RESUMEN
Triple-negative breast cancer is an aggressive subtype of breast cancer with low 5-year survival rates, high 3-year recurrence rates, and no known therapeutic targets. Recent studies have indicated that triple-negative breast cancers possess an altered metabolic state with higher rates of glycolysis, mitochondrial oxidative phosphorylation, and increased generation and utilization of tricarboxylic acid cycle intermediates. Here, we utilized label-free quantitative proteomics to gain insight into the anticancer mechanisms of a methanolic extract from the Central American plant Lippia origanoides on MDA-MB-231 triple-negative breast cancer cells. The L. origanoides extract dysregulated mitochondrial oxidative phosphorylation by suppressing the expression of several subunits of Complex I of the electron transport chain, and inhibited cellular metabolism by down-regulating key tricarboxylic acid cycle enzymes and mitochondrial lipid and amino-acid metabolic pathways. Our study also revealed that treatment with the extract activated the stress response and pathways related to cell-cycle progression and DNA repair. Overall, our results reveal compelling new evidence that the extract from L. origanodes triggers rapid irreversible apoptosis in MDA-MB-231 cells by effectively 'starving' the cells of metabolites and ATP. We continue to study the specific bioactive components of the extract in the search for novel, highly effective mitochondrial inhibitors to selectively target triple-negative breast cancer.
Asunto(s)
Lippia/química , Mitocondrias/efectos de los fármacos , Extractos Vegetales/farmacología , Proteómica/métodos , Neoplasias de la Mama Triple Negativas/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Complejo I de Transporte de Electrón/efectos de los fármacos , Complejo I de Transporte de Electrón/metabolismo , Femenino , Glucólisis/efectos de los fármacos , Humanos , Mitocondrias/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Triple negative breast cancer (TNBC) is difficult to treat due to lack of the three receptors, commonly used for treating breast cancers. Current standard of cure is either ineffective or refractive to many patients. Thus, there is a critical need for alternate, affordable therapies for TNBC cancers. Towards this, electrical pulse-mediated chemotherapy, known as electrochemotherapy is a viable option, because it uses the synergy of electrical pulses and the anticancer properties of chemo drug. Considering the cost and the harsh side effects of various commonly administered chemo drugs, in this study, low cost, yet effective, natural phytochemical curcumin is studied for its anticancer effect on MDA-MB-231, TNBC cells. We applied eight 10 µs, 2500 V/cm or 5000 V/cm pulses with 10 µM concentration of curcumin, and measured cell viability and cytotoxicity. Results indicate that cell survival, as low as 4% was induced by 5000 V/cm pulses, after 72 h, while it was 15% after 24 h. This demonstrates the potential of this treatment for TNBC and the transfer to clinical practice.
Asunto(s)
Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Curcumina/administración & dosificación , Curcumina/farmacología , Electroquimioterapia , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Curcumina/química , Curcumina/uso terapéutico , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Electroquimioterapia/efectos adversos , Humanos , Relación Estructura-Actividad , Factores de Tiempo , Neoplasias de la Mama Triple Negativas/patología , Células Tumorales CultivadasRESUMEN
DNA hypomethylation was previously implicated in cancer progression and metastasis. The purpose of this study was to examine whether stilbenoids, resveratrol and pterostilbene thought to exert anticancer effects, target genes with oncogenic function for de novo methylation and silencing, leading to inactivation of related signaling pathways. Following Illumina 450K, genome-wide DNA methylation analysis reveals that stilbenoids alter DNA methylation patterns in breast cancer cells. On average, 75% of differentially methylated genes have increased methylation, and these genes are enriched for oncogenic functions, including NOTCH signaling pathway. MAML2, a coactivator of NOTCH targets, is methylated at the enhancer region and transcriptionally silenced in response to stilbenoids, possibly explaining the downregulation of NOTCH target genes. The increased DNA methylation at MAML2 enhancer coincides with increased occupancy of repressive histone marks and decrease in activating marks. This condensed chromatin structure is associated with binding of DNMT3B and decreased occupancy of OCT1 transcription factor at MAML2 enhancer, suggesting a role of DNMT3B in increasing methylation of MAML2 after stilbenoid treatment. Our results deliver a novel insight into epigenetic regulation of oncogenic signals in cancer and provide support for epigenetic-targeting strategies as an effective anticancer approach.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas Nucleares/genética , Transportador 1 de Catión Orgánico/genética , Factores de Transcripción/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Cromatina/efectos de los fármacos , Islas de CpG/genética , ADN (Citosina-5-)-Metiltransferasas/biosíntesis , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genoma Humano , Humanos , Transportador 1 de Catión Orgánico/biosíntesis , Regiones Promotoras Genéticas , Receptores Notch/genética , Resveratrol , Transducción de Señal/efectos de los fármacos , Estilbenos/administración & dosificación , Transactivadores , Activación Transcripcional/genética , ADN Metiltransferasa 3BRESUMEN
Aim: Triple-negative breast cancer (TNBC) is a very aggressive subset of breast cancer, with limited treatment options, due to the lack of three commonly targeted receptors, which merits the need for novel treatments for TNBC. Towards this need, the use of metformin (Met), the most widely used type-2 diabetes drug worldwide, was explored as a repurposed anticancer agent. Cancer being a metabolic disease, the modulation of two crucial metabolites, glucose, and reactive oxygen species (ROS), is studied in MDA-MB-231 TNBC cells, using Met in the presence of electrical pulses (EP) to enhance the drug efficacy. Methods: MDA-MB-231, human TNBC cells were treated with Met in the presence of EP, with various concentrations Met of 1 mmol/L, 2.5 mmol/L, 5 mmol/L, and 10 mmol/L. EP of 500 V/cm, 800 V/cm, and 1,000 V/cm (with a pulse width of 100 µs at 1 s intervals) were applied to TNBC and the impact of these two treatments was studied. Various assays, including cell viability, microscopic inspection, glucose, ROS, and wound healing assay, were performed to characterize the response of the cells to the combination treatment. Results: Combining 1,000 V/cm with 5 mmol/L Met yielded cell viability as low as 42.6% at 24 h. The glucose level was reduced by 5.60-fold and the ROS levels were increased by 9.56-fold compared to the control, leading to apoptotic cell death. Conclusions: The results indicate the enhanced anticancer effect of Met in the presence of electric pulses. The cell growth is inhibited by suppressing glucose levels and elevated ROS. This shows a synergistic interplay between electroporation, Met, glucose, and ROS metabolic alterations. The results show promises for combinational therapy in TNBC patients.
RESUMEN
Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer lacks estrogen, progesterone, and HER2 receptors and hence, is therapeutically challenging. Towards this, we studied an alternate therapy by repurposing metformin (FDA-approved type-2 diabetic drug with anticancer properties) in a 3D-scaffold culture, with electrical pulses. 3D cell culture was used to simulate the tumor microenvironment more closely and MDA-MB-231, human TNBC cells, treated with both 5 mM metformin (Met) and 8 electrical pulses at 2500 V/cm, 10 µs (EP1) and 800 V/cm, 100 µs (EP2) at 1 Hz were studied in 3D and 2D. They were characterized using cell viability, reactive oxygen species (ROS), glucose uptake, and lactate production assays at 24 h. Cell viability, as low as 20 % was obtained with EP1 + 5 mM Met. They exhibited 1.65-fold lower cell viability than 2D with EP1 + 5 mM Met. ROS levels indicated a 2-fold increase in oxidative stress for EP1 + 5 mM Met, while the glucose uptake was limited to only 9 %. No significant change in the lactate production indicated glycolytic arrest and a non-conducive environment for MDA-MB-231 growth. Our results indicate that 3D cell culture, with a more realistic tumor environment that enhances cell death using metformin and electrical pulses could be a promising approach for TNBC therapeutic intervention studies.
Asunto(s)
Muerte Celular , Supervivencia Celular , Electroporación , Metformina , Especies Reactivas de Oxígeno , Humanos , Metformina/farmacología , Línea Celular Tumoral , Electroporación/métodos , Especies Reactivas de Oxígeno/metabolismo , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Glucosa/metabolismo , Técnicas de Cultivo Tridimensional de Células/métodos , Andamios del Tejido/química , Antineoplásicos/farmacología , Células MDA-MB-231RESUMEN
BACKGROUND: The major obstacles to the successful use of individual nutritional compounds as preventive or therapeutic agents are their efficacy and bioavailability. One approach to overcoming this problem is to use combinations of nutrients to induce synergistic effects. The objective of this research was to investigate the synergistic effects of two dietary components: docosahexaenoic acid (DHA), an omega-3 fatty acid present in cold-water fish, and curcumin (CCM), an herbal nutrient present in turmeric, in an in vivo model of DMBA-induced mammary tumorigenesis in mice. METHODS: We used the carcinogen DMBA to induce breast tumors in SENCAR mice on control, CCM, DHA, or DHA + CCM diets. Appearance and tumor progression were monitored daily. The tumors were harvested 15 days following their first appearance for morphological and immunohistological analysis. Western analysis was performed to determine expression of maspin and survivin in the tumor tissues. Characterization of tumor growth was analyzed using appropriate statistical methods. Otherwise all other results are reported as mean ± SD and analyzed with one-way ANOVA and Tukey's post hoc procedure. RESULTS: Analysis of gene microarray data indicates that combined treatment with DHA + CCM altered the profile of "PAM50" genes in the SK-BR-3 cell line from an ERâ»/Her-2⺠to that resembling a "normal-like" phenotype. The in vivo studies demonstrated that DHA + CCM treatment reduced the incidence of breast tumors, delayed tumor initiation, and reduced progression of tumor growth. Dietary treatment had no effect on breast size development, but tumors from mice on a control diet (untreated) were less differentiated than tumors from mice fed CCM or DHA + CCM diets. The synergistic effects also led to increased expression of the pro-apoptotic protein, maspin, but reduced expression of the anti-apoptotic protein, survivin. CONCLUSIONS: The SK-BR-3 cells and DMBA-induced tumors, both with an ERâ» and Her-2⺠phenotype, were affected by the synergistic interaction of DHA and CCM. This suggests that the specific breast cancer phenotype is an important factor for predicting efficacy of these nutraceuticals. The combination of DHA and CCM is potentially a dietary supplemental treatment for some breast cancers, likely dependent upon the molecular phenotype of the cancer.
Asunto(s)
Antineoplásicos/farmacología , Transformación Celular Neoplásica/efectos de los fármacos , Curcumina/farmacología , Ácidos Docosahexaenoicos/farmacología , Neoplasias Mamarias Experimentales/patología , 9,10-Dimetil-1,2-benzantraceno/efectos adversos , Animales , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Análisis por Conglomerados , Curcumina/administración & dosificación , Dieta , Ácidos Docosahexaenoicos/administración & dosificación , Sinergismo Farmacológico , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Neoplasias Mamarias Experimentales/inducido químicamente , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/terapia , Ratones , Receptor ErbB-2/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Serpinas/genética , Serpinas/metabolismo , Survivin , Carga Tumoral , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genéticaRESUMEN
Aim: Triple negative breast cancer (TNBC) is difficult to treat since it lacks all the three most commonly targeted hormone receptors. Patients afflicted with TNBC are treated with platinum core chemotherapeutics, such as cisplatin. Despite the initial effective anticancer effects of cisplatin, TNBC attenuates its effect and develops resistance eventually, which results in tumor reoccurrence. Hence, there is a critical demand for effective, alternative, and natural ways to treat TNBC. Towards this, a promising technique for inhibiting TNBC cell proliferation involves promoting the production of reactive oxygen species (ROS), which triggers pro-apoptotic caspases 9 and 3. Resveratrol (RESV), an active bio compound found in naturally available fruits, such as grapes, is utilized in this research for that. In addition, electrochemotherapy (ECT), which involves the application of electrical pulses (EP), was utilized to enhance the uptake of RESV. Methods: MDA-MB-231, human TNBC cells were treated with/out RESV, and eight 600-1,000 V/cm, 100 µs pulses at 1 Hz. The cells were characterized by using various assays, including viability assay, and ROS assay. Results: A TNBC cell viability of as low as 20% was obtained at 24 h (it was 13% at 60 h), demonstrating the potential of this novel treatment. ROS production was the highest in the combination of EP at 1,000 V/cm along with RESV at 100 µmol/L. Conclusions: Results indicate that RESV has the potential as an anti-TNBC agent and that EP + RESV can significantly enhance the cell death to reduce MDA-MB-231 cell viability by increasing ROS production and triggering apoptosis.
RESUMEN
Aim: The purpose of this research is to study the effect of electrical pulse mediated tomato lipophilic extract (TLE) on human breast cancer MCF-7 and non-tumorigenic MCF-10A cells. Materials and Methods: MCF-7 and MCF-10A cells were treated with 50 µg/mL TLE and eight 100 µs electric pulses of different electric field intensities (800, 1000, and 1200 V/cm), and the viability was studied using real time MT assay at 24 h of treatment. In addition, we studied cell viability of both the cells at 0 h using trypan blue assay and the ability to form colonies of both cells using colony forming unit (CFU) assay for all the treatments. We also imaged the cells at 24 h using microscope. Results: With 50 µg/mL TLE, the cell viability of MCF-7 and MCF-10A was same (84%). When the same concentration of TLE is combined with eight electrical pulses of 1200 V/cm, the cell viability of MCF-7 and MCF-10A was 2% and 87%, respectively. These results indicate that the effect of electrical pulses mediated TLE was higher on cancerous MCF-7 cells when compared to non-cancerous MCF-10A cells. Conclusion: The combination of electrical pulses with TLE is an effective strategy to selectively target cancer cells in the body.
Asunto(s)
Neoplasias de la Mama , Solanum lycopersicum , Humanos , Femenino , Neoplasias de la Mama/terapia , Células MCF-7 , Proliferación CelularRESUMEN
We investigated the combined potency of metformin and cisplatin on the MDA-MB-231, triple-negative breast cancer (TNBC) cells with the application of electrical pulses. There are no targeted therapies for this subset of breast cancer because of the absence of specific biomarkers. Cytotoxic chemotherapy is the mainstream mode of treatment for TNBC, and cisplatin is the most commonly used chemotherapeutic drug. While there is a good response initially, TNBC cells develop drug resistance eventually. Thus, there is a need for alternate therapies. Toward this, we studied the antiproliferation characteristics of electrical pulse-mediated combination therapy using metformin, the commonly used Type-2 diabetes drug, along with cisplatin. We used metformin, as it has various anticancer properties caused by repressing energy pathways in a cancer cell. Application of 8 pulses of 1000 V/cm, 100 µs, at 1 Hz frequency, enhanced the drug uptake leading to cell viability as low as 25.86% at 30 µM cisplatin and 5 mM metformin in a 24 h study. Also, the same studies were conducted on MCF10A, a non-cancerous human epithelial cell. It aided in comparing the result for both MDA-MB-231 and MCF10A cell lines while establishing a better understanding of the experimental outcomes. Overall, the various experimental results from colony-forming assay, reactive oxidative analysis, and the intracellular glucose metabolic assay indicate the possibility of the electrical pulses-based cisplatin and metformin drug combination as a potential alternative to TNBC treatment.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proliferación Celular/efectos de los fármacos , Electroquimioterapia , Neoplasias de la Mama Triple Negativas , Línea Celular Tumoral , Cisplatino/farmacología , Femenino , Humanos , Metformina/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
With the absence of the three most common receptor targets, and with high vascularity and higher-grade tumors, triple-negative breast cancer (TNBC) is the most aggressive of all breast cancer subtypes and is in need of additional/alternative/novel treatment strategies. With ~ 15% of the over 2 million new cases each year, there is an unmet need to treat TNBC. MDA-MB-231, human TNBC cells, were treated with neem leaf extract (Neem) and eight, 1200 V/cm, 100 µs electric pulses (EP), and their viability and proteomic profiles were studied. With EP + Neem, a lower viability of 37% was observed after 24 h, compared to 85% in the neem-only samples, indicating the efficacy of the combinational treatment. The proteomics results indicated significant upregulation of 525 proteins and downregulation of 572 proteins, with a number of different pathways in each case. These include a diverse group of proteins, such as receptors, heat shock proteins, and many others. The upregulated TCA cycle and OXPHOS pathways and the downregulated DNA replication and ubiquitin-mediated proteolytic pathways were associated with effective cell death, demonstrating the potency of this treatment. Viability results reveal the efficacious anticancer effects of the EP + Neem combination, via growth inhibition, on TNBC cells. Proteomics studies could readily identify the effected protein pathways, and their corresponding genes, that are responsible for cell death. This represents a potential therapeutic strategy against TNBC when patients are refractory to standard treatments.
Asunto(s)
Azadirachta/química , Electroquimioterapia , Proteínas de Neoplasias/metabolismo , Extractos Vegetales/farmacología , Hojas de la Planta/química , Neoplasias de la Mama Triple Negativas , Línea Celular Tumoral , Femenino , Humanos , Extractos Vegetales/química , Proteómica , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Heat shock proteins (HSPs) are available and/or induced for the survival of all organisms, including eukaryotic, prokaryotic, and plants, from higher temperature stresses. They are the chaperone proteins that protect all cells against heat, as the name implies. In addition to thermal stress, they also protect them from chemical, physical, and other stresses, including exposure to oxidative stress, nutritional deficiencies, ultraviolet radiation, ethanol, viral infection, ischemia-reperfusion injury, and cancer-related stresses. They are classified based on their molecular weights in kDa, such as HSP90 and HSP70. In our label-free, high-throughput, quantitative LC-MS/MS-based proteomic studies of MDA-MB-231, human, triple-negative breast cancer cells, treated with electrical pulses (EP) and cisplatin (CsP), we identified a number of HSPs, such as HSP90AA1, and others to be significantly downregulated in EP + CsP, compared to CsP alone. This indicates that cells will undergo apoptotic cell death and hence could cause effective cancer cure/treatment. Considering that over 2 million new cases and over 600,000 deaths in 2020, of which ~ 15% are TNBC, heat shock proteins could be the untapped resources, available for the next biomarkers and/or inhibitors for new/additional therapies.
Asunto(s)
Cisplatino/farmacología , Regulación hacia Abajo/efectos de los fármacos , Electroquimioterapia , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas HSP90 de Choque Térmico/biosíntesis , Proteínas de Neoplasias/biosíntesis , Neoplasias de la Mama Triple Negativas , Línea Celular Tumoral , Femenino , Humanos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
In the US, every 12 min, six women are diagnosed with breast cancer and one dies. This highlights a critical need for developing alternate therapies using natural compounds, which are cost effective and with less side effects. Curcumin, the yellow pigment of turmeric has been found to suppress initiation, progression, and metastasis of a variety of tumors. Multiple clinical trials highlight the efficacy of curcumin in treating breast cancer and other diseases. Our in vitro studies have demonstrated that the electrical pulse (EP) application can further enhance the effectiveness of curcumin against breast cancer cells in a therapy called electrochemotherapy (ECT). In a direct extension of these results, we studied the effect of ECT coupled with intratumoral curcumin administration (EP+Cur) on N-methyl-N-nitrosourea (MNU) induced mammary tumors in female Sprague Dawley rats. Beginning at the weaning and throughout the study, rats were fed either western diet (West) or western diet, supplemented with 1% curcumin (W+Cur). Our results showed that EP+Cur treatment led to a reduced growth rate in rats fed with W+Cur diet compared to West diet (57.14% vs. 16.67% in West diet). These results provide a foundation for further studies towards utilizing it in clinical practice.
RESUMEN
Curcumin (Cur), the yellow pigment of well-known turmeric (Curcuma longa L.) is effective in multiple cancers including triple negative breast cancer (TNBC). In combination with electrical pulses (EP), enhanced effects of curcumin (Curâ¯+â¯EP) are observed in TNBC cells. To gain insights into the mechanisms of enhanced anticancer effects of Curâ¯+â¯EP, we studied the proteins involved in the anticancer activity of Curâ¯+â¯EP in MDA-MB-231, human TNBC cells using high-throughput global proteomics. A curcumin dose of 50⯵M was applied with eight, 1200â¯V/cm, 100⯵s pulses, the most commonly used electrochemotherapy (ECT) parameter in clinics. Results show that the Curâ¯+â¯EP treatment reduced the clonogenic ability in MDA-MB-231 cells, with the induction of apoptosis. Proteomic analysis identified a total of 1456 proteins, of which 453 proteins were differentially regulated, including kinases, heat shock proteins, transcription factors, structural proteins, and metabolic enzymes. Eight key glycolysis proteins (ALDOA, ENO2, LDHA, LDHB, PFKP, PGM1, PGAM1 and PGK1) were downregulated in Curâ¯+â¯EP from Cur. There was a switch in the metabolism with upregulation of 10 oxidative phosphorylation pathway proteins and 8 tricarboxylic acid (TCA) cycle proteins in the Curâ¯+â¯EP sample, compared to curcumin. These results provide novel systematic insights into the mechanisms of ECT with curcumin.
Asunto(s)
Antineoplásicos/uso terapéutico , Curcumina/uso terapéutico , Electroquimioterapia/métodos , Proteínas de Neoplasias/metabolismo , Proteómica , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Curcumina/farmacología , Regulación hacia Abajo/efectos de los fármacos , Femenino , Glucólisis , Humanos , Fosforilación Oxidativa , Vía de Pentosa Fosfato/efectos de los fármacos , Reproducibilidad de los Resultados , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Triple negative breast cancer (TNBC) represents 15-20% of the over one million new breast cancer cases occurring each year. TNBC is an aggressive cancer phenotype, with low 5-year survival rates, high 3-year recurrence rates, and increased risk of metastasis. A lack of three commonly exploited hormone receptors renders TNBC resistant to endocrine therapies and lends to its critical absence of viable therapeutic targets. This necessitates the development of alternate and effective novel therapeutic strategies for TNBC. Towards this, our current work seeks to develop the technique of Electrical pulse (EP)-mediated Turmeric silver nanoparticles (TurNP) therapy, known as Electrochemotherapy (ECT), to effectively target TNBC cells. This technique involves the efficient delivery of natural bioactive molecules with anti-cancer effects via a biophysical means. In these experiments, the bioactive molecules are turmeric, a dried rhizome of Curcuma longa that has been used for centuries, both as a dietary supplement and as a medicine in Ayurveda (science of life) in the Indian subcontinent and in traditional Chinese medicine. Our results reveal the combined effect of TurNP + EP treatment in reducing MDA-MB-231 cell viability to as low as 9% at 12 h. Showing biological selectivity, this combination treatment has a substantially lower effect on non-tumorigenic mammary epithelial MCF10A cells (67% viability). To gain mechanistic insights into the actions of TurNP-based ECT treatment, we performed high-throughput, label-free quantitative proteomics studies. Proteomics results indicate that TurNP + EP treatment significantly influenced expression of a diverse list of proteins, including receptors, transcription factors, structural proteins, kinases, and metabolic enzymes. This include the downregulation of 25 proteins in PI3K-Akt signaling pathway (such as GRB2, EGFR, EPHA2, GNB1, GNB2, 14-3-3 family, and Integrin family proteins), and 12 proteins (AKR1A1, ALDOA, ALDOC, PGK1, PGM1, PGAM1, ENO1, ENO2, GAPDH, TPI1, LDHA, and LDHB) in the glycolytic pathway with concomitant reduction in metabolite levels (glucose uptake, and intracellular- lactate, glutamine, and glutamate). Compared to TurNP alone, TurNP + EP treatment upregulated 66 endoplasmic reticulum and 193 mitochondrial proteins, enhancing several processes and pathways, including Pyruvate Metabolism, Tricarboxylic acid (TCA) cycle, and Oxidative Phosphorylation (OXPHOS), which redirected the TNBC metabolism to mitochondria. This switch in the metabolism caused excessive production of H2O2 reactive oxygen species (ROS) to inflict cell death in MDA-MB-231 cells, demonstrating the potency of this treatment.
Asunto(s)
Antineoplásicos/uso terapéutico , Curcuma/química , Electroquimioterapia , Nanopartículas del Metal , Proteómica/métodos , Plata/química , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Línea Celular Tumoral , Supervivencia Celular , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Técnicas In Vitro , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Due to the lack of the three main receptors, triple negative breast cancer (TNBC) is refractive to standard chemotherapy. Hence, alternate therapies are needed. TNBCs utilize glycolysis, which heightens their growth, proliferation, invasiveness, chemotherapeutic resistance and poor therapeutic response. This calls for novel therapeutic strategies to target these metabolic vulnerabilities present in TNBC. Electroporation-mediated chemotherapy, known as electrochemotherapy (ECT) is gaining momentum as an attractive alternative. However, its molecular mechanisms need better understanding. Towards this, label-free quantitative proteomics is utilized to gain insight into the anticancer mechanisms of ECT using electrical pulses (EP) and Cisplatin (CsP) on MDA-MB-231, human TNBC cells. The results indicate that EP + CsP significantly downregulated 14 key glycolysis proteins (including ENO1, LDHA, LDHB, ACSS2, ALDOA, and PGK1), compared to CsP alone. EP + CsP caused a switch in the metabolism with upregulation of 34 oxidative phosphorylation pathway proteins and 18 tricarboxylic acid (TCA) cycle proteins compared to CsP alone, accompanied by the upregulation of proteins linked to several metabolic reactions, which produce TCA cycle intermediates. Moreover, EP + CsP promoted multiple pathways to cause 1.3-fold increase in the reactive oxygen species concentration and induced apoptosis. The proteomics results correlate well with cell viability, western blot, and qPCR data. While some effects were similar for EP, more comprehensive and long-lasting effects were observed for EP + CsP, which demonstrate the potential of EP + CsP against TNBC cells.
Asunto(s)
Antineoplásicos/administración & dosificación , Cisplatino/administración & dosificación , Electroquimioterapia/efectos adversos , Proteoma/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Apoptosis , Línea Celular , Línea Celular Tumoral , Ciclo del Ácido Cítrico , Glucólisis , Humanos , Fosforilación Oxidativa , Estrés Oxidativo , Proteoma/genéticaRESUMEN
Obesity is a highly prevalent and modifiable breast cancer risk factor. While the role of obesity in fueling breast cancer progression is well established, the mechanisms linking obesity to breast cancer initiation are poorly understood. A hallmark of breast cancer initiation is the disruption of apical polarity in mammary glands. Here we show that mice with diet-induced obesity display mislocalization of Par3, a regulator of cellular junctional complexes defining mammary epithelial polarity. We found that epithelial polarity loss also occurs in a 3D coculture system that combines acini with human mammary adipose tissue, and establish that a paracrine effect of the tissue adipokine leptin causes loss of polarity by overactivation of the PI3K/Akt pathway. Leptin sensitizes non-neoplastic cells to proliferative stimuli, causes mitotic spindle misalignment, and expands the pool of cells with stem/progenitor characteristics, which are early steps for cancer initiation. We also found that normal breast tissue samples with high leptin/adiponectin transcript ratio characteristic of obesity have an altered distribution of apical polarity markers. This effect is associated with increased epithelial cell layers. Our results provide a molecular basis for early alterations in epithelial architecture during obesity-mediated cancer initiation.
Asunto(s)
Neoplasias de la Mama/patología , Leptina/sangre , Glándulas Mamarias Animales/patología , Glándulas Mamarias Humanas/patología , Proteínas Adaptadoras Transductoras de Señales , Adipoquinas/metabolismo , Tejido Adiposo/metabolismo , Animales , Índice de Masa Corporal , Neoplasias de la Mama/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas de Ciclo Celular , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Humanos , Leptina/genética , Leptina/metabolismo , Glándulas Mamarias Humanas/metabolismo , Ratones Endogámicos BALB C , Obesidad/metabolismo , Obesidad/patología , Lesiones Precancerosas , Huso Acromático/metabolismo , Huso Acromático/patologíaRESUMEN
Leptin is an adipocyte-derived hormone that regulates energy expenditure and food intake. A significant role for leptin in breast cancer has also been indicated by the resistance of leptin knockout mice in development of mammary tumors. In vitro, leptin induces proliferation of MCF-7 cells by activating cellular signaling pathways (1, 11, 12, 16, 17, 56). As leptin is emerging as an important factor for tumor growth, and hormones can exert their actions via autocrine/paracrine mechanisms, we hypothesized leptin may act by regulating epithelial-derived proteins. To test this hypothesis, leptin-regulated proteins secreted from MCF-7 mammary tumor cells were identified using proteomics techniques. Treatment of MCF-7 cells with 500 ng/ml leptin for 24 hours resulted in a 40% increase in cell number and a 5-fold increase in protein secretion as compared to controls. Establishing the significance of leptin-induced secreted factors, the addition of conditioned media from leptin-treated MCF-7 cells to synchronized MCF-7 cells resulted in 40% increase in cell number. Identification of leptin-regulated secreted proteins was done by 2D gel electrophoresis coupled with MALDI-TOF mass spectrometry. Proteins identified using Pro Found software and NCBI database included KF10 Collagen Precursor, Serologically Defined Breast Cancer Antigen NY-BR-62 and Cortactin Isoform a. A Human Cytokine Antibody Array system was used to identify low abundant proteins in the media of control and 500 ng/ml leptin-stimulated MCF-7 cells. In leptin treated cells, levels of FGF-9 were increased while IGFBP-3 and TGF-beta3 levels were decreased. Many previous studies have focused on the regulation of distinct cellular proteins by leptin during mammary tumor cell proliferation. However, ours is the first study to identify leptin-regulated secreted proteins, many of which are known to play important roles in cancer. Our data support that leptin can influence mammary tumor growth and progression through regulation of autocrine/paracrine factors and by modulating the extracellular matrix composition.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Leptina/metabolismo , Proteínas de Neoplasias/metabolismo , Proteómica/métodos , Línea Celular Tumoral , Medios de Cultivo Condicionados/farmacología , Citocinas/metabolismo , Electroforesis en Gel Bidimensional , Citometría de Flujo , Humanos , Espectrometría de Masas , Modelos Biológicos , Proteínas/química , ARN Mensajero/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Treatments targeting hormone receptors typically fail to provide a positive clinical outcome against triple-negative breast cancers (TNBC), which lack expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2/neu). Towards identifying viable treatments for aggressive breast cancer, we have tested an extract of the tropical plant Lippia origanoides (LOE) on TNBC and normal cells lines to uncover its potential anticancer effects. Treatment with LOE reduced TNBC cell viability in a dose-dependent manner to a greater extent than in normal mammary epithelial MCF10A cells. In MDA-MB231 cells, LOE was found to halt the cell cycle in the G0/G1 phase via cyclin D1 and cIAP2 regulation, and induce apoptosis without promoting necrosis via caspase-8/-3 and PARP cleavage. Constitutive nuclear factor-κB (NF-κB) signaling has been shown to contribute to the heightened inflammatory state and survival in TNBC cells. Herein, we also provide evidence that LOE inhibits NF-κB signaling by reducing RIP1 protein levels in MDA-MB-231 cells. These studies reveal that LOE suppresses key features of the progression of aggressive breast cancer cells and provides a basis for further definition of its underlying mechanisms of action and anticancer potential.
Asunto(s)
Lippia/química , FN-kappa B/metabolismo , Extractos Vegetales/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Humanos , FN-kappa B/antagonistas & inhibidores , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , Fase de Descanso del Ciclo Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Obesity has been linked to breast cancer progression but the underlying mechanisms remain obscure. Here we report how leptin, an obesity-associated adipokine, regulates a transcriptional pathway to silence a genetic program of epithelial homeostasis in breast cancer stem-like cells (CSC) that promotes malignant progression. Using genome-wide ChIP-seq and RNA expression profiling, we defined a role for activated STAT3 and G9a histone methyltransferase in epigenetic silencing of miR-200c, which promotes the formation of breast CSCs defined by elevated cell surface levels of the leptin receptor (OBR(hi)). Inhibiting the STAT3/G9a pathway restored expression of miR-200c, which in turn reversed the CSC phenotype to a more differentiated epithelial phenotype. In a rat model of breast cancer driven by diet-induced obesity, STAT3 blockade suppressed the CSC-like OBR(hi) population and abrogated tumor progression. Together, our results show how targeting STAT3-G9a signaling regulates CSC plasticity during obesity-related breast cancer progression, suggesting a novel therapeutic paradigm to suppress CSC pools and limit breast malignancy.