Apoptosis Rate Of HepG2 Cells Research Articles

MiR-200a involvement in the biological behavior of hepatoma carcinoma cells by targeting the regulatory expression of mesenchymal-epithelial transition factor

Objective: To study the regulatory effect of miR-200a on mesenchymal-epithelial transition factor (MET) and its impact on the biological behavior of hepatoma carcinoma cells. Method: A luciferase reporter assay was used to determine miR-200a's regulatory impact on MET. Human hepatoma HepG2 cells were divided into a control group, a miR-200a group, a MET overexpression group, and a co-transfection group (miR-200a+MET). After culture, cell proliferation ability, cell migration ability, apoptosis, cell invasion ability, and the expression of MET and apoptosis-related (Bcl-2, Caspase-3, Bax) proteins were detected and observed by cell counting kit-8 (CCK-8), scratch assay, Annexin V-FITC staining, transwell chambers, and western blotting. The two groups were compared using the independent sample t-test. The multiple groups were statistically analyzed using one-way ANOVA. Results: The luciferase experiment showed that miR-200a had target MET. The proliferation rate, number of invasions in cells (55.00 ± 7.21, 85.00 ± 7.94, 164.67 ± 19.22, 104.00± 12.29), scratch healing rate (28.33% ± 5.03%, 61.67% ± 4.04%, 74.67% ± 7.02%, 49.33% ± 9.02%), and expression levels of MET, Bcl-2, and Caspase-3 proteins were lower in the miR-200a group than those in the control group, MET overexpression group, and co-transfection group, while the MET overexpression group had higher indexes than the other three groups, with statistically significant differences between the groups (P <0.05). The apoptosis rate of HepG2 cells and the expression level of Bax protein were higher in the miR-200a group than those in the control group, MET overexpression group, and co-transfection group (19.25% ± 2.98%, 6.80% ± 1.15%, 3.42% ±0.76%, 9.90% ± 2.72%), while the levels of various indexes in the MIF overexpression group were lower than those in the other three groups. The control group and co-transfection group were between the two groups, and the difference between the groups was statistically significant (P <0.05). Conclusion: HepG2 cell proliferation, migration, invasion, and cell apoptosis induction can be inhibited by miR-200a, and the functional mechanism for this may be associated with the miR-200a target's ability to down-regulate MET expression in HepG2 cells.

A new design of coculture microfluidic chip for HepG2 and LO2 cells

ObjectiveIn order to better screen targeted drugs, a microfluidic chip that can culture both diseased and normal cells is designed. MethodsWith Human hepatocellular carcinomas (HepG2) and Human normal liver cells (LO2) as the cell models, Polydimethylsiloxane-glass (PDMS-glass) chip as the carrier, and potential anticancer drug sanguinarine as the research object, two cells were co-cultured on the chip and the drug acted on both cells simultaneously in a diffusion manner. ResultsIn co-cultured cell chips, the apoptosis rate of HepG2 cells was significantly higher than that of LO2 cells under the action of sanguinarine. ConclusionThe chip diffusion perfusion form does not damage the cells, and can achieve cell staining and in situ observation more flexibly and conveniently, and more realistically reflects the selective effect of drugs on different cells, which has the advantages of simple operation and low cost.

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.

Downregulated lncRNA DRAIC enhances the radiotherapy sensitivity of human HCC cell line HepG2 by targeting miR-223-3p

Abstract Objective This study aims to investigate the effects of the long noncoding RNA (lncRNA) DRAIC on the proliferation, apoptosis, and radiosensitivity of hepatocellular carcinoma (HCC) cells and the molecular mechanisms involved. Methods Cancer tissues and their corresponding adjacent tissues from 30 patients with HCC were collected, and the expression levels of DRAIC and miR-223-3p were detected via RT-qPCR. DRAIC interference and miR-223-3p overexpression vectors were transfected into HepG2 cells. In addition, DRAIC and miR-223-3p interference vectors were co-transfected into HepG2 cells. The constructed cells were irradiated at 4 Gy. Cell colony formation assay, MTT assay, and flow cytometry were performed to detect the radiosensitivity, proliferation inhibition rate, and apoptosis rate of HepG2 cells, respectively. Dual luciferase reporter gene assay was performed to detect the targeted regulation of DRAIC on miR-223-3p expression. Results The expression level of DRAIC in HCC tissues was higher than that in paracancer tissues, whereas the expression level of miR-223-3p was lower in HCC tissues than that in paracancer tissues (P &lt; 0.05). Inhibition of DRAIC expression or overexpression of miR-223-3p increased the proliferation inhibition and apoptosis rates of HepG2 cells (P &lt; 0.05). After irradiation, cell survival fraction decreased and cell proliferation inhibition and apoptosis rates increased (P &lt; 0.05). DRAIC targeted the regulation of miR-223-3p expression, and interference of miR-223-3p expression reversed the effects of inhibiting DRAIC expression on the proliferation, apoptosis, and radiosensitivity of HepG2 cells. Conclusion Inhibition of DRAIC expression can inhibit the proliferation of HepG2 cells, promote cell apoptosis, and enhance the radiosensitivity of cells via upregulation of miR-223-3p.

Molecular docking and in vitro experiments verified that kaempferol induced apoptosis and inhibited human HepG2 cell proliferation by targeting BAX, CDK1, and JUN.

Hepatocellular carcinoma, as a common liver cirrhosis complication, has become the sixth most common cancer worldwide, and its increasing incidence has resulted in considerable medical and economic burdens. As a natural polyphenolic compound, kaempferol has exhibits a wide range of antitumor activities against multiple cancer targets. In this study, the Autodock software was used for molecular docking to simulate the interaction process between kaempferol and HCC targets and the PyMOL software was used for visualization. Proliferation of kaempferol HepG2 cells under the effect of kaempferol was detected using Cell Counting Kit-8 (CCK-8) assay, and the apoptosis rate of HepG2 cells was detected using flow cytometry. The expressions of proteins BAX, CDK1, and JUN protein expressions were detected by Western blot. Molecular docking found that the kaempferol ligand has 3 rotatable bonds, 6 nonpolar hydrogen atoms, and 12 aromatic carbon atoms, and can form complexes with the kaempferol targets P53, BAX, AR, CDK1, and JUN through electrostatic energy. GO (Gene Ontology) enrichment analysis suggests that kaempferol regulates the biological function of hepatocellular carcinoma cells and is related to apoptosis. Cell Counting Kit-8 assay suggested that Kaempferol can significantly inhibited HepG2 cell proliferation, and the inhibition rate increased with the increase in drug concentration and incubation time. Moreover, kaempferol can promoted HepG2 cell apoptosis in a dose-dependent manner. This compound upregulated BAX and JUN expression and downregulated CDK1 expression. Thus, Kaempferol can promote HepG2 cell apoptosis, and the regulatory mechanism may be related to the regulation of the expression levels of the apoptosis-related proteins BAX, CDK1, and JUN.

Curcumin-loaded zein/pectin nanoparticles: Caco-2 cellular uptake and the effects on cell cycle arrest and apoptosis of human hepatoma cells (HepG2)

Curcumin is a natural plant component with enormous in vitro anticancer effects. However, the bioavailability of curcumin is limited due to its poor solubility in water. Our previous studies showed that zein pectin core/shell nanoparticle-encapsulated curcumin (En-CUR) improved the solubility and bioaccessibility of curcumin. In this study, the transportation mechanism of En-CUR into Caco-2 cells and the inhibitory effects of En-CUR on HepG2 cell proliferation were investigated. The results showed that the uptake of En-CUR by Caco-2 cells required energy consumption. Caveolae-mediated endocytosis, clathrin-dependent endocytosis, and macropinocytosis were involved in the uptake of En-CUR by Caco-2 cells. The apoptosis rate of HepG2 cells after En-CUR treatment was 31.2%, which was approximately two-fold higher than that of the CUR-treated cells treated with curcumin at a concentration of 10 μg/mL, and the early apoptosis rate of En-CUR-treated cells was 2.7–3.3 times higher than that of CUR-treated cells. En-CUR-treated HepG2 cells exhibited more significant cell cycle arrest at cell cycle checkpoints S and G2/M than CUR-treated cells. En-CUR also decreased mitochondrial membrane potential. Western blot analysis revealed that En-CUR increased Bax protein expression, activated caspase-3 and caspase-9, and increased cytochrome c and p53 protein levels in HepG2 cells. Our results suggested that zein-pectin core/shell nanoparticles were desirable curcumin delivery systems and exhibited superior cancer cell antiproliferative effects.

Prediction of the potential mechanism of compound gingerol against liver cancer based on network pharmacology and experimental verification.

To explore gingerol's potential mechanism for treating liver cancer using network pharmacology and molecular docking technology and to conduct in-vitro experiments of human liver cancer cell HepG2 to verify important signalling pathways. We obtained potential targets of gingerol derivatives (6-gingerol, 8-gingerol and 10-gingerol) from PubChem and SwissTargetPrediction websites and collected related targets for liver cancer with the help of GeneCards. We performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on key targets using the DAVID data platform and combined with Cytoscape 3.7.1 software to construct a component-target-signal pathway interaction map to study its mechanism of action. Subsequently, the components and key proteins were molecularly docked through Autodock Vina software. Finally, the important signal pathways were verified by HepG2 cell in-vitro experiments. A total of 318 drug targets were screened for gingerol derivatives, and 2509 gene targets related to liver cancer were collected. The Venn diagram showed that there were 104 intersection targets between gingerol derivatives and liver cancer. Module analysis results show that these intersection targets can be divided into 5 modules and 49 nodes. Bioinformatics analysis found that GO obtained 20 important functional items including cancer cell proliferation, protein kinase activity, phosphotransferase activity and kinase activity; KEGG enrichment analysis yielded a total of 20 key signal pathways including the PI3K-Akt signalling pathway. The results of molecular docking show that the binding energy of gingerol derivatives has good binding activity with PI3K and Akt. In-vitro experimental results show that gingerol derivatives and compound gingerol (compound gingerol is composed of 6-gingerol, 8-gingerol and 10-gingerol in a ratio of 7:1.5:1.5) can produce HepG2 cell proliferation inhibition, and each administration group can significantly increase the apoptosis rate of HepG2 cells and the fluorescence intensity of the nucleus and block the cell cycle in the S phase; the results of Western Blot and real-time quantitative PCR show that gingerol derivatives and compound gingerol can down-regulate the expression of Akt and p-Akt and up-regulate the expression of Bax/Bcl-2. And the effect of compound gingerol is more obvious than that of gingerol derivatives. The results of network pharmacology and experimental validation suggest that gingerol derivatives and compound gingerol can act against liver cancer by acting on the PI3K-Akt signalling pathway.

RETRACTED ARTICLE]Isolation of curcumol from zedoary turmeric oil and its inhibitory effect on growth of human hepatocellular carcinoma xenografts in nude mice

This study investigated the inhibitory effect and mechanism of curcumol on growth of human hepatocellular carcinoma (HCC) HepG2 xenografts in nude mice. Curcumol was isolated from from zedoary turmeric oil. The HepG2 xenograft model in nude mice was established. Forty modeled nude mice were divided into model, cisplatin, curcumol and cisplatin+curcumol groups. The latter three groups were treated with 2 mg/kg cisplatin, 100 mg/kg curcumol and 2 mg/kg cisplatin combined with 100 mg/kg curcumol, respectively, once every two days, for total seven times. After treatment, compared with cisplatin group, in cisplatin+curcumol group the tumor weight and tumor inhibition rate were obviously increased, the apoptosis rate of HepG2 cells was obviously increased, the expression levels of Caspase-3 and B-cell lymphoma-2 associated X proteins in xenograft tumor were significantly increased, and the expression level of B-cell lymphoma-2 protein was significantly decreased. In addition, the thymus index and spleen index of animal had no significant difference between cisplatin group and cisplatin+curcumol group. In conclusion, the additional use of curcumol can obviously increase the inhibitory effect of cisplatin on growth of HCC HepG2 xenografts in nude mice. The mechanism may be related to the enhanced promotion of apoptosis of tumor.

The Effect of Icariin on Human Hepatoellular Carcinomas Cell Apoptosis and Cell Cycle Arrest

Background: Natural products often have novel frameworks and unique mechanisms, It is an important way to develop new anti-tumor drugs.The paper explored the effect of Icariin on HepG2 cell apoptosis and cell cycle arrest. Material and methods: Human liver cancer HepG2 cells were studied. The biological activity of Icariin on HepG2 cells was comprehensively investigated. The mechanism was preliminarily explored from the aspects of proliferation, cell cycle, and apoptosis. Results: Icariin showed significant inhibitory effect on cell proliferation after administration, and the effect was time-and-concentration-dependent. Annexin V-PI detection showed that, after 48 hours of administration of Icariin at different concentrations, the apoptosis rate of HepG2 cells increased in a concentration-dependent manner. The results of Hoechst 33342 staining showed that, after 48 hours of intervention with Icariin at different concentrations, HepG2 cells appeared densely stained and granular fluorescence, characterizing apoptosis. In the JC-1 mitochondrial membrane potential experiment, Icariin was found to destroy cell mitochondrial membrane potential and induce HepG2 cell apoptosis. After 48 h administration of Icariin at different concentrations, Bcl-2 and survivin proteins were down-regulated, and Bax was up-regulated, both in a concentration-dependent manner. PI single staining combined with flow cytometry to detect cell cycle results showed that, Icariin can induce G2/M phase arrest of HepG2 cells, and is time-and-concentration-dependent. Western Blot detection revealed that Icariin can down-regulate the cycle-related proteins Cyclin B and CDK1 in a concentration-dependent manner, and also significantly down-regulate the expressions of p-AKT, AKT, p-ERK and ERK proteins. Conclusion: Icariin is a selectively potential active compound that can treat liver cancer. The paper provided theoretical basis and experimental support for the clinical application of Icariin in the drug treatment of liver cancer. It is necessary to further study the antitumor effect of Icariin, explore and find the target, and provide higher selectivity for the treatment of liver cancer by Icariin.

OATP1B1 Plays an Important Role in the Transport and Treatment Efficacy of Sorafenib in Hepatocellular Carcinoma.

Background Sorafenib is an anticancer drug used in the treatment of unresectable hepatocellular carcinoma and advanced renal cell carcinoma. It is a substrate for the human OATP1B1. This study is aimed at assessing the role of OATP1B1 in transportation and uptake of sorafenib in hepatocellular carcinoma and how OATP1B1 affects the pharmacodynamics of sorafenib in vitro and in vivo. Methods Sorafenib transport was measured in HepG2, HepG2-OATP1B1∗1a, HepG2-OATP1B1∗1b, HepG2-OATP1B1∗15, LO2, LO2-OATP1B1∗1a, LO2-OATP1B1∗1b, and LO2-OATP1B1∗15 cells, as well as in HepG2 cells transfected with miR-148a mimics. The viability and apoptosis rate of cells treated with sorafenib were evaluated. A liver cancer rat model was established to explore the pharmacokinetics and pharmacodynamics of sorafenib after overexpression of Oatp2. Results Changes in expression and genetic mutations of OATP1B1 significantly affected the uptake of sorafenib in HepG2 and LO2 transgenic cells, and the uptake of sorafenib was higher in HepG2 than LO2. Genetic mutations of OATP1B1 significantly affected the cell viability and apoptosis rate of HepG2 cells after sorafenib treatment. Compared to control group, the uptake of sorafenib in miR-148a mimic-transfected HepG2 cells was decreased, and the cell viability was increased. PCN significantly increased the expression of Oatp2 and affected the pharmacokinetics of sorafenib. Vascular endothelial growth factor levels and microvascular density in tumor-adjacent tissues decreased significantly, suggesting that increased Oatp2 expression improves the treatment effect of sorafenib in a rat model of liver cancer. Conclusions OATP1B1 plays an important role in the pharmacokinetics and pharmacodynamics of sorafenib in hepatocellular carcinoma.

Effect of regulating TGF-β1/Smad signaling pathway on apoptosis of hepatocellular carcinoma HepG2 cells under endoplasmic reticulum stress

Objective: To investigate the effect of TGF-β1/Smad signaling pathway on the apoptosis of HepG2 cells under endoplasmic reticulum stress (ERS). Methods: An ERS model was established firstly. Human hepatocellular carcinoma HepG2 cells were treated with 3 μmol/L tunicamycin (TM) for 24 h to induce ERS. Cells were divided into 6 groups, each with 3 replicate holes, and the experiment was repeated 3 times. The 6 groups included untreated group, TM group (3 μmol/L TM treatment group), TM + NC group(3 μmol/L TM + si-TGF-β1 negative control group), TM + si-TGF-β1 group(3 μmol/L TM + si-TGF-β1 group), TM + pEX-3 group(3 μmol/L TM + plasmid control group), and TM + TGF-β1 pEX-3 group(3 μmol/L TM + TGF-β1 overexpressed plasmid group). HepG2 cells were transfected with TGF-β1 small interfering RNA (TGF-β1 si-RNA) and TGF-β1 overexpressed plasmids (TGF-β1 pEX-3) by Lipofectamine. Twenty-four hours after transfection, RT-qPCR and Western blot were used to detect the expression of TGF-β1 and p-Smad2 in HepG2 cells of each group. CCK-8 and flow cytometry were used to analyze changes in the proliferation inhibition rate and apoptosis rate of HepG2 cells in each group. Results: Compared with the untreated group, the expressions of TGF-β1 and p-Smad2 in TM group were significantly reduced (P＜0.05). Compared with the TM group, the expressions of TGF-β1 and p-Smad2, as well as the cell proliferation inhibition rate and apoptosis rate in TM + si-TGF-β1 group were obviously decreased (P＜ 0.01), while the expressions of TGF-β1 and p-Smad2, cell proliferation inhibition rate and apoptosis rate of TM + TGF-β1 pEX-3 group were significantly increased (P＜0.01). Conclusion: The TGF-β1/Smad signaling pathway was inhibited in hepatocellular carcinoma HepG2 cells under ERS, when this pathway was activated, the apoptosis rate of HepG2 cells under ERS was increased significantly.

Combination therapy with metformin and IL-12 to inhibit the growth of hepatic carcinoma by promoting apoptosis and autophagy in HepG2-bearing mice.

To investigate the effects and mechanism of metformin (Met) combined the interleukin-12 (IL-12) on inhibiting hepatoma HepG2 cell proliferation via in vitro and in vivo assays. MTT assay was used to detect inhibitory effects of Met, IL-12 alone or combination on HepG2 cells proliferation. Half inhibitory concentration (IC50) and combination index (CI) were also calculated. Anti-tumor effects of combination or monotherapy on the HepG2-bearing mice were investigated and protein expression levels of apoptosis, as well as the Akt/mTOR/STAT3 signaling pathway-related factors were detected by Western blot. MTT results showed that the inhibitory effect of Met combined with IL-12 on HepG2 cell proliferation was significantly enhanced (both p<0.01) compared with monomer therapy group with a significant synergistic effect (CI<1). The apoptosis rate of HepG2 cells treated with Met combined with IL-12 were 88.12±7.15% and significantly higher than the others (all p<0.01). Moreover, combination treatment significantly suppressed hepatoma growth and increased the survival rate of HepG2-bearing mice without evident body weight loss. Western blot analysis showed that Met combined with IL-12 significantly increased the expression of autophagy-related marker proteins, downregulated the protein expression levels of Bcl-2, p-Akt, p-mTOR, p-STAT3, upregulated the expression level of BAX in both HepG2 cells and tumor tissues. Met combined with IL-12 exhibited a synergistic antitumor effect on hepatoma HepG2 cells, and the mechanism may be related to its common inhibition of Akt/mTOR/STAT3 signaling pathway and increase of autophagy in HepG2-bearing mice.

The role of calcium regulation in the expression of PERK protein induced by sorafenib in hepatoma cells

AimThis study aimed to determine whether calcium signal participates in the sorafenib induced ER stress process and has an impact on the action of sorafenib for the hepatoma.MethodsLiver cancer HepG2 cell lines were maintained in DMEM with 10% fetal bovine serum and subcultured in plates or dishes at different densities for the following designed studies. HepG2 cells were basically treated with different doses of sorafenib (5–200 μM) for different time (0–48h) in the present or absent of trans‐Ned‐19 (a NAADP inhibitor, 100μM ) . The intracellular calcium signal was simultaneously recorded by confocal microscopy, and the expression level of PERK and caspase3 protein was detected by westernblot in HepG2 cells. The cell growth and apoptosis in these cells were analyzed by flow cytometry as well.ResultsConfocal microscopy showed that sorafenib (20–200 μM) induced substantial calcium release in HepG2 cells in a concentration‐dependent manner. Compared with the sorafenib intervention alone, trans‐Ned‐19 significantly attenuated sorafenib‐induced intracellular calcium elevation. Meanwhile, westernbolt assay showed that sorafenib time‐dependently but doses‐independently induced an increment in PERK expression in HepG2 cells. However, in the present of trans‐Ned‐19 was found to significantly enhance this sorafenib‐induced PERK and caspase3 levels, as well as apoptosis rate of HepG2 cells (P&lt;0.05). For further treatment with trans‐Ned‐19 for 24 h, microscopic observation showed that trans‐Ned‐19 significantly inhibited cell growth and caused abnormal cell morphology changes compared with the sorafenib alone group.ConclusionOur preliminary data showed that NAADP‐mediated intracellular calcium release was importantly involved in sorafenib‐induced perk protein expression and cell apoptosis. Blocking NAADP‐mediated calcium channels can significantly enhance the efficacy of sorafenib in the hepatoma cells, suggesting that the combination of NAADP analogues and sorafenib might be a potential strategy in the treatment of liver cancer.Support or Funding InformationSupport or Funding InformationSupported by NSFC Grants 81360077, 81860654, 30630145, 81172260 and GXNSFC Grants 2015GXNSFDA139022 to Guo Zhang

The antiproliferative effect of gambogic acid on HepG2 cells and its mechanism research

Objective To observe the inhibitory effect of gambogic acid on the proliferation of liver cancer cells HepG2 and explore its mechanism. Methods The vitro cultured liver cancer cells HepG2 were divided into the control group, low dose gambogic acid group, medium dose gambogic acid group and high dose gambogic acid group according to random number table method, 5 multiple holes in each group. The control group was cultured in normal medium and 0.5% DMSO was added as the solvent control. The 0.1, 1 and 10 μmol/L of gambogic acid were added to low, medium and high dose group of gambogic acid for intervention. MTT method was used to detect the inhibition rate of cell proliferation, flow cytometry was used to detect the apoptosis rate, Hoechst nuclear staining was used to observe the nucleus apoptosis, and Western blot was used to detect the expression of Bcl-2 and Bax protein. Results Compared with the control group, the HepG2 cells proliferation rate (68.00% ± 3.55%, 51.93% ± 4.36%, 47.16% ± 4.73% vs. 99.87% ± 4.53%) in low, medium and high dose gambogic acid group significantly decreased (P<0.01); apoptosis rate (23.00% ± 1.22%, 40.09% ± 4.65%, 70.32% ± 4.99% vs. 4.33% ± 0.57%) significantly elevated (P<0.01); Bcl-2 experssion (0.73 ± 0.10, 0.25 ± 0.10, 0.19 ± 0.08 vs. 0.97 ± 0.11) significantly decreased (P<0.01); and Bax expression (0.39 ± 0.14, 0.88 ± 0.15, 0.85 ± 0.13 vs. 0.22 ± 0.08) significantly elevated (P<0.01); Bax/Bcl-2 (0.34 ± 0.10, 1.87 ± 0.29, 1.63 ± 0.23 vs. 0.13 ± 0.06) significantly increased (P<0.01). Hoechst staining showed that the apoptosis rate of HepG2 cells increased in a dose-dependent manner with the increasing concentration of gambogic acid. Conclusions Gambogic acid could inhhibit HepG2 cells proliferation by regulating the expression of Bax and Bcl-2. Key words: Gambogic acid; Hep G2 cells; Cell proliferation; Apoptosis

Effect of metformin on mitochondrial pathway of apoptosis and oxidative stress in cell model of nonalcoholic fatty liver disease

Objective: To investigate the effects of metformin on mitochondrial pathway of apoptosis and oxidative stress in cell model of nonalcoholic fatty liver disease. Methods: An in vitro cell model of nonalcoholic fatty liver disease was established using 0.6 mmol/L oleic acid to induce lipid accumulation in HepG2 cells. HepG2 cells were divided into control (Con) group, oleic acid (OA) group, and metformin-low (1mmol/L) and high (10mmol/L) dose group. Oil Red O stain was used to detect intracellular lipid droplet distribution. The levels of alanine aminotransferase and aspartate aminotransferase in the culture supernatant were detected by assay kits. DCFH-DA method was used to detect the reactive oxygen species of HepG2 cells. Double staining flow cytometry was used to detect the apoptosis rate of HepG2 cells. Western blot was used to detect caspase-3, B-lymphocyte lymphoma-related protein, B-cell lymphoma 2, and cytochrome c protein. One-way analysis of variance was used to compare the data between groups. Results: Oleic acid-induced HepG2 cells were significantly increased with lipid droplets. Low and high-dose metformin had reduced intracellular lipid droplets accumulation. The effect of metformin in the high-dose group was more significant than that in the low-dose group. Aspartate aminotransferase and alanine aminotransferase in HepG2 cells of OA group were significantly increased, which were (43.41 ± 7.11) U/L and (29.56 ± 4.11) U/L, respectively. The intracellular aspartate aminotransferase and alanine aminotransferase were decreased significantly after the treatment with low and high-dose metformin, which were (32.44 ± 4.08)U/L, (19.31 ± 3.03) U/L, (26.00 ± 3.11) U/L and (15.11 ± 4.11) U/L, respectively and the differences were statistically significant (P < 0.05). DCFH-DA test results showed that the fluorescence intensity of reactive oxygen species in the oleic acid group was 41.21% ± 4.23%, while the fluorescence intensity of reactive oxygen species in the low and high-dose metformin groups were reduced to 27.44% ± 3.91%, and 17.55% ± 5.11%, respectively and the differences between the groups were statistically significant (P < 0.05). The results of flow cytometry analysis showed that the cell apoptosis rate of the OA group was significantly higher than that of the Con group (12.12% ± 0.72% vs. 3.04% ± 0.57%, P < 0.05).The apoptosis rate of HepG2 cells was significantly reduced after metformin treatment at low and high doses (8.71% ± 0.71%, 5.71% ± 0.61%, P < 0.05). Western blot results showed that compared with the Con group, the expressions of B-lymphocyte lymphoma-related protein, cytochrome c, and caspase-3 were increased in the OA group, while the B-cell lymphoma 2 were decreased (P < 0.05). The expression of B-lymphocyte lymphoma-related protein, cytochrome c, and caspase-3 protein in HepG2 cells was decreased after treatment with low and high-dose metformin, while B-cell lymphoma 2 was increased (P < 0.05). Conclusion: Metformin can effectively alleviate steatosis and improve the HepG2 function in cell model of nonalcoholic fatty liver disease. The mechanism of metformin may be related to the reduction of oxidative stress injury, the regulation of protein expression related to mitochondrial apoptosis pathway and the inhibition of cell apoptosis.

Poly(ethylene glycol) crosslinked multi-armed poly(ε-benzyloxycarbonyl-L-lysine)s as super-amphiphiles: Synthesis, self-assembly, and evaluation as efficient delivery systems for poorly water-soluble drugs

Polymeric micelles with high thermodynamic stability and loading capacity are of tremendous significance for their potential applications in drug delivery. In the present study, super-amphiphiles in the form of poly(ethylene glycol)-crosslinked multi-armed polyethylenimine-g-poly(ε-benzyloxycarbonyl-L-lysine)s (PEZ-alt-PEG) were designed, synthesized, and optimized as nanocarriers for hydrophobic drugs. In an aqueous solution, the copolymer PEZ-alt-PEG self-assembled into sub-100-nm spherical shell crosslinked micelles with low toxicity in vitro and in vivo. The crosslinked super-amphiphilic structure of PEZ-alt-PEG could not only enhance the thermodynamic stability of polymeric micelles, but it could also significantly improve the loading capacity of hydrophobic drugs, such as curcumin (CUR). CUR-loaded PEZ-alt-PEG micelles could mediate effective drug delivery with sustained and complete CUR release. The use of PEZ-alt-PEG micellar nanocarriers remarkably improved the cellular uptake of CUR and therefore exhibited effective inhibitory activity on the growth of human hepatoma (HepG2) cells. Compared to free CUR, CUR-loaded polymeric micelles significantly accelerated the apoptosis rate of HepG2 cells. Therefore, PEZ-alt-PEG polymeric micelles, with their high thermodynamic stability, high drug-loading capacity, enhanced drug uptake and improved pharmacodynamic effects, could serve as efficient and promising nanocarriers for poorly water-soluble drugs.

MiR-34a overexpression enhances the inhibitory effect of doxorubicin on HepG2 cells.

BACKGROUNDHepatocellular carcinoma (HCC) is the third leading cause of death from malignant tumors worldwide. More than 50% of HCC cases occur in China. The prognosis remains poor and overall efficacy is still unsatisfactory. Chemotherapy resistance is the most important reason for the poor outcome. Much progress has been made in the study of chemotherapy resistance of HCC; however, the specific mechanisms of progression of HCC have still only been partially established. Therefore, the mechanism of chemotherapy resistance in HCC requires more research.AIMTo investigate the effect of miR-34a expression on the growth inhibition of HepG2 cells by doxorubicin.METHODSA recombinant lentiviral vector containing miR-34a was constructed and transfected into HepG2 cells. The expression of miR-34a was detected by reverse transcription-polymerase chain reaction (commonly known as RT-PCR) before and after transfection. Cells were exposed to 2 μM doxorubicin or phosphate-buffered saline before and after transfection. Cell viability in each group was detected by MTT assay, and cell cycle and apoptosis were detected by flow cytometry. Changes in expression levels of phospho (p)-p53, sirtuin (SIRT) 1, cyclin D1, cyclin-dependent kinase (CDK) 4, CDK6, BCL-2, multidrug resistance protein (MDR) 1/P glycoprotein (P-gp), and AXL were detected by Western blotting.RESULTSRecombinant lentiviral vector LV-hsa-mir-34a was successfully constructed by restriction endonuclease digestion and sequencing. RT-PCR showed that expression of miR-34a in HepG2 cells was significantly upregulated after transfection (P < 0.01). MTT assay showed that growth of HepG2 cells was inhibited after upregulation of miR-34a, and viability was significantly decreased after combined treatment with doxorubicin (P < 0.01). Flow cytometry showed that the number of HepG2 cells in G1 phase increased, and G1 phase arrest was more obvious after intervention with doxorubicin (P < 0.01). The apoptosis rate of HepG2 cells was increased after upregulation of miR-34a, and became more obvious after intervention with doxorubicin (P < 0.01). Western blotting showed that upregulation of miR-34a combined with treatment with doxorubicin caused significant changes in the expression levels of p-p53, SIRT1, cyclin D1, CDK4, CDK6, BCL-2, MDR1/P-gp and AXL proteins (P < 0.01).CONCLUSIONMiR-34a may enhance the inhibitory effect of doxorubicin by downregulating MDR1/P-gp and AXL, which may be related to p53 expression.

Liraglutide promotes apoptosis of HepG2 cells by activating JNK signaling pathway.

To study the effect and mechanism of liraglutide on the apoptosis of human hepatocellular carcinoma HepG2 cells. HepG2 cell was treated with different concentrations of liraglutide at 0, 1, 10, 100, and 1000 nmol/L. The effect of liraglutide on HepG2 proliferation was detected by Cell Counting Kit-8 (CCK-8) method; the effect of liraglutide on the protein expression of c-Jun NH2-terminal Kinase (JNK) and phosphorylated JNK (p-JNK) was detected by Western blot; the degree of HepG2 apoptosis was observed by flow cytometry, and JNK pathway blocker SP600125 was used to further confirm that liraglutide promoted HepG2 apoptosis by regulating JNK signaling pathway. The proliferation inhibition rate of HepG2 cells increased with time and the increase in the concentration of liraglutide. The proliferation inhibition rate was the strongest when cultured for 48 h, and the IC50 (half maximal inhibitory concentration) was about 100 nmol/L of liraglutide. 100 nmol/L liraglutide was selected as the intervention condition for subsequent use of SP600165. The apoptosis rate of HepG2 cells increased with the increase of liraglutide's concentration. The apoptosis rate of HepG2 cells at blocker SP600125+100 nmol/L liraglutide was significantly lower than that at 100 nmol/L liraglutide alone (p<0.05). There was no significant difference in the expression of JNK protein in HepG2 cells at different concentrations of liraglutide (p>0.05). There was no significant difference in the expression of JNK protein in HepG2 cells using JNK pathway blocker SP600125 (p>0.05), while using JNK pathway blocker SP600125 significantly up-regulated the expression of p-JNK protein in HepG2 cells than 100 nmol/L of liraglutide alone (p<0.05). Liraglutide can promote the apoptosis of hepatocellular carcinoma HepG2 cells in a dose-dependent manner, and its mechanism may act by promoting the activation of the JNK signaling pathway.

LncRNA GAS5 enhanced the killing effect of NK cell on liver cancer through regulating miR-544/RUNX3.

This study aimed to explore the role of lncRNA GAS5 in the regulation of the killing effect of NK cells on liver cancer. Compared with a control group, lncRNA GAS5, RUNX3, and NCR1 were down-regulated in NK cells of patients with liver cancer, whereas miR-544 expression was up-regulated in NK cells of patients with liver cancer. Activated NK cells had higher IFN-γ level. Knockdown of GAS5 in activated NK cells decreased IFN-γ secretion, NK cell cytotoxicity, the percentage of CD107a+ NK cells, and the apoptosis rate of HepG2 and Huh7 cells. We also proved the interaction of GAS5 and miR-544, and the negative regulation role of GAS5 on miR-544. GAS5 overexpression in activated NK cells increased RUNX3 expression, IFN-γ secretion, the NK cell cytotoxicity, the percentage of CD107a+ NK cells, and the apoptosis rate of HepG2 cells, while miR-544 mimic abolished the promotion effect of GAS5 overexpression. Finally, in vivo experiments indicated an inhibition effect of GAS5 in tumor growth. LncRNA GAS5 overexpression enhances the killing effect of NK cell on liver cancer through regulating miR-544/RUNX3.

Cytisine induces endoplasmic reticulum stress caused by calcium overload in HepG2 cells.

Cytisine, a quinolizidine alkaloid, is one of the major bioactive components found in the small tree SophoraAlopecuraidesL., and is a traditional Chinese medicine that is used for treating hepatitis and liver cancer. In the 1960s, quinolizidine alkaloids were reported to exhibit inhibitory effects on tumour cell proliferation in several types of cancer cells. However, few studies have investigated the effect of cytisine on liver cancer. Our team confirmed that cytisine induced apoptosis in HepG2 cells via a mitochondrial pathway. The primary aim of the present study was to evaluate the endoplasmic reticulum (ER) stress caused by calcium overload in cytisine‑induced apoptosis in HepG2 cells and the molecular mechanisms of this phenomenon. In addition, the present study was undertaken to evaluate the expression of α7‑nAChR when apoptosis was induced by cytisine in HepG2 cells. In the present study, transmission electron microscopy was used to observe the morphological appearance of HepG2 cells. The apoptosis of the cells with cytoplasmic vacuolization was significant under electron microscopy. Apoptotic bodies, the expansion of the ER, and swelling of mitochondria were observed in the HepG2 cells after cytisine treatment. Flow cytometric analysis demonstrated that the apoptosis rate of HepG2 cells was upregulated. In addition, the intracellular calcium concentration was detected by laser confocal fluorescence microscopy. The laser confocal fluorescence microscopy showed that the calcium concentration was increased in a dose‑dependent manner. The activity of caspase‑4 was evaluated by an enzyme‑linked analyser, and the expression levels of CHOP, JNK, p‑JNK and α7‑nAChR were assessed via western blot analysis. In the present study, we observed that cytisine induced ER stress‑inducing factors and CHOP and p‑JNK1/2 protein expression, and it increased the JNK protein expression in the HepG2 cells. Furthermore, α7‑nAChR protein expression was promoted in a dose‑dependent manner after cytisine treatment. These findings suggest that cytisine induced the ER stress‑mediated apoptotic pathway via activation of CHOP, JNK and caspase‑4 in HepG2 cells, and cytisine is a potential new target compound for nAchRs (nicotinic acetylcholine receptors) to treat liver cancer.