Lai K. Leung

Stability of tea theaflavins and catechins

Abstract Green tea catechins (GTC) and theaflavins (TF) possess a variety of biological activities. The present study focused on stability of GTC and TF in various solutions and drinks. It was observed that both GTC and TF were vulnerable to degradation caused by elevation of temperature and pH of incubation media. In general, GTC was more stable than TF. When boiled in water, four GTCs showed similar rates of degradation, but in sodium phosphate buffer (pH7.4) at room temperature, four GTCs demonstrated varying stability, with epigallocatechin gallate (EGCG) and epigallocatechin (EGC) being completely degraded in 6 h of incubation, while epicatechin (EC) and epicatechin gallate (ECG) were only degraded by less than 35%. Four TFs also demonstrated varying stability, with theaflavin-3,3′-digallate (TF3) and theaflavin-3′-gallate- B (TF2B) in general being more stable than theaflavin-1 (TF1) and theaflavin-3-gallate-A (TF2A) in either boiling water or alkaline sodium phosphate buffer. When incubated in various solutions and soft drinks, both GTC and TF had poor long-term stability and decayed by at least 50% during the first month of storage at room temperature.

Difference in flavonoid and isoflavone profile between soybean and soy leaf

The present study was to compare the flavonoid profile between soybean and soy leaves. Soybean was most abundant in malonyl-genistin followed by malonyl-daidzin, genistin, daidzin, genistein and daidzein in a decreasing order. In contrast, soy leaves contained only trace amounts of malonyl-genistin and genistin, but they had the six unknown flavonoids that were absent in soybean. The six unknown compounds were isolated by using various chromatographic techniques and the structures were identified by studying their varying spectra of ultraviolet (UV), infrared (IR), Mass, 1H-NMR and 13C-NMR. It was found that the six unknown compounds were all kaempferol glycosides namely kaempferol-3-O-alpha-L-rhamnopyranosyl (1-->2)-beta-D-glucopyranosyl (1-->6)-beta-D-galactopyranoside, kaempferol-3-O-(2,6-di-O-alpha-rhamnopyranosyl)-beta-galactopyranoside, kaempferol-3-O-alpha-L-rhamnopyranosyl (1-->6)-beta-D-galactopyranoside, kaempferol-3-O-digalactopyranoside, kaempferol-3-O-diglucopyranoside and kaempferol-3-O-rutinoside. It was concluded that the flavonoids in soy leaves were mainly kaempferol glycosides, whereas those in soybean were mainly isoflavone glycosides and derivatives.

Differential effects of chemotherapeutic agents on the Bcl-2/Bax apoptosis pathway in human breast cancer cell line MCF-7

The present study explored the effects of three commonly used chemotherapeutic agents on the Bcl‐2/Bax apoptosis pathway and the interaction of these chemotherapeutic drugs with the estradiol‐mediated regulation of this pathway. Our results showed that: (1) Treatment of MCF‐7 cells with Adriamycin resulted in time‐ and concentration‐dependent decreases in Bcl‐2 and increases in Bax mRNA and protein levels. (2) Camptothecin elicited similar trends on Bcl‐2 and Bax as Adriamycin, while etoposide, at 50–100 fold (1–5 μM) the effective concentration of Adriamycin and camptothecin, only resulted in an increase in Bax mRNA levels. (3) Adriamycin and camptothecin, but not etoposide, were effective in suppressing estradiol‐stimulated increases in Bcl‐2 mRNA levels. Our study provides evidence that the Bcl‐2/Bax apoptosis pathway may be differentially regulated by chemotherapeutic agents. In addition, interaction between these agents and estradiol on the Bcl‐2/Bax apoptosis pathway may also exist.

A potential protective mechanism of soya isoflavones against 7, 12-Dimethylbenz [a] anthracene tumour initiation

Epidemiological studies indicate that Asian women have a lower breast cancer incidence compared with their counterparts in the West, and the difference has been related to soya consumption. Animal studies have suggested that soya may prevent dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis in the breast. In the present study a cell culture model was developed to address the effect of soya isoflavones on the DMBA-induced DNA damage. DMBA is metabolized into a DNA-attacking moiety by two phase I cytochrome P450 (CYP) enzymes CYP1A1 and CYP1B1. DNA mutation caused by this genotoxic agent is a crucial step in cancer initiation. Substances that interfere with the CYP1 enzyme activities can affect the initiation. In the present study, genistein was found to be an effective inhibitor of recombinant human CYP1A1 and CYP1B1 with Ki of 15.35 and 0.68 micromol/l. The other soya isoflavone daidzein, on the other hand, did not demonstrate any significant inhibition of the enzyme activities. At the transcriptional level, DMBA induced the CYP1 enzyme expressions by stimulating the xenobiotic response element (XRE)-dependent transactivation pathway. When genistein (25 micromol/l) was co-administered with DMBA, the XRE-Luc activity the CYP1 mRNA abundances were significantly suppressed. The present study illustrated that the soya isoflavone genistein, but not daidzein, protected against DMBA genotoxicity.

The red clover ( Trifolium pratense ) isoflavone biochanin A inhibits aromatase activity and expression

Biochanin A is an isoflavone isolated from red clover (Trifolium pratense), and is a commercially available nutraceutical for women suffering from postmenopausal symptoms. Isoflavones resemble the structure of oestrogen, and display agonistic and antagonistic interactions with the oestrogen receptor. Overexposure of oestrogen is a major contributing factor in the development of breast cancer, and cytochrome P450 (CYP) 19 enzyme, or aromatase, catalyses the reaction converting androgen to oestrogen. In the present study the effect of biochanin A on the gene regulation and enzyme activity of aromatase was investigated. By assaying MCF-7 cells stably transfected with CYP19, biochanin A inhibited aromatase activity and hampered cell growth attributing to the enzyme activity. In addition, 25 microm-biochanin A significantly reduced CYP19 mRNA abundance in the oestrogen receptor-negative breast cancer cells SK-BR-3. The transcriptional control of the CYP19 gene is exon-specific, and promoter regions I.3 and II have been shown to be responsible for CYP19 expression in SK-BR-3 cells. Luciferase reporter gene assays also revealed that biochanin A could repress the transcriptional control dictated by the promoter regulation. Interestingly, genistein did not inhibit aromatase but it might down regulate promoter I.3 and II transactivation. Since genistein is a major metabolite of biochanin A, it might contribute to biochanin As suppressive effect on CYP19 expression. The present study illustrated that biochanin A inhibited CYP19 activity and gene expression.

Bisphenol A downregulates CYP19 transcription in JEG-3 cells

Bisphenol A is an industrial contaminant and is considered to be an endocrine disruptor; its estrogenic property has been reported in many studies. Because of its ubiquitous existence in our environment, bisphenol A has drawn much discussion on its safety issues. Estrogen is important in the maintenance of human pregnancy, and the placenta is the major site of synthesis during this period of time. Aromatase or CYP19 catalyses the conversion of estrogen from its precursor, and is highly expressed in placental cells. In the present study, we examined the ability of the toxicant in suppressing the transcription of CYP19 in JEG-3 cells. Cells treated with bisphenol A displayed a reduced aromatase activity. Real-time PCR analysis indicated that 5muM of the compound significantly reduced the mRNA expression in these cells. As the transcriptional activity of CYP19 gene is controlled by the proximal promoter region of exon I.1 in placental cells, the promoter activity of this gene fragment and exon-I.1-spliced mRNA abundance were also evaluated. Both results indicated that bisphenol A repressed the transcriptional control of promoter I.1. The present study showed that bisphenol A potentially reduced estrogen synthesis by downregulating CYP of placental cells. This information could be useful for evaluating the exposure limit of bisphenol A.

The red clover (Trifolium pratense) isoflavone biochanin A modulates the biotransformation pathways of 7,12-dimethylbenz[a]anthracene

Several flavonoids have shown their anti-carcinogenic effects in various models. The soyabean isoflavone genistein was demonstrated earlier in our laboratory to be an effective inhibitor of dimethylbenz[a]anthracene (DMBA)-induced DNA damage in MCF-7 cells by curbing cytochrome P450 (CYP) 1 enzymes. The red clover (Trifolium pratense) isoflavone biochanin A is a methylated derivative of genistein, and its anti-mutagenic effect in bacterial cells has been shown previously. Because of its protection against chemical carcinogenesis in an animal model, biochanin A was selected for testing in our established MCF-7 cell system. From the results obtained in the semi-quantitative reverse transcription-polymerase chain reaction and xenobiotic response element (XRE)-luciferase reporter assays, biochanin A could reduce xenobiotic-induced CYP1A1 and -1B1 mRNA abundances through the interference of XRE-dependent transactivation. Enzyme kinetic studies also indicated that biochanin A inhibited both CYP1A1 and -1B1 enzymes with inhibition constant (Ki) values 4.00 and 0.59 microm respectively. Since the biotransformation of DMBA was dependent on CYP1 enzyme activities, biochanin A was able to decrease the DMBA-DNA lesions. The present study illustrated that the red clover isoflavone could protect against polycylic aromatic hydrocarbon-induced DNA damage.

Genistein-induced apoptosis in MCF-7 cells involves changes in Bak and Bcl-x without evidence of anti-oestrogenic effects

South-east Asian women have a lower rate of breast cancer compared with their counterparts in western countries and the difference in soyabean consumption has been claimed to be a major contributing factor. Genistein is the most studied phytochemical in the soyabean. An anti-oestrogenic effect is believed to play a crucial part in its chemopreventive mechanism. In the present study, we expressed oestrogen receptor (OR) in an OR-negative cell line, HepG2, to investigate the pro- and anti-oestrogenic effect of genistein on the OR transcriptional activity. Genistein by itself had an estimated concentration that induced 50 % of the maximum response (EC50) of 2.5 microm for the binding to OR-alpha. In these experiments, genistein concentration as high as 50 microm could not reduce the oestrogen response element-driven luciferase activities initiated by oestradiol. Instead, genistein potentiated the OR transactivational activity while cell death was detected. On the other hand, an increased Bak and a reduced Bcl-x(L) was observed at 50 microm-genistein by Western analysis. The combined effect of these two proteins could be important in the apoptotic process. Since plasma genistein >50 microm has never been documented following consuming of soyabean or soyabean products, the present study does not support the notion that dietary soyabean exerts its chemopreventive effect through antagonizing OR.

The soy isoflavone genistein induces estrogen synthesis in an extragonadal pathway.

Genistein is a phytoestrogen isolated from soyabean, and is a potential nutraceutical gearing for women suffering from perimenopausal symptoms. Because of its differential binding affinity to estrogen receptor (ER) isoforms, genistein is described as a selective estrogen receptor modulator (SERM). The ligand-receptor interaction is established, but the potential confounding factors have not been fully addressed. Alteration in estrogen metabolism is an important issue when determining the downstream effect of ER. Aromatase or CYP19 catalyzes the rate-limiting reaction of estrogen synthesis, and is highly expressed in the ovary. This organ is the source of estrogen in females. After menopause the ovaries cease to produce the hormone, and localized estrogen synthesis in extragonadal tissues could become physiologically significant. In the present study, effect of genistein on CYP19 regulation was investigated in the hepatic cells HepG2. The phytoestrogen induced aromatase activity in the cells. Increased mRNA expression with concurrent elevation in the usage of promoters I.3/II was also demonstrated. Luciferase reporter gene assays verified the transcriptional control dictated by the specific promoters under genistein treatment. Several protein kinases were examined, and PKC?, P38, ERK-1/2 appeared to be activated. Subsequent inhibition and expression experiments demonstrated the involvement of these kinases. The transcriptional factor CREB was ultimately activated in the gene regulation. The present study illustrated an extragonadal pathway by which genistein might increase estrogen synthesis.

Dietary flavones and flavonones display differential effects on aromatase (CYP19) transcription in the breast cancer cells MCF-7

Aromatase or cytochrome P450 (CYP19) enzyme catalyzes the rate-determining reaction in estrogen synthesis. Inhibiting aromatase is a major strategy in treating breast cancer patients. However, suppression on the transcriptional activity may be equally important in controlling aromatase. Dietary flavones and flavonones have been previously demonstrated to be the most potent aromatase-inhibitory flavonoids. In the present study we examined their effects on the transcription regulation of CYP19 in MCF-7 cells. Real-time PCR results indicated that luteolin suppressed CYP19 mRNA expression while hesperetin increased it. Reporter gene assays were employed to look into the transactivity of CYP19 driven by promoters I.3 and II, and the result was consistent with the observation in mRNA expression. Further investigation using truncation reporter gene and electrophoretic mobility shift assays suggested that luteolin and hesperetin differentially influenced AP-1- and C/EBP-binding on the CYP19 promoter. Western blot analysis indicated that signaling transduction pathways involving JNK and ERK could be the underlying mechanisms for their actions. The present study showed that dietary flavones and flavonones might differentially regulate aromatase transcription in breast cells in addition to the inhibition at the enzyme level.