BT-474 Human Breast Cancer Cells Research Articles

Nanocomplexes of Biodegradable Anticancer Macromolecules: Prolonged Plasma Half-life, Reduced Toxicity, and Increased Tumor Targeting.

Anticancer drug resistance is a large contributing factor to the global mortality rate of cancer patients. Anticancer macromolecules such as polymers were recently reported to overcome this issue. Anticancer macromolecules have unselective toxicity because they are highly positively charged. Herein, an anionic biodegradable polycarbonate carrier was synthesized and utilized to form nanocomplexes with an anticancer polycarbonate via self-assembly to neutralize its positive charges. Biotin was conjugated to the anionic carrier and served as a cancer cell-targeting moiety. The nanoparticles had sizes of <130nm with anticancer polymer loading levels of 38%-49%. Unlike the small molecular anticancer drug doxorubicin, the nanocomplexes effectively inhibited the growth of both drug-susceptible MCF7 and drug-resistant MCF7/ADR human breast cancer cell lines with low half maximal inhibitory concentration (IC50 ). The nanocomplexes increased the anticancer polymer's in vivo half-life from 1h to 6 - 8h, and rapidly killed BT474 human breast cancer cells primarily via an apoptotic mechanism. The nanocomplexes significantly increased the median lethal dose (LD50) and reduced the injection site toxicity of the anticancer polymer. They suppressed tumor growth by 32% - 56% without causing any damage to the liver and kidneys. These nanocomplexes may potentially be used for cancer treatment to overcome drug resistance. This article is protected by copyright. All rights reserved.

RKIP localizes to the nucleus through a bipartite nuclear localization signal and interaction with importin α to regulate mitotic progression

Raf kinase inhibitor protein (RKIP) is a multifunctional modulator of intracellular signal transduction. Although most of its functions have been considered cytosolic, we show here that the localization of RKIP is primarily nuclear in both growing and quiescent Madin-Darby canine kidney epithelial cells and in Cal-51 and BT-20 human breast cancer cells. We have identified a putative bipartite nuclear localization signal (NLS) in RKIP that maps to the surface of the protein surrounding a known regulatory region. Like classical NLS sequences, the putative NLS of RKIP is rich in arginine and lysine residues. Deletion of and point mutations in the putative NLS lead to decreased nuclear localization. Point mutation of all the basic residues in the putative NLS of RKIP particularly strongly reduces nuclear localization. We found consistent results in reexpression experiments with wildtype or mutant RKIP in RKIP-silenced cells. A fusion construct of the putative NLS of RKIP alone to a heterologous reporter protein leads to nuclear localization of the fusion protein, demonstrating that this sequence alone is sufficient for import into the nucleus. We found that RKIP interacts with the nuclear transport factor importin α in BT-20 and MDA-MB-231 human breast cancer cells, suggesting importin-mediated active nuclear translocation. Evaluating the biological function of nuclear localization of RKIP, we found that the presence of the putative NLS is important for the role of RKIP in mitotic checkpoint regulation in MCF-7 human breast cancer cells. Taken together, these findings suggest that a bipartite NLS in RKIP interacts with importin α for active transport of RKIP into the nucleus and that this process may be involved in the regulation of mitotic progression.

Synthesis and In Vitro Comparison of DOTA, NODAGA and 15-5 Macrocycles as Chelators for the 64Cu-Labelling of Immunoconjugates

The development of 64Cu-based immuno-PET radiotracers requires the use of copper-specific bifunctional chelators (BFCs) that contain functional groups allowing both convenient bioconjugation and stable copper complexes to limit in vivo bioreduction, transmetallation and/or transchelation. The excellent in vivo kinetic inertness of the pentaazamacrocyclic [64Cu]Cu-15-5 complex prompted us to investigate its potential for the 64Cu-labelling of monoclonal antibodies (mAbs), compared with the well-known NODAGA and DOTA chelators. To this end, three NODAGA, DOTA and 15-5-derived BFCs, containing a pendant azadibenzocyclooctyne moiety, were synthesised and a robust methodology was determined to form covalent bonds between them and azide-functionalised trastuzumab, an anti-HER2 mAb, using strain-promoted azide-alkyne cycloaddition. Unlike the DOTA derivative, the NODAGA- and 15-5-mAb conjugates were radiolabelled with 64Cu, obtaining excellent radiochemical yields, under mild conditions. Although all the radioimmunoconjugates showed excellent stability in PBS or mouse serum, [64Cu]Cu-15-5- and [64Cu]Cu-NODAGA-trastuzumab presented higher resistance to transchelation when challenged by EDTA. Finally, the immunoreactive fraction of the radioimmunoconjugates (88-94%) was determined in HER-2 positive BT474 human breast cancer cells, confirming that the bioconjugation and radiolabelling processes implemented had no significant impact on antigen recognition.

ONC201-Induced Mitochondrial Dysfunction, Senescence-like Phenotype, and Sensitization of Cultured BT474 Human Breast Cancer Cells to TRAIL.

ONC201, the anticancer drug, targets and activates mitochondrial ATP-dependent caseinolytic peptidase P (ClpP), a serine protease located in the mitochondrial matrix. Given the promise of ONC201 in cancer treatment, we evaluated its effects on the breast ductal carcinoma cell line (BT474). We showed that the transient single-dose treatment of BT474 cells by 10 µM ONC201 for a period of less than 48 h induced a reversible growth arrest and a transient activation of an integrated stress response indicated by an increased expression of CHOP, ATF4, and GDF-15, and a reduced number of mtDNA nucleoids. A prolonged exposure to the drug (>48 h), however, initiated an irreversible loss of mtDNA, persistent activation of integrated stress response proteins, as well as cell cycle arrest, inhibition of proliferation, and suppression of the intrinsic apoptosis pathway. Since Natural Killer (NK) cells are quickly gaining momentum in cellular anti-cancer therapies, we evaluated the effect of ONC201 on the activity of the peripheral blood derived NK cells. We showed that following the ONC 201 exposure BT474 cells demonstrated enhanced sensitivity toward human NK cells that mediated killing. Together our data revealed that the effects of a single dose of ONC201 are dependent on the duration of exposure, specifically, while short-term exposure led to reversible changes; long-term exposure resulted in irreversible transformation of cells associated with the senescent phenotype. Our data further demonstrated that when used in combination with NK cells, ONC201 created a synergistic anti-cancer effect, thus suggesting its possible benefit in NK-cell based cellular immunotherapies for cancer treatment.

Overcoming Drug Resistance in Breast Cancer Using Quercetin Nanodrugs

Background &amp; Objective: Breast cancer is one of the most common cancers in the world. Treatment methods for this disease have been with poor results. In recent years, more attention has been paid to stem cells due to their cytotoxic properties. According to previous results, the effect of nanoquercetin on breast cancer cells in this study was used concomitantly with doxorubicin, which is a chemotherapy drug and in some patients has therapeutic resistance.&#x0D; Materials &amp; Methods: The above study was performed on MCF7 and BT474 human breast cancer cells. Quercetin was synthesized, then different concentrations of 10, 20, 40, 80 and 160 of quercetin and 1, 2.5, 5 and 10 μg/ml of doxorubicin were prepared and two cell lines were treated for 24 h. MTT test was used to determine cell viability and flow cytometry test was used to determine the extent of apoptosis in both cell lines.&#x0D; Results: The results showed that MCF7 and BT474 cells that were affected by quercetin and doxorubicin, increasing the concentration of quercetin and doxorubicin decreased the survival of both cell lines within 24 h. The LC50 concentration was calculated for both cells treated with nanoquercetin 40 μg/ml and for doxorubicin (0.1 mg/mL). The results also showed that in both cell lines, an increase in cellular apoptosis occurred after incubation with quercetin and then incubation with doxorubicin.&#x0D; Conclusion: Comparison of doxorubicin (DOX), quercetin (QU) and quercetin and doxorubicin (QU-DOX) groups with controls shows that with the presence of quercetin-doxorubicin, it had a greater inhibitory effect on cancer cells.

HER2-Displaying M13 Bacteriophages induce Therapeutic Immunity against Breast Cancer.

Simple SummaryThe high incidence and death rates of breast cancer make the development of new therapies an urgent need. The introduction into the clinic of the anti-HER2 monoclonal antibody trastuzumab considerably improved the overall survival and time-to-disease progression of patients with HER2-positive breast cancer. However, many patients do not benefit from it because of resistance to therapy. Cancer vaccines, by inducing into the patient an anti-cancer specific immunity, might represent an alternative immunotherapeutic approach, but despite promises, so far no anti-HER2 cancer vaccine has been approved for human use. In this study, we propose therapeutic phage-based vaccines, against HER2 and its aggressive isoform Δ16HER2, able to elicit a protective immunity and potentially capable of preventing relapse in HER2-positive breast cancer patients, even in those who develop trastuzumab resistance.The advent of trastuzumab has significantly improved the prognosis of HER2-positive (HER2+) breast cancer patients; nevertheless, drug resistance limits its clinical benefit. Anti-HER2 active immunotherapy represents an attractive alternative strategy, but effective immunization needs to overcome the patient’s immune tolerance against the self-HER2. Phage display technology, taking advantage of phage intrinsic immunogenicity, permits one to generate effective cancer vaccines able to break immune tolerance to self-antigens. In this study, we demonstrate that both preventive and therapeutic vaccination with M13 bacteriophages, displaying the extracellular (EC) and transmembrane (TM) domains of human HER2 or its Δ16HER2 splice variant on their surface (ECTM and Δ16ECTM phages), delayed mammary tumor onset and reduced tumor growth rate and multiplicity in ∆16HER2 transgenic mice, which are tolerant to human ∆16HER2. This antitumor protection correlated with anti-HER2 antibody production. The molecular mechanisms underlying the anticancer effect of vaccine-elicited anti-HER2 antibodies were analyzed in vitro against BT-474 human breast cancer cells, sensitive or resistant to trastuzumab. Immunoglobulins (IgG) purified from immune sera reduced cell viability mainly by impairing ERK phosphorylation and reactivating retinoblastoma protein function in both trastuzumab-sensitive and -resistant BT-474 cells. In conclusion, we demonstrated that phage-based HER2 vaccines impair mammary cancer onset and progression, opening new perspectives for HER2+ breast cancer treatment.

Abstract 5380: Anticancer compound ONC201 induces prolonged stress and arrest of proliferation in BT474 human breast cancer cells without induction of cell death

Abstract Background and goals: ONC201 an experimental anticancer drug, targeting mitochondrial caseinolytic peptidase, ClpP is currently under investigation. Current data on the effect of ONC201 along with observations that ONC201 causes cytotoxic effect, there are data demonstrating that ONC201 is rather cytostatic and does not induce cell death. Recently, we demonstrated ONC201-induced arrest of proliferation and a decrease in the number of mitochondrial nucleoids. Here we extended our study to describe the effect of short-and long-term exposure of BT474 cells to ONC201. Observations: Proliferation and cell cycle arrest. ONC201 in a dose-dependent manner (0-50 µM) inhibited the proliferation of BT474 cells without induction of apoptotic cell death. Cells exposed to 10 µM ONC201 accumulated in G0/G1 phase (49.4 ± 11.5% vs 78.7 ± 5.1%) and declined in the S-phase of the cell cycle (39.2 ± 6.6% vs 9.0 ± 2.1%). In parallel, ONC201 exposure induced a decline in Cyclin E and Cdk2 protein levels. Nucleoids and mtDNA. Exposure of BT474 cells, to 10 µM ONC 201, decreased the number of mitochondrial nucleoids from 249 ± 52 to 155 ± 38 per cell after 24 h. Longer 72 h exposure further reduced the number of nucleoids to 84 ± 36 per cell. ONC201 exposure also induced reduction of mitochondrial size from 2.8 ± 0.5 µm2 to 1.5 ± 0.2 µm2 and depletion of mtDNA. Short-term and long-term consequences of cell exposure to ONC201. Short-term treatment (24 h exposure) demonstrated the reversible effect of ONC201 on the expression of stress proteins (ATF4, CHOP, and GDF-15), cell cycle regulatory proteins (cyclin E, Cdk2), and partial restoration of mitochondrial nucleoids (to 214 ± 46 nucleoids per cell). On the contrary, long-term treatment (72 h) resulted in the irreversible and persistent arrest of proliferation, decline of cell cycle regulatory proteins, and activation of stress proteins. Respiration. ONC201 exposure suppressed State 3, State 4, and uncoupled respiration in digitonin-permeabilized BT474 cells. Complex I activity was decreased from 3.3 ± 0.4 ng-atoms O/min/106 cells to 0.5 ± 0.1 and 0.4 ± 0.3 ng-atoms O/min/106 cells, in cells exposed to ONC201 for 24h and 72h, respectively. Similarly, ONC201 decreased the activity of Complex II from 3.5 ± 0.5 ng-atoms O/min/106 cells to 1.5 ± 0.6 and 0.3 ± 0.1 ng-atoms O/min/106. Conclusions: The consequences of ONC201 treatment were dependent on the duration of exposure to the drug. Short-term (24 h) exposure of BT474 cells resulted in reversible induction of stress proteins (ATF4, CHOP, and GDF-15), which were reversed following 120 h incubation in drug-free media. On the contrary, long-term (72 h) exposure of cells to ONC201 induced irreversible and persistent arrest of proliferation and elevation of expression of stress proteins. Thus, ONC201 can induce prolonged and irreversible elevation of stress proteins and persistent G1/S cell cycle arrest. Citation Format: Artem A. Mishukov, Irina V. Odinokova, Serazhutdin A. Abdullaev, Vitaly K. Zhalimov, Emily M. Fennel, Lucas J. Aponte-Collazo, Paul R. Graves, Lee M. Graves, Ekhson L. Holmuhamedov. Anticancer compound ONC201 induces prolonged stress and arrest of proliferation in BT474 human breast cancer cells without induction of cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5380.

The Synergistic Anticancer Traits of Graphene Oxide Plus Doxorubicin Against BT474 and MCF7 Breast Cancer Stem Cells In Vitro.

Breast cancer is among the leading causes of death due to cancers around the globe. Current therapeutic approaches towards healing of breast cancer have been associated with poor outcomes. Graphene and its derivatives have a two-dimensional flat structure, which is characterized by the ability to carry drugs and modify the surface, low cytotoxicity, and high biocompatibility. This study was performed on MCF7 and BT474 human breast cancer cells. Different concentrations of doxorubicin (DOX), graphene oxide (GO), and graphene oxide plus doxorubicin (GO-DOX) were subjected to both cell lines at specified intervals. At the end of the treatments, MTT test was applied to determine the viability of cells, and then flow cytometry, colony formation, and spheroid tests were implemented in both cell lines treated with DOX, GO, and GO-DOX components. We used DLS and TEM to confirm the GO properties. According to the MTT test results, 1 μL of DOX at 10mg/ml (equivalent to 0.1mg/ml) caused 50% survival of MCF7 cells at 24h. In both cell lines, an increase in apoptosis occurred after incubation with GO and DOX. Although a rate of mortality of MCF-7 cells was due to necrosis, the BT474 cell death was merely through the apoptosis. Furthermore, the results of the colony formation test outlined an enhancing inhibitory effect in the presence of GO-DOX as a comparison to the control. Additionally, spheroids formed following treatment with GO-DOX exhibited a significant decrease compared to their control group, with an increase in the number of spheroids in BT474 cells compared to those in the MCF-7. The decreasing effect of compounds against the migration and cell invasion potential was also observed, being higher in MCF7 than BT474 cells. The effects of cytotoxic GO were observed at higher concentrations.

Abstract 1078: Hypoxic microenvironment promotes resistance to targeted therapies in HER2-overexpressing breast cancer involving epithelial-to-mesenchymal transition features

Abstract Trastuzumab and trastuzumab emtansine (T-DM1) targeted therapies are the main choice for the treatment of HER2-overexpressing breast cancer patients. However, de novo or acquired resistance is still the major obstacle in clinical practice. It has been shown that several pathways, including HER2, can lead to HIF-α stabilization in breast cancer cells. Thus, the purpose of our study was to analyse the effect of hypoxia in acquired resistance to anti-HER2 therapies. We used HER2-overexpressing BT-474 and non-overexpressing MCF-7 human breast cancer cell lines. As an acute hypoxia model, we added CoCl2 (100 µM) to cell culture medium. The hypoxic status of the cells was evaluated by a Western blot analysis, showing a peak of HIF-1α expression after 6 hours of CoCl2 exposure. This result correlated with VEGF induction, as measured by RT-qPCR (p&amp;lt;0.05). Interestingly, under hypoxia, BT-474 cells treated with increasing concentrations of trastuzumab and T-DM1 for 72 hours presented a significantly higher viability compared with non-hypoxic cells (p&amp;lt;0.05). However, although the drugs decreased membrane HER2 protein expression (p&amp;lt;0.01), HER2 levels did not change under hypoxic conditions, as shown by flow cytometry and Western blot assays. Moreover, drug effects on MCF-7 cells did not change under hypoxic conditions. To further analyse this cytoprotective effect of hypoxia, proliferation-associated markers such as PCNA expression and AKT activation were evaluated by Western blot, while apoptosis frequency was measured by flow cytometry in BT-474 cells. PCNA and pAKT expression remained unaltered, while T-DM1-mediated apoptosis was significantly reduced under hypoxia (p&amp;lt;0.05). Then, using different open gene expression datasets, we identified a group of four genes (COL12A1, DKK1, CEACAM5 and ANTXR1) differentially expressed in T-DM1 resistant HER2-overexpressing breast cancer BT-474 and KPL-4 cell lines (p&amp;lt;0.05), which were coexpressed with hypoxia-related genes (HIF1A, EPAS1 and VEGFA) in HER2-overexpressing breast cancer tumours and cell lines from TCGA (p&amp;lt;0.01). Further in silico analyses revealed a direct association between those genes and the fibronectin gene (FN1, p&amp;lt;0.05). Remarkably, they were involved in TGF-β and angiogenesis pathways, as shown by a functional enrichment analysis (p&amp;lt;0.01). In addition, we found that BT-474 cells under hypoxia presented a higher mammosphere formation efficiency than non-hypoxic cells (p&amp;lt;0.05), showing a tendency towards a cancer stem cell phenotype. It is known that hypoxia represents a common feature of metastatic tumours and has a role in regulating stem cell behaviour. According to this, our results highlight a link between anti-HER2 therapy resistance and epithelial-to-mesenchymal transition characteristics, where hypoxia plays a key role which deserves further study. Citation Format: Virginia Judith Wolos, Ivana Jaqueline Tapia, Marianela Abrigo, Ezequiel Lacunza, Elisa Dora Bal de Kier Joffé, Gabriel León Fiszman. Hypoxic microenvironment promotes resistance to targeted therapies in HER2-overexpressing breast cancer involving epithelial-to-mesenchymal transition features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1078.

Abstract 2626: Exosomes derived from the BT-20 human breast cancer cell line express E-selectin ligands

Abstract Exosomes are small extracellular vesicles (40-150 nm in diameter) secreted by cells and initially proposed as a mechanism to eliminate cellular waste. More recently, exosomes have been reported to participate in cancer metastasis because of their ability to mediate local and distant cell-to-cell communication. Furthermore, exosomes create cancer-favored microenvironments, potentially enhancing cancer metastasis. However, it is unknown whether exosomes interact with E-selectin, an adhesion molecule expressed by vascular endothelium and shown to be involved in metastasis. Our lab has previously shown that BT-20 human breast cancer cells adhesively interact with endothelial E-selectin via several E-selectin ligands such as sialofucosylated CD44 variants, Mac-2BP, and gangliosides. Therefore, we hypothesize that exosomes secreted by the BT-20 human breast cancer cell line interact with E-selectin. To investigate this hypothesis, exosomes were generated by culturing cells in serum-depleted media and isolated by a series of ultracentrifugation steps. Molecular size analysis using dynamic light scattering revealed that the size range of the isolated particles was similar to the expected size of exosomes. To detect CD9 (an exosome marker) and E-selectin ligands, both breast cancer whole cell lysate and exosome lysate were tested by Western blot. The higher expression of CD9 in isolated particles than the whole cell lysates confirmed that the isolated particles were exosomes. When Western blots were labeled with recombinant E-selectin/Fc chimera with Ca2+, bands were detected at approximately 130 kDa, indicating the presence of E-selectin ligands. As a negative control, the divalent cation chelator EDTA was added to the primary labeling incubation. Bands were not detected in these negative control experiments, indicating no E-selectin interactions in the absence of Ca2+. Human IgG (hIgG) was also used as a negative control in Western blots and showed no signal with exosome lysates, as expected. In comparison, experiments were also performed with Hs578T breast cancer cells, a cell line that our lab has shown does not express E-selectin ligands. Exosomes isolated from Hs578T cells did not express E-selectin ligands, consistent with whole cells. Altogether, these results suggest that BT-20 human breast cancer exosomes express E-selectin ligands. Future work will focus on identifying and characterizing the exosome E-selectin ligands, as well as investigating other cancer cells that express E-selectin ligands. Ultimately, understanding the interaction between exosomes and E-selectin can lead to new diagnostic and therapeutic strategies for cancer metastasis. Citation Format: Yinan Huang, Christian A. Showalter, Monica M. Burdick. Exosomes derived from the BT-20 human breast cancer cell line express E-selectin ligands [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2626.

Abstract 6352: Potential anticancer role of secretion modification region (SMR) peptide reduces mortalin and HER2-overexpressing exosomes in human HER2-positive breast cancer cells

Abstract Background/Significance: Exosomes are 30-100 nm membrane vesicles secreted by many cell types. Tumors secrete large numbers of exosomes that transport oncoproteins and immune suppressive molecules, supporting tumor growth and metastasis. The role of exosomes in intercellular signaling is shown by human epidermal growth factor receptor type 2 (HER2)-expressing breast tumors, where exosomes with HER2 stimulate tumor growth and interfere with activity of the therapeutic antibody Herceptin, which binds to the extracellular domain of HER2 and prolongs the overall survival of HER2-positive breast cancer patients. Exosome release is modulated by growth factors EGF and heregulin, HER2 receptor-activating ligands naturally present in the surrounding tumor microenvironment. The SMRwt peptide reduces secretion of HER2 exosomes; thus, it can potentially be exploited as an anticancer agent. Methods: BT-474 and HCC1569 HER2-positive breast cancer cells overexpressing HER2 and mortalin were used in these experiments. Exosome size and concentration were measured by NanoSight. Purified tumor exosomes were prepared using paramagnetic beads coated with antibodies against tumor specific proteins and confirmed by flow cytometry and electron microscopy. To identify biological functions, we cultured tumor exosomes with breast cancer cells, which increased tumor cell proliferation. To determine SMRwt peptide inhibition effects, we assayed tumor exosome secretion and cell proliferation by NanoSight, Western blot and MTT. Results: We characterized exosomes secreted by mortalin and HER2-overexpressing breast cancer cell lines and analyzed SMRwt peptide's role in reducing tumor exosome release and cell proliferation. Exosome secretion increased in HER2-overexpressing tumor cell lines HCC1596 and BT474. Exosomes from HER2-positive tumor cell-conditioned supernatants stimulated tumor cell growth and tumor exosome release and inhibited Herceptin activity on HCC1569 and BT474 cell proliferation. Addition of SMRwt peptide reduced tumor cell proliferation and tumor exosome release. Conclusions: These data show the role of HER2-positive exosomes in modulating sensitivity to Herceptin and thus, to HER2-driven tumor aggressiveness; and indicate that the SMRwt peptide has potential anticancer functions in HER2-positive HCC1569 and BT474 human breast cancer cells. The data also indicate tumor exosomes affect the proliferation of parental cells. Tumor exosomes derived from BT-474 cells and HCC1569 cells stimulated the proliferation of BT-474 cells and HCC1569 cells, suggesting a biological function for tumor exosomes. Citation Format: Ming Bo Huang. Potential anticancer role of secretion modification region (SMR) peptide reduces mortalin and HER2-overexpressing exosomes in human HER2-positive breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6352.

Treatment of ErbB2 breast cancer by mitochondrial targeting

BackgroundErbB2 breast cancer still remains an unmet need due to primary and/or acquired resistance to current treatment strategies. MEDICA compounds consist of synthetic long-chain α,ω-dicarboxylic acids previously reported to suppress breast cancer in PyMT transgenic mice.MethodsMEDICA efficacy and mode of action in the ErbB2 context was studied in ErbB2 transgenic mice and human breast cancer cells.ResultsMEDICA treatment is shown here to suppress ErbB2 breast tumors and lung metastasis in ErbB2/neu MMTV transgenic mice, to suppress ErbB2/neu xenografts in nod/scid mice, and to suppress survival of AU565 and BT474 human ErbB2 breast cancer cells. Suppression of ErbB2 breast tumors by MEDICA is due to lipid raft disruption with loss of ErbB family members, including EGFR, ErbB2, and ErbB3. In addition, MEDICA inhibits mTORC1 activity, independently of abrogating the ErbB receptors and their signaling cascades. The double hit of MEDICA in abrogating ErbB and mTORC1 is partly accounted for by targeting mitochondria complex I.ConclusionsMitochondrial targeting by MEDICA suppresses ErbB2 breast tumors and metastasis due to lipid raft disruption and inhibition of mTORC1 activity. Inhibition of mTORC1 activity by MEDICA avoids the resistance acquired by canonical mTORC1 inhibitors like rapalogs or mTOR kinase inhibitors.

The novel camptothecin derivative, CPT211, induces cell cycle arrest and apoptosis in models of human breast cancer

ObjectiveBreast cancer is the second leading cause of cancer deaths in women worldwide and represents a highly aggressive nature with limited therapeutic options; thus, investigating novel therapeutic agents for breast cancer is much needed. In this study, we investigated the anticancer effects of a novel camptothecin derivative, CPT211, against human breast cancer. MethodsWe used hormone receptor-positive MCF-7, triple-negative (TNBC) MDA-MB-231, and HER2-positive BT-474 human breast cancer cells to examine cytotoxicity of CPT211. We measured cell viability with dose dependence of CPT211 treatments by an MTT assay and investigated the potential underlying mechanism through flow cytometric and Western blot methods. Furthermore, we evaluated the efficacy of the treatment combination of CPT211 and doxorubicin in a mouse model bearing MDA-MB-231 xenografts. ResultsCPT211 treatment led to dose-dependent decreases in cell viability of both MCF-7 and MDA-MB-231 cells, but not BT-474 cells. Analysis of the underlying molecular mechanism revealed that CPT211 activated p53-mediated apoptosis, by triggering intrinsic and extrinsic apoptotic pathways in MCF-7 cells. Additionally, CPT211 induced apoptosis and cell cycle arrest of MDA-MB-231 cells by activating Fas/FADD/caspase-8 signaling, suggesting that CPT211-mediated MDA-MB-231 cell apoptosis may occur through an extrinsic apoptosis pathway. CPT211 treatment with doxorubicin in mice bearing MDA-MB-231 xenografts was shown to enhance caspase-8 and caspase-7 activation, resulting in significant inhibition of tumor growth. ConclusionsThese results indicate that Fas/FADD/caspase-8 activation plays an important role in CPT211-mediated tumor growth suppression in TNBC, and the novel camptothecin derivative, CPT211, can be exploited for specific targeted therapies and potentially improve approaches to combination treatments for human breast cancer.

Dual-Receptor-Targeted (DRT) Radiation Nanomedicine Labeled with 177Lu Is More Potent for Killing Human Breast Cancer Cells That Coexpress HER2 and EGFR Than Single-Receptor-Targeted (SRT) Radiation Nanomedicines.

Resistance to HER2-targeted therapies in breast cancer (BC) is associated in some cases with an increased expression of epidermal growth factor receptors (EGFR). We describe a dual-receptor-targeted (DRT) radiation nanomedicine for local intratumoral (i.t.) treatment of BC composed of 15 nm sized gold nanoparticles (AuNPs) modified with trastuzumab (TmAb) to target HER2 and panitumumab (PmAb) to target EGFR. The AuNPs were modified with poly(ethylene glycol) (PEG3k) linked to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelators to complex the β-particle emitter, 177Lu. Our aim was to compare the properties of these DRT-AuNP-177Lu with single-receptor-targeted (SRT)-TmAb-AuNP-177Lu or PmAb-AuNP-177Lu or nontargeted (NT)-AuNP-177Lu using human BC cells that expressed HER2, EGFR, or both receptors. To construct these radiation nanomedicines, PEG5K was linked to TmAb or PmAb, while PEG3k was linked to DOTA. These polymers were conjugated to AuNP via two Au-thiol bonds using a terminal lipoic acid (LA) group on the polymers. NT-AuNP-177Lu were constructed without modification with TmAb or PmAb. MDA-MB-231-H2N, MDA-MB-468, and BT-474 human BC cells were designated as HER2mod/EGFRmod, EGFRhigh/HER2neg, and HER2high/EGFRlow, respectively, based on the expression of these receptors. Specific binding to HER2 and/or EGFR was assessed by incubating BC cells with DRT-AuNP-177Lu or TmAb-AuNP-177Lu or PmAb-AuNP-177Lu, or NT-AuNP-177Lu in the absence or presence of an excess of TmAb or PmAb or both competitors. Binding and internalization of AuNP by BC cells were assessed by dark-field microscopy. Cell fractionation studies were conducted to quantify AuNP-177Lu bound and internalized. The cytotoxicity of DRT-AuNP-177Lu was determined in clonogenic survival (CS) assays after an exposure of 5 × 105 BC cells to 3 MBq (1.4 × 1012 AuNP) for 16 h and then seeding and culturing the cells for 7-15 days. CS was compared to exposure to TmAb-AuNP-177Lu and PmAb-AuNP-177Lu or NT-AuNP-177Lu. The absorbed doses to the nucleus in these CS assays were estimated. DRT-AuNP-177Lu were specifically bound by BC cells that expressed HER2 or EGFR or both receptors. In contrast, SRT-TmAb-AuNP-177Lu and PmAb-AuNP-177Lu were bound and internalized only by BC cells that expressed HER2 or EGFR, respectively. NT-AuNP-177Lu exhibited very low binding to BC cells. DRT-AuNP-177Lu and SRT-TmAb-AuNP-177Lu or PmAb-AuNP-177Lu were internalized by BC cells in accordance with the receptor expression. Importantly, DRT-AuNP-177Lu were more potent in vitro than PmAb-AuNP-177Lu for killing MDA-MB-231-H2N cells that coexpress HER2 and EGFR (CS = 18.8 ± 1.0 vs 51.5 ± 10.4%; P = 0.006). Furthermore, DRT-AuNP-177Lu were more potent for killing BT-474 cells with high HER2 but low EGFR expression than TmAb-AuNP-177Lu (CS = 8.9 ± 3.3 vs 20.7 ± 2.4%; P = 0.007) or PmAb-AuNP-177Lu (CS = 63.9 ± 1.7%; P < 0.0001). Even for MDA-MB-468 cells that overexpress EGFR but have negligible HER2, DRT-AuNP-177Lu were more potent for cell killing than PmAb-AuNP-177Lu (CS = 3.2 ± 3.0 vs 7.5 ± 1.8%; P = 0.001) or TmAb-AuNP-177Lu (63.2 ± 3.2%; P = 0.0002). All targeted forms of AuNP-177Lu were more cytotoxic to BC cells than those of NT-AuNP-177Lu. High absorbed doses (36-119 Gy) were deposited in the nucleus of BC cells by DRT-AuNP-177Lu. We conclude that a DRT radiation nanomedicine is more potent for killing BC cells that coexpress HER2 and EGFR than SRT radiation nanomedicines. These results are promising for further evaluation of these DRT-AuNP-177Lu in vivo for the local radiation treatment of human BC tumors that coexpress HER2 and EGFR in mice following i.t. injection, especially tumors that are resistant to HER2-targeted therapies.

Development and Characterization of Nanoliposomal Hydroxyurea Against BT-474 Breast Cancer Cells.

Purpose: Hydroxyurea (HU) is a well-known chemotherapy drug with several side effects which limit its clinical application. This study was conducted to improve its therapeutic efficiency against breast cancer using liposomes as FDA-approved drug carriers. Methods: PEGylated nanoliposomes-containing HU (NL-HU) were made via a thin-film hydration method, and assessed in terms of zeta potential, size, morphology, release, stability, cellular uptake, and cytotoxicity. The particle size and zeta potential of NL-HU were specified by zeta-sizer. The drug release from liposomes was assessed by dialysis diffusion method. Cellular uptake was evaluated by flow cytometry. The cytotoxicity was designated by methyl thiazolyl diphenyl-tetrazolium bromide (MTT) test. Results: The size and zeta value of NL-HU were gotten as 85 nm and -27 mV, respectively. NL-HU were spherical.NL-HU vesicles were detected to be stable for two months. The slow drug release and Weibull kinetic model were obtained. Liposomes considerably enhanced the uptake of HU into BT-474 human breast cancer cells. The cytotoxicity of NL-HU on BT-474 cells was found to be significantly more than that of free HU. Conclusion: The results confirmed these PEGylated nanoliposomes containing drug are potentially suitable against in vitro model of breast cancer.

Amyloid precursor protein promotes the migration and invasion of breast cancer cells by regulating the MAPK signaling pathway.

To verify whether amyloid precursor protein (APP) affects the migration and invasion of breast cancer cell lines, and to understand its underlying mechanisms, epithelial-mesenchymal transition (EMT), the mitogen-activated protein kinase (MAPK) signaling pathway and the matrix metalloproteinase (MMP) family were investigated in MDA-MB-231, MCF-7 and BT474 human breast cancer cells. Breast cancer cell lines were transfected with plasmids containing APP coding sequences (pEGFP-n1-APP) and APP short hairpin RNA (pENTR APP shRNA). APP overexpres-sion efficiency, knockout efficiency and the expression levels of related genes were tested using reverse transcription-quantitative PCR (RT-qPCR) and western blot analyses. The effects of APP and mitogen-activated protein kinase kinase (MEK) inhibitor on cell migration and invasion were examined using Transwell assays. The results demonstrated that APP was significantly upregulated in the pEGFP-n1-APP group (P<0.05), and significantly downregulated in the pENTR APP shRNA group (P<0.05), compared with the control group. APP overexpression increased the migratory and invasive ability of human breast cancer cells (P<0.05), whereas APP silencing significantly inhibited cell migration and invasion (P<0.05). RT-qPCR and western blot analysis results suggested that APP overexpression significantly increased the expression of MMP-9, MMP-2, MMP-3, N-cadherin and vimentin (P<0.05). In addition, the enhanced expression of APP markedly affected the phosphorylation of mitogen-activated protein kinase kinase kinase 11 (MLK3), mitogen-activated protein kinase kinase 4 (MEK4) and mitogen-activated protein kinase 10 (JNK3; P<0.05). Additionally, APP overexpression had no effect on the total expression levels of MLK3, MEK4, and JNK3; however, APP overexpression significantly decreased the expression levels of E-cadherin and cytokeratin (P<0.05). Conversely, APP silencing had the opposite effects. When cells were treated with the MEK inhibitor PD0325901, the expression of APP was not altered, nor was the expression levels of MEK and its upstream signaling molecules. Taken together, the present findings suggested that APP could affect the migration and invasion of human breast cancer cells by mediating the activation of the MAPK signaling pathway, thereby promoting the EMT process.

Abstract 4614: Establishment of a HER2 positive breast cancer bone metastasis model for validation of novel therapies

Abstract Breast cancers with overexpression of human epidermal growth factor receptor 2 (HER2+) have aggressive clinical behavior, and at advanced stages are associated with increased risk for developing metastases to distant organs including bones, brain and lungs. The aim of this study was to establish a systemic metastasis model for HER2+ breast cancer with a special interest in bone metastasis. The effects of cell number, estrogen supplementation and mouse strain on metastasis formation were studied. In the study, 5-6 weeks old athymic nude and Rag2 mice (n=10-13 per group) were used. Half of the mice received estrogen supplementation (E2-releasing rods 5µg/day) one week before inoculation of the cancer cells. The mice were inoculated intracardially with 1 or 5 x 105 luciferase-labelled triple-positive (ER, PR positive and HER2 overexpressing) human BT-474 breast cancer cells. The formation of metastases was followed by bioluminescence imaging (BLI) at inoculation and once a week for the duration of the study. At sacrifice, X-ray imaging was performed and the bones were collected for histological analysis. The formation of bone metastases was observed in all study groups, and bone metastases were dominant in the model. Based on BLI, bone metastases developed in nude mice earlier than in Rag2 mice. However, detection of metastases in Rag2 mice was challenging due to dark fur hindering the signal transmittance. The bone metastases appeared between 7 to 36 days after inoculation of the cancer cells in nude mice and between 20 to 43 days in Rag2 mice. The number of bone metastases was higher in nude mice. E2 supplement accelerated the development of bone metastases in both mouse strains. In nude mice with E2 supplement, bone metastases appeared between 7 to 14 days compared to between 20 to 36 days in mice without E2 supplement. In Rag2 mice with E2 supplement bone metastases formed between 20 to 28 days compared to between 36 to 43 days without E2 supplement. The use of higher cell number accelerated the development of bone metastases but had no major effect on their incidence in both mouse strains. Take rate of bone metastases was 90-100% in nude mice with E2 supplement compared to 30-50% without E2 supplement. In Rag2 mice, the take rate of bone metastases was 50% with E2 supplement. X-ray imaging showed estrogen induced bone growth and large tumor-induced osteolytic lesions in the hind limbs in both strains. Due to the extensive bone lesions and occasional fractures, the first mice were sacrificed 50 days after inoculation of the cancer cells. Both mouse strains occasionally developed metastases in soft tissues including brain and ovaries. In conclusion, a high rate of bone metastasis was achieved in athymic nude mice supplemented with E2. This model can be used to study the efficacy of anti-cancer, such as HER2-targeted, compounds on tumor growth at metastatic locations or on the prevention of metastasis formation. Citation Format: Tiina E. Kähkönen, Mari I. Suominen, Jenni H. Mäki-Jouppila, Jussi M. Halleen, Jenni Bernoulli, Derek Grant. Establishment of a HER2 positive breast cancer bone metastasis model for validation of novel therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4614.

Human Immune System Increases Breast Cancer-Induced Osteoblastic Bone Growth in a Humanized Mouse Model without Affecting Normal Bone.

Bone metastases are prevalent in many common cancers such as breast, prostate, and lung cancers, and novel therapies for treating bone metastases are needed. Human immune system-engrafted models are used in immuno-oncology (IO) studies for subcutaneous cancer cell or patient-derived xenograft implantations that mimic primary tumor growth. Novel efficacy models for IO compounds on bone metastases need to be established. The study was performed using CIEA NOG (NOG) mice engrafted with human CD34+ hematopoietic stem cells (huNOG) and age-matched immunodeficient NOG mice. Bone phenotyping was performed to evaluate baseline differences. BT-474 human breast cancer cells were inoculated into the tibia bone marrow, and cancer-induced bone changes were monitored by X-ray imaging. Bone content and volume were analyzed by dual X-ray absorptiometry and microcomputed tomography. Tumor-infiltrating lymphocytes (TILs) and the expression of immune checkpoint markers were analyzed by immunohistochemistry. Bone phenotyping showed no differences in bone architecture or volume of the healthy bones in huNOG and NOG mice, but the bone marrow fat was absent in huNOG mice. Fibrotic areas were observed in the bone marrow of some huNOG mice. BT-474 tumors induced osteoblastic bone growth. Bone lesions appeared earlier and were larger, and bone mineral density was higher in huNOG mice. huNOG mice had a high number of human CD3-, CD4-, and CD8-positive T cells and CD20-positive B cells in immune-related organs. A low number of TILs and PD-1-positive cells and low PD-L1 expression were observed in the BT-474 tumors at the endpoint. This study reports characterization of the first breast cancer bone growth model in huNOG mice. BT-474 tumors represent a “cold” tumor with a low number of TILs. This model can be used for evaluating the efficacy of combination treatments of IO therapies with immune-stimulatory compounds or therapeutic approaches on bone metastatic breast cancer.

SYNTHESIS OF 2-((5-BENZYLIDENE-4-OXO-4,5-DIHYDROTHIAZOL-2-YL)-SUBSTITUTED AMINO ACIDS AS ANTICANCER AND ANTIMICROBIAL AGENTS

A series of rhodanine derivatives were synthesized by Knoevenagel condensation. All the synthesized compounds were tested for their in vitro anticancer activity against MCF-7 and BT-474 human breast cancer cell lines. All the synthesized compounds were characterized and screened for their antimicrobial activity against bacterial and fungal strain. Majority of the compounds showed good to moderate anticancer and antimicrobial activity. Among these compounds one showed promising activity against gram-positive bacteria B. subtilis and S. aureus when compared with ampicillin. Some of the most potent compounds possessed selective antimicrobial activity.

NEW THIAZOLONE DERIVATIVES: DESIGN, SYNTHESIS, ANTICANCER AND ANTIMICROBIAL ACTIVITY

In order to explore the anticancer and antimicrobial activity associated with the thiazolone framework, several new (Z)-2-((5-(3-fluorobenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl)amino)carboxylic acid derivatives have been synthesized in water as a solvent. All synthesized compounds were evaluated for anticancer and antimicrobial activity in vitro. Amongst these, the 3-methylbutanoic and the 3- or 4-methylpentanoic acid derivatives, the 3-hydroxy-, the 3-(1H-imidazol-4-yl) and the 3-(4-hydroxyphenyl)propanoic acid derivatives and the succinic acid derivative showed high antibacterial and antifungal activity. The unsubstituted propanoic acid derivative exhibited significant antibacterial activity against B. subtilis and significant antifungal activity against fungal strains, i.e., A. flavus . The in vitro anticancer studies revealed that the 3-(hydroxy)-, the 3-(1H-imidazol-4yl)- and the 3-(4-hydroxyphenyl)propanoic acid, or the succinic acid derivatives are the most active compounds against MCF-7 and BT-474 human breast cancer cell lines.