Gemcitabine-induced apoptosis in 5637 cell line: an in-vitro model for high-risk superficial bladder cancer.
Recent data suggest that new treatment options for superficial bladder cancer are necessary, owing to the high recurrence rate after conventional treatment, especially in T1G3 and Bacillus Calmette-Guerin-refractory patients. Phase I and II studies have demonstrated that gemcitabine may represent a candidate for intravesical therapy in superficial bladder cancer. Despite clinical trials, the in-vitro cytotoxic and proapoptotic effects of gemcitabine have been poorly investigated. In the present study, we investigated how gemcitabine affects apoptosis in bladder cancer cell line 5637, which has the same molecular features of high-risk superficial bladder cancer. Apoptosis was evaluated by DNA fragmentation, flow cytometry and caspase activation. bcl-2, bcl-X, bax, survivin and fas gene expression were also evaluated by reverse-transcriptase polymerase chain reaction. Nuclear factor-kappa B activation was assessed by immunofluorescence. Gemcitabine induced apoptosis in 5637 cells in a time-dependent manner, with activation of caspase-3, -8 and -9. Expression of bcl-2, bax, survivin and bcl-X was not affected by treatment, whereas fas strongly increased after 24 h of treatment. After treatment, we failed to find any nuclear localization of nuclear factor-kappa B. As gemcitabine-induced apoptosis involves fas upregulation, these results may encourage the investigation of intravesical gemcitabine in fas-negative bladder tumors. Furthermore, as nuclear factor-kappa B activation by cisplatin, doxorubicin and adriamycin may result in enhanced proliferation, migration, immortality and inhibition of apoptosis, the observation that gemcitabine does not activate nuclear factor-kappa B may have implications in intravesical therapy of high-risk superficial bladder cancer.
Gazzaniga
Paola
P
Department of Experimental Medicine and Pathology, University La Sapienza, Viale Regina Elena 324, 00161 Rome, Italy. paola.gazzaniga@uniroma1.it
Silvestri
Ida
I
Gradilone
Angela
A
Scarpa
Susanna
S
Morrone
Stefania
S
Gandini
Orietta
O
Gianni
Walter
W
Frati
Luigi
L
Aglianò
Anna Maria
AM
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002295:Carcinoma, Transitional Cell / Q000188:drug therapy* / Q000473:pathology; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000473:pathology; D051020:bcl-X Protein / Q000096:biosynthesis / Q000235:genetics; D000093542:Gemcitabine
D000964:Antimetabolites, Antineoplastic; D016328:NF-kappa B; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; D003841:Deoxycytidine; D020169:Caspases; D000093542:Gemcitabine