倍半萜类化合物Hirsutanols A抗肿瘤作用及作用机制研究
摘要
一研究背景与目的
癌症具有较高的发病率和死亡率,严重危害着人类的健康和生命。每年全世界约有700万人死于癌症,并且近几年癌症的死亡率呈明显的上升趋势。化疗是治疗肿瘤的主要手段之一,因此,寻找和开发高效低毒的抗肿瘤药物仍然是攻克肿瘤的主要途径。近年来,根据正常细胞与肿瘤细胞基因表达的不同,有目的地针对肿瘤特异性癌基因的靶向治疗已取得一定的进展,但是由于基因的不稳定性和耐药性问题,基因靶向治疗仍然面临很大的挑战。因此,针对肿瘤发生和发展的原因,寻找新的肿瘤治疗靶点已成为抗肿瘤药物研究开发的重点。近年来,针对肿瘤细胞特异生化环境的改变成为抗肿瘤药物研究的新靶点。
活性氧(ROS)是一类化学性质活泼的含氧功能基团物质的统称。它包括过氧化氢(H202)、羟基自由基(·OH)、超氧阴离子自由基(O2·)、单线态氧、臭氧(03)、氮氧化物(NOx)、次氯酸(HClO)等。在生理条件下ROS的产生对于细胞的生长是必需的,生理条件下的ROS可以调控细胞的增殖和分化,但是异常升高的ROS则可以促进肿瘤形成。由于肿瘤细胞长期处于ROS高水平的状态,肿瘤细胞的氧化还原体系相对正常细胞更为脆弱,对ROS的调控能力更低,当用ROS诱导剂诱导ROS升高可以选择性的杀死癌细胞而对正常细胞没有影响。细胞的氧化还原体系被认为是抗肿瘤药物研究的新靶点。
随着陆地药用生物资源开发范围的日益缩小,人们纷纷将目光投向海洋。海洋生物因生存环境的特殊性(高压、低氧、高盐等),往往含有大量结构新颖奇特、生理活性显著的代谢产物。因此,从海洋中寻求高效低毒的天然药物已引起国际上越来越浓厚的兴趣。Hirsutanol A是从南海珊瑚Sarcophyton tortuosum的内生菌Chondrostereum sp中分离得到的一个倍半萜类化合物,该化合物是从一个新的菌种中分离得到,且该化合物的化学结构仅报道过一次,药理活性未见报道。本研究对从海洋内生菌中分离出来的六个单体化合物进行细胞毒作用的筛选,发现一个倍半萜类化合物Hirsutanol A具有较强的细胞毒作用,于是我们对其抗肿瘤作用及其机制分别从体内和体外进行探讨。
二研究方法
MTT试验筛选具有抗肿瘤活性的化合物及检测Hirsutanol A对不同肿瘤细胞株的生长抑制作用。DHE. CM-H2DCF-DA染色流式细胞术检测ROS的超氧阴离子和过氧化氢的水平。AnnexinV/PI双染流式细胞术检测Hirsutanol A诱导肿瘤细胞的凋亡率。GFP-LC3转染细胞激光共聚焦检测细胞自噬。免疫印迹方法(Western Blot)检测JNK和Akt信号通路变化,凋亡相关蛋白caspase-3、PARP断裂带和自噬相关蛋白LC3Ⅰ-Ⅱ的转变。Akt激酶活性分析试剂盒检测Hirsutanol A对Akt活性的影响。用线粒体分离试剂盒将线粒体和胞浆分离,免疫印迹方法检测线粒体和胞浆中细胞色素C的表达。应用强抗氧化剂NAC检测ROS抑制对Hirsutanol A诱导的细胞增殖抑制和凋亡以及JNK,Akt磷酸化水平的影响。应用特异性小分子抑制剂SP600125和siRNA瞬时转染方法检测JNK信号通路抑制对于Hirsutanol A诱导的凋亡和ROS水平的影响。应用小分子抑制剂3-MA、Bafilomycin Al及siRNA瞬时转染方法抑制自噬,检测自噬在Hirsutanol A诱导的细胞死亡中的作用。用稳定转染的方法构建Hep3B/myr-Aktl, Hep3B-vector细胞株,MTT法检测Hirsutanol A对Hep3B/myr-Aktl, Hep3B-vector的生长抑制作用。裸鼠成瘤实验观察HirsutanolA对SW620裸鼠移植瘤生长的抑制作用。
三研究结果
1活性化合物的筛选及Hirsutanol A对不同肿瘤细胞株的细胞毒作用
我们对6种化合物进行了筛选,发现Hirsutanol A对多种肿瘤细胞均具有明显的细胞毒作用,并呈现明显的时间-剂量效应关系。
2 Hirsutanol A抗肿瘤作用分子机理
(1) Hirsutanol A对肿瘤细胞ROS水平的影响
我们分别用DHE和CM-H2DCF-DA染色检测Hirsutanol A对肿瘤细胞超氧阴离子和过氧化氢水平的影响,结果显示Hirsutanol A对超氧阴离子水平没影响但能显著增加肿瘤细胞过氧化氢水平,并呈现明显的时间-剂量效应关系。
(2) Hirsutanol A上调ROS诱导肿瘤细胞发生凋亡
①Hirsutanol A诱导SW620、MDA-MB-231、MCF-7肿瘤细胞发生凋亡
AnnexinV/PI双染色法检测Hirsutanol A诱导S W620、MDA-MB-231、MCF-7细胞发生凋亡的情况。结果显示Hirsutanol A能明显诱导SW620、MDA-MB-231、MCF-7细胞发生凋亡,其凋亡率分别为：SW620(2.8%、12.1%、45.0%、65.6%)、MDA-MB-231 (2.6%、10%、22.7%、30.6%)、MCF-7 (1.7%、10%、20、8%、31.3%)且呈明显的剂量依赖关系；Western blot法检测caspase-3、PARP的剪切片段,在17KD和85KD可见明显的断裂带。以上结果表明Hirsutanol A能诱导SW620、MDA-MB-231、MCF-7细胞发生凋亡。
②Hirsutanol A通过线粒体途径诱导凋亡
为了探讨Hirsutanol A能否通过线粒体途径诱导凋亡,我们分别检测了SW620、MDA-MB-231细胞线粒体膜电位改变以及线粒体和胞浆中细胞色素C的表达。结果显示：SW620、MDA-MB-231细胞线粒体膜电位均呈浓度依赖性升高。用Hirsutanol A处理细胞24h后,线粒体中细胞色素C的表达量明显减少而胞浆中细胞色素C的表达量明显增多。以上结果表明Hirsutanol A通过线粒体途径诱导凋亡。
③Hirsutanol A诱导凋亡的发生与ROS相关
我们在前面的研究中发现Hirsutanol A对多种肿瘤细胞均有增殖抑制作用,而且可以显著上调SW620、MDA-MB-231、MCF-7细胞的ROS水平,为了证明Hirsutanol A通过上调ROS而发挥抗肿瘤的作用。我们用强抗氧化剂NAC抑制Hirsutanol A对ROS的上调作用后,检测Hirsutanol A对细胞的增殖抑制作用和细胞凋亡的影响。结果显示NAC可明显减少Hirsutanol A诱导的增殖抑制作用和Hirsutanol A诱导的细胞凋亡。
④Hirsutanol A通过上调ROS激活JNK通路
研究发现ROS可以调控JNK、Akt、NF-κB等多条信号通路,接下来我们进一步探讨Hirsutanol A激活ROS后对JNK通路的影响。用HirsutanolA处理SW620细胞24 h, Western blot检测JNK和c-Jun蛋白磷酸化水平,可见JNK和c-Jun蛋白磷酸化水平明显升高。用ROS抑制剂NAC预处理细胞后,NAC可以抑制Hirsutanol A对JNK的激活作用。说明Hirsutanol A激活JNK通路的作用与ROS上调相关。
⑤JNK通路激活的作用
用JNK抑制剂SP600125抑制JNK通路后,分别用MTT检测细胞的活性和AnnexinV/PI双染色法检测细胞的凋亡,可见SP600125可明显增加Hirsutanol A诱导的细胞死亡；单用Hirsutanol A细胞的凋亡率为35.6%,用SP600125阻断JNK通路后,Hirsutanol A诱导的细胞凋亡率上升到48.3%,阻断JNK通路可以增加Hirsutanol A诱导的细胞凋亡。表明JNK通路的激活不仅不能诱导细胞发生凋亡,还能保护细胞免受死亡。
接下来我们进一步探讨了JNK通路激活对ROS的调控作用,用JNK抑制剂SP600125预处理细胞或siRNA阻断JNK通路后,流式细胞仪检测ROS的水平,结果显示阻断JNK通路能显著增加Hirsutanol A诱导的ROS水平。说明JNK通路的激活并不诱导细胞凋亡而是负反馈的调控ROS水平,是细胞的一种氧化应激保护机制。
(3) Hirsutanol A上调ROS诱导肿瘤细胞发生自噬
①Hirsutanol A通过上调ROS诱导肿瘤细胞发生自噬
LC3可作为自噬体膜标志性蛋白,我们先用与绿色荧光蛋白(GFP)结合的GFP-LC3来检测自噬,将Hep3B转染GFP-LC3质粒24 h后,用20μmol/L HirsutanolA处理24h,于激光共聚焦显微镜下观察,可见Hirsutanol A处理组出现大量LC3点状聚集,而对照组LC3呈弥散分布。接下来我们进一步用Western blot检测LC3蛋白表达,可见LC3由Ⅰ型向Ⅱ型转化增加,提示Hirsutanol A可以诱导肿瘤细胞发生自噬。
Hirsutanol A能够上调ROS水平和诱导自噬,接下来我们进一步探讨HirsutanolA上调ROS水平和诱导自噬之间的相关性。分别用Western blot检测LC3蛋白表达和激光共聚焦检测LC3点状聚集,结果显示加入NAC预处理细胞后,Hirsutanol A诱导的LC3由Ⅰ型向Ⅱ型转化明显减少,LC3点状聚集亦明显减少。以上结果表明Hirsutanol A通过上调ROS诱导肿瘤细胞发生自噬。
②Hirsutanol A通过上调ROS抑制Akt通路
Akt/mTOR是调控自噬的一条主要通路,我们在前面的研究中发现HirsutanolA可以诱导肿瘤细胞产生ROS,并诱导肿瘤细胞发生自噬。于是我们进一步探讨Hirsutanol A对Akt通路的影响。用Hirsutanol A处理细胞24 h后,Western blot检测Akt、P-Akt473和P-FKHR蛋白表达水平,发现Akt表达水平不变但P-Akt473和P-FKHR表达水平明显降低,说明Hirsutanol A可以抑制Akt通路。
接下来我们进一步探讨Hirsutanol A对Akt通路抑制作用与ROS上调的相关性,先用Akt激酶活性分析法检测Hirsutanol A在体外对Akt激酶活性的影响,结果显示Hirsutanol A在体外不能直接抑制Akt激酶的活性。用NAC预处理Hep3B细胞1h然后加入Hirsutanol A处理24 h, Western blot检测P-Akt473蛋白表达水平,可见NAC可部分逆转P-Akt473的表达水平。说明AKT通路抑制与ROS上调相关。
③Hirsutanol A诱导Hep3B细胞发生非凋亡性死亡
我们用AnnexinV/PI双染法检测细胞凋亡时发现,用5、10、20μmol/L HirsutanolA处理Hep3B细胞48 h后,AnnexinV单阳性率最高仅为0.5%,而PI阳性率分别为5.5%、15.9%、49.7%。Western blot检测Caspase-3, PARP表达,结果显示在17KD和85KD均未见明显的断裂带。该结果表明Hirsutanol A诱导Hep3B细胞发生非凋亡性死亡。
④Hirsutanol A诱导Hep3B细胞发生自噬性死亡
为了证明Hirsutanol A诱导Hep3B细胞发生自噬性死亡,我们分别用自噬抑制剂3-MA和siRNA-Beclinl阻断自噬。MTT法检测阻断自噬后Hirsutanol A对细胞生长抑制和死亡的影响。结果显示,阻断自噬可以明显降低Hirsutanol A诱导的细胞死亡。
⑤Hirsutanol A诱导Hep3B细胞自噬性死亡与ROS上调和Akt通路抑制相关
为了验证Hirsutanol A诱导Hep3B细胞自噬性死亡与ROS上调的关系,我们用1mmol/L的NAC预处理细胞1h,然后加入Hirsutanol A处理72 h,分别用MTT法和AnnexinV/PI双染法检测细胞增殖抑制和死亡情况,结果显示NAC可明显减少Hirsutanol A诱导的细胞增殖抑制和死亡。我们还构建了Hep3B/myr-Aktl, Hep3B-vector细胞株,检测Hirsutanol A诱导的细胞死亡与Akt通路之间的关系,结果显示Hirsutanol A对Hep3B/myr-Akt1细胞的生长抑制作用明显高于Hep3B-vector细胞。说明Hirsutanol A诱导Hep3B细胞自噬性死亡与ROS上调和Akt通路抑制相关。
⑥抑制自噬可以促进Hirsutanol A诱导的MCF-7细胞死亡
我们在研究中发现Hirsutanol A既可以诱导肿瘤细胞MCF-7发生凋亡也可以诱导发生自噬,那么Hirsutanol A诱导的自噬是起到保护作用还是促进细胞死亡的作用呢?我们分别用自噬抑制剂Bafilomycin Al和siRNA-Atg-7抑制自噬,MTT法检测细胞的死亡。结果显示,阻断自噬可以明显的增加细胞的死亡,说明Hirsutanol A诱导的自噬在MCF-7细胞中起保护细胞免受死亡的作用。
3 Hirsutanol A对裸鼠移植瘤的抑制作用
20 mg／kg剂量的Hirsutanol A能明显抑制裸鼠移植瘤的生长,抑瘤率31.5%而5 mg／kg的治疗剂量对瘤组织体积的增长无明显抑制作用。
四结论
1 Hirsutanol A通过上调ROS,引起线粒体损伤,细胞色素C释放从而诱导肿瘤细胞SW620、MDA-MB-231发生凋亡。
2 Hirsutanol A通过上调ROS，抑制Akt通路诱导肿瘤细胞Hep3B发生自噬性死亡。
3 Hirsutanol A既可诱导肿瘤细胞MCF-7发生凋亡也可诱导其发生自噬,自噬在MCF-7细胞中起到保护作用。
4 Hirsutanol A通过上调ROS水平激活JNK通路,JNK通路激活并不诱导凋亡而是负反馈调控ROS水平。
5 Hirsutanol A明显抑制裸鼠结肠癌SW620移植瘤的生长。
Background and objective
Cancer has become an increasing public health problem because of its high rates of morbidity and mortality. About 7 million people die from cancer each year all over the world. Recently, cancer significantly showed an increasing trend in mortality. Since chemotherapy is still one of the key therapeutic strategies against cancer, developing the novel anticancer drugs is implicated in cancer therapy. By exploiting the genetic differences between normal cells and cancer cells, targeting therapeutic strategies have acquired progress. However, it still faces significant challenges owing to acquired drug resistance and the genomic instability of cancer cells. Therefore, depending on the events involved in carcinogenesis and cancer progression, it is important to exploit new therapeutic target of anticancer drugs. Recently, targeting unique biochemical alternation has got extensively attentions as a new target of anticancer drugs
Reactive oxygen species(ROS) are a collective term of oxygen derived species, including hydrogen peroxide H2O2, hydroxyl radicals OH, superoxide anion radical O2, singlet oxygen, ozone, hypochloric acid etc. A basic level of intracellular ROS is essential for regulating cell proliferation and differentiation, whereas excessive amounts of ROS can contribute to the carcinogenesis and cancer progression. Compared to normal cells, cancer cells with increased intrinsic ROS are more vulnerable to damage by further ROS insults and more difficult to scavenge the increasing ROS induced by exogenous agents. Therefore, induction of ROS levels by redox modulation is a way to selectively kill cancer cells without significant toxicity to normal cells. So the redox system would be a new target of anticancer drugs.
As the biologic resources in the land are decreasing, researchers focus on marine resources. Since the particular living environments (high pressure, hypoxia and low salty), thousands of peculiar metabolites with physiological activities are found in marine lives, and have evoked great interests to find some natural drugs with high efficacy and low toxicity. Hirsutanol A is a novel sesquiterpene compound purified from Sarcophyton tortuosum. The pharmacologic effect of this compound is still unkown, though the chemcial structure has been reported. We found that Hirsutanol A showed potent cytotoxic effect via crude screen assay from six natural compounds extracted from Fungus Chondrostereum sp in Sarcophyton tortuosum. In this study we investigated the anticancer activity of Hirsutanol A and its mechanism.
Methods
MTT assay was used to screen compounds and evaluate the growth inhibition of various cancer cell lines induced by Hirsutanol A. Cells were stained with DHE or CM-H2DCF-DA to detect cellular superoxide or hydrogen peroxide level measured by flow cytometry. Annexin V-PI staining was used to assess the apoptosis. The transiently transfected GFP-LC3 cells were established by lipofectamine 2000 transfection. The punctuate dots of YFP-LC3 were detected by confocal microscopy. Western blot analysis was used to detect the expression of JNK and Akt signaling pathway -related proteins, cleavage fraction of caspase-3 and PARP, and conversion of LC3 I to LC3-II. Akt kinase activity assay in cell-free system was used to evaluate effect of Hirsutanol A on Akt kinase activity in vitro. Isolation of Mitochondrial and cytosol fraction was obtained by using Mitochondria isolation kit, then expression of cytochrome c in Mitochondria or cytosol was determined by Western blot analysis.SP600125,a small-molecule JNK inhibitor, and specific siRNA against JNK were used to block JNK activity or expression.3-MA and Bafilomycin A1 or siRNA against Beclinl and siRNA against Atg7 were employed to inhibit autophagy. Hep3B cells were stably transinfected with myr-Aktl plasmid to establish Hep3B/myr-Aktl which stably expressed constitutively activated Aktl. MTT assay was used to analysis the growth inhibition of Hep3B/myr-Aktl and Hep3B-vector cells induced by Hirsutanol A.Tumor xenografts were established by SW620 cells injected s.c into nude mice, the growth inhibitory effect was finally calculated after Hirsutanol A treatment.
Results
1 Screening compounds with cytotoxic activity and investigating the cytotoxic effect of Hirsutanol A on various cancer cell lines.
To explore compounds with anticancer activity,we detected the cytotoxic effect of six compounds by MTT assay, the results showed that Hirsutanol A exhibits potent cytotoxic effect on various human cancer cell lines in a dose-and time-dependent manner.
2 The anticancer activity of Hirsutanol A and its molecular mechanisms.
(1) Effect of Hirsutanol A on cellular ROS level in cancer cells:
To investigate the Effect of Hirsutanol A on cellular superoxide and hydrogen peroxide level in cancer cells, we analyzed the level of superoxide and hydrogen peroxide in cancer cells using flow cytometry. There was no significant change in DHE fluorescence but markedly increase in DCF-DA fluorescence after treatment with Hirsutanol A in a dose-and time-dependent fashion, suggesting that ROS induced by Hirsutanol A were mainly hydrogen peroxide but not superoxide.
(2) Hirsutanol A induces apoptosis via a ROS-mediated mechanism
①Hirsutanol A induces apoptosis in SW620, MDA-MB-231, MCF-7 cancer cells
To confirm that Hirsutanol A induced apoptosis, SW620,MDA-MB-231 and MCF-7 cells exposed to various concentrations of Hirsutanol A for 72h. The cells were analyzed by Annexin V-FITC/PI staining assay. The ratio of AnnexinV-positive cells was 2.8%、12.1%、45.0%、65.6% in SW620,2.6%、10%、22.7%、30.6% in MDA-MB-231, and 1.7%、10%、20.8%、31.3% in MCF-7. The results suggested that Hirsutanol A could induce apoptosis in a dose-dependent manner. Using immuno-blotting analysis, the cleavage of caspase-3 and PARP in both cancer cells was detected following treatment with Hirsutanol A. The results showed that pro-caspase-3 was cleaved to yield a 17KDa fragmentation and PARP was cleaved into 89KD fragmentation. These results revealed that Hirsutanol A remarkably induced apoptosis in SW620, MDA-MB-231 and MCF-7 cells.
②Hirsutanol A induced apoptosis through activation of mitochondria/ cytochrome c signaling pathway
To study whether Hirsutanol A induced apoptosis via activation of mitochondria/ cytochrome c signaliing pathway, we examined the change of mitochondrial membrane potential and the release of cytochrome c from mitochondria. mitochondrial membrane potential was elevated after treatment with various concentrations of Hirsutanol A. The expression of cytochrome c in mitochondrial was downregulated, whereas that in cytosol was upregulated after treatment with Hirsutanol A for 24h. These data revealed that Hirsutanol A induced apoptosis through activation of mitochondria/cytochrome c signaling pathway.
③Induction of apoptosis by Hirsutanol A associated with increased ROS generation
The evidences of apoptosis and upregulation of ROS levels by Hirsutanol A prompted us to investigate whether upregulation of ROS resulted in apoptosis. The increase of ROS levels induced by Hirsutanol A in cancer cells was prevented by preincubation with NAC, then growth inhibition and AnnexinV-positive cells were analyzed using MTT and AnnexinV/PI assay. The results showed that the induction of apotosis by Hirsutanol A was blocked when the cells were pretreated with NAC.
④Hirsutanol A activated JNK signaling pathway through upregulation of ROS
It has been reported that ROS can modulate several signaling pathways including JNK, Akt, NF-κB and so on. Therefore, we explored the effect of increasing ROS induced by Hirsutanol A on JNK signaling pathway. JNK and c-Jun phosphorylation were significantly activated in SW620 cells after treatment with Hirsutanol A for 24 h. Moreover, NAC prevented the activation of JNK induced by Hirsutanol A. It suggested that JNK may be a downstream target of increased ROS following Hirsutanol A treatment.
⑤The role of JNK signaling pathway in Hirsutanol A induced ROS increase
In order to explore the contribution of JNK signaling pathway to Hirsutanol A induced ROS, JNK signaling pathway was blocked using the small-molecule JNK inhibitor SP600125. Treated with Hirsutanol A only, the ratio of AnnexinV-positive cells was 35.6%, whereas in parallel treatment in combination with SP600125, the ratio of AnnexinV-positive cells was up to 48.3%, suggesting that blocking JNK signaling pathway promoted apoptosis induced by Hirsutanol A.
We further investigated the effect of activation of JNK signaling pathway on cellular ROS levels. Cellular ROS levels were remarkly increased in SW620 cells by SP600125 or JNK-siRNA to block JNK signaling pathway.These results suggest that activation of JNK is an oxidant stress which protect cell from death via regulation of ROS in a negative feedback manner but not a classic mechanism involved in apoptosis.
(3) Hirsutanol A induced autophagy through increasing ROS production
①Hirsutanol A induced autophagy via increase of ROS level
The lipidation of the protein LC3 during the process of autophagy can be used as a marker. In order to detect the autophagy induced by Hirsutanol A, we observed GFP-tagged LC3 puncta distribution in Hep3B cells. Hep3B cells showed diffuse distribuction of GFP-LC3 in the absence of Hirsutanol A, whereas Hirsutanol A-treated cells showed a marked increase in number and frequency of GFP-LC3 dots.To futher validate autophagy induced by Hirsutanol A, we measured the modification of LC3-Ⅰto LC3-Ⅱby Western blot. LC3 I to LC3-II conversion was remarkedly increased in a dose-and time-dependent manner indicating induction of autophagy by Hirsutanol A.
Since Hirsutanol A can increase cellular ROS levels and induce autophagy, we investigated the relationship between autophagy and upregulation of ROS induced by Hirsutaanol A. The increase of ROS level induced by Hirsutanol A was prevented by preincubation with NAC in Hep3B cells. NAC inhibited the LC3 I to LC3-II conversion and reduced the number of GFP-LC3 dots. These results demonstrated that activation of autophagy depended on the upregulation of ROS levels.
②Hirsutanol A inhibited Akt signaling pathway via increase of ROS levels
The above observations suggested that Hirsutanol A can induce autophagy and accumulation of cellular ROS. Since Akt/mTOR is a key modulator for autophagy, we futher detected the effect of Hirsutanol A on Akt signaling pathway. The results showed that the phosphorylated Akt and FKHR were inhibited by Hirsutanol A, suggesting Hirsutanol A could block Akt signaling pathway.
In order to futher study whether upregulation of ROS resulted in inhibition of Akt signaling pathway. We used Akt kinase assay kit to evalutate the effect of Hirsutanol A on the Akt kinase activity in vitro. Western blot analysis showed that Hirsutanol A could not inhibit phosphorylation of GSK-3a/β. However, NAC can prevent the inhibition of Akt and FKHR phosphorylation induced by Hirsutanol A. These results suggested that Hirsutanol A inhibited Akt signaling pathway not through targeting Akt kinase directly but through upregulation of ROS levels.
③Hirsutanol A induced no-apoptotic cell death in Hep3B cells
We detected the apoptosis in Hep3B cells after treated with 5,10,20μmol/L Hirsutanol A for 48 h using AnnexinV/PI assay.The AnnexinV positive cells were only 0.5%, whereas PI positive cells were up to 5.5%、15.9%%、49.7%respectively. Using immunoblotting analysis, the cleavage of caspase-3 in both cancer cells was not observed following treatment with Hirsutanol A. The cleavage of PARP was also undetectable. These data suggested that Hirsutanol A induced no-apoptotic cell death in Hep3B cells.
④Hirsutanol A induced autophagic cell death in Hep3B cells
In order to certify that Hirsutanol A induced autophagic cell death in Hep3B cells, we used the autophagy inhibitor 3-MA or Beclin-siRNA to inhibit Hirsutanol A-induced autophagy in Hep3B cells. Compared with control cells, cells treated with 3-MA or Beclin-siRNA reduced cell death induced by Hirsutanol A, indicating that Hirsutanol A induced autophagic cell death in Hep3B cells.
⑤Upregulation of ROS and inhibition of AKT pathway involved in autophagic cell death induced by Hirsutanol A
To investigate whether autophagic cell death induced by Hirsutanol A depended on increase of ROS in Hep3B cells, NAC was used to inhibit the ROS upregulation. The results showed that NAC reduced the cell death induced by Hirsutanol A. Furthermore, we established Hep3b/myr-Aktl cell line which stably expressed constitutively activated Akt1. Expressing constitutively active Akt increased growth inhibition in Hep3B cells after treatment with Hirsutanol A for 72h.These data together clarified that Hirsutanol A induced autophagic cell death via increase of ROS and inhibition of Akt signaling pathway.
⑥Autophagy protected cells from death induced by Hirsutanol A
To validate the contribution of autophagy in cell death induced by Hirsutanol A, we used autophagy inhibitor Bafilomycin A1 or Atg-7-siRNA to block autophagy induced by Hirsutanol A in MCF-7 cells. Blocking autophagy could remarkedly increase cell death induced by Hirsutanol A. These evidences suggested that Hirsutanol A-treated autophagy protected cells from death.
3 Effect of Hirsutanol A on tumor growth in mice bearing human colon cancer cells SW620
To detect antitumor activity of Hirsutanol A in vivo, we established SW620 xenografts. The results showed that Hirsutanol A at 20 mg/kg/d potently inhibited tumor growth and the tumor growth inhibition(T/C) was about 31.5%.
Conclusions
1 Hirsutanol A induced apoptosis via change of mitochondrial membrane potential and releasing of cytochrome c from mitochondria to cytosol modulated by increasing ROS production in SW620 and MDA-MB-231 cells.
2 Hirsutanol A induces autophagical programmed cell death through blockage of Akt signaling pathway and upregulation of ROS in Hep3B cells.
3 Hirsutanol A induces both autophagy and apoptosis in MCF-7 cells. Autophagy induced by Hirsutanol A protected MCF-7 cells from apoptosis.
4 Accumulation of ROS induced by Hirsutanol A resulted in activation of JNK signaling pathway. Blockage of JNK signaling pathway promoted apoptosis
5 Hirsutanol A significantly inhibited tumor growth of xenografts in nude mice.
癌症具有较高的发病率和死亡率,严重危害着人类的健康和生命。每年全世界约有700万人死于癌症,并且近几年癌症的死亡率呈明显的上升趋势。化疗是治疗肿瘤的主要手段之一,因此,寻找和开发高效低毒的抗肿瘤药物仍然是攻克肿瘤的主要途径。近年来,根据正常细胞与肿瘤细胞基因表达的不同,有目的地针对肿瘤特异性癌基因的靶向治疗已取得一定的进展,但是由于基因的不稳定性和耐药性问题,基因靶向治疗仍然面临很大的挑战。因此,针对肿瘤发生和发展的原因,寻找新的肿瘤治疗靶点已成为抗肿瘤药物研究开发的重点。近年来,针对肿瘤细胞特异生化环境的改变成为抗肿瘤药物研究的新靶点。
活性氧(ROS)是一类化学性质活泼的含氧功能基团物质的统称。它包括过氧化氢(H202)、羟基自由基(·OH)、超氧阴离子自由基(O2·)、单线态氧、臭氧(03)、氮氧化物(NOx)、次氯酸(HClO)等。在生理条件下ROS的产生对于细胞的生长是必需的,生理条件下的ROS可以调控细胞的增殖和分化,但是异常升高的ROS则可以促进肿瘤形成。由于肿瘤细胞长期处于ROS高水平的状态,肿瘤细胞的氧化还原体系相对正常细胞更为脆弱,对ROS的调控能力更低,当用ROS诱导剂诱导ROS升高可以选择性的杀死癌细胞而对正常细胞没有影响。细胞的氧化还原体系被认为是抗肿瘤药物研究的新靶点。
随着陆地药用生物资源开发范围的日益缩小,人们纷纷将目光投向海洋。海洋生物因生存环境的特殊性(高压、低氧、高盐等),往往含有大量结构新颖奇特、生理活性显著的代谢产物。因此,从海洋中寻求高效低毒的天然药物已引起国际上越来越浓厚的兴趣。Hirsutanol A是从南海珊瑚Sarcophyton tortuosum的内生菌Chondrostereum sp中分离得到的一个倍半萜类化合物,该化合物是从一个新的菌种中分离得到,且该化合物的化学结构仅报道过一次,药理活性未见报道。本研究对从海洋内生菌中分离出来的六个单体化合物进行细胞毒作用的筛选,发现一个倍半萜类化合物Hirsutanol A具有较强的细胞毒作用,于是我们对其抗肿瘤作用及其机制分别从体内和体外进行探讨。
二研究方法
MTT试验筛选具有抗肿瘤活性的化合物及检测Hirsutanol A对不同肿瘤细胞株的生长抑制作用。DHE. CM-H2DCF-DA染色流式细胞术检测ROS的超氧阴离子和过氧化氢的水平。AnnexinV/PI双染流式细胞术检测Hirsutanol A诱导肿瘤细胞的凋亡率。GFP-LC3转染细胞激光共聚焦检测细胞自噬。免疫印迹方法(Western Blot)检测JNK和Akt信号通路变化,凋亡相关蛋白caspase-3、PARP断裂带和自噬相关蛋白LC3Ⅰ-Ⅱ的转变。Akt激酶活性分析试剂盒检测Hirsutanol A对Akt活性的影响。用线粒体分离试剂盒将线粒体和胞浆分离,免疫印迹方法检测线粒体和胞浆中细胞色素C的表达。应用强抗氧化剂NAC检测ROS抑制对Hirsutanol A诱导的细胞增殖抑制和凋亡以及JNK,Akt磷酸化水平的影响。应用特异性小分子抑制剂SP600125和siRNA瞬时转染方法检测JNK信号通路抑制对于Hirsutanol A诱导的凋亡和ROS水平的影响。应用小分子抑制剂3-MA、Bafilomycin Al及siRNA瞬时转染方法抑制自噬,检测自噬在Hirsutanol A诱导的细胞死亡中的作用。用稳定转染的方法构建Hep3B/myr-Aktl, Hep3B-vector细胞株,MTT法检测Hirsutanol A对Hep3B/myr-Aktl, Hep3B-vector的生长抑制作用。裸鼠成瘤实验观察HirsutanolA对SW620裸鼠移植瘤生长的抑制作用。
三研究结果
1活性化合物的筛选及Hirsutanol A对不同肿瘤细胞株的细胞毒作用
我们对6种化合物进行了筛选,发现Hirsutanol A对多种肿瘤细胞均具有明显的细胞毒作用,并呈现明显的时间-剂量效应关系。
2 Hirsutanol A抗肿瘤作用分子机理
(1) Hirsutanol A对肿瘤细胞ROS水平的影响
我们分别用DHE和CM-H2DCF-DA染色检测Hirsutanol A对肿瘤细胞超氧阴离子和过氧化氢水平的影响,结果显示Hirsutanol A对超氧阴离子水平没影响但能显著增加肿瘤细胞过氧化氢水平,并呈现明显的时间-剂量效应关系。
(2) Hirsutanol A上调ROS诱导肿瘤细胞发生凋亡
①Hirsutanol A诱导SW620、MDA-MB-231、MCF-7肿瘤细胞发生凋亡
AnnexinV/PI双染色法检测Hirsutanol A诱导S W620、MDA-MB-231、MCF-7细胞发生凋亡的情况。结果显示Hirsutanol A能明显诱导SW620、MDA-MB-231、MCF-7细胞发生凋亡,其凋亡率分别为：SW620(2.8%、12.1%、45.0%、65.6%)、MDA-MB-231 (2.6%、10%、22.7%、30.6%)、MCF-7 (1.7%、10%、20、8%、31.3%)且呈明显的剂量依赖关系；Western blot法检测caspase-3、PARP的剪切片段,在17KD和85KD可见明显的断裂带。以上结果表明Hirsutanol A能诱导SW620、MDA-MB-231、MCF-7细胞发生凋亡。
②Hirsutanol A通过线粒体途径诱导凋亡
为了探讨Hirsutanol A能否通过线粒体途径诱导凋亡,我们分别检测了SW620、MDA-MB-231细胞线粒体膜电位改变以及线粒体和胞浆中细胞色素C的表达。结果显示：SW620、MDA-MB-231细胞线粒体膜电位均呈浓度依赖性升高。用Hirsutanol A处理细胞24h后,线粒体中细胞色素C的表达量明显减少而胞浆中细胞色素C的表达量明显增多。以上结果表明Hirsutanol A通过线粒体途径诱导凋亡。
③Hirsutanol A诱导凋亡的发生与ROS相关
我们在前面的研究中发现Hirsutanol A对多种肿瘤细胞均有增殖抑制作用,而且可以显著上调SW620、MDA-MB-231、MCF-7细胞的ROS水平,为了证明Hirsutanol A通过上调ROS而发挥抗肿瘤的作用。我们用强抗氧化剂NAC抑制Hirsutanol A对ROS的上调作用后,检测Hirsutanol A对细胞的增殖抑制作用和细胞凋亡的影响。结果显示NAC可明显减少Hirsutanol A诱导的增殖抑制作用和Hirsutanol A诱导的细胞凋亡。
④Hirsutanol A通过上调ROS激活JNK通路
研究发现ROS可以调控JNK、Akt、NF-κB等多条信号通路,接下来我们进一步探讨Hirsutanol A激活ROS后对JNK通路的影响。用HirsutanolA处理SW620细胞24 h, Western blot检测JNK和c-Jun蛋白磷酸化水平,可见JNK和c-Jun蛋白磷酸化水平明显升高。用ROS抑制剂NAC预处理细胞后,NAC可以抑制Hirsutanol A对JNK的激活作用。说明Hirsutanol A激活JNK通路的作用与ROS上调相关。
⑤JNK通路激活的作用
用JNK抑制剂SP600125抑制JNK通路后,分别用MTT检测细胞的活性和AnnexinV/PI双染色法检测细胞的凋亡,可见SP600125可明显增加Hirsutanol A诱导的细胞死亡；单用Hirsutanol A细胞的凋亡率为35.6%,用SP600125阻断JNK通路后,Hirsutanol A诱导的细胞凋亡率上升到48.3%,阻断JNK通路可以增加Hirsutanol A诱导的细胞凋亡。表明JNK通路的激活不仅不能诱导细胞发生凋亡,还能保护细胞免受死亡。
接下来我们进一步探讨了JNK通路激活对ROS的调控作用,用JNK抑制剂SP600125预处理细胞或siRNA阻断JNK通路后,流式细胞仪检测ROS的水平,结果显示阻断JNK通路能显著增加Hirsutanol A诱导的ROS水平。说明JNK通路的激活并不诱导细胞凋亡而是负反馈的调控ROS水平,是细胞的一种氧化应激保护机制。
(3) Hirsutanol A上调ROS诱导肿瘤细胞发生自噬
①Hirsutanol A通过上调ROS诱导肿瘤细胞发生自噬
LC3可作为自噬体膜标志性蛋白,我们先用与绿色荧光蛋白(GFP)结合的GFP-LC3来检测自噬,将Hep3B转染GFP-LC3质粒24 h后,用20μmol/L HirsutanolA处理24h,于激光共聚焦显微镜下观察,可见Hirsutanol A处理组出现大量LC3点状聚集,而对照组LC3呈弥散分布。接下来我们进一步用Western blot检测LC3蛋白表达,可见LC3由Ⅰ型向Ⅱ型转化增加,提示Hirsutanol A可以诱导肿瘤细胞发生自噬。
Hirsutanol A能够上调ROS水平和诱导自噬,接下来我们进一步探讨HirsutanolA上调ROS水平和诱导自噬之间的相关性。分别用Western blot检测LC3蛋白表达和激光共聚焦检测LC3点状聚集,结果显示加入NAC预处理细胞后,Hirsutanol A诱导的LC3由Ⅰ型向Ⅱ型转化明显减少,LC3点状聚集亦明显减少。以上结果表明Hirsutanol A通过上调ROS诱导肿瘤细胞发生自噬。
②Hirsutanol A通过上调ROS抑制Akt通路
Akt/mTOR是调控自噬的一条主要通路,我们在前面的研究中发现HirsutanolA可以诱导肿瘤细胞产生ROS,并诱导肿瘤细胞发生自噬。于是我们进一步探讨Hirsutanol A对Akt通路的影响。用Hirsutanol A处理细胞24 h后,Western blot检测Akt、P-Akt473和P-FKHR蛋白表达水平,发现Akt表达水平不变但P-Akt473和P-FKHR表达水平明显降低,说明Hirsutanol A可以抑制Akt通路。
接下来我们进一步探讨Hirsutanol A对Akt通路抑制作用与ROS上调的相关性,先用Akt激酶活性分析法检测Hirsutanol A在体外对Akt激酶活性的影响,结果显示Hirsutanol A在体外不能直接抑制Akt激酶的活性。用NAC预处理Hep3B细胞1h然后加入Hirsutanol A处理24 h, Western blot检测P-Akt473蛋白表达水平,可见NAC可部分逆转P-Akt473的表达水平。说明AKT通路抑制与ROS上调相关。
③Hirsutanol A诱导Hep3B细胞发生非凋亡性死亡
我们用AnnexinV/PI双染法检测细胞凋亡时发现,用5、10、20μmol/L HirsutanolA处理Hep3B细胞48 h后,AnnexinV单阳性率最高仅为0.5%,而PI阳性率分别为5.5%、15.9%、49.7%。Western blot检测Caspase-3, PARP表达,结果显示在17KD和85KD均未见明显的断裂带。该结果表明Hirsutanol A诱导Hep3B细胞发生非凋亡性死亡。
④Hirsutanol A诱导Hep3B细胞发生自噬性死亡
为了证明Hirsutanol A诱导Hep3B细胞发生自噬性死亡,我们分别用自噬抑制剂3-MA和siRNA-Beclinl阻断自噬。MTT法检测阻断自噬后Hirsutanol A对细胞生长抑制和死亡的影响。结果显示,阻断自噬可以明显降低Hirsutanol A诱导的细胞死亡。
⑤Hirsutanol A诱导Hep3B细胞自噬性死亡与ROS上调和Akt通路抑制相关
为了验证Hirsutanol A诱导Hep3B细胞自噬性死亡与ROS上调的关系,我们用1mmol/L的NAC预处理细胞1h,然后加入Hirsutanol A处理72 h,分别用MTT法和AnnexinV/PI双染法检测细胞增殖抑制和死亡情况,结果显示NAC可明显减少Hirsutanol A诱导的细胞增殖抑制和死亡。我们还构建了Hep3B/myr-Aktl, Hep3B-vector细胞株,检测Hirsutanol A诱导的细胞死亡与Akt通路之间的关系,结果显示Hirsutanol A对Hep3B/myr-Akt1细胞的生长抑制作用明显高于Hep3B-vector细胞。说明Hirsutanol A诱导Hep3B细胞自噬性死亡与ROS上调和Akt通路抑制相关。
⑥抑制自噬可以促进Hirsutanol A诱导的MCF-7细胞死亡
我们在研究中发现Hirsutanol A既可以诱导肿瘤细胞MCF-7发生凋亡也可以诱导发生自噬,那么Hirsutanol A诱导的自噬是起到保护作用还是促进细胞死亡的作用呢?我们分别用自噬抑制剂Bafilomycin Al和siRNA-Atg-7抑制自噬,MTT法检测细胞的死亡。结果显示,阻断自噬可以明显的增加细胞的死亡,说明Hirsutanol A诱导的自噬在MCF-7细胞中起保护细胞免受死亡的作用。
3 Hirsutanol A对裸鼠移植瘤的抑制作用
20 mg／kg剂量的Hirsutanol A能明显抑制裸鼠移植瘤的生长,抑瘤率31.5%而5 mg／kg的治疗剂量对瘤组织体积的增长无明显抑制作用。
四结论
1 Hirsutanol A通过上调ROS,引起线粒体损伤,细胞色素C释放从而诱导肿瘤细胞SW620、MDA-MB-231发生凋亡。
2 Hirsutanol A通过上调ROS，抑制Akt通路诱导肿瘤细胞Hep3B发生自噬性死亡。
3 Hirsutanol A既可诱导肿瘤细胞MCF-7发生凋亡也可诱导其发生自噬,自噬在MCF-7细胞中起到保护作用。
4 Hirsutanol A通过上调ROS水平激活JNK通路,JNK通路激活并不诱导凋亡而是负反馈调控ROS水平。
5 Hirsutanol A明显抑制裸鼠结肠癌SW620移植瘤的生长。
Background and objective
Cancer has become an increasing public health problem because of its high rates of morbidity and mortality. About 7 million people die from cancer each year all over the world. Recently, cancer significantly showed an increasing trend in mortality. Since chemotherapy is still one of the key therapeutic strategies against cancer, developing the novel anticancer drugs is implicated in cancer therapy. By exploiting the genetic differences between normal cells and cancer cells, targeting therapeutic strategies have acquired progress. However, it still faces significant challenges owing to acquired drug resistance and the genomic instability of cancer cells. Therefore, depending on the events involved in carcinogenesis and cancer progression, it is important to exploit new therapeutic target of anticancer drugs. Recently, targeting unique biochemical alternation has got extensively attentions as a new target of anticancer drugs
Reactive oxygen species(ROS) are a collective term of oxygen derived species, including hydrogen peroxide H2O2, hydroxyl radicals OH, superoxide anion radical O2, singlet oxygen, ozone, hypochloric acid etc. A basic level of intracellular ROS is essential for regulating cell proliferation and differentiation, whereas excessive amounts of ROS can contribute to the carcinogenesis and cancer progression. Compared to normal cells, cancer cells with increased intrinsic ROS are more vulnerable to damage by further ROS insults and more difficult to scavenge the increasing ROS induced by exogenous agents. Therefore, induction of ROS levels by redox modulation is a way to selectively kill cancer cells without significant toxicity to normal cells. So the redox system would be a new target of anticancer drugs.
As the biologic resources in the land are decreasing, researchers focus on marine resources. Since the particular living environments (high pressure, hypoxia and low salty), thousands of peculiar metabolites with physiological activities are found in marine lives, and have evoked great interests to find some natural drugs with high efficacy and low toxicity. Hirsutanol A is a novel sesquiterpene compound purified from Sarcophyton tortuosum. The pharmacologic effect of this compound is still unkown, though the chemcial structure has been reported. We found that Hirsutanol A showed potent cytotoxic effect via crude screen assay from six natural compounds extracted from Fungus Chondrostereum sp in Sarcophyton tortuosum. In this study we investigated the anticancer activity of Hirsutanol A and its mechanism.
Methods
MTT assay was used to screen compounds and evaluate the growth inhibition of various cancer cell lines induced by Hirsutanol A. Cells were stained with DHE or CM-H2DCF-DA to detect cellular superoxide or hydrogen peroxide level measured by flow cytometry. Annexin V-PI staining was used to assess the apoptosis. The transiently transfected GFP-LC3 cells were established by lipofectamine 2000 transfection. The punctuate dots of YFP-LC3 were detected by confocal microscopy. Western blot analysis was used to detect the expression of JNK and Akt signaling pathway -related proteins, cleavage fraction of caspase-3 and PARP, and conversion of LC3 I to LC3-II. Akt kinase activity assay in cell-free system was used to evaluate effect of Hirsutanol A on Akt kinase activity in vitro. Isolation of Mitochondrial and cytosol fraction was obtained by using Mitochondria isolation kit, then expression of cytochrome c in Mitochondria or cytosol was determined by Western blot analysis.SP600125,a small-molecule JNK inhibitor, and specific siRNA against JNK were used to block JNK activity or expression.3-MA and Bafilomycin A1 or siRNA against Beclinl and siRNA against Atg7 were employed to inhibit autophagy. Hep3B cells were stably transinfected with myr-Aktl plasmid to establish Hep3B/myr-Aktl which stably expressed constitutively activated Aktl. MTT assay was used to analysis the growth inhibition of Hep3B/myr-Aktl and Hep3B-vector cells induced by Hirsutanol A.Tumor xenografts were established by SW620 cells injected s.c into nude mice, the growth inhibitory effect was finally calculated after Hirsutanol A treatment.
Results
1 Screening compounds with cytotoxic activity and investigating the cytotoxic effect of Hirsutanol A on various cancer cell lines.
To explore compounds with anticancer activity,we detected the cytotoxic effect of six compounds by MTT assay, the results showed that Hirsutanol A exhibits potent cytotoxic effect on various human cancer cell lines in a dose-and time-dependent manner.
2 The anticancer activity of Hirsutanol A and its molecular mechanisms.
(1) Effect of Hirsutanol A on cellular ROS level in cancer cells:
To investigate the Effect of Hirsutanol A on cellular superoxide and hydrogen peroxide level in cancer cells, we analyzed the level of superoxide and hydrogen peroxide in cancer cells using flow cytometry. There was no significant change in DHE fluorescence but markedly increase in DCF-DA fluorescence after treatment with Hirsutanol A in a dose-and time-dependent fashion, suggesting that ROS induced by Hirsutanol A were mainly hydrogen peroxide but not superoxide.
(2) Hirsutanol A induces apoptosis via a ROS-mediated mechanism
①Hirsutanol A induces apoptosis in SW620, MDA-MB-231, MCF-7 cancer cells
To confirm that Hirsutanol A induced apoptosis, SW620,MDA-MB-231 and MCF-7 cells exposed to various concentrations of Hirsutanol A for 72h. The cells were analyzed by Annexin V-FITC/PI staining assay. The ratio of AnnexinV-positive cells was 2.8%、12.1%、45.0%、65.6% in SW620,2.6%、10%、22.7%、30.6% in MDA-MB-231, and 1.7%、10%、20.8%、31.3% in MCF-7. The results suggested that Hirsutanol A could induce apoptosis in a dose-dependent manner. Using immuno-blotting analysis, the cleavage of caspase-3 and PARP in both cancer cells was detected following treatment with Hirsutanol A. The results showed that pro-caspase-3 was cleaved to yield a 17KDa fragmentation and PARP was cleaved into 89KD fragmentation. These results revealed that Hirsutanol A remarkably induced apoptosis in SW620, MDA-MB-231 and MCF-7 cells.
②Hirsutanol A induced apoptosis through activation of mitochondria/ cytochrome c signaling pathway
To study whether Hirsutanol A induced apoptosis via activation of mitochondria/ cytochrome c signaliing pathway, we examined the change of mitochondrial membrane potential and the release of cytochrome c from mitochondria. mitochondrial membrane potential was elevated after treatment with various concentrations of Hirsutanol A. The expression of cytochrome c in mitochondrial was downregulated, whereas that in cytosol was upregulated after treatment with Hirsutanol A for 24h. These data revealed that Hirsutanol A induced apoptosis through activation of mitochondria/cytochrome c signaling pathway.
③Induction of apoptosis by Hirsutanol A associated with increased ROS generation
The evidences of apoptosis and upregulation of ROS levels by Hirsutanol A prompted us to investigate whether upregulation of ROS resulted in apoptosis. The increase of ROS levels induced by Hirsutanol A in cancer cells was prevented by preincubation with NAC, then growth inhibition and AnnexinV-positive cells were analyzed using MTT and AnnexinV/PI assay. The results showed that the induction of apotosis by Hirsutanol A was blocked when the cells were pretreated with NAC.
④Hirsutanol A activated JNK signaling pathway through upregulation of ROS
It has been reported that ROS can modulate several signaling pathways including JNK, Akt, NF-κB and so on. Therefore, we explored the effect of increasing ROS induced by Hirsutanol A on JNK signaling pathway. JNK and c-Jun phosphorylation were significantly activated in SW620 cells after treatment with Hirsutanol A for 24 h. Moreover, NAC prevented the activation of JNK induced by Hirsutanol A. It suggested that JNK may be a downstream target of increased ROS following Hirsutanol A treatment.
⑤The role of JNK signaling pathway in Hirsutanol A induced ROS increase
In order to explore the contribution of JNK signaling pathway to Hirsutanol A induced ROS, JNK signaling pathway was blocked using the small-molecule JNK inhibitor SP600125. Treated with Hirsutanol A only, the ratio of AnnexinV-positive cells was 35.6%, whereas in parallel treatment in combination with SP600125, the ratio of AnnexinV-positive cells was up to 48.3%, suggesting that blocking JNK signaling pathway promoted apoptosis induced by Hirsutanol A.
We further investigated the effect of activation of JNK signaling pathway on cellular ROS levels. Cellular ROS levels were remarkly increased in SW620 cells by SP600125 or JNK-siRNA to block JNK signaling pathway.These results suggest that activation of JNK is an oxidant stress which protect cell from death via regulation of ROS in a negative feedback manner but not a classic mechanism involved in apoptosis.
(3) Hirsutanol A induced autophagy through increasing ROS production
①Hirsutanol A induced autophagy via increase of ROS level
The lipidation of the protein LC3 during the process of autophagy can be used as a marker. In order to detect the autophagy induced by Hirsutanol A, we observed GFP-tagged LC3 puncta distribution in Hep3B cells. Hep3B cells showed diffuse distribuction of GFP-LC3 in the absence of Hirsutanol A, whereas Hirsutanol A-treated cells showed a marked increase in number and frequency of GFP-LC3 dots.To futher validate autophagy induced by Hirsutanol A, we measured the modification of LC3-Ⅰto LC3-Ⅱby Western blot. LC3 I to LC3-II conversion was remarkedly increased in a dose-and time-dependent manner indicating induction of autophagy by Hirsutanol A.
Since Hirsutanol A can increase cellular ROS levels and induce autophagy, we investigated the relationship between autophagy and upregulation of ROS induced by Hirsutaanol A. The increase of ROS level induced by Hirsutanol A was prevented by preincubation with NAC in Hep3B cells. NAC inhibited the LC3 I to LC3-II conversion and reduced the number of GFP-LC3 dots. These results demonstrated that activation of autophagy depended on the upregulation of ROS levels.
②Hirsutanol A inhibited Akt signaling pathway via increase of ROS levels
The above observations suggested that Hirsutanol A can induce autophagy and accumulation of cellular ROS. Since Akt/mTOR is a key modulator for autophagy, we futher detected the effect of Hirsutanol A on Akt signaling pathway. The results showed that the phosphorylated Akt and FKHR were inhibited by Hirsutanol A, suggesting Hirsutanol A could block Akt signaling pathway.
In order to futher study whether upregulation of ROS resulted in inhibition of Akt signaling pathway. We used Akt kinase assay kit to evalutate the effect of Hirsutanol A on the Akt kinase activity in vitro. Western blot analysis showed that Hirsutanol A could not inhibit phosphorylation of GSK-3a/β. However, NAC can prevent the inhibition of Akt and FKHR phosphorylation induced by Hirsutanol A. These results suggested that Hirsutanol A inhibited Akt signaling pathway not through targeting Akt kinase directly but through upregulation of ROS levels.
③Hirsutanol A induced no-apoptotic cell death in Hep3B cells
We detected the apoptosis in Hep3B cells after treated with 5,10,20μmol/L Hirsutanol A for 48 h using AnnexinV/PI assay.The AnnexinV positive cells were only 0.5%, whereas PI positive cells were up to 5.5%、15.9%%、49.7%respectively. Using immunoblotting analysis, the cleavage of caspase-3 in both cancer cells was not observed following treatment with Hirsutanol A. The cleavage of PARP was also undetectable. These data suggested that Hirsutanol A induced no-apoptotic cell death in Hep3B cells.
④Hirsutanol A induced autophagic cell death in Hep3B cells
In order to certify that Hirsutanol A induced autophagic cell death in Hep3B cells, we used the autophagy inhibitor 3-MA or Beclin-siRNA to inhibit Hirsutanol A-induced autophagy in Hep3B cells. Compared with control cells, cells treated with 3-MA or Beclin-siRNA reduced cell death induced by Hirsutanol A, indicating that Hirsutanol A induced autophagic cell death in Hep3B cells.
⑤Upregulation of ROS and inhibition of AKT pathway involved in autophagic cell death induced by Hirsutanol A
To investigate whether autophagic cell death induced by Hirsutanol A depended on increase of ROS in Hep3B cells, NAC was used to inhibit the ROS upregulation. The results showed that NAC reduced the cell death induced by Hirsutanol A. Furthermore, we established Hep3b/myr-Aktl cell line which stably expressed constitutively activated Akt1. Expressing constitutively active Akt increased growth inhibition in Hep3B cells after treatment with Hirsutanol A for 72h.These data together clarified that Hirsutanol A induced autophagic cell death via increase of ROS and inhibition of Akt signaling pathway.
⑥Autophagy protected cells from death induced by Hirsutanol A
To validate the contribution of autophagy in cell death induced by Hirsutanol A, we used autophagy inhibitor Bafilomycin A1 or Atg-7-siRNA to block autophagy induced by Hirsutanol A in MCF-7 cells. Blocking autophagy could remarkedly increase cell death induced by Hirsutanol A. These evidences suggested that Hirsutanol A-treated autophagy protected cells from death.
3 Effect of Hirsutanol A on tumor growth in mice bearing human colon cancer cells SW620
To detect antitumor activity of Hirsutanol A in vivo, we established SW620 xenografts. The results showed that Hirsutanol A at 20 mg/kg/d potently inhibited tumor growth and the tumor growth inhibition(T/C) was about 31.5%.
Conclusions
1 Hirsutanol A induced apoptosis via change of mitochondrial membrane potential and releasing of cytochrome c from mitochondria to cytosol modulated by increasing ROS production in SW620 and MDA-MB-231 cells.
2 Hirsutanol A induces autophagical programmed cell death through blockage of Akt signaling pathway and upregulation of ROS in Hep3B cells.
3 Hirsutanol A induces both autophagy and apoptosis in MCF-7 cells. Autophagy induced by Hirsutanol A protected MCF-7 cells from apoptosis.
4 Accumulation of ROS induced by Hirsutanol A resulted in activation of JNK signaling pathway. Blockage of JNK signaling pathway promoted apoptosis
5 Hirsutanol A significantly inhibited tumor growth of xenografts in nude mice.
引文
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