The iron-regulated metastasis suppressor, Ndrg-1: identification of novel molecular targets

A recently identified metastasis suppressor, N-myc downstream regulated gene-1 (Ndrg-1), has been shown to reduce the invasion and metastasis of breast, colon, prostate and pancreatic cancer. Among its many functions, Ndrg-1 is involved in modulating differentiation, proliferation and angiogenesis. However, knowledge of the molecular targets of Ndrg-1 is limited. The current study has focused on examining the functions of Ndrg-1 in a number of different cancer cell models including prostate, colon, lung and pancreatic cancer to elucidate the known pleiotropic nature of this protein. Furthermore, the potential gene targets of Ndrg-1 were analyzed using whole genome gene array revealing a substantial number of genes whose expression was affected by this metastasis suppressor. Significantly, Ndrg-1 up-regulated thiamine triphosphatase (Thtpa) expression in three of the four cell models. Thtpa is known to decrease the levels of the energy currency molecule, thiamine triphosphate, suggesting a potential pathway for the anti-proliferative effects of Ndrg-1. Furthermore, Ndrg-1 reduced the protein levels of cathepsin C which plays a role in invasion, indicating a potential mechanism of its anti-metastatic role in pancreatic cancer cells. These findings provide a potential link between the observed functions of Ndrg-1 and its molecular targets, further demonstrating its anti-metastatic effect.     

Polyphenolics from peach (Prunus persica var. Rich Lady) inhibit tumor growth and metastasis of MDA-MB-435 breast cancer cells in vivo

The tumor growth inhibition and anti-metastatic effects of peach polyphenolics were investigated in vivo using a xenograft model and MDA-MB-435 breast cancer cells. Results showed that tumor growth and lung metastasis were inhibited in vivo by peach polyphenolics in a dose range of 0.8–1.6 mg/day, and these effects were mediated by inhibition of metalloproteinases gene expression. Modulation of metalloproteinase-2, metalloproteinase-3 and metalloproteinase-13 gene expression may be some of the molecular targets for anti-metastatic activity of peach polyphenolics. Therefore, these compounds may constitute a novel chemopreventive tool to reduce the risk of metastasis in the combination therapy when primary cancer is diagnosed. Conversion to equivalent human intake for future clinical studies using the body surface area normalization method gave a dose of ~370.6 mg/day for a human adult of 60 kg, which can be supplied by consuming two to three peach fruit per day or alternatively using a dietary supplement peach polyphenol extract powder.     

Cancer spread and micrometastasis development: quantitative approaches for in vivo models

Death from cancer is usually due to metastasis. Fortunately, most cells that escape from a primary tumor fail to form metastases. Identifying reasons for this failure will help development of anti-metastatic therapies. Intravital videomicroscopy (IVVM) can be used to observe cancer cells injected into live animals. Co-injected microspheres can be used to assess cell survival. These techniques have been used to show that circulating tumor cells generally arrest in the microcirculation and may extravasate with high efficiency. While many tumor cells may survive in a secondary site, only a small subset form micrometastases and only a subset of these micrometastases persist to form vascularized macrometastases. Furthermore, solitary tumor cells may remain dormant for long periods of time in secondary sites. These findings suggest that metastatic growth and angiogenesis are prime targets for anti-metastatic therapy.     

Aiduqing formula suppresses breast cancer metastasis via inhibiting CXCL1-mediated autophagy

BackgroundMetastasis is the most common lethal cause of breast cancer-related death. Recent studies have implied that autophagy is closely implicated in cancer metastasis. Therefore, it is of great significance to explore autophagy-related molecular targets involved in breast cancer metastasis and to develop therapeutic drugs.PurposeThis study was designed to investigate the anti-metastatic effects and autophagy regulatory mechanisms of Aiduqing (ADQ) formula on breast cancer.Study Design/MethodsMultiple cellular and molecular experiments were conducted to investigate the inhibitory effects of ADQ formula on autophagy and metastasis of breast cancer cells in vitro. Meanwhile, autophagic activator/inhibitor as well as CXCL1 overexpression or interference plasmids were used to investigate the underlying mechanisms of ADQ formula in modulating autophagy-mediated metastasis. Furthermore, the zebrafish xenotransplantation model and mouse xenografts were applied to validate the inhibitory effect of ADQ formula on autophagy-mediated metastasis in breast cancer in vivo.ResultsADQ formula significantly inhibited the proliferation, migration, invasion and autophagy but induced apoptosis of high-metastatic breast cancer cells in vitro. Similar results were also observed in starvation-induced breast cancer cells which exhibited elevated metastatic ability and autophagy activity. Mechanism investigations further approved that either CXCL1 overexpression or autophagic activator rapamycin can significantly abrogated the anti-metastatic effects of ADQ formula, suggesting that CXCL1-mediated autophagy may be the crucial pathway of ADQ formula in suppressing breast cancer metastasis. More importantly, ADQ formula suppressed breast cancer growth, autophagy, and metastasis in both the zebrafish xenotransplantation model and the mouse xenografts.ConclusionOur study not only revealed the novel function of CXCL1 in mediating autophagy-mediated metastasis but also suggested ADQ formula as a candidate drug for the treatment of metastatic breast cancer.     

Baicalein Inhibits Progression of Gallbladder Cancer Cells by Downregulating ZFX

Baicalein, a widely used Chinese herbal medicine, has multiple pharmacological activities. However, the precise mechanisms of the anti-proliferation and anti-metastatic effects of baicalein on gallbladder cancer (GBC) remain poorly understood. Therefore, the aim of this study was to assess the anti-proliferation and anti-metastatic effects of baicalein and the related mechanism(s) on GBC. In the present study, we found that treatment with baicalein induced a significant inhibitory effect on proliferation and promoted apoptosis in GBC-SD and SGC996 cells, two widely used gallbladder cancer cell lines. Additionally, treatment with baicalein inhibited the metastasis of GBC cells. Moreover, we demonstrated for the first time that baicalein inhibited GBC cell growth and metastasis via down-regulation of the expression level of Zinc finger protein X-linked (ZFX). In conclusion, our studies suggest that baicalein may be a potential phytochemical flavonoid for therapeutics of GBC and ZFX may serve as a molecular marker or predictive target for GBC.     

Baohuoside i suppresses breast cancer metastasis by downregulating the tumor-associated macrophages/C-X-C motif chemokine ligand 1 pathway

BackgroundBreast cancer is the most common malignancy in women and metastasis is the leading cause of breast cancer-related deaths. Our previous studies have shown that XIAOPI formula, a newly approved drug by the State Food and Drug Administration of China (SFDA), can dramatically inhibit breast cancer metastasis by modulating the tumor-associated macrophages/C-X-C motif chemokine ligand 1 (TAMs/CXCL1) pathway. However, the bioactive compound accounting for the anti-metastatic effect of XIAOPI formula remains unclear.PurposeThis study was designed to separate the anti-metastatic bioactive compound from XIAOPI formula and to elucidate its action mechanisms.Study Design/MethodsTAMs/CXCL1 promoter activity-guided fractionation and multiple chemical structure identification approaches were conducted to screen the bioactive compound from XIAOPI formula. Breast cancer cells and TAMs were co-cultured in vitro or co-injected in vivo to simulate their coexistence. Multiple molecular biology experiments, zebrafish breast cancer xenotransplantation model and mouse breast cancer xenografts were applied to validate the anti-metastatic activity of the screened compound.ResultsBioactivity-guided fractionation identified baohuoside I (BHS) as the key bioactive compound of XIAOPI formula in inhibiting TAMs/CXCL1 promoter activity. Functional studies revealed that BHS could significantly inhibit the migration and invasion as well as the expression of metastasis-related proteins in both human and mouse breast cancer cells, along with decreasing the proportion of breast cancer stem cells (CSCs). Furthermore, BHS could suppress the M2 phenotype polarization of TAMs and therefore attenuate their CXCL1 expression and secretion. Notably, mechanistic investigations validated TAMs/CXCL1 as the crucial target of BHS in suppressing breast cancer metastasis as exogenous addition of CXCL1 significantly abrogated the anti-metastatic effect of BHS on breast cancer cells. Moreover, BHS was highly safe in vivo as it exhibited no observable embryotoxicity or teratogenic effect on zebrafish embryos. More importantly, BHS remarkably suppressed breast cancer metastasis and TAMs/CXCL1 activity in both zebrafish breast cancer xenotransplantation model and mouse breast cancer xenografts.ConclusionThis study not only provides novel insights into TAMs/CXCL1 as a reliable screening target for anti-metastatic drug discovery, but also suggests BHS as a promising candidate drug for metastatic breast cancer treatment.     

Rho kinase is required for CCR7-mediated polarization and chemotaxis of T lymphocytes

A chemokine receptor, CXCR4, and its endogenous ligand, stromal cell-derived factor-1 (SDF-1), have been recognized to be involved in the metastasis of several types of cancers. T140 analogs are peptidic CXCR4 antagonists composed of 14 amino acid residues that were previously developed as anti-HIV agents having inhibitory activity against HIV-entry through its co-receptor, CXCR4. Herein, we report that these compounds effectively inhibited SDF-1-induced migration of human breast cancer cells (MDA-MB-231), human leukemia T cells (Sup-T1) and human umbilical vein endothelial cells at concentrations of 10–100 nM in vitro. Furthermore, slow release administration by subcutaneous injection using an Alzet osmotic pump of a potent and bio-stable T140 analog, 4F-benzoyl-TN14003, gave a partial, but statistically significant (P≤0.05 (t-test)) reduction in pulmonary metastasis of MDA-MB-231 in SCID mice, even though no attempt was made to inhibit other important targets such as CCR7. These results suggest that T140 analogs have potential use for cancer therapy, and that small molecular CXCR4 antagonists could potentially replace neutralizing antibodies as anti-metastatic agents for breast cancer.     

β-Carotene inhibits neuroblastoma cell invasion and metastasis in vitro and in vivo by decreasing level of hypoxia-inducible factor-1α

Neuroblastoma is the most prevalent extracranial solid tumor in childhood and has poor clinical outcome due to its high potential for metastasis. Consequently, an understanding of the mechanisms that modulate cancer cell invasion, migration and metastasis is important for the development of more effective chemotherapeutic agents. While β-carotene is a vitamin A precursor that has been shown to exert antioxidant and anticancer effects, the anti-metastatic effects of β-carotene on neuroblastoma cells remain poorly understood. The aim of the present study was to investigate the anti-metastatic effects of β-carotene on highly malignant SK-N-BE(2)C neuroblastoma cells in vitro and in vivo. Treatment of SK-N-BE(2)C cells with β-carotene was found to attenuate the migratory and invasive capabilities of the cells. In addition, the enzymatic activity and expression of matrix metalloproteinase (MMP)-2 was suppressed following β-carotene treatment under both normoxia and hypoxia. To induce metastasis, immunodeficient nude mice were injected with SK-N-BE(2)C cells via the tail vein in vivo. The incidence of liver metastasis and mean tumor volume in mice that were administered β-carotene was decreased compared to controls. Furthermore, mRNA levels of MMPs, membrane-type (MT) 2 MMP and tissue inhibitors of metalloproteinases in liver tumor tissues were also lower following β-carotene treatment. Level of hypoxia-inducible factor-1α (HIF-1α) and its downstream targets, vascular endothelial growth factor and glucose transporter 1 (GLUT1), were lower both in vitro and in vivo following β-carotene treatment. In conclusion, the present study provides the first evidence that β-carotene may represent an effective chemotherapeutic agent by regulating the invasion and metastasis of neuroblastoma via HIF-1α.     

Reconstitution of a metastatic-resistant tumor microenvironment with cancer-associated fibroblasts enables metastasis

The tumor microenvironment is critical for metastasis to occur. Subcutaneous xenografts of tumors in immunodeficient mice are usually encapsulated and rarely metastasize as opposed to orthotopic tumors which metastasize if the original tumor was metastatic. In the present report, we were able to reconstitute a metastatic tumor microenvironment by subcutaneously co-transplanting a human cervical cancer cell line and human cervical cancer-associated fibroblasts (CAFs), in athymic mice, which resulted in lymph node metastasis in 40% of the animals. In contrast, no metastasis occurred from the cervical cancer without CAFs. These results suggest that CAFs can overcome an anti-metastatic tumor environment and are a potential target to prevent metastasis.     

A highly polymorphic CA/GT repeat in intron 3 of the human urokinase receptor gene (PLAUR)

We describe the first highly polymorphic microsatellite marker for the human urokinase plasminogen activator receptor gene (PLAUR). The urokinase receptor (uPAR) has a central role in cancer invasion and metastasis, which may enable the development of new anti-metastatic therapies. Analysis of the marker genotypes in colorectal cancer cell lines revealed three alleles that were not detected in a series of healthy control individuals, which encourages further genetic study of the role of uPAR in cancer.     

Deconvoluting the relationships between autophagy and metastasis for potential cancer therapy

Autophagy is a highly conserved lysosome-dependent degradation process that may digest some long-lived proteins and damaged organelles. As an essential homeostasis maintaining system in normal cells, autophagy plays a key role in several pathological settings, especially cancer. Metastasis, known as a crucial hallmark of cancer progression, is the primary cause of cancer lethality. The role of autophagy in metastasis is quite complex as supportive evidence has indicated both pro-metastatic and anti-metastatic functions of autophagy. Autophagy can inhibit metastasis by restricting necrosis and mediating autophagic cell death, whereas it may also promote metastasis by enhancing cancer cell fitness in response to stress. Moreover, the function of autophagy is context- and stage-dependent. Specifically, during the early steps of metastasis, autophagy mainly serves as a suppressor, while it plays a pro-metastatic role in the later steps. Here, we focus on highlighting the dual roles of autophagy in metastasis and address the molecular mechanisms involved in this process, which may provide a new insight into cancer biology. While, we also summarize several anti-metastatic agents manipulating autophagy, in the hope of shedding light on exploration of potential novel drugs for future cancer therapy.     

Translational approaches using metastasis suppressor genes

Cancer metastasis is a significant contributor to breast cancer patient morbidity and mortality. In order to develop new anti-metastatic therapies, we need to understand the biological and biochemical mechanisms of metastasis. Toward these efforts, we and others have studied metastasis suppressor genes, which halt metastasis in vivo without affecting primary tumor growth. The first metastasis suppressor gene identified was nm23, also known as NDP kinase. Nm23 represents the most widely validated metastasis suppressor gene, based on transfection and knock-out mouse strategies. The biochemical mechanism of metastasis suppression via Nm23 is unknown and likely complex. Two potential mechanisms include binding proteins and a histidine kinase activity. Elevation of Nm23 expression in micrometastatic tumor cells may constitute a translational strategy for the limitation of metastatic colonization in high risk cancer patients. To date, medroxyprogesterone acetate (MPA) has been identified as a candidate compound for clinical testing.     

Marine algal fucoxanthin inhibits the metastatic potential of cancer cells

Metastasis is major cause of malignant cancer-associated mortality. Fucoxanthin has effect on various pharmacological activities including anti-cancer activity. However, the inhibitory effect of fucoxanthin on cancer metastasis remains unclear. Here, we show that fucoxanthin isolated from brown alga Saccharina japonica has anti-metastatic activity. To check anti-metastatic properties of fucoxanthin, in vitro models including assays for invasion, migration, actin fiber organization and cancer cell–endothelial cell interaction were used. Fucoxanthin inhibited the expression and secretion of MMP-9 which plays a critical role in tumor invasion and migration, and also suppressed invasion of highly metastatic B16-F10 melanoma cells as evidenced by transwell invasion assay. In addition, fucoxanthin diminished the expressions of the cell surface glycoprotein CD44 and CXC chemokine receptor-4 (CXCR4) which play roles in migration, invasion and cancer–endothelial cell adhesion. Fucoxanthin markedly suppressed cell migration in wound healing assay and inhibited actin fiber formation. The adhesion of B16-F10 melanoma cells to the endothelial cells was significantly inhibited by fucoxanthin. Moreover, in experimental lung metastasis in vivo assay, fucoxanthin resulted in significant reduction of tumor nodules. Taken together, we demonstrate, for the first time, that fucoxanthin suppresses metastasis of highly metastatic B16-F10 melanoma cells in vitro and in vivo.     

In vitro and in vivo anti-metastatic effect of the alkaliod matrine from Sophora flavecens on hepatocellular carcinoma and its mechanisms

BackgroundsHepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancer with high metastasis and recurrence rates. Hypoxia-induced miRNAs and HIF-1α are demonstrated to play essential roles in tumor metastasis. Matrine (C₁₅H₂₄N₂O), an alkaloid extracted from Sophora flavescens Aiton, has been used as adjuvant therapy for liver cancer in China. The anti-metastasis effects of matrine on HCC and the underlying mechanisms remain poorly understood.PurposeWe aimed to investigate the effects of matrine on metastasis of HCC both in vitro and in vivo, and explored whether miR-199a-5p and HIF-1α are involved in the action of matrine.MethodsMTT method, colony formation, wound healing and matrigel transwell assays were performed to evaluate the effects of matrine on cell proliferation, migration and invasion. Nude mice xenograft model and immunohistochemistry (IHC) assay were employed to investigate the anti-metastatic action of matrine in vivo. Quantitative real-time PCR, western blot and dual luciferase reporter assay were conducted to determine the underlying mechanisms of matrine.ResultsMatrine exerted stronger anti-proliferative action on Bel7402 and SMMC-7721 cells under hypoxia than that in normoxia. Both matrine and miR-199a-5p exhibited significant inhibitory effects on migration, invasion and EMT in Bel7402 and SMMC-7721 cells under hypoxia. Further study showed that miR-199a-5p was downregulated in HCC cell lines, and this microRNA was identified to directly target HIF-1α, resulting in decreased HIF-1α expression. Matrine induced miR-199a-5p expression, decreased HIF-1α expression and inhibited metastasis of Bel7402 and SMMC-7721 cells, while miR-199a-5p knockdown reversed the inhibitory effects of matrine on cell migration, invasion, EMT and HIF-1α expression. In vivo, matrine showed significant anti-metastatic activity in the nude mouse xenograft model. H&E and IHC analysis indicated that lung and liver metastasis nodules were reduced, and the protein expression of HIF-1α and Vimentin were significantly decreased by i.p injection of matrine.ConclusionsMatrine exhibits significant anti-metastatic effect on HCC, which is attributed to enhanced miR-199a-5p expression and subsequently impaired HIF-1α signaling and EMT. These findings suggest that miR-199a-5p is a potential therapeutic target of HCC, and matrine may represent a promising anti-metastatic medication for HCC therapy.     

Dietary polyphenols in chemoprevention and synergistic effect in cancer: Clinical evidences and molecular mechanisms of action

BackgroundEpidemiological studies has revealed that a diet rich in fruits and vegetables could lower the risk of certain cancers. In this setting, natural polyphenols are potent anticancer bioactive compounds to overcome the non-target specificity, undesirable cytotoxicity and high cost of treatment cancer chemotherapy.PurposeThe review focuses on diverse classifications of the chemical diversity of dietary polyphenol and their molecular targets, modes of action, as well as preclinical and clinical applications in cancer prevention.ResultsThe dietary polyphenols exhibit chemo-preventive activity through modulation of apoptosis, autophagy, cell cycle progression, inflammation, invasion and metastasis. Polyphenols possess strong antioxidant activity and control multiple molecular events through activation of tumor suppressor genes and inhibition of oncogenes involved in carcinogenesis. Numerous in vitro and in vivo studies have evidenced that these dietary phytochemicals regulate critical molecular targets and pathways to limit cancer initiation and progression. Moreover, natural polyphenols act synergistically with existing clinically approved drugs. The improved anticancer activity of combinations of polyphenols and anticancer drugs represents a promising perspective for clinical applications against many human cancers.ConclusionThe anticancer properties exhibited by dietary polyphenols are mainly attributed to their anti-metastatic, anti-proliferative, anti-angiogenic, anti-inflammatory, cell cycle arrest, apoptotic and autophagic effects. Hence, regular consumption of dietary polyphenols as food or food additives or adjuvants can be a promising tactic to preclude adjournment or cancer therapy.     

Increase of anti-metastatic efficacy by selectivity- but not affinity-optimization of synthetic serine protease inhibitors

Although tumors frequently show elevated protease activities, the concept of anti-proteolytic cancer therapy has lost momentum after failure of clinical trials with broad-spectrum matrix metalloproteinase inhibitors. Thus we need to adapt our design strategies for protease inhibitors. Here, we employed a series of seven structurally fine-modulated and pharmacokinetically closely related synthetic 4-amidinobenzylamine-based inhibitors with distinct selectivity for prototypical serine proteases in a murine T cell lymphoma liver metastasis model. This in vivo screening revealed efficacy of urokinase inhibitors but no correlation between urokinase selectivity or affinity and anti-metastatic effect. In contrast, factor Xa-selective inhibitors were more potent, demonstrating factor Xa or a factor Xa-like serine protease likely to be more determinant in this model. Factor Xa selectivity, but not affinity, significantly improved anti-metastatic efficacy. For example, factor Xa inhibitors CJ-504 and CJ-510 exert similar affinity for factor Xa (K(i)=14 nM versus 8.8 nM) but CJ-504 was 70-fold more selective for factor Xa. This correlated with higher anti-metastatic efficacy (58.8% with CJ-504; 28.2% with CJ-510). Our results show that among the protease inhibitors employed that have affinities in the nanomolar range, the strategy of selectivity-optimization is superior to further improvement of affinity to significantly enhance anti-metastatic efficacy. This appreciation may be important for the future rational design of new anti-proteolytic agents for cancer therapy.     

Flt3 mutation activates p21WAF1/CIP1 gene expression through the action of STAT5

Metastin, a post-translationally modified variant of KiSS1, was recently identified as an endogenous peptide agonist for a novel G-protein coupled receptor, hOT7T175 (AXOR12, GPR54). In this study, we analyzed the role of KiSS1 and hOT7T175 in both pancreatic cancer tissues and pancreatic cancer cell lines. Furthermore, we synthesized novel short variant forms of metastin and tested the inhibitory effect of those variants on in vitro cell functions that are relevant to metastasis. Pancreatic cancer tissues showed significantly lower expression of KiSS1 mRNA than normal tissues (p=0.018), while cancer tissues showed significantly higher expression of hOT7T175 mRNA than normal pancreatic tissues (p=0.027). In human pancreatic cancer cell lines, KiSS1 mRNA was highly expressed in 2 out of 6 pancreatic cancer cell lines, while hOT7T175 mRNA was expressed in all cell lines at various degrees. PANC-1 cells showed the highest expression of hOT7T175. Exogenous metastin did not suppress cell proliferation but significantly reduced the in vitro migration of PANC-1 cells (p<0.01). Metastin induced activation of ERK1 in PANC-1 and AsPC-1 cells. Finally, we synthesized 3 novel short variant forms of metastin, FM053a2TFA, FM059a2TFA, and FM052a4TFA. These metastin variants significantly suppressed the migration of PANC-1 cells and activated ERK1. These data suggest that the metastin receptor, hOT7T175, is one of the promising targets for suppression of metastasis, and that small metastin variants could be an anti-metastatic agent to pancreatic cancer.