汉防己甲素衍生物W6和W18逆转肿瘤多药耐药及BrTet增强Bel7402细胞凋亡敏感性的作用机制研究
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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。
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.
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。
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.
引文
[1]Brawley, O.W.& Kramer, B.S. Cancer screening in theory and in Practice [J].J Clin Oncol,2005,23(2):293-300.
[2]Mimeault, M.& Batra, S.K. Recent advances on multiple tumorigenic cascadesinvolved in prostatic cancer progression and targeting therapies [J]. Carcinogenesis,2006,27(1):1-22.
[3]Shiraishi J, Abe H, Li F, Engelmann R, MacMahon H, Doi K. Computer-aided diagnosis for the detection and classification of lung cancers on chest radiographs ROC analysis of radiologists'performance [J]. Acad Radiol,2006,13(8):995-1003.
[4]Chatterton, K, Ray, E.& O'Brien, T.S. Fluorescence diagnosis of bladder cancer [J]. Br J Nurs,2006,15(11):595-597.
[5]Petignat, P, Du Bois, A., Bruchim, I., Fink, D.& Provencher, D.M. Should intraperitoneal chemotherapy be considered as standard first-line treatment in advanced stage ovarian cancer?[J], Crit Rev Oncol Hematol,2006,62(2):137-147.
[6]Mimeault, M., Brand, R.E., Sasson, A.A.& Batra, S.K. Recent advances on the molecular mechanisms involved in pancreatic cancer progression and therapies [J]. Pancreas,2005,31(4):301-316.
[7]Lock-Andersen, J., Horn, J.& Sjostrand, H. Prognosis after sentinel node biopsy in malignant melanoma [J]. Ugeskr Laeger,2006,168(25):2457-2462.
[8]Shabbits JA, Hu Y, Mayer LD. Tumor chemosensitization strategies based on apoptosis manipulations [J]. Mol Cancer Ther,2003,2 (8):805-813.
[9]Szakacs G, Paterson JK, Ludwig JA, Booth-Genthe C, Gottesman MM. Targeting multidrug resistance in cancer [J]. Nat Rev Drug Discov,2006,5(3):219-34 101.
[10]Mimeault M, Hauke R, Batra SK. Recent advances on the molecular mechanisms involved in the drug resistance of cancer cells and novel targeting therapies [J]. Clin Pharmacol Ther.2008,83(5):673-691.
[11]Raguz S, Yague E.Resistance to chemotherapy:new treatments and novel insights into an old problem [J]. Br J Cancer,2008,99(3):387-391.
[12]Dean M, Rzhetsky A, Allikmets R. The human ATP-binding cassette (ABC) transporter superfamily [J].Genome Res,2001,11 (7):1156-66.
[13]Chang G, Roth CB. Structure of MsbA from E. coli:a homolog of the multidrug resistance ATP binding cassette (ABC) transporter [J]. Science,2001, 293(5536):1793-1800.
[14]Dassa E, Bouige P.The ABC of ABCS:a phylogenetic and functional classification of ABC systems in living organisms [J].Res Microbiol,2001,152(3-4):211-29.
[15]Perez-Tomas R.Multidrug resistance:retrospect and prospects in anti-cancer drug treatment [J]. Curr Med Chem,2006,13(16):1859-1876.
[16]Eckford PD, Sharom FJ.ABC efflux pump-based resistance to chemotherapy drugs [J].Chem Rev.2009,109(7):2989-3011.
[17]Polgar O, Bates SE. ABC transporters in the balance:is there a role in multidrug resistance? [J]. Biochem Soc Trans.2005,33(Pt 1):241-245.
[18]Goldstein LJ. MDR1 gene expression in solid tumours. Eur J Cancer [J].1996, 32A(6):1039-1050.
[19]Penson RT, Oliva E, Skates SJ, Glyptis T, Fuller AF Jr, Goodman A, Seiden MV. Expression of multidrug resistance-1 protein inversely correlates with paclitaxel response and survival in ovarian cancer patients:a study in serial samples [J]. Gynecol Oncol,2004,93(1):98-106.
[20]Mimeault M, Hauke R, Batra SK. Recent advances on the molecular mechanisms involved in the drug resistance of cancer cells and novel targeting therapies [J]. Clin Pharmacol Ther,2008,83 (5):673-91.
[21]Wang G, Lemos JR.Tetrandrine:a new ligand to block voltage-dependent Ca2+and Ca(+)-activated K+channels [J]. Life Sci,1995,56(5):295-306.
[22]He QY,Meng FH, Zhang HQ.Reduction of doxorubicin resistance by tetrandrine and dauricine in harringtonine-resis-tant human leukemia (HL60) cells [J]. Zhongguo Yao Li Xue Bao,1996,17:179-18
[23]Tian H, Pan QC.A comparative study on e□ect of two bisbenzylisoquinolines, tetrandrine and berbamine, on reversa of multidrug resistance [J]. Yao Xue Xue Bao,1997,32:245-250
[24]Garattini S. Pharmacokinetics in cancer chemotherapy [J]. Eur J Cancer,2007, 43(2):271-282.
[25]Mansilla S, Bataller M, Portugal J. Mitotic catastrophe as a consequence of chemotherapy [J]. Anticancer Agents Med Chem,2006,6(6):589-602.
[26]Dimri GP. What has senescence got to do with cancer? [J]. Cancer Cell,2005, 7(6):505-512.
[27]Higgins CF. ABC transporters:from microorganisms to man [J]. Annu Rev Cell Biol, 1992,8:67-113.
[28]Frelet A, Klein M. Insight in eukaryotic ABC transporter function by mutation analysis [J]. FEBS Lett,2006,580(4):1064-1084.
[29]Dawson RJ, Locher KP.Structure of a bacterial multidrug ABC transporter [J]. Nature,2006,443(7108):180-185.
[30]Bakos E, Evers R, Szakacs G, Tusnady GE, Welker E, Szabo K, de Haas M, van Deemter L, Borst P, Varadi A, Sarkadi B. Functional multidrug resistance protein (MRP1) lacking the N-terminal transmembrane domain [J]. J Biol Chem,1998, 273(48):32167-75.
[31]Yang Y, Liu Y, Dong Z, Xu J, Peng H, Liu Z, Zhang JT. Regulation of function by dimerization through the amino-terminal membrane-spanning domain of human ABCC1/MRP1 [J]. J Biol Chem,2007,282(12):8821-8830.
[32]Sharom FJ. The P-glycoprotein efflux pump:how does it transport drugs? [J]. J Membr Biol,1997,160(3):161-175.
[33]Sharom FJ. ABC multidrug transporters:structure, function and role in chemoresistance [J]. Pharmacogenomics,2008,9(1):105-127.
[34]Sharom FJ, Yu X, DiDiodato G, Chu JW.Synthetic hydrophobic peptides are substrates for P-glycoprotein and stimulate drug transport [J]. Biochem J,1996, 320(Pt 2):421-428.
[35]Deeley RG, Cole SP. Substrate recognition and transport by multidrug resistance protein 1 (ABCC1) [J]. FEBS Lett,2006,580(4):1103-1111.
[36]Deeley RG, Westlake C, Cole SP. Transmembrane transport of endo-and xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins [J]. Physiol Rev, 2006,86(3):849-899.
[37]Imai Y, Asada S, Tsukahara S, Ishikawa E, Tsuruo T, Sugimoto Y. Breast cancer resistance protein exports sulfated estrogens but not free estrogens [J]. Mol Pharmacol,2003,64(3):610-618.
[38]Chen ZS, Robey RW, Belinsky MG, Shchaveleva I, Ren XQ, Sugimoto Y, Ross DD, Bates SE, Kruh GD. Transport of methotrexate, methotrexate polyglutamates, and 17beta-estradiol 17-(beta-D-glucuronide) by ABCG2:effects of acquired mutations at R482 on methotrexate transport [J]. Cancer Res,2003,63(14):4048-4054.
[39]Thiebaut F, Tsuruo T, Hamada H, Gottesman MM, Pastan I, Willingham MC. Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues [J]. Proc Natl Acad Sci U S A,1987,84(21):7735-7738.
[40]Croop JM, Raymond M, Haber D, Devault A, Arceci RJ, Gros P, Housman DE. The three mouse multidrug resistance (mdr) genes are expressed in a tissue-specific manner in normal mouse tissues [J]. Mol Cell Biol,1989,9(3):1346-1350.
[41]Liu R, Sharom FJ.Site-directed fluorescence labeling of P-glycoprotein on cysteine residues in the nucleotide binding domains [J].Biochemistry,1996, 35(36):11865-11873.
[42]Liu R, Siemiarczuk A, Sharom FJ. Intrinsic fluorescence of the P-glycoprotein multidrug transporter:sensitivity of tryptophan residues to binding of drugs and nucleotides [J]. Biochemistry,2000,39(48):14927-14938.
[43]Zaitseva J, Jenewein S, Jumpertz T, Holland IB, Schmitt L. H662 is the linchpin of ATP hydrolysis in the nucleotide-binding domain of the ABC transporter HlyB [J]. EMBO J,2005,24(11):1901-1910.
[44]Eytan GD, Regev R, Oren G, Assaraf YG The role of passive transbilayer drug movement in multidrug resistance and its modulation [J].J Biol Chem,1996, 271(22):12897-12902.
[45]Broxterman HJ, Pinedo HM, Kuiper CM, Kaptein LC, Schuurhuis GJ, Lankelma J. Induction by verapamil of a rapid increase in ATP consumption in multidrug-resistant tumor cells [J]. FASEB J,1988,2(7):2278-2282.
[46]Starling JJ, Shepard RL, Cao J, Law KL, Norman BH, Kroin JS, Ehlhardt WJ, Baughman TM, Winter MA, Bell MG, Shih C, Gruber J, Elmquist WF, Dantzig AH. Pharmacological characterization of LY335979:a potent cyclopropyldibenzosuberane modulator of P-glycoprotein [J].Adv Enzyme Regul, 1997,37:335-347.
[47]Sauna ZE, Peng XH, Nandigama K, Tekle S, Ambudkar SV.The molecular basis of the action of disulfiram as a modulator of the multidrug resistance-linked ATP binding cassette transporters MDR1 (ABCB1) and MRP1 (ABCC1) [J]. Mol Pharmacol,2004,65(3):675-684.
[48]Scotto KW.Transcriptional regulation of ABC drug transporters [J].Oncogene,2003, 22(47):7496-7511.
[49]Zhang Z, Wu JY, Hait WN, Yang JM.Regulation of the stability of P-glycoprotein by ubiquitination [J]. Mol Pharmacol,2004,66(3):395-403.
[50]Liu M, Aneja R, Wang H, Sun L, Dong X, Huo L, Joshi H, Zhou J. Modulation of multidrug resistance in cancer cells by the E3 ubiquitin ligase seven-in-absentia homologue 1[J]. J Pathol,2008,214(4):508-514.
[51]Katayama K, Yoshioka S, Tsukahara S, Mitsuhashi J, Sugimoto Y. Inhibition of the mitogen-activated protein kinase pathway results in the down-regulation of P-glycoprotein [J]. Mol Cancer Ther,2007,6(7):2092-20102.
[52]A. Korynevska, P. Heffeter, B. Matselyukh, L. Elbling, M. Micksche, R. Stoika, and W. Berger.Mechanisms underlying the anticancer activities of the angucycline landomycin E [J]. Biochem. Pharmacol,2007,74 (12):1713-1726.
[53]Ong RC, Lei J, Lee RK, Cheung JY, Fung KP, Lin C, Ho HP, Yu B, Li M, Kong SK. Polyphyllin D induces mitochondrial fragmentation and acts directly on the mitochondria to induce apoptosis in drug-resistant HepG2 cells [J].Cancer Lett, 2008,261(2):158-164.
[54]Shi Z, Peng XX, Kim IW, Shukla S, Si QS, Robey RW, Bates SE, Shen T, Ashby CR Jr, Fu LW, Ambudkar SV, Chen ZS. Erlotinib (Tarceva, OSI-774) antagonizes ATP-binding cassette subfamily B member 1 and ATP-binding cassette subfamily G member 2-mediated drug resistance [J].Cancer Res,2007,67(22):11012-11020.
[55]Tiwari AK, Sodani K, Wang SR, Kuang YH, Ashby CR Jr, Chen X, Chen ZS. Nilotinib (AMN107, Tasigna) reverses multidrug resistance by inhibiting the activity of the ABCB1/Pgp and ABCG2/BCRP/MXR transporters [J].Biochem Pharmacol,2009,78(2):153-161.
[1]Igney FH, Krammer PH. Death and anti-death:tumour resistance to apoptosis [J]. Nat Rev Cancer,2002,2 (4):277-288.
[2]Mimeault M, Hauke R, Batra SK. Recent advances on the molecular mechanisms involved in the drug resistance of cancer cells and novel targeting therapies [J]. Clin Pharmacol Ther,2008,83 (5):673-691.
[3]Shabbits JA, Hu Y, Mayer LD. Tumor chemosensitization strategies based on apoptosis manipulations [J]. Mol Cancer Ther,2003,2 (8):805-813.
[4]Green DR, Kroemer G. Pharmacological manipulation of cell death:clinical applications in sight? [J]. J Clin Invest,2005,115 (10):2610-2617.
[5]Galluzzi L, Larochette N, Zamzami N, Kroemer G. Mitochondria as therapeutic targets for cancer chemotherapy [J]. Oncogene,2006,25 (34):4812-4830.
[6]Kang MH, Reynolds CP. Bcl-2 inhibitors:targeting mitochondrial apoptotic pathways in cancer therapy [J]. Clin Cancer Res,2009,15 (4):1126-1132.
[7]Trauzold A, Schmiedel S, Oestern S, et al. Concerted deregulations of multiple apoptosis-controlling genes in pancreatic carcinoma cells [J]. Ann N Y Acad Sci, 2003,1010:510-513.
[8]谭君,祝连彩,王伯初.Survivin和Bcl-2调节槲皮素诱导的A549细胞凋亡[J].中国药理学通报,2008,24(9)：1220-1224
[9]Lu C, El-Deiry WS. Targeting p53 for enhanced radio-and chemo-sensitivity [J]. Apoptosis,2009,14 (4):597-606.
[10]Eischen CM, Lozano G. p53 and MDM2:antagonists or partners in crime? [J] Cancer Cell,2009,15 (3):161-162.
[11]Mazars A, Geneste O, Hickman J. The Bcl-2 family of proteins as drug targets [J]. J Soc Biol,2005,199 (3):253-265.
[12]de Thonel A, Eriksson JE. Regulation of death receptors-relevance in cancer therapies [J]. Toxicol Appl Pharmacol,2005,207 (2 Suppl):123-132.
[13]Schimmer AD. Novel therapies targeting the apoptosis pathway for the treatment of acute myeloid leukemia [J]. Curr Treat Options Oncol,2007,8 (4):277-286.
[14]LoPiccolo J, Blumenthal GM, Bernstein WB, Dennis PA. Targeting the PI3K/Akt/mTOR pathway:effective combinations and clinical considerations [J]. Drug Resist Updat,2008,11 (1-2):32-50.
[15]Lee CH, Jeon YT, Kim SH, Song YS. NF-kappaB as a potential molecular target for cancer therapy [J]. Biofactors,2007,29 (1):19-35.
[16]Jin J, Wang FP, Wei H, Liu G.T. Reversal of multidrug resistance of cancer through inhibition of P-glycoprotein by 5-bromotetrandrine [J].Cancer Chemother Pharmacol,2005,55(2):179-188.
[17]Kolitz JE, George SL, Dodge RK, Hurd DD, Powell BL, Allen SL, Velez-Garcia E, Moore JO, Shea TC, Hoke E, Caligiuri MA, Vardiman JW, Bloomfield CD, Larson RA; Cancer and Leukemia Group B.Dose escalation studies of cytarabine, daunorubicin, and etoposide with and without multidrug resistance modulation with PSC-833 in untreated adults with acute myeloid leukemia younger than 60 years: final induction results of Cancer and Leukemia Group B Study 9621 [J]. J Clin Oncol,2004,22(21):4290-4301.
[18]Garraway LA, Chabner B. MDR1 inhibition:less resistance or less relevance? [J]. Eur J Cancer,2002,38(18):2337-2340.
[19]Greenberg PL, Lee SJ, Advani R, Tallman MS, Sikic BI, Letendre L, Dugan K, Lum B, Chin DL, Dewald G, Paietta E, Bennett JM, Rowe JM. Mitoxantrone, etoposide, and cytarabine with or without valspodar in patients with relapsed or refractory acute myeloid leukemia and high-risk myelodysplastic syndrome:a phase III trial (E2995) [J].J Clin Oncol,2004,22(6):1078-1086.
[20]Eberle J, Fecker LF, Forschner T, Ulrich C, Rowert-Huber J, Stockfleth E.Apoptosis pathways as promising targets for skin cancer therapy [J].Br J Dermatol,2007,156 (Suppl 3):18-24.
[21]Jacobson MD, Weil M, Raff MC. Programmed cell death in animal development [J].Cell,1997,88(3):347-354.
[22]Ghavami S, Hashemi M, Ande SR, Yeganeh B, Xiao W, Eshraghi M, Bus CJ, Kadkhoda K, Wiechec E, Halayko AJ, Los M.Apoptosis and cancer:mutations within caspase genes [J].J Med Genet,2009,46(8):497-510.
[23]Rao RV, Ellerby HM, Bredesen DE.Coupling endoplasmic reticulum stress to the cell death program [J].Cell Death Differ,2004, 11(4):372-380.
[24]Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L. Anthracyclines:molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity [J].Pharmacol Rev,2004,56(2):185-229.
[25]Gewirtz DA. A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin [J].Biochem Pharmacol,1999,57(7):727-741.
[26]Boujrad H, Gubkina O, Robert N, Krantic S, Susin SA. AIF-mediated programmed necrosis:a highly regulated way to die [J].Cell Cycle,2007,6(21):2612-2619.
[27]Moretti L, Yang ES, Kim KW, Lu B. Autophagy signaling in cancer and its potential as novel target to improve anticancer therapy [J].Drug Resist Updat,2007, 10(4-5):135-143.
[28]Henry-Mowatt J, Dive C, Martinou JC, James D. Role of mitochondrial membrane permeabilization in apoptosis and cancer [J].Oncogene,2004,23(16):2850-2860.
[29]Green DR, Kroemer GThe pathophysiology of mitochondrial cell death.Science [J]. 2004,305(5684):626-629.
[30]Zamzami N, Kroemer G. The mitochondrion in apoptosis:how Pandora's box opens [J].Nat Rev Mol Cell Biol,2001,2(1):67-71.
[31]Liu H, Qin CK, Han GQ, Xu HW, Ren WH, Qin CY. Synthetic chenodeoxycholic acid derivative, HS-1200, induces apoptosis of human hepatoma cells via a mitochondrial pathway [J].Cancer Lett,2008,270(2):242-249.
[32]Arisan ED, Kutuk O, Tezil T, Bodur C, Telci D, Basaga H. Small inhibitor of Bcl-2, HA 14-1, selectively enhanced the apoptotic effect of cisplatin by modulating Bcl-2 family members in MDA-MB-231 breast cancer cells [J].Breast Cancer Res Treat, 2010,119(2):271-81.
[33]Chen L, Willis SN, Wei A, Smith BJ, Fletcher JI, Hinds MG, Colman PM, Day CL Adams JM, Huang DC.Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function [J]. Mol Cell,2005, 17(3):393-403.
[34]Reed JC. Apoptosis-targeted therapies for cancer [J].Cancer Cell,2003,3(1):17-22.
[35]Letai A.Pharmacological manipulation of Bcl-2 family members to control cell death [J]. J Clin Invest,2005,115(10):2648-2655.
[36]Korynevska A, Heffeter P, Matselyukh B, Elbling L, Micksche M, Stoika R, Berger W. Mechanisms underlying the anticancer activities of the angucycline landomycin E [J].Biochem Pharmacol,2007,74(12):1713-1726.
[37]Ong RC, Lei J, Lee RK, Cheung JY, Fung KP, Lin C, Ho HP, Yu B, Li M, Kong SK. Polyphyllin D induces mitochondrial fragmentation and acts directly on the mitochondria to induce apoptosis in drug-resistant HepG2 cells [J].Cancer Lett, 2008,261(2):158-164.
[38]Shi Z, Peng XX, Kim IW, Shukla S, Si QS, Robey RW, Bates SE, Shen T, Ashby CR Jr, Fu LW, Ambudkar SV, Chen ZS. Erlotinib (Tarceva, OSI-774) antagonizes ATP-binding cassette subfamily B member 1 and ATP-binding cassette subfamily G member 2-mediated drug resistance [J].Cancer Res,2007,67(22):11012-11020.
[39]Shimizu S, Konishi A, Kodama T, Tsujimoto Y BH4 domain of antiapoptotic Bcl-2 family members closes voltage-dependent anion channel and inhibits apoptotic mitochondrial changes and cell death [J]. Proc Natl Acad Sci U S A,2000, 97(7):3100-3105.
[40]Shimizu S, Tsujimoto Y. Proapoptotic BH3-only Bcl-2 family members induce cytochrome c release, but not mitochondrial membrane potential loss, and do not directly modulate voltage-dependent anion channel activity [J].Proc Natl Acad Sci US A,2000,97(2):577-582.
[41]Liu XD, Sun H, Liu GT.5-Bromotetrandrine enhances the sensitivity of doxorubicin-induced apoptosis in intrinsic resistant human hepatic cancer Be17402 cells [J].Cancer Lett,2010,292(1):24-31.
[2]Mimeault, M.& Batra, S.K. Recent advances on multiple tumorigenic cascadesinvolved in prostatic cancer progression and targeting therapies [J]. Carcinogenesis,2006,27(1):1-22.
[3]Shiraishi J, Abe H, Li F, Engelmann R, MacMahon H, Doi K. Computer-aided diagnosis for the detection and classification of lung cancers on chest radiographs ROC analysis of radiologists'performance [J]. Acad Radiol,2006,13(8):995-1003.
[4]Chatterton, K, Ray, E.& O'Brien, T.S. Fluorescence diagnosis of bladder cancer [J]. Br J Nurs,2006,15(11):595-597.
[5]Petignat, P, Du Bois, A., Bruchim, I., Fink, D.& Provencher, D.M. Should intraperitoneal chemotherapy be considered as standard first-line treatment in advanced stage ovarian cancer?[J], Crit Rev Oncol Hematol,2006,62(2):137-147.
[6]Mimeault, M., Brand, R.E., Sasson, A.A.& Batra, S.K. Recent advances on the molecular mechanisms involved in pancreatic cancer progression and therapies [J]. Pancreas,2005,31(4):301-316.
[7]Lock-Andersen, J., Horn, J.& Sjostrand, H. Prognosis after sentinel node biopsy in malignant melanoma [J]. Ugeskr Laeger,2006,168(25):2457-2462.
[8]Shabbits JA, Hu Y, Mayer LD. Tumor chemosensitization strategies based on apoptosis manipulations [J]. Mol Cancer Ther,2003,2 (8):805-813.
[9]Szakacs G, Paterson JK, Ludwig JA, Booth-Genthe C, Gottesman MM. Targeting multidrug resistance in cancer [J]. Nat Rev Drug Discov,2006,5(3):219-34 101.
[10]Mimeault M, Hauke R, Batra SK. Recent advances on the molecular mechanisms involved in the drug resistance of cancer cells and novel targeting therapies [J]. Clin Pharmacol Ther.2008,83(5):673-691.
[11]Raguz S, Yague E.Resistance to chemotherapy:new treatments and novel insights into an old problem [J]. Br J Cancer,2008,99(3):387-391.
[12]Dean M, Rzhetsky A, Allikmets R. The human ATP-binding cassette (ABC) transporter superfamily [J].Genome Res,2001,11 (7):1156-66.
[13]Chang G, Roth CB. Structure of MsbA from E. coli:a homolog of the multidrug resistance ATP binding cassette (ABC) transporter [J]. Science,2001, 293(5536):1793-1800.
[14]Dassa E, Bouige P.The ABC of ABCS:a phylogenetic and functional classification of ABC systems in living organisms [J].Res Microbiol,2001,152(3-4):211-29.
[15]Perez-Tomas R.Multidrug resistance:retrospect and prospects in anti-cancer drug treatment [J]. Curr Med Chem,2006,13(16):1859-1876.
[16]Eckford PD, Sharom FJ.ABC efflux pump-based resistance to chemotherapy drugs [J].Chem Rev.2009,109(7):2989-3011.
[17]Polgar O, Bates SE. ABC transporters in the balance:is there a role in multidrug resistance? [J]. Biochem Soc Trans.2005,33(Pt 1):241-245.
[18]Goldstein LJ. MDR1 gene expression in solid tumours. Eur J Cancer [J].1996, 32A(6):1039-1050.
[19]Penson RT, Oliva E, Skates SJ, Glyptis T, Fuller AF Jr, Goodman A, Seiden MV. Expression of multidrug resistance-1 protein inversely correlates with paclitaxel response and survival in ovarian cancer patients:a study in serial samples [J]. Gynecol Oncol,2004,93(1):98-106.
[20]Mimeault M, Hauke R, Batra SK. Recent advances on the molecular mechanisms involved in the drug resistance of cancer cells and novel targeting therapies [J]. Clin Pharmacol Ther,2008,83 (5):673-91.
[21]Wang G, Lemos JR.Tetrandrine:a new ligand to block voltage-dependent Ca2+and Ca(+)-activated K+channels [J]. Life Sci,1995,56(5):295-306.
[22]He QY,Meng FH, Zhang HQ.Reduction of doxorubicin resistance by tetrandrine and dauricine in harringtonine-resis-tant human leukemia (HL60) cells [J]. Zhongguo Yao Li Xue Bao,1996,17:179-18
[23]Tian H, Pan QC.A comparative study on e□ect of two bisbenzylisoquinolines, tetrandrine and berbamine, on reversa of multidrug resistance [J]. Yao Xue Xue Bao,1997,32:245-250
[24]Garattini S. Pharmacokinetics in cancer chemotherapy [J]. Eur J Cancer,2007, 43(2):271-282.
[25]Mansilla S, Bataller M, Portugal J. Mitotic catastrophe as a consequence of chemotherapy [J]. Anticancer Agents Med Chem,2006,6(6):589-602.
[26]Dimri GP. What has senescence got to do with cancer? [J]. Cancer Cell,2005, 7(6):505-512.
[27]Higgins CF. ABC transporters:from microorganisms to man [J]. Annu Rev Cell Biol, 1992,8:67-113.
[28]Frelet A, Klein M. Insight in eukaryotic ABC transporter function by mutation analysis [J]. FEBS Lett,2006,580(4):1064-1084.
[29]Dawson RJ, Locher KP.Structure of a bacterial multidrug ABC transporter [J]. Nature,2006,443(7108):180-185.
[30]Bakos E, Evers R, Szakacs G, Tusnady GE, Welker E, Szabo K, de Haas M, van Deemter L, Borst P, Varadi A, Sarkadi B. Functional multidrug resistance protein (MRP1) lacking the N-terminal transmembrane domain [J]. J Biol Chem,1998, 273(48):32167-75.
[31]Yang Y, Liu Y, Dong Z, Xu J, Peng H, Liu Z, Zhang JT. Regulation of function by dimerization through the amino-terminal membrane-spanning domain of human ABCC1/MRP1 [J]. J Biol Chem,2007,282(12):8821-8830.
[32]Sharom FJ. The P-glycoprotein efflux pump:how does it transport drugs? [J]. J Membr Biol,1997,160(3):161-175.
[33]Sharom FJ. ABC multidrug transporters:structure, function and role in chemoresistance [J]. Pharmacogenomics,2008,9(1):105-127.
[34]Sharom FJ, Yu X, DiDiodato G, Chu JW.Synthetic hydrophobic peptides are substrates for P-glycoprotein and stimulate drug transport [J]. Biochem J,1996, 320(Pt 2):421-428.
[35]Deeley RG, Cole SP. Substrate recognition and transport by multidrug resistance protein 1 (ABCC1) [J]. FEBS Lett,2006,580(4):1103-1111.
[36]Deeley RG, Westlake C, Cole SP. Transmembrane transport of endo-and xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins [J]. Physiol Rev, 2006,86(3):849-899.
[37]Imai Y, Asada S, Tsukahara S, Ishikawa E, Tsuruo T, Sugimoto Y. Breast cancer resistance protein exports sulfated estrogens but not free estrogens [J]. Mol Pharmacol,2003,64(3):610-618.
[38]Chen ZS, Robey RW, Belinsky MG, Shchaveleva I, Ren XQ, Sugimoto Y, Ross DD, Bates SE, Kruh GD. Transport of methotrexate, methotrexate polyglutamates, and 17beta-estradiol 17-(beta-D-glucuronide) by ABCG2:effects of acquired mutations at R482 on methotrexate transport [J]. Cancer Res,2003,63(14):4048-4054.
[39]Thiebaut F, Tsuruo T, Hamada H, Gottesman MM, Pastan I, Willingham MC. Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues [J]. Proc Natl Acad Sci U S A,1987,84(21):7735-7738.
[40]Croop JM, Raymond M, Haber D, Devault A, Arceci RJ, Gros P, Housman DE. The three mouse multidrug resistance (mdr) genes are expressed in a tissue-specific manner in normal mouse tissues [J]. Mol Cell Biol,1989,9(3):1346-1350.
[41]Liu R, Sharom FJ.Site-directed fluorescence labeling of P-glycoprotein on cysteine residues in the nucleotide binding domains [J].Biochemistry,1996, 35(36):11865-11873.
[42]Liu R, Siemiarczuk A, Sharom FJ. Intrinsic fluorescence of the P-glycoprotein multidrug transporter:sensitivity of tryptophan residues to binding of drugs and nucleotides [J]. Biochemistry,2000,39(48):14927-14938.
[43]Zaitseva J, Jenewein S, Jumpertz T, Holland IB, Schmitt L. H662 is the linchpin of ATP hydrolysis in the nucleotide-binding domain of the ABC transporter HlyB [J]. EMBO J,2005,24(11):1901-1910.
[44]Eytan GD, Regev R, Oren G, Assaraf YG The role of passive transbilayer drug movement in multidrug resistance and its modulation [J].J Biol Chem,1996, 271(22):12897-12902.
[45]Broxterman HJ, Pinedo HM, Kuiper CM, Kaptein LC, Schuurhuis GJ, Lankelma J. Induction by verapamil of a rapid increase in ATP consumption in multidrug-resistant tumor cells [J]. FASEB J,1988,2(7):2278-2282.
[46]Starling JJ, Shepard RL, Cao J, Law KL, Norman BH, Kroin JS, Ehlhardt WJ, Baughman TM, Winter MA, Bell MG, Shih C, Gruber J, Elmquist WF, Dantzig AH. Pharmacological characterization of LY335979:a potent cyclopropyldibenzosuberane modulator of P-glycoprotein [J].Adv Enzyme Regul, 1997,37:335-347.
[47]Sauna ZE, Peng XH, Nandigama K, Tekle S, Ambudkar SV.The molecular basis of the action of disulfiram as a modulator of the multidrug resistance-linked ATP binding cassette transporters MDR1 (ABCB1) and MRP1 (ABCC1) [J]. Mol Pharmacol,2004,65(3):675-684.
[48]Scotto KW.Transcriptional regulation of ABC drug transporters [J].Oncogene,2003, 22(47):7496-7511.
[49]Zhang Z, Wu JY, Hait WN, Yang JM.Regulation of the stability of P-glycoprotein by ubiquitination [J]. Mol Pharmacol,2004,66(3):395-403.
[50]Liu M, Aneja R, Wang H, Sun L, Dong X, Huo L, Joshi H, Zhou J. Modulation of multidrug resistance in cancer cells by the E3 ubiquitin ligase seven-in-absentia homologue 1[J]. J Pathol,2008,214(4):508-514.
[51]Katayama K, Yoshioka S, Tsukahara S, Mitsuhashi J, Sugimoto Y. Inhibition of the mitogen-activated protein kinase pathway results in the down-regulation of P-glycoprotein [J]. Mol Cancer Ther,2007,6(7):2092-20102.
[52]A. Korynevska, P. Heffeter, B. Matselyukh, L. Elbling, M. Micksche, R. Stoika, and W. Berger.Mechanisms underlying the anticancer activities of the angucycline landomycin E [J]. Biochem. Pharmacol,2007,74 (12):1713-1726.
[53]Ong RC, Lei J, Lee RK, Cheung JY, Fung KP, Lin C, Ho HP, Yu B, Li M, Kong SK. Polyphyllin D induces mitochondrial fragmentation and acts directly on the mitochondria to induce apoptosis in drug-resistant HepG2 cells [J].Cancer Lett, 2008,261(2):158-164.
[54]Shi Z, Peng XX, Kim IW, Shukla S, Si QS, Robey RW, Bates SE, Shen T, Ashby CR Jr, Fu LW, Ambudkar SV, Chen ZS. Erlotinib (Tarceva, OSI-774) antagonizes ATP-binding cassette subfamily B member 1 and ATP-binding cassette subfamily G member 2-mediated drug resistance [J].Cancer Res,2007,67(22):11012-11020.
[55]Tiwari AK, Sodani K, Wang SR, Kuang YH, Ashby CR Jr, Chen X, Chen ZS. Nilotinib (AMN107, Tasigna) reverses multidrug resistance by inhibiting the activity of the ABCB1/Pgp and ABCG2/BCRP/MXR transporters [J].Biochem Pharmacol,2009,78(2):153-161.
[1]Igney FH, Krammer PH. Death and anti-death:tumour resistance to apoptosis [J]. Nat Rev Cancer,2002,2 (4):277-288.
[2]Mimeault M, Hauke R, Batra SK. Recent advances on the molecular mechanisms involved in the drug resistance of cancer cells and novel targeting therapies [J]. Clin Pharmacol Ther,2008,83 (5):673-691.
[3]Shabbits JA, Hu Y, Mayer LD. Tumor chemosensitization strategies based on apoptosis manipulations [J]. Mol Cancer Ther,2003,2 (8):805-813.
[4]Green DR, Kroemer G. Pharmacological manipulation of cell death:clinical applications in sight? [J]. J Clin Invest,2005,115 (10):2610-2617.
[5]Galluzzi L, Larochette N, Zamzami N, Kroemer G. Mitochondria as therapeutic targets for cancer chemotherapy [J]. Oncogene,2006,25 (34):4812-4830.
[6]Kang MH, Reynolds CP. Bcl-2 inhibitors:targeting mitochondrial apoptotic pathways in cancer therapy [J]. Clin Cancer Res,2009,15 (4):1126-1132.
[7]Trauzold A, Schmiedel S, Oestern S, et al. Concerted deregulations of multiple apoptosis-controlling genes in pancreatic carcinoma cells [J]. Ann N Y Acad Sci, 2003,1010:510-513.
[8]谭君,祝连彩,王伯初.Survivin和Bcl-2调节槲皮素诱导的A549细胞凋亡[J].中国药理学通报,2008,24(9)：1220-1224
[9]Lu C, El-Deiry WS. Targeting p53 for enhanced radio-and chemo-sensitivity [J]. Apoptosis,2009,14 (4):597-606.
[10]Eischen CM, Lozano G. p53 and MDM2:antagonists or partners in crime? [J] Cancer Cell,2009,15 (3):161-162.
[11]Mazars A, Geneste O, Hickman J. The Bcl-2 family of proteins as drug targets [J]. J Soc Biol,2005,199 (3):253-265.
[12]de Thonel A, Eriksson JE. Regulation of death receptors-relevance in cancer therapies [J]. Toxicol Appl Pharmacol,2005,207 (2 Suppl):123-132.
[13]Schimmer AD. Novel therapies targeting the apoptosis pathway for the treatment of acute myeloid leukemia [J]. Curr Treat Options Oncol,2007,8 (4):277-286.
[14]LoPiccolo J, Blumenthal GM, Bernstein WB, Dennis PA. Targeting the PI3K/Akt/mTOR pathway:effective combinations and clinical considerations [J]. Drug Resist Updat,2008,11 (1-2):32-50.
[15]Lee CH, Jeon YT, Kim SH, Song YS. NF-kappaB as a potential molecular target for cancer therapy [J]. Biofactors,2007,29 (1):19-35.
[16]Jin J, Wang FP, Wei H, Liu G.T. Reversal of multidrug resistance of cancer through inhibition of P-glycoprotein by 5-bromotetrandrine [J].Cancer Chemother Pharmacol,2005,55(2):179-188.
[17]Kolitz JE, George SL, Dodge RK, Hurd DD, Powell BL, Allen SL, Velez-Garcia E, Moore JO, Shea TC, Hoke E, Caligiuri MA, Vardiman JW, Bloomfield CD, Larson RA; Cancer and Leukemia Group B.Dose escalation studies of cytarabine, daunorubicin, and etoposide with and without multidrug resistance modulation with PSC-833 in untreated adults with acute myeloid leukemia younger than 60 years: final induction results of Cancer and Leukemia Group B Study 9621 [J]. J Clin Oncol,2004,22(21):4290-4301.
[18]Garraway LA, Chabner B. MDR1 inhibition:less resistance or less relevance? [J]. Eur J Cancer,2002,38(18):2337-2340.
[19]Greenberg PL, Lee SJ, Advani R, Tallman MS, Sikic BI, Letendre L, Dugan K, Lum B, Chin DL, Dewald G, Paietta E, Bennett JM, Rowe JM. Mitoxantrone, etoposide, and cytarabine with or without valspodar in patients with relapsed or refractory acute myeloid leukemia and high-risk myelodysplastic syndrome:a phase III trial (E2995) [J].J Clin Oncol,2004,22(6):1078-1086.
[20]Eberle J, Fecker LF, Forschner T, Ulrich C, Rowert-Huber J, Stockfleth E.Apoptosis pathways as promising targets for skin cancer therapy [J].Br J Dermatol,2007,156 (Suppl 3):18-24.
[21]Jacobson MD, Weil M, Raff MC. Programmed cell death in animal development [J].Cell,1997,88(3):347-354.
[22]Ghavami S, Hashemi M, Ande SR, Yeganeh B, Xiao W, Eshraghi M, Bus CJ, Kadkhoda K, Wiechec E, Halayko AJ, Los M.Apoptosis and cancer:mutations within caspase genes [J].J Med Genet,2009,46(8):497-510.
[23]Rao RV, Ellerby HM, Bredesen DE.Coupling endoplasmic reticulum stress to the cell death program [J].Cell Death Differ,2004, 11(4):372-380.
[24]Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L. Anthracyclines:molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity [J].Pharmacol Rev,2004,56(2):185-229.
[25]Gewirtz DA. A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin [J].Biochem Pharmacol,1999,57(7):727-741.
[26]Boujrad H, Gubkina O, Robert N, Krantic S, Susin SA. AIF-mediated programmed necrosis:a highly regulated way to die [J].Cell Cycle,2007,6(21):2612-2619.
[27]Moretti L, Yang ES, Kim KW, Lu B. Autophagy signaling in cancer and its potential as novel target to improve anticancer therapy [J].Drug Resist Updat,2007, 10(4-5):135-143.
[28]Henry-Mowatt J, Dive C, Martinou JC, James D. Role of mitochondrial membrane permeabilization in apoptosis and cancer [J].Oncogene,2004,23(16):2850-2860.
[29]Green DR, Kroemer GThe pathophysiology of mitochondrial cell death.Science [J]. 2004,305(5684):626-629.
[30]Zamzami N, Kroemer G. The mitochondrion in apoptosis:how Pandora's box opens [J].Nat Rev Mol Cell Biol,2001,2(1):67-71.
[31]Liu H, Qin CK, Han GQ, Xu HW, Ren WH, Qin CY. Synthetic chenodeoxycholic acid derivative, HS-1200, induces apoptosis of human hepatoma cells via a mitochondrial pathway [J].Cancer Lett,2008,270(2):242-249.
[32]Arisan ED, Kutuk O, Tezil T, Bodur C, Telci D, Basaga H. Small inhibitor of Bcl-2, HA 14-1, selectively enhanced the apoptotic effect of cisplatin by modulating Bcl-2 family members in MDA-MB-231 breast cancer cells [J].Breast Cancer Res Treat, 2010,119(2):271-81.
[33]Chen L, Willis SN, Wei A, Smith BJ, Fletcher JI, Hinds MG, Colman PM, Day CL Adams JM, Huang DC.Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function [J]. Mol Cell,2005, 17(3):393-403.
[34]Reed JC. Apoptosis-targeted therapies for cancer [J].Cancer Cell,2003,3(1):17-22.
[35]Letai A.Pharmacological manipulation of Bcl-2 family members to control cell death [J]. J Clin Invest,2005,115(10):2648-2655.
[36]Korynevska A, Heffeter P, Matselyukh B, Elbling L, Micksche M, Stoika R, Berger W. Mechanisms underlying the anticancer activities of the angucycline landomycin E [J].Biochem Pharmacol,2007,74(12):1713-1726.
[37]Ong RC, Lei J, Lee RK, Cheung JY, Fung KP, Lin C, Ho HP, Yu B, Li M, Kong SK. Polyphyllin D induces mitochondrial fragmentation and acts directly on the mitochondria to induce apoptosis in drug-resistant HepG2 cells [J].Cancer Lett, 2008,261(2):158-164.
[38]Shi Z, Peng XX, Kim IW, Shukla S, Si QS, Robey RW, Bates SE, Shen T, Ashby CR Jr, Fu LW, Ambudkar SV, Chen ZS. Erlotinib (Tarceva, OSI-774) antagonizes ATP-binding cassette subfamily B member 1 and ATP-binding cassette subfamily G member 2-mediated drug resistance [J].Cancer Res,2007,67(22):11012-11020.
[39]Shimizu S, Konishi A, Kodama T, Tsujimoto Y BH4 domain of antiapoptotic Bcl-2 family members closes voltage-dependent anion channel and inhibits apoptotic mitochondrial changes and cell death [J]. Proc Natl Acad Sci U S A,2000, 97(7):3100-3105.
[40]Shimizu S, Tsujimoto Y. Proapoptotic BH3-only Bcl-2 family members induce cytochrome c release, but not mitochondrial membrane potential loss, and do not directly modulate voltage-dependent anion channel activity [J].Proc Natl Acad Sci US A,2000,97(2):577-582.
[41]Liu XD, Sun H, Liu GT.5-Bromotetrandrine enhances the sensitivity of doxorubicin-induced apoptosis in intrinsic resistant human hepatic cancer Be17402 cells [J].Cancer Lett,2010,292(1):24-31.