ABSTRACT

Harsh conditions within the tumor microenvironment, such as hypoxia and extracellular acidic pH (pH_e), inactivate some chemotherapies, which results in limited or no cytotoxicity. Standard MTT, ATPlite and protease assays that are used to determine the potency of newly developed drugs often give erroneous results when applied under hypoxic or acidic conditions. Therefore, development of a cytotoxicity assay that does not yield false positive or false negative results under circumstances of both hypoxia and acidic pH_e is needed. We evaluated currently used cell viability assays as well as neutral red staining to assess viability of ovarian and pancreatic cancer cells grown in an acidic pH_e microenvironment after treatment with carboplatin, gemcitabine or chloroquine. We validated cell viability using western blotting of pro-caspase-9 and cleaved-caspase-9, and LC3-I and – II. Standard cell viability assays indicated cell viability accurately at pH_e 7.4, but was not correlated with induction of apoptosis or autophagy at acidic pH_e. By contrast, our modified neutral red assay detected cell viability accurately over a range of pH_e as demonstrated by its correlation with induction of apoptosis and autophagy. Neutral red staining is effective for evaluating the effect of chemotherapeutic agents on cell viability under acidic pH_e or hypoxic conditions.

摘要

肿瘤微环境中的恶劣条件，例如缺氧和细胞外酸性 pH (pH _e )，会使一些化学疗法失活，从而导致细胞毒性有限或没有细胞毒性。用于确定新开发药物效力的标准 MTT、ATPlite 和蛋白酶测定在低氧或酸性条件下应用时通常会给出错误的结果。_{因此，需要开发在缺氧和酸性 pH e}的情况下不会产生假阳性或假阴性结果的细胞毒性测定。我们评估了目前使用的细胞活力测定以及中性红染色，以评估在酸性 pH 值_{e中生长的卵巢癌细胞和胰腺癌细胞的活力}用卡铂、吉西他滨或氯喹治疗后的微环境。我们使用 pro-caspase-9 和 cleaved-caspase-9 以及 LC3-I 和 – II 的蛋白质印迹验证了细胞活力。标准细胞活力测定在 pH _e 7.4 下准确显示细胞活力，但与酸性 pH _e下细胞凋亡或自噬的诱导无关。相比之下，我们改进的中性红测定法在一系列 pH _e范围内准确地检测到细胞活力，正如其与细胞凋亡和自噬诱导的相关性所证明的那样。中性红染色可有效评估化学治疗剂在酸性 pH _e或缺氧条件下对细胞活力的影响。