Modified C-type natriuretic peptide normalizes tumor vasculature, reinvigorates antitumor immunity, and improves solid tumor therapies.

Deficit of oxygen and nutrients in the tumor microenvironment (TME) triggers abnormal angiogenesis that produces dysfunctional and leaky blood vessels, which fail to adequately perfuse tumor tissues. Resulting hypoxia, exacerbation of metabolic disturbances, and generation of an immunosuppressive TME undermine the efficacy of anticancer therapies. Use of carefully scheduled angiogenesis inhibitors has been suggested to overcome these problems and normalize the TME. Here, we propose an alternative agonist-based normalization approach using a derivative of the C-type natriuretic peptide (dCNP). Multiple gene expression signatures in tumor tissues were affected in mice treated with dCNP. In several mouse orthotopic and subcutaneous solid tumor models including colon and pancreatic adenocarcinomas, this well-tolerated agent stimulated formation of highly functional tumor blood vessels to reduce hypoxia. Administration of dCNP also inhibited stromagenesis and remodeling of the extracellular matrix and decreased tumor interstitial fluid pressure. In addition, treatment with dCNP reinvigorated the antitumor immune responses. Administration of dCNP decelerated growth of primary mouse tumors and suppressed their metastases. Moreover, inclusion of dCNP into the chemo-, radio-, or immune-therapeutic regimens increased their efficacy against solid tumors in immunocompetent mice. These results demonstrate the proof of principle for using vasculature normalizing agonists to improve therapies against solid tumors and characterize dCNP as the first in class among such agents.

Fustin suppressed melanoma cell growth via cAMP/PKA-dependent mechanism.

Melanoma, a cancer arising from melanocytes, requires a novel treatment strategy because of the ineffectiveness of conventional therapies in certain patients. Fustin is a flavanonol found in young fustic (Cotinus coggygria). However, little is known about its antimelanoma effects. Our study demonstrates that fustin suppresses the growth of B16 melanoma cells. Phalloidin staining of cytoskeletal actin revealed that fustin induced a conformational change in the actin structure of melanoma cells, accompanied by suppressed phosphorylation of myosin regulatory light chain 2 (MLC2), a regulator of actin structure. Furthermore, the protein kinase A (cAMP-dependent protein kinase) inhibitor H89 completely attenuated fustin-induced downregulation of phosphorylated myosin phosphatase targeting subunit 1, which is involved in dephosphorylation of MLC2. In a mouse model, administration of fustin suppressed tumor growth in B16 melanoma cells without adverse effects. In conclusion, our findings suggest that fustin effectively suppresses melanoma cell growth both in vitro and in vivo.

Neuroprotective effect of isovaleraldehyde accompanied with upregulation of BDNF and CREB phosphorylation via the PKA pathway.

Recently, the number of patients diagnosed with dementia has increased. The World Health Organization (WHO) estimates that 50 million patients suffer from dementia. Although several therapeutic strategies have been proposed, currently, there is no curative approach for treating dementia. Neurodegeneration is an irreversible process. As this disease gradually progresses over 15-20 years, a low-cost and sustainable method for preventing these diseases is desired. Cacao nib is consumed in many countries, and a recent clinical study indicated that cocoa intake upregulates brain-derived neurotrophic factor (BDNF), which plays a significant role in memory formation and neuronal cell survival. In the present study, neural cells were treated with cacao nib extract or the 17 characteristic components of cacao nib. Treatment with Cacao nib extract upregulates BDNF mRNA expression. In addition, cacao nib extract elicits the phosphorylation of cAMP-response-element-binding protein (CREB), which regulates the transcription of BDNF. Among the 17 species screened, isovaleraldehyde (IVA), also known as an aroma component of cacao nibs extract, improved BDNF mRNA expression without SH-SY5Y cell toxicity. IVA also promoted CREB phosphorylation through a cAMP-dependent protein kinase (PKA)-dependent mechanism. In conclusion, IVA could be responsible for the BDNF upregulation effect of cacao nib, and IVA upregulated BDNF expression via the PKA-CREB axis.

The anti-cancer effect of epigallocatechin-3-O-gallate against multiple myeloma cells is potentiated by 5,7-dimethoxyflavone.

(-)-Epigallocatechin-3-O-gallate (EGCG) is one of the major components of green tea polyphenol. Previous studies have shown that EGCG induces cancer-specific cell death in vitro and in vivo without causing severe side effects. However, the anti-cancer effect of EGCG alone is limited. 5,7-dimethoxyflavone (5,7-DMF), one of the principal functional components of black ginger (Kaempferia parviflora), also exerts anti-cancer effects. Here, we show that 5,7-DMF synergistically enhances the anti-cancer effect of EGCG in multiple myeloma cells by potentiating EGCG-induced intracellular cyclic guanosine monophosphate (cGMP) production. Moreover, the combination of EGCG and 5,7-DMF induces apoptotic cell death in multiple myeloma cells, and this is accompanied by activation of the cGMP/acid sphingomyelinase (ASM)/cleaved caspase-3 pathway. In conclusion, we have shown that 5,7-DMF enhances the anti-cancer effect of EGCG by upregulating cGMP in multiple myeloma cells.

67-kDa laminin receptor mediates oolonghomobisflavan B-induced cell growth inhibition in melanoma.

BACKGROUND: Oolonghomobisflavans are unique polyphenols found in oolong teas. Oolonghomobisflavan B (OHBFB), a dimer of (-)-epigallocatechin-3-O-gallate (EGCG), is an active compound found in green tea. PURPOSE: OHBFB has been reported to exert an inhibitory effect on lipase enzyme activity. However, little is known regarding its intercellular signaling induction effect. Further, there are no reports describing the anti-cancer effects of OHBFB. METHODS: The effect of OFBFB on B16 melanoma cells was evaluated by cell counting, and its mechanisms were determined by western blot analysis with or without protein phosphatase 2A (PP2A) inhibitor treatment. Intracellular cyclic adenosine monophosphate (cAMP) levels were evaluated by time-resolved fluorescence resonance energy transfer analysis. Quartz crystal microbalance (QCM) analysis was performed to assess the binding of OHBFB to 67LR. RESULTS: Cell growth assay and western blot analyses showed that OHBFB inhibited melanoma cell growth, followed by myosin phosphatase target subunit 1 (MYPT1) and myosin regulatory light chain (MRLC) dephosphorylation via protein phosphatase 2A (PP2A)-dependent mechanisms. These effects are mediated by intracellular cAMP- and protein kinase A (PKA) A-dependent mechanisms. QCM analysis identified the 67-kDa laminin receptor (67LR) as an OHBFB receptor with a Kd of 3.7 µM. We also demonstrated for the first time that OHBFB intake suppresses tumor growth in vivo. CONCLUSIONS: Taken together, these results indicate that the cAMP/PKA/PP2A/MYPT1/MRLC pathway is a key mediator of melanoma cell growth inhibition following OHBFB binding to 67LR and that OHBFB suppresses tumor growth in vivo.

PPAR/PDK4 pathway is involved in the anticancer effects of cGMP in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a type of cancer with a high mortality rate. Current treatments for PDACs often have side effects, and drug resistance in cancer stem cells (CSCs) would be also a problem. Cyclic guanosine monophosphate (cGMP) suppresses the mitochondrial function of PDACs and inhibits their CSC properties. Metabolic regulation plays a crucial role in the maintenance of CSC phenotype, and we hypothesized that cGMP induction suppresses cancer stem cell properties in the cancer cell through energy-related signaling pathways. We demonstrated that induction of cGMP upregulated the PPARα/PDK4 pathway and suppressed CSC properties in PDAC, and patients with pancreatic cancer with high PDK4 gene expression had a better prognosis than those with low gene expression. Therefore, these mechanisms may provide new therapeutic targets for the eradication of pancreatic CSCs.

Plant miRNA osa-miR172d-5p suppressed lung fibrosis by targeting Tab1.

Lung fibrosis, including idiopathic pulmonary fibrosis, is an intractable disease accompanied by an irreversible dysfunction in the respiratory system. Its pathogenesis involves the transforming growth factorß (TGFß)-induced overproduction of the extracellular matrix from fibroblasts; however, limited countermeasures have been established. In this study, we identified osa-miR172d-5p, a plant-derived microRNA (miR), as a potent anti-fibrotic miR. In silico analysis followed by an in vitro assay based on human lung fibroblasts demonstrated that osa-miR172d-5p suppressed the gene expression of TGF-ß activated kinase 1 (MAP3K7) binding protein 1 (Tab1). It also suppressed the TGFß-induced fibrotic gene expression in human lung fibroblasts. To assess the anti-fibrotic effect of osa-miR172d-5p, we established bleomycin-induced lung fibrosis models to demonstrate that osa-miR172d-5p ameliorated lung fibrosis. Moreover, it suppressed Tab1 expression in the lung tissues of bleomycin-treated mice. In conclusion, osa-miR172d-5p could be a potent candidate for the treatment of lung fibrosis, including idiopathic pulmonary fibrosis.

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.

Phosphodiesterase 5 Inhibitor Potentiates Epigallocatechin 3-O-Gallate-Induced Apoptotic Cell Death via Activation of the cGMP Signaling Pathway in Caco-2 Cells.

Epigallocatechin 3-O-gallate (EGCG) is a predominant component in green tea with various health benefits. The 67 kDa laminin receptor (67LR) is a nonintegrin cell surface receptor that is overexpressed in various types of cancer; 67LR was identified a cell surface EGCG target that plays a pivotal role in tumor growth, metastasis, and resistance to chemotherapy. However, the plasma concentration of EGCG is limited, and its molecular mechanisms remain unelucidated in colon cancer. In this study, we found that the phosphodiesterase 5 (PDE5) inhibitor, vardenafil (VDN), potentiates EGCG-induced apoptotic cell death in colon cancer cells. The combination of EGCG and VDN induced apoptosis via activation of the endothelial nitric oxide synthase/cyclic guanosine monophosphate/protein kinase Cδ signaling pathway. In conclusion, the PDE5 inhibitor, VDN, may reduce the intracellular PDE5 enzyme activity that potentiates EGCG-induced apoptotic cell death in Caco-2 cells. These results suggest that PDE5 inhibitors can be used to elevate cGMP levels to induce 67LR-mediated, cancer-specific cell death. Therefore, EGCG may be employed as a therapeutic candidate for colon cancer.

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.

Inflammatory markers S100A8/A9 and metabolic alteration for evaluating signs of early phase toxicity of anticancer agent treatment.

Anticancer agents can cause various side effects, including tissue damages/inflammatory reactions. Drug-responsive biomarkers are essential for evaluating drug toxicity in disease processes. S100 calcium-binding proteins A8/A9 (S100A8/A9) are highly expressed in neutrophils and monocytes/macrophages accumulated at inflammatory sites and are known to be related to tissue damage/inflammation; however, their response to drug toxicity has not been reported. Herein, we investigated the effects of anticancer agents (doxorubicin, cisplatin, and docetaxel) on S100A8/A9 gene expression profiles in four representative tissues (heart, kidney, liver, and lung) in normal C57BL/6J mice. Both S100A8/A9 expression was transiently or time-dependently elevated in four tissues within 48 h after dosing of the three anticancer agents under toxicity-inducing conditions. S100A8/A9 patterns differed among agents and tissues. This result suggests that S100A8/A9 is useful for evaluating anticancer agent-induced tissue damage. Metabolomic analysis revealed that some metabolites showed temporal patterns similar to that of S100A8/A9 expression. The amounts of fumarate (doxorubicin-treated heart), tyrosine (cisplatin-treated kidney), acetylcarnosine (doxorubicin-treated liver), and 2-phosphoglycerate (docetaxel-treated lung) showed similar patterns to that of S100A8/A9 expression. Although these metabolites showed different behaviors between tissues and serum, they may be useful marker candidates for evaluating anticancer agent-induced tissue damage at an earlier stage after dosing.

67-kDa Laminin Receptor-Mediated Cellular Sensing System of Green Tea Polyphenol EGCG and Functional Food Pairing.

The body is equipped with a "food factor-sensing system" that senses food factors, such as polyphenols, sulfur-containing compounds, and vitamins, taken into the body, and plays an essential role in manifesting their physiological effects. For example, (-)-epigallocatechin-3-O-gallate (EGCG), the representative catechin in green tea (Camellia sinensi L.), exerts various effects, including anti-cancer, anti-inflammatory, and anti-allergic effects, when sensed by the cell surficial protein 67-kDa laminin receptor (67LR). Here, we focus on three representative effects of EGCG and provide their specific signaling mechanisms, the 67LR-mediated EGCG-sensing systems. Various components present in foods, such as eriodictyol, hesperetin, sulfide, vitamin A, and fatty acids, have been found to act on the food factor-sensing system and affect the functionality of other foods/food factors, such as green tea extract, EGCG, or its O-methylated derivative at different experimental levels, i.e., in vitro, animal models, and/or clinical trials. These phenomena are observed by increasing or decreasing the activity or expression of EGCG-sensing-related molecules. Such functional interaction between food factors is called "functional food pairing". In this review, we introduce examples of functional food pairings using EGCG.

Metabolic Profiling for Evaluating the Dipeptidyl Peptidase-IV Inhibitory Potency of Diverse Green Tea Cultivars and Determining Bioactivity-Related Ingredients and Combinations.

There are numerous cultivars of tea (Camellia sinensis L.), but the differences in their anti-hyperglycemic-related effects are largely unknown. The inhibition of the dipeptidyl peptidase (DPP)-IV enzyme plays an essential role in controlling hyperglycemia in diabetes by blocking the degradation of incretin hormones, which is necessary for insulin secretion. In this study, we examined the DPP-IV inhibitory activity of leaf extracts from diverse Japanese green tea cultivars. The inhibitory rates differed among tea extracts. Metabolic profiling (MP), using liquid chromatography-mass spectrometry, of all cultivars revealed compositional differences among cultivars according to their DPP-IV inhibitory capacity. Epigallocatechin-3-O-(3-O-methyl)gallate, kaempferol-3-O-rutinoside, myricetin-3-O-glucoside/galactoside, and theogallin were newly identified as DPP-IV inhibitors. The bioactivity of a tea extract was potentiated by adding these ingredients in combination. Our results show that MP is a useful approach for evaluating the DPP-IV inhibitory potency of green tea and for determining bioactivity-related ingredients and combinations.

Myricetin improves cognitive function in SAMP8 mice and upregulates brain-derived neurotrophic factor and nerve growth factor.

INTRODUCTION: Considering that neurodegeneration is an irreversible process, an efficient, low-burden approach to prevent dementia is strongly needed. Here, we show that the daily intake of myricetin normalised cognitive dysfunction in senescence-accelerated mouse prone 8 (SAMP8) mice. METHODS: SAMP8 mice were fed a diet supplemented with myricetin and novel object recognition tests and Y-maze tests were performed. Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in brains of SAMP8 mice were measured. The phosphorylation level of cAMP-response-element-binding protein (CREB) level in brains of SAMP8 mice were evaluated. Also, SH-SY5Y cells were treated with myricetin and cAMP levels were measured. RESULTS: In SAMP8 mice, neurotrophins, including BDNF and NGF, were downregulated relative to levels in their normal counterparts. In addition, myricetin intake upregulated the phosphorylation of CREB, the major transcription factor for BDNF and NGF. Also, myricetin induced cAMP upregulation, and CREB phosphorylation via a cAMP-dependent protein kinase-dependent manner in SH-SY5Y cells. CONCLUSION: Taken together, myricetin improves cognitive function in SAMP8 mice and upregulates BDNF and NGF.

Sesame lignans upregulate glutathione S-transferase expression and downregulate microRNA-669c-3p.

Oxidative stress is associated with aging and pathologies such as cardiovascular diseases, Alzheimer's disease, and cancer. Glutathione S-transferase (GST), a family of detoxification enzymes, plays a crucial role in countering oxidative stress. Therefore, there is a need for the development of physiologically functional foods and agricultural products, which enhance GST activity. Sesamin and episesamin are major lignans in refined sesame oil that exhibit beneficial properties including antioxidative stress effects. A previous study showed that sesamin upregulated GST activity. This study aimed to elucidate the mechanism underlying the GST activity enhancement elicited by sesame lignans. C57BL/6J mice were orally administered 20 mg/kg body weight sesame lignans (sesamin:episesamin=1:1) for 7 days. Oral administration of sesame lignans increased the GST activity in the mouse liver. Furthermore, the lignans upregulated GSTA1, GSTA4, and GSTM4 protein expression. Microarray analysis revealed that sesame lignans changed the expression of various microRNAs (miRNAs) (84 upregulated, 19 downregulated). We also found 16 miRNAs, including miR-669c-3p, that may negatively regulate GST expression among the 19 miRNAs with reduced expression caused by the sesame lignans. miR-669c is reportedly negatively correlated with GST. Additionally, we transfected NMuLi cells with an miR-669c-3p mimic and evaluated the effect of miR-669c-3p on GST mRNA and protein expressions. The results showed that the miR-669c-3p mimic suppressed the mRNA and protein levels of GSTA4 and GSTM4. In conclusion, sesame lignans increased GST protein expression and activity and downregulated miRNAs, including miR-669c-3p, which is a possible suppressor of GST.

Fustin, a Flavanonol, Synergically Potentiates the Anticancer Effect of Green Tea Catechin Epigallocatechin-3-O-Gallate with Activation of the eNOS/cGMP Axis.

Epigallocatechin-3-O-gallate (EGCG), a catechin present in green tea, selectively elicits apoptosis in multiple myeloma cells by activating the endothelial nitric oxide synthase (eNOS)/cyclic guanosine monophosphate (cGMP) axis. However, the effects of EGCG alone are limited. Herein, we revealed that fustin, a flavanonol, enhances the EGCG-elicited activation of the cGMP/eNOS axis in multiple myeloma cells. Isobologram analysis demonstrated that EGCG/fustin synergistically elicited cell death in multiple myeloma cells. Importantly, this chemical combination significantly promoted cell death without affecting the normal cells. To assess the effects of EGCG and fustin in vivo, female BALB/c mice were inoculated with multiple myeloma MPC11 cells and then treated with each compound. The combination of EGCG/fustin suppressed tumor growth in vivo without affecting alanine aminotransferase/aspartate aminotransferase levels, the dose-limiting toxicity of EGCG. Consistent with in vitro findings, this combination increased eNOS phosphorylation at Ser1177 in the tumor. Collectively, fustin amplified EGCG-induced activation of the eNOS/cGMP axis.

Methylated (-)-epigallocatechin 3-O-gallate potentiates the effect of split vaccine accompanied with upregulation of Toll-like receptor 5.

Split-virus vaccine serves as a major countermeasure against influenza virus, but its effectiveness and protective action are not complete. We previously demonstrated the effect of Benifuuki, a green tea cultivar in Japan, on enhancing the split-virus vaccine-elicited immune response. However, little is known about the detail mechanisms. Here, we show that EGCG3"Me intake significantly potentiated the vaccine-elicited hemagglutination inhibition titer increase. Flow cytometry analysis revealed the increased Toll-like receptor 5 (TLR5) expression after EGCG3"Me treatment in lamina propria dendritic cells (LPDCs) and macrophages, which play crucial roles in the humoral immune system. TLR5 expression correlated with the level of interleukin-6 (IL-6)/C-C chemokine type receptor 5, which are important mediators of the humoral immunity. Taken together, In vivo and ex vivo studies showed that EGCG3"Me potentiated the split-virus vaccine-elicited immune response accompanied with the upregulation of TLR5 in intestine and splenocyte macrophages.

The combined effect of green tea and α-glucosyl hesperidin in preventing obesity: a randomized placebo-controlled clinical trial.

Green tea, a widely consumed beverage in Asia, contains green tea catechins effective against obesity, especially epigallocatechin-3-O-gallate (EGCG), but must be consumed in an impractically huge amount daily to elicit its biological effect. Meanwhile, citrus polyphenols have various physiological effects that could enhance EGCG functionality. Here we investigated the antiobesity effect of a combination of EGCG and α-glucosyl hesperidin, a citrus polyphenol, at doses that have not been previously reported to exert antiobesity effects by themselves in any clinical trial. In a randomized, placebo-controlled, double-blinded, and parallel-group-designed clinical trial, 60 healthy Japanese males and females aged 30-75 years consumed green tea combined with α-glucosyl hesperidin (GT-gH), which contained 178 mg α-glucosyl hesperidin and 146 mg EGCG, for 12 weeks. Physical, hematological, blood biochemical, and urine examinations showed that GT-gH is safe to use. At week 12, GT-gH prevented weight gain and reduced body mass index (BMI) compared with the placebo. Especially in those aged < 50 years, triglyceride and body fat percentage decreased at week 6, visceral fat level and body fat percentage decreased at week 12; body weight, BMI, and blood LDL/HDL ratio also decreased. In conclusion, taking GT-gH prevents weight gain, and the antiobesity effect of GT-gH was more pronounced in people aged < 50 years.

Glucosyl-hesperidin enhances the cyclic guanosine monophosphate-inducing effect of a green tea polyphenol EGCG.

Animal and clinical studies have revealed that (-)-epigallocatechin-3-O-gallate (EGCG), one of the major bioactive polyphenols in green tea, showed several pharmacological effects including anti-obesity effect and anti-inflammatory effect. We previously reported that the second messenger cyclic guanosine monophosphate (cGMP) mediates its anti-inflammatory and anti-cancer properties. Here we demonstrated that glucosyl-hesperidin, enhances the cGMP-inducing effects of green tea extract in vivo. Moreover, glucosyl-hesperidin intake potentiated the green tea-elicited upregulation of the anti-inflammatory factor, toll-interacting protein.

Src Mediates Epigallocatechin-3-O-Gallate-Elicited Acid Sphingomyelinase Activation.

Epigallocatechin-3-O-gallate (EGCG) is one of the major bioactive compounds known to be present in green tea. We previously reported that EGCG shows selective toxicity through activation of the protein kinase B (Akt)/cyclic guanosine monophosphate (cGMP)/acid sphingomyelinase (ASM) axis via targeting its receptor 67-kDa laminin receptor (67LR), which is overexpressed in cancer. However, little is known about upstream mechanisms of EGCG-elicited ASM activation. In this study we show that the proto-oncogene tyrosine-protein kinase Src, also known as c-src, plays a crucial role in the anticancer effect of EGCG. We showed that EGCG elicits phosphorylation of Src at Tyr 416, a crucial phosphorylation site for its activity, and that the pharmacological inhibition of Src impedes the upstream events in EGCG-induced cell death signaling including upregulation of Akt activity, increase in cGMP levels, and activation of ASM. Moreover, focal adhesion kinase (FAK), which is involved in the phosphorylation of Src, is colocalized with 67LR. EGCG treatment enhanced interaction of FAK and 67LR. Consistent with these findings, pharmacological inhibition of FAK significantly neutralized EGCG-induced upregulation of Akt activity and activation of ASM. Taken together, FAK/Src play crucial roles in the upstream signaling of EGCG.