Nonenzymatic antioxidative and antiglycative effects of oleanolic acid and ursolic acid

Oleanolic acid and ursolic acid are two triterpenes presented in several herbs. This study analyzed the content of oleanolic acid and ursolic acid in eight locally available fresh herbs, also examined several nonenzymatic antioxidant activities of these two triterpenes, and used a liposome system to evaluate the influence of temperature and pH upon the antioxidant property of these two triterpenes. The impact of these two triterpenes on the production of nonenzymatic glycative products, pentosidine and carboxymethyllysine (CML), was also evaluated. alpha-Tocopherol was used for comparison. Results showed that the content of oleanolic acid and ursolic acid in glossy privet fruit and hawthorn fruit varied from season to season and was in the range of 200-650 microg/g of fresh weight. Both oleanolic acid and ursolic acid possessed greater antioxidant activity against 2,2'-azobis-(2-amidinopropane) dihydrochloride and less antioxidant activity against 2,2'-azobis(2,4-dimethylvaleronitrile) when compared with alpha-tocopherol at equal concentration (P <0.05). At 75 and 100 degrees C, oleanolic acid exhibited greater antioxidant activity than alpha-tocopherol and ursolic acid (P <0.05). At pH 2 and pH 4, oleanolic acid and ursolic acid showed greater antioxidant activity than alpha-tocopherol (P <0.05). These two triterpenes also exhibited a dose-dependent effect in superoxide anion scavenging activity, chelating effect, xanthine oxidase inhibition activity, and reducing power (P <0.05). Oleanolic acid significantly and dose-dependently inhibited pentosidine and CML formation (P <0.05). Ursolic acid also significantly suppressed CML formation (P <0.05). These data support that these two triterpenes possessed nonenzymatic antioxidative and antiglycative properties.     

Bioavailability, distribution, and antioxidative effects of selected triterpenes in mice

This study analyzed the content of eight triterpenes (oleanolic acid, ursolic acid, arjunolic acid, asiatic acid, boswellic acid, corosolic acid, madecassic acid, and maslinic acid) in ten vegetables and eight fruits. These compounds at 0.5% were supplied to mice for 4 or 8 weeks. The bioavailability, tissue distribution, and antioxidative protection of these triterpenes were examined. Results showed that triterpenes were detected in eight vegetables and six fruits. Basil and brown mustard contained seven test triterpenes, in the range of 14-102 mg/100 g dry weight. The level of each triterpene in plasma, brain, heart, liver, kidney, colon, and bladder increased as the feeding period was increased from 4 weeks to 8 weeks (P < 0.05). Renal homogenates from mice with triterpene intake had greater antioxidative effects against glucose-induced glutathione loss and malondialdehyde and oxidized glutathione production when compared with those from control groups (P < 0.05). These data support that these triterpenes were absorbed and deposited in their intact forms, which in turn exerted in vivo antioxidative protection.     

Triterpenic compounds from "orujo" olive oil elicit vasorelaxation in aorta from spontaneously hypertensive rats

There is currently a considerable amount of interest in the benefits of certain dietary elements, and in particular of olive oil, in endothelial function and thus in hypertension. "Orujo" or pomace olive oil is obtained from the residues of the olive by a novel centrifugation process, and it is a good dietary source of triterpenic compounds such as oleanolic and maslinic acid, erythrodiol, and uvaol. Until now, there was no information available regarding the properties of these triterpenoids on the vasculature of hypertensive animals. However, in this in vitro study, we have analyzed the vasorelaxation induced by these triterpenoids in isolated aorta from spontaneously hypertensive rats (SHR). The triterpenoids tested induced concentration-dependent vasorelaxation, mostly involving nitric oxide (NO). Indeed, the responses were attenuated by removal of the endothelium or following pretreatment with the NO synthase inhibitor L-NAME. Furthermore, the differences that were observed in the potency of relaxation, the selectivity, and the dependence on the endothelium were attributed to structural features of the triterpenoids. In conclusion, triterpenic components in pomace olive oil induce vasorelaxation of the aorta from SHR, and this effect generally involves endothelial NO.     

How heating affects extra virgin olive oil quality indexes and chemical composition

Two monovarietal extra virgin olive oils from Arbequina and Picual cultivars were subjected to heating at 180 degrees C for 36 h. Oxidation progress was monitored by measuring oil quality changes (peroxide value and conjugated dienes and trienes), fatty acid composition, and minor compound content. Tocopherols and polyphenols were the most affected by the thermal treatment and showed the highest degradation rate although their behavior was different for each cultivar. Alpha-tocopherol loss was more important in Arbequina oil whereas, total phenol content loss was greater in Picual oil. The later showed an important decrease in hydroxytyrosol (3,4-DHPEA) and its secoiridoid derivatives (3,4-DHPEA-EDA and 3,4-DHPEA-EA), while lignans decrease was lesser. For Arbequina oil these compounds remained stable, and a lowering tendency was observed for tyrosol (p-HPEA) and its derivatives (p-HPEA-EDA and p-HPEA-EA). In general, flavone content showed a decrease during heating, being higher for Arbequina oil. On the other hand, oleic acid, sterols, squalene, and triterpenic alcohols (erythrodiol and uvaol) and acids (oleanolic and maslinic) were quite constant, exhibiting a high stability against oxidation. From these results, we can conclude that despite the heating conditions, VOO maintained most of its minor compounds and, therefore, most of its nutritional properties.     

In vitro antioxidant and antiproliferative activities of selenium-containing phycocyanin from selenium-enriched Spirulina platensis

Both selenium and phycocyanin have been reported to show potent cancer chemopreventive activities. In this study, we investigated the in vitro antioxidant and antiproliferative activities of selenium-containing phycocyanin (Se-PC) purified from selenium-enriched Spirulina platensis. The antioxidant activity of Se-PC was evaluated by using four different free radical scavenging assays, namely, the 2,2'-azinobis-3-ethylbenzothiazolin-6-sulfonic acid (ABTS) assay, 1,1-diphenyl-2-picryhydrazyl (DPPH) assay, superoxide anion scavenging assay, and erythrocyte hemolysis assay. The results indicated that Se-PC exhibited stronger antioxidant activity than phycocyanin by scavenging ABTS, DPPH, superoxide anion, and 2,2'-azobis-(2-amidinopropane)dihydrochloride free radicals. Se-PC also showed dose-dependent protective effects on erythrocytes against H 2O 2-induced oxidative DNA damage as evaluated by the Comet assay. Moreover, Se-PC was identified as a potent antiproliferative agent against human melanoma A375 cells and human breast adenocarcinoma MCF-7 cells. Induction of apoptosis in both A375 and MCF-7 cells by Se-PC was evidenced by accumulation of sub-G1 cell populations, DNA fragmentation, and nuclear condensation. Further investigation on intracellular mechanisms indicated that depletion of mitochondrial membrane potential (DeltaPsi m) was involved in Se-PC-induced cell apoptosis. Our findings suggest that Se-PC is a promising organic Se species with potential applications in cancer chemoprevention.     

In vitro antioxidant properties, DNA damage protective activity, and xanthine oxidase inhibitory effect of cajaninstilbene acid, a stilbene compound derived from pigeon pea [Cajanus cajan (L.) Millsp.] leaves

The antioxidant properties, DNA damage protective activities, and xanthine oxidase (XOD) inhibitory effect of cajaninstilbene acid (CSA) derived from pigeon pea leaves were studied in the present work. Compared with resveratrol, CSA showed stronger antioxidant properties, DNA damage protective activity, and XOD inhibition activity. The IC(50) values of CSA for superoxide radical scavenging, hydroxyl radical scavenging, nitric oxide scavenging, reducing power, lipid peroxidation, and XOD inhibition were 19.03, 6.36, 39.65, 20.41, 20.58, and 3.62 μM, respectively. CSA possessed good protective activity from oxidative DNA damage. Furthermore, molecular docking indicated that CSA was more potent than resveratrol or allopurinol to interact with the active site of XOD (calculated free binding energy: -229.71 kcal mol(-1)). On the basis of the results, we conclude that CSA represents a valuable natural antioxidant source and may potentially be applicable in health food industry.     

Induction of apoptosis by 1-(2-hydroxy-5-methylphenyl)-3-phenyl-1,3-propanedione through reactive oxygen species production, GADD153 expression, and caspases activation in human epidermoid carcinoma cells

This study examined the growth inhibitory effects of the structurally related beta-diketones compounds in human cancer cells. Here, we report that 1-(2-hydroxy-5-methylphenyl)-3-phenyl-1,3-propanedione (HMDB) induces growth inhibition of human cancer cells and induction of apoptosis in A431 cells through modulation of mitochondrial functions regulated by reactive oxygen species (ROS). ROS generation occurs in the early stages of HMDB-induced apoptosis, preceding cytochrome c release, caspase activation, and DNA fragmentation. The changes occurred after single breaks in DNA were detected, suggesting that HMDB induced irreparable DNA damage, which in turn triggered the process of apoptosis. Up-regulation of Bad and p21; down-regulation of Bcl-2, Bcl-XL, Bid, p53, and fatty acid synthase; and cleavage of Bax were found in HMDB-treated A431 cells. Glutathione and N-acetylcysteine (NAC) suppress HMDB-induced apoptosis. HMDB markedly enhanced growth arrest DNA damage inducible gene 153 (GADD153) mRNA and protein in a time- and concentration-dependent manner. NAC prevented up-regulation of GADD153 mRNA expression caused by HMDB. These findings suggest that HMDB creates an oxidative cellular environment that induces DNA damage and GADD153 gene activation, which in turn helps trigger apoptosis in A431 cells.     

Antiangiogenic potential of three triterpenic acids in human liver cancer cells

Three triterpenic acids, oleanolic acid, ursolic acid and maslinic acid, at 2 or 4 μmol/L were used to study their antiangiogenic potential in human liver cancer Hep3B, Huh7 and HA22T cell lines. The effects of these compounds upon the level and/or expression of hypoxia-inducible factor (HIF)-1α, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), interleukin (IL)-8, urokinase plasminogen activator (uPA), reactive oxygen species (ROS), nitric oxide (NO) and cell invasion and migration were examined. Results showed that these triterpenic acids at 4 μmol/L significantly suppressed HIF-1α expression in three cell lines (P < 0.05); and these compounds at test doses failed to affect bFGF expression (P > 0.05). Three triterpenic acids dose-dependently decreased production and expression of VEGF and IL-8, retained glutathione level, lowered ROS and NO levels, and declined cell invasion and migration in test cell lines (P < 0.05). These compounds also dose-dependently reduced uPA production and expression in Hep3B and Huh7 cell lines (P < 0.05); but these agents only at 4 μmol/L significantly suppressed uPA production and expression in HA22T cells (P < 0.05). These findings suggest that these triterpenic acids are potent antiangiogenic agents to retard invasion and migration in liver cancer cells.     

5-Hydroxy-3,6,7,8,3',4'-hexamethoxyflavone induces apoptosis through reactive oxygen species production, growth arrest and DNA damage-inducible gene 153 expression, and caspase activation in human leukemia cells

This study examined the growth inhibitory effects of structurally related polymethoxylated flavones in human cancer cells. Here, we report that 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-OH-HxMF) induces growth inhibition of human cancer cells and induction of apoptosis in HL-60 cells through modulation of mitochondrial functions regulated by reactive oxygen species (ROS). ROS generation occurs in the early stages of 5-OH-HxMF-induced apoptosis, preceding cytochrome c release, caspase activation, and DNA fragmentation. The changes occurred after single breaks in DNA were detected, suggesting that 5-OH-HxMF induced irreparable DNA damage, which in turn triggered the process of apoptosis. Up-regulation of Bax was found in 5-OH-HxMF-treated HL-60 cells. In addition, a caspase-independent pathway indicated by endonuclease G also contributed to apoptosis caused by 5-OH-HxMF. Antioxidants suppress 5-OH-HxMF-induced apoptosis. 5-OH-HxMF markedly enhanced growth arrest DNA damage-inducible gene 153 (GADD153) protein in a time-dependent manner. N-acetylcysteine (NAC) and catalase prevented up-regulation of GADD153 expression caused by 5-OH-HxMF. These findings suggest that 5-OH-HxMF creates an oxidative cellular environment that induces DNA damage and GADD153 gene activation, which in turn helps trigger apoptosis in HL-60 cells. Meanwhile, ROS were proven an important inducer in this apoptotic process. The C-5 hydroxyl on the ring of 5-OH-HxMF was found to be essential for the antiproliferative and apoptosis-inducing activity. Our study identified the novel mechanisms of 5-OH-HxMF-induced apoptosis and indicated that these results have significant applications as potential chemopreventive and chemotherapeutic agents.     

Effect of dietary high-oleic-acid oils that are rich in antioxidants on microsomal lipid peroxidation in rats

In contrast to other metabolic functions, the role of dietary antioxidants and oil on microsomal lipid oxidation has been less extensively studied. This study examines ascorbate-Fe(2+) and NADPH-induced lipid peroxidation of hepatic microsomes of rats that were fed for three weeks high-oleic-acid oils (high-oleic sunflower oil, HOSO; olive oil, OO; or olive pomace oil, OPO) containing different concentrations of the antioxidants alpha-tocopherol, erythrodiol, and oleanolic acid. The fatty acid composition of hepatic microsomes of Wistar rats that were fed for three weeks with the above-mentioned oils had lower proportions of C16:0, C18:2n6, and C22:6n3 and higher proportions of C18:0 and C18:1n9 than rats fed the control diet. Light emission by hepatic microsomes increased, in the first 180 min, 2-fold after ascorbate-Fe(2+) addition compared with NADPH addition. Both increases were less pronounced in microsomes of OPO-fed rats and to a smaller extent in microsomes of OO-fed rats. Smaller increases in light emission were associated positively with higher concentrations of dietary alpha-tocopherol, erythrodiol, and oleanolic acid but were not associated with changes in fatty acid composition of hepatic microsomes. Addition of alpha-tocopherol, erythrodiol, or oleanolic acid decreased light emission of hepatic microsomes with a greater inhibition in microsomes of rats fed the control diet. Our data suggest that erythrodiol and oleanolic acids partly explain the protective effect of dietary OPO on microsomal lipid peroxidation in rats.     

Antcin B and its ester derivative from Antrodia camphorata induce apoptosis in hepatocellular carcinoma cells involves enhancing oxidative stress coincident with activation of intrinsic and extrinsic apoptotic pathway

The triterpenoids methylantcinate B (MAB) and antcin B (AB), isolated from the medicinal mushroom Antrodia camphorata , have been identified as strong cytotoxic agents against various type of cancer cells; however, the mechanisms of MAB- and AB-induced cytotoxicity have not been adequately explored. This study investigated the roles of caspase cascades, reactive oxygen species (ROS), DNA damage, mitochondrial disruption, and Bax and Bcl-2 proteins in MAB- and AB-induced apoptosis of hepatocellular carcinoma (HCC) HepG2 cells. Here, we showed that MAB and AB induced apoptosis in HepG2 cells, as characterized by increased DNA fragmentation, cleavage of PARP, sub-G1 population, chromatin condensation, loss of mitochondrial membrane potential, and release of cytochrome c. Increasing the levels of caspase-2, -3, -8, and -9 activities was involved in MAB- and AB-induced apoptosis, and they could be attenuated by inhibitors of specific caspases, indicating that MAB and AB triggered the caspase-dependent apoptotic pathway. Additionally, the enhanced apoptotic effect correlates with high expression of Fas, Fas ligand, as well as Bax and decreased protein levels of Bcl-(XL) and Bcl-2, suggesting that both the extrinsic and intrinsic apoptosis pathways were involved in the apoptotic processes. Incubation of HepG2 cells with antioxidant enzymes superoxide dismutase and catalase and antioxidants N-acetylcysteine and ascorbic acid attenuated the ROS generation and apoptosis induced by MAB and AB, which indicate that ROS plays a pivotal role in cell death. NADPH oxidase activation was observed in MAB- and AB-stimulated HepG2 cells; however, inhibition of such activation by diphenylamine significantly blocked MAB- and AB-induced ROS production and increased cell viability. Taken together, our results provide the first evidence that triterpenoids MAB and AB induced a NADPH oxidase-provoked oxidative stress and extrinsic and intrinsic apoptosis as a critical mechanism of cause cell death in HCC cells.     

Antioxidant activity, cytotoxicity, and DNA information of Glossogyne tenuifolia

This study investigates the antioxidant activity and cytotoxicity of Glossogyne tenuifolia extract on various cancer cell lines. The 5.8s DNA of G. tenuifolia was isolated, and the species of this plant was confirmed by NCBI's DNA database. G. tenuifolia was then extracted with ethanol and separated into several fractions using the partition procedure with water, n-butanol, and ethyl acetate (EA). Among these, the EA fraction most significantly affected the activity of DPPH(*) and superoxide anion scavenging. Additionally, only the EA fraction exhibited cytotoxicity on breast cancer cells (MCF-7 and MDA-MB-231) and liver cancer cells (Hep G2 and Hep 3B). Next, the EA fraction was further separated by column chromatography, and 15 fractions were obtained. Three effective components were isolated and identified separately from the active fractions: oleanolic acid (OA) from fraction 6, luteolin from fractions 8-10, and luteolin-7-glucoside from fraction 12. The test of these three compounds on scavenging activity of DPPH(*) and superoxide anion indicates that luteolin had the highest antioxidant activity, whereas the effect of OA was negligible. Additionally, a synergistic effect between luteolin and luteolin-7-glucoside was observed. Kick-out experiments showed that the activities were vanished or decreased. Especially on MDA-MB-231 and MCF-7 cells, the cytotoxicity completely disappeared when luteolin was eliminated from fractions 8-10. These findings demonstrate that luteolin plays a crucial role in the inhibition of the growth of hepatoma cancer cell lines. Fraction 3, which did not contain luteolin, luteolin-7-glucoside, and oleanolic acid, had cytotoxicity on MDA-MB-231, MCF-7, Hep G2, Hep 3B, and A549, which implies that this fraction contained some other effective ingredients and requires further study. The investigation is currently underway in our laboratory.     

Triterpenoids isolated from apple peels have potent antiproliferative activity and may be partially responsible for apple's anticancer activity

Bioactivity-guided fractionation of apple peels was used to determine the chemical identity of bioactive constituents. Thirteen triterpenoids were isolated, and their chemical structures were identified. Antiproliferative activities of the triterpenoids against human HepG2 liver cancer cells, MCF-7 breast cancer cells, and Caco-2 colon cancer cells were evaluated. Most of the triterpenoids showed high potential anticancer activities against the three human cancer cell lines. Among the compounds isolated, 2alpha-hydroxyursolic acid, 2alpha-hydroxy-3beta-{[(2E)-3-phenyl-1-oxo-2-propenyl]oxy}olean-12-en-28-oic acid, and 3beta-trans-p-coumaroyloxy-2alpha-hydroxyolean-12-en-28-oic acid showed higher antiproliferative activity toward HepG2 cancer cells. Ursolic acid, 2alpha-hydroxyursolic acid, and 3beta-trans-p-coumaroyloxy-2alpha-hydroxyolean-12-en-28-oic acid exhibited higher antiproliferative activity against MCF-7 cancer cells. All triterpenoids tested showed antiproliferative activity against Caco-2 cancer cells, especially 2alpha-hydroxyursolic acid, maslinic acid, 2alpha-hydroxy-3beta-{[(2E)-3-phenyl-1-oxo-2-propenyl]oxy}olean-12-en-28-oic acid, and 3beta-trans-p-coumaroyloxy-2alpha-hydroxyolean-12-en-28-oic acid, which displayed much higher antiproliferative activities. These results showed the triterpenoids isolated from apple peels have potent antiproliferative activity and may be partially responsible for the anticancer activities of whole apples.     

Biphasic effect of falcarinol on caco-2 cell proliferation, DNA damage, and apoptosis

The polyacetylene falcarinol, isolated from carrots, has been shown to be protective against chemically induced colon cancer development in rats, but the mechanisms are not fully understood. In this study CaCo-2 cells were exposed to falcarinol (0.5-100 microM) and the effects on proliferation, DNA damage, and apoptosis investigated. Low-dose falcarinol exposure (0.5-10 microM) decreased expression of the apoptosis indicator caspase-3 concomitantly with decreased basal DNA strand breakage. Cell proliferation was increased (1-10 microM), whereas cellular attachment was unaffected by <10 microM falcarinol. At concentrations above 20 microM falcarinol, proliferation of CaCo-2 cells decreased and the number of cells expressing active caspase-3 increased simultaneously with increased cell detachment. Furthermore, DNA single-strand breakage was significantly increased at concentrations above 10 microM falcarinol. Thus, the effects of falcarinol on CaCo-2 cells appear to be biphasic, inducing pro-proliferative and apoptotic characteristics at low and high concentrations of falcarinol, respectively.     

(-)-Anonaine induces DNA damage and inhibits growth and migration of human lung carcinoma h1299 cells

The anticancer effects of (-)-anonaine were investigated in this current study. (-)-Anonaine at concentration ranges of 50-200 μM exhibited significant inhibition to cell growth and migration activities on human lung cancer H1299 cells at 24 h, albeit cell cycle analyses showed that (-)-anonaine at the above concentration ranges did not cause any significant changes in cell-cycle distributions. Significant nuclear damages of H1299 cells were observed with 10-200 μM (-)-anonaine treatment in a comet assay, whereas higher concentrations (6 and 30 mM) of (-)-anonaine concentrations were required to cause DNA damages in an in vitro plasmid cleavage assay. In summary, our results demonstrated that (-)-anonaine exhibited dose-dependent antiproliferatory, antimigratory, and DNA-damaging effects on H1299 cells. We inferred that (-)-anonaine can cause cell-cycle arrest and DNA damage to hamper the physiological behavior of cancer cells at 72 h, and therefore, it can be useful as one of the potential herbal supplements for chemoprevention of human lung cancer.     

Differential effects of ferulic acid and p-coumaric acid on S phase distribution and length of S phase in the human colonic cell line Caco-2

Ferulic acid (FA) and para-coumaric acid (p-CA) may mediate the protective effects of whole-grain cereals against colon cancer. Therefore, the effects of FA and p-CA on the metabolic activity, proliferation, cell cycle phase distribution, and kinetics of the colonic endothelial tumor cell line Caco-2 was studied. Both compounds at 1500 microM decreased the number of cells to 43-75% of control after 2-3 days of treatment. Cell cycle phase distribution and cell cycle kinetics were determined by flow cytometric analysis after bromodeoxyuridine labeling. Each compound at 1500 microM decreased the proportion of cells in the G(1) phase and increased the proportion of cells in the S and G(2) phases. Treatment with 1500 microM FA significantly increased the length of the S phase, while p-CA did not. It was concluded that FA and p-CA inhibited cell proliferation by presumably affecting different cell cycle phases, and this warrants further investigations because this inhibition may be one explanation for the diet-related protection against cancer.     

Alkyl hydroxytyrosyl ethers show protective effects against oxidative stress in HepG2 cells

Alkyl hydroxytyrosyl ethers (methyl, ethyl, propyl, and butyl ethers) have been synthesized from hydroxytyrosol (HTy) in response to the increasing food industry demand of new lipophilic antioxidants. Having confirmed that these compounds reach portal blood partially unconjugated and thus are effectively absorbed, their potential antioxidant activity was evaluated in the human hepatocarcinoma cell line (HepG2). The effects of 0.5-10 μM alkyl hydroxytyrosyl ethers on HepG2 cell integrity and redox status were assessed as well as the protective effect against oxidative stress induced by tert-butylhydroperoxide (t-BOOH). Cell viability (Crystal violet) and cell proliferation (BrdU assay) were measured as markers of cell integrity, concentration of reduced glutathione (GSH), generation of reactive oxygen species (ROS), and activity of antioxidant enzymes glutathione peroxidase (GPx) and glutathione reductase (GR) as markers of redox status and determination of malondialdehyde (MDA) as a marker of lipid peroxidation. Direct treatment of HepG2 with alkyl hydroxytyrosyl ethers induced slight changes in cellular intrinsic antioxidants status, reducing ROS generation and inducing changes in GPx and GR activities. Pretreatment of HepG2 cells with alkyl hydroxytyrosyl ethers counteracted cell damage induced by t-BOOH, partially after 2 h and completely after 20 h, by increasing GSH and decreasing ROS generation, MDA levels, and antioxidant enzyme (GPx and GR) activity. According to these results the alkyl hydroxytyrosyl ethers show clear protective effects against oxidative stress, related to their lipophilic nature, that are similar to or even higher than those of their precursor, HTy.     

Attenuation of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced gap junctional intercellular communication (GJIC) inhibition in MCF-10A cells by c9,t11-conjugated linoleic acid

The protective effect of c9,t11-conjugated linoleic acid (CLA) on the inhibition of gap junctional intercellular communication (GJIC) was examined in a human mammary epithelial cell line (MCF-10A) treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), relative to t10,c12-CLA isomer. TPA inhibited GJIC in a dose-dependent and reversible manner and was associated with connexin 43 phosphorylation. Pretreatment of 20 μM c9,t11-CLA for 24 h prior to 60 nM TPA for 1 h prevented the inhibition of GJIC by reducing the phosphorylation of connexin 43 via suppressing extracellular signal-regulated kinases (ERK1/2) activation. Reactive oxygen species (ROS) accumulation by TPA was attenuated by c9,t11-CLA. The efficacy of c9,t11-CLA in protecting inhibition of GJIC, connexin 43 phosphorylation, and ROS production was superior to that of t10,c12-CLA. These results suggest that c9,t11-CLA, including t10,c12-CLA, prevents the carcinogenesis of MCF-10A cells by protecting down-regulation of GJIC during the cancer promotion stage, and lack of their toxicities could be an excellent indicator for the chemoprevention of breast cancer.