Y27632 attenuates the aristolochic acid-promoted invasion and migration of human urothelial cancer TSGH cells in vitro and inhibits the growth of xenografts in vivo
Abstract
Background. Aristolochic acid I (AAI) has been impli- cated in urothelial cell carcinoma (UCC) in humans. How- ever, whether AAI promotes invasion/migration of UCC has not been established.
Methods. A study of human UCC TSGH cells cultured with AAI was conducted. Cell viability, the effects of AAI on the activity of matrix metalloproteinase (MMP)- 9, the abilities of invasion/migration and the migration- related proteins (Ras, RhoA, ROCK1, PI-3K, pAkt and nuclear factor-kappaB) of the TSGH cells were assessed. The TSGH cells were subcategorized to 1-day or 30-day was demonstrated, especially in the 30-day AAI-exposed cells. Expressions of Ras/RhoA and other migration-related proteins were increased after AAI treatment, which could be inhibited by Y27632. The in vivo results demonstrated that Y27632 was able to attenuate the speed of growth of the inoculated tumors in nude mice.
Conclusion. Clinically, the patients with prolonged AAI exposure are highly associated UCC, our results provided in vitro and in vivo evidence that prolonged AAI exposure enhances invasion and migration of human TSGH cells.
Keywords: aristolochic acid; invasion; matrix metalloproteinase; migration; urothelial cell carcinoma
AAI exposure. An in vivo study using a nude mice xenograft model was employed to test the antitumor effects of Rho kinase inhibitor or Y27632.
Results. A time- and dose-dependent increase in both activity and messenger RNA (mRNA) level of MMP-9 were demonstrated. The mRNA level of urokinase-type plasminogen activator was increased and tissue inhibitor of metalloproteinase-1 was decreased in the cells with 30-day but not 1-day AAI exposure. A dose-dependent enhancement in wound-healing rate and cell migration
Introduction
Chinese herb nephrotoxicity has been traced to aristolochic acid (AA), which was the major alkaloid extracted from Aristolochia fangchi [1, 2]. AA is a mixture of structurally related nitrophenanthrene carboxylic acids, with aristolo- chic acid I (AAI) and AAII being major components.
Toxicological studies of AA have shown that AAI was a major component of the carcinogenic plant extract AA and exposure to AA was strongly associated with human urothelial malignancy [3, 4], which includes moderate atypia and atypical hyperplasia of the urothelium [5]. The prevalence rate of urothelial cell carcinoma (UCC) after exposure to AA has been reported to range from 39 to 46% [6–9] and, recently, the estimated cumulative dose of AA showed a statistically significant linear dose response [AA: at 151–250 mg, odds ratio (OR) ¼ 1.4, 95% confidence interval (CI) ¼ 1.1–1.8 and at >500 mg, OR ¼ 2.0, 95% CI ¼ 1.4–2.9)] with the risk of UCC (P < 0.001) [10]. The molecular changes that occur in human UCCs are numerous and can be categorized into several mechanisms: (i) chromosomal alterations leading to carcinogenesis, (ii) loss of cell cycle regulation accounting for cellular prolif- eration, (iii) metastasis, guided by events such as the extracellular matrix (ECM) and angiogenesis [11]. The mutagenic and carcinogenic properties of AA have been shown to be based on the formation of DNA adducts [12, 13], which were also demonstrated to be associated with cell proliferation, tumor induction [14, 15], activation of the H-ras oncogene and overexpression of the p53 [7, 16]. Activation of Ras proteins has been reported to induce the constitutive activation of downstream kinase cascades, which results in continuous mitogenic signaling and trans- formation of immortalized cells into human bladder cancer [17]. Recently, we reported that human bladder cancer tissue expressed increased Ras, PI-3K, Akt, nuclear factor-kappaB (NF-jB), RhoA and that human UCCs transfected with Ras would enhance expression of invasion/migration-promoting proteins [18]. Because UCC is the major cancer in the dialysis population and kidney transplant recipients in Taiwan [19, 20], we de- signed this study with the aim to test whether AA has a role enhancing invasion and migration of UCC cells and whether AA-associated urothelial tumorigenesis is also in- volved in the Ras/RhoA, PI-3K, Akt and NF-jB signaling [18]. Materials and methods Chemicals Tris (hydroxymethyl)-aminomethane, ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), phenylmethylsulfonyl fluoride, bovine serum albumin, leupeptin, Nonidet P-40, deoxycholic acid, sodium orthovanadate, phalloidin–fluorescein isothiocyanate (FITC) and AAI were purchased from Sigma–Aldrich (St Louis, MO). Phosphate buffer saline (PBS), trypsin–EDTA and powdered RPMI 1640 medium, fetal bovine serum (FBS), 100 U/mL penicillin G and 100 mg/mL streptomycin were purchased from Gibco/BRL (Gaithersburg, MD). Antibody against Akt and MAPK/ERK1/2, phosphorylated proteins were purchased from Cell Signaling Technology (Beverly, MA). PI-3K (p85), NF-jB (p65), Ras, RhoA and ROCK1 antibodies were purchased from BD Transduction Laboratories (San Diego, CA). Cell culture TSGH 8301 cells, established from a well-differentiated human UCC of the urinary bladder (Grade II, Stage A), were cultured in RPMI 1640 medium containing 10% (vol/vol) fetal bovine serum, 100 U/mL penicillin G and 100 mg/mL streptomycin at 37°C in a humidified atmosphere of 5% CO2 and 95% air. The medium was changed twice a week, and the cells were subcultured when confluence was achieved. Determination of cell viability [3-(4, 5-cimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay] Cells were seeded in a 100 mm dish at a density of 3.5 3 104 cells/dish and treated with concentrations of AAI from 0 to 50 lM at 37°C for 5 days. After the exposure period, the medium was removed and the cells were washed with PBS, then treated with AAI every day. At days 6, 11, 16, 21 and 26, the cells were subcultured and 3.5 3 104 cells were seeded. Then the cells were harvested to detect the viability using an [3-(4, 5-cimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay] assay after 30 days of AAI exposure. To evaluate the cytotoxicity of AAI, an MTT colorimetric assay was performed to determine the cell viability. Cells were seeded in 24-well plates at a density of 3.5 3 104 cells/well and treated with concentrations of AAI from 0 to 50 lM at 37°C for 24 h. After the exposure period, media were removed, followed by washing of the cells with PBS. Thereafter, the medium was changed and incubated with 20 lL MTT (5 mg/mL) for 4 h. The viable cell number per dish was directly proportional to the production of formazan, which, following solubilization with isopropanol, can be measured spectrophotometrically at 563 nm. Determination of matrix metalloproteinase-9 by zymography The levels of matrix metalloproteinase (MMP)-9 released in the cultured medium were detected by gelatin zymography assays as previously de- scribed [21]. First, serum-free conditioned medium was prepared with a 53 loading buffer containing 0.01% SDS without b-mercaptoethanol. The prepared samples were subjected to electrophoresis with 8% SDS poly- acrylamide gels containing 0.1% gelatin. Electrophoresis was performed at 140 V for 3 h in an ATTO apparatus. Gels were washed twice with 50 mL distilled water containing 2% Triton X-100 on a gyratory shaker for 30 min at room temperature to remove the SDS after electrophoresis. The gel was then incubated in 50 mL reaction buffer (40 mM Tris–HCl, pH 8.0, 10 mM CaCl2, 0.02% NaN3) overnight at 37°C, stained with Coomassie brilliant blue R-250 and destained with methanol–acetic acid–water (5, 7.5 and 87.5%, vol/vol/vol). Reverse transcription–polymerase chain reaction Total RNA was isolated from cells using a guanidinium chloride procedure and complementary DNA (cDNA) synthesis and polymerase chain reaction (PCR) amplification were performed as previously described [22]. For re- verse transcription, 4 lg of total cellular RNA was used as templates in the reaction buffer containing 4 lL deoxynucleoside triphosphate (2.5 mM), 5 lL oligo dT20 (10 pmol/mL) and 1 lL RTase (200 U/mL), and the reaction was performed at 42°C for 1 h. Then, the cDNA was stored at 4°C after reaction for 5 min at 99°C. Afterward, 10 lL cDNA product was amplified by PCR with the following primers: urokinase-type plasminogen acti- vator (uPA) (341 bp), 5#-CACGCAAGGGGAGATGAA-3# (sense) and 5#-ACAGCATTTTGGTGGTGACTT-3# (antisense); MMP-9 (263 bp),5#-CACTGTCCACCCCTCAGAGC-3# (sense) and 5#-GCCACTTGTCG- GCG-ATAAGG-3# (antisense); tissue inhibitor of metalloproteinase-1 (TIMP-1) (481 bp), 5#-CTGTTGTTGCTGTGGCTGATA-3# (sense) and 5#-CCGTCCACAAGCAATGAG T-3# (antisense) and glyceraldehyde 3- phosphate dehydrogenase (462 bp), 5#-GAAGGTGAAGGTCGGAGTC- 3# (sense) and 5#-GAAGATGGTGATGGGATTTC-3# (antisense). PCR products were analyzed by agarose gel electrophoresis and visualized by treatment with ethidium bromide. Wound-healing assay The TSGH 8301 cells were cultured in one well of a six-well culture dish. A line was drawn on the underside of the well with a yellow P200 pipette tip. These lines served as fiducial marks for the wound areas to be ana- lyzed. The growth medium was replaced by calcium-free PBS, then the PBS was removed and fresh medium added. The cells were observed using phase contrast microscopy on an inverted microscope. Invasion assay After 1- and 30-day incubation, the TSGH 8301 cells were trypsinized, and the in vitro invasion was tested in a Boyden chamber assay [23]. The cells were seeded in the Boyden chamber (Neuro Probe, Cabin John, MD). A medium containing 10% FBS was applied to the lower chamber as chemoattractant and then cells (1 3 105 cells/well in 50 lL of serum-free medium) were seeded on the upper chamber with 8 lm pore polycarbonate filters previously coated with metrigel. The cells that invaded the lower surface of the membranes were fixed with methanol and acetate (3:1), stained with Giemsa and counted under a light microscope. Electrophoresis and immunoblotting Analysis of Ras, PI-3K, pAkt, NF-jB, RhoA and ROCK1 was performed using SDS–polyacrylamide gel electrophoresis (PAGE) and immunoblot- ting. The medium was removed and washed with PBS. Then 0.5 mL of cold RIPA buffer (1% NP-40, 50 mM Tris–base, 0.1% SDS, 0.5% deox- ycholic acid, 150 mM NaCl, pH 7.5) with fresh leupeptin (17 lg/mL) and sodium orthovanadate (10 lg/mL) were added. Scraping of cells and trans- feral of the lysate into an Eppendorf tube were performed prior to a 30-min incubation on ice incubation with the addition of 5 lL of 10 mg/mL phenylmethlysulfonyl fluoride stock. The cell lysate was centrifuged (10 000 g) for 10 min at 4°C. Cell lysate (50 lg purified protein) was mixed with an equal volume of electrophoresis sample buffer and then boiled for 10 min, followed by analysis using SDS–PAGE and transfer of protein from the gel to nitrocellulose membranes (Millipore, Bedford, MA) using an electroblotting apparatus. Nonspecific binding was blocked by incubation of the membrane with Tris-buffered saline (TBS) containing 1% (wt/vol) nonfat dry milk and 0.1% (vol/vol) Tween-20 (TBST) for >2 h. Membranes were washed with TBST three times for 10 min and incubated with an appropriate dilution of primary antibody in TBST for 2 h. Membranes were then extensively washed with TBST before being incubated with an appropriate amount of horseradish peroxidase-conju- gated secondary antibody for 1 h. After washing the membrane three times for 10 min in TBST, detection was performed using ECL reagents for 1 min and exposed to ECL hyperfilm in a darkroom. Protein expression was determined by quantitative densitometry using an AlphaImager Series 2200 software.
Fluorescent phalloidin staining
Phalloidin staining is a useful tool for investigating the distribution of F- actin in cells [24]. Briefly, a 0.2 mg/mL stock solution of phalloidin–FITC was prepared in methanol. The cells were washed with PBS, fixed for 1 h with 3.7% formaldehyde in PBS and washed with PBS again. Next, the cells were dehydrated in acetone, rendered permeable with 0.1% Triton X-100 in PBS and washed again with PBS. Finally, the cells were stained with a 50 lg/mL fluorescent phalloidin conjugate solution in PBS (containing 1% dimethyl sulfoxide from the original stock solu- tion) for 1 h at room temperature. After at least four 1-min washes with PBS to remove the unbound phalloidin conjugate, the samples were then mounted in 50% PBS/50% glycerol (vol/vol) and examined in an Olympus fluorescence microscope with a 1003 oil immersion objective.
Preparation of nuclear fractions
Harvested cells were lysed with buffer A (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl2, 0.2% NP-40, 1 mM dithiothreitol (DTT) and 0.5 mM phenylmethylsulfonyl fluoride) and then centrifuged to shear off the cytoplasmic membranes. Nuclei were pelleted at 800 g for 30 s at 4°C in a microcentrifuge; nuclear proteins were extracted with high salt buffer B (20 mM HEPES, 25% glycerol, 1.5 mM MgCl2,0.1 mM EDTA, 420 mM NaCl, 1 mM DTT and 0.5 mM phenylme- thylsulfonyl fluoride).
Electrophoretic mobility shift assay
NF-jB-binding assay in nuclear extracts was performed with biotin- labeled double-stranded NF-jB oligonucleotide (Promega, Madison,WI). Electrophoretic mobility shift assay (EMSA) was carried out by a LightShift EMSA Optimization and Control Kit and Chemilumines- cent Nucleic Acid Detection Modules (Pierce, Rockford, IL). Binding reactions containing 10 lg of nuclear protein, 2 lL of 103 binding buffer, 1 lg poly-(dI·dC), 12.5 lg poly-L-lysine and 2 pmol of oligo- nucleotide probe were incubated for 20 min at room temperature. Pro- tein DNA complex was separated by electrophoresis on a 6% nondenaturing acrylamide gel, transferred to positively charged nylon membranes and then cross-linked in a Stratagene cross-linker. Gel shifts were visualized by streptavidin–horseradish peroxidase and followed by chemiluminescent detection.
Animal and tumor xenograft study
Balb/c nude mice (male, 5-week old) were purchased from the National Taiwan University Animal Center, Taiwan. The mice were randomly se- lected to be placed in three treatment groups (six in each group). The first group of mice (group T, controls) was subcutaneously (s.c.) injected with 2 3 106 TSGH cells mixed with an equal volume of Matrigel (BD Bio- sciences) in the left groin. The second and third groups (TA and TAY groups, respectively) were s.c. injected with 2 3 106 30-day 50 lM AAI- treated TSGH cells mixed with an equal volume of Matrigel in the left groin. Meanwhile, the TAY group was injected with Y27632 (25 mg/kg) in the left groin near the tumor cells three times a week. During the 4-week feeding period, all mice used were handled according to the guidelines of the Institu- tional Animal Care and Use Committee of Chung Shan Medical University (IACUC, CSMC) for the care and use of laboratory animals. The mice were housed with a regular 12 h light/12 h dark cycle. After 28 days, the mice were sacrificed for the assay of tumorigenicity (e.g. tumor weight and tumor inhibition).
Statistics
Data were shown as the mean SD. Statistical comparisons were per- formed using Student’s t-tests. P-values <0.05 were considered to be statistically significant. Results Effect of AAI on the viability of TSGH cells Prior to the investigation of the pharmacological potential of AAI on TSGH cell viability, we initially determined the time and dose dependence of the cytotoxic effects of AAI in TSGH cells by means of MTT assay. Unexpectedly, AAI showed a significant dose- and time-dependent inhibition, but no promotive effect on the growth of the TSGH cells (Figure 1a). Given that AAI, at a concentra- tion <50 lM and exposure for 1 day, had modest cyto- toxic effect on the cells, the cell viability of 30-day exposure to AAI was examined. TSGH cells in 30-day AAI exposure revealed a dose-dependent induction on the growth of TSGH cells, especially treatment with 50 lM AAI (P < 0.01) (Figure 1b). These results demon- strated that AAI increased cell survival after long-term incubation, whereas short-term AAI exposure resulted in loss of TSGH cell viability. Effect of AAI on MMP-9 activity using gelatin zymography assay and on MMP-9, up A, TIMP-1 messenger RNA level using reverse transcription–PCR of TSGH cells In addition to facilitating tumor migration, extracellular products such as MMPs can modulate migration, cancer cell proliferation and metastasis [25]. The levels of MMP-9 were assayed by gelatin zymography. TSGH cells with 30-day AAI exposure displayed a dose-dependent increase of MMP-9 (AAI 6.25 lM, P < 0.01; AAI 12.5, 25 and 50 lM, P < 0.001) (Figure 2). However, the expression of MMP-9 after 1-day AAI exposure was without significant variation. Furthermore, to explore whether AAI-regulated MMP-9, TIMP-1 and uPA on the transcriptional level, a semiquantitative reverse transcription (RT)–PCR analy- sis was performed. The 30-day AAI exposure stimulated uPA, MMP-9 messenger RNA (mRNA) levels but inhibited TIMP-1 mRNA level dose dependently (Figure 3), which indicated that TIMP-1 was inhibited and uPA was activated by 30-day AAI treatment in early stage and then promoted the formation of pro-MMP-9 to MMP-9. The wound-healing assay and the invasion ability of TSGH cells treated with AAI Cancer cell migration can be viewed as a process regulated by matrix-degrading proteinases, integrins, other cell adhe- sion molecules and the healing of a wound [26, 27]. The impact of AAI on wound healing and cell invasion was then studied (Figures 4 and 5). The results demonstrated that 30-day exposure of AAI exhibited prominent promo- tion on cell mobility/migration. In addition, the cell amount of invasion was dose-dependently increased when the cells were with 30-day AAI exposure compared with those with only 1-day exposure (AAI 12.5, 25 and 50 lM, P < 0.001) (Figure 5). Therefore, prolonged exposure with higher dose of AAI enhanced the capability of wound healing and in- vasion of TSGH cells. Effect of AAI on migration-related proteins of TSGH cells treated with or without Y27632 Stimulated Ras could promote many Ras-related effectors. We investigated whether different duration and concentra- tion of AAI exposure could affect the interaction of Ras and downstream Ras-dependent signalings. The TSGH cells with 1-day AAI exposure did not display significant changes in the levels of RhoA, ROCK1, PI-3K, pAkt and NF-jB com- pared to those of the control cells on different AAI concen- trations. However, after 30-day exposure, TSGH cells showed significantly dose-dependent increased levels in RhoA, ROCK1, PI-3K, pAkt, and NF-jB compared to those of the controls, suggesting that prolonged AAI exposure promoted both the PI-3K and RhoA pathways (Figure 6). Additionally, the addition of Y27632, a Rho kinase inhibitor, caused a significant reduction in the levels of ROCK1, PI-3K and PI-3K-related downstream proteins. A significant de- crease in the levels of PI-3K and PI-3K-related proteins were found after adding wortmannin, a PI-3K inhibitor. This dem- onstrated that ROCK1 was an intermediate factor regulating PI-3K and its related protein expression (Figure 7). Figure 8 demonstrated the increased phalloidin binding and cellular F-actin levels by AAI treatment in the TSGH cells. The amount of F-actin could be inhibited by the Rho kinase inhibitor or Y27632 but not PI-3K inhibitor. In addition, nuclear extracts were analyzed for NF-jB DNA-binding ability; AAI treatment increased NF-jB DNA binding ability which could be inhibited by Y27632 but not PI-3K inhibitor (Figure 9). Y27632 decreased the speed of growth of the inoculated tumors in nude mice To further test the antitumor effect of Y27632, xenograft assays were employed. After inoculating human bladder cancer AAI-treated TSGH cells into the nude mice s.c., Y27632 was simultaneously injected into the mice. After- ward, the same amount of Y27632 was injected into the mice three times a week. The tumor volume was calculated (1/2 3 length 3 height 3 width) every 4 days and the tumor weights were detected after sacrifice (Figure 10 and Table 1). The tumors in the Y27632-treated mice were much smaller than in the TA group. We also measured the diameters of the inoculated tumors 1 week after the inocu- lation when all of the mice started to show signs of the formation of solid tumor masses. The measurements were taken every 4 days for 28 days. At Day 28, the tumor masses from the mice inoculated with AAI-treated TSGH cells were ~18.5-fold or bigger than those treated with Y27632. A marked reduction (95.3%) in tumor formation was obtained after the s.c. inoculation of Y27632-treated TA cells as compared with the untreated TA cells. Interest- ingly, the tumor weight and tumor volume of the TAY group were also smaller than the TSGH-inoculated alone mice (P < 0.001). This in vivo study suggests that Y27632 is able to attenuate the speed of growth of inoculated tumors in nude mice. Taken together, a proposed model for the AAI-mediated invasion/migration of human bladder cancer cells is summarized in Figure 11. Discussion Our results demonstrated that AAI promoted invasion and migration of TSGH UCC cells via dual induction of MMP- 9 enzyme activity and gene transcription. The migration and invasion abilities of AAI on TSGH cells were signifi- cantly enhanced in accordance with longer duration and higher concentration of AAI. MMP-9 gene expression can be activated via a signal transduction pathway through Ras, RhoA, PI-3K and Akt, which were the upstream mod- ulators of NF-jB [28]. This study revealed that 30-day AAI exposure stimulated the Ras/RhoA signaling and expres- sions of Ras, RhoA, ROCK1, PI-3K/pAkt and NF-jB in TSGH cells, and that these promoting effects of invasion/ migration were time- and dose-dependent. The herbal drug AA, in which the major components are nitrophenanthrene carboxylic acids, after metabolic activa- tion via cytochrome P450 1A1, 1A2, NAD(P)H: quinine oxidoreductase (NQO1) [29, 30] and DT diaphorase [31], were genotoxic mutagens. Moreover, AA exerted genotox- icity via nitric oxide and its derivative peroxynitrite in a dose-dependent manner (0–200 lM) in human HepG2 cells [32]. After 48 h of treatment, mouse embryonic fibroblasts showed decreased cell survival, from 80% at 20 lM to <20% at 100 lM concentrations of AAI [33]. In rats, short-term toxicity (3 days) of AA could cause dysfunction of both kidney and liver [34, 35]. Oral or intra- venous administration of AA in rats and mice caused death from acute renal failure within 15 days with lethal dose, 50% ranged from 56 to 203 mg/kg orally or 38 to 83 mg/kg intravenously [36]. In mice, AA treatment in daily doses of 5.0 mg/kg for 3 weeks resulted in squamous cell carcinoma of the forestomach, adenocarcinoma of the glandular stom- ach, kidney adenomas, lung carcinomas and uterine he- mangiomas [37]. Big Blue rats treated with AAI at concentrations of 0.1, 1 and 10 mg/kg five times per week for 12 weeks disclosed a strong linear dose–response for mutation frequency inductions for AA-induced DNA ad- ducts, which suggested that the mutagenic effects of AA were associated with the formation of AA–DNA adducts [38]. The AA–DNA adducts had been identified and detected in experimental animals exposed to AA or bota- nical products containing AA and in urothelial tissues from AA nephropathy patients [6, 8, 37]. From 1998 to 2002, there were 949 new cases of end- stage renal disease in Taiwan in which the patients had consumed herbal products containing AA before the diag- nosis of chronic kidney disease, representing 3.7% of all new patients with end-stage renal disease [39]. Chang et al. [20] reported that chronic tubulointerstitial nephritis is the most likely underlying renal disease in hemodialysis pa- tients with UCC, and a high percentage of the chronic tubulointerstitial nephritis related to the usage of Chinese herbs or compound analgesics may contribute to the devel- opment of UCC. In addition, from 2001 to 2002, there were 118 new cases of UCC in Taiwan associated with the ingestion of >60 g of the Chinese herb Mu Tong (containing significant AA), representing 3% of all new patients with UCC [10]. Nortier et al. conducted regular cystoscopic examinations and the prophylactic removal of native kid- neys and ureters in patients with end-stage Chinese herb nephropathy. Among 39 patients who agreed to undergo prophylactic surgery, there were 18 cases of urothelial car- cinoma (prevalence, 46%; 95% CI, 29 to 62%), 17 cases of carcinoma of the ureter, renal pelvis or both and 1 case of a papillary bladder tumor [7]. The prevalence rate of UCC after exposure to AA has been reported to range from 39 to 46% [6–9]. AA correlated with a higher risk of UCC if total cumulative doses of AA were >200 g reported from Bel- gium [7] and 150 g reported from Taiwan [10].
Ras oncogenes played a key role in the initiation of human UCC carcinogenesis [40, 41]. AA-induced UCC was associated with the formation of DNA adducts and mutations in H-ras [42, 43] and p53 [12]. The carcino- genesis of human UCC was a multistep process and the rate-limiting step of cancer cell invasion was the break- down of connective tissue barriers, ECM, which com- prised collagens, proteoglycan, elastin, laminin and fibronectin [44]. The main groups of proteolytic enzymes involved in the ECM degradation were MMPs and zinc- dependent proteinases. The degradation of ECM by MMP-2 and -9 had been shown to be an important bio- logical process in the metastasis of cancer cells [45, 46]. MMP-9 had increased expression in UCC when compared to normal urothelium and also correlated with increased tumor stage [47].
A specific inhibitor of the Rho kinase, Y27632 [48, 49] had been reported to block both Rho-mediated activation of acto- myosin, the invasive activity of cultured rat MM1 hepatoma cells [50] and prevented Ras-induced migration-related pro- teins of TSGH human bladder cancer cells [18]. Continuous treatment with this inhibitor reduced dissemination of MM1 hepatoma cells implant into the peritoneal cavity of syngeneic rats [51]. In this study, Y27632 could inhibit RhoA, ROCK1, PI-3K, pAkt and NF-jB expression of AAI-treated TSGH cells and the growth of xenografts in vivo.
In summary, our study showed that 1-day AAI exposure caused cytotoxicity on TSGH cells and 30-day exposure of AAI promoted the expressions of Ras/RhoA, PI-3K, pAkt, Fig. 11. A proposed model for the effects of AAI on Ras- and RhoA- mediated invasion/migration of human bladder cancer cells. The dotted arrow represents the pathway, which has been published in the literature and the solid arrows represent the findings in this present study.NF-jB, ROCK1 and enhanced invasion/migration of TSGH cells. Moreover, RhoA kinase inhibitor could inhibit AAI-induced migration-related proteins. Our results pro- vide in vitro and in vivo evidence that prolonged and ac- cumulated exposure of AAI enhances UCC invasion and migration and the promotion was through Ras and/or RhoA pathways. This might offer hope with regard to the possi- bility of developing new treatment Aristolochic acid A strategies for interven- tions in AA-associated UCCs.