E1A基因对人肺腺癌细胞增殖和转移的影响

目的　探讨腺病毒 5型早期区 1A(Ad5E1A)基因对人肺腺癌细胞增殖和转移的影响 ,为人肺腺癌E1A基因治疗的可行性提供实验依据。方法　用本室构建的真核表达重组质粒pcDNA3 E1A ,通过脂质体介导将E1A基因转入人肺腺癌细胞系 (Anip 973) ,经G418筛选获得稳定表达E1A基因的转染细胞 (Anip 973 E1A)。通过体内、外实验 (生长速度、倍增时间、软琼脂集落形成、裸鼠致瘤性及转移能力等 ) ,观察E1A基因对人肺腺癌细胞增殖和转移的影响。结果　体外实验显示 ,Anip 973 E1A细胞生长速度减慢 ,倍增时间延长 ;软琼脂集落形成能力显著降低 ,亲本Anip 973、Anip 973 vect和Anip 973 E1A细胞集落形成率分别为 19.7%、17.2 %和 2 .3% ,集落形成抑制率为 86 .6 %。体内实验显示 ,Anip 973 E1A细胞组出瘤时间晚 ,肿瘤体积小 ,有 2只裸小鼠至实验结束未触及肿瘤 (2 /6 ) ,抑瘤率为 6 0 .1%。第 2 1天计数肺转移灶 ,亲本Anip 973、Anip 973 vect和Anip 973 E1A细胞组肺转移灶数目分别为 16 .1± 3.2、15 .4± 3.8和 7.3± 2 .3个 ,转移抑制率为 5 2 .6 %。结论　E1A基因能显著抑制人肺腺癌细胞的恶性表型 ,降低裸鼠致瘤性 ,减少肺转移灶形成 ,为人肺腺癌E1A基因治疗提供了实验依据。     

抗CD71人-鼠嵌合抗体的体外抗肿瘤效应及在肿瘤异种移植模型中的定位研究

Objective To investigate the anti-tumor effects in vitro and in vivo distribution of the human/murine chimeric antibody (D2C).Methods The CD71 positive target cells(K562,CEM and SMMC7721) and the effector cells ,freshly isolated human PBMC,with the ratio of target cells to effector cells 1:50,were incubated in various dilutions of D2C antibody(Ab).Antibody dependent cytotoxicity(AD-CC) was tested by using an LDH-release assay.Instead of effector cells,complement was added to the target cells (CEM,SMMC-7721) with various dilutions of D2C Ab.A method of counting death cells was used in complement dependent cytotoxicity(CDC) assay.Tumor localization and distribution of the chimeric antibody(D2C) were observed by labeling the chimeric Ab with radioiodine(^131I) and injecting in into nude mice(Balb/c nu/nu) transplanted with human hepatocellular carcinoma cells (SMMC-7721).Results A significant ADCC was observed with the increased concentration of the D2C Ab.Cytolysis of CD71-positive target cells by the D2C Ab was found in the presence of fresh rabbit complement.Labeled D2C administered by intraperitoneal as well as tumor regional in-jection,was visualized by SPECT. The distribution of D2C Ab in murine organs and tissues showed that non-specific binding was lower fol-lowing tumor regional administration than when the antibody was administered by an intraperitoneal injection.The human/murine chimeric antibody(D2C) has in vitro anti-tumor effects and can exert its effects in specific tumor localization.Its distribution and local effects in vi-vo can be detected by radioimmunoimaging. Conclusion CD71 human/murine chimeric antibody showed marked killing of tumor cells in vitro,and specific recognition and high affinity binding to tumor tissue in vivo.     

Enhanced anti-tumor and anti-angiogenic effects of metronomic cyclophosphamide combined with Endostar in a xenograft model of human lung cancer

Standard chemotherapy for advanced NSCLC has reached a therapeutic plateau. More effective strategies must be explored. The purpose of this study was to evaluate the role of metronomic chemotherapy combined with an angiogenesis inhibitor in non-small cell lung cancer (NSCLC). A total of 114 BALB/c nude mice were inoculated subcutaneously with human NSCLC cells (A549), and when xenograft tumors were palpable, mice were randomly injected with saline as controls (Ctrl), or treated with metronomic cyclophosphamide (MET CPA), recombinant human endostatin, Endostar (Endo), MET CPA combined with Endostar (MET CPA+Endo) or maximum tolerance dose of CPA (MTD CPA), respectively. The growth of xenograft tumors and mouse survival were monitored. The frequency of peripheral blood circulating endothelial cells (CECs), microvessel density (MVD) and pericyte coverage was determined using flow cytometry and immunofluorescence staining. In comparison with the controls, treatment with either drug significantly inhibited the growth of xenograft tumors in mice. Treatment with MET CPA or Endostar, but not with MTD CPA, significantly reduced the frequency of peripheral blood total and viable CECs and the value of MVD. Endostar also considerably reduced pericyte coverage in xenograft tumors. Moreover, MET CPA combined with Endostar further reduced the frequency of peripheral blood CECs, the value of MVD, and pericyte coverage, with concomitant delay in tumor growth and extension of mouse survival. Our results indicate that MET CPA combined with Endostar results in enhanced anti-tumor and anti-angiogenic effects in a xenograft model of human lung cancer. Combined therapy with metronomic chemotherapy and an angiogenesis inhibitor may serve as a promising treatment strategy for patients with advanced NSCLC.     

Transformation-defective adenovirus 5 E1A mutants exhibit antioncogenic properties in human BLM melanoma cells

Adenoviral E1 A proteins exhibit a strong tumor-suppressive activity in human tumor cells. However, E1 A is capable of transforming rodent and human cells in cooperation with other oncoproteins, such as activated RAS. Thus, the therapeutic use of wild-type E1A harbors the principal risk of enhancing tumor malignancy. This prompted us to construct E1A 13S cDNA-derived mutants that were unable to transform baby mouse kidney cells in cooperation with E1B and to test their tumor-suppressive activity in BLM human melanoma cells. Anchorage-independent growth in soft agar was reduced for those cell lines expressing the E1AdelCR2 mutant, which lacks the entire conserved region 2 (CR2) sequences, or for cells expressing the E1AcR3Ex2 mutant, which contains CR3 plus exon 2 sequences. In contrast, cell lines expressing the entire E1A wild-type (E1AWT) or only the exon 2 sequences (E1AEx2) grew like the parental BLM cells. Moreover, inoculation of nude mice with BLM cells or cells expressing E1AEx2 revealed large tumors after 2 weeks. In contrast, tumors derived from E1AdelCR2- or E1ACR3Ex2-expressing cells exhibited a substantial delay in tumor growth accompanied by a loss of E1A expression in the outgrown tumors. Cell lines expressing E1AWT showed an intermediate phenotype. Thus, expression of CR3 plus exon 2 sequences is sufficient to enhance both the antioncogenic properties and the therapeutic safety of E1A in our system.     

A HLA-A2-restricted CTL epitope induces anti-tumor effects against human lung cancer in mouse xenograft model

Cancer immunotherapy is attractive for antigen-specific T cell-mediated anti-tumor therapy, especially in induction of cytotoxic T lymphocytes. In this report, we evaluated human CTL epitope-induced anti-tumor effects in human lung cancer xenograft models. The tumor associated antigen L6 (TAL6) is highly expressed in human lung cancer cell lines and tumor specimens as compared to normal lung tissues. TAL6 derived peptides strongly inhibited tumor growth, cancer metastasis and prolonged survival time in HLA-A2 transgenic mice immunized with a formulation of T-helper (Th) peptide, synthetic CpG ODN, and adjuvant Montanide ISA-51 (ISA-51). Adoptive transfer of peptide-induced CTL cells from HLA-A2 transgenic mice into human tumor xenograft SCID mice significantly inhibited tumor growth. Furthermore, combination of CTL-peptide immunotherapy and gemcitabine additively improved the therapeutic effects. This pre-clinical evaluation model provides a useful platform to develop efficient immunotherapeutic drugs to treat lung cancer and demonstrates a promising strategy with benefit of antitumor immune responses worthy of further development in clinical trials.     

Triptolide augments the effects of 5-lipoxygenase RNA interference in suppressing pancreatic tumor growth in a xenograft mouse model

Purpose  

Pancreatic cancer has one of the highest fatality rates of all cancers, and new strategies or reagents to tackle this disease are needed. Triptolide (TL) is able to potently inhibit the growth of pancreatic tumor cells in vitro. On the other hand, blockage of 5-LOX pathway might be useful for treatment of pancreatic cancer. In the current study, we tested the effects of 5-LOX RNA interference and TL individually or in combination in suppressing human pancreatic tumor growth in xenograft mouse model.     

Development of a large-animal human brain tumor xenograft model in immunosuppressed cats

A large-animal model was developed to facilitate the noninvasive investigation of the effect on the human glioma-derived D-54 MG (glioblastoma multiforme) continuous cell line of a variety of therapeutic regimens. Twenty random-bred male cats were inoculated intracerebrally with 1 x 10(7) D-54 MG tumor cells after being initiated on one of three preparatory regimens of cyclosporin A p.o. Reproducible success of D-54 MG xenotransplantation (100%, 6 of 6 cats) was achieved only after pretreatment with 120 mg cyclosporin A p.o. (24-30 mg/kg) daily for greater than or equal to 10 days prior to tumor implantation. High-performance liquid chromatography-derived whole blood cyclosporin A 12-h trough levels of greater than or equal to 640 ng/ml were seen in successful implants. Lesions ranging from 2 to 20 mm in diameter were seen in cats sacrificed 27-44 days after implantation with no growth seen in control animals. Histopathological examination revealed the tumors to be well-circumscribed anaplastic intracerebral tumors with some invasion into surrounding host parenchyma. Perivascular lymphocytic cuffing was observed, but intratumoral lymphocytic infiltration was minimal. Gadolinium-EDTA-enhanced nuclear magnetic resonance imaging provided accurate tumor localization in T1-weighted images (TE 26 ms; TR 600 ms). Biochemical tests of kidney, liver, and hematological function were within normal limits, although 10% (2 of 20) of the animals developed gingival hyperplasia, and 5% (1 of 20) developed intussusception. The reproducible growth of the D-54 MG human glioblastoma cell line in a large-animal model eliminates many of the limitations associated with the standard nude mouse/rat model, thereby providing a novel test bed for a variety of imaging modalities as well as for drug immunoconjugate localization and toxicity studies.     

ProstaCaid inhibits tumor growth in a xenograft model of human prostate cancer

We have recently demonstrated that the dietary supplement ProstaCaid (PC) inhibits growth and invasive behavior of PC-3 human prostate cancer cells in vitro. In the present study, we evaluated toxicity and whether PC suppresses growth of prostate cancer in a xenograft model of human prostate cancer cells implanted in mice. Here, we show that an oral administration of PC (100, 200 and 400 mg/kg) did not affect body weight or activity of liver enzymes (ALT, AST) and did not show any sign of toxicity in liver, spleen, kidney, lung and heart tissues in mice. In addition, PC treatment resulted in the inhibition of tumor volumes (1024.6 ± 378.6 vs. 749.3 ± 234.3, P<0.001) in a xenograft model of prostate cancer with human hormone refractory (independent) PC-3 prostate cancer cells. Moreover, qRT-PCR analysis demonstrated significant upregulation of expression of CDKN1A (p21) and inhibition of expression of IGF2, NR2F2 and PLAU (uPA) genes by an oral administration of PC in prostate cancer xenografts. Our study demonstrates that the concentrations of the dietary supplement ProstaCaid tested did not show signs of toxicity, and its oral application has significant anticancer activity in vivo and can be considered as an alternative treatment for prostate cancer patients.     

Hypoxia-induced human endonuclease G expression suppresses tumor growth in a xenograft model

We have developed a hypoxia-inducible gene therapy approach for the expression of the mature form of human endonuclease G to facilitate cell death in hypoxic regions of the tumor. The chimeric therapeutic gene is placed under the control of a hypoxia response element based promoter and contains a translocation motif linked in frame to an oxygen-dependent degradation domain and the endonuclease G gene. Transient expression of the chimeric therapeutic gene in breast and prostate cancer cell lines resulted in efficient cell death under hypoxia-mimetic conditions. Stable MDA-MB-435 cells expressing the chimeric therapeutic gene under 1% O2 showed an increase in stable HIF-1alpha protein levels and synthesis of the endonuclease G protein in a time-dependent manner. In normoxic conditions, these stable transgenic cells exhibited no change in growth rate, invasion and motility when compared to parental cells. Moreover, xenografts generated using the transgenic cells exhibited highly significant suppression of tumor growth in a preclinical cancer model compared to the parental cell line. Thus, the hypoxia-modulated endonuclease G expression has the potential to be used as a gene-based-therapy system to kill malignant cells within hypoxic regions of tumors.     

Systemic gene therapy in human xenograft tumor models by liposomal delivery of the E1A gene

The adenovirus type 5 E1A protein has been demonstrated to elicit antitumor effects through the induction of apoptosis, inhibition of cell cycle progression, induction of differentiated epithelial phenotypes, repression of oncogene expression and function, and sensitization to chemotherapeutic agents and radiation. These unique properties have led to use of the E1A gene in adenoviral and lipid-based gene therapy systems, and it has demonstrated antitumor effects in tumor xenograft model systems. However, the delivery systems used in those studies are best suited for local or intratumoral delivery rather than systemic delivery. Because the effective treatment of many primary tumors as well as metastatic disease requires systemic delivery systems, a novel gene delivery system composed of liposome/protamine/DNA (LPD) was investigated for systemic delivery of the E1A gene. Athymic nude mice bearing human breast (MDA-MB-361) or head and neck (WSUHN-31) tumor xenografts were treated i.v. with LPD-E1A, and the expression of E1A protein and effects on tumor growth were assessed. In the MDA-MB-361 breast model, expression of E1A protein was detected in the tumors after LPD-E1A treatment, which was associated with down-regulation of HER-2/neu protein expression and the presence of apoptotic cells. Tumor volume was also smaller in mice treated with LPD-E1A than in controls in both of the xenograft models. Lastly, LPD-E1A in combination with paclitaxel was more effective than LPD-E1A or paclitaxel alone in the MDA-MB-361 model. Additional preclinical and clinical development of LPD-E1A is warranted for the treatment of advanced or metastatic cancer.     

Progenipoietin-generated dendritic cells exhibit anti-tumor efficacy in a therapeutic murine tumor model

Progenipoietin (ProGP-4) is a chimeric molecule, exhibiting both Flt-3 and granulocyte-colony stimulating factor (G-CSF) receptor agonist activities. Subcutaneous administration of ProGP-4 to BALB/c mice at a dose of 40-100 microg/day for up to 12 consecutive days induces both CD11c(+) dendritic cells (DCs) and CD11c(-)/CD11b(+) granulocytes in spleen, blood and lymph nodes of treated animals. Peak numbers of all cell populations were observed on day 7 of treatment, with CD11c(+) DCs representing approximately 8% of total splenocytes at that time. Approximately 40-50% of these CD11c(+) cells were also able to endocytose and process the exogenous fluorescent antigen DQ-BSA. As a test of their therapeutic utility, freshly prepared CD11c(+) DCs were pulsed with a defined tumor-associated peptide epitope (murine p53(232-240)) and injected as a vaccine into BALB/c mice bearing day 7 established CMS4 sarcomas. Similarly prepared DCs were injected again 1 week later. Based on our results, we conclude that (i) both peptide-pulsed CD11c(+) DCs (harvested directly from ProGP-4 treated mice) and pulsed bone marrow-derived DCs effectively slow the growth of or mediate the regression (in 25 of 89 [28%] cases) of CMS4 tumors, and (ii) nonpulsed DCs mediated minimal or no therapeutic effect. These data support the ability of ProGP-4 to enhance the peripheral frequencies of DCs that exhibit therapeutic efficacy when applied as a vaccine to treat tumor-bearing animals.     

Anti-tumor effects of human peripheral gammadelta T cells in a mouse tumor model

Peripheral gammadelta T cells derived from healthy donors were found to exhibit cytotoxicity against a variety of tumor cell lines in vitro, including CNE2, which was established from nasopharyngeal carcinoma (NPC). The anti-tumor effects were further studied in a mouse model. Control nude mice inoculated s.c. with 5 x 10(6) CNE2 cells regularly developed hypodermal tumors, which progressively increased in size, and animals had a mean survival of 35 +/- 3.4 days. Tumor growth was arrested and tumor size was reduced after animals were infused with 5 x 10(7) gammadelta T cells derived from a healthy donor. The anti-tumor effects were temporary, however, and tumor growth was resumed after about 1 week in a group of the animals that had been given a single dose of gammadelta T cells. In another group of animals given 2 doses of gammadelta cells 1 week apart, resumption of tumor growth was delayed for a further week. Mean survival of the 2 groups was increased to 61 +/- 15.7 and 74 +/- 12.9 days, respectively. Immunohistology revealed an accumulation of infused cells in tumors attended by focal tumor necrosis in specimens taken 2 days after infusion. Infiltrative cells virtually disappeared from tumor tissues 6 days after infusion, accompanied by increased mitotic indices of tumor cells. These temporal relationships suggested that the accumulation of infused gammadelta T cells in hypodermal tumors was responsible for the observed anti-tumor effects.     

Localization of radiolabeled antimyeloid antibodies in a human acute leukemia xenograft tumor model.

Acute myeloid leukemia is an attractive disease to treat with radiolabeled antibodies because it is radiosensitive and antibody has ready access to the marrow cavity. In order to evaluate potentially useful radiolabeled antibodies against human acute myeloid leukemia, we have developed a nude mouse xenograft model using the human acute leukemia cell line, HEL. Mice with s.c. xenografts of HEL cells received infusions of radioiodinated anti-CD33 antibody. Examination of the biodistribution of the antibody showed that uptake in the s.c. tumor was maximal [16.9% injected dose (ID)/g at 1 h after infusion] following infusion of 1-10 micrograms of antibody and decreased following infusion of 100 micrograms (6.5% ID/g at 1 h) presumably as a result of saturation of antigen sites. The radiolabel was poorly retained in tumor (4.5-8.2% ID/g at 24 h after infusion). These results were consistent with in vitro studies demonstrating rapid internalization and catabolism of the anti-CD33 antibody. Uptake in tumor could be improved by using either a radiolabel that is retained intracellularly, 111In-DTPA (18.5% ID/g at 24 h), or by targeting a surface antigen that does not internalize upon antibody binding, CD45 (20.5% ID/g at 24 h). These results indicate that this model system will be useful in evaluating the interaction of radiolabeled antibodies with human acute myeloid leukemia cells in an in vivo setting.     

人类DBC2基因重组腺病毒的构建及其在膀胱癌中的表达(英文)

Objective: The aim of the study was to construct recombinant type 5 adenovirus expressing the human DBC2(deleted in breast cancer 2) gene for in vitro and in vivo assay in human bladder cancer research. Methods: The human DBC2gene was first subcloned into a shuttle plasmid pAdTrack-CMV. After recombining with pAdEasy-1 vector in BJ5183 cells, thenew recombinant vector pAdEasy-DBC2-CMV was transfected into HEK-293 cells to produce adenovirus. The human bladdercancer cell line T24 was infected with DBC2-containing adenovirus particles. Both RNA and protein were collected from cellsharvested at 72 h after infection. Real time quantitative PCR (qPCR) and Western blot were used to examine mRNA and proteinlevels. Fluorescence microscopy was utilized to observe the expression of reporter green fluorescence protein. Results:Electrophoresis showed there was a 2.2 kb size band produced from high fidelity PCR. Pac I digest of the final producedrecombinant vector yielded band sizes of approximately 30 kb and 4.5 kb. After virus infection with the pAdEasy-DBC2-CMVvector, the T24 cell line was observed to highly express green fluorescence protein under a fluorescence microscope. qPCRand Western blot assay identified that the DBC2 gene was overexpressed at both the mRNA and protein levels in virustransfected cells. Conclusion: By using the pAdEasy adenovirus system, we successfully constructed an adenovirus thatcould highly overexpress the tumor suppressor DBC2 gene in a bladder cancer cell line. This viral construct would be widelyused for our further research in gene functional assays and gene therapy in bladder cancer.