Theanine combined with cisplatin inhibits the proliferation and metastasis of TNBC cells through Akt signaling pathway

Background:Theanine,was natural compounds,has been employed in antibacterial,antineoplastic,promotion of hair growth,therapy of poliosis,and removing pattogenic heat from the blood and toxic material from the body for centuries.However,few reports focused on the treatment by theanine combined with chemotherapy in triple negative breast cancer.Methods:Our study aimed to evaluate and compare the molecular and cellular effects of theanine in combination with cisplatin in triple negative breast cancer lines MDA-MB-231.The 50%inhibitory concentration and cell viability were determined by the cell counting kit-8 assay.Meanwhile,wound healing and transwell assays were used to evaluate the effect of theanine combination with cisplatin on triple negative breast cancer(TNBC)cell metastasis.Moreover,the label-free proteomic method was available to explore the molecular mechanism of theanine combination with cisplatin on TNBC cells.What’s more,reverse transcription-polymerase chain reaction and western blotting were carried out to evaluate the related protein and mRNA alteration in the Akt signaling pathway.Results:With theanine added,the inhibitory effect of cisplatin on cell proliferation and metastasis was significantly improved.Meanwhile,with the increase of theanine concentration,the anti-tumor effect of the combination group was also significantly improved.Afterward,label-free proteomics analysis showed that theanine combined with cisplatin could significantly activate the Akt signaling pathway,thus improving the anti-tumor effect of cisplatin.Finally,reverse transcription-polymerase chain reaction and western blotting experiment showed that the combination of cisplatin with theanine induced the greatest down-regulation of phosphorylated Akt expression.Conclusion:All in all,combining theanine with appears to be a promising strategy for the effective treatment of TNBC in the prospective application which merits further clinical investigation.

Extraction of Theanine from Waste Liquid of Tea Polyphenol Production in Aqueous Two-phase Systems with Cationic and Anionic Surfactants

Extraction of theanine from waste liquid of tea polyphenol production was studied in aqueous surfactanttwo-phase system （ASTP） with cationic surfactant （CTAB） and anionic surfactant （SDS）. Results indicate that the region of ASTP is narrow and there is only a two-phase region of cationic surfactant. The increase in concentrations of NaBr and Na2SO4 are beneficial to the formation of ASTP. Theanine concentration in the bottom phase increases with increasing concentration of theanine, whereas the Partition coefficient and extraction rate only change a littlewhen the concentration of theanine is above 0.2 g.L-＇. With the increase of SDS concentration, the phase ratio and the partition coefficient decrease, while the extraction efficiency of theanine increases and the concentration of theanine changes a little in the range from 2.4/7.5 to 2.8/7.2 for SDS/CTAB ratio. The temperature has a notable ef- fect on the concentration of theanine in the bottom phase, partition coefficient and extraction rate of theanine. The increase of waste liquid decreases the phase ratio, increases the concentration and extraction rate of theanine in the bottom ohase, since the orotein and the saccharide enter the bottom nhase with theanine.

Expression of the DNA-repair protein XRCC1 and DNA-repair enzyme Ku70 mRNA in the rat cerebral cortex following global cerebral ischemia/reperfusion Is this correlated with neuroprotective effects of theanine?

BACKGROUND： Numerous studies have shown that apoptosis, caused by cerebral ischemia/reperfusion injury, is a dynamic evolving process that occurs in a time-dependent fashion. Non-apoptotic cells can result in apoptosis, with a prolonged reperfusion period and an accumulation of DNA damage. Recent studies have indicated that theanine has a protective effect on the brain. Nonetheless, there is no research relating to whether theanine is neuroprotective following brain injury. OBJECTIVE： To investigate the effects of theanine on apoptosis in the cerebral cortex and mRNA expression of the DNA repair protein, XRCC1, and DNA repair enzyme, Ku70, at various time points following global cerebral ischemia/reperfusion in rats. DESIGN, TIME AND SETTING： A randomized, controlled animal study was performed at the Pharmacological Laboratory of Animal Experimental Center of Xi＇an Jiaotong University, China, from April to October 2007. MATERIALS： Theanine （Wuhan Remote Technology Co., Ltd., China）, XRCC1, Ku70 and GAPDH primers （Shanghai Sangon Biological Engineering Technology ＆ Services Co., Ltd., China） were used in this study. METHODS： A total of 108 healthy, male Sprague Dawley rats were randomly divided into sham operation, ischemia/reperfusion and theanine groups. Each group was further divided into 6 subgroups, with six rats in each, according to reperfusion time （2, 6, 12, 24, 48 and 72 hours）. Rat models of global cerebral ischemia/reperfusion were established by the four-vessel occlusion method. Theanine （1 000 mg/kg） was injected into the caudal vein of rats in the theanine group immediately after surgery. Saline （4 mL/kg） was used in the sham operation and ischemia/reperfusion groups. The same dose was administered every 24 hours. MAIN OUTCOME MEASURES： Flow cytometry was employed to determine apoptosis of cerebral cortical neurons. Real-time reverse transcdption-polymerase chain reaction was applied to quantify the expression of XRCC1 and Ku70 mRNA in the cerebral cortex. RESULTS： The apoptotic rate in the ischemia/reperfusion group was significantly greater compared with the sham operation group at various time points （P 〈 0.05）. The apoptotic rate in the theanine group was significantly diminished compared with the ischemia/reperfusion group, but significantly increased compared with the sham operation group at 6, 12, 24, 48 and 72 hours （P 〈 0.05）. Expression of XRCC1 and Ku70 mRNA was significantly decreased in the ischemia/reperfusion group compared with the sham operation group at 2, 6, 12, 24, 48 and 72 hours （P 〈 0.05）. The relative expression of XRCC1 mRNA in the theanine group was significantly increased compared with the ischemia/reperfusion group, but significantly reduced compared with the sham operation group at 6, 12, 24, 48 and 72 hours （P 〈 0.05）. The relative expression of Ku70 mRNA was significantly lower in the theanine group compared with the sham operation group at 2, 6 and 12 hours （P〈 0.05） and significantly greater than the ischemia/reperfusion group at 6, 12, 24, 48 and 72 hours （P 〈 0.05）. CONCLUSION： The apoptotic rate in rats following global cerebral ischemia/reperfusion injury reaches a peak at 24 hours after reperfusion; however, the levels of DNA repair protein began to decrease after only 2 hours of reperfusion. Theanine antagonizes the decrease in XRCC1 and Ku70 mRNA expression in the rat cerebral cortex during global cerebral ischemia/reperfusion injury.

Effect of Sunlight Exposure and Different Withering Durations on Theanine Levels in Tea (<i>Camellia sinensis</i>)

Theanine is a non-protein amino acid representing as much as 50% of the total amino acids in black tea and 1% - 2% of dry weight in green tea. It has been shown to be able to reduce high blood pressure, promote relaxation, and inhibit caffeine’s side effects among others. This current study explored the effects of sunlight and withering durations on theanine levels in tea shoots. Theanine content from three leaves and a bud, two leaves and a bud and internodes were detected and quantified by using High Performance Liquid chromatography (HPLC). Sunlight exposure experiment was started at dawn (0600 HRS, GMT + 3.00) when the light intensity was low and tea was collected at three-hour interval throughout the day to (1800 Hrs, GMT + 3.00) when the light intensity had dropped. At the start of the experiment, the theanine levels were significantly high but as the intensity of sunlight increased during the day there was a significant drop in theanine levels, and as the sun set the theanine levels increased significantly again in all samples. The results also showed that theanine levels were significantly increased after 15 hours of withering. Three leaves and a bud withered for 3 hours had mean theanine levels of 1.41% and those withered for 15 hours had mean theanine levels of 3.11%. Internodes exhibited higher mean theanine levels than those of leaves. In the light of these results, it’s evident that withering for a longer period of time and harvesting of tea when the light intensity is low ensure high amount of theanine in tea.

Effect of Seasons on Theanine Levels in Different Kenyan Commercially Released Tea Cultivars and Its Variation in Different Parts of the Tea Shoot

A study was conducted to determine the effect of seasons in 23 different Kenyan commercially released tea cultivars. This was done for three seasons viz: cold and wet, cold and humid, hot and dry. The cold and wet season was shown to be the best for obtaining high levels of theanine while the hot and dry season resulted in decrease in theanine levels in most of the test cultivars thus showing seasonal variation on the levels of theanine. Theanine content was determined using High Performance Liquid Chromatography (HPLC). On average, TRFK 31/8 contained 1.20% dw of theanine and highest in green tea while TRFK 56/89 contained 0.97% dw of theanine and highest in black tea. Green tea contained higher theanine levels than black tea and this has been shown in most of the test cultivars. To achieve the health benefits of theanine, one should drink 4 cups of tea brewed from cultivars with 0.63% dw and more of theanine. Also theanine levels were determined in the different partitions of the tea shoot (4 leaves + bud) and the internode contained the highest theanine levels up to 3.26% dw for both cultivars compared to the leafy partitions.

Theanine improves the function of dendritic cells via the downregulation of cyclooxygenase-2 expression

Background Tumor cells can reduce the number of dendritic cells （DCs） in the tumor environment and cause DC dysfunction through autocrine or paracdne pathways. We sought to measure cyclooxygenase-2 （COX-2） expression in bombesin-inhibited DCs treated with theanine in vitro and to explore the protection and activation effects of theanine on DCs. Methods Enzyme-linked immunosorbent assay （ELISA）, reverse transcription-polymerase chain reaction （RT-PCR）, and Western blotting were used to analyze the effects of theanine on COX-2 expression and interleukin （IL）-12/IL-10 secretion of bombesin-treated DCs. Results DCs acquired an impaired phenotype as a result of bombesin treatment. Theanine increased the expression of mature DC surface molecules. The number of cell apoptosis with the treatment of bombesin and theanine significantly decreased, accounting for 15.9%, compared with 26.1% of cell apoptosis with bombesin. COX-2 expression in bombesin- treated DCs was inhibited by theanine in a dose-dependent manner. Theanine promoted DC secretion of IL-12. IL-12 levels reached （137.4_＋4.9） pg/ml with theanine at 200 pmol/L. However, theanine inhibited the secretion of IL-10 in a dose-dependent manner. IL-10 levels were only （58.4_＋6.9） pg/ml with theanine at 200 IJmol/L. Conclusion Theanine inhibits the transcription and translation of COX-2 and regulates the balance of IL-10/IL-12 secretion in bombesin-inhibited DCs, leadincl to the recovery of a state of activation in DCs.

Tea Identification through Surface-Assisted Laser Desorption/Ionization Mass Spectrometry

We have applied surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) usingtitanium dioxide nanoparticles(TiO2NPs)as the matrix and captopril (CAP) as internal standard for the determination of the concentrations of theanine and four catechins—catechin, (_)-epigallocatechin (EGC), (_)-epicatechin gallate (ECG), and (_)-epigallocatechin gallate (EGCG).Under the optimal conditions (240 nM TiO2 NPs and 10 μM CAP), this SALDI-MS approach provides linearity of 0.3– 80 (r= 0.990), 1.2–100 (r= 0.987), 4–120 (r= 0.995), 6–120 (r= 0.983), and 2–120 μM (r= 0.991) for theanine, catechin, EGC, ECG, and EGCG, respectively. The limits of detection (LODs;S/N = 3)for theanine, catechin, EGC, ECG, and EGCG provided by this SALDI-MS approach are 0.1, 0.35, 1.0, 1.45, and 0.5 μM, respectively. This approach provides spot-to-spot and batch-to-batch variations of less than 10% and 13%, respectively, for the analysis of tea samples.With advantages of simplicity, accuracy, precision, and great reproducibility, we have applied the SALDI-MS approach for the analysis of tea samples, with identified peaks for theanine, catechin, EGC, ECG, and EGCG. Tea samples from Taiwan and four other areas have various SALDI-MS profiles, showing their potential for differentiation of tea samples from different sources. Our result also shows that tea samples harvested in different seasons and counties in Taiwan provide significantly different MS profiles. The amounts of theanine and EGC in the Oolong tea from Lishan are much higher than those in the other tea samples.

The cessation and detoxification effect of tea filters on cigarette smoke

To treat tobacco addiction,a tea filter was developed and studied for smoking cessation.This work reports the smoking cessation effect of tea when it was used as a component of cigarette filters.In one trial it was found that after using the tea filters for 2 months,the volunteer smokers decreased their cigarette consumption by 56.5%,and 31.7% of them stopped smoking.This work identified a new method and material,tea filter and theanine,which inhibit tobacco and nicotine addiction and provide an effective strategy for treating tobacco addiction.