduced mice ear edema at the same extent and like the results obtained with the standard drug (dexamethasone). The MPO activity was reduced in mice ear at doses of 1 and 3 mg/ear. Antinociceptive effect on the hot plate test was not observed, demonstrating that there is no analgesic activity.Our results suggest that the pectinolide-enriched fraction exhibits anti-inflammatory effects and that it is involved with inhibiting the release of the inflammatory mediators.

Simple Summary Triple-negative breast cancers (TNBC) represent approximately 15% of all breast cancers and lack the expression of a defined molecular target. This absence makes this subtype of cancer difficult to treat and control. Current chemotherapy drugs cause various side effects and toxicities that can jeopardize the quality of life of patients with TNBC cancer. Therefore, this research focuses on a new semi-synthetic compound derived from [6]-gingerol, where we demonstrate that it does not cause significant toxic effects in vivo and, more importantly, we demonstrate its antitumor and antimetastatic effects using preclinical xenograft models simulating two clinical scenarios of a woman with breast cancer. Abstract Breast cancer metastasis is the most common cause of cancer death in women worldwide. Triple-negative breast cancers (TNBC) form a heterogeneous group of tumors that have higher relapse rates and poorer survival compared to other breast cancer subtypes. Thus, this work reports the antitumor and antimetastatic activities of a [6]-gingerol-derived semi-synthetic compound named SSi6 on MDA-MB-231 TNBC cells using xenograft models. SSi6 did not cause toxic effects in vivo as demonstrated by body weight and hematological and histological evaluations. From the orthotopic xenograft model, we demonstrated that SSi6 slows and inhibits the growth of the primary tumor, as well as prevents metastatic spontaneous progression from lymph nodes to the lungs. Moreover, a second xenograft model with resection of the primary tumor showed that SSi6 also blocks the progression of metastases from the lymph nodes to other visceral organs. Taken together, our results demonstrate that SSi6 is a promising compound to be investigated in other preclinical and clinical models to be applied as a complementary therapy for TNBC.

OPTIMIZATION AND VALIDATION OF A SIMPLE AND LOW-COST SEMI-QUANTITATIVE ENZYMATIC METHOD TO SEARCH FOR CISTEINE PROTEASES INHIBITORS. Cysteine proteases are an important class of enzymes involved in several essential biological processes. For this reason, these enzymes are important therapeutic targets, and therefore can be used in enzymatic assays for the search of hits. The most widely used methods in the search of enzyme inhibitors are spectrophotometric, however they have relatively high operational costs. For this reason, simpler, and cheaper methods can be useful in the search for enzyme inhibitors. In this work, a simple and low-cost semi-quantitative method was optimized and validated using papain and skimmed powdered milk (casein) as a substrate. The ideal condition for the method was the one that used powdered milk, 0.250%, papain solution, 9.4 mg mL-1 and incubation time, 18 h. The method validation was performed using the inhibitor E64 and herb extracts, in different concentrations. After carrying out the experiments and statistical analyses, a high reproducibility can be observed, with relative standard deviations below 2.1%. Plant extracts were used to test the method and the results indicated that these extracts are promising for the search of cysteine protease inhibitors. The method developed here has simplicity and reproducibility and has great application, as preliminary study, in the search for inhibitors of cysteine proteases.

Triple negative breast cancer (TNBC) is very aggressive and lacks specific therapeutic targets, having limited treatment options and poor prognosis. [6]-gingerol is the most abundant and studied compound in ginger, presenting diverse biological properties such as antitumor activity against several types of cancer, including breast cancer. In this study, we show that the semi-synthetic analogue SSi6, generated after chemical modification of the [6]-gingerol molecule, using acetone-2,4-dinitrophenylhydrazone (2,4-DNPH) reagent, enhanced selective cytotoxic effects on MDA-MB-231 cells. Remarkably, unlike the original [6]-gingerol molecule, SSi6 enabled autophagy followed by caspase-independent apoptosis in tumor cells. We found a time-dependent association between SSi6-induced oxidative stress, autophagy and apoptosis. Initial SSi6-induced reactive oxygen species (ROS) accumulation (1h) led to autophagy activation (2-6h), which was followed by caspase-independent apoptosis (14h) in TNBC cells. Additionally, our data showed that SSi6 induction of ROS plays a key role in the promotion of autophagy and apoptosis. In order to investigate whether the observed cell death induction was dependent on preceding autophagy in MDA-MB-231 cells, we used siRNA to knock down LC3B prior to SSi6 treatment. Our data show that LC3B downregulation decreased the number of apoptotic cells after treatment with SSi6, indicating that autophagy is a key initial step on SSi6-induced caspase-independent apoptosis. Overall, the results of this study show that structural modifications of natural compounds can be an interesting strategy for developing antitumor drugs, with distinct mechanisms of actions, which could possibly be used against triple negative breast cancer cells that are resistant to canonical apoptosis-inducing drugs.

Hyptis pectinata is a herb popularly used in Brazil for the treatment of inflammations, pain, bacterial infections and cancer. In the present study, inflorescences (MPIn), leaves (MPL), branches (MPB), root (MPR) extracts and three compounds isolated from MPIn were assayed against breast tumor cell lines. The structures of the three compounds (pectinolide J, hyptolide and pectinolide E) were determined by means of spectroscopic analysis. Pectinolide J was isolated for the first time. The MPIn, MPL and MPR exhibited specific antiproliferative activity on tumor cell lines when compared to normal cell lines with IC50 of 52.01 ± 0.64, 45.91 ± 0.02 μg/mL and 82.84 ± 0.03 μg/mL, respectively. Although the isolated substances did not present good antiproliferative activity, when the three were associated, a greater biological effect was observed, suggesting a synergistic effect. Hyptolide (5.6 ± 0.4 μg/mL) showed IC50 sufficiently low to be considered as a drug prototype.

Six-gingerol, the most abundant secondary metabolite present in ginger oleoresin, is a bioactive compound with antioxidant, anti-inflammatory and anticancer activities. This compound presented great cytotoxic potential in several cancer cell lines, and an increment in its activity can be achieved through complexation with cyclodextrins. In addition, the 6G complexation can modify its physical state from liquid to solid and mask its pungent taste, unpleasant for some people. The [6]-gingerol/β-cyclodextrin binary inclusion complex (6G/βCD) and [6]-gingerol/β-cyclodextrin/chitosan (6G/βCD/chitosan) ternary inclusion complex were successfully prepared in the solid state with the coprecipitation method, and characterized by 1D and 2D Nuclear Magnetic Nuclear, Fourier transform-infrared spectroscopy, Differential Scanning Calorimetry, Scanning Electronic Microscopy and Molecular Modelling Studies. Analysis of phase solubility indicated that the inclusion complexes presented a B-type curve and stability constant values suitable for practical applications. NMR analysis, dissolution test and molecular modelling indicated that 6G can form total 1:2 inclusion complex with βCD, with the aromatic ring and alkyl chain of 6G located in the narrow side of the hydrophobic cavity of the two βCDs. The dissolution test showed a faster dissolution for inclusion complexes when compared to free 6G. Furthermore, the cytotoxicity activity of the 6G/βCD binary inclusion complex was examined to determine whether the bioactivities of 6G were affected by its inclusion. The cytotoxicity assays revealed that 6G/βCD had a slightly higher bioactivity than the free 6G, corroborating the slight increase in the solubility of 6G in the inclusion complex.

Scopoletin (7-hydroxy-6-methoxychromen-2-one, SC) is a phenolic coumarin commonly found in some medicinal plants. This compound presents relevant literature data demonstrating its anti-inflammatory, antioxidant, antitumor, hypocholesterolemic and hypoglycemic activities. SC was loaded in situ into the Zeolitic Imidazolate Framework-8 (ZIF-8), a slow-releasing material with pharmaceutical application. Both ZIF-8 and SC:ZIF-8 drug delivery systems (DDS) were characterized by x-ray diffraction (XRD), scanning electron microscopy (SEM), nuclear magnetic resonance (NMR), thermogravimetry (TG), differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR). The quantification of SC in the solution was performed by high-performance liquid chromatography with diode-array detection (HPLC-DAD) and the release profile at pHs 4.5 and 6.5 was compared under non-sink conditions of SC and SC:ZIF-8 DDS. Expectorant activity was also evaluated. The SC:ZIF-8 DDS showed an encapsulation efficiency of 22.5%. A pH-dependent release profile of SC present in the DDS was observed when compared to the pure SC. The most suitable kinetic model for pure SC at both pHs 4.5 and 6.5 was the Korsmeyer-Peppas model. For SC:ZIF-8 DDS at pH 4.5, the best model was the Korsmeyer-Peppas, but at pH 6.5 it was Peppas-Sahlin. SC also presented expectorant activity at 30 and 100 mg/kg doses, but the system did not show expectorant activity at 10 mg/kg dose.

Acute kidney injury (AKI) and metabolic dysfunction are critical complications in sepsis syndrome; however, their pathophysiological mechanisms remain poorly understood. Therefore, we evaluated whether the pharmacological properties of 6-gingerol (6G) and 10-gingerol (10G) could modulate AKI and metabolic disruption in a rat model of sepsis (faecal peritonitis). Animals from the sham and AKI groups were intraperitoneally injected with 6G or 10G (25 mg/kg). Septic AKI decreased creatinine clearance and renal antioxidant activity, but enhanced oxidative stress and the renal mRNA levels of tumour necrosis factor-α, interleukin-1β, and transforming growth factor-β. Both phenol compounds repaired kidney function through antioxidant activity related to decreased oxidative/nitrosative stress and proinflammatory cytokines. Metabolomics analysis indicated different metabolic profiles for the sham surgery group, caecal ligation and puncture model alone group, and sepsis groups treated with gingerols. 1H nuclear magnetic resonance analysis detected important increases in urinary creatine, allantoin, and dimethylglycine levels in septic rats. However, dimethylamine and methylsulfonylmethane metabolites were more frequently detected in septic animals treated with 6G or 10G, and were associated with increased survival of septic animals. Gingerols attenuated septic AKI by decreasing renal disturbances, oxidative stress, and inflammatory response through a mechanism possibly correlated with increased production of dimethylamine and methylsulfonylmethane.

A series of chalcone derivatives, 1-15, were prepared by Claisen-Schmidt condensation and evaluated for their cytotoxicities on tumor cell lines and also against proteolytic enzymes such as cathepsins B and K. Of the compounds synthesized, (E)-3-(3,4-dimethoxyphenyl)-1-phenylprop-2-en-1-one (12), (E)-3-(4-chlorophenyl)-1-phenylprop-2-en-1-one (13), (E)-3-(4-methoxyphenyl)-1-phenylprop-2-en-1-one (14), and (E)-3-(4-nitrophenyl)-1-phenylprop-2-en-1-one (15) showed significant cytotoxicities. The most effective compound was 15, which showed high cytotoxic activity with an IC50 value lower than 1 μg/ml, and no selectivity on the tumor cells evaluated. Substituents at C(4) of ring B were found to be essential for cytotoxicity. In addition, it was also demonstrated that some of these chalcones are moderate inhibitors of cathepsin K and have no activity against cathepsin B.

cope Ginger has long been used in traditional Asian medicine to treat osteoarthritis. Indeed, scientific research has reported that ginger derivatives (GDs) have the potential to control innate immune responses. Given the widespread use and demonstrated properties of GDs, we set out to study their anti-inflammatory and anticatabolic properties in chondrocytes. Methods and results 6-shogaol (6-S), the most active GD, was obtained from ginger. 6-S was not toxic as measured by MTT assay, and inhibited NO production and IL-6 and MCP-1 induced gene expression in LPSbut not in IL-1β-stimulated chondrocytes. 6-S also inhibited LPS-mediated ERK1/2 activation as well as NOS2 and MyD88 induced expression as determined by Western blot. Moreover, zymography revealed that 6-S inhibited matrix metalloproteinases (MMP) 2/9 induction in LPS-treated cells. Hydrated 6-S was modified to obtain a compound (SSi6) without 6-S potential anti-inflammatory properties. Both 6-S and SSi6 inhibited cathepsin-K activity. Conclusion 6-S blocked TLR4-mediated innate immune responses and MMP induction in chondrocytes. These results, together with GDs-mediated cathepsin-K inhibition, suggest the potential for GDs use against cartilage and bone degradation. Therefore, considering that clinical trials involving oral administration of ginger achieved relevant nontoxic GDs serum concentrations, we suggest that a ginger-supplemented diet might reduce OA symptoms.

Breast cancer is one of the most common malignant diseases in women worldwide. In developing countries, it is the second highest cause of death in women. Triple-negative breast cancer (TNBC) represents the approximately 15% of breast cancers that lack expression of estrogen (ER) and progesterone receptors (PR) and do not exhibit amplification of the human epidermal growth factor receptor 2 (HER2) gene. Metastatic process is described as a cascade of events. Normal cells are transformed into tumor cells due to mutations in genes that regulate critical pathways, producing an imbalance between proliferation and cell death that eventually leads to the formation of a primary tumor. Further interactions with the stromal microenvironment surrounding tumor cells and extracellular matrix (ECM) proteins contribute to the formation of new vessels. These interactions facilitate tumor cell invasion of surrounding tissues, intravasation through newly formed vessels, and dissemination to other tissues, to form secondary tumors. To treat metastasis from breast and many other cancer types, chemotherapy is one of the most extensively used methods. However, its efficacy and safety remain a primary concern, as well as its toxicity and other side effects. Thus, there is increasing interest in naturally occurring cancer antitumor agents. Ginger (Zingiber officinale Roscoe) is widely used worldwide as a food, spice, and herb. Recently we have demonstrated that [10]-gingerol is able to revert malignant phenotype in breast cancer cells in 3D culture and, moreover, to inhibit the dissemination of TNBC to multiple organs including to lung, bone, and brain, in spontaneous and experimental in vivo metastasis assays. The aim of this work was to investigate the effects of [10]-gingerol on the adhesion properties of MDA-MB-231 cells to the extracellular matrix (ECM). We hypothesized that, at least in part, these effects are mediated through [10]-gingerol interactions with αVβ3 integrin. Human MDA-MB-231 breast tumor cells were obtained from ATCC and maintained at 37°C, 5% CO2 in Dulbecco’s Modified Eagle Medium, containing FBS 10%). Ninety-six well plates were coated vitronectin (1µg/well) dissolved in adhesion buffer at 4ºC overnight, blocked for 2h with 1% BSA, and the wells washed with 100μL of adhesion buffer. MDA-MB-231 cells (5x105/mL) were pretreated for 30min with [10]-gingerol (1-100μM) in a humidified incubator with 5% CO2 at 37°C and 100µL of cell suspension plated into wells and incubated as above for a further 1 hour. Nonadherent cells were gently removed by washing and adherent cells were fixed with 100µl of 4% paraformaldehyde solution for 20min, stopped with PBS-Glycine, stained with DAPI, and counted in a HCS microscope. Data from docking were analyzed in AutoDock plugin for PyMOL and the two-dimensional scheme of interaction was obtained by LiPlot+.The electrochemical study of the interaction between [10]-gingerol and αVβ3 integrin was performed using disposable electrochemical cells (DCell), which are composed by a carbon working electrode, an Ag|AgCl pseudo-reference electrode, and a carbon counter electrode. Measurements were carried out employing the potentiostat DropSens 8000 and Dropview software 8400. The interaction of [10]-gingerol and αVβ3 integrin was evaluated by the changes in the [10]-gingerol electrochemical responses. For this, different amounts of standard αVβ3 integrin solution in PBS pH 7.0 were added and mixed homogeneously with a solution composed of 10µM [10]-gingerol. The differences of peak current were used to demonstrate the changes of electrochemical responses. We found that [10]-gingerol was able to inhibit TNBC cell adhesion to vitronectin ECM with more specificity compared to the other ECM components investigated, demonstrating that this compound interferes in this step of the metastatic process. We proposed that these effects could be, at least in part, resulting from the interaction between [10]-gingerol and αVβ3 integrin, as demonstrated by electrochemical and docking studies. Citation Format: Angelina Maria Fuzer, Ana Carolina B. M. Martin, Rebeka Tomasin, Carolina Venturini Uliana, Amanda Blanque Becceneri, Rafael L. Bressani Lino, James Almada da Silva, Paulo Cezar Vieira, Heloisa Sobreiro Selistre de Araújo, Rodolpho de Campos Braga, Ronaldo Censi Faria, Marcia Regina Cominetti. [10]-gingerol interferes with the adhesion of MDA-MB-231 tumor cells to extracellular matrix [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A53.

A number of known chalcone derivatives bearing different aromatic substituents is synthesized and tested for their biological activity.

Nephrotoxicity is the main complication of gentamicin (GM) treatment. GM induces renal damage by overproduction of reactive oxygen species and inflammation in proximal tubular cells. Phenolic compounds from ginger, called gingerols, have been demonstrated to have antioxidant and anti-inflammatory effects. We investigated if oral treatment with an enriched solution of gingerols (GF) would promote a nephroprotective effect in an animal nephropathy model. The following six groups of male Wistar rats were studied: (i) control group (CT group); (ii) gingerol solution control group (GF group); (iii) gentamicin treatment group (GM group), receiving 100 mg/kg of body weight intraperitoneally (i.p.); and (iv to vi) gentamicin groups also receiving GF, at doses of 6.25, 12.5, and 25 mg/kg, respectively (GM+GF groups). Animals from the GM group had a significant decrease in creatinine clearance and higher levels of urinary protein excretion. This was associated with markers of oxidative stress and nitric oxide production. Also, there were increases of the mRNA levels for proinflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-1β [IL-1β], IL-2, and gamma interferon [IFN-γ]). Histopathological findings of tubular degeneration and inflammatory cell infiltration reinforced GM-induced nephrotoxicity. All these alterations were attenuated by previous oral treatment with GF. Animals from the GM+GF groups showed amelioration in renal function parameters and reduced lipid peroxidation and nitrosative stress, in addition to an increment in the levels of glutathione (GSH) and superoxide dismutase (SOD) activity. Gingerols also promoted significant reductions in mRNA transcription for TNF-α, IL-2, and IFN-γ. These effects were dose dependent. These results demonstrate that GF promotes a nephroprotective effect on GM-mediated nephropathy by oxidative stress, inflammatory processes, and renal dysfunction.

This study describes an optimization of [6]-, [8]- and [10]-gingerol isolation and purification in semi-preparative HPLC scale and their anti-proliferative activity. The gingerols purification was carried out in HPLC system using a Luna-C18 and the best mobile phase evaluated was MeOH/H2O (75:25, v/v). This new methodology for the gingerols isolation was very effective, since considerable amounts (in the range of milligrams) with a good purity degree (∼98%) were achieved in 30 min of chromatographic run. [6]-, [8]- and [10]-Gingerol purified by this methodology inhibited the proliferation of MDA-MB-231 tumor cell line with IC50 of 666.2 ± 134.6 μM, 135.6 ± 22.6 μM and 12.1 ± 0.3 μM, respectively. These substances also inhibited human fibroblasts (HF) cell proliferation, however in concentrations starting from 500 μM. In conclusion, our results demonstrate an optimization of gingerols isolation and their specific anti-proliferative activities against tumor cells, suggesting their use as important models for drug design in an attempt to develop new compounds with fewer side effects when compared to conventional chemotherapy.

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.