scholarly journals In Vitro Effects of Selective COX and LOX Inhibitors and Their Combinations with Antineoplastic Drugs in the Mouse Melanoma Cell Line B16F10

2021 ◽  
Vol 22 (12) ◽  
pp. 6498
Author(s):  
Ines Da-Costa-Rocha ◽  
Jose M. Prieto
Keyword(s):  
Cell Line ◽  
Constitutive Expression ◽  
Fold Increase ◽  
Antineoplastic Drugs ◽  
Cox Inhibitors ◽  
B16f10 Cells ◽  
Lox Inhibitors ◽  
In Vitro Effects

The constitutive expression or overactivation of cyclooxygenase (COX) and lipoxygenase (LOX) enzymes results in aberrant metabolism of arachidonic acid and poor prognosis in melanoma. Our aim is to compare the in vitro effects of selective COX-1 (acetylsalicylic acid), COX-2 (meloxicam), 5-LOX (MK-886 and AA-861), 12-LOX (baicalein) and 15-LOX (PD-146176) inhibition in terms of proliferation (SRB assay), mitochondrial viability (MTT assay), caspase 3-7 activity (chemiluminescent assay), 2D antimigratory (scratch assay) and synthesis of eicosanoids (EIA) in the B16F10 cell line (single treatments). We also explore their combinatorial pharmacological space with dacarbazine and temozolomide (median effect method). Overall, our results with single treatments show a superior cytotoxic efficacy of selective LOX inhibitors over selective COX inhibitors against B16F10 cells. PD-146176 caused the strongest antiproliferation effect which was accompanied by cell cycle arrest in G1 phase and an >50-fold increase in caspases 3/7 activity. When the selected inhibitors are combined with the antineoplastic drugs, only meloxicam provides clear synergy, with LOX inhibitors mostly antagonizing. These apparent contradictions between single and combination treatments, together with some paradoxical effects observed in the biosynthesis of eicosanoids after FLAP inhibition in short term incubations, warrant further mechanistical in vitro and in vivo scrutiny.

Growth Suppressors IFI-204 and IFI-205 Inhibit Primitive Hematopoietic Cell Proliferation In Vitro and in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1691-1691
Author(s):  
Kimberly Klarmann ◽  
Daniel Gough ◽  
Benyam Asefa ◽  
Chris Clarke ◽  
Katie Renn ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Growth ◽  
Cell Line ◽  
Constitutive Expression ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Inhibit Cell Growth ◽  
Stem And Progenitor Cells

Abstract Members of the interferon inducible-200 (IFI-200) family of proteins inhibit cell growth and may be important mediators of differentiation. We examined IFI-204 and IFI-205 mRNA expression in purified populations of hematopoietic stem and progenitor cells at different stages of maturation using quantitative RT-PCR and found that their expression markedly increased during myeloid maturation. To evaluate the effect of IFI-205 and IFI-204 on hematopoietic stem cell (HSC) growth, we transduced these genes into mouse bone marrow cells (BMC) using retroviral vectors. The presence IFI-204 or IFI-205 resulted in a decrease in cell growth in response to hematopoietic growth factors. Further analysis revealed the infected cells were 98% c-Kit+ Sca-1+, indicative of the stem cell surface phenotype, suggesting they may be blocked in a primitive stage of maturation. When transplanted, BMC transduced with IFI-204 or IFI-205 failed to engraft lymphoid, myeloid, or erythroid lineages in both short and long term reconstitution assays, suggesting that constitutive expression of IFI-204 and IFI-205 inhibited HSC development both in vitro and in vivo. However, based on the quantitative RT-PCR results, which show that IFI-205 increased during myeloid differentiation, we know its endogenous, regulated expression must permit the cells to mature. Therefore, to study of the effects of these genes on differentiation we transduced the mulitpotential EML (erythroid, myeloid, lymphoid) cell line with IFI-204 and IFI-205 to circumvent severe growth inhibition caused by expression of IFI-204 and Ifi-205 in normal cells. Single cell analysis of EMLs transduced with IFI-205 demonstrated that expression of IFI-205 in this cell line did not significantly inhibit cell growth. We have isolated EML clones from the transduced cells and verified IFI-205 expression. In addition, we generated transgenic mice that express IFI-205 under control of the Vav and MRP8 promoters, and we identified transgenic lines that express IFI-205 at higher levels compared to wild type controls. Analysis of hematopoiesis in these animals is currently in progress. Altogether, our data demonstrate 3 findings: 1) IFI-204 and IFI-205 expression increases during myeloid development based on quantitative RT-PCR analysis, 2) constitutive expression of IFI-204 and -205 results in potent inhibition of growth and maturation of normal hematopoietic stem and progenitor cells in vivo and in vitro and 3) these genes did not significantly inhibit the proliferation of the EML cell line, which provides us with a means to study the mechanism by which these molecules regulate myeloid maturation. Finally, the considerable inhibitory effects of these family members on normal hematopoietic cell growth suggest their potential as therapeutic modalities for treatment of leukemia.

Heat Shock Protein 70 over Expression Is Associated to Imatinib Resistance in Chronic Myelogenous Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2005-2005 ◽  
Author(s):  
Marion Pocaly ◽  
Valérie Lagarde ◽  
Gabriel Etienne ◽  
Jean-Antoine Ribeil ◽  
Marc Bonneu ◽  
...  
Keyword(s):  
Heat Shock ◽  
Cell Line ◽  
Fold Increase ◽  
Hsp70 Expression ◽  
Hsp 70 ◽  
Imatinib Resistance ◽  
Over Expression ◽  
Imatinib Resistant

Abstract Imatinib is an effective therapy for chronic myeloid leukemia (CML), a myeloproliferative syndrome characterised by the expression of the recombinant oncoprotein Bcr-Abl. Imatinib inhibits Bcr-Abl tyrosine kinase activity leading to apoptosis of leukemic cells sparing normal hematopoiesis. Several mechanisms of resistance to imatinib have been identified both in vitro and in vivo: Bcr-Abl mutations, an over-expression of the Bcr-Abl kinase itself or other tyrosine kinase bypass. To identify unknown mechanism, we used an imatinib resistant cell line (K562-R) generated from the erythroblastic cell line K562 (K562-S) (Blood, 2000; 93: 1070–1079) for which all described mechanisms of resistance have been previously invalidated. Previous results from a proteomic study identified some chaperon proteins such as heat shock proteins with an increased expression level in K562-R. One of them, the heat shock protein 70, Hsp70, has a 3 fold increase expression level in K562-R cells, results which have been confirmed by western-blot analysis. To characterise the role of Hsp 70 in imatinib resistance, we inhibit Hsp 70 expression by RNA silencing (siRNA) in K562-R cells and over-express it in K562-S cells. Inhibition of Hsp70 protein expression by siRNA decrease Hsp70 expression rapidly over 90% at day 4 which is associated with a significant reduction of viability (66 ± 6%, n = 5, p < 0.03). Over expression of Hsp 70 in K562-S cells induced a significant increase of resistance to imatinib since the addition of imatinib only increases mortality by 27 ± 5 % in comparison to 52 ± 4 % for K562-S cells (n = 4, p < 0.001). Detection of HSF-1 phosphorylation, the major transcription factor involved in Hsp 70 expression, did not show significant differences between K562-S and K562-R cells although over a 3 fold increase is detected in the mRNA level of Hsp 70 in K562-R cells by quantitative PCR. Furthermore, the comparison of Hsp70 expression in mononuclear cells of 7 CML patients before imatinib treatment and at the relapse time shows that Hsp 70 is increased in imatinib resistant patients suggesting it could also play a role in resistance in vivo. Present study confirmed that over expression of Hsp 70 in the cell line K562-R is involved in the mechanism of imatinib resistance in vitro. Moreover, the correlation between the increase of Hsp 70 in CML patient cells and resistance suggests it could be an interesting marker and potentially a therapeutic target.

Constitutive Expression of the ABCG2 (BCRP), the Molecular Determinant of the Side Population, Increases the Proliferative Potential of Human Clonogenic Progenitors and Supports Human Myeloid Engraftment in the NOD/SCID Mouse Model.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 799-799
Author(s):  
Farid Ahmed ◽  
Natalia Arseni ◽  
Wolfgang Hiddemann ◽  
Christian Buske ◽  
Michaela Feuring-Buske
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Side Population ◽  
Myeloid Cells ◽  
Constitutive Expression ◽  
Fold Increase ◽  
Proliferative Potential ◽  
Molecular Determinant

Abstract Adult hematopoietic stem cells can be identified by the ability to rapidly efflux the Hoechst 33342 dye and consequently produce a characteristic side population (SP) phenotype. ABCG2 (Human Breast Cancer Resistance Protein, BCRP) is the molecular determinant of the SP phenotype. We have demonstrated previously that the SP phenotype together with the expression of CD34 and lack of CD38 distinguishes between normal and leukemic stem cells in patients with acute myeloid leukemia (AML), suggesting a role of this protein in early human hematopoiesis. To test this, normal highly purified human CD34+ cord blood cells were transduced retrovirally by ABCG2/YFP and analyzed for their in vitro and in vivo behaviour. In vitro constitutive expression of ABCG2 doubled the number of the most immature CFU-GEMM type colonies in the CFC assays (n=12; p&lt; 0.002). Furthermore, the protein enhanced the replating capacity of primary colonies with a mean 3.0 fold increase in the number of 2nd colonies (n=9; p&lt; 0.01), indicating a substantial enhancement of the proliferative potential of clonogenic progenitors by constitutive ABCG2 expression. In contrast, ABCG2 did not induce any major increase in the frequency of LTC-IC compared to the YFP control after 5 days as assessed by limiting dilution LTC-IC (1 LTC-IC per 3911 cells and 1 LTC-IC per 3641 cells, respectively). To study the impact of ABCG2 on human progenitor cells in vivo NOD/SCID mice were injected with highly purified ABCG2/YFP+ cells and analyzed 8 weeks after transplantation for human engraftment. Although mice in the ABCG2 group received less transduced cells than the control (on average 1.2 x 105 versus 3.7 x 105 per mouse, respectively), they showed significant higher engraftment compared to the control group (6.1 x 107 transduced cells (4.3–8.2) versus 4.2 x 107 (3.2–5.7) per mouse, respectively; p&lt;0.04). Mice that received ABCG2-transduced cells showed a 4.6fold increase in the number of engrafted CD34+ progenitor cells (1.4x 107 CD34+CD45+ vs 6.5x 106; p&lt;0.05). In addition, ABCG2 expression resulted in 2.2-fold increase of c-KIT+ cells (6.1x106 cells vs. 2.8 x 106 cells in the control arm; p&lt; 0.02) indicating that the constitutive expression of ABCG2 enhanced the number of human primitive progenitor cells. ABCG2 expression was also associated with an expansion in the CD15+ /CD33+ human myeloid compartment: in the control mice 1.1 x 107 human transduced myeloid cells (CD15+) were detected per mouse compared to 2.6 x 107 in the ABCG2 group 8 weeks post transplant (p&lt;0.05) whereas the human CD19+ lymphoid compartment was not changed. This resulted in an inversion of the ratio of engrafted CD19+/CD15+ human lymphoid/myeloid cells (mean of 0.5 for ABCG2 vs 1.1 in the control; p&lt;0.03). Furthermore, constitutive expression of ABCG2 promoted erythroid differentiation with a 3.6fold increase in glycophorin A expressing erythroid cells (9 x 106 vs 2.5 x 106 GlyA+ cells in the control; p &lt; 0.003). Taken together, our data characterize ABCG2 as a previously unrecognized potent positive regulator of primitive hematopoietic cell growth in vitro and in vivo and extend our so far limited knowledge about human stem cell regulation by this ABC transporter.

A preclinical model for skin sensitization prediction of antineoplasic drugs.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15643-e15643
Author(s):  
Jose Alejandro Perez-Fidalgo ◽  
Paula Montero Magallo ◽  
Ines Roger Laparra ◽  
Pilar Ribera Alba ◽  
Jesus Poveda Ferriols ◽  
...  
Keyword(s):  
Cell Line ◽  
Covalent Binding ◽  
Clinical Development ◽  
Preclinical Model ◽  
Antineoplastic Drugs ◽  
Skin Sensitization ◽  
Cutaneous Toxicity ◽  
In Vitro Techniques

e15643 Background: Skin side effects are common manifestations of antineoplastic drugs that are frequently observed in early clinical trials. Therefore, there is a need to identify skin toxic agents before clinical development in order to predict severe skin manifestations. In many cases, skin toxicity is due to sensitization, a key immunologic process mediating redness, swelling and itching that can lead to more severe skin alterations. Methods: We adapted three skin cellular in vitro techniques for cutaneous drug sensitization assessment of the Organization for Economic Cooperation and Development (OECD, 2012) in order to predict antineoplastic drug skin sensitization: 1) Direct peptide reactivity assay (DPRA) was assessed by high performance liquid chromatography (HPLC) coupled to ultraviolet detector, to detect covalent binding of antineoplastic to skin proteins (haptenation). 2) KeratinoSense skin sensitization assay was evaluated in keratinocyte cell line (HaCaT) transfected with a selectable plasmid to quantify luciferase gene induction by chemiluminescence as a measure of activation of Keap1-Nrf2-antioxidant response element involved in the cellular processes in skin sensitization and 3) The human cell line activation test (h-CLAT) represents the activation of dendritic cells by the increase of CD86 and CD54 surface markers measured by flow cytometry. An antineoplastic was considered sensitizing when at least two of these three tests were positive. Antineoplastic tested were paclitaxel, erlotinib, imatinib, sunitinib, cetuximab, olaparib, palbociclib and everolimus. Results: Paclitaxel was positive for all the three tests; the DPRA formation, keratinoSense activation and h-CLAT expression showing the highest level of sensitization. Moreover sunitinib, and imatinib were positive for two of the three in vitro cellular tests, being considered sensitizing drugs. In contrast, erlotinib, cetuximab, olaparib, palbociclib and everolimus were considered non-sensitizing antineoplastic agents. In the case of erlotinib a high proportion of apoptosis of the keratinocytes during the keratinosense test was observed, suggesting that cutaneous toxicity could be due to cytolysis instead of sensitization. Conclusions: Results obtained in this work suggest that pre-clinical assays may predict skin sensitization and be of potential value to predict skin sensitization of antineoplastic drugs before clinical development. Further assessment with in vivo tests will help to identify the cutaneous toxic mechanisms of those non-sensitizing drugs as cetuximab

scholarly journals Improved Activity against Acute Myeloid Leukemia with Chimeric Antigen Receptor (CAR)-NK-92 Cells Designed to Target CD123

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1365
Author(s):  
Michael A. Morgan ◽  
Arnold Kloos ◽  
Daniela Lenz ◽  
Nadine Kattre ◽  
Juliette Nowak ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Line ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Killer Cell ◽  
Constitutive Expression ◽  
Transfer Strategies ◽  
Acute Myeloid

Anti-cancer activity can be improved by engineering immune cells to express chimeric antigen receptors (CARs) that recognize tumor-associated antigens. Retroviral vector gene transfer strategies allow stable and durable transgene expression. Here, we used alpharetroviral vectors to modify NK-92 cells, a natural killer cell line, with a third-generation CAR designed to target the IL-3 receptor subunit alpha (CD123), which is strongly expressed on the surface of acute myeloid leukemia (AML) cells. Alpharetroviral vectors also contained a transgene cassette to allow constitutive expression of human IL-15 for increased NK cell persistence in vivo. The anti-AML activity of CAR-NK-92 cells was tested via in vitro cytotoxicity assays with the CD123+ AML cell line KG-1a and in vivo in a patient-derived xenotransplantation CD123+ AML model. Unmodified NK-92 cells or NK-92 cells modified with a truncated version of the CAR that lacked the signaling domain served as controls. Alpharetroviral vector-modified NK-92 cells stably expressed the transgenes and secreted IL-15. Anti-CD123-CAR-NK-92 cells exhibited enhanced anti-AML activity in vitro and in vivo as compared to control NK-92 cells. Our data (1) shows the importance of IL-15 expression for in vivo persistence of NK-92 cells, (2) supports continued investigation of anti-CD123-CAR-NK cells to target AML, and (3) points towards potential strategies to further improve CAR-NK anti-AML activity.

Anti-obesity role of Aster glehni extract: in vivo and in vitro effects

Planta Medica ◽  
2012 ◽  
Vol 78 (11) ◽  
Author(s):  
HM Lee ◽  
TG Ahn ◽  
CW Kim ◽  
HJ An
Keyword(s):  
In Vitro Effects

Comparison of Specific Antibody, D-Phe-Pro-Arg-CH2Cl and Aprotinin for Prevention of In Vitro Effects of Recombinant Tissue-Type Plasminogen Activator on Haemostasis Parameters

1987 ◽  
Vol 58 (03) ◽  
pp. 921-926 ◽  
Author(s):  
E Seifried ◽  
P Tanswell
Keyword(s):  
Specific Antibody ◽  
Plasma Concentrations ◽  
Fibrinolytic Therapy ◽  
Tissue Type ◽  
Control Value ◽  
Type Plasminogen Activator ◽  
In Vitro Effects ◽  
Fibrinolytic Parameters

SummaryIn vitro, concentration-dependent effects of rt-PA on a range of coagulation and fibrinolytic assays in thawed plasma samples were investigated. In absence of a fibrinolytic inhibitor, 2 μg rt-PA/ml blood (3.4 μg/ml plasma) caused prolongation of clotting time assays and decreases of plasminogen (to 44% of the control value), fibrinogen (to 27%), α2-antiplasmin (to 5%), FV (to 67%), FVIII (to 41%) and FXIII (to 16%).Of three inhibitors tested, a specific polyclonal anti-rt-PA antibody prevented interferences in all fibrinolytic and most clotting assays. D-Phe-Pro-Arg-CH2Cl (PPACK) enabled correct assays of fibrinogen and fibrinolytic parameters but interfered with coagulometric assays dependent on endogenous thrombin generation. Aprotinin was suitable only for a restricted range of both assay types.Most in vitro effects were observed only with rt-PA plasma concentrations in excess of therapeutic values. Nevertheless it is concluded that for clinical application, collection of blood samples on either specific antibody or PPACK is essential for a correct assessment of in vivo effects of rt-PA on the haemostatic system in patients undergoing fibrinolytic therapy.

A Novel Triazole Derivative Drug Presenting In Vitro and In Vivo Anticancer Properties

2018 ◽  
Vol 18 (17) ◽  
pp. 1483-1493
Author(s):  
Ricardo Imbroisi Filho ◽  
Daniel T.G. Gonzaga ◽  
Thainá M. Demaria ◽  
João G.B. Leandro ◽  
Dora C.S. Costa ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Cell Line ◽  
Cancer Cell ◽  
Cancer Cell Line ◽  
Hepatic Function ◽  
Anticancer Properties ◽  
Mcf 7

Background: Cancer is a major cause of death worldwide, despite many different drugs available to treat the disease. This high mortality rate is largely due to the complexity of the disease, which results from several genetic and epigenetic changes. Therefore, researchers are constantly searching for novel drugs that can target different and multiple aspects of cancer. Experimental: After a screening, we selected one novel molecule, out of ninety-four triazole derivatives, that strongly affects the viability and proliferation of the human breast cancer cell line MCF-7, with minimal effects on non-cancer cells. The drug, named DAN94, induced a dose-dependent decrease in MCF-7 cells viability, with an IC50 of 3.2 ± 0.2 µM. Additionally, DAN94 interfered with mitochondria metabolism promoting reactive oxygen species production, triggering apoptosis and arresting the cancer cells on G1/G0 phase of cell cycle, inhibiting cell proliferation. These effects are not observed when the drug was tested in the non-cancer cell line MCF10A. Using a mouse model with xenograft tumor implants, the drug preventing tumor growth presented no toxicity for the animal and without altering biochemical markers of hepatic function. Results and Conclusion: The novel drug DAN94 is selective for cancer cells, targeting the mitochondrial metabolism, which culminates in the cancer cell death. In the end, DAN94 has been shown to be a promising drug for controlling breast cancer with minimal undesirable effects.

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