Chondroitin Sulphate Enhances the Antitumor Activity of Gemcitabine and Mitomycin-C in Bladder Cancer Cells with Different Mechanisms
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ONCOLOGY REPORTS 27: 409-415, 2012 Chondroitin sulphate enhances the antitumor activity of gemcitabine and mitomycin-C in bladder cancer cells with different mechanisms MATTEO FERRO1, GAIA GIUBERTI2, SILVIA ZAPPAVIGNA2, SISTO PERDONÀ3, GAETANO FACCHINI3, PASQUALE SPERLONGANO4, STEFANIA PORTO2, GIUSEPPE DI LORENZO5, CARLO BUONERBA5, ALBERTO ABBRUZZESE2, VINCENZO ALTIERI1 and MICHELE CARAGLIA2 1Department of Urology, University Federico II; 2Department of Biochemistry and Biophysics, Second University of Naples; 3Urogynecological Department, Istituto Nazionale Tumori, Fondazione ‘G. Pascale’; 4Department of Anaesthesiological, Surgical and Emergency Sciences, Second University of Naples; 5Department of Endocrinology and Medical Oncology, Genitourinary Cancer Section, University Federico II, Naples, Italy Received July 22, 2011; Accepted September 9, 2011 DOI: 10.3892/or.2011.1526 Abstract. Non-muscle invasive bladder cancer is the most combination induced a strong potentiation of apoptosis with common type of bladder cancer in Western countries. The the consequent activation of caspases 9 and 3. On the other glycosaminoglycan (GAG) layer at the bladder surface hand, HT-1376 cells were necrotic if exposed to the CS/MMC non-specifically blocks the adherence of bacteria, ions and combination and no signs of caspase activation was observed. molecules to the bladder epithelium and bladder cancer In conclusion, in the human bladder cancer cell line HT-1376 cells express CD44 that binds GAG. Currently, there are pharmacological combination of CS with GEM or MMC few options other than cystectomy for the management of resulted in a strong synergism on cell growth inhibition. non-muscle invasive bladder cancer with intravesical chemo- therapy using several drugs such as gemcitabine (GEM) Introduction and mitomycin-C (MMC) with poor prophylactic activity. In this study, we investigated the effects of the GAG chon- Non-muscle invasive bladder cancer is the most common type droitin sulphate (CS) on the growth inhibition of human of bladder cancer in Western countries. There are multiple risk bladder cancer cell lines HT-1376 and effects of the combi- factors for bladder cancer, which include exposure to tobacco nation between GEM or MMC with CS. We have found that and industrial chemicals, ingestion of arsenic-laced water, CS, MMC and GEM induced 50% growth inhibition at 72 h. radiation therapy to organs adjacent to the bladder, therapeutic Therefore, we have evaluated the growth inhibition induced use of alkylating agents in chemotherapy regimens, and by different concentration of CS in combination with MMC infection with the trematode Schistosoma haematobium. The or GEM, respectively, at 72 h. We have observed, at Calcusyn glycosaminoglycan layer at the bladder surface non-specifi- analysis, a synergism when HT-1376 cells were treated with cally blocks the adherence of bacteria, ions and molecules CS in combination with MMC or GEM, respectively, if used to the epithelium. Therefore, it may be an important element at an equimolar ratio. We have also found that CS/GEM in the first line of defence against infection, calculi and even carcinogens for the transitional cells of the bladder. Qualitative or quantitative defects in the glycosaminoglycan(s) expression may influence an individual's susceptibility to the development of bladder tumors (1-3). Correspondence to: Dr Michele Caraglia or Dr Alberto Abbruzzese, At initial presentation 70% of bladder cancers are superfi- Department of Biochemistry and Biophysics, Second University of cial and include carcinoma in situ (CIS), Ta, and T1 disease. Naples, Via Costantinopoli 16, I-80138 Naples, Italy Transurethral resection (TUR) is the primary mode of clinical E-mail: [email protected] management for both diagnosis and treatment of superficial E-mail: [email protected] bladder cancer, but 60 to 70% of these cancers recur and 20% Abbreviations: BCG, Bacille Calmette-Guèrin; CIS, carcinoma in situ; progress to a higher stage (4). TUR is commonly followed by CS, chondroitin sulphate; GEM, gemcitabine; MMC, mitomycin-C; intravesical infusion of either chemotherapy or immune-modu- TUR, transurethral resection lating agents in order to reduce the incidence of recurrence and progression. Bacille Calmette-Guèrin (BCG) is the most Key words: bladder cancer, chondroitin sulphate, gemcitabine, effective agent in the prevention of recurrence resulting in a mitomycin-C, HT-1376 decrease in the rate of progression; however, only one third of patients respond to BCG an effect that can induce a range 410 FERRO et al: CHONDROITIN SULPHATE ENHANCES ANTITUMOR ACTIVITY of adverse effects from mild dysuria to systemic tuberculosis 100 µg/ml streptomycin, 1% L-glutamine and 1% sodium (5). Several conventional cytotoxic agents have been used pyruvate. The cells were grown in a humidified atmosphere of for prophylaxis of recurrences after resection (6). Adjuvant 95% air/5% CO2 at 37˚C. intravesical infusion in recurrent tumors with chemotherapy or immunotherapy is not yet clearly established. Patients Drug combination studies. For the study of the synergism in whom BCG fails are a challenge for both urologists and between CS and MMC or GEM on growth inhibition of oncologists, with the need for careful individualization of HT-1376 cells, the cells were seeded in 96-multiwell plates at therapy in experienced hands. Among the compounds used the density of 5x103 cells/well. After 24 h incubation at 37˚C in intravesical therapy, mitomycin C (MMC) is one of the the cells were treated with different concentrations of CS or most common. This anti-tumor antibiotic, according to the GEM or MMC and their combinations. Drug combination manufacturer's labeling, is indicated in intravesical infusion at studies were based on concentration-effect curves generated the dose of 40 mg (7). The European Urological Association as a plot of the fraction of unaffected (surviving) cells vs. drug guidelines recommend 20 to 40 mg as the standard dose of concentration after 72 h of treatment. Assessment of synergy MMC (7). MMC has been shown to be active in treating super- was performed quantitating drug interaction by the Calcusyn ficial bladder cancer, and given in multiple infusions produces computer program (Biosoft, Ferguson, MO). Combination response rates ranging between 40 and 50% (8). MMC is, at the index (CI) values of <1, 1, and >1 indicate synergy, additivity, present, one of the standard chemotherapy agents in the treat- and antagonism, respectively (15). Furthermore, we analyzed ment of superficial bladder cancer (9). In two recent studies, the specific contribution of CS, GEM and MMC on the cyto- MMC was compared with BCG showing a slightly decreased toxic effect of the combination by calculating the potentiation activity in the treatment of the disease and prevention of factor (PF), defined as the ratio of the IC50 of either CS or GEM recurrence (10,11). Multiple infusions of MMC, however, are or MMC alone to the IC50 of CS/GEM or CS/MMC combina- associated with an increased incidence of side effects (12). In tions, respectively, as described before; a higher PF indicates a fact, chemical cystitis and allergic reactions are quite common greater cytotoxicity. and disappear after cessation of therapy. The new pyrimidine analogue gemcitabine (GEM) exhibits anti-tumor activity Flow cytometric analysis of apoptosis. Annexin V-FITC against a variety of solid tumors including advanced bladder (fluorescein isothiocyanate) was used in conjunction with cancer. In fact, it is active and well-tolerated when used in a vital dye, propidium iodide (PI), to distinguish apoptotic the treatment of metastatic bladder cancer (13). The proven (Annexin V-FITC positive, PI negative) from necrotic efficacy of systemic therapy against advanced bladder cancer (Annexin V-FITC positive, PI positive) cells. Briefly, cells were led urologists to consider GEM as a potential new agent for the incubated with Annexin V-FITC (MedSystems Diagnostics) treatment of superficial transitional cell carcinoma by intra- and propidium iodide (Sigma) in a binding buffer (10 mM vesical administration (14). Recently, we have demonstrated HEPES, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, that GEM has better efficacy and lower toxicity than MMC in 2.5 mM CaCl2) for 10 min at room temperature, washed and a phase III randomized clinical trial in the prevention of the resuspended in the same buffer. Analysis of apoptotic cells was recurrence of superficial bladder cancer and correlates with performed by flow cytometry (FACScan, Becton-Dickinson). significantly lower side effects. For each sample, 2x104 events were acquired. Analysis was In this study, we investigated the effects of chondroitin carried out by triplicate determination on at least three separate sulphate (CS) (3) on the growth inhibition of human bladder experiments. cancer cell line HT-1376 and effects of the combination between GEM and MMC with chondroitin sulphate on the Western blot analysis. HT-1376 cells were grown for 48 h antitumor activity of these drugs in vitro. with or without CS and/or GEM or CS and/or MMC at 37˚C. For cell extract preparation, the cells were washed twice with Materials and methods ice-cold PBS/BSA, scraped, and centrifuged for 30 min at 4˚C in 1 ml of lysis buffer (1% Triton, 0.5% sodium deoxy- Materials. DMEM, BSA and FBS were purchased from Flow cholate, 0.1 M NaCl, 1 mM EDTA, pH 7.5, 10 mM Na2HPO4, Laboratories (Milan, Italy). Tissue culture plasticware was pH 7.4, 10 mM PMSF, 25 mM benzamidine, 1 mM leupeptin, from Becton-Dickinson (Lincoln Park, NJ, USA). Chondroitin 0.025 U/ml aprotinin). Equal amounts of cell proteins were sulphate (Uracyst®) was a gift of Stellar Pharmaceuticals, separated by SDS-PAGE, electrotransferred to nitrocellulose Inc., Gemcitabine (Gemzar®) was a gift of Lilly, mitomycin-C and reacted with the different antibodies. Blots were then was a gift of Kyowa Hakko Kirin. Caspase-3 Mab (31A1067) developed using enhanced chemiluminescence detection and caspase-9 pAb were purchased from Enzo Life Sciences. reagents (SuperSignal West Pico, Pierce) and exposed to X-ray Anti caspase-6 human Mab was purchased from Alexis.