De-Xing Hou

Potential mechanisms of cancer chemoprevention by anthocyanins.

Anthocyanins are the chemical components that give the intense color to many fruits and vegetables, such as blueberries, red cabbages and purple sweet potatoes. Epidemiological investigations have indicated that the moderate consumption of anthocyanin products such as red wine or bilberry extract is associated with a lower risk of cardiovascular disease and improvement of visual functions. Recently, there is increasing interesting in the pharmaceutical function of anthocyanins. This review summarizes current knowledge on the various molecular evidences of cancer chemoprevention by anthocyanins. These mechanisms can be subdivided into the following aspects: 1) the antioxidation; 2) the molecular mechanisms involved in anticarcinogenesis; 3) the molecular mechanisms involved in the apoptosis induction of tumor cells. Finally, the bioavailability and structure-activity relationship of anthocyanins are also summarized.

Molecular Mechanisms Behind the Chemopreventive Effects of Anthocyanidins

Anthocyanins are polyphenolic ring-based flavonoids, and are widespread in fruits and vegetables of red-blue color. Epidemiological investigations and animal experiments have indicated that anthocyanins may contribute to cancer chemoprevention. The studies on the mechanism have been done recently at molecular level. This review summarizes current molecular bases for anthocyanidins on several key steps involved in cancer chemoprevention: (i) inhibition of anthocyanidins in cell transformation through targeting mitogen-activated protein kinase (MAPK) pathway and activator protein 1 (AP-1) factor; (ii) suppression of anthocyanidins in inflammation and carcinogenesis through targeting nuclear factor kappa B (NF-κB) pathway and cyclooxygenase 2 (COX-2) gene; (iii) apoptotic induction of cancer cells by anthocyanidins through reactive oxygen species (ROS) / c-Jun NH2-terminal kinase (JNK)-mediated caspase activation. These data provide a first molecular view of anthocyanidins contributing to cancer chemoprevention.

Stabilization of p53 is involved in quercetin-induced cell cycle arrest and apoptosis in HepG2 cells

There is evidence for defects in the mechanisms that allow the activation of p53 in many of the cancers that retain wild-type p53. Reactivation of p53 has been suggested to be an effective strategy for cancer therapy in wild-type p53-retained tumor cells. In the present study, we attempted to reactivate p53 in HepG2 retaining wild-type p53 by quercetin, an ubiquitous bioactive plant flavonoid. Our results show that quercetin inhibited the proliferation of HepG2 cells through the induction of cell cycle arrest and apoptosis, as characterized by the cell cycle distribution and DNA fragmentation. Molecular data revealed that quercetin induced p53 phosphorylation and total p53 protein, but that it did not up-regulate p53 mRNA at the transcription level. Consequently, quercetin stimulated p21 expression and suppressed cyclin D1 expression in favor of cell cycle arrest. Quercetin also increased the ratio of Bax/Bcl-2 in favor of apoptosis with such treatment. Interestingly, quercetin inhibited p53 ubiquitination and extended the half-life (t 1⁄2) of p53 from 74 to 184 min. Quercetin also inhibited p53 mRNA degradation at the post-transcription stage. Silencing p53 with p53 small interfering RNA (siRNA) significantly abrogated the p53-dependent gene expression and apoptotic induction. Taken together, our data demonstrate that quercetin stabilized p53 at both the mRNA and protein levels to reactivate p53-dependent cell cycle arrest and apoptosis in HepG2 cells.

Myb controls G2/M progression by inducing cyclin B expression in the Drosophila eye imaginal disc

The c‐myb proto‐oncogene product (c‐Myb) is a transcriptional activator. Vertebrate c‐Myb is a key regulator of the G1/S transition in cell cycle, while Drosophila Myb (dMyb) is important for the G2/M transition. Here we report that dMyb induces expression of cyclin B, a critical regulator of the G2/M transition, in Drosophila eye imaginal disc. In the wild‐type eye disc, dmyb mRNA was expressed in the stripes both anterior and posterior to the morphogenetic furrow. Ectopic expression of C‐terminal‐truncated dMyb in the eye disc caused ectopic expression of cyclin B and the rough eye phenotype. This rough eye phenotype correlated with prolonged M phase, caused by overexpression of cyclin B. Cyclin B expression was lost in dmyb‐deficient clones. In Schneider cells, the activity of the cyclin B promoter was dramatically reduced by loss of dMyb using the RNA interference method. Mutations of the multiple AACNG sequences in the cyclin B promoter also abolished the promoter activity. These results indicate that dMyb regulates the G2/M transition by inducing cyclin B expression via binding to its promoter.

Akt is a direct target for myricetin to inhibit cell transformation

Akt, a serine/threonine kinase, is a critical regulator in many cellular processes including cell growth, proliferation, and apoptosis. In this study, we found that myricetin, a typical flavonol existing in many fruits and vegetables, could directly target Akt to inhibit cell transformation. Binding assay revealed that myricetin bound to Akt directly by competing with ATP. In vitro and ex vivo data confirmed that myricetin inhibited the phosphorylation and kinase activity of Akt. Molecular modeling suggested that myricetin easily docks to the ATP-binding site of Akt with hydrogen bonds. Signaling analysis data further demonstrated that myricetin inhibited Akt-mediated activator protein-1 (AP-1) transactivation, cyclin D1 expression and cell transformation. Overall, our results indicate that Akt is a direct target for myricetin to inhibit cell transformation.

Cosmid assembly and anchoring to human chromosome 21

A human chromosome 21-specific cosmid library from the Lawrence Livermore National Laboratory has been analyzed by two complementary methods, fingerprinting and hybridization; 40% coverage of the entire chromosome 21 has been achieved. To prepare a contig pool, approximately 9300 cosmid clones randomly selected from the library were fingerprinted and automatically assembled into 467 overlapping sets by the fluorescence-tagged restriction fragment method. The average size of the overlapping sets was 9.5 cosmids with minimal tiling paths consisting of 5.4 cosmids with a 10-kb extension each. However, as many as 10% of overlaps within members were estimated to be false. For regional localization, we hybridized gridded arrays of cosmids with inter-Alu-PCR probes obtained from YAC clones and somatic cell hybrids and assigned 592 cosmids to 26 subregions of 21q. Of these, 371 clones were incorporated into 139 contigs, anchoring the total 1864 cosmids to the subregion. The remaining 221 clones were mapped as orphans. To correlate the cytogenetic, YAC, and cosmid maps on 21q, the translocation breakpoints of the chromosomes contained in the somatic cell hybrids were mapped with respect to the STS content of the YACs. From the gene cluster regions, 176 ribosomal and 25 alphoid clones were isolated by hybridization. Together, these sets of anchored contigs and cosmids will provide a valuable resource for construction of a high-resolution map and for isolation of genes of interest from chromosome 21.

Phenolic composition and radical scavenging activity of sweetpotato-derived shochu distillery by-products treated with koji.

Phenolic composition and radical scavenging activity in the shochu distillery by-products of sweetpotato (Ipomoea batatas L.) treated with koji (Aspergillus awamori mut.) and cellulase (Cellulosin T2) were investigated to develop new uses. Koji and Cellulosin T2 treatment of shochu distillery by-products from sweetpotatoes, rice, and barley increased phenolic content. Caffeic acid was identified as a dominant phenolic component in the shochu distillery by-products of the sweetpotato. Adding koji and/or Cellulosin T2 to the shochu distillery by-product indicated that koji was involved in caffeic acid production. Caffeic acid was not detected in raw or steamed roots of “Koganesengan”, the material of sweetpotato for shochu production, suggesting that it is produced during shochu fermentation. The phenolic content and radical scavenging activity the shochu distillery by-product treated with koji and Cellulosin T2 were superior to those of commercial vinegar. These results suggest that koji treatment of sweetpotato-derived shochu distillery by-products has potential for food materials with physiological functions. Further koji treatment of sweetpotato shochu-distillery by-products may be applicable to mass production of caffeic acid.

Acetyl derivate of quercetin increases the sensitivity of human leukemia cells toward apoptosis

The hydroxyl groups of flavonoids are important for their bioactive functions and also prone to oxidation to quinones. To block the potential oxidation of quercetin, and generate a stronger bioactive compound, we synthesized acetyl and methyl derivatives of quercetin, 3,7,3′,4′‐O‐tetraacetylquercetin (4Ac‐Q) and 3,7,3′,4′‐O‐tetramethylquercetin (4Me‐Q), which substituted the hydroxyl groups of quercetin with acetyl or methyl groups at the 3,7,3′,4′ positions of quercetin, and then evaluated the ability to cause cell proliferation inhibition and apoptosis in HL‐60 cells. The results revealed that 4Ac‐Q and quercetin, but not 4Me‐Q, significantly inhibit cell proliferation by caspase‐mediated apoptosis when characterized by DNA fragmentation, activation of caspase‐3 and PARP cleavage while 4Me‐Q lost this ability. Interestingly, 4Ac‐Q revealed stronger apoptotic activity than parent quercetin via a ROS‐independent pathway. These findings provide a valuable strategy to increase the sensitivity of human leukemia HL‐60 cells toward apoptosis by modifying quercetin structure.

Expression Profiling of Genes Targeted by Bilberry (Vaccinium myrtillus) in Macrophages Through DNA Microarray

To evaluate the anti-inflammatory function and underlying genes targeted by bilberry, gene expression profiling through DNA microarray was performed on bilberry extract-treated macrophages. Among 22,050 oligonucleotides, the expression levels of 998 genes were increased by ≥ twofold in lipopolysaccharide (LPS)-activated RAW264 cells, 358 gene signals of which were attenuated by bilberry extract (≥ 1.5-fold). Expression levels of 2,086 genes were decreased by ≥ twofold in LPS-activated cells, of which 939 gene signals were enhanced by bilberry extract (≥ 1.5-fold). Utilizing Panther group analysis, 308 genes affected by bilberry extract were classified into 43 categories relating to biological processes (97), molecular functions (186), and signaling pathways (26) with ≥ 1.5-fold change. The genes categorized as “defense, inflammatory response, cytokines activities, and receptor activities” were further identified, and some of them were confirmed by real-time polymerase chain reaction. The DNA microarray results provide a molecular basis for the anti-inflammatory effects of bilberry.

Linoleic acid metabolite suppresses skin inflammation and tumor promotion in mice: possible roles of programmed cell death 4 induction

(+/-)-13-Hydroxy-10-oxo-trans-11-octadecenoic acid (13-HOA) is one of the lipoxygenase metabolites of linoleic acid (LA) from corn germ. Recently, we reported that this metabolite suppressed the expression of lipopolysaccharide-induced proinflammatory genes in murine macrophages by disrupting mitogen-activated protein kinases and Akt pathways. In this study, we investigated the inhibitory effects of 13-HOA on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in ears and skin, as well as tumor promotion in female ICR mice. Pretreatment with 13-HOA (1600 nmol) inhibited ear edema formation by 95% (P < 0.05) in an inflammation test and reduced tumor incidence and the number of tumors per mouse by 40 and 64% (P < 0.05 each), respectively, in a two-stage skin carcinogenesis model. Histological examinations revealed that it decreased epidermal thickness, the number of infiltrated leukocytes and cell proliferation index. Furthermore, 13-HOA (8-40 muM) suppressed TPA-induced anchorage-independent growth of JB6 mouse epidermal cells by 70-100%, whereas LA was virtually inactive. 13-HOA (40 muM) inhibited TPA-induced activator protein-1 transactivation but not extracellular signal-regulated kinase1/2 activation. Interestingly, 13-HOA (40 muM and 1600 nmol in JB6 cells and mouse skin, respectively) induced expression of programmed cell death 4 (Pdcd4), a novel tumor suppressor protein. To our knowledge, this is the first report of a food factor that is able to induce Pdcd4 expression. Collectively, our results indicate that 13-HOA may be a novel anti-inflammatory and antitumor chemopreventive agent with a unique mode of action.