Hepatocellular Cancer Cell Line HepG2 Research Articles

Rapid preparative isolation of cleistanthin A from the leaves of Cleistanthus collinus Using reverse-phase flash chromatography

Introduction:Cleistanthin A (CA) is an aryl naphthalene lignan, which has a potent anticancer activity by regulating the tumor microenvironment. The objective was to develop a new technique for the isolation of cleistanthin A from the acetone extract of Cleistanthus collinus utilizing reverse phase flash chromatography.Materials and Methods:Cleistanthus collinus leaves were shade dried, defatted using n-hexane and then macerated to obtain acetone extract which was further subjected to reverse phase flash chromatography for the isolation of cleistanthin A using the gradient mobile phase of 0.1% formic acid (v/v) in water and acetonitrile. Gradient elution of chromatographic run was performed for 80 min. The separated peaks that showed absorbance at λmax 254 nm were collected for the chemical characterization. Cell viability of the isolated cleistanthin A was studied on hepatocellular cancer cell line HePG2 and prostate cancer cell line PC3 using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.Results:The chemical characteristics of the isolated compound cleistanthin A was further characterized using spectral techniques such as 1H and 13C nuclear magnetic resonance (NMR), Fourier-transform infrared spectroscopy (FT-IR), and electron spray ionization-tandem mass spectrometry (ESI-MS/MS). Cleistanthin A has decreased the cell viability of the HePG2 cell line to 52.25% at 32 μg/ml and PC3 cell line to 51.82% at 16 μg/ml in a dose-dependent manner.Conclusion:Cleistanthin A was successfully isolated from the natural source using reverse phase flash chromatography and the MTT assay has shown that cleistanthin A has decreased the cell viability in both the HePG2 and PC3 cell lines in a dose-dependent manner.

A Lipidomic Signature Complements Stemness Features Acquisition in Liver Cancer Cells.

Lipid catabolism and anabolism changes play a role in stemness acquisition by cancer cells, and cancer stem cells (CSCs) are particularly dependent on the activity of the enzymes involved in these processes. Lipidomic changes could play a role in CSCs’ ability to cause disease relapse and chemoresistance. The exploration of lipid composition and metabolism changes in CSCs in the context of hepatocellular cancer (HCC) is still incomplete and their lipidomic scenario continues to be elusive. We aimed to evaluate through high-throughput mass spectrometry (MS)-based lipidomics the levels of the members of the six major classes of sphingolipids and phospholipids in two HCC cell lines (HepG2 and Huh-7) silenced for the expression of histone variant macroH2A1 (favoring stemness acquisition), or silenced for the expression of focal adhesion tyrosine kinase (FAK) (hindering aggressiveness and stemness). Transcriptomic changes were evaluated by RNA sequencing as well. We found definite lipidomic and transcriptomic changes in the HCC lines upon knockdown (KD) of macroH2A1 or FAK, in line with the acquisition or loss of stemness features. In particular, macroH2A1 KD increased total sphingomyelin (SM) levels and decreased total lysophosphatidylcholine (LPC) levels, while FAK KD decreased total phosphatidylcholine (PC) levels. In conclusion, in HCC cell lines knocked down for specific signaling/epigenetic processes driving opposite stemness potential, we defined a lipidomic signature that hallmarks hepatic CSCs to be exploited for therapeutic strategies.

Clinical significance and biological function of co-stimulation molecule B7 homolog 6 expression in human hepatocellular carcinoma

Objective To investigate the clinical significance and biological function of co-stimulatory molecule B7 homolog 6 (B7-H6) in human hepatocellular cancer. Methods The immunohistochemistry assay and the tissue micro-array were used to examine the B7-H6 expression in hepatocellular cancer tissues. The correlation between the expression level of B7-H6 and patients’ clinic pathological parameters was evaluated by using chi-square test. Down-regulation of B7-H6 was performed by using RNA interference (RNAi) method to investigate the role of B7-H6 in regulation of biological behaviors in human hepatocellular cancer cell lines HepG2 and SMMC-7721. The cell counting kit-8 (CCK-8) assay, wound healing assay, and Transwell assay were used to examine the cellular function after B7-H6 knockdown of these cells. Results The B7-H6 expression level in hepatocellular cancer tissues was significantly correlated with patients’ age (χ2=5.943, P<0.05) and tumor size (χ2=4.519, P<0.05). Next, to investigate the functions of B7-H6 in human hepatocellular cancer, we successfully constructed B7-H6 knockdown expression human hepatocellular cell lines using the RNA interference technology. CCK-8 assay showed that decreased B7-H6 expression in human hepatocellular cancer lines HepG2 and SMMC-7721 significantly attenuated cell proliferation (HepG2, 48 h: 0.413±0.020 vs. 0.572 ±0.025, t=4.951, P<0.05; 72 h: 0.503±0.014 vs. 0.691±0.018, t=8.372, P<0.01; SMMC-7721, 48 h: 0.534± 0.001 vs. 0.656±0.021, t=5.869, P<0.05; 72 h: 0.650± 0.044 vs. 0.856±0.025, t=4.069, P<0.05). The wound healing assay showed that decreased B7-H6 expression in human hepatocellular cancer lines HepG2 and SMMC-7721 significantly attenuated cell migration (HepG2, 24 h: t=4.581, P<0.05, 36 h: t=5.040, P<0.01; SMMC-7721, 36 h: t=5.118, P<0.01). The Transwell assay showed that decreased B7-H6 expression in human hepatocellular cancer lines HepG2 and SMMC-7721 significantly attenuated cell invasion (HepG2, 24 h: t=6.824, P<0.01, 36 h: t=11.030, P<0.001; SMMC-7721, 24 h: t=10.180, P<0.001, 36 h: t=10.340, P<0.001). Conclusion B7-H6 had important effect in initiation and development of human hepatocellular cancer. Key words: B7 homolog 6; Liver cancer; Immunohistochemistry; RNA interference; Tissue microarray

LncRNA CRNDE promotes hepatocellular carcinoma cell proliferation, invasion, and migration through regulating miR-203/ BCAT1 axis.

To investigate the impact of long noncodingRNA (lncRNA) colorectal neoplasia differentially expressed (CRNDE) on hepatocellular cancer (HCC) cell propagation, invasion, and migration by mediating miR-203/ BCAT1 axis. Microarray analysis was based on 25 pairs of HCC cancerous tissues and adjacent tissues. The expression levels of CRNDE, miR-203, and BCAT1 in HCC tissues were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The liver cell line L-02 and HCC cell lines HepG2 and Huh-7 were utilized to assess the regulatory effects of CRNDE and miR-203 on HCC progression in vitro. Western blot was used to qualify BCAT1 protein expression level. Cell proliferation and apoptosis were evaluated using CCK-8 and flow cytometry analysis, whereas cell invasion and migration assay were performed by the Transwell assay. The relationship among CRNDE, miR-203, and BCAT1 was validated by dual luciferase assay. Tumor Xenograft study was established to verify the pathological effect of CRNDE on HCC development in vivo. The expression levels of the CRNDE and BCAT1 were upregulated in HCC tissues and cells, whereas miR-203 was downregulated in HCC. Knockdown of CRNDE or miR-203 overexpression would inhibit HCC cell propagation and metastasis, and induced cell apoptosis. Moreover, miR-203 was negatively correlated with CRNDE, the same as miR-203 with BCAT1. Dual luciferase assay showed that miR-203 was an inhibitory target of CRNDE, and BCAT1 was directly targeted by miR-203 as well. LncRNA CRNDE could enhance HCC tumorgenesis by sponging miR-203 and mediating BCAT1. LncRNA CRNDE might facilitate HCC cell propagation, invasiveness, and migration through regulating miR-203/ BCAT1 axis.

The Protein Extract of Chlorella minutissima Inhibits The Expression of MMP-1, MMP-2 and MMP-9 in Cancer Cells through Upregulation of TIMP-3 and Down Regulation of c-Jun.

Objective Considering the bioactivities exhibited by microalgae, the effect of protein extract of Chlorella minutissimma (CP extract) was investigated on the expression of human matrix metalloproteinases-1 (MMP-1) in the breast cancer cell line MDA-MB231, and that of MMP-2 and -9 in hepatocellular cancer cell line HepG2 at different expression levels. The study aimed identification and analysis of inhibitory activity of microalgal components extracted from Chlorella minutissima against human MMPs. Materials and Methods In this experimental study, we analysed the effect of Chlorella extracts on MMP-1, -2, and -9 expression at various levels. Gelatin zymography was performed to study the inhibitory effect of Chlorella exracts on human gelatinases at the activity level, followed by western blotting to analyse the expression of all three MMPs at the protein level. The similar effect at the mRNA level along with the probable mechanism underlying inhibition of MMPs was assessed using real-time polymerase chain reaction (PCR). Results The results reveal that the treatment with CP extract decreased the mRNA expression of MMP-1, MMP-2, and MMP-9 by 0.26-, 0.29-, and 0.40-fold, respectively, at 20 μg/ml concentration as well as inhibited the activity of MMP-2 and MMP-9 by 37.56 and 42.64%, respectively, at 15 μg/ml concentration. Additionally, upregulated mRNA expression of tissue inhibitor of metalloproteinases-3 (TIMP-3) by 1.68-fold was seen in HepG2 cells at 20 μg/ml concentration treatment group. However, CP extract did not induce any change in the mRNA expression of the TIMP-1, -2 and -4 in HepG2 and TIMP-1, -2, -3 and -4 in MDA-MB231 cells. Activator protein-1 (AP-1)-dependent c-Jun-mediated transcriptional regulation of MMP-1, -2, and -9 was also studied to elucidate the appropriate mechanism involved in the inhibition of MMPs. ConclusionThe CP extract successfully inhibited MMP-1, -2, and -9 at different expression levels through TIMP-3 upregulation and c-Jun downregulation.

A c-Myc/miR-17-5p feedback loop regulates metastasis and invasion of hepatocellular carcinoma.

The molecular mechanisms that control metastasis of hepatocellular cancer (HCC) are still poorly understood. It has been determined that microRNA (miRNA) expression has tissue and cell specific, and decreased expression of specific miRNA could induce tumor genesis or metastasis. In this study, we identified that miR-17-5p was expressed lower in high metastatic capability HCC cell lines HCCLM3 and MHCC97H than low metastatic HCC cell line HepG2 by real-time (RT)-PCR. Restoration of miR-17-5p could significantly repress the invasiveness and metastasis of MHCC97H cell line. Furthermore, we validated c-Myc as a downstream and functional target of miR-17-5p using luciferase reporter assay. Immunohistochemical assay revealed that the expression of c-Myc protein levels was significantly increased in cancerous tissues compared with para-tumor tissues. After clinical data analysis, we observed that the higher level of c-Myc was significantly associated with a reduced overall survival (p = 0.0209). Consistent with previous research, we also demonstrated that c-Myc could upregulate the expression of miR-17-5p. Taken together, our data indicated that there is a regulatory feedback loop between miR-17-5p and c-Myc, in which miR-17-5p could suppress some of the distinguishing features, invasion, and metastasis, of oncogenic c-Myc in HCC cells, and meanwhile, miR-17-5p is upregulated by c-Myc role as a transcription factor, although further studies are still needed.

The modulating effect of ATM, ATR, DNA-PK inhibitors on the cytotoxicity and genotoxicity of benzo[a]pyrene in human hepatocellular cancer cell line HepG2.

The effect of inhibitors of phosphatidylinositol-3-kinase-related kinases (PIKK): ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK) on response of HepG2 human liver cancer cells to benzo[a]pyrene (BaP) was investigated. PIKK inhibitors: KU55933 (5 μM), NU7026 (10 μM) or caffeine (1 and 2mM) when used as single agents or in combinations (KU55933/NU7026 and caffeine/NU7026) did not significantly influence the BaP (3 μM) cytotoxicity (MTT reduction test). BaP induced a weak proapoptotic effect which was moderately enhanced by both inhibitor combinations. HepG2 cells exposed to BaP showed a strong S-phase arrest which was considerably diminished by both inhibitor combinations. The DNA damage (comet assay) induced after continuous 24h exposure to BaP was significantly diminished by both inhibitor combinations. Weak induction of reactive oxygen species by BaP was observed, which was not modulated by the inhibitor combinations. Similarly, no modulation of the glutathione levels was observed.

Anticancer effect of SN-38 combined with sorafenib on hepatocellular carcinoma in vitro and its mechanism

To investigate the anticancer effect and its mechanism of SN-38 combined with sorafenib on hepatocellular cancer cell lines HepG-2 and BEL-7402. SRB colorimetry was employed to measure the viability of HepG-2 and BEL-7402 cells after the treatment of SN-38 with sorafenib. Propidium iodide flow cytometric assay and DAPI staining were used to evaluate the apoptosis of HCC cells. Western blotting was conducted to detect the expression level of apoptosis-related and DNA damage-related proteins. SRB colorimetry showed the synergistic anticancer activities of SN-38 combined with sorafenib, with a combination index of <0.9. The apoptotic rates of HepG-2 cells in control, 60 nmol/L SN-38, 2.5μmol/L sorafenib and combination groups were 4.25%±2.45%, 28.95%±10.75%, 3.49%±2.49% and 53.19%±11.21%, respectively(P<0.05). Western blotting showed that the combination of these two drugs increased the enzymolysis of PARP, Caspase-8 and Caspase-3, and promoted the expression levels of p53, p21 and γ-H2AX significantly. SN-38 and sorafenib have synergistic anticancer activity on hepatocellular carcinoma cells in vitro with the augmentation of apoptosis.

The influence of ATM, ATR, DNA-PK inhibitors on the cytotoxic and genotoxic effects of dibenzo[def,p]chrysene on human hepatocellular cancer cell line HepG2

The effect of inhibitors of phosphatidylinositol-3-kinase related kinases (PIKK): ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK) on the response of HepG2 human liver cancer cells to dibenzo[def,p]chrysene (DBC) was investigated. High cytotoxicity of DBC (IC50=0.1μM) was observed after 72h incubation. PIKK inhibitors: KU55933 (5μM), NU7026 (10μM) or caffeine (1 and 2mM) when used alone did not significantly influence the cytotoxicity. However, two combinations: KU55933/NU7026 and caffeine/NU7026 significantly increased HepG2 viability (by 25%) after treatment with DBC at 0.5μM. The cytoprotective effect was confirmed by cell cycle and apoptosis/necrosis analysis. DNA damage level after exposure to DBC assessed by comet assay (single strand breaks) showed a long persistence and significant decrease after incubation of the cells in the presence the inhibitors (the combination of KU55933+NU7026 showed the strongest effect). Weak induction of reactive oxygen species (ROS) by DBC (0.5μM) was observed. Although, KU55933 and NU7026 when used alone did not increase ROS levels in the cells, their combination induced the ROS increase and moderately enhanced ROS generation by DBC. We propose a mechanism how cells with damaged DNA after exposure to DBC and under the condition of PIKK inhibition, may be at higher risk of undergoing malignant transformation.

Suppression of human hepatocellular cancer cell proliferation by Brucea javanica oil-loaded liposomes via induction of apoptosis.

IntroductionHepatocellular carcinoma (HCC) is a type of malignancy with high incidence and poor prognosis. Brucea javanica is extracted from Simaroubaceae plants. It is found to have low toxicity but high anti-cancer efficiency. The aim of this study is to determine the effects of Brucea javanica oil-loaded liposomes (BJOL) on human hepatocellular cancer cell line HepG2. The related molecular mechanisms were determined.Material and methodsMorphologic changes of HepG2 cells were observed by transmission electron microscope after treatment with BJOL in vitro. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after cell treatment with different doses of BJOL. Flow cytometry was performed. Nude mice were divided into 4 groups randomly and treated with different doses of BJOL. The apoptosis hepatocellular carcinoma was detected by TUNEL.ResultsProliferation of HepG2 was inhibited significantly by BJOL in a dose-dependent manner (2.5 mg/l or 5 mg/l). Compared with the animal models treated with the negative control, the animal models in the BJOL group had higher weight and lower metastasis rates (p < 0.01). The rate of apoptosis in hepatocellular carcinoma tissue of the BJOL groups was increased when compared with the control group (p < 0.05).ConclusionsBrucea javanica oil-loaded liposomes inhibits proliferation of HepG2. The effect appears to be dose-dependent, possibly by inducing apoptosis of cancer cells.

4,5-Diaryl imidazoles with hydroxamic acid appendages as anti-hepatoma agents.

Hepatocellular carcinoma (HCC) is the most abundant tumour of the liver with rising patient numbers in the Western world countries. Despite newly approved drugs like protein kinase inhibitors the survival rate is still poor. In order to identify potential new drugs for the treatment of HCC we investigated the real-time cell viability, apoptosis induction (sub-G1 cells), and HDAC (histone deacetylase) activity of two hepatocellular cancer cell lines HepG2 and Hep3B treated with new imidazole-tethered hydroxamates. The tested cinnamyl hydroxamates exhibited significant antiproliferative and cytotoxic activity in HCC cells as apparent from high sub-G1 cell levels in flow cytometric cell cycle analyses. In Hep3B cells HDAC inhibition was observed comparable in magnitude to that induced by the clinically applied HDAC inhibitor SAHA (Zolinza, Vorinostat). The new imidazolyl hydroxamic acids lend themselves as a possible alternative to SAHA in the therapy of HCC. Even more so since similar 4,5-diarylimidazoles lacking only the hydroxamate functionality were previously shown in animal studies to be well tolerated and orally applicable.

P-0070 Curcumin Inhibit the Epithelial–Mesenchymal Transition Induced by Hypoxia in Hepatocellular Carcinoma Cell Line HEPG2 in Vitro

IntroductionHepatocellular carcinoma is one of the most lethal cancers in the worlds due to the aggressive biological characteristics of the tumor. A critical event in the early stage of invasion and metastasis of tumor is the epithelial–mesenchymal transition. Evidences revealed that hypoxia could induce the epithelial–mesenchymal transition via the up-regulation of the hypoxia-inducible factor-1&agr; (HIF-1&agr;) and increase the invasion and metastasis of tumor cells ultimately. This study is aimed to investigate the effect of Curcumin, the principle active ingredient in turmeric which is derived from the rhizome of Curcuma longa, on epithelial–mesenchymal transition in hepatocellular cancer cell line HepG2 under the hypoxia condition induced by cobalt chloride (CoCl2). MethodsHepG2 cells were divided to 3 groups: normal control group, CoCl2 (10 µmol/L) group, and CoCl2 plus Curcumin (10 µmol/L) group. After 48h of treatment, proliferation of cell was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The invasion of HepG2 was detected by Transwell invasion assay. The mRNA expression of HIF-1&agr; was determined by real-time RT-PCR. The proteins of HIF-1&agr;, E-cadherin and vimentin were determined by Western blotting. ResultsCompared with the normal control group, the proliferation of HepG2 cell in CoCl2 group was significantly increased and in CoCl2 plus Curcumin group was decreased. Furthermore, the invasion of HepG2 cells were inhibited significantly by Curcumin compared with CoCl2 group. The difference of mRNA expressions of HIF-1&agr; was not observed. Meanwhile, HIF-1&agr; was obviously up-regulated, E-cadherin was increased and vimentin was deceased at protein levels in hypoxia induced by CoCl2 which is abolished by Curcumin. ConclusionCurcumin abolished the up-regulation of proliferation and migration of HepG2 cell in hypoxia which induced by CoCl2, through decreased the expression of HIF-1&agr; protein, and inhibited the epithelial–mesenchymal transition of HepG2.

PMO-092 TLR7 expression is increased in hepatocellular cancer (HCC) and its modulation is associated with alterations in tumour growth: a novel therapeutic target

IntroductionWe have previously described upregulation of TLR9, which is mainly located in the endosomes, in human HCC and cell lines. Their inhibition or stimulation was associated with alteration in tumour...

GLUT1 Expression Is Increased in Hepatocellular Carcinoma and Promotes Tumorigenesis

Accelerated glycolysis is one of the biochemical characteristics of cancer cells. The glucose transporter isoform 1 (GLUT1) gene encodes a key rate-limiting factor in glucose transport into cancer cells. However, its expression level and functional significance in hepatocellular cancer (HCC) are still disputed. Therefore, we aimed to analyze the expression and function of the GLUT1 gene in cases of HCC. We found significantly higher GLUT1 mRNA expression levels in HCC tissues and cell lines compared with primary human hepatocytes and matched nontumor tissue. Immunohistochemical analysis of a tissue microarray of 152 HCC cases revealed a significant correlation between Glut1 protein expression levels and a higher Ki-67 labeling index, advanced tumor stages, and poor differentiation. Accordingly, suppression of GLUT1 expression by siRNA significantly impaired both the growth and migratory potential of HCC cells. Furthermore, inhibition of GLUT1 expression reduced both glucose uptake and lactate secretion. Hypoxic conditions further increased GLUT1 expression levels in HCC cells, and this induction was dependent on the activation of the transcription factor hypoxia-inducible factor-1alpha. In summary, our findings suggest that increased GLUT1 expression levels in HCC cells functionally affect tumorigenicity, and thus, we propose GLUT1 as an innovative therapeutic target for this highly aggressive tumor.

The Effect of Doxorubicin on MEK-ERK Signaling Predicts Its Efficacy in HCC

Hepatocellular cancer (HCC) is a leading cause of cancer-related death worldwide. Historically, doxorubicin (DOX) has been widely used against unresectable HCC with variable response rates. We hypothesized that DOX combined with mitogen-activated protein kinase kinase-extracellular signal-regulated kinase (MEK-ERK) targeted therapy may provide enhanced anti-cancer effects. Human HCC cell lines (HepG2, Hep3B) were treated with DOX and MEK enzyme inhibitors, U0126 or PD184161, alone or in combination. Growth, apoptosis, and ERK expression/MEK activity were respectively determined by proliferation assay, DNA fragmentation enzyme-linked immunoassay or fluorochrome inhibitor of caspases, and Western blot. DOX (0.01-1 microM) decreased cell proliferation in Hep3B cells (IC(50) approximately 0.12 microM) at 48 to 72 h; DOX was less effective in HepG2 cells (IC(50) approximately 0.25 microM). At early time points (30 min) after DOX treatment of Hep3B cells, MEK activity was unchanged at low doses and decreased at higher doses; after 24 h, phospho-ERK levels increased at higher doses. Contrarily, in HepG2 cells, DOX caused a sustained, dose-dependent increase in phospho-ERK levels at early and late time points. The MEK inhibitor U0126 decreased phospho-ERK in both HCC lines. In contrast to DOX, HepG2 cells were more sensitive than Hep3B cells to U0126. The combination of DOX with U0126 (or PD184161) resulted in greater inhibition of proliferation in HepG2 but not in Hep3B cells. This effect may be mediated in part by enhanced apoptosis. The effect of DOX on early and late induction of MEK activity predicts its chemotherapeutic response in HCC. Furthermore, this effect may also determine the utility of MEK inhibitor combination treatment.

Expression of human telomerase reverse transcriptase gene in hepatocellular carcinoma and effect of antisense telomerase gene on apoptosis of hepatocellular cancer cell line HepG2

Expression of human telomerase reverse transcriptase gene in hepatocellular carcinoma and effect of antisense telomerase gene on apoptosis of hepatocellular cancer cell line HepG2