NIPA (Nuclear Interaction Partner of ALK) Is Crucial for Effective NPM-ALK Mediated Lymphomagenesis.

The NPM-ALK fusion kinase is expressed in 60% of systemic anaplastic large-cell lymphomas (ALCL). A Nuclear Interaction Partner of ALK (NIPA) was identified as a binding partner of NPM-ALK. To identify the precise role of NIPA for NPM-ALK-driven lymphomagenesis, we investigated various NPM-ALK+ cell lines and mouse models. Nipa deletion in primary mouse embryonic fibroblasts resulted in reduced transformation ability and colony formation upon NPM-ALK expression. Downregulating NIPA in murine NPM-ALK+ Ba/F3 and human ALCL cells decreased their proliferation ability and demonstrated synergistic effects of ALK inhibition and NIPA knockdown. Comprehensive in vivo analyses using short- and long-latency transplantation mouse models with NPM-ALK+ bone marrow (BM) revealed that Nipa deletion inhibited NPM-ALK-induced tumorigenesis with prolonged survival and reduced spleen colonies. To avoid off-target effects, we combined Nipa deletion and NPM-ALK expression exclusively in T cells using a lineage-restricted murine ALCL-like model resembling human disease: control mice died from neoplastic T-cell infiltration, whereas mice transplanted with Lck-CreTG/wtNipaflox/flox NPM-ALK+ BM showed significantly prolonged survival. Immunophenotypic analyses indicated a characteristic ALCL-like phenotype in all recipients but revealed fewer "stem-cell-like" features of Nipa-deficient lymphomas compared to controls. Our results identify NIPA as a crucial player in effective NPM-ALK-driven ALCL-like disease in clinically relevant murine and cell-based models.

Phosphorylation of BECLIN-1 by BCR-ABL suppresses autophagy in chronic myeloid leukemia.

Autophagy is a genetically regulated process of adaptation to metabolic stress and was recently shown to be involved in the treatment response of chronic myeloid leukemia (CML). However, in vivo data are limited and the molecular mechanism of autophagy regulators in the process of leukemogenesis is not completely understood. Here we show that Beclin-1 knockdown, but not Atg5 deletion in a murine CML model leads to a reduced leukemic burden and results in a significantly prolonged median survival of targeted mice. Further analyses of murine cell lines and primary patient material indicate that active BCR-ABL directly interacts with BECLIN-1 and phosphorylates its tyrosine residues 233 and 352, resulting in autophagy suppression. By using phosphorylation-deficient and phosphorylation-mimic mutants, we identify BCR-ABL induced BECLIN-1 phosphorylation as a crucial mechanism for BECLIN-1 complex formation: interaction analyses exhibit diminished binding of the positive autophagy regulators UVRAG, VPS15, ATG14 and VPS34 and enhanced binding of the negative regulator Rubicon to BCR-ABL-phosphorylated BECLIN-1. Taken together, our findings show interaction of BCR-ABL and BECLIN-1 thereby highlighting the importance of BECLIN-1-mediated autophagy in BCR-ABL+ cells.

Zebrafish Olfacto-Retinal Centrifugal Axon Projection and Distribution: Effects of Gonadotropin-Releasing Hormone and Dopaminergic Signaling.

The terminalis neurons (TNs) have been described in teleost species. In zebrafish, the TNs are located in the olfactory bulb. The TNs synthesize and release gonadotropin-releasing hormone (GnRH) as one of the major neurotransmitters. The TNs project axons to many brain areas, which include the neural retina. In the retina, the TN axons synapse with dopaminergic interplexiform cells (DA-IPCs) and retinal ganglion cells (RGCs). In this research, we examine the role of GnRH and dopaminergic signaling in TN axon projection to the retina using the transgenic zebrafish Tg(GnRH-3::GFP). While the TNs developed at 34 h postfertilization (hpf), the first TN axons were not detected in the retina until 48-50 hpf, when the first DA-IPCs were differentiated. In developing embryos, inhibition of retinal GnRH signaling pathways severely interrupted the projection of TN axons to the retina. However, inhibition of retinal dopaminergic signaling produced little effect on TN axon projection. In adult retinas, inactivation of GnRH receptors disrupted the patterns of TN axon distribution, and depletion of DA-IPCs abolished the TN axons. When DA-IPCs regenerated, the TN axons reappeared. Together, the data suggest that in developing zebrafish retinas GnRH signaling is required for TN axon projection, whereas in adult retinas activation of GnRH and dopaminergic signaling transduction is required for normal distribution of the TN axons.

Significantly inhibitory effects of low molecular weight heparin (Fraxiparine) on the motility of lung cancer cells and its related mechanism.

Low molecular weight heparin (LMWH) improving the cancer survival has been attracting attention for many years. Our previous study found that LMWH (Fraxiparine) strongly downregulated the invasive, migratory, and adhesive ability of human lung adenocarcinoma A549 cells. Here, we aimed to further identify the antitumor effects and possible mechanisms of Fraxiparine on A549 cells and human highly metastatic lung cancer 95D cells. The ability of cell invasion, migration, and adhesion were measured by Transwell, Millicell, and MTT assays. FITC-labeled phalloidin was used to detect F-actin bundles in cells. Chemotactic migration was analyzed in a modified Transwell assay. Measurement of protein expression and phosphorylation activity of PI3K, Akt, and mTOR was performed with Western blot. Our studies found that Fraxiparine significantly inhibited the invasive, migratory, and adhesive characteristics of A549 and 95D cells after 24 h incubation and showed a dose-dependent manner. Fraxiparine influenced the actin cytoskeleton rearrangement of A549 and 95D cells by preventing F-actin polymerization. Moreover, Fraxiparine could significantly inhibit CXCL12-mediated chemotactic migration of A549 and 95D cells in a concentration-dependent manner. Furthermore, Fraxiparine might destroy the interaction between CXCL12-CXCR4 axis, then suppress the PI3K-Akt-mTOR signaling pathway in lung cancer cells. For the first time, our data indicated that Fraxiparine could significantly inhibit the motility of lung cancer cells by restraining the actin cytoskeleton reorganization, and its related mechanism might be through inhibiting PI3K-Akt-mTOR signaling pathway mediated by CXCL12-CXCR4 axis. Therefore, Fraxiparine would be a potential drug for lung cancer metastasis therapy.

Targeting oncoprotein stability overcomes drug resistance caused by FLT3 kinase domain mutations.

FLT3 is the most frequently mutated kinase in acute myeloid leukemia (AML). Internal tandem duplications (ITDs) in the juxta-membrane region constitute the majority of activating FLT3 mutations. Several FLT3 kinase inhibitors were developed and tested in the clinic with significant success. However, recent studies have reported the development of secondary drug resistance in patients treated with FLT3 inhibitors. Since FLT3-ITD is an HSP90 client kinase, we here explored if targeting the stability of drug-resistant FLT3 mutant protein could be a potential therapeutic option. We observed that HSP90 inhibitor treatment resulted in the degradation of inhibitor-resistant FLT3-ITD mutants and selectively induced toxicity in cells expressing FLT3-ITD mutants. Thus, HSP90 inhibitors provide a potential therapeutic choice to overcome secondary drug resistance following TKI treatment in FLT3-ITD positive AML.

Synergistic effects of eukaryotic coexpression plasmid carrying LKB1 and FUS1 genes on lung cancer in vitro and in vivo.

PURPOSE: LKB1 and FUS1 are two kinds of new tumor suppressor genes as well as early-stage genes in lung cancer. Recent studies showed that LKB1 and FUS1 play important roles in lung carcinogenesis process. We hypothesized that combined gene therapy with LKB1 and FUS1 could inhibit lung cancer growth and development synergistically. METHODS: In this study, two kinds of tumor suppressor genes, LKB1 and FUS1, were constructed in an eukaryotic coexpression plasmid pVITRO(2), and then, we evaluated the synergistic effects of the two genes on anticancer activity and explored the relevant molecular mechanisms. RESULTS: We defined coexpression of LKB1 and FUS1 could synergistically inhibited lung cancer cells growth,invasion and migration and induced the cell apoptosis and arrested cell cycle in vitro. Intratumoral administration of liposomes: pVITRO(2)­LKB1­FUS1 complex (LPs­pVITRO(2)­LKB1­FUS1) into subcutaneous lung tumor xenograft resulted in more significant inhibition of tumor growth. Furthermore, intravenous injection of LPs­pVITRO(2)­LKB1­FUS1 into mice bearing experimental A549 lung metastasis demonstrated synergistic decrease in the number of metastatic tumor nodules. Finally, combined treatment with LKB1 and FUS1 prolonged overall survival in lung tumor-bearing mice. Further study showed tha tthe synergistic anti-lung cancer effects of coexpression ofLKB1 and FUS1 might be related to upregulation of p-p53, p-AMPK and downregulation of p-mTOR, p-FAK, MMPs, NEDD9, VEGF/R and PDGF/R. CONCLUSIONS: Our results suggest that combined therapy with eukaryotic coexpression plasmid carrying LKB1 and FUS1 genes may be a novel and efficient treatment strategy for human lung cancer.

Cationic liposome mediated delivery of FUS1 and hIL-12 coexpression plasmid demonstrates enhanced activity against human lung cancer.

FUS1 is one of the most important tumor suppressor genes in lung cancer, as well as an important immunomodulatory molecule. Interleukin (IL)-12 has attracted considerable interest as a potential anti-tumor cytokine. Cationic liposome has been shown to effectively deliver therapeutic genes to the lungs and control metastatic lung tumors when administered intravenously. Here we evaluated the enhanced efficacy of cationic liposome-mediated delivery of FUS1 and human IL (hIL)-12 eukaryotic coexpression plasmid (pVITRO2-FUS1-hIL-12) against the human lung cancer in HuPBL-NOD/SCID mice model by local and systemic administration, and explored the related molecular mechanism. Our study demonstrated that FUS1-hIL-12 coexpression could more sufficiently inhibit tumor growth and experimental lung metastasis, significantly prolong the survival of experimental lung metastasis mice. Moreover, FUS1-hIL-12 coexpression performed higher antitumor activity and lower toxicity in the inhibition of experimental lung metastatic tumor compared to cisplatin. We further identified that FUS1-hIL-12 coexpression could induce strong antitumor immune response by secreting much higher levels of human interferon-γ (hIFN-γ) and hIL-15, enhancing expression of MHC-I and Fas, increasing infiltration of activated human CD4+ and CD8+ T lymphocytes. FUS1-hIL-12 coexpression could also obviously induce tumor cell apoptosis and inhibit tumor cell proliferation partly by higher activation of STAT1 signal pathway and upregulation of p53. In addition, FUS1-hIL-12 coexpression also superiorly reduced the angiogenesis in tumors, which might be associated with downregulation of VEGF and VEGFR, and upregulation of human IP-10. Our results therefore suggest that cationic liposome-mediated FUS1-hIL-12 coexpression may be a new promising strategy for lung cancer treatment in clinical studies.

Cationic liposome-mediated nitric oxide synthase gene therapy enhances the antitumor effects of cisplatin in lung cancer.

Cisplatin is one of the most effective antitumor drugs for non-small cell lung carcinoma (NSCLC) patients. However, its efficacy has encountered a plateau due to its side effects and drug resistance. Inducible nitric oxide (NO) synthase (iNOS) gene therapy has been reported to have antitumor effects in several types of cancers and enhances sensitivity to cisplatin, but the effects of iNOS gene therapy alone or its combination with cisplatin in lung cancer remain unclear. In the current study, we evaluated the effects of cationic liposome (LP)-mediated iNOS gene transfection on enhancing low-dose cisplatin-mediated antitumor effects in the A549 human lung adenocarcinoma cell line in vitro. Furthermore, we examined whether iNOS gene therapy enhances the antitumor effects of low-dose cisplatin in two A549 human lung cancer cell xenograft mouse models. The results revealed that iNOS gene therapy may significantly enhance low-dose cisplatin-mediated inhibition of cell proliferation, invasion, migration and promotion of cell apoptosis in A549 cells. Intratumoral administration of the LP-pVAX-iNOS complex significantly enhanced low-dose cisplatin-mediated suppression of subcutaneous tumor growth. Moreover, intravenous injection of the LP-pVAX-iNOS complex greatly enhanced low-dose cisplatin-mediated inhibition of experimental lung metastasis and prolonged the life span of mice without significant organ-related toxicity in a nude mouse model of lung metastasis compared to the cisplatin alone-treated group. Furthermore, iNOS gene-mediated enhancement of cisplatin-mediated antitumor effects in lung cancer may be related to the attenuation of p-mTOR, MMP2 and the activation of p-p53. Thus, the combination treatment with iNOS gene therapy and cisplatin may be a novel and effective therapeutic strategy for lung cancer.

Antitumoral efficacy by systemic delivery of heparin conjugated polyethylenimine-plasmid interleukin-15 complexes in murine models of lung metastasis.

Gene therapy shows promising application in cancer therapy, but the lack of an ideal gene delivery system is still a tough challenge for cancer gene therapy. Previously, we prepared a novel cationic nanogel, heparin-polyethylenimine (HPEI), which had potential application in gene delivery. In the present study, we constructed a plasmid with high expression efficiency of interleukin-15 (IL15) and investigated the effects HPEI-plasmid IL15 (HPEI-pIL15) complexes on the distribution level of the lung. We then evaluated the anticancer effect of HPEI-pIL15 complexes on lung metastases of B16-F10 melanoma and CT26 colon carcinoma. These results demonstrated that intravenous injection of the HPEI-pIL15 complex exhibited the highest plasmid distribution level in the lung compared with that of PEI2K-pIL15 and PEI25K-pIL15, and mice treated with HPEI-pIL15 had a lower tumor metastasis index compared with other treatment groups. Moreover, the number of natural killer cells, which were intermingled among the tumor cells, and the level of tumor necrosis factor-α and interferon-γ in the serum also increased in the pIL15-treated mice. Furthermore, the cytotoxic activity of spleen cells also increased significantly in the HPEI-pIL15 group. In addition, induction of apoptosis and inhibition of cell proliferation in lung tumor foci in the HPEI-pIL15 group was observed. Taken together, treating lung metastasis cancer with the HPEI nanogels delivered by plasmid IL15 might be a new and interesting cancer gene therapy protocol.

[Study on characterization of the complexes of FUS1/hIL-12 with cationic liposome].

This study was aimed to shed light on the biological and pharmaceutical characterization of the complexes of FUS1/hIL-12 double gene with cationic liposome, and to assess such complexes' transfection efficiency, stability and cytotoxicity; for they have the potential for use as drugs in gene therapy of lung cancer. Gel retardation assay, diameter measurement, and surface charge by photon correlation spectroscopy (PCS) were employed to select the appropriate ratio of "cationic liposome to DNA" of the double-gene and liposome complexes. The plasmid EGFP and plasmid PVITO2-hIL12-FUS1 mediated by cationic liposome were transfected into A549 lung cancer cells respectively, and the expression levels of EGFP and FUS1 and hIL-12 were determined by inverted fluorescence microscope and immunohistochemical and enzyme linked immunosorbent assay (ELISA) respectively. Agarose gel electrophoresis was performed to detect the stability of the double-gene and liposome complexes, after they were incubated with serum and Dnase I respectively. After the erythrocytes being incubated with the complexes of FUS1/hIL-12 with cationic liposome, the morphology of erythrocyte was observed by microscopy. The result of this study provides a basis for the use of the complexes of FUS1/hIL-12 with cationic liposome in gene therapy of lung cancer.

Linear cyclen-based polyamine as a novel and efficient reagent in gene delivery.

Linear cyclen-based polyamine (LCPA, M(w) = 7392, M(w)/M(n) = 1.19) as a novel non-viral gene vector was designed and synthesized from 1,7-diprotected 1,4,7,10-tetraazacyclododecane (cyclen), bis(beta-hydroxylethyl)amine and epichlorohydrin. Agarose gel retardation and fluorescent titration using ethidium bromide showed the good DNA-binding ability of LCPA. It could retard pDNA at an N/P ratio of 4 and form polyplexes with sizes around 250-300 nm from an N/P ratio of 10 to 60 and relatively lower zeta-potential values (< +3 mV) even at the N/P ratio of 60. The cytotoxicity of LCPA assayed by MTT is much lower than that of 25 kDa PEI. In vitro transfection against A549 and 293 cells showed that the transfection efficiency of LCPA/DNA polyplexes is close to that of 25 kDa PEI at an N/P ratio of 10-15, indicating that the new material could be a promising non-viral polycationic reagent for gene delivery.

Effect of Fraxiparine, a type of low molecular weight heparin, on the invasion and metastasis of lung adenocarcinoma A549 cells.

Lung cancer is one of the most highly malignant tumors, and a significant threat to human health. Lung cancer patients often exhibit tumor cell invasion and metastasis, which often render current treatments ineffective. Recently, the beneficial effects of low molecular weight heparin (LMWH) on cancer metastasis were reported in pre-clinical research studies. LMWH may be a potential drug for cancer therapy. However, the mechanism of LMWH on the invasion and metastasis of cancer has yet to be determined. This study investigated the effects of Fraxiparine on the proliferation, invasion and metastasis of the human lung adenocarcinoma A549 cell line. MTT assay and flow cytometry showed that Fraxiparine slightly inhibited the cell viability dose- and time-dependently, but did not arrest the A549 cells in the G1 phase nor induce early apoptosis. The transwell chamber assay showed that Fraxiparine significantly suppressed the invasion and migration of the A549 cells in vitro. Fraxiparine also markedly inhibited the adhesion of the A549 cells to Matrigel. The RT-PCR assay demonstrated that the reduction in invasion and metastasis may be related to the up-regulation of nm23-H1 and the down-regulation of the heparanase expression. Moreover, the RT-PCR assay and Western blot analysis demonstrated that down-regulation of the expression of integrin ß1 and ß3, as well as that of matrix metalloproteinase-2 and -9 may be responsible for the inhibition of the invasion and metastasis of A549 cells by Fraxiparine.

[Effect of 20(R) ginsenoside Rg3 on protein expression of lung cancer cell line.].

BACKGROUND: Lung cancer is the most dangerous threating tumor to human's health and life. Metastasis is not only the malignant characteristics of lung cancer, but also the chief cause of failure to cure the disease and of high mortality. Ginsenoside Rg3 has been proved to have obvious effect against tumor. The molecular mechanism of Rg3 against lung cancer cell line will be investigated by using two-dimensional gel electrophoresis in this paper. METHODS: The IC50 of Rg3 against human high-metastatic large cell lung cancer cell line 95D was determined by MTT. Then 95D cells were treated with Rg3 at the concentration of 0.1*IC50 for 72 h. The total proteins of 95D cell line treated with Rg3 and not treated were separated and protein profiles were obtained by using immobilized PH gradient (IPG)-based two-dimensional gel electrophoresis. The differential expression proteins of 95D cell line treated with Rg3 and not treated were analyzed using image analysis software.15 of differentially expressed proteins were further identified using MALDI-TOF MS/MS analysis and LC-MS/MS analysis. Protein identification was performed by searching the protein database. RESULTS: The IC50 of Rg3 against 95D cell line was 100 mug/mL. There were 27 differently expressed protein spots through analysis by Image Master Microsoft.15 proteins were identified using mass spectrometry. Chloride intracellular channel protein 1, Ubiquitin-Conjugating Enzyme E2-25 Kda only expressed in control; 14-3-3 protein teta, SKI-interacting protein only expressed in 95D cell line treated by Rg3; Annexin A2, profilin 2 isoform b were downregulated in 95D cell line treated by Rg3; 14-3-3 protein zeta, Eukaryotic translation initiation factor 4H were upregulated in 95D cell line treated by Rg3. CONCLUSIONS: A significantly different expression of proteins were found in 95D cell line treated with Rg3 and those not treated. Most of identified proteins have been reported to be associated with tumor metastasis. The identified proteins will provide the basis for searching potential biomarkers and the molecular mechanism of Rg3 against the metastasis of lung cancer cells.

Mitogen-associated protein kinase- and protein kinase A-dependent regulation of rhodopsin promoter expression in zebrafish rod photoreceptor cells.

Mitogen-associated protein kinase (MAPK)- and protein kinase A (PKA)-dependent signal transductions play important roles in the regulation of gene expression. Both MAPK and PKA pathways can be activated by light exposure. In this study, we investigated the effect of light on MAPK and PKA signal transduction and their roles in the regulation of rhodopsin promoter expression by using transgenic zebrafish [Tg(rhod::GFP)]. The Tg(rhod::GFP) fish express short half-life GFP that is under the transcriptional control of the zebrafish rhodopsin promoter and can therefore be used for in vivo studies of rhodopsin gene transcription in live cells. Blue light plays a role in the regulation of rhodopsin promoter expression via an MAPK-mediated signal transduction cascade. Blue light excites cryptochromes (CRY), which activate the downstream PKC-dependent MAPK signal pathway. White light, on the other hand, regulates rhodopsin promoter expression via a G-protein-coupled cAMP-dependent PKA pathway. White light promotes dopamine release in the retina, which activates dopamine receptors and the downstream PKA pathway. Blocking MAPK signaling diminishes the blue light-induced increases in rhodopsin promoter expression, but this treatment has no effect on white light-mediated rhodopsin promoter expression. Conversely, blocking the PKA pathway diminishes the white light-induced rhodopsin promoter expression but does not affect rhodopsin promoter expression regulated by blue light. Together, the data suggest that MAPK and PKA regulate rhodopsin transcription through parallel signal transduction pathways.

Dopamine modulates voltage-activated potassium currents in zebrafish retinal on bipolar cells.

We report a study of the characterization of voltage-activated potassium (K+) currents in retinal ON bipolar cells in zebrafish. At single-channels levels, the open probability of the K+ channels increased when the membrane potential was increased. The maximal open proportion was 0.76+/-0.05 under our testing conditions. In whole-cell recordings, the K+ current displayed two exponential components with the activation time constants of 11-22 msec (tau1) and 0.8-4 msec (tau2). Dopamine modulated the K+ current. Dopamine reduced the time constant tau2 when the membrane potential was depolarized to high voltages. A decrease in K+ current was seen when dopamine D1 receptors were selectively activated by SKF38393 or when the D1 receptor-coupled G-proteins were activated by GTP-gamma-S. The activation of adenylate cyclase by forskolin or the increase of intracellular cAMP concentrations by 8-Br-cAMP or Sp-cAMPS also resulted in a decrease in K+ current. Together, the data suggest that dopamine modulates the K+ current via D1 receptor-coupled G-protein pathways.