Naringin Induces Lysosomal Permeabilization and Autophagy Cell Death in AGS Gastric Cancer Cells.

Autophagy is a process of active programmed cell death, where a dying cell induces autophagosomes and subsequently regulated by degradative machinery. The aim of this study was to investigate the mechanism behind induction of autophagic cell death by Naringin flavonoid in AGS cancer cells. Growth inhibition of AGS cells showed downregulation of PI3K/Akt/mTOR signaling by Naringin treatment. Transmission electron microscopy observation showed swollen mitochondria and lysosome near peri-nuclear zone fused with autophagic vacuoles. Rapamycin pre-treatment with Naringin showed significant decrease in mTOR phosphorylation and increase in LC3B activation in AGS cells. Decrease in mTOR phosphorylation is associated with lysosomal function activation was observed by time-dependent treatment of Naringin. Induction of lysosomal membrane permeabilization (LMP) was observed by LAMP1 activation leading lysosomal cell death by releasing Cathepsin D from lysosomal lumen to cytosol. Naringin treated AGS cells showed up-regulating BH3 domain Bad, down-regulating Bcl-xL, and Bad phosphorylation and significant mitochondrial fluorescence intensity expression. Significant localization of mitochondria and LC3B activation was examined by person coefficient correlation. Activation of ERK1/2-p38 MAPKs and production of intracellular ROS has been observed over Naringin treatment. It has also been elucidated that pre-treatment with NAC inhibited mitochondria-LC3B colocalization, where ROS acted as upstream of ERK1/2-p38 MAPKs activation. Lysosomal cell death involvement has been evaluated by BAF A1 pre-treatment, inhibiting LAMP1, Cathepsin D, ROS, and blocking autophagolysosome in AGS cell death. Taken together, these findings show that, Naringin induced autophagy cell death involves LMP mediated lysosomal damage and BH3 protein Bad activation in AGS cancer cells.

Korean Scutellaria baicalensis Georgi flavonoid extract induces mitochondrially mediated apoptosis in human gastric cancer AGS cells.

Korean Scutellaria baicalensis Georgi has been widely used in Korean folk medicines for its range of medicinal benefits, including its anticancer effect. The aim of the present study was to investigate the underlying molecular mechanism of action of a flavonoid extract from Korean Scutellaria baicalensis Georgi (FSB) on AGS human gastric cancer cells (gastric adenocarcinoma) in which FSB exhibits an anticancer effect. Treatment of AGS cells with FSB significantly inhibited cell viability in a concentration-dependent manner. Furthermore, FSB significantly increased the proportion of cells in sub-G1 phase, and Annexin V and Hoechst 33258 fluorescent staining confirmed the apoptotic cell death. Furthermore, western blotting results identified that treatment of AGS cells with FSB significantly downregulated the expression of caspase family members, namely procaspases 3 and 9, and poly(ADP-ribose) polymerase (PARP), and subsequently upregulated cleaved caspase 3 and cleaved PARP. It was observed that FSB treatment significantly decreased the mitochondrial membrane potential of AGS cells. In addition, the ratio of the mitochondrion-associated proteins B cell lymphoma 2-associated X protein and B cell lymphoma extra large was upregulated. The results of the present study provide novel insight into the underlying molecular mechanism of the anticancer effects of FSB on AGS human gastric cancer cells and indicate that FSB may be an alternative chemotherapeutic agent for the treatment of gastric cancer.

Flavone polyphenols dominate in Thymus schimperi Ronniger: LC-ESI-MS/MS characterization and study of anti-proliferative effects of plant extract on AGS and HepG2 cancer cells.

Thymus schimperi is a highly localized and a rare plant endemic to Ethiopia. An optimized and validated high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method was applied to characterize 23 polyphenolic compounds found in ethyl acetate extracts of the plant. From those, flavones dominated and luteolin was the major component contributing 21.83% of the total composition (or 46.05±0.59g/kg of fresh sample weight). Validation data showed a determination coefficient (R2)≥0.997. Limits of detection (LOD) and quantification (LOQ) were 0.03-0.97 and 0.11-3.23mg/L, while recovery values spiked at 5 and 50mg/L were between 70.89-115.39 and 67.65-120.19%, respectively. Except for caffeic acid and epicatechin gallate, the relative standard deviations (%RSDs) were far below 15%, showing acceptable precision values. The plant extracts inhibited cell proliferation and induced cell death in human gastric adenocarcinoma (AGS) and liver hepatocellular carcinoma (HepG2) cancer cells. This is the first report of polyphenolic components from T. schimperi being characterized using HPLC-ESI-MS/MS. Being components of many edible vegetables, fruits, and spices, the identified polyphenols suggest that T. schimperi could be a potential food with promising health benefits.

Korean Byungkyul - Citrus platymamma Hort.et Tanaka flavonoids induces cell cycle arrest and apoptosis, regulating MMP protein expression in Hep3B hepatocellular carcinoma cells.

Citrus platymamma Hort.et Tanaka is an indigenous fruit of Jeju island in Korea. In this study the bioactivity of C. platymamma flavonoids were evaluated on human hepatoma Hep3B cell lines. Eleven flavonoids were identified from the peels of C. platymamma Hort.et Tanaka through high-performance liquid chromatography-Tandem mass spectrometry and the anticancer effect of these C. platymamma flavonoids on human hepatoma Hep3B were studied. Chromatin condensation was observed in Hep3B cells treated with C. platymamma flavonoids. DNA fragmentation was confirmed through agarose gel electrophoresis and TUNEL assay. An increase in the total apoptotic cells and G2/M cell cycle arrest with decreased protein expression of CDC25C, CDK1, cyclin B1 and p21 were observed in Hep3B cells treated with flavonoids of C. platymamma. Further, protein expression of Bcl-XL, Bax, caspase-3 and -9 were also modulated by C. platymamma flavonoids treatment indicating that cell death is through intrinsic apoptotic pathway. Moreover, C. platymamma flavonoids also regulated the phosphorylation of MAPKs, PI3K, and Akt in Hep3B cells. Relevant to inhibiting metastasis, C. platymamma treatment reduced wound closure of Hep3B cells and the protein expression of matrix metalloproteinase-2 and -9 were reduced in C. platymamma treated cells. The results show that C. platymamma flavonoids induce cell cycle arrest and apoptosis following activation of MAPKs and suppression of PI3K/Akt pathway which eventually inhibits cell migration in Hep3B cells. The finding provides evidence on biochemical activities of C. platymamma Hort.et Tanaka, which would be an essential agent for hepatocellular carcinoma (HCC) treatment.

Flavonoids isolated from Citrus platymamma induced G2/M cell cycle arrest and apoptosis in A549 human lung cancer cells.

Citrus platymamma hort. ex Tanaka belongs to the Rutaceae family and is widely used in folk medicines in Korea due to its anti-proliferative, anti-cancer, anti-oxidant, anti-inflammatory and anti-diabetic activities. However, the molecular mechanism of its anti-cancer effect is not well understood. The present study was conducted to elucidate the anti-cancer effect and molecular mechanism of flavonoids from Citrus platymamma (FCP) on A549 cells. FCP displayed concentration-dependent inhibition on A549 cells proliferation. Further, flow cytometry revealed that FCP significantly increased the sub-G1 (apoptotic cell population) and G2/M phase population, and the total number of apoptotic cells, in a dose-dependent manner. Nuclear condensation and fragmentation were also observed upon staining with Hoechst 33342 in FCP-treated A549 cells. Immunoblotting demonstrated a dose-dependent downregulation of cyclin B1, cyclin-dependent kinase 1, cell division cycle 25c, pro-caspases -3, -6, -8 and -9, and poly (adenosine diphosphate-ribose) polymerase (PARP) in FCP-treated A549 cells. In addition, FCP induced caspase-3 activation and subsequent PARP cleavage, and increased the B-cell lymphoma (Bcl)-2-associated X protein/Bcl-extra large ratio in A549 cells. These findings suggest that FCP induced G2/M arrest and apoptosis of A549 cells. The present study provides evidence that FCP may be useful in the treatment of human lung cancer.

Flavonoids of Korean Citrus aurantium L. Induce Apoptosis via Intrinsic Pathway in Human Hepatoblastoma HepG2 Cells.

Korean Citrus aurantium L. has long been used as a medicinal herb for its anti-inflammatory, antioxidant, and anticancer properties. The present study investigates the anticancer role of flavonoids extracted from C. aurantium on human hepatoblastoma cell, HepG2. The Citrus flavonoids inhibit the proliferation of HepG2 cells in a dose-dependent manner. This result was consistent with the in vivo xenograft results. Apoptosis was detected by cell morphology, cell cycle analysis, and immunoblot. Flavonoids decreased the level of pAkt and other downstream targets of phosphoinositide-3-kinase/Akt pathway - P-4EBP1 and P-p70S6K. The expressions of cleaved caspase 3, Bax, and Bak were increased, while those of Bcl-2 and Bcl-xL were decreased with an increase in the expression of Bax/Bcl-xL ratio in treated cells. Loss of mitochondrial membrane potential was also observed in flavonoid-treated HepG2 cells. It was also observed that the P-p38 protein level was increased both dose and time dependently in flavonoid-treated cells. Collectively, these results suggest that flavonoid extracted from Citrus inhibits HepG2 cell proliferation by inducing apoptosis via an intrinsic pathway. These findings suggest that flavonoids extracted from C. aurantium L. are potential chemotherapeutic agents against liver cancer.

Flavonoids isolated from Citrus platymamma induce mitochondrial-dependent apoptosis in AGS cells by modulation of the PI3K/AKT and MAPK pathways.

Citrus platymamma hort. ex Tanaka (Rutaceae family) has been widely used in Korean folk medicine for its wide range of medicinal benefits including an anticancer effect. In the present study, we aimed to investigate the molecular mechanism of the anticancer effects of flavonoids isolated from Citrus platymamma (FCP) on AGS cells. FCP treatment significantly inhibited AGS cell growth in a dose­dependent manner. Furthermore, FCP significantly increased the percentage of cells in the sub-G1 phase (apoptotic cell population), and apoptosis was confirmed by Annexin V double staining. Chromatin condensation and apoptotic bodies were also noted in the FCP-treated AGS cells. Moreover, immunoblotting results showed that FCP treatment significantly decreased the expression of procaspase-3, -6, -8 and -9, and PARP and increased cleaved caspase-3, cleaved PARP and the Bax/Bcl-xL ratio in a dose-dependent manner. In addition, the phosphorylation of AKT was significantly decreased, whereas extracellular signal-related kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinases (MAPKs) were significantly increased in the FCP-treated AGS cells. Taken together, the cell death of AGS cells in response to FCP was mitochondrial-dependent via modulation of the PI3K/AKT and MAPK pathways. These findings provide new insight for understanding the mechanism of the anticancer effects of FCP. Thus, FCP may be a potential chemotherapeutic agent for the treatment of gastric cancer.

Proteome profiling of lipopolysaccharide induced L6 rat skeletal muscle cells response to flavonoids from Scutellaria baicalensis Georgi.

BACKGROUND: Scutellaria baicalensis Georgi is a commonly used medicinal herb in several Asian countries like Korea, China and Japan for thousands of years. It has been reported to have various medicinal properties such as anti-microbial, anti-inflammatory and anti-cancer effects. However, the anti-inflammatory mechanism of S. baicalensis G at proteome level has not yet been reported. Hence, we performed a proteome analysis to study differentially expressed proteins and its anti-inflammatory role in lipopolysaccharide (LPS) stimulated L6 skeletal muscle cells response to flavonoids isolated from S. baicalensis G. METHODS: For that, 150 µg of proteins from the L6 cells of the control (Vehicle only), LPS treated and flavonoid treated groups were separated using 18 cm, pH 4-7 IPG strips in the first dimension and resolved by 12% linear gradient SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The silver stained gels were analyzed by using progenesis SameSpots software and twenty six differentially expressed protein spots (≥ 2 fold, p < 0.05) were selected for matrix assisted laser desorption ionization- time of flight mass spectroscopy/mass spectrometry (MALDI-TOF/MS) analysis. Also, the expression of COX-2, iNOS and Annexin A2 proteins were analyzed by western blot. RESULTS: Totally, 12 differentially expressed proteins were successfully identified by MALDI-TOF/MS and database searching, that's involved in inflammatory responses such vimentin, T-box transcription factor TBX3, annexin A1, annexin A2 and annexin A5. In addition, flavonoids inhibited the expression of COX-2, iNOS and Annexin A2 proteins in LPS-stimulated L6 skeletal muscle cells. CONCLUSIONS: The findings revealed that the flavonoids from S. baicalensis G. directly protect the LPS stimulated inflammation process in L6 cells and, would be helpful to study further the muscle cell inflammatory mechanism. This is the first proteome study provide the anti-inflammatory mechanism of flavonoids from S. baicalensis G. in LPS stimulated L6 skeletal muscle cells.