The conformation-dependent activity of azobenzene combretastatin A4 (Azo-CA4) provides a unique approach to reduce the side-effects of chemotherapy, due to the light-triggered conformation transition of its azobenzene moiety. Under hypoxic tumor microenvironment, however, the high expression of azoreductase can reduce azobenzene to aniline. It was postulated that the Azo-CA4 might be degraded under hypoxia, resulting in the decrease of its anti-tumor activity. The aim of this study was to verify such hypothesis in HeLa cells in vitro. The quantitative drug concentration analysis shows the ratio-metric formation of degradation end-products, confirming the bioreduction of Azo-CA4. The tubulin staining study indicates that Azo-CA4 loses the potency of switching off microtubule dynamics under hypoxia. Furthermore, the cell cycle analysis shows that the ability of Azo-CA4 to induce mitotic arrest is lost at low oxygen content. Therefore, the cytotoxicity of Azo-CA4 is compromised under hypoxia. In contrast, combretastatin A4 as a positive control maintains the potency to inhibit tubulin polymerization and break down the nuclei irrespective of light irradiation and oxygen level. This work highlights the influence of hypoxic tumor microenvironment on the anti-tumor potency of Azo-CA4, which should be considered during the early stage of designing translational Azo-CA4 delivery systems.

偶氮苯康他汀A4（Azo-CA4）的依赖构象的活性提供了一种独特的方法来减少化学疗法的副作用，这是由于其偶氮苯部分的光触发了构象转变。然而，在缺氧的肿瘤微环境下，偶氮还原酶的高表达可以将偶氮苯还原为苯胺。据推测，缺氧条件下Azo-CA4可能会降解，导致其抗肿瘤活性下降。这项研究的目的是在体外验证HeLa细胞中的这种假设。定量药物浓度分析显示降解终产物的比例形成，证实了Azo-CA4的生物还原。微管蛋白染色研究表明，缺氧条件下Azo-CA4失去了关闭微管动力学的能力。此外，细胞周期分析表明，在低氧含量下，Azo-CA4诱导有丝分裂停滞的能力丧失了。因此，在缺氧条件下，Azo-CA4的细胞毒性受到损害。相反，康普他汀A4作为阳性对照，不管光照射和氧水平如何，都保持了抑制微管蛋白聚合和分解细胞核的能力。这项研究强调了缺氧肿瘤微环境对Azo-CA4抗肿瘤效力的影响，