乙酰紫草素通过ROS诱导激素非依赖性前列腺癌空泡化及LC3依赖的非自噬性死亡的分子机制

Prostate cancer (PCa) is the common malignant tumor and the second most frequently diagnosed cancer worldwide. Although the incidence of PCa in China was low, its incidence has recently increased substantially. Moreover, with the implementation of more comprehensive screening programmes, the incidence rate of PCa in China may rapidly increase in the future , which is becoming one of the malignant diseases affecting the health of Chinese old men. Acetyl shikonin is a derivative of shikonin, a naphthoquinone compound and one of the active components of Lithospermum erythrorhizon Sieb. et Zucc, a well known traditional herb in China. Its anti-tumor effects and the underlying mechanisms are still not known well. In the course of screening of antitumor activity, we discovered that acetyl shikonin (AS) not only remarkably abrogated proliferation in androgen independent prostate cancer (AIPC) cells, but also triggered extensive cytoplasmic vacuolation and LC3 expression through reactive oxgen species (ROS). Blockade of autophagy hardly decreased LC3 level as well as AS-indced cell death, however, inhibtion of LC3 expression by siRNA dramatically reversed cell death, which meaned AS induced LC3-dependent nonautophagic cell death. The acetyl shikonin-induced cytoplasmic vacuolation and LC3-dependent nonautophagic cell death in AIPC has not been reported before. Given that cytoplasmic vacuolation and LC3-dependent nonautophagic cell death was a novel nonapoptotic cell death. This project is aimed at elucidating the underlying mechanism of acetyl shikonin-mediated vacuolation and LC3-dependent nonautophagic cell death and validating it as a novel lead compound used in APIC therapy.

前列腺癌是男性常见的恶性肿瘤之一，发病率高居男性肿瘤第2位。近年来，我国前列腺癌的发病率，特别是晚期前列腺癌的发病率呈显著上升趋势。日益成为威胁我国老年男性健康的恶性疾病之一。乙酰紫草素是一种萘醌类化合物，也是中药紫草中主要分之一，其抗肿瘤作用的分子机制尚不十分清楚。本课题组研究发现，乙酰紫草素不仅抑制激素非依赖性前列腺癌细胞增殖，而且通过诱导ROS促进肿瘤细胞空泡化及自噬的标志蛋白LC3的表达。抑制自噬不仅不影响LC3的水平，更无法逆转乙酰紫草素诱导的细胞死亡，而阻断LC3的表达，细胞存活率显著增加。乙酰紫草素诱导前列腺癌细胞空泡化及LC3依赖的非自噬性死亡未见报道。鉴于细胞空泡化及LC3依赖的非自噬性死亡是一种新的非凋亡性细胞死亡，本项目拟以乙酰紫草素为代表研究诱导前列腺癌细胞空泡化及LC3依赖的非自噬性死亡的先导化合物，为研制针对前列腺癌的创新型药物提供依据。

近年来，我国前列腺癌的发病率和检出率呈现明显上升趋势，其发病率位居男性肿瘤泌尿肿瘤第1位。前列腺癌多发于60岁以上老年男性，正逐渐成为威胁我国老年男性健康的重要疾病之一。去势治疗一直是中晚期前列腺癌治疗的标准方法，但几乎所有患者都会进展为去势耐受性前列腺癌，治疗效果不佳，预后较差。因此寻找新的治疗靶点和新的潜在药物或通过新的机制诱导肿瘤细胞死亡对于去势耐受性前列腺癌治疗具有重要意义。乙酰紫草素是一种萘醌类化合物，也是传统中药紫草中主要成分之一，其抗肿瘤作用，尤其针对去势耐受性前列腺癌的作用机制有待进一步揭示。本项目通过抗肿瘤活性筛选发现，乙酰紫草素对去势耐受性前列腺癌细胞PC3和DU145的增殖具有显著的抑制作用，诱导细胞死亡并伴随细胞内大量空泡的产生，电镜下可见大量肿胀的内质网，抑制凋亡只能部分逆转细胞死亡。进一步研究发现，乙酰紫草素通过诱导线粒体超氧化物产生，抑制TRXR1活性导致细胞氧化应激耗竭细胞内GSH，并通过抑制蛋白酶体活性促进内质网应激。内质网应激激活PERK/eIF2α/ATF4信号通路上调ATF3的表达进而调控LC3B的表达。电镜下未见自噬泡和自噬溶酶体的显著增加，且抑制自噬不仅不影响LC3B的表达，也无法逆转乙酰紫草素诱导的细胞死亡，敲低LC3B的水平，细胞存活率显著增加，细胞空泡也明显减少。此外，乙酰紫草素还通过氧化应激上调PLIN2的表达，参与细胞空泡的形成。综合文献报道及本研究的结果，乙酰紫草素诱导细胞死亡符合旁凋亡的特征。体内抗肿瘤作用表明，乙酰紫草素可显著抑制肿瘤生长，降低瘤体重量，但对裸鼠体重无明显影响，免疫组化结果显示乙酰紫草素处理组肿瘤组织中ki67阳性细胞明显减少。本项目的研究结果为乙酰紫草素作为去势耐受型前列腺癌治疗的潜在药物提供转化医学依据，并为传统中药紫草的深加工和现代化提供了广阔的前景。