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.     

Inhibition of Proliferation by Omega-3 Fatty Acids in Chemoresistant Pancreatic Cancer Cells

Background Pancreatic cancer-gemcitabine (GEM) chemoresistance has been demonstrated to be associated with enhanced NF-kB activation and antiapoptotic protein synthesis. The well-known capacity of omega-3 fatty acids (n-3 FAs) to inhibit NF-kB activation and promote cellular apoptosis has the potential to restore or facilitate gemcitabine chemosensitivity. Methods Four pancreatic cancer cell lines (MIA PaCa-2, BxPC-3, PANC-1, and L3.6), each with distinct basal NF-kB and differing GEM sensitivity profiles, were administered: 100 uM of (1) n-3FA, (2) n-6FA, (3) GEM, (4) n-3FA + GEM, or (5) n-6FA + GEM for 24 and 48 hours. Proliferation was assessed using the WST-1 assay. To define the mechanism(s) of altered proliferation, electron mobility shift assay for NF-kB activity, western blots of phoshoStat3, phosphoIκB, and poly(ADP-ribose) polymerase (PARP) cleavage were performed in the MIA PaCa-2 cell line. Results All cell lines demonstrated a time/dose-dependent inhibition of proliferation in response to n-3FA. For MIA PaCa-2 cells, n-3FA and n-3FA + GEM treatment resulted in reduction of I-kB phosphorylation and NF-kB activation when compared with n-6FA control. n-3FA and combination treatment also significantly decreased Stat3 phosphorylation, whereas GEM alone had no effect. n-3FAs and n-3FA + GEM groups demonstrated increased PARP cleavage, mirroring NF-kB activity and Stat3 phosphorylation. Conclusions n-3 FA treatment is specifically associated with inhibition of proliferation in these four pancreatic cell lines irrespective of varied gemcitabine resistance. An experimental paradigm to screen for potential contributory mechanism(s) in altered pancreatic cancer cellular proliferation was defined, and using this approach the co-administration of n-3 FA with GEM inhibited GEM-induced NF-kB activation and restored apoptosis in the MIA PaCa-2 cell-line. Supported by: NIDDK K08 DK DK60778 (Espat)     

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.     

抗表皮生长因子受体单克隆抗体联合5-氟尿嘧啶、健择对胰腺癌的作用

目的 观察抗表皮生长因子受体(EGFR)单克隆抗体联合5-氟尿嘧啶(5-Fu)、健择对胰腺癌的作用.方法 将人胰腺癌肿瘤细胞悬液注射于裸鼠背部皮下,建立肿瘤模型.干预组腹腔注射鼠抗人EGFR单克隆抗体(MMAb-2)、5-Fu、健择及其配伍联合用药;对照组注射生理盐水.干预4周后处死裸鼠,评价干预效果.结果 MMAb-2单独应用对胰腺癌细胞增殖有抑制作用(P<0.05),MMAb-2联合应用5-Fu、健择对胰腺癌细胞增殖的抑制作用明显增强(P<0.05),MMAb-2组裸鼠体质量及其白细胞计数明显高于化疗组(P<0.05).结论 抗EGFR抗体单独应用有确切的抗肿瘤疗效;与化疗联合应用可以增效;对裸鼠机体免疫功能无明显毒性.     

Pancreatic Cancer Cell Genetics and Signaling Response to Treatment Correlate with Efficacy of Gemcitabine-Based Molecular Targeting Strategies

Introduction Pancreatic cancer is a deadly cancer with limited sensitivity to gemcitabine. Molecular targeting of critical signaling pathways [nuclear factor kappa-B (NF-κB), PI3K/AKT, and mitogen-activated protein kinase (MAPK)] in combination with gemcitabine may improve sensitivity. We hypothesize that pancreatic cancer cell genetics and signaling response to treatment correlate with efficacy of gemcitabine-based molecular targeting strategies. Materials and Methods PANC-1, PaCa-2, and BxPC-3 cells were treated with curcumin, LY294002, or PD325901 alone or in combination with gemcitabine. Proliferation was measured by cell counts and enzyme activity by Western blot and electrophoretic mobility shift assay. Results Each agent dose-dependently decreased proliferation. All cells decreased NF-κB activity with curcumin(24 h) except PaCa-2, MEK activity with PD325901(24 h), and PI3Kinase with LY294002(3 h). However, PI3K rebounded to(PaCa-2) or above (Panc-1,BxPC-3) basal in LY294002-treated cells (24 h). Combinations with gemcitabine resulted in at least additive effects on proliferative inhibition. For PANC-1, curcumin + gemcitabine was nearly synergistic, correlating with gemcitabine-induced NF-κB activity. LY294002 + gemcitabine was nearly synergistic in PaCa-2 cells, which showed a lower induction of PI3Kinase activity with LY294002. Finally, gemcitabine + PD325901 was only effective in BxPC-3, which exhibited increased MEK activity with gemcitabine. Conclusions These results demonstrate differences in treatment efficacy, which correlate with the cell’s signaling response to treatment. Signaling profiles of each tumor may be necessary to determine an optimal chemotherapy for pancreatic cancer. These findings were presented at the 2007 American Hepatopancreatico-biliary Association Meetings in Las Vegas, NV, USA.     

抗表皮生长因子受体单克隆抗体对人胰腺癌裸鼠模型的化疗增敏作用

目的 观察鼠抗人EGFR单克隆抗体联合5-氟尿嘧啶(5-Fu)、健择对裸鼠胰腺癌化疗效果的影响.方法 将人胰腺癌肿瘤细胞悬液注射于裸鼠背部皮下,建立肿瘤模型.干预组腹腔分别注射鼠抗人EGFR单克隆抗体(MMAb-2)、5-Fu、健择及其配伍联合用药;对照组腹腔注射生理盐水.干预4周后处死裸鼠,切取肿瘤,测量瘤体积,取肿瘤组织送病理学检查;免疫组化法检测肿瘤组织中bax、bcl-2的表达;免疫荧光法检测肿瘤组织中细胞凋亡指数,评价干预效果.结果 5-Fu、健择联合应用MMAb-2对裸鼠胰腺癌细胞增殖的抑制作用明显增强(P＜0.05),且能提高裸鼠胰腺癌细胞凋亡率(P＜0.05).结论 抗EGFR单克隆抗体能有效提高化疗药物5-Fu、健择对裸鼠胰腺癌细胞抑制作用的敏感性,可明显提高胰腺癌的化疗效果.     

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.     

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。     

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).     

Glial cell-derived neurotrophic factor upregulates the expression and activation of matrix metalloproteinase-9 in human pancreatic cancer

BACKGROUND: We have previously reported that the glial cell-derived neurotrophic factor (GDNF) promotes pancreatic cancer cell invasion in vitro. The purpose of this study was to determine whether GDNF regulates the expression and activation of matrix metalloproteinase-9 (MMP-9) in human pancreatic cancer cells. METHODS: We used human pancreatic cancer cell line MIA PaCa-2. The effect of GDNF on mRNA and protein expression was measured by Northern blot, Western blot and enzyme-linked immunosorbent assay. MMP proteolytic activity was detected by gelatin zymography. To determine which intracellular pathways were involved, we used the following inhibitors: tyrosine kinase inhibitor Genistein, MEK-1 inhibitor PD98059 and PI3-K inhibitor Wortmannin. RESULTS: GDNF increased MMP-9 mRNA and protein expression in MIA PaCa-2 cells in a dose-dependent manner. Treatment with GDNF enhanced gelatinolytic activity of the pro and active form of MMP-9. Inhibitor experiments showed that the expression and activity of pro MMP-9 was totally inhibited by Genistein and partially by Wortmannin, whereas PD98059 had no effect. All three compounds inhibited the activity of the active form of MMP-9. CONCLUSIONS: GDNF upregulates the expression and enzymatic activity of MMP-9 through different signaling pathways in MIA PaCa-2. These findings suggest that GDNF modulates MMP-9 expression and activation, and this may promote pancreatic cancer invasion.     

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.     

CaSm/gemcitabine chemo-gene therapy leads to prolonged survival in a murine model of pancreatic cancer

BACKGROUND: CaSm, the cancer-associated Sm-like oncogene, is overexpressed in greater than 80% of pancreatic tumors. We previously reported that an adenovirus expressing antisense RNA to CaSm (Ad-alpha CaSm) can decrease pancreatic tumor growth in vivo but is not curative. In the current study we investigated the mechanism of Ad-alpha CaSm's antitumor effect to rationally approach combinatorial therapy for improved efficacy. METHODS: AsPC-1 and Panc-1 human pancreatic cancer cells were treated with Ad-alpha CaSm and examined by MTT assay for in vitro proliferation changes. Flow cytometry determined the effect of CaSm down-regulation on the cell cycle, and then cells treated with Ad-alpha CaSm in combination with cisplatin, etoposide, or gemcitabine chemotherapies were reexamined by MTT assay. SCID-Bg mice bearing subcutaneous AsPC-1 tumors were treated with Ad-alpha CaSm, gemcitabine, or the combination and monitored for tumor growth and survival. RESULTS: Treatment with Ad-alpha CaSm reduced the proliferation of AsPC-1 and Panc-1 cells (59% and 44%, respectively; P <.05). The cell cycle revealed a cytostatic block with decreased G(1) phase and increased DNA content in treated cells. The combination of Ad-alpha CaSm with gemcitabine significantly reduced in vitro proliferation (66% vs 39% and 48% for controls), decreased in vivo AsPC-1 tumor growth by 71% (n = 10), and extended survival time from 57 to 100 days. CONCLUSIONS: Down-regulation of CaSm reduces the growth of pancreatic cancer cells by altering the cell cycle in a cytostatic manner. The combination of Ad-alpha CaSm with gemcitabine is more effective than either agent used separately.     

Dual-color imaging of nascent blood vessels vascularizing pancreatic cancer in an orthotopic model demonstrates antiangiogenesis efficacy of gemcitabine

BACKGROUND: The stem cell marker nestin recently has been shown to be expressed in nascent blood vessels in nestin-driven green fluorescent protein (ND-GFP) transgenic nude mice. MATERIALS AND METHODS: In the present study, we visualized by dual-color fluorescence imaging tumor angiogenesis in the ND-GFP transgenic nude mice after orthotopic transplantation of the MIA PaCa-2 human pancreatic cancer line expressing red fluorescent protein. Mice were treated with gemcitabine at 150 mg/kg/dose on days 3, 6, 10, and 13 after tumor implantation. At day 14, mice were sacrificed and mean nascent blood vessel density and tumor volume were calculated and compared to control mice. RESULTS: Nestin was highly expressed in proliferating endothelial cells and nascent blood vessels in the growing tumor. Results of immunohistochemical staining showed that CD31 co-localized in ND-GFP-expressing nascent blood vessels. The density of nascent blood vessels in the tumor was readily quantitated. Gemcitabine significantly decreased the mean nascent blood vessel density in the tumor as well as decreased tumor volume. CONCLUSION: The dual-color model of the ND-GFP nude mouse orthotopically implanted with RFP-expressing pancreatic tumor cells enabled the simultaneous visualization and quantitation of tumor angiogenesis and tumor volume. These results demonstrated for the first time that gemcitabine is an inhibitor of angiogenesis as well as tumor growth in pancreatic cancer. The results have important implications for the clinical application of gemcitabine in this disease.     

Efficacy of p120 antisense—Mediated therapy for pancreatic cancer

p120 Antisense oligodeoxynucleotides were used to determine whether they inhibited cell growth of MIA PaCa-2, a highly tumorigenic human pancreatic carcinoma cell line. Growth inhibition assays were determined in vitro by the ability of these oligomers to inhibit DNA synthesis and cell growth. For in vivo studies, nude mice were injected with cells and palpable tumors were found in 16 of 20 animals by day 14. Sixteen animals (8 in each group) were then treated daily (25 mg/kg intraperitoneally) for up to 40 days with nonsense control oligomers or p120 antisense oligomers. p120 Antisense oligomers inhibited the in vitro proliferation of MIA PaCa-2 cells in a dose-dependent manner, and optimal growth inhibition of greater than 90% was achieved at an antisense oligomer concentration of 100 μmol/L. The tumor volume was calculated for antisense- and nonsense-treated animals. Fifteen days after the beginning of treatment, control animals had a significantly greater (P=0.0035) tumor volume (425±244 mm³ above baseline) as compared to p120 antisense-treated animals (166±116 mm³). Seven of the eight control animals formed tumors that had a volume greater than 1200 mm³ 45 days after treatment was begun, whereas only three of eight p120 antisense-treated animals had tumors that were this large. Two of the latter three animals had relatively large, palpable tumors (>150 mm³) prior to treatment. Twenty days after treatment was stopped (day 60), all animals had tumors larger than 1200 mm³. p120 Antisense oligomers were effective for inhibiting in vitro growth of the pancreatic cancer cell line MIA PaCa-2. In preliminary studies, p120 antisense oligomers appeared to inhibit the rate of growth in nude mice; however, no cures were achieved. The most effective response was seen in animals with initial low tumor burden. Supported by grants from the Elsa U. Pardee Foundation and the McDowell Cancer Research Foundation. Presented at the Thirty-Seventh Annual Meeting of The Society for Surgery of the Alimentary Tract, San Francisco, Calif., May 19–22, 1996 (poster presentation).     

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.     

G2/M cell-cycle arrest and apoptosis by n-3 fatty acids in a pancreatic cancer model

BACKGROUND: n-3 fatty acids (n-3FA) have anti-inflammatory and anti-proliferative effects including modulation of pro-inflammatory cascade mediators and cytokine elaboration (i.e., TNF-alpha, IL-10 and PGE(2)) in many cell lines. However, mechanisms of anti-proliferative effects have not been clearly defined. MATERIALS AND METHODS: MIA PaCa-2 pancreatic cancer cells were treated either with n-3FA (treatment), media (control), or n-6FA (control) for all experiments. Cellular proliferation was evaluated with WST-1 reagent. Cells were stained with propidium iodide and analyzed by flow cytometry for cell-cycle arrest, which was further analyzed by cdc2 expression. Membrane and media lipid concentrations were analyzed by high-performance liquid chromatography. Apoptosis was evaluated by AnnexinV-FITC flow cytometry and reconfirmed by poly (ADP-ribose) polymerase (PARP) cleavage and B(cl)-2 expression. RESULTS: Propidium iodide flow cytometry of MIA PaCa-2 dosed with n-3FA showed a decrease in cells in G1 phase (11-17%) and an increase cells in G2 phase (7-13%) from controls. cdc2 expression was also decreased at 24 h compared to controls. Annexin-V staining of n-3FA-treated cells demonstrated time-dependent increased apoptosis and PARP cleavage was present only in the n-3FA treatment group. Phospho-B(cl)-2 was also decreased in the n-3FA-treated cells compared to controls. CONCLUSIONS: Co-incubation of MIA PaCa-2 cells with n-3FA results in both dose- and time-dependent cell-cycle arrest. Cells also progress to cell death via apoptosis. These data support the potential applicability for n-3FA as an antiproliferative and pro-apoptotic strategy.     

白细胞介素-13嵌合假单胞菌外毒素对胰腺癌细胞的靶向毒杀作用

目的：探讨白细胞介素（IL)-13嵌合假单胞菌外毒素（PE）对多种胰腺癌细胞系的靶向治疗作用。方法：用噻唑蓝（MTT）法分析IL-13嵌合假单胞菌外毒素（IL-13-PE38QQR）对胰腺癌细胞ASPC-1、CAPAN-1、COLO-357、MIA PaCa-2、PANC-1、T3M4的靶向毒杀作用,并分析IL-13对胰腺癌细胞系的增殖作用和与IL-13-PE38QQR的竞争结合作用。结果：IL-13对ASPC-1、CA-PAN-1、COLO-357有明显促增殖作用;对PANC-1作用次之;对MIA PaCa-2、T3M4不明显;IL-13-PE38QQR对6个胰腺癌细胞都有明显抑制作用;IL-13与IL-13-PE38QQR有竞争抑制作用。结论：IL-13-PE38QQR嵌合蛋白对胰腺癌有靶向治疗作用。