Synergistic antitumor activity of ZD6474, an inhibitor of vascular endothelial growth factor receptor and epidermal growth factor receptor signaling, with gemcitabine and ionizing radiation against pancreatic cancer.

PURPOSE: Standard treatments have modest effect against pancreatic cancer, and current research focuses on agents targeting molecular pathways involved in tumor growth and angiogenesis. This study investigated the interactions between ZD6474, an inhibitor of tyrosine kinase activities of vascular endothelial growth factor receptor-2 and epidermal growth factor receptor (EGFR), gemcitabine, and ionizing radiation in human pancreatic cancer cells and analyzed the molecular mechanisms underlying this combination. EXPERIMENTAL DESIGN: ZD6474, ionizing radiation, and gemcitabine, alone or in combination, were given in vitro to MIA PaCa-2, PANC-1, and Capan-1 cells and in vivo to MIA PaCa-2 tumor xenografts. The effects of treatments were studied by the evaluation of cytotoxicity, apoptosis, cell cycle, EGFR and Akt phosphorylation, modulation of gene expression of enzymes related to gemcitabine activity (deoxycytidine kinase and ribonucleotide reductase), as well as vascular endothelial growth factor immunohistochemistry and microvessel count. RESULTS: In vitro, ZD6474 dose dependently inhibited cell growth, induced apoptosis, and synergistically enhanced the cytotoxic activity of gemcitabine and ionizing radiation. Moreover, ZD6474 inhibited phosphorylation of EGFR and Akt and triggered cell apoptosis. PCR analysis showed that ZD6474 increased the ratio between gene expression of deoxycytidine kinase and ribonucleotide reductase. In vivo, ZD6474 showed significant antitumor activity alone and in combination with radiotherapy and gemcitabine, and the combination of all three modalities enhanced MIA PaCA-2 tumor growth inhibition compared with gemcitabine alone. CONCLUSIONS: ZD6474 decreases EGFR and Akt phosphorylation, enhances apoptosis, favorably modulates gene expression in cancer cells, and acts synergistically with gemcitabine and radiotherapy to inhibit tumor growth. These findings support the investigation of this combination in the clinical setting.     

In vitro synergistic cytotoxicity of gemcitabine and pemetrexed and pharmacogenetic evaluation of response to gemcitabine in bladder cancer patients

The present study was performed to investigate the capability of gemcitabine and pemetrexed to synergistically interact with respect to cytotoxicity and apoptosis in T24 and J82 bladder cancer cells, and to establish a correlation between drug activity and gene expression of selected genes in tumour samples. The interaction between gemcitabine and pemetrexed was synergistic; indeed, pemetrexed favoured gemcitabine cytotoxicity by increasing cellular population in S-phase, reducing Akt phosphorylation as well as by inducing the expression of a major gemcitabine uptake system, the human equilibrative nucleoside transporter-1 (hENT1), and the key activating enzyme deoxycytidine kinase (dCK) in both cell lines. Bladder tumour specimens showed an heterogeneous gene expression pattern and patients with higher levels of dCK and hENT1 had better response. Moreover, human nucleoside concentrative transporter-1 was detectable only in 3/12 patients, two of whom presented a complete response to gemcitabine. These data provide evidence that the chemotherapeutic activity of the combination of gemcitabine and pemetrexed is synergistic against bladder cancer cells in vitro and that the assessment of the expression of genes involved in gemcitabine uptake and activation might be a possible determinant of bladder cancer response and may represent a new tool for treatment optimization.     

Human pancreatic cancer cells express non-functional Fas receptors and counterattack lymphocytes by expressing Fas ligand; a potential mechanism for immune escape

The aim of this study was to investigate the expression and functional status of Fas ligand (FasL) and its receptor (Fas) in human pancreatic cancers. Using RT-PCR and Western blotting, Fas and FasL were expressed in seven surgically resected pancreatic cancer specimens and five cell lines; Capan-1, AsPC-1, BxPC-3, PANC-1, and MIA PaCa-2. In the resected specimens, pancreatic cancer cells induced apoptosis in the surrounding lymphoid cells. In coculture experiments of pancreatic cancer and Jurkat T cells, 50% of Jurkat T cells underwent apoptosis after 2 days, however, almost all pancreatic cancer cells remained viable. In addition, by testing Fas function using anti-Fas antibody (CH11), all cell lines were resistant to Fas-mediated apoptosis except Capan-1 cells which showed sensitivity similar to that of Jurkat T cells. These results suggest that pancreatic cancer cells evade immune surveillance by expression of FasL and non-functioning Fas that allow them to activated T-cells. These tumor escape mechanisms may contribute to the rapid fatal course of pancreatic cancer.     

CNT1 expression influences proliferation and chemosensitivity in drug-resistant pancreatic cancer cells

Overcoming the inherent chemoresistance of pancreatic cancers remains a major goal of therapeutic investigations in this disease. In this study, we discovered a role for the human concentrative nucleoside transporter-1 (hCNT1; SLC28A1), a high-affinity pyrimidine nucleoside transporter, in determining the chemosensitivity of human pancreatic cancer cells to gemcitabine, the drug used presently as a standard of care. Compared with normal pancreas and pancreatic ductal epithelial cells, hCNT1 expression was frequently reduced in pancreatic tumors and tumor cell lines. In addition, hCNT1-mediated (3)H-gemcitabine transport was lower in pancreatic cancer cell lines and correlated with cytotoxic IC(50) estimations of gemcitabine. In contrast to gemcitabine-sensitive pancreatic cancer cell lines, MIA PaCa-2, a gemcitabine-resistant pancreatic cancer cell line, exhibited relatively restrictive, cell cycle-dependent hCNT1 expression and transport. hCNT1 translation was suppressed in the late G1-enriched MIA PaCa-2 cell population possibly in an miRNA-dependent manner, which corresponded with the lowest hCNT1-mediated gemcitabine transport during this phase. Although hCNT1 protein was induced during G1/S transition, increased hCNT1 trafficking resulted in maximal cell surface recruitment and transport-overshoot in the G2/M phase-enriched cell population. hCNT1 protein was directed predominantly to proteasomal or lysosomal degradation in S or G2/M phase MIA PaCa-2 cells, respectively. Pharmacological inhibition of hCNT1 degradation moderately increased cell surface hCNT1 expression and cellular gemcitabine transport in MIA PaCa-2 cells. Constitutive hCNT1 expression reduced clonogenic survival of MIA PaCa-2 cells and steeply augmented gemcitabine transport and chemosensitization. In addition to supporting a putative tumor suppressor role for hCNT1, our findings identify hCNT1 as a potential candidate to render drug-resistant pancreatic cancer cells amenable to chemotherapy.     

PPARgamma ligand (thiazolidinedione) induces growth arrest and differentiation markers of human pancreatic cancer cells

The aim of this study was to examine whether a specific PPARgamma ligand can inhibit the growth of human pancreatic cancer cells through induction of terminal differentiation. PPARgamma was expressed in five human pancreatic cancer cell lines: Capan-1, AsPC-1, BxPC-3, PANC-1, and MIA PaCa-2. Treatment of these cells with a specific PPARgamma ligand, thiazolidinedione (TZD), resulted in inhibition of both cellular and clonogenic growth, and G1 cell cycle arrest. Finally, thiazolidinedione treatment resulted in induction of p21WAF-1 and increased expression of differentiation markers. These results suggest that thiazolidinedione treatment inhibits growth and induces cellular differentiation in pancreatic cancer cells and thereby reduces their development in favor of differentiated and stable cell phenotype.     

Enhancement of gemcitabine sensitivity in pancreatic cancer by co-regulation of dCK and p8 expression

The purpose of this study was to improve the gemcitabine sensitivity in pancreatic cancer by adenovirus-mediated co-regulation of dCK and p8 expression. Firstly, we analyzed the sensitivity of three human pancreatic tumor cell lines (Capan-2, Panc-1 and BxPc-3) to gemcitabine using MTT assays, and found Panc-1 to be relatively resistant to gemcitabine. Further, we investigated the expression of dCK and p8 in different pancreatic cancer cell lines using real-time PCR and Western blot analysis, and found that the expression levels of these two genes were related to the gemcitabine sensitivity of pancreatic cancer cells. We constructed recombinant adenovirus vectors, Ad-dCK and Ad-p8-siRNA, that overexpressed dCK and knocked down p8, respectively. Using MTT assays, we observed that combined infection using Ad-dCK and Ad-p8-siRNA in vitro led to a significant decrease in the gemcitabine IC50 with an increase in apoptosis and caspase-3 activity in Panc-1 cells, which are relatively resistant to gemcitabine. Furthermore, in established subcutaneous pancreatic cancer models in nude mice, the tumor inhibition was markedly enhanced accompanied by elevation of the apoptosis index after intratumoral injection of Ad-dCK and Ad-p8-siRNA on the basis of intraperitoneal gemcitabine chemotherapy. Taken together, the present findings suggest that, dCK and p8 may be the important factors in the regulation of gemcitabine sensitivity in pancreatic cancer cells. Moreover, co-regulation of the two factors achieved better effects than regulation of either one alone.     

Mesothelin regulates growth and apoptosis in pancreatic cancer cells through p53-dependent and -independent signal pathway

Mesothelin, a secreted protein, is overexpressed in some cancers, including pancreatic cancer. Rescent studies have shown that overexpression of mesothelin significantly increased tumor cell proliferation, and downregulation of mesothelin inhibited cell proliferation in pancreatic cancer cells, but its exact function and mechanism remains unclear. The aim of the present study was to evaluate the effects of mesothelin on proliferation and apoptosis in pancreatic cancer cells with different p53 status and to explore its signal pathway. Mesothelin levels were detected by western blot and RT-PCR assay in human pancreatic cancer AsPC-1, HPAC and Capan-2, Capan-1 and MIA PaCa-2 cell lines. Mesothelin was slienced by shRNA in AsPC-1, Capan-2 and Capan-1 cells with rich mesothelin level, and mesothelin was overexpressed in the HPAC and Capan-2 cells with less mesothelin level. We observed that in the AsPC-1 and Capan-1cells with mt-p53, and Capan-2 cells with wt-p53, shRNA mediated sliencing of the mesothelin significantly increased PUMA and Bax expression and caspase-3 activity, and decreased bcl-2 expression, followed by the reduced proliferation and colony forming capability and increased cell apoptosis. When PUMA was slienced by siRNA in the stable mesothelin shRNA transfected cells, proliferative capability was significantly increased, and apoptosis was decreased. However, in the Capan-2 cells with wt-p53, suppression of the mesothelin significantly increased wt-p53 levels. When p53 was blocked by siRNA in the stable mesothelin shRNA transfected Capan-2 cells, PUMA was inhibited, followed by increased proliferative capability and decreased cell apoptosis. In the HPAC and Capan-2 cells with wt-p53 and in the MIA PaCa-2 cells with mt-p53, overexpression of the mesothelin significantly decreased bax levels and increased bcl-2 levels, followed by increased proliferative and colony forming capability. Furthermore, mesothelin-shRNA-transfected cells exhibited a reduced rate of tumor growth under in vivo conditions. However, mesothelin-transfected cells exhibited a increased rate of tumor growth under in vivo conditions. Our data demonstrated that mesothelin promotes proliferation and inhibited apoptosis through p53-dependent pathway in pancreatic cancer cells with wt-p53, and p53-independent pathway in pancreatic cancer cells with mt-p53. Targeting mesothelin by shRNA is the important method for pancreatic cancer therapy.     

miR-130a对胰腺癌细胞系PANC-1细胞增殖和凋亡的调控作用及其 机制探讨

目的:研究miR-130a对胰腺癌细胞系PANC-1细胞增殖和凋亡的影响，并探讨其机制。方法:体外培养胰腺癌细胞PANC-1、SW 1990、MIA PaCa-2和正常胰腺上皮细胞HPDE6-C7，检测各细胞中miR-130a表达水平。将PANC-1细胞分为miR-130a低表达组（miR-130a-inhibitor组）、阴性对照组（miR-130a-NC组）和空白对照组（miR-130a-BC组）。转染48 h后，CCK-8试验检测细胞增殖情况；裸鼠皮下成瘤实验检测miR-130a对肿瘤体内生长的影响；流式细胞术检测细胞凋亡情况；TargetScan数据库预测miR-130a的靶基因，并采用蛋白免疫印迹（WB）和荧光素酶报告实验进行验证。结果:PANC-1、SW 1990、MIA PaCa-2人胰腺癌细胞中miR-130a表达水平均显著高于正常胰腺上皮细胞，差异有统计学意义（P<0.05）。转染miR-130a-inhibitor后，PANC-1细胞miR-130a相对表达量显著下调（P<0.05）；与miR-130a-NC和miR-130a-BC组比较，miR-130a-inhibitor组PANC-1细胞增殖能力显著下降（P<0.05）、裸鼠皮下肿瘤体积明显减小（P<0.05）、细胞凋亡率显著升高（P<0.05）。TargetScan数据库显示FOS样抗原1（FOSL1）是miR-130a潜在靶基因，WB和双荧光素酶报告实验证实FOSL1是miR-130a的作用靶点。结论:下调miR-130a表达通过作用于FOSL1基因抑制PANC-1细胞增殖，促进其凋亡，可能为胰腺癌的临床治疗提供新思路。     

BRG1 promotes chemoresistance of pancreatic cancer cells through crosstalking with Akt signalling

Gemcitabine is a standard chemotherapeutic agent for locally advanced and metastatic pancreatic cancer. However, the chemoresistance of pancreatic cancer is the major barrier to efficient chemotherapy. Here, we reported that BRG1, a chromatin modulator, was exclusively overexpressed in human pancreatic ductal adenocarcinoma tissues. BRG1 knockdown inhibited PANC-1 and MIA PaCa-2 cell growth in vitro and in vivo, reduced the phosphorylation/activation of Akt and p21^cip/waf, enhanced intrinsic and gemcitabine induced apoptosis and attenuated gemcitabine-induced downregulation of E-cadherin. Moreover, by establishing acquired chemoresistance of MIA PaCa-2 cells in vitro, we found that BRG1 knockdown effectively reversed the chemoresistance to gemcitabine. Surprisingly, inhibiting Akt phosphorylation resulted in BRG1 suppression in pancreatic cancer cells, indicating BRG1 as a new downstream target of Akt signalling. Taken together, our findings suggest that BRG1 promotes both intrinsic and acquired chemoresistance of pancreatic cancer cells, and BRG1 crosstalks with Akt signalling to form a positive feedback loop to promote pancreatic cancer development.     

Human pancreatic cancer cells disable function of Fas receptors at several levels in Fas signal transduction pathway

The aims of this study were to evaluate the functional expression of Fas receptors (Fas) in human pancreatic cancer cell lines; Capan-1, AsPC-1, BxPC-3, PANC-1, and MIA PaCa-2 and to search for the mechanisms of receptor-mediated inhibition of Fas signaling in these cells. Despite the expression of Fas receptors at considerable levels, exposure of these cells to agonistic Fas antibodies (500 ng/ml) induced only minimal apoptosis in 4 cell lines. The mechanisms allowing resistance to Fas-mediated apoptosis are complex. Using RT-PCR, we identified molecules which might counteract the apoptogenic signal at several levels of Fas signal transduction pathway. The most striking findings were the overexpression of Fas decoy receptors (DcR3), Fas associated phosphatase-1 (FAP-1), and FLICE-inhibitory protein (c-FLIP) in the resistant cell lines as well as in pancreatic cancer surgical specimens. In conclusion, pancreatic cancer cells express three molecules that can abrogate Fas function at different levels of Fas signaling cascade, resulting in resistance to Fas-mediated apoptosis, and this may promote the progression of this malignancy.     

Suppression of human pancreatic cancer cell proliferation by AGN194204, an RXR-selective retinoid

Retinoids may be useful agents for the treatment of pancreatic cancer. However, retinoic acid receptor (RAR)-selective retinoids produce unwanted side effects. In contrast, retinoid X receptor (RXR)-selective retinoids produce fewer side effects; however, it was not known whether RXR-selective retinoids could reduce pancreatic tumor cell proliferation. In the present study, the novel RXR-selective retinoid, AGN194204, was compared with that of other retinoids for the ability to suppress pancreatic cancer cell proliferation. We treated various pancreatic cancer cell lines with receptor-selective ligands and cytotoxic agents and monitored the effects on cell proliferation, markers of apoptosis and cell cycle. Our results indicate that AGN194204, at concentrations >10 nM, inhibits proliferation of MIA PaCa-2 and BxPC-3 cells but not the proliferation of AsPC-1 cells. Moreover, in BxPC-3 and MIA PaCa-2 cells, AGN194204 was 10-100 times more effective than RAR-selective retinoids. AGN194204-dependent suppression of MIA PaCa-2 cell proliferation is associated with reduced cyclin E and cyclin-dependent kinase 6 (cdk6) level, but cyclin D1, cdk2 and cdk4 content is not altered. In addition, p27 level increases 2-fold. The RXR-selective antagonist, AGN195393, reverses the AGN194204-dependent growth inhibition and the decline in cyclin E and cdk6 levels. In contrast, these changes are not reversed by treatment with the RAR antagonist, AGN193109. AGN194204 did not appear to alter cell apoptosis as measured by change in cleavage of procaspase-3, -8 or -9. We also examined the effects AGN194204 co-treatment with cytotoxic agents. Treatment of MIA PaCa-2 cells with AGN194204 + cisplatin, gemcitabine, 5-fluorouracil, interferon (IFN)alpha or IFNgamma resulted in an additive but not synergistic reduction in MIA PaCa-2 cell number. These results indicate that AGN194204, an RXR-selective retinoid, is a more effective inhibitor of pancreatic cell proliferation than the RAR-selective retinoids, and further indicate that AGN194204 produces an additive reduction in cell number when given with other agents. Our results suggest that RXR-selective ligands, which are less toxic than RAR-selective ligands, may be suitable agents for the treatment of pancreatic cancer.     

Interaction between gemcitabine and topotecan in human non-small-cell lung cancer cells: effects on cell survival, cell cycle and pharmacogenetic profile

The pyrimidine analogue gemcitabine is an established effective agent in the treatment of non-small-cell lung cancer (NSCLC). The present study investigates whether gemcitabine would be synergistic with the topoisomerase I inhibitor topotecan against the NSCLC A549 and Calu-6 cells. Cells were treated with gemcitabine and topotecan for 1 h and the type of drug interaction was assessed using the combination index (CI). Cell cycle alterations were analysed by flow cytometry, while apoptosis was examined by the occurrence of DNA internucleosomal fragmentation, nuclear condensation and caspase-3 activation. Moreover, the possible involvement of the PI3K-Akt signalling pathway was investigated by the measurement of Akt phosphorylation. Finally, quantitative, real-time PCR (QRT-PCR) was used to study modulation of the gemcitabine-activating enzyme deoxycytidine kinase (dCK) and the cellular target enzyme ribonucleotide reductase (RR). In results, it was found that simultaneous and sequential topotecan --> gemcitabine treatments were synergistic, while the reverse sequence was antagonistic in both cell lines. DNA fragmentation, nuclear condensation and enhanced caspase-3 activity demonstrated that the drug combination markedly increased apoptosis in comparison with either single agent, while cell cycle analysis showed that topotecan increased cells in S phase. Furthermore, topotecan treatment significantly decreased the amount of the activated form of Akt, and enhanced the expression of dCK (+155.0 and +115.3% in A549 and Calu-6 cells, respectively), potentially facilitating gemcitabine activity. In conclusion, these results indicate that the combination of gemcitabine and topotecan displays schedule-dependent activity in vitro against NSCLC cells. The gemcitabine --> topotecan sequence is antagonistic while drug synergism is obtained with the simultaneous and the sequential topotecan --> gemcitabine combinations, which are associated with induction of decreased Akt phosphorylation and increased dCK expression.     

Combination chemotherapy with gemcitabine and biotherapy with opioid growth factor (OGF) enhances the growth inhibition of pancreatic adenocarcinoma

Gemcitabine is the standard of care for advanced pancreatic neoplasia, and exerts its effect through inhibition of DNA synthesis. However, gemcitabine has limited survival benefits. Opioid growth factor (OGF) is an autocrine-produced peptide that interacts with the nuclear receptor, OGFr, to inhibit cell proliferation but is not cytotoxic or apoptotic. The present study was designed to examine whether a combination of chemotherapy with gemcitabine and biotherapy with OGF is more effective than either agent alone in inhibiting pancreatic cancer growth in vitro and in vivo. The combination of OGF (10^–6 M) and gemcitabine (10^–8 M) reduced MIA PaCa-2 cell number from control levels by 46% within 48 h, and resulted in a growth inhibition greater than that of the individual compounds. OGF in combination with 5-fluorouracil also depressed cell growth more than either agent alone. The action of OGF, but not gemcitabine, was mediated by a naloxone-sensitive receptor, and was completely reversible. OGF, but no other endogenous or exogenous opioids, altered pancreatic cancer growth in tissue culture. The combination of OGF and gemcitabine also repressed the growth of another pancreatic cancer cell line, PANC-1. MIA PaCa-2 cells transplanted into athymic mice received 10 mg/kg OGF daily, 120 mg/kg gemcitabine every 3 days; 10 mg/kg OGF daily and 120 mg/kg gemcitabine every 3rd day, or 0.1 ml of sterile saline daily. Tumor incidence, and latency times to tumor appearance, of mice receiving combined therapy with OGF and gemcitabine, were significantly decreased from those of the control, OGF, and gemcitabine groups. Tumor volumes in the OGF, gemcitabine, and OGF/gemcitabine groups were markedly decreased from controls beginning on days 14, 12, and 8, respectively, after tumor cell inoculation. Tumor weight and tumor volume were reduced from control levels by 36–85% in the OGF and/or gemcitabine groups on day 45 (date of termination), and the group of mice exposed to a combination of OGF and gemcitabine had decreases in tumor size of 70% and 63% from the OGF or the gemcitabine alone groups, respectively. This preclinical evidence shows that combined chemotherapy (e.g. gemcitabine) and biotherapy (OGF) provides an enhanced therapeutic benefit for pancreatic cancer.     

Ursolic acid inhibits growth and induces apoptosis in gemcitabine-resistant human pancreatic cancer via the JNK and PI3K/Akt/NF-κB pathways

Pancreatic cancer is one of the most deadly carcinomas worldwide. Although gemcitabine as the standard chemotherapy agent has been proven to be effective, the response rate remains at 5.4% and the 5-year survival rate is extremely poor. Ursolic acid (UA) is a small molecule compound extracted from Chinese herbs as well as edible vegetables and a well-known anti-inflammatory and immunosuppressive agent. Here, we show that UA has potential to be developed into an anti-neoplastic agent against gemcitabine-resistant pancreatic cancer and to explore its molecular mechanism of action. In vitro, we used three different malignancy grades of pancreatic resistant cancer cell lines including MIA PaCa-2, PANC-1 and Capan-1 to assess the antitumor effect of UA. We found that UA inhibited growth and induced apoptosis in a dose-dependent manner in all of the three pancreatic cancer cell lines. Both extrinsic and intrinsic pathways were found to be involved in apoptotic cascade. The potential signaling pathways are concerned with inactivation of the PI3K/Akt/NF-κB pathway and activation of the c-Jun-terminal kinase (JNK) pathway. The JNK inhibitor SP600125 partly abrogated the caspase-9 activation caused by UA. The Akt inhibitor LY294002 did not mimic the effect of UA on caspase-8 and -9, but inhibited the viability of MIA PaCa-2 cells to some extent. Equally, UA also overcame the chemoresistance in the chemoresistant endometrial and ovarian carcinoma cell lines (HEC-1A and OVCAR-3). Moreover, UA caused cytotoxicity to a nude mouse xenograft model in vivo. Therefore, our present data suggest that UA can act as a novel and potent therapeutic agent in gemcitabine-resistant pancreatic cancer and even as a promising candidate in other chemoresistant cancers.     

A preclinical evaluation of pemetrexed and irinotecan combination as second-line chemotherapy in pancreatic cancer

Gemcitabine (GEM)-based chemotherapy is regarded as the standard treatment of pancreatic adenocarcinoma, but yields a very limited disease control. Very few studies have investigated salvage chemotherapy after failure of GEM or GEM-containing chemotherapy and preclinical studies attempting to widen the therapeutic armamentarium, not including GEM, are warranted. MIA PaCa2, CFPAC-1 and Capan-1 pancreatic cancer cell lines were treated with GEM, fluouracil (5-FU), docetaxel (DCT), oxaliplatin (OXP), irinotecan (CPT-11), pemetrexed (PMX) and raltitrexed (RTX) as single agent. Pemetrexed, inducing apoptosis with IC50s under the Cmax in the three lines tested, appeared the most effective drug as single agent. Based on these results, schedule- and concentration-dependent drug interactions (assessed using the combination index) of PMX/GEM, PMX/DCT and PMX-CPT-11 were evaluated. The combinatory study clearly indicated the PMX and CPT-11 combination as the most active against pancreatic cancer. To confirm the efficacy of PMX-CPT-11 combination, we extended the study to a panel of 10 pancreatic cancer cell lines using clinically relevant concentrations (PMX 10 microM; CPT-11 1 microm). In eight of 10 lines, the PMX-CPT-11 treatment significantly reduced cell recovery and increased both the subG1 and caspase 3/7 fraction. After a 5-day wash out period, an increased fraction of subG1 and caspase3/7 persisted in PMX-CPT-11-pretreated cell lines and a significant reduction in the clonogenicity capacity was evident. Finally, in vivo, the PMX/CPT-11 combination showed the ability to inhibit xenograft tumours growth as second-line therapy after GEM treatment. The PMX and CPT-11 combination displays a strong schedule-independent synergistic cytotoxic activity against pancreatic cancer, providing experimental basis for its clinical testing as salvage chemotherapy in pancreatic cancer patients.     

Acquired resistance of pancreatic cancer cells towards 5-Fluorouracil and gemcitabine is associated with altered expression of apoptosis-regulating genes

OBJECTIVES: Resistance to chemotherapy is a major cause of treatment failure and poor prognosis in pancreatic cancer. Inasmuch as most effects of chemotherapeutic agents are mediated via the activation of apoptosis, the cytotoxic effects of gemcitabine and 5-fluorouracil (FU) in correlation with apoptosis-regulating genes in pancreatic cancer cell lines were analyzed. METHODS: The cytotoxic effects of 5-FU and gemcitabine in AsPC-1, Capan-1, Mia-PaCa-2 and T3M4 pancreatic cancer cell lines were assessed by growth assays, and mRNA expression levels of pro-apoptotic and anti-apoptotic genes of the Bcl-2 family were analyzed by RNAse protection assays. RESULTS: Pancreatic cancer cells displayed a wide range of responses towards 5-FU (IC(50) 0.22-4.63 microM) and gemcitabine (11.51-42.2 nM). After repeated treatment with 5-FU, the IC(50) values in Capan-1 and T3M4 cells increased 2.1- and 1.8-fold, respectively, compared to their parental cells. Following recurrent treatment with gemcitabine, the IC(50) values in Capan-1 cells increased significantly (1.5-fold, p < 0.01). RNase protection assay showed a negative correlation between bcl-x(L) and mcl-1 mRNA expression levels and the sensitivity to 5-FU and gemcitabine after 5-FU and gemcitabine treatment. The bax/bcl-2 ratio maintained relatively stable following 5-FU/gemcitabine treatment and reflected the chemotherapeutic sensitivity of these cell lines. CONCLUSIONS: These findings reveal that pancreatic cancer cell lines are generally resistant to 5-FU and are more sensitive towards gemcitabine. The bax/bcl-2 ratio is predictive of chemotherapy sensitivity, whereas bcl-x(L) and mcl-1 mRNA levels following repeated exposure to 5-FU or gemcitabine are associated with resistance to these drugs. These findings suggest that the activation of anti-apoptotic genes after repeated drug exposure contributes to chemoresistance of pancreatic cancer cells and that blockage of anti-apoptotic genes might enhance chemosensitivity in pancreatic cancer.     

Beta 2-microglobulin regulates amyloid precursor-like protein 2 expression and the migration of pancreatic cancer cells

Beta 2-microglobulin (β₂m) is a component of the major histocompatibility complex (MHC) class I molecule, which presents tumor antigens to T lymphocytes to trigger cancer cell destruction. Notably, β₂m has been reported as persistently expressed, rather than down regulated, in some tumor types. For renal cell and oral squamous cell carcinomas, β₂m expression has been linked to increased migratory capabilities. The migratory ability of pancreatic cancer cells contributes to their metastatic tendencies and lethal nature. Therefore, in this study, we examined the impact of β₂m on pancreatic cancer cell migration. We found that β₂m protein is amply expressed in several human pancreatic cancer cell lines (S2-013, PANC-1, and MIA PaCa-2). Reducing β₂m expression by short interfering RNA (siRNA) transfection significantly slowed the migration of the PANC-1 and S2-013 cancer cell lines, but increased the migration of the MIA PaCa-2 cell line. The amyloid precursor-like protein 2 (APLP2) has been documented as contributing to pancreatic cancer cell migration, invasiveness, and metastasis. We have previously shown that β₂m/HLA class I/peptide complexes associate with APLP2 in S2-013 cells, and in this study we also detected their association in PANC-1 cells but not MIA PaCa-2 cells. In addition, siRNA down regulation of β₂m expression diminished the expression of APLP2 in S2-013 and PANC-1 but heightened the level of APLP2 in MIA PaCa-2 cells, consistent with our migration data and co-immunoprecipitation data. Thus, our findings indicate that β₂m regulates pancreatic cancer cell migration, and furthermore suggest that APLP2 is an intermediary in this process.     

Antitumor effect of trastuzumab for pancreatic cancer with high HER-2 expression and enhancement of effect by combined therapy with gemcitabine.

PURPOSE: The purpose of the present study was to evaluate whether trastuzumab has antitumor effect against pancreatic cancer and whether this effect is concordant with levels of HER-2, which is reportedly overexpressed in pancreatic cancer. We also investigated whether the effect is potentiated in combined therapy with gemcitabine. EXPERIMENTAL DESIGN: Using immunohistochemistry and FACScan, we analyzed HER-2 expression in 16 pancreatic cancer cell lines. The in vitro antiproliferative effect of trastuzumab, alone and in combination with gemcitabine, was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The in vitro antibody-dependent cell-mediated cytotoxicity of trastuzumab was investigated by (51)Cr release assay. The in vivo antitumor effect of trastuzumab, alone and in combination with gemcitabine, was evaluated in nude mouse xenograft growth. The survival benefit was evaluated in a Capan-1 orthotopic implanted nude mouse model. RESULTS: HER-2 expression of 2+ or more was observed in 10 and of 3+ in 2 of the 16 cell lines. No in vitro growth-inhibitory effect of trastuzumab was found in any cell line, but trastuzumab induced antibody-dependent cell-mediated cytotoxicity in proportion to HER-2 expression level. Trastuzumab inhibited tumor growth in Capan-1 (HER-2: 3+) xenografts and prolonged survival in the orthotopic model. These effects were increased by combined therapy with gemcitabine. In contrast, trastuzumab exhibited no antitumor effect against PANC-1 (HER-2: 1+) or SW1990 (HER-2: 2+) xenografts. CONCLUSIONS: The antitumor effect of trastuzumab in pancreatic cancer with high HER-2 expression was shown in vitro and in vivo. Clinical application of trastuzumab is expected in pancreatic cancer with 3+ HER-2 expression.     

A novel humanized anti-human death receptor 5 antibody CS-1008 induces apoptosis in tumor cells without toxicity in hepatocytes.

BACKGROUND: The antitumor activity of CS-1008, a humanized agonistic anti-human death receptor (DR) 5 antibody, was investigated in preclinical models. Materials and methods: Cytotoxicity of CS-1008 was evaluated in a several human tumor cell lines as well as primary human hepatocytes in vitro. To evaluate antitumor efficacy, athymic nude mice were inoculated with human colorectal tumor COLO 205, pancreatic tumor MIA PaCa-2 or non-small-cell lung carcinoma NCI-H2122 and CS-1008 was i.v. administered. The combination effects of CS-1008 with gemcitabine or docetaxel (Taxotere) against MIA PaCa-2 or NCI-H2122 were evaluated in vivo, respectively. RESULTS: CS-1008 inhibited the growth of tumor cell lines with DR5 expression, including COLO 205, NCI-H2122, MIA PaCa-2 and renal cell adenocarcinoma ACHN in vitro with antibody cross-linkage. Using COLO 205, apoptosis induction was confirmed by annexin V staining. Weekly administration of CS-1008 resulted in the inhibition of COLO 205 tumor growth as well as MIA PaCa-2 in vivo. CS-1008 in combination with gemcitabine or docetaxel demonstrated enhanced antitumor activity against MIA PaCa-2 or NCI-H2122 cells, respectively. Unlike tumor necrosis factor-related apoptosis-inducing ligand, CS-1008 did not induce cell death in human primary hepatocytes. CONCLUSION: CS-1008 has a selective toxicity toward tumor cells expressing DR5 and the potential for antitumor efficacy in human malignancies.     

CDCA7通过与MCM3相互作用介导胰腺癌细胞增殖、侵袭及迁移

目的:探讨细胞分裂周期相关蛋白7(cell division cycle-associated protein 7,CDCA7)对人胰腺癌细胞增殖、侵袭及迁移能力的影响,及其可能的作用机制.方法:利用癌症基因组图谱(The Cancer Genome Atlas,TCGA)和基因表达谱数据动态分析(Gene Expre...