Sesquiterpene Lactones Downregulate G2/M Cell Cycle Regulator Proteins and Affect the Invasive Potential of Human Soft Tissue Sarcoma Cells

Soft tissue sarcomas (STS) represent a rare group of malignant tumors that frequently exhibit chemotherapeutic resistance and increased metastatic potential. Many studies have demonstrated the great potential of plant-derived agents in the treatment of various malignant entities. The present study investigates the effects of the sesquiterpene lactones costunolide and dehydrocostus lactone on cell cycle, MMP expression, and invasive potential of three human STS cell lines of various origins. Both compounds reduced cell proliferation in a time- and dose-dependent manner. Dehydrocostus lactone significantly inhibited cell proliferation, arrested the cells at the G2/M interface and caused a decrease in the expression of the cyclin-dependent kinase CDK2 and the cyclin-dependent kinase inhibitor p27^Kip1. In addition, accumulation of cells at the G2/M phase transition interface resulted in a significant decrease in cdc2 (CDK1) together with cyclin B1. Costunolide had no effect on the cell cycle. Based on the fact that STS tend to form daughter cell nests and metastasize, the expression levels of matrix metalloproteinases (MMPs), which play a crucial role in extracellular matrix degradation and metastasis, were investigated by Luminex® technology and real-time RT-PCR. In the presence of costunolide, MMP-2 and -9 levels were significantly increased in SW-982 and TE-671 cells. Dehydrocostus lactone treatment significantly reduced MMP-2 and -9 expression in TE-671 cells, but increased MMP-9 level in SW-982 cells. In addition, the invasion potential was significantly reduced after treatment with both sesquiterpene lactones as investigated by the HTS FluoroBlock™ insert system.     

Specific blockade of the ERK pathway inhibits the invasiveness of tumor cells: down-regulation of matrix metalloproteinase-3/-9/-14 and CD44

Elevated expression of matrix metalloproteinases (MMPs) is associated with increased metastatic potential in many tumor cells. As activation of the ERK pathway has been linked to the expression of MMP-9, we examined a possible correlation between ERK activation, MMP-9 expression, and invasive phenotype in human tumor cells. Activation state of the ERK pathway in tumor cells was well correlated with the invasive phenotype, which was determined by the ability of cells to invade through reconstituted extracellular matrix. Elevated expression of MMP-9 as well as of MMP-3, MMP-14, and CD44 was observed in tumor cells in which constitutive activation of the ERK pathway is detected. Blockade of the ERK pathway by treatment with PD184352, a specific and powerful inhibitor of mitogen-activated protein (MAP) kinase/ERK kinase (MEK), suppressed the expression of MMP-3, MMP-9, MMP-14, and CD44, and inhibited markedly the invasiveness of tumor cells. These results imply that, in addition to anti-proliferative effects, specific blockade of the ERK pathway is expected to result in anti-metastatic effects in tumor cells.     

The progression in the mouse skin carcinogenesis model correlates with ERK1/2 signaling

BACKGROUND: The ras family of proto-oncogenes encodes for small GTPases that play critical roles in cell-cycle progression and cellular transformation. ERK1/2 MAP kinases are major ras effectors. Tumors in chemically treated mouse skin contain mutations in the Ha-ras proto- oncogene. Amplification and mutation of Ha-ras has been shown to correlate with malignant progression of these tumors. Cell lines isolated from mouse skin tumors represent the stages of tumor development, such as the PDV:PDVC57 cell line pair and B9 squamous carcinoma and A5 spindle cells. PDVC57 cells were selected from PDV cells, which were transformed with dimethyl-benzanthracene (DMBA) in vitro and then transplanted in adult syngeneic mice. The PDV:PDVC57 pair contains ratio of normal:mutant Ha-ras 2:1 and 1:2, respectively. This genetic alteration correlates with more advanced tumorigenic characteristics of PDVC57 compared to PDV. The squamous carcinoma B9 cell clone was isolated from the same primary tumor as A5 spindle cell line. The mutant Ha-ras allele, also present in B9, is amplified and overexpressed in A5 cells. Therefore these cell line pairs represent an in vivo model for studies of Ha-ras and ERK1/2 signaling in mouse tumorigenesis. MATERIALS AND METHODS: The ERK1/2 status in the above mouse cell lines was examined by using various molecular techniques. For the study of the tumorigenic properties and the role of the ras/MEK/ERK1/2 pathway in the cell lines mentioned, phenotypic characteristics, colony formation assay, anchorage-independent growth, and gelatin zymography were assessed, after or without treatment with the MEK inhibitor, PD98059. RESULTS: ERK1/2 phosphorylation was found to be increased in PDVC57 when compared to PDV. This also applies to A5 spindle carcinoma cells when compared to squamous carcinoma and papilloma cells. The above finding was reproduced when transfecting human activated Ha-ras allele into PDV, thus demonstrating that Ha-ras enhances ERK1/2 signaling. To further test whether ERK1/2 activation was required for growth we used the MEK-1 inhibitor, PD98059. The latter inhibited cell proliferation and anchorage-independent growth of squamous and spindle cells. In addition, PD98059 treatment partially reverted the spindle morphology of A5 cells. CONCLUSIONS: These data suggest, for the first time, that oncogenicity and the degree of progression in the mouse skin carcinogenesis model correlates with ERK1/2 signaling.     

Matrix metalloproteinases and their endogenous regulators in squamous cervical carcinoma (A review of own results)

Own results of long-term studies of expression of matrix metalloproteinases (MMPs) and their endogenous regulators examined in fibroblasts transformed by oncogene E7 HPV16 (TF), immortalized fibroblasts (IF), cell lines associated with HPV16 and HPV18, and tumor tissue samples from patients with squamous cervical carcinoma (SCC) associated with HPV16 have been summarized. Transfection of fibroblasts with the E7 HPV16 oncogen was accompanied by induction of collagenase (MMP-1, MMP-14) and gelatinase (MMP-9) gene expression and the increase in catalytic activity of these MMP, while gelatinase MMP-2 expression remained unchanged. MMP expression correlated with the tumorigenic of transformed clones. Expression of MMP-9 was found only in TF. In TF expression mRNA TIMP-1 decreased, while expression of the genatinase inhibitor, TIMP-2, increased. Collagenase activity and expression of the MMP-14 (collagenase) mRNA increased, while gelatinase activity remained unchanged. The destructive potential of TF is associated with induction of collagenases, gelatinase MMP-9 and decreased levels of MMP inhibitors. MMP-9 may serve as a TF marker. Invasive potential of cell lines associated with HPV18 (HeLa and S4-1) was more pronounced than that of cell lines associated with HPV16 (SiHa and Caski). In most cell lines mRNA levels of collagenases MMP-1 and MMP-14 and the activator (uPA) increased, while gelatinase MMP-2 mRNA and tissue inhibitors mRNAs changed insignificantly. MMP-2 activity significantly increased in Caski and HeLa cell lines, while MMP-9 expression in these cell lines was not detected. The comparative study of expression MMP of and their endogenous regulators performed using SCC tumor samples associated with HPV16 has shown that the invasive and metastatic potentials of tumor tissue in SCC is obviously associated with increased expression of collagenases MMP-1, MMP-14 and gelatinase MMP-9, as well as decreased expression of inhibitors (TIMP-1 and TIMP-2), and to a lesser extent with increased expression of MMP-2. MMP-1 and MMP-9 can serve as markers of invasive and metastatic potential of the SCC tumor. The morphologically normal tissue adjacent to the tumor tissue is characterized by significant expression of MMP-1, MMP-2, and MMP-9. This also contributes to the increased destructive potential of the tumor.     

Mitogen-Activated Protein Kinase Kinase 4 (MAP2K4) Promotes Human Prostate Cancer Metastasis

Prostate cancer (PCa) is the second leading cause of cancer death in the US. Death from PCa primarily results from metastasis. Mitogen-activated protein kinase kinase 4 (MAP2K4) is overexpressed in invasive PCa lesions in humans, and can be inhibited by small molecule therapeutics that demonstrate favorable activity in phase II studies. However, MAP2K4''s role in regulating metastatic behavior is controversial and unknown. To investigate, we engineered human PCa cell lines which overexpress either wild type or constitutive active MAP2K4. Orthotopic implantation into mice demonstrated MAP2K4 increases formation of distant metastasis. Constitutive active MAP2K4, though not wild type, increases tumor size and circulating tumor cells in the blood and bone marrow. Complementary in vitro studies establish stable MAP2K4 overexpression promotes cell invasion, but does not affect cell growth or migration. MAP2K4 overexpression increases the expression of heat shock protein 27 (HSP27) protein and protease production, with the largest effect upon matrix metalloproteinase 2 (MMP-2), both in vitro and in mouse tumor samples. Further, MAP2K4-mediated increases in cell invasion are dependent upon heat shock protein 27 (HSP27) and MMP-2, but not upon MAP2K4''s immediate downstream targets, p38 MAPK or JNK. We demonstrate that MAP2K4 increases human PCa metastasis, and prolonged over expression induces long term changes in cell signaling pathways leading to independence from p38 MAPK and JNK. These findings provide a mechanistic explanation for human studies linking increases in HSP27 and MMP-2 to progression to metastatic disease. MAP2K4 is validated as an important therapeutic target for inhibiting human PCa metastasis.     

Metastatic function of BMP-2 in gastric cancer cells: the role of PI3K/AKT, MAPK, the NF-κB pathway, and MMP-9 expression

Bone morphogenetic proteins (BMPs) have been implicated in tumorigenesis and metastatic progression in various types of cancer cells, but the role and cellular mechanism in the invasive phenotype of gastric cancer cells is not known. Herein, we determined the roles of phosphoinositide 3-kinase (PI3K)/AKT, extracellular signal-regulated protein kinase (ERK), nuclear factor (NF)-κB, and matrix metalloproteinase (MMP) expression in BMP-2-mediated metastatic function in gastric cancer. We found that stimulation of BMP-2 in gastric cancer cells enhanced the phosphorylation of AKT and ERK. Accompanying activation of AKT and ERK kinase, BMP-2 also enhanced phosphorylation/degradation of IκBα and the nuclear translocation/activation of NF-κB. Interestingly, blockade of PI3K/AKT and ERK signaling using LY294002 and PD98059, respectively, significantly inhibited BMP-2-induced motility and invasiveness in association with the activation of NF-κB. Furthermore, BMP-2-induced MMP-9 expression and enzymatic activity was also significantly blocked by treatment with PI3K/AKT, ERK, or NF-κB inhibitors. Immunohistochemistry staining of 178 gastric tumor biopsies indicated that expression of BMP-2 and MMP-9 had a significant positive correlation with lymph node metastasis and a poor prognosis. These results indicate that the BMP-2 signaling pathway enhances tumor metastasis in gastric cancer by sequential activation of the PI3K/AKT or MAPK pathway followed by the induction of NF-κB and MMP-9 activity, indicating that BMP-2 has the potential to be a therapeutic molecular target to decrease metastasis.     

Calcium-induced matrix metalloproteinase 9 gene expression is differentially regulated by ERK1/2 and p38 MAPK in oral keratinocytes and oral squamous cell carcinoma

Matrix metalloproteinases (MMPs) play an important role in the invasive behavior of a number of cancers including oral squamous cell cancer (OSCC), and increased expression of MMP-9 is correlated with invasive and metastatic OSCC. Because calcium is an important regulator of keratinocyte function, the effect of modulating extracellular calcium on MMP-9 expression in OSCC cell lines was evaluated. Increasing extracellular calcium induced a dose-dependent increase in MMP-9 expression in immortalized normal and premalignant oral keratinocytes, but not in two highly invasive OSCC cell lines. Differential activation of MAPK signaling was also induced by calcium. p38 MAPK activity was down-regulated, whereas ERK1/2 activity was enhanced. Pharmacologic inhibition of p38 MAPK activity or expression of a catalytically inactive mutant of the upstream kinase MAPK kinase 3 (MKK3) increased the calcium induced MMP-9 gene expression, demonstrating that p38 MAPK activity negatively regulated this process. Interestingly blocking p38 MAPK activity enhanced ERK1/2 phosphorylation, suggesting reciprocal regulation between the ERK1/2 and p38 MAPK pathways. Together these data support a model wherein calcium-induced MMP-9 expression is differentially regulated by the ERK1/2 and p38 MAPK pathways in oral keratinocytes, and the data suggest that a loss of this regulatory mechanism accompanies malignant transformation of the oral epithelium.     

Collagenase expression and activity is modulated by the interaction of collagen types, hypoxia, and nutrition in human lung cells

Hypoxia not only controls organogenesis, embryogenesis, and wound repair, but also triggers tumor progression and metastasis. Matrix metalloproteinases (MMP), especially gelatinases (MMP-2, MMP-9) regulate the composition and stability of the extracellular matrix (ECM), which affects cell proliferation, migration, and differentiation. This study investigated the effect of hypoxia alone and in combination with ECM compounds and nutrition on MMP-2 and MMP-9 expression, activity, and synthesis in human lung fibroblasts and pulmonary vascular smooth muscle cells (VSMC). We also determined the expression of the tissue inhibitors of MMP (TIMP-1, -2). Cells were grown on plastic, collagen-I, collagen-IV, or gelatin and in either starving medium (0.1% serum) or growth medium (5% serum), and were subjected to normoxia or hypoxia (1% O(2)). Collagenases expression was determined by zymography. TIMP-1, -2 expression was assessed by Western blotting and RT-PCR. Depending on serum concentration human lung cells expressed pro-MMP-2 on all substrates. Hypoxia increased pro-MMP-2 expression, on collagen type I or type IV further via Erk1/2 and p38 MAP kinase signaling. MMP-9 was only expressed when cells were grown on collagen type IV and increased with serum concentration, and by hypoxia. TIMP-1 expression was only expressed when cells were grown on collagen type I and was significantly increased by hypoxia, while TIMP-2 expression was unchanged. We demonstrated that the hypoxia, ECM composition, and nutrition, rather than one of these conditions alone, modulate the expression and activity of collagenases and their inhibitors in primary human lung fibroblasts.     

Mixed lineage kinase 3 is required for matrix metalloproteinase expression and invasion in ovarian cancer cells

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates MAPK signaling pathways and regulates cellular responses such as proliferation, migration and apoptosis. Here we report high levels of total and phospho-MLK3 in ovarian cancer cell lines in comparison to immortalized nontumorigenic ovarian epithelial cell lines. Using small interfering RNA (siRNA)-mediated gene silencing, we determined that MLK3 is required for the invasion of SKOV3 and HEY1B ovarian cancer cells. Furthermore, mlk3 silencing substantially reduced matrix metalloproteinase (MMP)-1, -2, -9 and -12 gene expression and MMP-2 and -9 activities in SKOV3 and HEY1B ovarian cancer cells. MMP-1, -2, -9 and-12 expression, and MLK3-induced activation of MMP-2 and MMP-9 requires both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activities. In addition, inhibition of activator protein-1 (AP-1) reduced MMP-1, MMP-9 and MMP-12 gene expression. Collectively, these findings establish MLK3 as an important regulator of MMP expression and invasion in ovarian cancer cells.     

ROR2 promotes the epithelial-mesenchymal transition by regulating MAPK/p38 signaling pathway in breast cancer

Receptor tyrosine kinase-like orphan receptor 2 (ROR2) is a tyrosine-protein kinase receptor highly implicated in the growth plate and cartilage development, which may be involved in epithelial-mesenchymal transition (EMT) in breast cancer (BC) cells. Although ROR2 is known to promote the migration of BC cells, the detailed mechanism of this event is still not clear. Here, we found that ROR2 expression was significantly increased in BC lymphatic metastatic tissue as well as BC samples compared to normal adjacent breast tissues. A higher expression of ROR2 in MDA-MB-231 and a lower expression of ROR2 in MCF-7 cells were observed. MDA-MB-231-siROR2 cells with ROR2 knockdown inhibited MDA-MB-231 cell invasion, migration, and clonal formation, while MCF-7-OvROR2 cells with overexpression showed the opposite results. The underlying mechanisms involved in ROR2-induced EMT in MDA-MB-231 and MCF-7 cells were further investigated. ROR2 may activate EMT progression in BC cells by altering MAPK kinase 3/6 (MKK3/6) expression. The expressions of transforming growth factor-β, matrix metalloproteinase-2 (MMP-2), and MMP-9, which were related to tumor cell invasion activities, were notably increased in MCF-7-OvROR2 cells. The EMT markers, including snail, N-cadherin, tissue inhibitor of metalloproteinases-1, and vimentin, were significantly upregulated in MCF-7-OvROR2 cells. On the contrary, E-cadherin was obviously reduced expressed in MCF-7-OvROR2 cells. ROR2 may regulate the malignant phenotype of BC cells possibly via activation of mitogen-activated protein kinase (MAPK)/p38 signaling pathway. Collectively, ROR2 promotes BC carcinogenesis by mediating the MAPK/p38 pathway, which is independent of Wnt5α.     

Ras induces anchorage-independent growth by subverting multiple adhesion-regulated cell cycle events.

Anchorage-independent growth is a hallmark of transformed cells, but little is known of the molecular mechanisms that underlie this phenomenon. We describe here studies of cell cycle control of anchorage-independent growth induced by the ras oncogene, with the use of a somatic cell mutant fibroblast line (ER-1-2) that is specifically defective in oncogene-mediated, anchorage-independent growth. Control, nontransformed PKC3-F4 cells and ER-1-2 cells cannot proliferate in semisolid medium. Three important cell cycle events are dependent on adhesion of these cells to a substratum: phosphorylation of the retinoblastoma protein, pRB; cyclin E-dependent kinase activity; and cyclin A expression. PKC3-F4 cells that express ras (PKC3-F4/ras cells) proliferate in nonadherent cultures, and each of these three events occurs in the absence of adhesion in PKC3-F4/ras cells. Thus, ras can override the adhesion requirement of cellular functions that are necessary for cell cycle progression. ER-1-2 cells that express ras (ER-1-2/ras cells) possess hyperphosphorylated forms of pRB and cyclin E-dependent kinase activity in the absence of adhesion but remain adhesion dependent for expression of cyclin A. The adhesion dependence of pRB phosphorylation and cyclin E-dependent kinase activity is therefore dissociable from the adhesion dependence of cyclin A expression. Furthermore, ectopic expression of cyclin A is sufficient to rescue anchorage-independent growth of ER-1-2/ras cells but does not induce anchorage-independent growth of PKC3-F4 or ER-1-2 cells. However, like pRB phosphorylation and cyclin E-dependent kinase activity, the kinase activity associated with ectopically expressed cyclin A is dependent on cell adhesion, and this dependence is overcome by ras. Thus, the induction of anchorage-independent growth by ras may involve multiple signals that lead to both expression of cyclin A and activation of G1 cyclin-dependent kinase activities in the absence of cell adhesion.     

Phosphorylated c-Jun and Fra-1 induce matrix metalloproteinase-1 and thereby regulate invasion activity of 143B osteosarcoma cells

Osteosarcoma is the most common primary malignancy of bone and patients often develop pulmonary metastases. Despite the advances in surgical and medical management, the mechanisms underlying human osteosarcoma progression and metastasis remain to be elucidated. Gene expression profiles were compared by the cDNA microarray technique between two different human osteosarcoma sublines, MNNG/HOS and 143B, which differ greatly in spontaneous pulmonary metastatic potential. Here we report an enhanced expression of matrix metalloproteinase (MMP)-1 in the highly metastatic human osteosarcoma cell line 143B. Moreover, the in vitro invasion activity of 143B cells was MMP-1-dependent. The activator protein (AP)-1 binding site in the MMP-1 gene promoter was required for the constitutive expression of MMP-1 in 143B cells. Two AP-1 components, c-Jun and Fra-1, were phosphorylated, and bound to the AP-1 binding site of the MMP-1 promoter in 143B cells. Activated c-Jun and Fra-1 were essential for MMP-1 gene expression in 143B cells. Mitogen-activated protein kinase pathways including the c-Jun NH₂-terminal kinase and the extracellular signal-regulated kinase activate c-Jun and Fra-1 and thereby regulate c-Jun/Fra-1 mediated events, establishing the mitogen-activated protein kinase/AP-1/MMP-1 axis as important in 143B cells. These data suggest that MMP-1 plays a central role in osteosarcoma invasion. Accordingly, MMP-1 might be a biomarker and therapeutic target for invasive osteosarcomas and pulmonary metastases.     

p38 MAP kinase regulates rapid matrix metalloproteinase-9 release from eosinophils

Eosinophils constitutively produce and store matrix metalloproteinase-9 (MMP-9), a protease implicated in tissue remodeling observed in asthma. In this study, we examined the rapid release of stored MMP-9 from eosinophils following stimulation with either tumor necrosis factor-alpha (TNF-alpha or the bacterial product fMLP. TNF-alpha induced rapid and robust pro-MMP-9 release from eosinophils. MMP-9 could be detected in the cell-free supernatant as early as 15min after stimulation. Rapid MMP-9 release was similarly induced by fMLP. TNF-alpha stimulation activated the mitogen-activated protein (MAP) kinases p38 MAP kinase and extracellular signal-regulated kinase-2 (Erk-2) at times and concentrations similar to that observed for MMP-9 release. Using pharmacological inhibitors, we found that TNF-alpha-stimulated MMP-9 release was mediated by p38 MAP kinase, but not Erk-1/2. Signaling through p38 MAP kinase may represent a universal mechanism for MMP-9 release from eosinophils, as fMLP-induced MMP-9 release was also regulated by p38 MAP kinase.     

海参多糖抑制人肾癌细胞A498的生长转移作用机制

肾细胞癌(RCC)是最常见的恶性癌之一,癌症转移是目前导致肾癌患者死亡的主要原因之一。MMP-9被发现在许多具有侵袭性和转移能力的人类癌症中过表达,其表达和分泌受到NF-κB调控;VEGF在维持原发性癌和转移瘤生长所需的血管生成中发挥重要作用,其表达也受到活化的NF-κB调节。海参的多种活性物质在抗氧化、抗菌和抗癌方面都有出色的作用,而抗癌的主要机制则包括诱导癌细胞凋亡、抑制癌细胞生长、减少癌细胞转移等。本研究通过利用不同浓度的海参多糖处理人肾癌细胞A498,采用MTT细胞增殖实验、粘附实验、迁移实验和小室侵袭实验,研究了海参多糖对A498细胞的生长转移的影响;采用蛋白印记法检测了海参多糖对A498细胞内MMP-9、NF-κBp65和VEGF表达水平的影响。结果表明,海参多糖能够显著抑制A498细胞的增殖活力、粘附能力、迁移能力和侵袭能力,并且全都表现出明显的剂量依赖性;中浓度(100μg/mL)和高浓度(200μg/mL)的海参多糖能够显著下调A498细胞内MMP-9、NF-κBp65和VEGF的表达。这些结果说明海参多糖能有效抑制人肾癌细胞A498的生长、转移和侵袭,可能的机制是通过抑制NF-κB信号通路下调MMP-9和VEGF的表达,从而发挥抗癌细胞转移的作用。