Cancer Cell Lines AsPC-1 Research Articles

Cytotoxicity of gemcitabine enhanced by polyphenolics from Aronia melanocarpa in pancreatic cancer cell line AsPC-1

AimsExtending work with brain tumours, the hypothesis that micronutrients may usefully augment anticancer regimens, chokeberry (Aronia melanocarpa) extract was tested to establish whether it has pro-apoptotic effects in AsPC-1, an...

Effect of gemcitabine on proliferation, apoptosis and polo-like kinase-1 expression of pancreatic cancer cell line AsPC-1

Objective To investigate the effect of gemcitabine on proliferation,apoptosis and polo-like kinase-1 (PLK-1) expression of human pancreatic cancer cell line AsPC-1 cells.Methods AsPC-1 cells were treated with various concentrations of gemcitabine for different time.The effect of gemcitabine on survival of AsPC-1 cells was determined by methyl thiazol tetrazolium (MTT) assay.The effect of gemcitabine on apoptosis of AsPC-1 cells was mearsured by flow cytometry with various concentrations of 2 and 5 μmol/L,and at different time points (12,24,48,and 72 h).The expression levels of PLK-1 mRNA and protein were detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting,respectively.Results Gemcitabine decreased the proliferation of pancreatic cancer cells (P ＜ 0.05),and the apoptosis rate was significantly increased (P ＜ 0.05).After treatment with gemcitabine,the expression levels of PLK-1 mRNA (control:0.70 ±0.15,0.65 ±0.27,0.72 ±0.13,0.68 ±0.30;2 μmol/L:0.88 ±0.58,0.95 ±0.53,2.36 ±0.57,3.53 ±0.89;5 μmol/L:0.89 ± 0.60,1.02 ± 0.32,3.95 ± 1.84,4.52 ± 2.25) and protein (control:0.82 ± 0.41,0.78 ± 0.46,1.01 ±0.05,0.88 ±0.25 ;2 μmol/L:0.89 ±0.28,1.61 ±0.88,1.66 ±0.18,3.28 ± 1.33 ;5 μmol/L:0.85 ± 0.36,1.89 ± 0.18,2.73 ± 0.78,4.32 ± 2.13) were also increased significantly in pancreatic cancer cells (P ＜ 0.05).Conclusion Gemcitabine can influence the proliferation and apoptosis of AsPC-1 cells probably by the expression changes of PLK-1. Key words: Pancreatic carcinoma; Gemcitabine ; Apoptosis ; Polo-like kinase-1

Down-regulation of Notch1 by small interfering RNA enhances chemosensitivity to gemcitabine in pancreatic cancer cells through activating apoptosis activity

To investigate the effect of down-regulation of Notch1 by Notch1 small interfering RNA (siRNA) on chemosensitivity to gemcitabine in pancreatic cancer cells and its mechanism. Notch1 siRNA was transfected to pancreatic cancer cell lines AsPC-1, BxPC-3, MIAPaCa-2 and Panc-1. The transfected pancreatic cancer cells were treated with 10 μmol/L gemcitabine in vitro. The relative quantity of Notch1 mRNA of pancreatic cancer cells was detected by real-time PCR. The inhibition rates of gemcitabine-treated cells were evaluated by CCK-8 method. The expression of Bax protein was examined by Western blot, and the caspase 3 activity was detected by CaspACETM assay system kit. The relative quantity of Notch1 mRNA was the highest in BxPC-3 cell line and the lowest in Panc-1 cells. The inhibition rates of gemcitabine treated-cells were significantly higher in Notch1 siRNA transfection groups than in corresponding siRNA control groups (AsPC-1: 67.5±6.7 vs 47.5±6.8; BxPC-3: 90.5±4.4 vs 70.2±4.2; MIAPaCa-2: 80.9±5.7 vs 58.1±6.0; Ps<0.05), with the overexpression of protein Bax. The activity of caspase 3 was also significantly increased in Notch1 siRNA transfection groups compared with corresponding siRNA control groups (AsPC-1: 28.90±2.70 vs 12.82±3.44; BxPC-3: 59.87±6.77 vs 27.27±11.88; MIAPaCa-2: 29.34±4.06 vs 14.59±4.25; P<0.05). Inhibition of Notch signaling pathway by Notch1 siRNA can enhance chemosensitivity to gemcitabine in pancreatic cancer cells through activating apoptosis activity．

Effect of transfection of microRNA-10b and antisense microRNA-10b on the expression of RhoC in human pancreatic cancer cell line AsPC-1

Objective To observe the effect of microRNA (miR)-10b on the expression of RhoC in human pancreatic cancer cell line AsPC-1.Methods The recombinant eukaryotic expression plasmid vector with miR-10b sequence [pPG-CMV-enhanced green fluorescent protein (EGFP)-miR-10b] or the antisense sequence was transfected into human pancreatic cancer cell line (AsPC-1) with lipofectamine,and the efficiency of the instant expression was confirmed by real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and fluorescence microscope.The difference of the migration and invasion of the cells was evaluated by matrigel assay,and the expression of RhoC was evaluated by Western blotting.Results The expression of miR-10b in the sense group was increased by 10 folds,and in antisense group,the expression was decreased to 14％ compare to the control (P ＜ 0.05).The expression of RhoC protein in the sense group was (95.7 ± 8.5) unit,and that in the antisense group (28.5 ± 5.7) unit (P ＜0.05).As compared to the antisense group,the number of migrating and penerating cells was decreased from (85 ±7) and (35 ±9) to (32±5) and (8 ±3) in the sense group (P＜0.05) Conclusion miR10b can up-regulate the expression of RhoC,and promote the migration and invasion of the AsPC-1 cells. Key words: MicroRNA-10b; Pancreatic cancer ; Migration ; Invasion

TGF-β-Activated Kinase 1 (TAK1) Is an In Vivo Druggable Target for Reverting Pancreatic Cancer Chemoresistance

Context TAK1, a mitogen-activated protein kinase kinase kinase, functions in the activation of nuclear factor kappaB (NF-kappaB) and activator protein-1, which can suppress proapoptotic signaling pathways and thus promote resistance to chemotherapeutic drugs. However, it is not known if inhibition of TAK1 is effective in reducing chemoresistance to therapeutic drugs against pancreatic cancer. Methods : NF-kappaB activity was measured by luciferase reporter assay in human pancreatic cancer cell lines AsPc-1, PANC-1, and MDAPanc-28, in which TAK1 expression was silenced by small hairpin RNA. TAK1 kinase activity was targeted in AsPc-1, PANC-1, MDAPanc-28, and Colo357FG cells with exposure to increasing doses of a selective small-molecule inhibitor, LYTAK1, for 24 hours. To test the effect of LYTAK1 in combination with chemotherapeutic agents, AsPc-1, PANC-1, MDAPanc-28 cells, and control cells were treated with increasing doses of oxaliplatin, SN-38, or gemcitabine in combination with LYTAK1. In vivo activity of oral LYTAK1 was evaluated in an orthotopic nude mouse model (n=40, 5 per group) with luciferase-expressing AsPc-1 pancreatic cancer cells. Results AsPc-1 and MDAPanc-28 TAK1 knockdown cells had a statistically significantly lower NF-kappaB activity than did their respective control cell lines. TAK1 inhibitor LYTAK1 had potent in vitro cytotoxic activity in AsPc-1, PANC-1, MDAPanc-28, and Colo357FG cells, with IC 50 between 5 and 40 nM. LYTAK1 also potentiated the cytotoxicity of chemotherapeutic agents oxaliplatin, SN-38, and gemcitabine in AsPc-1, PANC-1, and MDAPanc-28 cells compared with control cells. In nude mice, oral administration of LYTAK1 plus gemcitabine statistically significantly reduced tumor burden and prolonged survival duration. Conclusions The results of this study suggest that genetic silencing or inhibition of TAK1 kinase activity in vivo is a potential therapeutic approach to reversal of the intrinsic chemoresistance of pancreatic cancer.

In vitro evaluation of photon and raster-scanned carbon ion radiotherapy in combination with gemcitabine in pancreatic cancer cell lines

Background: Pancreatic cancer is the fourth leading cause of cancer deaths, being responsible for 6% of all cancer-related deaths. Conventional radiotherapy with or without additional chemotherapy has been applied in the past in the context of neoadjuvant or adjuvant therapy concepts with only modest results, however new radiation modalities, such as particle therapy with promising physical and biological characteristics, present an alternative treatment option for patients with pancreatic cancer. Up until now the raster scanning technique employed at our institution for the application of carbon ions has been unique, and no radiobiological data using pancreatic cancer cells has been available yet. The aim of this study was to evaluate cytotoxic effects that can be achieved by treating pancreatic cancer cell lines with combinations of X-rays and gemcitabine, or alternatively with carbon ion irradiation and gemcitabine, respectively. Materials and Methods: Human pancreatic cancer cell lines AsPC-1, BxPC-3 and Panc-1 were irradiated with photons and carbon ions at various doses and treated with gemcitabine. Photon irradiation was applied with a biological cabin X-ray irradiator, and carbon ion irradiation was applied with an extended Bragg peak (linear energy transfer (LET) 103 keV/μm) using the raster scanning technique at the Heidelberg Ion Therapy Center (HIT). Responsiveness of pancreatic cancer cells to the treatment was measured by clonogenic survival. Clonogenic survival curves were then compared to predicted curves that were calculated employing the local effect model (LEM). Results: Cell survival curves were calculated from the surviving fractions of each combination experiment and compared to a drug control that was only irradiated with X-rays or carbon ions, without application of gemcitabine. In terms of cytotoxicity, additive effects were achieved for the cell lines Panc-1 and BxPC-3, and a slight radiosensitizing effect was observed for AsPC-1. Relative biological effectiveness (RBE) of carbon ion irradiation ranged from 1.5–4.5 depending on survival level and dose. Sensitizer enhancement ratio (SER) values calculated at 10% cell survival ranged from 1.24–1.66, depending on cell line, gemcitabine dose and irradiation modality. Experimentally ascertained survival curves matched those predicted by LEM-calculation. Conclusion: Our experiments have shown a combined treatment of irradiation and chemotherapy with gemcitabine to be a good means of achieving additive cytotoxic effects on pancreatic cancer cell lines. The data generated in this study will serve as radiobiological basis for further preclinical and clinical studies.

Effect of the combination of the dual mTOR/pI3K inhibitor NVP-BEZ235 with gemcitabine on growth inhibition in pancreatic cancer cells in vitro and in vivo.

e15070 Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignant tumours and is still associated with a very poor prognosis. Therefore new treatment strategies are needed. The PI3K/AKT and mTOR signaling pathways are frequently dysregulated in PDAC. Thus we investigated the effects of NVP-BEZ235, a novel dual PI3K/mTOR inhibitor, alone or in combination with gemcitabine first in vitro and after promising results also in vivo. Methods: We examined the effect of gemcitabine and NVP-BEZ235 (kindly provided by Novartis Pharma) on cell viability as single agents and in combination with sequential administrations in the four human pancreatic cancer cell lines MiaPaCa-2, Panc-1, AsPC-1 and BxPC-3. For in vivo experiments we used NOD SCID Mice, which were injected with BxPc3 into the right flank. Treatments consisted of Gemcitabine alone, NVP-BEZ235 alone, simultaneous application of both, first application of Gemcitabine followed by NVP-BEZ235 and NVP-BEZ235 followed by Gemcitabine. Results: Simultaneous incubation of gemcitabine and NVP-BEZ235 affected the PDAC cell lines significantly better than the single agent administration. But most effective was a sequential administration of gemcitabine followed by NVP-BEZ235. In vivo Gemcitabine and NVP-BEZ235 as single agents showed a slightly reduced tumor growth and the treatment in the sequence NVP-BEZ235 first, followed by Gemcitabine resulted in only a minimal reduction of tumor growth. The most effective results were obtained by simultaneous and even better in the sequence of Gemcitabine followed by NVP-BEZ235, respectively. Conclusions: The combination of gemcitabine with the dual PI3k/mTOR inhibitor NVP-BEZ235 enhanced the efficacy of PDAC treatment via down-regulation of the DDR related gene Survivin in vitro. This combination seems to be significantly more effective than single agent use in vitro and also in vivo. Furthermore we demonstrated that the sequence of administration of these agents could be a relevant issue. These promising results might offer a new and effective option for the treatment of pancreatic cancer in the future.

Metformin-mediated growth inhibition involves suppression of the IGF-I receptor signalling pathway in human pancreatic cancer cells

BackgroundEpidemiological studies have shown direct associations between type 2 diabetes and obesity, both conditions associated with hyperglycaemia and hyperinsulinemia, and the risk of pancreatic cancer. Up to 80% of pancreatic cancer patients present with either new-onset type 2 diabetes or impaired glucose tolerance at the time of diagnosis. Recent population studies indicate that the incidence of pancreatic cancer is reduced among diabetics taking metformin. In this study, the effects of exposure of pancreatic cancer cells to high glucose levels on their growth and response to metformin were investigated.MethodsThe human pancreatic cancer cell lines AsPC-1, BxPC-3, PANC-1 and MIAPaCa-2 were grown in normal (5 mM) or high (25 mM) glucose conditions, with or without metformin. The influence by metformin on proliferation, apoptosis and the AMPK and IGF-IR signalling pathways were evaluated in vitro.ResultsMetformin significantly reduced the proliferation of pancreatic cancer cells under normal glucose conditions. Hyperglycaemia however, protected against the metformin-induced growth inhibition. The anti-proliferative actions of metformin were associated with an activation of AMP-activated protein kinase AMPKThr172 together with an inhibition of the insulin/insulin-like growth factor-I (IGF-I) receptor activation and downstream signalling mediators IRS-1 and phosphorylated Akt. Furthermore, exposure to metformin during normal glucose conditions led to increased apoptosis as measured by poly(ADP-ribose) polymerase (PARP) cleavage. In contrast, exposure to high glucose levels promoted a more robust IGF-I response and Akt activation which correlated to stimulated AMPKSer485 phosphorylation and impaired AMPKThr172 phosphorylation, resulting in reduced anti-proliferative and apoptotic effects by metformin.ConclusionOur results indicate that metformin has direct anti-tumour activities in pancreatic cancer cells involving AMPKThr172 activation and suppression of the insulin/IGF signalling pathways. However, hyperglycaemic conditions enhance the insulin/IGF-I responses resulting in an altered AMPK activation profile and prevent metformin from fully switching off the growth promoting signals in pancreatic cancer cells.

Triterpenes from Poria cocos suppress growth and invasiveness of pancreatic cancer cells through the downregulation of MMP-7

Poria cocos is a medicinal mushroom that is widely used in traditional Asian medicine. Here, we show that a characterized mixture of triterpenes extracted from P. cocos (PTE) and three purified triterpenes: pachymic acid (PA), dehydropachymic acid (DPA) and polyporenic acid C (PPAC) suppress the proliferation of the human pancreatic cancer cell lines Panc-1, MiaPaca-2, AsPc-1 and BxPc-3. Moreover, the most effective compound, PA, only slightly affects the proliferation of HPDE-6 normal pancreatic duct epithelial cells. The anti-proliferative effects of PTE on BxPc-3 cells are mediated by the cell cycle arrest at G0/G1 phase. DNA microarray analysis demonstrated that PTE significantly downregulates the expression of KRAS and matrix metalloproteinase-7 (MMP-7) in BxPc-3 cells. In addition, PTE and PA suppress the invasive behavior of BxPc-3 cells. The inhibition of invasiveness by PTE and PA was associated with the reduction of MMP-7 at the protein level and the role of MMP-7 further confirmed by the gene silencing of MMP-7 which also suppressed the invasiveness of BxPc-3 cells. In conclusion, triterpenes from P. cocos demonstrate anticancer and anti-invasive effects on human pancreatic cancer cells and can be considered as new therapeutic agents in the treatment of pancreatic cancer.

Inhibition of in vitro pancreatic cancer cell migration by circulating anthocyanins and their metabolites from plasma of human subjects

Pancreatic tumor cell migration has important roles in cancer progression and is associated with poor prognosis. We investigated whether circulating plasma anthocyanins and their metabolites (PAM) deriving from fruit juice intake affect cell migration through modulation of the expression of matrix metalloproteinases MMP‐2 and MMP‐9. Ten healthy subjects consumed an anthocyanin‐rich fruit juice (840.9 mg/L). Before (PAM0min) and after (PAM60min) ingestion, blood samples were taken and PAM were prepared by centrifugation and subsequent solid phase extraction. PAM were subjected to the pancreatic cancer cell lines PANC‐1 and AsPC‐1 for 24 and 48 h. Finally, cell migration was determined in a Boyden chamber, the mRNA expression of MMP‐2, MMP‐9 as well as NF‐kB by real‐time PCR. Application of PAM60min lead to a reduced migration of PANC‐1 cells after 48h, whereas AsPC‐1 migration was not affected. Concomitantly, we observed reduced MMP‐2 and MMP‐9 as well as NF‐kB mRNA expression levels in PANC‐1. In contrary, AsPC‐1 showed lower level of NF‐kB which were not influenced by PAM60min. Also, MMP‐2 and ‐9 were not altered. Thus, it can be concluded that in dependency of the cancer phenotype anthocyanins and/or their metabolites may inhibit pancreatic cancer cell migration by reducing NF‐kB dependent MMP‐2 and ‐9 expressions in physiological concentrations.

Grape seed proanthocyanidins inhibit migration potential of pancreatic cancer cells by promoting mesenchymal-to-epithelial transition and targeting NF-κB

Here we explore the effect of grape seed proanthocyanidins (GSPs) on pancreatic cancer cell migration and the molecular mechanisms underlying these effects. Treatment of human pancreatic cancer cell lines Miapaca-2, PANC-1 and AsPC-1 with GSPs resulted in inhibition of cell migration (19–82%, P<0.01–0.001), which was associated with decreased phosphorylation of ERK1/2 and inactivation of NF-κB. Treatment of cells with UO126, an inhibitor of MEK, and caffeic acid phenethyl ester, an inhibitor of NF-κB, also inhibited the migration of cells (40–80%, P<0.01–0.001). Inhibition of cell migration by GSPs was associated with reversal of the epithelial-to-mesenchymal transition. This was associated with upregulation of E-cadherin and desmoglein-2 and down-regulation of fibronectin, N-cadherin and vimentin.

Semisynthetic Ursolic Acid Fluorolactone Derivatives Inhibit Growth with Induction of p21waf1 and Induce Apoptosis with Upregulation of NOXA and Downregulation of c‐FLIP in Cancer Cells

A series of ursolic acid ((1S,2R,4aS,6aR,6aS,6bR,8aR,10S,12aR,14bS)-10-hydroxy-1,2,6a,6b,9,9,12a-heptamethyl-2,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydro-1H-picene-4a-carboxylic acid) derivatives with a 12-fluoro-13,28β-lactone moiety were synthesized using the electrophilic fluorination reagent Selectfluor. The antiproliferative effects of these novel compounds were evaluated in AsPC-1 pancreatic cancer cells, and the structure-activity relationships (SARs) were evaluated. Of the compounds synthesized, ursolic acid derivatives carrying a heterocyclic ring, such as imidazole or methylimidazole, and cyanoenones were among the more potent inhibitors of AsPC-1 pancreatic cancer cell growth. 2-Cyano-3-oxo-12α-fluoro-urs-1-en-13,28β-olide, compound 20, was the most effective inhibitor with IC(50) values of 0.7, 0.9 and 1.8 μM in pancreatic cancer cell lines AsPC-1, MIA PaCa-2 and PANC-1, respectively. This compound also exhibited better antiproliferative activities against breast (MCF7), prostate (PC-3), hepatocellular (Hep G2) and lung (A549) cancer cell lines, with IC(50) values lower than 1 μM. The mechanism of action by which these compounds exert their biological effect was evaluated in AsPC-1 cells using the most potent inhibitor synthesized, compound 20. At 1 μM, the cell cycle arrested at the G1 phase with upregulation of p21(waf1). Apoptosis was induced at an inhibitor concentration of 8 μM with upregulation of NOXA and downregulation of c-FLIP. These data indicate that fluorolactone derivatives of ursolic acid have improved antiproliferative activity, acting through arrest of the cell cycle and induction of apoptosis.

Nicotine, IFN-γ and retinoic acid mediated induction of MUC4 in pancreatic cancer requires E2F1 and STAT-1 transcription factors and utilize different signaling cascades

BackgroundThe membrane-bound mucins are thought to play an important biological role in cell–cell and cell–matrix interactions, in cell signaling and in modulating biological properties of cancer cell. MUC4, a transmembrane mucin is overexpressed in pancreatic tumors, while remaining undetectable in the normal pancreas, thus indicating a potential role in pancreatic cancer pathogenesis. The molecular mechanisms involved in the regulation of MUC4 gene are not yet fully understood. Smoking is strongly correlated with pancreatic cancer and in the present study; we elucidate the molecular mechanisms by which nicotine as well as agents like retinoic acid (RA) and interferon-γ (IFN-γ) induce the expression of MUC4 in pancreatic cancer cell lines CD18, CAPAN2, AsPC1 and BxPC3.ResultsChromatin immunoprecipitation assays and real-time PCR showed that transcription factors E2F1 and STAT1 can positively regulate MUC4 expression at the transcriptional level. IFN-γ and RA could collaborate with nicotine in elevating the expression of MUC4, utilizing E2F1 and STAT1 transcription factors. Depletion of STAT1 or E2F1 abrogated the induction of MUC4; nicotine-mediated induction of MUC4 appeared to require α7-nicotinic acetylcholine receptor subunit. Further, Src and ERK family kinases also mediated the induction of MUC4, since inhibiting these signaling molecules prevented the induction of MUC4. MUC4 was also found to be necessary for the nicotine-mediated invasion of pancreatic cancer cells, suggesting that induction of MUC4 by nicotine and other agents might contribute to the genesis and progression of pancreatic cancer.ConclusionsOur studies show that agents that can promote the growth and invasion of pancreatic cancer cells induce the MUC4 gene through multiple pathways and this induction requires the transcriptional activity of E2F1 and STAT1. Further, the Src as well as ERK signaling pathways appear to be involved in the induction of this gene. It appears that targeting these signaling pathways might inhibit the expression of MUC4 and prevent the proliferation and invasion of pancreatic cancer cells.

Expression of the co-signaling molecules CD40-CD40L and their growth inhibitory effect on pancreatic cancer in vitro

We investigated the expression of the co-signalling molecule CD40 in pancreatic cancer and the growth inhibitory effect of the recombinant soluble human CD40 ligand (rshCD40L) in pancreatic cancer cell lines. Twenty-six cases of pancreatic cancer tissues and corresponding paratumoral normal tissues were immunohistochemically analyzed for CD40 expression. The association of CD40 expression with clinicopathological parameters, including clinical stage, pathological grade, invasion and metastasis, were statistically analyzed. The serum sCD40 levels in pancreatic cancer patients were examined by ELISA. The expression of CD40 in the pancreatic cancer cell lines Panc-1, Aspc-1 and Miapaca-2 was examined by RT-PCR and flow cytometry. The growth inhibitory activity of rshCD40L on pancreatic cancer cell lines was determined by MTT assay. Tumor cell apoptosis was detected by TUNEL and Annexin V/PI double staining method. CD40 was positive both on the membrane and in the cytoplasm of tumor cells, 69.2% (18/26) of the cases were positive for CD40. CD40 expression was significantly higher in pancreatic cancer tissues compared to adjacent normal tissues (P<0.05). High CD40 expression was associated with TNM stage and lymph node metastasis (both P<0.05). Patients with pancreatic cancer have higher serum sCD40L levels (3.53 ± 0.70 ng/ml) compared to healthy subjects (1.81 ± 0.48 ng/ml, P<0.05). rshCD40L significantly inhibited the proliferation of the pancreatic cancer cell lines and induced apoptosis in these cell lines. The co-signaling molecule CD40 is highly expressed in pancreatic cancer tissues and cell lines and rshCD40L is a potential tool for antitumor therapies.

Parecoxib inhibits cell proliferation and induces apoptosis in human pancreatic cancer cell lines BxPC-3 and AsPC-1

Parecoxib inhibits cell proliferation and induces apoptosis in human pancreatic cancer cell lines BxPC-3 and AsPC-1

Multiple Kinase Pathways Involved in the Different De Novo Sensitivity of Pancreatic Cancer Cell Lines to 17-AAG

17-Allylamino-17-demethoxygeldanamycin (17-AAG) specifically targets heat shock protein (HSP)90 and inhibits its chaperoning functions for multiple kinases involved in cancer cell growth and survival. To select responsive patients, the molecular mechanisms underlying the sensitivity of cancer cells to 17-AAG must be elucidated. We used cytotoxicity assays and Western blotting to explore the effects of 17-AAG and sorafenib on cell survival and expression of multiple kinases in the pancreatic cancer cell lines AsPC-1 and Panc-1. Gene cloning and transfection, siRNA silencing, and immunohistochemistry were used to evaluate the effects of mutant p53 protein on 17-AAG sensitivity. AsPC-1 and Panc-1 responded differently to 17-AAG, with half maximal inhibitory concentration (IC(50)) values of 0.12 and 3.18 μM, respectively. Comparable expression of HSP90, HSP70, and HSP27 was induced by 17-AAG in AsPC-1 and Panc-1 cells. P-glycoprotein and mutant p53 did not affect 17-AAG sensitivity in these cell lines. Multiple kinases are more sensitive to HSP90 inhibition in AsPC-1 than in Panc-1 cells. After 17-AAG treatment, p-Bad (S112) decreased in AsPC-1 cells and increased in Panc-1 cells. Sorafenib markedly increased p-Akt, p-ERK1/2, p-GSK-3β, and p-S6 in both cell lines. Accordingly, 17-AAG and sorafenib acted antagonistically in AsPC-1 and Panc-1 cells, except at high concentrations in AsPC-1 cells. Differential inhibition of multiple kinases is responsible for the different de novo sensitivity of AsPC-1 and Panc-1 cells to HSP90 inhibition. P-glycoprotein and mutant p53 protein did not play a role in the sensitivity of pancreatic cancer cells to 17-AAG.

Abstract B63: Synergistic combination between resveratrol and tanshinone I for effective prevention of pancreatic cancer

Abstract Pancreatic cancer is the fourth most common cause of cancer-related mortality in the United States. Pancreatic cancer is often dubbed the silent killer because it seldom causes symptoms until advanced. As a result, few pancreatic cancers are operable at diagnosis. Furthermore, pancreatic cancer is often resistant to conventional radiotherapy or chemotherapy, and the morbidity rate almost equals the mortality rate. Therefore, search for effective regimens with moderate side effects for pancreatic cancer therapy and prevention is an urgent necessity. Chinese herbal medicines have been used for centuries with demonstrated safety profiles, thus their bioactive ingredients may be effective candidates for the prevention and/or treatment of pancreatic cancer. Danshen (Salvia miltiorrhiza) has been widely adopted in the traditional Chinese medicinal preparations for treatment of cardiovascular disease with minimal side effects. In the present study, we evaluated the effect of several major Danshen components, tanshinone IIA (T2A), tanshinone I (T1) and cryptotanshinone (CT) on pancreatic cancer cells. We found that CT, T2A and T1 all showed inhibitory effects on the growth of human pancreatic cancer cell lines MIA PaCa-2 and ASPC-1 with IC50 around 30μM, 3–8μM and 3–5μM, respectively. The drug-resistant human pancreatic cancer cell line PANC-1 showed less sensitivity to CT and T2A treatment but was very sensitive to T1 treatment with IC50 around 3.5μM. The potent T1 activity against pancreatic cancer cells was associated with downregulation of several protein markers related to cell cycle (cyclin D and cyclin B) and anti-apoptosis (Bcl2 and survivin), c-myc and Aurora A and B kinases in both MIA PaCa-2 and PANC-1 cells. To further identify more potent synergistic combination effect of natural bioactive compounds against pancreatic cancer, we determined the anti-growth activity of the combination between T1 and resveratrol, a natural compound found in the skin of red grape and other fruits. T1 and resveratrol combinations consistently showed synergistic effect on inhibiting the growth of MIA PaCa-2, ASPC-1 and PANC-1 cells. Protein assays showed that the synergistic combination effect of T1 and resveratrol was associated with combined downregulation of c-myc and Aurora A protein levels, suggesting that T1 and resveratrol may inhibit the growth of pancreatic cancer cells in part via combined inhibition on the functions of c-myc and Aurora A. Aurora A is the kinase that plays a critical role in controlling mitosis and cell growth. We further confirmed if Aurora A was a functional target for the synergistic combination effect of T1 and resveratrol. Knockdown of Aurora A dramatically inhibited PANC-1 cell growth and decreased the T1 and resveratrol combination activity, suggesting that Aurora A could be one of the targets by the T1 and resveratrol combination effect. Aurora A gene promoter contains the c-myc binding site, suggesting that Aurora A may be regulated by c-myc and the inhibition of c-myc expression may be one of the causes of reduced Aurora A expression following T1 treatment. To confirm this, we treated PANC-1 cells with a c-myc inhibitor and found decreased Aurora A gene expression in a time-dependent manner, indicating the regulation of c-myc on Aurora A gene expression. Our results suggest that the T1 and resveratrol combination may serve as a novel chemopreventive regimen for effective prevention of pancreatic cancer. Supported in part by DOD (PC073988) and NCI/NIH (R21CA127794) Citation Information: Cancer Prev Res 2011;4(10 Suppl):B63.

Modulation of Pancreatic Cancer Chemoresistance by Inhibition of TAK1

TGF-β-activated kinase-1 (TAK1), a mitogen-activated protein kinase kinase kinase, functions in the activation of nuclear factor κB (NF-κB) and activator protein-1, which can suppress proapoptotic signaling pathways and thus promote resistance to chemotherapeutic drugs. However, it is not known if inhibition of TAK1 is effective in reducing chemoresistance to therapeutic drugs against pancreatic cancer. NF-κB activity was measured by luciferase reporter assay in human pancreatic cancer cell lines AsPc-1, PANC-1, and MDAPanc-28, in which TAK1 expression was silenced by small hairpin RNA. TAK1 kinase activity was targeted in AsPc-1, PANC-1, MDAPanc-28, and Colo357FG cells with exposure to increasing doses of a selective small-molecule inhibitor, LYTAK1, for 24 hours. To test the effect of LYTAK1 in combination with chemotherapeutic agents, AsPc-1, PANC-1, MDAPanc-28 cells, and control cells were treated with increasing doses of oxaliplatin, SN-38, or gemcitabine in combination with LYTAK1. In vivo activity of oral LYTAK1 was evaluated in an orthotopic nude mouse model (n = 40, 5 per group) with luciferase-expressing AsPc-1 pancreatic cancer cells. The results of in vitro proliferation were analyzed for statistical significance of differences by nonlinear regression analysis; differences in mouse survival were determined using a log-rank test. All statistical tests were two-sided. AsPc-1 and MDAPanc-28 TAK1 knockdown cells had a statistically significantly lower NF-κB activity than did their respective control cell lines (relative luciferase activity: AsPc-1, mean = 0.18, 95% confidence interval [CI] = 0.10 to 0.27; control, mean = 3.06, 95% CI = 2.31 to 3.80; MDAPanc-28, mean = 0.30, 95% CI = 0.13 to 0.46; control, mean = 4.53, 95% CI = 3.43 to 5.63; both P < .001). TAK1 inhibitor LYTAK1 had potent in vitro cytotoxic activity in AsPc-1, PANC-1, MDAPanc-28, and Colo357FG cells, with IC(50) between 5 and 40 nM. LYTAK1 also potentiated the cytotoxicity of chemotherapeutic agents oxaliplatin, SN-38, and gemcitabine in AsPc-1, PANC-1, and MDAPanc-28 cells compared with control cells (P < .001). In nude mice, oral administration of LYTAK1 plus gemcitabine statistically significantly reduced tumor burden (gemcitabine vs gemcitabine plus LYTAK1, P = .03) and prolonged survival duration (median survival: gemcitabine, 82 days vs gemcitabine plus LYTAK1, 122 days; hazard ratio = 0.334, 95% CI = 0.027 to 0.826, P = .029). The results of this study suggest that genetic silencing or inhibition of TAK1 kinase activity in vivo is a potential therapeutic approach to reversal of the intrinsic chemoresistance of pancreatic cancer.

Retinoic Acid–Induced Pancreatic Stellate Cell Quiescence Reduces Paracrine Wnt–β-Catenin Signaling to Slow Tumor Progression

Patients with pancreatic ductal adenocarcinoma are deficient in vitamin A, resulting in activation of pancreatic stellate cells (PSCs). We investigated whether restoration of retinol to PSCs restores their quiescence and affects adjacent cancer cells. PSCs and cancer cell lines (AsPc1 and Capan1) were exposed to doses and isoforms of retinoic acid (RA) in 2-dimensional and 3-dimensional culture conditions (physiomimetic organotypic culture). The effects of all-trans retinoic acid (ATRA) were studied in LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre mice, a model of human pancreatic ductal adenocarcinoma. After incubation with ATRA, PSCs were quiescent and had altered expression of genes that regulate proliferation, morphology, and motility; genes that encode cytoskeletal proteins and cytokines; and genes that control other functions, irrespective of culture conditions or dosage. In the organotypic model, and in mice, ATRA induced quiescence of PSCs and thereby reduced cancer cell proliferation and translocation of β-catenin to the nucleus, increased cancer cell apoptosis, and altered tumor morphology. ATRA reduced the motility of PSCs, so these cells created a "wall" at the junction between the tumor and the matrix that prevented cancer cell invasion. Restoring secreted frizzled-related protein 4 (sFRP4) secretion to quiescent PSCs reduced Wnt-β-catenin signaling in cancer cells and their invasive ability. Human primary and metastatic pancreatic tumor tissues stained strongly for cancer cell nuclear β-catenin but had low levels of sFRP4 (in cancer cells and PSCs). RA induces quiescence and reduces motility of PSCs, leading to reduced proliferation and increased apoptosis of surrounding pancreatic cancer cells. RA isoforms might be developed as therapeutic reagents for pancreatic cancer.

Omeprazole Inhibits Proliferation and Modulates Autophagy in Pancreatic Cancer Cells

BackgroundOmeprazole has recently been described as a modulator of tumour chemoresistance, although its underlying molecular mechanisms remain controversial. Since pancreatic tumours are highly chemoresistant, a logical step would be to investigate the pharmacodynamic, morphological and biochemical effects of omeprazole on pancreatic cancer cell lines.Methodology/Principal FindingsDose-effect curves of omeprazole, pantoprazole, gemcitabine, 5-fluorouracil and the combinations of omeprazole and 5-fluorouracil or gemcitabine were generated for the pancreatic cancer cell lines MiaPaCa-2, ASPC-1, Colo357, PancTu-1, Panc1 and Panc89. They revealed that omeprazole inhibited proliferation at probably non-toxic concentrations and reversed the hormesis phenomena of 5-fluorouracil. Electron microscopy showed that omeprazole led to accumulation of phagophores and early autophagosomes in ASPC-1 and MiaPaCa-2 cells. Signal changes indicating inhibited proliferation and programmed cell death were found by proton NMR spectroscopy of both cell lines when treated with omeprazole which was identified intracellularly. Omeprazole modulates the lysosomal transport pathway as shown by Western blot analysis of the expression of LAMP-1, Cathepsin-D and β-COP in lysosome- and Golgi complex containing cell fractions. Acridine orange staining revealed that the pump function of the vATPase was not specifically inhibited by omeprazole. Gene expression of the autophagy-related LC3 gene as well as of Bad, Mdr-1, Atg12 and the vATPase was analysed after treatment of cells with 5-fluorouracil and omeprazole and confirmed the above mentioned results.ConclusionsWe hypothesise that omeprazole interacts with the regulatory functions of the vATPase without inhibiting its pump function. A modulation of the lysosomal transport pathway and autophagy is caused in pancreatic cancer cells leading to programmed cell death. This may circumvent common resistance mechanisms of pancreatic cancer. Since omeprazole use has already been established in clinical practice these results could lead to new clinical applications.