Induced Breast Cancer Cell Death Research Articles

Abstract 4466: Omega-3 fatty acid-induced ferroptosis in 4T1 breast cancer through CD36/FABP5 axis

Abstract Mammalian cells utilize energy from many different sources, and fatty acids are one of these sources. Dietary fats contain various structures of fatty acids, each impacting cell lipid metabolism in unique ways. Omega-3 fatty acids are known to induce breast cancer cell death through ferroptosis. However, the specific molecular mechanisms by which omega-3 fatty acids trigger ferroptosis have yet to be fully elucidated. Fatty acid binding proteins (FABPs) constitute a family of evolutionarily conserved lipid chaperones, facilitating the transport and utilization of fatty acids within cells. Notably, FABP5 is highly expressed in breast cancer, especially in the most aggressive form, triple-negative breast cancer. We hypothesized that FABP5 acts as a lipid sensor, mediating the ferroptosis induced by omega-3 fatty acids in triple-negative breast cancer cells. To test this, we created Fabp5 stable knockout cell lines in 4T1 and MDA-MB-231, two commonly used triple-negative breast cancer cell lines, using CRISPR/Cas9 technology. When treated with docosapentaenoic acid (DPA), these Fabp5-knockout cells exhibited reduced ferroptosis compared to their wild-type counterparts. Interestingly, blocking the surface protein CD36, either with inhibitors or antibodies, also reduced DPA-induced ferroptosis. Furthermore, mutations in Fabp5 decreased maximal mitochondrial respiration in both 4T1 and MDA-MB-231 cells, suggesting a crucial role for FABP5 in mediating oxygen consumption and reactive oxygen species (ROS) production during DPA-induced ferroptosis. Additionally, mice fed a high-fat diet rich in omega-3 fatty acids showed reduced tumor size and lung metastasis. Collectively, our data indicate that FABP5 is a key molecular sensor mediating oxygen consumption and ROS production, thus contributing to ferroptosis induced by omega-3 fatty acids in triple-negative breast cancer. Citation Format: Anthony Avellino, Jiaqing Hao, Matthew Yorek, Jian yu, Bing Li. Omega-3 fatty acid-induced ferroptosis in 4T1 breast cancer through CD36/FABP5 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4466.

The bound polyphenols of foxtail millet (Setaria italica) inner shell inhibit breast cancer by promoting lipid accumulation-induced autophagic death.

Foxtail millet is a traditional excellent crop with high nutritional value in the world, belong to cereals. The bran of foxtail millet is rich in polyphenol that has antioxidant, anti-inflammatory, and anti-tumorigenic effects. Previously, we extracted bound polyphenols from the inner shell of foxtail millet bran (BPIS). Here, we report that BPIS specifically induced breast cancer cell death and elevated the autophagy level simultaneously. The addition of an autophagy inhibitor blocked BPIS-induced breast cancer cell death, indicating that excessive autophagy induced cell death. Furthermore, oil red O and BODIPY staining also confirmed that lipids, which are important inducers of autophagy, accumulated in breast cancer cells treated with BPIS. Lipidomics research revealed that glycerophospholipids were the main accumulated lipids induced by BPIS. Further study showed that elevated PCYT1A expression was responsible for glycerophospholipid accumulation, and BPIS contained ferulic acid and p-coumaric acid, which induced PCYT1A expression and breast cancer cell death. Collectively, our results revealed that BPIS resulted in autophagic death by enhancing lipid accumulation in breast cancer cells, and BPIS contains ferulic acid and p-coumaric acid, which provided new insights into developing nutraceuticals and drugs for breast cancer patients.

Quercetin Promotes TFEB Nuclear Translocation and Activates Lysosomal Degradation of Ferritin to Induce Ferroptosis in Breast Cancer Cells.

Objective. To investigate the antiproliferative efficacy of quercetin on breast cell lines and its mechanism of ferroptosis regulation. Cells (MCF-7 and MDA-231) were treated with quercetin at 0.1, 1, and 10 μM, respectively. The cell counting kit-8 (CCK-8) assay was applied to assess cell viability, and the intracellular iron level, malondialdehyde (MDA), and carbonylated protein were measured. After treating the cells with quercetin, western blot was applied to determine the level of transcription factor EB (TFEB) and lysosomal-associated membrane protein 1 (LAMP-1) in cells. Meanwhile, western blot was performed to assess the nuclear translocation of TFEB protein in cells. TFEB siRNA and autophagy lysosomal inhibitor, chloroquine, were used to block ferroptosis induced by quercetin. Quercetin induced breast cancer cell death and upregulated the level of iron, MDA, and carbonyl protein in a concentration-dependent manner. Meanwhile, TFEB was highly expressed in the nucleus and lowly expressed in the cytoplasm. The high expression of TFEB promoted the expression of lysosome-related gene LAMP-1, which in turn promoted the degradation of ferritin and the release of ferric ions. The above pharmacodynamic effects of quercetin can be blocked by TFEB siRNA or chloroquine. Quercetin promotes TFEB expression and nuclear transcription, induces the onset of iron death, and thus exerts a pharmacological effect on killing breast cancer cells.

A novel 8-hydroxyquinoline derivative induces breast cancer cell death through paraptosis and apoptosis.

Chemotherapy represents one of the main conventional therapies for breast cancer. However, tumor cells develop mechanisms to evade chemotherapeutic-induced apoptosis. Thus, it is of great significance to induce non-apoptotic cell death modes, such as paraptosis, in breast cancer. Herein, a novel 8-hydroxyquinoline derivative, 5,7-dibromo-8-(methoxymethoxy)-2-methylquinoline (HQ-11), was obtained and its potential anti-breast cancer mechanisms were investigated. Our results showed that extensive cytoplasmic vacuoles derived from the endoplasmic reticulum (ER) and mitochondria were appeared in MCF7 and MDA-MB-231 breast cancer cells by HQ-11 incubation, and pretreatment of cycloheximide was able to inhibit this vacuolation and HQ-11-induced cell death, showing the characteristics of paraptosis. ER stress was involved in HQ-11-caused paraptosis evidenced by the increase of glucose-regulated protein 78, C/EBP homologous protein and polyubiquitinated proteins. Molecular docking analysis revealed a favorable binding mode of HQ-11 in the active site of the chymotrypsin-like β5 subunit of the proteasome, indicative of proteasome dysfunction under HQ-11 treatment, which might result in further aggravated ER stress. Furthermore, treatment of HQ-11 resulted in increased phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase, and inhibition of ERK with U0126 significantly attenuated HQ-11-induced ER stress and paraptosis. In addition, exposure to HQ-11 also caused apoptosis in breast cancer cells partially through activation of ERK pathway. All these results conclusively indicate that HQ-11 triggers two distinct cell death modes via inhibition of proteasome and activation of ERK pathway in breast cancer cells, providing a promising candidate in future anti-breast cancer therapy.

The Ethyl Acetate Fraction of Marine Sponge Stylissa carteri Induces Breast Cancer Cell Death via Upregulation of Mcl-1S: an In vitro Study.

Objective:To evaluate the potency of the fraction of marine sponge Stylissa carteri in inducing cell death, inhibiting spheroid growth, and its impact on pro-apoptotic protein Mcl-1S in breast cancer cells. Methods: Stylissa carteri were collected from Pramuka Island followed by ethanol extraction and ethyl acetate fractionation. To evaluate the cytotoxic effect of fraction, the HCC-1954, MDA MB 231, and MCF-7 cells were treated with the fraction of Stylissa carteri and MTT assay was then performed. The effect on spheroid growth was evaluated in HCC-1954 cells. The combined effect of the ethyl acetate fraction and paclitaxel were analyzed using combination index (CI) and immunoblotting on the pro-apoptotic protein Mcl-1S. Furthermore, compounds in this fraction were identified using GC-MS. Results:Data showed that both the MDA MB 231 and HCC-1954 cells were interestingly more sensitive to the fraction as compared with MCF-7 cells. The IC50 of the ethyl acetate fraction on HCC-1954, MDA MB 231 and MCF-7 were 4.1 µg/ml, 3.9 µg/ml, and 123.8 µg/ml, respectively. In addition, the fraction triggered spheroid destruction within 10 days. The CI of paclitaxel and ethyl acetate fraction of Stylissa carteri were less than 0.52. Moreover, this combination induced upregulation of the Mcl-1S protein. Furthermore, some fatty acid-based structures were predicted as the major compounds in this fraction. Conclusion:The ethyl acetate fraction of Stylissa carteri induces cell death and spheroid destruction in aggressive breast cancer cells. It has a synergistic cytotoxic effect with paclitaxel on MDA MB 231 cell death and upregulates Mcl-1S protein.

Hydrogen phosphate selectively induces MDA MB 231 triple negative breast cancer cell death in vitro

Phosphate ions are the most abundant anions inside the cells, and they are increasingly gaining attention as key modulators of cellular function and gene expression. However, little is known about the effect of inorganic phosphate ions on cancer cells, particularly breast cancer cells. Here, we investigated the toxicity of different phosphate compounds to triple-negative human breast cancer cells, particularly, MDA-MB-231, and compared it to that of human monocytes, THP-1. We found that, unlike dihydrogen phosphate (H2PO4−), hydrogen phosphate (HPO42−) at 20 mM or lower concentrations induced breast cancer cell death more than immune cell death, mainly via apoptosis. We correlate this effect to the fact that phosphate in the form of HPO42− raises pH levels to alkaline levels which are not optimum for transport of phosphate into cancer cells. The results in this study highlight the importance of further exploring hydrogen phosphate (HPO42−) as a potential therapeutic for the treatment of breast cancer.

Combinatorial Effects of the Natural Products Arctigenin, Chlorogenic Acid, and Cinnamaldehyde Commit Oxidation Assassination on Breast Cancer Cells.

Major obstacles in current breast cancer treatment efficacy include the ability of breast cancer cells to develop resistance to chemotherapeutic drugs and the off-target cytotoxicity of these drugs on normal cells, leading to debilitating side effects. One major difference between cancer and normal cells is their metabolism, as cancer cells acquire glycolytic and mitochondrial metabolism alterations throughout tumorigenesis. In this study, we sought to exploit this metabolic difference by investigating alternative breast cancer treatment options based on the application of phytochemicals. Herein, we investigated three phytochemicals, namely cinnamaldehyde (CA), chlorogenic acid (CGA), and arctigenin (Arc), regarding their anti-breast-cancer properties. These phytochemicals were administered alone or in combination to MCF-7, MDA-MB-231, and HCC1419 breast cancer or normal MCF-10A and MCF-12F breast cells. Overall, our results indicated that the combination treatments showed stronger inhibitory effects on breast cancer cells versus single treatments. However, only treatments with CA (35 μM), CGA (250 μg/mL), and the combination of CA + CGA (35 μM + 250 μg/mL) showed no significant cytotoxic effects on normal mammary epithelial cells, suggesting that Arc was the driver of normal cell cytotoxicity in all other treatments. CA + CGA and, to a lesser extent, CGA alone effectively induced breast cancer cell death accompanied by decreases in mitochondrial membrane potential, increased mitochondrial superoxide, reduced mitochondrial and glycolytic ATP production, and led to significant changes in cellular and mitochondrial morphology. Altogether, the combination of CA + CGA was determined as the best anti-breast-cancer treatment strategy due to its strong anti-breast-cancer effects without strong adverse effects on normal mammary epithelial cells. This study provides evidence that targeting the mitochondria may be an effective anticancer treatment, and that using phytochemicals or combinations thereof offers new approaches in treating breast cancer that significantly reduce off-target effects on normal cells.

Combination of docetaxel and newly synthesized 9-Br-trimethoxybenzyl-noscapine improve tubulin binding and enhances antitumor activity in breast cancer cells

To strategically design and frame the novel 9-Br-Trimethoxybenzyl noscapine (BTN) with rigorous binding affinity with tubulin, the structure of noscapine (an antitussive plant alkaloid) was amended with a 3,4,5-trimethoxybenzyl group linked at the seventh position on the lower isobenzofuran unit. Molecular modelling and cellular studies were used to assess the single and combined effects of BTN and docetaxel (DOX). Based on MM-GBSA, the individual calculated free energies of binding (ΔGbind, pred) for BTN and DOX with tubulin was found to be −25.69 and −38.17 kcal/mol, respectively, and −29.11 and −36.60 kcal/mol based on MM-PBSA. Furthermore, the ΔGbind,pred of BTN was dramatically reduced (−30.02 and −33.54 kcal/mol using MM-GBSA and MM-PBSA) in presence of DOX on its binding pocket. Parenthetically, the ΔGbind,pred of DOX was substantially decreased (−39.17 and −35.80 kcal/mol using MM-GBSA and MM-PBSA) in the presence of BTN on its binding pocket. The synergistic activity of both compounds on tubulin dimmer was also analysed using purified tubulin, where a combined regimen of BTN and DOX attenuated tubulin intensity to a higher value (50%) particularly in comparison to the single regimen. In comparison to the single regimen, the combination of BTN and DOX effectively prevents cell cycle progression during the G2/M phase and induces breast cancer cell death. Female athymic nude mice were xenografted with MCF-7 cells and the efficacy of (150 mg/kg/day), DOX (1.5 mg/kg/week, i.v.), or in combination (BTN 300 mg/kg/day + DOX 1.0 mg/kg/week, i.v) were evaluated.

Type-3 Hyaluronan Synthase Attenuates Tumor Cells Invasion in Human Mammary Parenchymal Tissues.

The microenvironment for tumor growth and developing metastasis should be essential. This study demonstrated that the hyaluronic acid synthase 3 (HAS3) protein and its enzymatic product hyaluronic acid (HA) encompassed in the subcutaneous extracellular matrix can attenuate the invasion of human breast tumor cells. Decreased HA levels in subcutaneous Has3-KO mouse tissues promoted orthotopic breast cancer (E0771) cell-derived allograft tumor growth. MDA-MB-231 cells premixed with higher concentration HA attenuate tumor growth in xenografted nude mice. Human patient-derived xenotransplantation (PDX) experiments found that HA selected the highly migratory breast cancer cells with CD44 expression accumulated in the tumor/stroma junction. In conclusion, HAS3 and HA were detected in the stroma breast tissues at a high level attenuates effects for induced breast cancer cell death, and inhibit the cancer cells invasion at the initial stage. However, the highly migratory cancer cells were resistant to the HA-mediated effects with unknown mechanisms.

Canady Helios Cold Plasma Induces Breast Cancer Cell Death by Oxidation of Histone mRNA.

Breast cancer is the most common cancer among women worldwide. Its molecular receptor marker status and mutational subtypes complicate clinical therapies. Cold atmospheric plasma is a promising adjuvant therapy to selectively combat many cancers, including breast cancer, but not normal tissue; however, the underlying mechanisms remain unexplored. Here, four breast cancer cell lines with different marker status were treated with Canady Helios Cold Plasma™ (CHCP) at various dosages and their differential progress of apoptosis was monitored. Inhibition of cell proliferation, induction of apoptosis, and disruption of the cell cycle were observed. At least 16 histone mRNA types were oxidized and degraded immediately after CHCP treatment by 8-oxoguanine (8-oxoG) modification. The expression of DNA damage response genes was up-regulated 12 h post-treatment, indicating that 8-oxoG modification and degradation of histone mRNA during the early S phase of the cell cycle, rather than DNA damage, is the primary cause of cancer cell death induced by CHCP. Our report demonstrates for the first time that CHCP effectively induces cell death in breast cancer regardless of subtyping, through histone mRNA oxidation and degradation during the early S phase of the cell cycle.

Abstract 1494: IgE receptor-activated human mast cells have potent anti-cancer properties

Abstract The purpose of this study was to test the hypothesis that human mast cells (MC) have anti-tumor mediators that can be targeted to tumor specific antigens, induced to release these mediators, and cause cancer cell apoptosis. Breast cancer (BC) continues to be a significant cause of mortality for women in the United States. The oncogene HER2/neu is overexpressed in BC and is validated target for cancer therapeutics. New immunotherapy strategies and combinations of immune agents with non-redundant mechanisms of action are needed. The role of human tissue mast cells (MC) in the pathology of many cancers is not known. Nonetheless, high densities of MC are associated with favorable prognosis in certain types of cancer such as BC suggesting an anti-tumor effect. We previously reported that human adipose cells can be differentiated into autologous, fully functional MC (ADMC) capable of releasing mediators upon FcϵRI stimulation that induce BC cell death of cell suspensions of SK-BR3. Further data is presented demonstrating the anti-tumor effects of ADMC using BC tumor masses. The ADMC, sensitized with breast cancer-specific IgE anti-HER2/neu, bound to and were activated by, HER2/neu-positive human BC cells. The anti-HER2/neu IgE-sensitized ADMC bound to, infiltrated, and induced apoptosis of HER2/neu-positive BT-474 BC tumor masses as determined by time-lapse, live cell confocal imaging. Specific, HER2/neu IgE-mediated binding was also verified in vivo using immunocompromised xenograft models. Further studies are warranted to determine if tumor-targeted, autologous MC may be a new way to treat solid tumors. Citation Format: Chris Kepley, Mohammad Ferrydouni, Mona Motaghed, Kristen Dellinger. IgE receptor-activated human mast cells have potent anti-cancer properties [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 1494.

The Potent Cytotoxic and Oxidative Effects of β-2 Selective ICI-118,551 on Breast Adenocarcinoma Cell Lines with Different Aggressiveness

Objective: Beta-blockers are a group of drugs used in the treatment of cardiovascular ailments. On the other hand, the potential anticancer effects of these drugs have become increasingly important in recent two decades. In this paper, the effects of beta-1 selective esmolol, beta-2 selective ICI-118,551 and non-selective nadolol on breast cancer cell lines with different aggressiveness were investigated for the first time. Methods: A standard spectrophotometric MTT assay was used to determine cell viability. Catalase activities and malondialdehyde levels were measured spectrophotometrically based on the reduction of absorbance resulted from hydrogen peroxide decomposition and the formation of thiobarbituric acid- malondialdehyde product, respectively. Results: It was found that beta-2 selective ICI-118,551 was the most effective one among investigated blockers against MCF-7 and MDA-MB-231 cell lines. Additionally, it was seen that 50-150 µM ICI-118,551 treatment for 48 hours significantly changed catalase activities and malondialdehyde levels of both breast cancer cell lines in favour of radical production. Conclusion: The obtained results showed that beta-2 adrenergic receptor specific antagonism plays a significant role in beta-blocker induced breast cancer cell death. The outstanding suppression in catalase activities and concomitant increases in radical levels appear to contribute to potent cytotoxic effect of ICI-118,551 on breast adenocarcinoma. Consequently, it can be clearly interpreted that ICI-118,551 may be a valuable option in the treatment of breast cancer.

Selective AMPK activator leads to unfolded protein response downregulation and induces breast cancer cell death and autophagy

AimsAMPK plays a critical role regulating cell metabolism, growth and survival. Interfering with this enzyme activity has been extensively studied as putative mechanism for cancer therapy. The present work aims to identify a specific AMPK activator for cancer cells among a series of novel heterocyclic compounds. Materials and methodsA series of novel hybrid heterocyclic compounds, namely naphtoquinone-4-oxoquinoline and isoquinoline-5,8-quinone-4-oxoquinoline derivatives, were synthesized via Michael reaction and their structures confirmed by spectral data: infrared; 1H and 13C NMR spectroscopy (COSY, HSQC, HMBC); and high-resolution mass spectrometry (HRMS). The novel compounds were screened and tested for antitumoral activity and have part of their mechanism of action scrutinized. Key findingsHere, we identified a selective AMPK activator among the new hybrid heterocyclic compounds. This new compound presents selective cytotoxicity on breast cancer cells but not on non-cancer counterparts. We identified that by specifically activating AMPK in cancer cells, the drug downregulates unfolded protein response pathway, as well as inhibits mTOR signaling. SignificanceThese effects, that are selective for cancer cells, lead to activation of autophagy and, ultimately, to cancer cells death. Taken together, our data support the promising anticancer activity of this novel compound which is a strong modulator of metabolism.

Tagetes minuta leaf extracts triggered apoptosis in MCF-7 human breast cancer cell line

Tagetes minuta L. (Family: Asteraceae) has a history of human use as an ingredient in foods, therapeutics, and cosmetics. Although the in vitro cytotoxicity of Tagetes minuta (TM) has been reported in some human cancer cell lines, there is a paucity of information regarding its anticancer mechanisms of actions. In this study, we explored the mechanisms underlying the anticancer potential of TM in breast cancer. The apoptosis-inducing effect of TM leaf extracts was examined by Annexin V/propidium iodide (PI), Hoechst staining, flow cytometry, and fluorescence imaging using the MCF-7 human breast cancer cell line as a model. The activation of Caspase -3/7 was assessed using a luminogenic substrate (DEVD) consisting of a four amino acid peptide sequence conjugated to a fluorogenic compound (7-amino-4-trifluoromethyl coumarin). Also, the 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) and JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazoylcarbocyanine iodide) stains were used to determine cellular ROS generation and mitochondrial membrane depolarization respectively. The hexane and dichloromethane extracts of TM leaf exhibited moderate cytotoxicity against MCF-7 in a dose-dependent manner. These extracts induced reactive oxygen species (ROS) generation that was accompanied by the disruption of the mitochondrial transmembrane potential, resulting in caspase activation and eventually apoptotic cell death. The study revealed for the first time, that Tagetes minuta leaf extracts induce breast cancer cell death through mitochondrial caspase-dependent apoptosis. The conclusion, therefore, is that the hexane and dichloromethane extracts from Tagetes minuta leaf have the potential to be used as an anticancer agent.

Vanadium pentoxide nanoparticle mediated perturbations in cellular redox balance and the paradigm of autophagy to apoptosis

The redox-active transition metals such as copper, iron, chromium, vanadium, and silica are known for its ROS generation via mechanisms such as Haber-Weiss and Fenton-type reactions. Nanoparticles of these metals induce oxidative stress due to acellular factors owing to their small size and more reactive surface area, leading to various cellular responses. The intrinsic enzyme-like activity of nano vanadium has fascinated the scientific community. However, information concerning their cellular uptake and time-dependent induced effects on their cellular organelles and biological activity is lacking. This comprehensive study focuses on understanding the precise molecular interactions of vanadium pentoxide nanoparticles (VnNp) and evaluate their specific “nano” induced effects on MDA-MB-231 cancer cells. Understanding the mechanism behind NP-induced ROS generation could help design a model for selective NP induced toxicity, useful for cancer management. The study demonstrated the intracellular persistence of VnNp and insights into its molecular interactions with various organelles and its overall effects at the cellular level. Where triple-negative breast cancer MDA-MB-231 cells resulted in 59.6% cell death towards 48 h of treatment and the normal fibroblast cells showed only 15.4% cell death, indicating an inherent anticancer property of VnNp. It acts as an initial reactive oxygen species quencher, by serving itself as an antioxidant, while; it was also found to alter the cellular antioxidant system with prolonged incubation. The VnNp accumulated explicitly in the lysosomes and mitochondria and modulated various cellular processes including impaired lysosomal function, mitochondrial damage, and autophagy. At more extended time points, VnNp influenced cell cycle arrest, inhibited cell migration, and potentiated the onset of apoptosis.Results are indicative of the fact that VnNp selectively induced breast cancer cell death and hence could be developed as a future drug molecule for breast cancer management. This could override the most crucial challenge of chemo-resistance that still remain as the main hurdle to cancer therapy.

Erastin triggers autophagic death of breast cancer cells by increasing intracellular iron levels.

Erastin is a small molecular compound that induces ferroptosis by binding to voltage-dependent anion-selective channel protein (VDAC)2, VDAC3 and solute carrier family 7 member 5 inhibiting the cystine/glutamate antiporter. However, to the best of our knowledge, the mechanism of erastin-induced breast cancer cell death remains unclear. In present study aimed to explore the underlying mechanisms of the antitumor effects of erastin on breast cancer cells. Cellular viability was assessed using an MTT assay, a lactate dehydrogenase cytotoxicity assay kit was used to determine the cell death rate, the intracellular Fe2+ levels were determined using an iron colorimetric assay kit and western blotting was used to estimate the changes of autophagy-associated proteins levels. The present study demonstrated that erastin inhibited the viability of breast cancer cells and induced breast cancer cell death in a dose-dependent manner. Additionally, autophagy was activated by erastin, as demonstrated by upregulated expression levels of autophagy-associated proteins in breast cancer cells. Bafilomycin A1, 3-methyladenine and knockdown of autophagy related (ATG)5 with small interfering RNA prevented erastin-induced breast cancer cell death and inhibited the erastin-induced changes in the expression levels of the autophagy-associated proteins beclin1, ATG5, ATG12, microtubule-associated proteins 1A/1B light chain 3B (LC3B) and P62. Furthermore, erastin-induced breast cancer cell death was inhibited by an iron chelator, deferoxamine, which inhibited the increases of erastin-induced iron levels and inhibited the erastin-induced changes in the expression levels of the autophagy-related proteins beclin1, ATG5, ATG12, LC3B and P62. In summary, erastin triggered autophagic death in breast cancer cells by increasing intracellular iron levels.

Bishonokiol A Induces Multiple Cell Death in Human Breast Cancer MCF-7 Cells.

Objective:A dimeric neolignan, bishonokiol A (BHNKA) isolated from Magnolia grandiflora, significantly inhibits the proliferation of human breast cancer cells. However, the exact mechanism of BHNKA induced breast cancer cell death is unknown. In this study, we investigated the pharmacological mechanism underlying BHNKA induced MCF-7 cell death. Methods:Cell viability measurement was performed by the MTT assay. Flow cytometry with PI staining, DAPI staining, and electron microscopy were used to analyze cellular death modes. In addition, western blotting, siRNA transfection, ATP assay, and fluorescence microscopy were used to determine the mechanism of BHNKA induced MCF-7 cell death. Results:BHNKA induced cell death by apoptosis, necroptosis and autophagy at the same concentration and time in MCF-7 cells, and electron microscopy confirmed these results. The mechanism of BHNKA triggered apoptosis and autophagy in MCF-7 cells was primarily due to an increase in the Bax/Bcl-2 ratio and simultaneous up-regulation of LC3-II protein expression, respectively. BHNKA induced necroptosis by activation of the RIP1-RIP3-MLKL necroptosis cascade, up-regulation of cyclophilin D (CypD) protein expression to stimulate ROS generation. We further demonstrated that siRNA-mediated down-regulation of CypD protected against BHNKA induced cell death. Conclusions:These results suggest that BHNKA may be a potential lead compound for development as an anti-breast cancer agent for induction of multiple cell death pathways.

Wortmannolol Induces Breast Cancer Cell Death In Vitro and In Vivo by Targeting Phosphoinositide 3‐Kinaseα

Wortmannolol Induces Breast Cancer Cell Death In Vitro and In Vivo by Targeting Phosphoinositide 3‐Kinase<i>α</i>

Systems biology approaches based discovery of a small molecule inhibitor targeting both c-Met/PARP-1 and inducing cell death in breast cancer.

Breast cancer is the second most common types of cancer worldwide. Molecular strategies have developed rapidly; however, novel treatments strategies with high efficacy and lower toxicity are still urgently demanded. Notably, biological networks estimated from microarray data and functional activity network analysis could be utilized to identify and validate potential targets. In this study, two microarray data (GSE13477, GSE31192) were firstly selected, and analyzed by multi-functional activity network analysis to generate the core protein-protein-interaction (PPI) network. Several potential targets were subsequently identified and c-Met and poly (ADP-ribose) polymerase-1 (PARP-1) were manually chosen as the key targets in breast cancer. Furthermore, virtual screening and molecular dynamics (MD) simulations were utilized to recognize novel c-Met/PARP-1 inhibitors in Specs products database. Three small molecules, namely, ZINC19909930, ZINC20032678 and ZINC13562414 were selected. Additionally, these compounds were synthesized, and two breast cancer cell lines, MDA-MB-231 and MCF-7 cells were used to validate our bioinformatic findings in vitro. MTT assay and Hoechst staining showed that ZINC20032678 significantly induced breast cancer cell death, which was mediated through apoptosis by flow cytometry. Furthermore, ZINC20032678 was shown to target the active sites of the both targets and recruitment of downstream apoptotic signaling pathways, eventually inducing breast cancer cell apoptosis. Collectively, our findings not only offer systems biology approaches based drug target identification, but also provide the new clues for developing novel inhibitors for future breast cancer research.

Lovastatin-mediated MCF-7 cancer cell death involves LKB1-AMPK-p38MAPK-p53-survivin signalling cascade.

There is increasing evidence that statins, which are widely used in lowering serum cholesterol and the incidence of cardiovascular diseases, also exhibits anti‐tumour properties. The underlying mechanisms by which statins‐induced cancer cell death, however, remain incompletely understood. In this study, we explored the anti‐tumour mechanisms of a lipophilic statin, lovastatin, in MCF‐7 breast cancer cells. Lovastatin inhibited cell proliferation and induced cell apoptosis. Lovastatin caused p21 elevation while reduced cyclin D1 and survivin levels. Lovastatin also increased p53 phosphorylation, acetylation and its reporter activities. Results from chromatin immunoprecipitation analysis showed that p53 binding to the survivin promoter region was increased, while Sp1 binding to the region was decreased, in MCF‐7 cells after lovastatin exposure. These actions were associated with liver kinase B1 (LKB1), AMP‐activated protein kinase (AMPK) and p38 mitogen‐activated protein kinase (p38MAPK) activation. Lovastatin's enhancing effects on p53 activation, p21 elevation and survivin reduction were significantly reduced in the presence of p38MAPK signalling inhibitor. Furthermore, LKB1‐AMPK signalling blockade abrogated lovastatin‐induced p38MAPK and p53 phosphorylation. Together these results suggest that lovastatin may activate LKB1‐AMPK‐p38MAPK‐p53‐survivin cascade to cause MCF‐7 cell death. The present study establishes, at least in part, the signalling cascade by which lovastatin induces breast cancer cell death.