新型紫杉烷类化合物Lx2-32c的抗肿瘤作用及机制研究

【作者】 王洪波；

【导师】 陈晓光； 李燕； 程桂芳；

【作者基本信息】 中国协和医科大学 ， 药理学， 2008， 博士

【摘要】 癌症已经成为全球威胁人类健康的首要疾病之一,2007年美国癌症协会统计显示,发达国家癌症紧随心血管疾病位居死亡率第二位,在发展中国家死亡率位居心血管疾病与慢性感染性疾病之后的第三位。统计显示2007年全球新增癌症病例1200多万,2007年总癌症死亡病例大约760万。2007年我国卫生部卫生统计显示,城市、乡镇死亡率排名中癌症同样高举榜首,癌症正在威胁我国广大人民的生命与健康。迄今为止,临床针对癌症患者的治疗除了外科手术治疗、放射治疗外,化疗依然是治疗肿瘤患者最有效的方法。靶向微管动态平衡的抗有丝分裂药物目前在临床多种实体肿瘤治疗中处于重要的地位。按照对微管蛋白的作用方式分为抑制微管聚合的长春碱类、与促进微管聚合的紫杉烷类药物。紫杉醇（Paclitaxel）是第一个用于临床治疗的紫杉烷类药物,它是从红豆杉植物中发现的抗肿瘤天然药物,紫杉醇主要通过与肿瘤细胞微管蛋白β结合,促进微管聚合抑制微管解聚,使肿瘤细胞阻滞于G₂/M期,诱发肿瘤细胞凋亡或坏死而达到抗肿瘤的效果。经过漫长的临床前及临床研究后,1992年被美国FDA批准用于治疗复发的和难治的乳腺癌和卵巢癌,以及肺癌和头颈部肿瘤。尽管紫杉醇临床治疗中已获得巨大成功,但临床使用过程中亦发现一些缺点亟需克服。其一是紫杉醇的水溶性差,需要用蓖麻油作为助溶剂,这样就增加了紫杉醇使用时带来的不良反应。其二是易产生严重的耐药现象。主要表现为对某些肿瘤及继发性耐药肿瘤病人的化疗无效。基于上述原因,针对微管为治疗靶点的新型抗肿瘤药物的开发与研究一直都是抗肿瘤药物的研究热点,研究的主要目标是发现能够克服耐药,并具有较好溶解特性和更佳药理活性的新型药物。植物、微生物以及海洋生物等天然产物一直都是抗肿瘤药物发现的主要来源。分离提取天然产物中有效成分,进行相应的结构修饰是当前抗癌药物研究的热点,同时对现有化合物进行结构修饰也是抗癌药物研究的热点之一。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一系列全新紫杉烷类衍生物,其中以代号为Lx2-32c的化合物活性最强。我们已对这类化合物申请了中国化合物专利（申请号200610080890.7）与国际PCT专利（申请号PCT/CN2007/003235）,以便在国际上获得更大范围的保护。本文对Lx2-32c的体内外抗肿瘤作用及其主要作用机制进行了较深入的探讨,结果如下:体外研究中,Lx2-32c可抑制多种不同来源肿瘤细胞的生长,如人口腔上皮癌细胞KB及耐药株KB/V、人肺腺癌细胞A549及耐药株A549/Paclitaxel与AT1、人卵巢癌细胞A2780、人宫颈癌细胞Hela、人胃癌细胞BGC-803、BGC-823及MGC-803、人肝癌细胞Bel-7402及耐药株Bel-7402/5-Fu等,MTT法测得其在体外的半数抑制浓度IC₅₀为0.50～10 nmol/L。SRB法测得的GI₅₀为0.13～4.79 nmol/L。Lx2-32c可剂量依赖性地抑制A549、A2780及两株耐药的A549/Paclitaxel细胞的生长,并可显著抑制上述细胞集落形成（P＜0.05）。体内实验表明Lx2-32c能够抑制小鼠Lewis肺癌的生长。Lx2-32c（2.5、5、10mg/kg）对小鼠Lewis肺癌生长的抑制率分别为27.77%、32.46%及76.08%（P＜0.01）,呈现较好的剂量效应关系。体内裸鼠异体移植瘤模型中Lx2-32c对三种组织来源的肿瘤都呈现明显的生长抑制活性。其中Lx2-32c（7.5、15、30 mg/kg）对人胃癌BGC-823的生长抑制率分别为30.90%、57.99%和94.44%（P＜0.01）;相同剂量对人肺腺癌A549的生长抑制率分别为14.90%、41.70%和63.95%（P＜0.05）;同样剂量下对人卵巢癌A2780的抑制率分别为43.55%、42.47%和60.61%（P＜0.05）。体内实验中观察到Lx2-32c具有一定的毒性反应。采用DAPI细胞核染色及流式细胞术等技术观察了Lx2-32c对多种细胞周期的影响。流式细胞术显示,Lx2-32c能够诱导多种组织来源的肿瘤细胞发生G₂/M期阻滞,呈现较好的剂量效应关系及时间效应关系。特别对于耐药株A549/Paclitaxel细胞,Lx2-32c仍能够有效地抑制其细胞周期,表现出较强的周期抑制活性。DAPI细胞核染色结果发现,Lx2-32c能明显抑制A549细胞及其耐药株的有丝分裂,使细胞停滞在有丝分裂期。同时观察到部分细胞出现核碎裂现象,提示Lx2-32c能够诱导肿瘤细胞凋亡。利用相对提纯的微管蛋白,采用比浊法及纯化的微管蛋白用荧光探针的方法,探讨了Lx2-32c对微管蛋白动力学的影响。比浊法研究结果表明,Lx2-32c能明显地促进微管蛋白的聚合,同时能够明显地抑制聚合的微管蛋白发生解聚,两者都呈现较好的剂量效应关系。应用DAPI作为荧光探针研究显示Lx2-32c同样能明显地促进微管蛋白的聚合,作用的EC₅₀为2.45μmol/L,而紫杉醇的EC₅₀为10.26μmol/L,Docetaxel的EC₅₀为2.53μmol/L。两种实验中都能看到Lx2-02c诱导微管聚合的速率明显强于Docetaxel与Paclitaxel,Lx2-02c的最大作用效应亦强于后两者。应用微管蛋白间接免疫荧光法及Western Blot法检测细胞微管的原位聚合,以探讨Lx2-02c对细胞内微管动态平衡的影响。免疫荧光实验结果表明,Lx2-32c破坏肿瘤细胞的正常形态,阻碍细胞内纺锤丝的形成,诱导细胞内微管形成微管束,打破细胞的有丝分裂,作用方式与紫杉醇相似。同时对耐紫杉醇的耐药株具有较强的作用。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,结果同间接免疫荧光结果类似。说明Lx2-32c打破细胞内微管的正常动态平衡,使微管向着聚合态偏移。采用Flutax-1作为荧光探针以寻找Lx2-32c在微管蛋白上的结合位点。使用聚合好的微管应用Flutax-1作为探针竞争实验结果发现,Lx2-32c能够与Flutax-1竞争微管蛋白上的结合位点,表明Lx2-32c与Flutax-1具有相同的结合位点,即Lx2-32c的结合位点与Paclitaxel的结合位点相同,同时计算出Lx2-32c与微管蛋白的结合常数（KD）为7.38±0.16×10⁷mol/L。使用Hoechst 33258荧光染色及Western Blot方法分析Lx2-32c对A549细胞凋亡的影响。结果显示Lx2-32c作用24小时后,细胞出现核浓缩、核碎裂等典型的凋亡征象,Western blot分析显示Lx2-32c作用24 h后能够诱导凋亡促进蛋白P53、Bax表达增高。综上所述,Lx2-32c在体外可广泛抑制不同组织来源的肿瘤细胞的生长;在体内,Lx2-32c能够明显抑制小鼠Lewis肺癌的生长,同时还能抑制人肺腺癌A549细胞、人胃癌BGC-823及人卵巢癌A2780裸鼠异体移植瘤的生长。Lx2-32c的抗肿瘤作用与促进微管蛋白聚合,抑制微管解聚,抑制细胞内微管动力学平衡,阻滞细胞周期,诱导凋亡相关蛋白P53、Bax表达有关。同时研究发现Lx2-32c在微管蛋白的结合位点与Paclitaxel结合位点相同。目的:对人肺腺癌A549/Paclitaxel耐药细胞株的生物学性状进行鉴定并对其耐药机制进行初步探讨。方法:MTT法检测A549/Paclitaxel细胞对多种细胞毒类药物的耐药性。比较两株细胞生物学性状的差异;形态学、集落形成能力、生长曲线及细胞周期分布;采用间接免疫荧光方法观察细胞内微管存在状态;采用RT-PCR方法检测两株细胞内耐药相关基因MDR1及MRP mRNA的表达情况;同时采用间接免疫荧光法与Westernblot法检测两者药泵蛋白P-gp表达情况;采用Western blot法检测细胞内磷酸化表皮生长因子受体及磷酸化AKT的表达情况;使用Rodamine123及Flutax-1比较两者药泵功能;采用HPLC方法对两者胞内Paclitaxel进行定量分析;最后采用MTT法检测药泵抑制剂对A549/Paclitaxel细胞耐药性的影响。结果:A549/Paclitaxel细胞对Paclitaxel、Docetaxel、Vincristine、Topotecan、Adriamycin、Cephalomannine及Lx2-32c等多种细胞毒类化合物显示出不同程度的耐药性,表现出典型的多药耐药性状。A549/Paclitaxe细胞与A549细胞相比:形态无显著变化,集落形成能力降低（集落形成率分别为52%和78%）,细胞生长曲线与细胞周期分布基本相似,细胞内微管的存在状态偏向稳定态。RT-PCR检测发现A549/Paclitaxel细胞MDR1表达显著升高,MRP表达无明显的改变。间接免疫荧光法与Western Blot法皆发现A549/Paclitaxel细胞药泵蛋白（P-gp）的表达量明显高于A549细胞,同时发现p-EGFR及p-AKT表达较A549细胞有显著地增高。药泵功能检查证实A549/Paclitaxel细胞泵出Rodamine123及Flutax-1的能力强于A549细胞。HPLC检测显示A549/Paclitaxel胞内Paclitaxel含量明显低于A549胞内含量,说明耐药细胞对Paclitaxel的外排活性大大强于后者。MTT法检测显示同时加入药泵抑制剂后A549/Paclitaxel细胞恢复对Paclitaxel的敏感性。结论:A549/Paclitaxel细胞是一株获得性多药耐药细胞株,其耐药机制主要由MDR1mRNA的表达增高而致细胞表面P-gp蛋白表达增高,使细胞药物外排泵功能亢进,外排进入胞内的Paclitaxel,使胞内Paclitaxel有效浓度降低,从而引起细胞对Paclitaxel的耐药,同时A549/Paclitaxel的耐药机制也可能与EGFR及AKT的磷酸化水平增高有关。更多还原

【Abstract】 Cancer has been one of the top killers in the world and threatens many patients’ life. Global statistics shows that in 2007 worldwide,there are more than 12 million new cases; the estimatment for total cancer deaths are 7.6 million.Chinese Ministry of Health statistics shows that in 2007 cancer has been the top mortality rate both in the city and in the country.Besides surgery,radiotherapy and "biological" treatment,chemotherapy is still the most effective means to improve the patients’ life quality and prolong survival.Antimitosis agents targeting the dynamic equilibrium between the microtubule polymer and tubulin heterodimers are key components of chemotherapeutic regiments for various solid tumors.The strategy of using tubulin as a target for cancer chemotherapy was based on the decreased growth and division of cancer cells and the fact that drugs that interfered with mitosis such as the Vinca alkaloids that shifted the equilibrium to the depolymerized form of tubulin had proven effective in the treatment of cancer.Paclitaxel target tubulin but,unlike the Vinca alkaloids,shifted the equilibrium to the polymerized form,thus stabilizing microtubules.Paclitaxel,isolated from Taxus brevifolia,could stabilize microtubules and at stoichiometratic concentration enhance microtubules polymerization.From its introduction in 1992,paclitaxel has established itself as one of the most active antineoplastic agents against a wide spectrum of malignancies,including ovarian,breast,lung,and head and neck cancers and Kaposi’s sarcoma.Although paclitaxel is effective for management of different malignancies,resistance to paclitaxel frequently develops in some chemotherapy.In this case,there remains a significant unmeted medical need to develop new agents that overcome drug resistance and have improved pharmacology profiles.Natural products including plants,microorganisms and halobios provide rich resources for discovery of anticancer drugs.Cephalomannine is a natural congener of paclitaxel,and was isolated in the 1970s from Taxus wallichiana,which was erroneously assigned as Cephalotaxus manii at the time of its discovery.As part of our continuing effort to discover novel anticancer agents from natural products,we reorganized and qualified the structure of cephalomannine and got a series of new compounds. Lx2-32c was identified through the antiproliferation profiles screen in vitro.In the present study,we investigated the antineoplastic activities of Lx2-32c in vitro and in vivo.The results are as follows: In vitro,Lx2-32c was found to significantly inhibit the growth of cancer cells derived from different tissues,including human oral epidermoid carcinoma cells（KB） and its resistance cells（KB/V）,human hepatocellular carcinoma cells（Bel-7402） and its resistance cells （Bel-7402/5-Fu）,human lung adenocarcinoma cells（A549） and its resistance cells （A549/Paclitaxel）,human ovarian cancer cells（A2780）,human gastric cancer cells （BGC-823,BGC-803 and MGC-803）,human uterine cervix cancer cells（HeLa）.MTT assay showed that its IC₅₀ toward these tumor cells was 0.5～10.0 nmol/L.GI₅₀ evaluated by SRB assay was 0.13～4.79 nmol/L in four human cancer cell lines（A2780,KB,A549 and A549/Paclitaxel）.Additionally,the colony formation abilities in A2780,A549 and A549/Paclitaxel cells were inhibited significantly by Lx2-32c.In vivo,Lx2-32c administered by i.p.inhibited,the growth of Lewis lung cancer in C57/BL6 mice in a dose-dependent manner,.Lx2-32c administered at 2.5,5 and 10 mg/kg/day caused a 27.77%,32.46%and 76.08%inhibition in Lewis lung tumor growth, respectively.In addition,human cancer cell transplant models,BGC-823,A549 and A2780,in nude mice were used to evaluate the antitumor properties of Lx2-32c in vivo. Administered at 7.5,15 and 30 mg/kg every three days,Lx2-32c inhibited tumor growth 30.90%,57.99%and 94.44%in BGC-823 transplant tumor（p＜0.01 compared with vehicle-treated animals）.To further demonstrate the activity of this candidate,we tested it in other two tumor models（A549 and A2780）.In A549 tumor model,Lx2-32c given at 7.5,15 and 30 mg/kg every three days restrained the tumor growth at 23.08%,47.30% and 67.41%（p＜0.01 compared with vehicle-treated animals） respectively.Under the identical doses,Lx2-32c given caused a similar inhibition to the growth of A2780 tumor （43.55%,42.47 and 60.61%,respectively）.The effects of Lx2-32c on the cell cycle were determined by DAPI dye and flow cytometry（FCM） with PI staining to reveal the total amount of DNA.FCM analysis showed all the used cells（including A2780,BGC-823,A549 and A549/Paclitaxel）,which were treated with Lx2-32c for 12 h or 24 h,arrested in G₂/M phase in a time-and dose-dependent manner.The typical manners of cell cycle block were observed by DAPI staining after exposed Lx2-32c for 24 h.All the data indicated that Lx2-32c could induce G₂/M phase arrest.For in vitro tubulin polymerization assays,dog brain microtubule-associated protein （MAP）-rich tubulin and the MAP-free tubulin were prepared following the protocol modified from Williams and Lee.The turbidimetry assay showed that Lx2-32c enhanced tubulin polymerization in a dose-dependent manner without any apparent delay,and the effect of 5μmol/L Lx2-32c on tubulin polymerization was similar to that of 10μmol/L Paclitaxel.Using DAPI as fluorescent probe,the polymerization with MAP-free tubulin shown the similar result to the above,and the EC₅₀ value for Lx2-32c and Paclitaxel was 2.45μmol/L and 10.26μmol/L respectively.The EC₅₀ value for Docetaxel was 2.53μmol/L.All the data showed that Lx2-32c had potential profile in promoting the tubulin.The effects of Lx2-32c on microtubule morphology and dynamic balance in cells were tested by immunofluorescence assay and Western Blot analysis respectively.After 24 h treated by Lx2-32c,the normal metaphase plates with characteristic spindle poles were rarely observed,and cells were usually rounded.Microtubule bundle were easily found in the treated cells.Western Blot assay displayed that Lx2-32c could promote the microtubule state from "soluble" to "insoluble",and disrupted the normal function of the microtubule.To confirm the binding site of Lx2-32c on the tubulin,the competition assay was performed using Flutax-1 as fluorescent probe.The results showed that Lx2-32c could inhibit the binding of Flutax-1 to tubulin polymer like Paclitaxel,and the apparent binding constants obtained for Lx2-32c was 7.38±0.16×10⁷ mol/L.So it can be presumed that Lx2-32c could share the same binding site with Paclitaxel.The apoptosis induced by Lx2-32c in A549 cells was determined by Hoechst 33258 staining,and the influence of Lx2-32c on the expression of apoptosis related protein was assayed by Western blot assay.Treatment with Lx2-32c or Paclitaxel could significantly induce typical apoptosis characteristics in A549 cell line.Western Blotting analysis was performed to observe the expression of apoptosis related proteins,P53 and Bax.The results showed that the protein expression fo P53 and Bax increased.In summary,Lx2-32c,a novel taxane derivative semisynthesised from cephalomannine,inhibited growth of various cancer cells in vitro and in vivo.Lx2-32c binded to Beta-tubulin and disrupted microtubule function during mitosis which in turn lead to mitotic arrest,followed by cell death induction through apoptosis. AIM:To investigate the biological characteristics of A549/Paclitaxel cells,one paclitaxel-resistant lung adenocarcinama cell line,and its primary mechanism to resistance.METHODS:The resistance of A549/Paclitaxel cells against several cytotoxic compounds were determined by MTT assay.The biological characteristics of A549/Paclitaxel cells were compared with that of its parent cells-A549.They included the morphology,the clony formation rate,the growth curve,the cell cycle analysis and the microtubule.The expressions of MDR1 and MRP mRNA were assayed by RT-PCR. Then the expressions of P-gp was studied by indirect immunofluorescence assay and Western blot assay,and the p-EGFR and p-AKT protein were also determined by Western Blot assay.The function of drug flux pump was assayed by using Rodamine123 and Flutax-1.The concentration of Paclitaxel in cells was quantified by using HPLC analysis. At last,the response of A549/Paclitaxel to Paclitaxel was measured by MTT assay when co-incubation with P-gp inhibitor.RESULTS:A549/Paclitaxel cells displayed resistance against Paclitaxel,Docetaxel, Vincristine,Topotecan,Adriamycin,Cephalomannine and Lx2-32c,which was one novel taxane.There were no significant differences in cellular biology of the morphology,the cell cycle distributions and the cell growth curve.It was found that the clony formation rate of A549/Paclitaxel cells was lower than that of A549 cells,and the microtubule in A549/Paclitaxel cells was more ’stable’ than that in A549 cells.Assayed by RT-PCR,the expression of MDR1 mRNA in A549/Paclitaxel cells was much higher than that in A549 cells,but the expression of MRP mRNA was similar between two cell lines.The expression of P-gp in A549/Paclitaxel cells was found higher than that in A549 cells by Western Blot assay and indirect immunofluorescence assay.Using Rodamine123 and Flutax-1,the activity of ’drug flux pump’ in A549/Paclitaxel cells was stronger than that in A549 cells.Similarly,assayed by HPLC,the cellular residue of Paclitaxel in A549/Paclitaxel cells was lower than that in A549 cells,and the residue rate in A549/Paclitaxel cells after incubation in the drug-free media was lower than that in A549 cells.The response of A549/Paclitaxel to Paclitaxel was reversed by Verapamil,which was a P-gp inhibitor.CONCLUSION:A549/Paclitaxel cell line established by our lab was a multidrug resistance cell line against several cytotoxic agents.For the over-expression of MDR1 mRNA and the over-expression of P-gp,the function of ’drug flux pump’ was activated, as a result,the intercellular concentration of Paclitaxel was cut down and resulted in the obtained resistance.更多还原