Strong Cytotoxicity Research Articles

Immunogenic cell death: The cornerstone of oncolytic viro-immunotherapy.

According to the World Health Organization, cancer is one of the leading global health concerns, causing nearly 10 million deaths in 2020. While classical chemotherapeutics produce strong cytotoxicity on cancer cells, they carry limitations of drug resistance and off-target effects and sometimes fail to elicit adequate antitumor protection against tumor relapse. Additionally, most cancer cells have developed various ways to escape immune surveillance. Nevertheless, novel anticancer strategies such as oncolytic viro-immunotherapy can trigger immunogenic cell death (ICD), which can quickly grasp the attention of the host defense machinery, resulting in an ensuing antitumor immune response. Specifically, oncolytic viruses (OVs) can infect and destroy targeted cancer cells and stimulate the immune system by exposing pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) to promote inflammatory reactions, and concomitantly prime and induce antitumor immunity by the release of neoantigens from the damaged cancer cells. Thus, OVs can serve as a novel system to sensitize tumor cells for promising immunotherapies. This review discusses the concept of ICD in cancer, centralizing ICD-associated danger signals and their consequence in antitumor responses and ICD induced by OVs. We also shed light on the potential strategies to enhance the immunogenicity of OVs, including the use of genetically modified OVs and their combination with ICD-enhancing agents, which are helpful as forthcoming anticancer regimens.

Synthesis of biodegradable antimicrobial pH-sensitive silver nanocomposites reliant on chitosan and carrageenan derivatives for 5-fluorouracil drug delivery toward HCT116 cancer cells

The current research relies on a one-pot green biosynthesis of silver nanoparticles (SNPs) with various ratios of silver (Ag) in the existence of N, N, N-trimethyl chitosan chloride (TMC) and carboxymethyl kappa-carrageenan (CMKC), to investigate the effectiveness of the synthesized silver nanocomposites (SNCs) as pH sensitive biodegradable carrier for orally intestinal delivery of 5-fluorouracil (5-FU) drug. FTIR, XRD, TEM and FE-SEM/EDX methods were utilized to demonstrate the structure of the prepared polyelectrolyte complex PEC (TMC/CMKC) and SNCs (TMC/CMKC/Ag). The results showed that the 5-FU encapsulation effectiveness inside all of the prepared SNCs samples was improved by increasing the concentration of Ag, reaching 92.16 ± 0.57 % with 3 % Ag. In vitro release behavior of 5-FU loaded SNC 3 % (TMC/CMKC/Ag 3 %), displayed slow and sustained release reaching 96.3 ± 0.81 % up to 24 h into pH 7.4 medium. The successful release of 5-FU from the loaded SNC 3 % was confirmed through occurrence of strong cytotoxicity, with an IC50 value of 31.15 μg/ml, and high % of apoptotic cells (30.66 %) within the treated HCT116 cells. Besides, SNC 3 % showed good biodegradability and antimicrobial properties against different bacterial strains. Overall, SNC 3 % can be suggested as an effective system for both controlled drug delivery and antibacterial action.

Effect of Operating Parameters on the Properties of Carbon Dots from Spent Coffee Grounds

Carbon dots (nanosized materials) exhibit excellent properties such as strong fluorescence, low cytotoxicity, biocompatibility, and good biodegradability which can be synthesized from various carbon sources such as biomass and food waste etc. Due to their unique properties, they are considered as potential alternatives to replace conventional metal-based quantum dots in a number of applications from energy storage, biomedicine to water treatment. In this work, we demonstrate an intensified and green approach to synthesize carbon dots from waste coffee. The synthesized carbon dots have application in sensing e.g. detecting heavy metals, contaminants in water, drug delivery and bioimaging.

QUASSINOIDS FROM THE ROOTS OF Brucea javanica AND THEIR POTENTIAL CYTOTOXICITY

A phytochemical study of the roots of Brucea javanica was conducted and four alkaloids and three quassinoids were isolated. The structures of the compounds were verified using physical and spectroscopic data, which were compared to the literature. The cytotoxicity of the compounds was evaluated against KB (oral cavity carcinoma), NCI-H187 (small cell lung cancer), and MCF-7 (breast adenocarcinoma) cell lines. The results showed that the quassinoid, Bruceine A, exhibited very strong cytotoxicity against KB, and high potency against NCI-H187, with both being 581- and 100-fold more active than ellipticine, and 99- and 10-fold more active than doxorubicin, respectively. These results suggest the potential use of bruceine A as an anticancer agent

Synthesis, characterization, DFT, cytotoxicity evaluation and molecular docking of a new carbothioamide ligand and its coordination compounds

2-(2-hydroxybenzoyl)-N-phenylhydrazine-1-carbothioamide (H4L) and its Zn(II), Co(II), Cu(II), and Ni(II) coordination compounds have been synthesized. The results of IR, Mass spectra, UV–visible, 1H&13C-NMR, ESR, effective magnetic moments and elemental analyses suggested various structures of the synthesized coordination compounds. A square planar stereochemistry was suggested for Cu(II) and Ni(II) complexes, whereas octahedral stereochemistry was suggested for Co(II) complex. The structures were geometry optimized by DFT at exchange–correlation level (DFT/B3LYP) and 6-311++G(d,p) basis set. The electronic spectra were simulated and the optical band gap was calculated and compared with the experimental results. The determined optical band gap (Eg) represents direct electronic transitions with values in the range 4.42–4.45 eV. Molecular docking indicated the possible interactions of the compounds with the studied proteins. The cytotoxicity of the compounds were tested against HCT-116 and HePG2 cell lines and compared with Doxorubicin. H4L has a very strong cytotoxicity against the tested cell lines.

A2AR limits IL-15-induced generation of CD39+ NK cells with high cytotoxicity.

CD39-mediated inhibition of natural killer (NK) cell activity has been demonstrated, but the characteristics of CD39+ NK cells in humans are not known. We investigated the characteristics of human circulating CD39+ NK cells. In healthy donors, the proportion of circulating CD39+ NK cells in total NK cells was relatively low compared with that of CD39- NK cells. Nonetheless, a higher proportion of CD39+ NK cells expressed CD107a. Similarly, a higher proportion of CD39+ NK cells expressed CD107a in patients with hepatitis B virus or patients with hepatocellular carcinoma. Stimulation with NK-sensitive K562 cells or interleukin (IL)-12/IL-18 activated CD39+ NK cells to express higher levels of CD107a, IFN-γ and TNF-α, relative to CD39- NK cells. Importantly, IL-15 induced the generation of CD39+ NK cells. In contrast, A2A adenosine receptor (A2AR) ligation suppressed the generation of CD39+ NK cells by inhibiting IL-15 signaling. These data for the first time demonstrated that A2AR counteracts IL-15-induced generation of human CD39+ NK cells, which have a stronger cytotoxicity than CD39- NK cells. IL-15-induced human CD39+ NK cells might be better choice for immunotherapy based on adoptive transfer of NK cells.

Cr(vi) permanently binds to the lipid bilayer in an inverted hexagonal phase throughout the reduction process.

Cr(vi) is a harmful, carcinogenic agent with a high permeability rate throughout the lipid membranes. In an intracellular environment and during interactions with cellular membranes, it undergoes an instant reduction to lower oxidation states throughout radical states, recognized as the most dangerous factor for cells. The cellular membrane is the most visible cellular organelle in the interior and exterior of a cell. In this study, liposomes and non-lamellar inverted hexagonal phase lipid structures based on phosphoethanolamine (PE) were used as model cellular bilayers because of their simple composition, preparation procedure, and the many other properties of natural systems. The lipid membranes were subjected to 0.075 mM Cr(vi) for 15 min, after which the Cr content was removed via dialysis. This way, the remaining Cr content could be studied qualitatively and quantitatively. Using the combined XRF/XAS/EPR approach, we revealed that some Cr content (Cr(iii) and Cr(vi)) was still present in the samples even after long-term dialysis at a temperature significantly above the phase transition for the chosen liposome. The amount of bound Cr increased with increasing PE and -C[double bond, length as m-dash]C- bond content in lipid mixtures. Internal membrane order decreased in less fluid membranes, while in more liquified ones, internal order was only slightly changed after subjecting them to the Cr(vi) agent. The results suggest that the inverted hexagonal phase of lipid structures is much more sensitive to oxidation than the lamellar lipid phase, which can play an important role in the strong cytotoxicity of Cr(vi).

Effective combination of liposome-targeted chemotherapy and PD-L1 blockade of murine colon cancer.

Therapeutic cancer drug efficacy can be limited by insufficient tumor penetration, rapid clearance, systemic toxicity and (acquired) drug resistance. The poor therapeutic index due to inefficient drug penetration and rapid drug clearance and toxicity can be improved by using a liposomal platform. Drug resistance for instance against pemetrexed, can be reduced by combination with docetaxel. Here, we developed a specific liposomal formulation to simultaneously deliver docetaxel and pemetrexed to enhance efficacy and safety. Hydrophobic docetaxel and hydrophilic pemetrexed were co-encapsulated into pH-sensitive liposomes using a thin-film hydration method with high efficiency. The physicochemical properties, toxicity, and immunological effects of liposomes were examined in vitro. Biodistribution, anti-tumor efficacy, and systemic immune response were evaluated in vivo in combination with PD-L1 immune checkpoint therapy using two murine colon cancer models. In cellular experiments, the liposomes exhibited strong cytotoxicity and induced immunogenic cell death. In vivo, the treatment with the liposome-based drug combination inhibited tumor development and stimulated immune responses. Liposomal encapsulation significantly reduced systemic toxicity compared to the delivery of the free drug. Tumor control was strongly enhanced when combined with anti-PDL1 immunotherapy in immunocompetent mice carrying syngeneic MC38 or CT26 colon tumors. We showed that treatment with liposome-mediated chemotherapy of docetaxel and pemetrexed combined with anti-PD-L1 immunotherapy is a promising strategy for the treatment of colon cancers.

Quantitative determination of bromochloroacetamide in mice urine by gas chromatography combined with salting-out assisted dispersive liquid-liquid microextraction.

Bromochloroacetamide (BCAcAm) is the main haloacetamide (HAcAm) detected in drinking water in different regions and exhibits strong cytotoxicity and genotoxicity. However, there is no appropriate method for detecting BCAcAm in urine or other biological samples, and thus, the internal exposure level in the population cannot be accurately assessed. In this study, a gas chromatography-electron capture detector (GC-ECD) was combined with salting-out assisted dispersive liquid-liquid microextraction (SA-DLLME) to develop a rapid and robust method for BCAcAm detection in urine of mice continuously exposed to BCAcAm. The factors influencing the pre-treatment procedure, including the type and volume of extraction and disperser solvents, extraction and standing time, and the amount of salt, were evaluated systematically. Under the optimised conditions, the analyte achieved good linearity in the spiked concentration range of 1.00-400.00 μg L-1, and the correlation coefficient was higher than 0.999. The limit of detection (LOD) and the limit of quantification (LOQ) were 0.17 μg L-1 and 0.50 μg L-1, respectively. The recoveries ranged from 84.20% to 92.17%. The detection of BCAcAm at three different calibration levels using this method afforded an intra-day precision of 1.95-4.29%, while the inter-day precision range was 5.54-9.82% (n = 6). This method has been successfully applied to monitor the concentration of BCAcAm in mouse urine in toxicity experiments and can provide technical support for assessing human internal exposure levels and health risks in later studies.

High cytotoxicity of a degraded TBBPA, dibromobisphenol A, through apoptotic and necrosis pathways

Halogenated flame retardants comprising bisphenol A (BPA) derivatives, such as tetrabromobisphenol A (TBBPA), have been studied their adverse effects on human health. However, despite the fact that these halogenated BPAs are easily degraded in the environment, the risks to living organisms due to these degraded products have mostly been overlooked. To evaluate the potential toxicity of degraded TBBPAs and related compounds, we examined the cytotoxicity of halogenated bisphenol A derivatives possessing one to four halogen atoms in vitro. The results indicated that the degraded TBBPA derivatives exhibited strong cytotoxicity against HeLa cells than TBBPA. Interestingly, the di-halogenated BPA derivatives possessing two halogen atoms exhibited the strongest cytotoxicity among tested compounds. In addition, a lactate dehydrogenase release assay, fluorescence spectroscopy and flow cytometry results indicated that dibromo-BPA and diiodo-BPA induced both apoptotic and necrotic cell death by damaging the cell membranes of HeLa cells. Moreover, Escherichia coli growth was inhibited in the presence of dehalogenated TBBPA and related compounds. These findings suggest that halogenated BPA derivatives that leak from various flame-retardant-containing products require strict monitoring, as not only TBBPA but also its degraded products in environment can exert adverse effects to human health.

Synthesis, antiproliferative and anti-MDR activities of lathyrane diterpene derivatives based on configuration inversion strategy

A series of lathyrane-type Euphorbia diterpene derivatives featured 3R configuration (H-3β) were synthesized from natural rich Euphorbia factor L3via modified Mitsunobu reaction based on configuration inversion strategy. The antiproliferation activity and MDR reversal ability of the lathyrane derivatives were evaluated, and the most synthesized compounds showed moderate or strong potencies. Among them, diterpenes 21 (IC50 values of 2.6, 5.2 and 13.1 μM, respectively) and 25 (IC50 values of 5.5, 8.6 and 1.3 μM, respectively) presented the strong cytotoxicity against MCF-7, 4 T1 and HepG2 cells. Meanwhile, derivative 25 exhibited excellent MDR reversal ability with the reversal fold of 16.1 higher than that of verapamil. The cellular thermal shift assay and molecular docking proved direct engagement of diterpene 25 to ABCB1, suggesting 25 could be a promising MDR modulator. Furthermore, the preliminary SARs of these diterpenes were also discussed.

Low Dose of Green Synthesized Silver Nanoparticles is Sufficient to Cause Strong Cytotoxicity via its Cytotoxic Efficiency and Modulatory Effects on the Expression of PIK3CA and KRAS Oncogenes, in Lung and Cervical Cancer Cells

Low Dose of Green Synthesized Silver Nanoparticles is Sufficient to Cause Strong Cytotoxicity via its Cytotoxic Efficiency and Modulatory Effects on the Expression of PIK3CA and KRAS Oncogenes, in Lung and Cervical Cancer Cells

CD318 is a target of chimeric antigen receptor T cells for the treatment of colorectal cancer.

Colorectal cancer (CRC) currently has a poor prognosis with a 6.9-year median survival time; to relieve this malignant cancer, we proposed to establish CRC xenografts that can be used to evaluate the cytotoxicity of adoptive chimeric antigen receptor (CAR)-T cells and accelerate the clinical translation of CAR-T cells for use against CRC. We first verified that CD318 had a higher expression level in primary human CRC tissues than in normal tissues based on hundreds of clinical samples. Then, we redirected CAR-T cells containing anti-CD318 single-chain variable fragment (anti-CD318 scFv), CD3ζ, CD28, and Toll-like receptor 2 (TLR2) domains. Next, we evaluated the function of these CAR-T cells in vitro in terms of surface phenotype changes, cytotoxicity and cytokine secretion when they encountered CD318+ CRC cells. Finally, we established two different xenograft mouse models to assess in vivo antitumor activity. The results showed that CAR318 T cells were significantly activated and exhibited strong cytotoxicity and cytokine-secreting abilities against CRC cells in vitro. Furthermore, CAR318 T cells induced CRC regression in different xenograft mouse models and suppressed tumors compared with CAR19 T cells. In summary, our work demonstrates that CAR318 T cells possess strong antitumor capabilities and represent a promising therapeutic approach for CRC.

Role of Epidermal Growth Factor Receptor-Specific CAR-T Cells in the Suppression of Esophageal Squamous Cell Carcinoma.

ESCC is a highly malignant tumor, and its morbidity and mortality in China account for more than 50% of the world's total rates. As effective treatments are lacking, the 5-year survival rate of patients does not exceed 30%. CAR-T-cell-based immunotherapy has emerged as one of the most promising cancer treatments; however, there are relatively fewer reports regarding its application for ESCC. In this study, we conducted large-sample whole-genome sequencing (WGS) and RNA-seq analysis of patients with ESCC from China to examine the feasibility of EGFR-targeting CAR-T cells in the treatment of ESCC. We found much higher levels of EGFR gene amplification and overexpression in tumors than in the normal tissues, indicating that EGFR could be a promising target of CAR-T-cell-based immunotherapy in ESCC. Therefore, we tested EGFR-targeting CAR-T cells for lytic activity against ESCC cells as a model to establish cellular immunotherapy for ESCC. Five types of CAR-T cells targeting EGFR were constructed, two of which, CAR1-T and CAR2-T, showed a strong cytotoxicity against ESCC in in vitro and in vivo experiments. The results of this study suggest that CAR1-T and CAR2-T have the potential to be used for anti-ESCC immunotherapy in clinics.

Cytotoxicity, Phytochemical Screening and Genetic analysis of Ginger (Zingiber officinale Rosc.) Callus and Rhizome

• Ginger rhizome extracts displayed strong cytotoxic effect against HT29, HT116 and MCF-7 cancer cells lines with highest activity against HT29. • Ginger callus extracts recorded non-cytotoxic effect against all examined cancer cells with IC50 >100 µg/mL. • Ginger rhizome extracts contained more phenols and terpenes than the callus ones. • The principal components of ginger; 6-gingerol and 6-shogaol were not detected in callus extracts. • Incidence of genetic changes in DNA of ginger callus during in vitro culture in 2,4-D containing medium. • Most polymorphic bands (11 out of 12) were generated in callus during in vitro culture with total polymorphism 23.07% between callus and plant. Ginger ( Zingiber officinale Rosc.) is well known as distinctive spice with considered therapeutic values. In the current work we aimed to investigate cytotoxic effect, phytochemical screening and genetic analysis of ginger callus and their rhizome. Cytotoxicity of ginger rhizome and callus extracts was examined against some human cancer cell lines HT29, HT116 and MCF-7 by MTT method. Phytochemicals were screened by thin layer chromatography (TLC) and genetic analysis was performed by ISSR markers. Ginger rhizome principle components, 6-gingerol and 6-shogoal were used as standards to detect their presence in callus. Results showed that, rhizome extracts displayed a strong cytotoxicity towards all tested cancer cells with the highest activity (IC 50 20.4 ± 3.0 µg/mL; P< 0.05) against HT29. IC 50 of ginger callus extracts were >100 µg/mL in all examined cancer cell lines. TLC chromatograms of rhizome and callus extracts revealed the presence of phenols and terpenes, whereas 6-gingerol and 6-shogaol were only detected in rhizome extracts. Genetic analysis by ISSR markers revealed occurrence some genetic changes in DNA of callus compared to that of rhizome with total polymorphism 23.07%. In conclusion, ginger cytotoxicity to HT29, HT116 and MCF-7 cancer cells was mainly associated with the presence of 6-shogaol and 6-gingerol in extracts, which appeared as strong cytotoxic effect in rhizome compared to non-cytotoxic effect was evaluated in the callus extracts. Therefore, further studies on the callus elicitation and precursors feeding to induce biosynthesis of ginger bioactive molecules are recommended.

Anticancer effect of zinc oxide nanoparticles prepared by varying entry time of ion carriers against A431 skin cancer cells in vitro.

Although, zinc oxide nanoparticles (ZRTs) as an anti-cancer agent have been the subject of numerous studies, none of the reports has investigated the impact of the reaction entry time of ion-carriers on the preparation of ZRTs. Therefore, we synthesized variants of ZRTs by extending the entry time of NaOH (that acts as a carrier of hydroxyl ions) in the reaction mixture. The anti-proliferative action, morphological changes, reactive oxygen species (ROS) production, and nuclear apoptosis of ZRTs on human A431 skin carcinoma cells were observed. The samples revealed crystallinity and purity by X-ray diffraction (XRD). Scanning electron microscopy (SEM) images of ZRT-1 (5min ion carrier entry) and ZRT-2 (10min ion carrier entry) revealed microtubule like morphology. On prolonging the entry time for ion carrier (NaOH) introduction in the reaction mixture, a relative ascent in the aspect ratio was seen. The typical ZnO band with a slight shift in the absorption maxima was evident with UV-visible spectroscopy. Both ZRT-1 and ZRT-2 exhibited non-toxic behavior as evident by RBC lysis assay. Additionally, ZRT-2 showed better anti-cancer potential against A431 cells as seen by MTT assay, ROS generation and chromatin condensation analyses. At 25μM of ZRT-2, 5.56% cells were viable in MTT test, ROS production was enhanced to 166.71%, while 33.0% of apoptotic cells were observed. The IC50 for ZRT-2 was slightly lower (6μM) than that for ZRT-1 (8μM) against A431 cells. In conclusion, this paper presents a modest, economical procedure to generate ZRT nano-structures exhibiting strong cytotoxicity against the A431 cell line, indicating that ZRTs may have application in combating cancer.

Toward the total synthesis of scytophycins: Synthesis of the C7–C21 fragments of scytophycins A, B, and C

Scytophysins are cytotoxic macrolides with a 22-membered ring and are characterized by their strong cytotoxicities targeting cytoskeletal protein actin. Toward the comprehensive total synthesis of scytophycins, we have investigated the late-state functionalization using fragment compounds. We established stereoselective construction of the C7-C21 part in scytophycins A and B involving characteristic C16 epoxide by using Sharpless epoxidation. Although stereoselectivity remains unsatisfactory, reduction of the C16 exo-olefin for the synthesis of scytophycin C was achieved.

NNV024, a Humanized Anti-CD37 Antibody with Enhanced ADCC and Extended Plasma Half-Life for the Treatment of B-Cell Malignancies

Background: There is a significant unmet clinical need for new therapeutic approaches for patients with B-cell non-Hodgkin lymphomas (B-NHL) who relapse or are refractory to anti-CD20 therapies. Moreover, certain B-cell malignancies do not express CD20. High expression of the CD37 antigen on malignant B-cells associated with limited or no expression on other hematopoietic cells makes CD37 a highly attractive alternative target for B-cell depleting therapies. We have therefore developed a humanized anti-CD37 monoclonal antibody (NNV024) engineered to exhibit a strong antibody-dependent cellular cytotoxicity (ADCC). Methods: Generation of NNV024 was performed by combining in silico CDR-grafting and structure-based back mutation. Candidates were then selected for their affinity to CD37 expressing cell line, Ramos. In silico immunogenicity prediction tools were integrated in the selection process to support the generation of a candidate with low immunogenicity potential (NNV023). Last, to increase ADCC, the selected candidate was afucosylated at the N-linked bi-antennary glycan at N297 of the Fc region (NNV024). The NNV024 was characterized in vitro and in vivo. Binding to human Fc gamma receptors (FcγRs), neonatal Fc receptor (FcRn), and complement component 1q (C1q) was confirmed by ELISA and SPR. ADCC and antibody-dependent cellular phagocytosis (ADCP) induction were measured using cell-based reporter assays with eleven cell lines derived from B-NHL subtypes such as Burkitt's lymphoma, diffuse large B-Cell lymphoma (DLBCL), and mantel cell lymphoma (MCL). All cell lines express both CD20 and CD37 on their cell surface. In addition, B cell chronic lymphocytic leukaemia (B-CLL) cells isolated from patient samples were used to test induction of ADCC, ADCP and complement-dependent cytotoxicity (CDC) in vitro. NNV024 plasma pharmacokinetics were assessed in Tg32 transgenic mice expressing human FcRn, which is considered a key regulator of IgG plasma half-life. A therapy study comparing NNV024 (n=10) and obinutuzumab (n=10) was performed using a disseminated tumor (Daudi) model in CB17-SCID mice (107 cells/mouse, i.v.). The anti-CD20 Abs rituximab and obinutuzumab as well as the anti-CD37 Ab, Duohexabody-CD37, were used for benchmarking. Results: As expected, the glyco-engineering increased affinity of NNV024 to FcγRIIIa (KD before afucosylation: 460.8 ng/mL; KD after: 104.1 ng/mL). Binding of Abs to FcγRIIIa activates immunological effector cells such as Natural Killer cells, macrophages, monocytes, and eosinophils. NNV024 demonstrated ADCC superior to that observed with obinutuzumab, rituximab, and Duohexabody-CD37. NNV024 exhibited a 2-fold prolonged plasma half-life in mice (8.9±0.8 days) compared to obinutuzumab (4.4±1.3 days) and Duohexabody-CD37 (4.1±1.2 days). Mice survival was enhanced up to ~ 40% when treated with NNV024 (10 µg/mouse - 70.5 days; 50 µg/mouse - 84 days) compared to obinutuzumab at a dose of 10 µg (70.5 days vs. 53 days) or 50 µg (84 days vs. 62 days). Conclusion: These encouraging preclinical results support further development of NNV024 as a potential therapeutic mAb candidate for the treatment of B-cell malignancies and B-cell driven autoimmune disorders.

CD84 Is a Therapeutically Targetable Driver of Leukemogenesis Via Disruption of Energy Supply in Acute Myeloid Leukemia

CD84 is a member of the signaling lymphocyte activation molecule (SLAM) family; it forms homophilic dimers by self-association. CD84 is reported as an important survival receptor in chronic lymphocytic leukemia and its activation upregulates PD-L1 expression on leukemia cells. We and others recently reported that CD84 is highly expressed on myeloid derived suppressor cells, but its function in myeloid malignancies has yet to be investigated. Using publicly available microarray data (GSE13509), we found that CD84 is overexpressed on acute myeloid leukemia (AML) cells compared to normal hematopoietic counterparts (AML, n=542 vs. normal, n=73, p<0.0001). and that its upregulation is associated with poor prognosis in AML patients (GSE10358; p=0.0105). Knocking down CD84 expression in AML cell lines (THP1 and HEL) by shRNAs caused a substantial inhibition of cell viability and extended THP1-luciferase xenografted mice survival in vivo (median survival: shCtrl, 38 days vs shCD84, 53 days, p=0.0015, n=5). Moreover, we observed that CD84 depletion in AML primary patient cells dramatically induced cell apoptosis (shCtrl: 13.0±1.84% vs shCD84: 29.33±1.3%; n=3; p=0.02) and inhibited colony formation (shCtrl: 68.44±11.65 vs shCD84: 15.56±6.32; n=3; p=0.01). In light of highly expressed CD84 in MLL-r AML and inv(16) AML, we employed MLL-AF9 and CBFB-MYH11(CM) AML mouse models, and found that CD84 knockdown robustly dampened cell growth and decreased colony-forming units in vitro. In both models, bone marrow (BM) transplantation shown that CD84 deficiency significantly decreased leukemic engraftment in BM (MLL-AF9: shCtrl: 95.92±0.61% vs shCD84: 76.42±4.11%, p=0.0016, n=5/group; inv(16): shCtrl: 39.02±2.98% vs shCD84: 13.66±1.24%, p<0.0001, n=5/group), spleen, and peripheral blood (PB). In AML patient derived xenografts (PDX), we observed AML burden was markedly reduced in BM (shCtrl:51.22±10.29% vs shCD84: 2.304±0.16%; n=5/group, p=0.0014) and spleen (shCtrl: 8.790±2.989% vs shCD84: 0.8420±0.13%; n=5/group, p=0.0255) in the CD84 knockdown group. Mechanistically, CD84 deletion depressed mTOR signaling, induced mitochondrial stress, and disrupted energy supply chains based on RNA-seq analysis. We demonstrated CD84 deletion caused mitochondrial dysfunction as indicated by decreasing oxidation phosphorylation (shCtrl vs shCD84, p<0.0001) and fatty acid oxidation (shCtrl vs shCD84, p=0.0004). Next, we determined that CD84 deletion caused disruption of mitochondrial matrix morphology and loss of mitochondrial cristae in AML. We also observed a dramatic loss of mitochondrial membrane potential upon CD84 deletion. We generated a novel murine-human chimeric anti-human CD84 monoclonal antibody (mAb) with high affinity and specificity. In the presence of NK cells, anti-CD84 mAb induced strong antibody-dependent cell-mediated cytotoxicity in CD84-expressing AML lines (n=2) and primary samples (n=2), but not in healthy donor CD34+ cells (n=2). Next, the luciferase-expressing THP1 was transplanted into NSG mice. Mice with comparable tumor burden were randomized into 2 groups: control human IgG or anti-CD84 mAb (5mg/kg, i.v, bid, n=6/group), every week together with 3x106 human PB mononuclear cells (PBMCs) as effector cells (for 4 weeks). Anti-CD84 mAb significantly suppressed leukemia development and extended median survival (IgG: 30 days vs anti-CD84 mAb: undefined, p=0.0018). However, without PBMCs, human IgG or anti-CD84 mAb did not induce significant differences in tumor burden or median survival (n=5/group; p=0.5082). Finally, we developed two AML PDXs in NSG mice. Following engraftment >1% in PB, mice were divided into 2 groups (n=6/group). We maintained the same treatment strategy as applied above. In both AML PDXs, the BM engraftment of CD45+CD33+ cells markedly decreased in anti-CD84 mAb treated mice (AML #1: IgG: 94.25± 0.41% vs anti-CD84 mAb: 71.52±3.96%, p= 0.0002; AML #2: IgG: 88.74±0.7% vs anti-CD84 mAb: 53.98±5.61%, p=0.0003). Anti-CD84mAb treatments in secondary transplantation of BM harvested from the first treatment groups resulted in further significant reduction in BM engraftment (IgG: 86.48± 0.67% vs anti-CD84 mAb: 29.74±6.02%, n=5, p<0.0001), and extended survival (IgG, 28 days vs anti-CD84 mAbs, 36 days, p=0.0026, n=5) following third BM transplantation. The results indicated our pharmacological targeting of CD84 inhibits leukemogenesis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Preparation of Magnetized Mesenchymal Stem Cells Using Magnetic Liposomes to Enhance Their Retention in Targeted Tissue -Evaluation of Retention and Anti-inflammatory Efficiency in Skeletal Muscle

Magnetic nanoparticle-incorporated liposomes (magnetic liposomes) are considered a promising site-specific drug delivery carrier. Although there are many reports on the development of magnetic liposomes, most of them focus on the characteristics of magnetic nanoparticles, rather than liposomes. Therefore, we first evaluated the effect of the physicochemical properties of magnetic liposomes on their interaction with cells. The highest cellular uptake and retention under a magnetic field was observed using small magnetic cationic liposomes. However, magnetic cationic liposomes exhibited strong cytotoxicity. Based on these results, we constructed complexes of less toxic magnetic anionic liposomes (Mag-AL) and atelocollagen (ATCOL), a biocompatible cationic biomaterial. The cellular associated amount of Mag-AL under a magnetic field was significantly increased when Mag-AL was complexed with ATCOL, and it was comparable to that of magnetic cationic liposomes. Additionally, Mag-AL/ATCOL complexes produced no cytotoxic effect. Moreover, liver accumulation of Mag-AL/ATCOL complexes was significantly increased at a magnetic field-exposed region after intravenous injection in rats. These results indicate that Mag-AL/ATCOL complexes may be a safe and efficient magnetic responsive drug carrier. Next, we applied Mag-AL/ATCOL complexes to prepare magnetized cells for effective cell therapy. Mesenchymal stem cells (MSCs), which have the capacity to suppress tissue inflammation, were efficiently magnetized by incubation with Mag-AL/ATCOL complexes under a magnetic field. Intramuscularly injected magnetized MSCs were significantly retained in mouse skeletal muscle in the presence of a magnetic field and modulated tissue inflammatory responses. These results suggest that magnetized MSCs are useful for muscle regeneration.