血根碱抗宫颈癌作用及其机制的研究
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摘要
恶性肿瘤是当前危害人类健康的重要疾病之一，对人类生存构成最严重的威胁。目前较为成熟的治疗方法有手术、放疗、化疗及免疫治疗等。近年来，肿瘤化疗取得了很大的进步，但对危害人类生命健康最严重的、占恶性肿瘤90%以上的实体瘤的治疗效果不尽人意，并且绝大多数抗肿瘤药物在抑制或杀伤肿瘤细胞的同时对正常组织、器官不可避免地产生毒性作用，给患者带来痛苦，甚至导致死亡。据最新癌症研究统计，宫颈癌在世界范围发病率在女性恶性肿瘤中居第二位，仅次于乳腺癌，且近年来宫颈癌的年轻病例数呈逐年增多趋势。因此，开发与研究新型有效的抗宫颈癌药物是生物医药研究领域的重大课题和长期任务。
最近几年，使用中草药来预防和治疗宫颈癌已成为宫颈癌治疗的热点。寻求能够抑制肿瘤生长、引起细胞周期阻滞、诱导细胞凋亡和抑制肿瘤转移的中草药已成为当今宫颈癌研究的新领域。血根碱来源于罂粟科植物博落回的果实，早期的研究发现它具有抗炎、抗菌、抗氧化并对中枢神经有麻醉作用，最近的研究表明血根碱在人类上皮癌、前列腺癌、人黑色素瘤、结肠腺癌、乳腺癌等多种肿瘤中均有抗肿瘤作用，但通过文献检索发现，血根碱在宫颈癌方面的缺乏系统研究，对宫颈癌的作用机制尚不明确，因此本课题旨在研究血根碱抗宫颈癌作用及其机制，为更深入开展血根碱的基础和临床研究提供实验基础和理论依据。本课题的研究内容共分四个部分。
第一部分血根碱抑制宫颈癌细胞增殖、诱导凋亡及其机制的研究
目的：研究血根碱在体外对宫颈癌细胞HeLa和Siha生长、周期阻滞和凋亡的影响，并以正常GM和ECV304细胞作为对照，初步探讨其作用机制。
方法：采用倒置显微镜和活细胞工作站观察不同浓度血根碱作用后HeLa、Siha、GM和ECV304细胞形态的变化；应用MTT和克隆形成法检测不同浓度血根碱对细胞的生存率的影响；通过流式细胞术分析血根碱处理后细胞周期的变化；DNA凝胶电泳、TUNEL法、碱性“彗星”电泳、Annexin V和PI双染法检测、透射电镜等手段定性和定量探讨血根碱诱导宫颈癌细胞凋亡的状况。采用活性氧检测试剂盒和线粒体膜电位检测试剂盒测定血根碱处理后宫颈癌细胞中ROS的改变和线粒体膜电位的变化。最后用Western blot检测血根碱对细胞周期和凋亡相关蛋白表达的变化。
结果：(1)血根碱能够明显抑制宫颈癌HeLa细胞和Siha细胞的生长，并呈时间依赖和浓度依赖关系，且HeLa细胞对血根碱敏感性高于Siha细胞；(2)血根碱可以引起宫颈癌细胞周期G2/M期阻滞，并能明显诱导细胞凋亡，同时促发ROS的生成和引起线粒体膜电位的降低；(3)血根碱明显下调细胞周期调节蛋白CyclinB1、CyclinE和CDC2的表达水平，而上调了p21和p27的表达水平；(4)血根碱处理后，凋亡相关蛋白NF-кB、Bcl-2、Procaspase3、P-Erk、P-P38和P-JNK蛋白表达水平出现明显上调，而Cytochrome C、AIF、Cleaved PARP和P-JNK蛋白表达水平则出现明显下调。
第二部分血根碱体外诱导宫颈癌细胞自噬及其机制的研究
目的：研究血根碱在体外诱导宫颈癌细胞自噬及其可能的机制
方法：采用MTT比色法观察自噬抑制剂3-MA影响血根碱的宫颈癌细胞增殖抑制作用；通过透射电镜观察和MDC染色方法检测血根碱处理后宫颈癌细胞自噬的发生；应用Western blot方法检测自噬相关蛋白LC3和Beclin1表达水平。
结果：血根碱处理导致宫颈癌HeLa和Siha细胞中自噬囊泡明显增多，并呈浓度和时间依赖关系；自噬抑制剂3-MA可以逆转血根碱对宫颈癌细胞生长的抑制作用。血根碱处理使宫颈癌细胞中LC3II和Beclin1的蛋白表达水平出现了明显上调。
第三部分血根碱对体外宫颈癌细胞转移的影响及分子机制研究
目的：研究血根碱对宫颈癌细胞粘附、转移及侵袭的影响，并探讨其作用的初步机制。
方法：通过贴壁实验方法检测血根碱处理后宫颈癌细胞粘附能力的影响。采用划痕实验方法检测血根碱对肿瘤细胞体外迁移能力的影响。通过Boyden小室法检测血根碱对肿瘤细胞转移能力的影响；最后用Western blot方法检测侵袭和转移相关蛋白表达水平的变化。
结果：血根碱可以抑制宫颈癌HeLa和Siha细胞的粘附、迁徙、运动及侵袭转移能力，并呈浓度和时间依赖关系，而且在无毒性的剂量下也明显抑制细胞的侵袭转移。其机制与上调了钙粘附蛋白(E-cadherin)蛋白表达水平，下调α-catenin、β-catenin和γ-catenin的蛋白表达水平有关。另外也发现了基质金属蛋白酶MMP-2和MMP-9和VEFG的蛋白表达水平随着血根碱浓度的增加而下降；抑癌基因PTEN蛋白的表达水平则随着血根碱浓度的增加而增加。
第四部分血根碱体内对宫颈癌的抑制作用及其机制的研究
目的：建立裸鼠的宫颈癌移植瘤模型，观察血根碱在动物体内对宫颈癌生长的抑制作用，并初步探讨其作用机制。
方法：首先将HeLa细胞接种Babl/C裸鼠臀部，建立裸鼠宫颈癌移植瘤模型；按处理分组为：生理盐水对照组、顺铂阳性对照组(5mg/kg，1次/周，iP)、血根碱低浓度组(1.25mg/kg，1次/3天，iP)和血根碱高浓度组(2.5mg/kg，1次/3天，iP)，连续腹腔注射给药治疗28天，随后继续观察28天。期间观察动物一般生命体征的改变和肿瘤体积变化；实验结束时观察实验组和对照组肿瘤周围血管以及肿瘤侵袭转移情况，并取肿瘤进行HE染色观察肿瘤的病理学改变及瘤内和周边血管情况；采用免疫组化法检测肿瘤组织中CD34、Ki67、EGFR和VEGF的表达水平；Western blot方法检测肿瘤组织内Bcl-2、 Bax和VEGF蛋白表达水平；采用透射电镜检测肿瘤组织中细胞超微结构改变。
结果：血根碱在实验剂量下对荷瘤裸鼠无明显毒副作用，血根碱能够明显抑制宫颈癌HeLa细胞移植肿瘤的生长，并呈一定的浓度依赖性，且这种抑制作用与经典化疗药物顺铂的抑瘤疗效相当。其机制可能与血根碱能够抑制肿瘤的血管生成有关，发现在血根碱治疗的肿瘤组织中CD34、Ki67、EGFR和VEGF的表达均出现下调。电镜结果显示血根碱治疗组的肿瘤组织中出现了自噬和凋亡共存现象。血根碱治疗组肿瘤组织抑凋亡蛋白Bcl-2显现下调和促凋亡蛋白Bax则显现上调。
结论本课题分别以宫颈癌HeLa和Siha细胞为体外研究对象和裸鼠宫颈癌移植瘤模型为体内研究对象，分别从细胞，蛋白分子和动物研究水平探讨了血根碱抑制宫颈癌的抗肿瘤作用，研究结论总结如下：
1.血根碱可以抑制宫颈癌HeLa和Siha细胞生长，并呈剂量依赖和时间依赖关系。
2.血根碱可以诱导宫颈癌细胞发生G2/M期周期阻滞，其机制是可能是通过激活NF-κB，继而上调p21和p27表达，从而抑制Cdc2/CyclinB1复合物的活性有关。
3.血根碱可以诱导宫颈癌细胞发生凋亡，其机制是(1)通过上调Bax蛋白表达水平和下调Bcl-2、NF-кB的表达水平；(2)诱导宫颈癌细胞产生ROS，继而引起线粒体膜电位的下降，启动了Cytochrome C→Caspase-9→Caspase-3→PARP通路；(3)激活了MAPK信号通路。
4.血根碱可以诱导宫颈癌细胞发生自噬， LC3和Beclin1蛋白参与了自噬的调控。
5.血根碱可以抑制宫颈癌细胞的侵袭和转移能力，其机制可能包括(1)首先激活上游钙粘附蛋白(E-cadherin)、α-catenin、β-catenin和γ-catenin的表达，通过影响Catenins、E-Cadherin和细胞骨架之间信号传递的作用，继而抑制下游基质金属蛋白酶MMP-2和MMP-9活性抑制其侵袭和转移；(2)抑制VEFG和PTEN的表达从而抑制宫颈癌细胞的侵袭和转移能力。
6.血根碱在体内也能够明显抑制异体移植瘤宫颈癌的生长，其机制可能与抗血管生成、抑制肿瘤细胞增殖、诱导凋亡和自噬有关。
7.与肿瘤细胞的敏感性相比，血根碱对正常细胞的毒性明显要小，而且在明显抑制肿瘤生长的剂量条件下，血根碱对裸鼠未产生明显的毒副作用。这些实验结果表明血根碱对肿瘤细胞有较好的选择性，这为临床试验深入研究提供了非常好的实验基础和理论依据。
Purpose:
The aim of study was to investigate the ant-cancer activities of sanguinarine incervical cancer and its possible mechanisms via a series of in vitro and in vivo studies.
Methods:
In vitro studies, two human cervical cancer cell lines, HeLa and Siha, with thefollowing methods were employed, in some cases, normal cell lines GM and ECV304were used as control:1) Cell morphology was observed by inverted microscope and livecell imaging system;2) Cell viability was examined by MTT assay;3) Colonyformation assay was also used to study the cell growth inhibition of sanguinarine;4)Scratch healing assay and the trans-well assay were employed to study the influence ofsanguinarine on the cell migration and invasion;5) Flow cytometry was applied toanalyze cell cycle progression and apoptosis induction;6) Single cell gel electrophoresis(comet assay) was used to detect DNA damage;7) Apoptosis and necrosis weremeasured by TUNEL and Annexin V-FITC cell apoptosis kits;8) ROS content andmitochondrial membrane potential was determined by commercial kits;9) Alteration ofProtein expression level was detected by Western blot assay;10) Autophagy wasexamined by MDC (monodansylcadaverin) staining;11) Autophagy and apoptosisultrastructure changes of cells were observed by transmission electron microscope(TEM).
In vivo studies, a transplanted tumor model by injecting HeLa cells intosubcutaneous tissue of Babl/c mice was established. Mice were randomly assigned into4groups by intraperitoneal injection:1) saline solution as negative control;2)5mg/kgcisplatin as positive control (1/week);3)1.25mg/kg injection of sanguinarine (1/3days)and4)2.5mg/kg injection of sanguinarine (1/3days). The period time of therapies was28days, the tumor volume was measured once every other day upto another28days following sanguinarine treatment. At the end of experiments, tumors were carefullyremoved and weighted. Microvascular density in tumor tissues was measured by aninverted microscope and CD34immunostaining. The expression levels of Bcl-2, Baxand VEGF tumor tissues were detected by Western blot and the expression levels ofVEGF, EGFR and Ki67, by immunohistochemical assay, in tumor tissues. Changes ofcell ultrastructure related to autophagy and apoptosis in tumors were observed by TEM.
Results:
1) A dose-and time-dependent inhibition of cell proliferation by sanguinarine wasobserved in both cell lines. An IC50value for24h treatment of sanguinarine in HeLa andSiha cells was2.62±0.21μmol/L and3.07±0.23μmol/L, respectively, indicatingslightly more sensitivity in HeLa cells (P <0.05). While, sanguinarine showed lesstoxicity in normal GM and ECV304cells with an IC50value of4.93±0.22μmol/L and5.78±0.18μmol/L, respectively.
2) After treatment of0,1,2and3μmol/L sanguinarine for24h, the proportion ofG2/M phase in HeLa cells was (25.21±0.66)%,(29.97±0.84)%,(37.31±0.93)%and(57.72±0.13)%, respectively. Similar a dose-dependent arrest of G2/M phase wasobserved in Siha cells after sanguinarine treatment. Consistant with cell cycle arrest,sanguinarine caused a significant down-regulation of CyclinB1, CyclinE and CDC2protein expression and an up-regulation of P21and P27protein expression.
3) With24h treatment of sanguinarine, HeLa and Siha cells showed typicalapoptotic morphologic changes, including chromatin condensation and DNA fragment.Comet assay evidenced that sanguinarine increased the mean tail length and thepercentage of cells with comet tails. Sanguinarine decreased Bcl-2expression andincreased Bax expression via a dose-response manner, and consequently the ratio of Baxto Bcl-2was significantly increased (P<0.05). The expression of procaspase-3wasdecreased and AIF and Cleaved PARP were increased in HeLa and Siha cells treatedwith sanguinarine (P<0.05). The ROS content in HeLa cells treated with1μmol/Lsanguinarine markedly increased in0.5h (29.63±1.28)%compared with0h (38.4±2.90)%and reached peak in4h (58.37±6.03)%(P<0.01) following exposure tosanguinarine. Similar results were observed for ROS generation in Siha cells. On theother hand, mitochondrial transmembrane potential was decreased and the expression ofcytochrome c protein was increased in both of cell lines after sanguinarine treatment.
4) As for migration and invasion, sanguinarine significantly decreased theadherence rate and inhibited the ability to cover “cell wound” by scratch in both of celllines via a time-dependent fashion. Tran-well assay showed that HeLa cell number aftertreatment with sanguinarine at0,0.5and0.75mol/L was156±9.85,108±13.23and74.67±7.02, respectively (P <0.01). Similar observation was obtained in Siha cells withsanguinarine treatment. Additionally, a significant dose-dependent alteration of someproteins which are related to cell migration and invasion was found in both of cell linestreated with sanguinarine, i.e., a decrease level of VEGF, EGFR, PTEN, NF-κB,-catenin,-catenin and γ-catenin expression and an increased expression of E-cadherinprotein (P<0.05).
5) TEM observation indicated that autophagosomes and apoptotic bodies inducedby sanguinarine in both of cell lines. However, sanguinarine-induced autophagy wasreversed by emplying a specific autophagy inhibitor,3-methylademine (3-MA). Aquantitative method of MDC staining releaved that the cell number of autophagy was149±22.65and412.67±16.80in HeLa cells as well as138±17.47and371±20.84inSiha cells after4h treatment with1and2mol/L sanguinarine (P<0.01). Sanguinarinecaused a reduced expression of Beclin1protein. In addition, a decrease of LC3-I proteincomparing with an increase of LC3-II protein were detectable after treatment withsanguinarine.
6) A significant inhibition of tumor growth and volume in transplanted tumor micewere observed after therapy with sanguinarine via an intraperitoneal injection. At theend of experiment, the inhibition rate of tumor growth was29.40%,39.96%and45.16%in cisplatin,1.25mg/kg and2.5mg/kg sanguinarine, compared with that inun-treated control mice. Microvascular density in tumor tissues as evidenced by CD34immunostaining was77.33±7.04,25.00±4.90,28.33±1.70and15.67±2.05inun-treated controls, cisplatin,1.25mg/kg and2.5mg/kg sanguinarine, respectively(P<0.01). TEM observation also showed a significant change of apoptosis andautophagy in tumor tissues after therapy with sanguinarine. On the other hand,sanguinarine inhibited tumor angiogenesis possibly via down-regulating the expressionof VEGF, EGFR and NF-κB.
Conclusion:
In summary, a significant inhibitory proliferation in both of human cervical cancer cell lines, HeLa and Siha, was observed after treatment with sanguinarine in a dose-andtime-dependent manner. Sanguinarine-caused arrest of G2/M phase was accompaniedwith a down-regulation of CyclinB1, CyclinE, and CDC2protein and a up-regulation ofp21and p27expression. The occurrence of apoptosis and autophagy after sanguinarinetreatment was confirmed by a few methods, the underlying mechanisms may beinvolved in an increase of Bax/Bcl-2, an alteration of LC3II/LC3I expression, anactivation of caspase-3and MAPK pathway. Anti-cancer growth in vivo bysanguinarine may be related to an inhibition of tumor angiogenesis and an induction ofapoptosis and autophagy by down-regulating the expression of VEGF, EGFR andNF-κB. These novel findings indicate that sanguinarine inhibites cervical cancer growthin vitro and in vivo by multimechanisms, which suggesting that sanguinarine may serveas a potential therapeutic agent for cervical cancer.
最近几年，使用中草药来预防和治疗宫颈癌已成为宫颈癌治疗的热点。寻求能够抑制肿瘤生长、引起细胞周期阻滞、诱导细胞凋亡和抑制肿瘤转移的中草药已成为当今宫颈癌研究的新领域。血根碱来源于罂粟科植物博落回的果实，早期的研究发现它具有抗炎、抗菌、抗氧化并对中枢神经有麻醉作用，最近的研究表明血根碱在人类上皮癌、前列腺癌、人黑色素瘤、结肠腺癌、乳腺癌等多种肿瘤中均有抗肿瘤作用，但通过文献检索发现，血根碱在宫颈癌方面的缺乏系统研究，对宫颈癌的作用机制尚不明确，因此本课题旨在研究血根碱抗宫颈癌作用及其机制，为更深入开展血根碱的基础和临床研究提供实验基础和理论依据。本课题的研究内容共分四个部分。
第一部分血根碱抑制宫颈癌细胞增殖、诱导凋亡及其机制的研究
目的：研究血根碱在体外对宫颈癌细胞HeLa和Siha生长、周期阻滞和凋亡的影响，并以正常GM和ECV304细胞作为对照，初步探讨其作用机制。
方法：采用倒置显微镜和活细胞工作站观察不同浓度血根碱作用后HeLa、Siha、GM和ECV304细胞形态的变化；应用MTT和克隆形成法检测不同浓度血根碱对细胞的生存率的影响；通过流式细胞术分析血根碱处理后细胞周期的变化；DNA凝胶电泳、TUNEL法、碱性“彗星”电泳、Annexin V和PI双染法检测、透射电镜等手段定性和定量探讨血根碱诱导宫颈癌细胞凋亡的状况。采用活性氧检测试剂盒和线粒体膜电位检测试剂盒测定血根碱处理后宫颈癌细胞中ROS的改变和线粒体膜电位的变化。最后用Western blot检测血根碱对细胞周期和凋亡相关蛋白表达的变化。
结果：(1)血根碱能够明显抑制宫颈癌HeLa细胞和Siha细胞的生长，并呈时间依赖和浓度依赖关系，且HeLa细胞对血根碱敏感性高于Siha细胞；(2)血根碱可以引起宫颈癌细胞周期G2/M期阻滞，并能明显诱导细胞凋亡，同时促发ROS的生成和引起线粒体膜电位的降低；(3)血根碱明显下调细胞周期调节蛋白CyclinB1、CyclinE和CDC2的表达水平，而上调了p21和p27的表达水平；(4)血根碱处理后，凋亡相关蛋白NF-кB、Bcl-2、Procaspase3、P-Erk、P-P38和P-JNK蛋白表达水平出现明显上调，而Cytochrome C、AIF、Cleaved PARP和P-JNK蛋白表达水平则出现明显下调。
第二部分血根碱体外诱导宫颈癌细胞自噬及其机制的研究
目的：研究血根碱在体外诱导宫颈癌细胞自噬及其可能的机制
方法：采用MTT比色法观察自噬抑制剂3-MA影响血根碱的宫颈癌细胞增殖抑制作用；通过透射电镜观察和MDC染色方法检测血根碱处理后宫颈癌细胞自噬的发生；应用Western blot方法检测自噬相关蛋白LC3和Beclin1表达水平。
结果：血根碱处理导致宫颈癌HeLa和Siha细胞中自噬囊泡明显增多，并呈浓度和时间依赖关系；自噬抑制剂3-MA可以逆转血根碱对宫颈癌细胞生长的抑制作用。血根碱处理使宫颈癌细胞中LC3II和Beclin1的蛋白表达水平出现了明显上调。
第三部分血根碱对体外宫颈癌细胞转移的影响及分子机制研究
目的：研究血根碱对宫颈癌细胞粘附、转移及侵袭的影响，并探讨其作用的初步机制。
方法：通过贴壁实验方法检测血根碱处理后宫颈癌细胞粘附能力的影响。采用划痕实验方法检测血根碱对肿瘤细胞体外迁移能力的影响。通过Boyden小室法检测血根碱对肿瘤细胞转移能力的影响；最后用Western blot方法检测侵袭和转移相关蛋白表达水平的变化。
结果：血根碱可以抑制宫颈癌HeLa和Siha细胞的粘附、迁徙、运动及侵袭转移能力，并呈浓度和时间依赖关系，而且在无毒性的剂量下也明显抑制细胞的侵袭转移。其机制与上调了钙粘附蛋白(E-cadherin)蛋白表达水平，下调α-catenin、β-catenin和γ-catenin的蛋白表达水平有关。另外也发现了基质金属蛋白酶MMP-2和MMP-9和VEFG的蛋白表达水平随着血根碱浓度的增加而下降；抑癌基因PTEN蛋白的表达水平则随着血根碱浓度的增加而增加。
第四部分血根碱体内对宫颈癌的抑制作用及其机制的研究
目的：建立裸鼠的宫颈癌移植瘤模型，观察血根碱在动物体内对宫颈癌生长的抑制作用，并初步探讨其作用机制。
方法：首先将HeLa细胞接种Babl/C裸鼠臀部，建立裸鼠宫颈癌移植瘤模型；按处理分组为：生理盐水对照组、顺铂阳性对照组(5mg/kg，1次/周，iP)、血根碱低浓度组(1.25mg/kg，1次/3天，iP)和血根碱高浓度组(2.5mg/kg，1次/3天，iP)，连续腹腔注射给药治疗28天，随后继续观察28天。期间观察动物一般生命体征的改变和肿瘤体积变化；实验结束时观察实验组和对照组肿瘤周围血管以及肿瘤侵袭转移情况，并取肿瘤进行HE染色观察肿瘤的病理学改变及瘤内和周边血管情况；采用免疫组化法检测肿瘤组织中CD34、Ki67、EGFR和VEGF的表达水平；Western blot方法检测肿瘤组织内Bcl-2、 Bax和VEGF蛋白表达水平；采用透射电镜检测肿瘤组织中细胞超微结构改变。
结果：血根碱在实验剂量下对荷瘤裸鼠无明显毒副作用，血根碱能够明显抑制宫颈癌HeLa细胞移植肿瘤的生长，并呈一定的浓度依赖性，且这种抑制作用与经典化疗药物顺铂的抑瘤疗效相当。其机制可能与血根碱能够抑制肿瘤的血管生成有关，发现在血根碱治疗的肿瘤组织中CD34、Ki67、EGFR和VEGF的表达均出现下调。电镜结果显示血根碱治疗组的肿瘤组织中出现了自噬和凋亡共存现象。血根碱治疗组肿瘤组织抑凋亡蛋白Bcl-2显现下调和促凋亡蛋白Bax则显现上调。
结论本课题分别以宫颈癌HeLa和Siha细胞为体外研究对象和裸鼠宫颈癌移植瘤模型为体内研究对象，分别从细胞，蛋白分子和动物研究水平探讨了血根碱抑制宫颈癌的抗肿瘤作用，研究结论总结如下：
1.血根碱可以抑制宫颈癌HeLa和Siha细胞生长，并呈剂量依赖和时间依赖关系。
2.血根碱可以诱导宫颈癌细胞发生G2/M期周期阻滞，其机制是可能是通过激活NF-κB，继而上调p21和p27表达，从而抑制Cdc2/CyclinB1复合物的活性有关。
3.血根碱可以诱导宫颈癌细胞发生凋亡，其机制是(1)通过上调Bax蛋白表达水平和下调Bcl-2、NF-кB的表达水平；(2)诱导宫颈癌细胞产生ROS，继而引起线粒体膜电位的下降，启动了Cytochrome C→Caspase-9→Caspase-3→PARP通路；(3)激活了MAPK信号通路。
4.血根碱可以诱导宫颈癌细胞发生自噬， LC3和Beclin1蛋白参与了自噬的调控。
5.血根碱可以抑制宫颈癌细胞的侵袭和转移能力，其机制可能包括(1)首先激活上游钙粘附蛋白(E-cadherin)、α-catenin、β-catenin和γ-catenin的表达，通过影响Catenins、E-Cadherin和细胞骨架之间信号传递的作用，继而抑制下游基质金属蛋白酶MMP-2和MMP-9活性抑制其侵袭和转移；(2)抑制VEFG和PTEN的表达从而抑制宫颈癌细胞的侵袭和转移能力。
6.血根碱在体内也能够明显抑制异体移植瘤宫颈癌的生长，其机制可能与抗血管生成、抑制肿瘤细胞增殖、诱导凋亡和自噬有关。
7.与肿瘤细胞的敏感性相比，血根碱对正常细胞的毒性明显要小，而且在明显抑制肿瘤生长的剂量条件下，血根碱对裸鼠未产生明显的毒副作用。这些实验结果表明血根碱对肿瘤细胞有较好的选择性，这为临床试验深入研究提供了非常好的实验基础和理论依据。
Purpose:
The aim of study was to investigate the ant-cancer activities of sanguinarine incervical cancer and its possible mechanisms via a series of in vitro and in vivo studies.
Methods:
In vitro studies, two human cervical cancer cell lines, HeLa and Siha, with thefollowing methods were employed, in some cases, normal cell lines GM and ECV304were used as control:1) Cell morphology was observed by inverted microscope and livecell imaging system;2) Cell viability was examined by MTT assay;3) Colonyformation assay was also used to study the cell growth inhibition of sanguinarine;4)Scratch healing assay and the trans-well assay were employed to study the influence ofsanguinarine on the cell migration and invasion;5) Flow cytometry was applied toanalyze cell cycle progression and apoptosis induction;6) Single cell gel electrophoresis(comet assay) was used to detect DNA damage;7) Apoptosis and necrosis weremeasured by TUNEL and Annexin V-FITC cell apoptosis kits;8) ROS content andmitochondrial membrane potential was determined by commercial kits;9) Alteration ofProtein expression level was detected by Western blot assay;10) Autophagy wasexamined by MDC (monodansylcadaverin) staining;11) Autophagy and apoptosisultrastructure changes of cells were observed by transmission electron microscope(TEM).
In vivo studies, a transplanted tumor model by injecting HeLa cells intosubcutaneous tissue of Babl/c mice was established. Mice were randomly assigned into4groups by intraperitoneal injection:1) saline solution as negative control;2)5mg/kgcisplatin as positive control (1/week);3)1.25mg/kg injection of sanguinarine (1/3days)and4)2.5mg/kg injection of sanguinarine (1/3days). The period time of therapies was28days, the tumor volume was measured once every other day upto another28days following sanguinarine treatment. At the end of experiments, tumors were carefullyremoved and weighted. Microvascular density in tumor tissues was measured by aninverted microscope and CD34immunostaining. The expression levels of Bcl-2, Baxand VEGF tumor tissues were detected by Western blot and the expression levels ofVEGF, EGFR and Ki67, by immunohistochemical assay, in tumor tissues. Changes ofcell ultrastructure related to autophagy and apoptosis in tumors were observed by TEM.
Results:
1) A dose-and time-dependent inhibition of cell proliferation by sanguinarine wasobserved in both cell lines. An IC50value for24h treatment of sanguinarine in HeLa andSiha cells was2.62±0.21μmol/L and3.07±0.23μmol/L, respectively, indicatingslightly more sensitivity in HeLa cells (P <0.05). While, sanguinarine showed lesstoxicity in normal GM and ECV304cells with an IC50value of4.93±0.22μmol/L and5.78±0.18μmol/L, respectively.
2) After treatment of0,1,2and3μmol/L sanguinarine for24h, the proportion ofG2/M phase in HeLa cells was (25.21±0.66)%,(29.97±0.84)%,(37.31±0.93)%and(57.72±0.13)%, respectively. Similar a dose-dependent arrest of G2/M phase wasobserved in Siha cells after sanguinarine treatment. Consistant with cell cycle arrest,sanguinarine caused a significant down-regulation of CyclinB1, CyclinE and CDC2protein expression and an up-regulation of P21and P27protein expression.
3) With24h treatment of sanguinarine, HeLa and Siha cells showed typicalapoptotic morphologic changes, including chromatin condensation and DNA fragment.Comet assay evidenced that sanguinarine increased the mean tail length and thepercentage of cells with comet tails. Sanguinarine decreased Bcl-2expression andincreased Bax expression via a dose-response manner, and consequently the ratio of Baxto Bcl-2was significantly increased (P<0.05). The expression of procaspase-3wasdecreased and AIF and Cleaved PARP were increased in HeLa and Siha cells treatedwith sanguinarine (P<0.05). The ROS content in HeLa cells treated with1μmol/Lsanguinarine markedly increased in0.5h (29.63±1.28)%compared with0h (38.4±2.90)%and reached peak in4h (58.37±6.03)%(P<0.01) following exposure tosanguinarine. Similar results were observed for ROS generation in Siha cells. On theother hand, mitochondrial transmembrane potential was decreased and the expression ofcytochrome c protein was increased in both of cell lines after sanguinarine treatment.
4) As for migration and invasion, sanguinarine significantly decreased theadherence rate and inhibited the ability to cover “cell wound” by scratch in both of celllines via a time-dependent fashion. Tran-well assay showed that HeLa cell number aftertreatment with sanguinarine at0,0.5and0.75mol/L was156±9.85,108±13.23and74.67±7.02, respectively (P <0.01). Similar observation was obtained in Siha cells withsanguinarine treatment. Additionally, a significant dose-dependent alteration of someproteins which are related to cell migration and invasion was found in both of cell linestreated with sanguinarine, i.e., a decrease level of VEGF, EGFR, PTEN, NF-κB,-catenin,-catenin and γ-catenin expression and an increased expression of E-cadherinprotein (P<0.05).
5) TEM observation indicated that autophagosomes and apoptotic bodies inducedby sanguinarine in both of cell lines. However, sanguinarine-induced autophagy wasreversed by emplying a specific autophagy inhibitor,3-methylademine (3-MA). Aquantitative method of MDC staining releaved that the cell number of autophagy was149±22.65and412.67±16.80in HeLa cells as well as138±17.47and371±20.84inSiha cells after4h treatment with1and2mol/L sanguinarine (P<0.01). Sanguinarinecaused a reduced expression of Beclin1protein. In addition, a decrease of LC3-I proteincomparing with an increase of LC3-II protein were detectable after treatment withsanguinarine.
6) A significant inhibition of tumor growth and volume in transplanted tumor micewere observed after therapy with sanguinarine via an intraperitoneal injection. At theend of experiment, the inhibition rate of tumor growth was29.40%,39.96%and45.16%in cisplatin,1.25mg/kg and2.5mg/kg sanguinarine, compared with that inun-treated control mice. Microvascular density in tumor tissues as evidenced by CD34immunostaining was77.33±7.04,25.00±4.90,28.33±1.70and15.67±2.05inun-treated controls, cisplatin,1.25mg/kg and2.5mg/kg sanguinarine, respectively(P<0.01). TEM observation also showed a significant change of apoptosis andautophagy in tumor tissues after therapy with sanguinarine. On the other hand,sanguinarine inhibited tumor angiogenesis possibly via down-regulating the expressionof VEGF, EGFR and NF-κB.
Conclusion:
In summary, a significant inhibitory proliferation in both of human cervical cancer cell lines, HeLa and Siha, was observed after treatment with sanguinarine in a dose-andtime-dependent manner. Sanguinarine-caused arrest of G2/M phase was accompaniedwith a down-regulation of CyclinB1, CyclinE, and CDC2protein and a up-regulation ofp21and p27expression. The occurrence of apoptosis and autophagy after sanguinarinetreatment was confirmed by a few methods, the underlying mechanisms may beinvolved in an increase of Bax/Bcl-2, an alteration of LC3II/LC3I expression, anactivation of caspase-3and MAPK pathway. Anti-cancer growth in vivo bysanguinarine may be related to an inhibition of tumor angiogenesis and an induction ofapoptosis and autophagy by down-regulating the expression of VEGF, EGFR andNF-κB. These novel findings indicate that sanguinarine inhibites cervical cancer growthin vitro and in vivo by multimechanisms, which suggesting that sanguinarine may serveas a potential therapeutic agent for cervical cancer.
引文
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