Interferon-γ (IFN-γ) has been used to control cancers in clinical treatment. However, an increasing number of reports have suggested that in some cases effectiveness declines after a long treatment period, the reason being unclear. We have reported previously that long-term IFN-γ treatment induces malignant transformation of healthy lactating bovine mammary epithelial cells (BMECs) in vitro. In this study, we investigated the mechanisms underlying the malignant proliferation of BMECs under IFN-γ treatment. The primary BMECs used in this study were stimulated by IFN-γ (10 ng/mL) for a long term to promote malignancy. We observed that IFN-γ could promote malignant cell proliferation, increase the expression of cyclin D1/cyclin-dependent kinase 4 (CDK4), decrease the expression of p21, and upregulate the expression of cellular-abelsongene (c-Abl) and histone deacetylase 2 (HDAC2). The HDAC2 inhibitor, valproate (VPA) and the c-Abl inhibitor, imatinib, lowered the expression level of cyclin D1/CDK4, and increased the expression level of p21, leading to an inhibitory effect on IFN-γ-induced malignant cell growth. When c-Abl was downregulated, the HDAC2 level was also decreased by promoted proteasome degradation. These data suggest that IFN-γ promotes the growth of malignant BMECs through the c-Abl/HDAC2 signaling pathway. Our findings suggest that long-term application of IFN-γ may be closely associated with the promotion of cell growth and even the carcinogenesis of breast cancer.
Animals ; Carcinogenesis/pathology* ; Cattle ; Cell Cycle Proteins/metabolism* ; Cell Proliferation/drug effects* ; Cell Transformation, Neoplastic/pathology* ; Cells, Cultured ; Epithelial Cells/pathology* ; Female ; Histone Deacetylase 2/metabolism* ; Imatinib Mesylate/pharmacology* ; Interferon-gamma/pharmacology* ; Mammary Glands, Animal/pathology* ; Mammary Neoplasms, Experimental/pathology* ; Proto-Oncogene Proteins c-abl/metabolism* ; Signal Transduction ; Valproic Acid/pharmacology*

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

PURPOSE: Both genetic and epigenetic alterations can lead to abnormal expression of metastasis-regulating genes in tumor cells. Recent studies suggest that aberrant epigenetic alterations, followed by differential gene expression, leads to an aggressive cancer cell phenotype. We examined epigenetically regulated genes that are involved in ovarian cancer metastasis. MATERIALS AND METHODS: We developed SK-OV-3 human ovarian carcinoma cell xenografts in mice. We compared the mRNA expression and DNA methylation profiles of metastatic tissues to those of the original SK-OV-3 cell line. RESULTS: Metastatic implants showed increased mRNA expression of the carbonic anhydrase 9 (CA9) gene and hypomethylation at CpG sites in the CA9 promoter. Treatment of wild-type SK-OV-3 cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine reduced methylation of the CA9 promoter and increased CA9 mRNA expression. Eight CpGs, which were located at positions -197, -74, -19, -6, +4, +13, +40, and +86, relative to the transcription start site, were hypomethylated in metastatic tumor implants, compared to that of wild-type SK-OV-3. Overexpression of CA9 induced an aggressive phenotype, including increased invasiveness and migration, in SK-OV-3 cells. CONCLUSION: Alterations in the DNA methylation profile of the CA9 promoter were correlated with a more aggressive phenotype in ovarian cancer cells.
Animals ; Azacitidine/*analogs & derivatives/pharmacology ; Carbonic Anhydrases/metabolism ; *DNA Methylation ; Female ; Gene Expression Regulation, Neoplastic/*drug effects ; Humans ; Mice ; Neoplasm Invasiveness/genetics ; Neoplasm Metastasis/genetics/*pathology ; Neoplasms, Experimental ; Neoplasms, Glandular and Epithelial/genetics/*metabolism/pathology ; Ovarian Neoplasms/genetics/*metabolism/pathology ; Phenotype ; Promoter Regions, Genetic ; RNA, Messenger/metabolism

OBJECTIVETo evaluate the inhibitory effect of genistin combined with anastrozole on the growth and apoptosis of breast tumor tissue, and to study their anti-cancer mechanism by using the model of 7,12-dimethylbenz [alpha] anthracene (DMBA)-induced mammary tumors following ovariectomy in Sprague-Dawley (SD) rats.

METHODSThe DMBA induced postmenopausal SD rats were randomly divided into the control group, the genistein group, the anastrozole group, and the genistein combined with anastrozole group. The growth of tumors was observed in each group. The proliferation index and apoptosis index of tumor cells were determined. Moreover, estradiol (E2) and 17beta-HSD1 mRNA levels were determined by ELISA and RT-PCR respectively.

RESULTSThe tumor growth was inhibited in the genistein group and the anastrozole group. The inhibitory ratio was significantly higher in the genistein combined with anastrozole group (P < 0.05). Compared with the control group, levels of E2 and 17beta-HSD1 mRNA decreased more significantly in the genistein combined with anastrozole group (P < 0.05).

CONCLUSIONSGenistein could suppress the growth of mammary tumors in postmenopausal rats. It showed synergistic effect when combined with anastrozole, which resulted in reduced levels of E2 and 17beta-HSD1 mRNA. It had inhibitory effect on the growth of breast tumors.

17-Hydroxysteroid Dehydrogenases ; metabolism ; Animals ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Estradiol ; metabolism ; Female ; Genistein ; administration & dosage ; pharmacology ; Mammary Neoplasms, Experimental ; chemically induced ; pathology ; Nitriles ; administration & dosage ; pharmacology ; Ovariectomy ; Postmenopause ; Rats ; Rats, Sprague-Dawley ; Triazoles ; administration & dosage ; pharmacology

OBJECTIVETo explore the effect of ricin temperature response gel on breast cancer and its regulatory effect on immune function in rats.

METHODSRicin was purified by chromatography and identified by immunoblotting. The rat subcutaneously transplanted breast cancer model was established. Forty model rats with a tumor diameter of about 3.0 cm were subjected to the study. They were randomized into four groups equally: the model group and three treated groups (blank gel, ricin, ricin-gel) were administered with blank gel, ricin, and ricin temperature response gel via percutaneous intratumor injection, respectively. The tumor was isolated 10 days later for the estimation of tumor inhibition rate (TIR) by weighing, pathologic examination, and detection of tumor apoptosis-associated genes bcl-2 and bax with semiquantitative RT-PCR. Also, peripheral blood was obtained to test T-lymphocyte subsets, the killing function of lymphocytes, and the contents of tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2). The outcomes were compared between groups.

RESULTSThe TIR in the ricin-gel group was 61.8%, with the pathologic examination showing extensive tumor tissue necrosis. Compared with the model group, after ricin temperature response gel treatment, bcl-2 expression was down-regulated, bax expression was up-regulated, CD4+ lymphocytes and CD4+/CD8+ ratio in peripheral blood were increased, the killing function of lymphocytes was enhanced, and the contents of TNF-α and IL-2 were elevated (P < 0.05 or P < 0.01).

CONCLUSIONIntratumor injection of ricin temperature-responsive gel showed significant antitumor effect on breast cancer and could enhance the immune function in the tumor-bearing rat.

Animals ; Antineoplastic Agents ; administration & dosage ; Apoptosis ; drug effects ; CD4-CD8 Ratio ; Disease Models, Animal ; Female ; Gels ; therapeutic use ; Immunohistochemistry ; Immunomodulation ; drug effects ; Injections, Intralesional ; Interleukin-2 ; immunology ; metabolism ; Mammary Neoplasms, Experimental ; drug therapy ; immunology ; pathology ; Random Allocation ; Rats ; Rats, Wistar ; Ricin ; administration & dosage ; Sensitivity and Specificity ; Temperature ; Tumor Necrosis Factor-alpha ; immunology ; metabolism

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

In order to investigate the anti-proliferative effects of triptolide (TP) on 4T1 mice breast cancer cell line in vitro and in mouse model, as well as the possible mechanisms, we detected the effect of TP on cell proliferation by MTT assay or Crystal Violet Staining in our research. Flowcytometry combined with FITC-Annexin V/PI staining were used for detecting TP induced 4T1 cell apoptosis. The protein expression of ERalpha, p-ERalpha, ERbeta, p-ERbeta, ERK, p-ERK, p38, p-p38, SAPK/JNK, and p-SAPK/JNK was tested by western blotting. We also compare TP with chemotherapy drug doxorubicin in 4T1 tumor bearing BLAB/c mice model, the Xenogen bioluminescence imaging, H&E, and IHC result indicated that TP exhibits an anticancer proliferation activity. As a result, TP in 100, 10, 1, 0.1 micromol x L(-1), all inhibited the proliferation of 4T1 cells by MTT assay and Crystal Violet Staining. TP which concentrations is 10, 1, 0.1 micromol x L(-1) could induce the apoptosis of 4T1 cells and reduce the cell proliferation. TP in 200 microg x kg(-1) could inhibit the tumor growth in vivo. The anticancer proliferation of TP was involved in its effect on reducing expression of ERalpha, p-ERalpha, ERbeta, and p-ERbeta, but nothing to do with the activation of MAPK signaling pathway.
Animals ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; Diterpenes ; pharmacology ; therapeutic use ; Down-Regulation ; drug effects ; Epoxy Compounds ; pharmacology ; therapeutic use ; Female ; Lung Neoplasms ; secondary ; Mammary Neoplasms, Experimental ; drug therapy ; metabolism ; pathology ; Mice ; Mice, Inbred BALB C ; Phenanthrenes ; pharmacology ; therapeutic use ; Phosphorylation ; drug effects ; Receptors, Estrogen ; metabolism ; 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 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 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