[Levo-tetrahydropalmatine promotes apoptosis of human hepatocellular carcinoma through switching energy metabolism phenotype via upregulation of mitochondrial reactive oxygen species].

Mitochondrion is an important organelle that maintains cellular homeostasis and plays a crucial role in determining cell fate. The present study investigated the effect of levo-tetrahydropalmatine(THP) on autophagic flux and energy metabolism phenotype of human hepatocellular carcinoma(HCC) SMMC-7721 and BEL-7402 cells. SMMC-7721 and BEL-7402 cells were treated with THP(100 µmol·L~(-1)) with or without N-acetyl-L-cysteine(NAC, 10 µmol·L~(-1)) for 24 h. The mitochondrial reactive oxygen species(mtROS) was detected by flow cytometry(FCM) with MitoSOX probe and fluorescence microscopy, respectively. Thereafter, autophagic flux was detected by FCM with CYTO-ID probe, and the protein levels of microtubule-associated protein 1 A/1 B-light chain 3-Ⅰ(LC3Ⅰ), LC3Ⅱ, and phosphorylated AMP-activated protein kinase(p-AMPK)/AMPK were measured by Western blot. Mitochondrial respiration was examined by Seahorse XFp assay and cell proliferation by a system. Annexin V-FITC and PI/RNase staining was employed to detect apoptosis of SMMC-7721 and BEL-7402 cells treated with THP and/or NAC. Subsequently, membrane potential was measured with MitoTracker Red CMXRos. Compared with the control group, THP promoted mtROS production and THP combined with NAC attenuated the autophagic flux increase induced by THP alone in SMMC-7721 and BEL-7402 cells. When cells were co-treated with THP and chloroquine(CQ, an autophagy inhibitor), THP further increased mtROS and apoptosis. In addition, THP significantly reduced mitochondrial respiration in terms of mitochondrial basal respiration, ATP production, and maximal respiration. Meanwhile, THP significantly reduced the proliferation index and mitochondrial membrane potential of HCC cells accompanied by the increased apoptosis. This study demonstrates that the up-regulation of mtROS by THP significantly promotes HCC cell autophagy(protective autophagy) and impairs mitochondrial respiration through reprogramming energy metabolism, ultimately inducing the mitochondria-mediated apoptosis of SMMC-7721 and BEL-7402 cells.

Recombinant adenoviruses expressing apoptin suppress the growth of MCF­7 breast cancer cells and affect cell autophagy.

Autophagy and apoptosis both promote cell death; however, the relationship between them is subtle, and they mutually promote and antagonize each other. Apoptin can induce apoptosis of various tumor cells; however, tumor cell death is not only caused by apoptosis. Whether apoptin affects tumor cell autophagy is poorly understood. Therefore, the present study aimed to explore the potential mechanisms underlying the effects of apoptin using recombinant adenoviruses expressing apoptin. Reverse transcription­quantitative polymerase chain reaction, immunoblotting, flow cytometry, fluorescence microscopy and proteomics analyses revealed that apoptin could induce autophagy in MCF­7 breast cancer cells. The results also suggested that apoptin affected autophagy in a time­ and dose­dependent manner. During the early stage of apoptin stimulation (6 and 12 h), the expression levels of autophagy pathway­associated proteins, including Beclin­1, microtubule­associated protein 1A/1B­light chain 3, autophagy­related 4B cysteine peptidase and autophagy­related 5, were significantly increased, suggesting that apoptin promoted the upregulation of autophagy in MCF­7 cells. Conversely, after 12 h of apoptin stimulation, the expression levels of apoptosis­associated proteins were decreased, thus suggesting that apoptosis may be inhibited. Therefore, it was hypothesized that apoptin may enhance autophagy and inhibit apoptosis in MCF­7 cells at the early stage. In conclusion, apoptin­induced cell death may involve both autophagy and apoptosis. The induction of autophagy may inhibit apoptosis, whereas apoptosis may inhibit autophagy; however, occasionally both pathways operate at the same time and involve apoptin. This apoptin­associated selection between tumor cell survival and death may provide a potential therapeutic strategy for breast cancer.