Upregulating Noxa by ER Stress, Celastrol Exerts Synergistic Anti-Cancer Activity in Combination with ABT-737 in Human Hepatocellular Carcinoma Cells

The human hepatocellular carcinoma (HCC) represents biologically aggressive and chemo-resistant cancers. Owing to the low affinity with the apoptotic factor Mcl-1, the BH3 mimetic drug ABT-737 failed to exert potent cancer-killing activities in variety of cancer models including HCC. The current study demonstrated that combining ABT-737 and Celastrol synergistically suppressed HCC cell proliferation, and induced apoptosis which was accompanied with the activation of caspase cascade and release of cytochrome c from mitochondria. Further study revealed that the enhanced Noxa caused by Celastrol was the key factor for the synergy, since small interfering RNA-mediated knockdown of Noxa expression in HCC cells resulted in decreased apoptosis and attenuated anti-proliferative effects of the combination. In addition, our study unraveled that, upon Celastrol exposure, the activation of endoplasmic reticulum (ER) stress, specifically, the eIF2α-ATF4 pathway played indispensable roles in the activation of Noxa, which was validated by the observation that depletion of ATF4 significantly abrogated the Noxa elevation by Celastrol. Our findings highlight a novel signaling pathway through which Celastrol increase Noxa expression, and suggest the potential use of ATF4-mediated regulation of Noxa as a promising strategy to improve the anti-cancer activities of ABT-737.肝细胞癌（hepatocellular carcinoma, HCC）是一类生物学侵袭性强且兼具化疗耐药性的恶性肿瘤。由于与凋亡因子Mcl-1亲和力较低，BH3模拟类药物ABT-737无法在包括HCC在内的多种癌症模型中发挥强效的杀癌活性。本研究证实，将ABT-737与雷公藤红素（Celastrol）联合使用，可协同抑制肝癌细胞增殖并诱导细胞凋亡，该过程伴随半胱天冬酶级联激活以及线粒体细胞色素c的释放。进一步研究表明，雷公藤红素介导的Noxa表达上调是产生协同效应的关键因素：在肝癌细胞中通过小干扰RNA（small interfering RNA, siRNA）敲低Noxa的表达，会减弱联合用药诱导的细胞凋亡，并削弱其抗增殖作用。此外，本研究还揭示，在雷公藤红素处理后，内质网（endoplasmic reticulum, ER）应激的激活，尤其是eIF2α-ATF4通路，在Noxa的激活中发挥了不可或缺的作用；这一结论通过“敲低ATF4可显著逆转雷公藤红素对Noxa的上调作用”的实验结果得到了验证。本研究结果明确了一条雷公藤红素上调Noxa表达的全新信号通路，并提示以ATF4介导的Noxa调控作为潜在策略，可有效增强ABT-737的抗肿瘤活性。