氧化砷诱导胃癌细胞凋亡的实验研究

目的 研究三氧二化砷（氧化砷）对胃癌细胞的诱导凋亡作用。方法 应用ＴＵＮＥＬ染色、流式细胞仪技术研究氧化砷对胃癌细胞ＭＫＮ－４５、ＭＫＮ－２８的诱导凋亡作用。结果 氧化砷作用于不同分化程度的胃癌细胞后,可看到较为典型的细胞凋亡的形态学变化：细胞核固缩,染色质凝集,呈新月型紧贴于核膜周边,核碎裂,染色质片断化,凋 亡小体形成等。流式细胞仪ＤＮＡ直方图上出现典型的亚二倍体的“凋亡峰”。ＴＵＮＥＬ染色法     

The sensitivity of digestive tract tumor cells to As2O3 is associated with the inherent cellular level of reactive oxygen species

AIM: To explore the correlation of the inherent cellular ROS level with the susceptibility of the digestive tract tumor cells to apoptosis inducted by As2O3. METHODS: Two gastric carcinoma cell lines, SGC7901 and MKN45, and two esophageal carcinoma cell lines, EC/CUHK1(alternatively named EC1.71) and EC1867 with low concentration(2 micromol x L(-1))of As2O3 were cultured espectly, which confirmed the difference in apoptosis susceptibility between SGC7901 and MKN45, and between EC/CUHK1 and EC1867. The cells were incubated with dihydrogenrhodamine123 (DHR123), used as a ROS capture in absence of As2O3.The fluorescent intensity of rhodamine123, which was the product of cellular oxidation of DHR123, was detected by flow cytometry, and ROS was measured. RESULTS: Apoptosis induced by a low concentration of As2O3 was more readily to occur in SGC7901(22.4%+/-2.4%) and EC/CUHK1(27.0%+/-2.9%) than in MKN45(2.1%+/-0.5%) and EC1867(0.8%+/-0.5%).In other words, SGC7901 was more sensitive than MKN45 to As2O3, meanwhile EC/CUHK1 was more sensitive than EC1867 to As2O3. The level of inherent cellular ROS in SGC7901(650+/-37) was higher than that in MKN45(507+/-22)(P<0.01), and the level of inherent cellular ROS in EC/CUHK1(462+/-17) was higher than that in EC1867(187+/-12)(P<0.01).CONCLUSIONS: The cellular sensitivity to apoptosis induced by As2O3 is associated with the difference in cellular ROS level. The inherent ROS level might determinate the apoptotic sensitivity of tumor cells to As2O3.     

氧化砷诱发胃癌细胞株凋亡的初步研究

目的在三氧化二砷（Ａｓ２Ｏ３）治疗ＡＰＬ的基础上，进一步探讨Ａｓ２Ｏ３能否诱发胃癌细胞株凋亡。方法采用荧光标记法，经流式细胞仪和荧光显微镜观察Ａｓ２Ｏ３对ＭＫＮ４５和ＳＧＣ７９０１胃癌细胞株的凋亡诱发率和形态学改变。结果发现Ａｓ２Ｏ３诱发胃癌细胞凋亡率高于５－Ｆｕ的作用；形态学见凋亡细胞呈荧光标记阳性，细胞内出现斑块状荧光，体积缩小。结论Ａｓ２Ｏ３可诱发胃癌细胞凋亡，有必要进一步探索其对胃癌治疗的价值。     

Induction of apoptosis by arsenic trioxide and hydroxy camptothecin in gastriccancer cells in vitro

AIM To study the effects of arsenic trioxide and HCPT on different degrees of differentiated gastric cancer cells (SGC-7901, MKN-45, MKN-28)with respect to both cytotoxicity and induction of apoptosis in vitro. ~ODS The cytotoxicity of As2O3 and HCPT on gastric cancer cells was determined by MTTassay. Morphologic changes of apoptosis of gastric cancer cells were observed by light microscopy and transmission electron microscopy. Apoptosis and cell cycle changes of gastric cancer cells induced by HCPT and As2O3 were investigated by TUNEL method and flow cytometry. RESULTS As2O3 and HCPT had remarkable cytotoxic effects on different degrees of differentiated gastric cancer cells. The IC50 of As2O3 on well differentiated gastric cancer cell MKN-28, moderately differentiated gastric cancer cell SGC-7901, and poorly differentiated gastric cancer cell MKN-28 were 8. 91 μmol/L, 10. 57 μmol/L, and 11.65 μmol/L, respectively. The IC50 of HCPT on MKN-28, SGC-7901, and MKN-45 were 9. 35 rg/L, 10. 21 rg/L, and 12. 63 mg/L respectively after 48 h treatment. After 12 h of exposure to both drugs, gastric cancer cells exhibited morphologic features of apoptosis, including cell shrinkage, nuclear condensation,and formation of apoptotic bodies. A typical subdiploid peak before G0/G1 phase was observed by flow cytometry. The apoptotic rates of SGC7901, MKN-45, and MKN-28 were 13. 84%, 22.52%, and 9. 68%, respectively after 48 h exposure to 10 μmol/L As2O3. The apoptotic rates of SGC-7901, MKN-45, and MKN-28 were 21.88%, 12.35%, and 30. 26%, respectively after 48 h exposure to 10 mg/L HCPT. The apoptotic indice were 7% - 15% as assessed by TUNEL method. The effect of As2O3 on SGC-7901 showed remarkable cell cycle specificity, which induced cell death in G1 phase, and blocked G2/M phase. HCPT also showed a remarkable cell cycle specificity, by inducing cell death and apoptosis in G1 phase and arrest of proliferation at S phase. CONCLUSION AS2O3 and HCPT exhibit significant cytotoxicity on gastric cancer cells by induction of apoptosis. As2O3 and HCPT might have a promising prospect in the treatment of gastric cancer, which needs to be further studied.     

药物诱导胃癌细胞凋亡的初步探索

目的:了解羟基喜树碱和氧化砷体外诱导胃癌细胞凋亡的能力.探索最佳诱导时间和剂量.并初步阐明其作用机制。方法:利用HE染色法、流式细胞仪和DNA末端原位标记染色法(TUNEL)观察羟基喜树碱和氧化砷在体外对胃癌细胞MKN-28(高分化腺癌)。SGC-7901(中分化腺癌)。MKN-45(低分化腺癌)的作用 结果:药物作用48小时后,羟基喜树碱0.01mg/ml组胃癌细胞MKN-28、SGC-7901、MKN-45的凋亡率分别为30.26％、21.88％和12.35％,氧化砷10μmol/L组胃癌细胞MKN-28、SGC-7901、MKN-45的凋亡率分别为22.52％、13.83％和9.68％其中羟基喜树碱在细胞周期的S期诱导胃癌细胞发生凋亡,而氧化砷则主要作用于G2/M期。     

三氧化二砷诱导小细胞肺癌细胞凋亡及p53、bcl-2基因表达的研究

目的：研究三氧化二砷（As2O3）诱导小细胞肺癌细胞凋亡及其机制。方法：采用末端脱氧核苷酰转移（TUNEL）法检测细胞凋亡，免疫组织化学（SABC）法分析As2O3对小细胞肺癌细胞（NeI-H细胞）p53、bcl-2基因蛋白表达的影响。结果：As2O30.5μmol/L、1.0μmol/L、2.0μmol/L作用72h，NeI-H细胞均可呈现凋亡所特有的亚G1峰，凋亡比率随药物作用浓度增加和作用时间延长而增高。实验组p53基因蛋白表达较对照组明显增多，药物浓度越高表达越多（P=0.001）；bcl-2基因蛋白表达较对照组明显减少，药物作用浓度越高表达越少（P=0.001）。结论：As2O3抗肿瘤作用主要是通过诱导细胞凋亡实现的，其机制与上调p53基因表达及下调bcl-2基因表达有密切关系。     

Synergistic effect of cell differential agent-II and arsenic trioxide on induction of cell cycle arrest and apoptosis in hepatoma cells

AIM: To illustrate the possible role of cell differential agent-II (CDA-II) in the apoptosis of hepatoma cells induced by arsenic trioxide (As(2)O(3)). METHODS: Hepatoma cell lines BEL-7402 and HepG2 were treated with As(2)O(3) together with CDA-II. Cell surviving fraction was determined by MTT assay; morphological changes were observed by immunofluorescence staining of Hoechst 33,258; and cell cycle and the apoptosis index were determined by flow cytometry (FCM). RESULTS: Cytotoxicity of CDA-II was low. Nevertheless, CDA-II could strongly potentiate arsenic trioxide-induced apoptosis. At 1.0 g/L CDA-II, IC(50) of As(2)O(3) in hepatoma cell lines was reduced from 5.0 micromol/L to 1.0 micromol/L (P<0.01). The potentiation of apoptosis was dependent on the dosage of CDA-II. FCM indicated that in hepatoma, cell growth was inhibited by CDA-II at lower concentrations (<2.0 g/L) primarily by arresting at S and G(2) phase, and at higher concentrations (>2.0 g/L) apoptotic cell and cell cycle arresting at G(1) phase increased proportionally. The combination of two drugs led to much higher apoptotic rates, as compared with the either drug used alone. CONCLUSION: CDA-II can strongly potentiate As(2)O(3)-induced apoptosis in hepatoma cells, and two drugs can produce a significant synergic effect.     

Growth inhibition and apoptosis of gastric cancer cell lines by Anemarrhena asphodeloides Bunge

In this study, we aimed to determine the growth inhibition and the induction of apoptotic cell death brought about by the herb Anemarrhena asphodeloides Bunge in gastric cancer cell lines, and to clarify the mechanism of this apoptosis. Water-soluble ingredients of A. asphodeloides, and the gastric cancer cell lines, MKN45 and KATO-III, were used in vitro. Growth inhibition, induction of cell death, morphological features, the presence of DNA ladders, increases in caspase-3-like activity, the effects of a caspase-3 inhibitor on apoptotic cell death, and the release of cytochrome c by A. asphodeloides were analyzed. A. asphodeloides inhibited the growth and decreased the viability of the gastric cancer cell lines. The viability of normal skin fibroblasts in the presence of low concentrations of A. asphodeloides was higher than that of gastric cancer cells. Apoptotic bodies and DNA ladders were observed to be induced in MKN45 and KATO-III by A. asphodeloides. The caspase 3 inhibitor, Ac-DEVD-CHO, inhibited the apoptotic cell death of gastric cancer cells induced by A. asphodeloides. The caspase 3-like activity in MKN45 and KATO-III cells increased after the addition of A. asphodeloides. Cytochrome c was released from mitochondria into the cytosol 8 h after the addition of A. asphodeloides, and reached a peak at 16 h. The peak of cytochrome c release was earlier than that of caspase 3-like activity. We concluded that A. asphodeloides inhibited the growth of the gastric cancer cell lines MKN45 and KATO-III and induced apoptosis. The apoptosis of MKN45 and KATO-III cells induced by A. asphodeloides was associated with the release of cytochrome c from the mitochondria, followed by an increase in caspase 3-like activity. Received: December 10, 1999 / Accepted: September 1, 2000     

表没食子儿茶素没食子酸酯对人胃癌细胞株MKN45诱导凋亡作用的研究

目的了解表没食子儿茶素没食子酸酯（EGCG）是否诱导人胃癌细胞株MKN45凋亡及其凋亡信号传导途径，为其临床应用提供进一步的理论依据。方法用四甲基偶氮唑蓝（MTT）比色法检测EGCG对MKN45细胞生长的抑制作用；膜连蛋白V-异硫氰基荧光素（Annexin V-FITE）＋碘化丙啶（PI）方法测定EGCG作用后MKN45细胞的凋亡率；酶联免疫吸附测定（ELISA）检测EGCG对MKN45细胞内castmse-3活性的影响；Rhodamine123染色检测EGCG作用后MKN45细胞内线粒体膜电位的改变情况。结果EGCG作用后MKN45细胞发生凋亡，随着EGCG作用时间的延长及浓度的增加．凋亡率增加。在EGCG作用8h后MKN45细胞内的caspase-3的活性开始升高，并于作用12h后活性明显升高。线粒体的膜电位于EGCG作用4h后就开始明显下降，并与时间和浓度正相关。结论EGCG可以诱导人胃癌细胞株MKN45细胞凋亡，且与作用时间及浓度正相关。EGCG对MKN45细胞凋亡的诱导是通过线粒体途径发生的．     

叶酸对胃癌细胞凋亡的影响

目的胃癌的发生与发展中有细胞凋亡的变化,叶酸抗肿瘤的机理涉及到维持ＤＮＡ甲基化水平等,其与凋亡的关系尚未明了。方法以高低两种浓度的叶酸干预培养的人胃癌ＭＫＮ－４５和ＭＫＮ－２８细胞系７２小时后,以原位ＤＮＡ断端切口标记法和ＤＮＡ琼脂糖凝胶电泳检测凋亡形态学改变和ＤＮＡ断裂情况。结果未加干预者两种细胞系的凋亡指数均低于５％,不同分化细胞系间比较差异无显著性;低浓度叶酸可诱导ＭＫＮ－４５细胞凋亡,而高浓度者使ＭＫＮ－２８细胞凋亡率增加。结论胃癌细胞自然凋亡率较低。叶酸干预可影响凋亡的发生。     

Apoptotic effect of Epigallocatechin-3-gallate on the human gastric cancer cell line MKN45 via activation of the mitochondrial pathway

To investigate whether Epigallocatechin-3-gallate （EGCG） can induce apoptosis of the gastric cancer cell line MKN45 and its apoptotic pathway.METHODS： To determine this, apoptotic rates of MKN45 cells after EGCG treatment with or without caspase-3 inhibitor were evaluated by Annexin V-FITC ＋ PI staining The influence of EGCG on the activity of caspase-3 in the MKN45 cells was determined by ELISA. By Rhodamine123 staining, the membrane potential change of the mitochondrion was also investigated, and mRNAs and protein expression of the bcl-2 family were analyzed by RT-PCR and Western blot.RESULTS： EGCG can induce apoptosis of MKN45 cells in time- and dose-dependent manner. Eight hours after EGCG treatment, the activity of caspase-3 in the MKN45 increased, especially 12 h after treatment. The mitochondrial membrane potential was significantly weakened 4 h after EGCG insult. The mRNA and protein expression levels of pro-apoptotic members, such as Bax, Bid and Bad, were upregulated gradually as treated time increased. Moreover, the mRNA and protein expression levels of anti-apoptotic members, such as Bcl-xL and Bcl-2, were inhibited. CONCLUSION： These data support that EGCG can induce apoptosis of the human gastric cancer cell line MKN45, and the effect is in a time- and dose-dependent manner. The apoptotic pathway triggered by EGCG in MKN45 is mitochondrial-dependent.     

Effect of human noxa on irinotecan-induced apoptosis in human gastric carcinoma cell lines

BACKGROUND/AIMS: A protein BH3-only member bcl-2 family, Noxa is a proapoptotic mediator for p53-induced apoptosis. We analyzed the effect of Noxa on p53-induced apoptotic gastric carcinoma cell lines. METHODOLOGY: The expressions of human Noxa (hNoxa) mRNA on human gastric carcinoma cell lines were assessed with RT-PCR. Further, hNoxa antisense and sense S-oligodeoxynucleotide (ODN) were used to analyze the effect of hNoxa on p53-induced apoptotic gastric carcinoma cell lines. RESULTS: Various levels of hNoxa mRNA expression were detected in all gastric cell lines. MKN45 that has wild-type p53 showed severe inhibition by irinotecan compared with MKN28, which has mutated p53. Cell growth under hNoxa antisense S-ODN treatment did not differ from that under sense S-ODN treatment in MKN28. On the other hand, the suppression of cell growth in MKN45 decreased with hNoxa antisense S-ODN treatment as compared to hNoxa sense S-ODN treatment. MKN45 cells exhibited DNA fragmentation clearly after 24 hr of 3 mM hNoxa sense S-ODN treatment. The DNA fragmentation in MKN45 was inhibited by hNoxa antisense S-ODN treatment. CONCLUSIONS: It seems that hNoxa plays an important role in induction of apoptosis on p53 wild type gastric carcinoma cell lines.     

表没食子儿茶素没食子酸酯诱导细胞凋亡蛋白酶途径依赖的人胃癌细胞株MKN45凋亡

背景：表没食子儿茶素没食于酸酯（EGCG）可诱导人胃癌细胞株MKN45凋亡，但其凋亡信号的传导途径尚不清楚。目的：研究EGCG诱导人胃癌细胞株MKN45凋亡的作用是否通过细胞凋亡蛋白酶（caspsse-3）依赖途径，为其临床应用提供进一步的理论依据。方法：采用四甲摹偶氮唑蓝（MTT）比色法检测EGCG和caspase-3抑制剂z-DEVD-fmk作用后MKN45细胞的存活率：采用Annexin V-FITC＋PI双染色法检测EGCG和caspase-3抑制剂作用后MKN45细胞的凋亡率：采用酶联免疫吸附测定（ELISA）检测EGCG和caspase-3抑制剂作用后MKN45细胞内caspase-3活性的改变。结果：EGCG可诱导MKN45细胞凋亡，且细胞内caspase-3活性显著升高。而caspase-3抑制剂干预后，EGCG抑制MKN45细胞生长的作用明显减弱，细胞凋亡率下降，caspase-3活性显著下降。结论：EGCG可诱导MKN45细胞凋亡，该作用可被caspase-3抑制剂显著抑制，提示EGCG诱导MKN45细胞凋亡的作用是通过caspase-3依赖途径的。     

The sensitivity of digestive tract tumor cells to As2O3 is associated with the inherent cellular level of reactive oxygen species

AIM: To explore the correlation of the inherent cellular ROSlevel with the susceptibility of the digestive tract tumor cellsto apoptosis inducted by As2O3.METHODS: Two gastric carcinoma cell lines, SGC7901 andMKN45, and two esophageal carcinoma cell lines, EC/CUHK1 (alternatively named EC1. 71 ) and EC1867 with lowconcentration (2μmol@ L- 1)of As2O3 were cultured respectly,which confirmed the difference in apoptosis susceptibilitybetween SGC7901 and MKN45, and between EC/CUHK1 andEC1867. The cells were incubated withdihydrogenrhodamine123 (DHR123), used as a ROScapture, in absence of As2O3. The fluorescent intensity ofrhodamine123, which was the product of cellular oxidationof DHR123, was detected by flow cytometry, and ROS wasmeasured.RESULTS: Apoptosis induced by a low concentration ofAs2O3 was more readily to occur in SGC7901 (22.4% ± 2.4%)and EC/CUHK1 (27.0% ± 2.9%) than in MKN45 (2.1% ±0.5%) and EC1867(0.8% ± 0.5%). In other words, SGC7901was more sensitive than MKN45 to As2O3, meanwhile EC/CUHK1 was more sensitive than EC1867 to As2O3. The levelof inherent cellular ROS in SGC7901 (650 ± 37) was higherthan that in MKN45 (507 ± 22) ( P ＜ 0.01 ), and the level ofinherent cellular ROS in EC/CUHK1 (462 ± 17) was higherthan that in EC1867( 187 ± 12) ( P＜ 0.01).CONCLUSIONS: The cellular sensitivity to apoptosis inducedby As2O3 is associated with the difference in cellular ROSlevel. The inherent ROS level might determinate theapoptotic sensitivity of tumor cells to As2O3.     

Nitric oxide and calcium ions in apoptotic esophageal carcinoma cells induced by arsenite

AIM: To Quantitatively analyze the nitric oxide (NO) and Ca2+ in apoptosis of esophageal carcinoma cells induced by arsenic trioxide (As2O3). METHODS: The cell line SHEEC1, a malignant esophageal epithelial cell induced by HPV in synergy with TPA in our laboratory, was cultured in a serum-free medium and treated with As2O3. Before and after administration of As2O3, NO production in cultured medium was detected quantitatively using the Griess Colorimetric method. Intracellular Ca2+ was labeled by using the fluorescent dye Fluo3-AM and detected under confocal laser scanning microscope (CLSM), which was able to acquire data in real-time enabling Ca2+ dynamics of individual cells in vitro. The apoptotic cells were examined under electron microscopy. RESULTS: Intracellular concentration of Ca2+ increased from 1.00 units to 1.09-1.38 units of fluorescent intensity at As2O3 treatment and NO products subsequently released from As2O3-treated cells increased from 0.98-1.00 x10(-2)micromol x L(-1) up to 1.48-1.52 x10(-2)micromol x L(-1) and maintained in a high level continuously. Finally apoptosis of cells occurred,chromatin being agglutinated, cells shrunk,nuclei became round and mitochondria swelled. CONCLUSION: Ca2+ and NO increased with cell damage and apoptosis in cells treated by As2O3. The Ca2+ is an initial messenger to the apoptotic pathway. To investigate Ca2+ and NO will be a new direction for studying the apoptotic signaling messenger of the esophageal carcinoma cells induced by As2O3.     

三氧化二砷对正常肝细胞及肝癌细胞株的影响

目的观察不同浓度Ａｓ２Ｏ３作用不同时间对正常肝细胞及肝癌细胞株的影响。方法０．１２５～２μｍｏｌ／ＬＡｓ２Ｏ３与肝细胞及肝癌细胞株共孵育一定时间后,观察细胞的存活、形态学改变及细胞ＤＮＡ含量的分布。结果１μｍｏｌ／ＬＡｓ２Ｏ３处理的肝癌细胞第５天呈典型的凋亡特征性改变：在形态学上表现为细胞膜完整、染色质固缩、核碎裂、凋亡小体形成;流式细胞仪分析显示,在Ｇ１期细胞前出现亚二倍体峰,琼脂糖凝胶电泳呈凋亡特征性Ｌａｄｄｅｒ带;而正常肝细胞则未见明显改变。结论Ａｓ２Ｏ３可诱导肝癌细胞凋亡。     

羟基喜树碱诱导胃癌细胞凋亡的作用机制初步研究

目的：研究羟基喜树碱（HCPT）诱导胃癌细胞的凋亡作用及对凋亡相关基因p53,c-myc,bcl-2,bcl-xl和bcl-xs表达的影响,探讨其诱导胃癌细胞凋亡的作用机制。方法：应用TUNEL染色、流式仪、免疫组化和RT－PCR技术等研究HCPT对胃细胞SGC－7901和MKN－45的诱导凋亡作用和对凋亡相关有达的影响。结果：HCPT作用于细胞后,可看到较为典型的细胞凋亡的形态学变化;细胞核固缩,染色质凝集,呈新月型紧核膜周边,核碎裂,染色质片段化,凋亡小体形成等。流式细胞仪DNA直方图上出现典型的亚二倍体的“凋亡峰”。流式细胞仪计数显示,10μg/ml的HCPT诱导胃癌细胞SGC－7901和MKN－45的凋亡率为21．88％和12．34％。TUNEL染色法显示,细胞凋亡指数在1．865－9．54％之间。免疫组化和RT－PCR结果显示：HCPT能够明显下调SGC－7901细胞的P53和bcl-2基因的蛋白和mRNA表达,对SGC－7901细胞的c-myc,bcl-xl和bcl-xs基因的蛋白表达无影响。HCPT作用后MKN－45细胞的p53蛋白和mRNA的表达增加,对MKN－45细胞的bcl-2,c-myc,bcl-xl和bcl-xs基因的表达无影响。结论：HCPT能够诱导胃癌细胞凋亡,可能是通过调控胃癌细胞的P53和bcl-2的表达而诱导胃癌细胞凋亡。     

Growth inhibition and apoptosis induction of Sulindac on Human gastric cancer cells

AIM: To evaluate the effects of sulindac in inducing growth inhibition and apoptosis of human gastric cancer cells in comparison with human hepatocellular carcinoma (HCC) cells. METHODS: The human gastric cancer cell lines MKN45 and MKN28 and human hepatocellular carcinoma cell lines HepG(2) and SMMC7721 were used for the study. Anti-proliferative effect was measured by MTT assay, and apoptosis was determined by Hoechst-33258 staining, electronography and DNA fragmentation. The protein of cyclooxygenase-2 (COX-2) and Bcl-2 were detected by Western dot blotting. RESULTS: Sulindac could initiate growth inhibition and apoptosis of MKN45, MKN28, HepG(2) and SMMC7721 cells in a dose-and time-dependent manner. Growth inhibitory activity and apoptosis were more sensitive in HepG(2) cells than in SMMC7721 cells, MKN45 and MKN28 cells. After 24 hours incubation with sulindac at 2mmol x L(-1) and 4mmol x L(-1), the level of COX-2 and Bcl-2 protein were lowered in MKN45, SMMC7721 and HepG(2) cells but not in MKN28 cells. CONCLUSION: Sulindac could inhibit the growth of gastric cancer cells and HCC cells effectively in vitro by apoptosis induction, which was associated with regression of COX-2 and Bcl-2 expression. The growth inhibition and apoptosis of HCC cells were greater than that of human gastric cancer cells. The different effects of apoptosis in gastric cancer cells may be related to the differentiation of the cells.     

Effects of AZT and RNA-protein complex （FA-2-b-β） extracted from Liang Jin mushroom on apoptosis of gastric cancer cells

To investigate the synergistic effects of 3′-azido-3′- deoxythymidine （AZT） and FA-2-b-β extracted from Ling Jin mushroom on apoptosis of gastric cancer cells MKN45 in vitro. METHODS： Ml-I- analysis was made to examine the inhibition rate of MKN45 cells treated with AZT （2.5, 5, 10 and 20 mg/L） and FA-2-b-13 （5, 10, 20 and 40 mg/L） singly and combinatively for 24, 48 and 72 h. Apoptotic effects were evaluated by morphological methods, DNA agarose gel electrophoresis and flow cytometry, respectively. Telomerase activity was estimated by TRAP- ELISA. The mRNA expression of caspase-3 and Bcl-2 were detected by RT-PCR. RESULTS： AZT and FA-2-b-13 could significantly inhibit MKN45 cell proliferation and induce its apoptosis. MKN45 cells were inhibited in dose- and time- dependent manner. The inhibition effect of AZT combined with FA-2- b-β was obviously better than that used singly （0.469 ＋ 0.022 vs 1.075 4- 0.055, P 〈 0.05, 0.325 4- 0.029 vs 0.469 ＋ 0.022 P 〈 0.01）. AZT used singly and combination of FA-2-b-β could decrease the activity of tumor cell telomerase, and AZT has synergistic function with FA- 2-b-β. A certain concentration of AZT could up-regulate the expression of caspase-3 mRNA （r = 0.9969, P 〈 0.01）, which was positively related to apoptosis rate, and could down-regulate the expression of Bcl-2 mRNA, which was negatively related to apoptosis rate （r = 0.926, P 〈 0.01）. Furthermore, the effect of AZT combined with FA-2-b-13 was significantly higher than that used singly. CONCLUSION： Combination of AZT and FA-2-b-β has an obviously synergetic effect in the gastric cancer cells MKN45, which has provided a new approach to the treatment of gastric cancer clinically.     

Effects of AZT and RNA-protein complex (FA-2-b-β) extracted from Liang Jin mushroom on apoptosis of gastric cancer cells

AIM: To investigate the synergistic effects of 3'-azido-3'-deoxythymidine (AZT) and FA-2-b-β extracted from Ling Jin mushroom on apoptosis of gastric cancer cells MKN45 in vitro.METHODS: MTT analysis was made to examine the inhibition rate of MKN45 cells treated with AZT (2.5, 5,10 and 20 mg/L) and FA-2-b-β (5, 10, 20 and 40 mg/L)singly and combinatively for 24, 48 and 72 h. Apoptotic effects were evaluated by morphological methods,DNA agarose gel electrophoresis and flow cytometry,respectively. Telomerase activity was estimated by TRAPELISA. The mRNA expression of caspase-3 and Bcl-2 were detected by RT-PCR.RESULTS: AZT and FA-2-b-β could significantly inhibit MKN45 cell proliferation and induce its apoptosis. MKN45 cells were inhibited in dose- and time- dependent manner. The inhibition effect of AZT combined with FA-2-b-β was obviously better than that used singly (0.469 ±0.022 vs 1.075 ± 0.055, P ＜ 0.05, 0.325 ± 0.029 vs 0.469± 0.022 P ＜ 0.01). AZT used singly and combination of FA-2-b-β could decrease the activity of tumor cell telomerase, and AZT has synergistic function with FA-2-b-β. A certain concentration of AZT could up-regulate the expression of caspase-3 mRNA (r = 0.9969, P ＜ 0.01),which was positively related to apoptosis rate, and could down-regulate the expression of Bcl-2 mRNA, which was negatively related to apoptosis rate (r = 0.926, P ＜ 0.01).Furthermore, the effect of AZT combined with FA-2-b-β was significantly higher than that used singly.CONCLUSION: Combination of AZT and FA-2-b-β has an obviously synergetic effect in the gastric cancer cells MKN45, which has provided a new approach to the treatment of gastric cancer clinically.