Gene expression and apoptosis response in hepatocellular carcinoma cells induced by biocompatible polymer/magnetic nanoparticles containing 5-fluorouracil

BACKGROUND: Super-paramagnetic iron oxide nanoparticles (SPION) contain a chemotherapeutic drug and are regarded as a promising technique for improving targeted delivery into cancer cells. RESULTS: In this study, the fabrication of 5-fluorouracil (5-FU) was investigated with loaded Dextran (DEXSPION) using the co-precipitation technique and conjugated by folate (FA). These nanoparticles (NPs) were employed as carriers and anticancer compounds against liver cancer cells in vitro. Structural, magnetic, morphological characterization, size, and drug loading activities of the obtained FA-DEX-5-FUSPION NPs were checked using FTIR, VSM, FESEM, TEM, DLS, and zeta potential techniques. The cellular toxicity effect of FA-DEX-5-FU-SPION NPs was evaluated using the MTT test on liver cancer (SNU-423) and healthy cells (LO2). Furthermore, the apoptosis measurement and the expression levels of NF-1, Her-2/neu, c-Raf-1, and Wnt-1 genes were evaluated post-treatment using flow cytometry and RT-PCR, respectively. The obtained NPs were spherical with a suitable dispersity without noticeable aggregation. The size of the NPs, polydispersity, and zeta were 74 ± 13 nm, 0.080 and 45 mV, respectively. The results of the encapsulation efficiency of the nano-compound showed highly colloidal stability and proper drug maintenance. The results indicated that FA-DEX-5-FU-SPION demonstrated a sustained release profile of 5-FU in both phosphate and citrate buffer solutions separately, with higher cytotoxicity against SNU-423 cells than against other cells types. These findings suggest that FA-DEX-SPION NPs exert synergistic effects for targeting intracellular delivery of 5-FU, apoptosis induction, and gene expression stimulation. CONCLUSIONS: The findings proved that FA-DEX-5-FU-SPION presented remarkable antitumor properties; no adverse subsequences were revealed against normal cells.

Manuka honey enhanced sensitivity of HepG2, hepatocellular carcinoma cells, for Doxorubicin and induced apoptosis through inhibition of Wnt/ß-catenin and ERK1/2

BACKGROUND: Recently, there is increasing awareness focused on the identification of naturally occurring anticancer agents derived from natural products. Manuka honey (MH) has been recognized for its biological properties as antimicrobial, antioxidant, and anticancer properties. However, its antiproliferative mechanism in hepatocellular carcinoma is not investigated. The current study focused mainly on investigating the molecular mechanism and synergistic effect of anticancer properties of MH on Doxorubicin (DOX)-mediated apoptotic cell death, using two different p53 statuses (HepG2 and Hep3B) and one non-tumorigenic immortalized liver cell line. RESULTS: MH treatment showed a proliferative inhibitory effect on tested cells in a dose-dependent manner with IC50 concentration of (6.92 ± 0.005%) and (18.62 ± 0.07%) for HepG2 and Hep3B cells, respectively, and induced dramatic morphological changes of Hep-G2 cells, which considered as characteristics feature of apoptosis induction after 48 h of treatment. Our results showed that MH or combined treatments induced higher cytotoxicity in p53-wild type, HepG2, than in p53-null, Hep3B, cells. Cytotoxicity was not observed in normal liver cells. Furthermore, the synergistic effect of MH and Dox on apoptosis was evidenced by increased annexin-V-positive cells and Sub-G1 cells in both tested cell lines with a significant increase in the percentage of Hep-G2 cells at late apoptosis as confirmed by the flow cytometric analysis. Consistently, the proteolytic activities of caspase-3 and the degradation of poly (ADP-ribose) polymerase were also higher in the combined treatment which in turn accompanied by significant inhibitory effects of pERK1/2, mTOR, S6K, oncogenic ß-catenin, and cyclin D1 after 48 h. In contrast, the MH or combined treatment-induced apoptosis was accompanied by significantly upregulated expression of proapoptotic Bax protein and down-regulated expression of anti-apoptotic Bcl-2 protein after 48 h. CONCLUSIONS: Our data showed a synergistic inhibitory effect of MH on DOX-mediated apoptotic cell death in HCC cells. To our knowledge, the present study provides the first report on the anticancer activity of MH and its combined treatment with DOX on HCC cell lines, introducing MH as a promising natural and nontoxic anticancer compound.

Effect of lupeol on invasion and metastasis of human hepatoma HepG2 and SK-HEP-1 cells and its mechanism / 中国中药杂志

Epithelial-mesenchymal transformation(EMT) exists in embryonic development and is closely related to cell migration and invasion. The increased EMT level in tumors showed that E-cadherin was replaced by N-cadherin, and the expression of interstitial markers such as α-SMA and vimentin was up-regulated. It has been reported that lupeol can reduce the expression of matrix metalloproteinase-2(MMP-2), matrix metalloproteinase-9(MMP-9) and N-cadherin to inhibit the metastasis of osteoma cells. However lupeol has been less studied in liver cancer. Therefore, this paper investigated the effect of lupanol on invasion and metastasis of human hepatoma cell line HepG2 and SK-HEP-1 and its possible mechanism. MTT assay and Annexin V/PI double staining were used to investigate the effect of lupeol on activity and apoptosis of HepG2 cells and SK-HEP-1 cells. Moreover, the effect of lupeol on the invasion of HepG2 cells and SK-HEP-1 cells were evaluated by Transwell assay. The expressions of E-cadherin, N-cadherin, α-SMA, vimentin and MMP-9 were measured by Western blot. The model of subcutaneous transplantation of nude mice and the lung metastasis model of H22 hepatocellular carcinoma cells were established to evaluate the efficacy of lupeol in vivo on tumor growth and lung metastasis by HE staining combined with immunohistochemical assay. The results showed that lupeol inhibited the activity and invasion of HepG2 cells and SK-HEP-1 cells in a dose-dependent manner and induced apoptosis. Western blot showed that the expression of E-cadherin, a landmark protein for EMT, was induced by lupeol, and the expressions of N-cadherin, α-SMA, vimentin and MMP-9 were decreased. In vivo experiments showed that lupeol inhibited tumor growth in mice bearing xenograft. In addition, immunohistochemical experiments confirmed that lupeol could up-regulate the expression of E-cadherin in tumor tissues of nude mice, reduce the expression of N-cadherin, and inhibit the metastasis of liver cancer H22 cells in the lungs of mice. The above results indicated that the mechanism of lupeol inhibiting the invasion and metastasis of HCC cells may be related to the regulation of EMT process.

Ropivacaine promotes apoptosis of hepatocellular carcinoma cells through damaging mitochondria and activating caspase-3 activity

BACKGROUND: Recent evidences indicated that some local anaesthetic agents played a role in inhibiting the proliferation of cancer cells; Whether ropivacaine is able to promote apoptosis of hepatocellular carcinoma (HCC) cells is still unclear. The aim of this study was to investigate the effect of ropivacaine on the apoptosis of HCC cells. METHODS: In the present study, we treated the HCC cell lines, Bel7402 and HLE with ropivacaine. MTT, DAPI stain, trypan blue exclusion dye assay, flow cytometry, electron microscopy, computational simulation, laser confocal microscope, Western blotting, and enzyme activity analysis of caspase-3 were applied to detect the growth and apoptosis of HCC cells and to explore the role mechanism of ropivacaine. RESULTS: Ropivacaine was able to inhibit proliferation and promote apoptosis of HCC cells in a dose- and time-dependent manner. Ropivacaine also has a trait to inhibit the migration of HCC cells; ropivacaine damaged the mitochondria of HCC cells. The results also indicated that ropivacaine was able to interact with caspase-3, promote cytoplasmic caspase-3 migration into the nucleus, stimulate cleavage of caspase-3 and PARP-1, caspase-9 proteins, inhibit the expression of Bcl-2, promote expression of Apaf-1 and mitochondria release cytochrome C, and activate the activity of caspase-3. CONCLUSIONS: Ropivacaine has a novel role in promoting apoptosis of HCC cells; The role mechanism of ropivacaine maybe involve in damaging the function of mitochondria and activating the caspase-3 signalling pathway in HCC cells. Our findings provide novel insights into the local anaesthetic agents in the therapy of HCC patients.

Effect of Atractylodes macrocephala polysaccharide on proliferation and invasion of hepatocellular carcinoma cells / 南方医科大学学报

OBJECTIVE@#To investigate the inhibitory effect of polysaccharide of Atractylodes macrocephala (PAM) on the proliferation and invasion of hepatocellular carcinoma cells and the underlying mechanism.@*METHODS@#Hepatocellular carcinoma HepG2 cells were treated with different concentrations of PAM, and their proliferation and invasive ability were examined using CCK-8 assay and Transwell assay. Immunofluorescence assay was performed to detect the expression level of β-catenin, and real-time PCR and Western blotting were used to detect the mRNA and protein expressions of AKT, GSK-3β and MMP-2 in the cells. The changes in the proliferation, invasiveness and the expressions of pGSK-3β and MMP2 were examined in the cells following treatment with LiCl/PAM/LiCl plus PAM.@*RESULTS@#PAM treatment significantly reduced the cell viability, the number of migration cells, and the expression levels of β-catenin and MMP-2 ( < 0.05), and obviously inhibited the phosphorylation of AKT and GSK-3β in the cells ( < 0.05) in a dose-dependent manner. The rescue experiment showed that LiCl reversed the inhibition of cell proliferation, invasiveness, and the Wnt/β-catenin pathway induced by PAM.@*CONCLUSIONS@#PAM can inhibit the proliferation and invasion of hepatocellular carcinoma cells possibly by inhibiting the Wnt/β-catenin signaling pathway.

Aldolase a gene affects the growth of hepatocellular carcinoma cells / 国际药学研究杂志

Objective To preliminarily explore the effects of gene knock-out and overexpression of aldolase A on tumor cell proliferation, and then confirm the relationship between aldolase A and tumor growth during the process of anaerobic metabolism. Methods Through vector plasmid building and lentiviral infection, the cell lines stably overexpressing aldolase A and its gene knock- out cell lines were constructed respectively. The restructured cell lines were validated on gene and protein levels. Then, the changes of cell proliferation ratio were observed. Results Real time PCR results showed that aldolase A gene expression of the overexpressing cell line was about 3 times that of the normal cells. Among the five interfering targets of aldolase A designed according to aldolase A RNA sequences, aldolase A4 had more obvious interference effect on the target gene expression, the aldolase A mRNA level of which was about 1/50 of the normal cells. Compared with normal control group, the proliferation rate of aldolase A gene overexpression significantly increased by 40% in 48 h(P<0.05), while the proliferation rate of aldolase A gene knock out cells significantly de? creased about 86% in 24 h(P<0.05). Conclusion Aldolase A expressions have obvious effects on hepatocellular carcinoma cells.

Effect of lysophosphatidic acid on hepatocellular carcinoma cell migration and its involved mechanisms / 医用生物力学

Objective To investigate the effect of lysophosphatidic acid (LPA) on migration of hepatocellular carcinoma MHCC97H cells and its involved mechanisms. Methods Transwell was utilized to investigate the impact of LPA on cell migration of MHCC97H cells. Furthermore, the role of ROCK in the migration of MHCC97H cells through Y-27632 (a specific inhibitor of ROCK). Then, the expression of F-actin was observed with immunofluorescence staining and Western blotting. Atomic force microscopy (AFM) was employed to investigate elastic modulus of MHCC97H cells. Results LPA significantly promoted the migration of MHCC97H cells, while Y-27632 significantly blocked the migration of MHCC97H induced by LPA. Moreover, LPA up-regulated the expression of F-actin and decreased the elastic modulus of MHCC97H cells. Conclusions LPA promotes MHCC97H cell migration through decreasing the cell stiffness via ROCK/F-actin.

The effect of TRIM5αon the proliferation and apoptosis of hepatocellular carcinoma cells / 实用肿瘤学杂志

Ob jective To study the effect of Tripartite motif 5 alpha( TRIM5α) on the proliferation and apoptosis of hepatocellular carcinoma cells.Methods The TRIM5α construction was identified by enzyme diges-tion,PCR and sequencing.The TRIM5αplasmid was transfected into HepG2 cells and identified by RT-PCR and Western blotT.he cell proliferation was monitored by RTCA real -time instrument and cell apoptosis was an-alyzed by flow cytometry.The apoptosis related proteins were detected by Western blot.Results The TRIM5αvector was successfully constructed.The result of RT-PCR and Western blot showed that TRIM5αplasmid could enter HepG2 cells.TRIM5αcould inhibit HepG2 cells proliferation also increased their apoptosis through down-regulation of Bcl-2 expression and activation of Caspase-3 expression.TRIM5αalso could inhibit the tumor formation of HepG2 cells in vivo.Conclusion TRIM5αinhibited cell proliferation, promoted apoptosis and weakened the tumorigenic ability of HepG2 cells.This will establish the foundation for the mechanism study and the clinic use of TRIM5αfor tumor therapy in the future.

Aldolase A gene affects the growth of hepatocellular carcinoma cells / 国际药学研究杂志

Objective To preliminarily explore the effects of gene knock-out and overexpression of aldolase A on tumor cell proliferation,and then confirm the relationship between aldolase A and tumor growth during the process of anaerobic metabolism. Methods Through vector plasmid building and lentiviral infection,the cell lines stably overexpressing aldolase A and its gene knock-out cell lines were constructed respectively. The restructured cell lines were validated on gene and protein levels. Then ,the changes of cell proliferation ratio were observed. Results Real time PCR results showed that aldolase A gene expression of the overex?pressing cell line was about 3 times that of the normal cells. Among the five interfering targets of aldolase A designed according to aldol?ase A RNA sequences,aldolase A4 had more obvious interference effect on the target gene expression,the aldolase A mRNA level of which was about 1/50 of the normal cells. Compared with normal control group,the proliferation rate of aldolase A gene overexpression significantly increased by 40% in 48 h(P<0.05),while the proliferation rate of aldolase A gene knock out cells significantly de?creased about 86%in 24 h(P<0.05). Conclusion Aldolase A expressions have obvious effects on hepatocellular carcinoma cells.

Synthesis of novel gold nanoparticles and its radiosensitizing effect on HepG2 cells / 中华放射医学与防护杂志

Objective To synthesize novel gold nanoparticles of GAL-PEG-GNPs,study its radiation effect on hepatocellular carcinoma cells HepG2 cells in vitro,and investigate the underlying mechanisms.Methods GAL-PEG-GNPs were synthesized and characterized successfully.HepG2 cells were divided into three groups of control,GNPs and GAL-PEG-GNPs.The cytotoxicities of these compounds were tested by the CCK-8 assay and their IC50 values of HepG2 cells were calculated.Cell uptake of nanoparticles was detected by TEM and ICP-MS.The radiosensitization effect of nanoparticles was tested by the colony formation assay.Cell cycle distribution was detected by FCM.The expressions of CAT,SOD,and total GSH were detected with a microplate reader,and the expressions of apoptosis-related proteins were tested by Western blot.Results The GNPs and GAL-PEG-GNPs had absorption peaks at 520 and 530 nm,respectively,and their diameters were (22.6-±2.12) and (32.0 ± 1.41) nm detected by ICP-MS.The GAL-PEG-GNPs and GNPs had similar cytotoxicity profiles (P ＞ 0.05),while GAL-PEG-GNPs could be more effectively uptaken by HepG2 cells than GNPs.The sensitive enhancement ratio (SER) of GNPs and GAL-PEG-GNPs to HepG2 cells were 1.46 和 1.95,respectively.The percentage of cells at phase of G2/M in HepG2 population treated with GNP was higher than that of untreated cells (t =14.20,P ＜0.05).The protein expressions of Cytochrome C,Bax,Caspase-3,and Caspase-9 were upregulated while Bcl-2 expression was down-regulated in the cells treated with GNPs/radiation or GAL-PEGGNPs/radiation.The expressions of CAT,SOD and total GSH in the GNP treated groups were significantly decreased compared with the control group(t =12.34,29.39,12.85,P ＜ 0.05).Conclusions GALPEG-GNPs has obvious radiosensitization effect on HepG2 cells,which is related to the induction of cell apoptosis and the generation of free radicals.

Effect of siRNA on Wisp-1 gene expression, proliferation, migration and adhesion of mouse hepatocellular carcinoma cells

Objective: To study the inhibition effect of siRNA on the expression of Wisp-1 gene in Hca-F of mouse hepatocellular carcinoma cells strain and also its effect on the proliferation, migration and adhesion of hepatocellular carcinoma cells. Methods: Three expression vectors of siRNA were constructed. Lipo2000 was employed to transfect Hca-F cells and Western blot was used to detect the inhibition effect of siRNA on the expression of Wisp-1 gene. Afterward, CCK8 was adopted to detect the effect of Wisp-1 siRNA on the proliferation of Hca-F cells; Annexin V-FITC/PI double staining flow cytometry was used to detect the effect of Wisp-1 siRNA on the apoptosis of Hca-F cells; Transwell was used to detect the effect of Wisp-1 siRNA on the migration of Hca-F cells. The in vitro cell adhesion kit was used to detect of Wisp-1 siRNA on the change in the components of extracellular matrix to which Hca-F cells adhered. Western blot was used to detect the activation of protein kinase B (AKT)/glycogen synthase kinase-3β pathway and the expression of downstream target protein p53 and matrix metalloproteinases-2. Results: The siRNA showed interference effect on the expression of Wisp-1 gene. Compared with the control group, after being transfected to cells, Wisp-1 siRNA could significantly inhibit the proliferation, migration and adhesion of Hca-F cells and also promote the cell apoptosis, which was related to the down-regulated phosphorylation of AKT and glycogen synthase kinase-3β and the expression of p53 and matrix metalloproteinases-2 (P < 0.05). Conclusions: The inhibition of Wisp-1 expression can reduce the proliferation, migration and adhesion of mouse hepatocellular carcinoma cells, which is related to the AKT/glycogen synthase kinase-3β pathway. Wisp-1 gene may be the potential target to cure the hepatocellular carcinoma.

Mechanism study on apoptosis of hepatocellular carcinoma HepG2 cells induced by ursolic acid through inhibiting the expression of miR-21 / 中国药科大学学报

@#This study aimed at investigating the effect of miR-21 on the apoptosis of hepatocellular carcinoma HepG2 cells induced by ursolic acid(UA). MTT assay was used to determine the inhibition effect of ursolic acid on proliferation of hepatocellular carcinoma cells. The expression level of miR-21 in hepatocellular carcinoma cells and the regulation effect of ursolic acid on the expression of miR-21 in HepG2 cells were determined by qPCR. To up-regulate the expression of miR-21, miR-21 mimics were transfected into HepG2 cells. Then MTT assay, flowcytometry(Annexin V-FITC staining), and RT-PCR were used to detect the regulation effects of ursolic acid on the proliferation, apoptosis, and the expression of apoptosis-related genes after miR-21 over-expression. The results showed that the proliferation inhibition effect of ursolic acid on HepG2 cells and the expression level of miR-21 in HepG2 cells were higher than in hepatic cell L-02 and hepatocellular carcinoma SMCC-7721, Bel-7402 cells. So further study was performed in the HepG2 cells. Ursolic acid inhibited the expression of miR-21 in HepG2 cells. And the greatest inhibition effect was at 24 h after treatment with UA. Over-expression of miR-21 partially offset the effects of ursolic acid on the proliferation, apoptosis, and the expression of apoptosis-related genes such as Bcl-2, survivin and Bax after 24 h. The results suggested that apoptosis of hepatocellular carcinoma HepG2 cells could be induced by ursolic acid by down-regulating the expression of miR-21.

Effect of Oleanic Acid on Key Enzyme Activity in Insulin-Resistant HepG2 Cell Line / 医药导报

Objective To explore the effect of oleanic acid on key enzyme activity in insulin-resistant HepG2 cells. Methods The HepG2 cells were divided into normal control,model control,metformin,and oleanic acid groups.Glycogen content in insulin-resistant HepG2 cell model were detected by hepatic glycogen test kit upon treatment with oleanic acid.Activities of glucokinase ( GK) ,phosphoenolpyruvate carboxylase kinase (PEPCK),and glucose-6-phosphatase (G-6-Pase) were assayed by the glucose 6-phosphate dehydrogenase coupling colorimetric, lactate dehydrogenase coupling colorimetric and ammonium molybdate constant phosphorus methods. Results The oleanic acid enhanced glucose consumption,lowered the activity of G-6-Pase and PEPCK by 54.8% and 18.8%,respectively,and increased the activity of GK and glycogen content in also insulin-resistant HepG2 cells by 100.6% and 98.6%,respectively. Conclusion Aqueous extracts of shirako play a role in lowering PEPCK and G-6-Pase activities and inhibiting glucogenesis, resulting in the reduction of endogenous glucose in the cell. In addition,it can augment the activity of GK,accelerate the process of glucolysis,increase the glycogen content,and alleviate insulin resistance of HepG2.

Effect of siRNA on Wisp-1 gene expression, proliferation, migration and adhesion of mouse hepatocellular carcinoma cells

OBJECTIVE@#To study the inhibition effect of siRNA on the expression of Wisp-1 gene in Hca-F of mouse hepatocellular carcinoma cells strain and also its effect on the proliferation, migration and adhesion of hepatocellular carcinoma cells.@*METHODS@#Three expression vectors of siRNA were constructed. Lipo2000 was employed to transfect Hca-F cells and Western blot was used to detect the inhibition effect of siRNA on the expression of Wisp-1 gene. Afterward, CCK8 was adopted to detect the effect of Wisp-1 siRNA on the proliferation of Hca-F cells; Annexin V-FITC/PI double staining flow cytometry was used to detect the effect of Wisp-1 siRNA on the apoptosis of Hca-F cells; Transwell was used to detect the effect of Wisp-1 siRNA on the migration of Hca-F cells. The in vitro cell adhesion kit was used to detect of Wisp-1 siRNA on the change in the components of extracellular matrix to which Hca-F cells adhered. Western blot was used to detect the activation of protein kinase B (AKT)/glycogen synthase kinase-3β pathway and the expression of downstream target protein p53 and matrix metalloproteinases-2.@*RESULTS@#The siRNA showed interference effect on the expression of Wisp-1 gene. Compared with the control group, after being transfected to cells, Wisp-1 siRNA could significantly inhibit the proliferation, migration and adhesion of Hca-F cells and also promote the cell apoptosis, which was related to the down-regulated phosphorylation of AKT and glycogen synthase kinase-3β and the expression of p53 and matrix metalloproteinases-2 (P < 0.05).@*CONCLUSIONS@#The inhibition of Wisp-1 expression can reduce the proliferation, migration and adhesion of mouse hepatocellular carcinoma cells, which is related to the AKT/glycogen synthase kinase-3β pathway. Wisp-1 gene may be the potential target to cure the hepatocellular carcinoma.

Effect of calreticulin gene silencing on proliferation and invaison in human hepatocellular carcinoma cells / 中华肝胆外科杂志

Objective To explore the effect of calreticulin (CRT) on cell proliferation and invasion of human hepatocellular carcinoma cell line SMMC-7721 and HepG2.Methods SMMC-7721 and HepG2 cells were transfected with small interfering RNA (siRNA).The transfection rate was detected by immunoflurescence and western blot.The cell proliferation,invasion and apoptosis of SMMC-7721 and HepG2 cells were determined by using cell counting kit-8 (CCK-8) assays,transwell assays and flow cytometry,respectively.The p-Akt and Akt levels were detected by western blot.Results The growth inhibition rate in the siRNA experimental group of SMMC-7721 and HepG2 cells for 24,36 and 48 h were (41.0 ±2.2) ％,(46.5 ±1.6)％,(59.7 ±2.2)％ and (36.8 ±2.7)％,(47.3 ± 1.8)％,(61.5 ±3.2)％,respectively.The apoptosis rate after down-regulating the expression of CRT in SMMC-7721 and HepG2 cells for 36h were (45.2 ± 9.1) ％ and (48.9 ± 8.0) ％,respectively.Compared with the blank group and the negative control group,the growth inhibition rate in the siRNA experimental group was lower (P ＜0.05),but the apoptosis rate was significantly higher (P ＜ 0.05).Transwell experiments confirmed that the numbers of invaded SMMC-7721 and HepG2 cells in the blank group and the negative control group and siRNA experimental group were (96.8±7.3),(95.6±5.4),(34.0±4.2) and (124.0 ±9.9),(121.6 ±7.0),(70.4±9.5),respectively,indicating that cell invasion in the siRNA experimental group was significantly suppressed (P ＜ 0.05).The expression of p-Akt was decreased (P ＜ 0.05) after down-regulating the expression of CRT for 36h.Conclusion CRT gene silencing by siRNA can inhibit the SMMC-7721 and HepG2 cell proliferation and invasion,but increase the cell apoptosis by regulating PI3K/Akt signal pathway.

Metformin Promotes Apoptosis but Suppresses Autophagy in Glucose-Deprived H4IIE Hepatocellular Carcinoma Cells

BACKGROUND: Metformin, a well-known anti-diabetic drug, has gained interest due to its association with the reduction of the prevalence of cancer in patients with type 2 diabetes and the anti-proliferative effect of metformin in several cancer cells. Here, we investigated the anti-proliferative effect of metformin with respect to apoptosis and autophagy in H4IIE hepatocellular carcinoma cells. METHODS: H4IIE rat cells were treated with metformin in glucose-free medium for 24 hours and were then subjected to experiments examining the onset of apoptosis and/or autophagy as well as the related signaling pathways. RESULTS: When H4IIE cells were incubated in glucose-free media for 24 hours, metformin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) reduced the viability of cells. Inhibition of AMP-activated protein kinase (AMPK) by compound C significantly blocked cell death induced by metformin or AICAR. Pro-apoptotic events (nuclear condensation, hydrolysis of intact poly ADP ribose polymerase and caspase-3) were stimulated by metformin and then suppressed by compound C. Interestingly, the formation of acidic intracellular vesicles, a marker of autophagy, was stimulated by compound C. Although the deprivation of amino acids in culture media also induced apoptosis, neither metformin nor compound C affected cell viability. The expression levels of all of the autophagy-related proteins examined decreased with metformin, and two proteins (light chain 3 and beclin-1) were sensitive to compound C. Among the tested inhibitors against MAP kinases and phosphatidylinositol-3-kinase/mammalian target of rapamycin, SB202190 (against p38MAP kinase) significantly interrupted the effects of metformin. CONCLUSION: Our data suggest that metformin induces apoptosis, but suppresses autophagy, in hepatocellular carcinoma cells via signaling pathways, including AMPK and p38 mitogen-activated protein kinase.

Comparison of liver cancer models established by different injection routes of rat L2 hepato-cellular carcinoma cells / 临床与实验病理学杂志

Purpose To compare and investigate the oncogenic and metastatic phenotypes in nude mice by injection of rat hepatocellu-lar carcinoma ( HCC) cells L2 via two different routes. Methods Twenty of 7-week-old female BALB/cA mice were randomly divided into 2 groups. After the injections of L2 from liver or spleen lobe, the survival rate, tumor formation rate, carcinogenic features, and metastasis were comparatively studied. Results All of the liver orthotopic nude mice developed liver cancer (100%) with 60% lung metastasis rate, and exhibited an expansive growing pattern with surrounding invasion. In the spleen orthotopic model, 78% mice de-veloped HCC in spleen, with 67% liver metastatic rate and 11% lung metastatic rate, lower than the liver orthotopic model ( P <0. 05). But the microscopically hilar lymph node metastasis rate was 33%. Conclusion The direct liver injection of L2 in female nude mice is a better modeling method for studying the mechanism of both carcinogenesis and metastasis, as well as the evaluation of therapeutic effect of liver cancer.

Effects of different substrate stiffness on adhesion, spreading and migration of human hepatocellular carcinoma cells / 医用生物力学

Objective To investigate the effects of substrate stiffness on the adhesion, spreading and migration of hepatocellular carcinoma cells as well as the regulation of cytoskeleton assembly and integrinβ1 expression, and to explore the role of substrate mechanical properties in the metastasis of hepatocellular carcinoma cells. Methods The polyarcylamide gel with different stiffness was achieved by varying the relative ratio of acrylamide to bis acrylamide. The substrate surface was cross linked with extracellular matrix molecules for cell adhesion. The adhesion, spreading and migration of hepatocellular carcinoma cells on substrates with different stiffness were recorded by phase contrast microscope and made quantitative analysis by Image J software. The cytoskeleton assembly on substrates with different stiffness was detected by immunofluorences assay, and the expression of integrinβ1on different substrates was measured by flow cytometer. Results The rigid substrate enhanced the adhesion and spreading of hepatocellular carcinoma cells in shortened time. Neither the soft (1.1 kPa) nor over rigid (glass) substrate facilitated the migration of hepatocellular carcinoma cells, and the maximum migration velocity was found on the substrate with moderate stiffness（10.7 kPa）. The rigid substrate could promote cytoskeleton assembly and integrinβ1 expression. Conclusions The effects of substrate stiffness on adhesion, spreading and migration of hepatocellular carcinoma cells are regulated by the cytoskeleton assembly and integrin expression.

Inhibition of glucometabolism by a novel dehydroabielylamine derivative,DHAA-urea,in human hepatoma HepG2 cells / 中国药科大学学报

The effects of DHAA-urea,a novel dehydroabietylamine(DHAA) derivatives,on cell viability and glucose metabolism,in hypoxia and normoxia human hepatoma HepG2 cells were investigated.Hypoxia cells were achieved using DMEM containing high concentration of glucose without serum and pre-incubating of CoCl_2 (final concentration 150 μmol/L) for 24 h.The antiproliferation effect of DHAA-urea was measured by colorimetric MTT assay.The cellular ATP concentration,the lactate dehydrogenase(LDH) and glucose-6-phosphate dehydro genase (G6PD) activity were detected by their kits.It was shown that DHAA-urea markedly inhibited cell viability,cellular ATP level,LDH and G6PD activity in either aerobic or anaerobic circumstance in a dose-and time dependent manner.This suggested that DHAA-urea possibly inhibited HepG2 cells growth via the inhibition of glucolysis and glucolysis-dependent ATP depletion.DHAA-urea could be a promising candidate in the development of a novel class of agents used for human hepatocellular carcinoma.

Effects of p27mt gene transfection mediated by replication deficient adenovirus on the proliferation and apoptosis of human hepatocellular carcinoma cells / 肿瘤研究与临床

Objective To assess the effects of p27mt gene transfection on the proliferation and apoptosis of human hepatocellular carcinoma cell (HCC) lines SMMC-7721. Methods A replication deficient adenovirus vector encoding p27mt (Ad-p27mt) was used and p27mt cDNA was transfected into human SMMC-7721 cell lines in vitro. The synthesis of DNA in SMMC-7721 cells was determined by using 3H-thymidine incorporation; the cell apoptosis was determined by flow cytometry, TUNEL method and DNA fragmentation analysis. Results The virus titer was 7.95?1012 cfu/ml, the transduction efficiency was 100 % when multiplicity of infection ≥50, FCM analysis revealed a sub-G1 cell peak in Ad-p27mt transduced hepatocellular carcinoma cell lines. Agarose electrophoresis showed marked ladder .The difference of apoptotic index between the Ad-p27mt group and the control group was statistically significant (58.6?4.3, vs 4.5?1.6, P