EFFECTS OF BLEOMYCIN A5 COMBINED WITH CALMODU-LIN INHIBITOR ON THE PROLIFERATION OF S-180 CELLS IN VITRO

The effects of bleomycin A5 (BLM A5) alone and combined with calmodulin inhibitor N-(4-aminobutyl)-5-chloro-2-naphthalene sulfonamide (W-13) on the proliferation on S-180 cells in vitro were studied. IC50 of BLM used alone for the cells was about 2.63 μg/ml, but it was reduced to 1/3.8 and 1/9.5 of 2.63 μg/ml when plus W-13 1, 5 μg/ml respectively. The results indicated that nontoxic doses of W-13 enhanced the hinibition of cell proliferation under the condition of BLM 0.5 - 2.5 μg/ ml. In colony forming test, the survival fraction of S-180 cells treated with BLM plus W-13 was decreased to 1/87 - 240 of that of the cells treated with BLM alone. The results suggest that W-13 can enhance antitumor activity of BLM in vitro and may be used as an synergist of BLM A5 in vivo.

INDUCTION OF APOPTOSIS IN S-180 AND S-180R TUMOR CELLS BY ADRIAMYCIN IN VIVO

Apoptosis of tumor cells have become a new standard for chemotherapy. It is useful to demonstrate induction of apoptosis in tumor cells by anti-cancer drugs in vivo. We reported the results of apoptosis induction in murine tumor cell line S-180 and it's resistant cell line S-180R by adriamycin in different dose and different time. We found that apoptosis in S-180 cells could be induced by low dose of adriamycin, the apoptosis was started at 24 h. after the administration, and reached to 62.5% of the cells to apptosis until 72 h. Comparison with the parental cell line, only 13% of S-180R cells were apoptosed. At high dose, 20% of S-180R cells were apoptosed, whereas, almost all S-180 cells were killed in the same time. The lymphocytes were appeared in abdominal cavity of the mice after treatment of adriamycin for 24 h. It was very interested to find out that there was no lymphocyte left in the abdominal cavity of the mice with S-180R cells treated at high dose of adriamycin.

EFFECTS OF DIFFERENT DOSAGE OF MOXA CONE ON DRUG RESISTANCE IN S-180R CELLS

Drug resistance is a very important problem for cancer chemotherapy. The ma-jor type of drug resistance is due to the overexpression of P-glycoproteins (P-170) in cancer cells. P-170 could pump the drug out of the cells, so the drug could not be accumulated enough to kill the cell-s. We had developed the adriamycin resistant cell line S-180R in BABL/c mice. The cells overexpressP-170 stably. Different dosages of moxa cone were used on Guanyuan (CV 4) point of the mice.Stimulation of drug accumulation was found after administration of moxibustion for 30 min and a doseresponse manner was shown below 400 mg of moxa cone, but a little decrease of the stimulation wasobtained when using 600 mg of moxa cone. These results confirmed positive effects of moxibustion onovercoming the drug resistance through stimulation of drug accumulation in the cancer cells.

Saponin from Tupistra chinensis Bak Inhibits NF-κB Signaling in Sarcoma S-180 Cell Mouse Xenografts

This study examined the effect of saponins from Tupistra chinensis Bak （STCB） on the growth of sarcoma S-180 cells in vitro and in mouse xenografts as well as the underlying mechanisms. Cell proliferation was assessed by MTT assay. Cell cycle distribution was determined by flow cytometry. Sarcoma S-180 tumor-bearing mice were treated with different doses of STCB with 10 μg/mL 5-fluorouracil （5-Fu） as a positive control. The activity of nuclear factor （NF）-κB was detected by gel mobility shift assay. The mRNA level of NF-κB was determined by real-time quantitative RT-PCR. The results showed that in vitro STCB inhibited the growth of S-180 cells in a concentration-dependent manner, which was accompanied by cell cycle arrest at S-phase. In vivo STCB significantly inhibited the growth of S-180 tumor mouse xenografts in a dose-dependent manner with apparent induction of cell apoptosis. Moreover, STCB inhibited the activity of NF-rd3 p65 and reduced the expression of NF-κB p65 mRNA in mouse xenografts. It was concluded that STCB inhibits the proliferation and cell cycle progression of S- 180 cells by suppressing NF-κB signaling in mouse xenografts. Our findings suggest STCB is a promising agent for the treatment of sarcoma.

Preliminary Validation of Tumor Cell Attachment Inhibition Assay for Developmental Toxicants With Mouse S180 Cells

This study was designed to explore the possibility of using ascitic mouse sarcoma cell line (S180) to validate the mouse tumor cell attachment assay for developmental toxicants, and to test the inhibitory effects of various developmental toxicants. The results showed that 2 of 3 developmental toxicants under consideration, sodium pentobarbital and ethanol, significantly inhibited S180cells attachment to Concanavalin A-coaed surfaces. Inhibition was dependent on concentration, and the IC50 (the concentration tha reduced attachment by 50% ), of these 2 chemicals was 1.2×10-3mol/L and 1 .0 mol/L, respectively. Anoher developmental toxiant, hydmiortisone, did not show inhibitory activity. Two non-developmental toxicants, sodium chloride and glycine were also tested and these did not decrease attachment rates. The main results reported here were generally sindlar to those obtained with ascitic mouse ovdrian tumor cells as a model. Therefore, this study added further evidence to the conclusion that cell specificity does not lindt attachment inhibition to Con A-coated surfaces, so S180 cell may serve as an altemative cell model, especially when other cell lines are unavailable. Furthermore, after optimal validation, it can be suggested that an S180 cell attachment assay may be a candidate for a series of assays to detect developmental toxicants.

DEVELOPMENT OF AN ADRIAMYCIN RESISTANT MURINE TUMOR S-180 CELL LINE IN VIVO

Adriamycin resistant cells were obtained from low dotage treated BABL/c mice Inoculated with S-180 cells. Resistance of these cells for adriamycin was 66-fold more than their parental cells. The resistance for a typical DNA topoisomerase Ⅱ inhibitor VP16 (Etopcaide) was increased 9 times. Overexpression of multidrug resistant gene (MDR gene) products, P-glycoproteins (P-1 70), was also demonstrated by immunohistochemistry. Furthermore, the ability of the resistant cells to reduce net cellular drug accumulation measured by flow fluorescence cytometry was 89-fold higher than their parental cells. These results support the hypothesis that the resistance of S-180R cells to adriamycin was mainly due to the overexpression of P-glycoproteins. The S-180R cells will be useful to select drugs or some other therapeutic strategies to overcome multidrug resistance in vivo.

Effectiveness and safety of combined nimotuzumab and S-1 chemotherapy with concurrent radiotherapy for locally advanced esophageal cancer in malnourished and elderly patients:A prospective phaseⅡstudy

Objective:Definitive chemoradiotherapy(dCRT)is the standard treatment for unresectable locally advanced esophageal cancer.However,this treatment is associated with substantial toxicity,and most malnourished or elderly patients are unable to complete this therapy.Therefore,there is a need for a more suitable radiotherapy combination regimen for this population.This study was aimed to evaluate the efficacy and safety of a combination regimen comprising chemotherapy with nimotuzumab and S-1 and concurrent radiotherapy for patients with fragile locally advanced esophageal cancer with a high Nutritional Risk Screening 2002(NRS-2002)score.Methods:Eligible patients with unresectable esophageal carcinoma who had an NRS-2002 score of 2 or higher were enrolled.They were treated with S-1 and nimotuzumab with concurrent radiotherapy,followed by surgery or definitive radiotherapy.The primary endpoint was the locoregional control(LRC)rate.Results:A total of 55 patients who met the study criteria were enrolled.After completion of treatment,surgery was performed in 15 patients and radiotherapy was continued in 40 patients.The median follow-up period was 33.3[95%confidence interval(95%CI,31.4−35.1)]months.The LRC rate was 77.2%(95%CI,66.6%−89.4%)at 1 year in the entire population.The overall survival(OS)rate and event-free survival(EFS)rate were 57.5%and 51.5%at 3 years,respectively.Surgery was associated with better LRC[hazard ratio(HR)=0.16;95%CI,0.04−0.70;P=0.015],OS(HR=0.19;95%CI,0.04−0.80;P=0.024),and EFS(HR=0.25;95%CI,0.08−0.75;P=0.013).Most adverse events were of grade 1 or 2,and no severe adverse events occurred.Conclusions:For malnourished or elderly patients with locally advanced esophageal cancer,radiotherapy combined with nimotuzumab and S-1 is effective and has a good safety profile.

Fruiting increases total content of flavonoids and antiproliferative effects of Cereus jamacaru D.C. cladodes in sarcoma 180 cells in vitro

Objective: To evaluate the influence of fruiting phenological stage on total flavonoid content, antioxidant activity, and antiproliferative effects of Cereus jamacaru(C. jamacaru)(mandacaru) cladodes and fruit. Methods: Fruit and cladodes at vegetative and fruiting stage of C. jamacaru were collected. The fruit was dissected and bark, pulp, and seeds were separated. Vegetative and fruiting cladodes, together with bark, pulp, and seeds were used to obtain five hydroalcoholic extracts. The extracts were investigated for total flavonoid content, using AlCl3 colorimetric method, antioxidant activity by 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging capacity and Fe^(2+) ion chelating activity, and in vitro antiproliferative effects(sarcoma 180 cells) by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazolium bromide assay. Results: The extract of C. jamacaru cladodes at the fruiting stage showed higher flavonoid content compared to the other extracts. Seed extracts showed the highest antioxidant activity in 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) assays, and the extract of cladodes at vegetative stage showed better antioxidant activity in Fe^(2+) ion chelating activity. The extract of fruiting cladodes promoted higher antiproliferative effects compared to the other extracts. Conclusions: These findings suggest that fruiting increases the content of flavonoids and antiproliferative effects of C. jamacaru cladodes. Data reinforce the potential use of C. jamacaru cladodes and fruits as natural antioxidants and potent anticancer agent.

<i>Porphyromonas gingivalis</i>-Induced GEF Dock180 Activation by Src/PKC<i>δ</i>-Dependent Phosphorylation Mediates PLC<i>γ</i>2 Amplification in Salivary Gland Acinar Cells: Modulatory Effect of Ghrelin

Phospholipase Cγ2 (PLCγ2) plays a pivotal role in mediation of inflammatory reaction to bacterial lipopolysaccharide (LPS) as well as serves as a key target in modulatory influence of the hormone ghrelin. Here we explore the involvement of Rac1 and its activator, guanine nucleotide exchange factor (GEF), Dock180, in mediation of PLCγ2 activation in salivary gland acinar cells in response to P. gingivalis LPS and ghrelin. We show that stimulation of the acinar cells with the LPS leads to up-regulation in Dock and PLCγ2 activation, and is reflected in the membrane translocation of Rac1 and PLCγ2, while the effect of ghrelin is manifested by the suppression in Rac1 translocation. Further, we reveal that stimulation with the LPS leads to Dock180 phosphorylation on Tyr and Ser, while the modulatory influence of ghrelin, manifested by a drop in membrane Rac1-GTP, is asso-ciated with a distinct decrease in Dock180 phosphorylation on Ser. Moreover, we demonstrate that phosphorylation on Tyr remains under the control of Src kinase and is accompanied by Dock180 membrane translocation, while protein kinase Cδ(PKCδ) is involved in the LPS-induced phosphorylation of the membrane-recruited Dock180 on Ser. Thus, our findings underscore the role of Src/PKCδ-mediated GEF Dock180 phosphorylation on Tyr/Ser in modulation of salivary gland acinar cell PLCγ2 activation in response to P. gingivalis as well as ghrelin.

柴胡提取物对人肝癌细胞和小鼠S-180肉瘤的抑制作用

目的 :研究柴胡水提物对体外培养的人肝癌 SMMC-772 1细胞线粒体代谢活性、细胞增殖及小鼠移植性 S-1 80实体瘤的影响。方法 :中药柴胡 ( BCDC)经粉碎、浸提、超速离心、减压浓缩、冷冻干燥得柴胡提取物。人肝癌 SMMC-772 1细胞经不同浓度柴胡提取物处理不同时间后 ,检测了各实验组和对照组细胞的有丝分裂指数、细胞增殖及线粒体活性几方面的变化。S-1 80肉瘤小鼠连续 9d腹腔注射 3 3 .3 mg/ kg体重 BCDC提取物 ,停药 1 5d后 ,杀死小鼠称量瘤重。结果 :1经 3 55mg/ L BCDC提取物处理人肝癌 SMMC-772 1细胞 2 4 h,其线粒体活性为对照组的 50 .2 6% ,处理 72 h为 2 4 .82 %。2以 3 55mg/ L BCDC提取物处理 7d中 ,人肝癌细胞数分别是血清对照组( FCS)的为 57.5% ,4 5.88% ,3 7.0 6% ,2 8.3 7% ,2 0 .90 % ,1 2 .89% ,1 4.1 8% ;无血清对照组 ( NON)的 65.0 9% ,62 .2 4 % ,56.99% ,4 9.0 8% ,4 0 .78% ,2 6.78% ,2 8.4 1 %。 3经 BCDC提取物处理 2 4 h后 ,即使更换正常培养基 ,人肝癌细胞数在 96h的恢复培养中仍低于处理前的细胞数。 4有丝分裂指数实验组为 0 .3 0 ,对照组分别为 5.3和5.4 ( P<0 .0 0 1 )。 5BCDC提取物对小鼠 S-1 80实体瘤抑制率为 87.2 1 %。

盐酸洛拉曲克在体内、外对S-180细胞株的抗增殖作用

研究全合成新型胸苷合成酶抑制剂盐酸洛拉曲克在体内、外对S 1 80细胞株及正常人胚肾HEK2 93细胞的抗增殖作用 ;使用MTT法测定抑制率 ,以提高荷腹水瘤小鼠存活时间及荷实体瘤瘤重减轻情况为指标考察盐酸洛拉曲克对S 1 80所致肿瘤的治疗作用 .结果表明 :在体外盐酸洛拉曲克对S 1 80肿瘤细胞株有较强的细胞毒作用 ,对正常细胞HEK2 93抑制作用较弱 (P <0 .0 5 ) ;体内实验显示盐酸洛拉曲克可明显提高荷腹水瘤小鼠的存活时间 ,减轻荷实体瘤小鼠的瘤重 ,疗效与氟尿嘧啶 ( 1 0mg kg)相当 (P >0 .0 5 )或更好 (P <0 .0 5 ) .可见 ,盐酸洛拉曲克在体内、外对S 1 80肿瘤细胞有显著的抗增殖作用 。

美味牛肝菌多糖的生物活性及其抗S-180肿瘤的效应

美味牛肝菌（Boletusedulis）于实体，经水溶醇析法提取的多糖呈灰白色，其总精含量为73．21％，蛋白质含量为7．14％．经动物试验表明：该多糖（BP）能显著增加胸腺、脾脏器官质量，并可促进机体的免疫功能．采用ip，100μg（kg·d）剂量后，可使供试小白鼠外周血中的白细胞总数增加17％，同时红细胞中SOD酶活性显著提高，多达42．5％．还可显著促进牌淋巴细胞的转化．采用多糖液0．1mL／（只·d），剂量为10mg／（kg·d），腹腔注射已感染S-180肿瘤的小鼠，同样还能提高其生命延长率18．78％，具有明显的抗S-180肿瘤的效应．

11种食用菌的粗多糖抗S-180肉瘤的实验研究

用口磨 (Tricholomasp .)、松口蘑 (Tricholomamatsutake)、冬虫夏草C1 5(Cordycepssinensis)等 1 1种食用菌的粗多糖进行抗肿瘤药物实验。结果显示 :1 1种粗多糖能使带有S - 1 80腹水瘤的昆明小鼠生命延长率提高到 6 .60 %～ 31 .74% ,其中以口蘑多糖效果最佳 ,可达 31 .47% ,口蘑、冬虫夏草C1 5、香菇 (Lentinusedodes)三种多糖对带瘤小鼠的生命延长率达到了显著水平。该结果不定期表明 :虽属于同类冬虫夏草 ,但因菌株不同 ,以致抗S - 1 80腹水瘤效果差异悬殊 ,两者相差达 1 6 .75 %。以上菌种粗多糖抗S - 1 80肉瘤的最适注射剂量为 1 0mg·kg- 1 ·d- 1 。

电脉冲致离体S-180细胞电穿孔的观察与研究

扫描电镜下我们观察到了一定强度电脉冲致 S- 180细胞电穿孔的现象。同时分别改变电压、电容、脉冲个数 ,对 S- 180离体细胞进行电脉冲作用 ,通过苔盼蓝追踪 ,发现在一定的条件下 ,随着对 S- 180离体细胞电穿孔的电压越大、电容越小、脉冲个数越多 ,其穿孔百分率越大。

螺旋藻多糖对小鼠S-180肉瘤的免疫抑制作用

研究了螺旋藻多糖对小鼠S-180肉瘤的抑制作用。结果显示:螺旋藻多糖在50～200μg/g的剂量范围对小鼠S-180肉瘤均有一定的抑带作用,具有明显升高小鼠外用血白细胞的功能,促进荷瘤小鼠脾淋巴细胞转化作用,并有较好的剂量-效应关系,促进荷瘤小鼠脾NK细胞对靶细胞的细胞毒作用。提示螺旋藻多糖可通过全面调节机体的免疫功能,刺激T细胞、NK细胞等功能来达到控制和杀灭肿瘤细胞的目的。

山仙颗粒含药血清诱导S-180肉瘤细胞凋亡及其对bcl-2、Bax基因表达的影响

目的:探讨山仙颗粒(SXG)含药血清诱导体外培养S-180肉瘤细胞的凋亡作用及其对凋亡相关基因bcl-2及Bax表达的影响。方法:以不同浓度的山仙颗粒含药血清作用于体外培养的S-180肉瘤细胞,待24、48、72h后倒置显微镜下观察细胞形态变化并制备细胞涂片,采用TUNEL法检S-180肉瘤细胞凋亡率,免疫细胞化学(SABC)检测S-180肉瘤细胞中凋亡相关基因bcl-2、Bax表达的变化。结果:SXG含药血清可明显诱导S-180肉瘤细胞凋亡,导致其细胞形态改变,并能使bcl-2表达降低(P<0.01)、Bax表达增强(P<0.01),且呈浓度和时间依赖性。结论:SXG含药血清能有效诱体外培养的S-180肉瘤细胞凋亡,其机制可能与抑制bcl-2表达,促进Bax表达有关。

山仙颗粒对S-180荷瘤小鼠T细胞活性及Caspase-3的影响

目的:探讨山仙颗粒(SXG)对S-180荷瘤小鼠T细胞活性及Caspase-3的影响。方法:将60只昆明种小鼠随机选取12只作为空白对照组,其余48只建立S-180荷瘤小鼠模型后随机分为模型组及SXG低(0.025 g/mL)、中(0.05 g/mL)、高(0.1 g/mL)剂量组各12只,空白对照组、模型组每日早晚灌胃生理盐水0.4 mL,其余各组每日早晚灌胃相应剂量药物,连续30天。末次给药24小时后,采用颈椎脱臼法处死小鼠,游离脾脏,剥离肿瘤组织;应用MTT比色法及免疫组织化学法检测荷瘤小鼠脾脏T细胞毒效应及S-180实体瘤组织中Caspase-3的表达。结果:SXG低、中、高剂量组小鼠脾脏T细胞对于S-180荷瘤小鼠细胞的抑制率高于模型组(P<0.01),且与SXG浓度呈正相关;各组效应细胞与靶细胞的比例不同,对S-180荷瘤小鼠细胞的抑制作用也不同(P<0.01),且与T细胞浓度呈正相关。SXG低、中、高剂量组S-180荷瘤小鼠肿瘤组织中Caspase-3表达率高于模型组(P<0.01),且与SXG浓度呈正相关。结论:SXG具有抑制肿瘤生长的作用,其机制可能与其增强T细胞活性进而抑制肿瘤细胞增殖及促进Capase-3介导的肿瘤细胞凋亡有关。

横纹肌细胞培养液对S-180细胞生长抑制作用的体外实验研究

目的:观察横纹肌细胞微环境对肿瘤细胞生长的影响,探讨横纹肌内转移瘤罕见这一现象的发生机制。方法:采用酶解法原代培养新生Wistar大鼠心肌细胞和骨骼肌细胞,形态学和免疫酶染色方法检测横纹肌α-肌动蛋白进行细胞鉴定,免疫酶染色法进行细胞纯度鉴定,MTT法分析比较骨骼肌和心肌细胞细胞培养液对S-180细胞和正常肾小球系膜细胞的体外生长抑制作用,并运用胰酶消化法,热灭活法初步探讨横纹肌细胞培养液中抑瘤物质的理化性质。结果:S-180细胞与CMCM、MMCM共同培养后,细胞存活率明显下降,与DMEM相比差异均具有显著性(P<0.05),MCs细胞的细胞存活率无明显影响(P>0.05)。DDP对S-180细胞和MCs细胞均具有显著性抑制作用(P<0.05)。心肌细胞培养液和骨骼肌细胞培养液经胰酶消化后抑制活性仍然存在(P<0.05),经热灭活后活性消失(P>0.05)。结论:新生大鼠横纹肌细胞培养液对S-180细胞的增殖有明显的抑制作用;而对正常细胞的增殖无明显影响,与顺铂的抑制作用明显不同。横纹肌细胞培养液中有可能存在一种抑瘤物质,可能是横纹肌转移瘤罕见的关键因素。

SD大鼠山仙颗粒含药血清对S-180肉瘤细胞VEGF的表达及对细胞凋亡影响的实验研究

目的探讨山仙颗粒(SXG)含药血清对S-180肉瘤细胞VEGF表达水平及细胞凋亡的影响。方法将不同浓度的SD大鼠SXG含药血清作用于体外培养的S-180肉瘤细胞,24h、48h、72h后制备细胞涂片,用免疫细胞化学法(SABC)检测不同浓度、不同作用时间的含药血清对S-180肉瘤细胞中VEGF表达的影响,用TUNEL法检测S-180肉瘤细胞凋亡率。结果 SXG含药血清能下调VEGF表达水平、促进S-180肉瘤凋亡,且呈浓度和时间依赖性。结论 SD大鼠SXG含药血清能显著降低S-180肉瘤细胞中VEGF的表达水平,提高S-180肉瘤细胞凋亡率,其临床抑转移、抗肿瘤机制可能与其下调VEGF表达水平及促进细胞凋亡有关。

Effects of Low Dose Radiation on Tumor Apoptosis, Cell Cycle and Apoptosis-Related Protein Bcl-2 in Tumor-Bearing Mice

To study the effects of low dose radiation (LDR) on tumor apoptosis, cellcycle progression and changes of apoptosis-related protein Bcl-2 in tumor-bearing mice. Methods:Male mice of Kunming strain were implanted subcutaneously with S180 sarcoma cells in the left inguenas an in situ experimental animal model. Seven days later, the mice were subjected to 75 mGywhole-body γ-irradiation. At 24 and 48 h after the irradiation, all mice were sacrificed. The tumorsizes were measured, and tumor cell apoptosis and cell cycle progression were analyzed by flowcytometry. The expression of apoptosis-related protein Bcl-2 and the apoptotic rate of tumor cellswere observed by immunohistochemistry and electron microscopy. Results: Tumors grew significantlyslower after LDR (P 【 0.05). The tumor cells were arrested in G1 phrase and the expression of Bcl-2protein decreased at 24 h. Apoptotic rate of tumor cells was increased significantly at 48 h afterLDR (P 【 0.01). Conclusion: LDR could cause a G1-phase arrest and increase the apoptosis of tumorcells through the low level of apoptosis-related protein bcl-2 in the tumor-bearing mice. Theorganized immune function and anti-tumor ability are markedly increased after LDR. Our studyprovides practical evidence of clinical application to cancer treatment.