基于p53动力学的阿霉素PK/PD结合研究

A breakthrough in the field of p53 was the discovery of p53 dynamics. The tumor suppressor p53 displays distinct dynamic behaviors following different stimuli, which result in the expression of a different set of downstream genes and ultimately different cellular outcomes, such as cell cycle arrest, senescence and apoptosis. Although this has been well documented for DNA damage caused by γ-irradiation and UV radiation, it has rarely been determined for the stimulus of DNA breaks induced by genotoxic drugs. Our previous data show that variations in the two key elements of pharmacokinetics (PK), intracellular drug concentration and incubation time, led to distinct p53 dynamic patterns and subsequently altered apoptotic rates. Therefore, we propose a new hypothesis to explain our observation that cellular pharmacokinetics of genotoxic drugs could regulate p53 dynamic patterns evoked by the drug. We first choose Adriamycin as a probe drug, and design different treatment regimens by varying dosage, leading to p53 pulses of distinct amplitude and patterns and the subsequent cell fates. We will then modify cellular PK properties of Adriamycin by over-expression and silencing/inhibitions of two major components involved in PK of Adriamycin, the transporter p-glycoprotein and the metabolizing enzyme carbonyl reductase 1, and investigate the changes of p53 dynamics in response to the alterations. With the cellular concentrations of Adriamycin and p53 dynamics of protein level determined at selected time points upon administration, a cellular PK-pharmacodynamics (PD) model will be developed to assess how cellular PK properties of Adriamycin regulate the p53 dynamics, and subsequently control cell fates. The integration of cellular PK of Adriamycin with p53 dynamics firstly proposed in this study will elucidate the dynamic behaviors of p53 caused by chemotherapy drugs and shed light on the molecular mechanism how cell fates are determined secondary to this stimulus. Lastly, various dosing regimens of Adriamycin will be designed based on the PK-PD model, and given to tumor-bearing mice to reveal whether p53 dynamics could be employed to evaluate in vivo therapeutic effect. In summary, not only will this research uncover the underlying molecular mechanism of p53-dependent apoptosis caused by genotoxic drugs, it also demonstrates that regulation of cellular PK parameters of drugs might lead to desired cellular fates. Moreover, the PK-PD model developed in this study could be a game changer for current clinical design of dosing regimens by determining optimal chemotherapy treatment regiments based on p53-dependent apoptotic rate of tumor cells.

p53 蛋白分子动力学是新近发现的一种决定p53蛋白功能及细胞命运选择的重要机制。然而，化疗药物如何调控p53动力学进而诱导肿瘤细胞凋亡尚缺乏研究。根据我们的前期研究发现，化疗药物在肿瘤细胞内的曝露浓度与时间是决定其诱导细胞凋亡的重要决定因素。因此，本项目创新性提出细胞药代动力学与p53分子动力学结合研究的学术思想，以阿霉素为模型药，首先基于体外细胞模型，通过不同给药方式、化学干预与基因调控肿瘤细胞p-gp与代谢酶水平，诱导出不同特征与强度的p53分子动力学谱，通过细胞药代动力学参数与p53分子动力学参数的PK-PD结合研究，揭示在化疗药物刺激下细胞命运选择的分子机理；以此为引导，设计不同的整体给药方案，在荷瘤鼠模型考察 p53分子动力学与整体疗效的关联性，为临床化疗药物用药方案的优化设计提供参考依据。项目研究对于p53依赖化疗药物的分子作用机理及临床用药方案的优化设计具有普遍的借鉴意义。

DNA损伤化疗药物可诱发肿瘤细胞产生周期抑制、早衰和凋亡等不同现象，p53动力学在其中发挥着重要的角色。然而，化疗药物如何通过调控p53动力学进而调控细胞命运尚不明确。基于此，本研究首先通过调节阿霉素的暴露量（给药浓度和药物暴露时间），在体外建立细胞毒药物诱导的细胞周期抑制与修复、早衰和凋亡等不同细胞命运的模型。接着，基于0.1 μM阿霉素给药72 hrs与10 μM阿霉素给药8 hrs后继续培养所造成的相同比例的细胞凋亡的现象进行基于p53动力学的凋亡规律的探讨。首先由于低浓度给药方式诱导p53出现两相动力学现象，而高浓度给药方式造成p53持续累积的动力学过程，直接证明p53动力学模式本身并非决定细胞命运的标准。对两种不同形式的p53动力学进行计算，我们提出抉择细胞命运的标准为最低有效p53水平，在此水平以上的p53累积量为诱导细胞凋亡的有效累积量（E∫p53）；当E∫p53超过阈值，则细胞选择进入凋亡。这一理论经由扰动p53动力学、测定p53与下游命运相关基因结合规律，及建立多西环素可控p53表达系统等方法层层验证。我们进一步基于代谢组学与稳定同位素标记定量蛋白质组学的技术，研究阿霉素作用导致的MCF7在早衰或凋亡状态下的代谢改变。结果显示，在早衰细胞中三羧酸循环，磷酸戊糖通路和核酸合成通路都被激活以促使细胞增强抗氧化能力并促进DNA的部分修复，而脂肪酸合成通路下调，细胞无法增殖，并处于永久的周期阻滞；而在凋亡细胞中，大多数代谢通路均下调，细胞进入程序性死亡过程。抑制PPP通路关键酶G6PDH，可阻断ROS的清除与DNA修复，增加细胞凋亡率。目前，p53如何参与对代谢基因的调控的机制研究亟待开展。以上结果证明p53动力学及细胞代谢均参与细胞命运的调控中，通过合理的给药方案设计调控p53动力学行为，或以低剂量化疗药物联合干预代谢的药物有希望实现对癌细胞命运的精准调控，完成有效抑制肿瘤的同时避免“耐受剂量”化疗方案造成的毒副作用的治疗目标。