Cytotoxic Cells Research Articles

Anti-IL-1RAP scFv-mSA-S19-TAT fusion carrier as a multifunctional platform for versatile delivery of biotinylated payloads to myeloid leukemia cells

Acute myeloid leukemia (AML) is an aggressive blood cancer with frequently poor clinical outcomes. This heterogeneous malignancy encompasses genetically, molecularly, and even clinically different subgroups. This makes it difficult to develop therapeutic agents that are effective for all subtypes of the disease. Therefore, a selective, universal, and adaptable delivery platform capable of carrying various types of anti-neoplastic agents is an unmet requirement in this area. Two multifunctional fusion proteins were designed for the delivery of biotinylated cargoes to human myeloid leukemia cells by fusing an anti-IL-1RAP single-chain antibody with streptavidin (tetramer or monomer), a cell-penetrating peptide (CPP), and an endosomolytic peptide in a single biomacromolecule. The designed fusions were analyzed primarily in silico, and the biofunctionality of the selected fusion was fully characterized via several binding assays, hemolysis assay, confocal microscopy and cell cytotoxicity assay after production via the Escherichia coli (E. coli) system. The refolded protein exhibited desirable binding activity to leukemic cells, pure antigen and biotinylated BSA. Further analyses revealed efficient cellular uptake, endosomolytic activity, and nuclear penetration without any detectable cytotoxicity toward normal epithelial cells. The described platform seems to have great potential for targeted delivery of different therapeutics to malignant myeloid cells.

Processing-induced reduction in dianthrones content and toxicity of Polygonum multiflorum: Insights from ultra-high performance liquid chromatography triple quadrupole mass spectrometry analysis and toxicological assessment.

Polygonum multiflorum-induced liver injury (PM-DILI) has significantly hindered its clinical application and development. This study investigates the variation in content and toxicity of dianthrones, the toxic components of P. multiflorum, during different processing cycles. We employed the ultra-high-performance liquid chromatography triple quadrupole mass spectrometry method to quantify six dianthrones in raw P. multiflorum and formulations processed with a method called nine cycles of steaming and sunning. Additionally, toxicity assessments were conducted using human normal liver cell line L02 and zebrafish embryos. Results indicate a gradual reduction in dianthrones content with increasing processing cycles. Processed formulations exhibited significantly reduced cytotoxicity in L02 cells and hepatotoxicity in zebrafish embryos. Our findings elucidate the relationship between processing cycles and P. multiflorum toxicity, providing theoretical support for its safe use.

Development of FK506-loaded maleimide-functionalized cationic niosomes for prolonged retention and therapeutic efficacy in dry eye disease.

Tacrolimus (FK506) is widely used in ocular diseases such as corneal transplantation-host disease, uveitis, conjunctivitis, and dry eye disease (DED). However, its low aqueous solubility and poor ocular retention pose challenges for its application in the eye diseases. This study developed a novel FK506-loaded maleimide-functionalized cationic niosomes (FK506 M-CNS), aiming to prolong the retention time of FK506 in the eye and enhance its therapeutic efficacy. FK506 M-CNS had a particle size of 87.69 ± 1.05nm and zeta potential of 22.06 ± 1.01 mV. Results of histological evaluation through H&E staining and in vitro cytotoxicity of human corneal epithelial cells consistently revealed the excellent biocompatibility of FK506 M-CNS. FK506 M-CNS exhibited superior ocular retention compared to the market product Talymus®. FK506 M-CNS significantly alleviated the symptoms of DED and promoted the recovery of corneal epithelia. FK506 M-CNS group had the lowest expression levels of inflammatory factors associated with DED. These superiorities might be due to the electrostatic interaction between cationic niosomes and negatively charged mucin in the eye, and the covalent binding of maleimide with the thiol group in the mucin. The maleimide group improved the ocular retention and efficacy of FK506, but did not increase the toxicity. Results indicated that FK506 M-CNS had great potential as a nanopharmaceutical in the treatment of ocular diseases, and M-CNS could be a promising drug carrier for ophthalmic drug delivery systems.

Delivery of IL2RG mRNA via nanoparticles to enhance CD8+ T cell promotes anti-tumor effects against late-stage triple-negative breast cancer

BackgroundThe manipulation of CD8+ T cell functions through genetic modifications presents a novel approach to cancer immunotherapy. This study aimed to explore the effects of IL2RG-overexpressing CD8+ T cells and assess the efficacy of delivering IL2RG mRNA via nanoparticles (NPs) to the tumor microenvironment in triple-negative breast cancer (TNBC).MethodsSingle-cell RNA sequencing (scRNA-seq) was conducted on tissue from early and late-stage TNBC, followed by a meta-analysis of six breast cancer arrays from the GEO database to identify candidate genes. CRISPR/Cas9 was employed for gene knockout and overexpression in CD8+ T cells, which were then analyzed using flow cytometry, ELISA, immunofluorescence, and metabolic assays. A 3D cancer cell spheroid model was used for co-culture experiments. Lipid-coated calcium-phosphate (LCP)@IL2RG NPs were synthesized and injected into TNBC mouse models.ResultsIL2RG was identified as significantly associated with late-stage TNBC. Overexpression of IL2RG in CD8+ T cells led to increased cell activation, cytotoxicity, and glycolysis, while knockout reduced these effects. Overexpressing IL2RG in CD8+ T cells co-cultured with 3D cancer spheroids resulted in enhanced apoptosis and reduced cancer cell invasion. Injection of LCP@IL2RG NPs into TNBC mouse models significantly mitigated tumor growth.ConclusionsOverexpressing IL2RG in CD8+ T cells enhances tumor immunotherapy. Delivering IL2RG mRNA via NPs further amplifies this effect, offering a promising strategy for the treatment of late-stage TNBC.

Expression of Molecules Characterizing Metabolic and Cytotoxic Activity of Natural Killer Different Subpopulations of Peripheral Blood During Pregnancy

The functions of peripheral blood NK cells change significantly during pregnancy, which is mainly due to the inhibition of their cytotoxicity. The functional activity of NK cells is directly related to their metabolic status, but these changes in physiological pregnancy have not been studied. The aim of this work is to study the expression of Glut-1, CD94 and CD107a molecules characterizing metabolic and cytotoxic activity, as well as the mitochondrial mass of different subpopulations of peripheral blood NK cells in the I and III trimesters of physiological pregnancy. The object of the study was the peripheral blood of healthy women in the I and III trimesters of physiological pregnancy. The control group consisted of healthy non-pregnant women in the follicular phase of the menstrual cycle. The expression of Glut-1, CD94, CD107a molecules and the mitochondrial mass were analyzed by flow cytometry on regulatory (CD16–CD56bright), cytotoxic (CD16+CD56dim), minor cytotoxic (CD16hiCD56–) NK cells. It was found that in non-pregnant women, minor cytotoxic CD16hiCD56–NK have the highest expression of Glut-1, CD107a and the lowest expression of CD94 compared to other NK cell subpopulations. On regulatory CD16–CD 56bright and cytotoxic CD16+CD56dimNK, the expression of these molecules is comparable to each other. The mitochondrial mass is similar in all studied subpopulations. In the first trimester, the expression of Glut-1 increases on regulatory CD16–CD56brightNK, the mitochondrial mass and the expression of CD94, CD107a in all NK cells do not differ from non-pregnant ones. In the third trimester, the mitochondrial mass increases in cytotoxic CD16+CD56dimNK cells, but CD94 expression decreases compared to non-pregnant ones, and the expression CD94 in regulatory CD16–CD56brightNK increases compared to the first trimester. CD107a expression in minor cytotoxic CD16hiCD56–NK decreases, but in other subpopulations does not change compared to non-pregnant. The expression of Glut-1 does not change in all subpopulations. Thus, different subpopulations of peripheral blood NK cells are heterogeneous in the expression of Glut-1, CD107a, CD94. The expression of these molecules during physiological pregnancy varies by trimester. The obtained results are important for understanding the mechanisms of NK cell function regulations during pregnancy.

Intra- and Extrahepatic Cholangiocarcinomas Display Differing Sensitivities to NK Cell Lysis and Modulate NK Cell Function Through Shared and Distinct Pathways.

Cholangiocarcinoma (CCA) is a rare cancer that arises from the bile duct and is broadly classified by the location of the tumor as either intrahepatic (iCCA) or extrahepatic (eCCA). Immunotherapy has revolutionized cancer treatment, yet its utility in CCA has been limited as the tumor microenvironment (TME) in CCA is poorly understood compared to other common cancers. Utilizing previously published transcriptome data, our re-analysis has revealed that CCA has one of the highest relative levels of natural killer (NK) cells, a potent cytotoxic immune cell, compared to other cancers. However, despite iCCA and eCCA having comparable relative levels of NK infiltration, NK cell infiltration only correlated with survival in eCCA patients. Our subsequent investigation revealed that while iCCA and eCCA profoundly altered NK activity, eCCA had a significantly reduced impact on NK functionality. Whereas iCCA was resistant to long-term NK co-culture, eCCA was markedly more sensitive. Moreover, while both iCCA and eCCA dysregulated key NK activating receptors, eCCA co-culture did not impact NKp30 nor NKp44 expression. Furthermore, tumor transcriptome analysis of NKHigh CCA samples revealed modulation of multiple immune and non-immune cell types within the TME. Implications: These studies are the first to investigate how iCCA and eCCA impact NK cell functionality through shared and distinct mechanisms and how elevated NK cell infiltration could shape the CCA TME in a subtype-dependent manner.

Lesional senescent CD4+ T cells mediate bystander cytolysis and contribute to the skin pathology of human cutaneous leishmaniasis

Cytotoxic activity is a hallmark of the immunopathogenesis in human cutaneous leishmaniasis (CL). In this study, we identified accumulation of CD4+ granzyme B producing T cells with increased cytotoxic capacity in CL lesions. These cells showed enhanced expression of activating NK receptors (NKG2D and NKG2C), diminished expression of inhibitory NKG2A, along with the upregulation of the senescence marker CD57. Notably, CD4+ T cells freshly isolated from CL lesions demonstrated remarkable capacity to mediate NL-like bystander cytolysis. Phenotypic analyses revealed that lesional CD4+ T cells are mainly composed of late-differentiated effector (CD27-CD45RA-) and terminally differentiated (senescent) TEMRA (CD27-CD45RA+) subsets. Interestingly, the TEMRA CD4+ T cells exhibited higher expression of granzyme B and CD107a. Collectively, our results provide the first evidence that senescent cytotoxic CD4+ T cells may support the skin pathology of human cutaneous leishmaniasis and, together with our previous findings, support the notion that multiple subsets of cytotoxic senescent cells may be involved in inducing the skin lesions in these patients.

Nerolidol inhibits proliferation and triggers ROS-facilitated apoptosis in lung carcinoma cells via the suppression of MAPK/STAT3/NF-κB and P13K/AKT pathways.

Lung cancer (LC) is the leading cause of malignancy-related mortalities globally, and the existing treatment interventions are associated with harmful side effects. In the current study, we evaluated the anti-tumor efficiency of nerolidol (NRD) on human non-small cell lung cancer (NSCLC) cells. Nerolidol is a sesquiterpene alcohol extracted from the essential oils of aromatic flora with known anti-cancer activities. The latent action of NRD on antiproliferative and apoptotic effects in A549 cells is uncertain. Thus, our work is designed to explore the antiproliferative and apoptotic actions of NRD (20 and 25 μM/mL) against A549 cells. The activity of NRD on A549 cell cytotoxicity, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), apoptosis, anti-apoptotic proteins, and MAPK/TAT3/NF-κB and P13K/AKT signaling pathways were assessed using MTT tests, dichlorodihydrofluorescein diacetate (DCFH-DA), dual acridine orange/ethidium bromide (AO/EB), DAPI, Rh-123, reverse transciption polymerase chain reaction (RT-PCR), and western blot analyses. We found that NRD could inhibit NSCLC cell viability through elevated intracellular ROS and MMP loss and elicited apoptosis in a quantity-dependent manner. Similarly, NRD can reduce inflammatory cytokines and anti-apoptotic elements, as well as trigger apoptotic signaling pathways. Our data established that NRD decreases A549 cell proliferation through ROS-mediated apoptosis, triggering the MAPK/STAT3/NF-κB and P13K/AKT pathways, suggesting that NRD is a possible protective remedy for NSCLC.

Synthesis, Structural Characterization, Optical Properties, and Biological Potency of Co(II), Cu(II), and Cd(II) Complexes With Heterocyclic Thiosemicarbazide Ligand: Insights From Molecular Docking Studies

ABSTRACTA novel heterocyclic thiosemicarbazide ligand, [(Z)‐N‐(2‐oxoindolin‐3‐ylidene)nicotinohydrazide] (H2L), and its Co(II), Cu(II), and Cd(II) metal chelates were synthesized and characterized using a variety of analytical methods, including elemental analysis, FT‐IR, UV–Visible spectroscopy, EI‐mass, fluorescence spectroscopy, X‐ray powder diffraction, and TGA. The structures and geometries of the complexes were determined using these methods as [Co2(H2L)2Cl2(H2O)4]·2H2O, [Cu(H2L)2Cl2(H2O)2]·2H2O, and [Cd(H2L)2Cl2(H2O)2], all having octahedral geometry. The particulate diameters for [Co2(H2L)2Cl2(H2O)4]·2H2O, [Cu(H2L)2Cl2(H2O)2]·2H2O, and [Cd(H2L)2Cl2(H2O)2] were 192.68, 68.48, and 192.74 nm, respectively using Debye–Scherrer equation and FWHM approach. The corresponding FWHM values were 0.0078, 0.0217, and 0.0075, whereas the crystal strain was calculated as (ε) = 0.0018, 0.0056, and 0.0054, and the dislocation density (δ) as 2.69 × 10−5, 2.13 × 10−4, 2.69 × 10−5 Å−2. Quantum chemical calculations for the ligand and its solid metal complexes were carried out utilizing DFT with the DMOL3 module. Furthermore, the optical sensing response of the free ligand H2L to Co(II), Cu(II), and Cd(II) ions was investigated, revealing that the presence of these metal ions enhanced the fluorescence spectra of the ligand. Additionally, the antibacterial effects of these compounds, as well as their cytotoxicity in human cancer cells, were investigated. Compared to its metal complexes, the ligand, H2L exhibits increased antibacterial activity against various bacteria. Tests on HepG‐2 and MCF‐7 cells were conducted to evaluate the compounds' anticancer efficacy against the standard, Doxorubicin. The ligand, H2L, demonstrated greater cytotoxicity against the HepG‐2 cell line than its metal complexes. Docking experiments were conducted to assess the interaction of the ligand and its complexes with HepG‐2 (PDB ID: 2OH4) and MCF‐7 (PDB ID: 3W2S) cells, using the XP glide technique and Schrödinger suite software.

Anti-CD49d Ab treatment ameliorates age-associated inflammatory response and mitigates CD8+ T-cell cytotoxicity after traumatic brain injury

Patients aged 65 years and older account for an increasing proportion of patients with traumatic brain injury (TBI). Older TBI patients experience increased morbidity and mortality compared to their younger counterparts. Our prior data demonstrated that by blocking α4 integrin, anti-CD49d antibody (aCD49d Ab) abrogates CD8+ T-cell infiltration into the injured brain, improves survival, and attenuates neurocognitive deficits. Here, we aimed to uncover how aCD49d Ab treatment alters local cellular responses in the aged mouse brain. Consequently, mice incur age-associated toxic cytokine and chemokine responses long-term post-TBI. aCD49d Ab attenuates this response along with a T helper (Th)1/Th17 immunological shift and remediation of overall CD8+ T cell cytotoxicity. Furthermore, aCD49d Ab reduces CD8+ T cells exhibiting higher effector status, leading to reduced clonal expansion in aged, but not young, mouse brains with chronic TBI. Together, aCD49d Ab is a promising therapeutic strategy for treating TBI in the older people.

Antineoplastic activity of Artemisia annua bio-united Fe3O4/CeO2 on 4T1 breast cancer cells: in vivo and in vitro

Breast cancer has become the most prevalent malignancy among women, and it is estimated to comprise over 30% of all cancer cases diagnosed in women. This study evaluates the antitumor effects of a green-synthesized Fe3O4/CeO2 nanocomposite, utilizing Artemisia annua L. extract, against breast cancer. The viability assay was carried out using 100 to 1000 µg/mL of the as-prepared nanocomposites. The BALB/c mice were injected with 4T1 breast cancer cells and subsequently treated with 5 mg/kg body weight of the synthesized nanocomposites through intraperitoneal injection for two weeks. The in vitro studies on the cell viability and cytotoxicity of Fe3O4/CeO2 nanocomposites demonstrated superior effectiveness of the green-synthesized sample (AFC) compared to the chemically produced variant (FC) against 4T1 cells. Treatment with AFC significantly reduced tumor size in 4T1-injected BALB/c mice compared to untreated control. The IC50 results for FC (890.16 µg/mL) and AFC (490.21 µg/mL) corroborated these findings. Notably, the biointerface provided by the phytomolecules from A. annua extract played a crucial role in enhancing the cytotoxic effects of the nanocomposite. This research highlights the potential of green-synthesized Fe3O4/CeO2 nanocomposites as a safe and effective nanomedicine for targeting cancer cells while minimizing adverse effects on healthy cells.

Abstract A052: RNF43 p.G659fs mutation in MSI-high colorectal cancer leads to natural killer cell dysfunction

Abstract RNF43_p.G659fsV*41 mutation (RNF43659mut) is found in approximately 8% of colorectal cancers (CRCs) and is enriched in microsatellite-instability high (MSI-high) tumors. Previously, we reported that RNF43659mut promotes tumor growth independent of WNT signaling and activates PI3K/AKT signaling in CRC, conferring vulnerability to PI3K/MTOR inhibition. In addition, RNF43659mut was associated with decreased interferon signaling suggesting a link with anti-tumor immunity. Thus, in the current work we investigated the role of RNF43659mut in CRC immunomodulation. The immune-cell landscape was evaluated in RNF43659mut compared with RNF43-wild type (wt) MSI-high CRCs in two cohorts; single-cell (sc)-RNA-sequencing data from treatment-naïve primary tumors from 34 patients (cohort-1) and multiplex immunofluorescence / whole exome sequencing data from an independent cohort of 91 patients (cohort-2). Healthy donor blood-derived natural killer (NK) cell responses and cytotoxicity were investigated in co-cultures with RNF43659mut isogenic tumor lines and with patient-derived organoids harboring RNF43659mut or overexpressing RNF43wt. A third patient cohort with MSI-high CRCs treated with immune checkpoint blockade was analyzed to understand the impact of RNF43659mut on immunotherapy efficacy. Interrogation of sc-RNASeq data in cohort-1 revealed a significant enrichment of NK cells in the RNF43659mut patient samples compared to the RNF43wt ones. This finding was also confirmed by multiplex immunofluorescence in cohort-2. Single-cell transcriptomic analyses in cohort-1 showed increased inhibitory markers on NK cells despite their enrichment in RNF43659mut tumors. RNAseq analysis of sorted NK cells co-cultured with RNF43659mut isogenic tumor lines further supported their dysfunctional state. Flow cytometric analyses of NK cell maturation, activation, and inhibitory markers in NK cells co-cultured with RNF43659mut isogenic tumor lines and organoids highlighted decreased levels of NCAM1 (CD56)dim and NCAM1 (CD56)bright subsets, FCGR3A (CD16), and KLRK1 (NKG2D), along with increased levels of KLRC1 (NKG2A), KIR3DL1 (NKB1) and TIGIT inhibitory markers. There was reduced NK cell mediated tumor cell killing in RNF43659mut isogenic tumor lines and organoids. We postulated that aberrant signaling of the PI3K/AKT pathway with RNF43659mut could lead to NK cell dysfunction. Indeed, siRNA-mediated blocking of PI3K/AKT pathway restored the NK-cell dysfunctional phenotype and tumor cell killing in co-cultures with RNF43659mut isogenic tumor lines and organoids. Conversely, overexpression of WT RNF43 in RNF43659mut organoids downregulated PI3K/AKT signaling and increased NK cell cytotoxicity. Lastly, MSI-high CRC tumors harboring RNF43659mut in patients treated with immunotherapy showed less benefit from immune checkpoint blockade. PI3K/AKT activation due to RNF43659mut in MSI-high CRC leads to NK cell dysfunction and reduced NK cell cytotoxicity. This could be responsible for immunotherapy failure, warranting further investigation. Citation Format: Pushpamali De Silva, Samantha Fitzgerald, Jules Cazaubiel, Matan Hofree, Tomotaka Ugai, Juha P Väyrynen, Dane Ford-Roshon, Lishan Fang, Ana Garrido-Castro, Nir Hacohen, Kimmie Ng, Shuji Ogino, Jonathan A Nowak, Marios Giannakis. RNF43 p.G659fs mutation in MSI-high colorectal cancer leads to natural killer cell dysfunction [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A052.

RABV antigenic peptide loaded polymeric nanoparticle production, characterization, and preliminary investigation of its biological activity

Nanoparticle-based antigen carrier systems have become a significant area of research with the advancement of nanotechnology. Biodegradable polymers have emerged as particularly promising carrier vehicles due to their ability to address the limitations of existing vaccine systems. In this study, we successfully encapsulated the G5-24 linear peptide, located between amino acids 253 and 275 in the primary sequence of the rabies virus G protein, into biodegradable and biocompatible PLGA copolymer using the double emulsion solvent evaporation method. The resulting nanoparticles had a size of approximately 230.9 ± 0.9074 nm, with a PDI value of 0.168 ± 0.017 and a zeta potential value of -9.86 ± 0.132 mV. SEM images confirmed that the synthesized nanoparticles were uniform in size and distribution. Additionally, FTIR spectra indicated successful peptide loading into the nanoparticles. The encapsulation efficiency of the peptide-loaded nanoparticles was 73.3%, with a peptide loading capacity of 48.2% and a reaction yield of 30.4%. Peptide release studies demonstrated that 65.55% of the peptide was released in a controlled manner over 28 d, following a 'biphasic burst release' profile consistent with the degradation profile of PLGA. This controlled release is particularly beneficial for vaccine studies. Cytotoxicity tests revealed that the R-NP formulation did not induce cytotoxicity in fibroblast cells and enhanced NO production in macrophages, indicating its potential for vaccine development.

MicroRNA-130a-3p regulates osimertinib resistance by targeting runt-related transcription factor 3 in lung adenocarcinoma

Overcoming resistance to epidermal growth factor receptor tyrosine kinase inhibitors, including osimertinib, is urgent to improve lung cancer treatment outcomes. Extracellular vesicle (EV)-derived microRNAs (EV-miRNAs) play important roles in drug resistance and serve as promising biomarkers. In this study, we aimed to identify EV-miRNAs associated with osimertinib resistance and investigate their clinical relevance. The release of excess EVs was confirmed in the osimertinib-resistant lung adenocarcinoma cell line PC9OR. The exposure of PC9OR-derived EVs and EV-miRNAs to PC9 cells increased cell viability after osimertinib treatment. Microarray analysis revealed that miR-130a-3p was upregulated in EVs derived from PC9OR cells and another osimertinib-resistant cell line (H1975OR). Transfection with miR-130a-3p attenuated osimertinib-induced cytotoxicity and apoptosis in both PC9 and H1975 cells, whereas osimertinib resistance in PC9OR cells was reversed after miR-130a-3p inhibition. Bioinformatics analysis revealed that runt-related transcription factor 3 is a target gene of miR-130a-3p, and it induced osimertinib resistance in PC9 cells. Patients with lower baseline serum miR-130a-3p concentrations had longer progression-free survival. miR-130a-3p is a potential therapeutic target and a predictive biomarker of osimertinib resistance in adenocarcinomas.

Antibody-targeted T cells and natural killer cells for cancer immunotherapy

BackgroundAdoptive cell cancer therapies aim to re-engineer a patient’s immune cells to mount an anti-cancer response. Chimeric antigen receptor T and natural killer cells have been engineered and proved successful in treating some cancers; however, the genetic methods for engineering are laborious, expensive, and inefficient and can cause severe toxicities when they over-proliferate.ResultsWe examined whether the cell-killing capacity of activated T and NK cells could be targeted to cancer cells by anchoring antibodies to their cell surface. Using metabolic glycoengineering to introduce azide moieties to the cellular surface, we covalently attached a dibenzocyclooctyne-modified antibody using the strain-promoted alkyne azide cycloaddition reaction, creating antibody-conjugated T and NK cells. We targeted the immune cells to tumors possessing the xenoantigen, N-glycolyl neuraminic acid GM3 ganglioside, using the 14F7hT antibody. These activated T and NK cells are “armed” with tumour-homing capabilities that specifically lyses antigen-positive cancer cells without off-target toxicities. Moreover, when exposed to target cells, 14F7hT-conjugated T cells that are not preactivated exhibit increased perforin, granzyme, CD69, and CD25 expression and specific cell killing.ConclusionsThis research shows the potential for a non-genetic method for redirecting cytotoxic immune cells as a feasible and effective approach for tumor-targeted cell immunotherapy.Graphical

DST regulates cisplatin resistance in colorectal cancer via PI3K/Akt pathway.

Dystonin (DST), a potential tumor suppressor gene, plays a crucial role in regulating cancer cell proliferation and resistance to chemotherapy. However, DST's specific role in colorectal cancer (CRC) has not been thoroughly investigated, and this study aims to elucidate its molecular role in modulating cisplatin (DDP) resistance in CRC. DST expression was analyzed in CRC tumors, DDP-resistant CRC tissues, paracancer tissues, and normal tissues. Lentiviral overexpression and shRNA knockdown were conducted in advanced CRC and DDP-resistant cell lines to assess cell viability, apoptosis, invasion, migration, proliferation, and angiogenesis. Xenograft mouse models studied DST's impact on CRC tumor growth and DDP resistance in vivo. DST expression was significantly reduced in CRC tumor and DDP-resistant CRC tissues compared to paracancer and normal tissues (P < .001). Upregulating DST inhibited CRC and DDP-resistant cell viability, proliferation, invasion, and migration while promoting apoptosis. DST overexpression also reduced angiogenesis and attenuated DDP-induced cytotoxicity in CRC cells. Mechanistically, DST upregulation suppressed DDP resistance in CRC cells via the PI3K/Akt signaling pathway. DST upregulation reduced CRC tumor growth and mitigated DDP resistance, in vivo. DST plays a crucial role in limiting CRC progression and overcoming DDP resistance, suggesting potential for targeted CRC therapies.

GC-MS/HPLC Profiling and Sono-maceration Mediated Extraction of Osbeckia parvifolia Polyphenols: In Silico and In Vitro Analysis on Anti-Proliferative activity in Ovarian Cancer Cell Lines.

Osbeckia parvifolia, an endemic edible plant of Western Ghats, was investigated in the present study for its polyphenolic compounds, including content, constituents, extraction through an ultrasonic-assisted maceration technique and therapeutic potential in biomedical applications. The methanolic extract (OPM) exhibited an IC50 value of 1.25 µg/mL against 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radicals. Furthermore, the ethyl acetate and methanolic extracts also strongly inhibited 5-lipoxygenase, especially OPM (84.93%), which was comparable to standard curcumin. OPM also elicited cytotoxicity in SKOV3 ovarian cancer cells (93.80%), surpassing paclitaxel. Bio-accessibility analysis demonstrated that the release of phenolic compounds and antioxidant potential were very high (above 100%), revealing the possibility of synergistic efficacy of polyphenolic complexes in drug development. Gas Chromatography -Mass Spectrometry (GC-MS) analysis revealed 22 bioactive polyphenolic compounds in OPM, such as epicatechin, quercetin, and psoralidin. This was confirmed by High Performance Liquid Chromatography (HPLC) and High-Pressure Thin Layer Chromatography (HPTLC) analyses, which revealed a high quantity of catechin (37.45 mg/g). Molecular docking revealed the significant binding affinity of these proteins for the ovarian oncoproteins PI3K (-8.52 kcal/mol) and Casp-8 (-8.41 kcal/mol). Adsorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) profiling indicated the favorable pharmacokinetic properties of these compounds, supporting their candidacy in drug formulations against ovarian cancer.

Design, Synthesis, and Anti-Melanogenic Activity of 2-Mercaptomethylbenzo[d]imidazole Derivatives Serving as Tyrosinase Inhibitors: An In Silico, In Vitro, and In Vivo Exploration.

2-Mercaptomethylbenzo[d]imidazole (2-MMBI) derivatives were designed and synthesized as tyrosinase (TYR) chelators using 2-mercaptomethylimidazole scaffolds. Seven of the ten 2-MMBI derivatives exhibited stronger inhibition of mushroom TYR activity than kojic acid. Their ability to chelate copper ions was demonstrated through experiments using the copper chelator pyrocatechol violet and assays measuring TYR activity in the presence or absence of exogenous CuSO4. The inhibition mechanisms of derivatives 1, 3, 8, and 9, which showed excellent TYR inhibitory activity, were elucidated through kinetic studies and supported by the docking simulation results. Derivatives 3, 7, 8, and 10 significantly inhibited cellular TYR activity and melanin production in B16F10 cells in a dose-dependent manner, with stronger potency than kojic acid. Furthermore, in situ, derivatives 7 and 10 showed stronger inhibitory effects on B16F10 cell TYR activity than kojic acid. Six derivatives, including 8, showed highly potent depigmentation in zebrafish larvae, outpacing kojic acid even at 200-670 times lower concentrations. Additionally, all derivatives could scavenge for reactive oxygen species without causing cytotoxicity in epidermal cells. These results suggested that 2-MMBI derivatives are promising anti-melanogenic agents.

Thiazepine-based Hybrids as Promising Anti-colon Cancer Agents: Design, Synthesis, Computational and In Vitro Screening.

Novel thiazepine-based hybrids (9a-d) were designed and synthesized to create lead molecules with exceptional anti-colon cancer efficacy. Analytical methods, including IR, NMR, and HR-MS, characterized the synthesized compounds. The in vitro colorectal study was carried out to compare the biological activity of newly developed compounds with the computational data. The tested compounds induced cytotoxicity in HT-29 cells for both 24h and 48h in a dose-dependent manner. However, compound 9a induced cytotoxicity at much higher concentrations compared to the rest of the compounds. 9b and 9c caused 50% cell death (compared to the untreated cells) at a dose of ~ 50µM and 40 µM in case of 24-hour exposure, respectively. On the contrary, for 48h exposure, both 9b and 9c induced 50% cell death concerning untreated cells at a dose of around ~20 µM, whereas 9d exhibited 50% cell death at 5 µM in the case of 48 h exposure. In silico ADMET was also carried out to understand the pharmacokinetics and safety profiles of the drug candidates. We found some of the critical targets of these compounds, which eventually will be integral to exploring the mechanistic actions of these compounds in colon cancer.

Tanshinone IIA inhibits H2O2-induced ferroptosis in melanocytes through activating Nrf2 signaling pathway.

Melanocyte ferroptosis has been proven to contribute to the development of vitiligo. Tanshinone IIA (TSA), a Chinese herbal extract, has been shown to inhibit vitiligo progression. Whether TSA regulates ferroptosis in melanocytes remains unclear. Hydrogen peroxide (H2O2) was used to induce melanocytes to stimulate vitiligo cell model in vitro. Cell proliferation was examined by 5-ethynyl-2'-deoxyuridine assay. The levels of malondialdehyde (MDA), reactive oxygen species (ROS), glutathione peroxidase (GSH) and iron (Fe2+) were detected by corresponding commercial kit. The protein levels of ferroptosis-related markers and Nrf2 pathway-related markers were examined using western blot and immunofluorescence staining. Cell viability and cytotoxicity were analyzed using cell counting kit 8 assay and lactate dehydrogenase (LDH) detection. Mitochondrial morphology was examined using a transmission electron microscope. After H2O2 treatment, melanocyte proliferation was reduced, while oxidative stress and ferroptosis were enhanced. TSA treatment could inhibit ferroptosis in H2O2-induced melanocytes. Besides, TSA could activate Nrf2 pathway and promote Nrf2 nuclear translocation, and Nrf2 specific inhibitor (ML385) also reversed the inhibitory effect of TSA on H2O2-induced melanocyte ferroptosis. Our data showed that TSA alleviated H2O2-induced melanocyte ferroptosis via activating Nrf2 pathway.