Utility of Clostridium difficile Toxin B for Inducing Anti-Tumor Immunity

Clostridium difficile toxin B (TcdB) is a key virulence factor of bacterium and induces intestinal inflammatory disease. Because of its potent cytotoxic and proinflammatory activities, we investigated the utility of TcdB in developing anti-tumor immunity. TcdB induced cell death in mouse colorectal cancer CT26 cells, and the intoxicated cells stimulated the activation of mouse bone marrow-derived dendritic cells and subsequent T cell activation in vitro. Immunization of BALB/c mice with toxin-treated CT26 cells elicited potent anti-tumor immunity that protected mice from a lethal challenge of the same tumor cells and rejected pre-injected tumors. The anti-tumor immunity generated was cell-mediated, long-term, and tumor-specific. Further experiments demonstrated that the intact cell bodies were important for the immunogenicity since lysing the toxin-treated tumor cells reduced their ability to induce antitumor immunity. Finally, we showed that TcdB is able to induce potent anti-tumor immunity in B16-F10 melanoma model. Taken together, these data demonstrate the utility of C. difficile toxin B for developing anti-tumor immunity.     

Chemical characterization of cell-wall polysaccharides from tank-cultivated and wild plants ofDelesseria sanguinea (Hudson) Lamouroux (Ceramiales,delesseriaceae): culture patterns and potent anticoagulant activity

Tank cultivation ofDelesseria sanguinea was investigated in order to manipulate conditions for vegetative growth and to provide biomass for the analysis of cell wall polysaccharides. Seasonality is subject to short-day photoperiodic control. Night-break or long-day conditions prevented fertility in tetrasporophytes and gametophytes and triggered outgrowth of new blades. Long-day illuminations allowed a 1% daily growth rate. Seawater temperature below 13 °C was necessary for inducing formation of new blades. Both wild and cultivated ofD. sanguinea plants contained a non gelling sulfated heteropolysaccharide composed of a galactosyl backbone branched with xylosyl residues. The hot water extract at neutral pH displayed the highest anticoagulant activity (5 μg ml^-1 polysaccharide concentration in APTT clotting assay). No obvious differences were found in polysaccharide chemical composition and properties between gametophytes and sporophytes or between cultivated and wild plants.     

In vivo and in vitro anti-tumor and anti-metastasis effects of Coriolus versicolor aqueous extract on mouse mammary 4T1 carcinoma

Coriolus versicolor (CV), a medicinal mushroom widely consumed in Asian countries, has been demonstrated to be effective in stimulation of immune system and inhibition of tumor growth. The present study aimed to investigate the anti-tumor and anti-metastasis effects of CV aqueous extract in mouse mammary carcinoma 4T1 cells and in 4T1-tumor bearing mouse model. Our results showed that CV aqueous extract (0.125–2 mg/ml) did not inhibit 4T1 cell proliferation while the non-cytotoxic dose of CV extract (1–2 mg/ml) significantly inhibited cell migration and invasion (p < 0.05). Besides, the enzyme activities and protein levels of MMP-9 were suppressed by CV extract significantly. Animal studies showed that CV aqueous extract (1 g/kg, orally-fed daily for 4 weeks) was effective in decreasing the tumor weight by 36%, and decreased the lung metastasis by 70.8% against untreated control. Besides, micro-CT analysis of the tumor-bearing mice tibias indicated that CV extract was effective in bone protection against breast cancer-induced bone destruction as the bone volume was significantly increased. On the other hand, CV aqueous extract treatments resulted in remarkable immunomodulatory effects, which was reflected by the augmentation of IL-2, 6, 12, TNF-α and IFN-γ productions from the spleen lymphocytes of CV-treated tumor-bearing mice. In conclusion, our results demonstrated for the first time that the CV aqueous extract exhibited anti-tumor, anti-metastasis and immunomodulation effects in metastatic breast cancer mouse model, and could protect the bone from breast cancer-induced bone destruction. These findings provided scientific evidences for the clinical application of CV aqueous extract in breast cancer patients.     

Ruta graveolens L. Induces Death of Glioblastoma Cells and Neural Progenitors,but Not of Neurons,via ERK 1/2 and AKT Activation

Glioblastoma multiforme is a highly aggressive brain tumor whose prognosis is very poor. Due to early invasion of brain parenchyma, its complete surgical removal is nearly impossible, and even after aggressive combined treatment (association of surgery and chemo- and radio-therapy) five-year survival is only about 10%. Natural products are sources of novel compounds endowed with therapeutic properties in many human diseases, including cancer. Here, we report that the water extract of Ruta graveolens L., commonly known as rue, induces death in different glioblastoma cell lines (U87MG, C6 and U138) widely used to test novel drugs in preclinical studies. Ruta graveolens’ effect was mediated by ERK1/2 and AKT activation, and the inhibition of these pathways, via PD98058 and wortmannin, reverted its antiproliferative activity. Rue extract also affects survival of neural precursor cells (A1) obtained from embryonic mouse CNS. As in the case of glioma cells, rue stimulates the activation of ERK1/2 and AKT in A1 cells, whereas their blockade by pharmacological inhibitors prevents cell death. Interestingly, upon induction of differentiation and cell cycle exit, A1 cells become resistant to rue’s noxious effects but not to those of temozolomide and cisplatin, two alkylating agents widely used in glioblastoma therapy. Finally, rutin, a major component of the Ruta graveolens water extract, failed to cause cell death, suggesting that rutin by itself is not responsible for the observed effects. In conclusion, we report that rue extracts induce glioma cell death, discriminating between proliferating/undifferentiated and non-proliferating/differentiated neurons. Thus, it can be a promising tool to isolate novel drugs and also to discover targets for therapeutic intervention.     

Regulatory perspective on in vitro potency assays for human T cells used in anti-tumor immunotherapy

The adaptive immune system is known to play an important role in anti-neoplastic responses via induction of several effector pathways, resulting in tumor cell death. Because of their ability to specifically recognize and kill tumor cells, the potential use of autologous tumor-derived and genetically engineered T cells as adoptive immunotherapy for cancer is currently being explored. Because of the variety of potential T cell-based medicinal products at the level of starting material and manufacturing process, product-specific functionality assays are needed to ensure quality for individual products. In this review, we provide an overview of in vitro potency assays suggested for characterization and release of different T cell-based anti-tumor products. We discuss functional assays, as presented in scientific advices and literature, highlighting specific advantages and limitations of the various assays. Because the anticipated in vivo mechanism of action for anti-tumor T cells involves tumor recognition and cell death, in vitro potency assays based on the cytotoxic potential of antigen-specific T cells are most evident. However, assays based on other T cell properties may be appropriate as surrogates for cytotoxicity. For all proposed assays, biological relevance of the tests and correlation of the read-outs with in vivo functionality need to be substantiated with sufficient product-specific (non-)clinical data. Moreover, further unraveling the complex interaction of immune cells with and within the tumor environment is expected to lead to further improvement of the T cell-based products. Consequently, increased knowledge will allow further optimized guidance for potency assay development.     

Anti-tumor effects of canine adipose tissue-derived mesenchymal stromal cell-based interferon-β gene therapy and cisplatin in a mouse melanoma model

Background aimsAdipose tissue (AT)-derived mesenchymal stromal cells (MSC) (AT-MSC) represent a novel tool for delivering therapeutic genes to tumor cells. Interferon (IFN)-β is a cytokine with pleiotropic cellular functions, including anti-proliferative, immunomodulatory and anti-angiogenic activities. The purpose of this study was to engineer canine AT-MSC (cAT-MSC) producing IFN-β and to evaluate the anti-tumor effect of cAT-MSC–IFN-β combined with cisplatin in mouse melanoma modelMethodscAT-MSC engineered to express mouse IFN-β were generated using a lentiviral vector (cAT-MSC–IFN-β) and the secreted IFN-β-induced inhibition of tumor cell growth and apoptosis on B16F10 cells was investigated in vitro prior to in vivo studies. Melanoma-bearing mouse was developed by injecting B16F10 cells subcutaneously into 6-week-old C57BL/6 mice. After 14 days, cisplatin (10 mg/kg) was injected intratumorally, and 3 days later the engineered cAT-MSC were injected subcutaneously every 3 days to death. Tumor volume and survival times were measuredResultsThe combination treatment of cAT-MSC–IFN-β with cisplatin was more effective in inhibiting the growth of melanoma and resulted in significantly extended survival time than both an unengineered cAT-MSC–cisplatin combination group and a cisplatin-alone group. Interestingly, subcutaneously injected cAT-MSC–IFN-β were migrated to tumor sitesConclusionsOur data suggest that canine AT-MSC could serve as a powerful cell-based delivery vehicle for releasing therapeutic proteins to tumor lesions. Maximal anti-tumor effects were seen when this therapy was combined with a DNA-damaging chemotherapeutic agent. This study demonstrates the possible applicability of AT-MSC-mediated IFN-β in treating canine and human cancer patients.     

In vivo anti-tumor immunity detected by leukocyte adherence inhibition

Summary The immune response of mice to a transplacentally induced alveolar cell tumor was studied with the leukocyte adherence inhibition (LAI) assay. The lung tumor, designated 85, was induced in a C3HfB/HeN (C3Hf) mouse by l-ethyl-l-nitrosourea (ENU). While a dose of 10⁵ cells of this tumor does not grow in syngeneic C3Hf mice, it does grow readily in (A×C3Hf)F₁ hybrid mice. The tumor possesses a tumor associated transplantation antigen (TATA) which cross-reacts with a normal tissue alloantigen in strain A/HeN (A) mice. Normal mice, tumor-immunized C3Hf mice, and tumor-bearing (A×C3Hf)F₁ mice possessed peritoneal cells, the majority of which adhered rapidly to glass and resisted gentle washing. When incubated with an extract of the 85 tumor, peritoneal cells from tumor-immunized mice demonstrated marked inhibition of adherence (62.4%) compared to similarly incubated peritoneal cells of either normal mice (30.3%) or tumor bearing mice (37.1%). Specificity of the reactivity in the LAI assay was demonstrated with a neuroblastoma extract and peritoneal cells from neuroblastoma-immunized C3Hf mice. Peritoneal cells from lung tumor-immunized mice, but not tumor-bearing mice, responded to a lung extract from strain A mice. In contrast to the microcytotoxicity assay, the LAI assay is capable of distinguishing the effective anti-tumor response of tumor-immunized C3Hf mice from the ineffective immune response of tumor-bearing (A×C3Hf)F₁ mice.     

Ginseng Berry Extract Promotes Maturation of Mouse Dendritic Cells

Ginseng extract has been shown to possess certain anti-virus, anti-tumor and immune-activating effects. However, the immunostimulatory effect of ginseng berry extract (GB) has been less well characterized. In this study, we investigated the effect of GB on the activation of mouse dendritic cells (DCs) in vitro and in vivo. GB treatment induced up-regulation of co-stimulatory molecules in bone marrow-derived DCs (BMDCs). Interestingly, GB induced a higher degree of co-stimulatory molecule up-regulation than ginseng root extract (GR) at the same concentrations. Moreover, in vivo administration of GB promoted up-regulation of CD86, MHC class I and MHC class II and production of IL-6, IL-12 and TNF-α in spleen DCs. GB also promoted the generation of Th1 and Tc1 cells. Furthermore, Toll like receptor 4 (TLR4) and myeloid differentiation primary response 88 (MyD88) signaling pathway were essential for DC activation induced by GB. In addition, GB strongly prompted the proliferation of ovalbumin (OVA)-specific CD4 and CD8 T cells. Finally, GB induced DC activation in tumor-bearing mice and the combination of OVA and GB treatment inhibited B16-OVA tumor cell growth in C57BL/6 mice. These results demonstrate that GB is a novel tumor therapeutic vaccine adjuvant by promoting DC and T cell activation.     

Identification and analysis of the active phytochemicals from the anti-cancer botanical extract Bezielle

Bezielle is a botanical extract that has selective anti-tumor activity, and has shown a promising efficacy in the early phases of clinical testing. Bezielle inhibits mitochondrial respiration and induces reactive oxygen species (ROS) in mitochondria of tumor cells but not in non-transformed cells. The generation of high ROS in tumor cells leads to heavy DNA damage and hyper-activation of PARP, followed by the inhibition of glycolysis. Bezielle therefore belongs to a group of drugs that target tumor cell mitochondria, but its cytotoxicity involves inhibition of both cellular energy producing pathways. We found that the cytotoxic activity of the Bezielle extract in vitro co-purified with a defined fraction containing multiple flavonoids. We have isolated several of these Bezielle flavonoids, and examined their possible roles in the selective anti-tumor cytotoxicity of Bezielle. Our results support the hypothesis that a major Scutellaria flavonoid, scutellarein, possesses many if not all of the biologically relevant properties of the total extract. Like Bezielle, scutellarein induced increasing levels of ROS of mitochondrial origin, progressive DNA damage, protein oxidation, depletion of reduced glutathione and ATP, and suppression of both OXPHOS and glycolysis. Like Bezielle, scutellarein was selectively cytotoxic towards cancer cells. Carthamidin, a flavonone found in Bezielle, also induced DNA damage and oxidative cell death. Two well known plant flavonoids, apigenin and luteolin, had limited and not selective cytotoxicity that did not depend on their pro-oxidant activities. We also provide evidence that the cytotoxicity of scutellarein was increased when other Bezielle flavonoids, not necessarily highly cytotoxic or selective on their own, were present. This indicates that the activity of total Bezielle extract might depend on a combination of several different compounds present within it.     

Coupling programmed cell death 1-positive tumor-infiltrating T cells with anti-programmed cell death 1 antibody improves the efficacy of adoptive T-cell therapy

Background aimsAdoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) has shown great success in clinical trials. Programmed cell death 1 (PD-1)-expressing TILs show high specificity to autologous tumor cells. However, limited therapeutic efficiency is observed as a result of the tumor immune microenvironment (TIME).MethodsCoupling PD-1⁺ ex vivo-derived TILs with a monoclonal antibody against anti-PD-1 (aPD-1) reinvigorated the anti-tumor response of TILs against solid tumor without altering their high tumor targeting ability.ResultsUsing a melanoma-bearing mouse model, PD-1⁺ TILs blocked with aPD-1 (PD-1⁺ TILs-aPD-1) exhibited a high capability for tumor targeting as well as improved anti-tumor response in TIME. Tumor growth was substantially delayed in the mice treated with PD-1⁺ TILs-aPD-1.ConclusionsThe strategy utilizing TIL therapy coupled with immune checkpoint antibodies may extend to other therapeutic targets of ACT.     

IGF-1 receptor inhibition by picropodophyllin in medulloblastoma

The insulin-like growth factor-1 receptor (Igf1r) is a multifunctional membrane-associated tyrosine kinase associated with regulation of transformation, proliferation, differentiation and apoptosis. Increased IGF pathway activity has been reported in human and murine medulloblastoma. Tumors from our genetically-engineered medulloblastoma mouse model over-express Igf1r, and thus this mouse model is a good platform with which to study the role of Igf1r in tumor progression. We hypothesize that inhibition of IGF pathway in medulloblastoma can slow or inhibit tumor growth and metastasis. To test our hypothesis, we tested the role of IGF in tumor growth in vitro by treatment with the tyrosine kinase small molecule inhibitor, picropodophyllin (PPP), which strongly inhibits the IGF pathway. Our results demonstrate that PPP-mediated downregulation of the IGF pathway inhibits mouse tumor cell growth and induces apoptotic cell death in vitro in primary medulloblastoma cultures that are most reflective of tumor cell behavior in vivo.     

Synthesis and biological evaluation of amide derivatives of diflunisal as potential anti-tumor agents

To discover the new medicinal activity, the structure of diflunisal has been modified. Forty amide derivatives of diflunisal were synthesized starting from diflunisal in three steps. Their inhibition growth rate of human lung cancer cell (A549) and human endometrial adenocarcinoma cell (Ishikawa) in vitro was evaluated. The preliminary assay results showed that compounds 6j, 7o and 8c exhibited good anti-tumor activities and excellent selectivity for the Ishikawa cell, may be potential anti-tumor agents.     

One-step isolation of sappanol and brazilin from Caesalpinia sappan and their effects on oxidative stress-induced retinal death

Caesalpinia sappan is a well-distributed plant that is cultivated in Southeast Asia, Africa, and the Americas. C. sappan has been used in Asian folk medicine and its extract has been shown to have pharmacological effects. Two homoisoflavonoids, sappanol and brazilin, were isolated from C. sappan by using centrifugal partition chromatography (CPC), and tested for protective effects against retinal cell death. The isolated homoisoflavonoids produced approximately 20-fold inhibition of N-retinylidene-N-retinyl-ethanolamine (A2E) photooxidation in a dose-dependent manner. Of the 2 compounds, brazilin showed better inhibition (197.93 ± 1.59 μM of IC₅₀). Cell viability tests and PI/Hoechst 33342 double staining method indicated that compared to the negative control, sappanol significantly attenuated H₂O₂-induced retinal death. The compounds significantly blunted the up-regulation of intracellular reactive oxygen species (ROS), and sappanol inhibited lipid peroxidation in a concentration-dependent manner. Thus, both compounds represent potential antioxidant treatments for retinal diseases. [BMB Reports 2015; 48(5): 289-294]     

Corema album Leaves Mediate DNA Damage in Triple-Negative Breast Cancer Cells

Corema (C.) album is a shrub endemic to the Atlantic coast and has been described as yielding beneficial effects for human health. Nevertheless, studies concerning the bioactivity of C. album leaves are scarce. This study aims at investigating the anticancer potential and mode of action, of an hydroethanolic extract of C. album leaves (ECAL) on triple-negative breast cancer. This is a poor survival breast cancer subtype, owing to its high risk of distant reappearance, metastasis rates and the probability of relapse. The ECAL ability to prevent tumor progression through (i) the inhibition of cell proliferation (cell viability); (ii) the induction of apoptosis (morphological changes, TUNEL assay, caspase-3 cleaved) and (iii) the induction of DNA damage (PARP1 and γH2AX) with (iv) the involvement of NF-κB and of ERK1/2 pathways (AlphaScreen assay) was evaluated. ECAL activated the apoptotic pathway (through caspase-3) along with the inhibition of ERK and NF-κB pathways causing DNA damage and cell death. The large polyphenolic content of ECAL was presumed to be accountable for these effects. The extract of C. album leaves can target multiple pathways and, thus, can block more than one possible means of disease progression, evidencing the anticancer therapeutic potential from a plant source.     

Galectin-3C Inhibits Tumor Growth and Increases the Anticancer Activity of Bortezomib in a Murine Model of Human Multiple Myeloma

Galectin-3 is a human lectin involved in many cellular processes including differentiation, apoptosis, angiogenesis, neoplastic transformation, and metastasis. We evaluated galectin-3C, an N-terminally truncated form of galectin-3 that is thought to act as a dominant negative inhibitor, as a potential treatment for multiple myeloma (MM). Galectin-3 was expressed at varying levels by all 9 human MM cell lines tested. In vitro galectin-3C exhibited modest anti-proliferative effects on MM cells and inhibited chemotaxis and invasion of U266 MM cells induced by stromal cell-derived factor (SDF)-1α. Galectin-3C facilitated the anticancer activity of bortezomib, a proteasome inhibitor approved by the FDA for MM treatment. Galectin-3C and bortezomib also synergistically inhibited MM-induced angiogenesis activity in vitro. Delivery of galectin-3C intravenously via an osmotic pump in a subcutaneous U266 cell NOD/SCID mouse model of MM significantly inhibited tumor growth. The average tumor volume of bortezomib-treated animals was 19.6% and of galectin-3C treated animals was 13.5% of the average volume of the untreated controls at day 35. The maximal effect was obtained with the combination of galectin-3C with bortezomib that afforded a reduction of 94% in the mean tumor volume compared to the untreated controls at day 35. In conclusion, this is the first study to show that inhibition of galectin-3 is efficacious in a murine model of human MM. Our results demonstrated that galectin-3C alone was efficacious in a xenograft mouse model of human MM, and that it enhanced the anti-tumor activity of bortezomib in vitro and in vivo. These data provide the rationale for continued testing of galectin-3C towards initiation of clinical trials for treatment of MM.     

Nutrient regulation of bacterial growth and production of anti-tumor metabolites in <Emphasis Type="Italic">Stigmatella</Emphasis> WXNXJ-B fermentation

The metabolites produced by Stigmatella WXNXJ-B inhibited the growth of tumor cells. The aims of this research were to evaluate the inhibition potency to different tumor cell lines and to study the effects of ammonium, phosphate and iron salts on bacterial growth and production of bioactive metabolites in Stigmatella WXNXJ-B fermentation. The results showed that the chloroform extract (CE-ME) showed the strongest growth inhibition bioactivity on mouse melanoma cell line (B16), murine colon carcinoma cell line (CT-26), human liver carcinoma cell line (HepG2) and human breast cancer cell line (MDA-MB231) in vitro and the IC₅₀ values were 9.94, 7.33, 11.34 and 11.66 μg ml⁻¹ respectively. The IC₅₀ value was above 700 μg ml⁻¹ on normal mouse spleen cells. Morphology happened changes in B16 cells treated with CE-ME. The anti-tumor metabolites were mainly produced during the stationary phase of the bacterial growth. Cell growth was stimulated at the phosphate concentration below 5 mM, but it was inhibited partly with 10 mM phosphate. The production of bioactive substances was inhibited by the phosphate. Ammonium increased the cell growth by 250% at 5 mM addition. The inhibition rate to B16 cells was increased to 89% at the concentration of 40 mM ammonium. The bacteria showed the best growth with 4 mM iron. Iron had little effect on the production at 2 mM, but bigger inhibition effect at higher iron concentration.     

Suppression of Tumor Growth by Pleurotus ferulae Ethanol Extract through Induction of Cell Apoptosis,and Inhibition of Cell Proliferation and Migration

Cancer is the second leading cause of death worldwide. Edible medicinal mushrooms have been used in traditional medicine as regimes for cancer patients. Recently anti-cancer bioactive components from some mushrooms have been isolated and their anti-cancer effects have been tested. Pleurotus ferulae, a typical edible medicinal mushroom in Xinjiang China, has also been used to treat cancer patients in folk medicine. However, little studies have been reported on the anti-cancer components of Pleurotus ferulae. This study aims to extract bioactive components from Pleurotus ferulae and to investigate the anti-cancer effects of the extracts. We used ethanol to extract anti-cancer bioactive components enriched with terpenoids from Pleurotus ferulae. We tested the anti-tumour effects of ethanol extracts on the melanoma cell line B16F10, the human gastric cancer cell line BGC 823 and the immortalized human gastric epithelial mucosa cell line GES-1 in vitro and a murine melanoma model in vivo. Cell toxicity and cell proliferation were measured by MTT assays. Cell cycle progression, apoptosis, caspase 3 activity, mitochondrial membrane potential (MMP), migration and gene expression were studied in vitro. PFEC suppressed tumor cell growth, inhibited cell proliferation, arrested cells at G0/G1 phases and was not toxic to non-cancer cells. PFEC also induced cell apoptosis and necrosis, increased caspase 3 activity, reduced the MMP, prevented cell invasion and changed the expression of genes associated with apoptosis and the cell cycle. PFEC delayed tumor formation and reduced tumor growth in vivo. In conclusion, ethanol extracted components from Pleurotus ferulae exert anti-cancer effects through direct suppression of tumor cell growth and invasion, demonstrating its therapeutic potential in cancer treatment.     

MicroRNA-206 Targets notch3, Activates Apoptosis, and Inhibits Tumor Cell Migration and Focus Formation

MicroRNAs contribute to cancer development by acting as oncogenes or tumor suppressor genes. However, only a few microRNA target genes were determined. We identified a nearly perfect complementarity between miR-206 and the 3′-untranslated regions of both mouse and human notch3. Expression of miR-206 decreased the luciferase activity dose-dependently when cotransfected with the mouse or human notch3 3′-untranslated region-luciferase reporter containing the miR-206 target site in HeLa cells. This suppression was relieved by deletion and mutation of the miR-206-binding site and was partially recovered by expression of notch3 or by a specific inhibitor of miR-206. Interestingly, overexpression of miR-206 decreased the levels of both Notch3 protein and mRNA. Expression of miR-206 markedly induced apoptotic cell death and blocked the anti-apoptotic activity of Notch3. In addition, ectopic expression of miR-206 inhibited HeLa cell migration and focus formation. Therefore, we identified miR-206 as a pro-apoptotic activator of cell death, which was associated with its inhibition of notch3 signaling and tumor formation. The inhibition of cancer cell migration and focus formation by miR-206 strongly suggests that miR-206 may function as a novel tumor suppressor.