Ovarian cancer-associated fibroblasts contribute to epithelial ovarian carcinoma metastasis by promoting angiogenesis, lymphangiogenesis and tumor cell invasion

Cancer-associated fibroblasts (CAFs) are thought to play an essential role in cancer initiation and development. However, little research has been done to evaluate the role of CAFs in epithelial ovarian cancer (EOC) development. To address this issue, ninety-one specimens were immunostained with α-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP) antibodies to quantify CAFs, and antibodies D2-40 and CD34 to evaluate the lymphatic vessel density (LVD) and microvessel density (MVD) of the lesions. We found there were no α-SMA or FAP positive fibroblasts in normal ovary tissues. More CAFs were found in EOC than in borderline tumors and benign tumors (P<0.01). Abundant CAFs in EOC were associated with advanced-stage disease (P=0.002), the occurrence of lymph node metastases (P=0.02) and omentum metastases (P<0.0001), and increased LVD (P=0.002) and MVD (P=0.0004). CAFs isolated from EOC tissues induced more cancer cells to invade (P=0.003) and migrate (P=0.005) compared with normal fibroblasts (NFs) isolated from normal ovary tissues in vitro. Our data indicate that CAFs play a vital role in ovarian cancer progression and metastasis. Targeting CAFs as a therapeutic strategy against ovarian cancer is an intriguing concept that needs further study.     

Hepatocyte growth factor activates tumor stromal fibroblasts to promote tumorigenesis in gastric cancer

Cancer-associated fibroblasts (CAFs), as the activated fibroblasts in tumor stroma, are important modifiers of tumor progression. However, the mechanisms underlying stromal fibroblast activation and their promotion of tumor growth remain largely unknown in gastric cancer. Here, we show that normal fibroblasts (NFs) from non-cancerous regions of gastric cancer exhibit the traits of CAFs when grown together with gastric cancer cells in vivo. Activation of NFs can be induced by co-culture with gastric cancer cells, while deprivation of hepatocyte growth factor (HGF) using a neutralizing antibody inhibits the activation of NFs. Moreover, we identify HGF as an important factor from CAFs that acts in a paracrine manner to promote tumorigenesis in vitro and in vivo. Taken together, these results suggest that HGF may play a pivotal role in the regulatory circuit between gastric cancer cells and stromal fibroblasts, and neutralization of HGF inhibits both activation and tumor-promoting properties of CAFs.     

Ligand-independent activation of c-Met by fibronectin and α(5)β(1)-integrin regulates ovarian cancer invasion and metastasis

The role of the fibronectin receptor, α(5)β(1)-integrin, as an adhesion receptor and in angiogenesis is well established. However, its role in cancer cell invasion and metastasis is less clear. We describe a novel mechanism by which fibronectin regulates ovarian cancer cell signaling and promotes metastasis. Fibronectin binding to α(5)β(1)-integrin led to a direct association of α(5)-integrin with the receptor tyrosine kinase, c-Met, activating it in a hepatocyte growth factor/scatter factor (HGF/SF) independent manner. Subsequently, c-Met associated with Src, and activated Src and focal adhesion kinase (FAK). Inhibition of α(5)β(1)-integrin decreased the phosphorylation of c-Met, FAK and Src, both in vitro and in vivo. Independent activation of c-Met by its native ligand, HGF/SF, or overexpression of a constitutively active FAK in HeyA8 cells could overcome the effect of α(5)β(1)-integrin inhibition on tumor cell invasion, indicating that α(5)β(1)-integrin is upstream of c-Met, Src and FAK. Inhibition of α(5)β(1)-integrin on cancer cells in two xenograft models of ovarian cancer metastasis resulted in a significant decrease of tumor burden, which was independent of the effect of α(5)β(1)-integrin on angiogenesis. These data suggest that fibronectin promotes ovarian cancer invasion and metastasis through an α(5)β(1)-integrin/c-Met/FAK/Src-dependent signaling pathway, transducing signals through c-Met in an HGF/SF-independent manner.     

Use of a novel 3D culture model to elucidate the role of mesothelial cells, fibroblasts and extra-cellular matrices on adhesion and invasion of ovarian cancer cells to the omentum

The omentum is a major site of ovarian cancer metastasis. Our goal was to establish a three-dimensional (3D) model of the key components of the omental microenvironment (mesothelial cells, fibroblasts and extracellular matrices) to study ovarian cancer cell adhesion and invasion. The 3D model comprised of primary human fibroblasts extracted from normal human omentum, mixed with ECM and covered by a layer of primary human mesothelial cells, also from normal human omentum. After addition of ovarian cancer cells, the histological appearance of the 3D culture mimicked microscopic metastases to the omentum from patients with ovarian cancer. When ovarian cancer cells, SKOV3ip.1 and HeyA8, were applied to the 3D omental culture, 60% and 68% of all cells attached, respectively, but only 18% and 25% were able to invade. Ovarian cancer cells preferentially adhered to and invaded collagen I, followed by binding to collagen IV, fibronectin, vitronectin, laminin 10 and 1. Omental mesothelial cells significantly inhibited ovarian cancer cell adhesion and invasion, while omental fibroblasts induced adhesion and invasion. This effect is clearly mediated by direct cell-cell contact, since conditioned medium from mesothelial cells or fibroblasts has a minimal, or no, effect on ovarian cancer cell adhesion and invasion. In summary, we have established a 3D model to study the early steps of ovarian cancer metastasis to the human omentum, and found that omental mesothelial cells inhibit, while omental fibroblasts and the ECM enhance, the attachment and invasion of ovarian cancer cells.     

c-Ski activates cancer-associated fibroblasts to regulate breast cancer cell invasion

Aberrant expression of c-Ski oncoprotein in some tumor cells has been shown to be associated with cancer development. However, the role of c-Ski in cancer-associated fibroblasts (CAFs) of tumor microenvironment has not been characterized. In the current study, we found that c-Ski is highly expressed in CAFs derived from breast carcinoma microenvironment and this CAF-associated c-Ski expression is associated with invasion and metastasis of human breast tumors. We showed that c-Ski overexpression in immortalized breast normal fibroblasts (NFs) induces conversion to breast CAFs by repressing p53 and thereby upregulating SDF-1 in NFs. SDF-1 treatment or p53 knockdown in NFs had similar effects on the activation of NFs as c-Ski overexpression. The c-Ski-activated CAFs show increased proliferation, migration, invasion and contraction compared with NFs. Furthermore, c-Ski-activated CAFs facilitated the migration and invasion of MDA-MB-231 breast cancer cells. Our data suggest that c-Ski is an important regulator in the activation of CAFs and may serve as a potential therapeutic target to block breast cancer progression.     

癌相关成纤维细胞对喉癌细胞系Hep-2生物学行为影响的体外研究

目的 探讨癌相关成纤维细胞(CAFs)在体外的分泌功能及对喉癌细胞Hep-2生物学行为的影响.方法 提取江西省肿瘤医院2016-09-09 1例喉鳞状细胞癌手术患者的癌组织及癌旁组织,进行喉癌CAFs及正常成纤维细胞(NFs)的原代培养.ELISA试剂盒测定CAFs和NFs培养上清液中MMP-2、MMP-9的浓度.制备...     

Matrilysin (MMP-7) promotes invasion of ovarian cancer cells by activation of progelatinase

Although matrilysin (MMP-7) is overexpressed in various malignancies, few studies have evaluated its role in epithelial ovarian cancer (EOC) invasion and metastasis. We report that the secretion of MMP-7 in EOC is stimulated significantly by vascular endothelial growth factor (VEGF) and interlukin-8 (IL-8). We also examined the in vivo expression of MMP-7 in EOC and its effects on the in vitro invasion and progelatinase activation. We report that MMP-7 is overexpressed in ovarian cancer cell lines and EOC surgical specimens. DOV13 cells incubated with active rhMMP-7 significantly increased cellular invasion and proMMP-2 activation. RhMMP-7 also showed the ability to activate proMMP-2 and proMMP-9 in immortalized ovarian epithelial cell (IOSE-29) conditioned medium. In addition, rhMMP-7 was able to activate progelatinase in a concentration-dependent manner in vitro. TIMP-2 or the generic MMP inhibitor-GM6001 inhibited both the activation of proMMP-2 and the increased invasion of DOV13 cells promoted by rhMMP-7. By incubation of MMP2-TIMP-2 complex with equal molar rhMMP-7, MMP-2 was dissociated from the complex and activated in a time-dependent manner, suggesting that TIMP-2 helps to keep the latency of MMP-2. TIMP-2 also showed inhibitory effects on the MMP-7 induced increase of gelatinolytic activity in DOV13 and IOSE-29 conditioned media. A strong co-localization of MMP-7 and MMP-2 was observed in DOV13 cells and ovarian carcinoma permanent tissue sections. These results indicate MMP-7 is overexpressed in malignant ovarian epithelium and suggest MMP-7 may facilitate tumor cell invasion in vivo partly through the induction of progelatinase activation.     

CXCL12/CXCR4 activation by cancer‐associated fibroblasts promotes integrin β1 clustering and invasiveness in gastric cancer

Cancer‐associated fibroblasts (CAFs) are reportedly involved in invasion and metastasis in several types of cancer, including gastric cancer (GC), through the stimulation of CXCL12/CXCR4 signaling. However, the mechanisms underlying these tumor‐promoting effects are not well understood, which limits the potential to develop therapeutic targets against CAF‐mediated CXCL12/CXCR4 signaling. CXCL12 expression was analyzed in resected GC tissues from 110 patients by immunohistochemistry (IHC). We established primary cultures of normal fibroblasts (NFs) and CAFs from the GC tissues and examined the functional differences between these primary fibroblasts using co‐culture assays with GC cell lines. We evaluated the efficacy of a CXCR4 antagonist (AMD3100) and a FAK inhibitor (PF‐573,228) on the invasive ability of GC cells. High CXCL12 expression levels were significantly associated with larger tumor size, increased tumor depth, lymphatic invasion and poor prognosis in GC. CXCL12/CXCR4 activation by CAFs mediated integrin β1 clustering at the cell surface and promoted the invasive ability of GC cells. Notably, AMD3100 was more efficient than PF‐573,228 at inhibiting GC cell invasion through the suppression of integrin β1/FAK signaling. These results suggest that CXCL12 derived from CAFs promotes GC cell invasion by enhancing the clustering of integrin β1 in GC cells, resulting in GC progression. Taken together, the inhibition of CXCL12/CXCR4 signaling in GC cells may be a promising therapeutic strategy against GC cell invasion.     

钙黏素和整合素在卵巢上皮性癌中的研究进展

卵巢上皮性癌(EOC)是妇科癌症中最致命的恶性肿瘤。在过去的30年间,虽然诊断和治疗水平取得了进展,但是卵巢上皮性癌的发病率和死亡率却保持不变。卵巢上皮性癌腹膜转移癌晚期发病于附着的小簇癌细胞,这些癌细胞从初始部位脱落并经由腹水携带附着于腹部腹膜或网膜。这种行为表明细胞-细胞或细胞-基质黏附机制调节卵巢上皮性癌的生长和增殖。复杂的下游信号可能受黏附分子和共表达激活的信号蛋白之间的功能性串扰所影响,导致卵巢上皮性癌细胞的增殖/存活和迁移/侵袭。本文旨在阐述细胞与细胞机制的影响,该过程由钙黏素和细胞-细胞外基质黏附,由整合素对膜受体和细胞质蛋白质介导的信号级联放大,这些在卵巢上皮性癌细胞的增殖、迁移和侵袭中起着一定的作用。     

FGF‐1/‐3/FGFR4 signaling in cancer‐associated fibroblasts promotes tumor progression in colon cancer through Erk and MMP‐7

Cancer‐associated fibroblasts (CAFs), as the activated fibroblasts in the tumor stroma, are important modifiers of tumour progression. In the present study, we observed that azoxymethane and dextran sodium sulfate treatments induced increasingly severe colorectal mucosal inflammation and the intratumoural accumulation of CAFs. Fibroblast growth factor (FGF)‐1 and FGF‐3 were detected in infiltrating cells, and FGFR4, the specific receptor for FGF‐1 and FGF‐3, was detected in colon cancer tissues. The phosphorylation of FGFR4 enhanced the production of metalloproteinase (MMP)‐7 and mitogen‐activated protein kinase kinase (Mek)/extracellular signal‐regulated kinase (Erk), which was accompanied by excessive vessel generation and cell proliferation. Moreover, we separated CAFs, pericarcinoma fibroblasts (PFs), and normal fibroblasts (NFs) from human colon tissue specimens to characterize the function of CAFs. We observed that CAFs secrete more FGF‐1/‐3 than NFs and PFs and promote cancer cell growth and angiogenesis through the activation of FGFR4, which is followed by the activation of Mek/Erk and the modulation of MMP‐7 expression. The administration of FGF‐1/‐3‐neutralizing antibodies or the treatment of cells with FGFR4 siRNA or the FGFR4 inhibitor PD173074 markedly suppressed colon cancer cell proliferation and neovascularization. These observations suggest a crucial role for CAFs and FGF signaling in the initiation and progression of colorectal cancer. The inhibition of the FGF signaling pathway may be a useful strategy for the treatment of colon cancer.     

Omental cancer-associated fibroblast-derived exosomes with low microRNA-29c-3p promote ovarian cancer peritoneal metastasis

Ovarian cancer (OC) is characterized by frequent widespread peritoneal metastasis. Cancer-associated fibroblasts (CAFs) represent a critical stromal component of metastatic niche and promote omentum metastasis in OC patients. However, the role of exosomes derived from omental CAFs in metastasis remains unclear. We isolated exosomes from primary omental normal fibroblasts (NFs) and CAFs from OC patients (NF-Exo and CAF-Exo, respectively) and assessed their effect on metastasis. In mice bearing orthotopic OC xenografts, CAF-Exo treatment led to more rapid intraperitoneal tumor dissemination and shorter animal survival. Similar results were observed in mice undergoing intraperitoneal injection of tumor cells. Among the miRNAs downregulated in CAF-Exo, miR-29c-3p in OC tissues was associated with metastasis and survival in patients. Moreover, increasing miR-29c-3p in CAF-Exo significantly weakened the metastasis-promoting effect of CAF-Exo. Based on RNA sequencing, expression assays, and luciferase assays, matrix metalloproteinase 2 (MMP2) was identified as a direct target of miR-29c-3p. These results verify the significant contribution of exosomes from omental CAFs to OC peritoneal metastasis, which could be partially due to the relief of MMP2 expression inhibition mediated by low exosomal miR-29c-3p.     

Excessive activation of IL-33/ST2 in cancer-associated fibroblasts promotes invasion and metastasis in ovarian cancer

Ovarian cancer is highly prevalent and has high mortality rates due to metastasis and relapse. The cross communication between cancer-associated fibroblasts (CAFs) and cancer-associated macrophages (CAMs) in the ovarian tumor microenvironment leads to cancer cell invasion and metastasis. However, the role of overproduction of IL-33/ST2 in the CAFs of ovarian cancer is still unclear. The expression of IL-33, ST2, apoptosis-related proteins and epithelial-mesenchymal transition (EMT) markers was measured by western blotting. Primary normal fibroblasts and CAFs from ovarian cancerous tissue were isolated and cultured in vitro, and the medium was used to stimulate blood-derived monocytes. Flow cytometry analysis was used to detect the frequency of M2-like macrophages in blood-derived monocytes from patients with ovarian cancer. Cell invasion were evaluated using Transwell assays. A xenograft model was used to study tumor growth in ST2-knockout and wild-type NOD-SCID mice. The results demonstrated higher expression of IL-33 and ST2 in carcinoma tissues compared with in para-carcinoma tissues, and there was a survival improvement associated with elevated IL-33. IL-33 and culture supernatants from CAFs, rather than normal ovarian fibroblasts, led to a higher expression of M2 macrophage marker genes in human blood-derived monocytes. Invasion and migration were aggravated in COC1 cells co-cultured with CAF-induced CAMs, and the EMT marker genes were upregulated. It was reported that EMT marker genes were downregulated and tumor volumes were significantly reduced in ST2-deficient mice. Overall, the IL-33/ST2 axis in ovarian cancer might integrate IL-33-expressing CAFs with M2 type-like CAMs, which aggravated invasion and metastasis by promoting EMT.     

NPPB is a novel candidate biomarker expressed by cancer‐associated fibroblasts in epithelial ovarian cancer

Most solid tumors contain cancer‐associated fibroblasts (CAFs) that support tumorigenesis and malignant progression. However, the cellular origins of CAFs in epithelial ovarian cancers (EOCs) remain poorly understood, and their utility as a source of clinical biomarkers for cancer diagnosis has not been explored in great depth. Here, we report establishing in vitro and in vivo models of CAFs in ovarian cancer development. Normal ovarian fibroblasts and mesenchymal stem cells cultured in the presence of EOC cells acquired a CAF‐like phenotype, and promoted EOC cell migration in vitro. CAFs also promoted ovarian cancer growth in vivo in both subcutaneous and intraperitoneal murine xenograft assays. Molecular profiling of CAFs identified gene expression signatures that were highly enriched for extracellular and secreted proteins. We identified novel candidate CAF‐specific biomarkers for ovarian cancer including NPPB, which was expressed in the stroma of 60% primary ovarian cancer tissues (n = 145) but not in the stroma of normal ovaries (n = 4). NPPB is a secreted protein that was also elevated in the blood of 50% of women with ovarian cancer (n = 8). Taken together, these data suggest that the tumor stroma is a novel source of biomarkers, including NPPB, that may be of clinical utility for detection of EOC.     

Radiation to stromal fibroblasts increases invasiveness of pancreatic cancer cells through tumor-stromal interactions

Radiotherapy represents a major treatment option for patients with pancreatic cancer, but recent evidence suggests that radiation can promote invasion and metastasis of cancer cells. Interactions between cancer cells and surrounding stromal cells may play an important role in aggressive tumor progression. In the present study, we investigated the invasive phenotype of pancreatic cancer cells in response to coculture with irradiated fibroblasts. Using in vitro invasion assay, we demonstrated that coculture with nonirradiated fibroblasts significantly increased the invasive ability of pancreatic cancer cells and, surprisingly, the increased invasiveness was further accelerated when they were cocultured with irradiated fibroblasts. The hepatocyte growth factor (HGF) secretion from fibroblasts remained unchanged after irradiation, whereas exposure of pancreatic cancer cells to supernatant from irradiated fibroblasts resulted in increased phosphorylation of c-Met (HGF receptor) and mitogen-activated protein kinase activity, possibly or partially via increased expression of c-Met. We also demonstrated that scattering of pancreatic cancer cells was accelerated by the supernatant from irradiated fibroblasts. The enhanced invasiveness of pancreatic cancer cells induced by coculture with irradiated fibroblasts was completely blocked by NK4, a specific antagonist of HGF. These data suggest that invasive potential of certain pancreatic cancer cells is enhanced by soluble mediator(s) released from irradiated fibroblasts possibly through up-regulation of c-Met expression/phosphorylation and mitogen-activated protein kinase activity in pancreatic cancer cells. Our present findings further support the potential use of NK4 during radiotherapy for patients with pancreatic cancer.     

Engagement of collagen-binding integrins promotes matrix metalloproteinase-9-dependent E-cadherin ectodomain shedding in ovarian carcinoma cells

Reversible modulation of cell-cell adhesion, cell-matrix adhesion, and proteolytic activity plays a critical role in remodeling of the neoplastic ovarian epithelium during metastasis, implicating cadherins, integrins, and proteinases in i.p. metastatic dissemination of epithelial ovarian carcinoma (EOC). Aberrant epithelial differentiation is an early event in ovarian carcinogenesis; thus, in contrast to most carcinomas that lose E-cadherin expression with progression, E-cadherin is abundant in primary EOC. Metastasizing EOCs engage in integrin-mediated adhesion to submesothelial interstitial collagens and express matrix metalloproteinases (MMP) that facilitate collagen invasion, thereby anchoring secondary lesions in the submesothelial matrix. As metalloproteinases have also been implicated in E-cadherin ectodomain shedding, the current study was undertaken to model the effects of matrix-induced integrin clustering on proteinase-catalyzed E-cadherin ectodomain shedding. Aggregation of collagen-binding integrins induced shedding of an 80-kDa E-cadherin ectodomain [soluble E-cadherin (sE-cad)] in a MMP- and Src kinase-dependent manner, and sE-cad was prevalent in ascites from ovarian cancer patients. Expression of MMP-9 was elevated by integrin aggregation, integrin-mediated ectodomain shedding was inhibited by a MMP-9 function blocking antibody, and incubation of cells with exogenous MMP-9 catalyzed E-cadherin ectodomain shedding. In contrast to other tumors wherein sE-cad is released into the circulation, EOC tumors maintain direct contact with sE-cad-rich ascites at high concentration, and incubation of EOC cells with physiologically relevant concentrations of recombinant sE-cad disrupted adherens junctions. These data support a novel mechanism for posttranslational modification of E-cadherin function via MMP-9 induction initiated by cell-matrix contact and suggest a mechanism for promotion of EOC metastatic dissemination.     

CAFs对乳腺癌细胞生物学特性的影响及机制探讨

目的：通过乳腺癌间质成纤维细胞（ carcinoma-associated fibroblasts,CAFs）与乳腺癌细胞系MDA-MB-231共培养的体外细胞实验及裸鼠接种的在体动物实验,观察CAFs对乳腺癌细胞增殖、凋亡、侵袭和转移活性的影响,并探讨其作用机制。方法：体外实验：分离培养浸润性导管癌组织中CAFs和正常成纤维细胞（ normal fibroblasts,NFs）,然后分别与乳腺癌细胞MDA-MB-231体外共培养,采用MTT法、流式细胞仪、Ma-trigel人工模拟基底膜法分别检测乳腺癌细胞的增殖、凋亡、细胞黏附和侵袭能力。动物在体实验：选择乳腺癌细胞系MDA-MB-231、CAFs和NFs,结合生理盐水（NS）、基质细胞衍生因子-1（SDF-1）及其配体拮抗剂AMD3100,组成不同的组别并接种于裸鼠（共6组）。观察肿瘤的大小,有无淋巴结、肺、肝脏转移。留取血标本及肿瘤组织行SDF-1表达水平的检测。结果：MDA-MB-231与CAFs和NFs共培养后乳腺癌细胞的增殖活性显著增强,其中CAFs的作用较NFs更强（P＝0.011）;CAFs组的黏附能力（34.70±4.84个/视野）明显强于NFs组（20.16±3.09个/视野）,P＝0.000;而CAFs组的侵袭性（89.0±4.62个/视野）也明显强于NFs组（81.6±6.08个/视野,P＝0.045）。CAFs组中的MDA-MB-231的早期凋亡率（2.9±2.4）较NFs组（5.0±4.2）明显降低（P＝0.026）;MDA231﹢CAFs ﹢NS 组的种植肿瘤平均体积最大（9.092±2.662cm3, P＝0.000）;此外,该组共有4只（66.6％）,MDA231﹢NS组有2只（33.3％）存在腋窝淋巴结转移,未见肝肺转移灶。在MDA231﹢CAFs﹢NS组中,血标本 SDF -1值（75.25±16.23pg/ml）、肿瘤组织标本中 SDF -1mRNA值（11.686±8.926）、组织中SDF-1蛋白表达水平（1.006±0.327）均为最高,与其他各组相比均有统计学差异（ P＝0.000）。结论：CAFs可影响乳腺癌肿瘤细胞的生物学特性,具有促进肿瘤细胞增殖,增强其黏附、侵袭及转移能力。其机制可能是通过乳腺癌间质成纤维细胞分泌SDF-1与其特定的受体CXCR4结合这一信号通路来实现的。     

Curcumin Inhibits TGF-β1-Induced MMP-9 and Invasion through ERK and Smad Signaling in Breast Cancer MDAMB- 231 Cells

Objective: To evaluate the effects of curcumin on matrixmetalloproteinase-9 (MMP-9) and invasion abilityinduced by transforming growth factor-β1 (TGF-β1) in MDA-MB-231 cells and potential mechanisms. Methods:Human breast cancer MDA- MB-231 cells were used with the CCK-8 assay to measure the cytotoxicity ofcurcumin. After treatment with 10 ng/ml TGF-β1, with or without curcumin (≤10 μM), cell invasion was checkedby transwell chamber. The effects of curcumin on TGF-β1-stimulated MMP-9 and phosphorylation of Smad2,extracellular-regulated kinase (ERK), and p38 mitogen activated protein kinases (p38MAPK) were examined byWestern blotting. Supernatant liquid were collected to analyze the activity of MMP-9 via zymography. Followingtreatment with PD98059, a specific inhibitor of ERK, and SB203580, a specific inhibitor of p38MAPK, Westernblotting and zymography were employed to examine MMP-9 expression and activity, respectively. Results: Lowdose curcumin (≤10 μM) did not show any obvious toxicity to the cells, while 0~10 μmol/L caused a concentration–dependent reduction in cell invasion provoked by TGF-β1. Curcumin also markedly inhibited TGF-β1-regulatedMMP-9 and activation of Smad2, ERK1/2 and p38 in a dose- and time-dependent manner. Additionally, PD98059,but not SB203580, showed a similar pattern of inhibition of MMP-9 expression. Conclusion: Curcumin inhibitedTGF-β1-stimulated MMP-9 and the invasive phenotype in MDA-MB-231 cells, possibly associated with TGF-β/Smad and TGF-β /ERK signaling.