新型Eg5蛋白抑制剂S（MeO）TLC治疗膀胱肿瘤的实验研究
摘要
研究背景
膀胱癌主要指膀胱移行上皮细胞癌,又称膀胱尿路上皮癌。其中约75％是局限于膀胱粘膜层(Ta)和粘膜下层(T1)的非肌层浸润癌；尽管可以通过经尿道膀胱镜切除并配合术后腔内化疗灌注治疗,约2／3的患者会出现复发,并且复发患者中15％～30％会进展为更高的病理分期和／或伴有分级升高。除了肿瘤分期和细胞分级,虽然以往的研究已经报道了大量的肿瘤分子标记物可用以预测膀胱癌的预后,但是目前尚未发现一种可靠的预测指标。
肌层浸润性膀胱癌占初发膀胱癌的25％,预后较差,5年生存率仅为10％～50％。对伴有转移的晚期浸润性膀胱癌的传统化疗方法是甲氨蝶呤、长春碱、多柔比星和顺铂联合用药的MVAC方案。该联合化疗方案虽然可以取得46％～60％治疗反应率,但也伴有严重的毒性反应,而且患者经过数疗程治疗后可能出现获得性耐药。近年来,尽管毒性较低的吉西他滨和顺铂新型GC联合方案开始逐渐成为一线的晚期膀胱癌化疗措施,然而该方案治疗反应率并不比传统的MVAC高。因此,寻找新的治疗晚期膀胱癌的化疗方法已经成为近期研究的热点,以求在减低化疗毒性的基础上提高疗效,为患者延长生存时间并争取更好的生活质量。
纺锤体驱动蛋白Eg5在细胞有丝分裂过程中发挥关键作用,近年来成为了靶向抗癌治疗的研究热点。Eg5在增殖组织细胞中表达,其主要功能与双极纺锤体的形成和分离有关,在细胞有丝分裂早期中起到了关键作用。
研究已经证实Eg5蛋白在恶性组织中存在高表达,但是在有丝分裂后分化稳定的成熟组织细胞中不表达,如中枢神经系统细胞。研究结果证实Eg5作为新的特异性抗有丝分裂药物的靶点进行抗癌治疗时,不会对神经细胞造成直接损害而出现类似于抗微管药物的神经病变副作用。研究也表明Eg5与肿瘤的发生和发展密切相关,而且最近的临床研究还提示Eg5在恶性肿瘤组织中的表达水平与化疗疗效以及患者的预后密切相关。
1999年研究发现了第一个Eg5蛋白小分子抑制剂monastrol, monastrol能特异地与驱动蛋白Eg5结合并抑制Eg5功能,从而使细胞有丝分裂停滞,具有强烈抑制哺乳动物细胞分裂增殖的作用。Monastrol不抑制其他的驱动蛋白活性功能,从而具有较少的副作用和广泛的抗肿瘤作用。以上的特点均促使近年来大量研究集中在研制Eg5抑制剂作为新的抗有丝分裂化疗药物应用上,并取得了很多令人兴奋的结果,临床试验也已经用来检验Eg5抑制剂的抗癌效果。
2004年Dimitrios A等首先证实了STLC是作用于Eg5靶点的强有力的小分子抑制剂,与monastrol具有相同的Eg5结合位点,且结合牢固、特异性高,对细胞生长的抑制能力提高了36倍。日本京都大学对STLC的衍生物进行研究后首先报道了S(MeO)TLC是一种强有力的小分子细胞渗透性Eg5抑制剂,抑制细胞生长的能力比STLC提高了10倍,而且S(MeO)TLC与STLC一样具有特异性强的特点。此外,京都大学药学部最近又研制了多种联芳类Eg5抑制剂。
如上所述,Eg5与恶性肿瘤的发生、发展有关,已经有多种Eg5小分子抑制剂成为靶向抗癌药物的研究热点,但是目前还没有Eg5蛋白表达与膀胱癌生物学行为以及临床预后的相关性研究,也没有Eg5蛋白抑制剂治疗膀胱癌的系统研究。所以,在本次研究中,研究者通过山东海外留学联合培养项目,在山东大学附属省立医院和日本京都大学医学部对Eg5蛋白表达与膀胱癌关系以及5种Eg5抑制剂(STLC、S(MeO)TLC、KPYC10665、KPYC1066和KPYC 10728)治疗膀胱癌的疗效进行了研究。从而为膀胱肿瘤的临床诊断、预后判断以及化学治疗提供有效地新型方法。
目的
本研究首先探讨Eg5蛋白表达水平与与膀胱癌生物学行为以及非肌层浸润癌患者术后预后间关系的探讨；并进一步研究新型的Eg5蛋白抑制剂对膀胱癌的治疗效果,尤其是针对晚期转移癌的疗效。
方法
(1)采用免疫组织化学染色的方法检测了193例膀胱癌临床标本中的Eg5表达(分级：1级32例,2级98例,3级63例：分期：Ta期49例,T1期114例,T2～T4期30例),分析Eg5表达水平与肿瘤生物学行为指标之间的关系,并通过生存分析研究了Eg5表达水平与163例非肌层浸润膀胱癌患者经膀胱镜电切治疗后的预后问关系。
(2)培养膀胱癌细胞并通过免疫印迹方法检测各种细胞株中Eg5蛋白表达水平,包括RT112、KU7、RT4、EJ细胞等；同时通过免疫印迹方法检测正常膀胱粘膜、高分级和低分级膀胱癌组织标本中Eg5表达水平。
(3)体外实验：采用MTT及台盼兰染色细胞活性实验对比S(MeO)TLC及其他4种Eg5抑制剂(KPYC 10665、KPYC 10666、KPYC 10728和STLC)对膀胱癌细胞增殖的抑制能力,并进一步通过Heochst细胞凋亡实验和流式细胞仪检测药物诱导细胞凋亡的效果和机制。采用细胞免疫荧光技术和免疫印迹技术检测Caspase凋亡通路蛋白,探讨S(MeO)TLC诱导膀胱癌凋亡的分子通路机制。
(4)体内实验：采用KU7细胞株建立裸鼠膀胱癌模型,包括皮下肿瘤模型与全身多发转移癌模型,研究药物的体内抗癌疗效；通过经腹腔注射Eg5抑制剂,观察药物抑制膀胱癌皮下肿瘤生长情况,并通过活体体内成像系统(IVIS)检测S(MeO)TLC治疗晚期转移癌的疗效。
结果
(1)Eg5的表达与肿瘤床分级(P=0.002)和分期(P=0.021)显著相关。163例非肌层浸润膀胱癌患者术后平均随访32.52m(6～72m),Kaplan-Meier单因素变量分析表明Eg5强表达患者无复发生存期显著降低(P=0.012),而对肿瘤复发时临床进展时间的影响未达显著水平(P=0.070)。但是针对高风险的T1期膀胱癌患者,单因素变量分析表明Eg5强表达复发患者无肿瘤进展生存期呈显著降低(P=0.041)。随后的Cox风险模型多因素分析显示膀胱癌细胞分级(P=0.045)以及Eg5表达水平(P=0.029)是预测非肌层浸润癌患者术后复发时间的独立预后因素。
(2)蛋白免疫印迹检测各种膀胱癌细胞株均表达Eg5,且浸润性来源细胞株表达较强；正常膀胱粘膜不表达Eg5蛋白,高分级肿瘤表达高于低分级肿瘤。(P<0.05)
(3)体外实验表明S(MeO)TLC在5种被检测的Eg5抑制剂具有最强的细胞增殖抑制作用,导致细胞有丝分裂停滞形成单极纺锤体细胞,进而同时激活内源性和外源性Caspase依赖性凋亡途径诱导细胞发生凋亡。
(4)体内试验结果表明S(MeO)TLC能有效地抑制皮下肿瘤的生长(P<0.05),20mg/kg S(MeO)TLC治疗组肿瘤可出现明显坏死。S(MeO)TLC能有效抑制晚期膀胱癌转移灶的生长；与对照组小鼠相比,治疗组小鼠的生存时间显著延长(P<0.001)。结论
(1)膀胱癌Eg5强表达患者肿瘤分化较差且高表达T1期患者可能具有较早的进展风险,Eg5表达水平是预测非肌层浸润膀胱癌患者术后早期复发的独立预后因素。
(2) S(MeO)TLC作为新型的Eg5蛋白靶向抑制剂,能显著抑制体内膀胱肿瘤生长,尤其是对已经出现转移的晚期膀胱癌具有很好的治疗效果。创新及意义
(1)国内外首次研究了Eg5蛋白表达水平与膀胱癌生物学行为和非肌层浸润膀胱癌术后预后之间关系。为膀胱癌临床诊断以及预后判断提供依据。
(2)国内外首次证实了Eg5蛋白可以作为膀胱癌治疗的有效靶点；并首次对S(MeO)TLC治疗恶性肿瘤的实验疗效进行了研究；首次研究了Eg5抑制剂治疗膀胱癌的抗癌疗效,尤其是晚期转移癌的治疗作用。可为膀胱癌晚期转移提供创新性的靶向化疗方案。
(3)国内外首次通过活体体内成像系统(IVIS)评估Eg5蛋白抑制剂治疗晚期转移癌疗效,有助于晚期转移癌治疗相关实验研究方法的开展。
Background
Bladder urothelial carcinoma (UC) is the fifth most common malignancy worldwide, and the large majority (75%) present as non-muscle invasive tumorsl, which mainly confined to the mucosa (Ta) or subepithelial layer (T1). Despite received standard transurethral resection (TUR) and adjuvant intravesical chemotherapeutic instillations, about two-thirds of patients recur and 15% to 30% will progress to a higher stage and/or grade with greater mortality. Besides the clinical parameter (e.g. grade, stage), a number of markers were expected to predict the prognosis of non-muscle invasive UC, but few of them has been recognized as reliable method till now. Thus, there is a considerable need for investigating effective markers that can accurately identify non-muscle invasive cases with a high risk of recurrence or progression, which could contribute to not only the improvement of the prognosis by treating such individual patients more aggressively but also development of novel anticancer target.
Invasive bladder cancer, which accounts for 25% of all bladder cancers, has a poor prognosis with low 5-year survival levels of 10～50%. Traditionally, the combination of methotrexate, vinblastine, doxorubicin and cisplatin (MVAC) has been adopted for such advanced cases with systemic spreading of disease. However, these chemotherapy modalities only deliver a 46～60% response rate. Although new combination of gemcitabine and cisplatin has been identified as first line chemotherapy for less toxicity, no better response rate was found. To overcome the limited efficacy of these conventional chemotherapies for advanced bladder cancer, novel therapeutic approaches are required.
The mitotic kinesin Eg5, a member of the kinesin-5 family which plays a crucial role in the formation and maintenance of the bipolar spindle during mitosis, has been identified as an attractive target for cancer therapies. Since the discovery of the first Eg5 inhibitor, monastrol, many Eg5 inhibitors have been demonstrated to have anticancer efficacy.
In consistent with the fact that Eg5 is only expressed in mitotic cells, especially in malignant cells, Eg5 expression levels appeared to be directly proportional to the mitotic population in both cancer cell lines and clinical tumors. Furthermore, Eg5 has been shown to correlate with oncogenesis, proliferative rate and clinical outcomes in cancers. However, to our knowledge, there was no data on the correlation between Eg5 expression and the prognosis of UC.
Among the recognized Eg5 inhibitors, DeBonis et al. first identified (S)-trityl-L-cystein (STLC) as an effective Eg5 inhibitor. This inhibitor showed antiproliferative activity that was 36 times more potent than monastrol. Recently, it has been found by ourselves and other investigators that S(MeO)TLC, a (S)-trytyl-L-cystein derivative, is 10-fold more potent than STLC in cytotoxic activity. Furthermore, we also identified KPYC10665, KPYC10666 and KPYC10728, which are functionalized carbazoles prepared by one-pot N-arylation-oxidative biaryl coupling of anilines and phenyl triflates. Although there have been no reports concerning bladder tumors, the expression of Eg5 have been found to be induced in several kinds of tumors, including malignant melanoma, lung cancers and squamous cell carcinoma of the head and neck.
In the present study we investigated the potency and specificity of several Eg5 inhibitors of bladder cancer cell proliferation. Furthermore, the antitumor effect of the most prominent inhibitor was examined in xenograft models to assess whether it could have potential in a clinical setting. Besides, we analyzed the expression of Eg5 in clinical bladder UC specimens by immunohistochemistry, and evaluated the correlation between Eg5 expression and clinicopathological characteristics, aiming at identifying the evidence for its usefulness as a prognostic marker in patients with non-muscle invasive UCs.
Objective
We investigate the relationship between Eg5 expression and prognosis of patients with bladder cancer, and then examine the anticancer activity of a novel Eg5 inhibitor for bladder cancer, with particular reference to metastatic diseases.
Methods
Eg5 expression was examined by immunohistochemistry in bladder cancer specimens (Grade:Grade 1,32 cases; Grade 2,98 cases and Grade 3,63 cases. Stage: pTa 49 cases; pTl,114 cases; pT2-T4,30 cases), and the correlation between clinicopathological characteristics and Eg5 expression was evaluated. The prognostic significance of Eg5 immunoreactivity was analyzed via survival analysis in 163 non-muscle invasive bladder cases that were treated with transurethral resection and adjuvant intravesical instillations.
Bladder cancer cell lines were also examined for Eg5 expression, including RT112, KU7, RT4, EJ cell lines etc. The anti-proliferative activity of 5 Eg5 inhibitors was analyzed in cell lines using cell viability assay. The anticancer efficacy of the most potent Eg5 inhibitor was investigated in vitro using an apoptosis assay with Hoechst nuclear staining and flow cytometry. Immunofluorescence and immunostaining were used to elucidate the inhibitory mechanism. Furthermore, the inhibitory effect was evaluated in vivo using subcutaneous xenografts and metastatic cancer models.
Results
The expression of Eg5 was significantly associated with tumor grade (P= 0.002) and stage (P= 0.021).163 patients with non-muscle invasive bladder cancer were regularly followed up with the mean of 32.52 (from 6 to 72) months. Univariate analysis revealed Eg5 overexpression exhibited unfavorable influence on the intravesical recurrence with a significance (P= 0.012) while a marginal statistical significance on progression (P= 0.070), and the Eg5 overexpression had significant adverse impact on the progression free survival in T1 tumors (P= 0.041). Subsequent Cox hazard multivariate analysis revealed that both grade (P= 0.045) and Eg5 expression(P= 0.029) were identified as independent predictors for intravesical recurrence with adverse significance.
Eg5 expressed in bladder cancer cell lines. (S)-methoxy-trityl-L-cystein, S(MeO)TLC, exhibited the strongest antiproliferative activity of the 5 Eg5 inhibitors, and induced cell death after mitotic arrest via the Caspase-dependent apoptotic pathway. In vivo, S(MeO)TLC effectively suppressed tumor growth in both subcutaneous and metastatic xenograft models. Moreover, the survival times of S(MeO)TLC-treated nude mice with metastases were significantly longer than those of untreated mice (P< 0.001).
Conclusion
Eg5 overexpression correlates with poor differentiation and progression of bladder cancer, and has an independent validity in predicting the intravesical recurrence in patients with non-muscle invasive cases. We conclude that S(MeO)TLC is a promising novel anticancer agent for the treatment of bladder cancer. Importantly, our data indicates the potential of S(MeO)TLC as an effective therapy for metastatic bladder cancers. Additionally, this study was the first to use IVIS in investigating efficacy of novel chemotherapy in vivo metastatic models.
膀胱癌主要指膀胱移行上皮细胞癌,又称膀胱尿路上皮癌。其中约75％是局限于膀胱粘膜层(Ta)和粘膜下层(T1)的非肌层浸润癌；尽管可以通过经尿道膀胱镜切除并配合术后腔内化疗灌注治疗,约2／3的患者会出现复发,并且复发患者中15％～30％会进展为更高的病理分期和／或伴有分级升高。除了肿瘤分期和细胞分级,虽然以往的研究已经报道了大量的肿瘤分子标记物可用以预测膀胱癌的预后,但是目前尚未发现一种可靠的预测指标。
肌层浸润性膀胱癌占初发膀胱癌的25％,预后较差,5年生存率仅为10％～50％。对伴有转移的晚期浸润性膀胱癌的传统化疗方法是甲氨蝶呤、长春碱、多柔比星和顺铂联合用药的MVAC方案。该联合化疗方案虽然可以取得46％～60％治疗反应率,但也伴有严重的毒性反应,而且患者经过数疗程治疗后可能出现获得性耐药。近年来,尽管毒性较低的吉西他滨和顺铂新型GC联合方案开始逐渐成为一线的晚期膀胱癌化疗措施,然而该方案治疗反应率并不比传统的MVAC高。因此,寻找新的治疗晚期膀胱癌的化疗方法已经成为近期研究的热点,以求在减低化疗毒性的基础上提高疗效,为患者延长生存时间并争取更好的生活质量。
纺锤体驱动蛋白Eg5在细胞有丝分裂过程中发挥关键作用,近年来成为了靶向抗癌治疗的研究热点。Eg5在增殖组织细胞中表达,其主要功能与双极纺锤体的形成和分离有关,在细胞有丝分裂早期中起到了关键作用。
研究已经证实Eg5蛋白在恶性组织中存在高表达,但是在有丝分裂后分化稳定的成熟组织细胞中不表达,如中枢神经系统细胞。研究结果证实Eg5作为新的特异性抗有丝分裂药物的靶点进行抗癌治疗时,不会对神经细胞造成直接损害而出现类似于抗微管药物的神经病变副作用。研究也表明Eg5与肿瘤的发生和发展密切相关,而且最近的临床研究还提示Eg5在恶性肿瘤组织中的表达水平与化疗疗效以及患者的预后密切相关。
1999年研究发现了第一个Eg5蛋白小分子抑制剂monastrol, monastrol能特异地与驱动蛋白Eg5结合并抑制Eg5功能,从而使细胞有丝分裂停滞,具有强烈抑制哺乳动物细胞分裂增殖的作用。Monastrol不抑制其他的驱动蛋白活性功能,从而具有较少的副作用和广泛的抗肿瘤作用。以上的特点均促使近年来大量研究集中在研制Eg5抑制剂作为新的抗有丝分裂化疗药物应用上,并取得了很多令人兴奋的结果,临床试验也已经用来检验Eg5抑制剂的抗癌效果。
2004年Dimitrios A等首先证实了STLC是作用于Eg5靶点的强有力的小分子抑制剂,与monastrol具有相同的Eg5结合位点,且结合牢固、特异性高,对细胞生长的抑制能力提高了36倍。日本京都大学对STLC的衍生物进行研究后首先报道了S(MeO)TLC是一种强有力的小分子细胞渗透性Eg5抑制剂,抑制细胞生长的能力比STLC提高了10倍,而且S(MeO)TLC与STLC一样具有特异性强的特点。此外,京都大学药学部最近又研制了多种联芳类Eg5抑制剂。
如上所述,Eg5与恶性肿瘤的发生、发展有关,已经有多种Eg5小分子抑制剂成为靶向抗癌药物的研究热点,但是目前还没有Eg5蛋白表达与膀胱癌生物学行为以及临床预后的相关性研究,也没有Eg5蛋白抑制剂治疗膀胱癌的系统研究。所以,在本次研究中,研究者通过山东海外留学联合培养项目,在山东大学附属省立医院和日本京都大学医学部对Eg5蛋白表达与膀胱癌关系以及5种Eg5抑制剂(STLC、S(MeO)TLC、KPYC10665、KPYC1066和KPYC 10728)治疗膀胱癌的疗效进行了研究。从而为膀胱肿瘤的临床诊断、预后判断以及化学治疗提供有效地新型方法。
目的
本研究首先探讨Eg5蛋白表达水平与与膀胱癌生物学行为以及非肌层浸润癌患者术后预后间关系的探讨；并进一步研究新型的Eg5蛋白抑制剂对膀胱癌的治疗效果,尤其是针对晚期转移癌的疗效。
方法
(1)采用免疫组织化学染色的方法检测了193例膀胱癌临床标本中的Eg5表达(分级：1级32例,2级98例,3级63例：分期：Ta期49例,T1期114例,T2～T4期30例),分析Eg5表达水平与肿瘤生物学行为指标之间的关系,并通过生存分析研究了Eg5表达水平与163例非肌层浸润膀胱癌患者经膀胱镜电切治疗后的预后问关系。
(2)培养膀胱癌细胞并通过免疫印迹方法检测各种细胞株中Eg5蛋白表达水平,包括RT112、KU7、RT4、EJ细胞等；同时通过免疫印迹方法检测正常膀胱粘膜、高分级和低分级膀胱癌组织标本中Eg5表达水平。
(3)体外实验：采用MTT及台盼兰染色细胞活性实验对比S(MeO)TLC及其他4种Eg5抑制剂(KPYC 10665、KPYC 10666、KPYC 10728和STLC)对膀胱癌细胞增殖的抑制能力,并进一步通过Heochst细胞凋亡实验和流式细胞仪检测药物诱导细胞凋亡的效果和机制。采用细胞免疫荧光技术和免疫印迹技术检测Caspase凋亡通路蛋白,探讨S(MeO)TLC诱导膀胱癌凋亡的分子通路机制。
(4)体内实验：采用KU7细胞株建立裸鼠膀胱癌模型,包括皮下肿瘤模型与全身多发转移癌模型,研究药物的体内抗癌疗效；通过经腹腔注射Eg5抑制剂,观察药物抑制膀胱癌皮下肿瘤生长情况,并通过活体体内成像系统(IVIS)检测S(MeO)TLC治疗晚期转移癌的疗效。
结果
(1)Eg5的表达与肿瘤床分级(P=0.002)和分期(P=0.021)显著相关。163例非肌层浸润膀胱癌患者术后平均随访32.52m(6～72m),Kaplan-Meier单因素变量分析表明Eg5强表达患者无复发生存期显著降低(P=0.012),而对肿瘤复发时临床进展时间的影响未达显著水平(P=0.070)。但是针对高风险的T1期膀胱癌患者,单因素变量分析表明Eg5强表达复发患者无肿瘤进展生存期呈显著降低(P=0.041)。随后的Cox风险模型多因素分析显示膀胱癌细胞分级(P=0.045)以及Eg5表达水平(P=0.029)是预测非肌层浸润癌患者术后复发时间的独立预后因素。
(2)蛋白免疫印迹检测各种膀胱癌细胞株均表达Eg5,且浸润性来源细胞株表达较强；正常膀胱粘膜不表达Eg5蛋白,高分级肿瘤表达高于低分级肿瘤。(P<0.05)
(3)体外实验表明S(MeO)TLC在5种被检测的Eg5抑制剂具有最强的细胞增殖抑制作用,导致细胞有丝分裂停滞形成单极纺锤体细胞,进而同时激活内源性和外源性Caspase依赖性凋亡途径诱导细胞发生凋亡。
(4)体内试验结果表明S(MeO)TLC能有效地抑制皮下肿瘤的生长(P<0.05),20mg/kg S(MeO)TLC治疗组肿瘤可出现明显坏死。S(MeO)TLC能有效抑制晚期膀胱癌转移灶的生长；与对照组小鼠相比,治疗组小鼠的生存时间显著延长(P<0.001)。结论
(1)膀胱癌Eg5强表达患者肿瘤分化较差且高表达T1期患者可能具有较早的进展风险,Eg5表达水平是预测非肌层浸润膀胱癌患者术后早期复发的独立预后因素。
(2) S(MeO)TLC作为新型的Eg5蛋白靶向抑制剂,能显著抑制体内膀胱肿瘤生长,尤其是对已经出现转移的晚期膀胱癌具有很好的治疗效果。创新及意义
(1)国内外首次研究了Eg5蛋白表达水平与膀胱癌生物学行为和非肌层浸润膀胱癌术后预后之间关系。为膀胱癌临床诊断以及预后判断提供依据。
(2)国内外首次证实了Eg5蛋白可以作为膀胱癌治疗的有效靶点；并首次对S(MeO)TLC治疗恶性肿瘤的实验疗效进行了研究；首次研究了Eg5抑制剂治疗膀胱癌的抗癌疗效,尤其是晚期转移癌的治疗作用。可为膀胱癌晚期转移提供创新性的靶向化疗方案。
(3)国内外首次通过活体体内成像系统(IVIS)评估Eg5蛋白抑制剂治疗晚期转移癌疗效,有助于晚期转移癌治疗相关实验研究方法的开展。
Background
Bladder urothelial carcinoma (UC) is the fifth most common malignancy worldwide, and the large majority (75%) present as non-muscle invasive tumorsl, which mainly confined to the mucosa (Ta) or subepithelial layer (T1). Despite received standard transurethral resection (TUR) and adjuvant intravesical chemotherapeutic instillations, about two-thirds of patients recur and 15% to 30% will progress to a higher stage and/or grade with greater mortality. Besides the clinical parameter (e.g. grade, stage), a number of markers were expected to predict the prognosis of non-muscle invasive UC, but few of them has been recognized as reliable method till now. Thus, there is a considerable need for investigating effective markers that can accurately identify non-muscle invasive cases with a high risk of recurrence or progression, which could contribute to not only the improvement of the prognosis by treating such individual patients more aggressively but also development of novel anticancer target.
Invasive bladder cancer, which accounts for 25% of all bladder cancers, has a poor prognosis with low 5-year survival levels of 10～50%. Traditionally, the combination of methotrexate, vinblastine, doxorubicin and cisplatin (MVAC) has been adopted for such advanced cases with systemic spreading of disease. However, these chemotherapy modalities only deliver a 46～60% response rate. Although new combination of gemcitabine and cisplatin has been identified as first line chemotherapy for less toxicity, no better response rate was found. To overcome the limited efficacy of these conventional chemotherapies for advanced bladder cancer, novel therapeutic approaches are required.
The mitotic kinesin Eg5, a member of the kinesin-5 family which plays a crucial role in the formation and maintenance of the bipolar spindle during mitosis, has been identified as an attractive target for cancer therapies. Since the discovery of the first Eg5 inhibitor, monastrol, many Eg5 inhibitors have been demonstrated to have anticancer efficacy.
In consistent with the fact that Eg5 is only expressed in mitotic cells, especially in malignant cells, Eg5 expression levels appeared to be directly proportional to the mitotic population in both cancer cell lines and clinical tumors. Furthermore, Eg5 has been shown to correlate with oncogenesis, proliferative rate and clinical outcomes in cancers. However, to our knowledge, there was no data on the correlation between Eg5 expression and the prognosis of UC.
Among the recognized Eg5 inhibitors, DeBonis et al. first identified (S)-trityl-L-cystein (STLC) as an effective Eg5 inhibitor. This inhibitor showed antiproliferative activity that was 36 times more potent than monastrol. Recently, it has been found by ourselves and other investigators that S(MeO)TLC, a (S)-trytyl-L-cystein derivative, is 10-fold more potent than STLC in cytotoxic activity. Furthermore, we also identified KPYC10665, KPYC10666 and KPYC10728, which are functionalized carbazoles prepared by one-pot N-arylation-oxidative biaryl coupling of anilines and phenyl triflates. Although there have been no reports concerning bladder tumors, the expression of Eg5 have been found to be induced in several kinds of tumors, including malignant melanoma, lung cancers and squamous cell carcinoma of the head and neck.
In the present study we investigated the potency and specificity of several Eg5 inhibitors of bladder cancer cell proliferation. Furthermore, the antitumor effect of the most prominent inhibitor was examined in xenograft models to assess whether it could have potential in a clinical setting. Besides, we analyzed the expression of Eg5 in clinical bladder UC specimens by immunohistochemistry, and evaluated the correlation between Eg5 expression and clinicopathological characteristics, aiming at identifying the evidence for its usefulness as a prognostic marker in patients with non-muscle invasive UCs.
Objective
We investigate the relationship between Eg5 expression and prognosis of patients with bladder cancer, and then examine the anticancer activity of a novel Eg5 inhibitor for bladder cancer, with particular reference to metastatic diseases.
Methods
Eg5 expression was examined by immunohistochemistry in bladder cancer specimens (Grade:Grade 1,32 cases; Grade 2,98 cases and Grade 3,63 cases. Stage: pTa 49 cases; pTl,114 cases; pT2-T4,30 cases), and the correlation between clinicopathological characteristics and Eg5 expression was evaluated. The prognostic significance of Eg5 immunoreactivity was analyzed via survival analysis in 163 non-muscle invasive bladder cases that were treated with transurethral resection and adjuvant intravesical instillations.
Bladder cancer cell lines were also examined for Eg5 expression, including RT112, KU7, RT4, EJ cell lines etc. The anti-proliferative activity of 5 Eg5 inhibitors was analyzed in cell lines using cell viability assay. The anticancer efficacy of the most potent Eg5 inhibitor was investigated in vitro using an apoptosis assay with Hoechst nuclear staining and flow cytometry. Immunofluorescence and immunostaining were used to elucidate the inhibitory mechanism. Furthermore, the inhibitory effect was evaluated in vivo using subcutaneous xenografts and metastatic cancer models.
Results
The expression of Eg5 was significantly associated with tumor grade (P= 0.002) and stage (P= 0.021).163 patients with non-muscle invasive bladder cancer were regularly followed up with the mean of 32.52 (from 6 to 72) months. Univariate analysis revealed Eg5 overexpression exhibited unfavorable influence on the intravesical recurrence with a significance (P= 0.012) while a marginal statistical significance on progression (P= 0.070), and the Eg5 overexpression had significant adverse impact on the progression free survival in T1 tumors (P= 0.041). Subsequent Cox hazard multivariate analysis revealed that both grade (P= 0.045) and Eg5 expression(P= 0.029) were identified as independent predictors for intravesical recurrence with adverse significance.
Eg5 expressed in bladder cancer cell lines. (S)-methoxy-trityl-L-cystein, S(MeO)TLC, exhibited the strongest antiproliferative activity of the 5 Eg5 inhibitors, and induced cell death after mitotic arrest via the Caspase-dependent apoptotic pathway. In vivo, S(MeO)TLC effectively suppressed tumor growth in both subcutaneous and metastatic xenograft models. Moreover, the survival times of S(MeO)TLC-treated nude mice with metastases were significantly longer than those of untreated mice (P< 0.001).
Conclusion
Eg5 overexpression correlates with poor differentiation and progression of bladder cancer, and has an independent validity in predicting the intravesical recurrence in patients with non-muscle invasive cases. We conclude that S(MeO)TLC is a promising novel anticancer agent for the treatment of bladder cancer. Importantly, our data indicates the potential of S(MeO)TLC as an effective therapy for metastatic bladder cancers. Additionally, this study was the first to use IVIS in investigating efficacy of novel chemotherapy in vivo metastatic models.
引文
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21. Inoue T, Yoshida T, Shimizu Y, et al:Requirement of androgen-dependent activation of protein kinase C zeta for androgen-dependent cell proliferation in LNCaP Cells and its roles in transition to androgen-independent cells. Mol Endocrinol 2006; 20:3053.
22. Hayashi N, Koller E, Fazli L, et al:Effects of Eg5 knockdown on human prostate cancer xenograft growth and chemosensitivity. Prostate 2008; 68: 1283.
23. Tao W, South VJ, Zhang Y, et al:Induction of apoptosis by an inhibitor of the mitotic kinesin KSP requires both activation of the spindle assembly checkpoint and mitotic slippage. Cancer Cell 2005; 8:49.
1. Jemal A, Murray T, Ward E,et al. Cancer statistics. CA. Cancer J. Clin. 2005;55:10-30.
2. Hassen W, Droller MJ. Current concepts in assessment and treatment of bladder cancer. Curr. Opin. Urol.2000; 10:291-9.
3. Theodoropoulos VE, Lazaris AC, Kastriotis I, et al. Evaluation of hypoxia-inducible factor lalpha overexpression as a predictor of tumour recurrence and progression in superficial urothelial bladder carcinoma. BJU. Int. 2005;95:425-31.
4. Stavropoulos NE, Filiadis I, Ioachim E, et al. Prognostic significance of p53, bcl-2 and Ki-67 in high risk superficial bladder cancer. Anticancer Res. 2002;22:3759-64.
5. Kapitein LC, Peterman EJ, Kwok BH, Kim JH, Kapoor TM,Schmidt CF. The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks. Nature 2005;435:114-8.
6. Tao W, South VJ, Diehl RE, et al. An inhibitor of the kinesin spindle protein activates the intrinsic apoptotic pathway independently of p53 and de novo protein synthesis. Mol. Cell Biol.2007;27:689-98.
7. Sakowicz R, Finer JT, Beraud C, et al. Antitumor activity of a kinesin inhibitor. Cancer Res.2004;64:3276-80.
8. Hayashi N, Koller E, Fazli L,Gleave ME. Effects of Eg5 knockdown on human prostate cancer xenograft growth and chemosensitivity. Prostate 2008;68:1283-95.
9. Marcus Al, Peters U, Thomas SL, Garrett S, Zelnak A, Kapoor TM,Giannakakou P. Mitotic kinesin inhibitors induce mitotic arrest and cell death in Taxol-resistant and-sensitive cancer cells. J. Biol. Chem. 2005;280:11569-77.
10. Hansen GM, Justice MJ. Activation of Hex and mEg5 by retroviral insertion may contribute to mouse B-cell leukemia. Oncogene 1999; 18:6531-9.
11. Saijo T, Ishii G, Ochiai A, et al. Eg5 expression is closely correlated with the response of advanced non-small cell lung cancer to antimitotic agents combined with platinum chemotherapy. Lung Cancer 2006;54:217-25.
12. Vaughn DJ. Moving forward in advanced bladder cancer. J. Clin. Oncol. 2007;25:2162-3.
13. Park J, Song C, Hong JH, Park BH, Cho YM, Kim CS, Ahn H. Prognostic significance of non-papillary tumor morphology as a predictor of cancer progression and survival in patients with primary T1G3 bladder cancer. World J. Urol.2009;27:277-83.
14. Kausch I, Bohle A. Molecular aspects of bladder cancer III. Prognostic markers of bladder cancer. Eur. Urol.2002;41:15-29.
15. Birkhahn M, Mitra AP, Williams AJ, et a. Predicting Recurrence and Progression of Noninvasive Papillary Bladder Cancer at Initial Presentation Based on Quantitative Gene Expression Profiles. Eur. Urol. Published online:9 Sep 2009; doi:10.1016/j.eururo.2009.09.013
16. Nakanishi R, Oka N, Nakatsuji H, et al. Effect of vascular endothelial growth factor and its receptor inhibitor on proliferation and invasion in bladder cancer. Urol. Int.2009;83:98-106.
17. Nakai R, Iida S, Takahashi T, Tsujita T, Okamoto S, Takada C, et al. K858, a novel inhibitor of mitotic kinesin Eg5 and antitumor agent, induces cell death in cancer cells. Cancer Res.2009;69:3901-9.
18. Tang PA, Siu LL, Chen EX, et al. Phase II study of ispinesib in recurrent or metastatic squamous cell carcinoma of the head and neck. Invest. New Drugs 2008;26:257-64.
19. Nadaoka J, Horikawa Y, Saito M, et al. Prognostic significance of HIF-1 alpha polymorphisms in transitional cell carcinoma of the bladder. Int. J. Cancer 2008; 122:1297-302.
20. Noguchi M, Kikuchi H, Ishibashi M,Noda S. Percentage of the positive area of bone metastasis is an independent predictor of disease death in advanced prostate cancer. Br. J. Cancer.2003;88:195-201.
21. Katayama A, Bandoh N, Kishibe K, et al. Expressions of matrix metalloproteinases in early-stage oral squamous cell carcinoma as predictive indicators for tumor metastases and prognosis. Clin. Cancer Res. 2004; 10:634-40.
22. Turashvili G, Leung S, Turbin D, et al. Inter-observer reproducibility of HER2 immunohistochemical assessment and concordance with fluorescent in situ hybridization (FISH):pathologist assessment compared to quantitative image analysis. BMC. Cancer 2009;9:165.
23. Zieger K, Wiuf C, Jensen KM, Orntoft TF, Dyrskjot L. Chromosomal imbalance in the progression of high-risk non-muscle invasive bladder cancer. BMC. Cancer 2009;9:149.
24. Rodriguez-Alonso A, Pita-Fernandez S, Gonzalez-Carrero J,Nogueira-March JL. Multivariate analysis of survival, recurrence, progression and development of mestastasis in T1 and T2a transitional cell bladder carcinoma. Cancer 2002;94:1677-84.
25. Ali-El-Dein B, Sarhan O, Hinev A, Ibrahiem el-HI, Nabeeh A, Ghoneim MA. Superficial bladder tumours:analysis of prognostic factors and construction of a predictive index. BJU. Int.2003;92:393-9.
26. Sylvester RJ, van der Meijden AP, Oosterlinck W, et al. Predicting recurrence and progression in individual patients with stage Ta T1 bladder cancer using EORTC risk tables:a combined analysis of 2596 patients from seven EORTC trials. Eur. Urol.2006;49:466-77.
2 Hassen W, Droller MJ. Current concepts in assessment and treatment of bladder cancer. Curr Opin Urol,2000,10(4):291-9.
3 Theodoropoulos VE, Lazaris AC, Kastriotis I, et al. Evaluation of hypoxia-inducible factor lalpha overexpression as a predictor of tumour recurrence and progression in superficial urothelial bladder carcinoma. BJU Int, 2005,95(3):425-31.
4 Stavropoulos NE, Filiadis I, Ioachim E, et al. Prognostic significance of p53, bcl-2 and Ki-67 in high risk superficial bladder cancer. Anticancer Res, 2002,22(6B):3759-64.
5 Miki H, Okada Y,Hirokawa N. Analysis of the kinesin superfamily:insights into structure and function. Trends Cell Biol,2005,15(9):467-76.
6 Turner J, Anderson R, Guo J, et al. Crystal structure of the mitotic spindle kinesin Eg5 reveals a novel conformation of the neck-linker. J Biol Chem, 2001,276(27):25496-502.
7 Kapitein LC, Peterman EJ, Kwok BH, et al. The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks. Nature,2005,435(7038):114-8.
8王伦善,王保龙,任维华.驱动蛋白Eg5及其靶向治疗药物的抗肿瘤机制.生命的化学,2008,28(2)：190-193.
9 Sakowicz R, Finer JT, Beraud C, et al. Antitumor activity of a kinesin inhibitor. Cancer Res,2004,64(9):3276-80.
10 Hansen GM, Justice MJ. Activation of Hex and mEg5 by retroviral insertion may contribute to mouse B-cell leukemia. Oncogene,1999,18(47):6531-9.
11 Castillo A, Morse HC 3rd, Godfrey VL, et al. Overexpression of Eg5 causes genomic instability and tumor formation in mice. Cancer Res, 2007,67(21):10138-47.
12 Marcus Al, Peters U, Thomas SL, et al. Mitotic kinesin inhibitors induce mitotic arrest and cell death in Taxol-resistant and-sensitive cancer cells. J Biol Chem,2005,280(12):11569-77.
13 Hayashi N, Koller E, Fazli L, et al. Effects of Eg5 knockdown on human prostate cancer xenograft growth and chemosensitivity. Prostate, 2008,68(12):1283-95.
14 Saijo T, Ishii G, Ochiai A, et al. Eg5 expression is closely correlated with the response of advanced non-small cell lung cancer to antimitotic agents combined with platinum chemotherapy. Lung Cancer,2006,54(2):217-25.
15那彦群.2007年中国泌尿外科疾病诊疗指南.第l版.北京：人民卫生出版社,2007.87-129.
16 Sylvester RJ, van der Meijden AP, Oosterlinck W, et al. Predicting recurrence and progression in individual patients with stage Ta T1 bladder cancer using EORTC risk tables:a combined analysis of 2596 patients from seven EORTC trials. Eur Urol,2006,49(3):466-5; discussion 475-7.
17 Grossman HB, Liebert M, Antelo M, et al. p53 and RB expression predict progression in T1 bladder cancer. Clin Cancer Res,1998,4(4):829-34.
18 Nakanishi R, Oka N, Nakatsuji H, et al. Effect of vascular endothelial growth factor and its receptor inhibitor on proliferation and invasion in bladder cancer. Urol Int,2009,83(1):98-106.
19 Tang PA, Siu LL, Chen EX, et al. Phase II study of ispinesib in recurrent or metastatic squamous cell carcinoma of the head and neck. Invest New Drugs, 2008,26(3):257-64.
20 Nadaoka J, Horikawa Y, Saito M, et al. Prognostic significance of HIF-1 alpha polymorphisms in transitional cell carcinoma of the bladder. Int J Cancer, 2008,122(6):1297-302.
21 Turashvili G, Leung S, Turbin D, et al. Inter-observer reproducibility of HER2 immunohistochemical assessment and concordance with fluorescent in situ hybridization (FISH):pathologist assessment compared to quantitative image analysis. BMC Cancer,2009,9:165.
22 Katayama A, Bandoh N, Kishibe K, et al. Expressions of matrix metalloproteinases in early-stage oral squamous cell carcinoma as predictive indicators for tumor metastases and prognosis. Clin Cancer Res, 2004,10(2):634-40.
23叶应妩,王毓三.全国临床检验操作规程,第2版.南京：东南大学出版社,1997.163-166.
24 Zieger K, Wiuf C, Jensen KM, et al. Chromosomal imbalance in the progression of high-risk non-muscle invasive bladder cancer. BMC Cancer, 2009,9:149.
25 Rodriguez-Alonso A, Pita-Fernandez S, Gonzalez-Carrero J, et al. Multivariate analysis of survival, recurrence, progression and development of mestastasis in T1 and T2a transitional cell bladder carcinoma. Cancer,2002,94(6):1677-84.
26 Ali-El-Dein B, Sarhan O, Hinev A, et al. Superficial bladder tumours:analysis of prognostic factors and construction of a predictive index. BJU Int, 2003,92(4):393-9.
27 Park J, Song C, Hong JH, et al. Prognostic significance of non-papillary tumor morphology as a predictor of cancer progression and survival in patients with primary T1G3 bladder cancer. World J Urol,2009,27(2):277-83.
1 Jemal A, Siegel R, Ward E, et al. Cancer statistics,2007. CA Cancer J Clin, 2007,57(1):43-66.
2 von der Maase H, Hansen SW, Roberts JT, et al. Gemcitabine and cisplatin versus methotrexate, vinblastine, doxorubicin, and cisplatin in advanced or metastatic bladder cancer:results of a large, randomized, multinational, multicenter, phase III study. J Clin Oncol,2000,18(17):3068-77.
3 Mayer TU, Kapoor TM, Haggarty SJ, et al. Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science, 1999,286(5441):971-4.
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5 Maliga Z, Kapoor TM,Mitchison TJ. Evidence that monastrol is an allosteric inhibitor of the mitotic kinesin Eg5. Chem Biol,2002,9(9):989-96.
6 Sakowicz R, Finer JT, Beraud C, et al. Antitumor activity of a kinesin inhibitor. Cancer Res,2004,64(9):3276-80.
7 Duhl DM, Renhowe PA. Inhibitors of kinesin motor proteins--research and clinical progress. Curr Opin Drug Discov Devel,2005,8(4):431-6.
8 DeBonis S, Skoufias DA, Lebeau L, et al. In vitro screening for inhibitors of the human mitotic kinesin Eg5 with antimitotic and antitumor activities. Mol Cancer Ther,2004,3(9):1079-90.
9 Skoufias DA, DeBonis S, Saoudi Y, et al. S-trityl-L-cysteine is a reversible, tight binding inhibitor of the human kinesin Eg5 that specifically blocks mitotic progression. J Biol Chem,2006,281(26):17559-69.
10 Carter BZ, Mak DH, Shi Y, et al. Regulation and targeting of Eg5, a mitotic motor protein in blast crisis CML:overcoming imatinib resistance. Cell Cycle, 2006,5(19):2223-9.
11 Ogo N, Oishi S, Matsuno K, et al. Synthesis and biological evaluation of L-cysteine derivatives as mitotic kinesin Eg5 inhibitors. Bioorg Med Chem Lett, 2007,17(14):3921-4.
12 Debonis S, Skoufias DA, Indorato RL, et al. Structure-activity relationship of S-trityl-L-cysteine analogues as inhibitors of the human mitotic kinesin Eg5. J Med Chem,2008,51 (5):1115-25.
13 Watanabe T, Oishi S, Fujii N, et al. Palladium-catalyzed direct synthesis of carbazoles via one-pot N-arylation and oxidative biaryl coupling:synthesis and mechanistic study. J Org Chem,2009,74(13):4720-6.
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15 Nakai R, Iida S, Takahashi T, et al. K858, a novel inhibitor of mitotic kinesin Eg5 and antitumor agent, induces cell death in cancer cells. Cancer Res, 2009,69(9):3901-9.
16 Okumura H, Nakazawa J, Tsuganezawa K, et al. Phenothiazine and carbazole-related compounds inhibit mitotic kinesin Eg5 and trigger apoptosis in transformed culture cells. Toxicol Lett,2006,166(1):44-52.
17 Kenji Matsuno, Jun-ichi Sawada, Akira Asai. Therapeutic potential of mitotic kinesin inhibitors in cancer. Expert Opin. Ther. Patents,2008,18(3):1-9.
18 Matsuno K, Sawada J, Sugimoto M, et al. Bis(hetero)aryl derivatives as unique kinesin spindle protein inhibitors. Bioorg Med Chem Lett,2009,19(4):1058-61.
19 Tao W, South VJ, Diehl RE, et al. An inhibitor of the kinesin spindle protein activates the intrinsic apoptotic pathway independently of p53 and de novo protein synthesis. Mol Cell Biol,2007,27(2):689-98.
20尹芳,张端莲,熊彦娥,等.高糖诱导小鼠囊胚Caspase-3的表达及其意义.中国组织化学与细胞化学杂志,2005,14(5)：544-546.
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23 Tao W, South VJ, Zhang Y, et al. Induction of apoptosis by an inhibitor of the mitotic kinesin KSP requires both activation of the spindle assembly checkpoint and mitotic slippage. Cancer Cell,2005,8(1):49-59.
24 Marcus AI, Peters U, Thomas SL, et al. Mitotic kinesin inhibitors induce mitotic arrest and cell death in Taxol-resistant and-sensitive cancer cells. J Biol Chem,2005,280(12):11569-77.
25 Shi J, Orth JD,Mitchison T. Cell type variation in responses to antimitotic drugs that target microtubules and kinesin-5. Cancer Res,2008,68(9):3269-76.
26王伦善,王保龙,任维华.驱动蛋白Eg5及其靶向治疗药物的抗肿瘤机制.生命的化学,2008,28(2)：190-193.
27 Koller E, Propp S, Zhang H, et al. Use of a chemically modified antisense oligonucleotide library to identify and validate Eg5 (kinesin-like 1) as a target for antineoplastic drug development. Cancer Res,2006,66(4):2059-66.
28 Sudo T, Nitta M, Saya H, et al. Dependence of paclitaxel sensitivity on a functional spindle assembly checkpoint. Cancer Res,2004,64(7):2502-8.
29 Chin GM, Herbst R. Induction of apoptosis by monastrol, an inhibitor of the mitotic kinesin Eg5, is independent of the spindle checkpoint. Mol Cancer Ther, 2006,5(10):2580-91.
30 Bharadwaj R, Yu H. The spindle checkpoint, aneuploidy, and cancer. Oncogene, 2004,23(11):2016-27.
31 Tang PA, Siu LL, Chen EX, et al. Phase II study of ispinesib in recurrent or metastatic squamous cell carcinoma of the head and neck. Invest New Drugs, 2008,26(3):257-64.
32 Wetterwald A, van der Pluijm G, Que I, et al. Optical imaging of cancer metastasis to bone marrow:a mouse model of minimal residual disease. Am J Pathol,2002,160(3):1143-53.
33 Nogawa M, Yuasa T, Kimura S, et al. Monitoring luciferase-labeled cancer cell growth and metastasis in different in vivo models. Cancer Lett, 2005,217(2):243-53.
1. Jemal A, Siegel R, Ward E, et al:Cancer statistics,2007. CA Cancer J Clin JT-CA:a cancer journal for clinicians 2007; 57:43.
2. Juffs HG, Moore MJ, Tannock IF:The role of systemic chemotherapy in the management of muscle-invasive bladder cancer. Lancet Oncol 2002; 3:738.
3. von der Maase H, Hansen SW, Roberts JT, et al:Gemcitabine and cisplatin versus methotrexate, vinblastine, doxorubicin, and cisplatin in advanced or metastatic bladder cancer:results of a large, randomized, multinational, multicenter, phase III study. J Clin Oncol 2000; 18:3068.
4. Loehrer PJ Sr, Einhorn LH, Elson PJ, et al:A randomized comparison of cisplatin alone or in combination with methotrexate, vinblastine, and doxorubicin in patients with metastatic urothelial carcinoma:a cooperative group study. J Clin Oncol 1992; 10:1066.
5. Grossman HB, Natale RB, Tangen CM, et al:Neoadjuvant chemotherapy plus cystectomy compared with cystectomy alone for locally advanced bladder cancer. N Engl J Med 2003; 349:859.
6. Sharp DJ, Rogers GC,Scholey JM:Microtubule motors in mitosis. Nature 2000; 407:41.
7. Sakowicz R, Finer JT, Beraud C, et al:Antitumor activity of a kinesin inhibitor. Cancer Res 2004; 64:3276.
8. Kinoshita M, Watanabe N:Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Tanpakushitsu Kakusan Koso 2007; 52:1796.
9. Mayer TU, Kapoor TM, Haggarty SJ, et al:Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science 1999; 286: 971.
10. Ogo N, Oishi S, Matsuno K, et al:Synthesis and biological evaluation of L-cysteine derivatives as mitotic kinesin Eg5 inhibitors. Bioorg Med Chem Lett 2007; 17:3921.
11. Duhl DM, Renhowe PA:Inhibitors of kinesin motor proteins--research and clinical progress. Curr Opin Drug Discov Devel 2005; 8:431.
12. DeBonis S, Skoufias DA, Lebeau L, et al:In vitro screening for inhibitors of the human mitotic kinesin Eg5 with antimitotic and antitumor activities. Mol Cancer Ther 2004; 3:1079.
13. Debonis S, Skoufias DA, Indorato RL, et al:Structure-activity relationship of S-trityl-L-cysteine analogues as inhibitors of the human mitotic kinesin Eg5. J Med Chem 2008; 51:1115.
14. Watanabe T, Oishi S, Fujii N, et al:Palladium-catalyzed direct synthesis of carbazoles via one-pot N-arylation and oxidative biaryl coupling:synthesis and mechanistic study. J Org Chem 2009; 74:4720.
15. Lee CW, Belanger K, Rao SC, et al:A phase II study of ispinesib (SB-715992) in patients with metastatic or recurrent malignant melanoma:a National Cancer Institute of Canada Clinical Trials Group trial. Invest New Drugs 2008; 26: 249.
16. Saijo T, Ishii G, Ochiai A, et al:Eg5 expression is closely correlated with the response of advanced non-small cell lung cancer to antimitotic agents combined with platinum chemotherapy. Lung Cancer 2006; 54:217.
17. Tang PA, Siu LL, Chen EX, et al:Phase II study of ispinesib in recurrent or metastatic squamous cell carcinoma of the head and neck. Invest New Drugs 2008; 26:257.
18. Matsui Y, Watanabe J, Ding S, et al:Dicoumarol enhances doxorubicin-induced cytotoxicity in p53 wild-type urothelial cancer cells through p38 activation. BJU Int,2009; Jul 6.
19. Nogawa M, Yuasa T, Kimura S, et al:Monitoring luciferase-labeled cancer cell growth and metastasis in different in vivo models. Cancer Lett 2005,217:243.
20. Toda Y, Kono K, Abiru H, et al:Application of tyramide signal amplification system to immunohistochemistry:a potent method to localize antigens that are not detectable by ordinary method. Pathol Int 1999; 49:479.
21. Inoue T, Yoshida T, Shimizu Y, et al:Requirement of androgen-dependent activation of protein kinase C zeta for androgen-dependent cell proliferation in LNCaP Cells and its roles in transition to androgen-independent cells. Mol Endocrinol 2006; 20:3053.
22. Hayashi N, Koller E, Fazli L, et al:Effects of Eg5 knockdown on human prostate cancer xenograft growth and chemosensitivity. Prostate 2008; 68: 1283.
23. Tao W, South VJ, Zhang Y, et al:Induction of apoptosis by an inhibitor of the mitotic kinesin KSP requires both activation of the spindle assembly checkpoint and mitotic slippage. Cancer Cell 2005; 8:49.
1. Jemal A, Murray T, Ward E,et al. Cancer statistics. CA. Cancer J. Clin. 2005;55:10-30.
2. Hassen W, Droller MJ. Current concepts in assessment and treatment of bladder cancer. Curr. Opin. Urol.2000; 10:291-9.
3. Theodoropoulos VE, Lazaris AC, Kastriotis I, et al. Evaluation of hypoxia-inducible factor lalpha overexpression as a predictor of tumour recurrence and progression in superficial urothelial bladder carcinoma. BJU. Int. 2005;95:425-31.
4. Stavropoulos NE, Filiadis I, Ioachim E, et al. Prognostic significance of p53, bcl-2 and Ki-67 in high risk superficial bladder cancer. Anticancer Res. 2002;22:3759-64.
5. Kapitein LC, Peterman EJ, Kwok BH, Kim JH, Kapoor TM,Schmidt CF. The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks. Nature 2005;435:114-8.
6. Tao W, South VJ, Diehl RE, et al. An inhibitor of the kinesin spindle protein activates the intrinsic apoptotic pathway independently of p53 and de novo protein synthesis. Mol. Cell Biol.2007;27:689-98.
7. Sakowicz R, Finer JT, Beraud C, et al. Antitumor activity of a kinesin inhibitor. Cancer Res.2004;64:3276-80.
8. Hayashi N, Koller E, Fazli L,Gleave ME. Effects of Eg5 knockdown on human prostate cancer xenograft growth and chemosensitivity. Prostate 2008;68:1283-95.
9. Marcus Al, Peters U, Thomas SL, Garrett S, Zelnak A, Kapoor TM,Giannakakou P. Mitotic kinesin inhibitors induce mitotic arrest and cell death in Taxol-resistant and-sensitive cancer cells. J. Biol. Chem. 2005;280:11569-77.
10. Hansen GM, Justice MJ. Activation of Hex and mEg5 by retroviral insertion may contribute to mouse B-cell leukemia. Oncogene 1999; 18:6531-9.
11. Saijo T, Ishii G, Ochiai A, et al. Eg5 expression is closely correlated with the response of advanced non-small cell lung cancer to antimitotic agents combined with platinum chemotherapy. Lung Cancer 2006;54:217-25.
12. Vaughn DJ. Moving forward in advanced bladder cancer. J. Clin. Oncol. 2007;25:2162-3.
13. Park J, Song C, Hong JH, Park BH, Cho YM, Kim CS, Ahn H. Prognostic significance of non-papillary tumor morphology as a predictor of cancer progression and survival in patients with primary T1G3 bladder cancer. World J. Urol.2009;27:277-83.
14. Kausch I, Bohle A. Molecular aspects of bladder cancer III. Prognostic markers of bladder cancer. Eur. Urol.2002;41:15-29.
15. Birkhahn M, Mitra AP, Williams AJ, et a. Predicting Recurrence and Progression of Noninvasive Papillary Bladder Cancer at Initial Presentation Based on Quantitative Gene Expression Profiles. Eur. Urol. Published online:9 Sep 2009; doi:10.1016/j.eururo.2009.09.013
16. Nakanishi R, Oka N, Nakatsuji H, et al. Effect of vascular endothelial growth factor and its receptor inhibitor on proliferation and invasion in bladder cancer. Urol. Int.2009;83:98-106.
17. Nakai R, Iida S, Takahashi T, Tsujita T, Okamoto S, Takada C, et al. K858, a novel inhibitor of mitotic kinesin Eg5 and antitumor agent, induces cell death in cancer cells. Cancer Res.2009;69:3901-9.
18. Tang PA, Siu LL, Chen EX, et al. Phase II study of ispinesib in recurrent or metastatic squamous cell carcinoma of the head and neck. Invest. New Drugs 2008;26:257-64.
19. Nadaoka J, Horikawa Y, Saito M, et al. Prognostic significance of HIF-1 alpha polymorphisms in transitional cell carcinoma of the bladder. Int. J. Cancer 2008; 122:1297-302.
20. Noguchi M, Kikuchi H, Ishibashi M,Noda S. Percentage of the positive area of bone metastasis is an independent predictor of disease death in advanced prostate cancer. Br. J. Cancer.2003;88:195-201.
21. Katayama A, Bandoh N, Kishibe K, et al. Expressions of matrix metalloproteinases in early-stage oral squamous cell carcinoma as predictive indicators for tumor metastases and prognosis. Clin. Cancer Res. 2004; 10:634-40.
22. Turashvili G, Leung S, Turbin D, et al. Inter-observer reproducibility of HER2 immunohistochemical assessment and concordance with fluorescent in situ hybridization (FISH):pathologist assessment compared to quantitative image analysis. BMC. Cancer 2009;9:165.
23. Zieger K, Wiuf C, Jensen KM, Orntoft TF, Dyrskjot L. Chromosomal imbalance in the progression of high-risk non-muscle invasive bladder cancer. BMC. Cancer 2009;9:149.
24. Rodriguez-Alonso A, Pita-Fernandez S, Gonzalez-Carrero J,Nogueira-March JL. Multivariate analysis of survival, recurrence, progression and development of mestastasis in T1 and T2a transitional cell bladder carcinoma. Cancer 2002;94:1677-84.
25. Ali-El-Dein B, Sarhan O, Hinev A, Ibrahiem el-HI, Nabeeh A, Ghoneim MA. Superficial bladder tumours:analysis of prognostic factors and construction of a predictive index. BJU. Int.2003;92:393-9.
26. Sylvester RJ, van der Meijden AP, Oosterlinck W, et al. Predicting recurrence and progression in individual patients with stage Ta T1 bladder cancer using EORTC risk tables:a combined analysis of 2596 patients from seven EORTC trials. Eur. Urol.2006;49:466-77.