Cell cycle arrest and induction of apoptosis in pancreatic cancer cells exposed to eicosapentaenoic acid in vitro.

Eicosapentaenoic acid (EPA) has been shown to have an inhibitory effect on the growth of several pancreatic cancer cell lines in vitro. This study investigates the mechanism of growth inhibition and cytotoxicity of EPA on the pancreatic cancer cell line MIA PaCa-2. Cells were analysed for cell count, viability, cell cycle distribution and ultrastructural changes. There was a time- and dose-dependent decrease in cell count and viability in cultures of pancreatic cancer cells supplemented with EPA. Flow cytometric DNA analysis of MIA PaCa-2 cells incubated with EPA demonstrated the presence of sub G1 populations corresponding to the presence of apoptotic cells and the blockade of cell cycle progression in S-phase and G2/M-phase. The presence of apoptosis in EPA-supplemented cultures was further confirmed by DNA fragmentation and ultrastructural changes associated with apoptosis. Therefore, we conclude that EPA mediates its effect on the pancreatic cancer cell line MIA PaCa-2, at least in part, via cell cycle arrest and the induction of apoptosis.     

Arsenic trioxide inhibits proliferation and induces apoptosis in pancreatic cancer cells

Because of the poor therapeutic responsiveness of pancreatic cancer patients, new chemotherapeutic agents for pancreatic cancer would be extremely beneficial. The effects of arsenic trioxide in pancreatic cancer have not been explored. To evaluate the anti-pancreatic cancer effects of arsenic trioxide, three human pancreatic cell lines, HPAF, MiaPaCa-2 and PANC-1, were tested. Arsenic trioxide caused dose- and time dependent inhibition of pancreatic cancer cell proliferation. In parallel with inhibition of cell proliferation, arsenic trioxide induced significant morphological changes, including shrunken cytoplasm, membrane blebbing, and nuclear condensation consistent with apoptosis. Propidium iodide DNA staining showed an increase of the sub-G0/G1 cell population. The DNA fragmentation induced by arsenic trioxide in these three cell lines was confirmed by the TUNEL assay. Furthermore, Western blotting analysis indicated that caspase-3 was activated following arsenic trioxide as measured by procaspase-3 cleavage and PARP cleavage. These findings show that arsenic trioxide has potent anti-proliferative effects on human pancreatic cancer cells with induction of apoptosis in vitro.     

OSU03012 activates Erk1/2 and Cdks leading to the accumulation of cells in the S-phase and apoptosis

OSU03012, a Celecoxib derivative, has been shown to inhibit proliferation and induce apoptosis in human cancer cell lines. However, its underlying mechanisms are not completely understood. In our study, the relationship between cell cycle inhibition and apoptosis induced by OSU03012 was investigated in human oral cancer cell lines. In the premalignant and malignant cell lines, OSU03012-induced growth inhibition, S-phase arrest, and apoptosis were accompanied by a marked increase in the activity of Erk1/2 and Cdk2/cyclin A. Inhibition of Cdks by roscovitine partially blocked OSU03012-induced growth inhibition and apoptosis. Although the activity of cdc2/cyclin B was reduced, expression of constructively active cdc2AF did not reverse OSU03012-induced S-phase arrest. When Erk1/2 was inhibited by U0126 before addition of OSU03012, growth inhibition and apoptosis induced by OSU03012 were attenuated. The levels of the Cdk2/cyclin A were reduced and cells accumulated in the G(0)/G(1) phase. When cells were allowed to accumulate in S-phase before addition of U0126, apoptosis also was attenuated suggesting that Erk1/2 is required for both progression of cells into the S-phase and apoptosis. Expression of constructively active MEK enhanced OSU03012-induced apoptosis. OSU03012 selectively inhibited the proliferation in premalignant and malignant, but not normal human oral cell lines. In conclusion, we show that OSU03012 has potent anti-proliferative and apoptotic activity against premalignant and malignant human oral cells through activation of Erk1/2, and Cdks. OSU0312 may provide unique opportunities for cancer prevention and sensitization of cancer cells to S-phase modalities.     

Blockade of cyclooxygenase-2 inhibits proliferation and induces apoptosis in human pancreatic cancer cells

Cyclooxygenase (COX), also referred to as prostaglandin endoperoxide synthase, is a key enzyme in the conversion of arachidonic acid to prostaglandins and other eicosanoids. Epidemiologic, animal and in vitro observations show a positive correlation between the expression of COX (especially COX-2) and colonic cancer development, growth and apoptosis. Constitutive expression of COX-2 in human pancreatic cancer cells was recently reported. To evaluate the potential role of COX in pancreatic cancer, RT-PCR was used to determine the constitutive expression of COX-2 in four pancreatic cancer cell lines. MiaPaCa2, PANC-1, HPAF, ASPC-1. The effect of COX blockade with either the general COX inhibitor, indomethacin, or the specific COX-2 inhibitor, NS-398, on [3H]-thymidine incorporation and cell number was investigated in these four pancreatic cancer cell lines. In addition, the effects of these COX inhibitors on pancreatic cancer cell apoptosis was evaluated by DNA propidium iodide staining and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. All four human pancreatic cancer cell lines expressed COX-2 and their proliferation was concentration- and time-dependently inhibited by both indomethacin andNS398. Substantial apoptosis was also induced by treatment of pancreatic cancer cells with either indomethacin or NS398, as indicated by both DNA propidium iodide staining and the TUNEL assay. Furthermore, indomethacin and NS398 were equipotent for growth inhibition and induction of apoptosis, indicating that eicosanoid synthesis via COX-2 is involved in pancreatic cancer cell proliferation and survival. In conclusion, these findings suggest that the COX pathway, especially COX-2, contributes to the growth and apoptosis of pancreatic cancer. Specific COX-2 inhibitors are likely to be valuable for the treatment and prevention of this deadly cancer.     

Natural retinoids inhibit proliferation and induce apoptosis in pancreatic cancer cells previously reported to be retinoid resistant

BACKGROUND: The anticancer ability of natural retinoids on pancreatic adenocarcinoma, an aggressive tumor, is still controversial. This investigation tested the hypothesis that all-trans retinoic acid can inhibit proliferation and induce apoptosis in pancreatic cancer cell lines. MATERIALS AND METHODS: Using our previously optimized conditions, the effect of all-trans retinoic acid (atRA, 0.001-10 microM) was tested in ten human pancreatic adenocarcinoma cell lines with various degrees of differentiation. Proliferation was monitored by cell number, [3H]-thymidine incorporation and cell cycle arrest. Apoptosis was investigated morphologically by light and electron microscopy and biochemically by tissue transglutaminase activity (TGase), mitochondrial membrane potential, cell cycle analysis of sub-G1 cells and detection of fragmented DNA (fragmentation of prelabeled DNA, agarose electrophoresis and TUNEL assays). RESULTS: Retinoic acid caused potent concentration- and time-dependent inhibition of proliferation of all cell lines studied. Cell cycle was arrested at G1 or G2 with extensive reduction of number of cells at S-phase after 24 hours of treatment with apoptotic concentration of atRA. Complete inhibition of proliferation was followed by apoptosis as indicated by the progressive accumulation of sub-G1 apoptotic cells which was confirmed by the more specific DNA fragmentation assays. There were extensive apoptosis-indicative light and electron microscopic changes preceded by phenotypic redifferentiation. TGase was induced between 3-5-fold the control level and its inhibition partially reversed the antiproliferative effect of atRA. Cellular viability during the preapoptotic stage was confirmed by normal mitochondrial membrane potential in the first two days of treatment with the maximum atRA concentration used. However, the potential was progressively reduced with time as a preapoptotic change. Caspase 3-like activity was induced by the apoptotic concentrations of atRA at late time points. However, the redifferentiation indicative changes were not prevented by cotreatment with Ac-DEVE-CHO caspase 3 inhibitor. CONCLUSIONS: Together, our results demonstrated the efficient anticancer ability of natural retinoids on human pancreatic cancer cell lines tested, even those previously reported to be retinoid resistant.     

Actinomycin D induces apoptosis and inhibits growth of pancreatic cancer cells

Pancreatic cancer cells are usually resistant to apoptosis induced by cytotoxic drugs, by activation of surface receptors such as Fas and TNF receptor or by serum or growth factor withdrawal. Actinomycin D (actD) is an inhibitor of RNA synthesis and acts as a potent inducer of apoptosis in several cell lines. In the present study, we investigated the effects of actD on PANC-1 pancreatic cancer cells. ActD caused apoptosis in PANC-1 cells in a dose-dependent manner, as determined by cell growth assays, DNA laddering and TUNEL assays. Induction of apoptosis correlated with activation of the JNK/SAPK pathway and increased expression of Bax but not Bad or p53. PANC-1 cells were completely resistant to Fas antibody and TNF-alpha. In contrast, TRAIL decreased the growth of PANC-1 cells by 22%. Low concentrations of actD (10 ng/ml) enhanced the cytotoxic effects of all 3 cytokines. EGF, FGF-2 and IGF-I did not protect PANC-1 cells from actD-mediated apoptosis. ActD (10 ng/ml) also inhibited the growth of CAPAN-1 and T3M4 pancreatic cancer cells but not MiaPaCa-2 cells. Our observations suggest that actD may act via JNK/SAPK and Bax to promote apoptosis in PANC-1 cells and that it may inhibit the growth of other pancreatic cancer cell lines.     

CBHA is a family of hybrid polar compounds that inhibit histone deacetylase, and induces growth inhibition, cell cycle arrest and apoptosis in human endometrial and ovarian cancer cells

OBJECTIVES: We investigated the effect of a novel synthesized histone deacetylase inhibitor (HDACI), CBHA, on three endometrial cancer cell lines, two ovarian cancer cell lines, and normal human endometrial epithelial cells. METHODS: Endometrial and ovarian cancer cells were treated with various concentrations of CBHA, and its effect on cell growth, cell cycle, apoptosis, and related measurements was investigated. RESULTS: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that all endometrial and ovarian cancer cell lines were sensitive to the growth-inhibitory effect of CBHA, although normal endometrial epithelial cells were viable after treatment with the same doses of CBHA that induced growth inhibition in endometrial and ovarian cancer cells. Cell cycle analysis indicated that their exposure to CBHA decreased the proportion of cells in the S-phase and increased the proportion in the G(0)/G(1) phases of the cell cycle. Induction of apoptosis was confirmed by annexin V staining of externalized phosphatidylserine and loss of the transmembrane potential of mitochondria. This induction occurred in concert with altered expression of genes related to cell growth, malignant phenotype, and apoptosis. Furthermore, CBHA treatment of these cell lines increased acetylation of H3 and H4 histone tails. CONCLUSIONS: These results raise the possibility that CBHA may prove particularly effective in the treatment of endometrial and ovarian cancers.     

Pancreatic cancer cells resistance to gemcitabine: the role of MUC4 mucin

Background:

A major obstacle to the successful management of pancreatic cancer is to acquire resistance to the existing chemotherapeutic agents. Resistance to gemcitabine, the standard first-line chemotherapeutic agent for advanced and metastatic pancreatic cancer, is mainly attributed to an altered apoptotic threshold in the pancreatic cancer. The MUC4 transmembrane glycoprotein is aberrantly overexpressed in the pancreatic cancer and recently, has been shown to increase pancreatic tumour cell growth by the inhibition of apoptosis.

Methods:

Effect of MUC4 on pancreatic cancer cells resistance to gemcitabine was studied in MUC4-expressing and MUC4-knocked down pancreatic cancer cell lines after treatment with gemcitabine by Annexin-V staining, DNA fragmentation assay, assessment of mitochondrial cytochrome c release, immunoblotting and co-immunoprecipitation techniques.

Results:

Annexin-V staining and DNA fragmentation experiment demonstrated that MUC4 protects CD18/HPAF pancreatic cancer cells from gemcitabine-induced apoptosis. In concert with these results, MUC4 also attenuated mitochondrial cytochrome c release and the activation of caspase-9. Further, our results showed that MUC4 exerts anti-apoptotic function through HER2/extracellular signal-regulated kinase-dependent phosphorylation and inactivation of the pro-apoptotic protein Bad.

Conclusion:

Our results elucidate the function of MUC4 in imparting resistance to pancreatic cancer cells against gemcitabine through the activation of anti-apoptotic pathways and, thereby, promoting cell survival.     

Interleukin-4 enhances proliferation of human pancreatic cancer cells: evidence for autocrine and paracrine actions

Interleukin-4 (IL-4) is an immunomodulatory cytokine, which can inhibit the growth of tumour cells. Pancreatic cancer cells and tissues express high levels of IL-4 receptors. The aim of this study was to characterise the effects of IL-4 on the growth and signalling pathways of pancreatic cancer cells. Cell growth was determined by cell counting and MTT assays in association with fluorescence-activated cell sorter analysis, IL-4 expression using ELISA and real-time PCR techniques, and signal transduction using immunoprecipitation or immunoblot analysis. We now report for the first time that IL-4 significantly enhanced the growth of five out of six cultured pancreatic cancer cell lines in a dose-dependent manner in association with an increased fraction of cells in S-phase. Surprisingly, all six cell lines expressed endogenous IL-4, and IL-4 was detectable in the supernatant. Incubating cells with neutralising IL-4 antibodies resulted in a significant inhibition of basal growth in three cell lines, including IL-4-unresponsive MIA PaCa-2 cells, which however expressed the highest endogenous IL-4 levels. Interleukin-4 enhanced activity of MAPK, Akt-1, and Stat3 in IL-4-responsive, but not in IL-4-unresponsive MIA PaCa-2 cells; however, IL-4 enhanced tyrosine phosphorylation of insulin receptor substrate-1 and -2 in all cell lines. Our results demonstrate for the first time that pancreatic cancer cells produce IL-4 and that IL-4 can act as a growth factor in pancreatic cancer cells. Together with the observation that neutralising IL-4 antibodies can inhibit the growth of these cells, our results suggest that IL-4 may act as an autocrine growth factor in pancreatic cancer cells and also give rise to the possibility that cancer-derived IL-4 may suppress cancer-directed immunosurveillance in vivo in addition to its growth-promoting effects, thereby facilitating pancreatic tumour growth and metastasis.     

Induction of apoptosis by taxol and cisplatin and effect on cell cycle-related proteins in cisplatin-sensitive and -resistant human ovarian cells.

The effect of taxol (TX) and cisplatin (CDDP), singly or in association, was assessed on two human ovarian cancer cell lines, one sensitive (A2780) and one resistant (A2780 cp8) to CDDP. Cell lines showed a similar sensitivity to TX, whereas different cytotoxicity results were obtained in the two cell lines as a function of TX and CDDP sequence. Specifically, TX followed by CDDP induced simply additive effects in both cell lines, whereas the opposite sequence produced antagonistic effects in A2780 cells and synergistic effects in A2780 cp8 cells. TX, with or without CDDP, induced oligonucleosomal DNA fragmentation typical of the apoptotic process, but the biochemical mechanisms undergoing apoptosis were different in the two cell lines. In fact, in A2780 cells, TX (with or without CDDP) treatment markedly increased p53 as well as p21waf1 protein expression. In A2780 cp8 cells, drug treatment enhanced p53 levels, whereas the expression of p21waf1 was always undetectable at mRNA and protein levels. In the latter cell line, a premature activation of p34cdc2 kinase was observed in correspondence with the drug-induced increase in the S-phase cell fraction. Such an activation was not ascribable to an increase in the overall expression of p34cdc2 or cyclin B1 proteins, but to a dephosphorylation of p34cdc2 kinase. Overall, our results indicate that TX-induced apoptosis in human ovarian cancer cells may be sustained by different events at the cell cycle-control level.     

Effects of heavy water (D2O) on human pancreatic tumor cells

BACKGROUND: Pancreatic cancer constitutes an entity which is difficult to treat and, therefore, mostly fatal. Since heavy water (deuterium oxide, D2O) was shown to be active in various cancer cell lines in vitro and in vivo, we now investigated its effects in human pancreatic tumor cells. MATERIALS AND METHODS: The cytotoxic effects of D2O were examined in three pancreatic cancer cell lines (AsPC-1, BxPC-3 and PANC-1). Induction of apoptosis was determined by Hoechst/propidium iodide double staining and cell cycle distribution was investigated by FACS analysis. RESULTS: Employing a clonogenic assay, D2O yielded IC50 values of 15%, 18% and 27% in AsPC-1, PANC-1 and BxPC-3 cells, respectively, and led to the induction of apoptosis when compared to untreated controls. Moreover, D2O caused a cell cycle arrest in the G2-M-phase (BxPC-3, PANC-1) or in the S-phase (AsPC-1). CONCLUSION: It is hoped that D2O might offer an additional option for the treatment of pancreatic carcinomas.     

K-Ras-independent effects of the farnesyl transferase inhibitor L-744,832 on cyclin B1/Cdc2 kinase activity, G2/M cell cycle progression and apoptosis in human pancreatic ductal adenocarcinoma cells

Pancreatic ductal adenocarcinoma is a highly lethal malignancy that is resistant to traditional cytotoxic therapy. High rates of activating codon 12 K-Ras mutations in this disease have generated considerable interest in the therapeutic application of novel farnesyl transferase inhibitors (FTIs). However, a comprehensive analysis of the effects of FTI treatment on pancreatic cancer cells has not been performed. Treatment of five different human pancreatic cancer cell lines with FTI L-744,832 resulted in inhibition of anchorage-dependent growth, with wide variation in sensitivity among different lines. Effective growth inhibition by L-744,832 correlated with accumulation of cells with a tetraploid (4N) DNA content and high levels of cyclin B1/cdc2 kinase activity, implying cell cycle arrest downstream from the DNA damage-inducible G2/M cell cycle checkpoint. In addition, sensitive cell lines underwent apoptosis as evidenced by changes in nuclear morphology and internucleosomal DNA fragmentation. L-744,832 at a concentration of 1 µM additively enhanced the cytotoxic effect of ionizing radiation, apparently by overriding G2/M checkpoint activation. The effects of FTI treatment on cell growth and cell cycle regulation were associated with changes in posttranslational processing of H-Ras and N-Ras, but not K-Ras. The results confirm the potential therapeutic efficacy of FTI treatment in pancreatic cancer, and suggest that farnesylated proteins other than K-Ras may act as important regulators of G2/M cell cycle kinetics.     

Increased cyclooxygenase-2 expression in human squamous cell carcinomas of the head and neck and inhibition of proliferation by nonsteroidal anti-inflammatory drugs

BACKGROUND: Cyclooxygenase-2 (COX-2) has been found to be up-regulated in several types of human cancers and its role in the carcinogenic process has been proposed The aim of this study was to examine the expression of COX-2 in human squamous cell carcinoma of the head and neck (SCCHN) and to find out the effects of COX-2 inhibitors on the growth of cultured cells. MATERIALS AND METHODS: We investigated the effect of indomethacin and NS-398 at various concentrations on the growth of SCCHN cell lines using cell proliferation assay, cell cycle analysis and quantification of apoptosis. RESULTS: Immunostaining revealed a significantly increased COX-2 expression in tumor tissues compared with normal controls (p<0.05). Western blotting analysis using a COX-2 antibody, indicated that seven SCCHN cell lines tested constitutively expressed COX-2 protein. Treatment of head and neck cancer cells with NS-398 (10-200 microM) or indomethacin (50-1000 microM) for 72 hours showed a significant dose-dependent inhibition of cell growth (p<0.01) and a significant increase in the number of cells in the G0/G1-phases of the cell cycle with a concomitant reduction at the S-phase in a dose-dependent manner (p<0.05). NS-398 was more effective in cell cycle arrest and growth inhibition than indomethacin (p<0.05) and induced significant apoptosis in two out of three SCCHN cell lines tested at the concentration of 100 microM. CONCLUSION: Our study showed that COX-2 could be a participant in carcinogenesis of SCCHN and that COX-2 inhibitors would be a potential tool for the treatment and prevention of SCCHN.     

Selective cyclooxygenase-2 inhibitors inhibit growth and induce apoptosis of bladder cancer

Selective COX-2 inhibitors such as celecoxib and NS-398 are being evaluated as chemopreventive and therapeutic agents for bladder and other cancers. We investigated the effects of these nonsteroidal anti-inflammatory agents on a panel of bladder cancer cell lines, and assessed their effects on anchorage-dependent and -independent growth, cell cycle, apoptosis and morphology. The human bladder cancer cell lines UM-UC-1, -3, and -6 were assayed for COX-2 expression by Western analysis using a monoclonal antibody to COX-2. UM-UC-1, -3, and -6 cells were grown in the presence of increasing concentrations of NS-398 and celecoxib, and cell growth was quantitated over 7 days by crystal violet elution. The cell lines were treated with NS-398 and celecoxib for 48 h and analyzed by flow cytometry with propidium iodide staining and Br-dUTP staining for apoptosis. Anchorage-independent growth was assessed using an agarose growth assay. Western analysis demonstrated that COX-2 expression in UM-UC-1, -6, and -3 was high, low, and undetectable, respectively. NS-398 and celecoxib produced dose-dependent growth inhibition of UM-UC-1 and -6. Both NS-398 and celecoxib also inhibited anchorage-dependent and -independent growth of UM-UC-3 in a dose-dependent fashion, despite the low basal expression of COX-2 in this cell line. Cell cycle analyses of UM-UC-1 and -6 revealed a 50% reduction in S-phase in the presence of 100 microM NS-398 whereas a smaller reduction in S-phase was noted in UM-UC-3 cells. Furthermore, treatment with 100 microM celecoxib resulted in significant apoptosis in all three cell lines, which was associated with downregulation of Bcl-2. COX-2 selective inhibitors NS-398 and celecoxib produced dose-dependent growth inhibition of bladder cancer cells associated with a significant reduction in S-phase. Induction of apoptosis in all three cell lines by celecoxib was associated with downregulation of Bcl-2. These changes occur independently of COX-2 expression levels suggesting the presence of a COX-2 independent pathway.     

Medroxyprogesterone acetate inhibits human pancreatic carcinoma cell growth by inducing apoptosis in association with Bcl-2 phosphorylation

BACKGROUND: A previous study found that medroxyprogesterone acetate (MPA) delayed the in vivo growth of three (AsPC-1, Capan-2, and MiaPaCa-2) of nine human pancreatic carcinoma cell lines transplanted into nude mice (Cancer 1995; 75:1263-72). The current study was undertaken to evaluate the basis for this inhibitor. METHODS: The estrogen receptor (ER) and progesterone receptor (PgR) status in nine human pancreatic carcinoma cell lines, AsPC-1, BxPC-3, Capan-1, Capan-2, Hs-700T, Hs-766T, MiaPaCa-2, PANC-1, and SUIT-2, was assessed using an enzyme immunoassay (EIA). The authors tested the growth inhibitory activity of MPA and the morphologic changes in these nine pancreatic carcinoma cell lines. Cell cycle progression and DNA fragmentation also were evaluated in these cell lines. Immunoblot analysis was used to determine bcl-2 expression and phosphorylation. RESULTS: In the EIA assay, ER was detected in three cell lines (BxPC-3, Capan-2, and MiaPaCa-2), and PgR was also detected in three (AsPC-1, Capan-2, and MiaPaCa-2). Medroxyprogesterone acetate inhibited the growth of three cell lines (AsPC-1, Capan-2, and MiaPaCa-2) with IC50 values ranging from 2.3 x 10(7) to 6.1 x 10(-7) M. In these three responsive cell lines, MPA caused cell detachment and decreased cell density. The nuclei of the MPA-treated cells were condensed and often fragmented. Cell cycle analysis of these three cell lines showed that MPA induced the appearance of a sub-G1 peak, which is characteristic of early apoptotic cells. DNA degradation assay after MPA treatment showed a typical DNA ladder pattern consistent with apoptosis. Immunoblot analysis of MPA-treated cells that overexpressed bcl-2 revealed a pattern consistent with bcl-2 phosphorylation. CONCLUSIONS: Clinically attainable concentrations of MPA can inhibit the growth of some human pancreatic carcinoma cells in vitro by inducing apoptosis, probably through their PgR, in association with the phosphorylation of bcl-2. This agent may be useful for treating patients with pancreatic carcinoma.     

Overexpression of neuropilin-1 promotes constitutive MAPK signalling and chemoresistance in pancreatic cancer cells

Neuropilin-1 (NRP-1) is a novel co-receptor for vascular endothelial growth factor (VEGF). Neuropilin-1 is expressed in pancreatic cancer, but not in nonmalignant pancreatic tissue. We hypothesised that NRP-1 expression by pancreatic cancer cells contributes to the malignant phenotype. To determine the role of NRP-1 in pancreatic cancer, NRP-1 was stably transfected into the human pancreatic cancer cell line FG. Signal transduction was assessed by Western blot analysis. Susceptibility to anoikis (detachment induced apoptosis) was evaluated by colony formation after growth in suspension. Chemosensitivity to gemcitabine or 5-fluorouracil (5-FU) was assessed by MTT assay in pancreatic cancer cells following NRP-1 overexpression or siRNA-induced downregulation of NRP-1. Differential expression of apoptosis-related genes was determined by gene array and further evaluated by Western blot analysis. Neuropilin-1 overexpression increased constitutive mitogen activated protein kinase (MAPK) signalling, possibly via an autocrine loop. Neuropilin-1 overexpression in FG cells enhanced anoikis resistance and increased survival of cells by > 30% after exposure to clinically relevant levels of gemcitabine and 5-FU. In contrast, downregulation of NRP-1 expression in Panc-1 cells markedly increased chemosensitivity, inducing > 50% more cell death at clinically relevant concentrations of gemcitabine. Neuropilin-1 overexpression also increased expression of the antiapoptotic regulator, MCL-1. Neuropilin-1 overexpression in pancreatic cancer cell lines is associated with (a) increased constitutive MAPK signalling, (b) inhibition of anoikis, and (c) chemoresistance. Targeting NRP-1 in pancreatic cancer cells may downregulate survival signalling pathways and increase sensitivity to chemotherapy.     

TFPI-2基因对Panc-1细胞凋亡的影响

[目的]研究TFPI-2基因对胰腺癌细胞系Panc-1细胞增殖及凋亡的影响。[方法]测定Panc-1-TFPI-2、Panc-1-V和Panc-1-P三组细胞的生长曲线．DNA片段化实验检测细胞凋亡，并用流式细胞仪分析三组细胞的细胞增殖、细胞周期情况。[结果]Panc-1-TFPI-2细胞同Panc-1-V和Panc-1-P细胞相比，细胞生长缓慢，细胞生长受到抑制，被阻滞于G0～G1期；DNA片段化实验显示Panc-1-TFPI-2细胞呈现凋亡细胞特有的“梯状”条带．而Panc-1-V和Panc-1-P细胞无明显“梯状”条带：流式细胞仪分析显示早期Panc-1-TFPI-2细胞凋亡率（6．93％± 0．53％）较Panc-1-V（3．01％± 0．39％）和Panc-1-P细胞凋亡率（3．52％±0．41％）增加，有显著性差异（P〈0．05）。[结论]TFPI-2能够抑制胰腺癌细胞生长、诱导胰腺癌细胞凋亡．可能具有抑制肿瘤细胞增殖的作用。     

The kinase Mirk/Dyrk1B mediates cell survival in pancreatic ductal adenocarcinoma

Ductal adenocarcinoma of the pancreas is almost uniformly lethal as this cancer is invariably detected at an advanced stage and is resistant to treatment. The serine/threonine kinase Mirk/Dyrk1B has been shown to be antiapoptotic in rhabdomyosarcomas. We have now investigated whether Mirk might mediate survival in another cancer in which Mirk is widely expressed, pancreatic ductal adenocarcinoma. Mirk was an active kinase in each pancreatic cancer cell line where it was detected. Mirk knockdown by RNA interference (RNAi) reduced the clonogenicity of Panc1 pancreatic cancer cells 4-fold and decreased tumor cell number, showing that Mirk mediates survival in these cells. Mirk knockdown by synthetic duplex RNAis in Panc1, AsPc1, and SU86.86 pancreatic cancer cells induced apoptosis and enhanced the apoptosis induced by gemcitibine. Mirk knockdown did not increase the abundance or activation of Akt. However, four of five pancreatic carcinoma cell lines exhibited either elevated Mirk activity or elevated Akt activity, suggesting that pancreatic cancer cells primarily rely on Mirk or Akt for survival signaling. Mirk protein was detected by immunohistochemistry in 25 of 28 cases (89%) of pancreatic ductal adenocarcinoma, with elevated expression in 11 cases (39%). Increased expression of Mirk was seen in pancreatic carcinomas compared with primary cultures of normal ductal epithelium by serial analysis of gene expression and by immunohistochemistry. Thus, Mirk is a survival factor for pancreatic ductal adenocarcinoma. Because knockout of Mirk does not cause embryonic lethality, Mirk is not essential for normal cell growth and may represent a novel therapeutic target.