Abstract
Indolizine derivatives are a class of compounds with excellent biological activity. In this study, a series of indolizine derivatives, compound 1 (C1), compound 2 (C2), compound 3 (C3), and compound 4 (C4), were synthesized. 3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium bromide (MTT) assay was used to evaluate their cytotoxicity against HepG2 (p53-wild), A549, and HeLa cell lines. HepG2 cells apoptosis induced by C3 was determined using Hoechst staining and acridine orange/ethidium bromide staining. Cells’ apoptotic ratio was measured by Annexin V–FITC/PI double staining. Changes in mitochondrial membrane potential and intracellular reactive oxygen species (ROS) in HepG2 cells after C3 treatment were determined. Immunofluorescence staining and Western blot analysis were carried out to detect p53 levels and analyze the apoptosis-associated proteins, respectively. Moreover, the cytotoxic activity of C3 was examined in two other hepatocellular carcinoma (HCC) cell lines with different p53 status including Huh-7 cells (p53-mutant) and Hep3B cells (p53-null). The results indicated that C3 showed stronger inhibition towards HepG2 cells than other cell lines. Fluorescent staining and flow cytometry analysis confirmed that C3 induced apoptosis of HepG2 cells. C3 could also increase intracellular ROS and cause a decrease in the mitochondrial membrane potential. C3 promoted p53 activation and increased p53 accumulation in nuclei. The expression of p53 and Bax was increased with the down-regulation of Bcl-2, which promoted the release of cytochrome c and caspase-3 activation. Collectively, the study demonstrated that C3 caused HepG2 cell apoptosis via the mitochondria p53 pathway. These results inspired us to further develop indolizine derivatives as potential potent inhibitors against liver cancer.
Highlights
MATERIALS AND METHODSIndolizine, an important class of heterocycles, has attracted critical attention of chemists for its application value
The reaction was allowed to stir at room temperature under Ar atmosphere for 2 h
compound 3 (C3) treatment (20 and 40 μg/ml) in HepG2 cells augmented the green fluorescent monomer population to 7.44% and 13.28% at 24 h, indicating that C3 could decrease the MMP. These results indicated that C3 might induce HepG2 cells apoptosis through the mitochondria-mediated apoptosis pathway
Summary
Indolizine, an important class of heterocycles, has attracted critical attention of chemists for its application value. They are found in a large number of natural products such as (±)-homocrepidine A (Hu et al, 2015), tashiromine (Marsden and McElhinney, 2008), swainsonine (Lee et al, 2013), Pandalisines A and B (Cheng et al, 2015), Flueggedine (Zhao et al, 2013), and so on. Four novel indolizine derivatives were synthesized (Figure 1) in a brand-new way. Their anti-tumor activities and the underlying mechanisms have not been fully elucidated. The aim of this study was to investigate the optimal activity of four new indolizine derivatives synthesized on a variety of cancer cells and to explore the related mechanisms
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