RESUMEN
Ewing sarcoma family tumors (ESFTs) are a group of aggressive tumors mainly affecting children and young people. A compound derived from Curcuma wenyujin plant or lemon grass, ß-elemene, has exhibited antitumor effects to ESFT cells, the mechanism of which remains to be clarified further. Autophagy is involved in the antitumor effects of various drugs, whereas the role of autophagy in the antitumor effects of ß-elemene persists controversial. Herein we found that ß-elemene treatment inhibited the viability of ESFT cells in a dose-dependent manner. The increase of LC3-II level and the decrease of p62 level were observed in ß-elemene-treated cells, as well as the increase of autolysosomes, which indicated the promotion of autophagic flux. Sequentially the autophagy inhibition using 3-MA treatment or ATG5 depletion significantly reversed the viability repression and apoptosis induction by ß-elemene treatment. In addition, autophagy was found to be important in the toxic effects induced by the combination treatment of ß-elemene and IGF1R inhibition in ESFT cells. Our data suggested an essential role of autophagy in ß-elemene-induced apoptosis in ESFT cells, which is anticipated to provide novel insights to the development of ESFT treatments.
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Sarcoma de Ewing , Sesquiterpenos , Niño , Humanos , Adolescente , Sarcoma de Ewing/tratamiento farmacológico , Sesquiterpenos/farmacología , Autofagia , ApoptosisRESUMEN
Melanoma is a highly aggressive skin cancer and accounts for most of the skin cancer-related deaths. The efficacy of current therapies for melanoma remains to be improved. The isopropanolamine derivative of ß-elemene LXX-8250 was reported to present better water solubility and stronger toxicity to tumor cells than ß-elemene. Herein, LXX-8250 treatment showed 4-5-fold more toxicity to melanoma cells than the well-known anti-melanoma drug, Dacarbazine. LXX-8250 treatment induced apoptosis remarkably, which was caused by the impairment of autophagic flux. To clarify the molecular mechanism, microarray analyses were conducted, and PFKFB4 expression was found to be suppressed by LXX-8250 treatment. The cells overexpressed with PFKFB4 exhibited resistance to apoptosis induction and autophagic flux inhibition by LXX-8250 treatment. Moreover, LXX-8250 treatment suppressed glycolysis, to which the cells overexpressed with PFKFB4 were tolerant. LXX-8250 treatment inhibited the growth of melanoma xenografts and suppressed PFKFB4 expression and glycolysis in vivo. Taken together, LXX-8250 treatment induced apoptosis through inhibiting autophagic flux and glycolysis in melanoma cells, which was mediated by suppression of PFKFB4 expression. The study provides a novel strategy to melanoma treatment.
RESUMEN
The clinical management of bladder cancer (BCa) is hindered by the lack of reliable biomarkers. We aimed to investigate the potential of lamprey immunity protein (LIP), a lectin that specifically binds to multi-antennary sialylated N-glycolylneuraminic acid (Neu5Gc) structures on UMOD glycoproteins in the urine of BCa patients. Primary BCa patients had higher levels of LIP-bound Neu5Gc in urine than healthy participants and patients receiving postoperative treatment did. In addition, lectin chip assay and mass spectrometry were used to analyze the glycan chain structure, which can recognize the UMOD glycoprotein decorated with multi-antennary sialylated Neu5Gc structures. Furthermore, compared with urine samples from healthy patients (N = 2821, T/C = 0.12 ± 0.09) or benign patients (N = 360, T/C = 0.11 ± 0.08), the range of the urine T/C ratio detected using LIP test paper was 1.97 ± 0.32 in patients with bladder cancer (N = 518) with significant difference (P < 0.0001). Our results indicate that LIP may be a tool for early BCa identification, diagnosis, and monitoring. Neu5Gc-modified UMOD glycoproteins in urine and Neu5Gc-modified N-glycochains and sialyltransferases may function as potential markers in clinical trials.
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Neoplasias de la Vejiga Urinaria , Animales , Biomarcadores , Glicoproteínas , Humanos , Lampreas/metabolismo , Lectinas/metabolismo , Polisacáridos/química , Sialiltransferasas , Neoplasias de la Vejiga Urinaria/diagnóstico , UromodulinaRESUMEN
The abundance of proteins in human urine is low and easily to be masked by high-abundance proteins during mass spectrometry analysis. Development of efficient and highly selective enrichment methods is therefore a prerequisite for achieving deep coverage of urine protein markers. Notably, different experimental methods would affect the urine protein enrichment efficacy and the coverage of urine proteome. In this study, ultrafiltration, nitrocellulose membrane enrichment and saturated ammonium sulfate precipitation were used to process 10 mL urine samples from five healthy volunteers and five bladder cancer patients. The urine proteins were enriched and separate by SDS-PAGE to compare the purification efficiency of different methods. Moreover, the peptide identification effects of different purification methods were analyzed by mass spectrometry to determine the best method for enriching urine protein histones. Saturated ammonium sulfate precipitation method outperformed the ultrafiltration and the nitrocellulose membrane enrichment methods in terms of the protein enrichment efficacy and quality. The interference of highly abundant albumin was reduced, whereas the amount of low-abundance protein was increased, and the sensitivity of mass spectrometry identification was increased. The saturated ammonium sulfate precipitation method may be applied for large-scale urine processing for screening clinical diagnostic markers through proteomics.
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Proteoma , Proteómica , Histonas , Humanos , Espectrometría de Masas , UrinálisisRESUMEN
The reissner lamprey Lethenteron reissneri, belonging to the class Cyclostomata, serves as a bridge between invertebrates and jawed vertebrates, and is considered the sister group of jawed vertebrates. However, despite this evolutionary significance, the genetic mechanisms underlying the adaptive evolution of the lamprey lineage remain unclear. Here, we assembled a 1.06 Gb chromosome-level draft genome of L. reissneri, with 72 chromosomes (ranging in length from 4.5 Mb to 25.9 Mb) and a scaffold N50 length of 13.23 Mb. Genome quality comparisons revealed that the reissner lamprey genome has higher completeness and contiguity than the previously published sea lamprey and Japanese lamprey genomes. Moreover, reissner lamprey, sea lamprey, and Japanese lamprey species share similar transposable element profiles and Hox gene cluster compositions, suggesting that a burst of transposable element activity and whole genome duplication occurred before their divergence. Additionally, the Lip gene copy numbers, which have been studied for their functions in the host defence system, were found to be expanded uniquely in lamprey lineages, suggesting key roles for these genes in lamprey evolution and adaptation. We also identified two neural-related genes, Nrn1 and Unc13a, with copy number expansions in jawed vertebrates, which may be functionally relevant to the origin of lamprey brains. Hence, this study not only provides the first chromosome-level reference genome for Cyclostomata, but also highlights features of the unique biology and adaptive evolution of the lamprey lineage.
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Evolución Molecular , Lampreas , Animales , Cromosomas/genética , Genoma , Lampreas/genética , FilogeniaRESUMEN
BACKGROUND: In previous research, we found that lamprey immune protein (LIP) possessed cytocidal activity against tumor cells, but the mechanism of the selective recognition and killing of tumor cells by LIP was not identified. METHODS: Superresolution microscopy, crystallographic structural analysis, glycan chip assay, SPR experiments, FACS assays, computational studies and mass spectrometric analysis firmly establish the mode of action of LIP, which involves dual selective recognition and efficient binding. RESULTS: We determined the overall crystallographic structure of LIP at a resolution of 2.25 Å. LIP exhibits an elongated structure with dimensions of 105 Å × 30 Å × 30 Å containing an N-terminal lectin module and a C-terminal aerolysin module. Moreover, the Phe209-Gly232 region is predicted to insert into the lipid bilayer to form a transmembrane ß-barrel, in which the hydrophobic residues face the lipid bilayer, and the polar residues constitute the hydrophilic lumen of the pore. We found that LIP is able to kill various human cancer cells with minimal effects on normal cells. Notably, by coupling biochemical and computational studies, we propose a hypothetical mechanism that involves dual selective recognition and efficient binding dependent on both N-linked glycans on GPI-anchored proteins (GPI-APs) and sphingomyelin (SM) in lipid rafts. Furthermore, specific binding of the lectin module with biantennary bisialylated nonfucosylated N-glycan or sialyl Lewis X-containing glycan structures on GPI-APs triggers substantial conformational changes in the aerolysin module, which interacts with SM, ultimately resulting in the formation of a membrane-bound oligomer in lipid rafts. CONCLUSIONS: LIP holds great potential for the application of a marine protein towards targeted cancer therapy and early diagnosis in humans.
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Antineoplásicos/química , Citotoxinas/química , Proteínas de Peces/química , Lampreas/metabolismo , Microdominios de Membrana/efectos de los fármacos , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Citotoxinas/farmacología , Proteínas de Peces/farmacología , Proteínas Ligadas a GPI/metabolismo , Humanos , Lectinas/metabolismo , Microdominios de Membrana/metabolismo , Simulación de Dinámica Molecular , Unión Proteica , Esfingomielinas/metabolismoRESUMEN
Caveolin-1 is the main structural and functional component of caveolin, and it is involved in the regulation of cholesterol transport, endocytosis, and signal transduction. Moreover, changes in caveolin-1 play an important role in tumorigenesis and inflammatory processes. Previous studies have demonstrated that human caveolin-1 is mainly located in the cell membrane and exhibits cell type- and stage-dependent functional differences during cancer development and inflammatory responses. However, the role of Lamprey-caveolin-like (L-caveolin-like) in lamprey remained unknown. Here, we demonstrated that L-caveolin-like performs anti-inflammation and oncogenic functions and the function of caveolin-1 diverged during vertebrate evolution. Moreover, the results reveal the mechanism underlying the antiapoptotic effects of L-caveolin-like. An L-caveolin-like gene from Lampetra japonica (L. japonica) was identified and characterized. L-Caveolin-like was primarily distributed in the leukocytes, intestines and supraneural bodies (Sp-bodies) immune organs as indicated by Q-PCR and immunohistochemistry assays. The mRNA and protein expression levels of L-caveolin exhibited consistent increases in expression at 2 and 72â¯h in adult tissues after exposure to lipopolysaccharide (LPS) and in leukocytes stimulated by Vibrio anguillarum (V. anguillarum), Staphylococcus aureus (S. aureus), and Poly I:C. Furthermore, the overexpression of pEGFP-N1-L-caveolin-like was associated with a distinct localization in mitochondria, with decreased cytochrome C (Cyt C) and mitochondrial Cyt C oxidase subunit I (CO I) expression. In addition, increased cellular ATP levels suggested that this protein prevented mitochondrial damage. The overexpression of pEGFP-N1-L-caveolin-like led to the altered expression of factors related to apoptosis, such as decreased Caspase-9, Caspase-3, p53, and Bax expression and increased Bcl-2 expression. In addition, the overexpression of pEGFP-N1-L-caveolin-like promoted cell proliferation associated with upregulated EGF, bFGF, and PDGFB expression. Together, these findings indicated that the L-caveolin-like protein from L. japonica induced the activation of antiapoptotic effects via the mitochondrial Cyt C-mediated Caspase-3 signaling pathway. Our analysis further suggests that L-caveolin-like is an oncogene protein product and anti-inflammatory molecule from lamprey that evolved early in vertebrate evolution.
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Proteínas Reguladoras de la Apoptosis/inmunología , Caveolina 1/inmunología , Evolución Molecular , Proteínas de Peces/inmunología , Lampreas/fisiología , Animales , Apoptosis/genética , Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis/genética , Caspasa 3/metabolismo , Caveolina 1/genética , Proliferación Celular/genética , Citocromos c/metabolismo , Femenino , Proteínas de Peces/genética , Células HeLa , Humanos , Masculino , Mitocondrias/inmunología , Mitocondrias/metabolismo , Transducción de Señal/inmunologíaRESUMEN
Tumor local invasion is the first step of metastasis cascade which remains the key obstacle for cancer therapy. Collective cell migration plays a critical role in tumor invading into surrounding tissues. In vitro assays fail to assess collective invasion in a real time manner. Herein we aim to develop a three-dimensional (3D) microfluidic cell invasion model to determine the dynamic process. In this model, collective invasion of breast cancer cells is induced by the concentration gradient of fetal bovine serum. We find that breast cancer cells adopt a collective movement rather than a random manner when the cells invade into extracellular matrix. The leading cells in the collective movement exhibit an increased expression of an Aurora kinase family protein - AURKA compared with the follower cells. Inhibition of AURKA kinase activity by VX680 or AKI603 significantly reduces the phosphorylation of ERK1/2 (Thr202/Tyr204) and collective cohort formation. Together, our study illustrates that AURKA acts as a potential therapeutic target for suppressing the process of tumor collective invasion. The 3D microfluidic cell invasion model is a reliable, measurable and dynamic platform for exploring potential drugs to inhibit tumor collective invasion.
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Aurora Quinasa A/antagonistas & inhibidores , Aurora Quinasa A/metabolismo , Movimiento Celular/efectos de los fármacos , Microfluídica , Inhibidores de Proteínas Quinasas/farmacología , Aurora Quinasa A/genética , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Movimiento Celular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Microfluídica/instrumentación , Microfluídica/métodosRESUMEN
Alzheimer's disease (AD) is a chronic neurodegenerative disease which contributes to memory loss and cognitive decline in the elderly. Fucoidan, extracted from brown algae, is a complex sulfated polysaccharide and potential bioactive compound. In this study, we investigated whether fucoidan protects PC12 cells from apoptosis induced by a combination of beta-amyloid 25-35 (Aß25-35) and d-galactose (d-Gal), and improves learning and memory impairment in AD model mice. The results indicated that fucoidan could inhibit the release of cytochrome c from the mitochondria to cytosol and activation of caspases, and increase the expression of apoptosis inhibitor proteins (IAPs), including livin and X-linked IAP (XIAP) in PC12 cells damaged by Aß25-35 and d-Gal-induction. Fucoidan reversed the decreased activity of acetylcholine (ACh) and choline acetyl transferase (ChAT), as well as the increased activity of acetylcholine esterase (AChE), in AD model mice induced by infusion of d-Gal. Furthermore, fucoidan improved antioxidant activity in vitro and in vivo by activation of superoxide dismutase (SOD) and glutathione (GSH). These results suggested that fucoidan could protect PC12 cells from apoptosis and ameliorate the learning and memory impairment in AD model mice, which appeared to be due to regulating the cholinergic system, reducing oxidative stress, and inhibiting the caspase-dependent apoptosis pathway.
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Péptidos beta-Amiloides/farmacología , Disfunción Cognitiva/inducido químicamente , Disfunción Cognitiva/tratamiento farmacológico , Galactosa/farmacología , Fármacos Neuroprotectores/farmacología , Síndromes de Neurotoxicidad/tratamiento farmacológico , Fragmentos de Péptidos/farmacología , Polisacáridos/farmacología , Acetilcolina/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Animales , Antioxidantes/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Colina O-Acetiltransferasa/metabolismo , Disfunción Cognitiva/metabolismo , Modelos Animales de Enfermedad , Glutatión/metabolismo , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Síndromes de Neurotoxicidad/metabolismo , Estrés Oxidativo/efectos de los fármacos , Células PC12 , Ratas , Superóxido Dismutasa/metabolismoRESUMEN
Lymphangiogenesis is one of the promoters of tumor lymphatic metastasis. Fucoidan which is a fucose-enriched sulfated polysaccharide has effect on various pharmacological activities including anti-metastasis activity. However, the inhibitory effect of fucoidan on lymphangiogenesis remains unclear. Here, fucoidan extracted from U. pinnatifida sporophylls suppressed HLECs proliferation, migration and tube-like structure formation, and had inhibitory effect of tumor-induced lymphangiogenesis in vitro. Additionally, we found that fucoidan had a dose-dependent depressive effect on the expressions of PROX1, vascular endothelial growth factor receptor 3 (VEGFR3), NF-κB, phospho-PI3K and phospho-Akt in HLECs. Moreover, anti-lymphangiogenesis effect of fucoidan was assessed by using mouse tumor model. In summary, fucoidan inhibit tumor lymphangiogenesis and lymphatic metastasis by suppressing the NF-κB/PI3K/Akt signaling pathway through reduced levels of PROX1 and VEGFR3.
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Antineoplásicos/farmacología , Células Endoteliales/efectos de los fármacos , Proteínas de Homeodominio/biosíntesis , Linfangiogénesis/efectos de los fármacos , Polisacáridos/farmacología , Proteínas Supresoras de Tumor/biosíntesis , Receptor 3 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Humanos , Metástasis Linfática , Masculino , Ratones , Polisacáridos/administración & dosificación , Distribución Aleatoria , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Metastasis, the greatest clinical challenge associated with cancer, is closely connected to multiple biological processes, including invasion and adhesion. The hypoxic environment in tumors is an important factor that causes tumor metastasis by activating HIF-1α. Fucoidan, extracted from brown algae, is a sulfated polysaccharide and, as a novel marine biological material, has been used to treat various disorders in China, Korea, Japan and other countries. In the present study, we demonstrated that fucoidan derived from Undaria pinnatifida sporophylls significantly inhibits the hypoxia-induced expression, nuclear translocation and activity of HIF-1α, the synthesis and secretion of VEGF-C and HGF, cell invasion and lymphatic metastasis in a mouse hepatocarcinoma Hca-F cell line. Fucoidan also suppressed lymphangiogenesis in vitro and in vivo. In addition, accompanied by a reduction in the HIF-1α nuclear translocation and activity, fucoidan significantly reduced the levels of p-PI3K, p-Akt, p-mTOR, p-ERK, NF-κB, MMP-2 and MMP-9, but increased TIMP-1 levels. These results indicate strongly that the anti-metastasis and anti-lymphangiogenesis activities of fucoidan are mediated by suppressing HIF-1α/VEGF-C, which attenuates the PI3K/Akt/mTOR signaling pathways.
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Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Polisacáridos/farmacología , Undaria/química , Animales , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Carcinoma Hepatocelular/patología , Hipoxia de la Célula , Línea Celular Tumoral , Neoplasias Hepáticas/patología , Linfangiogénesis/efectos de los fármacos , Metástasis Linfática/prevención & control , Masculino , Ratones , Polisacáridos/aislamiento & purificación , Transducción de Señal/efectos de los fármacosRESUMEN
Metastasis is one of the major causes of cancer-related death. It is a complex biological process involving multiple genes, steps, and phases. It is also closely connected to many biological activities of cancer cells, such as growth, invasion, adhesion, hematogenous metastasis, and lymphatic metastasis. Fucoidan derived from Undaria pinnatifida sporophylls (Ups-fucoidan) is a sulfated polysaccharide with more biological activities than other fucoidans. However, there is no information on the effects of Ups-fucoidan on tumor invasion and metastasis. We used the mouse hepatocarcinoma Hca-F cell line, which has high invasive and lymphatic metastasis potential in vitro and in vivo, to examine the effect of Ups-fucoidan on cancer cell invasion and metastasis. Ups-fucoidan exerted a concentration- and time-dependent inhibitory effect on tumor metastasis in vivo and inhibited Hca-F cell growth, migration, invasion, and adhesion capabilities in vitro. Ups-fucoidan inhibited growth and metastasis by downregulating vascular endothelial growth factor (VEGF) C/VEGF receptor 3, hepatocyte growth factor/c-MET, cyclin D1, cyclin-dependent kinase 4, phosphorylated (p) phosphoinositide 3-kinase, p-Akt, p-extracellular signal regulated kinase (ERK) 1/2, and nuclear transcription factor-κB (NF-κB), and suppressed adhesion and invasion by downregulating L-Selectin, and upregulating protein levels of tissue inhibitor of metalloproteinases (TIMPs). The results suggest that Ups-fucoidan suppresses Hca-F cell growth, adhesion, invasion, and metastasis capabilities and that these functions are mediated through the mechanism involving inactivation of the NF-κB pathway mediated by PI3K/Akt and ERK signaling pathways.
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Antineoplásicos Fitogénicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Polisacáridos/farmacología , Undaria/química , Animales , Antineoplásicos Fitogénicos/aislamiento & purificación , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ciclina D1/antagonistas & inhibidores , Ciclina D1/genética , Ciclina D1/metabolismo , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Factor de Crecimiento de Hepatocito/antagonistas & inhibidores , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Metástasis Linfática , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , FN-kappa B/antagonistas & inhibidores , FN-kappa B/genética , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Extractos Vegetales/química , Polisacáridos/aislamiento & purificación , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Fucoidans, fucose-enriched sulfated polysaccharides isolated from brown algae and marine invertebrates, have been shown to exert anticancer activity in several types of human cancer, including leukemia and breast cancer and in lung adenocarcinoma cells. In the present study, the anticancer activity of the fucoidan extracted from the brown seaweed Undaria pinnatifida was investigated in human hepatocellular carcinoma SMMC-7721 cells, and the underlying mechanisms of action were investigated. SMMC-7721 cells exposed to fucoidan displayed growth inhibition and several typical features of apoptotic cells, such as chromatin condensation and marginalization, a decrease in the number of mitochondria, and in mitochondrial swelling and vacuolation. Fucoidan-induced cell death was associated with depletion of reduced glutathione (GSH), accumulation of high intracellular levels of reactive oxygen species (ROS), and accompanied by damage to the mitochondrial ultrastructure, depolarization of the mitochondrial membrane potential (MMP, Δψm) and caspase activation. Moreover, fucoidan led to altered expression of factors related to apoptosis, including downregulating Livin and XIAP mRNA, which are members of the inhibitor of apoptotic protein (IAP) family, and increased the Bax-to-Bcl-2 ratio. These findings suggest that fucoidan isolated from U. pinnatifida induced apoptosis in SMMC-7721 cells via the ROS-mediated mitochondrial pathway.