Inhibited Cancer Invasion Research Articles

Black Tea Polyphenols Reverse Epithelial-to-Mesenchymal Transition and Suppress Cancer Invasion and Proteases in Human Oral Cancer Cells

Epithelial-to-mesenchymal transition (EMT) in cancer cells is considered to be a prerequisite for acquiring invasive/migratory phenotype and subsequent metastasis. This study provides molecular evidence associated with the antimetastatic effect of black tea polyphenol extracts (BTE), which contain polyphenols including gallic acid, gallocatechin, catechin, epigallocatechin-3-gallate, epicatechin-3-gallate, and theaflavin 3,3'-digallate, in an an oral squamous cell culture system by showing a nearly complete inhibition on the invasion (p < 0.001) of SCC-4 cells via reduced activities of MMP-2 (p < 0.001) and u-PA (p < 0.001). Immunoblot was performed to find that BTE could induce up-regulation of epithelial markers such as E-cadherin and inhibit mesenchymal markers such as snail-1 and vimentin. BTE inhibited p-FAK and p-paxillin, indicating the anti-EMT effect of BTE in oral squamous cell carcinoma. BTE was evidenced by its inhibition of the tumor growth of SCC-4 cells via cancer cell xenografted nude mice mode. These results suggested that BTE could reduce invasion by reversing EMT in human oral cancer cells.

Impact of miRNA-23a-p21-activated kinase 6 signaling on the migration and invasion activities of prostate cancer cells

Objective To investigate the role of the p21-activated kinase 6 (PAK6) and its target microRNA (miRNA) on prostate cancer cells migration and invasion.Methods MiRNA candidates which potentially target PAK6 was predicted by target prediction programs.The expression of PAK6 were measured by real-time polymerase chain reaction ( Real-time PCR ) and Western blotting after PC-3 cells were transfected with miR-23a mimics or inhibitor oligonucleotides.Luciferase reporter assay was used to determine whether PAK6 was the direct target of miR-23a.Cell migration and invasion were detected hy matrigel invasion assay and transwell migration assay.Results MiRNA 23a was identified by target prediction programs. Exogenetic overexpression of miR-23a resulted in a remarkable decrease of PAK6 expression (79％),whereas miR-23a inhibitor oligonucleotides induced pronounced increase of PAK6 expression (40％).The luciferase activities were significantly inhibited 32％ in wild-type PAK6 group ( P＜0.05 ),while that were no significant difference in the mutation group (P＞0.05). PAK6 mRNA levels had no changed as detected by real-time PCR( P＞0.05). Matrigel invasion assay and transwell migration assay demonstrated that exogenetic overexpression of miR-23a markedly reduced migration and invasion of PC-3 cells (57％,91％ ).Conclusion MiRNA-23a inhibited prostate cancer cell migration and invasion by repressing PAK6. Key words: MicroRNA; p21-activated kinase 6; Prostate cancer; Migration; Invasion

Abstract 3432: HLJ1 serves as an inhibitor of epithelial-mesenchymal transition by inactivating β-catenin signaling

Abstract HLJ1 (DnaJ-like heat shock protein), also known as DNAJB4, was classified as a tumor suppressor gene. The expression of HLJ1 inversely associates with invasive ability and can predict the clinical outcomes of non-small-cell lung cancer (NSCLC) patients. However, the role of HLJ1 in suppressing cancer metastasis is still unclear. Here, we identified HLJ1 as a novel inhibitor of epithelial-mesenchymal transition (EMT). Our functional assays in vitro and in vivo demonstrated that Knock-down of HLJ1 promotes invasion capabilities of lung cancer cells and causes liver macrometastasis; meanwhile, the crucial domain of HLJ1 in suppression of cancer invasion was also confirmed. We found that the HLJ1-overexpressing cells maintain the well-organized cell adhesion and polarity. Inversely, an increase in actin polymerization and the formation of actin stress fibers were observed in the HLJ1-silencing cells. HLJ1 down-regulates the transcriptional activities of slug promoter and facilitates E-cadherin transcription, leading to the inhibition of epithelial to mesenchymal transition. Further, we identified the Wnt/β-catenin signalling pathway in which HLJ1 involved by cDNA microarray and pathway analysis. The subcellular distribution of β-catenin is predominant in the nucleus and the tyrosine-phosphorylated levels of β-catenin are up-regulated in the HLJ1-silenced clones. Finally, we showed a decreased accumulation of β-catenin and an enhanced degradation of β-catenin in absence of HLJ1 expression. Taken together, our study reveals that HLJ1 has anti-metastatic activity by repressing β-catenin signaling and provides a molecular model for the development of new anticancer agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3432. doi:1538-7445.AM2012-3432

Abstract B74: EGCG inhibits cancer invasion by regulating tumor-stromal crosstalk in oral squamous cell carcinoma

Abstract Introduction: Epigallocatechin-3-O-gallate (EGCG), a major constituent polyphenol of green tea, has been shown to have suppressive effects on the invasion of various cancer cells, whereas the exact mechanisms have not yet been fully elucidated. In this study, the effects of EGCG on the invasion of oral squamous cell carcinoma (OSCC) were investigated where the new perspective focused on tumor-stromal crosstalk was emphasized, unlikely other previous studies focused on cancer cell mainly. Materials and Methods: OSCC were cultured with or without carcinoma-associated fibroblasts (CAF) in the dose dependent treatment of EGCG. In order to investigate the invasiveness, invasion assay and zymography were examined. Based on our previous study found that several cytokines are concerned with tumor-stromal crosstalk in invasion of OSCC, RT-PCR and ELISA assay were employed to examine the influence of EGCG on the expression levels of cytokines. Results: The invasiveness of OSCC was reduced by EGCG treatment, and the 50% reduction was demonstrated at the half concentration of EGCG (25 μM) in co-cultured group with CAF, compared to OSCC mono-cultured (50 μM). Zymography showed that EGCG treatment reduced MMP-9 expression in co-cultured condition. In addition, EGCG resulted in a noticeable decrease in the expression of cytokines, especially GRO-α/CXCL1, concerned with tumor-stromal crosstalk in invasion of OSCC. Conclusions: In this study, inhibitory effect of EGCG on cancer invasion was found at which its concentration is lower than previously known. Taking these results into consideration, EGCG may play an effective role in preventing invasion of OSCC by regulating tumor-stromal crosstalk. Acknowledgement: This work was supported by Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010–0029702). Citation Information: Cancer Prev Res 2011;4(10 Suppl):B74.

Abstract 2360: Dimethyl sulfoxide induces upregulation of tumor suppressor HLJ1 through activator protein-1 activation in human lung adenocarcinoma CL1-5 cells

Abstract Dimethyl sulfoxide (DMSO) is an amphipathic molecule and has a diversity of antitumor activities. Previous studies demonstrated that DMSO could modulate AP-1 activity and lead to cell cycle arrest at G1 phase. HLJ1 is a newly identified tumor and invasion suppressor that inhibits tumorigenesis and cancer metastasis. Its transcriptional activity is also regulated by transcription factor AP-1. However, the effect of DMSO on HLJ1 is still unknown. In this study, low-HLJ1 expressing highly invasive CL1-5 lung adenocarcinoma cells were subjected to DMSO treatment. We found that DMSO can significantly inhibit cancer cell invasion and migration capabilities through up-regulation of HLJ1 in a concentration-dependent manner. In addition, knockdown of HLJ1 expression by siRNA was able to block the effects of DMSO-induction. The HLJ1 promoter and enhancer reporter assay revealed that DMSO transcriptionally upregulates HLJ1 expression through an AP-1 site within the HLJ1 enhancer. Furthermore, AP-1 subfamily members, JunD and JunB, were significantly upregulated by DMSO in a concentration-dependent manner. In conclusion, our results suggest that DMSO could be an important stimulator of the tumor suppressor protein, HLJ1, through AP-1 activation in highly invasive lung adenocarcinoma CL1-5 cells. Our findings provide some important information for further investigations to develop new drugs such as DMSO-derived analogues which may inhibit cancer invasion and progression by targeting HLJ1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2360. doi:10.1158/1538-7445.AM2011-2360

Methylation of an intronic region regulates miR-199a in testicular tumor malignancy

In the testicular cancer cell line, NT2, we previously demonstrated that differentially methylated regions were located in introns or intergenic regions, and postulated these might regulate non-coding RNAs. Three microRNAs and three small nucleolar RNAs were differentially methylated; one, miR-199a, was associated with the progression and prognosis of gastric and ovarian cancers. In this report we document, by epigenomic profiling of testicular tissue, that miR-199a is transcribed as antisense of dynamin 3 (chromosome 1q24.3), and hypermethylation of this region is correlated with miR-199a-5p/3p repression and tumor malignancy. Re-expression of miR-199a in testicular cancer cells led to suppression of cell growth, cancer migration, invasion and metastasis. The miR-199a-5p, one of two mature miRNA species derived from miR-199a, is associated with tumor malignancy. We further identified the embryonal carcinoma antigen podocalyxin-like protein 1 (PODXL), an anti-adhesive protein expressed in aggressive tumors, as a target of miR-199a-5p. We demonstrated PODXL is overexpressed in malignant testicular tumor, and cellular depletion of PODXL resulted in suppression of cancer invasion. The inverse relationship between PODXL and miR-199a-5p expression suggests PODXL is a downstream effector mediating the action of miR199a-5p. This report identifies DNA methylation, miR-199a dysregulation and PODXL as critical factors in tumor malignancy.

RECK in osteosarcoma

Targeted therapy in osteosarcoma (OS) is needed to improve patient outcomes. Human RECK may have a role because it inhibits cancer invasion and regulates angiogenesis. This study aimed to characterize RECK expression in human OS, to examine in vitro effects of RECK on vascular endothelium and OS cell behavior, and to analyze the effect of RECK on OS grown orthotopically in nude mice. RECK was examined in human OS samples. Interactions between RECK and VEGF were studied in tissue and cells. RECK transfection was used to study its effects on vascular endothelial (HMEC-1) and OS (SaOS-2) cell behavior in vitro and in vivo. SaOS-2 co-culture with RAW 246.7-derived osteoclasts on osteoslides was used to assess effects on osteoclast activity. RECK was absent from OS cells but was expressed in tumor vessel endothelium. Via microarray analysis, RECK mRNA was elevated in samples with low proliferative activity, a trend most evident in poorly differentiated samples. VEGF induced RECK expression in HMEC-1. RECK transfection inhibited HMEC-1 invasion and induced thicker, although more numerous, tube formation. RECK inhibited SaOS-2 invasion, proliferation, colony formation, and osteoclast activity but supported SaOS-2 adhesion to collagen I. In vivo, RECK inhibited SaOS-2 tumor growth, bone destruction, and consequent metastasis. RECK expression is downregulated in highly proliferative OS but is present in tumor vessels and upregulated in endothelium by VEGF. RECK inhibits invasion and tumorigenic properties in SaOS-2, as confirmed in vivo. Further testing of RECK delivery in OS is warranted.

Abstract MS1-2: Long Noncoding RNAs in Breast Cancer Progression

Abstract Long intergenic noncoding RNAs (lincRNAs) are pervasively transcribed in the genome, yet their potential involvement in human disease is not well understood. Recent studies of dosage compensation, imprinting, and homeotic gene expression suggest that individual lincRNAs can function as the interface between DNA and specific chromatin remodeling activities. We found that many lincRNAs become systematically dysregulated during breast cancer progression. A lincRNA termed HOTAIR is increased in expression in primary breast tumors and metastases, and HOTAIR expression level in primary tumors is a powerful predictor of eventual metastasis and death. Enforced expression of HOTAIR in epithelial cancer cells induced genome-wide re-targeting of Polycomb Repressive Complex 2 (PRC2) to an occupancy pattern more resembling embryonic fibroblasts, leading to altered histone H3 lysine 27 methylation, gene expression, and increased cancer invasiveness and metastasis in a manner dependent on PRC2. Conversely, loss of HOTAIR can inhibit cancer invasiveness, particularly in cells that possess excessive PRC2 activity. A second long noncoding RNA, named PANDA, is part of the DNA damage response mediated by p53 and controls the balance between cell death and cell cycle arrest. Collectively, these findings suggest that lincRNAs play active roles in modulating the cancer epigenome and may be important targets for cancer diagnosis and therapy. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr MS1-2.

Autotaxin: A protein with two faces

Autotaxin (ATX) is a catalytic protein, which possesses lysophospholipase D activity, and thus involved in cellular membrane lipid metabolism and remodeling. Primarily, ATX was thought as a culprit protein for cancer, which potently stimulates cancer cell proliferation and tumor cell motility, augments the tumorigenicity and induces angiogenic responses. The product of ATX catalyzed reaction, lysophosphatidic acid (LPA) is a potent mitogen, which facilitates cell proliferation and migration, neurite retraction, platelet aggregation, smooth muscle contraction, actin stress formation and cytokine and chemokine secretion. In addition to LPA formation, later ATX has been found to catalyze the formation of cyclic phosphatidic acid (cPA), which have antitumor role by antimitogenic regulation of cell cycle, inhibition of cancer invasion and metastasis. Furthermore, the very attractive information to the scientists is that the LPA/cPA formation can be altered at different physiological conditions. Thus the dual role of ATX with the scope of product manipulation has made ATX a novel target for cancer treatment.

Enzastaurin inhibits invasion and metastasis in lung cancer by diverse molecules.

Background:Enzastaurin (Enz) is a serine/threonine kinase inhibitor blocking protein kinase C (PKC)β/AKT pathway. However, an ability of this compound to inhibit cancer invasion and metastasis is not yet clearly elucidated.Methods:The ability of Enz to inhibit invasion and metastasis, and to target molecules was investigated in non-small cell lung cancer (NSCLC) by RT–PCR validated microarray, Matrigel, and in vivo chorionallantoic membrane (CAM) assays.Results:Enzastaurin significantly reduced migration, invasion, and in vivo metastasis to lungs and liver (CAM assay) of diverse NSCLC cell lines. Genes promoting cancer progression (u-PAR, VEGFC, and HIF1α) and tumour suppression (VHL, RASSF1, and FHIT) of NSCLC were significantly (P<0.05) down- or upregulated after Enz treatment in H460, A549, and H1299 cells, respectively. Luciferase/chromatin immunoprecipitation analysis showed that Enz transcriptionally controls urokinase-type plasminogen activator receptor (u-PAR) expression by promoter inhibition through Sp1, Sp3, and c-Jun(AP-1). Moreover, siRNA knockdown of u-PAR re-sensitised Enz-resistant cells and induced apoptosis, suggesting u-PAR as a marker of Enz resistance.Conclusion:This study shows that Enz inhibits migration, invasion, and in vivo metastasis by targeting u-PAR, besides further targeting progression-related and tumour-suppressor genes in NSCLC. Together with u-PAR being a novel putative marker of Enz response, these data encourage molecularly tailored clinical studies on Enz in NSCLC therapy.

Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis

Large intervening noncoding RNAs (lincRNAs) are pervasively transcribed in the genome1, 2, 3 yet their potential involvement in human disease is not well understood4,5. Recent studies of dosage compensation, imprinting, and homeotic gene expression suggest that individual lincRNAs can function as the interface between DNA and specific chromatin remodeling activities6,7,8. Here we show that lincRNAs in the HOX loci become systematically dysregulated during breast cancer progression. The lincRNA termed HOTAIR is increased in expression in primary breast tumors and metastases, and HOTAIR expression level in primary tumors is a powerful predictor of eventual metastasis and death. Enforced expression of HOTAIR in epithelial cancer cells induced genome-wide re-targeting of Polycomb Repressive Complex 2 (PRC2) to an occupancy pattern more resembling embryonic fibroblasts, leading to altered histone H3 lysine 27 methylation, gene expression, and increased cancer invasiveness and metastasis in a manner dependent on PRC2. Conversely, loss of HOTAIR can inhibit cancer invasiveness, particularly in cells that possess excessive PRC2 activity. These findings suggest that lincRNAs play active roles in modulating the cancer epigenome and may be important targets for cancer diagnosis and therapy.

Anti-Invasive and Anti-Angiogenic Effects of Xanthohumol and Its Synthetic Derivatives

Abstract － Invasion and metastasis is the main cause of cancer mortality. Angiogenesis is a prerequisite for the tumor growth and metastasis. Matrix metalloproteinases (MMPs) are the key enzymes playing in the invasive growth and metastasis of cancer as well as angiogenesis. Xanthohumol, a prenylated chalcone of the Hop plant (Humulus lupulus L ), has been reported to suppress cancer invasion and angiogenesis. In the present study, we investigated the antiinvasive effects of xanthohumol (1) and its synthetic derivatives, 4'- O -methylxanthohumol SEM ether (2), xanthohumol C (3), and xanthohumol C MOM ether (4) in relation to MMP expression in HT-1080 human fibrosarcoma cells. The compound 1 and its derivative, 3 and 4, significantly inhibited serum-induced HT-1080 cell invasion, and 12- O -tetradecanoylphorbol-13-acetate (TPA)-enhanced activity and expression level of MMP-2 and MMP-9 in a concentration-dependant manner. In addition, they inhibited TPA-enhanced expression of MT1-MMP with relatively weak inhibition in tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 level. The compound 1 significantly decreased the cell viability, whereas the derivatives, 2 and 3 showed no cytotoxicity, and compound 4 showed slight cytotoxicity in the cells. Furthermore, in a chick chorioallantoic membrane (CAM) assay, the derivatives 3 and 4 dose-dependently suppressed vascular endothelial growth factor (VEGF)-induced angiogenesis, which is similar to that of compound 1. Taken together, the results indicate that compounds 3 and 4 may be valuable anti-angiogenic agents in the treatment of chronic diseases such as cancer and inflammation working through suppression of MMP-2 and MMP-9.Keywords: Xanthohumol, Xanthohumol C, Cancer cell invasion, Angiogenesis, Matrix metalloproteinase

Characterization of LMX‐1A as a metastasis suppressor in cervical cancer

DNA methylation is important in cancer development and is a promising biomarker for cancer detection. An epigenomic approach used in our previous work showed that LMX-1A is methylation-silenced in cervical cancer. LMX-1A, a LIM-homeobox gene, is known to participate in developmental events; however, there are at present no data on the role of LMX-1A in cancers. In this study, we characterized the function of this transcription factor by examining cell lines, animal models and human cervical neoplastic tissues, and found that over-expression of LMX-1A does not affect cell proliferation or the cell cycle of cervical cancer cell lines but significantly inhibits colony formation and invasion in vitro. Analysis of changes in epithelial-mesenchymal transition (EMT) markers, such as CDH1, CDH2, VIMENTIN, SNAIL, SLUG and TWIST, revealed involvement of the EMT in LMX-1A-mediated cancer invasion; this result was validated in a stable transfectant over-expressing LMX-1A with RNA interference. Xenograft studies using immunocompromised mice confirmed the suppressor effects of LMX-1A on tumour formation and distant metastasis in cervical cancer cell lines. LMX-1A immunohistochemical staining of tissue arrays containing the full spectrum of cervical neoplasms, including normal cervix, low-grade cervical intra-epithelial neoplasia (CIN), high-grade CIN, locally invasive and distant metastatic cancers, demonstrated the critical role of LMX-1A in invasion and metastasis. Furthermore, we found by analysing TGFbeta-BMP signalling that BMP4 and BMP6 are down-regulated by LMX-1A. The results of this study suggest that LMX-1A suppresses cancer invasion and metastasis in cervical cancer through an incomplete EMT.

P53 controls cancer cell invasion by inducing the MDM2-mediated degradation of Slug

The tumour suppressor p53 is known to prevent cancer progression by inhibiting proliferation and inducing apoptosis of tumour cells. Slug, an invasion promoter, exerts its effects by repressing E-cadherin transcription. Here we show that wild-type p53 (wtp53) suppresses cancer invasion by inducing Slug degradation, whereas mutant p53 may stabilize Slug protein. In non-small-cell lung cancer (NSCLC), mutation of p53 correlates with low MDM2, high Slug and low E-cadherin expression. This expression profile is associated with poor overall survival and short metastasis-free survival in patients with NSCLC. wtp53 upregulates MDM2 and forms a wtp53-MDM2-Slug complex that facilitates MDM2-mediated Slug degradation. Downregulation of Slug by wtp53 or MDM2 enhances E-cadherin expression and represses cancer cell invasiveness. In contrast, mutant p53 inactivates Slug degradation and leads to Slug accumulation and increased cancer cell invasiveness. Our findings indicate that wtp53 and p53 mutants may differentially control cancer invasion and metastasis through the p53-MDM2-Slug pathway.

Transmembrane Interactions Are Needed for KAI1/CD82-Mediated Suppression of Cancer Invasion and Metastasis

In transmembrane (TM) domains, tetraspanin KAI1/CD82 contains an Asn, a Gln, and a Glu polar residue. A mutation of all three polar residues largely disrupts the migration-, invasion-, and metastasis-suppressive activities of KAI1/CD82. Notably, KAI1/CD82 inhibits the formation of microprotrusions and the release of microvesicles, while the mutation disrupts these inhibitions, revealing the connections of microprotrusion and microvesicle to KAI1/CD82 function. The TM polar residues are needed for proper interactions between KAI1/CD82 and tetraspanins CD9 and CD151, which also regulate cell movement, but not for the association between KAI1/CD82 and alpha3beta1 integrin. However, KAI1/CD82 still efficiently inhibits cell migration when either CD9 or CD151 is absent. Hence, KAI1/CD82 interacts with tetraspanin and integrin by different mechanisms and is unlikely to inhibit cell migration through its associated proteins. Moreover, without significantly affecting the glycosylation, homodimerization, and global folding of KAI1/CD82, the TM interactions maintain the conformational stability of KAI1/CD82, evidenced by the facts that the mutant is more sensitive to denaturation and less associable with tetraspanins and supported by the modeling analysis. Thus, the TM interactions mediated by these polar residues determine a conformation either in or near the tightly packed TM region and this conformation and/or its change are needed for the intrinsic activity of KAI1/CD82. In contrast to immense efforts to block the signaling of cancer progression, the perturbation of TM interactions may open a new avenue to prevent cancer invasion and metastasis.

DNA methylation and histone modification regulate silencing of epithelial cell adhesion molecule for tumor invasion and progression

Epithelial cell adhesion molecule (Ep-CAM) is believed to have a critical role in carcinogenesis and cell proliferation. However, the association of Ep-CAM with cancer invasion and progression is less clear. We found that Ep-CAM was highly expressed on low-invasive cells compared with highly invasive cells. Forced expression of Ep-CAM decreased cancer invasiveness, and silencing Ep-CAM expression elevated cancer invasiveness. Ep-CAM expression was associated with promoter methylation. Treatment with a demethylating agent, and/or the histone deacetylase inhibitor reactivated Ep-CAM expression in Ep-CAM-negative cells and inhibited cancer invasiveness. Using a promoter-reporter construct, we demonstrated methylation of the promoter fragment drive Ep-CAM-silenced transcription. Additionally, silenced Ep-CAM gene in cancer cells was enriched for hypermethylated histone 3 lysine 9. When unmethylated and active, this promoter was associated with acetylated histone 3 lysine 9. Furthermore, we observed an increased association of Ep-CAM promoter with repression components as tumor invasiveness increased. In cancer tissues, Ep-CAM expression significantly correlated with tumor progression and associated with promoter methylation. Our data support the idea that modulation of Ep-CAM plays a pivotal role in tumor invasion and progression. Moreover, aberrant DNA methylation of Ep-CAM is implicated in enhancing invasive/metastatic proclivity of tumors.

Adenovirus-Mediated Calponin h1 Gene Therapy Directed against Peritoneal Dissemination of Ovarian Cancer: Bifunctional Therapeutic Effects on Peritoneal Cell Layer and Cancer Cells

Calponin h1 (CNh1), one of the family of actin-binding proteins, stabilizes the filaments of actin and modulates various cellular biological phenotypes. Recent studies revealed the close correlation between the invasive tumor spread and the reduced expression of CNh1 and alpha-smooth muscle actin in the surrounding stromal cells. The purpose of this study is to evaluate the efficacy of i.p. CNh1 gene therapy against peritoneal dissemination of ovarian cancer. We used an adenoviral vector to induce the CNh1 gene into peritoneal cells and ovarian cancer cells as a means of enhancing or inducing the expression of alpha-smooth muscle actin as well as CNh1. The efficacy of gene transfer was examined by in vitro cell culture and in vivo animal experiments. The formation of longer and thicker actin fibers was observed in each transfected cell line, and the localization of these fibers coincided with that of externally transducted CNh1. With respect to changes in cell behavior, the CNh1-transfected peritoneal cells acquired an ability to resist ovarian cancer-induced shrinkage in cell shape; thus, cancer cell invasion through the monolayer of peritoneal cells was inhibited. In addition, CNh1-transfected ovarian cancer cells showed suppressed anchorage-independent growth and invasiveness, the latter of which accompanied impaired cell motility. The concomitant CNh1 transfection into both peritoneal cells and ovarian cancer cells produced an additive inhibitory effect with respect to cancer cell invasion through the peritoneal cell monolayer. By in vivo experiments designed to treat nude mice that had been i.p. inoculated with ovarian cancer cells, we found that the i.p. injected CNh1 adenovirus successfully blocked cancer-induced morphologic changes in peritoneal cell surface and significantly prolonged the survival time of tumor-bearing mice. Moreover, CNh1 adenovirus could successfully enhance the therapeutic effect of an anticancer drug without increase in side effects. Thus, CNh1 gene therapy against peritoneal dissemination of ovarian cancer is bifunctionally effective (i.e., through inhibitory effects on the infected peritoneal cell layers that suppress cancer invasion and through direct antitumor effects against invasion and growth properties of cancer cells).

Intrathecal Administration of Y-27632, a Specific Rho-Associated Kinase Inhibitor, for Rat Neoplastic Meningitis

The small GTP-binding protein Rho and its target Rho-associated kinase trigger an intracellular signaling cascade that controls actin cytoskeleton and plays an essential role in cell motility and adhesion. A specific Rho-associated kinase inhibitor, Y-27632, has been reported to inhibit cancer invasion. Clinically, disseminated tumor cells in the cerebrospinal fluid invade the intraparenchymal region, damaging the brain and nerves, resulting in fatal brain stem dysfunction, despite intrathecal chemotherapy. To expand therapeutic options for this devastating neoplastic meningitis, we evaluated the potential use of intrathecal Y-27632 administration by employing Walker 256 cells, a rat mammary cancer cell line. Y-27632 dose-dependently inhibited chemotactic and invasive activity of Walker 256 cells. Y-27632 also inhibited the phosphorylation level of regulatory myosin light chain in vitro, but the effect was temporary and was considerably diminished within 16 hours. Y-27632 induced striking morphologic changes in Walker 256 cells, as evidenced by decreased cell-matrix adhesion in culture dishes and three-dimensional collagen I gels, and slightly inhibited colony formation in soft agar. Nevertheless, this drug treatment did not affect Walker 256 cell growth rate. We were able to administer continuous delivery of this inhibitor using an osmotic pump and maintaining drug concentration of 10 mumol/L within the brain. Importantly, this concentration of Y-27632 showed minimal neurotoxicity both in vitro and in vivo. We found that an intrathecal therapy, combining 5-fluoro-2'-deoxyuridine with Y-27632, significantly increased the survival time of rats bearing meningeal carcinomatosis in comparison with animals treated with 5-fluoro-2'-deoxyuridine alone. Taken together, our findings indicate that continuous intrathecal administration of Y-27632 could be a promising therapeutic method when combined with chemotherapy for treating human neoplastic meningitis.

Inhibition of urokinase receptor gene expression and cell invasion by anti‐uPAR DNAzymes in osteosarcoma cells

The urokinase-type plasminogen activator (uPA) receptor (uPAR) has been implicated in signal transduction and biological processes including cancer metastasis, angiogenesis, cell migration, and wound healing. It is a specific cell surface receptor for its ligand uPA, which catalyzes the formation of plasmin from plasminogen, thereby activating the proteolytic cascade that contributes to the breakdown of extracellular matrix, a key step in cancer metastasis. We have synthesized three different DNA enzymes (Dz372, Dz483 and Dz720) targeting uPAR mRNA at three separate purine (A or G)-pyrimidine (U or C) junctions. Two of these DNAzymes, Dz483 and Dz720, cleaved uPAR transcript in vitro with high efficacy and specificity at a molar ratio (uPAR to Dz) as low as 1 : 0.2. When analyzed over 2 h with a 200-fold molar excess of DNAzymes to uPAR transcript, Dz720 and Dz483 were able to decrease uPAR transcript in vitro by approximately 93% and approximately 84%, respectively. They also showed an ability to cleave uPAR mRNA in the human osteosarcoma cell line Saos-2 after transfection. The DNAzyme Dz720 decreased uPAR mRNA within 4 h of transfection, and inhibited uPAR protein concentrations by 55% in Saos-2 cells. The decrease in uPAR mRNA and protein concentrations caused by Dz720 significantly suppressed Saos-2 cell invasion as assessed by an in vitro Matrigel assay. The use of DNAzyme methodology adds a new potential clinical agent for decreasing uPAR mRNA expression and inhibiting cancer invasion and metastasis.

Hyperforin, a bio‐active compound of St. John's Wort, is a new inhibitor of angiogenesis targeting several key steps of the process

Hyperforin, a phloroglucinol derivative found in St. John's wort related mainly to its antidepressant effects, has been reported recently to induce apoptosis in tumour cells and to inhibit cancer invasion and metastasis. We show that hyperforin inhibits angiogenesis in vitro in bovine aortic endothelial cells and in vivo in the chorioallantoic membrane assay. In a variety of experimental systems representing the sequential events of the angiogenic process, hyperforin treatment of endothelial cells resulted in strong inhibitory effects. Hyperforin inhibited the growth of endothelial cells in culture. Capillary tube formation on Matrigel was abrogated completely by addition of hypeforin at the low micromolar range. Hyperforin also exhibited a clear inhibitory effect on the invasive capabilities of endothelial cells. Zymographic assays showed that hyperforin treatment produced a complete inhibition of urokinase and a remarkable inhibition of matrix metalloproteinase 2. Our data indicates that hyperforin is a compound that interferes with key events in angiogenesis, confirming the recent and growing evidence about a potential role of this compound in cancer and metastasis inhibition and making it a promising drug for further evaluation in the treatment of angiogenesis-related pathologies.