MCF-7 ADR Cells Research Articles

Annexin-A5 Overexpression Increases Sensitivity of MCF-7 and MCF-7/ADR Cells to Epirubicin.

Multi-drug resistance is an important challenge in the chemotherapy of cancer. The role of annexin A5 (ANXA5) in the biology of cancer has been the focus of many studies. Breast Cancer (BC) is frequent cancer in women with high morbidity and mortality rate. The present study aimed to investigate the effects of ANXA5 overexpression on the anti-tumor activity of Epirubicin (EPI) in MCF-7 and MCF-7/ADR cells. MCF-7 and MCF-7/ADR cells were transfected with the pAdenoVator-CMV-ANXA5-IRES-GFP plasmid or mock plasmid. The overexpression of ANXA5 was evaluated using qPCR. The effects of ANXA5 overexpression and EPI on the cell viability of MCF-7 and MCF-7/ADR cells were measured using an MTT assay. Cell apoptosis was measured by annexin V/7-AAD flow cytometry assay. Following the overexpression of ANXA5, the viability of MCF-7 and MCF-7/ADR was significantly decreased. Furthermore, the overexpression of ANXA5 in MCF-7 cells increased the cytotoxic effects of EPI in all doses and reduced the IC50 of EPI from 17.69 μM to 4.07 μM. Similarly, the overexpression of ANXA5 in MCF7-ADR cells reduced the IC50 of EPI from 27.3 μM to 6.69 μM. ANXA5 overexpression alone or combined with EPI treatment increased the apoptosis of MCF7 and MCF7-ADR cells. The results of the present study demonstrate that ANXA5 overexpression increases the sensitivity of MCF-7 and MCF-7/ADR to EPI, suggesting a possible beneficial role of ANXA5 in the therapy of BC.

Jatrophone: a cytotoxic macrocylic diterpene targeting PI3K/AKT/NF-κB pathway, inducing apoptosis and autophagy in resistant breast cancer cells

BackgroundBreast cancer is a prevalent malignant tumor that affects women worldwide. The primary challenge in treating breast cancer is combating drug resistance, which contributes to relapse and metastasis. Jatrophone is a unique macrocyclic jatrophane diterpene found in various Jatropha and Euphorbia species. It possesses diverse biological and pharmacological activities, including anticancer activity. However, it is unclear whether jatrophone can overcome drug resistance in breast cancer.MethodsThis study includes the investigation of the cytotoxicity of jatrophone on doxorubicin-resistant breast cancer cells (MCF-7ADR) and the underlying molecular mechanisms. The effects of jatrophone on cell viability were determined using the sulforhodamine B (SRB) assay, while flow cytometry was used to evaluate cell cycle progression, apoptosis, and autophagy. A scratch assay was conducted to observe cell migration, and western blotting was used to measure downstream protein levels (PI3K, AKT, and NF-κB). Unpaired Student’s t-tests were used for comparison between the two groups and the results were analyzed by one-way ANOVA with Tukey- Kremer post hoc test.ResultsIt was shown that jatrophone exhibited potent cytotoxic activity on MCF-7ADR cells in a dose-dependent manner, with an IC50 value of 1.8 µM. It also significantly induced cell cycle S and G/M phase arrest. Interestingly, jatrophone induced both early and late apoptotic cell death, as well as autophagic cell death, with negligible necrosis. Furthermore, jatrophone treatment diminished the migration of MCF-7ADR cells. At the molecular level, jatrophone treatment significantly down-regulated the expression levels of PI3K, AKT, and NF-κB. β.ConclusionsThe results of the study suggest that jatrophone decreases the proliferation of MCF-7/ADR cells at a low micromolar concentration; induces cell cycle arrest; promotes apoptotic, and autophagic cell death; inhibits migration and EMT; and works on resistance by a mechanism involving the inhibition of the PI3K/Akt/ NF-κB pathway. These findings provide evidence of the potential of jatrophone to be a promising lead compound for targeting doxorubicin-resistant breast cancer cells and could be further investigated for its clinical application as a chemotherapy adjuvant.

Variability in the volatile constituents and biological activities of Achillea millefolium L. essential oils obtained from different plant parts and by different solvents

Achillea millefolium Linn. is an aromatic edible plant used traditionally for the remediation of wounds, liver disorders, and as an herbal tea for the treatment of various gastrointestinal disorders. The plant's volatile oils have economical value in cosmetic preparation and as an insect repellant. The essential oils obtained from different plant parts, i.e., leaves, stems, and aerial portions, by hydrodistillation and by non-polar solvents, i.e., n-hexane and diethyl ether, were phytochemically analyzed and biologically investigated from the plant species cultivated in Saudi Arabia for their potential antioxidant and anticancer activities. The GC-analysis revealed substantial variations among the major constituents and classes of volatile oils in all batches of the plant, characterized by the significant high percentages of monoterpenes, e.g., myrcene, 1,8 cineole, camphor, α-thujone, and β-thujone, in the distilled oils and sesquiterpenes in the non-polar extracts of the plant. Furthermore, the ketonic monoterpenes α-thujone and β-thujone were found to be the most representative compounds in the distilled and extracted batches of the plant. Variable antioxidant activity of different batches of the plant was recognized; however, distilled batches of leaves, stems, and aerial parts exhibited better total antioxidant activity (TAA) compared to the plant’s non-polar extracts. With the little cytotoxic activity of distilled batches, the non-polar extracts exhibited remarkable cytotoxic effects against Panc-1 and MCF-7-Adr (35 and 43.3 μg/mL and 41.3 and 57.1, respectively, in the n-hexane and diethyl ether extracts), which were attributed to their comparatively higher contents of germacrene D, viridiflorol, and caryophyllene oxide. The n-hexane extract induced a concentration- and time-dependent apoptotic effect in Panc-1 and MCF-7 Adr cells, as observed by staining with FITC/PI and flow cytometry analysis. The docking simulation of the major n-hexane extract constituents indicated the prospective mechanism behind the ability of the extract to inhibit MCF-7-Adr cells by inhibiting P-glycoprotein/multidrug resistance 1/adenosine triphosphate-binding cassette.

Reversal Effect of Saikosaponin A and Saikosaponin B on Doxorubicin-resistant Breast Cancer Cells and its Mechanism

Background One of the key factors limiting the effectiveness of chemotherapy treatment for malignancies is multidrug resistance (MDR). The MDR phenotype is related to P-glycoprotein (P-gp) expression and function. The main triterpenoid saponins generated from Bupleurum chinense DC (BCDC), saikosaponin A (SSa), has been found to have anti-tumor potential. Saikosaponin B (SSb) has the potential for utility in combination with anticancer drugs as the secondary saikosaponins. Objective In this study, we looked into the impact of SSa and SSb on doxorubicin (Dox)-resistant breast cancer cells and its underlying mechanisms. Materials and Methods Dox-resistant breast cancer cells (MCF-7ADR) and MCF-7 cells were used in the study. The experimental cells were divided into a different concentration SSa administration group, a different concentration SSb administration group, and a control group, and the related biochemical parameters of MCF-7 and MCF-7ADR cells were detected. Results We discovered that SSa and SSb both suppressed MCF-7 and MCF-7ADR cell proliferation in a dose-dependent manner. Additionally, SSa at 2.5 and 5.0 µg/mL and SSb at 3.0 and 7.0 µg/mL could significantly enhance the cytotoxicity of Dox and reverse MDR in MCF-7ADR cells. The combination of Dox and SSa or SSb induced obvious synergistic effects. SSa and SSb could increase the sensitivity of MCF-7ADR cells to Dox by decreasing P-gp expression, increasing intracellular accumulation, and delaying the drug efflux of rhodamine 123 (Rh123, a P-gp substrate). Additionally, SSa and SSb both induced G1-phase arrest in MCF-7ADR cells in the presence of Dox. Conclusion According to the study, SSa and SSb may be novel MDR reversal medicines for breast cancer chemotherapy and have significant therapeutic significance for MDR during tumor therapy.

Compound 968 reverses adriamycin resistance in breast cancer MCF-7ADR cells via inhibiting P-glycoprotein function independently of glutaminase

Adriamycin (ADR) is a chemotherapeutic drug widely utilized to treat multiple types of cancers; however, the clinical efficacy of ADR is compromised due to the development of drug resistance in patients. The combination of drugs with ADR may provide a better therapeutic regimen to overcome this obstacle. Glutaminase (GLS) has been explored as a therapeutic cancer target, and its inhibition also results in increased sensitivity of tumor cells to chemotherapeutic agents. This study aimed to investigate whether GLS inhibition could reverse ADR resistance. We treated the ADR-resistant MCF-7 (MCF-7ADR) cells with a GLS inhibitor, compound 968 or CB-839, in combination with ADR. We found that compound 968, rather than CB-839, together with ADR synergistically inhibited the cell viability. These results indicated that compound 968 reversed ADR resistance in MCF-7ADR cells independently of GLS. Moreover, we modified the structure of compound 968 and finally obtained a compound 968 derivative, SY-1320, which was more potent than compound 968 in eliminating the drug resistance in MCF-7ADR cells. Furthermore, using drug affinity responsive target stability and streptavidin–biotin immunoprecipitation assays, we demonstrated that SY-1320 could specifically target P-glycoprotein (P-gp) and increase ADR accumulation through inhibition of P-gp, thereby resulting in cell death in MCF-7ADR cells. Together, our findings indicate that compound 968 or SY-1320 might be a promising drug for new combination chemotherapy in breast cancer to overcome the drug resistance.

Folic Acid-Terminated Poly(2-Diethyl Amino Ethyl Methacrylate) Brush-Gated Magnetic Mesoporous Nanoparticles as a Smart Drug Delivery System.

Currently, chemotherapy is an important method for the treatment of various cancers. Nevertheless, it has many limitations, such as poor tumour selectivity and multi-drug resistance. It is necessary to improve this treatment method by incorporating a targeted drug delivery system aimed to reduce side effects and drug resistance. The present work aims to develop pH-sensitive nanocarriers containing magnetic mesoporous silica nanoparticles (MMSNs) coated with pH-responsive polymers for tumour-targeted drug delivery via the folate receptor. 2-Diethyl amino ethyl methacrylate (DEAEMA) was successfully grafted on MMSNs via surface initiated ARGET atom transfer radical polymerization (ATRP), with an average particle size of 180 nm. The end groups of poly (2-(diethylamino)ethyl methacrylate) (PDEAEMA) brushes were converted to amines, followed by a covalent bond with folic acid (FA) as a targeting agent. FA conjugated to the nanoparticle surface was confirmed by X-ray photoelectron spectroscopy (XPS). pH-Responsive behavior of PDEAEMA brushes was investigated by Dynamic Light Scattering (DLS). The nanoparticles average diameters ranged from ca. 350 nm in basic media to ca. 650 in acidic solution. Multifunctional pH-sensitive magnetic mesoporous nanoparticles were loaded with an anti-cancer drug (Doxorubicin) to investigate their capacity and long-circulation time. In a cumulative release pattern, doxorubicin (DOX) release from nano-systems was ca. 20% when the particle exposed to acidic media, compared to ca. 5% in basic media. The nano-systems have excellent biocompatibility and are minimally toxic when exposed to MCF-7, and -MCF-7 ADR cells.

Type of pH sensitive linker reveals different time-dependent intracellular localization, in vitro and in vivo efficiency in alpha-fetoprotein receptor targeted doxorubicin conjugate

Despite the presence of a variety of modern anticancer drugs at the market, doxorubicin (Dox) is still widely used in antineoplastic therapy, although its administration causes severe side effects. To enhance specific activity of such molecules, various approaches have been exploited: targeted moieties like monoclonal antibodies, onco-specific proteins and peptides are utilized as specific vector molecules; environment sensitive linkers are exploited to facilitate transported drug release at the target point etc. Acid-labile linkers are frequently used in synthesis due to the ability to be cleaved inside specific cellular compartments with acidic environment, avoiding possible recycling mechanisms. Two types of conjugates containing different acid-labile linkers have been synthesized. In vitro efficiency of doxorubicin conjugates with recombinant receptor-binding domain of human alpha-fetoprotein (3dAFPpG) synthesized with use of cis-aconitic anhydride (CAA) and linker based on succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and 3-(2-pyridyldithio)propionic acid hydrazide (PDPH) was compared. The 3dAFPpG-SPDP-PDPH-Dox revealed a comparable with unmodified doxorubicin cytotoxic effect against the Dox sensitive MCF7 cell line and greater cytotoxicity against the anthracycline resistant MCF7Adr cells. Meanwhile the 3dAFPpG-CAA-Dox cytotoxic effect was significantly lower, although doxorubicin’s pH-dependent release profiles and intracellular accumulation rates were similar. These differences in cytotoxic activity were arguably explained by the dissimilarities in intracellular doxorubicin localization, which may originate from thiol reductase activity in lysosomes and late endosomes.

Redox-responsive hyaluronic acid nanogels for hyperthermia- assisted chemotherapy to overcome multidrug resistance

Although chemotherapy has been widely used in the treatment of many kinds of cancer, drug resistance and side effects are the main obstacles in the cancer chemotherapy that result in an inferior therapeutic outcome. For the design of drug delivery system, extracellular stability and intracellular effective release are also a pair of contradictions. In this research, gold nanorods (AuNRs) loaded hyaluronic acid (HA) nanogels with reduction sensitivity were prepared for the efficient intracellular delivery of doxorubicin (DOX). The aforementioned HA-CysNG@AuNR nanogels with cystamine (Cys) as crosslinker could remain stable in the physiological condition and release DOX rapidly in the mimic intracellular glutathione (GSH) condition. Meanwhile, the cellular uptake efficiency by the human breast carcinoma (MCF-7) cells was enhanced because of the highly expressed HA receptor (CD44) on the cytomembrane. However, further cell experiments verified that it was difficult to achieve desired results for drug-resistant human breast cancer (MCF-7 ADR) cells due to the reduced drug uptake and enhanced drug efflux. Interestingly, this multidrug resistance of MCF-7 ADR cells could be reversed after treated with near-infrared (NIR) light. This might ascribe to the hyperthermia generated by AuNRs under NIR, which suspended drug efflux process and led to excellent hyperthermia-assisted chemotherapy outcome. Overall, our studies suggested that AuNRs loaded reduction-sensitive HA nanogels were excellent candidates of drug carriers to reverse the drug-resistance and induce severe apoptosis of drug-resistant MCF-7 ADR cells.

Nanostructured lipid carriers co-delivering lapachone and doxorubicin for overcoming multidrug resistance in breast cancer therapy.

BackgroundMultidrug resistance is responsible for the poor outcome in breast cancer therapy. Lapa is a novel therapeutic agent that generates ROS through the catalysis of the NAD(P) H:quinone oxidoreductase-1 (NQO1) enzyme which significantly facilitate the intracellular accumulation of the co-delivered DOX to overcome MDR in cancer cells.PurposeHerein, in our study, nanostructured lipid carrier (NLC) co-delivering β-lapachone (Lapa) and doxorubicin (DOX) was developed (LDNLC) with the aim to overcome the multidrug resistance (MDR) in breast cancer therapy.Patients and methodsLapa and DOX were loaded into NLC to prepare LDNLC using melted ultrasonic dispersion method.ResultsThe well designed LDNLC was nanoscaled particles that exhibited preferable stability in physiological environment. In vitro cell experiments on MCF-7 ADR cells showed increased DOX retention as compared to DOX mono-delivery NLC (DNLC). In vivo anti-cancer assays on MCF-7 ADR tumor bearing mice model also revealed significantly enhanced efficacy of LDNLC than mono-delivery NLCs (DNLC and LNLC).ConclusionLDNLC might be a promising platform for effective breast cancer therapy.

Development of a polymer system for the delivery of daunorubicin to tumor cells to overcome drug resistance

Method for the synthesis of polymeric nanoparticles (NP) with encapsulated daunorubicin (DNR) was developed on the basis of double emulsion solvent evaporation technique using biodegradable poly(lactide-co-glycolide) (PLGA), which is aimed at customization of pharmacokinetic properties of the preparation, enhanced accumulation of DNR in tumor cells and prolongation of its action. The obtained polymer nanoparticles (DNR-PLGA) had average size ranging around 138±36 nm, with zeta-potential of –25.3 mV and the polydispersity index (PDI) of 0.072. The release kinetics of DNR from polymer nanoparticles at pH 7.4 and 5.0 has been studied. In vitro studies showed similar specific activity of DNR- PLGA in K562 and MCF-7 cancer cell lines together with an increase in activity in K562 Adr and MCF-7 Adr cell lines, which are anthracycline resistant, by 1.6 and 3.4 times. The study demonstrated the efficacy of the developed PLGA-based DNR delivery system in the improvement of antitumor effect of DNR, overcoming multidrug resistance in cancer cells, and also in the decrease in nonspecific toxicity of the preparation.

Mitochondrial autophagosomes as a mechanism of drug resistance in breast carcinoma

ABSTRACTWe have previously described the process by which mitochondria donate their membranes for the formation of autophagosomes, and in this study we show that the same process could be involved in drug sequestration and exocytosis resulting in multidrug-resistant cancerous cells. We examine the implications of mitochondrial vesicle formation of mitoautophagosomes (MAPS) in response to the cytotoxic drug MKT-077, which targets mortalin, in a drug-resistant breast carcinoma cell line overexpressing P-glycoprotein (P-gp). The breast cancer cell line MCF-7Adr is derived from MCF-7, but differs from its ancestral line in tolerance of MKT-077-induced mitochondrial toxicity. Our ultrastructural observations suggest that autophagy in the MCF-7Adr cells entails regional sequestration of MKT077 in multilamellar LC3-labeled MAPS, which then separate from their mitochondria, and fuse with or engulf each other. MAPS appeared to be migrating through the cytoplasm and fusing with the plasma membrane, thus carrying out exocytotic secretion. This mechanism, which seems ineffective in the ancestral cell line, provides a resistance mechanism for MKT-077 by enhancing the efflux process of the cells. After 8 hr of MKT-077 exposure, a fraction of the resistant cells appeared viable and contained larger number of smaller sized mitochondria. Mitoautophagosomes, therefore, provide a potentially novel model for multidrug resistance in cancerous cells and may contribute to the P-gp efflux process.

A glutathione-responsive sulfur dioxide polymer prodrug as a nanocarrier for combating drug-resistance in cancer chemotherapy

Multidrug resistance (MDR) in cancer remains a significant challenge for curing cancer by chemotherapy. In this work, a kind of glutathione (GSH)-responsive polymer prodrug of SO2 was designed and synthesized, which presented synergistic effect with doxorubicin (DOX) for combating MCF-7 ADR human breast cancer cell. Firstly, a small molecular prodrug of SO2, N-(3-azidopropyl)-2,4-dinitrobenzenesulfonamide (AP-DNs), was chemically conjugated onto the side chain of methoxy poly (ethylene glycol)-block-poly (γ-propargyl-l-glutamate) (mPEG-PPLG) block copolymer to generate an amphiphilic polymer prodrug of SO2, mPEG-PLG (DNs). The obtained mPEG-PLG (DNs) prodrug could self-assemble into micelles in aqueous media and release SO2 rapidly in response to thiol compounds. Then, DOX was loaded into mPEG-PLG (DNs) nanoparticles with ultrahigh drug-loading efficiency (97.3%). In vitro drug release tests indicated that the DOX-loaded nanoparticles could simultaneously release SO2 and DOX by GSH triggering. Moreover, the effective cellular uptake of the DOX-loaded nanoparticles and subsequent intracellular release of SO2 and DOX were verified by confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) analyses. The released SO2 could promote the reactive oxygen species (ROS) level in tumor cells, which thereby resulted in oxidative damages of cancer cells, together with restoration of MCF-7 ADR cells sensitivity to DOX. As a result, the released DOX and SO2 showed synergistic therapeutic effect against MCF-7 ADR cells. In vivo antitumor evaluation further indicated that, compared with free DOX, the DOX-loaded nanoparticles exhibited better antitumor effect in a MCF-7 ADR-xenografted nude mice model while had lower system toxicity. Overall, we demonstrated, for the first time, that a SO2 polymer prodrug, acting as a stimuli-responsive nanocarrier to codeliver DOX, can efficiently inhibit the proliferation of MDR tumor cells, which may offer a new weapon for combating MDR in cancer therapy.

Abstract 1205: Rubrofusarin and toralactone sensitize resistant MCF-7adr cell line to paclitaxel via inhibiting P-glycoprotein efflux activity

Abstract The use of anti-cancer adjuvant therapy is rationalized by potentiating the efficacy and/or protecting from major side effects of chemotherapeutics. Paclitaxel is an effective spindle toxin anticancer agent used for the treatment of breast cancer. Rubrofusarin and toralactone are naturally occurring structurally related naphthopyrones with potent antioxidant and hence chemoprotective activity. The purpose of this study is to investigate the influence of rubrofusarin and toralactone on the cytotoxic profile of paclitaxel (PTX) against chemo-resistant breast cancer cell line (MCF-7adr) compared to parent MCF-7 cells. MCF-7adr was prepared by continuous culturing of MCF-7 cells in media containing subcytotoxic concentration of doxorubicin (IC10%) for two months duration. Toralactone and rubrofusarin showed moderate cytotoxic effects against MCF-7 cells with IC50’s of 32.4±4.2 µM and 13.9±6.1 µM, respectively. Interestingly, toralactone and rubrofusarin showed stronger cytotoxicity against MCF-7adr cells with IC50’s of 7.3±0.7 µM and 7.8±0.6 µM, respectively. PTX treatment was opposed by significant resistance from MCF-7adr cells compared to parent MCF-7 cells having IC50’s of 4.9±0.9 µM and 57.7±4.5 nM, respectively. Combination of torolactone or rubrofusarin with PTX did not significantly improve its cytotoxicity against MCF-7; IC50’s were 24.2±2.2 nM and 69.7±6.6 nM, respectively compared to 57.7±4.5 nM for PTX alone. Surprisingly, combination with either torolactone or rubrofusarin abolished the resistance of MCF-7adr to PTX resulting in IC50’s of 3.4±0.2 nM and 4.9±0.5 6 nM, respectively compared to 4.9±0.9 µM for PTX alone. Using flowcytometric analysis, rubrofusarin and toralactone increased median intracellular fluorescence signal count of rhodamine dye (non-cytotoxic P-gp substrate) within MCF-7adr from 1794.7±21.5 to 2003.3±12.5 and 2113±10.5 cellular events, respectively. Using purified membrane bound humanized recombinant P-gp molecules; it was found that both rubrofusarin and toralactone might be inhibiting P-gp efflux activity via dual interaction with both P-gp ATPase enzyme subunit and P-gp active binding site. In both MCF-7 and MCF-7adr cells, PTX exerted significant cell cycle block at G2/M-phase with significant increase in the pre-G dead cell fraction from 3.4±0.9% to 23.6±2.1% and from 1.4±0.6% to 17.2±3.2%, respectively. Neither rubrofusarin nor toralactone exerted any significant influence for PTX induced cell cycle interruption in both normal (MCF-7) and resistant (MCF-7adr) cell lines. In conclusion, rubrofusarin and toralactone can enhance the cytotoxic profile of P-gp substrates, such as PTX, via inhibiting P-gp efflux activity and enhancing the intracellular entrapment of P-gp substrates. Citation Format: Salwa D. Alqahtani, Hanan A. Assiri, Fahad A. Al-Abbasi, Ali M. El-Halawany, Ahmed M. Al-Abd. Rubrofusarin and toralactone sensitize resistant MCF-7adr cell line to paclitaxel via inhibiting P-glycoprotein efflux activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1205. doi:10.1158/1538-7445.AM2017-1205

An in vitro demonstration of overcoming drug resistance in SKOV3 TR and MCF7 ADR with targeted delivery of polymer pro-drug conjugates

Drug resistance is a common phenomenon that occurs in cancer chemotherapy. Delivery of chemotherapeutic agents as polymer pro-drug conjugates (PPDCs) pretargeted with bispecific antibodies could circumvent drug resistance in cancer cells. To demonstrate this approach to overcome drug resistance, Paclitaxel (Ptxl)-resistant SKOV3 TR human ovarian- and doxorubicin (Dox)-resistant MCF7 ADR human mammary-carcinoma cell lines were used. Pre-targeting over-expressed biotin or HER2/neu receptors on cancer cells was conducted by biotinylated anti-DTPA or anti-HER2/neu affibody – anti-DTPA Fab bispecific antibody complexes. The targeting PPDCs are either D-Dox-PGA or D-Ptxl-PGA. Cytotoxicity studies demonstrate that the pretargeted approach increases cytotoxicity of Ptxl or Dox in SKOV3 TR or MCF7 ADR resistant cell lines by 5.4 and 27 times, respectively. Epifluorescent microscopy – used to track internalization of D-Dox-PGA and Dox in MCF7 ADR cells – shows that the pretargeted delivery of D-Dox-PGA resulted in a 2- to 4-fold increase in intracellular Dox concentration relative to treatment with free Dox. The mechanism of internalization of PPDCs is consistent with endocytosis. Enhanced drug delivery and intracellular retention following pretargeted delivery of PPDCs resulted in greater tumor cell toxicity in the current in vitro studies.

Abstract 2168: Optimizing doxorubicin derivatives delivery using temperature sensitive biopolymers in multidrug resistant breast cancer cells

Abstract The anticancer agent doxorubicin is an anthracycline compound that shows high potency in treating cancer, and it is one of the most widely used chemotherapeutics. However, efficiency of doxorubicin treatment is limited by low blood plasma solubility, poor blood pharmacokinetics, and non-selective cell killing that results in serious toxicity to healthy tissues. Additionally, cancer cells often develop drug resistance, which is a significant limiting factor to the drug's effectiveness. Motivated by these problems, we have designed a drug delivery system that can specifically deliver drug to the tumor site while overcoming doxorubicin resistance in breast cancer cell lines. This drug delivery system consists of: ELP - Elastin like polypeptide, CPP - Cell penetrating peptide, a cleavable linker – to enable doxorubicin release in the targeted low pH environment, and a derivative of the anticancer agent Doxorubicin (modified by a 6-maleimidocaproyl moiety for conjugation to a terminal cysteine residue on ELP). ELP is thermally responsive and improves the complex's pharmacokinetics by prolonging its clearance rate while the CPP mediates cellular uptake of large macromolecules. The linker is an acid sensitive amino acid sequence (Gly-Phe-Leu-Gly) that serves as a substrate for lysosomal enzymes. In this study, we compared cytotoxicity of a cleavable (cDox) and non-cleavable (ncDox) doxorubicin derivative delivered by ELP biopolymer in the breast cancer cell lines MCF-7 and MCF7/ADR (doxorubicin resistant cell line). We showed that cDox had two fold higher cytotoxicity than ncDox in both cell lines. When ncDox, however, was conjugated to the ELP biopolymer containing a lysosomally degradable GFLG spacer, the drug delivery construct was equally cytotoxic to both sensitive and resistant cell lines, indicating that the construct delivers doxorubicin into cells by a mechanism that bypasses doxorubicin resistance. Confocal fluorescence microscopy showed that after two hours of exposure to the doxorubicin derivatives, cDox was predominantly localized in the nucleus in both cell lines. However, ncDox was localized in the plasma membrane and cytosol. Intracellular doxorubicin accumulation examined by flow cytometry indicated 3 fold higher uptake of ELP-cDox in sensitive MCF 7 cells compared to resistant MCF7 ADR cells. Cellular uptake of ELP-cDox was further enhanced two fold when conjugated with CPP. In conclusion, our current results indicate that ELP-doxorubicin conjugates may successfully overcome drug resistance in breast cancer cells, providing a promising approach for the use of chemotherapeutic agents such as doxorubicin in patients with drug resistant breast cancers. Citation Format: Sonja Dragojevic, Jung Su Ryu, Felix Kratz, Drazen Raucher. Optimizing doxorubicin derivatives delivery using temperature sensitive biopolymers in multidrug resistant breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2168.

A44: Detection of HER2 mRNA splice variants in exosomes of breast cancer cells

Background HER2 is a known driver of breast cancer progression in 15–30% of primary breast carcinomas and an important target for therapy. More recently, it was established that transcript splice variants of HER2, including delta16-HER2, herstatin, p100 participate in malignant progression and drug resistance. Exosomes are small vesicles of endocytic origin, which are released by many different cell types and can be used as potential novel biomarkers, since they contain DNA and RNA derived from cancer cells. The aim of this study is to explore the use of exosomes for detecting HER2 splice variants and compare levels HER2 splice variants in cells and their exosomes. Material and methods Breast cancer cell lines MDA-MB-361, MDA-MB- 231, MCF7, MCF7-Adr and BT549 were grown as monolayer cultures as recommended by American Type Culture Collection. The exosomes were isolated by an ultracentrifugation protocol. Total RNA was isolated and cDNA was generated to be used for classic PCR and SYBR Green I quantitative ( q ) PCR. Results We determined HER2 splice variants levels in MDA-MB-361, MDA-MB-231 breast cancer cell lines and in exosomes derived from MDA-MB-36. MDA-MB-361 cells expressed high levels of full length HER2 mRNA, delta16-HER2 mRNA, whereas no expression was detected in other cell lines. Exosomes derived from MDA-MB-231 cells line were negative for Herstatin, delta16-HER2 and p100 but positive for mRNA of full length HER2. Conclusion The absence of HER2 mRNA and splice variants in MCF7, MCF7-Adr and BT549 cells lines corresponded to their absence in exosomes. Although the other cell lines remain to be examined, we speculate that exosomes will reflect the HER2 splice variants content present in the breast cancer cells.

Effect of Pin1 inhibitor juglone on proliferation, migration and angiogenic ability of breast cancer cell line MCF7Adr

This study aimed to evaluate the effects of Pin1 inhibitor Juglone on proliferation, migration and the angiogenic ability of breast cancer cell line MCF7Adr. MCF7Adr cells were cultured and separately treated with Pin1 inhibitor Juglone (treatment group) and DMEM without drug (control group). The cell cycle was examined by flow cytometry. Cell migration was measured by wound-healing assay. Cyclin E protein content was detected by Western blotting. The angiogenesis factor vascular endothelial growth factor (VEGF) in cell media was determined by enzyme linked immunosorbent assay. The results showed that the percentage of cells in G2/M phase in treatment group was significantly higher than that in control group (25.5% vs. 10.1%, P<0.05), and that in G0/G1 phase and S stage in treatment group was significantly lower than that in control group (40.5% vs. 48.2%, and 33.7% vs. 41.7%, P<0.05). Cyclin E protein content in treatment group was significantly lower than that in control group (39.2 ± 7.4 vs. 100 ± 23.1, P<0.05). (A0-A24)/A0 value in treatment group was significantly lower than that in control group (23.9 ± 3.8 vs. 100 ± 14.4, P<0.05). VEGF-A, -B, and -C contents in cell media of treatment group were significantly lower than those in control group (P<0.05). It was suggested that Pin1 inhibitor Juglone can effectively inhibit the proliferation, migration and the angiogenic ability of MCF7Adr cells, and can be used as an alternative drug therapy for breast cancer.

Sigma‐2 Receptor Agonists as Possible Antitumor Agents in Resistant Tumors: Hints for Collateral Sensitivity

With the aim of contributing to the development of novel antitumor agents, high-affinity σ2 receptor agonists were developed, with 6,7-dimethoxy-2-[4-[1-(4-fluorophenyl)-1H-indol-3-yl]butyl]-1,2,3,4-tetrahydroisoquinoline (15) and 9-[4-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-yl)butyl]-9H-carbazole (25) showing exceptional selectivity for the σ2 subtype. Most of the compounds displayed notable antiproliferative activity in human MCF7 breast adenocarcinoma cells, with similar activity in the corresponding doxorubicin-resistant MCF7adr cell line. Surprisingly, a few compounds, including 25, displayed enhanced activity in MCF7adr cells over parent cells, recalling the phenomenon of collateral sensitivity, which is under study for the treatment of drug-resistant tumors. All of the compounds showed interaction with P-glycoprotein (P-gp), and 15 and 25, with the greatest activity, were able to revert P-gp-mediated resistance and reestablish the antitumor effect of doxorubicin in MCF7adr cells. We therefore identified a series of σ2 receptor agonists endowed with intriguing antitumor properties; these compounds deserve further investigation for the development of alternate strategies against multidrug- resistant cancers.

Metabolomic approach to evaluating adriamycin pharmacodynamics and resistance in breast cancer cells

Continuous exposure of breast cancer cells to adriamycin induces high expression of P-gp and multiple drug resistance. However, the biochemical process and the underlying mechanisms for the gradually induced resistance are not clear. To explore the underlying mechanism and evaluate the anti-tumor effect and resistance of adriamycin, the drug-sensitive MCF-7S and the drug-resistant MCF-7Adr breast cancer cells were used and treated with adriamycin, and the intracellular metabolites were profiled using gas chromatography mass spectrometry. Principal components analysis of the data revealed that the two cell lines showed distinctly different metabolic responses to adriamycin. Adriamycin exposure significantly altered metabolic pattern of MCF-7S cells, which gradually became similar to the pattern of MCF-7Adr, indicating that metabolic shifts were involved in adriamycin resistance. Many intracellular metabolites involved in various metabolic pathways were significantly modulated by adriamycin treatment in the drug-sensitive MCF-7S cells, but were much less affected in the drug-resistant MCF-7Adr cells. Adriamycin treatment markedly depressed the biosynthesis of proteins, purines, pyrimidines and glutathione, and glycolysis, while it enhanced glycerol metabolism of MCF-7S cells. The elevated glycerol metabolism and down-regulated glutathione biosynthesis suggested an increased reactive oxygen species (ROS) generation and a weakened ability to balance ROS, respectively. Further studies revealed that adriamycin increased ROS and up-regulated P-gp in MCF-7S cells, which could be reversed by N-acetylcysteine treatment. It is suggested that adriamycin resistance is involved in slowed metabolism and aggravated oxidative stress. Assessment of cellular metabolomics and metabolic markers may be used to evaluate anti-tumor effects and to screen for candidate anti-tumor agents.Electronic supplementary materialThe online version of this article (doi:10.1007/s11306-013-0517-x) contains supplementary material, which is available to authorized users.

Identification of Glycan Structure Alterations on Cell Membrane Proteins in Desoxyepothilone B Resistant Leukemia Cells

Resistance to tubulin-binding agents used in cancer is often multifactorial and can include changes in drug accumulation and modified expression of tubulin isotypes. Glycans on cell membrane proteins play important roles in many cellular processes such as recognition and apoptosis, and this study investigated whether changes to the glycan structures on cell membrane proteins occur when cells become resistant to drugs. Specifically, we investigated the alteration of glycan structures on the cell membrane proteins of human T-cell acute lymphoblastic leukemia (CEM) cells that were selected for resistance to desoxyepothilone B (CEM/dEpoB). The glycan profile of the cell membrane glycoproteins was obtained by sequential release of N- and O-glycans from cell membrane fraction dotted onto polyvinylidene difluoride membrane with PNGase F and β-elimination respectively. The released glycan alditols were analyzed by liquid chromatography (graphitized carbon)-electrospray ionization tandem MS. The major N-glycan on CEM cell was the core fucosylated α2-6 monosialo-biantennary structure. Resistant CEM/dEpoB cells had a significant decrease of α2-6 linked sialic acid on N-glycans. The lower α2-6 sialylation was caused by a decrease in activity of β-galactoside α2-6 sialyltransferase (ST6Gal), and decreased expression of the mRNA. It is clear that the membrane glycosylation of leukemia cells changes during acquired resistance to dEpoB drugs and that this change occurs globally on all cell membrane glycoproteins. This is the first identification of a specific glycan modification on the surface of drug resistant cells and the mechanism of this downstream effect on microtubule targeting drugs may offer a route to new interventions to overcome drug resistance.