Reversal of breast cancer resistance protein-mediated drug resistance by estrogen antagonists and agonists

Breast cancer resistance protein (BCRP), an ATP-binding cassette transporter, confers resistance to a series of anticancer agents such as SN-38, mitoxantrone, and topotecan. In a previous study, we found that estrogens reverse drug resistance of BCRP-expressing cells. In this study, estrogen antagonists, estrogen agonists, and their derivatives were evaluated for BCRP-reversing activity. First, compounds were tested for effects on the cellular accumulation of topotecan in BCRP-transduced K562 cells (K562/BCRP). Next, these compounds were examined for their ability to reverse SN-38 and mitoxantrone resistance in K562/BCRP cells. Among commercially available estrogen antagonists and agonists tested, diethylstilbestrol showed the strongest BCRP-reversing activity. Diethylstilbestrol increased the cellular accumulation of topotecan and reversed drug resistance in K562/BCRP cells but showed marginal or no effect in parental K562 cells. The reversal activities of estrone and diethylstilbestrol were more prominent for mitoxantrone than for SN-38. Tamoxifen and toremifene were also found to enhance topotecan uptake in K562/BCRP cells. Next, various tamoxifen derivatives were screened for anti-BCRP activity. In the first cycle of screening with 14 compounds, TAG-11 showed the strongest effect. In the second cycle of screening of 25 TAG-11-related compounds, TAG-139 showed the strongest effect. Reversal of SN-38 and mitoxantrone resistance in K562/BCRP cells by TAG-139 was 5-fold stronger than that by estrone. Dose-dependent characteristics of drug resistance reversal with estrone and TAG-139 were very similar, suggesting that estrone and tamoxifen derivatives interact with the same drug-binding site of BCRP. Derivatives of antiestrogens that exhibit no other biological effects promise to be useful in overcoming BCRP-mediated drug resistance.     

Taxane-based reversal agents modulate drug resistance mediated by P-glycoprotein, multidrug resistance protein, and breast cancer resistance protein

Overexpression of ATP-binding cassette transport proteins, including P-glycoprotein (Pgp), multidrug resistance (MDR) protein (MRP-1), and breast cancer resistance protein (BCRP), is a well-characterized mechanism of MDR in tumor cells. Although the cytotoxic taxanes paclitaxel and docetaxel are substrates for Pgp-mediated efflux, the semisynthetic taxane analogue ortataxel inhibits drug efflux mediated by Pgp as well as, as we recently demonstrated, MRP-1 and BCRP. Nevertheless, ortataxel is not optimal for development as a clinical MDR modulator because of its cytotoxicity [corrected]. We sought to identify noncytotoxic taxane-based broad-spectrum modulators from a library of noncytotoxic taxane-based reversal agents (tRAs) designed by eliminating the C-13 side chain of the taxane molecule, which inhibits microtubule depolymerization. Twenty tRAs, selected based on modulation of paclitaxel cytotoxicity in Pgp-overexpressing MDA435/LCC6(mdr1) cells, were studied for modulation of retention and cytotoxicity of substrates of MRP-1 and BCRP as well as Pgp in established cell lines overexpressing each of these transporters. Four tRAs modulated MRP-1 and 17 modulated BCRP in addition to Pgp. The four broad-spectrum tRAs strongly modulated daunorubicin and mitoxantrone efflux and enhanced their cytotoxicity in cell lines overexpressing the three MDRs, decreasing IC(50) values by as much as 97% [corrected]. These tRAs, especially tRA 98006, have promise for development as clinical broad-spectrum MDR modulators and warrant more preclinical analysis to determine pharmacokinetic interactions and efficacy.     

Contribution of the drug transporter ABCG2 (breast cancer resistance protein) to resistance against anticancer nucleosides

We have studied the potential contribution of ABCG2 (breast cancer resistance protein) to resistance to nucleoside analogues. In cells transfected with DNA constructs resulting in overexpression of human or mouse ABCG2, we found resistance against cladribine, clofarabine, fludarabine, 6-mercaptopurine, and 6-mercaptopurine riboside in both MDCKII and HEK293 cells and against gemcitabine only in HEK293 cells. With Transwell studies in MDCK cells and transport experiments with vesicles from Sf9 and HEK293 cells, we show that ABCG2 is able to transport not only the nucleotide CdAMP, like several other ATP-binding cassette transporters of the ABCC (multidrug resistance protein) family, but also the nucleoside cladribine itself. Expression of ABCG2 in cells results in a substantial decrease of intracellular CdATP, explaining the resistance against cladribine. The high transport rate of cladribine and clofarabine by ABCG2 deduced from Transwell experiments raises the possibility that this transporter could affect the disposition of nucleoside analogues in patients or cause resistance in tumors.     

Conquering drug resistance in lung cancer

The emergence of drug resistance of the tumors is the critical problem in lung cancer. The multifactorial mechanisms of cisplatin resistance are major problems. The reversal agents against MDR have been evaluated clinically. Increased adverse effects and modulation of pharmacokinetics in combined with cytotoxic drugs are the problems in clinical setting. The new ABC transporters related with MDR were cloned and characterized. Modulators for these transporters could be evaluated their clinical efficacy. High-frequent mutations of beta-tubulin in clinical samples are reported as a predictive marker for paclitaxel. Target-based drugs might conquer the resistance to conventional drugs. Prediction of the drug sensitivity and resistance in each patient will be expected using high-throghput gene screening system.     

肿瘤相关巨噬细胞诱导乳腺癌耐药的实验研究

目的探索肿瘤相关巨噬细胞(TAM)在乳腺肿瘤细胞耐药中的作用及其耐药机制。方法通过佛波酯(PMA)、白细胞介素4(IL-4)、白细胞介素13(IL-13)刺激人单核细胞白血病细胞系THP-1,建立TAM模型在体外与乳腺癌细胞共培养,运用流式细胞术(FCM)检测TAM表面分子CD14、CD204;使用噻唑蓝(MTT)比色法检测紫杉醇对乳腺癌细胞系T47D、BT-549的有效杀伤浓度和时间,以及乳腺癌细胞单独培养、乳腺癌细胞与TAM/TAM上清共培养下乳腺癌细胞的杀伤;运用Western blot检测肿瘤细胞的磷酸化信号转导与转录激活因子3(p-STAT3)、磷酸化c-jun氨基末端激酶(p-JNK)信号通路变化。结果 THP-1细胞经PMA、IL-4、IL-13诱导分化为TAM,其细胞表面表达CD14、CD204(表达率分别为31.52%±9.39%、48.21%±8.76%)。TAM/TAM上清与肿瘤细胞共培养均可显著削弱紫杉醇对肿瘤细胞的杀伤(T47D相对存活率由43.52%±9.40%提高至92.68%±2.32%/92.20%±2.10%,BT-549相对存活率由63.08%±2.71%提高至77.96%±2.64%/79.55%±2.35%,P0.05),明显下调其凋亡信号p-JNK(P0.05),同时显著上调p-STAT3(P0.05)。结论成功建立TAM模型,TAM介导乳腺肿瘤细胞耐药,其机制可能与TAM分泌的细胞因子影响肿瘤细胞STAT3、JNK信号分子磷酸化有关。     

Involvement of microRNA-451 in resistance of the MCF-7 breast cancer cells to chemotherapeutic drug doxorubicin

Many chemotherapy regiments are successfully used to treat breast cancer; however, often breast cancer cells develop drug resistance that usually leads to a relapse and worsening of prognosis. We have shown recently that epigenetic changes such as DNA methylation and histone modifications play an important role in breast cancer cell resistance to chemotherapeutic agents. Another mechanism of gene expression control is mediated via the function of small regulatory RNA, particularly microRNA (miRNA); its role in cancer cell drug resistance still remains unexplored. In the present study, we investigated the role of miRNA in the resistance of human MCF-7 breast adenocarcinoma cells to doxorubicin (DOX). Here, we for the first time show that DOX-resistant MCF-7 cells (MCF-7/DOX) exhibit a considerable dysregulation of the miRNAome profile and altered expression of miRNA processing enzymes Dicer and Argonaute 2. The mechanistic link of miRNAome deregulation and the multidrug-resistant phenotype of MCF-7/DOX cells was evidenced by a remarkable correlation between specific miRNA expression and corresponding changes in protein levels of their targets, specifically those ones that have a documented role in cancer drug resistance. Furthermore, we show that microRNA-451 regulates the expression of multidrug resistance 1 gene. More importantly, transfection of the MCF-7/DOX-resistant cells with microRNA-451 resulted in the increased sensitivity of cells to DOX, indicating that correction of altered expression of miRNA may have significant implications for therapeutic strategies aiming to overcome cancer cell resistance.     

A new mechanism of drug resistance in breast cancer cells: fatty acid synthase overexpression-mediated palmitate overproduction

Multidrug resistance is a major problem in successful cancer chemotherapy. Various mechanisms of resistance, such as ABC transporter-mediated drug efflux, have been discovered using established model cancer cell lines. While characterizing a drug-resistant breast cancer cell line, MCF7/AdVp3000, we found that fatty acid synthase (FASN) is overexpressed. In this study, we showed that ectopic overexpression of FASN indeed causes drug resistance and that reducing the FASN expression increased the drug sensitivity in breast cancer cell lines MCF7 and MDA-MB-468 but not in the normal mammary epithelial cell line MCF10A1. Use of FASN inhibitor, Orlistat, at low concentrations also sensitized cells with FASN overexpression to anticancer drugs. The FASN-mediated drug resistance appears to be due to a decrease in drug-induced apoptosis from an overproduction of palmitic acid by FASN. Together with previous findings of FASN as a poor prognosis marker for breast cancer patients, our results suggest that FASN overexpression is a new mechanism of drug resistance and may be an ideal target for chemosensitization in breast cancer chemotherapy.     

Recently identified drug resistance biomarkers in ovarian cancer

Ovarian cancer, consisting mainly of ovarian carcinoma, is the most lethal gynecologic malignancy. Improvements in outcome for patients with advanced-stage disease are limited by intrinsic and acquired chemoresistance and by tumor heterogeneity at different anatomic sites and along disease progression. Molecules and cellular pathways mediating chemoresistance appear to be different for the different histological types of ovarian carcinoma, with most recent research focusing on serous and clear cell carcinoma. This review discusses recent data implicating various biomarkers in chemoresistance in this cancer, with focus on studies in which clinical specimens have been central.     

Gefitinib therapy for advanced non-small-cell lung cancer

OBJECTIVE: To review the pharmacology, pharmacokinetics, clinical efficacy, and toxicity of gefitinib in non-small-cell lung cancer (NSCLC). DATA SOURCES: Primary literature search through MEDLINE and CANCERLIT, and abstract presentations (1966-May 2003). STUDY SELECTION AND DATA EXTRACTION: All published trials and abstracts citing gefitinib were evaluated, and all information deemed relevant was included in this article. DATA SYNTHESIS: NSCLC is known to overexpress epidermal growth factor receptor (EGFR). Gefitinib is a selective EGFR tyrosine kinase inhibitor. Based on the Phase I/II trial results, the optimal dose is 250 mg/d orally. It is well tolerated, with minimal and reversible toxicity. Skin rash and diarrhea are the most common adverse effects. Recent trials have shown that gefitinib provided a 10% tumor response rate and improved disease-related symptoms in patients with refractory, advanced NSCLC. CONCLUSIONS: Gefitinib, with a unique mechanism of action and favorable toxicity profile, has demonstrated clinical activity in NSCLC patients with chemotherapy-refractory disease. It provides a valuable addition to the treatment options as monotherapy in patients with advanced NSCLC after failure of both platinum-based and docetaxel chemotherapies. Further research is required to evaluate the use of gefitinib in different clinical settings.     

微乳丹参酮逆转肿瘤多药耐药的观察

背景:肿瘤多药耐药的出现,增加了肿瘤治疗的难度。肿瘤多药耐药的形成机制复杂,涉及多个环节,选择不同作用机制的药物联合应用,对肿瘤多药耐药的临床效果可能更有效。目的:观察中药新剂型微乳丹参酮对人白血病细胞株及其阿霉素耐药株的多药耐药性逆转效果。设计:观察对比实验。单位:吉林大学附属第一医院中心实验室。材料:人红白血病细胞株及其阿霉素耐药株,由中科院天津血研所提供。丹参酮、微乳丹参酮,由吉林大学药学院制剂室提供。阿霉素(辉瑞法玛西亚药业),P糖蛋白-PE荧光抗体、B细胞淋巴瘤/白血病-2基因-FITC荧光抗体(Immunotech公司),二甲基亚砜(北京化工厂),噻唑蓝(华美生物)。方法:实验于2003-03/2004-01在吉林大学附属一院中心实验室完成。①将人红白血病敏感细胞株与含100g/L小牛血清、100U/mL青霉素和链霉素的RMPI1640培养液中,在37℃,饱和湿度及体积分数为0.05的CO2细胞培养箱内传代;阿霉素耐药株连续培养于阿霉素上述培养液中,培养条件相同。②将中药制剂微乳丹参酮作为逆转剂,丹参酮和微乳体系作为对照,作用于人红白血病细胞株和阿霉素耐药株。③采用四甲基偶氮唑盐法检测微乳丹参酮,丹参酮及微乳的细胞毒性及抗药性逆转倍数。④分别在对数生长期的细胞株中加入10,20,40,60mg/L的阿霉素,采用荧光分光光度法检测微乳丹参酮,丹参酮及微乳对人红白血病细胞株及阿霉素耐药株中细胞内阿霉素浓度的影响。⑤流式细胞术检测给予10mg/L阿霉素的人红白血病细胞株及加入微乳丹参酮,丹参酮及微乳的阿霉素耐药株中P糖蛋白和B细胞淋巴瘤/白血病-2基因蛋白表达及肿瘤细胞凋亡碎片百分比。主要观察指标:①微乳丹参酮,丹参酮及微乳的细胞毒性及抗药性逆转倍数。②微乳丹参酮,丹参酮及微乳对人红白血病细胞株及阿霉素耐药株中细胞内阿霉素浓度的影响。③给予10mg/L阿霉素的人红白血病细胞株及加入微乳丹参酮,丹参酮及微乳的阿霉素耐药株中P糖蛋白和B细胞淋巴瘤/白血病-2基因蛋白表达及肿瘤细胞凋亡碎片百分比。结果:①微乳丹参酮,微乳体系,中药丹参酮均可显著降低阿霉素耐药株的耐药性,以浓度为0.2mg/L、0.5mg/L的微乳丹参酮的逆转倍数显著高于其他两组(P<0.05)。②微乳丹参酮,微乳体系,中药丹参酮均可使细胞内药物浓度升高,微乳丹参酮作用后的阿霉素耐药株细胞中阿霉素的浓度最高,差异有显著性(P<0.05)。③微乳丹参酮,微乳体系,丹参酮均可降低阿霉素耐药株的P糖蛋白和B细胞淋巴瘤/白血病-2基因表达水平,使肿瘤细胞凋亡比率上升,微乳丹参酮的作用最强,可显著降低P糖蛋白和B细胞淋巴瘤/白血病-2基因表达水平,使肿瘤细胞在ADM作用下的凋亡比率增加(P<0.05)。结论:微乳丹参酮0.5mg/L可引起阿霉素耐药株内药物浓度增加,与P糖蛋白、B细胞淋巴瘤/白血病-2基因表达下调的结论相印证;微乳丹参酮对多药耐药逆转作用较丹参酮和微乳逆转效果更显著。     

微乳丹参酮逆转肿瘤多药耐药的观察

背景：肿瘤多药耐药的出现，增加了肿瘤治疗的难度。肿瘤多药耐药的形成机制复杂，涉及多个环节，选择不同作用机制的药物联合应用，对肿瘤多药耐药的临床效果可能更有效。 目的：观察中药新剂型微乳丹参酮对人白血病细胞株及其阿霉素耐药株的多药耐药性逆转效果。 设计：观察对比实验。 单位：吉林大学附属第一医院中心实验室。 材料：人红白血病细胞株及其阿霉素耐药株，由中科院天津血研所提供。丹参酮、微乳丹参酮，由吉林大学药学院制剂室提供。阿霉素（辉瑞法玛西亚药业），P糖蛋白-PE荧光抗体、B细胞淋巴瘤／白血病-2基因-FITC荧光抗体（Immunotech公司），二甲基亚砜（北京化工厂），噻唑蓝（华美生物）。 方法：实验于2003—03／2004—01在吉林大学附属一院中心实验室完成。①将人红白血病敏感细胞株与含100g／L小牛血清、100U／mL青霉素和链霉素的RMPI1640培养液中，在37℃，饱和湿度及体积分数为0．05的CO2细胞培养箱内传代；阿霉素耐药株连续培养于阿霉素上述培养液中，培养条件相同。②将中药制剂微乳丹参酮作为逆转剂，丹参酮和微乳体系作为对照，作用于人红白血病细胞株和阿霉素耐药株。③采用四甲基偶氮唑盐法检测微乳丹参酮，丹参酮及微乳的细胞毒性及抗药性逆转倍数。④分别在对数生长期的细胞株中加入10，20，40，60mg／L的阿霉素，采用荧光分光光度法检测微乳丹参酮，丹参酮及微乳对人红白血病细胞株及阿霉素耐药株中细胞内阿霉素浓度的影响。⑤流式细胞术检测给予10mg／L阿霉素的人红白血病细胞株及加入微乳丹参酮，丹参酮及微乳的阿霉素耐药株中P糖蛋白和B细胞淋巴瘤／白血病-2基因蛋白表达及肿瘤细胞凋亡碎片百分比。 主要观察指标：①微乳丹参酮，丹参酮及微乳的细胞毒性及抗药性逆转倍数。②微乳丹参酮，丹参酮及微乳对人红白血病细胞株及阿霉素耐药株中细胞内阿霉素浓度的影响。③给予10mg／L阿霉素的人红白血病细胞株及加入微乳丹参酮，丹参酮及微乳的阿霉素耐药株中P糖蛋白和B细胞淋巴瘤／白血病-2基因蛋白表达及肿瘤细胞凋亡碎片百分比。 结果：④微乳丹参酮，微乳体系，中药丹参酮均可显著降低阿霉素耐药株的耐药性，以浓度为0．2mg／L、0．5mg／L的微乳丹参酮的逆转倍数显著高于其他两组（P〈0．05）。②微乳丹参酮，微乳体系，中药丹参酮均可使细胞内药物浓度升高，微乳丹参酮作用后的阿霉素耐药株细胞中阿霉素的浓度最高，差异有显著性（P〈0．05）。③微乳丹参酮，微乳体系，丹参酮均可降低阿霉素耐药株的P糖蛋白和B细胞淋巴瘤／白血病-2基因表达水平，使肿瘤细胞凋亡比率上升，微乳丹参酮的作用最强，可显著降低P糖蛋白和B细胞淋巴瘤／白血病-2基因表达水平，使肿瘤细胞在ADM作用下的凋亡比率增加（P〈0．05）。 结论：微乳丹参酮0．5mg／L可引起阿霉素耐药株内药物浓度增加，与P糖蛋白、B细胞淋巴瘤／白血病-2基因表达下调的结论相印证；微乳丹参酮对多药耐药逆转作用较丹参酮和微乳逆转效果更显著。     

Trastuzumab causes antibody-dependent cellular cytotoxicity-mediated growth inhibition of submacroscopic JIMT-1 breast cancer xenografts despite intrinsic drug resistance

Trastuzumab is a recombinant antibody drug that is widely used for the treatment of breast cancer. Despite encouraging clinical results, some cancers are primarily resistant to trastuzumab, and a majority of those initially responding become resistant during prolonged treatment. The mechanisms of trastuzumab resistance have not been fully understood. We examined the role of antibody-dependent cellular cytotoxicity (ADCC) using JIMT-1 cells that are ErbB2 positive but intrinsically resistant to trastuzumab in vitro. Unexpectedly, in experiments mimicking adjuvant therapy of submacroscopic disease in vivo (JIMT-1 cells inoculated s.c. in severe combined immunodeficiency mice), trastuzumab was able to inhibit the outgrowth of macroscopically detectable xenograft tumors for up to 5-7 weeks. The effect is likely to be mediated via ADCC because trastuzumab-F(ab')(2) was ineffective in this model. Moreover, in vitro ADCC reaction of human leukocytes was equally strong against breast cancer cells intrinsically sensitive (SKBR-3) or resistant (JIMT-1) to trastuzumab or even against a subline of JIMT-1 that was established from xenograft tumors growing despite trastuzumab treatment. These results suggest that ADCC may be the predominant mechanism of trastuzumab action on submacroscopic tumor spread. Thus, measuring the ADCC activity of patient's leukocytes against the tumor cells may be a relevant predictor of clinical trastuzumab responsiveness in vivo.     

An overview of drug development for metastatic breast cancer

The prevalence of breast cancer is increasing as more women are living with the disease. Outcomes have improved as a result of progress in all major aspects of multidisciplinary care. These include surgery, radiotherapy, hormonal therapy, chemotherapy and newer targeted drugs. Two aspects merit particular attention here. First, there is an understanding now that cancer is a heterogenous disease and a 'one-size-fits-all' approach is becoming redundant, albeit slowly. Second, basic science and an appreciation of cellular molecular targets in those different types of breast cancer is being translated into the clinic and has led to the development of exciting new drugs for both triple negative and HER2-positive relapsed disease. An improved understanding of endocrine resistance remains an unmet need in drug development and here, it appears worthwhile to adopt less conventional approaches. Better trial design with a focus on biomarkers should lower barriers to regulatory approval as well as increase cost effectiveness.     

吉非替尼治疗老年晚期非小细胞肺癌不良反应的护理

目的探讨吉非替尼单药治疗老年晚期非小细胞肺癌的疗效及护理方法。方法选择27例老年晚期非小细胞肺癌患者吉非替尼单药治疗90d后,评价其疗效及不良反应,并对用药期间的护理进行回顾性分析,总结护理经验。结果本组患者治疗有效率为29.63%(8/27),疾病控制率为74.07%(20/27);不良反应有皮疹13例(48.15%),恶心呕吐12例(44.44%),腹泻9例(33.33%),肝功能异常3例(11.11%),口腔溃疡2例(7.41%)。结论用药前进行宣教,用药期间加强观察,及时发现不良反应,并进行护理干预和对症处理,可保证用药安全,提高患者的舒适度和治疗依从性,改善生活质量。