神经酰胺诱导人乳腺癌细胞凋亡的研究

目的 :观察神经酰胺对人乳腺癌细胞株MCF 7及耐多柔比星细胞株MCF 7/ADR的诱导凋亡作用及其对bcl 2基因表达的影响 ,探讨神经酰胺诱导凋亡的机制。方法 :采用MTT法检测细胞生存率 ,用流式细胞术检测凋亡率 ,运用流式细胞术、RT PCR方法检测bcl 2基因表达。结果 :神经酰胺对MCF 7、MCF 7/ADR细胞有明显的抑制生长作用。 2 5 μmol/LC6 神经酰胺作用48h后 ,MCF 7及MCF 7/ADR细胞凋亡率分别为( 2 0 70± 2 66) %、( 12 3 9± 1 76) % ,前者凋亡率高于后者 ,P <0 0 1。流式细胞术检测 ,MCF 7、MCF 7/ADR细胞bcl 2蛋白阳性率分别为 ( 2 7 5 9± 2 94) %、( 79 5 6±3 2 0 ) %。MCF 7/ADR细胞经神经酰胺作用后 ,bcl 2蛋白及mRNA水平均降低。结论 :神经酰胺对MCF 7、MCF 7/ADR细胞有抑制生长及诱导凋亡作用 ,神经酰胺诱导凋亡可能是通过下调bcl 2基因表达而实现的     

紫杉醇诱发人乳癌细胞凋亡的机制研究

探讨紫杉醇诱发人乳腺癌细胞凋亡的发生机制。方法应用细胞形态观察、琼脂糖凝胶电泳、流式细胞仪、活细胞视频观察和蛋白印迹免疫法进行检测和观察。结果癌细胞在紫杉醇作用下,细胞分裂阻滞在分裂期的中期,并诱导细胞发生凋亡。凋亡细胞表现为细胞固缩,核染色质凝聚或者断裂。细胞ＤＮＡ裂解片段呈现典型的“阶梯状”排列的条带。凋亡抑制基因Ｂｃｌ－２在细胞凋亡过程中呈低表达并发生修饰反应,而凋亡诱导基因Ｂａｘ先呈高表达然后表达降低。结论紫杉醇诱发的人乳癌细胞的凋亡与细胞分裂期阻滞密切相关,Ｂｃｌ－２和Ｂａｘ在紫杉醇诱发的人乳癌细胞凋亡中可能起着重要的调控作用     

MECHANISM OF TAXOL-INDUCED APOPTOSIS IN HUMAN BREAST CANCER CELLS

ＭＥＣＨＡＮＩＳＭＯＦＴＡＸＯＬＩＮＤＵＣＥＤＡＰＯＰＴＯＳＩＳＩＮＨＵＭＡＮＢＲＥＡＳＴＣＡＮＣＥＲＣＥＬＬＳＣｈｅｎＬｉｒｏｎｇ陈丽荣ＺｈｅｎｇＳｈｕ郑树ＭＣＷｉｌｉｎｇｈａｍＦａｎＷｅｉｍｉｎ范伟民ＣａｎｃｅｒＩｎｓｔｉｔｕｔｅ，Ｚｈ...     

Synthesis and characterization of novel ceramide analogs for induction of apoptosis in human cancer cells

A variety of anti-cancer drugs elevate endogenous ceramide, thereby inducing apoptosis in tumor cells. Recently, we have introduced novel ceramide analogs of the beta-hydroxy alkyl amide type, which trigger pro-apoptotic signaling pathways without prior elevation of endogenous ceramide. They induce apoptosis specifically in rapidly dividing neuroblastoma cells, but not in resting or differentiated cells. We characterize new ceramide mimics that have been derived from N-acylation of serinol (S), diethanolamine (B), propanolamine (P), and tris(hydroxy-methyl)methylamine (T) with myristic (14), palmitic (16), or oleic (18) acid. The water solubility of these compounds exceeds that of ceramide by more than 100-fold (up to 5 mM). Apoptosis of human neuroblastoma, glioma, medulloblastoma, and adenocarcinoma cells is induced by N-(2-hydroxy-1-(hydroxymethyl)ethyl)-palmitoylamide, C16-serinol (S16), N-(2-hydroxy-1-(hydroxymethyl)ethyl)-oleoylamide, C18-serinol (S18), N-bis(2-hydroxyethyl)-myristoyl-amide (B16), and N-tris(hydroxymethyl)methyl-oleoylamide (T18) within 60 min of incubation, and is completed even after removal of the compound from the medium. This is most likely due to a rapid uptake of the analogs followed by their slow release from the cells. Alteration of the acyl chain length to less than 14 methylene units, removal of the amino group, or reducing the number of hydroxyalkyl residues to less than two significantly lowers or eliminates the pro-apoptotic potential of these compounds. The target specificity of novel ceramide analogs for tumor cells, their water solubility, and fast pro-apoptotic mechanism indicates a high therapeutic potential for cancer treatment.     

Lactoferricin-induced apoptosis in estrogen-nonresponsive MDA-MB-435 breast cancer cells is enhanced by C6 ceramide or tamoxifen

Bovine lactoferricin (LfcinB) is a cationic peptide that selectively induces caspase-dependent apoptosis in human leukemia and carcinoma cell lines. Ceramide is a second messenger in apoptosis signaling that has been shown to increase the cytotoxicity of various anti-cancer drugs. In this study, we determined whether manipulation of intracellular ceramide levels enhanced LfcinB-induced apoptosis of estrogen-nonresponsive MDA-MB-435 breast carcinoma cells. LfcinB caused DNA fragmentation and morphological changes consistent with apoptosis in MDA-MB-435 breast cancer cell cultures, but did not affect the viability of untransformed mammary epithelial cells. MDA-MB-435 breast cancer cells also exhibited DNA fragmentation and morphological changes consistent with apoptosis following exposure to the cell-permeable ceramide analog C6. An additive increase in DNA fragmentation was observed when both LfcinB and C6 ceramide were added to MDA-MB-435 breast cancer cell cultures. A greater than additive increase in DNA fragmentation was seen when LfcinB was used in combination with tamoxifen, which prevents the metabolism of endogenous ceramide to glucosylceramide by glucosylceramide synthase, as well as blocking estrogen receptor signaling. However, a selective inhibitor of glucosylceramide synthase,1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, failed to further increase DNA fragmentation by LfcinB, suggesting that tamoxifen enhanced LfcinB-induced apoptosis in breast cancer cells via a mechanism that did not involve glucosylceramide synthase inhibition. We conclude that combination therapy with LfcinB and tamoxifen warrants further investigation for possible use in the treatment of breast cancer.     

JNK pathway regulates estradiol-induced apoptosis in hormone-dependent human breast cancer cells

Estrogen is known to stimulate breast cancer development in humans. Ironically, high doses of estrogen can induce regression of hormone-dependent breast cancer in postmenopausal women. The mechanism by which estrogen induces tumour regression in breast cancer is still unknown. We found that under low growth-stimulated conditions, high concentrations of 17-β-estradiol (estradiol) induces apoptosis and concomitantly increases phosphorylation of c-jun in estrogen receptor (ER)-positive breast cancer cell line, MCF-7, but not in ER-negative breast cancer cell line MDA-MB 231 suggesting an ER-mediated event. Interestingly, when the c-jun NH₂-terminal kinase (JNK) signalling pathway was disrupted by the JNK inhibitor SP600125, the ability of estradiol to inhibit the growth of MCF-7 cells and to induce apoptosis was completely blocked. These data suggest that JNK plays a central role in mediating the anticancer effect of high concentrations of estradiol in MCF-7 cells. Our data showing the apoptotic effect of estradiol in low growth-stimulated conditions suggest potential implications for the pharmacological control of breast cancer with high dose estrogen in postmenopausal women. Furthermore, our results indicate that augmenting JNK activity could be an efficient novel approach for treating breast cancer.     

Gold nanoparticles induce apoptosis in MCF-7 human breast cancer cells

Background: Gold nanoparticles have recently been investigated with respect to biocompatibility accordingto their interactions with cells. The purpose of this study was to examine cytotoxicity and apoptosis induction bywell-characterized gold nanoparticles in human breast epithelial MCF-7 cells. Methods: Apoptosis was assessedby TUNEL, cytotoxicity by MTT assay and caspase 3, 9, p53, Bax and Bcl expression by real-time PCR assays.Results: Gold nanoparticles at up to 200 μg/mL for 24 hours exerted concentration-dependent cytotoxicity andsignificant upregulation of mRNA expression of p53, bax, caspase-3 & caspase-9, whereas expression of antiapoptoticbcl-2 was down-regulated. Conclusion: To the best of our knowledge this is the first report showingthat gold nanoparticles induce apoptosis in MCF-7cells via p53, bax/bcl-2 and caspase pathways.     

Hsp27 overexpression inhibits doxorubicin-induced apoptosis in human breast cancer cells

Previously we demonstrated that heat shock protein 27 (hsp27) overexpression confers resistance to the chemotherapeutic agent doxorubicin in MDA-MB-231 breast cancer cells. Since induction of apoptosis is one underlying mechanism of chemotherapeutic drug action, we investigated the effect of hsp27 overexpression on doxorubicin-induced apoptosis, finding that hsp27 protects MDA-MB-231 cells from apoptosis. We also examined expression of the doxorubicin target, topoisomerase II (topo II), in control and hsp27-overexpressing stable transfectants, as topo II expression is important for both drug sensitivity and the initiation of apoptosis by doxorubicin. The relative levels of both topo IIalpha and beta were higher in the controls than the hsp27-overexpressing clones, suggesting that the apoptotic protective effect of hsp27 overexpression in MDA-MB-231 cells is associated with altered topo II expression.     

Mechanism of 2-methoxyestradiol-induced apoptosis and growth arrest in human breast cancer cells

2-Methoxyestradiol, a well-known nonpolar endogenous metabolite of 17beta-estradiol, has been shown to selectively induce apoptosis in a number of cancer cell lines, but not in normal cells. The mechanism of 2-methoxyestradiol-induced apoptosis appears to vary considerably in different cell lines examined. In the present study, we systematically analyzed the mechanisms of 2-methoxyestradiol-induced apoptosis in the estrogen receptor-negative MDA-MB-435s human breast cancer cells. We found that 2-methoxyestradiol induced the activation of JNK, ERK, and p38 MAPKs. 2-methoxyestradiol-induced JNK activation was associated with the induction of apoptosis through the mitochondrial pathways as a result of increased phosphorylation (inactivation) of the anti-apoptotic Bcl-2 and Bcl-xL proteins. In comparison, 2-methoxyestradiol-induced activation of ERK and p38 in these cells was found to have a protective effect against 2-MeO-E(2)-induced apoptosis. Consistent with this observation, the presence of pharmacological inhibitor of ERK or p38 enhanced 2-methoxyestradiol-induced apoptosis. Mechanistically, inhibition of ERK and p38 activity was associated with activation of various caspases and PARP cleavage, and it also stabilized the pro-apoptotic proteins Bax and Bim, thereby preventing them from degradation during 2-methoxyestradiol treatment. These results suggest that ERK and p38 MAPKs may serve as viable targets for the sensitization of human breast cancer cells to 2-methoxyestradiol-induced apoptosis.     

维生素E琥珀酸酯通过酸性神经磷脂酶-神经酰胺诱导胃癌细胞凋亡的机制研究

目的：探讨维生素E琥珀酸酯（VES）通过酸性神经磷脂酶（ASmase）-神经酰胺（Cer）诱导人胃癌细胞凋亡过程中对死亡受体信号通路以及氧化应激反应的影响。方法：将人胃癌SGC-7901细胞分为对照组、ASMase抑制剂地昔帕明（DES）组（12.5 μmol/L）、VES处理组（20 μg/mL）以及VES+DES组（12.5 μmol/L的DES预处理细胞2 h后加入20 μg/mL的VES）。在不同时间点用ELISA法测定ASMase活性,免疫荧光法检测Cer表达情况;处理24 h时用DAPI染色检测细胞凋亡率,Western blot法检测死亡受体信号通路蛋白Fas、死亡受体5（DR5）、caspase-8、caspase-9和PARP的蛋白表达水平,流式细胞术检测活性氧簇（ROS）水平。结果：VES处理细胞后ASMase活性在1.5 h时开始增加,同时Cer开始在细胞膜上的聚集增加,两者的表达水平分别在3、6 h时达到高峰,24 h的细胞凋亡率为（41.00±1.00）%;抑制ASMase活性显著降低了VES处理组细胞的凋亡率,Fas、DR5、c-caspase-8、c-caspase-9、c-PARP蛋白表达水平和氧化应激水平（P均 < 0.01）。结论：ASMase/Cer可能是VES通过死亡受体信号通路及氧化应激反应促进胃癌细胞发生凋亡的上游因子。     

Desferri-exochelin induces death by apoptosis in human breast cancer cells but does not kill normal breast cells

A major goal of cancer chemotherapy is the identification of cytotoxic compounds that are highly selective for cancer cells. We describe here one such compound – a novel iron chelator, desferri-exochelin 772SM. This desferri-exochelin has unique chemical and pharmacological properties, including extremely high iron binding affinity, the capacity to block iron-mediated redox reactions, and lipid solubility which enables it to enter cells rapidly. At low concentrations, this desferri-exochelin kills T47D-YB and MCF-7 human breast cancer cells by inducing apoptosis, but only reversibly arrests the growth of normal human mammary epithelial cells without cytotoxicity. Since iron-loaded exochelin is ineffective, iron chelation accounts for the efficacy of desferri-exochelin. For both the killing of breast cancer cells and the growth arrest of normal breast epithelial cells, desferri-exochelin was effective at much lower concentrations than the lipid-insoluble iron chelator deferoxamine, which has shown only limited potential as an anti-cancer agent. Growth arrest of progesterone receptor positive T47D-YB cells with the progestin R5020 transiently protects them from the cytotoxic effects of desferri-exochelin, but the cells are killed after cell growth resumes. Similarly, MCF-7 cells arrested with the estrogen antagonist ICI182780 are transiently resistant to killing by desferri-exochelin. Thus the desferri-exochelin is cytotoxic only to actively growing tumor cells. Since desferri-exochelin 772SM can selectively and efficiently destroy proliferating cancer cells without damaging normal cells, it may prove useful for the treatment of cancer.     

Bortezomib-induced apoptosis in cultured pancreatic cancer cells is associated with ceramide production

Purpose

The proteasome inhibitor bortezomib (PS-341) has displayed significant efficiency against pancreatic cancer cells. However, the underlying mechanisms are not fully understood. Here, we tested if ceramide production was involved in the bortezomib’s effect.

Methods

Two transformed pancreatic cancer cell lines (PANC-1 and Mia) and the primary pancreatic cancer cells were used. Cell death was analyzed by MTT viability assay and trypan blue staining. Cell apoptosis was analyzed by Histone DNA-ELISA assay and Annexin V FACS. Western blots were used to test signal protein changes. The cellular ceramide level after bortezomib treatment was also determined.

Results

In cultured pancreatic cancer cells, bortezomib increased cellular ceramide production to promote cell apoptosis. The ceramide de novo synthase inhibitor fumonisin B1 (F-B1) suppressed bortezomib-induced ceramide production and apoptosis, while exogenously added C6-ceramide facilitated bortezomib-induced pancreatic cancer cell death. Meanwhile, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), the inhibitor of glucosylceramide synthetase as well as the sphingosine kinase 1 inhibitors (SKI-II and SKI-IV), facilitated bortezomib-induced ceramide production and subsequent cell apoptosis. Further, bortezomib-induced pro-apoptotic c-Jun N-terminal kinase (JNK) activation was also associated with ceramide production. JNK activation by bortezomib was suppressed by F-B1, but was enhanced by SKI-II and PDMP in pancreatic cancer cells. Finally, C6-ceramide, SKI-II, and PDMP dramatically enhanced bortezomib-induced cytotoxicity in primary cultured pancreatic cancer cells.

Conclusions

We found that bortezomib-induced apoptosis was associated with ceramide production in primary and transformed pancreatic cancer cells.     

Buthionine sulfoximine sensitizes antihormone-resistant human breast cancer cells to estrogen-induced apoptosis

Introduction  

Estrogen deprivation using aromatase inhibitors is one of the standard treatments for postmenopausal women with estrogen receptor (ER)-positive breast cancer. However, one of the consequences of prolonged estrogen suppression is acquired drug resistance. Our group is interested in studying antihormone resistance and has previously reported the development of an estrogen deprived human breast cancer cell line, MCF-7:5C, which undergoes apoptosis in the presence of estradiol. In contrast, another estrogen deprived cell line, MCF-7:2A, appears to have elevated levels of glutathione (GSH) and is resistant to estradiol-induced apoptosis. In the present study, we evaluated whether buthionine sulfoximine (BSO), a potent inhibitor of glutathione (GSH) synthesis, is capable of sensitizing antihormone resistant MCF-7:2A cells to estradiol-induced apoptosis.     

Recombinant Apoptin gene retrovirus induces apoptosis in human breast cancer cells 435

Objective  To construct recombinant Apoptin gene (vp3) retrovirus pLVP3 and to study its apoptosis inducing effect on human breast cancer cells 435 as well as to discuss its mechanism in vitro and in vivo. Methods   vp3 gene was cloned and recombinated into retrovirus vector pLP-LNCX-VP3 (pLVP3) at loxP site, which was transformed into package cell line PT67 and then into NIH3T3 cells for titer assay. The human breast cancer cell line 435 was infected with retrovirus pLVP3, and then MTT assay and Western blotting were adopted to detect cellular proliferation and Apoptin protein expression. Forty-eight hours after infection flow cytometry (FCM) was used for apoptosis detection and Surface Enhanced Laser Desorption Ionization-Time of Flight-Mass Spectrometry (SELDI-TOF-MS) was used for protein profile assay. Nude mice model of human breast cancer cells 435 was set up to observe the tumor inhibiting rates of pLVP3, and TUNEL assay was used to detect tumor apoptosis as well as real-time PCR for vp3 gene expression. Results  Recombinant plasmid pLVP3 was successfully constructed. Virus titer reached to 5×10⁸ pfu/ml in the PT67 culture supernatant. Forty-eight hours after infection, cellular inhibition rate was 65.1% in MTT assay, higher than that in blank control (P<0.05) and Apoptin protein expressed more in test group in Western blotting. FCM assay showed apoptotic peaks with a percentage of 15.42%. SELDI-TOF-MS findings suggested that two protein peaks, M_2544.1+H and M_3712.4+H, were statistically different between infection group and control group (P<0.05). The tumor inhibition rates in pLVP3 group were 65.52% and 68.23%, much higher than that of control group (t=4.06, P<0.01). TUNEL assay findings showed that positive yellow stains were seen in pLVP3 retrovirus group and 5-FU positive control group without difference (t1=1.05, t2=0.84, P>0.05). Conclusion  The experiment demonstrated that vp3 could induce apoptosis in tumor cells in vivo and in vitro, which laid a basis for further study on molecular mechanism of tumor cell apoptosis induced by Apoptin and provided valuable reference for tumor gene therapy. Contributed equally to this work.     

MYB suppresses differentiation and apoptosis of human breast cancer cells

Introduction  

MYB is highly expressed in estrogen receptor positive (ER + ve) breast tumours and tumour cell lines. We recently demonstrated that MYB is essential for the proliferation of ER + ve breast cancer cells, and have now investigated its role in mammary epithelial differentiation.     

Curcumin mediates ceramide generation via the de novo pathway in colon cancer cells

A wealth of evidence supports the notion that curcumin, a phytochemical present in turmeric, is a potent chemopreventive agent for colon cancer. Its mechanism of action remains incompletely understood. Here we report that curcumin's apoptosis-inducing effects in colon cancer cell lines are accompanied by robust ceramide generation. This occurs through de novo synthesis as the increase in ceramide could be attenuated by pre-incubation of the cells with myriocin, and no changes were observed in sphingomyelin levels, or in either acidic or neutral sphingomyelinase activities. Furthermore, cell death could in part be reversed by myriocin, indicating, for the first time, that endogenous ceramide generation by this agent contributes towards its biological activity. We then investigated the role of reactive oxygen species (ROS) in this phenomenon and demonstrated that curcumin induced robust oxidant generation in the cell lines tested, and its reversal by N-acetylcysteine, completely attenuated apoptosis. We next confirmed that curcumin could activate c-jun N-terminal kinase (JNK) and that its modulation could reverse cell death; however, this intervention could not block ceramide generation, or ROS production. Conversely, however, the inhibition of ROS using N-acetylcysteine led to an inhibition of JNK activation. Hence, we conclude that curcumin induces apoptosis via a ROS-associated mechanism that converges on JNK activation, and to a lesser extent via a parallel ceramide-associated pathway.