Gazzaniga Paola, Silvestri Ida, Gradilone Angela, Scarpa Susanna, Morrone Stefania, Gandini Orietta, Gianni Walter, Frati Luigi, Aglianò Anna Maria
Department of Experimental Medicine and Pathology, University La Sapienza, Viale Regina Elena 324, 00161 Rome, Italy.
Anticancer Drugs. 2007 Feb;18(2):179-85. doi: 10.1097/CAD.0b013e328010ef47.
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.
近期数据表明，由于传统治疗后浅表性膀胱癌的复发率较高，尤其是在T1G3和卡介苗难治性患者中，因此需要新的治疗方案。I期和II期研究表明，吉西他滨可能是浅表性膀胱癌膀胱内灌注治疗的一个候选药物。尽管有临床试验，但吉西他滨的体外细胞毒性和促凋亡作用尚未得到充分研究。在本研究中，我们研究了吉西他滨如何影响具有高危浅表性膀胱癌相同分子特征的膀胱癌细胞系5637中的细胞凋亡。通过DNA片段化、流式细胞术和半胱天冬酶激活来评估细胞凋亡。还通过逆转录聚合酶链反应评估bcl-2、bcl-X、bax、生存素和fas基因的表达。通过免疫荧光评估核因子-κB的激活。吉西他滨以时间依赖性方式诱导5637细胞凋亡，同时激活半胱天冬酶-3、-8和-9。bcl-2、bax、生存素和bcl-X的表达不受治疗影响，而治疗24小时后fas强烈增加。治疗后，我们未发现核因子-κB有任何核定位。由于吉西他滨诱导的细胞凋亡涉及fas上调，这些结果可能会促使人们对fas阴性膀胱肿瘤进行膀胱内灌注吉西他滨的研究。此外，由于顺铂、阿霉素和多柔比星激活核因子-κB可能导致增殖、迁移增强、永生化和细胞凋亡抑制，吉西他滨不激活核因子-κB这一观察结果可能对高危浅表性膀胱癌的膀胱内治疗有影响。
