RESUMO
Our previous findings have shown that the chlorophyllides composites have anticancer activities to breast cancer cell lines (MCF-7 and MDA-MB-231). In the present study, microarray gene expression profiling was utilized to investigate the chlorophyllides anticancer mechanism on the breast cancer cells lines. Results showed that chlorophyllides composites induced upregulation of 43 and 56 differentially expressed genes (DEG) in MCF-7 and MDA-MB-231 cells, respectively. In both cell lines, chlorophyllides composites modulated the expression of annexin A4 (ANXA4), chemokine C-C motif receptor 1 (CCR1), stromal interaction molecule 2 (STIM2), ethanolamine kinase 1 (ETNK1) and member of RAS oncogene family (RAP2B). Further, the KEGG annotation revealed that chlorophyllides composites modulated DEGs that are associated with the endocrine system in MCF-7 cells and with the nervous system in MDA-MB-231 cells, respectively. The expression levels of 9 genes were validated by quantitative reverse transcription PCR (RT-qPCR). The expression of CCR1, STIM2, ETNK1, MAGl1 and TOP2A were upregulated in both chlorophyllides composites treated-MCF-7 and MDA-MB-231 cells. The different expression of NLRC5, SLC7A7 and PKN1 provided valuable information for future investigation and development of novel cancer therapy.
Assuntos
Neoplasias da Mama , Clorofilídeos , Sistema y+L de Transporte de Aminoácidos , Mama , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Detecção Precoce de Câncer , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células MCF-7 , Proteínas rap de Ligação ao GTPRESUMO
The purity of chlorophylls plays one of the key role for the production of chlorophyllides. We have designed a facile method for chlorophyll purification by twice solvent extraction. Twice extraction causes the loss of chlorophylls, but the purity of total chlorophylls can be enhanced 182%. Then, the purified chlorophylls can be converted to relatively pure chlorophyllides facilely. The results show that higher purity of chlorophyllides could be obtained when purified chlorophylls (ethanol-hexane extract) was used as starting materials than that of crude chlorophylls (ethanol-only extract). In biocompatibility test, the results showed that the prepared chlorophyllides can be applied as biomaterials. When the prepared chlorophyllides were applied to anticancer tests, they were active both in MCF7 and MDA-MB-231 (multidrug resistant breast cancer cells) cell lines. In addition, the results suggested that the prepared chlorophyllides could be a potential candidate of combination therapy with doxorubicin to breast cancers.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Clorofila/isolamento & purificação , Clorofilídeos/farmacologia , Resistência a Múltiplos Medicamentos/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Clorofila/química , Clorofila/farmacologia , Clorofilídeos/biossíntese , Clorofilídeos/química , Doxorrubicina/efeitos adversos , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Células MCF-7 , Linfócitos T Citotóxicos/efeitos dos fármacosRESUMO
Chlorophyllide (chlide) is a natural catabolic product of chlorophyll (Chl), produced through the activity of chlorophyllase (chlase). The growth inhibitory and antioxidant effects of chlide from different plant leaf extracts have not been reported. The aim of this study is to demonstrate that chlide in crude extracts from leaves has the potential to exert cytotoxic effects on cancer cell lines. The potential inhibitory and antioxidant effects of chlide in crude extracts from 10 plant leaves on breast cancer cells (MCF7 and MDA-MB-231), hepatocellular carcinoma cells (Hep G2), colorectal adenocarcinoma cells (Caco2), and glioblastoma cells (U-118 MG) were studied using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and DPPH (1,1-diphenyl-2-picrylhydrazyl) assays. The results of the MTT assay showed that chlide in crude extracts from sweet potato were the most effective against all cancer cell lines tested. U-118 MG cells were the most sensitive, while Caco2 cells were the most resistant to the tested crude extracts. The cytotoxic effects of chlide and Chl in crude extracts from sweet potato and of commercial chlorophyllin (Cu-chlin), in descending order, were as follows: chlide > Chl > Cu-chlin. Notably, the IC50 of sweet potato in U-118 MG cells was 45.65 µg/mL while those of Chl and Cu-chlin exceeded 200 µg/mL. In the DPPH assay, low concentrations (100 µg/mL) of chlide and Cu-chlin from crude extracts of sweet potato presented very similar radical scavenging activity to vitamin B2. The concentration of chlide was negatively correlated with DPPH activity. The current study was the first to demonstrate that chlide in crude extracts from leaves have potential cytotoxicity in cancer cell lines. Synergism between chlide and other compounds from leaf crude extracts may contribute to its cytotoxicity.
RESUMO
Tanshinone IIA, one of the most pharmacologically bioactive phytochemicals isolated from Salvia miltiorrhiza Bunge, possesses several biological activities such as anti-inflammation, anti-cancer, neuroprotection and hypolipidemic activities. In this study, we aim to investigate the hypocholesterolemic effect of tanshinone IIA in hepatic cells. We demonstrated that tanshinone IIA significantly increased the amount of low-density lipoprotein receptor (LDLR) and LDL uptake activity in HepG2 cells at the post-transcriptional regulation. We further demonstrated that tanshinone IIA inhibited the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA and mature protein, which may lead to an increase the cell-surface LDLR in hepatic cells. We further identified a regulatory DNA element involved in the tanshinone IIA-mediated PCSK9 down-regulation, which is located between the -411 and -336 positions of the PCSK9 promoter. Moreover, we found that tanshinone IIA markedly increased the nuclear forkhead box O3a (FoxO3a) level, enhanced FoxO3a/PCSK9 promoter complexes formation and decreased the PCSK9 promoter binding capacity of hepatocyte nuclear factor 1α (HNF-1α), resulting in suppression of PCSK9 gene expression. Finally, we found that the statin-induced PCSK9 overexpression was attenuated and the LDLR activity was elevated in a synergic manner by combination of tanshinone IIA treatment in HepG2 cells. Overall, our results reveal that the tanshinone IIA modulates LDLR level and activity via down-regulation of PCSK9 expression in hepatic cells. Our current findings provide a molecular basis of tanshinone IIA to develop PCSK9 inhibitors for cholesterol management.
Assuntos
Abietanos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Lipoproteínas LDL/metabolismo , Pró-Proteína Convertase 9/genética , Células Hep G2 , Humanos , Regiões Promotoras Genéticas , Receptores de LDL/genéticaRESUMO
Luteolin (3',4',5,7-tetrahydroxyflavone), a flavonoid found in several vegetables and fruits, has been reported to possess neurotrophic activities that are associated with its capacity to promote neuronal survival and differentiation. In the present study, we report for the first time a genomewide screen for microRNAs (miRNAs) regulated during the luteolin-mediated neurite outgrowth of PC12 cells. We found that after luteolin treatment, the abundance of 16 miRNAs was markedly up-regulated and that of 3 miRNAs was down-regulated in PC12 cells. The induction of miR-34a by luteolin was the most pronounced among these differentially expressed miRNAs. The correlation between miR-34a down-regulation and decreased luteolin-mediated neurite outgrowth may indicate a mechanism by which miR-34a may act as a modulator of neuronal differentiation. Furthermore, we found that luteolin enhanced the phosphorylation of p53 at Ser15, which was associated with the promotion of miR-34a transcription and neurite outgrowth. Moreover, the level of sirtuin 1 (SIRT1), a known miR-34a target, was reduced during luteolin-induced neurite outgrowth. In turn, the level of acetylated p53, a substrate of SIRT1, was correspondingly increased in luteolin-treated PC12 cells. In addition to p53 activation, we further identified that luteolin-induced miR-34a transcription and neurite outgrowth involved the activation of the JNK and p38 MAPK pathways. However, the inhibition of JNK and p38 MAPK activation did not block luteolin-induced p53 activation in PC12 cells. Our findings suggested that the activation of both p53-dependent and p53-independent miR-34a/SIRT1 pathways plays a critical role in the mechanisms underlying luteolin-induced neuritogenesis.
Assuntos
Luteolina/farmacologia , MicroRNAs/genética , Neuritos/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Animais , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , MicroRNAs/metabolismo , Neuritos/metabolismo , Células PC12 , Ratos , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Fisetin (3,7,3',4'-tetrahydroxyflavone), a flavonol compound of flavonoids, exhibits a broad spectrum of biological activities including anti-oxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The aim of this study is to investigate the cytoprotective effect of fisetin and the underlying molecular mechanism against hypoxia-induced cell death in PC12 cells. The results of this study showed that fisetin significantly restored the cell viability of PC12 cells under both cobalt chloride (CoCl2)- and low oxygen-induced hypoxic conditions. Treatment with fisetin successfully reduced the CoCl2-mediated reactive oxygen species (ROS) production, which was accompanied by an increase in the cell viability of PC12 cells. Furthermore, we found that treatment of PC12 cells with fisetin markedly upregulated hypoxia-inducible factor 1α (HIF-1α), its nuclear accumulation and the hypoxia-response element (HRE)-driven transcriptional activation. The fisetin-mediated cytoprotection during CoCl2 exposure was significantly attenuated through the administration of HIF-1α siRNA. Moreover, we demonstrated that MAPK/ERK kinase 1/2 (MEK1/2), p38 MAPK and phosphatidylinositol 3-kinase (PI3 K) inhibitors significantly blocked the increase in cell survival that was induced by fisetin treatment under hypoxic conditions. Consistently, increased phosphorylation of ERK, p38 and Akt proteins was observed in PC12 cells treated with fisetin. However, the fisetin-induced HRE-driven transcription was not affected by inhibition of these kinase signaling pathways. Current results reveal for the first time that fisetin promotes cell survival and protects against hypoxia-induced cell death through ROS scavenging and the activation of HIF1α-, MAPK/ERK-, p38 MAPK- and PI3 K/Akt-dependent signaling pathways in PC12 cells.
Assuntos
Flavonoides/farmacologia , Sequestradores de Radicais Livres/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/agonistas , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Compostos Fitoquímicos/farmacologia , Espécies Reativas de Oxigênio/antagonistas & inibidores , Animais , Hipóxia Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cobalto/toxicidade , Flavonoides/antagonistas & inibidores , Flavonóis , Sequestradores de Radicais Livres/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neurônios/citologia , Neurônios/metabolismo , Fármacos Neuroprotetores/antagonistas & inibidores , Células PC12 , Compostos Fitoquímicos/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , Ratos , Espécies Reativas de Oxigênio/metabolismo , Elementos de Resposta/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Monocyte recruitment and invasion play critical roles in the initiation and progression of atherosclerosis. The reduction in monocyte adhesion and infiltration is thought to exert antiatherosclerotic effects. Curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC) are the major active components of curcuminoids and exhibit several biological activities, including anti-inflammatory, anticarcinogenic, and hypocholesterolemic activities. The aim of this study was to investigate the antiatherogenic effects and mechanisms of curcuminoids during monocyte to macrophage differentiation. The results showed that curcumin, DMC, and BDMC (20 µM) suppressed matrix invasion from 100.0 ± 5.0% to 24.8 ± 1.4%, 26.6 ± 2.9%, and 33.7 ± 1.7%, respectively, during PMA-induced THP-1 differentiation. We found that curcuminoids significantly reduced PMA-induced CD11b and MMP-9 expression by THP-1 cells. Production of reactive oxygen species (ROS) induced by PMA (126.7 ± 2.1%) was markedly attenuated by curcumin, DMC, and BDMC to 99.5 ± 7.8%, 87.8 ± 8.2%, and 89.8 ± 7.6%, respectively, resulting in the down-regulation of CD11b and MMP-9 expression. We demonstrated that curcuminoids inhibited NADPH oxidase through the down-regulation of NOX2 expression and the reduction of p47phox membrane translocation. Moreover, we found involvement of PKCδ in the PMA-induced NOX2, CD11b, and MMP-9 mRNA expression. Curcumin, DMC, and BDMC decreased the active form of PKCδ protein stimulated by PMA in THP-1 cells. Overall, our results reveal that curcuminoids suppress matrix invasion through the inhibition of the PKCδ/NADPH oxidase/ROS signaling pathway during monocyte-macrophage differentiation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Curcumina/farmacologia , Macrófagos/citologia , Monócitos/citologia , NADPH Oxidases/metabolismo , Proteína Quinase C-delta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , Monócitos/metabolismo , NADPH Oxidases/genética , Proteína Quinase C-delta/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
SCOPE: Curcumin has been demonstrated as having numerous desirable characteristics, such as antioxidant, anti-inflammatory, and antiatherogenic activities. We report the hypocholesterolemic effect and molecular mechanism of curcumin. METHODS AND RESULTS: We found that curcumin enhanced LDL receptor (LDLR) level on the cell surface, as well as LDLR activity; however, LDLR transcription and mRNA stability were not affected. Furthermore, we found that proprotein convertase subtilisin/kexin type 9 (PCSK9) gene was downregulated at the transcriptional level by curcumin, leading to an increase in LDL uptake in HepG2 cells. The curcumin-responsive element of the PCSK9 promoter, a binding site for hepatocyte nuclear factor 1α (HNF-1α), was also identified. We demonstrated that curcumin reduced the nuclear abundance of hepatocyte nuclear factor 1α, resulting in its attenuated interaction with the PCSK9 promoter and leading to a downregulation of PCSK9 expression. Finally, we showed that curcumin decreased the statin-induced PCSK9 expression and potentially synergized with statin administration. CONCLUSION: Current results indicate that curcumin suppression of PCSK9 expression is associated with increases in cell-surface LDLR and LDLR activity in hepatic cells and it acts in a molecular mechanism that is distinct from the statins. Curcumin exhibits hypolipidemic activity and may serve as a useful supplement to statin treatment for hypercholesterolemia.
Assuntos
LDL-Colesterol/metabolismo , Curcumina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Pró-Proteína Convertases/metabolismo , Receptores de LDL/metabolismo , Serina Endopeptidases/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Curcumina/química , Sinergismo Farmacológico , Células Hep G2 , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Hipercolesterolemia/tratamento farmacológico , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de LDL/genética , Serina Endopeptidases/genética , Transcrição GênicaRESUMO
Polymethoxyflavones (PMFs) exhibit a broad spectrum of biological properties, including anticancer, antiatherogenic, and neuroprotective effects. The aim of this study is to investigate the neurotrophic effects of 5-demethylnobiletin, a hydroxylated PMF found in citrus plants, and gardenin A, a synthetic PMF analogue, on neurite outgrowth and neuronal differentiation in PC12 cells. The results of this study showed that 5-demethylnobiletin and gardenin A (10-20 µM) potently induce neurite outgrowth in PC12 cells, accompanied by the expression of neuronal differentiation and synapse formation marker proteins, growth-associated protein-43 (GAP-43), and synaptophysin. We observed that the addition of K252a, a TrKA antagonist, significantly inhibited NGF-induced neurite outgrowth in PC12 cells, but 5-demethylnobiletin- or gardenin A-induced neurite outgrowth was not affected. Treatment with 5-demethylnobiletin and gardenin A markedly induced the phosphorylation of both cyclic AMP response element-binding protein (CREB) and CRE-mediated transcription, which was suppressed through the administration of the inhibitor 2-naphthol AS-E phosphate (KG-501) or using CREB siRNA. Furthermore, our results showed that MAPK/ERK kinase 1/2 (MEK1/2), protein kinase A (PKA), and protein kinase C (PKC) inhibitors blocked the CRE transcription activity and neurite outgrowth induced through 5-demethylnobiletin or gardenin A. Consistently, increased ERK phosphorylation and PKA and PKC activities were observed in PC12 cells treated with 5-demethylnobiletin or gardenin A. These results reveal for the first time that 5-demethylnobiletin and gardenin A promote neuritogenesis through the activation of MAPK/ERK-, PKC-, and PKA-dependent, but not TrkA-dependent, CREB signaling pathways in PC12 cells.