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Aim To explore the signaling pathway of matrine derivative ZS10 in inhibiting proliferation and inducing apoptosis of BEL-7402 cells. Methods ZS10 was synthesized by organic synthesis. The inhibitory effect of ZS10 on the proliferation of BEL-7402 cells was analyzed by MTT method at the time of 24 h, 48 h and 72 h, respectively, and IC50 was calculated. DAPI staining was used to observe the state of BEL-7402 cells. Clone formation method was used to observe the colony formation of BEL-7402 cells, flow cytometry was used to observe the cell cycle arrest and apoptosis of BEL7402 cells, and Western blot was used to detect the expression level of PI3K/AKT pathway and related proteins. Results MTT results showed that the IC50 was(6.62±1.11)μmol·L-1; DAPI staining showed that the cell state changed significantly with the increase of drug concentration, and the results of colony formation showed that ZS10 significantly inhibited the colony formation of BEL-7402 cells. The results of flow cytometry showed that ZS10 induced S phase arrest and cycle apoptosis of BEL-7402 cells. Western blot showed that ZS10 at the concentration of 08 μ mol·L-1 could regulate the PI3K/AKT pathway and its related proteins in a dose-dependent manner. Compared with the control group, the expression of PI3K, AKT, P-AKT and anti-apoptotic protein Bcl-2 significantly decreased, the expression of pro-apoptotic protein Bax significantly increased, the expression of Cyclin D1 and CDK2 significantly decreased, and the expression of EGFR and N-cadherin, Vimentin significantly decreased in the treatment group. The expression of E-cadherin increased. Conclusions Matrine derivative ZS10 can inhibit the growth and proliferation of hepatocellular carcinoma cell line BEL-7402.
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@#[摘 要] 目的：分析环氧合酶2（cyclooxygenase-2，COX-2）的表达与肝癌患者临床病理特征及预后的关系，探讨刺芒柄花素（formononetin，FOR）在肝癌发生和发展中的作用及其机制。方法：收集2021年1~5月河北医科大学第四医院20例手术治疗肝癌患者的癌和相应的癌旁组织、60例手术治疗肝癌患者的临床资料，以及人肝癌细胞系HepG2和Bel-7402。用qPCR法和免疫组织化学染色法检测肝癌组织中COX-2的表达，分析其表达与患者临床病理特征和预后的关系。构建小鼠二乙基亚硝胺诱导肝癌模型，验证FOR对肝癌发病的影响。用0.003、0.006 μmol/ml的FOR分别处理肝癌细胞HepG2和Bel-7402 48 h后，用CCK-8法和流式细胞术检测FOR对肝癌细胞增殖和周期的影响，qPCR和WB法检测细胞中COX-2、CDK4、CDK6和cyclin D1表达的变化。结果：肝癌组织中COX-2 mRNA表达水平显著高于癌旁组织（P<0.01），COX-2蛋白表达阳性率为68.3%（41/60）；COX-2表达阳性与患者TNM分期晚、肿瘤大、生存期短有关（均P<0.05）。诱导小鼠肝癌模型中，FOR处理组小鼠肝癌的发生率显著降低、肝癌组织中COX-2表达显著降低（均P<0.05）。FOR处理可显著抑制HepG2和Bel-7402细胞的增殖能力，增加G0/G1期细胞比例、降低S和G2期细胞比例（均P<0.05），抑制细胞中COX-2和cyclin D1的表达（均P<0.01）。结论：COX-2表达阳性肝癌患者临床分期较晚且预后较差，FOR可通过抑制COX-2和cyclin D1表达来降低肝癌的发生和发展。
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Objective: To investigate the effects and the underlying mechanisms of evodiamine (EVO) on apoptosis of hepatocellular carcinoma (HCC) BEL-7402 cells. Methods: Cell counting kit-8 (CCK-8) assay was used to detect the effect of EVO on proliferation activity of BEL-7402 cells. Hoechst 33258 staining was used for observing morphological changes of apoptosis. The cell apoptosis and cycle distribution were analyzed by flow cytometry. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting assay were used to detect the expression levels of key genes from Hippo-YAP pathway in HL-7702 and BEL-7402 cells, including mammalian STE20-like protein kinase 1/2 (MST1/2), large tumor suppressor 1 (LATS1), and Yes-associated protein (YAP), then to examine the effect of EVO on the expression levels of these genes in BEL-7402 cells. The effect of EVO on the expression of YAP in hepatocellular carcinoma BEL-7402 cells was observed by immunofluorescence assay. Results: The proliferation of BEL-7402 cells were significantly inhibited by EVO in a dose- and time-dependent manners. Hoechst 33258 staining showed that EVO induced BEL-7402 cell typical apoptotic morphology, such as nuclear chromatin concentration and edge accumulation. Besides, flow cytometry tests showed that BEL-7402 cell apotosis were increased and cell cycle arrested in G2/M phase after being treated with EVO (P < 0.01). qRT-PCR and Western blotting showed that the expression level of MST2 and LATS1 were lower in BEL-7402 cell (P < 0.05), while the transcription and protein levels of YAP were significantly higher (P < 0.05). EVO could activate Hippo signal pathway, upregulate the expression of MST2 and LATS1 and then inhibit the expression of YAP in BEL-7402 cell (P < 0.05). Immunofluorescence assay also validated that EVO would significantly inhibit the overexpression of YAP in BEL-7402 cell (P < 0.01). Conclusion: EVO can induce the apoptosis of BEL-7402 cells, which may be through activating Hippo signal pathway and then down-regulate the expression of YAP.
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Aim To investigate the effect of miR-320a up-regulation on the apoptosis and migration of Bel7402 cells induced by ribonucleic acid Ⅱ.Methods The different expression levels of miR-320a in normal liver cells and hepatocellular carcinoma (HCC) cells were detected by qRT-PCR.Bel-7402 cell was transfected with miR-320a mimic,and the miR-320a expression levels were measured by qRT-PCR.The effect of ribonucleic acid Ⅱ on proliferation of Bel-7402 and Bel-7402-miR-320a cells was measured by CCK-8 assay,and cell cycle and apoptosis were detected by flow cytometry.The migration and invasion ability of ribonucleic acid Ⅱ on Bel-7402 cells were tested by Transwell method.The expression of p53,Cyclin D1,Bax,Bcl-2,MMP-3 proteins were examined by Western blot.Results miR-320a expression levels in HCC cell line Bel-7402 were significantly lower than those in normal cell line HL-7702.Bel-7402 cells were successfully transfected with miR-320a mimic,named Bel-7402-miR-320a.CCK-8 showed that ribonucleic acid Ⅱ could effectively inhibit the proliferation of Bel7402 and Bel-7402-miR-320a cells in vitro in a dosedependent manner at the range of 100,200,300,400,500 mg · L-1.The IC50 of ribonucleic acid Ⅱexposure on Bel-7402 and Bel-7402-miR-320a cells for 12 h and 24 h was 250,200 mg · L-t and 150,120 mg · L-1,respectively;flow cytometric analysis indicated that over-expression of miR-320a could arrest Bel-7402 and Bel-7402-miR-320a cells induced by ribonucleic acid Ⅱ in G0/G1 phase,and promote the apoptosis of HCC cells.Transwell method showed that Bel-7402-miR-320a + Ribonucleic acid Ⅱ group could significantly inhibit the migration of HCC cells compared with control group and Bel-7402 + Ribonucleic acid Ⅱ group.Western blot results showed that the expression of p53,Bax proteins increased,while the Cyclin D1,Bcl-2,MMP-3 proteins were down-regulated in Bel-7402 and Bel-7402-miR-320a cells induced by ribonucleic acid Ⅱ.Conclusions The expression of miR-320a is lower in HCC cells than that in normal cell line.While ribonucleic acid Ⅱ could promote the apoptosis of liver cancer cells by arresting the cell cycle protein expression of Cyclin D1,activating p53 signaling pathway,down-regulating Bcl-2,up-regulating Bax and destroying Bcl-2/Bax proportions,and inhibiting the migration and invasion of HCC cells by downregulating MMP-3.Overexpression of miR-320a could increase the sensitivity and boost the pharmacological effects of ribonucleic acid Ⅱ on HCC cells.
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Objective:To investigate effects of miR-503 on cisplatin sensitivity in BEL-7402 cells by targeting of bcl-2.Methods:MiR-503 and bcl-2 mRNA expression levels in hepatocellular carcinoma cells were measured by real-time quantitative (qRT)-PCR;Bcl-protein level was detected by Western blot;miR-503 mimics were transiently transfected to the BEL-7402 cells by liposome transfection;potential target genes of miR-503 were predicted by Bioinformatics software;miR-503 potential targets were validated by dual luciferase activity;and the cell viability was measured by MTT assay.Results:MiR-503 level was down-regulated and Bcl-2 protein expression level was up-regulated in BEL-7402 cells compared with HL-7702 cells.MiR-503 could interact with bcl-2 and inhibit its expression.Cell vitality with miR-503 transfection was significantly reduced compared to that in the negative control.Conclusion:MiR-503 may enhance the sensitivity of BEL-7402 cells to cisplatin and inhibit the cell proliferation by targeting bcl-2.
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OBJECTIVE:To study the targeting of folic acid(FA)-modified docetaxel(DOC)nano-liposome(L-DOC-FA)to hepatocellular carcinoma Bel-7402 cells in vivo and in vitro. METHODS:The cell viability and survival rate of Bel-7402 cells was tested by CCK-8 kit after treated with 0,1,2,5,10 and 20 μg/ml DOC,L-DOC and L-DOC-FA for 24 h. And then,the fluores-cein isothiocyanate was used to label L-DOC and L-DOC-FA nano-liposome,and the rate of L-DOC and L-DOC-FA absorbed by hepatocellular carcinoma Bel-7402 cells were detected. 125I was used to label L-DOC and L-DOC-FA nano-liposome,and then the contents of them in the subcutaneous tumor tissues were detected. 28 Balb/c naked mice were selected and given liver cell suspen-sion via back ih to induce tumor model. After modeling,naked mice were divided into blank control group(normal saline),DOC group(3 mg/kg),L-DOC(3 mg/kg,by DOC)and L-DOC-FA(3 mg/kg,by DOC). They were given relevant medicine intrave-nously once a day for consecutive 30 d. The relative tumor volume in naked mice was detected. RESULTS:DOC,L-DOC and L-DOC-FA all inhibited the cell viability of Bel-7402 cells,the survival rate of cells decreased in concentration-dependant manner;compared with DOC and L-DOC,the cell viability decreased after treated with L-DOC-FA,the survival rate of cells decreased (PL-DOC (31.2%),with statistical significance (P<0.01). The content of L-DOC-FA in tumor was significantly more than that of L-DOC (P<0.01). In addition,3 mg/kg L-DOC-FA showed better inhibitory effect than 3 mg/kg L-DOC and DOC on tumor,and the rela-tive tumor volume was smaller(P<0.01). CONCLUSIONS:L-DOC-FA has obvious targeting to Bel-7402 cells in vivo and in vi-tro,and shows good inhibitory effect on tumor in vivo and in vitro.
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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 investigate the effects of Pulsatilla saponin D and sorafenib on the metastasis of human hepa?toma cell line. Methods The human hepatoma cell line BEL-7402 cells were divided into Pulsatilla saponin D group (con?centration of 11.9 mg/L), sorafenib group (concentration of 2.15μmol/L), the combined group (Pulsatilla saponin D 11.9 mg/L+Sorafenib 2.15μmol/L) and the control group (ordinary broth). The inhibition effects of Pulsatilla saponin D and sorafenib monotherapy and combination therapy on BEL-7402 cell migration were detected by MTT assay, Transwell chamber experi?ment and cell scratch experiment. Western blot assay was used to detect the expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 gene protein. Results MTT assay showed that Pulsatilla saponin D (11.9 mg/L), sorafenib (2.15μmol/L) monotherapy and combination therapy had inhibitory effects on BEL-7402 cell proliferation, and the 24-h inhibi?tion rate was<15%. Results of Transwell chamber experiment and cell scratch test showed that the migration inhibitory rate was significantly higher in combination group than that of monotherapy group (P<0.01). The combined effect of madicine was the addition (0.85≤Q≤1.15). Western blot detection showed that there was a higher effect of down-regulation on MMP-2 and MMP-9 in combined group than that of monotherapy group. Conclusion Pulsatilla saponin D and sorafenib synergis?tically inhibit the metastasis of BEL-7402 cells. The joint effects are superior to monotherapy.
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OBJECTIVE To evaluate the effect of single traditional Chinese medicine(TCM) herb extracts on hepatoma and normal fibroblast cells using high-throughput screening in order to obtain extracts with specific anti-hepatoma effect. METHODS 242 commonly used TCM herbs were extracted by petroleum ether,ethanol and water,respectively. The total number of TCM extracts was 554. The cyto?toxicity of samples was evaluated by MTT in human hepatoma cells Bel7402 and mice normal fibroblasts NIH3T3. RESULTS 7.4%of the total extracts had an inhibitory effect greater than 50%for Bel7402,but 14.8% for fibroblasts NIH3T3 cells. Extracts with an inhibitory effect above 50% on both Bel7402 and NIH3T3 cells accounted for 4.4%of the total extracts. Our results showed that the sample DF173 had preferable cytotoxicity effect on hepatoma carcinoma cells in a good dose-effect relationship. DF173 is an ethanol extract from Stephania tetrandra,which is a commonly used herb in TCM. The cytotoxic IC50 of DF173 against Bel7402 was 8.27 mg·L-1,and 19.48 mg·L-1 on NIH3T3. CONCLUSION The components of TCM herbs are highly complicated. The combination of tumor cells with normal fibroblast cells to evaluate the cytotoxicity effect during anti-tumor drug screening will contribute much to the discovery of TCM drugs with high anti-tumor efficiency and lower toxicity.
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OBJECTIVE: To investigate the apoptosis of the auction and mechanism of the hepatoma BEL-7402 cells induced by the ginseng polysaccharides (GPS). METHODS: The hematoma cells BEL-7402 were incubated with GPS, cell viability was measured by CCK8, cell cycle distribution was assessed by fluorescence-activated cell sorting (FACS), the cell morphological changes were traced with TUNEL and scanning electron microscope, TNFRl, BCL-2 and Bax protein expression and ERK phosphorylation are tested by Western blot. RESULTS: CCK8 results showed that GPS inhibited cells growth of BEL-7402 in dose-dependent and time-dependent. Flow cytometry found that S phase arrest was increased upon GPS concentrations. Obviously apoptotic sub-g peak was also found. And the peak was gradually enhanced with the concentration increased. TUNEL staining and SEM results showed that GPS led to significant cell morphology changes in hepatoma cells BEL-7402. And the apoptosis effect was increased upon the GPS concentrations. Western blot showed that level of apoptosis related protein Bax was increased, the expression of apoptosis-antagonizing protein Bcl-2 was decreased and the expression of death receptor TNFRl appeared with GPS concentrations increased gradually, raise ERK phosphorylation. CONCLUSION: GPS can induce apoptosis of hepatoma cells BEL-7402 by the mitochondrial pathway and the death receptor dependent pathway and ERK pathway.
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OBJECTIVE@#To investigate the antitumor activity of the compound HS-4 and the action mechanism.@*METHODS@#MTT method was used to test in vitro antitumor activity of the compound HS-4. Orthotopic xenotransplantation tumor model of liver cancer was established in nude mice, and, in vivo antitumor activity of compound HS-4 was tested with a small animal in-vivo imaging system. Sequencing of small RNA library and RNA library was performed in HS-4 treated tumor cell group and control group to investigate the anti-cancer mechanism of HS-4 at level of functional genomics, using high-throughput sequencing technology.@*RESULTS@#HS-4 was found to have relatively high in-vitro antitumor activity against liver cancer cells, gastric cancer cells, renal cancer cells, lung cancer cells, breast cancer cells and colon cancer cells. The IC50 values against SMMC-7721 and Bel-7402 of liver cancer cells were 0.14 and 0.13 nmol/L respectively, while the IC50 values against MGC-803 and SGC-7901 of gastric cancer cells were 0.19 and 0.21 nmol/L, respectively. It was demonstrated that HS-4 possessed a better therapeutic effect in liver cancer.@*CONCLUSIONS@#A new reliable orthotopic xenotransplantation tumor model of liver cancer in nude mice is established. The new compounds HS-4 was found to possess relatively high in vivo and in vitro antitumor activity against liver cancer cells.
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Objective:To investigate the antitumor activity of the compound HS-4 and the action mechanism.Methods:MTT method was used to testin vitroantitumor activity of the compound HS-4. Orthotopic xenotransplantation tumor model of liver cancer was established in nude mice, and,in vivoantitumor activity of compound HS-4 was tested with a small animal in-vivo imaging system. Sequencing of small RNA library and RNA library was performed in HS-4 treated tumor cell group and control group to investigate the anti-cancer mechanism of HS-4 at level of functional genomics, using high-throughput sequencing technology. Results:HS-4 was found to have relatively highin-vitro antitumor activity against liver cancer cells, gastric cancer cells, renal cancer cells, lung cancer cells, breast cancer cells and colon cancer cells. The IC50 values against SMMC-7721 and Bel-7402 of liver cancer cells were 0.14 and 0.13 nmol/L respectively, while the IC50 values against MGC-803 and SGC-7901 of gastric cancer cells were 0.19 and 0.21 nmol/L, respectively. It was demonstrated that HS- 4 possessed a better therapeutic effect in liver cancer.Conclusions: A new reliable orthotopic xenotransplantation tumor model of liver cancer in nude mice is established. The new compounds HS-4 was found to possess relatively highin vivo andin vitroantitumor activity against liver cancer cells.
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Objective: To investigate the antitumor activity of the compound HS-4 and the action mechanism. Methods: MTT method was used to test in vitro antitumor activity of the compound HS-4. Orthotopic xenotransplantation tumor model of liver cancer was established in nude mice, and, in vivo antitumor activity of compound HS-4 was tested with a small animal in-vivo imaging system. Sequencing of small RNA library and RNA library was performed in HS-4 treated tumor cell group and control group to investigate the anti-cancer mechanism of HS-4 at level of functional genomics, using high-throughput sequencing technology. Results: HS-4 was found to have relatively high in-vitro antitumor activity against liver cancer cells, gastric cancer cells, renal cancer cells, lung cancer cells, breast cancer cells and colon cancer cells. The IC50 values against SMMC-7721 and Bel-7402 of liver cancer cells were 0.14 and 0.13 nmol/L respectively, while the IC50 values against MGC-803 and SGC-7901 of gastric cancer cells were 0.19 and 0.21 nmol/L, respectively. It was demonstrated that HS-4 possessed a better therapeutic effect in liver cancer. Conclusions: A new reliable orthotopic xenotransplantation tumor model of liver cancer in nude mice is established. The new compounds HS-4 was found to possess relatively high in vivo and in vitro antitumor activity against liver cancer cells.
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Background and purpose:Recently, studies showed that non-steroidal anti-inlfammatory drugs (NSAIDs) could reduce the incidence of cancer. Whether ibuprofen could inhibit the growth of hepatocellular carcinoma cells had not been reported yet. In the current study, we investigated the effects of ibuprofen on hepatoma carcinoma BEL-7402 cells and the relevant mechanisms. Methods: Hepatocellular carcinoma BEL-7402 cells were randomly divided into 7 groups:the control group and the ibuprofen groups (0.1, 0.5, 1.0, 2.0, 3.0 and 4.0 mmol/L). The effect of ibu-profen on BEL-7402 HCC cells was measured by MTT method, the cell cycles were analyzed by flow cytometry (FCM), cell vitality and apoptosis were determined by cell analyzer. PCNA, Cyclin D1, Bcl-2 and COX-2 protein levels were examined by Western blot, and the expressions of prostaglandin E2 (PGE2) were measured by ELISA. Results:After the exposure to ibuprofen, the suppression ratio of BEL-7402 cells was increased (P<0.05). BEL-7402 cell vitality was decreased by degrees significantly (P<0.05), early apoptosis of BEL-7402 cells was increased (P<0.05), and the G0/Gl phase ratio was increased significantly compared with control group (P<0.05). Ibuprofen effectively decreased PCNA, Cyclin D1, Bcl-2 and COX-2 expressions in BEL-7402 cells (P<0.05), and decreased PGE2 protein expression in cell culture supernatants sig-nificantly (P<0.05). Conclusion:Ibuprofen is effective for inhibiting the proliferations, increasing apoptosis and blocking cell cycles of BEL-7402 HCC cells. The anti-tumor mechanisms of ibuprofen may be related with the inhibition of COX-2 and PGE2 expressions.
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Objective: To investigate the regulatory effect of C-Jun N-terminal kinase (JNK) signal transduction pathway on multi-resistance in the human hepatic cancer Bel-7402/5-fluorouracil (FU) cells, and provide a possible novel target for study on the mechanism of multidrug-resistance in human hepatic cancer cells. Methods: The protein expression levels of JNK and phospho-JNK (p-JNK) in parental human hepatic cancer Bel-7402 cells and the drug-resistant Bel-7402/FU cells were detected by Western blotting. After the inhibition of JNK pathway induced by specific inhibitior SP600125, the expressions of multi-drug resistance 1 (MDR1) mRNA and P-glycoprotein (P-gp) in Bel-7402/FU cells were detected by RT-PCR and immunocytochemistry, respectively. The accumulation and efflux of rhodanmine 123 (Rh123) in the cells were examined by flow cytometry, and the sensitivity to 5-FU of Bel-7402/FU cells was detected by MTT method. Results: The expression of JNK protein was not significantly different between the Bel-7402/FU cells and the parental Bel-7402 cells, but the expression of p-JNK protein in the Bel-7402/FU cells was significantly increased. After inhibition of JNK pathway, the expressions of MDR1 mRNA and P-gp protein were obviously decreased, with a markedly increased accumulation and decreased efflux of Rh123, leading to enhhanced sensitivity to 5-FU of Bel-7402/FU cells. Conclusion: JNK signal transduction pathway is involved in the expressions of MDR1 gene and P-gp in drug-resistant human hepatic cancer Bel-7402/FU cells, and it can regulate the sensitivity to 5-FU of Bel-7402/FU cells.
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Objective: To investigate the antirumour activity of saponins from Pulsatilla chinensis against the Bel-7402 human hepatocellular carcinoma xenograft in nude mice, and to explore the mechanism of energy metabolism. Methods: The antitumor activity in vivo was estimated using a Bel-7402 xenograft model. The tumor volume was measured, the tumor inhibitory rate was calculated, the levels of lactic acid and ATP were assessed in Bel-7402 xenograft in nude mice, energy metabolic enzymes in Bel-7402 xenograft in nude mice were detected by ELISA assay, and hypoxia inducible factor-1 (HIF-1α) was detected by Western blotting assay. Results: The sapoinins from P. chinensis could inhibit the growth of Bel-7402 xenograft in a dose-depedent manner. The tumor inhibitory rates at the dose of 400, 200, and 100 mg/kg were 42.8%, 31.5%, and 14.9%. They had no side effects on white blood cell (WBC) in blood; The saponins from P. chinensis could decrease the contents of lactic acid and ATP. They also could decrease the levels of glycolytic enzymes phosphofructokinase (PFK), hexokinase-II (HK-II), and pyruvate kinase (PK), while increase the level of succinatedehydrogenase (SDH) that was the key enzyme during tricarboxylic acid cycle. Western blotting indicated that the expression of HIF-1α protein was significantly decreased in Bel-7402 xenograft in nude mice. Conclusion: The saponins from P. chinensis could inhibit the growth of Bel-7402 xenograft in nude mice in vivo, the mechanism is related with regulating energy metabolism through the HIF-1α pathway.
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Objective To explore the inhibition effect and the possible mechanism of resveratrol on human hepatocellular carcinoma cell line Bel-7402 both in vitro and vivo.Methods Four Res drugs in the experiment group,the final concentrations were 12.5,25,50,100μmol/L,the control group at the same time set not containing Res drugs,MTT assay was used to measure the inhibition of resveratrol on Bel-7402.The expression of Bcl-2 was detected by RT-PCR and Western Blot.The levels of IL-2,IL-6,IL-12 and TNF-αwere detected by ELISA.Results Resveratrol inhibited Bel-7402 cell proliferation in dose and time manner,and influenced the expression of Bcl-2 mRNA and protein.At the same time,resveratrol inhibited the growth of tumor and improved the levels of IL-2,IL-6,IL-12 and TNF-α.Conclusion Resveratrol could inhibit Bel-7402 cell proliferation both in vitro and vivo, the possible mechanism may be that resveratrol could low down the expression of Bcl-2 and improve the levels of IL-2,IL-6,IL-12 and TNF-α.
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OBJECTIVE To validate the anticancer effect of Angong Niuhuang pill (AGNH) and pinpoint associated molecular mechanisms using human cancer cells.METHODS Human MGC-803 gastric carcinoma and human BEL-7402 hepatocarcinoma cells were incubated with AGNH 9,30,90,300 and 900 mg·L-1 for 24,48and 72 h,respectively.Cell viability was detected with 3-(4,5-dimethylthiazol-2-yl) -5-( 3-carboxymethoxyphe-nyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and colony formation assay.Apoptosis was measured with flow cytometry and Hoechst 33258/PI staining.Change in mitochondrial membrane potential (△qψ) was detected by spectrofluorophotometer.RESULTS AGNH inhibited MGC-803 cell proliferation ( for 48 h,r =0.996,P =0.002; for72 h,r=0.756,P=0.024 ) and BEL-7402 cells (for 48 h,r =0.732,P=0.030; for72 h,r=0.702,P =0.037) in a concentration-dependent manner,as showed by MTS assay.AGNH inhibited colony formation on MGC-803 cells (r =0.914,P =0.011 ) and BEL-7402 cells (r =0.871,P =0.024) in a concentration-dependent manner for 24 h.Hoechst 33258/PI staining and flow cytometry assay showed that AGNH 900 mg·L-1 for 24 h induced apoptosis of MGC-803 and BEL-7402 cells,and the apoptosis rate was 27.2％ and 19.7％,respectively.Compared with normal control group,AGNH 900 mg·L-1 for 3 min decreased the mitochondrial membrane potential of MGC-803 and BEL-7402 cells to 15.9％ and 15.0％ of control group.CONCLUSION AGNH inhibits proliferation of human cancer cells.Apoptosis and depolarization of mitochondrial transmembrane potential are probablly its mechanism.
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Objective To investigate the mechanism of extremely low frequency electromagnetic field (ELF-EMF) and X-ray irradiation on liver carcinoma cell lines BEL-7402. Methods Liver carcinoma cell lines BEL-7402 had been incubated with ELF-EMF (100 Hz, 0.7 mT, 30 min, 3 days) after X-ray irradiation at different doses (0, 2, 4, 6, 8,10 Gy). The cells were observed on morphologic changes with scanning electron microscope. Flow cytometry and gene microarray were used to investigate the mechanism of cell apeptosis. Results ELF-EMF plus X-ray induced apoptosis on BEL-7402 was observed under scanning electron microscope.When X-irradiation was 2, 4, 6, 8 and 10 Gy, the apoptosis ratios of combined group and only X-irradiation group were 10.0%, 14.5%, 4.3%, 5.1%, 7.1% and 0.1%, 8.1%, 0.1%, 0.4%, 2.2% (P < 0.05) on flow cytometry. The result of microarray indicated that 1465 genes were up-regulated and 1108 down*regulated in the ELF-EMF plus X-ray group in comparison with the control group. The change rates of 110 apoptosis related genes were above 2 times, which including 71 up-regulated and 39 down-regulated. Gene microarray showed that ELF-EMF and X-ray irradiation had a mainly effect on different gene of apoptosis paths (CDC25 and CHKI, ATM, p38, PTEN, p53, G1/S, Fas, G2/M, Cell Cycle, Apoptosis and Caspase). The same genes of ELF-EMF plus X-ray group were showed 13 in ELF-EMF group and 42 in X-ray group. Conclusions Apoptosis paths were significantly different between ELF-EMF and X-irradiation. ELF-EMF cooperates with X-irradiation on inducing BEL-7402 cell apoptosis.
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Objective: Bel-7402 cells were stably transfected with a vector constructed with multiple copies of the hypoxia response- element (HRE) sequence of the human vascular endothelial growth factor (VEGF) gene and with the enhanced green fluorescent protein (EGFP) to establish a human hepatoma cell line Bel-7402/5HRE-EGFP. This paper aimed to study the responses of HRE in human liver cancer cells to different microenvironments by observing the changes in hypoxia-inducible factor 1 (HIF-1α) and vascular endothelial growth factor (VEGF) expression in Bel-7402/5HRE-EGFP cell lines under hypoxic conditions. Methods: The expression vector was constructed with 5 copies of HRE sequence and a minimal cytomegalovirus (CMV) as promoter and green fluorescent protein (GFP) as a reporter gene. The effect of different microenvironments such as hypoxia, H2 O2 or acidic pH on the activity of HRE in Bel-7402 cells and the changes in the expression levels of HIF-1α and VEGF under hypoxic condition were determined by using flow cytometry and immunohistochemical staining method. The association of the staining intensity and the distribution of pimonidazole, a hypoxic probe, with the expression and the distribution of HIF-1α, VEGF, and GFP were analyzed. The influence of hypoxia in tumor tissues of nude mice on the activity of HRE and expression of related genes were observed. Results: The HRE in liver cancer cells was very sensitive to hypoxia, which induces up-regulation of HIF-1α and VEGF expressions in tumor cells or in tumor tissues. Both distribution regions of HIF-1α and VEGF were almost the same. Conclusion: Hypoxia plays a pivotal role in controlling the expression of angiogenesis-related factors in human liver cancer cells.