Effects of tanshinone IIA on proliferation,apoptosis and expression of HIF-1α, VEGF and wild-type P53 in human hepatoma HepG2 cells under hypoxia

AIM:To investigate the effects of tanshinone IIA (Tan IIA) on proliferation, apoptosis and its molecular mechanism in human hepatoma HepG2 cells under hypoxic condition. METHODS:Hypoxia model was established by treatment with cobalt chloride (CoCl2). The cells were divided into normoxia control group, hypoxia control group and hypoxia combined at different concentrations of Tan IIA groups. After HepG2 cells were incubated with different concentrations of Tan IIA (0.5, 1.0, 2.0, 5.0 and 10.0 mg/L) for 24 h, 48 h and 72 h under hypoxic condition, the cell proliferation was determined by MTT assay. After Tan IIA was added to the media at different concentrations for 24 h and 48 h, the apoptotic cells were observed by Hoechst 33258 staining. The protein levels of hypoxia-inducible factor 1 alpha (HIF-1α), vascular endothelial growth factor (VEGF) and wild-type P53 were detected by Western blotting after cultured with different concentrations of Tan IIA for 48 h. RESULTS:Tan IIA inhibited the proliferation of HepG2 cells in a dose- and time-dependent manner. Tan IIA induced the typical morphology of apoptotic cells and increased the apoptotic rate in a dose- and time-dependent manner after treatment with 1.0 mg/L~5.0 mg/L for 24 h and 48 h under hypoxic condition. The protein levels of HIF-1α and VEGF were weakly expressed in HepG2 cells under normoxia but up-regulated after incubated under hypoxia for 48 h. The protein expression of HIF-1α and VEGF were decreased with the increase in the concentration of Tan IIA under hypoxia. The protein expression of wild-type P53 was increased with the increase in the concentrations of Tan IIA under hypoxia. CONCLUSION: Tan IIA significantly inhibits the proliferation and induces the apoptosis of human hepatoma HepG2 cells under hypoxia, which may be related to the down-regulation of HIF-1α and VEGF and up-regulation of wild-type P53.     

Ginsenoside Rh4 induces apoptosis of human hepatocellular carcinoma HepG2 cells

AIM: To investigate the apoptosis and molecular mechanism of human hepatocellular carcinoma HepG2 cells induced by ginsenoside Rh4. METHODS: Human hepatocellular carcinoma HepG2 cells were treated with ginsenoside Rh4 at doses of 10, 20 and 40 μmol/L, and the inhibitory effect of ginsenoside Rh4 on HepG2 cell viability was measured by MTT assay. The apoptotic rate of HepG2 cells was analyzed by flow cytometry. The morphological changes of the HepG2 cells were observed by Hoechst 33258 and TUNEL staining. The expression of apoptosis-related proteins Bax, Bcl-2, caspase-3 and caspase-9 was determined by Western blot. RESULTS: Ginsenoside Rh4 promoted apoptosis of HepG2 cells in a dose-dependent manner. TUNEL and Hoechst 33258 staining showed that the cells appeared obvious shrinking, swelling and rupture after treated with ginsenoside Rh4 for 24 h. The results of Western blot showed that with the increasing concentrations of ginsenoside Rh4, the expression of pro-apoptotic proteins Bax, cleaved caspase-3 and caspase-9 increased, while anti-apoptotic protein Bcl-2 decreased gradually. CONCLUSION: Ginsenoside Rh4 induces apoptosis of human hepatocellular carcinoma HepG2 cells, and the main mechanism may be related to down-regulation of Bcl-2 and up-regulation of Bax, cleaved caspase-3, and caspase-9.     

Effects of shRNA targeting SIAH2 gene on proliferation and apoptosis of human hepatoma cell line HepG2

AIM：To investigate the effects of shRNA-mediated seven in absentia homolog 2 (SIAH2; one of ubiquitin ligases) gene silencing on cell cycle and apoptosis of human hepatoma cell line HepG2. METHODS：The specific recombinant vector pGenesil-SIAH2 was transiently transfected into HepG2 cells with Lipofectamine 2000. RT-PCR and Western blotting were performed to detect the mRNA and protein expression levels of SIAH2. MTS assay was employed to evaluate the cell proliferation. Flow cytometry was used to determine the cell cycle and apoptosis of the transfected cells. RESULTS：Compared with control groups, the mRNA and protein levels of SIAH2 were reduced by pGenesil-SIAH2 transfection in HepG2-SIAH2 group. The proliferation of HepG2-SIAH2 cells was significantly inhibited. The percentage of G1-phase cells and the early apoptotic rate were significantly higher in HepG2-SIAH2 cells. CONCLUSION: Tansfection of pGenesil-SIAH2 effectively inhibits the proliferation of HepG2 cells, arrests the cells in G1 phase and induces apoptosis, indicating an experimental basis of SIAH2-targeting gene therapy for hepatocellular carcinoma.     

Oxidative stress induces apoptosis in HepG2 cells

AIM: Direct exposure of cells to reactive oxygen species can induce apoptosis. In this study we investigate how oxidative stress induces cell death in HepG2 cells and characterize the molecular events involved.METHODS: Oxidative stress was created by exposing HepG2 cells to 2 mmol/L H2O2. Apoptosis was determined by analysis of DNA fragmentation by agarose gel electorphoresis. The mitochondrial membrane potential was analyzed using DePsipher fluorescent staining and the expression of cytochrome c in the cytosolic fraction was measured by Western blotting analysis. The caspase activity was detected using fluorometric assay kit by a fluorescence microplate reader.RESULTS: When HepG2 cells were treated with 2 mmol/L H2O2, the cells displayed DNA fragmentation, a typical feature of apoptosis, after 12 h. The mitochondrial membrane potential appeared different in two group of cells. H2O2-treated cells appeared green fluorescence as early as 4 h, which represents de-energized mitochondria, the untreated cells appeared red fluorescence, a feature of mitochondria with intact membrane potential. In treated cells, the expression of cytochrome c increased and accumulated in cytosolic fraction with treatment time, caspase-3 activity increased by 6.7-fold (P<0.01) at 8 h and caspase-9 activity increased by 3.6-fold (P<0.01) at 12 h, respectively, however, the activity of caspase-8 remained unchanged.CONCLUSION: These findings suggest that oxidative stress can induce apoptotic cell death in HepG2 cells, and the mechanism is related to mitochondrial pathway, which activates caspase-9 and-3, but not caspase-8.     

Effects of artesunate on proliferation,apoptosis and chemotherapeutic drug sensitivity of human hepatocellular carcinoma HepG2 cells

AIM: To investigate the effects of artesunate on proliferation and apoptosis in human hepatocelluar carcinoma cell line HepG2 and to study the sensitizing effect of artesunate on HepG2 cells to chemotherapeutic drugs. METHODS: The proliferation of HepG2 cells was determined by the assay of cell counting kit-8 (CCK-8) and the colony formation test. The morphology of HepG2 cells with Hoechst 33258 staining was observed under fluorescent microscope. Annexin V/propidium iodide (PI) was used to analyze the apoptosis and the cell cycle. The sensitizing effects of artesunate on HepG2 cells to chemotherapeutic drugs were determined by CCK-8 assay. RESULTS: When treated with artesunate for 48 h, the proliferation of HepG2 cells was significantly inhibited in a dose-dependent manner. The IC₅₀ was 19.2 μmol/L. Compared with the control cells, the colony formation of HepG2 cells treated with artesunate for 7 days was significantly inhibited. The nuclear fragmentation, karyopyknosis, chromosomal condensation, cell shrinkage, and attachment loss in HepG2 cells treated with artesunate were observed. The cells in G₂ phase increased obviously, and the percentages of hypodiploid cells and early apoptotic rates were significantly higher in artesunate treatment groups than those in control group. The IC₅₀ of 5-FU, carboplatin and epirubicin combined with artesunate was 3.33, 2.02 and 1.71 times sensitized as compared with control group, respectively. CONCLUSION: Artesunate effectively inhibits proliferation and induces apoptosis of HepG2 cells. Aresunate also sensitizes HepG2 cells to chemotherapeutic drugs.     

Effect of low expression of miR-21 on apoptosis of HepG2 cells induced by matrine

AIM: To explore whether miR-21 low expression enhances the effect of matrine (MAT) on the apoptosis of hepatocellular carcinoma cells.METHODS: Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the expression of miR-21 in the HepG2 cells treated with different concentrations of MAT. The effect of miR-21 on MAT-induced HepG2 cell apoptosis was analyzed by flow cytometry. The mRNA and protein expression of Bcl-2 and Bax in the HepG2 cells treated with MAT was determined by RT-qPCR and Western blot.RESULTS: The expression of miR-21 increased with the increasing concentration of MAT. Low expression of miR-21 promoted MAT-induced apoptosis, and enhanced the expression of Bax at mRNA and protein levels (P<0.05), while inhibited the expression of Bcl-2 at mRNA and protein levels (P<0.05).CONCLUSION: Low expression of miR-21 enhances MAT-induced HepG2 cell apoptosis by inhibiting the expression of Bcl-2 and promoting Bax expression.     

Inmbition of nodosin on cell proliferation and expression of Bcl-2 and Bax in HepG2 cells

AIM:To investigate the effects of nodosin extracted from Chinese traditional medicine on the proliferation of HepG2 cells cultured in vitroand to detect the protein expression of Bcl-2 and Bax in HepG2 cells. METHODS:HepG2 cells were treated with different concentrations (1.25, 2.5, 5, 10 and 20 μmol/L) of nodosin for 24 h. The morphological changes of HepG2 cells were observed under inverted microscope. The inhibitory rates of HepG2 cell growth were detected by MTT assay. The apoptotic rates and the protein expression of Bcl-2 and Bax were analyzed by flow cytometry. RESULTS:Shrunken and suspended HepG2 cells increased with the increases in the concentrations of nodosin. The apoptotic rates and the expression of Bax increased with the increases in the doses of nodosin, while the expression of Bcl-2 decreased. CONCLUSION: Nodosin inhibits the growth of HepG2 cells in a dose-dependent manner. The inhibition of HepG2 cell growth is induced by decreasing Bcl-2 and increasing Bax, thus promoting cell apoptosis.     

Effect of GPNMB on proliferation,apoptosis and invasion of human hepatoma cells

AIM：To investigate the effect of glycoprotein nonmetastatic melanoma protein B (GPNMB) on the proliferation, apoptosis and invasion of human hepatoma HepG2 cells and its molecular mechanisms. METHODS：The GPNMB siRNA and GPNMB-overexpressing vector were constructed, and then transfected into HepG2 cells. MTT assay, flow cytometry and Transwell chamber were used to determine the effects of GPNMB down-regulation and up-regulation on the proliferation, apoptosis and invasive ability of HepG2 cells. RESULTS：The proliferation of HepG2 cells was obviously promoted by the up-regulation of GPNMB. No effect of GPNMB on the apoptosis of HepG2 cells was observed. The invasion of HepG2 cells was also significantly promoted by the up-regulation of GPNMB. When integrin β1 was silenced by siRNA, the promoting effect of GPNMB on the proliferation and invasive ability of HepG2 cells was significantly suppressed. CONCLUSION: GPNMB may promote the proliferation and invasion of HepG2 cells by the interaction with integrin β1, and may be used as a potential therapeutic target in liver cancer.     

Harmine induces apoptosis of HepG2 cells via JNK signaling pathway and enhances their chemosensitivity to 5-fluorouracil and cisplatin

AIM: To investigate the proliferation-inhibitory and apoptosis-inducing effects of harmine on human hepatocarcinoma HepG2 cells. METHODS: The proliferation of HepG2 cells was determined in the absence or presence of a JNK inhibitor SP600125 by Cell Counting Kit-8 (CCK-8) assay and colony formation test. The morphology of HepG2 cells was observed by Hoechst 33258 staining under fluorescence microscope. The cell apoptosis was analyzed by Annexin V-PI staining. The expression of apoptosis-regulated proteins,poly(ADP-ribose) polymerase (PARP),c-Jun N-terminal kinase (JNK) and p-JNK, was detected by Western blotting. The sensitizing effects of harmine on HepG2 cells to chemotherapeutic drugs 5-fluorouracil (5-FU) and cisplatin were determined by CCK-8 assay. RESULTS: Harmine inhibited the proliferation of HepG2 cells and induced apoptosis in a dose-dependent manner. After the JNK signaling pathway was blocked by SP600125, the cell apoptotic rate decreased significantly. Hoechst 33258 staining revealed that the nuclear fragmentation, chromosomal condensation, cell shrinkage and attachment loss appeared in the HepG2 cells treated with harmine. The percentage of the sub-G1 fraction was increased and the population of early apoptotic cell death was observed. Apoptosis of HepG2 cells with harmine treatment was associated with the activation of JNK. Combined with harmine, the IC50 values of 5-FU and cisplatin for the tumor cells were 1.47 and 5.78 times sensitized as compared with the correspon-ding single drug treatment groups, respectively. CONCLUSION: Harmine exhibits an anti-proliferative effect on HepG2 cells by inducing apoptosis. The JNK signaling pathway is involved in the apoptosis of HepG2 cells. Harmine enhances the chemosensitivity of the cells to 5-FU and cisplatin.     

Effects of TKTL1 silencing by siRNA on growth microenvironment of human hepatocellular carcinoma HepG2 cells

AIM:To explore the relationship between the expression of transketolase-like 1 protein(TKTL1) and the metastasis of hepatocellular carcinoma by investigating the change of some indicators in growth microenvironment including lactate production, reduced glutathione(GSH) level and ratio of NADPH/NADP⁺ in hepatocellular carcinoma cell line HepG2 after silencing of TKTL1 expression by siRNA. METHODS:Specific siRNA expression vector targeting TKTL1 gene was constructed and transfected into HepG2 cell line. The effect of TKTL1 silencing was evaluated by detecting the mRNA level and the activity of transketolase(TKTL1). The changes of lactate production, GSH level, the ratio of NADPH/NADP⁺ and glucose-6-phosphate dehydrogenase(G-6-PD) activity were observed in transfected HepG2 cells compared with the untransfected control cells. RESULTS:Compared with the untransfected control cells, the mRNA expression of TKTL1 and the TKT activity decreased significantly(P<0.01). Meanwhile, the lactate production, GSH level and ratio of NADPH/NADP⁺ also decreased significantly(P<0.01). However, no change of the G-6-PD activity was observed. CONCLUSION:Carcinoma cells switch the glucose metabolism by overexpression of TKTL1 to modify the lactate production and the levels of reactive oxygen species, thus changing the growth microenvironment in favor of tumor metastasis.     

Effect of insulin resistance on biological function of HepG2 cells and sensitivity to cisplatin

AIM: To investigate the effect of insulin resistance (IR) on the biological function of hepatocellular carcinoma (HCC) and sensitivity to cisplatin. METHODS: IR was induced in HepG2 cells via incubation with a high concentration of insulin. Afterwards, the effects of IR on adhesion, migration, invasion and sensitivity to cisplatin of the cells were detected.RESULTS: The results indicated that glucose consumption was reduced in the IR cells. The expression of the insulin receptor and glucose transporter 2 was down-regulated. Furthermore, HepG2/IR cells displayed markedly enhanced adhesion, migration, and invasion. These cells exhibited a lower sensitivity to cisplatin. On the contrary, HepG2/IR cells exhibited decreased adhesion and invasion after treatment with the insulin sensitizer pioglitazone hydrochloride.CONCLUSION: IR is closely related to drug resistance, adhesion, migration and invasion in HepG2 cells. These findings may help explain the clinical observation of the limited efficacy of chemotherapy on a background of IR.     

Effects of 2-deoxy-D-glucose and oxaliplatin on proliferation and apoptosis of human hepatoma cell line SMMC-7721

AIM: To investigate the effects of 2-deoxy-D-glucose (2-DG) or oxaliplatin (L-OHP) alone and the combination of both on the proliferation and apoptosis of hepatoma cell line SMMC-7721 in vitro. METHODS: Human hepatoma cell line SMMC-7721 was treated with 2-DG or L-OHP alone, or both. The inhibitory effect on the proliferation of SMMC-7721 cells was estimated by MTT method. The q value, which represents synergistic effect, was determined. Apoptotic rate and cell cycle were assayed by flow cytometry. The activity of caspase-3 was detected by a caspase-3 activity assay kit. RESULTS: 2-DG or L-OHP at different concentrations inhibited the growth of SMMC-7721 cells obviously and the inhibitory effect on SMMC-7721 cell growth strongly depended on the exposure time and dose. When the 2 drugs worked together, the inhibitory effect was improved (P<0.05). 2-DG induced cell apoptosis and arrested the cells at G₂/M phase. When combined with L-OHP,the 2 drugs induced more severe apoptosis and arrested the cells at S and G₂/M phase. Meanwhile, the activity of caspase-3 increased when the 2 drugs used together. CONCLUSION: 2-DG inhibits the growth of hepatoma cell line SMMC-7721. The combination of 2-DG and L-OHP improves the ability of L-OHP to attack the tumor cells. The mechanism might be related to increasing the activity of caspase-3.     

Effect of miR-181b on cell proliferation and expression of Bcl-2 and SP1 in HepG2 cells

AIM: To investigate the role of miR-181b in the expression of Bcl-2 and SP1 at mRNA and protein levels in the human hepatoma G2 cells (HepG2), and to explore the effect of miR-181b on the regulation of HepG2 cell proliferation. METHODS: The synthetic double-strand complementary DNA based on the sequence of miR-181b was inserted into the vector of miRNASelect^TM pEGP-miR. The microRNA high-expression plasmid was cloned, and the sequences were identified. The miR-181b plasmid was transfected into HepG2 cells with liposomes. The stable cell line was screened by puromycin. The mRNA and protein levels of Bcl-2 and SP1 were measured by RT-PCR and Western blotting, respectively. Methyl thiazolyl tetrazolium (MTT) method was used to analyze the proliferation of HepG2 cells. RESULTS: The Western blotting results showed that miR-181b inhibited the protein expression of Bcl-2 and SP1. The result of RT-PCR also indicated that the mRNA expression of Bcl-2 and SP1 was suppressed. Compared with the control, the growth rate of HepG2 with high expression of miR-181b was significantly decreased.CONCLUSION: miR-181b inhibits the proliferation of HepG2, which may be related to the down-regulation of Bcl-2 and SP1.     

Effect of Rip2 overexpression on apoptosis in human pancreatic cancer cell line Panc-1

AIM: To observe the effect of receptor-interacting protein 2 (Rip2) overexpression on human pancreatic cancer cell line Panc-1. METHODS: pEGFP-C2 and pEGFP-Rip2 plasmids were respectively transfected into the Panc-1 cells using JetPRIME reagent. The cells were divided into control group, pEGFP-C2 group and pEGFP-Rip2 group. The apoptosis in the cells was detected 48 h after transfection by flow cytometry. Rip2 level and the expression of apoptosis-related proteins, Bax, cytoplasmic cytochrome c (Cyt-c) and Bcl-2, were analyzed by Western blot. The activity of caspase-3 was measured by colorimetric method. RESULTS: Rip2 protein expression significantly increased in the cells transfected with control and pEGFP-C2 plasmids. The apoptotic rate in pEGFP-Rip2 group was higher than that in control group and pEGFP-C2 group, whereas no significant difference of apoptotic rate was observed between control group and pEGFP-C2 group. The protein expression of Bax and cytoplasmic Cyt-c was remarkably increased and the protein expression of Bcl-2 was obviously decreased in pEGFP-Rip2 group as compared with control group and pEGFP-C2 group. The activity of caspase-3 in pEGFP-Rip2 group was obviously increased as compared with control group and pEGFP-C2 group. CONCLUSION: Overexpression of Rip2 is able to induce apoptosis in the Panc-1 cells, and the mechanism may be related to the up-regulation of Bax and cytoplasmic Cyt-c protein expression, down-regulation of Bcl-2 protein expression and enhancement of caspase-3 activity, thus activating intrinsic apoptotic pathway.     

Effects of NS-398 on the proliferation and apoptosis of HepG2 cells

AIM: To evaluate the effects of NS-398, a cyclooxygenase-2(COX-2) inhibitor, on the proliferation and apoptosis in HepG2 cells. METHODS: The effects of NS-398 on the proliferation of HepG2 cells was evaluated by MTT. DNA fragmentation gel analysis was used to analyze the apoptotic cells; DNA ploidy and apoptotic cell percentage were examined by flow cytometry. Furthermore, the expression of COX-2 and Bcl-2 mRNA was identified by competitive RT-PCR. RESULTS: NS-398 inhibited cell proliferation and induced apoptosis in HepG2 in a concentration-dependent manner. DNA ploidy analysis showed that S phase cells were significantly decreased with NS-398 concentration increasing. The quiescent G₀/G₁ phase was accumulated with decreasing of Bcl-2 mRNA. Whereas NS-398 had no effect on the expression of COX-2 mRNA, no correlations were found between COX-2 mRNA and the HepG2 cell proliferation and apoptosis induced by NS-398 (r=0.056 and r=0.119, respectively). CONCLUSION: NS-398 significantly inhibits the proliferation and induces apoptosis in HepG2. Mechanisms may be involved in accumulation of quiescent G₀/G₁ phase and decrease in Bcl-2 mRNA expression, but independent to COX-2 mRNA expression.     

Effects of XPD on expression of Rb and MAD2 in human hepatoma HepG2 cells

AIM：To investigate the effects of xeroderma pigmentosum group D (XPD) protein on the growth of human hepatoma HepG2 cells and the expression of retinoblastoma (Rb) and mitotic arrest deficient 2 (MAD2) proteins. METHODS：The recombinant plasmid pEGFP-N2-XPD and empty plasmid pEGFP-N2 were transfected into HepG2 cells by LipofectamineTM 2000. The cells were divided into 4 groups including blank control group, liposome group, pEGFP-N2 group (N2 group) and pEGFP-N2-XPD group (XPD group). The expression of XPD, Rb and MAD2 at mRNA and protein levels was detected by RT-PCR and Western blotting. The cell growth was measured by MTT assay. The cell apoptosis and cell cycle were analyzed by flow cytometry. RESULTS：Overexpression of XPD up-regulated the expression of Rb, and down-regulated the expression of MAD2 at mRNA and protein levels. XPD inhibited the proliferation of HepG2 cells and exacerbated the apoptosis. XPD prevented the hepatoma cells from G1 stage to S stage. CONCLUSION：XPD suppresses the growth of hepatoma cells, up-regulates the expression of Rb, and down-regulates the expression of MAD2.     

Effect of human xeroderma pigmentosum D on expression of Mdm2 and Mdm4 in HepG2 cells

AIM：To investigate the effects of human xeroderma pigmentosum D (XPD) on the expression of murine double minute 2 (Mdm2) and murine double minute 4 (Mdm4) in human hepatoma cells. METHODS：Recombinant plasmid pEGFP-N2/XPD and vacant plasmid pEGFP-N2 were transfected into HepG2 cells using liposome, and the cells were divided into blank control group, pEGFP-N2 group and pEGFP-N2/XPD group. The cell growth was detected by MTT assay. The cell cycle and apoptotic rate were examined by flow cytometry. The mRNA and protein expression levels of XPD, Mdm2, Mdm4 and P53 were determined by RT-PCR and Western blotting. RESULTS：The results of MTT assay showed that the cell growth was inhibited by the transfection of pEGFP-N2/XPD. The results of flow cytometry showed that the transfection of pEGFP-N2/XPD increased the cell number in G 1 phase, decreased the cell number in S phase and increased the apoptotic rate of HepG2 cells. The results of RT-PCR and Western blotting showed that the transfection of pEGFP-N2/XPD increased the expression of XPD, decreased the expression of Mdm2 and Mdm4, and increased the expression of P53. CONCLUSION：XPD down-regulates Mdm2 and Mdm4 expression and up-regulates P53 expression in hepatoma cells. Moreover, the proliferation of hepatoma cells can be inhibited and the apoptosis can be induced by XPD.     

Effect of extracellular HSP70/HSP70-PCs on epithelial-mesenchymal transition of human hepatocellular carcinoma HepG2 cells

AIM：To investigate the effect of extracellular heat-shock protein 70 (HSP70)/HSP70-peptide complexes (HSP70-PCs) on epithelial-mesenchymal transition (EMT) of human hepatocellular carcinoma HepG2 cells and its probable mechanism. METHODS：HepG2 cells were divided into 3 groups: control group, HSP70/HSP70-PCs (2 mg/L) group and LY294002+HSP70/HSP70-PCs group. The mRNA and protein expression of epithelial cell surface marker E-cadherin, mesenchymal cell surface marker α-smooth muscle actin (α-SMA), phosphatidylinositol 3-kinase (PI3K) and hypoxia-inducible factor 1α (HIF-1α) was examined by real-time RT-PCR and Western blotting. RESULTS：Extracellular HSP70/HSP70-PCs promoted the initiation of EMT of HepG2 cells. The expression of HIF-1α and PI3K significantly increased in the process of EMT of HepG2 cells. After PI3K was blocked by LY294002, EMT did not occur and HIF-1α was not up-regulated in HepG2 cells. CONCLUSION：Extracellular HSP70/HSP70-PCs may promote EMT of hepatocellular carcinoma cells via PI3K/HIF-1α signaling pathway.     

Et-DHA induces apoptosis of HepG2 cells through activation of mitochondrial pathway and induction of granzyme B expression in T cells

AIM：To investigate the effect of ethyl docosahexaenoate (Et-DHA) on the apoptosis of human hepatocarcinoma HepG2 cells. METHODS：HepG2 cells were used to test the anticarcinogenicity of Et-DHA. The direct inhibition of HepG2 cells by Et-DHA was detected by MTT. Nuclear morphological features of the HepG2 cells were observed under fluorescence microscope after staining with Hochest 33258. The levels of Bax, Bak, Bid, Bcl-2, Smac and cytochrome C (Cyt C) in mitochondria and cytosol, the cleaved caspase-8, cleaved caspase-9, and cleaved caspase-3 in cytosol, as well as the release of reactive oxygen species (ROS), total superoxide dismutase (SOD) and caspase-9 activity in the Et-DHA-treated HepG2 cells were determined by Western blotting and ELISA. Furthermore, by co-culturing the HepG2 cells with T cells, the effects of proliferation of Et-DHA-treated T cells on the activity of HepG2 cells were observed, and the level of granzyme B was detected. RESULTS：Et-DHA significantly inhibited the growth of HepG2 cells in a concentration- and time-dependent manner. The ROS release and caspase-9 activity increased markedly in Et-DHA-treated HepG2 cells, and no significant change of the total SOD activity was observed. The levels of the pro-apoptotic proteins Bax, Bak and Bid in mitochondria increased, the anti-apoptotic protein Bcl-2 as well as mitochondrial Cyt C and Smac levels decreased, and the cytoplasmic Cyt C, Smac, cleaved caspase-8, cleaved caspase-9, cleaved caspase-3 and cleaved Bid levels showed dose-dependent increases. Additionally, the degree of Et-DHA-induced apoptosis in HepG2 cells in the co-culture group (T cells+HepG2 cells) showed a further increase as compared with the HepG2 cells treated with Et-DHA alone. Due to Et-DHA inducing elevation of granzyme B level in the T cells, the granzyme B released into HepG2 cells was significantly increased. CONCLUSION:Et-DHA might induce the apoptosis of HepG2 cells through activation of caspase-3 mainly via a mitochondrial intrinsic pathway and a caspase-8 pathway, and promote the increase in granzyme B indirectly by activating T cells, thus enhancing the cytotoxic effect on HepG2 cells.