Induction of biliary cholangiocarcinoma cell apoptosis by 103Pd cholangial radioactive stent γ-rays

Background In recent years, interventional tumor therapy, involving implantation of intra-cholangial metal stents through percutaneous trans-hepatic punctures, has provided a new method for treating cholangiocarcinoma, ^103Pd cholangial radioactive stents can concentrate high radioactive dosages into the malignant tumors and kill tumor cells effectively, in order to prevent re-stenosis of the lumen caused by a relapsed tumor. The aim of the present study was to investigate the efficacy of y-rays released by the ^103pd biliary duct radioactive stent in treating cholangiocarcinoma via induction of biliary cholangiocarcinoma cell apoptosis.
Methods A group of biliary duct cancer cells was collectively treated with a dose of y-rays. Cells were then examined by the 3-（4,5-dimethyl thiazol-2-yl）-2,5-diphenyl terazolium-bromide （MTF） technique for determining the inhibition rate of the biliary duct cancer cells, as well as with other methods including electron microscopy, DNA agarose gel electrophoresis, and flow cytometry were applied for the evaluation of their morphological and biochemical characteristics. The growth curve and the growth inhibition rate of the cells were determined, and the changes in the ultrastructure of the cholangiocarcinoma cells and the DNA electrophoresis bands were examined under a UV-lamp.
Results The y-ray released by ^103Pd inhibited cholangiocarcinoma cell growth, as demonstrated when the growth rate of the cells was stunned by a y-ray with a dosage larger than 197.321 MBq. Typical features of cholangiocarcinoma cell apoptosis were observed in the 197.321 MBq dosage group, while cell necrosis was observed when irradiated by a dosage above 245.865 MBq. DNA agarose gel electrophoresis results were different between the 197.321 MBq irradiation dosage group, the 245.865 MBq irradiation dosage group, and the control group.
Conclusions ^103pd radioactive stents which provide a radioactive dosage of 197.321 MBq are effective in the treatment of cholangiocaminoma; ^103pd radioactive     

INHIBITION OF ANGIOSTATIN TO THE GROWTH AND METASTASIS OF GASTRIC CANCER IN NUDE MICE

Objective To study the inhibition effect on tumor angiogenesis and metastasis of angionstatin, which generated from human plasminogen. Methods Plasminogen was isolated metastasis of angio-from human plasma by Sepharose chromatography arm then catalyzed by elastase. Angiostatin was isolated by Sepharose 4B-Lysine chromatography. Nude mice model of metastatic gastric cancer was set up by intact tumor tissue implantation orthotopically. From the day of operation, mice received daily intraperitoneal injections of human angiostatin,intact plasminogen, or saline, respectively. 24μg ( 1.2mg/kg) of angiostatin or plasminogen was given on the day of operation, followed by a daily dose of 12μg( 0. 6mg /kg ) via intraperitoneal injection for three weeks.Ten weeks after implantation, mice were sacrificed and autopsied. Microvascular density was measured by im.munohistochemistry. Results Molecular weight of plasminogen isolated from plasma was 94KD. Plaanino-gen was catalyzed into two fragment peptides by elastase , which were 41～43KD and 51～53KD in molecular weight. Growth of the orthotopically implanted tumor was significantly reduced in size in the mice treated with angiostastin with an inhibition rate of 54.0%. Tumor metastasis to the liver and peritoneum was also signifi-cantly inhibited by angiostatin with inhibition rate of 61.9% and 55.6% respectively. The microvascular den-sity was also decreased significantly in the angiostatin treated mice. Conclusion Angiostatin may be generat-ed from plasma, and has inhibitory effect both on tumor growth and metastasis in nude mice model of human gastric cancer.     

EFFECT OF“LIUWEI DIHUANG DECOCTION六味地黄汤” ON PREVENTIONANDTREATMENTOFTUMOR

Clinical and experimental investigations ofLiuwei Dihuang decoction(LWDH,an ancientrecipe)were made in our laboratory.LWDHdecreased the incidence of tumor of anteriorstomach or lung produced by N-nitrososarcosineor urethan in mice,prolonged the life of tumor-bearing animals,and increased the cAMP con-tent in murine uterocervical carcinoma(U_)cells.The recipe enhanced the phagocytic func-tion of the reticuloendothelial system,promot-ed the proliferation of hemogenic stem cells andlymphoid tissue,maintained thyroid function,and decreased the catabolism of protein in U_-bearing mice.LWDH also strengthened the mus-cles and increased body weight in normal mice.Patients with epithelial dysplasia of esopha-gus,a preneoplastic lesion of esophageal car-cinoma,were treated by using this recipe.Thecanceration rate within 1 year was 2.2%(2/92)in the treated,and 12.4%(11/89)in an untreatedgroup.Within 5 years these rates were 9%(5/57)and 26%(12/47)respectively(P<0.025).     

Anti-tumor effect of bufalin on the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice

Objective: To investigate anti-tumor effect of bufalin on the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice. Methods: BEL-7402 cells of human hepatocellular carcinoma were inoculated to form subcutaneous tumors in nude mice by subcutaneous injection. Then the subcutaneous tumors were implanted into the liver of nude mice, and the orthotopic transplantation tumor models of human hepatocellular carcinoma were established. Seventy-five models were randomized into 5 groups ( n = 15) . Bufalin was injected intraperitoneally into the 3 groups at dose of 1.5,1 and 0.5 mg/kg for day 15 - 24, respectively. NS group were injected equal volume saline as above and adriamycin were injected intraperitoneally into ADM group at dose of 8.0 mg/kg for day 15. Ten mice in each group were killed at day 25 and detected on morphological and ultrastructural changes in myocardium, brain, liver, kidney and tumor tissues by pathology and electron microscope. The survival time in each group     

Antitumorigenic potential of diallyl sulfide in Ehrlich ascites tumor bearing mice

To study the effects of diallyl sulfide (DAS), an organosulfur compound present in garlic (Allium sativum),on the life span ofehrlich ascites (EA) tumor bearing Swiss albino mice, cytotoxicity and angiogenesis. Methods EA tumor cells were maintained by serial transplantation in peritoneal cavity of male Swiss albino mice. EA tumor cells were inoculated at concentrations of 1 × 106EA cells, 2.5 × l06EAcells and 5 × 106 EA cells. DAS was given in 0.2 mi normal saline i. p., daily for seven days followed one hour later by inoculation with EA cells in respective groups. Results The results revealed that administration of DAS increased the life span of EA tumor bearing animals by more than 25 percent. A significant dose dependant cytotoxic response of DAS was also observed on EA tumor cells. DAS was also found to inhibit the angiogenesis in EA tumor bearing mice in a dose dependent manner. Conclusion It is suggested that DAS may exert its anticarcinogenic effects by more than one mechanism and is a useful chemopreventive and chemotherapeutic agent.     

Effect of Allicin in Antagonizing Mice's Bladder Cancerin vitro and in vivo

Objective: To explore anti-tumor effect and mechanism of Allicin in treating murine bladder tumor. Methods: To observe Allicin's effect on MBT-2 tumor cells in vitro, 100 μg/ml Allicin was added to the tumor cell culture, and the morphology of tumor cells was observed by phase contrast microscope 6 hrs later. The direct effects of Allicin on tumor cell growth in vitro in the MTT Assay was also evaluated. To determine anti-tumor effect of Allicin in vivo, C3H/He mice were randomly grouped prior to initiation of experiment. The mice received 1×105 MBT-2 cells administered subcutaneously into the right posterior flank on the Day 0 the experiment started. Allicin was injected at the site near tumor transplantation on the Day 1. The mice were examined for tumor development and the tumors were measured in two dimensions with calipers twice a week. On Day 21 the tumors were resected and examined pathologically to see the immune response. Results: The observation of morphology of MBT-2 cells in vitro and MTT a     

Heparanase antisense oligodeoxynucleotide inhibits angiogenesis and metastasis of human mammary carcinoma cell xenografts in nude mice

Objective： To evaluate the inhibitory effect of heparanase antisense oligodeoxynucleotide （AS-ODN） on the angiogenesis and metastasis of human mammary carcinoma cell xenografts in nude mice. Methods： The AS-ODN complementary to the start codon region of heparanase mRNA and its control, scrambled nonsense oligodeoxynucleotide （NS-ODN） were designed and synthesized. A subcutaneous growth model and an acute hematogenous metastasis model of human mammary carcinoma were established in nude mice and were treated with ODNs. The heparanase expression in tumor was evaluated by RT-PCR and Western blot. The microvessel density （MVD） was measured by immunohistochemistry for factor VS. The tumor volume was calculated and lung metastatic nodules were counted. Results ： The heparanase expression, MVD, tumor volume and lung metastatic nodules in AS-ODN treated group were significantly decreased compared with that in NS-ODN treated group and that in PBS group （P〈0.01）. Conclusion ： Heparanase AS-ODN has significant inhibitory effect on the angiogenesis and metastasis of human mammary carcinoma cell xenografts in nude mice.     

Simvastatin attenuates bleomycin-induced pulmonary fibrosis in mice

Background Bleomycin-induced fibrosis is extensively used to model aspects of the pathogenesis of interstitial pulmonary fibrosis. This study aimed to determine the benefic effects and mechanisms of simvastatin on bleomycininduced pulmonary fibrosis in mice. Methods Bleomycin-induced pulmonary fibrosis mice were administered with simvastatin in different doses for 28 days. We measured inflammatory response, fibrogenic cytokines and profibrogenic markers in both bleomycin-stimulated and control lungs, and correlated these parameters with pulmonary fibrosis. Results Simvastatin attenuated the histopathological change of bleomycin-induced pulmonary fibrosis and prevented the increase of lung hydroxyproline content and collagen （Ⅰ and Ⅲ） mRNA expression induced by bleomycin. Moreover, simvastatin down-regulated the increased expression of transforming growth factor-β1 （TGF-β1） and connective tissue growth factor （CTGF） induced by bleomycin at both gene and protein levels. Simultaneously, the accumulation of neutrophils and lymphocytes and the increased production of tumor necrosis factor-a （TNF-α） in bronchial alveolar lavage fluid were inhibited by simvastatin in early inflammatory phase after bleomycin infusion. The higher dose of simvastatin was associated with a more significant reduction in these inflammatory and fibrotic parameters. Furthermore, the inactivation of p38, RhoA and Smad2/3 signaling pathways was observed during simvastatin administration. Conclusions Simvastatin attenuated bleomycin-induced pulmonary fibrosis, as indicated by decreases in Ashcroft score and lung collagen accumulation. The inhibitory effect of simvastatin on the progression of pulmonary fibrosis may be demonstrated by reducing inflammatory response and production of TGF-β1 and CTGR These findings indicate that simvastatin may be used in the treatment of pulmonary fibrosis.     

Induction of protective antitumor activity of tumor lysate-pulsed dendritic cells vaccine in RM-1 prostate cancer model

Objective： To investigate the antitumor activity of tumor lysate-pulsed dendritic cells vaccine in RM-1 prostate cancer mice model with the survival time of mice calculated and the tumor size measured in DC vaccine therapy. Methods： C57BL/6 mice were immunized on the dorsal flank by s.c. inoculation of Lysate-DC, ova-DC, and non-DC on day -7. On day 0, 2× 10^6cells of RM-1 tumor cells （H-2b） were injected s.c. in C57BL/6 mice pre-treated by s.c. inoculation of modified DCs, correspondingly. DTH assay was performed with modified DCs. In partial test, for the determination of which immune cells were required for antitumor activity, mice were immunodepleted of CD4, CDS, or natural killer （NK） NK1.1 cells with the corresponding monoclonal antibodies. The survival time of nude mice loaded with tumor cells was calculated and the size of tumor measured. Results： In RM-1 mice prostate cancer model, immunized with lysate-DC, compared with ova-DC and non-DC, the pre-infection vaccine resulted in 100% clearance of primary tumors, whereas on day 0 of injection vaccine cleared 40-60% of primary tumors. On day 0, C57BL/6 mice （H-2b） were immunized with Lysate-DC, compared with ova-DC and non-DC by caudal vein injection, then on day 15, RM-1 cells were inoculated. On day 30, average diameters of tumor in different groups of modified DC were 23.7±5.4 mm, 22.1±4.9 mm, 4.3±2.6 mm, respectively. Lysate-DC, compared with ova-DC and non-DC, can greatly depressed RM-1 tumor cell growth （P〈0.01）. The mean survival time of C57BL/6 mice in Lysate-DC, ova-DC and non-DC groups were 15.8±2.6, 16.6±3.2, 39.0±5.6, respectively, and there was a significant difference in the mean survival time in lysate-DC group between ova-DC and non-DC group （P〈0.01）. DTH test showed that lysate-DC could prime T lymphocyte and elicit tumor antigen specific immune response, and over 80% mice in groups of lysate-DC showed obvious swelling in their foot pad. This response was strengthened with repeating inoculation, whereas DTH response was not seen in control group. In vivo depletion of NK cells resulted in a 40-60% reduction in growth suppression within the primary tumor, and depletion of CD4^＋ cells resulted in a 20% reduction in growth suppression. Conclusion： The minor lysate-pulsed dendritic cells vaccine could elicit antitumor activity in RM-1 loaded C57BL/6 mice, and prolong the duration of RM-1 loaded C57BL/6 mice. So DC-based immunotherapy with hormone-refractory prostate carcinoma yielded protective immunity, generated efficient cellular antitumor responses, thereby providing further preclinical support for feasible immunotherapy approaches for prostate cancer.     

Growth suppression of human lung cancer cells and implanted tumors by adenovirus-mediated transfer of the PTEN gene

This study examined the effects of a recombinant adenovirus AdTEN-EGFP on the proliferation of A549 cells, a human lung carcinoma cell line, in vitro and on the growth of the implanted tumors in the nude mice in vivo, explored the underlying mechanisms and evaluated the in vitro transfection efficiency of Ad-PTEN-EGFP into A549 cells. The expression of Ad-PTEN-EGFP in the A549 cells was determined. The proliferation and the apoptosis rates of the A549 cells with Ad-PTEN-EGFP transfection or not was detected by MTT and flow cytometry. Ad-PTEN-EGFP at different doses was injected intratumorally to the tumor-bearing mice induced by the A549 cells. Tumor sizes were measured on an alternate day. After all the mice were sacrificed, the implanted tumors were removed for routine histological examination, weight test, HE staining and immunohistochemical staining. The expressions of Bax, P16 and P53 in the tumor tissues and those of caspase-3, CD34 and VEGF in the mouse sera were detected. Tumor cell apoptosis was measured by TUNEL method. The results showed that the vitality of the A549 cells after transfection with Ad-PTEN-EGFP declined. The expression of green fluorescent protein was observed under fluorescent microscope. The transfection rate was in excess of 50%. The mRNA and protein expression of PTEN in the transfected cells was confirmed. The proliferation rate of the transfected cells was significantly decreased when compared with that of the non-transfected cells （P〈0.05）. The number of the apoptosi＇s cells was increased in the transfected cells （P〈0.05）. The models of implanted tumors were successfully estab- lished by injection of the A549 cells in the flank of Balb/c nude mice. Administration of Ad-PTEN-EGFP to the tumor-bearing nude mice resulted in a suppression of tumor growth. There were statistically significant differences in the tumor weight and tumor volume between the Ad-PTEN-EGFP-treated group and the control groups （P〈0.05）. In contrast to those in the control groups, tumor tissues in the Ad-PTEN-EGFP-treated group were shown to have typical extensive vacuolar degeneration and massive hemorrhagic necrosis. Apoptotic bodies were also observed in the tumor cells. The expressions of Bax, caspase-3 and P16 were increased （P〈0.05） while those of CD34, VEGF and P53 decreased （P〈0.05） in the Ad-PTEN-EGFP-treated group. It is concluded that Ad-PTEN-EGFP could induce the apoptosis of the A549 cells and inhibit their proliferation. And it could also substantially suppress the tumor growth in the tumor-bearing nude mice and induce apoptosis of the tumor cells as well. These findings carry significant implications for adenovirus vector-based PTEN gene therapies for lung cancers.     

Screening of specific binding peptide targeting to blood vessel of human esophageal cancer using phage display C7C peptide library in vivo

AIM: Identify the candidate peptides used for anti-angiogenic therapy of esophageal cancer by in vivo screening C7C peptide library for peptides binding specifically to blood vessels of human esophageal cancer. METHODS: The phage displayed C7C peptide library was injected intravenously into mice bearing human esophageal tumor xenografts under renal capsule. After 5 rounds of screening, 13 clones were picked up individually and sequenced. During each round of screening, titers of phage recovery were calculated from tumor xenograft and control tissues. Homing of these 9 peptides to tumor vessel was detected by calculating phage titers in the tumor xenograft and control tissues(lung and spleen) after each phage was injected into mice model, and compared with the distribution of phage M13 and Ⅷ-related antigen in tumor xenograft by immuno- histochemical staining. Comparisons among groups of data were made using one-way analysis of variance (ANOVA), followed by the Bonferroni multiple comparisons test. RESULTS: The number of phage recovered from tumor tissue of each round increased gradually and the number of the fifth round was about 10.63 folds than it is in the first round (P<0.01). At the same time, the titers of spleen and lung used as control tissues decreased (P<0.01in spleen & P<0.05 in lung) by using analysis of variance of repeated measurement data. Immunohistochemical staining showed similar staining pattern with M13 antibody or Ⅷ-related antigen antibody, suggesting that phages displaying the selected peptides could home to blood vessel of human esophageal cancer. According to their DNA, 9 corresponding peptide sequences were deduced. And the homing ability to blood vessel of phages displaying the selected peptides was confirmed by comparing with their recovery in tumor and control tissues There was significant statistical difference in sequence YSFNSWM,PNPNNST,YSINDWH,LPAMPNS,YPTPYDI ,PMNADNL,SRHDLNS and STVATSQ between tumor group and the two control group as using analysis of variance. Two motifs,YSXNXW and PXNXXN,were also obtained by analyzing the homology of these peptide sequences. The staining distribution of phage with the sequence of PNPNNST was similar to that of the blood vessel marker Factor Ⅷ-related antigen staining. After sequencing, each phage with the selected peptide of PNPNNST with 1011 pfu was injected intravenously into mice. The homing ability to tumor vessel of these 9 kinds of peptides in the xenograft was higher than control tissues(lung and spleen). CONCLUSION: Nine peptides obtained from in vivo screening homed to the blood vessel of human esophageal cancer, and the two motifs of YSXNXW and PXNXXN are the possible biochemical recognition units binding to vascular endothelial cells of esophageal cancer. CONCLUSION: Nine peptides obtained from in vivo screening homed to the blood vessel of human esophageal cancer, and the two motifs of YSXNXW and PXNXXN are the possible biochemical recognition units binding to vascular endothelial cells of esophageal cancer.     

Endogenous porphyrins in murine skin and transplanted PAM-212 squamous cell carcinoma tissues after injection of delta-aminolevulinic acid.

After intraperitoneal (IP) injection of delta-aminolevulinic acid (ALA), the endogenous porphyrins in murine skin and tumor tissues were determined by a method involving solvent and acid extractions. The results showed that the total amount of porphyrins in the tumor tissues after ALA injection was much higher than that in the skin from the same mice, although the amount of porphyrins in the skin from the ALA-injected mice was higher than that from the saline-injected (control) mice. The porphyrins in the tumor were mostly protoporphyrin and coproporphyrin, with only a small amount of uroporphyrin. The optimum period for porphyrin accumulation in the tumor as well as in the skin was 1 hour after the injection of ALA. As the period was extended to 3 and 6 hours, the amount of porphyrins in these tissues decreased considerably. These findings could be valuable for further application of ALA in the photodynamic therapy of skin cancer.

     

Effects of Ad-p27mt gene transfer on the expression of Bax,Bcl-2, VEGF and MMP-9 in the transplanted liver tumors in nude mice

In this study, the mechanism by which Ad-p27mt inhibits the growth, invasion and metastasis of transplanted liver tumor was studied by examining the effects of Ad-27mt gene transfer on the expression of Bax, Bcl-2, VEGF and MMP-9 in the transplanted liver tumors in nude mice.The model of transplanted hepatic tumor was established in nude mice.The mice were then divided into three groups, which were injected with PBS, Ad-LacZ and Ad-p27mt and the growth of the transplanted liver tumor was observed.The expressions of P27, Bax and Bcl-2 proteins were detected by Western blotting and the expressions of VEGF and MMP-9 were immunohistochemically determined.Our result showed that the tumor size, expressions of Bax, Bcl-2 proteins, VEGF and MMP-9 were all lower than those in PBS and Ad-LacZ groups and the differences were statistically significant (P<0.05).Our study suggested that Ad-p27mt could inhibit the growth, invasion and metastasis of hepatic cancer by lowering the expressions of VEGF and MMP-9.     

Screening of specific binding peptide targeting blood vessel of human esophageal cancer in vivo in mice

Background  Cancer of the esophagus and gastroesophageal junction remains a virulent malignancy with poor prognosis. Rapid progresses were made in chemotherapeutic agents and the development of molecular markers allowed better identification of candidates for targeted therapy. This study aimed to identify the candidate peptides used for anti-angiogenic therapy of esophageal cancer by in vivo screening C7C peptide library for peptides binding specifically to blood vessels of human esophageal cancer.

Methods  The phage displayed C7C peptide library was injected intravenously into mice bearing human esophageal tumor xenografts under renal capsule. After 5 rounds of screening, 13 clones were picked up individually and sequenced. During each round of screening, titers of phage recovery were calculated from tumor xenograft and control tissues. Homing of these 9 peptides to tumor vessel was detected by calculating phage titers in the tumor xenograft and control tissues (lung and spleen) after each phage was injected into mice model, and compared with the distribution of phage M13 and VIII-related antigen in tumor xenograft by immunohistochemical staining. Comparisons among groups of data were made using one-way analysis of variance (ANOVA), followed by the Bonferroni multiple comparisons test.

Results  The number of phage recovered from tumor tissue of each round increased gradually in tumor group while decreased in control groups (P <0.01 in tumor and spleen, P <0.05 in lung). Immunohistochemical staining showed similar staining pattern with M13 antibody or VIII-related antigen antibody, suggesting that phages displaying the selected peptides could home to blood vessel of human esophageal cancer. According to their DNA, 9 corresponding peptide sequences were deduced. And the homing ability to blood vessel of phages displaying the selected peptides was confirmed by comparing with their recovery in tumor and control tissues. Two motifs, YSXNXW and PXNXXN, were also obtained by analyzing the homology of these peptide sequences. The staining distribution of phage with the sequence of PNPNNST was similar to that of the blood vessel marker factor VIII-related antigen staining. After sequencing, each phage with the selected peptide of PNPNNST with 1.0×10¹¹ pfu/ml was injected intravenously into mice. The homing ability to tumor vessel of these 9 kinds of peptides in the xenograft was higher than control tissues (lung and spleen).

Conclusion  Nine peptides obtained from in vivo screening homed to the blood vessel of human esophageal cancer, and the two motifs of YSXNXW and PXNXXN are the possible biochemical recognition units binding to vascular endothelial cells of esophageal cancer. 

     

Hepatoprotective activity of the ethanol extract of Sarcopyramis Nepalensis

The present study examined the protective effect of the ethanol extract of Sarcopyramis nepalensis (EESN) on agents-induced hepatotoxicity in mice and the possible mechanism. Acute liver injury was induced by administration of either CCl4 or D-GalN. The animals were divided into 5 groups in terms of different treatment: normal group, CCl4 or D-GalN group, silymarin or bifendate group, low dose EESN group (10 mg/kg) and high dose EESN group (30 mg/kg). Liver function was evaluated by detecting the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The oxidize stress markers were measured, including malondialdehyde (MDA), glutathione peroxidase (GSH) and superoxide dismutase (SOD). Liver tissues were histopathologically examined by hematoxy-lin-eosin (H&E) staining. The acute toxicity study revealed that there was no toxicity of EESN at the dose of 5 g/kg in mice. The levels of ALT and AST in serum, and the MDA level in live tissues were significantly increased and the activities of SOD and GSH substantially decreased in mice after CCl4 or D-GalN treatment. These biochemical and oxidize stress markers were profoundly improved after treatment with EESN at different doses, which was similar to the results of silymarin or bifendate treatment. The histophathological examination revealed the significant improvement in the pathological changes of the liver in EESN-treated mice as compared to those in CCl4 or D-GalN group. It was concluded that EESN possesses potential antioxidant and hepatoprotective properties and has therapeutic potential for liver diseases.     

Anti-tumor Immunity Elicited by Adenovirus Encoding AdhTrp2 or AdmTrp2 without Vitiligo

To compare the difference in tumor immunity and autoimmunity elicited by adenovirus(Ad) encoding human or murine tyrosinase-related protein 2(AdhTRP2 or AdmTRP2),and to find the most effective way to induce immunity by AdhTRP2 or AdmTRP2,C57BL/6 mice were im-munized with AdhTRP2 or AdmTRP2 intramuscularly at different doses of 105,106,107 and 108 separately(10 mice for each dose).Two weeks after the immunization,in vivo CTL assay and in-tracellular staining(ICS) of IFN-γ were carried out to analyze the dose-effect relationship.Tumor growth and vitiligo(as an sign of autoimmunity) were observed until 3 months after challenge with 105 B16F10 tumor cells.The results showed that Ad encoding AdmTrp2 induced weak tumor im-mune response.Similar immunization with AdhTrp-2 elicited stronger protective immunity.CTL activity and IFN-γ-produced CD8 T cells were directly proportional to dose of AdhTrp2 or AdmTrp2.Moreover,AdhTrp2 group showed tumor rejection in 100% of challenged mice till the end of 3rd month while 60% of mice immunized with AdmTrp2 were protected against tumor.In the whole process of this experiment,no vitiligo was observed in mice immunized either with AdhTrp2 or AdmTrp2.It is concluded that anti-melanoma responses induced by genetic vaccina-tion expressing xenoantigens breaks immune tolerance effectively and is able to elicit strong anti-gen-specific cytotoxic T cell response without vitiligo.     

Effects of Ning Shen Ling Granule (宁神灵冲剂) and dehydroepiandrosterone on cognitive function in mice undergoing chronic mild stress

Objective： To investigate the changes of spontaneous and cognitive behavior, and cholinergic M receptors in the brain of mice subjected to chronic mild stress （CMS）, and to determine the effect of Ning Shen Ling Granule （宁神灵冲剂, NSL） and dehydroepiandrosterone （DHEA） on them. Methods：CMS model mice were established by applying stress every day for 3 consecutive weeks with 7 kinds of unforeseeable stress sources, and they were medicated for 1 week beginning at the 3rd week of modeling. The changes in behavior were determined by Morris Water Maze and spontaneous movement test, and M-receptor binding activity in cerebral cortex, hippocampus and hypothalamus were measured by radioactive ligand assay with 3H-QNB. Results： （1） The spontaneous movement in CMS model mice was significantly reduced, with the latency for searching platform in Morris Water Maze obviously prolonged （P〈0.01）, and these abnormal changes in behavior were improved in those treated with NSL and DHEA. （2） The binding ability of M-receptor in cerebral cortex and hippocampus of CMS mice was significantly decreased as compared with those in the control group （P〈0.05）, but could be restored to the normal level after intervention with NSL or DHEA. Conclusion： The decline of spontaneous movement and spatial learning and memory ability could be induced in animals by chronic mild stress, and that may be related to the low activity of central cholinergic M-receptors. Both NSL and DHEA could effectively alleviate the above-mentioned changes.     

Therapeutic antitumor response to cervical cancer in mice immunized with U14 v accines transfected with costimulatory B7 gene

Objective To investigate the effect of U14 vaccine transfected with the B7 gene in inducin g antitumor immune response to murine cervical carcinoma in Chinese 615-strain mice.Methods A recombinant retroviral plasmid vector expressing mouse B7-1 gene (pLNSX-mB7) was transfected into 615-strain mouse cervical carcinoma cell line No. 14 (U1 4) by electroporation to set up a highly-expressed mB7-1 U14 cell clonal strai n (B7(+)U14). In vivo experiments: (1) B7(+)U14 vaccine was primed to protect t he 615-strain mice against U14 re-challenge. (2) B7(+)U14 vaccine was injecte d into tumor-bearing mice with different tumor sizes. Lifetimes and tumor s izes were recorded. In vitro cytotoxicity assay: Mice were immunized with B 7(+)U14 or U14 vaccine and 2 weeks later, spleen cells of those mice were cultur ed for 2 days. The cytotoxicity of these cells against U14 was detected by 5-d iphenyl tetrazolium bromide assay.Results We obtained several B7-1 high expression clonal U14 lines. In vivo experiment, we did not find tumor growing in 3 of the 6 mice primed by B7(+)U14 vaccine during their entire life after re-challenge with U14. The other 3 mice develo ped tumors and their average survival time was longer than that of the control g roup (P&lt;0.01). All 6 mice grew tumors in the control group. When the transplanted tumors became palpable, the mice were randomly divided into 3 group s to be injected with B7(+)U14 vaccine. It was effective for tumor-bearing mic e only when the tumor diameters were &lt;3 mm. When the diameters were ≥3 mm, it was not efficacious to inject B7(+)U14 vaccine (P&lt;0.05). In vitro cytotoxicity assay, cytotoxic T lymphocytes induced by B7(+)U14 vaccine h ad a high er cytotoxicity against U14 than that induced by U14 vaccine (F=310.8, P &lt;0.001).Conclusions Vaccines of cervical cancer cells transfected with the costimulatory molecule B7 gene can induce antitumor immune protection in host mice against U14 re-challe nge. This treatment may cure part of the tumor-bearing mice but be restricted by tumor size. The results suggest that transfecting the B7 gene into cervical cancer as a cell vaccine may be an efficient supplementary method to treat cervi cal cancer after operation.     

Effect and Mechanism of Superantigen Staphylococcal Enterotoxin Therapy for Mouse Gastric Tumor

Summary:The anti-tumor effect and mechanism of the staphylococcal enterotoxin A(SEA)werestudied.The mouse gastric tumor model was produced by subcutaneously inoculating gastric tumorcells(MGC80-3).The experimental group was treated with SEA,and the control group was treatedwith normal saline.The percentage of tumor generation and tumor mass was measured.The resultsshowed that the percentage of the tumor tumor generation in the SEA-treated mice was lower than in thecontrol group,but there was no significant difference(P>0.05).However,the tumor mass in theexperimental group was significantly lighter than in the control group,with the difference bing verysignificant(P<0.001).There were more CD_4~+ T cells and CD_8~+ T cells in the tumor of the micetreated with SEA than those of the control group.SEA has an obvious anti-tumor effect on mice gas-tric tumor.The mechanism might be that SEA induces the effect of superantigen-dependent cell me-diated cytotoxicity to the tumor cells.     

Morinda citrifolia (Noni) Juice Suppresses A549 Human Lung Cancer Cells via Inhibiting AKT/Nuclear Factor-κ B Signaling Pathway

Objective: To study the mechanism of the anti-tumor effect of Morinda citrifolia (noni). Methods: The influences of noni juice on cell proliferation, apoptosis, invasion, migration and the activity of AKT/nuclear factor-κ B (NF-κ B) signaling pathway in A549 human lung cancer cells were detected by MTT, cell counting kit-8, colony formation, Annexin V/PI double labeling, transwell, scratch test and immunoblotting assay, respectively. A549 cells were inoculated into the right axilla of nude mice, followed by noni juice treatment. The body weight of the nude mice was weighed, and the tumor volume and weight were measured. Cell proliferation and expression of apoptosis-related proteins were measured by immunohistochemistry, and the activity of NF-κ B signaling pathway was measured by immunoblotting. Results: The in vitro studies showed that noni juice inhibited the A549 cells proliferation, migration and invasion. Noni juice also promoted cells apoptosis in A549 cells. Immunoblotting assay showed that the phosphorylation level of AKT, p50, and STAT3 proteins was inhibited to different extents after noni juice treatment. The in vivo studies showed that noni juice effectively suppressed tumor formation of A549 cells in nude mice. Noni juice treatment inhibited the expression of Ki67, PCNA, and Bcl-2 protein in the tumor; while promoted the expression of caspase-3 protein. Additionally, we also found that noni juice treatment could restrain the activity of AKT/NF-κ B signaling pathway in the tumor tissue. Conclusion: Noni juice inhibited the proliferation of A549 lung cancer cells, induced apoptosis, and inhibited cell invasion and migration via regulating AKT/NF-κ B signaling pathway.