Novel polyphenol molecule isolated from licorice root (Glycrrhiza glabra) induces apoptosis, G2/M cell cycle arrest, and Bcl-2 phosphorylation in tumor cell lines

Herbal therapies are commonly used by patients with cancer, despite little understanding about biologically active chemical derivatives. We recently demonstrated that the herbal combination PC-SPES, which contains licorice root, had anti-prostate cancer activity attributable to estrogen(s) that produced a chemical castration. A recent study also demonstrated that licorice root alone decreased circulating testosterone in men. Other studies demonstrated antitumor activity of PC-SPES in vitro associated with decreased expression of the anti-apoptotic protein Bcl-2 and in patients independent of chemical castration, suggesting that other mechanisms of antitumor activity exist separate from chemical castration. In the present study, we assessed licorice root extract for effects on Bcl-2 to identify novel cytotoxic derivatives. Licorice root extract induced Bcl-2 phosphorylation as demonstrated by immunoblot and G2/M cell cycle arrest, similarly to clinically used antimicrotubule agents such as paclitaxel. Bioassay-directed fractionations resulted in a biologically active fraction for Bcl-2 phosphorylation. HPLC separation followed by mass spectrometry and NMR identified 6 compounds. Only one molecule was responsible for Bcl-2 phosphorylation; it was identified as 1-(2,4-dihydroxyphenyl)-3-hydroxy-3-(4'-hydroxyphenyl) 1-propanone (beta-hydroxy-DHP). The effect on Bcl-2 was structure specific, because alpha-hydroxy-DHP, 1-(2,4-dihydroxyphenyl)-2-hydroxy-3-(4'-hydroxyphenyl) 1-propanone, in contrast to beta-hydroxy-DHP, was not capable of Bcl-2 phosphorylation. Pure beta-hydroxy-DHP induced Bcl-2 phosphorylation in breast and prostate tumor cells, G2/M cell cycle arrest, apoptosis demonstrated by Annexin V and TUNEL assay, decreased cell viability demonstrated by a tetrazolium (MTT) assay, and altered microtubule structure. Therefore, these data demonstrate that licorice root contains beta-hydroxy-DHP, which induced Bcl-2 phosphorylation, apoptosis, and G2/M cell cycle arrest, in breast and prostate tumor cells, similarly to the action of more complex (MW >800) antimicrotubule agents used clinically.     

Berry phenolic extracts modulate the expression of p21(WAF1) and Bax but not Bcl-2 in HT-29 colon cancer cells

Previous studies have shown that anthocyanin-rich berry extracts inhibit the growth of cancer cells in vitro. The objective of this study was to compare the effects of berry extracts containing different phenolic profiles on cell viability and expression of markers of cell proliferation and apoptosis in human colon cancer HT-29 cells. Berry extracts were prepared with methanol extraction, and contents of the main phenolic compounds were analyzed using HPLC. Anthocyanins were the predominant phenolic compounds in bilberry, black currant, and lingonberry extracts and ellagitannins in cloudberry extract, whereas both were present in raspberry and strawberry extracts. Cells were exposed to 0-60 mg/mL of extracts, and the cell growth inhibition was determined after 24 h. The degree of cell growth inhibition was as follows: bilberry > black currant > cloudberry > lingonberry > raspberry > strawberry. A 14-fold increase in the expression of p21WAF1, an inhibitor of cell proliferation and a member of the cyclin kinase inhibitors, was seen in cells exposed to cloudberry extract compared to other berry treatments (2.7-7-fold increase). The pro-apoptosis marker, Bax, was increased 1.3-fold only in cloudberry- and bilberry-treated cells, whereas the pro-survival marker, Bcl-2, was detected only in control cells. The results demonstrate that berry extracts inhibit cancer cell proliferation mainly via the p21WAF1 pathway. Cloudberry, despite its very low anthocyanin content, was a potent inhibitor of cell proliferation. Therefore, it is concluded that, in addition to anthocyanins, also other phenolic or nonphenolic phytochemicals are responsible for the antiproliferative activity of berries.     

Evaluation of the estrogenic effects of legume extracts containing phytoestrogens

Seven legume extracts containing phytoestrogens were analyzed for estrogenic activity. Methanol extracts were prepared from soybean (Glycine max L.), green bean (Phaseolus vulgaris L.), alfalfa sprout (Medicago sativa L.), mung bean sprout (Vigna radiata L.), kudzu root (Pueraria lobata L.), and red clover blossom and red clover sprout (Trifolium pratense L.). Extracts of kudzu root and red clover blossom showed significant competitive binding to estrogen receptor beta (ERbeta). Estrogenic activity was determined using an estrogen-dependent MCF-7 breast cancer cell proliferation assay. Kudzu root, red clover blossom and sprout, mung bean sprout, and alfalfa sprout extracts displayed increased cell proliferation above levels observed with estradiol. The pure estrogen antagonist, ICI 182,780, suppressed cell proliferation induced by the extracts, suggesting an ER-related signaling pathway was involved. The ER subtype-selective activities of legume extracts were examined using transiently transfected human embryonic kidney (HEK 293) cells. All seven of the extracts exhibited preferential agonist activity toward ERbeta. Using HPLC to collect fractions and MCF-7 cell proliferation, the active components in kudzu root extract were determined to be the isoflavones puerarin, daidzin, genistin, daidzein, and genistein. These results show that several legumes are a source of phytoestrogens with high levels of estrogenic activity.     

Induction of apoptosis by the Anthocyanidins through regulation of Bcl-2 gene and activation of c-Jun N-terminal kinase cascade in hepatoma cells

Anthocyanidins that are reddish pigments widely distributed in fruit and vegetables have been reported to possess antioxidant and anticancer activities. To understand the molecular basis of the putative anticancer activity of anthocyanidins, we investigated the antiproliferation effects of anthocyanidins in human hepatoma cell lines. Delphinidin, cyanidin, and malvidin exhibited strong growth inhibitory effects against human hepatoma HepG(2), but were less effective against Hep3B. According to the appearance of the caspase-3 fragments and stimulated proteolytic cleavage of poly (ADP-ribose) polymerase (PARP) in time-dependent studies, delphinidin induced apoptotic cell death characterized by internucleosomal DNA fragmentation and caused a rapid induction of caspase-3 activity. RT-PCR and Western blot data revealed that delphinidin stimulated an increase in the c-Jun and JNK phosphorylation expression at mRNA and protein levels, respectively. Moreover, delphinidin-induced apoptotic cell death was accompanied by up-regulation of Bax and down-regulation of Bcl-2 protein. Dephinidin-induced DNA fragmentation was blocked by N-acetyl-l-cysteine and catalase, suggesting that the death signaling was triggered by oxidative stress. Our experiments provide evidence that delphinidin is an effective apoptosis inducer in HepG(2) cells through regulation of Bcl-2 family moleculars and activation of c-Jun N-terminal kinase cascade. The results suggest that induction of apoptosis by anthocyanidins is a pivotal mechanism of their cancer chemopreventive functions.     

Induction of apoptosis by hydroxydibenzoylmethane through coordinative modulation of cyclin D3, Bcl-X(L), and Bax,release of cytochrome c,and sequential activation of caspases in human colorectal carcinoma cells

DBM (dibenzoylmethane) is a minor constituent of licorice that has antimutagenic activity. However, its other biological activities are not well-known. The structurally related beta-diketones hydroxydibenzoylmethane (HDB) and hydroxymethyldibenzoylmethane (HMDB) were able to induce apoptosis in colorectal carcinoma COLO 205 cells. Thus, the effect of structurally related beta-diketones on cell viability, DNA fragmentation, and caspase activity was assessed. The potency of these compounds on these features of apoptosis were in the order of HDB > HMDB > DBM in colorectal carcinoma COLO 205 cells. Here, we found that HDB-induced apoptotic cell death was accompanied by upregulation of cyclin D3, Bax, and p21 and down-regulation of Bcl-X(L), while HDB had no effect on the levels of Bcl-2 and Bad protein. These results indicate that HDB allows caspase-activated deoxyribonuclease to enter the nucleus and degrade chromosomal DNA and induces DFF-45 degradation. It is suggested that HDB-induced apoptosis is triggered by the release of cytochrome c into cytosol, procaspase-9 processing, activation of caspase-3 and caspase-2, degradation of PARP, and DNA fragmentation caused by the caspase-activated deoxyribonuclease through the digestion of DFF-45. The induction of apoptosis by HDB may provide a pivotal mechanism for its cancer chemopreventive action.     

Antioxidant and antiproliferative activities of loach ( Misgurnus anguillicaudatus ) peptides prepared by papain digestion

Loach protein hydrolysates (LPH) prepared by papain digestion were fractionated into four fractions, LPH-I (MW > 10 kDa), LPH-II (MW = 5-10 kDa), LPH-III (MW = 3-5 kDa), LPH-IV (MW < 3 kDa), and the in vitro antioxidant and antiproliferative (anticancer) activities of all fractions were determined. LPH-IV showed the lowest IC(50) value (16.9 ± 0.21 mg/mL) for hydroxyl radical scavenging activity and the highest oxygen radical scavenging capacity (ORAC) value (reaching 215 ± 5.9 mM Trolox/100 g loach peptide when the concentration was 60 μg/mL). Compared with other fractions, LPH-IV also exhibited stronger antiproliferative activity for human liver (HepG2), breast (MCF-7), and colon (Caco-2) cancer cell lines in a dose-dependent manner. When the protein concentration was 40 mg/mL, the HepG2 and MCF-7 cell proliferation of LPH-IV reached 7 and 4%, respectively, with no significant difference from those of LPH (8 and 7%, p > 0.05), with significantly less growth than those of LPH-I, LPH-II, and LPH-III, respectively (p < 0.05). The Caco-2 colon cell proliferation of LPH-IV was 12.8- and 8.7-fold smaller than those of LPH-I and LPH-II, respectively (p < 0.05). All of the fractions had a greater ability to inhibit Caco-2 colon cancer cell proliferation than to inhibit HepG2 liver cancer and MCF-7 breast cancer cell proliferation. The ORAC values of most of the fractions correlated (R(2) > 0.86, p < 0.01) with the antiproliferative activity of the three cancer cell lines, suggesting that higher antioxidant activity leads to better antiproliferative activity. However, further mechanistic and human clinical studies of the anticancer activity of loach protein hydrolysate fractions are needed.     

岩藻黄质对人红白血病HEL细胞的凋亡作用及其机制

为探讨岩藻黄质对人红白血病HEL细胞株增殖抑制作用及其诱导凋亡机制,以岩藻黄质处理人红白血病细胞(HEL细胞),采用四甲基偶氮唑蓝(MTT)法检测细胞增殖,流式细胞仪检测细胞凋亡率,分析细胞周期,测定细胞线粒体膜电位,并应用实时荧光定量PCR(RT-qPCR)和Western blot法检测凋亡相关基因及蛋白表达的变化。结果表明,岩藻黄质呈剂量依赖性抑制HEL细胞增殖(P<0.01)。流式细胞术检测显示,岩藻黄质作用24 h后,HEL细胞早期和晚期凋亡的比率极显著增高(P<0.01),G₀/G₁细胞比例增多,S期和G₂/M期细胞比例减少,线粒体膜电位降低;岩藻黄质通过上调促凋亡基因和下调抑凋亡基因的表达引起细胞的凋亡,Bcl-xL蛋白的表达变化不显著(P >0.05),Bcl-2蛋白的表达下调,Caspase-3和Bax蛋白的表达极显著上调(P<0.01)。由此可见,岩藻黄质能诱导HEL细胞凋亡,这为新型抗白血病功能食品的开发及白血病的治疗提供了理论依据。     

Inhibition of cancer cell proliferation in vitro by fruit and berry extracts and correlations with antioxidant levels

The effects of 10 different extracts of fruits and berries on cell proliferation of colon cancer cells HT29 and breast cancer cells MCF-7 were investigated. The fruits and berries used were rosehips, blueberries, black currant, black chokeberries, apple, sea buckthorn, plum, lingonberries, cherries, and raspberries. The extracts decreased the proliferation of both colon cancer cells HT29 and breast cancer cells MCF-7, and the effect was concentration dependent. The inhibition effect for the highest concentration of the extracts varied 2-3-fold among the species, and it was in the ranges of 46-74% (average = 62%) for the HT29 cells and 24-68% (average = 52%) for the MCF-7 cells. There were great differences in the content of the analyzed antioxidants in the extracts. The level of the vitamin C content varied almost 100-fold, and the content of total carotenoids varied almost 150-fold among the species. Also in the composition and content of flavonols, hydroxycinnamic acids, anthocyanins, and phenolics were found great differences among the 10 species. The inhibition of cancer cell proliferation seen in these experiments correlated with levels of some carotenoids and with vitamin C levels, present at levels that can be found in human tissues. The same inhibition of cell proliferation could not be found by ascorbate standard alone. This correlation might indicate a synergistic effect of vitamin C and other substances. In MCF-7 cells, the anthocyanins may contribute to the inhibition of proliferation.     

Estrogenic effects of extracts from cabbage, fermented cabbage, and acidified brussels sprouts on growth and gene expression of estrogen-dependent human breast cancer (MCF-7) cells

Cruciferous vegetable extracts from freeze-dried cabbage (FDC), freeze-dried fermented cabbage (FDS), and acidified Brussels sprouts (ABS) were prepared by exhaustive extraction with ethyl acetate. Estrogenic and antiestrogenic effects of these extracts were analyzed. To identify whether the extracts are potential estrogen receptor (ER) ligands that can act as agonists or antagonists, the binding affinity of extracts for the ER was measured using a competitive radiometric binding assay. The extracts bound with low affinity to the ER, and the relative binding affinity is estradiol > FDS > FDC > ABS. These extracts were evaluated for their estrogenic and antiestrogenic activities in estrogen-dependent human breast cancer (MCF-7) cells using as endpoints proliferation and induction of estrogen-responsive pS2 gene expression, which was analyzed using Northern blot assay. At low concentrations (5-25 ng/mL) all of the extracts reduced 1 nM estradiol-induced MCF-7 cell proliferation. Extracts at 25 ng/mL also inhibited estradiol-induced pS2 mRNA expression. At higher extract concentrations (50 ng/mL-25 microg/mL), however, increased proliferation in MCF-7 cells was observed. Similarly, expression of the pS2 gene was induced by higher extract concentrations (0.25-25 microg/mL). The pure estrogen antagonist, ICI 182,780, suppressed the cell proliferation induced by the extracts as well as by estradiol and also the induction of pS2 expression by the extracts. The ER subtype-selective activities of FDC and FDS were analyzed using a transfection assay in human endometrial adenocarcinoma (HEC-1) cells. FDS acted as an ERalpha-selective agonist while FDC fully activated both ER-alpha and ER-beta. Growth of the ER-negative MDA-231 cells was not affected by the extracts or by estradiol. This study demonstrates that cruciferous vegetable extracts act bifunctionally, like an antiestrogen at low concentrations and an estrogen agonist at high concentrations.     

Mycelia from Antrodia camphorata in Submerged culture induce apoptosis of human hepatoma HepG2 cells possibly through regulation of Fas pathway

The objective of this study was to investigate the antiproliferative effect and the mechanism of the methanol extracts of mycelia (MEM) form Antrodia camphorata in submerged culture toward HepG2 cells. The results showed that MEM-induced cell apoptosis involved up-regulation of Fas and down-regulation of Bcl-2, DR3, DR4, TNFRI, and TNFRII in HepG2 cells, while no changes on the levels of Bax, Bid, Bad, and Bak protein were observed. On the basis of these results, the involvement of the Fas/Fas ligand (FasL) death-receptor pathway, in MEM-induced apoptosis in HepG2 cells, was investigated. The apoptosis inducing activity was significantly enhanced by a Fas activator and inhibited by a Fas antagonist. To know about the effect of MEM on the activation of the apoptotic pathway, the adenovirus transfected with Bcl-2 was infected on HepG2 cells. The data showed that the percentage of apoptotic cells induced by MEM in Bcl-2-infected HepG2 (Bcl-2 overexpression) was not significantly different from that of uninfected HepG2. These results demonstrate that MEM induces HepG2 apoptosis through inhibition of cell growth and up-regulation of Fas/FasL to activate the pathway of caspase-3 and -8 cascades.     

A mixture of trans, trans conjugated linoleic acid induces apoptosis in MCF-7 human breast cancer cells with reciprocal expression of Bax and Bcl-2

The growth inhibitory effect of a mixture of trans, trans conjugated linoleic acid isomers (t, t CLA) was investigated in a human breast cancer cell line, MCF-7, with references to c9, t11 CLA, t10, c12 CLA, and linoleic acid. The t, t CLA treatment effectively induced a cytotoxic effect in a time-dependent (0-6 days) and concentration-dependent (0-40 microM) manner, as compared to the reference and control treatments. The apoptotic parameters were measured on cells treated with 40 microM t, t CLA for 4 days. The occurrence of the characteristic morphological changes and DNA fragmentation confirmed apoptosis. The t, t CLA treatment led to an increase in the level of p53 tumor suppressor protein and Bax protein, but suppressed the expression of Bcl-2 protein. In addition, cytochrome c was released from the mitochondria into the cytosol, and the activation of caspase-3 led to the cleavage of poly(ADP-ribose) polymerase (PARP). Moreover, the composition of the linoleic and arachidonic acids was decreased in cellular membranes. These findings suggest that incorporation of t, t CLA in the membrane induces a mitochondria-mediated apoptosis that can enhance the antiproliferative effect of t, t CLA in MCF-7 cells.     

Growth inhibition and induction of apoptosis in NB4 promyelocytic leukemia cells by trypsin inhibitor from sweet potato storage roots

The objective of this study was to investigate the antiproliferative effect and the mechanism of trypsin inhibitor (TI) from sweet potato [Ipomoea batatas (L.) Lam. 'Tainong 57'] storage roots on NB4 promyelocytic leukemia cells. The results showed that TI inhibited cellular growth of NB4 promyelocytic leukemia cells in a time-dependent and dose-dependent manner, and treatment for 72 h induced a marked inhibition of cellular growth, showing an IC50 of 57.1 +/- 8.26 microg/mL. TI caused cell cycle arrest at the G1 phase as determined by flow cytometric analysis and apoptosis as shown by DNA laddering. TI-induced cell apoptosis involved p53, Bcl-2, Bax, and cytochrome c protein in NB4 cells. P53 and Bax proteins were accumulated, and antiapoptotic molecule Bcl-2 was decreased in the tested cells in a time-dependent manner during TI treatment. TI also induced a substantial release of cytochrome c from the mitochondria into the cytosol. Hence, TI induced apoptosis in NB4 cells through a mitochondria-dependent pathway, which was associated with the activation of caspase-3 and -8. These results demonstrated that TI induces NB4 cell apoptosis through the inhibition of cell growth and the activation of the pathway of caspase-3 and -8 cascades.     

Evaluation of the phytoestrogenic activity of Cyclopia genistoides (honeybush) methanol extracts and relevant polyphenols

Unfermented C. genistoides methanol extracts of different harvestings and selected polyphenols were evaluated for phytoestrogenic activity by comparing binding to both ER subtypes, transactivation of an ERE-containing promoter reporter, proliferation of MCF-7-BUS and MDA-MB-231 breast cancer cells, and binding to SHBG. The extracts from one harvesting of C. genistoides (P104) bound to both ER subtypes. All extracts transactivated ERE-containing promoter reporters via ERbeta but not via ERalpha. All extracts, except P122, caused proliferation of the estrogen-sensitive MCF-7-BUS cells. Proliferation of MCF-7-BUS cells was ER-dependent as ICI 182,780 reversed proliferation. Physiologically more relevant, extracts antagonized E2-induced MCF-7-BUS cell proliferation. Furthermore, all extracts, except P122, induced proliferation of the estrogen-insensitive MDA-MB-231 cells, suggesting that the extracts are able to induce ER-dependent and ER-independent cell proliferation. Binding to SHBG by extracts was also demonstrated. These results clearly show that C. genistoides methanol extracts display phytoestrogenic activity and act predominantly via ERbeta. HPLC and LC-MS analysis, however, suggests that the observed phytoestrogenic activity cannot be ascribed to polyphenols known to be present in other Cyclopia species.     

Tea polyphenol (-)-epigallocatechin 3-gallate suppresses heregulin-beta1-induced fatty acid synthase expression in human breast cancer cells by inhibiting phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase cascade signaling

Tumor-associated fatty acid synthase (FAS) is implicated in tumorigenesis and connected to HER2 (human epidermal growth factor receptor 2) by systemic analyses. Suppression of FAS in cancer cells may lead to growth inhibition and cell apoptosis. Our previous study demonstrated that (-)-epigallocatechin 3-gallate (EGCG), the green tea catechin, could down-regulate FAS expression by suppressing EGFR (epidermal growth factor receptor) signaling and downstream phosphatidylinositol 3-kinase (PI3K)/Akt activation in the MCF-7 breast cancer cell line. Herein, we examined the effects of EGCG on FAS expression modulated by another member of the erbB family, that is, HER2 or HER3. We identified that heregulin-beta1 (HRG-beta1), a HER3 ligand, stimulated dose-dependent FAS expression in breast cancer cell lines MCF-7 and AU565, but not MDA-MB-453. The time-dependent increase in FAS expression after HRG-beta1 stimulation was also observed in MCF-7 cells, and this up-regulation was de novo RNA synthesis dependent. Treatment of MCF-7 cells with EGCG markedly inhibited HRG-beta1-dependent induction of mRNA and protein of FAS. EGCG also decreased the phosphorylation of Akt and extracellular signal-regulated kinase 1/2 that were demonstrated as selected downstream HRG-beta1-responsive kinases required for FAS expression using dominant-negative Akt, PI3K inhibitors (LY294002 and wortmannin), or MEK inhibitor (PD98059). FAS induction by HRG-beta1 was also blocked by AG825, a selective HER2 inhibitor, and by genistein, a selective tyrosine kinase inhibitor, indicating the formation of a heterodimer between HER2 and HER3, and their tyrosine kinase activities are essential for HRG-beta1-mediated elevation of FAS. Additionally, growth inhibition of HRG-beta1-treated cells was parallel to suppression of FAS by EGCG. Taken together, these findings extend our previous study to indicate that EGCG may be useful in the chemoprevention of breast carcinoma in which FAS overexpression results from HER2 or/and HER3 signaling.     

Acacetin induces apoptosis in human gastric carcinoma cells accompanied by activation of caspase cascades and production of reactive oxygen species

Acacetin (5,7-dihydrocy-4'-methoxy flavone), which is a flavonoid compound, possesses anti-peroxidative and anti-inflammatory effects. The effects of acacetin on cell viability in human gastric carcinoma AGS cells were investigated. This study demonstrated that acacetin was able to inhibit cell proliferation and induce apoptosis in a concentration- and time-dependent manner. Acacetin-induced cell death was characterized with changes in nuclear morphology, DNA fragmentation, and cell morphology. The molecular mechanism of acacetin-induced apoptosis was also investigated. Treatment with acacetin caused induction of caspase-3 activity in a time-dependent manner, but not caspase-1 activity, and induced the degradation of DNA fragmentation factor (DFF-45) and poly(ADP-riobse) polymerase. Cell death was completely prevented by a pancaspase inhibitor, Z-Val-Ala-Asp-fluoromethyl ketone. Furthermore, treatment with acacetin caused a rapid loss of mitochondrial transmembrane potential, stimulation of reactive oxygen species (ROS), release of mitochondrial cytochrome c into cytosol, and subsequent induction of procaspase-9 processing. Antioxidants such as N-acetylcysteine and catalase, but not superoxide dismutase, allopurinol, or pyrrolidine dithiocarbamate, significantly inhibited acacetin-induced cell death. In addition, it was found that acacetin promoted the up-regulation of Fas and FasL prior to the processing and activation of pro-caspase-8 and cleavage of Bid, suggesting the involvement of a Fas-mediated pathway in acacetin-induced apoptosis. On the other hand, the results showed that acacetin-induced apoptosis was accompanied by up-regulation of Bax and p53, down-regulation of Bcl-2, and cleavage of Bad. Taken together, these results suggest that ROS production and a certain intimate link might exist between receptor- and mitochondria-mediated death signalings that committed to acacetin-induced apoptosis in AGS cells. The induction of apoptosis by acacetin may provide a pivotal mechanism for its cancer chemopreventive action.     

Blackberry, black raspberry, blueberry, cranberry, red raspberry, and strawberry extracts inhibit growth and stimulate apoptosis of human cancer cells in vitro

Berry fruits are widely consumed in our diet and have attracted much attention due to their potential human health benefits. Berries contain a diverse range of phytochemicals with biological properties such as antioxidant, anticancer, anti-neurodegerative, and anti-inflammatory activities. In the current study, extracts of six popularly consumed berries--blackberry, black raspberry, blueberry, cranberry, red raspberry and strawberry--were evaluated for their phenolic constituents using high performance liquid chromatography with ultraviolet (HPLC-UV) and electrospray ionization mass spectrometry (LC-ESI-MS) detection. The major classes of berry phenolics were anthocyanins, flavonols, flavanols, ellagitannins, gallotannins, proanthocyanidins, and phenolic acids. The berry extracts were evaluated for their ability to inhibit the growth of human oral (KB, CAL-27), breast (MCF-7), colon (HT-29, HCT116), and prostate (LNCaP) tumor cell lines at concentrations ranging from 25 to 200 micro g/mL. With increasing concentration of berry extract, increasing inhibition of cell proliferation in all of the cell lines were observed, with different degrees of potency between cell lines. The berry extracts were also evaluated for their ability to stimulate apoptosis of the COX-2 expressing colon cancer cell line, HT-29. Black raspberry and strawberry extracts showed the most significant pro-apoptotic effects against this cell line. The data provided by the current study and from other laboratories warrants further investigation into the chemopreventive and chemotherapeutic effects of berries using in vivo models.     

Mechanisms of cancer chemoprevention by hop bitter acids (beer aroma) through induction of apoptosis mediated by Fas and caspase cascades

The bitter acids of hops (Humulus lupulus L.) mainly consist of alpha-acids, beta-acids, and their oxidation products that contribute the unique aroma of the beer beverage. Hop bitter acids displayed a strong growth inhibitory effect against human leukemia HL-60 cells, with an estimated IC(50) value of 8.67 microg/mL, but were less effective against human histolytic lymphoma U937 cells. Induction of apoptosis was confirmed in HL-60 cells by DNA fragmentation and the appearance of a sub-G1 DNA peak, which were preceded by dissipation of mitochondrial membrane potential, cytochrome c release, and subsequent induction of pro-caspase-9 and -3 processing. Cleavages of PARP and DFF-45 were accompanied with activation of caspase-9 and -3 triggered by hop bitter acids in HL-60 cells. The change in the expression of Bcl-2, Bcl-X(L), and Bax in response to hop bitter acids was studied, and the Bcl-2 protein level slightly decreased; however, the Bcl-X(L) protein level was obviously decreased, whereas the Bax protein level was dramatically increased, indicating that the control of Bcl-2 family proteins by hop bitter acids might participate in the disruption of mitochondrial integrity. In addition, the results showed that hop bitter acids promoted the up-regulation of Fas and FasL prior to the processing and activation of pro-caspase-8 and cleavage of Bid, suggesting the involvement of a Fas-mediated pathway in hop bitter acids-induced cells. Taken together, these findings suggest that a certain intimate link might exist between receptor- and mitochondria-mediated death signalings that committed to cell death induced by hop bitter acids. The induction of apoptosis by hop bitter acids may offer a pivotal mechanism for their chemopreventive action.     

Effect of extrusion on isoflavone content and antiproliferative bioactivity of soy/corn mixtures

The present studies were conducted to determine changes in the quantities of select isoflavones and in the bioactivity (ability to inhibit proliferation of human breast cancer cell lines) of extracts from blends of soy protein and cornmeal during extrusion processing. The extrusion of samples resulted in an average 24% decrease in the concentration of total isoflavones for all samples. Although the amounts of specific genistein-derived and daidzein-derived forms changed following extrusion, the content of the aglycones genistein and daidzein per g sample generally did not change. The extrusion of samples generally resulted in decreased antiproliferative action toward breast cancer cells, although antiproliferative activity was not eliminated. Therefore, extrusion of soy protein/cornmeal-containing foods are likely to retain a considerable portion of their isoflavone content and some of the health benefits associated with soy.     

Structure-activity relationships of tea compounds against human cancer cells

The content of the biologically active amino acid theanine in 15 commercial black, green, specialty, and herbal tea leaves was determined as the 2,4-dinitrophenyltheanine derivative (DNP-theanine) by a validated HPLC method. To define relative anticarcinogenic potencies of tea compounds and teas, nine green tea catechins, three black tea theaflavins, and theanine as well as aqueous and 80% ethanol/water extracts of the same tea leaves were evaluated for their ability to induce cell death in human cancer and normal cells using a tetrazolium microculture (MTT) assay. Compared to untreated controls, most catechins, theaflavins, theanine, and all tea extracts reduced the numbers of the following human cancer cell lines: breast (MCF-7), colon (HT-29), hepatoma (liver) (HepG2), and prostate (PC-3) as well as normal human liver cells (Chang). The growth of normal human lung (HEL299) cells was not inhibited. The destruction of cancer cells was also observed visually by reverse phase microscopy. Statistical analysis of the data showed that (a) the anticarcinogenic effects of tea compounds and of tea leaf extracts varied widely and were concentration dependent over the ranges from 50 to 400 microg/mL of tea compound and from 50 to 400 microg/g of tea solids; (b) the different cancer cells varied in their susceptibilities to destruction; (c) 80% ethanol/water extracts with higher levels of flavonoids determined by HPLC were in most cases more active than the corresponding water extracts; and (d) flavonoid levels of the teas did not directly correlate with anticarcinogenic activities. The findings extend related observations on the anticarcinogenic potential of tea ingredients and suggest that consumers may benefit more by drinking both green and black teas.