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.

Inhibitory effect of endostatin expressed by human liver carcinoma SMMC7721 on endothelial cell proliferation in vitro

AIM: To construct a stable transfectant of human liver carcinoma cell line SMMC7721 that could secret human endostatin and to explore the effect of human endostatin expressed by the transfectant on endothelial cell proliferation. METHODS: Recombinant retroviral plasmid pLncx-Endo containing the cDNA for human endostatin gene together with rat albumin signal peptide was engineered and transferred into SMMC7721 cell by lipofectamine. After selection with G418, endostatin-transfected SMMC7721 cells were chosen and expanded. Immunohistochemical staining and Western blot were used to detect the expression of human endostatin in transfected SMMC7721 cells and its medium. The conditioned medium of endostatin-transfected and control SMMC7721 cells were collected to cultivate with human umbilical vein endothelial cells for 72 hours. The inhibitory effect of endostatin, expressed by transfected SMMC7721 cells, on endothelial proliferation in vitro was observed by using MTT assay. RESULTS: A 550 bp specific fragment of endostatin gene was detected from the PCR product of endostatin-transfected SMMC7721 cells. Immunohistochemistry and Western blot analysis confirmed the expression and secretion of foreign human endostatin protein by endostatin-transfected SMMC7721 cells. In vitro endothelial proliferation assay showed that 72 hours after cultivation with human umbilical vein endothelial cells, the optical density (OD) in group using the medium from endostatin-transfected SMMC7721 cells was 0.51 +/- 0.06, lower than that from RPMI 1640 group (0.98 +/- 0.09) or that from control plasmid pLncx-transfected SMMC7721 cells (0.88 +/- 0.11). The inhibitory rate for medium from endostatin-transfected SMMC7721 cells was 48%, significantly higher than that from empty plasmid pLncx-transfected SMMC7721 cells (10.2%, P【0.01). CONCLUSION: Human endostatin can be stably expressed by SMMC7721 cell transferred with human endostatin gene and its product can significantly inhibit the proliferation of human umbilical vein endothelial cell in vitro.

Effects of Meloxicam on Vascular Endothelial Growth Factor and Angiopoietin-2 Expression in Colon Carcinoma Cell Line HT-29

To investigate the effect of meloxicam, a selected NSAIDs, on cell growth, expression of VEGF and angiopointin-2 （Ang-2） protein in HT-29 cell line, cultured HT-29 cells were treated with meloxicam of various concentrations for various lengths of time. The proliferation of HT-29 was detected by cell counting kit-8 （CCK-8）, the cell cycle was determined by flow cytometer and the levels of VEGF and Ang-2 protein in supernatants were examined by enzyme linked immunosorbent assay （ELISA）. The mRNA expressions of VEGF and Ang-2 in cultured HT-29 were determined by real-time quantitative reverse-transcription polymerase chain reaction. Our results showed that treatment of meloxicam of different concentrations and for various lengths of time had a cytotoxicic effect on the cell proliferation of HT-29 cells in a concentration-dependant and time-dependant manner. Cell cycle analysis showed that the cells were mainly blocked in G0/G1 phase. The VEGF and Ang-2 protein levels in supernatants of the culture medium were decreased gradually in a concentration-dependent or time-dependent fashion. The mRNA expression of cox-2, VEGF and Ang-2 showed a gradual and concentration-dependent reduction. It is concluded that meloxicam can reduce the expression of VEGF and Ang-2 at the protein and mRNA level in colon carcinoma cell line.

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.

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.

Establishment and characterization of four human hepatocellular carcinoma cell lines containing hepatitis B virus DNA

AIM To investigate the characteristics of newly established four hepatocellular carcinoma cell lines (SNU 739, SNU 761, SNU 878 and SNU 886) from Korean hepatocellular cancer patients. METHODS Morphologic and genetic studies were done. RESULTS All four lines grew as a monolayer with an adherent pattern, and their doubling times ranged from 20 to 29 hours. The viability rate was relatively high (88%-94%). Neither mycoplasmal nor bacterial contamination was present. The lines showed different patterns in fingerprinting analysis. The hepatitis B virus (HBV) DNA was integrated in the genomes of all four lines, and in all of them HBx, HBc and HBs transcripts were detected by reverse transcriptase PCR methods. Among the three cell lines used as control (Hep 3B, SK Hep1 and Hep G2), only Hep 3B showed HBx expression, and this line was used as a HBV integrated control. The RNA of albumin was detected in three lines (SNU 761, SNU 878 and SNU 886), that of transferrin in two lines (SNU 878, SNU 886), and that of IGF Ⅱ was detected in none of the cell lines. CONCLUSION These well characterized cell lines may be very useful for studying the biology of hepatocellular carcinoma in association with the hepatitis B virus.

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.

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.

Reversing effect of Tanshinone on malignant phenotypes of human hepatocarcinoma cell line

AIM To study the reversing effect of Chinese drug tanshinone on malignant phenotype of cancer cells.METHODS Human hepatocarcinoma cell line (SMMC-7721) was treated in vitro with 0.5mg/L tanshinone for 4 days, and variation in cell differentiation was detected.RESULTS The morphology of cancer cells was tended toward well differentiation and cell growth was markedly inhibited. BrdU uptake assay and immunohistochemical stain of PCNA showed that the BrdU labeling rate and PCNA positive rate were lower than the controls, but no difference was found statistically as compared with all transretinoic acid. Flow cytometric assay demonstrated that S phase cells decreased and G0/G1 phase cells increased. Expression of c-myc oncogene protein decreased but the c-fos oncogene protein markedly increased.CONCLUSION Tanshinone could reverse the inducing differentiation in human hepatocarcinoma cells (SMMC-7721). It may become a new prospective inducer of cell differentiation to treat cancers.

Pro-apoptotic effects of tectorigenin on human hepatocellular carcinoma HepG2 cells

AIM:To investigate the effects of tectorigenin on human hepatocellular carcinoma(HCC)HepG2 cells.METHODS:Tectorigenin,one of the main components of rhizome of Iris tectorum,was prepared by simple methods,such as extraction,filtration,concentration,precipitation and recrystallization.HepG2 cells were incubated with tectorigenin at different concentrations,and their viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Apoptosis was detected by morphological observation of nuclear change,agarose gel electrophoresis of DNA ladder,and flow cytometry with Hoechst 33342,Annexin V-EGFP and propidium iodide staining.Generation of reactive oxygen species was quantified using DCFH-DA.Intracellular Ca2+was monitored by Fura 2-AM.Mitochondrial membrane potential was monitored using Rhodamine 123.Release of cytochrome c from mitochondria to cytosol was detected by Western blotting.Activities of caspase-3,-8 and-9 were investigated by Caspase Activity Assay Kit.RESULTS:The viability of HepG2 cells treated by tectorigenin decreased in a concentration-and timedependent manner.The concentration that reduced the number of viable HepG2 cells by 50%(IC50)after 12,24 and 48 h of incubation was 35.72 mg/L,21.19 mg/L and 11.06 mg/L,respectively.However,treatment with tectorigenin at 20 mg/L resulted in a very slight cytotoxicity to L02 cells after incubation for 12,24 or 48 h.Tectorigenin at a concentration of 20 mg/L greatly inhibited the viability of HepG2 cells and induced the condensation of chromatin and fragmentation of nuclei.Tectorigenin induced apoptosis of HepG2 cells in a time-and dose-dependent manner.Compared with the viability rate,induction of apoptosis was the main mechanism of the anti-proliferation effect of tectorigenin in HepG2 cells.Furthermore,tectorigenininduced apoptosis of HepG2 cells was associated with the generation of reactive oxygen species,increased intracellular[Ca2+]i,loss of mitochondrial membrane potential,translocation of cytochrome c,and activation of caspase-9 and-3.CONCLUSION:Tectorigenin induces apoptosis of HepG2 cells mainly via mitochondrial-mediated pathway,and produces a slight cytotoxicity to L02 cells.

Autophagy in anti-apoptotic effect of augmenter of liver regeneration in HepG2 cells

AIM:To investigate the role of autophagy in the antiapoptotic effect of augmenter of liver regeneration(ALR).METHODS:Autophagy was induced through serum deprivation.An ALR-expressing plasmid was transfected into HepG2 cells,and autophagic flux was determined using fluorescence microscopy,electron microscopy,Western blot and quantitative polymerase chain reaction(q PCR) assays.After ALR-expressing plasmid transfection,an autophagy inhibitor [3-methyladenine(3-MA)] was added to HepG2 cells,and apoptosis was observed using fluorescence microscopy and flow cytometry.RESULTS:Autophagy was activated in HepG2 cells,peaking at 24 h after serum deprivation.Microtubuleassociated protein light chain three-II levels were higher in HepG2 cells treated with ALR than in control cells,fluorescence microscopy,electron microscopy and q PCR studies showed the similar trend,and p62 levels showed the opposite trend,which indicated that ALR may play an important role in increasing autophagy flux.The numbers of apoptotic cells were substantially higher in HepG2 cells treated with both ALR and 3-MA than in cells treated with ALR alone.Therefore,the protective effect of ALR was significantly attenuated or abolished when autophagy was inhibited,indicating that the anti-apoptotic effect of ALR may be related to autophagy.CONCLUSION:ALR protects cells from apoptosis partly through increased autophagy in HepG2 cells and may be valuable as a new therapeutic treatment for liver disease.

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.

Construction of cell lines with CD44 cDNA and its application in hepatocellular carcinoma

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Optimization of the microwave-assisted extraction of phlorotannins from Saccharina japonica Aresch and evaluation of the inhibitory effects of phlorotannin-containing extracts on HepG2 cancer cells

The use of a microwave-assisted extraction （MAE） method for the extraction ofphlorotannins from Saccharinajaponica Aresch （S.japonica） has been evaluated with particular emphasis on the influential parameters, including the ethanol concentration, solid/liquid ratio, extraction time, extraction temperature, and microwave power. The MAE procedure was optimized using single-factor design and orthogonal array design （OAD）. The content of total phlorotannins in S. japonica was determined using a Folin-Ciocalteu （FC） assay. A maximum total phlorotannin content of 0.644 mg of phloroglucinol equivalent per gram of dry weight plant （mg PGE/g DW） was obtained using the optimized model, which included an ethanol concentration of 55%, solid/liquid ratio of 1：8, extraction time of 25 min, irradiation power of 400 W, and temperature of 60~C. Under similar conditions, the application of a conventional extraction method led to a lower phlorotarmin yield of 0.585 mg PGE/g WD. These results demonstrated that the MAE approach provided better results for the extraction ofphlorotarmins from S.japonica and was a promising technique for the extraction of phenolic compounds from S. japonica and other materials. In addition, screening tests for the inhibitory activity showed that the phlorotannin-containing extracts significantly inhibited the growth of human hepatocellular carcinoma cells （HepG2） by inducing their apoptosis. The morphological changes that occurred during cell apoptosis were characterized using Hoechst33258 staining.

siRNA of ADAM17 gene induces apoptosis,proliferation inhibition and enhances the effects of genistein on HepG2 cells

Objective：To investigate the effects of siRNA of ADAM17 gene and genistein on apoptosis and the inhibition of proliferation in HepG2 cells in an attempt to seek an effective therapy for hepatocellular carinoma. Methods：Cells were divided into control groups and experimental groups and siRNA was used to silence the ADAM17 gene, alone and in combination with genistein. Cells were harvested at several time periods and assessed for proliferation and apoptosis. Proliferation was assayed by MTT at 24, 48, 72 and 96 hours following treatment and apoptosis was assessed by flow cytometric analysis at 48 hours. Results：In siRNA groups, proliferation of cells was significantly inhibited compared to the control groups at 24, 48 and 72 hours（P 〈 0.05）, and apoptosis was significantly increased at 48 hours（P〈 0.01）; In genistein groups, proliferation was inhibited at 24, 48, 72 and 96 hours, and the apoptosis ratio was significantly increased at 48 hours（P〈 0.01）; while in the groups that received the combination of siRNA transfection and genistein treatment, there was a further significant decrease of proliferation and increase in apoptosis compared with either treatment alone. Conclusion：The ADAM17 gene could be an effective target, and genistein could be a useful agent, in the treatment of hepatocellular carcinoma, siRNA of ADAM17 gene and genistein both inhibited HepG2 cells proliferation and promoted apoptosis, and further, the combination of these treatments had a greater effect than either treatment alone.

Non-islet cell tumor hypoglycemia as an initial presentation of hepatocellular carcinoma coupled with end-stage liver cirrhosis: A case report and review of literature

BACKGROUND Non-islet cell tumor hypoglycemia(NICTH)is a rare cause of persistent hypoglycemia seen in patients with hepatocellular carcinoma(HCC).It is likely to be underdiagnosed especially in the patients with poor hepatic function and malnutrition.Herein,we report a rare case of NICTH as the initial presentation of HCC in a patient with chronic hypoglycemia due to end-stage liver cirrhosis.CASE SUMMARY A 62-year-old male with chronic fasting hypoglycemia secondary to end-stage hepatitis C-related cirrhosis,presented with altered mental status and dizziness.He was found to have severe hypoglycemia refractory to glucose supplements.Imaging studies and biopsy discovered well differentiated HCC without metastasis.Further evaluation showed low insulin,C-peptide and betahydroxybutyrate along with a high insulin-like growth factor-2/insulin-like growth factor ratio,consistent with the diagnosis of NICTH.As patient was not a candidate for surgical resection or chemotherapy,he was started on prednisolone with some improvements in the glucose homeostasis,but soon decompensated after a superimposed hospital acquired pneumonia.CONCLUSION NICTH can occur as the sole initial presentation of HCC and is often difficult to correct without tumor removal.Clinicians should maintain high clinical suspicion for early recognition of paraneoplastic NICTH in patients at risk for HCC,even those with chronic fasting hypoglycemia in the setting of severe hepatic failure and malnutrition.

Review of the treatment of metastatic non small cell lung carcinoma:A practical approach

In recent years,as we have a better knowledge and understanding of the biology of non small cell lung carcinoma(NSCLC),which leads us to targeting biomarkers driving the NSCLC carcinogenesis and metastatic potential,we now have an increased number of options to offer our patients with NSCLC.We also realize the importance of distinguishing squamous and non squamous histology to guide our treatment decisions of NSCLC.The palliative care concomitant with therapies from the very start of the treatment also showed an impact on survival.This review examines the treatment options in all lines of therapy for metastatic NSCLC that have been approved in Canada,the United States,or Europe.

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.

Protective effect of brain and muscle arnt-like protein-1 against ethanol-induced ferroptosis by activating Nrf2 in mice liver and HepG2 cells

Alcohol abuse has recently become a serious health concern worldwide,and the incidence of alcoholic liver disease(ALD)is rapidly increasing with high morbidity and mortality.Ferroptosis is a newly recognized form of regulated cell death caused by the iron-dependent accumulation of lipid peroxidation.Here we showed that the circadian clock protein brain and muscle arnt-like protein-1(BMAL1)in hepatocytes is both necessary and sufficient to protect against ALD by mitigating ferroptosis.U pon exposure to alcohol(5%Lieber-DeCarli liquid alcohol diet for 10 days before binged alcohol with 5 g/kg body weight in vivo,300 mmol/L for 12 h in vitro,respectively),the content of iron,reactive oxygen species(ROS)and malondialdehyde(MDA)was boosted signifi cantly while glutathione(GSH)was decreased that mainly based on the downregulated protein expression of ferritin heavy chain(FTH),ferroportin(FPN),heme oxygenase1(HO-1)and anti-cystine/glutamate antiporter(SLC7A11),while these changes could be abolished by ferroptosis inhibitor Ferrostatin-1[Fer-1(5 mg/kg body weight for 10 days in vivo,10μmol/L for 2 h in vitro,respectively)].Further study indicated that the alcohol could activate the protein expression of BMAL1 which exerts a protective effect against ferroptosis through promoting nuclear factor erythroid 2-related factor 2(Nrf2)translocation into nuclear and subsequently stimulating its downstream proteins FTH,FPN,glutathione peroxidase 4 activity(GPX4),HO-1,SLC7A11,while knockdown of BMAL1 and Nrf2 by RNA interference further downregulated the expression of these protein and thus promoting ferroptosis in response to alcohol.Collectively,our results unveiled that the protective action of BMAL1 during alcohol challenge depends on its ability to activate Nrf2-ARE antiferroptosis pathway and targeting hepatic BMAL1 to dampen hepatic ferroptosis signaling may have therapeutic potential for ALD.

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.