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

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

骨髓增生异常综合征的表观遗传改变

表观遗传改变是指不涉及DNA序列变化但影响细胞基因表达的一些可遗传的改变,主要包括DNA启动子的甲基化和组蛋白的各种共价修饰。表观遗传改变与肿瘤的发生密切相关,在多种表观遗传相关分子共同参与下,组蛋白氨基酸残基去乙酰化或甲基化,抑癌基因启动子甲基化,使各种转录因子无法与DNA结合而导致抑癌基因失活。目前已知,骨髓增生异常综合征(MDS)存在多种抑癌基因的表观遗传改变,也存在着表观遗传调节蛋白PcG的表达异常,这些都与MDS的进展和预后有着密切的关系,提示了它们可作为新的疾病标志物应用于临床。本文就MDS的存在的表观遗传改变分子机制、抑癌基因失活和PcG蛋白异常作一综述。     

Tamoxifen guided liposomes for targeting encapsulated anticancer agent to estrogen receptor positive breast cancer cells: In vitro and in vivo evaluation

Tamoxifen (TMX), an estrogen receptor (ER) antagonist, incorporated at surface of liposomes loaded with Doxorubicin (DOX), was hypothesized to serve as ligand for targeting overexpressed ERs on surface and cytosol of breast cancer cells, in addition to its synergism with DOX in killing MCF-7 cells. The TMX-DOX liposomes demonstrated mean size of 188.8 ± 2.2 nm and positive potential of +47 mV, both suitable for better cellular interaction. TMX-DOX liposomes sustained DOX release in vitro (25.9%) in pH 7.4 at 48 h, in comparison with 64.5% DOX release at pH 5.5. In vitro cell line studies demonstrated that TMX-DOX liposomes were more cytotoxic to ER +ve MCF-7 cells as compared to DOX liposomes, DOX solution and TMX-DOX solution (P < 0.05). However, there was no statistical difference in cyto-toxicity of TMX-DOX liposomes and DOX liposomes towards ER –ve MDA-MB-231 cells. Flow cytometry and confocal studies in MCF-7 cells revealed greater cell and nuclear uptake of DOX, with TMX guided liposomes as compared to DOX liposomes and DOX solution. TMX-DOX liposomes demonstrated significantly increased inhibition of MCF-7 cell based tumor growth in nude mice (P < 0.05) in comparison to DOX solution and DOX liposomes, indicative of target specificity and higher DOX accumulation at tumor site.     

Amelioration of doxorubicin-induced cardiotoxicity by an anticancer-antioxidant dual-function compound, HO-3867

Doxorubicin (DOX) is a drug commonly used for the treatment of cancer. The development of resistance to DOX is common, and high cumulative doses cause potentially lethal cardiac side effects. HO-3867 (3,5-bis(4-fluorobenzylidene)-1-[(2,2,5,5-tetramethyl-2,5-dihydro-1-hydroxy-pyrrol-3-yl)methyl]piperidin-4-one), a synthetic curcumin analog, has been shown to exhibit both anticancer and cardioprotective effects. However, its cardioprotection in the setting of a conventional cancer therapy has not been established. This work investigated the use of HO-3867 and DOX to achieve a complementary outcome, i.e., increased toxicity toward cancer cells, and reduced cardiac toxicity. Combination treatment was investigated using DOX-resistant MCF-7 breast cancer cells [MCF-7 multidrug-resistant (MDR)] and BALB/c mice. Lower doses of HO-3867 and DOX (5 and 2.5 μM, respectively) reduced viability of MCF-7 MDR cells to an extent significantly greater than that when either drug was used alone, an effect equivalent to that induced by exposure to 50 μM DOX. In normal cardiac cells, the loss of viability from combination treatment was significantly lower than that induced by 50 μM DOX. Increases in apoptotic markers, e.g., cleaved caspase-3, and decreases in fatty acid synthase and pAkt expressions were observed by Western blotting. Mice treated with both HO-3867 and DOX showed significant improvement in cardiac functional parameters compared with mice treated with DOX alone. Reduced expression of Bcl-2 and pAkt was observed in mice treated with DOX alone, whereas mice given combination treatment showed levels similar to control. The study indicates that combination treatment of HO-3867 and DOX is a viable option for treatment of cancer with reduced cardiotoxic side effects.     

MCF-7 breast cancer cells transfected with protein kinase C-alpha exhibit altered expression of other protein kinase C isoforms and display a more aggressive neoplastic phenotype.

Increased protein kinase C (PKC) activity in malignant breast tissue and positive correlations between PKC activity and expression of a more aggressive phenotype in breast cancer cell lines suggest a role for this signal transduction pathway in the pathogenesis and/or progression of breast cancer. To examine the role of PKC in the progression of breast cancer, human MCF-7 breast cancer cells were transfected with PKC-alpha, and a group of heterogenous cells stably overexpressing PKC-alpha were isolated (MCF-7-PKC-alpha). MCF-7-PKC-alpha cells expressed fivefold higher levels of PKC-alpha as compared to parental or vector-transfected MCF-7 cells. MCF-7-PKC-alpha cells also displayed a substantial increase in endogenous expression of PKC-beta and decreases in expression of the novel delta- and eta-PKC isoforms. MCF-7-PKC-alpha cells displayed an enhanced proliferative rate, anchorage-independent growth, dramatic morphologic alterations including loss of an epithelioid appearance, and increased tumorigenicity in nude mice. MCF-7-PKC-alpha cells exhibited a significant reduction in estrogen receptor expression and decreases in estrogen-dependent gene expression. These findings suggest that the PKC pathway may modulate progression of breast cancer to a more aggressive neoplastic process.     

Epigenetic biomarkers in colorectal cancer diagnostics

Colorectal cancer (CRC) is a significant health burden worldwide. Despite advancements in treatment options, improvements in CRC patient survival have been limited owing to lack of early detection and limited capacity for optimal therapeutic decision-making. Biomarkers to improve CRC diagnosis, prognosis and prediction of treatment response therefore represent opportunities to improve patient outcome. In addition to genetic alterations and genomic instability, it is now clear that epigenetic alterations play dramatic roles in driving tumor onset and progression in CRC. A recent surge in investigation of epigenetic biomarkers including DNA methylation, miRNA expression and histone modifications has demonstrated that these alterations may be enticing translational biomarker candidates in CRC. In particular, methylation kits have already been incorporated into clinical practice for a handful of cancers, including CRC. This review will aim to summarize the established and emerging roles of epigenetic modifications in CRC detection, prognostication and prediction of treatment response.     

Modulation of P-glycoprotein function and reversal of multidrug resistance by (-)-epigallocatechin gallate in human cancer cells.

Multidrug resistance (MDR) is a major obstacle in the chemotherapeutic treatment of many human cancers. In this study, the reversal of P-glycoprotein (P-gp) mediated multidrug resistance by (-)-epigallocatechin gallate (EGCG) and its molecular mechanism were investigated. A three-dimensional model of carboxyl-terminal nucleotide binding domain (NBD2) from P-gp was built by homology modeling. The structural model of the complex indicates that EGCG was tightly bound to the ATP-binding site of NBD2. EGCG modulated the function of P-gp and increased the intracellular accumulation of chemotherapeutic agent doxorubicin (DOX) in drug-resistant KB-A1 cells. When KB-A1 cells were exposed to 10 microg/ml DOX combined with 10, 30, 50 microM EGCG for 4 h, the intracellular concentrations of DOX were increased 1.5, 1.9, 2.3 times, respectively compared with DOX alone treatment. In vitro EGCG potentiated the cytotoxicity of DOX to drug-resistant KB-A1 cells. In KB-A1 cell xenograft model, EGCG could also enhance the efficacy of DOX and increased the DOX concentration in the resistant tumors. Thus, these results suggest that EGCG modulated the function of P-gp and reversed P-gp mediated multidrug resistance in human cancer cells.     

Epigenomic reactivation screening to identify genes silenced by DNA hypermethylation in human cancer

Promoter DNA hypermethylation is the key epigenetic mechanism by which tumor suppressor genes are inactivated in human cancer. The discovery of the importance of this mechanism has spurred the development of a number of approaches to identify genes silenced by DNA hypermethylation in cancer. One of the most useful of these approaches is an epigenomic reactivation screening strategy that combines treatment of cancer cells in vitro with DNA methyltransferase and/or histone deacetylase inhibitors, followed by global gene expression analysis using microarrays, to identify upregulated genes. This approach is most effective when complemented by microarray analyses to identify genes repressed in primary tumors. Epigenomic reactivation screening has a number of key advantages over direct analysis of cancer-associated DNA methylation changes; most notably, it directly identifies genes in which epigenetic changes lead to altered gene expression. An increasing number of studies have utilized epigenomic reactivation screening to discover novel tumor suppressor genes in cancer. The results of some of the most recent studies are highlighted in this review.     

Epigenetics in pain and analgesia: An imminent research field

Heritable phenotypes resulting from environment‐caused changes in a chromosome without alterations in the DNA sequence are increasingly recognized as a basis of personalized therapy. Epigenetic mechanisms include covalent modifications of the DNA (methylation) or of the DNA‐packaging histones (e.g., deacetylation or phosphorylation). In addition, regulatory non‐coding RNA molecules (micro‐RNAs) exert epigenetic actions. This leads to disruption or otherwise modified expression of genes. Environmental influences such as nutritional factors, exposure to chemicals or drugs, but also social factors appear to exert epigenetic actions. Histone modifications and DNA methylation are associated with the subject's age. Epigenetic mechanisms can silence the expression of pro‐ or antinociceptive genes. To the epigenetic control of nociception adds its control of the pharmacodynamics or pharmacokinetics of analgesics by epigenetic control of drug targets and analgesics metabolizing enzymes. Although epigenetics‐based strategies for pain therapy are not yet available, experiments in rodents suggest that RNA interference may become a new therapy approach for neuropathic and other pain. Another epigenetic approach to analgesic treatment employs inhibitors of histone deacetylase that act on the epigenome by indirectly remodeling the spatial conformation of the chromatin. Finally, epigenetic techniques such as RNA interference have been employed in pain research to proof the contribution of certain proteins to nociception. Thus, the new field of epigenetics becomes increasingly used in research and management of pain and will complement genetics. This article introduces epigenetics to pain and summarizes the current and future utility.     

Doxorubicin loaded iron oxide nanoparticles overcome multidrug resistance in cancer in vitro

Multidrug resistance (MDR) is characterized by the overexpression of ATP-binding cassette (ABC) transporters that actively pump a broad class of hydrophobic chemotherapeutic drugs out of cancer cells. MDR is a major mechanism of treatment resistance in a variety of human tumors, and clinically applicable strategies to circumvent MDR remain to be characterized. Here we describe the fabrication and characterization of a drug-loaded iron oxide nanoparticle designed to circumvent MDR. Doxorubicin (DOX), an anthracycline antibiotic commonly used in cancer chemotherapy and substrate for ABC-mediated drug efflux, was covalently bound to polyethylenimine via a pH sensitive hydrazone linkage and conjugated to an iron oxide nanoparticle coated with amine terminated polyethylene glycol. Drug loading, physiochemical properties and pH lability of the DOX-hydrazone linkage were evaluated in vitro. Nanoparticle uptake, retention, and dose-dependent effects on viability were compared in wild-type and DOX-resistant ABC transporter over-expressing rat glioma C6 cells. We found that DOX release from nanoparticles was greatest at acidic pH, indicative of cleavage of the hydrazone linkage. DOX-conjugated nanoparticles were readily taken up by wild-type and drug-resistant cells. In contrast to free drug, DOX-conjugated nanoparticles persisted in drug-resistant cells, indicating that they were not subject to drug efflux. Greater retention of DOX-conjugated nanoparticles was accompanied by reduction of viability relative to cells treated with free drug. Our results suggest that DOX-conjugated nanoparticles could improve the efficacy of chemotherapy by circumventing MDR.     

Breast cancer cells with acquired antiestrogen resistance are sensitized to cisplatin-induced cell death

Antiestrogens are currently used for treating breast cancer patients who have estrogen receptor-positive tumors. However, patients with advanced disease will eventually develop resistance to the drugs. Therefore, compounds effective on antiestrogen-resistant tumors will be of great importance for future breast cancer treatment. In this study, we have investigated the effect of the chemotherapeutic compound cisplatin using a panel of antiestrogen-resistant breast cancer cell lines established from the human breast cancer cell line MCF-7. We show that the antiestrogen-resistant cells are significantly more sensitive to cisplatin-induced cell death than antiestrogen-sensitive MCF-7 cells and we show that cisplatin induces cell death by activating both the caspase and lysosomal death pathways. The antiestrogen-resistant cell lines express lower levels of antiapoptotic Bcl-2 protein compared with parental MCF-7 cells. Our data show that Bcl-2 can protect antiestrogen-resistant breast cancer cells from cisplatin-induced cell death, indicating that the reduced expression of Bcl-2 in the antiestrogen-resistant cells plays a role in sensitizing the cells to cisplatin treatment.     

Overexpression of glucosylceramide synthase and P-glycoprotein in cancer cells selected for resistance to natural product chemotherapy

Resistance to natural product chemotherapy drugs is a major obstacle to successful cancer treatment. This type of resistance is often acquired in response to drug exposure; however, the mechanisms of this adverse reaction are complex and elusive. Here, we have studied acquired resistance to Adriamycin, Vinca alkaloids, and etoposide in MCF-7 breast cancer cells, KB-3-1 epidermoid carcinoma cells, and other cancer cell lines to determine if there is an association between expression of glucosylceramide synthase, the enzyme catalyzing ceramide glycosylation to glucosylceramide, and the multidrug-resistant (MDR) phenotype. This work shows that glucosylceramide levels increase concomitantly with increased drug resistance in the KB-3-1 vinblastine-resistant sublines KB-V.01, KB-V.1, and KB-V1 (listed in order of increasing MDR). The levels of glucosylceramide synthase mRNA, glucosylceramide synthase protein, and P-glycoprotein (P-gp) also increased in parallel. Increased glucosylceramide levels were also present in Adriamycin-resistant KB-3-1 sublines KB-A.05 and KB-A1. In breast cancer, detailed analysis of MCF-7 wild-type and MCF-7-AdrR cells (Adriamycin-resistant) demonstrated enhanced glucosylceramide synthase message and protein, P-gp message and protein, and high levels of glucosylceramide in resistant cells. Similar results were seen in vincristine-resistant leukemia, etoposide-resistant melanoma, and Adriamycin-resistant colon cancer cell lines. Cell-free glucosylceramide synthase activity was higher in lysates obtained from drug-resistant cells. Lastly, glucosylceramide synthase promoter activity was 15-fold higher in MCF-7-AdrR compared with MCF-7 cells. We conclude that selection pressure for resistance to natural product chemotherapy drugs selects for enhanced ceramide metabolism through glucosylceramide synthase in addition to enhanced P-gp expression. A possible connection between glucosylceramide synthase and P-gp in drug resistance biology is suggested.     

Reversal of doxorubicin-resistance by multifunctional nanoparticles in MCF-7/ADR cells

The efficacy of many chemotherapeutic agents is reduced in cells that have developed multiple drug resistance (MDR). To address this important problem, a biodegradable polymer was coupled to a photosensitizer and the resulting photosensitizer-nanoparticles were loaded with the chemotherapeutic agent doxorubicin. The combination of photosensitizer and chemotherapeutic agent had a synergistic action on a doxorubicin-resistant breast cancer MCF-7 cell line. To increase the effectiveness of this combination, d-alpha-tocopheryl poly(ethylene glycol) 1000 succinate (TPGS), an inhibitor of the multidrug transporter overproduced in these resistant cells, was added during the formation of the nanoparticles. The insertion of TPGS decreased the P-glycoprotein activity, increased the intracellular accumulation doxorubicin, and also increased the therapeutic efficacy of the resulting nanoparticles. Both TPGS and irradiation of the photoreactive nanoparticles caused doxorubicin to move from the cytoplasm to the nucleus. This combination of photodynamic activity in a powerful nanocarrier loaded with the chemotherapeutic agent doxorubicin can be used to deliver two types of cancer therapy simultaneously, and the addition of TPGS can further enhance the entry of doxorubicin into the nucleus. Therefore, this innovative delivery system can act as a potential nanomedicine for both drug-sensitive and drug-resistant cancer therapy.     

Doxycycline induces expression of P glycoprotein in MCF-7 breast carcinoma cells

P-glycoprotein (P-gp) overexpression by tumor cells imparts resistance to multiple antineoplastic chemotherapeutic agents (multiple drug resistance). Treatment of tumor cells with chemotherapeutic agents such as anthracyclines, epipodophyllotoxins, and Vinca alkaloids results in induction of P-gp expression. This study was performed to determine if clinically relevant antimicrobial drugs (i.e., drugs that are used to treat bacterial infections in cancer patients) other than antineoplastic agents can induce expression of P-gp in MCF-7 breast carcinoma cells. Expression of P-gp and MDR1 mRNA was determined in samples from MCF-7 cells that were treated in culture with doxorubicin (positive control) and the antimicrobial drugs doxycycline, piperacillin, and cefoperazone. The functional status of P-gp was assessed using laser cytometry to determine intracellular doxorubicin concentrations. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was used to determine if the cytotoxicity of experimental drugs was related to their ability to induce P-gp expression. MCF-7 cells treated with doxycycline (MCF-7/doxy) were stimulated to overexpress P-gp, whereas cells treated with piperacillin and cefoperazone did not overexpress P-gp. MCF-7/doxy cells were compared to a positive-control subline, MCF-7/Adr, previously selected for doxorubicin resistance, and to MCF-7 cells treated with doxorubicin (MCF-7/doxo). All three sublines overexpressed P-gp and MDR1 mRNA and accumulated less intracellular doxorubicin than did control MCF-7 cells. P-gp expression was induced only by experimental drugs that were cytotoxic (doxorubicin and doxycycline). Doxycycline, a drug that has been used for treatment of bacterial infections in cancer patients, can induce functional P-gp expression in cancer cells, resulting in multidrug resistance.     

Defective Acidification in Human Breast Tumor Cells and Implications for Chemotherapy

Multidrug resistance (MDR) is a significant problem in the treatment of cancer. Chemotherapeutic drugs distribute through the cyto- and nucleoplasm of drug-sensitive cells but are excluded from the nucleus in drug-resistant cells, concentrating in cytoplasmic organelles. Weak base chemotherapeutic drugs (e.g., anthracyclines and vinca alkaloids) should concentrate in acidic organelles. This report presents a quantification of the pH for identified compartments of the MCF-7 human breast tumor cell line and demonstrates that (a) the chemotherapeutic Adriamycin concentrates in acidified organelles of drug-resistant but not drug-sensitive cells; (b) the lysosomes and recycling endosomes are not acidified in drug-sensitive cells; (c) the cytosol of drug-sensitive cells is 0.4 pH units more acidic than the cytosol of resistant cells; and (d) disrupting the acidification of the organelles of resistant cells with monensin, bafilomycin A1, or concanamycin A is sufficient to change the Adriamycin distribution to that found in drug-sensitive cells, rendering the cell vulnerable once again to chemotherapy. These results suggest that acidification of organelles is causally related to drug resistance and is consistent with the hypothesis that sequestration of drugs in acidic organelles and subsequent extrusion from the cell through the secretory pathways contribute to chemotherapeutic resistance.     

A novel strategy for the diagnosis,prognosis, treatment,and chemoresistance of hepatocellular carcinoma: DNA methylation

The cancer mortality rate of hepatocellular carcinoma (HCC) is the second highest in the world and the therapeutic options are limited. The incidence of this deadly cancer is rising at an alarming rate because of the high degree of resistance to chemo- and radiotherapy, lack of proper, and adequate vaccination to hepatitis B, and lack of consciousness and knowledge about the disease itself and the lifestyle of the people. DNA methylation and DNA methylation-induced epigenetic alterations, due to their potential reversibility, open the access to develop novel biomarkers and therapeutics for HCC. The contribution to these epigenetic changes in HCC development still has not been thoroughly summarized. Thus, it is necessary to better understand the new molecular targets of HCC epigenetics in HCC diagnosis, prevention, and treatment. This review elaborates on recent key findings regarding molecular biomarkers for HCC early diagnosis, prognosis, and treatment. Currently emerging epigenetic drugs for the treatment of HCC are summarized. In addition, combining epigenetic drugs with nonepigenetic drugs for HCC treatment is also mentioned. The molecular mechanisms of DNA methylation-mediated HCC resistance are reviewed, providing some insights into the difficulty of treating liver cancer and anticancer drug development.