Min-Hsiung Pan

Comparative studies on the suppression of nitric oxide synthase by curcumin and its hydrogenated metabolites through down-regulation of IκB kinase and NFκB activation in macrophages

Nitric oxide (NO) plays an important role in inflammation and in the multiple stages of carcinogenesis. In this study, we investigated the inhibitory effects of curcumin and its metabolites, tetrahydrocurcumin, hexahydrocurcumin, and octahydrocurcumin, on the induction of NO synthase (NOS) in RAW 264.7 cells activated with lipopolysaccharide (LPS). Western blotting and northern blotting analyses demonstrated that curcumin strongly reduced 130-kDa protein and 4.5-kb mRNA levels of iNOS in LPS-activated macrophages compared with its metabolites, tetrahydrocurcumin, hexahydrocurcumin, and octahydrocurcumin. Moreover, electrophoretic mobility shift assay (EMSA) experiments indicated that curcumin blocked the LPS-induced binding of nuclear factor-kappaB (NFkappaB), a transcription factor necessary for iNOS induction to its (32)P-labeled double-stranded oligonucleotide probe. The inhibition of NFkappaB activation occurred through the prevention of inhibitor kappaB (IkappaB) degradation. Transient transfection experiments also showed that curcumin inhibited NFkappaB-dependent transcriptional activity. Curcumin blocked the disappearance of inhibitory kappaBalpha (IkappaBalpha) and p65 from the cytosolic fraction, and inhibited the phosphorylation of IkappaBalpha. Furthermore, we showed that curcumin could inhibit the IkappaB kinase 1 (IKK1) and IkappaB kinase 2 (IKK2) activities induced by LPS, but tetrahydrocurcumin, hexahydrocurcumin, and octahydrocurcumin were less active. These results suggest that curcumin may exert its anti-inflammatory and anti-carcinogenic properties by suppressing the activation of NFkappaB through inhibition of IKK activity.

Modulation of Inflammatory Genes by Natural Dietary Bioactive Compounds

Several epidemiologic studies have shown that chronic inflammation predisposes individuals to various types of cancer. Many cancers arise from sites of infection, chronic irritation, and inflammation. Conversely, an oncogenic change induces an inflammatory microenvironment that promotes the development of tumors. Natural bioactive compounds in dietary plant products including fruits, vegetables, grains, legumes, tea, and wine are claimed to help prevent cancer, degenerative diseases, and chronic and acute inflammation. Modern methods in cell and molecular biology allow us to understand the interactions of different natural bioactive compounds with basic mechanisms of inflammatory response. The molecular pathways of this cancer-related inflammation are now unraveled. Natural bioactive compounds exert anti-inflammatory activity by modulating pro-inflammatory gene expressions have shown promising chemopreventive activity. This review summarizes current knowledge on natural bioactive compounds that act through the signaling pathways and modulate inflammatory gene expressions, thus providing evidence for these substances in cancer chemopreventive action.

6‐Shogaol suppressed lipopolysaccharide‐induced up‐expression of iNOS and COX‐2 in murine macrophages

Ginger, the rhizome of Zingiber officinale, is a traditional medicine with carminative effect, antinausea, anti-inflammatory, and anticarcinogenic properties. In this study, we investigated the inhibitory effects of 6-shogaol and a related compound, 6-gingerol, on the induction of nitric oxide synthase (NOS) and cyclooxygenase-2 (COX-2) in murine RAW 264.7 cells activated with LPS. Western blotting and reverse transcription-PCR analyses demonstrated that 6-shogaol significantly blocked protein and mRNA expression of inducible NOS (iNOS) and COX-2 in LPS-induced macrophages. The in vivo anti-inflammatory activity was evaluated by a topical 12-O-tetradecanoylphorbol 13-acetate (TPA) application to mouse skin. When applied topically onto the shaven backs of mice prior to TPA, 6-shogaol markedly inhibited the expression of iNOS and COX-2 proteins. Treatment with 6-shogaol resulted in the reduction of LPS-induced nuclear translocation of nuclear factor-kappaB (NF kappaB) subunit and the dependent transcriptional activity of NF kappaB by blocking phosphorylation of inhibitor kappaB (I kappaB)alpha and p65 and subsequent degradation of I kappaB alpha. Transient transfection experiments using NF kappaB reporter constructs indicated that 6-shogaol inhibits the transcriptional activity of NF kappaB in LPS-stimulated mouse macrophages. We found that 6-shogaol also inhibited LPS-induced activation of PI3K/Akt and extracellular signal-regulated kinase 1/2, but not p38 mitogen-activated protein kinase (MAPK). Taken together, these results show that 6-shogaol downregulates inflammatory iNOS and COX-2 gene expression in macrophages by inhibiting the activation of NF kappaB by interfering with the activation PI3K/Akt/I kappaB kinases IKK and MAPK.

Molecular mechanisms for chemoprevention of colorectal cancer by natural dietary compounds

Colorectal cancer is one of the major causes of cancer-related mortality in both men and women worldwide. This review focuses on preventing the initiation and promotion of neoplastic growth in colorectal cancer, particularly with natural dietary compounds. Chemoprevention is defined as the use of natural dietary compounds and/or synthetic substances that can delay, prevent, or even reverse the development of adenomas, as well as the progression from adenoma to carcinoma. The molecular mechanisms of their chemopreventive action are associated with the modulation of signaling cascades, gene expressions involved in the regulation of cell proliferation, differentiation, and apoptosis and the suppression of chronic inflammation, metastasis, and angiogenesis. Here, we summarize the currently known targets and signaling pathways whereby natural dietary compounds interfere with the development of colorectal cancer, and thus providing evidence for these substances in colonic cancer chemopreventive action.

6‐Shogaol induces apoptosis in human colorectal carcinoma cells via ROS production, caspase activation, and GADD 153 expression

Ginger, the rhizome of Zingiber officinale, is a traditional medicine with anti-inflammatory and anticarcinogenic properties. This study examined the growth inhibitory effects of the structurally related compounds 6-gingerol and 6-shogaol on human cancer cells. 6-Shogaol [1-(4-hydroxy-3-methoxyphenyl)-4-decen-3-one] inhibits the growth of human cancer cells and induces apoptosis in COLO 205 cells through modulation of mitochondrial functions regulated by reactive oxygen species (ROS). ROS generation occurs in the early stages of 6-shogaol-induced apoptosis, preceding cytochrome c release, caspase activation, and DNA fragmentation. Up-regulation of Bax, Fas, and FasL, as well as down-regulation of Bcl-2 and Bcl-X(L )were observed in 6-shogaol-treated COLO 205 cells. N-acetylcysteine (NAC), but not by other antioxidants, suppress 6-shogaol-induced apoptosis. The growth arrest and DNA damage (GADD)-inducible transcription factor 153 (GADD153) mRNA and protein is markedly induced in a time- and concentration-dependent manner in response to 6-shogaol.

Pterostilbene inhibited tumor invasion via suppressing multiple signal transduction pathways in human hepatocellular carcinoma cells

Pterostilbene, a natural dimethylated analog of resveratrol, is known to have diverse pharmacologic activities including anticancer, anti-inflammation, antioxidant, apoptosis, anti-proliferation and analgesic potential. However, the effects of pterostilbene in preventing invasion of cancer cells have not been studied. Here, we report our finding that pterostilbene significantly suppressed 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced invasion, migration and metastasis of human hepatoma cells (HepG(2) cells). Increase in the enzyme activity, protein and messenger RNA levels of matrix metalloproteinase (MMP)-9 were observed in TPA-treated HepG(2) cells, and these were blocked by pterostilbene. In addition, pterostilbene can inhibit TPA-induced expression of vascular endothelial growth factor, epidermal growth factor and epidermal growth factor receptor. Transient transfection experiments also showed that pterostilbene strongly inhibited TPA-stimulated nuclear factor kappa B (NF-kappaB) and activator protein-1 (AP-1)-dependent transcriptional activity in HepG(2) cells. Moreover, pterostilbene can suppress TPA-induced activation of extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, c-Jun N-terminal kinases 1/2 and phosphatidylinositol 3-kinase/Akt and protein kinase C that are upstream of NF-kappaB and AP-1. Significant therapeutic effects were further demonstrated in vivo by treating nude mice with pterostilbene (50 and 250 mg/kg intraperitoneally) after inoculation with HepG(2) cells into the tail vein. Presented data reveal that pterostilbene is a novel, effective, anti-metastatic agent that functions by downregulating MMP-9 gene expression.

Increased Growth Inhibitory Effects on Human Cancer Cells and Anti-Inflammatory Potency of Shogaols from Zingiber officinale Relative to Gingerols

Ginger, the rhizome of the plant Zingiber officinale , has received extensive attention because of its antioxidant, anti-inflammatory, and antitumor activities. Most researchers have considered gingerols as the active principles and have paid little attention to shogaols, the dehydration products of corresponding gingerols during storage or thermal processing. In this study, we have purified and identified eight major components, including three major gingerols and corresponding shogaols, from ginger extract and compared their anticarcinogenic and anti-inflammatory activities. Our results showed that shogaols ([6], [8], and [10]) had much stronger growth inhibitory effects than gingerols ([6], [8], and [10]) on H-1299 human lung cancer cells and HCT-116 human colon cancer cells, especially when comparing [6]-shogaol with [6]-gingerol (IC50 of approximately 8 versus approximately 150 microM). In addition, we found that [6]-shogaol had much stronger inhibitory effects on arachidonic acid release and nitric oxide (NO) synthesis than [6]-gingerol.

Pterostilbene is more potent than resveratrol in preventing azoxymethane (AOM)-induced colon tumorigenesis via activation of the NF-E2-related factor 2 (Nrf2)-mediated antioxidant signaling pathway.

Inflammatory bowel diseases have been a risk factor of colorectal cancer (CRC). The reactive oxygen species (ROS) generated by inflammatory cells create oxidative stress and contribute to neoplastic transformation, proliferation, and even metastasis. Previously, resveratrol (RS) and pterostilbene (PS) had been reported to prevent chemical-induced colon carcinogenesis by anti-inflammatory and pro-apoptotic properties. In this study, we investigated whether RS and PS could prevent the azoxymethane (AOM)-induced colon tumorigenesis via antioxidant action and to explore possible molecular mechanisms. Male BALB/c mice were injected with AOM (5 mg/kg of body weight) with or without RS or PS, and at the end of the protocol, all of the mice were euthanized and colons were analyzed. Administrations of PS can be more effective than RS in reducing AOM-induced formation of aberrant crypt foci (ACF), lymphoid nodules (LNs), and tumors. We also find that PS is functioning more effectively than RS to reduce nuclear factor-κB (NF-κB) activation by inhibiting the phosphorylation of protein kinase C-β2 (PKC-β2) and decreasing downstream target gene expression, including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and aldose reductase (AR) in mouse colon stimulated by AOM. Moreover, administration of RS and PS for 6 weeks significantly enhanced expression of antioxidant enzymes, such as heme oxygenase-1 (HO-1) and glutathione reductase (GR), via activation of NF-E2-related factor 2 (Nrf2) signaling. When the above findings are taken together, they suggest that both stilbenes block cellular inflammation and oxidative stress through induction of HO-1 and GR, thereby preventing AOM-induced colon carcinogenesis. In comparison, PS was a more potent chemopreventive agent than RS for the prevention of colon cancer. This is also the first study to demonstrate that PS is a Nrf2 inducer and AR inhibitor in the AOM-treated colon carcinogenesis model.

EGCG inhibits protein synthesis, lipogenesis, and cell cycle progression through activation of AMPK in p53 positive and negative human hepatoma cells.

In the previous studies, (-)-epigallocatechin-3-gallate (EGCG) has been shown to have anticarcinogenic effects via modulation in protein expression of p53. Using p53 positive Hep G2 and p53 negative Hep 3B cells, we found that treatment of EGCG resulted in dose-dependent inhibition of cellular proliferation, which suggests that the interaction of EGCG with p53 may not fully explain its inhibitory effect on proliferation. Caloric restriction (CR) reduces the incidence and progression of spontaneous and induced tumors in laboratory rodents. EGCG has multiple beneficial activities similar to those associated with CR. One key enzyme thought to be activated during CR is AMP-activated kinase (AMPK), a sensor of cellular energy levels. Here, we showed that EGCG activated AMPK in both p53 positive and negative human hepatoma cells. The activation of AMPK suppressed downstream substrates, such as mammalian target of rapamycin (mTOR) and eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) and a general decrease in mRNA translation. Moreover, EGCG activated AMPK decreases the activity and/or expression of lipogenic enzymes, such as fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC). Interestingly, the decision between apoptosis and growth arrest following AMPK activation is greatly influenced by p53 status. In p53 positive Hep G2 cells, EGCG blocked the progression of cell cycle at G1 phase by inducing p53 expression and further up-regulating p21 expression. However, EGCG inducted apoptosis in p53 negative Hep 3B cells. Based on these results, we have demonstrated that EGCG has a potential to be a chemoprevention and anti-lipogenesis agent for human hepatoma cells.

Anti-inflammatory activity of lipophilic epigallocatechin gallate (EGCG) derivatives in LPS-stimulated murine macrophages

Epigallocatechin gallate (EGCG), the major polyphenol in green tea and the main bioactive compound responsible for the health benefits of tea consumption, has been proposed as a functional ingredient for food and natural health products. However, EGCG is hydrophilic with poor cellular absorption and thus compromised bioefficiency in vivo. In order to enhance the lipophilicity of EGCG for improved bioefficiency and to take advantage of the health beneficial omega 3 fatty acids, the EGCG molecule was esterified with docosapentaenoic acid (DPA), upon which a mixture of ester derivatives with different degrees of substitution was produced. The EGCG-DPA esters were evaluated for their anti-inflammatory activity in LPS (lipopolysaccharides)-stimulated murine RAW 264.7 macrophages. The production of pro-inflammatory mediators nitric oxide (NO) and prostaglandin (PGE(2)) was significantly inhibited by treatment of EGCG-DPA esters, and the inhibition was largely due to their down-regulatory effect on iNOS (inducible NO synthase) and COX (cyclooxygenase)-2 gene expression at transcriptional level. The EGCG-DPA esters effectively suppressed the expression of iNOS and COX -2 proteins as well as their mRNA, as observed with western blotting and RT-PCR analyses. Ester derivatives of EGCG with other fatty acids (stearic acid, SA; eicosapentaenoic acid, EPA; and docosahexaenoic acid, DHA) were also prepared in the form of pure tetraesters, which also exhibited anti-inflammatory effect in the macrophages. The results suggest that EGCG ester derivatives with anti-inflammatory potentials may be useful in preventing/treating inflammation-mediated diseases and health conditions.