AsPC-1 Pancreatic Cancer Cells Research Articles

A novel activity from an old compound: Manzamine A reduces the metastatic potential of AsPC-1 pancreatic cancer cells and sensitizes them to TRAIL-induced apoptosis

Pancreatic cancer is the fourth leading cause of cancer death in the United States, and new drugs to treat the disease are needed. Pancreatic cancer cells are highly metastatic and exhibit resistance to apoptosis. Small molecules that can restore sensitivity to apoptosis or reduce metastasis would have therapeutic potential against this disease. Manzamine A is an alkaloid isolated from marine sponges that was suspected to have inhibitory activity against the mitogen activated kinase kinase (MEK). Because of this, the effects of Manzamine A were studied in pancreatic cancer cells. AsPC-1 cells were treated for 48 h in the presence of various concentrations of Manzamine A and their phenotype, cytotoxicity, cell invasion and susceptibility to apoptosis were observed. Manzamine A decreased single cell formation, abrogated cell migration and restored the susceptibility of the cells to TRAIL-induced apoptosis in AsPC-1 cells. Its mechanism of action remains unknown, as manzamine A does not inhibit MEK. Manzamine A appears to have a formerly unrecognized activity in blocking tumor cell invasion as well as in restoring cancer cell susceptibility to apoptosis in vitro and therefore has the potential to be used as an adjuvant to existing cancer therapies.

Inhibition of oligopeptide transporter suppress growth of human pancreatic cancer cells

Oligopeptide transporters are abundantly expressed in various types of cancer cells. We here synthesized two novel dipeptides, l-phenylalanyl sarcosine (Phe-Sar) and 4-(4-methoxyphenyl)-l-phenylalanyl sarcosine (Bip(OMe)-Sar), and examined their effect on the growth of human pancreatic cancer AsPC-1 cells, which are known to highly express oligopeptide transporter PEPT1/SLC15A1. Growth of AsPC-1 cells was inhibited by these two peptides and a typical PEPT1/SLC15A1 substrate Gly-Sar. Growth inhibition by Gly-Sar, Phe-Sar and Bip(OMe)-Sar was concentration-dependent with half-maximal inhibitory concentration of 50, 0.91 and 0.55mM, respectively. These peptides also inhibited PEPT1-mediated [(3)H]Gly-Sar uptake with half-maximal inhibitory concentration of 2.6, 0.81 and 0.27mM, respectively. Thus, the rank order of the tumor cell growth inhibition by these three peptides was the same as that of PEPT1-inhibitory activity. Growth of AsPC-1 cells was also inhibited by 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH), which is a typical inhibitor of amino acid transporter system L. The growth inhibition by BCH and Gly-Sar was additive, suggesting that these compounds act at distinct loci. Oligopeptide transporters thus appear to be a promising target for inhibition of pancreatic cancer progression. These results also proposed the idea that oligopeptide transporter is required for growth of AsPC-1 cells.

Lin28- let7 Modulates Radiosensitivity of Human Cancer Cells With Activation of K-Ras

To evaluate the potential of targeting Lin28-let7 microRNA regulatory network for overcoming the radioresistance of cancer cells having activated K-Ras signaling. A549 lung carcinoma cells and ASPC1 pancreatic cancer cells possessing K-RAS mutation were transfected with pre-let7a microRNA or Lin28 siRNA, respectively. Clonogenic assay, quantitative reverse transcription polymerase chain reaction, and Western analysis were performed. The effects of Lin28 on SQ20B cells having wild-type K-RAS, and a normal fibroblast were also assessed. The overexpression of let-7a decreased expression of K-Ras and radiosensitized A549 cells. Inhibition of Lin28, a repressor of let-7, attenuated K-Ras expression and radiosensitized A549 and ASPC1 cells. Neither SQ20B cells expressing wild-type K-RAS nor HDF, the normal human fibroblasts, were radiosensitized by this approach. The Lin28-let7 regulatory network may be a potentially useful therapeutic target for overcoming the radioresistance of human cancers having activated K-Ras signaling.

Growth inhibitory effects of dihydroartemisinin on pancreatic cancer cells: involvement of cell cycle arrest and inactivation of nuclear factor-κB

In a recent publication, we have shown that dihydroartemisinin (DHA), a derivative of antimalaria drug artemisinin, inhibits growth of pancreatic cancer cells in vitro and in vivo mediated by its anti-proliferative and pro-apoptotic effects. As it has been shown that the apoptosis might be induced due to cell cycle arrest, and that transcriptional factor nuclear factor-kappa B (NF-kappaB) plays vital roles in the apoptosis of pancreatic cancer cells, we extend our study to investigate the effects of DHA on cell cycle progression and NF-kappaB activity in pancreatic cancer cells to further reveal the anticancer effects of DHA on pancreatic cancer. Cell cycle progression was determined by propidium iodide staining and flow cytometry. Changes in the expression of cell cycle-associated proteins were detected using Western blot analysis. Measurement of NF-kappaB activity was performed with immunoblot analyzing the nuclear protein expression of NF-kappaB/p65 and ELISA detecting the NF-kappaB DNA-binding activity. The treatment with DHA resulted in a dose-dependent G(0)/G(1) cell cycle arrest and regulated the expression of some cyclins, cdks and cdk inhibitors that involved in the G(0)/G(1) cell cycle progression such as cyclin E, cdk2, cdk4 and p27(Kip1) in pancreatic cancer BxPC-3 and AsPC-1 cells. The translocation and DNA-binding activity of NF-kappaB were inhibited in DHA-treated cells in a dose-dependent manner, indicated the inactivation effects of DHA in pancreatic cancer cells. Together with our previous observations, our data show that DHA induces cell cycle arrest and apoptosis in pancreatic cancer cells, and this effect might be due to inhibition of NF-kappaB signaling. We suggest that DHA could be developed as a novel agent against pancreatic cancer.

Intratumoral delivery of shRNA targeting cyclin D1 attenuates pancreatic cancer growth

The aim of this to study was to assess the biological consequences of cyclin D1 silencing in pancreatic cancer cells. A replication-defective lentivirus based small hairpin RNA (shRNA) system targeting cyclin D1 caused a marked reduction in cyclin D1 protein levels in ASPC-1 and BxPC3 pancreatic cancer cell lines in conjunction with decreased cell growth and invasiveness in vitro. Moreover, a single intratumoral injection of the recombinant lentivirus targeting cyclin D1 attenuated the growth of pre-existing tumors arising from two distinct cell lines. This attenuated growth correlated with decreased proliferation and angiogenesis, and attenuated VEGF expression. It is concluded that lentivirus-delivered shRNA targeting cyclin D1 suppresses the growth, invasiveness, tumorigenicity and pro-angiogenic potential of human pancreatic cancer cells, raising the possibility that intra-tumoral injections of viruses targeting cyclin D1 could provide a novel therapeutic approach in pancreatic ductal adenocarcinoma.

Increased CXCR4 expression in AsPC1 pancreatic carcinoma cells with RNA interference-mediated knockdown of DNMT1 and DNMT3B

The effect of DNA methylation on CXCR4 expression has been demonstrated in pancreatic cancer and melanoma cells, but little is known about the effect of DNA methyltransferases 1 and 3 (DNMT1 and DNMT3B) on CXCR4 expression. Employing lentiviral vectors, we created stable RNA interference-mediated knockdown of DNMT1 and DNMT3B in AsPC1 pancreatic cancer cells. Using reverse transcription real-time quantitative PCR and flow cytometric analysis, we evaluated the increase in the expression of CXCR4 transcript and protein levels in these cells. Bisulfite sequencing analysis showed that the level of promoter demethylation appeared more effective in cells with knockdown of DNMT1 than in those with DNMT3B knockdown. Furthermore, the combined RNA interference knockdown of both DNMT1 and DNMT3B increased promoter demethylation, leading to a slight increase in CXCR4 expression. However, the demethylating agent 5-Aza-2’-deoxycytidine exhibited the strongest effect on promoter demethylation, which correlated with the highest production of CXCR4 transcript and protein in AsPC1 cells. Our results indicate that DNMT1 plays the main role in maintenance of methylation of CXCR4 promoter, while DNMT3B may function as an accessory DNA methyltransferase to modulate CXCR4 expression in AsPC1 cells.

Combined treatment with Denbinobin and Fas ligand has a synergistic cytotoxic effect in human pancreatic adenocarcinoma BxPC‐3 cells

Human pancreatic carcinoma is a highly malignant cancer. Previous studies have shown that the decoy receptor 3 (DcR3) for Fas ligand (FasL) plays significant roles in tumour progression and immune suppression. In the present study, we evaluated the anti-cancer activity of a natural compound, denbinobin (5-hydroxy-3,7-dimethoxy-1,4-phenanthraquinone), through decreasing DcR3 levels in human pancreatic adenocarcinoma cell lines. We used immunoprecipitation and ELISA assays to examine DcR3 levels, and used FACS to determine the percentage of cells with a sub-G1 DNA content. AsPC-1 and BxPC-3 human pancreatic cancer cells express high levels of DcR3. Denbinobin concentration-dependently decreased DcR3 levels in BxPC-3 cells. MTT and flow cytometry assays indicated that BxPC-3 was FasL-resistant because high concentrations (100 ng.mL(-1)) of soluble FasL did not inhibit cell growth. However, combinations of denbinobin (3 micromol.L(-1)) with lower concentrations of soluble FasL (10, 30 and 50 ng.mL(-1)) or membrane-bound FasL, were synergistic on cell growth inhibition and apoptosis. Exogenous excess DcR3 reversed this synergistic effect. We observed no significant increase in the levels of surface Fas, cleaved forms of caspase-8, -3, -9, Bax, Bid, Bcl-xL, cytochrome c or mitochondrial membrane potentials following denbinobin treatment. However, denbinobin treatment increased the levels of apoptosis-inducing factor. Denbinobin and FasL trigger a synergistic cytotoxic effect in human pancreatic adenocarcinoma cells. Denbinobin mediated a decrease in levels of DcR3, which played a major role in this synergistic effect, and also increased caspase-independent apoptosis, via apoptosis-inducing factor.

Study on anticancer effect of dihydroartemisinin on pancreatic cancer

To investigate the anti-tumor activity of dihydroartemisinin in pancreatic cancer in vitro and in vivo. For cultured cells, cell growth was determined by the MTT assay and apoptosis was evaluated by flow cytometry analysis stained with Annexin V-FITC/PI. The protein expression in BxPC-3 cells was analyzed by Western blot assay. BxPC-3 cells were injected subcutaneously into nude mice to establish pancreatic xenograft tumors and the tumor volume was monitored after exposure to dihydroartemisinin. Ki-67 staining and TUNEL assay were used to assess tumor cell proliferation and apoptosis in tumor tissue. After treatment by dihydroartemisinin in vitro, the proliferative inhibition rates of pancreatic cancer cells BxPC-3 and AsPC-1 reached up to (76.2 +/- 3.5)% and (79.5 +/- 2.9)%, and the apoptosis rates were up to (55.5 +/- 3.2)% and (40.0 +/- 3.5)%, the differences were significantly (P < 0.01) compared with control [(2.0 +/- 1.3)% and (0.9 +/- 0.4)%]. Dihydroartemisinin inhibited the growth of pancreatic xenograft tumors in nude mice. The proliferation index and apoptosis index were (49.1 +/- 3.9)% and (50.2 +/- 4.4)% respectively in dihydroartemisinin 50 mg/kg treatment group, compared to those of (72.1 +/- 3.3)% and (9.4 +/- 2.9)% in control, the differences were significantly (P < 0.01). Western blot assay indicated that dihydroartemisinin up-regulates expression of proliferation-associated protein p21(WAF1) and down-regulates expression of PCNA, increases expression of apoptosis-associated protein Bax and decreases expression of Bcl-2 and activates caspase-9 in BxPC-3 cells. Dihydroartemisinin exerts anti-tumor activity in pancreatic cancer both in vitro and in vivo by proliferation inhibition and apoptosis induction. Dihydroartemisinin can be used as a potential anti-tumor drug in pancreatic cancer.

Ceramide Regulates Gemcitabine-Induced Senescence and Apoptosis in Human Pancreatic Cancer Cell Lines

Bioactive sphingolipids are potent intracellular signaling molecules having profound effects on cell death, growth, and differentiation. Pharmacologic manipulation of sphingolipid levels could have a significant effect on the induction of apoptosis by anticancer agents, and thus, improve treatment efficacy. We observed that gemcitabine cannot completely kill AsPc1 and Panc1 human pancreatic cancer cells in culture; even at high concentrations of gemcitabine, 30% to 40% of the cells remain viable. By adding sphingomyelin to the culture medium, gemcitabine-induced cell death increased synergistically to >90%. Panc1 cells that survived high concentrations of gemcitabine had an increase in beta-galactosidase activity, a marker of senescence. The inclusion of sphingomyelin with gemcitabine reduced beta-galactosidase activity, as compared with cells treated with gemcitabine alone. Expression of p21(waf1/cip1) in both cell lines exposed to sphingomyelin, gemcitabine, and gemcitabine + sphingomyelin varied relative to the untreated group. C(8)-ceramide induced both cell death and senescence in a dose-dependent manner. These results indicate that gemcitabine induces senescence in pancreatic cancer cells and that sphingomyelin-enhanced chemosensitivity is achieved through reducing the induction of senescence by redirecting the cell to enter the apoptotic pathway. Ceramide levels seem to be critical to this decision, with cell cycle progression being uninhibited at low ceramide levels, senescence induced at moderate levels, and apoptosis initiated at high levels. Our results provide further evidence that targeting the sphingolipid metabolism is a means of enhancing the efficacy of chemotherapeutic agents.

The Flavonoid Apigenin Potentiates the Growth Inhibitory Effects of Gemcitabine and Abrogates Gemcitabine Resistance in Human Pancreatic Cancer Cells

The aim of the study was to evaluate the effect of combination therapy of apigenin and gemcitabine on cell proliferation, the cell cycle, and gemcitabine resistance in human pancreatic cancer cells. Cell counting was used to assess the effect of single-agent and combination treatment on the proliferation of CD18 and AsPC-1 pancreatic cancer cells. Flow cytometry was performed to assess the effect of combination treatment on cell cycle progression and induction of apoptosis. Western blot analysis was used to evaluate phosporylated AKT (pAkt) and cell cycle proteins. The effect of apigenin on gemcitabine-resistant AsPC-1 cells was assessed via thymidine incorporation. Apigenin in combination with gemcitabine inhibited pancreatic cancer cell proliferation more than either agent alone. Combination treatment induced both S and G2/M phase arrest and increased apoptosis. Apigenin down-regulated pAkt expression and abrogated gemcitabine-mediated pAkt induction. In gemcitabine-resistant AsPC-1 cells, apigenin significantly inhibited cell proliferation in a dose-dependent manner. Combination treatment with apigenin and gemcitabine inhibited pancreatic cancer cell growth via cell cycle arrest, down-regulation of the prosurvival factor pAkt, and induction of apoptosis. Combination therapy may prove useful for the treatment of pancreatic cancer.

Crocetin inhibits pancreatic cancer cell proliferation and tumor progression in a xenograft mouse model

Crocetin, a carotenoid compound derived from saffron, has long been used as a traditional ancient medicine against different human diseases including cancer. The aim of the series of experiments was to systematically determine whether crocetin significantly affects pancreatic cancer growth both in vitro and/or in vivo. For the in vitro studies, first, MIA-PaCa-2 cells were treated with crocetin and in these sets of experiments, a proliferation assay using H(3)-thymidine incorporation and flow cytometric analysis suggested that crocetin inhibited proliferation. Next, cell cycle proteins were investigated. Cdc-2, Cdc-25C, Cyclin-B1, and epidermal growth factor receptor were altered significantly by crocetin. To further confirm the findings of inhibition of proliferation, H(3)-thymidine incorporation in BxPC-3, Capan-1, and ASPC-1 pancreatic cancer cells was also significantly inhibited by crocetin treatment. For the in vivo studies, MIA-PaCa-2 as highly aggressive cells than other pancreatic cancer cells used in this study were injected into the right hind leg of the athymic nude mice and crocetin was given orally after the development of a palpable tumor. The in vivo results showed significant regression in tumor growth with inhibition of proliferation as determined by proliferating cell nuclear antigen and epidermal growth factor receptor expression in the crocetin-treated animals compared with the controls. Both the in vitro pancreatic cancer cells and in vivo athymic nude mice tumor, apoptosis was significantly stimulated as indicated by Bax/Bcl-2 ratio. This study indicates that crocetin has a significant antitumorigenic effect in both in vitro and in vivo on pancreatic cancer.

Dihydroartemisinin inhibits growth of pancreatic cancer cells in vitro and in vivo

Dihydroartemisinin (DHA), a semisynthetic derivative of artemisinin, has recently shown antitumor activity in various cancer cells. Its effect on pancreatic cancer is, however, unknown and the mechanism is unclear. The study aims to investigate its antitumor activity and underlying mechanisms in human pancreatic cancer BxPC-3 and AsPC-1 cells in vitro and subcutaneous BxPC-3 xenograft tumors in mice. The MTT assay was used to evaluate cell viability, and flow cytometry and laser scanning confocal microscopy were used to detect apoptosis, for cultured cells. Pancreatic tumors were established by subcutaneous injection of BxPC-3 cells in nude BALB/c mice, and DHA was administered intraperitoneally to the mice. The size of tumors was monitored and they were harvested after the mice had been killed. Tumor sections were immunostained with an anti-Ki-67 Ab to assess the proliferation index, or stained with TUNEL to evaluate in-situ cell apoptosis. The gene expression in cells and tumors was evaluated by western blot analysis. In the cultured cells, DHA inhibited cell viability, downregulated the expression of proliferating cell nuclear antigen and cyclin D1, and upregulated p21(WAF1/CIP1); and induced apoptosis by reducing the ratio of Bcl-2/Bax and increasing the activation of caspase-9, in a dose-dependent manner. Similarly, in mice bearing BxPC-3 xenograft tumors, administration of DHA inhibited tumor growth in a dose-dependent manner, and modulated tumoral gene expression consistent with the in-vitro observations. This study indicates that DHA may be a potent and promising agent to combat pancreatic cancer.

Nicotine induces cell proliferation, invasion and epithelial‐mesenchymal transition in a variety of human cancer cell lines

Cigarette smoking is strongly correlated with the onset of nonsmall cell lung cancer (NSCLC). Nicotine, an active component of cigarettes, has been found to induce proliferation of lung cancer cell lines. In addition, nicotine can induce angiogenesis and confer resistance to apoptosis. All these events are mediated through the nicotinic acetylcholine receptors (nAChRs) on lung cancer cells. In this study, we demonstrate that nicotine can promote anchorage-independent growth in NSCLCs. In addition, nicotine also induces morphological changes characteristic of a migratory, invasive phenotype in NSCLCs on collagen gel. These morphological changes were similar to those induced by the promigratory growth factor VEGF. The proinvasive effects of nicotine were mediated by alpha7-nAChRs on NSCLCs. RT-PCR analysis showed that the alpha7-nAChRs were also expressed on human breast cancer and pancreatic cancer cell lines. Nicotine was found to promote proliferation and invasion in human breast cancer. The proinvasive effects of nicotine were mediated via a nAChR, Src and calcium-dependent signaling pathway in breast cancer cells. In a similar fashion, nicotine could also induce proliferation and invasion of Aspc1 pancreatic cancer cells. Most importantly, nicotine could induce changes in gene expression consistent with epithelial to mesenchymal transition (EMT), characterized by reduction of epithelial markers like E-cadherin expression, ZO-1 staining and concomitant increase in levels of mesenchymal proteins like vimentin and fibronectin in human breast and lung cancer cells. Therefore, it is probable that the ability of nicotine to induce invasion and EMT may contribute to the progression of breast and lung cancers.

Antitumor effects of KITC, a new resveratrol derivative, in AsPC-1 and BxPC-3 human pancreatic carcinoma cells

Pancreatic cancer is a very aggressive malignant disease due to lack of early diagnosis and chemotherapeutic resistance of the tumor cells. There is distinct evidence that food derived polyphenols possess chemopreventive effects in the development of several cancers including pancreatic carcinoma. Resveratrol is one of those phenolic compounds found in grape skins and other fruits with known anticancer activity. Various polymethoxylated resveratrol derivatives showed stronger antiproliferative effects than resveratrol in tumor cell lines. The aim of our study was to evaluate the cytotoxic and biochemical effects of a newly synthesized polymethoxylated resveratrol analogue, N-hydroxy-N'-(3,4,5-trimethoxphenyl)-3,4,5-trimethoxy-benzamidine (KITC) in two human pancreatic cancer cell lines. The human pancreatic cancer cell lines, AsPC-1 and BxPC-3 were used to test the potential inhibitory effect of the resveratrol derivative on cell proliferation and the underlying mechanisms of this effect. After 7 days of incubation, KITC inhibited the growth of AsPC-1 and BxPC-3 cells with IC(50) values of 9.6 and 8.7 microM, respectively. KITC (40 microM) arrested cells in the G0/G1 phase and depleted cells in the S phase of the cell cycle (-105% and -35% of control, respectively). KITC induced dose-dependent apoptosis in both pancreatic cancer cell lines and was found to significantly reduce the in situ activity of ribonucleotide reductase, the key enzyme of DNA synthesis. Employing growth inhibition assays, KITC acted synergistically with gemcitabine in both cell lines. In summary, we found that KITC exerted considerable antitumor activity against human pancreatic cancer cells and could be a promising candidate for further investigations to establish a new chemotherapeutic regimen.

FGF10/FGFR2 signal induces cell migration and invasion in pancreatic cancer

Pancreatic cancer has one of the highest mortalities among all malignancies and there is an urgent need for new therapy. This might be achieved by resolving the detailed biological mechanism, and in this study we examined how pancreatic cancer cells develop aggressive properties by focusing on signalling through the fibroblast growth factor (FGF)10 and FGF receptor (FGFR)2, which play important roles in pancreatic organogenesis. Immunostaining of pancreatic cancer tissues showed that FGFR2 was expressed in cancer cells, whereas FGF10 was expressed in stromal cells surrounding the cancer cells. Patients with high FGFR2 expression in cancer cells had a shorter survival time compared to those with low FGFR2 expression. Fibroblast growth factor 10 induced cell migration and invasion of CFPAC-1 and AsPC-1 pancreatic cancer cells through interaction with FGFR2-IIIb, a specific isoform of FGFR2. Fibroblast growth factor 10 also induced expression of mRNA for membrane type 1-matrix metalloproteinase (MT1-MMP) and transforming growth factor (TGF)-β1, and increased secretion of TGF-β1 protein from these cell lines. These data indicate that stromal FGF10 induces migration and invasion in pancreatic cancer cells through interaction with FGFR2, resulting in a poor prognosis. This suggests that FGF10/FGFR2 signalling is a promising target for new molecular therapy against pancreatic cancer.

IGFBPs contribute to survival of pancreatic cancer cells under severely hypoxic conditions

Protein synthesis in general is suppressed under hypoxic conditions in both cancer cells and normal cells. In human pancreatic cancer AsPC-1 cells, which are resistant to low oxygen tension, protein synthesis at day 4 under hypoxia (1% O 2) was about 20% of that under normoxia. Secretion of some proteins actually increased in spite of a general reduction in protein synthesis; two of these proteins were insulin-like growth factor binding protein-1 (IGFBP-1) and IGFBP-3. Hypoxia also induced transcription of multiple IGFBPs. The IGFBPs contributed to reduced IGF signaling and to the survival of cancer cells under conditions of low oxygen tension.

INTERLEUKIN-13 EXERTS AUTOCRINE GROWTH PROMOTING EFFECTS IN HUMAN PANCREATIC CANCER AND ITS EXPRESSION CORRELATES WITH A PROPENSITY FOR LYMPH NODE METASTASES

Purpose: Pancreatic cancer cells express receptors for interleukin-13 (IL-13). It is not known, however, whether pancreatic cancer cell produce endogenous IL-13 and whether IL-13 can modulate pancreatic cancer cell growth and lymph node metastases. Experimental Design: Cell growth and signaling were analyzed by cell counting, colorimetric proliferation assays, FACS, and kinase activity assays. IL-13 expression and secretion were determined by Northern blot analysis and ELISA, respectively. Localization of IL-13 and IL-13 receptors (IL-4R and IL-13R) in primary pancreatic ductal adenocarcinoma (PDAC) was characterized by immunohistochemistry. Results: IL-13 significantly enhanced the growth of ASPC-1, CAPAN-1, and COLO-357 pancreatic cancer cells. This was associated with enhanced p44/42 MAPK phoshorylation. In contrast to p44/42 MAPK, PI-3 kinase activity was also induced in IL-13 unresponsive MIA PaCa-2, PANC-1, and T3M4 cells. All tested cell lines expressed and secreted IL-13 at various levels. Neutralizing IL-13 antibodies inhibited the growth of ASPC-1 and CAPAN-1 cells. Immunohistochemical analysis of resected primary PDAC specimen revealed high IL-13 and IL-4R immunoreactivity was present in 30 of 70 and 28 of 70 cases, respectively. Fifteen of 16 specimen (94%) exhibiting high IL-13 and IL-4R levels displayed lymph node metastases, while only 30 of the remaining 54 samples (56%) had positive lymph nodes (p = 0.0134). Conclusion: IL-13 can act as autocrine growth factor in PDAC. Endogenous expression of IL-13 in conjunction with IL-4R in the cancer cells may facilitate lymph node metastasis by modulating the interactions between tumor cells and immune cells.

The Survival Kinase Mirk/Dyrk1B Is a Downstream Effector of Oncogenic K-ras in Pancreatic Cancer

The kinase Mirk is overexpressed in many resected pancreatic adenocarcinomas and is amplified in a subset of pancreatic cancer cell lines. Depletion of Mirk has been shown to lead to apoptosis in pancreatic cancer cell lines, and thus to inhibit their clonogenic growth. Mirk is activated by signaling from activated Rac1 to MKK3 in MDCK cells, but the mechanism of activation of Mirk in pancreatic cancers is unknown. In this report, Mirk is shown to be a novel effector of K-ras, a gene mutated in approximately 90% of pancreatic cancers. Activation of Mirk signaling from oncogenic K-ras through Rac1 was shown in transient expression systems and reporter assays. Mirk activation in pancreatic cancer cells was blocked by RNA interference using three different synthetic duplex RNAis to K-ras, or two RNAis to Rac1, by pharmacologic inhibition of Rac1, or by expression of dominant negative K-rasS17N. Rac1 was activated in four out of five pancreatic cancer cell lines, and was activated by signaling from oncogenic K-ras. Mirk knockout does not induce embryonic lethality, and depletion of Mirk had no effect on the survival of normal diploid fibroblasts. In contrast, the clonogenic ability of Panc1 and AsPc1 pancreatic cancer cell lines was reduced 8- to 12-fold by the depletion of Mirk, with a greater reduction seen following the depletion of K-ras or both genes. Mirk is a novel downstream effector of oncogenic K-ras and mediates some of the survival signals activated by ras signaling.

Molecular targeting of IGF-I receptor for human pancreatic cancer

14051 Background: Pancreatic cancer is one of the worst prognostic diseases. Continuous tumor growth depends on the altered regulation of the cell cycle that is modulated by signals from growth factors and their receptors, including insulin-like growth factors (IGF) and IGF-I receptor (IGF-Ir). It have been reported that blockade of IGF-Ir can suppress tumorigenicity and induce regression of established tumors. Recently several approaches of targeting IGF-Ir have been reported, including monoclonal antibodies and tyrosine kinase inhibitors. Methods: To study the mechanism of this effect and develop potential targeted therapeutics, we have constructed truncated IGF-Irs that function as dominant negative inhibitor (IGF-Ir/dn) and short hairpin RNA for IGF-Ir (shIGF-Ir). Those were cloned into recombinant adenoviruses (ad-IGF-Ir/dn and ad-shIGF-Ir). The effects of these adenoviruses were studied in human pancreatic cancer cell lines, BxPC3, Panc1, and AsPC1. We assessed efficiency of IGF-Ir signaling blockade, the effect of the adenoviruses on cell growth, steady-state, radiation, and chemotherapy induced apoptosis, and in vivo therapeutic efficacy in xenografts. Results: The blockade for IGF-Ir suppressed tumorigenicity of cancer cells both in vitro and in vivo and increased the amount of steady-state apoptosis in cancer cells. IGF-Ir/dn effectively blocked both IGF-I and IGF-II signaling through Akt-1 and shIGF-Ir reduced both IGF-Ir expression and ligands inducing phosphorylaion. The blockade for IGF-Ir increased chemotherapy and radiation induced apoptosis in vitro and the combination therapy with chemotherapy was very effective against established tumors in mice. Conclusions: These data suggest that the blocking of IGF-Ir signals, by ad-IGF-Ir/dn or ad-shIGF-Ir, may be a useful therapeutic strategy for pancreatic cancers. No significant financial relationships to disclose.

Treatment with rAD001 improves the efficacy of siRNA mediated down-regulation of survivin/BIRC5 in pancreatic carcinoma cell lines

15146 The rapamycin derived mTOR inhibitor RAD001 (everolimus) is cytotoxic to a number of cell lines. Because survivin, an important mediator of apoptosis and cell survival, is one of the targets down-regulated by RAD001, we tested the ability of a combination of RAD001 and siRNA mediated inhibition of survivin to enhance the cytotoxic effects. We have used four pancreatic cancer cell lines, MiaPaCa-2, BxPC-3, AsPC-1 and Panc-1. We found that the cytotoxic effect was enhanced in all cell lines after treatment with RAD001 and survivin siRNA. An efficient way of treatment was the initial down-regulation of survivin with RAD001 and a subsequent incubation with survivin siRNA. We conclude, that the transformation efficiency of siRNA, still a limiting factor in using this method, is more sufficiently combining both ways to down-regulate survivin. Our data indicate that a combination of mTOR inhibitors like RAD001 and survivin-targeted down-regulation with siRNA may improve the efficacy of therapy in pancreatic cancer. No significant financial relationships to disclose.