Chromosome-Scale Genome Assembly and Characterization of Top-Quality Japanese Green Tea Cultivar 'Seimei'.

Japanese green tea, an essential beverage in Japanese culture, is characterized by the initial steaming of freshly harvested leaves during production. This process efficiently inactivates endogenous enzymes such as polyphenol oxidases, resulting in the production of sencha, gyokuro and matcha that preserves the vibrant green color of young leaves. Although genome sequences of several tea cultivars and germplasms have been published, no reference genome sequences are available for Japanese green tea cultivars. Here, we constructed a reference genome sequence of the cultivar 'Seimei', which is used to produce high-quality Japanese green tea. Using the PacBio HiFi and Hi-C technologies for chromosome-scale genome assembly, we obtained 15 chromosome sequences with a total genome size of 3.1 Gb and an N50 of 214.9 Mb. By analyzing the genomic diversity of 23 Japanese tea cultivars and lines, including the leading green tea cultivars 'Yabukita' and 'Saemidori', it was revealed that several candidate genes could be related to the characteristics of Japanese green tea. The reference genome of 'Seimei' and information on genomic diversity of Japanese green tea cultivars should provide crucial information for effective breeding of such cultivars in the future.

Influence of steaming duration, chlorophyll-a and -b content and ratio, and pH on the color of green tea processed from multiple tea (Camellia sinensis L.) cultivars.

BACKGROUND: The color of green tea is an important quality indicator. In recent years, shading of tea (Camellia sinensis L.) plants has been widely adopted for green tea production to enhance its green color and umami taste. In this study, we identified factors that influence green tea color by (i) examining variation in the chlorophyll content of fresh new tea shoots among cultivars, cropping seasons, and the degree of shading, (ii) investigating the rate of conversion of chlorophyll to pheophytin during the tea manufacturing process, specifically with steaming duration, and (iii) analyzing the effects of the new tea shoot properties and the steaming process on colorimetric values of the steamed new tea shoots. RESULTS: Multiple regression analysis revealed that three factors contributed to the rate of conversion of each chlorophyll type to pheophytin in steamed new tea shoots (ranked by importance): steaming duration > each chlorophyll type (chlorophyll-a and chlorophyll-b) content of fresh new tea shoots > pH. The colorimetric hue angle (h) value of steamed new tea shoots was influenced by four factors (ranked by importance): steaming duration > total chlorophyll (chlorophyll-a + chlorophyll-b) content in fresh new tea shoots > pH > chlorophyll-a/chlorophyll-b ratio in fresh new tea shoots. CONCLUSION: Differences in the color of new tea shoots can be explained by the aforementioned four factors. The findings will be useful for cultivar selection, and determining the appropriate degree of shading and steaming duration, to produce high-quality green teas with a good appearance. © 2024 Society of Chemical Industry.

Circulating miRNA profiles in mice plasma following flavonoid intake.

BACKGROUND: Polyphenols, including flavonoids, have been the focus of numerous studies that have revealed diverse health benefits. MicroRNAs (miRNAs) constitute a class of small non-coding RNAs that function as posttranscriptional regulators of gene expression. miRNAs can be detected in the blood and these so-called circulating miRNAs are potential biomarkers of various diseases. This study aimed to explore circulating miRNAs in plasma as a means to predict the biological effects of functional food ingredients. METHODS AND RESULTS: We used miRNA microarray analysis to compare plasma miRNA levels in mice orally administered three flavonoids (daidzein, quercetin, and delphinidin). Several miRNAs were differentially expressed in plasma from mice in each treatment group compared with the vehicle-treated group. The plasma levels of miR-25-5p, miR-146b-5p, and miR-501-3p were increased in the flavonoid-treated and the plasma levels of miR-148b-3p, miR-669e-5p, and miR-3962 were decreased. CONCLUSIONS: Our findings suggested that flavonoids alter miRNA expression in plasma and identified promising plasma miRNAs for assessing the functionality of flavonoids.

Green Tea Polyphenol EGCG Upregulates Tollip Expression by Suppressing Elf-1 Expression.

TLR signaling is critical to innate immune system regulation; however, aberrant TLR signaling is involved in several diseases, including insulin resistance, Alzheimer's disease, and tumor metastasis. Moreover, a recent study found that TLR-4 signaling pathway inhibition might be a target for the suppression of chronic inflammatory disorders. In this article, we show that the green tea polyphenol epigallocatechin-3-O-gallate (EGCG) increases the expression of Toll interacting protein, a strong inhibitor of TLR4 signaling, by suppressing the expression of E74-like ETS transcription factor 1 (Elf-1). A mechanistic study revealed that EGCG suppressed Elf-1 expression via protein phosphatase 2A/cyclic GMP (cGMP)-dependent mechanisms. We also confirmed that orally administered EGCG and a cGMP inducer upregulated Toll interacting protein expression, increased intracellular levels of cGMP in macrophages, and suppressed Elf-1 expression. These data support EGCG and a cGMP inducer as potential candidate suppressors of TLR4 signaling.

Green Tea Polyphenol Epigallocatechin-3-gallate Suppresses Toll-like Receptor 4 Expression via Up-regulation of E3 Ubiquitin-protein Ligase RNF216.

Toll-like receptor 4 (TLR4) plays an essential role in innate immunity through inflammatory cytokine induction. Recent studies demonstrated that the abnormal activation of TLR4 has a pivotal role in obesity-induced inflammation, which is associated with several diseases, including hyperinsulinemia, hypertriglyceridemia, and cardiovascular disease. Here we demonstrate that (-)-epigallocatechin-3-O-gallate, a natural agonist of the 67-kDa laminin receptor (67LR), suppressed TLR4 expression through E3 ubiquitin-protein ring finger protein 216 (RNF216) up-regulation. Our data indicate cyclic GMP mediates 67LR agonist-dependent RNF216 up-regulation. Moreover, we show that the highly absorbent 67LR agonist (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3″Me) significantly attenuated TLR4 expression in the adipose tissue. EGCG3″Me completely inhibited the high-fat/high-sucrose (HF/HS)-induced up-regulation of tumor necrosis factor α in adipose tissue and serum monocyte chemoattractant protein-1 increase. Furthermore, this agonist intake prevented HF/HS-induced hyperinsulinemia and hypertriglyceridemia. Taken together, 67LR presents an attractive target for the relief of obesity-induced inflammation.

The FOXO3/PGC-1ß signaling axis is essential for cancer stem cell properties of pancreatic ductal adenocarcinoma.

In 95% of patients with pancreatic ductal adenocarcinoma, recurrence is observed following chemotherapy. Findings from several studies have indicated that cancer stem cells (CSCs) are resistant to anticancer agents and may be involved in cancer recurrence and metastasis. The CD44 protein is a major CSC marker, and CD44 also plays an indispensable role in the CSC properties in several cancers, including pancreatic cancer; however, no clinical approach exists to inhibit CD44 activity. Here, we have performed knock-in/knockdown experiments, and we demonstrate that the forkhead box O3 (FOXO3)/liver kinase B1 (LKB1)/AMP-activated protein kinase/peroxisome proliferator-activated receptor-γ co-activator-1ß (PGC-1ß)/pyruvate dehydrogenase-A1 pathway is essential for CD44 expression and CSC properties. We observed that patients exhibiting high pyruvate dehydrogenase-A1 expression have a poor prognosis. Systemic PGC-1ß knock-out mice are fertile and viable and do not exhibit an overt phenotype under normal conditions. This suggests that cGMP induction and PGC-1ß inhibition represent potential strategies for treating patients with pancreatic ductal adenocarcinoma.

γ-Tocotrienol upregulates aryl hydrocarbon receptor expression and enhances the anticancer effect of baicalein.

Previous studies have identified biomolecules that mediate the physiological actions of food factors, such as amino acids, vitamins, fatty acids, minerals, plant polyphenols, and lactobacilli, suggesting that our bodies are equipped with an innate system that senses which food factors are required to maintain our health. However, the effects of environmental factors on food factor sensing (FFS) remains largely unknown. Tocotorienols (T3s), which belongs to the vitamin E family, possess several physiological functions, including cholesterol lowering and neuroprotective effects. Here, we investigated the effects of naturally abundant γ-T3 on FFS-related gene expressions in melanoma using a DNA chip. Our results showed that γ-T3 increased the expression level of aryl hydrocarbon receptor (AhR), a sensing molecule to plant polyphenol baicalein. The co-treatment with γ-T3 and baicalein enhanced the anti-proliferative activity of baicalein, accompanied by the downstream events of AhR-activation induced by baicalein. These data suggest that γ-T3 upregulates AhR expression and enhances its sensitivity to baicalein.

Delphinidin prevents disuse muscle atrophy and reduces stress-related gene expression.

Delphinidin is a member of the anthocyanidin class of plant pigments. We examined the effects of delphinidin on muscle atrophy. Oral administration of delphinidin suppressed the muscle weight loss induced by mechanical unloading. Microarray analysis showed that delphinidin suppresses the upregulation of oxidative stress-related gene expression, including the expression of Cbl-b. Thus, delphinidin may prevent unloading-mediated muscle atrophy.

67-kDa laminin receptor-dependent protein phosphatase 2A (PP2A) activation elicits melanoma-specific antitumor activity overcoming drug resistance.

The Ras/Raf/MEK/ERK pathway has been identified as a major, druggable regulator of melanoma. Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, resulting in constitutive melanoma hyperproliferation. A selective BRAF inhibitor showed remarkable clinical activity in patients with mutated BRAF. Unfortunately, most patients acquire resistance to the BRAF inhibitor, highlighting the urgent need for new melanoma treatment strategies. Green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) inhibits cell proliferation independently of BRAF inhibitor sensitivity, suggesting that increased understanding of the anti-melanoma activity of EGCG may provide a novel therapeutic target. Here, by performing functional genetic screening, we identified protein phosphatase 2A (PP2A) as a critical factor in the suppression of melanoma cell proliferation. We demonstrated that tumor-overexpressed 67-kDa laminin receptor (67LR) activates PP2A through adenylate cyclase/cAMP pathway eliciting inhibitions of oncoproteins and activation of tumor suppressor Merlin. Activating 67LR/PP2A pathway leading to melanoma-specific mTOR inhibition shows strong synergy with the BRAF inhibitor PLX4720 in the drug-resistant melanoma. Moreover, SET, a potent inhibitor of PP2A, is overexpressed on malignant melanoma. Silencing of SET enhances 67LR/PP2A signaling. Collectively, activation of 67LR/PP2A signaling may thus be a novel rational strategy for melanoma-specific treatment.

IL-4 receptor α in non-lipid rafts is the target molecule of strictinin in inhibiting STAT6 activation.

Strictinin has been shown to suppress interleukin (IL)-4-induced signal transducer and activator of transcription (STAT)-6 phosphorylation, which is a critical event for IgE class switching. However, it is unclear how strictinin inhibits STAT6 activation. Strictinin inhibited STAT6 phosphorylation by suppressing IL-4 receptor α (IL-4Rα) activation. Strictinin was bound to the cell surface and only localized in non-lipid raft fraction of the cells where IL-4Rα was also located. In addition, strictinin directly bound to IL-4Rα and inhibited binding of IL-4 to IL-4Rα. These results suggest that IL-4Rα locating in non-lipid raft region is a target molecule for strictinin in inhibiting STAT6 activation.

Green tea polyphenol EGCG induces lipid-raft clustering and apoptotic cell death by activating protein kinase Cδ and acid sphingomyelinase through a 67 kDa laminin receptor in multiple myeloma cells.

EGCG [(-)-epigallocatechin-3-O-gallate], the major polyphenol of green tea, has cancer chemopreventive and chemotherapeutic activities. EGCG selectively inhibits cell growth and induces apoptosis in cancer cells without adversely affecting normal cells; however, the underlying molecular mechanism in vivo is unclear. In the present study, we show that EGCG-induced apoptotic activity is attributed to a lipid-raft clustering mediated through 67LR (67 kDa laminin receptor) that is significantly elevated in MM (multiple myeloma) cells relative to normal peripheral blood mononuclear cells, and that aSMase (acid sphingomyelinase) is critical for the lipid-raft clustering and the apoptotic cell death induced by EGCG. We also found that EGCG induces aSMase translocation to the plasma membrane and PKCδ (protein kinase Cδ) phosphorylation at Ser664, which was necessary for aSMase/ceramide signalling via 67LR. Additionally, orally administered EGCG activated PKCδ and aSMase in a murine MM xenograft model. These results elucidate a novel cell-death pathway triggered by EGCG for the specific killing of MM cells.

Quercetin up-regulates the expression of tumor-suppressive microRNAs in human cervical cancer.

Quercetin, a flavonol present in many vegetables and fruits, has been identified as a chemoprevention agent in several cancer models. However, the molecular mechanism of quercetin's anticancer activity is not entirely understood. MicroRNAs (miRNAs), small noncoding RNAs, have been reported to play key roles in various biological processes by regulating their target genes. We hypothesized that quercetin can exert an anticancer effect through the regulation of miRNAs. To test this hypothesis, we investigated the effects of quercetin on the expression of tumor-suppressive miRNAs in cervical cancer. Quercetin up-regulated the in vivo and in vitro expression of tumor-suppressive miRNAs miR-26b, miR-126, and miR-320a. Quercetin suppressed the level of ß-catenin, encoded by catenin beta 1 (CTNNB1), by up-regulating miR-320a in HeLa cells. Moreover, quercetin increased the expression of mir-26b, mir-126, and mir-320a precursors in HeLa cells. The results from this study show that quercetin has the potential to prevent cervical cancer by regulating the expression of tumor-suppressive miRNAs.

Soy isoflavone metabolite equol inhibits cancer cell proliferation in a PAP associated domain containing 5-dependent and an estrogen receptor-independent manner.

Isoflavone is a species of polyphenol found mainly in soy and soy products. Many studies have demonstrated its estrogen receptor (ER)-dependent action. Equol is an intestinal metabolite of a major soy isoflavone daidzein. We aimed to elucidate the mechanism for ER-independent actions of equol. Equol has been shown to inhibit proliferation of HeLa human cervical cancer cells and mouse melanoma B16 cells in an ER-independent manner. Using functional genetic screening, PAP associated domain containing 5 (PAPD5), which is a non-canonical poly(A) polymerase, was identified as an essential molecule in the ER-independent action. While peroral administration of equol inhibited tumor growth of control B16 cells subcutaneously inoculated in mice, it had little effect on the growth of PAPD5-ablated B16 cells. Intriguingly, equol progressed tumor growth of the PAPD5-ablated human breast cancer MCF-7 cells, which have high ERα expression. Equol has been found to induce polyadenylation of snoRNAs in a PAPD5-depdendent manner. Furthermore, peroral equol administration increased microRNA miR-320a expression in tumors. Together, these results suggest that equol may have a dual effect on ER-positive cancer cells, acting with, antiproliferative activity through PAPD5 and exhibiting proliferative activity via ERα and the former could be associated with miR-320a.

Effects of a Tea Cultivar "MK5601" on Behaviors and Hippocampal Neurotrophin-3 Levels in Middle-Aged Mice.

Dietary factors are thought to play an important role in the prevention of cognition diseases and depression in late life. In the present study, we compared the effects between the theogallin-rich tea cultivar, "MK5601" and a common Japanese tea cultivar, "Yabukita" on behaviors and hippocampal neurotrophin levels in experimental animals. Middle-aged mice (aged 8 mo) were given either of the tea infusions or water ad libitum for 4 mo. In the novel object location test, the middle-aged mice drinking water or "Yabukita" performed worse than young mice (aged 2-3 mo) although the middle-aged mice drinking "MK5601" retained spatial memory at the same level as the young mice. We also found that the middle-aged mice drinking "MK5601" showed high levels of neurotrophin-3 in the hippocampus. In conclusion, the "MK5601" tea infusion appears to be effective in preventing age-related changes in cognitive function, as compared with a common Japanese tea cultivar.

Plasma Homocysteine Concentration is Associated with the Expression Level of Folate Receptor 3.

Folic acid and folate receptors (FOLRs) play an important role in the downregulation of homocysteine (Hcy), a risk factor of Alzheimer's disease, thrombosis, neuropsychiatric illness and fractures. While several studies have reported that FOLR1 and FOLR2 import folic acid into cells, the role of FOLR3 remains unknown. In this study, we evaluated the impact of FOLR3 on the metabolism of Hcy alongside its protective effect against homocysteine-induced neurotoxicity. To reveal the role of FOLR3, we constructed FOLR3-overexpressed HEK293 cells (FOLR3+ cells) and evaluated cell growth, folic acid intake and Hcy-induced neurotoxicity. Subjects with a high expression of FOLR3 exhibited low levels of plasma homocysteine. The ectopic expression of FOLR3 enhanced cell growth, and the enhanced effect was neutralised by folic acid-deficient media. The Western blot analysis revealed that FOLR3 is secreted into cell supernatant. The folic acid intake of FOLR3+ cells was higher than that of wild-type cells. Supernatant from FOLR3+ cells showed a protective effect on Hcy-induced cytotoxicity. FOLR3 expression in plasma is negatively correlated with plasma homocysteine. Our study emphasizes the role of FOLR3 in the intake of folic acid into cells on the one hand and its protective role in Hcy-induced cytotoxicity on the other.

Saturated fatty acid attenuates anti-obesity effect of green tea.

Green tea and its major polyphenol epigallocatechin-3-O-gallate (EGCG) have suppressive effect on dietary obesity. However, it remains unsolved what type of diet on which they exhibit high or low anti-obesity effect. In the present study, we investigated whether anti-obesity effect of green tea differs depending on composition of fats or fatty acids that consist high-fat (HF) diet in mouse model. Green tea extract (GTE) intake dramatically suppressed weight gain and fat accumulation induced by olive oil-based HF diet, whereas the effects on those induced by beef tallow-based HF diet were weak. GTE also effectively suppressed obesity induced by unsaturated fatty acid-enriched HF diet with the stronger effect compared with that induced by saturated fatty acid-enriched HF diet. These differences would be associated with the increasing action of GTE on expression of PPARδ signaling pathway-related genes in the white adipose tissue. Expressions of genes relating to EGCG signaling pathway that is critical for exhibition of physiological effects of EGCG were also associated with the different effects of GTE. Here, we show that anti-obesity effect of GTE differs depending on types of fats or fatty acids that consist HF diet and could be attenuated by saturated fatty acid.

Hydrogen sulphide donors selectively potentiate a green tea polyphenol EGCG-induced apoptosis of multiple myeloma cells.

Hydrogen sulphide (H2S) is a colourless gas with the odour of rotten eggs and has recently been recognized as a signal mediator in physiological activities related with the regulation of homeostasis, the vascular system and the inflammatory system. Here we show that H2S donors, including sodium hydrogen sulphide (NaHS), GYY 4137 and diallyltrisulfide (DATS), synergistically enhanced the anti-cancer effect of a green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) against multiple myeloma cells without affecting normal cells. NaHS significantly potentiated the anti-cancer effect of EGCG and prolonged survival in a mouse xenograft model. In this mechanism, H2S enhanced apoptotic cell death through cyclic guanosine monophosphate (cGMP)/acid sphingomyelinase pathway induced by EGCG. Moreover, NaHS reduced the enzyme activity of cyclic nucleotide phosphodiesterase that is known as cGMP negative regulator. In conclusion, we identified H2S as a gasotransmitter that potentiates EGCG-induced cancer cell death.

Delphinidin Prevents Muscle Atrophy and Upregulates miR-23a Expression.

Delphinidin, one of the major anthocyanidins, shows protective effects against a variety of pathologies, including cancer, inflammation, and muscle atrophy. The purpose of this study was to determine the preventive mechanism of delphinidin on disuse muscle atrophy. In vitro and in vivo models were used to validate the effects of delphinidin on the expression of MuRF1, miR-23a, and NFATc3. Delphinidin suppressed the upregulation of MuRF1 (1.77 ± 0.05 vs 1.03 ± 0.17, P < 0.05) expression and inhibited the downregulation of miR-23a (0.56 ± 0.05 vs 0.94 ± 0.06, P < 0.05) and NFATc3 (0.61 ± 0.02 vs 1.02 ± 0.08, P < 0.01) expression in dexamethasone-treated C2C12 cells. In gastrocnemius, muscle weight loss was prevented by oral administration of delphinidin. Moreover, delphinidin suppressed MuRF1 (3.35 ± 0.13 vs 2.26 ± 0.3, P < 0.01) expression and promoted miR-23a (0.58 ± 0.15 vs 2.25 ± 0.29, P < 0.001) and NFATc3 (0.85 ± 0.17 vs 1.54 ± 0.13, P < 0.001) expressions. Delphinidin intake may prevent disuse muscle atrophy by inducing miR-23a expression and suppressing MuRF1 expression.

Oligomer formation of a tea polyphenol, EGCG, on its sensing molecule 67 kDa laminin receptor.

Green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) has been attributed to the activation of its cell surface sensing receptor 67 kDa laminin receptor (67LR). However, the action of EGCG to activate 67LR remains unknown. Here we show that EGCG undergoes oligomer formation on its surface receptor 67LR.