【作者】 刘小东；

【导师】 刘耕陶； 陈晓光； 程桂芳； 张建军；

【作者基本信息】 中国协和医科大学 ， 药理学， 2010， 博士

【摘要】 肿瘤多药耐药(multidrug resistance, MDR)是肿瘤治疗中面临的一大难题。肿瘤对化疗药物产生耐药性的机制十分复杂,主要包括：药物外排泵ABC转运蛋白(P-glycoprotein, Pgp等)过量表达,药物作用靶点的改变,DNA损伤修复活性增强,药物解毒、消除酶活性增强或过量表达及肿瘤细胞凋亡敏感性的降低等。本文探讨了两种汉防己甲素衍生物抑制P-glycoprotein逆转MDR及5-溴代汉防己甲素增强内在耐药的肝癌细胞Bel7402对阿霉素诱导凋亡的敏感性的作用机理。采用MTT方法检测两种汉防己甲素衍生物的细胞毒性,W6和W18发现对耐药的肿瘤细胞及其对应的亲本细胞(KBv200和KB细胞、MCF-7/Dox和MCF-7细胞、A549/Taxol和A549细胞)的细胞毒作用无显著性差异,提示W6和W18不是药物外排泵ABC转运蛋白(P-glycoprotein, Pgp等)的底物。Western blot检测与MDR关系最为密切的三种药物外排泵Pgp、MRP 1和BCRP在上述六种细胞和内在耐药的Bel7402细胞中的表达水平,发现Pgp在获得性耐药KBv200和MCF-7/Dox细胞中表达水平显著升高,在获得性耐药的A549/Taxol细胞中略有升高；MRP1蛋白在获得性耐药的A549/Taxl细胞及其对应的亲本细胞A549中表达水平显著升高,在获得性耐药的KBv200和MCF-7/Dox细胞中略有升高；BCRP蛋白在上述7种细胞中的表达水平都较低,在MCF-7/Dox和A549细胞中的表达水平高于其他细胞。无毒浓度的W6或W18 (0.25μM、0.5μM、1.OμM)能剂量依赖性的提高获得性耐药肿瘤细胞KBv200和MCF-7/Dox对Pgp底物长春新碱、阿霉素和紫杉醇的敏感性,1.0gM浓度下几乎完全逆转KBv200和MCF-7/Dox对上述三种抗肿瘤药物的耐药性,逆转活性强于10gM的Pgp底物竞争性抑制剂维拉帕米,W6或W18(0.25μM、0.5μM、1.OμM)也能提高获得性耐药的A549/Taxol对上述三种Pgp底物的敏感性,但逆转活性较低；W6或W18在1.0gM浓度下对过量表达MRP1蛋白的A549细胞对MRP1底物长春新碱和阿霉素的敏感性没有影响,提示W6和W18的MDR逆转活性与Ppg的过量表达水平关系密切,与MRP1蛋白和BCRP蛋白的过表达水平不相关,两种化合物W6和W18很可能通过抑制Pgp逆转多种耐药肿瘤细胞的耐药性。内在耐药的Bel7402细胞Pgp、MRP1和BCRP的表达水平都很低,但W6或W18 (0.25μM、0.5μM、1.0μM)仍能剂量依赖性的提高其对上述三种抗肿瘤药物的敏感性,提示Bel7402细胞内在耐药的作用机制很可能与Pgp、MRP1和BCRP的过表达无关,两种化合物W6和W18通过其它作用机制逆转Bel7402细胞MDR表型。采用阿霉素蓄积实验检测W6和W18对Pgp功能的影响,W6或W18(0.25μM、0.5μM、1.0μM)能剂量依赖性的增加阿霉素在Pgp过表达的KBv200和MCF-7/Dox细胞中浓度,抑制Pgp对阿霉素的外排作用。采用化学发光方法进一步检测W6和W18抑制Pgp功能的作用机制,W6 (12.5μM,25μM,50μM)能剂量依赖性的抑制基础水平的Pgp ATPase活性,也能抑制Pgp底物维拉帕米(200μM)刺激引起的升高的Pgp ATPase活性；W18 (12.5μM,25μM,50μM)对生理水平的Pgp ATPase活性没有影响,但是能抑制Pgp底物维拉帕米(200μM)刺激引起的升高的PgP ATPase活性,提示W6和W18不是Pgp的竞争性转运底物,而是通过抑制Pgp结合或水解ATP进而抑制其转运功能。Western blot检测两种汉防己甲素衍生物W6和W18对Pgp表达的影响,W6或W18(1.0gM)能时间依赖性的降低KBv200细胞Pgp的表达水平。RT-PCR检测W6和W18对mdr1基因转录水平的影响,W6或W18(1.0μM)对KBv200细胞Pgp的mRNA水平没有影响,对MRP1、BCRP和LRP的mRNA水平也没有影响,提示两种汉防己甲素衍生物W6和W18通过其他作用机制下调Pgp表达。鉴于肿瘤细胞凋亡敏感性降低在MDR中的作用,采用Western blot检测两种汉防己甲素衍生物W6和W18对凋亡存活通路中关键蛋白的影响,与敏感的KB细胞相比,耐药的KBv200细胞中PI3K/Akt存活通路活性显著降低,MAPK/Erk1/2存活通路活性显著升高,促凋亡蛋白p53表达水平略有降低,促凋亡蛋白Bax表达水平显著降低,抗凋亡蛋白XIAP表达显著升高,Bcl-2水平没有变化,W6或W18(1.0μM)能抑制Erk1／2存活通路活性,但随着时间的推移,抑制作用有所减弱；对PI3K/Akt存活通路、p53、Bax、XIAP和Bcl-2均没有影响。本研究还讨论了5-溴代汉防己甲素增强内在耐药肝癌细胞Bel7402对阿霉素诱导的凋亡的敏感性的作用机理。早期研究发现5-溴代汉防己甲素(BrTet)通过影响PgP的表达和功能在体外、体内有很强的肿瘤多药耐药(MDR)逆转作用。在对BrTet逆转MDR作用机制的进一步研究过程中发现BrTet能增强耐药肿瘤细胞Bel7402凋亡的敏感性,乃对BrTet增强凋亡敏感性的机制做进一步探讨。Bel7402细胞与不同浓度BrTet和阿霉素(Dox) (3μM)共培养,或者与Dox (3μM)或BrTet单独培养24h,以维拉帕米(Vrp) (10μM)作为阳性对照。采用Hochest33258染色法观察细胞形态学变化,PI染色流式细胞技术分析细胞周期和凋亡百分率,琼脂糖凝胶电泳分析细胞DNA片段化状态,caspase-3荧光底物Ac-DEVD-pNA检测caspase-3活性,Western blot检测凋亡相关蛋白Fas、FasL、caspase-8、caspase-3、cleaved caspase-3、Bax、Bcl-2的表达变化及线粒体膜间隙蛋白细胞色素C和AIF的释放,JC-1染色检测线粒体膜电位变化情况。Dox (3μM), BrTet (4μM)或Vrp (10μM)单独作用Bel7402细胞24 h,细胞均未发生凋亡。当BrTet (lμM,2μM,4μM)、Vrp (10μM)培养细胞24 h,去除药物,再加入DOX (3μM)继续培养24 h, Bel7402细胞出现明显凋亡,且凋亡百分率与BrTet的浓度呈剂量依赖性关系。相同的给药方案,BrTet (4μM),Vrp (10能明显增强DOX (3μM)诱导的caspase-3的活化,促使线粒体细胞色素C和AIF的释放,降低线粒体膜电位,提高Bax与Bcl-2相对比例；但对Fas、FasL和caspase-8的表达无影响。提示BrTet能增强Bel7402细胞对Dox诱导凋亡的敏感性,其机制可能是提高Bax与Bcl-2蛋白的比例,降低线粒体膜电位,进而影响线粒体的功能,激活细胞内源性凋亡通路,诱导凋亡综上所述,本研究中两种汉防己甲素衍生物W6和W18在体外能逆转多种耐药肿瘤细胞的MDR表型,作用机理是抑制P-glycoprotein的药物外排功能和过量表达,抑制MAPK/Erk1/2通路活性,它们的其它作用机制和体内活性值得进一步深入研究。此外,本研究中BrTet能增强Bel7402细胞对Dox诱导凋亡的敏感性,其机制可能是提高Bax与Bcl-2蛋白的比例,降低线粒体膜电位,进而影响线粒体的功能,激活细胞内源性凋亡通路。BrTet已完成Ⅰ期临床试验,正拟申请Ⅱ期临床试验,这为BrTet的临床研究提供了新的实验依据。更多还原

【Abstract】 MDR (multidrug-resistance) is a major obstacle in the chemotheraputy of cancer. Numerous mechanisms are known to contribute to MDR, including overexpression of drug efflux pumps, increased activity of DNA repair mechanisms, alteration of drug target enzymes, overexpression of enzymes involved in drug detoxification and elimination, and alterations at the level of apoptosis control of cancer cells. The present study focused on the reversing MDR effect of two compounds by inhibiting P-glycoprotein, and the mechanism of 5-bromotetrandrine enhancing the sensitivity of doxorubicin-induced apoptosis in intrinsic resistant human hepatic cancer Bel7402 cells.The cytotoxity of two derivatives of Tetrandrine W6 and W18 on resistant cells and their parent cells (such as KBv200 and KB, MCF-7/Dox and MCF-7, A549/Taxol and A549) was determined by MTT assay. Resistant cell lines and their parent cell lines had similar sensitivity to W6和W18, which shown that the two compounds seems not to be the substrates of drug efflux pumps. The expression of P-glycoprotein, MRP land BCRP in the six cell lines mentioned above and intrinsic resistant Bel7402 cells were detected by Western blot analysis. P-glycoprotein significantly overexpressed in KBv200 and MCF-7/Dox cells, and slightly overexpressed in A549/Taxol cells compared with other cell lines. MRP1 significantly overexpressed in required resistant A549/Taxol and its parent A549 cells compared with other cells. The expression of BCRP was relatively low in all cell lines, but the level of BCRP was higher in MCF-7/Dox and A549/Taxol than that of other cells. Nontoxic concentrations of W6 or W18 (0.25μM、0.5μM、1.OμM) obviously enhanced the sensitivity of KBv200 and MCF-7/Dox cells to vincristine (VCR), doxorubicin (Dox) and taxol which were the substrates of P-glycoprotein in a dose-dependent manner. 1.0μM W6 or W18 nearly completely reversed the KBv200 and MCF-7/Dox cells to the three anticancer drugs. The MDR reversal effect of W6 or W18 at 1.0μM was better than that of 10μM verapamil (Vrp). Though W6 and W18 also enhanced the sensitivity of A549/Taxol to the three anticancer drugs, the MDR reversal activity was low. However, 1.0μM W6 or W18 had no chemosensitising effect on A549 cells to VCR and Dox which are the substrates of MRP 1 and BCRP (R482G and R482T) respectively. It seems that the MDR reversal activities of W6 and W18 depended on the levels of over-expressing P-glycoprotein. Therefore the MDR reversal effect of the two derivatives based on Tetrandrine was possibly through the inhibition of P-glycoprotein.Flow cytometry determined the effect of the two compounds on the drug efflux function of P-glycoprotein. W6 or W18 (0.25μM、0.5μM、1.0μM) were able to inhibit the drug efflux function of P-glycoprotein, and significantly increased the accumulation of Dox in KBv200 and MCF-7 cells in a concentration dependent manner. Chemiluminescence method further determined the mechanism of the two compounds inhibiting function of P-glycoprotein. W6 (12.5μM、25μM、50μM) inhibited basal activity of recombinant human Pgp ATPase in a concentration dependent manner, and also inhibited the increased activity of recombinant human Pgp ATPase stimulated by Vrp (200μM) which is the reversible competitive substrate of P-glycoprotein. W18 (12.5μM、25μM、50μM) had no effect on the basal activity of recombinant human Pgp ATPase, but also inhibited the increased activity of recombinant human Pgp ATPase stimulated by Vrp (200μM) too. These results further suggest that the two compounds were not the substrates of P-glycoprotein, and they inhibited the drug efflux effect by inhibiting the activity of Pgp ATPase. Western blot analysis detected the effect of the two derivatives based on Tetrandrine on the expression of P-glycoprotein in KBv200 cells. W6 or W18 (1.OμM) was able to reduce the expression of P-glycoprotein in a time dependent manner. RT-PCR determined whether the two compounds reduced the expression of P-glycoprotein by regulating its transcription. W6 or W18 (1.OμM) had no effect on the levels of mRNA of Pgp as well as MRP1, BCRP and LRP. The mechanism of the two compounds reducing the over-expression might be through other pathways.Because alterations at the level of apoptosis control of cancer cells play a very important roll in MDR, Western blot analysis detected the effect of the two compounds on the key proteins involved in apoptosis regulation. Compared with its parent KB cells, PI3K/Akt pathway was low active and MAPK/Erkl/2 pathway was highly active in resistant KBv200 cells. W6 or W18 (1.OμM)had no effect on the PI3K/Akt pathway, but reduced the level of p-Erk1/2 to inhibit the activation MAPK/Erk1/2 survival pathway. Compared with its parent KB cells, pro-apoptotic protein p53 slightly reduced, pro-apoptotic protein Bax significantly reduced, anti-apoptotic protein XIAP obviously increased, and anti-apoptotic protein Bcl-2 did not change in KBv200 cells. W6 or W18 (1.0μM) had no effect on these proteins. It is concluded that the two derivatives of Tetrandrine W6 and W18 reversed MDR in vitro by inhibiting P-glycoprotein and MAPK/Erk1/2 survival pathway.The present study also investigated the mechanism of 5-bromotetrandrine enhancing the sensitivity of doxorubicin-induced apoptosis in intrinsic resistant human hepatic cancer Bel7402 cells.5-bromotetrandrine (BrTet) was shown to overcome multi-drug resistance (MDR) in vitro and in vivo by inhibiting the overexpression and efflux function of P-glycoprotein in our previous study. The purpose of the present study was to evaluate the effect of BrTet on the sensitivity of doxorubicin (Dox) induced apoptosis in intrinsic resistant human hepatic cancer Bel7402 cells. The cells were treated with non-cytotoxic concentrations of BrTet (1μM,2μM,4μM) or the positive control drug verapamil (Vrp) (10μM) for 24 h followed by a low dose Dox (3μM) for 24 h. The results showed that BrTet pretreatment followed by Dox led to typical apoptotic characters as indicated by morphologic changes, DNA fragmentation and changes in cell cycle, while the same dose of BrTet, Vrp and Dox alone did not induce apoptosis in Bel7402 cells. In addition, the pretreatment of BrTet or Vrp followed by Dox induced activation of caspase-3, release of cytochrome c and AIF from mitochondria into cytosol, loss of mitochondrial transmembrane potential(ΔΨm) and elevation of Bax/Bcl-2 ratio, with no effect on activation of caspase-8 and the expression of Fas/FasL. In conclusion, BrTet pretreatment enhanced the sensitivity of Dox to induce apoptosis by causing loss ofΔΨm and elevating the ratio of Bax/Bcl-2, eventually activated mitochondrial apoptotic pathway.In summary, the present study evaluated the MDR reversal activity of two Tetrandrine derivatives W6 and W18. They were shown to inhibit drug efflux function and overexpression of P-glycoprotein, and inhibit the highly activated MAPK/Erk1/2 survival pathway to sensitize several resistant cancer cells to anti-cancer drugs in vitro. Other mechanism and MDR reversal activity would be worthy to be further investigated. In addition, the present study also shown that BrTet pretreatment enhanced the sensitivity of Dox to induce apoptosis by causing loss ofΔΨm and elevating the ratio of Bax/Bcl-2, eventually activated mitochondrial apoptotic pathway. These findings further support the potential of BrTet to be used in clinical trail of cancer treatment.更多还原