Epigenetics/epigenomics and prevention by curcumin of early stages of inflammatory-driven colon cancer.

Colorectal cancer (CRC) is associated with significant morbidity and mortality in the US and worldwide. CRC is the second most common cancer-related death in both men and women globally. Chronic inflammation has been identified as one of the major risk factors of CRC. It may drive genetic and epigenetic/epigenomic alterations, such as DNA methylation, histone modification, and non-coding RNA regulation. Current prevention modalities for CRC are limited and some treatment regimens such as use the nonsteroidal anti-inflammatory drug aspirin may have severe side effects, namely gastrointestinal ulceration and bleeding. Therefore, there is an urgent need of developing alternative strategies. Recently, increasing evidence suggests that several dietary cancer chemopreventive phytochemicals possess anti-inflammation and antioxidative stress activities, and may prevent cancers including CRC. Curcumin (CUR) is the yellow pigment that is found in the rhizomes of turmeric (Curcuma longa). Many studies have demonstrated that CUR exhibit strong anticancer, antioxidative stress, and anti-inflammatory activities by regulating signaling pathways, such as nuclear factor erythroid-2-related factor 2, nuclear factor-κB, and epigenetics/epigenomics pathways of histones modifications, and DNA methylation. In this review, we will discuss the latest evidence in epigenetics/epigenomics alterations by CUR in CRC and their potential contribution in the prevention of CRC.

Toxicogenomics of resveratrol in rat liver.

Resveratrol, a polyphenolic compound found in grape skin and peanuts has been shown to prevent many diseases including cardiovascular diseases and cancer. To better understand resveratrol's potential in vivo toxicity, we studied the dose response using cDNA stress arrays coupled with drug metabolizing enzymatic (DME) assays to investigate the expression of stress-responsive genes and Phase I and II detoxifying enzymes in rat livers. Male and female CD rats were treated with high doses of resveratrol (0.3, 1.0 and 3.0 gm/kg/day) for a period of 28 days. Total RNA from rat liver was reverse-transcribed using gene-specific primers and hybridized to stress-related cDNA arrays. Among female rats, Phase I DME genes were repressed at 0.3 and 1.0 gm/kg/day doses, while genes such as manganese superoxide dismutase, cytochrome P450 reductase, quinone oxidoreductase and thiosulfate sulfurtransferase demonstrated a dose-dependent increase in gene expression. The modulation of these liver genes may implicate the potential toxicity as observed among the rats at the highest dose level of resveratrol. Real-Time PCR was conducted on some of the Phase II DME genes and anti-oxidant genes to validate the cDNA array data. The gene expression from real-time PCR demonstrated good correlation with the cDNA array data. UGT1A genes were amongst the most robustly induced especially at the high doses of resveratrol. We next performed Phase I and Phase II enzymatic assays on cytochrome P450 2E1 (CYP2E1), cytochrome P450 1A1 (CYP1A1), NAD(P)H:quinone oxidoreductase (NQO1), glutathione S-transferase (GST) and UDP-glucuronosyl transferase (UGT). Induction of Phase II detoxifying enzymes was most pronounced at the highest dose of resveratrol. CYP1A1 activity demonstrated a decreasing trend among the 3 dose groups and CYP2E1 activity increased marginally among female rats over controls. In summary, at lower doses of resveratrol there are few significant changes in gene expression whereas the modulation of liver genes at the high dose of resveratrol may implicate the potential toxicity observed.

Induction of detoxifying enzymes by garlic organosulfur compounds through transcription factor Nrf2: effect of chemical structure and stress signals.

Garlic organosulfur compounds (OSCs) are recognized as a group of potential chemopreventive compounds. It is known that garlic OSCs can modulate drug metabolism systems, especially various phase II detoxifying enzymes, though the mechanism underlying their inductive effect on these enzymes remains largely unknown. In the present study, we investigated the transcriptional levels of NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO1) genes, the reporter activity mediated by antioxidant response element (ARE), and the protein level of transcription factor nuclear factor E2-related factor 2 (Nrf2), after administration of three major garlic OSCs--diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS)--in human hepatoma HepG2 cells. Our results showed that ARE activation and Nrf2 protein accumulation were well correlated with phase II gene expression induction. The structure-activity relationship study indicated that the third sulfur in the structure of OSCs contributed substantially to their bioactivities, and that allyl-containing OSCs were more potent than propyl-containing OSCs. To better understand the signaling events involved in the upregulation of detoxifying enzymes by DATS, ARE activity and Nrf2 protein levels were examined after transient transfection of HepG2 cells with mutant Nrf2, cotreatment with antioxidants, and pretreatment with protein kinase inhibitors. DATS-induced ARE activity was inhibited by dominant-negative Nrf2 Kelch-like ECH-associating protein 1 and constructs. Cotreatment with thiol antioxidants decreased the ARE activity and Nrf2 protein level induced by DATS. Three major mitogen-activated protein kinases (MAPKs)--extracellular signal-regulated protein kinase, c-Jun N-terminal kinase, and p38--were activated by DATS treatment. However, the inhibition of these MAPKs did not affect DATS-induced ARE activity. Pretreatment with various upstream protein kinase inhibitors showed that the protein kinase C pathway was not directly involved in DATS-induced ARE activity, but instead the calcium-dependent signaling pathway appeared to play a role in the DATS-induced cytoprotective effect.

Dietary chemopreventive compounds and ARE/EpRE signaling.

Chemoprevention comprises multiple intervention methods using either pharmacological or dietary agents to impede, arrest, or reverse carcinogenesis at various stages. Development of dietary compounds as potential cancer chemopreventive agents is highly desirable, due to their safety, low toxicity, and general acceptance as dietary supplements. In this review, potential application of the dietary detoxifying enzyme inducers for chemoprevention and their relevant signaling events are discussed. Overall, the detoxifying enzyme system plays an important role in determining the final fate of carcinogens/procarcinogens and their subsequent impact on carcinogenesis. Among those positive regulators, phenolic and sulfur-containing compounds are two major classes of dietary detoxifying enzyme inducers. Regulation of many detoxifying enzymes by dietary chemopreventive compounds is mediated by the antioxidant response element (ARE)/electrophile response element (EpRE), which is located in the promoter region of related genes. Transcription factor nuclear factor E2-related factor 2 (Nrf2) binds to the ARE sequence to initiate gene expression. In response to treatments of various detoxifying enzyme inducers, several signal transduction pathways, including the oxidative stress, mitogen-active protein kinase, protein kinase C, and phosphatidylinositol 3-kinase pathways, are activated. The consequences of the activation of these signaling cascades, whether directly or indirectly, lead to the dissociation of Nrf2 from its cytosolic sequester Kelch-like ECH associating protein 1, nuclear translocation, and accumulation of Nrf2 protein in the nucleus, and ultimately increase the expression level of detoxifying enzymes through transcriptional activation of ARE/EpRE in those responsible genes.

Quinacrine induces cytochrome c-dependent apoptotic signaling in human cervical carcinoma cells.

Quinacrine (QU), a phospholipase-A2 (PLA-2) inhibitor has been used clinically as a chemotherapeutic adjuvant. To understand the mechanisms leading to its chemotherapeutic effect, we have investigated QU-induced apoptotic signaling pathways in human cervical squamous carcinoma HeLa cells. In this study, we found that QU induced cytochrome c-dependent apoptotic signaling. The release of pro-apoptotic cytochrome c was QU concentration- and time-dependent, and preceded activation of caspase-9 and -3. Flow cytometric FACScan analysis using fluorescence intensities of DiOC6 demonstrated that QU-induced cytochrome c release was independent of mitochondrial permeability transition (MPT), since the concentrations of QU that induced cytochrome c release did not alter mitochondrial membrane potential (delta pai(m)). Moreover, kinetic analysis of caspase activities showed that cytochrome c release led to the activation of caspase-9 and downstream death effector, caspase-3. Caspase-3 inhibitor (Ac-DEVD-CHO) partially blocked QU-induced apoptosis, suggesting the importance of caspase-3 in this apoptotic signaling mechanism. Supplementation with arachidonic acid (AA) sustained caspase-3 activation induced by QU. Using inhibitors against cellular arachidonate metabolism of lipooxygenase (Nordihydroxyguaiaretic Acid, NDGA) and cyclooxygenase (5,8,11,14-Eicosatetraynoic Acid, ETYA) demonstrated that QU-induced apoptotic signaling may be dependent on its role as a PLA-2 inhibitor. Interestingly, NDGA attenuated QU-induced cytochrome c release, caspase activity as well as apoptotic cell death. The blockade of cytochrome c release by NDGA was much more effective than that attained with cyclosporin A (CsA), a MPT inhibitor. ETYA was not effective in blocking cytochrome c release, except under very high concentrations. Caspase inhibitor z-VAD blocked the release of cytochrome c suggesting that this signaling event is caspase dependent, and caspase-8 activation may be upstream of the mitochondrial events. In summary, we report that QU induced cytochrome c-dependent apoptotic signaling cascade, which may be dependent on its role as a PLA-2 inhibitor. This apoptotic mechanism induced by QU may contribute to its known chemotherapeutic effects.

Activation of antioxidant-response element (ARE), mitogen-activated protein kinases (MAPKs) and caspases by major green tea polyphenol components during cell survival and death.

Green tea polyphenols (GTP) have been demonstrated to suppress tumorigenesis in several chemical-induced animal carcinogenesis models, and predicted as promising chemopreventive agents in human. Recent studies of GTP extracts showed the involvement of mitogen-activated protein kinases (MAPKs) in the regulation of Phase II enzymes gene expression and induction of apoptosis. In the current work we compared the biological actions of five green tea catechins: (1) induction of ARE reporter gene, (2) activation of MAP kinases, (3) cytotoxicity in human hepatoma HepG2-C8 cells, and (4) caspase activation in human cervical squamous carcinoma HeLa cells. For the induction of phase II gene assay, (-)-epigallocatechin-3-gallate (EGCG) and (-)-epicatechin-3-gallate (ECG) potently induced antioxidant response element (ARE)-mediated luciferase activity, with induction observed at 25 microM with EGCG. The induction of ARE reporter gene appears to be structurally related to the 3-gallate group. Comparing the activation of MAPK by the five polyphenols, only EGCG showed potent activation of all three MAPKs (ERK, JNK and p38) in a dose- and time-dependent manner, whereas EGC activated ERK and p38. In the concentration range of 25 microM to 1 mM, EGCG and ECG strongly suppressed HepG2-ARE-C8 cell-growth. To elucidate the mechanisms of green tea polyphenol-induced apoptosis, we measured the activation of an important cell death protein, caspase-3 induced by EGCG, and found that caspase-3 was activated in a dose- and time-dependent manner. Interestingly, the activation of caspase-3 was a relatively late event (peaked at 16 h), whereas activation of MAPKs was much earlier (peaked at 2 h). It is possible, that at low concentrations of EGCG, activation of MAPK leads to ARE-mediated gene expression including phase II detoxifying enzymes. Whereas at higher concentrations of EGCG, sustained activation of MAPKs such as JNK leads to apoptosis. These mechanisms are currently under investigation in our laboratory. As the most abundant catechin in GTP extract, we found that EGCG potently induced ARE-mediated gene expression, activated MAP kinase pathway, stimulated caspase-3 activity, and induced apoptosis. These mechanisms together with others, may contribute to the overall chemopreventive function of EGCG itself as well as the GTP

Activation of mitogen-activated protein kinases by green tea polyphenols: potential signaling pathways in the regulation of antioxidant-responsive element-mediated phase II enzyme gene expression.

Green tea polyphenols, major constituents of green tea, are potent chemopreventive agents in a number of experimental models of cancer in animals. The mechanisms of cancer protection by these agents are not clear, but may involve modulation of the enzyme systems responsible for the detoxification of chemical carcinogens. The present studies show that a green tea polyphenol extract (GTP) induces chloramphenicol acetyltransferase (CAT) activity in human heptoma HepG2 cells transfected with a plasmid construct which contains an antioxidant-responsive element (ARE) and a minimal glutathione S-transferase Ya promoter linked to the CAT reporter gene. This indicates that GTP stimulates the transcription of Phase II detoxifying enzymes through the ARE. To explore the upstream signaling pathways leading to gene expression, we studied the involvement of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 2 (ERK2) and c-Jun N-terminal kinase 1 (JNK1). Potent activation of ERK2 was seen following treatment of HepG2 cells with different concentrations of GTP. Similar to ERK2, JNK1 was also strongly activated by treatment with GTP, although to a lesser extent and in a different dose-dependent fashion. Kinetic studies revealed that GTP activation of JNK1 was delayed and sustained, whereas ERK2 activation was rapid and transient. Furthermore, GTP treatment also increased mRNA levels of the immediate-early genes c-jun and c-fos, as determined by reverse transcriptase-coupled polymerase chain reaction. Taken together, these studies provide insights into the action of GTP and suggest that the stimulation MAPKs may be the potential signaling pathways utilized by GTP to activate ARE-dependent genes.