Antitumor Activity of the Novel Flavonoid Oncamex in Preclinical Breast Cancer Models. Homo sapiens

Introduction: The flavonoid myricetin has been shown to induce cell cycle arrest and mitochondrial-dependent apoptosis in preclinical cancer models. We hypothesised that myricetin-derived flavonoids with redox properties and improved physicochemical attributes that enhance cell uptake and mitochondrial targeting might have increased potential as antitumour agents, since mitochondria are the main site of production of reactive oxygen species (ROS) and redox status is known to regulate the development and advancement of certain cancers. In this study we assessed a small library of novel flavonoids, then focussed on the lead compound, second-generation analogue OncamexTM, its mechanism of action and structure-activity relationships as an anti-proliferative agent. Methods: With this aim, we studied the effect of OncamexTM in a panel of 7 breast cancer cell lines using proliferation, cytotoxicity and apoptosis assays. The redox properties and mitochondrial delivery of the drug were studied using cyclic voltammetry and fluorescence microscopy, respectively. The mechanism of action was further studied using western blotting, gene expression analysis and immunohistochemistry (IHC) of treated xenograft tissue from in vivo mice models. Results: Sulforhodamine B (SRB) proliferation assays demonstrated strong, anti-proliferative properties of OncamexTM, with IC50 values in the low micromolar range. Treatment for 8 h exerted concentration-dependent reduction in cell viability and induction of cytotoxicity and apoptosis, with increased caspase activation. Microarray analysis suggested that OncamexTM regulates changes in cell cycle and apoptosis at gene expression level. Fluorescence microscopy was used to investigate mitochondrial targeting and ROS regulation in treated cells. OncamexTM was found to induce production of superoxide at concentrations which exerted anti-proliferative effects. Initial in vivo studies in mice implanted with a MDA-MB-231 breast cancer xenograft showed that OncamexTM inhibited tumour growth, reducing tissue viability and Ki-67 proliferation, with no overall systemic toxicity. Conclusions: OncamexTM is a novel flavonoid capable of specific delivery to the mitochondria and induction of ROS production. We have shown its antitumor activity in preclinical models of breast cancer, both in vitro and in initial in vivo models, where tumour growth was arrested without inducing toxicity, support the potential of this novel drug for its continued development as an anticancer agent. Overall design: Total RNA was obtained from 5 breast cancer cell lines subjected to 6 hours of 10µM OncamexTM and matched untreated controls (1% DMSO)

引言：黄酮类化合物杨梅素（myricetin）在临床前癌症模型中已被证实可诱导细胞周期阻滞与线粒体依赖性细胞凋亡。我们提出假说：具备氧化还原特性、且理化性质优化以增强细胞摄取与线粒体靶向能力的杨梅素衍生黄酮类化合物，有望成为更具潜力的抗肿瘤药物——因线粒体是活性氧（reactive oxygen species, ROS）的主要产生位点，而氧化还原状态已被证实可调控部分癌症的发生与进展。本研究首先对小型新型黄酮类化合物文库进行筛选，随后聚焦于第二代先导类似物Oncamex™，探究其作为抗增殖剂的作用机制与构效关系。
方法：为此，我们采用增殖、细胞毒性与细胞凋亡检测方法，评估了Oncamex™在7株乳腺癌细胞系组成的细胞库中的作用效果。分别通过循环伏安法与荧光显微镜技术，研究该药物的氧化还原特性与线粒体递送能力。进一步通过蛋白质印迹（western blotting）、基因表达分析，以及对经药物处理的小鼠体内异种移植瘤组织的免疫组织化学（immunohistochemistry, IHC）检测，深入探究其作用机制。
结果：磺酰罗丹明B（sulforhodamine B, SRB）增殖检测结果显示，Oncamex™具备显著的抗增殖活性，其半数抑制浓度（IC50）处于低微摩尔浓度范围。给药8小时后，细胞存活率呈浓度依赖性降低，同时诱导细胞毒性与细胞凋亡，并伴随半胱天冬酶（caspase）激活水平升高。基因芯片分析结果表明，Oncamex™可在基因表达层面调控细胞周期与细胞凋亡相关的变化。采用荧光显微镜技术，探究经药物处理的细胞中线粒体靶向性与ROS调控情况。研究发现，在发挥抗增殖作用的浓度下，Oncamex™可诱导超氧阴离子产生。针对植入MDA-MB-231乳腺癌异种移植瘤的小鼠开展的初步体内研究显示，Oncamex™可抑制肿瘤生长，降低肿瘤组织存活率与Ki-67增殖标记物表达，且未引发明显的全身性毒性反应。
结论：Oncamex™是一种新型黄酮类化合物，可特异性靶向递送至线粒体并诱导ROS产生。我们已证实，该药物在乳腺癌临床前模型（包括体外与初步体内模型）中具备抗肿瘤活性，可阻滞肿瘤生长且未引发毒性反应，这为其作为抗癌药物持续开发的潜力提供了支持。
整体实验设计：从5株乳腺癌细胞系中提取总RNA，这些细胞系分别经10μM Oncamex™处理6小时，以及匹配的未处理对照组（含1%二甲基亚砜，DMSO）