RESUMEN
Breast cancer is the first cause of cancer death in women. Many patients are resistant to current therapies, and even those were sensitive at first may eventually become resistant later. Thiosemicarbazones (TSCs) are synthetic compounds that exhibit several pharmacological activities. In this study, we investigated the potential anti-tumor activity of a set of N4 -arylsubstituted TSCs (N4 -TSCs) on human breast cancer cell lines. Studies on the effect of N4 -TSCs (T1, T2, and T3) were carried on MCF-7, MDA-MB 231, and BT 474 cell lines which differ in their expression of ER, PR, and Her2/neu. Non-transformed MCF-10A breast cell line were used as normal cells. Action of N4 -TSCs were evaluated by proliferation assay, quantification of apoptosis and cell cycle analysis. Modulation of clonogenic efficiency and migratory capacity by N4 -TSCs were also evaluated. We further investigated the effects of N4 -TSCs on ROS level and Ribonucleotide Reductase (RR) activity. We analyzed the action of these compounds on cellular mammosphere-forming capacity. We found that T1 and T2 had specific anti-tumor effect on all breast cancer cell lines based on their pro-apoptotic action and inhibitory effect on clonogenic efficiency and cell migration capacity. We also showed that both compounds increased ROS level and inhibited RR activity. Finally, we found that all N4 -TSCs diminished mammospehere-forming capacity of MCF-7 and BT 474 cells. N4 -TSCs showed specific anti-tumor action on human breast cancer cells independently their biomarkers expression pattern. Our results place these compounds as promising novel anti-tumor drugs with potential therapeutic application against different types of breast cancer.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Indanos/farmacología , Tiosemicarbazonas/farmacología , Antineoplásicos/síntesis química , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Indanos/síntesis química , Células MCF-7 , Necrosis , Especies Reactivas de Oxígeno/metabolismo , Ribonucleótido Reductasas/antagonistas & inhibidores , Ribonucleótido Reductasas/metabolismo , Transducción de Señal/efectos de los fármacos , Tiosemicarbazonas/síntesis químicaRESUMEN
We investigated activity and mechanism of action of two AhR ligand antitumor agents, AFP 464 and 5F 203 on human renal cancer cells, specifically examining their effects on cell cycle progression, apoptosis, and migration. TK-10, SN12C, Caki-1, and ACHN human renal cancer cell lines were treated with AFP 464 and 5F 203. We evaluated cytotoxicity by MTS assays, cell cycle arrest, and apoptosis by flow cytometry and corroborated a mechanism of action involving AhR signal transduction activation. Changes in migration properties by wound healing assays were investigated: 5F 203-sensitive cells show decreased migration after treatment, therefore, we measured c-Met phosphorylation by Western blot in these cells. A 5F 203 induced a decrease in cell viability which was more marked than AFP 464. This cytotoxicity was reduced after treatment with the AhR inhibitor α-NF for both compounds indicating AhR signaling activation plays a role in the mechanism of action. A 5F 203 is sequestered by TK-10 cells and induces CYP1A1 expression; 5F 203 potently inhibited migration of TK-10, Caki-1, and SN12C cells, and inhibited c-Met receptor phosphorylation in TK-10 cells. AhR ligand antitumor agents AFP 464 and 5F 203 represent potential new candidates for the treatment of renal cancer. A 5F 203 only inhibited migration of sensitive cells and c-Met receptor phosphorylation in TK-10 cells. c-Met receptor signal transduction is important in migration and metastasis. Therefore, we consider that 5F 203 offers potential for the treatment of metastatic renal carcinoma. J. Cell. Biochem. 118: 4526-4535, 2017. © 2017 Wiley Periodicals, Inc.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Células Renales/tratamiento farmacológico , Flavonoides/farmacología , Neoplasias Renales/tratamiento farmacológico , Proteínas de Neoplasias/agonistas , Receptores de Hidrocarburo de Aril/agonistas , Tiazoles/farmacología , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Humanos , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Proteínas de Neoplasias/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Aminoflavone (AFP 464, NSC 710464), an antitumor agent which recently entered phase II clinical trials, acts against estrogen-positive breast cancer (ER+). AFP 464, which has a unique mechanism of action by activating aryl hydrocarbon receptor (AhR) signaling pathway, decreased tumor size, and growth rate in the estrogen dependent, Tamoxifen-sensitive spontaneous M05 mouse model. Considering that AhR has recently emerged as a physiological regulator of the innate and adaptive immune responses, we investigated whether AFP 464 modulates the immune response in our mouse model. Studies on the effect of AFP 464 on the immune system were carried in BALB/c mice bearing M05 semi-differentiated mammary adenocarcinomas that express estrogen and progesterone receptors. Splenic cells and tumor inflammatory infiltrates were studied by cytometric analyses. The modulation of splenocytes cytotoxic activity by AFP 464 was also evaluated. We further investigated the effects of AFP 464 on peritoneal macrophages by evaluating metalloproteinase, arginase, and iNOS activities. We found that AFP 464 increased splenic cytotoxic activity, diminished the number of systemic and local Treg lymphocytes, and MDSCs, and induced a M1 phenotype in peritoneal macrophages of M05 tumor bearing mice. Therefore, we conclude that AFP 464 modulates immune response which collaborates with its anti-tumor activity. Our results place the immune system as a novel target for this anti-cancer agent to strengthen the rationale for its inclusion in breast cancer treatment regimens. J. Cell. Biochem. 118: 2841-2849, 2017. © 2017 Wiley Periodicals, Inc.
Asunto(s)
Antineoplásicos/farmacología , Flavonoides/farmacología , Inmunidad Celular/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Neoplasias Mamarias Animales/tratamiento farmacológico , Linfocitos T Reguladores/inmunología , Animales , Femenino , Macrófagos Peritoneales/patología , Neoplasias Mamarias Animales/inmunología , Neoplasias Mamarias Animales/patología , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico Sintasa de Tipo II/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
Traditional chemotherapies debulk tumors but fail to produce long-term clinical remissions due to their inability to eradicate tumor-initiating cells (TICs). This necessitates therapy with activity against the TIC niche. Αlpha6-integrin (α6-integrin) promotes TIC growth. In contrast, aryl hydrocarbon receptor (AhR) signaling activation impedes the formation of mammospheres (clusters of cells enriched for TICs). We investigated the ability of AhR agonist Aminoflavone (AF) and AF pro-drug (AFP464) to disrupt mammospheres derived from breast cancer cells and a M05 mammary mouse model of breast cancer respectively. We further examined the capacity of AF and AFP464 to exhibit anticancer activity and modulate the expression of 'stemness' genes including α6-integrin using immunofluorescence, flow cytometry and qRT-PCR analysis. AF disrupted mammospheres and prevented secondary mammosphere formation. In contrast, AF did not disrupt mammospheres derived from AhR ligand-unresponsive MCF-7 cells. AFP464 treatment suppressed M05 tumor growth and disrupted corresponding mammospheres. AF and AFP464 reduced the expression and percentage of cells that stained for 'stemness' markers including α6-integrin in vitro and in vivo respectively. These data suggest AFP464 thwarts bulk breast tumor and TIC growth via AhR agonist-mediated α6-integrin inhibition.
Asunto(s)
Antineoplásicos/farmacología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/agonistas , Neoplasias de la Mama/tratamiento farmacológico , Flavonoides/farmacología , Integrina alfa6/metabolismo , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Profármacos/farmacología , Receptores de Hidrocarburo de Aril/agonistas , Transporte Activo de Núcleo Celular , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Integrina alfa6/genética , Ligandos , Células MCF-7 , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones Endogámicos BALB C , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fenotipo , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de Señal/efectos de los fármacos , Esferoides Celulares , Factores de TiempoRESUMEN
Aminoflavone (AF; NSC 686288, AFP464, NSC710464) is a new anticancer drug that has recently entered phase II clinical trials. It has demonstrated antiproliferative effects in MCF-7 human breast cancer cells mediated by the aryl hydrocarbon receptor (AhR). AF also exhibits noteworthy evidence of antitumor activity in vitro and in vivo against neoplastic cells of renal origin. AF treatment of sensitive renal cells, in contrast to resistant cells, promotes the induction of CYP1A1, the covalent binding of AF-reactive intermediates and apoptosis. Based on this evidence, the aim of this study was to evaluate the role of AhR, the main transcriptional regulator of CYP1A1, in the antiproliferative effects of AF in human renal cancer cells. AF-cytoxicity in human renal cell lines and a renal cancer cell strain was assessed by MTS assay in the presence or absence of an Ahr inhibitor. Drug-induced AhR nuclear translocation was evaluated by western blotting of AhR in cytosolic and nuclear fractions and by measuring xenobiotic response element-driven luciferase activity. Apoptosis induced by the drug was evaluated by 4,6-diamidino-2-phenylindole and acridine orange/ethidium bromide staining and by measuring phosphorylated P53 (p-P53) and P21 levels, caspase 3 activation and poly(ADP-ribose) polymerase cleavage. AF inhibited cell growth in a dose-dependent manner in TK-10, Caki-1, SN12-C and A498 human renal cells but not in ACHN cells. The antiproliferative effect of AF was abrogated by pre-incubation of TK-10, Caki-1 and SN12-C cells with the AhR antagonist, α-naphthoflavone. AF treatment also induced apoptosis in TK-10, Caki-1 and SN12-C cells, which was not observed in ACHN cells. AF induced time-dependent AhR nuclear translocation and AhR transcriptional activity in sensitive renal cancer cell lines. A renal cell strain derived from a human papillary tumor also showed sensitivity to AF, as well as AhR pathway activation and drug-induced apoptosis. AhR translocation could be included as a marker of sensitivity to AF in sensitive renal tumor cells of different histological origin, in ongoing phase II clinical trials.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma Papilar/tratamiento farmacológico , Carcinoma de Células Renales/tratamiento farmacológico , Flavonoides/farmacología , Neoplasias Renales/tratamiento farmacológico , Receptores de Hidrocarburo de Aril/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Hidrocarburo de Aril Hidroxilasas/genética , Carcinoma Papilar/patología , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1 , Genes Reporteros , Humanos , Neoplasias Renales/patología , Luciferasas/biosíntesis , Luciferasas/genética , Terapia Molecular Dirigida , Regiones Promotoras Genéticas , Activación Transcripcional/efectos de los fármacosRESUMEN
Many estrogen-receptor- (ER-) expressing breast cancers become refractory to ER-based therapies. New antitumor drugs like aminoflavone (AF) and benzothiazoles (Bzs) have been developed and have exquisite antitumor activity in ER+MCF-7 and T47D cells and in a MCF-7 nude mouse model. ER(-) breast cancer cells like MDA-MB-231 are less susceptible. We previously found in MCF-7 cells that these drugs activate the aryl hydrocarbon receptor (AhR) via translocation to the nucleus, induction of AhR-specific DNA binding activity, and expression of CYP1A1, whose transcription is controlled by the AhR-ARNT transcription factor. CYP1A1 metabolizes AF and Bz to a species which directly or after further metabolism damages DNA. In contrast an AhR-deficient variant of MCF-7 or cells with predominantly nuclear AhR expression, such as MDA-MB 231, are resistant. Thus, these drugs, unlike other neoplastic agents, require AhR-mediated signaling to cause DNA damage. This is a new treatment strategy for breast cancers with intact AhR signaling.
RESUMEN
2-(4-Amino-3-methylphenyl)-5-fluoro-benzothiazole (5F 203) potently inhibits MCF-7 breast cancer cell growth in part by activating the aryl hydrocarbon receptor (AhR) signaling pathway. Ligands for the AhR (i.e. dioxin) have also been shown to modulate the NF-kappaB signaling cascade, affecting physiological processes such as cellular immunity, inflammation, proliferation and survival. The objective of this study was to investigate the effect of 5F 203 treatment on the NF-kappaB signaling pathway in breast cancer cells. Exposure of MCF-7 cells to 5F 203 increased protein-DNA complex formation on the NF-kappaB-responsive element as determined by electrophoretic mobility shift assay, but this effect was eliminated in MDA-MB-435 cells, which are resistant to the antiproliferative effects of 5F 203. An increase in NF-kappaB-dependent transcriptional activity was confirmed by a significant increase in NF-kappaB-dependent reporter activity in sensitive MCF-7 cells, which was absent in resistant MDA-MB-435 cells and AhR-deficient subclones of MCF-7 cells. Inhibition of NF-kappaB activation enhanced the increase in xenobiotic response element-dependent reporter activity in MCF-7 cells when treated with 5F 203. The drug candidate 5F 203 also induced mRNA levels of IL-6, an NF-kappaB-responsive gene, in MCF-7 cells, but not in MDA-MB-435 cells, as determined by quantitative RT-PCR. These findings suggest that 5F 203 activation of the NF-kappaB signaling cascade may contribute to 5F 203-mediated anticancer activity in human breast cancer MCF-7 cells.
Asunto(s)
Antineoplásicos/farmacología , FN-kappa B/agonistas , Tiazoles/farmacología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Ensayo de Cambio de Movilidad Electroforética , Femenino , Genes Reporteros , Humanos , Interleucina-6/biosíntesis , Interleucina-6/genética , Luciferasas/genética , FN-kappa B/genética , Receptores de Hidrocarburo de Aril/genética , Elementos de Respuesta , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción GenéticaRESUMEN
Aminoflavone (4H-1-benzopyran-4-one, 5-amino-2-(4-amino-3-fluorophenyl)-6,8-difluoro-7-methyl; NSC 686288) demonstrates differential antiproliferative activity in the National Cancer Institute's anticancer drug screen. We demonstrate here that MCF-7 human breast cancer cells are sensitive to aminoflavone both in vitro and when grown in vivo as xenografts in athymic mice. As previous work has indicated that aminoflavone requires metabolic activation by cytochrome P450 1A1 (CYP1A1), we investigated the effect of aminoflavone on CYP1A1 expression and on the aryl hydrocarbon receptor (AhR), a transcriptional regulator of CYP1A1. In aminoflavone-sensitive but not aminoflavone-resistant cells, the drug caused a 100-fold induction of CYP1A1 mRNA and a corresponding increase in ethoxyresorufin-O-deethylase activity. An AhR-deficient variant of the MCF-7 breast carcinoma, AH(R100), with diminished CYP1A1 inducibility, exhibits cellular resistance to aminoflavone and is refractory to CYP1A1 mRNA induction by the drug. The increase in CYP1A1 mRNA in the aminoflavone-sensitive MCF-7 breast tumor cell results from transcriptional activation of xenobiotic-responsive element (XRE)-controlled transcription. Aminoflavone treatment causes a translocation of the AhR from the cytoplasm to the nucleus with subsequent formation of AhR-XRE protein DNA complexes. In contrast to the aminoflavone-sensitive MCF-7 cells, the resistant cell lines (MDA-MB-435, PC-3, and AH(R100)) demonstrated constitutive nuclear localization of AhR. Additionally, aminoflavone failed to induce ethoxyresorufin-O-deethylase activity, CYP1A1 transcription, AhR-XRE complex formation, and apoptosis in aminoflavone-resistant cells. These results suggest that the cytotoxicity of aminoflavone in a sensitive breast tumor cell line is the result of the engagement of AhR-mediated signal transduction.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Flavonoides/farmacología , Receptores de Hidrocarburo de Aril/metabolismo , Animales , Antineoplásicos/metabolismo , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Hidrocarburo de Aril Hidroxilasas/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1 , Inducción Enzimática/efectos de los fármacos , Flavonoides/metabolismo , Flavonoides/toxicidad , Humanos , Ratones , Ratones Desnudos , Regiones Promotoras Genéticas/genética , Transporte de Proteínas/efectos de los fármacos , Receptores de Hidrocarburo de Aril/genética , Elementos de Respuesta/genética , Transcripción Genética/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
2-(4-Amino-3-methylphenyl) benzothiazole (NSC 674495; DF 203) demonstrates drug uptake and metabolism by tumor cells sensitive to the antiproliferative activity of the drug [J Med Chem 1999;42:4172-4184]. In insensitive cells, little metabolism occurs. Because CYP1A1 can metabolize DF 203, the aryl hydrocarbon receptor (AhR) may mediate drug action. We demonstrate here that DF 203 increases CYP1A1 and CYP1B1 transcription in sensitive MCF-7 cells, accompanied by AhR translocation to the nucleus, increase in xenobiotic-responsive element (XRE)-driven luciferase activity, and induction of protein/DNA complexes on the XRE sequence of the CYP1A1 promoter. MDA-MB-435 and PC3 cells, resistant to DF 203, did not show drug-induced CYP1A1 and CYP1B1 gene expression. AhR was observed to be constitutively localized in the nucleus, with no induction of XRE-driven luciferase activity in transiently transfected cells and weak or no induction of protein/DNA complexes on the XRE sequence of CYP1A1. Taken together, these data elucidate a novel basis for antitumor drug action: induction in sensitive cells of a metabolizing system for the drug itself. These results suggest that clarification of the basis for differential engagement of AhR-related signaling in different tumor cell types may aid in further preclinical development and perhaps early clinical studies.