Weiqun Wang

Dietary agents in cancer prevention: flavonoids and isoflavonoids

Flavones and isoflavones may play a prominent role in cancer prevention since these compounds are found in numerous plants that are associated with reduced cancer rates. This article reviews recent epidemiological and animal data on isoflavones and flavones and their role in cancer prevention. It covers aspects of the bioavailability of these dietary constituents and explores their mechanism of action. Human epidemiology data comes primarily from studies in which foods rich in isoflavones or flavones are associated with cancer rates. This approach has been particularly useful with isoflavones because of their abundance in specific foods, including soy foods. The bioavailability of flavones and isoflavones has been shown to be influenced by their chemical form in foods (generally glycoside conjugates), their hydrophobicity, susceptibility to degradation, the microbial flora of the consumer, and the food matrix. Some information is available on how these factors influence isoflavone bioavailability, but the information on flavones is more limited. Many mechanisms of action have been identified for isoflavone/flavone prevention of cancer, including estrogenic/antiestrogenic activity, antiproliferation, induction of cell-cycle arrest and apoptosis, prevention of oxidation, induction of detoxification enzymes, regulation of the host immune system, and changes in cellular signaling. It is expected that some combination of these mechanisms will be found to be responsible for cancer prevention by these compounds. Compelling data suggest that flavones and isoflavones contribute to cancer prevention; however, further investigations will be required to clarify the nature of the impact and interactions between these bioactive constituents and other dietary components.

Cell-Cycle Arrest at G2/M and Growth Inhibition by Apigenin in Human Colon Carcinoma Cell Lines

Apigenin, a common dietary flavonoid, has been shown to induce cell cycle arrest in both epidermal and fibroblast cells and inhibit skin tumorigenesis in murine models. The present study assessed the influence of apigenin on cell growth and the cell cycle in the human colon carcinoma cell lines SW480, HT‐29, and Caco‐2. Treatment of each cell line with apigenin (0–80 μM) resulted in a dose‐dependent reduction in both cell number and cellular protein content, compared with untreated control cultures. DNA flow cytometric analysis indicated that treatment with apigenin resulted in G2/M arrest in all three cell lines in a time‐ and dose‐dependent manner. Apigenin treatment (80 μM) for 48 h produced maximum G2/M arrest of 64%, 42%, and 26% in SW480 cells, HT‐29 cells, and Caco‐2 cells, respectively, in comparison with control cells (15%). The proportion of S‐phase cells was not altered by apigenin treatment in each of the three cell lines. The G2/M arrest was reversible after 48 h of apigenin treatment in the most sensitive cell line SW480. The degree of G2/M arrest by apigenin was inversely correlated with the corresponding inhibition of cell growth measurements in all three cell lines (r = −0.626 to −0.917, P≤0.005). Moreover, an immune complex kinase assay demonstrated an inhibition of p34cdc2 kinase activity, a critical enzyme in G2/M transition, in each cell line after treatment with apigenin (50–80 μM). Western blot analyses indicated that both p34cdc2 and cyclin B1 proteins were also decreased after apigenin treatment. These results indicate that apigenin inhibits colon carcinoma cell growth by inducing a reversible G2/M arrest and that this arrest is associated, at least in part, with inhibited activity of p34cdc2 kinase and reduced accumulation of p34cdc2 and cyclin B1 proteins. Differences in induction of G2/M arrest by apigenin in the three colon carcinoma cell lines suggest that dietary apigenin may be differentially effective against tumors with specific mutational spectra. Mol. Carcinog. 28:102–110, 2000.

Individual and Interactive Effects of Apigenin Analogs on G2/M Cell-Cycle Arrest in Human Colon Carcinoma Cell Lines

Apigenin has been previously shown to induce G2/M cell-cycle arrest in human colon cancer cell lines. The present study assessed the individual and interactive influence of seven apigenin analogs on cell cycle, cell number, and cell viability in human SW480 and Caco-2 colonic carcinoma cells. Cellular concentration of selected apigenin analogs was further assessed by high-performance liquid chromatography to assess cellular availability. The apigenin analogs studied were acacetin, chrysin, kampherol, luteolin, myricetin, naringenin, and quercetin. DNA flow cytometric analysis indicated that treatment with either chrysin or acacetin at 0 to 80 μM for 48 h resulted in cell-cycle arrest at the G2/M phase in a dose-dependent manner in the SW480 cells but not in the Caco-2 cells. The percentage of SW480 cells at G2/M also increased when cells were treated with kampherol, luteolin, or quercetin between 5 and 30 μM, but the percentage of cells in G2/M decreased at doses greater than 40 μM. Cell number was significantly decreased in a time- and dose-dependent manner following the treatments with each analog except for naringenin and myricetin. The interactive effects of these analogs with apigenin were further assessed by combining each analog at doses from 0 to 80 μM with apigenin at 20 μM, a dose at which apigenin was found to double the proportion of SW480 cells in G2/M. When either acacetin, chrysin, luteolin, kampherol, or quercetin at doses between 5 and 30 μM were combined with apigenin at 20 μM, there was an increase of 22% in the proportion of G2/M cells over that observed with 20 μM apigenin alone (P < 0.05). At doses higher than 40 μM, however, the interaction became antagonistic, and the proportion of cells in G2/M decreased below that observed with apigenin alone. Cell viability, as assessed by Trypan blue exclusion assay, significantly decreased by treatments with high doses of each agent or each agent combined with apigenin. Cellular concentration of apigenin, chrysin, or naringenin in SW480 cells significantly increased at doses of 40 μM or greater, but it was not correlated with their impact on G2/M cell-cycle arrest. The induction of cell-cycle arrest by five of seven tested apigenin analogs and the additive induction by the combination of flavonoids at low doses suggest that apigenin-related flavonoids may cooperatively protect against colorectal cancer through conjoint blocking of cell-cycle progression.

Identification of molecular targets for dietary energy restriction prevention of skin carcinogenesis: An idea cultivated by Edward Bresnick

Dietary energy restriction (DER) has long been known to strikingly inhibit carcinogenesis in many animal models. The animal data has been corroborated by recent and ongoing epidemiological studies demonstrating the importance of energy balance, physical exercise and obesity in human cancer. Dr. Edward Bresnick provided key insights into this important area of research and pivotal direction for the authors research while he served as Director of the Eppley Institute for Research in Cancer, Omaha, NE. These insights moved this research toward demonstrating that DER reduced the expression of key protein kinase C isoforms in mouse skin. More recent studies have uncovered downstream events that are inbibited by DER including blockage of tumor promoter activation of Raf‐1, ERK 1,2 and AP‐1 expression. Parallel studies have demonstrated the DER inhibition of these key cellular signaling events in mouse skin carcinogenesis are dependent upon an intact adrenal gland because adrenalectomized mice fed DER diet did not have reduced tumor burden or inhibited signaling and blocked AP‐1 activation as was observed in DER mice with intact adrenal glands. In addition, the DER inhibition of tumorigenesis and AP‐1 signaling was restored in adrenalectomized mice that were given corticosterone in the drinking water. This showed that in mice in the chemical carcinogenesis protocol glucocorticoid hormone plays a major role in mediating DER prevention of cancer. Studies are ongoing to further assess the mechanism of DER modulation of skin cancer by assessing impacts on transcriptional regulation and expression of genes that are critical in skin carcinogenesis.

Effect of Dietary Apigenin on Colonic Ornithine Decarboxylase Activity, Aberrant Crypt Foci Formation, and Tumorigenesis in Different Experimental Models

Abstract: The efficacy of dietary apigenin, a dietary flavonoid, in colon cancer prevention was investigated by evaluating the inhibition of the ornithine decarboxylase (ODC) activity and the formation of aberrant crypt foci (ACF) and by studying the ability of apigenin to block colon carcinogenesis in two mouse models. First, the activity of ODC was measured in colon cancer cells (Caco-2) and in the colon epithelium of CF-1 mice. Apigenin at 10 and 30 μM significantly inhibited the ODC activity of Caco-2 cells by 26% and 57%, respectively. Colonic ODC activity in CF-1 mice was reduced with 0.1% dietary apigenin by 42% compared with the control, but this difference was not statistically significant. Second, ACF formation was evaluated in azoxymethane (AOM)-induced CF-1 mice. Female CF-1 mice at 6 wk of age were i.p. injected with 5 mg/kg body weight (BW) AOM once to induce ACF. ACF formation in CF-1 mice was reduced by 50% (P < 0.05) with 0.1% dietary apigenin fed for 6 wk when compared with the control. Dietary apigenin inhibited ACF only in the distal region of the CF-1 mouse colon. Finally, tumorigenesis studies were conducted using two different mouse models: AOM-induced CF-1 mice and Min mice with mutant adenomatous polyposis coli (APC) gene. Female CF-1 mice at 6 wk of age were i.p. injected with 10 mg/kg BW AOM weekly for 6 (AOM Study I) or 4 (AOM Study II) wk to induce tumors. CF-1 mice were fed diets containing 0.025% or 0.1% apigenin for 23-25 wk. Female Min mice were fed diets for 10 wk beginning at 5 wk of age. In two AOM-treated mouse colon tumor studies 0.025% and 0.1% dietary apigenin modestly reduced tumors in the group fed 0.025% apigenin (25% incidence in comparison with 65% in the controls) in a non-dose response manner. Apigenin failed to inhibit adenoma formation in the Min mouse study. These results suggest that dietary apigenin showed promise in cancer prevention by reducing the ODC activity and ACF formation, however, clear evidence of cancer prevention was not obtained in mouse tumor studies. Further investigation of the potential chemopreventive effect of apigenin in carcinogenesis is warranted.

Dietary energy restriction, in part through glucocorticoid hormones, mediates the impact of 12-O-tetradecanoylphorbol-13-acetate on jun D and fra-1 in sencar mouse epidermis

Dietary energy restriction (DER, 40% calorie reduction from fat and carbohydrate) inhibited mouse skin carcinogenesis and decreased 12‐O‐tetradecanoyl‐13‐phorbol acetate (TPA)‐induced activator protein‐1 (AP‐1):DNA binding previously. This study measured protein levels of c‐jun, jun B, jun D, c‐fos, fra‐1, and fra‐2 and examined their contribution to AP‐1:DNA binding by electrophoretic mobility shift assay (EMSA) with supershift analysis in the epidermis of control and DER Sencar mice exposed to TPA. TPA significantly increased c‐jun, jun B, c‐fos, fra‐1, and fra‐2 and decreased jun D within 3–6 h after treatment. AP‐1:DNA binding reached a maximum 2.5‐fold induction over controls 4 h after TPA treatment and antibodies to jun B, jun D, and fra‐2 in the EMSA binding reaction resulted in supershifts in both acetone‐ and TPA‐treated mice 1–6 h after treatment. The effect of corticosterone (CCS) and DER on the AP‐1 proteins and on the composition of the AP‐1:DNA complex was measured in adrenalectomized (adx) mice. DER reduced the TPA impact on jun D and enhanced the induction of fra‐1. In addition, CCS‐supplemented groups had significantly lower jun D and higher fra‐2 than adx groups and sham groups. While sham animals treated with either acetone or TPA contained jun B, jun D, and fra‐2 proteins in the AP‐1:DNA complex by supershift analysis, fra‐2 was no longer seen in adx DER animals. In summary, our study supports potential roles for jun D, jun B, and fra‐1 in the DER regulation of AP‐1 function in the Sencar mouse skin carcinogenesis model.