Antimetastatic Agents Research Articles

Praeruptorin-B Inhibits 12-O-Tetradecanoylphorbol-13-Acetate-Induced Cell Invasion by Targeting AKT/NF-κB via Matrix Metalloproteinase-2/-9 Expression in Human Cervical Cancer Cells.

Praeruptorins, a seselin-type coumarin, possess anti-inflammatory and antitumor promoting properties. However, molecular mechanisms through which Praeruptorin-B (Pra-B) exerts an antimetastatic effect on cervical cancer cells remain unclear. Cell viability was examined using the MTT assay, whereas cell migration and invasion were examined using the Boyden chamber assay. Western blotting and RT-PCR were performed to investigate the inhibitory effect of Pra-B on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced matrix metalloproteinase-2/-9 (MMP-2/-9) expression in HeLa cells. The findings of the luciferase assay confirmed the inhibitory effect of Pra-B on TPA-induced transcriptional activity of MMP2/-9 in HeLa cells. Pra-B inhibited TPA-induced metastatic ability of human cervical cancer cells without any significant toxicity. Pra-B suppressed TPA-induced mRNA and protein expression and transcriptional activity of MMP-2/-9 in HeLa cells. Furthermore, Pra-B inhibited AKT phosphorylation but did not affect the MAPK pathway. Cotreatment of HeLa cells with TPA plus Pra-B or LY294002 (a PI3K inhibitor) reduced cell invasion and MMP-2/-9 expression and transcriptional activity. In addition, Pra-B attenuated TPA-induced nuclear translocation of NF-κB-p65/-p50, which reduced Ikk-α phosphorylation in HeLa cells. Cotreatment of HeLa cells with TPA plus Pra-B or LY294002 reduced NF-κB nuclear translocation. These results suggested that Pra-B-mediated inhibition of TPA-induced cell metastasis involved the suppression of p-AKT/NF-κB via MMP-2/-9 expression in HeLa cells. Pra-B can be a potential antimetastatic agent against cervical cancer.

Modification and Biological Evaluation of a Series of 1,5-Diaryl-1,2,4-triazole Compounds as Novel Agents against Pancreatic Cancer Metastasis through Targeting Myoferlin.

Pancreatic cancer is one of the most common cancers with an extremely low survival rate. Metastasis, as one of the key reasons of cancer-related death, is found in more than 50% pancreatic cancer patients at diagnosis. Novel therapeutic targets and drugs blocking cancer metastasis are urgently needed. Herein, we report a series of 1,5-diaryl-1,2,4-triazole derivatives as potent antimetastatic agents. Lead compound 6y displayed effective antimetastatic activities in pancreatic cancer in vitro and in vivo. Concomitant studies indicated that 6y probably binds with myoferlin (MYOF), a novel potential antitumor metastasis target, which regulates vesicle trafficking and metastasis-related proteins. Subsequent biophysical and biochemical methods verified that 6y bound to MYOF. Mechanism studies revealed that 6y inhibited pancreatic cancer metastasis through reversing the epithelial mesenchymal transition, inhibiting the secretions of matrix metalloproteinase and blocking the receptor tyrosine kinases. Our findings suggest that targeting MYOF with 6y may be a promising therapeutic strategy to prevent pancreatic cancer metastasis.

Discovery of a natural small-molecule compound that suppresses tumor EMT, stemness and metastasis by inhibiting TGF\u03b2/BMP signaling in triple-negative breast cancer

BackgroundThe transforming growth factor β (TGFβ) and bone morphogenetic protein (BMP) signaling pathways are both constitutively activated in triple-negative breast cancer (TNBC). We are interested in isolating the naturally-derived small-molecule inhibitor that could simultaneously targeting TGFβ/BMP pathways and further studying its anti-proliferative/−metastatic effects as well as the underlying mechanisms in multiple tumor models.MethodsMultiple in vitro cell-based assays are used to examine the compound’s inhibitory efficacy on TNBC cell growth, stemness, epithelial-mesenchymal transition (EMT), invasion and migration by targeting TGFβ/BMP signaling pathways. Transgenic breast cancer mouse model (MMTV-PyMT), subcutaneous xenograft and bone metastasis models are used to examine ZL170’s effects on TNBC growth and metastasis potentials in vivo.ResultsZL170 dose-dependently inhibits cell proliferation, EMT, stemness, invasion and migration in vitro via specifically targeting canonical TGFβ/BMP-SMADs pathways in TNBC cells. The compound significantly hinders osteolytic bone metastasis and xenograft tumor growth without inflicting toxicity on vital organs of tumor-bearing nude mice. ZL170 strongly inhibits primary tumor growth and lung metastases in MMTV-PyMT transgenic mice. ZL170-treated tumors exhibit impaired TGFβ/BMP signaling pathways in both epithelial and stromal compartments, thereby creating a suppressive tumor microenvironment characterized by reduced extracellular matrix deposition and decreased infiltration of stromal cells.ConclusionsZL170 inhibits tumor EMT, stemness and metastasis and could be further developed as a potent anti-metastatic agent used in combination with cytotoxic drugs for treatment of TNBC and other advanced metastatic cancers.

Anti-metastatic effect of ranolazine in an in vivo rat model of prostate cancer, and expression of voltage-gated sodium channel protein in human prostate.

Voltage-gated Na+ channels (VGSCs) are functionally upregulated in rat and human prostate cancer (PCa) where channel activity promotes cellular invasiveness in vitro and metastasis in vivo. Ranolazine is a clinically used VGSC inhibitor/anti-anginal drug, which has been shown previously to inhibit breast cancer metastasis in vivo. Using the Dunning model of rat PCa, the effect of ranolazine applied systemically (by gavage) was tested on the development of primary tumours and metastases following subcutaneous inoculation of Mat-LyLu cells into Copenhagen rats. In addition, human prostate tissue microarrays were used to determine VGSC protein expression in cancerous versus non-cancerous tissue. Several public databases were searched to compare Nav1.7/ SCN9A expression levels in 'normal' vs. PCa tissues. Ranolazine (2.5 and 5 µM) decreased the number of lung metastases by up to 63%. In contrast, primary tumourigenesis was not affected. Ranolazine also reduced the percentage of cells in the metastases expressing Nav1.7, the main VGSC subtype expressed in PCa, but the expression level was higher. In prostate tissue microarrays, VGSC protein expression was significantly higher in cancerous versus non-cancerous tissue. There was no correlation between the VGSC expression and either prostate-specific antigen or Gleason score. In public databases, little information could be found on Nav1.7 protein expression in PCa. In addition, the database information on Nav1.7 mRNA (SCN9A) expression levels did not correlate with previously reported upregulation in PCa cells and tissues. The main conclusions were (i) ranolazine inhibited metastasis and (ii) it was a subpopulation of cells with particularly high levels of Nav1.7 protein that reached the metastatic sites. These data extend earlier studies and suggest that Nav1.7 expression could serve as a functional biomarker of metastatic PCa and that VGSC blockers may be useful as anti-metastatic agents.

Ampelopsins A and C Induce Apoptosis and Metastasis through Downregulating AxL, TYRO3, and FYN Expressions in MDA-MB-231 Breast Cancer Cells.

Ampelopsins A and C are resveratrol oligostilbenes whose role in cancer development remains unknown. This study evaluated the antimetastatic and apoptosis-inducing properties of ampelopsins A and C in MDA-MB-231 cells. The IC50 values of ampelopsins A and C against MDA-MB-231 cells at 72 h were 38.75 ± 4.61 and 2.71 ± 0.21 μM, respectively. However, at 24 h, ampelopsins A and C decreased cell metastasis significantly. Among the 71 proteins present on the human phosphoreceptor tyrosin kinase array, ampelopsin C decreased the phosphorylated protein level of AXL, Dtk (TYRO3), EphA2, EphA6, Fyn, Hck, and SRMS. Additionally, antiproliferation effects of ampelopsin C were enhanced when combined with luteolin and chrysin compared to either two or a single agent in MDA-MB-231 cells. Overall, ampelopsins A and C extracted from Vitis thunbergii are both novel antimetastatic agents and potential therapeutic targets in patients with breast cancer.

Yifei Tongluo, a Chinese Herbal Formula, Suppresses Tumor Growth and Metastasis and Exerts Immunomodulatory Effect in Lewis Lung Carcinoma Mice.

This study was aimed to investigate the anti-tumor, anti-metastasis and immunomodulatory effects of Yifei Tongluo (YFTL), a Chinese herbal formula, in Lewis lung carcinoma mice and to explore the underlying mechanisms. LLC cells were inoculated subcutaneously in C57BL/6 mice to establish the Lewis lung carcinoma model. We observed that YFTL effectively inhibited tumor growth and prolonged the overall survival of tumor-bearing mice. Additionally, YFTL treatment resulted in a significantly decreased number of surface lung metastatic lesions compared with the model control group. Meanwhile, TUNEL staining confirmed that the tumors from YFTL-treated mice exhibited a markedly higher apoptotic index. The results suggest that Akt and mitogen-activated protein kinase (MAPKs) pathways may be involved in YFTL-induced apoptosis. The results show that YFTL also inhibited the vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMP)-2, MMP-9, N-cadherin, and Vimentin expression, but increased E-cadherin expression. Mechanistic studies indicated that YFTL could suppress the angiogenesis and the epithelial-mesenchymal transition (EMT) of the tumor through Akt/ERK1/2 and TGFβ1/Smad2 pathways. In addition, YFTL also showed immunomodulatory activities in improving the immunosuppressive state of tumor-bearing mice. Therefore, our findings could support the development of YFTL as a potential antineoplastic agent and a potentially useful anti-metastatic agent for lung carcinoma therapy.

A polysaccharide from Hedyotis diffusa interrupts metastatic potential of lung adenocarcinoma A549 cells by inhibiting EMT via EGFR/Akt/ERK signaling pathways.

In this study we investigated the potential effects of a polysaccharide (HDP) from Hedyotis diffusa on the metastasis in human lung adenocarcinoma A549 cells. HDP (25, 50 and 100μg/ml) significantly suppressed the cell adhesion, invasion and migration of A549 cells in a dose dependent manner by downregulation of matrix metalloproteinase (MMP-2 and MMP-9) and upregulation of tissue inhibitors of metalloproteinase (TIMP-2 and TIMP-9). Moreover, HDP effectively downregulated the protein expressions of epithelial-mesenchymal transition (EMT) markers (N-cadherin and vimentin), and upregulated E-cadherin protein expression, which is involved in interrupting EGFR/Akt/ERK signaling pathways, as well as inhibiting COX-2 protein expression. All these results demonstrated that HDP might be a novel anti-metastatic agent for NSCLC treatment.

Actinomycetes as a Paramount Source of Biologically Important Enzyme Inhibitors – “A Boon to Mankind”

Background:Bioactive compounds from microorganisms have been widely studied for several biological, therapeutic and pharmaceutical importances. Bacterial secondary metabolites have proven their worth as a prolific source of antibiotics, antifungal, antiviral, anticholesterol and immunosuppressant. The majority of inhibitors are secondary metabolites of varying chemical moieties produced by microorganisms among which actinomycetes are most important due to their tremendous diversity. Actinomycetes are most economically and commercially important prokaryotes known for their metabolic versatility. They have gained attention due to their ability to produce novel bioactive compounds with many applications. This review provides an overview on well-established actinobacterial bioactive compounds used as enzyme inhibitors for the treatment and management of diseases and their future perspectives.Objective:We focused on actinobacterial bioactive compounds which were reported to possess enzyme inhibition activity. An extensive search on well-acknowledged enzyme inhibitors was done by referring to peer-reviewed research papers. The papers were screened on the basis of the significance of research work done.Results:The research papers referred in this review article suggest the potential of bioactive compounds as therapeutically important enzyme inhibitors. The actinobacterial compounds were found to possess enzyme inhibition potential and could be developed into an antibacterial, antifungal, antimetastatic, antidiabetic and antihypertensive agent. These inhibitors were structurally elucidated and belonged to the class of peptides, proteins and pseudotrisaccharides.Conclusion:The findings of this review paper highlight the enormous potential of actinomycetes and bioactive compounds as enzyme inhibitors of therapeutic and pharmaceutical importance.

Benproperine, an ARPC2 inhibitor, suppresses cancer cell migration and tumor metastasis

Metastasis is the leading cause of cancer mortality and cancer cell migration is an essential stage of metastasis. We identified benproperine (Benp, a clinically used antitussive drug) as an inhibitor of cancer cell migration and an anti-metastatic agent. Benp selectively inhibited cancer cell migration and invasion, which also suppressed metastasis of cancer cells in animal models. Actin-related protein 2/3 complex subunit 2 (ARPC2) was identified as a molecular target of Benp by affinity column chromatography with Benp-tagged Sepharose beads. Benp bound directly to ARPC2 in cells, which was validated by pull-down assay using Benp-biotin and label-free biochemical methods such as the drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA). Benp inhibited Arp2/3 function, showing disruption of lamellipodial structure and inhibition of actin polymerization. Unlike Arp2/3 inhibitors, Benp selectively inhibited the migration of cancer cells but not normal cells. ARPC2-knockdown cancer cells showed defective cell migration and suppressed metastasis in an animal model. Therefore, ARPC2 is a potential target for anti-metastatic therapy, and Benp has the clinical potential to block metastasis. Furthermore, Benp is a useful agent for studying the functions of the Arp2/3 complex in cancer cell migration and metastasis.

Structure-based design, synthesis and biological evaluation of a novel series of isoquinolone and pyrazolo[4,3-c]pyridine inhibitors of fascin 1 as potential anti-metastatic agents

Fascin is an actin binding and bundling protein that is not expressed in normal epithelial tissues but overexpressed in a variety of invasive epithelial tumors. It has a critical role in cancer cell metastasis by promoting cell migration and invasion. Here we report the crystal structures of fascin in complex with a series of novel and potent inhibitors. Structure-based elaboration of these compounds enabled the development of a series with nanomolar affinities for fascin, good physicochemical properties and the ability to inhibit fascin-mediated bundling of filamentous actin. These compounds provide promising starting points for fascin-targeted anti-metastatic therapies.

Adenylate kinase 4 modulates oxidative stress and stabilizes HIF-1\u03b1 to drive lung adenocarcinoma metastasis

BackgroundAdenylate kinase 4 (AK4) has been identified as a biomarker of metastasis in lung cancer. However, the impacts of AK4 on metabolic genes and its translational value for drug repositioning remain unclear.MethodsIngenuity upstream analyses were used to identify potential transcription factors that regulate the AK4 metabolic gene signature. The expression of AK4 and its upstream regulators in lung cancer patients was examined via immunohistochemistry. Pharmacological and gene knockdown/overexpression approaches were used to investigate the interplay between AK4 and its upstream regulators during epithelial-to-mesenchymal transition (EMT). Drug candidates that reversed AK4-induced gene expression were identified by querying a connectivity map. Orthotopic xenograft mouse models were established to evaluate the therapeutic efficacy of drug candidates for metastatic lung cancer.ResultsWe found that HIF-1α is activated in the AK4 metabolic gene signature. IHC analysis confirmed this positive correlation, and the combination of both predicts worse survival in lung cancer patients. Overexpression of AK4 exaggerates HIF-1α protein expression by increasing intracellular ROS levels and subsequently induces EMT under hypoxia. Attenuation of ROS production with N-acetylcysteine abolishes AK4-induced invasion potential under hypoxia. Pharmacogenomics analysis of the AK4 gene signature revealed that withaferin-A could suppress the AK4-HIF-1α signaling axis and serve as a potent anti-metastatic agent in lung cancer.ConclusionsOverexpression of AK4 promotes lung cancer metastasis by enhancing HIF-1α stability and EMT under hypoxia. Reversing the AK4 gene signature with withaferin-A may serve as a novel therapeutic strategy to treat metastatic lung cancer.

Novel Hybrid Compound 4-[(E)-2-phenylethenesulfonamido]-N-hydroxybutanamide with Antimetastatic and Cytotoxic Action: Synthesis and Anticancer Screening.

One of the most promising strategies to develop multi-targeted anticancer therapeutics is to introduce to the structure of a potential drug two or more pharmacophores (functional groups or structural fragments), which have antiproliferative, proapoptotic or antimetastatic properties acting via different mechanisms. To design, synthesize and perform screening of a novel hybrid anticancer compound. A novel hybrid compound 4-[(E)-2-phenylethenesulfonamido]-N-hydroxybutanamide, combining butanehydroxamate and styrenesulfonamide moieties, was designed, synthesized and investigated as a potent antimetastatic and antiproliferative agent. The structure and purity of the synthesized compound were confirmed by 1H NMR, 13C NMR, LC/MS spectroscopy and elemental analysis. The compound was screened for the anticancer activity in vitro against HeLa and in vivo against Lewis lung carcinoma tumor, using an antitumor metalloenzyme inhibitor GM6001 (Ilomastat, Galardin) and Pifithrin-μ as control anticancer agents. It was found that the application of our compound resulted in a high fraction of apoptotic cells in the cell population, along with disruption in the cell cycle profile manifested as arrest of proliferative phases. Furthermore, changes of the morphological properties (i.e., an enhancement of adhesive properties and reduction of the nuclear-to-cytoplasm ratio) were found. The in vivo screening revealed that the compound significantly inhibited the metastasizing process that was manifested by a reduction in the number and volume of metastases. The obtained results demonstrate that our compound can serve as a base for further structure optimization in order to design new highly-effective antimetastatic and antitumor agents.

Ethanol Extracts of Dietary Herb, Alpinia nantoensis, Exhibit Anticancer Potential in Human Breast Cancer Cells.

Recent advances in mammography screening, chemotherapy, and adjuvant treatment modalities have improved the survival rate of women with breast cancer. Nevertheless, the breast tumor with metastatic progression is still life-threatening. Indeed, combination therapy with Ras-ERK and PI3K inhibitors is clinically effective in malignant breast cancer treatment. Constituents from genus Alpinia plants have been implicated as potent anticancer agents in terms of their efficacy of inhibiting tumor cell metastasis. In this study, we tested the effects of ethanol extracts of Alpinia nantoensis (rhizome, stem, and leaf extracts) in cultured human breast cancer cells and particularly focused on the Ras-ERK and PI3K/AKT pathways. We found that the rhizome and leaf extracts from A nantoensis inhibited cell migration, invasion, and sphere formation in MCF-7 and MDA-MB-231 cells. The potency was extended with the inhibition of serum-induced PI3K/AKT and Ras-ERK activation and epidermal growth factor (EGF)-mediated EGFR activation in MDA-MB-231 cells. These results indicate that extracts of A nantoensis could inhibit signal transduction at least involved in EGFR as well as the PI3K/AKT and Ras-ERK pathways, which are crucial players of tumor cell migration and invasion. Our study strongly supports that the extracts of A nantoensis could be a novel botanical drug lead for the development of an antimetastatic agent for the treatment of human malignant breast cancer.

Bee Venom Suppresses EGF-Induced Epithelial-Mesenchymal Transition and Tumor Invasion in Lung Cancer Cells.

Bee venom of Apis mellifera is a traditional medicine in Asia. It has been used with promoting results for the treatment of pain, rheumatoid, and cancer disease. The purpose of this study was to investigate the effects of bee venom on epidermal growth factor (EGF)-induced epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC) and determine possible signaling pathway affected in EGF-induced EMT in A549 cells. Bee venom inhibited EGF-induced F-actin reorganization and cell invasion, and suppressed EGF-induced EMT, processes associated with tumor metastasis in NSCLC. Bee venom enhanced the upregulation of E-cadherin and the downregulation of vimentin and inhibited EGF-induced ERK, JNK, FAK, and mTOR phosphorylation in A549 cells. However, the inhibition of JNK phosphorylation by bee venom was not related to the inhibitory effects of EMT. Furthermore, we found that bee venom suppressed the EMT-related transcription factors ZEB2 and Slug by blocking EGF-induced ERK, FAK and mTOR phosphorylation. Bee venom inhibits EGF-induced EMT by blocking the phosphorylation of ERK, FAK, and mTOR, resulting in the suppression of ZEB2 and Slug. These data suggest bee venom as a potential antimetastatic agent for NSCLC.

Anti-metastatic effect of curcumin analog pentagamaboronon-0-fructose (PGB-0-F) against 4T1 breast cancer cells

Development of a chemotherapeutic agent and boron carrying pharmaceutical based on triple-negative breast cancer is important due to its metastatic progression. Metastases are often more dangerous than the primary tumor and they are responsible for 90% of all cancer deaths. The purpose of this study was to explore the anti-metastatic activities of the PGB-0 complex with fructose (PGB-0-F) against 4T1 breast cancer cells. A scratch wound healing assay was carried out to determine the migration inhibition ability of PGB-0-F, while MMP-9 expression was analysed using gelatin zymography. The testing of anti-migration activity showed that PGB-0-F inhibited in 4T1 cells, whereas the gelatin zymography assay revealed a suppression of MMP-9 expression. PGB-0-F inhibited closure on 4T1 metastatic breast cancer cells line compared with the control. PGB-0-F decreased the MMP-9 expression level compared with the control. Based on these results, PGB-0-F has the potential to be developed as a chemotherapeutic agent, and especially as an anti-metastatic agent.

Shedding Light on the Hydrolysis Mechanism of cis, trans-[Ru(dmso)4Cl2] Complexes and Their Interactions with DNA-A Computational Perspective.

Using several computational tools such as density functional theory analysis, docking, and MD simulations, we performed a study on cis, trans-[Ru(II)(dmso)4Cl2] complexes, which have therapeutic potential as antimetastatic agents, and their association with DNA. Kohn-Sham energy decomposition analysis reveals that dmso ligands have much smaller interaction energies compared to the chlorido ligands, and their substitution by aquo ligands induces an extra stabilization of the other metal-ligand bonds. Once the complex is hydrolyzed, the aquo ligands have the weakest interactions to the metallic center and therefore are more labile for substitution by a DNA atom. Molecular docking and molecular dynamics were employed to understand the complex preassociation to DNA, pointing to a higher affinity of the hydrolyzed complexes, as well as showing spontaneous binding events during the simulations. Our results are consistent with the experimentally available data that suggest a mechanism in which the complexes are quickly hydrolyzed in solution, before forming cross-links with the DNA molecule. We present a set of methods that could be used to optimize these complexes computationally, aiding in the development of new drugs based on transition metals.

Actein Inhibits the Proliferation and Adhesion of Human Breast Cancer Cells and Suppresses Migration in vivo.

Background and purpose: Metastasis is an important cause of death in breast cancer patients. Anti-metastatic agents are urgently needed since standard chemotherapeutics cannot diminish the metastatic rate. Actein, a cycloartane triterpenoid, has been demonstrated to exhibit anti-angiogenic and anti-cancer activities. Its anti-metastatic activity and underlying mechanisms were evaluated in the present study.Methods: The effects of actein on the proliferation, cell cycle phase distribution, migration, motility and adhesion were evaluated using two human breast cancer cell lines, MDA-MB-231 (estrogen receptor-negative) and MCF-7 cells (estrogen receptor-positive) in vitro. Western blots and real-time PCR were employed to examine the protein and mRNA expression of relevant signaling pathways. A human metastatic breast cancer cell xenograft model was established in transparent zebrafish embryos to examine the anti-migration effect of actein in vivo.Results: In vitro results showed that actein treatment significantly decreased cell proliferation, migration and motility. Furthermore, actein significantly caused G1 phase cell cycle arrest and suppressed the protein expression of matrix metalloproteinases of MDA-MB-231 cells. In addition, actein inhibited breast cancer cell adhesion to collagen, also reduced the expression of integrins. Actein treatment down-regulated the protein expression of epidermal growth factor receptor (EGFR), AKT and NF-κB signaling proteins. In vivo results demonstrated that actein (60 μM) significantly decreased the number of zebrafish embryos with migrated cells by 74% and reduced the number of migrated cells in embryos.Conclusion: Actein exhibited anti-proliferative, anti-adhesion and anti-migration activities, with the underlying mechanisms involved the EGFR/AKT and NF-kappaB signalings. These findings shed light for the development of actein as novel anti-migration natural compound for advanced breast cancer.

Anti-metastatic and anti-proliferative activity of eugenol against triple negative and HER2 positive breast cancer cells

BackgroundEugenol is a natural phenolic compound and possesses anticancer and antibacterial activities. Breast cancer is a major global health problem, and most of the chemotherapeutic agents are highly toxic with long-term side effects. Therefore, this study aimed to explore the possibility of using eugenol as an anti-metastatic and anti-proliferative agent against MDA-MB-231 and SK-BR-3 breast cancer cells.MethodsBreast cancer cell lines MDA-MB-231 and SK-BR-3 were treated with eugenol and cell proliferation was measured using a real-time cell electronic sensing system. Annexin V analysis with flow cytometry was used to detect the effect of eugenol on cell death. In MDA-MB-231 and SK-BR-3 cells, metastatic potential after eugenol treatment was examined using a wound-healing assay. Real-time PCR was used to study the effect of eugenol on the expression of anti-metastatic genes such as MMP2, MMP9, and TIMP-1, and genes involved in apoptosis including Caspase3, Caspase7, and Caspase9.ResultsTreatment with 4 μM and 8 μM eugenol for 48 h significantly inhibited cell proliferation of MDA-MB-231, with an inhibition rate of 76.4%, whereas 5 μM and 10 μM of eugenol for 48 h significantly inhibited the proliferation of SK-BR-3 cells with an inhibition rate of 68.1%. Eugenol-treated cells showed significantly decreased MMP2 and MMP9 expression and an insignificant increase in TIMP1 expression in HER2 positive and triple negative breast cancer cells. Eugenol significantly increased the proportion of MDA-MB-231 and SK-BR-3 cells in late apoptosis and increased the expression of Caspase3, Caspase7, and Caspase9.ConclusionTo the best of our knowledge, this is the first study to describe the anti-metastatic effect of eugenol against MDA-MB-231 and SK-BR-3 breast cancer cell lines.

Design and synthesis of a native heparin disaccharide grafted poly‑2‑aminoethyl methacrylate glycopolymer for inhibition of melanoma cell metastasis

Numerous studies have proved that heparin, a sub-group of glycosaminoglycan, possesses great potential as anti-metastasis agent. However, the native strong anti-coagulant activity which causes serious side effects, such as bleeding, has limited its clinical applications for safety concern. To overcome this problem, we synthesized a panel of novel glycopolymers that mimic heparin structure with substantially reduced anti-coagulant activity by a simple grafted-on strategy. The influence of molecular weight & distribution, substituting degree and sulfonic density on cytotoxicity were determined by systematic MTT analysis to select the candidates with highly bio-compatibility. Among these glycopolymers, a sulfated poly‑2‑aminoethyl methacrylate grafted with heparin disaccharide (abbreviated as SGPHD) has shown potent efficacy in inhibition of heparanase activity and microvascular endothelial cell proliferation in vitro. Further experiments demonstrated that SGPHD inhibited B16 murine melanoma cell migration, invasion and adhesion to platelets or microvascular endothelial cells, thus, presented as a possible anti-metastatic agent by providing a whole course protection during tumor metastasis. The results will have significant impacts for the further rational design of glycopolymer medicine.

Blocking Dimeric Pyruvate Kinase M2 Transformation and Nuclear Translocation Leading to Suppression of Aerobic Glycolysis Mediates Beta-Elemene Inhibition of Breast Cancer Metastasis

Pyruvate kinase M2 (PKM2) plays a central role in regulating aerobic glycolysis and is considered as a potential target for cancer therapy. However, its role in cancer metastasis is rarely known. Here, we found a tight relationship between PKM2 and breast cancer metastasis, which is demonstrated by the finding that beta-elemene (β-elemene), an approved drug for complementary cancer therapy, exerted distinct anti-metastatic activity dependent on PKM2. The results indicated thatβ-elemene inhibited breast cancer cell migration, invasion and angiogenesis. β-elemene also inhibited the process of glycolysis, and decreased the utilization of glucose and the production of pyruvate and lactate through suppressing pyruvate kinase activity by modulating the transformation of dimeric and tetrameric forms of PKM2, which was reversed in part by fructose-1,6-bisphosphate (FBP) or L-cysteine. Our further analysis revealed that β-elemene suppressed aerobic glycolysis through inhibiting the nuclear transportation of PKM2 and the expression of GLUT1, MCT1, MCT4 and LDHA by influencing the expression of importin α5. Additionally, β-elemene remarkably inhibited the breast cancer metastases in lung and liver and selectively influenced the glycolysis of the lung and liver metastatic foci in mouse models. Taken together, tetrameric transformation and nuclear translocation of PKM2 are essential for cancer metastasis, and β-elemene inhibits breast cancer metastasis via blocking aerobic glycolysis induced by dimeric PKM2 transformation and EGFR-importin α5 mediated PKM2 nuclear translocation, being a promising anti-metastatic agent. Funding Statement: The work was supported by National Natural Science Foundation of China (No. 81673648, 81673725, 81573859, 81603339), Natural Science Foundation of Higher School of Jiangsu Province (17KJA360003), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: All animal studies were approved by the Animal Care and Use Committee of Nanjing University of Chinese Medicine. All mouse experimental procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of People’s Republic of China.