Genistein Inhibits Proliferation of BRCA1 Mutated Breast Cancer Cells: The GPR30-Akt Axis as a Potential Target.

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.

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.

Pro-apoptotic and migration-suppressing potential of EGCG, and the involvement of AMPK in the p53-mediated modulation of VEGF and MMP-9 expression.

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 suppresses migration and proliferation of Hep3B hepatocarcinoma cells through inhibition of the Wnt signaling pathway.

Curcumin, the major phytochemical in turmeric, exerts anti­proliferative, anticancer and anti­inflammatory 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. ß­catenin and GSK3ß 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. 12­O­tetradecanoylphorbol­13­acetate (TPA)­induced cell migration was observed to be suppressed by curcumin treatment. In addition, curcumin suppressed TPA­induced activation of Wnt signaling. These results indicate that curcumin induces anti­migratory activity, which functions via the Wnt signaling pathway.

Soybean isoflavone genistein regulates apoptosis through NF-κB dependent and independent pathways.

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.

Cryptotanshinone induces ER stress-mediated apoptosis in HepG2 and MCF7 cells.

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.

Anti-tumor effect of luteolin is accompanied by AMP-activated protein kinase and nuclear factor-κB modulation in HepG2 hepatocarcinoma cells.

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.

Anthocyanins are novel AMPKα1 stimulators that suppress tumor growth by inhibiting mTOR phosphorylation.

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.

Regulation of mutual inhibitory activities between AMPK and Akt with quercetin in MCF-7 breast cancer cells.

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.

Cryptotanshinone sensitizes DU145 prostate cancer cells to Fas(APO1/CD95)-mediated apoptosis through Bcl-2 and MAPK regulation.

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.

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.

Characterization of a profile of the anthocyanins isolated from Vitis coignetiae Pulliat and their anti-invasive activity on HT-29 human colon cancer cells.

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.

Induction of apoptosis by quercetin is mediated through AMPKalpha1/ASK1/p38 pathway.

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.

Anti-inflammatory and anticarcinogenic effect of genistein alone or in combination with capsaicin in TPA-treated rat mammary glands or mammary cancer cell line.

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.

Regulatory effect of the AMPK-COX-2 signaling pathway in curcumin-induced apoptosis in HT-29 colon cancer cells.

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.

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).

Effects of cotreatment of 12-O-tetradecanoylphorbol-13-acetate and H2O2 on apoptotic regulation via AMP-activated protein kinase-cyclooxygenase-2 signals.

Colorectal cancer displays elevated cyclooxygenase-2 (COX-2) expression, and several studies have suggested that COX-2 expression is associated with parameters of aggressive colon cancer. AMP-activated protein kinase (AMPK) is a sensor of cellular energy status, and recent studies indicate that AMPK activation strongly suppresses cell proliferation in nonmalignant cells as well as in tumor cells. As a metabolic sensing signal, AMPK is involved in cancer cell apoptosis. In HT-29 colon cancer cells, the regulation of COX-2 expression by treating with TPA (12-O-tetradecanoylphorbol-13-acetate), low-level H(2)O(2), high-level H(2)O(2), and finally the combinations of TPA and low H(2)O(2) or high H(2)O(2) was investigated. We found that COX-2 expression levels with treatment reacted as follows: with TPA alone > TPA and low H(2)O(2) > low H(2)O(2) > high H(2)O(2) > TPA and high H(2)O(2). COX-2 regulation by these agents was accompanied by the alteration of AMPK control. The apoptotic bodies were detected as follows: high level of H(2)O(2) > TPA > low level of H(2)O(2). The present findings suggest that both COX-2 stimulators (TPA and H(2)O(2)) might have differential effects on COX-2 and AMPK regulation and further apoptotic regulation.

AMP kinase/cyclooxygenase-2 pathway regulates proliferation and apoptosis of cancer cells treated with quercetin.

AMPK (AMP-activated protein kinase) is highly conserved in eukaryotes, where it functions primarily as a sensor of cellular energy status. Recent studies indicate that AMPK activation strongly suppresses cell proliferation in non-malignant cells as well as in tumor cells. In this study, quercetin activated AMPK in MCF breast cancer cell lines and HT-29 colon cancer cells, and this activation of AMPK seemed to be closely related to a decrease in COX-2 expression. The application of a COX-2 inhibitor or cox-2-/- cells supported the idea that AMPK is an upstream signal of COX-2, and is required for the anti-proliferatory and pro-apoptotic effects of quercetin. The suppressive or growth inhibitory effects of quercetin on COX-2 were abolished by treating cancer cells with an AMPK inhibitor Compound C. These results suggest that AMPK is crucial to the anti-cancer effect of quercetin and that the AMPK-COX-2 signaling pathway is important in quercetin-mediated cancer control.