Discovery of the Novel 1H-Pyrrolo[2,3-b]pyridine Derivative as a Potent Type II CDK8 Inhibitor against Colorectal Cancer.

Few targeted drugs were approved for treatment of colorectal cancer (CRC). Cyclin-dependent kinase 8 played a vital role in regulating transcription and was a key colorectal oncogene associated to colorectal cancer. Here, through de novo drug design and in depth structure-activity relationship analysis, title compound 22, (3-(3-(1H-pyrrolo[2,3-b]pyridin-5-yl)phenyl)-N-(4-methyl-3-(trifluoromethyl)phenyl)propenamide), was discovered as a potent type II CDK8 inhibitor, which exhibited potent kinase activity with an IC50 value of 48.6 nM and could significantly inhibit tumor growth in xenografts of CRC in vivo. Further mechanism studies indicated that it could target CDK8 to indirectly inhibit ß-catenin activity, which caused downregulation of the WNT/ß-catenin signal and inducing cell cycle arrest in G2/M and S phases. More importantly, the title compound exhibited low toxicity with good bioavailability (F = 39.8%). These results could provide the reference for design of new type II CDK8 inhibitors against colorectal cancer.

PTK7, a Catalytically Inactive Receptor Tyrosine Kinase, Increases Oncogenic Phenotypes in Xenograft Tumors of Esophageal Squamous Cell Carcinoma KYSE-30 Cells.

Protein tyrosine kinase 7 (PTK7), a catalytically defective receptor protein tyrosine kinase, is upregulated in tumor tissues and cell lines of esophageal squamous cell carcinoma (ESCC). We showed that PTK7 plays an oncogenic role in various ESCC cell lines. However, its role as an oncogene has not been demonstrated in vivo. Here, we examined the influence of PTK7 on the tumorigenic potential of ESCC KYSE-30 cells, which are known to establish xenograft tumors. Overexpression of PTK7 enhanced the proliferation, adhesion, wound healing, and migration of KYSE-30 cells, and these effects were reversed by the knockdown of PTK7. PTK7 overexpression and knockdown, respectively, increased and decreased the tyrosine phosphorylation of cellular proteins and the phosphorylation of ERK, AKT, and FAK, which are important for cell proliferation, survival, adhesion, and migration. Additionally, PTK7 overexpression and silencing, respectively, increased and decreased the weight, volume, and number of Ki-67-positive proliferating cells in xenograft tumors of KYSE-30 cells. Therefore, we propose that PTK7 plays an important role in the tumorigenesis of ESCC cells in vivo and is a potential therapeutic target for ESCC.

Radiosynthesis of a novel antisense imaging probe targeting LncRNA HOTAIR in malignant glioma.

BACKGROUND: Long non-coding RNA (LncRNA) HOTAIR was amplified and overexpressed in many human carcinomas, which could serve as a useful target for cancer early detection and treatment. The 99mTc radiolabeled antisense oligonucleotides (ASON) could visualize the expression of HOTAIR and provide a diagnostic value for malignant tumors. The aim of this study was to evaluate whether liposome-coated antisense oligonucleotide probe 99mTc-HYNIC-ASON targeting HOTAIR can be used in in vivo imaging of HOTAIR in malignant glioma xenografts. METHODS: The ASON targeting LncRNA HOTAIR as well as mismatched ASON (ASONM) were designed and modified. The radiolabeling of 99mTc with two probes were via the conjugation of bifunctional chelator HYNIC. Then probes were purified by Sephadex G25 and tested for their radiolabeling efficiency and purity, as well as stability by ITLC (Instant thin-layer chromatography) and gel electrophoresis. Then the radiolabeled probes were transfected with lipofectamine 2000 for cellular uptake test and the next experimental use. Furthermore, biodistribution study and SPECT imaging were performed at different times after liposome-coated 99mTc-HYNIC-ASON/ASONM were intravenously injected in glioma tumor-bearing mice models. All data were analyzed by statistical software. RESULTS: The labeling efficiencies of 99mTc-HYNIC-ASON and 99mTc-HYNIC-ASONM measured by ITLC were (91 ± 1.5) % and (90 ± 0.6) %, respectively, and both radiochemical purities were more than 89%. Two probes showed good stability within 12 h. Gel electrophoresis confirmed that the oligomers were successfully radiolabeled no significant degradation were found. Biodistribution study demonstrated that liposome-coated antisense probes were excreted mainly through the kidney and bladder and has higher uptake in the tumor. Meanwhile, the tumor was clearly shown after injection of liposome coated 99mTc-HYNIC-ASON, and its T/M ratio was higher than that in the non-transfection group and mismatched group. No tumor was seen in mismatched and blocking group. CONCLUSION: The liposome encapsulated 99mTc-HYNIC-ASON probe can be used in the in vivo, real-time imaging of LncRNA HOTAIR expression in malignant glioma.

Targeting Folate Metabolism Is Selectively Cytotoxic to Glioma Stem Cells and Effectively Cooperates with Differentiation Therapy to Eliminate Tumor-Initiating Cells in Glioma Xenografts.

Glioblastoma (GBM) is one of the deadliest of all human cancers. Developing therapies targeting GBM cancer stem cells or glioma stem cells (GSCs), which are deemed responsible for the malignancy of GBM due to their therapy resistance and tumor-initiating capacity, is considered key to improving the dismal prognosis of GBM patients. In this study, we found that folate antagonists, such as methotrexate (MTX) and pemetrexed, are selectively cytotoxic to GSCs, but not to their differentiated counterparts, normal fibroblasts, or neural stem cells in vitro, and that the high sensitivity of GCSs to anti-folates may be due to the increased expression of RFC-1/SLC19A1, the reduced folate carrier that transports MTX into cells, in GSCs. Of note, in an in vivo serial transplantation model, MTX alone failed to exhibit anti-GSC effects but promoted the anti-GSC effects of CEP1347, an inducer of GSC differentiation. This suggests that folate metabolism, which plays an essential role specifically in GSCs, is a promising target of anti-GSC therapy, and that the combination of cytotoxic and differentiation therapies may be a novel and promising approach to effectively eliminate cancer stem cells.

MicroRNA-99a-3p/GRP94 axis affects metastatic progression of human papillary thyroid carcinoma by regulating ITGA2 expression and localization.

Papillary thyroid cancer (PTC) usually has favorable prognosis; however, distant metastasis is a leading cause of death associated with PTC. MicroRNA-99a-3p (miR-99a-3p) is a member of the miR-99 family that is shown to be a tumor suppressor in various human cancers including the anaplastic thyroid cancer, another type of thyroid cancer. The Cancer Genome Atlas database and our previous study reported that miR-99a-3p is downregulated in human PTC tissues as well as human papillary thyroid carcinoma B-CPAP and TPC-1 cell lines. However, its pathological role in PTC remains unclear, especially its impact on PTC metastasis. In the present study, the role of miR-99a-3p in PTC metastasis was molecularly evaluated in in vitro and in vivo models. Our functional study revealed that overexpressing miR-99a-3p significantly suppresses epithelial-mesenchymal transition (EMT) and anoikis resistance as well as migration and invasion of B-CPAP and TPC-1 cells. The mechanical study indicated that glucose-regulated protein 94 (GRP94) is the direct target of miR-99a-3p. Moreover, GRP94 overexpression reverses the inhibitory effect of miR-99a-3p on PTC metastasis. In addition, the miR-99a-3p/GRP94 axis exerts its effect via inhibiting the expression and cytoplasmic relocation of integrin 2α (ITGA2). Furthermore, in vivo experiments confirmed that miR-99a-3p significantly inhibits tumor growth and lung metastasis in PTC xenograft mice. Overall, our findings suggested that the miR-99a-3p/GRP94/ITGA2 axis may be a novel therapeutic target for the prevention of PTC metastasis.

An immune-humanized patient-derived xenograft model of estrogen-independent, hormone receptor positive metastatic breast cancer.

BACKGROUND: Metastatic breast cancer (MBC) is incurable, with a 5-year survival rate of 28%. In the USA, more than 42,000 patients die from MBC every year. The most common type of breast cancer is estrogen receptor-positive (ER+), and more patients die from ER+ breast cancer than from any other subtype. ER+ tumors can be successfully treated with hormone therapy, but many tumors acquire endocrine resistance, at which point treatment options are limited. There is an urgent need for model systems that better represent human ER+ MBC in vivo, where tumors can metastasize. Patient-derived xenografts (PDX) made from MBC spontaneously metastasize, but the immunodeficient host is a caveat, given the known role of the immune system in tumor progression and response to therapy. Thus, we attempted to develop an immune-humanized PDX model of ER+ MBC. METHODS: NSG-SGM3 mice were immune-humanized with CD34+ hematopoietic stem cells, followed by engraftment of human ER+ endocrine resistant MBC tumor fragments. Strategies for exogenous estrogen supplementation were compared, and immune-humanization in blood, bone marrow, spleen, and tumors was assessed by flow cytometry and tissue immunostaining. Characterization of the new model includes assessment of the human tumor microenvironment performed by immunostaining. RESULTS: We describe the development of an immune-humanized PDX model of estrogen-independent endocrine resistant ER+ MBC. Importantly, our model harbors a naturally occurring ESR1 mutation, and immune-humanization recapitulates the lymphocyte-excluded and myeloid-rich tumor microenvironment of human ER+ breast tumors. CONCLUSION: This model sets the stage for development of other clinically relevant models of human breast cancer and should allow future studies on mechanisms of endocrine resistance and tumor-immune interactions in an immune-humanized in vivo setting.

IFN-γ Licensing Does Not Enhance the Reduced Immunomodulatory Potential and Migratory Ability of Differentiation-Induced Porcine Bone Marrow-Derived Mesenchymal Stem Cells in an In Vitro Xenogeneic Application.

IFN-γ licensing to mesenchymal stem cells (MSCs) is applied to enhance the therapeutic potential of MSCs. However, although the features of MSCs are affected by several stimuli, little information is available on changes to the therapeutic potential of IFN-γ-licensed differentiated MSCs during xenogeneic applications. Therefore, the present study is aimed at clarifying the effects of adipogenic/osteogenic differentiation and IFN-γ licensing on the in vitro immunomodulatory and migratory properties of porcine bone marrow-derived MSCs in xenogeneic applications using human peripheral blood mononuclear cells (PBMCs). IFN-γ licensing in differentiated MSCs lowered lineage-specific gene expression but did not affect MSC-specific cell surface molecules. Although indoleamine 2,3 deoxygenase (IDO) activity and expression were increased after IFN-γ licensing in undifferentiated MSCs, they were reduced after differentiation. IFN-γ licensing to differentiated MSCs elevated the reduced IDO expression in differentiated MSCs; however, the increase was not sufficient to reach to the level achieved by undifferentiated MSCs. During a mixed lymphocyte reaction with quantification of TNF-α concentration, proliferation and activation of xenogeneic PBMCs were suppressed by undifferentiated MSCs but inhibited to a lesser extent by differentiated MSCs. IFN-γ licensing increasingly suppressed proliferation of PBMCs in undifferentiated MSCs but it was incapable of elevating the reduced immunosuppressive ability of differentiated MSCs. Migratory ability through a scratch assay and gene expression study was reduced in differentiated MSCs than their undifferentiated counterparts; IFN-γ licensing was unable to enhance the reduced migratory ability in differentiated MSCs. Similar results were found in a Transwell system with differentiated MSCs in the upper chamber toward xenogeneic PBMCs in the lower chamber, despite IFN-γ licensing increased the migratory ability of undifferentiated MSCs. Overall, IFN-γ licensing did not enhance the reduced immunomodulatory and migratory properties of differentiated MSCs in a xenogeneic application. This study provides a better understanding of the ways in which MSC therapy can be applied.

Knockdown of DEAD-box RNA helicase 52 (DDX52) suppresses the proliferation of melanoma cells in vitro and of nude mouse xenografts by targeting c-Myc.

The ATP-dependent protein DEAD-box RNA helicase 52 (DDX52) is an important regulator in RNA biology and has been implicated in the development of prostate and lung cancer. However, its biological functions and clinical importance in malignant melanoma (MM) are still unclear. Understanding the potential mechanism underlying the regulation of MM progression by DDX52 might lead to novel therapeutic strategies. The aim of the present study was to investigate the role of DDX52 in the regulation of MM progression and its clinical relevance. DDX52 expression in normal and MM tissues was evaluated by GEO analysis and immunohistochemistry. The effects of DDX52 on cell growth were evaluated in MM cells with downregulated DDX52 expression. In this study, we found that DDX52 was markedly overexpressed in MM tissues compared with nontumor tissues and was associated with shorter overall survival in patients; therefore, DDX52 might be a prognostic marker in MM. Downregulation of DDX52 expression in the MM cell lines A2058 and MV3 markedly inhibited cell proliferation and colony formation. Additionally, knockdown of DDX52 in MM cells caused significant regression of established tumors in nude mice and delayed the onset time. Moreover, downregulation of DDX52 markedly suppressed c-Myc mRNA and protein expression, and an RNA immunoprecipitation assay confirmed the association between DDX52 and c-Myc. Restoration of c-Myc expression partly rescued the effects of DDX52 deficiency in MM cells. In conclusion, our study found that DDX52 mediated oncogenesis by promoting the transcriptional activity of c-Myc and could be a therapeutic target in MM.

MICAL1 regulates actin cytoskeleton organization, directional cell migration and the growth of human breast cancer cells as orthotopic xenograft tumours.

The Molecule Interacting with CasL 1 (MICAL1) monooxygenase has emerged as an important regulator of cytoskeleton organization via actin oxidation. Although filamentous actin (F-actin) increases MICAL1 monooxygenase activity, hydrogen peroxide (H2O2) is also generated in the absence of F-actin, suggesting that diffusible H2O2 might have additional functions. MICAL1 gene disruption by CRISPR/Cas9 in MDA MB 231 human breast cancer cells knocked out (KO) protein expression, which affected F-actin organization, cell size and motility. Transcriptomic profiling revealed that MICAL1 deletion significantly affected the expression of over 700 genes, with the majority being reduced in their expression levels. In addition, the absolute magnitudes of reduced gene expression were significantly greater than the magnitudes of increased gene expression. Gene set enrichment analysis (GSEA) identified receptor regulator activity as the most significant negatively enriched molecular function gene set. The prominent influence exerted by MICAL1 on F-actin structures was also associated with changes in the expression of several serum-response factor (SRF) regulated genes in KO cells. Moreover, MICAL1 disruption attenuated breast cancer tumour growth in vivo. Elevated MICAL1 gene expression was observed in invasive breast cancer samples from human patients relative to normal tissue, while MICAL1 amplification or point mutations were associated with reduced progression free survival. Collectively, these results demonstrate that MICAL1 gene disruption altered cytoskeleton organization, cell morphology and migration, gene expression, and impaired tumour growth in an orthotopic in vivo breast cancer model, suggesting that pharmacological MICAL1 inhibition could have therapeutic benefits for cancer patients.

Ex Vivo and In Vivo Analysis of a Novel Porcine Aortic Patch for Vascular Reconstruction.

(1) Background: The aim of the present study was the biocompatibility analysis of a novel xenogeneic vascular graft material (PAP) based on native collagen won from porcine aorta using the subcutaneous implantation model up to 120 days post implantationem. As a control, an already commercially available collagen-based vessel graft (XenoSure®) based on bovine pericardium was used. Another focus was to analyze the (ultra-) structure and the purification effort. (2) Methods: Established methodologies such as the histological material analysis and the conduct of the subcutaneous implantation model in Wistar rats were applied. Moreover, established methods combining histological, immunohistochemical, and histomorphometrical procedures were applied to analyze the tissue reactions to the vessel graft materials, including the induction of pro- and anti-inflammatory macrophages to test the immune response. (3) Results: The results showed that the PAP implants induced a special cellular infiltration and host tissue integration based on its three different parts based on the different layers of the donor tissue. Thereby, these material parts induced a vascularization pattern that branches to all parts of the graft and altogether a balanced immune tissue reaction in contrast to the control material. (4) Conclusions: PAP implants seemed to be advantageous in many aspects: (i) cellular infiltration and host tissue integration, (ii) vascularization pattern that branches to all parts of the graft, and (iii) balanced immune tissue reaction that can result in less scar tissue and enhanced integrative healing patterns. Moreover, the unique trans-implant vascularization can provide unprecedented anti-infection properties that can avoid material-related bacterial infections.

Molecular Imaging and Preclinical Studies of Radiolabeled Long-Term RGD Peptides in U-87 MG Tumor-Bearing Mice.

The Arg-Gly-Asp (RGD) peptide shows a high affinity for αvß3 integrin, which is overexpressed in new tumor blood vessels and many types of tumor cells. The radiolabeled RGD peptide has been studied for cancer imaging and radionuclide therapy. We have developed a long-term tumor-targeting peptide DOTA-EB-cRGDfK, which combines a DOTA chelator, a truncated Evans blue dye (EB), a modified linker, and cRGDfK peptide. The aim of this study was to evaluate the potential of indium-111(111In) radiolabeled DOTA-EB-cRGDfK in αvß3 integrin-expressing tumors. The human glioblastoma cell line U-87 MG was used to determine the in vitro binding affinity of the radiolabeled peptide. The in vivo distribution of radiolabeled peptides in U-87 MG xenografts was investigated by biodistribution, nanoSPECT/CT, pharmacokinetic and excretion studies. The in vitro competition assay showed that 111In-DOTA-EB-cRGDfK had a significant binding affinity to U-87 MG cancer cells (IC50 = 71.7 nM). NanoSPECT/CT imaging showed 111In-DOTA-EB-cRGDfK has higher tumor uptake than control peptides (111In-DOTA-cRGDfK and 111In-DOTA-EB), and there is still a clear signal until 72 h after injection. The biodistribution results showed significant tumor accumulation (27.1 ± 2.7% ID/g) and the tumor to non-tumor ratio was 22.85 at 24 h after injection. In addition, the pharmacokinetics results indicated that the 111In-DOTA-EB-cRGDfK peptide has a long-term half-life (T1/2λz = 77.3 h) and that the calculated absorbed dose was safe for humans. We demonstrated that radiolabeled DOTA-EB-cRGDfK may be a promising agent for glioblastoma tumor imaging and has the potential as a theranostic radiopharmaceutical.

Landscapes of cellular phenotypic diversity in breast cancer xenografts and their impact on drug response.

The heterogeneity of breast cancer plays a major role in drug response and resistance and has been extensively characterized at the genomic level. Here, a single-cell breast cancer mass cytometry (BCMC) panel is optimized to identify cell phenotypes and their oncogenic signalling states in a biobank of patient-derived tumour xenograft (PDTX) models representing the diversity of human breast cancer. The BCMC panel identifies 13 cellular phenotypes (11 human and 2 murine), associated with both breast cancer subtypes and specific genomic features. Pre-treatment cellular phenotypic composition is a determinant of response to anticancer therapies. Single-cell profiling also reveals drug-induced cellular phenotypic dynamics, unravelling previously unnoticed intra-tumour response diversity. The comprehensive view of the landscapes of cellular phenotypic heterogeneity in PDTXs uncovered by the BCMC panel, which is mirrored in primary human tumours, has profound implications for understanding and predicting therapy response and resistance.

MLL5 improves ATRA driven differentiation and promotes xenotransplant engraftment in acute promyelocytic leukemia model.

Although the mixed lineage leukemia 5 (MLL5) gene has prognostic implications in acute promyelocyte leukemia (APL), the underlying mechanism remains to be elucidated. Here, we demonstrate the critical role exerted by MLL5 in APL regarding cell proliferation and resistance to drug-induced apoptosis, through mtROS regulation. Additionally, MLL5 overexpression increased the responsiveness of APL leukemic cells to all-trans retinoic acid (ATRA)-induced differentiation, via regulation of the epigenetic modifiers SETD7 and LSD1. In silico analysis indicated that APL blasts with MLL5high transcript levels were associated with retinoic acid binding and downstream signaling, while MLL5low blasts displayed decreased expression of epigenetic modifiers (such as KMT2C, PHF8 and ARID4A). Finally, APL xenograft transplants demonstrated improved engraftment of MLL5-expressing cells and increased myeloid differentiation over time. Concordantly, evaluation of engrafted blasts revealed increased responsiveness of MLL5-expressing cells to ATRA-induced granulocytic differentiation. Together, we describe the epigenetic changes triggered by the interaction of MLL5 and ATRA resulting in enhanced granulocytic differentiation.

Presence of complete murine viral genome sequences in patient-derived xenografts.

Patient-derived xenografts are crucial for drug development but their use is challenged by issues such as murine viral infection. We evaluate the scope of viral infection and its impact on patient-derived xenografts by taking an unbiased data-driven approach to analyze unmapped RNA-Seq reads from 184 experiments. We find and experimentally validate the extensive presence of murine viral sequence reads covering entire viral genomes in patient-derived xenografts. The existence of viral sequences inside tumor cells is further confirmed by single cell sequencing data. Extensive chimeric reads containing both viral and human sequences are also observed. Furthermore, we find significantly changed expression levels of many cancer-, immune-, and drug metabolism-related genes in samples with high virus load. Our analyses indicate a need to carefully evaluate the impact of viral infection on patient-derived xenografts for drug development. They also point to a need for attention to quality control of patient-derived xenograft experiments.

Structure-Based Design of a Selective Class I Histone Deacetylase (HDAC) Near-Infrared (NIR) Probe for Epigenetic Regulation Detection in Triple-Negative Breast Cancer (TNBC).

Abnormally high levels of class I histone deacetylases (HDACs) are associated with triple-negative breast cancer (TNBC) proliferation, malignant transformation, and poor prognosis of patients. Herein, we report a near-infrared imaging probe for TNBC detection via visualizing class I HDACs. Conjugating Cy5.5 to a cyclic depsipeptide inhibitor, we obtained the probe (20-Cy5.5) that retained desirable class I HDAC affinity and selectivity. Then, this probe could visualize epigenetic changes by class I HDACs in TNBC MDA-MB-231 cells and in xenograft tumor models in real time. Treatment with suberoylanilide hydroxamic acid (SAHA) significantly reduced the uptake of the probe in tumors, suggesting its potential use in evaluation of therapeutic responses of HDACi-mediated therapy. Moreover, 20-Cy5.5 could detect class I HDAC expression in TNBC lung metastasis. This novel NIR probe that achieves tumor class I HDAC imaging not only leads to a better understanding of epigenetic regulation in tumors but also has great potential for improving the TNBC diagnosis and treatment.

Imaging of Fibroblast Activation Protein in Cancer Xenografts Using Novel (4-Quinolinoyl)-glycyl-2-cyanopyrrolidine-Based Small Molecules.

Fibroblast activation protein (FAP) has become a favored target for imaging and therapy of malignancy. We have synthesized and characterized two new (4-quinolinoyl)-glycyl-2-cyanopyrrolidine-based small molecules for imaging of FAP, QCP01 and [111In]QCP02, using optical and single-photon computed tomography/CT, respectively. Binding of imaging agents to FAP was assessed in six human cancer cell lines of different cancer types: glioblastoma (U87), melanoma (SKMEL24), prostate (PC3), NSCLC (NCIH2228), colorectal carcinoma (HCT116), and lung squamous cell carcinoma (NCIH226). Mouse xenograft models were developed with FAP-positive U87 and FAP-negative PC3 cells to test pharmacokinetics and binding specificity in vivo. QCP01 and [111In]QCP02 demonstrated nanomolar inhibition of FAP at Ki values of 1.26 and 16.20 nM, respectively. Both were selective for FAP over DPP-IV, a related serine protease. Both enabled imaging of FAP-expressing tumors specifically in vivo. [111In]QCP02 showed high uptake at 18.2 percent injected dose per gram in the U87 tumor at 30 min post-administration.

A Xenograft Model for Venous Malformation.

Xenograft models allow for an in vivo approach to monitor cellular functions within the context of a host microenvironment. Here we describe a protocol to generate a xenograft model of venous malformation (VM) based on the use of human umbilical vein endothelial cells (HUVEC) expressing a constitutive active form of the endothelial tyrosine kinase receptor TEK (TIE2 p.L914F) or patient-derived EC containing TIE2 and/or PIK3CA gene mutations. Hyperactive somatic TIE2 and PIK3CA mutations are a common hallmark of VM in patient lesions. The EC are injected subcutaneously on the back of athymic nude mice to generate ectatic vascular channels and recapitulate histopathological features of VM patient tissue histology. Lesion plugs with TIE2/PIK3CA-mutant EC are visibly vascularized within 7-9 days of subcutaneous injection, making this a great tool to study venous malformation.

LINC00460 Enhances Bladder Carcinoma Cell Proliferation and Migration by Modulating miR-612/FOXK1 Axis.

INTRODUCTION: LincRNA (long intergenic noncoding RNA) has been indicated as a mediator in tumorigenesis of bladder carcinoma. This study was performed to evaluate the role of LINC00460 in bladder carcinoma progression. METHODS: Expression levels of LINC00460 in bladder carcinoma tissues and cell lines were analyzed via qRT-PCR. MTT, EdU (5-ethynyl-2'-deoxyuridine) staining, and colony formation assays were utilized to evaluate cell viability and proliferation. The wound healing assay was performed to evaluate bladder cancer cell migration, and the transwell assay was used to evaluate cell invasion. The microRNA (miRNA) target of LINC00460 and the corresponding target gene were validated via the dual luciferase activity assay. The tumorigenic function of LINC00460 was determined via establishment of a xenotransplanted tumor model. RESULTS: LINC00460 was elevated in bladder carcinoma tissues and cell lines. Elevated LINC00460 was associated with shorter overall survival of bladder carcinoma patients. Overexpression of LINC00460 promoted cell viability, proliferation, invasion, and migration, while silencing of LINC00460 indicated the opposite effect on bladder carcinoma progression. LINC00460 could directly bind to miR-612 and inhibit miR-612 expression. Moreover, LINC00460 expression was negatively correlated with miR-612 in patients with bladder carcinoma. FOXK1 (Forkhead Box K1) was identified as the target of miR-612 and upregulated in patients with bladder carcinoma. Overexpression of FOXK1 attenuated interference of LINC00460-inhibited bladder carcinoma progression. Knockdown of LINC00460 suppressed in vivo bladder carcinoma growth. CONCLUSIONS: LINC00460 promoted bladder carcinoma progression via sponging miR-612 to facilitate FOXK1 expression, suggesting that LINC00460 might have the potential of being explored as a therapeutic target for treatment of bladder carcinoma.

125I seeds irradiation inhibits tumor growth and induces apoptosis by Ki-67, P21, survivin, livin and caspase-9 expression in lung carcinoma xenografts.

BACKGROUND: Lung cancer is a fatal disease and a serious health problem worldwide. Patients are usually diagnosed at an advanced stage, and the effectiveness of chemotherapy for such patients is very limited. Iodine 125 seed (125I) irradiation can be used as an important adjuvant treatment for lung carcinoma. The purpose of this study was to examine the role of irradiation by 125I seeds in human lung cancer xenograft model and to determine the underlying mechanisms involved, with a focus on apoptosis. METHODS: 40 mice with A549 lung adenocarcinoma xenografts were randomly divided into 4 groups: control group (n = 10), sham seed (0 mCi) implant group (n = 10), 125I seed (0.6 mCi) implant group (n = 10) and 125I seed (0.8 mCi) implant group (n = 10), respectively. The body weight and tumor volume, were recorded every 4 days until the end of the study. Apoptotic cells were checked by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and activities of caspase-3 and caspase-8 enzyme were tested. Expression of P21, survivin, livin, caspase-9 and proliferating cell nuclear antigen (Ki-67) was detected with immunohistochemical staining. RESULTS: The results of TUNEL staining assays showed that 125I seed irradiation suppresses the growth of lung cancer xenografts in nude mice and induced apoptosis. The activity of caspase-3 and caspase-8 was significantly higher. The expression levels Ki67, survivin and livin were substantially downregulated, while P21 and caspase-9 protein expression were significantly increased following 125I seed irradiation. This study revealed that 125I seed irradiation could significantly change apoptosis-related protein in human lung cancer xenografts. CONCLUSIONS: Overall, our study demonstrates that radiation exposure by 125I seeds could be a new treatment option for lung cancer.