Potent antitumor effects of CD154 transduced tumor cells in experimental bladder cancer

PURPOSE: Current intravesical immunotherapy for bladder cancer with bacillus Calmette-Guerin instillations is standard treatment for patients with high risk superficial tumors but relapses are common. We evaluated the tumor vaccine concept in murine bladder cancer by comparing tumor cell transduction with genes coding for the immunostimulatory molecules CD154, interleukin (IL)-12 and CD80 to design a novel vaccination strategy. MATERIALS AND METHODS: Adenoviral vectors were used to transduce murine bladder cancer MB-49 cells with genes coding for CD154, IL-12 and CD80. Parental or transduced MB-49 cells were injected subcutaneously into syngeneic mice. The effects of transgene expression on tumorigenicity and the generation of protective immunological memory against challenge with parental tumor were studied. RESULTS: All 76 animals injected with parental MB-49 cells had tumors within 8 to 12 days. Tumor cell expression of CD154 combined with IL-12 completely inhibited tumor outgrowth with all 21 mice tumor-free and CD154 transduction alone was almost as effective with 33 of 35 tumor-free. IL-12 production by tumor cells delayed tumor outgrowth and 4 of 10 mice remained tumor-free. Over expression of CD80 had no effect on tumorigenicity. CD154 expressing tumors were rapidly infiltrated with large numbers of CD4+ and CD8+ T cells. Mice vaccinated 4 times with adenoviral CD154 transduced MB-49 cells were completely protected against challenge with parental tumor. Co-injection of CD154 modified cells with parental MB-49 cells retarded tumor growth. CONCLUSIONS: Our experimental results suggest that the potent antitumor effects of CD154 gene transduction should be considered for immunostimulatory gene therapy for bladder cancer.     

Effect of polyphenols from the Mediterranean diet on proliferation and mediators of in vitro invasiveness of the MB-49 murine bladder cancer cell line

ObjectiveTo evaluate the effect of different polyphenols on the proliferation and invasive capacity of MB-49 murine bladder tumor cell lines and to identify the mediators involved in this process.Materials and MethodsMB-49 murine bladder cancer cells were cultured in media supplemented with resveratrol, rutin, morin, quercetin, gallic acid and tannic acid (all of them are polyphenols usually present in Mediterranean diet) for periods of 24, 48 and 72 hours to quantify the expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in the culture medium, as well as of metalloproteinase-9 (MMP-9) and cell proliferation.ResultsAll the polyphenols studied significantly inhibited proliferation of MB-49 cells, varying according to the time periods and doses used. The cells in the media supplemented with the nutrients to study did not show inhibition of mRNA expression of urokinase-type plasminogen activator (uPA) or its high affinity receptor (uPAR). It was even slightly increased in certain cases. However, mRNA expression of metalloproteinase-9 was strongly inhibited.ConclusionsThe polyphenols present in our usual diet exert an effect on the proliferation and mediators of bladder tumor invasiveness in MB-49 cells.     

Oncolytic Vaccinia Virus as an Adjuvant Treatment to Cytoreductive Surgery for Malignant Peritoneal Mesothelioma

Background

Malignant peritoneal mesothelioma (MPM) is an aggressive cancer with a dismal prognosis. Oncolytic viruses are a promising new therapy for cancer because of their ability to kill tumor cells with minimal toxicity to normal tissues. This experimental study aimed to examine the potential of modified vaccinia virus (VV) to treat MPM when administered alone or as an adjuvant treatment to surgery.

Methods

Two aggressive murine mesothelioma cell lines (AC29, AB12), were used. Cell viability and viral cytopathic effects were assessed using MTS and crystal violet assays. Immunocompetent mice were injected intraperitoneally with MPM cells and treated with intraperitoneal VV. Tumor-bearing mice also underwent cytoreductive surgery (CRS) followed by VV (or control) therapy.

Results

The cytotoxic effects of VV on MPM cell lines was significantly increased compared with the control non-cancer cell line. In both orthotopic models, VV induced tumor regression, prolonging median and long-term survival. VV treatment after incomplete CRS was not superior to VV alone; however, when mice with microscopic disease were treated with VV, further prolongation of median and long-term survivals was observed.

Conclusions

VV selectively kills MPM cells in vitro and leads to improved survival and cures in immunocompetent murine models. Higher efficacy of the virus in the microscopic disease context suggests the use of the virus as an adjuvant treatment to complete surgical resection. These promising results justify further studies of VV in humans as a novel treatment for MPM.     

A novel dendritic cell-based cancer vaccine produces promising results in a syngenic CC-36 murine colon adenocarcinoma model

BACKGROUND: This study was conducted to test the efficacy of a new cancer vaccine, composed of dendritic cells (DCs) pulsed with an interleukin-2 gene-encoded vaccinia virus tumor oncolysate (DC-IL-2VCO) in a CC-36 murine colon adenocarcinoma model. MATERIALS AND METHODS: CC-36 tumor cells were injected subcutaneously into the left flank of four- to six-week old male BALB/c mice. The mice were divided into three groups, each of which received one of the following treatments: (1) DCs pulsed with the IL-2 gene-encoded vaccinia oncolysate (DC-IL-2VCO), (2) DCs pulsed with the tumor oncolysate alone (DC-CO), or (3) no treatment (control). Tumor incidence was measured, and survival rates were compared using a paired Student's t-test. Cytolytic T cell activity was measured in peripheral blood lymphocytes (PBL) and splenic lymphocytes using a (51)Cr-release assay. Lastly, mice were depleted of either CD4+ or CD8+ lymphocytes prior to receiving the vaccine to test the mechanism of tumor immunity in these mice. RESULTS: Mice treated with DC-IL-2VCO demonstrated decreased tumor burden, increased survival, and greater cytolytic activity compared with control mice and mice receiving DC-CO. In addition, mice depleted of CD8+ T cells prior to immunization with IL-2VV + DC-IL-2VCO had a significant increase in the incidence of tumor, similar to the untreated control mice. CONCLUSIONS: DCs pulsed with an IL-2 gene-encoded vaccinia virus tumor oncolysate (DC-IL-2VCO) produced safe and effective immune responses in a murine CC-36 colon adenocarcinoma model. This vaccine (DC-MelVac; Patent no. 11221/5) has the potential to treat humans with cancer, and has received FDA approval for use in Phase I clinical trials.     

Transgenic mouse with human mutant p53 expression in the prostate epithelium

BACKGROUND: Apoptosis is disrupted in prostate tumor cells, conferring a survival advantage. p53 is a nuclear protein believed to regulate cancer progression, in part by inducing apoptosis. To test this possibility in future studies, the objective of the present study was to generate a transgenic mouse model expressing mutant p53 in the prostate (PR). METHODS: Transgene incorporation was tested using Southern analysis. Expression of mutant p53 protein was examined using immunofluorescence microscopy. Apoptosis in the PR was evaluated using the Tunnel method. RESULTS: A construct, consisting of the rat probasin promoter and a mutant human p53 fragment, was prepared and used to generate transgenic mice. rPB-mutant p53 transgene incorporation, as well as nuclear accumulation of mutant human p53 protein, was demonstrated. Prostatic intraepithelial neoplasia (PIN) III and IV were found in PR of 52-week old transgenic mice, whereas no pathological changes were found in the other organs examined. PR ability to undergo apoptosis following castration was reduced in rPB-mutant p53 mice as compared to non transgenic littermates. CONCLUSIONS: Transgenic rPB-mutant p53 mice accumulate mutant p53 protein in PR, resulting in neoplastic lesions and reduced apoptotic potential in the PR. Breeding rPB-mutant p53 mice with mice expressing an oncogene in their PR will be useful in examining interactions of multiple genes that result in progression of slow growing prostate tumors expressing oncogenes alone to metastatic cancer.     

Role of p53-dependent activation of caspases in chronic obstructive uropathy: evidence from p53 null mutant mice

Chronic obstructive uropathy (COU) created by unilateral ureteric ligation is associated with increased renal cell apoptosis and p53 expression. Genetically engineered mice were used to examine the role of p53 in renal cell apoptosis in COU and the involved molecular pathways. Obstructed kidneys in p53+/+, p53+/-, and p53-/- mice were examined at days 4, 7, 15, 20, and 30 for apoptosis, and mRNA were examined for p53, members of the bcl-2 family, the death receptor family, and the common effectors of apoptosis. Obstructed kidneys in p53+/- and p53-/- mice exhibited equal attenuation of tubular and interstitial cell apoptosis (70 and 50%, respectively), compared with p53+/+ mice. However, p53 gene deficiency did not confer complete protection from apoptosis. Obstructed kidneys from p53-/- mice did not express p53 mRNA, whereas those from p53+/- and p53+/+ mice displayed mild and marked increase in their expression, respectively. Obstructed kidneys in p53+/+, p53+/-, and p53-/- mice displayed upregulation of mRNA for members of the bcl-2 family and most of the death receptor family, except for a lower level of tumor necrosis factor receptor-1, TRAIL, and FAP in p53+/+ mice. Obstructed kidneys in p53-/- and p53+/- mice showed virtual absence of caspase 11 and marked attenuation of caspases 1 and 12, contrasted with their strong expression in p53+/+ kidneys. These data suggest that apoptosis in obstructed kidneys involves p53-dependent as well as p53-independent pathways. The p53-dependent pathway may involve activation of caspases 1, 11, and 12, whereas the p53-independent pathway may involve activation of members of the bcl-2 and death receptor families.     

Urothelial susceptibility to tumor cell implantation: comparison of cauterization with N-methyl-N-nitrosourea

To determine whether or not transitional cell carcinoma cells will preferentially implant and grow on an altered urothelial surface, cauterization and instillation of N-methyl-N-nitrosourea (MNU) were used to alter the murine bladder urothelium. Transplantable tumor cells (2.09 X 10(6) ) were placed into the bladders of 53 mice. Tumor cell implantation occurred in only 6 per cent of the mice with a normal bladder, whereas tumors were present in 28 per cent of mice pretreated with intravesical MNU and in 67 per cent of mice which had a portion of the bladder cauterized prior to insertion of tumor cells (p less than 0.005). This study not only establishes the optimal technique for implantation in this experimental model, but also suggests that seeding may be a contributory factor in the high recurrence rate following endoscopic treatment of bladder tumors in man.     

p53重组腺病毒联合顺铂或三氧化二砷对人膀胱癌EJ细胞的作用

目的：探讨p53重组腺病毒（Ad-p53）与顺铂（CDDP）或三氧化二砷（As2O3）联合应用提高膀胱癌疗效的可能性及其机制。方法：将Ad-p53[100感染强度（MOI）]与CDDP（0．5mg/L)或As2O3(2.0μmol/L）联合应用，通过细胞生长抑制实验、克隆形成实验、细胞周期分析、免疫组织化学分析以及裸鼠皮下移植瘤模型，观察其对膀胱癌EJ细胞的作用及机制。结果：与单独应用相比，Ad-p53与低剂量的CDDP或As2O3联合可明显抑制EJ细胞体外生长，诱导EJ细胞凋亡，EJ细胞G2／M期阻滞更明显；裸鼠体内肿瘤发生时间延迟，4周后肿瘤体积与单独应用时相比，差异有显著性（P＜0．05）。结论：基因治疗与化疗联合应用，可进一步提高膀胱癌的疗效。     

p53 protein transduction therapy: successful targeting and inhibition of the growth of the bladder cancer cells

INTRODUCTION: Virus-mediated gene therapy for bladder cancer has some problems, such as efficiency of gene delivery and safety issues. We have reported that poly-arginine peptide (11R) has the ability to increase protein transduction in cells. Here, we show that p53 protein transduction using 11R is useful for targeting to bladder tumors and suppressing the growth of bladder cancer cells. MATERIALS AND METHODS: An 11R-fused p53 protein (11R-p53) was transduced in bladder cancer cell lines (J82 and T24) to evaluate the anti-tumor effect. Cell viability was assessed by performing the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST) assay. To investigate whether 11R-p53 enhanced the effect on anti-cancer drug-dependent apoptosis of bladder cancer cells, the cell lines were cotreated with 11R-p53 and cis-diaminedichloroplatinum (CDDP). Apoptotic cells were identified using Hoechst staining. To investigate the efficiency of protein transduction mediated by 11R in bladder tumors in vivo, SCID mice were transplanted with J82 cells in the bladder and 11R-GFP was transurethrally transduced into the bladder. The transduction of 11R-GFP in the tumor was examined by confocal microscopy. RESULTS: 11R-p53 inhibited the growth of both J82 and T24 cells in a dose-dependent manner. The transduction of 11R-p53 enhanced CDDP-dependent induction of apoptosis. Transurethral application of 11R-GFP resulted in transduction of GFP in bladder tumors but not in the normal bladder epithelium or subepithelial tissues. CONCLUSION: The present results suggest that p53 protein transduction therapy may be a promising method for the treatment of bladder cancer.     

Exposure to Vinorelbine Inhibits in Vitro Proliferation and Invasiveness of Transitional Cell Bladder Carcinoma

Purpose

To study the effect of vinorelbine (VNR) on in vitro cell proliferation, invasiveness, cell adhesion to substrate, cell motility and metalloproteinase secretion of MB-49, a murine transitional cell carcinoma of the bladder (TCC).

Materials and Methods

The colorimetric MTS assay, which depends upon viable versus nonviable mitochondria, was used to evaluate the effect of graded concentrations of VNR on in vitro MB-49 cell growth. Chemoinvasion and cell motility were studied in TCC cells exposed for 24 hours to a noncytotoxic dose of VNR, through their ability to migrate across Matrigel -coated or Type IV collagen-coated 8-micrometer. pore filters. Zymographic studies in gelatin-embedded polyacrylamide gels were done to investigate gelatinolytic activity in conditioned media from treated and untreated MB-49 cells.

Results

Vinorelbine inhibited MB-49 cell growth in a dose-dependent manner (IC₅₀ 40 ng./ml.). In vitro cell invasive capacity of MB-49 cells pretreated for 24 hours with VNR at noncytotoxic doses (1 and 10 ng./ml.) was significantly lower than that of untreated cells. The decreased invasion of VNR-treated cells was not accompanied by a diminished adhesion to Matrigel (R) or type IV collagen nor by a significant reduced secretion of gelatinolytic metalloproteinases. Instead, motility of MB-49 cells exposed to noncytotoxic concentrations of VNR was inhibited in a dose-response fashion similar to that of invasion.

Conclusion

Vinorelbine proved to be an effective drug to inhibit tumor cell growth and invasion in a transitional cell bladder carcinoma model. The results obtained would justify preclinical studies to evaluate the effectiveness of VNR as a potential treatment of TCC.     

重组人干扰素-α-2b-BCG小鼠原位膀胱肿瘤灌注治疗模型的构建

目的 建立适合膀胱内灌注BCG的膀胱癌实验动物模型,观察重组Hifn-α-2b-BCG对其治疗效果.方法 利用亚硝酸银化学损伤、MB49细胞移植改进方法 ,构建C57BL/6原位膀胱肿瘤小鼠模型,进行重组BCG和野生BCG灌注治疗,观察荷瘤小鼠生存率和苏木素.伊红(HE)染色后病理组织学变化,免疫组织化学检测p53.结果 p53在荷瘤小鼠膀胱肿瘤表达强阳性.重组BCG和野生BCG各组灌注治疗后各重要器官组织未见肿瘤和结核结节形成.重组BCG灌注治疗组的小鼠平均膀胱重量(140.8±33.2)mg显著低于磷酸盐缓冲液(PBS)对照组的251.6±38.4,与野生型BCG治疗组的150.1±42.1、野生BCG+IFN治疗组的144.6±39.7比较.差异无统计学意义(P＞0.05),但重组BCG治疗组的小鼠生存率高于野生型BCG治疗组.结论 重组BCG小鼠原位膀胱肿瘤灌注治疗模型造模成功、肿瘤浸润迅速,恶性度高.重组hWN-α-2b-BCG对小鼠体内膀胱肿瘤具有抑制肿瘤生长,影响肿瘤预后.     

IMMUNOTHERAPY OF BLADDER CANCER TARGETING P53

PURPOSE: Superficial bladder cancer is often responsive to immunotherapy with bacillus Calmette-Guerin (BCG). However, some tumors progress despite BCG treatment, and most of these have mutations in the p53 tumor suppressor gene resulting in its over-expression. Overexpressed p53 is therefore a potential target for immunotherapy. The objective of this study was to demonstrate whether human bladder cancer xenografts in SCID mice could be eliminated by cytotoxic T cells (CTL) which recognize over-expressed p53. MATERIALS AND METHODS: Murine CTL which are specific for human p53 were previously generated in our laboratory by peptide immunization of HLA A2.1 transgenic mice. These CTL recognize and lyse human tumor cell lines which over-express p53 in the context of HLA A2.1. The p53 over-expressing HLA A2.1+ human bladder cancer cell line J82 was used to establish subcutaneous or intravesicular tumors in SCID mice. The mice were then administered tail vein injections of 5 x 10(7) p53-specific CTL, control CTL, or phosphate buffered saline (PBS). RESULTS: The subcutaneous tumor mean volume at 5 weeks for the p53-specific CTL treatment group was significantly lower than for both the control CTL or the PBS group (32 mm.3 versus 185 mm.3, p = 0.04 and 32 mm.3 versus 418 mm.3, p = 0.0001). In the mice with intravesicular tumors, a reduction to nonpalpable tumor size in vivo was seen with specific CTL therapy (14% palpable) versus control CTL treatment (86% palpable), the final tumor volume at necropsy was 127 mm.3 versus 246 mm.3 (N.S.). CONCLUSION: The overall response of the human bladder tumors in the SCID mouse model suggests the possibility of targeting p53 in patients with bladder cancer.     

奥曲肽对人胆囊癌裸鼠皮下种植瘤的抑制作用

目的：探讨奥曲肽对人胆囊癌裸鼠皮下种植瘤生长的影响及其机制。方法：建立人原发性胆囊癌皮下种植瘤裸鼠模型。将荷瘤裸鼠随机分成实验组和对照组各9只,分别自腹腔注射奥曲肽100 μg/(kg·d)和生理盐水,用药共6周。观察两组种植瘤生长情况。流式细胞术测定肿瘤细胞凋亡率,免疫组化法检测p53,bcl-2,Ki-67的表达。结果：实验组裸鼠皮下种植瘤的生长明显受到抑制,实验组肿瘤重量显著低于对照组［(0.99±0.54)g vs. (1.58±0.51)g,t=2.38,P<0.05］,抑瘤率达37.3%,肿瘤细胞的凋亡率显著高于对照组［(7.76±2.62)% vs. (4.27±1.50)%,P<0.01］;实验组的p53,bcl-2,Ki-67阳性细胞百分率均低于对照组［(79.48±5.22)%vs. (87.13±8.26)%,(46.72±6.40)%vs.(53.85±7.72)%,(37.56±6.67)%vs(45.45±8.73)%,P均<0.05］。结论：奥曲肽能抑制人胆囊癌裸鼠皮下种植瘤的生长,可能系通过抑制肿瘤细胞增殖、诱导凋亡的机制而实现的。     

端粒酶启动子调控p53基因表达对T24细胞的影响

目的 探讨端粒酶催化亚基（hTERT）启动子调控p53基因在膀胱肿瘤细胞T24中表达对细胞的影响。方法 将成功构建的phTERT-p53载体，瞬时转染膀胱肿瘤T24细胞；应用流式细胞仪观察细胞凋亡率变化，透射电镜下观察细胞凋亡形态学改变。结果 hTERT启动子调控p53基因表达使细胞凋亡率明显升高[24、48h凋亡率：实验组15．42％、36．57％；转染绿色荧光蛋白基因（GFP）组5．16％、8．19％；阴性对照组4．49％、7．18％]，电镜下观察到典型凋亡改变。凋亡指数达25％。结论 hTERT启动子能够激活phTERT-p53载体表达p53，使肿瘤细胞凋亡增加。     

Phase i study of intravesical vaccinia virus as a vector for gene therapy of bladder cancer

PURPOSE: Vaccinia virus is a DNA poxvirus previously used as a vaccine to eradicate smallpox. The virus has a high efficiency of infection, replicates in the cytoplasm without chromosomal integration and can transport a large amount of recombinant DNA without losing infectivity. Therefore, it is an excellent choice as a vector for gene delivery in vivo. Large quantities of vaccinia have been injected into dermal, subcutaneous and peripheral lymph node melanoma metastases without significant side effects, and with efficient infection of the tumor cells and recombinant gene transfection. To determine if vaccinia, when given intravesically, can effectively infect bladder mucosa and tumor with acceptable toxicity, we performed a phase I trial of intravesical vaccinia in patients with muscle invasive transitional cell carcinoma before radical cystectomy. MATERIALS AND METHODS: After documenting immune competence and demonstration of a major reaction after revaccination, patients received 3 increasing doses of intravesical Dryvax vaccinia virus (Wyeth-Ayerst Laboratories, Philadelphia, Pennsylvania) that was provided by the Centers for Disease Control. Approximately 24 hours after the third dose, cystectomy was performed and the tissue was examined microscopically. RESULTS: There were 4 patients who were treated. The 3 patients who received the highest doses (100 x 106 plaque forming units) had significant mucosal and submucosal inflammatory infiltration by lymphocytes, eosinophils, and plasma cells into tumor and normal tissue. Dendritic cells were recruited to the site after exposure to the vaccinia. Significant mucosal edema and vascular ectasia were seen. Tumor and normal urothelial cells showed evidence of viral infection, including enlarged vacuolated cells with cytoplasmic inclusions. There were no clinical or laboratory manifestations of vaccinia related toxicity except mild dysuria. Of the 4 patients 3 survived and were free of disease at 4-year followup. CONCLUSIONS: Our study demonstrates that vaccinia virus can be administered safely into the bladder with recruitment of lymphocytes and induction of a brisk local inflammatory response. To our knowledge, this is the first report of direct delivery of live virus into the human bladder. The role of wild type vaccinia as immunotherapy for bladder cancer warrants further study. Furthermore, these data support the exploration of recombinant vaccinia as a putative gene therapy vector for intravesical infection and transfection of bladder tumor cells with cytokine or other genes, an approach that our group pioneered and most recently studied in patients with superficial melanoma.     

C57BL／6小鼠原位膀胱肿瘤模型的建立

目的建立C57BL／6小鼠原位膀胱肿瘤模型。方法硝酸银膀胱黏膜处理、MB49膀胱灌注法建立C57BL／6小鼠原位膀胱肿瘤模型。结果三组小鼠中,7d组10只小鼠有9只成瘤,10d组和15d组20只小鼠全部成瘤。总成瘤率96．7％。结论硝酸银膀胱黏膜处理、MB49膀胱灌注法建立的CS7BL／6小鼠原位膀胱肿瘤模型操作简单,成瘤率高,是一种理想的、有实际应用前景的小鼠原位膀胱肿瘤模型。