Antitumor effects of IL-12 and GM-CSF co-expressed in an engineered oncolytic HSV-1.

Oncolytic viruses selectively replicate and destroy cancer cells while sparing normal cells, prompting their recognition as promising antitumor agents. Herpes simplex virus (HSV) is suitable as an anticancer agent, given its considerable therapeutic gene capacity and excellent safety profile in clinical trials. Interleukin (IL)-12 induces a Th1-type immune response that mediates interferon (IFN)-γ release from natural killer (NK), CD4+ and CD8+ T cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces the generation of antigen-presenting cells and promotes dendritic cell differentiation. We established a novel oncolytic HSV-1 (∆6/GM/IL12) co-expressing IL-12 and GM-CSF and tested its effects against a B16-F10 murine melanoma model. ∆6/GM/IL12 administration diminished tumor growth and prolonged survival compared to treatment with ∆6/GM or ∆6/IL12 expressing each individual cytokine. Flow cytometry and histological analysis showed increased activation of CD4+ and CD8+ T cells in ∆6/GM/IL12-treated mice. Enzyme-linked immunosorbent spot assay showed an increase in the phenotypically characterized IFN-γ-producing cell population in ∆6/GM/IL12-treated mice. Moreover, ∆6/GM/IL12 induced a B16-F10-specific cytotoxic immune response that enhanced IFN-γ production by CD3+CD8+ T cells. Therefore, IL-12 and GM-CSF from an engineered oncolytic HSV have a synergistic effect, boosting the immune response to increase their antitumor effects.

Theragnosis-based combined cancer therapy using doxorubicin-conjugated microRNA-221 molecular beacon.

Recently, microRNA (miRNA or miR) has emerged as a new cancer biomarker because of its high expression level in various cancer types and its role in the control of tumor suppressor genes. In cancer studies, molecular imaging and treatment based on target cancer markers have been combined to facilitate simultaneous cancer diagnosis and therapy. In this study, for combined therapy with diagnosis of cancer, we developed a doxorubicin-conjugated miR-221 molecular beacon (miR-221 DOXO MB) in a single platform composed of three different nucleotides: miR-221 binding sequence, black hole quencher 1 (BHQ1), and doxorubicin binding site. Imaging of endogenous miR-221 was achieved by specific hybridization between miR-221 and the miR-221 binding site in miR-221 DOXO MB. The presence of miR-221 triggered detachment of the quencher oligo and subsequent activation of a fluorescent signal of miR-221 DOXO MB. Simultaneous cancer therapy in C6 astrocytoma cells and nude mice was achieved by inhibition of miRNA-221 function that downregulates tumor suppressor genes. The detection of miR-221 expression and inhibition of miR-221 function by miR-221 DOXO MB provide the feasibility as a cancer theragnostic probe. Furthermore, a cytotoxic effect was induced by unloading of doxorubicin intercalated into miR-221 DOXO MB inside cells. Loss of miR-221 function and cytotoxicity induced by the miR-221 DOXO MB provides combined therapeutic efficacy against cancers. This method could be used as a new theragnostic probe with enhanced therapy to detect and inhibit many cancer-related miRNAs.

Electro-hyperthermia inhibits glioma tumorigenicity through the induction of E2F1-mediated apoptosis.

PURPOSE: Modulated electro-hyperthermia (mEHT), also known as oncothermia, shows remarkable treatment efficacies for various types of tumours, including glioma. The aim of the present study was to investigate the molecular mechanism underlying phenotypic changes in oncothermic cancer cells. MATERIALS AND METHODS: U87-MG and A172 human glioma cells were exposed to mEHT (42 °C/60 min) three times with a 2-day interval and subsequently tested for growth inhibition using MTS, FACS and microscopic analysis. To obtain insights into the molecular changes in response to mEHT, global changes in gene expression were examined using RNA sequencing. For in vivo evaluation of mEHT, we used U87-MG glioma xenografts grown in nude mice. RESULTS: mEHT inhibited glioma cell growth through the strong induction of apoptosis. The transcriptomic analysis of differential gene expression under mEHT showed that the anti-proliferative effects were induced through a subset of molecular alterations, including the up-regulation of E2F1 and CPSF2 and the down-regulation of ADAR and PSAT1. Subsequent Western blotting revealed that mEHT increased the levels of E2F1 and p53 and decreased the level of PARP-1, accelerating apoptotic signalling in glioma cells. mEHT significantly suppressed the growth of human glioma xenografts in nude mice. We also observed that mEHT dramatically reduced the portion of CD133(+) glioma stem cell population and suppressed cancer cell migration and sphere formation. CONCLUSIONS: These findings suggest that mEHT suppresses glioma cell proliferation and mobility through the induction of E2F1-mediated apoptosis and might be an effective treatment for eradicating brain tumours.

Molecular Targeted Therapy for Hepatocellular Carcinoma: Present Status and Future Directions.

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most lethal neoplasm, causing an estimated 700000 deaths annually. Currently HCC has only one systemic molecular targeted therapy, the multi-kinase inhibitor, sorafenib. The standard-of-care for advanced liver cancer is limited because sorafenib can expand the median life expectancy of patients for only 1 year. Thus there is an urgent need to develop a novel molecular targeted therapy to improve therapeutic outcomes for HCC. HCCs are phenotypically and genetically heterogeneous tumors driven by diverse molecular mechanisms. However, HCCs exhibit certain common traits selected through genetic and epigenetic alterations. The identification of common molecular alterations may provide an opportunity to develop more effective anticancer treatment through targeted therapy. Recent studies in liver cancer biology have revealed a limited number of molecular targets responsible for initiating and maintaining dysregulated cell proliferation, including vascular endothelial growth factor receptor (VEGFR), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR), c-mesenchymal-epithelial transition factor-1 (c-Met), mammalian target of rapamycin (mTOR) and histone deacetylases (HDACs). New treatments involving inhibitors targeting several of these critical pathways are in development. This review describes the current understanding of target pathways, ongoing clinical trials using HCC-targeted agents, and future directions in the treatment of HCC.

Signal transducer and activator of transcription 3-mediated CD133 up-regulation contributes to promotion of hepatocellular carcinoma.

UNLABELLED: Enhanced expression of the cancer stem cell (CSC) marker, CD133, is closely associated with a higher rate of tumor formation and poor prognosis in hepatocellular carcinoma (HCC) patients. Despite its clinical significance, the molecular mechanism underlying the deregulation of CD133 during tumor progression remains to be clarified. Here, we report on a novel mechanism by which interleukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3) signaling up-regulates expression of CD133 and promotes HCC progression. STAT3 activated by IL-6 rapidly bound to CD133 promoter and increased protein levels of CD133 in HCC cells. Reversely, in hypoxic conditions, RNA interference silencing of STAT3 resulted in decrease of CD133 levels, even in the presence of IL-6, with a concomitant decrease of hypoxia-inducible factor 1 alpha (HIF-1α) expression. Active STAT3 interacted with nuclear factor kappa B (NF-κB) p65 subunit to positively regulate the transcription of HIF-1α providing a mechanistic explanation on how those three oncogenes work together to increase the activity of CD133 in a hypoxic liver microenvironment. Activation of STAT3 and its consequent induction of HIF-1α and CD133 expression were not observed in Toll-like receptor 4/IL-6 double-knockout mice. Long-term silencing of CD133 by a lentiviral-based approach inhibited cancer cell-cycle progression and suppressed in vivo tumorigenicity by down-regulating expression of cytokinesis-related genes, such as TACC1, ACF7, and CKAP5. We also found that sorafenib and STAT3 inhibitor nifuroxazide inhibit HCC xenograft formation by blocking activation of STAT3 and expression of CD133 and HIF-1α proteins. CONCLUSION: IL-6/STAT3 signaling induces expression of CD133 through functional cooperation with NF-κB and HIF-1α during liver carcinogenesis. Targeting STAT3-mediated CD133 up-regulation may represent a novel, effective treatment by eradicating the liver tumor microenvironment.

Sperm DNA-mediated reduction of nonspecific fluorescence during cellular imaging with quantum dots.

Salmon sperm DNA was used as a blocking agent to reduce background fluorescence signals from gelatin-coated cell culture dishes.

Antitumor effects in hepatocarcinoma of isoform-selective inhibition of HDAC2.

Histone deacetylase 2 (HDAC2) is a chromatin modifier involved in epigenetic regulation of cell cycle, apoptosis, and differentiation that is upregulated commonly in human hepatocellular carcinoma (HCC). In this study, we show that specific targeting of this HDAC isoform is sufficient to inhibit HCC progression. siRNA-mediated silencing of HDAC inhibited HCC cell growth by blocking cell-cycle progression and inducing apoptosis. These effects were associated with deregulation of HDAC-regulated genes that control cell cycle, apoptosis, and lipid metabolism, specifically, by upregulation of p27 and acetylated p53 and by downregulation of CDK6 and BCL2. We found that HDAC2 silencing in HCC cells also strongly inhibited PPARγ signaling and other regulators of glycolysis (ChREBPα and GLUT4) and lipogenesis (SREBP1C and FAS), eliciting a marked decrease in fat accumulation. Notably, systemic delivery of HDAC2 siRNA encapsulated in lipid nanoparticles was sufficient to blunt the growth of human HCC in a murine xenograft model. Our findings offer preclinical proof-of-concept for HDAC2 blockade as a systemic therapy for liver cancer.

Soil microbial community analysis of between no-till and tillage in a controlled horticultural field.

The present study evaluated the changes of soil microbial communities that were subjected to no-till and compared the results to those subject to tillage for organic farming in a controlled horticultural field by fatty acid methyl ester. Fungi (P < 0.001), gram-positive bacteria (P < 0.001), arbuscular mycorrhizal fungi (P < 0.01), and actinomycetes (P < 0.01) in the no-till soils were significantly larger than those in the tillage soils. The no-till in the subsoil had a significantly lower ratio of cy17:0 to 16:1ω7c compared to that of tillage, indicating that microbial stress decreased because the soils were not disturbed (P < 0.05). Fungi should be considered as a potential factor responsible for the obvious microbial community differentiation that was observed between the no-till and tillage areas in a controlled horticultural field.

Molecular imaging of a cancer-targeting theragnostics probe using a nucleolin aptamer- and microRNA-221 molecular beacon-conjugated nanoparticle.

MicroRNAs (miRNA, miR) have been reported as cancer biomarkers that regulate tumor suppressor genes. Hence, simultaneous detecting and inhibiting of miRNA function will be useful as a cancer theragnostics probe to minimize side effects and invasiveness. In this study, we developed a cancer-targeting therangostics probe in a single system using an AS1411 aptamer - and miRNA-221 molecular beacon (miR-221 MB)-conjugated magnetic fluorescence (MF) nanoparticle (MFAS miR-221 MB) to simultaneously target to cancer tissue, image intracellularly expressed miRNA-221 and treat miRNA-221-involved carcinogenesis. AS1411 aptamer-conjugated MF (MFAS) nanoparticles displayed a great selectivity and delivery into various cancer cell lines. The miR-221 MB detached from the MFAS miR-221 MB in the cytoplasm of C6 cells clearly imaged miRNA-221 biogenesis and simultaneously resulted in antitumor therapeutic effects by inhibiting miRNA function, indicating a successful astrocytoma-targeting theragnostics. MFAS miRNA MB can be easily applied to other cancers by simply changing a targeted miRNA highly expressed in cancers.

Optimizing DC vaccination by combination with oncolytic adenovirus coexpressing IL-12 and GM-CSF.

Dendritic cell (DC)-based vaccination is a promising strategy for cancer immunotherapy. However, clinical trials have indicated that immunosuppressive microenvironments induced by tumors profoundly suppress antitumor immunity and inhibit vaccine efficacy, resulting in insufficient reduction of tumor burdens. To overcome these obstacles and enhance the efficiency of DC vaccination, we generated interleukin (IL)-12- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-coexpressing oncolytic adenovirus (Ad-ΔB7/IL12/GMCSF) as suitable therapeutic adjuvant to eliminate immune suppression and promote DC function. By treating tumors with Ad-ΔB7/IL12/GMCSF prior to DC vaccination, DCs elicited greater antitumor effects than in response to either treatment alone. DC migration to draining lymph nodes (DLNs) dramatically increased in mice treated with the combination therapy. This result was associated with upregulation of CC-chemokine ligand 21 (CCL21(+)) lymphatics in tumors treated with Ad-ΔB7/IL12/GMCSF. Moreover, the proportion of CD4(+)CD25(+) T-cells and vascular endothelial growth factor (VEGF) expression was decreased in mice treated with the combination therapy. Furthermore, combination therapy using immature DCs also showed effective antitumor effects when combined with Ad-ΔB7/IL12/GMCSF. The combination therapy had a remarkable therapeutic efficacy on large tumors. Taken together, oncolytic adenovirus coexpressing IL-12 and GM-CSF in combination with DC vaccination has synergistic antitumor effects and can act as a potent adjuvant for promoting and optimizing DC vaccination.

A multimodal nanoparticle-based cancer imaging probe simultaneously targeting nucleolin, integrin αvß3 and tenascin-C proteins.

Molecular imaging of cancers has been characterized based on the sensitivity and selectivity of a single cancer probe targeting a cancer biomarker of a specific cancer cell line. Here, we designed a multimodal nanoparticle-based Simultaneously Multiple Aptamers and RGD Targeting (SMART) cancer probe targeting multiple cancer biomarkers to enhance the specificity and signal sensitivity for various cancers. Transmission electron microscopy revealed that the multimodal SMART cancer probe was spheric and well dispersed. Fluorescence, radioisotope, and magnetic resonance analysis demonstrated that the SMART cancer probe simultaneously targeting the nucleolin, integrin α(v)ß(3) and Tnc proteins had dramatically enhanced specificity and signal intensity when used to target cancers from C6, NPA, DU145, HeLa and A549 cells when compared with single cancer probes conjugated with AS1411, RGD or TTA1 targeting a single cancer biomarker. The results demonstrated that the SMART cancer probe will be useful for the diagnosis of different cancers as a cancer master probe.

Molecular beacon-based bioimaging of multiple microRNAs during myogenesis.

MicroRNAs (miRNAs, miR) are associated with multiple cellular processes and diseases. Here, we designed fluorescent DNA probes composed of stem loop-structured DNA complementary to miRNAs and fluorophore-quencher pairs [molecular beacon (MB)] to simultaneously monitor the biogenesis of miR-206 and miR-26a, which are highly expressed during myogenic differentiation. C2C12 cells were transfected with an MB targeting miR-26a and containing a 6-FAM-BHQ1 pair (miRNA-26a MB) or an MB targeting miR-206 with a Texas Red-BHQ2 pair (miRNA-206 MB). In vitro and in vivo fluorescence analysis revealed that, only in differentiated single C2C12 cell, significantly increased fluorescence signals of miRNA-26a MB, miRNA-206 MB or simultaneous incubation of both beacons were detected due to the hybridization of miR-206 or miR-26a with their respective beacons, resulting in activation of fluorescence. Our MB-based miRNA imaging system may serve as a new imaging probe for monitoring multiple miRNAs during various cellular or disease processes associated with miRNAs.

Therapeutic and tumor-specific immunity induced by combination of dendritic cells and oncolytic adenovirus expressing IL-12 and 4-1BBL.

Recently, gene-based cytokine treatment has been actively pursued as a new promising approach in treating cancer. In an effort to augment the efficiency of antitumor effect by cytokine-mediated immunotherapy, we selected both interleukin (IL)-12 and 4-1BB ligand (4-1BBL) as suitable cytokines to fully activate the type-1 immune response. Coexpression of IL-12 and 4-1BBL mediated by oncolytic adenovirus (Ad) greatly enhanced the antitumor effect. Further, synergistic enhancement in interferon (IFN)-gamma levels were seen in mice treated with oncolytic Ad expressing both IL-12 and 4-1BBL. Next, to improve the overall antitumor immune response, we coadministered IL-12- and 4-1BBL-coexpressing oncolytic Ad with dendritic cells (DCs). Combination treatment of IL-12- and 4-1BBL-coexpressing oncolytic Ad and DCs elicited greater antitumor and antimetastatic effects than either treatment alone. Moreover, enhanced type-1 antitumor immune response and higher migratory abilities of DCs in tumors were also observed in the combination arms. The nature of the enhanced antitumor immune response seems to be mediated through the enhanced cytolytic activity of cytotoxic T lymphocytes (CTLs) and IFN-gamma-releasing immune cells. Taken together, these data highlight the potential therapeutic benefit of combining IL-12- and 4-1BBL-coexpressing oncolytic Ad with DCs and warrants further evaluation in the clinic.

E1A- and E1B-Double mutant replicating adenovirus elicits enhanced oncolytic and antitumor effects.

Gene-modified replication-competent adenoviruses (Ads) are emerging as a promising new modality for the treatment of cancer. We have previously shown that E1B 19kDa and E1B 55kDa gene-deleted Ad (Ad-DeltaE1B19/55) exhibits improved tumor-specific replication and cell lysis, leading to an enhanced antitumor effect. In an effort to increase cancer cell selectivity of a replicating adenovirus, we first generated 11 E1A mutant Ads (Ad-E1mt1 to Ad-E1mt11) with deletion or substitution in retinoblastoma (pRb)-binding sites of E1A. Of these, Ad-E1mt7 demonstrated significant improvement in cytopathic effect (CPE) and viral replication in a cancer cell-specific manner. To further enhance the cancer cell specificity of Ad-E1mt7, Ad-DeltaE1Bmt7 was generated, in which both the E1B 19kDa and E1B 55kDa genes were deleted. As assessed in CPE assay and immunoblot analysis for Ad fiber expression, Ad-DeltaE1Bmt7 exerted marked enhancement in cancer cell-specific killing as well as viral replication in comparison with its comparative controls (Ad-E1mt7, Ad-DeltaE1B55). Furthermore, the growth of established human cervical carcinoma in nude mice was significantly suppressed by intratumoral injection of Ad-DeltaE1Bmt7. In summary, we have developed an oncolytic adenovirus with a significantly improved therapeutic profile for cancer treatment.

Enhanced antitumor effect of oncolytic adenovirus expressing interleukin-12 and B7-1 in an immunocompetent murine model.

PURPOSE: We investigated whether an armed viral platform, where lytic property of a viral infection is coupled to viral-mediated delivery of therapeutic genes, could increase the therapeutic potential of a viral-based therapy. EXPERIMENTAL DESIGN: We generated interleukin (IL)-12-expressing oncolytic adenovirus (YKL-IL-12) and IL-12- and B7-1-expressing (YKL-IL12/B7) oncolytic adenovirus. Therapeutic efficacy of these newly engineered adenoviruses was then evaluated in vivo using an immunocompetent mouse bearing murine melanoma B16-F10 tumors. Overall survival was assessed with the Kaplan-Meier method. The induction of immune cell cytotoxicity was assessed by CTL assay, IFN-gamma enzyme-linked immunospot assay, and immunohistochemical studies. RESULTS: YKL-IL12/B7 oncolytic adenovirus, expressing both IL-12 and B7-1, showed a higher incidence of complete tumor regression compared with the analogous oncolytic adenovirus, YKL-1, or IL-12-expressing, YKL-IL12. Significant survival advantage was also seen in response to YKL-IL12/B7. Moreover, IL-12 and IFN-gamma levels produced in tumors treated with YKL-IL12/B7 was significantly greater than those treated with YKL-IL12. The enhanced survival advantage was mediated by the induction of immune cell cytotoxicity. In agreement with these results, massive infiltration of CD4(+) and CD8(+) T cells into tissues surrounding the necrotic area of the tumor was observed following in situ delivery of YKL-IL12/B7. CONCLUSION: Combination of oncolysis and the enhancement of antitumor immune response by oncolytic adenovirus expressing both IL-12 and B7-1 elicits potent antitumor effect and survival advantage.