Apoptosis In Hepatocellular Carcinoma Cells Research Articles

CENPL accelerates cell proliferation, cell cycle, apoptosis, and glycolysis via the MEK1/2-ERK1/2 pathway in hepatocellular carcinoma

Centromere protein L (CENPL) is involved in the mitotic process of eukaryotic cells and the development of various types of cancer. However, its role in hepatocellular carcinoma (HCC) remains unclear. This study aimed to investigate the expression and clinical significance of CENPL in HCC, and explore its involvement in regulating HCC cell proliferation, apoptosis, cell cycle, and glycolysis both in vivo and in vitro. CENPL expression was analyzed in HCC and normal liver tissues using The Cancer Genome Atlas, Gene Expression Omnibus mining, real-time quantitative polymerase chain reaction, and immunohistochemistry. Functional assays were used to assess the role of CENPL in HCC cell proliferation, apoptosis, cell cycle, and glycolysis. The potential pathways underlying the regulatory effects of CENPL, as well as the expression of mitogen-activated protein kinase (MAPK) signaling pathway-related molecules and markers of proliferation and glycolysis were investigated. CENPL was significantly upregulated in HCC tissue and associated with multiple clinicopathological features and poor patient prognosis. Univariate and multivariate analyses demonstrated that CENPL may serve as an independent prognostic factor for HCC. Upregulation of CENPL in HCC regulated tumor proliferation and glycolytic processes. Mechanistic studies revealed that differentially expressed genes between the CENPL-overexpressing and control groups were mainly concentrated in the MAPK signaling pathway. Pathway inhibition analysis indicated that CENPL activated the MEK1/2-ERK1/2 signaling pathway to promote proliferation and glycolysis in HCC cells. This study elucidated the role of CENPL in regulating cell proliferation, apoptosis, cell cycle, and glycolysis in HCC. CENPL may represent a therapeutic target and prognostic biomarker for HCC.

Tumor-Promoting Effects of Microrna-421/4-Aminobutyrate Aminotransferase Axis in Hepatocellular Carcinoma.

Background: MicroRNA-421 (miR-421) has been implicated in hepatocellular carcinoma (HCC), but its potential mechanism in HCC remains unclear. Objectives: The study aimed to study the potential mechanism of miR-421 in HCC which is necessary. Methods: The downstream target genes of miR-421 were screened in HCC tissues and cells using miDIP, Targetscan, and starBase databases. Differential analysis, survival analysis, and Pearson correlation analysis were performed between miR-421 and its downstream target genes. Quantitative reverse transcription polymerase chain reaction and western blot were used to assay RNA and protein levels of 4-aminobutyrate aminotransferase (ABAT) and epithelial-mesenchymal transition (EMT)-related proteins. Cell-based assays, including CCK-8, wound healing, transwell, flow cytometry, and metabolic measurements, were implemented to assess proliferation, migration, invasion, cell cycle, and apoptosis of HCC cells with different treatments. Dual-luciferase assay was utilized to detect the targeting relationship between miR-421 and ABAT. Results: miR-421 level was elevated in HCC tissues and cells, and low miR-421 expression hindered phenotype progression of HCC cells. ABAT was identified as a direct target of miR-421 in HCC cells, and miR-421 could inhibit ABAT expression. Rescue assay revealed that miR-421 promoted HCC cell tumorigenesis progress and affected cell metabolic remodeling through down-regulating ABAT. Conclusion: The miR-421/ABAT regulatory axis promoted HCC cell tumorigenesis progress, highlighting its potential as a therapeutic target for HCC. (REV INVEST CLIN. 2023;75(5):233-48).

Curcumin Induces Apoptosis by Suppressing XRCC4 Expression in Hepatocellular Carcinoma

Curcumin is a chemical with various pharmacological activities used for cancer treatment. It inhibits hepatocellular carcinoma (HCC) by inducing apoptosis. Here, the mechanism underlying the effect of curcumin on the apoptosis of HCC cells was studied. Cell counting kit-8 and plate cloning assays were used to assess the proliferation of HCC cells, and acridine orange/ethidium bromide and Annexin V/PI staining were used to analyze their apoptosis. HCC xenograft tumor models were established to validate anti-cancer effects of curcumin. Expression levels of XRCC4 protein in tumor tissues were assessed by immunohistochemistry. Correlation between XRCC4 expression and the prognosis of patients with HCC was analyzed by integrating publicly available gene expression data. Curcumin inhibited HCC cells proliferation in a dose-dependent manner. Compared with the control group, curcumin significantly promoted the apoptosis of HCC cells in vitro and in vivo. Immunohistochemical analysis revealed that curcumin downregulated XRCC4 expression levels in HCC tissues. Prognosis of HCC patients with high XRCC4 expression was poorer than that of patients with low XRCC4 expression. Therefore, curcumin exerts anti-cancer effects by inhibiting cell proliferation and promoting cell apoptosis in HCC. This may be due to curcumin interference in the repair process of the nonhomologous DNA terminal link of HCC cells by downregulating XRCC4 expression.

Astragaloside IV Attenuates Programmed Death-Ligand 1-Mediated Immunosuppression during Liver Cancer Development via the miR-135b-5p/CNDP1 Axis.

Astragaloside IV (AS-IV) is a pivotal contributor to anti-tumour effects and has garnered extensive attention in research. Tumour cell immune suppression is closely related to the increase in Programmed Death-Ligand 1 (PD-L1). Hepatocellular carcinoma (HCC) is a malignant tumour originating from hepatic epithelial tissue, and the role of AS-IV in regulating PD-L1 in anti-HCC activity remains unclear. Various concentrations of AS-IV were administered to both human liver immortalised cells (THEL2) and HCC (Huh-7 and SMMC-7721), and cell growth was assessed using the CCK-8 assay. HCC levels and cell apoptosis were examined using flow cytometry. Mice were orally administered AS-IV at different concentrations to study its effects on HCC in vivo. Immunohistochemistry was employed to evaluate PD-L1 levels. Western blotting was employed to determine PD-L1 and CNDP1 protein levels. We carried out a qRT-PCR to quantify the levels of miR-135b-3p and CNDP1. Finally, a dual-luciferase reporter assay was employed to validate the direct interaction between miR-135b-3p and the 3'UTR of CNDP1. AS-IV exhibited a dose-dependent inhibition of proliferation in Huh-7 and SMMC-7721 while inhibiting PD-L1 expression induced by interferon-γ (IFN-γ), thus attenuating PD-L1-mediated immune suppression. MiR-135b-5p showed significant amplification in HCC tissues and cells. AS-IV mitigated PD-L1-mediated immune suppression through miR-135b-5p. MiR-135b-5p targeted CNDP1, and AS-IV mitigated PD-L1-induced immunosuppression by modulating the miR-135b-5p/CNDP1 pathway. AS-IV decreases cell surface PD-L1 levels and alleviates PD-L1-associated immune suppression via the miR-135b-5p/CNDP1 pathway. AS-IV may be a novel component for treating HCC.

Escin-sorafenib synergy up-regulates LC3-II and p62 to induce apoptosis in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a common solid cancer and the leading cause of cancer deaths worldwide. Sorafenib is the first drug used to treat HCC but its effectiveness needs to be improved, and it is important to find ways to treat cancer that combine sorafenib with other drugs. Synergistic therapies lower effective drug doses and side effects while enhancing the anticancer effect. In the present study, the therapeutic potential of sorafenibin combination with escin and its underlying mechanism in targeting liver cancer has been established. The IC50 of sorafenib and escin against HepG2, PLC/PRF5 and Huh7 cell lines were determined using MTT assay. The combination index, dose reduction index, isobologram and concentrations producing synergy were evaluated using the Chou-Talaly algorithm. The sub-effective concentration of sorafenib and escin was selected to analyze cytotoxic synergistic potential. Cellular ROS, mitochondrial membrane potential, annexin V and cell cycle were evaluated using a flow-cytometer, and autophagy biomarkers were determined using western blotting. Moreover, autophagy was knocked down using ATG5 siRNA to confirm its role. A DEN-induced liver cancer rat model was developed to check the synergy of sorafenib and escin. Different concentrations of escin reduced the IC50 of sorafenib in HepG2, PLC/PRF5 and Huh7 cell lines. Chou-Talaly algorithm determined cytotoxic synergistic concentrations of sorafenib and escin in these cell lines. Mechanistically, this combination over-expressed p62 and LC-II, reflecting autophagy block and induced late apoptosis, further reconfirmed by ATG5 knockdown. Sorafenib and escin combination reduced HCC serum biomarker α-feto protein (α-FP) by 1.5 folds. This combination restricted liver weight, tumor number and size, also, conserved morphological features of liver cells. The combination selectively targeted the G0 /G1 phase of cancer cells. Escin and sorafenib combination potentially up-regulates p62 to block autophagy to induce late apoptosis in liver cancer cells.

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.

LINC01343 targets miR-526b-5p to facilitate the development of hepatocellular carcinoma by upregulating ROBO1

Long noncoding RNAs (lncRNAs) contribute to hepatocellular carcinoma (HCC) progression and development. However, the function and molecular mechanisms of action of LINC01343 in HCC remain unclear. qRT-PCR and western blotting were performed to assess miR-526b-5p, LINC01343, and ROBO1 levels in HCC cell lines and tissue samples. Flow cytometry, transwell, and cell counting kit-8 assays were conducted in vitro to assess how LINC01343 influences the apoptosis, migration, and proliferation of HCC cells. In addition, the role of LINC01343 in the growth of tumors was verified using an in vivo xenograft tumor assay. Specific binding of miR-526b-5p to LINC01343/ROBO1 was validated using RNA immunoprecipitation and dual-luciferase reporter experiments. LINC01343 was upregulated in HCC cells and tissues. In vitro, LINC01343-knockdown Hep3B and Huh-7 cells exhibited enhanced apoptosis and suppressed proliferation and migration. An in vivo study further validated that LINC01343-knockdown repressed tumor growth. In terms of mechanisms, LINC01343 directly sponged miR-526b-5p, negatively modulating its expression. Moreover, further experiments revealed that inhibiting miR-526b-5p could counteract the tumor-suppressive effects of LINC01343-knockdown in Hep3B and Huh-7 cells. ROBO1 was identified as a direct target of miR-526b-5p. ROBO1 knockdown weakens the migratory and proliferative abilities of Hep3B and Huh-7 cells. Nonetheless, the inhibition of miR-526b-5p mitigated this effect. These findings revealed that LINC01343 serves as a vital oncogene in HCC. Moreover, the LINC01343/miR-526b-5p/ROBO1 axis may be a prospective target for HCC treatment.

Andrographolide anti-proliferation and metastasis of hepatocellular carcinoma through LncRNA MIR22HG regulation.

Hepatocellular carcinoma (HCC) treatment is a major challenge. Although andrographolide (Andro) has an anti-proliferation effect on HCC, its underlying mechanism is not yet elucidated, and whether Andro can inhibit HCC metastasis has not been reported. The present study aimed to clarify whether Andro inhibits SK-Hep-1 cell proliferation and HCC metastasis, and the mechanisms. The results showed that Andro significantly reduced the survival of HCC cells and tumor weight and volume in tumor-bearing nude mice. Andro also triggered apoptosis of HCC cells and upregulated MIR22HG, Cleaved Caspase 9/7/3 expression levels, and downregulated BCL-2 mRNA, BCL-2 expression levels. Knockdown of MIR22HG or overexpression of HuR attenuated the effects of Andro on the signal transduction of mitochondrial apoptotic pathway and proliferation ability in HCC cells. Moreover, Andro significantly reduced the invasive ability of the cells and the level of HCC cell lung metastasis, upregulated miR-22-3p expression level and downregulated HMGB1 and MMP-9 expression levels. MIR22HG or miR-22-3p knockdown attenuated the effects of Andro on the signaling of HMGB1/MMP-9 pathway and invasive ability in HCC cells, while the overexpression of HMGB1 attenuated the inhibitory effects of Andro on the MMP-9 expression level and invasive ability in HCC cells. Thus, the regulation of MIR22HG-HuR/BCL-2 and MIR22HG/HMGB1 signaling pathways is involved in the anti-HCC proliferation and metastasis effects of Andro. This study provided a new pharmacological basis for Andro in HCC treatment and, for the first time, identified a natural product molecule capable of positively regulating MIR22HG, which has a robust biological function.

Family with sequence similarity 111 member B contributes to tumor growth and metastasis by mediating cell proliferation, invasion, and EMT via transforming acidic coiled-coil protein 3/PI3K/AKT signaling pathway in hepatocellular carcinoma.

As a complex systemic disease, primary liver cancer ranks third in death rate for solid tumors worldwide. Family with sequence similarity 111 member B (FAM111B), which was found to be aberrantly mutated in multiple cancers, is a candidate oncogene. We aimed to determine the function and mechanism of FAM111B in hepatocellular carcinoma (HCC). The expression of FAM111B was evaluated in HCC tissues, adjacent tissues, HCC cell lines. The impact of FAM111B on proliferation, invasion, apoptosis and EMT of HCC cells were detected by CCK-8, Transwell, flow cytometry and Western blot assays. The relationship between FAM111B and transforming acidic coiled-coil protein 3 (TACC3) was assessed by CoIP and Immunofluorescence (IF) staining assays. The effect of FAM111B on tumor growth was detected by using xenograft model of nude mice. The expression of FAM111B was upregulated in HCC tissues and cell lines, and the prognosis of HCC patients was worse in the high FAM111B expression group, and its expression level was associated with the TNM stage of HCC. FAM111B silencing inhibited HCC cell proliferation and invasion, EMT and induced apoptosis. Besides, TACC3 served as an interactor for FAM111B, which could enhance TACC3 expression, thus activing PI3K/AKT pathway. Rescue experiments revealed that elevated of TACC3 restored the inhibitory effect of FAM111B overexpression on the cell functions via PI3K/AKT pathway. In vivo, FAM111B inhibition hampered tumor growth and metastasis of HCC. This study highlighted a key player of FAM111B in modulating the malignant biological progression of HCC via TACC3/PI3K/AKT signaling pathway, displaying a potential therapeutic target for HCC.

Hsa_circ_0119412 is a tumor promoter in hepatocellular carcinoma by inhibiting miR-526b-5p to upregulate STMN1

ABSTRACT Hepatocellular carcinoma (HCC) is always deemed a deadly malignancy worldwide. Non-coding RNAs, including circRNAs, are becoming more widely recognized as essential regulators of the malignant development of HCC. Thus, we elaborated the regulating role of hsa_circ_0119412 in HCC advancement. The qRT-PCR was done to estimate the expressions of hsa_circ_0119412, miR-526b-5p, and Stathmin 1 (STMN1) in HCC (clinical samples and cell lines), and immunoblotting was used to detect STMN1 protein level in HCC cell lines. The stability of the circRNA was checked by processing with ribonuclease R. The proliferative potential of HCC cells was examined via the CCK-8 assay and the migratory potential by the wound healing assay. Immunoblotting was done to examine Bax and Bcl-2 (apoptosis-related proteins). Luciferase and RIP assays were employed to establish the direct interactions among miR-526b-5p and hsa_circ 0119412/STMN1. In vivo tumor growth was measured by doing a xenograft tumor experiment. In the tissues of HCC patients and cell lines derived from HCC cells, hsa_circ_0119412 was distinctly over-expressed. Knocking down hsa_circ_0119412 impeded proliferation and migration while inducing apoptosis in HCC cells. Moreover, silencing hsa_circ_0119412 diminished tumor weight and volume in vivo. Interestingly, miR-526b-5p inhibition partially restored the anti-tumor effects of silencing hsa_circ_0119412. STMN1 expression was also abundant in HCC, suggesting that it play a tumor-promoting role. Mechanistically, hsa_circ_0119412 sponged miR-526b-5p, resulting in STMN1 upregulation and thus facilitating the progression of HCC. In conclusion, this study reveals that hsa_circ_0119412 knockdown attenuates the progression of HCC by targeting miR-526b-5p/STMN1 axis.

B-364 Inhibition of Hypoxia-Inducible Factor-1a (HIF-1 a) Provides a Therapeutic Approach for Hepatocellular Carcinoma (HCC)

Abstract Background The most frequent type of primary liver cancer in adults is hepatocellular carcinoma (HCC) which is the third-highest cause of cancer-related death worldwide. Globally, people die from liver cancer each year, with HCC accounting for around 90% of those deaths, making it a serious public health concern. Cirrhosis, hepatitis B, hepatitis C, and non-alcoholic fatty liver disease are all high-risk factors for HCC development. In addition, HCC is one of the most frequent solid tumors found in humans. Due to insufficient blood supply, these solid tumors usually are devoid of oxygen and nutrients. A reduction in oxygen concentration results in the formation of a hypoxic environment that downregulates the metabolic process and is fatal to cancer cells. To circumvent this barrier to growth, cancer cells produce hypoxia-inducible factor-1 (HIF-1), which alters their metabolic pathways and allows them to survive. HIF-1alpha (HIF-1α), a cytoplasmic subunit of HIF-1 can be overexpressed in hypoxic conditions. HCC cells cannot proliferate under hypoxia without a high level of HIF-1α expression, making it as a potential cancer treatment target. Using the LOPAC (Library of Pharmacologically Active Compounds) drug library, we have identified multiple drug molecules that inhibit the expression of HIF-1α. Methods We constructed specific SK-HEP-1 cells, a human liver cancer cell line, expressing the luciferase reporter gene under the promoter of HIF-1α. By using the cells, we screened the (LOPAC) library. Other bioanalytical methods were used to confirm and analyze identified candidates. Results We successfully identified several compounds that are able to inhibit HIF-1α expression by using luciferase activity assays and Western blot analysis (WB). Further investigation was conducted to determine the molecular mechanism and function for one of the drug candidates, termed as UZ-1, in inhibiting HIF-1α in HCC by using the MTT assay and WB. UZ-1, an antirheumatic agent, showed a strong inhibitory effect on the expression of HIF-1α in SK-HEP-1 cells. In addition, it suppressed the signaling pathways associated with HIF-1α, leading to the apoptosis of HCC cells. Conclusion The UZ-1 molecule inhibits HIF-1α expression, as a result, and displays anticancer function in HCC cells through inducing apoptosis. Our result suggests that UZ-1 may be a potential drug candidate for the treatment of HCC.

Milk thistle nano-micelle formulation promotes cell cycle arrest and apoptosis in hepatocellular carcinoma cells through modulating miR-155-3p /SOCS2 /PHLDA1 signaling axis

BackgroundHepatocellular Carcinoma (HCC) is a prevalent form of liver cancer that causes significant mortality in numerous individuals worldwide. This study compared the effects of milk thistle (MT) and nano-milk thistle (N-MT) on the expression of the genes that participate in apoptosis and cell cycle pathways in Huh-7 and HepG2 cells.MethodsIC50 values of MT and N-MT were determined using the MTT assay. Huh-7 and HepG2 cell lines (containing mutant and wild-type TP53 gene, respectively) were incubated with MT and N-MT for 24h and 48h and the impact of MT and N-MT on the proliferation of these cell lines was evaluated through a comparative analysis. Cell cycle and apoptosis were assessed by flow cytometry after 24h and 48h treatment in the cell lines mentioned. Real-time PCR was used to analyze miR-155-3p, PHLDA1, SOCS2, TP53, P21, BAX, and BCL-2 expression in the cell lines that were being treated.ResultsN-MT reduces cancer cell growth in a time and concentration-dependent manner, which is more toxic compared to MT. Huh-7 was observed to have IC50 values of 2.35 and 1.7 μg/ml at 24h and 48h, and HepG2 was observed to have IC50 values of 3.4 and 2.6 μg/ml at 24 and 48h, respectively. N-MT arrested Huh-7 and HepG2 cells in the Sub-G1 phase and induced apoptosis. N-MT led to a marked reduction in the expression of miR-155-3p and BCL-2 after 24h and 48h treatments. Conversely, PHLDA1, SOCS2, BAX, and P21 were upregulated in the treated cells compared to untreated cells, which suggests that milk thistle has the potential to regulate these genes. N-MT reduced the expression of TP53 in Huh-7 cells after mentioned time points, while there was a significant increase in the expression of the TP53 gene in HepG2 cells. No gene expression changes were observed in MT-treated cells after 24h and 48h.ConclusionN-MT can regulate cancer cell death by arresting cell cycle and inducing apoptosis. This occurs through the alteration of apoptotic genes expression. A reduction in the expression of miR-155-3p and increase in the expression of SOCS2 and PHLDA1 after N-MT treatment showed the correlation between miR-155-3p and PHLDA1/SOCS2 found in bioinformatics analysis. While N-MT increased TP53 expression in HepG2, reduced it in Huh-7. The findings indicate that N-MT can function intelligently in cancer cells and can be a helpful complement to cancer treatment.

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.

Low glucose metabolite 3-phosphoglycerate switches PHGDH from serine synthesis to p53 activation to control cell fate.

Glycolytic intermediary metabolites such as fructose-1,6-bisphosphate can serve as signals, controlling metabolic states beyond energy metabolism. However, whether glycolytic metabolites also play a role in controlling cell fate remains unexplored. Here, we find that low levels of glycolytic metabolite 3-phosphoglycerate (3-PGA) can switch phosphoglycerate dehydrogenase (PHGDH) from cataplerosis serine synthesis to pro-apoptotic activation of p53. PHGDH is a p53-binding protein, and when unoccupied by 3-PGA interacts with the scaffold protein AXIN in complex with the kinase HIPK2, both of which are also p53-binding proteins. This leads to the formation of a multivalent p53-binding complex that allows HIPK2 to specifically phosphorylate p53-Ser46 and thereby promote apoptosis. Furthermore, we show that PHGDH mutants (R135W and V261M) that are constitutively bound to 3-PGA abolish p53 activation even under low glucose conditions, while the mutants (T57A and T78A) unable to bind 3-PGA cause constitutive p53 activation and apoptosis in hepatocellular carcinoma (HCC)cells, even in the presence of high glucose. In vivo, PHGDH-T57A induces apoptosis and inhibits the growth of diethylnitrosamine-induced mouse HCC, whereas PHGDH-R135W prevents apoptosis and promotes HCC growth, and knockout of Trp53 abolishes these effects above. Importantly, caloric restriction that lowers whole-body glucose levels can impede HCC growth dependent on PHGDH. Together, these results unveil a mechanism by which glucose availability autonomously controls p53 activity, providing a new paradigm of cell fate control by metabolic substrate availability.

Interplay between SUMO1-related SUMOylation and phosphorylation of p65 promotes hepatocellular carcinoma progression

The nuclear factor kappaB (NF-κB) subunit p65, plays an important role in the progression of hepatocellular carcinoma (HCC). Phosphorylation of p65 is considered as an important mechanism for the positive regulation of NF-κB activity. According to our previous data, p65 can be SUMOylated by small ubiquitin-related modifier 1 (SUMO1) protein, and SUMO1 promotes p65 nuclear import and HCC progression. However, the effect of SUMO1-related p65 SUMOylation on NF-κB transcriptional activity and the relationship between phosphorylation and SUMOylation of p65 remain obscure. Here, we found that phosphorylated p65 level was increased in cancer tissues of HCC patients, and similar phenomenon was found for SUMO1 expression but not for SUMO2/3. Further clinical data showed a positive correlation between SUMO1 and phosphorylated p65. We also verified that overexpression of SUMO1 upregulated phosphorylated p65 levels. Next, we verified SUMO1-related p65 SUMOylation with in vitro SUMOylation assay, constructed mutants of p65 SUMOylation and phosphorylation, and found that SUMO1-related p65 SUMOylation promoted p65 nuclear import and increased NF-κB activity. Both SUMO1-related p65 SUMOylation and p65 phosphorylation (especially at S276 site) increased the viability and invasion of hepatoma cells, and decreased the apoptosis of hepatoma cells. At last, we found that the phosphorylation of p65 promoted the level of SUMO1-related p65 SUMOylation, and SUMO1-related p65 SUMOylation upregulated phosphorylated p65 (at S276 site). Our study contributes to the exploration of the oncogenic mechanism of p65, which is the important protein in HCC.

Cellular senescence throws new insights into patient classification and pharmacological interventions for clinical management of hepatocellular carcinoma.

Cellular senescence, a state of stable growth arrest, is intertwined with human cancers. However, characterization of cellular senescence-associated phenotypes in hepatocellular carcinoma (HCC) remains unexplored. To address this issue, we delineated cellular senescence landscape across HCC. We enrolled two HCC datasets, TCGA-LIHC and International Cancer Genome Consortium (ICGC). Unsupervised clustering was executed to probe tumor heterogeneity based upon cellular senescence genes. Least absolute shrinkage and selection operator algorithm were utilized to define a cellular senescence-relevant scoring system. TRNP1 expression was measured in HCCs and normal tissues through immunohistochemistry, immunoblotting and quantitative real-time polymerase chain reaction. The influence of TMF-regulated nuclear protein (TRNP)1 on HCC senescence and growth was proven via a series of experiments. TCGA-LIHC patients were classified as three cellular senescence subtypes, named C1-3. The robustness and reproducibility of these subtypes were proven in the ICGC cohort. C2 had the worst overall survival, C1 the next, and C3 the best. C2 presented the highest levels of immune checkpoints, abundance of immune cells, and immunogenetic indicators. Thus, C2 might possibly respond to immunotherapy. C2 had the lowest somatic mutation rate, while C1 presented the highest copy number variations. A cellular senescence-relevant gene signature was generated, which can predict patient survival, and chemo- or immunotherapeutic response. Experimentally, it was proven that TRNP1 presented the remarkable upregulation in HCCs. TRNP1 knockdown induced apoptosis and senescence of HCC cells and attenuated tumor growth. These findings provide a systematic framework for assessing cellular senescence in HCC, which decode the tumor heterogeneity and tailor the pharmacological interventions to improve clinical management.

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.

CircELMOD3 increases and stabilizes TRIM13 by sponging miR-6864-5p and direct binding to inhibit HCC progression

Many circular RNAs (circRNAs) have been identified to be associated with hepatocellular carcinoma (HCC) progression. We aim to explore the diagnostic potential, functions, and mechanism of circELMOD3 in HCC. Differentially expressed circRNAs in HCC and its paired adjacent tissues were identified by RNA sequencing. circELMOD3 was downregulated in HCC tissues and was related to clinicopathological characteristics of HCC patients. Additionally, plasma circELMOD3 was shown to be a highly sensitive and non-invasive biomarker to distinguish HCC from healthy controls. Functional assays showed that circELMOD3 inhibited proliferation and induced apoptosis of HCC cells both invitro and invivo. Mechanistically, RNA antisense purification (RAP) and luciferase reporter assays verified that circELMOD3 functioned as a sponge for miR-6864-5p leading to increased expression of its target gene TRIM13. Interestingly, RNA stability test demonstrated that circELMOD3 overexpression led to enhanced stability of its directly bound TRIM13 mRNA, which in turn co-activated the p53 signaling pathway.

A Novel Gene Pair CSTF2/DPE2A Impacts Prognosis and Cell Cycle of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC), one of the commonest cancers at present, possesses elevated mortality. This study explored the predictive value of CSTF2/PDE2A for HCC prognosis. In this study, clinical information and RNA sequencing expression profiles of HCC patients were acquired from common databases. Kaplan-Meier curve compound with time-dependent ROC curve, nomogram model, and univariate/multivariate Cox analysis were carried out to access the prediction capacity of CSTF2/PDE2A. The immune status, tumor microenvironment, drug sensitivity, biological function and pathway between HCC and adjacent non-tumorous tissue were analyzed and compared. Finally, RT-qPCR, Western blot, and apoptosis assays were performed to verify the effect on HCC cells of CSTF2/PDE2A. The optimal cut-off value of CSTF2, PDE2A and CSTF2/PDE2A was 6.95, 0.95 and 3.63, respectively. In TCGA and ICGC cohorts, the high group of CSTF2/PDE2A presented higher OS compared to low group. The area under the curve (AUC) for OS at 1-, 2-, and 3-years predicted by CSTF2/PDE2A were 0.731/0.695, 0.713/0.732 and 0.689/0.755, higher than the counterparts of the single gene CSTF2 and PDE2A. Multivariate Cox analysis revealed that CSTF2/PDE2A (HR = 1.860/3.236, 95% CI = 1.265-2.733/1.575-6.645) was an independent prognostic factor for HCC. The OS nomogram model created according to five independent factors including CSTF2/PDE2A showed excellent capacity for HCC prognosis. Furthermore, the immune status of the CSTF2/PDE2A high group was deleted, cell cycle-related genes and chemotherapy resistance were increased. Finally, cell experiments revealed distinct differences in the proliferation, apoptosis, protein and mRNA expression of HCC cells after si-CSTF2 transfection compared with the negative control. Taken together, the gene pair CSTF2/PDE2A is able to forecast the prognosis of HCC and regulates cell cycle, which is promising as a novel prognostic predictor of HCC.

Inhibitory effect and molecular mechanism of sinomenine on human hepatocellular carcinoma HepG2 and SK-HEP-1 cells

This study aimed to investigate the effect and molecular mechanism of sinomenine on proliferation, apoptosis, metastasis, and combination with inhibitors in human hepatocellular carcinoma HepG2 cells and SK-HEP-1 cells. The effect of sinomenine on the growth ability of HepG2 and SK-HEP-1 cells were investigated by CCK-8 assay, colony formation assay, and BeyoClick~(TM) EdU-488 staining. The effect of sinomenine on DNA damage was detected by immunofluorescence assay, and the effect of sinomenine on apoptosis of human hepatocellular carcinoma cells was clarified by Hoechst 33258 staining and CellEvent~(TM) Cystein-3/7Green ReadyProbes~(TM) reagent assay. Cell invasion assay and 3D tumor cell spheroid invasion assay were performed to investigate the effect of sinomenine on the invasion ability of human hepatocellular carcinoma cells in vitro. The effect of sinomenine on the regulation of protein expression related to the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/signal transducer and activator of transcription 3(STAT3) signaling pathway in HepG2 and SK-HEP-1 cells was examined by Western blot. Molecular docking was used to evaluate the strength of affinity of sinomenine to the target cysteinyl aspartate specific proteinase-3(caspase-3) and STAT3, and combined with CCK-8 assay to detect the changes in cell viability after combination with STAT3 inhibitor JSI-124 in combination with CCK-8 assay. The results showed that sinomenine could significantly reduce the cell viability of human hepatocellular carcinoma cells in a concentration-and time-dependent manner, significantly inhibit the clonogenic ability of human hepatocellular carcinoma cells, and weaken the invasive ability of human hepatocellular carcinoma cells in vitro. In addition, sinomenine could up-regulate the cleaved level of poly ADP-ribose polymerase(PARP), a marker of apoptosis, and down-regulate the protein levels of p-Akt, p-mTOR, and p-STAT3 in human hepatocellular carcinoma cells. Molecular docking results showed that sinomenine had good affinity with the targets caspase-3 and STAT3, and the sensitivity of sinomenine to hepatocellular carcinoma cells was diminished after STAT3 was inhibited. Therefore, sinomenine can inhibit the proliferation and invasion of human hepatocellular carcinoma cells and induce apoptosis, and the mechanism may be attributed to the activation of caspase-3 signaling and inhibition of the Akt/mTOR/STAT3 pathway. This study can provide a new reference for the in-depth research and clinical application of sinomenine and is of great significance to further promote the scientific development and utilization of sinomenine.