Autophagy In Hepatocellular Carcinoma Cells Research Articles

Connections Between Endoplasmic Reticulum Stress and Prognosis of Hepatocarcinoma

Endoplasmic reticulum (ER) stress is a state in which misfolded or unfolded proteins accumulate in the lumen of the ER as a result of some exogenous or endogenous factors. It plays a crucial role in the pathogenesis of malignancies, affecting cell survival, proliferation, and metastasis in cancer. ER stress genes could provide new ideas for potential therapeutic targets in cancer. In our study, we aimed to construct an ER stress-related genes (ERGs) model for hepatocellular carcinoma (HCC). ERGs with differential expression and significant survival were screened to construct a prognostic model. The effectiveness of the model was successfully validated by external datasets. High and low-risk groups were classified based on risk scores. Functional analysis showed risk groups involved in the unfolded protein response, DNA repair, and other differential pathways. When compared to patients with low risk, the prognosis for HCC patients in the high-risk group might be worsened by disruptions in these pathways. Importantly, we considered genomic druggability and predicted drugs. Sorafenib-induced autophagy in HCC cells through an ES stress mechanism. Sorafenib was more sensitive for high-risk patients. In brief, our model predicted the prognosis of HCC and provided novel treatment strategies for the study of other cancers.

Amentoflavone regulates the miR-124-3p/CAPN2 axis to promote mitochondrial autophagy in HCC cells.

Hepatocellular carcinoma (HCC) is a disease with poor prognosis and high mortality. Amentoflavone (AF) possesses the characteristics of marginal toxicity, stable curative effect, and good anti-HCC activity. This study aimed to evaluate the molecular mechanism of AF inhibiting HCC and provide a new idea for HCC treatment. Clinical tissue of HCC was collected. AF was given with HCC cells, and transfected with corresponding vectors. MiR-124-3p expression in HCC clinical samples and cells was ascertained by qRT-PCR assay. HCC cells viability was identified by CCK-8 assay. LC3 protein expression was ascertained by immunofluorescence assay. The expressions of CAPN2, β-catenin and mitochondrial autophagy-related proteins were detected by western blot. Dual-luciferase reporter gene assay confirmed the targeting relationship of miR-124-3p and CAPN2. MiR-124-3p expression was inhibited and CAPN2 expression was increased in HCC tissues and cells. AF decreased HCC cell viability, up-regulated miR-124-3p expression, and inhibited CAPN2 expression and β-catenin nuclear transcription. Moreover, AF could activate the mitochondrial autophagy of HCC cells. MiR-124-3p specifically regulated CAPN2 expression. This study found that CAPN2 could promote β-catenin nuclear translocation, thus activating wnt/β-catenin pathway to inhibit mitochondrial autophagy in HCC cells. MiR-124-3p mimics enhanced AF function in promoting mitochondrial autophagy in HCC cells. However, CAPN2 overexpression, miR-124-3p inhibitor and SKL2001 attenuated the effectiveness of AF. This study confirmed that AF regulated miR-124-3p/CAPN2 axis to restraint β-catenin nuclear translocation and then inhibit the wnt/β-catenin pathway, thereby promoting mitochondrial autophagy in HCC.

ONC206 targeting ClpP induces mitochondrial dysfunction and protective autophagy in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the most common form of liver cancer, accounting for approximately 90 % of all cases. ONC201, a member of the imipridone drug family, has shown promising therapeutic potential and a good safety profile in both malignant pediatric central nervous system tumors (diffuse midline glioma [DMG]) and hematologic malignancies. ONC206 is a more potent analog of ONC201. However, the ONC206 potential and mechanism of action in HCC remain to be elucidated. We found that ONC206 hindered HCC growth by suppressing cell proliferation and inducing apoptosis. Moreover, ONC206 induced cytoprotective autophagy, and blocking autophagy enhanced the proapoptotic effect of ONC206. Additionally, ONC206 induced mitochondrial swelling, reduced the mitochondrial membrane potential (MMP), and led to the accumulation of mitochondrial ROS in HCC cells, ultimately resulting in mitochondrial dysfunction. The HCC patient samples exhibited notably elevated levels of caseinolytic protease proteolytic subunit (ClpP), which serves as a mediator of ONC206-induced mitochondrial dysfunction and the activation of protective autophagy. knockdown of ClpP reversed the cytotoxic effects of ONC206 on HCC cells. In summary, our results provide the first insight into the mechanism by which ONC206 exerts its anti-HCC effects and induces protective autophagy in HCC cells through ClpP.

SPRED2 Is a Novel Regulator of Autophagy in Hepatocellular Carcinoma Cells and Normal Hepatocytes.

Sprouty-related enabled/vasodilator-stimulated phosphoprotein homology 1 domain containing 2 (SPRED2) is an inhibitor of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway and has been shown to promote autophagy in several cancers. Here, we aimed to determine whether SPRED2 plays a role in autophagy in hepatocellular carcinoma (HCC) cells. The Cancer Genome Atlas (TCGA) Liver Cancer Database showed a negative association between the level of SPRED2 and p62, a ubiquitin-binding scaffold protein that accumulates when autophagy is inhibited. Immunohistochemically, accumulation of p62 was detected in human HCC tissues with low SPRED2 expression. Overexpression of SPRED2 in HCC cells increased the number of autophagosomes and autophagic vacuoles containing damaged mitochondria, decreased p62 levels, and increased levels of light-chain-3 (LC3)-II, an autophagy marker. In contrast, SPRED2 deficiency increased p62 levels and decreased LC3-II levels. SPRED2 expression levels were negatively correlated with translocase of outer mitochondrial membrane 20 (TOM20) expression levels, suggesting its role in mitophagy. Mechanistically, SPRED2 overexpression reduced ERK activation followed by the mechanistic or mammalian target of rapamycin complex 1 (mTORC1)-mediated signaling pathway, and SPRED2 deficiency showed the opposite pattern. Finally, hepatic autophagy was impaired in the liver of SPRED2-deficient mice with hepatic lipid droplet accumulation in response to starvation. These results indicate that SPRED2 is a critical regulator of autophagy not only in HCC cells, but also in hepatocytes, and thus the manipulation of this process may provide new insights into liver pathology.

GDF11 inhibits the malignant progression of hepatocellular carcinoma via regulation of the mTORC1‑autophagy axis.

Hepatocellular carcinoma (HCC) is a common malignant tumor, which is associated with a poor prognosis and high mortality rate. It is well known that growth differentiation factor 11 (GDF11) acts as a tumor suppressor in various types of cancer, including HCC. The present study aimed to determine the tumor-suppressive properties of GDF11 in HCC and to assess the intrinsic mechanisms. In the present study, the human hepatoma cell line Huh-7 was transfected with the GDF11 overexpression plasmid (Oe-GDF11) for gain-of-function experiments to investigate the effects of GDF11 on the biological behaviors of HCC cells, including proliferation, colony formation, apoptosis, cell cycle arrest, migration, invasion, epithelial-mesenchymal transition (EMT) and angiogenesis. The proliferation, colony formation, apoptosis, cell cycle, migration, invasion and angiogenesis of HCC cells were assessed by CCK-8, EdU staining, colony formation, flow cytometry, wound healing, Transwell and tube formation assays, respectively. Apoptosis-, cell cycle-, EMT-related key factors were also determined by western blot assay. Furthermore, Oe-GDF11-transfected Huh-7 cells were treated with the mammalian target of rapamycin (mTOR) activator MHY1485 for rescue experiments to explore whether GDF11 could exert antitumor effects against HCC via mediating the mTOR complex 1 (mTORC1)-autophagy axis. In the present study, GDF11 was verified to be lowly expressed in HCC cells. Overexpression of GDF11 inhibited the proliferation, colony formation, migration, invasion, EMT and angiogenesis of HCC cells, and facilitated the apoptosis and cell cycle arrest of HCC cells. Additionally, it was verified that overexpression of GDF11 inactivated the mTORC1 signaling pathway to enhance autophagy in HCC cells. Treatment with the mTOR activator MHY1485 partially reversed the tumor-suppressive effects of GDF11 overexpression on HCC. In conclusion, GDF11 may exert tumor-suppressive properties in HCC cells through inactivating the mTORC1 signaling pathway to strengthen autophagy.

Ent-8(14),15-Pimaradiene-2β,19-diol, a diterpene from Aleuritopteris albofusca, inhibits growth and induces protective autophagy in hepatocellular carcinoma cells.

A new pimarane-type diterpene, ent-8(14),15-pimaradiene-2β,19-diol (JXE-23), was isolated from the fern plant Aleuritopteris albofusca by our previous work; however, the biological activity of this diterpene remains unclear. In the present study, the anti-cancer potential of JXE-23 in various cancer cells was investigated. Among MCF-7 breast cancer cells, A549 lung cancer cells, and HepG2 liver cancer cells, JXE-23 displayed significant cytotoxicity to HepG2 cells with an IC50 value of 17.20 ± 1.73 µM, while showing no obvious toxicity in normal hepatocytes HL7702. JXE-23 inhibited cell growth and colony formation in HepG2 cells. A cell cycle distribution analysis showed that JXE-23 caused G2/M cell cycle arrest. Besides, JXE-23 also suppressed the migration of HepG2 cells. Interestingly, an increase of light chain 3 II (LC3II) and Beclin 1 and a decrease of P62 have occurred in JXE-23-treated cells, as well as the formation of GFP-LC3 dots, indicative of autophagy induction by JXE-23. When combined with autophagy inhibitor 3-methyladenine and chloroquine, the cell viability was significantly reduced, suggesting that JXE-23 triggered protective autophagy in hepatoma cells. Further study showed that JXE-23 inactivated the CIP2A/p-AKT/c-Myc signaling axis in HepG2 cells. Our data provided evidence that JXE-23 inhibited cell growth, arrested cells at the G2/M phase, and induced protective autophagy in HepG2 hepatocellular carcinoma cells. JXE-23 may be a potential lead compound for anti-cancer drug development, and autophagy inhibitor treatment may provide an effective strategy for improving its anti-cancer effect.

Autophagy inhibition potentiates energy restriction-induced cell death in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) significantly contributes to cancer-related mortality due to the limited response of HCC to current anticancer therapies, thereby necessitating more effective treatment approaches. Energy restriction mimetic agents (ERMAs) have emerged as potential therapies in targeting the Warburg effect, a unique metabolic process in cancer cells. However, ERMAs exhibit limited efficacy when used as monotherapy. Additionally, ERMAs have been found to induce autophagy in cancer cells. The role of autophagy in cancer survival remains a subject of debate. Thus, it is crucial to ascertain whether ERMA-induced autophagy is a mechanism for cell survival or cell death in HCC. Our study aims to investigate the effect of autophagy inhibition on the survival of HCC cells treated with ERMAs while also examining the potential of combining an autophagy inhibitor such as spautin-1 with ERMAs to enhance HCC cell death. Our results suggest a cytoprotective role for ERMA-induced autophagy in HCC cells, as combining the autophagy inhibitor spautin-1 with ERMAs effectively suppressed ERMA-induced autophagy and synergistically enhanced their antitumor activity. The treatment combination promoted HCC death through apoptosis, cell cycle arrest, and inhibition of AKT and ERK activation, which are known to play a key role in cellular proliferation. Collectively, our findings highlight a potential strategy to combat HCC by combining energy restriction with autophagy inhibition.

Sorcin regulate pyroptosis by interacting with NLRP3 inflammasomes to facilitate the progression of hepatocellular carcinoma

A high recurrence rate and easy metastasis are two prominent clinical features of hepatocellular carcinoma (HCC), which is also the most common cause of cancer-related death. However, the molecular pathogenesis of HCC remains unclear. Soluble resistance-related calcium-binding protein (Sorcin) is highly expressed in a variety of tumor cell lines and multidrug-resistant cell lines and participates in the malignant progression of tumors by regulating apoptosis. Pyroptosis is also a form of programmed cell death that plays a crucial role in exerting tumor suppression function and evoking anti-tumor immune responses. However, there is no consensus that Sorcin promotes HCC progression by regulating pyroptosis. Our study manifested that Sorcin was considerably upregulated, whereas pyroptosis-associated proteins were significantly decreased in HCC tissues and cells. Sorcin silencing attenuated the proliferation, migration, and invasion of HCC cells. Knockdown of Sorcin activates pyroptosis, and overexpression of Sorcin inhibits pyroptosis, yet has no significant effect on apoptosis, ferroptosis, and autophagy in HCC cells. Furthermore, coimmunoprecipitation and immunofluorescence assays revealed that Sorcin interacted with NLRP3 inflammasome to regulate pyroptosis in HCC cells. Then, the NLRP3 inhibitor MCC950 inhibited the activation of Sorcin knockdown-induced pyroptosis and reversed the effect of Sorcin silencing-induced weakening of malignant biological behavior in HCC. Similarly, suppression of Caspase-1 reversed the inhibitory effect of Sorcin knockdown on the malignant progression of HCC via knockdown of Caspase-1 or the inhibitor VX765. Consistent with the in vitro results, the nude mouse experiment showed that Sorcin knockdown inhibited the growth of HCC by activating pyroptosis, while Caspase-1 knockdown partially restored the growth inhibition caused by Sorcin knockdown. Collectively, high Sorcin expression in HCC negatively regulates pyroptosis by interacting with the NLRP3 inflammasome to promote HCC proliferation, migration, and invasion. The results of this study provide a scientific basis for Sorcin as a new biomarker and potential therapeutic target for HCC.

Upregulation of FAM134B inhibits endoplasmic reticulum stress-related degradation protein expression and promotes hepatocellular carcinogenesis.

Endoplasmic reticulum (ER) stress can stimulate the proliferation and metastasis of hepatocellular carcinoma (HCC) cells while hindering apoptosis and immune system function, but the molecular mechanism of ER stress in HCC has yet to be fully studied. We aim to investigate the molecular mechanism by which FAM134B inhibits autophagy of HCC cells by reducing the expression of ER stress-related degradation proteins. Clinical samples were collected for this study. Normal liver cell lines HL7702 and Hep3B and Huh7 HCC cell lines were cultured. Construction of FAM134B knockdown cell line. Cell proliferation was measured using the CCK-8 assay, while cell migration and invasion capabilities were detected using the plate colony formation assay. Flow cytometry was used to detect the apoptosis rate. Transmission electron microscopy was used to observe the formation of autophagosomes. qRT-PCR and WB detective expression changes related to autophagy proteins. Finally, the expression of the relevant proteins was observed by immunohistochemistry. The expression of FAM134B was significantly increased in human liver cancer tissue and HCC cell lines Hep3B and Huh7. After the lentiviral vector was transfected into Hep3B cells with sh-FAM134B, results showed that sh-FAM134B could effectively inhibit Hep3B cell proliferation and promote HCC cell apoptosis. Meanwhile, sh-FAM134B could effectively induce the autophagy of Hep3B liver cancer cells. Immunohistochemistry results showed that sh-FAM134B could effectively induce ER stress. FAM134B inhibits HCC cell autophagy and promotes the progression of liver cancer by inhibiting the expression of ER stress-related degradation factors such as DERL2, EDEM1, SEL1L and HRD1.

Autophagy inhibition mediated by trillin promotes apoptosis in hepatocellular carcinoma cells via activation of mTOR/STAT3 signaling.

Apoptosis and autophagy have been shown to act cooperatively and antagonistically in self-elimination process. On the one side, apoptosis and autophagy can act as partners to induce cell death in a coordinated or cooperative manner; on the flip side, autophagy acts as an antagonist to block apoptotic cell death by promoting cell survival. Our previous research indicated that trillin could induce apoptosis of PLC/PRF/5 cells, but the effects of trillin on autophagy as well as its functional relationship to apoptosis have not been elucidated. Here, the running study aims to investigate the function and molecular mechanism of trillin on autophagy with hepatocellular carcinoma (HCC) cells. The objective of this study is to investigate the molecular mechanism of trillin on autophagy in HCC cells. Protein levels of autophagy markers beclin1, LC3B, and p62 were detected by western blotting. 6-Hydroxyflavone and stattic were used to test the role of trillin regulation of autophagy via serine threonine kinase (AKT)/extracellular-regulated protein kinases (ERK) 1/2/mammalian target of rapamycin (mTOR)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Flow cytometry was used to detect caspase 3 activity and apoptosis in PLC/PRF/5 cells treated with trillin for 24 h with or without rapamycin, stattic, and 6-hydroxyflavone. The protein level of autophagy marker beclin1 was decreased, whilst the protein level of p62 was significantly increased by trillin treatment, indicating trillin treatment led to inhibition of autophagy in HCC cells. Trillin treatment could reduce the protein levels of p-AKT and p-ERK1/2, but enhance the protein levels of mTOR and p-mTOR, suggesting that trillin could inhibit AKT/ERK rather than mTOR. The AKT/ERK activator 6-hydroxyflavone could reverse the loss of AKT and ERK1/2 phosphorylation induced by trillin, implying that trillin impairs autophagy through activated mTOR rather than AKT/ERK. STAT3 and p-STAT3 were significantly upregulated by the trillin treatment with an increase in dose from 0 to 50 μM, suggesting that autophagy inhibition is mediated by trillin via activation of STAT3 signaling. The STAT3 inhibitor stattic significantly reversed the increased STAT3 phosphorylation at tyrosine 705 induced by trillin. The mTOR signaling inhibitor rapamycin reversed the trillin-induced mTOR phosphorylation enhancement but exerted no effects on total mTOR levels, suggesting trillin treatment led to inhibition of autophagy in HCC cells through activating mTOR/STAT3 pathway. Furthermore, caspase 3 activities and the total rate of apoptosis were increased by trillin treatment, which was reversed by rapamycin, stattic, and 6-hydroxyflavone, proving that trillin promotes apoptosis via activation of mTOR/STAT3 signaling. Trillin induced autophagy inhibition and promoted apoptosis in PLC/PRF/5 cells via the activation of mTOR/STAT3 signaling. Trillin has the potential to be a viable therapeutic option for HCC treatment.

Low expression of GOT2 promotes tumor progress and predicts poor prognosis in hepatocellular carcinoma.

Background: To explore the biological function and the underlying mechanisms of GOT2 in hepatocellular carcinoma (HCC). Materials & methods: The expression level and prognostic value of GOT2 were examined using International Cancer Genome Consortium and International Cancer Proteogenome Consortium databases. The cell counting kit-8 method, clone formation, Transwell® assays and western blotting were used to evaluate the effects of GOT2 on the biological function and autophagy of HCC cells. Results: The expression of GOT2 was downregulated in HCC tissues and correlated with poor prognosis of HCC patients. Knockdown of GOT2 promoted proliferation, migration and invasion of HCC cells and promoted cells' proliferation by inducing autophagy. Conclusion: GOT2 plays a tumor-inhibitory role in HCC and may be a potential therapeutic target for HCC.

Piezoeletric cold atmospheric plasma induces apoptosis and autophagy in human hepatocellular carcinoma cells through blocking glycolysis and AKT/mTOR/HIF-1α pathway

Hepatocellular carcinoma (HCC) is the sixth most prevalent cancer and the fourth leading cause of cancer-related death worldwide. Advanced or metastatic HCC is currently managed using systemic drug therapy with unsatisfactory patient survival. Cold atmospheric plasma has emerged as a promising, physicochemical, and broad-spectrum oncotherapy. In this preclinical study, we investigated the anti-neoplastic functions and mechanism of piezoelectric direct discharge technology-based CAP, Piezo-CAP, on HCC in vitro and in vivo. Various HCC cells lines, such as SMMC7721, HepG2 and LM3, were used as in vitro cancer model for the phenotypic and mechanistic studies. Specifically, the cell counting Kit-8 and colony formation assay, flow cytometry, Transwell assay, Western blot, reactive oxygen species (ROS) assay, and glutathione to oxidized glutathione ratio (GSH/GSSG) assay were used to demonstrate plasma-induced changes in HCC cell proliferation, cell cycle progression, migration and invasion, epithelial-to-mesenchymal transition, intracellular ROS, and antioxidant capacity, respectively. In addition, the Acridine orange and ethidium bromide (AO/EB) staining and transmission electron microscopy were performed for cellular and subcellular assessment of HCC cell apoptosis. The Ad-mCherry-RFP-LC3B fluorescent double-labeled lentiviral system was used to detect autophagic flux. On the other hand, RNA-sequencing, quantitative real-time PCR, and Western blot were used to demonstrate plasma-induced metabolic and molecular disruption of tumor glycolysis and oncogenic proliferation, respectively. In vivo experiments using a human cell-line-derived xenograft model and immunohistochemistry (IHC) were utilized to investigate the mechanism. Piezo-CAP exerted anti-neoplastic functions through inhibiting cell proliferation, migration and invasion, and promote cell apoptosis and autophagy. Treatment of Piezo-CAP could suppress proliferation and induce autophagy of HCC cells through simultaneously disrupts cancer survival pathways of redox deregulation, glycolytic pathway, and PI3K/AKT/mTOR/HIF1α pathway signaling. Moreover, upon translation of these in vitro results into the tissue level, Piezo-CAP significantly suppressed in situ tumor growth. These findings collectively suggest that Piezo-CAP-induced apoptosis and autophagy of HCC cells though a multitargeted blockade of major cancer survival pathways of deregulated redox balance, glycolysis, and PI3K/AKT/mTOR/HIF-1α signaling.

LncRNA XXYLT1-AS2 promotes tumor progression via autophagy inhibition through ubiquitinated degradation of TFEB in hepatocellular carcinoma.

There is compelling evidence that long-stranded non-coding RNAs (lncRNAs) play an important role in the progression of hepatocellular carcinoma (HCC). The aim of this study was to investigate the role of lncRNA XXYLT1 antisense-2 (XXYLT1-AS2) in HCC progression. Real-time PCR was used to assess the levels of XXYLT1-AS2 in plasma from HCC and normal patients. Cell proliferation, apoptosis, migration, and invasion were monitored, and tumor xenografts were established to investigate the biological functions of XXYLT1-AS2 by gain-of-function and loss-of-function studies in vitro and in vivo, the expression of autophagy biomarkers and transcriptional factor EB (TFEB) was examined by immunoprecipitation, ubiquitination assays, and western blotting. Autophagy inhibitor, 3-methyladenine (3MA), and proteasome inhibitor, MG132, were used to verify the role of autophagy in HCC progression and the effect of XXYLT1-AS2 on TFEB ubiquitination, respectively. In this study, we identified that lncRNA XXYLT1-AS2 is highly expressed in HCC plasma and promotes tumor growth in vivo. In functional studies, it was found that silent expression of XXYLT1-AS2 inhibited HCC proliferation, migration, invasion, and activated autophagy of HCC cells, which were attenuated by autophagy inhibitor, 3MA. Mechanistically, XXYLT1-AS2 decreased the protein level of TFEB through promoting its degradation by ubiquitin proteasome pathway. XXYLT1-AS2 plays an oncogenic role in HCC progression through inhibition of autophagy via promoting the degradation of TFEB, and thus could be a novel target for HCC treatment.

Morin induces autophagy and apoptosis in hepatocellular carcinoma cells through Akt/mTOR/STAT3 pathway

This study investigated the effect and mechanism of morin in inducing autophagy and apoptosis in hepatocellular carcinoma cells through the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/signal transducer and activator of transcription protein 3(STAT3) pathway. Human hepatocellular carcinoma SK-HEP-1 cells were stimulated with different concentrations of morin(0, 50, 100, 125, 200, and 250 μmol·L~(-1)). The effect of morin on the viability of SK-HEP-1 cells was detected by Cell Counting Kit-8(CCK-8). The effect of morin on the proliferation and apoptosis of SK-HEP-1 cells was investigated using colony formation assay, flow cytometry, and BeyoClick~(TM) EdU-488 with different concentrations of morin(0, 125, and 250 μmol·L~(-1)). The changes in the autophagy level of cells treated with morin were examined by transmission electron microscopy and autophagy inhibitors. The impact of morin on the expression levels of proteins related to the Akt/mTOR/STAT3 pathway was verified by Western blot. Compared with the control group, the morin groups showed decreased viability of SK-HEP-1 cells in a time-and concentration-dependent manner, increased number of apoptotic cells, up-regulated expression level of apoptosis marker PARP, up-regulated phosphorylation level of apoptosis-regulating protein H2AX, decreased number of positive cells and the colony formation rate, an upward trend of expression levels of autophagy-related proteins LC3-Ⅱ, Atg5, and Atg7, and decreased phosphorylation levels of Akt, mTOR, and STAT3. These results suggest that morin can promote apoptosis, inhibit proliferation, and induce autophagy in hepatocellular carcinoma cells, and its mechanism of action may be related to the Akt/mTOR/STAT3 pathway.

Exosomal circTGFBR2 promotes hepatocellular carcinoma progression via enhancing ATG5 mediated protective autophagy

Exosomes contribute substantially to the communication between tumor cells and normal cells. Benefiting from the stable structure, circular RNAs (circRNAs) are believed to serve an important function in exosome-mediated intercellular communication. Here, we focused on circRNAs enriched in starvation-stressed hepatocytic exosomes and further investigated their function and mechanism in hepatocellular carcinoma (HCC) progression. Differentially expressed circRNAs in exosomes were identified by RNA sequencing, and circTGFBR2 was identified and chosen for further study. The molecular mechanism of circTGFBR2 in HCC was demonstrated by RNA pulldown, RIP, dual-luciferase reporter assays, rescue experiments and tumor xenograft assay both in vitro and vivo. We confirmed exosomes with enriched circTGFBR2 led to an upregulated resistance of HCC cells to starvation stress. Mechanistically, circTGFBR2 delivered into HCC cells via exosomes serves as a competing endogenous RNA by binding miR-205-5p to facilitate ATG5 expression and enhance autophagy in HCC cells, resulting in resistance to starvation. Thus, we revealed that circTGFBR2 is a novel tumor promoter circRNA in hepatocytic exosomes and promotes HCC progression by enhancing ATG5–mediated protective autophagy via the circTGFBR2/miR-205-5p/ATG5 axis, which may be a potential therapeutic target for HCC.

Potential of curcumin and niacin-loaded targeted chitosan coated liposomes to activate autophagy in hepatocellular carcinoma cells: An in vitro evaluation in HePG2 cell line

The objective of this study is to activate autophagy in hepatocellular carcinoma for the enhancement of its cellular degradation. Liposomes incorporated chitosan in the core used to improve the stability of lecithin and increase the niacin loading efficiency. Additionally, curcumin as a hydrophobic molecule entrapped into liposomal layers and used as a face layer to minimize the release of niacin in physiological pH 7.4. Folic acid-conjugated chitosan was used to facilitate the delivery of liposomes into a specific location of cancer cells. TEM, UV Visible spectrophotometer, and FTIR confirmed the successful liposomal formation and good encapsulation efficiency. Based on the cellular proliferation of HePG2, the results revealed that there was a significant inhibition of growth rate of HePG2 after 48 h of incubation at a concentration of 100 μg/mL by 91 % ± 1 %, P ≤ 0.002 (pure niacin), 55 % ± 3 %, P ≤ 0.001 (pure curcumin), 83 % ± 1.5 %, P ≤ 0.001 (niacin NPs), and 51 % ± 1.5 % P ≤ 0.0001 (curcumin-niacin NPs) of relative to the control. Increasingly, The expression of mRNA of mTOR was significantly increased by 0.72 ± 0.08 P ≤ 0.001, 1 ± 0.1, 0. P ≤ 0.001, 5 ± 0.07 P ≤ 0.01, and 1.3 ± 0.02 P ≤ 0.001 folds) in pure niacin, pure curcumin, niacin NPs and curcumin -niacin NPs, respectively, relative to the control with an expression of 0.3 ± 0.08. Additionally, the expression of p62 mRNA was significantly increased by 0.92 ± 0.07 P ≤ 0.05, 1.7 ± 0.07 P ≤ 0.0001, 0.72 ± 0.08 P ≤ 0.5, and 2.1 ± 0.1 P ≤ 0.0001 folds relative to that of the control with an expression of 0.72 ± 0.08. The results highlight the efficient therapies of biomaterials derived from natural sources that can be used in cancer therapies instead of traditional chemotherapies.

High expression of GPR50 promotes the proliferation, migration and autophagy of hepatocellular carcinoma cells in vitro.

G protein-coupled receptors (GPCRs) play important roles in tumorigenesis and the development of hepatocellular carcinoma (HCC). GPR50 is an orphan GPCR. Previous studies have indicated that GPR50 could protect against breast cancer development and decrease tumor growth in a xenograft mouse model. However, its role in HCC remains indistinct. To detect the role and the regulation mechanism of GPR50 in HCC, GPR50 expression was analyzed in HCC patients (gene expression omnibus database (GEO) (GSE45436)) and detected in HCC cell line CBRH-7919, and the results showed that GPR50 was significantly up-regulated in HCC patients and CBRH-7919 cell line compared to the corresponding normal control. Gpr50 cDNA was transfected into HCC cell line CBRH-7919, and we found that Gpr50 promoted the proliferation, migration, and autophagy of CBRH-7919. The regulation mechanism of GPR50 in HCC was detected by isobaric tags for relative and absolute quantification (iTRAQ) analysis, and we found that GPR50 promoted HCC was closely related to CCT6A and PGK1. Taken together, GPR50 may promote HCC progression via CCT6A-induced proliferation and PGK1-induced migration and autophagy, and GPR50 could be an important target for HCC.

FOXA2 plays a critical role in hepatocellular carcinoma progression and lenvatinib-associated drug resistance.

Hepatic forkhead box protein A2 (FOXA2) is a crucial transcription factor for liver development and metabolic homeostasis. However, its role in hepatocellular carcinoma (HCC) progression and lenvatinib-related drug resistance remains unknown. In this study, the level of FOXA2 expression was found to be lower in HCC tissues than in paired adjacent tumor tissues. A low level of FOXA2 expression was associated with aggressive tumor characteristics (vascular invasion and poor differentiation). A low level of FOXA2 expression was found to be an independent risk factor for tumor recurrence (hazard ratio (HR): 1.899, P < 0.001) and long-term survival (HR: 2.011, P = 0.003) in HCC patients after hepatectomy. In xenograft animal models, FOXA2 overexpression significantly inhibited tumor growth. Moreover, FOXA2 overexpression was found to enhance the inhibitory effect of lenvatinib on HCC cells by upregulating the adenosine monophosphate-activated protein kinase-mechanistic target of rapamycin (AMPK-mTOR) pathway. Conversely, inhibition of adenosine monophosphate-activated protein kinase (AMPK) or stimulation of mechanistic target of rapamycin (mTOR) attenuated the sensitization of cells overexpressing FOXA2 to lenvatinib. Similarly, FOXA2 overexpression augmented the antitumor effect of lenvatinib in animal models with xenograft tumors. FOXA2 overexpression increased autophagy in HCC cells treated with lenvatinib. Lenvatinib treatment activated the platelet-derived growth factor receptor-extracellular regulated protein kinase (PDGFR-ERK) pathway in HCC. FOXA2 overexpression further downregulated the PDGFR-ERK pathway through the activation of the AMPK-mTOR axis. In conclusion, FOXA2 was identified as an independent risk factor for HCC after hepatectomy. FOXA2 was found to be closely associated with the biological progression of HCC. By modulating the AMPK-mTOR-autophagy signaling pathway, FOX2 significantly augmented antitumor effect of lenvatinib in HCC.

MiR-375 attenuates sorafenib resistance of hepatocellular carcinoma cells by inhibiting cell autophagy.

Sorafenib is the first-line treatment for hepatocellular carcinoma (HCC), but its efficacy is limited by the drug resistance of HCC cells. MiR-375 has been shown to be an inhibitor of autophagy that contributes to sorafenib resistance of HCC cells. In this context, this study probed into the unaddressed molecular target of miR-375 in inhibiting the autophagy of HCC cells under sorafenib treatment. Western blotting and qRT-PCR (quantitative reverse transcription-polymerase chain reaction) have been applied to measure the expressions of miR-375 and SIRT5 in parental HCC cells (HepG2 and Huh7) and sorafenib-resistant HCC cells (HepG2/so and Huh7/so). HepG2/so cells were accordingly transfected with miR-375 mimic, miR-375 inhibitor, sh-SIRT5, pcDNA3.1-SIRT5 or negative control. Expressions of p62, LC3I and LC3II in HCC cells have been measured by Western blotting. Viability and apoptosis of HCC cells have been assessed by CCK-8 (cell counting kit 8) and flow cytometry respectively. Bioinformatics techniques and dual-luciferase reporter assay have been used to predict and verify the targeting relationship between miR-375 and SIRT5. MiR-375 was under-expressed and SIRT5 was over-expressed in HCC cells. An autophagy inhibitor impaired the survival of HepG2/so cells transfected with miR-375 inhibitor. An autophagy activator enhanced the drug resistance of HepG2/so cells transfected with miR-375 mimic. MiR-375 suppressed the drug resistance of HepG2/so cells by inhibiting autophagy. SIRT5 enhanced the drug resistance of HepG2/so cells by promoting autophagy and it could be targeted by miR-375. MiR-375 suppresses autophagy to attenuate the sorafenib resistance of HCC cells by regulating SIRT5. The findings of this study may provide new therapeutic targets for treating HCC.

Autologous blood transfusion promotes autophagy and inhibits hepatocellular carcinoma progression through HIF-1α signalling pathway.

To explore the molecular mechanism of autologous blood transfusion promoting autophagy of hepatocellular carcinoma (HCC) cells and inhibiting the HCC progression through HIF-1α signalling pathway. This is a research paper. Rat hepatocellular carcinoma model and HepG2 cell model were built. The rats with HCC were conducted a surgery, and their blood was collected for detection to detect the recurrence and metastasis of the rats. Western blot was used to analysed the expression of HIF-1α, TP53, MDM2, ATG5 and ATG14 protein. The apoptosis rate of HepG2 cells was detected by flow cytometry, and autophagosomes were observed by transmission electron microscopy. HIF-1α expression was measured by immunofluorescence assay. The expressions of HIF-1α, TP53, MDM2, ATG5 and ATG14 protein were highest in model + autoblood group compared with the model group. HIF-1α content of model group was higher, but content of TP53, MDM2, ATG5 and ATG14 in the model group is the second. The highest apoptosis rate was found in HepG2 + autoblood group. The number of autophagosomes in HepG2 + autoblood was obviously larger than that of HepG2 + autoblood + inhibitor. HIF-1α expression of immunofluorescence assay showed that high expression of HIF-1α was clearly observed in HepG2 and HepG2 + autoblood group from confocal observation. However, there was no HIF-1α protein expression in HepG2 + autoblood + inhibitor group. The migration rate in HepG2 group, HepG2 + autoblood group and HepG2 + autoblood + inhibitor group was 85.71 ± 7.38%, 14.36 ± 6.54% and 61.25 ± 5.39%, respectively. Autologous blood transfusion promotes autophagy of HCC cells through HIF-1α signalling pathway, which further inhibits HCC migration and erosion.