Suchismita Raha

Naringin induces autophagy-mediated growth inhibition by downregulating the PI3K/Akt/mTOR cascade via activation of MAPK pathways in AGS cancer cells

Naringin, one of the major bioflavonoid of Citrus, has been demonstrated as potential anticancer agent. However, the underlying anticancer mechanism still needs to be explored further. This study investigated the inhibitory effect of Naringin on human AGS cancer cells. AGS cell proliferation was inhibited by Naringin in a dose- and time-dependent manner. Naringin did not induce apoptotic cell death, determined by no DNA fragmentation and the reduced Bax/Bcl-xL ratio. Growth inhibitory role of Naringin was observed by western blot analysis demonstrating downregulation of PI3K/Akt/mTOR cascade with an upregulated p21CIPI/WAFI. Formation of cytoplasmic vacuoles and autophagosomes were observed in Naringin-treated AGS cells, further confirmed by the activation of autophagic proteins Beclin 1 and LC3B with a significant phosphorylation of mitogen activated protein kinases (MAPKs). Collectively, our observed results determined that anti-proliferative activity of Naringin in AGS cancer cells is due to suppression of PI3K/Akt/mTOR cascade via induction of autophagy with activated MAPKs. Thus, the present finding suggests that Naringin induced autophagy- mediated growth inhibition shows potential as an alternative therapeutic agent for human gastric carcinoma.

Mitochondrial Dysfunction and Ca(2+) Overload Contributes to Hesperidin Induced Paraptosis in Hepatoblastoma Cells, HepG2.

Paraptosis is a programmed cell death which is morphologically and biochemically different from apoptosis. In this study, we have investigated the role of Ca2+ in hesperidin‐induced paraptotic cell death in HepG2 cells. Increase in mitochondrial Ca2+ level was observed in hesperidin treated HepG2 cells but not in normal liver cancer cells. Inhibition of inositol‐1,4,5‐triphosphate receptor (IP3R) and ryanodine receptor also block the mitochondrial Ca2+ accumulation suggesting that the release of Ca2+ from the endoplasmic reticulum (ER) may probably lead to the increase in mitochondrial Ca2+ level. Pretreatment with ruthenium red (RuRed), a Ca2+ uniporter inhibitor inhibited the hesperidin‐induced mitochondrial Ca2+ overload, swelling of mitochondria, and cell death in HepG2 cells. It has also been demonstrated that mitochondrial Ca2+ influxes act upstream of ROS and mitochondrial superoxide production. The increased ROS production further leads to mitochondrial membrane loss in hesperidin treated HepG2 cells. Taken together our results show that IP3R and ryanodine receptor mediated release of Ca2+ from the ER and its subsequent influx through the uniporter into mitochondria contributes to hesperidin‐induced paraptosis in HepG2 cells. J. Cell. Physiol. 231: 1261–1268, 2016.

Vitamin D2 suppresses amyloid-β 25–35 induced microglial activation in BV2 cells by blocking the NF-κB inflammatory signaling pathway

AIMS Present emerging world is emphasizing the implication of vitamin D deficiency associated with development of inflammation and neurodegenerative disorder like Alzheimers disease (AD). The chief neuropathological hallmark of AD is aggregation of amyloid-beta (Aβ) peptides surrounding microglial cells in human brain. Microglial activation plays a key role in inflammatory response and neuronal injury. Naturally abundant vitamin D2 (VD2) exhibiting anti-inflammatory activities are yet to explore more. This study has investigated the inhibitory effect of VD2 on inflammatory activities of BV2 microglial cells. MAIN METHODS Cellular compatibility of VD2 and Aβ25-35 protein in treated BV2 microglial cells were measured by CCK-8 assay. Induction of iNOS, COX-2 and NF-κB signaling cascade were measured by western blotting, whereas pro-inflammatory cytokines were measured by ELISA. In addition, generation of ROS was detected by fluorescence intensity. KEY FINDINGS Morphological observations showed that Aβ25-35 induced BV2 cells stimulation noticeably got reduced in VD2 pre-treated group at 24h time period. Anti-inflammatory activities of VD2 was observed demonstrating the inhibition of up-regulated iNOS and COX-2 protein expression further confirmed by attenuating the activated microglia released pro-inflammatory cytokines IL-1β, IL-6, TNF- α and ROS, while blocking the phosphorylation of NF-κB p65 in nucleus by preventing IκB-α degradation and phosphorylation in cytosol. SIGNIFICANCE The present study revealed that VD2 blocked the phosphorylation of NF-κB inflammatory signaling pathway in Aβ25-35 induced activated BV2 microglial cells by suppressing ROS generation and inflammatory cytokines. Our finding suggests that vitamin D2 has therapeutic potential against inflammation and Alzheimers disease.

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.

Poncirin Induces Apoptosis in AGS Human Gastric Cancer Cells through Extrinsic Apoptotic Pathway by up-Regulation of Fas Ligand

Poncirin, a natural bitter flavanone glycoside abundantly present in many species of citrus fruits, has various biological benefits such as anti-oxidant, anti-microbial, anti-inflammatory and anti-cancer activities. The anti-cancer mechanism of Poncirin remains elusive to date. In this study, we investigated the anti-cancer effects of Poncirin in AGS human gastric cancer cells (gastric adenocarcinoma). The results revealed that Poncirin could inhibit the proliferation of AGS cells in a dose-dependent manner. It was observed Poncirin induced accumulation of sub-G1 DNA content, apoptotic cell population, apoptotic bodies, chromatin condensation, and DNA fragmentation in a dose-dependent manner in AGS cells. The expression of Fas Ligand (FasL) protein was up-regulated dose dependently in Poncirin-treated AGS cells Moreover, Poncirin in AGS cells induced activation of Caspase-8 and -3, and subsequent cleavage of poly(ADP-ribose) polymerase (PARP). Inhibitor studies’ results confirm that the induction of caspase-dependent apoptotic cell death in Poncirin-treated AGS cells was led by the Fas death receptor. Interestingly, Poncirin did not show any effect on mitochondrial membrane potential (ΔΨm), pro-apoptotic proteins (Bax and Bak) and anti-apoptotic protein (Bcl-xL) in AGS-treated cells followed by no activation in the mitochondrial apoptotic protein caspase-9. This result suggests that the mitochondrial-mediated pathway is not involved in Poncirin-induced cell death in gastric cancer. These findings suggest that Poncirin has a potential anti-cancer effect via extrinsic pathway-mediated apoptosis, possibly making it a strong therapeutic agent for human gastric 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. Copyright

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 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.

Inhibition of IAP’s and activation of p53 leads to caspase-dependent apoptosis in gastric cancer cells treated with Scutellarein

Gastric cancer is the fifth most common cancer and the third leading cause of cancer deaths worldwide. South Korea is in first place with 9,180 death alone attributed to gastric cancer in 2013. Plenty of literature suggests the evasion of apoptosis is implicated in neurodegeneration, autoimmune diseases, and tumors development due to dysregulation in the apoptotic mechanism. Reduced apoptosis or its resistance in cancer cells plays a significant role in carcinogenesis. It’s imperative to understand apoptosis, which provides the basis for novel targeted therapies that can induce cancer cell death or sensitize them to cytotoxic agents by regulating key factors like IAPs, MDM2, p53, caspases and much more. Studies have demonstrated that Scutellarein have the ability to inhibit several cancer cells by inducing apoptosis with both: Scutellarein monomers as well as scutellarein containing flavonoids. MTT results revealed that scutellarein inhibited cell viability in both dose and time dependent manner. Flow cytometry and western blot analysis showed that scutellarein induces apoptosis in both AGS and SNU-484 human gastric cancer cells and G2/M phase cell cycle arrest in SNU-484 cells. This study demonstrated that the Scutellarein on AGS and SNU-484 cells significantly inhibits cell proliferation and induces apoptotic cell death via down regulating MDM2 and activated the tumor suppresser protein p53, subsequently down regulating the IAP family proteins (cIAP1, cIAP2, and XIAP) leading to caspase-dependent apoptosis in AGS and SNU-484 cells.

Identification of a novel biomarker in tangeretin‑induced cell death in AGS human gastric cancer cells

Proteomic analysis serves as an important biological tool for identifying biological events. Novel biomarkers of a specific disease such as cancer may be identified using these promising techniques. The aim of the present study was to investigate the effect of tangeretin and to identify potential biomarkers in AGS gastric cancer cells using a proteomics approach. The results of the present study revealed that tangeretin inhibited AGS cell viability dose-dependently with a half-maximal inhibitory concentration of 100 µM. Two-dimensional gel electrophoresis was performed to determine the potential biomarker between control and tangeretin (100 µM)-treated AGS cells. A total of 16 proteins was identified from 36 significant protein spots using matrix-assisted laser-desorption/ionization time-of-flight-mass spectrometry using peptide fingerprinting. The bioinformatics tools Protein ANalysis THrough Evolutionary Relationships (PANTHER) and Database for Annotation, Visualization and Integrated Discovery (DAVID) were used to identify the functional properties and association of the proteins obtained. Using western blot analysis, the regulatory pattern of four selected proteins, protein kinase Cε, mitogen-activated protein kinase 4, phosphoinositide 4-kinase and poly(ADP-ribose) polymerase 14, were successfully verified in replicate sample sets. These selected proteins are primarily involved in apoptosis signaling, angiogenesis, cell cycle regulation, receptor kinase binding, intracellular cytoplasmic and nuclear alterations. Therefore, aim of the present study was to identify potential diagnostic biomarkers from the functional categories of altered protein expression in tangeretin-inhibited AGS gastric cancer cell viability.