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.

Polymethoxyflavones activate Ca2+-dependent apoptotic targets in adipocytes.

Induction of apoptosis is an emerging strategy for the prevention and treatment of obesity because removal of adipocytes via apoptosis will result in reducing body fat and may help to maintain a long-lasting weight loss. Our previous studies have shown that a sustained increase in intracellular Ca(2+) triggers apoptosis in various cell types via activation of Ca(2+)-dependent proteases and that the apoptosis-inducing effect of polymethoxyflavones (PMFs) in cancer cells is mediated through Ca(2+) signaling. This paper reports that PMFs induce apoptosis in mature mouse 3T3-L1 adipocytes via activation of Ca(2+)-dependent calpain and Ca(2+)/calpain-dependent caspase-12. Treatment of adipocytes with PMFs evoked, in a concentration- and time-dependent fashion, sustained increase in the basal level of intracellular Ca(2+). The increase in Ca(2+) was associated with induction of apoptosis and activation of mu-calpain and caspase-12. Apoptosis-inducing activity of hydroxylated PMFs was significantly higher than that of the corresponding nonhydroxylated compounds. These results demonstrate that the apoptotic molecular targets activated by PMFs in adipocytes are Ca(2+)-dependent calpain and caspase-12. The findings obtained provide rationale for evaluating the role of PMFs in the prevention and treatment of obesity.

Anti-inflammatory and antitumor promotional effects of a novel urinary metabolite, 3',4'-didemethylnobiletin, derived from nobiletin.

We reported previously that 3',4'-didemethylnobiletin (DDMN) is the major metabolite of nobiletin in mouse urine. In this study, we examined DDMN's molecular mechanism of action and its anti-inflammatory and antitumor properties. We demonstrated that topical application of DDMN effectively inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated transcription of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and ornithine decarboxylase (ODC) messenger RNA and protein expression in mouse skin. Pretreatment with DDMN has resulted in the reduction of TPA-induced nuclear translocation of the nuclear factor-kappa B (NF-kappaB) subunit. DDMN also reduced DNA binding by blocking phosphorylation of inhibitor kappaB (IkappaB) alpha and p65 and caused subsequent degradation of IkappaBalpha. DDMN inhibited TPA-induced phosphorylation and nuclear translocation of the signal transducer and activator of transcription 3. Moreover, DDMN suppressed TPA-induced activation of extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt and protein kinase C that are upstream of NF-kappaB and activator protien-1. We also found that DDMN significantly inhibited TPA-induced mouse skin inflammation by decreasing inflammatory parameters. Furthermore, DDMN significantly inhibited 7,12-dimethylbenz[a]anthracene/TPA-induced skin tumor formation measured by the tumor multiplicity of papillomas at 20 weeks. Presented data for the first time reveal that DDMN is an effective antitumor agent that functions by downregulating inflammatory iNOS, COX-2 and ODC gene expression in mouse skin. It is suggested that DDMN is a novel functional agent capable of preventing inflammation-associated tumorigenesis.

Antitumor activity of 3,5,4'-trimethoxystilbene in COLO 205 cells and xenografts in SCID mice.

Resveratrol (R-3), a trihydroxy trans-stilbene from grape, inhibits multistage carcinogenesis in animal models. Here we report that 3,5,4'-trimethoxystilbene (MR-3), the permethylated derivative of R-3 was more potent against the growth of human cancer cells (HT-29, PC-3, COLO 205) with estimated IC(50) values of 81.31,42.71, and 6.25 microM, respectively. We further observed that MR-3 induced apoptosis in COLO 205 cells through modulation of mitochondrial functions regulated by reactive oxygen species (ROS). ROS generation occurs in the early stages of MR-3-induced apoptosis, preceding cytochrome-c release, caspase activation, and DNA fragmentation. Significant therapeutic effects were demonstrated in vivo by treating severe combined immune deficiency (SCID) mice bearing COLO 205 tumor xenografts with MR-3 (50 mg/kg ip). Assays on DNA fragmentation and caspase activation were performed and demonstrated that apoptosis occurred in tumor tissues treated with MR-3. The appearance of apoptotic cells, as shown by Hematoxylin and Eosin (H&E) staining, and an increase in p21 and decrease in proliferating cell nuclear antigen (PCNA) protein by immuno-histochemistry were observed in tumor tissues under MR-3 treatment. Our study identifies the novel mechanisms of the antitumor effects of MR-3 and indicates that these results may have significant applications for cancer chemotherapy.

Apoptosis-inducing activity of hydroxylated polymethoxyflavones and polymethoxyflavones from orange peel in human breast cancer cells.

Sweet orange (Citrus sinensis L.) peel is a rich resource of flavonoids, especially polymethoxyflavones (PMFs). Citrus flavonoids exert a broad spectrum of biological activity, including antiproliferative and proapoptotic effects in cancer cells. We have recently shown that individual PMFs from orange peel induce Ca(2+)-mediated apoptosis in human breast cancer cells and that hydroxylation of PMFs is critical for enhancing their proapoptotic activity. Here, we report that the fraction of orange peel extract containing a mixture of non-hydroxylated PMFs (75.1%) and hydroxylated PMFs (5.44%) and the fraction containing only hydroxylated PMFs (97.2%) induce apoptosis in those cells as well. Treatment of MCF-7 breast cancer cells with these fractions inhibited growth and induced apoptosis associated with an increase in the basal level of intracellular Ca(2+). Effective concentrations of the hydroxylated PMFs fraction in inhibiting growth, inducing apoptosis, and increasing intracellular Ca(2+) were lower than those of the non-hydroxylated PMFs fraction. Our results strongly imply that bioactive PMFs from orange peel exert proapoptotic activity in human breast cancer cells, which depends on their ability to induce an increase in intracellular Ca(2+ )and thus, activate Ca(2+)-dependent apoptotic proteases.

5-Hydroxy-3,6,7,8,3',4'-hexamethoxyflavone induces apoptosis through reactive oxygen species production, growth arrest and DNA damage-inducible gene 153 expression, and caspase activation in human leukemia cells.

This study examined the growth inhibitory effects of structurally related polymethoxylated flavones in human cancer cells. Here, we report that 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-OH-HxMF) induces growth inhibition of human cancer cells and induction of apoptosis in HL-60 cells through modulation of mitochondrial functions regulated by reactive oxygen species (ROS). ROS generation occurs in the early stages of 5-OH-HxMF-induced apoptosis, preceding cytochrome c release, caspase activation, and DNA fragmentation. The changes occurred after single breaks in DNA were detected, suggesting that 5-OH-HxMF induced irreparable DNA damage, which in turn triggered the process of apoptosis. Up-regulation of Bax was found in 5-OH-HxMF-treated HL-60 cells. In addition, a caspase-independent pathway indicated by endonuclease G also contributed to apoptosis caused by 5-OH-HxMF. Antioxidants suppress 5-OH-HxMF-induced apoptosis. 5-OH-HxMF markedly enhanced growth arrest DNA damage-inducible gene 153 (GADD153) protein in a time-dependent manner. N-acetylcysteine (NAC) and catalase prevented up-regulation of GADD153 expression caused by 5-OH-HxMF. These findings suggest that 5-OH-HxMF creates an oxidative cellular environment that induces DNA damage and GADD153 gene activation, which in turn helps trigger apoptosis in HL-60 cells. Meanwhile, ROS were proven an important inducer in this apoptotic process. The C-5 hydroxyl on the ring of 5-OH-HxMF was found to be essential for the antiproliferative and apoptosis-inducing activity. Our study identified the novel mechanisms of 5-OH-HxMF-induced apoptosis and indicated that these results have significant applications as potential chemopreventive and chemotherapeutic agents.

Isolation and syntheses of polymethoxyflavones and hydroxylated polymethoxyflavones as inhibitors of HL-60 cell lines.

Fifteen polymethoxyflavones (PMFs) and hydroxylated PMFs were isolated from sweet orange (Citrus sinensis) peel extract and synthesized to investigate their biological activity. All obtained compounds were tested in HL-60 cancer cell proliferation and apoptosis induction assays. While some PMFs and hydroxylated PMFs had moderate anti-carcinogenic activities, 5-hydroxy-6,7,8,3',4'-pentamethoxyflavone and 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone showed strong inhibitory activities against the proliferation and induced apoptosis of HL-60 cell lines.

Polymethoxylated flavones induce Ca(2+)-mediated apoptosis in breast cancer cells.

Flavonoids, polyphenolic phytochemicals which include flavones and isoflavones, are present in the common human diet. It has been suggested that these compounds may exert anticancer activity; however, the mechanisms involved remain unknown. We have recently shown (Sergeev, 2004, Biochem Biophys Res Commun 321: 462-467) that isoflavones can activate the novel apoptotic pathway mediated by cellular Ca(2+). Here, we report that polymethoxyflavones (PMFs) derived from sweet orange (Citrus sinensis L.) inhibit growth of human breast cancer cells via Ca(2+)-dependent apoptotic mechanism. The treatment of MCF-7 breast cancer cells with 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-OH-HxMF) and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone (3'-OH-TtMF) induced a sustained increase in concentration of intracellular Ca(2+) ([Ca(2+)](i)) resulting from both depletion of the endoplasmic reticulum Ca(2+) stores and Ca(2+) influx from the extracellular space. This increase in [Ca(2+)](i) was associated with the activation of the Ca(2+)-dependent apoptotic proteases, mu-calpain and caspase-12, as evaluated with the calpain and caspase-12 peptide substrates and antibodies to active (cleaved) forms of the enzymes. Corresponding non-hydroxylated PMFs, 3,5,6,7,8,3',4'-heptamethoxyflavone (HpMF) and 5,6,7,3',4'-pentamethoxyflavone (PtMF), were dramatically less active in inducing Ca(2+)-mediated apoptosis. Our results strongly suggest that the cellular Ca(2+) modulating activity of flavonoids underlies their apoptotic mechanism and that hydroxylation of PMFs is critical for their ability to induce an increase in [Ca(2+)](i) and, thus, activate Ca(2+)-dependent apoptotic proteases.

Inhibitory effects of black tea theaflavin derivatives on 12-O-tetradecanoylphorbol-13-acetate-induced inflammation and arachidonic acid metabolism in mouse ears.

Tea has been shown to possess several health beneficial properties primarily due to its polyphenolic content. The major polyphenolic compounds in black tea leaves are theaflavins (TFs) formed by oxidative coupling of catechins in tea leaves during its processing. In this paper, we report the characterization of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear inflammatory model and the inhibitory effects of major black tea TFs derivatives on this inflammation. In addition, the effect on inflammatory biomarkers, such as proinflammatory cytokines and arachidonic acid metabolites, are reported as well. A single topical application of TPA to ears of CD-1 mice induced a time- and dose-dependent increase in edema as well as formation of proinflammatory cytokine proteins interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) in mouse ears. A single topical application of equimolar of black tea constituents (TF, theaflavin-3-gallate, theaflavin-3'-gallate, and theaflavin-3,3'-digallate) strongly inhibited TPA-induced edema of mouse ears. Application of TFs mixture to mouse ears 20 min prior to each TPA application once a day for 4 days inhibited TPA-induced persistent inflammation, as well as TPA-induced increase in IL-1beta and IL-6 protein levels. TFs also inhibited arachidonic acid (AA) metabolism via both cyclooxygenase (COX) and lipoxygenase pathways. This observation was substantiated by decreased amounts of AA metabolites prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) levels. Combined application of TF and sulindac, a nonsteroidal anti-inflammatory drug resulted a significant synergetic anti-inflammatory effect. Oral administration of TFs or the hot water extract of black tea leaves also significantly inhibited TPA-induced edema in mouse ears. In conclusion, proinflammatory cytokines, IL-1beta and IL-6, as well as the intermediated metabolites of AA, PGE2, and LTB4 are good biomarkers for inflammation. Black tea constituents, TF and its derivatives, had strongly anti-inflammatory activity in vivo which may be due to their ability to inhibit AA metabolism via lipoxygenase and COX pathways.

Optimizing phytoremediation of heavy metal-contaminated soil by exploiting plants' stress adaptation.

Soil phytoextraction is based on the ability of plants to extract contaminants from the soil. For less bioavailable metals, such as Pb, a chelator is added to the soil to mobilize the metal. The effect can be significant and in certain species, heavy metal accumulation can rapidly increase 10-fold. Accumulation of high levels of toxic metals may result in irreversible damage to the plant. Monitoring and controlling the phytotoxicity caused by EDTA-induced metal accumulation is crucial to optimize the remedial process, i.e. to achieve maximum uptake. We describe an EDTA-application procedure that minimizes phytotoxicity by increasing plant tolerance and allows phytoextraction of elevated levels of Pb and Cd. Brassica juncea is tested in soil with typical Pb and Cd concentrations of 500 mg kg-1 and 15 mg kg-1, respectively. Instead of a single dose treatment, the chelator is applied in multiple doses, that is, in several small increments, thus providing time for plants to initiate their adaptation mechanisms and raise their damage threshold. In situ monitoring of plant stress conditions by chlorophyll fluorescence recording allows for the identification of the saturating heavy metal accumulation process and of simultaneous plant deterioration.

Bioengineering of a phytoremediation plant by means of somatic hybridization.

Phytoremediation is a technology that exploits a plant's ability to remove contaminants from the environment or render toxic compounds harmless. An efficient metal phytoremediating plant must combine high biomass production and established cultivation methods with high tolerance to a specific contaminant and ability for root uptake, translocation, and compartmentalization of contaminants in the above-ground biomass. Symmetric and asymmetric somatic hybridizations were used to introduce toxic metal-resistant traits from Thlaspi caerulescens into Brassica juncea. B. juncea hypocotyl protoplasts were fused with T. caerulescens mesophyll protoplasts. The hypocotyl protoplasts of B. juncea were stained with CFDA before fusion and thus fluoresced green under UV, whereas the mesophyll protoplasts of T. caerulescens had red autofluorescense. Heteroplasmic fusion products were identified and selected by flow cytometry and cell sorting. All putative hybrids grown in the greenhouse had morphological characteristics of B. juncea. A Thlaspi-specific repetitive sequence was hybridized to total DNA of plants, including the parental species. All plants from both symmetric and asymmetric fusions showed Thlaspi-specific hybridization patterns while B. juncea did not exhibit any hybridization signal. Hybrid plants, produced by asymmetric somatic hybridization between the two species, demonstrated high metal accumulation potential, tolerance to toxic metals, and good biomass production.