Neuronal nitric oxide synthase protects neuroblastoma cells from oxidative stress mediated by garlic derivatives

In this study, we further examined the effects of diallyl disulfide (DADS), one of the major components of oil-soluble garlic extracts (GE) and of raw water GE on SH-SY5Y and NSC34 neuronal cell lines. Both treatments with DADS and GE were able to induce growth arrest and apoptosis, and we observed an increased flux of reactive oxygen and nitrogen species as early signs of cytotoxicity. We demonstrated that the content of neuronal nitric oxide synthase (nNOS) increased as early as 1 h of treatment demonstrating to be a very early sensor of DADS and GE cytotoxicity. Treatments with L-nitropropyl-arginine, an inhibitor of nNOS, increased the rate of apoptosis whereas the overexpression of nNOS significantly reduced cell death by inhibiting DNA damage, protein oxidation, and the activation of the JNK/c-Jun apoptotic signaling cascade. Overall these results demonstrate that garlic derivatives may modulate nNOS and suggest an important contribution of nitric oxide in counteracting their reactive oxygen species-mediated cytotoxicity.     

Molecular mechanism of diallyl disulfide in cell cycle arrest and apoptosis in HCT‐116 colon cancer cells

Diallyl disulfide (DADS) is the most prevalent oil‐soluble sulfur compound in garlic and inhibits cell proliferation in many cancer cell lines. Here we examined DADS cytotoxicity in a redox‐mediated process, involving reactive oxygen species (ROS) production. In the present study, p53‐independent cell cycle arrest at G2/M phase was observed with DADS treatment, along with time‐dependent increase of cyclin B1. In addition, apoptosis was also observed upon 24‐h DADS treatment accompanied by activation of p53. In HCT‐116 cells, DADS application induced a dose‐dependent increase and time‐dependent changes in ROS production. Scavenging of DADS‐induced ROS by N‐acetyl cysteine or reduced glutathione inhibited cell cycle arrest, apoptosis and p53 activation by DADS. These results suggest that ROS trigger the DADS‐induced cell cycle arrest and apoptosis and that ROS are involved in stress‐induced signaling upstream of p53 activation. Transfection of p53 small interfering RNA prevents the accumulation of cleaved poly(ADP‐ribose) polymerase and sub‐G1 cell population by 65% and 35%, respectively. Moreover, DADS‐induced apoptosis was also prevented by treatment with oligomycin, which is known to prevent p53‐dependent apoptosis by reducing ROS levels in mitochondria. These results suggest that mitochondrial ROS may serve as second messengers in DADS‐induced apoptosis, which requires activation of p53. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:71–79, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20266     

The Role of Ca2+ on the DADS-induced Apoptosis in Mouse–Rat Hybrid Retina Ganglion Cells (N18)

Diallyl disulfide (DADS), a component of garlic, has been shown to induce growth inhibition and apoptosis in human cancer cell types. The present studies were designed to investigate the effects of DADS on mouse–rat hybrid retina ganglion cells (N18) to better understand its effect on apoptosis and apoptosis-related genes in vitro. Cell viability, cell cycle analysis, reactive oxygen species (ROS), Ca²⁺ production, mitochondria membrane potential, apoptosis induction, associated gene expression and caspases-3 activity were examined by flow cytometric assay and/or Western blot. After 24-h treatment with DADS, a dose- and time-dependent decrease in the viability of N18 cells was observed and the approximate IC₅₀ was 27.6 μM. The decreased percentage of viable cells are associated with the production of ROS then followed by the production of Ca²⁺ which is induced by DADS. DADS induced apoptosis in N18 cells via the activation of caspase-3. DADS increased the protein levels of p53, cytochrome c and phosphated JNK within 24 h of treatment and it decreased the levels of Bcl-2 and those factors may have led to the mitochondria depolarization of N18 cells. DADS induced apoptosis were accompanied by increased levels of Ca²⁺ and decreased mitochondrial membrane potential which then led to release the cytochrome c, cleavage of pro-caspase-3. Deleted levels of Ca²⁺ by BAPTA-AM 10 μM (intracellular calcium chelator) then led to decrease DADS-induced apoptosis. Inhibition of caspase-3 activation by inhibitor (z-VAD-fmk) completely blocked DADS-induced apoptosis on N18 cells. The results indicated that oxidative stress modulates cell proliferation and Ca²⁺ modulates the cell death induced by DADS.     

Caudatin induces cell cycle arrest and caspase-dependent apoptosis in HepG2 cell

In the present study, we investigate the anti-cancer activity and mechanism of caudatin, the C-21 steroidal glycosides, on human hepatoma cell line HepG2. The MTT assay and flow cytometry were used to evaluate HepG2 cell proliferation and cell cycle. Annexin-V/PI and DAPI staining were used to investigate cell apoptosis. Western blotting analysis was used to evaluate the expression levels of proteins. It is found that caudatin inhibits HepG2 cell growth and induces of G₀/G₁ phase arrest in a dose dependent manner, which is associated with a decreased in the expression of cyclinD₁ and increased the levels of p21 and p53. HepG2 cells dealing with caudatin showed typical characteristics of apoptosis. Western blotting analysis indicated that the levels of Bcl-2 were down-regulated after caudatin treatment, whereas the expression of Bax was up-regulated. Furthermore, caudatin-induced apoptosis was accompanied by activation of caspase-3, -9, and poly(ADP-Ribose) Polymerase (PARP). Treatment with caudatin also induced phosphorylation of extracellular-signal regulating kinase (ERK) and c-Jun N-terminal kinase (JNK). These results demonstrate that caudatin inhibits cell proliferation via DNA synthesis reduction and induces caspase-dependent apoptosis in HepG2 cell. Activation of ERK and JNK may be involved in caudatin-induced hepatoma cell apoptosis.     

The role of reactive oxygen species (ROS) production on diallyl disulfide (DADS) induced apoptosis and cell cycle arrest in human A549 lung carcinoma cells

Diallyl disulfide (DADS), an oil soluble constituent of garlic (Allium sativum), has been reported to cause antimutagentic and anticarcinogenic effects in vitro and in vivo by modulating phases I and II enzyme activities. In recent years, several studies suggested that the chemopreventive effects of DADS can also be attributed to induction of cell cycle arrest and apoptosis in cancer cells. In the present study, we reported that DADS-induced cell cycle arrest at G2/M and apoptosis in human A549 lung cancer cells in a time- and dose-dependent manner. Additionally, a significant increase of intracellular reactive oxygen species (ROS) was induced in A549 cells less than 0.5 h after DADS treatment, indicating that ROS may be an early event in DADS-modulated apoptosis. Treatment of A549 cells with N-acetyl cysteine (NAC) completely abrogated DADS-induced cell cycle arrest and apoptosis. The result indicated that oxidative stress modulates cell proliferation and cell death induced by DADS.     

Antiproliferative effect of diallyl disulfide (DADS) on prostate cancer cell line LNCaP

Garlic has been used throughout the world to treat coughs, toothache, earache, dandruff, hypertension, hysteria, diarrhoea, dysentery, diptheria, vaginitis and many other conditions. Garlic contains a complex mixture of oil and water-soluble organosulfur compounds. Diallyl disulfide (DADS), an oil-soluble constituent of garlic seems to be effective in reducing tumour cells originating from colon, lung and skin. Hence our present study focuses on the dose-dependent effect of DADS on an androgen-dependent prostate cancer cell line. Various concentrations of DADS ranging from 25 to 100 microM were given to LNCaP cells and the activity of lactate dehydrogenase (LDH) prostatic acid phosphatase (PAcP) and the level of prostate specific antigen were studied. DADS reduced the secretory activity of LNCaP cells with the gradual increase in dosage. DADS was found to act as a good antiproliferative agent, which was confirmed by proliferation assay. DADS also induced apoptosis and nuclear segmentation in the higher doses.     

蓖麻根提取物对HepG2,NCI-H460和SGC-7901细胞增殖及凋亡作用的影响

探讨蓖麻根不同提取物对肝癌HepG2细胞株、肺癌NCI-H460细胞株和胃癌SGC-7901细胞株增殖及其凋亡的影响.采用MTT法检测蓖麻根不同提取物处理48h、72h对HepG2细胞、NCI-H460细胞和SGC-7901细胞增殖的抑制率;Hoechst 33258荧光染料染色法观察HepG2细胞凋亡,流式细胞术检测...     

BBR induces apoptosis in HepG2 cell through an Akt‐ASK1‐ROS‐p38MAPKs‐linked cascade

Berberine (BBR) has indicated significant antimicrobial activity against a variety of organisms including bacteria, viruses, and fungi. The mechanism by which BBR initiates apoptosis remains poorly understood. In the present study, we demonstrated that BBR exhibited significant cytotoxicity in human hepatoma HepG2 cells. Herein, we investigated cytotoxicity mechanism of BBR in HepG2 cells. The results showed that the induction of apoptosis in HepG2 cells by BBR was characterized by DNA fragmentation, an increased percentage of annexin V, and the activation of caspase‐3. The expressions of Bcl‐2 protein and pro‐caspase‐3 were reduced by BBR in HepG2 cells. However, Bax protein was increased in the cells. BBR‐induced apoptosis was preceded by increased generation of reactive oxygen species (ROS). NAC treatment, a scavenger of ROS, reversed BBR‐induced apoptosis effects via inhibition of Bax activation and Bcl‐2 inactivation. BBR‐induced, dose‐dependent induction of apoptosis was accompanied by sustained phosphorylation of MAP Kinases (JNK and p38 MAPK), ASK1, Akt, and p53. Furthermore, SB203580, p38 inhibitor, reduced the apoptotic effect of BBR, and blocks the generation of ROS and NO as well as activation of Bax. We found that the treatment of HepG2 cells with BBR triggers generation of ROS through Akt phosphorylation, resulting in dissociation of the ASK1‐mediated activation of JNK and p38 pathways. J. Cell. Biochem. 109: 329–338, 2010. © 2009 Wiley‐Liss, Inc.     

二烯丙基二硫通过激活Caspase3和释放Cyt-c诱导Raji细胞凋亡

二烯丙基二硫（diallyl disulfide,DADS）作为天然植物大蒜中的提取物,能抑制多种肿瘤细胞生长,但其抑瘤的分子机制还不十分清楚。在该研究中,作者采用CCK-8（cell counting kit）技术检测发现,DADS能有效地抑制人淋巴瘤Raji细胞增殖,形态学观察、DNA琼脂糖凝胶电泳和流式细胞仪检测证实DADS呈时间和浓度依赖性诱导Raji细胞凋亡, DADS处理细胞24 h后, MCL1和Bcl-2蛋白表达下降,而Bax 和Bak蛋白表达水平无变化,Bid和Caspase3被激活,线粒体中Cyt-c释放增多,用Caspase 抑制剂Z-VAD-FMK能部分阻断DADS诱导人淋巴瘤Raji细胞凋亡, 提示DADS诱导的Raji细胞凋亡作用通过Bcl-2/MCL1-线粒体-caspase3通路介导。     

Garlic Compound, Diallyl Disulfide Induces Cell Cycle Arrest in Prostate Cancer Cell Line PC-3

Prostate cancer is the most predominant cancer in men and related death rate increases every year. Till date, there is no effective therapy for androgen independent prostate cancer. Previous studies reported that aged garlic extract suppresses cancer growth. In the present study, diallyl disulfide [DADS], oil soluble organosulfur compound of garlic, was studied for its antiproliferative and induction of cell cycle arrest on prostate cancer cells in vitro. The suppression of cell growth was assessed by MTT assay. Induction of cell cycle arrest was assessed and confirmed by propidium iodide staining in flowcytometric analysis and western blotting analysis of major cell cycle regulator proteins. The results showed that DADS inhibited the growth of prostate cancer cells in a dose dependent manner, compared to the control. At 25 μM and 40 μM concentrations, DADS induced cell cycle arrest at G2/M transition in PC-3 cells. Western blotting analysis of cyclin A, B₁ and cyclin dependent kinase 1 [CDK1] revealed that DADS inhibited the cell cycle by downregulating CDK1 expression. It is concluded that DADS, inhibits proliferation of prostate cancer cells through cell cycle arrest. Dose dependent effect of DADS on PC-3 cell line was observed in the present study.     

JNK- and p38 kinase-mediated phosphorylation of Bax leads to its activation and mitochondrial translocation and to apoptosis of human hepatoma HepG2 cells

Mitochondrial translocation of pro-apoptotic Bax prior to apoptosis is well established after treatment with many cell death stimulants or under apoptosis-inducing conditions. The mechanism of mitochondrial translocation of Bax is, however, still unknown. The aim of this work was to investigate the mechanism of Bax activation and mitochondrial translocation to initiate apoptosis of human hepatoma HepG2 and porcine kidney LLC-PK1 cells exposed to various cell death agonists. Phosphorylation of Bax by JNK and p38 kinase activated after treatment with staurosporine, H(2)O(2), etoposide, and UV light was demonstrated by the shift in the pI value of Bax on two-dimensional gels and confirmed by metabolic labeling with inorganic [(32)P]phosphate in HepG2 cells. Specific inhibitors of JNK and p38 kinase significantly inhibited Bax phosphorylation and mitochondrial translocation and apoptosis of HepG2 cells. A specific small interfering RNA to MAPKK4 (the upstream protein kinase of JNK and p38 kinase) markedly decreased the levels of MAPKK4 and MAPKK3/6, blocked the activation of JNK or p38 kinase, and inhibited Bax phosphorylation. However, the negative control small interfering RNA did not cause these changes. Confocal microscopy of various Bax mutants showed differential rates of mitochondrial translocation of Bax before and after staurosporine treatment. Among the Bax mutants, T167D did not translocate to mitochondria after staurosporine exposure, suggesting that Thr(167) is a potential phosphorylation site. In conclusion, our results demonstrate, for the first time, that Bax is phosphorylated by stress-activated JNK and/or p38 kinase and that phosphorylation of Bax leads to mitochondrial translocation prior to apoptosis.     

Anti-proliferative and pro-apoptotic effect of carvacrol on human hepatocellular carcinoma cell line HepG-2

Carvacrol is one of the members of monoterpene phenol and is present in the volatile oils of Thymus vulgaris, Carum copticum, origanum and oregano. It is a safe food additive commonly used in our daily life, and few studies have indicated that carvacrol has anti-hepatocarcinogenic activities. The rationale of the study was to examine whether carvacrol affects apoptosis of human hepatoma HepG2 cells. In this study, we showed that carvacrol inhibited HepG2 cell growth by inducing apoptosis as evidenced by Hoechst 33258 stain and Flow cytometric (FCM) analysis. Incubation of HepG2 cells with carvacrol for 24 h induced apoptosis by the activation of caspase-3, cleavage of PARP and decreased Bcl-2 gene expression. These results demonstrated that a significant fraction of carvacrol treated cells died by an apoptotic pathway in HepG2 cells. Moreover, carvacrol selectively altered the phosphorylation state of members of the MAPK superfamily, decreasing phosphorylation of ERK1/2 significantly in a dose-dependent manner, and activated phosphorylation of p38 but not affecting JNK MAPK phosphorylation. These results suggest that carvacrol may induce apoptosis by direct activation of the mitochondrial pathway, and the mitogen-activated protein kinase pathway may play an important role in the antitumor effect of carvacrol. These results have identified, for the first time, the biological activity of carvacrol in HepG2 cells and should lead to further development of carvacrol for liver disease therapy.     

Cryptotanshinone induces G1 cell cycle arrest and autophagic cell death by activating the AMP-activated protein kinase signal pathway in HepG2 hepatoma

AMP-activated protein kinase (AMPK) performs a pivotal function in energy homeostasis via the monitoring of intracellular energy status. Once activated under the various metabolic stress conditions, AMPK regulates a multitude of metabolic pathways to balance cellular energy. In addition, AMPK also induces cell cycle arrest or apoptosis through several tumor suppressors including LKB1, TSC2, and p53. LKB1 is a direct upstream kinase of AMPK, while TSC2 and p53 are direct substrates of AMPK. Therefore, it is expected that activators of AMPK signal pathway might be useful for treatment or prevention of cancer. In the present study, we report that cryptotanshinone, a natural compound isolated from Salvia miltiorrhiza, robustly activated AMPK signaling pathway, including LKB1, p53, TSC2, thereby leading to suppression of mTORC1 in a number of LKB1-expressing cancer cells including HepG2 human hepatoma, but not in LKB1-deficient cancer cells. Cryptotanshinone induced HepG2 cell cycle arrest at the G1 phase in an AMPK-dependent manner, and a portion of cells underwent apoptosis as a result of long-term treatment. It also induced autophagic HepG2 cell death in an AMPK-dependent manner. Cryptotanshinone significantly attenuated tumor growth in an HCT116 cancer xenograft in vivo model, with a substantial activation of AMPK signal pathways. Collectively, we demonstrate for the first time that cryptotanshinone harbors the therapeutic potential for the treatment of cancer through AMPK activation.     

Abieslactone Induces Cell Cycle Arrest and Apoptosis in Human Hepatocellular Carcinomas through the Mitochondrial Pathway and the Generation of Reactive Oxygen Species

Abieslactone is a triterpenoid lactone isolated from Abies plants. Previous studies have demonstrated that its derivative abiesenonic acid methyl ester possesses anti-tumor-promoting activity in vitro and in vivo. In the present study, cell viability assay demonstrated that abieslactone had selective cytotoxicity against human hepatoma cell lines. Immunostaining experiments revealed that abieslactone induced HepG2 and SMMC7721 cell apoptosis. Flow cytometry and western blot analysis showed that the apoptosis was associated with cell cycle arrest during the G₁ phase, up-regulation of p53 and p21, and down-regulation of CDK2 and cyclin D1. Furthermore, our results revealed that induction of apoptosis through a mitochondrial pathway led to upregulation of Bax, down-regulation of Bcl-2, mitochondrial release of cytochrome c, reduction of mitochondrial membrane potential (MMP), and activation of caspase cascades (Casp-9 and -3). Activation of caspase cascades also resulted in the cleavage of PARP fragment. Involvement of the caspase apoptosis pathway was confirmed using caspase inhibitor Z-VAD-FMK pretreatment. Recent studies have shown that ROS is upstream of Akt signal in mitochondria-mediated hepatoma cell apoptosis. Our results showed that the accumulation of ROS was detected in HepG2 cells when treated with abieslactone, and ROS scavenger partly blocked the effects of abieslactone-induced HepG2 cell death. In addition, inactivation of total and phosphorylated Akt activities was found to be involved in abieslactone-induced HepG2 cell apoptosis. Therefore, our findings suggested that abieslactone induced G₁ cell cycle arrest and caspase-dependent apoptosis via the mitochondrial pathway and the ROS/Akt pathway in HepG2 cells.     

Increased ROS generation and p53 activation in α-lipoic acid-induced apoptosis of hepatoma cells

α-lipoic acid (α-LA) is an antioxidant used for the treatment of a variety of diseases, including liver cirrhosis, heavy metal poisoining, and diabetic polyneuropathy. In addition to its protective effect against oxidative stress, α-LA induces apoptosis in different cancer cells types. However, whether α-LA acid induces apoptosis of hepatoma cells is unknown. Herein, we investigated whether α-LA induces apoptosis in two different hepatoma cell lines FaO and HepG2. The results showed that α-LA inhibits the growth of both cell lines as indicated by the reduction in cell number, the reduced expression of cyclin A and the increased levels of the cyclin/CDKs inhibitors, p27^Kip1 and p21^Cip1. Cell cycle arrest was associated with cell loss, and DNA laddering indicative of apoptosis. Apoptosis was preceded by increased generation of reactive oxygen species, and associated with p53 activation, increased expression of Bax, release of cytochrome c from mitochondria, caspases activation, decreased levels of survivin, induction of pro-apoptotic signaling (i.e JNK) and inhibition of anti-apoptotic signaling (i.e. PKB/Akt) pathways. In conclusion, this study provides evidence that α-LA induces apoptosis in hepatoma cells, describes a possible sequence of molecular events underlying its lethal effect, and suggests that it may prove useful in liver cancer therapy.     

Docosahexaenoic acid induces apoptosis in CYP2E1-containing HepG2 cells by activating the c-Jun N-terminal protein kinase related mitochondrial damage

Docosahexaenoic acid (DHA) causes apoptosis of various cancer cells, but the mechanism of DHA-induced cell death is still unclear. We hypothesized that the early signaling of apoptosis may be important in causing cell death as well as the production of free radical metabolites. DHA caused time- and dose-dependent cell death in human HepG2 hepatoma cells transduced with CYP2E1 (E47) but not in C34 (without CYP2E1), suggesting an important role of CYP2E1 in the DHA-mediated damage. DHA increased the c-Jun N-terminal protein kinase (JNK) activity until 8 h without activating other mitogen-activated protein kinases. The contents of proapoptotic Bad and FasL at 4 h and cytochrome c and caspase 3 activity at 8 h were increased and accompanied by the JNK activation in a successive manner. In contrast, Bax and Bcl-2 were not changed. Levels of lipid peroxides (LPOs) were elevated three- and fivefold at 8 and 24 h, respectively, in DHA-induced E47 cells. However, pretreatment with chlormethiazole (CMZ), a specific inhibitor of CYP2E1, significantly reduced the levels of LPO, CYP2E1, JNK activity and the rate of cell death. In addition, pretreatment with quercetin (one as a JNK inhibitor and one as an antioxidant) significantly reduced the cell death rate and JNK and SEK-1 activities. Our results indicated that DHA-mediated apoptosis in E47 cells was induced through the activation of the JNK-related cell death pathway, which may be involved in the production of LPO or reactive oxygen species during the CYP2E1 catalytic cycle, followed by mitochondrial injury and apoptosis.     

Apelin 通过调节ROS 水平抑制TNF-α 诱导的HepG2 细胞凋亡

目的:探讨apelin在肿瘤坏死因子(tumor necrosis factor-α,TNF-α)诱导的肝细胞凋亡中的作用及可能机制。方法:PCR检测HepG2细胞和原代小鼠肝细胞中APJ受体的表达;采用Hoechst 33342染色检测TNF-α诱导的HepG2细胞凋亡;用活性氧(ROS)检测试剂盒结合流式细胞术测定细胞内ROS水平;通过Western blot检测信号分子JNK的磷酸化水平;比较给予apelin处理对上述指标的影响。结果:HepG2细胞和原代小鼠肝细胞均表达APJ受体;apelin可抑制TNF-α导致的细胞内ROS生成增多和JNK磷酸化水平升高并减少TNF-α诱导的HepG2细胞凋亡。结论:Apelin可能通过拮抗TNF-α诱导的细胞内ROS水平升高,使JNK信号失活,从而抑制HepG2细胞凋亡。     

维甲酸对肝癌细胞HepG2糖基化磷脂酰肌醇特异性磷酯酶D表达及细胞生物学特性的影响

应用MTT、流式细胞仪、免疫印迹法检测全反式维甲酸(ATRA)单独或联合糖基化磷脂酰肌醇特异性磷酯酶D(GPI-PLD)特异性抑制剂1,10-二氮杂菲对肝癌细胞HepG2生物学特性的改变.ATRA使肝癌细胞HepG2 GPI-PLD基因表达及酶活性上调,并呈现剂量和时间依赖性.ATRA可抑制肝癌细胞HepG2增殖,使肝癌细胞Caspase-3表达水平显著增加,Bcl-2表达水平下调,促进肝癌细胞凋亡(P<0.05).ATRA联合1,10-二氮杂菲诱导组细胞,Bcl-2、细胞增殖活性较ATRA单独诱导组显著增强,Caspase-3、凋亡率显著下降.维甲酸可促进肝癌细胞HepG2 GPI-PLD基因表达上调,高活性的GPI-PLD有助于维甲酸抑制肝癌细胞增殖,促进肝癌细胞凋亡.