【作者】 宣嫣艳；

【导师】 胡汛；

【作者基本信息】 浙江大学 ， 肿瘤学， 2009， 博士

【摘要】 肿瘤耐药性是临床化疗的主要障碍。从细胞学层面看,其机理主要包括细胞表面耐药蛋白高表达介导的多药耐药及抗凋亡因子过表达导致的凋亡耐受。目前临床上还没有针对耐药肿瘤的有效药物。2007年,本研究小组首次报道紫草素能诱导肿瘤细胞发生一种非凋亡的程序性死亡,即Necroptosis,并以此绕开了肿瘤细胞的耐药机制,对药敏和耐药肿瘤细胞都有同等程度的杀伤作用。但是,仅有一个紫草素的发现不仅没有普遍性意义,更不足以为开发这一类新药提供理论基础:如果紫草素结构的微小变化就会使其丧失这种特性,那将无望开发出一类基于诱导necroptosis的新型小分子药物。因此,本论文以紫草素为切入点,对六种新的天然紫草萘醌类化合物,即去氧紫草素、乙酰紫草素、异丁酰紫草素、β,β-二甲基丙烯酰紫草素、异戊酰紫草素及2-甲基-正丁酰紫草素是否也具有诱导肿瘤细胞发生necroptosis从而克服肿瘤耐药性的特点进行探讨。MTT实验结果表明,该类化合物对八对药敏和耐药肿瘤细胞具有相似的IC₅₀值,证明其在药敏和耐药细胞中有相似的杀伤作用,并发现2-甲基-正丁酰紫草素具有比紫草素更强的细胞毒作用。其次,Necrostatin-1能有效抑制这六种化合物引起的细胞死亡,初步证明该死亡模式为necroptosis。以2-甲基-正丁酰紫草素为代表的进一步死亡模式鉴定实验显示,死亡细胞出现典型的坏死样形态特征,如细胞膜破裂、胞浆空泡化、线粒体极度肿胀等,并发现有双层膜包裹的自噬小体,提示自噬参与该细胞死亡。同时,Nec-1能部分抑制2-甲基-正丁酰紫草素引起的线粒体膜电位下降,死亡细胞没有caspase-3,8,9的激活,不出现凋亡亚二倍体峰,也没有检测到DNA梯带,提示2-甲基-正丁酰紫草素诱导肿瘤细胞发生necroptosis。体内毒性试验显示,一周内灌胃给予三次100mg/kg的2-甲基-正丁酰紫草素引起小鼠体重的适度减轻,但无其他明显毒性表现,不引起小鼠死亡。同样剂量的紫草素对小鼠没有明显毒性。体内抑瘤实验表明,2-甲基-正丁酰紫草素、紫草素及阿霉素对K562肿瘤均无明显抑制效果;2-甲基-正丁酰紫草素对K562/ADR耐药肿瘤有很好的抑制效果,肿瘤大小与对照组相比有显著性差异,而紫草素和阿霉素无明显抑瘤效果。小鼠乳腺癌细胞4T1在BALB/c小鼠中自发肺转移的动物模型表明,紫草素对原位4T1肿瘤的抑制效果与对照组相比有极显著性差异;对4T1肺转移无明显抑制作用。本研究表明天然紫草萘醌类化合物是一类能诱导肿瘤细胞发生necroptosis从而克服肿瘤耐药性的小分子化合物,提示其具有一定的临床应用价值和开发前景。更多还原

【Abstract】 Cancer drug resistance is the major obstacle in chemotherapy.Cellular factors that contribute to drug resistance include drug transporters mediated multidrug resistance and anti-apoptotic factors mediated blocked apoptosis.Currently,there are no clinically available drugs against drug-resistant tumors.In the year 2007,we first reported that shikonin could circumvent cancer drug resistance by induction of necroptosis,a non-apoptotic programmed cell death.It exhibited similar cytotoxic effect against drug-resistant tumor cells and their drug-sensitive parental cells.However,the findings based on a single molecule-shikonin may not have general scientific significance.The reason is straightforward.If only shikonin could induce necroptosis and if a slight chemical modification of shikonin could alter its nature to induce necroptosis,there is no future to develop the drugs based on necroptotic induction.We therefore extended our study from one molecule shikonin to a series of naturally-occurring naphthoquinone analogues,namely,Deoxyshikonin,Acetylshikonin,Isobutyrylshikonin,β,β-Dimethylacrylshikonin, Isovalerylshikonin and（2-Methyl-n-butyl）shikonin,and further investigated if these compounds could also circumvent cancer drug resistance by induction of necroptosis.The MTT assay revealed that all these compounds had similar IC₅₀ values against eight pairs of drug-sensitive and drug-resistant tumor cells in vitro, among which（2-Methyl-n-butyl）shikonin displayed the strongest cytotoxic effect. Necrostatin-1 efficiently inhibited the cell death induced by these analogues,indicating that they induced necroptosis in tumor cells.We further examined if （2-Methyl-n-butyl）shikonin-induced cell death was necroptosis.Cells treated with （2-Methyl-n-butyl）shikonin showed a necrotic morphology such as rupture of cell membrane,vacuolation of cytoplasm and swelling of mitochondria.Autophagosomes were observed in dead cells,indicating the involvement of autophagy in cell death. Nec-1 partially blocked the decrease of mitochondrial membrane potential induced by （2-Methyl-n-butyl）shikonin.Furthermore,no caspase-3,8,9 activation,no obvious hypodiploid peak and no apoptotic DNA ladder were observed in dead cells.The results thus demonstrated that（2-Methyl-n-butyl）shikonin induced necroptosis in tumor cells. The in vivo acute toxicity study revealed that（2-Methyl-n-butyl）shikonin caused a moderate decrease of mice body weight at 100mg/kg dosed intragastrically three times a week.No other obvious toxicity was observed and no mouse died during the experiment. Shikonin administered at the same dosage did not cause any apparent side effect in mice. We further evaluated the antitumor activity of（2-Methyl-n-butyl）shikonin,shikonin and Dox in vivo.The result showed that all three drugs did not obviously inhibit the growth of K562 tumor.On the other hand,（2-Methyl-n-butyl）shikonin,but not shikonin or Dox, displayed a strong inhibitory effect against K562/ADR tumor.We further established a tumor metastasis model in BALB/c mice inoculated with mouse 4T1 breast cancer cells. We found that shikonin significantly inhibited the growth of primary 4T1 tumor,in comparison to controls,although it did not show apparent inhibitory effect on lung metastasis.To sum up,we demonstrated that the naturally-occurring naphthoquinone analogues were a class of necroptotic inducers that could circumvent cancer drug resistance,suggesting their potential applications in clinical cancer therapy.更多还原