Investigation of prunetrin induced G2/M cell cycle arrest and apoptosis via Akt/mTOR/MAPK pathways in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) stands as a leading cause of mortality, and despite recent advancements in the overall survival rates, the prognosis remains dismal. Prunetin 4-O-glucoside (Prunetrin or PUR), an active compound derived from Prunus sp., was explored for its impact on HepG2 and Huh7 cells. The cytotoxicity assessment revealed a notable reduction in cell viability in both cell lines, while exhibiting non-toxicity towards HaCaT cells. Colony formation studies underscored PUR's inhibitory effect on cell proliferation, dose-dependently. Mechanistically, PUR downregulated cell cycle proteins (CDC25c, Cdk1/CDC2, and Cyclin B1), inducing G2/M phase arrest, corroborated by flow cytometry. Western blot analyses exhibited dose-dependent cleavages of PARP and caspase 3, indicative of apoptosis. Treatment with the apoptotic inhibitor z-vmd-fmk provided evidence of PUR-induced apoptosis. Annexin V and PI flow cytometry further affirmed apoptotic induction. Enhanced expression of cleaved-caspase 9 and the pro-apoptotic protein Bak, coupled with reduced anti-apoptotic Bcl-xL, and affirmed PUR's induction of intrinsic apoptosis. Additionally, PUR activated the MAPK pathway, evidenced by elevated phospho p38 and phospho ERK expressions in both cell lines. Notably, a concentration-dependent decrease in mTOR and Akt expressions indicated PUR's inhibition of the Akt/mTOR pathway in HepG2 and Huh7 cells. These findings illuminate PUR's multifaceted impact, revealing its potential as a promising therapeutic agent against HepG2 and Huh7 cells through modulation of cell cycle, apoptosis, and key signaling pathways.

Potential Antioxidant and Anti-Inflammatory Properties of Polyphenolic Compounds from Cirsium japonicum Extract.

Cirsium japonicum is a medicinal plant that has been used due to its beneficial properties. However, extensive information regarding its therapeutic potential is scarce in the scientific literature. The antioxidant and anti-inflammatory potential of polyphenols derived from the Cirsium japonicum extracts (CJE) was systematically analyzed. High-performance liquid chromatography (HPLC) with mass spectrometry (MS) was used to examine the compounds in CJE. A total of six peaks of polyphenol compounds were identified in the extract, and their MS data were also confirmed. These bioactive compounds were subjected to ultrafiltration with LC analysis to assess their potential for targeting cyclooxygenase-2 (COX2) and DPPH. The outcomes showed which primary compounds had the highest affinity for binding both COX2 and DPPH. This suggests that components that showed excellent binding ability to DPPH and COX2 can be considered significant active substances. Additionally, in vitro analysis of CJE was carried out in macrophage cells after inducing inflammation with lipopolysaccharide (LPS). As a result, it downregulated the expression of two critical pro-inflammatory cytokines, COX2 and inducible nitric oxide synthase (iNOS). In addition, we found a solid binding ability through the molecular docking analysis of the selected compounds with inflammatory mediators. In conclusion, we identified polyphenolic compounds in CJE extract and confirmed their potential antioxidant and anti-inflammatory effects. These results may provide primary data for the application of CJE in the food and pharmaceutical industries with further analysis.

Antioxidant effects of phenolic compounds in through the distillation of Lonicera japonica & Chenpi extract and anti-inflammation on skin keratinocyte.

The phenolic compounds in Lonicera japonica & Chenpi distillation extract (LCDE) were thoroughly examined for their antioxidant and anti-inflammatory properties. Phenolic compounds in LCDE were analyzed for five peaks using high-performance liquid chromatography (HPLC) combined with mass spectrometry (MS) and determined. Five phenolic compounds were identified from the samples and MS data. Ultrafiltration with LC analysis was used to investigate the ability of bioactive compounds to target DPPH. As a result, it was confirmed that the major compounds exhibited a high binding affinity to DPPH and could be regarded as antioxidant-active compounds. In addition, the anti-inflammatory effect of LCDE was confirmed in vitro, and signal inhibition of anti-inflammation cytokines, MAPK and NF-kB pathways was confirmed. Finally, Molecular docking analysis supplements the anti-inflammatory effect through the binding affinity of selected compounds and inflammatory factors. In conclusion, the phenolic compounds of the LCDE were identified and potential active compounds for antioxidant and anti-inflammatory activities were identified. Additionally, this study will be utilized to provide basic information for the application of LCDE in the pharmaceutical and pharmaceutical cosmetics industries along with information on efficient screening techniques for other medicinal plants.

Potential Joint Protective and Anti-Inflammatory Effects of Integrin αvß3 in IL-1ß-Treated Chondrocytes Cells.

In osteoarthritis (OA), the articular cartilage covering the articular surface of the bone wears out, exposing the subchondral bone, and the synovial membrane surrounding the joint becomes inflamed, causing pain and deformity. OA causes pain, stiffness, and swelling, and discomfort in the knee when climbing stairs is a typical symptom. Although drug development studies are conducted to treat these inflammatory joint diseases, it is difficult to find conclusive research results which could reduce inflammation and slow cartilage tear. The development of drugs to relieve inflammatory pain often utilizes inflammatory triggers. Interleukins, one of the proteins in the limelight as pro-inflammatory factors, are immune-system-stimulating factors that promote the body's fight against harmful factors such as bacteria. In this study, inflammation was induced in Chondrocytes cells (Chon-001 cells) with IL-1ß and then treated with integrin αvß3 to show anti-inflammatory and chondrogenesis effects. Integrin αvß3 was not toxic to Chon-001 cells in any concentration groups treated with or without IL-1ß. COX-2 and iNOS, which are major markers of inflammation, were significantly reduced by integrin αvß3 treatment. Expressions of p-ERK, p-JNK, and p-p38 corresponding to the MAPKs signaling pathway and p-IκBα and p-p65 corresponding to the NF-κB signaling pathway were also decreased in a dose-dependent manner upon integrin αvß3 treatment, indicating that inflammation was inhibited, whereas treatment with integrin αvß3 significantly increased the expression of ALP, RUNX2, BMP2, BMP4, Aggrecan, SOX9, and COL2A1, suggesting that osteogenesis and chondrogenesis were induced. These results suggest that integrin αvß3 in-duces an anti-inflammatory effect, osteogenesis, and chondrogenesis on IL-1ß-induced Chon-001 cells.

Potential Antioxidant and Anti-Inflammatory Effects of Lonicera japonica and Citri Reticulatae Pericarpium Polyphenolic Extract (LCPE).

Dermatitis is an inflammatory condition of the outer layer of the skin that causes itching, blisters, redness, swelling, and often exudation, scabs, and peeling. Among them, purulent inflammation is a symptom that often occurs on the skin and appears in the form of boils and acne. Various studies are being conducted to treat these inflammatory diseases. Accordingly, Lonicera japonica and Citri Reticulatae Pericarpium Polyphenolic Extract (LCPE), which uses herbal preparations such as Lonicera japonica, Citri Reticulatae Pericarpium, and Glycyrrhiza uralensis, has been used to suppress inflammation since ancient times, and its anti-inflammatory effect can be observed in skin keratinocytes after inducing inflammation. In this study, the major polyphenolic compounds in LCPE were quantitatively determined by analyzing the data through peak values using high-performance chromatography (HPLC-MS/MS) coupled with mass spectrometry. Additionally, bioactive compounds targeting 2,2-diphenyl-1-picrylhydrazyl (DPPH) were analyzed by ultrafiltration integrated with LC. Several compounds with the most significant effects were selected (chlorogenic acid, narirutin, and isorhamnetin). Skin keratinocytes induced by lipopolysaccharide (LPS) were treated with LCPE to show its anti-inflammatory effects. After LCPE treatment, inflammation-mediating cytokines such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were decreased. In addition, nuclear factor kappa (NF-кB) and mitogen-activated protein kinase (MAPK) were inhibited in important pathways related to inflammation. Lastly, molecular modeling was performed to determine binding scores with inflammation-related proteins using molecular docking for the selected compounds. According to these results, LCPE is effective in treating keratinocytes induced by LPS and reducing inflammation and has potential antioxidant effects, and the polyphenol components have been identified.

Mechanistic Action of Cell Cycle Arrest and Intrinsic Apoptosis via Inhibiting Akt/mTOR and Activation of p38-MAPK Signaling Pathways in Hep3B Liver Cancer Cells by Prunetrin-A Flavonoid with Therapeutic Potential.

Hepatocellular carcinoma (HCC) has a poor prognosis and a low survival rate. Drugs without side effects are desperately needed since chemotherapy has a negative effect on the host cells. Previous research has firmly established that plant-based compounds have significant bioactivities without a negative impact on the host. Flavonoids, in particular, are a class of compounds with both anti-inflammatory and anti-cancer properties. Prunetrin (PUR) is a glycosyloxyisoflavone (Prunetin 4'-O-glucoside) derived from Prunus sp., and its other form, called prunetin, showed optimistic results in an anti-cancerous study. Hence, we aimed to discover the anti-cancer ability of prunetrin in liver cancer Hep3B cells. Our cytotoxicity results showed that PUR can decrease cell viability. The colony formation assay confirms this strongly and correlates with cell cytotoxicity results. Prunetrin, in a dose-dependent manner, arrested the cell cycle in the G2/M phase and decreased the expression of cyclin proteins such as Cyclin B1, CDK1/CDC2, and CDC25c. Prunetrin treatment also promoted the strong cleavage of two important apoptotic hallmark proteins called PARP and caspase-3. It also confirms that apoptosis occurs through the mitochondrial pathway through increased expression of cleaved caspase-9 and increased levels of the pro-apoptotic protein Bak. Bak was significantly increased with the declining expression of the anti-apoptotic protein Bcl-xL. Next, it inhibits the mTOR/AKT signaling pathways, proving that prunetrin includes apoptosis and decreases cell viability by suppressing these pathways. Further, it was also observed that the activation of p38-MAPK was dose-dependent. Taken together, they provide evidence that prunetrin has an anti-cancerous ability in Hep3B liver cancer cells by arresting the cell cycle via p38 and inhibiting mTOR/AKT.

Inhibition of Cell Proliferation and Cell Death by Apigetrin through Death Receptor-Mediated Pathway in Hepatocellular Cancer Cells.

Epidemiologic research recommends using flavonoids in the diet due to their overall health benefits. Apigetrin (Apigenin 7-O-glucoside) is a glycoside phytonutrient found in fruits and vegetables and known for different biological activities such as antioxidant and anti-inflammatory properties. Hepatocellular cancer (HCC) is a major health concern because of its adverse prognosis and side effects of chemotherapeutic agents. In the present study, we determine the impact of apigetrin on HepG2 cells and its cell death mechanism. Apigetrin reduced HepG2 cell proliferation with morphological changes and floating cells in treated cells. Colony formation and wound healing assays showed a reduced cell number in treatment groups. Further, we checked for the cell cycle through flow cytometry to understand the cell death mechanism. Apigetrin induced G2/M phase arrest in HepG2 cells by regulating Cyclin B1 and CDK1 protein levels in HepG2 cells. Annexin V and propidium iodide (PI) staining was performed to confirm the apoptotic cell population in treated groups. At the higher concentration, apigetrin showed a late apoptotic population in HepG2 cells. Chromatin condensation was also found in the treatment groups. Western blot analysis showed an increased expression of extrinsic apoptotic proteins such as FasL, Cleaved caspase 8, Cleaved caspase 3, and cleavage of PARP. In comparison, intrinsic apoptotic pathway markers showed no changes in Bax, Bcl-xL, and Cleaved caspase 9. Altogether, these findings strongly indicate that apigetrin causes cell death in HepG2 cells through the extrinsic apoptotic pathway, and that the intrinsic/mitochondrial pathway is not involved.

Flavones: The Apoptosis in Prostate Cancer of Three Flavones Selected as Therapeutic Candidate Models.

Cancer is a widespread but dangerous disease that can strike anyone and is the second 1leading cause of death worldwide. Prostate cancer, in particular, is a prevalent cancer that occurs in men, and much research is being done on its treatment. Although chemical drugs are effective, they have various side effects, and accordingly, anticancer drugs using natural products are emerging. To date, many natural candidates have been discovered, and new drugs are being developed as drugs to treat prostate cancer. Representative candidate compounds that have been studied to be effective in prostate cancer include apigenin, acacetin and tangeretin of the flavone family among flavonoids. In this review, we look at the effects of these three flavones on prostate cancer cells via apoptosis in vitro and in vivo. Furthermore, in addition to the existing drugs, we suggest the three flavones and their effectiveness as natural anticancer agents, a treatment model for prostate cancer.

Apigetrin Promotes TNFα-Induced Apoptosis, Necroptosis, G2/M Phase Cell Cycle Arrest, and ROS Generation through Inhibition of NF-κB Pathway in Hep3B Liver Cancer Cells.

Apigetrin (7-(ß-D-glucopyranosyloxy)-4',5-dihydroxyflavone), a glycoside bioactive dietary flavonoid derived from Taraxacum officinale and Teucrium gnaphalodes, is known to possess anticancer, antioxidant, and anti-inflammatory effects on numerous cancers. In the present study, we examined the effect of apigetrin in Hep3B hepatocellular cancer cell line (HCC). Apigetrin inhibited cell growth and proliferation of Hep3B cells, as confirmed by MTT and colony formation assay. We used apigetrin at concentrations of 0, 50, and 100 µM for later experiments. Of these concentrations, 100 µM of apigetrin showed a significant effect on cell inhibition. In apigetrin-treated Hep3B cells, cell cycle arrest occurred at the G2/M phase. Apoptosis and necroptosis of Hep3B cells treated with apigetrin were confirmed by Annexin V/propidium iodide (PI) staining and flow cytometry results. Morphological observation through 4',6-diamidino-2-phenylindole (DAPI) staining showed intense blue fluorescence representing chromatin condensation. Hematoxylin staining showed necroptotic features such as formation of vacuoles and swelling of organelles. Apigetrin increased reactive oxygen species (ROS) levels in cells, based on fluorescence imaging. Furthermore, the underlying mechanism involved in the apoptosis and necroptosis was elucidated through western blotting. Apigetrin up-regulated TNFα, but down-regulated phosphorylation of p-p65, and IκB. Apigetrin inhibited the expression of Bcl-xl but increased Bax levels. Up-regulation of cleaved PARP and cleaved caspase 3 confirmed the induction of apoptosis in apigetrin-treated Hep3B cells. Additionally, necroptosis markers RIP3, p-RIP3, and p-MLKL were significantly elevated by apigetrin dose-dependently, suggesting necroptotic cell death. Taken together, our findings strongly imply that apigetrin can induce apoptosis and necroptosis of Hep3B hepatocellular cancer cells. Thus, apigetrin as a natural compound might have potential for treating liver cancer.

Flavones: Six Selected Flavones and Their Related Signaling Pathways That Induce Apoptosis in Cancer.

Cancer is a horrific disease that, to date, has no cure. It is caused by various factors and takes many lives. Apoptosis is a programmed cell death mechanism and if it does not function correctly in cancer cells, it can lead to severe disease. There are various signaling pathways for regulating apoptosis in cancer cells. Flavonoids are non-artificial natural bioactive compounds that are gaining attention as being capable of for inducing apoptosis in cancer cells. Among these, in this study, we focus on flavones. Flavones are a subclass of the numerous available flavonoids and possess several bioactive functions. Some of the most reported and well-known critical flavones, namely apigenin, acacetin, baicalein, luteolin, tangeretin, and wogonin, are discussed in depth in this review. Our main aim is to investigate the effects of the selected flavones on apoptosis and cell signaling pathways that contribute to death due to various types of cancers.

Cellular Regulation of Kynurenic Acid-Induced Cell Apoptosis Pathways in AGS Cells.

Kynurenic acid was included in the three compounds (caffeic acid, chlorogenic acid, and kynurenic acid) that showed high antioxidant and anti-inflammatory potential among the phenolic compounds contained in Gynura procumbens. In this study, the mechanism of cancer cell death induced by kynurenic acid (KYNA), which has the highest molecular binding affinity, in the gastric cancer cell line AGS was confirmed in molecular docking analysis. KYNA showed the most cancer cell death effect on AGS cells among several gastric cancer cell lines (MKN, AGS, and SNU). AGS cells were used for later experiments, and KYNA concentrations of 0, 150, 200, and 250 µM were used. KYNA inhibited cell migration and proliferation in AGS cells in a concentration-dependent manner. G2/M phase cell cycle arrest and reduction of related proteins (Cdc25C, CDK1 and CyclinB1) were confirmed in KYNA-treated AGS cells. Apoptosis of KYNA-treated AGS cells was confirmed by Annexin V/propidium iodide (PI) staining flow cytometry analysis. As a result of morphological chromatin condensation through DAPI (4',6-diamidino-2-phenylindole), intense blue fluorescence was confirmed. The mechanism of apoptosis induction of KYNA-treated AGS cells was confirmed by western blotting. In the extrinsic pathway, apoptosis induction markers FasL, Fas, and Caspase-3 and -8 were increased in a concentration-dependent manner upon KYNA treatment. In the intrinsic pathway, the expression of anti-apoptotic factors PI3K, AKT, and Bcl-xL was down-regulated, and the expression of apoptosis-inducing factors BAD, Bak, Bax, Cytochrom C, and Caspase-9 was up-regulated. Therefore, in the present study, we strongly imply that KYNA induces apoptosis in AGS gastric cancer cells. This suggests that KYNA, a natural compound, could be the basis for drug for the treatment of gastric cancer.

Function and Application of Flavonoids in the Breast Cancer.

Breast cancer is one of the top causes of death, particularly among women, and it affects many women. Cancer can also be caused by various factors, including acquiring genetic alteration. Doctors use radiation to detect and treat breast cancer. As a result, breast cancer becomes radiation-resistant, necessitating a new strategy for its treatment. The approach discovered by the researchers is a flavonoid, which is being researched to see if it might help treat radiation-resistant breast cancer more safely than an approved medicine already being used in the field. As a result, this study focuses on the role of flavonoids in breast cancer suppression, breast cancer gene anomalies, and the resulting apoptotic mechanism.

Scutellarein Inhibits LPS-Induced Inflammation through NF-κB/MAPKs Signaling Pathway in RAW264.7 Cells.

Inflammation is a severe topic in the immune system and play a role as pro-inflammatory mediators. In response to such inflammatory substances, immune cells release cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). Lipopolysaccharide (LPS) is known as an endotoxin in the outer membrane of Gram-negative bacteria, and it catalyzes inflammation by stimulating the secretion of inflammatory-mediated cytokines such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) by stimulated immune cells. Among the pathways involved in inflammation, nuclear factor kappa (NF-кB) and mitogen-activated protein kinases (MAPKs) are important. NF-kB is a diploid composed of p65 and IkBα and stimulates the pro- gene. MAPKs is a family consisting of the extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38, JNK and p38 play a role as proinflammatory mediators. Thus, we aim to determine the scutellarein (SCU) effect on LPS stimulated RAW264.7 cells. Furthermore, since scutellarein has been shown to inhibit the SARS coronavirus helicase and has been used in Chinese medicine to treat inflammatory disorders like COVID-19, it would be required to examine scutellarein's anti-inflammatory mechanism. We identified inflammation-inducing substances using western blot with RAW264.7 cells and SCU. And we discovered that was reduced by treatment with SCU in p-p65 and p-IκBα. Also, we found that p-JNK and p-ERK were also decreased but there was no effect in p-p38. In addition, we have confirmed that the iNOS was also decreased after treatment but there is no change in the expression of COX-2. Therefore, this study shows that SCU can be used as a compound to treat inflammation.

Prunetinoside Inhibits Lipopolysaccharide-Provoked Inflammatory Response via Suppressing NF-κB and Activating the JNK-Mediated Signaling Pathway in RAW264.7 Macrophage Cells.

Inflammation is a multifaceted response of the immune system at the site of injury or infection caused by pathogens or stress via immune cells. Due to the adverse effects of chemical drugs, plant-based compounds are gaining interest in current research. Prunetinoside or prunetin-5-O-glucoside (PUG) is a plant-based active compound, which possesses anti-inflammatory effects on immune cells. In this study, we investigate the effect of PUG on mouse macrophage RAW264.7 cells with or without stimulation of lipopolysaccharide (LPS). Cytotoxicity results showed that PUG is non-cytotoxic to the cells and it reversed the cytotoxicity in LPS-stimulated cells. The levels of nitric oxide (NO) and interleukin-6 (IL-6) were determined using a NO detection kit and IL-6 ELISA kit, respectively, and showed a significant decrease in NO and IL-6 in PUG-treated cells. Western blot and qRT-PCR were performed for the expression of two important pro-inflammatory cytokines, COX2 and iNOS, and found that their expression was downregulated in a dose-dependent manner. Other pro-inflammatory cytokines, such as IL-1ß, IL-6, and TNFα, had reduced mRNA expression after PUG treatment. Furthermore, a Western blot was performed to calculate the expression of NF-κB and MAPK pathway proteins. The results show that PUG administration dramatically reduced the phosphorylation of p-Iκbα, p-NF-κB 65, and p-JNK. Remarkably, after PUG treatment, p-P38 and p-ERK remain unchanged. Furthermore, docking studies revealed that PUG is covalently linked to NF-κB and suppresses inflammation. In conclusion, PUG exerted the anti-inflammatory mechanism by barring the NF-κB pathway and activating JNK. Thus, prunetinoside could be adopted as a therapeutic compound for inflammatory-related conditions.

Identification of Kynurenic Acid-Induced Apoptotic Biomarkers in Gastric Cancer-Derived AGS Cells through Next-Generation Transcriptome Sequencing Analysis.

Understanding the triggers and therapeutic targets for gastric cancer, one of the most common cancers worldwide, can provide helpful information for the development of therapeutics. RNA sequencing technology can be utilized to identify complex disease targets and therapeutic applications. In the present study, we aimed to establish the pharmacological target of Kynurenic acid (KYNA) for gastric cancer AGS cells and to identify the biological network. RNA sequencing identified differentially expressed genes (DEGs) between KYNA-treated and untreated cells. A total of 278 genes were differentially expressed, of which 120 genes were up-regulated, and 158 genes were down-regulated. Gene ontology results confirmed that KYNA had effects such as a reduction in genes related to DNA replication and nucleosome organization on AGS cells. Protein-protein interaction was confirmed through STRING analysis, and it was confirmed that cancer cell growth and proliferation were inhibited through KEGG, Reactome, and Wiki pathway analysis, and various signaling pathways related to cancer cell death were induced. It was confirmed that KYNA treatment reduced the gene expression of cancer-causing AP-1 factors (Fos, Jun, ATF, and JDP) in AGS cell lines derived from gastric cancer. Overall, using next-generation transcriptome sequencing data and bioinformatics tools, we confirmed that KYNA had an apoptosis effect by inducing changes in various genes, including factor AP-1, in gastric cancer AGS cells. This study can identify pharmacological targets for gastric cancer treatment and provide a valuable resource for drug development.