RESUMEN
Surgically unresectable colorectal and pancreatic carcinomas have a high rate of mortality as current therapeutic options are limited. One common chemotherapeutic used to broadly treat both cancers is 5-flurouracil (5-Fu); however, treatment serves only to slow progression of the disease and comes with many side effects due to 5-Fu's intrinsic toxicity. Thus, strategies to decrease the dose of 5-Fu utilized therapeutically as well as reduce 5-Fu's off-target toxicity are paramount. Using cell models of colorectal and pancreatic cancers, we show that cotreatment with Achyrocline B (3,5 dihydroxy-6,7,8-trimethoxyflavone, AcB), a natural flavone from Achyrocline bogotensis, allows for four-fold reduction in 5-Fu dosage without loss of efficacy. We further show that the action of AcB is due to continued cell cycle progression despite 5-Fu pressure to synchronize at the G1/S threshold. In addition to AcB's effect on cancer cells, we found that AcB can directly reduce toxicity of 5-Fu in cells mimicking non-cancerous tissues. These in vitro results are then supported by xenograft modeling. AcB was shown to increase apoptosis in tumors leading to degeneration of the outer tumoral boundary. Furthermore, in 5-Fu treated animals it was found that AcB provided protection to the intestinal tract as indicated by preserved histological and immunohistochemical features. These results show promise for a new adjuvant therapy for colorectal and pancreatic carcinomas that not only reduces tumor progression, but more importantly has the potential to improve patient quality of life.
Asunto(s)
Achyrocline , Carcinoma , Neoplasias del Colon , Neoplasias Colorrectales , Neoplasias Pancreáticas , Animales , Humanos , Fluorouracilo/toxicidad , Reducción Gradual de Medicamentos , Calidad de Vida , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Colorrectales/tratamiento farmacológico , Carcinoma/tratamiento farmacológico , Neoplasias del Colon/patología , Neoplasias PancreáticasRESUMEN
BACKGROUND: Antineoplastic activity has been previously shown for two isomeric flavones, 5,7-dihydroxy-3,6,8-trimethoxy flavone (flavone A) and 3,5-dihydroxy-6,7,8-trimethoxy flavone (flavone B), against colon cancer cell lines (Thomas et al. in PLoS ONE 7:e39806, 5). Here, we present modified methods for the extraction and quantification of flavones A and B in rat colon tissue after intravenous dosing via high performance liquid chromatography, from the originally described procedure for extraction and quantification in rat plasma (Whitted et al. in J Chromatogr B Analyt Technol Biomed Life Sci 1001:150-155, 7). RESULTS: Modifications included tissue homogenization (1 g tissue: 2 mL water), filtration of the supernatant with a PVDF membrane, and the use of only one calibration curve to determine the concentration of each flavone in colon tissue. Good separation was achieved and representative equations were linear with r 2 ≥ 0.99 for both flavones. Precision and accuracy for flavone A ranged from 0.88-24.03 and 109-116%. Precision and accuracy for flavone B ranged from 1.62-33.56 and 98-113%. Concentrations of 1639 ± 601 ng/g flavone A and 5975 ± 2480 ng/g of flavone B were detected in rat colon tissue 6 h post dosing. CONCLUSIONS: Modifications to the extraction methods for flavone A and flavone B from rat colon tissue had good separation, precision, and accuracy.
Asunto(s)
Cromatografía de Fase Inversa , Colon/química , Flavonas/química , Animales , Calibración , Línea Celular Tumoral , Masculino , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los ResultadosRESUMEN
Apoptosis can be triggered in two different ways, through the intrinsic or the extrinsic pathway. The intrinsic pathway is mediated by the mitochondria via the release of cytochrome C while the extrinsic pathway is prompted by death receptor signals and bypasses the mitochondria. These two pathways are closely related to cell proliferation and survival signaling cascades, which thereby constitute possible targets for cancer therapy. In previous studies we introduced two plant derived isomeric flavonoids, flavone A and flavone B which induce apoptosis in highly tumorigenic cancer cells of the breast, colon, pancreas, and the prostate. Flavone A displayed potent cytotoxic activity against more differentiated carcinomas of the colon (CaCo-2) and the pancreas (Panc28), whereas flavone B cytotoxic action is observed on poorly differentiated carcinomas of the colon (HCT 116) and pancreas (MIA PaCa). Apoptosis is induced by flavone A in better differentiated colon cancer CaCo-2 and pancreatic cancer Panc 28 cells via the intrinsic pathway by the inhibition of the activated forms of extracellular signal-regulated kinase (ERK) and pS6, and subsequent loss of phosphorylation of Bcl-2 associated death promoter (BAD) protein, while apoptosis is triggered by flavone B in poorly differentiated colon cancer HCT 116 and MIA PaCa pancreatic cancer cells through the extrinsic pathway with the concomitant upregulation of the phosphorylated forms of ERK and c-JUN at serine 73. These changes in protein levels ultimately lead to activation of apoptosis, without the involvement of AKT.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Flavonas/farmacología , Apoptosis/efectos de los fármacos , Caspasa 10/metabolismo , Caspasa 8/metabolismo , Caspasa 9/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Flavonas/química , Flavonas/aislamiento & purificación , Humanos , Fosforilación , Extractos Vegetales/química , Extractos Vegetales/farmacología , Proteínas Quinasas S6 Ribosómicas/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína Letal Asociada a bcl/metabolismoRESUMEN
Isomers 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5,7-dihydroxy-3,6,8 trimethoxy flavone) (flavone A) and 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxy flavone) (flavone B) have recently demonstrated differential antineoplastic activities against pancreatic cancer in vitro. These studies also indicated that these compounds target highly tumorigenic cells while sparing normal cells. The in vivo antitumor activities of these flavones have not been determined, and detection protocols for these compounds are needed to conduct pre-clinical assays following intravenous dosing. Here, we report methods developed using acetonitrile to extract two flavone isomers and corresponding internal standards, celecoxib and diclofenac, from rat plasma. Separation was achieved using a Shimadzu liquid chromatography system with a C18 column and mobile phase acetonitrile/water (60:40 and 70:30 for flavones A and B, respectively) containing 0.2% acetic acid and 0.05% triethylamine at a flow rate of 0.4mL/min and detection at 245nm. Calibration curves ranging from 250 to 2500ng/mL and 2500 to 100,000ng/mL for both flavones were linear (r(2)≥0.99) with the lower limits of quantification being 250ng/mL. Recovery of concentrations 250, 1000, 2500, 5000, and 100,000ng/mL ranged from 87 to 116% and 84 to 103% (n=3) for flavone A and B, respectively. Stability of both flavones after a freezing/thawing cycle yielded a mean peak ratio ≥0.92 when compared to freshly extracted samples. Intravenous administration of a 20mg/kg dose in rats yielded half-lives of 83.68±56.61 and 107.45±53.31min with clearance values of 12.99±13.78 and 80.79±35.06mL/min/kg for flavones A and B, respectively.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Cromatografía de Fase Inversa/métodos , Flavonas/análisis , Animales , Flavonas/farmacocinética , Isomerismo , Límite de Detección , Masculino , Ratas , Ratas Sprague-Dawley , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
Flavones, found in nature as secondary plant metabolites, have shown efficacy as anti-cancer agents. We have examined the binding of two flavones, 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5,7-dihydroxy-3,6,8-trimethoxy flavone; FlavA) and 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxy flavone; FlavB), to phiX174 RF DNA using restriction enzyme activity assays employing the restriction enzymes Alw44, AvaII, BssHII, DraI, MluI, NarI, NciI, NruI, PstI, and XhoI. These enzymes possess differing target and flanking sequences allowing for observation of sequence specificity analysis. Using restriction enzymes that cleave once with a mixture of supercoiled and relaxed DNA substrates provides for observation of topological effects on binding. FlavA and FlavB show differing sequence specificities in their respective binding to phiX. For example, with relaxed DNA, FlavA shows inhibition of cleavage with DraI (reaction site (5') TTTAAA) but not BssHII ((5') GCGCGC) while FlavB shows the opposite results. Evidence for tolological specificity is also observed, Molecular modeling and conformational analysis of the flavones suggests that the phenyl ring of FlavB is coplanar with the flavonoid ring while the phenyl ring of FlavA is at an angle relative to the flavonoid ring. This may account for aspects of the observed sequence and topological specificities in the effects on restriction enzyme activity.
Asunto(s)
ADN , Flavonas , Secuencia de Bases , ADN/metabolismo , Enzimas de Restricción del ADN , Modelos Moleculares , Datos de Secuencia Molecular , Especificidad por SustratoRESUMEN
Over 4000 flavonoids have been identified so far and among these, many are known to have antitumor activities. The basis of the relationships between chemical structures, type and position of substituent groups and the effects these compounds exert specifically on cancer cells are not completely elucidated. Here we report the differential cytotoxic effects of two flavone isomers on human cancer cells from breast (MCF7, SK-BR-3), colon (Caco-2, HCT116), pancreas (MIA PaCa, Panc 28), and prostate (PC3, LNCaP) that vary in differentiation status and tumorigenic potential. These flavones are derived from plants of the family Asteraceae, genera Gnaphalium and Achyrocline reputed to have anti-cancer properties. Our studies indicate that 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5,7-dihydroxy-3,6,8-trimethoxy flavone) displays potent activity against more differentiated carcinomas of the colon (Caco-2), and pancreas (Panc28), whereas 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxy flavone) cytototoxic action is observed on poorly differentiated carcinomas of the colon (HCT116), pancreas (Mia PaCa), and breast (SK-BR3). Both flavones induced cell death (>50%) as proven by MTT cell viability assay in these cancer cell lines, all of which are regarded as highly tumorigenic. At the concentrations studied (5-80 µM), neither flavone demonstrated activity against the less tumorigenic cell lines, breast cancer MCF-7 cells, androgen-responsive LNCaP human prostate cancer line, and androgen-unresponsive PC3 prostate cancer cells. 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5,7-dihydroxy-3,6,8-trimethoxy flavone) displays activity against more differentiated carcinomas of the colon and pancreas, but minimal cytotoxicity on poorly differentiated carcinomas of these organs. On the contrary, 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3,5-dihydroxy-6,7,8-trimethoxy flavone) is highly cytotoxic to poorly differentiated carcinomas of the colon, pancreas, and breast with minimal activity against more differentiated carcinomas of the same organs. These differential effects suggest activation of distinct apoptotic pathways. In conclusion, the specific chemical properties of these two flavone isomers dictate mechanistic properties which may be relevant when evaluating biological responses to flavones.
Asunto(s)
Achyrocline/química , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Flavonas/química , Flavonas/farmacología , Gnaphalium/química , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Etiquetado Corte-Fin in Situ , Isomerismo , Masculino , FitoterapiaRESUMEN
The flavonoids 3,5-dihydroxy-7-methoxy-flavanone, 3,5-dihydroxy-7-methoxyflavone and 3,5,7-trihydroxy-6-methoxyflavone were isolated from the leaves of C. leivensis. Preliminary observations in K562 cells (human erythroleukemia) using the trypan blue test, showed a 90% viability at a concentration of 100 microg/mL; however, further testing of the flavonoids at concentrations of 25, 50 and 100 microg/mL showed toxicity affecting the morphology of human erythroleukemia cells (K562) and human melanoma cells (A375). Induction of apoptosis was produced by 3,5-dihydroxy-7-methoxyflavone at 72 hours after treatment with arrest in the G2 / M phase of the cell cycle. The A375 cells treated with 50 microg/mL of 3,5-dihydroxy-7-methoxy-flavanone for 24, 48 and 72 hours, display effects on the behavior of the cell cycle. The flavonoid 3,5-dihydroxy-7-methoxyflavone has activity on the mitochondrial membrane at concentrations of 25, 50 and 100 microg/mL, at time intervals of 8 to 12 hours. The flavonoids 3,5-dihydroxy-7-methoxy-flavanone and 3,5-dihydroxy-7-methoxyflavone at a concentration of 25 microg/mL increased the expression of costimulatory molecules corresponding to the phenotype presented by mature dendritic cells with differentiation markers CD40, CD83, CD86 and HLA-DR. The two flavonoids at concentrations between 0.39 and 100 microg/mL slightly increased the proliferation of peripheral blood mononuclear cells in the presence and in the absence of phytohemagglutinin. These flavonoids at concentrations of 50 and 100 microg/mL slightly increased the proliferation of fibroblasts.
Asunto(s)
Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Chromolaena/química , Flavonas/aislamiento & purificación , Flavonas/farmacología , Extractos Vegetales/farmacología , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Colombia , Flavonas/química , Citometría de Flujo , Humanos , Células K562 , Leucocitos Mononucleares/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Hojas de la Planta/químicaRESUMEN
Foi demonstrada a ação antifúngica do extrato clorofórmico e de duas substâncias isoladas da superfície foliar de Pentacalia ledifolia (H.B.K.) Cuatr. e P. corymbosa (Benth) Cuatr. frente aos fungos fitopatógenos Fusarium oxysporum e Botrytis cinerea, cultivados em BDA (batata-dextrose-ágar). Destes extratos foram isolados, além de cumarinas já identificadas em estudos anteriores, dois derivados quinóides: (1-hidroxi-4-oxo-2,5-ciclohexadienil) acetato de metila ou jacaranona e (1-hidroxi-4-oxo-2,5-ciclohexadienil) acetato de etila ou metiljacaranona. Para o (1-hidroxi-4-oxo-2,5-ciclohexadienil) acetato de etila foi calculado CI50 de 650 μg/mL para os dois tipos de fungos e o (1-hidroxi-4-oxo-2,5-ciclohexadienil) acetato de metila teve um CI50 de 660 μg/mL.
Quinols identified in the surface waxes of Pentacalia ledifolia (H.B.K.) Cuatr and P. corymbosa (Benth) Cuatr. leaves, possess antifungal activity against Fusarium oxysporum and Botrytis cinerea, cultured on PDA (potato-dextrose-agar) medium. These extracts were prepared by dipping fresh leaves in chloroform for 5 min, and afforded ethyl-(1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl) acetate and methyl-(1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl) acetate, the major surface compounds.