Effects of berberine on arylamine N-acetyltransferase activity and 2-aminofluorene-DNA adduct formation in human leukemia cells

Berberine is an alkaloid occurring in the plant genera Berberis and Coptis. Although berberine had been demonstrated to have antineoplastic function by inhibiting DNA-synthesis in activated lymphocytes, there is no available information to address berberine affects on human leukemia cell N-acetyltransferase (NAT) activity and 2-aminofluorene (AF)-DNA adduct formation. Thus, berberine was tested for inhibition of arylamine NAT activity and AF-DNA adduct formation in human leukemia cells. The NAT activity was measured by a high performance liquid chromatography assaying for the amounts of N-acetyl-2-aminofluorene (AAF) and N-acetyl-p-aminobenzoic acid (N-Ac-PABA) and the remaining AF and p-aminobenzoic acid (PABA). The NAT activity and AF-DNA adduct formation in human leukemia cells were inhibited by berberine in a dose-dependent manner, i.e. the higher the concentration of berberine, the higher the inhibition of NAT activity and AF-DNA adduct. The data also indicate that berberine decreased the apparent values of Km and Vmax from human leukemia cells in both cytosol and intact cells.     

Effect of norcantharidin on N-acetyltransferase activity in HepG2 cells

The inhibition ofarylamine N-acetyltransferase (NAT) activity by norcantharidin (NCTD), the demethylated form of cantharidin, in human hepatocellular carcinoma HepG2 cells was investigated. By using high performance liquid chromatography, NAT activity on acetylation of 2-aminofluorene (AF) and p-aminobenzoic acid (PABA) were examined. Two assay systems were performed, one with cellular cytosols, the other with intact HepG2 cell suspensions. The NAT activity in HepG2 cell line was inhibited by norcantharidin in a dose-dependent manner in both types of examined systems: i.e. the greater the concentration of norcantharidin in the reaction, the greater the inhibition of NAT activities. This report is the first to show that norcantharidin has an inhibitory effect on NAT activity in HepG2 cell.     

Effects of (-)-menthol on arylamine N-acetyltransferase activity in human liver tumor cells.

To evaluate whether or not (-)-menthol affects arylamine N-acetyltransferase (NAT) activity, we selected human liver tumor cell line (J 5) for examination. By using high performance liquid chromatography, NAT activity for acetylation of 2-aminofluorene (AF) was determined. (-)-Menthol displayed a dose-dependent inhibition to cytosolic NAT activity. Time-course experiments showed that NAT activity measured from intact human liver tumor cells was inhibited by (-)-menthol for up to 24 hrs. But in human liver tumor intact cells, the low doses (0.0032 and 0.032 mM) of (-)-menthol promoted the NAT activity and the high doses (3.2 and 32 mM) of (-)-menthol inhibited NAT activity and the 0.32 mM (-)-menthol did not show any significant differences between control and (-)-menthol treated groups. Using standard steady-state kinetic analysis, it was demonstrated that (-)-menthol was a possible uncompetitive inhibitor (decrease Km and Vmax) to NAT activity in cytosols. This report is the first demonstration which showed (-)-menthol affect on human liver tumor cells NAT activity.     

Effects of garlic components diallyl sulfide and diallyl disulfide on arylamine N-acetyltransferase activity and 2-aminofluorene-DNA adducts in human promyelocytic leukemia cells

Two components of garlic, diallyl sulfide (DAS) and diallyl disulfide (DADS), inhibited arylamine N-acetyltransferase (NAT) activity and 2-aminofluorene-DNA adduct in human promyelocytic leukemia cells (HL-60). The NAT activity was measured by high performance liquid chromatography assaying for amounts of N-acetyl-2-aminofluorene (2-AAF) and remaining 2-aminofluorene (2-AF). Cellular cytosols and intact cell suspensions were assayed. The inhibition of NAT activity and 2-AF-DNA adduct formation in human leukemia cells by DAS and DADS were dose-dependent and were directly proportional. The data also indicated that DAS and DADS decrease the apparent values of Km and Vmax from human leukemia cells in both assays. This is the first report of garlic components affecting human leukemia cell NAT activity and 2-AF-DNA adduct formation.     

龙葵碱对HepG2细胞NAT1酶活性及动力学影响的研究

 目的 探讨龙葵碱诱导HepG2细胞凋亡的芳香胺N-乙酰化转移酶（NAT1的影响。方法 采用高效液相色谱法(HPLC,以对氨基苯甲酸(PABA为底物,以PABA被NAT1乙酰化为乙酰对氨基苯甲酸(Ac-PABA的量反应NAT1酶的活性。观察不同浓度、不同时间龙葵碱对完整HepG2细胞NAT1酶活性的影响;龙葵碱对HepG2细胞细胞质中NAT1酶活性的影响;通过改变底物PABA浓度,采用双倒数作图法,以底物PABA浓度的倒数(1/S对NAT1反应速率的倒数(1/V作直线,得出回归方程,计算K_m和V_max。结果 在NAT1酶活性测定中,龙葵碱能显著降低HepG2完整细胞NAT1的活性;龙葵碱能够降低HepG2细胞质内NAT1的活性;随着作用时间的增加Ac-PABA生成的量逐渐增加,但在相同作用时间段龙葵碱能显著降低Ac-PABA生成的量。动力学研究表明,以PABA为底物,对于HepG2完整细胞,阴性对照组的K_m和V_max分别为(1.04×10-3±8.36×10-5mmol·L -1、(1.64×10-4±9.57×10-6 nmol·106 cells-1,龙葵碱组的K_m和V_max分别为(1.06×10-3±6.97×10-5 mmol·L-1和(1.48×10-4±4.28×10-6 nmol·106 cells-1·h-1。对于HepG2细胞质,阴性对照组的K_m和V_max分别为(3.32×10-1±2.35×10-4mmol·L -1、(2.60×10-3±6.79×10-6 nmol·h-1·mg pro-1,龙葵碱组的K_m和V_max分别为(3.35×10-1±1.66×10-4 mmol·L-1和(2.22×10-3±8.12×10-6 nmol·h-1·mg（Pro-1,经统计学处理表明,对于HepG2完整细胞和细胞质,阴性对照组和龙葵碱组的K_m没有差异,而V_max差异显著。结论 龙葵碱是HepG2细胞NAT1酶的非竞争性抑制剂。龙葵碱通过作用于NAT1与PABA结合位点以外的其他位点抑制NAT1酶的活性而诱导HepG2细胞凋亡。     

葎草酮对人胃癌细胞SGC-7901 N-乙酰基转移酶1活性及基因表达的抑制作用

目的探讨葎草酮抗肿瘤作用以及其与N-乙酰基转移酶1(NAT1酶)之间的关系。方法采用HPLC法,以对氨基苯甲酸(PABA)为底物,测定PABA被NAT1乙酰化为乙酰化对氨基苯甲酸(Ac-PABA)的量,间接反映NAT1酶的活性。采用RT-PCR法,测定葎草酮对NAT1mRNA表达的影响。结果葎草酮能够显著降低SGC-7901完整细胞和细胞质中Ac-PABA的生成量;随着作用时间的增加,Ac-PABA的生成量逐渐增加,但与其相同时间点的阴性对照组比较,葎草酮组Ac-PABA的生成量明显减少;葎草酮能够降低SGC-7901细胞中NAT1 mRNA的表达。结论葎草酮通过抑制NAT1的活性和基因表达两个方面减少芳香胺类化合物代谢为乙酰化的芳香胺类致癌物的量,从而预防癌症的发生,防止癌症的进一步恶化。     

小檗碱衍生物CPU 86017抑制KV1.5电流（IKur）及对抗SD大鼠房颤作用

目的：研究小檗碱衍生物CPU86017对抗房颤作用及其离子通道机制。方法：运用膜片钳技术在稳定表达人心房肌Kv1.5钾通道细胞系（HEK293）及豚鼠心房肌细胞上观察CPU86017对超速整流钾通道（IKur）的作用,观察CPU86017（1.25或2.5mg·kg-1）对比阳性药azimilide（3mg·kg^-1）对乙酰胆碱（ACh）和CaCl2诱发的大鼠房颤的治疗作用。结果：CPU86017依浓度抑制IKur,对豚鼠心房肌细胞和培养的HEK293细胞上IKur的半数抑制浓度（IC50）分别是4.56和0.21μmol·L^-1。在乙酰胆碱（ACh）和CaCl2诱发的房颤模型上,CPU86017有效缩短房颤持续时间,逆转房颤所致心房有效不应期（AERP）的缩短。结论：CPU86017能抑制KV1.5编码的电流IKur,改善房颤大鼠心房ERP的病变,有效对抗房颤。     

龙葵碱对人肝癌HepG2细胞N-乙酰基转移酶活性的影响

目的探讨龙葵碱对HepG2细胞N-乙酰基转移酶(NAT)活性的影响。方法采用HPLC方法,以2-氨基芴(2-AF)为底物,以2-AF被NAT乙酰化为2-乙酰氨基芴(2-AAF)的量来反应NAT的活性。结果龙葵碱能显著降低HepG2完整细胞NAT的活性;龙葵碱能够降低HepG2细胞质内NAT的活性,作用具有剂量依赖性。结论龙葵碱通过抑制HepG2细胞NAT的活性发挥细胞毒作用。     

吴茱萸碱和盐酸小檗碱对大肠癌HT29细胞端粒酶活性的影响

目的:探讨左金丸的主要成分吴茱萸碱和盐酸小檗碱对结肠癌HT29细胞凋亡及端粒酶催化亚基(hTERT)表达的影响,为临床应用左金丸治疗大肠癌提供实验依据。方法:体外培养人结肠癌HT29细胞,加入不同浓度的吴茱萸碱及盐酸小檗碱进行培养,采用活细胞计数试剂盒(CCK-8)检测细胞增殖,TRAP法检测端粒酶活性,RT-PCR法检测hTERT的表达。结果:吴茱萸碱和盐酸小檗碱作用HT29细胞后,增殖明显受到抑制,呈剂量和时间依赖性;能够降低HT29端粒酶活性,但二者无协同作用;并且端粒酶hTERT表达降低,具有剂量依赖关系。结论:吴茱萸碱和盐酸小檗碱对结肠癌HT29细胞具有显著生长抑制作用,该抑制作用可能通过下调端粒酶hTERT基因表达,降低端粒酶的活性,为临床治疗大肠癌提供实验依据。     

龙葵生物碱体外抑制肿瘤细胞增殖作用的实验研究

目的:研究龙葵生物碱提取物对人肺癌A549细胞株增殖的抑制作用。方法:采用盐酸-乙醇混合溶剂加热回流法从龙葵中提取分离生物碱。以MTT法考察龙葵生物碱不同浓度对人肺癌A549细胞株增殖的抑制作用,采用倒置显微镜观察药物对肿瘤细胞株形态的影响。结果:龙葵生物碱提取物对人肺癌A549细胞株具有显著的细胞增殖抑制作用,且呈剂量依赖关系,并可使肿瘤细胞形态发生显著变化。结论:龙葵生物碱具有对肺癌细胞的抑制作用。     

健脾化瘀方含药血清对人结肠癌细胞LoVo凋亡的影响

目的:探讨健脾化瘀方含药血清对人结肠癌细胞LoVo凋亡的影响。方法:①应用MTT法检测健脾化瘀方含药血清对人结肠癌细胞LoVo的增殖抑制作用;②运用TUNEL法和AnnexinV/PI法检测LoVo细胞的凋亡率。结果:①健脾化瘀方含药血清高浓度(20%)可抑制LoVo细胞的增殖(P<0.01);②健脾化瘀方含药血清作用细胞48h后可以诱导细胞凋亡。结论:健脾化瘀方含药血清能抑制结肠癌细胞LoVo的增殖能力,并诱导细胞凋亡。     

Abietane diterpenes from Salvia miltiorrhiza inhibit the activation of hypoxia-inducible factor-1

The hypoxia-inducible factor-1 (HIF-1) has been known to be correlated to the adaptation and proliferation of tumor cells; therefore HIF-1 has become an important target in the development of anticancer drugs. A phytochemical study of the CHCl3-soluble fraction of Salvia miltiorrhiza, which strongly inhibited hypoxia-induced reporter gene expression, led to the isolation of 12 abietane-type diterpenes. Of these compounds, sibiriquinone A (1), sibiriquinone B (2), cryptotanshinone (3), and dihydrotanshinone I (4) potently inhibited hypoxia-induced luciferase expression with IC50 values of 0.34, 3.36, 1.58, and 2.05 microM on AGS cells, a human gastric cancer cell line, and 0.28, 3.18, 1.36, and 2.29 microM on Hep3B cells, a human hepatocarcinoma cell line, respectively. Consistently, 1 and 4 dose-dependently suppressed the HIF-1alpha accumulation and 1 inhibited mRNA expression of vascular endothelial growth factor (VEGF) under hypoxia. These results suggest that the anticancer activity of tanshinones is likely at least in part associated with their inhibition of HIF-1 accumulation.     

复方浙贝浸膏联合奥沙利铂对人结肠癌耐奥沙利铂移植瘤及细胞膜耐药相关蛋白表达的影响

目的观察复方浙贝浸膏(CZBE)联合奥沙利铂(L-OHP)对人结肠癌耐奥沙利铂细胞株HCT116/L-OHP移植瘤小鼠的抑瘤作用,以及对移植瘤细胞耐药相关蛋白MDR1、MRP1及LRP5表达的影响。方法将人结肠癌耐奥沙利铂细胞株HCT116/L-OHP细胞接种于Balb/C裸鼠右前腋皮下构建人结肠癌耐L-OHP移植瘤模型,成模后按随机数字表法将移植瘤小鼠分成模型组、L-OHP组(L-OHP 3 mg/kg)、CZBE高剂量(CZBE 10 g/kg)联合L-OHP组、CZBE中剂量(CZBE 5 g/kg)联合L-OHP组、CZBE低剂量(CZBE 2.5 g/kg)联合L-OHP组。分组当日开始给药,CZBE灌胃给药,每日1次;L-OHP腹腔注射,隔日1次。连续给药2周。实验结束后处死小鼠,称重后完整剥离瘤体并称瘤质量,计算肿瘤抑制率。将瘤块组织制成切片,免疫组化检测HCT116/L-OHP移植瘤细胞膜耐药相关蛋白表达。结果各剂量CZBE联合L-OHP均可有效抑制HCT116/L-OHP裸鼠移植瘤的增长。L-OHP组肿瘤抑制率为27.239%,CZBE高剂量联合L-OHP组肿瘤抑制率为45.031%,与L-OHP组比较差异有统计学意义(P<0.05),CZBE中剂量联合L-OHP组肿瘤抑制率为37.669%,CZBE低剂量联合L-OHP组肿瘤抑制率为32.147%。与模型组比较,L-OHP组移植瘤细胞MDR1、MRP1、LRP5的IOD/Area值差异无统计学意义(P>0.05);与模型组及L-OHP组比较,各剂量CZBE联合L-OHP组移植瘤细胞MDR1、MRP1、LRP5蛋白表达量均不同程度降低,其中,CZBE中、高剂量联合L-OHP组移植瘤细胞MDR1、MRP1蛋白表达量明显降低,差异有统计学意义(P<0.05),CZBE高剂量联合L-OHP组移植瘤细胞LRP5蛋白表达明显降低,差异有统计学意义(P<0.05)。结论CZBE联合L-OHP可提高人结肠癌耐L-OHP细胞株移植瘤的肿瘤抑制率,其机制可能是通过减少人结肠癌耐L-OHP细胞膜耐药相关蛋白表达,增加结肠癌细胞对化疗药物的敏感性,从而起到协同增效作用。     

Inhibition of pro-inflammatory mediators and tumor cell proliferation by Anisomeles indica extracts

Anisomeles indica (L.) Kuntze (Labiatae), is a traditional anti-inflammatory herb used in Taiwan. The aqueous and methanolic extracts of whole plants, leaves, flowers and stems; and chloroform and n-butanol fractions of methanol extract, from A. indica were investigated for their anti-inflammatory activity on murine peritoneal macrophages. In addition, the tumor cells proliferation inhibition activities of these extracts were also evaluated against a panel of tumor cell lines such as Colon 205, PC 3, HepG2 and MCF 7. Treatment with A. indica extracts did not reduce cell viability at any dose used. However, all the extracts significantly inhibited the enhanced production of NO radicals, and pro-inflammatory cytokines (TNF-alpha, and IL-12) induced by LPS/IFN-gamma in a dose-dependent manner. Furthermore, methanolic extracts of leaves and flowers significantly and dose-dependently arrest mitogen-stimulated spleen cells in G0/G1 stage, in addition to their cell proliferation inhibition against Colon 205, MCF 7 and PC 3 by 94, 82; 98, 71; 82, 98%, respectively, at 200 microg/mL concentration. This is the first report on A. indica extracts for their growth inhibitory activities, against inflammatory mediator production, and human tumor cell lines, colon, prostate, hepatoma and breast cells proliferation.     

小檗碱增强K562/DOX细胞对多柔比星敏感性的研究

 目的 探讨小檗碱增强耐药K562/DOX细胞对化疗药多柔比星的敏感性的作用。方法 四甲基偶氮唑蓝法检测小檗碱的细胞毒性及其对多柔比星抗肿瘤活性的增强作用;高内涵活细胞成像系统检测无毒剂量小檗碱作用后,多柔比星在K562/DOX细胞内的蓄积量;PI/Hoechst33342双染法检测小檗碱对多柔比星诱导的K562/DOX细胞凋亡的影响;罗丹明123蓄积实验检测小檗碱对P-糖蛋白外排功能的影响。结果 1 μmol·L^-1为小檗碱的无毒剂量,在此无毒剂量下,小檗碱使多柔比星对K562/DOX细胞的IC₅₀降低了1.5倍;1 μmol·L^-1小檗碱可使多柔比星在K562/DOX细胞内的蓄积量增加,增强多柔比星诱导的K562/DOX细胞凋亡, 增加K562/DOX细胞内罗丹明123的蓄积量,从而抑制P-糖蛋白的外排功能。结论 小檗碱可通过抑制K562/DOX细胞膜上P-糖蛋白的外排功能,增加K562/DOX细胞内多柔比星浓度,促进多柔比星对耐药细胞的诱导凋亡作用,逆转K562/DOX细胞的多药耐药性。     

Extracts from the roots of Lindera strychifolia induces apoptosis in lung cancer cells and prolongs survival of tumor-bearing mice

Lindera strychifolia, a scandent shrub Lauraceous medicinal plant, has been used in Chinese traditional medicine as a palliative and an anti-spasmodic. It also shows cytotoxic effects against several tumor cell lines and inhibits marcromolecule biosynthesis. This study investigated the anti-tumor effects of L. strychifolia extract against lung cancer cells using in vitro and in vivo models. Two human lung cancer cell lines A549 (adenocarcinoma) and SBC-3 (small cell carcinoma), and a non-tumor cell line 3T3-L1 (mice fibroblasts) were subjected to L. strychifolia extract treatment. On lung cancer cells, L. strychifolia induced cell growth inhibition in a dose-dependent manner. Conversely, the extract did not show any significant cytotoxic effect on 3T3-L1 cells. Therefore, the extract is specific for tumor cells. Tumor cells treated with L. strychifolia extract showed typical morphological appearance of apoptosis including nuclei fragmentation and cell condensation. The in vivo effects of L. strychifolia extract were investigated in C57BL/6 mice transplanted with Lewis lung cancer (LL-2) cells, and in BALB/c nude mice transplanted with A549 or SBC-3 human lung cancer cells. Oral administration of L. strychifolia extract prolonged survival time and inhibited tumor growth in a dose-dependent manner by inducing apoptosis in the LL-2 cell mice model. Furthermore, in A549 or SBC-3 cell nude mice models, oral administration of L. strychifolia extract also significantly inhibited tumor growth at the 5.0 mg/ml concentration. These findings suggested that the components of L. strychifolia have anticancer activity and may contribute to clinical applications in the prevention and treatment of lung cancer.     

青蒿酿液体外抗肿瘤活性研究

目的研究青蒿酿液的体外抗肿瘤活性。方法采用四甲基偶氮唑盐法测定青蒿酿液体外抑制人肺癌A549、MDA231细胞,结肠癌SW620、LOVO细胞,胃癌AGS细胞以及肝癌Hep-2细胞的活性。结果青蒿酿液对以上六株肿瘤细胞均有明显的抑制作用。结论青蒿酿液在体外对人肺癌A549、MDA231细胞,结肠癌SW620、LOVO细胞,胃癌AGS细胞以及肝癌Hep-2细胞均有明显的抗肿瘤作用。     

小檗碱对HeLa细胞凋亡及其凋亡相关蛋白表达的影响

目的 研究小檗碱对人宫颈癌HeLa细胞株增殖及凋亡的影响及其发生机制.方法 采用MTT法观察小檗碱对HeLa细胞增殖的影响,DNA ladder、流式细胞仪等方法进行细胞凋亡检测,用Western blotting方法检测凋亡过程中相关蛋白Bel-2和Bax表达的变化.结果 小檗碱在体外试验中能明显抑制HeLa细胞的增殖,在一定的时间、剂量范围内呈时间-剂量依赖关系.20、40 mg/L小檗碱作用48 h后,细胞DNA片段分析可看到凋亡时降解形成的梯带,流式细胞仪检测到了细胞凋亡峰,各组的凋亡率分别是(16.7±2.8)%、(29.6±4.4)%,与对照组(1.9±0.6)%和5 mg/L小檗碱组(2.3±0.8)%相比差异显著(P＜0.01).在凋亡过程中,Bax蛋白表达明显增高,而Bcl-2蛋白表达下凋.结论 小檗碱体外能抑制HeLa细胞增殖并诱导其发生凋亡,其机制可能与Bax蛋白表达明显增高,而Bcl-2蛋白表达下调有关.