Regulatory Network of Mitomycin C Action in Human Colon Cancer Cells

A network composed of activation and inactivation pathways to regulate mitomycin C (MMC) action is suggested to exist in human cancer cells. COLO201 colon cancer cells were stably transfected with human NQO1 cDNA that encodes NAD(P)H: quinone oxidoreductase (DT-diaphorase, DTD), and a clonal cell line with about 57-fold elevated DTD activity was obtained. Northern analysis revealed that expression of the NADPH:cytochrome P450 reductase (P450 reductase) gene was decreased in the transfectant, COLO201/NQO1, associated with the increase of NQO1 expression. Biochemical characterization of the cells showed a significant increase of the glutathione (GSH) content concomitantly with the decrease of the P450 reductase activity. As a result of these coordinated modulations, sensitivity of COLO201/NQO1 to MMC was not increased as compared to the parent cells. Analyses of inhibition by specific inhibitors of DTD, P450 reductase and glutathione S -transferase (GST) in 5 human colon cancer cell lines including the transfectant showed that DTD and P450 reductase play significant roles in MMC activation in cells with sufficiently high DTD activity and with marginal DTD activity, respectively. In contrast, GST appeared to participate in MMC inactivation in cells with a high level of GST activity. These results indicated that DTD, P450 reductase, GSH and GST may act together compensatively or competitively, depending on their levels in cells, to determine the cellular sensitivity to MMC.     

紫檀芪对卵巢癌SKOV3细胞凋亡和糖酵解的影响及机制研究

目的 探讨紫檀芪对卵巢癌SKOV3细胞凋亡和糖酵解的影响及机制研究。方法 分别采用0、25、50、100、150 μmol/L的紫檀芪处理SKOV3细胞24、48、72 h,用CCK-8检测紫檀芪对SKOV3细胞增殖的影响,根据CCK-8结果选择后续实验组紫檀芪浓度;采用流式细胞术检测紫檀芪对细胞凋亡的影响;采用葡萄糖氧化酶法和化学比色法检测紫檀芪对SKOV3细胞葡萄糖消耗和乳酸生成的影响;Western blot法检测信号转导与转录激活因子3(STAT3)、磷酸化的STAT3(p-STAT3)、己糖激酶2(HK2)蛋白的表达;qRT-PCR法检测葡萄糖转运蛋白1(GLUT1)、M2型丙酮酸激酶(PKM2)mRNA的表达。结果 CCK-8结果显示,紫檀芪对SKOV3细胞的增殖有抑制作用,且呈时间剂量依赖性,根据CCK-8结果,选择100 μmol/L紫檀芪处理组作为后续实验组,0 μmol/L为对照组;流式细胞术结果显示,紫檀芪可明显促进SKOV3的凋亡,浓度越大,凋亡作用越明显,差异有统计学意义(P＜0.05)。此外,100 μmol/L组紫檀芪作用SKOV3细胞后,葡萄糖消耗和乳酸生成水平均较0 μmol/L组降低(P＜0.01)。Western blot结果显示,与0 μmol/L组相比,100 μmol/L组p-STAT3、HK2蛋白的表达明显降低(P＜0.001)。qRT-PCR结果显示,100 μmol/L组GLUT1、PKM2 mRNA的表达水平也较0 μmol/L组降低(P＜0.01)。结论 紫檀芪可抑制SKOV3细胞的增殖,促进凋亡,并可能通过STAT3/HK2途径抑制卵巢癌的糖酵解。     

NGX6基因联用5-Fu对人结肠癌细胞凋亡的影响

目的:探讨候选抑瘤基因NGX6联用5-Fu对结肠癌细胞凋亡的影响.方法:以稳定转染并表达NGX6基因的HT-29细胞与5-Fu联用作为实验组.以PDTC与5-Fu联用的HT-29细胞作为对照组.通过EMSA检测各组结肠癌HT-29细胞核转录因子-κB(NF-κB)的激活情况,利用MTT比色法检测各组细胞增殖的情况.吖啶橙(AO)/溴化乙啶(EB)双染法显微镜观测以及PI/Annexin-V双染流式细胞仪检测各组细胞凋亡情况.结果:稳定转染并表达NGX6基因的HT-29细胞以及应用了PDTC的HT-29细胞NF-κB的激活均明显受到抑制;与5-Fu作用的HT-29细胞组比较,5-Fu联合PDTC作用于HT-29细胞后,HT-29细胞增殖受到明显抑制,5-Fu诱导HT-29细胞凋亡作用增强;与5-Fu联合PDTC作用于HT-29细胞的对照组比较,在诱导细胞凋亡以及抑制细胞增殖方面,稳定转染并表达NGX6基因的HT-29细胞与5-Fu联用组和对照组所取得一致的效果,NGX6基因增强5-Fu对HT-29细胞增殖抑制的能力及诱导HT-29细胞凋亡的能力.结论:NGX6基因抑制了肿瘤细胞NF-κB的激活,具有增强5-Fu诱导结肠癌细胞凋亡的能力,其机制可能是抑制肿瘤细胞NF-κB的激活,NGX6基因对肿瘤的治疗及预后起积极作用.     

Glutathione S-Transferases in Small Intestinal Mucosa of Patients with Coeliac Disease

Patients with villous atrophy due to coeliac disease have an increased risk of developing small intestinal malignancies. Intestinal glutathione (GSH) and glutathione S‐transferases (GST) are involved in the protection against carcinogenesis. The aim of this study was to evaluate GSH content and GST enzyme activity in small intestinal mucosa of untreated coeliacs compared to controls. We evaluated GSH content and GST enzyme activity, including the levels of GST classes α, μ, π, θ in small intestinal biopsies of untreated coeliacs (flat mucosa, Marsh IHC, n=12) compared to normal subjects (n=23). Next, we evaluated GSH and GST's in coeliacs in remission (Marsh 0‐1, n=11), coeliacs with persisting villous atrophy while on a gluten‐free diet (partial villous atrophy, Marsh IIIA (n=5); subtotal villous atrophy, Marsh IIIB (n=6) and patients with infiltrative/crypt‐hyperplastic Marsh II lesions (n=4). Total GST enzyme activity and content of GSTa are markedly suppressed in Marsh IIIC lesions compared to controls (resp. 220±79 vs. 4641189 nmol/mg protein‐min (P<0.001) and 2.79±2.46 vs. 6.47±2.29 μg/mg protein (P<0.001). In coeliacs in remission these levels normalized. Total GST enzyme activity and GSTα levels are proportionately lowered according to the degree of mucosal pathology in Marsh II, IIIA and IIIB. (Spearman's σ correlation coefficient for total GST, ‐0.596, P<0.001; GSTα, ‐0.620, P<0.001). GSTμ, π and θ and GSH levels are not significantly different in the selected study groups of mucosal pathology compared to controls. Total GST enzyme activity and content of GSTα in small intestinal mucosa are significantly lower in untreated coeliac disease compared to controls. In Marsh II, IIIA and IIIB, GST enzyme activity and GSTα content are proportionally lower according to the degree of mucosal pathology. Normal values are seen in coeliacs in remission. This correlation between coeliac disease and a suppressed GSH/GST detoxification system may explain in part the carcinogenic risk in untreated coeliac disease.     

Regulatory network of mitomycin C action in human colon cancer cells.

A network composed of activation and inactivation pathways to regulate mitomycin C (MMC) action is suggested to exist in human cancer cells. COLO201 colon cancer cells were stably transfected with human NQO1 cDNA that encodes NAD(P)H:quinone oxidoreductase (DT-diaphorase, DTD), and a clonal cell line with about 57-fold elevated DTD activity was obtained. Northern analysis revealed that expression of the NADPH:cytochrome P450 reductase (P450 reductase) gene was decreased in the transfectant, COLO201/NQO1, associated with the increase of NQO1 expression. Biochemical characterization of the cells showed a significant increase of the glutathione (GSH) content concomitantly with the decrease of the P450 reductase activity. As a result of these coordinated modulations, sensitivity of COLO201/NQO1 to MMC was not increased as compared to the parent cells. Analyses of inhibition by specific inhibitors of DTD, P450 reductase and glutathione S-transferase (GST) in 5 human colon cancer cell lines including the transfectant showed that DTD and P450 reductase play significant roles in MMC activation in cells with sufficiently high DTD activity and with marginal DTD activity, respectively. In contrast, GST appeared to participate in MMC inactivation in cells with a high level of GST activity. These results indicated that DTD, P450 reductase, GSH and GST may act together compensatively or competitively, depending on their levels in cells, to determine the cellular sensitivity to MMC.     

NO-donating aspirin induces phase II enzymes in vitro and in vivo

Modulation of drug metabolizing enzymes, leading to facilitated elimination of carcinogens represents a successful strategy for cancer chemoprevention. Nitric oxide-donating aspirin (NO-ASA) is a promising agent for the prevention of colon and other cancers. We studied the effect of NO-ASA on drug metabolizing enzymes in HT-29 human colon adenocarcinoma and Hepa 1c1c7 mouse liver adenocarcinoma cells and in Min mice treated with NO-ASA for 3 weeks. In these cell lines, NO-ASA induced the activity and expression of NAD(P)H:quinone oxireductase (NQO) and glutathione S-transferase (GST). Compared with untreated Min mice, NO-ASA increased in the liver the activity (nmol/min/mg; mean+/-SEM for all) of NQO (85+/-6 versus 128+/-11, P<0.05) and GST (2560+/-233 versus 4254+/-608, P<0.005) and also in the intestine but not in the kidney; the expression of NQO1 and GST P1-1 was also increased. NO-ASA had only a marginal effect on P450 1A1 and P450 2E1, two phase I enzymes. The release of NO from NO-ASA, determined with a selective microelectrode was paralleled by the induction of NQO1 and abrogated by NO scavengers; an exogenous NO donor also induced the expression of NQO1. NO-ASA induced concentration-dependently the translocation of Nrf2 into the nucleus as documented by immunofluorescence and immunoblotting; this paralleled the induction of NQO1 and GST P1-1. Thus NO-ASA induces phase II enzymes, at least in part, through the action of NO that it releases and by modulating the Keap1-Nrf2 pathway; this effect may be part of its mechanism of action against colon and other cancers.     

Eradication of Helicobacter pylori Restores Glutathione S-Transferase Activity and Glutathione Levels in Antral Mucosa

Glutathione S-transferases (GST) and glutathione peroxidases (GPO) are important in detoxification. GST activity in the mucosa of the gastrointestinal tract is inversely correlated with the development of gastrointestinal cancer. Helicobacter pylori (H. pylori) infection has been associated with gastric cancer. We studied GST activity and the substrate glutathione (GSH) in patients with H. pylori-associated gastritis. GST activity and isoenzyme levels, GPO activity and GSH levels were studied in antral biopsies of 38 H. /pyfori-positive patients, before and after eradication treatment. In 31 patients in whom H. pylori was successfully eradicated, antral GST enzyme activity before therapy was 532 (465–598) nmol/mg protein-min (mean and 95% confidence interval) and that after therapy was 759 (682–836) nmol/mg protein-min ( P <0.0001). Correspondingly, levels of GST α and GST-P1 were higher after eradication ( P <0.001). GSH concentration significantly increased: 21.2 (16.2–26.2) nmol/mg protein before and 27.1 (23.6–30.6) nmol/mg protein after therapy ( P <0.05). In 7 patients in whom H. pylori was not eradicated, GST activity was 671 (520–823) nmol/mg protein min and 599 (348–850) nmol/mg protein before and after treatment respectively ( P =0.32). GSH levels were 17.4 (9.0–25.7) nmol/mg protein and 18.2 (9.1–27.3) nmol/mg protein, respectively ( P =0.84). No differences in antral GPO enzyme activity, both of selenium (Se)-dependent and total GPO, before and after successful treatment were found. Eradication of H. pylori infection increases GST activity and GSH levels in antral mucosa. Low GST activity and GSH concentration due to H. pylori infection might play a role in gastric carcinogenesis.     

Isozyme-specific glutathione S-transferase inhibitors potentiate drug sensitivity in cultured human tumor cell lines

 Novel glutathione (GSH) analogs, previously shown to inhibit glutathione S-transferase (GST) activity at about 1 μM in vitro, were tested for their ability to potentiate the killing of cultured tumor cells by chemotherapeutic drugs. When tested at doses up to 200 μM, the analogs were neither toxic nor capable of potentiating drug toxicity unless the diethyl ester (DEE) form was used for treatment of the cells. HPLC analysis revealed rapid internalization of the DEE and intracellular conversion to a monoethyl ester form that accumulated in the cell, followed by a more gradual loss of the second ester to generate the active parent form. For the four GSH analogs tested, the ability of the DEE forms to potentiate chlorambucil (CMB) toxicity in HT-29 human colon adenocarcinoma cells strongly correlated with the in vitro ability of the parent form to inhibit recombinant human P1-1. This isozyme is the dominant form of GST present in HT-29 cells. Of the four analog DEEs tested, γ-glutamyl-S-(benzyl)cysteinyl-R(−)-phenyl glycine (TER 117) DEE was the most effective in potentiating CMB toxicity in several cell lines: HT-29, HT4-1 (HT-29 subclone), SKOV-3 ovarian carcinoma, and SK VLB (vinblastine-resistant variant of SKOV-3) cells. γ-Glutamyl-S-(octyl)cysteinyl-glycine (TER 143) DEE potentiated mitomycin C (MTC) toxicity in HT4-1 and SK VLB cells while TER 117 DEE did not. TER 117 DEE enhanced melphalan effects on xenografts of HT4-1 in mice to a similar extent as that achieved with the previously described nonspecific GST inhibitor, ethacrynic acid. Taken together, our results indicate that cell-permeable analogs of GSH can potentiate cytotoxicity of common chemotherapeutic drugs and this effect has a strong positive correlation with the ability of the analogs to inhibit specific GST isozymes. Received: 18 October 1994, Accepted: 14 May 1995     

Resveratrol Affects Protein Kinase C Activity and Promotes Apoptosis in Human Colon Carcinoma Cells

Background: Resveratrol has been reported to have potential chemopreventive and apoptosis-inducing properties in a variety of tumor cell lines. Objective: In this study, to investigate the effects of resveratrol on protein kinase C (PKC) activity and apoptosis in human colon carcinoma cells, we used HT-29 cells and examined the PKCα and ERK1/2 signaling pathways. Methods: To test the effects of resveratrol on the growth of HT- 29 cells, the cells were exposed to varying concentrations and assessed with the the MTT cell-viability assay.Fluorescence-activated cell sorter (FACS) analysis was applieded to determine the effects of resveratrol on cell apoptosis. Western blotting was performed to determine the protein levels of PKCα and ERK1/2. In inhibition experiments, HT-29 cells were treated with Gő6976 or PD98059 for 30 min, followed by exposure to 200 μM resveratrol for 72 h. Results: Resveratrol had a significant inhibitory effect on HT-29 cell growth. FACS revealed that resveratrol induced apoptosis. Western blotting showed that e phosphorylation of PKCα and ERK1/2 was significantly increased in response to resveratrol treatment. Pre-treatment with PKCα and ERK1/2 inhibitors (Gő6976 and PD98059) promoted apoptosis. Conclusion: Resveratrol has significant anti-proliferative effects on the colon cancer cell line HT-29. The PKC- ERK1/2 signaling pathway can partially mediate resveratrol-induced apoptosis of HT-29 cells.     

紫檀芪的抗肿瘤活性研究进展

紫檀芪是白藜芦醇的衍生物之一,对多种肿瘤细胞增殖具有显著抑制作用.其抗肿瘤机制包括诱导肿瘤细胞凋亡、引起细胞周期阻滞及阻断肿瘤细胞生长和增殖信号转导等.紫檀芪有望发展成为新一代的抗肿瘤药物,逐渐受到人们的关注.     

Prognostic Significance of Altered Blood and Tissue Glutathione Levels in Head and Neck Squamous Cell Carcinoma Cases

Glutathione is a thiol compound that plays an important role in the antioxidant defense system of the celland its deficiency leads to an increased susceptibility to oxidative stress and, thus, progression of many diseasestates including head and neck cancer. In the present study, alterations of glutathione levels were investigatedin study cohort of 500 samples (cohort 1 containing 200 head and neck cancer blood samples along with 200healthy controls and cohort II with 50 head and neck squamous cell carcinoma tissue samples along with 50control tissues) by high performance liquid chromatography. The results indicated that mean blood glutathionelevels were significantly reduced in head and neck cancer patients (p<0.001) compared to respective controls.In contrast, the levels of glutathione total (p<0.05) and glutathione reduced (p<0.05) were significantly elevatedin head and neck squamous cell carcinoma tissues compared to the adjacent cancer-free control tissues. Inaddition to this, pearson correlation performed to correlate different tissue glutathione levels (GSH) with clinical/pathological parameters demonstrated a significant negative correlation between pT-stage and GSH level (r=-0.263**; p<0.01), C-stage and GSH level (r=-0.335**; p<0.01), grade and GSH (r=-0.329**; p<0.01) and gradeversus redox index (r=-0.213**; p<0.01) in HNSCC tissues. Our study suggests that dysregulation of glutathionelevels in head and neck cancer has the potential to predict metastasis, and may serve as a prognostic marker.     

Induction of NAD(P)H quinone: oxidoreductase1 inhibits carcinogen-induced aberrant crypt foci in colons of Sprague-Dawley rats.

Phase II detoxifying enzymes like NAD(P)H (quinone acceptor)oxidoreductase1 (NQO1), glutathione S-transferases (GST), and UDP-glucuronyltransferases (UGT) may play an important role in preventing carcinogen-induced cancers. Inducers of these enzymes have been shown to inhibit carcinogen-induced colon tumors in rat and mouse models. However, it has not been clearly demonstrated that NQO1 contributes to this effect. We examined the effect of NQO1 inducers on colon carcinogenesis using an aberrant crypt foci (ACF) rat model. Sprague-Dawley rats were fed control diet or diet containing 400 ppm dimethyl fumarate or 200 ppm oltipraz for 7 days, and Phase II enzymes in rat colon and liver were measured. Dimethyl fumarate significantly increased NQO1 and GST activities in colon and liver but did not increase UGT activities in these tissues. In contrast, oltipraz significantly increased NQO1 activities in colon and liver and produced a small increase in GST activity in the liver but did not increase GST activity in the colon or UGT activities in the liver or colon. Sprague Dawley rats were fed control diet or diet containing 200 ppm oltipraz and then treated with the carcinogens azoxymethane or methyl nitrosourea. Both carcinogens produced ACF in all of the rat colons, but rats fed oltipraz diet had significantly fewer ACF than those fed control diet. This protective effect was reversed in rats treated with the NQO1 inhibitor, dicoumarol. However, treatment with oltipraz did not alter the distribution of crypt multiplicities in the ACF. These studies demonstrated that induction of NQO1 plays a significant role in inhibiting initiation of carcinogen-induced ACF in Sprague-Dawley rats. This provides the first direct evidence that NQO1 may play a role in preventing colon cancer. The study also found that oltipraz added to the diet of Sprague-Dawley rats selectively increased NQO1 activity in colon mucosa with no increase in GST and UGT activities in these tissues. Thus, this model will be useful for further investigating the role of NQO1 in prevention of colon cancer.     

Prevention of N-methyl-N'-nitro-N-nitrosoguanidine and saturated sodium chloride-induced gastric carcinogenesis in Wistar rats by lycopene.

We investigated "the "chemopreventive potential of lycopene against gastric carcinogenesis induced in male Wistar rats by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and saturated sodium chloride (S-NaCl). Administration of lycopene inhibited MNNG+S-NaCl-induced gastric carcinogenesis as revealed by the absence of carcinomas. Lipid peroxidation, reduced glutathione (GSH), glutathione peroxidase (GPx), glutathione S-transferase (GST) and glutathione reductase (GR) were used to monitor the chemopreventive potential of lycopene. The extent of lipid peroxidation was significantly lower, whereas GSH, GPx, GST and GR were markedly enhanced in the gastric mucosa of tumour-bearing animals. Our data suggest that lycopene may exert its inhibitory effects by modulating the oxidant and antioxidant status in the gastric mucosa.     

血液肿瘤细胞对氧化砷的敏感性与其抗氧化能力的关系

目的　探讨血液肿瘤细胞对三氧化二砷 (As2 O3 )的敏感性和细胞抗氧化能力的关系。方法　应用 9个血液肿瘤细胞系 ,通过细胞活力、形态学和流式细胞仪检测细胞凋亡 ,并测定细胞系的谷胱甘肽 (GSH)含量和 4种抗氧化酶的活性。结果　除了HL 6 0、U937、K5 6 2和Jurkat细胞外 ,其他5个细胞对As2 O3 诱导的凋亡敏感。与敏感细胞系比较 ,As2 O3 耐受细胞系存在较高的GSH含量和(或 )过氧化氢酶活性。谷胱甘肽过氧化物酶、谷胱甘肽S转移酶和超氧化物歧化酶活性与细胞对As2 O3 诱导凋亡效应敏感性无明显相关。结论　细胞内GSH水平和过氧化氢酶的活性是决定血液肿瘤细胞对As2 O3 敏感性的重要因素。     

Effects of garlic on 7,12-Dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis

The inhibitory effect of garlic (Allium sativum Linn) on 7, 12-dimethylbenz[a]anthracene (DMBA)-induced buccal pouch carcinogenesis was investigated in male Syrian hamsters. Measurement of lipid peroxidation, reduced glutathione (GSH), glutathione peroxidase (GPx), and glutathione S-transferase (GST) was used to monitor the chemopreventive potential of garlic. All hamsters painted on their buccal pouches with DMBA alone for 14 weeks developed well-differentiated squamous cell carcinomas. Diminished lipid peroxidation in the oral tumor tissue was accompanied by a significant increase in the levels of GSH, GPx, and GST. Administration of 250 mg/kg body weight aqueous garlic extract three times a week for 14 weeks effectively suppressed DMBA-induced oral carcinogenesis as revealed by the reduced incidence of neoplasms. The results of the present study suggest that garlic may exert its chemopreventive effects by modulating lipid peroxidation and enhancing the levels of GSH, GPx, and GST.     

Low Glutathione and Glutathione S-Transferase Levels in Barrett's Esophagus as Compared to Normal Esophageal Epithelium

Patients with Barrett's esophagus, wherein squamous epithelium has been replaced by columnar epithelium, have an increased risk for developing esophageal adenocarcinoma as compared to the general population. Glutathione S-transferase (GST), a family of detoxification enzymes consisting of class α, μ, π, and θ isoforms, is involved in detoxification of carcinogens and low levels of these enzymes correlated with high cancer risk. We have now compared GST enzyme activity, GST isoenzyme composition and glutathione (GSH) content of Barrett's mucosa with that of adjacent normal squamous epithelium. Biopsy specimens of 98 patients with Barrett's esophagus were taken from both Barrett's and adjacent normal squamous epithelium. GST enzyme activity towards 1–chloro-2,4–dinitrobenzene was measured, and GST isoenzyme levels were determined by densitometrical analyses of western blots after immunodetection with monoclonal antibodies. Total GSH content was determined by high-performance liquid chromatography after conjugation with monobromobimane. Wilcoxon's signed rank test and Spearman correlation analyses were used for statistical evaluation. As compared with adjacent normal squamous epithelium, GST enzyme activity in Barrett's epithelium was reduced by 35%, and GST μ, GST π and GSH levels were reduced by 24%, 30%, and 63%, respectively. However, the minor GST α and GST θ levels were higher in Barrett's epithelium (by 625% and 33%, respectively). High levels of GSH and GSTs in general are correlated with protection against cellular or cytogenetic damage. The observed reduction in GSTs and GSH in Barrett's epithelium may therefore contribute to the increased cancer risk in this tissue.     

Pterostilbene, an active constituent of blueberries, suppresses aberrant crypt foci formation in the azoxymethane-induced colon carcinogenesis model in rats.

PURPOSE: Epidemiologic studies have linked the consumption of fruits and vegetables to reduced risk of several types of cancer. Laboratory animal model studies have provided evidence that stilbenes, phenolic compounds present in grapes and blueberries, play a role in inhibiting the risk of certain cancers. Pterostilbene, a naturally occurring stilbene from blueberries, was tested for its preventive activity against colon carcinogenesis. EXPERIMENTAL DESIGN: Experiments were designed to study the inhibitory effect of pterostilbene against the formation of azoxymethane-induced colonic aberrant crypt foci (ACF) preneoplastic lesions in male F344 rats. Beginning at 7 weeks of age, rats were treated with azoxymethane (15 mg/kg body weight s.c., once weekly for 2 weeks). One day after the second azoxymethane treatment, rats were fed experimental diets containing 0 or 40 ppm of pterostilbene. At 8 weeks after the second azoxymethane treatment, all rats were sacrificed, and colons were evaluated for ACF formation and for inhibition of inducible nitric oxide synthase (iNOS) and proliferating cell nuclear antigen. Effects on mucin MUC2 were also determined. RESULTS: Administration of pterostilbene for 8 weeks significantly suppressed azoxymethane-induced formation of ACF (57% inhibition, P < 0.001) and multiple clusters of aberrant crypts (29% inhibition, P < 0.01). Importantly, dietary pterostilbene also suppressed azoxymethane-induced colonic cell proliferation and iNOS expression. Inhibition of iNOS expression by pterostilbene was confirmed in cultured human colon cancer cells. CONCLUSIONS: The results of the present study suggest that pterostilbene, a compound present in blueberries, is of great interest for the prevention of colon cancer.     

p53-independent G1 cell cycle arrest of human colon carcinoma cells HT-29 by sulforaphane is associated with induction of p21CIP1 and inhibition of expression of cyclin D1

Isothiocyanate sulforaphane (SFN) is a potent cancer chemopreventive agent. We investigated the mechanisms underlying the anti-proliferative effects of SFN in the human colon carcinoma cell line, HT-29. We demonstrate that SFN inhibits the growth of HT-29 cells in a dose- and time-dependent manner. Treatment of serum-stimulated HT-29 cells with SFN suppressed the re-initiation of cell cycle by inducing a G₁ phase cell cycle arrest. At high doses (>25 μM), SFN dramatically induces the expression of p21^CIP1 while significantly inhibits the expression of the G₁ phase cell cycle regulatory genes such as cyclin D1, cyclin A, and c-myc. This regulation can be detected at both the mRNA and protein levels as early as 4 h post-treatment of SFN at 50 μM. Additionally, SFN activates MAPKs pathways, including ERK, JNK and p38. Exposure of HT-29 cells with both SFN and an antioxidant, either NAC or GSH, completely blocked the SFN-mediated activation of these MAPK signaling cascades, regulation of cyclin D1and p21^CIP1 gene expression, and G₁phase cell cycle arrest. This finding suggests that SFN-induced oxidative stress plays a role in these observed effects. Furthermore, the activation of the ERK and p38 pathways by SFN is involved in the upregulation of p21^CIP1 and cyclin D1, whereas the activation of the JNK pathway plays a contradictory role and may be partially involved in the downregulation of cyclin D1. Because cyclin D1 and p21^CIP1 play opposing roles in G₁ phase cell cycle progression regulation, blocking the activation of each MAPK pathway with specific MAPK inhibitors, is unable to rescue the SFN-induced G₁ phase cell cycle arrest in HT-29 cells.     

紫檀芪诱导人视网膜母细胞瘤WERI-Rb-1细胞的细胞凋亡及其机制研究

背景与目的：紫檀芪是一种天然抗氧化剂,其抗视网膜母细胞瘤的效果仍不明确。拟探讨紫檀芪对人视网膜母细胞瘤WERI-Rb-1细胞的细胞增殖、细胞凋亡及细胞自噬的作用,并初步阐明其作用机制。方法：采用细胞计数试剂盒(cell counting kit-8,CCK-8)法测定细胞的增殖活力,Annexin V/PI流式细胞术检测细胞凋亡,吖啶橙染色观察细胞内自噬囊泡的变化,蛋白［质］印迹法(Western blot)检测LC3B及P62蛋白的表达。结果：紫檀芪显著抑制WERI-Rb-1细胞的增殖活力(P<0.01),以25、50和100 μmol/L的药物浓度作用细胞24 h时,细胞增殖活力分别为(93.02±0.47)%、(55.10±2.04)%和(30.33±1.45)%;50 μmol/L紫檀芪处理细胞12、24和48 h时,细胞增殖活力分别为(88.38±3.70)%、(53.37±1.17)%和(29.60±1.05)%。紫檀芪显著诱导细胞凋亡(P<0.01),对照组、24和48 h细胞的凋亡率分别为(4.08±0.79)%、(13.44±2.12)%和(23.49±2.01)%。紫檀芪能够激活WERI-Rb-1细胞发生细胞自噬,上调LC3蛋白的表达,下调P62蛋白的表达,增加细胞内自噬囊泡的数量(P<0.01)。3-MA及Beclin1 siRNA抑制细胞自噬后能够部分逆转紫檀芪的抗肿瘤作用(P<0.01)。3-MA抑制细胞自噬后,紫檀芪处理组的细胞凋亡率为(12.97±2.09)%,3-MA+紫檀芪组为(8.35±1.11)%。siRNA敲低Beclin1后,紫檀芪处理组和siRNA+紫檀芪组的细胞凋亡率分别为(13.80±2.19)%和(9.62±0.52)%。结论：紫檀芪可以显著抑制WERI-Rb-1细胞的细胞增殖并激活细胞自噬促进细胞凋亡。     

Glutathione, glutathione S-transferases, and related redox enzymes in Adriamycin-resistant cell lines with a multidrug resistant phenotype

Friend erythroleukemia cells (FLC) selected by exposure to Adriamycin (doxorubicin) express an approximate 2.5-fold (ARN1) or 13-fold (ARN2) resistance to the drug with various degrees of cross-resistance to other anthracyclines, vinca alkaloids, and epipodophyllotoxins. Because the redox cycling of the quinone moiety of Adriamycin is known to produce oxidative stress, however, an analysis of glutathione (GSH) and related enzyme systems was undertaken in the wild-type and selected resistant cells. In ARN1 and ARN2, superoxide dismutase (SOD) and catalase activities were slightly decreased, intracellular GSH and GSH reductase were essentially unchanged, and total GSH peroxidase, glutathione S-transferase (GST), and DT-diaphorase activities were slightly elevated. In each case there was no stoichiometric relationship between degree of resistance and level of activity. GST isozymes were purified from each cell line by HPLC GSH affinity column chromatography. Two-dimensional gel electrophoresis and western blot immunoreactivity against a battery of GST isozyme polyclonal antibodies determined that both the resistant and sensitive cells expressed isozymes of the alpha, pi, and mu classes (alternative murine nomenclature: M1, M2, M3). Of significance, both ARN1 and ARN2 cell lines expressed a unique alpha subunit which was absent from the parent FLC cell line. This isozyme presumably accounted for the increased GSH peroxidase activity (cumene hydroperoxide as substrate) found in ARN1 and ARN2 and may play a role in the small incremental resistance to melphalan found for both resistant lines. Expression of the isozyme was not stoichiometric with respect to degree of resistance. The presence of this isozyme may contribute to the resistant phenotype or may be the consequence of a more general cellular response to oxidative stress.