RESUMO
The honey mushroom, Armillaria mellea, is known to have medicinal qualities and has been used in recent years as a health food and dietary supplement worldwide. In Asia, it is commonly consumed as an herbal medicine, being a key component of the Chinese preparation "Tien-ma". Here, we examined the antitumor effects of armillaridin, a bioactive compound isolated from A. mellea, on human hepatocellular carcinoma (HCC) cells. Armillaridin inhibited the growth of human Huh7, HepG2, and HA22T HCC cells, and its cytotoxicity was confirmed by observations of its induction of mitochondrial transmembrane potential collapse. However, armillaridin treatment did not result in large numbers of cells with fragmented chromosomal DNA, suggesting that apoptosis was not responsible for these effects. We therefore tested for signs of autophagic cell death following armillaridin administration. Armillaridin induced LC3 aggregation in green fluorescent protein-LC3-overexpressing cells. Moreover, flow cytometry and immunoblotting revealed that it increased the number of acridine orange-positive cells and upregulated autophagy-related proteins, respectively. Furthermore, armillaridin cytotoxicity was suppressed by the autophagy inhibitor 3-methyladenine. In summary, our results indicated that armillaridin induces HCC cell death by autophagy, and demonstrated the potential of armillaridin as an antihepatoma agent.
Assuntos
Antineoplásicos Fitogênicos , Armillaria/química , Morte Celular Autofágica/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Sesquiterpenos/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Carcinoma Hepatocelular/fisiopatologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/fisiopatologia , Necrose Dirigida por Permeabilidade Transmembrânica da Mitocôndria/efeitos dos fármacos , Sesquiterpenos/antagonistas & inibidores , Sesquiterpenos/isolamento & purificaçãoRESUMO
Ets-1 is a prototype of the ETS protein family. Members of the ETS protein family contain a unique ETS domain. Ets-1 is associated with cancer progression and metastasis in many types of cancer. Many studies have shown a link between elevated expression of Ets-1 in cancer biopsies and poor survival. CCR7 is a chemokine that binds to specific ligand CCL21/CCL19. CCR7 expression is associated with tumor metastasis and infiltration into lymph nodes. The objective of this study was to test whether Ets-1 could regulate CCR7 expression and enhance tumor metastasis. Our data showed that CCR7 expression was downregulated in Ets-1-deficient T cells upon T-cell stimulation. Overexpression of Ets-1 increased CCR7 expression in breast cancer cell lines. In contrast, knockdown of Ets-1 reduced CCR7 expression. Ets-1 could directly bind to CCR7 promoter and mediate CCR7 expression in luciferase reporter assays and chromatin immunoprecipitation assays. Transactivation activity of Ets-1 was independent of the Pointed domain of Ets-1. Ets-1 could also enhance NF-κB and CBP transactivation of CCR7 promoter. Our results also showed that Ets-1 could modulate cancer cell transmigration by altering CCR7 expression in transwell assay and wound healing assay. Taken together, our data suggest that Ets-1 can enhance CCR7 expression and contribute to tumor cell migration. [BMB Reports 2019; 52(9): 548-553].
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Proteína Proto-Oncogênica c-ets-1/metabolismo , Receptores CCR7/genética , Receptores CCR7/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Quimiocina CCL19/genética , Quimiocina CCL19/metabolismo , Quimiocina CCL21/genética , Quimiocina CCL21/metabolismo , Feminino , Humanos , NF-kappa B/metabolismo , Proteína Proto-Oncogênica c-ets-1/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Traditional Chinese herb Tithonia diversifolia, belonging to the Compositae family, has long been applied for the treatment of liver diseases. In recent years, many reports also indicated that it possesses hepato-protective, anti-inflammatory, and anti-cancer activities. OBJECTIVE: In this study, we evaluated whether T. diversifolia is an effective therapy for hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Dry leaves of T. Diversifolia were first extracted in ethyl acetate, then further fractionated by different ratio of n-hexane-ethyl acetate (8:2â0:1) or methanol as fractions 1-6 (Td-F1 to Td-F6), respectively. We first showed that the ethyl acetate extracts of T. diversifolia leaves (Td-L-EA) exhibits growth inhibition on human hepatoma HepG2 cells. To further check the extracts-induced apoptosis, microscopic observation, fragmented chromosomal DNA electrophoresis, apoptotic DNA-detection ELISA assay, flow cytometry, and Western blot analysis were performed. RESULTS: After isolating the effective fractions from Td-L-EA, we found strong cytotoxic effects of fraction-2 (Td-F2). By further analyzing the mechanisms of cytotoxic activities using microscopic observation, fragmented chromosomal DNA electrophoresis, apoptotic DNA-detection ELISA assay, and flow cytometry, we found that induction of apoptosis such as DNA fragmentation increased the apoptosis rate and the apoptosis sub-G1 populations in Td-F2-treated HepG2 cells. In addition, we also confirmed Td-F2-induced degradation of caspase-8, caspase-9, caspase-3, and caspase-3 substrate PARP. Besides, Td-F2 also increased the Bcl-2 proapoptotic family protein Bax expression. CONCLUSION: In short, our results clearly showed the induction of apoptosis by ethyl acetate extracts of T. diversifolia leaves in human hepatoma HepG2 cells, suggesting its potential application as an antitumor agent. SUMMARY: T. Diversifolia leaves were first extracted in ethyl acetate, then further fractionated by different ratio of n-hexane/ethyl acetate (8:2â0:1) or methanol.These extracts exhibit growth inhibition on human hepatoma (HCC) HepG2 cells.n-Hexane/ethyl acetate (6:4) extract (Td-F2) induces apoptosis of HCC. Abbreviations used:T. diversifolia, Tithonia diversifolia; HCC, Hepatocellular carcinoma DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; HRP, horseradish peroxidase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; OD, optical density; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; PARP, Poly (ADP-ribose) polymerase; PBS, phosphate buffered saline; PI, propidium iodide.
RESUMO
Armillaridin (AM) is an aromatic ester compound isolated from Armillaria mellea. Treatment with AM markedly reduced the viability of human chronic myelogenous leukemia K562, chronic erythroleukemia HEL 92.1.7, and acute monoblastic leukemia U937 cells, but not normal human monocytes, in a dose- and time-dependent manner. Treatment of K562 cells with AM caused changes characteristic of autophagy. Only a small amount of AM-treated K562 cells exhibited apoptosis. By contrast, AM treatment resulted in extensive apoptotic features in U937 and HEL 92.1.7 cells without evident autophagy. The autophagy of K562 cells induced by AM involved autophagic flux, including autophagosome induction, the processing of autophagosome-lysosome fusion and downregulation of BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3). By bcr-abl knockdown, the growth inhibition of K562 cells caused by AM was partially blocked, suggesting that AM-induced cell death might be a bcr-abl-dependent mode of autophagy-associated cell death. In conclusion, AM is capable of inhibiting growth and inducing autophagy-associated cell death in K562 cells, but not in normal monocytes. It may have potential to be developed as a novel therapeutic agent against leukemia.
Assuntos
Autofagia/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Sesquiterpenos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Citometria de Fluxo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Células Tumorais CultivadasRESUMO
Lapatinib is an oral-form dual tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR or ErbB/Her) superfamily members with anticancer activity. In this study, we examined the effects and mechanism of action of lapatinib on several human leukemia cells lines, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and acute lymphoblastic leukemia (ALL) cells. We found that lapatinib inhibited the growth of human AML U937, HL-60, NB4, CML KU812, MEG-01, and ALL Jurkat T cells. Among these leukemia cell lines, lapatinib induced apoptosis in HL-60, NB4, and Jurkat cells, but induced nonapoptotic cell death in U937, K562, and MEG-01 cells. Moreover, lapatinib treatment caused autophagic cell death as shown by positive acridine orange staining, the massive formation of vacuoles as seen by electronic microscopy, and the upregulation of LC3-II, ATG5, and ATG7 in AML U937 cells. Furthermore, autophagy inhibitor 3-methyladenine and knockdown of ATG5, ATG7, and Beclin-1 using short hairpin RNA (shRNA) partially rescued lapatinib-induced cell death. In addition, the induction of phagocytosis and ROS production as well as the upregulation of surface markers CD14 and CD68 was detected in lapatinib-treated U937 cells, suggesting the induction of macrophagic differentiation in AML U937 cells by lapatinib. We also noted the synergistic effects of the use of lapatinib and cytotoxic drugs in U937 leukemia cells. These results indicate that lapatinib may have potential for development as a novel antileukemia agent.
RESUMO
Armillaria mellea is a honey mushroom often used in the traditional Chinese medicine "Tianma". Currently, this medicinal mushroom is also used as a dietary supplement in numerous Western and Eastern countries. Armillarikin was isolated from A. mellea, and we previously discovered that it induced cytotoxicity in human leukemia cells. In this study, we further investigated the cytotoxicity of armillarikin against liver and intrahepatic bile duct cancer cells. Armillarikin was cytotoxic against human hepatocellular carcinoma Huh7, HA22T, and HepG2 cells based on the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and alamarBlue(®) assays. Armillarikin treatment also induced the collapse of the mitochondrial transmembrane potential of these cells. Furthermore, armillarikin-induced apoptotic cell death was demonstrated by sub-G1 chromosomal DNA formation by using flow cytometry. In addition, the apoptosis was inhibited by the pan-caspase inhibitor, Z-VAD-fmk. Immunoblotting also revealed the armillarikin-induced activation of procaspase-3, -8, and -9 and upregulation of the apoptosis- and cell cycle arrest-related phospho-histones 2 and 3, respectively. Moreover, reactive oxygen species scavengers also inhibited the armillarikin-induced apoptosis in human hepatocellular carcinoma, suggesting that reactive oxygen species formation played an important role in the armillarikin-induced apoptosis of human hepatocellular carcinoma. In conclusion, our study indicates the potential of armillarikin as an effective agent for hepatoma or leukemia therapies.
RESUMO
Atractylodes macrocephula Koidz (A. macrocephula, also known as Baizhu) is an important ingredient in several traditional Chinese herb complexes for the treatment of abdominal pain and gastroenterology diseases for thousands of years. We previously demonstrated the induction of ROS-mediated apoptosis by methanol extract of A. macrocephula in human leukemia cells. After purification and assessment of those active compounds from A. macrocephula ethanol extracts, in this study, we focused on the major active compound, atractylenolide I (ATL-I). Through MTT assay and morphology observation, we found cytotoxic effect of ATL-I in human K562 chronic myeloblastic leukemia (CML), U937 acute myeloblastic leukemia (AML) and Jurkat T lymphoma cells. In addition, ATL-I-induced apoptosis was demonstrated by sub G1 and fragmented chromosomal DNA detection using flow cytometry, enzyme-linked immunosorbent assay (ELISA) and agarose electrophoresis. Finally, we found ATL-I also induced caspase-3 and caspase-9 activation through the detection of procaspase-3, procaspase-9 and caspase-3 substrate poly(ADP-ribose) polymerase (PARP) by immunoblotting. Interestingly, we found that ATL-I induced not only apoptosis but also differentiation, as upregulation of CD14 and CD68 surface markers and increase of phagocytosis ability were discovered in ATL-I-treated K562 CML and U937 AML cells. Our study thus suggests the potential of developing new leukemia therapies by using ATL-I for leukemia treatment in the future.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Atractylodes/química , Diferenciação Celular/efeitos dos fármacos , Lactonas/farmacologia , Leucemia/patologia , Sesquiterpenos/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Lactonas/química , Lactonas/isolamento & purificação , Estrutura Molecular , Sesquiterpenos/química , Sesquiterpenos/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
Lindera megaphylla has been traditionally used as an antineoplastic and wound healing remedy. We previously demonstrated the antitumor effects of D-dicentrine, a natural aporphine alkaloid from the root of L. megaphylla. To generate analogues, series of phenanthrene alkaloids from D-dicentrine were synthesized by degradation with ethyl chloroformate in pyridine, base hydrolysis, and N-alkylation. In this study, we demonstrated that one of the synthesized D-dicentrine analogues (here after designated as analogue 1) exhibited more potent cytotoxic effects than D-dicentrine in colon adenocarcinoma, hepatoma, leukemia, and epidermoid carcinoma cells. We performed cell cycle and apoptotic analysis by flow cytometry, an apoptotic DNA detection ELISA assay, and topoisomerase II activity by the kinetoplast DNA concatenation assay for studying their cytotoxic mechanisms. We found that both D-dicentrine and analogue 1 induced apoptosis and G2/M arrest in HL-60 leukemia cells. The percentage of apoptotic cells induced by analogue 1 was 4.5-fold higher than that induced by D-dicentrine as evident from measuring the amount of histone-bound DNA fragments. Moreover, we found that analogue 1 was 28-fold more potent than D-dicentrine for inhibition of topoisomerase II activity by the kinetoplast DNA concatenation assay. Our findings indicate that D-dicentrine analogue 1 is very promising as a potential antitumor agent for future study.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Aporfinas/química , Inibidores da Topoisomerase II/farmacologia , Animais , Aporfinas/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Fibroblastos/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Camundongos , Estrutura Molecular , Inibidores da Topoisomerase II/químicaRESUMO
Lapatinib, an orally administered small-molecule tyrosine kinase inhibitor targeting epidermal growth factor receptors (EGFR) and Her2/Neu, has been widely accepted in the treatment of breast cancer. In this study, we found that lapatinib induced cytotoxicity in human hepatoma Huh7, HepG2 and HA22T cells. For the mode of cell death, we found lapatinib induced a higher percent of dead cells and a lower percent of hypodiploid cells, suggesting non-apoptotic cell death in lapatinib-treated hepatoma cells. Moreover, lapatinib-induced autophagy in hepatoma cells was confirmed by the detection of autophagic LC3-II conversion, the up-regulation of autophagy-related proteins, and the down-regulation of p62 by immunoblotting. Autophagic cell death was demonstrated by images of punctuated LC3 patterns, a higher percent of acridine orange positive cells, as well as a partial rescue of cell death by autophagy inhibitor 3-methyladenine or chloroquine. We also found massive vacuoles in lapatinib-treated hepatoma cells by electronic microscopy. In addition, the shRNA of knocked-down autophagy-related proteins rescued the hepatoma cells from lapatinib-induced growth inhibition. We also demonstrated a reduction of tumorigenesis by lapatinib in vivo. In conclusion, lapatinib induced autophagic cell death and the growth of human hepatoma cells. Our study provides potential cancer therapies by using lapatinib as a treatment for hepatoma.
Assuntos
Autofagia/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Quinazolinas/farmacologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/genética , Proteína 5 Relacionada à Autofagia , Proteína 7 Relacionada à Autofagia , Proteína Beclina-1 , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Relação Dose-Resposta a Droga , Células Hep G2 , Humanos , Immunoblotting , Lapatinib , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Carga Tumoral/efeitos dos fármacos , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
TNFα stimulation triggers both cell death and survival programs. Since dysregulated apoptosis or cell growth can cause inflammatory diseases, cancer, or autoimmune disorders, it is important to understand the molecular mechanism of controlling cell death and survival by TNFR downstream signaling molecules. In this study, we used normal diploid cells, mouse embryonic fibroblasts (MEFs), to mimic the general TNFα-resistant phenomenon seen under physiological conditions.We elucidated the TNFα-induced death signaling complexes in TNF α-resistant WT MEFs and TNFα-sensitive MEFs that were cFLIP-, RelA-, TRAF2- or RIP1-deficient. Consistent with TNFα-mediated killing, we detected TNFα-induced high molecular weight complexes containing caspase-8 and FADD by gel filtration in the deficient MEFs, especially in those devoid of cFLIP. In addition to the presence of caspase-8-FADD in the TNFα-induced-death complex in the deficient MEFs, we also detected an intermediate protein complex containing RIP1, TRAF2 and caspase-8.Moreover, we demonstrated a correlation between TNFα-sensitivity and death-inducing complex ability in two transformed cell lines, E1A- and Ras- transformed MEFs and PDGF-B-transformed NIH-3T3 cells with PDGF-B signaling inhibited by the tyrosine kinase inhibitor STI571. Taken together, our results suggest the involvement of cFLIP-, RelA-, RIP1-, or TRAF2-related mechanisms for preventing FADD-caspase-8 interaction in wild-type MEFs.
Assuntos
Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais , Animais , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/deficiência , Caspase 8/metabolismo , Morte Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Proteína de Domínio de Morte Associada a Fas/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas Ativadoras de GTPase/deficiência , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Células NIH 3T3 , Fator 2 Associado a Receptor de TNF/deficiência , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Lapatinib is an oral, small-molecule, dual tyrosine kinase inhibitor of epidermal growth factor receptors (EGFR, or ErbB/Her) in solid tumors. Little is known about the effect of lapatinib on leukemia. Using human chronic myelogenous leukemia (CML) K562 cells as an experimental model, we found that lapatinib simultaneously induced morphological changes resembling apoptosis, autophagy, and megakaryocytic differentiation. Lapatinib-induced apoptosis was accompanied by a decrease in mitochondrial transmembrane potential and was attenuated by the pancaspase inhibitor z-VAD-fmk, indicating a mitochondria-mediated and caspase-dependent pathway. Lapatinib-induced autophagic cell death was verified by LC3-II conversion, and upregulation of Beclin-1. Further, autophagy inhibitor 3-methyladenine as well as autophagy-related proteins Beclin-1 (ATG6), ATG7, and ATG5 shRNA knockdown rescued the cells from lapatinib-induced growth inhibition. A moderate number of lapatinib-treated K562 cells exhibited features of megakaryocytic differentiation. In summary, lapatinib inhibited viability and induced multiple cellular events including apoptosis, autophagic cell death, and megakaryocytic differentiation in human CML K562 cells. This distinct activity of lapatinib against CML cells suggests potential for lapatinib as a therapeutic agent for treatment of CML. Further validation of lapatinib activity in vivo is warranted.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Megacariócitos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Western Blotting , Citometria de Fluxo , Humanos , Células K562 , Lapatinib , Megacariócitos/citologiaRESUMO
Tumor necrosis factor receptor 1-associated death domain protein (TRADD) is the core adaptor recruited to TNF receptor 1 (TNFR1) upon TNFalpha stimulation. In cells from TRADD-deficient mice, TNFalpha-mediated apoptosis and TNFalpha-stimulated NF-kappaB, JNK, and ERK activation are defective. TRADD is also important for germinal center formation, DR3-mediated costimulation of T cells, and TNFalpha-mediated inflammatory responses in vivo. TRADD deficiency does not enhance IFNgamma-induced signaling. Importantly, TRADD has a novel role in TLR3 and TLR4 signaling. TRADD participates in the TLR4 complex formed upon LPS stimulation, and TRADD-deficient macrophages show impaired cytokine production in response to TLR ligands in vitro. Thus, TRADD is a multifunctional protein crucial both for TNFR1 signaling and other signaling pathways relevant to immune responses.
Assuntos
Transdução de Sinais , Proteína de Domínio de Morte Associada a Receptor de TNF/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Apoptose , Centro Germinativo , Inflamação , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Linfócitos T , Proteína de Domínio de Morte Associada a Receptor de TNF/fisiologia , Receptor 3 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Lipid rafts are involved in many cell biology events, yet the molecular mechanisms on how rafts are formed are poorly understood. In this study we probed the possible requirement of reactive oxygen species (ROS) for T-cell receptor (TCR)-induced lipid raft formation. Microscopy and biochemical analyses illustrated that blockage of ROS production, by superoxide dismutase-mimic MnTBAP, significantly reduced partitioning of LAT, phospho-LAT, and PLC-gamma in lipid rafts. Another antioxidant N-acetylcysteine (NAC) displayed a similar suppressive effect on the entry of phospho-LAT into raft microdomains. The involvement of ROS in TCR-mediated raft assembly was observed in T-cell hybridomas, T leukemia cells, and normal T cells. Removal of ROS was accompanied by an attenuated activation of LAT and PKCtheta, with reduced production of IL-2. Consistently, treating T cells with the ROS-producer tert-butyl hydrogen peroxide (TBHP) greatly enhanced membrane raft formation, distribution of phospho-LAT into lipid rafts, and increased IL-2 production. Our results indicate for the first time that ROS contribute to TCR-induced membrane raft formation.
Assuntos
Espécies Reativas de Oxigênio/metabolismo , Linfócitos T/metabolismo , Humanos , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
In this study, we examined the antitumor effect of marine algae extracts on human hepatoma and leukemia cells. Ethyl acetate extracts from Colpomenia sinuosa (Cs-EA), Halimeda discoidae (Hd-EA), and Galaxaura oblongata (Go-EA) directly inhibited the growth of human hepatoma HuH-7 cells and leukemia U937 and HL-60 cells in a time- and dose-dependent manner. Specifically, these algae extracts induced apoptosis of U937 and HL-60 cells as evaluated by detection of hypodiploid cells using flow cytometry and observation of condensed and fragmented nuclei in algae extract-treated cells. Intracellular reactive oxygen species (ROS), especially hydrogen peroxide and superoxide anion, were increased about 2-3-fold in U937 cells treated with Cs-EA for 3-5 h. Interestingly, antioxidant N-acetylcysteine effectively blocked Cs-EA-, Hd-EA-, and Go-EA-induced apoptosis, suggesting that ROS is a key mediator in the apoptotic signaling pathway. In conclusion, our results show that algae extracts induce apoptosis in human leukemia cells through generation of ROS.
Assuntos
Apoptose/efeitos dos fármacos , Eucariotos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/farmacologia , Carcinoma Hepatocelular , Clorófitas/metabolismo , Células HL-60 , Humanos , Leucemia Mieloide , Neoplasias Hepáticas , Phaeophyceae/metabolismo , Rodófitas/metabolismo , Células Tumorais CultivadasRESUMO
Baizhu (Atractylodes macrocephala Koidz) has traditionally been used as an important ingredient of several Chinese herbal medicines, which have been used for abdominal pain and gastroenterology diseases for thousands of years. Despite its popularity in herbal therapies, little is known about the anticancer effect of Baizhu. In this study, the anticancer potential of Baizhu on human hepatoma and leukemia cell lines was evaluated. Baizhu methanol extract induced apoptosis in human lymphoma Jurkat T cells, leukemia U937, and HL-60 cells. This was confirmed by several methods, including hypodiploid cells detection using flow cytometry, the examination of apoptotic bodies containing cells using confocal laser scanning microscopy, and hypodiploid cell population inhibition using the broad spectrum caspase inhibitor z-VAD. Finally, the intracellular reactive oxygen species (ROS), especially hydrogen peroxide (H(2)O(2)) and superoxide anion (O(2)(-)), were found to be elevated after treatment of these cells with Baizhu extracts. Antioxidant N-acetyl cysteine (NAC) pretreatment almost completely inhibited Baizhu-induced apoptosis, suggesting that ROS are the key mediators for Baizhu-induced apoptosis. All these data indicate that Baizhu is a possible anti-tumor agent that induces apoptosis of human leukemia cells through ROS generation.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Atractylodes , Espécies Reativas de Oxigênio/metabolismo , Antineoplásicos/isolamento & purificação , Apoptose/fisiologia , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Células HL-60 , Humanos , Células Jurkat , Células U937RESUMO
DNA-damaging reagents may kill tumor cells through the generation of reactive oxygen species (ROS). Cytotoxic reagents may also induce apoptosis of cancer cells in Fas-FADD-dependent manners. In this study, we explored the possible link between these two apparently distinct pathways in T leukemia cell Jurkat. Our results demonstrated that gamma-irradiation, similar to cisplatin, induced apoptosis by triggering Fas aggregation and activating FADD-caspase-8 apoptotic cascade. The absence of caspase-8 or Fas greatly reduced the sensitivity to apoptosis mediated by DNA-damaging agents. In addition, apoptosis induced by cisplatin and gamma-irradiation, but not by Fas, was inhibited by ROS scavengers, including N-acetyl cysteine, MnTBAP, and C60. Importantly, these ROS scavengers effectively prevented the clustering of Fas receptor induced by cisplatin and gamma-irradiation. Our results suggest that cisplatin and gamma-irradiation promote ROS production, which in turn contributes to Fas receptor aggregation and cell death. The novel coupling between ROS and Fas clustering likely plays a significant role in apoptosis triggered by DNA-damaging reagents in Fas-expressing leukemia cells.