OBJECTIVES: To investigate the inhibitory effect of Zheng GanDecoction (ZGF) on tumor progression in a rat model of diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) and explore the possible mechanism. METHODS: Seventy SD rats were subjected to regular intraperitoneal injections of DEN (50 mg/kg) for 12 weeks to induce HCC tumorigenesis, with another 10 rats receiving saline injections as the normal control. After successful modeling, the rats were randomized into 5 groups (n=10) for daily treatment with distilled water ( model group), Huaier Granules (4 g/kg; positive control group), or ZGF at low, medium, and high doses (2, 4, and 8 g/kg, respectively) via gavage for 17 weeks. Body weight changes of the rats were monitored, and after completion of the treatments, the rats were euthanized for measurement of liver, spleen and thymus indices and morphological and histopathological examinations of the liver tissues using HE staining. The expressions of YAP, p-YAP, MST1, LATS1 and p-LATS1 in the liver tissues were detected using immunohistochemistry and Western blotting. RESULTS: Compared with the normal control rats, the rat models with DEN-induced HCC exhibited much poorer general condition with a significantly reduced survival rate, increased body weight and liver and spleen indices, and a lowered thymus index. ZGF treatment obviously reduced liver and spleen indices, increased the thymus index, and improved pathologies of the liver tissues of the rat models. Immunohistochemistry and Western blotting showed a dose-dependent reduction of YAP expression and an increment of p-YAP expression in ZGF-treated rats, which also exhibited significantly upregulated hepatic expressions of MST1, LATS1 and p-LATS1. CONCLUSIONS ZGF inhibits DEN-induced HCC in rats by activating the Hippo/YAP pathway via upregulating MST1 and LATS1 expression, which promotes YAP phosphorylation and degradation to suppress proliferation and induce apoptosis of the tumor cells.
Animals ; Drugs, Chinese Herbal/pharmacology* ; Diethylnitrosamine ; Rats, Sprague-Dawley ; Rats ; Signal Transduction/drug effects* ; Protein Serine-Threonine Kinases/metabolism* ; Carcinoma, Hepatocellular/drug therapy* ; YAP-Signaling Proteins ; Liver Neoplasms/drug therapy* ; Hippo Signaling Pathway ; Male ; Liver Neoplasms, Experimental/metabolism* ; Transcription Factors/metabolism* ; Adaptor Proteins, Signal Transducing/metabolism*

OBJECTIVE: To observe the micromorphological changes of ultrastructure, apoptosis-related proteins expression and tumor cell apoptosis after ablation with the high-intensity focused ultrasound (HIFU), and to explore the mechanisms responsible for the thermal and non-thermal effect.
 METHODS: Forty rabbits with hepatic VX2 tumors were randomly divided into a thermal group (n=20) and a non-thermal group (n=20), and were subjected to HIFU ablation with thermal or non-thermal condition, respectively. Five animals in each group were sacrificed on the 1st, 3rd, 7th or 14th day after the ablation. The changes of ultrastructure, apoptosis-related proteins expression and tumor cell apoptosis were detected.
 RESULTS: The results of transmission electron microscope (TEM) revealed more severe injury on tissue and cells in the non-thermal group than that in the thermal group. The changes of apoptosis-related proteins expression and tumor cell apoptosis in transient zone were significantly different in comparison with that in the ablated area or peripheral area between the two groups. The expression of vascular endothelial growth factor (VEGF) was at low level on the 1st and 3rd day and elevated gradually on the 7th and 14th day, with no significant difference (all P>0.05). The expression of caspase-3 reached peak on the 3rd day and decreased on the 7th and 14th day. It was significantly higher in the non-thermal group than that in the thermal group on the 3rd and 7th day (all P<0.05). The expression of NF-κB was elevated from the 3rd day and reached peak on the 7th day while decreased on the 14th day. There was no significant difference at every time point between the 2 groups (all P>0.05). The apoptosis index in the non-thermal group and the thermal group on the 3rd and 7th day were (28.60±1.14)% vs (21.80±1.92)% and (21.00±1.58)% vs (14.80±1.48)%, respectively. It was higher in the non-thermal group than that in the thermal group (both P<0.01).
 CONCLUSION Both the thermal and the non-thermal effect of HIFU can induce apoptosis in transient zone, but the latter have a stronger effect.
Animals ; Apoptosis ; Caspase 3 ; metabolism ; High-Intensity Focused Ultrasound Ablation ; Liver Neoplasms ; pathology ; ultrastructure ; NF-kappa B ; metabolism ; Neoplasms, Experimental ; pathology ; ultrastructure ; Rabbits ; Vascular Endothelial Growth Factor A ; metabolism

Alkaloids ; pharmacology ; Animals ; Liver Neoplasms, Experimental ; enzymology ; prevention & control ; Quinolizines ; pharmacology ; Rats ; Selenium ; pharmacology ; Telomerase ; metabolism ; Yeast, Dried ; pharmacology

OBJECTIVETo investigate the efficacy of treatment with siRNA targeting Bcl-2 in combination with HCPT against H₂₂ hepatoma transplanted in mice.

METHODSsiRNA targeting Bcl-2 mRNA was successfully designed and synthesized. Then, the Bcl-2 siRNA was transfected into H₂₂ hepatoma transplanted in mice in combination with HCPT for treatment. The changes of tumor volume, body weight and survival rate were observed. Tumor tissues were processed into paraffin blocks and sections were stained with hematoxylin and eosin (HE) to investigate the morphological changes of the tumor cells. RT-polymerase chain reaction (PT-PCR) was used to assess the expression of Bcl-2 mRNA in tumors and cells. Cell cycle and apoptosis of H₂₂ hepatoma cells transplanted in mice were further determined by flow cytometry.

RESULTSAfter treatment for 21 days, the tumor volume was around (571.47 ± 67.31)mm³ in the group of siRNA in combination with HCPT, which was significant smaller than that of the groups of HCPT [(880.47 ± 107.31) mm³, P < 0.05], siRNA interfere [(1119.55 ± 158.60)mm³, P < 0.01] and saline (1357.64 ± 197.92)mm³, P < 0.01]. The median survival time of the group receiving siRNA in combination with HCPT treatment was 26 days, which was significantly longer than that of the group receiving HCPT (14 day, P < 0.05), siRNA interfere (21 day, P < 0.05) and saline (12 day, P < 0.05). Larger necrotic area, lower expression of Bcl-2 mRNA, less cells at S phase and more apoptotic cells could be obviously seen in tumor tissues in the group of siRNA in combination with HCPT treatment.

CONCLUSIONBcl-2 siRNA in combination with HCPT has good synergetic antitumor efficacy in H₂₂ hepatoma-bearing mice.

Animals ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Camptothecin ; analogs & derivatives ; pharmacology ; Cell Cycle ; drug effects ; Drug Synergism ; Liver Neoplasms, Experimental ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Random Allocation ; Transfection ; Tumor Burden ; drug effects

5-Flucytosine (5-FC) could be changed to 5-fluorouracil (5-FU) by cytosine deaminase (CD), the latter is able to kill cancer cells. However, there is no efficient method to deliver the CD gene into the tumor cells, which hampers the application of the suicide gene system. In this experiment, for the first time, the NDV has been utilized as a vector to deliver the CD gene into the cancer cells, the virus can infect the cancer cells specifically, replicate and assemble, while the cytosine deaminase is expressed. Then the CD converts the prodrug 5-FC into 5-FU to achieve the purpose of inhibiting tumor. Firstly, the whole genome of E. coli JM109 was extracted, and the CD gene was obtained by cloning method. Then the CD and IRES-EGFP were ligated into the pEE12.4 expression vector to become a recombinant pEE12.4IE-CD eukaryotic expression plasmid. The human liver cancer cells were transfected with the plasmid. The cells were treated with different concentrations of 5-FC, MTT method was used to determine the killing effect of CD/5-FC system on the human liver cancer cells. The cell deaths were 18.07%, 42.98% and 62.20% respectively when the concentrations of prodrug were at 10, 20 and 30 mmol x L(-1). In 5-FC acute toxicity experiment, Kunming mice were injected with different concentrations of 5-FC at intervals of 1:0.5. The LD50 of 5-FC through iv injection was determined by improved Karber's method, the LD50 was 507 mg x kg(-1) and the 95% confidence limit was 374-695 mg x kg(-1). According to the maximum LD0 dose of the LD50, the maximum safe dose was 200 mg x kg(-1). Body weight and clinic symptoms of the experimental animals were observed. These results laid the foundation to verify the antitumor effect and safety of CD/5-FC system in animal models. The CD gene was ligated into the NDV (rClone30) carrier, then the tumor-bearing animal was established to perform the tumor inhibiting experiment. The result showed that the recombinant rClone30-CD/5-FC system has a high antitumor activity in vivo. To summarize, CD gene has been cloned and its bioactivity has been confirmed in the mammalian cells. It is the first time in this study to utilize the recombinant NDV to deliver the CD gene into the tumor cells; our result proves the rClone30-CD/5-FC system is a potential method for cancer therapy.
Animals ; Antimetabolites, Antineoplastic ; metabolism ; pharmacology ; Cell Death ; drug effects ; Chick Embryo ; Cytosine Deaminase ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Flucytosine ; metabolism ; pharmacology ; Fluorouracil ; metabolism ; pharmacology ; Genetic Vectors ; Hep G2 Cells ; Humans ; Lethal Dose 50 ; Liver Neoplasms, Experimental ; pathology ; Mice ; Newcastle disease virus ; genetics ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Transfection ; Tumor Burden ; drug effects

OBJECTIVETo explore the mechanism of Xiaoai Jiedu Recipe (XJR) for fighting against tumors by detecting tumor gene expression profiles of H22 tumor-bearing mice.

METHODSH22 tumor-bearing mice were randomly divided into the normal control group, the low dose XJR group, the medium dose XJR group, the high dose XJR group, and the Cisplatin group. The differentially expressed genes of tumor tissues in H22 tumor-bearing mice were detected by using gene chip technique. The antitumor mechanism of XJR associated signaling pathways and gene expressions were found out by pathway analysis. The chemokine signaling pathways were analyzed.

RESULTSXJP could significantly affect multiple signaling pathways associated with tumor growth, apoptosis, and immunity. XJP also could decrease expressions of CCL3 and CXCL2 in the chemokine signaling pathway.

CONCLUSIONXJP could inhibit the growth and invasion of tumor cells possibly by affecting expressions of some genes in the chemokine signaling pathway.

Animals ; Cell Line, Tumor ; Chemokine CCL3 ; metabolism ; Chemokine CXCL2 ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Profiling ; Liver Neoplasms, Experimental ; genetics ; metabolism ; Mice ; Mice, Inbred ICR ; Signal Transduction ; drug effects

OBJECTIVETo investigate the effect of enoyl coenzyme A hydratase-1 (Ech1) on the proliferation and invasion ability of mouse hepatocarcinoma Hca-P cells in vitro.

METHODSRecombinant pcDNA3.1(+)-Ech1 gene and pcDNA3.1(+) were transfected into Hca-P cells by cationic liposomes introduction. Clone of PEch1 cells that stably expressing Ech1 and clone of control Pvector cells were screened by G418. The Ech1 expression was identified subsequently by reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The malignant behaviors of the cell lines were compared by proliferation, invasion and migration test.

RESULTSThe cell line Hca-P cells stably expressing Ech1 gene was constructed. The relative expression of Ech1 mRNA in the PEch1 group was 3.21 ± 0.43 and in the Pvector group was 1.44 ± 0.03, with a significant difference between the two groups (P = 0.029). The results of ELISA revealed that the expression of Ech1 protein was 0.140 ± 0.005 in the PEch1 group, 0.088 ± 0.003 in the Pvector group, and 0.078 ± 0.006 in the Hca-P group, showing a significant difference between the PEch1 group and the Pvector and Hca-P groups (P < 0.05). Transwell migration test showed that the number of penetrated cells in the PEch1 group was 143.00 ± 7.25 cells, significantly higher than that of the Pvector group (95.73 ± 3.88 cells) and un-treated Hca-1 group (106.67 ± 3.54 cells, both P < 0.05). The Transwell invasion assay showed that the number of penetrated cells was 77.20 ± 5.46 cells in the PEch1 group, significantly higher than 46.34 ± 4.35 cells in the Pvector group and 49.80 ± 5.21 cells in the un-treated Hca-1 group (both P < 0.05).

CONCLUSIONSThe results showed that overexpressed Ech1 in Hca-P cells may significantly increase the cell proliferation in a time-dependent manner. The up-regulation of Ech1 may increase to some extent the migration and invasion capacity of Hca-P cells. The efforts aiming at up-regulation of Ech1 expression may become a therapeutic target in the treatment of hepatocarcinoma.

Animals ; Carbon-Carbon Double Bond Isomerases ; genetics ; metabolism ; Cell Movement ; Cell Proliferation ; Liver Neoplasms, Experimental ; enzymology ; pathology ; Mice ; Neoplasm Invasiveness ; Plasmids ; RNA, Messenger ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Transfection ; Tumor Cells, Cultured ; Up-Regulation

To evaluate the changes induced in tumor tissue, the feeding artery, and neovascularization upon pingyangmycin-lipiodol emulsion treatment via transcatheter arterial chemoembolization (TACE) using the rabbit VX2 liver cancer model. The VX2 liver tumor model was established in 28 rabbits, and baseline tumor volume (V1, in mm3) was measured by spiral scan computed tomography (CT). Then, the rabbits were randomly divided into four groups (n = 7 each) and administered intraarterial therapies of: ultrafluid lipoidol embolization (group A); pingyangmycin (group B); pingyangmycin-lipiodol emulsion (group C); or saline (group D). All rabbits were sacrificed seven days later, and the response to therapy was determined by measuring the tumor volume (V2, in mm3), calculating the tumor growth rate, detecting expression of the vascular endothelial growth factor (VEGF) tumor biomarker, and performing histological analysis of the microvessel density (MVD) in the liver. Prior to therapy, the average V1 of the groups was statistically similar (A: 389.8+/-167.3, B: 404.1+/-184.9, C: 355.1+/-158.3, D: 378.1+/-189.0; (F = 0.257, P more than 0.05). In contrast, after therapy the average V2 of the groups was significantly different (A: 922.6+/-32.9, B: 665.9+/-99.9, C: 349.5+/-177.8, D: 1403.5+/-411.2; F = 26.23, P less than 0.05), as was the tumor growth ratio (A: 1.4, B: 0.6, C: -0.02, D: 2.7) and the mean positive ratio of VEGF (A: 57.1%, B: 42.9%, C: 28.6%, D: 100%; F = 8.407, P less than 0.05). MVD was highest in group D and lowest in group C (all, P less than 0.05). Bivariate correlation analysis revealed a positive correlation between VEGF expression and MVD (r = 0.743, P less than 0.01). Pingyangmycin exerts anti-tumor effects in the rabbit VX2 liver cancer model, but is more effective when administered as the combination therapy of pingyangmycin-lipiodol emulsion with TACE.
Animals ; Antibiotics, Antineoplastic ; administration & dosage ; therapeutic use ; Bleomycin ; administration & dosage ; analogs & derivatives ; therapeutic use ; Chemoembolization, Therapeutic ; methods ; Emulsions ; Ethiodized Oil ; administration & dosage ; therapeutic use ; Female ; Iodized Oil ; administration & dosage ; therapeutic use ; Liver Neoplasms, Experimental ; blood supply ; drug therapy ; pathology ; Male ; Microvessels ; Neoplasm Transplantation ; Neovascularization, Pathologic ; Rabbits ; Random Allocation ; Tumor Burden ; drug effects ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo establish a syngeneic mouse model of liver tumor stably expressing hepatitis B virus (HBV) antigens.

METHODSMelanoma cell line B16 cells were transfected with pLXSN-2HBV. Cells (named B16/HBV) stably and persistently expressing HBV surface (HBsAg) and core (HBcAg) antigens were identified. The cells were injected into the hepatic subcapsular space of fifteen C57BL/6J mice. The mice were divided into 3 groups, receiving 100, 1000 or 5000 cells in a total volume of 5 µl per mouse, respectively, five mice in each group. Two weeks after the tumor cell inoculation, serum samples from the mice were collected weekly and the serum concentration of HBsAg and anti-HBs was quantified by ELISA. The tumor growth in the mouse liver was monitored by a high-resolution ultrasound system. Expression of HBsAg and HBcAg in the tumor tissues was determined by immunohistochemistry.

RESULTSLiver tumors were formed in all the mice receiving 1000 and 5000 B16/HBV cells per mouse, and in 80% of the mice receiving 100 B16/HBV cells. HBsAg and anti-HBs were detectable in their sera from 2 weeks after tumor cell inoculation. The mice receiving 100 cells per mouse began to die 4 weeks, those receiving 1000 cells per mouse began to die 3 - 4 weeks and those receiving 5000 cells began to die 2 - 3 weeks after the cell inoculation. All the tumor cells expressed HBsAg and HBcAg.

CONCLUSIONSThe B16/HBV cells stably and persistently express HBV antigens both in vitro and in vivo. A mouse model of transplanted liver tumor stably expressing HBV antigens has been successfully established by inoculation of those cells into the hepatic subcapsular space.

Animals ; Cell Line, Tumor ; Disease Models, Animal ; Female ; Hepatitis B Core Antigens ; metabolism ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; metabolism ; Liver Neoplasms, Experimental ; immunology ; virology ; Melanoma, Experimental ; metabolism ; pathology ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Transfection

To investigate the influence of bear bile on rat hepatocarcinoma induced by diethylnitrosamine (DEN), a total of 40 rats were randomly divided into 4 groups: normal control group, model group, and two bear bile treatment groups. The rat liver cancer model was induced by breeding with water containing 100 mg x L(-1) DEN for 14 weeks. The rats of the bear bile groups received bear bile powder (200 or 400 mg x kg(-1)) orally 5 times per week for 18 weeks. The general condition and the body weight of rats were examined every day. After 18 weeks the activities of serum alanine transaminase (ALT), aspartate transaminase (AST) and total bilirubin (TBIL) were detected. Meanwhile, the pathological changes of liver tissues were observed after H&E staining. The expression of proliferative cell nuclear antigen (PCNA) and a-smooth muscle actin (alpha-SMA) in liver tissue were detected by immunohistochemical method. After 4 weeks the body weights of rats in normal group were significantly more than that in other groups (P < 0.05); and that in the two bile groups was significantly more than that in the model group. Compared with normal group, the level of serum glutamic-pyruvic transaminase and total bilirubin increased significantly in other groups; compared with model group, these two indexes decreased significantly in two bile groups. Hepatocellular carcinoma occurred in all rats except for normal group; there were classic cirrhosis and cancer in model group while there were mild cirrhosis and high differentiation in two bile groups. There were almost no expressions of PCNA and alpha-SMA in normal group while there were high expressions in model group; the two bile groups had some expressions but were inferior to the model group, and alpha-SMA reduced markedly. It indicated that bear bile restrained the development of liver cancer during DEN inducing rat hepatocarcinoma, which may be related to its depressing hepatic stellate cell activation and relieving hepatic lesion and cirrhosis.
Actins ; metabolism ; Alanine Transaminase ; blood ; Animals ; Antineoplastic Agents ; pharmacology ; Aspartate Aminotransferases ; blood ; Bile ; chemistry ; Bilirubin ; blood ; Body Weight ; drug effects ; Carcinoma, Hepatocellular ; blood ; chemically induced ; pathology ; Diethylnitrosamine ; Liver ; metabolism ; pathology ; Liver Cirrhosis ; chemically induced ; pathology ; Liver Neoplasms, Experimental ; blood ; chemically induced ; pathology ; Male ; Powders ; pharmacology ; Proliferating Cell Nuclear Antigen ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Ursidae