Efficacy In Breast Cancer Cells Research Articles

Enhanced efficacy of anti-miR-191 delivery through stearylamine liposome formulation for the treatment of breast cancer cells

MicroRNAs are gaining rapid attention as promising targets for cancer treatment; however, efficient delivery of therapeutic miRNA or anti-miRNA into cancer cells remains a major challenge. Our previous work identified miR-191 as an oncogenic miRNA overexpressed in breast cancer that assists in progression of malignant transformation. Thus, inhibition of miR-191 using antisense miR-191 (anti-miR-191) has immense therapeutic potential. Here, we have developed a stearylamine (SA) based cationic liposome for delivery of miR-191 inhibitor (anti-miR-191), and studied its efficacy in breast cancer cells (MCF-7 and ZR-75-1) in culture. SA liposomes alone inhibited cancer cell growth with lesser IC50s (50% inhibitory concentration) values as compared to normal mouse fibroblast cells (L929). The efficient delivery of anti-miR-191 in SA liposome complex was found to be highly effective in killing the cancer cells than a comparable dose of SA free anti-miR-191 liposome complex. The formulation also showed negligible cytotoxicity in human erythrocytes. Combined treatment of SA liposome with anti-miR-191 markedly enhanced apoptotic cell death and suppressed the migration of cancer cells in vitro. Notably, anti-miR-191 loaded SA liposome complex increased chemosensitivity of breast cancer cells to currently used anti-cancer drugs (doxorubicin or cisplatin) in free form. Our work demonstrates that anti-miR-191 loaded in SA liposome complex has promising clinical application for breast cancer therapy.

Targeted co-delivery of polypyrrole and rapamycin by trastuzumab-conjugated liposomes for combined chemo-photothermal therapy

Trastuzumab is a therapeutic monoclonal antibody that selectively recognizes HER2/neu receptor for targeting breast cancers. In this study, we aimed to present a strategy to combine chemo and phototherapy and targeted delivery via monoclonal antibody for enhanced anticancer effects. We co-loaded a chemotherapeutic agent, rapamycin, and a photosensitizer, polypyrrole, in trastuzumab-conjugated liposomes (LRPmAb) for combined chemo-photothermal therapy. LRPmAb had small size (172.2±9.6nm), narrow distribution, and negative ζ-potential (−12.0±0.3mV). In addition, LRPmAb showed pH- and temperature-dependent release profiles. LRPmAb showed significantly enhanced uptake in BT-474 cells, a natural HER2/neu expressing cell line. We found that these LRPmAb were effective in delivering rapamycin and showed higher therapeutic efficacy in breast cancer cells overexpressing HER2/neu receptors compared with cells that did not overexpress these receptors. Furthermore, LRPmAb showed synergistic activity against rapamycin-sensitive and resistant cell lines in vitro. These findings indicated that LRPmAb-mediated drug delivery could improve the therapeutic efficacy against breast cancer and overcome drug resistance.

Co-delivery of IL17RB siRNA and doxorubicin by chitosan-based nanoparticles for enhanced anticancer efficacy in breast cancer cells.

Overexpression of IL17RB is associated with poor prognosis and short survival of the breast cancer patients.IL17RB/IL17B signaling triggers a substantial increase in the cell growth, proliferation and migration through the activation of NF-κB as well as the up-regulation of the Bcl-2. In this study we designed carboxymethyl dextran (CMD) Chitosan nanoparticles (ChNPs) to encapsulated IL17RB siRNA and doxorubicin (DOX) as an anticancer drug. Then we investigated the efficiency of the simultaneous delivery of drug/siRNA on viability and gene expression of MDA-MB361 cell lines. Furthermore the efficacy of dual agent nanoparticles to induce apoptosis and inhibit migration of breast cancer cells was assessed by Annexin-V and wound healing assays respectively. Our results showed that DOX-siRNA-CMD-ChNPs had about 114nm size; with polydispersity index and zeta potential about 0.3 and 10.1mV respectively. Fourier transform infrared spectroscopy (FTIR) confirmed the formation of DOX-siRNA-CMD-ChNPs complex. In addition IL17RB siRNA had significant effect on DOX-induced cytotoxicity in MDA-MB361 cells. Furthermore treatment with dual agent nanoparticles resulted in a significant silencing of NF-κB and Bcl-2 relative gene expression, apoptosis induction and migration inhibition in MDA-MB361 cells. In conclusion, co-delivery of IL17RB siRNA and DOX can be considered as an effective system for the treatment of breast cancer.

Dual EZH2 and EHMT2 histone methyltransferase inhibition increases biological efficacy in breast cancer cells.

BackgroundMany cancers show aberrant silencing of gene expression and overexpression of histone methyltransferases. The histone methyltransferases (HKMT) EZH2 and EHMT2 maintain the repressive chromatin histone methylation marks H3K27me and H3K9me, respectively, which are associated with transcriptional silencing. Although selective HKMT inhibitors reduce levels of individual repressive marks, removal of H3K27me3 by specific EZH2 inhibitors, for instance, may not be sufficient for inducing the expression of genes with multiple repressive marks.ResultsWe report that gene expression and inhibition of triple negative breast cancer cell growth (MDA-MB-231) are markedly increased when targeting both EZH2 and EHMT2, either by siRNA knockdown or pharmacological inhibition, rather than either enzyme independently. Indeed, expression of certain genes is only induced upon dual inhibition. We sought to identify compounds which showed evidence of dual EZH2 and EHMT2 inhibition. Using a cell-based assay, based on the substrate competitive EHMT2 inhibitor BIX01294, we have identified proof-of-concept compounds that induce re-expression of a subset of genes consistent with dual HKMT inhibition. Chromatin immunoprecipitation verified a decrease in silencing marks and an increase in permissive marks at the promoter and transcription start site of re-expressed genes, while Western analysis showed reduction in global levels of H3K27me3 and H3K9me3. The compounds inhibit growth in a panel of breast cancer and lymphoma cell lines with low to sub-micromolar IC50s. Biochemically, the compounds are substrate competitive inhibitors against both EZH2 and EHMT1/2.ConclusionsWe have demonstrated that dual inhibition of EZH2 and EHMT2 is more effective at eliciting biological responses of gene transcription and cancer cell growth inhibition compared to inhibition of single HKMTs, and we report the first dual EZH2-EHMT1/2 substrate competitive inhibitors that are functional in cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0118-9) contains supplementary material, which is available to authorized users.

Co-delivery of doxorubicin and P-glycoprotein siRNA by multifunctional triblock copolymers for enhanced anticancer efficacy in breast cancer cells.

Combined treatment of chemotherapeutics and small interfering RNAs (siRNAs) is a promising therapy strategy for breast carcinoma via their synergetic effects. In this study, to improve the therapeutic effect of doxorubicin (DOX), novel triblock copolymers, folate/methoxy-poly(ethylene glycol)-block-poly(l-glutamate-hydrazide)-block-poly(N,N-dimethylaminopropyl methacrylamide) (FA/m-PEG-b-P(LG-Hyd)-b-PDMAPMA), were synthesized and used as a vehicle for the co-delivery of DOX and P-glycoprotein (P-gp) siRNA into breast cancer cells. The triblock copolymers were synthesized by a combination of ring-opening polymerization of γ-benzyl-l-glutamate-N-carboxyanhydride using cystamine-terminated heterotelechelic PEG derivatives possessing folate or methoxy end groups (FA/m-PEG-Cys) as initiators and reversible addition-fragmentation chain transfer polymerization of N,N-dimethylaminopropyl methacrylamide followed by hydrazinolysis. The successful synthesis of the copolymers was confirmed by 1H NMR and gel permeation chromatography. DOX was covalently conjugated onto the poly(l-glutamate-hydrazide) blocks via a pH-labile hydrazone linkage, and the DOX-conjugated triblock copolymers could self-assemble into nanoparticles in aqueous solutions. P-glycoprotein (P-gp) siRNA was then bound to the cationic poly(N,N-dimethylaminopropyl methacrylamide) (PDMAPMA) blocks through an electrostatic interaction, resulting in the formation of spherical nanocomplexes with an average diameter of 196.8 nm and a zeta potential of +28.3 mV. The in vitro release behaviors of DOX and siRNA from the nanocomplexes were pH- and reduction-dependent, and the release rates were much faster under a reductive acidic condition (pH 5.0, glutathione: 10 mM) simulating the intracellular endo-lysosomal environment of cancer cells compared to physiological conditions. The fast payload release rates were closely related to both the glutathione-triggered detachment of PEG blocks from the nanocomplex surface and the pH-sensitive cleavage of hydrazone linkages. FA-decorated nanocomplexes showed higher cellular uptake efficiency and cytotoxicity against MCF-7 cells than FA-free nanocomplexes, as confirmed by confocal laser scanning microscopy, transmission electron microscopy, MTT and flow cytometry analyses. Our results demonstrated that the multifunctional triblock copolymer-mediated co-delivery of DOX and P-gp siRNA might be a new promising therapeutic strategy for breast cancer treatment.

Abstract 16201: Combined Treatment of the PI3k/mTOR Inhibitor BEZ235 With Doxorubicin Does Not Increase Cardiotoxicity While Improving Efficacy in Breast Cancer Cells

Background: Cardiotoxicity is a major clinical limitation with doxorubicin (DOX). Also, the PI3K/Akt survival pathway is one of the most commonly mutated pathways in cancer. Therefore, the combination of kinase inhibitors and chemotherapy, such as DOX, are necessary for achieving cancer control. The PI3K pathway is also critical for protecting the heart from stress. Therefore, we examined the combined effect of BEZ235 (BEZ), a dual PI3k/mTOR inhibitor, with DOX on cardiac function and killing of breast cancer cells. Methods and results: c57 mice were treated with BEZ (40 mg/kg daily oral gavage) and DOX (3 IV injections of 5 mg/kg) either alone or in combination. Systolic function was assessed 12 weeks after initial treatment using echocardiography. Decreased ejection fraction was observed after DOX treatment. Interestingly, the combined treatment with BEZ and DOX did not exacerbate DOX cardiotoxicity (Fig. A). Western blot analysis showed significant increase in AKT (thr 308) and ERK phosphorylation with BEZ and DOX as compared individual treatment with BEZ or DOX in the heart (Fig. B). To address the anti-cancer effects of these drugs, MDA-MB-231 breast cancer cells were treated with BEZ and DOX either alone or in combination for 48 hours. Necrosis, measured using the trypan blue assay, showed a significant increase in dead cells compared to either drug alone (Fig. C). Conclusion: Concurrent treatment with DOX and BEZ did not exacerbate cardiac toxicity as demonstrated by similar level of deficit in ejection fraction between DOX as well as DOX plus BEZ. This could be due to enhanced survival signaling in the combination group. BEZ also sensitized breast cancer cells to DOX resulting in increased cell death. We propose that this combination strategy might lead to improved long term outcomes for patients with breast cancer without additional complications related to cardiac dysfunction.

554 Dual EZH2 and EHMT2 histone methyltransferase inhibition increases biological efficacy in breast cancer cells

554 Dual EZH2 and EHMT2 histone methyltransferase inhibition increases biological efficacy in breast cancer cells

Loss of Protein Kinase Cδ/HuR Interaction Is Necessary to Doxorubicin Resistance in Breast Cancer Cell Lines

The protein kinase Cδ (PKCδ) interacts with and phosphorylates HuR, dictating its functionality. We show here that the genotoxic stimulus induced by doxorubicin triggers PKCδ interaction with HuR and leads to HuR phosphorylation on serines 221 and 318 and cytoplasmic translocation. This series of events is crucial to elicit the death pathway triggered by doxorubicin and is necessary to promote HuR function in post-transcriptional regulation of gene expression, because genetic ablation of PKCδ caused the inability of HuR to bind its target mRNAs, topoisomerase IIα (TOP2A) included. In in vitro select doxorubicin-resistant human breast cancer cell lines upregulating the multidrug resistance marker ABCG2, PKCδ, and HuR proteins were coordinately downregulated together with the doxorubicin target TOP2A protein whose mRNA was HuR-regulated. Therefore, we show here that PKCδ, HuR, and TOP2A constitute a network mediating doxorubicin efficacy in breast cancer cells. The importance of these molecular events in cancer therapy is suggested by their being profoundly suppressed in cells selected for doxorubicin resistance.

Small Activating RNA Restores the Activity of the Tumor Suppressor HIC-1 on Breast Cancer

HIC-1 is a gene that is hypermethylated in cancer, and commonly downregulated in human breast cancer. However, the precise mechanisms and molecular pathways regulated by HIC-1 remain unclear. We assessed HIC-1 expression on a tissue microarray containing 80 cases of breast cancer. We also analyzed its biological function by restoring HIC-1 expression using 5-aza-2′ deoxycytidine (5-CdR) and small-activating RNAs for the reversal of HIC-1 tumor suppressive effects on MCF-7 and MDA-MB-231 cell lines. An Agilent Q44h global expressing microarray was probed after restoring the expression of HIC-1. Data demonstrated that HIC-1 expression was reduced significantly in breast cancer tissues. HIC-1 immunohistochemistry resulted in mean staining scores in cancer tissue and normal ductal epithelia of 3.54 and 8.2, respectively (p<0.01). 5-CdR partially reversed HIC-1 expression, and modulated cell growth and apoptosis. dsHIC1-2998, an saRNA, showed activating efficacy in breast cancer cells. A group of differentially expressed genes were characterized by cDNA microarray. Upon saRNA treatment, genes upregulated included those involved in immune activation, cell cycle interference, the induction of apoptosis, anti-metastasis, and cell differentiation. Downregulated genes included oncogenes and those that play roles in cell invasion, cell growth, and cell division. Our findings may provide valuable resources not only for gene functional studies, but also for potential clinical applications to develop novel drug targets.

β-sitosterol induces G1 arrest and causes depolarization of mitochondrial membrane potential in breast carcinoma MDA-MB-231 cells

BackgroundsIt is suggested that dietary phytosterols, such as β-sitosterol (ST), have cancer chemopreventive effects; however, studies are limited to support such claims. Here, we evaluated the efficacy of ST on three different human cancer cell lines including skin epidermoid carcinoma A431 cells, lung epithelial carcinoma A549 cells and breast adenocarcinoma MDA-MB-231.MethodsCell growth assay, cell cycle analysis, FACS, JC-1 staining, annexin V staining and immunoblotting were used to study the efficacy of ST on cancer cells.ResultsST (30–90 μM) treatments for 48 h and 72 h did not show any significant effect on cell growth and death in A431 cells. Whereas similar ST treatments moderately inhibited the growth of A549 cells by up to 13% (p ≤ 0.05) in 48 h and 14% (p ≤ 0.05-0.0001) in 72 h. In MDA-MB-231 cells, ST caused a significant dose-dependent cell growth inhibition by 31- 63% (p ≤ 0.0001) in 48 h and 40-50% (p ≤ 0.0001) in 72 h. While exploring the molecular changes associated with strong ST efficacy in breast cancer cells, we observed that ST induced cell cycle arrest as well as cell death. ST caused G0/G1 cell cycle arrest which was accompanied by a decrease in CDK4 and cyclin D1, and an increase in p21/Cip1and p27/Kip1 protein levels. Further, cell death effect of ST was associated with induction of apoptosis. ST also caused the depolarization of mitochondrial membrane potential and increased Bax/Bcl-2 protein ratio.ConclusionsThese results suggest prominent in vitro anti-proliferative and pro-apoptotic effects of ST in MDA-MB-231 cells. This study provides valuable insight into the chemopreventive efficacy and associated molecular alterations of ST in breast cancer cells whereas it had only moderate efficacy on lung cancer cells and did not show any considerable effect on skin cancer cells. These findings would form the basis for further studies to understand the mechanisms and assess the potential utility of ST as a cancer chemopreventive agent against breast cancer.

Abstract 5659: Surface modification of epirubicin-loaded PLGA nanoparticle with biotinylated chitosan enhances anti-cancer efficacy in breast cancer cells.

Abstract Nanoparticle (NP) delivery systems in cancer therapies provide better penetration of therapeutic and diagnostic substances within the body at a reduced risk in comparison with conventional cancer therapies. This is to continue our earlier work on the poly(D,L-lactide-co-glycolide) (PLGA) NPs with biotinylated chitosan (Bio-CS) modification, the modified PLGA NPs were further used as an epirubicin (EPB) carrier to enhance cancer targeting activity and to overcome multidrug resistance (MDR) in adriamycin-resistance human breast cancer cell line (MCF-7/ADM). In this study, Bio-CS were synthesized and characterized. The degree of substitution (DS), as defined as the number of biotin per 100 anhydroglucose units of CS, was determined by 1H-NMR and ICP. EPB-loaded PLGA NPs were prepared by a solvent evaporation technique (W1/O/W2). The PLGA NPs surface was modified by Bio-CS via covalent binding. PLGA NPs of (231.4±21.0) nm in diameter characterized by laser light scattering technique, scanning electron microscopy are spherical and its drug encapsulation efficiency is (84.1±3.4)%. Zeta potential of unmodified NP was measured to be negative -(21.21±2.13) mV. The positive zeta potential of modified NPs represented the presence of CS on the surface of the modified NPs. Modified NPs were characterized for surface chemistry by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FT-IR). N region corresponding to the primary amide of CS and S region corresponding to the biotin meant that PLGA NPs were successfully surface-modified with Bio-CS. The 31% of surface DS biotin of Bio-CS modified PLGA NPs were determined and the value was (1.36 ± 0.34) μmol/100mg. In vitro drug release studies showed that Bio-CS modified NPs had many advantages, such as prolonged drug release property and decreased burst release, in comparison to the unmodified NPs, and the modified NPs achieved relatively constant release kinetics. For cytotoxicity evaluation in vitro, the MCF-7/ADM cells were treated with free EPB, EPB-loaded PLGA NP and EPB-loaded NP with Bio-CS modification, respectively, at a same concentration of 150μg/mL, for 4, 24, 48 and 72 h. We found that Bio-CS modified NPs markedly increased EPB anti-cancer activity, compared to the unmodified NPs. Flowcytometry and confocal laser scanning microscopy revealed that Bio-CS-modified NPs exhibited greater extent of cellular uptake than unmodified NP and free EPB at 48h. Moreover, the uptakes of CS-modified NPs were significantly increased and cell viabilities were dramatically decreased in the MCF-7/ADM cells, compared to the free EPB and PLGA NPs. In summary, the NPs by surface modification with Bio-CS significantly enhanced anti-cancer drug EPB delivery, increased drug efficacy, and thus this system will have great potential for cancer chemotherapy. Citation Format: Hongli Chen, Rui Liu, Wenbin Nan, Yongxue Wang, Yinsong Wang, Wancai Yang, Wancai Yang. Surface modification of epirubicin-loaded PLGA nanoparticle with biotinylated chitosan enhances anti-cancer efficacy in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5659. doi:10.1158/1538-7445.AM2013-5659 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Detection of trastuzumab efficacy using 1H MRI ex vivo of breast cancer cells

This study is aimed on Magnetic resonance imaging (MRI) modalities for monitoring Herceptin (trastuzumab) efficacy in breast cancer cells ex vivo. The efficacy profile of trastuzumab (0.010 μg/ml) was examined using T2 relaxivity measurements prior to and after 24, 48, and 72 h treatment. The untreated MCF-7 cells were considered as a control. MRI results show early tumor response to treatment while comparing pre- and post-treatment T2. The changes of T2 MRI correspond to tumor growth inhibition. The study shows that MRI can be used for the non-invasive monitoring of cancer cells’ death caused by immuno-targeting in preclinical testing of efficacy and treatment regimens.

Silk Fibroin-Derived Nanoparticles for Biomedical Applications

The treatment of disease in the future will be influenced by the ability to produce therapeutic formulations that have high availability at the disease site, sustained and long-term release, with minimal to no toxicity to healthy tissues. Biologically derived delivery systems offer promise in this regard owing to minimization of adverse effects while increasing the efficacy of the entrapped therapeutic. Silk fibroin nanoparticles overcome barriers set by synthetic nondegradable nanoparticles made of silicone, polyethylene glycol and degradable polylactic acid-polyglycolic acid polymers. Silk fibroin-mediated delivery has demonstrated high efficacy in breast cancer cells. While the targeting is associated with the specificity of entrapped therapeutic for the diseased cells, silk fibroin-derived particles enhance intracellular uptake and retention resulting in downmodulation of more than one pathway due to longer availability of the therapeutic. The mechanism of targeting for the nanoparticle is based on the silk fibroin composition, beta-sheet structure and self-assembly into beta-barrels.

An update of the mechanisms of resistance to EGFR-tyrosine kinase inhibitors in breast cancer: Gefitinib (Iressa™)-induced changes in the expression and nucleo-cytoplasmic trafficking of HER-ligands (Review)

Intrinsic resistance to the epidermal growth factor receptor (EGFR; HER1) tyrosine kinase inhibitor (TKI) gefitinib, and more generally to EGFR TKIs, is a common phenomenon in breast cancer. The availability of molecular criteria for predicting sensitivity to EGFR-TKIs is, therefore, the most relevant issue for their correct use and for planning future research. Though it appears that in non-small-cell lung cancer (NSCLC) response to gefitinib is directly related to the occurrence of specific mutations in the EGFR TK domain, breast cancer patients cannot be selected for treatment with gefitinib on the same basis as such EGFR mutations have been reported neither in primary breast carcinomas nor in several breast cancer cell lines. Alternatively, there is a general agreement on the hypothesis that the occurrence of molecular alterations that activate transduction pathways downstream of EGFR (i.e., MEK1/MEK2 right curved arrow ERK1/2 MAPK and PI-3'K right curved arrow AKT growth/survival signaling cascades) significantly affect the response to EGFR TKIs in breast carcinomas. However, there are no studies so far addressing a role of EGF-related ligands as intrinsic breast cancer cell modulators of EGFR TKI efficacy. We recently monitored gene expression profiles and sub-cellular localization of HER-1/-2/-3/-4 related ligands (i.e., EGF, amphiregulin, transforming growth factor-alpha, beta-cellulin, epiregulin and neuregulins) prior to and after gefitinib treatment in a panel of human breast cancer cell lines. First, gefitinib-induced changes in the endogenous levels of EGF-related ligands correlated with the natural degree of breast cancer cell sensitivity to gefitinib. While breast cancer cells intrinsically resistant to gefitinib (IC50 > or =15 microM) markedly up-regulated (up to 600 times) the expression of genes codifying for HER-specific ligands, a significant down-regulation (up to 10(6) times) of HER ligand gene transcription was found in breast cancer cells intrinsically sensitive to gefitinib (IC50 < or =1 microM). Second, loss of HER1 function differentially regulated the nuclear trafficking of HER-related ligands. While gefitinib treatment induced an active import and nuclear accumulation of the HER ligand NRG in intrinsically gefitinib-resistant breast cancer cells, an active export and nuclear loss of NRG was observed in intrinsically gefitinib-sensitive breast cancer cells. In summary, through in vitro and pharmacodynamic studies we have learned that, besides mutations in the HER1 gene, oncogenic changes downstream of HER1 are the key players regulating gefitinib efficacy in breast cancer cells. It now appears that pharmacological inhibition of HER1 function also leads to striking changes in both the gene expression and the nucleo-cytoplasmic trafficking of HER-specific ligands, and that this response correlates with the intrinsic degree of breast cancer sensitivity to the EGFR TKI gefitinib. The relevance of this previously unrecognized intracrine feedback to gefitinib warrants further studies as cancer cells could bypass the antiproliferative effects of HER1-targeted therapeutics without a need for the overexpression and/or activation of other HER family members and/or the activation of HER-driven downstream signaling cascades.