Celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, may elicit anti-tumor results in various malignancies. Cisplatin-based chemotherapy is the standard treatment for patients with metastatic UC [3], [4]; however, despite regimens such as the cisplatin, gemcitabine or paclitaxel combination, the overall response rates vary between 40% and 65% [5], [6]. Most patients eventually die of metastatic disease and the overall median survival is about 1 year [2]. Moreover, patients suffer chemotherapy-related side effects or toxicities during the process [4], [6]. Intense efforts have focused on the development of active agents to improve the therapeutic efficacy. It is imperative to develop new therapeutic strategy to prolong survival and minimize chemotherapy-related morbidity [2]. Celecoxib is a selective inhibitor of cyclooxygenase-2 (COX-2) and is widely used for anti-inflammation or pain control. Selective COX-2 inhibitor has been reported to elicit anti-proliferative response in various tumors [7], [8], [9], [10], [11] including urinary bladder cancer [8], [9], [12], [13], [14]; however, the detail effects and mechanisms of celecoxib on UC cells have not been fully explored. Several mechanisms have been proposed in other tumor models such as induction of cell cycle arrest, mitochondria-mediated pathway, Akt phosphorylation inhibition, endoplasmic reticulum (ER) stress, and autophagy [15], [16], [17], [18], [19], [20]. Autophagy is a process of cell destruction whereby cytoplasmic proteins and organelles are sequestered in vacuoles and delivered to lysosomes for degradation, which supports metabolism for tumor growth [21], [22]. Some anti-cancer drugs have been reported to induce autophagy and apoptosis [23], [24]. Targeting autophagy to sensitize cancers may be an effective therapeutic strategy to conquer drug resistance (+)-Bicuculline IC50 [23]. Therefore, we hypothesize the interference of autophagy can enhance the celecoxib-induced cytotoxicity in bladder UC cells. In Rabbit Polyclonal to RAD18 this study, we try to investigate the role of autophagy in celecoxib-induced cytotoxicity in human bladder UC cells. Materials and Methods Cell Culture NTUB1 cell line, kindly provided from Dr. Yeong-Shiau Pu (Department of Urology, National Taiwan University Hospital, Taipei, Taiwan), was derived from the surgical specimen of a 70-year-old female patient with high grade transitional bladder cell carcinoma and were reported to be tumorigenic in a xenograft model [16], [25], [26], [27], [28], [29], [30], [31], [32]. T24 cell line, obtained (+)-Bicuculline IC50 from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan), was derived from a highly malignant grade III human urinary bladder carcinoma [33]. The (+)-Bicuculline IC50 cells were maintained at 37C with 5% CO2 in RPMI-1640 medium (NTUB1 cells) or Dulbecco’s Modified Eagle Medium (T24 cells) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 1 mg/ml streptomycin. The cell culture media and supplements were purchased from Invitrogen (Carlsbad, CA, USA). Reagents and Antibodies Celecoxib pure compound was provided by Pfizer (New York, NY, USA). ZVAD-FMK (Z-Val-Ala-Asp(OMe)-CH2F), 3-MA, rapamycin, bafilomycin A1 and LM-1685 were obtained from Merck Calbiochem (Darmstadt, Germany). Antibodies against cleaved caspase-3, cleaved caspase-7, cleaved PARP, phospho-SAPK/JNK (Thr183/Tyr185), phospho-c-Jun (Ser73), ATF-4, phospho-eIF2 (Ser51), autophagy-related protein 5, 12 (Atg5, 12), and microtubule-associated protein light chain 3 B (LC3B), for immunoblotting analysis or immunofluorescence staining were purchased from Cell Signaling Technology (Danvers, MA, USA). Moreover, -tubulin antibody was purchased from GeneTex (Irvine, CA, USA), GAPDH and -actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Other chemicals and reagents all obtained from Sigma-Aldrich (St. Louis, MO, USA) or Serva (Heidelberg, Germany). Cell Viability and Flow Cytometry (FACS) for Apoptosis Assay Celecoxib, ZVAD-FMK, 3-MA, bafilomycin A1, rapamycin, LM-1685 or DMSO (Mock, as non-treated control) were diluted in the culture media promptly before exposing to cells. Following the treatments, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT, Sigma-Aldrich) assay were performed to detect cell viabilities according to the methods described previously [4], [6]. For apoptosis assay, the cells were harvested as described earlier and analyzed with Becton Dickinson.