Induction of apoptosis in malignant pleural mesothelioma cells by activation of the Fas (Apo-1/CD95) death-signal pathway

OBJECTIVE: Although well characterized in several solid tumors, the effects of Fas/Fas ligand interactions in malignant pleural mesothelioma cells have not been defined. The present study was undertaken to examine the functional status of the Fas/Fas ligand pathway in malignant pleural mesothelioma cells and to determine the feasibility of targeting this death-signal pathway for molecular intervention in patients with mesotheliomas. METHODS: Fas expression in primary normal human bronchial epithelial cells and 6 malignant pleural mesothelioma cell lines was quantified by means of flow cytometry. The caspase components of the Fas-mediated apoptotic pathway were evaluated by means of Western blot techniques. Soluble Fas ligand-mediated cytotoxicity and apoptosis were evaluated by means of MTS and TUNEL assays, respectively. Cisplatin (3 microg/mL) and lymphokine-activated killer cells were used to enhance mesothelioma sensitivity to soluble Fas ligand. An H2373 nude mouse xenograft model of malignant pleural mesothelioma was established to assess the in vivo effects of soluble Fas ligand. RESULTS: Four of 6 malignant pleural mesothelioma lines exhibited high levels of Fas expression, and 2 of 4 were inherently susceptible to soluble Fas ligand-mediated cytotoxicity (soluble Fas ligand 50% inhibitory concentration, < 15 ng/mL). Two soluble Fas ligand refractory cell lines (H2052 and H513) exhibited high levels of Fas receptor. Pretreatment with cisplatin resulted in a reduction of 50% inhibitory concentration from infinity to 4.17 +/- 0.14 ng/mL and 10.23 +/- 1.58 ng/mL, respectively. Two additional soluble Fas ligand refractory cell lines (H2595 and REN) expressed low levels of Fas. Exposure of these cells to lymphokine-activated killer cells or lymphokine-activated killer cell-conditioned medium followed by a 24-hour treatment with cisplatin resulted in a significant reduction in 50% inhibitory concentration of soluble Fas ligand and pronounced induction of apoptosis. Intraperitoneally administered soluble Fas ligand mediated regression of H2373 xenografts. CONCLUSION: The Fas/Fas ligand pathway in mesothelioma cells is either intrinsically intact or can be rendered functional with chemotherapeutic agents or immune effector cells. These preclinical data support further evaluation of strategies to enhance Fas-mediated apoptosis in mesotheliomas.     

Sunitinib can enhance BCG mediated cytotoxicity to transitional cell carcinoma through apoptosis pathway

Bacillus Calmette-Guerin (BCG)-refractory generated a high risk to patients with bladder cancer during treatment. Tyrosine kinase receptor (TKR) and TKR-mediated signal transduction pathways play an important role in tumor initiation, maintenance, angiogenesis, and vascular proliferation. Theoretically, it is helpful in adjuvant treatment for transitional cell carcinoma (TCC). Hence, we proposed that sunitinib, a endothelial growth factor receptor (VEGFR) inhibitor, may have a synergistic effect with BCG in enhancing its cytotoxicity to bladder cancer. The level of VEGF in various TCC cell lines was quantified by real time PCR. High grade TCC-T24 cell line with high level of VEGF expression was selected as representative tumor cells for further study. The single drug and combined inhibitory effects of BCG and sunitinib in T24 cells were determined by MTT method. The drug mediated cell apoptosis in T24 cells was characterized by flow cytometry with PI and annexin V stain. Bcl-2 apoptotic pathway induction by BCG and sunitinib treatment was evaluated by Western blotting method. Inhibitory ability of sunitinib in BCG induced cell migration was verified by cell migration assay. The results shown that expression level of VEGF mRNA in high grade T24 cells was higher than low grade J82, TSGH 8301, and TCC 9202 cell lines. Both BCG and sunitinib treatment presented cytotoxic effect to T24 cells in a dose-dependent manner. Combination of BCG and sunitinib revealed superior cytotoxicity effect than single agent when cells were pretreated with low dosage BCG before sunitinib treatment. By Annexin V analysis it was observed that cell death associated with increased early and late apoptosis process individually. Furthermore, the bcl-2 expression was significant reduced in T24 cells in metachronous BCG and sunitinib combination treatment than single agent. Tumor cell migration activity was also markedly inhibited with BCG and sunitinib combination treatment. In conclusion, these results suggested that during BCG and sunitinib combination treatment both reagents interacted with each other and caused TCC cells apoptosis in addition to direct cytotoxicity. This combination therapeutic model may have the potential for future clinical application to bladder cancer treatment.     

A study of an effective sunitinib–chemotherapeutic combination regimen for bladder cancer treatment using a mouse model

ObjectiveTo determine if tyrosine kinase receptor inhibitor, sunitinib malate, combined with chemotherapeutic drugs may present synergistic enhancement of cytotoxicity to transitional cell carcinoma cells (TCC).MethodsThe mRNA and protein contents of vascular endothelial growth factor-α (VEGFα) in various TCC cell lines were detected individually by quantitative-polymerase chain reaction and Western blot. The inhibitory concentrations of various chemotherapeutic drugs, including gemcitabine, doxorubicin, and cisplatin, and their combination with sunitinib to TCC cancer cells were determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The synergist efficacy was measured using the observed/expected ratio calculation method. Finally, the synergistic effect of sunitinib and selected anticancer drug gemcitabine was elucidated in C3H/MBT-2 animal tumor model with monitoring tumor growth volume and survival rate.ResultsThe mRNA of VEGFα had high expression in high-grade TCC cell lines (T24, TCC 8701, and TCC 8702) when compared with low-grade TCC cell lines (TCC 8301 and TSGH 9201). The expression of VEGFα protein level was closely correlated with the mRNA content in each individual cell line. Sunitinib, combined with gemcitabine, has shown the highest synergistic cytotoxic effect to TCC cells in an MTT assay. In the xenografted tumor model, MBT-2 bearing mice treated by sunitinib and gemcitabine combination had the lower mean tumor volume (265 ± 95 mm³ vs. 2605 ± 320 mm³) and higher survival rate (100% vs. 56%) at 30 days follow-up when compared with control mice.ConclusionCombination of the tyrosine kinase receptor inhibitor sunitinib with gemcitabine chemotherapy synergistically enhances tumor cytotoxicity and may provide a new treatment modality for advanced bladder cancer.     

Omega-6 fatty acids can inhibit Fas-mediated apoptosis in a human colorectal carcinoma cell line: a potential mechanism for escape from immune surveillance

The Fas/Fas ligand pathway may play an important role in the pathogenesis of colorectal carcinoma by allowing tumor cells to evade host immune defenses. Since dietary fats, in particular omega-6 fatty acids, facilitate tumor development their influence on the Fas/Fas ligand pathway needs to be elucidated. The purpose of this study was to determine the effect of membrane free fatty acid (FFA) alterations on Fas expression and sensitivity. Two human colorectal carcinoma cell lines (CX-1 and CCL-188) were grown in cell culture media supplemented with omega-3 (docosahexanoic acid) and omega-6 (linoleic acid) fatty acids. Membrane alterations were confirmed by gas chromatography (GC). Cell surface Fas expression was determined with flow cytometry using an anti-Fas monoclonal antibody. Sensitivity to Fas mediated apoptosis was measured by now cytometric measurement of fragmented DNA stained with propidium iodide. Appropriate changes in the membrane FFA composition were found by GC. Cell surface Fas expression was unaffected in either cell line. Omega-3 fatty acids did not alter Fas sensitivity for either cell line compared to control (CX-1: 59.7%, +/- 5.4 vs 51.4% +/- 7.1 for control, CCL-188: 54.3% +/- 8.6 vs 51.2% +/- 4.8 for control). Omega-6 fatty acids produced a significant decrease in Fas-mediated apoptosis (CX-1: 34.2% +/- 4.8 and CCL-188: 22% +/- 6.0, p < 0.05 vs control). These data indicate that although membrane FFA alterations did not affect Fas expression, omega-6 fatty acids significantly decreased Fas-mediated apoptosis. This inhibitory effect may protect colorectal carcinoma cells from lymphocyte Fas-mediated cell death.     

Inflammatory cytokines and lipopolysaccharide induce Fas-mediated apoptosis in renal tubular cells

BACKGROUND/AIMS: Increased susceptibility of the kidney to acute renal failure (ARF) in the setting of sepsis even in the absence of systemic hypotension is well known. In the hypothesis that the proinflammatory cytokines and lipopolysaccharide (LPS) in gram-negative sepsis can directly cause renal tubular cell apoptosis via Fas- and caspase-mediated pathways, we examined apoptosis and Fas, Fas ligand, FADD expression, as well as PARP cleavage in cultured human proximal tubular cells under the cytokine and LPS-stimulated conditions. METHODS: HK-2 cell, immortalized human proximal tubular cell lines, were treated with 5 and 30 ng/ml of tumor necrosis factor-alpha (TNF-alpha), 5 and 20 ng/ml of interleukin-1beta (IL-1beta) and 30 ng/ml LPS for 24 h. Fas expression was examined by RT-PCR and Fas ligand, Fas-associated protein with death domain (FADD) and poly ADP ribose polymerase (PARP) cleavage were examined by Western blot analysis. Apoptosis was assessed by flow cytometer using Annexin V-FITC and propidium iodide (PI) staining and also by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) methods. RESULTS: Fas mRNA expression (ratio of Fas/L-19) increased in the TNF-alpha 5, 30 ng/ml and LPS treated group (p < 0.01, p < 0.01, p = 0.02), but there was no difference between the low- and high-dose TNF-alpha groups. Fas ligand protein expression did not increase in the low-dose TNF-alpha treated group, but it increased significantly in the high-dose TNF-alpha treated group (p < 0.01), IL-1beta- and LPS-treated groups (p < 0.01, p = 0.01, p < 0.01, p = 0.02). The intracellular adaptor protein, FADD expression also increased significantly in the high-dose TNF-alpha- and IL-beta-treated groups (p = 0.04, p = 0.04), but in the low-dose TNF-alpha and IL-beta treated group, it did not show statistically significant differences. In the LPS group, FADD expression also showed an increased tendency, but it was not statistically significant (p = 0.09). Western blot for PARP, a DNA repair enzyme mainly cleaved by caspase 3, showed increased 89- and 24-kD PARP cleavage products in TNF-alpha, IL-1beta and LPS treated cells. The degree of apoptosis examined by DNA fragmentation and translocation of membrane phosphatidyl serine significantly increased in cytokines and LPS treated groups. CONCLUSION: These results suggest that Fas- and caspase-mediated apoptosis of tubular cells by inflammatory cytokines and LPS can be one of the possible mechanisms of renal dysfunction in endotoxemia.     

Transitional cell carcinoma expresses high levels of Fas ligand in vivo

OBJECTIVE: To characterize the Fas-Fas ligand system, a main apoptotic pathway, in transitional cell carcinoma (TCC) of the urinary bladder, by analysing the expression of Fas and Fas ligand (FasL) in TCC samples. MATERIALS AND METHODS: Archival paraffin-embedded tissues from 37 patients with TCC were analysed by immunohistochemistry to determine Fas and FasL expression. RESULTS: Fas and FasL were detected on the cell surface and cytoplasm of respectively 34 (92%) and all cases analysed. The expression of Fas and FasL did not differ with the cytological grade of TCC. CONCLUSION: The high expression of FasL in TCC, reported for the first time in the present study, suggests that FasL may contribute to the immune escape of TCC through killing Fas-bearing lymphocytes. Co-expression of Fas with FasL also suggests that TCC may have pathways resistant to Fas-mediated autocrine cell suicide.     

阿霉素上调肿瘤细胞Fas基因表达并诱导凋亡的实验研究

目的 研究化疗药物阿霉素(ADM)对肿瘤细胞Fas凋亡基因的调控，并探讨其致凋亡机制。方法 逆转录一聚合酶链反应(RT-PCR)比较经。ADM处理前后肿瘤细胞胃癌ADM细胞系SGG-7901、MGC-803和肺癌细胞系A-549的Fas mRNA的表达水平，同时观察其对抗Fas抗体的敏感性，并用流式细胞术检测肿瘤细胞凋亡率。结果 所有3种肿瘤细胞在ADM作用下Fas表达水平显著增强，差异有统计学意义(P＜0．05)，同时肿瘤细胞凋亡率明显增加(凋亡率分别为40．3％、43．6％、54．4％)。ADM能增加肿瘤细胞对Fas抗体的敏感性。结论 ADM上调肿瘤细胞Fas的表达，ADM与Fas抗体联合应用将增强肿瘤细胞的凋亡率，有一定的抗肿瘤临床应用前景。     

Mechanism of T cell-mediated endothelial apoptosis

BACKGROUND: Cytotoxic T lymphocyte (CTL)-mediated destruction of allogeneic vascular endothelium is important in the pathogenesis of both acute and chronic allograft rejection. Despite the importance of this phenomenon, the effector mechanisms responsible for endothelial cell killing are not well defined, and conflicting conclusions have been reached based on variation in experimental methodology. METHODS: We used a recently described method for isolating mouse vascular endothelium to evaluate endothelial cell lysis by CTLs. Endothelial cell destruction was assessed in vitro both by 51Cr release and DNA fragmentation using wild-type and lpr (Fas deficient) endothelium of C3H/HeJ (H2(k)) mice by MHC alloantigen-specific T cells of wild-type, gld (Fas ligand deficient), and perforin-deficient mice on a C57BL/6 (H2(b)) background. RESULTS: Although maximal lysis of 56.6+/-0.8% was seen when using wild-type targets and effectors, only a moderate decrease in apoptosis to 37.6+/-4.0% was detected when the Fas/Fas ligand death receptor pathway was eliminated. This decrease in cytotoxicity occurred despite the preserved functional capacity of this pathway. Alternatively, a significant decrease in cytotoxicity to 17.4+/-4.7% was seen when the perforin/granzyme exocytosis pathway was eliminated. CONCLUSIONS: These data indicate that CTLs destroy vascular endothelium primarily by the perforin/granzyme exocytosis pathway with only a minor contribution to apoptosis by the Fas/Fas ligand death receptor pathway. These data are critical for the proper interpretation of studies evaluating acute and chronic allograft rejection and for the design of rational strategies to ameliorate vascular injury concomitant to the rejection process.     

肿瘤坏死因子α对人肝癌多药耐药逆转作用的实验研究

目的研究肿瘤坏死因子α（TNF-α）对体外培养的人肝癌耐阿霉素细胞系（HepG2／ADM）多药耐药现象的逆转作用。方法不同浓度（100、500及2500U／ml）TNF-α作用于HepG2／ADM细胞72h后进行以下试验：用实时荧光定量聚合酶链反应技术检测各组多药耐药相关基因（MDR1）及脂质过氧化物酶体增殖物激活受体α（PPAR-α）基因的mRNA表达情况;用罗丹明外排法检测各组P-糖蛋白活性;用Annexin V检测0．5mg／L阿霉素诱导的各组细胞凋亡情况;利用MTF法检测各组耐药性的改变。结果TNF-α能诱导HepG2／ADM细胞的MDR1基因表达下调,PPAR-α基因表达上调,且能增加阿霉素诱导的凋亡细胞的比例及细胞毒作用。结论TNF-α可能分别通过抑制MDR1表达及促进PPAR-α表达而逆转HepG2／ADM细胞的耐药性。     

Pressure and endothelial coculture upregulate myocytic Fas-FasL pathway and induce apoptosis by way of direct and paracrine mechanisms

BACKGROUND: Pressurized endothelial cell (EC)-smooth muscle cell (SMCs) coculture significantly increases the apoptosis of SMCs. Our current hypothesis was that in EC-SMC coculture, pressure upregulates SMC apoptosis SMCs through EC-derived paracrine factors and that SMC apoptosis is induced through Fas-Fas ligand (FasL) activation. METHODS: Conditioned media (CM) from ECs and SMCs exposed to ambient or high pressure was transferred to recipient SMCs. SMCs were stained with terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling. Fas and FasL expression was assessed in SMC grown in monoculture, coculture with EC, pressurized monoculture, and pressurized coculture with EC. RESULTS: CM from pressurized ECs caused a 30% increase in SMC apoptosis compared with CM from control ECs (P < .05). Pressure increased Fas and FasL expression in monocultured and cocultured SMCs (1.6-fold and 2.3-fold for Fas [P < .05] and 1.65-fold and 1.7-fold for FasL [P < or = .05]). Coculture had synergistic effect on Fas expression and no effect on FasL expression. CONCLUSIONS: Pressure plays significant role in EC-SMC interaction, SMC apoptosis, and vascular remodeling.     

Differential involvement of the Fas receptor/ligand system in p53-dependent apoptosis in human prostate cancer cells

BACKGROUND: The objective of this study was to characterize the involvement of the Fas receptor/ligand system in p53-dependent apoptosis in human prostate cancer cells. METHODS: The effects of adenovirus-mediated p53 gene transfer (Ad5CMV-p53) into human prostate cancer LNCaP, DU145, and PC3 cells on their growth, apoptosis and Fas receptor/ligand expression were examined by the MTT assay, DNA fragmentation assay, and Northern blot analysis, respectively. The sensitivity of these cells to an agonistic anti-Fas receptor antibody (CH11) and the effects of an antagonistic anti-Fas ligand antibody (4H9) on Ad5CMV-p53-induced apoptosis were analyzed by the MTT assay and DNA fragmentation assay. RESULTS: Ad5CMV-p53 treatment resulted in substantial growth inhibition, induction of apoptosis and up-regulation of Fas receptor as well as Fas ligand mRNA expression in LNCaP, DU145 and PC3 cells. Despite the abundant expression of Fas receptor in all of these cells, CH11 induced apoptosis only in PC3 cells. Furthermore, 4H9 partially blocked the apoptosis induced by Ad5CMV-p53 in PC3 cells, but not in LNCaP and DU145 cells. CONCLUSIONS: The Fas receptor/ligand system is differentially involved in p53-dependent apoptosis in prostate cancer cells; therefore, reintroduction of wild-type p53 into prostate cancer cells may induce apoptosis through Fas receptor/ligand interaction as well as through an alternative pathway.     

Induction of apoptosis in chondrocytes by tumor necrosis factor-alpha.

Tumor necrosis factor alpha (TNF-alpha) induces apoptosis in a number of cell types and plays an essential role in bone remodeling, both stimulating the proliferation of osteoblasts and activating osteoclasts. During endochondral ossification, apoptosis of chondrocytes occurs concurrently with new bone formation and the resorption and replacement of mineralized cartilage with woven bone. In the present study, the role of TNF-alpha in promoting chondrocyte apoptosis was examined. Chondrocyte cell populations, enriched in either hypertrophic or non-hypertrophic cells, were isolated from the cephalic and caudal portions of 17-day chick embryo sterna, respectively, and treated in vitro with 0.1-10 nM recombinant human TNF-alpha. As a positive control, apoptosis was also induced by Fas receptor antibody binding. Dye exclusion assays of the live/dead ratios of cells showed that TNF-alpha caused a dose-dependent 1.5- and 2.0-fold increase in the number of dead cells in both hypertrophic and non-hypertrophic chondrocytes. Induction of apoptosis was independently assayed by measurement of interleukin-1beta-converting enzyme (ICE) activity, and analyzed by a semi-quantitative determination of DNA fragmentation. When compared to untreated cells, these analyses also showed dose-dependent increases in TNF-alpha induced apoptosis in both chondrocyte populations, with increases in the levels of ICE activity for all doses of TNF-alpha (from approximately 5 to approximately 20 fold). Osteoblasts, however, were not affected by treatment with TNF-alpha or by Fas antibody/protein G induction. Immunostaining of chondrocytes for Fas receptor and caspase-2 protein expression showed that most of the chondrocytes expressed these two markers of apoptosis after treatment with TNF-alpha. Although cell killing and ICE induction were higher in the more hypertrophic cells, TNF-alpha induced apoptosis in both hypertrophic and non-hypertrophic chondrocyte populations. These results demonstrate that apoptosis may be induced in both hypertrophic and non-hypertrophic chondrocytes through both Fas and TNF-alpha receptor mediated signaling, and suggest that chondrocytes are more sensitive to apoptotic effects of TNF-alpha within the skeletal lineage than are osteoblasts.     

CD4+ cells play a major role in xenogeneic human anti-pig cytotoxicity through the Fas/Fas ligand lytic pathway

BACKGROUND: In this study, the role of cell-mediated cytotoxicity by human leukocytes against pig endothelial cells was examined in vitro. The aim was to determine which cell subsets were responsible for this phenomenon and which pathways were involved in cell lysis. METHODS: Primed human peripheral blood mononuclear cells (PBMC) or purified CD4+ or CD8+ T cells were used in a cell-mediated cytotoxicity assay in which cytotoxicity of an SV40 transformed porcine endothelial cell (EC) line (SVAP) was determined by Annexin V binding. RESULTS: Human PBMC demonstrated specific lysis of porcine EC that was proportional to the effector: target ratio. CD4+ T cells accounted for >60% of this lysis, whereas CD8+ T cells accounted for <20%. CD4+ T cell-mediated lysis depended on direct recognition of porcine major histocompatibility complex class II molecules as inhibition of swine leukocyte antigen class II on porcine EC-inhibited CD4+ T cell cytotoxicity. This lysis was mediated through the Fas/FasL pathway as addition of anti-Fas and/or anti-FasL antibody profoundly inhibited antiporcine lysis. In addition, FasL gene expression was detected in primed PBMC and CD4+ T cells by RT-PCR, whereas granzyme B gene expression was not. Primed CD4+ T cells demonstrated high level FasL protein by Western blotting and two-color FACS analysis, whereas NK cells and CD8+ T cells did not. Finally, recombinant human FasL induced apoptosis in Fas expressing porcine EC cells, demonstrating that human FasL interacted with and activated Fas on porcine EC cells. CONCLUSIONS: In conclusion, human to pig cell-mediated cytotoxicity was mediated predominantly by CD4+ T cells through the Fas/FasL pathway of apoptosis. These results suggest that direct cytotoxicity by xenoreactive CD4+ T cells may be one of several effector mechanisms involved in cellular xenograft rejection.     

Sensitization of human renal cell carcinoma cell lines to TRAIL-induced apoptosis by anthracyclines

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new member of the tumor necrosis factor family. The present study investigated whether anthracyclines enhance TRAIL-induced apoptosis and cytotoxicity in renal cell carcinoma (RCC) cells. METHODS: Cytotoxicity was measured using the microtiter assay. Apoptosis was monitored using DNA ladder analysis. Caspase activity was determined using a quantitative colorimetric assay. RESULTS: Treatment of ACHN and Caki-1 human RCC lines with TRAIL, in combination with subtoxic concentrations of epirubicin (EPI) or pirarubicin (THP), enhanced induction of apoptosis and cytotoxicity. Sequential treatment with EPI followed by TRAIL induced significantly more cytotoxicity than the inverse treatment. The combined cytotoxicity of TRAIL and EPI was significantly inhibited by the TRAIL-neutralizing fusion protein DR5:Fc, although EPI did not affect the mRNA expression of DR4, DR5, DcR1 or DcR2. The combination treatment with TRAIL and EPI activated caspase-6 and -3, which were downstream molecules of the death receptor. Furthermore, the combined cytotoxicity of TRAIL and EPI was almost completely inhibited by Z-VAD-FMK, and partly inhibited by Ac-DMQD-CHO. CONCLUSION: These findings indicate that anthracyclines sensitize RCC cells to TRAIL-induced apoptosis and cytotoxicity through activation of caspases, suggesting that TRAIL, in combination with anthracyclines, has a therapeutic potential in the treatment of RCC.     

The essential role of interferon-gamma during interleukin-12 therapy for murine transitional cell carcinoma of the bladder

PURPOSE: Recombinant interleukin (IL)-12 and adenoviral IL-12 gene therapy have been shown to be potent therapeutic interventions for murine transitional cell carcinoma (TCC) of the bladder in vivo. We investigated the mechanisms through which IL-12 induces antibladder cancer immunity. MATERIALS AND METHODS: The ability of IL-12 to enhance interferon-gamma (IFN-gamma) expression, a major T-helper type 1 cytokine, was analyzed in murine serum, urine and splenocyte cultures. MB49, a murine TCC line, was treated with IFN-gamma and evaluated for its proliferation, surface molecule expression and sensitivity to splenocyte mediated cytotoxicity. Neutralizing antiIFN-gamma antibody was applied to test the role of IFN-gamma in the IL-12 therapy of MB49 tumor. RESULTS: IL-12 was observed to significantly increase IFN-gamma concentrations in serum and urine as well as in splenocyte cultures. While IL-12 had no direct activity against TCC in vitro, IFN-gamma showed potent dose dependent antiproliferative and pro-apoptotic activity, which was further enhanced by supplementation of tumor necrosis factor-alpha. In addition, IFN-gamma substantially up-regulated the expression of surface immune molecules on TCC cells, including MHC-I, MHC-II, ICAM-I, B7.1, B7.2 and Fas. Maximum splenocyte mediated cytotoxicity against TCC was enhanced by pretreatment of target bladder cancer cells with IFN-gamma plus tumor necrosis factor-alpha. Furthermore, IL-2 in combination with IL-12 further enhanced splenocyte mediated cytotoxicity. The in vivo antibladder cancer activity of IL-12 was abolished by concurrent treatment with antibodies to IFN-gamma. CONCLUSIONS: This study strongly suggests that IFN-gamma has an essential role in IL-12 induced antibladder tumor immunity. Activation of host effector immune cells by IL-12 is also required for induction of optimal tumor destruction in IL-12 therapy.     

人肿瘤坏死因子α联合溴隐亭对人肝癌裸鼠耐药模型耐药性逆转的研究

目的探讨人肿瘤坏死因子α(TNFα)联合溴隐亭(BCT)对人肝癌裸鼠耐药模型耐药性的逆转作用。方法将人肝癌细胞系HepG2及其经阿霉素(ADM)诱导建立的耐药细胞系HepG2ADM和转染TNFα基因后的耐药细胞系HepG2ADMTNFα分别原位种植BALB/C裸鼠肝脏,建立裸鼠原位肝移植瘤模型。64只裸鼠分为4组:HepG2组(HepG2细胞系种植瘤裸鼠),ADM组(HepG2ADM细胞系种植瘤裸鼠),TNFα组(HepG2ADMTNFα细胞系种植瘤裸鼠)和BCT组(HepG2ADMTNFα细胞系种植瘤裸鼠同时口服BCT),成瘤后均给以每天腹腔内注射0.15g/kg的氟脲嘧啶+1.5mg/kg的丝裂霉素+10mg/kg的ADM,连续3d;BCT组化疗的同时另行BCT灌胃治疗(6.25mg·kg-1·d-1)。B超观察种植瘤的大小变化,病理观察组织学结构及裸鼠生长状况和对化疗药物的敏感性。采用免疫组织化学和逆转录聚合酶链反应(RTPCR)检测各组种植瘤的多药耐药相关基因(MDR1)和肺耐药相关蛋白(LRP)在mRNA水平、蛋白水平的变化,末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法(TUNEL法)检测化疗后肿瘤组织凋亡指数情况。结果细胞系原位种植裸鼠肝脏成功率100%,种植瘤组织学特点符合人肝癌特征;TNFα组和BCT组种植瘤生长速度慢,与HepG2和ADM两组比较差异有统计学意义(P<0.05)。化疗14d后,BCT组重量抑瘤率(67%)最明显,与HepG2、TNFα和ADM3组比较差异有统计学意义(P<0.05)。4组均有MDR1和LRPmRNA表达,组间差异具有统计学意义(P<0.05);免疫组化显示TNFα和BCT组肿瘤组织MDR1蛋白表达比ADM组低,差异具有统计学意义(P<0.01),与HepG2组比较差异无统计学意义(P>0.05);BCT、TNFα组凋亡指数比ADM组高(P<0.05),且TNFα和BCT两组之间差异亦有统计学意义(P<0.05),但与HepG2组之间比较差异均无统计学意义(P>0.05)。结论TNFα基因能下调MDR1和LRPmRNA及蛋白表达,联合BCT能加强对化疗药物的敏感性。     

Fas—L与P53在膀胱肿瘤中的表达及临床意义

目的 探讨膀胱肿瘤中免疫逃避作用引起的细胞凋亡与P53的关系。方法 采用免疫组织化学方法检测33例移行细胞癌,8例膀胱腺癌及11例膀胱炎组织石蜡切片中Fas-L与P53的表达。结果 P53及Fas-L在膀胱炎及肿瘤中的表达具有显著性差异(P<0.05)。在膀胱腺癌与移行细胞癌中P53、Fas-L的表达无显著性差异(P>0.05)。C_1、G_2、G_3级中P53表达具有显著性差异(P<0.05),Fas-L的表达无显著性差异(P=0.07)。在膀胱肿瘤中,20例P53阳性患者中14例Fas-L阳性(70％),P53与Fas-L表达无显著性差异(p=0.238)及相关性(p=0.393)。结论 P53可能通过免疫逃避作用使肿瘤细胞凋亡下降,但不是唯一因素。     

阿霉素诱导人胆囊癌细胞凋亡的研究

目的　观察阿霉素 (ADM )对胆囊癌细胞凋亡的影响。方法　在体外培养的胆囊癌细胞中加入不同浓度的阿霉素 ,应用光镜、电镜、DNA凝胶电泳及流式细胞仪检测胆囊癌细胞凋亡的形态学特征、生化学特征、细胞凋亡百分率及细胞周期的变化。结果　在阿霉素作用下 ,凋亡的胆囊癌细胞出现膜小泡、凋亡小体等特征性改变 ;DNA电泳呈现典型的“梯状”条带 ;流式细胞仪测定 ,出现典型的凋亡峰 ,其凋亡百分率随着药物浓度的提高而明显升高 ,分别与对照组比较 ,差异有显著性 (P <0 .0 5 ) ;同时 ,S期细胞含量下降 ,而G2 /M期细胞含量上升。非凋亡细胞则无此表现。结论　ADM可诱导胆囊癌细胞发生凋亡 ,并可使胆囊癌细胞生长受阻于G2 /M期 ,这可能是其杀伤肿瘤的一种重要机制