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PDF file - 30K, The cytotoxicity of IAA against normal melanocytes.

Licorice root is known to possess various bioactivities, including anti-inflammatory and anticancer effects. Glycyrrhizin, a triterpene compound, is the most abundant constituent of dried licorice root. However, high intake or long-term consumption of glycyrrhizin causes several side effects, such as hypertension, hypertensive encephalopathy, and hypokalemia. Therefore, finding additional active compounds other than glycyrrhizin in licorice that exhibit anticancer effects is worthwhile. We found that isoangustone A (IAA), a novel flavonoid from licorice root, suppressed proliferation of human melanoma cells. IAA significantly blocked cell-cycle progression at the G1-phase and inhibited the expression of G1-phase regulatory proteins, including cyclins D1 and E in the SK-MEL-28 human melanoma cell line. IAA suppressed the phosphorylation of Akt, GSK-3β, and JNK1/2. IAA also bound to phosphoinositide 3-kinase (PI3K), MKK4, and MKK7, strongly inhibiting their kinase activities in an ATP-competitive manner. Moreover, in a xenograft mouse model, IAA significantly decreased tumor growth, volume, and weight of SK-MEL-28 xenografts. Collectively, these results suggest that PI3K, MKK4, and MKK7 are the primary molecular targets of IAA in the suppression of cell proliferation. This insight into the biologic actions of IAA provides a molecular basis for the potential development of a new chemotherapeutic agent. Cancer Prev Res; 6(12); 1293–303. ©2013 AACR.

PDF file - 24K, The effect of IAA on tumor weight in a xenograft mouse model.

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Supplementary Figures and Legends - PDF file 2854K, Supplementary Figure 1. Effect of USP8 knockdown on cell viability Supplementary Figure 2. Effect of USP8 knockdown on EGFR wildtype cells and apoptosis of NSCLC and normal cells. Supplementary Figure 3. Effect of USP8 knockdown on mRNA levels of RTKs. Supplementary Figure 4. Effect of USP8 inhibitor treatment on apoptosis of NSCLC and normal cells. Supplementary Figure 5. Fluorescence Intensity Profiling Supplementary Figure 6. Effect of USP8 knockdown or an USP8 inhibitor on insulin receptor (IR) expression. Supplementary Figure 7. An USP8 inhibitor reduces ERKs and STAT3 phosphorylation levels in tumors

Purpose: Common treatment modalities for non–small cell lung cancer (NSCLC) involve the EGF receptor-tyrosine kinase inhibitors (EGFR-TKIs) like gefitinib and erlotinib. However, the vast majority of treated patients acquire resistance to EGFR-TKIs, due, in large part, to secondary mutations in EGFR or amplification of the MET gene. Our purpose was to test ubiquitin-specific peptidase 8 (USP8) as a potential therapeutic target for gefitinib-resistant and -sensitive non–small cell lung cancer (NSCLC).Experimental Design: Testing the effect of knockdown of USP8 and use of a synthetic USP8 inhibitor to selectively kill gefitinib-resistant (or -sensitive) NSCLCs with little effect on normal cells in cell culture and a xenograft mouse model.Results: Knockdown of ubiquitin-specific peptidase 8 (USP8) selectively kills gefitinib-resistant NSCLCs while having little toxicity toward normal cells. Genetic silencing of USP8 led to the downregulation of several receptor tyrosine kinases (RTK) including EGFR, ERBB2, ERBB3, and MET. We also determined that a synthetic USP8 inhibitor markedly decreased the viability of gefitinib-resistant and -sensitive NSCLC cells by decreasing RTK expression while having no effect on normal cells. Moreover, treatment with a USP8 inhibitor led to significant reductions in tumor size in a mouse xenograft model using gefitinib-resistant and -sensitive NSCLC cells.Conclusions: Our results show for the first time that the inhibition of USP8 activity or reduction in USP8 expression can selectively kill NSCLC cells. We propose USP8 as a potential therapeutic target for gefitinib-resistant and -sensitive NSCLC cells. Clin Cancer Res; 19(14); 3894–904. ©2013 AACR.

Supplementary Data from Cocarcinogenic Effect of Capsaicin Involves Activation of EGFR Signaling but Not TRPV1

Arsenite is a well-known human carcinogen that especially targets skin. The tumor progression locus 2 (Tpl2) gene encodes a serine/threonine protein kinase that is overexpressed in various cancer cells. However, the relevance of Tpl2 in arsenite-induced carcinogenesis and the underlying mechanisms remain to be explored. We show that arsenite increased Tpl2 kinase activity and its phosphorylation in mouse epidermal JB6 P+ cells in a dose- and time-dependent manner. Exposure to arsenite resulted in a marked induction of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2), important mediators of inflammation and tumor promotion. Treatment with a Tpl2 kinase inhibitor or Tpl2 short hairpin RNA suppressed COX-2 expression and PGE2 production induced by arsenite treatment, suggesting that Tpl2 is critical in arsenite-induced carcinogenesis. We also found that arsenite-induced phosphorylation of extracellular signal-regulated kinases (ERK) or c-Jun NH2-terminal kinases (JNK) was markedly suppressed by Tpl2 kinase inhibitor or Tpl2 short hairpin RNA. Inhibition of arsenite-induced ERK or JNK signaling using a pharmacologic inhibitor of ERK or JNK substantially blocked COX-2 expression. Furthermore, inhibition of Tpl2 reduced the arsenite-induced promoter activity of NF-κB and activator protein-1 (AP-1), indicating that NF-κB and AP-1 are downstream transducers of arsenite-triggered Tpl2. Our results show that Tpl2 plays a key role in arsenite-induced COX-2 expression and PGE2 production and further elucidate the role of Tpl2 in arsenite signals that activate ERK/JNK and NF-κB/AP-1 in JB6 P+ cells. [Cancer Res 2009;69(20):8043–9]

Epidemiologic and animal studies revealed that capsaicin can act as a carcinogen or cocarcinogen. However, the molecular mechanisms of the cancer-promoting effects of capsaicin are not clear. Here, we report that capsaicin has a cocarcinogenic effect on 12-O-tetradecanoylphorbol-13-acetate (TPA)–promoted skin carcinogenesis in vivo and is mediated through the epidermal growth factor receptor (EGFR), but not the transient receptor potential vanilloid subfamily member 1 (TRPV1). Topical application of capsaicin on the dorsal skin of 7,12-dimetylbenz(a)anthracene–initiated and TPA-promoted TRPV1 wild-type (WT) and TRPV1 knockout (KO) mice induced more and larger skin tumors in TRPV1/KO mice, suggesting a TRPV1-independent mechanism. Cyclooxygenase-2 (COX-2) was highly elevated by capsaicin treatment in tumors and murine embryonic fibroblasts from TRPV1/KO mice. Inhibitors of EGFR/MEK signaling suppressed TPA/capsaicin-induced COX-2 expression in TRPV1/KO cells, indicating that activation of EGFR and its downstream signaling is involved in COX-2 elevation. Capsaicin induced a further induction of TPA-increased COX-2 expression in EGFR/WT cells, but not in EGFR/KO cells. TPA/capsaicin cotreatment caused EGFR tyrosine phosphorylation and activated EGFR downstream signaling, including ERKs and Akt in EGFR/WT, but not EGFR/KO cells. Specific inhibition of EGFR and TRPV1 indicated that capsaicin-induced ERK activation in A431 cells was dependent on EGFR, but not TRPV1. Together, these findings suggest that capsaicin might act as a cocarcinogen in TPA-induced skin carcinogenesis through EGFR-dependent mechanisms. Cancer Res; 70(17); 6859–69. ©2010 AACR.

Naturally occurring dietary phytochemicals have been recognized as possessing many health-promoting effects. Recent stu- dies suggest that inflammation is closely related to chronic disease, including cancer, atherosclerosis, neurodegeneration, obesity, diabetes, asthma, articular rheumatism, and skin-aging. Extracellular stimuli transmit signals into a cell by activating the target kinases involved in inflammation and the onset of chronic diseases. Phytochemicals can directly bind to specific proteins in- volved in intracellular signaling networks and regulate their activity, leading to diverse physiological effects. A better under- standing of the direct interactions between phytochemicals and target proteins would contribute to development of nutritional strategies for preventing or delaying the development of chronic diseases. In this review, recently identified molecular targets and signaling pathways regulated by phytochemicals will be discussed.

Erucin is a natural isothiocyanate found in rocket salad and broccoli which are believed to have health benefits including weight control effect and used as a functional food globally. Though anti-cancer effect of erucin has been intensively studied, anti-obesity effect of erucin and its molecular mechanisms have not yet been reported. In this study, we investigated anti-adipogenic effect of erucin in 3T3-L1 preadipocytes and its underlying molecular mechanisms. Erucin inhibited adipogenesis of 3T3-L1 preadipocytes and reduced adipogenic gene expressions. This anti-adipogenic activity of erucin was most effective at early stage of adipogenesis including mitotic clonal expansion (MCE). Fluorescence-activated cell sorter (FACS) analysis and trypan blue assay results showed that erucin induced G1/S arrest and inhibited cell proliferation at MCE stage. Western blot analysis results showed that erucin inhibited extracellular signal-regulated kinase (ERK) signaling pathway by blocking Ras activity. Taken together, these results indicated that erucin inhibits adipogenesis of 3T3-L1 preadipocytes by blocking Ras activity at early stage of adipogenesis.

Bioactive phytochemicals can suppress the growth of malignant cells, and investigation of the mechanisms responsible can assist in the identification of novel therapeutic strategies for cancer therapy. Ginger has been reported to exhibit potent anti-cancer effects, although previous reports have often focused on a narrow range of specific compounds. Through a direct comparison of various ginger compounds, we determined that gingerenone A selectively kills cancer cells while exhibiting minimal toxicity toward normal cells. Kinase array screening revealed JAK2 and S6K1 as the molecular targets primarily responsible for gingerenone A-induced cancer cell death. The effect of gingerenone A was strongly associated with relative phosphorylation levels of JAK2 and S6K1, and administration of gingerenone A significantly suppressed tumor growth in vivo. More importantly, the combined inhibition of JAK2 and S6K1 by commercial inhibitors selectively induced apoptosis in cancer cells, whereas treatment with either agent alone did not. These findings provide rationale for dual targeting of JAK2 and S6K1 in cancer for a combinatorial therapeutic approach.

Insulin resistance is a major characteristic of obesity and type 2 diabetes, but the underlying mechanism is unclear. Recent studies have shown a metabolic role of capsaicin that may be mediated via the transient receptor potential vanilloid type-1 (TRPV1) channel. In this study, TRPV1 knockout (KO) and wild-type (WT) mice (as controls) were fed a high-fat diet (HFD), and metabolic studies were performed to measure insulin and leptin action. The TRPV1 KO mice became more obese than the WT mice after HFD, partly attributed to altered energy balance and leptin resistance in the KO mice. The hyperinsulinemic-euglycemic clamp experiment showed that the TRPV1 KO mice were more insulin resistant after HFD because of the ∼40% reduction in glucose metabolism in the white and brown adipose tissue, compared with that in the WT mice. Leptin treatment failed to suppress food intake, and leptin-mediated hypothalamic signal transducer and activator of transcription (STAT)-3 activity was blunted in the TRPV1 KO mice. We also found that the TRPV1 KO mice were more obese and insulin resistant than the WT mice at 9 mo of age. Taken together, these results indicate that lacking TRPV1 exacerbates the obesity and insulin resistance associated with an HFD and aging, and our findings further suggest that TRPV1 has a major role in regulating glucose metabolism and hypothalamic leptin’s effects in obesity.—Lee, E., Jung, D. Y., Kim, J. H., Patel, P. R., Hu, X., Lee, Y., Azuma, Y., Wang, H.-F., Tsitsilianos, N., Shafiq, U., Kwon, J. Y., Lee, H. J., Lee, K. W., Kim, J. K. Transient receptor potential vanilloid type-1 channel regulates diet-induced obesity, insulin resistance, and leptin resistance.

We evaluated the effects of the two main kiwifruit cultivars (gold kiwifruit (GOK) and green kiwifruit (GRK)) and their active phenolic compound, quercetin, on H2O2-induced inhibition of gap-junction intercellular communication (GJIC) in WB-F344 rat liver epithelial cells. We found that both GOK and GRK protect WB-F344 cells from H2O2-induced inhibition of GJIC. The extracellular signal-regulated protein kinase 1/2 (ERK1/2)–connexin 43 (Cx43) signalling pathway is crucial for the regulation of GJIC, and both GOK and GRK blocked the H2O2-induced phosphorylation of Cx43 and ERK1/2 in WB-F344 cells. Quercetin alone attenuated the H2O2-mediated ERK1/2–Cx43 signalling pathway and consequently reversed H2O2-mediated inhibition of GJIC in WB-F344 cells. A free radical-scavenging assay using 1,1-diphenyl-2-picrylhydrazyl showed that the scavenging activity of quercetin was higher than that of a synthetic antioxidant, butylated hydroxytoluene, per mol, suggesting that the chemopreventive effect of quercetin on H2O2-mediated inhibition of ERK1/2–Cx43 signalling and GJIC may be mediated through its free radical-scavenging activity. Since the carcinogenicity of reactive oxygen species such as H2O2 is attributable to the inhibition of GJIC, GOK, GRK and quercetin may have chemopreventive potential by preventing the inhibition of GJIC.

Dehydroglyasperin D (DHGA-D), a compound present in licorice, has been found to exhibit anti-obesity, antioxidant and anti-aldose reductase effects. However, the direct molecular mechanism and molecular targets of DHGA-D during skin inflammation remain unknown. In the present study, we investigated the effect of DHGA-D on inflammation and its mechanism of action on UVB-induced skin inflammation in HaCaT human keratinocytes and SKH-1 hairless mice. DHGA-D treatment strongly suppressed UVB-induced COX-2 expression, PGE2 generation and AP-1 transactivity in HaCaT cells without affecting cell viability. DHGA-D also inhibited phosphorylation of the mitogen-activated protein kinase kinase (MKK) 3/6/p38, MAPK/Elk-1, MKK4/c-Jun N-terminal kinase (JNK) 1/2/c-Jun/mitogen, and stress-activated protein kinase (MSK), whereas phosphorylation of the mixed-lineage kinase (MLK) 3 remained unaffected. Kinase and co-precipitation assays with DHGA-D Sepharose 4B beads showed that DHGA-D significantly suppressed MLK3 activity through direct binding to MLK3. Knockdown of MLK3 suppressed COX-2 expression as well as phosphorylation of MKK4/p38 and MKK3/6/JNK1/2 in HaCaT cells. Furthermore, Western blot assay and immunohistochemistry results showed that DHGA-D pre-treatment significantly inhibits UVB-induced COX-2 expression in vivo. Taken together, these results indicate that DHGA-D may be a promising anti-inflammatory agent that mediates suppression of both COX-2 expression and the MLK3 signalling pathway through direct binding and inhibition of MLK3.

Moisture determination in cheese is a critical test for regulatory compliance, functionality, and economic reasons. Common methods for moisture determination in cheese rely upon the thermal volatilization of water from cheese and calculation of moisture content based on the resulting loss of mass. Residual sugars, such as lactose and galactose, are commonly present in cheeses at levels ranging from trace amounts to 5%. These sugars are capable of reacting with other compounds in cheese, especially under the thermal conditions required for moisture determination, to yield volatile reaction products. The hypothesis of this work is that residual sugars in cheese will be converted into volatile compounds over the course of moisture determination at a level sufficient to result in overestimated cheese moisture. A full-factorial statistical design was used to evaluate the effects of cheese type, sugar type, sugar level, method type, and all interactions. Cheddar and low-moisture, part-skim (LMPS) Mozzarella cheeses were prepared with 1, 3, and 5% added lactose or galactose, and subjected to either vacuum oven or microwave-based moisture determination methods. Browning index and colorimetry were measured to characterize the color and extent of browning. Volatile analyses were performed to provide chemical evidence of the reactions proposed. The presence of residual sugars altered moisture calculations as a function of cheese type, sugar type, sugar level, method type, and numerous interactions. At higher concentrations of residual sugar, the percentage moisture determinations were increased by values of up to 1.8. Measures of browning reactions, including browning index, colorimetry, and volatile profiles demonstrate that the proposed browning reactions played a causative role. This work establishes the need to consider cheese type, sugar type, sugar levels, and method type as a means of more accurately determining moisture levels.

Licorice root is known to possess various bioactivities, including anti-inflammatory and anticancer effects. Glycyrrhizin, a triterpene compound, is the most abundant constituent of dried licorice root. However, high intake or long-term consumption of glycyrrhizin causes several side effects, such as hypertension, hypertensive encephalopathy, and hypokalemia. Therefore, finding additional active compounds other than glycyrrhizin in licorice that exhibit anticancer effects is worthwhile. We found that isoangustone A (IAA), a novel flavonoid from licorice root, suppressed proliferation of human melanoma cells. IAA significantly blocked cell-cycle progression at the G1-phase and inhibited the expression of G1-phase regulatory proteins, including cyclins D1 and E in the SK-MEL-28 human melanoma cell line. IAA suppressed the phosphorylation of Akt, GSK-3β, and JNK1/2. IAA also bound to phosphoinositide 3-kinase (PI3K), MKK4, and MKK7, strongly inhibiting their kinase activities in an ATP-competitive manner. Moreover, in a xenograft mouse model, IAA significantly decreased tumor growth, volume, and weight of SK-MEL-28 xenografts. Collectively, these results suggest that PI3K, MKK4, and MKK7 are the primary molecular targets of IAA in the suppression of cell proliferation. This insight into the biologic actions of IAA provides a molecular basis for the potential development of a new chemotherapeutic agent. Cancer Prev Res; 6(12); 1293–303. ©2013 AACR.

Background: Resistance of ovarian cancer cells to chemotherapy is a major therapeutic problem. Results: Hirsutenone induces cisplatin sensitivity via p53, X-linked inhibitor of apoptosis protein, and apoptosis-inducing factor. Conclusion: Hirsutenone sensitizes resistant ovarian cancer cells to cisplatin. Significance: Co-treatment with hirsutenone may have the potential to overcome chemoresistance., Cisplatin (CDDP) and its derivatives are considered first-line treatments for ovarian cancer (OVCA). However, despite initial results that often appear promising, in most cases patients will return with recurrent disease that fails to respond to further chemotherapy. We assayed a number of food phytochemicals with reported PI3K inhibitory ability to identify candidates that can influence CDDP treatment outcomes in chemoresistant OVCA cell lines. A direct comparison revealed that the diarylheptanoid hirsutenone from the tree bark of Alnus hirsuta var. sibirica was superior at inducing CDDP sensitivity in a number of chemoresistant cancer cell lines. Whereas hirsutenone treatment activated p53, its modest efficacy in p53-mutant and -null cell lines suggested the existence of a p53-independent mode of action. Further investigation revealed that hirsutenone causes CDDP-dependent apoptosis in chemoresistant cells by ubiquitin-proteasome-dependent X-linked inhibitor of apoptosis degradation and by enhancing the translocation of apoptosis-inducing factor from the mitochondria to the nucleus. This was found to be, at least in part, under the influence of upstream Akt activity, linking hirsutenone-dependent PI3K inhibition with downstream effects on apoptosis-inducing factor, X-linked inhibitor of apoptosis, and apoptosis. Our findings provide rationale for further investigation of the effects of hirsutenone on chemoresistant OVCA in clinical studies.

Atopic dermatitis (AD) is a common allergic disease, imposing large social and economic burdens worldwide. Atopic dermatitis is characterized by eczematous skin lesions and immunoglobulin E (IgE) hypersecretion. We investigated the role of JNK1 on the development of AD in mice. The vitamin D3 analogue MC903, a psoriasis therapeutic drug, was used to induce AD-like symptoms in wild-type (WT) and JNK1-/- mice. The symptoms of AD were less severe in JNK1-/- mice compared with WT mice. JNK1-/- mice showed less ear thickening and infiltration of eosinophils and mast cells in AD-like lesions than did WT mice when treated with MC903. MC903-treated JNK1-/- mice also showed significantly lower level of serum IgE, which was elevated in MC903-treated WT mice. Splenocytes isolated from MC903-treated WT and JNK1-/- mice were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies. Splenocytes from JNK1-/- mice produced lower levels of T-helper (Th2) cytokines (interleukin-4 and -13) and transcription factor GATA-binding protein 3, and produced increased levels of the Th1 cytokines interferon-γ and transcription factor T-box expressed in T cells. Our results indicate that JNK1 plays an important role in the pathogenesis of AD and may be a useful target for therapies to ameliorate AD.

Metastasis is one of the most threatening features of the oncogenic process and the main cause of cancer-related mortality. Several studies have demonstrated that matrix metalloproteinases (MMPs) are critical for tumor invasion and metastasis. Resveratrol (3,5,4'-trihydroxystilbene), a phenolic compound of red wine, has been reported to be a natural chemopreventive agent. However, the cancer preventive effects of piceatannol (3,5,3',4'-tetrahydroxystilbene), a metabolite of resveratrol and the underlying molecular mechanisms have not yet been fully elucidated. In this study, we report that piceatannol inhi-bits H-ras-induced MMP-2 activity and the invasive phenotype of MCF10A human breast epithelial cells harboring mutated H-ras (H-ras MCF10A cells) more effectively than resveratrol. Piceatannol attenuated the H-ras-induced phosphorylation of Akt in a time- and dose-dependent manner, whereas resveratrol, at the same concentrations, did not exert an inhibitory effect. In vitro kinase assays demonstrated that piceatannol significantly inhibited phosphatidylinositol 3-kinase (PI3K) activity and suppressed phospha-tidylinositol (3,4,5)-trisphosphate (PIP3) expression in the H-ras MCF10A cells. Ex vivo pull-down assays revealed that piceatannol directly bound to PI3K, inhibiting PI3K activity. Data from molecular docking suggested that piceatannol is a more tight-binding inhibitor than resveratrol due to the additional hydrogen bond between the hydroxyl group and the backbone amide group of Val882 in the ATP-binding pocket of PI3K.

A variety of nuruk were collected from various provinces in Korea, and their microflora profiles were analyzed at the species level. A total of 42 nuruk samples were collected and when the viable cell numbers in these nuruk were enumerated, the average cell numbers of bacteria, fungi, yeast, and lactic acid bacteria from all nuruk were 7.21, 7.91, 3.49, and 4.88 log CFU/10 g, respectively. There were no significant differences in viable cell numbers of bacteria or fungi according to regions collected. Bacillus amyloliquefaciens and B. subtilis were the predominant bacterial strains in most samples. A significant portion, 13 out of 42 nuruk, contained foodborne pathogens such as B. cereus or Cronobacter sakazakii. There were various species of lactic acid bacteria such as Enterococcus faecium and Pediococcus pentosaceus in nuruk. It was unexpectedly found that only 13 among the 42 nuruk samples contained Aspergillus oryzae, the representative saccharifying fungi in makgeolli, whereas a fungi Lichtheimia corymbifera was widely distributed in nuruk. It was also found that Pichia jadinii was the predominant yeast strain in most nuruk, but the representative alcohol fermentation strain, Saccharomyces cerevisiae, was isolated from only 18 out of the 42 nuruk. These results suggested that a variety of species of fungi and yeast were distributed in nuruk and involved in the fermentation of makgeolli. In this study, a total of 64 bacterial species, 39 fugal species, and 15 yeast species were identified from nuruk. Among these strains, 37 bacterial species, 20 fungal species, and 8 yeast species were distributed less than 0.1%.

Ovarian cancer is the most lethal gynecological malignancy. Cisplatin and its derivatives are first-line chemotherapeutics, and their resistance is a major hurdle in successful ovarian cancer treatment. Understanding the molecular dysregulation underlying chemoresistance is important for enhancing therapeutic outcome. Here, we review two established pathways in cancer chemoresistance. p53 is a major tumor suppressor regulating proliferation and apoptosis, and its mutation is a frequent event in human malignancies. The PI3K/Akt axis is a key oncogenic pathway regulating survival and tumorigenesis by controlling several tumor suppressors, including p53. The interplay between these pathways is well established, although the oncogenic phosphatase PPM1D adds a new layer to this intricate relationship and provides new insights into the processes determining cell fate. Inhibition of the PI3K/Akt pathway by functional food compounds as an adjunct to chemotherapeutics may tip the balance in favor of apoptosis rather than survival, enhancing therapeutic efficacy, and reducing side effects.

Myricetin is a widely distributed flavonol that is found in many plants, including tea, berries, fruits, vegetables, and medicinal herbs. Abundant sources provide interesting insights into the multiple mechanisms by which myricetin mediates chemopreventive effects on skin cancer. Myricetin strongly inhibited tumor promoter-induced neoplastic cell transformation by inhibiting MEK, JAK1, Akt, and MKK4 kinase activity directly. In a mouse skin model, myricetin attenuated the ultraviolet B (UVB)-induced COX-2 expression and skin tumor formation by regulating Fyn. Myricetin-mediated inactivation of Akt in the UVB response plays a role in regulating UVB-induced carcinogenesis. Recently, myricetin was found to inhibit UVB-induced angiogenesis by targeting PI3-K in an SKH-1 hairless mouse skin tumorigenesis model. Raf kinase is a critical target for myricetin in inhibiting the UVB-induced formation of wrinkles and suppression of type I procollagen and collagen levels in mouse skin. Accumulated data suggest that myricetin acts as a promising agent for the chemoprevention of skin cancer.

IL-32 was recently identified as a proinflammatory cytokine that is induced by IL-18 in natural killer (NK) cells and is highly correlated with inflammatory disorders. However, the relationship between IL-32 and tumor progression is still unknown. In this study, we investigated whether overexpression of IL-32 affects susceptibility of chronic myeloid leukemia (CML) cells to NK cells. Interestingly, IL-32α-overexpressing CML cell lines, K562, Kcl22, and BV173, showed higher NK cell-mediated killing. Flow cytometry analysis revealed that overexpression of IL-32α induced increased expression of Fas and UL16-binding protein 2 (ULBP2) in CML cells. The direct relationship between overexpression of surface molecules by IL-32α and increased NK cell-mediated killing was confirmed by Fas or ULBP2 siRNA transfection. IL-32α-induced Fas and ULBP2 expression are regulated p38 MAPK. In addition, the transcription factor Ets1 plays a key role in ULBP2 specific expression by IL-32α overexpression in ULBP family members. Taken together, these data show that IL-32α stimulates Fas and ULBP2 expression via activation of p38 MAPK, which increases NK susceptibility of CML cells. Enhanced NK cell susceptibility of CML cells by IL-32α overexpression may improve the efficiency of NK cell-based immunotherapy.

Nonmelanoma skin cancer is one of the most frequently occurring cancers in the United States. Chronic exposure to UVB irradiation is a major cause of this cancer. Daidzein, along with genistein, is a major isoflavone found in soybeans; however, little is known about the chemopreventive effects of daidzein and its metabolites in UVB-induced skin cancer. Here, we found that 7,3′,4′-trihydroxyisoflavone (THIF), a major metabolite of daidzein, effectively inhibits UVB-induced cyclooxygenase 2 (COX-2) expression through the inhibition of NF-κB transcription activity in mouse skin epidermal JB6 P+ cells. In contrast, daidzein had no effect on COX-2 expression levels. Data from Western blot and kinase assays showed that 7,3′,4′-THIF inhibited Cot and MKK4 activity, thereby suppressing UVB-induced phosphorylation of mitogen-activated protein kinases. Pull-down assays indicated that 7,3′,4′-THIF competed with ATP to inhibit Cot or MKK4 activity. Topical application of 7,3′,4′-THIF clearly suppressed the incidence and multiplicity of UVB-induced tumors in hairless mouse skin. Hairless mouse skin results also showed that 7,3′,4′-THIF inhibits Cot or MKK4 kinase activity directly, resulting in suppressed UVB-induced COX-2 expression. A docking study revealed that 7,3′,4′-THIF, but not daidzein, easily docked to the ATP binding site of Cot and MKK4, which is located between the N- and C-lobes of the kinase domain. Collectively, these results provide insight into the biological actions of 7,3′,4′-THIF, a potential skin cancer chemopreventive agent.

Numerous in vitro and in vivo studies have shown that isoflavones exhibit anti-proliferative activity against epidermal growth factor (EGF) receptor-positive malignancies of the breast, colon, skin, and prostate. 7,3',4'-Trihydroxyisoflavone (7,3',4'-THIF) is one of the metabolites of daidzein, a well known soy isoflavone, but its chemopreventive activity and the underlying molecular mechanisms are poorly understood. In this study, 7,3',4'-THIF prevented EGF-induced neoplastic transformation and proliferation of JB6 P+ mouse epidermal cells. It significantly blocked cell cycle progression of EGF-stimulated cells at the G(1) phase. As shown by Western blot, 7,3',4'-THIF suppressed the phosphorylation of retinoblastoma protein at Ser-795 and Ser-807/Ser-811, which are the specific sites of phosphorylation by cyclin-dependent kinase (CDK) 4. It also inhibited the expression of G(1) phase-regulatory proteins, including cyclin D1, CDK4, cyclin E, and CDK2. In addition to regulating the expression of cell cycle-regulatory proteins, 7,3',4'-THIF bound to CDK4 and CDK2 and strongly inhibited their kinase activities. It also bound to phosphatidylinositol 3-kinase (PI3K), strongly inhibiting its kinase activity and thereby suppressing the Akt/GSK-3beta/AP-1 pathway and subsequently attenuating the expression of cyclin D1. Collectively, these results suggest that CDKs and PI3K are the primary molecular targets of 7,3',4'-THIF in the suppression of EGF-induced cell proliferation. These insights into the biological actions of 7,3',4'-THIF provide a molecular basis for the possible development of new chemoprotective agents.

Kaempferol (KF), which is a natural dietary flavonoid, has potential beneficial effects as a chemopreventive agent for critical health conditions, such as cancer. However, the molecular mechanisms underlying the activity of KF remain unknown. We report on the inhibition of neoplastic cell transformation by KF through the suppression of phosphatidylinositol 3-kinase (PI3K) activity. Epidermal growth factor (EGF)-induced neoplastic transformation of mouse epidermal JB6 P+ cells was inhibited by 40 microM KF. The activation of activator protein-1 and nuclear factor-kappaB induced by EGF was also attenuated by KF. The EGF-induced phosphorylation of Akt (protein kinase B) was completely suppressed by KF, although extracellular signal-regulated kinase, p38, c-Jun N-terminal kinase and p90 ribosomal S6 kinase were unaffected by KF. Kinase assay data revealed that KF bound directly to PI3K, which is upstream of Akt, and suppressed its activity. Furthermore, KF inhibited ultraviolet B (UVB)-induced PI3K activity and attenuated UVB-induced phosphorylation of Akt. Our results suggest that KF docks at the adenosine triphosphate-binding site of PI3K, which is located between the N-lobe and C-lobe of the kinase domain. Inhibition by KF of PI3K, which is an important factor in carcinogenesis, and its downstream effects may explain the chemopreventive action of KF.

Chronic exposure to solar ultraviolet (UV) light causes skin photoaging. Many studies have shown that naturally occurring phytochemicals have anti-photoaging effects, but their direct target molecule(s) and mechanism(s) remain unclear. We found that myricetin, a major flavonoid in berries and red wine, inhibited wrinkle formation in mouse skin induced by chronic UVB irradiation (0.18J/cm(2), 3 days/week for 15 weeks). Myricetin treatment reduced UVB-induced epidermal thickening of mouse skin and also suppressed UVB-induced matrix metalloproteinase-9 (MMP-9) protein expression and enzyme activity. Myricetin appeared to exert its anti-aging effects by suppressing UVB-induced Raf kinase activity and subsequent attenuation of UVB-induced phosphorylation of MEK and ERK in mouse skin. In vitro and in vivo pull-down assays revealed that myricetin bound with Raf in an ATP-noncompetitive manner. Overall, these results indicate that myricetin exerts potent anti-photoaging activity by regulating MMP-9 expression through the suppression of Raf kinase activity.

Aims Abnormal migration and proliferation of human aortic smooth muscle cells (HASMCs) to the intima causes intimal thickening of the aorta, which is strongly related to the development of atherosclerosis. Previous studies have suggested that red wine polyphenols, particularly resveratrol, have great protective effects against cardiovascular diseases. Here, we compared the anti-atherosclerotic effect of piceatannol, a metabolite of resveratrol, and its underlying mechanisms. Methods and results We demonstrated that piceatannol inhibited platelet-derived growth factor (PDGF)-BB-induced cell migration using a modified Boyden chamber assay and wound healing assay. Western blot analysis showed that PDGF-BB-induced phosphorylation of Akt, p70S6K, and p38 was inhibited by piceatannol, but not resveratrol. In vitro and ex vivo phosphoinositide 3-kinase (PI3K) assays demonstrated that piceatannol suppressed PI3K activity more effectively than resveratrol. PDGF-BB-induced migration and proliferation of HASMCs were inhibited by treatment with a commercial PI3K inhibitor, LY294002. Both in vitro and ex vivo pull-down assays revealed that piceatannol directly binds with sepharose 4B-PI3K beads in an ATP-competitive manner. Conclusion The results of the present study demonstrate that piceatannol directly binds with PI3K in an ATP-competitive manner and suppresses PI3K activity with anti-atherosclerotic effects.

Recent studies suggest that anthocyanidins play a pivotal role in the chemopreventive effects of fruits and vegetables. However, the underlying molecular mechanisms and cellular targets remain unknown. Neoplastic transformation of cells and inflammation are considered to be major events contributing to carcinogenesis. Here, we report that delphinidin, a major dietary anthocyanidin, inhibits tumor promoter–induced transformation and cyclooxygenase-2 (COX-2) expression in JB6 promotion-sensitive mouse skin epidermal (JB6 P+) cells by directly targeting Raf and mitogen-activated protein kinase kinase (MEK). Delphinidin inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced neoplastic transformation and COX-2 expression at both the protein and transcriptional levels. The activation of activator protein-1 and nuclear factor-κB induced by TPA was dose dependently inhibited by delphinidin treatment. Delphinidin strongly suppressed Raf1 and MEK1 kinase activities and subsequently attenuated TPA-induced phosphorylation of MEK, extracellular signal-regulated kinase (ERK), p90RSK, and MSK. Although delphinidin suppressed ERK and c-Jun NH2-terminal kinase activities, it was more effective at inhibiting Raf1 or MEK1 activities. Pull-down and competition assays revealed that delphinidin binds with Raf1 or MEK1 noncompetitively with ATP. Delphinidin also dose dependently suppressed JB6 P+ cell transformation induced by epidermal growth factor and H-Ras, both of which are involved in the activation of Raf/MEK/ERK signaling. Together, these findings suggested that the targeted inhibition of Raf1 and MEK activities and COX-2 expression by delphinidin contribute to the chemopreventive potential of fruits and vegetables.

Cocoa was shown to inhibit chemically induced carcinogenesis in animals and exert antioxidant activity in humans. However, the molecular mechanisms of the chemopreventive potential of cocoa and its active ingredient(s) remain unknown. Here we report that cocoa procyanidins inhibit neoplastic cell transformation by suppressing the kinase activity of mitogen-activated protein kinase kinase (MEK). A cocoa procyanidin fraction (CPF) and procyanidin B2 at 5 μg/ml and 40 μm, respectively, inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ mouse epidermal (JB6 P+) cells by 47 and 93%, respectively. The TPA-induced promoter activity and expression of cyclooxygenase-2, which is involved in tumor promotion and inflammation, were dose-dependently inhibited by CPF or procyanidin B2. The activation of activator protein-1 and nuclear factor-κB induced by TPA was also attenuated by CPF or procyanidin B2. The TPA-induced phosphorylation of MEK, extracellular signal-regulated kinase, and p90 ribosomal s6 kinase was suppressed by CPF or procyanidin B2. In vitro and ex vivo kinase assay data demonstrated that CPF or procyanidin B2 inhibited the kinase activity of MEK1 and directly bound with MEK1. CPF or procyanidin B2 suppressed JB6 P+ cell transformation induced by epidermal growth factor or H-Ras, both of which are known to be involved in MEK/ERK signal activation. In contrast, theobromine (up to 80 μm) had no effect on TPA-induced transformation, cyclooxygenase-2 expression, the transactivation of activator protein-1 or nuclear factor-κB, or MEK. Notably, procyanidin B2 exerted stronger inhibitory effects compared with PD098059 (a well known pharmacological inhibitor of MEK) on MEK1 activity and neoplastic cell transformation.

Background: Ginseng (Panax ginseng C.A. Meyer) has been used as a traditional herb in the treatment of many medical disorders. Ginsenosides, which are triterpene derivatives that contain sugar moieties, are the main pharmacological ingredients in ginseng. This study was designed to investigate the effect of ginsenoside Rg3-enriched ginseng extract (Rg3GE) on scopolamine-induced memory impairment in mice. Methods: Rg3GE (50 and 100 mg/kg) were administered to C57BL/6 mice by oral gavage for 14 days (days 1-14). Memory impairment was induced by scopolamine (1 mg/kg, intraperitoneal injection) for 6 days (days 914). The Morris water maze test was used to assess hippocampus-dependent spatial memory. The effects of scopolamine with or without Rg3GE on acetylcholinesterase and nuclear factor-kappa B (NF-kappa B) in the hippocampus were also examined. Results: Mice with scopolamine treatment alone showed impairments in the acquisition and retention of spatial memory. Mice that received Rg3GE and scopolamine showed no scopolamine-induced impairment in the acquisition of spatial memory. Oral administration of Rg3GE suppressed the scopolamine-mediated increase in acetylcholinesterase activity and stimulation of the NF-kappa B pathway (i.e., phosphorylation of p65) in the hippocampus. Conclusion: These findings suggest that Rg3GE may stabilize scopolamine-induced memory deficits through the inhibition of acetylcholinesterase activity and NF-kappa B signaling in the hippocampus.

Numerous studies have shown that the levels of matrix metalloproteinase (MMP)-2 and/or MMP-9 are associated with the invasive phenotypes of cancer cells. This study investigated the effects of caffeic acid phenethyl ester (CAPE), a chemopreventive phytochemical derived from honeybee propolis, on the invasive phenotype of SK-Hep1 human hepatocellular carcinoma cells (SK-Hep1 cells). CAPE effectively suppressed SK-Hep1 cell invasion in a dose-dependent manner. The constitutive expression of MMP-2 and MMP-9 in SK-Hep1 cells was almost completely abolished by treatment with 12.5 muM CAPE. CAPE also significantly inhibited nuclear factor kappa B (NF-kappaB) DNA-binding activity in SK-Hep1 cells. These results taken together suggest that CAPE exerts antimetastatic potential through inhibition of MMP-2 and MMP-9 expression, possibly by targeting NF-kappaB in hepatocellular carcinoma.

Daidzein and genistein are isoflavones found in soybean. Genistein is known to exhibit anticarcinogenic activities and inhibit tyrosine kinase activity. However, the underlying molecular mechanisms of the chemopreventive activities of daidzein and its metabolite, equol, are not understood. Here we report that equol inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ mouse epidermal cells by targeting the MEK/ERK/p90RSK/activator protein-1 signaling pathway. TPA-induced neoplastic cell transformation was inhibited by equol, but not daidzein, at noncytotoxic concentrations in a dose-dependent manner. Equol dose-dependently attenuated TPA-induced activation of activator protein-1 and c-fos, whereas daidzein did not exert any effect when tested at the same concentrations. The TPA-induced phosphorylation of ERK1/2, p90RSK, and Elk, but not MEK or c-Jun N-terminal kinase, was inhibited by equol but not by daidzein. In vitro kinase assays revealed that equol greatly inhibited MEK1, but not Raf1, kinase activity, and an ex vivo kinase assay also demonstrated that equol suppressed TPA-induced MEK1 kinase activity in JB6 P+ cell lysates. Equol dose-dependently inhibited neoplastic transformation of JB6 P+ cells induced by epidermal growth factor or H-Ras. Both in vitro and ex vivo pull-down assays revealed that equol directly bound with glutathione S-transferase-MEK1 to inhibit MEK1 activity without competing with ATP. These results suggested that the antitumor-promoting effect of equol is due to the inhibition of cell transformation mainly by targeting a MEK signaling pathway. These findings are the first to reveal a molecular basis for the anticancer action of equol and may partially account for the reported chemopreventive effects of soybean.

Evidence suggests that mitogen-activated protein kinase kinase (MEK) plays a role in cell transformation and tumor development and might be a significant target for chemoprevention. 3,5,4'-Trihydroxy-trans-stilbene (resveratrol), a non-flavonoid polyphenol found in various foods and beverages, including red wines, is reported to be a natural chemopreventive agent. However, the concentrations required to exert these effects might be difficult to achieve by drinking only one or two glasses of red wine a day. On the other hand, the flavonol content of red wine is approximately 30 times higher than that of resveratrol. Here we demonstrated that 3,3',4',5,5',7-hexahydroxyflavone (myricetin), one of the major flavonols in red wine, is a novel inhibitor of MEK1 activity and transformation of JB6 P+ mouse epidermal cells. Myricetin (10 microM) inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA) or epidermal growth factor (EGF)-induced cell transformation by 76 or 72%, respectively, compared with respective reductions of 26 or 19% by resveratrol (20 microM). A combination of myricetin and resveratrol exerted additive but not synergistic effects on either TPA- or EGF-induced transformation. Myricetin, but not resveratrol, attenuated tumor promoter-induced activation of c-fos or activator protein-1. Myricetin strongly inhibited MEK1 kinase activity and suppressed TPA- or EGF-induced phosphorylation of extracellular signal-regulated kinase (ERK) or p90 ribosomal S6 kinase, downstream targets of MEK. Moreover, myricetin inhibited H-Ras-induced cell transformation more effectively than either PD098059, a MEK inhibitor, or resveratrol. Myricetin directly bound with glutathione S-transferase-MEK1 but did not compete with ATP. Overall, these results indicated that myricetin has potent anticancer-promoting activity and mainly targets MEK signaling, which may contribute to the chemopreventive potential of several foods including red wines.

Lycopene, which is the predominant carotenoid in tomatoes and tomato-based foods, may protect humans against various cancers. Effects of lycopene on the adhesion, invasion, migration, and growth of the SK-Hep1 human hepatoma cell line were investigated. Lycopene inhibited cell growth in dose-dependent manners, with growth inhibition rates of 5% and 40% at 0.1 microM and 50 microM lycopene, respectively, after 24 hrs of incubation. Similarly, after 48 hrs of incubation, lycopene at 5 microM and 10 microM decreased the cell numbers by 30% and 40%, respectively. Lycopene decreased the gelatinolytic activities of both matrix metalloproteinase (MMP)-2 and MMP-9, which were secreted from the SK-Hep1 cells. Incubation of SK-Hep1 cells with 110 microM of lycopene for 60 mins significantly inhibited cell adhesion to the Matrigel-coated substrate in a concentration-dependent manner. To study invasion, SK-Hep1 cells were grown either on Matrigel-coated Transwell membranes or in 24-well plates. The cells were treated sequentially for 24 hrs with lycopene before the start of the invasion assays. Cell growth and death were assessed under the same conditions. The invasion of SK-Hep1 cells treated with lycopene was significantly reduced to 28.3% and 61.9% of the control levels at 5 microM and 10 microM lycopene, respectively (P < 0.05). In the migration assay, lycopene-treated cells showed lower levels of migration than untreated cells. These results demonstrate the antimetastatic properties of lycopene in inhibiting the adhesion, invasion, and migration of SK-Hep1 human hepatoma cells.

A bifunctional alcohol/acetaldehyde dehydrogenase (AdhE) gene (adhE) was cloned from Leuconostoc mesenteroides C7 (LMC7), which is the dominant lactic acid bacterium produced during heterofermentation of kimchi. The nucleotide sequence of the DNA fragment containing putative adhE, which is 2685 bp long and encodes an 886 amino acid polypeptide, exhibits 99% homology with Leu. mesenteroides sp. cremoris. The deduced AdhE comprises two conserved domains: alcohol dehydrogenase (Adh) and acetaldehyde dehydrogenase (Aldh). Moreover, two NAD-binding sites were observed, based on the presence of the GXGXXG motif. A pADHE containing the adhE gene expressed AdhE at the translational level in Escherichia coli BL21, which was at a higher level than in E. coli DH5alpha and E. coli JM109. The AdhE of LMC7 showed Adh and Aldh activities that, when expressed in E. coli. BL21, were 7.5 and 5.7 U mg(-1) , respectively.

To examine a potential role for soybean phytoestrogens in postmenopausal bone loss, twenty-four 12-week-old Sprague-Dawley rats were divided randomly into 4 groups and given controlled diets for 16 weeks. The treatment groups were as followed: sham operated, ovariectomized (OVX) control, OVX + isoflavone extract (6.25 g/kg), and OVX + 17beta-estradiol (4 mg/kg). OVX treatments reduced femoral and fourth lumbar vertebral bone density and mineral content (p0.01), decreased uterine weight (p0.01), accelerated body weight increases (p0.05), and increased the activities (p0.01) of both serum alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP). Supplementation with isoflavone prevented the losses of bone density and mineral content caused by OVX (p0.01). Although both isoflavone and 17beta-estradiol exhibited similar bone-sparing ability on the OVX-induced bone loss, the effect of isoflavone was not the same as that of 17beta-estradiol on the serum ALP and TRAP, body weight increase, and uterine weight change. We concluded that dietary supplementation with soybean isoflavone can prevent postmenopausal bone loss via a different mechanism of estrogen in OVX rats.

The new integration vector for Lactobacillus, pJC4, was developed using the extracellular endoglucanase A gene (celA) of Clostridium thermocellum as a screening marker. pJC4 was transformed into four Lactobacillus species, Lb. johnsonii, Lb. gasseri, Lb. bulgaricus, and Lb. plantarum. In each species, the pJC4 integrants were easily and accurately detected by the appearance of a clear halo on a cellulase screening plate without any false transformants. Polymerase chain reaction and Southern hybridization indicated that all transformants with clear halos contained pJC4 in their chromosomal DNAs. The celA gene could be a useful screening marker for other lactic acid bacteria.

A DNA fragment, which complemented the growth of E. coli both on M9 medium containing raffinose and on LB medium containing ampicillin, IPTG and 5-bromo-4-chloro-3-indoxyl-α-d-galactoside, was isolated from the genomic library of Bifidobacterium longum SJ32, which had been digested with EcoRI. In the cloned DNA fragment, a gene encoding a sucrose phosphorylase (splP) and a partially cloned putative sucrose regulator gene (splR) were identified using the deletion analysis and sequence analysis. A 56 kDa protein was synthesized in E. coli and partially purified by DEAE-ion exchange chromatography. The partially purified enzyme did not react with melibiose, melezitoze and raffinose but did with sucrose. It had transglucosylation activity in addition to hydrolytic activity.

Combination chemotherapy for the treatment of pancreatic cancer commonly employs gemcitabine with an EGFR inhibitor such as erlotinib. Here, we show that the retinoic acid derivative, ABPN, exhibits more potent anticancer effects than erlotinib, while exhibiting less toxicity toward noncancerous human control cells. Low micromolar concentrations of ABPN induced apoptosis in BxPC3 and HPAC pancreatic cancer cell lines, concomitant with a reduction in phosphorylated EGFR as well as decreased ErbB3, Met and BRUCE protein levels. The degradation of ErbB3 is a result of proteasomal degradation, possibly due to the ABPN-dependent upregulation of Nrdp1. Administration of ABPN showed significant reductions in tumor size when tested using a mouse xenograft model, with higher potency than erlotinib at the same concentration. Analysis of the tumors demonstrated that ABPN treatment suppressed ErbB3 and Met and induced Nrdp1 in vivo. The data suggest that ABPN may be more suitable in combination chemotherapy with gemcitabine than the more widely used EGFR inhibitor, erlotinib.

Whole cells, cytoplasms and peptidoglycans of ten different lactic acid bacteria (LAB) were tested for in vitro cytotoxicity on diverse cancer cell lines using the 3H-thymidine incorporation assay. The peptidoglycans and cytoplasm fractions, as well as heat-killed whole cells of LAB, had significant antiproliferative activities against several cancer cell lines. In particular, the cytoplasm fractions exhibited marked direct antiproliferative activities against colon and gastric cancer cell lines, whereas the peptidoglycans retarded growth of colon and bladder cancer cell lines. The cytoplasm fractions of Bifidobacterium longum and Lactococcus lactis ssp. lactis inhibited proliferation of two cancer cell lines by 50% at 33 and 23 μg ml−1 for SNUC2A (a human colon adenocarcinoma cell line) and 17 and 11 μg ml−1 for SNU-1 (a human gastric cancer cell line), respectively.

Zanthoxylum piperitum (prickly ash) was grown as a suspension culture in Schenk and Hildebrandt medium supplemented with 50 g sucrose l−1 and 0.5 mg 2,4-dichlorophenoxyacetic acid l−1 for 21 days with elicitation by pectin added at day 15. Volatile compounds were extracted from the culture and 3-hydroxy-2-butanone was identified by GC-MS as the most abundant compound, followed by γ-butyrolactone and 2,3-butanedione. 2,3-Butanedione, ethyl 3-methylbutyrate, and ethyl 2-methylpropanoate were identified as the most intense aroma-active compounds and represented the characteristic aroma of the culture.