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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY
Institutes of Pharmacology (H.-L.L., L.-H.C., C.-W.C.) and Clinical Medicine (S.-H.C., Y.-P.Y.), School of Medicine (C.-W.W.), National Yang-Ming University, Taipei, Taiwan; Departments of Medical Research and Education (S.-H.C., W.-B.L., Y.-P.Y., M.-L.T., C.-W.C.) and General Surgery (C.-W.W.), Taipei Veterans General Hospital, Taipei, Taiwan; Chi Mei Medical Center, Tainan, Taiwan (Y.-H.U.); and Department of Medicinal Chemistry, School of Pharmacy, Taipei Medical University, Taipei, Taiwan (J.-P.L.)
Received April 26, 2007; accepted July 19, 2007.
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Abstract |
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). According to statistics, early detection of stage I gastric cancer patients have a 5-year survival rate over 88%. The overall incidences of lymph node metastasis, liver metastasis, and peritoneal metastasis are 47.8, 4.5, and 11.5%, respectively (Thompson et al., 1993; Ferlay et al., 2001). Peritoneal dissemination is often noted in patients at recurrence after primary treatment of the gastric cancer (Soga, 2005). Meanwhile, tumor resection could also induce increased expression of cytokines, such as hepatocyte growth factor, leading to tissue repair and reorganization. Hepatocyte growth factor facilitates migration of tumor cells to distant sites by AKT activation in gastric cancer cells (Trusolino et al., 2001). AKT functions as a signaling transducer of another important metastatic-related receptor protein (HER2) in breast cancer (Benovic and Marchese, 2004). Thus, an increasing number of studies is focusing on targeting AKT-related pathways to restrain tumor dissemination (Larue and Bellacosa, 2005; Yoeli-Lerner and Toker, 2006).
Combretastatin A4 (CA4), isolated from the African tree Combretum caffrum, demonstrated to inhibit tubulin polymerization at colchicines-binding site of 
-tubulin (Woods et al., 1995; Liou et al., 2004). Using CA4 alone or combined with radiation or chemotherapeutic agents is the remedy for a variety of tumors (Tozer et al., 1999; Boehle et al., 2001; Young and Chaplin, 2004; Badn et al., 2006). The proposed action mechanism of CA4 is focused on tumor vasculature shrinkage and reducing tumor perfusion after treatment for 30 min to 6 h (Dziba et al., 2002; Anderson et al., 2003). Recent study demonstrated that CA4 inhibits endothelial cell migration and capillary tube formation through disruption of vascular endothelial-cadherin, -catenin, and AKT signaling pathway, thereby leading to rapid vascular collapse and tumor necrosis (Vincent et al., 2005). However, whether the treatment of CA4 can directly inhibit AKT activity in gastric tumor cells and subsequently lead to decreased tumor cell growth and reduce tumor cell dissemination was still unclear.
In this study, we attempted to elucidate the possible action mechanism of CA4 on gastric cancer cell metastasis rather than vascular endothelial cells. Our results demonstrated that CA4 inhibited the growth of nine human gastric cancer cell lines with submicromolar IC50. Interestingly, a trend that gastric cancer cells with the phosphorylated serine 473 on AKT (p-AKT) expression tend to be more sensitive to CA4 treatment in terms of cell viability is emerging. We found that CA4 inhibited AKT activation, and the differential cytotoxicity correlated well with p-AKT in positive and negative cell lines. Furthermore, inhibition of p-AKT by CA4 resulted in decreased cell proliferation, cell cycle arrest, and reduced in vitro migration/invasiveness and in vivo metastatic ability. These results suggest that activation of p-AKT is an important molecular event in the metastasis of gastric cancers, and inhibition of this oncogenic pathway by CA4 reduces metastasis.
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Materials and Methods |
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). Human gastric cancer cell lines SC-M1 (from Dr. C. L. Meng), NUGC-3 (from Dr. M. Sekiguchi), MKN1, MKN45, and MKN74 (Japanese Collection of Research Bioresources, Tokyo, Japan), and AGS (American Type Culture Collection, Manassas, VA) were cultured in RPMI 1640 (Invitrogen) containing 10% fetal bovine serum (Invitrogen) and 10 µg/ml gentamycin in a CO2 incubator at 37°C, with 5% CO2 and 95% filtered air. Establishment of Metastatic Human Gastric Cancer Cells with Stable Green Fluorescence Protein Expression in Nude Mice. AGS cells were transfected with the cDNA plasmid of green fluorescence protein (GFP) and selected for stably expressing GFP cells. Briefly, AGS cells were cultured in RPMI 1640 medium for 24 h at a cell density of 1 x 105 in a six-well plate. GFP DNA plasmid (2 µg; pEGFP-C1 vector; BD Biosciences Clontech, Palo Alto, CA) formulated with Lipofectamine 2000 was delivered to the AGS cells. G418 sulfate was applied (400–2000 µg/ml) to select GFP-positive AGS cells. Two metastatic AGS-GFP (AGS-GFPM1 and AGS-GFPM2) cell clones were selected from the liver metastatic foci of AGS-GFP cells i.v. injected in nude mice.
Gastric Cancer Cell Viability and Mobility Evaluation. Cells were cultured in a 96-well cell culture cluster at a density of 3 x 103 cells/well in 100 µl of medium. After the drug treatment for 24 h, the medium was discarded and replaced with an equal volume (100 µl) of fresh medium containing 0.2 mg/ml 3-(4,5-dimethylthiazo l-2-yl)-5-(3-carboxy methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and incubated for an additional 1.5 h. Cell growth was proportional to optical density (490 nm) value that was measured by colorimetric assay. The IC50 of each compound that inhibits 50% of cell growth activity was then determined. To evaluate the mobility of cancer cells, cells were seeded on a 24-well plate with 2 x 105 cells/well for 24 h, and then a sterile plastic scraper was used. To quantitatively analyze the fluorescence intensity of the scratched area, the percentage of fluorescence signal per photographed field was analyzed by Image process software (Image-Pro Plus; Media Cybernetics, Inc., Silver Spring, MD).
In Vitro Cell Invasion Analysis. The 24-well plate Transwell system with a polycarbonate filter membrane of 8-µm pore size was used. The filter membrane was coated with Matrigel (BD Biosciences, San Jose, CA) and 400 µg/ml serum-free RPMI 1640 medium 100 µl/well and incubated overnight at 37°C. Cells were seeded to the upper compartment of the Transwell chamber at a cell density of 2 x 105 in 100 µl of serum-free RPMI 1640 medium. The lower chamber was filled with 10% FBS-containing RPMI 1640 medium with or without LY294002 (Sigma-Aldrich, St. Louis, MO). After a 24-h incubation period, the cells remaining on the upper surface of the filter membrane were removed, and the cells on the opposite surface of the filter membrane were stained with hematoxylin for 1 h. The migrated cells were then visualized and counted from five different viewing areas of 100-fold magnification under an inverted microscope.
Cell Cycle Analysis. Both floating and adhesive cells were collected. Around 1 x 106 cells were added with 500 µl of lysing buffer (0.5% Triton X-100, 0.2 µg/ml Na2EDTA·2H2O, and 1% bovine serum albumin in phosphate-buffered saline), and cells were stained with a DNA staining solution (50 µg/ml propidium iodide and 5 kU/ml RNase A). The DNA content of the stained cells was measured using a FACSCalibur flow cytometer (BD Biosciences). Cell cycle data from flow cytometry were obtained and analyzed by CellQuest and Modfit LT software, respectively.
Western Blot Analysis. The cells were lysed in the lysis buffer (20 mM Tris buffer, pH 7.5, 1 mM EDTA, 100 µM phenylmethylsulfonyl fluoride, 2 µg/ml aprotonin, 2 µg/ml pepstatin, and 2 µg/ml leupeptin) for 30 min at 4°C. The protein level was quantified using the BCA protein assay kit. Cell lysates containing an equal amount (25 µg) of total protein were separated by 10 or 12.5% SDS-polyacrylamide gel electrophoresis. The loaded protein samples were then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore Corporation, Billerica, MA). The PVDF membrane with transferred proteins was blocked with 5% nonfat dry milk in Tris-buffered saline for 1 h and incubated overnight with anti-AKT (1:2500, SKB1; Upstate Biotechnology, Lake Placid, NY) and p-AKT (1:500; Cell Signaling Technology, Beverly, MA). After washing, anti-mouse or anti-rabbit secondary antibody conjugated with horseradish peroxidase was added (1:5000; Pierce Biotechnology) for incubation for 1 h. The horseradish peroxidase activity was detected by incubating the membrane with enhanced chemiluminescence reagent (Pierce ECL WBS). The image was visualized by developing on BioMax X-ray film. Cellular -actin (1:5000, AC-15; Sigma-Aldrich) was also immunodetected on PVDF membrane and served as an internal standard of each sample.
In Vivo Analysis of the Effect of Combretastatin A4 on Tumor Metastasis. All the animal practices were in accordance with the institutional animal welfare guideline of Taipei Veterans General Hospital. AGS-GFPM2 cells were injected into the s.c. and gastric sites of BALB/c nude mice aged 8 weeks. Each nude mouse received 5 x 106 and 1 x 107 cells on s.c. and abdominal gastric sites, respectively. After the s.c. tumors grew to around 3 x 3 mm (in diameter) in size and were examined by GFP signals, CA4 phosphate (CA4-P) was administered by i.p. injection (100 or 200 mg/kg) at day 0 in a volume of 300 ml. The CA4-P treatment was then given to the mice on days 3 and 6 for a total of three times. In vivo GFP imaging was visualized and measured by an illuminating device [LT-9500 Illumatool TLS equipped with excitation illuminating source (470 nm) and filter plate (515 nm)]. The tumor size was measured by a caliper, and the volume was calculated according to the formula: (length x width2)/2. The integrated optical density of green fluorescence intensity was captured and then analyzed by Image-Pro Plus software. Phosphorylated AKTs were analyzed in the tumor from control and CA4-P-treated groups using immunohistochemical assay. The anti-phosphorylated AKT (Cell Signaling Technology, Inc.) antibody was used. Procedures of deparaffinization, rehydration, antigen retrieval, and immunohistochemistry were performed as described previously (Chiou et al., 2006).
Statistical Analysis. The results were reported as mean ± S.D. Statistical analysis was performed using Student's t test or the one- or two-way analysis of variance test followed by Tukey's test, as appropriate. p < 0.05 was considered to be statistically significant.
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Results |
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). Figure 1, A and B, shows a significant level of p-AKT expression in U87MG cells. Among the 10 gastric cancer cell lines, it was found that MKN45, MKN45-GFP, AGS, AGS-GFP, and SC-M1 had low or nondetectable p-AKT expression, whereas MKN1, MKN74, NUGC-3, AGS-GFPM1, and AGS-GFPM2 were found to have relatively higher p-AKT expression. We further evaluated the cytotoxic and antiproliferation activities of CA4 on nine human gastric cancer cell lines. Given the results of p-AKT expression (Fig. 1, A and B), the nine cell lines were separated into p-AKT-positive and p-AKT-negative groups. From the analysis of dose response of CA4 treatments on different p-AKT expression cell lines, it was observed that the most sensitive p-AKT-positive cell lines were NUGC-3 and AGS-GFPM1/2, with as low as a 0.02 µM growth-inhibitory concentration after 24-h CA4 treatment (Fig. 1C). In contrast to the p-AKT-positive group, there was no significant inhibitory effect on the growth of p-AKT-negative cells at 0.02 µMof 24-h CA4 treatment (Fig. 1D). It was found that all cell lines with p-AKT-positive expression appeared significantly more sensitive to 0.02 µM CA4 than p-AKT-negative cell lines (Fig. 1, C and D).
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Combretastatin A4 Affected Human Gastric Cancer Cell Attachment and Mobility. To examine the status of tumor cells attachment in vitro, a cultured system of cell attachment was employed. We have found that the morphology of AGS-GFP, AGS-GFPM1, and AGS-GFPM2 cells was rounded up as the CA4 dose increased to 0.02 µM at 24-h after CA4 treatment, but a low dose (0.002 µM) had no effect compared with the spread-out and well attached vehicle-treated cell lines (data not shown). At the same concentration (0.02 µM) treatment, less morphological changes were observed in 5-fluorouracil-, cisplatin-, and cyclophosphamide-treated cells (Fig. 1E). To further quantitatively evaluate the effect of CA4 on the gastric cancer cell mobility, GFP-expressed AGS-GFPM1/2 and other cell lines were employed on the scratch experiments (Fig. 2). Cell migration ability was calculated as the ratio of integrated optical area over the dark field of the scratch site. The ratio of fluorescent signal was 0.2 at 0 h (Fig. 2, A and B) at the generation of scratch. After 6 h of incubation with 10% FBS medium, GFP-positive cells began to migrate from the scratch (Fig. 2I). At 24 h, the gap was closed at control group (Fig. 2D), but the gap junction at the CA4-treated cells had a dose-dependent inhibitory effect (Fig. 2, E–H). Our results demonstrated that CA4 could effectively inhibit AGS-GFPM1 and AGS-GFPM2 migration as compared with cisplatin, 5-fluorouracil, or cyclophosphamide treatment at a dose range of 0.02 to 2 µM. However, there was no significant difference for all compounds at the 0.002 µM treatment dosage (Fig. 2, J and K). The other four wild-type cell lines were also investigated for CA4-induced antimigration effect. Figure 2L shows that CA4 reduced AGS and NUGC-3 cells migration at the concentration of 0.02 µM, but CA4 had only a minor effect on the less migratory MKN45 and SC-M1 cells.
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Combretastatin A4 Inhibited Human Gastric Cancer Cell Invasion in Vitro. As shown in Fig. 3, the data revealed that 24-h CA4 treatment significantly decreased the invasion of the p-AKT-positive cell lines (AGS-GFPM1, AGS-GFPM2, and MKN1) on the Transwell with a dose-responsive effect (Fig. 3, A and B). Our results also showed that LY294002 at 0.2 to 20 µM concentration significantly decreased tumor invasiveness of both AGS-GFPM1 and AGS-GFPM2 cells (Fig. 3C). To determine whether blocking AKT expression or reducing p-AKT levels would affect the activity of CA4 treatment, the specific AKT inhibitor (catalog no. 124005; Calbiochem) was used to inhibit AKT activity. The results showed that similar treatment effects were observed between CA4 and AKT inhibitor on AGS-GFPM1, AGS-GFPM2, and MKN1 cells (Fig. 3, B and D). More importantly, our results demonstrated that CA4 combined with the AKT inhibitor did not have significant or additive effects (Fig. 3E). These findings supported that CA4 inhibited the invasion ability of human gastric cells mainly through inhibition of the activation of AKT pathway.
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Combretastatin A4 Inhibited p-AKT Expression in Human Gastric Cancer Cells. Figure 4A shows that CA4 inhibited p-AKT expression in a dose-dependent manner on AGS-GFPM1 and AGS-GFPM2 cells (Fig. 4A). To investigate the time sequence of CA4 effects on the reduction of p-AKT expression, two different doses (1 nM and 1 µM) of CA4 were applied on AGS-GFPM1 and AGS-GFPM2 cells. It was observed that the high dose (1 µM) inhibited p-AKT expression initially after 2 h of incubation and sustained its effect for 24 h (Fig. 4B). The low dose (1 nM) of CA4 decreased the p-AKT expression on AGS-GFPM2 cells after a 24-h treatment (Fig. 4B). Similar results were observed in AGS-GFPM1 cells (data not shown). When the PI3 kinase inhibitor, LY294002, was applied on AGS-GFPM2 cells, it was observed that 1 to 20 µM LY294002 effectively reduced the level of p-AKT (Fig. 4C). Moreover, in an attempt to examine whether the observation of p-AKT inhibition by CA4 could also be found on other p-AKT-positive human gastric cancer cell lines, MKN74 and NUGC-3 were employed. As shown in Fig. 4D, it was apparent that CA4 was equally effective in reducing the level of p-AKT in MKN74 and NUGC-3 cells. In contrast, one p-AKT-negative human gastric cancer cell line (AGS), which showed no signal of p-AKT in different concentrations of CA4 treatments, was included as negative control (Fig. 4D).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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CA4 has been reported to be more effective with a nanomolar IC50 on cultured and primary tumor cell lines (Young and Chaplin, 2004). However, there are still some conflicting results regarding the extent of tumor vessel shrinkage in animal models and clinical trials using the different concentration of CA4 treatment (Badn et al., 2006). With respect to administering safe dosages of 52 and 68 mg/m2 in clinical trials, it has been revealed that peak plasma concentrations (Cmax) of CA4 are 1.89 and 2.26 µM, respectively (Dowlati et al., 2002). In this study, our data showed that a 24-h treatment of CA4 on p-AKT-positive gastric cancer cells resulted in a dose-dependent (0.02–20 µM) inhibition effect on cells growth, migration, and invasiveness as well as G2/M phase accumulation. In addition, these effects corresponded well with the level of p-AKT in CA4-treated p-AKT-positive cells (Figs. 1, 2, 3, 4, 5). Meanwhile, we further found that the p-AKT-positive cell lines are more sensitive to CA4 (0.2–20 µM) after a 48-h treatment, and a dose-dependent increase of cell detachment and apoptosis was observed (data not shown). This finding is consistent with the previous report that a 24-h incubation with CA4P did not induce endothelial cell death but significantly decreased the cell viability after a 48-h incubation with CA4P (Vincent et al., 2006). These results together suggest that CA4-inhibitory activities on AKT phosphorylation in human gastric cancer cells (0.2–2 µM) may be pharmacologically accessible clinically.
PI3 kinase/AKT pathway is well known to play many critical functions on tumorigenesis and tumor cell dissemination (Bader et al., 2005; Koul et al., 2005). PI3 kinase and AKT protein molecules have been drawing attention in targeting therapy (Brazil et al., 2004). Moreover, the mutations on PI3 kinase resulted in differential cytotoxicity toward LY294002 on human colorectal tumor cell lines (HCT116 and DLD1), suggesting cell lines with higher PI3 kinase/AKT activity were more susceptible to compounds targeting on this pathway (Kang et al., 2005). In this study, 10 human gastric cancer cell lines with different expression levels of p-AKT were correlated with the treatment effect of CA4 in the modulation of the expression level of p-AKT (Figs. 1, 2, 3, 4). Using the phosphoinositide 3-kinase inhibitor (LY294002) and specific AKT inhibitor as control treatment, CA4 displayed a similar toxic response on p-AKT-positive cells (Fig. 3, B and D). These results supported that CA4 can be an effective antitumor drug as mediated by targeting on the PI3 kinase/AKT pathway. In addition, the upstream tyrosine kinase receptors of PI3 kinase/AKT pathway included epidermal growth factor receptor and hepatocyte growth factor receptor. It has been reported that attenuated hepatocyte growth factor receptor function by a small molecule-specific inhibitor (PHA-665752) led to reduced gastric cancer cell migration through abolishing AKT activity in gastric cancer cells (GTL-16) (Christensen et al., 2003). Thus, whether the p-AKT expression pattern could be considered as another patient recruitment criteria or surrogate marker of therapeutic response needs further investigation.
In conclusion, our study is the first to reveal a novel mechanism of CA4 on targeting and inhibiting AKT activity and improving the treatment in human gastric cancer cells. Our results implicated that this AKT activity correlated with antitumor activities and reduced cell metastatic functions in vitro and in vivo under CA4 treatment. Given the more selective inhibition of the PI3 kinase-AKT pathway by new agents, it is promising to investigate other compounds with improved solubility and/or oral bioavailability in terms of chemical scaffold of CA4.
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Footnotes |
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H.-L.L. and S.-H.C. contributed equally to this work.
J.-P.L. and C.-W.C. contributed equally to this work.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: CA4, combretastatin A4; p-AKT, phosphorylated serine 473 on AKT; GFP, green fluorescence protein; FBS, fetal bovine serum; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride; PVDF, polyvinylidene difluoride; PI3 kinase, phosphoinositide 3-kinase; CA4-P, combretastatin A4-phosphate; PHA-665752, (3Z)-5-[(2,6-dichlorobenzyl)sulfonyl]-3-[(3,5-dimethyl-4-{[(2R)-2-(pyrrolidin-1-yl methyl)pyrrolidin-1-yl]carbonyl}-1H-pyrrol-2-yl)methylene]-1,3-dihydro-2H-indol-2-one.
Address correspondence to: Dr. Chin-Wen Chi, Department of Medical Research and Education, Taipei Veterans General Hospital, 201, Section 2, Shih-Pai Road, Taipei 11217, Taiwan. E-mail: cwchi{at}vghtpe.gov.tw
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