Incomplete radiofrequency ablation promotes the development of CD133+cancer stem cells in hepatocellular carcinoma cell line HepG2 via inducing SOX9 expression

Background: Cancer stem cells(CSCs) accelerate the growth of hepatocellular carcinoma(HCC) residual after incomplete radiofrequency ablation(In-RFA). The present study aimed to detect the effects of In-RFA on stemness transcription factors(STFs) expression which are important for the production and function of CSCs, and to find which STFs promote HCC stemness after In-RFA. Methods: HepG2 cells were used for in vitro and in vivo studies. Flow cytometry and sphere-formation assays were used to detect the level and function of CD133~+ CSCs in the models. PCR array and ELISA were applied to analyze the altered expression of 84 STFs in CD133~+ CSCs in two models. Specific lentiviral shRNA was used to knockdown STFs expression, followed by detecting In-RFA’s effects on the levels and function of CD133~+ CSCs. Results: In-RFA was identified to induce CD133~+ CSCs and increase their tumorigenesis ability in vitro and in vivo. The mRNA levels of 84 STFs in CD133~+ CSCs were detected by PCR array, showing that 15 and 22 STFs were up-regulated in two models, respectively. Meanwhile, the mRNA levels of seven common STFs were up-regulated in both models. ELISA assay demonstrated that only the protein of sex determining region Y-box 9(SOX9) was up-regulated in both models, the protein levels of the other 6 common STFs did not increase in both models. Finally, SOX9 was identified to play an important role in inducing, maintaining stemness and promoting tumorigenesis ability of CD133~+ CSCs in both models. Conclusion: In-RFA-induced SOX9 stimulates CD133~+ CSCs proliferation and increases their tumorigenesis ability, suggesting that SOX9 may be a good target for HCC treatment.

Modulating effects of survivin antisense oligonucleotide on changes of apoptosis and cell cycle of human hepatocellular carcinoma cell line SMMC-7721

To investigate the modulating effects of survivn antisense oligonucletode (ASODN) on the cell cycle and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721 and explore its mechanism.Methods Survivin ASODN was transfected into SMMC-7721 cells mediated by DOTAP liposomal reagent.Electron microscopy,flow cytometry and RT-PCR were used to detect the changes in cell ultrastructure,apoptosis,cell cycle and the expression of cyclinB1 mRNA,respectively.Results After transfection of survivin ASODN,the expression of cyclinB1 mRNA in the cells significantly increased and increase in G2-M arrest and apoptosis appeared.Meanwhile,the cell ultrastructure had apoptotic changes such as chromatin condensation and apoptotic body formation.Conclusion Survivin ASODN can induce the expression of cyclinB1 that may result in G2-M arrest.Consequently,apoptosis is triggered.Survivin ASODN transfection might be an improtant new treatment for HCC.14 refs,2 figs,1 tab.

Possible mechanisms associated with immune escape and apoptosis on anti-hepatocellular carcinoma effect of Mu Ji Fang granules

BACKGROUND Hepatocellular carcinoma(HCC)is one of the most common digestive system cancers with high mortality rates worldwide.The main ingredients in Mu Ji Fang Granules(MJF)are alkaloids,flavonoids,and polysaccharides.MJF has been used in the clinical treatment of hepatitis,cirrhosis and HCC for more than 30 years.Few previous studies have focused on the mechanism of MJF on tumor immunology in the treatment of HCC.AIM To explore the mechanism of action of MJF on tumor immunology in the treatment of HCC.METHODS The absorbable ingredients of MJF were identified using Molecule Network related to High Performance Liquid Chromatography-Electron Spray Ionization-Time of Flight-Mass Spectrometry,and hub potential anti-HCC targets were screened using network pharmacology and pathway enrichment analysis.Forty male mice were randomly divided into the Blank,Model,and MJF groups(1.8,5.4,and 10.8 g/kg/d)following 7 d of oral administration.Average body weight gain,spleen and thymus indices were calculated,tumor tissues were stained with hematoxylin and eosin,and Interferon gamma(IFN-γ),Tumor necrosis factorα(TNF-α),Interleukin-2,aspartate aminotransferase,alanine aminotransferase,alpha-fetoprotein(AFP),Fas,and FasL were measured by Enzyme-linked Immunosorbent Assay.Relevant mRNA expression of Bax and Bcl2 was evaluated by Real Time Quantitative PCR(RTqPCR)and protein expression of Transforming growth factorβ1(TGF-β1)and Mothers against decapentaplegic homolog(SMAD)4 was assessed by Western blotting.The HepG2 cell line was treated with 10 mg/mL,20 mg/mL,30 mg/mL,40 mg/mL of MJF,and another 3 groups were treated with TGF-β1 inhibitor(LY364947)and different doses of MJF.Relevant mRNA expression of TNF-α,IFN-γ,Bax and Bcl2 was evaluated by RT-qPCR and protein expression of TGF-β1,SMAD2,p-SMAD2,SMAD4,and SMAD7 was assessed by Western blotting.RESULTS It was shown that MJF improved body weight gain and tumor inhibition rate in H22 tumorbearing mice,protected immune organs and liver function,reduced the HCC indicator AFP,affected immunity and apoptosis,and up-regulated the TGF-β1/SMAD signaling pathway,by increasing the relative expression of TGF-β1,SMAD2,p-SMAD2 and SMAD4 and decreasing SMAD7,reducing immune factors TNF-αand IFN-γ,decreasing apoptosis cytokines Fas,FasL and Bcl2/Bax,and inhibiting the effect of LY364947 in HepG2 cells.CONCLUSION MJF inhibits HCC by activating the TGF-β1/SMAD signaling pathway,and affecting immune and apoptotic cytokines,which may be due to MJF adjusting immune escape and apoptosis.

Effects of suppressing glucose transporter-1 by an antisense oligodeoxynucleotide on the growth of human hepatocellular carcinoma cells

BACKGROUND:The glucose transporter-1(Glut-1),a key ratelimiting factor in the transport and metabolism of glucose in cancer cells,is over-expressed in many human cancer cells and this overexpression is correlated with poor biological behavior. The increased levels of Glut-1 expression in hepatocellular carcinoma(HCC)cells functionally affect tumorigenicity.This study was undertaken to investigate effects of suppressing Glut-1 by an antisense oligodeoxynucleotide(AS-ODN)on the growth of human hepatocellular carcinoma(HepG-2)cells. METHODS:We used AS-ODN targeting against the Glut-1 gene in a HepG-2 cell line.There were four experimental groups: empty pcDNA3.1 vector(mock transfection),pcDNA3.1-anti-Glut(+),pcDNA3.1-Glut(+),and non-transfected HepG-2 cells. The Glut-1 mRNA expression was detected by RT-PCR and the Glut-1 protein expression by Western blotting after cell culture, and the glucose uptake was detected after glucose stimulation in each group. RESULTS:Compared with non-transfected HepG-2 or Glut-1 pcDNA3.1,a down-regulation of Glut-1 mRNA in HepG-2 cells transfected with anti-Glut-1 pcDNA3.1 was noted(P<0.05).Glut-1 protein in HepG-2 cells transfected with Glut-1 AS-ODN was decreased compared with non-transfected HepG-2,Glut-1 pcDNA3.1,or empty vectors. Glucose uptake by the HepG-2 cells transfected with AS-ODN was decreased at 1 hour after glucose stimulation.CONCLUSIONS:The application of Glut-1 AS-ODN can down-regulate the expression of Glut-1 at mRNA and protein,and inhibit glucose uptake partially in HepG-2 cells.The Glut-1 gene maybe a potential therapeutic target for HCC.

The receptor for β2GPⅠon membrane of hepatocellular carcinoma cell line SMMC-7721 is annexin Ⅱ

AIM: To evaluate the receptor protein which can specifically bind to β2GPⅠon the membrane of hepatocellular carcinoma (HCC) cell line SMMC-7721, and to study the biological function of the receptor.METHODS: Through β2GPⅠ-affinity chromatography column, the peptid-polysome-mRNA complex, which can specially bind to β2GPⅠ, stayed with the column and was separated from the whole polysome of liver cells, and then eluted and collected. Using cDNA synthesis kit and cDNA PCR kit, the corresponding cDNA was obtained and sequenced. RT-PCR was used to amplify annexinⅡ, and flow cytometry was used to study the competitive binding of annexinⅡ with β2GPⅠto SMMC-7721.RESULTS: A total of 1.1 kb of the cDNA fragment of the specific binding protein of β2GPⅠon liver cell membrane was obtained. The sequence of cDNA shared high homology with human annexinⅡ (98%). AnnexinⅡ was expressed on the membrane of SMMC-7721, and could compete with β2GPⅠfor combining with SMMC-7721.CONCLUSION: The receptor for β2GPⅠon membrane of SMMC-7721 cells is annexinⅡ, which might bridge HBV to infect hepatocytes.

STUDY ON THE EXPRESSION OF CYCLOOXYGENASE-2 IN HEPATOCELLULAR CARCINOMA CELL LINES AND ON THE GROWTH INHIBITION EFFECT OF NS-398

Objective： To investigate the expression of cyclooxygenase -2 （COX-2） in hepatocellular carcinoma cell lines and to explore the effect of NS-398, a selective inhibitor for COX-2, on HepG-2 cell line. Methods： lmmunohistochemistry and RT-PCR were used to investigate COX-2 expression in 6 HCC cell lines. MTT and Flowcytometry were used to evaluate the effect of the selective inhibitor of COX-2, NS-398, on HepG-2 cell lines. Results： All six HCC cell lines showed COX-2 expression at protein level. Five out of 6 cell lines showed COX-2 expression at mRNA level. NS-398 could suppress the growth of HepG-2 cell line, in a time and dose dependant manner. Conclusion： NS-398, a selective inhibitor of COX-2, showed inhibition effect on HepG-2 HCC cell line. The efficacy of inhibition was time and dose dependent, providing a new evidence for chemoprovention of hepatocellular carcinorma with COX-2 selective inhibitors.

EFFECT OF MATRINE ON EXPRESSION OF HCCR1 AND HCCR2 PROTEINS IN CULTURAL HUMAN HEPATOCELLULAR CARCINOMAS CELLS

Objective： To explore the effects of matrine on HCCR1 and HCCR2 expression in cultural human hepatocellular carcinomas （HCC） cells at the level of gene and protein. Methods： Three methods, representational difference analysis （RDA） of cDNA, microarrays and fluorescence in situ hybridization （FISH） were used to detect levels of mRNA and protein expression of HCCR1 and HCCR-2 before and after treatment of matrine. Results： Matrine had inhibitory effect on the mRNA and protein expression of HCCR1 and HCCR2 in cultural HCC cells. Conclusion： Matrine has inhibitory effect on gene transcription, protein expression of HCCR 1 and HCCR2 in cultural HCC cells.

Cyclin L2, a novel RNA polymerase Ⅱ-associated cyclin, is involved in pre-mRNA splicing and induces apoptosis of human hepatocellular carcinoma cells

We report the cloning and functional characterization of human cyclin L2, a novel member of the cyclin family. Human cyclin L2 shares significant homology to cyclin L1, K, T1, T2, and C, which are involved in transcriptional regulation via phosphorylation of the C-terminal domain of RNA polymerase Ⅱ. The cyclin L2 protein contains an N-terminal "cyclin box" and C-terminal dipeptide repeats of alternating arginines and serines, a hallmark of the SR family of splicing factors. A new isoform and the mouse homologue of human cyclin L2 have also been cloned in this study. Human cyclin L2 is expressed ubiquitously in normal human tissues and tumor cells. We show here that cyclin L2 co-localizes with splicing factors SC-35 and 9G8 within nuclear speckles and that it associates with hyperphosphorylated, but not hypophosphorylated, RNA polymerase Ⅱ and CDK p110 PITSLRE kinase via its N-terminal cyclin domains. It can also associate with the SC-35 and 9G8 through its RS repeat region. Recombinant cyclin L2 protein can stimulate in vitro mRNA splicing. Overexpression of human cyclin L2 suppresses the growth of human hepatocellular carcinoma SMMC 7721 cells both in vitro and in vivo, inducing cellular apoptosis. This process involves up-regulation of p53 and Bax and decreased expression of Bcl-2. The data suggest that cyclin L2 represents a new member of the cyclin family, which might regulate the transcription and RNA processing of certain apoptosis-related factors, resulting in tumor cell growth inhibition and apoptosis.

Initial study on apoptosis in HepG-2 Human heptocarcinoma cell line by CSS

Objective To discuss on mechanism of the killing and apoptosis inducing effect induced by total alkaloid in the CSS(Capparis spinosa L.saponin,CSS)on human hepatocarcinoma cell Line HepG-2.Methods The killing effect of the CSS on human hepatocarcinoma cell Line HepG-2 was observed by MTT method.Morphological observation of the HepG-2 cells was completed by fluorescence microscope.This test was signed to observe the changes of the cell cycle of HepG-2 cells affected by the CSS by PI single-staining,and to observe if there were typical apoptosis peaks.The apoptosis inducing effect and changing of mitochondria membrane potential of the CSS on the HepG-2 cells were studied by flow cytometry.The effect of intracellular Ca2+ level of CSS on the HepG-2 cells was measured by laser confocal microscope.Results CSS has growth inhibiting on the HepG-2 and seems to be enhanced with the increasing concentration of CSS,and its IC50 value was 46.16 μg·mL-1.The HepG-2 cells are characteristic apoptosis morphologic changed,and the apoptosis percentage is increased to 66.652% in the 50 μg·mL-1 dosage group.The cells cycle has been changed obviously that the progresses of cells cycle of G1 period and G2 period in high dosage group have been blocked,and the cellular proportion in G2 period is decreased by the function of CSS for 24 h.The mitochondria membrane potential of HepG-2 cells induced by CSS is decreased in various degrees.In addition,the intracellular Ca2+ level is increased by the function of CSS in the middle and high dose groups.Conclusions The CSS has obviously killing and apoptosis inducing effect on human hepatocarcinoma cell Line HepG-2 by the mechanism of decreasing the mitochondria membrane potential and increasing the intracellular Ca2+ level.

Expression and interaction of CDC25A and IL-6 in hepatocellular carcinoma HepG2 cells

Objective:This study was to further investigate the effect of CDC25A gene on proliferation, invasion and metastasis of hepatocellular carcinoma HepG2 cells, and to explore whether CDC25A and IL6 interact in liver cancer.Methods: HepG2 cells were transfected with lentiviral CDC25A-shRNA to specifically block the expression of CDC25A in the experimental group (KD group), transfected with lentiviral-negative shRNA as a negative control group (NC group), and the conventionally cultured HepG2 cells were blank control group (Control group). Real-time quantitative PCR and Western blot were used to detect the mRNA and protein expression levels of CDC25A and IL6.Results:The expression level of IL6 mRNA in the experimental group was 0.4650.026, which was significantly lower than that of the negative control group of 1.0940.179 and the blank control group of 1.0020.311. The difference was statistically significant(P<0.05). The protein expression level of IL6 was 0.4990.061, which was significantly lower than the negative control group of 1.3110.034 and the blank control group of 1.2530.035. The difference was statistically significant (P<0.05). Conclusions: The CDC25A gene in hepatoma HepG2 cells was silenced, while the expression of IL6 was down-regulated. CDC25A may play a role in the development and invasion of liver cancer by regulating the expression of IL6.

Inhibitory effect of metformin on the proliferation of human hepatoma HepG2 cells and its potential mechanism

Objective： This work aimed to study the inhibitory effect and the related mechanism of metformin （MET） on the proliferation of human hepatoma HepG2 cells. Methods： Human hepatoma HepG2 cells were treated with MET （0, 2, 10, and 50 mM）. The inhibitory effect of MET on the proliferation of HepG2 cells was determined by MTT method. The apoptosis of HepG2 cells was detected by flow cytornetry. The expression of cyclin D1 in HepG2 cells was examined by Western blot. ROS-DHE fluorescence probe was used to stain the reactive oxygen species （ROS） generated by HepG2 cells after treat- ment. Results： MET could inhibit the proliferation of HepG2 cells in a dose and time dependent manner. MET promoted the apoptosis of HepG2 cells. In addition, MET suppressed the expression of cell cycle protein cyclin D1 and induced the produc- tion of ROS in HepG2 cells. Conclusion： MET can inhibit the proliferation of human hepatoma HepG2 cells and induce cell apoptosis. Meanwhile, MET has the ability to decrease the expression of cyclin D1 and induce ROS generation, which may be involved in the mechanism of inhibiting hepatoma cells proliferation.

Propofol inhibits the adhesion of hepatocellular carcinoma cells by upregulating microRNA-199a and downregulating MMP-9 expression

BACKGROUND: Propofol is one of the extensively and commonly used intravenous anesthetics and has the ability to influence the proliferation, motility, and invasiveness of many cancer cells. In this study, the effects of propofol on hepatocellular carcinoma cells invasion ability were examined. METHODS: We assessed the invasion ability of HepG2 cells in vitro by determining enzyme activity and protein expression of MMP-9 using gelatin zymography assay and Western blot. The real-time PCR was used to evaluate the effect of propofol on microRNA-199a (miR-199a) expression, and miR-199a-2 precursor to evaluate whether over-expression of miR-199a can affect MMP-9 expression. Finally, the effect of miR-199a on propofol-induced anti-tumor activity using anti-miR-199a was assessed. RESULTS: Propofol significantly elevated the expression of miR-199a and inhibited the invasiveness of HepG2 cells. Propofol also efficiently decreased enzyme activity and protein expression of MMP-9. Moreover, the over-expression of miR-199a decreased MMP-9 protein level. Interestingly, the neutralization of miR-199a by anti-miR-199a antibody reversed the effect of propofol on alleviation of tumor invasiveness and inhibition of MMP-9 activity in HepG2 cells. CONCLUSION: Propofol decreases hepatocellular carcinoma cell invasiveness, which is partly due to the down-regulation of MMP-9 expression by miR-199a.

Effects of SAHA on proliferation and apoptosis of hepatocellular carcinoma cells and hepatitis B virus replication

AIM: To investigate the effects of suberoylanilide hydroxamic acid(SAHA) on proliferation and apoptosis of a human hepatocellular carcinoma cell line(HepG2.2.15) and hepatitis B virus(HBV) replication.METHODS: HepG2.2.15 cells were treated with different concentrations of SAHA.Cell morphology was examined by confocal laser scanning microscopy,and cell proliferation was determined using a MTT colorimetric assay.Flow cytometry was used to detect apoptosis and determine cell cycle phase,while hepatitis B surface antigen and hepatitis B e antigen content were measured using chemiluminescence.Reverse transcription polymerase chain reaction was performed to measure HBV DNA in cell lysate.RESULTS: Cell proliferation rates were significantly reduced by the addition of SAHA.The inhibitory effect of SAHA on cell proliferation was both time-and dosedependent.After 24 h of treatment with SAHA,the early cell apoptotic rate increased from 3.25% to 21.02%(P = 0.041).The proportion of G0 /G1 phase cells increased from 50.3% to 65.3%(P = 0.039),while that of S phase cells decreased from 34.9% to 20.6%(P = 0.049).After 48 h of treatment,hepatitis B surface antigen and hepatitis B e antigen content increased from 12.33 ± 0.62 to 25.42 ± 2.67(P = 0.020) and 28.92 ± 1.24 to 50.48 ± 1.85(P = 0.026),respectively.Furthermore,HBV DNA content increased from 4.54 ± 0.46 to 8.34 ± 0.59(P = 0.029).CONCLUSION: SAHA inhibits HepG2.2.15 cell proliferation,promotes apoptosis,and stimulates HBV replication.In combination with anti-HBV drugs,SAHA may potentially be used cautiously for treatment of hepatocellular carcinoma.

Effect of 5-Aza-2'-deoxycytidine on the expression of p16 in hepatocellular carcinoma cells in vitro

Objective: To study the effect of 5-Aza-2’ -deoxycytidine (5-Aza-cdR) on tumour suppressor gene p16 expres- sion in hepatocellular carcinoma cells. Method: Expression of pl6 mRNA and protein in hepatocellular carcinoma cell lines SMMC-7721 and HePG2 before and after treatment with 5-Aza-cdR were analyzed via reverse transcriptase polymerase chain reaction(RT-PCR) and immunohistochemistrty Results: The expression levels of p16 mRNA and protein were increased dramatically after treatment with 5-Aza-cdR. Conclusion: Our data show that, 5-Aza-2’ -deoxycytidine can increase the expression of pl6 gene both at transcription and translation. The findings suggested that 5-Aza-cdR may reactivate the pl6 gene by demethylation.

Brucea javanica oil inhibits proliferation of hepatocellular carcinoma cells and induces apoptosis via the PI3K/AKT pathway

Background:Brucea javanica oil(BJO),distributed primarily in Southeast Asia,has long been utilized as a therapeutic agent for treating malignancies.However,its anticancer mechanisms are not clearly understood.The objective of this study was to examine the mechanisms underlying its treatment of hepatocellular carcinoma cells.Methods:CCK8 assay was used to evaluate cell viability.Hoechst33342 staining and flow cytometry analyses were used to examine apoptosis.Mito-Tracker Red CMXRos kit was used to measure the membrane potential of mitochondria.ATP assay kit was used to evaluate ATP levels.Western blots were used to assess the presence of AKT,adenosine monophosphate-activated protein kinase,Caspase3,Caspase9,Bax,and Bcl-2.Results:BJO inhibited the proliferation of hepatocellular carcinoma cells HepG2 in a time-and dose-dependent manner.It induced apoptosis,with the percentage of cells treated with 50–150μg/mL BJO increasing from 8.01%to 28.02%in a concentration-dependent manner(P<0.05,when 50μg/mL of BJO group compared with the control group;P<0.001,when 100 or 150μg/mL of BJO group compared with the control group).After exposed to BJO,the expression of C-caspase3,C-caspase9 and Bax upregulated while that of Bcl-2 downregulated.BJO suppressed the PI3K/AKT pathway and promoted phosphorylation of adenosine monophosphate-activated protein kinase,while repressing the phosphorylation of mechanistic target of rapamycin.Compared with treatment by BJO alone,the PI3K/AKT agonist 740Y-P increased the survival rate of HepG2 cells(P<0.01)and attenuated the inhibitory effect of BJO on cell apoptosis(P<0.05).Conclusion:BJO is capable of inhibiting proliferation of HepG2 cells and inducing apoptosis via the PI3K/AKT pathway.

Inhibitory effects of apogossypolone on subcutaneous implants of human LNCaP prostatic carcinoma cells

Objective:The aim of this study was to investigate the inhibitory effect of apogossypolone (ApoG2) on subcutaneous implants of human LNCaP prostatic carcinoma cells, and explore its mechanism. Methods:To establish human LNCaP prostatic carcinoma cell line subcutaneous xenograft models and observe the inhibitory effect of ApoG2 on the tumor model. Immunohistochemistry was employed to observe the expression of Bcl-2, PCNA, CD31, caspase-3 and-8 in tumor tissues. The microvessel density was calculated. Results:ApoG2 could obviously inhibit the growth of subcutaneous prostatic carcinoma implant. ApoG2 decreased the expression of PCNA and CD31, and increased the expression of caspases-3,-8 in tumor tissues. Conclusion:ApoG2 has an inhibitory effect on prostatic carcinoma implants.

Relation between the Expression of K-ras in Hep-2 Cells and Development of Laryngeal Carcinoma~*

Objective： To investigate the expression of K-ras in human laryngeal squamous cell carcinoma cell lines （Hep-2） and its significance for establishing a solid foundation for further study of the relationship between human laryngeal squamous cell carcinoma and K-ras gene point mutations. Methods： The expression of K-ras in human laryngeal squamous cell carcinoma cell lines （Hep-2） and human pancreatic carcinoma cell lines （MIAPaCa-2） was detected by using RT-PCR. Results： The expression of K-ras mRNA in Hep-2 and MIAPaCa-2 was strong and positive. Conclusion： The expression of K-ras mRNA in human laryngeal squamous cell carcinoma cell lines （Hep-2） is positive. Development of laryngeal carcinoma might be related to the activation of K-ras gene point mutation.

Oncolytic Virus Senecavirus A Inhibits Hepatocellular Carcinoma Proliferation and Growth by Inducing Cell Cycle Arrest and Apoptosis

Background and Aims:Hepatocellular carcinoma(HCC)isa highly aggressive tumor with limited treatment options andhigh mortality.Senecavirus A(SVA)has shown potential inselectively targeting tumors while sparing healthy tissues.This study aimed to investigate the effects of SVA on HCCcells in vitro and in vivo and to elucidate its mechanisms ofaction.Methods:The cell counting kit-8 assay and colonyformation assay were conducted to examine cell proliferation.Flow cytometry and nuclear staining were employed toanalyze cell cycle distribution and apoptosis occurrence.Asubcutaneous tumor xenograft HCC mouse model was createdin vivo using HepG2 cells,and Ki67 expression in thetumor tissues was assessed.The terminal deoxynucleotidyltransferase dUTP nick end labeling assay and hematoxylinand eosin staining were employed to evaluate HCC apoptosisand the toxicity of SVA on mouse organs.Results:In vitro,SVA effectively suppressed the growth of tumor cells by inducingapoptosis and cell cycle arrest.However,it did nothave a notable effect on normal hepatocytes(MIHA cells).In an in vivo setting,SVA effectively suppressed the growthof HCC in a mouse model.SVA treatment resulted in a significantdecrease in Ki67 expression and an increase in apoptosisof tumor cells.No notable histopathological alterationswere observed in the organs of mice during SVA administration.Conclusions:SVA inhibits the growth of HCC cells byinducing cell cycle arrest and apoptosis.It does not causeany noticeable toxicity to vital organs.

Effect of Nimesulide on proliferation and apoptosis of human hepatoma SMMC-7721 cells

AIM: Cyclooxygenase-2 (COX-2) has been suggested to be associated with carcinogenesis. We sought to investigate the effect of the selective COX-2 inhibitor, Nimesulide on proliferation and apoptosis of SMMC-7721 human hepatoma cells.METHODS: This study was carried out on the culture of hepatic carcinoma SMMC-7721 cell line. Various concentrations of Nimesulide (0, 200 micromol/L, 300 micromol/L, 400 micromol/L) were added and incubated. Cell proliferation was detected with MTT colorimetric assay, cell apoptosis by electron microscopy, flow cytometry and TUNEL.RESULTS: Nimesulide could significantly inhibit SMMC-7721 cells proliferation dose-dependent and in a dependent manner compared with that of the control group. The duration lowest inhibition rate produced by Nimesulide in SMMC-7721 cells was 19.06%, the highest inhibition rate was 58.49%. After incubation with Nimesulide for 72 h, the most highest apoptosis rate and apoptosis index of SMMC-7721 cells comparing with those of the control were 21.20%+/-1.62% vs 2.24%+/-0.26% and 21.23+/-1.78 vs 2.01+/-0.23 (P【0.05). CONCLUSION:The selective COX-2 inhibitor, Nimesulide can inhibit the proliferation of SMMC-7721 cells and increase apoptosis rate and apoptosis index of SMMC-7721 cells. The apoptosis rate and the apoptosis index are dose-dependent. Under electron microscope SMMC-7721 cells incubated with 300 micromol and 400 micromol Nimesulide show apoptotic characteristics. With the clarification of the mechanism of selective COX-2 inhibitors, These COX-2 selective inhibitors can become the choice of prevention and treatment of cancers.

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

BACKGROUND Hepatocellular 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.AIM To investigate the effect of miR-34 a expression on the growth inhibition of HepG2 cells by doxorubicin.METHODS A recombinant lentiviral vector containing miR-34 a was constructed and transfected into HepG2 cells. The expression of miR-34 a 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 phosphatebuffered 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.RESULTS Recombinant lentiviral vector LV-hsa-mir-34 a was successfully constructed by restriction endonuclease digestion and sequencing. RT-PCR showed that expression of miR-34 a 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-34 a, 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-34 a, and became more obvious after intervention with doxorubicin(P < 0.01). Western blotting showed that upregulation of miR-34 a 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).CONCLUSION MiR-34 a may enhance the inhibitory effect of doxorubicin by downregulating MDR1/P-gp and AXL, which may be related to p53 expression.