昆明山海棠根部水提液诱导HL-60细胞凋亡及对c-Myc基因表达的调控

目的 研究昆明山海棠Tripterygium hypoglaucum(Celastraceae)(THH)根部水提液对早幼粒白血病HL-60细胞凋亡的诱导作用及机制。方法 通过特征性的形态学观察，Annexin—V标记，以及流式细胞仪检测中次G1峰的形成确定THH的诱导细胞凋亡作用；应用Western Blotting研究THH对c—Myc蛋白表达的影响。结果 THH根部水提液可诱导早幼粒白血病HL-60细胞凋亡。在浓度高于18μg／mL时，THH对HL-60细胞的诱导凋亡作用呈现浓度与时间的相关性。在THH处理2～4h后，癌基因蛋白c—Myc表达降低99％以上。结论 THH抑制了c—Myc蛋白的翻译或后翻译过程，从而降低了c—Myc在细胞周期运转中的作用，导致大量细胞凋亡．     

Morin enhances auranofin anticancer activity by up‐regulation of DR4 and DR5 and modulation of Bcl‐2 through reactive oxygen species generation in Hep3B human hepatocellular carcinoma cells

Evidence suggests that auranofin (AF) exhibits anticancer activity by inhibiting thioredoxin reductase (TrxR). Here, in this study, we have investigated the synergistic effects of AF and morin and their mechanism for the anticancer effects focusing on apoptosis in Hep3B human hepatocellular carcinoma cells. We assessed the anticancer activities by annexin V/PI double staining, caspase, and TrxR activity assay. Morin enhances the inhibitory effects on TrxR activity of AF as well as reducing cell viability. Annexin V/PI double staining revealed that morin/AF cotreatment induced apoptotic cell death. Morin enhances AF‐induced mitochondrial membrane potential (ΔΨm) loss and cytochrome c release. Further, morin/AF cotreatment upregulated death receptor DR4/DR5, modulated Bcl‐2 family members (upregulation of Bax and downregulation of Bcl‐2), and activated caspase‐3, ‐8, and ‐9. Morin also enhances AF‐induced reactive oxygen species (ROS) generation. The anticancer effects results from caspase‐dependent apoptosis, which was triggered via extrinsic pathway by upregulating TRAIL receptors (DR4/DR5) and enhanced via intrinsic pathway by modulating Bcl‐2 and inhibitor of apoptosis protein family members. These are related to ROS generation. In conclusion, this study provides evidence that morin can enhance the anticancer activity of AF in Hep3B human hepatocellular carcinoma cells, indicating that its combination could be an alternative treatment strategy for the hepatocellular carcinoma.     

Anticancer activity of isoobtusilactone A from Cinnamomum kotoense: involvement of apoptosis, cell-cycle dysregulation, mitochondria regulation, and reactive oxygen species

In this study, we investigate the anticancer effect of isoobtusilactone A (IOA), a constituent isolated from the leaves of Cinnamomum kotoense, on human non-small cell lung cancer (NSCLC) A549 cells. IOA was found to induce the arrest of G2-M phase, induce apoptosis, increase sub-G1, and inhibit the growth of these cells. Further investigation revealed that IOA's blockade of the cell cycle was associated with increased levels of p21/WAF1, p27 (kip1), and p53. In addition, IOA triggered the mitochondrial apoptotic pathway, as indicated by an increase in Bax/Bcl-2 ratios, resulting in a loss of mitochondrial membrane potential, release of cytochrome c, activation of caspase-9 and caspase-3, and cleavage of PARP. We also found the generation of reactive oxygen species (ROS) to be a critical mediator in IOA-induced inhibition of A549 cell growth. In antioxidant and NO inhibitor studies, we found that by pretreating A549 cells with either N-acetylcystenine (NAC), catalase, mannitol, dexamethasone, trolox, or L-NAME we could significantly decrease IOA production of ROS. Moreover, using NAC to block ROS, we could significantly suppress IOA-induced antiproliferation, antimigration, and anti-invasion. Finally, we found that IOA inhibited the migration and invasion of A549 cell migration and invasion. Taken together, these results suggest that IOA has anticancer effects on A549 cells.     

Tanshinone IIA isolated from Salvia miltiorrhiza BUNGE induced apoptosis in HL60 human premyelocytic leukemia cell line

Apoptosis is a new therapeutic target of cancer research. Tanshinone IIA isolated from Salvia miltiorrhiza BUNGE, a traditional oriental medical herb, was observed to induce apoptosis in HL60 human premyelocytic leukemia cell line. Tanshinone IIA induced DNA fragmentation into the multiples of 180 bp and increased the percentage of hypodiploid cells in flow cytometry after propidium iodide (PI) staining. Tanshinone IIA-induced apoptosis is accompanied by the specific proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) and the activation of caspase-3, a major component in apoptotic cell death mechanism.     

西兰花中异硫氰酸盐诱导人肝癌细胞HepG-2凋亡作用及其机制的研究

目的：探讨西兰花中异硫氰酸盐(isothiocyanates,ITCS)诱导人肝癌细胞HepG-2凋亡过程中的作用及其可能机制。方法：不同浓度ITCS处理人肝癌细胞HepG-2,应用MTT法检测ITCS对HepG-2细胞增殖的影响；流式细胞仪检测活性氧、线粒体膜电位(Δψm)和细胞亚二倍体凋亡峰变化情况。结果：ITCS可明显抑制HepG-2细胞增殖；15,30,60,120,240 μg·mL^-1的ITCS作用于HepG-2细胞24 h后,细胞内活性氧分别为(23.1±1.8)%,(53.3±3.3)%,(57.9±2.0)%,(79.9±3.4)%,(93.4±2.6)%；Δψm分别为(94.8±5.5)%,(91.8±5.4)%,(66.0±5.6)%,(65.5±6.6)%,(44.3±2.7)%；60,120,240 μg·mL^-1的ITCS作用于HepG-2细胞48 h后可见细胞凋亡率分别为(16.6±2.8)%,(21.9±4.4)%,(70.2±5.3)%。结论：ITCS能够通过刺激HepG-2细胞内活性氧的产生,损伤线粒体,使其膜电位下降,引起HepG-2细胞凋亡。     

A flavonoid glycoside isolated from Smilax china L. rhizome in vitro anticancer effects on human cancer cell lines

The anticancer activity of eight crude extracts of Smilax china L. rhizome (SCR) against HeLa cells was assessed by MTT assay and clonogenic assay, the fraction rich in flavonoids had show good activity against HeLa cells. A bioassay-guided separation on this extract lead to the detection of kaempferol-7-O-beta-D-glucoside (KG), which belongs to flavonoid glycoside, displayed marked anticancer activity. We evaluated its in vitro cytotoxicity and antiproliferative effect in a panel of established cancer cell lines by MTT assay and clonogenic assay. KG induces A375 and HL60 cells apoptosis, which was demonstrated by morphological changes, DNA fragmentation and flow cytometric analysis. Fluorescent staining with Hoechst 33258 showed fragmentation and condensation of chromatin in the A375 and HL60 cells. Flow cytometric analysis shown that A375 and HL60 cells treated with KG resulted in the appearance of a hypodiploid peak (A0 region), probably due to the presence of apoptosing cells and/or apoptotic bodies with DNA content less than 2n. Quantitation of the hypodiploid cells shows a dose-dependent response to KG, and this result is in good accordance with that of the DNA fragmentation assay by agarose gel electrophoresis. Our results suggested that cell cycle arrest at G(1) phase and induce apoptosis as a mechanism by which KG exerts an antiproliferative effect.     

Escin sodium induces apoptosis of human acute leukemia Jurkat T cells

Escin sodium has been used in the clinic as an antioedematous, antiexudative and vasoprotective agent for many years and has shown excellent tolerability. However, little is known about its anticancer activity. This is a report for the first time that escin sodium exerts a cytotoxic effect on human acute leukemia Jurkat T cells via the induction of apoptosis rather than cell cycle arrest. Escin sodium activated the initiator caspase-8, -9, and the effector caspase-3, degraded poly (ADP-ribose) polymerase (PARP) and attenuated the expression of Bcl-2. In addition, escin sodium inhibited the growth of cancer cells in a selective manner with Jurkat cells most sensitive to it. Taken together, the data show that escin sodium possesses potent apoptogenic activity toward human acute leukemia Jurkat T cells.     

Dickkopf (DKK1) ���˰�Ѫ��ϸ�������ĵ��ػ����о�

??OBJECTIVE To explore the regulation of Dickkopf (DKK1) on apoptosis of human acute leukemia U937 cell. METHODS The reactive oxygen species(ROS) was assessed with DCFH-DA fluorescence probe; the mitochondria membrane potential was assessed with JC-1 staining method, apoptosis rate was detected by AnnexinV - FITC/ PI double staining, Western-blot was used to analyse the expression of related protein. RESULTS Dickkopf can rise the ROS and reduce the mitochondria membrane potential, meanwhile up-regulate the expressing of BAX, down-regulating expressing of Mcl-1 and Bcl-2. CONCLUSION Dickkopf can promote apoptosis of human acute leukemia U937 cell.     

Xanthohumol induces apoptosis in human malignant glioblastoma cells by increasing reactive oxygen species and activating MAPK pathways

The effect of the biologically active prenylated chalcone and potential anticancer agent xanthohumol (1) has been investigated on apoptosis of the T98G human malignant glioblastoma cell line. Compound 1 decreased the viability of T98G cells by induction of apoptosis in a time- and concentration-dependent manner. Apoptosis induced by 1 was associated with activation of caspase-3, caspase-9, and PARP cleavage and was mediated by the mitochondrial pathway, as exemplified by mitochondrial depolarization, cytochrome c release, and downregulation of the antiapoptotic Bcl-2 protein. Xanthohumol induced intracellular reactive oxygen species (ROS), an effect that was reduced by pretreatment with the antioxidant N-acetyl-L-cysteine (NAC). Intracellular ROS production appeared essential for the activation of the mitochondrial pathway and induction of apoptosis after exposure to 1. Oxidative stress due to treatment with 1 was associated with MAPK activation, as determined by ERK1/2 and p38 phosphorylation. Phosphorylation of ERK1/2 and p38 was attenuated using NAC to inhibit ROS production. After treatment with 1, ROS provided a specific environment that resulted in MAPK-induced cell death, with this effect reduced by the ERK1/2 specific inhibitor PD98059 and partially inhibited by the p38 inhibitor SB203580. These findings suggest that xanthohumol (1) is a potential chemotherapeutic agent for the treatment of glioblastoma multiforme.     

芦荟大黄素对Jurkat T细胞增殖和凋亡的作用及机制

目的 研究芦荟大黄素对Jurkat T细胞的增殖抑制和凋亡诱导作用,并探讨可能的机制.方法 应用细胞计数测定增殖,Hoechst/PI双染法及DNA片断化分析细胞凋亡特征,流式细胞术检测细胞周期分布.进一步应用二氢乙锭荧光染色测量活性氧产物,Western blotting检测胞浆细胞色素C(Cyt-c)的量,比色法检测caspase-3和caspase-9活性.结果 芦荟大黄素剂量和时间依赖性地抑制Jurkat T细胞增殖.芦荟大黄素诱发细胞核皱缩,核DNA片断化,细胞周期出现亚G1期凋亡峰,且停滞于G2/M期.芦荟大黄素介导细胞内活性氧水平显著提高,线粒体释放Cyt-c量显著增加,caspase-3和caspase-9活性显著增强.结论 芦荟大黄素剂量依赖性地抑制Jurkat T细胞增殖并诱导其凋亡.诱导凋亡的机制中,包括增加活性氧产物和线粒体损伤途径.     

JR6, a new compound isolated from Justicia procumbens, induces apoptosis in human bladder cancer EJ cells through caspase-dependent pathway

Ethnopharmacological relevance

Numerous efforts have been conducted in searching for effective agents against cancer, in particular from herbal medicines. Justicia procumbens is a traditional herbal remedy which was produced in the south-western and southern provinces of China and Taiwan province used to treat fever, pain, and cancer. Here, we identified a new compound 6′-hydroxy justicidin A (JR6) from Justicia procumbens, which showed obvious anti-cancer effects.

Materials and methods

The cytotoxicity activity was assayed using MTT and SRB. Intracellular ROS visualization and quantification were acquired by using a laser scanning confocal microscopy. Apoptosis was measured using a propidium iodide (PI) apoptosis detection kit by flow cytometry. Activation of caspases (caspase-3, caspase-8, and caspase-9) was evaluated respectively using GloMax luminescence detector and Caspase-Glo 3,8,9 assay kits. Loss of mitochondrial membrane potential was observed by microscopy using JC-1 dye. Quantitative real-time PCR analysis was employed to detect the expression of protein associated with cell death.

Results

JR6 remarkably inhibited growth in human bladder cancer EJ cells by decreasing cell proliferation, reduced the SOD activity, increased the content of reactive oxygen species (ROS), and induced apoptosis. Activation of caspase-8, caspase-9, and the subsequent activation of caspase-3 indicated that JR6 may be inducing intrinsic and extrinsic apoptosis pathways. Caspase-3, caspase-8, and caspase-9 inhibition rendered this extract ineffective, thus JR6-induced apoptosis is caspase-dependent. JR6 also disrupted the mitochondrial membrane potential (Δψm) and unregulated the Bax and p53 expressions in EJ cells.

Conclusion

These observations suggest that JR6 induce apoptosis through caspase-dependent pathway in human bladder cancer EJ cells, emphasizing the importance of this traditional medicine and thus presents a potential novel alternative to bladder cancer therapy.     

Neferine,an alkaloid from lotus seed embryo targets HeLa and SiHa cervical cancer cells via pro‐oxidant anticancer mechanism

Apoptosis and autophagy are important processes that control cellular homeostasis and have been highlighted as promising targets for novel anticancer drugs. This study aims to investigate the inhibitory effects and mechanisms of Neferine (Nef), an alkaloid from the lotus seed embryos of Nelumbo nucifera (N. nucifera), as a dual inducer of apoptosis and autophagy through the reactive oxygen species (ROS) activation in cervical cancer cells. Nef and N. nucifera extract suppressed the cell viability of HeLa and SiHa cells in a dose‐dependent manner. Importantly, Nef showed minimal toxicity to normal cells. Furthermore, Nef inhibited anchorage‐independent growth, colony formation and migration ability of cervical cancer cells. Nef induces mitochondrial apoptosis by increasing pro‐apoptotic protein bax, cytochrome‐c, cleaved caspase‐3 and caspase‐9, poly‐ADP ribose polymerase (PARP) cleavage, DNA damage (pH₂AX) while downregulating Bcl‐2, procaspase‐3 and procaspase‐9, and TCTP. Of note, apoptotic effect by Nef was significantly attenuated in the presence of N‐acetylcysteine (NAC), suggesting pro‐oxidant activity of this compound. Nef also promoted autophagy induction through increasing beclin‐1, atg‐4, atg‐5 and atg‐12, LC‐3 activation, and _P62/SQSTM1 as determined by western blot analysis. Collectively, these results demonstrate that Nef is a potent anticancer compound against cervical cancer cells through inducing apoptosis and autophagic pathway involving ROS.     

Fucoidan from Undaria pinnatifida induces apoptosis in A549 human lung carcinoma cells

Fucoidan, a sulfated polysaccharide, has various biological activities, such as anticancer, antiangiogenic and antiinflammatory effects; however, the mechanisms of action of fucoidan on anticancer activity have not been fully elucidated. The anticancer effects of fucoidan from Undaria pinnatifida on A549 human lung carcinoma cells were examined. Treatment of A549 cells with fucoidan resulted in potent antiproliferative activity. Also, some typical apoptotic characteristics, such as chromatin condensation and an increase in the population of sub-G1 hypodiploid cells, were observed. With respect to the mechanism underlying the induction of apoptosis, fucoidan reduced Bcl-2 expression, but the expression of Bax was increased in a dose-dependent manner compared with the controls. Furthermore, fucoidan induced caspase-9 activation, but decreased the level of procaspase-3. Cleavage of poly-ADP-ribose polymerase (PARP), a vital substrate of effector caspase, was found. The study further investigated the role of the MAPK and PI3K/Akt pathways with respect to the apoptotic effect of fucoidan, and showed that fucoidan activates ERK1/2 in A549 cells. Unlike ERK1/2, however, treatment with fucoidan resulted in the down-regulation of phospho-p38 expression. In addition, fucoidan resulted in the down-regulation of phospho-PI3K/Akt. Together, these results indicate that fucoidan induces apoptosis of A549 human lung cancer cells through down-regulation of p38, PI3K/Akt, and the activation of the ERK1/2 MAPK pathway.     

Induction of apoptosis in HL-60 cells through the ROS-mediated mitochondrial pathway by ramentaceone from Drosera aliciae

Ramentaceone (1) is a naphthoquinone constituent of Drosera aliciae that exhibits potent cytotoxic activity against various tumor cell lines. However, its molecular mechanism of cell death induction has still not been determined. The present study demonstrates that 1 induces apoptosis in human leukemia HL-60 cells. Typical morphological and biochemical features of apoptosis were observed in 1-treated cells. Compound 1 induced a concentration-dependent increase in the sub-G1 fraction of the cell cycle. A decrease in the mitochondrial transmembrane potential (ΔΨm) was also observed. Furthermore, 1 reduced the ratio of anti-apoptotic Bcl-2 to pro-apoptotic Bax and Bak, induced cytochrome c release, and increased the activity of caspase 3. The generation of reactive oxygen species (ROS) was detected in 1-treated HL-60 cells, which was attenuated by the pretreatment of cells with a free radical scavenger, N-acetylcysteine (NAC). NAC also prevented the increase of the sub-G1 fraction induced by 1. These results indicate that ramentaceone induces cell death through the ROS-mediated mitochondrial pathway.     

中医药基于B细胞淋巴瘤-2/Bax基因表达对白血病细胞凋亡调控影响的研究进展

细胞凋亡调控异常是白血病发生及病情进展的重要原因之一,其中,抗凋亡蛋白B细胞淋巴瘤-2（Bcl-2）/白血病蛋白和促凋亡蛋白Bax对于白血病细胞凋亡的调控起着关键作用。相对于经典的化学药物治疗,中医药治疗白血病具有不可替代的优越性,许多单味中药、中药有效成分和中药复方都有很强的抗肿瘤活性,近年来中医药对于白血病Bcl-2和Bax基因表达影响机制的探究成为了医学领域的研究热点。     

Effect of Orostachys japonicus on cell growth and apoptosis in human hepatic stellate cell line LX2

Orostachys japonicus (O. japonicus), used to treat diseases such as various cancers, gastric ulcers, fever, hepatitis, arthritis, eczema, for hemostasis, and intoxication in folk medicine, has been an important constituent in many herbal formulae. We demonstrated that the water extract of O. japonicus led to growth inhibition of LX2 cells by inducing apoptosis through the caspase activation, related to the MAPK pathway. O. japonicus inhibited proliferation of LX2 cells in a dose- and time-dependent manner, increased the apoptosis fraction at cell cycle progression with an accompanying DNA fragmentation, and resulted in a significant decrease in Bcl-2 and an increase in Bax mRNA levels. Exposure of LX2 cells to O. japonicus induced caspase-3 activation, however when the LX2 cells were also treated with the pan-caspase inhibitor Z-VAD-FMK and the caspase-3 inhibitor Z-DEVE-FMK, apoptosis was blocked. O. japonicus inhibited anti-apoptotic Mcl-1 protein and MEK/ERK phosphorylation in LX2 cells. The results indicate that O. japonicus inhibits the cell growth of LX2 cells by inducing apoptosis through caspase activity. O. japonicus down-regulated Mcl-1 protein levels and inhibited the phosphorylation of MEK/ERK, suggesting that it mediates cell death in LX2 cells through the down-regulation of Mcl-1 protein via a MEK/ERK-independent pathway.     

Qian-Kun-Nin, a Chinese herbal medicine formulation, attenuates mitochondrial oxidant stress in cardiomyocytes

Qian-Kun-Nin is a Chinese herbal medicine formulation used for several indications, including the treatment of cardiovascular diseases. This formulation contains herbs which possess antioxidant properties. In this study, Qian-Kun-Nin's ability to confer protection to cardiomyocytes against reactive oxygen species (ROS) generated during mitochondrial electron transport inhibition was tested. The intracellular fluorescent probe 2',7'-dichlorofluorescin diacetate (DCFH-DA, sensitive to H(2)O(2) and hydroxyl radicals) was used to assess intracellular ROS, and propidium iodide (PI) was used to assess viability in cultured chick embryonic cardiomyocytes. Qian-Kun-Nin significantly attenuated oxidation of DCFH in cells exposed to the mitochondrial site III inhibitor, antimycin A, consistent with a decrease in oxidative stress. These attenuated oxidant levels were associated with improved cell survival. After antimycin A exposure, Qian-Kun-Nin decreased cell death from 51. 6+/-3.3% in untreated cells to 27.3+/-3.8% in treated cells at 2 h. We conclude that Qian-Kun-Nin attenuates oxidant stress and protects cells from lethal oxidant damage during mitochondrial electron transport inhibition, and thus its therapeutic potential in treating cardiovascular diseases may relate to its antioxidant properties.     

三棱针点刺放血结合运动疗法治疗中风后遗症66例

目的探讨三棱针点刺放血治疗中风后遗症疗效。方法从2003年3月～2007年12月,在本院门诊及住院收治的例中风后遗症的患者进行分析,采用随机抽取的方法对患者进行分组,治疗组用中医三棱针点刺放血治疗、运动疗法综合治疗照组用康复训练治疗,2个月为一个疗程,参照中医诊疗规范,经医学统计学处理评定疗效,并用2年～6个月对其中的66例进随访观察疗效。结果治疗组疗效优于对照组,其结果令人满意。结论三棱针点刺放血具有开窍泻热、活血化瘀、疏通经络用,对中风后遗症治疗有显著疗效。     

Neuroprotective effects of Hypericum perforatum on trauma induced by hydrogen peroxide in PC12 cells

The standard extracts of Hypericum perforatum L. (SEHP), a well-known medicinal plant, are used for the treatment of depression, exhibited upgrading and significant protective effects on the trauma of PC12 cells induced by 200 microM H2O2 in a dose-dependent manner within 24-hour treatment. Cell viability was assessed by the MTT method, and in situ cellular hydrogen peroxide (H2O2)-induced oxidative stress was examined by measurement of reactive oxygen species (ROS) formation using CDCFH procedures. Intra- and extra-cellular ROS levels decreased significantly to 71.9% and 50.0% of the control at a moderate concentration of 20 microg/ml, respectively, suggesting that SEHP could easily enter the cells and play important roles in reducing ROS levels. Our results were proved by detection of DNA fragmentation and inspection of cell morphology of PC12 cells. SEHP can obviously block DNA fragmentation and prevent the cells from shrinking and turning round of H2O2-induced apoptosis in PC12 cells at concentrations of 10 approximately 100 microg/ml. This data suggests SEHP may be a candidate for application in neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease.     

In Vitro culture studies of FlorEssence on human tumor cell lines

FlorEssence (FE) is an herbal tea widely used by patients to treat chronic conditions in North America, particularly cancer patients during chemo- and radiation therapy. Although individual components of FE have antioxidant, antiestrogenic, immunostimulant and antitumor properties, in vitro evidence of anticancer activity for the herbal tea itself is still lacking. We studied the antiproliferative effect of FE on MCF7 and MDA-MB-468 human breast cancer, and Jurkat and K562 leukemia cell lines. We found that FE significantly inhibited the proliferation of both breast and leukemia cells in vitro only at high concentrations, with 50% inhibition of MDA-MB-468 cells at about 1[sol ]20 dilution, Jurkat cells at about 1[sol ]10 dilution and MCF7 and K562 cells at less than 1[sol ]10 dilution. Flow cytometry analysis showed that treatment with a high concentration of FE induced G2[sol ]M arrest in MCF7 and Jurkat cells, with also an increased SubG0[sol ]G1 fraction in MCF7 cells. MDA-MB-468 cells showed a significantly increased Sub G0[sol ]G1 fraction after treatment with 1[sol ]10 dilution of FE while the cell cycle of K562 was unaffected. When MCF7 and MDA-MB-468 breast cancer cells were treated with a combination of FE with either paclitaxel or cisplatin, results showed that only the combination of 1[sol ]20 dilution of FE with 0.5 microM cisplatin resulted in a small but significantly higher MCF7 cell survival than 0.5 microM cisplatin treatment alone. FE at 1[sol ]20 and 1[sol ]50 dilutions did not affect the antiproliferative properties of these two commonly used chemotherapeutic agents. The results suggest that FE at high concentrations show differential inhibitory effect on different human cancer cell lines. Further studies are needed to assess the biological activities of FE.