Development Of Resistance In Cancer Cells Research Articles

Noncoding RNAs as novel immunotherapeutic tools against cancer.

Immunotherapy is implemented as an important treatment strategy in various malignancies. In cancer, immunotherapy is employed for successful killing of tumor cells with high specificity and greater efficacy, with minimum side effects. Despite various available strategies, cellular immunotherapy including innate (NK cells, macrophages, dendritic cells) and adaptive (B cells and T cells) immune cells plays a critical role in tumor microenvironment. Since past few years, many drugs targeting immune checkpoint proteins including CTLA-4 and PD-1/PD-L1 have been investigated as immunotherapy approach against cancer but complete effectiveness still remains a question, as diverse mechanisms involved in tumorigenesis may result in the development of cancer cell resistance. Number of evidences have highlighted the significant role of non-coding RNAs (ncRNAs) in regulating multiple stages of cancer initiation, progression & immunity. ncRNAs comprises 98% human transcriptome and are basically considered as dark genome. Among ncRNAs, miRNAs and lncRNAs have been extensively studied in regulating diverse processes of cancer tumorigenesis. Upregulation of oncogenic and downregulation of tumor suppressive miRNAs/lncRNAs has been reported to facilitate the cancer progression and invasiveness. This chapter summarizes how an interplay between ncRNAs and immune cells in cancer pathogenesis can be therapeutically targeted to improve current treatment regimen. Strategies should be employed to improve the efficacy and reduce off-target effects of ncRNA based immunotherapy. Henceforth, combination of ncRNAs and available immunotherapy can be argued to enhance the efficacy of existing immunotherapeutic approaches against cancer to improve patient's survival.

Sensitivity of Cells with Various Levels of Ppm1d Expression to Classical Chemotherapeutic Drugs for Colorectal Cancer Treatment

Mutations and amplifications of the Ppm1d gene encoding Wip1 phosphatase have been found in various tumors. Recent studies have shown that the presence of Wip1 stable mutant after chemotherapy suggests that Wip1 is involved in the resistance of malignant cells to chemotherapeutic drugs. In the present work, we investigated the role of Wip1 in the response of colon cancer cells to the antitumor drugs 5-fluorouracil and oxaliplatin. Cell lines with an increased level of Ppm1d expression were obtained with acquired using lentiviral transduction. It has been shown that Wip1 overexpression maintains the cell viability of cells exposed to oxaliplatinum, while deletion of the Ppm1d gene decreases the viability and clonogenicity both under the combined and single exposure to these drugs. These findings suggest that an increased Wip1 level in cancer cells during chemotherapy may contribute to the development of cancer cell resistance to antitumor therapy. Methods aimed at reducing the Wip1 level will increase the efficiency of colon cancer treatment.

Overexpression of CASC11 in ovarian squamous cell carcinoma mediates the development of cancer cell resistance to chemotherapy

LncRNA CASC11 promotes gastric cancer and colon cancer. Our study analyzed the role of CASC11 in ovarian squamous cell carcinoma (OSCC). In the present study we showed that plasma CASC11 was upregulated in OSCC, and the upregulation of CASC11 distinguished OSCC patients from control group. Plasma levels of CASC11 were further increased after chemotherapy. Treatment with oxaliplatin, tetraplatin, cisplatin, and carboplatin mediated the upregulation of CASC11 in cells of OSCC cell line. In addition, overexpression of CASC11 led to increased cancer cell viability under oxaliplatin, tetraplatin, cisplatin, and carboplatin treatment, while CASC11 siRNA silencing played an opposite role. Therefore, overexpression of CASC11 in OSCC mediated the development of cancer cell resistance to chemotherapy.

Pharmacophore-Based Virtual Screening of Novel GSTP1-1 Inhibitors

Glutathione transferase enzymes play an important role in metabolism and detoxification of numerous xenobiotics, electrophilic drugs, environmental carcinogens, and products of oxidative stress in living organisms. Human GST P1-1 is the most prevalent isoform of the mammalian cytosolic GSTs and this enzyme participates in a particular role in one of the mechanisms of the development of resistance in cancer cells toward the administration of anticancer agents in chemotherapy. Herein, pharmacophore analysis were performed using bioactive conformation of the known inhibitor of GSTP1-1, ethacrynic acid (pdb ID:2GSS). Phase module of the Schrödinger suite was used to generate pharmacophore hypothesis. Molecules with same pharmacophoric features were screened from among the commercially available compounds in the ZINC database and ligand filtration was also done to obtain an efficient collection of hit molecules by employing Lipinski “rule of five” using Qikprop module. As a result, some of the compounds obtained from this study, could be the promising inhibitors of hGSTP1-1 enzyme.

P-304 - Glutathione in redox regulation of the development of cancer cell resistance

The ratio of reduced glutathione to its oxidized form (GSH/GSSG) is essential for cell viability including its role in redox-dependent regulation of gene expression. The aim of our study was to estimate GSH/GSSG ratio and expression of redox-dependent genes (glutaredoxin, thioredoxin, peroxiredoxin) under development of cancer cell resistance to cisplatin (CDDP) possessed pro-oxidant action. Under development of resistance of human erythroleukemia K562 and ovarian carcinoma SKOV-3 cells to CDDP co-ordinative enhanced expression of genes encoding glutathione synthetase (GS), subunits of γ-glutamylcysteine synthetase (γ-GCSH, γ-GCSL) was found. In addition, the growth of GSH/GSSG, enhanced expression of glutaredoxin (GLRX1, GLRX2), thioredoxin (TRX2), peroxiredoxin (PRDX6) genes as well as elevated level of transcription factor Nrf2 were observed in both types of resistant cells. We suggest that mechanism of development of cancer cell resistance to CDDP can include activation of GSH synthesis de novo, redox-dependent increase in expression of GLRX1, GLRX2, TRX2, PRDX6 genes which are co-ordinative regulated by redox-dependent transcription factor Nrf2 and elevated level of GSH/GSSG. The publication was prepared with the support of the “RUDN University Program 5–100”.

P-16 - Role of peroxiredoxins in the development of drug resistance in cancer cells

Peroxiredoxins (Prxs) regulate cell signaling and proliferation, protect protein structure and inhibit redox-dependent pathways of apoptosis activation, which attests to their important role in cell protection from oxidative stress. Here we studied the role of Prxs in the formation of cancer cell resistance to cisplatin (cis-diamine dichloroplatinum, CDDP); its cytotoxic effect, apart from alkylating mechanism, is associated with activation of ROS generation. It was found that drug resistance formation was accompanied by a significant increase in the expression of PRDX1, PRDX3, PRDX6 genes in human mammary adenocarcinoma (MCF -7/CDDP) and ovarian carcinoma (SKOV-3/CDDP) cells. A more pronounced rise was noted for Prx6 mRNA. It is known that oxidized forms of Prx1 and Prx3 are reduced by thioredoxin1 (Trx1) while glutathione repairs the oxidized Prx6. Our study found that siRNA knockdown of Trx1 increased CDDP-induced death of resistant cells which was significantly rose under additively using of siRNA knockdown of Prx6. It can be concluded that the data obtained confirm the important contribution of Prxs into redox-dependent mechanisms in the development of cancer cell resistance to CDDP. The publication was prepared with the support of the “RUDN University Program 5–100”.

Evaluation of Potential Mechanisms Controlling the Catalase Expression in Breast Cancer Cells.

Development of cancer cell resistance against prooxidant drugs limits its potential clinical use. MCF-7 breast cancer cells chronically exposed to ascorbate/menadione became resistant (Resox cells) by increasing mainly catalase activity. Since catalase appears as an anticancer target, the elucidation of mechanisms regulating its expression is an important issue. In MCF-7 and Resox cells, karyotype analysis showed that chromosome 11 is not altered compared to healthy mammary epithelial cells. The genomic gain of catalase locus observed in MCF-7 and Resox cells cannot explain the differential catalase expression. Since ROS cause DNA lesions, the activation of DNA damage signaling pathways may influence catalase expression. However, none of the related proteins (i.e., p53, ChK) was activated in Resox cells compared to MCF-7. The c-abl kinase may lead to catalase protein degradation via posttranslational modifications, but neither ubiquitination nor phosphorylation of catalase was detected after catalase immunoprecipitation. Catalase mRNA levels did not decrease after actinomycin D treatment in both cell lines. DNMT inhibitor (5-aza-2′-deoxycytidine) increased catalase protein level in MCF-7 and its resistance to prooxidant drugs. In line with our previous report, chromatin remodeling appears as the main regulator of catalase expression in breast cancer after chronic exposure to an oxidative stress.

Increased vimentin mRNA expression in MCF-7 breast cancer cell line after repeated endoxifen-treatment

Background: Epithelial mesenchymal transition (EMT) plays a significant role in the development of cancer cell resistance to drugs. Vimentin, a type III intermediate filament protein, is a marker of EMT. Vimentin's over-expression in cancer correlates well with increased tumor growth, change in cell shape and poor prognosis. Endoxifen is an active metabolite of tamoxifen and has become a new potent agent in the treatment of breast cancer. This is a study that aimed to investigate the effect of endoxifen exposure with or without estradiol on cell viability, cell morphology and EMT progression through the analysis of vimentin mRNA expression after 4-week treatment.
 Methods: Endoxifen, 100 nM or 1,000 nM, with or without beta-estradiol were given repeatedly to MCF-7 cells. Cells treated with dimethyl sulfoxide (DMSO) 0.001% were used as control. After 2- and 4-week exposure, the cells were counted, analyzed for mRNA vimentin expression, and observed for morphological changes.
 Results: Compared to control, there were significant decreases in vimentin mRNA expressions in endoxifen and endoxifen+β-estradiol treated cells after 2-weeks, which then significantly increased after 4-week compared with the 2-week exposure. We found no change in morphology of MCF-7 cells.
 Conclusion: Repeated exposure of endoxifen might induce EMT progression through increased expression of vimentin in MCF-7 breast cancer cell line.

Exosomes and development of cancer cell resistance to metformin: pilot study

Objective : to study the role of the intercellular interactions in the progression of the cancer cells resistance to metformin, a biguanide antidiabetic drug exhibited the marked anti-tumor activity. Results . Earlier we have demonstrated the effect of horizontal transferring of hormonal resistance of breast cancer cells from cell to cell, and showed the key role of exosomes on the transferring of the resistance. Here we have shown the effect of the horizontal transferring of metformin resistance in breast cancer cells – similar to the progression of hormonal resistance. We found that horizontal transferring of the metformin resistance is mediated via exosomes secreted by the resistant cells. The proteome analysis of the exosomes revealed several proteins differentially expressed in the exosomes of metformin-resistant cells and associated with the regulation of cell response to apoptotic drugs. Conclusions. Totally, the data presented demonstrate the new mechanism of the development of the cancer cell resistance based on the intercellular interactions, opening the new insights in the target therapy of breast cancer.

Expression of mitochondrial antioxidant enzymes, Bcl-2, Bcl-xl and ROS generation under development of cancer cell resistance

Expression of mitochondrial antioxidant enzymes, Bcl-2, Bcl-xl and ROS generation under development of cancer cell resistance

Abstract 3592: Macrophages confer resistance to cisplatin in MTLn3 and MDA-MB 231 breast cancer cells

Abstract Drug resistance presents a clinical challenge to the treatment of breast cancer. Stromal cells such as macrophages may play a role in drug resistance. The purpose of this study is to examine the interaction between breast cancer cells and macrophages and its effects on drug therapy resistance. The five-year survival for localized hormone sensitive disease is 98% and as low as 15% for triple negative (ER/PR/Her2) and metastatic disease. Most targeted anticancer therapies, as well as cytotoxic therapies, are burdened by the development of cancer cell resistance. It is hypothesized that the tumor microenvironment may confer innate resistance to drug therapy. It is well established that chemotactic factors found in the tumor microenvironment can promote motility and invasion. We hypothesize that tumor associated macrophages can confer drug resistance to breast cancer cells via overexpression of epidermal growth factor (EGF). EGF is overexpressed in many tumor types and is clinically correlated with a poor overall prognosis. Previous studies have shown that EGF is produced by non-tumor cells in the microenvironment; in particular tumor-associated macrophages (TAM) and endothelial cells. We have previously shown that macrophages and tumor cells, via a paracrine loop, are necessary and sufficient for comigration and invasion. Breast cancer cells produce colony stimulating factor 1 (CSF-1) which positively feeds back to increase macrophage expression of EGF. Increased EGF expression in turn leads to tumor growth and invasion. We selected two triple negative breast cancer cell lines, MDA-MB 231 (human mammary adenocarcinoma) and MTLn3 (rat mammary adenocarcinoma) that were transfected to express GFP. Co-culture cell survival assays were performed with macrophages, fibroblasts, and endothelial cells. All tissue culture was completed in DMEM (HyClone) plus 10% FBS. Cells were grown for 24 hours in co-culture, treated with chemotherapy (doxorubicin or cisplatin at a range of concentrations) for 24 hours, and then analyzed at 72 hours using the DXP-10 Calibur analyzer. Prior to analysis, cells were again replated to measure “replating efficiency” i.e. chemoresistant cell survival. We found that coculture with macrophages increased tumor cell survival upon exposure to cisplatin. We conclude that macrophages are an important component of the tumor microenvironment that may play a role in chemotherapy resistance. Citation Format: Joshua K. Sabari, Ramon Cabrera, Jeffrey Segall. Macrophages confer resistance to cisplatin in MTLn3 and MDA-MB 231 breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3592. doi:10.1158/1538-7445.AM2014-3592

In vitro Modulation of P-glycoprotein, MRP-1 and BCRP Expression by Mangiferin in Doxorubicin-Treated MCF-7 Cells

The multidrug resistance phenotype is one of the major problems in development of cancer cell resistance to chemotherapy. Some natural compounds from medicinal plants have demonstrated promising capacity in enhancing anticancer effects in drug resistant cancer cells. We aimed to investigate whether mangiferin might have an ability to re-sensitize MCF-7 breast cancer cells previously treated with short-term doxorubicin in vitro, through the modulation of efflux transporters, P-glycoprotein (P-gp), MRP1 and BCRP. We exposed MCF-7 breast cancer cells pretreated with doxorubicin for 10 days to mangiferin (10, 25 or 50 μM) for 96 hours. Afterwards, we evaluated influence on cell viability and level of mRNA expression of P-gp, MRP1 and BCRP. Doxorubicin given in combination with mangiferin at low concentrations (10 and 25 μM) failed to give significant reduction in cell viability, while at the highest concentrations, the combination significantly reduced cell viability. The mRNA expression analysis of P-gp, MRP1 and BCRP showed that mangiferin had inhibitory effects on P-gp but no effects on MRP1 and BCRP. In conclusion, we suggest that mangiferin at high concentrations can be used as chemosensitizer for doxorubicin therapy. This effect might be attributed by inhibitory effects of mangiferin on P-glycoprotein expression.

Mechanisms of cell uptake and toxicity of the anticancer drug cisplatin.

Two major issues which hamper the use of the anticancer drug cisplatin are the development of cancer cell resistance and its nephrotoxicity. One possible mechanism by which resistance is reported to develop is a reduction in drug uptake across the cell membrane. While the passive uptake of cisplatin has long been cited as an important contribution, far greater attention has been given to active modes of uptake, particularly in recent research. Using unilamellar lipid vesicles together with the stopped-flow kinetic method we show here that the permeability coefficient of cisplatin increases significantly with the chloride concentration of the medium. This supports the hypothesis that cisplatin can enter cells via passive permeation through the lipid phase of the membrane, but becomes trapped within the cytoplasm because dissociation of chloride ligands yields a membrane-impermeant positively-charged aqua derivative. This is important evidence for a major role of passive membrane diffusion in the uptake of cisplatin, and suggests that reduced cell uptake is unlikely to be a significant mechanism leading to the development of drug resistance. Studies of rubidium ion uptake into the cytoplasm of Xenopus oocytes via the Na(+),K(+)-ATPase show significant inhibition of this ion pump when cisplatin is present in the cytoplasm. Because Na(+),K(+)-ATPase activity is essential to the survival of all animal cells, e.g. via maintenance of cell volume, and the Na(+),K(+)-ATPase is expressed at particularly high levels within the membranes of kidney tubules where it plays a crucial role in nutrient reabsorption, these results suggest that cisplatin-induced inhibition of the Na(+),K(+)-ATPase is a likely contributing cause for the nephrotoxicity of cisplatin.

The role of ABC transporters in progression and clinical outcome of colorectal cancer

Worldwide, colorectal cancer (CRC) is the third most common cancer, with the highest mortality rates occurring in Central Europe. The use of chemotherapy to treat CRC is limited by the inter-individual variability in drug response and the development of cancer cell resistance. ATP-binding cassette (ABC) transporters play a crucial role in the development of resistance by the efflux of anticancer agents outside of cancer cells. The aim of this study was to explore transcript levels of all human ABCs in tumours and non-neoplastic control tissues from CRC patients collected before the first line of treatment by 5-fluorouracil (5-FU)-containing regimen. The prognostic potential of ABCs was evaluated by the correlation of transcript levels with clinical factors. Relations between transcript levels of ABCs in tumours and chemotherapy efficacy were also addressed. The transcript profile of all known human ABCs was assessed using real-time polymerase chain reaction with a relative standard curve. The majority of the studied ABCs were down-regulated or unchanged between tumours and control tissues. ABCA12, ABCA13, ABCB6, ABCC1, ABCC2 and ABCE1 were up-regulated in tumours versus control tissues. Transcript levels of ABCA12, ABCC7 and ABCC8 increased in direction from colon to rectum. Additionally, transcript levels of ABCB9, ABCB11, ABCG5 and ABCG8 followed the reverse significant trend, i.e. a decrease in direction from colon to rectum. The transcript level of ABCC10 in tumours correlated with the grade (P = 0.01). Transcript levels of ABCC6, ABCC11, ABCF1 and ABCF2 were significantly lower in non-responders to palliative chemotherapy in comparison with responders. The disease-free interval of patients treated by adjuvant chemotherapy was significantly shorter in patients with low transcript levels of ABCA7, ABCA13, ABCB4, ABCC11 and ABCD4. In conclusion, ABCC11 may be a promising candidate marker for a validation study on 5-FU therapy outcome.

Resistance to paclitaxel increases the sensitivity to other microenvironmental stresses in prostate cancer cells

The microenvironment is central to many aspects of cancer pathobiology and has been proposed to play a role in the development of cancer cell resistance to therapy. To examine the response to microenvironmental conditions, two paclitaxel resistant prostate cancer (PCa) cell lines (stable and reversible) and one reversible heat resistant cell line were studied. In comparison to their parental cell lines, both paclitaxel resistant cell lines (stable and reversible) were more sensitive to microenvironmental heat, potentially yielding a synergistic therapeutic opportunity. In the two phenotypic cells repopulated after acute heat or paclitaxel treatments, there was an inverse correlation between paclitaxel and heat resistance: resistance to paclitaxel imparted sensitivity to heat; resistance to heat imparted sensitivity to paclitaxel. These studies indicate that as cancer cells evolve resistance to single microenvironmental stress they may be more sensitive to others, perhaps allowing us to design new approaches for PCa therapy.

Inhibition of lung tumor growth by complex pulmonary delivery of drugs with oligonucleotides as suppressors of cellular resistance

Development of cancer cell resistance, low accumulation of therapeutic drug in the lungs, and severe adverse treatment side effects represent main obstacles to efficient chemotherapy of lung cancer. To overcome these difficulties, we propose inhalation local delivery of anticancer drugs in combination with suppressors of pump and nonpump cellular resistance. To test this approach, nanoscale-based delivery systems containing doxorubicin as a cell death inducer, antisense oligonucleotides targeted to MRP1 mRNA as a suppressor of pump resistance and to BCL2 mRNA as a suppressor of nonpump resistance, were developed and examined on an orthotopic murine model of human lung carcinoma. The experimental results show high antitumor activity and low adverse side effects of proposed complex inhalatory treatment that cannot be achieved by individual components applied separately. The present work potentially contributes to the treatment of lung cancer by describing a unique combinatorial local inhalation delivery of drugs and suppressors of pump and nonpump cellular resistance.

Role of apoptosis in the immune system.

Apoptosis is becoming recognized as a fundamental component of the immune system, i.e., in its development and its regulation. It is involved in many diverse areas that fall into two broad categories: firstly, the development and shaping of the immune receptor repertoire and, secondly, immune effector mechanisms. We have employed several model systems for analysing the development of the immune system, e.g., haemopoietic progenitor cell development and thymocyte development and selection, as well as the role of apoptosis in CD4+ T-cell-mediated cytotoxicity. These studies have helped illustrate the fundamental role of active cell death in the physiological functioning of the immune system. Failure of such a fundamental process would be expected to have serious consequences and we have been particularly involved in analysing the role of inappropriate suppression of apoptosis by the BCL2 family of genes both in oncogenesis in the immune system and in the development of cancer cell resistance to therapy. It seems likely that this is only one of many mechanisms by which apoptosis may be disrupted with pathological consequences.

Nitric oxide donors: Novel cancer therapeutics (Review)

The development of cancer cell resistance to various cytotoxic stimuli continues to be a major challenge in oncology and novel therapeutic approaches are urgently needed. Nitric oxide (NO) is emerging as a potential anti-oncogenic agent to overcome tumor cell resistance to conventional therapeutic agents. NO is a ubiquitous, water-soluble, free radical gas that exerts a wide range of biological effects. The actions of nitric oxide are highly variable in oncology with reports in the literature on both sides of the spectrum as an anti-neoplastic vs. a pro-neoplastic agent. The final activity of NO in oncology is dependent on its working microenvironment, including the type of cell exposed to the compound, the redox state of the reaction, as well as the final intracellular concentration and the duration of intracellular exposure to nitric oxide. There is, however, no unifying mechanistic explanation for the biphasic role of nitric oxide in oncology. Nitric oxide donors mimic continuous production of NO in a wide range of time intervals (seconds to days). Thus, multiple biological and (pro- vs. anti-) neoplastic responses are elicited from NO donors depending on the half-life and the type of cell exposed to the compound. The large variety of nitric oxide donors may serve as a tool to explore the wide range of oncologic properties of NO in cancer. In the present report, we discuss classic nitric oxide donors and their potential therapeutic roles as cytotoxic agents or chemo-radio or -immune-sensitizing compounds in the treatment of drug-resistant cancers.