Fluoropyrimidine resistance in colon cancer

A significant obstacle for the management of patients with colorectal cancer is intrinsic drug resistance or in patients that respond to chemotherapy, acquired drug resistance. Drug resistance can occur through a variety of mechanisms. These include alterations in drug influx, drug efflux, intracellular metabolic activation, intracellular catabolism, through alterations in the drug’s target or through numerous changes downstream of the target including alterations in genes involved in the regulation of the cell cycle, apoptosis or in DNA damage repair. In this article, the mechanisms of action and the mechanisms of resistance to the fluoropyrimidines are reviewed focusing on newer studies using tumor samples obtained from patients. Clinical trials that can potentially overcome the relevant mechanisms of resistance are described.     

Fluoropyrimidine resistance in colon cancer

A significant obstacle for the management of patients with colorectal cancer is intrinsic drug resistance or in patients that respond to chemotherapy, acquired drug resistance. Drug resistance can occur through a variety of mechanisms. These include alterations in drug influx, drug efflux, intracellular metabolic activation, intracellular catabolism, through alterations in the drug's target or through numerous changes downstream of the target including alterations in genes involved in the regulation of the cell cycle, apoptosis or in DNA damage repair. In this article, the mechanisms of action and the mechanisms of resistance to the fluoropyrimidines are reviewed focusing on newer studies using tumor samples obtained from patients. Clinical trials that can potentially overcome the relevant mechanisms of resistance are described.     

大肠癌耐药机制的研究进展

化疗药物的耐药较大程度地限制了大肠癌化疗的效果，其中约90％的转移性病例治疗失败可以归因于药物耐药。即使在辅助化疗中，耐药性微转移灶的存在也可能降低化疗的疗效。目前的研究表明，许多因素可以影响化疗药物的敏感性，包括药物的药代动力学、药物的激活或失活、药物作用靶点的变化、（药物诱导的DNA损伤修复、细胞凋亡的逃逸等。本文试从药物代谢、药物靶点、DNA损伤修复及细胞凋亡等方面叙述大肠癌对常规化疗药物的耐药机制，并对新的靶向药物可能的耐药机制作一简要介绍。     

Molecular mechanisms of chemoresistance in gastric cancer

Gastric cancer is the fourth most common cancer and the second leading cause of cancer deaths worldwide. Chemotherapy is one of the major treatments for gastric cancer, but drug resistance limits the effectiveness of chemotherapy, which results in treatment failure. Resistance to chemotherapy can be present intrinsically before the administration of chemotherapy or it can develop during chemotherapy. The mechanisms of chemotherapy resistance in gastric cancer are complex and multifactorial. A variety of factors have been demonstrated to be involved in chemoresistance, including the reduced intracellular concentrations of drugs, alterations in drug targets, the dysregulation of cell survival and death signaling pathways, and interactions between cancer cells and the tumor microenvironment. This review focuses on the molecular mechanisms of chemoresistance in gastric cancer and on recent studies that have sought to overcome the underlying mechanisms of chemoresistance.     

Molecular determinants of response to 5-fluorouracil-based chemotherapy in colorectal cancer: The undisputable role of micro-ribonucleic acids

5-flurouracil (5-FU)-based chemotherapy is the main pharmacological therapy for advanced colorectal cancer (CRC). Despite significant progress in the treatment of CRC during the last decades, 5-FU drug resistance remains the most important cause of failure in CRC therapy. Resistance to 5-FU is a complex and multistep process. Different mechanisms including microsatellite instability, increased expression level of key enzyme thymidylate synthase and its polymorphism, increased level of 5-FU-activating enzymes and mutation of TP53 are proposed as the main determinants of resistance to 5-FU in CRC cells. Recently, micro-ribonucleic acids (miRNA) and their alterations were found to have a crucial role in 5-FU resistance. In this regard, the miRNA-mediated mechanisms of 5-FU drug resistance reside among the new fields of pharmacogenetics of CRC drug response that has not been completely discovered. Identification of the biological markers that are related to response to 5-FU-based chemotherapy is an emerging field of precision medicine. This approach will have an important role in defining those patients who are most likely to benefit from 5-FU-based chemotherapy in the future. Thereby, the identification of 5-FU drug resistance mechanisms is an essential step to predict and eventually overcome resistance. In the present comprehensive review, we will summarize the latest knowledge regarding the molecular determinants of response to 5-FU-based chemotherapy in CRC by emphasizing the role of miRNAs.     

Drug resistance reversal--are we getting closer?

Clinical drug resistance is a major barrier to overcome before chemotherapy can become curative for most patients presenting with metastatic cancer. Rational attempts to tackle clinical drug resistance need to be based on an understanding of the mechanisms involved; these are likely to be complex and multifactorial, and may be due to inadequate drug exposure or alterations in the cancer cell itself. This article reviews a number of strategies used to tackle drug resistance, focussing on work in our institution related to the treatment of ovarian cancer and resistance to platinum and taxane-based chemotherapy. Further progress towards drug resistance reversal will require a three-pronged approach, namely: the development of novel cytotoxics which exploit selectively expressed targets; modulation of resistance to conventional agents and, most importantly, a serious attempt to understand resistance mechanisms in tumour samples taken both pre- and post-chemotherapy.     

Intrinsic and acquired resistance to HER2-targeted therapies in HER2 gene-amplified breast cancer: mechanisms and clinical implications

Approximately 25% of human breast cancers overexpress the HER2 (ErbB2) proto-oncogene, which confers a more aggressive tumor phenotype and associates with a poor prognosis in patients with this disease. Two approved therapies targeting HER2, the monoclonal antibody trastuzumab and the tyrosine kinase inhibitor lapatinib, are clinically active against this type of breast cancer. However, a significant fraction of patients with HER2+ breast cancer treated with these agents eventually relapse or develop progressive disease. This suggests that tumors acquire or possess intrinsic mechanisms of resistance that allow escape from HER2 inhibition. This review focuses on mechanisms of intrinsic and/or acquired resistance to HER2-targeted therapies that have been identified in preclinical and clinical studies. These mechanisms involve alterations to HER2 itself, coexpression or acquisition of bypass signaling through other receptor or intracellular signaling pathways, defects in mechanisms of cell cycle regulation or apoptosis, and host factors that may modulate drug response. Emerging clinical evidence already suggests that combinations of therapies targeting HER2 as well as these resistance pathways will be effective in overcoming or preventing resistance.     

The role of UDP-glucuronosyltransferases and drug transporters in breast cancer drug resistance

One of the major limitations of chemotherapy is that often, over time, tumor cells become either inherently resistant or develop multidrug resistance to the treatment. Another limitation of chemotherapy is toxicity to normal tissues and adverse side effects. The reasons for the failure of some cancers to respond to chemotherapeutic drugs are not clear but have been attributed to alterations in many molecular pathways, which include drug metabolizing enzymes and drug transporter genes. Alterations in the energy-dependent ATP-binding cassette (ABC) transporter genes have been suggested to confer a drug-resistant phenotype by decreasing the intracellular accumulation of chemotherapeutic drugs via efflux mechanisms. In addition, polymorphisms in UDP-glucuronosyltransferases (UGTs) have been reported to correlate with clinical outcome and drug resistance. In this review, we provide an overview of known polymorphisms within UGTs and ABC transporter genes that have been reported to have altered expression and/or activity in breast cancer. Those polymorphic variants that affect the clinical efficacy and confer drug resistance of chemotherapeutic agents, including hormonal therapies, taxanes, anthracyclines, and alkylating agents, in breast cancer.     

Genetic predictors for drug resistance in soft tissue sarcoma: a review of publications in 2004

PURPOSE OF REVIEW: Sarcomas are generally managed through a multidisciplinary approach including surgery, radiotherapy, and chemotherapy. Metastatic tumor cells frequently develop resistance to cytotoxic agents. Several molecular mechanisms of drug resistance have been uncovered recently. RECENT FINDINGS: Among genes governing the apoptosis pathway, overexpression of the bcl2 family or mutations of p53 have recently been reported to be involved in drug resistance in sarcoma. The multidrug resistance genes are involved in the efflux of chemotherapeutic agents. Identification of members of this family of genes may predict resistance for developing new strategies. Several histologic subtypes of sarcomas have specific mechanisms of resistance. Methotrexate is one of the four active drugs for osteosarcoma and penetrates in tumor cell through specific carrier (reduced folate carrier) whose levels of expression or changes in amino acid sequence correlate to resistance to methotrexate. Specific molecular alteration defining nosological entities among sarcoma also correlates to drug resistance. Thirty percent of human sarcomas are characterized by specific translocations that may predict, for specific subtypes, survival and response to treatment. Finally, gastrointestinal tumors harbor specific activating mutations in KIT or PDGFRA genes, which are responsive to imatinib. Specific mutations of the KIT and PDGFRA genes have now repeatedly been found correlated to response or resistance to imatinib. SUMMARY: The molecular alterations present in tumor cells predict influence sensitivity or resistance to chemotherapy. Ultimately, this may delineate specific therapeutic strategies for both cytotoxic and targeted therapies in sarcomas.     

Characterization of non-small-cell lung cancer cell lines established before and after chemotherapy

We established several in vitro drug-resistant cell lines after continuous, long-term exposure of each drug to elucidate mechanisms of drug resistance. Whether drug resistance in these in vitro resistant cell lines reflects clinical drug resistance still remains unanswered. In this study, a pair of lung cancer cell lines was established from one patient with squamous cell carcinoma of the lung, with one line being established before and one line after combination chemotherapy (cisplatin/ifosfamide/vindesine). Combination chemotherapy selected resistant EBC-2/R cells, which showed cross-resistance to 4-hydroxyifosfamide (3.2-fold), cisplatin (2.3-fold), and methotrexate (3.7-fold) and collateral sensitivity to vindesine (0.77-fold) compared with parent EBC-2 cells. EBC-2/R cells showed decrease in intracellular accumulation of cisplatin, increase in intracellular concentration of glutathione (GSH), and overexpression of multidrug resistance-associated protein (MRP) 3 when compared with EBC-2 cells. A single cycle of chemotherapy was not sufficient to select other mechanisms of drug resistance, such as multidrug resistance-1/P-glycoprotein, MRPs 1, 2, 4, and 5, lung resistance-related protein, metallothionein IIa, glutathione S-transferase pi, gamma-glutamylcysteine synthetase (light and heavy chain), and excision repair cross complementing 1. Sequentially we established two cell lines, which cell lines showed the differences of the cisplatin resistance, expression level of MRP3, intracellular GSH level and intracellular accumulation of cisplatin. A pair of cell lines will be useful to elucidate resistant mechanisms of cisplatin in heterogeneous lung cancer cells.     

Multiple mechanisms confer drug resistance to mitoxantrone in the human 8226 myeloma cell line

Selection for in vitro drug resistance can result in a complex phenotype with more than one mechanism of resistance emerging concurrently or sequentially. We examined emerging mechanisms of drug resistance during selection with mitoxantrone in the human myeloma cell line 8226. A novel transport mechanism appeared early in the selection process that was associated with a 10-fold resistance to mitoxantrone in the 8226/MR4 cell line. The reduction in intracellular drug concentration was ATP-dependent and ouabain-insensitive. The 8226/MR4 cell line was 34-fold cross-resistant to the fluorescent aza-anthrapyrazole BBR 3390. The resistance to BBR 3390 coincided with a 50% reduction in intracellular drug concentration. Confocal microscopy using BBR 3390 revealed a 64% decrease in the nuclear:cytoplasmic ratio in the drug-resistant cell line. The reduction in intracellular drug concentration of both mitoxantrone and BBR 3390 was reversed by a novel chemosensitizing agent, fumitremorgin C. In contrast, fumitremorgin C had no effect on resistance to mitoxantrone or BBR 3390 in the P-glycoprotein-positive 8226/DOX6 cell line. Increasing the degree of resistance to mitoxantrone in the 8226 cell line from 10 to 37 times (8226/MR20) did not further reduce the intracellular drug concentration. However, the 8226/MR20 cell line exhibited 88 and 70% reductions in topoisomerase II beta and alpha expression, respectively, compared with the parental drug sensitive cell line. This decrease in topoisomerase expression and activity was not observed in the low-level drug-resistant, 8226/MR4 cell line. These data demonstrate that low-level mitoxantrone resistance is due to the presence of a novel, energy-dependent drug efflux pump similar to P-glycoprotein and multidrug resistance-associated protein. Reversal of resistance by blocking drug efflux with fumitremorgin C should allow for functional analysis of this novel transporter in cancer cell lines or clinical tumor samples. Increased resistance to mitoxantrone may result from reduced intracellular drug accumulation, altered nuclear/cytoplasmic drug distribution, and alterations in topoisomerase II activity.     

Molecular basis of antifolate resistance

Folates play a key role in one-carbon metabolism essential for the biosynthesis of purines, thymidylate and hence DNA replication. The antifolate methotrexate has been rationally-designed nearly 60 years ago to potently block the folate-dependent enzyme dihydrofolate reductase (DHFR) thereby achieving temporary remissions in childhood acute leukemia. Recently, the novel antifolates raltitrexed and pemetrexed that target thymidylate synthase (TS) and glycineamide ribonucleotide transformylase (GARTF) were introduced for the treatment of colorectal cancer and malignant pleural mesothelioma. (Anti)folates are divalent anions which predominantly use the reduced folate carrier (RFC) for their cellular uptake. (Anti)folates are retained intracellularly via polyglutamylation catalyzed by folylpoly-γ-glutamate synthetase (FPGS). As the intracellular concentration of antifolates is critical for their pharmacologic activity, polyglutamylation is a key determinant of antifolate cytotoxicity. However, anticancer drug resistance phenomena pose major obstacles towards curative cancer chemotherapy. Pre-clinical and clinical studies have identified a plethora of mechanisms of antifolate-resistance; these are frequently associated with qualitative and/or quantitative alterations in influx and/or efflux transporters of (anti)folates as well as in folate-dependent enzymes. These include inactivating mutations and/or down-regulation of the RFC and various alterations in the target enzymes DHFR, TS and FPGS. Furthermore, it has been recently shown that members of the ATP-binding cassette (ABC) superfamily including multidrug resistance proteins (MRP/ABCC) and breast cancer resistance protein (BCRP/ABCG2) are low affinity, high capacity ATP-driven (anti)folate efflux transporters. This transport activity is in addition to their established facility to extrude multiple cytotoxic agents. Hence, by actively extruding antifolates, overexpressed MRPs and/or BCRP confer antifolate resistance. Moreover, down-regulation of MRPs and/or BCRP results in decreased folate efflux thereby leading to expansion of the intracellular folate pool and antifolate resistance. This chapter reviews and discusses the panoply of molecular modalities of antifolate-resistance in pre-clinical tumor cell systems in vitro and in vivo as well as in cancer patients. Currently emerging novel strategies for the overcoming of antifolate-resistance are presented. Finally, experimental evidence is provided that the identification and characterization of the molecular mechanisms of antifolate-resistance may prove instrumental in the future development of rationally-based novel antifolates and strategies that could conceivably overcome drug-resistance phenomena.     

Down-regulation of mitochondrial F1F0-ATP synthase in human colon cancer cells with induced 5-fluorouracil resistance

5-Fluorouracil (5-FU) is widely used for treatment of advanced colorectal cancer. However, it is common for such patients to develop resistance to 5-FU, and this drug resistance becomes a critical problem for chemotherapy. The mechanisms underlying this resistance are largely unknown. To screen for proteins possibly responsible for 5-FU resistance, cells resistant to 5-FU were derived from human colon cancer cell lines and two-dimensional gel electrophoresis-based comparative proteomics was done. Two-dimensional gel electrophoresis data showed there was lower expression of the alpha subunit of mitochondrial F(1)F(0)-ATP synthase (ATP synthase) in 5-FU-resistant cells compared with parent cells. Western blotting showed that expression of other ATP synthase complex subunits was also lower in 5-FU-resistant cell lines and that these resistant cells also showed decreased ATP synthase activity and reduced intracellular ATP content. The ATP synthase inhibitor, oligomycin A, strongly antagonized 5-FU-induced suppression of cell proliferation. When 5-FU sensitivity was compared with ATP synthase activity in six different human colon cancer cell lines, a positive correlation has been found. Furthermore, suppressed ATP synthase d-subunit expression by siRNA transfection increased cell viability in the presence of 5-FU. Bioenergetic dysfunction of mitochondria has been reported as a hallmark of many types of cancers (i.e., down-regulation of ATP synthase beta-subunit expression in liver, kidney, colon, squamous oesophageal, and lung carcinomas, as well as in breast and gastric adenocarcinomas). Our findings show that ATP synthase down-regulation may not only be a bioenergetic signature of colorectal carcinomas but may also lead to cellular events responsible for 5-FU resistance.     

Apoptosis and chemo-resistance in colorectal cancer

Systemic chemotherapy plays an integral part in treating advanced colorectal cancer. However 50% of patients respond poorly or have disease progression due to resistance to chemotherapeutic agents. This article reviews the pathways that regulate apoptosis, apoptotic mechanisms through which chemotherapeutic agents mediate their effect and how deregulation of apoptotic proteins may contribute to chemo-resistance. Also discussed are potential therapeutic strategies designed to target these proteins and thereby improve response rates to chemotherapy in colorectal cancer.     

Use of comparative proteomics to identify potential resistance mechanisms in cancer treatment

Drug resistance is a major problem in successful cancer chemotherapy. Many molecular mechanisms that are responsible for drug resistance are known whereas others have yet to be discovered. Determining the exact mechanism activated in a particular case (clinical or laboratory) is a difficult task. Recently, proteomics has been applied to investigate drug resistance mechanisms in model cancer cell lines. As a result, novel mechanisms of resistance have been discovered and known mechanisms of resistance confirmed. In this paper, we wish to review recent developments and progresses in the application of proteomic tools to identify known and novel drug resistance mechanisms in drug-selected model cancer cell lines. Our combined analyses of multiple proteomic studies of various drug resistant cancer cell lines revealed that many mechanisms of resistance likely exist in any given drug-selected cancer cell line and that common mechanisms of resistance may be selected in a spectrum of cancer cell lines. These observations suggest that combination therapies targeting multiple mechanisms to sensitize drug resistant cancers may be necessary to eradicate cancers in the future.     

ABCG2 overexpression in colon cancer cells resistant to SN38 and in irinotecan-treated metastases

Overcoming drug resistance has become an important issue in cancer chemotherapy. Among all known mechanisms that confer resistance, active efflux of chemotherapeutic agents by proteins from the ATP-binding cassette family has been extensively reported. The aim of the present study was to determine the involvement of ABCG2 in resistance to SN38 (the active metabolite of irinotecan) in colorectal cancer. By progressive exposure to increasing concentrations of SN38, we isolated 2 resistant clones from the human colon carcinoma cell line HCT116. These clones were 6- and 53-fold more resistant to SN38 than the HCT116-derived sensitive clone. Topoisomerase I expression was unchanged in our resistant variants. The highest resistance level correlated with an ABCG2 amplification. This overexpression was associated with a marked decrease in the intracellular accumulation of SN38. The inhibition of ABCG2 function by Ko143 demonstrated that enhanced drug efflux from resistant cells was mediated by the activity of ABCG2 protein and confirmed that ABCG2 is directly involved in acquired resistance to SN38. Furthermore, we show, for the first time in clinical samples, that the ABCG2 mRNA content in hepatic metastases is higher after an irinotecan-based chemotherapy than in irinotecan-naive metastases. In conclusion, this study supports the potential involvement of ABCG2 in the development of irinotecan resistance in vivo.     

干预自噬影响DPD的表达促进5-FU抗结直肠癌疗效的实验研究

背景与目的：结直肠癌作为常见的消化系统恶性肿瘤之一,其化疗与耐药一直以来备受关注,5-氟尿嘧啶（5-fluorouracil,5-FU）是结直肠癌的一线化疗药物,其疗效常因耐药或不良反应而受到影响。二氢嘧啶脱氢酶（dihydropyrimidine dehydrogenase,DPD）是5-FU代谢的关键限速酶,其表达或降解可能成为影响5-FU疗效的因素。自噬是细胞内蛋白质代谢的重要途径,其在化疗诱导的细胞死亡或增殖抑制中的作用仍存在争议。本研究旨在探讨自噬在结直肠癌化疗过程中的作用以及干预自噬影响5-FU耐药的机制。方法：体外细胞培养人结肠癌HCT-8、COLO205、LOVO和SW480细胞系,观察不同细胞系中DPD的表达对5-FU敏感性的影响,通过药物敏感性实验筛选出5-FU敏感细胞,检测5-FU敏感细胞株及其相对应的耐药细胞株中DPD的表达、自噬水平及自噬关键因子微管相关蛋白轻链3（light chain 3,LC3）、P62的表达,并通过诱导/抑制细胞自噬,观察调控自噬状态对DPD的表达变化以及肿瘤细胞生物学行为和化疗抵抗能力的影响,通过UbiBrowser数据库筛选及免疫共沉淀实验（co-inmunoprecipitation,Co-IP）实验验证DPD降解过程中的E3连接酶,探讨自噬降解DPD逆转5-FU耐药的分子机制。结果：DPD低表达的细胞系对5-FU的敏感性更强,其中COLO205细胞系在4种细胞中DPD表达量最高,并对5-FU的耐药性最强,而HCT-8细胞DPD表达最低并对5-FU最为敏感。与HCT-8细胞相比,HCT-8/FU耐药细胞表达较高水平的DPD,以及较低的基础自噬水平;雷帕霉素（rapamycin,RAPA）介导的自噬激活增强了细胞的自噬水平,降低了DPD表达,同时降低了细胞增殖、侵袭和5-FU耐药性;3甲基腺嘌呤（3-methyladenine,3-MA）及羟氯喹（hydroxychloroquine,HCQ）介导的自噬抑制减弱了细胞的自噬水平,增加了DPD表达,增强了5-FU耐药性。5-FU与自噬激活剂联合应用对细胞的抑效果要远远强于单用5-FU及与自噬抑制剂联合应用的效果;DPD的降解需要完整的自噬流的参与,其中自噬溶酶体的形成是DPD降解的关键步骤之一,而单一的自噬体无法对DPD进行降解。E3连接酶NEDD4在HCT-8/5-FU细胞中与DPD和P62蛋白免疫共沉淀,在DPD的自噬降解中发挥作用。结论：自噬参与DPD降解影响结直肠癌细胞对化疗药物的敏感性,激活自噬可促进DPD降解和抑制5-FU的分解代谢,可能成为逆转结直肠癌5-FU耐药的新途径。