Genetic variation in APE1 gene promoter is associated with noise-induced hearing loss in a Chinese population.

OBJECTIVE: To investigate whether the apurinic/apyrimidinic endonuclease 1 (APE1) 1349 T>G and -656 T>G polymorphisms were associated with the risk of noise-induced hearing loss (NIHL) in a Chinese population. METHODS: The two APE1 polymorphisms were analyzed among 613 NIHL workers and 613 normal hearing workers using the minor groove binder TaqMan probe assay. RESULTS: We found that the APE1 -656 TT genotype was associated with a increased risk of NIHL [adjusted odds ratio (OR) 1.46, 95% confidence interval (CI) 1.05-2.06]. This increased risk was more pronounced in the stratification analysis. Furthermore, we found that subjects with two risk genotypes (hOGG1 Cys/Cys, APE1 -656 TT) had a significantly increased risk of NIHL (adjusted OR 1.91, 95% CI 1.27-2.88). CONCLUSION: Our study identified that the APE1 -656 T>G polymorphism may contribute to the susceptibility of NIHL.

New Insights into Macrophage Polarization and its Prognostic Role in Patients with Colorectal Cancer Liver Metastasis.

Background: As liver metastasis is the most common cause of mortality in patients with colorectal cancer, studying colorectal cancer liver metastasis (CLM) microenvironment is essential for improved understanding of tumor biology and to identify novel therapeutic targets. Methods: We used multiplex immunofluorescence platform to study tumor associated macrophage (TAM) polarization and adaptive T cell subtypes in tumor samples from 105 CLM patients (49 without and 56 with preoperative chemotherapy). Results: CLM exhibited M2 macrophage polarization, and helper T cells were the prevalent adaptive T cell subtype. The density of total, M2 and TGFß-expressing macrophages, and regulatory T cells was lower in CLM treated with preoperative chemotherapy. CLM with right-sided primary demonstrated enrichment of TGFß-expressing macrophages, and with left-sided primary had higher densities of helper and cytotoxic T cells. In multivariate analysis, high density of M2 macrophages correlated with longer recurrence-free survival (RFS) in the entire cohort [hazard ratio (HR) 0.425, 95% CI 0.219-0.825, p=0.011) and in patients without preoperative chemotherapy (HR 0.45, 95% CI 0.221-0.932, p=0.032). High pSMAD3-expressing macrophages were associated with shorter RFS in CLM after preoperative chemotherapy. Conclusions: Our results highlight the significance of a multi-marker approach to define the macrophage subtypes and identify M2 macrophages as a predictor of favorable prognosis in CLM.

Zyflamend suppresses growth and sensitizes human pancreatic tumors to gemcitabine in an orthotopic mouse model through modulation of multiple targets.

Agents that can potentiate the efficacy of standard chemotherapy against pancreatic cancer are of great interest. Because of their low cost and safety, patients commonly use a variety of dietary supplements, although evidence of their efficacy is often lacking. One such commonly used food supplement is Zyflamend, a polyherbal preparation with potent anti-inflammatory activities and preclinical efficacy against prostate and oral cancer. Whether Zyflamend has any efficacy against human pancreatic cancer alone or in combination with gemcitibine, a commonly used agent, was examined in cell cultures and in an orthotopic mouse model. In vitro, Zyflamend inhibited the proliferation of pancreatic cancer cell lines regardless of p53 status and also enhanced gemcitabine-induced apoptosis. This finding correlated with inhibition of NF-κB activation by Zyflamend and suppression of cyclin D1, c-myc, COX-2, Bcl-2, IAP, survivin, VEGF, ICAM-1 and CXCR4. In nude mice, oral administration of Zyflamend alone significantly inhibited the growth of orthotopically transplanted human pancreatic tumors, and when combined with gemcitabine, further enhanced the antitumor effects. Immunohistochemical and Western blot analyses of tumor tissue showed that the suppression of pancreatic cancer growth correlated with inhibition of proliferation index marker (Ki-67), COX-2, MMP-9, NF-κB and VEGF. Overall, these results suggest that the concentrated multiherb product Zyflamend alone can inhibit the growth of human pancreatic tumors and, in addition, can sensitize pancreatic cancers to gemcitabine through the suppression of multiple targets linked to tumorigenesis.

Protein kinase D1 promotes anchorage-independent growth, invasion, and angiogenesis by human pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal diseases. Novel molecularly targeted therapies are urgently needed. Here, we extended our studies on the role of protein kinase D1 (PKD1) in PDAC cell lines. Given that Panc-1 express moderate levels of PKD1, we used retroviral-mediated gene transfer to create a Panc-1 derivative that stably over-expresses PKD1 (Panc-1-PKD1). Reciprocally, we used shRNA targeting PKD1 in Panc-28 to produce a PKD1 under-expressing Panc-28 derivative (Panc-28-shPKD1). Our results demonstrate that Panc-1-PKD1 cells exhibit significantly increased anchorage-independent growth in soft agar and increased in vitro invasion compared with Panc-1-mock. Reciprocally, Panc-28-shPKD1 cells show a significant decrease in anchorage-independent growth and invasiveness, as compared with Panc-28-mock cells. The selective PKD family inhibitor CRT0066101 markedly decreased colony-forming ability and invasiveness by either Panc-1-PKD1 or Panc-28-mock cells. Secretion of the pro-angiogenic factors vascular endothelial growth factor (VEGF) and CXC chemokines (CXCL8) was significantly elevated by PKD1 over-expression in Panc-1 cells and reduced either by depletion of PKD1 via shRNA in Panc-28 cells or by addition of CRT0066101 to either Panc-1-PKD1 or Panc-28-mock cells. Furthermore, human umbilical vein endothelial cell (HUVEC) tube formation was significantly enhanced by co-culture with Panc-1-PKD1 compared with Panc-1-mock in an angiogenesis assay in vitro. Conversely, PKD1 depletion in Panc-28 cells decreased their ability to induce endotube formation by HUVECs. PDAC-induced angiogenesis in vitro and in vivo was markedly inhibited by CRT0066101. Our results lend further support to the hypothesis that PKD family members provide a novel target for PDAC therapy.

Epidermal growth factor down-regulates the expression of neutrophil gelatinase-associated lipocalin (NGAL) through E-cadherin in pancreatic cancer cells.

BACKGROUND: The authors previously reported that neutrophil gelatinase-associated lipocalin (NGAL) overexpression significantly blocked invasion and angiogenesis of pancreatic ductal adenocarcinoma (PDAC). They also demonstrated a loss of NGAL expression in the advanced stages of PDAC. However, little is known regarding the mechanisms of NGAL regulation in PDAC. Because the epidermal growth factor (EGF)-EGF receptor (EGFR) axis is up-regulated significantly in PDAC, they examined EGF-mediated NGAL regulation in these cells. METHODS: The NGAL-positive cell lines AsPC-1 and BxPC-3 were used as a model system. Quantitative real-time polymerase chain reaction (RT-PCR), Western blot analysis, and immunofluorescence studies were used to investigate EGF-mediated effects on NGAL expression. E-cadherin expression was manipulated using lentiviral overexpression or small hairpin RNA constructs. NGAL promoter activity was assessed by luciferase-reporter assay and electrophoretic mobility shift assay. RESULTS: NGAL expression was positively associated with tumor differentiation and was down-regulated significantly after EGF treatment along with a concomitant reduction of E-cadherin expression in PDAC cells. E-cadherin down-regulation was partly through the EGFR-dependent mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) (MEK-ERK) signaling pathway. In addition, E-cadherin down-regulation reduced NGAL expression in PDAC cells, whereas overexpression of E-cadherin led to increased NGAL expression and partly rescued the inhibition of NGAL expression by EGF. Furthermore, EGF, in part through E-cadherin, reduced NGAL promoter activity by blocking nuclear factor κB (NF-κB) activation. CONCLUSIONS: The current study demonstrated for the first time that EGF potently blocked NGAL expression in PDAC cells. This effect was mediated in part through activation of the EGFR-MEK-ERK signaling pathway, which, in turn, down-regulated E-cadherin with a subsequent reduction in NF-κB activation. These findings illustrate a novel mechanism by which EGF regulates NGAL expression in PDAC.

Development and Validation of a Gene Signature Classifier for Consensus Molecular Subtyping of Colorectal Carcinoma in a CLIA-Certified Setting.

PURPOSE: Consensus molecular subtyping (CMS) of colorectal cancer has potential to reshape the colorectal cancer landscape. We developed and validated an assay that is applicable on formalin-fixed, paraffin-embedded (FFPE) samples of colorectal cancer and implemented the assay in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory. EXPERIMENTAL DESIGN: We performed an in silico experiment to build an optimal CMS classifier using a training set of 1,329 samples from 12 studies and validation set of 1,329 samples from 14 studies. We constructed an assay on the basis of NanoString CodeSets for the top 472 genes, and performed analyses on paired flash-frozen (FF)/FFPE samples from 175 colorectal cancers to adapt the classifier to FFPE samples using a subset of genes found to be concordant between FF and FFPE, tested the classifier's reproducibility and repeatability, and validated in a CLIA-certified laboratory. We assessed prognostic significance of CMS in 345 patients pooled across three clinical trials. RESULTS: The best classifier was weighted support vector machine with high accuracy across platforms and gene lists (>0.95), and the 472-gene model outperforming existing classifiers. We constructed subsets of 99 and 200 genes with high FF/FFPE concordance, and adapted FFPE-based classifier that had strong classification accuracy (>80%) relative to "gold standard" CMS. The classifier was reproducible to sample type and RNA quality, and demonstrated poor prognosis for CMS1-3 and good prognosis for CMS2 in metastatic colorectal cancer (P < 0.001). CONCLUSIONS: We developed and validated a colorectal cancer CMS assay that is ready for use in clinical trials, to assess prognosis in standard-of-care settings and explore as predictor of therapy response.

CXCL8/IL-8 and CXCL12/SDF-1alpha co-operatively promote invasiveness and angiogenesis in pancreatic cancer.

CXC-chemokines are involved in the chemotaxis of neutrophils, lymphocytes and monocytes. However, role of these chemokines in tumorigenesis, especially with regard to interaction between tumor and its microenvironment, has not been clearly elucidated. The purpose of this study was to analyze the co-operative role of CXCL8 and CXCL12 in the tumor-stromal interaction in pancreatic cancer (PaCa). Using enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR), we initially confirmed the expression of ligands and receptors, respectively, of CXC-chemokines in PaCa and stromal cells. We examined the co-operative role of CXCL8 and CXCL12 in proliferation/invasion of PaCa and human umbilical vein endothelial cells (HUVECs), and in HUVEC tube-formations through tumor-stromal interaction by MTS, Matrigel invasion, and angiogenesis assays, respectively. We detected expression of CXCR4, but not CXCR2, in all PaCa cells and fibroblasts. PaCa cells secreted CXCL8, and fibroblast cells secreted CXCL12. CXCL8 production in PaCa was significantly enhanced by CXCL12, and CXCL12 production in fibroblasts was significantly enhanced by co-culturing with PaCa. CXCL8 enhanced proliferation/invasion of HUVECs but did not promote proliferation/invasion of PaCa. Both recombinant and PaCa-derived CXCL8 enhanced tube formation of HUVECs that were co-cultured with fibroblast cells. CXCL12 enhanced the proliferation/invasion of HUVECs and the invasion of PaCa cells but had no effect on tube formation of HUVEC. We showed that PaCa-derived CXCL8 and fibroblast-derived CXCL12 cooperatively induced angiogenesis in vitro by promoting HUVEC proliferation, invasion, and tube formation. Thus, corresponding receptors CXCR2 and CXCR4 are potential antiangiogenic and antimetastatic therapeutic targets in PaCa.

CXC-chemokine/CXCR2 biological axis promotes angiogenesis in vitro and in vivo in pancreatic cancer.

Angiogenesis is essential for tumor growth and metastasis. Although ELR(+)-CXC-chemokines and their corresponding receptor, CXC-receptor 2 (CXCR2), are known mediators of angiogenesis, little is known about their role in pancreatic cancer (PaCa). The aim of our study was to determine the role of ELR(+)-CXC-chemokine/CXCR2 biological axis in promoting PaCa angiogenesis. We prospectively collected secretin-stimulated exocrine pancreatic secretions (SSEPS) from normal individuals (NP) and PaCa patients. We showed that summed concentrations of ELR(+)-CXC-chemokines in SSEPS from PaCa patients were significantly higher than in those from NP (p = 0.002). We measured ELR(+)-CXC-chemokine levels in supernatants from multiple PaCa cell lines and confirmed that BxPC-3, Colo-357 and Panc-28 had significantly higher expression compared with an immortalized human pancreatic ductal epithelial (HPDE) cell line. After confirming lack of autocrine effects of ELR(+)-CXC-chemokines on PaCa cells (due to absence of CXCR2 expression), we investigated paracrine effects of these chemokines on human umbilical vein endothelial cells (HUVEC). Both recombinant ELR(+)-CXC-chemokines and co-culturing with BxPC-3 significantly enhanced proliferation, invasion, and tube formation of HUVEC (p < 0.05). These biological effects were significantly inhibited by treatment with a neutralizing antibody against CXCR2 (anti-CXCR2 Ab) (p < 0.05). Finally, anti-CXCR2 Ab significantly reduced tumor volume (p < 0.05), Ki-67 proliferation index (p = 0.043) and Factor VIII(+) microvessel density (p = 0.004) in an orthotopic nude mouse PaCa model. Our results show that ELR(+)-CXC-chemokines promote PaCa tumor-associated angiogenesis through CXCR2, suggesting that CXCR2 is an anti-angiogenic target in PaCa.

A novel patient-derived orthotopic xenograft model of esophageal adenocarcinoma provides a platform for translational discoveries.

Mouse models of gastroesophageal junction (GEJ) cancer strive to recapitulate the intratumoral heterogeneity and cellular crosstalk within patient tumors to improve clinical translation. GEJ cancers remain a therapeutic challenge due to the lack of a reliable mouse model for preclinical drug testing. In this study, a novel patient-derived orthotopic xenograft (PDOX) was established from GEJ cancer via transabdominal surgical implantation. Patient tumor was compared to subcutaneously implanted patient-derived tumor xenograft (PDX) and PDOX by Hematoxylin and Eosin staining, immunohistochemistry and next-generation sequencing. Treatment efficacy studies of radiotherapy were performed. We observed that mechanical abrasion of mouse GEJ prior to surgical implantation of a patient-derived tumor in situ promotes tumor engraftment (100%, n=6). Complete PDOX engraftment was observed with rapid intra- and extraluminal tumor growth, as evidenced by magnetic resonance imaging. PDOXs contain fibroblasts, tumor-associated macrophages, immune and inflammatory cells, vascular and lymphatic vessels. Stromal hallmarks of aggressive GEJ cancers are recapitulated in a GEJ PDOX mouse model. PDOXs demonstrate tumor invasion into vasculature and perineural space. Next-generation sequencing revealed loss of heterozygosity with very high allelic frequency in NOTCH3, TGFB1, EZH2 and KMT2C in the patient tumor, the subcutaneous PDX and the PDOX. Immunohistochemical analysis of Her2/neu (also known as ERBB2), p53 (also known as TP53) and p16 (also known as CDKN2A) in PDX and PDOX revealed maintenance of expression of proteins found in patient tumors, but membranous EGFR overexpression in patient tumor cells was absent in both xenografts. Targeted radiotherapy in this model suggested a decrease in size by 61% according to Response Evaluation Criteria in Solid Tumors (RECIST), indicating a partial response to radiation therapy. Our GEJ PDOX model exhibits remarkable fidelity to human disease and captures the precise tissue microenvironment present within the local GEJ architecture, providing a novel tool for translating findings from studies on human GEJ cancer. This model can be applied to study metastatic progression and to develop novel therapeutic approaches for the treatment of GEJ cancer.This article has an associated First Person interview with the first author of the paper.

Targeting CDK9 and MCL-1 by a new CDK9/p-TEFb inhibitor with and without 5-fluorouracil in esophageal adenocarcinoma.

BACKGROUND: CDK9 inhibitors are antitumorigenic against solid tumors, including esophageal adenocarcinoma (EAC). However, efficacy of a CDK9 inhibitor combined with 5-fluorouracil (5-FU) and target proteins that are targeted by these agents in EAC are unknown. METHODS: The anti-EAC efficacy of a new CDK9 inhibitor, BAY1143572, with and without 5-FU was assessed in vitro and in xenograft models in athymic nu/nu mice. Synergy between BAY1143572 and 5-FU in inhibiting cell proliferation was analyzed by calculating the combination index using CompuSyn software. Potential targets of BAY1143572 and 5-FU were identified by reverse-phase protein array. The effects of BAY1143572 and 5-FU on MCL-1 in vitro were analyzed by Western blotting, quantitative real-time polymerase chain reaction, and chromatin immunoprecipitation assay. MCL-1 protein expression in tumors from patients with locoregional EAC treated with chemoradiation and surgery was assessed by immunohistochemistry. RESULTS: BAY1143572 had dose-dependent antiproliferative and proapoptotic effects and demonstrated synergy with 5-FU against EAC in vitro. The median volumes of FLO-1 and ESO-26 xenografts treated with 5-FU plus BAY114352 were significantly smaller than those of xenografts treated with either agent alone (p < 0.05). BAY1143572 downregulated MCL-1 by inhibiting HIF-1α binding to the MCL-1 promoter. 5-FU enhanced BAY1143572-induced MCL-1 downregulation and stable MCL-1 overexpression reduced the apoptosis induced by BAY1143572 and 5-FU in vitro. High patients' tumor MCL-1 expression was correlated with shorter overall and recurrence-free survival. CONCLUSIONS: BAY1143572 and 5-FU have synergistic antitumorigenic effects against EAC. MCL-1 is a downstream target of CDK9 inhibitors and a predictor of response to neoadjuvant chemoradiation in EAC.

Targeting cyclin-dependent kinase 9 by a novel inhibitor enhances radiosensitization and identifies Axl as a novel downstream target in esophageal adenocarcinoma.

Cyclin-dependent kinase 9 (CDK9) transcriptionally regulates several proteins and cellular pathways central to radiation induced tissue injury. We investigated a role of BAY1143572, a new highly specific CDK9 inhibitor, as a sensitizer to radiation in esophageal adenocarcinoma. In vitro synergy between the CDK9 inhibitor and radiation was evaluated by clonogenic assay. In vivo synergy between the CDK9 inhibitor and radiation was assessed in multiple xenograft models including a patient's tumor derived xenograft (PDX). Reverse phase protein array (RPPA), western blotting, immunohistochemistry, and qPCR were utilized to identify and validate targets of the CDK9 inhibitor. The CDK9 inhibitor plus radiation significantly reduced growth of FLO-1, SKGT4, OE33, and radiation resistant OE33R xenografts and PDXs as compared to the cohorts treated with either single agent CDK9 inhibitor or radiation alone. RPPA identified Axl as a candidate target of CDK9 inhibition. Western blot and qPCR demonstrated reduced Axl mRNA (p = 0.02) and protein levels after treatment with CDK9 inhibitor with or without radiation in FLO-1 and SKGT4 cells. Axl protein expression in FLO-1 xenografts treated with combination of CDK9 inhibitor and radiation was significantly lower than the xenografts treated with radiation alone (p = 0.003). Clonogenic assay performed after overexpression of Axl in FLO-1 and SKGT4 cells enhanced radiosensitization by the CDK9 inhibitor, suggesting dependency of radiosensitization effects of the CDK9 inhibitor on Axl. In conclusion, these findings indicate that targeting CDK9 by BAY1143572 significantly enhances the effects of radiation and Axl is a novel downstream target of CDK9 in esophageal adenocarcinoma.

Antitumor effects of cyclin dependent kinase 9 inhibition in esophageal adenocarcinoma.

Role of cyclin dependent kinase 9(CDK9) as a potential target in esophageal adenocarcinoma (EAC) is unknown. We investigated CDK9 protein expression in EAC and Barrett's esophagus and role of CDK9 in oncogenic processes of EAC in vitro and in murine xenografts. The CDK9 expression was significantly higher in EAC as compared to Barrett's esophagus in patient samples. Stable shCDK9 in SKGT4 reduced proliferation by 37% at day 4, increased apoptosis at 48 hours and induced G1 cell cycle arrest at 48 hours (58.4% vs. 45.8%) compared to controls SKGT4 cells. SKGT4-shCDK9 cell-derived tumors were significantly smaller than control SKGT4-derived tumors in xenografts (72.89mm3 vs. 270mm3). Pharmaceutical inhibition of CDK9 by Flavopiridol (0.1µm for 48 hours) and CAN508 (20 and 40µm for 72 hours) induced significant reduction in proliferation and 2-fold increase in apoptosis in SKGT4, FLO1 and OE33 cells. In xenograft models, CAN508 (60 mg/kg/dayx10 days) and Flavopiridol (4mg/kg/dayx10 days) caused 50.8% and 63.1% reduction in xenograft tumors as compared to control on post-treatment day 21. Reduction of MCL-1 and phosphorylated RNA polymerase II was observed with transient shCDK9 in SKGT4 cells but not with stable shCDK9. CAN508 (20 and 40 µm) and Flavopiridol (0.1, 0.2 and 0.3 µm) for 4 hours showed reduction in MCL-1 mRNA (84% and 96%) and protein. Mcl-1 overexpression conferred resistance to Flavopiridol (0.2 µm or 0.4 µm for 48 hours) and CAN 508 (20 or 40µm for 72 hours). Chromatin immunoprecipitation demonstrated significant reduction of binding of transcriptional factor HIF-1α to MCL-1 promoter in FLO-1 cells by CDK9 inhibitors.

Cytotoxicity of a non-cyclooxygenase-2 inhibitory derivative of celecoxib in non-small-cell lung cancer A549 cells.

Lung cancer is one of the most common causes of cancer death worldwide. Although recent advances in chemotherapy and radiation therapy have yielded modest improvements in patient outcomes, overall survival remains poor. Therefore, new therapeutic targets are needed. Phosphoinositide-dependent kinase-1 (PDK1) is one potential target. The aim of the present studies was to investigate the potential of a celecoxib-derived PDK1 inhibitor (OSU03013), that does not inhibit cyclooxygenase-2, to kill lung cancer cells in vitro. Using human non-small-cell lung cancer A549 cells, OSU03013 dose-dependently induced apoptosis. After 6 h of treatment with 7.5 microM OSU03013, 26% of the cells were apoptotic, compared to 4% of the control cells as determined by measuring the sub-G1 peak of propidium iodide stained cells with flow cytometry. A similar increase in apoptosis was evident using the Cell Death ELISA assay. OSU03013-induced apoptosis was accompanied by a reduction in the mitochondrial membrane potential, the release of cytochrome c and the cleavage of caspase-3. Surprisingly, the phosphorylation of Akt at serine 473 was increased in A549 cells treated with 7.5 microM OSU03013. However, the toxicity of OSU03013 was reduced in A549 cells expressing a constitutively active form of Akt. These data demonstrate that OSU03013 induces apoptosis in A549 cells via the mitochondrial pathway. Inhibition of the Akt pathway appears uninvolved in this toxicity, although Akt can provide protection. These results also suggest the potential of celecoxib-derived agents to treat some forms of lung cancer.

Neutrophil gelatinase-associated lipocalin as a survival factor.

NGAL (human neutrophil gelatinase-associated lipocalin) and its mouse analogue 24p3 are members of the lipocalin family of small secreted proteins. These proteins are up-regulated in a number of pathological conditions, including cancers, and may function as transporters of essential factors. Although previous publications have suggested that 24p3 has pro-apoptotic functions, other data are more suggestive of a survival function. The current study was designed to determine whether NGAL is pro- or anti-apoptotic. Apoptosis induced in human adenocarcinoma A549 cells by the 5-lipoxygenase-activating-protein inhibitor MK886, or several celecoxib-derived PDK1 (phosphoinositide-dependent kinase 1) inhibitors that are devoid of cyclo-oxygenase-2 inhibitory activity, was accompanied by a dose- and time-dependent increase of NGAL mRNA levels, as was reported previously with 24p3. A similar induction of NGAL mRNA was observed in human breast cancer MCF7 cells treated with MK886, indicating this was not a cell-specific effect. Treatment of A549 cells with up to 150 mug/10(6) cells of purified recombinant NGAL protein had no effect on viability, whereas antisera against the full-length NGAL protein induced apoptosis in these cells. The stable overexpression of NGAL in A549 cells had no effect on proliferation or viability. However, the cell death induced by a PDK1 inhibitor was reduced by 50% in NGAL-overexpressing cells. Decreasing NGAL mRNA and protein expression with siRNA (small interfering RNA) in A549 cells increased the toxicity of a PDK1 inhibitor by approx. 45%. These data indicate that, although the induction of NGAL correlates with apoptosis, this induction represents a survival response. Because NGAL is a secreted protein, it may play an extracellular role in cell defence against toxicants and/or facilitate the survival of the remaining cells.

Sustained activation of the extracellular signal-regulated kinase pathway protects cells from photofrin-mediated photodynamic therapy.

Photodynamic therapy (PDT) is a cancer therapy in which a photosensitizer selectively accumulates in tumor cells and is subsequently activated by light of a specific wavelength. The activation of the photosensitizer leads to cytotoxic photoproducts that result in tumor regression. PDT can lead to several cellular responses including cell cycle arrest, necrosis, and apoptosis, as well as trigger many signaling pathways. It has been suggested that extracellular signal-activated protein kinases (ERKs), one subfamily of mitogen-activated protein kinases, play a crucial role in the cellular response to radiation therapy and chemotherapy. However, the role of ERKs in the cell survival after PDT is less clear. We have examined the response of the extracellular signal-regulated kinase ERK1/2 in PDT-resistant (LFS087) and PDT-sensitive (GM38A) cells after Photofrin-mediated PDT. ERK1/2 activity was induced rapidly in both cell types after PDT. The PDT-induced ERK1/2 activity was transient in GM38A cells and by 3 h had returned to a level significant lower than basal levels, whereas the induction of ERK1/2 was sustained in LFS087 cells and lasted for at least 11 h. Blocking of the sustained ERK activity with PD98059, an inhibitor of mitogen-activated protein/ERK kinase, significantly decreased cell survival of LFS087 after PDT. PDT also induced the expression of mitogen-activated protein kinase phosphatase, MKP-1, but reduced Raf-1 protein levels in both cell types. In GM38A cells, the substantially induced levels of MKP-1 correlated with the transient activation of ERK1/2 by PDT, and both basal and induced levels of MKP-1 were substantially greater in GM38 compared with Li Fraumeni syndrome cells. These observations suggest that sustained ERK1/2 activation protects cells from Photofrin-mediated phototoxicity and that the duration of ERK1/2 activation is regulated by MKP-1. In addition, the activation of ERK1/2 by Photofrin-mediated PDT is Raf-1 independent.

Response of mouse thymic cells to radiation after transfusion of mesenchymal stem cells.

Thymic lymphoma is a highly invasive and even metastatic cancer. This study investigated the effects of mesenchymal stem cells (MSCs) transfusion on cell cycle, cell proliferation, CD3 expression, mutation frequency of T cell receptor using mouse model of thymic lymphoma.C57BL/6J young mouse models of thymoma were injected with MSCs. Six months later, the thymus was taken for pathological examination and flow cytometry studies. The cells were labeled with anti-CD4, CD8, CD3, propidium iodide, or CFDA-SE, cell cycle, proliferation kinetics, and mutation frequency of T cell receptor, respectively.Pathologic results showed that control had clear corticomedular structure with regularly shaped lymphocytes. After radiation, the thymus structure was completely destroyed, with lymphoid tumor cells diffusely distributed and heavily stained, and large nuclei. Transfusion of MSCs resulted in normal thymus structure. Cytometry studies showed that there were more CD4-/CD8- T cells in the thymus of irradiated mice than in control; transfusion of MSCs led to reduced CD4-/CD8- T cells. In irradiated mice, there were less CD4+/CD8+ T cells than in control and MSCs transfusion groups. It was observed that there were more cells arrested in G1 phase in the thymus cells and CD4-/CD8- T cells in irradiated mice than in other 2 groups, whereas there were more cells arrested in S phase in CD4+/CD8+ and CD4+/CD8- T cells in irradiated mice than in the other mice. In the thymus cells, and CD4+/CD8+ and CD4+/CD8- T cells, irradiated mice group had significantly less parent, G2, G3, and G4 cells, and more cells at higher generations, and also higher proliferation index. In CD4-/CD8- T cells, irradiated mice had significantly more parent, G2, and G3 cells, and less G4, G5, G6, and propidium iodide, as compared with the other 2 groups. The expression of CD3 in CD4/CD8 T cells was significantly higher than in control. MSCs transfusion improved CD3 expression, but was still less than the control. Irradiation resulted in very high mutation frequency of T cell receptor, which was barely affected by MSCs transfusion.Mesenchymal stem cell transfusion is able to restore the cell cycle and cell proliferation, but not CD3 expression and mutation frequency of T cell receptor in irradiated mice to control level.

Genetic Variations in the Promoter of the APE1 Gene Are Associated with DMF-Induced Abnormal Liver Function: A Case-Control Study in a Chinese Population.

Acute or long-term exposure to N,N-dimethylformamide (DMF) can induce abnormal liver function. It is well known that DMF is mainly metabolized in the liver and thereby produces reactive oxygen species (ROS). The base excision repair (BER) pathway is regarded as a very important pathway involved in repairing ROS-induced DNA damage. Several studies have explored the associations between GSTM1, GSTT1, CYP2E1 polymorphisms and DMF-induced abnormal liver function; however, little is known about how common hOGG1, XRCC1 and APE1 polymorphisms and DMF induce abnormal liver function. The purpose of this study was to investigate whether the polymorphisms in the hOGG1 (rs159153 and rs2072668), XRCC1 (rs25487, rs25489, and rs1799782), APE1 (rs1130409 and 1760944) genes in the human BER pathway were associated with the susceptibility to DMF-induced abnormal liver function in a Chinese population. These polymorphisms were genotyped in 123 workers with DMF-induced abnormal liver function and 123 workers with normal liver function. We found that workers with the APE1 rs1760944 TG/GG genotypes had a reduced risk of abnormal liver function, which was more pronounced in the subgroups that were exposed to DMF for <10 years, exposed to ≥10 mg/m³ DMF, never smoked and never drank. In summary, our study supported the hypothesis that the APE1 rs1760944 T > G polymorphism may be associated with DMF-induced abnormal liver function in the Chinese Han population.

Activation of the stress-activated JNK and p38 MAP kinases in human cells by Photofrin-mediated photodynamic therapy.

We have examined the possible role of the stress-activated JNK and p38 protein kinases in cellular sensitivity following Photofrin-mediated photodynamic therapy (PDT). Previously we reported that immortalized Li-Fraumeni syndrome (LFS) cells are more resistant to Photofrin-mediated PDT compared to normal human fibroblasts (NHF) at equivalent cellular Photofrin levels. In the current work we report that Photofrin-mediated PDT increased the activity of JNK1 and p38 within 30 min in both cell types. However, the increased activity of JNK1 and p38 was transient in the sensitive NHF cells and returned back to near basal levels by 3 h after PDT. In contrast, the resistant LFS cells exhibited a more prolonged activation of JNK and p38, which lasted for at least 11 h and 7 h after PDT, respectively. Blocking of the p38 pathway in LFS cells by transient infection with a recombinant adenovirus expressing a dominant negative mutant of p38 or in HeLa cells by stable transfection with a dominant negative mutant of p38 had no effect on cell survival following PDT. These data suggest that although Photofrin-mediated PDT is able to induce JNK1 and p38 in human cells, the p38 pathway alone does not play a major role in the sensitivity of LFS cells to Photofrin-mediated PDT.

Target-specific agents imaging ectopic and orthotopic human pancreatic cancer xenografts.

OBJECTIVES: This study aimed to develop target-specific binding agents for in vitro and in vivo imaging of human pancreatic cancer. METHODS: A monoclonal neutrophil gelatinase-associated lipocalin (NGAL)-specific antibody and a peptide specific for matrix metalloproteinase (MMP) were labeled with a near-infrared dye for in vitro and in vivo imaging studies. Fluorescence or confocal microscopy was used to determine antibody or peptide binding and internalization of agents into human AsPC-1, Panc-1, and MiaPaCa pancreatic cancer cell lines and in mice bearing ectopic or orthotopic pancreatic tumor transplants. RESULTS: Both the NGAL-specific antibody and MMP peptide bound to pancreatic cancer cells with high specificity; most NGAL-specific antibody localized to the cytosol. In vivo imaging results demonstrated high signal intensity of both agents bound to the tumor. The average tumortr-to-background ratio of antibody and peptide was 1.29 and 2.86, respectively. Signal was also detectable in the liver, kidneys, and bladder. CONCLUSIONS: Both NGAL-specific antibody and MMP peptide bound to cancer cells, and the labeled antibody was internalized. These results demonstrate that both agents can be used to enhance detection of human pancreatic cancer xenografts. However, the biodistribution patterns of these agents might limit their use in research and clinical practice.