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.

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.

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.

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.

Inhibitory activity of polysaccharide extracts from three kinds of edible fungi on proliferation of human hepatoma SMMC-7721 cell and mouse implanted S180 tumor

AIM To determine the activities ofpolysaccharide extracts from Flammulina velutipes (Curt. ex Fr. ) Sing (FV), Lentinusedodes (LE) and Agaricus bisporus Sing (AB)on the proliferation of human hepatoma SMMC-7721 cells in vitro and on mouse implanted S-180tumors in vivo.METHODS The polysaccharide extracts were isolated from the fruit bodies of FV, LE and AB by the methods of hot-water extraction, Sevag’sremoval of proteins, ethanol precipitation,trypsin digestion and ethanol fractionalprecipitation. Human hepatoma SMMC-7721 cells were treated with 50 mg/L Polysaccharide extracts, and the mitosis index, mitochondria activity and cell proliferation were detected at different times in both control and experimental groups. The mice with S-180 implanted tumors were injected with the polysaccharide extracts at 24 mg/ kg body weight for 9 d and the tumorweight was measured on the 15th day.RESULTS The mitosis index of hepatoma cells in vitro could be significantly decreased by treatment with the polysaccharide extracts fromthe three kinds of edible fungi (P < 0 .005 ). Thecell numbers and mitochondria activity of SMMC7721 cells treated with polysaccharide extracts were lower than those in control groups (P <0.005). The inhibition rates of polysaccharide extracts against implanted S-180 tumors in mice were 52.8%, 56.6% and 51 .9% respectivelycompared with that in c0ntrol gr0ups.CONCLUSI0N The POIysaccharide extractsfrom the three kinds of edible fungi could inhibitnot only the Cultured malignant cells in vitfO butalso impIanted Sl80 tum0r i0 vivo.

Antitumor effect and mechanism of Gecko on human esophageal carcinoma cell lines in vitro and xenografted sarcoma 180 in Kunming mice

AIM: To investigate the anti-tumor effect of Chinese medicine Gecko on human esophageal carcinoma cell lines and xenografted sarcoma 180 in Kunming mice and its mechanism. METHODS: The serum pharmacological method was used in vitro . The growth rates of the human esophageal carcinoma cells (EC9706 or EC1) were measured by a modifi ed 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The transplanted tumor model of the mouse S180 sarcoma was established. Fifty mice were randomly divided into fi ve groups (n = 10). Three Gecko groups were treated respectively with oral administration of Gecko powder at a daily dose of 13.5 g/kg, 9 g/kg, and 4.5 g/kg. The negative group (NS group) was treated with oral administration of an equal volume of saline and the positive group (CTX group) was treated with 100 mg/kg Cytoxan by intraperitoneal injection at the fi rst day. After 2 wk of treatment, the anti-tumor activity was evaluated by tumor tissue weighing. The impact on immune organ was detected based on the thymus index, spleen index, phagocytic rate and phagocytic index. The protein expression of vascular endothelingrowth factor (VEGF) and basic fibroblast growth factor (bFGF) were detected by immunohistochemistry. The cell apoptotic rate was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay. RESULTS: The A value in each group treated with Gecko after 72 h was reduced signif icantly in EC9706 and in EC1. The tumor weight in each group of Gecko was decreased signifi cantly (1.087 ± 0.249 vs 2.167 ± 0.592; 1.021 ± 0.288 vs 2.167 ± 0.592; 1.234 ± 0.331 vs 2.167 ± 0.592; P < 0.01, respectively). However, the thymus index and Spleen index of mice in Gecko groups had no significant difference compared with the NS group. The immunoreactive score of VEGF and bFGF protein expression of each Gecko group by immunohistochemical staining were lowered signifi cantly. The apoptosis index (AI) of each group was increased progressively with increase of dose of Gecko by TUNEL. CONCLUSION: Gecko has anti-tumor effects in vitro and in vivo; induction of tumor cell apoptosis and the down-regulation of protein expression of VEGF and bFGF may be contributed to anti-tumor effects of Gecko.

Neural cell adhesion molecule-180 expression as a prognostic criterion in colorectal carcinoma:Feasible or not?

AIM： To evaluate the frequency of neural cell adhesion molecule （NCAM）-180 expression in fresh tumor tissue samples and to discuss the prognostic value of NCAM-180 in routine clinical practice.METHODS： Twenty-six patients （16 men, 10 women） with colorectal cancer were included in the study. Fresh tumor tissue samples and macroscopically healthy proximal margins of each specimen were subjected to flow-cytometric analysis for NCAM-180 expression.RESULTS： Flow-cytometric analysis determined NCAM-180 expression in whole tissue samples of macroscopically healthy colorectal tissues. However, NCAM-180 expression was positive in only one case （3.84%） with well-differentiated Stage Ⅱ disease who experienced no active disease at 30 months follow-up. CONCLUSION： As a consequence of the limited number of cases in our series, it might not be possible to make a generalisation, nevertheless the routine use of NCAM-180 expression as a prognostic marker for colorectal carcinoma seems to be unfeasible and not cost-effective in clinical practice due to its very low incidence.

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.

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.

绞股蓝总皂甙对小鼠S_(180)肉瘤及K_(562)细胞的抑制作用

目的 绞股蓝总皂甙 (GP)对小鼠S180 肉瘤及K562 细胞的抑制作用。方法 通过动物实验观察绞股蓝总皂甙对小鼠S180 肉瘤生长状况、肿瘤坏死面积 (TNA)与肿瘤总面积 (TTA)的比率、瘤周瘤内免疫活性细胞浸润状况及荷瘤小鼠脾脏的影响 ;通过细胞培养观察绞股蓝总皂甙对K562 细胞生长的抑制作用。结果 经重复实验证实 ,GP能显著抑制小鼠S180 肉瘤的生长 ,TNA与TTA的比率显著增加 ,瘤周尤其是瘤内淋巴细胞、巨噬细胞浸润数量明显增加 ,荷瘤小鼠脾重增加、脾白髓数目增多、体积增大。同时证实 ,GP对K562 细胞株具有明显的生长抑制作用。结论 GP的抑瘤作用主要是直接杀伤瘤细胞 。

复方抗瘤冲剂抑制小鼠肉瘤180的生长及对p53和增殖细胞核抗原表达的影响

探讨复方抗瘤冲剂对小鼠肉瘤 180的作用及机制。通过体内实验 ,观察复方抗瘤冲剂对小鼠肉瘤180的抑瘤作用和免疫组化法检测p5 3和增殖细胞核抗原 (PCNA)的表达水平。结果显示 ,经复方抗瘤冲剂及其不同组合和CTX治疗 10天后 ,对小鼠肉瘤 180有不同程度的抑制作用 ,抑瘤率分别为 2 0 31%、6 5 6 %、12 5 %、19 0 6 %和 30 3%。同时各组药物对p5 3和PCNA的表达水平也有不同程度的影响。提示复方抗瘤冲剂对小鼠肉瘤 180的生长具有一定的抑制作用 ,其机制可能与抑制突变型p5

五味子多糖对S180荷瘤小鼠红细胞免疫抗肿瘤的作用

目的:探讨五味子多糖(FSCP)增强荷瘤小鼠红细胞结构稳定性与红细胞免疫黏附功能的抗肿瘤作用及其红细胞免疫调节机制。方法:实验动物随机分为正常对照组、生理盐水组、环磷酰胺(CP)组及FSCP高、中、低3个剂量组。用药7d后,眼球取血,获得红细胞悬液。采用激光共聚焦技术检测荷瘤小鼠红细胞Ca2+含量的变化;采用分光光度法检测红细胞膜唾液酸(SA)含量、红细胞膜封闭度;采用荧光偏振法检测红细胞膜脂流动性(LFU);采用花环法检测红细胞天然免疫黏附能力。结果:与生理盐水组比较,FSCP各组能降低荷瘤小鼠红细胞Ca2+的浓度(P<0.01),增加红细胞膜表面SA含量(P<0.01)以及红细胞膜自我封闭度(P<0.05或P<0.01)。高剂量FSCP可提高S180荷瘤小鼠红细胞膜流动性(P<0.05);增强S180荷瘤小鼠红细胞免疫黏附肿瘤细胞的能力(P<0.05),FSCP高剂量组免疫花环率达到24.17%。结论:FSCP可能通过改善S180荷瘤小鼠红细胞膜的功能状态,提高膜的稳定性,增强红细胞免疫黏附肿瘤细胞的能力,从而发挥其抗肿瘤作用。

阿霉素在小鼠体内诱导S－180细胞株抗药性的实验研究

利用ＢＡＢＬ／ｃ小鼠，腹腔接种Ｓ－１８０瘤细胞，阿霉素腹腔注射治疗１５个周期培育传代，得到抗阿霉素的Ｓ－１８０细胞株（Ｓ－１８０Ｒ）。此抗药细胞对阿霉素的抗药性比亲本细胞提高６６倍。对典型的ＤＮＡ拓扑异构酶Ⅱ抑制刻ＶＰ１６（Ｅｔｏｐｏｓｉｄｅ）抗药性增加９倍。经免疫组化进一步证实，抗药细胞显示多药抗药基因（ＭＤＲｇｅｎｅ）产物Ｐ－１７０糖蛋白过表达。流式萤光细胞仪测试结果也表明抗药细胞比亲本细胞排出药物能力提高８９倍。可以肯定这是ＭＤＲ基因过表达产物所致。本工作为筛选有效逆转抗药功能的药物及其他治疗手段提供了有用的动物模型。

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.

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 。

有机锗多酸衍生物对S_(180)肿瘤细胞DNA合成的抑制作用

目的 探讨有机锗多酸衍生物对S1 80 肿瘤细胞DNA合成的抑制作用。方法 用3H TdR掺入法检测有机锗多酸衍生物在体外对S1 80 肿瘤细胞DNA合成的抑制作用 ,观察不同剂量有机锗多酸衍生物对S1 80 实体瘤的抑制作用和对免疫器官的影响。结果 体外实验表明 ,有机锗多酸衍生物对S1 80 细胞的生长具有明显的抑制作用 ,而且浓度越高 ,抑制作用越强。体内实验也表明 ,有机锗多酸衍生物对S1 80 实体瘤的生长具有抑制作用 ,而且剂量越大 ,抑制作用越强。且有机锗多酸衍生物可防止荷瘤鼠脾脏重量的下降。结论 有机锗多酸衍生物可抑制肿瘤细胞生长 。

壳寡糖对S180肉瘤细胞凋亡、细胞周期以及凋亡相关蛋白Bcl-2、Bax的影响

目的探讨壳寡糖对S180肉瘤细胞的生物学效应,为其治疗肿瘤提供新的理论基础。方法体外实验:用流式细胞仪测壳寡糖作用下S180肉瘤细胞周期、细胞凋亡情况。体内实验:建立S180肉瘤小鼠皮下移植瘤模型,给予壳寡糖后,测瘤体积,并用免疫组化法测瘤组织Bax、Bcl-2蛋白的表达情况。结果体外实验:高浓度壳寡糖作用后S180肉瘤细胞阻滞于G0/G1期,同时S180肉瘤细胞凋亡增加。体内实验:壳寡糖对小鼠S180肉瘤有明显的抑制作用并能提高促凋亡基因Bax的表达,降低抑凋亡基因Bcl-2的表达。结论壳寡糖可抑制S180肉瘤细胞的生长,使S180细胞阻滞于G0/G1期并诱导其发生凋亡,其机制可能与Bcl-2蛋白下调而Bax蛋白上调有关。

聚焦超声结合原卟啉对S180肿瘤细胞杀伤作用的研究

采用频率为2.2MHz及不同强度的聚焦超声激活原卟啉对小鼠腹水型S180肿瘤细胞的杀伤作用进行研究。台盼蓝拒染法检测了不同处理后细胞的相对存活率,通过扫描电镜和透射电镜观察细胞超微结构的变化。实验结果表明:单纯原卟啉对S180肿瘤细胞几乎没有影响,单纯超声和超声结合原卟啉对细胞均有一定的损伤,肿瘤细胞受损程度随着声照强度的增加而加剧,并且在一定范围内随着原卟啉剂量浓度的加大,细胞受损也逐渐增强。当原卟啉浓度为120μM时,超声激活原卟啉对腹水型S180肿瘤细胞的协同杀伤效应表现最为明显。