Autophagy In Liver Cancer Cells Research Articles

Autophagy plays a pro-apoptotic role in arsenic trioxide-induced cell death of liver cancer

ObjectiveThe effects of arsenic trioxide (As2O3) on hepatocellular carcinoma have been documented widely. Autophagy plays dual roles in the survival and death of cancer cells. Therefore, we investigated the exact role of autophagy in As2O3-induced apoptosis in liver cancer cells. MethodsThe viability of hepatoma cells was determined using the MTT assay with or without fetal bovine serum. The rate of apoptosis in liver cancer cells treated with As2O3 was evaluated using flow cytometry, Hoechst 33258 staining, and TUNEL assays. The rate of autophagy among liver cancer cells treated with As2O3 was detected using immunofluorescence, Western blot assay and transmission electron microscopy. ResultsUpon treatment with As2O3, the viability of HepG2 and SMMC-7721 cells was decreased in a time- and dose-dependent manner. The apoptosis rates of both liver cancer cell lines increased with the concentration of As2O3, as shown by flow cytometry. Apoptosis in liver cancer cells treated with As2O3 was also shown by the activation of the caspase cascade and the regulation of Bcl-2/Bax expression. Furthermore, As2O3 treatment induced autophagy in liver cancer cells; this finding was supported by Western blot, immunofluorescence of LC3-II and beclin 1, and transmission electron microscopy. In liver cancer cells, As2O3 inhibited the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signal pathway that plays a vital role in both apoptosis and autophagy. The PI3K activator SC-79 partially reversed As2O3-induced autophagy and apoptosis. Furthermore, inhibiting autophagy with 3-methyladenine partially reversed the negative effects of As2O3 on cell viability. Serum starvation increased autophagy and amplified the effect of As2O3 on cell death. ConclusionAs2O3 induces apoptosis and autophagy in liver cancer cells. Autophagy induced by As2O3 may have a proapoptotic effect that helps to reduce the viability of liver cancer cells. This study provides novel insights into the effects of As2O3 against liver cancer.Please cite this article as: Deng ZT, Liang SF, Huang GK, Wang YQ, Tu XY, Zhang YN, Li S, Liu T, Cheng BB. Autophagy plays a pro-apoptotic role in arsenic trioxide-induced cell death of liver cancer. J Integr Med. 2024; 22(3): 295–302.

MiR-339-5p Inhibits Ferroptosis by Promoting Autophagic Degradation of FTH1 Through Targeting ATG7 in Liver Cancer Cells.

Liver cancer has a high incidence and mortality rate worldwide, and there is an urgent need to identify new therapeutic strategies and predictive targets to improve the clinical outcomes of advanced liver cancer. Ferroptosis holds promise as a novel strategy for cancer therapy. Epigenetic dysregulation is a hallmark of cancer, and noncoding RNAs are tightly involved in cell fate determination. Therefore, we aimed to identify a novel ferroptosis regulator from aberrantly expressed microRNAs that may serve as a novel biomarker and therapeutic target for liver cancer. The expression signature and prognostic value of miR-339 was assessed using TCGA data set. The role of miR-339/ATG7/FTH1 axis in liver cancer cells were evaluated through growth curve, colony formation, 7-AAD staining. The role of miR-339 in regulation of ferroptosis was determined by immunofluorescence staining, flow cytometry, and Elisa kits. Here, we showed that miR-339 is aberrantly overexpressed in patients with liver cancer. In addition, miR-339 inhibition dramatically suppresses liver cancer progression. Furthermore, miR-339 silencing drives cell death and inhibits liver cancer progression, indicating that miR-339 may serve as a novel ferroptosis suppressor. Mechanistically, we demonstrated that miR-339 targets ATG7 to facilitate the autophagic degradation of FTH1 and prevent ferroptosis in liver cancer cells. We provide important evidence that the miR-339 inhibition activates of the autophagy pathway to promote ferroptosis by degrading FTH1 in liver cancer cells. We found that miR-339 regulates the balance between ferroptosis and autophagy in liver cancer cells.

Functionalized SWCNTs@Ag–TiO2 nanocomposites induce ROS-mediated apoptosis and autophagy in liver cancer cells

Abstract Hybrid nanomaterials with unique physiochemical properties have received a lot of attention, making them attractive for application in different fields like cancer treatment. This study was designed to investigate the combined effects of single-walled carbon nanotubes (SWCNTs) hybridized with silver titanium dioxide composite (SWCNTs@Ag–TiO2). Transmission electron microscopy and field emission scanning electron microscopy images demonstrated the accumulation of SWCNTs with Ag–TiO2 due to an increased main grain size with functionalization to 40 nm. The D and G bands in SWCNTs @Ag–TiO2 shifted to 1,366 and 1,534 cm−1, respectively. SWCNTs@Ag-TiO2 were assessed for their cytotoxicity and autophagy induction in liver cancer cells (Hep-G2) using the lactate dehydrogenase assay, MTT assay, and flow cytometry methods. The results showed that SWCNTs and SWCNTs@Ag–TiO2 exhibited strong anti-cancer activity in vitro against Hep-G2 cells by inducing apoptosis and autophagy in liver cancer cells via controlling the AKT and JNK mitogen-activated protein kinase pathways. The results show that SWCNTs and SWCNTs coated with silver/titanium dioxide (SWCNTs@Ag–TiO2) reduce the cells’ viability and proliferation. It was shown that an excessive amount of reactive oxygen species was a crucial mediator of both the cell death caused by SWCNTs and the cell death caused by SWCNTs combined with Ag–TiO2. Based on these findings, it appears that SWCNTs and SWCNTs@Ag–TiO2 have the potential to be developed as nanotherapeutics for the treatment of liver cancer cells.

SH3BGRL Suppresses Liver Tumor Progression through Enhanced ATG5-Dependent Autophagy.

SH3BGRL, an adaptor protein, is upregulated in breast cancers and indicates its tumorigenic role. But the function of SH3BGRL in other types of cancers is largely unknown. Here, we modulate SH3BGRL expression level in two liver cancer cells and conduct both in vitro and in vivo analyses of SH3BGRL in cell proliferation and tumorigenesis. Results demonstrate that SH3BGRL notably inhibits cell proliferation and arrests the cell cycle in both LO2 and HepG2 cells. Molecularly, SH3BGRL upregulates the expression of ATG5 from proteasome degradation as well as the inhibitions of Src activation and its downstream ERK and AKT signaling pathways, which eventually enhance autophagic cell death. The xenograft mouse model reveals that SH3BGRL overexpression can efficiently suppress tumorigenesis in vivo, while the additional silencing ATG5 in SH3BGRL-overexpressing cells attenuates the inhibitory effect of SH3BGRL on both hepatic tumor cell proliferation and tumorigenicity in vivo. The relevance of SH3BGRL downregulation in liver cancers and their progression is validated based on the large-scale tumor data. Taken together, our results clarify the suppressive role of SH3BGRL in tumorigenesis of liver cancer, which would be of help to the diagnosis of liver cancer, while either promoting the autophagy of liver cancer cells or inhibiting the downstream signaling induced from SH3BGRL downregulation would be a promising therapy.

Apoptin mediates mitophagy and endogenous apoptosis by regulating the level of ROS in hepatocellular carcinoma

BackgroundApoptin, as a tumor-specific pro-apoptotic protein, plays an important anti-tumoral role, but its mechanism of autophagy activation and the interaction between autophagy and apoptosis have not been accurately elucidated. Here, we studied the mechanism of apoptin-induced apoptosis and autophagy and the interaction between two processes.MethodsUsing crystal violet staining and the CCK-8 assay, we analyzed the effect of apoptin in the inhibition of liver cancer cells in vitro and analyzed the effect of inhibiting liver cancer in vivo by establishing a nude mouse tumor model. Flow cytometry and fluorescence staining were used to analyze the main types of apoptin-induced apoptosis and autophagy. Subsequently, the relationship between the two events was also analyzed. Flow cytometry was used to analyze the effect of ROS on apoptin-mediated apoptosis and autophagy mediated by apoptin. The effect of ROS on two phenomena was analyzed. Finally, the role of key genes involved in autophagy was analyzed using gene silencing.ResultsThe results showed that apoptin can significantly increase the apoptosis and autophagy of liver cancer cells, and that apoptin can cause mitophagy through the increase in the expression of NIX protein. Apoptin can also significantly increase the level of cellular ROS, involved in apoptin-mediated autophagy and apoptosis of liver cancer cells. The change of ROS may be a key factor causing apoptosis and autophagy.ConclusionThe above results indicate that the increase in ROS levels after apoptin treatment of liver cancer cells leads to the loss of mitochondrial transmembrane potential, resulting in endogenous apoptosis and mitophagy through the recruitment of NIX. Therefore, ROS may be a key factor connecting endogenous apoptosis and autophagy induced by apoptin in liver cancer cells.Graphical abstractF4GajfVYHPvFTjahqy1NffVideo abstract

Significance of lncRNA CDKN2B-AS1 in Interventional Therapy of Liver Cancer and the Mechanism under Its Participation in Tumour Cell Growth via miR-199a-5p.

Methods Totally 34 LC patients admitted to our hospital between January 2020 and March 2021 (Obs group) and 32 healthy individuals over the same time span (Con group) were enrolled. CDKN2B-AS1 and miR-199a-5p in the two groups were PCR quantified, and their association and value for the diagnosis and therapy of LC were analyzed. In addition, purchased LC cells were adopted for in vitro assays, and the influences of CDKN2B-AS1 and miR-199a-5p on biological behaviours of LC cells were assessed through CCK-8, Transwell, and flow cytometry experiment, and their regulatory association was verified by the dual luciferase reporter (DLR) assay and rescue assay. And the autophagic protein expression was tested by the western blot to confirm the effect of both on the autophagic capacity of LC cells. Results CDKN2B-AS1 in LC cases presented high expression and dropped after therapy (P < 0.05), and the opposite situation of miR-199a-5p was found in the LC cases (P < 0.05). In vitro assays, after silencing of CDKN2B-AS1 and upregulation of miR-199a-5p, LC cells presented weaker viability, invasion and migration activities, and stronger apoptotic activity (all P < 0.05). The DLR assay revealed suppressed fluorescence activity of CDKN2B-AS1-WT by miR-199a-5p (P < 0.05). Moreover, according to the rescue assay, the impacts of silencing CDKN2B-AS1 on LC cells could be completely offset by silencing miR-199a-5p (P < 0.05). According to the clone formation and WB assay, the growth and autophagy of LC cells were under the regulation of CDKN2B-AS1 targeting miR-199a-5p (P < 0.05). Conclusion With high expression in LC cases, CDKN2B-AS1 is implicated in the development and progression of LC by suppressing cell autophagy through targeting miR-199a-5p.

Purified PTEN-Long Induces Liver Cancer Cells to Undergo Autophagy and Apoptosis.

BackgroundPTEN-Long is a translational variant of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). This tumor suppressor is frequently lost or mutated and even it has been shown as the determinant in several human tumors. Therefore, we will determine the significant roles of PTEN-Long in the development of liver cancer.MethodsIn the present study, we characterized the antitumor effects of PTEN-Long and PTEN in proliferation, migration of HepG2 cells, apoptosis and autophagy in liver cancer cells. To extends, we have also measured the effects of purified PTEN and PTEN-Long in the above index of HepG2 cells.ResultsPTEN and PTEN-Long were ectopic-expressed in HepG2 cells, and their phenotypic effects were recorded. As expected, there was less expression of PTEN-Long and PTEN in liver cancer samples than in paired normal tissues. Ectopic expression of PTEN-Long or PTEN significantly decreased the proliferation and migration of HepG2 cells and increased apoptosis. PTEN ectopic-expression increased the number of GFP-/RFP+-LC3 puncta and levels of beclin-1 and LC3BII/LC3BI, suggesting autophagy induction. Purified PTEN-Long freely entered cells, decreased proliferation, and increased autophagy and apoptosis, while purified PTEN did not.ConclusionsOur results identify an antitumor function of purified PTEN-Long and suggest its potential utility for liver cancer treatment.

Alisol B 23-Acetate Increases the Antitumor Effect of Bufalin on Liver Cancer through Inactivating Wnt/β-Catenin Axis.

Objective Liver cancer seriously threatens the health of people. Meanwhile, it has been reported that bufalin could act as an inhibitor in liver cancer. In addition, alisol B 23-acetate is a natural product derived from Alisma plantago-aquatica Linn which has an antitumor effect. In this study, we aimed to explore whether alisol B 23-acetate could increase the antitumor effect of bufalin on liver cancer. Methods In order to detect the effect of alisol B 23-acetate in combination with bufalin on liver cancer, human liver cancer SMMC-7721 and MHCC97 cells were used as subjects. Bufalin and alisol B 23-acetate were performed on cells. Cell viability was tested by MTT assay. In addition, flow cytometry was performed to assess the cell apoptosis. Autophagy-related protein levels were tested by western blotting. Results The data revealed that bufalin significantly decreased the viability of liver cancer cells, and the inhibitory effect was further increased by alisol B 23-acetate. In addition, alisol B 23-acetate notably enhanced the apoptotic effect of bufalin on liver cancer cells through mediation of Mcl-1, Bax, Bcl-2, and cleaved caspase-3. Meanwhile, alisol B 23-acetate in combination with bufalin induced the autophagy in liver cancer cells through mediation of Beclin-1 and p62. Furthermore, alisol B 23-acetate in combination with bufalin significantly downregulated the level of GSK-3β and increased the expression of β-catenin in liver cancer cells. Conclusion In summary, these findings provide the first evidence that alisol B 23-acetate improves the anticancer activity of bufalin on liver cancer through activation of the Wnt/β-catenin axis, and these outcomes might shed new lights on exploring the new methods against liver cancer.

High expression of H2A histone family member Y promotes the proliferation and autophagy of hepatocellular carcinoma cells

ABSTRACT Hepatocellular carcinoma is a common malignant tumor and the third most common cause of cancer-related deaths. In this study, we selected H2AFY as a potential oncogene from three online databases, and verified differential expression between normal and liver cancer tissues. Moreover, H2AFY expression was significantly correlated with the clinical characteristics and the survival of liver cancer patients. H2AFY expression was correlated with poor prognosis of liver cancer patients. H2AFY expression was also significantly higher in liver cancer cells. Knockdown and overexpression of H2AFY in liver cancer cells showed that H2AFY promoted the proliferation and clone formation of liver cancer cells but had no significant effects on the migration and invasion ability of liver cancer cells. Western blot analysis, immunohistochemistry, and immunofluorescence double staining confirmed that H2AFY upregulated LC3 and p62 expression in liver cancer tissues and cells. In conclusion, H2AFY is highly expressed in liver cancer cells and tissues, and promotes the proliferation and autophagy of liver cancer cells. H2AFY is a potential target for liver cancer therapy. Abbreviations: APLF: aprataxin pnk-like factor; HCC: Hepatocellular carcinoma; H2AFY: H2A histone family member Y

LncRNA RP11-295G20.2 regulates hepatocellular carcinoma cell growth and autophagy by targeting PTEN to lysosomal degradation

PTEN is a crucial tumor suppressor and loss of PTEN protein is involved in various cancers. However, the detailed molecular mechanisms of PTEN loss in cancers remain elusive, especially the involvement of lncRNAs. Here, lncRNA RP11-295G20.2 is found to be significantly upregulated in hepatocellular carcinoma (HCC) and promotes the growth of liver cancer cells both in vitro and in vivo. Furthermore, RP11-295G20.2 inhibits autophagy in liver cancer cells. Interestingly, RP11-295G20.2 directly binds to the PTEN protein and leads to its degradation. RP11-295G20.2 expression is inversely correlated with PTEN protein expression in 82 TCGA/TCPA-LIHC samples. Surprisingly, RP11-295G20.2-induced PTEN degradation occurs through the lysosomal pathway instead of the proteasome pathway. RP11-295G20.2 binds to the N terminus of PTEN and facilitates the interaction of p62 with PTEN. Thus, PTEN is translocated into lysosomes and degraded. RP11-295G20.2 also influences AKT phosphorylation and forkhead box O 3a (FOXO3a) translocation into the nucleus, in turn regulating the transcription of autophagy-related genes. Collectively, RP11-295G20.2 directly binds to PTEN and enables its lysosomal degradation. This newly identified RP11-295G20.2/PTEN axis reveals an unexplored molecular mechanism regarding PTEN loss in liver cancer and might provide new therapeutic benefits for liver cancer patients.

Sorafenib prevents the proliferation and induces the apoptosis of liver cancer cells by regulating autophagy and hypoxia-inducible factor-1.

Sorafenib has been approved as a systemic drug for advanced liver cancer; however, the underlying mechanisms remain unclear. The present study aimed to investigate the effects of sorafenib on the proliferation, autophagy and apoptosis of HepG2 cells under hypoxia. Briefly, reverse transcription-quantitative PCR and western blotting was performed to quantify HIF-1, LC3II/I, mTOR and p70s6K expression levels. Cell proliferation was determined using the Cell Counting Kit-8 assay and the cell apoptosis rate was evaluated using flow cytometry. The results demonstrated that autophagy and apoptosis were induced by hypoxia, and that sorafenib further enhanced hypoxia-induced autophagy and apoptosis in HepG2 cells in a dose-dependent manner. Furthermore, the mechanism of sorafenib-mediated autophagy in liver cancer cell were investigated by using chloroquine (CQ). The results showed that CQ significantly inhibited autophagy by decreasing LC3II/LC3I ratio in HepG2 cells treated with sorafenib and/or hypoxia. By contrast, sorafenib could increase the expression of hypoxia-inducible factor-1 (HIF-1) and of the autophagy marker (LC3II/I) and decrease the expression of mammalian target of rapamycin and p70 ribosomal S6 kinase in HepG2 cells under normoxia and hypoxia conditions, suggesting that sorafenib could induce hypoxia and autophagy in liver cancer cells. In addition, sorafenib was demonstrated to prevent proliferation and induce apoptosis of HepG2 cells under normoxia and hypoxia. Sorafenib could also prevent the malignant behavior of HepG2 by inducing hypoxia and autophagy. In summary, the findings from the present study suggested that sorafenib may inhibit liver cancer progression by activating autophagy and HIF-1 signaling pathway.

Research progress of neural precursor cells-expressed developmentally down-regulated protein-8 in liver diseases

Neural precursor cells-expressed developmentally down-regulated protein-8 (NEDD8) is one of the important members of the ubiquitin family, which plays an important role in maintaining cell stability, cell cycle regulation, signal transduction, transcription, and translation, DNA repair, and tumorigenesis through covalently bound substrates (also known as neddylation modification). In recent years, studies have found that the dysfunction of NEDD8 and its related enzymes is common in liver diseases, and is widely involved in the biological processes of hepatitis, liver fibrosis, proliferation, invasion, apoptosis and autophagy of liver cancer cells. This article focuses on the research progress of NEDD8 in liver diseases.

MiR-155-5p increases the sensitivity of liver cancer cells to adriamycin by regulating ATG5-mediated autophagy.

Liver cancer is the sixth most prevalent cancer worldwide and the third leading cause of cancer-related deaths. Adriamycin (ADR) resistance, which often leads to the progression of malignant tumors, is a major treatment obstacle for liver cancer. It has been confirmed that miR-155-5p could reverse drug resistance in human breast cancer. However, the biological function of miR-155-5p in ADR-resistant liver carcinoma (HepG2/ADR) cells remains unclear. miR-155-5p and ATG5 expression was determined by RT-qPCR and western blot. In addition, MTT, flow cytometry, immunofluorescence staining, and western blotting were performed to evaluate the proliferation, apoptosis, and autophagy of liver cancer cells. Finally, the effect of miR-155-5p on the expression of autophagy-related 5 (ATG5) was analyzed by luciferase activity assay, western blot, and RT-qPCR. Our results showed that miR-155-5p was downregulated in HepG2/ADR cells. Increasing the expression of miR-155-5p enhanced the sensitivity of liver carcinoma cells to ADR and promoted apoptosis through inhibition of autophagy in vitro. In addition, the binding site between miR-155-5p and ATG5 was identified, and miR-155-5p could directly regulate ATG5. Finally, ATG5 partially rescued the effect of miR-155-5p on autophagy and the apoptosis of HepG2/ADR cells. In conclusion, our findings showed that miR-155-5p could reverse ADR resistance in liver cancer by targeting ATG5, which may function as a potential target for liver cancer treatment.

Gomisin N Exerts Anti-liver Cancer Effects and Regulates PI3K-Akt and mTOR-ULK1 Pathways in Vitro.

Primary liver cancer is a lethal cancer. The phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway has been implicated in the pathogenesis of liver cancer. Gomisin N (GN), a lignan isolated from the dried fruits of Schisandra chinensis (Turca.) Baill., has been reported to reduce viability of, and induce apoptosis in, HepG2 liver cancer cells. In preadipocytes, GN was found to inhibit Akt activity. In the present study, Akt signaling-related anti-liver cancer mechanisms of GN were investigated. We confirmed that GN reduces cell viability of, and triggers apoptosis in, more liver cancer cell lines. Mechanistic studies revealed that GN lowers protein levels of phospho-PI3K (p85 tyrosine (Tyr)458), phospho-Akt (serine (Ser)473), and Akt downstream molecules Mcl-1 in HepG2 and HCCLM3 cells. Meanwhile, GN activates mTOR and inhibits ULK1 (a negative downstream effector of mTOR) activities. Activation of mTOR has been reported to suppress ULK1 activity and repress autophagy. Indeed, we observed that GN inhibits autophagy in liver cancer cells. In summary, we for the first time demonstrated that GN inhibits the PI3K-Akt pathway and regulates the mTOR-ULK1 pathway in liver cancer cells.

Inhibitory mechanism of muscone in liver cancer involves the induction of apoptosis and autophagy.

Traditionally, musk has been used as an analgesic to treat pain associated with cancer. Hepatocellular carcinoma (HCC) is an aggressive tumor; however, patients with liver cancer that received musk were reported to live longer and have a higher quality of life. Thus, the present study aimed to investigate whether muscone, a macrocyclic compound of musk, demonstrated potential as an anti-liver cancer drug for the non-surgical treatment of advanced liver cancer. Briefly, liver cancer cells were treated with muscone and the rates of cellular apoptosis and autophagy were investigated using staining techniques and western blotting. The underlying molecular mechanisms of muscone were evaluated using high-throughput sequencing and the in vitro effects of muscone were subsequently validated in vivo using a nude mouse model. Muscone increased the rates of apoptosis and autophagy in liver cancer cells; the increase in cellular apoptosis was observed to occur through endoplasmic reticulum stress responses, whereas muscone-induced autophagy was closely associated with the AMP kinase/mTOR complex 1 signaling pathway. These findings were verified in vivo. Notably, sestrin-2 expression levels were also significantly decreased in liver cancer tissues compared with paracancerous tissues. In conclusion, the present study suggests that muscone demonstrates potential as an anticancer drug, and the findings of the present study provide the basis for the development of effective anticancer drugs derived from natural compounds.

Histone deacetylase inhibitor MGCD0103 causes cell cycle arrest, apoptosis, and autophagy in liver cancer cells.

Background: Liver cancer is a common cause of cancer-related death all over the world. MGCD0103, a histone deacetylase inhibitor, exerts antitumor effect on various cancers. However, its role in liver cancer remains unclear.Methods: The effect of MGCD0103 on HepG2 and Huh7 cells was verified by several experiments such as cell viability assay, colony formation assay, cell cycle analysis, apoptosis analysis, reactive oxygen species (ROS) assay, western blotting, immunohistochemistry, and xenograft assay.Results: Cell viability and colony formation assays showed that MGCD0103 inhibited the proliferation of liver cancer cells in vitro. Flow cytometry and western blotting analysis demonstrated that MGCD0103 induced G2/M phase arrest and mitochondrial-related apoptosis. A pan-caspase inhibitor and ROS scavenger inhibited apoptosis induced by MGCD0103. What's more, MGCD0103 led to autophagy associated with cell death and an autophagy inhibitor inhibited apoptosis and autophagy induced by MGCD0103. Ultimately, MGCD0103 attenuated tumor growth but did not show significant systemic toxicity in animal model.Conclusions: MGCD0103 suppressed the growth of liver cancer cells in vitro and in vivo. It could serve as a novel therapeutic approach for liver cancer.

Nitric oxide inhibits autophagy and promotes apoptosis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the second most common cause of cancer‐related mortality worldwide. The expression of nitric oxide synthase (NOS) and the inhibition of autophagy have been linked to cancer cell death. However, the involvement of serum nitric oxide (NO), the expression of NOS and autophagy have not been investigated in HCC. In the present study, we first established that the NO level was significantly higher in hepatitis B virus‐related HCC than in the liver cirrhosis control (53.60 ± 19.74 vs 8.09 ± 4.17 μmol/L, t = 15.13, P < 0.0001). Using immunohistochemistry, we found that the source of NO was at least partially attributed to the expression of inducible NOS and endothelial NOS but not neuronal NOS in the liver tissue. Furthermore, in human liver cancer cells, NO‐induced apoptosis and inhibited autophagy. Pharmacological inhibition of autophagy also induced apoptosis, whereas the induction of autophagy could ameliorate NO‐induced apoptosis. We also found that NO regulates the switch between apoptosis and autophagy by disrupting the Beclin 1/Vps34 association and by increasing the Bcl‐2/Beclin 1 interaction. Overall, the present findings suggest that increased NOS/NO promotes apoptosis through the inhibition of autophagy in liver cancer cells, which may provide a novel strategy for the treatment of HCC.

Role of IL-6-mediated expression of NS5ATP9 in autophagy of liver cancer cells.

This study aimed to investigate the relationship between interleukin-6 (IL-6) and NS5ATP9 in autophagy of liver cancer cells. Autophagy is one of the important regulators of the replication of hepatitis C virus and the survival of tumors. IL-6 is a multifunctional cytokine that plays an important role in autophagy and development of many kinds of tumors. However, the role of IL-6 in autophagy has not been fully explored. A previous study had shown that a novel gene, NS5ATP9, could modulate autophagy. The present study demonstrated that human IL-6 recombinant protein induced autophagy of HepG2 cells. Conversely, autophagy decreased after IL-6 was silenced or neutralized with monoclonal antibody against human IL-6. In addition, NS5ATP9 was upregulated by IL-6 via nuclear factor-kappaB activation, as detected by Western blot. Further studies indicated that the induction of autophagy by IL-6 could be attenuated by silencing NS5ATP9. Interestingly, the expression of NS5ATP9, in turn, resulted in the upregulation of IL-6. In conclusion, IL-6 could induce autophagy by expressing NS5ATP9, while NS5ATP9 upregulated IL-6 levels in turn, which further induced autophagy.

The biological role of autophagy in regulating and controlling the proliferation of liver cancer cells induced by bufalin.

Autophagy is an important mechanism which regulates the processes of cell growth and death. The biological role of autophagy in regulating and controlling the proliferation of liver cancer cells by bufalin remains unknown. In the present study we investigated the effect of bufalin on autophagy of liver cancer cells. The growth inhibition and autophagy of liver cancer cells were detected using acridine orange fluorescence staining, flow cytometry and transmission electron microscopy. Combined with autophagy inhibitors (3‑MA and CQ), CCK8 staining and western blot analysis were used to detect the effect of bufalin on the proliferation and autophagy‑related protein expression in HCC‑LM3 cells at the indicated time‑points. Results indicated that combined with autophagy inhibitors 3‑MA and CQ, the inhibitive effect of bufalin on the proliferation of HCC‑LM3 cells was significantly enhanced. When combined with autophagy inhibitors 3‑MA or CQ, bufalin significantly reduced the autophagosome and acidic vesicles in HCC‑LM3 cells. When combined with autophagy inhibitors 3‑MA and/or CQ for 12h, bufalin significantly inhibited the expression of LC3‑I and Beclin‑1 in HCC‑LM3 cells, and upregulated the expression of LC3‑II and P62 in HCC‑LM3 cells. When combined with autophagy inhibitors 3‑MA and/or CQ for 24h, bufalin significantly inhibited the LC3‑II expression in HCC‑LM3 cells, and upregulated the LC3‑I, P62 and Beclin‑1 expression in HCC‑LM3 cells. When combined with autophagy inhibitors 3‑MA and/or CQ for 48h, bufalin significantly inhibited the expression of LC3‑II and Beclin‑1 in HCC‑LM3 cells, and upregulated the expression of LC3‑I and P62 in HCC‑LM3 cells. These findings indicated that bufalin induced cell autophagy and inhibited proliferation of liver cancer cells by influencing the expression of autophagy related proteins including LC3‑I, LC3‑II, P62 and Beclin‑1 in liver cancer cells. The autophagic state of liver cancer cells affected the inhibitory effect of bufalin on the proliferation of liver cancer cells.

Isovitexin (IV) induces apoptosis and autophagy in liver cancer cells through endoplasmic reticulum stress

Liver cancer is a leading cause of cancer death worldwide, and novel chemotherapeutic drugs to suppress liver cancer are urgently required. Isovitexin (IV), a glycosylflavonoid, is extracted from rice hulls of Oryza sativa, and has various biological activities. However, the anti-tumor effect of IV against liver cancer has not yet been demonstrated in vitro or in vivo. In the present study, we showed that IV significantly suppressed the growth of liver cancer cells. Mechanistic studies indicated that IV induced apoptosis by the mitochondrial apoptotic pathway, as evidenced by the increase of Bax, cleaved Caspase-3, poly (ADP-ribose) polymerase (PARP), and cytoplasm Cyto-c released from mitochondria. In addition, IV resulted in autophagy in liver cancer cells, supported by the enhancement of LC3II, autophagy-related protein (Atg) 3, Atg5 and Beclin1. Suppressing autophagy using bafilomycin A1 (BFA) or siRNA Atg-5 reduced apoptotic cells in IV-treated cells, demonstrating that autophagy induction regulated apoptosis. Moreover, IV was found to cause endoplasmic reticulum (ER) stress in liver cancer cells, along with the promotion of ER stress-related molecules, including inositol-requiring enzyme 1α (IRE1α), X-box-binding protein-1s (XBP-1s), C/EBP homologous protein (CHOP) and glucose-regulated protein (GRP)-78. Of note, inhibition of ER stress by use of its inhibitor, tauroursodeoxycholate (TUDCA), significantly reversed IV-induced apoptosis and autophagy. In vivo, IV treatment showed significant tumor growth inhibition compared to the non-treated group. IV could therefore be a strong candidate for liver cancer prevention.