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Clinical Cancer Research Vol. 11, 3455-3464, May 1, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

Antimetastatic Effect of Salvicine on Human Breast Cancer MDA-MB-435 Orthotopic Xenograft Is Closely Related to Rho-Dependent Pathway

Jing-Yu Lang1,3, Hua Chen4, Jin Zhou1,3, Yi-Xiang Zhang1,3, Xiong-Wen Zhang1, Mei-Hong Li1,3, Li-Ping Lin1, Jin-Sheng Zhang2, Michael P. Waalkes4 and Jian Ding1

Authors' Affiliations: 1 Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, 2 Division of Phytochemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, P.R. China, 3 Graduate School of Chinese Academy of Sciences, 4 Laboratory of Comparative Carcinogenesis, National Cancer Institute at National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina

Requests for reprints: Jian Ding, Division of Antitumor Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu-chong-zhi Road, Shanghai, 201203, P.R. China. Phone: 86-21-50806722; Fax: 86-21-50806722; E-mail: jding{at}mail.shcnc.ac.cn.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Salvicine is a novel DNA topoisomerase II inhibitor with potent anticancer activity. In present study, the effect of salvicine against metastasis is evaluated using human breast carcinoma orthotopic metastasis model and its mechanism is further investigated both in animal and cellular levels.

Experimental Design: The MDA-MB-435 orthotopic xenograft model was applied to detect the antimetastatic effect of salvicine. Potential target candidates were detected and analyzed by microarray technology. Candidates were verified and explored by reverse transcription-PCR and Western blot. Salvicine activities on stress fiber formation, invasion, and membrane translocation were further investigated by immunofluorescence, invasion, and ultracentrifugal assays.

Results: Salvicine significantly reduced the lung metastatic foci of MDA-MB-435 orthotopic xenograft, without affecting primary tumor growth obviously. A comparison of gene expression profiles of primary tumors and lung metastatic focus between salvicine-treated and untreated groups using the CLOTECH Atlas human Cancer 1.2 cDNA microarray revealed that genes involved in tumor metastasis, particularly those closely related to cell adhesion and motility, were obviously down-regulated, including fibronectin, integrin {alpha}3, integrin ß3, integrin ß5, FAK, paxillin, and RhoC. Furthermore, salvicine significantly down-regulated RhoC at both mRNA and protein levels, greatly inhibited stress fiber formation and invasiveness of MDA-MB-435 cells, and markedly blocked translocation of both RhoA and RhoC from cytosol to membrane.

Conclusion: The unique antimetastatic action of salvicine, particularly its specific modulation of cell motility in vivo and in vitro, is closely related to Rho-dependent signaling pathway.

Key Words: salvicine • metastasis • cell motility • Rho • MDA-MB-435

Metastasis refers to the dissemination of cancer cells from initial tumor to distant sites and involves a series of processes, including loss of adhesion, acquisition of cell motility, extracellular proteolysis, and angiogenesis (1). Cell motility plays a fundamental role in the onset and progression of cancer and is particularly important in tumor invasion and metastasis. Tumor cell lines that are more highly invasive and metastatic exhibit a higher degree of motility than their lower metastatic counterparts.

The dynamic organization of the actin cytoskeleton that provides the force for cell motility is regulated by Rho GTPases (2–4). The Rho GTPases, including Rac, cdc42, and Rho, are key regulators of the actin cytoskeleton (4). Rho, including RhoA, RhoB, and RhoC, controls actin stress fibers and focal adhesion contact formation, whereas Rac and cdc42 are responsible for the formation of lamellipodia and filopodia, respectively (4). Recent evidence shows that Rho is an essential regulator of cell motility and metastasis, (5–7) and its overexpression is intimately correlated with the invasive and/or metastatic phenotypes of numerous carcinomas (5, 7–17). Several upstream pathways that activate Rho as well as the downstream targets of activated Rho have been identified (18–22). Integrins, which directly or indirectly bind to talin, {alpha}-actinin, vinculin, paxillin, and focal adhesion kinase (FAK), mediated rearrangement of actin cytoskeleton in focal adhesion and focal adhesion complexes through a Rho-dependent pathway (23). Lysophosphatidic acid (LPA), a completely effective substitute for serum, (24) induces membrane translocation of Rho via specific G-protein–coupled receptors on the cell surface, which drives cytoskeletal contraction, leading to the activation of downstream effectors, and formation of focal adhesion and stress fibers promoting cell migration and invasion (25–28). Dominant inhibition of Rho function eliminates integrin clustering and LPA-induced migration and invasion activities in vitro, (4, 29) and is markedly attributed to tumor metastasis reduction of mammary and lung carcinomas in vivo (5, 7, 29–31). Consistent with these results, agents targeting cell motility via in vivo inhibition of Rho/ROCK also results to substantial suppression of tumor metastasis without affecting the tumorigenicity (31–33). These findings suggest a potential therapeutic cure for tumor metastasis via blockage of Rho-dependent pathways (31, 34, 35).

Salvicine [4,5-seco-5,10-friedo-abieta-3,4-dihydroxy-5(10),6,8,13-tetraene-11,12-dione], a novel diterpenoid quinone compound, is a structurally modified derivative of a natural product lead from the traditional Chinese herb, Salvia prionitis Hance (Labiatae). This compound is a novel DNA topoisomerase II inhibitor with evident anti-multiple drug–resistant activity, (36–38) and has entered a phase I clinical trial in China. Previous studies by our group highlighted the antiangiogenetic potential of salvicine5 and prompted us to hypothesize that this compound may additionally exert antimetastatic activity. Accordingly, in this study, the antimetastatic effect of salvicine and its underlying molecular mechanisms of action were investigated at both the animal and cellular level.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell line. The human breast cancer MDA-MB-435 cell line was obtained from American Type Culture Collection (Rockville, MD). The culture was maintained in DMEM (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, 100 units/mL penicillin sodium, 100 µg/mL streptomycin sulfate, and 0.25 µg/mL amphotericin B (Life Technologies). Cells were cultured in a humidified atmosphere of 5% CO2 and 95% air at 37°C.

Animals. Female athymic nude mice (BALB/cA nu/nu) ages 4 to 5 weeks were obtained from the Shanghai Institute of Materia Medica (Shanghai, China), housed in sterile cages under laminar airflow hoods in a specific pathogen-free room with a 12-hour light and 12-hour dark schedule and fed autoclaved chow and water ad libitum. All experiments were done according to institutional ethical guidelines on animal care.

Chemicals. Salvicine was structurally modified from the lead compound isolated from the Chinese medicinal plant S. prionitis by the Phytochemistry Department of Shanghai Institute of Materia Medica, Chinese Academy of Sciences. The end product was purified by column chromatography on a silica gel eluted with a cyclohexane/ethyl acetate mixture (4:1, v/v) to produce salvicine (65% yield). Purity was >99.8%, as determined by high-performance liquid chromatography. Salvicine was dissolved in DMSO at 0.1 mol/L (used in vitro) or a mixture of ethanol, ethane-1,2-diol, and saline (8:1:1; used in vivo) as a stock solution and maintained at –20°C in the dark. LPA and fibronectin were purchased from Sigma (Saint Louis, MO). Y27632, C3 exoenzyme, and rabbit polycolonal antibodies against G12 and G13 were obtained from Calbiochem (La Jolla, CA). Goat polyclonal antibodies against RhoC and ß-actin and rabbit polyclonal antibodies against RhoA, Rac1, and cdc42 were acquired from Santa Cruz Biotechnology (Santa Cruz, CA). Texas Red-X phalloidin and ProLong Antifade Kit were obtained from Molecular Probes, Inc. (Eugene, OR). Fluorescein-conjugated goat anti-rabbit IgG and rabbit anti-goat immunoglobulin G were purchased from KPL (Gaithersburg, MD) and Zhongshan (Beijing, China), respectively. Matrigel was obtained from BD Biosciences (San Jose, CA). cDNA expression arrays were purchased from Clontech (Palo Alto, CA). [{alpha}-32P]dATP was from Amersham Pharmacia Biotech (Piscataway, NJ).

Spontaneous metastasis assay. Human breast cancer MDA-MB-435 cells were orthotopically injected into mammary fat pads of female athymic nude mice ages 4 to 5 weeks. Mice were anesthetized with chloral hydrate, and a 5-mm incision was made in the skin over the lateral thorax, as described previously (39). The mammary fat pad was exposed and an inoculum of 1 x 106 cells/0.2 mL was implanted into the tissue through a 27-gauge needle. Skin incisions were closed with wound clips that were removed 1 week later. At a volume of ~100 to 200 mm3, mice were divided into five experimental groups after balancing tumor volumes, specifically: (a) untreated (n = 18); (b) Adriamycin (5 mg/kg) or etoposide (15 mg/kg; n = 6); (c) salvicine (6 mg/kg; n = 12); (d) salvicine (12 mg/kg; n = 12); and (e) salvicine (24 mg/kg; n = 12). Salvicine was i.v. administrated through the tail vein weekly for 10 weeks thereafter. Tumors were measured individually twice per week with microcalipers. Tumor volumes were calculated according to the formula: length x width x width x 0.5 and presented as RTV = Tumor Volume (day after initial treatment) / Tumor volume (day of initial treatment). "Tumor growth delay" was the difference in days for treated groups versus the control group to reach the same volume, and calculated as T/C, where T and C represent the median times (in days) required for the treatment group or control group tumors to reach the same predetermined size, respectively. Body weights of the animals were measured on the days of initial injection and autopsy. Mice were sacrificed 5 days later by cervical dislocation after the final therapy. Tumor weights were measured, and lungs removed. Samples of two of three mice in each group were fixed with Bouin's solution for 24 hours. Metastasis lesions on lungs were counted under a dissecting microscope. The other samples in each group were either stored in RNAlater solution (Ambion, Austin, TX) at –80°C, or total RNA or proteins extracted immediately.

RNA extraction and reverse transcription-PCR. Total RNA was extracted from frozen tissue samples using Trizol (Life Technologies), following the manufacturer's instructions. The quantity and quality of RNA were assessed by spectrophotometry at 260 and 280 nm. Total RNA (1-3 µg) was subjected to reverse transcription. Next, cDNA was amplified using 2.5 units of Taq DNA Polymerase (Sino-American Biotechnology Co., Luoyang, China) and 0.2 µmol/L of specific oligonucleotide primers in a final reaction volume of 50 µL containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 2 mmol/L MgCl2, and 0.2 mmol/L each deoxynucleotide triphosphate (Sangon, Shanghai, China). The primers used in this experiment as following: sense 5'-CTGGTGATTGTTGGTGATGG-3' and antisense 5'-GCGATCATAATCTTCCTGCC -3' for RhoA, 183 bp; sense 5'-ACCATGGCTGCAATCCGAAAGAAG -3' and antisense 5'-AAGGGAGGGGGCATGTAGGAAAAG -3' for RhoC, 626 bp; sense 5'-ATGCAGGCCATCAAGTGTGTGGTG-3' and antisense 5'-TTACAACAGCAGGCATTTTCTCTTCC-3' for Rac1, 600 bp; sense 5'-TCTCCTGAATGATGGTCTGG G-3' and antisense 5'-GATAGAGTGGAAAAGGGAGTAGG-3' for cdc42, 250 bp; sense 5'-GAAGGTGAAGGTCGGAGTCA-3' and antisense 5'-GAAGATGGTGATGGGATTTC-3' for glyceraldehyde-3-phosphate dehydrogenase, 250 bp. Reverse transcription-PCR for target genes was done using a thermal cycler (MJ Research, Inc., Waltham, MA) according to a set program. The number of cycles was determined in preliminary experiments to be within the exponential range of PCR. Negative controls were run in parallel to confirm that samples were not contaminated with genomic DNA. PCR products (10 µL) were electrophoresed on a 2% agarose gel. Bands were visualized by ethidium bromide staining, and recorded using a UVP GDS8000 Gel Documentation System (UVP, Upland, CA).

Microarray analysis. We compared initial primary tumors versus metastases using the CLONTECH Atlas human Cancer 1.2 cDNA microarray (1,176 genes). Total RNA in tissue samples from three mice of each salvicine-treated or untreated group was isolated using Trizol reagent (Invitrogen, Carlsbad, CA), according to manufacturer's instructions. Microarray analysis was done using the manufacturer's protocol. Briefly, 1 µg total RNA was converted to 32P-labeled cDNA probes using Moloney murine leukemia virus reverse transcriptase and [{alpha}–32P]dATP with the CLONTECH Atlas human CDS primer mix. The 32P-labeled cDNA probe was purified using CHROMA SPIN-200 (Clontech) columns and denatured in 0.1 mol/L NaOH, 10 mmol/L EDTA at 68°C for 20 minutes followed by neutralization with an equal volume of 1 mol/L NaH2PO4 for another 10 minutes. Microarray membranes were prehybridized with ExpressHyb (Clontech) containing sheared salmon testes DNA (100 µg/mL) for 30 to 60 minutes at 68°C followed by hybridization overnight at 68°C with the cDNA probes. Array membranes were washed four times in 2x SSC/1% SDS for 30 minutes each and twice in 0.1x SSC/0.5% SDS for 30 minutes. Membranes were sealed in plastic bags, and exposed to a PhosphorImage screen (Molecular Dynamics, Sunnyvale, CA). Images were analyzed densitometrically using AtlasImage software (ver.1.5; Clontech). Genecluster 2.0 (MIT, Cambridge, MA) was employed for further analysis. Four relatively consistent housekeeping genes (i.e., 40S ribosomal protein, ß-actin, myosin heavy chain, and phospholipase A2 precursor) were used to normalize the hybrid intensity of each gene of interest.

Western blot. Cells (5 x 105) were lysed in loading buffer, boiled for 10 minutes, subjected to 12% SDS-PAGE, and blotted on a polyvinylidene difluoride membrane (Hybond-PVDF, Amersham Pharmacia Biotech). The membrane was blocked for 1 hour with 5% skimmed milk in TTBS containing 150 mmol/L NaCl, 20 mmol/L Tris-HCl (pH 7.2), and 0.1% Tween 20 and further incubated overnight at 4°C with primary antibody (1:500 to 1:1,000 dilution). After incubation with horseradish peroxidase–conjugated secondary antibody (1:1,000 to 1:2,000 dilution) for 1 hour, immunoreactive bands were stained with the Super Signal West Pico Chemiluminescent kit (Pierce, Rockford, IL).

Matrigel invasion assay. Transwell chamber membranes (6.5 mm diameter, 8 µm pore size; Costar, Corning, NY) were coated with 100 µL of 1 mg/mL Matrigel (dissolved in serum-free DMEM medium; Becton Dickinson Sciences, Franklin Lakes, NJ). LPA (10 µmol/L) and fibronectin (5 µg/mL), or 20% FCS were added to the lower chambers. Various concentrations of salvicine, C3 exoenzmye, or Y27632 were added to the upper chambers at the same time. Cells (1 x 105) were added to the upper chamber and allowed to invade for 20 hours at 37°C in a CO2 incubator. Cells that had not migrated were removed from the upper chamber with a cotton swab. The remaining cells were fixed, stained with staining buffer [0.1 mol/L borate acid, 0.1% (w/v) crystal violet, and 2% (v/v) ethanol] for 10 minutes at room temperature, and measured at 595 nm after extraction with 10% acetic acid for 10 minutes.

Immunofluorescence assay. For fluorescence staining, cells were plated on 1% gelatin-coated glass culture slides at a density of 1 x 105 cells/mL in DMEM containing 10% FCS. The medium was replaced with serum-free DMEM containing 0.5% fatty acid–free bovine serum albumin for 24 hours. Various concentrations of salvicine, C3 exoenzyme, or Y27632 were added to the culture medium, and incubation was done at 37°C in a CO2 incubator for the indicated times. LPA (10 µmol/L) was added to the culture medium and incubated for 10 minutes. Fixed preparations were obtained by exposing cells on culture slides to 4% paraformaldehyde in PBS for 10 minutes at 25°C followed by washing thrice with PBS containing 0.2% Triton X-100 for 5 minutes. For stress fibers staining, cells were stained with both Texas Red-X phalloidin (Molecular Probes) and 4',6-diamidino-2-phenylindole for visualization of filamentous actin and nuclear acid, respectively, followed by washing thrice with PBS containing 0.2% Triton X-100 for 5 minutes. For Rho proteins staining, cells were incubated with primary antibodies of RhoA (1:200 dilution) and RhoC (1:100 dilution) for 60 minutes followed by incubation of FITC-conjugated second antibody (1:200 dilution) for 20 minutes. Pictures were obtained using a fluorescence microscope (Olympus BX51, Olympus, Tokyo, Japan) with a digital camera.

Separation of particulate and cytosolic fractions. Cells were grown to subconfluency (60-70%) in DMEM containing 10% FCS. The medium was replaced with a serum-free DMEM containing 0.5% fatty acid–free bovine serum albumin for 24 hours. Various concentrations of salvicine were added to culture medium followed by incubation at 37°C in a CO2 incubator for the indicated times. LPA (10 µmol/L) was added to the culture medium and incubated for 10 minutes. Cells (2 x 106) were lysed by freeze-thawing in 300 µL ice-cold lysis buffer [50 mmol/L HEPES (pH 7.5), 50 mmol/L NaCl, 1 mmol/L MgCl2, 2 mmol/L EDTA, 10 mmol/L NaF, 1 mmol/L DTT, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL aprotinin, and 10 µg/mL leupeptin] and centrifuged at 100,000 x g for 30 minutes at 4°C (HITACHI GX series himac CS150GXL microultracentrifuge, Tokyo, Japan), and the supernatant was collected as the cytosolic fraction. Pellets were resuspended, and membrane proteins homogenized in 150 µL lysis buffer containing 2% Triton X-114. The homogenate was centrifuged at 800 x g for 10 minutes. The supernatant (particulate fraction) and pellet (detergent-insoluble particulate fraction) were collected separately. Whole cell, cytosolic, and particulate fraction proteins were separated by SDS-PAGE.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Salvicine suppresses lung metastasis of MDA-MB-435 orthotopic xenograft in athymic mice but does not affect primary tumor growth. We evaluated the efficacy of salvicine against tumor metastasis of the human breast cancer MDA-MB-435 orthotopic xenograft model in athymic mice. With weekly i.v. drug administration for 10 weeks, the number of pulmonary metastatic nodules of human breast cancer MDA-MB-435 orthotopic xenograft of control, etoposide 15 mg/kg, Adriamycin 5 mg/kg, salvicine 6 mg/kg, salvicine 12 mg/kg, and salvicine 24 mg/kg groups were 13.6 ± 4.8, 13.8 ± 8.3, 6.3 ± 1.5, 9.1 ± 3.9, 6.0 ± 4.2, and 2.5 ± 1.0, respectively. Salvicine (LD10iv = 57.0 mg/kg) at doses of 6, 12, and 24 mg/kg significantly decreased lung metastasis of human breast cancer MDA-MB-435 orthotopic xenograft with inhibition rates of 33.0%, 54.0%, and 81.6%, respectively (Fig. 1A). However, salvicine did not noticeably affect primary tumor growth [T/C (%) values at the last day of treatment in the first experiment were 122.7%, 162.4%, and 125.3%, respectively (Fig. 1B), and 115.3%, 82.0%, and 68.4%, respectively, in the second experiment (Fig. 1C)]. Etoposide, a DNA topoisomerase II inhibitor, given at a dose of 15 mg/kg (LD10iv = 34 mg/kg), displayed no effect on tumor metastasis and proliferation (Fig. 1A and B). Adriamycin (5 mg/kg; LD10iv = 10 mg/kg), another DNA topoisomerase II inhibitor, exhibited a 46.4% inhibitory effect against tumor metastasis (Fig. 1A), possibly due to its obvious blockage of primary tumor growth [T/C (%) at the last day of treatment was 17.4% (Fig. 1C)]. All mice treated with drugs survived with healthy appearance and no loss of body weight.



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  		Fig. 1. Efficacy of salvicine on lung metastasis and primary tumor proliferation of MDA-MB-435 breast carcinoma orthotopic xenografts in nude mice. Animals were divided into five experimental groups and were given drugs i.v. weekly thereafter. The experiment was terminated 10 weeks after the initiation of therapy. The tumors were measured individually twice per week with microcalipers, and tumor volumes were calculated and represented as RTV, and lungs were removed and fixed with Bouin's solution for 24 hours. Metastatic lesions on the lungs were counted under a dissecting microscope. A, efficacies of salvicine, etoposide, and Adriamycin against tumor metastasis of MDA-MB-435 breast carcinoma orthotopic xenografts. Columns, means of a typical experiment; bars, ±SE. B, efficacies of salvicine and etoposide against tumor proliferation of MDA-MB-435 primary tumors. C, efficacies of salvicine and Adriamycin against tumor proliferation of MDA-MB-435 primary tumors. Similar results were obtained from at least three separate experiments.

 
The Rho-dependent signaling pathway is essentially involved in the in vivo antimetastatic efficacy of salvicine. The gene transcript expression profiling of primary and lung metastatic tumors of human breast cancer MDA-MB-435 orthotopic xenograft after salvicine treatment was evaluated with a CLONTECH Altas human cancer 1.2 array containing 1,176 tumor-related genes. We set Min = 500, Max = 16,000, Max/Min ≥ 3, and Max-Min ≥ 100 as the filtering conditions. Following normalization and filtering, 872 genes were further analyzed with Genecluster 2.0 software by self-organized mapping, unsupervised hierarchical clustering, and function classification analysis. Based on 2x self-organized mapping analysis, all groups were divided automatically into two clusters. In cluster 0, the metastatic tumor groups treated with salvicine (6, 12, and 24 mg/kg) displayed distance/similarity values of 0.041, 0.074, and 0.113, respectively. In cluster 1, the distance/similarity observed in negatively controlled primary tumor group, 12 mg/kg salvicine-treated primary tumor group, and negatively controlled metastatic tumor group were 0.044, 0.050, 0.033, and 0.035, respectively (Fig. 2). Little or no differences were observed between the negatively controlled metastatic tumor group and the negatively controlled primary tumor group. The results indicate that gene expression profiles of metastatic tumors were significantly affected by salvicine, whereas those of the primary tumor were less affected.



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  		Fig. 2. Molecular profiling of human breast cancer MDA-MB-435 carcinomas by salvicine treatment in vivo by self-organized mapping analysis.

 
Expressed genes were further grouped by detailed application of CLONTECH gene function classification analysis. Salvicine altered the expression of 14 in 125 genes implicated in the cell cycle (11.2%; e.g., increased expression of cyclin 1, CDK3, CDK9, MTA-1, and cdc-like kinase 2 and decreased the expression of growth inhibitory factor, CDK6, G1-S transition protein 1, and PRL-1). The compound additionally altered the levels of 4 in 66 genes involved in DNA synthesis and damage repair (6.1%; e.g., increased expression of BRCA2 and PCNA and decreased expression of GSH and MSH6). Importantly, salvicine affected the expression of 15 of 80 genes related to cell motility (18.8%; e.g., up-regulated tight junction protein 1, RAP1A, N-Ras, RalB, cdc42 homologue and down-regulated cytokeratin 8, cytokeratin 12, BIGH3, RhoC, Rac1, and motility-related protein) and influenced 13 in 64 genes implicated in cell adhesion (20.3%; e.g., increased expression of CD49, cadherin 4, plexin-related protein and decreased expression of Integrin {alpha}3, Integrin {alpha}6, Integrin {alpha}E, Integrin ß3, Integrin ß5, Integrin ß8, paxillin, and FAK). Moreover, treatment with salvicine reduced the expression of 14 in 86 genes involved in proteolysis and extracellular matrix proteins (16.3%; e.g., collegen 8{alpha}1, collegen 16{alpha}1, fibronectin, tenascin, osteonectin, MMP11, TIMP1, TIMP3, t-PA). Those results show that genes involved in tumor metastasis are strongly influenced by salvicine.

We further analyzed those genes by the properties of tumor metastasis, including cell adhesion, motility, proteolysis, and angiogenesis. It was found that salvicine greatly influenced the mRNA expression of genes implicated in cell adhesion, such as fibronectin, osteonectin, Integrin {alpha}3, Integrin {alpha}6, Integrin {alpha}E, Integrin ß3, Integrin ß5, Integrin ß8, paxillin, and FAK and markedly reduced the transcript expression of cell motility-related genes, including cytokeratin 8, cytokeratin 12, BIGH3, RhoC, Rac1, and motility-related protein. Among the genes identified, integrins and fibronectin, FAK, paxillin, and RhoC played important roles in cell adhesion and motility, and were essentially implicated in Integrin-Rho signal pathway. As determined by unsupervised hierarchical clustering analysis, RhoC was one of the most significantly down-regulated genes (data not shown). Based on these results, we propose that genes related to cell motility and adhesion, particularly those involved in the Integrin-Rho signal pathway, might be greatly involved in salvicine's antimetastasis processes (Table 1).


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  		Table 1. The gene expression profiles of primary and metastatic tumors of MDA-MB-435 orthotopic xenografts after salvicine treatment in vivo

 
Salvicine inhibits both mRNA and protein expression of RhoC. To determine whether salvicine exerts effects on Rho GTPases, we further assessed expression at both mRNA and protein levels by reverse transcription-PCR and immunoblotting. MDA-MB-435 cells were cultured in DMEM with 10% fetal bovine serum and exposed to salvicine, either at 20 µmol/L for specific time periods or various concentrations for 16 hours. Salvicine down-regulated both RhoC mRNA and protein expression in a dose- and time-dependent manner but had no obvious effect on the levels of RhoA, Rac, and cdc42 in MDA-MB-435 cells (Fig. 3A, C, and D). Notably, the level of RhoC protein in primary tumors of MDA-MB-435 orthotopic xenografts was additionally down-regulated in the presence of salvicine (Fig. 3B).



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  		Fig. 3. Salvicine inhibited RhoC, without affecting RhoA, Rac1, and cdc42 mRNA and protein expression in MDA-MB-435 breast cancer cells. Cells (5 x 105) were cultured in DMEM containing 10% FCS. Various concentrations of salvicine was added to the culture medium for 16 hours or treated by 20 µmol/L salvicine for different time periods, and incubation was carried out at 37°C in a CO2 incubator. The mRNA expression level of RhoA, RhoC, Rac1, and cdc42 were detected by reverse transcription-PCR, compared with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The protein expression level of RhoA, RhoC, Rac1, and cdc42 were detected by immunoblotting, compared with actin. A, dose course of the effect of salvicine on mRNA expression of Rho GTPases for 16 hours. B, dose course of the effect of salvicine on protein expression of RhoC in primary tumor of human breast cancer MDA-MB-435 orthotopic xenografts in athymic mice. C, dose course of the effect of salvicine on protein expression of Rho GTPases for 16 hours. D, time course of the effect of 20 µmol/L salvicine on protein expression of Rho GTPases. Similar results were obtained from at least three separate experiments.

 
Salvicine disrupts Rho-induced stress fiber formation. LPA is a serum phospholipid with growth factor–like activities for many cell types, expressed with significant levels (>1 µmol/L) in various human body fluids. LPA can activate Rho function, trigger cytoskeletal reorganization, induce stress fiber formation, and alter cell morphology. Accordingly, we determined whether salvicine affects Rho-induced stress fiber formation of human breast cancer MDA-MB-435 cells with or without specific Rho activator stimulation. As shown in Fig. 4A, serum-starved cells displayed low levels of stress fibers, as evaluated from the Texas Red-X phalloidin (combining filamentous actin) immunostaining assay (Fig. 4A, a). Typical shapes of MDA-MB-435 cells cultured in DMEM with 10% fetal bovine serum were flat, well spread, and rich in stress fibers (Fig. 4A, b). In contrast, exposure of MDA-MB-435 cells to 10 µmol/L LPA for 10 minutes induced a dramatic increase in stress fiber formation (Fig. 4A, c). Importantly, treatment of 15 µmol/L salvicine for 16 hours resulted in a obvious reduction of stress fiber formation in LPA-stimulated MDA-MB-435 cells, accompanied by cell retraction from the substratum, rounding up, and loss of contact between neighboring cells (Fig. 4A, i). In addition, salvicine also markedly disrupted the formation of stress fibers in serum-stimulated MDA-MB-435 cells (Fig. 4A, h). C3 exoenzyme, a Rho-specific inhibitor (50 µg/mL) displayed similar results as salvicine, regardless LPA treatment, whereas Y27632, a ROCK-specific inhibitor (20 µmol/L), induced reduction in stress fiber formation, but didn't change the elongated cell shape (Fig. 4A, d, e, f, g).



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  		Fig. 4. Effect of salvicine on cell morphology and Rho translocation in MDA-MB-435 cells. MDA-MB-435 cells were incubated for 16 h in DMEM medium with 10%FCS (A: b, d, f, h) or serum-free (A: a, c, e, g, i; B, C, and D) in the absence or presence of drug. For immunofluorescence staining, cells were fixed by 4% paraformaldehyde after drug treatment and were stained with Texas Red-X phalloidin (Molecular Probes) or fluorescein (KPL) for visualization of filamentous actin and Rho, respectively. For membrane protein separation assay, cells were incubated for 16 hours in serum-free DMEM containing 0.1% bovine serum albumin with various concentration of salvicine and incubated for 10 minutes in the presence of LPA (10 µmol/L). Proteins were extracted and separated into whole cell, cytosolic, and membrane fractions, and RhoA, RhoC, G12, and G13 were detected by immunoblotting. A, efficacies of salvicine, C3, and Y27632 on the formation of stress fibers in MDA-MB-435 cells. a, serum-starved cells; b, cells with 10% FCS; c, cells treated with 10 µM LPA only; d, cells treated with C3 exoenzyme 50 µg/mL only; e, cells treated with 50 µg/mL C3 exoenzyme and 10 µmol/L LPA; f, cells treated with 20 µmol/L Y27632 only; g, cells treated with Y27632 20 and 10 µmol/L LPA; h, cells treated with 15 µmol/L salvicine; i, cells treated with salvicine 15 and 10 µmol/L LPA. B, efficacy of salvicine on Rho subcellular localization in MDA-MB-435 cells. Salvicine (15 µmol/L) strongly blocked RhoA and RhoC clustering in the membrane fraction in LPA-stimulated MDA-MB-435 cells, respectively. C, time course of the effect of salvicine on the LPA-induced translocation of RhoA, RhoC, G12, and G13. D, dose course of the effect of salvicine on the LPA-induced translocation of RhoA, RhoC, G12, and G13. Similar results were obtained from at least three separate experiments.

 
Salvicine antagonizes the lysophosphatidic acid–induced in vitro invasive capacity of human breast cancer MDA-MB-435 cells. Next, we designed an experiment to determine the effect of salvicine on the in vitro invasion capacity stimulated by LPA or serum using the Matrigel invasion assay. In the presence of 10 µmol/L LPA, the invasion capacity of MDA-MB-435 cells was increased to ~200% (Fig. 5A and B). Migration of MDA-MB-435 cells through the Matrigel-coated polycarbonate membrane was inhibited by salvicine in a dose-dependent manner, with half-maximal inhibition at about 7.5 µmol/L, and ~100% inhibition at 20 µmol/L. C3 exoenzyme 50 µg/mL also displayed inhibitory effects (Fig. 5A and B). In addition, salvicine, C3 exoenzyme, and Y27632 showed similar inhibitory effects against the in vitro invasiveness of 20% FCS-stimulated MDA-MB-435 cells (data not shown). Interestingly, 20 µmol/L salvicine did not block MDA-MB-435 cell attachment to Matrigel-coated membranes 4 hours after seeding or affect cell growth during the 20 hours of incubation period. Our results confirm that salvicine inhibits the Rho-activated migration of cells towards the chemoattractant, LPA, which is located in the lower chamber.



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  		Fig. 5. Effect of salvicine on LPA-induced invasion of MDA-MB-435 cells. Cells were resuspended in serum-free medium containing 0.1% bovine serum albumin with the indicated concentration of salvicine, C3 exoenzyme, or Y27632. The cells (1 x 105 cells per well) were added to the upper compartment, and the lower compartment was immediately filled with the same medium in the absence or presence of 10 µmol/L LPA. In vitro invasion was assessed after 20 hours and recorded under microscope. Columns, means of three separate experiments in triplicate wells; bars, ±SE. A, efficacies of salvicine, C3 exoenzyme, and Y27632 inhibited in vitro invasion of LPA-stimulated MDA-MB-435 breast cancer cells. B, efficacies of salvicine, C3 exoenzyme, and Y27632 against in vitro invasion capacity of MDA-MB-435 cells. a, untreated groups; b, stimulated with 10 µmol/L LPA for 20 hours; c, treated with 20 µmol/L Y27632 and 10 µmol/L LPA for 20 hours; d, treated with 50 µg/mL C3 exoenzyme and 10 µmol/L LPA for 20 hours; e, treated with 10 µmol/L salvicine and 10 µmol/L LPA for 20 hours; f, treated with 20 µmol/L salvicine and 10 µmol/L LPA for 20 hours.

 
Salvicine blocks lysophosphatidic acid–induced translocation of RhoA and RhoC from the cytosol to membrane. Rho proteins must be targeted to the plasma membrane for activation and full function, which depends on lipid modification. LPA can promote the translocation of Rho from cytosol to the membrane via G12/G13 proteins; thus, we detected whether salvicine exerts the inhibitory effect against translocation of RhoA and RhoC from cytosol to membrane. In subcellular localization assay, Rho protein was mainly distributed in the cell cytosol in serum-starved condition and greatly clustered in cell membrane after LPA stimulation (Fig. 4B). Salvicine effectively blocked LPA-stimulated translocation of both RhoA and RhoC from cytosol to membrane in MDA-MB-435 cells (Fig. 4B). In membrane protein isolation experiment, it was revealed that stimulation of MDA-MB-435 cells with 10 µmol/L LPA for 10 minutes led to a dramatic increase in the amount of RhoA and RhoC in the membrane fraction, compared with control (Fig. 4C and D). Salvicine inhibited LPA-induced cell membrane translocation of RhoA and RhoC significantly in a time- and dose-dependent manner but had no obvious influence on the translocation of G12 and G13 (Fig. 4C and D). Half-maximal inhibition by salvicine was estimated at about 7.5 µmol/L for 16 hours or about 20 µmol/L at <8 hours. Additionally, treatment with salvicine 20 µmol/L for 16 hours led to a less expression of active RhoA and RhoC (in the membrane fraction) than that of the nonstimulated group (Fig. 4C and D).


   Discussion
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 Abstract
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 Discussion
References
 
The panel of antitumor drugs, mostly discovered by chance or semiempirical procedures, is largely inefficient for treating disseminate diseases, and there is currently a pressing need for antimetastasis drugs. "True" antimetastatic agents should only include those compounds that repeatedly display the capacity to selectively interfere with metastasis formation, with marginal or no effect on primary tumor growth (40). To elucidate mechanisms of those compounds will be great helpful for understanding metastasis itself and finding potential targets for metastasis prevention.

In the present study, we show that the topoisomerase II inhibitor salvicine reduces the number of pulmonary metastatic colonies of human breast cancer MDA-MB-435 orthotopic xenograft in a dose-dependent manner without obviously affecting primary tumor growth. However, two other topoisomerase II inhibitors, Adriamycin and etoposide, display different modes of action. Etoposide fails to combat tumor metastasis, whereas Adriamycin suppresses tumor metastasis, which may be attributable to its marked inhibition of primary tumor growth. In view of these findings, we propose that salvicine combats tumor metastasis via a unique mechanism distinguishable from that of antitumor cytotoxicity.

In the following study, we compare transcript expression profilings of primary and metastatic tumors after salvicine treatment to find potential targets involved in its antimetastasis processes. The self-organized mapping and gene function classification analysis indicate significant influence of salvicine on genes involved in tumor metastasis. To further dissect the signal pathway mainly influenced by salvicine treatment, we find that components of Integrin-Rho pathway are mostly affected in the extent and range, such as integrin {alpha}6, integrin ß3, integrin ß5, fibronectin (integrin ligand), FAK, paxillin, and RhoC. Integrins play an important role in organizing the actin cytoskeleton at sites of adhesion to the extracellular matrix, such as focal complexes and focal adhesions. Integrins, which directly or indirectly bind to talin, {alpha}-actinin, vinculin, paxillin, and FAK, mediated rearrangement of actin cytoskeleton in focal adhesion and focal adhesion complexes through a Rho-dependent pathway (23). In the present study, we show that salvicine greatly disrupts Rho-induced stress fiber formation and blocks the in vitro invasiveness, accompanied by the inhibition of RhoC at both the protein and mRNA levels in MDA-MB-435 cells. In addition, we also observe that salvicine inhibits tyrosine phosphorylation of FAK and paxillin in fibronectin-stimulated MDA-MB-435 cells.6 Because inhibition or loss of components of Integrin-Rho pathway (e.g., dominant-negative Rho or Rho inhibitors) severely restricts adhesion complex turnover and inhibits cell motility, (18, 41–44); thus, down-regulated expressions of genes in this pathway, such as integrins, fibronectin, FAK, paxillin, and RhoC, all contributed to blockage of cell motility and tumor metastasis by salvicine treatment. These findings also provide a reasonable explanation for the suppression of tumor metastasis by salvicine in vivo.

LPA is a serum phospholipid with growth factor–like activities for many cell types, expressed with significant levels (>1 µmol/L) in various human body fluids (25). Its aberrant expression and signaling probable contribute to cancer initiation, progression and metastasis. LPA can promote Rho translocation from cytosol to the membrane via specific G-protein–coupled receptors on the cell surface, leading to activation of many downstream effectors and formation of cell adhesion and stress fibers, and plays a dominant role in tumor cell migration and invasion (25). The membrane translocation of Rho GTPases, which maybe required for ATP ribosylation, (45) is necessary to trigger cascades that lead to their subsequent full function (46). In the present study, salvicine significantly blocks the LPA-induced translocation of RhoA and RhoC from cytosol to the membrane fraction in a time- and dose-dependent manner in LPA-stimulated MDA-MB-435 cells, detected by both subcellular localization immunofluorescence staining and ultracentrifugal membrane protein isolation assays. It also markedly disrupts Rho-induced stress fiber formation and blocks the in vitro invasiveness of LPA-stimulated MDA-MB-435 cells, characterized as the blockage of the translocation of RhoA and RhoC from the cytosol to the membrane. The blockage of membrane translocation of Rho leads to significant reduction of stress fiber formation and invasion induced by integrin clustering and LPA (29, 46–48). Cumulatively, the data imply that inhibition of Rho translocation from cytosol to membrane plays important role in salvicine's antimetastasis processes in vivo. In addition, LPA can promote the Integrin-Rho signaling via the positive feedback loop between integrins and Rho GTPases, leading to clustering of integrins, tyrosine phosphorylation of FAK and paxillin, and formation of focal adhesions and stress fibers (44, 49). Inhibition of Rho function can also block LPA-induced integrin clustering, phosphorylation of FAK and paxillin, and stress fibers formation (26, 29). Taken together, it signifies the strict involvement of Rho-dependent signaling pathways in regulating antimetastatic processes of salvicine.

The C3 exoenzyme from Clostridium botulinum, the prototype protein for this family of toxins, was the first bacterial toxin shown to catalyze covalent modification of a Rho GTPases (33). The enzyme efficiently ADP-ribosylates RhoA, RhoB, and RhoC, with marginal or no modification of Rac or cdc42, and often serves as a useful tool to characterize the pharmacologic and cell biological functions of Rho Proteins (33). The biological changes after salvicine treatment, such as cell retraction from the substratum, rounding up, loss of contacts between neighboring cells, stress fiber reduction, and blockage of invasiveness, were similar as that of C3 in MDA-MB-435 cells.

Having established that the antimetastatic activities of salvicine correlate with cell membrane translocation of the Rho protein family, we further attempted to characterize the detailed regulatory mechanisms. Recent exciting evidence shows that LPA-stimulated translocation processes of RhoA and RhoC are regulated by G12/13, major upstream regulators of Rho function. In contrast to the positive effects of LPA on G12/13 expression, salvicine had a negative effect. This finding indicates that the antagonizing potency of salvicine against cell membrane translocation is independent of G12/13, and the compound might affect other downstream effectors, such as Rho guanine nucleotide exchange factors. Related studies on Rho guanine nucleotide exchange factors and isoprenylation are currently under way.

It is also interesting to investigate how salvicine affects the expression of Rho proteins. The three isoforms of Rho share 85% amino acid sequence identity, but in the present study, we found that salvicine inhibited the expression of RhoC, whereas not affecting RhoA. It might be due to that RhoA, RhoB, and RhoC had preferential interactions with different regulators and effectors in signal transduction context, and their gene expression depended on tissue types very significantly and were regulated by different mechanism (50).

In conclusion, this is the first study to disclose a unique underlying mechanism of salvicine against tumor metastasis, which is one of few compounds interfering with metastasis formation with marginal or no effect on primary tumor growth. The inhibition of Rho-dependent signaling pathways is at least partially responsible for salvicine's antimetastatic effects, both in vitro and in vivo. These findings support that Rho-dependent pathway, particularly Rho, might be a therapeutic potential target for tumor metastasis cure. Careful elucidation of the molecular mechanisms underlying the antimetastatic action of salvicine may widen its clinical applications, and provide novel structural data for targeting Rho-associated proteins in antimetastasis drug development.


   Footnotes
 
Grant support: National Natural Science Foundation of China grant 30200324 and Ministry of Science and Technology of China grant 2002AA2Z3461.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

5 Unpublished data. Back

6 Unpublished data. Back

Received 10/ 4/04; revised 1/30/05; accepted 2/ 9/05.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Vial E, Sahai E, Marshall CJ. ERK-MAPK signaling coordinately regulates activity of Rac1 and RhoA for tumor cell motility. Cancer Cell 2003;4:67–79.[CrossRef][Medline]
  2. Wittmann T, Waterman-Storer CM. Cell motility: can Rho GTPases and microtubules point the way? J Cell Sci 2001;114:3795–803.[Abstract/Free Full Text]
  3. Lauffenburger DA, Horwitz AF. Cell migration: a physically integrated molecular process. Cell 1996;84:359–69.[CrossRef][Medline]
  4. Hall A. Rho GTPases and the actin cytoskeleton. Science 1998;279:509–14.[Abstract/Free Full Text]
  5. Clark EA, Golub TR, Lander ES, Hynes RO. Genomic analysis of metastasis reveals an essential role for RhoC. Nature 2000;406:532–5.[CrossRef][Medline]
  6. Kamai T, Tsujii T, Arai K, et al. Significant association of Rho/ROCK pathway with invasion and metastasis of bladder cancer. Clin Cancer Res 2003;9:2632–41.[Abstract/Free Full Text]
  7. Ikoma T, Takahashi T, Nagano S, et al. A definitive role of RhoC in metastasis of orthotopic lung cancer in mice. Clin Cancer Res 2004;10:1192–200.[Abstract/Free Full Text]
  8. Shinto E, Tsuda H, Matsubara O, Mochizuki H. Significance of RhoC expression in terms of invasion and metastasis of colorectal cancer. Nippon Rinsho 2003;61 Suppl 7:215–9.
  9. Kleer CG, van Golen KL, Zhang Y, Wu ZF, Rubin MA, Merajver SD. Characterization of RhoC expression in benign and malignant breast disease: a potential new marker for small breast carcinomas with metastatic ability. Am J Pathol 2002;160:579–84.[Abstract/Free Full Text]
  10. Wang W, Yang LY, Yang ZL, Huang GW, Lu WQ. Expression and significance of RhoC gene in hepatocellular carcinoma. World J Gastroenterol 2003;9:1950–3.[Medline]
  11. Kondo T, Sentani K, Oue N, Yoshida K, Nakayama H, Yasui W. Expression of RHOC is associated with metastasis of gastric carcinomas. Pathobiology 2004;71:19–25.[CrossRef][Medline]
  12. Shikada Y, Yoshino I, Okamoto T, Fukuyama S, Kameyama T, Maehara Y. Higher expression of RhoC is related to invasiveness in non-small cell lung carcinoma. Clin Cancer Res 2003;9:5282–6.[Abstract/Free Full Text]
  13. Kamai T, Arai K, Tsujii T, Honda M, Yoshida K. Overexpression of RhoA mRNA is associated with advanced stage in testicular germ cell tumour. BJU Int 2001;87:227–31.[CrossRef][Medline]
  14. Yoshioka K, Nakamori S, Itoh K. Overexpression of small GTP-binding protein RhoA promotes invasion of tumor cells. Cancer Res 1999;59:2004–10.[Abstract/Free Full Text]
  15. Suwa H, Ohshio G, Imamura T, et al. Overexpression of the rhoC gene correlates with progression of ductal adenocarcinoma of the pancreas. Br J Cancer 1998;77:147–52.[Medline]
  16. Fritz G, Just I, Kaina B. Rho GTPases are over-expressed in human tumors. Int J Cancer 1999;81:682–7.[CrossRef][Medline]
  17. Horiuchi A, Imai T, Wang C, et al. Up-regulation of small GTPases, RhoA and RhoC, is associated with tumor progression in ovarian carcinoma. Lab Invest 2003;83:861–70.[Medline]
  18. Clark EA, King WG, Brugge JS, Symons M, Hynes RO. Integrin-mediated signals regulated by members of the rho family of GTPases. J Cell Biol 1998;142:573–86.[Abstract/Free Full Text]
  19. Amano M, Chihara K, Kimura K, et al. Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase. Science 1997;275:1308–11.[Abstract/Free Full Text]
  20. Fujisawa K, Madaule P, Ishizaki T, et al. Different regions of Rho determine Rho-selective binding of different classes of Rho target molecules. J Biol Chem 1998;273:18943–9.[Abstract/Free Full Text]
  21. Fukata Y, Oshiro N, Kinoshita N, et al. Phosphorylation of adducin by Rho-kinase plays a crucial role in cell motility. J Cell Biol 1999;145:347–61.[Abstract/Free Full Text]
  22. Maekawa M, Ishizaki T, Boku S, et al. Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase. Science 1999;285:895–8.[Abstract/Free Full Text]
  23. Fukata M, Nakagawa M, Kuroda S, Kaibuchi K. Cell adhesion and Rho small GTPases. J Cell Sci 1999;112:4491–500.[Abstract]
  24. Ridley AJ, Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 1992;70:389–99.[CrossRef][Medline]
  25. Mills GB, Moolenaar WH. The emerging role of lysophosphatidic acid in cancer. Nat Rev Cancer 2003;3:582–91.[CrossRef][Medline]
  26. Imamura F, Mukai M, Ayaki M, Akedo H. Y-27632, an inhibitor of rho-associated protein kinase, suppresses tumor cell invasion via regulation of focal adhesion and focal adhesion kinase. Jpn J Cancer Res 2000;91:811–6.[CrossRef][Medline]
  27. Imamura F, Shinkai K, Mukai M, et al. rho-Mediated protein tyrosine phosphorylation in lysophosphatidic-acid-induced tumor-cell invasion. Int J Cancer 1996;65:627–32.[CrossRef][Medline]
  28. Hirakawa M, Oike M, Karashima Y, Ito Y. Sequential activation of RhoA and FAK/paxillin leads to ATP release and actin reorganization in human endothelium. J Physiol 2004;558:479–88.[Abstract/Free Full Text]
  29. O'Connor KL, Shaw LM, Mercurio AM. Release of cAMP gating by the {alpha}6ß4 integrin stimulates lamellae formation and the chemotactic migration of invasive carcinoma cells. J Cell Biol 1998;143:1749–60.[Abstract/Free Full Text]
  30. Bouzahzah B, Albanese C, Ahmed F, et al. Rho family GTPases regulate mammary epithelium cell growth and metastasis through distinguishable pathways. Mol Med 2001;7:816–30.[Medline]
  31. Collisson EA, Kleer C, Wu M, et al. Atorvastatin prevents RhoC isoprenylation, invasion, and metastasis in human melanoma cells. Mol Cancer Ther 2003;2:941–8.[Abstract/Free Full Text]
  32. Itoh K, Yoshioka K, Akedo H, Uehata M, Ishizaki T, Narumiya S. An essential part for Rho-associated kinase in the transcellular invasion of tumor cells. Nat Med 1999;5:221–5.[CrossRef][Medline]
  33. Barbieri JT, Riese MJ, Aktories K. Bacterial toxins that modify the actin cytoskeleton. Annu Rev Cell Dev Biol 2002;18:315–44.[CrossRef][Medline]
  34. Lozano E, Betson M, Braga VM. Tumor progression: small GTPases and loss of cell-cell adhesion. Bioessays 2003;25:452–63.[CrossRef][Medline]
  35. Sahai E, Marshall CJ. Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis. Nat Cell Biol 2003;5:711–9.[CrossRef][Medline]
  36. Meng LH, Zhang JS, Ding J. Salvicine, a novel DNA topoisomerase II inhibitor, exerting its effects by trapping enzyme-DNA cleavage complexes. Biochem Pharmacol 2001;62:733–41.[CrossRef][Medline]
  37. Miao ZH, Ding J. Transcription factor c-Jun activation represses mdr-1 gene expression. Cancer Res 2003;63:4527–32.[Abstract/Free Full Text]
  38. Miao ZH, Tang T, Zhang YX, Zhang JS, Ding J. Cytotoxicity, apoptosis induction and downregulation of MDR-1 expression by the anti-topoisomerase II agent, salvicine, in multidrug-resistant tumor cells. Int J Cancer 2003;106:108–15.[CrossRef][Medline]
  39. Price JE, Polyzos A, Zhang RD, Daniels LM. Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice. Cancer Res 1990;50:717–21.[Abstract/Free Full Text]
  40. Sava G, Bergamo A. Drug control of solid tumour metastases: a critical view. Anticancer Res 1999;19:1117–24.[Medline]
  41. O'Connor KL, Nguyen BK, Mercurio AM. RhoA function in lamellae formation and migration is regulated by the {alpha}6ß4 integrin and cAMP metabolism. J Cell Biol 2000;148:253–8.[Abstract/Free Full Text]
  42. Leng L, Kashiwagi H, Ren XD, Shattil SJ. RhoA and the function of platelet integrin {alpha}IIbß3. Blood 1998;91:4206–15.[Abstract/Free Full Text]
  43. Martin KH, Slack JK, Boerner SA, Martin CC, Parsons JT. Integrin connections map: to infinity and beyond. Science 2002;296:1652–3.[Abstract/Free Full Text]
  44. Schwartz MA, Shattil SJ. Signaling networks linking integrins and rho family GTPases. Trends Biochem Sci 2000;25:388–91.[CrossRef][Medline]
  45. Molnar G, Dagher MC, Geiszt M, Settleman J, Ligeti E. Role of prenylation in the interaction of Rho-family small GTPases with GTPase activating proteins. Biochemistry 2001;40:10542–9.[CrossRef][Medline]
  46. Bourne HR, Sanders DA, McCormick F. The GTPase superfamily: a conserved switch for diverse cell functions. Nature 1990;348:125–32.[CrossRef][Medline]
  47. Kusama T, Mukai M, Ayaki M, et al. Inhibition of lysophosphatidic acid-induced RhoA activation and tumor cell invasion by 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors. Int J Oncol 2003;23:1173–8.[Medline]
  48. Shao F, Vacratsis PO, Bao Z, Bowers KE, Fierke CA, Dixon JE. Biochemical characterization of the Yersinia YopT protease: cleavage site and recognition elements in Rho GTPases. Proc Natl Acad Sci U S A 2003;100:904–9.[Abstract/Free Full Text]
  49. Ren XD, Kiosses WB, Schwartz MA. Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J 1999;18:578–85.[CrossRef][Medline]
  50. Wheeler AP, Ridley AJ. Why three Rho proteins? RhoA, RhoB, RhoC, and cell motility. Exp Cell Res 2004;301:43–9.[CrossRef][Medline]



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