AMPK interacts with ß-catenin in the regulation of hepatocellular carcinoma cell proliferation and survival with selenium treatment.

Selenium has received much attention as an anticancer agent, although the mechanisms of action underlying its pro-apoptotic properties remain unclear. Tumors that respond well to antioxidant treatments, such as hepatocellular carcinoma (HCC), may benefit from treatment with selenium as this compound also has antioxidant properties. Furthermore, a major oncogenic driver in HCC is the nuclear transcription co-activator, ß-catenin. In the present study, we examined the mechanism by which selenium reduces survival of HCC cells, and whether this was associated with modulation of the ß-catenin pathway. Hep3B cell lines and cancer cell xenografted animals were treated with selenium, and apoptotic events or signals such as AMPK, ß-catenin and GSK3ß were determined. Further interactions among ß-catenin, glycogen synthase kinase 3ß (GSK3ß), and AMPK were explored by applying AMPK small interfering RNA (siRNA) or GSK3ß siRNA with western blotting or immunofluorescence microscopic observation. Selenium activated AMPK, which in turn suppressed ß-catenin. Selenium induced the translocation of AMPK into the nucleus and prevented the accumulation of ß-catenin therein. Upon inactivation of AMPK by AMPK siRNA, selenium no longer modulated ß-catenin, implying that AMPK is an upstream signal for ß-catenin. We found that the binding between AMPK and ß-catenin occurs in the cytosolic fraction, and therefore concluded that the cancer cell antiproliferative effects of selenium are mediated by a GSK3ß-independent AMPK/ß-catenin pathway, although AMPK-mediated GSK3ß regulation was also observed. We primarily discovered that AMPK is a crucial regulator initiating selenium-induced inhibition of ß-catenin expression. Taken together, these novel findings help to illuminate the molecular mechanisms underlying the anticancer effect of selenium and highlight the regulation of ß-catenin by selenium.

The involvement of AMPK/GSK3-beta signals in the control of metastasis and proliferation in hepato-carcinoma cells treated with anthocyanins extracted from Korea wild berry Meoru.

BACKGROUND: Activation of the Wnt pathway is known to promote tumorigenesis and tumor metastasis, and targeting Wnt pathway inhibition has emerged as an attractive approach for controlling tumor invasion and metastasis. The major pathway for inhibiting Wnt is through the degradation of ß-catenin by the GSK3-beta/CK1/Axin/APC complex. It was found that Hep3B hepato-carcinoma cells respond to anthocyanins through GSK3-beta-induced suppression of beta-catenin; however, they cannot dephosphorylate GSK3-beta without AMPK activation. METHODS: We tested the effects of anthocyanins on proliferation and apoptosis by MTT and Annexin V-PI staining in vitro. Mouse xenograft models of hepato-carcinomas were established by inoculation with Hep3B cells, and mice were injected with 50 mg/kg/ml of anthocyanins. In addition, protein levels of p-GSK3-beta, beta-catenin, p-AMPK, MMP-9, VEGF, and Ang-1 were also analyzed using western blot. RESULTS: Anthocyanins decrease phospho-GSK3-beta and beta-catenin expression in an in vivo tumor xenograft model, increase AMPK activity in this model, and inhibit cell migration and invasion, possibly by inhibiting MMP-2 (in vitro) and the panendothelial marker, CD31 (in vivo). To elucidate the role of the GSK3-beta/beta-catenin pathway in cancer control, we conditionally inactivated this pathway, using activated AMPK for inhibition. Further, we showed that AMPK siRNA treatment abrogated the ability of anthocyanins to control cell proliferation and metastatic potential, and Compound C, an AMPK inhibitor, could not restore GSK3-beta regulation, as exhibited by anthocyanins in Hep3B cells. CONCLUSION: These observations imply that the AMPK-mediated GSK3-beta/beta-catenin circuit plays crucial roles in inhibiting cancer cell proliferation and metastasis in anthocyanin-treated hepato-carcinoma cells of Meoru origin.

Suppression of mTOR via Akt-dependent and -independent mechanisms in selenium-treated colon cancer cells: involvement of AMPKalpha1.

Activation of the mammalian target of rapamycin (mTOR) pathway promotes tumorigenesis, and inhibiting the mammalian target of rapamycin complex 1 (mTORC1) has emerged as an attractive target for suppressing tumor growth. We found that selenium treatment of HT-29 colon cancer cells suppressed mTORC1 through Akt-independent and -dependent pathways. In Akt-independent mTORC1 inhibition in selenium-treated colon cancer cells, adenosine monophosphate-activated protein kinase (AMPK) alpha(1) was crucial for suppression of mTORC1 activity. In contrast, the Akt-dependent mTORC1 inhibition by selenium did not require AMPKalpha(1). The importance of the AMPKalpha(1)-mTORC1 pathway in mediating the antiproliferative action of selenium was examined in xenograft tumors, and the suppression of mTORC1 as well as Akt was concomitant with an increase in AMPKalpha(1) activity. These findings suggest that the antiproliferative effect of selenium is mediated by an Akt-independent AMPKalpha(1)/mTORC1 pathway or by the Akt/tuberous sclerosis complex 2 /mTORC1 pathway.

Kidney bean husk extracts exert antitumor effect by inducing apoptosis involving AMP-activated protein kinase signaling pathway.

In this study, we investigated the molecular basis of Korean kidney bean husk extract, with emphasis on its ability to control intracellular signaling cascades of AMP-activated protein kinase (AMPK) responsible for inducing antitumor activities in colon cancer cells. Recently, the evolutionarily conserved serine/threonine kinase, AMPK, has emerged as a possible target molecule of tumor control. We investigated the effects of Korean kidney bean husk extract on apoptosis regulation and the activation of AMPK. Korean kidney bean husk extract exhibited a series of antitumor effects such as cell death and apoptotic body appearance. These antitumor potentials were accompanied by the increase in p-AMPK and p-Acc as well as antitumor proteins p53 and p21. The stimulation of AMPK by this extract was blocked with the synthetic AMPK inhibitor Compound C at 10 micromol/L, and the combined treatment of Compound C and the AMPK activator AICAR (5-aminoimiazole-4-carboxamide-1-beta-D-ribofuranoside) showed that Compound C could inhibit the activation of AMPK at the concentration of 20 micromol/L. In conclusion, the ability of carcinogenesis control by Korean kidney bean husk extract with high potency suggests its value as an antitumor agent in colon cancer therapy.

Green tea catechin controls apoptosis in colon cancer cells by attenuation of H2O2-stimulated COX-2 expression via the AMPK signaling pathway at low-dose H2O2.

This study investigated the apoptotic regulation by green tea catechin epigallcatechin-3-gallate (EGCG) on colon cancer cells in the presence of low-dose H(2)O(2) known to exert the activation of signal pathways leading to cell proliferation. In the presence of low-dose H(2)O(2), EGCG induced apoptosis and abolished the cell-proliferative effect exhibited by low-dose H(2)O(2). This reduction of growth was accompanied by an activation of AMP-activated kinase (AMPK), a decrease in cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) levels, and the induction of apoptotic markers such as p53 and poly(ADP-ribose) polymerase (PARP) cleavage. The low-dose H(2)O(2) stimulated COX-2 expression, and treating cells with synthetic AMPK activator AICAR (5-aminoimiazole-4-carboxamide-1-beta-d-ribofuranoside) resulted in greater suppression of COX-2 expression and PGE(2). By treating cells with high concentrations of the reactive oxygen species (ROS) scavenger NAC (N-acetyl-1-cysteine), the apoptotic effect of EGCG was abolished and led to suppression of AMPK and COX-2, indicating that the liberation of excessive ROS might be the upstream signal of the AMPK-COX-2 signaling pathway even in the presence of low-dose H(2)O(2).

Attenuation of Abeta-induced apoptosis of plant extract (Saengshik) mediated by the inhibition of mitochondrial dysfunction and antioxidative effect.

Recently, considerable attention has been focused on dietary manipulation of oxidative and/or nitrosative damage on neuronal cells. In this article, a neuroprotective effect of plant (Saengshik) extracts was investigated. Rat pheochromocytoma (PC12), cells treated with beta-amyloid underwent apoptotic death as determined by positive in situ terminal end-labeling (TUNEL staining), decreased mitochondrial transmembrane potential, and elevated caspase-3 activity co-occurring with enhanced MDA accumulation and the reduction of GSH levels. Saengshik pretreatment attenuated beta-amyloid-induced apoptosis in PC12 cells possibly by inhibiting mitochondrial dysfunction and exerting antioxidant properties. Saengshik pretreatment inhibited the loss of mitochondrial membrane potentials and reduced the activation of caspase-3. The in vitro antioxidant activities of Saengshik extracts were verified by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method and superoxide dismutase (SOD) mimetic activity. In beta-amyloid-challenged PC12 cells, Saengshik prevented the production of ROS, decreased the level of MDA, and elevated GSH. The potential of Saengshik as one of the neuroprotective regimens has been suggested through this article, and the combination with defined pharmaceuticals or other dietary antioxidants may provide a better therapeutic or preventive advantage for the management of Alzheimer's disease.

Possible link between NO concentrations and COX-2 expression in systems treated with soy-isoflavones.

The production of nitric oxide (NO) emerges as an essential determinant in auto- and paracrine signaling. NO is known to be generated under inflammatory conditions, carcinogenesis, and circulatory shock. The large amount of NO produced in response to cytokines plays an important role in inflammatory conditions. Cyclooxygenase (COX), the central enzyme in prostanoid biosynthesis, is involved in the first step of prostanoid synthesis from arachidonic acid. The reported studies to evaluate the relationship between NO and COX-2 have revealed both inhibitory and stimulatory effects of NO on COX-2 expression. Genistein, one of soy-isoflavones, is a polyphenolic flavonoid and a potent antioxidant and anti-inflammatory agent. In the present article, the effect of soy-isoflavones on NO production and COX-2 gene expression was examined. NO production by soy-isoflavones was greatly increased even though eNOS and iNOS expression were not different from nontreated control. The increment of NO was accompanied with the elevated expression of COX-2 and the concentrations of PGE2. The COX-2 stimulatory effect of soy-isoflavones appeared to be modulated by ERK-1 and -2 and p38. In mammalian cancer system, incubation with the NO donor sodium nitroprusside (SNP) resulted in a slight upregulation of COX-2, and cotreatment with genistein decreased COX-2 expression possibly by the activation of AMP-activated protein kinase (AMPK).

Soy isoflavone supplementation alleviates oxidative stress and improves systolic blood pressure in male spontaneously hypertensive rats.

The aim of this study was to investigate the protective effect of isoflavone against hypertension, via the mitigation of oxidative stress and prevention of nitric oxide (NO, a potent vasodilator) reduction, in spontaneously hypertensive rats (SHR). The 8 wk-old male SHR were divided into two groups, and fed a casein-based high fat diet (120 g fat, 1 g cholesterol/kg diet) for 30 d, either with or without 10 g of soy powder (containing 31.2% of isoflavones)/kg. During the 30-d study period, tail systolic blood pressures (BP) in the control SHR group increased, from 162.4 +/- 2.3 to 177.9 +/- 5.4 mmHg (p<0.05), while the isoflavone-supplemented group benefited from a clear antihypertensive effect (160.1 +/- 1.8 to 160.2 +/- 4.9 mmHg). The serum NO and total radical trapping antioxidant potential (TRAP) were elevated in the isoflavone group. The isoflavone group also experienced a significant decrease in oxidative DNA damage in leukocytes, using comet assay. DNA damage correlated positively with incremental BP during the study, and systolic BP at the end of the study (p<0.01). Our results indicate that soy isoflavone has an antihypertensive effect, possibly through the amelioration of oxidative stress, and the augmentation of NO production, in SHR.

Chemopreventive potential of epigallocatechin gallate and genistein: evidence from epidemiological and laboratory studies.

A wide array of antioxidative and anti-inflammatory substances derived from edible plants have been reported to possess chemopreventive and chemoprotective activities. Among the most extensively investigated and well-defined dietary chemopreventives are epigallocatechin gallate (EGCG), a principal antioxidant derived from green tea and genistein, a major pharmacologically active isoflavone widely present in soy products. Multiple lines evidence from epidemiologic studies indicate that frequent consumption of green tea is inversely associated with the risk of several types of human cancer, and studies with animal and in vitro cell culture models have revealed EGCG as a major chemopreventive ingredient of green tea. The lower frequencies of breast and prostate cancer in Asian population in general, compared to those in Western societies have been attributed to their consumption of relatively large amounts of soy products. Genistein, as a principal chemopreventive components of soy, exerts a wide array of chemopreventive activities in each stage of multistep carcinogenesis. The purpose of this review is to provide perspectives on the molecular basis of chemopreventive activities of EGCG and geneistein as representative functional food phytochemicals with emphasis on their ability to control intracellular signaling cascades responsible for regulating cell growth and differentiation.

Effect of vitamin E supplementation on antioxidant defense systems and humoral immune responses in young, middle-aged and elderly Korean women.

Free radical-mediated oxidative stress has been implicated in the pathogenesis of numerous chronic diseases. Vitamin E is known to play an important role in the free-radical quenching process. However, clinical trials with vitamin E have yielded contrasting results in the prevention of several diseases related to oxidative stress. This study was undertaken to investigate the antioxidative and humoral immunologic effects of vitamin E supplementation in three different age groups: young (mean age 32.7 +/- 5.7 y), middle-aged (mean age 47.0 +/- 5.0 y) and elderly (67.6 +/- 4.7 y) women. Volunteer subjects were given a supplement of 400 IU dl-alpha-tocopherol acetate for 6 wk. Thiobarbituric acid reacting substances (TBARS) in the plasma significantly decreased with vitamin E supplementation. In addition, the radical scavenger activities (RSA) of red blood cells significantly increased with vitamin E supplementation in all age groups. However, humoral immune response modulation was not observed following vitamin E supplementation. Even though there is no clear indication that vitamin E supplementation is necessary to improve the humoral immune functions, vitamin E supplementation may be beneficial to all adult age groups as a preventive measure for complications related to oxidative damage.