Park Shin Young, Kim Ki Yun, Jun Do Youn, Hwang Su-Kyeong, Kim Young Ho
Laboratory of Immunobiology, School of Life Sciences, BK21 FOUR KNU Creative BioResearch Group, Kyungpook National University, Daegu 41566, Korea.
Astrogen Inc., Techno-Building 313, Kyungpook National University, Daegu 41566, Korea.
Cancers (Basel). 2020 Dec 19;12(12):3845. doi: 10.3390/cancers12123845.
In vitro antitumor activity of the CDK7 inhibitor BS-181 against human T-ALL Jurkat cells was determined. Treatment of Jurkat clones (JT/Neo) with BS-181 caused cytotoxicity and several apoptotic events, including TRAIL/DR4/DR5 upregulation, c-FLIP down-regulation, BID cleavage, BAK activation, ΔΨ loss, caspase-8/9/3 activation, and PARP cleavage. However, the BCL-2-overexpressing Jurkat clone (JT/BCL-2) abrogated these apoptotic responses. CDK7 catalyzed the activating phosphorylation of CDK1 (Thr161) and CDK2 (Thr160), and CDK-directed retinoblastoma phosphorylation was attenuated in both BS-181-treated Jurkat clones, whereas only JT/BCL-2 cells exhibited G cell cycle arrest. The G-blocker hydroxyurea augmented BS-181-induced apoptosis by enhancing TRAIL/DR4/DR5 upregulation and c-FLIP down-regulation. BS-181-induced FITC-annexin V-positive apoptotic cells were mostly in the sub-G and G phases. BS-181-induced cytotoxicity and mitochondrial apoptotic events (BAK activation/ΔΨ loss/caspase-9 activation) in Jurkat clones I2.1 (FADD-deficient) and I9.2 (caspase-8-deficient) were significantly lower than in A3 (wild-type). Exogenously added recombinant TRAIL (rTRAIL) markedly synergized BS-181-induced apoptosis in A3 cells but not in normal peripheral T cells. The cotreatment cytotoxicity was significantly reduced by the DR5-blocking antibody but not by the DR4-blocking antibody. These results demonstrated that the BS-181 anti-leukemic activity is attributed to extrinsic TRAIL/DR5-dependent apoptosis preferentially induced in G-arrested cells, and that BS-181 and rTRAIL in combination may hold promise for T-ALL treatment.
测定了CDK7抑制剂BS-181对人T细胞急性淋巴细胞白血病Jurkat细胞的体外抗肿瘤活性。用BS-181处理Jurkat克隆（JT/Neo）会导致细胞毒性和多种凋亡事件，包括TRAIL/DR4/DR5上调、c-FLIP下调、BID裂解、BAK激活、线粒体膜电位丧失、半胱天冬酶-8/9/3激活以及PARP裂解。然而，过表达BCL-2的Jurkat克隆（JT/BCL-2）消除了这些凋亡反应。CDK7催化CDK1（苏氨酸161）和CDK2（苏氨酸160）的激活磷酸化，并且在两个经BS-181处理的Jurkat克隆中，CDK介导的视网膜母细胞瘤磷酸化均减弱，而只有JT/BCL-2细胞表现出G期细胞周期停滞。G期阻滞剂羟基脲通过增强TRAIL/DR4/DR5上调和c-FLIP下调来增强BS-181诱导的凋亡。BS-181诱导的FITC-膜联蛋白V阳性凋亡细胞大多处于亚G期和G期。BS-181在Jurkat克隆I2.1（FADD缺陷型）和I9.2（半胱天冬酶-8缺陷型）中诱导的细胞毒性和线粒体凋亡事件（BAK激活/线粒体膜电位丧失/半胱天冬酶-9激活）明显低于A3（野生型）。外源性添加的重组TRAIL（rTRAIL）显著增强了BS-181在A3细胞中诱导的凋亡，但在正常外周T细胞中无此作用。DR5阻断抗体可显著降低联合处理的细胞毒性，而DR4阻断抗体则无此作用。这些结果表明，BS-181的抗白血病活性归因于在G期停滞细胞中优先诱导的外源性TRAIL/DR5依赖性凋亡，并且BS-181与rTRAIL联合应用可能对T细胞急性淋巴细胞白血病的治疗具有前景。
