Tumor cell populations have been recently proposed to be composed of two compartments: tumor-initiating cells characterized by a slow and asymmetrical growth, and the "differentiated" cancer cells ...with a fast and symmetrical growth. Cancer stem cells or cancer-initiating cells (CICs) play a crucial role in tumor recurrence. The resistance of CICs to drugs and irradiation often allows them to survive traditional therapy. NK cells are potent cytotoxic lymphocytes that can recognize tumor cells. In this study, we have analyzed the NK cell recognition of tumor target cells derived from the two cancer cell compartments of colon adenocarcinoma lesions. Our data demonstrate that freshly purified allogeneic NK cells can recognize and kill colorectal carcinoma-derived CICs whereas the non-CIC counterpart of the tumors (differentiated tumor cells), either autologous or allogeneic, is less susceptible to NK cells. This difference in the NK cell susceptibility correlates with higher expression on CICs of ligands for NKp30 and NKp44 in the natural cytotoxicity receptor (NCR) group of activating NK receptors. In contrast, CICs express lower levels of MHC class I, known to inhibit NK recognition, on their surface than do the "differentiated" tumor cells. These data have been validated by confocal microscopy where NCR ligands and MHC class I molecule membrane distribution have been analyzed. Moreover, NK cell receptor blockade in cytotoxicity assays demonstrates that NCRs play a major role in the recognition of CIC targets. This study strengthens the idea that biology-based therapy harnessing NK cells could be an attractive opportunity in solid tumors.
This study describes the biological investigations of methanolic extract of leaves and stem of Jatropha gossypiifolia, a plant belonging to the family Euphorbiaceae, to reveal their possible ...contribution as an antiemetic, antinociceptives, cytotoxic and thrombolytic factor. The leaves and stems of Jatropha gossypiifolia were extracted with methanol. The leaves extracts were used for the observation of thrombolytic potential. Result reveals that the leaves at a concentration of 2.5mg/mL, showed maximum activity (27.255 ± 0.90%), whereas 20 mg/mL sample solution showed the lowest activity (7.722 ± 0.26%), when comparing positive control streptokinase. The extracts were used for the investigation of cytotoxic activity considering vincristine sulfate as a positive control. The leaves and the stems portion have considerable cytotoxic property (LC50 are 0.00532 µ...g/ml for stems and 0.000109 µg/ml for leaves, respectively) when comparing with positive control. The methanolic extract showed potent antiemetic activity at the highest concentration both at leaves and stem extracts as compared to positive control metoclopramide. Where metoclopramide shows 73% of inhibition, there leaves and the stem portion of the plant shows 90% and 91% of emesis inhibition respectively. By using a standard protocol, the crude methanolic stem and leaves extract of J. gossypiifolia was conducted to attempt for antinociceptives activities, where Diclofenac Na is used as a standard. The crude methanolic extract of leaves and stem exhibit 31.07% and 30.79% writhing inhibition in test animals respectively, which are statistically significant. The results of the study showed that the plant extract has potential thrombolytic, cytotoxic, antiemetic and antinociceptive activities.
Two known triterpenoids, friedelin and aglaitriol, and a new dammarane triterpenoid, aglaitriol 3-caffeate, have been isolated and identified in the chloroform bark extract of Drypetes acuminata P.I. ...Forst. (Putranjivaceae) from Paluma, north Queensland, Australia. The compounds were screened for antimicrobial and cytotoxic activity. Aglaitriol showed potent cytotoxic activity against three human tumor cell lines (MCF-7, MDA-MB-231, and 5637). Aglaitriol 3-caffeate showed moderate antibacterial activity against Staphylococcus aureus and Escherichia coli. A molecular docking analysis suggests topoisomerase II may be a likely protein target for aglaitriol.
This study evaluated for the first time the antibacterial activity, cell viability and migration ability on 3T3 murine fibroblast cells of extracts and isolated compounds lupeol (1), ...hexamethylcoruleoellagic acid (2) and a mixture of 1 and betulinaldehyde (3) of Myrciaria ferruginea. In antibacterial assays extracts were susceptible only against S. aureus (MIC 500 μg/mL) and S. epidermidis (MIC ranging from 7.8 to 500 μg/mL) and compounds 1-3have shown no significant activity. In trials for c ell viability, with exception of MeOH-H 2O fraction from leaves (viable cells > 90%), both the crude extract and other fractions showed inhibition of cell growth (viable cells ≤ 80% at 15.625 and 31.25 μg/mL); while the samples from stems, with the exception of CHCl 3 fraction that showed strong cytotoxic effect at the lowest concentration tested (15.625 μg/mL), the other fractions were not cytotoxic. Compounds (1-3) inhibited cell viability in dose dependent manner (15.625 to 500 μg/mL). Mixture containing 1 and 3 showed inhibitions only in concentrations greater than 62.5 μg/mL while compound 2 decreased from the lowest concentration tested. In scratch wound assay, these compoundsnot increased the population of fibroblasts at concentrations less than 62.5 μg/mL.
Immunotherapy using chimeric antigen receptor (CAR)‐engineered lymphocytes has shown impressive results in leukemia. However, for solid tumors such as colorectal cancer (CRC), new preclinical models ...are needed that allow to test CAR‐mediated cytotoxicity in a tissue‐like environment. Here, we developed a platform to study CAR cell cytotoxicity against 3‐dimensional (3D) patient‐derived colon organoids. Luciferase‐based measurement served as a quantitative read‐out for target cell viability. Additionally, we set up a confocal live imaging protocol to monitor effector cell recruitment and cytolytic activity at a single organoid level. As proof of principle, we demonstrated efficient targeting in diverse organoid models using CAR‐engineered NK‐92 cells directed toward a ubiquitous epithelial antigen (EPCAM). Tumor antigen‐specific cytotoxicity was studied with CAR‐NK‐92 cells targeting organoids expressing EGFRvIII, a neoantigen found in several cancers. Finally, we tested a novel CAR strategy targeting FRIZZLED receptors that show increased expression in a subgroup of CRC tumors. Here, comparative killing assays with normal organoids failed to show tumor‐specific activity. Taken together, we report a sensitive in vitro platform to evaluate CAR efficacy and tumor specificity in a personalized manner.
Synopsis
Establishment of a preclinical in vitro organoid model allows testing the efficacy and safety of solid tumor cancer immunotherapy with chimeric antigen receptor (CAR)‐engineered lymphocytes in a tissue‐like environment. 3D live‐imaging of single colorectal cancer (CRC) organoids is highly sensitive and allows to validate treatment assumptions to minimize therapy side effects.
Co‐culture of human natural killer (NK) cells with normal or CRC organoids on an ECM layer allows stable effector‐target cell interaction.
Luciferase‐based cytotoxicity assessment is benchmarked with EPCAM‐directed effector NK cells.
Low expression of EGFRvIII neoantigen is sufficient to render CRC organoids susceptible to lysis.
CAR targeting of FRIZZLED receptors in intestinal organoids reveals off‐target cytotoxicity against wild‐type epithelia of human origin.
A preclinical organoid co‐culture allows testing of immunotherapy with chimeric antigen receptor (CAR)‐engineered lymphocytes in a personalised tissue‐like environment.
BackgroundIn exercising their hospital activity, the pharmacist is faced with multiple tasks that can compromise, for security reasons, a positive trend in the health status of patients.There are ...areas that are traditionally regarded as critical (preparation of non-sterile formulations, handling cytotoxic or other sterile mixtures).The Cytotoxic Preparation Manual, by the Portuguese Council in Hospital Pharmacy Specialty, states: “double checking should be implemented in the critical steps of the preparation process. Double checking should be carried out independently by a second person or by a computerised system”. Compliance with this recommendation is not uniform in the various hospitals due to a shortage of human resources.PurposeTo create conditions for the fulfilment of the double validation process by eliminating the actual and permanent physical presence of a second element in the preparation of sterile room mixtures, keeping the final quality of the process.Material and methodsMultiple image capture methods in handling the environment in the laminar air flow chamber were tested, after consultation with the national Data Protection Authority, which enabled such viewing. The final solution was a system composed of special glasses with a high definition camera which enables real time recording with up to 30 images per second and marking of critical points that can be downloaded to a computer for a verification process.ResultsThe test phase was successfully passed, after correct viewing images in the real work environment. The ocular device allows the use of a visor and does not interfere with the manipulation. It allows identification of the drug, solvent validation and identification of a reconstituted final volume for the patient and medical prescription. The validation can be done elsewhere from the pharmaceutical services, outside the clean room, and consists of the display of marked critical points and, in doubtful cases, the full view of the event. This validation reduces by at least 75% the time allocated to the second element.ConclusionThe possibility of implementation/maintenance of the double validation process, reducing by more than 75% of the associated workload and elimination of sterile equipment required for entry into the clean room, enables compliance with the rules of the Cytotoxic Preparation Manual, with rationalisation of associated resources.References and/or AcknowledgementsManual de Preparação de CitotóxicosNo conflict of interest.
Manganese has been known to induce neurological disorders. In the present study, we determined the effect of manganese on the expression of a-synuclein in PC12 cells and its role in manganese-induced ...cytotoxicity. We also investigated the relationship between a-synuclein expression and the change of ERK1/2 MAPK activity. In our research, manganese exposure induced the overexpression of a-synuclein, while siRNA knockdown of a-synuclein reversed manganese-induced cytotoxicity. Furthermore, manganese induced the activation of ERK1/2 MAPK. The MEK1 inhibitor PD98059, which inhibits the activation of ERK MAPK, attenuated the overexpression of a-synuclein and the cytotoxicity induced by manganese. In conclusion, our studies show that manganese may induce the overexpression of a-synuclein via ERK1/2 activation, which may play a role in manganese-induced cytotoxicity. a-ordmManganese induced the overexpression of a-synuclein though an ERK1/2M APK dependent manner. a-ordmBoth the inhibition of ERK1/2 MAPK and the knockdown of a-synuclein alleviated manganese-induced cytotoxicity. a-ordmThese findings suggest that manganese induces toxicity through an ERK1/2 MAPK/a-synuclein-dependent pathway.
The study was aimed to evaluate the cytotoxic and thrombolytic activity of methanol extract of Musa paradisiaca. The cytotoxic activity of the crude extract was determined by using brine shrimp ...lethality bioassay and LC50 values of the sample were 22.336 ± 0.41μg/ml whereas for standard vincristine sulfate was 8.50 ± 0.16μg/ml as a positive control. In thrombolytic activity using in vitro clot lysis method, the plant’s extract showed (46.26 ±1.54%) clot lysis as compared to standard streptokinase (67.32±0.34%).
NK cells directly kill mycobacteria through a contactdependent mechanism leading to the release of perforin and granulysin.
Although the mechanisms underlying the cytotoxic effect of NK cells on ...tumor cells and intracellular bacteria have been studied extensively, it remains unclear how these cells kill extracellular bacterial pathogens. In this study, we examine how human NK cells kill Mycobacterium kansasii and M.tb. The underlying mechanism is contact dependent and requires two cytolytic proteins: perforin and granulysin. Mycobacteria induce enhanced expression of the cytolytic proteins via activation of the NKG2D/NCR cell‐surface receptors and intracellular signaling pathways involving ERK, JNK, and p38 MAPKs. These results suggest that NK cells use similar cellular mechanisms to kill both bacterial pathogens and target host cells. This report reveals a novel role for NK cells, perforin, and granulysin in killing mycobacteria and highlights a potential alternative defense mechanism that the immune system can use against mycobacterial infection.
Previously, CD8+ T cells were found to be a sensitive target for suppression by Δ9-tetrahydrocannabinol (Δ9-THC) in a murine model of influenza infection. To study the effect of Δ9-THC on CD8+ ...cytotoxic T lymphocytes (CTL), an allogeneic model of MHC I mismatch was used to elicit CTL. In addition, to determine the requirement for the cannabinoid receptors 1 (CB1) and 2 (CB2) in Δ9-THC-mediated CTL response modulation, mice null for both receptors were used (CB1 −/−CB2 −/−). Δ9-THC suppressed CTL function independent of CB1 and CB2 as evidenced by reduction of 51Cr release by CTL generated from CB1 −/−CB2 −/− mice. Furthermore, viability in CD4+ and CD8+ cells was reduced in a concentration-dependent manner with Δ9-THC, independent of CB1 and CB2, but no effect of Δ9-THC on proliferation was observed, suggesting that Δ9-THC decreases the number of T cells initially activated. Δ9-THC increased expression of the activation markers, CD69 in CD8+ cells and CD25 in CD4+ cells in a concentration-dependent manner in cells derived from WT and CB1 −/−CB2 −/− mice. Furthermore, Δ9-THC synergized with the calcium ionophore, ionomycin, to increase CD69 expression on both CD4+ and CD8+ cells. In addition, without stimulation, Δ9-THC increased CD69 expression in CD8+ cells from CB1 −/−CB2 −/− and WT mice. Overall, these results suggest that CB1 and CB2 are dispensable for Δ9-THC-mediated suppression and that perturbation of Ca2+ signals during T cell activation plays an important role in the mechanism by which Δ9-THC suppresses CTL function.
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