RI基因真核表达载体的构建及其对C6胶质瘤细胞生长的影响

目的通过构建核糖核酸酶抑制因子(ribonuclease inhibitor, RI)基因的真核表达载体——pLNCX-ri, 并转染C6神经胶质瘤细胞,探讨RI抑制肿瘤生长的作用机制.方法用Nde I / Xho从已构建的pET-ri上切下1.4 kb的RI基因片段,再构建到pLNCX上,获得真核表达载体(pLNCX-ri),采用Lipofect AMINE辅助转染大鼠C6神经胶质瘤细胞,经G418筛选获得稳定转染的细胞克隆,用Western blotting 检测RI基因的表达水平.将转染阳性的C6神经胶质瘤细胞接种于大鼠皮下,观察肿瘤的生长情况.结果在转染的C6神经胶质瘤细胞中,RI基因的表达量明显高于未转染的C6神经胶质瘤细胞,转染阳性的C6神经胶质瘤细胞在大鼠体内的成瘤潜伏期23±5.7天(对照组14±3.5天),瘤组织重量转染组1.35±0.43g比对照组2.40±0.61g(P＜0.01)明显下降,瘤组织的血管密度转染组27.2±4.31比对照组47±6.54(P＜0.01)明显减少.结论RI基因的转染对肿瘤的生长有明显抑制作用,其作用机制可能是抑制肿瘤组织血管的形成.     

神经干细胞分化与神经节细胞胶质瘤恶化相关分子研究

目的神经干细胞分化障碍是否是神经胶质瘤发生的原因尚未定论,主要是分子变化规律还未阐明.本研究旨在探讨神经干细胞与神经节细胞胶质瘤分化的相关基因,为阐明胶质瘤起源的细胞分子病因创造条件.方法对自建的神经节细胞胶质瘤恶变为多形性胶质母细胞瘤基因表达谱中的分化发育相关基因进行生物信息学聚类筛选,所得基因用RT-PCR方法检测其在体外培养神经干细胞分化过程中的表达变化.结果基因谱中的CXCR4、TN-C、GLT1、IL1-RI、EGFR-8、CDC2、Ndr3和MAPKK4共8条基因的表达变化与神经干细胞分化相关,其中GLT1、CDC2和MAPKK4基因随神经干细胞分化而表达量下降,随神经节细胞胶质瘤恶性进展而表达量增高.结论源于神经干细胞分化障碍所致的神经节细胞胶质瘤的进一步恶变(去分化)与神经干细胞分化过程中的基因GLT1、CDC2和MAPKK4等表达量呈负相关,在延伸研究中可作为胶质瘤起源细胞的分子病因的目标基因作诸如RNA干扰、基因转染或敲除等功能研究.     

银杏叶提取物通过调控NF-κB信号通路抑制神经胶质瘤U87细胞增殖和侵袭

[目的]评价银杏叶提取物(EGb761)对神经胶质瘤U87细胞的生长与侵袭的影响,并进一步分析EGb761对U87细胞作用的分子机制。[方法]体外培养人神经胶质瘤U87细胞,给予不同浓度(0、100、200、400mg/L)银杏叶提取物干预(24h、48h、72h)后,采用CCK-8技术检测EGb761对U87细胞增殖的影响。Transwell小室和流式细胞凋亡技术分析EGb761对U87细胞侵袭和凋亡的影响;Western blot检测EGb761处理后U87细胞内NF-κB与cyclin D1、 iNOS和COX-2蛋白的表达变化。[结果]银杏叶提取物可有效抑制神经胶质瘤U87细胞的增殖和侵袭,促进细胞凋亡;Western blot提示EGb761干预的U87细胞内NF-κB与cyclin D1、 iNOS、COX-2蛋白的表达均明显下调。[结论] EGb761抑制神经胶质瘤U87细胞的生长增殖和侵袭能力,其可能是通过NF-κB信号通路而发挥抗肿瘤作用的。     

Ad.RGD-ING4-PTEN对神经胶质瘤U87细胞增殖、凋亡及侵袭的影响

目的:探讨经RGD修饰的生长抑制因子4(inhibitor of growth 4,ING4)基因与第10染色体缺失与张力蛋白同源的磷酸酶基因(phosphatase and tensin homologue deleted on chromosome ten,PTEN)双基因共表达的腺病毒载体(Ad.RGD-ING4-PTEN)体外对神经胶质瘤U87细胞的增殖、凋亡及侵袭的影响.方法:以Ad.RGD-ING4-PTEN为实验组,Ad.RGD-ING4/-PTEN为单基因对照组,PBS、Ad.RGD/Ad-GFP为空白对照组,分别体外感染U87神经胶质瘤细胞.Western blotting检测目的基因ING4和PTEN在U87细胞中的表达,MTT法检测实验组病毒感染对U87细胞增殖的影响,流式细胞术及Real-time PCR法检测神经胶质瘤细胞凋亡及凋亡相关基因(Bcl-2、Bax、caspase-3、HIF-1α)表达变化,划痕实验及Transwell实验检测实验组病毒感染对U87细胞迁移及侵袭能力的影响,Real-time PCR法检测侵袭相关基因(MMP-2、MMP-9)表达变化.结果:成功检测到ING4和PTEN仅在实验组及相应单基因对照组中表达.实验组第5天细胞抑制率可达(83.1±4.6)％、凋亡率可达(40.7±4.3)％,与单基因组及空白对照组相比差异均有统计学意义(P＜0.05);实验组能明显上调U87细胞中Bax、caspase-3和下调HIF-1α、Bcl-2等细胞凋亡相关蛋白的表达(均P＜0.05),而且肿瘤侵袭相关分子MMP-2、MMP-9的表达也明显下调(均P＜0.05);实验组细胞迁移距离[(70.1±6.2)μm]和穿膜细胞数[(26.6±3.5)个]均明显减少,与单基因组及空白对照组比较差异有统计学意义(均P＜0.05).结论:与单基因腺病毒相比,Ad.RGD-ING4-PTEN双基因具有更显著的抑制U87神经胶质瘤细胞增殖、诱导其凋亡,并抑制其迁移及侵袭能力.     

Midkine对神经母细胞瘤TNB1细胞生长与分化的影响

目的:通过对中期因子(midkine,MK)在神经母细胞瘤TNB1细胞中的生物学作用进行研究,揭示该因子在肿瘤细胞生长和分化中相关分子机制.方法:1)利用神经母细胞瘤临床患者数据库,明确MK的表达与神经母细胞瘤患者生存率之间的关系.在体外细胞实验中,利用携带MKshRNA载体的慢病毒体外感染神经母细胞瘤TNB1细胞,分...     

辛伐他汀诱导神经胶质瘤细胞凋亡的实验研究

目的探讨胆固醇合成抑制剂辛伐他汀(SIM)对人神经胶质瘤U251细胞抗增殖和诱导凋亡作用,并探讨其可能的分子机制。方法用不同浓度的SIM干预U251细胞,四甲基偶氮唑盐(MTT)测定细胞增殖,流式细胞分析检测细胞周期和凋亡率、survivin和Bax蛋白表达,Hoechst33258荧光染色法观察细胞形态学改变,半定量逆转录-聚合酶链反应(RT-PCR)检测survivin和Bax mRNA表达的变化。结果①SIM能有效抑制U251细胞增殖,培养48h抑制率可达93.12%,IC_(50)为(11.33±0.16)μg/ml。②SIM诱导U251细胞凋亡,影响细胞周期进程,细胞阻滞于G_1-S期。SIM诱导U251细胞凋亡过程中随作用时间延长,survivin蛋白表达水平逐渐下降,Bax蛋白表达水平逐渐升高。③经SIM处理后,U251细胞核固缩、边集,核浆比例减小并见有凋亡小体形成。④survivin mRNA表达逐渐降低,Bax mRNA表达逐渐升高。结论SIM对人神经胶质瘤U251细胞有明显的抑制增殖和诱导凋亡作用,survivin基因表达下降、Bax基因表达升高可能为其分子机制之一。     

顺铂增加人脑胶质瘤细胞U251对TRAIL敏感性的实验研究

目的 研究顺铂在增加人脑胶质瘤细胞U251对肿瘤坏死因子相关凋亡诱导配体(TRAIL)敏感性的作用,并探讨其分子机制.方法 通过倒置荧光显微镜下观察重组腺病毒载体携带的绿色荧光蛋白的表达确定最适感染复数MOI;运用MTT法检测细胞增殖活性;倒置荧光显微镜下观察Hoechst33342染色细胞凋亡形态;PI染色后通过流式细胞术检测诱导凋亡作用,RT-PCR法检测凋亡相关基因.结果 感染后TRAIL基因的表达明显上调.顺铂增敏TRAIL组与单独组相比,有显著抑制细胞增殖作用(P<0.05),Hoechst33342染色观察有明显的核固缩,核碎裂.流式细胞术检测细胞有凋亡峰的出现,增敏组与单独组有显著性差异(P<0.05).RT-PCR检测到TRAIL、DB5、caspase-3基因上调,survivin基因下调,DB4表达无明显变化.结论 顺铂可以增加人脑胶质瘤细胞U251对TRAIL的敏感性,其分子机制可能与DR5、caspase-3基因上调,survivin基因下调有关.     

神经胶质瘤中microRNA的研究进展

近年来神经胶质瘤的治疗正寻找新的途径,研究发现非编码小RNA(microRNA,miRNA)参与了与肿瘤相关的所有过程,包括增殖、凋亡、转移、血管生成、免疫应答等,通过抑制信号网络中特定分子表达,发挥促癌或抑癌作用;某些miRNA可以分泌至外周血血清参与循环,这些为神经胶质瘤分子靶向治疗及诊断预后提供基础.本文就近几年来神经胶质瘤中新发现的、且报道较多的miRNA进行综述.     

依托咪酯对神经胶质瘤细胞缝隙连接通讯的影响

背景与目的：细胞缝隙连接(gap junction,GJ)通讯能增强放疗或化疗药物对肿瘤细胞的细胞毒性作用。以往研究发现部分麻醉药物能够改变GJ功能从而影响肿瘤细胞对放疗的敏感性。该研究拟观察依托咪酯对神经胶质瘤细胞由缝隙连接蛋白Cx43组成的缝隙连接功能的影响,为麻醉药对化疗敏感性影响的机制研究提供线索。方法：采用磺酰罗丹明B法观察依托咪酯对神经胶质瘤细胞的抑制作用;细胞接种荧光法观察依托咪酯对神经胶质瘤细胞GJ功能的影响。结果：0.1、0.5、1和5μmol/L依托咪酯在作用4 h时间内均对细胞生长无抑制作用,将不影响细胞缝隙的数量;取药物浓度接近血药浓度的依托咪酯作用细胞4 h,与对照组相比,0.1μmol/L依托咪酯作用细胞4 h后,神经胶质瘤细胞GJ通讯的荧光传递功能无明显变化;而0.5和1μmol/L依托咪酯作用细胞4 h后,神经胶质瘤细胞的荧光传递功能明显减弱。结论：依托咪酯能抑制神经胶质瘤细胞Cx43组成的GJ功能。     

Rab31对神经胶质瘤细胞增殖迁移侵袭的影响及机制研究

目的:研究Rab31对人神经胶质瘤细胞增殖迁移侵袭的影响及相关分子机制。方法:Western blot检测正常人胶质细胞NHA和3株人神经胶质瘤细胞SHG-44、TJ905和LN229中Rab31表达;将3种Rab31沉默siRNA及其对照分别转染至神经胶质瘤SHG-44细胞中,分别记为si-Rab31-1组、si-Rab31-2组、si-Rab31-3组和si-NC组,Western blot验证转染效率,CCK-8法检测细胞增殖,Transwell实验检测细胞迁移侵袭,Western blot检测PI3K/AKT信号通路蛋白PI3K p101、p-AKT和AKT表达水平。结果:与正常人胶质细胞NHA相比,神经胶质瘤细胞SHG-44、TJ905和LN229中Rab31蛋白表达显著增加（P＜0.01）;与si-NC组比较,si-Rab31-1、si-Rab31-2和si-Rab31-3组神经胶质瘤细胞SHG-44中Rab31蛋白表达显著降低（P＜0.01）,其中si-Rab31-2组SHG-44细胞中Rab31表达最低;抑制Rab31表达可显著抑制SHG-44细胞增殖、迁移和侵袭,并抑制SHG-44细胞中PI3K/AKT信号通路转导（P＜0.01）。结论:抑制Rab31可通过抑制PI3K/AKT信号通路转导抑制胶质瘤SHG-44细胞增殖、迁移和侵袭。     

Nogo-A/NgR signaling regulates stemness in cancer stem-like cells derived from U87MG glioblastoma cells

Neurite outgrowth inhibitor A (Nogo-A), a member of the reticulon 4 family, is an axon regeneration inhibitor that is negatively associated with the malignancy of oligodendroglial tumors. It has been suggested that the Nogo-A/Nogo Receptor (NgR) pathway plays a promoting effect in regulating cancer stem-like cells (CSCs) derived from glioblastoma, indicating that Nogo-A could exert different roles in CSCs than those in parental cancer cells. In the present study, CSCs were generated from the human Uppsala 87 malignant glioma (U87MG) cell line. These U87MG-CSCs were characterized by the upregulation of CD44 and CD133, which are two markers of stemness. The expression levels of Nogo-A and the differentiation of U87MG-CSCs were investigated. In addition, the proliferation, invasion and colony formation U87MG-CSCs were examined. Using culture in serum-containing medium, U87MG-CSCs were differentiated into neuron-like cells specifically expressing MAP2, β-III-tubulin and nestin. Nogo-A was upregulated in U87MG-CSCs compared with parental cells. Knockdown of Nogo-A and inhibition of the Nogo-A/NgR signaling pathway in U87MG-CSCs markedly decreased cell viability, cell cycle entry, invasion and tumor formation, indicating that Nogo-A could regulate U87MG-CSC function. Moreover, Nogo-A was involved in intracellular ATP synthesis and scavenging of accumulated reactive oxygen species. Nogo-A/NgR pathway exerted protective effects against hypoxia-induced non-apoptotic and apoptotic cell death. These results suggest that Nogo-A plays an important role in regulating U87MG-CSCs via the Nogo-A/NgR signaling pathway. Nogo-A may also different roles in U87MG-CSCs compared with their parental cells.     

Glycine- and proline-rich glycoprotein isolated from Solanum nigrum Linne activates caspase-3 through cytochrome c in HT-29 cells

This study was carried out to investigate apoptotic effects of the glycoprotein (SNL glycoprotein, 150 kDa) isolated from Solanum nigrum Linne, which has been used as an anti-pyretic and anti-cancer agent in folk medicine. We found that the SNL glycoprotein consists of carbohydrate (69.74%) and protein content (30.26%), which has >50% hydrophobic amino acids containing glycine and proline. LDH assay indicated that the SNL glycoprotein has obvious cytotoxic and apoptotic effects (>50% cell death) at 40 microg/ml SNL glycoprotein for 2 h in HT-29 cells. The results showed that the SNL glycoprotein has a stimulatory effect on the release of mitochondrial cytochrome c, cleavages of pro-caspase-9, pro-caspase-3, and poly(ADP-ribose) polymerase (PARP) proteins in HT-29 cells. However, the SNL glycoprotein did not significantly stimulate or change the levels of intracellular reactive oxygen species (ROS). The results of this experiment suggest that the SNL glycoprotein activates caspase-3 in HT-29 cells, independent of ROS.     

糖蛋白在胆囊良恶性肿瘤中的表达

目的检测Ｐ糖蛋白在不同胆囊组织中的表达,以探讨该蛋白与胆囊癌抗化疗特性的关系及作为胆囊癌生物标志物的潜在价值。方法采用碱性磷酸酶抗碱性磷酸酶（ＡＰＡＡＰ）的免疫组化方法,用二种决定簇不同的抗人Ｐ糖蛋白的单克隆抗体ＪＳＢ１和ＵＩＣ２,检测２６例胆囊癌、１４例胆囊良性肿瘤及９例正常胆囊组织中Ｐ糖蛋白的表达情况,并分析其表达程度与不同临床资料的相关关系。结果单抗ＪＳＢ１测得Ｐ糖蛋白在胆囊癌中表达的阳性率为７６．９％,显著高于胆囊良性肿瘤组及正常胆囊组（阳性率分别为３５．７％和３３．３％,Ｐ＜０．０５）。单抗ＵＩＣ２测得Ｐ糖蛋白在胆囊癌中表达的阳性率为６９．２％,也显著高于胆囊良性肿瘤组及正常胆囊组（阳性率分别为２１．４％和１１．１％,Ｐ＜０．０１）。Ｐ糖蛋白的表达阳性率与胆囊癌的ＴＮＭ分期及胆囊良恶性肿瘤的病理类型均无相关关系。结论Ｐ糖蛋白在胆囊恶性肿瘤中的过表达,提示其在胆囊癌对化疗不敏感的特性中起着重要作用,同时Ｐ糖蛋白在不同胆囊组织中表达的显著差异性,也提示其作为胆囊恶性肿瘤的生物标志物的价值     

UDN glycoprotein regulates activities of manganese-superoxide dismutase, activator protein-1, and nuclear factor-kappaB stimulated by reactive oxygen radicals in lipopolysaccharide-stimulated HCT-116 cells

This study was carried out to investigate the anti-inflammatory effects of glycoprotein (UDN glycoprotein, 116-kDa) isolated from Ulmus davidiana Nakai, which has been used to heal inflammatory diseases in Korean herbal medicine. We found that UDN glycoprotein has strong scavenging effect on the production of intracellular superoxide anion (O(2)(-)), hydrogen peroxides (H(2)O(2)), and nitric oxide (NO) without any cytotoxicity, and that the glycoprotein also selectively normalizes the aberrant activation of manganese-superoxide dismutases (Mn-SOD) activity in lipopolysaccaride (LPS)-treated cancerous human colon epithelial cells (HCT-116 cells). The results obtained from electrophoretic mobility shift assay (EMSA) and Western blot analysis showed that UDN glycoprotein blocks the DNA binding activities of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1), and attenuates the activities of NF-kappaB subunits (p50 and p65), and AP-1 subunits (c-Jun and c-Fos), respectively. To further verify the anti-inflammatory effect of UDN glycoprotein, we investigated the activity of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and matrix metalloproteinases-9 (MMP-9) in LPS-treated HCT-116 cells, using Western blot analysis and gelatin zymographic assay. Results in this study indicated that 200mug/ml of UDN glycoprotein has inhibitory effects on the activations of iNOS, COX-2, and MMP-9. Therefore, UDN glycoprotein, a natural antioxidant, is a potential modulator of inflammatory signal pathways in LPS-treated HCT-116 cells.     

Substrate-dependent bidirectional modulation of P-glycoprotein-mediated drug resistance by erlotinib

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‐TKIs) inhibit the function of certain adenosine triphosphate (ATP)‐binding cassette transporters, including P‐glycoprotein/ABCB1 and breast cancer resistance protein (BCRP)/ABCG2. We previously reported an antagonistic activity of gefitinib towards BCRP. We have now analyzed the effects of erlotinib, another EGFR‐TKI, on P‐glycoprotein and BCRP. As with gefitinib, erlotinib effectively reversed BCRP‐mediated resistance to SN‐38 (7‐ethyl‐10‐hydroxycamptothecin) and mitoxantrone. In contrast, we found that erlotinib effectively suppressed P‐glycoprotein‐mediated resistance to vincristine and paclitaxel, but did not suppress resistance to mitoxantrone and doxorubicin. Conversely, erlotinib appeared to enhance P‐glycoprotein‐mediated resistance to mitoxantrone in K562/MDR cells. This bidirectional activity of erlotinib was not observed with verapamil, a typical P‐glycoprotein inhibitor. Flow cytometric analysis showed that erlotinib co‐treatment restored intracellular accumulation of mitoxantrone in K562 cells expressing BCRP, but not in cells expressing P‐glycoprotein. Consistently, erlotinib did not inhibit mitoxantrone efflux in K562/MDR cells although it did vincristine efflux in K562/MDR cells and mitoxantrone efflux in K562/BCRP cells. Intravesicular transport assay showed that erlotinib inhibited both P‐glycoprotein‐mediated vincristine transport and BCRP‐mediated estrone 3‐sulfate transport. Intriguingly, Lineweaver‐Burk plot suggested that the inhibitory mode of erlotinib was a mixed type for P‐glycoprotein‐mediated vincristine transport whereas it was a competitive type for BCRP‐mediated estrone 3‐sulfate transport. Collectively, these observations indicate that the pharmacological activity of erlotinib on P‐glycoprotein‐mediated drug resistance is dependent upon the transporter substrate. These findings will be useful in understanding the pharmacological interactions of erlotinib used in combinational chemotherapy. (Cancer Sci 2009; 100: 1701–1707)     

Preparation of monoclonal antibodies against pseudorabies virus glycoprotein gC by adenovirus immunization alone or as a boost following DNA priming

The objective of the present study was to demonstrate the usefulness of recombinant adenoviral vector in the generation of monoclonal antibodies (MAb) against natural epitopes of proteins using the glycoprotein gC of pseudorabies virus (PRV) as the target antigen. The recombinant adenovirus expressing the glycoprotein gC (Ad-gC) was constructed according to the AdMax method. Three immunization protocols consisting of various combinations of intramuscular injection of Ad-gC and a plasmid DNA expressing gC (pcDNA-gC) were conducted in BALB/c mice at 2-week intervals. The two groups with the highest antibody levels (Ad-gC/Ad-gC and pcDNA-gC/pcDNA-gC/Ad-gC) were selected for fusion following a final protein boost. Nine MAbs against the glycoprotein gC of PRV were subsequently developed and characterized to be isotypes of IgG1, IgG2a, and IgG2b with ascitic titers ranging from 1:2 x 10(5) to 1:5 x 10(6). Immunofluorescence assay (IFA) and Western blotting analysis confirmed that these MAbs could recognize linear epitopes on the glycoprotein gC of PRV. Our results provide a new strategy for preparation of specific MAb against viral protein.     

Effects of homoharringtonine on protein glycosylation in human bladder carcinoma cell T-24

Rates of [3H]glucosamine and mannose incorporation into glycoproteins and dolichol-linked oligosaccharides in exponentially growing T-24 bladder cancer cells were examined after exposure to homoharringtonine (HHT). Two-h treatment of HHT (10 ng/ml) reduced [3H]glucosamine and mannose incorporation into the glycoproteins to 61% and 32% of controls. Concomitantly, respective sugar incorporation into dolichol-linked oligosaccharides was elevated 29% and 30% above control. The maximal inhibition of glycoprotein biosynthesis and stimulation of the lipid-linked oligosaccharides occurred within 2 to 4 h after exposure to 50 ng/ml of the drug. Prolonged drug exposure (greater than 8 h) resulted in generalized suppression of glycoprotein biosynthesis and lipid-linked oligosaccharide formation. The kinetic study indicated that the time course on reduction of glycoprotein biosynthesis and accumulation of dolichol-linked oligosaccharides paralleled the decline in protein synthesis. Further, the inhibition of glycoprotein synthesis and stimulation of dolichol-linked oligosaccharides were reversible 4 h after drug withdrawal. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographic analysis of the [3H]mannose-labeled glycoprotein revealed no pronounced difference between HHT-treated and control cells. These data suggest that the inhibition of glycosylation results from combined decrease of acceptors for glycoprotein biosynthesis with a simultaneous accumulation of the dolichol-linked oligosaccharides. Collectively these data may account for many of the HHT-induced bioresponses.     

Detection and comparison of a 90-kd glycoprotein tumor-associated antigen-specific immune-complexes with cea and ca15-3 in breast-cancer

Serum samples selected randomly from 106 patients that had histopathologically proven breast cancer, and from 107 self-proclaimed and apparently healthy females were analyzed for the presence of a 90 kD subunit containing glycoprotein TAA-specific immune complexes (IC) by a murine monoclonal antibody based ELISA. The incidence of the glycoprotein antigen specific IC in breast cancer patients was 63% (67/106), as indicated by the normalized ELISA value above 0.410 OD405nm. On the contrary, only 3 (2.8%) of 107 apparently healthy controls had positive ELISA value (p<0.05). Comparison of the glycoprotein TAA-specific IC results in breast cancer patients with evidence of disease with the results of CEA and CA15-3 revealed that the incidence of abnormal values was increased to 91%. Thus, use of the glycoprotein TAA specific-IC marker in conjunction with CEA and/or CA15-3 may prove to be more sensitive than when used alone for immunodiagnosis and immunoprognosis.     

Development and characterization of an adriamycin-resistant sw480 human colon adenocarcinoma cell-line

An adriamycin resistant Sw480 human colon adenocarcinoma cell line (Sw480 Adr) was developed. Characteristics of the Sw480 Adr cells were compared with that of the parent Sw480 (Sw480 WT) cell line. Sw480 Adr cells were cross-resistant to mitoxantrone but not to cisplatin. Glutathione S-transferase enzyme activity as well as glutathione S-transferase pi content were increased 1.5 fold in the resistant cells, whereas P-170 glycoprotein content was increased several hundredfold. Sw480 Adr cells could be partly resensitised towards adriamycin by prior incubation with verapamil (1.65-3.3 mug/ml) and completely resensitised by cyclosporin A (2.5-5.0 mug/ml). Incubation of Sw480 Adr cells with various concentrations of adriamycin (200-1600 nM) during one week, did not change the P-170 glycoprotein levels. In addition, Sw480 Adr cells cultured in adriamycin-free medium for ten weeks (and passaged each week), showed no significant change in P-170 glycoprotein content. These results indicate that the most important mechanism for adriamycin resistance in Sw480 Adr human colon adenocarcinoma cells is overexpression of the P-170 glycoprotein, an ATP-driven drug efflux pump located in the plasma membrane. This overexpression of the P-170 glycoprotein in Sw480 Adr cells does not seem to be reversible.     

Apoptosis induced by glycoprotein (150-kDa) isolated from Solanum nigrum L. is not related to intracellular reactive oxygen species (ROS) in HCT-116 cells

This study was carried out to investigate the apoptotic effects of glycoprotein [Solanum nigrum L. (SNL) glycoprotein, 150-kDa] isolated from Solanum nigrum L., which has been used as an antipyretic and anticancer agent in folk medicine. With the purified SNL glycoprotein, we evaluated the cytotoxic and apoptotic effects of SNL glycoprotein on HCT-116 cells, DNA fragmentation and nuclear staining assays, respectively. SNL glycoprotein has an apparent cytotoxic and apoptotic effect at a concentration of 40 μg/ml after 4 h. To further verify the apoptotic effect, we investigated the changes in activity of the apoptotic-related proteins [Bid, cytochrome c, caspases and poly(ADP-ribose)polymerase (PARP)] triggered by SNL glycoprotein, using a western blot analysis. The results in this study indicated that SNL glycoprotein has a stimulatory effect on Bid activation, resulting in the release of cytochrome c, the stimulation of caspase-8, -9 and -3 activities, and the cleavage of PARP in HCT-116 cells. However, SNL glycoprotein did not significantly stimulate an increase in levels of intracellular reactive oxygen species (ROS). From the results in this experiment, it is suggested that SNL glycoprotein induces apoptosis through the mitochondrial apoptotic signal pathway in HCT-116 cells, rather than through intracellular ROS.