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Epidemiologic and experimental evidences indicate that selenium, an essential trace element, can reduce the risk of a variety of cancers. Protection against certain types of cancers, particularly colorectal cancers, is closely associated with pathways involving cyclooxygenase-2 (COX-2). We found that AMP-activated protein kinase (AMPK), which functions as a cellular energy sensor, mediates critical anticancer effects of selenium via a COX-2/prostaglandin E2 signaling pathway. Selenium activated AMPK in tumor xenografts as well as in colon cancer cell lines, and this activation seemed to be essential to the decrease in COX-2 expressions. Transduction with dominant-negative AMPK into colon cancer cells or application of cox-2−/−-negative cells supported the evidence that AMPK is an upstream signal of COX-2 and inhibits cell proliferation. In HT-29 colon cancer cells, carcinogenic agent 12-O-tetradecanoylphorbol-13-acetate (TPA) activated extracellular signal-regulated kinase (ERK) that led to COX-2 expression and selenium blocked the TPA-induced ERK and COX-2 activation via AMPK. We also showed the role of a reactive oxygen species as an AMPK activation signal in selenium-treated cells. We propose that AMPK is a novel and critical regulatory component in selenium-induced cancer cell death, further implying AMPK as a prime target of tumorigenesis. (Cancer Res 2006; 66(20): 10057-63)

Supplementary Figure Legends 1-2 from Selenium Regulates Cyclooxygenase-2 and Extracellular Signal-Regulated Kinase Signaling Pathways by Activating AMP-Activated Protein Kinase in Colon Cancer Cells

Supplementary Figure 2 from Selenium Regulates Cyclooxygenase-2 and Extracellular Signal-Regulated Kinase Signaling Pathways by Activating AMP-Activated Protein Kinase in Colon Cancer Cells

Supplementary Figure 1 from Selenium Regulates Cyclooxygenase-2 and Extracellular Signal-Regulated Kinase Signaling Pathways by Activating AMP-Activated Protein Kinase in Colon Cancer Cells

Background BRCA1 mutated breast cancer cells exhibit the elevated cell proliferation and the higher metastatic potential. G protein-coupled receptor 30 (GPR30) has been shown to regulate growth of hormonally responsive cancers, such as ovarian and breast cancers, and high expression of GPR30 is found in estrogen receptor (ER)-negative breast cancer cells. ER-negative breast cancer patients often have a mutation in the tumor suppressor gene, BRCA1. This study explored antiproliferative effects of genistein, a chemopreventive isoflavone present in legumes, and underlying molecular mechanisms in triple negative breast cancer cells with or without functionally active BRCA1. Methods Expression of BRCA1, GPR30 and Nrf2 was measured by Western blot analysis. Reactive oxygen species (ROS) accumulation was monitored by using the fluorescence-generating probe, 2',7'-dichlorofluorescein diacetate. The effects of genistein on breast cancer cell viability and proliferation were assessed by the MTT, migration and clonogenic assays. Results The expression of GPR30 was dramatically elevated at both transcriptional and translational levels in BRCA1 mutated breast cancer cells compared to cells with wild-type BRCA1. Notably, there was diminished Akt phosporylation in GPR30 silenced cells. Treatment of BRCA1 silenced breast cancer cells with genistein resulted in the down-regulation of GPR30 expression and the inhibition of Akt phosphorylation as well as the reduced cell viability, migration and colony formation. Genistein caused cell cycle arrest at the G2/M phase in BRCA1-mutant cells through down-regulation of cyclin B1 expression. Furthermore, BRCA1-mutant breast cancer cells exhibited higher levels of intracellular ROS than those in the wild-type cells. Genistein treatment lowered the ROS levels through up-regulation of Nrf2 expression. Conclusions Lack of functional BRCA1 activates GPR30 signaling, thereby stimulating Akt phosphorylation and cell proliferation. Genistein induces G2/M phase arrest by down-regulating cyclin B1 expression, which is attributable to its suppression of GPR30 activation and Akt phosphorylation in BRCA1 impaired breast cancer cells.

Suppressing the mammalian target of rapamycin (mTOR) pathway has emerged as an attractive method for controlling cancer growth and preventing cancers. Epigallocatechin-3-gallate (EGCG) is a well-known chemopreventive polyphenol, and its effects on AMP-activated protein kinase (AMPK) activation were previously reported. In this study the regulatory mechanisms of EGCG on mTOR and Akt, 2 cancer survival signals, and the interrelationships among mTORC1, Akt, and AMPK were examined. It was found that the suppression of mTORC1 by EGCG requires signals from AMPK, however, the inhibition of Akt with EGCG seems to be AMPK independent. Further, there was no clear indication of Akt as an upstream regulator of mTOR in EGCG treated HT-29 colon cancer cells.

Cyclooxygenase-2 (COX-2), the key enzyme of the conversion of arachidonic acid to prostaglandins is an important regulator of inflammation and perhaps apoptosis. Genistein is an active component of legumes and other related food associated with prevention of degenerative diseases possibly through modulating certain signaling pathways. It was investigated whether the induction of apoptosis with genistein was carried out via COX-2 suppression through the regulation of NF-κB. The cox-2 positive and negative cells were used to compare the effect of genistein on the modulation of NF-κB in COX-2 expressed or non-expressed genotypic systems. Suppression of COX-2 as well as decreasing NF-κB DNA binding activity was accompanied with the induction of apoptosis in genistein-treated COX-2 expressed cells. However, in cox-2 negative cells, apoptosis occurred without any involvement of NF-κB with genistein treatement. Genistein induced apoptosis through the generation of reactive oxygen species (ROS) both of cox-2 positive and negative cells. These results suggested that genistein is capable of exihibiting NF-κB-dependent and NF-κB-independent apoptotic control via ROS generation depending on genetic cell types.

Akt is known to play an important role in cell proliferation and differentiation, and is also over-expressed in several types of cancer cells. In this study, we explored the anti-proliferative effects of selenium in HT-29 colon cancer cells, mediated through effects on Akt and COX-2. Selenium treatments at different concentrations and for different durations inhibited proliferation of HT-29 colon cancer cells and increased apoptotic cell death. Selenium treatment decreased Akt phosphorylation and COX-2 expression. Treatment with LY294002 (an Akt inhibitor) decreased proliferation of HT-29 cells, while a combined treatment with LY294002 and selenium resulted in even further decreases in cell proliferation. Inactivation of Akt by Akt siRNA treatment abolished these inhibitory effects on cell growth. COX-2 expression decreased in Akt transfected cells compared to non-transfected cells. These results suggest that selenium induced both anti-proliferative and apoptotic effects by inhibiting Akt phosphorylation and COX-2 expression. Selenium treatment also appeared to induce synergistic anti-proliferative effects by inhibition of Akt in HT-29 colon cancer cells.

Resveratrol, a kind of phytochemical, is presented in grape skins. Resveratorl exerts antiproliferative, anti-cancer and pro-apoptotic activities in cancer cells. Mammalian target of rapamycin (mTOR) is a critical regulator of cellular growth and proliferation, and it is known to be a strategic target for anti-cancer therapeutic uses. mTOR is a major downstream of the PI3K/Akt pathway, which is activated in various cancer cells. It also plays an important role in the survival, proliferation and angiogenesis of cells. Cyclooxygenase-2 (COX-2) is an important protein that mediates inflammatory processes. It plays an important role in various tumors by affecting cell proliferation, mitosis, apoptosis and angiogenesis. In this study, we have investigated the effects of resveratrol on apoptosis through mTOR and COX-2 expression in MCF-7 breast cancer cells. The treatment of resveratrol with different concentrations inhibited proliferation of MCF-7. The data showed that resveratrol induced apoptotic cell death of cancer cells and decreased mTOR and COX-2 expression. These results suggest that resveratrol induces apoptosis of MCF-7 breast cancer cells by inhibiting mTOR and COX-2 expression.

Quercetin has been reported to possess therapeutic effects in the treatment of cancers. In this study, the molecular action of quercetin, with emphasis on its ability to control the intracellular signaling cascades of hypoxia inducible factor-1α (HIF-1α), mammalian target of rapamycin (mTOR), and AMP-activated protein kinase (AMPK), responsible for survival or induction of apoptosis in hypoxic MCF-7 cells, was investigated. The effects of quercetin on apoptosis in relation to its ability to prevent HIF-1α induction were investigated. The involvement of HIF-1α reduction in quercetin-based cancer control was clearly shown in conditions of mTOR inhibition by rapamycin, an mTOR inhibitor. Surprisingly, quercetin induced an AMPK-suppressed state in a CoCl2-induced hypoxic condition. It is speculated that quercetin is capable of inhibiting AMPK to decrease HIF-1α, which is a critical survival factor in hypoxia. A complex control of HIF-1α, mTOR, and AMPK is necessary in inducing apoptosis of MCF-7 breast cancer cells under hypoxia.

파이토케미컬의 일종인 EGCG는 녹차의 카테킨 성분으로, 세포 내 신호 경로 조절을 통하여 항산화, 항암효과를 나타내는 것으로 알려져 있다. 본 연구에서는 hypoxia 상태인 B16F10 피부암 세포에서 HIF- $1{\alpha}$ 를 포함한 AMPK의 신호경로를 통하여 EGCG의 apoptosis 유도 효과를 규명하였다. AMPK는 hypoxia, 영양분 결핍, 운동, heat shock 등, 세포 내 ATP의 결핍에 의해서 활성화되며 암세포의 증식을 억제하고 apoptosis를 유도한다. 세포에서 중요한 에너지 센서로서 작용하는 AMPK가 hypoxia 상태의 암세포 내에서는 HIF- $1{\alpha}$ 의 전사 활성을 유도하는데, HIF- $1{\alpha}$ 는 hypoxia 상태에서 산소 결핍에 반응하는 첫 번째 전사 조절인자로서 암세포의 생존을 위한 세포내 산소공급과 혈관신생형성을 조절한다. Hypoxia 상태가 아닌 B16F10 세포에서와 hypoxia 상태에서의 B16F10 세포에서 EGCG에 의한 apoptosis 효과를 관찰하였다. 실험 결과, hypoxia 상태에서 EGCG는 더 강한 apoptosis를 유도하며, 혈관신생형성을 조절할 수 있는 HIF- $1{\alpha}$ 의 전사 활성을 억제시킨다. 이러한 관찰을 통해 EGCG가 hypoxia 상태의 피부암 세포에서 암의 성장과 신생혈관형성을 저해하는 것으로 보인다. 이와 같은 연구는 향후 식품에 첨가된 파이토케미컬을 이용하여 암을 예방하는 연구에서 있어서, 도움이 될 것으로 여겨진다. 【EGCG, catechins in green tea, is a kind of phytochemical. Through the regulation of signal pathways, EGCG has been known to show anti-oxidant and anti-tumor effects in cells. In this study, we investigated the apoptotic effects of EGCG through AMP-activated protein kinase (AMPK) signal pathways, including hypoxia inducible factor-1 alpha (HIF- $1{\alpha}$ ). The experiments were performed in B16F10 melanoma cells in a hypoxic state. AMPK is activated by ATP consumption such as nutrient deficiency, exercise, heat shock, etc. The activated AMPK that plays an important role as an energy sensor inhibits proliferation of cancer cells, as well as inducing apoptosis. HIF- $1{\alpha}$ , the primary transcriptional regulator of the response to oxygen deprivation, plays a critical role in modulating tumor growth and angiogenesis in a hypoxic state. The apoptotic effects of EGCG were studied in B16F10 cells in a hypoxic state. The results show that EGCG inhibits the transcriptional activity of HIF- $1{\alpha}$ and induces apoptosis. These observations suggest that EGCG may exert inhibitory effects of angiogenesis and control tumor cell growth in hypoxic melanoma cells.】

In this study, the potential of cherry silver berry (Elaeagnus multiflora) as a cancer preventive agent through regulating inflammatory signals including cyclooxygenase-2 (COX-2) and Akt was examined. Cherry silver berry has reported to exert anti-oxidative, anti-inflammatory, and anti-proliferative effects. The extracts from seed and flesh of cherry silver berry were obtained, and analyzed COX-2 and Akt activities in cherry silver berry extract treated HT-29 colon cancer cells. The results showed that the treatment of seed extracts reduced cell viability at the concentrations above 1,600 mg/mL, effectively reduced COX-2 and p-Akt expression. In addition, the anti-inflammatory effects seemed to be related to cancer cell death. Both of seed and flesh extracts inhibited cell growth and induced apoptosis in HT-29 cells. These results suggest that extracts of cherry silver berry may contribute to suppressing cancer growth through its anti-inflammatory and anti-proliferative effects, and natural products used as oriental medicine have the possibility to control tumor cell growth.

It has been well known that resveratrol inhibits the proliferation of various cancer cells. AMP-activated protein kinase (AMPK), a sensor of cellular energy status, has emerged as a potent target for cancer prevention and/or treatment. It has been found that the activation of AMPK by resveratrol was crucial for the inhibition of HT-29 colon cancer cells. Resveratrol strongly inhibited phosphorylation of Akt. The possibility whether AMPK activation was essential to the inhibition of p-Akt was investigated in resveratrol-treated cancer cells. The inhibitory effect of resveratrol on Akt was not observed when AMPK activities were blocked by the treatment with AMPK siRNA at a relatively lower level of resveratrol. However, the higher concentrations of resveratrol inhibited Akt without the activation of AMPK. Therefore, it was concluded that resveratrol could modulate Akt AMPK-dependently or AMPK-independently. The inhibition of Akt along with the activation of AMPK may contribute to the unraveling anti-cancer mechanism of resveratrol.

Effective strategies for cancer prevention and treatment can be identified by understanding the mechanism of apoptotic pathways. In this study, we investigated the regulatory mechanism of quercetin-induced apoptosis through apoptosis signal-regulating kinase (ASK)-1 and mitogen-activated protein kinase pathways. Our results showed that quercetin increased apoptotic cell death through reactive oxygen species (ROS) generation and was responsible for ASK1 activation. Increasing ASK1 activity was accompanied by p38 activation. Interestingly, AMP-activated protein kinase (AMPK) seemed to be a critical controller of quercetin-regulated ASK1/p38 activation. Blocking AMPKalpha1 activity using Compound C, a synthetic inhibitor or siRNA showed that quercetin-activated ASK1 could not stimulate p38 activity. Thus, we suggested that quercetin-exerted apoptotic effects involve ROS/AMPKalpha1/ASK1/p38 signaling pathway, and AMPKalpha1 is a necessary element for apoptotic event induced by ASK1.

We isolated anthocyanins from fruits of Vitis coignetiae Pulliat, characterized the anthocyanin profile, and investigated the anti-invasive effects of the anthocyanins on human colon cancer cells. The anthocyanins inhibited cell invasion in a dose-dependent manner, as measured by Matrigel invasion assays, by suppression of matrix metalloproteinase (MMP)-2 and MMP-9 expression. The anti-invasive activity of the anthocyanins was associated with modulation of constitutive nuclear factor kappaB (NF-kappaB) activation. The activation of NF-kappaB triggered by tumor necrosis factor-alpha was also inhibited by the anthocyanins through suppression IkappaBalpha phosphorylation. AIMs inhibited the expression of NF-kappaB-regulated proteins. In conclusion, this study suggested that the anthocyanins isolated from fruits of V. coignetiae Pulliat should have anti-invasive activities on human colon cancer cells and the activities should be related to the inhibition of NF-kappaB-regulated proteins such as MMP-2 and MMP-9 expression through the inhibition of NF-kappaB activation.

The topical application of TPA (12-O-tetradecanoylphorbol-13-acetate) to animal skin or direct treatment of TPA to cell cultures leads to inflammatory responses by enhancing cyclooxygenase 2 (COX-2) expression, and specific COX-2 inhibitors counteract this kind of inflammatory response. Furthermore, suppression of these inflammatory events by dietary-origin chemopreventive agents can provide a potential strategy to control carcinogenesis. In this in vivo study, the mammary glands of mature female rats were treated with TPA, and then the effects of genistein alone or in combination with capsaicin on suppression of inflammatory responses were examined. The combined effects of genistein and capsaicin on COX-2, pJNK, pERK, and pp38 expressions were additive or nonadditive, depending on signals tested. In vitro MCF-7 breast cancer cells, the apoptotic bodies as shown with Hoechst 33342 dye, exhibited a synergistic effect between genistein and capsaicin. The abilities of genistein alone or in combination with capsaicin in inhibiting breast cancer cell proliferation through the modulation of AMPK and COX-2 were tested. AMPK activation by genistein in combination with capsaicin is critical for inhibiting COX-2. We propose that genistein in combination with capsaicin exerts anti-inflammatory and anticarcinogenic properties through the modulation of AMPK and COX-2 and possibly various mitogen-activated protein kinases synergistically or nonsynergistically.

Photodynamic therapy (PDT) is a treatment for cancer involving three key components — a sensitizing compound (light) tissue, and oxygen. In this study we applied phototreatment to cancer cells with 2 J.cm-2of red light after sensitizing with 9-hydroxypheophorbide-α (9-HpbD-α), a new chlorophyll-derived photosensitizer. We have investigated the cytotoxic and apoptotic effects of 9-HpbD-α-induced PDT in cervical cancer cells, the enhancing effect of genistein in PDT, and explored the molecular mechanisms of E6 or E7 oncogenes, apoptotic signaling molecules, and ER stress. Co-treatment downregulated the transcripts of the E6*I, E6*II, and E7 oncogenes. Combined treatment with PDT and genistein showed typical apoptotic features, i.e. apoptotic bodies. To elucidate the mechanism of combination treatment-induced apoptosis, various mediators of apoptosis were investigated. Activation of caspase-8, caspase-3, and PARP were distinct after combination treatment. Furthermore, ER stress-related proteins, such as CHOP and GRP78, were activated after combination treatment. We conclude that genistein sensitizes CaSki cells to apoptosis treated with PDT by 9-HpbD-α (0.59 μg/mL) through mechanisms that involve downregulation of the E6*I, E6*II, and E7 oncogenes, activation of caspase-8 or caspase-3, and ER stress.

Selenium was investigated using human origin preadipocytes to see whether it possesses preventive or therapeutic effects for obesity. Unveiling the potential of selenium in the reduction of adipogenesis can help predict the therapeutic capabilities of selenium in obesity. In the present study, the molecular mechanism of the inhibition of adipogenesis by selenium was explored to unravel the involvement of the AMP-activated protein kinase. There is emerging evidence that AMPK, a sensor of cellular energy status, is a possible molecular target of controlling adipocyte differentiation on the basis of discovery that AMPK is responsible for the major metabolic responses to exercise, and integration of nutritional and hormonal signals to modulate feeding behavior or energy expenditure in the hypothalamus. Treatment of selenium resulted in inhibition of the adipocyte differentiation process and induction of mature apoptosis in 3T3-L1 adipocytes. We hypothesized that selenium may exert anti-adipogenic potential though modulating AMPK. We have found that selenium significantly activated AMPK and phosphorylated its substrate acetyl-CoA carboxylase (ACC-serine??) during the inhibitory process of adipocytes. Also, the inhibition process of adipocyte differentiation by selenium was comparable to either reveratrol or a synthetic AMPK activator, AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside). To evaluate the involvement of AMPK in anti-lipogensis, we applied AICAR and Compound C, an AMPK inhibitor, to 3T3-L1-adipocytes and found that AMPK is required for the adipocyte differentiation blocking process. These results suggest that selenium has a potential to control adipogenesis and that this effect is mediated by AMPK, an essential kinase for both inhibition of adipocyte differentiation and apoptosis of mature adipocytes.

Genistein is an active component of legumes and other related food shown to be associated with prevention of degenerative diseases such as cancer through inducing signaling pathways. Treatment of genistein resulted in the induction of apoptosis in the cultured cancer cells. This induction of apoptosis was demonstrated by the Tunel assay in these cells. Unveiling the potential of genistein in cytotoxicity via apoptosis when it is treated with estrogen can predict the therapeutic capability of genistein in breast cancers in the presence of endogenous estrogen. We have found that apoptosis induced by genistein treatment in the presence of estrogen is agonistic or antagonistic depending on the concentrations and treatment periods applied in MCF-7 breast cancer cells. For the suppression of cell survival, 24 hr of treatment was required to induce a synergistic agonistic response between estrogen and genistein at low concentrations of genistein. After this period, the agonistic pattern of genistein to estrogen disappeared. The decrement of COX-2 expression in MCF-7 cells treated with genistein was accompanied with the activation of AMPK only at a high concentration of genistein. This association between AMPK activation and down-regulation of COX-2 by genistein was dampened in the presence of estrogen. It was also demonstrated that genistein and estrogen regulate cell survival and apoptosis by modulating p53 and caspase-3 in the opposite direction. These results suggest that genistein has the potential to control breast cancer development, and co-treatment with estrogen can cause agonistic or antagonistic action on breast cancer cell control.

EGCG [(-)epigallocatechin-3-gallate], a green tea-derived polyphenol, has been shown to suppress cancer cell proliferation, and interfere with the several signaling pathways and induce apoptosis. Practically, there is emerging evidence that EGCG has a potential to increase the efficacy of chemotherapy in patients. We hypothesized that EGCG may exert cell cytotoxicity through modulating AMPK (AMP-activated protein kinase) followed by the decrease in COX-2 expression. EGCG treatment to colon cancer cells resulted in a strong activation of AMPK and an inhibition of COX-2 expression. The decreased COX-2 expression as well as prostaglandin E(2) secretion by EGCG was completely abolished by inhibiting AMPK by an AMPK inhibitor, Compound C. Also, the activation of AMPK was accompanied with the reduction of VEGF (vascular endothelial growth factor) and glucose transporter, Glut-1 in EGCG-treated cancer cells. These findings support the regulatory role of AMPK in COX-2 expression in EGCG-treated cancer cells. Furthermore, we have found that reactive oxygen species (ROS) is an upstream signal of AMPK, and the combined treatment of EGCG and chemotherapeutic agents, 5-FU or Etoposide, exert a novel therapeutic effect on chemo-resistant colon cancer cells. AMPK, a molecule of newly defined cancer target, was shown to control COX-2 in EGCG-treated colon cancer cells.

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).

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, β-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 β-catenin pathway. Hep3B cell lines and cancer cell xenografted animals were treated with selenium, and apoptotic events or signals such as AMPK, β-catenin and GSK3β were determined. Further interactions among β-catenin, glycogen synthase kinase 3β (GSK3β), and AMPK were explored by applying AMPK small interfering RNA (siRNA) or GSK3β siRNA with western blotting or immunofluorescence microscopic observation. Selenium activated AMPK, which in turn suppressed β-catenin. Selenium induced the translocation of AMPK into the nucleus and prevented the accumulation of β-catenin therein. Upon inactivation of AMPK by AMPK siRNA, selenium no longer modulated β-catenin, implying that AMPK is an upstream signal for β-catenin. We found that the binding between AMPK and β-catenin occurs in the cytosolic fraction, and therefore concluded that the cancer cell antiproliferative effects of selenium are mediated by a GSK3β-independent AMPK/β-catenin pathway, although AMPK-mediated GSK3β regulation was also observed. We primarily discovered that AMPK is a crucial regulator initiating selenium-induced inhibition of β-catenin expression. Taken together, these novel findings help to illuminate the molecular mechanisms underlying the anticancer effect of selenium and highlight the regulation of β-catenin by selenium.

Phytochemicals such as soy isoflavone genistein have been reported to possess therapeutic effects for obesity, diabetes, and cardiovascular diseases. In the present study, the molecular basis of selective phytochemicals with emphasis on their ability to control intracellular signaling cascades of AMP-activated kinase (AMPK) responsible for the inhibition of adipogenesis was investigated. Recently, the evolutionarily conserved serine/threonine kinase, AMPK, emerges as a possible target molecule of anti-obesity. Hypothalamic AMPK was found to integrate nutritional and hormonal signals modulating feeding behavior and energy expenditure. We have investigated the effects of genistein, EGCG, and capsaicin on adipocyte differentiation in relation to AMPK activation in 3T3-L1 cells. Genistein (20-200muM) significantly inhibited the process of adipocyte differentiation and led to apoptosis of mature adipocytes. Genistein, EGCG, and capsaicin stimulated the intracellular ROS release, which activated AMPK rapidly. We suggest that AMPK is a novel and critical component of both inhibition of adipocyte differentiation and apoptosis of mature adipocytes by genistein or EGCG or capsaicin further implying AMPK as a prime target of obesity control.

In mammary carcinogenesis, hormonal effects have been reported to be important factors. Estrogens are known to regulate the proliferation of breast cancer cells, whereas genistein has been shown to induce apoptosis in mammary tumor cells. This study examined genistein-induced apoptosis through the regulation of bcl-2 and bax expression in the presence of estrogen. MCF-7 cells were treated with either genistein (25, 50, and 100 microM) or in the presence of 17beta-estradiol (12.5, 25, and 50 nM) for 48 h. DNA ladder analysis and Western blot analysis of bcl-2, bax, cyclin B(1), p21, and p53 were carried out. For comparison, the in vivo system was employed using estrogen-deficient and estrogen-sufficient female rats at two different concentrations of genistein. In MCF-7 cells, DNA fragmentation was evident by the treatment of genistein in the absence and presence of estrogen. Downregulation of bcl-2 and upregulation of bax by genistein were observed. However, genistein showed no proapoptotic properties in the presence of estrogen except with the lowest concentration of estrogen. In the presence of estrogen, p21 and p53 protein expression were upregulated by high concentrations of genistein. Bcl-2/bax ratios were decreased by genistein treatment in the presence or absence of estrogen in female rats. These results demonstrate that the proapoptotic property of genistein might be influenced greatly by the concentration of estrogen in vitro, but that this influence by estrogen is not evident in vivo.

To examine the effect of two types of resistant starch on blood glucose and insulin levels, colonic events, hypolipidemic actions and humoral immune responses, Sprague-Dawley streptozotocin-induced diabetic rats were fed diet containing resistant starch from corn or rice. The marked body weight loss by inducing diabetes was not recovered by feeding resistant starch, even though there are no differences in food intakes compared to the non-diabetic control rats. No significant effect of resistant starch feeding on blood glucose and insulin was found. Even though the length of small intestines, and cecum, colon and rectum together with the tissue weight of cecum were not affected by feeding resistant starch, the intestinal transit time was markedly shortened by both types of resistant starch and resistant starch from corn had a more pronounced effect. The short chain fatty acids in the intestinal contents did not appear to be different among the groups. Nonetheless, both of resistant starch from corn and rice significantly lowered plasma total lipid and cholesterol concentrations compared to the diabetic control. The total liver cholesterol lowering effect was observed with resistant starch from rice. Neither immunoglobulin G nor C(3) were influenced by resistant starch.

The changes in amino acid concentrations and transsulfuration enzyme activities in liver were investigated after 4-week fed on 23% casein diet (control group) and 5% casein diet without (protein–calorie malnutrition, PCM group) or with (PCMC group) oral administration of cysteine, 250 mg/kg (twice daily, starting from the fourth week) using rats as an animal model. By supplementation with cysteine in PCM rats (PCMC group), cysteine level was elevated almost close to the control level, and glutathione (GSH), aspartic acid and serine levels were restored greater than the control levels. The measurement of transsulfuration enzyme activities exhibited that γ-glutamylcysteine ligase (γ-GCL) activity was up-regulated in rats with protein restriction (PCM group), and cysteine supplementation (PCMC group) down-regulated to the control level. One-week supplementation of cysteine (PCMC group) significantly down-regulated the cysteine sulfinate decarboxylase activity. These results indicate that the availability of sulfur amino acid(s) especially cysteine appears to play a role in determining the flux of cysteine between cysteine catabolism and GSH synthesis.

Nutritional status, iron-deficiency-related biochemical indices, and immunologic patterns of female Judo athletes and control subjects were evaluated. The subjects' 3-d food records showed that 41.0 kcal/kg of energy was consumed daily and the contributions of protein, fat, and carbohydrate to total energy intake were 12.5%, 29.2%, and 58.3%, respectively. The reported vitamin intakes of athletic subjects were above those of the recommended daily allowance, however, calcium and iron intakes were less than 100% of the recommended daily allowance. Intakes of energy, protein, phosphate, vitamin B1, and vitamin B2 were higher in the athletes than in the control subjects. Analysis using the Nutrient Adequacy Ratio and the Index of Nutritional Quality showed that athletic subjects had more desirable patterns than the control subjects. There was no any indication of anemia, which often occurs as a result of hemodilution in strenuously trained athletes. The subjects' immunologic patterns showed a slight immunosuppression. Iron, vitamin B1, niacin intakes were positively correlated with immunoglobulin (Ig) G levels in the athletes. The relation between nutrient intakes and the immune systems of endurance-trained athletes needs further investigation.

The present study investigated the regulatory mechanisms by which epigallocatechin-3-gallate (EGCG) exerts vascular endothelial growth factor (VEGF)-, p53- and AMP-activated protein kinase (AMPK)-associated pro-apoptotic and migration-suppressing effects on colon cancer cells. EGCG decreased the expression levels of VEGF and matrix metalloproteinase (MMP)-9. EGCG treatment induced apoptosis in the presence of wild-type and mutant p53, indicating that a p53-independent pathway may contribute to EGCG-induced apoptosis in these cells. EGCG showed migration-suppressing effects, suggesting that this activity may also have p53-dependent and -independent components. The interaction between p53 and VEGF in the EGCG-treated cells was investigated using pifithrin-α. Notably, the suppression of p53 activity blocked the ability of EGCG to inhibit VEGF and MMP-9 in the cells expressing wild-type p53, but not mutant p53, indicating that the effects of EGCG on VEGF may be p53-dependent or -independent. Finally, although AMPK and VEGF did not appear to co-localize, the results indicated that AMPK controls VEGF in EGCG-treated cells regardless of the p53 status.

Curcumin, the major phytochemical in turmeric, exerts anti‑proliferative, anticancer and anti‑inflammatory activities in various types of cancer cells. Curcumin has been demonstrated to induce apoptosis through multiple signaling pathways; however, its association with survival pathways, including the Wnt signaling pathway, is not fully understood. The Wnt signaling pathway is involved in diverse functions, including cell development, growth and proliferation. This pathway is important for cancer cell survival and metastasis. β‑catenin and GSK3β play a key role in the Wnt signaling pathway and therefore, various members of the Wnt signaling pathway have been hypothesized to represent potential targets for anticancer therapy. In the present study, the effect of curcumin on the suppression of migration and proliferation of Hep3B hepatocarcinoma cells was investigated via suppression of Wnt signaling in vitro and in vivo. 12‑O‑tetradecanoylphorbol‑13‑acetate (TPA)‑induced cell migration was observed to be suppressed by curcumin treatment. In addition, curcumin suppressed TPA‑induced activation of Wnt signaling. These results indicate that curcumin induces anti‑migratory activity, which functions via the Wnt signaling pathway.

Oxidative stress has been reported to be associated with cellular injury. Here, we investigated the antioxidant ability of a novel peptide mixture purified from black soybean (Rhynchosia volubilis) that exhibited significant antioxidant ability (IC50 = 119.8 ± 1.2 µg/ml) under in vitro ABTS assay conditions. To confirm this result, we next examined the antioxidant ability of the peptide mixture using cell culture systems. Black soybean peptide (BSP) significantly reduced the H2O2-induced cell death in C2C12 myocyte. Moreover, extracellular signal-regulated kinase (ERK) signaling was activated by BSP treatment. Under the same conditions, inhibition of the ERK signaling pathway with PD98059 significantly reduced the antioxidant ability of BSP in C2C12 cells. Finally, we also assessed the antithrombotic ability of BSP using a rat model; we found that the BSP also exerted antithrombotic effects in rat blood. Collectively, these results indicate that BSP purified from black soybean exerts an inhibitory effect on both oxidative stress and thrombosis. Key words: Black soybean peptide (BSP), anti-oxidant, thrombosis, extra cellular signal regulated protein kinase, oxidative stress.

The endoplasmic reticulum (ER) is a central organelle in eukaryotic cells that functions in protein synthesis and maturation, and also functions as a calcium storage organelle. Perturbation of ER functions leads to ER stress, which has been previously associated with a broad variety of diseases. ER stress is generally regarded as compensatory, but prolonged ER stress can activate apoptotic pathways in damaged cells. For this reason, pharmacological interventions that effectively enhance tumor death through ER stress have been the subject of a great deal of attention for anti-cancer therapy. Cryptotanshinone, the major active constituent isolated from the root of Salvia miltiorrhiza Bunge, has been recently evaluated for its anti-cancer activity, but the molecular mechanisms underlying these activities remain poorly understood. In particular, it remains completely unknown as to whether or not cryptotanshinone can induce ER stress. Herein, we identify cryptotanshinone as a potent stimulator of ER stress, leading to apoptosis in many cancer cell lines, including HepG2 hepatoma and MCF7 breast carcinoma, and also demonstrate that mitogen-activated protein kinases function as mediators in this process. Reactive oxygen species generated by cryptotanshinone have been shown to play a critical role in ER stress-induced apoptosis. Cryptotanshinone also evidenced sensitizing effects to a broad range of anti-cancer agents including Fas/Apo-1, TNF-α, cisplatin, etoposide or 5-FU through inducing ER stress, highlighting the therapeutic potential in the treatment of human hepatoma and breast cancer.

Luteolin, a plant-derived flavonoid, is thought to inhibit tumor growth. However, the precise molecular mechanisms by which luteolin inhibits cancer cell growth remain unclear. In the present study, we evaluated the role of AMP-activated protein kinase (AMPK) in the inhibition of cancer cell growth by luteolin in HepG2 hepatocarcinoma cells. AMPK is a metabolic sensor and may prevent carcinogenesis via modulation of signaling networks. We found that luteolin strongly induced cell death in HepG2 cells and dramatically reduced the tumor volume in a tumor xenograft model; both effects were accompanied by AMPK activation by luteolin. Luteolin also had a strong inhibitory effect on nuclear factor (NF)-κB. To determine the relationship between AMPK and NF-κB signaling, we used Compound C, a pharmacological AMPK inhibitor, and a dominant-negative form of AMPK. Our results indicated that inhibition of AMPK activity restored luteolin-inhibited NF-κB DNA-binding activity. These results suggest that AMPK activity is critical for the inhibition of cancer cell growth, possibly via modulation of NF-κB activity. We also showed that luteolin treatment causes the release of reactive oxygen species (ROS) and that these intracellular ROS in turn mediate AMPK-NF-κB signaling in HepG2 hepatocarcinoma cells. In conclusion, we propose that AMPK is a novel regulator of NF-κB in luteolin-induced cancer cell death. Furthermore, our results suggest that AMPK is an attractive target for cancer prevention by flavonoids.

AMP-activated protein kinase (AMPK) has emerged as a therapeutic target of cancer. AMPK functions as an upstream regulator of proliferative signals such as mammalian target of rapamycin (mTOR), tuberous sclerosis complex (TSC), p70S6 and elongation factor-2, indicating that AMPK can be applied for the inhibition of cancer cell proliferation via modulating the proliferative signaling network. The Akt/mTOR signaling pathway is activated in colon cancer. The well known mTOR inhibitor rapamycin has a disadvantage of feedback stimulation of Akt. Anthocyanins are naturally-occurring mTOR inhibitor possessing Akt inhibitory activities. We have investigated the mTOR inhibitory effect of anthocyanins through the activation of AMPK. In this study, anthocyanins were applied to colon cancer cells and tumor-bearing xenograft models to investigate their anti-proliferative and pro-apoptotic effects, and elucidate the mechanisms that link AMP-activated protein kinase (AMPK) α1 activation to the survival signal of mTOR. Our results indicated that anthocyanins significantly decreased phospho-mTOR comparable to rapamycin, a synthetic mTOR inhibitor, and this inhibitory effect of anthocyanins on mTOR was completely abrogated by inactivating AMPKα1. Furthermore, suppression of cell growth with anthocyanins was also alleviated in the absence of noticeable AMPKα1 activities. For the first time we have found anthocyanins as novel AMPKα1 activators, and in conditions of AMPKα1 inactivation, anthocyanins lost their ability to inhibit mTOR in HT-29 colon cancer cells. The activation of AMPKα1, and the deactivation of mTOR and Akt were observed in anthocyanins-treated tumor-bearing xenograft models. The results from this study suggest that there is a complex interaction between AMPKα1 and mTOR signaling, and anthocyanins are powerful AMPKα1 activators that inhibit cancer cell growth by inhibiting mTOR phosphorylation.

In lieu of elucidating bidirectional connecting mechanism between AMP-activated protein kinase (AMPK) and survival signal Akt we applied MCF-7 breast cancer cells to determine whether AMPK modulation alters Akt signals and vice versa. Suppression of Akt activities with a synthetic Akt inhibitor alleviated AMPK activities suggesting that Akt is capable of inhibiting AMPK. Also the activation of AMPK with quercetin strongly abrogated Akt activities. Treating cancer cells with AMPK siRNA or Compound C resulted in marked increment of Akt dephosphorylation indicating that AMPK has antagonistic activities towards Akt. However, quercetin exerted Akt inhibitory activities in the absence of AMPK activation. Quercetin induced partial co-localization of phospho-Akt and phospho-AMPK in the nucleus even though their interaction seems to be indirect since the immunoprecipitation data indicate there was no direct binding between total Akt and AMPK. These results suggest there is a mutual suppressive interaction between AMPK and Akt. The investigation of mutual suppression between Akt and AMPK by chemo-preventive agents such as quercetin may provide a mechanistic rational for controlling breast tumor cell growth.

AMP-activated kinase, a sensor of cellular energy status, has emerged as a potent target for cancer prevention and/or treatment. Thus, the application of dietary origin AMPK activators could link t...

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.

Fas/APO-1/CD95, a member of the tumor necrosis factor (TNF) receptor superfamily, is a potential anti-cancer factor as it can induce apoptosis in tumor cells. However, despite the fact that many cancer cells express Fas on the membrane, some tumors such as prostate cancer display resistance to Fas-induced apoptosis. In these cases, combination therapy using chemotherapeutic agents and Fas may be more suitable than therapy using Fas alone. In the present study, we demonstrate that the apoptosis inhibitory protein, Bcl-2, was highly expressed in response to Fas in DU145 prostate cancer cells, thereby conferring resistance to apoptosis. We have screened a number of naturally occurring products that may overcome this resistance. Here we report that cryptotanshinone, the major tanshinone isolated from Salvia miltiorrhiza Bunge, can suppress Bcl-2 expression and augment Fas sensitivity in DU145 cells. We further show that JNK and p38 MAPK act upstream of Bcl-2 expression in Fas-treated DU145 cells, and that cryptotanshinone significantly blocked activation of these kinases. Moreover, cryptotanshinone sensitized several tumor cells to a broad range of anti-cancer agents. Collectively, our data suggest that cryptotanshinone has therapeutic potential in the treatment of human prostate cancer.

AMP-activated protein kinase (AMPK), a highly conserved protein in eukaryotes, functions as a major metabolic switch to maintain energy homeostasis. It also intrinsically regulates the mammalian cell cycle. Moreover, the AMPK cascade has emerged as an important pathway implicated in cancer control. In this study we investigated the effects of curcumin on apoptosis and the regulatory effect of the AMPK-cyclooxygenase-2 (COX-2) pathway in curcumin-induced apoptosis. Curcumin has shown promise as a chemopreventive agent because of its in vivo regression of various animal-model colon cancers. This study focused on exploiting curcumin to apply antitumorigenic effects through modulation of the AMPK-COX-2 cascade. Curcumin exhibited a potent apoptotic effect on HT-29 colon cancer cells at concentrations of 50 micromol/L and above. These apoptotic effects were correlated with the decrease in pAkt and COX-2, as well as the increase in p-AMPK. Cell cycle analysis showed that curcumin induced G(1)-phase arrest. Further study with AMPK synthetic inhibitor Compound C has shown that increased concentrations of Compound C would abolish AMPK expression, accompanied by a marked increase in COX-2 as well as pAkt expression in curcumin-treated HT-29 cells. By inhibiting AMPK with Compound C, we found that curcumin-treated colon cancer cells were no longer undergoing apoptosis; rather, they were proliferative. These results indicate that AMPK is crucial in apoptosis induced by curcumin and further that the pAkt-AMPK-COX-2 cascade or AMPK-pAkt-COX-2 pathway is important in cell proliferation and apoptosis in colon cancer cells.

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).

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.

Resveratrol, one of polyphenols derived from red wine, has been shown to protect against cell death, possibly through the association with several signaling pathways. Currently numerous studies indicate that cardiovascular diseases are linked to the release of intracellular reactive oxygen species (ROS) often generated in states such as ischemia/reperfusion injury. In the present study, we investigated whether resveratrol has the capability to control intracellular survival signaling cascades involving AMP-activated kinase (AMPK) in the inhibitory process of cardiac injury. We hypothesized that resveratrol may exert a protective effect on damage to heart muscle through modulating of the AMPK signaling pathway. We mimicked ischemic conditions by inducing cell death with H(2)O(2) in H9c2 muscle cells. In this experiment, resveratrol induced strong activation of AMPK and inhibited the occurrence of cell death caused by treatment with H(2)O(2). Under the same conditions, inhibition of AMPK using dominant negative AMPK constructs dramatically abolished the effect of resveratrol on cell survival in H(2)O(2)-treated cardiac muscle cells. These results indicate that resveratrol-induced cell survival is mediated by AMPK in H9c2 cells and may exert a novel therapeutic effect on oxidative stress induced in cardiac disorders.

Resveratrol has been reported to possess therapeutic effects for various cancers including colon cancers. In this article, the molecular basis of resveratrol with emphasis on its ability to control intracellular signaling cascades of adenosine monophosphate (AMP)-activated protein kinase (AMPK) responsible for inducing apoptosis in drug-resistant cancer cells was investigated. Recently, the evolutionarily conserved serine/threonine kinase, AMPK, emerges as a possible target molecule of cancer control. We have investigated the effects of resveratrol on apoptosis in relation to AMPK in HT-29 cells shown chemoresistant to a cancer chemotherapeutic drug, etoposide. Resveratrol exhibited a variety of molecular events in etoposide-based combination therapy in HT-29 colon cancer cells including the AMPK activation, inhibition of cell growth, induction of apoptosis, and reactive oxygen species (ROS) generation. The involvement of AMPK signaling cascade in resveratrol-based cancer therapy was clearly shown by comparing the conditions of AMPK activated states and inactivated states. We have identified ROS as an upstream regulator of AMPK. Further investigation warrants to elucidate the mechanism by which resveratrol generates ROS and AMPK activation.

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.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is activated during ATP-depleting metabolic states, such as hypoxia, heat shock, oxidative stress, and exercise. As a highly conserved heterotrimeric kinase that functions as a major metabolic switch to maintain energy homeostasis, AMPK has been shown to exert as an intrinsic regulator of mammalian cell cycle. Moreover, AMPK cascade has emerged as an important pathway implicated in cancer control. In this article, we have investigated the effects of capsaicin on apoptosis in relation to AMPK activation in colon cancer cell. Capsaicin-induced apoptosis was revealed by the presence of nucleobodies in the capsaicin-treated HT-29 colon cancer cells. Concomitantly, the activation of AMPK and the increased expression of the inactive form of acetyl-CoA carboxylase (ACC) were detected in capsaicin-treated colon cancer cells. We showed that both capsaicin and 5'-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR), an AMPK activator possess the AMPK-activating capacity as well as apoptosis-inducing properties. Evidence of the association between AMPK activation and the increased apoptosis in HT-29 colon cancer cells by capsaicin treatment, and further findings of the correlation of the activated AMPK and the elevated apoptosis by cotreatment of AICAR and capsaicin support AMPK as an important component of apoptosis, as well as a possible target of cancer control.

This study was undertaken to examine the effect of low and high concentrations of H2O2 on cancer cell proliferation and apoptosis, and AMPK signaling pathways in HT-29 human colon cancer cells. Nontoxic doses of H2O2 (10 microM) induced cancer cell proliferation, whereas the toxic level of 1,000 microM H2O2 induced apoptosis. The stimulation of cell proliferation was accompanied with an increase in cyclooxygenase-2 (COX-2), and apoptosis induced by high-dose H2O2 was correlated with the activation of AMPK and negatively correlated with COX-2 expression. These results suggest that ROS at nontoxic levels can stimulate cancer cell growth by regulating AMP-activated protein kinase (AMPK) and/or COX-2, and the abundant exogenous ROS linked to the growth inhibition through modulating AMPK signaling pathways.

Epidemiologic and experimental evidences indicate that selenium, an essential trace element, can reduce the risk of a variety of cancers. Protection against certain types of cancers, particularly colorectal cancers, is closely associated with pathways involving cyclooxygenase-2 (COX-2). We found that AMP-activated protein kinase (AMPK), which functions as a cellular energy sensor, mediates critical anticancer effects of selenium via a COX-2/prostaglandin E2 signaling pathway. Selenium activated AMPK in tumor xenografts as well as in colon cancer cell lines, and this activation seemed to be essential to the decrease in COX-2 expressions. Transduction with dominant-negative AMPK into colon cancer cells or application of cox-2−/−-negative cells supported the evidence that AMPK is an upstream signal of COX-2 and inhibits cell proliferation. In HT-29 colon cancer cells, carcinogenic agent 12-O-tetradecanoylphorbol-13-acetate (TPA) activated extracellular signal-regulated kinase (ERK) that led to COX-2 expression and selenium blocked the TPA-induced ERK and COX-2 activation via AMPK. We also showed the role of a reactive oxygen species as an AMPK activation signal in selenium-treated cells. We propose that AMPK is a novel and critical regulatory component in selenium-induced cancer cell death, further implying AMPK as a prime target of tumorigenesis. (Cancer Res 2006; 66(20): 10057-63)

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