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Keywords:

  • Lewis (y) antigen;
  • ovarian cancer;
  • drug resistance;
  • topoisomerase;
  • integrin αvβ3

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITEATURE CITED

Lewis (y) antigen, a difucosylated oligosaccharide, has been shown to be associated with malignant properties of ovarian carcinomas. In this study, we have investigated the potential role of Lewis (y) antigen, which was stably transfected into ovarian cancer RMG-1 cells, on carboplatin-induced apoptosis. Overexpression of Lewis (y) antigen effectively protected vitronectin-adherent RMG-1 cells from carboplatin-induced apoptosis as assessed by Hoechst 33258 staining and flow cytometry. Treatment with anti-Lewis (y) antigen, anti-integrin αv, or anti-integrin β3 antibody partially abolished the protective effect on apoptosis and markedly inhibited the expression of Topo-II β in cells overexpressing Lewis (y) antigen (all P < 0.01). Moreover, elevated expression of Topo-I and Topo-II β was found in Lewis (y) antigen-overexpressing cells (P < 0.01). However, no obvious changes in Topo-II α were observed throughout the study (P > 0.05). Taken together, these data suggest that the overexpression of Lewis (y) antigen confers cell adhesion-mediated drug resistance to apoptosis in ovarian cancer cells by the upregulation of Topo-I and Topo-II β. Therefore, the inhibition of Lewis (y) antigen may be a novel strategy of cancer chemotherapy. Anat Rec, 2011. © 2011 Wiley-Liss, Inc.

Ovarian cancer is the most lethal malignancy of the female reproductive system. Despite significant advances in multidisciplinary treatment approaches, such as surgery, chemotherapy, and radiotherapy, the 5-year survival rate for stage III and IV disease is reported to be around 30% (Jemal et al., 2007). Drug resistance is one of the major reasons for the low 5-year survival rate. Therefore, a better understanding of the molecular mechanisms underlying drug resistance may provide novel strategies to improve cancer chemotherapy.

Anticancer drug resistance is a multifactorial phenomenon involving several major mechanisms, such as increased energy-dependent efflux of chemotherapeutic drugs, increased repair of DNA damage, avoidance of apoptosis, and cell adhesion-mediated drug resistance (CAM-DR) (Damiano et al., 1999; Gottesman, 2002; Luqmani, 2005). The Lewis (y) antigen belongs to a family of blood group-related difucosylated oligosaccharides that is overexpressed on the surface of 60%–90% of human epithelial cancer cells, including breast, ovary, prostate, and colon (Hellstrom et al., 1990; Saleh et al., 2000; Madjd et al., 2005). In our previous studies, we have successfully established a stable human ovarian cancer cell line (RMG-1-hFUT) constitutively expressing Lewis (y) antigen by transfection with cDNA encoding part of the human α1,2-fucosyltransferase (α1,2-FUT) gene into the ovarian cancer cell line RMG-1 (Hao et al., 2008). Our further experiments demonstrated that the RMG-1-hFUT cells not only exhibited increased proliferation and invasion capacity but also showed high tolerance to common chemotherapy drugs for ovarian cancer, such as carboplatin, 5-fluorouracil, and taxol (Liu et al., 2009; Sato et al., 2009; Liu et al., 2010; Gao et al., 2010). More recently, we found that the transfection of the α1,2-FUT gene into RMG-1 cells leads to elevated expression of integrin αvβ1 with Lewis (y) antigen and thereby enhances the adhesion and spreading potentials of cells through the integrin-fibronection interaction, suggesting a potential role of Lewis (y) antigen in the CAM-DR of human ovarian cancer cells (Yan et al., 2010). Therefore, these findings lead us to explore whether overexpression of Lewis (y) antigen is involved in CAM-DR in ovarian cancer cells.

In this study, we examined the effects of overexpression of Lewis (y) antigen on carboplatin-induced apoptosis in Lewis (y) antigen-overexpressing cells that adhered to vitronectin (VN) and determined the mechanisms responsible. Our findings may reveal some new mechanisms involved in Lewis (y) antigen-induced carboplatin resistance in tumor treatment.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITEATURE CITED

Cell Culture

The human ovarian cancer cell line, RMG-1, was kindly provided by Professor Iwamori Masao (Tokyo University, Japan). As previously described, RMG-1-hFUT cell line, highly expressing Lewis (y) antigen, was established by transfecting the pcDNA3.1 (-)-HFUT-H expression vector (containing a1,2-FUT gene) into RMG-1 cells, and RMG-1 cells stably transfected with pcDNA3.1 (RMG-1-pcDNA3.1) were also established and used as the control (Gao et al., 2010; Yan et al., 2010). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT) and 200 μg/mL G418 (geneticin, Gibco) at 37°C in a humidified 5% CO2 atmosphere. The cells were routinely passaged, and cells at logarithmic growth phase were used for the experiments.

Preparation of a VN-Coated Cell Adhesion Model

Each well of a 96-well plate was coated with 3 μg/mL VN (Gibco) and allowed to dry at room temperature. Subsequently, the plate was maintained at 4°C overnight, and then cells were seeded. At the end of the 2-hr incubation at 37°C, 5% CO2, unattached cells (∼10% of initial seeding) were removed by washing, and adherent cells were used for further experiments.

MTT Assay

Cells (3 × 103)/well were seeded into 96-well plates and allowed to attach for 24 h. Subsequently, these cells were exposed to increasing concentrations of carboplatin (10, 30, 60, and 120 μg/mL, Qilu Pharmaceutical, Jinan, China) for 48 hr. Twenty microliters of 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (5 mg/mL; MTT, Amresco, Solon, OH) was then added to each well, and cells were incubated continuously at 37°C for 4 hr. After removal of medium, the crystals were dissolved in DMSO, and absorbance was assessed at 490 nm with a microplate reader. The inhibition rates (IR) of cell proliferation were calculated as follows: IR = (AcontrolAtreatment)/Acontrol.

Flow Cytometry Analysis

Apoptosis of cells induced by carboplatin was determined by flow cytometry using the Annexin V: propidium iodide Apoptosis Detection Kit (Jingmei Biotech, Shenzhen, China) according to the manufacturer's instruction. Briefly, cells at logarithmic phase were transferred to a 25 cm2 culture flask. After adherence, varying concentrations of carboplatin (30 and 60 μg/mL) were added and allowed to incubate at 37°C for 48 hr. Following this incubation, cells were harvested and apoptotic cells were detected.

Staining the Nuclei with Hoechst 33258

Cells at logarithmic phase were transferred to a six-well plate and cultured for 48 hr. After washing with cold phosphate buffered saline (PBS) twice, cells were fixed with 4% paraformaldehyde. Then 500-μL Hoechst 33258 (2 μg/mL, Beyotime Institute of Biotechnology, Haimen, China) was added to each well and incubated at room temperature for 10 min. After washing with PBS for 5 min, the morphology of nuclei was observed and photographed through a fluorescence microscope.

Western Blot Analysis

Total proteins were isolated from treated cells, and protein concentrations were determined using a bovine serum albumin standard line. Equal amounts of protein were separated by SDS-PAGE and then electrotransferred to PVDF membranes. Membranes were blocked with 5% skim milk, incubated at 37°C with mouse anti-human Topo-I (1:10,000), Topo-II α (1:2,000), or Topo-II β (1:4,000) monoclonal antibody (all from BD Biosciences, San Jose, CA) for 2 hr, followed by horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized with an ECL plus chemiluminescence kit (Beyotime Institute of Biotechnology, Haimen, China).

Anti-Lewis (y) Antigen, Anti-integrin αv, and Anti-integrin β3 Antibodies Blocking Test

Cultured RMG-1-hFUT cells at the exponential growth phase were harvested to prepare single cell suspensions, and mouse anti-human Lewis (y) monoclonal antibodies (Abcam, UK), and rabbit anti-integrin αv and anti-integrin β3 polyclonal antibodies (Boster Biological Technology, Wuhan, China) were then added. After incubation at 37°C for 4 hr, suspended cells were inoculated into culture dishes (35 mm) at a density of 3 × 105 cells/dish and allowed to attach. At 48 hr after treatment with 60 μg/mL carboplatin, cells were harvested for flow cytometry or Western blot analysis.

Statistical Analysis

All experiments were performed in triplicate, and all data were expressed as means ± SD. Raw data were analyzed by the unpaired Student's t test using SPSS 11.0 software (SPSS, Chicago, IL). A P-value < 0.05 was considered to be statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITEATURE CITED

Overexpression of Lewis (y) Antigen Protects RMG-1 Cells from Carboplatin-Induced Growth Inhibition

Considering the fact that the overexpression of Lewis (y) antigen could enhance the adhesion and spreading potentials of cells through the integrin-fibronection interaction, we attempted to address whether Lewis (y) antigen overepression plays a role in the CAM-DR in RMG-1 cells. To this end, we examined the effects of carboplatin on the proliferation of VN-adherent and non-VN-adherent RMG-1-pcDNA3.1 and RMG-1-hFUT cells using MTT assays. As shown in Fig. 1, all cells displayed a dose-dependent reduction in cell proliferation. However, no significant differences of IR were observed between VN-adherent and nonadherent RMG-1-pcDNA3.1 at any concentration of carboplatin (P > 0.05). Notably, we found that the VN-adherent RMG-1hFUT cells have a significant survival advantage over those nonadherent cells (P < 0.01). These results indicated that the overexpression of Lewis (y) antigen protects RMG-1 cells from the growth inhibitory effects of carboplatin.

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Figure 1. Carboplatin-induced growth inhibition of RMG-1 cells. Cell culture plate was coated with VN or uncoated. RMG-1-pcDNA3.1 or RMG-1-hFUT cells (3 × 103)/96-well plates were plated and exposed to increasing concentrations of carboplatin for 48 hr. Then MTT assays were performed to determine the inhibition rates. Depicted are the mean values of three independent determinations.

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Protection of RMG-1 Cells Overexpressing Lewis (y) Antigen from Apoptosis Induced by Carboplatin

Next, we performed Hoechst 33258 staining to detect the apoptosis of VN-adherent and nonadherent RMG-1-pcDNA3.1 and RMG-1-hFUT cells induced by carboplatin. As illustrated in Fig. 2A, after exposure to carboplatin for 48 hr and staining with Hoechst 33258, both nonadherent RMG-1-pcDNA3.1 and RMG-1-hFUT cells exhibited more prominent nuclei and contained more cells in the later morphological stages of apoptosis (e.g., severe membrane blebbing and dense chromatin) than their control cells. When the cells were counted, we observed an increased percentage of apoptotic cells compared with their controls (P < 0.01, Fig. 2B). In contrast, RMG-1-hFUT adhered to VN cells displayed more cells with normal morphology than those nonadherent RMG-1-pcDNA3.1 cells. To confirm the apoptotic changes, we used Annexin V staining and flow cytometric analysis to examine the apoptosis induced by carboplatin. Consistently, VN-adherent RMG-1-hFUT cells had a significantly lower percentage of apoptotic cells compared with nonadherent controls (Fig. 2C,D). These observations demonstrated that the overexpression of Lewis (y) antigen in RMG-1 cells increases their CAM-DR to apoptosis induced by carboplatin.

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Figure 2. (A) Hoechst 33258 staining showing the changes in the morphology of VN-adherent or nonadherent RMG-1-pcDNA3.1 and RMG-1-hFUT cells treated or untreated with carboplatin. (B) Quantitative data of Hoechst 33258 staining expressed as mean ± SD. (C) Results of flow cytometry showing the percent apoptosis of VN-adherent or nonadherent RMG-1-pcDNA3.1 and RMG-1-hFUT cells exposed to carboplatin. (D) Mean values of three independent flow cytometry experiments.

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Contribution of Integrin αvβ3 in Lewis (y) Antigen-Mediated CAM-DR to Apoptosis

After the RMG-1-pcDNA3.1 and RMG-1-hFUT cells were treated with carboplatin and different antibodies (anti-Lewis (y) antigen, anti-integrin αv, and anti-integrin β3 antibodies), the apoptosis rates were determined by Annexin V staining and flow cytometric analysis. As shown in Fig. 3, anti-Lewis (y) antigen antibody treatment significantly increased the apoptosis in RMG-1-hFUT cells (P < 0.01). Moreover, a smaller, but statistically different (P < 0.01), increase in apoptosis was observed in VN-adherent RMG-1-pcDNA3.1 cells treated with anti-integrin αv or anti-integrin β3 antibody. These results revealed that the integrin αv and integrin β3 are involved in the Lewis (y) antigen-mediated CAM-DR.

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Figure 3. Flow cytometry analysis of carboplatin-induced apoptosis in VN-adherent or nonadherent RMG-1-pcDNA3.1 and RMG-1-hFUT cells treated with anti-Lewis (y) antigen, anti-integrin αv or anti-integrin β3 antibody. (A) Graphical representation of apoptosis for all treatments of VN-adherent or nonadherent RMG-1-pcDNA3.1 and RMG-1-hFUT cells. (B) Bar graph of all conditions. Control, irrelevant isotype-matched control.

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The Expression of Topo-I and Topo-II β in Cells Overexpressing Lewis (y) Antigen

To further dissect the possible mechanisms by which Lewis (y) antigen contributes to the CAM-DR, we examined the expression levels of Topo-I, Topo-II, α and Topo-II β proteins in RMG-1-pcDNA3.1 and RMG-1-hFUT cells using Western blot analysis. As shown in Fig. 4, elevated expression of Topo-I and Topo-II β was found in RMG-1-hFUT cells compared with RMG-1-pcDNA3.1 cells (P < 0.01), but no significant changes of Topo-II α were observed (P > 0.05). In addition, the expression of Topo-II β was upregulated, but no obvious changes of Topo-I in VN-adherent RMG-1-hFUT cells compared with nonadherent RMG-1-hFUT cells. Furthermore, the expression of Topo-II β was inhibited by treatment with anti-Lewis (y) antigen, anti-integrin αv, or anti-integrin β3 antibody (Fig. 5). Collectively, our data suggest that the overexpression of Lewis (y) antigen confers CAM-DRto apoptosis in ovarian cancer cells by the upregulation of Topo-I and Topo-II β.

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Figure 4. (A) Western blot analysis of Topo-I, Topo-II α, and Topo-II β protein levels in VN-adherent or nonadherent RMG-1-pcDNA3.1 and RMG-1-hFUT cells. A representative experiment out of three performed is shown and protein size is expressed in kDa. (B) Densitometric quantification data were expressed as the intensity ratio of Topo-I, Topo-II α, or Topo-II β to β-actin (mean ± SD).

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Figure 5. Western blot analysis of Topo-II β protein levels in VN-adherent or nonadherent RMG-1-pcDNA3.1 and RMG-1-hFUT cells treated with anti-Lewis (y) antigen, anti-integrin αv, or anti-integrin β3 antibody. (A) Three independent and reproducible experiments. Quantitative data were expressed as the intensity ratio Topo-II β to β-actin (B).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITEATURE CITED

Lewis (y) antigen is a difucosylated oligosaccharide that belongs to the A, B, H, Lewis blood group family with specific fucosylation of the terminal end of carbohydrate structure catalyzed by the α1,2-FUT. Its abnormally high expression has been found in a wide range of human cancers, and the high expression level is associated with tumor grade and prognosis (Saleh et al., 2000). In particular, varying degrees of Lewis (y) antigen overexpresion have been shown in 75% ovarian carcinoma tissues and the increasing expression of Lewis (y) antigen correlates with a highly malignant phenotype in these cases (Federici et al., 1999). In our previous studies, a cell model overexpressing Lewis (y) antigen was successfully established by introducing the α1,2-FUT gene into human ovarian cancer cell line RMG-1 through gene transfection. We further demonstrated that the Lewis (y) antigen is able to promote the proliferation, enhances the adhesive and spreading potentials, and increases resistance to chemotherapy drugs of ovarian carcinoma RMG-1 cells (Sato et al., 2009; Yan et al., 2010). However, the molecular mechanisms by which Lewis (y) antigen enhances these malignant behaviors of ovarian cancer cells are still not fully understood.

It is currently known that the drug resistance in cancer involves multiple mechanisms, such as increased drug efflux, enhanced drug detoxification, mutation at drug target sites, and attenuated apoptosis (Zheng et al., 2010). Our previous study indicated that the overexpression of Lewis (y) antigen may enhance the gene expression of multiple drug resistance-associated proteins, including MRP1, MRP2, protein kinase C-α, and multidrug resistance gene-1 (MDR-1), thereby inducing the development of drug resistance in tumor cells (Gao et al., 2010). However, this mechanism alone cannot account for all drug resistance induced by Lewis (y) antigen in RMG-1 cells. CAM-DR is a de novo drug-resistant mechanism that may allow for initial tumor cell survival and the eventual emergence of acquired drug resistance (Hazlehurst and Dalton, 2001). Integrin αvβ3 has been reported to be associated with the CAM-DR in cancer cells (Sethi et al., 1999; Hodkinson et al., 2007). In addition, our recent study also found increased expression of integrin αvβ3 in RMG-1-hFUT cells compared with RMG-1 cells (Yan et al., 2010). Therefore, we surmised that the CAM-DR may represent one underlying mechanism for the Lewis (y) antigen-induced drug resistance in ovarian cancer cells. In the current study, we found that the RMG-1-hFUT adhered to VN cells have a significant survival advantage over those nonadherent cells. Furthermore, anti-Lewis (y) antigen, anti-integrin αv, or anti-integrin β3 antibody treatment significantly increased the apoptosis rates in RMG-1-hFUT cells. These results strongly suggest that the Lewis (y) antigen contributes to the CAM-DR-medicated by integrin αvβ3. It has been a subject of debate about the role of integrin αvβ3 in the growth of cancer cells. Consistent with our results, Maubant et al. (2002) also found that the adherent IGROV1-R10 cells, a cisplatin-resistant ovarian cancer cell line, could draw proliferative advantage from the expression of integrin αvβ3 at their cell surface. Additionally, integrin αvβ3 has been demonstrated to confer a survival advantage to human glioma cell lines treated with topotecan, and this chemoresistance was associated with an increase in the expression of antiapoptotic proteins, Bcl-2, and BclxL (Uhm et al., 1999). Conversely, integrin αvβ3 has been reported to be able to promote apoptosis in human intestinal carcinoma cells and hypopharyngeal squamous carcinomas cells (Kozlova et al., 2001; Lu et al., 2009). Thus, it is probable that the integrin αvβ3 functions in a cell-type-specific manner.

Numerous studies have demonstrated that Topo I, Topo-II α, and Topo-II β are involved in the CAM-DR (Hazlehurst et al., 2001). In the current study, we detected the protein levels of Topo I, Topo-II α, and Topo-II β in RMG-1-pcDNA3.1 and RMG-1-hFUT cells. We found elevated expression of Topo-I and Topo-II β in RMG-1-hFUT cells compared with RMG-1-pcDNA3.1 cells, but no changes of Topo-II α were observed. In agreement with these results, our preliminary work showed that the Topo I mRNA was significantly upregulated in RMG-1-hFUT cells compared with RMG-1 cells (Gao et al., 2010). We further examined the protein levels of Topo-II α and Topo-II β in RMG-1-hFUT cells treated with anti-Lewis (y) antigen, anti-integrin αv, or anti-integrin β3 antibody. The results showed that the expression levels of Topo-II β were decreased by treatment with the antibodies. These results are consistent with a previous study in a lymphoma cell line (Hazlehurst et al., 2006). Notably, no obvious change of Topo-II α was observed throughout the study. Nonetheless, our work could not exclude the potential role of Topo-II α in the Lewis (y) antigen-induced CAM-DR, because we did not detect the cellular distribution and activity of Topo-II α in the current study. Therefore, the precise mechanisms of Topo I, Topo-II α, and Topo-II β in the Lewis (y) antigen-induced CAM-DR need to be further elucidated. Additionally, the anti-Lewis (y) antigen antibody only partially reversed the reduced susceptibility to apoptosis in VN-adherent RMG-1-hFUT cells, suggesting that other adhesive systems must be implicated. Anti-integrin αv or anti-integrin β3 antibody also reversed this reduced susceptibility to apoptosis, but their effects were weaker than that of anti-Lewis (y) antigen antibody. These findings again indicated that the Lewis (y) antigen contributes to the CAM-DR-mediated by integrin αvβ3.

In conclusion, our study demonstrated that the overexpression of Lewis (y) antigen confers CAM-DR to apoptosis in ovarian cancer cells by the upregulation of Topo-I and Topo-II β. Therefore, the inhibition of Lewis (y) antigen may be a novel strategy of cancer chemotherapy.

LITEATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. LITEATURE CITED
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