Therapy Of Human Pancreatic Cancer Research Articles

Salidroside inhibits proliferation, migration and invasion of human pancreatic cancer PANC1 and SW1990 cells through the AKT and ERK signaling pathway.

Salidroside is the main compounds extracted from the Chinese medicine Rhodiola rosea and has many pharmacological effects, including anti-tumor effects. However, the role of salidroside in human pancreatic cancer remains poorly known. Thus, the focus of this study was to evaluate the inhibitory effect of purified salidroside on human pancreatic cancer cells and its underlying molecular mechanisms. PANC1 and SW1990 cells were incubated with various concentrations of salidroside, and CCK-8 assay, colony formation, apoptosis, migration and invasion, western blot were conducted. As a result, it was found that salidroside significantly inhibited pancreatic cancer cells viability, proliferation, migration and invasion, and also induced cell apoptosis. Furthermore, we also detected that salidroside inhibited pancreatic cancer cells by downregulating the AKT and ERK signaling pathways. In conclusion, these findings suggest that salidroside may be a promising candidate for the development of a therapy of human pancreatic cancer.

The flavonoid p-hydroxycinnamic acid exhibits anticancer effects in human pancreatic cancer MIA PaCa-2 cells in vitro: Comparison with gemcitabine.

Pancreatic cancer is a highly aggressive malignancy with a notoriously dismal prognosis. A major contributor to this poor clinical outcome is pancreatic cancer's prominent chemoresistance. The present study was undertaken to determine whether the flavonoid p‑hydroxycinnamic acid (HCA), which is a botanical factor, possesses anticancer effects on cloned human pancreatic cancer MIA PaCa‑2 cells that possess resistance to radiation therapy in vitro. Proliferation of MIA PaCa‑2 cells was suppressed after culture with HCA (10‑1,000 nM). Such an effect was also noted in human pancreatic cancer Pt45P1 cells. In the MIA PaCa‑2 cells, HCA induced G1 and G2/M phase cell cycle arrest in the cells. The suppressive effects of HCA on proliferation were suggested to be mediated through the inhibition of various signaling pathways related to nuclear factor‑κB (NF‑κB), extracellular signal‑regulated kinase (ERK), protein kinase C, phosphatidylinositol 3‑kinase (PI3K) or nuclear transcription activity. Moreover, HCA was found to stimulate cell death in the MIA PaCa‑2 and Pt45P1 cells in vitro. The anticancer effects of HCA on MIA PaCa‑2 cells were exhibited at a lower concentration than gemcitabine, a potent cancer drug. The flavonoid HCA may be a useful tool in the therapy of human pancreatic cancer in vivo.

Abstract 3317: Efficacy of Salmonella typhimurium A1-R treatment on human patient pancreatic tumorgrafts.

Abstract We have previously developed the genetically-modified Salmonella typhimurium A1-R (A1-R) strain that selectively targets tumors and demonstrated its efficacy for various types of cancer cell lines in nude mouse models. The aim of this study is to determine the efficacy A1-R treatment for human-patient pancreatic tumorgrafts. Pancreatic-cancer-patient tumor specimens were initially established subcutaneously in NOD/SCID mice immediately after surgery. The patient tumors were then harvested from NOD/SCID mice and passed orthotopically in transgenic nude mice, expressing fluorescent protein in order for the tumors to acquire fluorescent stroma for imaging. After confirmation of tumor growth by fluorescence imaging, the nude mice were treated in the following groups: (1) 5-fluorouracil (5FU) (10 mg/kg, ip); (2) cisplatin (CDDP) (10 mg/kg, ip); (3) gemcitabine (GEM) (150 mg/kg, ip); (4) A1-R (A1-R) (1.5x108 cfu/body, ip); and (5) PBS (vehicle/control) (ip). Control, 5FU, CDDP, GEM and A1-R injections were performed on a weekly basis from day-21 after tumor implantation for 4 weeks. Animals were sacrificed at 7 weeks, and tumors were harvested for analysis. Each treatment arm involved 5 tumor-bearing mice. No significant effects on body weight, morbidity, or severe toxicities were observed in any treatment arm. The tumor weight of each group was as follows: (1) 5FU, 0.044 ± 0.027g; (2) CDDP, 0.04 ± 0.032g; (3) GEM, 0.058 ± 0.051g; (4) A1-R, 0.106 ± 0.038g; and (5) PBS, 0.258 ± 0.209g. S. typhimurium A1-R treatment significantly reduced the tumor weight compared to control treatment (p = 0.011), as did the other treatments including 5FU (p = 0.005), CDDP (p = 0.004), and GEM (p = 0.001). S. typhimurium A1-R treatment was effective for human pancreatic tumorgrafts and could be a promising therapy for human pancreatic cancer. Citation Format: Ming Zhao, Matthew H.G. Katz, Jason B. Fleming, Atsushi Suetsugu, Yong Zhang, Ali Maawy, Takashi Chishima, Kuniya Tanaka, Michael Bouvet, Yukihiko Hiroshima, Itaru Endo, Robert M. Hoffman. Efficacy of Salmonella typhimurium A1-R treatment on human patient pancreatic tumorgrafts. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3317. doi:10.1158/1538-7445.AM2013-3317

An RNA aptamer that specifically binds pancreatic adenocarcinoma up-regulated factor inhibits migration and growth of pancreatic cancer cells

Previously, we reported that a novel secretory protein, pancreatic adenocarcinoma up-regulated factor (PAUF), which is highly expressed in pancreatic cancer and mediates the growth and metastasis of pancreatic cancer cells. In this study, we generated and characterized a 2′-fluoropyrimidine modified RNA aptamer (P12FR2) directed against human PAUF. P12FR2 binds specifically to human PAUF with an estimated apparent K D of 77 nM. P12FR2 aptamer inhibits PAUF-induced migration of PANC-1, human pancreatic cancer cells, in a wound healing assay. Moreover, intraperitoneal injection of P12FR2 decreased tumor growth by about 60% in an in vivo xenograft model with CFPAC-1 pancreatic cancer cells, without causing a loss of weight in the treated mice. Taken together, we propose here that PAUF-specific RNA aptamer, P12FR2, has the potential to be effective in the therapy of human pancreatic cancer.

Abstract 2458: MicroRNA miR-34 as a natural regulator and potential therapeutic for pancreatic cancer stem cells

Abstract MicroRNAs (miRNAs) have been implicated in cancer initiation and progression and are involved in cancer stem cell dysregulation. We show that miR-34 directly regulates Notch and Bcl-2, suggesting the possibility of a role for miR-34 in the maintenance and survival of cancer stem cells. We examined the roles of miR-34 in p53-mutant human pancreatic cancer cell lines and the potential link to cancer stem cells. Restoration of miR-34 expression in the cancer cells downregulated Bcl-2 and Notch1/2. miR-34 restoration significantly inhibited clonogenic cell growth and invasion, induced apoptosis and G1 and G2/M arrest in cell cycle, and sensitized the cancer cells to chemotherapy and radiation. We identified that CD44+/CD133+ cancer cells are enriched with tumorsphere-forming and tumor-initiating cells or cancer stem/progenitor cells with high levels of Notch/Bcl-2 and loss of miR-34. More significantly, miR-34 restoration led to significant reduction of the tumor-initiating cell population, accompanied by inhibition of tumorsphere growth in vitro and tumor-initiation in vivo. Our results demonstrate that miR-34 may restore, at least in part, the tumor suppressing function of the p53 in p53-deficient human pancreatic cancer. Our data support that miR-34 may be involved in cancer stem cell self-renewal, potentially via the direct modulation of downstream targets Bcl-2 and Notch, implying that miR-34 may play an important role in cancer stem cell self-renewal and/or cell fate determination. Currently, we are exploring the nanoparticle-targeted delivery of miR-34 to cancer stem cells as a novel molecular therapy for human pancreatic cancer, based on our patented nanobiotechnology which has been approved by FDA for clinical trials. Taken together, our results demonstrate that restoration of miR-34 may hold significant promise as a novel molecular therapy for human pancreatic cancer with loss of p53-miR34, potentially via inhibiting cancer stem cells. Molecularly targeted miRNA therapy could be a powerful tool to correct the cancer stem cell dysregulation. Supported in part by NIH grants CA121830(S1) and CA134655. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2458. doi:10.1158/1538-7445.AM2011-2458

Antiproliferative and antimetastatic effects of emodin on human pancreatic cancer

Emodin (1, 3, 8-trihydroxy-6-methylanthraquinone) is an active constituent isolated from the root of Rheum palmatum L and is the main effective component of some Chinese herbs and plants. Pharmacological studies have demonstrated that emodin exhibits anti-cancer effects on several human cancers. However, the molecular mechanisms of emodin-mediated tumor regression have not been fully defined. This study was performed to investigate the antiproliferative and antimetastatic effects of emodin on pancreatic cancer in vitro and in vivo. Our results showed that emodin induced a higher percentage of growth inhibition and apoptosis in the pancreatic cancer cell line SW1990 compared to that of control, and emodin suppressed the migration and invasion of SW1990 cells in a dose-dependent manner. To investigate the possible mechanisms involved in these events, we performed electrophoretic mobility shift assay (EMSA) and Western blot analysis, and found that emodin significantly down-regulated NF-κB DNA-binding activity, survivin and MMP-9 in SW1990 cells. Moreover, the expression of cleaved caspase-3 was up-regulated in SW1990 cells after treatment with emodin. In addition, a metastatic model simulating human pancreatic cancer was established by orthotopic implantation of histologically intact human tumor tissue into the pancreatic wall of nude mice. Oral administration of emodin significantly decreased tumor weight and metastasis compared to control. Furthermore, the expression of NF-κB, survivin and MMP-9 were also suppressed in tumor tissues after treatment with emodin. Collectively, our results indicated that emodin exerts antiproliferative and antimetastatic activity on pancreatic cancer both invitro and invivo, which may be related to down-regulation of NF-κB and its regulated molecules such as survivin and MMP-9 proteins. Consequently, these results provide important insights into emodin as an anti-invasive agent for the therapy of human pancreatic cancer.

Abstract 2084: Tumor suppressor microRNA miR-34 inhibits human pancreatic cancer stem cells

Abstract MicroRNAs (miRNAs) have been implicated in cancer initiation and progression via their ability to affect expression of genes and proteins that regulate cell proliferation and/or cell death. Transcription of the three miRNA miR-34 family members was recently found to be directly regulated by p53. We show that miR-34 directly regulates Notch and Bcl-2, suggesting the possibility of a role for miR-34 in the maintenance and survival of cancer stem cells. We examined the roles of miR-34 in p53-mutant human pancreatic cancer cell lines and the potential link to cancer stem cells. Restoration of miR-34 expression in the cancer cells by miR-34 mimics or lentiviral miR-34 downregulated Bcl-2 and Notch1/2. miR-34 restoration significantly inhibited clonogenic cell growth and invasion, induced apoptosis and G1 and G2/M arrest in cell cycle, and sensitized the cancer cells to chemotherapy and radiation. We identified that CD44+/CD133+ cancer cells are enriched with tumorsphere-forming and tumor-initiating cells or cancer stem/progenitor cells with high levels of Notch/Bcl-2 and loss of miR-34. More significantly, miR-34 restoration led to an 87% reduction of the tumor-initiating cell population, accompanied by significant inhibition of tumorsphere growth in vitro and tumor-initiation in vivo. Our results demonstrate that miR-34 may restore, at least in part, the tumor suppressing function of the p53 in p53-deficient human pancreatic cancer. Our data support that miR-34 may be involved in cancer stem cell self-renewal, potentially via the direct modulation of downstream targets Bcl-2 and Notch, implying that miR-34 may play an important role in cancer stem cell self-renewal and/or cell fate determination. Currently, we are exploring the nanoparticle-targeted delivery of miR-34 to cancer stem cells as a novel molecular therapy for human pancreatic cancer, based on our patented nanobiotechnology which has been approved by FDA for clinical trials. Taken together, our results demonstrate that restoration of miR-34 may hold significant promise as a novel molecular therapy for human pancreatic/gastric cancer with loss of p53-miR34, potentially via inhibiting cancer stem cells. The project is funded by NIH grants CA121830, CA128220 and CA134655 (to L. Xu.). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2084.

Preclinical Evaluation of Monoclonal Antibody 14C5 for Targeting Pancreatic Cancer

The use of radiolabeled antibodies that are able to target primary tumors as well as metastatic tumor sites with minimal reactivity to normal tissues is a promising approach for treating pancreatic cancer. In this study, the integrin alpha(v)beta(5) is studied as a target for the diagnosis of and potential therapy for human pancreatic cancer by using the radiolabeled murine monoclonal antibody (mAb) 14C5. Biopsy specimens from human pancreatic tumors were examined for the expression of the integrin alpha(v)beta(5). The pancreatic tumor cell line Capan-1 was used to test the in vitro targeting potency of mAb 14C5 labeled with 125/131-iodine and 111-indium. Internalization, retention, and metabolism were investigated in cellular radioimmunoassays. Biodistribution and tumor-targeting characteristics were studied in Capan-1 xenografts. All tumor sections were positive for the integrin alpha(v)beta(5), with an extensive positive staining of the stroma. Saturation binding experiments showed high affinity with comparable K(d)s. In vitro internalization experiments showed a longer intracellular retention of (111)In-p-benzyl isothiocyanate-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bz-DOTA)-14C5 in comparison to (125)I-14C5 and (111)In-p-isothiocyanatobenzyl diethylenetriaminepentaacetic acid (p-SCN-Bz-DTPA)-14C5. In vivo radioisotope tumor uptake was maximum at 48-72 hours, with the uptake of (111)In-p-SCN-Bz-DOTA-14C5 (35.84 +/- 8.64 percentage of injected dose per g [%ID/g]) being 3.9- and 2.2-folds higher than (131)I-14C5 (12.16 +/- 1.03%ID/g) and (111)In-p-SCN-Bz-DTPA-14C5 (14.30 +/- 3.76%ID/g), respectively. Planar gamma imaging with mAb 14C5 indicated clear localization of the pancreatic tumors versus minimal normal tissue uptake. mAb 14C5 is a promising new antibody for targeting the integrin alpha(v)beta(5) for the diagnosis of and potential therapy for pancreatic cancer.

MicroRNA miR-34 inhibits human pancreatic cancer tumor-initiating cells.

BackgroundMicroRNAs (miRNAs) have been implicated in cancer initiation and progression via their ability to affect expression of genes and proteins that regulate cell proliferation and/or cell death. Transcription of the three miRNA miR-34 family members was recently found to be directly regulated by p53. Among the target proteins regulated by miR-34 are Notch pathway proteins and Bcl-2, suggesting the possibility of a role for miR-34 in the maintenance and survival of cancer stem cells.Methodology/Principal FindingsWe examined the roles of miR-34 in p53-mutant human pancreatic cancer cell lines MiaPaCa2 and BxPC3, and the potential link to pancreatic cancer stem cells. Restoration of miR-34 expression in the pancreatic cancer cells by either transfection of miR-34 mimics or infection with lentiviral miR-34-MIF downregulated Bcl-2 and Notch1/2. miR-34 restoration significantly inhibited clonogenic cell growth and invasion, induced apoptosis and G1 and G2/M arrest in cell cycle, and sensitized the cells to chemotherapy and radiation. We identified that CD44+/CD133+ MiaPaCa2 cells are enriched with tumorsphere-forming and tumor-initiating cells or cancer stem/progenitor cells with high levels of Notch/Bcl-2 and loss of miR-34. More significantly, miR-34 restoration led to an 87% reduction of the tumor-initiating cell population, accompanied by significant inhibition of tumorsphere growth in vitro and tumor formation in vivo.Conclusions/SignificanceOur results demonstrate that miR-34 may restore, at least in part, the tumor suppressing function of the p53 in p53-deficient human pancreatic cancer cells. Our data support the view that miR-34 may be involved in pancreatic cancer stem cell self-renewal, potentially via the direct modulation of downstream targets Bcl-2 and Notch, implying that miR-34 may play an important role in pancreatic cancer stem cell self-renewal and/or cell fate determination. Restoration of miR-34 may hold significant promise as a novel molecular therapy for human pancreatic cancer with loss of p53–miR34, potentially via inhibiting pancreatic cancer stem cells.

Recent advances in gene therapy for human pancreatic cancer

Recent advances in gene therapy for human pancreatic cancer

Inhibition of PDGFR phosphorylation and Src and Akt activity by GN963 leads to therapy of human pancreatic cancer growing orthotopically in nude mice

GN963 is a tyrosine kinase inhibitor with activity against platelet-derived growth factor receptor (PDGFR) and Src kinases. We determined whether oral administration of GN963, alone or in combination with gemcitabine produces therapy against L3.6pl human pancreatic cancer cells growing orthotopically in nude mice. The optimal biological dosage of oral GN963 was determined to be 100 mg/kg every 48 h. Seven days after injection of L3.6pl cells into the pancreas of nude mice, mice (n=10) were treated with vehicle (control), thrice-weekly oral GN963 (100 mg/kg), twice-weekly intraperitoneal gemcitabine (100 mg/kg), or GN963 plus gemcitabine. Treatment with gemcitabine did not significantly differ from control. In contrast, treatment with GN963 (100 mg/kg) or GN963 plus gemcitabine produced a 52% and 81% decrease in tumor volume, respectively. GN963 plus gemcitabine completely inhibited the incidence of liver metastasis. Administration of GN963 inhibited PDGFR phosphorylation in both tumor and tumor-associated endothelial cells, decreased Src and Akt kinase activity in tumor cells, decreased microvessel density, and decreased tumor cell proliferation, while increasing apoptosis of tumor and tumor-associated endothelial cells. Collectively, these data indicate that targeting PDGFR, Src, and Akt on tumor and tumor-associated endothelial cells may be an effective therapy for human pancreatic carcinoma.

Combination therapy of human pancreatic cancer implanted in nude mice by oral fluoropyrimidine anticancer agent (S-1) with interferon-alpha

We evaluated the antitumor and antiangiogenic activities of human natural interferon-alpha (IFN-alpha) alone or in combination with S-1 against human pancreatic cancer cells. Three days after the subcutaneous (s.c.) implantation of tumor cells, mice (n = 12) were received s.c. injection with IFN-alpha alone (10,000 U six times a week), oral administration with S-1 alone (8 mg/kg six times a week), or both with IFN-alpha and S-1 (8, 10, 12 mg/kg six times a week). Administration of IFN-alpha in combination with S-1 significantly decreased progressive growth and angiogenesis of human pancreatic cancer cells. The combination therapy produced more significant inhibition in expression of the representative proangiogenic molecules, vascular endothelial growth factor and basic fibroblast growth factor than individual treatment either IFN-alpha or S-1 alone did. These treatments also decreased the staining of proliferating cell nuclear antigen, induced apoptosis and decreased microvessel density. In order to better understand the precise molecular mechanisms by which IFN-alpha and S-1 exert its effects, we have utilized cDNA microarray including 124 known genes to determine the gene expression profile altered by IFN-alpha and S-1 treatment. We found a total of seven genes which showed a twofold change after IFN-alpha and S-1 treatment in addition to VEGF, bFGF, CD31, MMP-2, MMP-7 and MMP-9. Among these genes, we found down-regulation of six genes and up-regulation of one gene, which are related to angiogenesis, tumor cell invasion and metastasis. These data suggest that administration of IFN-alpha in combination with S-1 may provide a novel and effective approach to the treatment of human pancreatic cancer.

Signal therapy of human pancreatic cancer and NF1-deficient breast cancer xenograft in mice by a combination of PP1 and GL-2003, anti-PAK1 drugs (Tyr-kinase inhibitors)

The majority of cancers are caused by mutations of a few signal transducers such as the GTPase RAS, the kinase Src and the tumor suppressor p53. Thus, a group of specific chemical compounds called ‘signal therapeutics’, that block or reverse selectively these abnormally activated signaling pathways would be very useful for the treatment of these signally disordered cancers. More than 90% of human pancreatic cancers are associated with oncogenic mutations of RAS, in particular K-RAS at codon 12. We have previously shown that, PAK1, the Rac/CDC42-dependent Ser/Thr kinase, is essential for RAS/estrogen-induced transformation and neurofibromatosis (NF). Furthermore, we and others have demonstrated that the growth of mouse RAS-induced sarcomas allografts in mice is almost completely suppressed by either FK228 or a combination of two complimentary Tyr-kinase inhibitors, PP1 and AG 879, all of which block the RAS-induced activation of PAK1. Since, so far no effective therapeutic is available for the treatment of pancreatic cancer patients, we have examined the therapeutic potential of either FK228, the combination of these two Tyr-kinase inhibitors or GL-2003, a water-soluble derivative of AG 879, on human pancreatic cancer (Capan-1) xenograft in mice. Among these PAK1-blocking approaches, the PP1/GL-2003 combination is the most effective in the therapy of this cancer xenograft model. Its therapeutic potential is equivalent to those of gemcitabine and kigamicin D which suppress by 70–80% the growth of a similar human pancreatic cancer xenograft model. Also, this PP1/GL-2003 combination therapy has been proven to be very effective to suppress the estrogen-independent growth of an NF1-deficient multidrug/FK228-resistant human breast cancer (MDA-MB-231) xenograft in mice.

Transcriptional anti-angiogenesis therapy of human pancreatic cancer

Pancreatic cancer angiogenesis has been attributed to genetic and epigenetic alterations (e.g., oncogene activation and suppressor inactivation) and a chaotic tumor microenvironment (e.g., hypoxia, acidosis, free radical stress and imbalanced growth factor production). Those diverse “upstream signal” factors appear to converge their signaling pathways on limited sets of nuclear transcription factors (e.g., Sp1, Stat3 and NF-κB). Aberrant activities of these factors confer a tremendous survival and growth advantage to existing and/or emerging malignant cells through alteration of the expression and functions of their diverse “downstream effector” factors (e.g., VEGF and IL-8). Therefore, targeting a single transcription factor can affect the malignant phenotype more profoundly than just targeting any single upstream signal and/or downstream effector factor.

Modulation of drug cytotoxicity by reintroduction of wild-type p53 gene (Ad5CMV-p53) in human pancreatic cancer.

Chemotherapy does not significantly improve prognosis in pancreatic cancer. New therapeutical approaches involving p53 gene replacement appear to be very encouraging due to the key role of p53 in the cell response to DNA damage. Here, we have evaluated the effectiveness of combining wild-type p53 (wt-p53) gene reintroduction (Ad5CMV-p53) and exposure to two genotoxic drugs, gemcitabine and cisplatin, in several human pancreatic cell lines. The efficiency of the combinations was clearly dependent upon timing, as assessed by cell survival determinations. Although wt-p53 transduction before drug treatment induced chemoresistance, p53 transduction in cells treated previously with gemcitabine increased cytotoxicity. Cell cycle profiles showed significant decreases in the percentage of cells in the S phase as a consequence of arrests provoked by the expression of exogenous p53, reducing the number of cells susceptible to the drug. The sensitivity of cells to cisplatin, which has a lower degree of S-phase specificity, was not modified as much by p53 gene replacement. In contrast, the recognition of the previous drug-induced DNA damage by the newly expressed wt-p53 elicited increases in sub-G1 populations, consistent with the annexin determinations and bax/bcl-2 ratios observed. Experiments on subcutaneous pancreatic xenografts corroborated the effectiveness of this approach in vivo. Thus, the combination of p53 transduction and chemotherapy, under a correct schedule of administration, appears to be a very promising therapy for human pancreatic cancer.

26S Proteosome inhibition induces apoptosis and activation of p21 Cip1-Waf-1 in human pancreatic cancer

Intact 26S proteosome activity promotes the transcription of anti-apoptotic genes. New highly selective 26S proteosome inhibitors are becoming available for testing against human malignancies. We examined whether selective inhibition of the 26S proteosome alone alters proliferation and apoptosis in human pancreatic cancer. Methods:BxPC-3 human pancreatic cancer cells were serum starved for 60 hours. Cells were repleted with media containing 10% serum (FCS) alone or combined with the boronic acid inhibitor PS-341 (loonM; Leukosite, Inc., Cambridge, MA). Proliferation was measured by MTT assay. Apoptosis was determined by FACS analysis (sub-Go peak) and nuclear morphology (Pap stain). Data were comgared by two-tailed t-test (*p<0.05). Activation of caspase-3 and p21 ,pl-Waf-I by lOOnM PS-341 were determined by Western blot. Results: Proteosome inhibition with PS-341 significantly blocked mitogen (FCS) induced BxPC-3 cell proliferation. PS-341 alone caused apoptosis to occur as indicated by FACS analysis and nuclear morphology. This corresponds with activation of p21 within 3 hours (Fig), and subsequent activation of caspase-3 within 12 hours (Western not shown). Conclusions:In BxPC-3 pancreatic cancer cells, targeted inhibition (PS-341) of the 26S proteosome alone blocks proliferation and induces apoptosis as pro-apoptotic kinases become activated. These novel findings support further study of 26S proteosome inhibition in the therapy of human pancreatic cancer.

Adenovirus-mediated wild-type p53 tumor suppressor gene therapy induces apoptosis and suppresses growth of human pancreatic cancer [seecomments

The p53 tumor suppressor gene is mutated in up to 70% of pancreatic adenocarcinomas. We determined the effect of reintroduction of the wild-type p53 gene on proliferation and apoptosis in human pancreatic cancer cells using an adenoviral vector containing the wild-type p53 tumor suppressor gene. Transduction efficiencies of six p53-mutant pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, CFPAC-1, MIA PaCa-2, and PANC-1) were determined using the reporter gene construct Ad5/CMV/beta-gal. Cell proliferation was monitored using a 3H-thymidine incorporation assay, Western blot analysis for p53 expression was performed, and DNA laddering and fluorescence-activated cell sorter analysis were used to assess apoptosis. p53 gene therapy was tested in vivo in a subcutaneous tumor model. The cell lines varied in transduction efficiency. The MIA PaCa-2 cells had the highest transduction efficiency, with 65% of pancreatic tumor cells staining positive for beta-galactosidase (beta-gal) at a multiplicity of infection (MOI) of 50. At the same MOI, only 15% of the CFPAC-1 cells expressed the beta-gal gene. Adenovirus-mediated p53 gene transfer suppressed growth of all human pancreatic cancer cell lines in a dose-dependent manner. Western blot analysis confirmed the presence of the p53 protein product at 48 hours after infection. DNA ladders demonstrated increased chromatin degradation, and fluorescence-activated cell sorter analysis demonstrated a four-fold increase in apoptotic cells at 48 and 72 hours following infection with Ad5/CMV/p53 in the MIA PaCa-2 and PANC-1 cells. Suppression of tumor growth mediated by induction of apoptosis was observed in vivo in an established nude mouse subcutaneous tumor model following intratumoral injections of Ad5/CMV/p53. Introduction of the wild-type p53 gene using an adenoviral vector in pancreatic cancer with p53 mutations induces apoptosis and inhibits cell growth. These data provide preliminary support for adenoviral mediated p53 tumor suppressor gene therapy of human pancreatic cancer.

Effects of new bombesin antagonists given singly or in combination with a somatostatin analog on nitrosamine-induced pancreatic cancers in hamsters

In three experiments, hamsters with N-nitroso-bis(2-oxopropyl)amine-induced pancreatic cancers were treated for two months with bombesin/GRP antagonists RC-3095 [D-Tpi(6),Leu(13)psi(CH2NH)Leu(14)-bombesin(6-14)], RC-3910-II [D-Tpi(6),Leu(13)psi(CH2N)Tac(14)-bombesin(6-14)], RC-3940-II [Hca(6),Leu(13)psi(CH2N)Tac(14)-bombesin(6-14)], RC-3950-II [D-Phe(6),Leu(13)psi(CH2N)Tac(14)-bombesin(6-14)], somatostatin analog RC-160 (D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2), or the combination of RC-3095 with RC160. All peptides inhibited pancreatic cancers to various degrees, reducing the number of tumorous animals, lowering the weight of tumorous pancreata by 40-55% and decreasing AgNOR numbers which are indicators of cell proliferation rate. Combination therapy with RC-3095 and RC-160 did not inhibit tumors better than single peptides. Among new bombesin/GRP antagonists, RC-3940-II had the strongest inhibitory effect. RC-3950-II and RC-3095 caused similar inhibition, but RC-3910-II was less effective. Tumor inhibitory activity of the bombesin/GRP antagonists was correlated with their binding affinities to bombesin receptors on tumor cells. RC-3940-II caused 50% inhibition of specific binding of [I-125-Tyr(4)]bombesin to tumor cell membranes at 0.96 nM concentration, while the IC50 for RC-3950-II was 5.27 nM and 12.94 nM for RC-3095. Our findings suggest that in addition to RC-3095, other bombesin/GRP antagonists such as RC-3950-II and especially RC-3940-II could be further developed for therapy of human pancreatic cancer.

The effect of the somatostatin analogue SMS 201-995 on experimental pancreatic carcinogenesis in the Syrian golden hamster

Somatostatin analogues have been suggested as possible therapy for human pancreatic cancer. This paper investigates the effect of the somatostatin analogue SMS 201-995 (Sandoz) in the Syrian golden hamster model of nitrosamine-induced pancreatic carcinogenesis. Step-wise increasing doses of i.v. SMS 201-995 suppressed pancreatic juice output from a median basal value of 212 mg/kg body wt/h (Q1:Q3 = 121:334) to a median basal value of 70 mg/kg body wt/h during infusion of 5 micrograms/kg body wt/h of SMS 201-995 (Q1:Q3 = 64:102, P less than 0.05). Chronic s.c. injection of 5 and 10 micrograms/kg body wt SMS 201-995 twice daily for 3 days each week, did not affect pancreatic wet wt or pancreatic total DNA content after 1 or 6 weeks of treatment when compared to controls. The most interesting and unexpected finding in our study was that SMS 201-995 seemed to promote pancreatic carcinogenesis when administered in low dosage. More SMS 201-995 treated animals receiving 5 micrograms/kg body wt developed invasive pancreatic adenocarcinoma than controls after 15 weeks of carcinogen (4/10 animals versus 0/10, P less than 0.05, Fisher's exact test) and pancreatic involvement by tumour was more extensive (17/75 pancreatic blocks affected versus 0/71, P less than 0.001). When carcinoma in situ and microcarcinomata were analysed with invasive lesions, animals injected with 5 micrograms/kg body wt SMS 201-995 were still significantly more affected than controls (33/75 blocks versus 9/71, P less than 0.001). Hamsters injected with the higher SMS 201-995 dose (10 micrograms/kg body wt) did not show any increase in malignancy over the controls. These results suggest that the effect of SMS 201-995 on pancreatic carcinogenesis in the Syrian hamster is complex and varies with dose administered. Further work is required before its use on man can be justified.