Combination Of Epigallocatechin-3-gallate Research Articles

Combination of 5-Florouracil and polyphenol EGCG exerts suppressive effects on oral cancer cells exposed to radiation

ObjectiveNatural compounds such as epigallocatechin-3-gallate (EGCG) have previously shown chemotherapeutic properties with few side-effects. In our study, we evaluated the effects of combining EGCG with 5-fluorouracil (5-FU) and radiotherapy on oral squamous cell cancer. We evaluated whether the combination of lower doses of 5-FU with EGCG could be equally or more effective than the use of higher doses of 5-FU alone. MethodsCell viability, migration and cell cycles were assayed in oral cancer cell lines treated with 5-FU, 5-FU + EGCG and radiation (0, 2.5 and 5 Gy). ResultsThis study found that the combination of EGCG with 5-FU reduced cell viability and migration distance compared to control samples and the same dose of 5-FU alone. Addition of EGCG increased the number of cells in the G2/M phase, while 5-FU arrested the cell cycle in phase S. Moreover, cell exposure to 5 Gy radiation decreased the effects of combining with EGCG. ConclusionsIn summary, the combination of EGCG and 5-FU reduced both cell viability and migration as well as altered the cell cycle to a greater extent than 5-FU alone.

Diet supplemented with phytochemical epigallocatechin gallate and probiotic Lactobacillus fermentum confers second generation synbiotic effects by modulating cellular immune responses and antioxidant capacity in aging mice.

In the present study, we systematically identified and evaluated a synbiotic combination of phytochemical epigallocatechin gallate (EGCG) and probiotic bacteria in amelioration of immunosenescence and oxidative stress in aged mice. Inhibitory effects of EGCG against different bacterial species were evaluated in vitro, followed by analysis to identify potential combination of EGCG and probiotic bacteria against alleviation of oxidative and inflammatory stress ex vivo. The best synbiotic combination, vis-à-vis prebiotic and probiotic supplementation alone, was then evaluated in aged Swiss albino mice for modulation of various immunological and antioxidative parameters. EGCG strongly inhibited the growth of pathogenic microbes as compared to probiotic bacteria. A combination of EGCG with probiotic Lactobacillus fermentum (LF) provided evidence of additive effects in the amelioration of oxidative and inflammatory stress-induced cell death. In vivo study revealed that combined supplementation of LF and EGCG significantly enhanced neutrophil oxidative index, CD3+ cell numbers and activation status, Th1/Th2 cytokines in splenic supernatants as well as liver Nrf-2 expression in comparison with treatments with LF or EGCG alone. The combined application of EGCG and LF did not simply result in additive or synergistic effects in relation with individual treatments. These observations suggest that EGCG could be considered as a potential prebiotic that can offer second generation synbiotic health beneficial effects for the alleviation of some of the deleterious aspects of immunosenescence and aging.

Assessment of diets containing curcumin, epigallocatechin-3-gallate, docosahexaenoic acid and α-lipoic acid on amyloid load and inflammation in a male transgenic mouse model of Alzheimer's disease: Are combinations more effective?

Increasingly, evidence is accumulating pointing at a protective role of a healthy diet at decreasing the risk of Alzheimer's disease. To test the effectiveness of nutritional components, the following food-derived compounds: curcumin alone (curcumin), curcumin combined with (−)epigallocatechin-3-gallate (EGCG), docosahexaenoic acid (DHA) and α-lipoic acid (ALA) (curcumin + EDA), or a combination of EGCG, DHA and ALA (EDA) were assessed in male Tg2576 transgenic mice on amyloid plaque load, amyloid levels (Aβ40/Aβ42, but not oligomers due to tissue limitations), microglial activation and memory using the contextual and cued fear conditioning test. The combination diet EDA, resulted in the strongest reduction of amyloid plaque load in both the cortical (p < .0001) and hippocampal (p < .0001) areas of the Tg2576 mouse brain, along with lower Aβ40/Aβ42 levels in the frontal cortex (p = .000129 and p = .000039, respectively) and Aβ42 levels in the temporal lobe (p = .000082). A curcumin only diet was shown to lower amyloid plaque load (p = .028), but when combined with EGCG, DHA and ALA did not result in further decreases in amyloid plaque load. The EDA combination group showed the most prominent decrease in microglial activation (number of microglia around plaques: p < .05 and p < .0001, respectively, for the cortex and hippocampus). Analysing the hippocampal associated contextual fear conditioning revealed that both the curcumin+EDA (p < .0001) and EDA groups (p = .001) spent increased time on freezing compared to the control group. In addition, the curcumin+EDA group showed a significant increase in time spent freezing compared with the curcumin only group. In the amygdala associated cued test, all mice demonstrated the ability to associate the conditioned stimulus with the unconditioned stimulus as evidenced by a significant increase in freezing behaviour in response to the presentation of the cue (p < .0001). Post-hoc analysis showed that only curcumin+EDA (p < .0001) and EDA groups (p < .0001) developed a significant increase in freezing during the cue presentation. The results from this study show that the combination of EGCG, DHA and ALA (EDA) appeared to have the most potent anti-inflammatory and neuroprotective effect. Our results also demonstrate that interactions between nutraceutical products might result in counterproductive outcomes, highlighting the fact that manufacturers of nutraceuticals containing multiple compounds should be careful not to claim additive or synergistic effects of their combination products in vivo without having tested it in animal models and/or human clinical trials.

Shining a Light on the Effects of the Combination of (-)-Epigallocatechin-3-gallate and Tapentadol on the Growth of Human Triple-negative Breast Cancer Cells.

Breast cancer is characterized by a high rate of mortality and is considered one of the deadliest types of cancer. It is of note that (-)-epigallocatechin-3-gallate (EGCG), the principal catechin of green tea, is able to hinder the growth of MDA-MB-231 breast cancer cells by influencing different signaling pathways, including apoptosis. Furthermore, EGCG is also used in the treatment of bone cancer pain. Tapentadol, an opioid drug acting at the level of noradrenaline (norepinephrine) reuptake inhibition and μ-opioid receptor, is able to modulate bone cancer pain and influence cancer cell viability by regulating apoptosis. In vitro assays were performed on triple-negative MDA-MB-231 cells treated with tapentadol (1, 5, 10, 20, 40 and 80 μg/ml) and EGCG (1, 10, 20, 40, 80, 160 μmol/l), alone and in combination. The effects of EGCG and TAP on viability were determined by wound-healing and MTT assays, while cell migration was assessed by transwell migration. Cell proliferation, viability and apoptosis of MDA-MB-231 cells were impaired by the combination of EGCG and tapentadol. Specifically, our data show that EGCG and TAP reduced the proliferation of MDA-MB-231 cells by impairing cell-cycle progression (p<0.05). These findings suggest that the combination of these substances may represent a new strategy for the treatment of patients suffering from triple-negative breast cancer.

Elucidating the Non-Catalytic Function of Fatty Acid Synthase and Its Autophagy-Dependent Degradation in Acute Myelocytic Leukemia Differentiation Therapy

Acute myeloid leukemia (AML) is characterized by a block in myeloid differentiation and an aberrant cell proliferation of leukemia blasts. While other AML therapies cure only 30% of the patients, acute promyelocytic leukemia (APL) is successfully treated by differentiation therapy using all-trans retinoic acid (ATRA) in combination with chemotherapy or arsenic trioxide. A better understanding of the molecular mechanisms underlying ATRA therapy in APL may offer new perspectives in the treatment of additional AML subtypes.Fatty acid synthase (FASN) is the only human lipogenic enzyme able of de novo fatty acid synthesis. Its expression is very low in healthy adult tissues, whereas increased expression is commonly found in a variety of cancerous tissues. Therefore, FASN represents an attractive potential drug target.To investigate if a similar expression pattern is found in normal versus malignant myeloid cells, we determined FASN expression in fresh leukemic blast cells from untreated AML patients obtained at the Inselspital Bern (Switzerland) (n=68) and expression data available online from the Bloodspot server (n=203). In both AML cohorts, we found that FASN expression is significantly higher in AML patients than in healthy granulocytes (p<0.05). Accordingly, FASN protein levels are reduced in NB4 and HT93 APL cells upon ATRA-induced granulocyte differentiation. Additionally, silencing FASN in NB4 cells led to an acceleration of differentiation upon ATRA treatment. Surprisingly, solely inhibiting the catalytic function of FASN using C75 or Orlistat did not affect granulocyte differentiation nor FASN expression. To validate that FASN levels and not activity attenuate granulocyte differentiation, we took advantage of the green tea polyphenol epigallocatechin-3-gallate (EGCG) that reduces FASN protein expression. Indeed, similar to knocking down FASN, EGCG treatment of NB4 cells resulted in increased differentiation paralleled by decreased FASN protein levels.We and others demonstrated the importance of autophagy for successful ATRA-induced APL differentiation. Autophagy is an intracellular degradation system that ensures a dynamic recycling of cytoplasmic contents. This process is regulated by the so-called autophagy related genes or ATGs. ATG1, or ULK1, is a key autophagy gene of the initiation complex that is inhibited by mTOR-mediated phosphorylation leading to reduced autophagy activity.FASN expression correlates with mTOR activity in several solid cancers, therefore we hypothesized that FASN activates mTOR resulting in decreased autophagy in AML cells. Indeed, lowering FASN protein expression by either shRNA or EGCG resulted in increased autophagy together with a decreased mTOR-mediated phosphorylation of ULK1.Next, we aimed at understanding how FASN expression is regulated during ATRA treatment. We found a negative correlation between KLF5 and FASN expression in both AML patient cohorts (r=-0.3) and 4 potential KLF binding sites on the FASN promoter, suggesting that KLF5 inhibits FASN transcription.However, since the protein half-life of FASN is around 24 hours, transcriptional inhibition is not sufficient to fully explain the reduced protein levels upon ATRA treatment. We therefore tested if FASN protein itself is degraded via autophagy. Co-treatment of NB4 cells with ATRA and the lysosome inhibitor Bafilomycin A1 led to a dose dependent accumulation of FASN protein suggesting that FASN is an autophagy substrate during ATRA treatment. Furthermore, using mCherry-LC3B expressing NB4 cells, we were able to show FASN and LC3B co-localization by fluorescence microscopy.Finally, we asked whether inhibiting FASN expression levels would increase ATRA sensitivity in non-APL AML cells. To this end, we treated KG-1, OCI-AML2 and MOLM-13 AML cells with EGCG in combination with ATRA. Importantly, combining ATRA with EGCG resulted in increased differentiation of these non-APL cells paralleled by reduced FASN levels, decreased mTOR activity and ULK1 phosphorylation.Together, our data suggest that EGCG treatment is beneficial for differentiation therapy of non-APL AML cells by activating autophagy via degradation of FASN and thereby inactivating mTOR. Furthermore, high FASN expression in AML is likely due to low expression of its positive transcriptional regulator KLF5 and low autophagy activity. DisclosuresNo relevant conflicts of interest to declare.

Theaflavin and epigallocatechin-3-gallate synergistically induce apoptosis through inhibition of PI3K/Akt signaling upon depolymerizing microtubules in HeLa cells.

Theaflavin (TF) and epigallocatechin-3-gallate (EGCG) both have been reported previously as microtubule depolymerizing agents that also have anticancer effects on various cancer cell lines and in animal models. Here, we have applied TF and EGCG in combination on HeLa cells to investigate if they can potentiate each other to improve their anticancer effect in lower doses and the underlying mechanism. We found that TF and EGCG acted synergistically, in lower doses, to inhibit the growth of HeLa cells. We found the combination of 50 µg/mL TF and 20 µg/mL EGCG to be the most effective combination with a combination index of 0.28. The same combination caused larger accumulation of cells in the G 2 /M phase of the cell cycle, potent mitochondrial membrane potential loss, and synergistic augmentation of apoptosis. We have shown that synergistic activity might be due to stronger microtubule depolymerization by simultaneous binding of TF and EGCG at different sites on tubulin: TF binds at vinblastine binding site on tubulin, and EGCG binds near colchicines binding site on tubulin. A detailed mechanistic analysis revealed that stronger microtubule depolymerization caused effective downregulation of PI3K/Akt signaling and potently induced mitochondrial apoptotic signals, which ultimately resulted in the apoptotic death of HeLa cells in a synergistic manner.

Epigallocatechin-3-gallate Enhances Radiation Sensitivity in Colorectal Cancer Cells Through Nrf2 Activation and Autophagy.

Epigallocatechin-3-gallate (EGCG) is a major polyphenolic component of green tea. EGCG plays a potential role in radio-sensitizing cancer cells. The combined effect of EGCG and radiation was investigated in a colorectal cancer cell line, focusing on nuclear factor (erythroid-derived 2)-like 2 (Nrf2) autophagy signalling. HCT-116 cells were treated with 12.5 μM EGCG for different periods of time, 2 Gy radiation, or both. Cell viability was determined with the WST-8 assay. The number of colonies was determined with the colony formation assay. mRNA expression of LC3 and caspase-9 was analyzed with quantitative real-time polymerase chain reaction. Combination treatment with EGCG and radiation significantly decreased the growth of HCT-116 cells. The number of colonies was reduced to 34.2% compared to the control group. Immunofluorescence microscopy images showed that nuclear translocation of Nrf2 was significantly increased when cells were treated with the combination of EGCG and radiation compared to the control and single-treatment groups. Combined treatment with EGCG and radiation significantly induced LC3 and caspase-9 mRNA expression. EGCG increased the sensitivity of colorectal cancer cells to radiation by inhibiting cell proliferation and inducing Nrf2 nuclear translocation and autophagy.

Epigallocatechin-3-gallate affects the proliferation, apoptosis, migration and invasion of tongue squamous cell carcinoma through the hippo-TAZ signaling pathway

The purpose of the present study was to investigate the mechanism by which epigallocatechin-3-gallate (EGCG) inhibits the biological behaviors of the tongue squamous cell carcinoma (TSCC) through the Hippo-tafazzin (TAZ) signaling pathway and to provide insights into molecular targeted therapy in TSCC. CAL27 and SCC15 cells were treated with different concentrations of EGCG for 24 h. Cell proliferation was determined using Cell-Counting Kit-8 and EdU assays. Cell apoptosis was evaluated by flow cytometry. Cell migration and invasion were measured using scratch and Transwell assays, respectively. Furthermore, protein levels of associated target genes were detected using a western blot assay. It was demonstrated that EGCG affected biological behaviors of CAL27 and SCC15 cells in concentration- and time-dependent manners. In addition, EGCG decreased the protein levels of TAZ, LATS1, MOB1 and JNK. Overexpression of TAZ alleviated the effect of EGCG on CAL27 cells. Furthermore, the combination of EGCG and simvastatin inhibited the proliferation, migration and invasion, and promoted apoptosis significantly compared with single treatment in CAL27 cells. The results of the present study suggested that EGCG affects proliferation, apoptosis, migration and invasion of TSCC through the Hippo-TAZ signaling pathway.

Synergic Inhibition of Lung Carcinoma 95-D Cell Proliferation and Invasion by Combination with (−)-Epigallocatechin-3-Gallate and Ascorbic Acid

In this study, the anti-invasion effects of (−)-epigallocatechin- 3-gallate (EGCG) mixed with ascorbic acid (Vc) on human lung carcinoma 95-D cells in vitro were examined and the synergism of the combination of EGCG and Vc was evaluated. Soft agar colony formation assay, cell migration assay, invasion assay, western blot analysis of NF-κB, in situ detection of cellular oxidative stress, and statistical analysis were assessed. The results showed that combining EGCG with Vc could inhibit clone forming rate of 95-D cell by 73.2%, reduce the migration ability of 95-D cell by 65.9%, and decrease the intracellular reactive oxygen species(ROS) level by 76.8%. The results of western blot proved that Vc enhanced the activity of EGCG in inhibiting NF-κB localization. It is speculated that the combination of EGCG and Vc can strongly suppress the proliferation and metastasis of lung carcinoma cells in a synergic manner, possibly with a mechanism associated with the scavenging of reactive oxygen species.

Combination of Epigallocatechin Gallate and Sulforaphane Counteracts In Vitro Oxidative Stress and Delays Stemness Loss of Amniotic Fluid Stem Cells.

Amniotic fluid stem cells (AFSCs) are characterized in vivo by a unique niche guarantying their homeostatic role in the body. Maintaining the functionality of stem cells ex vivo for clinical applications requires a continuous improvement of cell culture conditions. Cellular redox status plays an important role in stem cell biology as long as reactive oxygen species (ROS) concentration is finely regulated and their adverse effects are excluded. The aim of this study was to investigate the protective effect of two antioxidants, sulforaphane (SF) and epigallocatechin gallate (EGCG), against in vitro oxidative stress due to hyperoxia and freeze-thawing cycles in AFSCs. Human AFSCs were isolated and characterized from healthy subjects. Assays of metabolic function and antioxidant activity were performed to investigate the effect of SF and EGCG cotreatment on AFSCs. Real-time PCR was used to investigate the effect of the cotreatment on pluripotency, senescence, osteogenic and adipogenic markers, and antioxidant enzymes. Alkaline phosphatase assays and Alizarin Red staining were used to confirm osteogenic differentiation. The cotreatment with SF and EGCG was effective in reducing ROS production, increasing GSH levels, and enhancing the endogenous antioxidant defences through the upregulation of glutathione reductase, NAD(P)H:quinone oxidoreductase-1, and thioredoxin reductase. Intriguingly, the cotreatment sustained the stemness state by upregulating pluripotency markers such as OCT4 and NANOG. Moreover, the cotreatment influenced senescence-associated gene markers in respect to untreated cells. The cotreatment upregulated osteogenic gene markers and promoted osteogenic differentiation in vitro. SF and EGCG can be used in combination in AFSC culture as a strategy to preserve stem cell functionality.

Synergistic effects of combination treatment using EGCG and suramin against the chikungunya virus

Chikungunya is a severe disease that results from infection with the chikungunya virus (CHIKV), an arbovirus. Thus, we (1) explored a new approach to combining previously researched drugs that have shown the potential to inhibit CHIKV infection; and (2) demonstrated the antiviral effects of (−)-Epigallocatechin-3-gallate (EGCG) and the underlying mechanisms. Specifically, we used U2OS cells infected with CHIVK to assess the synergistic antiviral activities of EGCG and suramin. EGCG presented the ability to inhibit the viral RNA, progeny yield, and cytopathic effect (CPE) of CHIKV and also demonstrated the ability to protect against virus entry, replication, and release. Moreover, the results confirmed that EGCG and suramin can have synergistic effects against CHIKV strain S27 infection and two other clinical isolates of CHIKV. Our findings suggest that treatment with a combination of EGCG and suramin could provide a basis for the development of novel stretages against CHIKV infection.

Epigallocatechin-3-gallate enhances ER stress-induced cancer cell apoptosis by directly targeting PARP16 activity

Poly(ADP-ribose) polymerases (PARPs) are ADP-ribosylating enzymes and play important roles in a variety of cellular processes. Most small-molecule PARP inhibitors developed to date have been against PARP1, a poly-ADP-ribose transferase, and suffer from poor selectivity. PARP16, a mono-ADP-ribose transferase, has recently emerged as a potential therapeutic target, but its inhibitor development has trailed behind. Here we newly characterized epigallocatechin-3-gallate (EGCG) as a potential inhibitor of PARP16. We found that EGCG was associated with PARP16 and dramatically inhibited its activity in vitro. Moreover, EGCG suppressed the ER stress-induced phosphorylation of PERK and the transcription of unfolded protein response-related genes, leading to dramatically increase of cancer cells apoptosis under ER stress conditions, which was dependent on PARP16. These findings newly characterized EGCG as a potential inhibitor of PARP16, which can enhance the ER stress-induced cancer cell apoptosis, suggesting that a combination of EGCG and ER stress-induced agents might represent a novel approach for cancer therapy or chemoprevention.

EGFR inhibition by (-)-epigallocatechin-3-gallate and IIF treatments reduces breast cancer cell invasion.

Epidermal growth factor receptor (EGFR) expression is an important marker in breast carcinoma pathology and is considered a pivotal molecule for cancer cell proliferation, invasion and metastasis. We investigated the effects of epigallocatechin-3-gallate (EGCG), the most active green tea catechin, in combination with 6-OH-11-O-hydroxyphenanthrene (IIF), a synthetic retinoid X receptor-γ (RXRγ) agonist, on three breast carcinoma cell lines: MCF-7, MCF-7TAM and MDA-MB-231. EGFR and AKT activation and molecular markers of cell motility and migration (CD44, extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN), MMP-2, MMP-9 and tissue inhibitor of metalloproteinases (TIMPs)) were studied after EGCG and IIF treatments. The EGCG + IIF treatment was the most active in down-regulating EGFR phosphorylation at Tyr1068 in all the investigated cell lines; p473AKT was also down-regulated in MCF-TAM cells. EGCG + IIF was also the most active treatment in reducing the expression of markers of invasion and migration in all the three cell lines: CD44, EMMPRIN, MMP-2 and -9 expression decreased, whereas TIMPs were up-regulated. Zymography and scratch assay also confirmed the reduced invasion tendency. We considered that EGCG and IIF treatments could alter the molecular network based on EGFR, CD44 and EMMPRIN expression interdependence and reduced the migration tendency in MCF-7, MCF-7TAM and MDA-MB-231 cells. These events only occurred in association with AKT inactivation in MCF-7TAM cells. In conclusion, the combination of EGCG and IIF significantly attenuated the invasive behaviour of breast carcinoma cells.

In vitro evaluation of combination of EGCG and Erlotinib with classical chemotherapeutics on JAR cells.

Gestational Trophoblastic Neoplasia (GTN) is a term used for a group of malignant gynecological tumors including choriocarcinoma. Low-risk neoplasias can be cured using single agents Methotrexate (MTX) and actinomycin-D (ACD), but in certain cases, decreased responsiveness and serious side effects occur. Therefore, researchers have been attempting to find new treatment modalities. One of the most popular way for increasing cancer patient survival rates is supporting treatment with adjuvant molecules or chemosensitizers. For this purpose, we investigated epigallocatechin-3-gallate (EGCG), a green tea cathecin, and Erlotinib, an EGFR tyrosine kinase inhibitor, as single agents and combined with MTX or ACD. In accordance with this, JAR (human placenta choriocarcinoma) cell line was used as an in vitro model and MTT, LDH, caspase-3 activation, RT-PCR, and Western Blot analyses were performed to investigate the effects of the test materials. Our studies demonstrate that combination of Erlotinib and EGCG with MTX and ACD decreases JAR cell proliferation and metastatic HER2 protein synthesis and increases caspase-3 activation compared to ACD or MTX alone. In addition, significant increase was observed in the apoptotic Bax gene, but no notable protein synthesis occurred in the Western Blot analysis, which suggests that combination of Erlotinib and EGCG with classical chemotherapeutics ACD or MTX may lead the JAR cells to apoptosis, but not by a mitochondrial pathway. All the results indicate that the synergetic effect of Erlotinib and EGCG with classical chemotherapeutics may help to increase patient survival rates of choriocarcinoma, but the detailed mechanism needs further investigation.

The regulation of steroid receptors by epigallocatechin-3-gallate in breast cancer cells.

It has been reported that phytoestrogen epigallocatechin gallate (EGCG) suppresses cancer cell proliferation and may have antitumor properties. In this study, we analyzed the effects of EGCG on estrogen receptor α (ERα) and progesterone receptor in hormone-dependent T-47D breast cancer cells. Western blot analysis revealed EGCG induced a concentration-dependent decrease in ERα protein levels, with a 56% reduction occurring with 60 µM EGCG when compared to controls. Downregulation of ERα protein levels was observed after 24-hour co-treatment of T-47D cells with 60 µM EGCG and 10 nM 17β-estradiol (E2). The proliferative effect of E2 on cell viability was reversed when treated in combination with EGCG. In contrast, the combination of EGCG with the pure ER antagonist, ICI 182, 780, showed no further reduction in cell number as only 5% of the cells were viable after 6 days of treatment. These studies may provide further understanding of the interactions among flavonoids and steroid receptors in breast cancer cells.

Down-regulation of histone deacetylase 4, −5 and −6 as a mechanism of synergistic enhancement of apoptosis in human lung cancer cells treated with the combination of a synthetic retinoid, Am80 and green tea catechin

(−)-Epigallocatechin gallate (EGCG), a green tea catechin, acts as a synergist with various anticancer drugs, including retinoids. Am80 is a synthetic retinoid with a different structure from all-trans-retinoic acid: Am80 is now clinically utilized as a new drug for relapsed and intractable acute promyelocytic leukemia patients. Our experiments showed that the combination of EGCG and Am80 synergistically induced both apoptosis in human lung cancer cell line PC-9 and up-regulated expressions of growth arrest and DNA damage-inducible gene 153 (GADD153), death receptor 5, and p21waf1 genes in the cells. To understand the mechanisms of synergistic anticancer activity of the combination, we gave special attention to the lysine acetylation of proteins. Proteomic analysis using nanoLC-ESI-MS/MS revealed that PC-9 cells treated with the combination contained 331 acetylated proteins, while nontreated cells contained 553 acetylated proteins, and 59 acetylated proteins were found in both groups. Among them, the combination increased acetylated-p53 and acetylated-α-tubulin through reduction of histone deacetylase (HDAC) activity in cytosol fraction, although the levels of acetylation in histones H3 or H4 did not change, and the combination reduced protein levels of HDAC4, −5 and −6 by 20% to 80%. Moreover, we found that a specific inhibitor of HDAC4 and −5 strongly induced p21waf1 gene expression, and that of HDAC6 induced both GADD153 and p21waf1 gene expression, which resulted in apoptosis. All results demonstrate that EGCG in combination with Am80 changes levels of acetylation in nonhistone proteins via down-regulation of HDAC4, −5 and −6 and stimulates apoptotic induction.

Metformin Sensitizes Non-small Cell Lung Cancer Cells to an Epigallocatechin-3-Gallate (EGCG) Treatment by Suppressing the Nrf2/HO-1 Signaling Pathway.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. (-)-Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, is widely studied as a cancer chemopreventive agent with potential anti-cancer effects. The NF-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway is considered to mediate cellular resistance to EGCG. Metformin, a classical antidiabetic drug, has been shown to prevent cancer progression. Researchers have not reported whether metformin potentiates the anti-cancer efficacy of EGCG. In this study, metformin inhibited HO-1 expression and augmented the anti-tumor effect of EGCG. Metformin also enhanced ROS (reactive oxygen species) generation induced by EGCG (100 μM), subsequently resulting in apoptosis. Based on the results of the in vivo study, size of xenografts treated with the combination of metformin and EGCG was smaller than other groups. Mechanistically, metformin modulated the EGCG-activated Nrf2/HO-1 pathway through Sirtuin 1 (SIRT1)-dependent deacetylation of Nrf2. Moreover, metformin upregulated SIRT1 expression partially through the NF-kB pathway. Comparatively, the combination of EGCG and metformin showed little impact on normal lung epithelial BEAS-2B cells. Based on our findings, metformin sensitized NSCLC cells to the EGCG treatment by suppressing the Nrf2/HO-1 signaling pathway.

Antibacterial activity of epigallocatechin-3-gallate (EGCG) and its synergism with β-lactam antibiotics sensitizing carbapenem-associated multidrug resistant clinical isolates of Acinetobacter baumannii

BackgroundInfections caused by Acinetobacter baumannii were responsive to conventional antibiotic therapy. However, recently, carbapenem-associated multidrug resistant isolates have been reported worldwide and present a major therapeutic challenge. Epigallocatechin-3-Gallate (EGCG) extracted from green tea exhibits antibacterial activity. PurposeWe evaluated the antibacterial activity of EGCG and possible synergism with antibiotics in carbapenem-associated multidrug resistant A. baumannii. A potential mechanism for synergism was also explored. Materials and methodsSeventy clinical isolates of A. baumannii collected from geographically different areas were analyzed by minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of EGCG. Checkerboard and time-killing assays were performed to exam the synergism between EGCG and antibiotics. The effects of EGCG on a multidrug efflux pump inhibitor (1-[1-naphthylmethyl] piperazine; NMP) and β-lactamase production were also examined in A. baumannii. ResultsSixty-three of 70 clinical isolates of A. baumannii carried carbapenemase-encoding genes with carbapenem-associated multidrug resistance. Levels of MIC and MBC of EGCG ranged from 64 to 512µg/ml and from 128 to ≥1024µg/ml, respectively among the clinical isolates. MIC90 and MBC86 levels were 256µg/ml and 512µg/ml of EGCG, respectively. Subinhibitory concentration of EGCG in combination with all antibiotics tested, including carbapenem, sensitized (MICs fall≤1.0µg/ml) all carbapenem-associated multidrug resistant isolates. Checkerboard and time-killing assays showed synergism between EGCG and meropenem (or carbenicillin) counted as fractional inhibitory concentration of < 0.5 and cell numbers’ decrease per ml of >2log10 within 12h, respectively. EGCG significantly increased the effect of NMP but was unrelated to β-lactamase production in A. baumannii, suggesting EGCG may be associated with inhibition of efflux pumps. ConclusionOverall we suggest that EGCG-antibiotic combinations might provide an alternative approach to treat infections with A. baumannii regardless of antibiotic resistance.

Competitive binding of (−)-epigallocatechin-3-gallate and 5-fluorouracil to human serum albumin: A fluorescence and circular dichroism study

Combination therapy with more than one therapeutic agent can improve therapeutic efficiency and decrease drug resistance. In this study, the interactions of human serum albumin (HSA) with individual or combined anticancer drugs, (−)-epigallocatechin-3-gallate (EGCG) and 5-fluorouracil (FU), were investigated by fluorescence and circular dichroism (CD) spectroscopy. The results demonstrated that the interaction of EGCG or FU with HSA is a process of static quenching and EGCG formed a more stable complex. The competitive experiments of site markers suggested that both anti-carcinogens mainly bound to site I (subdomain IIA). The interaction forces which play important roles in the binding process were discussed based on enthalpy and entropy changes. Moreover, the competition binding model for a ternary system was proposed so as to precisely calculate the binding parameters. The results demonstrated that one drug decreased the binding affinity of another drug with HSA, resulting in the increasing free drug concentration at the action sites. CD studies indicated that there was an alteration in HSA secondary structure due to the binding of EGCG and FU. It can be concluded that the combination of EGCG with FU may enhance anticancer efficacy. This finding may provide a theoretical basis for clinical treatments.

Y-27632 Increases Sensitivity of PANC-1 Cells to EGCG in Regulating Cell Proliferation and Migration.

BackgroundThe study aimed to investigate the inhibitory effect of (1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl) cyclohexanecarboxamide (Y-27632) and (−)-epigallocatechin-3-gallate (EGCG) on the proliferation and migration of PANC-1 cells. EGCG, found in green tea, has been previously shown to be one of the most abundant and powerful catechins in cancer prevention and treatment. Y-27632, a selective inhibitor of rho-associated protein kinase 1, is widely used in treating cardiovascular disease, inflammation, and cancer.Material/MethodsPANC-1 cells, maintained in Dulbecco’s Modified Eagle’s Medium, were treated with dimethyl sulfoxide (control) as well as different concentrations (20, 40, 60, and 80 μg/mL) of EGCG for 48 h. In addition, PANC-1 cells were treated separately with 60 μg/mL EGCG, 20 μM Y-27632, and EGCG combined with Y-27632 (60 μg/mL EGCG + 20 μM Y-27632) for 48 h. The effect of EGCG and Y-27632 on the proliferation and migration of PANC-1 cells was evaluated using Cell Counting Kit-8 and transwell migration assays. The expression of peroxisome proliferator–activated receptor alpha (PPARα) and Caspase-3 mRNA was determined by Quantitative real-time polymerase chain reaction (RT-qPCR).ResultsEGCG (20–80 μg/mL) inhibited cell viability in a dose-dependent manner. Y-27632 enhanced the sensitivity of PANC-1 cells to EGCG (by increasing the expression of PPARα and Caspase-3 mRNA) and suppressed cell proliferation. PANC-1 cell migration was inhibited by treatment with a combination of EGCG and Y-27632.ConclusionsY-27632 increases the sensitivity of PANC-1 cells to EGCG in regulating cell proliferation and migration, which is likely to be related to the expression of PPARα mRNA and Caspase-3 mRNA.