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This manuscript aims to investigate the effects of resibufogenin on the proliferation, migration and invasion of human hepatocellular carcinoma cells and its related mechanisms. MTT assay was used to determine the inhibitory effect of resibufogenin on the growth of four hepatocellular carcinoma cells in vitro. Wound-healing assay and Transwell assay were used to evaluate the migration and invasion ability of resibufogenin on MHCC-97H cells. Western blot assay was used to detect the expression of migration and invasion related proteins in MHCC-97H cells treated with different concentrations of resibufogenin. The results showed that resibufogenin significantly inhibited the proliferation of hepatocellular carcinoma cells in vitro. The half maximal inhibitory concentration (IC50) values on MHCC-97H, HepG2, SK-Hep-1 and Huh-7 cells were 0.55 ± 0.06, 2.83 ± 0.24, 5.25 ± 0.49, 14.89 ± 2.28 μmol·L-1, respectively. Resibufogenin also suppressed the migration and invasion of MHCC-97H cells in a concentration-dependent manner. The protein expression of integrin α2, integrin α6, integrin β1, N-cadherin, matrix metalloproteinase 2 (MMP2) and transcription factor Twist in MHCC-97H cells were decreased significantly with the increase of the concentration of resibufogenin, while the protein expression of E-cadherin increased. In addition, we found that p-PI3K/PI3K and p-AKT/AKT ratios were significantly reduced after treatment with resibufogenin. In conclusion, resibufogenin can inhibit the proliferation, migration and invasion of hepatocellular carcinoma MHCC-97H cells in vitro, which is related to the regulation of intracellular migration and invasion protein expression and PI3K/AKT signaling pathway.
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Aim To investigate the synergy effects of deoxyschizandrin and gemcitabine (GEM) on the proliferation of HepG2 human hepatocellular carcinoma cells in vitro and the underlying mechanism. Methods CCK8 method and colony formation assay were used to detect the effects of deoxyschizandrin monotherapy, GEM monotherapy and combination therapy on the proliferation of HepG2 cells. Flow cytometry was used to detect the change of apoptosis rate of HepG2 cells after treatment with single drug or the combination use of two drugs. Western blot was performed to detect the expression of BCL2, BAX, pro-caspase3, caspase3, pro-caspase9, caspase9, ß-catenin and TCF-4. Results Deoxyschizandrin, GEM and combination group significantly inhibited the proliferation of HepG2 cells and promoted cell apoptosis. The effects of the combination group on HepG2 cells were significantly stronger than those of single-drug groups (P < 0. 05). Western blot results showed the expression of pro-caspase3 and pro-caspase9 was changed slightly within deoxyschizandrin, GEM and combination groups compared with that of normal control, while the expression of B C L 2, ß-catenin and TCF-4 protein expression was down-regulated significantly (P < 0. 05). The expression of B A X, cleaved-caspase3 and cleaved-caspase 9 protein increased significantly after treatment with deoxyschizandrin, GEM and combination group (P < 0. 05). Specially, the increasing effect of the expression of the protein in combined group was more significant than that of single drug groups (P < 0. 05). Conclusions The combination of deoxyschizandrin and GEM significantly inhibited the proliferation of HepG2 cells and induced cell apoptosis, as well as suppressed the ß-catenin/TCF-4 pathway.
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Objective: To detect the effects of dicentrine on the migration and invasion of human hepatocellular carcinoma MHCC97-H cells and potential mechanisms. Methods: MHCC97-H cells were cultured in vitro and cultured at logarithmic growth stage. The cells were treated with 5, 10, 25, 50, 100 μmol/L dicentrine for 24, 48, 72 h, and cell proliferation was determined by MTT assay. The cells were treated with 5, 10, 25 μmol/L dicentrine for 24 h, the cell adhesion, cell migration, cell invasion, gene expression of VEGF, MMP-2 and MMP-9, and protein expression of p-JAK2 and p-STAT3 were determined by MTT, scratch, Transwell, real-time qPCR, and western blot assay, respectively. Results: The cell viability of MHCC-97H cells treated with 25 μmol/L dicentrine at 48 and 72 h, and 50 and 100 μmol/L dicentrine at 24, 48, and 72 h was decreased significantly, which was significantly different from that of the control group (P < 0.05 or P < 0.01). Compared with control group, cell adhesion ability, wound healing ability, cell penetration modulus, gene expression of VEGF, MMP-2 and MMP-9, and protein expression of p-JAK2 and p-STAT3 of MHCC-97H cells treated with 5, 10 and 25 μmol/L dicentrine at 24 h were decreased significantly (P < 0.05 or P < 0.01). Conclusion: Dicentrine can inhibit the migration and invasion of human hepatocellular carcinoma MHCC97-H cells, which is related to the down-regulation of VEGF, MMP-2, and MMP-9 gene expression and inhibition of JAK2/STAT3 signaling pathway activation.
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Objective To study the inhibitory effect of aspirin on proliferation of human hepatocellular carcinoma HepG2 cell line and its possible mechanismt. Methods MTT assay and plate cloning experiments was used to detect proliferation of human hepatoma HepG2 cells. Effects of aspirin on autophagosomes in HepG2 cells were detected by acridine orange fluorescence staining. The expression of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) protein in human hepatocellular carcinoma HepG2 cells was detected by Western blot. Results 10 mmol/L concentration of aspirin could inhibit the proliferation of HepG2 cells, but increase the number of autophagosomes of HepG2 cells, increase AMPK expression, decrease mTOR expression. After combination treatemnt with 40 μmol/L autophagy inhibitor chloroquine (CQ), CQ could enhance the inhibitory effect of 10 mmol/L aspirin on proliferation of human hepatoma HepG2 cells. Conclusion Combination treatment with autophagy inhibitor CQ attenuates 10 mmol/L aspirin-induced autophagy thus enhance its anti-HepG2 effect.
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Objective:To explore the effects of hypoxia-inducible factor 2α genes on under hypoxia on proliferation,apoptosis, cell cycle distribution and migration of invasiveness of human hepatocellular carcinoma cell HepG2.Methods: Human hepatoma cell line HepG2 induced by cobalt chloride (CoCl2) was selected as the research object,construction of siRNA specific carrier HIF-2α, transfection of HepG2 cells under hypoxia.Real-time PCR,Western blot method in the detection of before and after transfection in each group of HIF-2α mRNA and protein expression;MTT method to detect the proliferation of HepG2 cells before and after transfection;apoptosis rate and distribution of cell cycle of HepG2 cells before and after transfection were detected by flow cytometry;Transwell test was used to detect the invasion and migration ability of HepG2 cells before and after transfection.Results: Under hypoxia,significant increased HIF-2α expression in hepatocellular carcinoma HepG2 cells.Specific transfection of HIF-2α siRNA in HepG2 cells after HIF-2α mRNA and protein expression levels were significantly inhibit cell proliferation decreased,apoptosis rate increased in the ratio of G0/G1 phase cells increased synthesis phase (S) and late (G2/M) synthesis cell ratio decreased,which in vitro invasion and migration of cells was inhibited.Conclusion:Expression of HIF-2α increases in hepatocellular carcinoma HepG2 cells under hypoxia. Specific siRNA can be cut by HIF-2α gene expression in HepG2 cells under hypoxia,to inhibit HepG2 cell proliferation,invasion, migration,and change the distribution of cell cycle and induce its apoptosis.
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Increasing evidence suggests that hepatocellular carcinomas (HCCs) are sustained by a distinct subpopulation of self-renewing cells known as cancer stem cells (CSC). However, our understanding of their regulation is limited. Rapid reversible changes of CSC-like cells within tumors may result from the effect of biological mediators found in the tumor microenvironment. This paper aims to explore how nitrite, a key cellular modulator whose level is elevated in many tumors, affects CSC-like phenotypes of human hepatoma cells SMMC-7721 cells. The SMMC-7721 cell line was cultured under serum-free conditions to produce floating spheres. The distribution of cell cycle was analyzed by flow cytometry, the capability of cells self-renew was detected by colony-forming capabilities and spheroid-formation assay, the expression of stemness protein such as CD133, CD90 and EpCAM were determined by flow cytometry and Western blot, cell invasion was analyzed by transwell assay, and viability of SMMC-7721 parental cells and spheroids cancer cells was determined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Xenograft tumor models were established by subcutaneously injecting SMMC-7721 spheroids cancer cells, the transplanted tumor tissue ROS levels was detected by reactive oxygen species (ROS) test kits, the expression of HIF-1α was observed by immunofluorescence. Our results showed that the SMMC-7721 spheroid cells were enriched with CSCs properties, indicated by the ability to self-renew, increased expression of CSCs markers, and increased resistance to chemotherapeutic drugs. Additionally, SMMC-7721 parental cells and spheroids cancer cells were treated with 150 μmol·L-1 sodium nitrite for 6 days, compared with control cells, an increased accumulation of G0/G1 phase cells was observable in treatment cells. Indeed, our data demonstrated that in parent cells and spheres cells that were treated with sodium nitrite for different time, the cells' ability to chemoresistance and invasion, clone-forming efficiencies and the spheres forming ability were significantly higher than that of control cells. Exposure of sodium nitrite regulated CSC-like phenotype, indicated by increased expression of known CSC markers, CD133, CD90 and EpCAM in the exposed parental cells, as well as in dormant spheroids cancer cells. Compared with the parent cells, the above effects of nitrite on the spheres cells were significantly enhanced. In vivo data also presented a more significant promotion of tumor xenograft growth from the nitrite treatment than from either of the control. Mechanistic analysis indicated that nitrite induced the upregulation of HIF-1α as well as the downregulation of ROS in the tumor microenvironment. These results suggest that nitrite increases the invasiveness of SMMC-7721 cells through up-regulation of tumor stemness.
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Objective The objective of this study was to investigate effects of lycopene(LP) on the proliferation and apoptosis of human hepatocellular carcinoma HepG2 cells and to explore its mechanism.Methods HepG2 cells in logarithmic growth phase were treated with 0,5,10,20 μg/mL of LP and 40 μg/mL of Cisplatin for 48 h.Ten replicates in each dose were designed in this study.After treatments,the cell viability was measured by MTT colorimetric assay.The distribution of cell cycle was detected by flow cytometry(FCM).The mRNA expression of Bax and Bcl-2 were measured by RT-PCR.The expression of Caspase-3 protein was explored by Western blot.Results The inhibition rate of HepG2 cells was significantly increased by 10 μg/mL and 20μ g/mL of LP or 40 μg/mL of cisplatin when compared to the negative control group.The cell cycle of HepG2 cells were arrested at the G0/G1 phase and the apoptosis rate were significantly increased in comparison with the negative control group.The level of Bax mRNA expression was significantly increased and decreased in the expression of Bcl-2 mRNA.They were shown an increasing ratio of Bax/Bcl-2 and up-regulated Caspase-3 protein in HepG2 cells treated with LP.All effects in this study show a dose-dependent manner.Conclusion LP can inhibit the proliferation and promote the apoptosis in HepG2 cells.This mechanism may be contributed to arresting cell cycle and regulating gene expression related to apoptosis.
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Objective To investigate the impact of 5-fluorouracil (5-FU) and cisplatin on miR-449b expression in human hepatocellular carcinoma (HCC) and elucidate the molecular mechanism of 5-FU and cisplatin inhibiting the migration of HCC cells.Methods Real-time qPCR analysis was conducted to determine the expression of miR-449b in 50 HCC tissues.RT-PCR assay was performed to detect the expression of miR-449b in HCC cells with 5-FU and cisplatin treatment.The migration of HCC cells with the overexpression of miR-449b was determined by wound-healing assay;Rescue assay was employed to investigate the correlation between 5-FU & cisplatin, miR-449b and the migration capacity of HCC cells;The putative targets of miR-449b were predicted and validated using target prediction programs and immunoblots.Results The expression of miR-449b decreased in HCC tissues (P<0.0001).miR-449b expression increased in HCC cells upon the treatment of 5-FU and cisplatin (P<0.001).The overexpression of miR-449b inhibited the migration of HCC cells (P<0.001).Rescue assay revealed that inhibition of miR-449b to prevent 5-FU and cisplatin induction resulted in suppressed migration in SMMC7721 cells(P<0.05).Catenin-δ was a functional target of miR-449b.Conclusions 5-FU and cisplatin inhibit the migration of HCC cells at least partly via inducing the expression of miR-449b.
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[Objective]This study was conducted to examine the effects of dexmedetomidine on the proliferation and angiogenesis of MHCC97H and SMCC7721 human hepatocellular carcinoma(HCC)cell lines cultured in hypoxia condition in vitro,and investigated the possible mechanism involved.[Methods]MHCC97H and SMCC7721 human HCC cell lines under hypoxia culture condition were treated with presence or absence of dexmedetomidine(100 μmol/L). Cell viability,colony formation,vasculogenic mimicry(VM) formation were assessed. The effects of dexmedetomidine on α-2A adrenergic receptor(α2A),hypoxia induced factor-1a(HIF-1a),and vascular endothelial growth factor(VEGF)protein expression were evaluated with Western blot analysis.[Results]Cell proliferation assay and colony formation assay indicated that hypoxia obviously promoted the proliferation of MHCC 97H and SMCC7721 cells(CoCl2 group vs corresponding control group,the proliferation rate of MHCC97H and SMCC7721:Day 3,142.2%and 133.8%;Day 4,134.7%and 131.0%;Day 5,133.5%and 136.2%;all P<0.05),and VM formation assay suggested that hypoxia increased angiogenesis of MHCC97H and SMCC7721 cells. Whereas dexmedetomidine significantly inhibited the proliferation(Dex+CoCl2 group vs CoCl2 group,the proliferation rate of MHCC97H and SMCC7721:Day 3,55.7%vs 60.7%;Day 4,46.9%vs 58.1%;Day 5,46.4%vs 57.0%,all P<0.05)and angiogenesis of MHCC97H,SMCC7721 cells induced by hypoxia. Dexmedetomidine may exert these functions by activating α-2A adrenergic receptor,causing an decrease in HIF-1a and VEGF protein,while hypoxia activated HIF-1a and VEGF protein to promote the growth and angiogenesis of cells.[Conclusion]The findings provide evidence that hypoxia could promote the proliferation and angiogenesis of MHCC97H and SMCC7721 cells,while dexmedetomidine might inhibit these effects by down-regulating HIF-1a and VEGF protein expression through activatingα-2A adrenergic receptor.
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Objective To explore the apoptosis and mechanisms of HepG-2 cells induced by total flavonoids of Verbena offici?nalis L.(TFV). Methods HepG-2 cells were cultured with different concentrations of TFV. The apoptosis of HepG-2 cells was evalu?ated by flow cytometry and DNA ladder. Reactive oxygen species(ROS)and membrane potential were measured by flow cytometry. Ex?pression of Caspase-3,Caspase-8,Caspase-9,and P53 was analyzed by Western blotting. Results TFV 50,100 and 200 mg/L in?creased the apoptosis rate(P<0.01),increased ROS levels of HepG-2 cells(P<0.01),and decreased the mitochondria membrane potential(P<0.05,P<0.01). TFV(50,100 and 200 mg/L)increased the proteins′ expression of Caspase-3,Caspase-9 and P53 (P<0.05,P<0.01). Conclusion TFV may induce the apoptosis of HepG-2 cells via increasing ROS levels,decreasing mitochon?dria membrane potential,and up-regulateing the expression of Caspase-3,Caspase-9 and P53 protein in HepG-2 cells.
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Nitrites play multiple characteristic functions in invasion and metastasis of hepatic cancer cells, but the exact mechanism is not yet known. Cancer cells can maintain the malignant characteristics via clearance of excess mitochondria by mitophagy. The purpose of this article was to determine the roles of nitrite, reactive oxygen species (ROS) and hypoxia inducing factor 1 alpha (HIF-1α) in mitophagy of hepatic cancer cells. After exposure of human hepatocellular carcinoma SMMC-7721 cells to a serial concentrations of sodium nitrite for 24 h under normal oxygen, the maximal cell vitality was increased by 16 mg·L-1 sodium nitrite. In addition, the potentials of migration and invasion for SMMC-7721 cells were increased significantly at the same time. Furthermore, sodium nitrite exposure displayed an increase of stress fibers, lamellipodum and perinuclear mitochondrial distribution by cell staining with Actin-Tracker Green and Mito-Tracker Red, which was reversed by N-acetylcysteine (NAC, a reactive oxygen scavenger). DCFH-DA staining with fluorescent microscopy showed that the intracellular level of ROS concentration was increased by the sodium nitrite treatment. LC3 immunostaining and Western blot results showed that sodium nitrite enhanced cell autophagy flux. Under the transmission electron microscopy (TEM), more autolysosomes formed after sodium nitrite treatment and NAC could prevent autophagosome degradation. RT-PCR results indicated that the expression levels of COXⅠ and COXⅣ mRNA were decreased significantly after sodium nitrite treatment. Meanwhile, laser scanning confocal microscopy showed that sodium nitrite significantly reduced mitochondrial mass detected by Mito-Tracker Green staining. The expression levels of HIF-1α, Beclin-1 and Bnip3 (mitophagy marker molecular) increased remarkably after sodium nitrite treatment, which were reversed by NAC. Our results demonstrated that sodium nitrite (16 mg·L-1) increased the potentials of invasion and migration of hepatic cancer SMMC-7721 cells through induction of ROS and HIF-1α mediated mitophagy.
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Aim To investigate the effect of neferine on proliferation and invasion of human hepatocellular car-cinoma. Methods HepG2 and Bel-7402 cells were cultured in vitro with different concentrations of nefer-ine, then cell proliferation was observed by CCK-8 as-say; cell invasion was observed by transwell invasion assay; the protein expression of RhoA, RhoC and ROCK was detected by Western blot. Results CCK-8 results showed that neferine could significantly inhibit cell proliferation in a dose-dependent manner compared with the control group. Transwell invasion assay showed that cell invasion was significantly decreased with neferine 3 μmol · L-1 . Western blot results showed that RhoA, RhoC and ROCK protein expres-sion was decreased when neferine was co-incubated with hepatocellular carcinoma cells. Conclusion Nef-erine can inhibit proliferation and invasion of HepG2 and Bel-7402 cells, which is mediated mainly by the inhibition of RhoA, RhoC and ROCK protein expres-sion.
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OBJECTIVE:To study the inhibitory effects of emodin on the proliferation of human hepatocellular carcinoma SMMC7721 cells. METHODS:SMMC7721 cells were treated with 0(negative control),25,37.5,50,62.5,75,87.5,100μmol/L emodin solution and 100 μmol/L 5-FU for 24 h,48 h,72 h. The optical density value of cells was detected,and inhibition rate was calculated. SMMC7721 cells were treated with 0 (negative control),25,50,75 μmol/L emodin solution and 100 μmol/L 5-FU for 48 h,and cell apoptosis rate,cell cycle and the expression of Bax and Bcl-2 gene were detected. RESULTS:Compared with negative control,the rate of cell proliferation inhibition increased after treated with 25,37.5,50,62.5,75,87.5,100 μmol/L emodin and 100 μmol/L 5-FU,which was positively associated with the concentration and duration. Compared with negative con-trol,the rate of cell apoptosis increased after treated with 25,50,75 μmol/L emodin solution and 100 μmol/L 5-FU;the expres-sion of Bax increased and that of Bcl-2 dereased;50,75 μmol/L emodin solution and 100 μmol/L 5-FU could arrested cells at G0/G1 phase(P<0.05 or P<0.01). CONCLUSIONS:Emodin can inhibit the proliferation of SMMC7721,promote cell apoptosis and in-hibit cell growth.
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Aim To investigate the effect of plumbagin on invasion and apoptosis of human hepatocellular car-cinoma ( HepG2 ) . Methods HepG2 cells were cul-tured in vitro with different concentrations of plumba-gin, then cell proliferation was observed by MTT as-say; cell invasion was observed by transwell invasion assay; cell apoptosis was detected by flow cytometry, and the protein expression of Bax, Bcl-2 was detected by immunocytochemistry. Results MTT results showed that plumbagin could significantly inhibit cell proliferation compared with the control group, and in a dose-dependent manner ( P ( P < 0. 01 ) . Flow cytometry showed that apoptosis rate was significantly higher in 4 , 8 μmol · L-1 group of plumbagin compared with that of the control group ( P<0. 01 ) . Immunocytochemistry showed that, with the increasing concentration of plumbagin, Bax protein expression increased, Bcl-2 protein expression was de-creased, both in a dose-dependent manner ( P <0. 01 ) . Conclusion Plumbagin can inhibit HepG2 cell proliferation and accelerate apoptosis of HepG2 cells, but also has the ability to inhibit HepG2 cell in-vasion.
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OBJECTIVE: To investigate the anti-tumor mechanisms of a new oleanolic acid derivative (the test substance, 3β- hydroxyolea-12-en-28-oic acid-3, 5, 6-trimethylpyrazin-2-methyl ester) on the human hepatocellular carcinoma SMMC-7721 cells. METHODS: MTT assay and morphology observation were used to determine the effects of the new oleanolic acid derivative on human hepatocellular carcinoma SMMC-7721 cells. Cell membrane integrity was assessed with AV/PI uptake by fluorescence microscope. The loss of mitochondrial membrane potential (Δψm) was detected by using JC-1 assay. Western blotting was used to detect the apoptosisrelated proteins Bcl-2, Bax, caspase-9 and caspase-3. RESULTS: The new oleanolic acid derivative reduced the cell viability of SMMC-7721 cells in dose-and time-dependent manner. After treatment of the new oleanolic acid derivative on cells for 48 h, cell nucleus pycnosis, cell lysis and the generation apoptosis body and so on were observed, the cell membrane integrity was damaged, the mitochondrial membrane potential (Δψm) was decreased, the protein levels of Bax, pro-caspase-9, cleaved caspase-9, pro-caspase-3 and cleaved caspase-3 were upregulated, and the protein level of Bcl-2 was downregulated. CONCLUSION: The growth inhibition and proapoptotic effects of this new oleanolic acid derivative on human SMMC-7721 cells associate with its damage on mitochondrial function.
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Objective The cancer risk of patients with diabetes mellitus who are treated by metformin declines remarkably in comparison to patients receiving other drug therapies.The article was to investigate the relationship between antineopastic activity and fatty acid synthase (FASN) of metformin in human hepatocellular carcinoma cell(HCC) line HepG2. Methods HepG2 cells were treated with various concentrations of metformin( 0, 1, 5, 10, 15 mmol/L) for 24, 48 and 72 h respectively and cell growth was assessed by CCK-8 assay.Positive control(paclitaxel 10μg/mL) and negative control(metformin 0mmol/L) were set up simultaneously.After being treated with doses of metformin(0, 5, 10,15mmol/L) for 72h, protein expression levels of AMPKα、P-AMPKα、FASN、P-mTOR and P-Akt were measured by western blotting analysis and FASN mRNA expression levels were measured by RT-PCR. Results Being treated with vari-ous doses of metformin(1, 5, 10, 15 mmol/L) for 24, 48 and 72 h, the growth of HepG2 cells were inhibited by metformin in dose-dependent and time-dependent manner( P0.05) .FASN mRNA expression levels decreased significantly in all metformin-treated groups( P<0.05) . Conclusion Met-formin actitiviates AMPK, inhibits mTOR and downregulates FASN, which are implicated in its antineopastic activity on HCC.Although metformin inhibits mTOR activation, it is not involved in Akt upregulation through a negative loop.
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BACKGROUND: Ubiquitin Specific Peptidase 39 (USP39) is a 65 kDa SR-related protein involved in RNA splicing. Previous studies showed that USP39 is related with tumorigenesis of human breast cancer cells. RESULTS: In the present study, we investigated the functions of USP39 in human hepatocellular carcinoma (HCC) cell line SMMC-7721. We knocked down the expression of USP39 through lentivirus mediated RNA interference. The results of qRT-PCR and western blotting assay showed that both the mRNA and protein levels were suppressed efficiently after USP39 specific shRNA was delivered into SMMC-7721 cells. Cell growth was significantly inhibited as determined by MTT assay. Crystal violet staining indicated that colony numbers and sizes were both reduced after knock-down of USP39. Furthermore, suppression of USP39 arrested cell cycle progression at G2/M phase in SMMC-7721cells. In addition, Annexin V showed that downregulation of USP39 significantly increased the population of apoptotic cells. CONCLUSIONS: All our results suggest that USP39 is important for HCC cell proliferation and is a potential target for molecular therapy of HCC.
Subject(s)
Humans , Cell Cycle , Carcinoma, Hepatocellular/pathology , Lentivirus/genetics , RNA Interference/physiology , Cell Proliferation , Ubiquitin-Specific Proteases/metabolism , Liver Neoplasms/pathology , Neoplasm Proteins/metabolism , In Vitro Techniques , Gene Expression Regulation, Neoplastic/genetics , Cell Cycle/genetics , Blotting, Western , Apoptosis , Gene Transfer Techniques , Carcinoma, Hepatocellular/enzymology , Gene Silencing , Cell Line, Tumor , Cell Proliferation/genetics , Gene Knockdown Techniques , Real-Time Polymerase Chain Reaction , Ubiquitin-Specific Proteases/genetics , Liver Neoplasms/enzymology , Neoplasm Proteins/geneticsABSTRACT
Objective To investigate the effect of B7-H3 on human hepatocellular carcinoma cell line HepG2 mediating regulation on human peripheral blood CD8+T cell activation,cell cycle and secretion of IL-17.Methods The expression of the B7-H3 on HepG2 cells was detected by RT-PCR and FCM respectively.B7-H3 was silenced by PGPU6/GFP/neo-B7-H3shRNA plasmid via cathodolyte liposome transfection method.CD8+T cells were sorted from healthy human peripheral blood with immunomagetic beads.The effect of HepG2 cells on activation,cell cycle and cytokine secretion of CD8+T cells which was stimulated by PHA or PMA respectively were analyzed by FCM.Results B7-H3 was highly expressed on HepG2 cells,and PGPU6/GFP/neo-B7-H3shRNA plasmid could effectively block down its expression.Otherwise,HepG2 cells could inhibit the expression of CD69,the early activation phenotype of T cell,blockade B7-H3 on HepG2 cells could significantly attenuate the inhibitory effects.Likewise,blockade B7-H3 on HepG2 cells apparently reversed the inhibitory effects of HepG2 cells on CD8+T cell cycle through down-regulating the cell number in G0/G1 phase and up-regulating the cell number in S phase;Moreover,HepG2 cells caused a sharp increase of IL-17 which was secreted by CD8+T cells and the level of IL-17 was further up-regulated after blocking down B7-H3.Conclusion HepG2 cells highly expressed B7-H3 that could promote the inhibitory the effect of HepG2 on expression of CD69 and cell cycle of CD8+T cells.HepG2 cells were able to up-regulate the level of IL-17 secreted by CD8+T cells,in which B7-H3 played an inhibitory role.
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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.
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Objective:To establish proteomic technique system of human hepatocellular carcinoma cell line QCY-7701. Method:Two different lysis buffer were taken to extract cell proteins. After two-dimensional gel electrophuresis (2-DE) and PDQuest analysis, 10 good-matched protein spots were chosen to be identified by MALDI-TOF-MS. Results:A steady 2-DE electrophregram of hepatocellular carcinoma cell line QGY-7701 was established. Compared with lysis bufferⅠ,protein quantities extracted from lysis bufferⅡincreased by 25%;protein spots in 2-DE gel increased by 32%. 9 out of 10 candidate protein spots were successfully identified (P