RESUMO
Gambogic acid (GA) is an anticancer agent in phase IIb clinical trial in China. In HeLa cells, GA inhibited cell proliferation, induced cell cycle arrest at G2/M phase and apoptosis, as showed by results of MTT assay and flow cytometric analysis. Possible target-related proteins of GA were searched using comparative proteomic analysis (2-DE) and nine proteins at early (3 h) stage together with nine proteins at late (24 h) stage were found. Vimentin was the only target-related protein found at both early and late stage. Results of both 2-DE analysis and Western blotting assay suggested cleavage of vimentin induced by GA. MS/MS analysis of cleaved vimentin peptides indicated possible cleavage sites of vimentin at or near ser51 and glu425. Results of targeted proteomic analysis showed that GA induced change in phosphorylation state of the vimentin head domain (aa51-64). Caspase inhibitors could not abrogate GA-induced cleavage of vimentin. Over-expression of vimentin ameliorated cytotoxicity of GA in HeLa cells. The GA-activated signal transduction, from p38 MAPK, heat shock protein 27 (HSP27), vimentin, dysfunction of cytoskeleton, to cell death, was predicted and then confirmed. Results of animal study showed that GA treatment inhibited tumor growth in HeLa tumor-bearing mice and cleavage of vimentin could be observed in tumor xenografts of GA-treated animals. Results of immunohistochemical staining also showed down-regulated vimentin level in tumor xenografts of GA-treated animals. Furthermore, compared with cytotoxicity of GA in HeLa cells, cytotoxicity of GA in MCF-7 cells with low level of vimentin was weaker whereas cytotoxicity of GA in MG-63 cells with high level of vimentin was stronger. These results indicated the important role of vimentin in the cytotoxicity of GA. The effects of GA on vimentin and other epithelial-to-mesenchymal transition (EMT) markers provided suggestion for better usage of GA in clinic.
Assuntos
Proteoma/metabolismo , Proteômica/métodos , Neoplasias do Colo do Útero/tratamento farmacológico , Vimentina/metabolismo , Xantonas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Eletroforese em Gel Bidimensional , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Células HeLa , Humanos , Células MCF-7 , Camundongos Nus , Microscopia Confocal , Proteoma/genética , Interferência de RNA , Espectrometria de Massas em Tandem , Fatores de Tempo , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Vimentina/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The formation and metastasis of tumor cells are closely related to the gene regulation. It is critical to elucidate the molecular mechanism of a compound using in cancer therapy. In this article, we reviewed the anti-cancer molecular mechanism of arsenic trioxide and artemisinin. Its anti-cancer function mainly includes: regulation of cell cycle regulatory proteins to inhibit tumor cell proliferation, cell apoptosis signal transduction pathway to promote apoptosis in tumor cells, immortalization associated genes to reduce the life of tumor cells, angiogenesis/invasion/metastasis gene to block the spread of tumor cells, promoter methylation and protein ubiquitination gene to enhance anti-oncogene expression and ubiquitin- mediated protein degradation, micro RNA to inhibit proliferation or induce apoptosis in tumor cells, DNA synthesis and repair of DNA damage and repair gene to decrease the DNA synthesis of tumor cells, signal transduction pathways of cell proliferation/apoptosis and invasion/metastasis etc., the expression of hormone receptors and so on. We indicated the problems existing in current studies and also prospected the future of using the compound to fight cancer.
Assuntos
Antineoplásicos/farmacologia , Arsenicais/farmacologia , Artemisininas/farmacologia , Neoplasias/tratamento farmacológico , Óxidos/farmacologia , Apoptose , Trióxido de Arsênio , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs , Transdução de SinaisRESUMO
AIM: Ginger rhizome is used worldwide as a spicy flavor agent. This study was designed to explore the potential effects of pungent ginger components, 6-, 8-, and 10-gingerol, on human cytochrome P450 (CYP450) enzymes that are responsible for the metabolism of many prescription drugs. METHODS: The activities of human CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were analyzed using Vivid P450 assay kits. The mRNA expression of CYP3A4 in human hepatocellular carcinoma cell line HepG2 was measured using quantitative real-time PCR assay. RESULTS: All three gingerols potently inhibited CYP2C9 activity, exerted moderate inhibition on CYP2C19 and CYP3A4, and weak inhibion on CYP2D6. 8-Gingerol was the most potent in inhibition of P450 enzymes with IC50 values of 6.8, 12.5, 8.7, and 42.7 µmol/L for CYP2C9, CYP2C19, CYP3A4, and CYP2D6, respectively. By comparing the effects of gingerols on CYP3A4 with three different fluorescent substrate probes, it was demonstrated that the inhibition of gingerols on CYP3A4 had no substrate-dependence. In HepG2 cells, 8-gingerol and 10-gingerol inhibited, but 6-gingerol induced mRNA expression of CYP3A4. CONCLUSION: 6-, 8-, and 10-gingerol suppress human cytochrome P450 activity, while 8- and 10-gingerol inhibit CYP3A4 expression. The results may have an implication for the use of ginger or ginger products when combined with therapeutic drugs that are metabolized by cytochrome P450 enzymes.
Assuntos
Catecóis/farmacologia , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/fisiologia , Álcoois Graxos/farmacologia , Zingiber officinale , Relação Dose-Resposta a Droga , Células Hep G2 , HumanosRESUMO
In the present study, we found that celastrol, a natural compound with well-known apoptosis-inducing effect, could also induce paraptosis-like cytoplasmic vacuolization in cancer cell lines including HeLa cells, A549 cells and PC-3 cells derived from cervix, lung and prostate, respectively. Further study using HeLa cells indicated that the vacuoles induced by celastrol might be derived from dilation of endoplasmic reticulum. And, in celastrol-treated cells, markers of autophagy such as transformation of microtubule-associated protein 1 light chain 3 (LC3)I to LC3II and LC3 punctates formation were identified. Interestingly, autophagy inhibitors could not interrupt but enhance the induction of cytoplasmic vacuolization. Furthermore, MAPK pathways were activated by celastrol and inhibitors of MEK and p38 pathways could prevent the formation of cytoplasmic vacuolization. Celastrol treatment also induced G2/M cell cycle arrest and apoptosis in HeLa cells. In conclusion, celastrol induced a kind of paraptosis accompanied by autophagy and apoptosis in cancer cells. The coincidence of apoptosis and autophagy together with paraptosis might contribute to the unique characteristics of paraptosis in celastrol-treated cells such as the dependence of paraptosis on MAPK pathways and dynamic change of LC3 proteins. Both paraptosis and apoptosis could contribute to the cell death induced by celastrol while autophagy might serve as a kind of survival mechanism. The potency of celastrol to induce paraptosis, apoptosis and autophagy at the same dose might be related to its capability to affect a variety of pathways including proteasome, ER stress and Hsp90.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Triterpenos/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Retículo Endoplasmático/ultraestrutura , Ativação Enzimática , Inibidores Enzimáticos/metabolismo , Células HeLa , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Triterpenos Pentacíclicos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Vacúolos/efeitos dos fármacos , Vacúolos/ultraestruturaRESUMO
There is no effective treatment for acute lung injury (ALI) at present. Some studies have reported the anti-inflammatory effect of Ejiao, but no study has addressed the underlying action mechanism. In this study, the CCK8 assay displayed Ejiao had a protective effect against LPS-elicited inflammatory lung epithelial Beas 2B cells (LILEB 2B cells). Beas 2B cells treated with LPS and Ejiao were challenged with NFκB inhibitor Bay11-7082 and ROS scavenger N-acetyl cysteine (NAC) alone and in combination. The results of qRT-PCR, Western blotting and fluorescence labeling experiments using Bay11-7082 and NAC demonstrated Ejiao could significantly decrease the expression of p-p65 and p-IκBα in NFκB signaling pathway and its downstream NLRP3, ASC, Caspase-1 and IL-1ß related to pyroptosis of LILEB 2B cells. Moreover, Ejiao reduced the production of mitochondrial ROS and reversed the change of mitochondrial membrane potential of LILEB 2B cells. Then, HE staining demonstrated Ejiao had a protective effect against the LPS-elicited ALI mouse model (LAMM). Ejiao also dramatically decreased the cell amount and the overall protein concentration of bronchoalveolar lavage fluid in LAMM. Immunohistochemical staining showed Ejiao remarkably reduced the expression of p-p65 and p-IκBα in NFκB signaling pathway and its downstream NLRP3, ASC, Caspase-1 and IL-1ß. The ELISA of IL-1ß revealed Ejiao could dose-dependably decrease the concentration of IL-1ß in lung tissues, serum and BALF of LAMM. Finally, fluorescence labeling demonstrated Ejiao significantly reduced the mitochondrial ROS generation in the lung tissue of LAMM. This finding may afford a novel strategy for the precaution and therapy of ALI.
Assuntos
Lesão Pulmonar Aguda , Pneumonia , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Animais , Caspase 1/metabolismo , Gelatina , Inflamassomos/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Inibidor de NF-kappaB alfa , NF-kappa B , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pneumonia/induzido quimicamente , Pneumonia/tratamento farmacológico , Pneumonia/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer death worldwide. Amygdalin, a natural compound commonly distributed in plants of the Rosaceae species, owns anticancer activity, less side effects, wide source, and relatively low price. Although the apoptosis is a central process activated by amygdalin in cancer cells, the underlying molecular mechanisms through which amygdalin induces the apoptosis of lung cancer cells remain poorly understood. In this research work, amygdalin could suppress the proliferation of lung cancer A549 and PC9 cells by CCK8 assay. Amygdalin significantly promoted the apoptosis of lung cancer A549 and PC9 cells stained with Annexin V-FITC/PI by flow cytometry assay. Furthermore, amygdalin dose-dependently decreased the mitochondrial membrane potential (MMP) with JC-1 dye by flow cytometry. To investigate the underlying molecular mechanisms through which amygdalin induced mitochondria-mediated apoptosis of cancer cells, the differentially-expressed genes with a fold change >2.0 and p < 0.05 were acquired from the cDNA microarray analysis. The results of qRT-PCR further confirmed that the differentially-expressed level of the NF[Formula: see text]B-1 gene was most obviously enhanced in lung cancer cells treated with amygdalin. The results of immunofluorescence staining, Western blotting and siRNA knockdown indicated that amygdalin induced mitochondria-mediated apoptosis of lung cancer cells via enhancing the expression of NF[Formula: see text]B-1 and inactivating NF[Formula: see text]B signaling cascade and further changing the expressions of proteins (Bax, Bcl-2, cytochrome C, caspase 9, caspase 3 and PARP) related to apoptosis, which were further checked by in vivo study of the lung cancer cell xenograft mice model accompanying with immunohistochemical staining and TUNEL staining. Our results indicated that amygdalin might be a potential activator of NF[Formula: see text]B-1, which sheds more light on the molecular mechanism of anticancer effects of amygdalin. These results highlighted amygdalin as a potential therapeutic anticancer agent, which warrants its development as a therapy for lung cancer.
Assuntos
Amigdalina , Neoplasias Pulmonares , Amigdalina/metabolismo , Amigdalina/farmacologia , Amigdalina/uso terapêutico , Animais , Apoptose , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Mitocôndrias/metabolismo , NF-kappa B/metabolismoRESUMO
As an antioxidant, α-lipoic acid (LA) has attracted much attention to cancer research. However, the exact mechanism of LA in cancer progression control and prevention remains to be unclear. In this study, we demonstrated that α-lipoic acid has inhibitory effects on the proliferation, migration, and proapoptotic effects of non-small-cell lung cancer (NSCLC) cell lines A549 and PC9. LA-induced NSCLC cell apoptosis was mediated by elevated mitochondrial reactive oxygen species (ROS). Further study confirmed that it is by downregulating the expression of PDK1 (the PDH kinase), resulted in less phospho-PDH phenotype which could interact with Keap1, the negative controller of NRF2, directly leading to NRF2 decrease. Thus, by downregulating the NRF2 antioxidant system, LA plays a role in promoting apoptosis through the ROS signaling pathway. Moreover, LA could enhance other PDK inhibitors with the proapoptosis effect. In summary, our study shows that LA promotes apoptosis and exerts its antitumor activity against lung cancer by regulating mitochondrial energy metabolism enzyme-related antioxidative stress system. Administration of LA to the tumor-bearing animal model further supported the antitumor effect of LA. These findings provided new ideas for the clinical application of LA in the field of cancer therapy.
Assuntos
Neoplasias Pulmonares/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Ácido Tióctico/metabolismo , Apoptose , HumanosRESUMO
Triterpenes isolated from Ganoderma lucidum could inhibit the growth of numerous cancer cell lines and were thought to be the basis of the anticancer effects of G. lucidum. Ganoderic acid D (GAD) is one of the major components in Ganoderma triterpenes. GAD treatment for 48 h inhibited the proliferation of HeLa human cervical carcinoma cells with an IC(50) value of 17.3 +/- 0.3 microM. Flow cytometric analysis and DNA fragmentation analysis indicated that GAD induced G(2)/M cell cycle arrest and apoptosis. To identify the cellular targets of GAD, two-dimensional gel electrophoresis was performed, and proteins altered in expressional level after GAD exposure of cells were identified by MALDI-TOF MS/MS. The regulation of proteins was also confirmed by Western blotting. The cytotoxic effect of GAD was associated with regulated expression of 21 proteins. Furthermore these possible GAD target-related proteins were evaluated by an in silico drug target searching program, INVDOCK. The INVDOCK analysis results suggested that GAD could bind six isoforms of 14-3-3 protein family, annexin A5, and aminopeptidase B. The direct binding affinity of GAD toward 14-3-3 zeta was confirmed in vitro using surface plasmon resonance biosensor analysis. In addition, the intensive study of functional association among these 21 proteins revealed that 14 of them were closely related in the protein-protein interaction network. They had been found to either interact with each other directly or associate with each other via only one intermediate protein from previous protein-protein interaction experimental results. When the network was expanded to a further interaction outward, all 21 proteins could be included into one network. In this way, the possible network associated with GAD target-related proteins was constructed, and the possible contribution of these proteins to the cytotoxicity of GAD is discussed in this report.
Assuntos
Antineoplásicos/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Proteoma/metabolismo , Proteômica , Triterpenos/farmacologia , Proteínas 14-3-3/análise , Proteínas 14-3-3/metabolismo , Antineoplásicos/química , Apoptose , Western Blotting , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Células HeLa , Humanos , Proteoma/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Triterpenos/químicaRESUMO
Natural products, especially microtubule-binding natural products, play important roles in the war against cancer. From the clinical use of vinblastine in 1961, paclitaxel in 1992, to ixabepilone in 2007, microtubule-binding natural products have continually contributed to the development of cancer therapy. The present review summarizes the development of representative microtubule-binding natural products including agents binding to the colchicine-binding site, the VINCA alkaloid-binding site, the taxane-binding site and other binding sites. Future directions for the development of new anticancer microtubule-binding natural products are discussed. Finding new formulations, new targets and new sources of microtubule-binding natural products may enable more members of this kind of agent to be introduced into the clinic for cancer therapy.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Microtúbulos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Moduladores de Tubulina/uso terapêutico , Antineoplásicos Fitogênicos/efeitos adversos , Antineoplásicos Fitogênicos/farmacologia , Sítios de Ligação , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Ensaios Clínicos como Assunto , Colchicina/metabolismo , Colchicina/farmacologia , Epotilonas/metabolismo , Epotilonas/farmacologia , Epotilonas/fisiologia , Humanos , Microtúbulos/química , Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Neoplasias/ultraestrutura , Taxoides/metabolismo , Taxoides/farmacologia , Moduladores de Tubulina/efeitos adversos , Moduladores de Tubulina/farmacologia , Alcaloides de Vinca/metabolismo , Alcaloides de Vinca/farmacologiaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Lung cancer is the leading cause of cancer incidence and mortality worldwide. Arenobufagin (Arg), a representative natural bufadienolide compound, is one of the major bioactive components isolated from toad venom ("Chan Su"named in Chinese to treat multifarious clinical neoplasms in China). However, the underlying molecular mechanisms that Arg inhibited the metastasis of lung cancer cells remain poorly understood. MATERIALS AND METHODS: The mobility capacities of lung cancer cells treated with Arg were evaluated using wound healing assay. The anti-migratory and anti-invasive effects of Arg on lung cancer cells were investigated by transwell invasion assay and matrigel invasion assay. iTRAQ-labeled LC-MS proteomics was used to analyze the potential proteins related to metastasis in lung cancer cells treated with Arg and differentially-expressed proteins related to EMT and NFκB signaling cascade were further confirmed by Western blotting assay. The changed subcellular localization of p65 in lung cancer A549 and H1299 cells treated with Arg was detected by immunofluorescence staining. Molecular docking and molecular dynamic (MD) simulation assay were performed to verify the binding between Arg and IKKα/IKKß. siRNA knockdown was used to check whether Arg inhibited EMT of lung cancer cells via targeting NFκB signaling cascade, which was further verified by in vivo study of lung cancer cell xenograft mice model and pulmonary metastasis mice model accompanying with immunohistochemical and hematoxylin-eosin (HE) staining. RESULTS: Arg suppressed the wound closure of lung cancer cells using wound healing assay. Moreover, Arg significantly inhibited the migration and invasion of lung cancer cells by transwell invasion assay and matrigel invasion assay. 24 unique differentially-expressed proteins related to metastasis in lung cancer cells treated with Arg were identified using iTRAQ-labeled LC-MS proteomics and 14 differentially-expressed proteins related to EMT were further confirmed by Western blotting assay. Arg significantly decreased the phosphorylation of IKKß, IκBα and p65 in the cytoplasm of lung cancer cells by Western blotting assay, and remarkably reduced the release of p65 from the cytoplasm to the nucleus. Arg could be bound in the ATP binding pocket of IKKα and IKKß by molecular docking assay, and MD simulation assay further demonstrated that Arg binding to the ATP-binding pocket of IKKß was very stable in 300 ns MD simulation, compared with the binding of Arg and IKKα. IKKß/NFκB signaling cascade was also involved in the inhibitory effect of Arg on EMT of lung cancer cells by siRNA knockdown assay. The study of lung cancer cell xenograft mice model and pulmonary metastasis mice model in vivo indicated that Arg inhibited EMT and suppressed migration and invasion of lung cancer cells via downregulating IKKß/NFκB signaling cascade. CONCLUSION: In the present study, we explored the molecular mechanism of Arg prohibiting the metastasis of lung cancer cells in vitro and in vivo, which displayed Arg could target IKKß to inactive NFκB signaling cascade and further change the expression of proteins related to EMT. These results highlight the potential of toad venom as a potential chemotherapeutic agent and warrant its development as the clinical therapy for lung cancer.
Assuntos
Venenos de Anfíbios/química , Bufanolídeos/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Células A549 , Animais , Bufanolídeos/isolamento & purificação , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Humanos , Quinase I-kappa B/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Simulação de Acoplamento Molecular , NF-kappa B/metabolismo , Invasividade Neoplásica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Twelve new xanthones (1-12), a pair of new natural products (13 and 14), and 18 known related compounds were isolated from the resin of Garcinia hanburyi. The structures of 1-14 were elucidated by detailed spectroscopic analyses. A cytotoxic assay of the isolated compounds revealed that, with the exception of 2, these compounds were active against the HeLa tumor cell line.
Assuntos
Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Garcinia/química , Plantas Medicinais/química , Xantonas/isolamento & purificação , Xantonas/farmacologia , Antineoplásicos Fitogênicos/química , China , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Estrutura Molecular , Resinas Vegetais/química , Xantonas/químicaRESUMO
A pair of new flavanol racemates (1a and 1b) and a new flavanol racemic mixture (2) were isolated from crude propolis from Henan Province, People's Republic of China. Also obtained were nine known compounds, including two flavones, four flavonols, two flavanols, and isoferulic acid. Spectroscopic analysis was employed to assign the structures of these new compounds and the absolute configurations of 1a and 1b. Cytotoxicity of the isolated compounds against the HeLa human cervical carcinoma cancer cell line was evaluated, with only compounds 1a, 1b, 2, and rhamnetin (3) being active.
Assuntos
Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Flavonóis/isolamento & purificação , Flavonóis/farmacologia , Própole/química , Quercetina/análogos & derivados , Antineoplásicos Fitogênicos/química , Ensaios de Seleção de Medicamentos Antitumorais , Medicamentos de Ervas Chinesas/química , Feminino , Flavonóis/química , Humanos , Estrutura Molecular , Quercetina/química , Quercetina/isolamento & purificação , Quercetina/farmacologia , EstereoisomerismoRESUMO
Triterpenes are the main components with cytotoxicity in Ganoderma lucidum, which is used popularly as a complementary treatment for cancer therapy in traditional Chinese medicine. To investigate the possible interaction between chemotherapeutic agents and triterpenes extracted from G. lucidum, the cytotoxicity of doxorubicin (DOX) combined with Ganoderma triterpenes (GTS) or lucidenic acid N (LCN), a purified compound, was examined in HeLa cells. The combinations targeting DOX with GTS or LCN resulted in a synergistic interaction in HeLa cells. Moreover, to identify the molecular targets of GTS, two-dimensional gel electrophoresis-based comparative proteomics was carried out and proteins with altered expression levels after GTS treatment in HeLa cells were identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. The results of our proteomic study indicated that the GTS treatment caused regulated expression of 14 proteins, which play important roles in cell proliferation, the cell cycle, apoptosis, and oxidative stress. Flow cytometric analysis confirmed that GTS could induce weak G(0)-G(1) phase arrest and combined use of GTS with DOX could induce apoptosis in cells. Furthermore, GTS enhanced the reactive oxygen species (ROS)-producing effect of DOX, and a ROS scavenger could affect the synergism between GTS and DOX. In cells with high Ku80 protein expression, the synergism between GTS and DOX was also partly affected. Importantly, in cells with high Ku80 expression that were treated with a ROS scavenger, the synergism between GTS and DOX totally disappeared. These results suggest that the synergism between GTS and DOX might be based on GTS-induced sensitization of cells to chemotherapeutics through enhanced oxidative stress, DNA damage, and apoptosis.
Assuntos
Doxorrubicina/farmacologia , Ganoderma/química , Proteínas de Neoplasias/análise , Proteômica , Triterpenos/farmacologia , Antígenos Nucleares/análise , Western Blotting , Ciclo Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/análise , Sinergismo Farmacológico , Células HeLa , Humanos , Autoantígeno Ku , Proteína Fosfatase 2/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Pancreatic cancer is a type of common malignant tumors with high occurrence in the world. Most patients presented in clinic had pancreatic cancer at advanced stages. Furthermore, chemotherapy or radiotherapy had very limited success in treating pancreatic cancer. Complementary and alternative medicines, such as natural products/herbal medicines, represent exciting adjunctive therapies. In this review, we summarize the recent advances of using natural products/herbal medicines, such as Chinese herbal medicine, in combination with conventional chemotherapeutic agents to treat pancreatic cancer in preclinical and clinical trials.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Produtos Biológicos/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Antineoplásicos/uso terapêutico , Quimioterapia Adjuvante , Ensaios Clínicos como Assunto , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Humanos , GencitabinaRESUMO
To develop traditional medicines as modern pharmacotherapies, understanding their molecular mechanisms of action can be very helpful. We have recently reported that Artemisinin and its derivatives, which are clinically used anti-malarial drugs, have significant effects against ovarian cancer, but the direct molecular targets and related combination therapy have been unclear. Herein, we report that dihydroartemisinin, one of the most active derivatives of Artemisinin, directly targets platelet-derived growth factor receptor-alpha (PDGFRα) to inhibit ovarian cancer cell growth and metastasis. Dihydroartemisinin directly binds to the intercellular domain of PDGFRα, reducing its protein stability by accelerating its ubiquitin-mediated degradation, which further inactivates downstream phosphoinositide 3-Kinase and mitogen-activated protein kinase pathways and subsequently represses epithelial-mesenchymal transition, inhibiting cell growth and metastasis of PDGFRα-positive ovarian cancer in vitro and in vivo. A combinational treatment reveals that dihydroartemisinin sensitizes ovarian cancer cells to PDGFR inhibitors. Our clinical study also finds that PDGFRα is overexpressed and positively correlated with high grade and metastasis in human ovarian cancer. Considering that Artemisinin compounds are currently clinically used drugs with favorable safety profiles, the results from this study will potentiate their use in combination with clinically used PDGFRα inhibitors, leading to maximal therapeutic efficacy with minimal adverse effects in PDGFRα-positive cancer patients. These findings also shed high light on future development of novel Artemisinin-based targeted therapy.
RESUMO
Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K-ATPase α1 and α3 subunits and increased the expression of WEE1 in HeLa cells. Antibodies against Na, K-ATPase α1 and α3 subunits alone or combinated with arenobufagin also inhibited the activity of proteasome. Furthermore, the expression of the possible intermediate proteins ataxin-1 and translationally-controlled tumor protein was increased in HeLa cells treated with arenobufagin by flow cytometry analysis, respectively. These results indicated that arenobufagin might directly bind with Na, K-ATPase α1 and α3 subunits and the inhibitive effect of arenobufagin on proteasomal activity of HeLa cells might be related to its binding with Na, K-ATPase.
Assuntos
Antineoplásicos/farmacologia , Bufanolídeos/farmacologia , Inibidores de Proteassoma/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico , Venenos de Anfíbios/química , Antineoplásicos/química , Bufanolídeos/química , Colo do Útero/efeitos dos fármacos , Colo do Útero/metabolismo , Colo do Útero/patologia , Feminino , Células HeLa , Humanos , Simulação de Acoplamento Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Mapas de Interação de Proteínas/efeitos dos fármacos , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologiaRESUMO
Gamabufotalin (CS-6) is a major bufadienolide of Chansu, which shows desirable metabolic stability and less adverse effect in cancer therapy. CS-6 treatment inhibited the proliferation of NSCLC in a nanomolar range. And CS-6 could induce G2/M cell cycle arrest and apoptosis in A549 cells. However, its molecular mechanism in antitumor activity remains poorly understood. We employed a quantitative proteomics approach to identify the potential cellular targets of CS-6, and found 38 possible target-related proteins. Among them, 31 proteins were closely related in the protein-protein interaction network. One of the regulatory nodes in key pathways was occupied by Hsp90. Molecular docking revealed that CS-6 interacted with the ATP-binding sites of Hsp90. In addition, CS-6 inhibited the chaperone function of Hsp90 and reduced expression of Hsp90-dependent client proteins. Moreover, CS-6 markedly down-regulated the protein level of Hsp90 in tumor tissues of the xenograft mice. Taken together, our results suggest that CS-6 might be a novel inhibitor of Hsp90, and the possible network associated with CS-6 target-related proteins was constructed, which provided experimental evidence for the preclinical value of using CS-6 as an effective antitumor agent in treatment of NSCLC.
Assuntos
Antineoplásicos/química , Bufanolídeos/química , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Neoplasias Pulmonares/metabolismo , Proteômica , Relação Quantitativa Estrutura-Atividade , Animais , Antineoplásicos/farmacologia , Bufanolídeos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Camundongos , Modelos Moleculares , Conformação Molecular , Proteômica/métodos , Espectrometria de Massas em TandemRESUMO
Gambogic acid (GA) is an anticancer agent in phase âb clinical trial in China but its mechanism of action has not been fully clarified. The present study was designed to search the possible target-related proteins of GA in cancer cells using proteomic method and establish possible network using bioinformatic analysis. Cytotoxicity and anti-migration effects of GA in MDA-MB-231 cells were checked using MTT assay, flow cytometry, wound migration assay, and chamber migration assay. Possible target-related proteins of GA at early (3 h) and late stage (24 h) of treatment were searched using a proteomic technology, two-dimensional electrophoresis (2-DE). The possible network of GA was established using bioinformatic analysis. The intracellular expression levels of vimentin, keratin 18, and calumenin were determined using Western blotting. GA inhibited cell proliferation and induced cell cycle arrest at G2/M phase and apoptosis in MDA-MB-231 cells. Additionally, GA exhibited anti-migration effects at non-toxic doses. In 2-DE analysis, totally 23 possible GA targeted proteins were found, including those with functions in cytoskeleton and transport, regulation of redox state, metabolism, ubiquitin-proteasome system, transcription and translation, protein transport and modification, and cytokine. Network analysis of these proteins suggested that cytoskeleton-related proteins might play important roles in the effects of GA. Results of Western blotting confirmed the cleavage of vimentin, increase in keratin 18, and decrease in calumenin levels in GA-treated cells. In summary, GA is a multi-target compound and its anti-cancer effects may be based on several target-related proteins such as cytoskeleton-related proteins.
Assuntos
Antineoplásicos/farmacocinética , Neoplasias da Mama/tratamento farmacológico , Biologia Computacional/métodos , Proteômica/métodos , Xantonas/farmacocinética , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Tumoral , Ensaios de Migração Celular , Inibição de Migração Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citoesqueleto/metabolismo , Eletroforese em Gel Bidimensional , Citometria de Fluxo , Expressão Gênica , Humanos , Queratina-18/genética , Oxirredução , Biossíntese de Proteínas/efeitos dos fármacos , Transporte Proteico , Transcrição Gênica/efeitos dos fármacos , Proteases Específicas de Ubiquitina/farmacocinética , Vimentina/genéticaRESUMO
Artemisinin and related compounds (artemisinins), as a frontline treatment for malaria, have been used to save millions of lives. Their potential application in cancer treatment is promising. Nevertheless, the precise mechanisms of action of artemisinins are still controversial. In particular, the system-level influence of artemisinins on protein interactions and regulatory networks remains unknown, limiting progress in development of this class of compounds as anticancer drugs. In the present study, we investigated the mechanism of action of artemisinins in cancer therapy through an analysis based on biological networks. According to experimental evidence from more than 400 literature studies, 558 key proteins were derived and the artemisinins-rewired protein interaction network was constructed. Topological properties were analyzed to show that the protein network was a scale-free biological system. And the modularity analysis and pathway identification were performed. Five key pathways including PI3K-Akt, T cell receptor, Toll-like receptor, TGF-beta and insulin signaling pathways were involved in artemisinins-mediated anticancer effects; their identification was confirmed by microarray data. Based on these results, predictions were made about the targets of artemisinins in various pathways. These results provide a deeper understanding of the molecular mechanisms of action of artemisinins and will contribute to the development and application of this class of compounds in cancer treatment.