TPM4对人胰腺癌细胞侵袭和迁移的影响

目的 探讨原肌球蛋白4(TPM4)对人胰腺癌细胞侵袭和迁移的影响。方法 采用qRT-PCR检测人正常胰腺导管上皮细胞(HPDE)和人胰腺癌细胞系(ASPC-1、BXPC-3、MIA PaCa-2、PANC-1、SW1990)中TPM4 RNA表达量。采用siRNA或过表达慢病毒感染胰腺癌细胞(MIA PaCa-2、PANC-1),通过qRT-PCR、Western blotting检测各组细胞TPM4的表达,划痕实验及Transwell实验检测各组胰腺癌细胞侵袭和迁移能力。结果 GEPIA数据库中胰腺癌组织TPM4表达明显高于正常胰腺组织(P<0.05),qRT-PCR分析TPM4 RNA在胰腺癌细胞系中表达均高于HPDE(P<0.0001)。与对照组相比,在MIA Paca-2和PANC-1中过表达TPM4促进了胰腺癌细胞侵袭迁移能力(均P<0.01),而敲低TPM4胰腺癌细胞侵袭迁移能力则明显减弱(均P<0.01)。结论 TPM4在胰腺癌细胞中呈现高水平表达,可能在胰腺癌中发挥着癌基因的作用,其并可促进胰腺癌细胞迁移及侵袭能力。     

白细胞介素-1α、6对胰腺癌细胞分泌血管内皮生长因子A、C的调节作用

目的探讨细胞活素白细胞介素-1α、6(IL-1α、IL-6)对胰腺癌细胞产生和分泌血管内皮生长因子(VEGF)A、C的调节。方法用Northern杂交和Western杂交法分别分析人胰腺癌细胞株(ASPC-1、CAPAN-1、MIA—PαCα-2、P／MNC-1、COLO-357和T3M4)中VEGF-A、C基因和蛋白的表达；以细胞活索IL-1α(10pg／L)或IL-6(100pg／L)刺激细胞株(CAPAN-1和COLO-357)后用逆转录一聚合酶链式反应技术(RT—PCR)分析其VEGF—A、C基因的表达。结果Northern杂交法显示6种胰腺癌细胞株均有4．1kbVEGF-A基因和2．4kbVEGF—C基因的表达；Western杂交法显示这6种胰腺癌细胞株均有分子量为43kDα的VEGF—A蛋白质和分子量为55kDα的VEGF—C蛋白质的表达。RT—PCR分析法显示IL-1α使细胞株COLO-357产生VEGF—A、C基因分别增加1～2倍、1倍，但对细胞株CAPAN-1无明显刺激作用；而IL-6刺激细胞株CAPAN1产生VEGF—A、C基因分别增加2～5倍、1倍，但对细胞株COLO-357无明显刺激作用。结论胰腺癌中血管内皮生长因子A、C和细胞活素IL-1α、IL-6间的相互作用导致了癌细胞的血行和淋巴转移，促进胰腺癌的进展恶化。     

胰腺星状细胞对胰腺癌细胞增殖能力的影响及机制

目的 观察胰腺星状细胞(PSC)对人胰腺癌细胞株增殖能力的影响,探讨其分子机制.方法 用免疫印迹实验检测成纤维细胞生长因子(FGFs)在胰腺癌细胞及PSC中的表达及分泌;噻唑蓝(MTT)比色法检测PSC细胞条件培养基(CDM)对胰腺癌细胞体外增殖的影响,以及除去FGFs之后影响的变化;MTT法检测PSC细胞CDM对Cyclopamine抑制胰腺癌细胞增殖作用的影响;体内实验观察PSC对胰腺癌细胞成瘤能力的影响.结果 PSC主要表达并分泌FGF2,而胰腺癌MIA PaCa-2和PANC-1细胞则主要表达FGF5;PSC细胞CDM刺激48 h后,胰腺癌MIA PaCa-2和PANC-1细胞体外增殖能力分别为对照组的(1.2993±0.1170)倍和(1.2447±0.0123)倍(P＜0.05);中和CDM中FGFs后,FGFs(-)CDM对胰腺癌细胞增殖的刺激作用显著削弱;与单纯注射胰腺癌PANC-1细胞的裸鼠皮下种植瘤比较,PSC与PANC-1细胞混合注射组的平均肿瘤体积、质量均明显增加,分别由380.13 mm3和170 g增加至601.31 mm3和349 g(P＜0.05);PSC细胞CDM处理后,Cyclopamine对胰腺癌MIA PaCa-2和PANC-1细胞的增殖抑制作用显著下降(P＜0.01).结论 PSC在体内、体外均显著刺激胰腺癌细胞的增殖,增强其成瘤能力,其机制与PSC旁分泌FGF2作用于癌细胞相关;PSC显著降低了胰腺癌细胞对Hedgehog信号通路特异性抑制剂Cyclopamine治疗的敏感性.

Abstract：

Objective To assess the effects of pancreatic stellate cells (PSC) on proliferation of pancreatic cancer cells and to reveal its possible mechanism. Methods Expression and secretion of fibroblast growth factor (FGF) ligands in both pancreatic cancer cells and stellate cells were determined by immunoblotting analysis. Mmethyl thiazol tetrazolium (MTT) assay was used to examine the effects of conditioned medium (CDM) of PSC on the proliferation of pancreatic cancer cells. An in vivo tumorigenicity assay was used to evaluate the effect of PSC on xenograft formation in cancer cells. Results FGF2 was predominantly expressed and secreted by PSC. Yet MIA PaCa-2 and PANC-1 pancreatic cancer cells mainly expressed FGF5. CDM of PSC enhanced the proliferation of MIA PaCa-2 and PANC-1 cells for ( 1. 2993 ±0. 1170 ,P ＜0. 05 ) times and ( 1. 2447 ±0. 0123 ,P ＜0. 05 ) times, respectively. Neutralizing the effects of FGFs in the CDM by heparin-sepharose precipitation abolished this effect. The volume and weight of subcutaneous tumors in nude mice by injection of combination of pancreatic cancer cells with PSC were significantly greater than those by injection of PANC-1 cells alone (380. 13 mm3 and 170 g versus 601.31 mm3and 349 g,P ＜0. 05, respectively). The CDM of PSC reduced the antiproliferative effect by cyclopamine on pancreatic cancer cells ( P ＜ 0. 01, respectively). Conclusion We identified in this study a mechanism based on stroma-tumor interactions involving PSC that can contribute to enhance the proliferation of human pancreatic cancer cells.     

表没食子儿茶素没食子酸酯通过PI3K/Akt通路抑制胰腺癌细胞糖酵解的实验研究

目的：探讨表没食子儿茶素没食子酸酯（EGCG）对胰腺癌细胞的增殖及糖代谢的影响。方法：用CCK8试剂盒检测不同浓度EGCG对胰腺癌PANC-1细胞增殖的影响，用葡萄糖乳酸试剂盒检测EGCG对糖酵解的影响，Western blot法检测糖酵解酶及PI3K/Akt通路蛋白的表达。结果：CCK8结果显示，EGCG可以抑制PANC-1细胞的增殖，其抑制作用具有剂量依赖性和时间依赖性（P <0.05）。葡萄糖乳酸检测实验表明，EGCG可以抑制PANC-1细胞的葡萄糖消耗和乳酸生成（P <0.05）。Western blot结果表明，EGCG可以抑制糖酵解酶己糖激酶-2(HK-2)、磷酸果糖激酶（PFK）和乳酸脱氢酶（LDH）及磷酸化PI3K和Akt的表达，且EGCG浓度越高，抑制作用越明显（P <0.05）。结论：EGCG抑制人胰腺癌细胞系PANC-1的增殖和糖酵解，其抑制作用可能与抑制PANC-1细胞中PI3K/Akt通路有关。     

以白细胞介素4-受体为靶标的胰腺癌靶向治疗研究

目的 检测白细胞介素4受体(IL-4R)在胰腺癌组织及人胰腺癌细胞株中的表达,观察以抗白细胞介素-4受体单抗(MAIL4R)为载体构建的免疫毒素MAIL4R-CTX对胰腺癌细胞的靶向杀伤作用.方法 应用免疫组织化学检测IL-4R在26例胰腺癌组织、15例胰腺正常组织中的表达;免疫细胞化学染色检测IL-4R在人胰腺癌PANC-1、BxPC-3细胞和人肺腺癌H1299细胞中的表达;采用噻唑蓝(MTT)法观察MAIL4R、眼镜蛇毒细胞毒素(CTX)、免疫毒素MAIL4R-CTX对体外培养PANC-1,BxPC-3细胞和H1299细胞生长的影响.结果 IL-4R在26例胰腺癌组织中均呈不同程度的阳性表达,而15例胰腺正常组织中仅1例呈弱阳性表达,余均为阴性表达;人胰腺癌PANC-1,BxPC-3细胞均表达IL-4R,H1299细胞不表达IL-4R;CTX对PANC-1,BxPC-3和H1299细胞均有明显抑制作用,但对3株细胞的抑制率差异无统计学意义(P＞0.05);MAIL4R对PANC-1、BxPC-3和H1299细胞均无明显抑制作用;MAIL4R-CTX对过表达IL-4R的BxPC-3和PANC-1细胞的生长具有显著的抑制作用,并且为剂量依赖性,而对低表达IL-4R的H1299细胞不敏感,在浓度为18.75 mg/L,作用4h时PANC-1和BxPC-3细胞分别有86.4%和95.2%被杀伤,H1299细胞仅死亡26.8%(P＜0.01).结论 IL-4R过表达于胰腺癌组织和胰腺癌细胞株,而低表达于正常胰腺组织中,免疫毒素MAIL4R-CTX在体外对过表达IL-4R的细胞株PANC-1、BxPC-3有靶向性杀伤作用.     

RasTG基因与胰腺癌的相关性研究

目的体外观察高尔基嵌合Ras基因(RasTG基因)在胰腺癌组织中的表达情况和对人胰腺癌细胞株PANC-1的生长、增殖及细胞成瘤能力的影响,并探讨其作用机理。方法以人胰腺癌细胞株PANC-1为研究对象,构建含RasTG基因的慢病毒转染人胰腺癌细胞株PANC-1,利用RNA干扰技术干扰细胞内RasTG基因的表达,观察干扰后PANC-1细胞的增殖、转化和成瘤情况,并通过组织芯片技术检测胰腺导管癌及其癌旁组织中RasTG蛋白的表达情况。结果 ①RasTG无十分特异的组织表达谱,在脑、肝脏及肾上腺中表达相对较高;②RasTG在胰腺导管癌组织中的表达明显高于癌旁组织(P<0.05);③干扰RasTG基因后的人胰腺癌细胞株PANC-1的生长、增殖和转化能力均下降;④干扰RasTG基因后的人胰腺癌细胞株PANC-1的成瘤能力明显减弱,转染组的成瘤体积明显小于未转染组(P<0.05)。结论 RasTG基因在动物体内广泛分布;RasTG在胰腺癌组织中的表达比在癌旁组织中高;干扰RasTG基因的表达后对人胰腺癌细胞株PANC-1的增殖、转化和成瘤能力均有抑制作用,RasTG基因是正调节因子。     

成纤维细胞生长因子受体1在胰腺癌细胞中的表达和调控

目的 研究成纤维细胞生长因子受体1(FGFR1)蛋白和mRNA在胰腺癌细胞系中的表达和调控机制.方法 采用Western免疫印迹实验、Northern印迹分析和RT-PCR检测FGFR1在胰腺癌细胞中的表达.使用外源性生长因子刺激细胞并使用激酶抑制剂阻断细胞内信号转导通路,观察FGFR1蛋白和mRNA在胰腺癌细胞中表达的变化情况.结果 FGFR1蛋白和mRNA在胰腺癌细胞系中均有不同程度的表达.生长因子刺激可上调FGFR1蛋白和mRNA的表达水平.其中IGF-1、EGF和FGF2显著增加Mia PaCa-2细胞FGFR1的表达,EGF和FGF2显著增加PANC-1细胞FGFR1的表达(P＜0.05).FGF2对FGFR1表达的调节具有时间依赖性.ERK1/2抑制剂UO126和p38 MAPK抑制剂SB203580降低了PANC-1细胞中FGFR1的蛋白和mRNA的表达水平. 结论生长因子可上调FGFR1在胰腺癌细胞中的表达水平,MAPK信号转导通路中的ERK1/2和p38 MAPK亚通路参与FGFR1表达的调节.     

南瓜蛋白对人胰腺癌细胞系PANC-1的细胞毒性及化疗增敏作用

目的：观察南瓜蛋白（CUS）对人胰腺癌细胞系PANC-1的细胞毒性和化疗增敏作用。方法：采用四甲基偶氮唑蓝（MTT）比色法,观察不同浓度（量效）的CUS及作用不同时间（时效）对胰腺癌细胞PANC-1的细胞毒性;采用 Chou Talalay 联合指数法判断CUS增加胰腺癌细胞对吉西他滨(GEM)的化疗敏感性。结果：CUS对胰腺癌PANC-1细胞具有显著的增殖抑制作用,0.3125, 0.625, 1.25, 2.5, 5.0, 10.0, 20.0, 40.0, 80.0 μg/mL的CUS作用于胰腺癌细胞24 h,其抑制率分别为9.16％,11.9％,14.4％,20.2％,32.0％,36.4％,44.1％,50.3％和58.6％,呈现明显的剂量依赖性;随着作用时间的延长,其抑制率明显增加, 24,48,72 h的半数抑制浓度（IC 50）分别为40.24,8.24,1.17 μg/mL,呈现明显的时间依赖性。两药在不同效应时各自所需浓度之间相互作用均为协同作用（CI<1）, 单吉西他滨用于胰腺癌细胞的IC 50为36.76 nmol/L, CUS与其合用时的IC 50为12.14 nmol/L,合用比单用时吉西他滨的剂量减少67.0%。结论：CUS对胰腺癌的细胞毒性作用呈明显的剂量依赖性和时间依赖性,与吉西他滨联合使用可增加其细胞毒作用,起到化疗增敏效应。     

胰腺星状细胞通过白细胞介素-22影响胰腺癌侵袭转移及化疗耐药的研究

目的探讨具有不同白细胞介素-22(IL-22)表达水平的胰腺星状细胞(PSC)对胰腺癌细胞侵袭转移及化疗耐药的影响。方法将人原代胰腺癌间质PSC细胞与胰腺癌PANC-1细胞共培养后检测IL-22的表达及胰腺癌细胞的生长增殖能力、迁移和侵袭能力变化;构建稳定敲低或者过表达IL-22的人原代胰腺癌间质PSC细胞并与胰腺癌PANC-1细胞进行体外共培养, 检测PANC-1细胞的生长增殖、迁移和侵袭能力;利用稳定敲低或者过表达IL-22的人原代胰腺癌间质PSC细胞并与胰腺癌PANC-1细胞进行体外共培养后检测E-钙黏蛋白(E-cadherin)和波形蛋白(Vimentin)的表达情况;将含有不同表达水平的胰腺癌间质PSC细胞与胰腺癌PANC-1细胞体外共培养并加入不同浓度梯度的吉他西滨培养72 h后检测细胞存活率, 计算细胞耐受吉他西滨的半数抑制浓度(IC50);将稳定敲低或者过表达IL-22的人原代胰腺癌间质PSC细胞并与胰腺癌PANC-1细胞进行混合后种植于裸鼠皮下, 40 d后处理裸鼠取出肿瘤并绘制肿瘤生长曲线, 比较两组细胞的成瘤能力。采用t检验分析各组间差异的显著性。结果单纯PANC...     

miR-7-5p抑制胰腺癌细胞放疗后加速再增殖的体外实验研究

目的 研究放疗后濒死胰腺癌细胞（PANC-1）释放的微小RNA-7-5p（miR-7-5p）对存活PANC-1加速再增殖效应的相关机制。方法 通过慢病毒感染,建立荧光素酶标记的PANC-1-LUC胰腺癌细胞放疗后加速再增殖模型。通过RT-qPCR检测miRNA-7-5p的表达水平,流式细胞仪检测细胞周期及凋亡,双荧光素酶法验证miR-7-5p靶基因,Western blotting检测γH2A.X与DDIT4 蛋白的表达情况。结果 miR-7-5p在受辐照PANC-1（10 Gy）濒死细胞上清中表达下调,受辐照PANC-1细胞与存活PANC-1-LUC细胞共培养,可促进放疗后存活PANC-1-LUC细胞加速再增殖及其DNA损伤修复,而上调miR-7-5p可抑制放疗后胰腺癌细胞PANC-1-LUC再增殖。进一步研究表明,miR-7-5p在胰腺癌细胞中通过靶向下调DDIT4通路促进凋亡信号。结论 受辐照胰腺癌细胞可通过下调miR-7-5p促进DDIT4表达,从而抑制细胞凋亡及调控细胞周期再分布来加速再增殖,表明提高miR-7-5p的水平能够提高胰腺癌细胞的放疗敏感性,这将为改善胰腺癌放疗抵抗提供了一种新的思路和方法。     

Inhibition of pancreatic cancer cell growth and induction of apoptosis with novel therapies directed against protein kinase A

BACKGROUND: Pancreatic cancer is the most lethal abdominal malignancy. Expression of the RIalpha subunit of protein kinase A (PKA) has been associated with neoplastic transformation and mitogenic signaling. The effect of PKA inhibition on pancreatic cancer cell growth and apoptosis is unknown. In pancreatic cancer cells, we sought to determine (1) whether inhibition of PKA can inhibit growth or induce apoptosis, and (2) whether growth can be inhibited by silencing of RIalpha expression. METHODS: Human pancreatic cancer cells (PANC-1, MIA PaCa-2, and SUIT-2) were treated with inhibitors of PKA (H89 or PKI) and cell growth, kinase activity, and induction of apoptosis measured. Small inhibitory RNA (siRNA) directed against the RIalpha subunit was synthesized and transfected into PANC-1 cells. RESULTS: H89 decreased PKA activity and inhibited pancreatic cancer cell growth. Apoptosis was also induced by H89 in PANC-1 and MIA PaCa-2 cells. PANC-1 cells express high levels of the RIalpha subunit; transfection of siRNA decreased RIalpha protein expression and inhibited growth. CONCLUSIONS: Inhibition of PKA in pancreatic cancer cells induces growth arrest and apoptosis; similar effects are noted in cells with siRNA used to block RIalpha expression. Inhibition of PKA may represent a novel therapeutic strategy for the adjuvant treatment of pancreatic cancer.     

Specific gene expression and therapy for pancreatic cancer using the cytosine deaminase gene directed by the rat insulin promoter

Suicide gene therapy has been shown to be an effective means of destroying pancreatic cancer cells, but cell-specific delivery of the gene is required to limit host toxicity. The objective of this study is to determine whether the rat insulin promoter (RIP) will permit cell-specific gene delivery and subsequent cell death in human pancreatic cancer cells. The RIP DNA was amplified using polymerase chain reaction (PCR), and the purified fragment was inserted into pCR-Blunt II-TOPO plasmid at the SpeI site, which contains the coding sequence of yeast cytosine deaminase (CD). Transfection assays were carried out using both RIP-lacZ and RIP-CD DNA constructs in two human pancreatic cancer cell lines, PANC-1 and MIA PaCa-2. Reporter assays using X-gal staining were performed, and the in vitro cytotoxicity was examined in RIP-CD-transfected cells treated with 5-.ucytosine for 5 days. The expression levels of CD protein in the transfected cells were determined 2 days after transfection by Western blot analysis. The expression levels of insulin promoter factor (IPF-1/PDX-1) in these human pancreatic cell lines, as well as in freshly isolated human pancreatic cancer specimens, were determined using in situ immunohistochemistry analysis. After transfection with RIP-lacZ, only PANC-1 cells, but not MIA PaCa-2 cells, were positive for RIP-lacZ expression, indicating that RIP-directed reporter gene expression occurred only in PANC-1 cells. After transfection with RIP-CD and treatment with 5-flucytosine, PANC-1 cells had a significantly increased cell death rate compared with that of MIA PaCa-2 cells, suggesting that RIP-directed suicide gene expression occurred only in PANC-1 cells. Western blot analysis demonstrated that only PANC-1 cells were able to express the CD protein and that significantly increased levels of PDX-1 were found in PANC-1 but not in Mia PaCa-2 cells. In situ immunohistochemical analysis of both cell lines showed that PDX-1 was only expressed in the nuclei of PANC-1 cells and not in MIA PaCa-2 cells. Furthermore, two freshly isolated human pancreatic cancer specimens had significantly increased levels of PDX-1. The RIP is activated in PANC-1 cells, but not in Mia PaCa-2 cells, and the mechanism of activation is via PDX-1. Pancreatic cancer-specific cytotoxicity can be achieved with the use of RIP-CD and 5-flucytosine treatment in vitro. Significantly increased levels of PDX-1 have been found in human pancreatic cancer specimens. These results suggest that RIP could be used for cell-specific suicide gene therapy to target human pancreatic tumors. Presented at the Forty-Fourth Annual Meeting of The Society for Surgery of the Alimentary Tract, Orlando, Florida May 18–21, 2003 (oral presentation). Supported by an Advances in Technology Transfer Grant (ATP1176) from the State of Texas Department of Education and by grant NCI R01 (CA95731).     

The cytotoxic effects of bile acids in crude bile on human pancreatic cancer cell lines

Pancreatic cancer frequently causes extrahepatic cholestasis. To identify the direct effects of bile acids in jaundiced serum on pancreatic cancer, the proliferation of PANC-1 and MIA PaCa-2 cells as well as the ultrastructural alteration of PANC-1 cells cultured in crude bile modified media were studied. The growth of these cells in the RPMI-1640 media with or without 1%, 2%, and 4% of the refined crude bile was assessed after 48 and 96 h of incubation. The ultrastructure of PANC-1 cells was investigated by scanning and transmission electron microscopy after 24 and 48 h of incubation. The proliferation of both cell lines in the bile-treated media was greatly inhibited. The inhibitory rates of bile on PANC-1 ranged from 24.1% ± 3.3% to 66.9% ± 6.6% (P < 0.01) and those on MIA PaCa-2 ranged from 16.7% ± 3.8% to 50.7% ± 5.5%. (P < 0.01). When the bile-added media were replaced, the cells were able to restore their proliferating ability. The PANC-1 cells incubated in the bile-supplied media indicated that the mirovilli, mitochondria, and other organelles had thus been injured. These results suggest that bile acids appear to inhibit the proliferation of PANC-1 and MIA PaCa-2 cells, and the probable inhibitory mechanism is mainly considered to be due to the cytotoxicity of such bile acids. Received: November 2, 1999 / Accepted: May 30, 2000     

Dimethylamino Parthenolide Enhances the Inhibitory Effects of Gemcitabine in Human Pancreatic Cancer Cells

Introduction

Gemcitabine is standard treatment for pancreatic cancer but has limited clinical benefit due to chemoresistance. Nuclear factor-kappaB (NF-??B) can promote chemoresistance and is therefore an attractive therapeutic target. We hypothesize that NF-??B suppression with the novel, orally bioavailable inhibitor dimethylamino parthenolide (DMAPT) will sensitize pancreatic cancer cells to gemcitabine.

Methods

BxPC-3, PANC-1, and MIA PaCa-2 human pancreatic cancer cell lines were treated with gemcitabine and/or DMAPT. Effects on the NF-??B pathway were determined by electrophoretic mobility shift assay, ELISA, or Western blot. Proliferation and apoptosis were measured by cell counts and ELISA, respectively. The effect of gemcitabine in vivo was determined using a MIA PaCa-2 heterotopic xenograft model.

Results

Gemcitabine induced NF-??B activity in BxPC-3, PANC-1, and MIA PaCa-2 cells and decreased the level of the NF-??B inhibitor I??B?? in BxPC-3 and PANC-1 cells. DMAPT prevented the gemcitabine-induced activation of NF-??B. The combination of DMAPT/gemcitabine inhibited pancreatic cancer cell growth more than either agent alone. Gemcitabine also induced intratumoral NF-??B activity in vivo.

Conclusions

DMAPT enhanced the anti-proliferative effects of gemcitabine in association with NF-??B suppression in pancreatic cancer cells in vitro. Furthermore, gemcitabine induced NF-??B activity in vivo, thus supporting the evaluation of NF-??B-targeted agents to complement gemcitabine-based therapies.     

Treatment with gemcitabine and TRA-8 anti-death receptor-5 mAb reduces pancreatic adenocarcinoma cell viability in vitro and growth in vivo

Gemcitabine is a first line agent for pancreatic cancer, but yields minimal survival benefit. This study evaluated in vitro and in vivo effects of a monoclonal antibody (TRA-8) to human death receptor 5, combined with gemcitabine, using two human pancreatic cancer cell lines, S2VP10 and MIA PaCa-2. A subcutaneous model of pancreatic cancer was employed to test in vivo efficacy. S2VP10 and MIA PaCa-2 cells were treated with varying doses of gemcitabine and TRA-8. Cell viability and apoptosis were determined with an adenosine triphosphate assay and annexin V staining, respectively. Mitochondrial membrane destabilization was evaluated with fluorescence-activated cell sorting analysis of JC-1 stained cells. Caspase activation was evaluated by Western blot analysis. MIA PaCa-2 subcutaneous xenografts in athymic nude mice were evaluated for response to treatment with 200 μg of TRA-8 (intraperitoneal on days 9, 13, 16, 20, 23, and 27 postimplant) and 120 mg/kg gemcitabine (I.P. on days 10, 17, and 24). Tumor growth was measured with calipers. MIA PaCa-2 and S2VP10 cells receiving combination treatment with TRA-8 and gemcitabine demonstrated enhanced cytotoxicity, annexin V staining, and mitochondrial destabilization compared to either agent alone. Combination treatment produced enhanced caspase-3 and -8 activation in both cell lines compared with either agent alone. In vivo studies demonstrated mean subcutaneous tumor surface area (produce of two largest diameters) doubling times of 38 days untreated, 32 days gemcitabine, 49 days TRA-8, and 64 days combination treatment. TRA-8 is an apoptosis-inducing agonistic monoclonal antibody that produced synergistic cytotoxicity in combination with gemcitabine in vitro through enhanced caspase activation. These findings, with substantial inhibition of tumor growth in a mouse pancreatic cancer xenograft model receiving combination therapy, are encouraging for anti-death receptor therapy in the treatment of pancreatic cancer. Presented at the Forty-Seventh Annual Meeting of The Society for Surgery of the Alimentary Tract, Los Angeles, California, May 20–24, 2006 (poster presentation). Supported by the National Institutes of Health/NRSA T32 CA91078 Research Training Program in Surgical Oncology Training Grant (Dr. Kirby Bland P.I.) and NIH SPORE in Pancreatic Cancer 1 P20 CA10195-01.     

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.     

Neurotensin regulates growth of human pancreatic cancer.

OBJECTIVE: The effect of neurotensin (NT) on in vitro-growth of human pancreatic cancer cells (MIA PaCa-2) was examined. Furthermore, the intracellular signal-transduction pathways by which neurotensin regulates growth of MIA PaCa-2 cells were determined. SUMMARY BACKGROUND DATA: NT is trophic for normal rat pancreas, but the effect of NT on growth of human pancreatic cancer is not known. METHODS: Effects of NT (10(-12) to 10(-6) mol/l) on growth of MIA PaCa-2 cells were determined by both count of cell numbers and 3H-thymidine incorporation. Action of NT on phosphatidylinositol (PI) hydrolysis, cyclic AMP production, and intracellular calcium level were determined by conventional methods. The effects of 8-bromo-cyclic AMP and prostaglandin E2 on cell growth were determined. RESULTS: Low concentrations of NT (10(-12) to 10(-9) mol/l) stimulated growth in a dose-dependent manner, but higher concentrations of NT (10(-8) to 10(-6) mol/l) did not stimulate growth of MIA PaCa-2 cells. NT (10(-12) to 10(-6) mol/l) stimulated PI hydrolysis and increased intracellular calcium levels in a dose-dependent manner. High concentrations of NT (10(-8) to 10(-6) mol/l) stimulated production of cyclic AMP in a dose-dependent manner. 8-bromo-cyclic AMP inhibited growth of MIA PaCa-2 cells; prostaglandin E2 did not affect growth of MIA PaCa-2 cells. CONCLUSIONS: NT stimulates growth of MIA PaCa-2 cells through stimulation of PI hydrolysis and mobilization of calcium. Stimulation of the cyclic AMP pathway by high concentrations of NT abolishes the growth-stimulatory effect of NT that is mediated through PI hydrolysis or calcium mobilization.     

Defining the role of the epidermal growth factor receptor in pancreatic cancer grown in vitro

BACKGROUND: The purpose of this study was to determine the effect of a novel epidermal growth factor (EGF) receptor tyrosine kinase inhibitor, Erlotinib, on pancreatic cancer cell lines of varying differentiation in vitro. METHODS: Six pancreatic cancer cell lines (AsPc-1, CAPAN-1, HPAC, HPAF-II, Mia PaCa-2, PANC-1) were grown in the presence of 50 microM or 100 microM of Erlotinib or recombinant EGF. Cell proliferation was determined using the MTT assay over 72 hours. The EGF receptor gene and protein expression were determined by polymerase chain reaction and immunohistochemistry respectively. RESULTS: All cell lines demonstrated the presence of the EGF receptor gene and its gene product. Five of six cell lines showed significant growth inhibition at 72 hours compared with controls (P <0.05). The EGF augmentation increased proliferation of each cell line but this increase was only significant in AsPc-1. CONCLUSIONS: Inhibition of EGF receptor is a valid therapeutic strategy in pancreatic cancer.