Lymphokine-activated Killer Research Articles

Oxidized-Desialylated Low-Density Lipoprotein Inhibits the Antitumor Functions of Lymphokine Activated Killer Cells.

Elevated concentrations of circulating low density lipoprotein (LDL) that is abnormally oxidized and desialylated is both a precursor to and a hallmark of atherosclerosis. Peripheral blood mononuclear cells (PBMCs) treated in vitro with interleukin-2 (IL-2) become lymphokine activated killer (LAK) cells, the primary effectors of which are NK cells and NKT cells. LAK cells display antitumor functions such as increased cytotoxicity and IFN-γ production, and they have been evaluated as a potential cancer therapeutic. Atherosclerotic processes may influence innate immunity against cancer. Because prior studies have shown that low density lipoprotein (LDL) reduces T-cell and NK cell antitumor functions, we asked whether oxidized-desialylated LDL affects the functionality of LAK cells in vitro. We show here that LAK cells take up oxidized-desialylated LDL to a significantly greater extent than native LDL over a period of 72 hours. This resulted in a significant downregulation of LAK cell cytotoxicity against K562 cells. In particular, the expression of IFN-γ, CD56, and NKG2D were reduced upon oxidized-desialylated LDL treatment of LAK cells and, conversely, their expression was enhanced with native LDL. It was also observed that as the number of CD56 and NKG2D positive cells decreased upon treatment with oxidized-desialylated LDL, the number of CD3 positive cells increased in proportion. Additionally, only a slight inhibition of LAK cell cytotoxicity was observed with desialylation alone of LDL, and no significant inhibition was observed with oxidation alone of LDL. Thus, this study describes a new role of oxidized-desialylated LDL as an inhibitor of the antitumor functions of LAK cells. These observations have implications for how atherosclerosis processes, namely oxidation and desialylation of LDL, may influence LAK cell antitumor activity.

PPARγ induces PD-L1 expression in MSS+ colorectal cancer cells

ABSTRACT Only a small subset of colorectal cancer (CRC) patients benefits from immunotherapies, comprising blocking antibodies (Abs) against checkpoint receptor “programmed-cell-death-1” (PD1) and its ligand (PD-L1), because most cases lack the required mutational burden and neo-antigen load caused by microsatellite instability (MSI) and/or an inflamed, immune cell-infiltrated PD-L1+ tumor microenvironment. Peroxisome proliferator-activated-receptor-gamma (PPARγ), a metabolic transcription factor stimulated by anti-diabetic drugs, has been previously implicated in pre/clinical responses to immunotherapy. We therefore raised the hypothesis that PPARγ induces PD-L1 on microsatellite stable (MSS) tumor cells to enhance Ab-target engagement and responsiveness to PD-L1 blockage. We found that PPARγ-agonists upregulate PD-L1 mRNA/protein expression in human gastrointestinal cancer cell lines and MSS+ patient-derived tumor organoids (PDOs). Mechanistically, PPARγ bound to and activated DNA-motifs similar to cognate PPARγ-responsive-elements (PPREs) in the proximal −2 kb promoter of the human PD-L1 gene. PPARγ-agonist reduced proliferation and viability of tumor cells in co-cultures with PD-L1 blocking Ab and lymphokine-activated killer cells (LAK) derived from the peripheral blood of CRC patients or healthy donors. Thus, metabolic modifiers improved the antitumoral response of immune checkpoint Ab, proposing novel therapeutic strategies for CRC.

Journal-related Activities and Other Special Activities at the 2020 American Society of Anesthesiologists Meeting.

Journal-related Activities and Other Special Activities at the 2020 American Society of Anesthesiologists Meeting.

Immunostimulatory Action of High-Content Active Arabinoxylan in Rice Bran.

Immunostimulatory activity comprises specific and nonspecific immune responses stimulated by internal and external factors. Arabinoxylan is well known for its immunostimulatory activity in vivo and in vitro, although the biological activities of arabinoxylan oligosaccharides depend on their structural features. In this study, we aimed to evaluate in vitro and in vivo the immunostimulatory activity of high-content active arabinoxylan (HCAA) obtained from rice bran through bioconversion by microorganisms and acid hydrolysis. Three microorganisms, Penicillium rocheforti, Aspergillus oryzae, and Pleurotus osteatus, and three different acid concentrations of hydrochloric acid (5, 10, and 20%) and acetic acid (25, 50, and 75%) were used for producing HCAA. HPLC analysis of arabinose and xylose content revealed that fermentation with P. rocheforti followed by hydrolysis with 5% hydrochloric acid was the most efficient to produce HCAA. GPC analysis of HCAA indicates that HCAA is a complex of various forms of saccharides and shows an average molecular weight of 625. Further, in vitro evaluation disclosed that exposure to HCAA (10–200 μg/mL) increased cell viability in mice splenic cells and RAW 264.7 cells. Additionally, exposure of mice to oral administration of HCAA (100 mg/kg) for 4–7 days increased lymphokine-activated killer (LAK)- and macrophage-mediated cytotoxic activity in cancer cells (YAC-1). Furthermore, in vitro exposure to HCAA and oral administrations in mice revealed increased interferon-γ (IFN-γ) and interleukin-10 (IL-10) protein expression through western blot analysis in RAW 264.7 cells and isolated splenic cells. Our results suggest that HCAA developed by bioconversion and acid hydrolysis may enhance immune responses in vivo and in vitro.

Interleukin-8 drives CD38 to form NAADP from NADP+ and NAAD in the endolysosomes to mobilize Ca2+ and effect cell migration.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+ mobilizing second messenger whose formation has remained elusive. In vitro, CD38-mediated NAADP synthesis requires an acidic pH and a nonphysiological concentration of nicotinic acid (NA). We discovered that CD38 catalyzes synthesis of NAADP by exchanging the nicotinamide moiety of nicotinamide adenine dinucleotide phosphate (NADP+ ) for the NA group of nicotinic acid adenine dinucleotide (NAAD) inside endolysosomes of interleukin 8 (IL8)-treated lymphokine-activated killer (LAK) cells. Upon IL8 stimulation, cytosolic NADP+ is transported to acidified endolysosomes via connexin 43 (Cx43) and gated by cAMP-EPAC-RAP1-PP2A signaling. CD38 then performs a base-exchange reaction with the donor NA group deriving from NAAD, produced by newly described endolysosomal activities of NA phosphoribosyltransferase (NAPRT) and NMN adenyltransferase (NMNAT) 3. Thus, the membrane organization of endolysosomal CD38, a signal-mediated transport system for NADP+ and luminal NAD+ biosynthetic enzymes integrate signals from a chemokine and cAMP to specify the spatiotemporal mobilization of Ca2+ to drive cell migration.

Generation of Distinct Patterns of Rheumatoid Arthritis Autoantigens by Peptidylarginine Deiminase Types 2 and 4 During Perforin-Induced Cell Damage.

To address the independent roles of peptidylarginine deiminase type 2 (PAD2) and PAD4 in generating rheumatoid arthritis (RA) autoantigens by using a system that mimics intracellular citrullination in the RA joint. PAD2- or PAD4-expressing 293T cells and mock-transfected cells were used as targets in cytotoxic assays using lymphokine-activated killer cells, cytotoxic YT cell granule contents, or purified human perforin. Protein citrullination and autoantigen production were determined by immunoblotting using the anti-modified citrulline-Senshu method and RA sera (n = 30), respectively. RA sera recognized at least 3 categories of autoantigens in PAD-expressing target cells killed by the cytotoxic lymphocyte granule-induced death pathway. These included: 1) autoantigens targeted in their native form, 2) citrullinated antigens, and 3) antigens cleaved by cytotoxic proteases (e.g., granzymes). Interestingly, although target cells expressing PAD2 or PAD4 showed prominent hypercitrullination of a broad range of proteins during cytotoxic granule-induced cell damage, autoantibodies in RA sera targeted only a very limited number of antigens in hypercitrullinated cells. Furthermore, RA sera showed distinct reactivities to autoantigens generated by PAD2 or PAD4. The cytotoxic granule-induced death pathway has the capacity to modify antigens by inducing hypercitrullination and antigen cleavage in target cells. Interestingly, among a large number of citrullinated proteins generated by PAD2 and PAD4 in cells, only a few are likely involved in the production of autoantibodies in RA.

Effect of administering activated lymphocytes originated from the dam on the immune cell reaction in Holstein calves.

Injection of lymphokine activated killer (LAK) cells is known as useful for activation of cellular immune system. Although the effect of LAK cells has been clarified in human or mice, this effect on function of immune cells has not been examined in calves. Healthy ten Holstein calves were injected with the LAK cells 2 days after birth (LAK Group), and another eight calves were observed as controls (Control Group). All calves received the colostrum formulation on the day of birth, and then, were inoculated with a live attenuated vaccine of bovine herpesvirus (BHV)-1 at 2 (the first vaccination) and 6 (the second vaccination) weeks after birth. Peripheral blood of their dam obtained 3 weeks before calving was used for preparation of LAK cells. Blood samples were taken prior to vaccine inoculation and 3 days after the first inoculation, as well as 3 and 6 days after the second vaccination from all calves. Numbers of CD8+ and CD21+ cells increased significantly after the second vaccination in the LAK Group compared with Control Group. The present study suggested the improved effect of injecting LAK cells originated from dams on immune cells function of young calves after BHV-1 live vaccine.

CAR T cells: continuation in a revolution of immunotherapy.

The recent clinical successes of immunotherapy, as a result of a broader and more profound understanding of cancer immunobiology, and the leverage of this knowledge to effectively eradicate malignant cells, has revolutionised the field of cancer therapeutics. Immunotherapy is now considered the fifth pillar of cancer care, alongside surgery, chemotherapy, radiotherapy, and targeted therapy. Recently, the success of genetically modified T cells that express chimeric antigen receptors (CAR T cells) has generated considerable excitement. CAR T-cell therapy research and development has built on experience generated by laboratory research and clinical investigation of lymphokine-activated killer cells, tumour-infiltrating lymphocytes, and allogeneic haemopoietic stem-cell transplantation for cancer treatment. This Review aims to provide a background on the field of adoptive T-cell therapy and the development of genetically modified T cells, most notably CAR T-cell therapy. Many challenges exist to optimise efficacy, minimise toxicity, and broaden the application of immunotherapies based on T cells.

Assessment of the efficacy of passive cellular immunotherapy for glioma patients.

To evaluate the therapeutic efficacy of passive cellular immunotherapy for glioma, a total of 979 patients were assigned to the meta-analysis. PubMed and the Cochrane Central Register of Controlled Trials were searched initially from February 2018 and updated in April 2019. The overall survival (OS) rates and Karnofsky performance status (KPS) values of patients who underwent passive cellular immunotherapy were compared to those of patients who did not undergo immunotherapy. The proportion of survival rates was also evaluated in one group of clinical trials. Pooled analysis was performed with random- or fixed-effects models. Clinical trials of lymphokine-activated killer cells, cytotoxic T lymphocytes, autologous tumor-specific T lymphocytes, chimeric antigen receptor T cells, cytokine-induced killer cells, cytomegalovirus-specific T cells, and natural killer cell therapies were selected. Results showed that treatment of glioma with passive cellular immunotherapy was associated with a significantly improved 0.5-year OS (p = 0.003) as well as improved 1-, 1.5-, and 3-year OS (p ≤ 0.05). A meta-analysis of 206 patients in one group of clinical trials with 12-month follow-up showed that the overall pooled survival rate was 37.9% (p = 0.003). Analysis of KPS values demonstrated favorable results for the immunotherapy arm (p < 0.001). Thus, the present meta-analysis showed that passive cellular immunotherapy prolongs survival and improves quality of life for glioma patients, suggesting that it has some clinical benefits.

Mannan-Binding Lectin Regulates Inflammatory Cytokine Production, Proliferation, and Cytotoxicity of Human Peripheral Natural Killer Cells.

Natural killer (NK) cells represent the founding members of innate lymphoid cells (ILC) and play critical roles in inflammation and the immune response. NK cell effector functions are regulated and fine-tuned by various immune modulators. Mannan (or mannose)-binding lectin (MBL), a soluble C-type lectin, is traditionally recognized as an initiator of the complement pathway. Recently, it is also considered as an immunomodulator by its interaction with kinds of immune cells. However, the effect of MBL on NK cell function remains unexplored. In this study, we found that human plasma MBL could interact directly with peripheral NK cells partially via its collagen-like region (CLR). This MBL binding markedly suppressed the interleukin-2- (IL-2-) induced inflammatory cytokine tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) production but increased the IL-10 production in NK cells. In addition, the expression of activation surface markers such as CD25 and CD69 declined after MBL treatment. Also, MBL impaired the proliferation and lymphokine-activated killing (LAK) of NK cells. Moreover, we demonstrated that MBL inhibited IL-2-induced signal transducers and activators of transcription 5 (STAT5) activation in NK cells. In conclusion, we have uncovered a far unknown regulatory role of MBL on NK cells, a new clue that could be important in the immunomodulatory networks of immune responses.

Battle of Thermopylae: 300 Spartans (natural killer cells plus obinutuzumab) versus the immortal warriors (chronic lymphocytic leukemia cells) of Xerxes' army.

Aim:To analyze the effects of subcutaneous or intravenous rituximab + lymphokine-activated killer cells, obinutuzumab or ibrutinib on natural killer (NK) cell levels in chronic lymphocytic leukemia and follicular lymphoma patients.Patients & methods:The distribution of peripheral blood NK cells of 31 patients was analyzed by flow cytometry.Results:We detected a decrease of NK cells in peripheral blood below normal range after obinutuzumab treatment. During maintenance treatment with subcutaneous rituximab, an NK cell reduction was less pronounced than after intravenous rituximab treatment, despite lymphokine-activated killer cell infusions.Conclusion:After one dose of obinutuzumab, each NK cell in peripheral blood destroys 25 leukemic cells.

TOPKi-NBD: a fluorescent small molecule for tumor imaging.

OTS514 is a highly specific inhibitor targeting lymphokine-activated killer T cell-originated protein kinase (TOPK). A fluorescently labeled TOPK inhibitor could be used for tumor delineation or intraoperative imaging, potentially improving patient care. Fluorescently labeled OTS514 was obtained by conjugating the fluorescent small molecule NBD to the TOPK inhibitor. HCT116 colorectal cancer cells were used to generate tumors in NSG mice for in vivo studies. Images were generated in vitro using confocal microscopy and ex vivo using an IVIS Spectrum. OTS514 was successfully conjugated to a fluorescent sensor and validated in vitro, in vivo, and ex vivo. The labeling reaction led to TOPKi-NBD with 67% yield and 97% purity after purification. We were able to test binding properties of TOPKi-NBD to its target, TOPK, and compared them to the precursor inhibitor. EC50s showed similar target affinities for TOPKi-NBD and the unlabeled OTS514. TOPKi-NBD showed specific tumor uptake after systemic administration and was microscopically detectable inside cancer cells ex vivo. Blocking controls performed with an excess of the unlabeled OTS514 confirmed specificity of the compound. Overall, the results represent a first step toward the development of a class of TOPK-specific fluorescent inhibitors for in vivo imaging and tumor delineation. TOPK has the potential to be a new molecular target for cancer-specific imaging in a large variety of tumors. This could lead to broad applications in vitro and in vivo.

Application Of Adoptive Immunotherapy In Ovarian Cancer.

Ovarian cancer (OC) has been the most fatal gynecological disease that threatens women’s health. Surgery and platinum-based chemotherapy are the basic ovarian cancer treatments that can improve survival, but the five-year survival rate has not improved because of delayed diagnosis, drug resistance, and recurrence. Novel treatments are needed to improve the prognosis and survival rate of ovarian cancer patients. In recent years, adoptive cell therapy (ACT) has received increasing attention as an emerging therapeutic strategy in the treatment of solid tumors including OC. ACT has shown promising results in many preclinical and clinical trials of OC. The application of ACT depends on different effector cells, such as lymphokine-activated killer (LAK) cells, tumor-infiltrating lymphocytes (TILs), and genetically modified T cells. In this review, we focus on adoptive immunotherapies in ovarian cancer and summarize completed and ongoing preclinical/clinical trials. The future development directions and obstacles for ACT in OC treatment are discussed.

Staccato pulse interleukin-2 (IL-2) in metastatic melanoma (MM) previously treated with checkpoint inhibitors (CPI).

e21022 Background: The CPI ipilimumab, nivolumab, and pembrolizumab have been adopted into common use for patients (pts) with MM. While their clinical activity is undeniable, a substantial fraction of pts treated with these agents experience disease progression. Additional approaches are therefore needed. Intravenous IL-2 given via daily single intravenous infusions (pulses) has been developed to mitigate toxicity while maintaining anticancer activity against MM. Staccato pulse IL-2 is based on three prior observations. Daily IL-2 schedules have previously been demonstrated to induce Lymphokine Activated Killer cell (LAK) activity [Mitchell, 1989]. LAK generated by IL-2 then subsequently exposed to more IL-2 display enhanced cytotoxicity in vitro [Hank, 1990]. Increased numbers of LAK are seen in pts with melanoma treated with daily IL-2 [Quan, 2011]. Methods: In this retrospective study, ten pts with MM were treated with IL-2 18 Million IU/M2 intravenously over 15-30 minutes on days 1-3 and 21.6 Million IU/M2 intravenously over 15-30 minutes on day 5 on an outpatient basis. Cycles were repeated every 3 weeks. Results: Characteristics: 8 females/ 2 males, median age-56 (range: 21-74), median ECOG-1 (0-1); common disease sites: lymph nodes (12), subcutaneous (6), lungs (5), soft tissue (4). Prior CPI: Ipilimumab (8); Pembrolizumab (5); Nivolumab (2). Common toxicities: nausea/emesis (8), myalgia/arthralgia (5), sinus/catarrhal symptoms (5), fatigue (5), and hypotension requiring intravenous fluids (5). No pts required hospitalization for toxicity related to therapy. Median number of cycles: 3 (2-8). Four pts have had partial responses (total response rate = 40%; 95% CI: 10-70%). The median duration of response exceeds 7.4+ months. One pt having had resolution of prior lung, lymph node, and peritoneal metastases (partial response of 24.5+ months) underwent surgical resection of the residual intraabdominal disease focus and is free of disease at 36.5+ months. Overall, responses have occurred in lung, lymph nodes, subcutaneous, bones, peritoneum, small bowel, and soft tissue sites. Conclusions: Staccato pulse intravenous IL-2 has activity in melanoma pts previously treated with CPI.

Targeting a Novel COX2/MET/TOPK Signaling Axis Overcomes gefitinib Resistance in NSCLC

Background: MET overactivation is one of the crucial reasons for tyrosine kinase inhibitors (TKIs) resistance. Cyclooxygenase2 (COX2), a rate-limiting enzyme that converts arachidonic acid to prostaglandins (PGs), and T lymphokine-activated killer cell-originated protein kinase (TOPK) are involved in many neoplastic tissues. Coincidently, we found that COX2 and TOPK were highly expressed in the gefitinib-resistant HCC827 (HCC827GR) cells with MET overactivation. But the relationship between COX2, TOPK and MET in gefitinib-resistance patients has not been explored. Methods: COX2, TOPK and MET expression were examined in EGFR activating mutated NSCLC by immunohistochemical (IHC) analysis. The relationship between COX2, TOPK and MET were explored in vitro and ex vivo. And the inhibition of HCC827GR cell growth by combinating of the COX2 inhibitor (celecoxib), the TOPK inhibitor (pantoprazole) and gefitinib was verified in vitro and in vivo. Findings: We found that COX2 and TOPK were highly expressed in EGFR activating mutated NSCLC; and the progression-free survival (PFS) of COX2, MET, and TOPK positive patients was shorter than that of negative patients. Next, we observed that COX2-TXA2 signaling pathway modulating MET through AP-1 promoted gefitinib resistance. Furthermore, we demonstrated that MET could phosphorylate TOPK at Tyr74 and then induced gefitinib resistance. At last the combination of celecoxib, pantoprazole and gefitinib could inhibit the tumor growth. Interpretation: Our work reveals a novel COX2/MET/TOPK signaling axis inducing gefitinib resistance and suggests a triple combination of the FDA-approved drugs would provide a low-cost and practical strategy to overcome gefitinib resistance. Funding Statement: This work was supported by the national Natural Science Foundation of China (No. 81672936, 81672739, 81503105 & 81472602), the Independent innovation project of Huazhong University of Science and Technology (No. 2018KFYYXJJ084, 2016YXZD034, 2015QN151) and Hubei Province Health and Family Planning Scientific Research Project (No. WJ2019M109, WJ2017M050). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: Ethical approval was obtained from medical ethics committee of Tongji Medical College, Huazhong University of Science and Technology.

Ex vivo-expanded highly purified natural killer cells in combination with temozolomide induce antitumor effects in human glioblastoma cells in vitro.

Glioblastoma is the leading malignant glioma with a poor prognosis. This study aimed to investigate the antitumor effects of natural killer cells in combination with temozolomide as the standard chemotherapeutic agent for glioblastoma. Using a simple, feeder-less, and chemically defined culture method, we expanded human peripheral blood mononuclear cells and assessed the receptor expression, natural killer cell activity, and regulatory T cell frequency in expanded cells. Next, using the standard human glioblastoma cell lines (temozolomide-sensitive U87MG, temozolomide-resistant T98G, and LN-18), we assessed the ligand expressions of receptors on natural killer cells. Furthermore, the antitumor effects of the combination of the expanded natural killer cells and temozolomide were assessed using growth inhibition assays, apoptosis detection assays, and senescence-associated β-galactosidase activity assays in the glioblastoma cell lines. Novel culture systems were sufficient to attain highly purified (>98%), expanded (>440-fold) CD3−/CD56+ peripheral blood-derived natural killer cells. We designated the expanded population as genuine induced natural killer cells. Genuine induced natural killer cells exhibited a high natural killer activity and low regulatory T cell frequency compared with lymphokine-activated killer cells. Growth inhibition assays revealed that genuine induced natural killer cells inhibited the glioblastoma cell line growth but enhanced temozolomide-induced inhibition effects in U87MG. Apoptosis detection assays revealed that genuine induced natural killer cells induced apoptosis in the glioblastoma cell lines. Furthermore, senescence-associated β-galactosidase activity assays revealed that temozolomide induced senescence in U87MG. Genuine induced natural killer cells induce apoptosis in temozolomide-sensitive and temozolomide-resistant glioblastoma cells and enhances temozolomide-induced antitumor effects in different mechanisms. Hence, the combination of genuine induced natural killer cells and temozolomide may prove to be a promising immunochemotherapeutic approach in patients with glioblastoma if the antitumor effects in vivo can be demonstrated.

A Review of Immunotherapeutic Strategies in Canine Malignant Melanoma.

In dogs, melanomas are relatively common tumors and the most common form of oral malignancy. Biological behavior is highly variable, usually aggressive, and frequently metastatic, with reported survival times of three months for oral or mucosal melanomas in advanced disease stages. Classical clinical management remains challenging; thus, novel and more efficacious treatment strategies are needed. Evidence-based medicine supports the role of the immune system to treat neoplastic diseases. Besides, immunotherapy offers the possibility of a precise medicinal approach to treat cancer. In recent years, multiple immunotherapeutic strategies have been developed, and are now recognized as a pillar of treatment. In addition, dogs represent a good model for translational medicine purposes. This review will cover the most relevant immunotherapeutic strategies for the treatment of canine malignant melanoma, divided among five different categories, namely, monoclonal antibodies, nonspecific immunotherapy activated by bacteria, vaccines, gene therapy, and lymphokine-activated killer cell therapy.

Probable Mechanisms Involved in Immunotoxin Mediated Capillary Leak Syndrome (CLS) and Recently Developed Countering Strategies.

Antibody-toxin fused agents or immunotoxins, are a newly engineered class of cytotoxic agents consisting of a bacterial or plant toxin moiety hooked up either to a monoclonal antibody or a specific growth factor. Nevertheless, acquiring a full potency in clinic is mostly restricted due to the Capillary leak syndrome (CLS), a serious immune provoked, life-threatening side effect, subsequent to the endothelial damage, resulting in fluid escape from the bloodstream into tissues including lungs, muscle and brain, developing organ failure and eventually death. Proposed underlying mechanisms include direct damage to endothelial cells, acute inflammation, Lymphokine-activated killer (LAK) cells engagement, alteration in cell-cell/cell-matrix connectivities and cytoskeletal dysfunction. Very poor biodistribution and heterogeneous extravasation pattern in tumor site result in accumulation of ITs close to the extravasation site, gradual toxin release and initiation of nearby endothelial cells lysis, secretion of pro-inflammatory cytokines, development of acute inflammation and engagement of Lymphokine-activated killer (LAK) cells. Intrinsic immunogenicity of applied toxin moiety is another important determinant of CLS incidence. Toxins with more intrinsic immunogenicity possess more probability for CLS development. Recently, development of new generations of antibodies and mutated toxins with conserved cytotoxicity has partly tapered risk of CLS development. Here, we describe probable mechanisms involved in CLS and introduce some of the recently applied strategies for lessening incidence of CLS as much as possible.

Oxidative activation of type III CD38 by NADPH oxidase-derived hydrogen peroxide in Ca2+ signaling.

Reactive oxygen species (ROS) derived from NADPH oxidase (Nox) has been shown to activate ADP-ribosyl cyclase (ARC), which produces the Ca2+ mobilizing second messenger, cyclic ADP-ribose (cADPR). In the present study, we examined how ROS activates cluster of differentiation (CD)38, a mammalian prototype of ARC. CD38 exists in type II and III forms with opposing membrane orientation. This study showed the coexpression of type II and III CD38 in lymphokine-activated killer (LAK) cells. The catalytic site of the constitutively active type II CD38 faces the outside of the cell or the inside of early endosomes (EEs), whereas the basally inactive type III CD38 faces the cytosol. Type III CD38 interacted with Nox4/phosphorylated-p22phox (p-p22phox) in EEs of LAK cells upon IL-8 treatment. H2O2 derived from Nox4 activated type III CD38 by forming a disulfide bond between Cys164 and Cys177, resulting in increased cADPR formation. Our study identified the mechanism by which type III CD38 is activated in an immune cell (LAK), in which H2O2 generated by Nox4 oxidizes and activates type III CD38 to generate cADPR. These findings provide a novel model of cross-talk between ROS and Ca2+ signaling.-Park, D.-R., Nam, T.-S., Kim, Y.-W., Bae, Y. S., Kim, U.-H. Oxidative activation of type III CD38 by NADPH oxidase-derived hydrogen peroxide in Ca2+ signaling.

Design, synthesis and biological evaluation of novel 1-phenyl phenanthridin-6(5H)-one derivatives as anti-tumor agents targeting TOPK

T–lymphokine-activated killer cell–originated protein kinase (TOPK) is a serine-threonine mitogen-activated protein kinase that is highly expressed in many types of human cancer. Due to its important role in cancer progression, TOPK is becoming an attractive target in chemotherapeutic drug design. In this study, a series of 1-phenyl phenanthridin-6(5H)-one derivatives have been identified as a novel chemical class of TOPK inhibitors. Some of them displayed very potent anti-cancer activity with IC50s less than 100 nM, superior than reference compound OTS964. The most potent compound, 9g suppressed the growth of cancer cells by apoptosis and specifically inhibited the activities of TOPK. Oral administration of 9g effectively suppressed tumor growth with TGI >79.7% in colorectal cancer xenograft models, demonstrating superior efficacy compared to OTS964. Pharmacokinetic studies reveal its good oral bioavailability. Our findings therefore show that 9g is a specific inhibitor of TOPK both in vitro and in vivo that may be further developed as a potential therapeutic agent against colorectal cancer.