KRIBB3 (5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl) isoxazole) inhibited cancer cell growth in vitro and in vivo. Flow cytometry studies showed that KRIBB3 caused cell cycle arrest at the G(2)/M phase and subsequent apoptosis. This was confirmed as accumulation of Cyclin B1 and cleavage of poly(ADP-ribose) polymerase (PARP) were detected. While transient inhibition by KRIBB3 led to reversible mitotic arrest, prolonged exposure to KRIBB3-induced apoptosis. Co-immunoprecipitation experiments showed that KRIBB3 initially induced association of inhibitory Mad2 with p55CDC (mammalian homologue of CDC20), an activator of APC/C (anaphase-promoting complex/cyclosome), suggesting that the mitotic spindle checkpoint was activated by KRIBB3. However, the level of this inhibitory complex of Mad2 with p55CDC was gradually decreased 24 h after KRIBB3 treatment, and was hardly detectable after 48 h, indicating some slipping of the mitotic checkpoint. Consistent with these observations, KRIBB3 activated the mitotic spindle checkpoint by disrupting the microtubule cytoskeleton. KRIBB3 was proven to be a tubulin inhibitor using in vitro polymerization assays and in vivo indirect immunofluorescence staining. The temporal pattern of Bax activation by KRIBB3 was similar to PARP cleavage, suggesting that Bax is a mediator of KRIBB3-dependent apoptosis. Furthermore, when KRIBB3 was administered intraperitoneally into nude mice at 50 mg/kg or 100 mg/kg, it inhibited 49.5 or 70.3% of tumor growth, respectively. These results suggest that KRIBB3 is a good drug candidate for cancer therapy.

中文翻译：

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新型微管抑制剂KRIBB3诱导人癌细胞中的有丝分裂停滞和凋亡。

KRIBB3（5-（5-乙基-2-羟基-4-甲氧基苯基）-4-（4-甲氧基苯基）异恶唑）在体内外抑制癌细胞的生长。流式细胞仪研究表明KRIBB3导致细胞周期停滞在G（2）/ M期和随后的细胞凋亡。证实这是由于细胞周期蛋白B1的积累和聚（ADP-核糖）聚合酶（PARP）的裂解被检测到。虽然KRIBB3的瞬时抑制导致可逆的有丝分裂阻滞，但长时间暴露于KRIBB3诱导的细胞凋亡。免疫共沉淀实验表明，KRIBB3最初诱导抑制性Mad2与APC / C的激活剂p55CDC（CDC20的哺乳动物同源物）（后期促进复合物/环体）的缔合，表明KRIBB3激活了有丝分裂纺锤体检查点。然而，在KRIBB3处理后24小时，这种抑制Mad2与p55CDC的复合物的水平逐渐降低，并且在48小时后几乎检测不到，表明有丝分裂检查点有些滑落。与这些观察结果一致，KRIBB3通过破坏微管细胞骨架激活了有丝分裂纺锤体检查点。使用体外聚合测定和体内间接免疫荧光染色证明KRIBB3是微管蛋白抑制剂。KRIBB3激活Bax的时间模式类似于PARP裂解，表明Bax是KRIBB3依赖性细胞凋亡的介体。此外，当以50 mg / kg或100 mg / kg腹膜内给裸鼠腹膜内施用KRIBB3时，它分别抑制了49.5％或70.3％的肿瘤生长。这些结果表明，KRIBB3是用于癌症治疗的良好药物候选物。并且在48小时后几乎无法检测到，这表明有丝分裂检查点有所滑落。与这些观察结果一致，KRIBB3通过破坏微管细胞骨架激活了有丝分裂纺锤体检查点。使用体外聚合测定和体内间接免疫荧光染色证明KRIBB3是微管蛋白抑制剂。KRIBB3激活Bax的时间模式类似于PARP裂解，表明Bax是KRIBB3依赖性细胞凋亡的介体。此外，当以50 mg / kg或100 mg / kg腹膜内给裸鼠腹膜内施用KRIBB3时，它分别抑制了49.5％或70.3％的肿瘤生长。这些结果表明，KRIBB3是用于癌症治疗的良好药物候选物。并且在48小时后几乎无法检测到，这表明有丝分裂检查点有所滑落。与这些观察结果一致，KRIBB3通过破坏微管细胞骨架激活了有丝分裂纺锤体检查点。使用体外聚合测定和体内间接免疫荧光染色证明KRIBB3是微管蛋白抑制剂。KRIBB3激活Bax的时间模式类似于PARP裂解，表明Bax是KRIBB3依赖性细胞凋亡的介体。此外，当以50 mg / kg或100 mg / kg腹膜内给裸鼠腹膜内施用KRIBB3时，它分别抑制了49.5％或70.3％的肿瘤生长。这些结果表明，KRIBB3是用于癌症治疗的良好药物候选物。KRIBB3通过破坏微管细胞骨架激活有丝分裂纺锤体检查点。使用体外聚合测定和体内间接免疫荧光染色证明KRIBB3是微管蛋白抑制剂。KRIBB3激活Bax的时间模式类似于PARP裂解，表明Bax是KRIBB3依赖性细胞凋亡的介体。此外，当将KRIBB3以50 mg / kg或100 mg / kg腹膜内给予裸鼠时，分别抑制了49.5％或70.3％的肿瘤生长。这些结果表明，KRIBB3是用于癌症治疗的良好药物候选物。KRIBB3通过破坏微管细胞骨架激活有丝分裂纺锤体检查点。使用体外聚合测定和体内间接免疫荧光染色证明KRIBB3是微管蛋白抑制剂。KRIBB3激活Bax的时间模式类似于PARP裂解，表明Bax是KRIBB3依赖性细胞凋亡的介体。此外，当以50 mg / kg或100 mg / kg腹膜内给裸鼠腹膜内施用KRIBB3时，它分别抑制了49.5％或70.3％的肿瘤生长。这些结果表明，KRIBB3是用于癌症治疗的良好药物候选物。KRIBB3激活Bax的时间模式类似于PARP裂解，表明Bax是KRIBB3依赖性细胞凋亡的介体。此外，当以50 mg / kg或100 mg / kg腹膜内给裸鼠腹膜内施用KRIBB3时，它分别抑制了49.5％或70.3％的肿瘤生长。这些结果表明，KRIBB3是用于癌症治疗的良好药物候选物。KRIBB3激活Bax的时间模式类似于PARP裂解，表明Bax是KRIBB3依赖性细胞凋亡的介体。此外，当以50 mg / kg或100 mg / kg腹膜内给裸鼠腹膜内施用KRIBB3时，它分别抑制了49.5％或70.3％的肿瘤生长。这些结果表明，KRIBB3是用于癌症治疗的良好药物候选物。