MCF-7 Breast Cancer Stem Cells Research Articles

Investigation of the effect of belinostat on MCF-7 breast cancer stem cells via the Wnt, Notch, and Hedgehog signaling pathway.

To evaluate belinostat's (PXD101) activity on MCF-7 breast cancer stem cells (CSCs) via Wnt, Notch, and Hedgehog. This research study was carried out at the Department of Medical Biology, Necmettin Erbakan University, Konya, Turkey, from June 2017 to July 2019. The effect of PXD101 on MCF-7 cell viability was determined by cell proliferation kit (XTT). Following belinostat treatment, CD44+/CD24- MCF-7 CSCs were isolated by FACS. Ribonucleic acid isolation and copy-deoxyribonucleic acid synthesis were carried out using HEK-293 cells, MCF-7 cells, and MCF-7 CSCs. Expression changes of metastasis-related genes, Wnt, Hedgehog, Notch, and stem cell markers were analysed by quantitative polymerase chain reaction. The IC50 in MCF-7 cancer cells was 5 μM for 48 hours. The FACS analysis indicated that 2% of the MCF-7 cancer cells were CSCs. Following belinostat treatment, the MCF-7 cell count decreased by 44%, and the MCF-7 CD44+/CD24- CSC count decreased by 66%. Belinostat treatment reduced the expression of metastasis, Wnt, Notch, Hedgehog, and stem cell marker genes. Belinostat has a potential effect on the differentiation and self-renewal of breast CSCs.

Effects of Combined Exposure to Carbon Ions and Protons on the Pool of MCF-7 Breast Cancer Stem Cells In Vitro.

We studied the effects of single and combined action of protons and carbon ions 12C6+ on the pool of MCF-7 human breast cancer stem cells. Single irradiation with a beam of protons or carbon ions had no significant effects on the relative number of cancer stem cells (CSC). The effects of combined irradiation in a total equieffective dose of 4 Gy depended on the sequence of exposure to ionizing radiations: the relative number of CSC did not change after irradiation with carbon ions and then with protons, but increased in the case of the reverse sequence. The most favorable result, i.e. a decrease in the CSC pool, was observed in the case of sequential irradiation with carbon ions and protons and their equal contribution to total equieffective dose. In this case, the absolute number of CSC decreased by on average 2.1 times in comparison with the control (p<0.05). The revealed regularities are of interest for the further development of new methods of radiation therapy.

Effects of DNA-binding ligands from dimeric bisbenzimidazoles series DBA(n) and DBPA( n) in combination with γ-radiation on epithelial-mesenchymal transition and pool size of MCF-7 breast cancer stem cells

Radiation therapy is one of the main treatments for malignant neoplasms, including breast cancer. However, it is known, that radiation therapy can lead to an increase in the number of cancer stem cells, which are resistant to traditional antitumor effects, and are believed to be responsible for the development of recurrences and metastases. Therefore, the development of cancer stem cells elimination means is of considerable interest, especially in combination with ionizing radiation. The effects of single and combined exposure to new series of minor-groove DNA ligands - dimeric bisbenzimidazoles - DBA(n) and DBPA(n) (where n is the number of methylene groups between two bisbenzimidazole blocks) and Y-radiation on human Breast cancer cells were studied in our work for MCF-7 line in vitro. Compounds with maximum cytotoxic effect and cell binding were selected, and then the effects of the latter on the CD44+CD24-/low cancer stem cells population and radiation-induced epithelial-mesenchymal transition were studied by the criterion of vimentin expression. An increase in the expression level of this protein and simultaneously in the relative number of cancer stem cells after a single exposure of Y-radiation at a dose of 4 Gy were shown. DBPA(1,4) in combination with irradiation blocked the radiation-induced vimentin expression and decreased the relative number of cancer stem cells by 1.7 and 4.1 times compared with irradiation (p = 0.041 and p = 0.005), respectively. At the same time, the absolute number of cancer stem cells decreased by 2.8 and 12.0 times compared to irradiation (p = 0.029 and p = 0.004), respectively. In contrast, DBA(5,7) by itself or in combination with Y-irradiation increased the level of vimentin expression and the same compounds when combined with irradiation increased the relative number of cancer stem cells by 3.1 and 3.6 times (p = 0.006 and p = 0.005), respectively, compared with irradiation. The absolute number of cancer stem cells increased by 2.2 and 1.5 times (p = 0.017 and p = 0.032), respectively. The data obtained show a close relationship between epithelial-mesenchymal transition and formation of the cancer stem cells pool after radiation exposure, and also indicate the prospects for further study of DBPA(1,4) as a means of cancer stem cells elimination under in vivo conditions.

The Effect of DNA-Binding Ligands from Dimeric Bisbenzimidazoles of the DBA(n) and DBPA(n) Series in Combination with γ-Radiation on Epithelial–Mesenchymal Transition and Pool Size of MCF-7 Breast Cancer Stem Cells

The Effect of DNA-Binding Ligands from Dimeric Bisbenzimidazoles of the DBA(n) and DBPA(n) Series in Combination with γ-Radiation on Epithelial–Mesenchymal Transition and Pool Size of MCF-7 Breast Cancer Stem Cells

3D bioprinted drug-resistant breast cancer spheroids for quantitative in situ evaluation of drug resistance

Drug-resistant cancer spheroids were fabricated by three-dimensional (3D) bioprinting for the quantitative evaluation of drug resistance of cancer cells, which is a very important issue in cancer treatment. Cancer spheroids have received great attention as a powerful in vitro model to replace animal experiments because of their ability to mimic the tumor microenvironment. In this work, the extrusion printing of gelatin-alginate hydrogel containing MCF-7 breast cancer stem cells successfully provided 3D growth of many single drug-resistant breast cancer spheroids in a cost-effective 3D-printed mini-well dish. The drug-resistant MCF-7 breast cancer spheroids were able to maintain their drug-resistant phenotype of CD44high/CD24low/ALDH1high in the gelatin-alginate media during 3D culture and exhibited higher expression levels of drug resistance markers, such as GRP78 chaperon and ABCG2 transporter, than bulk MCF-7 breast cancer spheroids. Furthermore, the effective concentration 50 (EC50) values for apoptotic and necrotic spheroid death could be directly determined from the 3D printed-gelatin-alginate gel matrix based on in situ 3D fluorescence imaging of cancer spheroids located out of the focal point and on the focal point. The EC50 values of anti-tumor agents (camptothecin and paclitaxel) for apoptotic and necrotic drug-resistant cancer spheroid death were higher than those for bulk cancer spheroid death, indicating a greater drug resistance. Statement of significanceThis study proposed a novel 3D bioprinting-based drug screening model, to quantitatively evaluate the efficacy of anticancer drugs using drug-resistant MCF-7 breast cancer spheroids formed within a 3D-printed hydrogel. Quantitative determination of anticancer drug efficacy using EC50, which is extremely important in drug discovery, was achieved by 3D printing that enables concurrent growth of many single spheroids efficiently. This study verified whether drug-resistant cancer spheroids grown within 3D-printed gelatin-alginate hydrogel could maintain and present drug resistance. Also, the EC50 values of the apoptotic and necrotic cell deaths were directly acquired in 3D-embedded spheroids based on in situ fluorescence imaging. This platform provides a single-step straightforward strategy to cultivate and characterize drug-resistant spheroids to facilitate anticancer drug screening.

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.

Influence of high-molecular hyaluronic acid on MCF-7 breast cancer stem cells

In recent years, more and more data have been accumulating on the high radio- and chemo-resistance of tumor stem cells (CSCs), confirming the assumption that these cells play an important role in the recurrence of the tumor process in some cancer patients. Therefore it is of considerable interest to elucidate the patterns and mechanisms of the formation of the CSC pool under the influence of various factors, including one of the main components of the extracellular matrix, hyaluronic acid (HA). The purpose of the study was to elucidate the effects of HA with a molecular weight of (1-3) x 106 Dа on the amount of CSCs in MCF-7 breast cancer cell culture in vitro. Material and methods. Using the MTT test, the viability of MCF-7 cancer cells was determined after incubation with HA at a concentration of 0.3-5.0 mg/ml. Using flow cytometry, we evaluated the change in the relative and absolute amount of CSCs under the influence of HA (0.6 mg/ml). CSCs were detected by CD44+CD24-/low immunophenotype, as well as by their ability to efficiently efflux Hoechst 33342 fluorescent dye from cells and form so-called side population (SP). Results. Viability of cancer cells in the general population decreased with increasing concentration of HA (R=-0.94, p=0.006). At the same time the incubation of cells with HA led to an increase in both the relative and absolute amounts of CSCs (SP) compared with the control. Thus, the relative number of SP cells increased by 2.0 times (p = 0.002), CD44 + CD24- / low cells - by 6.1 times (p = 0.005). The absolute number of these cells changed to a lesser extent compared to the control: the increase was 1.6 (p = 0.004) and 2.9 times (p = 0.02), respectively. Conclusion. Thus, the effect of high-molecular weight HA on breast cancer cells of MCF-7 line leads to an increase in amount of CSCs, while the viability of non-stem cancer cells decreases under the same conditions.

Abstract 3795: Investigate the mechanism of actions of flavonoid dimers in reversing doxorubicin resistance in breast cancer stem cells

Abstract Drug resistance, radioresistance and metastasis are the biggest hurdle in effective cancer treatment. Recent studies suggest that these problems might be caused by the presence of cancer stem cells (CSCs). CSCs are a small group of cells which can self-renew, remain undifferentiated, resist to cancer drugs and radiotherapy. It can be predicted that selective killing of CSCs may lead to tutor regression and may be a new therapeutic solution. Naturally occurring flavonoids are weakly active in inhibiting ABC transporter leading to drug accumulation in cancer cells. We have been working on the design, synthesis and characterisation of novel flavonoid dimers that can be used to reverse drug resistance in cancer cells. We recently discovered that a new class of triazole-linked flavonoid dimers 13_9 and 15_2 were effective in reversing drug resistance in CD44+CD24-/low MCF7 breast cancer stem cells at an effective concentration of less than 32 nM. Our studies suggested that a novel mechanism was involved which is independent of ABC transporters. To determine the mechanism behind, we have designed and synthesised a photoactivatable derivative of 13_9 (13_9X2) to identify the protein target(s). 13_9X2 contains a photoactivating functional group which can capture the target protein by forming a covalent bond under UV irradiation and a clickable functional group which can be used for pull-down and further identification using mass spectrometry. One candidate target is peroxiredoxin 1 (PRDX-1) which is a reactive oxygen species (ROS) scavenger. We hypothesise that CSCs are resistant to doxorubicin-induced ROS damage by overexpressing PRDX-1. By binding and disrupting PRDX-1, 13_9 and 15_2 reduce ROS scavenging ability of PRDX-1, resulting in the reversal of drug resistance in CSCs. This work provides new insights into the mechanisms of cell signalling regulation of cancer cell resistance in breast CSCs and provides a better understanding of how flavonoid dimers reverse drug resistance in CSCs. Additionally, this work contributes to the development of new anticancer drugs which can sensitize CSCs to current chemotherapy. Citation Format: Gege Sun, Iris L. Wong, Jiahua Cui, Zhen Liu, Bill T. Chan, Larry M. Chow. Investigate the mechanism of actions of flavonoid dimers in reversing doxorubicin resistance in breast cancer stem 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 3795.

C-Src functionality controls self-renewal and glucose metabolism in MCF7 breast cancer stem cells.

Deregulation of Src kinases is associated with cancer. We previously showed that SrcDN conditional expression in MCF7 cells reduces tumorigenesis and causes tumor regression in mice. However, it remained unclear whether SrcDN affected breast cancer stem cell functionality or it reduced tumor mass. Here, we address this question by isolating an enriched population of Breast Cancer Stem Cells (BCSCs) from MCF7 cells with inducible expression of SrcDN. Induction of SrcDN inhibited self-renewal, and stem-cell marker expression (Nanog, Oct3-4, ALDH1, CD44). Quantitative proteomic analyses of mammospheres from MCF7-Tet-On-SrcDN cells (data are available via ProteomeXchange with identifier PXD017789, project DOI: 10.6019/PXD017789) and subsequent GSEA showed that SrcDN expression inhibited glycolysis. Indeed, induction of SrcDN inhibited expression and activity of hexokinase, pyruvate kinase and lactate dehydrogenase, resulting in diminished glucose consumption and lactate production, which restricted Warburg effect. Thus, c-Src functionality is important for breast cancer stem cell maintenance and renewal, and stem cell transcription factor expression, effects linked to glucose metabolism reduction.

Targeting Tumorigenicity of Breast Cancer Stem Cells Using SAHA/Wnt-b Catenin Antagonist Loaded Onto Protein Corona of Gold Nanoparticles.

BackgroundAmong various theories for the origin of cancer, the “stemness phenotype model” suggests a dynamic feature for tumor cells in which non-cancer stem cells (non-CSCs) can inter-convert to CSCs. Differentiation with histone-deacetylase inhibitor, vorinostat (SAHA), can induce stem cells to differentiate as well as enforces non-CSCs to reprogram to CSCs. To avoid this undesirable effect, one can block the Wnt-βcatenin pathway. Thus, a dual delivery system of SAHA and a Wnt-βcatenin blocker will be beneficial in the induction of differentiation of CSCs. Protein corona (PC) formation in nanoparticle has a biologic milieu, and despite all problematic properties, it can be employed as a medium for dual loading of the drugs.Materials and MethodsWe prepared sphere gold nanoparticles (GNPs) with human plasma protein corona loaded with SAHA as differentiating agent and PKF118-310 (PKF) as a Wnt-βcatenin antagonist. The MCF7 breast cancer stem cells were treated with NPs and the viability and differentiation were evaluated by Western blotting and sphere formation assay.ResultsWe found that both drugs loaded onto corona-capped GNPs had significant cytotoxicity in comparison to bare GNP-corona. Data demonstrated an increase in stem cell population and upregulation of mesenchymal marker, Snail by SAHA-loaded GNPs treatment; however, the combination of PKF loaded GNPs along with SAHA-loaded GNPs resulted in a reduction of stem cell populations and Snail marker. We have shown that in MCF7 and its CSCs simultaneous treatment with SAHA and PKF118-310 induced differentiation and inhibition of Snail induction.ConclusionOur study reveals the PC-coated GNPs as a biocompatible career for both hydrophilic (PKF) and hydrophobic (SAHA) agents which can decrease breast cancer stem cell populations along with reduced stemness state regression.

Dimeric bisbenzimidazoles DB(n) in combination with ionizing radiation decrease number and clonogenic activity of MCF-7 breast cancer stem cells

Purpose of the study was search for agents to eliminate the pool of cancer stem cells (CSCs) and increase their radiosensitivity among DNA minor groove binding ligands, synthetic dimeric bisbenzimidazoles of the DB(n) series, where n is the number of methylene groups in the linker connecting two bisbenzimidazole residues (n = 1, 3, 5, 7, 9, 11). The investigation was concerned with MCF-7 breast cancer cells in vitro. Six compounds were synthesized, and their binding to cells and subcellular distribution were studied using flow cytometry and laser scanning microscopy. DB(5) and DB(7) demonstrated the highest binding and accumulation in cell nuclei and were selected for studying single and combined effects with γ-radiation at a dose of 4 Gy on number, clonogenic activity of CSCs and expression of vimentin, one of the major markers of epithelial to mesenchymal transition (EMT). Radiation exposure of MCF-7 cells increased the relative and absolute numbers of CD44+СD24−/low CSCs by a factor of 1.7 (р = 0.04) and 1.6 (р = 0.008), respectively, compared with the control. The combined exposure to DB(5) or DB(7) and γ-radiation led to a considerable reduction of CSC pool compared to control and single irradiation. Particularly, the CSC absolute number decreased by a factor of 16.6 and 14.1, respectively, after combined exposure compared to the radiation alone (р = 0.006 in both cases). The combined effects on the clonogenic activity were synergistic in case of CSCs, and additive or subadditive in case of non-CSCs. The combined exposure to DB(5) or DB(7) and γ-radiation resulted in inhibiting the radiation-induced EMT. Thus, DB(5, 7) were capable of removing the stimulating effect of γ-radiation on the CSC population. The suppression of radiation-induced EMT under the influence of these compounds caused a considerable reduction of CSC pool compared to both radiation and control.

Effects of Dimeric Bisbenzimidazoles and Ionizing Radiation on MCF-7 Breast Cancer Stem Cells

Abstract—This study aimed to determine how to eliminate and/or increase the radiosensitivity of a population of cancer stem cells that are responsible for radioresistance in malignant neoplasms. The single and combined effects of γ-radiation at a dose of 4 Gy and dimeric bisbenzimidazoles of the DB(n) series on the cancer stem cells have been studied using the MCF-7 human breast cancer cell line. The DB(n) molecules contain n (five or seven) methylene groups in the linker between two benzimidazoles. It has been shown that DB(5) significantly reduces the relative and absolute numbers of the cancer stem cells by factors of 5.8 and 7.5, respectively, compared to the control (p = 0.001 in both cases), and DB(7) reduces the above cell numbers by factors of 3.6 (p = 0.003) and 5.2 (p = 0.004), respectively. Irradiation leads to an increase of the relative and absolute numbers of cancer stem cells by factors of 1.7 and 1.6 (p < 0.05) compared to the control, respectively. The combined effects of DB(5, 7) and ionizing radiation led to a significant decrease in the relative number of cancer stem cells by factors of 8.2 and 4.1, respectively, compared to only the single irradiation (p = 0.003 and p = 0.004). Even more pronounced outcomes of the combined action of DB(5, 7) and radiation have been found with regard to the absolute number of cancer stem cells, which decreases by factors of 16.6 and 14.1, respectively, compared to those after only single irradiation (p = 0.006 in both cases). The combined effect of DB(5, 7) and ionizing radiation led to the inhibition of the radiation-induced epithelial-mesenchymal transition as shown by the expression of vimentin, which is one of the markers of this process that plays an important role in the formation of cancer stem cells pool.

Targeting breast cancer stem cells by a self-assembled, aptamer-conjugated DNA nanotrain with preloading doxorubicin.

BackgroundCancer relapse and metastasis is an obstacle to the treatment of breast cancer. Breast cancer stem cells (BCSCs), which can evade the killing effect of traditional chemotherapies, such as doxorubicin (DOX), may contribute to cancer development. Therefore, it is necessary to develop novel drugs that can target and eliminate BCSCs. While multiple strategies have been conceived, they are normally limited by the low drug loading capacity.PurposeAn aptamer-conjugated DNA nanotrain TA6NT-AKTin-DOX, which consists of a CD44 aptamer TA6, DNA building blocks M1 and M2 conjugated with an AKT inhibitor peptide AKTin individually and DOX, was designed.MethodsThis DNA nanotrain was prepared through hybridization chain reactionand this highly ordered DNA duplex has plenty of sites where DOX and AKTin can be intercalated or anchored. By performing on MCF-7 BCSCs and tumors by xenografting BCSCs into nude mice, efficacy of the newly prepared drug was evaluated and compared with that of free DOX and various DNA nanotrains.ResultsTA6NT-AKTin-DOX showed better efficacy both in vitro and in vivo. To some extent, the enhanced efficacy could be attributed to the targeting effect of TA6 and the high drug loading capacity of the nanotrain (~20 DOX molecules). Besides, a synergistic response was demonstrated by combining DOX with AKTin, probably due to that the anchored AKTin can reverse the drug resistance of BCSCs including apoptosis resistance and ABC transporters overexpression via the AKT signaling pathway.ConclusionThe aptamer-conjugated DNA nanotrain TA6NT-AKTin-DOX demonstrated its targeting capability to BCSCs.

Ribosome biogenesis mediates antitumor activity of flavopiridol in CD44+/CD24‑ breast cancer stem cells

Flavopiridol is a synthetically produced flavonoid that potently inhibits the proliferation of human tumor cell lines. Flavopiridol exerts strong antitumor activity via several mechanisms, including the induction of cell cycle arrest and apoptosis, and the modulation of transcriptional regulation. The aim of the present study was to determine the effect of flavopiridol on a subpopulation of cluster of differentiation (CD)44+/CD24− human breast cancer MCF7 stem cells. The CD44+/CD24− cells were isolated from the MCF7 cell line by fluorescence-activated cell sorting and treated with 100, 300, 500, 750 and 1,000 nM flavopiridol for 24, 48 and 72 h. Cell viability and proliferation assays were performed to determine the inhibitory effect of flavopiridol. Gene expression profiling was analyzed using Illumina Human HT-12 v4 Expression BeadChip microarray. According to the results, the half maximal inhibitory concentration (IC50) value of flavopiridol was 500 nM in monolayer cells. Flavopiridol induced growth inhibition and cytotoxicity in breast cancer stem cells (BCSCs) at the IC50 dose. The present study revealed several differentially regulated genes between flavopiridol-treated and untreated cells. The result of the pathway analysis revealed that flavopiridol serves an important role in translation, the ribosome biogenesis pathway, oxidative phosphorylation, the electron transport chain pathway, carbon metabolism and cell cycle. A notable result from the present study is that ribosome-associated gene expression is significantly affected by flavopiridol treatment. The data of the present study indicate that flavopiridol exhibits antitumor activity against CD44+/CD24− MCF7 BCSCs through different mechanisms, mainly by inhibiting translation and the ribosome biogenesis pathway, and could be an effective chemotherapeutic molecule to target and kill BCSCs.

Antibiotics target MCF-7 breast cancer stem cells in hypoxic environment.

e14068 Background: Deregulated cellular metabolism is characteristic for cancer cells which heavily rely on glycolysis to meet metabolic demands. Studies have shown that increased glucose metabolism promotes tumor cell survival through mechanisms related to mitochondrial activities. Recently, mitochondrial inhibitors have been proposed as a potential anticancer therapeutics. In 2015 Lamb et al. presented anti-cancer activity of several antibiotics targeting mitochondrial function. Interestingly, these drugs have the highest inhibitory effect on cancer stem cells (CSCs), a small population of cancer initiating cells. It is believed that tumor microenvironment plays significant role in maintaining CSC population. The hypoxia is a major feature of the tumor microenvironment contributing into CSC phenotype, tumorigenicity and metastasis. Therefore, we sought to determine the effect of five antibiotics targeting mitochondrial function on CSC survival in hypoxia conditions. Methods: MCF-7 cells were cultured in normoxic (20% O2) and hypoxic (4% O2) conditions for 14 days in a presence of azithromycin, doxycycline, tetracycline, erythromycin and chloramphenicol. The ability to form mammospheres was used to determine CSC survival. Results: Incubation of CSCs with erythromycin, doxycycline, tetracycline and chloramphenicol reduced the number of mammospheres, the decrease in mammosphere number was comparable for MCF-7 cells incubated under normoxic and hypoxic conditions [erythromycin (62.5 % ± 10.8% vs. 58.9% ± 2.2% ), doxycycline (75.2% ± 5.9% vs. 68.5% ± 5.4% ), tetracycline (56.0% ± 3.2% vs. 65.0% ± 15.0%) and chloramphenicol (71.5% ± 6.7% vs. 73.7% ± 5.5%)]. Interestingly, azithromycin did not show inhibiting activity on sphere formation under hypoxic condition, while number of spheres was reduced in normoxic conditions Conclusions: These data indicate that four out of five antibiotics in our study can be potentially applied for targeted eradication of breast CSCs in hypoxiс conditions. Acknowledgements: The study was funded by RFBR, according to the research project No. 16-34-60210 mol_а_dk, and by Russian Government Program of Competitive Growth of Kazan Federal University.

Изменение популяции стволовых клеток рака молочной железы линии MCF-7 при одиночном и комбинированном действии ионизирующего излучения и конъюгатов дендритных полимеров с доксорубицином

Изменение популяции стволовых клеток рака молочной железы линии MCF-7 при одиночном и комбинированном действии ионизирующего излучения и конъюгатов дендритных полимеров с доксорубицином

Monobenzyltin Complex C1 Induces Apoptosis in MCF-7 Breast Cancer Cells through the Intrinsic Signaling Pathway and through the Targeting of MCF-7-Derived Breast Cancer Stem Cells via the Wnt/β-Catenin Signaling Pathway.

Monobenzyltin Schiff base complex, [N-(3,5-dichloro-2-oxidobenzylidene)-4-chlorobenzyhydrazidato](o-methylbenzyl)aquatin(IV) chloride, C1, is an organotin non-platinum metal-based agent. The present study was conducted to investigate its effects on MCF-7 cells with respect to the induction of apoptosis and its inhibitory effect against MCF-7 breast cancer stem cells. As determined in a previous study, compound C1 revealed strong antiproliferative activity on MCF-7 cells with an IC50 value of 2.5 μg/mL. Annexin V/propidium iodide staining coupled with flow cytometry indicated the induction of apoptosis in treated cells. Compound C1 induced apoptosis in MCF-7 cells and was mediated through the intrinsic pathway with a reduction in mitochondrial membrane potential and mitochondrial cytochrome c release to cytosol. Complex C1 activated caspase 9 as a result of cytochrome c release. Subsequently, western blot and real time PCR revealed a significant increase in Bax and Bad expression and a significant decrease in the expression levels of Bcl2 and HSP70. Furthermore, a flow cytometric analysis showed that treatment with compound C1 caused a significant arrest of MCF-7 cells in G0/G1 phase. The inhibitory analysis of compound C1 against derived MCF-7 stem cells showed a significant reduction in the aldehyde dehydrogenase-positive cell population and a significant reduction in the population of MCF-7 cancer stem cells in primary, secondary, and tertiary mammospheres. Moreover, treatment with C1 down-regulated the Wnt/β-catenin self-renewal pathway. These findings indicate that complex C1 is a suppressive agent of MCF-7 cells that functions through the induction of apoptosis, cell cycle arrest, and the targeting of MCF-7-derived cancer stem cells. This work may lead to a better treatment strategy for the reduction of breast cancer recurrence.

Tunicamycin-induced endoplasmic reticulum stress reduces in vitro subpopulation and invasion of CD44+/CD24- phenotype breast cancer stem cells

Tunicamycin is an inhibitor of glycosylation that disturbs protein folding machinery in eukaryotic cells. Tunicamycin causes accumulation of unfolded proteins in cell endoplasmic reticulum (ER) and induces ER stress. ER stress is an essential mechanism for cellular homeostasis has role in cell death via reprogramming of protein processing, regulation of autophagy and apoptosis. In this study we show effect of tunicamycin on subpopulation and invasion of CD44+/CD24- MCF7 breast cancer stem cells. CD44+/CD24- cells were isolated from MCF7 cell line by fluorescence activated cell sorting (FACS) and treated with tunicamycin. ER stress was monitored by evaluation of X-box binding protein 1(XBP-1) mRNA splicing, cleaved activating transcription factor 6 (ATF6) nuclear translocation and CCAAT/enhancer-binding protein homologous protein (CHOP) expression. CD44+/CD24- subpopulation was analyzed using flow cytometry. Invasion was investigated by scratch assay, trypan blue staining, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation and in vitro migration assays. Increased level of spliced XBP-1, ATF6 nuclear translocation and CHOP protein expression were detected in CD44+/CD24- and original MCF7 cells treated with tunicamycin. Also, a significant decline in CD44+/CD24- cell subpopulation was determined in the cells treated with tunicamycin. The results also showed inhibited invasion, increased cell death, suppressed proliferation and reduced migration in the CD44+/CD24- and CD44+/CD24- rich MCF7 cell culture, under effect of tunicamycin. Our results indicate that CD44+/CD24- phenotype MCF7 cells are susceptible to tunicamycin. The results showed that tunicamycin-induced ER stress suppresses CD44+/CD24- phenotype cell subpopulation and in vitro invasion and accelerates tumorosphore formation. These results suggest that tunicamycin-induced ER stress inhibits CD44+/CD24- phenotype MCF7 breast cancer stem cells. We conclude that using ER-targeting chemicals like tunicamycin is an interesting approach to target breast cancer stem cells inside tumor.

Increased activity of CHK enhances the radioresistance of MCF-7 breast cancer stem cells.

The resistance of breast cancer to radiotherapy remains a major obstacle to successful cancer management. Radiotherapy may result in DNA damage and activate breast cancer stem cells. DNA damage may lead to activation of the checkpoint kinase (CHK) signaling pathway, of which debromohymenialdisine (DBH) is a specific inhibitor. Radiotherapy also increases the expression of phosphorylated CHK1/2 (pCHK1/2) in the breast cancer cell line, MCF-7, in vitro in a dose-dependent manner. DBH is a relatively stable effective inhibitor that significantly reduces pCHK1/2 expression and MCF-7 proliferation. Low-dose radiotherapy combined with DBH resulted in a higher MCF-7 inhibition rate compared with high-dose radiation alone. This result indicates that the inhibition of the CHK1/2 signal pathway may significantly reduce DNA damage within radiated cells. Radiotherapy may also regulate the proportion of CD44+/CD24− MCF-7 cancer stem cells in a dose- and time-dependent manner. However, the stem cell proportion of MCF-7 cells was significantly reduced by treatment with DBH. The inhibition is relatively stable and time dependent. Significant reductions were observed after 3 days of culture (P<0.01). The results of the present study indicate that the DBH-induced downregulation of CHK may provide a novel method of enhancing the effect of radiotherapy and reducing stem cell survival in the MCF-7 cell line.

Koenimbin, a natural dietary compound of Murraya koenigii (L) Spreng: inhibition of MCF7 breast cancer cells and targeting of derived MCF7 breast cancer stem cells (CD44(+)/CD24(-/low)): an in vitro study.

BackgroundInhibition of breast cancer stem cells has been shown to be an effective therapeutic strategy for cancer prevention. The aims of this work were to evaluate the efficacy of koenimbin, isolated from Murraya koenigii (L) Spreng, in the inhibition of MCF7 breast cancer cells and to target MCF7 breast cancer stem cells through apoptosis in vitro.MethodsKoenimbin-induced cell viability was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Nuclear condensation, cell permeability, mitochondrial membrane potential, and cytochrome c release were observed using high-content screening. Cell cycle arrest was examined using flow cytometry, while human apoptosis proteome profiler assays were used to investigate the mechanism of apoptosis. Protein expression levels of Bax, Bcl2, and heat shock protein 70 were confirmed using Western blotting. Caspase-7, caspase-8, and caspase-9 levels were measured, and nuclear factor kappa B (NF-κB) activity was assessed using a high-content screening assay. Aldefluor™ and mammosphere formation assays were used to evaluate the effect of koenimbin on MCF7 breast cancer stem cells in vitro. The Wnt/β-catenin signaling pathway was investigated using Western blotting.ResultsKoenimbin-induced apoptosis in MCF7 cells was mediated by cell death-transducing signals regulating the mitochondrial membrane potential by downregulating Bcl2 and upregulating Bax, due to cytochrome c release from the mitochondria to the cytosol. Koenimbin induced significant (P<0.05) sub-G0 phase arrest in breast cancer cells. Cytochrome c release triggered caspase-9 activation, which then activated caspase-7, leading to apoptotic changes. This form of apoptosis is closely associated with the intrinsic pathway and inhibition of NF-κB translocation from the cytoplasm to the nucleus. Koenimbin significantly (P<0.05) decreased the aldehyde dehydrogenase-positive cell population in MCF7 cancer stem cells and significantly (P<0.01) decreased the size and number of MCF7 cancer stem cells in primary, secondary, and tertiary mammospheres in vitro. Koenimbin also significantly (P<0.05) downregulated the Wnt/β-catenin self-renewal pathway.ConclusionKoenimbin has potential for future chemoprevention studies, and may lead to the discovery of further cancer management strategies by reducing cancer resistance and recurrence and improving patient survival.