An investigation of the influence of reactive oxygen species produced from riboflavin-5'-phosphate by blue or violet light on the inhibition of WiDr colon cancer cells.

Riboflavin-5'-phosphate (FMN), an innocuous product of riboflavin (RF) phosphorylation, is vital for humans. FMN is sensitive to light illumination, as indicated by reactive oxygen species (ROS) formation. This investigation was undertaken to evaluate the influence of blue light illumination (BLI) and violet light illumination (VLI) upon FMN to develop a method to inhibit WiDr colon cancer cells by FMN photolysis. When FMN is subjected to BLI and VLI, it inhibits WiDr colon cancer cells by generating superoxide radical anions (O2•-). The respective reduction rates are 42.6 and 81.9 % in WiDr colon cancer cells for FMN treated with BLI and VLI at 20 W/m2 for 0.5 h. FMN treated with VLI inhibits WiDr colon cancer cells more effectively than BLI. Propidium iodide (PI) is a fluorescent dye that is used to detect abnormal DNA due to cell death by apoptosis or necrosis. The PI-positive count for nuclei increased significantly for the WiDr colon cancer cells that were treated with FMN under VLI at 20 W/m2 for 0.5 h. FMN photolysis achieved using VLI allows efficient photodynamic therapy (PDT) by triggering the cytotoxicity of FMN on WiDr colon cancer cells.

A flexible liposomal polymer complex as a platform of specific and regulable immune regulation for individual cancer immunotherapy.

BACKGROUND: The applicability and therapeutic efficacy of specific personalized immunotherapy for cancer patients is limited by the genetic diversity of the host or the tumor. Side-effects such as immune-related adverse events (IRAEs) derived from the administration of immunotherapy have also been observed. Therefore, regulatory immunotherapy is required for cancer patients and should be developed. METHODS: The cationic lipo-PEG-PEI complex (LPPC) can stably and irreplaceably adsorb various proteins on its surface without covalent linkage, and the bound proteins maintain their original functions. In this study, LPPC was developed as an immunoregulatory platform for personalized immunotherapy for tumors to address the barriers related to the heterogenetic characteristics of MHC molecules or tumor associated antigens (TAAs) in the patient population. Here, the immune-suppressive and highly metastatic melanoma, B16F10 cells were used to examine the effects of this platform. Adsorption of anti-CD3 antibodies, HLA-A2/peptide, or dendritic cells' membrane proteins (MP) could flexibly provide pan-T-cell responses, specific Th1 responses, or specific Th1 and Th2 responses, depending on the host needs. Furthermore, with regulatory antibodies, the immuno-LPPC complex properly mediated immune responses by adsorbing positive or negative antibodies, such as anti-CD28 or anti-CTLA4 antibodies. RESULTS: The results clearly showed that treatment with LPPC/MP/CD28 complexes activated specific Th1 and Th2 responses, including cytokine release, CTL and prevented T-cell apoptosis. Moreover, LPPC/MP/CD28 complexes could eliminate metastatic B16F10 melanoma cells in the lung more efficiently than LPPC/MP. Interestingly, the melanoma resistance of mice treated with LPPC/MP/CD28 complexes would be reversed to susceptible after administration with LPPC/MP/CTLA4 complexes. NGS data revealed that LPPC/MP/CD28 complexes could enhance the gene expression of cytokine and chemokine pathways to strengthen immune activation than LPPC/MP, and that LPPC/MP/CTLA4 could abolish the LPPC/MP complex-mediated gene expression back to un-treatment. CONCLUSIONS: Overall, we proved a convenient and flexible immunotherapy platform for developing personalized cancer therapy.

Transdermal nanolipoplex simultaneously inhibits subcutaneous melanoma growth and suppresses systemically metastatic melanoma by activating host immunity.

Benefit for clinical melanoma treatments, the transdermal neoadjuvant therapy could reduce surgery region and increase immunotherapy efficacy. Using lipoplex (Lipo-PEG-PEI-complex, LPPC) encapsulated doxorubicin (DOX) and carrying CpG oligodeoxynucleotide; the transdermally administered nano-liposomal drug complex (LPPC-DOX-CpG) would have high cytotoxicity and immunostimulatory activity to suppress systemic metastasis of melanoma. LPPC-DOX-CpG dramatically suppressed subcutaneous melanoma growth by inducing tumor cell apoptosis and recruiting immune cells into the tumor area. Animal studies further showed that the colonization and growth of spontaneously metastatic melanoma cells in the liver and lung were suppressed by transdermal LPPC-DOX-CpG. Furthermore, NGS analysis revealed IFN-γ and NF-κB pathways were triggered to recruit and activate the antigen-presenting-cells and effecter cells, which could activate the anti-tumor responses as the major mechanism responsible for the therapeutic effect of LPPC-DOX-CpG. Finally, we have successfully proved transdermal LPPC-DOX-CpG as a promising penetrative carrier to activate systemic anti-tumor immunity against subcutaneous and metastatic tumor.

Lipoplex-based targeted gene therapy for the suppression of tumours with VEGFR expression by producing anti-angiogenic molecules.

BACKGROUND: The anti-angiogenic fusion protein RBDV-IgG1 Fc (RBDV), which comprises the receptor-binding domain of vascular endothelial growth factor-A (VEGF-A), has shown antitumour effects by reducing angiogenesis in vivo. This study used the cationic lipoplex lipo-PEG-PEI-complex (LPPC) to simultaneously encapsulate both the RBDV targeting protein and the RBDV plasmid (pRBDV) without covalent bonds to assess VEGFR targeting gene therapy in mice with melanoma in vivo. RESULTS: LPPC protected the therapeutic transgene from degradation by DNase, and the LPPC/RBDV complexes could specifically target VEGFR-positive B16-F10 cells both in vitro and in vivo. With or without RBDV protein-targeting direction, the pRBDV-expressing RBDV proteins were expressed and reached a maximal concentration on the 7th day in the sera after transfection in vivo and significantly elicited growth suppression against B16-F10 melanoma but not IgG1 control proteins. In particular, LPPC/pRBDV/RBDV treatment with the targeting molecules dramatically inhibited B16-F10 tumour growth in vivo to provide better therapeutic efficacy than the treatments with gene therapy with IgG1 protein targeting or administration of a protein drug with RBDV. CONCLUSIONS: The simultaneous combination of the LPPC complex with pRBDV gene therapy and RBDV protein targeting might be a potential tool to conveniently administer targeted gene therapy for cancer therapy.

Blockade of ITGA2 Induces Apoptosis and Inhibits Cell Migration in Gastric Cancer.

BACKGROUND: Gastric cancer is currently the fourth leading cause of cancer-related death worldwide. Gastric cancer is often diagnosed at advanced stages and the outcome of the treatment is often poor. Therefore, identifying new therapeutic targets for this cancer is urgently needed. Integrin alpha 2 (ITGA2) subunit and the beta 1 subunit form a heterodimer for a transmembrane receptor for extracellular matrix, is an important molecule involved in tumor cell proliferation, survival and migration. Integrin α2ß1 is over-expressed on a variety of cancer cells, but is low or absent in most normal organs and resting endothelial cells. RESULTS: In this report, we assessed the ITGA2 as the potential therapeutic target with the bioinformatics tools from the TCGA dataset in which composed of 375 gastric cancer tissues and 32 gastric normal tissues. According to the information from the Cancer Cell Line Encyclopedia (CCLE) database, the AGS cell line with ITGA2 high expression and the SUN-1 cell line with low expression were chosen for the further investigation. Interestingly, the anti-ITGA2 antibody (at 3 µg/ml) inhibited approximately 50% survival of the AGS cells (over-expressed ITGA2), but had no effect in SNU-1 cells (ITGA2 negative). The extents of antibody-mediated cancer inhibition positively correlated with the expression levels of the ITGA2. We further showed that the anti-ITGA2 antibody induced apoptosis by up-regulating the RhoA-p38 MAPK signaling to promote the expressions of Bim, Apaf-1 and Caspase-9, whereas the expressions of Ras and Bax/Bcl-2 were not affected. Moreover, blocking ITGA2 by the specific antibody at lower doses also inhibited cell migration of gastric cancer cells. Blockade of ITGA2 by a specific antibody down-regulated the expression of N-WASP, PAK and LIMK to impede actin organization and cell migration of gastric cancer cells. CONCLUSIONS: Here, we showed that the mRNA expression levels of ITGA2 comparing to normal tissues significantly increased. In addition, the results revealed that targeting integrin alpha 2 subunit by antibodies did not only inhibit cell migration, but also induce apoptosis effect on gastric cancer cells. Interestingly, higher expression level of ITGA2 led to significant effects on apoptosis progression during anti-ITGA2 antibody treatment, which indicated that ITGA2 expression levels directly correlate with their functionality. Our findings suggest that ITGA2 is a potential therapeutic target for gastric cancer.

The inhibitory effect of 7,7″-dimethoxyagastisflavone on the metastasis of melanoma cells via the suppression of F-actin polymerization.

7,7″-Dimethoxyagastisflavone (DMGF), a biflavonoid isolated from Taxus × media cv. Hicksii, induces apoptotic and autophagic cell death. However, whether DMGF suppresses tumor metastasis is unclear. The aim of this study was to investigate the anti-metastatic activities of DMGF on the metastatic processes of melanoma cells in vivo and in vitro. A transwell assay showed that DMGF could effectively attenuate the motility of B16F10 cells, and the results of real-time PCR revealed that DMGF also suppressed the expressions of matrix metalloproteinase-2 (MMP-2). Moreover, DMGF did not influence tube formation but inhibited the migration of endothelial cells. Furthermore, animal models were used to monitor the effects of DMGF on tumor metastasis, and all models showed that DMGF significantly suppressed the metastatic behaviors of B16F10 cells, including intravasation, colonization, and invasion of the lymphatic duct. In addition, DMGF could also reduce the densities of the blood vessels in the tumor area in vivo. Further investigation of the molecular mechanisms of anti-metastatic activity revealed that DMGF can down-regulate the levels of key modulators of the Cdc42/Rac1 pathway to interfere in F-actin polymerization and suppress the formation of lamellipodia by reducing the phosphorylation of CREB. These data suggested that DMGF presents anti-metastatic activities in B16F10 melanoma cells. Here, we demonstrated that DMGF can inhibit the metastasis of highly invasive melanoma cancer cells through the down-regulation of F-actin polymerization. Considering these findings, DMGF may be further developed to serve as a chemoprevention drug for patients with metastatic melanoma.

miRTarBase 2016: updates to the experimentally validated miRNA-target interactions database.

MicroRNAs (miRNAs) are small non-coding RNAs of approximately 22 nucleotides, which negatively regulate the gene expression at the post-transcriptional level. This study describes an update of the miRTarBase (http://miRTarBase.mbc.nctu.edu.tw/) that provides information about experimentally validated miRNA-target interactions (MTIs). The latest update of the miRTarBase expanded it to identify systematically Argonaute-miRNA-RNA interactions from 138 crosslinking and immunoprecipitation sequencing (CLIP-seq) data sets that were generated by 21 independent studies. The database contains 4966 articles, 7439 strongly validated MTIs (using reporter assays or western blots) and 348 007 MTIs from CLIP-seq. The number of MTIs in the miRTarBase has increased around 7-fold since the 2014 miRTarBase update. The miRNA and gene expression profiles from The Cancer Genome Atlas (TCGA) are integrated to provide an effective overview of this exponential growth in the miRNA experimental data. These improvements make the miRTarBase one of the more comprehensively annotated, experimentally validated miRNA-target interactions databases and motivate additional miRNA research efforts.