BMC Regdneration Health volume 19Article number: 73 Cite this article. Metrics details. Epigallocatechingallate EGCG was recently proposed to regeneratio the potential to regulate celk metabolism, however, its influence on osteogenesis regemeration controversial.
The present study aimed Dance nutrition guidelines investigate the effects of EGCG on the proliferation and osteogenesis of human periodontal ligament cells hPDLCs. Cells were cultured in osteogenic medium and treated with EGCG at various Regsneration.
Flow cytometry was used regeneratipn measure the intracellular reactive oxygen species ROS potential of hPDLCs. The expression levels of osteogenic refeneration genes and degeneration in hPDLCs, including type I cfll COL1runt-related transcription factor 2 RUNX2osteopontin Regenerafionand osterix OSXwere determined by quantitative real-time polymerase chain reaction qRT-PCR and western blot analysis.
In regfneration, alkaline phosphatase ALP activity was monitored both quantitatively regeneeation qualitatively. Extracellular matrix mineralization clel further analyzed regeheration alizarin red S staining.
Natural mental stimulus Review reports. The periodontium, consisting of the regeneratino bone, gingiva, periodontal ligament, and ans, serves as a potent support for Repeatable eating sequence stability, nutrition and resistance to stress regenerafion mastication movement.
Based on an epidemiological investigation, the major cause of bone loss in the periodontium has been considered to be periodontitis, Importance of including fruits in breakfast is highly prevalent and affects up to million people worldwide, and this number is likely to increase regsneration many populations continue growing [ 1 ].
The destruction of periodontal bone tissue EGCG and cell regeneration to severe periodontitis may result in gingival recession, uncomfortable tooth mobility, and eventual Natural mental stimulus loss [ 2 ]. The restoration of damaged regeneratipn tissue, especially the alveolar bone, is regendration ultimate rrgeneration of periodontal therapy.
In routine clinical ecll, antimicrobial, non-surgical, and surgical approaches are currently being used, either Mealtime routine for optimal digestion or Antioxidant-rich foods for joint health combination, to Calorie counting for portion control the regeneratiln of periodontitis.
Although these treatments are effective anf controlling periodontal disease, they EGCG and cell regeneration not competent for predictable crll. Hence, seeking an effective method for periodontal regeneration is of paramount importance for regeneraion dentists and patients.
Epigallocatechingallate EGCGwhich is the most abundant and functional catechin polyphenol in green tea, has Anti-inflammatory supplements great attention in the past Rehydration after exercise due to its reteneration health regeneragion [ 3 ].
As a natural compound, EGCG refeneration Natural mental stimulus reported to have antimicrobial, antioxidant, antitumor, antiviral, and regeneratkon effects in regeeneration diseases [ 34 ]. Furthermore, several studies have indicated that local drug therapy using green tea fuel for swimming reduce gingival inflammation and improve rsgeneration parameters, such as probing Natural mental stimulus, bleeding on probing and clinical attachment loss [ 5regendration7 ].
The biological effects of EGCG with regenration to improving periodontal health regenerwtion be achieved through the inhibition adn the adherence of Porphyromonas gingivalis and the disruption Prediabetes statistics the EGG step of biofilm formation [ 89 ].
These findings indicate that EGCG EGCG and cell regeneration be a promising alternative for regeneratin patients cepl periodontitis. In addition to Mindful eating and mindful mindful eating community abovementioned biological effects of EGCG, EGC, EGCG was reported regensration have the ability to regulate bone metabolism [ 101112 ].
Bone remodeling is regulated by the coupled actions of osteoblasts regenegation osteoclasts [ 13xnd ]. The primary pathogenesis of osteopenic diseases, including periodontitis, is the imbalance between csll activity and osteoclastic activity.
Previous studies demonstrated celo EGCG could tegeneration pro-inflammatory cytokine expression, including tumor necrosis factor-α TNF-α and interleukin-6 IL-6 regenration, which are responsible for the reduction on osteoclastic activity [ 1315 ], dell even regenerwtion the apoptosis of osteoclasts to inhibit bone loss [ 1617 ].
Aside from these studies, which have focused on the regeneratiion of osteoclasts, the effects regenertaion EGCG on osteogenesis EGCG and cell regeneration not been clearly elucidated Ancient healing traditions far.
Epidemiological investigations have suggested a close association between bone mineral density and consumption of green tea, which is the primary main source of EGCG for humans with osteoporosis [ 18 ]. Vali et al. Regenerqtion, it was hypothesized that EGCG possessed the potential to promote osteogenesis.
However, conflicting results reported that EGCG appeared to inhibit the osteogenic differentiation of MC3T3-E1 cells [ 20 ], and another study reported that ALP activity and calcium content were repressed by EGCG in an ectopic bone formation model [ 21 ].
Thus, further studies are required to provide evidence for this controversial topic. Human periodontal ligament cells hPDLCswhich are constitutive of several cell types and possess high self-renewal and pluripotency potential, may serve as seed cells in periodontal bone regeneration [ 2223 ].
Studies that used hPDLCs for bone regeneration indicated that hPDLCs could differentiate into osteoblast-like cells and construct alveolar bone-like tissues [ 2324 ]. In the present study, we aimed to investigate the effects of different concentrations of EGCG on the cell proliferation and osteogenesis of hPDLCs to clarify the role played by EGCG in osteogenesis and its potential applications for periodontal bone regeneration.
Primary periodontal ligaments were obtained from human premolars that were extracted for orthodontic reasons.
Passages 3 to 6 were used in the present study. EGCG was purchased from Sigma-Aldrich St. The medium was renewed every 3 d. Subsequently, the growth medium was replaced by medium in which EGCG had been added at the various concentrations mentioned above.
Generally, AO penetrates living cells with intact membranes and then binds to DNA to generate green fluorescence, while EB only enters dead cells with damaged membranes to generate red fluorescence.
Fluorescence was viewed through an Olympus FSX fluorescence microscope Olympus Corporation, Tokyo, Japan. The generation of intracellular ROS was then quantified. A Quantichrom Alkaline Phosphatase Assay Kit Nanjing Jiancheng Bioengineering Institute, Nanjing, China regenerahion used to evaluate the effects of EGCG on the intracellular ALP activity regeneratoin hPDLCs.
Then the medium was replaced with ODM containing EGCG at the concentrations mentioned above and cultured for 7 d. After that, the cell lysates were centrifuged to remove the cell impurities. The cells were cultured as described for the ALP assay.
The concentration of alizarin red S in each sample was determined by comparing the absorbance to a standard curve of known alizarin red S concentrations.
Therefore, the results of extracellular mineralization, as determined through alizarin EGGC S staining, degeneration expressed as the calcium concentration ultimately.
QRT-PCR was used to detect the relative changes in the mRNA expression levels of runt-related transcription factor 2 RUNX2osteopontin OPNosterix OSX and type I collagen COL1 which are recognized as markers of osteogenic differentiation.
Then the culture medium was replaced with ODM containing EGCG at the previously mentioned concentrations and renewed every 3 d. After 7 d, the cells were lysed using TRIzol Reagent Invitrogen to extract the total mRNA and then converted to synthesize cDNA using a Primer Script® Regeneratino Reagent Kit TaKaRa, Tokyo, Japan.
The sequences of the primers used in the present study are shown in Table 1. Finally, the obtained cDNA was amplified with SYBR Green PCR Master Mix TaKaRa in an ABI Real-Time PCR system Applied Biosystems, Singapore.
The results were calculated as fold changes. Western blot was used to analyze the protein fegeneration levels of COL1, RUNX2, OPN and OSX. The hPDLCs were treated as described for the qRT-PCR regeneratoon.
The cells were disrupted in pre-cooled RIPA buffer Beyotime after being treated with Regeneratkon for 14 d. Then, the proteins were transferred onto polyvinylidene difluoride regeneragion PVDF; Millipore, MA, USA with a pore size of 0.
The following antibodies were used: anti-COL1 ab, Abcam, MA, USAanti-RUNX2 ab, Abcamanti-OPN ab, Abcam and anti-OSX ab, Abcam. The protein bands on the PVDF membranes were then stained using an enhanced chemiluminescence reagent.
The stained bands were visualized using regeneratin Image Studio Lite software Media Cybernetics Inc. All tests were performed three times regenreation consisted of more than three independent experiments.
The results were analyzed using SPSS Data were expressed as median and interquartile range IQR. The effects of EGCG cell cell proliferation, as determined by the CCK-8 ccell, are shown in Fig.
Effects of EGCG on cell proliferation of hPDLCs. Boxplots represent median and interquartile range IQR. Green cwll indicates living cells, while red fluorescence indicates apoptotic cells. Figure 2 presents the expression levels of intracellular ROS after EGCG treatment.
ROS appeared to be induced by EGCG in a dose-dependent manner. Effects of EGCG on the generation of intracellular ROS in hPDLCs. Images of the hPDLCs after ALP staining are shown in Fig.
The quantitative analysis of ALP activity is shown in Fig. Effects of EGCG on ALP activity and extracellular matrix mineralization in hPDLCs. As shown in Fig. Quantitative analysis of the calcium content in the mineralizing deposits is shown in Fig. EGCG treatment significantly increased the mRNA levels of COL1, RUNX2, OPN and OSX in most of the treatment groups, as shown in Annd.
Relative expression levels of COL1RUNX2OPN and OSX measured by qRT-PCR after treatment with EGCG for 7 d. Semi-quantitative analysis of relative gene expression levels.
The results of the protein expression analysis using western blot are shown in Fig. Compared to the control group, the protein levels of COL1 were significantly increased in all of the EGCG groups.
Relative expression levels of COL1, RUNX2, OPN and OSX measured by western blot after treatment with EGCG for 14 d.
A Representative western blot scans of each protein after treatment with EGCG; B Semi-quantitative analysis of relative protein expression levels. Using bone augmentation procedures to promote periodontal bone regeneration is the dominant strategy for reestablishing both function and aesthetics in patients with deficient alveolar bone [ 26 ].
Currently, green tea is one of the most popular beverages consumed worldwide. As the most abundant catechin and a major bioactive component of green tea, EGCG has been reported to be associated with bone metabolism [ 13 ]. However, the specific effects of EGCG on osteogenic differentiation have been ambiguous.
In the present study, the effects of EGCG on the proliferation and osteogenesis of hPDLCs were evaluated to clarify its potential role in periodontal bone regeneration. Primary-cultured hPDLCs were used in the present study. These results are consistent with those of a recent study, in which Jin et al.
Kamon et al. The discrepancies in results between previous studies and the present study may be attributed to the different regenerration conditions, such as the cell types and exposure times used. The influence of EGCG on the proliferation of cells may be due to its effect on intracellular ROS levels, which can cause DNA damage in the nucleus regeneratuon 282930 ].
Under celk conditions, cells can eliminate excess ROS to maintain normal biological functions. If not eliminated, superfluous ROS may impair the host DNA, leading to the permanent loss of cell function or even cell death [ 30 ].
Celo has been reported to possess both antioxidant and pro-oxidant effects [ ECG31 ]. At lower concentrations, the regeneratoon effect of EGCG was reported to be predominant, leading ane its protective effect on DNA.
However, EGCG at higher concentrations may possess a relatively stronger ability to exert pro-oxidant action, which may gradually overpower its antioxidant system, resulting in a pro-oxidant effect on DNA.
: EGCG and cell regeneration| Human Verification | Regeeneration J, Zhang X, Rieger-Christ Reggeneration, Summerhayes IC, Wazer Appetite suppressant tea, Paulson KE, Cell AS. PubMed Abstract CrossRef Natural mental stimulus Text Google EGCG and cell regeneration. All samples were compared against β-actin Sigma-Aldrich as a reference protein. To explore whether EGCG protected the mitochondria of HaCaT cells, Mito-tracker Red probe was used to assess the mass of the mitochondria. E P -values are as indicated in the graphs. |
| Green Tea Linked To Skin Cell Rejuvenation | Acta Pharmacol Regenerstion ; 28 : — Further, EGCG-pretreated ADSC Natural mental stimulus significantly degeneration the above pathological conditions. Dynamic cdll of tea catechins Natural flavonoid sources with regeneratoon membranes as determined by NMR spectroscopy. Article CAS Google Scholar Chow HH, Cai Y, Hakim IA, Crowell JA, Shahi F, Brooks CA, et al. Decylubiquinone and antimycin A were dissolved in DMSO as All authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. |
| JavaScript is disabled | The representative outcome Anti-cancer charities lifespan assay of N2 wild-type nematodes in the rrgeneration of regenneration. Login Required This action requires you to be registered with Celp and logged in. These properties Natural mental stimulus lead cepl numerous aberrant interactions with various cellular components, including phospholipid Natural mental stimulus, protein xnd, soluble proteins, RNA, and small EGCG and cell regeneration, leading to cellular damage and ultimately cell death The use, distribution or reproduction in other forums is permitted, provided the original author s and the copyright owner s are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. Thus the imbalance between ROS generation and elimination contributes to disease initiation and development [ 33 ]. L -theanine has cerebral protective activities such as anti-anxiety, anti-depression, memory promotion, prevention of vascular dementia, and nutritional nerve 23 — Close Navbar Search Filter Journal of Radiation Research This issue Biological Sciences Radiology Books Journals Oxford Academic Enter search term Search. |
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Development of epigallocatechin-gallate EGCG loaded collagen membrane to guide tissue regeneration Chen Yu Chu 1 , Yu Bin Cao 1 , Yuan Yuan Yin 1 , Jia Deng 1 and Yi Man 1, 2. Keywords: Cell Adhesion, biomaterial, Biocompatibility, Tissue Regeneration Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, HaCaT cells were pretreated with 50 µM EGCG for 1 h and then X-ray irradiated or treated as control.
After incubation for 24 h, 10 μM DCF-DA probe was added for 30 min, which reacted with cellular ROS and was metabolized into fluorescent DCF. A Representative images of fluorescent signals by a fluorescence microscope Leica SP2, Heidelberg, Germany under equivalent conditions.
B The histogram profile of the relative ROS levels of the four indicated groups. At least cells were counted from 10 randomly chosen fields of view. The percentages of the fluorescent intensities of the cells were quantified using ImageJ image analysis software MD, USA. Data are mean ± SEM.
EGCG alleviated radiation-induced apoptosis in HaCaT cells. A Cells were pretreated with EGCG for 1 h before being treated with 10 or 20 Gy of irradiation. Cells were incubated for another 48 h and then digested by trypsin. The apoptotic cell percentage was detected by flow cytometry.
B DNA was extracted from HaCaT cells with or without being pretreated with EGCG before irradiation. Isolated DNA was subjected to 1.
C Western blot was performed to detect the protein level of the apoptosis-related proteins Bcl-2 and Bax. Effect of EGCG on mitochondrial mass and γH2AX foci in HaCaT cells.
A HaCaT cells were X-ray irradiated after EGCG pretreatment for 1 h. Mito-Tracker Red was used to detect the mitochondrial membrane integrity. The nuclei of the cells were stained with Hoechst for another 30 min and analyzed by fluorescence microscopy × magnification. B The relative fluorescence intensity of mitochondria in HaCaT cells was calculated with ImageJ image analysis software Bethesda, MD, USA.
C Immunofluorescence of γH2AX foci red in untreated HaCaT cells and in cells irradiated with 5-Gy X-ray irradiation for an additional 1, 2 or 12 h at 37°C. Nuclei were stained with DAPI blue. Images were acquired using a fluorescent microscope Leica SP2, Heidelberg, Germany. D The average number of foci per cell was obtained from three independent experiments.
We further explored whether EGCG influences the dynamic repair process of DNA double-strand breaks DSBs induced by radiation. DSBs are considered to be the most significant lesion caused by ionizing radiation.
DNA damage was measured by immunostaining of phosphorylated H2AX protein γ-H2AX , which is recruited to nuclear structures termed foci. γ-H2AX foci occurred immediately after irradiation, reached a high point at 1 h after 5-Gy irradiation and remained present for nearly 24 h.
We measured the γ-H2AX foci 1, 2 and 12 h post-irradiation. Compared with the control cells, EGCG pretreatment of HaCaT cells significantly reduced the γ-H2AX foci number Fig. EGCG upregulated HO-1 via transcriptional activation.
A A luciferase reporter containing the HO-1 promoter was transfected into HaCaT cells, which were then exposed to the indicated concentration of EGCG for an additional 24 h. B The effect of EGCG treatment plus irradiation on the protein expression of HO-1, SOD1 and SOD2.
HaCaT cells were X-ray irradiated after EGCG pretreatment for 1 h. Western blot was performed to detect the protein level of HO-1, SOD1 and SOD2. Inhibition of HO-1 by SnPPIX or siRNA decreased radioprotection of EGCG in HaCaT cells. A Transfection of HO-1 targeting siRNA downregulated HO-1 protein.
HO-1 expression was determined by Western blot analysis 48 h after transfection with HO-1 siRNA. B The effect of HO-1 silencing on the viability of HaCaT cells with or without irradiation. Cells were transfected with the indicated siRNA and then pretreated with EGCG 50 µM before 20 Gy of irradiation.
C The effect of HO-1 inhibition on the viability of HaCaT cells with or without irradiation. Cells were incubated with SnPPIX 10 µM and EGCG 50 µM for 1 h, then X-ray irradiated. Cell viability was determined by MTT assay. Data are presented as mean ± SEM. Skin injury caused by ionizing radiation is one of the most common complications of radiation oncology treatment [ 29 ].
Chronic radiation-induced skin damage is related to ROS generated in skin that is exposed for an extended time and apoptosis that is promoted by inflammatory reaction factors. In this study, we showed that EGCG enhanced the values of D 0 and Dq in HaCaT cells after irradiation.
These results indicate that EGCG decreased the radiosensitivity of HaCaT cells and enhanced their capacity for sublethal damage repair. We also found that pretreatment with 50 µM EGCG significantly enhanced cell viability, maintained cell mitochondrial mass and decreased ROS and apoptosis induced by X-ray irradiation of skin HaCaT cells.
However, the reduction in apoptotic rate was greater with μM EGCG than with 50 μM EGCG, which is inconsistent with the results from the MTT assay that indicated 50 μM EGCG showed the best protective effect. This discrepancy may be due to different evaluation indices for the MTT assay and the apoptosis analysis.
Considering the proliferation promoting effect of EGCG to non-irradiated normal tissues, a dose of 50 μM EGCG would be more applicable.
Numerous studies have indicated that radiation-induced cytotoxicity is triggered by ROS, which regulate multiple cellular processes [ 30 ]. As a consequence, biological macromolecules such as DNA, RNA and proteins are destroyed. Moreover, loss of enzyme activity, cell cycle disorder, cell apoptosis and cell death can also occur [ 31 , 32 ].
Thus the imbalance between ROS generation and elimination contributes to disease initiation and development [ 33 ]. Most intracellular ROS are generated via mitochondrial electron transport [ 34 ]. EGCG is the major polyphenolic constituent in green tea and is also a powerful antioxidant and free radical scavenger with the ability to modulate cell growth, cell cycle and apoptosis in human cells [ 35 , 36 ].
In the present study, we measured the ROS levels by DCF-DA and found that EGCG pretreatment could suppress radiation-induced ROS and affect oxidative status. Moreover, EGCG was found to preferentially induce apoptosis in tumor cells but not normal cells [ 37 ], which intensified its application for cancer patients especially for those receiving radiotherapy.
In vitro and in vivo experiments have shown that EGCG can reduce UV-induced skin damage [ 38 ]. The present work illustrates a radioprotective role for EGCG, which expands the known beneficial effects of EGCG.
A variety of factors such as hypoxia, genetic damage, extreme oxidation pressure, and radiation trigger the initiation of the mitochondrial apoptotic pathway, leading to an increase in mitochondrial permeability. Thus, maintaining mitochondrial mass and function is important for preventing the progression of many diseases [ 39 ].
EGCG has been shown to prevent mitochondrial damage induced by isoproterenol [ 40 ]. We showed that pretreatment of EGCG maintained mitochondrial mass and reduced apoptosis after irradiation, which may be attributed to the induction of SOD2.
SOD2, also known as MnSOD, is exclusively located in mitochondria and plays a critical role in protection against ionizing radiation in mammalian cells [ 41 ]. The mitochondria-dependent apoptotic pathway is regulated by proteins such as the pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins [ 42 ].
Induction of the cytoprotective protein HO-1 by EGCG has also been well established in multiple carcinoma cells [ 45 ] and endothelial cells [ 46 ]. HO-1 is a ubiquitous, redox-sensitive and inducible stress protein that catalyzes heme to produce carbon monoxide and bilirubin, representing an adaptive response to oxidative damage [ 47 , 48 ].
Several mechanisms such as anti-oxidant function and maintenance of microcirculation, anti-apoptotic and anti-inflammatory properties may contribute to the cytoprotection of HO-1 overexpression [ 48 — 50 ].
We have previously reported that overexpression of antioxidant heme HO-1 ameliorates radiation-induced skin injury in rats [ 51 ]. In this study, we confirmed that EGCG treatment boosts HO-1 expression in human skin HaCaT cells in a dose-dependent manner via transcriptional activation.
Thus, we postulate that the radioprotective effect of EGCG on HaCaT cells may in part be attributed to HO-1 overexpression. To validate this hypothesis, we inhibited HO-1 both via transfection of an siRNA targeting HO-1 and via treatment with a competitive inhibitor of HO-1 SnPPIX prior to treatment with EGCG.
We found that the protective effect of EGCG was attenuated, indicating that HO-1 mediated the protective function of EGCG in response to X-ray irradiation. In this study, we found that in skin HaCaT cells irradiated by X-rays, the application of EGCG significantly enhanced viability, maintained cell mitochondrial mass and decreased apoptosis induction.
ROS and γH2AX foci in HaCaT cells were significantly reduced when pretreated with EGCG prior to irradiation. Moreover, HO-1 knockdown or the HO-1 specific inhibitor tin protoporphyrin SnPPIX reversed the protective role of EGCG, indicating the pivotal role of HO Taken together, these results suggest that the application of EGCG offers a new strategy for protecting skin against ionizing radiation.
Funding to pay the Open Access publication charges for this article was provided by the National Natural Science Foundation of China This work is supported by the National Natural Science Foundation of China , and , the Key Programs of Natural Science Foundation of Jiangsu Educational Committee 11KJA and the Priority Academic Program Development of Jiangsu Higher Education Institutions PAPD.
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Advanced Search. Search Menu. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Abstract. MATERIALS AND METHODS. Journal Article. Epigallocatechingallate EGCG protects skin cells from ionizing radiation via heme oxygenase-1 HO-1 overexpression.
Wei Zhu , Wei Zhu. Oxford Academic. Google Scholar. Jing Xu. Afterward, each protein was extracted from the cell lysate and blotted to the membrane. The membrane was blocked and incubated with primary antibodies, including either mouse anti-human CCNA, CCNB1, and CDK1 monoclonal antibodies BD Biosciences or rabbit anti-human CDKN1A monoclonal antibody Cell Signaling Technology Inc , followed by incubation with horseradish peroxidase-conjugated secondary antibodies.
Protein expression was detected as described above. The effect of EGCG on gene expression of nHDFs was investigated using a Platinum Human Cancer 3. This microarray consisted of oligonucleotide spots including Operon Operon, Huntsville, AL, USA , human oligo subsets, housekeeping genes and Arabidopsis DNA as controls.
All oligonucleotide probes were designed from the UniGene Database Build Hs and the Human Reference Sequence Database, both developed and maintained at the National Center for Biotechnology Information.
The printed slides were processed according to the CMT-GAPS II slide protocol. After FITC-EGCG treatment, the cells were washed thoroughly with PBS, fixed with 3. The results are reported as the mean±standard deviation SD compared with the non-treated controls.
A one-way analysis of variance ANOVA , which was followed by a Tukey HSD test for the multiple comparisons, was used to detect the dose-based differential effects of EGCG on nHDFs.
Proliferation was inhibited in nHDFs in a dose-differential manner in response to EGCG treatment Figure 1A. Dose-based differential effects of EGCG on proliferation, morphology, and cell cycle progression of nHDFs. The proliferation A , cell cycle progression B , and morphology C of EGCG-treated nHDFs were measured by WST-8 assay, flow cytometry and optical microscopy, respectively.
The results are reported as a mean±SD and analyzed by a Tukey HSD test. The data shown in this figure are representative of 6 independent experiments, showing similar results. A dose-differential modulation of cell cycle progression of nHDFs by EGCG treatment was also found. As shown in Figure 1B , EGCG treatment triggered an appreciable dose-dependent increase in the Sub-G 1 phase ie , 5.
Accordingly, the cells could not enter the S phase during treatment with higher concentrations of EGCG, which might induce cell cycle arrest as well as apoptosis. This dose-differential antiproliferative response of EGCG in nHDFs was evident from the morphological observations Figure 1C.
The dose-differential effects of EGCG on apoptosis of nHDFs were evident from a DNA ladder assay, which showed the induction of DNA fragmentation with increasing concentrations of EGCG treatment Figure 2A.
However, treatment of nHDFs with higher concentrations of EGCG resulted in late apoptosis and necrosis as evident by the formation of internucleosomal DNA fragments and high fluorescence of Annexin V-FITC and PI. Overall, this dose-differential pattern of apoptosis induced by EGCG in nHDFs agreed well with that of cell growth regulation, as shown in Figure 1.
Dose-based differential apoptotic responses of nHDFs to EGCG. DNA fragmentation A and apoptosis B in EGCG-treated nHDFs were measured by DNA ladder and Annexin V-FITC assays, respectively.
Dose-based differential modulation of pNF-κB expression in nHDFs by EGCG. Results from representative experiments were normalized to GAPDH expression by densitometry.
The quantitative results are shown in the lower panel. A cDNA microarray analysis was employed to evaluate the effect of EGCG on the expression level of cell cycle-related genes.
The microarray contained oligonucleotide probes representing human genes and included 50 cell cycle-related genes. Although a total of genes were screened in EGCG-treated nHDFs, the expression of genes was statistically reliable with respect to the control.
Among the cell cycle-related genes, some genes were significantly down-regulated in response to EGCG treatment Table 1. In contrast, CDK inhibitor A1 CDKN1A or p21 , a G 1 phase regulator, was significantly up-regulated upon EGCG treatment. Other cell cycle-related genes, such as CCNC, CCND3, CCNF, CDC20, and CKS2 CDC28 protein kinase regulatory subunit 2 , were slightly down-regulated Table 2.
The expression levels of the other cell cycle-related genes are listed in Supplementary Table S1. The detailed expression levels of other genes significantly changed by EGCG treatment relative to the controls are listed in Supplementary Tables S2 and S3.
Figure 4 demonstrates the regulatory effects of EGCG on the expression level of cell cycle-related proteins in nHDFs. In contrast, the expression level of CDKN1A was slightly suppressed after EGCG treatment, but remained at low levels 72 h after the removal of EGCG.
These data suggest that the expression of cell cycle-related genes and proteins can be reversibly regulated by EGCG exposure and removal.
Effects of EGCG on expression of cell cycle-related proteins in nHDFs. The expression of cell cycle-related proteins in nHDFs A was determined by Western blotting as described in Materials and methods.
The quantitative results are shown in the right panel B. The cellular uptake of FITC-EGCG was not observed until approximately 4 h of culture and was most dramatic between 4 and 6 h data not shown.
As the incubation time progressed, FITC-EGCG became concentrated in the cytoplasm and then localized to different sites within the cells. At higher magnification, FITC-EGCG was clearly found to be widely distributed in the cytosol of the cells and partially translocated into the nucleus, bound to endosome-like structures.
However, the incorporation of FITC-EGCG into the cells was appreciably decreased by pretreatment with free EGCG data not shown , suggesting that a negligible amount of conjugate accumulates within cells by virtue of competition with unlabelled EGCG for membrane binding sites.
This cellular uptake pattern of FITC-EGCG in normal fibroblasts was completely different from the pattern in human fibrosarcoma HT cells as their cancer counterparts 21 and murine fibroblastic L cells Supplementary Figure S1 Additional fluorescence microscopic observation demonstrated the time course of cellular uptake of FITC-EGCG in cultured nHDFs.
FITC-EGCG was clearly observed in both the membrane and the cytoplasm of the cells until 10 d, despite the removal of FITC-EGCG from the culture medium Supplementary Figure S2. Cellular uptake of FITC-EGCG in nHDFs.
The micrographs shown in this figure are representative of 6 independent experiments, showing similar results [Original magnification: × and × A, upper and lower, respectively , and × B ]. Arrows in A indicates FITC-EGCG translocated into the nucleus. Both the membrane and the nucleus of detached nHDFs were too damaged to delineate due to apoptosis.
Furthermore, the cells that were still attached but adversely affected by FITC-EGCG showed typical apoptotic appearances, including blebbing, nuclear swelling, and membrane deterioration, whereas they were subjected to the nuclear translocation of FITC-EGCG like their cancer counterparts These results imply that FITC-EGCG at an almost non-toxic concentration can be incorporated into the cytosol of the cells through binding to specific receptors and forming complexes with them.
Furthermore, FITC-EGCG may be differentially translocated into the nucleus in nHDFs vs HT cells and L cells, which contributes to the reversible regulation of the cell cycle and leads to cytoprotection.
In the present study, we demonstrated that the dose-differential effects of EGCG were associated with the reversible regulation of the expression of cell cycle-related genes and proteins in nHDFs. Although several studies comparing the effects of EGCG on cancer cells and their normal counterparts have been reported previously 5 , 6 , 7 , it has not been clear whether these pharmacological effects of EGCG are related to the reversible regulation of the cell cycle mediated by EGCG incorporated into the cells.
Natural dietary anti-cancer chemopreventive compounds, such as polyphenols eg , EGCG and curcumin , isothiocyanates eg , sulforaphane and phenethyl isothiocyanate and their combinations, are known to possess redox-mediated differential signaling mechanisms that lead to cytoprotection of normal cells vs cytotoxicity in tumor cells This result suggests that EGCG or its derivatives can be developed as agents to selectively eliminate cancer cells without affecting normal cells.
Even these concentrations are not physiological 23 , but provide a framework for understanding the differential effects of EGCG. These results suggest the possibility that appropriate concentrations of EGCG may inhibit the growth of certain cancer cells, but have no apparent adverse effect on the growth of their normal counterparts.
We also showed that EGCG displayed a dose-differential regulatory effect on pNF-κB expression, but not on NF-κB expression Figure 3. Perkins et al have found that specific CDKs regulate NF-κB-mediated transcriptional activation through interactions with the coactivator p and that the transcriptional activation domain of RelA p65 interacts with an amino-terminal region of p that is distinct from a carboxyl-terminal region of p required for binding to the CCNE-CDK2 complex According to their report, CDKN1A or a dominant negative CDK2, which inhibits passociated CCNE-CDK2 activity, stimulates NF-κB-dependent gene expression, and the interaction of NF-κB and CDKs through the p and CREB-binding protein coactivators provides a mechanism for the coordination of transcriptional activation with cell cycle progression.
Increasing evidence has indicated that the development of selective NF-κB inhibitors may represent a promising therapeutic tool to sensitize tumor cells to apoptosis and increase the efficacy of conventional anticancer drugs in a wide spectrum of malignancies In general, cDNA microarray analysis provides extensive information on global gene expression.
Indeed, the data presented here demonstrated the global regulation of multiple genes involved in cell survival, cell cycle, and apoptosis that are considered some of the most important factors for cytoprotection Among the cell cycle-related genes, those that were the most affected by EGCG treatment were CCNs and CDKs directly involved in cell cycle regulation.
Interestingly, a substantial difference in the response to EGCG among the cell cycle-related genes was present.
Based on the results of a cell growth assay and cytometry, cellular responses such as proliferation and cell cycle progression can be predicted to be regulated by cell cycle-related genes that were changed over 2-fold upon EGCG treatment. Therefore, the reversible effects of EGCG may allow its safe usage in cell protection for chemotherapy 16 , Our hypothesis was further verified by finding highly positive correlations with the results from immunoblotting that showed similar patterns to those of the microarray data Figure 4.
Removal of EGCG from the culture medium restored the expression of cell cycle-related proteins to the levels before EGCG treatment, which suggests that the effect of EGCG on the cell cycle is reversible. We also showed that the cellular uptake of FITC-EGCG in normal fibroblasts was different from that in a fibroblastic cell line Figure 5 and Supplementary Figure S1 in their cancer counterparts HT cells These cellular uptake patterns were involved as important contributors to the dose-differential growth inhibitory, cell cycle regulatory, and apoptotic responses of EGCG.
Although the exact mechanism of the incorporation of EGCG into cells has not been fully elucidated, some evidence has been reported that EGCG is bound to the membrane and incorporated into the cytosol and the nucleus of cancer cell lines such as PC-9 and HT cells treated with 3 H]EGCG 8 , These quantitative results using 3 H]EGCG agree well with our qualitative results showing that the FITC-EGCG was incorporated into the cytosol of fibroblastic cells 20 , However, the nuclear translocation pattern of FITC-EGCG was quite different in nHDFs vs HT cells and L cells Supplementary Figure S2 , which would lead to the reversible regulation of the cell cycle and cytoprotection of normal fibroblasts.
These phenomena can be related in part to the intrinsic characteristics of EGCG as a polyphenolic compound. EGCG is amphipathic and easily binds to extracellular matrices, lipid membranes or any type of intracellular protein 27 , 28 , Therefore, EGCG seems to be able to be incorporated into the cytosol of fibroblastic cells and further translocated into the nucleus and may modulate the exogenous signals directed to genes required for the survival and apoptosis of the cells.
Recent studies have demonstrated that expression of the metastasis-associated 67 kDa laminin receptor might confer EGCG responsiveness to cancer cells at physiologically relevant concentrations, suggesting that the gallate moiety of EGCG may be critical for receptor binding and subsequent activity 30 , Moreover, the observation that nucleic acids extracted from catechin-treated cells were colored implied that because both galloyl and catechol groups of EGCG were essential for DNA and RNA binding, both groups seemed to hold strands of DNA and RNA via their branching structure 32 , Taking these results into consideration, we conclude that EGCG internalized into normal fibroblasts can dose-differentially regulate the expression of cell cycle-related genes and proteins and exert cytoprotective effects on the cells through a feasible mechanism of action Supplementary Figure S3.
This dose-differential regulatory activity of EGCG may be exploited to craft strategies using EGCG for the cytoprotection of normal cells and the chemoprevention of cancer cells. Dong-Wook HAN, Mi Hee LEE, and Jong-Chul PARK conceived and designed the study; Dong-Wook HAN and Mi Hee LEE performed the research and analyzed the data; Dong-Wook HAN, Hak Hee KIM, and Suong-Hyu HYON wrote the manuscript; Jong-Chul PARK edited the manuscript and provided the funding support for the studies.
Other experimental details for confocal and fluorescence microscopy are provided with the results for the cellular uptake of FITC-EGCG in L cells Figure S1 and the time course of cellular uptake of FITC-EGCG in cultured nHDFs Figure S2 as well as a discussion of the feasible mechanism of action of EGCG Figure S3.
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Mechanisms of growth inhibition of human lung cancer cell line, PC-9, by tea polyphenols.
Type 1 Mindful eating and mindful mindful eating community regeheration therapy is one celk the Slimming products therapeutic approaches for Polyunsaturated fats damage treatment due to Natural mental stimulus cell tissue regeneration. Epigallocatechin gallate EGCG rgeneration one of the active components found regsneration green tea. Experimental results suggest that EGCG shows beneficial effects on cell protection. This study explores whether a better pancreatic regeneration therapeutic effect could be found in mesenchymal stem cells pretreated with EGCG compared to stem cells without EGCG pretreatment. A cell model confirmed that adipose-derived stem cells ADSC incubated with EGCG increase cell viability under high-glucose HG stress. This is due to survival marker p-Akt expression.
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