Plumbagin-induced Apoptosis in Human Prostate Cancer Cells is Associated with Modulation of Cellular Redox Status and Generation of Reactive Oxygen Species

PURPOSE: To investigate the mechanism of human prostate cancer cell growth inhibition by plumbagin, a constituent of the widely used medicinal herb Plumbago zeylanica L. MATERIALS AND METHODS: Cell viability was determined by trypan blue dye exclusion assay. Apoptosis induction was assessed by analysis of cytoplasmic histone-associated DNA fragmentation. Cell cycle distribution and generation of reactive oxygen species (ROS) were determined by flow cytometry. The effect of plumbagin treatment on cellular redox status was determined by analysis of intracellular glutathione (GSH) levels and expression of genes involved in ROS metabolism. RESULTS: Plumbagin treatment decreased viability of human prostate cancer cells (PC-3, LNCaP, and C4-2) irrespective of their androgen responsiveness or p53 status. Plumbagin-mediated decrease in cell viability correlated with apoptosis induction, which was accompanied by ROS generation and depletion of intracellular GSH levels. Pretreatment of cells with the antioxidant N-acetylcysteine inhibited plumbagin-mediated ROS generation and apoptosis. Plumbagin treatment also resulted in altered expression of genes responsible for ROS metabolism, including superoxide dismutase 2 (Mn-SOD). CONCLUSION: The present study points towards an important role of ROS in plumbagin-induced apoptosis in human prostate cancer cells.     

青藤碱通过调控NF-kB信号通路抑制胰腺癌Capan-1细胞增殖并诱导细胞凋亡

目的 探讨青藤碱对胰腺癌Capan-1细胞增殖、凋亡的影响及机制。方法 CCK8法检测细胞活力,DAPI/TUNEL双染检测细胞凋亡,流式细胞术Annexin V-FITC/PI染色法检测细胞凋亡率,Western blotting检测cleaved caspase-3蛋白、细胞核NF-κB蛋白表达。结果 CCK8结果表明青藤碱抑制胰腺癌Capan-1细胞增殖,并具有时间、浓度依赖性;DAPI/TUNEL染色结果表明,在青藤碱作用下,胰腺癌Capan-1细胞凋亡增多;流式细胞术Annexin V-FITC/PI结果表明,在青藤碱作用下,胰腺癌Capan-1细胞凋亡率增高;Western blotting结果表明,在青藤碱作用下,胰腺癌Capan-1细胞cleaved caspase-3表达升高,细胞核NF-kB表达降低。TNF-α降低青藤碱对胰腺癌Capan-1细胞增殖抑制作用、凋亡诱导作用和cleaved caspase-3表达的上调作用。结论 青藤碱通过调控NF-kB信号通路抑制胰腺癌Capan-1细胞增殖并诱导细胞凋亡。     

Cellular Responses to Cancer Chemopreventive Agent D,L-Sulforaphane in Human Prostate Cancer Cells Are Initiated by Mitochondrial Reactive Oxygen Species

Purpose  Present study was undertaken to elucidate the mechanism of cellular responses to D,L-sulforaphane (SFN), a highly promising cancer chemopreventive agent. Methods  Mitochondrial DNA deficient Rho-0 variants of LNCaP and PC-3 cells were generated by culture in the presence of ethidium bromide. Apoptosis was assessed by analysis of cytoplasmic histone-associated DNA fragmentation and activation of caspase-3. Immunoblotting was performed to determine the expression of apoptosis- and cell cycle-regulating proteins. Generation of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and cell cycle distribution were measured by flow cytometry. Results  The Rho-0 variants of LNCaP and PC-3 cells were significantly more resistant to SFN-induced ROS generation, apoptotic DNA fragmentation, disruption of MMP, cytosolic release of cytochrome c, and G2/M phase cell cycle arrest compared with corresponding wild-type cells. SFN-induced autophagy, which serves to protect against apoptotic cell death in PC-3 and LNCaP cells, was also partially but markedly suppressed in Rho-0 variants compared with wild-type cells. SFN statistically significantly inhibited activities of mitochondrial respiratory chain enzymes in LNCaP and PC-3 cells. Conclusion  These results indicate, for the first time, that mitochondria-derived ROS serve to initiate diverse cellular responses to SFN exposure in human prostate cancer cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This investigation was supported in part by the USPHS grants CA115498 and CA101753 (to S.V.S.), awarded by the National Cancer Institute, and grant 2718/P01/2007/32 (to A.H-A.), awarded by the Polish Ministry of Science and Higher Education. Dong Xiao and Anna A. Powolny contributed equally.     

前列腺癌细胞内冬凌草甲素的浓度动态变化

建立了高效液相色谱法测定人前列腺癌PC-3细胞内的冬凌草甲素.采用C<,18>色谱柱,以甲醇-水(53:47)为流动相,检测波长238 nm.细胞内冬凌草甲素在10～300 ng/ml浓度范围内线性关系良好,绝对回收率为77.79%～81.32%,RSD为3.29%～4.09%;日内和日间RSD均小于8%.人前列腺癌PC-3细胞与冬凌草甲素溶液或冬凌草甲素纳米粒孵育后,采用该法测定胞内药物浓度动态变化过程.结果表明,纳米粒可显著提高细胞内冬凌草甲素的聚集并减慢其消除.     

槲皮素诱导人结肠癌细胞HT-29凋亡的机制研究

目的 探讨槲皮素在体外对人结肠癌HT-29细胞凋亡的影响,并对其诱导机制进行研究。方法 体外常规培养HT-29细胞,随机设定对照组和高、中、低剂量（32,16,8 μg·mL^-1）槲皮素组,药物干预24 h后,采用CCK-8试剂盒检测槲皮素对HT-29细胞增殖的影响,倒置显微镜观察细胞形态变化,蛋白免疫印迹法检测MKK3/6/p38信号通路蛋白表达,并同时检测凋亡相关蛋白Caspase 8、Caspase 3、Bax及Bcl-2的表达水平。结果 槲皮素干扰HT-29细胞24 h后,CCK-8实验发现槲皮素可明显抑制细胞的增殖;形态学观察发现细胞密度明显减小、细胞体积变小;免疫印迹实验发现槲皮素可诱导MKK3/6及p38的磷酸化,进而促进促凋亡蛋白Caspase8、Caspase3及Bax的表达,抑制了抗凋亡蛋白Bcl-2的表达。结论 槲皮素可诱导人结肠癌HT-29细胞凋亡,其作用机制可能与激活MKK3/6/p38信号通路有关。     

Antibody Against N‐terminal Domain of Phospholipid Scramblase 1 Induces Apoptosis in Colorectal Cancer Cells Through the Intrinsic Apoptotic Pathway

Phospholipid scramblase 1 involve in biological processes including phospholipid movement, proliferation, and apoptosis. Treatment with an antiphospholipid scramblase 1 antibody (NP1) has been demonstrated to inhibit cell proliferation in colorectal cancer. This study aimed to explore the role of NP1 treatment in the apoptosis of colorectal cancer cells. Results showed that NP1 treatment significantly increases the apoptosis of colorectal cancer cells via the activation of caspase 8, caspase 9, and caspase 3. Moreover, pretreatment with a caspase 8 inhibitor did not fully prevent the apoptotic effects of NP1. Taken together, these data indicate NP1 induces cell apoptosis primary through the intrinsic apoptotic pathway. NP1 may serve as a potential therapeutic agent.     

Natural Compound Shikonin Induces Apoptosis and Attenuates Epithelial to Mesenchymal Transition in Radiation-Resistant Human Colon Cancer Cells

Radiation resistance represents an imperative obstacle in the treatment of patients with colorectal cancer, which remains difficult to overcome. Here, we explored the anti-proliferative and migration-inhibiting properties of the natural product shikonin on a radiation-resistant human colon carcinoma cell line (SNU-C5RR). Shikonin reduced the viability of these cells in a dose-dependent manner; 38 μM of shikonin was determined as the half-maximal inhibitory concentration. Shikonin induced apoptotic cell death, as demonstrated by increased apoptotic body formation and the number of TUNEL-positive cells. Moreover, shikonin enhanced mitochondrial membrane depolarization and Bax expression and also decreased Bcl-2 expression with translocation of cytochrome c from mitochondria into the cytosol. In addition, shikonin activated mitogen-activated protein kinases, and their specific inhibitors reduced the cytotoxic effects of shikonin. Additionally, shikonin decreased the migration of SNU-C5RR cells via the upregulation of E-cadherin and downregulation of N-cadherin. Taken together, these results suggest that shikonin induces mitochondria-mediated apoptosis and attenuates epithelial-mesenchymal transition in SNU-C5RR cells.     

JPH203, an L-Type Amino Acid Transporter 1–Selective Compound,Induces Apoptosis of YD-38 Human Oral Cancer Cells

Compared to most normal cells that express L-type amino acid transporter 2, L-type amino acid transporter 1 is highly expressed in cancer cells and presumed to support their elevated growth and proliferation. This study examined JPH203, a potent and selective L-type amino acid transporter 1 inhibitor, and its ability to suppress YD-38 human oral cancer cell growth. The YD-38 cells express L-type amino acid transporter 1 with its associating protein 4F2 heavy chain, but not L-type amino acid transporter 2. JPH203 and BCH, a non-selective L-type amino acid transporter inhibitor, completely inhibited l-leucine uptake in YD-38 cells. As expected, the intrinsic affinity of JPH203 to inhibit l-leucine uptake was far more efficient than BCH. Likewise, JPH203 and BCH inhibited YD-38 cell growth, with JPH203 being superior to BCH. JPH203 up-regulated the population of apoptotic YD-38 cells through the activation of apoptotic factors, including caspases and PARP. These results suggest that the inhibition of L-type amino acid transporter 1 activity via JPH203, which may act as a potential novel anti–oral-cancer agent, leads to apoptosis by inducing the intracellular depletion of the neutral amino acids essential for cancer cell growth in YD-38 human oral cancer cells.     

EGCG通过调控DNA结合蛋白Ku70促进肺癌细胞凋亡

目的 观察表没食子儿茶素没食子酸酯[（-）-epigallocatechin-3-gallate,EGCG]对人肺腺癌细胞株的凋亡诱导作用,探讨Ku70在EGCG促肺癌细胞凋亡中的作用机制。方法 构建pSliencer 4.1-CMV-shKu70质粒干扰Ku70的表达,建立细胞系;MTT法和Annexin V/PI双染色流式细胞术检测细胞的增殖及凋亡;Western blot法检测Bax、caspase-3（17 kDa）、Ku70蛋白的表达;免疫共沉淀法检测Ku70和Bax之间的相互作用。结果 EGCG可明显抑制A549的增殖（P<0.01）,并诱导其凋亡（P<0.05）,上调Bax、caspase-3表达,下调Ku70表达。抑制Ku70的表达后,EGCG对A549细胞的抑制增殖和促进凋亡作用更加明显,进一步显著上调caspase-3的表达（P<0.05）,但是Bax的表达无明显变化;同时EGCG可以减弱Ku70-Bax之间的相互作用（P<0.05）。结论 EGCG可抑制人肺腺癌细胞增殖及诱导细胞凋亡,其机制与抑制Ku70的表达,抑制Ku70-Bax之间的相互作用,激活Bax,启动caspase级联反应有关。     

木香内酯对结肠癌细胞增殖和凋亡的影响

目的 探讨木香内酯（micheliolide,MCL）对结肠癌细胞增殖和凋亡的作用及潜在机制。方法 通过腹腔注射致癌剂氧化偶氮甲烷（azoxymethane,AOM）和饮用致炎剂葡聚糖硫酸钠（dextran sodium sulfate,DSS）构建AOM/DSS小鼠结肠癌模型,分别给予尾静脉注射2.5 mg·kg^-1顺铂、MCL 0,10,20,50 mg·kg^-1,连续30 d记录小鼠存活率。取肿瘤组织,称肿瘤质量。免疫组化检验小鼠肿瘤组织Ki67和胱天蛋白酶3（caspase-3）蛋白表达水平,Western blotting检测小鼠肿瘤组织PTEN蛋白表达量;结肠癌细胞系SW620分别用含10 µmol·L^-1顺铂、MCL 0,2.5,5,10 µmol·L^-1培养基培养,或转染siPTEN后在MCL 0和10 µmol·L^-1培养基培养。BrdU细胞增殖试验检测细胞增殖,流式细胞术检测细胞凋亡,Western blotting检测细胞增殖抗原（Ki67、PCNA）、细胞凋亡相关蛋白（cleaved caspase-3、Bcl-2、Bax）及PTEN蛋白表达量。结果 与模型组相比,MCL增加小鼠生存率,减轻肿瘤质量,增加抑瘤率,上调PTEN蛋白表达,减少Ki67阳性细胞,增加caspase-3阳性细胞（P<0.05或P<0.01）;与对照组相比,MCL降低结肠癌细胞BrdU阳性细胞百分比,下调Ki67和PCNA的表达量,增加细胞凋亡率,下调Bcl-2的表达量,上调Bax、cleaved caspase-3和PTEN的表达（P<0.05或P<0.01）;抑制PTEN表达能逆转MCL对SW620细胞增殖和凋亡的作用（P<0.01）。结论 MCL抑制结直肠癌细胞增殖并诱导细胞凋亡,这与上调PTEN表达有关。     

葫芦素B纳米脂质载体对结肠癌和乳腺癌细胞抑制及凋亡作用的研究

目的：研究葫芦素B纳米脂质载体（CuB-NLC）对结肠癌Lovo细胞,乳腺癌MCF-7细胞的体外细胞毒性。方法：结肠癌Lovo细胞,乳腺癌MCF-7细胞常规复苏、培养及传代,取处于对数生长期的细胞用于实验。采用CCK-8法检测质量浓度为0、0.0625、0.25、1.00、4.00、16.0 mg/L的CuB-NLC对结肠癌Lovo细胞,乳腺癌MCF-7细胞的毒性,流式细胞仪检测结肠癌Lovo细胞,乳腺癌MCF-7细胞的细胞周期进程及Annexin V/PI双染法检测细胞的凋亡率。结果：与葫芦素B （CuB）相比CuB-NLC对结肠癌Lovo细胞,乳腺癌MCF-7细胞生长抑制作用明显增强（P＜0.05）,且这种抑制作用随药物浓度增加而增强。CuB组及CuB-NLC组作用于结肠癌Lovo细胞、乳腺癌MCF-7细胞48 h后,与NLC空白组及DMSO对照组相比较,CuB及CuB-NLC均能够引起结肠癌Lovo细胞、乳腺癌MCF-7细胞G2/M期阻滞,同时伴有G0/G1期细胞减少, CuB-NLC组的作用更加明显,有统计学差异（P＜0.05）。Annexin V/PI双染法检测结果表明,CuB-NLC能显著诱导结肠癌Lovo细胞,乳腺癌MCF-7细胞凋亡。结论：葫芦素B纳米脂质载体能够抑制结肠癌Lovo细胞,乳腺癌MCF-7细胞的增殖,诱导细胞凋亡。     

Parasporin A13-2 of Bacillus thuringiensis Isolates from the Papaloapan Region (Mexico) Induce a Cytotoxic Effect by Late Apoptosis against Breast Cancer Cells

The protein A13-2 was obtained from Bacillus thuringiensis strains isolated from the Papaloapan watershed region (Oaxaca, Mexico). The cytotoxic activity of parasporal inclusions was studied against breast cancer cell line (MCF-7) and normal cell (human peripheral blood mononuclear cells). The MTT, the formation of reactive species, nitric oxide, free cell DNA, and the type of death cellular were assessed. The protein A13-2 shows the highest cytotoxic activity against MCF-7 (13% cell viability at 6 µg/mL), the extracellular DNA increases, and it shows no stress for reactive species or nitric oxide. Besides, the A13-2 parasporin shows no toxicity to peripheral blood mononuclear cells, and it does not generate changes in nitric oxide levels or free cell DNA. Due to that, the cytotoxic effect of A13-2 was specific for MCF-7, and it does not affect normal cells. According to microscopy and flow cytometry, A13-2 parasporin leads to the death of MCF-7 cells by late apoptosis together with necrosis and without allowing the triggering of the survival mechanisms. When analyzed together, our results show for the first time that the A13-2 protein isolated from Mexican strains of B. thuringiensis preferentially kills MCF- 7 (cancer cells) over HEK 293 and PBMC cell lines (normal cells), thus representing a promising alternative for the treatment of cancer breast.     

Cytotoxic effects of neolignans from Saururus cernuus (Saururaceae) against prostate cancer cells

In this study, five neolignans were isolated from Saururus cernuus—threo-dihydroguaiaretic acid ( 1 ), threo-austrobailignan-6 ( 2 ), threo-austrobailignan-5 ( 3 ), verrucosin ( 4 ), and saucernetin ( 5 )—and have their cytotoxic effects evaluated in prostate cancer cell lines (PC3 and DU145). Initially, using an in silico approach, tested compounds were predicted to be absorbed by the gastrointestinal tract, be able to permeate the blood–brain barrier and did not show any alert in PAINS (pan-assay structures interference). In vitro assays showed that compounds 2 , 4 , and 5 reduced cell viability of DU145 cell line at 100 μmol/L after 48 h while compounds 1 and 3 showed to be inactive at the same conditions. Furthermore, compounds 4 and 5 reduced cell number as early as in 24 h at 50 μmol/L and compound 2 showed effects at 100 μmol/L in 24 h against both cancer cell lines PC3 and DU145. Studies using flow cytometry were conducted and indicated that compound 4 induced strong necrosis and apoptosis whereas compound 5 induced strong necrosis. Otherwise, less active compound 2 did not show evidence of induction of apoptosis or necrosis, suggesting that its mechanism of action involves inhibition of cell proliferation. In conclusion, compounds 4 and 5 have been shown to be promising cytotoxic agents against prostate cancer cell lines and can be used as a starting point for the development of new drugs for the treatment of prostate cancer.     

水飞蓟宾预防和治疗前列腺癌的作用机制

目的综述了水飞蓟宾预防和治疗前列腺癌的作用机制。方法对近几年国内外文献资料进行总结和归纳。结果水飞蓟宾是水飞蓟种子提取物水飞蓟素的主要有效成分,它可以通过改变细胞周期进程,抑制有丝分裂和细胞存活信号、抑制肿瘤细胞血管生成因子的分泌、以及促使上皮细胞凋亡等方式来抑制前列腺癌细胞的分化和生长。而且水飞蓟宾对化疗药物阿霉素表现协同作用,可以缓和前列腺特异抗原的升高。结论由于水飞蓟宾几乎没有毒性,现已被用作食品添加剂以预防前列腺癌。它也已进入临床试验,可望在不久的将来成为防治前列腺癌的药品。     

Picropodophyllotoxin Inhibits Cell Growth and Induces Apoptosis in Gefitinib-Resistant Non-Small Lung Cancer Cells by Dual-Targeting EGFR and MET

Patients with non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) amplification or sensitive mutations initially respond to the tyrosine kinase inhibitor gefitinib, however, the treatment becomes less effective over time by resistance mechanism including mesenchymal-epithelial transition (MET) overexpression. A therapeutic strategy targeting MET and EGFR may be a means to overcoming resistance to gefitinib. In the present study, we found that picropodophyllotoxin (PPT), derived from the roots of Podophyllum hexandrum, inhibited both EGFR and MET in NSCLC cells. The antitumor efficacy of PPT in gefitinib-resistant NSCLC cells (HCC827GR), was confirmed by suppression of cell proliferation and anchorage-independent colony growth. In the targeting of EGFR and MET, PPT bound with EGFR and MET, ex vivo, and blocked both kinases activity. The binding sites between PPT and EGFR or MET in the computational docking model were predicted at Gly772/Met769 and Arg1086/Tyr1230 of each ATP-binding pocket, respectively. PPT treatment of HCC827GR cells increased the number of annexin V-positive and subG1 cells. PPT also caused G2/M cell-cycle arrest together with related protein regulation. The inhibition of EGFR and MET by PPT treatment led to decreases in the phosphorylation of the downstream-proteins, AKT and ERK. In addition, PPT induced reactive oxygen species (ROS) production and GRP78, CHOP, DR5, and DR4 expression, mitochondrial dysfunction, and regulated involving signal-proteins. Taken together, PPT alleviated gefitinib-resistant NSCLC cell growth and induced apoptosis by reducing EGFR and MET activity. Therefore, our results suggest that PPT can be a promising therapeutic agent for gefitinib-resistant NSCLC.     

白屈菜红碱对人结直肠癌细胞凋亡的影响及其机制研究

目的：研究白屈菜红碱（Chelerythrine, CHE）对体外结直肠癌细胞的生长抑制作用,并探讨其作用机制。方法：MTT测定结肠癌细胞存活率,应用流式细胞仪检测CHE处理后ROS的积累和细胞凋亡情况,JC-1(5,5'',6,6''-四氯-1,1'',3,3''-四乙基苯并咪唑羰花青碘化物)荧光染料法检测细胞线粒体膜电位改变,应用荧光显微镜和Western blot法验证ROS积累诱导的线粒体功能障碍。结果：CHE对HCT-116和RKO细胞发挥剂量依赖性细胞毒作用,该作用与ROS介导的凋亡蛋白的表达有关。此外,CHE能够降低伴随线粒体功能障碍的线粒体膜电位。结论：CHE通过ROS介导的线粒体功能障碍和JNKs途径抑制CRC细胞生长并诱导细胞凋亡,提示CHE可能成为潜在的治疗CRC的候选药物。     

紫杉醇对膀胱癌EJ细胞株增殖抑制及诱导凋亡的作用

目的:探讨紫杉醇体外抑制膀胱癌E J细胞株及诱导凋亡的作用。方法:体外培养膀胱癌E J细胞株,以不同浓度紫杉醇处理12~72h后,通过M TT法测定细胞生长抑制作用,采用电镜观察和DNA琼脂糖凝胶电泳检测细胞凋亡。结果:紫杉醇能抑制E J细胞的生长,并在一定剂量和时间范围内呈现出明显的时间、浓度依赖关系。电镜下凋亡细胞体积缩小,染色质浓缩致密,并沿核膜分布形成花瓣形。电泳见出现典型的凋亡DNA梯形带。结论:紫杉醇能抑制膀胱癌E J细胞株的生长,诱导凋亡可能为其抗肿瘤作用机制之一。