RESUMEN
Analogs of 1α,25-dihydroxyvitamin D3 (S1) with 20-epi modification (20-epi analogs) possess unique biological properties. We previously reported that 1α,25-dihydroxy-20-epi-vitamin D3 (S2), the basic 20-epi analog is metabolized into less polar metabolites (LPMs) in rat osteosarcoma cells (UMR-106) but not in a perfused rat kidney. Furthermore, we also noted that only selective 20-epi analogs are metabolized into LPMs. For example, 1α,25-dihydroxy-16-ene-20-epi-vitamin D3 (S4), but not 1α,25-dihydroxy-16-ene-23-yne-20-epi-vitamin D3 (S5) is metabolized into LPMs. In spite of these novel findings, the unequivocal identification of LPMs has not been achieved to date. We report here on a thorough investigation of the metabolism of S4 in UMR-106 cells and isolated two major LPMs produced directly from the substrate S4 itself and two minor LPMs produced from 3-epi-S4, a metabolite of S4 produced through C-3 epimerization pathway. Using GC/MS, ESI-MS and 1H NMR analysis, we identified all the four LPMs of S4 as 25-hydroxy-16-ene-20-epi-vitamin D3-1-stearate and 25-hydroxy-16-ene-20-epi-vitamin D3-1-oleate and their respective C-3 epimers. We report here for the first time the elucidation of a novel pathway of metabolism in UMR-106 cells in which both 1α,25(OH)2-16-ene-20-epi-D3 and 1α,25(OH)2-16-ene-20-epi-3-epi-D3 undergo C-1 esterification into stearic and oleic acid esters.
Asunto(s)
Colecalciferol/metabolismo , Animales , Calcitriol/química , Calcitriol/metabolismo , Línea Celular Tumoral , Colecalciferol/química , Ésteres/química , Ésteres/metabolismo , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Espectroscopía de Resonancia Magnética , Osteosarcoma/metabolismo , Ratas , Espectrometría de Masa por Ionización de Electrospray , Estereoisomerismo , Vitamina D/análogos & derivados , Vitamina D/química , Vitamina D/metabolismoRESUMEN
Tumor-initiating cells (also known as cancer stem cells) are the subpopulation of cells shown to be responsible for tumor initiation, maintenance and recurrence. In breast cancer, CD44(+)/CD24(-/low) cells were identified as tumor-initiating cells. We previously reported that a Gemini vitamin D analog, 1,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol (BXL0124), reduced CD44(+)/CD24(-/low) cells in MCF10DCIS basal-like breast cancer cells. Since Notch has been identified as one of the key signaling pathways involved in breast cancer stem cells, the effect of BXL0124 on the Notch signaling pathway was investigated in breast cancer. The CD44(+)/CD24(-/low) subpopulation of MCF10DCIS cells showed elevated Notch1 signaling and increased cell proliferation compared to the CD44(+)/CD24(high) subpopulation. Treatment with the Gemini vitamin D analog BXL0124 decreased the level of activated Notch1 receptor. In addition, mRNA and protein levels of the Notch ligands, Jagged-1, Jagged-2 and DLL1, were significantly reduced by treatment with BXL0124, which was followed by repression of c-Myc, a key downstream target of Notch signaling. Interestingly, HES1, a known downstream target of Notch signaling, was rapidly induced by treatment with BXL0124. The inhibitory effect of BXL0124 on Notch signaling was reversed by knockdown of HES1. Overexpression of HES1 inhibited Notch1 signaling and reduced the CD44(+)/CD24(-/low) subpopulation, confirming a role of HES1 in Notch1 signaling. In conclusion, the Gemini vitamin D analog, BXL0124, represses the tumor-initiating subpopulation by HES1-mediated inhibition of Notch1 signaling. The present study demonstrates BXL0124 as a potent inhibitor of Notch signaling to target tumor-initiating cells in basal-like breast cancer. This article is part of a Special Issue entitled "17th Vitamin D Workshop".
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Antígeno CD24/metabolismo , Calcitriol/análogos & derivados , Carcinoma Basocelular/tratamiento farmacológico , Proteínas de Homeodominio/metabolismo , Receptores de Hialuranos/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Receptor Notch1/metabolismo , Apoptosis/efectos de los fármacos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Calcitriol/farmacología , Carcinoma Basocelular/metabolismo , Carcinoma Basocelular/patología , Proliferación Celular/efectos de los fármacos , Femenino , Citometría de Flujo , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/genética , Humanos , Microscopía Fluorescente , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor Notch1/antagonistas & inhibidores , Receptor Notch1/genética , Receptores de Calcitriol/antagonistas & inhibidores , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción HES-1 , Células Tumorales CultivadasRESUMEN
Breast cancer stem cells (BCSCs) are a subset of tumor cells that are believed to be the cells responsible for the establishment and maintenance of tumors. Moreover, BCSCs are suggested to be the main cause of progression to metastasis and recurrence of cancer because of their tumor-initiating abilities and resistance to conventional therapies. Ductal carcinoma in situ (DCIS) is an early precursor in breast carcinogenesis which progresses to invasive ductal carcinoma (IDC). We have previously reported that a vitamin D compound, BXL0124, inhibits the progression of DCIS to IDC. In the present study we sought to determine whether this effect was mediated through an influence on BCSCs. In MCF10DCIS cells treated with vitamin D compounds (1α25(OH)2D3 or BXL0124), the breast cancer stem cell-like population, identified by the CD44(+)/CD24(-/low) and CD49f(+)/CD24(-/low) subpopulations, was reduced. To determine the effects of vitamin D compounds on cancer stem cell activity, the MCF10DCIS mammosphere cell culture system, which enriches for mammary progenitor cells and putative BCSCs, was utilized. Untreated MCF10DCIS mammospheres showed a disorganized and irregular shape. When MCF10DCIS cells were treated with 1α25(OH)2D3 or BXL0124, the mammospheres that formed exhibited a more organized, symmetrical and circular shape, similar to the appearance of spheres formed by the non-malignant, normal mammary epithelial cell line, MCF10A. The mammosphere forming efficiency (MFE) was significantly decreased upon treatment with 1α25(OH)2D3 or BXL0124, indicating that these compounds have an inhibitory effect on mammosphere development. Treatment with 1α25(OH)2D3 or BXL0124 repressed markers associated with the stem cell-like phenotype, such as CD44, CD49f, c-Notch1, and pNFκB. Furthermore, 1α25(OH)2D3 and BXL0124 reduced the expression of pluripotency markers, OCT4 and KLF-4 in mammospheres. This study suggests that vitamin D compounds repress the breast cancer stem cell-like population, potentially contributing to their inhibition of breast cancer. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Mama/efectos de los fármacos , Células Madre Neoplásicas/efectos de los fármacos , Vitamina D/farmacología , Vitaminas/farmacología , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/genética , Western Blotting , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Femenino , Citometría de Flujo , Humanos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esferoides Celulares/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Ductal carcinoma in situ (DCIS) is a nonmalignant lesion of the breast with the potential to progress to invasive ductal carcinoma (IDC). The disappearance and breakdown of the myoepithelial cell layer and basement membrane in DCIS have been identified as major events in the development of breast cancer. The MCF10DCIS.com cell line is a well-established model, which recapitulates the progression of breast cancer from DCIS to IDC. We have previously reported that a novel Gemini vitamin D analog, 1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol (BXL0124) is a potent inhibitor of the growth of MCF10DCIS.com xenografted tumors without hypercalcemic toxicity. In this study, we utilized the MCF10DCIS.com in vivo model to assess the effects of BXL0124 on breast cancer progression from weeks 1 to 4. Upon DCIS progression to IDC from weeks 3 to 4, tumors lost the myoepithelial cell layer and basement membrane as shown by immunofluorescence staining with smooth muscle actin and laminin 5, respectively. Administration of BXL0124 maintained the critical myoepithelial cell layer as well as basement membrane, and animals treated with BXL0124 showed a 43% reduction in tumor volume by week 4. BXL0124 treatment decreased cell proliferation and maintained vitamin D receptor levels in tumors. In addition, the BXL0124 treatment reduced the mRNA levels of matrix metalloproteinases starting at week 3, contributing to the inhibition of invasive transition. Our results suggest that the maintenance of DCIS plays a significant role in the cancer preventive action of the Gemini vitamin D BXL0124 during the progression of breast lesions.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Calcitriol/análogos & derivados , Carcinoma Ductal de Mama/prevención & control , Carcinoma Intraductal no Infiltrante/tratamiento farmacológico , Carcinoma Intraductal no Infiltrante/patología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Animales , Calcitriol/química , Calcitriol/uso terapéutico , Carcinoma Ductal de Mama/patología , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Femenino , Humanos , Ratones , Ratones Desnudos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The potency of 25-hydroxyvitamin D3 (25(OH)D3) is increased by several fold through its metabolism into 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) by cytochrome P450 27B1 (CYP27B1). Thus, the pivotal role of 1α-hydroxylation in the activation of vitamin D compounds is well known. Here, we examined the metabolism of 25-hydroxy-16-ene-23-yne-vitamin D3 (25(OH)-16-ene-23-yne-D3), a synthetic analog of 25(OH)D3 in a cell-free system and demonstrated that 25(OH)-16-ene-23-yne-D3 is neither activated by CYP27B1 nor inactivated by cytochrome P450 24A1 (CYP24A1). These findings were also confirmed in immortalized normal human prostate epithelial cells (PZ-HPV-7) which are known to express both CYP27B1 and CYP24A1, indicating that the structural modifications featured in 25(OH)-16-ene-23-yne-D3 enable the analog to resist the actions of both CYP27B1 and CYP24A1. To provide intelligible structure-function information, we also performed molecular docking analysis between the analog and CYP27B1. Furthermore, 25(OH)-16-ene-23-yne-D3 was found to suppress the growth of PZ-HPV-7 cells with a potency equivalent to 1α,25(OH)2D3. The antiproliferative activity of 25(OH)-16-ene-23-yne-D3 was found to be vitamin D receptor (VDR)-dependent as it failed to inhibit the growth of mammary tumor cells derived from VDR-knockout mice. Furthermore, stable introduction of VDR into VDR-knockout cells restored the growth inhibition by 25(OH)-16-ene-23-yne-D3. Thus, we identified 25-hydroxy-16-ene-23-yne-vitamin D3 as a novel non-1α-hydroxylated vitamin D analog which is equipotent to 1α,25(OH)2D3 in its antiproliferative activity. We now propose that the low potency of the intrinsic VDR-mediated activities of 25(OH)D3 can be augmented to the level of 1α,25(OH)2D3 without its activation through 1α-hydroxylation by CYP27B1, but by simply preventing its inactivation by CYP24A1.
Asunto(s)
25-Hidroxivitamina D3 1-alfa-Hidroxilasa/metabolismo , Colecalciferol/análogos & derivados , Neoplasias de la Próstata/tratamiento farmacológico , Vitamina D3 24-Hidroxilasa/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/química , Animales , Catálisis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colecalciferol/administración & dosificación , Colecalciferol/química , Resistencia a Antineoplásicos/genética , Humanos , Masculino , Ratones , Simulación del Acoplamiento Molecular , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Receptores de Calcitriol/química , Receptores de Calcitriol/metabolismo , Vitamina D3 24-Hidroxilasa/químicaRESUMEN
We have synthesized 39 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] analogs having two side chains attached to carbon-20 (Gemini) with various modifications and compared their anticancer activities. Five structure-function rules emerged to identify analogs with enhanced anticancer activity. One of these active analogs, BXL-01-0126, was more potent than 1,25(OH)2D3 in mediating 50% clonal inhibition of cancer cell growth. Murine studies found that BXL-01-0126 and 1,25(OH)2D3 had nearly the same potency to raise serum calcium levels. Taken together, BXL-01-0126 when compared to 1,25(OH)2D3 has greater anticancer potency, but similar toxicity causing hypercalcemia. We focused on the effect of these compounds on the stimulation of expression of human cathelicidin antimicrobial peptide (CAMP) whose gene has a vitamin D response element in its promoter. Expression of CAMP mRNA and protein increased in a dose-response fashion after exposure of acute myeloid leukemia (AML) cells to the Gemini analog, BXL-01-126, in vitro. A xenograft model of AML was developed using U937 AML cells injected into NSG-immunodeficient mice. Administration of vitamin D3 compounds to these mice resulted in substantial levels of CAMP in the systemic circulation. This suggests a unique prophylactic treatment at diagnosis or during induction chemotherapy for AML patients to provide them with protection against various microbial infections through CAMP induction.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Calcitriol/análogos & derivados , Catelicidinas/biosíntesis , Colecalciferol/farmacología , Animales , Péptidos Catiónicos Antimicrobianos , Antineoplásicos/química , Calcitriol/síntesis química , Calcitriol/química , Calcitriol/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colecalciferol/análogos & derivados , Colecalciferol/síntesis química , Citometría de Flujo , Xenoinjertos , Humanos , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Relación Estructura-ActividadRESUMEN
HER2 (or ErbB2), a member of ErbB receptor tyrosine kinases, is overexpressed in approximately 20% of human breast cancer, and the ErbB2 signaling pathway is a critical therapeutic target for ErbB2-overexpressing breast cancer. We investigated the inhibitory effects of the Gemini vitamin D analog BXL0124, the synthetic triterpenoid CDDO-Im and the combination on the tumorigenesis of ErbB2-overexpressing breast cancer. MMTV-ErbB2/neu transgenic mice were treated with BXL0124, CDDO-Im, or the combination from three months of age until the end of the experiment. Formation and growth of MMTV-ErbB2/neu mammary tumors were monitored every week, and all three treatments delayed the development of mammary tumors without significant toxicity. Decreased activation of ErbB2 as well as other ErbB receptors, ErbB1 and ErbB3, in MMTV-ErbB2/neu mammary tumors was shown by all treatments. Protein levels of downstream targets of the ErbB2 signaling pathway, including activated-Erk1/2, activated-Akt, c-Myc, CycD1, and Bcl2, were repressed by all three treatments, with the combination treatment exhibiting the strongest effects. To investigate therapeutic efficacy, the combination of BXL0124 and CDDO-Im was given to MMTV-ErbB2/neu mice after mammary tumors were established between 23 and 30 weeks of age. Short-term treatment with the combination did not show effects on tumor growth nor the ErbB2 signaling pathway. The present study shows BXL0124, CDDO-Im, and the combination as potential agents for prevention, but not treatment, against the tumorigenesis of ErbB2-overexpressing breast cancer.
Asunto(s)
Calcitriol/análogos & derivados , Transformación Celular Neoplásica/efectos de los fármacos , Imidazoles/administración & dosificación , Neoplasias Mamarias Animales/prevención & control , Ácido Oleanólico/análogos & derivados , Receptor ErbB-2/fisiología , Transducción de Señal/efectos de los fármacos , Administración Oral , Animales , Protocolos de Quimioterapia Combinada Antineoplásica , Western Blotting , Calcitriol/administración & dosificación , Calcitriol/farmacología , Transformación Celular Neoplásica/patología , Femenino , Humanos , Imidazoles/farmacología , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Virus del Tumor Mamario del Ratón/genética , Ratones , Ratones Transgénicos , Microscopía Fluorescente , Ácido Oleanólico/administración & dosificación , Ácido Oleanólico/farmacología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Changing the geminal methyl groups on 1α,25-dihydroxyvitamin D3 and its analogues to the deuterio versions generally improves the bioactivity. Derivatives of 1α,25-dihydroxyvitamin D3 with two chains emanating at C20, commonly referred to as gemini, are subject to the same phenomenon. Additionally, gemini with different side chains are susceptible to bioactivity differentials where the C17-C20 threo configuration usually imparts higher activity than the corresponding erythro arrangement. In an effort to analyze the deuterium effect on gemini with minimal diastereotopic distortion, we synthesized gemini with equal side chains but introduced deuterium diastereospecifically on either chain. We solved the crystal structures of these compounds in the zebra fish zVDR ligand binding domain as complexes with NCoA-2 coactivator peptide and correlated the findings with growth inhibition in a breast cancer cell line.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Calcitriol/análogos & derivados , Deuterio/química , Animales , Calcitriol/química , Calcitriol/farmacología , Línea Celular Tumoral , Cromatografía en Capa Delgada , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Indicadores y Reactivos , Ligandos , Modelos Moleculares , Conformación Molecular , Receptores de Calcitriol/efectos de los fármacos , Espectrofotometría Ultravioleta , Estereoisomerismo , Pez CebraRESUMEN
BACKGROUND: CD44, a transmembrane glycoprotein, is a major receptor for extracellular proteins involved in invasion and metastasis of human cancers. We have previously demonstrated that the novel Gemini vitamin D analog BXL0124 [1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluro-cholecalciferol] repressed CD44 expression in MCF10DCIS.com basal-like human breast cancer cells and inhibited MCF10DCIS xenograft tumor growth. In the present study, we investigated potential factors downstream of CD44 and the biological role of CD44 repression by BXL0124 in MCF10DCIS cells. METHODS AND FINDINGS: The treatment with Gemini vitamin D BXL0124 decreased CD44 protein level, suppressed STAT3 signaling, and inhibited invasion and proliferation of MCF10DCIS cells. The interaction between CD44 and STAT3 was determined by co-immunoprecipitation. CD44 forms a complex with STAT3 and Janus kinase 2 (JAK2) to activate STAT3 signaling, which was inhibited by BXL0124 in MCF10DCIS cells. The role of CD44 in STAT3 signaling and invasion of MCF10DCIS cells was further determined by the knockdown of CD44 using small hairpin RNA in vitro and in vivo. MCF10DCIS cell invasion was markedly decreased by the knockdown of CD44 in vitro. The knockdown of CD44 also significantly decreased mRNA expression levels of invasion markers, matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA), in MCF10DCIS cells. In MCF10DCIS xenograft tumors, CD44 knockdown decreased tumor size and weight as well as invasion markers. CONCLUSIONS: The present study identifies STAT3 as an important signaling molecule interacting with CD44 and demonstrates the essential role of CD44-STAT3 signaling in breast cancer invasion. It also suggests that repression of CD44-STAT3 signaling is a key molecular mechanism in the inhibition of breast cancer invasion by the Gemini vitamin D analog BXL0124.
Asunto(s)
Neoplasias de la Mama , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Receptores de Hialuranos/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Calcitriol/análogos & derivados , Calcitriol/farmacología , Línea Celular Tumoral , Femenino , Humanos , Receptores de Hialuranos/genética , Ratones , Invasividad Neoplásica , Metástasis de la Neoplasia/tratamiento farmacológico , Factor de Transcripción STAT3/genética , Transducción de Señal , Trasplante Heterólogo , Vitamina D/análogos & derivados , Vitamina D/farmacologíaRESUMEN
Derivatives of vitamin D(3) containing a second side-chain emanating at C-20 are known as gemini and act as vitamin D receptor agonists. Recently, two of these, namely Gemini-0072 and the epimeric Gemini-0097, were selected for further studies in view of their high biological activities and lack of hypercalcemic effects. We now show that the two analogs recruit coactivator SRC-1 better than the parental gemini and act as VDR superagonists. The crystal structures of complexes of zVDR with Gemini-0072 and Gemini-0097 indicate that these ligands induce an extra cavity within the ligand-binding pocket similar to gemini and that their superagonistic activity is due to an increased stabilization of helix H12.
RESUMEN
We examined the metabolism of two synthetic analogs of 1α,25-dihydroxyvitamin D3 (1), namely 1α,25-dihydroxy-16-ene-23-yne-vitamin D3 (2) and 1α,25-dihydroxy-16-ene-23-yne-26,27-dimethyl-vitamin D3 (4) using rat cytochrome P450 24A1 (CYP24A1) in a reconstituted system. We noted that 2 is metabolized into a single metabolite identified as C26-hydroxy-2 while 4 is metabolized into two metabolites, identified as C26-hydroxy-4 and C26a-hydroxy-4. The structural modification of adding methyl groups to the side chain of 1 as in 4 is also featured in another analog, 1α,25-dihydroxy-22,24-diene-24,26,27-trihomo-vitamin D3 (6). In a previous study, 6 was shown to be metabolized exactly like 4, however, the enzyme responsible for its metabolism was found to be not CYP24A1. To gain a better insight into the structural determinants for substrate recognition of different analogs, we performed an in silico docking analysis using the crystal structure of rat CYP24A1 that had been solved for the substrate-free open form. Whereas analogs 2 and 4 docked similar to 1, 6 showed altered interactions for both the A-ring and side chain, despite prototypical recognition of the CD-ring. These findings hint that CYP24A1 metabolizes selectively different analogs of 1, based on their ability to generate discrete recognition cues required to close the enzyme and trigger the catalytic mechanism.
Asunto(s)
Esteroide Hidroxilasas/metabolismo , Vitamina D/análogos & derivados , Animales , Cromatografía de Gases y Espectrometría de Masas , Modelos Moleculares , Unión Proteica , Ratas , Esteroide Hidroxilasas/química , Vitamina D/química , Vitamina D/metabolismo , Vitamina D3 24-HidroxilasaRESUMEN
CD44 is a multifunctional transmembrane protein involved in cell proliferation, angiogenesis, invasion, and metastasis. CD44 is identified as a cancer stem cell marker, and the CD44-positive breast cancer cells are enriched in residual breast cancer cell populations after conventional therapies, suggesting that CD44 may be an important target for cancer prevention and therapy. Therefore, we investigated for the inhibitory effect of a novel Gemini vitamin D analog, 1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol (BXL0124), on mammary tumor growth and CD44 expression in MCF10DCIS.com human breast cancer in vitro and in vivo. MCF10DCIS.com cells were injected into mammary fat pads in immunodeficient mice, and BXL0124 was then administered intraperitoneally (0.1 µg/kg body weight) or orally (0.03 or 0.1 µg/kg body weight) 6 days a week for 5 weeks. At necropsy, mammary tumors and blood were collected for evaluating tumor growth, CD44 expression, and serum calcium level. BXL0124 suppressed mammary tumor growth and markedly decreased the expression of CD44 protein in MCF10DCIS xenograft tumors without causing hypercalcemic toxicity. BXL0124 also inhibited the expression of CD44 protein and mRNA as well as the transcriptional activity of the CD44 promoter in cultured MCF10DCIS.com cells. The repression of CD44 expression induced by BXL0124 was blocked by siRNA vitamin D receptor (VDR), indicating that the regulation of CD44 expression by BXL0124 is a VDR-dependent event. The novel Gemini vitamin D analog, BXL0124, represses CD44 expression in MCF10DCIS.com cells in vitro and in xenograft tumors, suggesting an inhibitory role of a Gemini vitamin D derivative on breast cancer stem cells.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Calcitriol/análogos & derivados , Receptores de Hialuranos/biosíntesis , Animales , Western Blotting , Neoplasias de la Mama/química , Neoplasias de la Mama/tratamiento farmacológico , Calcitriol/farmacología , Calcio/sangre , Línea Celular Tumoral , Femenino , Citometría de Flujo , Humanos , Receptores de Hialuranos/análisis , Neoplasias Mamarias Experimentales/química , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones SCID , Microscopía Fluorescente , Trasplante de Neoplasias , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Transcripción Genética/efectos de los fármacosRESUMEN
Numerous synthetic vitamin D analogs have been studied for their effects on the prevention and treatment of breast cancer. However, the inhibitory effects of naturally occurring 1alpha,25-dihydroxyvitamin D3 or its synthetic analogs on ErbB2 overexpressing mammary tumorigenesis have not been reported. Gemini vitamin D analogs are novel synthetic vitamin D derivatives with a unique structure of two six-carbon chains at C-20. We have previously shown that Gemini vitamin D analogs significantly inhibited carcinogen-induced estrogen receptor (ER)-positive mammary tumorigenesis and reduced ER-negative MCF10DCIS.com xenograft tumor growth without hypercalcemic toxicity. In the present study, we have determined the inhibitory effect of a potent Gemini vitamin D analog BXL0124 (1alpha,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-cholecalciferol) on the ErbB2/Her-2/neu overexpressing mammary tumorigenesis. The Gemini BXL0124 inhibits ErbB2-positive mammary tumor growth and down-regulates the phosphorylation of ErbB2, ERK and AKT in tumors of MMTV-ErbB2/neu transgenic mice. These effects of Gemini BXL0124 in vivo were confirmed by using the ErbB2 overexpressing tumor cells derived from the mammary tumors of MMTV-ErbB2/neu mice. In conclusion, the Gemini vitamin D analog BXL0124 inhibits the growth of ErbB2 overexpressing mammary tumors through regulating the ErbB2/AKT/ERK signaling pathways, suggesting that Gemini vitamin D analog may be considered for translational studies.
Asunto(s)
Calcitriol/análogos & derivados , Colecalciferol/análogos & derivados , Neoplasias Mamarias Animales/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-2/metabolismo , Animales , Calcitriol/farmacología , Calcio/metabolismo , Carcinógenos , Línea Celular Tumoral , Proliferación Celular , Ratones , Ratones Transgénicos , Microscopía Fluorescente/métodos , Transducción de SeñalRESUMEN
Induction of terminal differentiation is a conceptually attractive approach for the therapy of neoplastic diseases. Although vitamin D derivatives (deltanoids) can induce differentiation of AML cells in vitro, so far deltanoids have not been successfully brought to the clinic, due to the likelihood of life-threatening hypercalcemia. Here, we incubated freshly obtained blood cells from patients with AML with a plant antioxidant (PAOx), silibinin (SIL), alone or together with a deltanoid. Twenty patients with AML (all subtypes except M3) were available for this study, and in 14 (70%), SIL (60 µM) either induced differentiation ex vivo, or enhanced differentiation induced by deltanoids, or both. Interestingly, SIL acting alone induced differentiation only in cases in which chromosome aberrations could not be detected. In eleven samples sufficient material was available for a limited analysis of the underlying events. Quantitative RT-PCR showed that differentiation markers were upregulated at the mRNA level by both SIL and deltanoids, suggesting that intracellular signaling pathways upstream of transcription factors (TFs) were activated by these agents. Western analysis for proteins which function as TFs in deltanoid-induced monocytic differentiation, such as members of Jun and C/EBP families, surprisingly demonstrated that SIL upregulated all these TFs in the cases tested. This suggests that although the presence of SIL may not always be sufficient to induce differentiation, it can serve as a differentiation enabling factor for blasts obtained from a large proportion of patients with AML. Thus, SIL/deltanoid combinations warrant further consideration as preventive/therapeutic regimens in human leukaemia.
Asunto(s)
Colecalciferol/análogos & derivados , Colecalciferol/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Silimarina/farmacología , Adulto , Anciano , Diferenciación Celular/efectos de los fármacos , Femenino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Masculino , Persona de Mediana Edad , Monocitos/efectos de los fármacos , Monocitos/patología , Receptores de Calcitriol/biosíntesis , Receptores de Calcitriol/genética , Receptor alfa X Retinoide/biosíntesis , Receptor alfa X Retinoide/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Silibina , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Calcitriol is implicated in many cellular functions including cellular growth and differentiation, thus explaining its antitumor effects. It was shown that gemini, the calcitriol derivative containing two side chain at C20, is also active in gene transcription with enhanced antitumor activity. We have now further optimized both the A-ring and the two side chains. The chemical structures of the resulting 18 geminis were correlated with biological activities. Those containing the 1alpha-fluoro A-ring are the least active. Those featuring 23-yne and 23(E) side-chains are generally more active in human breast cancer cell growth inhibition and human leukemia cell differentiation induction than their 23(Z) counterparts. On the basis of these evaluations, we selected as lead compound a 20(R) gemini, related to calcitriol in terms of it is A-ring, where one side chain was modified by introduction of a 23-yne function and replacement of the geminal methyl groups with trifluoromethyl groups, the other created by extension of C21 with a 3-hydroxy-3-trideuteromethyl-4,4,4-trideutero-butyl moiety.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Neoplasias de la Mama/patología , Calcitriol/análogos & derivados , Calcitriol/farmacología , Diferenciación Celular/efectos de los fármacos , Leucemia/patología , Animales , Antineoplásicos/síntesis química , Calcitriol/síntesis química , Línea Celular Tumoral , Humanos , Concentración 50 Inhibidora , Ratones , Modelos Moleculares , Conformación MolecularRESUMEN
1alpha,25(OH)(2)-16-ene-20-cyclopropyl-vitamin D(3) (13) is several fold more potent than the natural hormone 1alpha,25-dihydroxyvitamin D(3) (1) as an anti-inflammatory agent. Here, we have further analyzed the anti-inflammatory properties of 13, confirming it as the most potent analogue tested within this family. We then determined the structures of all the natural metabolites of 13, including the 24-oxo metabolite 14, and carried out its synthesis. A comparison of 13 with 14 showed a similar induction of the primary VDR target genes CYP24A1 and CAMP and comparable anti-inflammatory properties as revealed by a similar inhibition of TNF-alpha, IL-12/23p40, IL-6, and IFN-gamma production. Interestingly, 14 displays a 3-fold lower calcemic activity in vivo compared to 13. Collectively, these findings indicate that the strong potency of 13 can be explained by the accumulation of its stable 24-oxo metabolite, which shows immunoregulatory and anti-inflammatory properties superimposable to those exerted by 13 itself.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Calcitriol/análogos & derivados , Calcio/sangre , Animales , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/metabolismo , Péptidos Catiónicos Antimicrobianos/biosíntesis , Péptidos Catiónicos Antimicrobianos/genética , Calcitriol/síntesis química , Calcitriol/química , Calcitriol/metabolismo , Calcitriol/farmacología , Catelicidinas , Línea Celular Tumoral , Células Cultivadas , Citocinas/antagonistas & inhibidores , Humanos , Factores Inmunológicos/síntesis química , Factores Inmunológicos/química , Factores Inmunológicos/metabolismo , Factores Inmunológicos/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Ratas , Receptores de Calcitriol/fisiología , Esteroide Hidroxilasas/biosíntesis , Esteroide Hidroxilasas/genética , Relación Estructura-Actividad , Vitamina D3 24-HidroxilasaRESUMEN
The active form of vitamin D(3), 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], modulates proliferation and induces differentiation of many cancer cells. A new class of analogs of vitamin D(3) has been synthesized, having two side-chains attached to carbon-20 (Gemini) and deuterium substituted on one side-chain. We have examined six of these analogs for their ability to inhibit growth of myeloid leukemia (HL-60), prostate (LNCaP, PC-3, DU145), lung (H520), colon (HT-29), and breast (MCF-7) cancer cell lines. Dose-response clonogenic studies showed that all six analogs had greater antiproliferative activities against cancer cells than 1,25(OH)(2)D(3). Although they had similar potency, the most active of these analogs was BXL-01-0120. BXL-01-0120 was 529-fold more potent than 1,25(OH)(2)D(3) in causing 50% clonal growth inhibition (ED(50)) of HL-60 cells. Pulse-exposure studies demonstrated that exposure to BXL-01-120 (10(-9)M, 48h) resulted in 85% clonal inhibition of HL-60 growth. BXL-01-0120 (10(-11)M, 4 days) induced the differentiation marker, CD11b. Also, morphologically differentiation was more prominent compared to 1,25(OH)(2)D(3). Annexin V assay showed that BXL-01-0120 (10(-10)M, 4 days) induced significantly (p<0.05) more apoptosis than 1,25(OH)(2)D(3). In summary, these analogs have a unique structure resulting in extremely potent inhibition of clonal proliferation of various types of cancer cells, especially HL-60 cells.
Asunto(s)
Antineoplásicos/farmacología , Colecalciferol/análogos & derivados , Colecalciferol/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , HumanosRESUMEN
OBJECTIVE: Differentiation therapy with the hormonal form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (1,25D(3)), is a promising approach to treatment of acute myeloid leukemia (AML); however, 1,25D(3) induces hypercalcemia at pharmacologically active doses. We investigated the in vitro and in vivoantileukemic efficacy of combined treatment with non-toxic doses of a low-calcemic 1,25D(3) analogue, 1,25-dihydroxy-21(3-hydroxy-3-methyl-butyl)-19-nor-cholecalciferol (19-nor-Gemini; Ro27-5646), and rosemary plant agents in a mouse model of AML. METHODS: Proliferation and differentiation of WEHI-3B D- (WEHI) murine myelomonocytic leukemia cellsin vitro were determined by standard assays. Reactive oxygen species, glutathione and protein expression levels were measured by flow cytometry, enzymatic assay and Western blotting, respectively. Systemic AML was developed by intravenous injection of WEHI cells in syngeneic Balb/c mice. RESULTS: 19-nor-Gemini had a higher potency than its parent compounds, Gemini (Ro27-2310) and 1,25D(3), in the induction of differentiation (EC(50) = 0.059 +/- 0.011, 0.275 +/- 0.093 and 0.652 +/- 0.085 nM, respectively) and growth arrest (IC(50) = 0.072 +/- 0.018, 0.165 +/- 0.061 and 0.895 +/- 0.144 nM, respectively) in WEHI cells in vitro, and lower in vivo toxicity. Combined treatment of leukemia-bearing mice with 19-nor-Gemini (injected intraperitoneally) and standardized rosemary extract (mixed with food) resulted in a synergistic increase in survival (from 42.2 +/- 2.5 days in untreated mice to 66.5 +/- 4.2 days, n = 3) and normalization of white blood cell and differential counts. This was consistent with strong cooperative antiproliferative and differentiation effects of low concentrations of 19-nor-Gemini or 1,25D(3) combined with rosemary extract or its major polyphenolic component, carnosic acid, as well as with the antioxidant action of rosemary agents and vitamin D derivatives in WEHI cell cultures. CONCLUSION: Combined effectiveness of 1,25D(3) analogues and rosemary agents against mouse AML warrants further exploration of this therapeutic approach in translational models of human leukemia.
Asunto(s)
Abietanos/uso terapéutico , Calcitriol/análogos & derivados , Modelos Animales de Enfermedad , Leucemia Experimental/tratamiento farmacológico , Leucemia Mieloide Aguda/tratamiento farmacológico , Extractos Vegetales/uso terapéutico , Animales , Antioxidantes/uso terapéutico , Médula Ósea/efectos de los fármacos , Calcitriol/uso terapéutico , Diferenciación Celular/efectos de los fármacos , Proliferación Celular , Colecalciferol/análogos & derivados , Colecalciferol/uso terapéutico , Sinergismo Farmacológico , Humanos , Immunoblotting , Ratones , Ratones Endogámicos BALB C , Especies Reactivas de Oxígeno/metabolismo , Rosmarinus/química , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
Inducible cyclooxygenase-2 (COX-2) has been implicated to play a role in inflammation and carcinogenesis and selective COX-2 inhibitors have been considered as anti-inflammatory and cancer chemopreventive agents. 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), the active hormonal form of vitamin D3 also has been considered to be a cancer chemopreventive agent in addition to its important role in maintaining calcium homeostasis. Based on these observations, we studied the direct effect of 1alpha,25(OH)2D3 and one of its less calcemic synthetic analogs, 1alpha,25(OH)2-16-ene-23-yne-D3 on the activity of both COX-1 and COX-2 in an in vitro enzyme assay. Preliminary data indicated that both 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-23-yne-D3 inhibited selectively the activity of COX-2 with no effect on the activity of COX-1. Out of the two compounds, 1alpha,25(OH)2-16-ene-23-yne-D3 was found to be more effective with an IC50 of 5.8 nM. Therefore, the rest of the experiments were performed using 1alpha,25(OH)2-16-ene-23-yne-D3 only. 1alpha,25(OH)2-16-ene-23-yne-D3 inhibited the proliferation of lipopolysaccharide (LPS) stimulated mouse macrophage cells (RAW 264.7) with a reduction in the expression of COX-2 along with other inflammatory mediators like inducible nitric oxide synthase (iNOS) and interleukin-2 (IL-2). Furthermore, 1alpha,25(OH)2-16-ene-23-yne-D3 also inhibited carrageenan induced inflammation in an air pouch of a rat and effectively reduced the expression of COX-2, iNOS, and IL-2 in the tissues of the same air pouch. In both cases, 1alpha,25(OH)2-16-ene-23-yne-D3 did not show any effect on the expression of COX-1. In summary, our results indicate that 1alpha,25(OH)2-16-ene-23-yne-D3, a less calcemic vitamin D analog, exhibits potent anti-inflammatory effects and is a selective COX-2 inhibitor.
Asunto(s)
Calcitriol/análogos & derivados , Inhibidores de la Ciclooxigenasa 2/farmacología , Ciclooxigenasa 2/metabolismo , Vitamina D/análogos & derivados , Vitamina D/farmacología , Animales , Western Blotting , Calcitriol/farmacología , Carragenina , Recuento de Células , Línea Celular , Proliferación Celular/efectos de los fármacos , Ciclooxigenasa 1/metabolismo , Humanos , Inflamación/enzimología , Concentración 50 Inhibidora , Interleucina-2/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Masculino , Ratones , Óxido Nítrico Sintasa de Tipo II/metabolismo , Ratas , Ratas WistarRESUMEN
Numerous preclinical, epidemiologic, and clinical studies have suggested the benefits of vitamin D and its analogues for the prevention and treatment of cancer. However, the hypercalcemic effects have limited the use of 1alpha,25(OH)(2)D(3), the hormonally active form of vitamin D. To identify vitamin D analogues with better efficacy and low toxicity, we have tested >60 novel Gemini vitamin D analogues with a unique structure of two side chains for growth inhibition of breast cancer cells. Our initial studies found that some Gemini analogues are 5-15 times more active than 1alpha,25(OH)(2)D(3) in growth inhibition assay. In vivo experiments were designed to study the inhibitory effect of selected Gemini vitamin D analogues against mammary carcinogenesis by using (a) an N-methyl-N-nitrosourea-induced estrogen receptor (ER)-positive mammary tumor model and (b) an MCF10DCIS.com xenograft model of ER-negative mammary tumors. Among vitamin D analogues we tested, Gemini 0072 [1alpha,25-dihydroxy-20S-21(3-trideuteromethyl-3-hydroxy-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-19-nor-cholecalciferol] and Gemini 0097 [1alpha,25-dihydroxy-20R-21(3-trideuteromethyl-3-hydroxy-4,4,4-trideuterobutyl)-23-yne-26,27-hexafluoro-19-nor-cholecalciferol] administration inhibited by 60% the NMU-induced mammary tumor burden compared with the NMU-treated control group, but these compounds were devoid of hypercalcemia toxicity. In an ER-negative xenograft model, Gemini 0097 significantly suppressed tumor growth without hypercalcemia toxicity. We found that the inhibitory effect of Gemini 0097 was associated with an increased level of cyclin-dependent kinase inhibitor p21 and the insulin-like growth factor binding protein 3 in both ER-positive and ER-negative mammary tumors. Our results suggest that Gemini vitamin D analogues may be potent agents for the prevention and treatment of both ER-positive and ER-negative breast cancer without hypercalcemia toxicity.