RESUMO
Doublecortin like kinase 1 (DCLK1) plays a crucial role in several cancers including colon and pancreatic adenocarcinomas. However, its role in squamous cell carcinoma (SCC) remains unknown. To this end, we examined DCLK1 expression in head and neck SCC (HNSCC) and anal SCC (ASCC). We found that DCLK1 is elevated in patient SCC tissue, which correlated with cancer progression and poorer overall survival. Furthermore, DCLK1 expression is significantly elevated in human papilloma virus negative HNSCC, which are typically aggressive with poor responses to therapy. To understand the role of DCLK1 in tumorigenesis, we used specific shRNA to suppress DCLK1 expression. This significantly reduced tumor growth, spheroid formation, and migration of HNSCC cancer cells. To further the translational relevance of our studies, we sought to identify a selective DCLK1 inhibitor. Current attempts to target DCLK1 using pharmacologic approaches have relied on nonspecific suppression of DCLK1 kinase activity. Here, we demonstrate that DiFiD (3,5-bis [2,4-difluorobenzylidene]-4-piperidone) binds to DCLK1 with high selectivity. Moreover, DiFiD mediated suppression of DCLK1 led to G2/M arrest and apoptosis and significantly suppressed tumor growth of HNSCC xenografts and ASCC patient derived xenografts, supporting that DCLK1 is critical for SCC growth.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Humanos , Apoptose , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Quinases Semelhantes a Duplacortina , Pontos de Checagem da Fase G2 do Ciclo Celular , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , AnimaisRESUMO
Phytochemicals modulate key cellular signaling pathways and have proven anticancer effects. Alcea rosea(AR; Hollyhock) is an ornamental plant with known anti-inflammatory properties. This study explored its role as an anticancer agent. The AR seed extract (AR extract) inhibited proliferation and colony formation in a dose- and time-dependent manner and promoted apoptosis as was evidenced by cleavage of PARP and increased expression of Bax accompanying reduced levels of BCL-xl protein in HCT116 and SW480 cells, respectively. In addition, AR extract-arrested cells at Go/G1 phase of cell cycle and exhibited decreases in Cyclin D1. AR extract-treated cells exhibited reduced number and size of colonospheres in a dose-dependent manner concomitant with decreases in cancer stem cell (CSC) markers ALDH1A1 and Dclk1. Relative levels of ß-catenin, Notch-ICD, Hes1 and EZH2 were also attenuated by AR extract. TOP-flash reporter activity, a measure of Wnt signaling, decreased significantly in response to treatment while overexpression of wild type but not mutant EZH2, reversed the inhibitory effects. Moreover, WIF1 (a Wnt antagonist) promoter activity increased dramatically following treatment with AR extract which phenocopied increases in WIF1 reporter activity following EZH2 knockdown.In vivo, AR extract attenuated tumor growth due probably to reduced levels of EZH2, ß-catenin, CyclinD1 and Ki-67 along with reduced levels of CSC markers. Since partial purification via HPLC yielded a prominent peak, efforts are underway to identify the active ingredient(s). Taken together, the results clearly suggest that AR extract/active component(s) can be an effective preventative/therapeutic agent to target colon cancer.
Assuntos
Neoplasias do Colo/patologia , Epigênese Genética , Células-Tronco Neoplásicas/patologia , Plantas , Transdução de Sinais , HumanosRESUMO
Formulation development of protein therapeutics using polymeric nanoparticles has found very little success in recent years. Major formulation challenges include rapid denaturation, susceptibility to lose bioactivity in presence of organic solvents and poor encapsulation in polymeric matrix. In the present study, we have prepared hydrophobic ion pairing (HIP) complex of lysozyme, a model protein, using dextran sulfate (DS) as a complexing polymer. We have optimized the process of formation and dissociation of HIP complex between lysozyme and DS. The effect of HIP complexation on enzymatic activity of lysozyme was also studied. Nanoparticles were prepared and characterized using spontaneous emulsion solvent diffusion method. Furthermore, we have also investigated release of lysozyme from nanoparticles along with its enzymatic activity. Results of this study indicate that nanoparticles can sustain the release of lysozyme without compromising its enzymatic activity. HIP complexation using a polymer may also be employed to formulate sustained release dosage forms of other macromolecules with enhanced encapsulation efficiency.
Assuntos
Química Farmacêutica/métodos , Portadores de Fármacos/síntese química , Desenho de Fármacos , Interações Hidrofóbicas e Hidrofílicas , Muramidase/síntese química , Nanopartículas/química , Portadores de Fármacos/administração & dosagem , Avaliação Pré-Clínica de Medicamentos/métodos , Estabilidade de Medicamentos , Substâncias Macromoleculares/administração & dosagem , Substâncias Macromoleculares/síntese química , Micrococcus/efeitos dos fármacos , Micrococcus/enzimologia , Muramidase/administração & dosagem , Nanopartículas/administração & dosagem , Eletricidade Estática , TermodinâmicaRESUMO
The availability of clinical pharmacology and safety data regarding the use of prescription medications in pregnant and lactating individuals has been historically limited, despite significant efforts to improve the quantity and quality of the information in labeling. The Food and Drug Administration's (FDA) Pregnancy and Lactation Labeling Rule took effect on June 30, 2015, and updated information in labeling to more clearly describe available data to assist health care providers in counseling pregnant and lactating individuals. Additionally, the FDA published a revised draft guidance, "Clinical Lactation Studies: Considerations for Study Design," to provide pharmaceutical companies and investigators with information about how and when to conduct lactation studies. Clinical pharmacology information derived from lactation studies is important in determining the presence of medications in breast milk and counseling lactating individuals regarding the potential of medication exposure in the breast milk and its attendant risks to the breastfed infant. Examples of Pregnancy and Lactation Labeling Rule labeling changes that resulted from dedicated clinical lactation studies for certain neuropsychiatric medications are described in this publication. These medications are discussed because neuropsychiatric conditions commonly affect women of reproductive potential, including lactating individuals. As the FDA guidance and these studies illustrate, bioanalytical method validation, study design, and data analysis considerations are essential for obtaining quality lactation data. Well-designed clinical lactation studies play an important role in informing product labeling that ultimately is useful to health care providers in making prescribing decisions with lactating individuals.
Assuntos
Farmacologia Clínica , Medicamentos sob Prescrição , Estados Unidos , Lactente , Gravidez , Feminino , Humanos , Lactação , Aleitamento Materno , Rotulagem de Produtos , Medicamentos sob Prescrição/efeitos adversos , Leite HumanoRESUMO
The aim of this study was to characterize and utilize MDCK cell line expressing CYP3A4 and P-glycoprotein as an in vitro model for evaluating drug-herb and drug-drug of abuse interactions. MDCK cell line simultaneously expressing P-gp and CYP3A4 (MMC) was developed and characterized by using expression and activity studies. Cellular transport study of 200 µM cortisol was performed to determine their combined activity. The study was carried across MDCK-WT, MDCK-MDR1 and MMC cell lines. Similar studies were also carried out in the presence of 50 µM naringin and 3 µM morphine. Samples were analyzed by HPLC for drug and its CYP3A4 metabolite. PCR, qPCR and Western blot studies confirmed the enhanced expression of the proteins in the transfected cells. The Vivid CYP3A4 assay and ketoconazole inhibition studies further confirmed the presence of active protein. Apical to basal transport of cortisol was found to be 10- and 3-fold lower in MMC as compared to MDCK-WT and MDCK-MDR1 respectively. Higher amount of metabolite was formed in MMC than in MDCK-WT, indicating enhanced expression of CYP3A4. Highest cortisol metabolite formation was observed in MMC cell line due to the combined activities of CYP3A4 and P-gp. Transport of cortisol increased 5-fold in the presence of naringin in MMC and doubled in MDCK-MDR1. Cortisol transport in MMC was significantly lower than that in MDCK-WT in the presence of naringin. The permeability increased 3-fold in the presence of morphine, which is a weaker inhibitor of CYP3A4. Formation of 6ß-hydroxy cortisol was found to decrease in the presence of morphine and naringin. This new model cell line with its enhanced CYP3A4 and P-gp levels in addition to short culture time can serve as an invaluable model to study drug-drug interactions. This cell line can also be used to study the combined contribution of efflux transporter and metabolizing enzymes toward drug-drug interactions.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico/genética , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Interações Medicamentosas/genética , Animais , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Linhagem Celular , Linhagem Celular Tumoral , Cães , Flavanonas/farmacologia , Células Hep G2 , Humanos , Hidrocortisona/farmacologia , Cetoconazol/farmacologia , Células Madin Darby de Rim Canino , Morfina/farmacologia , Permeabilidade , Transfecção/métodosRESUMO
Honokiol (HNK) is a biphenolic compound that has been used in traditional medicine for treating various ailments, including cancers. In this study, we determined the effect of HNK on colon cancer cells in culture and in a colitis-associated cancer model. HNK treatment inhibited proliferation and colony formation while inducing apoptosis. In addition, HNK suppressed colonosphere formation. Molecular docking suggests that HNK interacts with reserve stem cell marker protein DCLK1, with a binding energy of -7.0 Kcal/mol. In vitro kinase assays demonstrated that HNK suppressed the DCLK1 kinase activity. HNK also suppressed the expression of additional cancer stem cell marker proteins LGR5 and CD44. The Hippo signaling pathway is active in intestinal stem cells. In the canonical pathway, YAP1 is phosphorylated at Ser127 by upstream Mst1/2 and Lats1/2. This results in the sequestration of YAP1 in the cytoplasm, thereby not allowing YAP1 to translocate to the nucleus and interact with TEAD1-4 transcription factors to induce gene expression. However, HNK suppressed Ser127 phosphorylation in YAP1, but the protein remains sequestered in the cytoplasm. We further determined that this occurs by YAP1 interacting with PUMA. To determine if this also occurs in vivo, we performed studies in an AOM/DSS induced colitis-associated cancer model. HNK administered by oral gavage at a dose of 5mg/kg bw for 24 weeks demonstrated a significant reduction in the expression of YAP1 and TEAD1 and in the stem marker proteins. Together, these data suggest that HNK prevents colon tumorigenesis in part by inducing PUMA-YAP1 interaction and cytoplasmic sequestration, thereby suppressing the oncogenic YAP1 activity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Compostos de Bifenilo/farmacologia , Carcinogênese/patologia , Neoplasias do Colo/patologia , Lignanas/farmacologia , Células-Tronco Neoplásicas/patologia , Fatores de Transcrição/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinogênese/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colite/complicações , Quinases Semelhantes a Duplacortina , Regulação para Baixo/efeitos dos fármacos , Via de Sinalização Hippo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos Endogâmicos ICR , Modelos Biológicos , Células-Tronco Neoplásicas/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaio Tumoral de Célula-Tronco , Proteínas de Sinalização YAPRESUMO
HEPES has been widely employed as an organic buffer agent in cell culture medium as well as uptake and transport experiments in vitro. However, concentrations of HEPES used in such studies vary from one laboratory to another. In this study, we investigated the effect of HEPES on the uptake and bidirectional transport of P-gp substrates employing both Caco-2 and MDCK-MDR1 cells. ATP-dependent uptake of glutamic acid was also examined. ATP production was further quantified applying ATP Determination Kit. An addition of HEPES to the growth and incubation media significantly altered the uptake and transport of P-gp substrates in both Caco-2 and MDCK-MDR1 cells. Uptake of P-gp substrates substantially diminished as the HEPES concentration was raised to 25 mM. Bidirectional (A-B and B-A) transport studies revealed that permeability ratio of P(appB-A) to P(appA-B) in the presence of 25 mM HEPES was significantly higher than control. The uptake of phenylalanine is an ATP-independent process, whereas the accumulation of glutamic acid is ATP-dependent. While phenylalanine uptake remained unchanged, glutamic acid uptake was elevated with the addition of HEPES. Verapamil is an inhibitor of P-gp mediated uptake; elevation of cyclosporine uptake in the presence of 5 muM verapamil was compromised by the presence of 25 mM HEPES. The results of ATP assay indicated that HEPES stimulated the production of ATP. This study suggests that the addition of HEPES in the medium modulated the energy dependent efflux and uptake processes. The effect of HEPES on P-gp mediated drug efflux and transport may provide some mechanistic insight into possible reasons for inconsistencies in the results reported from various laboratories.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Aminoácidos Neutros/metabolismo , Transporte Biológico/efeitos dos fármacos , HEPES/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Células CACO-2 , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Ciclosporina/metabolismo , Cães , Ácido Glutâmico/metabolismo , Humanos , Lopinavir , Fenilalanina/metabolismo , Pirimidinonas/metabolismo , Ritonavir/metabolismo , Verapamil/farmacologiaRESUMO
Pancreatic cancer is the fourth leading cause of cancer deaths in the US and no significant treatment is currently available. Here, we describe the effect of crocetinic acid, which we purified from commercial saffron compound crocetin using high performance liquid chromatography. Crocetinic acid inhibits proliferation of pancreatic cancer cell lines in a dose- and time-dependent manner. In addition, it induced apoptosis. Moreover, the compound significantly inhibited epidermal growth factor receptor and Akt phosphorylation. Furthermore, crocetinic acid decreased the number and size of the pancospheres in a dose-dependent manner, and suppressed the expression of the marker protein DCLK-1 (Doublecortin Calcium/Calmodulin-Dependent Kinase-1) suggesting that crocetinic acid targets cancer stem cells (CSC). To understand the mechanism of CSC inhibition, the signaling pathways affected by purified crocetinic acid were dissected. Sonic hedgehog (Shh) upon binding to its cognate receptor patched, allows smoothened to accumulate and activate Gli transcription factor. Crocetinic acid inhibited the expression of both Shh and smoothened. Finally, these data were confirmed in vivo where the compound at a dose of 0.5 mg/Kg bw suppressed growth of tumor xenografts. Collectively, these data suggest that purified crocetinic acid inhibits pancreatic CSC, thereby inhibiting pancreatic tumorigenesis.
Assuntos
Carotenoides/química , Proteínas Hedgehog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Cromatografia Líquida de Alta Pressão , Crocus/química , Relação Dose-Resposta a Droga , Quinases Semelhantes a Duplacortina , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Fosforilação , Extratos Vegetais/química , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Transdução de Sinais , Receptor Smoothened , Esferoides Celulares/metabolismo , Proteína 2 de Transformação que Contém Domínio 2 de Homologia de Src , Vitamina A/análogos & derivadosRESUMO
The main goal of this study is to investigate the expression of sodium dependent vitamin C transport system (SVCT2). Moreover, this investigation has been carried out to define uptake mechanism and intracellular regulation of ascorbic acid (AA) in human breast cancer cells (MDA-MB231, T47D and ZR-75-1). Uptake of [(14)C] AA was studied in MDA-MB231, T47D and ZR-75-1 cells. Functional parameters of [(14)C] AA uptake were delineated in the presence of different concentrations of unlabeled AA, pH, temperature, metabolic inhibitors, substrates and structural analogs. Molecular identification of SVCT2 was carried out with reverse transcription-polymerase chain reaction (RT-PCR). Uptake of [(14)C] AA was studied and found to be sodium, chloride, temperature, pH and energy dependent in all breast cancer cell lines. [(14)C] AA uptake was found to be saturable, with Km values of 53.85 ± 6.24, 49.69 ± 2.83 and 45.44 ± 3.16 µM and Vmax values of 18.45 ± 0.50, 32.50 ± 0.43 and 33.25 ± 0.53 pmol/min/mg protein, across MDA-MB231, T47D and ZR-75-1, respectively. The process is inhibited by structural analogs (l-AA and d-iso AA) but not by structurally unrelated substrates (glucose and PAHA). Ca(++)/calmodulin and protein kinase pathways appeared to play a crucial role in modulating AA uptake. A 626 bp band corresponding to a vitamin C transporter (SVCT2) based on the primer design was detected by RT-PCR analysis in all breast cancer cell lines. This research article describes AA uptake mechanism, kinetics, and regulation by sodium dependent vitamin C transporter (SVCT2) in MDA-MB231, T47D and ZR-75-1 cells. Also, MDA-MB231, T47D and ZR-75-1 cell lines can be utilized as a valuable in vitro model to investigate absorption and permeability of AA-conjugated chemotherapeutics.
Assuntos
Ácido Ascórbico/metabolismo , Neoplasias da Mama/metabolismo , Transportadores de Sódio Acoplados à Vitamina C/metabolismo , Neoplasias da Mama/patologia , Feminino , Humanos , Concentração de Íons de Hidrogênio , Transportadores de Sódio Acoplados à Vitamina C/genética , Temperatura , Fatores de Tempo , Células Tumorais CultivadasRESUMO
BACKGROUND: Malignant melanoma is an aggressive form of skin cancer with limited effective therapeutic options. Melanoma research concentrates on maximizing the effect on cancer cells with minimal toxicity to normal cells. AMP-activated protein kinase (AMPK) is an important regulator of cellular energy homeostasis and has been shown to control tumor progression regulating the cell cycle, protein synthesis, and cell growth and/or survival. Honokiol (HNK) is a biphenolic compound derived from Magnolia officinalis, a plant that has been used in traditional Chinese and Japanese medicine for the treatment of various pathological conditions. Recent studies have shown that HNK has antitumor activity with relatively low toxicity. In this study, we demonstrated that the growth inhibitory effects of HNK on melanoma and melanoma cancer stem cells were mediated through the activation of AMPK and hence AMPK signaling in melanoma cells. METHODS: We determined the effects of HNK treatment on various melanoma cell lines. HNK-induced cell growth inhibitory effects were determined using hexosaminidase assay. Protein expression studies were done by immunoblotting. Primary spheroid assay was used to assess stemness by growing single suspension cells in ultralow attachment plates. RESULTS: HNK is highly effective in inhibiting melanoma cells by attenuating protein kinase B/mammalian target of rapamycin and AMPK signaling. HNK showed significant inhibition of the spheroid-forming capacity of melanoma cells and, hence, stemness. HNK significantly decreased the number and size of melanospheres in a dose-dependent manner. Western blot analyses showed enhanced phosphorylation of AMPK in melanoma cells. Furthermore, HNK decreased the cellular adenosine triphosphate pool in a dose-dependent manner with maximum effects observed at 48 hours. CONCLUSIONS: The results suggest that HNK can target melanoma cells and mark them for cell death through AMPK signaling. Further studies are warranted for developing HNK as an effective chemopreventive/therapeutic agent in melanoma.
Assuntos
Proteínas Quinases Ativadas por AMP/efeitos dos fármacos , Antineoplásicos Fitogênicos/farmacologia , Compostos de Bifenilo/farmacologia , Lignanas/farmacologia , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Immunoblotting , Melanoma/enzimologia , Microscopia Eletrônica de Varredura , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/enzimologia , Melanoma Maligno CutâneoRESUMO
BACKGROUND: The purpose of this study is to identify the effect of binary and ternary combinations of anti-HIV protease inhibitors (PIs) on the expression of metabolizing enzyme (CYP3A4) and efflux transporters [multidrug resistance-associated protein 2 (MRP2), P-glycoprotein (P-gp) and breast cancer resistant protein (BCRP)] in a model intestinal cell line (LS-180). METHODS: LS-180 cells were treated with various combinations of PIs (amprenavir, indinavir, saquinavir and lopinavir), and the mRNA expression levels of metabolizing enzyme and efflux transporters were measured using quantitative reverse transcription polymerase chain reaction. The alteration of gene expression was further correlated to the expression of nuclear hormone receptor PXR. Uptake of fluorescent and radioactive substrates was carried out to study the functional activity of these proteins. Cytotoxicity and adenosine triphosphate (ATP) assays were carried out to measure stress responses. RESULTS: Binary and ternary combinations of PIs appeared to modulate the expression of CYP3A4, MRP2, P-gp and BCRP in a considerable manner. Unlike the individual PIs, their binary combinations showed much greater induction of metabolizing enzyme and efflux proteins. However, such pronounced induction was not observed in the presence of ternary combinations. The observed trend of altered mRNA expression was found to correlate well with the change in expression levels of PXR. The gene expression was found to correlate with activity assays. Lack of cytotoxicity and ATP activity was observed in the treatment samples, suggesting that these alterations in expression levels were probably not stress responses. CONCLUSIONS: In the present study, we demonstrated that combinations of drugs can have serious consequences toward the treatment of HIV infection by altering their bioavailability and disposition.
Assuntos
Citocromo P-450 CYP3A/genética , Expressão Gênica/efeitos dos fármacos , Inibidores da Protease de HIV/farmacologia , Disponibilidade Biológica , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocromo P-450 CYP3A/metabolismo , Sinergismo Farmacológico , Quimioterapia Combinada , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Infecções por HIV/metabolismo , Inibidores da Protease de HIV/administração & dosagem , Inibidores da Protease de HIV/efeitos adversos , Inibidores da Protease de HIV/farmacocinética , Humanos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Distribuição TecidualRESUMO
Recently, we demonstrated that extracts of bitter melon (BME) can be used as a preventive/therapeutic agent in colon cancers. Here, we determined BME effects on anticancer activity and bioavailability of doxorubicin (DOX) in colon cancer cells. BME enhanced the effect of DOX on cell proliferation and sensitized the cells toward DOX upon pretreatment. Furthermore, there was both increased drug uptake and reduced drug efflux. We also observed a reduction in the expression of multidrug resistance conferring proteins (MDRCP) P-glycoprotein, MRP-2, and BCRP. Further BME suppressed DOX efflux in MDCK cells overexpressing the three efflux proteins individually, suggesting that BME is a potent inhibitor of MDR function. Next, we determined the effect of BME on PXR, a xenobiotic sensing nuclear receptor and a transcription factor that controls the expression of the three MDR genes. BME suppressed PXR promoter activity thereby suppressing its expression. Finally, we determined the effect of AMPK pathway on drug efflux because we have previously demonstrated that BME affects the pathway. However, inhibiting AMPK did not affect drug resistance, suggesting that BME may use different pathways for the anticancer and MDR modulating activities. Together, these results suggest that BME can enhance the bioavailability and efficacy of conventional chemotherapy.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Cucurbitaceae/química , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Antineoplásicos/farmacocinética , Disponibilidade Biológica , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Doxorrubicina/farmacocinética , Humanos , Proteína 2 Associada à Farmacorresistência Múltipla , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificaçãoRESUMO
The objective of this study is to delineate whether overexpression of human efflux transporters (P-gp, MRP2, and BCRP) in transfected MDCK cells affect the functional activities, and gene and protein expression of endogenous influx peptide transporter system (PepT). Real-time PCR, immunoblotting, uptake and permeability studies of [(3)H]Gly-Sar were conducted on transfected MDCKII and wild-type cells to investigate functional differences. Cellular [(3)H]Gly-Sar accumulation was significantly lower in transfected MDCKII cell lines compared to wild-type cells. Transport efficiency of apical peptide transporters was markedly reduced to around 25%, 30%, and 40% in P-gp-, MRP2-, and BCRP-overexpressed MDCK cell lines, respectively. With ascending cell-passage, transport efficiency was enhanced. A significantly higher Gly-Sar permeability was observed across parental cell-monolayers over transfected cells at all pHs. Levels of mRNA for both canine PepT1 and PepT2 were substantially reduced when efflux transporters overexpressed but enhanced when mRNA-levels of efflux genes diminished with ascending cell-passage of transfected cells. An inverse correlation was evident between endogenous PepT and exogenous efflux transporters in transfected MDCKII cells. Results of protein expression also supported these findings. Overexpression of MDR genes can affect endogenous PepT function which might be due to the phenomenon of transporter-compensation resulting in down-regulation of endogenous genes.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas de Neoplasias/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Transporte Biológico , Western Blotting , Dipeptídeos/metabolismo , Cães , Regulação para Baixo , Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Células Madin Darby de Rim Canino , Proteína 2 Associada à Farmacorresistência Múltipla , Transportador 1 de Peptídeos , Reação em Cadeia da Polimerase em Tempo Real , Simportadores/genética , TransfecçãoRESUMO
Bitter melon fruit is recommended in ancient Indian and Chinese medicine for prevention/treatment of diabetes. However its effects on cancer progression are not well understood. Here, we have determined the efficacy of methanolic extracts of bitter melon on colon cancer stem and progenitor cells. Both, whole fruit (BMW) and skin (BMSk) extracts showed significant inhibition of cell proliferation and colony formation, with BMW showing greater efficacy. In addition, the cells were arrested at the S phase of cell cycle. Moreover, BMW induced the cleavage of LC3B but not caspase 3/7, suggesting that the cells were undergoing autophagy and not apoptosis. Further confirmation of autophagy was obtained when western blots showed reduced Bcl-2 and increased Beclin-1, Atg 7 and 12 upon BMW treatment. BMW reduced cellular ATP levels coupled with activation of AMP activated protein kinase; on the other hand, exogenous additions of ATP lead to revival of cell proliferation. Finally, BMW treatment results in a dose-dependent reduction in the number and size of colonospheres. The extracts also decreased the expression of DCLK1 and Lgr5, markers of quiescent, and activated stem cells. Taken together, these results suggest that the extracts of bitter melon can be an effective preventive/therapeutic agent for colon cancer.
RESUMO
A majority of studies involving prodrugs are directed to overcome low bioavailability of the parent drug. The aim of this study is to increase the bioavailability of acyclovir (ACV) by designing a novel prodrug delivery system which is more lipophilic, and at the same time site specific. In this study, a lipid raft has been conjugated to the parent drug molecule to impart lipophilicity. Simultaneously a targeting moiety that can be recognized by a specific transporter/receptor in the cell membrane has also been tethered to the other terminal of lipid raft. Targeted lipid prodrugs i.e., biotin-ricinoleicacid-acyclovir (B-R-ACV) and biotin-12hydroxystearicacid-acyclovir (B-12HS-ACV) were synthesized with ricinoleicacid and 12hydroxystearicacid as the lipophilic rafts and biotin as the targeting moiety. Biotin-ACV (B-ACV), ricinoleicacid-ACV (R-ACV) and 12hydroxystearicacid-ACV (12HS-ACV) were also synthesized to delineate the individual effects of the targeting and the lipid moieties. Cellular accumulation studies were performed in confluent MDCK-MDR1 and Caco-2 cells. The targeted lipid prodrugs B-R-ACV and B-12HS-ACV exhibited much higher cellular accumulation than B-ACV, R-ACV and 12HS-ACV in both cell lines. This result indicates that both the targeting and the lipid moiety act synergistically toward cellular uptake. The biotin conjugated prodrugs caused a decrease in the uptake of [(3)H] biotin suggesting the role of sodium dependent multivitamin transporter (SMVT) in uptake. The affinity of these targeted lipid prodrugs toward SMVT was studied in MDCK-MDR1 cells. Both the targeted lipid prodrugs B-R-ACV (20.25 ± 1.74 µM) and B-12HS-ACV (23.99 ± 3.20 µM) demonstrated higher affinity towards SMVT than B-ACV (30.90 ± 4.19 µM). Further, dose dependent studies revealed a concentration dependent inhibitory effect on [(3)H] biotin uptake in the presence of biotinylated prodrugs. Transepithelial transport studies showed lowering of [(3)H] biotin permeability in the presence of biotin and biotinylated prodrugs, further indicating a carrier mediated translocation by SMVT. Overall, results from these studies clearly suggest that these biotinylated lipid prodrugs of ACV possess enhanced affinity towards SMVT. These prodrugs appear to be potential candidates for the treatment of oral and ocular herpes virus infections, because of higher expression of SMVT on intestinal and corneal epithelial cells. In conclusion we hypothesize that our novel prodrug design strategy may help in higher absorption of hydrophilic parent drug. Moreover, this novel prodrug design can result in higher cell permeability of hydrophilic therapeutics such as genes, siRNA, antisense RNA, DNA, oligonucleotides, peptides and proteins.
Assuntos
Aciclovir/farmacocinética , Antivirais/farmacocinética , Sistemas de Liberação de Medicamentos , Aciclovir/administração & dosagem , Animais , Antivirais/administração & dosagem , Disponibilidade Biológica , Biotina/química , Biotina/farmacocinética , Células CACO-2 , Linhagem Celular , Membrana Celular/metabolismo , Cães , Relação Dose-Resposta a Droga , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipídeos/química , Permeabilidade , Pró-Fármacos , Ácidos Ricinoleicos/química , Ácidos Esteáricos/química , Distribuição TecidualRESUMO
Fluoroquinolones are broad spectrum antibiotics widely indicated in the treatment of both human and animal diseases. The primary objective of this study was to assess short and long term affinities of gemifloxacin towards efflux transporters (P-gp, MRP2) and nuclear hormone receptor (PXR). Uptake and dose dependent inhibition studies were performed with [(14)C] erythromycin (0.25 µCi/ml) on MDCKII-MDR1 and MDCKII-MRP2 cells. Cellular accumulation of calcein-AM was further determined to confirm the affinity of gemifloxacin towards P-gp and MRP2. Transport studies were conducted to determine bi-directional permeability and to assess efflux ratio of gemifloxacin. LS-180 cells were treated with three different concentrations of gemifloxacin for 72 h and real-time PCR analysis was performed to study the quantitative gene expression levels of PXR, MDR1 and MRP2. Further, [(14)C] erythromycin uptake was also performed on LS-180 treated cells to better delineate the functional activity of efflux transporters. Results from our study suggest that gemifloxacin may be a substrate of both the efflux transporters studied. This compound inhibited both P-gp and MRP2 mediated efflux of [(14)C] erythromycin in a dose dependent manner with IC(50) values of 123 ± 2 µM and 16 ± 2 µM, respectively. The efflux ratio of [(14)C] erythromycin lowered from 3.56 to 1.63 on MDCKII-MDR1 cells and 4.93 to 1.26 on MDCKII-MRP2 cells. This significant reduction in efflux ratio further confirmed the substrate specificity of gemifloxacin towards P-gp and MRP2. Long term exposure significantly induced the expression of PXR (18 fold), MDR1 (6 fold) and MRP2 (6 fold). A decrease (20%) in [(14)C] erythromycin uptake further confirmed the elevated functional activity of P-gp and MRP2. In conclusion, our studies demonstrated that gemifloxacin is effluxed by both P-gp and MRP2. Long term exposure induced their gene expression and functional activity. This substrate specificity of gemifloxacin towards these efflux transporters may be one of the major factors accounting for low oral bioavailability (71%). Better understanding of these mechanistic interactions may aid in the development of newer strategies to achieve adequate therapeutic levels and higher bioavailability.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antibacterianos/metabolismo , Fluoroquinolonas/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Naftiridinas/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/metabolismo , Animais , Antibacterianos/farmacologia , Ligação Competitiva , Transporte Biológico , Linhagem Celular , Permeabilidade da Membrana Celular , Cães , Relação Dose-Resposta a Droga , Eritromicina/metabolismo , Fluoresceínas/metabolismo , Fluoroquinolonas/farmacologia , Gemifloxacina , Regulação da Expressão Gênica , Humanos , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Naftiridinas/farmacologia , Receptor de Pregnano X , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Esteroides/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
Sutherlandia frutescens (sutherlandia), an African herbal supplement was recommended by the South African Ministry of Health for the treatment of AIDS patients. However, no reports yet exist delineating the effect of sutherlandia on pharmacokinetics of antiretroviral agents. Therefore, this investigation aimed at screening the effects of short term and chronic exposure of sutherlandia on oral bioavailability and pharmacokinetics of nevirapine (NVP), a non-nucleoside reverse transcriptase inhibitor, in Sprague Dawley rats. NVP (6 mg/kg) was administered orally alone (control) and with co-administration of sutherlandia; short term (12 mg/kg single dose) and long term (12 mg/kg, once a day for 5 days). No significant difference in the pharmacokinetic parameters of NVP was found upon short-term co-administration of Sutherlandia. However, there was a 50% decrease (p<0.05) in the AUC and C(max) values of NVP after 5 days of chronic exposure with Sutherlandia. In addition, quantitative RT-PCR studies demonstrated a 2-3-fold increase in the hepatic and intestinal mRNA expression of CYP3A2, relative to vehicle control. To further confirm, if this could translate into a clinically relevant pharmacokinetic interaction in patients, we tested this hypothesis employing LS-180 cells as an in vitro induction model for human CYP3A4. Ninety-six hours post treatment, similar to positive control rifampicin (25 µM), sutherlandia extract (300 µg/mL) resulted in elevated m-RNA expression levels and functional activity of CYP3A4 (human homologue of rodent CYP3A2) in LS-180 cells. Taken together, these results suggest that a potential drug-herb interaction is possible when NVP is co-administered with S. frutescens, although this hypothesis still remains to be investigated in a clinical setting.
Assuntos
Fabaceae , Interações Ervas-Drogas , Nevirapina/farmacocinética , Extratos Vegetais/farmacocinética , Inibidores da Transcriptase Reversa/farmacocinética , Administração Oral , Animais , Linhagem Celular Tumoral , Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A , Avaliação Pré-Clínica de Medicamentos , Formazans , Humanos , Masculino , Nevirapina/administração & dosagem , Nevirapina/metabolismo , Nevirapina/farmacologia , Extratos Vegetais/administração & dosagem , Extratos Vegetais/metabolismo , Extratos Vegetais/farmacologia , Ratos , Ratos Sprague-Dawley , Inibidores da Transcriptase Reversa/administração & dosagem , Inibidores da Transcriptase Reversa/metabolismo , Inibidores da Transcriptase Reversa/farmacologia , Sais de Tetrazólio , Fatores de TempoRESUMO
Multidrug regimens and corresponding drug interactions cause many adverse reactions and treatment failures. Drug efflux transporters: P-gp, MRP, BCRP in conjunction with metabolizing enzymes (CYPs) are major factors in such interactions. Most effective combination antiretrovirals (ARV) therapy includes a PI or a NNRTI or two NRTI. Coadministration of such ARV may induce efflux transporters and/or CYP3A4 resulting in sub-therapeutic blood levels and therapeutic failure due to reduced absorption and/or increased metabolism. A similar prognosis is true for ARV-compounds and drugs of abuse combinations. Morphine and nicotine enhance CYP3A4 and MDR1 expression in vitro. A 2.5 fold rise of cortisol metabolite was evident in smokers relative to nonsmokers. Altered functions of efflux transporters and CYPs in response to ARV and drugs of abuse may result in altered drug absorption and metabolism. Appropriate in vitro models can be employed to predict such interactions. Influence of genetic polymorphism, SNP and inter-individual variation in drug response has been discussed. Complexity underlying the relationship between efflux transporters and CYP makes it difficult to predict the outcome of HAART as such, particularly when HIV patients taking drugs of abuse do not adhere to HAART regimens. HIV(+) pregnant women on HAART medications, indulging in drugs of abuse, may develop higher viral load due to such interactions and lead to increase in mother to child transmission of HIV. A multidisciplinary approach with clear understanding of mechanism of interactions may allow proper selection of regimens so that desired therapeutic outcome of HAART can be reached without any side effects.
Assuntos
Fármacos Anti-HIV/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Transtornos Relacionados ao Uso de Substâncias/complicações , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Analgésicos Opioides/metabolismo , Fármacos Anti-HIV/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Benzodiazepinas/metabolismo , Cocaína/metabolismo , Citocromo P-450 CYP3A/efeitos dos fármacos , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Interações Medicamentosas , Feminino , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Humanos , Metanfetamina/metabolismo , Proteínas de Neoplasias/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Gravidez , Ritonavir/metabolismo , Ritonavir/uso terapêuticoRESUMO
The purpose of the study is to screen the interactions of fourth generation fluoroquinolone-gatifloxacin with efflux pumps, i.e., P-gp, MRP2 and BCRP. Mechanism of gatifloxacin interaction with efflux transporters may explain its acquired resistance. Such clarification may lead to the development of strategies to overcome efflux and enhance its bioavailability at target site. This process will aid in the reduction of dose volume, further eliminating the chances of systemic toxicity from topical gatifloxacin eye drops. MDCK cell lines transfected with the targeted efflux transporters were used for this study. [(14)C] Erythromycin was selected as a model substrate for P-gp and MRP2 whereas Hoechst 33342 was employed as a substrate for BCRP. Uptake and transport studies of these substrates were performed in the presence of gatifloxacin to delineate its interaction with efflux transporters. Further the efflux ratio in the presence of gatifloxacin was calculated from bidirectional transport studies. The concentration of [(14)C] erythromycin and Hoechst 33342 was measured using scintillation counter and fluorescence plate reader, respectively. A concentration dependent inhibition effect in the presence of gatifloxacin was revealed on [(14)C] erythromycin uptake. The efflux ratio (BL-AP/AP-BL) of substrates was found to approach unity at higher gatifloxacin concentrations. Increased concentration of gatifloxacin did not elevate uptake of Hoechst 33342. All these studies were validated with known inhibitors as positive control. Uptake and transport studies support the hypothesis that gatifloxacin is a substrate for P-gp, MRP2 but not for BCRP. Possible interactions of gatifloxacin with P-gp and MRP2 may be a possible mechanism for acquired resistance of gatifloxacin. This information can be further extended to design prodrugs or formulations in order to prevent development of acquired resistance and improve therapeutic efficacy with its reduction in side effects.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Fluoroquinolonas/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Antibacterianos/metabolismo , Benzimidazóis/metabolismo , Ligação Competitiva , Transporte Biológico , Linhagem Celular , Polaridade Celular , Dicetopiperazinas , Cães , Relação Dose-Resposta a Droga , Eritromicina/metabolismo , Fluoroquinolonas/metabolismo , Gatifloxacina , Compostos Heterocíclicos de 4 ou mais Anéis , Humanos , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Propionatos/farmacologia , Quinidina/farmacologia , Quinolinas/farmacologia , TransfecçãoRESUMO
Breast cancer resistance protein (BCRP), a 72 kDa protein belongs to the subfamily G of the human ATP-binding cassette transporter superfamily. Overexpression of BCRP was found to play a major role in the development of resistance against various chemotherapeutic agents. BCRP plays an important role in absorption, distribution and elimination of several therapeutic agents. BCRP expression and functional activity across human bronchial epithelium and its impact on pulmonary drug accumulation has not been established. The objective of this study was to identify and characterize the BCRP efflux transporter across human bronchial epithelium. Calu-3, a human bronchial epithelial cell line was employed as a model for this study. Reverse transcription-polymerase chain reaction (RT-PCR), Western blot and immunocytochemical studies were performed to identify and characterize the expression of BCRP. RT-PCR studies detected ABCG2 mRNA levels in Calu-3 cells. A strong band for BCRP with a molecular weight of approximately 72 kDa was observed in Western blot analysis. Immunocytochemical studies confirmed the presence of BCRP on the apical membrane of human bronchial epithelium. Functional activity of BCRP was determined by performing uptake of radioactive substrate [3H]-mitoxantrone in the presence and absence of BCRP inhibitors. Uptake of [3H]-mitoxantrone was elevated significantly in the presence of GF120918 and fumitremorgin C. An increase in the accumulation of Hoechst 33342, a fluorescent dye was also detected in the presence of BCRP inhibitors when compared to control. In summary, this study provides evidence for the presence of an ATP dependent, membrane bound efflux transporter BCRP across human bronchial epithelial cell line, Calu-3.