绿茶和硒抗促癌作用的初步研究

本文采用细胞培养技术，用刻痕标记荧光扩散试验对绿茶和硒及其联合抗TPA抑制NIH 3T3细胞间隙连接通讯的作用进行了初步研究．结果表明。绿茶水浸液、亚硒酸钠和硒酸钠均具有明显拮抗TPA的作用，且在一定范围内与作用浓度和时间呈明显的正相关关系。其中亚硒酸钠的拮抗作用显著大于硒酸钠的作用．绿茶水浸液和硒的联合抗TPA作用也很明显。     

一组初筛抗促癌物方法的探讨

抗促癌物的研究和应用对肿瘤预防有重要意义。由于促癌作用的机制复杂,目前还缺乏比较成熟的筛选促癌物和抗促癌物快速方法。本研究选用文献已报道的三种经动物证明具有抗促癌作用的化合物或提取物,即姜黄素(Curcumin),表儿茶素复合物(Epi-catechin Compounds),迷迭香抗氧化成分(Rosemary antioxdants),观察其对促癌物TPA所诱导的生物学反应的抑制作用。结果表明,这三种抗促癌物均可以明显抑制TPA     

没食子酰表没食子儿茶素对促癌物TPA抑制作用的研究

本研究首次证明,绿茶多酚类的主要成分没食子酰表没食子儿茶素(EGCG)具有很强的抑制促癌物TPA诱导的皮肤炎性水肿和增生反应,以及抑制TPA促进经甲基胆蒽启动的BALB/3T3细胞的转化作用。本实验结果表明,EGCG可能是绿茶多酚提取物中抑制TPA促癌作用的重要成分。     

Bhas 42细胞转化试验高通量检测方法的建立及应用

目的: 建立Bhas 42细胞转化试验高通量检测方法,并应用于染料木黄酮(GEN)潜在致癌性的检测中。方法:建立Bhas 42细胞转化试验方法,比较细胞灶法和H2O2法对结果判定的影响。细胞灶法试验组在第21天直接固定染色。H2O2法试验组在细胞接种后第19天采用H2O2处理,染色并测定D(450)以确定细胞转化率。计算转化率以验证两种方法的相关性。并通过细胞生长试验选择适宜的GEN浓度进行细胞转化试验,H2O2法判定转化试验结果。结果:在细胞灶法中,启动试验3-甲基胆蒽(3-MCA)组、促癌试验佛波醇酯(TPA)组的转化灶个数分别与启动、促癌试验DMSO组相比明显升高(P2O2法中,启动试验3-MCA组、促癌试验TPA组的转化灶个数、D(450)分别与启动、促癌试验DMSO组相比均明显升高(PD(450)相对于阴性组有显著差异(P结论:成功建立了Bhas 42细胞转化试验的H2O2法并实现了高通量检测,该法进一步缩短了实验周期并提高了结果的客观性,适用于非遗传毒性致癌物的体外早期筛选。GEN在促癌试验中呈阳性,但在启动试验中呈阴性,提示其可能是非遗传毒性致癌物。     

12-0-十四酰佛波-13-乙酸酯对培养细胞和小鼠皮肤脂质过氧化的影响

研究了促癌剂12—0—十四酰佛波—13—乙酸酯(TPA)对脂质过氧化的影响。经TPA处理的结肠癌细胞共脂质过氧化明显降低;经TPA处理的小鼠皮肤其脂质过氧初期升高,其后降低。γ—亚麻酸抵消TPA对小鼠皮肤脂质过氧化的影响。实验结果支持吞噬细胞,如中性粒细胞和巨噬细胞,对佛波酯的反应方式是产生超氧化物阴离子和氧自由基,但不表明氧自由基在促癌作用中起重要作用。推测促癌剂TPA引起脂质过氧化减弱的促癌过程。     

肿瘤促进物TPA对三株大鼠食管细胞的作用及其与细胞致瘤能力关系的研究

肿瘤促进物TPA(12—0—四癸酰基佛波—13—乙酸酯)是从巴豆中提取的。我国传统医学曾用它作泻药;现有研究证明,TPA是小鼠皮肤肿瘤促进物。本文研究TPA对三株大鼠食管上皮细胞株(RE149、REB_2和REB_2T)的作用,以及这一作用与细胞致瘤能力的关系。三株大鼠食管细胞中的RE149是非致瘤性细胞株,REB_2是前致瘤性细胞株,REB_2T是致瘤性细胞株。本研究比较了这三株细胞对TPA的不同反应性。结果表明,0.1—1200ng/ml范围内的TPA的作用不提高三株细胞的克隆形成率。10ng/ml TPA可诱导RE149细胞经历终末期分化和完全抑制增殖。与此相反,400—800ng/ml的TPA不能明显抑制细胞生长。由此可见,三株细胞对TPA的不同反应性与它们的致瘤能力密切相关。TPA可否用于临床鉴别肿瘤细胞和正常细胞值得进一步研究探讨。     

雌性激素体外诱发小鼠胚胎细胞肿瘤性转化的进一步研究

本文进一步观察戊酸雌二醇(E)和已酸孕酮(P)两种成分单独应用是否对小鼠胚胎细胞有致癌作用,同时与促进剂TPA联合应用是否会增强其致癌性。 EP、E、P、EP TPA、E TPA、P TPA组细胞均有转化灶形成。抗乌哇巴因抑制生长试验阴性,说明细胞无Na~ /K~ ATP酶位点突变。转化细胞在软琼脂培养基有细胞集落形成。未见TPA明显增强E和/或P的致癌性。上述结果表明雌性激素诱发细胞转化过程,不是通过细胞突变,而可能是通过阻断核分裂中期,形成多倍体,导致细胞转化。     

促癌剂TPA促进离体培养的鸡胚水晶体过度生长的实验研究

为了研究间隙连接在肿瘤促进中的可能作用,本室新近发展了一种水晶体离体培养技术,我们利用这种技术进行了促癌剂TPA促进水晶体过度生长的实验研究,即把20期的鸡胚水晶体培养于含有80ng/ml TPA的DMEM中,39℃下培养24小时。实验结果指出,实验组的水晶体非常明显地大于对照组(P<0.01)。已报道这个浓度的TPA可使间隙连接通讯安全抑制,表明以上结果可能与细胞间通讯的被抑制密切相关。联系到我们过去利用单克隆抗体所作的实验研究,本文结果进一步证明了间隙连接在生长控制中起重要作用;间隙连接通讯的抑制可能正是肿瘤促进的一种细胞机制。此外,本文报道的鸡胚水晶体离体培养似乎也可作为检测有害物质对生长发育影响的实验系统。     

88. 六种中草药抗突变及抗肿瘤活性的试验报告

目的探讨山楂、香菇、杏仁、大枣、板蓝根、仙鹤草等6种中草药的抗突变抗肿瘤作用及机制,以期进一步进行用于人群的应用研究,为肿瘤的预防提供科学依据。方法采用抗突变和致突变同步快速试验研究了6种中草药的抗突变和致突变性,以维生素C(VitC)做抗突变阳性对照剂,丝裂霉素阴性对照剂,并做加大鼠肝脏微粒体酶(S9)和不加S9的两种试验。采用体外抑瘤试验研究了6种中草药的4个浓度组对4种肿瘤细胞(人T细胞白血病细胞株MT-Ⅱ、小鼠纤维母细胞株L929、人卵巢癌细胞株SK、人巨噬细胞白血病细胞株K562)的抑制作用,以磷酸盐缓冲液做阴性对照,顺铂做阳性对照,培养细胞加受试物37℃,培养48h后,加氚-脱氧胸腺嘧啶核苷(3H-TdR),再继续培养24h后,收集细胞,测3H-TdR掺入的放射性强度。结果经加和不加S9两种试验,6种中草药均未显示有致突变毒性,山楂、杏仁、大枣和香菇显示对MMC引起的致突变作用有拮抗效应。山楂、香菇和仙鹤草水溶性提取液的各剂量组对4种肿瘤细胞的3H-TdR掺入的作用(P<0.01),板蓝根水溶性提取液仅高浓度组(4mg/ml)对L929、SK细胞有抑制3H-TdR掺入的作用(P<0.05),大枣水溶性提取液的各浓度组对4种细胞均未显示明显的抑制3H-TdR掺入的作用(P>0.05)。结论山楂、香菇、杏仁和大枣具有抗突变活性,山楂、香菇、仙鹤草对MT-Ⅱ、L929、SK、K5624种细胞的DNA合成有明显的抑制作用。     

Inhibition of tumor promoter activity toward mouse fibroblasts and their in vitro transformation by tissue inhibitor of metalloproteinases- 1 (TIMP-1)

Tissue inhibitor of metalloproteinases-1 (TIMP-1), a natural inhibitor of matrix metalloproteinases (MMPs), is known to inhibit invasion and metastasis of tumor cells. In the present study we examined anti-tumor promoter activity of TIMP-1 and its effect on in vitro cell transformation using BALB/3T3 cells in low serum culture medium. In the dye transfer assay the tumor promoter 12-O-tetradecanoylphorbol-13- acetate (TPA) continuously blocked gap-junctional intercellular communication (GJIC) of BALB/3T3 cells in confluent phase. TIMP-1 did not prevent transient inhibition of GJIC induced by TPA, but it quickly restored the reduced GJIC level to the control level. The recovery of GJIC was dependent on the concentration of TIMP-1 from 1 to 1000 ng/ml. In an in vitro two-stage transformation assay in which BALB/3T3 cells were treated with 0.5 microg/ml N-metyl-N'-nitro-N-nitrosoguanidine as initiator and 100 ng/ml TPA as promoter, TIMP-1 at concentrations > 10 ng/ml inhibited the focus formation of transformed cells by approximately 60%. TIMP-2 and a synthetic MMP inhibitor showed a similar inhibitory activity on in vitro cell transformation. Furthermore, zymographyic analysis showed that TPA treatment of BALB/3T3 cells induced secretion of gelatinase B and stromelysin-1 into the culture medium. These results indicate that TIMP-1 and TIMP-2 have inhibitory activity on in vitro transformation of cells. It seems likely that TPA-inducible MMPs are involved in carcinogenesis and TIMPs have a protective role against carcinogenesis in vivo.      

Preventive effect of epicatechin and ginsenoside Rb(2) on the inhibition of gap junctional intercellular communication by TPA and H(2)O(2)

The anticarcinogenic effects of epicatechin (EC) and ginsenoside Rb(2) (Rb(2)), which are major components of green tea and Korea ginseng, respectively, were investigated using a model system of gap junctional intercellular communication (GJIC) in WB-F344 rat liver epithelial cells. 12-O-tetradecanoylphorbol-13-acetate (TPA) and hydrogen peroxide, known as cancer promoters, inhibited GJIC in the epithelial cells as determined by the scrape loading/dye transfer assay, fluorescence redistribution assay after photobleaching, and immunofluorescent staining of connexin 43 using a laser confocal microscope. The inhibition of GJIC by TPA and H(2)O(2) was prevented with treatment of Rb(2) or EC. The effect of EC on GJIC was stronger in TPA-treated cells than in H(2)O(2)-treated cells, while the effect of Rb(2) was opposite to that of EC. EC, at the concentration of 27.8 microg/ml, prevented the TPA-induced GJIC inhibition by about 60%. Rb(2,) at the concentration of 277 microg/ml, recovered the H(2)O(2)-induced GJIC inhibition by about 60%. These results suggest that Rb(2) and EC may prevent human cancers by preventing the down-regulation of GJIC during the cancer promotion phase and that the anticancer effect of green tea and Korea ginseng may come from the major respective components, EC and Rb(2).     

Effects of peroxisome proliferators and 12-O-tetradecanoyl phorbol-13-acetate on intercellular communication and connexin43 in two hamster fibroblast systems

Effects of 12-O-tetradecanoyl phorbol 13-acetate (TPA) and the hepatic peroxisome proliferators (HPPs) clofibrate, methyl clofenapate (MCP), di(2-ethylhexyl)phthalate (DEHP) and mono(2-ethylhexyl)phthalate (MEHP) were studied in 2 gap junctional intercellular communication (GJIC) systems, metabolic cooperation in V79 cells and microinjection/dye transfer in Syrian hamster embryo (SHE) cells and V79 cells. TPA inhibited GJIC in both systems but was considerably more potent in V79 cells. SHE cells showed a rapid and transient inhibition of GJIC after exposure to HPPs, with maximal inhibition occurring at 5–15 min. The transient inhibition could be caused by metabolization of the compounds. Clofibrate and MEHP produced strong inhibition of metabolic cooperation in V79 cells at high concentrations, while the effect of MCP and DEHP was lower. However, DEHP, MEHP and clofibrate strongly inhibited dye transfer in V79 cells after a 30 min exposure. Clofibrate also showed a dose- and time-dependent effect on dye transfer in V79 cells. The phosphorylation status of the gap junction protein connexin43 (Cx43) changed minimally in SHE cells after exposure to TPA or HPPs. Cx43 from V79 cells was strongly affected by TPA, but not by HPPs. Immunofluorescence of Cx43 disappeared in both cell types when they were exposed to TPA and MEHP, but not to the other HPPs. Thus, there is no direct correlation between the inhibition of GJIC and changes in the phosphorylation status of Cx43 or the appearance of Cx43 in immunofluorescence experiments. The discrepancies may partly be explained by binding of accessory proteins to Cx43. We point out sequences that may be involved in such binding. Int. J. Cancer 73:240–248, 1997. © 1997 Wiley-Liss, Inc.     

Prevention of the down-regulation of gap junctional intercellular communication by green tea in the liver of mice fed pentachlorophenol

Much evidence has been documented supporting the hypothesis that the down-regulation of gap junctional intercellular communication (GJIC) is a cellular event underlying the tumor promotion process and that treatment to prevent the down-regulation or to up-regulate GJIC is important in preventing tumor promotion. We explored the potential preventive effects of green tea against the promoting action of pentachlorophenol (PCP) in mouse hepatocarcinogenesis, examining whether drinking green tea prevents the down-regulation of GJIC inhibition in the liver caused by tumorigenic doses of PCP. We used a modified in vivo GJIC assay, the incision loading/dye transfer method. Male B6C3F1 mice were given a green tea infusion for 1 week and then PCP was fed at a dose of 300 or 600 p.p.m. in the diet for the following 2 weeks, along with green tea treatment. A dose-related inhibition of GJIC in the hepatocytes was evident in the mice treated with PCP alone that was associated with a reduction in connexin32 (Cx32) plaques in the plasma membrane and an increase in the cell proliferation index. Drinking green tea significantly protected mice against GJIC inhibition, the reduction in Cx32 and the elevation of the labeling index. These findings suggest that green tea might act as an anti-promoter against PCP-induced mouse hepatocarcinogenesis via its ability to prevent down-regulation of GJIC.     

Apigenin and tangeretin enhance gap junctional intercellular communication in rat liver epithelial cells

Two flavones, apigenin and tangeretin, were studied for theirability to modulate gap junctional intercellular communication(GJIC) in the rat liver epithelial cell line REL. Their cytotoxicitywas first determined by cell density and neutral red uptakeassays: neither apigenin nor tangeretin are cytotoxic at 10and 25 µM, the concentrations used in our experiments.We then studied GJIC using the dye transfer assay and we observedthat both apigenin and tangeretin enhance it, the maximum stimulation(x 1.7–1.8) being achieved at 25 µM for 24 h. Whenthe dye transfer was enhanced, the amount of connexin 43 increased,which was demonstrated by Western blot and immunofluorescenceanalysis. For apigenin only, Northern blot analysis showed anaccumulation of connexin 43 mRNA. In addition, the incubationof REL cells with the two compounds, for 1 or 24 h, preventedthe inhibition of dye transfer by 12-O-tetradecanoylphorbol-13-acetate(1or 10 ng/ml). The enhancement of GJIC by apigenin could be oneof the major mechanisms responsible for apigenin's anti-tumourpromoting action in vivo. As for tangeretin, its capacity toenhance GJIC completes its potential protective properties towardsthe post-initiation process.     

Rapid inhibition of intercellular communication between BALB/c 3T3 cells by diacylglycerol, a possible endogenous functional analogue of phorbol esters

Intercellular communication between cultured cells is reversibly inhibited by phorbol ester tumor promoters, which have been shown to activate protein kinase C directly, replacing the role of diacylglycerol. In order to see whether a presumed endogenous functional analogue, a diacylglycerol, could inhibit intercellular communication in the same way as phorbol esters, we compared the effects of 1-oleoyl-2-acetyl-glycerol (OAG) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on intercellular communication between BALB/c 3T3 cells, using a fluorescent dye transfer method. When cells were treated with OAG, dose-dependent inhibition of dye transfer between cells was observed, which was almost complete with OAG at 50 micrograms/ml. The effect was rapid, a maximal effect occurring within 30 min after addition. The inhibitory effect of both compounds was maintained for at least for 4 h when the cells were in the growing phase; thereafter, the capacity to transfer dye recovered gradually and then returned to the control level after 8 or 12 h of treatment with OAG or TPA, respectively. Further additions of OAG or TPA had no effect. When OAG was added to cultures during a refractory period produced by TPA, the culture was also refractory to OAG; however, TPA could induce at least 60% inhibition of dye transfer in cultures that had been made refractory to OAG. However, when cultures that had been made refractory to TPA were washed and then OAG was added, it induced extensive inhibition of dye transfer at any time after removal of TPA, whereas addition of TPA to the culture caused no significant reinhibition by 6 h and was detectable only 9 h after removal of TPA. These results indicate that OAG can inhibit dye transfer in a similar manner to TPA, suggesting that activation of protein kinase C may be a mechanism by which phorbol esters inhibit intercellular communication. Our results also suggest that there is some difference between the mechanisms by which OAG and TPA inhibit intercellular communication.     

Reduced gap junctional intercellular communication and altered biological effects in mouse osteoblast and rat liver oval cell lines transfected with dominant-negative connexin 43

Gap junctional intercellular communication (GJIC) maintains normal growth and differentiation of cells in a tissue. The intercellular molecules traversing gap junctions are largely unknown, but the molecular weight (MW) cutoff is normally 1200 Da. No differences in dye transfer were observed in normal or vector controls of WB-F344 rat liver epithelial or mouse osteoblastic MC3T3-E1 cells with either Lucifer Yellow (LY) with a MW of 457 Da (LY-457) or LY with a MW of 649 Da (LY-649). Transfection of a dominant negative-connexin 43 (Cx43) gene decreased GJIC (>50%) when LY-649 was used, however, normal GJIC was observed in both cell lines when LY-457 was used. Therefore, the MW cut off in these clones was considerably less than the wild type. The dominant negative clones of the MC3T3-E1 cells exhibited over 90% less alkaline phosphatase (ALPase) activity and calcium deposition after the induction of differentiation. Similarly, dominant negative Cx43 inhibited gene expression of ALPase and bone sialoprotein but not osteocalcin in MC3T3-E1. WB-F344 cells normally exhibit a biphasic response to 12-O-tetradecanoylphorbol-13-acetate (TPA) where inhibition of GJIC recovers after 2 h, but the dominant negative clones showed no recovery from inhibition of GJIC by TPA. Dominant negative Cx43 also inhibited the formation of network-like structures by WB-F344 cells on Matrigel. These results demonstrate that the dominant negative gene transfected into cell types containing the wild-type connexins result in diminished channel sizes, thus allowing the determination of whether distinct biological endpoints, i.e., differentiation, are dependent upon either small or high MW intercellular signals.     

Diallyl disulfide (DADS) enhances gap-junctional intercellular communication by both direct and indirect mechanisms in rat liver cells

Diallyl disulfide (DADS), a sulfur compound from garlic has been shown to exert many biological effects: induction of carcinogen detoxication, inhibition of tumor cell proliferation, etc. These effects are consistent with its anticarcinogenic properties in animal models and could account for garlic protective effects in humans. Our study demonstrates that DADS can improve gap-junctional intercellular communication (GJIC) in vitro. In rat liver epithelial cells (REL cells), using the dye transfer assay, we observe a time-dependent stimulation of GJIC by DADS at non-cytotoxic concentrations. In addition, incubation of cells with DADS for 1 h prevents the inhibition of GJIC induced by 3,5-di-tertio-butyl-4-hydroxytoluene (BHT). We have studied the direct effects of DADS on the regulation of GJIC, and especially on the expression and localization of the connexin expressed in these cells (Cx43): the enhancement of dye transfer (x1.6) by DADS from 1 to 50 micro M is associated with an increase (x1.3-1.8) in the amount of Cx43 protein (western blotting) with no alteration of its localization in the cell-cell contact regions of the plasma membrane (immunofluorescence analysis). We have also explored the possibility that DADS might act indirectly on GJIC. On one hand, DADS does not change the amount of E-cadherin, the adhesion molecule expressed in epithelial cells. On the other hand, it induces rapid inhibition of protein glycosylation. The data suggest that DADS could reduce local constraints imposed by glycoproteins, thus facilitating dye transfer. In conclusion, DADS can be included with other plant microconstituents, which have been demonstrated to improve GJIC. Its effect on REL cells can be explained by its ability to enhance the amount of Cx43 and also to diminish the level of glycosylated proteins.     

Okadaic Acid and Phorbol Esters: Comparative Effects of These Tumor Promoters on Cell Transformation, Intercellular Communication and Differentiation in vitro

Okadaic acid is a non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoter in the mouse skin carcinogenesis system. Here we report on the in vitro activity of okadaic acid in 3 assay systems: BALB/c 3T3 cell transformation, gap junctional intercellular communication (GJIC) in various cell types, and inhibition of induction of differentiation of Friend virus-transformed murine erythroleukemia (MEL) cells. The activity of okadaic acid was compared to that of the phorbol ester tumor promoters TPA and phorbol-12,13-didecanoate (PDD). In a test system involving a 2-week exposure of BALB/c 3T3 cells following 3-methylcholanthrene initiation, okadaic acid at a concentration of 10 ng/ml was equipotent to PDD as a promoter of cell transformation (4.9 and 3.7 foci/dish, respectively). Longer exposures to okadaic acid resulted in cytotoxicity. Okadaic acid-generated as well as PDD-generated transformed foci displayed a selective lack of GJIC between focus cells and surrounding normal cells, i.e., transformed cells communicate among themselves but not with surrounding cells. However, in contrast to TPA, there was no inhibition by okadaic acid, except at toxic doses, of homologous GJIC in BALB/c 3T3 cells or human and mouse keratinocytes. Furthermore, okadaic acid, unlike TPA, did not inhibit MEL cell differentiation. Together, these results indicate that okadaic acid acts as a promoter of cell transformation but that its mechanism of action is different from that of the phorbol ester tumor promoters.     

Okadaic acid and phorbol esters: comparative effects of these tumor promoters on cell transformation, intercellular communication and differentiation in vitro

Okadaic acid is a non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoter in the mouse skin carcinogenesis system. Here we report on the in vitro activity of okadaic acid in 3 assay systems: BALB/c 3T3 cell transformation, gap junctional intercellular communication (GJIC) in various cell types, and inhibition of induction of differentiation of Friend virus-transformed murine erythroleukemia (MEL) cells. The activity of okadaic acid was compared to that of the phorbol ester tumor promoters TPA and phorbol-12,13-didecanoate (PDD). In a test system involving a 2-week exposure of BALB/c 3T3 cells following 3-methylcholanthrene initiation, okadaic acid at a concentration of 10 ng/ml was equipotent to PDD as a promoter of cell transformation (4.9 and 3.7 foci/dish, respectively). Longer exposures to okadaic acid resulted in cytotoxicity. Okadaic acid-generated as well as PDD-generated transformed foci displayed a selective lack of GJIC between focus cells and surrounding normal cells, i.e., transformed cells communicate among themselves but not with surrounding cells. However, in contrast to TPA, there was no inhibition by okadaic acid, except at toxic doses, of homologous GJIC in BALB/c 3T3 cells or human and mouse keratinocytes. Furthermore, okadaic acid, unlike TPA, did not inhibit MEL cell differentiation. Together, these results indicate that okadaic acid acts as a promoter of cell transformation but that its mechanism of action is different from that of the phorbol ester tumor promoters.