Therapeutic effects of 2B8T2M, a novel fusion of ALT-803, an IL-15 superagonist, with 4 single-chains of anti-CD20 antibody in combination with expanded natural killer cells against rituximab sensitive and resistant Burkitt lymphoma (BL).

2018 ◽  
Vol 36 (5_suppl) ◽  
pp. 32-32
Author(s):  
Yaya Chu ◽  
Nang Kham Su ◽  
Sarah Alter ◽  
Emily Jeng ◽  
Peter R. Rhode ◽  
...  
Keyword(s):  
Nk Cells ◽  
Granzyme B ◽  
Treatment Options ◽  
Binding Activity ◽  
In Vitro Cytotoxicity ◽  
Patient Treatment ◽  
Therapeutic Effects ◽  
Comparison Results

32 Background: Patients retreated with rituximab often relapse which limit patient treatment options (Goldman/Cairo, Leukemia, 2013). Our group has successfully expanded functional and active peripheral blood NK cells (exPBNK) to target BL (Chu/Cairo, et al, Can Imm Res, 2015). 2B8T2M was generated by fusing ALT-803, an IL-15 superagonist, to four single-chains of rituximab (Liu/Wong, et al, JBC, 2016). 2B8T2M displayed tri-specific CD20 binding activity, activated NK cells to enhance antibody-dependent cellular cytotoxicity, and induced apoptosis of B-lymphoma cells (Liu/Wong, et al, JBC, 2016). Methods: ALT-803 and 2B8T2M were generously provided by Altor BioScience Corporation. NK expansion, NK receptors expression and cytotoxicity were examined as we previous described (Chu/Cairo, et al, Can Imm Res 2015). IFNg and granzyme B levels were examined by ELISA assays. Equal doses of IgG, Rituximab, ALT-803, Rituximab+ALT-803, obinutuzumab (obinu, generously provided by Christian Klein, PhD from Roche) were used for comparison. Results: 2B8T2M significantly enhanced exPBNK cytotoxicity against rituximab-sensitive Raji cells compared to the controls IgG, Rituximab, ALT-803, Rituximab+ALT-803, obinu (p < 0.001, E:T = 1:1). 2B8T2M also significantly enhanced exPBNK cytotoxicity against rituximab-resistant Raji-2R cells (p < 0.001, E:T = 1:1) and resistant Raji-4RH cells (p < 0.001, E:T = 1:1). Furthermore, 2B8T2M significantly enhanced IFN-g and granzyme B production from exPBNK against Raji, Raji-2R and Raji-4RH compared to IgG (p < 0.001), rituximab (p < 0.001), ALT-803 (p < 0.001), Rituximab+ALT-803 (p < 0.001), and obinutuzumab (p < 0.001). Conclusions: 2B8T2M compared to rituximab, ALT-803 or obinutuzumab significantly enhanced exPBNK in vitro cytotoxicity against rituximab-sensitive and –resistant BL cells. The in vivo functions of 2B8T2M with exPBNK using humanized NSG models are under investigation.

scholarly journals RTID-07. HUMAN PLACENTAL HEMATOPOIETIC STEM CELL DERIVED NATURAL KILLER CELLS (CYNK-001) FOR TREATMENT OF RECURRENT GLIOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii194-ii195
Author(s):  
Nazanin Majd ◽  
Maha Rizk ◽  
Solveig Ericson ◽  
Kris Grzegorzewski ◽  
Sharmila Koppisetti ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
In Vitro Cytotoxicity ◽  
Orthotopic Mouse Model ◽  
Hematopoietic Stem ◽  
Dismal Prognosis

Abstract Glioblastoma (GBM) is the most aggressive primary brain tumor with dismal prognosis. Recent advances of immunotherapy in cancer have sparked interest in the use of cell therapy for treatment of GBM. Active transfer of Natural Killer (NK) cells is of particular interest in GBM because NK cells are capable of exerting anti-tumor cytotoxicity without the need for antigen presentation and sensitization, processes that are impaired in GBM. CYNK-001 is an allogeneic, off-the-shelf product enriched for CD56+/CD3- NK cells expanded from placental CD34+ cells manufactured by Celularity. Here, we demonstrate in vitro cytotoxicity of CYNK-001 against several GBM lines and its in vivo anti-tumor activity in a U87MG orthotopic mouse model via intracranial administration resulting in 94.5% maximum reduction in tumor volume. We have developed a phase I window-of-opportunity trial of CYNK-001 in recurrent GBM via intravenous (IV) and intratumoral (IT) routes. In the IV cohort, subjects receive cyclophosphamide for lymphodepletion followed by 3-doses of IV CYNK-001 weekly. In the IT cohort, subjects undergo placement of an IT catheter with an ommaya reservoir followed by 3-doses of IT CYNK-001 weekly. Patients are monitored for 28-days after last infusion for toxicity. Once maximum safe dose (MSD) is determined, patients undergo IV or IT treatments at MSD followed by surgical resection and the tumor tissue will be analyzed for NK cell engraftment and persistence. We will utilize a 3 + 3 dose de-escalation design (maximum n=36). Primary endpoint is safety and feasibility. Secondary endpoints are overall response rate, duration of response, time to progression, progression free survival and overall survival. Main eligibility criteria include age ≥18, KPS ≥60, GBM at first or second relapse with a measurable lesion on ≤2mg dexamethasone. This is the first clinical trial to investigate CYNK-001 in GBM and will lay the foundation for future NK cell therapy in solid tumors.

scholarly journals Novel and Modified Neutrophil Elastase Inhibitor Loaded in Topical Formulations for Psoriasis Management

Pharmaceutics ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 358 ◽  
Author(s):  
Andreia Nunes ◽  
Joana Marto ◽  
Lídia Maria Gonçalves ◽  
Sandra Simões ◽  
Rita Félix ◽  
...  
Keyword(s):  
Serine Protease ◽  
Neutrophil Elastase ◽  
In Vitro Cytotoxicity ◽  
Human Neutrophil Elastase ◽  
Therapeutic Effects ◽  
Neutrophil Elastase Inhibitor ◽  
Analytical Methodology ◽  
Elastase Inhibitor

Human neutrophil elastase (HNE) is a serine protease that degrades matrix proteins. An excess of HNE may trigger several pathological conditions, such as psoriasis. In this work, we aimed to synthesize, characterize and formulate new HNE inhibitors with a 4-oxo-β-lactam scaffold with less toxicity, as well as therapeutic index in a psoriasis context. HNE inhibitors with 4-oxo-β-lactam scaffolds were synthesized and characterized by NMR, FTIR, melting point, mass spectrometry and elemental analysis. In vitro cytotoxicity and serine protease assays were performed. The compound with the highest cell viability (AAN-16) was selected to be incorporated in an emulsion (AAN-16 E) and in a microemulsion (AAN-16 ME). Formulations were characterized in terms of organoleptic properties, pH, rheology, droplet size distribution, in vitro drug release and in vivo psoriatic activity. All compounds were successfully synthesized according to analytical methodology, with good yields. Both formulations presented suitable physicochemical properties. AAN-16 E presented the most promising therapeutic effects in a murine model of psoriasis. Overall, new HNE inhibitors were synthesized with high and selective activity and incorporated into topical emulsions with potential to treat psoriasis.

scholarly journals The role of granzyme B in murine models of acute graft-versus-host disease and graft rejection

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1232-1237 ◽  
Author(s):  
TA Graubert ◽  
JH Russell ◽  
TJ Ley
Keyword(s):  
Nk Cells ◽  
Graft Versus Host Disease ◽  
Graft Rejection ◽  
Granzyme B ◽  
Cytotoxic Lymphocytes ◽  
Host Disease ◽  
Graft Versus Host ◽  
Hybrid Resistance

A complete molecular description of the syndromes of graft-versus-host disease (GVHD) and graft rejection could have a significant impact on clinical bone marrow transplantation. Recent in vitro experiments (Heusel et al, Cell 76:977, 1994 and Shresta et al, Proc Natl Acad Sci USA 92:5679, 1995) have shown that the putative mediators of these two syndromes, cytotoxic lymphocytes (CTL) and natural killer (NK) cells, respectively, initiate a program of cell death (apoptosis) in susceptible target tissues in a manner critically dependent on the serine protease Granzyme B (gzm B). In the present study, we have analyzed the phenotype of gzm B-deficient mice using experimental transplant models designed to isolate their CD8+ CTL, CD4+ CTL, and NK compartments. We found a significant impairment in class I-dependent GVHD mediated by gzm B -/- CD8+ CTL, whereas class II-dependent GVHD was not altered using gzm B -/- CD4+ effectors. In a hybrid resistance model, gzm B -/- hosts rejected haplo-identical marrow grafts as efficiently as did their wild-type littermates. This result is surprising in light of a severe defect in the ability of gzm B -/- NK cells to induce apoptosis in susceptible targets in vitro. These in vivo data define significant role for gzm B in cytotoxicity mediated by CD8+ CTL, but not by CD4+ CTL. Furthermore, these results do not support a model of hybrid resistance in which NK cells play a pivotal role.

Genetically Reengineered K562 Cells Significantly Expand Cord Blood (CB) Natural Killer (NK) Cells Ex-Vivo Expanded with Increased NK Cell Activation and Cytolytic Activity Both In-Vitro and In-Vivo: Potential Use In Adoptive Cellular Immunotherapy (ACI)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3928-3928
Author(s):  
Michele Levin ◽  
Janet Ayello ◽  
Frances Zhao ◽  
Andrew Stier ◽  
Lauren Tiffen ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Granzyme B ◽  
K562 Cells ◽  
Cytolytic Activity ◽  
Scid Mice ◽  
Activation Marker

Abstract Abstract 3928 Background: NK cells play a role in reducing relapse in hematological malignancy following AlloSCT (Dunbar et al, Haematologica, 2008). NK cell limitations include lack of tumor recognition and/or limited numbers of viable and functional NK cells (Shereck/Cairo et al, Ped Bld Can, 2007). NK ACI provide safe and effective therapy against tumor relapse; yet NK cells are limited to specific cancer types and not all patients demonstrate optimal response (Ruggieri et al. Science, 2002; Ljunggren et al. Nat Rev Immuno, 2007). To circumvent these limitations, methods to expand and activate PBMNCs with genetically engineered K562 cells expressing membrane bound IL-15 and 41BB ligand (K562-mbIL15-41BBL [modK562]; Imai/Campana et al, Blood, 2005) have shown to significantly increase NK cells in number and maintain heterogeneous KIR expression (Fusaki/Campana et al BJH, 2009). We have shown that CB NK cells can be activated/expanded and exhibit enhanced cytolytic activity when cultured in a cytokines/antibody cocktail (Ayello/Cairo et al, BBMT, 2006; Exp Heme, 2009). Objective: To evaluate CBNK expansion, activation, cytolytic mechanism and function against Burkitt lymphoma (BL) tumor target and its influence on NK cell mediated in-vitro and in-vivo cytotoxicity in NOD-SCID mice following stimulation with modK562 cells (generously supplied by D.Campana, St Jude's Children's Hospital, Memphis, Tx). Methods: Following 100GY irradiation, modK562cells were incubated 1:1 with CBMNCs in RPMI+IL-2 (10IU/ml) for 7 days in 5%CO2, 37°C. NK activation marker (LAMP-1), perforin and granzyme B were determined by flow cytometry. Cytotoxicty was determined via europium assay at 20:1 E:T ratio with Ramos (BL) tumor targets (ATCC). The mammalian expression construct (ffLucZeo-pcDNA (generously supplied by L.Cooper, MD, PhD) was transfected to BL cells using lipofectin and selected by zeocin for stable transfection. Six week old NOD-SCID mice received 5×106 BL cells subcutaneously. Upon engraftment, xenografted NOD-SCID mice were divided in 5 groups: injected with PBS (control), BL only, 5×106 wildtype (WT) K562 expanded (E) CBNK cells, modK562 expanded (E) CB NK cells (5×106) and modK562 expanded (E) CBNK cells (5×107). Ex-vivo ECBNK cells were injected weekly for 5 weeks and xenografted NOD-SCID mice were monitored by volumetric measurement of tumor size (Tomayko/Reynolds, Can Chemother Pharmac, 1989), bioluminescent imaging (Inoue et al Exp Heme, 2007) and survival. The survival distribution for each group was estimated using the Fisher exact test. Results: On Day 0, NK cells (CD56+/3-) population was 3.9±1.3%. After 7 days, modK562 expanded CBNK cells was significantly increased compared to WTK562 and media alone (72±3.9 vs 43±5.9 vs 9±2.4%, p<0.01). This represented a 35-fold or 3374±385% increase of the input NK cell number. This was significantly increased compared to WTK562 (1771±300%, p<0.05). ModK562 ECBNK cells demonstrated increased perforin and granzyme B expression compared to WTK562 (42±1.5 vs 15±0.5%,p<0.001; 22±0.5 vs 11±0.3%,p<0.001, respectively). Cytotoxicity was against BL tumor targets was significantly increased (42±3 vs 18±2%,p<0.01), along with NK activation marker expression, CD107a (p<0.05). At 5 weeks, in-vivo studies demonstrated increased survival of NOD-SCID mice receiving both 5×106 and 5×107 modK562 ECBNK cells when compared to those with no treatment (p=0.05, p=0.0007, respectively). There was no difference in survival when comparing mice that received 5×106 vs 5×107 modK562 ECBNK cells (p=0.0894) at 5 weeks. Tumor volume of mice receiving either dose of modK562 ECBNK cells was significantly less than those receiving WTK562 ECBNK cells (1.92±0.57 and 0.37±0.05 vs 3.41±0.25, p=0.0096 and p=0.0001, respectively). Conclusions: CBMNCs stimulated and expanded with modK562 cells results in significant expansion of CBNK cells with enhanced in-vitro cytotoxicity, significant receptor expression of NK activation marker (LAMP-1), and perforin and granzyme B. Furthermore, modK562 ECBNK cells leads to increased survival and lower tumor burden of NOD-SCID mice xenografted with BL. Future directions include modK562 ECBNK cells to be genetically modified to express chimeric antigen receptor CD20 (MSCV-antiCD20-41BB-CD3 ζ) against CD20+ hematologic malignancies for future studies to evaluate whether targeting enhances in-vitro and in-vivo cytotoxicity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Capsaicin Exerts Therapeutic Effects by targeting tNOX-SIRT1 Axis and Augmenting ROS-Dependent Cytotoxic Autophagy in Melanoma Cancer Cells

2020 ◽  
Author(s):  
Atikul Islam ◽  
Pei-Fang Hsieh ◽  
Jou-Chun Chou ◽  
Jiunn-Wang Liao ◽  
Ming-Kun Hsieh ◽  
...  
Keyword(s):  
Malignant Melanoma ◽  
Cancer Cells ◽  
Nadh Oxidase ◽  
Treatment Options ◽  
Melanoma Cells ◽  
Therapeutic Effects ◽  
Melanoma Cancer ◽  
Anticancer Properties

Abstract Background: Although considered a rare form of skin cancer, malignant melanoma has steadily increased internationally and is a main cause of cancer-associated death worldwide. The treatment options for malignant melanoma are very limited. Accumulating data suggest that the natural compound, capsaicin, exhibits preferential anticancer properties to act as a nutraceutical agent. Here, we explored the underlying molecular events involved in the inhibitory effects of capsaicin on the growth of melanoma cells.Methods: The cellular thermal shift assay (CETSA) and isothermal dose response fingerprint (ITDRFCETSA) were utilized to validate the binding of capsaicin with the tumor-associated NADH oxidase, tNOX (ENOX2) in melanoma cells. We also assessed the cellular impact of capsaicin-targeting of tNOX on A375 cells by flow cytometry and protein analysis. The essential role of tNOX in tumor- and melanoma-growth limiting abilities of capsaicin was evaluated in C57BL/6 mice.Results: Our data show that capsaicin directly targets cellular tNOX to inhibit its enzymatic activity and enhance protein degradation capacity. The inhibition of tNOX by capsaicin is accompanied by the attenuation of SIRT1, a NAD+-dependent deacetylase that enhances ULK1 acetylation to induce ROS-dependent autophagy in melanoma cells. Capsaicin treatment of mice implanted with melanoma cancer cells suppressed tumor growth by down-regulating tNOX and SIRT1, which was also seen in an in vivo xenograft study with tNOX-depleted melanoma cells. Conclusions: Together, our findings suggest that tNOX expression is important for the growth of melanoma cancer cells both in vitro and in vivo, and that inhibition of the tNOX-SIRT1 axis contributes to inducting cytotoxic ROS-dependent autophagy in melanoma cells.

scholarly journals Nano-Mediated Photodynamic Therapy for Cancer: Enhancement of Cancer Specificity and Therapeutic Effects

2018 ◽  
Author(s):  
Ivan Mfouo Tynga ◽  
Heidi Abrahamse
Keyword(s):  
Photodynamic Therapy ◽  
Cancer Cells ◽  
Treatment Options ◽  
Therapeutic Effects ◽  
Drug Resistant ◽  
Severe Condition ◽  
Cell Niche ◽  
Extended Exposure

Deregulation of cell growth and development lead to cancer, a severe condition that claims millions of lives worldwide. Targeted or selective approaches used during cancer treatment determine the efficacy and outcome of the therapy. In order to enhance specificity and targeting and better treatment options for cancer, novel and alternative modalities are currently under development. Photodynamic therapy has the potential to eradicate cancer and combination therapy would yield even greater outcomes. Nanomedicine-aided cancer therapy shows enhanced specificity for cancer cells and minimal side-effects coupled with effective cancer destruction both in vitro and in vivo. Nanocarriers used in drug-delivery systems are well able to penetrate cancer stem cell niche, simultaneously killing cancer cells and eradicate drug-resistant cancer stem cells, yielding therapeutic efficiency up to 100 fold against drug-resistant cancer in comparison with free drugs. Safety precautions should be considered when using Nano-mediated therapy as the effects of extended exposure to biological environments are still to be determined.

scholarly journals NF-kB c-Rel is dispensable for the development but is required for the cytotoxic function of NK cells

2021 ◽  
Author(s):  
Y Vicioso ◽  
K Zhang ◽  
Parameswaran Ramakrishnan ◽  
Reshmi Parameswaran
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Granzyme B ◽  
Conditional Knockout ◽  
Cytotoxic Lymphocytes ◽  
And Control ◽  
Cytotoxic Function

AbstractNatural Killer (NK) cells are cytotoxic lymphocytes critical to the innate immune system. We found that germline deficiency of NF-kB c-Rel results in a marked decrease in cytotoxic function of NK cells, both in vitro and in vivo, with no significant differences in the stages of NK cell development. We found that c-Rel binds to the promoters of perforin and granzyme B, two key proteins required for NK cytotoxicity, and controls their transactivation. We generated a NK cell specific c-Rel conditional knockout to study NK cell intrinsic role of c-Rel and found that both global and conditional c-Rel deficiency leads to decreased perforin and granzyme B expression and thereby cytotoxic function. We also confirmed the role of c-Rel in perforin and granzyme B expression in human NK cells. c-Rel reconstitution rescued perforin and granzyme B expressions in c-Rel deficient NK cells and restored their cytotoxic function. Our results show a previously unknown role of c-Rel in transcriptional regulation of perforin and granzyme B expressions and control of NK cell cytotoxic function.

Effect of activating chimeric receptor on IL-15 armored NK cell on providing in vitro and in vivo antigen specific tumor response.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15016-e15016
Author(s):  
Louis F. Chai ◽  
Prajna Guha ◽  
Sarah Wadsworth ◽  
Denise Gonzalez ◽  
Nafees Rahman ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
In Vitro Cytotoxicity ◽  
Cell Delivery ◽  
Tumor Control ◽  
Chimeric Receptor ◽  
Cytokine Release ◽  
Regional Delivery

e15016 Background: Colorectal cancer liver metastases (CRCLM) are a major source of morbidity and mortality. Historically, curative therapy has been limited to surgical resection, but only a small fraction of patients are eligible. Cellular immunotherapy has shown promise in hematologic cancers, but challenges related to solid tumor therapy remain with optimal cell trafficking, elevated interstitial fluid pressures (IFP), and immunosuppression. We hypothesized that engineered natural killer (NK) cells expressing a natural killer group 2, member D (NKG2D) activating chimeric receptor (ACR) and membrane bound IL-15 (NKX101) would increase anti-tumor activity in vitro and in vivo utilizing our established regional delivery strategies. Methods: In vitro cytotoxicity and cytokine release of NKX101 cells or non-transduced NK cells (NT-NK) derived from the same donor were determined by co-culture systems with HCT116 cells that endogenously express NKG2D ligands. CRCLM-bearing NSG™ mice were treated with NKX101, NT-NK, or vehicle (CTRL) via portal vein (PV) for regional delivery (RD) or tail vein (TV) for systemic delivery (SD). Tumor burden (TB) was measured via tumor bioluminescence (TBL) and histopathology (HP). Flow cytometry (FC) determined the quantity of cells delivered. Student’s t-test and Mann-Whitney tests were performed for statistical comparisons. Results: NKX101 transduction efficiencies ranged between 63.5 – 75.6% across 3 separate healthy donors. EC50 values derived from a 4-hour cytotoxicity assay for NKX101 vs. NT-NK were 3-4 fold lower with the greatest difference observed at the 1:1 effector-to-target (E:T) ratio (mean percent cytotoxicity: 72% vs. 20%, p = 0.001). In vitro cytokine assessment revealed 2.0-2.6 fold increases in IFN-γ, GM-CSF, and TNF-α levels compared to NT-NK cells (p < 0.0001 across all groups). In vivo, FC showed 2.89-fold increase in cell delivery using RD vs. SD on PTD1 (n = 3, p = 0.006). TBL was improved with 5 x 106 cells via PV vs. TV (n = 6) from post-treatment day (PTD) 1-7, with greatest difference seen on PTD7 (12.9 vs. 42.6, p = 0.07). HP analysis showed reduction of TB at PTD7 with PV treatment. Conclusions: NKX101 demonstrated improvements in in vitro cytotoxicity and pro-inflammatory cytokine release. RD techniques in vivo revealed increased cell delivery and improved tumor control. Further studies are underway to confirm our initial findings and understand NKX101 cellular kinetics and susceptibility to immunosuppression in the liver, along with planned clinical evaluation in Phase 1 trials.

Murine NK Cells Require Activation-Dependent Expression of Granzyme B and Perforin To Become Potent Cytotoxic Effectors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 920-920
Author(s):  
Todd A. Fehniger ◽  
Sheng F. Cai ◽  
Xuefang Cao ◽  
Andrew J. Bredemeyer ◽  
Rachel M. Presti ◽  
...  
Keyword(s):  
Cell Death ◽  
Nk Cells ◽  
Target Cell ◽  
Post Infection ◽  
Nk Cell ◽  
Granzyme B ◽  
Poly I:C ◽  
Wild Type

Abstract NK cells predominantly utilize the granule exocytosis pathway to kill virus-infected and malignant target cells. Current paradigms suggest that resting NK cells have pre-formed granules containing granzymes A, B, and perforin and are ready to kill targets immediately upon proper recognition by NK receptors. Here, we report that resting murine NK cells in the spleen exhibit poor cytotoxicity (5.4±1.6% target cell death, 20:1 E:T ratio and 4 hour incubation), compared with cytokine-activated (IL-15, 48 hours) splenic NK cells (59.7±10.6% target cell death), against the RMAS tumor cell line in vitro as measured by a flow-based killing assay. In addition, using intracellular flow cytometric analysis with monoclonal antibodies specific for granzymes A, B, and perforin, we find that resting murine NK cells express abundant granzyme A (86.2±1.9% positive), but little or no granzyme B (4.4±5.4% positive) or perforin (2.6±1.8% positive). Activation of murine NK cells with IL-15 induces robust expression of both perforin (59.1±2.0% positive) and granzyme B (91.5±7.9% positive), which correlates with increased cytotoxicity. Further, granzyme B cluster −/− (26±6.7% target cell death) and perforin −/− (5.7±1.3% target cell death) NK cells have poor cytotoxicity in vitro despite IL-15 activation. Poly I:C simulates RNA virus infection and activates NK cell cytotoxicity in vivo through TLR3 and cytokine cascades. NK cell granzyme B and perforin expression is induced in vivo 24 hours after poly I:C injection, correlating with increased in vitro NK killing of tumor targets. In wild type mice infected with murine cytomegalovirus (MCMV), NK cell expression of both perforin (83.5±4.9% positive) and granzyme B (89.3±2.1% positive) is upregulated in the spleen, peaking 2–4 days post-infection and returning to baseline by 8 days post-infection. In addition, MCMV titers are significantly elevated at day 3 post-infection in both granzyme B cluster −/− (P&lt;0.01) and perforin −/− (P&lt;0.01) mice, compared to wild type mice. Moreover, survival following MCMV infection was significantly lower in granzyme B cluster −/− and perforin −/− mice, compared with wild type mice (P&lt;0.001, see survival curve). Thus, our findings show that murine NK cells require the activation of granzyme B and perforin to become potent cytotoxic effectors. We also demonstrate for the first time that granzyme B is critical for early host defense against MCMV. These findings explain the long-standing observation that murine NK cells require prior activation for potent natural killing of tumor targets in vitro. Further, this requirement for activation-dependent granzyme B and perforin expression in NK cells may influence outcomes in murine models of innate immune anti-tumor and anti-viral responses. Figure Figure

scholarly journals Anticancer Efficacy of Cordyceps militaris Ethanol Extract in a Xenografted Leukemia Model

2017 ◽  
Vol 2017 ◽  
pp. 1-7 ◽  
Author(s):  
Jae Gwang Park ◽  
Young-Jin Son ◽  
Tae Ho Lee ◽  
Nam Joon Baek ◽  
Deok Hyo Yoon ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Caspase 3 ◽  
In Vitro Cytotoxicity ◽  
Cordyceps Militaris ◽  
C6 Glioma Cells ◽  
Control Group ◽  
Therapeutic Effects ◽  
Anticancer Activities

Cordyceps militaris is used widely as a traditional medicine in East Asia. Although a few studies have attempted to elucidate the anticancer activities of C. militaris, the precise mechanism of C. militaris therapeutic effects is not fully understood. We examined the anticancer activities of C. militaris ethanolic extract (Cm-EE) and its cellular and molecular mechanisms. For this purpose, a xenograft mouse model bearing murine T cell lymphoma (RMA) cell-derived cancers was established to investigate in vivo anticancer mechanisms. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, immunoblotting analysis, and flow cytometric assay were employed to check in vitro cytotoxicity, molecular targets, and proapoptotic action of Cm-EE. Interestingly, cancer sizes and mass were reduced in a C. militaris-administered group. Levels of the phosphorylated forms of p85 and AKT were clearly decreased in the group administered with Cm-EE. This result indicated that levels of phosphoglycogen synthase kinase 3β (p-GSK3β) and cleaved caspase-3 were increased with orally administered Cm-EE. In addition, Cm-EE directly inhibited the viability of cultured RMA cells and C6 glioma cells. The number of proapoptotic cells was significantly increased in a Cm-EE treated group compared with a control group. Our results suggested that C. militaris might be able to inhibit cancer growth through regulation of p85/AKT-dependent or GSK3β-related caspase-3-dependent apoptosis.

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