多药耐药相关蛋白及其逆转剂的研究进展

多药耐药相关蛋白(MRP)是一种ATP依赖型跨膜蛋白,是谷胱甘肽(GSH)-S-共轭物运转泵,在GSH参与下,转运共轭的有机阴离子,起到药物外排泵的作用,是继P-170糖蛋白后发现的又一肿瘤多药耐药(MDR)机制.在一些肿瘤组织中,MRP的表达显著增高.它可能是肿瘤细胞发生耐药的重要机制.化学逆转剂可能具有逆转由MRP介导的MDR,从而增加肿瘤细胞对化疗药物的敏感性,克服耐药,提高化疗效果.     

肿瘤化疗增敏剂研究进展

化疗增敏剂通过逆转肿瘤组织耐药而提高化疗疗效。其作用机制包括：抑制药物泵功能；针对谷胱甘肽代谢相关酶、拓扑异构酶Ⅱ、DNA修复相关酶、蛋白激酶C等靶点逆转耐药；促凋亡；干扰肿瘤细胞与组织微环境之间耐药信号转导等。现按其作用机制,分类介绍化疗增敏剂的研发现状。     

多药耐药相关蛋白及其逆转剂的研究进展

多药耐药相关蛋白(MRP)是一种ATP依赖型跨膜蛋白，是谷胱甘肽(GSH)-S-共轭物运转泵，在GSH参与下，转运共轭的有机阴离子，起到药物外排泵的作用，是继P-170糖蛋白后发现的又一肿瘤多药耐药(MDR)机制。在一些肿瘤组织中，MRP的表达显著增高。它可能是肿瘤细胞发生耐药的重要机制。化学逆转剂可能具有逆转由MRP介导的MDR，从而增加肿瘤细胞对化疗药物的敏感性，克服耐药，提高化疗效果。     

肿瘤化疗增敏剂研究进展

化疗增敏剂通过逆转肿瘤组织耐药而提高化疗疗效。其作用机制包括：抑制药物泵功能；针对谷胱甘肽代谢相关酶、拓扑异构酶Ⅱ、DNA修复相关酶、蛋白激酶C等靶点逆转耐药；促凋亡；干扰肿瘤细胞与组织微环境之间耐药信号转导等。现按其作用机制，分类介绍化疗增敏剂的研发现状。     

血液肿瘤细胞对氧化砷的敏感性与其抗氧化能力的关系

目的　探讨血液肿瘤细胞对三氧化二砷 (As2 O3 )的敏感性和细胞抗氧化能力的关系。方法　应用 9个血液肿瘤细胞系 ,通过细胞活力、形态学和流式细胞仪检测细胞凋亡 ,并测定细胞系的谷胱甘肽 (GSH)含量和 4种抗氧化酶的活性。结果　除了HL 6 0、U937、K5 6 2和Jurkat细胞外 ,其他5个细胞对As2 O3 诱导的凋亡敏感。与敏感细胞系比较 ,As2 O3 耐受细胞系存在较高的GSH含量和(或 )过氧化氢酶活性。谷胱甘肽过氧化物酶、谷胱甘肽S转移酶和超氧化物歧化酶活性与细胞对As2 O3 诱导凋亡效应敏感性无明显相关。结论　细胞内GSH水平和过氧化氢酶的活性是决定血液肿瘤细胞对As2 O3 敏感性的重要因素。     

硫氧还蛋白与肿瘤转移

 硫氧还蛋白（Thioredoxin,Trx）是一类广泛存在于生物体内的抗氧化二硫还原蛋白,参与调节细胞内氧化还原平衡。近年研究发现,Trx在多种肿瘤组织中过表达,与肿瘤细胞增殖和凋亡以及细胞周期调控有关。Trx促进缺氧诱导因子（HIF）-1α的合成和稳定,与氧自由基的调控密切相关,参与肿瘤细胞的化疗耐药,在肿瘤转移中发挥重要作用,可能成为抑制肿瘤转移的新靶点。     

肿瘤细胞中染色质修饰与代谢的相互调控

肿瘤细胞中表观遗传的改变可影响癌基因和抑癌基因的表达水平;此外,肿瘤细胞中伴有的葡萄糖消耗增加、氧化磷酸化减少以及大量乳酸生成等代谢重编程现象,为生物合成提供了更多的中间代谢产物,并重新平衡了肿瘤细胞的氧化还原状态。而表观遗传的改变和代谢重编程均是肿瘤细胞的重要标志。随着研究的不断深入,发现肿瘤细胞代谢和表观遗传（主要是染色质修饰）之间存在着重要的双向调节机制。代谢重编程可影响染色质修饰酶所需的辅助因子,产生的肿瘤代谢产物作为染色质修饰酶的激动剂或拮抗剂,影响染色质修饰;反之,染色质修饰可以直接调控代谢酶的表达,或者改变参与细胞代谢控制的级联信号转导来调节细胞代谢。因此,代谢与表观遗传通过相互调控影响着细胞增殖、转移和多能性,并在肿瘤发生、发展中发挥关键作用。在这篇综述中,我们总结了肿瘤细胞中染色质修饰和代谢相关的最新发现及相互调控机制,以期揭示其在肿瘤发生、发展中的潜在作用,为发展基于肿瘤表观遗传修饰和代谢的靶向治疗方案提供新策略。     

SREBP-1 及其抑制剂调节肿瘤代谢的研究进展

脂类作为三大营养物质之一,参与能量的供应和储存、细胞的构建和作为活性分子参与细胞生命活动。脂类代 谢异常是肿瘤细胞的重要特征之一,主要表现为脂肪酸从头合成及氧化代谢活跃,这些改变与肿瘤信号通路增强,相关代 谢酶改变等密切相关。目前,脂代谢通路中相关酶及转录因子成为肿瘤治疗的潜在药物靶点。SREBP-1 是细胞内调控脂类 代谢的关键转录因子,主要调控胆固醇和脂肪酸合成中关键酶基因的表达,调节脂质新生。SREBP-1 及其调控的脂肪酸合 成途径通常在正常组织和细胞中的表达和活性较低,在肿瘤细胞中,SREBP-1 被激活,其下游基因的表达也升高,在加快 胞内脂肪酸和胆固醇合成的同时,还通过细胞内各信号通路影响肿瘤细胞脂代谢、糖代谢以及氨基酸代谢,为维持肿瘤细 胞的增殖和转移提供额外的能源和物质。运用遗传和药理手段抑制肿瘤细胞中SREBP-1 的表达和活化,可显著抑制体内 和体外多种肿瘤细胞的增殖和迁移。因此,SREBP-1 将是一个有潜力的针对肿瘤细胞脂代谢的肿瘤治疗靶标。本文综述了 SREBP-1 在肿瘤中的异常表达对肿瘤信号通路和物质代谢的影响以及 SREBP-1 抑制剂在肿瘤中的研究进展。     

肿瘤与机体抗氧化系统

机体针对反应性氧族 (reactiveoxygenspecies ,ROS)产生的氧化损伤形成了一套抗氧化酶系统 ,包括超氧化物歧化酶 (superoxidedismutase ,SOD)、过氧化氢酶 (catalase ,CAT)、还原型谷胱甘肽 (reducedglutathione ,GSH)、谷胱甘肽过氧化物酶 (glutathioneperoxidase ,GPx)、谷胱甘肽 -S -转移酶 ( glutathionetransferase ,GST)等。它们在肿瘤的发生和发展中起到一定的作用 ,而肿瘤患者也常表现出抗氧化酶系统的失衡。就肿瘤与抗氧化系统的关系予以综述 ,为肿瘤的治疗提出一条新的思路     

核糖体蛋白与恶性肿瘤治疗研究进展

核糖体蛋白在多种肿瘤中存在异常表达,通过多种机制影响肿瘤细胞的凋亡、衰老、生长、侵袭、药物和放射治 疗抵抗等。核糖体蛋白在肿瘤的表达水平与肿瘤的病理分级,临床分期密切相关,成为肿瘤诊断,治疗和预后判断的潜在指标。 核糖体蛋白可以通过磷酸化等修饰,改变其在肿瘤发生发展中的活性和作用方式。核糖体蛋白与化疗药物的作用机制不同, 能够调节肿瘤细胞对药物的敏感性,从而以一种低毒、有效的方式逆转肿瘤细胞的多药耐药。通过调控核糖体蛋白进行细胞 周期的再分布,将肿瘤的细胞周期阻滞在G0~G1 期,减少G2~M 期细胞的比例。以参与核糖体蛋白合成的RNA 聚合酶Ⅰ蛋 白酶等作为靶点间接调控核糖体蛋白的治疗方式,在动物模型中显示出显著的抗肿瘤活性。作用于核糖体蛋白的药物,由于 不直接对基因组产生影响,没有明显的细胞毒性,对分化成熟的正常组织细胞的遗传毒性很低,具有更好的靶向性。显而易见, 针对核糖体蛋白的肿瘤治疗具有高度特异性,能够有效逆转多药耐药和放射增敏,提供了一种潜在低毒的治疗方式。     

Stromal cell-mediated mitochondrial redox adaptation regulates drug resistance in childhood acute lymphoblastic leukemia

Despite the high cure rates in childhood acute lymphoblastic leukemia (ALL), relapsed ALL remains a significant clinical problem. Genetic heterogeneity does not adequately explain variations in response to therapy. The chemoprotective tumor microenvironment may additionally contribute to disease recurrence. This study identifies metabolic reprogramming of leukemic cells by bone marrow stromal cells (BMSC) as a putative mechanism of drug resistance. In a BMSC-extracellular matrix culture model, BMSC produced chemoprotective soluble factors and facilitated the emergence of a reversible multidrug resistant phenotype in ALL cells. BMSC environment induced a mitochondrial calcium influx leading to increased reactive oxygen species (ROS) levels in ALL cells. In response to this oxidative stress, drug resistant cells underwent a redox adaptation process, characterized by a decrease in ROS levels and mitochondrial membrane potential with an upregulation of antioxidant production and MCL-1 expression. Similar expanded subpopulations of low ROS expressing and drug resistant cells were identified in pre-treatment bone marrow samples from ALL patients with slower response to therapy. This suggests that the bone marrow microenvironment induces a redox adaptation in ALL subclones that protects against cytotoxic stress and potentially gives rise to minimal residual disease. Targeting metabolic remodeling by inhibiting antioxidant production and antiapoptosis was able to overcome drug resistance. Thus metabolic plasticity in leukemic cell response to environmental factors contributes to chemoresistance and disease recurrence. Adjunctive strategies targeting such processes have the potential to overcome therapeutic failure in ALL.     

Vasodilator oxyfedrine inhibits aldehyde metabolism and thereby sensitizes cancer cells to xCT‐targeted therapy

The major cellular antioxidant glutathione (GSH) protects cancer cells from oxidative damage that can lead to the induction of ferroptosis, an iron‐dependent form of cell death triggered by the aberrant accumulation of lipid peroxides. Inhibitors of the cystine‐glutamate antiporter subunit xCT, which mediates the uptake of extracellular cystine and thereby promotes GSH synthesis, are thus potential anticancer agents. However, the efficacy of xCT‐targeted therapy has been found to be diminished by metabolic reprogramming that affects redox status in cancer cells. Identification of drugs for combination with xCT inhibitors that are able to overcome resistance to xCT‐targeted therapy might thus provide the basis for effective cancer treatment. We have now identified the vasodilator oxyfedrine (OXY) as a sensitizer of cancer cells to GSH‐depleting agents including the xCT inhibitor sulfasalazine (SSZ). Oxyfedrine contains a structural motif required for covalent inhibition of aldehyde dehydrogenase (ALDH) enzymes, and combined treatment with OXY and SSZ was found to induce accumulation of the cytotoxic aldehyde 4‐hydroxynonenal and cell death in SSZ‐resistant cancer cells both in vitro and in vivo. Microarray analysis of tumor xenograft tissue showed cyclooxygenase‐2 expression as a potential biomarker for the efficacy of such combination therapy. Furthermore, OXY‐mediated ALDH inhibition was found to sensitize cancer cells to GSH depletion induced by radiation therapy in vitro. Our findings thus establish a rationale for repurposing of OXY as a sensitizing drug for cancer treatment with agents that induce GSH depletion.     

Intercellular transfer of drug resistance

The effect of L-phenylalanine mustard (L-PAM) on heterogeneous cell populations containing sensitive and resistant cells was evaluated by flow cytometric analysis of DNA damage. Cell cultures were treated with L-PAM for 1 h, fixed, and stained with anti-DNA monoclonal antibody which detects DNA damage induced by alkylating agents. DNA damage was significantly lower in sensitive A2780 cells cocultured with resistant A549 or A2780/PAM cells than in A2780 cells grown separately. Decrease of DNA damage in sensitive cells did not occur when sensitive and resistant cells were grown in common medium without direct contact. Transfer of drug resistance in cocultures was prevented by phorbol ester which is known to inhibit metabolic cooperation via cell junctions. Treatment of cocultures with buthionine sulfoximine increased DNA damage in resistant cells and prevented decrease of DNA damage in sensitive cells. Glutathione (GSH) content in A2780 cells cocultured with A549 cells was significantly higher than GSH content in A2780 cells grown separately. We conclude that decreased response of sensitive cells in cocultures was induced by contact transfer of GSH from GSH-rich resistant cells to sensitive cells. Intercellular transfer of drug resistance demonstrated by analysis of DNA damage was confirmed by colony formation assay. Treatment with L-PAM and Adriamycin killed all cells in A2780/MDR and A549 cultures. Coculture of these lines survived combination treatment because transfer of GSH to multidrug-resistant cells from GSH-rich A549 cells induced resistance to L-PAM and Adriamycin in a single cell. The presence of 2% A549 cells increased resistance of A2780/MDR cells to L-PAM. Phorbol ester eliminated resistance of coculture to combination treatment. Metabolic cooperation between cell subsets with different mechanisms of drug resistance induced resistance to treatment with drugs of different classes (multiclass drug resistance). Inhibition of cell cooperation may improve the response of tumors to combination chemotherapy.     

Role of glutathione in cancer pathophysiology and therapeutic interventions

Glutathione (GSH) is an important intracellular antioxidant that instills several vital roles within a cell including maintenance of the redox state, drug detoxification, and cellular protection from damage by free radicals, peroxides and toxins. Molecular alterations in the components of the GSH system in various tumors can lead to increased survival and enhanced tumor drug resistance. Early identification of the importance of intracellular GSH to detoxification reactions has now led to investigating the potential importance that GSH chemistry has on signal transduction, molecular regulation of cellular physiology and regulation of apoptosis pathway. Several therapeutic agents that target this system have been developed and used experimentally and clinically in an attempt to improve cancer chemotherapy. This review highlights different roles played by GSH that finally regulate tumor growth and advances in the use of GSH-based drugs to specifically target this detoxifying system in cancer treatment as a means to increase therapeutic response and decrease chemotherapeutic drug resistance.     

A study of cross-resistance pattern and expression of molecular markers of multidrug resistance in a human small-cell lung-cancer cell line selected with doxorubicin

A doxorubicin-resistant variant of the human small-cell lung-cancer cell line N592 was selected by in vitro continuous exposure to increasing drug concentrations. The aim of this study was to examine the cross-resistance pattern, cellular pharmacokinetics of doxorubicin and expression of molecular factors of resistance. The sub-line N592/DX exhibited a multidrug-resistance phenotype, which was somewhat atypical, since it included cisplatin. Development of doxorubicin resistance could not be attributed to differential doxorubicin uptake or retention. Verapamil partially reverted doxorubicin resistance without affecting cellular pharmacokinetics. These findings are consistent with undetectable levels of mdr-1 -gene expression in these cells. A molecular analysis of other putative mechanisms of multidrug resistance indicated no alterations in GSH levels or GSH-related enzymes, but a marginal reduction of topoisomerase II α expression in the resistant sub-line. This reduction, which was associated with an increase in topoisomerase I, does not explain the high degree of resistance. This study supports the view that alternative, unidentified mechanisms, which may be of clinical relevance, must be involved in the development of multidrug resistance of small-cell lung cancer.     

Regulation of cellular glutathione modulates nuclear accumulation of daunorubicin in human MCF7 cells overexpressing multidrug resistance associated protein

Multidrug resistance (MDR) is frequently associated with the overexpression of P-glycoprotein (Pgp) and/or multidrug resistance associated protein (MRP1), both members of the ABC superfamily of transporters. Pgp and MRP1 function as ATP-dependent efflux pumps that extrude cytotoxic drugs from tumour cells. Glutathione (GSH) has been considered to play an important role in the MRP1-mediated MDR. In our study, we examined the effects of buthionine sulphoximine (BSO), an inhibitor of GSH biosynthesis, on the nuclear accumulation of daunorubicin (DNR), in etoposide (VP16) and doxorubicin (ADR) resistant MCF7 cell lines, overexpressing respectively MRP1 (MCF7/VP) and Pgp (MCF7/ADR). The study of DNR transport was carried out using scanning confocal microspectrofluorometry. This technique allows the determination of the nuclear accumulation of anthracyclines in single living tumour cells. Treatment of MCF7/VP cells with BSO increased the sensitivity of these cells to DNR whilst the cytotoxicity of the drug in MCF7/ADR cells remained unchanged. In MCF7 resistant cells treated with BSO, their GSH level decreased as observed by confocal microscopy. DNR nuclear accumulation in MCF7/VP cells was increased by BSO whereas in MCF7/ADR cells BSO was unable to significantly increase the DNR nuclear accumulation. These data suggest a requirement for GSH in MRP1-mediated resistance whilst the nuclear efflux of GSH conjugates is probably not the primary mechanism of Pgp-mediated MDR. Finally, BSO might be a useful agent in clinical assays for facilitating detection of MRP1 expression.     

Do glutathione and related enzymes play a role in drug resistance in small cell lung cancer cell lines?

Small cell lung cancer (SCLC) is treated primarily with combination chemotherapy. Despite high initial response rates, most patients eventually die with drug resistant disease. In some tumours, resistance to multiple chemotherapeutic agents is attributed to overexpression of P-glycoprotein (P-gp). However, this does not appear to be a frequent occurrence in drug resistant SCLC. Increased levels of glutathione (GSH) and related enzymes may play a role in resistance to alkylating agents as well as natural product drugs. We measured levels of GSH, glutathione S-transferase (GST), glutathione reductase (GSH Red), glutathione peroxidase (GSH Px), and gamma-glutamyl transpeptidase (gamma-GT) in a panel of 20 SCLC cell lines. Most of these lines were established from patients treated at this centre. Each cell line had a characteristic and reproducible profile of GSH and related enzyme levels. Immunoblot analysis indicated that the predominant GST in the cell lines was the anionic pi isoenzyme. The relative sensitivity of each of these cell lines to 16 different chemotherapeutic agents was measured using a modified MTT assay. Spearman rank correlation analysis was used to determine the relationships between the relative chemosensitivity of these cell lines and the levels of GSH and related enzymes. The number of positive correlations was no greater than expected by chance alone. Furthermore, there was no correlation with the treatment history of the patients from whom the cell lines were derived. These data suggest that alterations in glutathione metabolism do not play a major role in resistance to chemotherapeutic agents in these human SCLC cell lines.     

Effect of buthionine sulfoximine on toxicity of verapamil and doxorubicin to multidrug resistant cells and to mice

Resistance of tumor cells to chemotherapeutic drugs may be due to several mechanisms within a single cell line. Resistance to doxorubicin in the human multidrug resistant breast cancer cell line, MCF-7 AdrR, has been attributed to increased glutathione (GSH) S-transferase and GSH peroxidase activity, as well as to increased expression of the mdr1 gene product, P-glycoprotein. We studied the potentiation of doxorubicin activity in these cells by buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, and by verapamil and trans-flupenthixol, agents which interact with P-glycoprotein. Treatment with BSO enhanced the effect of doxorubicin by 1.5-fold, while verapamil or transflupenthixol caused a greater reversal of drug resistance. The combination of BSO with trans-flupenthixol produced no further potentiation of doxorubicin activity. However, the combination of BSO with verapamil and doxorubicin caused up to a 10-fold increment in antiproliferative effect. To explore the mechanism by which BSO interacted with this drug combination, we determined whether or not BSO might potentiate the effects of verapamil. These studies demonstrated that the effects of BSO were predominantly due to an increase in verapamil toxicity rather than to doxorubicin toxicity. In addition, when mice received concentrations of BSO in their drinking water sufficient to deplete GSH and were treated with verapamil, the calcium channel blocker was lethal to 9 of 12 mice receiving BSO compared to 1 of 10 control animals receiving verapamil alone. These studies demonstrate that BSO does not markedly increase the pharmacological effect of doxorubicin against MCF-7 AdrR cells and suggest that alterations in GSH and related enzymes are not a major factor in drug resistance in this cell line. Furthermore, BSO can increase the toxicity of verapamil, a finding which may have important implications for clinical trials.     

Sensitization of drug resistant human ovarian cancer cells to cyanomorpholino doxorubicin (MRA-CN) by modulation of glutathione metabolism.

MRA-CN, the alkylating cyanomorpholino derivative of doxorubicin (DOX), is extremely potent (100 to 1000 fold increase in cytotoxicity in vitro and in vivo), more lipophilic, non-cardiotoxic, and non-cross-resistant in multidrug resistant cells compared to DOX. We have developed an ovarian carcinoma cell line ES-2R that is 4-fold resistant to MRA-CN, compared to the parental ES-2 cells. This resistant cell line exhibits cross-resistance to alkylators and ionizing radiation. Glutathione (GSH) and GSH-dependent enzymes were found to be altered in the resistant cells with 1.5-fold increase in GSH, and 2- to 3-fold increase in the pi-class glutathione-s-transferase (GST) protein. Both D,L buthionine-S,R-sulfoximine (BSO) and ethacrynic acid (EA), inhibitors of GSH biosynthesis and pi-class GST activity, respectively, could sensitize the ES-2R cells to MRA-CN. These findings implicate a role for GSH metabolism in resistance of ES-2R cells to MRA-CN. The data also indicates the potential utility of EA to modulate GST activity and sensitize tumor cells toward alkylators.