The In Vivo Therapeutic Efficacy of the Oncolytic Adenovirus Delta24-RGD Is Mediated by Tumor-Specific Immunity

The oncolytic adenovirus Delta24-RGD represents a new promising therapeutic agent for patients with a malignant glioma and is currently under investigation in clinical phase I/II trials. Earlier preclinical studies showed that Delta24-RGD is able to effectively lyse tumor cells, yielding promising results in various immune-deficient glioma models. However, the role of the immune response in oncolytic adenovirus therapy for glioma has never been explored. To this end, we assessed Delta24-RGD treatment in an immune-competent orthotopic mouse model for glioma and evaluated immune responses against tumor and virus. Delta24-RGD treatment led to long-term survival in 50% of mice and this effect was completely lost upon administration of the immunosuppressive agent dexamethasone. Delta24-RGD enhanced intra-tumoral infiltration of F4/80+ macrophages, CD4+ and CD8+ T-cells, and increased the local production of pro-inflammatory cytokines and chemokines. In treated mice, T cell responses were directed to the virus as well as to the tumor cells, which was reflected in the presence of protective immunological memory in mice that underwent tumor rechallenge. Together, these data provide evidence that the immune system plays a vital role in the therapeutic efficacy of oncolytic adenovirus therapy of glioma, and may provide angles to future improvements on Delta24-RGD therapy.     

HDAC Inhibitors Increase NRF2-Signaling in Tumour Cells and Blunt the Efficacy of Co-Adminstered Cytotoxic Agents

The NRF2 signalling cascade provides a primary response against electrophilic chemicals and oxidative stress. The activation of NRF2-signaling is anticipated to have adverse clinical consequences; NRF2 is activated in a number of cancers and, additionally, its pharmacological activation by one compound can reduce the toxicity or efficiency of a second agent administered concomitantly. In this work, we have analysed systematically the ability of 152 research, pre-clinical or clinically used drugs to induce an NRF2 response using the MCF7-AREc32 NRF2 reporter. Ten percent of the tested drugs induced an NRF2 response. The NRF2 activators were not restricted to classical cytotoxic alkylating agents but also included a number of emerging anticancer drugs, including an IGF1-R inhibitor (NVP-AEW541), a PIM-1 kinase inhibitor (Pim1 inhibitor 2), a PLK1 inhibitor (BI 2536) and most strikingly seven of nine tested HDAC inhibitors. These findings were further confirmed by demonstrating NRF2-dependent induction of endogenous AKR genes, biomarkers of NRF2 activity. The ability of HDAC inhibitors to stimulate NRF2-signalling did not diminish their own potency as antitumour agents. However, when used to pre-treat cells, they did reduce the efficacy of acrolein. Taken together, our data suggest that the ability of drugs to stimulate NRF2 activity is common and should be investigated as part of the drug-development process.     

Combination Therapy with the Histone Deacetylase Inhibitor LBH589 and Radiation Is an Effective Regimen for Prostate Cancer Cells

Radiation therapy (RT) continues to be one of the most popular treatment options for localized prostate cancer (CaP). The purpose of the study was to investigate the in vitro effect of LBH589 alone and in combination with RT on the growth and survival of CaP cell lines and the possible mechanisms of radiosensitization of this combination therapy. The effect of LBH589 alone or in combination with RT on two CaP cell lines (PC-3 and LNCaP) and a normal prostatic epithelial cell line (RWPE-1) was studied by MTT and clonogenic assays, cell cycle analysis, western blotting of apoptosis-related and cell check point proteins, and DNA double strand break (DSB) repair markers. The immunofluorescence staining was used to further confirm DSB expression in treated CaP cells. Our results indicate that LBH589 inhibited proliferation in both CaP and normal prostatic epithelial cells in a time-and-dose-dependent manner; low-dose of LBH589 (IC₂₀) combined with RT greatly improved efficiency of cell killing in CaP cells; compared to RT alone, the combination treatment with LBH589 and RT induced more apoptosis and led to a steady increase of sub-G1 population and abolishment of RT-induced G2/M arrest, increased and persistent DSB, less activation of non-homologous end joining (NHEJ)/homologous recombination (HR) repair pathways and a panel of cell cycle related proteins. These results suggest that LBH589 is a potential agent to increase radiosensitivity of human CaP cells. LBH589 used either alone, or in combination with RT is an attractive strategy for treating human CaP.     

Combined Therapy with Cytokine-Induced Killer Cells and Oncolytic Adenovirus Expressing IL-12 Induce Enhanced Antitumor Activity in Liver Tumor Model

Both adoptive immunotherapy and gene therapy hold a great promise for treatment of malignancies. However, these strategies exhibit limited anti-tumor activity, when they are used alone. In this study, we explore whether combination of cytokine-induced killer (CIK) adoptive immunotherapy with oncolytic adenovirus-mediated transfer of human interleukin-12 (hIL-12) gene induce the enhanced antitumor potency. Our results showed that oncolytic adenovirus carrying hIL-12 (AdCN205-IL12) could produce high levels of hIL-12 in liver cancer cells, as compared with replication-defective adenovirus expressing hIL-12 (Ad-IL12). AdCN205-IL12 could specifically induce cytotoxocity to liver cancer cells. Combination of CIK cells with AdCN205-IL12 could induce higher antitumor activity to liver cancer cells in vitro than that induced by either CIK or AdCN205-IL12 alone, or combination of CIK and control vector AdCN205-GFP. Furthermore, treatment of the established liver tumors with the combined therapy of CIK cells and AdCN205-IL12 resulted in tumor regression and long-term survival. High level expression of hIL-12 in tumor tissues could increase traffic of CIK cells to tumor tissues and enhance their antitumor activities. Our study provides a novel strategy for the therapy of cancer by the combination of CIK adoptive immunotherapy with oncolytic adenovirus-mediated transfer of immune stimulatory molecule hIL-12.     

Induction of cell cycle arrest and DNA damage by the HDAC inhibitor panobinostat (LBH589) and the lipid peroxidation end product 4-hydroxynonenal in prostate cancer cells

Histone deacetylase inhibitors (HDACIs) are promising antineoplastic agents for the treatment of cancer. Here we report that the lipid peroxidation end product 4-hydroxynonenal (HNE) significantly potentiates the anti-tumor effects of the HDAC inhibitor panobinostat (LBH589) in the PC3 prostate cancer cell model. Panobinostat and HNE inhibited proliferation of PC3 cells and the combination of the two agents resulted in a significant combined effect. Cell cycle analysis revealed that both single agents and, to a greater extent, their combined treatment induced G2/M arrest, but cell death occurred in the combined treatment only. Furthermore, HNE and, to a greater extent, the combined treatment induced dephosphorylation of Cdc2 leading to progression into mitosis as confirmed by α-tubulin/DAPI staining and phospho-histone H3 (Ser10) analysis. To evaluate possible induction of DNA damage we utilized the marker phosphorylated histone H2A.X. Results showed that the combination of panobinostat and HNE induced significant DNA damage concomitant with the mitotic arrest. Then, by using androgen receptor (AR)-expressing PC3 cells we observed that the responsiveness to HNE and panobinostat was independent of the expression of functional AR. Taken together, our data suggest that HNE potentiates the antitumoral effect of the HDACI panobinostat in prostate cancer cells.     

雷帕霉素联合ZD55-TRAIL病毒对肿瘤抑制及其机制探讨

溶瘤腺病毒的肿瘤靶向性研究一直是一个热点。目前已有商业化的ONYX-015、H101溶瘤腺病毒。在此基础上,科学家又进一步发展形成基因-病毒治疗方案,如文中应用的ZD55-TRAIL病毒。本研究利用刘新垣实验室提供的携带TRAIL(TNF-related apoptosis-inducing ligand,TNF相关的凋亡诱导配体)的溶瘤腺病毒ZD55-TRAIL联合雷帕霉素杀伤肿瘤     

Combination Treatment with the GSK-3 Inhibitor 9-ING-41 and CCNU Cures Orthotopic Chemoresistant Glioblastoma in Patient-Derived Xenograft Models

Resistance to chemotherapy remains a major challenge in the treatment of human glioblastoma (GBM). Glycogen synthase kinase-3β (GSK-3β), a positive regulator of NF-κB–mediated survival and chemoresistance of cancer cells, has been identified as a potential therapeutic target in human GBM. Our objective was to determine the antitumor effect of GSK-3 inhibitor 9-ING-41 in combination with chemotherapy in patient-derived xenograft (PDX) models of human GBM. We utilized chemoresistant PDX models of GBM, GBM6 and GBM12, to study the effect of 9-ING-41 used alone and in combination with chemotherapy on tumor progression and survival. GBM6 and GBM12 were transfected by reporter constructs to enable bioluminescence imaging, which was used to stage animals prior to treatment and to follow intracranial GBM tumor growth. Immunohistochemical staining, apoptosis assay, and immunoblotting were used to assess the expression of GSK-3β and the effects of treatment in these models. We found that 9-ING-41 significantly enhanced 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) antitumor activity in staged orthotopic GBM12 (no response to CCNU) and GBM6 (partial response to CCNU) PDX models, as indicated by a decrease in tumor bioluminescence in mouse brain and a significant increase in overall survival. Treatment with the combination of CCNU and 9-ING-41 resulted in histologically confirmed cures in these studies. Our results demonstrate that the GSK-3 inhibitor 9-ING-41, a clinical candidate currently in Investigational New Drug (IND)-enabling development, significantly enhances the efficacy of CCNU therapy for human GBM and warrants consideration for clinical evaluation in this difficult-to-treat patient population.     

MET Gene Amplification and MET Receptor Activation Are Not Sufficient to Predict Efficacy of Combined MET and EGFR Inhibitors in EGFR TKI-Resistant NSCLC Cells

Epidermal growth factor receptor (EGFR), member of the human epidermal growth factor receptor (HER) family, plays a critical role in regulating multiple cellular processes including proliferation, differentiation, cell migration and cell survival. Deregulation of the EGFR signaling has been found to be associated with the development of a variety of human malignancies including lung, breast, and ovarian cancers, making inhibition of EGFR the most promising molecular targeted therapy developed in the past decade against cancer. Human non small cell lung cancers (NSCLC) with activating mutations in the EGFR gene frequently experience significant tumor regression when treated with EGFR tyrosine kinase inhibitors (TKIs), although acquired resistance invariably develops. Resistance to TKI treatments has been associated to secondary mutations in the EGFR gene or to activation of additional bypass signaling pathways including the ones mediated by receptor tyrosine kinases, Fas receptor and NF-kB. In more than 30–40% of cases, however, the mechanisms underpinning drug-resistance are still unknown. The establishment of cellular and mouse models can facilitate the unveiling of mechanisms leading to drug-resistance and the development or validation of novel therapeutic strategies aimed at overcoming resistance and enhancing outcomes in NSCLC patients. Here we describe the establishment and characterization of EGFR TKI-resistant NSCLC cell lines and a pilot study on the effects of a combined MET and EGFR inhibitors treatment. The characterization of the erlotinib-resistant cell lines confirmed the association of EGFR TKI resistance with loss of EGFR gene amplification and/or AXL overexpression and/or MET gene amplification and MET receptor activation. These cellular models can be instrumental to further investigate the signaling pathways associated to EGFR TKI-resistance. Finally the drugs combination pilot study shows that MET gene amplification and MET receptor activation are not sufficient to predict a positive response of NSCLC cells to a cocktail of MET and EGFR inhibitors and highlights the importance of identifying more reliable biomarkers to predict the efficacy of treatments in NSCLC patients resistant to EGFR TKI.     

Terpene Conjugates of the Nigella sativa Seed‐Oil Constituent Thymoquinone with Enhanced Efficacy in Cancer Cells

Thymoquinone (TQ; 1 ) is a weak anticancer constituent of black seed oil. Derivatives bearing terpene‐terminated 6‐alkyl residues were tested in cells of human HL‐60 leukemia, 518A2 melanoma, multidrug‐resistant KB‐V1/Vbl cervix, and MCF‐7/Topo breast carcinomas, as well as in non‐malignant human foreskin fibroblasts. Derivatives with a short four‐atom spacer between quinone and cyclic monoterpene moieties were more antiproliferative than analogues with longer spacers. 6‐(Menthoxybutyryl)thymoquinone ( 3a ) exhibited single‐digit micromolar IC₅₀ (72 h) values in all four cell lines. It was seven times more active than TQ ( 1 ) in 518A2 melanoma cells and four times in KB‐V1/Vbl cervix carcinoma cells, while only half as toxic in the fibroblasts. Compound 3a was also not a substrate for the P‐gp and BCRP drug transporters of the resistant cancer cells. The caryophyllyl and germacryl conjugates 3e and 3f specifically inhibited the growth of the resistant MCF‐7 breast carcinoma cells. Conjugation of TQ with the triterpene betulinic acid via the OH group as in 3g led to a loss in activity, while conjugation via the carboxylic acid afforded compound 4 with nanomolar IC₅₀ (72 h) activity against HL‐60 cells. All anticancer‐active derivatives of TQ ( 1 ) induced apoptosis associated with DNA laddering, a decrease in mitochondrial membrane potential and a slight increase in reactive oxygen species.     

Mutant Fusion Proteins with Enhanced Fusion Activity Promote Measles Virus Spread in Human Neuronal Cells and Brains of Suckling Hamsters

Subacute sclerosing panencephalitis (SSPE) is a fatal degenerative disease caused by persistent measles virus (MV) infection in the central nervous system (CNS). From the genetic study of MV isolates obtained from SSPE patients, it is thought that defects of the matrix (M) protein play a crucial role in MV pathogenicity in the CNS. In this study, we report several notable mutations in the extracellular domain of the MV fusion (F) protein, including those found in multiple SSPE strains. The F proteins with these mutations induced syncytium formation in cells lacking SLAM and nectin 4 (receptors used by wild-type MV), including human neuronal cell lines, when expressed together with the attachment protein hemagglutinin. Moreover, recombinant viruses with these mutations exhibited neurovirulence in suckling hamsters, unlike the parental wild-type MV, and the mortality correlated with their fusion activity. In contrast, the recombinant MV lacking the M protein did not induce syncytia in cells lacking SLAM and nectin 4, although it formed larger syncytia in cells with either of the receptors. Since human neuronal cells are mainly SLAM and nectin 4 negative, fusion-enhancing mutations in the extracellular domain of the F protein may greatly contribute to MV spread via cell-to-cell fusion in the CNS, regardless of defects of the M protein.     

雷贝拉唑联合莫沙必利治疗反流性食管炎的临床疗效观察

目的:治疗雷贝拉唑联合莫沙必利治疗返流性食管炎的临床疗效。方法:108例反流性食管炎患者随机分成两组,治疗组56例和奥美拉唑对照组52例,治疗组应用雷贝拉唑和莫沙必利,对照组应用奥美拉唑和莫沙必利,观察治疗前后患者的临床症改善和内镜变化。结果:治疗8周,治疗组症状积分明显优于对照组(P<0.05),治疗组疗效明显优于对照组(P<0.05),胃镜检查结果示治疗组食管黏膜愈合情况优于对照组(P<0.5)。结论:雷贝拉唑和莫沙必利联用治疗反流性食管炎临床症状明显改善,联合用药临床疗效优于奥美拉唑。     

Expression of p24 gag Protein of Bovine Leukemia Virus in Insect Cells and Its Use in Immunodetection of the Disease

Bovine leukemia is a common retroviral infection of cattle. The disease is characterized by a strong immunological response to several viral proteins, but the antibodies against p24 and gp51 are predominant. In this study, a recombinant baculovirus containing the gag gene p24 was constructed and the protein, used as antigen, analyzed by western blot and an indirect in-house rp24-ELISA test. This allowed detecting the presence of antibodies for bovine leukemia virus in a panel of cattle sera. The authentication of the protein expands its potential use for different medical applications, from improved diagnosis of the disease to source of antigens to be included in a subunit vaccine.     

Combined autophagy and HDAC inhibition: A phase I safety,tolerability, pharmacokinetic,and pharmacodynamic analysis of hydroxychloroquine in combination with the HDAC inhibitor vorinostat in patients with advanced solid tumors

We previously reported that inhibition of autophagy significantly augmented the anticancer activity of the histone deacetylase (HDAC) inhibitor vorinostat (VOR) through a cathepsin D-mediated mechanism. We thus conducted a first-in-human study to investigate the safety, preliminary efficacy, pharmacokinetics (PK), and pharmacodynamics (PD) of the combination of the autophagy inhibitor hydroxychloroquine (HCQ) and VOR in patients with advanced solid tumors. Of 27 patients treated in the study, 24 were considered fully evaluable for study assessments and toxicity. Patients were treated orally with escalating doses of HCQ daily (QD) (d 2 to 21 of a 21-d cycle) in combination with 400 mg VOR QD (d one to 21). Treatment-related adverse events (AE) included grade 1 to 2 nausea, diarrhea, fatigue, weight loss, anemia, and elevated creatinine. Grade 3 fatigue and/or myelosuppression were observed in a minority of patients. Fatigue and gastrointestinal AE were dose-limiting toxicities. Six-hundred milligrams HCQ and 400 mg VOR was established as the maximum tolerated dose and recommended phase II regimen. One patient with renal cell carcinoma had a confirmed durable partial response and 2 patients with colorectal cancer had prolonged stable disease. The addition of HCQ did not significantly impact the PK profile of VOR. Treatment-related increases in the expression of CDKN1A and CTSD were more pronounced in tumor biopsies than peripheral blood mononuclear cells. Based on the safety and preliminary efficacy of this combination, additional clinical studies are currently being planned to further investigate autophagy inhibition as a new approach to increase the efficacy of HDAC inhibitors.     

Enhanced Production of Virus-Inhibiting Factor (Interferon) in Human Diploid Cells by Ultraviolet Irradiation and Temperature Shift-Down after Stimulation with Newcastle Disease Virus

The production of the virus-inhibiting factor or interferon (IF) was highest in cells incubated at 37 C after inoculation with Newcastle disease (ND) virus and decreased as the incubation temperature was lowered. Shift-down of incubation temperature to 32 C or 34 C after incubation at 37 C for 4–7 hr enhanced IF production in cell cultures stimulated with ND virus, as compared with cultures incubated continuously at 37 C. Shift-down to 32 C after incubation at 37 C for 6 hr. was optimal for this enhancement of IF yield. Enhanced IF production was also observed in cell cultures irradiated by ultraviolet light 4–7 hr after stimulation with ND virus.     

Metabolic Requirements for the Development of Hemadsorption Activity and Virus Formation in BHK-21 Cells Infected with Newcastle Disease Virus

Differential effect of various metabolic inhibitors on the development of hemadsorption activity and virus formation in cells infected with Newcastle disease virus (NDV) was investigated. It was found that, in BHK-21 cells infected with NDV, cycloheximide did not prevent the development of hemadsorption activity, whereas protein synthesis and virus formation by the cell were rapidly inhibited by the drug. When the drug was added to the culture at 4.5 h after infection or later, hemadsorption activity of the cell continued to develop normally for about 1 h. Similar increase in hemadsorption activity was found in cells which were treated with anti-NDV serum (to neutralize their hemadsorption activity) and then washed and incubated with cycloheximide. However, when cells were treated with the drug early in the infection (1.5 or 3.0 h), they did not show any detectable hemadsorption reaction throughout the infection. In contrast to cycloheximide, iodoacetate added to the culture together with sodium azide inhibited completely both the development of hemadsorption activity and the formation of progeny virus. These results suggest that the change of cell surface to become hemadsorptive may depend upon the energy generating system but not upon de novo synthesis of protein, whereas production of infectious virus may require continuous synthesis of protein.