Synthesis of matrine derivative ZS10 and its mechanism of apoptosis induced by PI3K/AKT signaling pathway in human hepatocellular carcinoma cell line BEL-7402 / 中国药理学通报

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.

Tertiary sulphonamide derivatives as dual acting small molecules that inhibit LSD1 and suppress tubulin polymerisation against liver cancer.

A series of tertiary sulphonamide derivatives were synthesised and evaluated for their antiproliferative activity against liver cancer cell lines (SNU-475, HepG-2, and Bel-7402). Among these tertiary sulphonamides, compound 17a displayed the best anti-liver cancer activity against Bel-7402 cells with an IC50 value of 0.32 µM. Compound 17a could effectively inhibit tubulin polymerisation with an IC50 value of 1.27 µM. Meanwhile, it selectively suppressed LSD1 with an IC50 value of 63 nM. It also concentration-dependently inhibited migration against Bel-7402 cells. Importantly, tertiary sulphonamide 17a exhibited the potent antitumor activity in vivo. All these findings revealed that compound 17a might be a tertiary sulphonamide-based dual inhibitor of tubulin polymerisation and LSD1 to treat liver cancer.

lncRNA MAGI2-AS3 overexpression had antitumor effect on Hepatic cancer via miRNA-23a-3p/PTEN axis.

The present study aimed to evaluate the antitumor effects of MAGI2-AS3 and its mechanism in liver cancer. Cancer tissues and adjacent nontumor tissues were collected, and lncRNAs were analyzed via chip assay. The correlation between MAGEI2-AS3 and patient pathology and prognosis was then analyzed. Bel-7402 and Huh-7 cell lines were also used in our study. For the in vitro study, MTT assay, flow cytometry, transwell assay, and wound healing assay were conducted to evaluate hepatic cancer cell (Bel-7402 and Huh-7) proliferation, apoptosis, invasion, and migration. The relative mechanisms were evaluated by Western blot (WB) and cellular immunofluorescence. The correlation among MAGI2-AS3, miRNA-23a-3p, and PTEN was determined by a dual-luciferase reporter assay. The expression of lncRNA MAGI2-AS3 was significantly downregulated in tumor tissues. MAGI2-AS3 expression was closely correlation with HCC patient's clinicopathology and prognosis and prognosis. In the cell experiment, compared with the negative control (NC) group, MAGI2-AS3 overexpression reduced cell proliferation, invasion, and migration and increased cell apoptosis in Bel-7402 and Huh-7 cell lines. However, when Bel-7402 and Huh-7 cells were transfected with miRNA-23a-3p, their biological activities (proliferation, invasion, and migration) were significantly increased. Through WB assay, MAGI2-AS3 could increase PTEN and depress p-AKT and MMP-9 protein expressions via miRNA-23a-3p suppression. The dual-luciferase reporter assay revealed that MAGI2-AS3 directly targeted miRNA-23a-3p and that miRNA-23a-3p could target PTEN. MAGI2-AS3 might be a potential therapeutic target for liver cancer owing to its regulation by the miRNA-23a-3p/PTEN axis.

Formononetin affects the pathogenesis of liver cancer by inhibiting the COX-2/cyclin D1 axis / 中国肿瘤生物治疗杂志

@#[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。

Long Noncoding RNA (lncRNA) MT1JP Suppresses Hepatocellular Carcinoma (HCC) in vitro.

INTRODUCTION: The purpose of this study was to evaluate the effects and mechanisms of the long noncoding RNA (lncRNA) MT1JP on hepatocellular carcinoma (HCC) in vitro. PATIENTS AND METHODS: Thirty pairs of tumor and adjacent normal tissues were collected from HCC patients. Tissue pathology and MT1JP expression were evaluated by hematoxylin and eosin staining and in situ hybridization (ISH), respectively. The correlation between MT1JP and HCC prognosis was investigated. MTT assays, cloning, flow cytometry, transwell assays, and wound-healing assays were used to evaluate the effects of MT1JP on HCC cell lines. RT-qPCR and Western blot were used to measure the relative mRNA and protein expression levels. RESULTS: The expression of MT1JP was downregulated in HCC tumor tissues compared with that in adjacent normal tissues, while the percent survival was significantly greater in the high MT1JP expression group than in the low MT1JP expression group (P=0.0238). In vitro, overexpression of MT1JP suppressed the proliferation, invasion, and migration, reduced colony cell number, increased cell apoptosis, and induced G1-phase cell cycle arrest in Bel-7402 and Huh-7 cells. Meanwhile, the mRNA and protein expression levels of RUNX3 and P21 were significantly upregulated, whereas those of MMP2 and MMP9 were significantly downregulated, in Bel-7402 and Huh-7 cells overexpressing MT1JP (all P<0.001). CONCLUSION: LncRNA MT1JP may function as a tumor suppressor in HCC. Overexpression of MT1JP suppressed HCC cell biological activities through the regulation of RUNX3.

Compositional characterization of Pyrus ussuriensis Maxim and their antioxidant activities and induction of apoptosis in Bel-7402 cell.

In this work, a comparison study on active sites (the total phenolic, total flavonoids, and total triterpenes contents) and antioxidant activities (DPPH, ABTS, and FRAP assays) of different fractions from Pyrus ussuriensis Maxim was evaluated. Moreover, the inhibition capability on human hepatocarcinoma cells Bel-7402 cells and the mechanism was also discussed. Results showed that the ethyl acetate fraction significantly scavenged DPPH and ABTS+ radicals, exhibited ferric ion reducing antioxidant, and inhibited Bel-7402 cells proliferation. In addition, oleanolic acid was the dominant compound act on the Bel-7402 cells in the extract and it induced apoptosis by the caspase pathway and induced cell cycle arrest at the G0/G1 phase by inhibiting the cyclin D1/CDK4 pathway. The extracts of P. ussuriensis Maxim were confirmed to have anti-oxidative and antiproliferative effects against Bel-7402 cell in vitro. PRACTICAL APPLICATIONS: Fruits and vegetables which contain high levels of antioxidants can help to reduce the risk of chronic diseases such as cancer. Pyrus ussuriensis Maxim is a kind of edible and medical fruit with multiple bioactivities, whereas the capability to anti-lung cancer activity has not been investigated. The extracts of P. ussuriensis Maxim were revealed to have anti-oxidative and antiproliferative effects against Bel-7402 cell in vitro. Accordingly, it is the first time to verify that oleanolic acid was the main chemical components of P. ussuriensis with antitumor potential.

lncRNA RMRP knockdown suppress hepatocellular carcinoma biological activities via regulation miRNA-206/TACR1.

Long noncoding RNA, RNA component of mitochondrial RNA processing endoribonuclease (RMRP) plays an important role in cancer development and is closely correlated with prognosis in cancer patients. However, whether RMRP affects prognosis in patients with hepatocellular carcinoma (HCC) remains unclear. The aim of the present study was to investigate the expression level of RMRP in HCC and its correlation with prognosis in patients with HCC and explain the effects and associated mechanisms by conducting an in vitro study. The high expression level of RMRP was correlated with poor prognosis in patients with HCC. Using in vitro analysis, RMRP knockdown suppressed HCC cell proliferation, invasion, and migration (P < .05). miRNA-206 overexpression had similar effects in HCC cell lines (Bel-7402 and Huh-7). Using Western blot analysis and cellular immunofluorescence detection, RMRP downregulation significantly suppressed TACR1/Erk1/2 pathway, while miRNA-206 was significantly upregulated (P < .05). RMRP downregulation inhibits HCC-related biological activities by the regulation of miRNA-206/TACR1.

Injectable peptide hydrogel as intraperitoneal triptolide depot for the treatment of orthotopic hepatocellular carcinoma.

Chemotherapy is among the limited choices approved for the treatment of hepatocellular carcinoma (HCC) at intermediate and advanced stages. Preferential and prolonged drug exposure in diseased sites is required to maximize the therapeutic index of the drug. Here, we report an injectable supramolecular peptide hydrogel as an intraperitoneal depot for localized and sustained release of triptolide for the treatment of orthotopic HCC. We chose peptide amphiphile C16-GNNQQNYKD-OH-based nanofibers as gelators and carriers for triptolide. Sustained triptolide release from the hydrogel was achieved over 14 days in vitro, with higher accumulation in and cytotoxicity against human HCC Bel-7402 in comparison with L-02 fetal hepatocytes. After intraperitoneal injection, the hydrogel showed prolonged retention over 13 days and preferential accumulation in the liver, realizing HCC growth inhibition by 99.7 ± 0.1% and animal median survival extension from 19 to 43 days, without causing noticeable pathological changes in the major organs. These results demonstrate that injectable peptide hydrogel can be a potential carrier for localized chemotherapy of HCC.

Inhibitory effect of taspine derivative TAD1822-7 on tumor cell growth and angiogenesis via suppression of EphrinB2 and related signaling pathways.

The aim of this study was to investigate the inhibitory effect of TAD1822-7, a synthesized taspine derivative, on cancer through its effects on tumor cell growth and angiogenesis via suppression of EphrinB2. The obtained data showed that TAD1822-7 decreased Bel-7402 cell viability and colony formation ability and suppressed cell migration. TAD1822-7 effectively inhibited blood vessel formation in an aortic ring assay to examine angiogenesis. Moreover, it also down regulated the expression of VEGFR2, VEGFR3, CD34, PLCγ, Akt, MMP2, MMP9, and CXCR4, and suppressed the expression of EphrinB2 and its PDZ protein, PICK1, in Bel-7402 cells. These results indicate that TAD1822-7 is a potential anti-angiogenic agent that can inhibit the viability and migration of Bel-7402 cells via suppression of EphrinB2 and the related signaling pathways.

Angiotensin II and tumor necrosis factor-α stimulate the growth, migration and invasion of BEL-7402 cells via down-regulation of GRK2 expression.

PURPOSE: To investigate the effects of angiotensin II (Ang II) and tumor necrosis factor-α (TNF-α) on the biological characteristics of hepatocellular carcinoma (HCC) cells and the associated changes in G protein-coupled receptor kinase 2 (GRK2) expression. METHODS: The mean serum levels of Ang II and TNF-α in normal subjects and patients with benign liver tumors (BLTs) and HCC were evaluated by enzyme-linked immunosorbent assay (ELISA), and liver samples from the patients with HCC and HCC mice were used to assess the protein levels of both cytokines, their major receptors and GRK2. In addition, the dynamics of Bel-7402 cells were determined with cell counting kit-8 (CCK-8) and Transwell experiments, while the levels of the primary cytokine receptors Ang II type-1 receptor (AT1R) and type-2 receptor (AT2R) as well as TNF receptor 1 (TNFR1) were detected by flow cytometry (FCM). The effects of Ang II and TNF-α on the GRK2 levels in Bel-7402 cells and on the dynamics of GRK2-knockdown HCC cells were also investigated. RESULTS: Both cytokines independently enhanced Bel-7402 cell growth, migration and invasion by decreasing the GRK2 level. In contrast, down-regulating the GRK2 level in Bel-7402 cells suppressed these effects. No synergistic effects were discovered when Ang II and TNF-α were administered together. Furthermore, increased AT1R and TNFR1 levels stimulated HCC initiation and progression, whereas AT2R overexpression produced the opposite effect. CONCLUSIONS: The present results suggested that Ang II and TNF-α promote Bel-7402 cell growth, migration and invasion by down-regulating GRK2 expression, and that the associated receptors AT1R, AT2R and TNFR1 participate in HCC initiation and progression.

Evodiamine induces apoptosis of human hepatoma cell line BEL-7402 by Hippo-YAP pathway / 中草药

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.

Tegillarca granosa extract Haishengsu inhibits tumor activity via a mitochondrial­mediated apoptotic pathway.

Haishengsu (HSS) is an active natural extract isolated from Tegillarca granosa, which has previously been demonstrated to inhibit the proliferation of several types of cancer cells in vitro. Our previous study indicated that HSS may induce apoptosis to suppress growth of human hepatocellular carcinoma BEL­7402 cells by activating Fas pathway. The present study demonstrated that HSS treatment induces the in vitro apoptosis of BEL­7402 cells via the mitochondrial­mediated apoptotic pathway detected by DNA fragmentation assay, caspase activity assay and transmission electron microscopy assay, and inhibits tumor xenograft growth in vivo. Alterations in apoptotic regulatory proteins were detected, including decreased expression of B­cell lymphoma2 (Bcl­2), upregulation of Bcl­2­associated X protein and mitochondrial cytochrome c release, and downstream activation of apoptotic signaling. Furthermore, apoptotic induction was caspase­dependent, as indicated by cleavage of the caspase substrate, poly (ADP­ribose) polymerase. Oral administration of 62.5­250 mg/kg HSS markedly educed the growth of hepatocellular carcinoma tumor xenografts in nude mice. In addition, immunohistochemical staining for caspase­3 protein and transmission electron microscopy further indicated the induction of apoptosis in these tumor tissues. Taken together, the present study demonstrated that HSS may effectively induce apoptosis to suppress the growth of BEL­7402 cells in vitro and in vivo, and therefore may hold promise for further development as a novel cancer therapy.

[Differences of Therapeutic Efficacy Between Different Kinds of Somatostatin Analogue for Primary Hepatocellular Carcinoma].

OBJECTIVE: To explore the effects of SOM230, octreotide and lanreotide on hepatocellular carcinoma cell line Bel-7402 In vivoand In vitro, and to analyze the differences of their therapeutic efficacy with relevant mechanisms. METHODS: At different time points (24, 48, 72 h), the cell counting kit-8 (CCK8) was used to evaluate cell proliferation (drug concentration 1×10-10-1×10-5mol/L) and the Annexin Ⅴ-FITC/PI staining was used to assess cell apoptosis (drug concentration 1×10-5mol/L), while the real-time quantitative PCR was used to detect cellular SSTRexpression changes before and after interventions. A transplanted tumor model was set up, and the tumor-bearing nude mice were treated by three drugs with a common dose of 100 µg/ (kg·d) or same volume of normal saline, respectively. After a treatment period of 6 weeks, real-time quantitative PCR and Western blot test were performed to detect the expression changes ofSSTR in tumor tissues from the level of gene and protein. RESULTS: All three drugs could inhibit the cell proliferation of Bel-7402. However, they were unable to promote the cell apoptosis. In vitro, the expressions ofSSTR1, SSTR4genes did not change over time in each group. The expressions ofSSTR2gene were decreased in three intervention groups while the expressions ofSSTR5gene were increased first and then decreased. Compared with the control group, all differences have statistical significance (P<0.05). All three drugs could improve the survival rate and quality of life for nude mice bearing hepatoma. In vivo, the expression ofSSTR1gene in SOM230 group was increased when compared with that of the other groups; the expressions ofSSTR2gene in three intervention groups were increased when compared with that of the control group; the expressions of SSTR5gene in SOM230 group and lanreotide group were increased when compared with that of the octreotide group and the control group, and all differences have statistical significance (P<0.05). The Western blot test confirmed these results from the protein level. CONCLUSION: In a certain concentration range, the long-term treatment of SSTA with high affinities to SSTR2 and SSTR5 could inhibit the growth of HCC.

Synthesis and biological evaluation of novel podophyllotoxin-NSAIDs conjugates as multifunctional anti-MDR agents against resistant human hepatocellular carcinoma Bel-7402/5-FU cells.

Currently, multi-drug resistance (MDR) to chemotherapy agents is a major hindrance to the treatment of hepatocellular carcinoma. Development of novel antineoplastic drug with anti-MDR activity is an effectively way to overcome cancer resistance. In present study, novel podophyllotoxin-NSAIDs conjugates were synthesized, and their antiproliferative activities were evaluated in vitro. The most potent conjugate, A1, displayed selective cytotoxicity against resistant Bel-7402/5-FU cells with an IC50 value of 0.065 ± 0.016 µM and a lower resistant factor value of 0.32. In addition, all conjugate molecules efficiently triggered cell cycle arrest at S + G2 phase, induced apoptosis, disrupted the microtubule network and showed antimigratory activity in Bel-7402/5-FU cells. Finally, three conjugates regulated the levels of cell cycle arrest-, apoptosis-, migratory-, inflammatory- and MDR-related proteins, as well as three signaling in Bel-7402/5-FU cells by some but not all similar molecular mechanisms. Together, these findings highlighted the cytotoxic and multifunctional anti-MDR properties of podophyllotoxin-NSAIDs conjugates for the first time, which may be promising candidates for intervention of resistant hepatocellular carcinoma.

MiR-503 sensitizes human hepatocellular carcinoma cells to cisplatin by targeting bcl-2 / 中南大学学报(医学版)

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.

Effect of miR-320a up-regulation on apoptosis and migration of Bel-7402 cells induced by ribonucleic acid Ⅱ / 中国药理学通报

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.

The anti-tumor effects of cordycepin-loaded liposomes on the growth of hepatoma 22 tumors in mice and human hepatoma BEL-7402 cells in culture.

Liposomes have successfully been used for decades to encapsulate and protect drugs that are prone to deactivation in the body. The present study aimed to demonstrate the use of liposomes to encapsulate cordycepin, an adenosine analog that quickly loses its activity in vivo. The cordycepin-loaded liposomes were prepared by the ammonium sulfate gradient approach, and its in vitro and in vivo antitumour activities were evaluated using BEL-7402 cells and hepatocellular carcinoma H22 transplanted tumors, respectively. An MTT assay was used to observe the cytotoxicity of cells treated with cordycepin and cordycepin-loaded liposomes in vitro. High-content screening (HSC) was carried out using Hoechst 33342 to detect apoptotic cells and the ratio of cells in different cell cycle stages. The data demonstrated that both the cordycepin and the cordycepin-loaded liposomes resulted in clear cytotoxicity with IC50 values of 18.97 and 29.39 µg/mL, respectively. The latter showed significantly strong inhibitory effects on H22 tumor growth in mice, while the former did not show any inhibitory effects on tumor growth. In addition, the HSC assay showed that the cordycepin-loaded liposomes resulted in a higher rate of apoptosis than the cordycepin alone in BEL-7402 cells. Further data analysis revealed that the cells treated with cordycepin-loaded liposomes were predominately arrested at the G2/M phase (p < 0.05), while those treated with cordycepin alone were arrested in the G0/G1 phase (p < 0.05). In conclusion, these results suggest that liposomes can enhance and maintain the in vivo anti-tumor activity of cordycepin.

Effect of neferine on proliferation and invasion of human hepatocellular carcinoma cell line HepG2 and Bel-7402 / 中国药理学通报

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.

Apoptosis induction and its mechanism of ginseng polysaccharide on human heratoma BEL-7402 cells / 中国药学杂志

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.