Toxicogenomics of A375 human malignant melanoma cells

Toxicogenomics applications are increasingly applied to the evaluation of preclinical drug safety, and to explain toxicities associated with compounds at the mechanism level. In this review, we aim to describe the application of toxicogenomics tools for studying the genotoxic effect of active compounds on the gene-expression profile of A375 human malignant melanoma cells, through the other molecular functions of target genes, regulatory pathways and mechanisms of malignant melanomas. It also includes the current systems biology approaches, which are very useful for analyzing the biological system and understanding the entire mechanisms of malignant melanomas. We believe that this review would be very potent and useful for studying the toxicogenomics of A375 melanoma cells, and for further diagnostic and therapeutic applications.     

异甘草素诱导人黑色素瘤A375细胞凋亡研究

目的研究异甘草素诱导人黑色素瘤(A375)细胞凋亡及其初步机制。方法采用硫氰酸盐B(SRB)法测定异甘草素对A375细胞生长的抑制效应,AO/EB和Hoechst33258荧光染色法观察细胞凋亡形态,流式细胞技术检测A375细胞凋亡率;荧光染料二乙酸-2’,7’-二氯荧光素(DCFH-DA)测定A375细胞内活性氧的变化。5,5',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑羰花青碘化物(JC-1)测定线粒体膜电势的变化,利用试剂盒检测异甘草素处理后的A375细胞内ATP和培养液中的乳酸、葡萄糖含量。结果异甘草素能抑制A375细胞的增殖,并呈浓度依赖性;在荧光显微镜下发现,异甘草素处理后的A375细胞出现明显的凋亡形态;细胞凋亡率随异甘草素浓度的增加而升高;异甘草素处理后的细胞内活性氧有明显上升趋势,细胞线粒体膜电位明显下降;异甘草素作用后胞内ATP含量下降(P<0.05或P<0.01),培养液中的乳酸含量下降,葡萄糖含量上升(P<0.05或P<0.01)。结论异甘草素能够抑制A375细胞的恶性增殖,最终诱导细胞凋亡。推测异甘草素通过活性氧升高,引起线粒体膜电势下降,从而影响A375细胞的糖酵解途径而导致细胞凋亡。     

Regulation of IL-1-induced selective IL-6 release from human mast cells and inhibition by quercetin

Mast cells are involved in allergic reactions, but also in innate immunity and inflammation. Crosslinkage of mast cell Fc immunoglobulin E receptors (FcvarepsilonRI) by multivalent antigen triggers secretion of granule-stored mediators, as well as de novo synthesis of cytokines, including interleukin (IL)-6. We showed recently that the proinflammatory cytokine IL-1 stimulates human leukemic mast cells (HMC-1) and human umbilical cord blood-derived cultured mast cells (hCBMCs) to release newly synthesized IL-6 without tryptase in the absence of degranulation. Here, we investigated several signal-transduction pathways activated by IL-1 leading to IL-6 production by HMC-1 and hCBMCs. We also investigated the effect of the flavonol quercetin that was recently shown to strongly inhibit IL-6 secretion in response to allergic stimulation from hCBMCs.IL-1 stimulated p38, but did not activate extracellular signal-regulated kinase (ERK) or c-jun N-terminal kinase (JNK); it also did not activate protein kinase C (PKC) isozymes alpha, beta, mu and zeta, except for PKC-theta, which was phosphorylated. The p38 inhibitor SB203580 and the PKC inhibitors Calphostin C and G?6976 completely inhibited IL-1-induced IL-6 production. Quercetin 1-100 microM inhibited IL-1-induced IL-6 secretion, p38 and PKC-theta phosphorylation in a dose-dependent manner. These results indicate that IL-1-stimulated IL-6 production from human mast cells is regulated by biochemical pathways distinct from IgE-induced degranulation and that quercetin can block both IL-6 secretion and two key signal transduction steps involved.     

Endothelin-1-induced arachidonic acid release by cytosolic phospholipase A2 activation in rat vascular smooth muscle via extracellular signal-regulated kinases pathway

The present study investigates whether endothelin-1 (ET-1), like noradrenaline (NA), stimulates the release of arachidonic acid (AA) via cytosolic phospholipase A2 (cPLA2) in rat tail artery. In tail artery segments labelled with [3H]AA, ET-1-induced AA release in a concentration-dependent manner with an EC50 of 1.3 nM. The effect of ET-1 was inhibited by bosentan and was insensitive to BQ788, suggesting the involvement of ETA receptor. The stimulation of AA release induced by ET-1 was prevented by arachydonyl trifluoromethyl ketone (AACOCF3), a selective inhibitor of cPLA2 and not by RHC80267, a diacylglycerol lipase inhibitor. Furthermore, PD98059, inhibitor of mitogen-activated protein kinase kinase (MEK) cascade and calphostin C, a protein kinase C (PKC) inhibitor, prevented the stimulation of AA release induced by ET-1 and NA. Immunoblotting of the cytosolic fraction of rat tail arteries stimulated with ET-1 or NA showed an increase in extracellular signal-regulated kinases (ERKs) phosphorylation and this effect was abolished by calphostin C treatment. These findings show that in rat tail artery ET-1 and NA induce a sequential activation of protein kinase C and extracellular signal-regulated kinases that results in stimulation of AA release via cPLA2 activation. This may represent a general pathway by which G-proteins coupled receptors stimulate AA release and its metabolites in vascular smooth muscle.     

Activation of extracellular signal-regulated kinases is essential for pressure-induced proliferation of vascular smooth muscle cells

In hypertension, increased transmural pressure directly influences vascular smooth muscle cells and causes cell proliferation. However, the mechanisms of transmural pressure-induced proliferation of vascular smooth muscle cells are unknown. We investigated the role of various protein kinases in pressure-induced proliferation of vascular smooth muscle cells. Pressure was applied to quiescent rat vascular smooth muscle cells in culture by compressed helium gas in a loading apparatus. Pressure application increased [3H]thymidine incorporation in a time- and pressure-dependent manner and significantly increased the cell number. The pressor response was significantly suppressed by various protein kinase inhibitors for protein kinase C (bisindolylmaleimide I), tyrosine kinase (genistein), extracellular signal-regulated kinase kinase (PD98059; 2'-amino-3'-methoxyflavone) and p38 mitogen-activated protein kinases (MAPK) (SB203580; 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole). Pressure rapidly increased the phosphorylation and activity of extracellular signal-regulated kinases (ERK). Pressure also caused increment of phosphorylation level of p38 MAPK but not that of c-JUN N-terminal protein kinase (JNK). In ERK-deficient cells prepared by transfection of an antisense oligonucleotide for ERK, pressure-induced DNA synthesis was almost abolished. Our results suggest that activation of ERK is essential for pressure-induced DNA synthesis in rat vascular smooth muscle cells, in addition to activation of protein kinase C, tyrosine kinase and p38 MAPK. These processes could be involved in the pathogenesis of hypertension-related atherosclerosis.     

赖氨匹林对人恶性黑色素瘤A375细胞凋亡的诱导作用

目的 研究赖氨匹林（Aspisol）对人恶性黑色素瘤A375细胞凋亡的诱导作用。方法采用四甲荃偶氮唑蓝（MTr）比色法检测赖氨匹林对A375细胞增殖的抑制作用,倒置显微镜下观察A375细胞形态学变化,流式细胞仪（FCM）检测不同浓度的赖氨匹林诱导A375细胞的早期凋亡率。结果不同浓度的赖氨匹林对A375细胞均有明显的生长抑制作用,且其作用具有时间和浓度依赖性,差异具有非常显著性（P〈0．01）。不同浓度的赖氨匹林对A375细胞作用24h后,均可诱导其发生早期凋亡,并且呈浓度依赖性,差异具有显著性（P〈0．05）。结论赖氨匹林能抑制A375的增殖,诱导其发生凋亡。     

文冠果壳苷诱导人恶性黑色素瘤A375.S2细胞的凋亡及机制

目的观察文冠果壳苷对人恶性黑色素瘤A375.S2细胞增殖抑制作用及诱导凋亡的机理。方法采用四甲基噻唑蓝法(MTT法)检测化合物对细胞的生长抑制作用,光学显微镜及荧光显微镜观察细胞形态学变化,免疫印迹法(Western blotting)检测p-p38、p38、核转录因子-κB(nuclear factor kappa B,NF-κB)p65及核转录抑制因子-κB(nuclear factor kappa B inhibitor,I-κB)蛋白表达水平,检测细胞核内NF-κB p65表达水平。结果文冠果壳苷可浓度依赖性地抑制A375.S2细胞增殖,并优于5-氟尿嘧啶(5-FU);10μmol·L-1文冠果壳苷作用于A375.S2细胞,形态学观察发现明显的凋亡小体和核固缩;Western blotting检测发现文冠果壳苷可时间依赖性地降低p-p38、p38、NF-κB p65及I-κB的蛋白表达水平;文冠果壳苷抑制NF-κB p65自细胞浆到细胞核的转位。结论文冠果壳苷可能通过抑制NF-κB和p38的活化诱导人恶性黑色素瘤A375.S2细胞凋亡。     

Physalin B from Physalis angulata triggers the NOXA-related apoptosis pathway of human melanoma A375 cells

Melanoma is a lethal form of skin cancer that can metastasize rapidly. While surgery and radiation therapy provide palliative therapy for local tumor growth, systemic therapy is the mainstay of treatment for metastatic melanoma. However, limited chemotherapeutic agents are available for melanoma treatment. In this study, we investigated the anti-melanoma effect of physalin B, the major active compound from a widely used herb medicine, Physalis angulata L.This study demonstrated that physalin B exhibits cytotoxicity towards v-raf murine sarcoma viral oncogene homolog B1 (BRAF)-mutated melanoma A375 and A2058 cells (the IC₅₀ values are lower than 4.6 μg/ml). Cytotoxicity is likely resulted from apoptosis since the apoptotic marker phosphatidylserine are detected immediately under physalin B treatment and apoptotic cells formation. Further examination revealed that physalin B induces expression of the proapoptotic protein NOXA within 2 h and later triggers the expression of Bax and caspase-3 in A375 cells. These results indicate that physalin B can induce apoptosis of melanoma cancer cells via the NOXA, caspase-3, and mitochondria-mediated pathways, but not of human skin fibroblast cells and myoblastic cells. Thus, physalin B has the potential to be developed as an effective chemotherapeutic lead compound for the treatment of malignant melanoma.     

吴茱萸碱在诱导人黑色素瘤A375-S2细胞凋亡过程中对SIRT1和p53蛋白的影响

目的研究吴茱萸碱(evodiamine)在诱导A375-S2细胞凋亡过程中对SIRT1和p53蛋白表达的调控。方法电镜观察吴茱萸碱诱导细胞凋亡过程中细胞形态的变化。Western blot方法分析药物作用后对SIRT1蛋白和p53及其下游p21蛋白表达的影响。结果经15μmol.L-1吴茱萸碱处理24 h的A375-S2细胞表现出典型的凋亡特征,即细胞表面微绒毛消失、胞质空泡化、染色质浓集、边聚。在吴茱萸碱诱导A375-S2细胞凋亡过程中,SIRT1蛋白的表达下降,p53和p-p53的表达有所上升,其下游p21蛋白也被激活。结论在吴茱萸碱诱导的A375-S2细胞凋亡中,p53及其下游p21蛋白被活化,SIRT1蛋白表达被下调。     

Oridonin induces human melanoma A375-S2 cell death partially through inhibiting insulin-like growth factor 1 receptor signaling

Our previous studies indicated that oridonin, a diterpenoid isolated from Rabdosia rubescens, induced human melanoma A375-S2 cell apoptosis. In this study, we investigated whether the proapoptotic effect of oridonin on A375-S2 cells would depend on an interference with function of the insulin-like growth factor 1 (IGF-1) receptor, a plasma membrane receptor critical for the survival or antiapoptotic ability in melanoma cells. We found that IGF-1 receptor (IGF-1R) signaling was a potential survival pathway against a low concentration of 20 μmol/L oridonin-induced apoptosis in A375-S2 cells. The activation of Ras or its downstream effector p38 mitogen-activated protein kinase (p38 MAPK) was shown to be necessary for IGF-1-mediated protection, but the activation of phosphatidylinositol-3-OH kinase (PI3 kinase) or extracellular signal-regulated kinase (ERK) did not correlate with the regulation of survival. However, in the presence of 40 μmol/L (IC₅₀ at 24 h) oridonin, A375-S2 cells could not be protected by IGF-1 from apoptosis, accompanied by a severe impairment of IGF-1R expression. Therefore, we concluded that the proapoptotic activity of oridonin was partially attributed to its repression of IGF-1R signaling. In addition, p53 was supposed to be a pivotal transducer of proapoptotic and survival signaling pathway in this system.     

Timosaponin AIII induces apoptosis and autophagy in human melanoma A375-S2 cells

Timosaponin AIII (AIII), a steroidal saponin isolated from Anemarrhena asphodeloides Bge. Our study showed that AIII induced both apoptosis and autophagy, and autophagy inhibited apoptosis in A375S2 cells. Furtherly, this study was carried out to investigate what kind of cytokines plays an important role in this process. The results revealed that AIII induced apoptosis through activating c-Jun N-terminal protein kinase (JNK) or extracellular signal related kinase (ERK) signaling pathway and generating NO. However, JNK or ERK inhibited autophagy, while NO had no effect on autophagy. Therefore, JNK, ERK or NO regulates two programmed death processes in different ways. AIII did not show obvious cytotoxic effect on human peripheral blood mononuclear cells, which indicated that AIII has less side effects on normal cells, and could be considered as a leading compound for developing novel anticancer drug.     

Contribution of extracellular signal-regulated kinase to UTP-induced interleukin-6 biosynthesis in HaCaT keratinocytes

UTP causes interleukin (IL)-6 production via mRNA expression through P2Y(2)/P2Y(4) receptors in human HaCaT keratinocytes. In the present study, we analyzed the mechanism of UTP-induced IL-6 production in these cells. UTP, an agonist of P2Y(2)/P2Y(4) receptors, induced phosphorylation of extracellular signal-regulated kinase (ERK) in a concentration- and time-dependent manner. PD98059, a MEK (mitogen-activated protein kinase kinase) inhibitor, and BAPTA-AM [O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester], an intracellular Ca(2+) chelator, reduced UTP-induced ERK phosphorylation and IL-6 mRNA expression. 2-APB [(2-aminoethoxy)diphenylborane], an inositol 1,4,5-trisphosphate (IP(3))-receptor antagonist, inhibited UTP-induced IL-6 mRNA expression; and the action of A23187, a Ca(2+) ionophore, resembled the action of UTP. In contrast, protein kinase C (PKC) downregulation and pertussis toxin did not affect UTP-induced IL-6 mRNA expression, suggesting that PKC and G(i) are not involved in the UTP-induced IL-6 production. However, AG1478, an epidermal growth factor (EGF)-receptor inhibitor, partially decreased UTP-induced ERK phosphorylation and IL-6 expression. These results suggest that UTP-induced IL-6 production is in part mediated via phosphorylation of ERK through G(q/11)/IP(3)/[Ca(2+)](i) and transactivation of the EGF receptor.     

Oridonin induces human melanoma A375-S2 cell death partially through inhibiting insulin-like growth factor 1 receptor signaling

Our previous studies indicated that oridonin, a diterpenoid isolated from Rabdosia rubescens, induced human melanoma A375-S2 cell apoptosis. In this study, we investigated whether the proapoptotic effect of oridonin on A375-S2 cells would depend on an interference with function of the insulin-like growth factor 1 (IGF-1) receptor, a plasma membrane receptor critical for the survival or antiapoptotic ability in melanoma cells. We found that IGF-1 receptor (IGF-1R) signaling was a potential survival pathway against a low concentration of 20 micromol/L oridonin-induced apoptosis in A375-S2 cells. The activation of Ras or its downstream effector p38 mitogen-activated protein kinase (p38 MAPK) was shown to be necessary for IGF-1-mediated protection, but the activation of phosphatidylinositol-3-OH kinase (PI3 kinase) or extracellular signal-regulated kinase (ERK) did not correlate with the regulation of survival. However, in the presence of 40 micromol/L (IC50 at 24 h) oridonin, A375-S2 cells could not be protected by IGF-1 from apoptosis, accompanied by a severe impairment of IGF-1R expression. Therefore, we concluded that the proapoptotic activity of oridonin was partially attributed to its repression of IGF-1R signaling. In addition, p53 was supposed to be a pivotal transducer of proapoptotic and survival signaling pathway in this system.     

Oridonin induces human melanoma A375-S2 cell death partially through inhibiting insulin-like growth factor 1 receptor signaling

Our previous studies indicated that oridonin, a diterpenoid isolated from Rabdosia rubescens, induced human melanoma A375-S2 cell apoptosis. In this study, we investigated whether the proapoptotic effect of oridonin on A375-S2 cells would depend on an interference with function of the insulin-like growth factor 1 (IGF-1) receptor, a plasma membrane receptor critical for the survival or antiapoptotic ability in melanoma cells. We found that IGF-1 receptor (IGF-1R) signaling was a potential survival pathway against a low concentration of 20 mumol/L oridonin-induced apoptosis in A375-S2 cells. The activation of Ras or its downstream effector p38 mitogen-activated protein kinase (p38 MAPK) was shown to be necessary for IGF-1-mediated protection, but the activation of phosphatidylinositol-3-OH kinase (PI3 kinase) or extracellular signal-regulated kinase (ERK) did not correlate with the regulation of survival. However, in the presence of 40 mumol/L (IC(50) at 24 h) oridonin, A375-S2 cells could not be protected by IGF-1 from apoptosis, accompanied by a severe impairment of IGF-1R expression. Therefore, we concluded that the proapoptotic activity of oridonin was partially attributed to its repression of IGF-1R signaling. In addition, p53 was supposed to be a pivotal transducer of proapoptotic and survival signaling pathway in this system.     

人参皂苷Rh2对人黑色素瘤A375—S2细胞的促凋亡作用

目的;研究人参皂苷G－Rh2诱导人黑色素肿瘤细胞A375－S2凋亡的分子生物学机制。方法：用结晶紫染色的方法测定细胞的死亡率。用置显微镜观察细胞形态学的变化。用琼脂糖凝胶电泳检测核酸片断。用流式细胞仪检测细胞凋亡和细胞周期。结果：G－Rh2抑制A375－S2细胞增殖并在20μmol/L可以诱导A375－S2细胞产生凋亡,Caspase抑制剂z-VAD-fmk(caspase家族）,z-DEVD-fmk(caspase-3)或z-IETD-fmk(caspase-8)能部分抑制细胞凋亡,但是Ac-YVAD-cmk(caspase-1)不能抑制A375－S2细胞凋亡。结论：GRh2在体外抑制A375－S2细胞的增殖,通过细胞形态学和核酸片断分析,G－Rh2能够诱导A375－S2细胞产生凋亡。这种作用是通过细胞内caspase一类半胱氨酸蛋白酶进行传导的,G－Rh2对A375－S2细胞的周期没有影响。     

Antiproliferative and apoptosis-inducing effects of lipophilic vitamins on human melanoma A375 cells in vitro

The effects of six lipophilic vitamins: tretinoin (ATRA), vitamin D(3) (VD(3)), VE, VK(1), VK(3), and VK(5) on cell proliferation and apoptosis in human A375 melanoma cells were investigated. VD(3), VK(3), and VK(5) were found to inhibit cell proliferation significantly at concentration ranges of 10-100?μmol/L (p<0.01), while the other vitamins did not show inhibitory effects at 100?μmol/L. VK(3) and VK(5) showed the strongest effects with IC(50) values of less than 10?μmol/L. Dacarbazine slightly inhibited the proliferation of A375 cells at a concentration range of 25-100?μmol/L, but the effects were not statistically significant. VK(3) and VK(5) increased annexin-V positive apoptotic cells, as well as activating caspase-3, in A375 cells. Our findings showed that VD(3), VK(3,) and VK(5) inhibited the growth of dacarbazine resistant human melanoma cells, while ATRA, VE, and VK(1) had little effect on the cell growth. The effects of VK(3) and VK(5) were observed at concentrations lower than 10?μmol/L, which are suggested to have resulted from apoptosis-induction in the melanoma cells.     

EGCG downregulates IL-1RI expression and suppresses IL-1-induced tumorigenic factors in human pancreatic adenocarcinoma cells

Human pancreatic cancer is currently one of the fifth-leading causes of cancer-related mortality with a 5-year survival rate of less than 5%. Since pancreatic carcinoma is largely refractory to conventional therapies, there is a strong medical need for the development of novel and innovative therapeutic strategies. Increasing evidence suggests an association of carcinogenesis and chronic inflammation. Because IL-1 plays a crucial role in inflammation-associated carcinogenesis, we analyzed the biological effects of IL-1 and its modulation by the chemopreventive green tea polyphenol (−)-epigallocatechin-3-gallate (EGCG) in the human pancreatic adenocarcinoma cell line Colo357. Proinflammatory IL-6 and PGHS-2 as well as proangiogenic IL-8 and VEGF were induced by IL-1, whereas the secretion of invasion-promoting MMP-2 remained unaffected. IL-1 responsiveness and constitutive MMP-2 release in Colo357 were downregulated by EGCG in a dose- and time-dependent manner. Moreover, EGCG reduced cell viability via induction of apoptosis in Colo357. Since EGCG effects on cytokine production precede reduction in cell viability, we hypothesize that these findings are not only a result of cell death but also depend on alterations in the IL-1 signaling cascade. In this context, we found for the first time an EGCG-induced downregulation of the IL-1RI expression possibly being caused by NF-κB inhibition and causative for its inhibitory action on the production of tumorigenic factors. Thus, our data might have future clinical implications with respect to the development of novel approaches as an adjuvant therapy in high-risk patients with human pancreatic carcinoma.     

Cytochrome c release from oridonin-treated apoptotic A375-S2 cells is dependent on p53 and extracellular signal-regulated kinase activation

We have reported that oridonin isolated from Rabdosia rubescens induces apoptosis of human melanoma A375-S2 cells within 12 h. In this study, TUNEL assay and flow cytometric analysis also indicate that one of the causes of A375-S2 cell death induced by oridonin was apoptosis. The cell death was preceded by the release of cytochrome c from the mitochondria. Twelve hours after treatment with oridonin, the ratio of Bax/Bcl-xL protein expression was increased and release of cytochrome c was decreased by an extracellular signal-regulated kinase (ERK) MAPK inhibitor (PD98059) and a phosphoinositide 3-kinases (PI3-K) inhibitor (wortmannin). A mitochondrial permeability transition (MPT) inhibitor, decylubiquinone, suppressed the release of cytochrome c without affecting Bax expression. The activation of p53 by oridonin was also blocked by wortmannin. In addtion, PD98059 and wortmannin significantly decreased oridonin-induced DNA fragmentation, but the p38 MAPK inhibitor (SB203580) did not after DNA fragmentation. Oridonin induced A375-S2 cell apoptosis by activating parallel p53 and ERK pathways, increasing the ratio of Bax/Bcl-xL protein expression, and promoting the release of cytochrome c into the cytosol, resulting in apoptotic cell death.