Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced increase in vascular permeability in mouse skin by lipoxygenase inhibitors

The painting of mouse dorsal skin with 12-O-tetradecanoylphorbol-13-acetate (TPA) (0.2-2.5 nmol/mouse) induced a dose-related increase in vascular permeability, which was determined by pontamine sky blue exudation into the skin 5 hr after the TPA treatment. Marked infiltration of neutrophils in the dermal interstitium was also observed 5 hr after TPA treatment. Treatment of mice with nordihydroguaiaretic acid (NDGA) (10 mumol/mouse). 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861) (10 mumol/mouse) and quercetin (3 mumol/mouse) significantly inhibited the TPA-induced dye exudation. However, indomethacin (250-1000 nmol/mouse) tended to inhibit the TPA-induced dye exudation, but the inhibition was not statistically significant. Treatment with AA861 (10 mumol/mouse) also caused a marked inhibition of TPA-induced neutrophil infiltration. Quercetin, NDGA and AA861 inhibited epidermal lipoxygenase activity, but indomethacin failed to inhibit it. On the other hand, indomethacin inhibited epidermal cyclooxygenase, but quercetin, NDGA and AA861 failed to inhibit it. The present study suggests involvement of a lipoxygenase product(s) in the mechanism of the TPA-induced increase in vascular permeability in the dorsal skin of mice.     

葛根素下调基质金属蛋白酶-16表达影响乳腺癌细胞的生物学行为

目的：探讨葛根素通过下调基质金属蛋白酶-16（matrix metalloproteinase-16,MMP-16）表达抑制MCF-7乳腺癌细胞的增殖并促进细胞凋亡行为的调控及其机制。方法：qPCR检测不同浓度的葛根素对乳腺癌细胞的活性影响情况;分析葛根素对乳腺癌细胞增殖行为的影响情况;流式细胞术实验检测葛根素对乳腺癌细胞周期的影响;Western blotting实验检测葛根素对乳腺癌细胞中基质金属蛋白酶-16表达情况的影响。结果：1 μmol·L^-1的葛根素对乳腺癌细胞的活力的抑制作用最佳;随着时间的进展,葛根素对乳腺癌细胞的增殖能力的直接抑制作用,可以在一定程度上干扰乳腺癌细胞的生长进程;葛根素对乳腺癌细胞的凋亡行为有一定的促进作用;合适浓度葛根素对乳腺癌细胞中MMP-16蛋白有直接的干扰作用。结论：葛根素通过下调基质金属蛋白酶-16表达抑制MCF-7乳腺癌细胞的增殖并促进细胞凋亡行为。     

Inhibitory effect of echinocystic acid on 12-O-tetradecanoylphorbol-13-acetate-induced dermatitis in mice

The rhizome of Codonopsis lanceolata (family Campanulaceae), which contains lancemaside A as a main constituent, is frequently used in the traditional Chinese medicine for the treatment of inflammatory diseases. Lancemaside A exhibits anti-inflammatory effect in vitro and in vivo. However, orally administered lancemaside A is metabolized to echinocystic acid by the intestinal microflora and the metabolite is absorbed into the blood. Therefore, to understand whether echinocystic acid is effective against skin inflammatory diseases, we assessed its inhibitory effect against 12-O-tetra decanoylphorbol-13-acetate (TPA)-induced ear inflammation in mice. Topically administered echinocystic acid potently suppressed TPA-induced ear swelling. The suppression rates at 0.05 and 0.10 % concentrations were 65 and 73 %, respectively. Echinocystic acid also inhibited TPA-induced myeloperoxidase activity, as well as COX-2, iNOS, TNF-α and IL-1β expressions. Echinocystic acid inhibited NF-κB in TPA-treated mouse ears, as well as in lipopolysaccharide-stimulated peritoneal macrophages. Its potency is comparable with that of dexamethasone. These findings indicate that echinocystic acid may ameliorate inflammatory diseases, such as dermatitis.     

A subpopulation of 12-O-tetradecanoylphorbol-13-acetate-induced papillomas is not inhibited by retinoic acid.

The inhibitory effect of retinoic acid (RA) on 12-O-tetradecanoylphorbol-13-acetate- (TPA) induced mouse skin tumors was studied. Two subpopulations of tumors, small (< 2 mm) and large (> or = 2 mm) appeared after 12 weeks of cutaneous promotion by TPA (10 nmol), following initiation by application of 2 x 100 nmol of 7,12-dimethylbenz[a]anthracene (DMBA) to the skin. RA in the doses of 17 and 34 nmol, prior to each TPA treatment inhibited (P < 0.05) the formation of small tumors at 12 weeks of promotion. However, RA in either dose did not inhibit the formation of large (> or = 2 mm) tumors. Ten weeks following withdrawal of all treatments, the number of large tumors persisted in a significantly (P < 0.05) higher number as compared to small tumors in all groups. Our results provide evidence for the existence of tumor subpopulations with a differential response to RA. In addition, elevated levels of metallothionein (MT) expression were demonstrated in papillomas induced by TPA, 72 h after the last TPA treatment. Comparing papillomas treated with RA prior to each TPA treatment and papillomas treated with TPA only, demonstrated that the elevated MT expression in papillomas was unaffected by RA. This indicated that RA did not affect the expression of a protein that showed elevated level in TPA-induced papillomas.     

氧化苦参碱诱导人乳腺癌细胞MCF-7凋亡的实验研究

目的　研究氧化苦参碱对MCF 7细胞的诱导凋亡作用机制。方法　用光学显微镜、电子显微镜、激光共聚焦显微镜、流式细胞仪和DNA凝胶电泳等技术观察细胞凋亡。结果　实验显示氧化苦参碱作用于体外培养的MCF 7细胞可诱导发生凋亡 ,凋亡细胞表现为细胞固缩 ,核染色质聚集或碎裂、胞质空泡化等 ;DNA电泳可见DNA梯形条带 ;激光共聚焦显微镜示DNA含量下降 ,流式细胞仪检测sub G1峰在G1期前出现 ,S期细胞比例增高。结论　氧化苦参碱对体外培养MCF 7细胞生长有抑制作用 ,机制与通过阻止细胞周期的进程 ,启动细胞自身调控程序 ,诱导肿瘤细胞凋亡有关     

Inhibition of mitogen-activated protein kinase kinase enhances apoptosis induced by arsenic trioxide in human breast cancer MCF-7 cells

Arsenic trioxide (As2O3) has recently been used to treat acute promyelocytic leukaemia and has activity in vitro against several solid tumour cell lines where the induction of differentiation and apoptosis are the prime effects. The mechanism of As2O3-induced cell death has yet to be clarified, especially in solid cancers. In the present study, the human breast cancer cell line MCF-7 was examined as a cellular model for As2O3 treatment. The involvement of extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) was investigated in As2O3-induced cell death. 3. It was found that As2O3 activates the prosurvival mitogen-activated protein kinase kinase (MEK)/ERK pathway in MCF-7 cells, which, conversely, may compromise the efficacy of As2O3. Hence, a combination treatment of As2O3 and MEK inhibitors was investigated to determine whether this treatment could lead to enhanced growth inhibition and apoptosis in MCF-7 cells. 4. Inhibition of MEK/ERK with the pharmacological inhibitors U0126 (10 micromol/L) or PD98059 (20 micromol/L) together with As2O3 (2 and 5 micromol/L) resulted in a significant enhancement of growth inhibition in breast cancer MCF-7 cells as determined by the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay and [Methyl-3H]-thymidine incorporation. Furthermore, the results demonstrated that combined treatment with As2O3 and the MEK1/2 inhibitor U0126 could augment breast cancer MCF-7 cell apoptosis approximately twofold compared with the effects of the two drugs alone, as determined by Hoechst 33258 or annexin V/propidium iodide (PI) staining and flow cytometry. 5. In addition, As2O3 activated p38 in a dose-dependent manner, but had no effect on JNK1/2. Treatment with a p38 inhibitor did not prevent As2O3-induced apoptosis. 6. In conclusion, the results of the present study showed that enhanced apoptosis is detected in breast cancer MCF-7 cells in the presence of As2O3 and an MEK inhibitor, which may be a new promising adjuvant to current breast cancer treatments.     

Autophagy preceded apoptosis in oridonin-treated human breast cancer MCF-7 cells

Recent studies have shown that MCF-7 cells undergo autophagy under some conditions, such as tamoxifen treatment and starvation. In this study, we investigated autophagy in MCF-7 cells under oridonin treatment and further examined the relationship between autophagy and apoptosis. After 3-MA (the specific inhibitor of autophagy) pre-culture, MCF-7 cells were exposed to oridonin, and the growth inhibitory ratio, morphologic changes, DNA fragmentation, proteins expression, autophagic ratio and apoptotic ratio were evaluated. Oridonin inhibited the proliferation of MCF-7 cells and induced autophagy in vitro. MDC (a specific dye for autophagosome) recruitment and typical apoptotic features, including apoptotic bodies, membrane blebbing as well as nuclear condensation, were induced by oridonin. Oridonin downregulated the phosphorylation of ERK, whereas those of JNK and P38 kinase were upregulated. In the condition of oridonin treatment, 3-MA significantly reduced the autophagic level, and the apoptotic cell ratio was also declined. Furthermore, combined treatment with oridonin and 3-MA upregulated ERK phosphorylation and downregulated JNK and P38 kinases phosphorylation compared with oridonin alone treatment groups, indicating that autophagy facilitated apoptosis in oridonin-induced MCF-7 cells. In addition, 3-MA application downregulated DNA ladder and Bax expression but upregulated Bcl-2 expression, compared with oridonin alone treatment. Taken together, oridonin simultaneously induced MCF-7 cells both apoptosis and autophagy, and in this settings, inhibition of autophagy induced lowered apoptotic level, therefore, autophagy participated in upregulation of apoptosis.     

Phthalates inhibit tamoxifen-induced apoptosis in MCF-7 human breast cancer cells

Environmental estrogens represent a class of compounds that can mimic the function or activity of the endogenous estrogen 17 -estradiol (E2). Phthalates including butyl benzyl phthalate (BBP), di(n-butyl) phthalate (DBP), and di(2-ethylhexyl) phthalate (DEHP) are used as plasticizers, and also widely used in food wraps and cosmetic formulations. Phthalates have been shown to mimic estrogen and are capable of binding to the estrogen receptor (ER). It has been demonstrated that estrogen promotes drug resistance to tamoxifen (TAM) in breast cancer. In order to further evaluate the potential role of the phthalates as environmental estrogens, the effect of phthalates was investigated on TAM-induced apoptosis in MCF-7 human breast cancer cells. Our results show that phthalates, BBP (100 M), DBP (10 M), and DEHP (10 M), significantly increased cell proliferation in MCF-7, but not in MDA-MB-231 cells. In addition, BBP, DBP, and DEHP mimicked estrogen in the inhibition of TAM-induced apoptosis in MCF-7 cells. Our data suggest that the inhibitory effect of phthalates on TAM-induced apoptosis involves an increase in intracellular Bcl-2 to Bax ratio. Given that the phthalates are widely used in cosmetics mainly for women, our findings that revealed the promoting effect of BBP, DBP, and DEHP on chemotherapeutic drug resistance to TAM in breast cancer may be of biological relevance.     

路路通酸对乳腺癌MCF-7细胞和宫颈癌C-33A细胞增殖的影响

目的研究路路通酸(BTA)对乳腺癌MCF-7细胞及宫颈癌C-33A细胞增殖的影响。方法采用MTT法检测不同浓度BTA作用不同时间后对MCF-7细胞、C-33A细胞的增殖抑制作用;流式细胞仪检测BTA处理后细胞周期的变化。结果 BTA作用于乳腺癌MCF-7细胞24、48、72 h后的IC50分别为(37.62±1.72)、(27.32±0.99)、(19.19±0.90)μmol/L。BTA作用于宫颈癌C-33A细胞24、48、72 h后的IC50分别为(34.55±0.88)、(27.20±1.03)、(16.74±0.79)μmol/L,BTA对2种细胞增殖有明显抑制作用,且呈浓度时间依赖性(P<0.05),流式结果显示,BTA将MCF-7细胞阻滞在S期,并诱导其凋亡;BTA将C-33A细胞阻滞在G1-S期。结论BTA对乳腺癌MCF-7细胞和宫颈癌C-33A细胞具有较强的增殖抑制作用,其机制与细胞周期阻滞和诱导细胞凋亡有关。     

Fate of 3,3'-diindolylmethane in cultured MCF-7 human breast cancer cells

3,3'-Diindolylmethane (DIM) is a major in vivo product of the cancer preventative agent indole-3-carbinol that is found in vegetables of the genus Brassica. Here, we report on the metabolic fate of radiolabeled DIM in MCF-7 cells. DIM was slowly metabolized to several sulfate conjugates of oxidized DIM products that were primarily detected in the medium. The radioactivity detected in cells was predominantly unmodified DIM (81-93%) at all time intervals up to 72 h treatment. Co-treatment of MCF-7 cells with quercetin slowed the rate that oxidized DIM products accumulated in the medium, while indole[3,2-b]carbazole (ICZ) co-treatment accelerated their production. ICZ is an inducer of P450 1A2, while quercetin is a specific inhibitor of this isoform, suggesting that P450 1A2 is primarily responsible for the oxidation of DIM, probably through 2,3-epoxidation similar to 3-methylindole. Sulfate conjugates of oxidized DIM metabolites were cleaved by sulfatase digestion and identified by LC/MS as 3-(1H-indole-3-ylmethyl)-2-oxindole (2-ox-DIM), bis(1H-indol-3-yl)methanol (3-methylenehydroxy-DIM), 3-[hydroxy-(1H-indol-3-yl)-methyl]-1,3-dihydro-2-oxindole (3-methylenehydroxy-2-ox-DIM), and 3-hydroxy-3-(1H-indole-3-ylmethyl)-2-oxindole (3-hydroxy-2-ox-DIM). Derivatives of 2-ox-DIM represented greater than 30% of the radioactivity in the sulfatase-digested medium. Although oxindole formation was the primary metabolic pathway in MCF-7 cells, synthetic 2-ox-DIM was inactive in a 4-ERE-luciferase reporter assay and, therefore, probably not responsible for the estrogenic activity previously observed for DIM. Unmodified DIM rapidly accumulated in the nuclear membranes representing approximately 35-40% of the radioactivity after 0.5-2 h treatment. Uptake of radiolabeled DIM appeared to be a passive partitioning into the nuclear membranes and was not dependent upon the cell cytosol. The nuclear uptake of DIM was not saturable and could not be blocked by pretreatment with unlabeled DIM (100 microM). Further, treatments in serum-free medium increased the uptake of radiolabeled DIM by the MCF-7 cells. These findings show that the uptake of DIM by membranes significantly increases its localized concentration, which may contribute to its biological activities.     

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced epidermal ornithine decarboxylase activity and tumor promotion by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) in mouse skin

N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited epidermal ornithine decarboxylase (ODC) induction caused either by 12-O-tetradecanoylphorbol-13-acetate (TPA) or teleocidin in CD-1 mice. Inhibitory effect of W-7 on TPA-caused ODC induction was also observed in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated skin and even after repetitive TPA treatment. TPA-induced skin tumor promotion was also suppressed by W-7. Meanwhile, W-7 showed only slight inhibitory effects on calcium-activated, phospholipid-dependent protein kinase (protein kinase C) activity of mouse epidermis stimulated either by Ca2+ or TPA in the presence of phosphatidylserine. Thus, it is unlikely that the anti-ODC-inducing and anti-tumor-promoting actions of W-7 are due to its inhibitory effect on protein kinase C. It may be possible that a calmodulin-mediating process is involved in the mechanism of epidermal ODC induction and tumor promotion caused by tumor promoters such as TPA and teleocidin.     

姜黄素诱导乳腺癌MCF-7细胞凋亡

目的 研究姜黄素对人乳腺癌细胞株MCF-7细胞增殖抑制和诱导凋亡作用.方法 MTT法检测姜黄素对MCF-7细胞的增殖抑制作用;流式细胞术(FCM)PI单染检测细胞周期;Annexin V/PI双染法检测细胞凋亡;Western blot法检测Bcl-2和Bax蛋白的表达.结果 姜黄素对MCF-7细胞生长有明显抑制作用,并呈剂量、时间依赖性;姜黄素能使MCF-7细胞阻滞在G1/S期,可以诱导细胞凋亡,Bax蛋白表达上调,而Bcl-2的表达减少.结论 姜黄素对人乳腺癌MCF-7细胞的增殖具有显著的抑制作用并可诱导细胞凋亡.其分子作用机制可能与其上调Bax基因表达水平的同时下调Bcl-2基因表达水平,从而诱导细胞凋亡有关.     

Broccoli and watercress suppress matrix metalloproteinase-9 activity and invasiveness of human MDA-MB-231 breast cancer cells

A high dietary intake of cruciferous vegetables has been associated with a reduction in numerous human pathologies particularly cancer. In the current study, we examined the inhibitory effects of broccoli (Brassica oleracea var. italica) and watercress (Rorripa nasturtium aquaticum) extracts on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cancer cell invasion and matrix metalloproteinase-9 activity using human MDA-MB-231 breast cancer cells. Aberrant overexpression of matrix metalloproteinases, including metalloproteinase-9, is associated with increased invasive potential in cancer cell lines. Our results demonstrate that extracts of broccoli and Rorripa suppressed TPA-induced MMP-9 activity and invasiveness in a concentration dependent manner as determined by zymographic analysis. Furthermore, fractionation of individual extracts followed by liquid chromatography mass spectroscopy analysis (LC-MS) revealed that the inhibitory effects of each vegetable were associated with the presence of 4-methysulfinylbutyl (sulforaphane) and 7-methylsulphinylheptyl isothiocyanates. Taken together, our data indicate that isothiocyanates derived form broccoli and Rorripa inhibit metalloproteinase 9 activities and also suppress the invasive potential of human MDA-MB-231 breast cancer cells in vitro. The inhibitory effects observed in the current study may contribute to the suppression of carcinogenesis by diets high in cruciferous vegetables.     

保护性自噬对顺铂诱导人乳腺癌 MCF-7细胞凋亡的抑制作用探讨

目的：探讨顺铂对乳腺癌 MCF-7细胞自噬的影响及自噬在顺铂诱导凋亡中的作用。方法顺铂处理乳腺癌 MCF-7细胞,MTT 检测细胞增殖的能力,Hoechst 33342染分析细胞的凋亡,吖啶橙染色分析细胞的自噬,Western blot 分析自噬蛋白 LC3Ⅰ／Ⅱ和 p62的表达和凋亡蛋白多聚 ADP-核糖聚合酶 PARP 表达。结果顺铂呈时间和剂量依赖性抑制乳腺癌 MCF-7细胞的增殖,并且凋亡细胞数量随顺铂浓度的递增而增加;同时顺铂能诱导微管相关蛋白轻链3-Ⅱ（LC3Ⅱ）蛋白的增加,p62蛋白的减少以及酸性自噬溶酶体的增加,顺铂联合氯喹明显增加了 LC3Ⅱ和 p62的蛋白的表达;与单药顺铂相比,自噬抑制剂氯喹明显降低细胞存活率（89．17％±2．56％）vs （74．63％±1．51％）,（P ＜0．05）,而且 PARP 蛋白发生了明显的裂解（P ＜0．05）。结论顺铂诱导乳腺癌 MCF-7细胞保护性自噬,抑制自噬可以增加顺铂诱导乳腺癌 MCF-7细胞凋亡,自噬抑制剂联合顺铂为乳腺癌提供了新的治疗策略。     

Metabolism of equilenin in MCF-7 and MDA-MB-231 human breast cancer cells

Sulfate conjugates of the B-ring unsaturated estrogens, equilin, equilenin, and 8-dehydroestrone, and their 17alpha- and 17beta-dihydro analogues, constitute about 54% of Premarin (Wyeth-Ayerst), the most commonly prescribed estrogen formulation in estrogen replacement therapy. Despite the wide clinical use of Premarin, there have been very few studies on the metabolism of the B-ring unsaturated estrogens in humans and there is no information regarding the fate of these compounds in breast tissue or tumors. In this study, we investigated the metabolism of equilenin in two lines of human breast-cancer cells, MCF-7 and MDA-MB-231. MCF-7 cells respond to treatment with Ah-receptor agonists with induction of cytochromes P450 1A1 and 1B1, whereas in MDA-MB-231 cells P450 1B1 is predominantly induced. Metabolites of equilenin were identified and quantified by GC/MS utilizing a series of synthetic metabolite standards and deuterium-labeled analogues as internal standards. In the two cell lines, the same pathways of equilenin metabolism were observed. Equilenin was reduced at C-17 to the 17beta-dihydro form, with minimal production of the 17alpha-dihydro isomer. Both equilenin and 17beta-dihydroequilenin were hydroxylated at the C-4 position, and the resultant catechol metabolites were methylated to form 4-methoxyequilenin and 4-methoxy-17beta-dihydroequilenin. Rates of equilenin metabolism were markedly elevated in cultures exposed to the Ah-receptor agonists, 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3,4,4',5-tetrachlorobiphenyl, implicating the activities of P450s 1A1 and 1B1 in the metabolism. The 2-hydroxylation pathways of equilenin and 17beta-dihydroequilenin metabolism were not observed. In microsomal reactions with cDNA-expressed human enzymes, both P450s 1A1 and 1B1 catalyzed the 4-hydroxylation of 17beta-dihydroequilenin, whereas with 17beta-estradiol as substrate P450 1A1 catalyzes predominantly 2-hydroxylation and P450 1B1 predominantly 4-hydroxylation. Since P450 1B1 is constitutively expressed and both P450s 1A1 and 1B1 are inducible in many extrahepatic tissues including the mammary epithelium, these results indicate the potential for 4-hydroxylation of equilenin and 17beta-dihydroequilenin in extrahepatic, estrogen-responsive tissues.