Activation of P38 mitogen-activated protein kinase (MARK) pathway increases the sensitivity to gemcitabine in human pancreatic cancer cells

GEM. Apoptosis was analyzed on now cytometry and shown up as the sub-G1 population. GEM increased p38 activity in both parental cells, but had no effect on the activation of JNK, Akt, and ERK. The blockade of p38 MAPK with SB203580 prevented apoptosis in the presence of GEM. In contrast, GEM-resistant cells showed the decreased p38 activation and reduced apoptosis in the presence of GEM. Pretreatment with anisomycin increased p38 MAPK activation and induced apoptosis in GEM-resistant ceils. Only a small increase of apoptosis was observed in the cells treated with anisomycin alone. These results suggest that p38 MAPK plays a key role in GEM-induced apoptosis, and a combination with other drugs that activate the p38 MAPK pathway may enhance the efficacy of the treatment for pancreatic cancer.

resulted in dose dependent elevations of serum endostatin 4-8 weeks later. A dose of 1 x 109 i.u. resulted in serum endostatin levels of -35-40 ng/ml at 6 weeks. These endostatin levels were sufficient to inhibit tumor induced angiogenesis m vivo. They also suppressed both the initiation of HT29 xenograft formation following tumor eel! injection and the growth of established macroscopic tumors. These findings indicate that the delivery of human endostatin via rAAV vectors may provide an effective anticancer therapy.

M1001 S100P is Expressed in Pancreatic Adenocarcinoma and Stimulates Pancreatic Cancer Cell Proliferation and Survival Thiruvengadam Arumugam, Diane Simeone, Craig Logsdon

MI004

Background: Pancreatic adenocarcinoma is the most lethal of major cancers, due in part to its resistance to therapy. The mechanisms underlying this resistance are not well understood. In the current study, we investigated the possible rote of SLOOPas a mediator of pancreatic cancer cell survival. Methods: The expression of $100 proteins was assessed using Affymetrix microarrays, Q-RT-PCR, Western blotting, and immunocytochemistry. The secretion of S100P into the culture media by pancreatic cancer cells was assessed using an ELISA. The effects of exogenous SI00P, prepared as a fusion protein, or endogenous expression of S 100P, were studied in assays of cell transformation, prohferation, and resistance to apoptotic insults (5-FU and suspension) in normal (NIH3T3, HPDE) and pancreatic cancer (Panel) cell lines. Activation of Erk was analyzed by Western blotting with anti-phospho-Erk antibodies. NFKB activation was assessed by EMSA Results: S100P was highly expressed in the neoplastic epithelium of pancreatic adenocarcinoma and was secreted by pancreatic cancer cell lines. In cell models, S 100P stimulated Erk and NFKB activity, increased cell proliferation, and inhibited apoptotic responses to 5-FU or suspension. Conclusions: These data indicate that S100P may act in an autocrine manner to stimulate growth and survival of pancreatic cancer cells. Therefore, this molecule may be a useful target for the development of new therapies for this deadly tumor.

All-trans Retinoid Acid-induced Apoptosis in Pancreatic Cancer Cells is Mediated by bcb2/bax and p53 Expression Lu Xia, Yin Bao, Yaozong Ynan, Xuejun Zhang Aims: Pancreatic carcinoma cells are extremely resistant to apoptosis induction by chemotherapy. The therapeutic and preventive effects of retinoids in cancer are due to their ability to modulate the differentiation, and survival or apoptosis of cancer ceils. The present study using moderate-differentiated pancreatic adenocarcinoma cell line Patu-8988 to observe the effect of All-trans retinoid acid(ATRA) on cell growth in vitro and the pathway in apoptosis of pancreatic cancer induced by ATRA. Methods: Cells were cultured in low glucose DMEM, and treated by ATRA(Sigma,lp.M) or both ATRA and caspase inhibitor Z-VAD-FMK(Promega,20p.M) for 12hr,24hr,48hr,72hr and 96hr separately. Apoptosis was detected by flow cytometry, DNA degradatiori assay and TUNEL method respectively, p53 and Bcl-2/Bax mRNA level were assessed by RT-PCR method, the protein level of Bcl-2, Bax, phosphop53 and the expression of cleaved caspase-3 were detected by Western-blot analysis. Use caspase-3 colorimetric assay to determine the increased enzymatic activity of the caspase-3 in apoptotic cells. Results: After the treatment of ATRA, the apoptotic cells were detected by flow cytometry at the rate of 8.70, 15.19, 16.68, 28.28 and 38.79% at 0,12hr,24hr,48hr and 72hr respectively. Compare to ATRA-treated cells, the treatment of ATRA and Z-VADFMK in combination did not change the rate of apoptosis (36.93%/38.79%, p>0.05). Caspase-3 co[orimetric assay indicated that during the time course, the activities of caspase3 were parallel to the rate of apoptosis rate, while in Z-VAD-FMK treated ceils, the activity of caspase-3 was greatly decreased (0.042/0.078, p<0.05). TUNEL assay showed strong positive reactions after the treatment at 72hr. DNA degradation assay also showed a typical DNA ladder pattern consistent with apoptosis. Apoptosis was accompanied by upregnlation of cleaved caspase-3 activity, and increased 8ax and phospho-p53(Ser 46) expression, but the expression of Bcl-2 is down-regulated compare with the untreated cells. During the ATRA treatment, the expression of phosho-p53(Ser 392) remained unchanged. Conclusions: These data show that ATRA is able to induce apoptosis in pancreatic cancer cells, and the expression of bcl-2/bax and p53 is contributed to the apoptosis induced by ATRA, but the phenomenon that the inhibition of caspases did not inhibit the apoptosis accordingly suggest some other proteases may have also played an important role in apoptosis pathways of pancreatic cancer cells.

MlOO2 Hes-I And Cox-2 Expression In Intraductal Papillary-Mucinous Tumor Of The Pancreas Ryo Hosutani, Michihiko Wada, Tateo Kajiwara, Yoshiharu Miyamoto, Toshihiko Masui, Koji Fujimoto, Ryuichiro Doi, Masayuki lmamura Adenoma-careinoma sequence and associated genetic changes in pancreatic intraepithelial neoplasia (PanlN) and intraductal papillary-mucinous tumor of the pancreas (IPMT) have been well investigated. Several molecular markers, such as p16INK4A and smad4/DPC4, are known to be related to the carcinogenesis of IPMT; however, markers specifically expressed in the earlier stages of this tumor are poorly understood. Notch signahng is the pathway which controls cell fate in pancreatic developmem, and Hes-1, a notch signaling product, inhibits the endocrine differentiation of precursor ceils. In this study, Hes-1 and Cox-2 expression were immunohistochemically examined in IPMT and compared with the changes in p16 and smad4. Methods: Archival paraffin-embedded samples from six surgically obtained tumors were used. All of the tumors were histologically diagnosed as carcinoma in situ (CIS). Since differentiation of neoplastic epithelium of ]PMT varies within the same tumor, a total of 142 intraducta[ lesions (an average of 24 lesions per case) were identified; 35 were CIS, 34 borderline malignancies, 37 adenomas and 36 hyperplasias. IHC for Hes1 (PoAb donated by Dr.Sudo), Cox-2 (CX229; Caman), p16 (13251A; Pharmingen) and smad4 (B-8; Santa Cruz) was performed on each sample. Advanced cancer samples served as positive controls for Hes-1 and Cox-2 staining, and normal duct epithelium or islet as the internal positive control for smad4 and p16. Results are summraized in the table. Conclusions: Stepwlse expression of molecular markers was found in 1PMT, with loss of p16 predominant in the step from borderline malignancy to carcinoma in situ. Hes-1 and Cox-2 over-expression may be involved in the earlier and low dyspfastic changes of [PMT, such as hyperpfasia and adenoma.

MI005 Non-Mitochondrial NAD(P)H Oxidase Mediates Growth Factor-induced ROS Production and Suppression of Apoptosis in Pancreatic Cancer Cells Eva C. Vaquero, Kyung J. Nam, Ilya Gukovsky, Stephen J. Pandol, Anna S. Gukovskaya

Background& Aims: Generation of reactive oxygen species (ROS) is a common characteristic of cancer cells and was recently linked to tumor progression. We previously showed that ROS protect pancreatic cancer cells from apoptosis. The aim of the present study was to determine the source of ROS induced by growth factors and its role in apoptosis of pancreatic cancer cells. Methods:Human pancreatic carcinoma MIA PaCa-2 cells were cultured without serum, with 15% fetal bovine serum (FBS), or with insulin-like growth factor I (IGF-I; 100 ng/ml). Apoptosis was determined by measuring internucleosomal DNA fragmentation with ELISA and phosphatidylserine externalization with Annexin V; intracellufar ROS, by using the redox-sensitive probe DCF; and NADPH oxidase activity, by chemiluminescence. Mitochondrial (mt)DNA-depleted cells were generated by long term culture with ethidium bromide. Results: FBS and 1GF-I markedly and time-dependently increased ROS in MIA PaCa2 cells, which was prevented by antioxidants tiron, N-acetylcysteine (NAC) and the inhibitor of NAD(P)H oxidases, diphenylene iodonium (DPI). In contrast, inhibitors of nitric oxide synthase (L-NAME), xanthine oxidase (allopurinol), and phospholipase A2 (AACOCF3) had no effect. These data indicate that the source of ROS induced by growth factors is a NAD(P)H-dependent oxidase. The main types of NAD(P)H oxidases inhibited by DPI are the mitochondrial NAD(P)H:ubiquinone oxidoreductase and the plasma membrane nonphagocytic oxidase, Nox. To determine the contribution of mitochondrial oxidoreductase, we depleted MIA PaCa-2 cells of mtDNA which codes for this enzyme. The extent of ROS stimulation by growth factors was the same in parental (Rho +) and mtDNA-depleted (Rho~ cells, and it was similarly inhibited by DP1 in both types of cells. Thus, the source of ROS induced by growth factors is a non-mitochondrial NAD(P)H oxidase. With RT-PCR and Western blotting we showed the expression of Nox-i and -2, but not Nox-5, in MIA PaCa2 cells. NAD(P)H oxidase activity in cell homogenates was stimulated 2-3 fold by growth factors, which was blocked by tiron and DPI. The anti-oxidants stimulated apoptosis to the same extent in Rho + and Rho~ cells, and prevented the inhibition of apoptosis caused by FBS or IGF-I. Conclusion: Non-mitochondrial NAD(P)H oxidase mediates growth factorinduced ROS and has a pro-survival role in pancreatic cancer cells.

Table 1. Immunostainingratios in each IPMI"lesion. Hes.l positive Cox-2 po~Uve Loss of p16 Lon of smad4

hyperphnda 67 % 30 % 0% 5%

adenoma 97 % 83 % 0% 6%

borderline 84 % 87 % 9% 25%

ClS 86 % 100 % 64% 29%

M1003 Activation of P38 Mitogen-Activated Protein Kinase (MAPK) Pathway Increases The Sensitivity to Gemcitabine in Human Pancreatic Cancer Cells Atsuya Habiro, Satoshi Tanno, Kazuya Koizumi, Yasuhiro Nakano, Manabu Osanai, Yutaka Kohgo Gemcitabine (GEM) is presently recommended as a first-line treatment in pancreatic cancer, however, even with this drug, the objective response rate is less than 20% in the clinic. Although a combination with other drugs raises the possibility to improve the prognosis of pancreatic cancer patients, complicated mechanism of action with multiple cellular targets may be involved in the sensitivity to GEM. Recent evidences show that p38 MAPK, a newly discovered stress-activated protein kinase, is activated by various genotoxic stresses, resulting in the induction of apoptosis. We investigated the role of p38 MAPK signaling in the GEMinduced apoptosis of human pancreatic cancer cells and whether activation of p38 MAPK overcomes the resistance to GEM in GEM-resistant cells. Using human pancreatic cancer cell hnes PK-1 and PC1-43, a resistance to GEM was induced by exposing those cells to stepwtse increasing GEM concentrations. To modulate the p38 MAPK activation, the selective inhibitor SB203580 and potential activator anisomycin were used. p38 MAPK activity was assessed by determining the phosphorylation of p38 using polyclonal phospho-specific antibody. SB203580 or anisomycin was added as a single agent or in combination with

AGA

Abstracts

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