RESUMO
Bone is the most favored site for metastasis for each major subtype of breast cancer. Therapeutic modalities for alleviation of clinical symptoms associated with bone metastasis include surgical resection, radiation, and bone-targeted therapies, including bisphosphonates (e.g., zoledronic acid; ZA) and a humanized antibody against receptor activator of nuclear factor-κB ligand (denosumab). However, the bone-targeted therapies are expensive, and have poor pharmacokinetic attributes and/or serious adverse effects. Therefore, novel strategies are needed for treatment of bone metastasis or to increase effectiveness of existing bone-targeted therapies. We have shown previously that benzyl isothiocyanate (BITC) is a novel inhibitor of osteoclast differentiation in vitro and bone metastasis in vivo. The present study shows that BITC + ZA combination synergistically inhibits osteoclast differentiation induced by addition of conditioned media from breast cancer cells. These effects were associated with a significant increase in levels of several antiosteoclastogenic cytokines, including interferons, interleukin (IL)-3, IL-4, and IL-27. Kyoto Encyclopedia of Genes and Genomes pathway analysis of RNA-seq data from BITC and/or ZA-treated cells revealed downregulation of genes of many pathways (e.g., actin cytoskeleton, Hippo signaling, etc.) by treatment with BITC + ZA combination, but not by BITC alone or ZA alone. Confocal microscopy confirmed severe disruption of actin cytoskeleton upon treatment of MCF-7 and MDA-MB-231 cells with the BITC + ZA combination. This combination also decreased the nuclear level of yes-associated protein, a core component of Hippo signaling. In conclusion, the present study offers a novel combination for prevention or treatment of bone metastasis of breast cancer.
Assuntos
Neoplasias Ósseas , Neoplasias da Mama , Isotiocianatos , Humanos , Feminino , Ácido Zoledrônico/farmacologia , Ácido Zoledrônico/uso terapêutico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Osteoclastos/metabolismo , Osteoclastos/patologia , Transformação Celular Neoplásica , Neoplasias Ósseas/tratamento farmacológicoRESUMO
Bone is the most prone to metastatic spread of breast cancer cells for each subtype of the disease. Bone metastasis-related complications including severe pain and pathological fractures affect patients' quality of life. Current treatment options including surgery, radiation, and bone-targeted therapies (e.g., bisphosphonates) are costly or have serious adverse effects such as renal toxicity and osteonecrosis of the jaws. Therefore, a safe, inexpensive, and efficacious agent for prevention of breast cancer bone metastasis is urgently needed. Our previously published RNA sequencing analysis revealed that many genes implicated in bone remodeling and breast cancer bone metastasis were significantly downregulated by treatment with withaferin A (WA), which is a promising cancer chemopreventive agent derived from a medicinal plant (Withania somnifera). The present study investigated whether WA inhibits breast cancer induction of osteoclast differentiation. At plasma achievable doses, WA treatment inhibited osteoclast differentiation (osteoclastogenesis) induced by three different subtypes of breast cancer cells (MCF-7, SK-BR-3, and MDA-MB-231). WA and the root extract of W. somnifera were equally effective for inhibition of breast cancer induction of osteoclast differentiation. This inhibition was accompanied by suppression of interleukin (IL)-6, IL-8, and receptor activator of nuclear factor-κB ligand, which are pivotal osteoclastogenic cytokines. The expression of runt-related transcription factor 2, nuclear factor-κB, and SOX9 transcription factors, which positively regulate osteoclastogenesis, was decreased in WA-treated breast cancer cells as revealed by confocal microscopy and/or immunoblotting. Taken together, these data suggest that WA could be a promising agent for prevention of breast cancer-induced bone metastasis.
Assuntos
Neoplasias Ósseas , Neoplasias da Mama , Vitanolídeos , Humanos , Feminino , Neoplasias da Mama/genética , Osteoclastos/metabolismo , Osteoclastos/patologia , Qualidade de Vida , Apoptose , Vitanolídeos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Diferenciação CelularRESUMO
Forkhead Box Q1 (FoxQ1) transcription factor is overexpressed in luminal-type and basal-type human breast cancers when compared to normal mammary tissue. This transcription factor is best known for its role in promotion of breast cancer stem-like cells and epithelial to mesenchymal transition. The present study documents a novel function of FoxQ1 in breast cancer cells. Overexpression of FoxQ1 in basal-like SUM159 cells and luminal-type MCF-7 cells resulted in increased conversion of microtubule-associated protein light chain 3 beta-I (LC3B-I) to LC3B-II, which is a hallmark of autophagy. Autophagy induction by FoxQ1 overexpression was confirmed by visualization of LC3B puncta as well as by transmission electron microscopy. Expression profiling for genes implicated in autophagy regulation revealed upregulation of many genes, including ATG4B, ATG16L1, CTSS, CXCR4 and so forth but downregulation of BCL2L1, DRAM1, TNF, ULK2 and so forth by FoxQ1 overexpression in SUM159 cells. Western blot analysis confirmed upregulation of ATG4B and CXCR4 proteins by FoxQ1 overexpression in both SUM159 and MCF-7 cells. Chromatin immunoprecipitation assay revealed recruitment of FoxQ1 at the promoter of ATG4B. Pharmacological inhibition of ATG4B using S130 significantly increased apoptosis induction by DOX in empty vector transfected as well as FoxQ1 overexpressing SUM159 and MCF-7 cells but this effect was statistically significantly lowered by FoxQ1 overexpression indicating the protective role of FoxQ1 on apoptosis. Treatment of SUM159 cells with S130 and DOX enhanced LC3B-II level in both empty vector transfected cells and FoxQ1 overexpressing SUM159 cells but not in FoxQ1 overexpressing MCF-7 cells. In conclusion, FoxQ1 is a novel regulator of autophagy.
Assuntos
Neoplasias da Mama , Feminino , Humanos , Autofagia/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Fatores de TranscriçãoRESUMO
The FoxQ1 is an oncogenic transcription factor that is overexpressed in basal-like and luminal-type human breast cancers when compared to the normal mammary tissue. The FoxQ1 is implicated in mammary tumor progression. However, the mechanism by which FoxQ1 promotes mammary tumorigenesis is not fully understood. In this study, we present experimental evidence for a novel function of FoxQ1 in the regulation of complex I activity of the electron transport chain. The RNA-seq data from FoxQ1 overexpressing basal-like SUM159 cells revealed a statistically significant increase in the expression of complex I subunits NDUFS1 and NDUFS2 when compared to the empty vector (EV) transfected control cells. Consistent with these results, the basal and ATP-linked oxygen consumption rates were significantly increased by FoxQ1 overexpression in SUM159 and luminal-type MCF-7 cells. The FoxQ1 overexpression in both cell lines resulted in increased intracellular levels of pyruvate, lactate, and ATP that was associated with overexpression of pyruvate dehydrogenase and pyruvate carboxylase proteins. Activity and assembly of complex I were significantly enhanced by FoxQ1 overexpression in SUM159 and MCF-7 cells that correlated with increased mRNA and/or protein levels of complex I subunits NDUFS1, NDUFS2, NDUFV1, and NDUFV2. The chromatin immunoprecipitation assay revealed the recruitment of FoxQ1 at the promoters of both NDUFS1 and NDUFV1. The cell proliferation of SUM159 and MCF-7 cells was increased significantly by overexpression of NDUFS1 as well as NDUFV1 proteins. In conclusion, we propose that increased complex I-linked oxidative phosphorylation is partly responsible for oncogenic role of FoxQ1 at least in human breast cancer cells.
Assuntos
Neoplasias da Mama , Trifosfato de Adenosina/metabolismo , Neoplasias da Mama/metabolismo , Transporte de Elétrons , Complexo I de Transporte de Elétrons/genética , Feminino , Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Ácido PirúvicoRESUMO
Diallyl trisulfide (DATS) is a promising small molecule phytochemical that exhibits in vitro and in vivo activity in multiple preclinical solid tumor models including breast cancer, but the underlying mechanism is not fully understood. We have shown previously that forkhead box Q1 (FoxQ1) transcription factor is a novel target for breast cancer stem-like cells (bCSC) inhibition by DATS. Analysis of the breast TCGA (The Cancer Genome Atlas) data revealed that FoxQ1 expression was positively associated with that of SLC16A1/monocarboxylate transporter 1 (MCT1). Western blot analysis confirmed increased expression of MCT1 protein in SUM159 (basal-like) and MCF-7 cells (luminal-type) stably transfected to overexpress FoxQ1. Furthermore, FoxQ1 was recruited to the promoter of SLC16A1/MCT1. Treatment of SUM159 and MCF-7 cell lines with DATS resulted in suppression of MCT1 protein level that was accompanied by a decrease in intracellular and secreted levels of lactate. Overexpression or knockdown of MCT1 protein failed to alter DATS-mediated inhibition of colony formation or cell migration when compared to corresponding control cells. On the other hand, overexpression of MCT1 protein conferred partial but statistically significant protection against DATS-mediated inhibition of bCSC fraction (CD49fhigh /CD44high and aldehyde dehydrogenase 1 activity). The size of the mammospheres was relatively smaller in the DATS-treated group compared to control group. Inhibition of bCSC upon DATS treatment was augmented by knockdown of the MCT1 protein. In conclusion, the present study reveals that MCT1 is a novel target for bCSC inhibition by DATS treatment.
Assuntos
Compostos Alílicos , Neoplasias da Mama , Transportadores de Ácidos Monocarboxílicos/metabolismo , Simportadores/metabolismo , Compostos Alílicos/farmacologia , Apoptose , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Fatores de Transcrição Forkhead/genética , Humanos , Sulfetos/farmacologiaRESUMO
Withaferin A (WA) exhibits cancer chemopreventive efficacy in preclinical models representative of two different subtypes of breast cancer. However, the mechanism(s) underlying breast cancer chemoprevention by WA is not fully elucidated. We performed RNA-seq analyses using a non-tumorigenic mammary epithelial cell line (MCF-10A) and human breast cancer cells (BCC) belonging to the luminal-type (MCF-7), HER2-enriched (SK-BR-3), and basal-like subtype (MDA-MB-231) to identify novel cancer-selective mechanistic targets of WA. The WA-regulated transcriptome was strikingly different between MCF-10A versus BCC. The Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed downregulation of genes associated with cellular senescence in WA-treated BCC. Consequently, the number of senescence-associated ß-galactosidase positive cells was decreased significantly in WA-treated BCC but not in the MCF-10A cells. WA treatment caused upregulation of senescence marker p21 more robustly in BCC than in MCF-10A. Breast cancer prevention by WA in rats was also associated with upregulation of p21 protein expression. The Reactome pathway analyses indicated upregulation of genes associated with cellular response to stress/external stimuli in WA-treated BCC but not in MCF-10A. Two proteins represented in these pathways (HSPA6 and NRF2) were further investigated. While HSPA6 was dispensable for WA-mediated apoptosis and autophagy or inhibition of cell migration, the NRF2 knockout cells were more resistant to apoptosis resulting from WA treatment than control cells. Finally, expression of some glycolysis-related proteins was decreased by WA treatment both in vitro and in vivo. In summary, this study provides novel insights into cancer-selective pathways affected by WA that may contribute to its chemopreventive efficacy in breast cancer.
Assuntos
Anticarcinógenos/farmacologia , Neoplasias da Mama/prevenção & controle , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Vitanolídeos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Células MCF-7 , RNA-Seq , Ratos , Transcriptoma/efeitos dos fármacosRESUMO
Withaferin A (WA) is a promising phytochemical exhibiting in vitro and in vivo anticancer activities against prostate and other cancers, but the mechanism of its action is not fully understood. In this study, we performed RNA-seq analysis using 22Rv1 human prostate cancer cell line to identify mechanistic targets of WA. Kyoto Encyclopedia of Genes and Genomes pathway analysis of the differentially expressed genes showed most significant enrichment of genes associated with metabolism. These results were validated using LNCaP and 22Rv1 human prostate cancer cells and Hi-Myc transgenic mice as models. The intracellular levels of acetyl-CoA, total free fatty acids and neutral lipids were decreased significantly following WA treatment in both cells, which was accompanied by downregulation of mRNA (confirmed by quantitative reverse transcription-polymerase chain reaction) and protein levels of key fatty acid synthesis enzymes, including ATP citrate lyase, acetyl-CoA carboxylase 1, fatty acid synthase and carnitine palmitoyltransferase 1A. Ectopic expression of c-Myc, but not constitutively active Akt, conferred a marked protection against WA-mediated suppression of acetyl-CoA carboxylase 1 and fatty acid synthase protein expression, and clonogenic cell survival. WA was a superior inhibitor of cell proliferation and fatty acid synthesis in comparison with known modulators of fatty acid metabolism including cerulenin and etomoxir. Intraperitoneal WA administration to Hi-Myc transgenic mice (0.1 mg/mouse, three times/week for 5 weeks) also resulted in a significant decrease in circulating levels of total free fatty acids and phospholipids, and expression of ATP citrate lyase, acetyl-CoA carboxylase 1, fatty acid synthase and carnitine palmitoyltransferase 1A proteins in the prostate in vivo.
Assuntos
Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Metaboloma , Neoplasias da Próstata/patologia , RNA-Seq/métodos , Vitanolídeos/farmacologia , Animais , Apoptose , Proliferação de Células , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Células Tumorais CultivadasRESUMO
The transcription factor forkhead box Q1 (FoxQ1) is overexpressed in different solid tumors including breast cancer, but the mechanism underlying its oncogenic function is still not fully understood. In this study, we compared RNA-seq data from FoxQ1 overexpressing SUM159 cells with that of empty vector-transfected control cells to identify novel mechanistic targets of this transcription factor. Analysis of The Cancer Genome Atlas (TCGA) data set revealed significantly higher expression of FoxQ1 in black breast cancer patients compared with white women with this disease. In contrast, expression of FoxQ1 was comparable in ductal and lobular carcinomas in the breast cancer TCGA data set. Complementing our published findings in basal-like subtype, immunohistochemistry revealed upregulation of FoxQ1 protein in luminal-type human breast cancer tissue microarrays when compared with normal mammary tissues. Many previously reported transcriptional targets of FoxQ1 (eg, E-cadherin, N-cadherin, fibronectin 1, etc) were verified from the RNA-seq analysis. FoxQ1 overexpression resulted in the downregulation of genes associated with cell cycle checkpoints, M phase, and cellular response to stress/external stimuli as evidenced from the Reactome pathway analysis. Consequently, FoxQ1 overexpression resulted in mitotic arrest in basal-like SUM159 and human mammary epithelial cell line, but not in luminal-type MCF-7 cells. Finally, we show for the first time that FoxQ1 is a direct transcriptional regulator of interleukin (IL)-1α, IL-8, and vascular endothelial growth factor in breast cancer cells as evidenced by chromatin immunoprecipitation assay. In conclusion, the present study reports novel mechanistic targets of FoxQ1 in human breast cancer cells.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Apoptose , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Ciclo Celular , Movimento Celular , Proliferação de Células , Feminino , Fatores de Transcrição Forkhead/genética , Perfilação da Expressão Gênica , Humanos , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Prognóstico , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Cancer chemoprevention, a scientific term coined by Dr. Sporn in the late seventies, implies use of natural or synthetic chemicals to block, delay or reverse carcinogenesis. Phytochemicals derived from edible and medicinal plants have been studied rather extensively for cancer chemoprevention using preclinical models in the past few decades. Nevertheless, some of these agents (e.g., isothiocyanates from cruciferous vegetables like broccoli and watercress) have already entered into clinical investigations. Examples of widely studied and highly promising phytochemicals from edible and medicinal plants include cruciferous vegetable constituents (phenethyl isothiocyanate, benzyl isothiocyanate, and sulforaphane), withaferin A (WA) derived from a medicinal plant (Withania somnifera) used heavily in Asia, and an oriental medicine plant component honokiol (HNK). An interesting feature of these structurally-diverse phytochemicals is that they target mitochondria to provoke cancer cell-selective death program. Mechanisms underlying cell death induction by commonly studied phytochemicals have been discussed rather extensively and thus are not covered in this review article. Instead, the primary focus of this perspective is to discuss experimental evidence pointing to mitochondrial dysfunction in cancer chemoprevention by promising phytochemicals.
Assuntos
Quimioprevenção , Suplementos Nutricionais , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Neoplasias/prevenção & controle , Compostos Fitoquímicos/administração & dosagem , Plantas Medicinais/química , Animais , Complexo de Proteínas da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Humanos , Mitocôndrias/genética , Dinâmica Mitocondrial/efeitos dos fármacosRESUMO
Increased de novo synthesis of fatty acids is a rather unique and targetable mechanism of human prostate cancer. We have shown previously that oral administration of sulforaphane (SFN) significantly inhibits the incidence and/or burden of prostatic intraepithelial neoplasia and well-differentiated adenocarcinoma in TRansgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice. The present study used cellular models of prostate cancer and archived plasma/adenocarcinoma tissues and sections from the TRAMP study to demonstrate inhibition of fatty acid synthesis by SFN treatment in vitro and in vivo. Treatment of androgen-responsive (LNCaP) and castration-resistant (22Rv1) human prostate cancer cells with SFN (5 and 10 µM) resulted in downregulation of protein and mRNA levels of acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FASN), but not ATP citrate lyase. Protein and mRNA levels of carnitine palmitoyltransferase 1A (CPT1A), which facilitates fatty acid uptake by mitochondria for ß-oxidation, were also decreased following SFN treatment in both cell lines. Immunohistochemistry revealed a significant decrease in expression of FASN and ACC1 proteins in prostate adenocarcinoma sections of SFN-treated TRAMP mice when compared with controls. SFN administration to TRAMP mice resulted in a significant decrease in plasma and/or prostate adenocarcinoma levels of total free fatty acids, total phospholipids, acetyl-CoA and ATP. Consistent with these results, number of neutral lipid droplets was lower in the prostate adenocarcinoma sections of SFN-treated TRAMP mice than in control tumors. Collectively, these observations indicate that prostate cancer chemoprevention by SFN in TRAMP mice is associated with inhibition of fatty acid metabolism.
Assuntos
Anticarcinógenos/farmacologia , Ácidos Graxos/metabolismo , Isotiocianatos/farmacologia , Neoplasias da Próstata/prevenção & controle , Adenocarcinoma/metabolismo , Adenocarcinoma/prevenção & controle , Animais , Quimioprevenção/métodos , Ácido Graxo Sintases/efeitos dos fármacos , Ácido Graxo Sintases/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Próstata/efeitos dos fármacos , Próstata/metabolismo , Neoplasia Prostática Intraepitelial/metabolismo , Neoplasia Prostática Intraepitelial/prevenção & controle , Neoplasias da Próstata/metabolismo , SulfóxidosRESUMO
Diallyl trisulfide (DATS), a metabolic byproduct of garlic, is known to inhibit the growth of breast cancer cells in vitro and in vivo This study demonstrates that DATS targets breast cancer stem cells (bCSC). Exposure of MCF-7 and SUM159 human breast cancer cells to pharmacological concentrations of DATS (2.5 and 5 µm) resulted in dose-dependent inhibition of bCSC, as evidenced by a mammosphere assay and flow cytometric analysis of aldehyde dehydrogenase 1 (ALDH1) activity and the CD44(high)/CD24(low)/epithelial specific antigen-positive fraction. DATS-mediated inhibition of bCSC was associated with a decrease in the protein level of FoxQ1. Overexpression of FoxQ1 in MCF-7 and SUM159 cells increased ALDH1 activity and the CD49f(+)/CD24(-) fraction. Inhibition of ALDH1 activity and/or mammosphere formation upon DATS treatment was significantly attenuated by overexpression of FoxQ1. In agreement with these results, stable knockdown of FoxQ1 using small hairpin RNA augmented bCSC inhibition by DATS. Expression profiling for cancer stem cell-related genes suggested that FoxQ1 may negatively regulate the expression of Dachshund homolog 1 (DACH1), whose expression is lost in invasive breast cancer. Chromatin immunoprecipitation confirmed recruitment of FoxQ1 at the DACH1 promoter. Moreover, inducible expression of DACH1 augmented DATS-mediated inhibition of bCSC. Expression of FoxQ1 protein was significantly higher in triple-negative breast cancer cases compared with normal mammary tissues. Moreover, an inverse association was observed between FoxQ1 and DACH1 gene expression in breast cancer cell lines and tumors. DATS administration inhibited ALDH1 activity in vivo in SUM159 xenografts. These results indicate that FoxQ1 is a novel target of bCSC inhibition by DATS.
Assuntos
Compostos Alílicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Fatores de Transcrição Forkhead/metabolismo , Isoenzimas/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Retinal Desidrogenase/antagonistas & inibidores , Sulfetos/farmacologia , Família Aldeído Desidrogenase 1 , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Células MCF-7 , Camundongos , Camundongos Nus , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/patologia , Retinal Desidrogenase/genética , Retinal Desidrogenase/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The present study is the first to report inhibition of breast cancer cell growth in vitro and in vivo and suppression of self-renewal of breast cancer stem cells (bCSC) by a pine bark component (leelamine). Except for a few recent publications in melanoma, anticancer pharmacology of this interesting phytochemical is largely elusive. Leelamine (LLM) dose-dependently inhibited viability of MDA-MB-231 (triple-negative), MCF-7 (estrogen receptor-positive), and SUM159 (triple-negative) human breast cancer cells in association with apoptotic cell death induction. To the contrary, a normal mammary epithelial cell line derived from fibrocystic breast disease and spontaneously immortalized (MCF-10A) was fully resistant to LLM-mediated cell growth inhibition and apoptosis induction. LLM also inhibited self-renewal of breast cancer stem cells. Apoptosis induction by LLM in breast cancer cells was accompanied by a modest increase in reactive oxygen species production, which was not due to inhibition of mitochondrial electron transport chain complexes. Nevertheless, ectopic expression of manganese superoxide dismutase conferred partial protection against LLM-induced cell death but only at a lower yet pharmacologically relevant concentration. Exposure of breast cancer cells to LLM resulted in (a) induction and/or activation of multidomain proapoptotic proteins Bax and Bak, (b) caspase-9 activation, and (c) cytosolic release of cytochrome c. Bax and Bak deficiency in immortalized fibroblasts conferred significant protection against cell death by LLM. Intraperitoneal administration of LLM (7.5 mg/kg; 5 times/wk) suppressed the growth of orthotopic SUM159 xenografts in mice without any toxicity. In conclusion, the present study provides critical preclinical data to warrant further investigation of LLM. © 2016 Wiley Periodicals, Inc.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Mama/efeitos dos fármacos , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos Nus , Pinus/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
The present study offers novel insights into the molecular circuitry of accelerated in vivo tumor growth by Notch2 knockdown in triple-negative breast cancer (TNBC) cells. Therapeutic vulnerability of Notch2-altered growth to a small molecule (withaferin A, WA) is also demonstrated. MDA-MB-231 and SUM159 cells were used for the xenograft studies. A variety of technologies were deployed to elucidate the mechanisms underlying tumor growth augmentation by Notch2 knockdown and its reversal by WA, including Fluorescence Molecular Tomography for measurement of tumor angiogenesis in live mice, Seahorse Flux analyzer for ex vivo measurement of tumor metabolism, proteomics, and Luminex-based cytokine profiling. Stable knockdown of Notch2 resulted in accelerated in vivo tumor growth in both cells reflected by tumor volume and/or latency. For example, the wet tumor weight from mice bearing Notch2 knockdown MDA-MB-231 cells was about 7.1-fold higher compared with control (P < 0.0001). Accelerated tumor growth by Notch2 knockdown was highly sensitive to inhibition by a promising steroidal lactone (WA) derived from a medicinal plant. Molecular underpinnings for tumor growth intensification by Notch2 knockdown included compensatory increase in Notch1 activation, increased cellular proliferation and/or angiogenesis, and increased plasma or tumor levels of growth stimulatory cytokines. WA administration reversed many of these effects providing explanation for its remarkable anti-cancer efficacy. Notch2 functions as a tumor growth suppressor in TNBC and WA offers a novel therapeutic strategy for restoring this function.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Receptor Notch2/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Vitanolídeos/administração & dosagem , Animais , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Receptor Notch1/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Vitanolídeos/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Withaferin A (WA), a C5,C6-epoxy steroidal lactone derived from a medicinal plant (Withania somnifera), inhibits growth of human breast cancer cells in vitro and in vivo and prevents mammary cancer development in a transgenic mouse model. However, the mechanisms underlying the anticancer effect of WA are not fully understood. Herein, we report that tubulin is a novel target of WA-mediated growth arrest in human breast cancer cells. The G2 and mitotic arrest resulting from WA exposure in MCF-7, SUM159, and SK-BR-3 cells was associated with a marked decrease in protein levels of ß-tubulin. These effects were not observed with the naturally occurring C6,C7-epoxy analogs of WA (withanone and withanolide A). A non-tumorigenic normal mammary epithelial cell line (MCF-10A) was markedly more resistant to mitotic arrest by WA compared with breast cancer cells. Vehicle-treated control cells exhibited a normal bipolar spindle with chromosomes aligned along the metaphase plate. In contrast, WA treatment led to a severe disruption of normal spindle morphology. NMR analyses revealed that the A-ring enone in WA, but not in withanone or withanolide A, was highly reactive with cysteamine and rapidly succumbed to irreversible nucleophilic addition. Mass spectrometry demonstrated direct covalent binding of WA to Cys(303) of ß-tubulin in MCF-7 cells. Molecular docking indicated that the WA-binding pocket is located on the surface of ß-tubulin and characterized by a hydrophobic floor, a hydrophobic wall, and a charge-balanced hydrophilic entrance. These results provide novel insights into the mechanism of growth arrest by WA in breast cancer cells.
Assuntos
Neoplasias da Mama/metabolismo , Regulação para Baixo/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Vitanolídeos/farmacologia , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Regulação para Baixo/genética , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Fuso Acromático/genética , Fuso Acromático/metabolismo , Fuso Acromático/patologia , Tubulina (Proteína)/genética , Vitanolídeos/farmacocinéticaRESUMO
We showed previously that garden cress constituent benzyl isothiocyanate (BITC) inhibits self-renewal of breast cancer stem cells (bCSC) in vitro and in vivo. The present study offers novel insights into the mechanism by which BITC inhibits bCSC. Flow cytometry and mammosphere assay were performed to quantify bCSC fraction. Protein expression was determined by western blotting. Apoptosis was assessed by flow cytometry using Annexin V-propidium iodide method. Cell migration was determined by Boyden chamber assay. BITC treatment resulted in a marked decrease in protein level of polycomb group protein B-lymphoma Moloney murine leukemia virus insertion region-1 (Bmi-1) in cultured human breast cancer cells (MCF-7, SUM159, MDA-MB-231, and MDA-MB-361) and MDA-MB-231 xenografts in vivo. Overexpression (MCF-7) or knockdown (SUM159, and MDA-MB-231) of Bmi-1 protein had no meaningful impact on the BITC's ability to inhibit cell viability and cell migration and/or induce apoptosis. On the other hand, inhibition of bCSC markers (aldehyde dehydrogenase 1 activity and mammosphere frequency) resulting from BITC exposure was significantly altered by Bmi-1 overexpression and knockdown. BITC was previously shown to cause activation of Notch1, Notch2, and Notch4 in association with induction of γ-secretase complex component Nicastrin, which are also implicated in maintenance of cancer stemness. BITC-mediated inhibition of bCSC was augmented by knockdown of Notch4 and Nicastrin, but not by RNA interference of Notch1 or Notch2. The present study highlights important roles for Bmi-1 and Notch4 in BITC-mediated suppression of bCSC.
Assuntos
Neoplasias da Mama/genética , Proliferação de Células/efeitos dos fármacos , Isotiocianatos/administração & dosagem , Complexo Repressor Polycomb 1/genética , Proteínas Proto-Oncogênicas/genética , Receptores Notch/genética , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Isotiocianatos/química , Lepidium sativum/química , Células MCF-7 , Células-Tronco Neoplásicas/efeitos dos fármacos , Complexo Repressor Polycomb 1/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Interferência de RNA , Receptor Notch4 , Receptores Notch/biossínteseRESUMO
Despite advances in therapeutic approaches, the five-year survival rate for head and neck squamous cell carcinoma (HNSCC) patients is still less than fifty percent. Research has indicated that the consumption of Allium vegetables or processed garlic containing diallyl trisulfide (DATS) can lower the risk of multiple types of cancer. Nevertheless, the effectiveness and underlying mechanisms of DATS against HNSCC have not been thoroughly explored until the current study. In this research, it was found that DATS notably curtailed the growth and viability of HNSCC cells. Additionally, DATS triggered a significant G2/M cell cycle arrest in these cells, accumulating cyclin B1, Cip1/p21, and Ser-10 phospho-histone H3-this was indicative of mitotic arrest attenuated by NAC pretreatment, suggesting the role of reactive oxygen species (ROS) induction. The production of ROS induced by DATS led to DNA damage and apoptosis, a process associated with elevated levels of cleaved caspase-3 and cleaved PARP, along with reduced XIAP. When HNSCC cells were exposed to pharmacological concentrations of DATS, it resulted in the suppression of cancer stem cell (CSC) populations, as indicated by a decrease in the CD133high/CD44high cell fraction, reduced aldehyde dehydrogenase 1 (ALDH1) activity, inhibited spheroid formation and downregulated SOX2 and Oct4 expression. Furthermore, the administration of DATS to tumor xenografts demonstrated its in vivo capacity to hinder CSCs. Further, DATS treatment inhibited the growth of UMSCC-22B head and neck cancer tumor xenograft in immunocompromised mice. Overall, DATS inhibited cell proliferation; induced cell cycle mitotic arrest and apoptosis involving DNA damage through ROS generation; reduced the CSC fraction and spheroid formation; and downregulated SOX2 and Oct4 expression. More importantly, DATS inhibited HNSCC tumor growth and CSC fraction in vivo. Thus, DATS could be a potential anticancer agent that can be used against head and neck cancer.
RESUMO
We showed previously that breast cancer chemoprevention with benzyl isothiocyanate (BITC) in MMTV-neu mice was associated with induction of E-cadherin protein in vivo. Loss of E-cadherin expression and induction of mesenchymal markers (e.g. vimentin) are biochemical hallmarks of epithelial-mesenchymal transition (EMT), a developmental process implicated in progression of cancer to aggressive state. This study offers novel insights into the mechanism by which BITC inhibits EMT. Exposure of MDA-MB-231, SUM159 and MDA-MB-468 human breast cancer cells to BITC (2.5 and 5 µM) resulted in transcriptional repression of urokinase-type plasminogen activator (uPA) as well as its receptor (uPAR). However, ectopic expression of uPAR in MDA-MB-468 cells failed to confer protection against induction of E-cadherin and inhibition of cell invasion/migration resulting from BITC treatment. The BITC-mediated induction of E-cadherin and inhibition of cell migration was sustained in MDA-MB-231 and SUM159 cells transiently transfected with an uPAR-targeted small interfering RNA. Overexpression of Forkhead Box Q1 (FOXQ1), whose protein and messenger RNA levels were decreased by BITC treatment in cells and MDA-MB-231 xenografts, conferred marked protection against BITC-mediated inhibition of EMT and cell migration. In conclusion, this study implicates FOXQ1 suppression in BITC-mediated inhibition of EMT in human breast cancer cells.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fatores de Transcrição Forkhead/metabolismo , Isotiocianatos/farmacologia , Animais , Neoplasias da Mama/genética , Caderinas/biossíntese , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Progressão da Doença , Feminino , Fatores de Transcrição Forkhead/genética , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transplante Heterólogo , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Withaferin A (WA), which is a small molecule derived from a medicinal plant (Withania somnifera), inhibits growth of human breast cancer xenografts and mammary tumor development in rodent models without any toxicity. However, the mechanism underlying inhibition of mammary cancer development by WA administration is not fully understood. Herein, we demonstrate that the fatty acid synthesis pathway is a novel target of WA in mammary tumors. Treatment of MCF-7 and MDA-MB-231 cells with WA resulted in suppression of fatty acid metabolizing enzymes, including ATP-citrate lyase (ACLY), acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FASN), and carnitine palmitoyltransferase 1A (CPT1A). Expression of FASN and CPT1A was significantly higher in N-methyl-N-nitrosourea-induced mammary tumors in rats when compared with normal mammary tissues. WA-mediated inhibition of mammary tumor development in rats was associated with a statistically significant decrease in expression of ACC1 and FASN and suppression of plasma and/or mammary tumor levels of total free fatty acids and phospholipids. WA administration also resulted in a significant increase in percentage of natural killer cells in the spleen. The protein level of sterol regulatory element binding protein 1 (SREBP1) was decreased in MDA-MB-231 cells after WA treatment. Overexpression of SREBP1 in MDA-MB-231 cells conferred partial but significant protection against WA-mediated downregulation of ACLY and ACC1. In conclusion, circulating and/or mammary tumor levels of fatty acid synthesis enzymes and total free fatty acids may serve as biomarkers of WA efficacy in future clinical trials. PREVENTION RELEVANCE: The present study shows that breast cancer prevention by WA in rats is associated with suppression of fatty acid synthesis.
Assuntos
Neoplasias da Mama , Neoplasias Mamárias Animais , Vitanolídeos , Ratos , Humanos , Animais , Feminino , Ácidos Graxos não Esterificados/uso terapêutico , Apoptose , Vitanolídeos/farmacologia , Vitanolídeos/uso terapêutico , Neoplasias da Mama/patologia , Neoplasias Mamárias Animais/tratamento farmacológico , Ácidos GraxosRESUMO
Cathode active materials and conductive additives for thermal batteries operating at high temperatures have attracted research interest, with a particular focus on compounds offering high thermal stability. Recently, FeF3 has been proposed as a candidate for high-voltage cathode materials; however, its commercialization is hindered by its low conductivity. In this study, conductive additives, such as Ni-coated carbon composites (multi-walled carbon nanotubes (MWCNTs) and carbon black (CB)), were utilized to enhance the thermal stability and conductivity of FeF3. The incorporation of metal-carbon conductive additives in the FeF3 composite increased the thermal stability by more than 10 wt.% and ensured high capacity upon conductivity enhancement. The FeF3@Ni/MWCB 15 wt.% composite containing 30 wt.% Ni exhibited a discharge capacity of â¼86% of the theoretical capacity of 712 mAh/g. The use of Ni-coated carbon-based conductive additives will allow the application of FeF3 as an effective high-temperature cathode material for thermal batteries.
RESUMO
Considerable research is being conducted on the use of FeF3 as a cathode replacement for FeS2 in thermal batteries. However, FeF3 alone is inefficient as a cathode active material because of its low electrical conductivity due to its wide bandgap (5.96 eV). Herein, acetylene black and multi-walled carbon nanotubes (MWCNTs) were combined with FeF3, and the ratio was optimized. When acetylene black and MWCNTs were added separately to FeF3, the electrical conductivity increased, but the mechanical strength decreased. When acetylene black and MWCNTs were both added to FeF3, the FeF3/M1AB4 sample (with 1 wt.% MWCNTs and 4% AB) afforded a discharge capacity of approximately 74% of the theoretical capacity (712 mAh/g) of FeF3. Considering the electrical conductivity and mechanical strength, this composition was confirmed to be the most suitable.