1、新型紫杉烷类化合物Lx2-32c抗肿瘤耐药药效学及机制研究 2、紫杉醇耐药的人胃腺癌BGC-823/TA细胞株及BGC-823/Paclitaxel裸鼠异种移植瘤模型的建立及耐药机制的探讨
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摘要
紫杉醇(paclitaxel)是1971年从美国西部短叶红豆杉中分离提取出来的一种天然抗肿瘤药物,具有独特的抗癌机制。紫杉醇的主要靶点是细胞内微管系统,通过促进微管聚合抑制微管解聚,使细胞阻滞于G2/M期,并最终导致细胞死亡。目前紫杉醇己成为包括乳腺癌、卵巢癌、非小细胞性肺癌等多种实体瘤的一线治疗药物,应用前景十分广阔。然而,紫杉醇的开发也面临着诸多问题,如来源有限,水溶性差等,而获得性耐药现象的出现则是制约其临床疗效的最主要问题。紫杉醇耐药的产生机制十分复杂,主要包括：药物外排泵ABC转运蛋白过表达和功能上调导致细胞内药物浓度降低；药物作用靶点改变；凋亡信号通路调控异常以及紫杉醇耐药相关基因TRAG-3过表达等。
目前,解决耐药问题主要有两条思路：一是利用肿瘤耐药逆转剂抑制转运蛋白的功能或表达,以恢复耐药肿瘤细胞对化疗药物的敏感性；二是开发本身具有抗耐药活性的化合物直接杀伤或抑制耐药肿瘤的生长。近年来,抗耐药研究越来越受到关注。我国具有丰富的天然产物资源,从天然产物中分离提取有效成分并进行相应的结构修饰是当前抗耐药研究的一大热点。在本研究的第一部分,我们以对紫杉醇耐药的细胞株及体内耐药裸鼠异体移植瘤模型为研究对象,考察了一种紫杉烷类衍生物Lx2-32c体内外的抗肿瘤耐药活性,并对其可能的抗耐药机制作出研究。
为研究肿瘤多药耐药的机制,或筛选耐药逆转剂及具有抗耐药活性的化合物,建立多药耐药细胞系及体内裸鼠异体移植瘤模型是主要手段之一。在本研究的第二部分,我们建立了对紫杉醇耐药的人胃腺癌BGC-823细胞亚系和体内耐药模型,并对这两个模型进行了耐药性的鉴定,以及主要耐药机制的探讨。
第一部分新型紫杉烷类化合物Lx2-32c抗肿瘤耐药药效学及机制研究
本部分实验主要研究了Lx2-32c体内外抗肿瘤耐药作用,并对其主要的耐药机制进行了较深入的探讨。Lx2-32c是由中国医学科学院药物研究所天然药物化学研究室方唯硕研究员课题组通过对三尖杉宁碱(Cephalomannine)进行结构修饰半合成得到的一类全新紫杉烷类衍生物中活性最强的一个。此类类化合物已申请中国化合物专利(申请号200610080890.7)与国际PCT专利(申请号PCT/CN2007/003235)
体外研究中,Lx2-32c可抑制多种不同组织来源的耐药细胞的生长,如人乳腺癌多药耐药细胞株MX-1/T及亲本细胞株MX-1,人肺腺癌多药耐药细胞株A549/T及其亲本细胞株A549,人乳腺癌多药耐药细胞株MCF-7/T及其亲本细胞株MCF-7,人胃腺癌多药耐药细胞株BGC-823/TA及亲本细胞株BGC-823,人口腔上皮癌多药耐药细胞株KB/V及其亲本细胞株KB,以及人肝癌多药耐药细胞株Bel-7402/5-Fu及亲本细胞株Bel-7402。MTT法测得其在体外的半数抑制浓度IC50为29.61±5.76nmol/L(从1.02至98.92nmol/L)。接下来,我们选择人乳腺癌多药耐药细胞株MX-1/T及亲本细胞株MX-1作为研究对象,以Paclitaxel作为阳性对照药,探讨Lx2-32c较确切的抗耐药作用机制。
SRB法测得Lx2-32c对MX-1/T细胞的GI50为67.35nM,集落形成实验测定Lx2-32c对MX-1/T细胞的IC50为5.33nM,均显著低于Paclitaxel的相应值。
体内实验表明Lx2-32c能够抑制对紫杉醇耐药的人乳腺癌MX-1裸鼠异体移植瘤的生长。Lx2-32c(15、30、45mg/kg)对裸鼠移植瘤生长的抑制率分别为40.11%、63.46%及77.67%,呈现较好的剂量效应关系,而Paclitaxel30mg/kg组的抑瘤率仅为22.85%。对移植瘤组织的TUNEL检测亦显示Lx2-32c可剂量依赖性诱导耐药瘤组织产生凋亡现象。
接下来我们研究了Lx2-32c对MX-1/T细胞内微管蛋白的作用。首先,我们利用微管蛋白间接免疫荧光法及Western Blot法检测细胞内微管的原位聚合,以探讨Lx2-32c对耐药细胞微管动态平衡的影响。免疫荧光实验结果表明,Lx2-32c的作用方式呈典型的紫杉醇样作用,可促进细胞内微管聚合,诱导细胞内微管形成微管束,阻碍细胞内纺锤丝的形成,从而干扰细胞的有丝分裂。细胞内原位微管聚合实验表明,Lx2-32c能够明显地将细胞内微管的动态平衡由“可溶”态向“不溶”态推进,使微管向着聚合态偏移,结果同间接免疫荧光结果类似。然后我们检测了细胞内Tau蛋白的表达,结果表明,与亲本MX-1细胞相比,耐药的MX-1/T细胞Tau蛋白表达明显增高,Lx2-32c可剂量依赖性地降低Tau蛋白的表达。
P-gp蛋白与肿瘤的多药耐药现象关系密切,首先,我们利用RT-PCR及Western Blot法检测了P-gp在细胞内的表达,结果表明,MX-1/T细胞是一株典型的P-gp高表达细胞株,这可能是其具有多药耐药性的主要原因,而Lx2-32c对MX-1/T细胞内P-gp的表达无明显影响。接下来,我们考察了MX-1/T细胞内的药物蓄积情况。质谱分析表明,相同剂量的Lx2-32c在细胞内的蓄积量明显高于Paclitaxel的蓄积量,脱药后Lx2-32c的外排亦明显少于Paclitaxel,其脱药后的6h蓄积率为脱药时的32.63%,显著多于Paclitaxel的6h蓄积率(1.74%)。Rhodamine123蓄积实验显示,与MX-1/T细胞的基础荧光强度相比,Paclitaxel可使细胞内的荧光强度有一定程度的回升,提示此时P-gp在转运Rhodamine123的同时参与Paclitaxel的外排,故P-gp对Rhodamine123的转运减少,胞内荧光增强。Lx2-32c对细胞内荧光强度影响不大,说明此时P-gp对Lx2-32c的外排较少,仍主要参与Rhodamine123的外排,此结果间接反映了Lx2-32c在MX-1/T细胞内的蓄积多于Paclitaxel的蓄积。10uMVerapamil与不同剂量Paclitaxel合用72h后,可明显逆转MX-1/T细胞对Paclitaxel的耐药性,逆转倍数达38.89倍,而其与不同剂量Lx2-32c合用72h后,对Lx2-32c的IC50值影响不大,结果提示P-gp在MX-1/T细胞对Paclitaxel产生耐药的过程具有重要地位,而对Lx2-32c在耐药细胞内的蓄积影响不大。
药物与P-gp作用的另一个重要方面是二者的结合能力。计算机辅助设计提示,Lx2-32c与P-gp的结合力较弱,Fitness评分为31.41,显著低于Paclitaxel与P-gp的Fitness评分(36.90)。P-gp ATPase的测定也表明与Paclitaxel处理组相比,Lx2-32c三个剂量组(20,100,500nM)产生的荧光强度均明显增加,说明ATP的消耗量减少,由于P-gp对底物的转运有赖于ATP的水解,故此结果间接反映了P-gp对Lx2-32c的外排较Paclitaxel组减少,与前述实验结果吻合。另外,由于Lx2-32c处理组产生的荧光强度呈剂量依赖性降低,说明Lx2-32c在耐药细胞内增加的蓄积并不是由于其具有抑制P-gp功能的作用。结合其他实验,我们推测,Lx2-32c在细胞内的蓄积较Paclitaxel增加的原因在于其与P-gp亲和力较低,故P-gp对其外排亦降低,因此Lx2-32c可在P-gp高表达的耐药细胞中仍保持有效浓度,这是其具有抗耐药活性的最主要机制。
解除耐药细胞的凋亡抑制是抗耐药药物研究的一大思路,我们也考察了Lx2-32c对MX-1/T细胞的凋亡诱导作用。流式细胞术显示不同剂量的Lx2-32c可使MX-1/T细胞产生G2/M期阻滞,染色体数目不稳定性及凋亡峰(亚G1峰)的出现。Annexin V-FITC/PI双染法定量地检测了Lx2-32c(20、100、500nM)作用48h产生的凋亡百分比,分别为14.09%、30.67%和63.52%。利用DAPI染色及AO/EB染色均可观察到Lx2-32c作用后MX-1/T细胞呈现的典型凋亡形态,如核固缩,核碎裂等。DNA ladder实验可见Lx2-32c诱导细胞DNA片段化,产生明显的梯形条带。JC-1染色及DiOC6染色均显示Lx2-32c对细胞线粒体膜电位的降低作用。Western Blot结果显示,Lx2-32c可剂量依赖性地升高Bax/Bcl-2比例,使细胞色素C、AIF从线粒体释放到胞浆,进而活化Caspase-9,-3,和PARP,启动内源性凋亡通路,而对Fas, FasL、Caspase-8的表达无影响。
最后,我们检测了Lx2-32c对肿瘤细胞侵袭转移及血管生成的影响。划痕-愈合试验结果表明,50nM Lx2-32c持续作用24h,明显抑制MX-1/T细胞侧向迁移能力；粘附试验结果表明,10nM及50nM Lx2-32c预处理30min,可明显抑制MX-1/T细胞与人工基底膜成分Matrigel的粘附；明胶酶谱法结果表明,Lx2-32c作用24h,可在一定程度上抑制人乳腺癌细胞MDA-MB231细胞分泌MMP-9及MMP-2,人纤维肉瘤细胞HT-1080分泌aMMP-9,以及人黑色素瘤细胞分泌MMP-2。另外,10nM及50nM Lx2-32c可显著减弱人脐静脉内皮细胞融合细胞EA-hy926的铺展能力及体外形成血管环的能力,显示出一定的抗血管生成作用。
综上所述,Lx2-32c是一种具有确切的体内外抗耐药活性的新型紫杉烷类化合物,其抗耐药机制可能与促进微管聚合,降低Tau蛋白表达,较少被P-gp外排从而能保持较高的细胞内药物蓄积浓度,以及诱导耐药细胞凋亡有关,同时,Lx2-32c亦显示出一定的体外抗肿瘤侵袭转移及抗血管生成的作用。
第二部分紫杉醇耐药的人胃腺癌BGC-823/TA细胞株及BGC-823/Paclitaxel裸鼠异种移植瘤模型的建立及耐药机制的探讨
本部分研究主要是建立并鉴定了对紫杉醇耐药的人胃腺癌BGC-823/TA细胞株及体内耐药的BGC一823/Paclitaxel裸鼠异种移植瘤模型,并对其耐药机制进行了探讨。
本研究选用人肺腺癌细胞株BGC-823作为亲本细胞,以紫杉醇对BGC-823细胞的IC1。为初始诱导剂量,经紫杉醇小剂量逐步增加剂量诱导和克隆选择,18个月后,成功建立了能在含2.00×10-8rnol/L紫杉醇的培养基中正常生长和传代的耐药株BGC-823/TA,耐药倍数为92.31倍。MTT法检测显示BGC-823/TA细胞对Docetaxel. Vincristine.Adriamycin及Lx2-32c等多种细胞毒类化合物也具有出不同程度的交叉耐药性,表现出典型的多药耐药特征。与亲本BGC-823细胞相比,BGC-823/TA细胞的细胞形态、细胞核形态、细胞周期分布、运动能力,粘附能力等基本生物学特征无明显改变,但生长速率较亲本细胞略微减慢,集落形成能力也有所降低,集落形成率分别为62.88%和74.38%。间接免疫荧光检测显示两株细胞内微管蛋白形态无明显改变,Western Blot法检测显示,BGC-823/TA细胞内p微管蛋白亚型(βⅠ、βⅡ、βⅢ、βⅣ微管蛋白)的表达与亲本BGC-823细胞无明显差异,两株细胞内Tau蛋白的表达量也差别不大。
P-gp.MRP及LRP等耐药蛋白的表达及活性是细胞多药耐药性形成的重要机制。RT-PCR检测发现BGC-823/TA细胞MDR1表达显著升高,间接免疫荧光法与Western Blot法均显示BGC-823/TA细胞P-gp高表达,Western Blot法还显示BGC-823/TA细胞内其它药泵蛋白如MRP.BCRP.LRP表达未见变化。
P-gp蛋白的功能研究证实BGC-823/TA细胞泵出Rodaminel23.阿霉素及Flutax-1的能力强于BGC-823细胞。质谱检测显示BGC-823/TA细胞内Paclitaxel含量明显低于BGC-823胞内含量,且脱药2h后BGC-823/TA细胞内Paclitaxel含量下降更为明显。MTT法检测显示同时加入药泵抑制剂Verapamil后BGC-823/TA细胞可恢复对Paclitaxel的敏感性。
上述研究结果表明,BGC-823/TA细胞是一株获得性多药耐药细胞株,其耐药机制主要由于MDR1mRNA的表达增高导致细胞表面P-gp蛋白过表达,从而使P-gp的药物外排泵功能亢进,最终使胞内Paclitaxel有效浓度降低,出现耐药现象。
与体外模型相对应,我们同时建立了对紫杉醇耐药的人胃腺癌BGC-823裸鼠异种移植瘤模型,该裸鼠模型经历15代传代,紫杉醇的诱导剂量由1Omg/kg提高至30mg/kg,最终得到体内耐药模型BGC-823/Paclitaxel。裸鼠异体移植瘤实验证实,耐药的BGC-823/Paclitaxel肿瘤生长速率比亲本BGC-823肿瘤略快,但无显著性差异。给予20mg/kg Paclitaxel治疗后,BGC-823/Paclitaxel肿瘤的抑制率及RTV均明显低于BGC-823肿瘤,体现出一定的耐药性。对BGC-823/Paclitaxel瘤块进行WesternBlot检测,耐药肿瘤出现P-gp表达增高,且随传代次数的增加,药物对肿瘤不断诱导,耐药肿瘤与亲本肿瘤间的P-gp表达差异愈明显,说明P-gp蛋白的过表达可能是导致BGC-823/Paclitaxel肿瘤对紫杉醇耐药的一个重要原因。
Paclitaxel, fistly isolated from Taxus brevifolia in1971, was a natural product with anti-cancer activity and unique mechanism of action. Paclitaxel binds to and stabilizes microtubules against depolymerization and inhibits cell proliferation by disrupting normal mitotic spindle formation. From its introduction in1992, paclitaxel has been crowned as one of the most active antineoplastic agents against a a board spectrum of malignancies, including ovarian, breast, lung, head and neck cancers and Kaposi' Ssarcoma. However, there are some problems delay the pace of its development, such as poor solubility, limitied resource, and resistance to paclitaxel which is frequently observed in patients and poses a major impediment to successful therapy. Therefore, researches on paclitaxel resistance arouse wide concern and the development of novel agents with definite anti-resistance activity is of great importance. Resistance to paclitaxel has been attributed to several mechanisms including overexpression of the efflux pump P-glycoprotein, alterations in the expression of (3-tubulin isotypes and mutations at the taxane binding site in microtubule, alterations in the apoptosis signaling pathway, as well as overexpression of TRAG-3.
There are two major strategies to combat tumor resistance.The first one is the development of MDR modulators whicn could restore the sensitivity of MDR tumor cells by inhibiting the function of ABC transporter proteins.In contrast, the other method is the development of anti-resisitance agents which could directly eradicate the tumor cells by themselves.Recently, the anti-resistance research attract more and more attention.Since China is a country with abundant natural resourse, endeavous to discover novel anti-resistance agent from natural products become major focus of research.
This article consists of two parts:Part I Studies on anti-drug-resistance activities and mechanisms of action of Lx2-32c, a novel taxane; Part Ⅱ The establishment of paclitaxel-resistant BGC-823in vitro cell line and in vivo xenograft model and the research on their mechanisms of resistance.
For our unremitting pursuit of novel agents with anti-drug-resistance activities, we reorganized and qualified the structure of cephalomannine, which is a natural congener of paclitaxel, and obtained a series of new compounds.Lx2-32c,2-debenzoyl-2-(3-azido-benzoyl)-7-propi-onyl-10-deacetylcephalo mannine (fig.1), was screened out from these compounds for its outstanding cytotoxicity. This part is to evaluate the anti-resistance activity of Lx2-32c, in vitro and in vivo, and to explore its possible mechanisms.
In vitro, Lx2-32c exerted extensive cytotoxicity to both sensitive and resistant cells from different origins, including human oral epidermoid carcinoma cell line(KB)and its resistant cell line(KB/V), human hepatocellular carcinomacell line(Bel-7402) and its resistant cell line(Bel-7402/5-Fu), human lung adenocarcinoma cell line(A549) and its resistant cell line (A549/T), human breast cancer cell lines(MX-1,MCF-7) and their resistant cell lines (MX-1/T,MCF-7/T), human gastric cancer cell line(BGC-823) and its resistant cell line(BGC-823/T),with an IC50value of29.61±5.76nmol/L (ranging from1.02to98.92nM). For further study, we selected MX-1/T cell line due to its stable resistance.The GI50of Lx2-32c in MX-1/T cell was67.35nM by SRB assay and IC50of Lx2-32c was5.33nM by clone formation assay, showing that the growth of MX-1/T cell was inhibited significantly by Lx2-32c.
In vivo, Lx2-32c administration inhibited the growth of tumors of Paclitaxel-resistant MX-1nude mice model in a dose-dependent manner.Lx2-32c administered at15,30and45mg/kg/day resulted in a40.11%、63.46%and77.67%inhibition in tumor growth, respectively. What is more, Lx2-32c showed great efficacy in the induction of apoptosis in MX-1/T tumor by TUNEL assay.
The effect of Lx2-32c on microtubule morphology and dynamic balance in MX-1/T cells were checked by immunofluorescence assay and Western Blot analysis. Lx2-32c (20,100nM) showed a typical taxane-like effect on the network with rare characteristic spindle poles and formation of thick microtubule bundles. Multinucleated cells were also observed by PI staining. Western blot assay showed that Lx2-32c promote the microtubule state from soluble to insoluble, thereby stabilizing microtubule. Meanwhile, Lx2-32c could also inhibit the expression of Tau in MX-1/T cells.
P-gp plays an important role in the development of drug resistance. RT-PCR and western blot analysis displayed no difference in the expression of MDRland P-gp upon Lx2-32c treatment. Then, we evaluated the accumulation of Lx2-32c in MX-1/T cells by MS and Rhodamine123accumulation assay. The results indicated the much greater accumulation of Lx2-32c than Paclitaxel. Verapamil reverse assay showed that the inhibition of P-gp has little effect on the cytotoxicity of Lx2-32c, suggesting a slighter influence of P-gp on Lx2-32c.
Then, we explore the interaction of Lx2-32c and P-gp. The result of molecular docking showed that Lx2-32c could bind with ATP pocket of P-gp by hydrophobic ineraction, but the binding is much weaker than the one between Paclitaxel and P-gp. Pgp ATPase assay reflected indirectly the less efflux of Lx2-32c, in contrast to the efflux of Paclitaxel.From these results, we speculated that the difference of efflux between Lx2-32c and Paclitaxel lied in the difference of their affinity toward P-gp.
Strategies in trying to combat resistance by modulating aberrant apoptosis pathways would prove effective and is attracting extensive attention. In this study,we also evaluated the induction of apoptosis by Lx2-32c.The results showed that Lx2-32c treatment led to dramatic cell cycle arrest in G2/M phase and typical apoptosis characters indicated by morphological changes and DNA fragmentation Apoptosis induced by Lx2-32c was associated with loss of mitochondrial membrane potential, enhancement of mitochondrial cytochrome c and AIF release, elevation of the Bax/Bcl-2ratio, activation of caspase-9,-3, but not caspase-8, and degradation of PARP.
Finally, we evaluated the effect of Lx2-32c on the metastasis and angiogenesis. A wound healing assay showed that Lx2-32c treatment (50nM) could inhibit the migration of MX-1/T cells. Adhesion assay showed that Lx2-32c reduced the adhesion of MX-1/T cells to Matrigel. What is more,to reveal the mechanisms of the inhibition role of Lx2-32c on tumor cell metastasis, the activity of MMPs protein was investigated by a Gelatin zymography assay. Results showed that Lx2-32c could significantly block the secretions of MMPs in MDA-MB231cells, HT1080cells and A375cells. Meanwhile, Lx2-32c also displayed impressive activity in anti-angiogenesis by in vitro tube forming assay.
In conclusion, our study demonstrated that Lx2-32c is a microtubule-stabilizing agent with gorgeous efficacy in suppressing the proliferation of MX-1/T cells, and other drug-resistant cell lines. Moreover, Lx2-32c displayed potent antitumor activity on paclitaxel-resistant MX-1in vivo model. Its mechanism of action may be associated with improved activity in promoting microtubule polymerization, inhibition of Tau expression, weaker affinity towards P-gp and induction of apoptosis via intrinsic apoptotic pathway.
Part Ⅱ is about the establishment of a Paclitaxel-resistant human gastric adenocareinoma cell line BGC-823/TA, as well as Paclitaxel-resisitant BGC-823/Paclitaxel in vivo nude mice model, and the exploration of their possible mechanism of resistance.
For in vivo part, BGC-823/TA was selected by continuous exposure to increasing Paclitaxel concentration from the IC10of Paclitaxel to2×10-8mol/L. The IC50of Paclitaxel to BGC-823and BGC-823/TA were1.79nM and165.24nM,respectively. The resistance index was92.31fold. This cell line also showed muti-drug resistance to many other chemotherapy agents such as Docetaxel、Vincristine、Adriamycin and Lx2-32c.
In contrast to parent BGC-823cell line, there were little changes of BGC-823/TA in morphorlogy、cell cycle distribution、motility and adhesion ability, but the proliferation of BGC-823/TA cell was relatively slow. The result of growth curve showed a delay of time for reaching peak value and lower peak value. In clony formation assay. The rate of clone formation were62.88%and74.38%respectively. Meanwhile, little difference in the state of microtubule, expression of Tau and β-tubulin isotypes was detected by indirect immunofluorescence assay and Western blot analysis, respectively.
Overexpression of P-gp, MRP and LRP is the major mechanism underlying MDR. The results by RT-PCR assay indicated the overexpressionl of MDR1in BGC-823/TA cells. Western Blot showed that the level of P-gp was significantly higher in BGC-823/TA cells while the level of MRP、LRP、BCRP remain the same when compared with their expression in BGC-823cells.
Using Rodamine123、ADR and Flutax-1accumulation assay, we found that the activity of drug flux pump in BGC-823/TA cells was stronger than the one in BGC-823cells. Similar results were obtained by MS assay, which indicating the cellular residue of Paclitaxel in BGC-823/TA cells was lower than that in BGC-823cells, and the residue rate in BGC-823/TA cells after incubation without Paclitaxel was also less than that in BGC-823cells. What is more,the sensitivity of BGC-823/TA cells lto Paclitaxel could be restored by Vempamil, which could inhibit the efflux of Paclitaxel by P-gp.
In conclusion, the BGC-823/TA cell line established by myself was a typical What is more, Lx2-32c also displayed definite activity of anti-metastasis and anti-angiogenesis. multi-drug resistance cell line against several cytotoxic agents. The mechanism of its resistance was implicated with the overexpression of MDR1mRNA and the overexpression of P-gP as well as the hyperfunction of drug efflux pump. As a result, the intercellular concentration of Paclitaxel decreased and led to the acquired resistance.
Accordingly, we established the Paclitaxel-resistant BGC-823nude mice model. For this in vivo model, tumors developed resistance by continuous paclitaxel induction from generation to generation after fifteen passages, rather than a single injection of resistant cell lines which had gained resistance by in vitro culture. The induction dose of Paclitaxel was from15mg/kg to30mg/kg. After Paclitaxel treantment of20mg/kg, the inhibitory rate of the growth of BGC-823/Paclitaxel tumor was significantly lower than that of BGC-823tumors, suggesting the resistant phenotype of BGC-823/Paclitaxel tumor. Western blot assay indicated that the expression of P-gp was higher in BGC-823/Paclitaxel tumors than that in BGC-823tumors, and the difference of expression increased by the passage, indicating that P-gp played an important role in the resistance of BGC-823/Paclitaxel tumors.
目前,解决耐药问题主要有两条思路：一是利用肿瘤耐药逆转剂抑制转运蛋白的功能或表达,以恢复耐药肿瘤细胞对化疗药物的敏感性；二是开发本身具有抗耐药活性的化合物直接杀伤或抑制耐药肿瘤的生长。近年来,抗耐药研究越来越受到关注。我国具有丰富的天然产物资源,从天然产物中分离提取有效成分并进行相应的结构修饰是当前抗耐药研究的一大热点。在本研究的第一部分,我们以对紫杉醇耐药的细胞株及体内耐药裸鼠异体移植瘤模型为研究对象,考察了一种紫杉烷类衍生物Lx2-32c体内外的抗肿瘤耐药活性,并对其可能的抗耐药机制作出研究。
为研究肿瘤多药耐药的机制,或筛选耐药逆转剂及具有抗耐药活性的化合物,建立多药耐药细胞系及体内裸鼠异体移植瘤模型是主要手段之一。在本研究的第二部分,我们建立了对紫杉醇耐药的人胃腺癌BGC-823细胞亚系和体内耐药模型,并对这两个模型进行了耐药性的鉴定,以及主要耐药机制的探讨。
第一部分新型紫杉烷类化合物Lx2-32c抗肿瘤耐药药效学及机制研究
本部分实验主要研究了Lx2-32c体内外抗肿瘤耐药作用,并对其主要的耐药机制进行了较深入的探讨。Lx2-32c是由中国医学科学院药物研究所天然药物化学研究室方唯硕研究员课题组通过对三尖杉宁碱(Cephalomannine)进行结构修饰半合成得到的一类全新紫杉烷类衍生物中活性最强的一个。此类类化合物已申请中国化合物专利(申请号200610080890.7)与国际PCT专利(申请号PCT/CN2007/003235)
体外研究中,Lx2-32c可抑制多种不同组织来源的耐药细胞的生长,如人乳腺癌多药耐药细胞株MX-1/T及亲本细胞株MX-1,人肺腺癌多药耐药细胞株A549/T及其亲本细胞株A549,人乳腺癌多药耐药细胞株MCF-7/T及其亲本细胞株MCF-7,人胃腺癌多药耐药细胞株BGC-823/TA及亲本细胞株BGC-823,人口腔上皮癌多药耐药细胞株KB/V及其亲本细胞株KB,以及人肝癌多药耐药细胞株Bel-7402/5-Fu及亲本细胞株Bel-7402。MTT法测得其在体外的半数抑制浓度IC50为29.61±5.76nmol/L(从1.02至98.92nmol/L)。接下来,我们选择人乳腺癌多药耐药细胞株MX-1/T及亲本细胞株MX-1作为研究对象,以Paclitaxel作为阳性对照药,探讨Lx2-32c较确切的抗耐药作用机制。
SRB法测得Lx2-32c对MX-1/T细胞的GI50为67.35nM,集落形成实验测定Lx2-32c对MX-1/T细胞的IC50为5.33nM,均显著低于Paclitaxel的相应值。
体内实验表明Lx2-32c能够抑制对紫杉醇耐药的人乳腺癌MX-1裸鼠异体移植瘤的生长。Lx2-32c(15、30、45mg/kg)对裸鼠移植瘤生长的抑制率分别为40.11%、63.46%及77.67%,呈现较好的剂量效应关系,而Paclitaxel30mg/kg组的抑瘤率仅为22.85%。对移植瘤组织的TUNEL检测亦显示Lx2-32c可剂量依赖性诱导耐药瘤组织产生凋亡现象。
接下来我们研究了Lx2-32c对MX-1/T细胞内微管蛋白的作用。首先,我们利用微管蛋白间接免疫荧光法及Western Blot法检测细胞内微管的原位聚合,以探讨Lx2-32c对耐药细胞微管动态平衡的影响。免疫荧光实验结果表明,Lx2-32c的作用方式呈典型的紫杉醇样作用,可促进细胞内微管聚合,诱导细胞内微管形成微管束,阻碍细胞内纺锤丝的形成,从而干扰细胞的有丝分裂。细胞内原位微管聚合实验表明,Lx2-32c能够明显地将细胞内微管的动态平衡由“可溶”态向“不溶”态推进,使微管向着聚合态偏移,结果同间接免疫荧光结果类似。然后我们检测了细胞内Tau蛋白的表达,结果表明,与亲本MX-1细胞相比,耐药的MX-1/T细胞Tau蛋白表达明显增高,Lx2-32c可剂量依赖性地降低Tau蛋白的表达。
P-gp蛋白与肿瘤的多药耐药现象关系密切,首先,我们利用RT-PCR及Western Blot法检测了P-gp在细胞内的表达,结果表明,MX-1/T细胞是一株典型的P-gp高表达细胞株,这可能是其具有多药耐药性的主要原因,而Lx2-32c对MX-1/T细胞内P-gp的表达无明显影响。接下来,我们考察了MX-1/T细胞内的药物蓄积情况。质谱分析表明,相同剂量的Lx2-32c在细胞内的蓄积量明显高于Paclitaxel的蓄积量,脱药后Lx2-32c的外排亦明显少于Paclitaxel,其脱药后的6h蓄积率为脱药时的32.63%,显著多于Paclitaxel的6h蓄积率(1.74%)。Rhodamine123蓄积实验显示,与MX-1/T细胞的基础荧光强度相比,Paclitaxel可使细胞内的荧光强度有一定程度的回升,提示此时P-gp在转运Rhodamine123的同时参与Paclitaxel的外排,故P-gp对Rhodamine123的转运减少,胞内荧光增强。Lx2-32c对细胞内荧光强度影响不大,说明此时P-gp对Lx2-32c的外排较少,仍主要参与Rhodamine123的外排,此结果间接反映了Lx2-32c在MX-1/T细胞内的蓄积多于Paclitaxel的蓄积。10uMVerapamil与不同剂量Paclitaxel合用72h后,可明显逆转MX-1/T细胞对Paclitaxel的耐药性,逆转倍数达38.89倍,而其与不同剂量Lx2-32c合用72h后,对Lx2-32c的IC50值影响不大,结果提示P-gp在MX-1/T细胞对Paclitaxel产生耐药的过程具有重要地位,而对Lx2-32c在耐药细胞内的蓄积影响不大。
药物与P-gp作用的另一个重要方面是二者的结合能力。计算机辅助设计提示,Lx2-32c与P-gp的结合力较弱,Fitness评分为31.41,显著低于Paclitaxel与P-gp的Fitness评分(36.90)。P-gp ATPase的测定也表明与Paclitaxel处理组相比,Lx2-32c三个剂量组(20,100,500nM)产生的荧光强度均明显增加,说明ATP的消耗量减少,由于P-gp对底物的转运有赖于ATP的水解,故此结果间接反映了P-gp对Lx2-32c的外排较Paclitaxel组减少,与前述实验结果吻合。另外,由于Lx2-32c处理组产生的荧光强度呈剂量依赖性降低,说明Lx2-32c在耐药细胞内增加的蓄积并不是由于其具有抑制P-gp功能的作用。结合其他实验,我们推测,Lx2-32c在细胞内的蓄积较Paclitaxel增加的原因在于其与P-gp亲和力较低,故P-gp对其外排亦降低,因此Lx2-32c可在P-gp高表达的耐药细胞中仍保持有效浓度,这是其具有抗耐药活性的最主要机制。
解除耐药细胞的凋亡抑制是抗耐药药物研究的一大思路,我们也考察了Lx2-32c对MX-1/T细胞的凋亡诱导作用。流式细胞术显示不同剂量的Lx2-32c可使MX-1/T细胞产生G2/M期阻滞,染色体数目不稳定性及凋亡峰(亚G1峰)的出现。Annexin V-FITC/PI双染法定量地检测了Lx2-32c(20、100、500nM)作用48h产生的凋亡百分比,分别为14.09%、30.67%和63.52%。利用DAPI染色及AO/EB染色均可观察到Lx2-32c作用后MX-1/T细胞呈现的典型凋亡形态,如核固缩,核碎裂等。DNA ladder实验可见Lx2-32c诱导细胞DNA片段化,产生明显的梯形条带。JC-1染色及DiOC6染色均显示Lx2-32c对细胞线粒体膜电位的降低作用。Western Blot结果显示,Lx2-32c可剂量依赖性地升高Bax/Bcl-2比例,使细胞色素C、AIF从线粒体释放到胞浆,进而活化Caspase-9,-3,和PARP,启动内源性凋亡通路,而对Fas, FasL、Caspase-8的表达无影响。
最后,我们检测了Lx2-32c对肿瘤细胞侵袭转移及血管生成的影响。划痕-愈合试验结果表明,50nM Lx2-32c持续作用24h,明显抑制MX-1/T细胞侧向迁移能力；粘附试验结果表明,10nM及50nM Lx2-32c预处理30min,可明显抑制MX-1/T细胞与人工基底膜成分Matrigel的粘附；明胶酶谱法结果表明,Lx2-32c作用24h,可在一定程度上抑制人乳腺癌细胞MDA-MB231细胞分泌MMP-9及MMP-2,人纤维肉瘤细胞HT-1080分泌aMMP-9,以及人黑色素瘤细胞分泌MMP-2。另外,10nM及50nM Lx2-32c可显著减弱人脐静脉内皮细胞融合细胞EA-hy926的铺展能力及体外形成血管环的能力,显示出一定的抗血管生成作用。
综上所述,Lx2-32c是一种具有确切的体内外抗耐药活性的新型紫杉烷类化合物,其抗耐药机制可能与促进微管聚合,降低Tau蛋白表达,较少被P-gp外排从而能保持较高的细胞内药物蓄积浓度,以及诱导耐药细胞凋亡有关,同时,Lx2-32c亦显示出一定的体外抗肿瘤侵袭转移及抗血管生成的作用。
第二部分紫杉醇耐药的人胃腺癌BGC-823/TA细胞株及BGC-823/Paclitaxel裸鼠异种移植瘤模型的建立及耐药机制的探讨
本部分研究主要是建立并鉴定了对紫杉醇耐药的人胃腺癌BGC-823/TA细胞株及体内耐药的BGC一823/Paclitaxel裸鼠异种移植瘤模型,并对其耐药机制进行了探讨。
本研究选用人肺腺癌细胞株BGC-823作为亲本细胞,以紫杉醇对BGC-823细胞的IC1。为初始诱导剂量,经紫杉醇小剂量逐步增加剂量诱导和克隆选择,18个月后,成功建立了能在含2.00×10-8rnol/L紫杉醇的培养基中正常生长和传代的耐药株BGC-823/TA,耐药倍数为92.31倍。MTT法检测显示BGC-823/TA细胞对Docetaxel. Vincristine.Adriamycin及Lx2-32c等多种细胞毒类化合物也具有出不同程度的交叉耐药性,表现出典型的多药耐药特征。与亲本BGC-823细胞相比,BGC-823/TA细胞的细胞形态、细胞核形态、细胞周期分布、运动能力,粘附能力等基本生物学特征无明显改变,但生长速率较亲本细胞略微减慢,集落形成能力也有所降低,集落形成率分别为62.88%和74.38%。间接免疫荧光检测显示两株细胞内微管蛋白形态无明显改变,Western Blot法检测显示,BGC-823/TA细胞内p微管蛋白亚型(βⅠ、βⅡ、βⅢ、βⅣ微管蛋白)的表达与亲本BGC-823细胞无明显差异,两株细胞内Tau蛋白的表达量也差别不大。
P-gp.MRP及LRP等耐药蛋白的表达及活性是细胞多药耐药性形成的重要机制。RT-PCR检测发现BGC-823/TA细胞MDR1表达显著升高,间接免疫荧光法与Western Blot法均显示BGC-823/TA细胞P-gp高表达,Western Blot法还显示BGC-823/TA细胞内其它药泵蛋白如MRP.BCRP.LRP表达未见变化。
P-gp蛋白的功能研究证实BGC-823/TA细胞泵出Rodaminel23.阿霉素及Flutax-1的能力强于BGC-823细胞。质谱检测显示BGC-823/TA细胞内Paclitaxel含量明显低于BGC-823胞内含量,且脱药2h后BGC-823/TA细胞内Paclitaxel含量下降更为明显。MTT法检测显示同时加入药泵抑制剂Verapamil后BGC-823/TA细胞可恢复对Paclitaxel的敏感性。
上述研究结果表明,BGC-823/TA细胞是一株获得性多药耐药细胞株,其耐药机制主要由于MDR1mRNA的表达增高导致细胞表面P-gp蛋白过表达,从而使P-gp的药物外排泵功能亢进,最终使胞内Paclitaxel有效浓度降低,出现耐药现象。
与体外模型相对应,我们同时建立了对紫杉醇耐药的人胃腺癌BGC-823裸鼠异种移植瘤模型,该裸鼠模型经历15代传代,紫杉醇的诱导剂量由1Omg/kg提高至30mg/kg,最终得到体内耐药模型BGC-823/Paclitaxel。裸鼠异体移植瘤实验证实,耐药的BGC-823/Paclitaxel肿瘤生长速率比亲本BGC-823肿瘤略快,但无显著性差异。给予20mg/kg Paclitaxel治疗后,BGC-823/Paclitaxel肿瘤的抑制率及RTV均明显低于BGC-823肿瘤,体现出一定的耐药性。对BGC-823/Paclitaxel瘤块进行WesternBlot检测,耐药肿瘤出现P-gp表达增高,且随传代次数的增加,药物对肿瘤不断诱导,耐药肿瘤与亲本肿瘤间的P-gp表达差异愈明显,说明P-gp蛋白的过表达可能是导致BGC-823/Paclitaxel肿瘤对紫杉醇耐药的一个重要原因。
Paclitaxel, fistly isolated from Taxus brevifolia in1971, was a natural product with anti-cancer activity and unique mechanism of action. Paclitaxel binds to and stabilizes microtubules against depolymerization and inhibits cell proliferation by disrupting normal mitotic spindle formation. From its introduction in1992, paclitaxel has been crowned as one of the most active antineoplastic agents against a a board spectrum of malignancies, including ovarian, breast, lung, head and neck cancers and Kaposi' Ssarcoma. However, there are some problems delay the pace of its development, such as poor solubility, limitied resource, and resistance to paclitaxel which is frequently observed in patients and poses a major impediment to successful therapy. Therefore, researches on paclitaxel resistance arouse wide concern and the development of novel agents with definite anti-resistance activity is of great importance. Resistance to paclitaxel has been attributed to several mechanisms including overexpression of the efflux pump P-glycoprotein, alterations in the expression of (3-tubulin isotypes and mutations at the taxane binding site in microtubule, alterations in the apoptosis signaling pathway, as well as overexpression of TRAG-3.
There are two major strategies to combat tumor resistance.The first one is the development of MDR modulators whicn could restore the sensitivity of MDR tumor cells by inhibiting the function of ABC transporter proteins.In contrast, the other method is the development of anti-resisitance agents which could directly eradicate the tumor cells by themselves.Recently, the anti-resistance research attract more and more attention.Since China is a country with abundant natural resourse, endeavous to discover novel anti-resistance agent from natural products become major focus of research.
This article consists of two parts:Part I Studies on anti-drug-resistance activities and mechanisms of action of Lx2-32c, a novel taxane; Part Ⅱ The establishment of paclitaxel-resistant BGC-823in vitro cell line and in vivo xenograft model and the research on their mechanisms of resistance.
For our unremitting pursuit of novel agents with anti-drug-resistance activities, we reorganized and qualified the structure of cephalomannine, which is a natural congener of paclitaxel, and obtained a series of new compounds.Lx2-32c,2-debenzoyl-2-(3-azido-benzoyl)-7-propi-onyl-10-deacetylcephalo mannine (fig.1), was screened out from these compounds for its outstanding cytotoxicity. This part is to evaluate the anti-resistance activity of Lx2-32c, in vitro and in vivo, and to explore its possible mechanisms.
In vitro, Lx2-32c exerted extensive cytotoxicity to both sensitive and resistant cells from different origins, including human oral epidermoid carcinoma cell line(KB)and its resistant cell line(KB/V), human hepatocellular carcinomacell line(Bel-7402) and its resistant cell line(Bel-7402/5-Fu), human lung adenocarcinoma cell line(A549) and its resistant cell line (A549/T), human breast cancer cell lines(MX-1,MCF-7) and their resistant cell lines (MX-1/T,MCF-7/T), human gastric cancer cell line(BGC-823) and its resistant cell line(BGC-823/T),with an IC50value of29.61±5.76nmol/L (ranging from1.02to98.92nM). For further study, we selected MX-1/T cell line due to its stable resistance.The GI50of Lx2-32c in MX-1/T cell was67.35nM by SRB assay and IC50of Lx2-32c was5.33nM by clone formation assay, showing that the growth of MX-1/T cell was inhibited significantly by Lx2-32c.
In vivo, Lx2-32c administration inhibited the growth of tumors of Paclitaxel-resistant MX-1nude mice model in a dose-dependent manner.Lx2-32c administered at15,30and45mg/kg/day resulted in a40.11%、63.46%and77.67%inhibition in tumor growth, respectively. What is more, Lx2-32c showed great efficacy in the induction of apoptosis in MX-1/T tumor by TUNEL assay.
The effect of Lx2-32c on microtubule morphology and dynamic balance in MX-1/T cells were checked by immunofluorescence assay and Western Blot analysis. Lx2-32c (20,100nM) showed a typical taxane-like effect on the network with rare characteristic spindle poles and formation of thick microtubule bundles. Multinucleated cells were also observed by PI staining. Western blot assay showed that Lx2-32c promote the microtubule state from soluble to insoluble, thereby stabilizing microtubule. Meanwhile, Lx2-32c could also inhibit the expression of Tau in MX-1/T cells.
P-gp plays an important role in the development of drug resistance. RT-PCR and western blot analysis displayed no difference in the expression of MDRland P-gp upon Lx2-32c treatment. Then, we evaluated the accumulation of Lx2-32c in MX-1/T cells by MS and Rhodamine123accumulation assay. The results indicated the much greater accumulation of Lx2-32c than Paclitaxel. Verapamil reverse assay showed that the inhibition of P-gp has little effect on the cytotoxicity of Lx2-32c, suggesting a slighter influence of P-gp on Lx2-32c.
Then, we explore the interaction of Lx2-32c and P-gp. The result of molecular docking showed that Lx2-32c could bind with ATP pocket of P-gp by hydrophobic ineraction, but the binding is much weaker than the one between Paclitaxel and P-gp. Pgp ATPase assay reflected indirectly the less efflux of Lx2-32c, in contrast to the efflux of Paclitaxel.From these results, we speculated that the difference of efflux between Lx2-32c and Paclitaxel lied in the difference of their affinity toward P-gp.
Strategies in trying to combat resistance by modulating aberrant apoptosis pathways would prove effective and is attracting extensive attention. In this study,we also evaluated the induction of apoptosis by Lx2-32c.The results showed that Lx2-32c treatment led to dramatic cell cycle arrest in G2/M phase and typical apoptosis characters indicated by morphological changes and DNA fragmentation Apoptosis induced by Lx2-32c was associated with loss of mitochondrial membrane potential, enhancement of mitochondrial cytochrome c and AIF release, elevation of the Bax/Bcl-2ratio, activation of caspase-9,-3, but not caspase-8, and degradation of PARP.
Finally, we evaluated the effect of Lx2-32c on the metastasis and angiogenesis. A wound healing assay showed that Lx2-32c treatment (50nM) could inhibit the migration of MX-1/T cells. Adhesion assay showed that Lx2-32c reduced the adhesion of MX-1/T cells to Matrigel. What is more,to reveal the mechanisms of the inhibition role of Lx2-32c on tumor cell metastasis, the activity of MMPs protein was investigated by a Gelatin zymography assay. Results showed that Lx2-32c could significantly block the secretions of MMPs in MDA-MB231cells, HT1080cells and A375cells. Meanwhile, Lx2-32c also displayed impressive activity in anti-angiogenesis by in vitro tube forming assay.
In conclusion, our study demonstrated that Lx2-32c is a microtubule-stabilizing agent with gorgeous efficacy in suppressing the proliferation of MX-1/T cells, and other drug-resistant cell lines. Moreover, Lx2-32c displayed potent antitumor activity on paclitaxel-resistant MX-1in vivo model. Its mechanism of action may be associated with improved activity in promoting microtubule polymerization, inhibition of Tau expression, weaker affinity towards P-gp and induction of apoptosis via intrinsic apoptotic pathway.
Part Ⅱ is about the establishment of a Paclitaxel-resistant human gastric adenocareinoma cell line BGC-823/TA, as well as Paclitaxel-resisitant BGC-823/Paclitaxel in vivo nude mice model, and the exploration of their possible mechanism of resistance.
For in vivo part, BGC-823/TA was selected by continuous exposure to increasing Paclitaxel concentration from the IC10of Paclitaxel to2×10-8mol/L. The IC50of Paclitaxel to BGC-823and BGC-823/TA were1.79nM and165.24nM,respectively. The resistance index was92.31fold. This cell line also showed muti-drug resistance to many other chemotherapy agents such as Docetaxel、Vincristine、Adriamycin and Lx2-32c.
In contrast to parent BGC-823cell line, there were little changes of BGC-823/TA in morphorlogy、cell cycle distribution、motility and adhesion ability, but the proliferation of BGC-823/TA cell was relatively slow. The result of growth curve showed a delay of time for reaching peak value and lower peak value. In clony formation assay. The rate of clone formation were62.88%and74.38%respectively. Meanwhile, little difference in the state of microtubule, expression of Tau and β-tubulin isotypes was detected by indirect immunofluorescence assay and Western blot analysis, respectively.
Overexpression of P-gp, MRP and LRP is the major mechanism underlying MDR. The results by RT-PCR assay indicated the overexpressionl of MDR1in BGC-823/TA cells. Western Blot showed that the level of P-gp was significantly higher in BGC-823/TA cells while the level of MRP、LRP、BCRP remain the same when compared with their expression in BGC-823cells.
Using Rodamine123、ADR and Flutax-1accumulation assay, we found that the activity of drug flux pump in BGC-823/TA cells was stronger than the one in BGC-823cells. Similar results were obtained by MS assay, which indicating the cellular residue of Paclitaxel in BGC-823/TA cells was lower than that in BGC-823cells, and the residue rate in BGC-823/TA cells after incubation without Paclitaxel was also less than that in BGC-823cells. What is more,the sensitivity of BGC-823/TA cells lto Paclitaxel could be restored by Vempamil, which could inhibit the efflux of Paclitaxel by P-gp.
In conclusion, the BGC-823/TA cell line established by myself was a typical What is more, Lx2-32c also displayed definite activity of anti-metastasis and anti-angiogenesis. multi-drug resistance cell line against several cytotoxic agents. The mechanism of its resistance was implicated with the overexpression of MDR1mRNA and the overexpression of P-gP as well as the hyperfunction of drug efflux pump. As a result, the intercellular concentration of Paclitaxel decreased and led to the acquired resistance.
Accordingly, we established the Paclitaxel-resistant BGC-823nude mice model. For this in vivo model, tumors developed resistance by continuous paclitaxel induction from generation to generation after fifteen passages, rather than a single injection of resistant cell lines which had gained resistance by in vitro culture. The induction dose of Paclitaxel was from15mg/kg to30mg/kg. After Paclitaxel treantment of20mg/kg, the inhibitory rate of the growth of BGC-823/Paclitaxel tumor was significantly lower than that of BGC-823tumors, suggesting the resistant phenotype of BGC-823/Paclitaxel tumor. Western blot assay indicated that the expression of P-gp was higher in BGC-823/Paclitaxel tumors than that in BGC-823tumors, and the difference of expression increased by the passage, indicating that P-gp played an important role in the resistance of BGC-823/Paclitaxel tumors.
引文
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