Gracillin Isolated from Reineckia carnea Induces Apoptosis of A549 Cells via the Mitochondrial Pathway

Abstract

Background: Reineckia carnea is commonly used to treat cough, pneumonia and other diseases in China. In our previous study, it was found that the ethanol extracts of Reineckia carnea have a strong inhibitory effect on the proliferation of human lung cancer A549 cells. Here, we isolated gracillin from ethanol extracts for the first time. Purpose: Clarify the antiproliferation effect of gracillin on A549 cells and further explore its mechanisms via the mitochondrial pathway. Methods: Gracillin was isolated and purified by silica gel, D-101 macroporous resin and preparative RP-HPLC, then identified by NMR and HR-MS. The inhibitory effects of gracillin on the proliferation of A549 cells were detected by the MTS method. Its mechanisms were further explored by flow cytometry and Western blot. Results: A steroid saponin, gracillin, was isolated and identified from Reineckia carnea for the first time. In a concentration-dependent and time-dependent manner, gracillin significantly inhibited the proliferation of A549 cells with an IC 50 value at 2.54 μmol/L and induced morphological changes. The results of flow cytometry analysis showed that the apoptosis rate of A549 cells was significantly increased (p < 0.05), and the cells proportion was obviously arrested in S phase. The concentration of intracellular calcium was raised (p < 0.01), and the mitochondrial membrane potential was greatly decreased (p < 0.01). In addition, the expression levels of Bax, caspase-3, cleaved caspase-3, and cytochrome C were dramatically up-regulated while Bcl-2 was down-regulated (p < 0.05) in A549 cells. Conclusion:This study confirmed that gracillin has a significant antiproliferative effect on A549 cells. Gracillin could induce the apoptosis of A549 cells through the mitochondrial pathway, which might be associated with regulation of the concentration of intracellular calcium, the mitochondrial membrane potential and the expression levels of Bax, Bcl-2, caspase-3, cleaved caspase-3, and cytochrome C.

Identification and vitro verification of the potential drug targets of active ingredients of Chonglou in the treatment of lung adenocarcinoma based on EMT-related genes

“…The expression differences of m 6 A-related genes, including ALKBH5, HNRNPC, METTL14, METTL3, YTHDF2, IGF2BP1, IGF2BP3, METTL16, RBMX, IGF2BP2, and HNRNPA2BA, were analyzed between the low-risk and the high-risk groups mentioned above in LUAD patients (Huang et al, 2020). (Tao et al, 2018;Ding et al, 2020;Wu et al, 2020), gracillin (Liu et al, 2021;Yang J. et al, 2021;, polyphyllin I (Chang et al, 2017;Tian et al, 2020;Lai et al, 2021;Shen et al, 2021), polyphyllin II (Niu et al, 2020), polyphyllin III (Zhou et al, 2021), polyphyllin VI (Liu et al, 2020;Teng et al, 2020), polyphyllin VII (He et al, 2020;Zhao et al, 2021) and polyphyllin H) were collected by using the online tools (Su et al, 2019), including PubChem (https://pubchem. The overlapping gene set between the drug targets of Chonglou active ingredients with the EMT-LUAD related genes were enquired in the STRING database (version 11.5) (EMT-LUAD- Frontiers in Genetics frontiersin.org Chonglou related genes), which was used for the following proteinprotein interactions (PPI) network, hub genes and enrichment analyses.…”

“…), is a natural medicine. The active ingredients of Chonglou have been reported to effectively inhibit various cancers, such as Dioscin and Gracillin promote the apoptosis and cell cycle arrest in lung cancer, colorectal cancer and osteosarcoma cells ( Ding et al, 2020 ; Wu et al, 2020 ; Yang J. et al, 2021 ) Meanwhile, polyphyllin Ⅰ inhibits osteosarcoma development through inactivating Wnt signaling pathway ( Chang et al, 2017 ), and polyphyllin Ⅱ inhibits the metastasis of bladder cancer cells by regulating EMT ( Niu et al, 2020 ). Chonglou is a traditional Chinese anti-tumour herbal medicine and is often used as one of the ingredients in many patented Chinese anti-tumour drugs in modern clinical treatment to reduce toxicity and increase the efficiency of treatment, such as Gan-Fu-Le capsules for the adjuvant treatment of primary hepatocellular carcinoma and Bo-Er-Ning capsules for the adjuvant treatment in many cancers ( Wang et al, 2018 ).…”

Common methods in mitochondrial research (Review)

“…Furthermore, although the mitochondrial extraction method is suitable for most tissues and cells, the extraction efficiency and quantity of mitochondria in different tissues and cells are significantly different. This is determined by the number of mitochondria in the tissue or cell and the energy consumption of muscles and liver; larger tissues contain more mitochondria, so these tissues and cells have higher mitochondrial extraction efficiency than other tissues, such as the lungs (18)(19)(20).…”

Dichloromethane fractions of Calystegia soldanella induce S‑phase arrest and apoptosis in HT‑29 human colorectal cancer cells

“…Since MMP changes are also associated with apoptosis (29)(30)(31), the effects of dcM fraction treatment on the MMP in HT-29 cells were investigated. The proportions of live and dead cells with depolarized mitochondria were markedly increased following dcM fraction treatment compared with those in the 0 µg/ml group.…”