Resistance Phenotype In Cancer Cells Research Articles

CRISPR/Cas9 system: a novel approach to overcome chemotherapy and radiotherapy resistance in cancer.

Cancer presents a global health challenge with rising incidence and mortality. Despite treatment advances in cancer therapy, radiotherapy and chemotherapy remained the most common treatments for all types of cancers. However, resistance phenotype in cancer cells leads to unsatisfactory results in the efficiency of therapeutic strategies. Therefore, researchers strive to propose effective solutions to overcome treatment failure, which requires a deep knowledge of treatment-resistant mechanisms. The progression and occurrence of tumors can be attributed to gene mutation. Over the past decade, the emergence of clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) genome editing has revolutionized cancer research. This versatile technology enables cancer modeling, manipulation of specific DNA sequences, and genome-wide screening. CRISPR/Cas9 is an effective tool for identifying radio- and chemoresistance genes and offering potential adjunctive treatments to overcome tumor recurrence after chemo- and radiotherapy. This article aims to explain the potential of the CRISPR/Cas9 system in improving the effectiveness of chemo- and radiotherapy and ultimately overcoming treatment failure.

Increased expression of the pathological O-glycosylated form of oncofetal fibronectin in the multidrug resistance phenotype of cancer cells

Changes in protein glycosylation are a hallmark of transformed cells and modulate numerous phenomena associated with cancer progression, such as the acquisition of multidrug resistance (MDR) phenotype. Different families of glycosyltransferases and their products have already been described as possible modulators of the MDR phenotype. Among the glycosyltransferases intensively studied in cancer research, UDP-N-acetyl-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase-6 (pp-GalNAc-T6), which is widely expressed in many organs and tissues, stands out. Its influence in several events associated with kidney, oral, pancreatic, renal, lung, gastric and breast cancer progression has already been described. However, its participation in the MDR phenotype has never been studied. Here, we demonstrate that human breast adenocarcinoma MCF-7 MDR cell lines, generated by chronic exposure to doxorubicin, in addition to exhibiting increased expression of proteins belonging to the ABC superfamily (ABCC1 and ABCG2), and anti-apoptotic proteins (Blcl-2 and Bcl-xL), also present high expression of pp-GalNAc-T6, the enzyme currently proposed as the main responsible for the biosynthesis of oncofetal fibronectin (onf-FN), a major extracellular matrix component expressed by cancer cells and embryonic tissues, but absent in healthy cells. Our results show that onf-FN, which is generated by the addition of a GalNAc unit at a specific threonine residue inside the type III homology connective segment (IIICS) domain of FN, is strongly upregulated during the acquisition of the MDR phenotype. Also, the silencing of pp-GalNAc-T6, not only compromises the expression of the oncofetal glycoprotein, but also made the MDR cells more sensitive to all anticancer drugs tested, partially reversing the MDR phenotype. Taken together, our results demonstrate for the first time the upregulation of the O-glycosylated oncofetal fibronectin, as well as the direct participation of pp-GalNAc-T6 during the acquisition of a MDR phenotype in a breast cancer model, giving credence to the hypothesis that in transformed cells, glycosyltransferases and/or their products, such as unusual extracellular matrix glycoproteins can be used as potential therapeutic targets for the treatment of cancer.

Multidrug Resistance of Cancer Cells and the Vital Role of P-Glycoprotein.

P-glycoprotein (P-gp) is a major factor in the multidrug resistance phenotype in cancer cells. P-gp is a protein that regulates the ATP-dependent efflux of a wide range of anticancer medicines and confers resistance. Due to its wide specificity, several attempts have been made to block the action of P-gp to restore the efficacy of anticancer drugs. The major goal has been to create molecules that either compete with anticancer medicines for transport or function as a direct P-gp inhibitor. Despite significant in vitro success, there are presently no drugs available in the clinic that can “block” P-gp–mediated resistance. Toxicity, unfavourable pharmacological interactions, and a variety of pharmacokinetic difficulties might all be the reason for the failure. On the other hand, P-gp has a significant effect in the body. It protects the vital organs from the entry of foreign bodies and other toxic chemicals. Hence, the inhibitors of P-gp should not hinder its action in the normal cells. To develop an effective inhibitor of P-gp, thorough background knowledge is needed in this field. The main aim of this review article was to set forth the merits and demerits of the action of P-gp on cancer cells as well as on normal cells. The influence of P-gp on cancer drug delivery and the contribution of P-gp to activating drug resistance were also mentioned.

Assessing the ABCG2 Protein Interactome: a Potential Strategy to Disrupt ABCG2 Function

The ATP‐binding cassette sub‐family G member 2, ABCG2, is an important plasma membrane transporter that removes potentially harmful endogenous toxins and xenobiotics from cells. ABCG2 was identified as an important contributor to the multidrug resistance phenotype in cancer cells because it exported a wide variety of structurally diverse chemotherapeutic agents, including mitoxantrone, topotecan and the tyrosine kinase inhibitors. High ABCG2 expression in a variety of tumours is associated with poor clinical outcomes. In our present study, we hypothesized that proteins that interact with ABCG2 modulate its function. This knowledge would allow us to discover novel therapeutic targets that could be exploited to alter ABCG2 expression and/or function.To identify interacting proteins for ABCG2, we employed a site‐specific biotinylation system utilizing a 15 amino acid peptide tag known as AviTag, fused to ABCG2. In the presence of the E. coli biotin ligase (BirA), biotin can be covalently attached to the AviTag peptide and the complex can be purified using the non‐covalent streptavidin‐biotin interaction. A plasmid harboring both AviTag‐ABCG2 and an IRES directed BirA was constructed. We stably expressed it in HEK293 cells and confirmed that AviTag ABCG2 was expressed at the plasma membrane and retained typical ABCG2 transport activity. Importantly, the cells expressing the AviTag‐ABCG2 and BirA readily labelled AviTag‐ABCG2 with biotin. The AviTag‐ABCG2 protein complexes captured by streptavidin agarose beads were purified and eluted according to an empirically developed protocol.Using a proteomic approach with mass spectrometry, we identified 39 proteins (both known and novel ABCG2 interacting proteins) at a p‐value threshold of 0.05 in the AviTag‐ABCG2 protein complex compared to the negative control. As expected, ABCG2 was one of the top 10 proteins in abundance (high spectral counts). Among the 39 interacting proteins, we confirmed by immunoblotting that several important cancer‐related proteins interact with ABCG2. These proteins appear capable of regulating ABCG2 function in cancer cells.Future studies will elucidate how these interactions affect ABCG2 function. These findings might aid in the development of new strategies for blocking ABCG2 function in multidrug resistant cancers, which could have important implications in improving patient’s response to chemotherapy.Support or Funding InformationThis work was supported by NIH and ALSAC

Abstract B46: Role of TNF-α and transcriptional factors YB-1 and NFκB in P-glycoprotein expressing cancer cells

Abstract Introduction and Objective: Multidrug resistance phenotype (MDR) is characterized by overexpression of P-glycoprotein (Pgp/ABCB1) and related to chemotherapy cancer treatment failure. However, MDR is considered a multifactorial phenotype associated with molecular pathways deregulation, such as changes in apoptosis-associated proteins or transcriptional regulators expression. Y-box protein 1 (YB-1) and NFκB may regulate Pgp expression, acting as regulators of MDR1/ABCB1 gene. Studies have shown that Pgp and apoptosis-associated proteins expression may also contribute to multifactorial cancer resistance. Tumor necrosis factor-alpha (TNF-α) is an important cytokine that presents ambiguous function on cancer development, since it may act as death signaling or tumor growth factor. Also, TNF-α is a main activator of NFκB pathway. Therefore, the aim of this study was to investigate the role of Pgp expression, the proapoptotic protein TNF-α, and YB-1 and NFκB transcriptional factors in the contribution of multifactorial resistance phenotype in cancer cells. Materials and Methods: In this study we used two cervical cancer cell lines: KB-3-1, parental cell line, and KB-C1, Pgp-positive cell line selected from KB-3-1 through increased doses of colchicine. KB-3-1 and KB-C1 cell lines were treated with doxorubicin, cisplatin, and colchicine for 24, 48, or 72 h and cell viability was analyzed by MTT assay. KB-3-1 and KB-C1 cell lines were treated with recombinant TNF-α (rTNF-α) for 30 min or 24 h and apoptosis index was measured by Annexin-V/PI staining using flow cytometry. Pgp expression, function, and subcellular localization were analyzed by Western blot, flow cytometry, and immunofluorescence, respectively. TNF-α, YB-1, and NFκB expression and subcellular localization were also investigated. Results and Conclusion: Our data showed that KB-3-1 cells were sensitive to drug treatment while KB-C1 cells were resistant to doxorubicin and colchicine drugs, and it is probably related to a functional overexpression of Pgp. However, both cell lines showed sensitivity to high doses of cisplatin, a non-Pgp substrate. Then, we observed that KB-3-1 cells showed higher expression of YB-1 and NFκB/p65 subunit than KB-C1, but lower expression of NFκB/p105 subunit. We observed a perinuclear, nuclear, and cytoplasmatic subcellular distribution of NFκB in both cell lines. Also, YB-1 was detected in cytoplasm and nuclear foci in both cell lines, but apparently wide larger in KB-C1 than KB-3-1. KB-C1 cells exhibited cytoplasmatic and nuclear TNF-α distribution, while KB-3-1 cells showed mostly nuclear localization. Further, we observed low apoptosis rate following rTNF-α treatment in both cell lines. Besides that, Pgp expression was increased and showed strong membrane staining in KB-C1 cells treated with rTNF-α. Also, KB-3-1 and KB-C1 showed higher expression of TNF-α and cytoplasmatic localization after treatment with rTNF-α. In summary, our results suggest that Pgp expression in KB-C1 cell line might be regulated by YB-1 pathway. In addition, rTNF-α treatment changed Pgp expression in resistant cells, suggesting a possible role of TNF-α in supporting resistance phenotype. More data are required to understand the role of TNF-α protein in MDR phenotype. Citation Format: Tandressa Berguetti, Paloma Souza, Raquel Maia. Role of TNF-α and transcriptional factors YB-1 and NFκB in P-glycoprotein expressing cancer cells [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B46.

MiR-503 regulates cisplatin resistance of human gastric cancer cell lines by targeting IGF1R and BCL2

Background Studies have shown that the drug resistance of gastric cancer cells can be modulated by abnormal expression of microRNAs (miRNAs). We investigated the role of miR-503 in the development of cisplatin resistance in human gastric cancer cell lines. Methods MiR-503 expression was measured by quantitative real-time PCR. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and clonogenic assays were used to examine changes in cell viability and the drug resistance phenotype of cancer cells associated with upregulation or downregulation of the miRNA. A dual-luciferase activity assay was used to verify target genes of miR-503. Immunohistochemistry, Western blotting analysis, and a flow cytometric apoptosis assay were used to elucidate the mechanism by which miR-503 modulates drug resistance in cancer cells. Results MiR-503 was significantly downregulated in gastric cancer tissues and several gastric cancer cell lines. Additionally, downregulation of miR-503 in the cisplatin (DDP)-resistant gastric cancer cell line SGC7901/DDP was concurrent with the upregulation of insulin-like growth factor-1 receptor (IGF1R) and B-cell lymphoma 2 (BCL2) expression compared with the parental SGC7901 cell line. An in vitro drug sensitivity assay showed that overexpression of miR-503 sensitized SGC7901/DDP cells to cisplatin. The luciferase activity of reporters driven by IGF1R and BCL2 3′-untranslated regions in SGC7901/DDP cells suggested that IGF1R and BCL2 were both direct target genes of miR-503. Enforced miR-503 expression in SGC7901/DDP cells reduced expression of the target proteins, inhibited proliferation, and sensitized the cells to DDP-induced apoptosis. Conclusion Our findings suggest that hsa-miR-503 modulates cisplatin resistance of human gastric cancer cells at least in part by targeting IGF1R and BCL2.

Effect of efflux pumps expression and cell bioenergetics in the multidrug resistance phenotype in cancer cells

Effect of efflux pumps expression and cell bioenergetics in the multidrug resistance phenotype in cancer cells

NEURONAL MIMICRY AS A RESISTANCE PHENOTYPE IN CANCER CELLS

You have accessJournal of Urology1 Apr 2009NEURONAL MIMICRY AS A RESISTANCE PHENOTYPE IN CANCER CELLS Gustavo E Ayala, Yi Ding, Chad Creighton, and David R Rowley Gustavo E AyalaGustavo E Ayala More articles by this author , Yi DingYi Ding More articles by this author , Chad CreightonChad Creighton More articles by this author , and David R RowleyDavid R Rowley More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(09)60607-8AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "NEURONAL MIMICRY AS A RESISTANCE PHENOTYPE IN CANCER CELLS." The Journal of Urology, 181(4S), p. 211 © 2009 by American Urological AssociationFiguresReferencesRelatedDetails Volume 181Issue 4SApril 2009Page: 211 Advertisement Copyright & Permissions© 2009 by American Urological AssociationMetricsAuthor Information Gustavo E Ayala More articles by this author Yi Ding More articles by this author Chad Creighton More articles by this author David R Rowley More articles by this author Expand All Advertisement PDF DownloadLoading ...

Troglitazone reverses the multiple drug resistance phenotype in cancer cells

A major problem in treating cancer is the development of drug resistance. We previously demonstrated doxorubicin (DOX) resistance in K562 human leukemia cells that was associated with upregulation of glyoxalase 1 (GLO-1) and histone H3 expression. The thiazolidinedione troglitazone (TRG) downregulated GLO-1 expression and further upregulated histone H3 expression and post-translational modifications in these cells, leading to a regained sensitivity to DOX. Given the pleiotropic effects of epigenetic changes in cancer development, we hypothesized that TRG may downregulate the multiple drug resistance (MDR) phenotype in a variety of cancer cells. To test this, MCF7 human breast cancer cells and K562 cells were cultured in the presence of low-dose DOX to establish DOX-resistant cell lines (K562/DOX and MCF7/DOX). The MDR phenotype was confirmed by Western blot analysis of the 170 kDa P-glycoprotein (Pgp) drug efflux pump multiple drug resistance protein 1 (MDR-1), and the breast cancer resistance protein (BCRP). TRG markedly decreased expression of both MDR-1 and BCRP in these cells, resulting in sensitivity to DOX. Silencing of MDR-1 expression also sensitized MCF7/DOX cells to DOX. Use of the specific and irreversible peroxisome proliferator-activated receptor gamma (PPARgamma) inhibitor GW9662 in the nanomolar range not only demonstrated that the action of TRG on MCF/DOX was PPARgamma-independent, but indicated that PPARgamma may play a role in the MDR phenotype, which is antagonized by TRG. We conclude that TRG is potentially a useful adjunct therapy in chemoresistant cancers.

Ceramide and glucosylceramide upregulate expression of the multidrug resistance gene MDR1 in cancer cells

In the present study we used human breast cancer cell lines to assess the influence of ceramide and glucosylceramide (GC) on expression of MDR1, the multidrug resistance gene that codes for P-glycoprotein (P-gp), because GC has been shown to be a substrate for P-gp. Acute exposure (72 h) to C8-ceramide (5 μg/ml culture medium), a cell-permeable ceramide, increased MDR1 mRNA levels by 3- and 5-fold in T47D and in MDA-MB-435 cells, respectively. Acute exposure of MCF-7 and MDA-MB-231 cells to C8-GC (10 μg/ml culture medium), a cell-permeable analog of GC, increased MDR1 expression by 2- and 4- fold, respectively. Chronic exposure of MDA-MB-231 cells to C8-ceramide for extended periods enhanced MDR1 mRNA levels 45- and 390-fold at passages 12 and 22, respectively, and also elicited expression of P-gp. High-passage C8-ceramide-grown MDA-MB-231 (MDA-MB-231/C8cer) cells were more resistant to doxorubicin and paclitaxel. Incubation with [1- 14C]C6-ceramide showed that cells converted short-chain ceramide into GC, lactosylceramide, and sphingomyelin. When challenged with 5 μg/ml [1- 14C]C6-ceramide, MDA-MB-231, MDA-MB-435, MCF-7, and T47D cells took up 31, 17, 21, and 13%, respectively, and converted 82, 58, 62, and 58% of that to short-chain GC. Exposing cells to the GCS inhibitor, ethylenedioxy-P4, a substituted analog of 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol, prevented ceramide's enhancement of MDR1 expression. These experiments show that high levels of ceramide and GC enhance expression of the multidrug resistance phenotype in cancer cells. Therefore, ceramide's role as a messenger of cytotoxic response might be linked to the multidrug resistance pathway.

551 POSTER Clinical significance of MDR1/ABCB1 single nucleotide polymorphism (SNP) in the breast cancer patients receiving neoadjuvant chemotherapy

The breast cancer resistance protein, BCRP/ABCG2, is a half-molecule ATP-binding cassette transporter that facilitates the efflux of various anticancer agents from the cell, including 7-ethyl-10-hydroxycamptothecin, topotecan and mitoxantrone. The expression of BCRP can thus confer a multidrug resistance phenotype in cancer cells, and its transporter activity is involved in the in vivo efficacy of chemotherapeutic agents. Thus, the elucidation of the substrate preferences and structural relationships of BCRP is essential to understanding its in vivo functions during chemotherapeutic treatments. Single nucleotide polymorphisms (SNPs) have also been found to be key factors in determining the efficacy of chemotherapeutics, and those therapeutics that inhibit BCRP activity, such as the SNP that results in a C421A mutant, may result in unexpected side effects of the BCRP- anticancer drugs interaction even at normal dosages. In order to modulate the BCRP activity during chemotherapy, various compounds have been tested as inhibitors of this protein. Estrogenic compounds including estrone, several tamoxifen derivatives in addition to phytoestrogens and flavonoids have been shown to reverse BCRP-mediated drug resistance. Intriguingly, recently developed molecular targeted cancer drugs, such as the tyrosine kinase inhibitors imatinib mesylate, gefitinib and others, can also interact with BCRP. Since both functional SNPs and inhibitory agents of BCRP modulate the in vivo pharmacokinetics and pharmacodynamics of its substrate drugs, BCRP activity is an important consideration in the development of molecular targeted chemotherapeutics.

Inhibition of the multidrug resistance P-glycoprotein activity by green tea polyphenols

Many beneficial proprieties have been associated with polyphenols from green tea, such as chemopreventive, anticarcinogenic, antiatherogenic and antioxidant actions. In this study, we investigated the effects of green tea polyphenols (GTPs) and their principal catechins on the function of P-glycoprotein (P-gp), which is involved in the multidrug resistance phenotype of cancer cells. GTPs (30 μg/ml) inhibit the photolabeling of P-gp by 75% and increase the accumulation of rhodamine-123 (R-123) 3-fold in the multidrug-resistant cell line CHRC5, indicating that GTPs interact with P-gp and inhibit its transport activity. Moreover, the modulation of P-gp transport by GTPs was a reversible process. Among the catechins present in GTPs, EGCG, ECG and CG are responsible for inhibiting P-gp. In addition, EGCG potentiates the cytotoxicity of vinblastine (VBL) in CHRC5 cells. The inhibitory effect of EGCG on P-gp was also observed in human Caco-2 cells, which form an intestinal epithelial-like monolayer. Our results indicate that, in addition to their anti-cancer properties, GTPs and more particularly EGCG inhibit the binding and efflux of drugs by P-gp. Thus, GTPs or EGCG might be potential agents for modulating the bioavailability of P-gp substrates at the intestine and the multidrug resistance phenotype associated with expression of this transporter in cancer cells.

The (patho)physiological functions of the MRP family.

The identification of certain members of the large superfamily of ATP binding cassette transport proteins such as MDR1 -P-glycoprotein and the multidrug resistance protein MRP1 as ATP-dependent drug efflux pumps has been a major contribution in our understanding of the multidrug resistance phenotype of cancer cells. Importantly, both transport proteins that exhibit only low structural homology have a very different substrate specificity but confer resistance to a similar spectrum of natural product chemotherapeutic drugs. In contrast to the drug transporter MDR1, MRP1 mainly transports anionic Phase II-conjugates. In addition MRP1-mediated drug resistance is highly dependent on high intracellular glutathione levels which may be linked to the apparent physiological involvement of MRP1 in glutathione-related cellular processes. This review summarizes the current knowledge about functional aspects of MRP1 and its five recently cloned homologues MRP2-MRP6 and discusses their substrate specificities and cellular localization with emphasis on drug resistance. Copyright 2000 Harcourt Publishers Ltd.

Transport of glutathione-conjugates in human erythrocytes.

The last step of detoxification of both endogenous and environmental toxicants is typically a conjugation that produces a bulky hydrophilic molecule. The excretion of such conjugates out of cells is of sufficient biological importance to have led to the evolution of ATP-driven export pumps for this purpose. The substrate specificity of such transporters is broad, and in some cases it has been shown to include not only anionic conjugates but also neutral or weakly cationic drugs. In the present article, we review the molecular identity, functional and structural characteristics of these pumps, mainly on the example of human erythrocytes, and discuss their physiological role in detoxification and in the multidrug resistance phenotype of cancer cells.

P-glycoprotein—A mediator of multidrug resistance in tumour cells

toxic drugs (multidrug resistance, MDR) is a major limitation to the successful chemotherapeutic treatment of cancer. The genetic and biochemical alterations responsible for the multidrug resistance phenotype of cancer cells have been the subject of intense investigation for more than 25 years and dramatic progress has been made in the understanding of multidrug resistance-associated genes, proteins and their mechanisms of action. Because of the inherent difficulty of studying drug resistance in Z&J (e.g. due to heterogeneity of human cancer, or difficulty in obtaining several clinical specimens from the same tumour during the course of chemotherapeutic