Green tea (-)-epigallocatechin-3-gallate inhibits HGF-induced progression in oral cavity cancer through suppression of HGF/c-Met

Hepatocyte growth factor (HGF) and c-Met have recently attracted a great deal of attention as prognostic indicators of patient outcome, and they are important in the control of tumor growth and invasion. Epigallocatechin-3-gallate (EGCG) has been shown to modulate multiple signal pathways in a manner that controls the unwanted proliferation and invasion of cells, thereby imparting cancer chemopreventive and therapeutic effects. In this study, we investigated the effects of EGCG in inhibiting HGF-induced tumor growth and invasion of oral cancer in vitro and in vivo. We examined the effects of EGCG on HGF-induced cell proliferation, migration, invasion, induction of apoptosis and modulation of HGF/c-Met signaling pathway in the KB oral cancer cell line. We investigated the antitumor effect and inhibition of c-Met expression by EGCG in a syngeneic mouse model (C3H/HeJ mice, SCC VII/SF cell line). HGF promoted cell proliferation, migration, invasion and induction of MMP (matrix metalloproteinase)-2 and MMP-9 in KB cells. EGCG significantly inhibited HGF-induced phosphorylation of Met and cell growth, invasion and expression of MMP-2 and MMP-9. EGCG blocked HGF-induced phosphorylation of c-Met and that of the downstream kinases AKT and ERK, and inhibition of p-AKT and p-ERK by EGCG was associated with marked increases in the phosphorylation of p38, JNK, cleaved caspase-3 and poly-ADP-ribose polymerase. In C3H/HeJ syngeneic mice, as an in vivo model, tumor growth was suppressed and apoptosis was increased by EGCG. Our results suggest that EGCG may be a potential therapeutic agent to inhibit HGF-induced tumor growth and invasion in oral cancer.     

Oxygen-mediated regulation of gelatinase and tissue inhibitor of metalloproteinases-1 expression by invasive cells.

The relative expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) is an important determinant in trophoblast invasion of the uterus and tumor invasion and metastasis. Our previous studies have shown that low oxygen levels increase the in vitro invasiveness of trophoblast and tumor cells. The present study examined whether changes in oxygen levels affect TIMP and MMP expression by cultured trophoblast and breast cancer cells. Reverse zymographic analysis demonstrated reduced TIMP-1 protein secretion by HTR-8/SVneo trophoblast cells as well as MDA-MB-231 and MCF-7 breast carcinoma cells cultured in 1% vs 20% oxygen for 24 h. While gelatin zymography revealed no changes in the levels of MMP-9 secreted by HTR-8/SVneo trophoblasts cultured under various oxygen concentrations for 24 h, human MDA-MB-231 breast carcinoma cells displayed increased MMP-9 secretion and human MCF-7 breast cancer cells exhibited reduced secretion of this enzyme when cultured under similar conditions. In contrast, MMP-2 levels remained unchanged in all cultures incubated under similar conditions. Western blot analysis of MMP-9 protein in cell extracts confirmed the results of zymography. To assess the contribution of enhanced MMP activity to hypoxia-induced invasion, the effect of an MMP inhibitor (llomastat) on the ability of MDA-MB-231 cells to penetrate reconstituted extracellular matrix (Matrigel) was examined. Results showed that MMP inhibition significantly decreased the hypoxic upregulation of invasion by these cells. These findings indicate that the increased cellular invasiveness observed under reduced oxygen conditions may be due in part to a shift in the balance between MMPs and their inhibitors favoring increased MMP activity.     

Arsenic trioxide (As2O3) inhibits invasion of HT1080 human fibrosarcoma cells: role of nuclear factor-kappaB and reactive oxygen species

In order to define the role of As2O3 in regulating the tumor cell invasiveness, the effects of As2O3 on secretion of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA), and in vitro invasion of HT1080 human fibrosarcoma cells were examined. As2O3 inhibited cell adhesion to the collagen matrix in a concentration dependent manner, whereas the same treatment enhanced cell to cell interaction. In addition, As2O3 inhibited migration and invasion of HT1080 cells stimulated with phorbol 12-myristate 13-aceate (PMA), and suppressed the expression of MMP-2, -9, membrane type-1 MMP, uPA, and uPA receptor (uPAR). In contrast, As2O3 increased the expression of tissue inhibitor of metalloproteinase (TIMP)-1 and PA inhibitor (PAI)-1, and reduced the MMP-2, -9, and uPA promoter activity in the presence and absence of PMA. Furthermore, the promoter stimulating and DNA binding activity of nuclear factor-kappaB (NF-kappaB) was blocked by As2O3, whereas the activator protein-1 activity was unchanged. Pretreatment of the cells with N-acetyl-L-cysteine (NAC) significantly prevented suppression of MMPs and uPA secretion, DNA binding activity of NF-kappaB, and in vitro invasion of HT1080 cells by As2O3, suggesting a role of reactive oxygen species (ROS) in this process. These results suggest that As2O3 inhibits tumor cell invasion by modulating the MMPs/TIMPs and uPA/uPAR/PAI systems of extracellular matrix (ECM) degradation. In addition, the generation of ROS and subsequent suppression of NF-kappaB activity by As2O3 might partly be responsible for the phenomena. Overall, As2O3 shows potent activity controlling tumor cell invasiveness in vitro.     

KAI1/CD82 suppresses tumor invasion by MMP9 inactivation via TIMP1 up-regulation in the H1299 human lung carcinoma cell line

We conducted a study on the mechanism of KAI1/CD82-mediated suppression of tumor invasiveness and metastasis, and examined its effect on MMP-9 activity and the TIMP1 levels in H1299 human non-small cell lung carcinoma cells. The H1299 human lung carcinoma cells were transfected with pcDNA3.1-CD82 and stable transfectant clones that had a high KAI1/CD82 expression were obtained. We performed Western blot analysis, cell invasion assay, gelatin zymography, and RT-PCR to assess the KAI1/CD82 expression and tumor invasiveness, the MMP-9 activity, the MMP-9 mRNA and protein levels, and the TIMP1 levels in the H1299/CD82 transfectant cells and compared the results with those of the control groups. The H1299/CD82 transfectants exhibited significant suppression of cell invasion, reduced MMP9 enzyme activity, elevated MMP9 mRNA and MMP-9 protein levels, and elevated TIMP1 levels. It may be postulated that KAI1/CD82 over-expression in the H1299 non-small cell lung carcinoma cells suppresses the tumor invasiveness and metastatic potential by inducing MMP9 inactivation via the up-regulation of TIMP1.     

Rac1 mediates type I collagen-dependent MMP-2 activation. role in cell invasion across collagen barrier

Cell migration and proteolysis are two essential processes during tumor invasion and metastasis. Matrix metalloproteinase (MMP)-2 (type IV collagenase; gelatinase A), is implicated in tumor metastasis as well as in primary tumor growth. The Rho family of small GTPases regulates the dynamics of actin cytoskeleton associated with cell motility. In this report, we provide evidence that Rac1, one member of Rho-related small GTPases, is a mediator of MMP-2 activation in HT1080 fibrosarcoma cells cultured in three-dimensional collagen gel (3D-col) and that MMP-2 activation is required for Rac1-promoted cell invasion through collagen barrier. Stable expression of dominant negative (Rac1V12N17) and constitutively active Rac1 (Rac1V12), respectively, in HT1080 cells demonstrates that Rac1 promoted cell invasiveness across type I collagen and collagen-dependent MMP-2 activation. Active Rac1 is sufficient to induce MMP-2 activation in cells cultured in fibrin gel, an extracellular matrix component that does not support MMP-2 activation. The Rac1-dependent MMP-2 activation occurred in a cell-associated fashion and required MMP activities. Because the cell membrane-mediated MMP-2 activation requires MT1-MMP and low amount of issue inhibitor of matrix metalloproteinase-2 (TIMP-2), their expression was examined. Rac1 modulated MT1-MMP mRNA level and the accumulation of a 43-kDa form of MT1-MMP protein, in correlation with MMP-2 activation profile. However, TIMP-2 expression was independent of Rac1 activity. The coordinate modulation of MMP-2 activity and MT1-MMP expression/processing by Rac1 is consistent with cell collagenolytic activity. The C-terminal hemopexin-like domain of MMP-2, which interferes with the cell membrane activation of MMP-2, reduced Rac1-promoted cell invasiveness as monitored by collagen invasion assay. These results suggest that collagen-dependent MMP-2 activation and MT1-MMP expression/processing contribute to Rac-promoted tumor cell invasion through interstitial collagen barrier.     

Protease Nexin-1 affects the migration and invasion of C6 glioma cells through the regulation of urokinase Plasminogen Activator and Matrix Metalloproteinase-9/2

Protease Nexin-1 (PN-1) or Serpine2 is a physiological regulator of extracellular proteases as thrombin and urokinase (uPA) in the brain. Besides, PN-1 is also implicated in some human cancers and further identified as a substrate for Matrix Metalloproteinase (MMP)-9, a key enzyme in tumor invasiveness. Our aim was to study the role of PN-1 in the migration and invasive potential of glioma cells, using the rat C6 glioma cell line as stable clones transfected with pAVU6 + 27 vector expressing PN-1 short-hairpin RNA. We find that PN-1 knockdown enhanced the in vitro migration and invasiveness of C6 cells which also showed a strong gelatinolytic activity by in situ zymography. PN-1 silencing did not alter prothrombin whereas increased uPA, MMP-9 and MMP-2 expression levels and gelatinolytic activity in a conditioned medium from stable C6 cells. Selective inhibitors for MMP-9 (Inhibitor I), MMP-2 (Inhibitor III) or exogenous recombinant PN-1 added to the culture medium of C6 silenced cells restored either the migration and invasive ability or gelatinolytic activity thus validating the specificity of PN-1 silencing strategy. Phosphorylation levels of extracellular signal-related kinases (Erk1/2 and p38 MAPK) involved in MMP-9 and MMP-2 signaling were increased in PN-1 silenced cells. This study shows that PN-1 affects glioma cell migration and invasiveness through the regulation of uPA and MMP-9/2 expression levels which contribute to the degradation of extracellular matrix during tumor invasion.     

Inhibition of melanoma growth and metastasis by combination with (-)-epigallocatechin-3-gallate and dacarbazine in mice.

(-)-Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, was shown to have cancer chemopreventive activity. In this study, we examined the antimetastatic effects of EGCG or the combination of EGCG and dacarbazine on B16-F3m melanoma cells in vitro and in vivo. First, the antimetastatic potentials of five green tea catechins were examined by soft agar colony formation assay, and the results show that EGCG was more effective than the other catechins in inhibiting soft agar colony formation. Second, EGCG dose-dependently inhibited B16-F3m cell migration and invasion by in vitro Transwell assay. Third, EGCG significantly inhibited the spread of B16-F3m cells on fibronectin, laminin, collagen, and Matrigel in a dose-dependent manner. In addition, EGCG significantly inhibited the tyrosine phosphorylation of focal adhesion kinase (FAK) and the activity of matrix metalloproteinase-9 (MMP-9). In animal experiments, EGCG alone reduced lung metastases in mice bearing B16-F3m melanomas. However, a combination of EGCG and dacarbazine was more effective than EGCG alone in reducing the number of pulmonary metastases and primary tumor growths, and increased the survival rate of melanoma-bearing mice. These results demonstrate that combination treatment with EGCG and dacarbazine strongly inhibits melanoma growth and metastasis, and the action mechanisms of EGCG are associated with the inhibition of cell spreading, cell-extracellular matrix and cell-cell interactions, MMP-9 and FAK activities.     

Sulindac and its metabolites inhibit invasion of glioblastoma cells via down-regulation of Akt/PKB and MMP-2

Non-steroidal anti-inflammatory drug (NSAID), sulindac has chemopreventive and anti-tumorigenic properties, however, the molecular mechanism of this inhibitory action has not been clearly defined. The Akt/protein kinase B, serine/threonine kinase is well known as an important mediator of many cell survival signaling pathways. In the present study, we demonstrate that down-regulation of Akt is a major effect of anti-invasiveness property of sulindac and its metabolites in glioblastoma cells. Myristoylated Akt (MyrAkt) transfected U87MG glioblastoma cells showed increase invasiveness, whereas DN-Akt transfected cells showed decrease invasiveness indicating that Akt potently promoted glioblastoma cell invasion. MMP-2 promoter and enzyme activity were up-regulated in Akt kinase activity dependent manner. Sulindac and its metabolites down-regulated Akt phosphorylation, inhibited MMP-2 production, and significantly inhibited invasiveness of human glioblastoma cells. In addition, sulindac and LY294002, a selective inhibitor of phosphoinositide 3-kinase (PI3K), synergistically inhibited the invasion of glioblastoma cells. Furthermore, only celecoxib showed Akt phosphorylation reduction and an anti-invasivness in glioblastoma cells, whereas aspirin, ketoprofen, ketorolac, and naproxen did not. In conclusion, our results provide evidence that down-regulation of Akt pathway and MMP-2 may be one of the mechanisms by which sulindac and its metabolites inhibit glioblastoma cell invasion.     

TGF-β1 facilitates MT1-MMP-mediated proMMP-9 activation and invasion in oral squamous cell carcinoma cells

Matrix metalloproteinase (MMP)-2 and MMP-9, also known as gelatinases or type IV collagenases, are recognized as major contributors to the proteolytic degradation of extracellular matrix during tumor invasion. Latent MMP-2 (proMMP-2) is activated by membrane type 1 MMP (MT1-MMP) on the cell surface of tumor cells. We previously reported that cell-bound proMMP-9 is activated by the MT1-MMP/MMP-2 axis in HT1080 cells treated with concanavalin A in the presence of exogenous proMMP-2. However, the regulatory mechanism of proMMP-9 activation remains largely unknown. Transforming growth factor (TGF)-β1 is frequently overexpressed in tumor tissues and is associated with tumor aggressiveness and poor prognosis. In this study, we examined the role of TGF-β1 on MT1-MMP-mediated proMMP-9 activation using human oral squamous cell carcinoma cells. TGF-β1 significantly increased the expression of MMP-9. By adding exogenous proMMP-2, TGF-β1-induced proMMP-9 was activated during collagen gel culture, which was suppressed by the inhibition of TGF-β1 signaling or MT1-MMP activity. This MT1-MMP-mediated proMMP-9 activation was needed to facilitate TGF-β1-induced cell invasion into collagen gel. Thus, TGF-β1 may facilitate MT1-MMP-mediated MMP-9 activation and thereby stimulate invasion of tumor cells in collaboration with MT1-MMP and MMP-2.     

The novel estrogen receptor GPER regulates the migration and invasion of ovarian cancer cells

G protein-coupled estrogen receptor (GPER) was identified as a new member of the estrogen receptor family in recent years. It has become apparent that GPER mediates the non-genomic signaling of 17β-estradiol (E₂) in a variety of estrogen-related cancers. Our previous study has found that GPER was overexpressed in human epithelial ovarian cancer and was positively correlated with the expression of matrix metalloproteinase 9 (MMP-9), which suggested GPER might promote the metastasis of ovarian cancer. However, the mechanisms underlying GPER-dependent metastasis of ovarian cancer are still not clear. In the present study, estrogen receptor α (ERα)-negative/GPER-positive OVCAR5 ovarian cancer cell line was used to investigate the role of GPER in the migration and invasion of ovarian cancer. Wound healing assay and transwell matrigel invasion assay were performed to determine the potentials of cell migration and invasion, respectively. The production and activity of MMP-9 in OVCAR5 cells were examined by Western blot and gelatin zymography analysis. The results showed that E₂ and selective GPER agonist G-1 increased cell motility and invasiveness, and upregulated the production and proteolytic activity of MMP-9 in OVCAR5 cells. Small interfering RNA (siRNA) targeting GPER and G protein inhibitor pertussin toxin (PTX) inhibited the migration and invasion of OVCAR5 cells, and also reduced the expression and activity of MMP-9. Our data suggested that GPER promoted the migration and invasion of ovarian cancer cells by increasing the expression and activity of MMP-9. GPER might play an important role in the progression of ovarian cancer.     

Haishengsu, a Protein from Shellfish Tegillarca L. granosa, Inhibits the Growth and the Activity of Matrix Metalloproteinases-2 and -9 in Human Lung Carcinoma

Haishengsu (HSS) is a seashell protein extracted from Tegillarca L. granosa, a type of Malaysian shellfish. Previous in vitro studies showed that HSS might possess biological anticancer activity. In this combined in vitro and in vivo study, we investigated the inhibitory effects of HSS on tumor growth, invasion, and metastasis using human lung carcinoma cell lines A549 and NCI-H292, both intensely positive for matrix metalloproteinases-2 (MMP-2) and MMP-9. HSS significantly inhibited the proliferation of A549 and NCI-H292 as estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The transwell chamber assay showed that HSS effectively blocked the invasion and migration of the carcinoma cells through the reconstituted extracellular matrix (Matrigel). Gelatin zymography analysis revealed that the secretion and activity of MMP-2 and MMP-9 in the supernatants of the cultured cells A549 and NCI-H292 were decreased after treatment with HSS. The levels of MMP-2 and MMP-9 in these cancer cells were further examined by Western blot assay in which a significant decrease of MMP-2 and MMP-9 was observed in A549 and NCI-H292 cells after 24 h of exposure to HSS. The anticancer activity of HSS was verified in a mouse model in which HSS delayed the growth of A549 xenografts after 3 weeks of oral administration. Inhibition of MMP-2 and MMP-9 expression was also demonstrated in the A549 xenografts as determined by Western blot analysis. These results suggest that HSS is a novel seashell protein that cannot only inhibit tumor growth but also prevent tumor invasion and metastasis through suppressing the activity of MMP-2 and MMP-9.     

EGCG inhibited bladder cancer SW780 cell proliferation and migration both in vitro and in vivo via down-regulation of NF-κB and MMP-9

Epigallocatechin-3-gallate (EGCG), the bioactive polyphenol in green tea, has been demonstrated to have various biological activities. Our study aims to investigate the antiproliferation and antimigration effects of EGCG against bladder cancer SW780 cells both in vitro and in vivo. Our results showed that treatment of EGCG resulted in significant inhibition of cell proliferation by induction of apoptosis, without obvious toxicity to normal bladder epithelium SV-HUC-1 cells. EGCG also inhibited SW780 cell migration and invasion at 25–100 μM. Western blot confirmed that EGCG induced apoptosis in SW780 cells by activation of caspases-8, -9 and -3, Bax, Bcl-2 and PARP. Besides, animal study demonstrated that EGCG [100 mg/kg, intraperitoneal (i.p.) injection daily for 3 weeks] decreased the tumor volume significantly in mice bearing SW780 tumors, as well as the tumor weight (decreased by 68.4%). In addition, EGCG down-regulated the expression of nuclear factor-kappa B (NF-κB) and matrix metalloproteinase (MMP)-9 in both protein and mRNA level in tumor and SW780 cells. When NF-κB was inhibited, EGCG showed no obvious effect in cell proliferation and migration. In conclusion, our study demonstrated that EGCG was effective in inhibition SW780 cell proliferation and migration, and presented first evidence that EGCG inhibited SW780 tumor growth by down-regulation of NF-κB and MMP-9.     

基质金属蛋白酶MMP-26能有效促进人绒毛膜上皮癌细胞JEG-3浸润能力

胚胎植入过程中,滋养层细胞浸润与肿瘤的迁移过程非常相似,但显著的区别在于前者是受严格调控的有节制的浸润,基质金属蛋白酶(MMPs)的许多成员在其中起重要的作用.MMP-26是近年来发现的MMPs家族的新成员,它在滋养层细胞中的作用所知甚少.利用国际常用的人滋养层细胞模型——人绒毛膜上皮癌细胞系(JEG-3)作为体外实验模型,探讨MMP-26在人滋养层细胞浸润调节中的作用.将含有MMP-26全长cDNA的pCR3.1质粒转染到JEG-3细胞中,获得过量表达MMP-26基因的稳定细胞系JEG-3/MMP-26;细胞浸润分析表明JEG/MMP-26细胞的浸润能力较母本细胞明显增强;RT-PCR和明胶酶谱分析显示JEG-3/MMP-26细胞中MMP-9的表达和分泌水平提高;双荧光免疫细胞化学进一步显示MMP-26和MMP-9蛋白在细胞中有共定位现象.上述结果表明MMP-26能有效促进人滋养层细胞浸润,其作用可能是通过与其他MMP分子(如MMP-9)的协调来实现的.     

Tumor-associated microglia/macrophages enhance the invasion of glioma stem-like cells via TGF-β1 signaling pathway

The invasion of malignant glioma cells into the surrounding normal brain tissues is crucial for causing the poor outcome of this tumor type. Recent studies suggest that glioma stem-like cells (GSLCs) mediate tumor invasion. However, it is not clear whether microenvironment factors, such as tumor-associated microglia/macrophages (TAM/Ms), also play important roles in promoting GSLC invasion. In this study, we found that in primary human gliomas and orthotopical transplanted syngeneic glioma, the number of TAM/Ms at the invasive front was correlated with the presence of CD133(+) GSLCs, and these TAM/Ms produced high levels of TGF-β1. CD133(+) GSLCs isolated from murine transplanted gliomas exhibited higher invasive potential after being cocultured with TAM/Ms, and the invasiveness was inhibited by neutralization of TGF-β1. We also found that human glioma-derived CD133(+) GSLCs became more invasive upon treatment with TGF-β1. In addition, compared with CD133(-) committed tumor cells, CD133(+) GSLCs expressed higher levels of type II TGF-β receptor (TGFBR2) mRNA and protein, and downregulation of TGFBR2 with short hairpin RNA inhibited the invasiveness of GSLCs. Mechanism studies revealed that TGF-β1 released by TAM/Ms promoted the expression of MMP-9 by GSLCs, and TGFBR2 knockdown reduced the invasiveness of these cells in vivo. These results demonstrate that TAM/Ms enhance the invasiveness of CD133(+) GSLCs via the release of TGF-β1, which increases the production of MMP-9 by GSLCs. Therefore, the TGF-β1 signaling pathway is a potential therapeutic target for limiting the invasiveness of GSLCs.     

锌离子存在下EGCG对前列腺癌细胞生长的影响

本文研究了锌离子存在下EGCG对前列腺癌细胞PC-3生长的影响．研究发现Zn^2＋可以增强EGCG抗癌活性,Zn^2＋存在下。EGCG处理后前列腺癌细胞PC-3克隆形成率显著下降。以RT—PCR、免疫组化方法研究Zn^2＋、EGCG对67kD层粘连蛋白受体（67kD Laminin Receptor,67LR）表达调控,结果表明Znn可通过上调67LR的表达,为EGCG提供更多作用的靶位点,增强EGCG对前列腺癌细胞PC-3的毒性作用。MMP-9是肿瘤侵袭转移过程中关键的基质金属蛋白酶。MMP-9活性与癌细胞的转移潜能密切相关。本文研究发现Zn^2＋、EGCG处理可通过抑制MMP-9活性,降低前列腺癌细胞PC-3的迁移率．其中80umol／LEGCG＋80umol／L Zn^2＋处理24h后显著抑制了PC-3细胞的迁移率。     

Kaposi's sarcoma-associated herpesvirus infection promotes invasion of primary human umbilical vein endothelial cells by inducing matrix metalloproteinases

Matrix metalloproteinases (MMPs) play important roles in cancer invasion, angiogenesis, and inflammatory infiltration. Kaposi's sarcoma is a highly disseminated angiogenic tumor of proliferative endothelial cells linked to infection by Kaposi's sarcoma-associated herpesvirus (KSHV). In this study, we showed that KSHV infection increased the invasiveness of primary human umbilical vein endothelial cells (HUVEC) in a Matrigel-based cell invasion assay. KSHV-induced cell invasion was abolished by an inhibitor of MMPs, BB-94, and occurred in both autocrine- and paracrine-dependent fashions. Analysis by zymography and Western blotting showed that KSHV-infected HUVEC cultures had increased secretion of MMP-1, -2, and -9. KSHV increased the secretion of MMP-2 within 1 h following infection without upregulating its mRNA expression level. In contrast, the secretion of MMP-1 and -9 was not increased until 6 h after KSHV infection and was correlated with the upregulation of their mRNA expression levels. Promoter analysis by reporter assays and electrophoretic mobility shift assays identified an AP-1 cis-element as the dominant KSHV-responsive site in the MMP-1 promoter. Together, these results suggest that KSHV infection modulates the production of multiple MMPs to increase cell invasiveness and thus contributes to the pathogenesis of KSHV-induced malignancies.     

Autocrine/paracrine prostaglandin E2 production by non-small cell lung cancer cells regulates matrix metalloproteinase-2 and CD44 in cyclooxygenase-2-dependent invasion

Tumor cyclooxygenase-2 (COX-2) expression is known to be associated with enhanced tumor invasiveness. In the present study, we evaluated the importance of the COX-2 product prostaglandin E2 (PGE2) and its signaling through the EP4 receptor in mediating non-small cell lung cancer (NSCLC) invasiveness. Genetic inhibition of tumor COX-2 led to diminished matrix metalloproteinase (MMP)-2, CD44, and EP4 receptor expression and invasion. Treatment of NSCLC cells with exogenous 16,16-dimethylprostaglandin E2 significantly increased EP4 receptor, CD44, and MMP-2 expression and matrigel invasion. In contrast, anti-PGE2 decreased EP4 receptor, CD44, and MMP-2 expression in NSCLC cells. EP4 receptor signaling was found to be central to this process, because antisense oligonucleotide-mediated inhibition of tumor cell EP4 receptors significantly decreased CD44 expression. In addition, agents that increased intracellular cAMP, as is typical of EP4 receptor signaling, markedly increased CD44 expression. Moreover, MMP-2-AS treatment decreased PGE2-mediated CD44 expression, and CD44-AS treatment decreased MMP-2 expression. Thus, PGE2-mediated effects through EP4 required the parallel induction of both CD44 and MMP-2 expression because genetic inhibition of either MMP-2 or CD44 expression effectively blocked PGE2-mediated invasion in NSCLC. These findings indicate that PGE2 regulates COX-2-dependent, CD44- and MMP-2-mediated invasion in NSCLC in an autocrine/paracrine manner via EP receptor signaling. Thus, blocking PGE2 production or activity by genetic or pharmacological interventions may prove to be beneficial in chemoprevention or treatment of NSCLC.