大鼠胰腺癌微环境诱导未成熟树突状细胞功能的变化

目的 观察胰腺癌微环境对树突状细胞(DC)成熟的影响及功能变化并探讨胰腺肿瘤细胞免疫逃逸的机制.方法 培养树突状细胞,加入粒细胞巨噬细胞集落刺激因子(rmGM-CSF)40μg/L、白细胞介素(IL)-4 40μg/L,培养到第6天时得到大量的未成熟树突状细胞(imDC),加入大鼠胰腺癌细胞(AR42J cell)培养上清液诱导,流式细胞术检测DC的表面分子CD86、CD80的表达(n=6),观察能否延缓或阻断imDC的成熟及其在脂多糖(LPS)刺激后这种作用能否被逆转.并观察AR42J细胞培养上清诱导的Dc对同种异体混合淋巴细胞增殖.结果 加入胰腺癌癌细胞上清液培养的DC,与正常成熟的DC比较,CD80+CD86+阳性率由(70.88±3.60)%降至(7.15±0.71)%,LPS刺激后DC细胞的表面分子CD80+CD86+表达仍然较低(7.43±1.05)%,表明胰腺癌细胞培养上清液对DC的成熟有阻断作用.AR42J细胞上清诱导培养的imDC组刺激同种异体混合淋巴细胞增殖的强度显著低于正常培养的imDC组刺激同种异体混合淋巴细胞增殖的强度.结论 体外大鼠胰腺癌细胞培养上清液可以诱导DC处于不成熟状态,且这种不成熟状态不容易被逆转.     

TNF-alpha protects dendritic cells from prostate cancer-induced apoptosis

We have recently shown that human prostate cancer (PCa) cells induced apoptotic death of the most potent antigen-presenting cells, dendritic cells (DC), which are responsible for the induction of specific antitumor immune responses. Here we have evaluated the effect of murine PCa cells RM-1 on the survival of immature and tumor necrosis factor-alpha (TNF-alpha)-stimulated mature DC. PCa cells and DC were co-incubated for 24-48 h and DC apoptosis was assessed by morphologic criteria, Annexin V assay, and TUNEL staining. We have shown that co-incubation of RM-1 cells with DC is accompanied by an increased level of DC apoptosis, which was mediated by decreased expression of anti-apoptotic protein Bcl-2. Stimulation of DC maturation by TNF-alpha resulted in increased resistance of DC to PCa-induced apoptosis. In TNF-alpha treated mature DC, but not in immature DC, the expression of Bcl-2 was not blocked after exposure to RM-1-derived factors. Thus, these data suggest that TNF-alpha-induced maturation of DC increases their resistance to PCa induced apoptosis. This is likely to be due to the stabilizing of the expression of anti-apoptotic protein Bcl-2. The difference in the sensitivity of mature and immature DC to PCa-induced cell death should be considered during the design of DC-based clinical trials for PCa patients.Prostate Cancer and Prostatic Diseases (2001) 4, 221-227.     

MAGE-1抗原肽致敏DC活化淋巴细胞对裸鼠肝癌移植瘤的免疫防护作用

目的　观察黑色素瘤 1基因 (MAGE 1)抗原肽致敏树突状细胞 (DC)所活化的淋巴细胞(CTL)对人肝癌HCC移植瘤的抑制和消退作用 ,评估临床治疗HCC的可行性和有效性。　方法BEL 74 0 2HCC细胞于 30只裸鼠背部皮下接种 ,建立裸鼠HCC移植瘤模型 ,其中 2 2只成瘤 ;用MAGE 1九肽致敏DC所活化的淋巴细胞 (1× 10 6)注入肿瘤部位皮下 (治疗组A ,n =5 ) ,其余 17只随机分成 5组 (B、C、D、E、F) ,用其他不同性质的细胞治疗 ,观察各组肿瘤生长情况并进行病理学分析和统计学处理 ,阐明特异性CTL对肿瘤的作用机制。　结果　 (1)A组HCC移植瘤均停止生长并趋于缩小 ,荷瘤裸鼠观察期内无死亡 ;而其他 5组肿瘤均快速生长 ,大部分荷瘤裸鼠 2周内死亡。MAGE 1九肽致敏DC所活化淋巴细胞能显著抑制HCC移植瘤生长 ,促使肿瘤消退 (P <0 0 1)。 (2 )A组移植肿瘤广泛坏死 ,肿瘤细胞广泛凋亡。　结论　MAGE 1九肽致敏DC有抑制HCC生长 ,促进HCC消退 ,防止肿瘤转移、复发的作用。肿瘤细胞凋亡增强是DC肿瘤免疫的可能机制。MAGE 1九肽联合DC可作为治疗HCC的新型疫苗。     

Induction of cytolytic T lymphocytes against pediatric solid tumors in vitro using autologous dendritic cells pulsed with necrotic primary tumor

Purpose

Effective and generally applicable methods for generating cancer vaccines in children have not been defined. Dendritic cells (DCs) are the most potent professional antigen-presenting cells capable of activating primary cytolytic T cells. We tested the ability of DCs generated from pediatric patients' peripheral blood monocytes and pulsed with a necrotic tumor to activate autologous tumor-specific cytolytic T cells.

Methods

Tumor and peripheral blood cells were obtained from pediatric patients undergoing biopsy or resection for advanced solid tumors according to an institutional research board-approved protocol and after acquiring informed consent from them. To generate DCs, we treated peripheral blood monocytes with granulocyte-macrophage colony stimulating factor and interleukin (IL)-4. Maturation was induced with a cytokine cocktail (CC) containing tumor necrosis factor-α, IL-6, IL-1β, and prostaglandin E₂. The DC phenotype was assayed using flow cytometry. Tumor necrosis was induced by exposure to UV-B irradiation (1000 mJ). Dendritic cells pulsed with a UV-B-treated primary tumor and matured with CC were used to stimulate autologous peripheral blood lymphocytes weekly. Tumor-specific cytolytic activity was assayed using 4-hour ⁵¹Cr release.

Results

Peripheral blood monocytes isolated from pediatric patients differentiated into immature DCs (CD14⁻, MHCII⁺ [major histocompatibility complex], CD80^low, CD86^low) in the presence of granulocyte-macrophage colony stimulating factor and IL-4. Cytokine cocktail induced maturation of DCs, as characterized by increased expressions of MHCII, CD83, CD80, and CD86. Patients' peripheral blood lymphocytes stimulated in vitro with DCs loaded with a necrotic primary tumor and matured with CC specifically lysed autologous neuroblastoma in 7 of 9 patients.

Conclusion

Dendritic cells generated from the peripheral blood of children with advanced solid tumors and pulsed with a necrotic primary tumor undergo maturation and effectively stimulate autologous tumor-specific cytolytic T cells in vitro. We describe a simple method for generating a vaccine capable of activating cytotoxic T cells against pediatric solid tumors that does not require the genetic identification of tumor-associated antigens.     

以树突状细胞为基础个体化抗胃癌过继免疫治疗的研究

目的探讨负载自体肿瘤细胞裂解物的成熟树突状细胞(ATLs-mDCs)体外诱导个体化抗胃癌过继免疫治疗的效应。方法建立短期培养的原代胃癌细胞系。用ATLs-mDCs激活自体T细胞,制备肿瘤特异性细胞毒性T细胞(CTLs)。自体树突状细胞(DCs)均分为未成熟DCs、成熟DCs和ATLs-mDCs 3组,分别应用流式细胞仪及混合淋巴细胞增殖反应方法,检测DCs的免疫功能状态;应用细胞毒杀伤试验验证肿瘤特异性CTLs的杀伤活性;应用酶联免疫吸附试验(ELISA)测定DCs培养上清中白细胞介素12(IL-12)和CTLs上清中γ干扰素(IFN-γ)的分泌水平。结果ATLs-mDCs上调HLA-DR、CD80、CD83和CD86的表达水平,同时获得高效刺激自体T细胞增殖的能力。ATLs-mDCs诱导产生的CTLs对自体胃癌细胞的杀伤率为(84±11)%,显著高于对两株异体胃癌细胞的杀伤率[(19±7)%和(19±11)%(t=54.18和56.46,P值均<0.01)]。ATLs-mDCs上清液中IL-12的浓度显著高于单纯成熟DCs(t=15.47,P<0.01)及未成熟DCs(t=28.44,P<0.01)。3组DCs分别激活自体T细胞产生的CTLs上清液中INF-γ的浓度ATLs-mDCs组高于单纯成熟DCs组(t=4.84,P<0.05),并显著高于未成熟DCs组(t=13.74,P<0.01)。结论ATLs-mDCs在体外诱导产生的CTLs能有效特异性杀伤自体胃癌细胞。     

7-Hydroxystaurosporine-induced apoptosis in 9L glioma cells provides an effective antigen source for dendritic cells and yields a potent vaccine strategy in an intracranial glioma model

OBJECTIVE: On the basis of recent studies indicating that tumoral apoptotic bodies may provide a potent source of antigen for delivery to antigen-presenting cells, as well as observations that signal transduction modulation may constitute a promising approach for inducing glioma cell apoptosis, we explored the efficacy of vaccination with glioma apoptotic body-pulsed dendritic cells (DCs) for inhibiting tumor growth in the syngeneic 9L glioma/Fischer rat model. METHODS: For induction of apoptosis, 7-hydroxystaurosporine (UCN-01) (200-300 ng/ml), a selective protein kinase C inhibitor, was co-incubated with 9L cells in vitro for 72 or 96 hours. After this pretreatment period, glioma cells and DCs were mixed, and the interaction between DCs and apoptotic 9L tumor cells was assessed using two-color flow cytometry. In a series of experiments, the efficacy of vaccination strategies using DCs co-cultured with apoptotic 9L cells was then examined in animals harboring intracranial tumors. RESULTS: Pretreatment of 9L cells with UCN-01 resulted in approximately 50% of cells' being observed to undergo apoptosis as compared with less than 3% of controls. After subsequent co-culture, two-color flow cytometry demonstrated a time-dependent physical association of DCs with the apoptotic glioma cells. Survival in animals harboring intracranial tumors was significantly longer for the animals treated with a glioma apoptotic body-pulsed DC vaccine than in the animals that received apoptotic glioma cells and DCs alone or vehicle (i.e., the controls), especially those that underwent a sequential vaccination strategy (P < 0.0001). Long-term survival (>90 d) was demonstrated in 6 (75%) of 8 animals that underwent this vaccination approach versus 0 (0%) of 16 controls. In contrast, no survival benefit was observed in animals that received DCs that were co-cultured with vehicle-treated (non-apoptotic) 9L cells. Three of four long-term survivors that were rechallenged intracranially with tumor cells also survived over the long term. CONCLUSION: These studies suggest that induction of apoptosis in glioma cells by use of UCN-01 may promote the uptake of tumor antigens by DCs. This finding is important because apoptotic body-stimulated DCs may hold promise in promoting a host response against an established intracranial glioma, particularly if the parameters for apoptotic induction, duration of co-culture, and vaccination can be optimized.     

PI-3' kinase and NF-kappaB cross-signaling in human pancreatic cancer cells.

Because tumor necrosis factor-alpha (TNF-alpha) and some chemotherapeutic agents activate both apoptosis and NF-kappaB-dependent antiapoptotic genes, they may neutralize their own antitumor effects. The cell-signaling mechanisms for such chemoresistance are not clear but may involve phosphotidylinositol-3' kinase (PI3K). To clarify this we examined whether cross-signaling between PI3K and NF-kappaB enhances the antitumor effect of TNF-alpha in human pancreatic cancer cells. Quiescent pancreatic cancer cells (Panc-1, MiaPaCa-2) with TNF-alpha, Ly294002 (PI3K inhibitor), alone or combined, were restimulated with mitogen (10% fetal calf serum [FCS] to induce cell cycle entry). Proliferation (monotetrazolium), cell cycle progression (ApoBrDU and fluorescence-activated cell sorter analysis), and apoptosis (PARP cleavage; caspase-3 activation) were measured. Akt activation (Akt kinase assay) and IkappaBalpha degradation were determined by Western blot analysis. Translocation of NF-kappaB into the nucleus was examined by EMSA, whereas an NF-kappaB/luciferase reporter gene was used to quantify NF-kappaB-dependent gene expression. Statistical analysis was carried out by means of two-tailed t test (P <0.05). PI3K inhibition significantly enhanced the antiproliferative and proapoptotic effects of TNF-alpha in both cell lines, Ly294002 also blocked TNF-alpha-induced Akt activation but failed to alter cytoplasmic IkappaBalpha degradation or subsequent NF-kappaB nuclear translocation. NF-kappaB-dependent gene expression, however, was ultimately suppressed by Ly294002, suggesting that PI3k-dependent activation of NF-kappaB is IkappaBalpha independent. PI3K inhibition can block NF-kappaB-dependent gene expression regardless of cytoplasmic IkappaBalpha/NF-kappaB activation. Because it also regulates the antitumor effects of TNF-alpha, PI3K may in part determine NF-kappaB-induced chemoresistance in human pancreatic cancer.     

Mycophenolic acid inhibits cell proliferation and extracellular matrix synthesis in rat vascular smooth muscle cells through direct and indirect inhibition of cellular reactive oxygen species

BACKGROUND: Kaempferol is one of the most important constituents in ginkgo flavonoids. Recent studies indicate kaempferol may have antitumor activities. The objective of this study was to determine the effect and mechanisms of kaempferol on pancreatic cancer cell proliferation and apoptosis. MATERIALS AND METHODS: Pancreatic cancer cell lines MIA PaCa-2 and Panc-1 were treated with kaempferol, and the inhibitory effects of kaempferol on pancreatic cancer cell proliferation were examined by direct cell counting, 3H-thymidine incorporation, and MTS assay. Lactate dehydrogenase release from cells was determined as an index of cytotoxicity. Apoptosis was analyzed by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay. RESULTS: Upon the treatment with 70 microm kaempferol for 4 days, MIA PaCa-2 cell proliferation was significantly inhibited by 79% and 45.7% as determined by direct cell counting and MTS assay, respectively, compared with control cells (P < 0.05). Similarly, the treatment with kaempferol significantly inhibited Panc-1 cell proliferation. Kaempferol treatment also significantly reduced 3H-thymidine incorporation in both MIA PaCa-2 and Panc-1 cells. Combination treatment of low concentrations of kaempferol and 5-fluorouracil showed an additive effect on the inhibition of MIA PaCa-2 cell proliferation. Furthermore, kaempferol had significantly less cytotoxicity than 5-fluorouracil in normal human pancreatic ductal epithelial cells (P = 0.029). In both MIA PaCa-2 and Panc-1 cells, apoptotic cell population was increased when treated with kaempferol in a concentration-dependent manner. CONCLUSIONS: Ginkgo biloba extract kaempferol effectively inhibits pancreatic cancer cell proliferation and induces cancer cell apoptosis, which may sensitize pancreatic tumor cells to chemotherapy. Kaempferol may have clinical applications as adjuvant therapy in the treatment of pancreatic cancer.     

肿瘤坏死因子相关细胞凋亡诱导配体受体-4在人胰腺癌组织中的表达

目的：通过检测肿瘤坏死因子相关细胞凋亡诱导配体受体-4(TRAIL-R4)在胰腺癌组织中的表达，探讨胰腺癌细胞抵抗细胞凋亡的机理。方法：应用mRNA印迹法(Northern blotting)、蛋白质印迹法(Western blotting)和免疫组织化学技术，定性、定位分析TRAIL-R4在正常胰腺组织和胰腺癌组织中的表达。结果：TRAIL-R4 mRNA和蛋白在正常胰腺组织中呈弱表达或不表达，而在胰腺癌组织中呈高表达；免疫组织化学检测显示，在胰腺癌细胞中TRAIL-R4蛋白呈强染色。结论：TRAIL-R4表达水平在正常胰腺组织与胰腺癌组织中存在显著差异，提示胰腺癌细胞中可能存在对TRAIL介导的细胞凋亡抵抗新机理。     

辅助性T细胞和细胞毒性T淋巴细胞双表位修饰的树突状细胞肿瘤疫苗治疗胃癌的实验研究

目的研究辅助性T细胞（Th）表位和细胞毒性T淋巴细胞（CTL）双表位修饰的树突状细胞（DCs）肿瘤疫苗用于胃癌免疫治疗的效果。方法用CTL表位MAGE-341-49和Th表位MAGE-322-36混合多肽冲击DCs，每周刺激脾脏T细胞1次，4周后收集多肽特异性T细胞。流式仪分析T细胞亚群分布，测定CD4^＋T细胞识别抗原细胞因子分泌及CD8^＋T细胞杀伤肿瘤细胞效能，观察双表位修饰的DCs肿瘤疫苗治疗胃癌的保护性免疫效应。结果双表位致敏的DCs体外可同时活化CD4^＋和CD8^＋T细胞，其中CD4^＋T细胞识别肿瘤细胞小鼠前胃癌细胞株MFC后分泌大量Th1型细胞因子[干扰素（IFN）-γ，白介素（IL）-2]，CD8^＋T细胞强效杀伤MFC。双表位修饰的DCs肿瘤疫苗小鼠体内免疫治疗获得抵抗后继胃癌细胞MFC的免疫保护能力，并显著高于单一表位（CTL或Th）修饰的DC8疫苗。结论Th和CTL双表位修饰的DCs肿瘤疫苗可同时激活CD4^＋Th1细胞和CD8^＋CTL抗肿瘤免疫，有效清除胃癌细胞。     

Effect of the XIAP inhibitor Embelin on TRAIL-induced apoptosis of pancreatic cancer cells

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in a wide variety of tumor cells, while it has no toxicity for the majority of normal cells.Therefore, TRAIL may be a suitable agent for anticancer therapy. We previously reported that a number of pancreatic cancer cell lines show resistance to TRAIL-induced apoptosis via overexpression of XIAP and FLIP. The present study was conducted to further examine TRAIL-based therapeutic strategies by aiming to restore functional apoptotic pathways in resistant pancreatic cancer cells. METHODS: In various pancreatic cancer cell lines, TRAIL-induced apoptosis was evaluated in the presence or absence of an XIAP-inhibitor (Smac peptide). Second, TRAIL-induced apoptosis was evaluated in TRAIL-resistant AsPC-1 cells with or without FLIP antisense. Third, the combined effect of Smac peptide and FLIP antisense was tested, and the activation of apoptosis-related caspases and poly (ADP-ribose) polymerase was evaluated. Finally, TRAIL-induced apoptosis was evaluated in the presence or absence of FLIP antisense and an XIAP inhibitor (embelin). RESULTS: Smac peptide enhanced TRAIL-induced apoptosis in a dose-dependent manner for several pancreatic cancer cell lines, but showed no effect on TRAIL-resistant AsPC-1 cells. Smac peptide alone had no influence on cell viability. TRAIL-induced apoptosis was restored in TRAIL-resistant AsPC-1 cells by exposure to FLIP antisense, which suppressed the expression of FLIP. The effect of TRAIL was augmented by the combination of FLIP antisense and Smac peptide. Similarly, TRAIL-induced apoptosis was restored by the combination of FLIP antisense and embelin. Activation of apoptotic caspases and cleavage of poly (ADP-ribose) polymerase was observed after sensitization of TRAIL-resistant pancreatic cancer cells. CONCLUSIONS: Pancreatic cancer cells gain resistance to TRAIL-induced apoptosis via expression of the antiapoptotic proteins XIAP and FLIP. Smac peptide and FLIP antisense could restore the apoptotic effect of TRAIL. An XIAP inhibitor, embelin, enhanced the effect of TRAIL in the presence of FLIP antisense. These findings may provide useful information for the development of TRAIL-based therapeutic strategies by restoring functional apoptotic pathways in resistant pancreatic cancer cells. In addition, a low molecular weight XIAP inhibitor like embelin could be a lead compound for the development of effective XIAP inhibitors.     

Impact of psoralen/UVA-treatment on survival, activation, and immunostimulatory capacity of monocyte-derived dendritic cells

BACKGROUND: Extracorporeal Photopheresis (ECP) has been shown to be an effective treatment of graft-versus-host disease, solid organ graft rejection, and other T-cell-mediated diseases. The mechanisms of action of ECP include lymphocyte apoptosis, cytokine modulation, and the induction of regulatory T cells. It has been suggested that dendritic cells (DCs) are more resistant to ECP-induced apoptosis and might be directly modulated by ECP. We tested this hypothesis using in vitro Psoralen/UVA (PUVA) treatment as an in vitro model of ECP. METHODS: Monocyte-derived DCs (mo-DCs) were treated with 8-methoxypsoralen /UVA and analyzed for surface molecule expression, apoptosis markers, endocytosis, and migratory and immunostimulatory capacity. Mo-DC phenotype and cytokine secretion was tested after CD40L stimulation. Naive T cells stimulated with PUVA-treated mo-DCs were tested for Th1/Th2 cytokine secretion and associated chemokine receptor patterns. RESULTS: DCs underwent apoptosis after in vitro PUVA and in vivo ECP. In vitro, the induction of apoptosis was preceded by partial maturation of immature mo-DCs. PUVA-treated immature mo-DCs also exhibited enhanced migratory and immunostimulatory capacity. However, mo-DCs stimulation through CD40 ligation was abrogated and interleukin (IL)-12 secretion was abolished 24 hr after PUVA treatment. PUVA-treated mo-DCs skewed naive T cells toward a Th2 response as defined by increased IL-4, IL-10, and IL-13 and decreased interferon-gamma levels, and the expression of the Th2-associated chemokine receptors CCR4 and CCR10. The observed Th2 shift was partially reversed by exogenous IL-12. CONCLUSION: These data suggest that direct modulation of DC function as well as apoptosis contribute to the immunoregulatory effects of ECP.     

人胰腺癌细胞株放射敏感性的体外研究

目的通过体外实验探讨人胰腺癌细胞株的放射敏感性。方法培养人胰腺癌细胞株SW1990、Capan-1、AsPC-1、MIAPaCa-2、PANC-1、P3，应用集落形成实验测定胰腺癌细胞在6MVX线不同剂量照射后的存活分数，计算各株细胞的放射生物学参数。结果胰腺癌细胞株MIAPaCa-2对放射治疗最敏感，SF2为0．388，而Capan-1对放射治疗最不敏感，SF2为0．758，MIAPaCa-2与As-PC-1、AsPC-1与SW1990之间的放射敏感性无显著差异，其余各株胰腺癌细胞之间存在显著差异。结论不同胰腺癌细胞株的放射敏感性存在差异。     

Angiogenesis inhibitor TNP-470 reduces human pancreatic cancer growth

In this study we investigated the effects of the angiogenesis inhibitor TNP-470 on human pancreatic cancer cells in vitro and in vivo. The action of TNP-470 on vascular endothelial growth factor (VEGF) was also assessed. In vitro human pancreatic cancer cells (MIAPaCa-2, AsPC-1, and Capan-1), and human umbilical vein endothelial cells (HUVEC) were exposed to increasing concentrations (1 pg/ml to 100 (μg/ml) of TNP-470. Cell proliferation was assessed after 3 days by cell count and MTT assay. In vivo, 5 Χ 10⁶ pancreatic cancer cells were injected subcutaneously into nude mice. Four weeks later, 1 mm³ fragments of the resulting tumors were implanted into the pancreas of other mice. Animals received either TNP-470 (30 mg/kg every other day) or vehicle subcutaneously for 14 weeks. The volume of the primary tumor and metastatic spread were determined at autopsy. Concentrations of VEGF were determined in serum (VEGF_S) and ascites (VEGF_A) by enzyme-linked immunosorbent assay. Microvessel density was analyzed by immunohistochemistry in CD31 -stained tumor sections. In vitro, proliferation and viability of the human pancreatic cancer cell lines were significantly inhibited at high concentrations of TNP-470 (>1 μg/ml). In contrast, TNP-470 effectively decreased the growth of HUVEC at 100 pg/ml. In vivo, tumor volume and dissemination scores were significantly lower in all three pancreatic cancer cell lines. VEGF_S and VEGF_A were not different between treated groups. Treatment with TNP-470 significantly reduced neoangiogenesis in tumors of all three human pancreatic cancer cell lines: MIAPaCa-2 = 74.8 ±7.8/0.74 mm² vs. 24.8 ±3.7/0.74 mm²; AsPC-1 = 65.3 ±5.0/0.74 mm² vs. 26.0 ±3.4/0.74 mm²; and Capan-1 = 82.2 ±5.8/0.74 mm² vs. 26.9 ±2.5/0.74 mm² (P <0.001). However, survival was not statistically different between groups. TNP-470 reduced tumor growth and metastatic spread of pancreatic cancer in vivo. This was probably due to the antiproliferative effect of the agent on endothelial cells rather than to the direct inhibition of pancreatic cancer cell growth. TNP-470 activity was not associated with alteration of VEGF secretion. Supported by the R.S. Hirshberg Foundation and the Deutsche Forschungsgemeinschaft (grant HO 1843-1). Presented at the Forty-First Annual Meeting of The Society for Surgery of the Alimentary Tract, San Diego, Calif., May 21–24, 2000.     

Restoration of E-cadherin/beta-catenin expression in pancreatic cancer cells inhibits growth by induction of apoptosis

BACKGROUND: beta-Catenin is a component of the E-cadherin/catenin adhesion complex that maintains epithelial cell integrity. We have previously observed decreased beta-catenin expression in both human pancreatic cancer cell lines and primary tumors. To determine the significance of this finding with respect to pancreatic carcinogenesis, this study evaluated the effects of restoring expression of beta-catenin with and without E-cadherin in pancreatic cancer cells. METHODS: MiaPaca-2 cells were stably transfected with full-length cDNAs for beta-catenin, E-cadherin, or a mutated E-cadherin lacking the beta-catenin-binding domain. Doubly transfected cell clones containing beta-catenin and either E-cadherin or deleted E-cadherin were also selected. Assays for cell adhesion, cell cycle profile, motility, and apoptosis were performed. RESULTS: Cell clones expressing beta-catenin alone or beta-catenin and deleted E-cadherin did not differ significantly from the parental cell lines in any of the assays performed. In contrast, MiaPaca-2 cell clones expressing both beta-catenin and E-cadherin showed tight adhesion, decreased cell growth, and a significantly increased apoptotic index as compared to the parental line or singly transfected clones. CONCLUSIONS: MiaPaca-2 cells undergo apoptosis at a significantly increased rate after restoration of the E-cadherin/beta-catenin adhesion complex. This increase in apoptosis is dependent on the ability of E-cadherin to bind beta-catenin. Loss of beta-catenin expression may therefore provide pancreatic cancer cells with a growth advantage that contributes to tumor progression.     

Salicylates inhibit NF-kappaB activation and enhance TNF-alpha-induced apoptosis in human pancreatic cancer cells

INTRODUCTION: Tumor necrosis factor (TNF-alpha)-induced apoptosis is limited by its coactivation of nuclear factor kappa B (NF-kappaB)-dependent anti-apoptotic genes. Sodium salicylate (NaSal) inhibits NF-kappaB activation by limiting phosphorylation and degradation of its bound inhibitor protein, IkappaB-alpha. We examined whether NaSal enhances TNF-alpha-induced apoptosis in cultured human pancreatic cancer cell lines. METHODS: Two cultured human pancreatic cancer cell lines were studied. PANC-1 and BxPC-3 cells were serum-starved for 12 h, pretreated or not for 1 h with NaSal (5-20 mM), and then stimulated with recombinant human TNF-alpha (400 units/ml). Western blots of cytoplasmic lysates were performed to demonstrate IkappaB-alpha phosphorylation and degradation. Western blots of nuclear extracts were performed to assess nuclear translocation of NF-kappaB. In separate cultures, apoptosis was measured 4.5 h after TNF-alpha stimulation by both ELISA detection of interhistone DNA fragments and flow cytometry with propidium iodide staining. RESULTS: TNF-alpha induced IkappaB-alpha phosphorylation and degradation, which was inhibited by NaSal in both cell lines. TNF-alpha-induced apoptosis (DNA fragmentation) increased significantly when BxPC-3 cells were pretreated with NaSal. Flow cytometry confirmed this, demonstrating increases in apoptotic cell fractions: 8.5% (untreated), 9.3% (TNF-alpha alone), 14.9% (15 mM NaSal), and 22.9% (NaSal and TNF-alpha). In contrast, no increases in apoptosis were measured in the PANC-1 cell line among the various treatment groups. CONCLUSIONS: NaSal enhances TNF-alpha-induced apoptosis while inhibiting IkappaB-alpha phosphorylation and degradation in BxPC-3 human pancreatic cancer cells.     

树突状细胞融合瘤诱导抗结肠转移癌免疫应答

目的 检测结肠转移癌细胞SW620和树突状细胞（DC）融合构建的肿瘤疫苗对结肠转移癌细胞SW620及其同源结肠癌细胞SW480的杀伤作用。方法 应用促融合剂50％聚乙二醇（PEG）对SW620细胞和从外周血单个核细胞（PBMC）诱生的DC进行融合,用流式细胞仪（FCM）分析其表型并检测融合效率,电镜及免疫细胞化学观察DC、SW620和融合细胞（DC／SW620）形态;^51Cr释放法检测DC／SW620致敏CTL对SW620及SW480细胞的杀伤作用。结果 从PBMC成功诱生出高表达HLA-ABC、HLA-DR、CD80、CD86、CD83的成熟DC,DC／SW620的融合效率达到27．12％。融合细胞兼具DC与肿瘤细胞的结构特点及免疫表型。^51Cr释放法检测DC／SW620激活的CTL对SW620及SW480的杀伤作用强于各对照组（P〈0．01）。结论 DC与SW620细胞融合体外致敏自体T淋巴细胞能生成抗原特异CTL,对结肠转移癌细胞SW620及同源结肠癌细胞SW480有一定的杀伤作用。     

The Penetratin Sequence in the Anticancer PNC-28 Peptide Causes Tumor Cell Necrosis Rather Than Apoptosis of Human Pancreatic Cancer Cells

Background  PNC-27 and PNC-28 are p53-derived peptides from the human double minute (hdm-2) binding domain attached to penetratin. These peptides induce tumor cell necrosis of cancer cells, but not normal cells. The anticancer activity and mechanism of PNC-28 (p53 aa17–26-penetratin) was specifically studied against human pancreatic cancer. Methods  MiaPaCa-2 cells were treated with PNC-28. Necrosis was determined by measuring lactate dehydrogenase (LDH) and apoptosis as assayed for measuring elevation of proapoptotic proteins. PNC-29, an unrelated peptide, and hdm-2-binding domain p53 aa12-26 without penetratin (PNC-26) were used as controls. Since there is evidence that penetratin is required for tumor cell necrosis, we tested “naked” p53 peptide without penetratin by transfecting a plasmid that encodes p53 aa17–26 segment of PNC-28 into MiaPaCa-2 and an untransformed rat pancreatic acinar cell line, BMRPA1. Time-lapse electron microscopy was employed to further elucidate anticancer mechanism. Results  Treatment with PNC-28 does not result in the elevation of proapoptotic proteins found in p53-induced apoptosis, but elicits rapid release of LDH, indicative of tumor cell necrosis. Accordingly, we observed membrane pore formation and dose-dependent killing. In direct contrast, transfected MiaPaCa-2 cells underwent apoptosis, and not necrosis, as evidenced by expression of high levels of caspases-3 and 7 and annexin V with background levels of LDH. Conclusion  These results suggest that PNC-28 may be effective in treating human pancreatic cancer. The penetratin sequence appears to be responsible for the fundamental change in the mechanism of action, inducing rapid necrosis initiated by membrane pore formation. Cancer cell death by apoptosis was observed in the absence of penetratin. Wilbur B. Bowne, Kelley A. Sookraj, Michael Vishnevetsky, and Victor Adler contributed equally to this work. Presented in Part at the Society of Surgical Oncology, 61^st Annual Cancer Symposium Chicago, IL, March 13-16, 2008.