Objective To investigate the autophagy activation effect of hernandezine (HER) on hepatocellular carcinoma (HCC) cells and its role in inducing autophagic cell death. Methods The inhibitory effects of HER on the proliferation of HCC cell lines Hep 3B and Huh-7 were assessed using CCK-8 assay. HCC cells were treated with HER at different concentrations and time points gradient. Western blot analysis was performed to evaluate the conversion of autophagy marker LC3 from type Ⅰ to type Ⅱ and the levels of SQSTM1/p62. Stable red fluorescent protein(RFP)-LC3-expressing cell lines were established through lentiviral infection, and the accumulation of RFP-LC3 puncta following HER treatment was observed under confocal microscope. Autophagosomes in HER-treated cells were visualized using transmission electron microscopy. The effects of autophagy inhibitors, bafilomycin A1 (BafA1) or hydroxychloroquine (HCQ), in combination with HER were investigated by analyzing cell death rates via flow cytometry. The phosphorylation levels of adenosine 5′-monophosphate(AMP)-activated protein kinase(AMPK) and mammalian target of rapamycin(mTOR) were assessed by Western blot.Results HER inhibited the proliferation of HCC cells Hep 3B and Huh-7, promoted the conversion of LC3-Ⅰ to LC3-Ⅱ, and activated autophagy in a dose- and time-dependent manner. Confocal microscopy and transmission electron microscopy revealed a significant increase in autophagic vesicles in HER-treated cells. HER-induced cell death was attenuated by the autophagy inhibitors BafA1 or HCQ. HER treatment increased AMPK phosphorylation levels while decreasing mTOR phosphorylation levels.Conclusions HER induces autophagy and promotes autophagic cell death in HCC cells via the AMPK/mTOR signaling pathway.

OBJECTIVES: To investigate the expression of cartilage acidic protein 1 (CRTAC1) in gastric cancer (GC) and its effect on biological behaviors and immune cell infiltration of GC. METHODS: Transcriptomic, GO and KEGG analyses were conducted to investigate the association of CRTAC1 expression with prognosis of GC patients and its involvement in cell function and signaling pathways. ESTIMATE algorithm was used to analyze the effect of CRTAC1 expression on the tumor microenvironment and the tumor mutation load. In two GC cell clines (HGC-27 and MKN-74), CCK8, EdU and clone formation assays, flow cytometry, and Hoechst staining were used to examine the effects of CRTAC1 knockdown on cell proliferation, cell cycle changes and apoptosis. Wound healing assay, Transwell assay, and Western blotting were performed to analyze the effect of CRTAC1 knockdown on GC cell migration and the underlying mechanism. RESULTS: Bioinformatics analysis showed significantly higher expression of CRTAC1 in GC tissues than in adjacent tissues (P<0.05). Age and tumor stage were both prognostic risk factors in GC patients with high CRTAC1 expression (P<0.001). Analysis using ESTIMATE algorithm showed that CRTAC1 expression increased immune cell infiltration and decreased tumor mutational load in GC (P<0.001). In HGC-27 and MKN-74 cells, CRTAC1 knockdown significantly inhibited cell proliferation and migration and promoted cell apoptosis. Western blotting demonstrated that CRTAC1 knockdown significantly increased E-cadherin expression and reduced the expression levels of vimentin, p-PI3K, AKT2, p-AKT and p-mTOR in GC cells. CONCLUSIONS High expression of CRTAC1 in GC tissues affects immunotherapeutic efficacy and prognosis of the patients, possibly by promoting epithelial-mesenchymal transition via modulating tumor mutational load, tumor microenvironment, and the PI3K/AKT signaling pathway.
Stomach Neoplasms/metabolism* ; Humans ; Cell Proliferation ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Signal Transduction ; Cell Movement ; Cell Line, Tumor ; Prognosis ; Apoptosis ; Tumor Microenvironment ; Female ; Male ; Epithelial-Mesenchymal Transition/genetics*