The effects of PDTC plus leflunomide and cyclosporine on the NF-kappaB signaling pathway in mouse-to-rat cardiac xenografts.

In this study, the effects of pirrolidine dithiocarbamate (PDTC) plus leflunomide (Lef) and cyclosporine (CsA) on the NF-kappaB signaling pathway in mouse-to-rat cardiac xeno-transplantation models were investigated. NIH mice and Wistar rats served as donors and recipients respectively. Mouse-to-rat cardiac xenotransplantation was performed. The recipients were divided into 5 groups: group A (the control group), group B (PDTC group), group C (PDTC plus CsA group), group D (PDTC plus Lef group) and group E (PDTC plus Lef and CsA group). The expressions of IKKalpha/beta, NF-kappaB-P65, IkappaBalpha, ICAM-1 and NF-kappaB DNA binding activity in xenograft tissues were determined by immunohistochemistry and Western blot as well as electrophoretic mobility shift assay (EMSA). The median survival time of cardiac xenografts in the control group, PDTC group, PDTC plus CsA group, PDTC plus Lef group and PDTC plus Lef and CsA group was (2.17+/-0.41), (2.33+/-0.52), (4.67+/-1.21), (7.00+/-1.79) and (9.00+/-1.41) days respectively. The survival time of xenografts in the PDTC plus Lef and CsA group was significantly longer than that in other four groups (P<0.05 for all), that in the PDTC plus Lef group longer than that in the control group, PDTC group and PDTC plus CsA group (P<0.05 for all), that in PDTC plus CsA group longer than the control group and PDTC group (P<0.05 for all). The expressions of IKKalpha/beta, NF-kappaB-P65, IkappaBalpha and ICAM-1 and NF-kappaB DNA binding activity were notably increased in mouse-to-rat cardiac xenografts. The expressions were decreased in the control group, PDTC group, PDTC plus CsA group, PDTC plus Lef and PDTC plus Lef and CsA group in turn. It was concluded that PDTC plus Lef and CsA can significantly suppress the expressions of IKKalpha/beta, NF-kappaB-P65, IkappaBalpha, ICAM-1 and NF-kappaB DNA binding activity, thereby prolonging the survival of the cardiac xenografts.

The Effects of PDTC plus Leflunomide and Cyclosporine on the NF-кB Signaling Pathway in Mouse-to-rat Cardiac Xenografts / 华中科技大学学报（医学）（英德文版）

In this study,the effects of pirrolidine dithiocarbamate (PDTC) plus leflunomide (Lef) models were investigated.NIH mice and Wistar rats served as donors and recipients respectively.Mouse-to-rat cardiac xenotransplantation was performed.The recipients were divided into 5 groups:group A (the control group),group B (PDTC group),group C (PDTC plus CsA group),group D (PDTC plus Lef group) and group E (PDTC plus Lef and CsA group).The expressions of IKKα/β,by immunohistochemistry and Western blot as well as electrophoretic mobility shift assay (EMSA).The median survival time of cardiac xenografts in the control group,PDTC group,PDTC plus CsA group,PDTC plus Lef group and PDTC plus Lef and CsA group was (2.17±0.41),(2.33±0.52),(4.67±1.21),(7.00±1.79) and (9.00±1.41) days respectively.The survival time of xenografts in the PDTC plus Lef and CsA group was significantly longer than that in other four groups (P<0.05 for all),that in the PDTC plus Lef group longer than that in the control group,PDTC group and PDTC plus CsA group (P<0.05 for all),that in PDTC plus CsA group longer than the control group and PDTC binding activity were notably increased in mouse-to-rat cardiac xenografts.The expressions were decreased in the control group,PDTC group,PDTC plus CsA group,PDTC plus Lef and PDTC plus Lef and CsA group in turn.It was concluded that PDTC plus Lef and CsA can significantly suppress prolonging the survival of the cardiac xenografts.

An oligonucleotide decoy for Stat3 activates the immune response of macrophages to breast cancer.

Tumor-associated macrophages (TAMs) have the potential to induce both immune activation and immune tolerance. Recent studies have indicated that in breast cancers the pro-tumor role of TAMs is dominant. We induced rat peritoneal macrophages with rat breast cancer cell-conditioned medium and analyzed signal transducer and activators of transcription 3 (Stat3) activities of the cells. Then these cells were transfected with Stat3 decoy oligonucleotides (ODNs) and were stimulated by lipopolysaccharide (LPS). The results demonstrate that induced macrophages displayed a reduction of cytotoxicity and antigen-presenting function in comparison with control but transfection with Stat3 decoy ODNs enhanced cytotoxicity and antigen-presenting function of the macrophages. Furthermore, injection of induced macrophages promoted tumor growth accompanied by immunosuppression in the rat tumor models, but injection of induced macrophages transfected with Stat3 decoy ODNs led to retarded tumor growth accompanied by immune activation. The data suggest that immunosuppressive activities of TAMs correlate with over-activated Stat3 signaling of the cells and disruption of Stat3 activity of TAMs can enhance rat immune response to breast cancer.

[The effect of selective cyclooxygenase-2 inhibitor nimesulide on breast cancer induced by dimethylbenzoic acid in rats].

OBJECTIVE: To study the effect of nimesulide (NIM) on the tumorigenesis of mammary tumors induced by dimethylbenzoic acid (DMBA), and to investigate possible mechanisms of NIM against tumors. METHODS: The studied rats were randomly divided into four groups: experimental control group, NIM group, diet and drug of NIM control group. The incidence of mammary tumor was observed. RT-PCR, Western blot were used to detect 8 cancerous tissues in every group, randomly. The expressions of cylooxygenase-2 (COX-2) were assessed by immunohistochemistry. The levels of prostaglandin E(2) (PGE(2)) in blood plasma and tumor tissues were determined by means of radio-immunity assay. The apoptosis index and the proliferation index were evaluated by TUNEL assay, immunohistochemical staining for proliferating cell nuclear antigen (PCNA), respectively. RESULTS: The incidence of mammary tumor was 69.2% in experimental control group, 40.3% in NIM group. There was obviously decreased incidence in NIM group; The expressions of COX-2 mRNA and protein were significantly down-regulated in NIM group compared with experimental control group. The increased levels of PGE(2) synthesis in blood plasma and tumor tissues were significantly decreased by administering NIM (P < 0.05). The apoptosis index was obviously higher, the proliferation index was markedly less in NIM group than experimental control group. CONCLUSIONS: Nimesulide could inhibit the tumorigenesis and development of DMBA-induced mammary tumors by inhibition of proliferation and induction of apoptosis. COX-2 and COX-2-mediated PGE(2) synthesis may play an important role in rat DMBA-induced breast cancer.

[Effect of tamoxifen plus octreotide on DMBA-induced mammary tumors in rats].

OBJECTIVE: To further study the antitumor effects of TAM and somatostatin (SST) analog octreotide (OCT) in vivo. METHODS: Eight weeks following dimethylbenzanthracene (DMBA, a single dose 100 mg/kg by subcutaneous injection) administration, 96 Wistar rats were randomly divided into four groups: control group, OCT (100 microg/kg bid for 14 weeks by subcutaneous injection) group, TAM (1 mg/kg 5 times weekly for 14 weeks by subcutaneous injection) group and OCT + TAM group. The mean latent phases of mammary tumorigenesis and incidence of mammary tumor-positive rats were observed. The number and volumes of tumors per animal were measured. The histological structures and ultrastructures of mammary samples were observed by using a light microscopy and a transmission electron microscopy. RESULTS: (1) The latent phases of mammary tumorigenesis in the TAM group and the OCT + TAM group were significantly longer than those in the control or OCT group (all P < 0.05). (2) The incidence of mammary tumor-positive rats were 70% in the control group, 52.4% in the OCT group, 45.5% in TAM group and 23.8% in the OCT + TAM group respectively, significantly longer in the three treated groups (P < 0.05 or P < 0.01), and the differences between the OCT + TAM group and the OCT group or TAM group were significant (both P < 0.05). (3) The numbers of mammary tumors per rat were markedly less in the three treated groups than in the control group (P < 0.05 or P < 0.01), and there were significantly differences between the OCT + TAM group and the OCT group or TAM group (all P < 0.05). (4) The mean volumes of mammary tumors per rat were significantly greater in the control group (6434 mm(3)) than in the three treated groups group (P < 0.05 or P < 0.01), but the tumor volume in the OCT + TAM group (1285 mm(3)) was less than those in the OCT group (4366 mm(3)) or TAM group (4138 mm(3)) (P < 0.01). At 10th week of treatment the mean volume was obviously smaller in TAM group than in OCT group (P < 0.05), but at 14th week of treatment the difference was not significant (P > 0.05). (5) Histopathological examination revealed that the mammary tumors in the OCT + TAM group were more differentiated and exhibited a less aggressive phenotype, compared with the tumors growing in the control group. CONCLUSION: Both OCT and TAM inhibit the tumorigenesis and development of DMBA-induced mammary tumors, however, resistance to TAM may appear during TAM treatment. Combination of OCT and TAM has significant synergetic antitumor effect. TAM in combination with OCT may become be an efficient hormone therapy means for breast cancer patients.

Effects of antisense oligonucleotides of PKC-alpha on proliferation and apoptosis of HepG2 in vitro.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignancies in China. The long-term survival rate of patients with HCC after prevention and management remains unsatisfactory. In order to provide a novel strategy to cure HCC, we investigated the effects of antisense oligonucleotides of PKC-alpha on proliferation and apoptosis of human hepatoma cell line HepG2 in vitro. METHODS: The human hepatocellular carcinoma cell line HepG2 was cultured and subcultured in RPMI1640 medium in vitro. PKC-alpha antisense oligonucleotides(asODN) of different concentrations with a random sequence as a control were transfected into HepG2 cells by lipofectin(LP). The cell growth index (GI) and the clone formation rate of HepG2 were detected by MTT colorimetric assay and soft agar assay, respectively. The apoptosis rate of HepG2 treated with PKC-alpha asODN was assayed by flow cytometry(FCM). The results were analyzed by SPSS 10.0 software. RESULTS: The GI of HepG2 transfected by PKC-alpha asODN with concentrations ranging from 0.10 micromol to 1.00 micromol were lower significantly than those of control groups (P<0.05). The clone formation rates of HepG2 transfected by PKC-alpha asODN from 0.05 micromol to 1.00 micromol were lower significantly than those of the control groups (P<0.01), and there was a dose-dependent relationship among them. The apoptosis rates of HepG2 treated with PKC-alpha asODN from 0.50 micromol to 1.00 micromol were significantly higher than those of the control groups. CONCLUSION: PKC-alpha asODN could inhibit the growth and proliferation of HepG2 and induce its apoptosis by blocking the cell signal transduction related to PKC-alpha in vitro, and may be potentially used in the prevention and management of recurrent and metastatic HCC.

Effects of tumor necrosis factor, endothelin and nitric oxide on hyperdynamic circulation of rats with acute and chronic portal hypertension.

AIM: To evaluate the effect of tumor necrosis factor (TNF), endothelin (ET) and nitric oxide (NO) on hyperdynamic circulation (HC) of rats with acute and chronic portal hypertension (PHT). METHODS: Chronic portal hypertension was induced in Wistar rats by injection of carbon tetrachloride. After two weeks of cirrhosis formation, L-NMMA (25 mg/kg) was injected into one group of cirrhotic rats via femoral vein and the experiment was begun immediately. Another group of cirrhotic rats was injected with anti-rat TNFalpha (300 mg/kg) via abdominal cavity twice within 48 h and the experiment was performed 24 h after the second injection. The blood concentrations of TNFalpha, ET-1 and NO in portal vein and the nitric oxide synthase (NOS) activity in hepatic tissue were determined pre-and post-injection of anti-rat TNFalpha or L-NMMA. Stroke volume (SV), cardiac output (CO), portal pressure (PP), superior mesenteric artery blood flow (SMA flow) and iliac artery blood flow (IAflow) were measured simultaneously. Acute portal hypertension was established in Wistar rats by partial portal-vein ligation (PVL). The parameters mentioned above were determined at 0.5 h, 24 h, 48 h, 72 h and 120 h after PVL. After the formation of stable PHT, the PVL rats were injected with anti-rat TNFalpha or L-NMMA according to different groups, the parameters mentioned above were also determined. RESULTS: In cirrhotic rats, the blood levels of TNFalpha, NO in portal vein and the liver NOS activity were significantly increased (P<0.05) while the blood level of ET-1 was not statistically different (P>0.05) from the control animals (477.67+/-83.81 pg/mL vs 48.87+/-32.79 pg/mL, 278.41+/-20.11 micromol/L vs 113.28+/-14.51 micromol/L, 1.81+/-0.06 u/mg.prot vs 0.87+/-0.03 u/mg.prot and 14.33+/-4.42 pg/mL vs 8.72+/-0.79 pg/mL, respectively). After injection of anti-rat TNFalpha, the blood level of TNFalpha was lower than that in controls (15.17+/-18.79 pg/mL vs 48.87+/-32.79 pg/mL). The blood level of NO and the liver NOS activity were significantly decreased, but still higher than those of the controls. The blood level of ET-1 was not significantly changed. PP, SV, CO, SMAflow and IAflow were ameliorated. After injection of L-NMMA, the blood level of NO and the liver NOS activity were recovered to those of the controls. PP and CO were also recovered to those of the controls. SV, SMAflow and IAflow were ameliorated. In PVL rats, the blood levels of TNFalpha, NO in portal vein and the liver NOS activity were gradually increased and reached the highest levels at 48 h after PVL. The blood level of ET-1 among different staged animals was not significantly different from the control animals. PP among different staged animals (2.4+/-0.18 kPa at 0.5 h, 1.56+/-0.08 kPa at 24 h, 1.74+/-0.1 kPa at 48 h, 2.38+/-0.05 kPa at 72 h, 2.39+/-0.16 kPa at 120 h) was significantly higher than that in controls (0.9+/-0.16 kPa). After injection of anti-rat TNFalpha in 72 h PVL rats, the blood level of TNFalpha was lower than that in controls (14+/-14 pg/mL vs 48.87+/-32.79 pg/mL). The blood level of NO and the liver NOS activity were significantly decreased, but still higher than those of the controls. The blood level of ET-1 was not significantly changed. PP was decreased from 2.38+/-0.05 kPa to 1.68+/-0.12 kPa, but significantly higher than that in controls. SV, CO, SMAflow and IAflow were ameliorated. After injection of L-NMMA in 72 h PVL rats, the blood level of NO and the liver NOS activity were recovered to those of the controls. PP, SV, CO, SMAflow and IAflow were also recovered to those of the controls. CONCLUSION: NO plays a critical role in the development and maintenance of HC in acute PHT and is a key factor for maintenance of HC in chronic PHT. TNFalpha may not participate in the hemodynamic changes of HC directly, while play an indirect role by inducing the production of NO through activating NOS. No evidence that circulating ET-1 plays a role in both models of portal hypertension has been found.

[Interleukin-18 antisense oligodeoxynucleotide promotes hepatocyte regeneration of partial liver allograft].

OBJECTIVE: To study the effect of interleukin (IL)-18 ASPODN on regeneration of allogeneic partial liver graft in rats. METHODS: Ninety donor SD rats and ninety recipient LEW rats were randomly divided into 3 groups: 50% partial liver transplantation group (PLT group); PLT+IL-18 antisense phosphorothioate oligodeoxynucleotide (ASPODN) treatment group (IL-18 ASPODN group) and PLT+IL-18 SPODN treatment group (IL-18 SPODN group) in which liposomes encapsulated IL-18 ASPODN or IL-18 SPODN were intravenous injection every day after PLT. BrdU labeling of hepatocytes, expression of IL-18 protein and IFN-gamma mRNA in liver graft, and serum level of IFN-gamma were measured with immunohistochemistry analysis, Western blotting, semi-quantification RT-PCR, and ELISA, respectively. RESULTS: Although regeneration of liver graft from each group peaked 72 hour after transplantation, BrdU labeling of hepatocytes in IL-18 ASPODN group (58.3%+/-7.5%) were significantly higher than those of PLT group (31.6%+/-6.7%) (t=6.503, P<0.001) and IL-18 SPODN group (33.4%+/-5.5%) (t=6.558, P<0.001). Expression of IL-18 protein and IFN-gamma mRNA in liver graft, and serum level of IFN-gamma in IL-18 ASPODN group from 48 hour, 72 hour and 96 hour after transplantation were significantly suppressed compared with PLT group (IL-18protein: t=2.950, t=5.916, t=7.947, P<0.05, P<0.001; INF-gamma mRNA: t=2.558, t=6.292, t=8.925, P<0.05, P<0.001; IFN-gamma level: t=16.998, t=15.483, t=54.723, P<0.001) and IL-18 SPODN group (IL-18 protein: t=2.845, t=6.062, t=6.973, P<0.05, P<0.001; INF-gamma mRNA: t=3.117, t=6.154, t=8.738, P<0.05, P<0.001; IFN-gamma level: t=14.531, t=18.139, t=46.924, P<0.001). CONCLUSION: IL-18 ASPODN could promote hepatocyte regeneration of allogeneic partial liver graft by the suppression of IL-18 and IFN-gamma production.

[The clinical significance of expression of cyclooxygenase-2 gene in breast cancer].

OBJECTIVE: To investigate the expressions of Cyclooxygenase-2 (COX-2) gene and protein in breast cancer, and understand its clinical significance. METHODS: Reverse transcription-PCR, immunohistochemistry were used to assess the expression of COX-2 in 30 cancerous tissues, paired tissues adjacent to breast cancers and 6 normal tissues far from neoplasm. RESULTS: Strong expression of COX-2 mRNA was detected in 86% of breast cancers with range of 0 - 1.180 with reference to the expression of beta-actin gene, and increased expression in paired tissues adjacent to cancers with range of 0 - 0.652, but nearly no expression in normal tissues. There were significant difference expression of COX-2 mRNA between cancers and paired tissues adjacent to cancers or normal tissues. Immunohistochemical analysis showed that COX-2 were expressed in 80% malignant epithelial cells, and 58% of paired tissues adjacent to cancers, no expression in normal tissues. CONCLUSION: Expressions of COX-2 gene and protein elevated not only in most human breast cancers, but also in paired tissues adjacent to cancers. Up-regulation of COX-2 is relatively early event in mammary carcinogenesis, nonsteroidal anti-inflammatory drugs and selective COX-2 inhibitors may be useful in the chemoprevention and therapy of human breast cancer.

Relationship between expression of somatostatin receptors subtype 2 mRNA and estrogen and progesterone receptors in breast cancer.

OBJECTIVES: To observe the expression of somatostatin receptor subtype 2 (SSTR2) mRNA, and investigate the relationship between the expression of SSTR2 mRNA and the expressions of estrogen and progesterone receptors (ERs and PRs) in benign and malignant breast tissues. METHODS: Samples from a total of 23 breast carcinomas, 16 mammary hyperplasias, and 9 mammary fibroadenomas were analyzed. SSTR2 mRNA expression was examined by in situ hybridization using multiphase oligoprobes. ER and PR expressions were detected by immunohistochemical staining. A computerized image analysis system was utilized to estimate the relative content of SSTR2 mRNA. RESULTS: The rate of expression (87.0%) and relative content (0.47) of SSTR2 mRNA in breast cancer were higher than those in benign breast tissue (64%, 0.26) (P < 0.05). SSTR2 mRNA expression was closely correlated with ER and PR expressions in breast cancer (P < 0.05). SSTR2 mRNA was also positively correlated with ER expression in benign breast tissues. CONCLUSIONS: SSTR2 mRNA expression is higher or in benign breast tissues than in malignant ones. There is a significant positive correlation between SSTR2 mRNA and ER and PR expressions. Combined antiestrogen and somatostatin analogue in treatment of ER-positive breast cancers should be further investigated.

Functional changes of dendritic cells derived from allogeneic partial liver graft undergoing acute rejection in rats.

AIM: To investigate functional change of dendritic cells (DCs) derived from allogeneic partial liver graft undergoing acute rejection in rats. METHODS: Allogeneic (SD rat to LEW rat) whole and 50 % partial liver transplantation were performed. DCs from liver grafts 0 hr and 4 days after transplantation were isolated and propagated in the presence of GM-CSF in vitro. Morphological characteristics of DCs propagated for 4 days and 10 days were observed by electron microscopy. Phenotypical features of DCs propagated for 10 days were analyzed by flow cytometry. Expression of IL-12 protein and IL-12 receptor mRNA in DCs propagated for 10 days was also measured by Western blotting and semiquantitative RT-PCR, respectively. Histological grading of rejection were determined. RESULTS: Allogeneic whole liver grafts showed no features of rejection at day 4 after transplantation. In contrast, allogeneic partial liver grafts demonstrated moderate to severe rejection at day 4 after transplantation. DCs derived from allogeneic partial liver graft 4 days after transplantation exhibited typical morphological characteristics of DC after 4 days' culture in the presence of GM-CSF. DCs from allogeneic whole liver graft 0 hr and 4 days after transplantation did not exhibit typical morphological characteristics of DC until after 10 days' culture in the presence of GM-CSF. After 10 days' propagation in vitro, DCs derived from allogeneic whole liver graft exhibited features of immature DC, with absence of CD40, CD80 and CD86 surface expression, and low levels of IL-12 proteins (IL-12 p35 and IL-12 p40) and IL-12 receptor (IL-12Rbeta(1) and IL-12Rbeta(2)) mRNA, whereas DCs from allogeneic partial liver graft 4 days after transplantation displayed features of mature DC, with high levels of CD40, CD80 and CD86 surface expression, and as a consequence, higher expression of IL-12 proteins (IL-12 p35 and IL-12 p40) and IL-12 receptors (IL-12Rbeta1 and IL-12Rbeta2) mRNA than those of DCs both from partial liver graft 0 hr and whole liver graft 4 days after transplantation (P<0.001) was observed. CONCLUSION: DCs derived from allogeneic partial liver graft undergoing acute rejection display features of mature DC.