Pectolinarigenin (PEC), a natural flavonoid present in Cirsium chanroenicum and in some species of Citrus fruits, has various pharmacological benefits such as anti-inflammatory and anti-cancer activities. In the present study, we investigated the anti-cancer mechanism of PEC induced cell death caused by autophagy and apoptosis in AGS and MKN28 human gastric cancer cells. The PEC treatment significantly inhibited the AGS and MKN28 cell growth in a dose-dependent manner. Further, PEC significantly elevated sub-G1 phase in AGS cells and G2/M phase cell cycle arrest in both AGS and MKN28 cells. Apoptosis was confirmed by Annexin V and Hoechst 33342 fluorescent staining. Moreover, Immunoblotting results revealed that PEC treatment down-regulated the inhibitor of apoptosis protein (IAP) family protein XIAP that leads to the activation of caspase-3 thereby cleavage of PARP (poly-ADP-ribose polymerase) in both AGS and MKN28 cells in a dose-dependent manner. The autophagy-inducing effect was indicated by the increased formation of acidic vesicular organelles (AVOs) and increased protein levels of LC3-II conversion in both AGS and MKN28 cells. PEC shows the down regulation of PI3K/AKT/mTOR pathway which is a major regulator of autophagic and apoptotic cell death in cancer cells that leads to the down-regulation of p-4EBP1, p-p70S6K, and p-eIF4E in PEC treated cells when compared with the untreated cells. In conclusion, PEC treatment might have anti-cancer effect by down-regulation of PI3K/AKT/mTOR pathway leading to G2/M phase cell cycle arrest, autophagic and apoptotic cell death in human gastric cancer cells. Further studies of PEC treatment can support to develop as a potential alternative therapeutic agent for human gastric carcinoma.

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-1β (IL-1β). 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.

Despite widespread use and prospective biomedical applications of copper nanoparticles (Cu NPs), their biosafety issues and kinetics remain unclear. Thus, the aim of this study was to compare the detailed in vivo toxicity of Cu NPs and cupric ions (CuCl₂; Cu ions) after a single oral dose. We determined the physicochemical characteristics of Cu NPs, including morphology, hydrodynamic size, zeta potential, and dissolution in gastric (pH 1.5), vehicle (pH 6.5), and intestinal (pH 7.8) conditions. We also evaluated the kinetics of Cu following a single equivalent dose (500 mg/kg) of Cu NPs and Cu ions. Cu NPs had highest dissolution (84.5%) only in gastric conditions when compared with complete dissolution of Cu ions under various physiological milieus. Kinetic analysis revealed that highest Cu levels in blood and tested organs of Cu NP-treated rats were 15%–25% lower than that of Cu ions. Similar to the case of Cu ions, Cu levels in the tested organs (especially liver, kidney, and spleen) of Cu NP-treated rats increased significantly when compared with the vehicle control. However, delay in reaching the highest level and biopersistence of Cu were observed in the blood and tested organs of Cu NP-treated rats compared with Cu ions. Extremely high levels of Cu in feces indicated that unabsorbed Cu NPs or absorbed Cu ions were predominantly eliminated through liver/feces. Cu NPs exerted apparent toxicological effects at higher dose levels compared with Cu ions and showed sex-dependent differences in mortality, biochemistry, and histopathology. Liver, kidney, and spleen were the major organs affected by Cu NPs. Collectively, the toxicity and kinetics of Cu NPs are most likely influenced by the release of Cu dissociated from Cu NPs under physiological conditions.

Scutellarein (SCU), a flavone found in the perennial herb Scutellaria baicalensis, is known for a wide range of biological activities. In the present study, we investigated the effects of treatment with SCU flavonoids on inducing apoptosis via the extrinsic pathway in Hep3B cells. SCU treatment significantly inhibited Hep3B cell proliferation and induced G2/M phase cell cycle arrest by inhibiting the expression level of the proteins Cdc25C, cdk1 and Cyclin B1. Allophycocyanin (APC)/Annexin V and propidium iodide (PI) double-staining showed upregulation of apoptotic cell death fraction. We further confirmed apoptosis by 4′-6-diamidino-2-phenylindole (DAPI) fluorescent staining and observed DNA fragmentation with agarose gel electrophoresis. Further, immunoblotting results showed that treatment with SCU showed no changes in Bax and Bcl-xL protein levels. In addition, SCU treatment did not affect the mitochondrial membrane potential (MMP) in Hep3B cells. On the contrary, treatment with SCU increased the expression of Fas and Fas ligand (FasL), which activated cleaved caspase-8, caspase-3, and polymeric adenosine diphosphate ribose (PARP), whereas the expression level of death receptor 4 (DR4) decreased. We confirmed that the proteins expressed upon treatment with SCU were involved in the Fas-mediated pathway of apoptosis in Hep3B cells. Thus, our findings in the current study strongly imply that SCU can be a basic natural source for developing potent anti-cancer agents for hepatocellular carcinoma (HCC) treatment.

Apigetrin (7-(β-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.