Panax notoginseng Inhibits Tumor Growth through Activating Macrophage to M1 Polarization

2018 ◽  
Vol 46 (06) ◽  
pp. 1369-1385 ◽  
Author(s):  
Bosung Kim ◽  
Eun-Yeong Kim ◽  
Eun-Ji Lee ◽  
Jung Ho Han ◽  
Chung-Hwan Kwak ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Culture Media ◽  
Tumor Model ◽  
Annexin V ◽  
Panax Notoginseng ◽  
Raw264.7 Cells ◽  
M1 Polarization ◽  
Chemotactic Protein ◽  
M1 Phenotype

Among the herbal ingredients of HangAmDan-B, a medicinal formula that redirects macrophages to become tumoricidal effectors, we found that Panax notoginseng (Burk.) F. H. Chen is the active component responsible for its macrophage-mediated antitumor activity. The water extracted roots of P. notoginseng (PN) did not affect the viability of RAW264.7 murine macrophage-like cells and murine Lewis lung carcinoma (LLC) cells up to a concentration of 100[Formula: see text][Formula: see text]g/mL. However, the transfer of culture media from PN-treated RAW264.7 cells suppressed the growth of LLC cells. The expression of classically activated (M1) markers, such as interleukin (IL)-1[Formula: see text], monocyte chemotactic protein (MCP)-1, tumor necrosis factor (TNF)-[Formula: see text], and inducible nitric oxide synthase (iNOS), was increased by PN treatment. The expression of alternatively activated (M2) markers including CD206, IL-10, and [Formula: see text]-[Formula: see text]-acetylhexosaminidases (YM-1) was reduced by PN treatment in the presence of IL-4. Flow cytometry also revealed that PN drives M1 activation of RAW264.7 cells. The transfer of culture media from PN-treated RAW264.7 cells induced the apoptosis of LLC cells as measured by flow cytometry using Annexin-V staining and western blot analysis for caspase cascade-related proteins. In addition, the results from in vivo tumor allograft model demonstrated that PN reduced both tumor volume and weight. The activation of macrophages toward an M1 phenotype was confirmed in the tumor allograft tumor model. These results collectively show that PN can serve as a potent anticancer agent through reeducation of macrophages toward an M1 phenotype.

Endothelial Cell-Derived Microparticles in the Pathogenesis of Chemotherapy-Associated Hypercoagulability.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1457-1457
Author(s):  
Daniel Lechner ◽  
Marietta Kollars ◽  
Sabine Eichinger ◽  
Paul Alexander Kyrle ◽  
Ansgar Weltermann
Keyword(s):  
Thrombin Generation ◽  
Culture Media ◽  
Annexin V ◽  
Membrane Vesicles ◽  
Procoagulant Activity ◽  
Dependent Manner ◽  
Thrombin Generation Assay ◽  
Apoptotic Effect ◽  
Mitochondrial Dehydrogenase Activity

Abstract Background: Cisplatin-based chemotherapy is a risk factor of venous thromboembolism in cancer patients. The underlying pathogenesis remains unclear. We hypothesized an apoptotic effect of cisplatin on endothelial cells (EC) inducing a release of small membrane vesicles, so-called microparticles (MP) which are known to cause hemostasis activation. Objectives: To quantify the release of MP from EC following administration of cisplatin and to investigate MP-associated procoagulant mechanisms. Methods: Two EC lines (HUVEC, HMVEC-L) were exposed to cisplatin (1, 2.5, 5, 10, and 20 μM) for up to 120 h. Cell viability was assessed by quantification of mitochondrial dehydrogenase activity, counts and procoagulant activity of MP were measured by flow cytometry and a thrombin generation assay, respectively. Tissue factor (TF) antigen levels were determined by ELISA. Results: EC viability decreased in a dose- and time-dependent manner and was accompanied by an increasing release of MP into culture media (maximum: HUVEC + 544%; HMVEC-L + 1738%). In parallel, procoagulant activity of media increased by up to 150% (HUVEC) and 493% (HMVEC-L), respectively. The procoagulant activity was almost abolished by annexin V but was not suppressed by a monoclonal TF-antibody. TF antigen levels on MP were persistently low even at high cisplatin concentrations. Conclusion: At pharmacologically relevant concentrations, cisplatin induced a marked release of procoagulant MP from EC. Negatively charged phospholipids but not TF on MP were decisive for total thrombin generation. Further studies are warranted to investigate the cisplatin-induced release of EC-derived MP in vivo.

Combined Inhibition of Wee1 and Poly(ADP Ribose) Polymerase1/2 Synergistically Inhibits Acute Myeloid Leukemia Cells By Enhancing DNA Damage and the Induction of Apoptosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3621-3621 ◽  
Author(s):  
Jonathan C Snedeker ◽  
Tamara M Burleson ◽  
Raoul Tibes ◽  
Christopher C. Porter
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
Dna Damage ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Combination Index ◽  
Annexin V ◽  
Concomitant Treatment ◽  
Western Blots

Abstract Introduction: Successful treatment of AML remains dependent upon cytotoxic chemotherapy. However, traditional regimens are not well tolerated by older patients who are at highest risk of disease, and salvage rates after relapse are low, necessitating novel therapeutic strategies. Our groups identified Wee1 as a potential therapeutic target in AML, particularly in the context of concomitant treatment with cytarabine (Tibes et al, Blood, 2012; Porter et al, Leukemia, 2012). Wee1 inhibits CDK1&2 via phosphorylation thereby stalling cell cycle progression. One consequence of Wee1 inhibition/CDK1 activation is impairment of DNA repair via homologous recombination (Krajewska et al, Oncogene, 2013). Cells in which HR is impaired are dependent upon Parp1/2 function, and HR deficient cells are particularly sensitive to Parp1/2 inhibition. Therefore, we hypothesized that combined Wee1 and Parp1/2 inhibition may result in greater inhibition of AML cell proliferation and survival than either alone. Methods: Human AML cell lines, MV4-11 and Molm-13, and a mouse AML that expresses MLL-ENL/FLT3-ITD were cultured with various concentrations of a Wee1 inhibitor (AZ1775) and a Parp1/2 inhibitor (olaparib) and counted 72 hours later by propidium iodide exclusion and flow cytometry. In some experiments, cells were split into fresh media to recover for 72 more hours. Combination Index (CI) values were calculated by the method of Chou and Talalay. Apoptosis was measured using Annexin V/7AAD and flow cytometry. Western blots were used to confirm inhibition of CDK1/2 phosphorylation and to measure DNA damage induction (gamma-H2AX). Results: Combined inhibition of Wee1 and Parp1/2 was synergistic, as measured by cell numbers at 72 hours, in all 3 cell lines tested, with combination index values ranging from 0.3 to 0.9. When cells were allowed to recover after treatment, those treated by single agents were able to continue proliferating. However, those treated with the combination did not recover as well or at all, indicating greatly impaired proliferative capacity. Combined inhibition of Wee1 and Parp1/2 also resulted in a significant increase in apoptosis greater than either drug alone. Western blots for gamma-H2AX confirmed that the combination of Wee1 and Parp1/2 resulted in more DNA damage than either drug alone. Discussion: Combined inhibition of Wee1 and Parp1/2 results in greater inhibition of AML cell proliferation, DNA damage and apoptosis than either drug alone. Future studies will include experiments with primary patient samples, as well as in vivo trials combining Wee1 inhibition with Parp1/2 inhibition. These preliminary studies raise the possibility of rational combinations of targeted agents for leukemia in those for whom conventional chemotherapeutics may not be well tolerated. Disclosures No relevant conflicts of interest to declare.

scholarly journals Wnt/β-catenin signaling regulates lipopolysaccharide-altered polarizations of RAW264.7 cells and alveolar macrophages in mouse lungs

2021 ◽  
Vol 19 ◽  
pp. 205873922110593
Author(s):  
Jiali Yang ◽  
Ying Wang ◽  
Dandan Yang ◽  
Jia Ma ◽  
Shuang Wu ◽  
...  
Keyword(s):  
Alveolar Macrophage ◽  
Alveolar Macrophages ◽  
Macrophage Polarization ◽  
Raw264.7 Cells ◽  
M1 Polarization ◽  
Inflammatory Phenotype ◽  
Inflammatory State ◽  
Anti Inflammatory

Introduction Macrophages are capable of exerting both proinflammatory and anti-inflammatory functions in response to distinct environmental stimuli, by polarizing into classically inflammatory state (M1) and anti-inflammatory phenotype (M2), respectively. The Wnt/β-catenin signaling plays an important role in the tissue homeostasis and immune regulations, including the macrophage polarizations. However, the molecular mechanism of Wnt/β-catenin signaling in regulating alveolar macrophage polarization in an inflammatory state remains unclear. Methods The Wnt/β-catenin signaling-altered phenotypes of murine macrophage-like RAW264.7 cells in vitro and alveolar macrophage in vivo in both of naïve and lipopolysaccharide-induced inflammation states were accessed by immunoblotting and immunostaining assays. Results The activation of Wnt/β-catenin signaling inhibited macrophage M1 polarization, but promoted alternative M2 polarization in murine RAW264.7 cells under a naïve state. Interestingly, in an LPS-induced inflammation condition, the enhanced Wnt/β-catenin activity suppressed both M1 and M2 polarizations in RAW264.7 cells in vitro, and primary alveolar macrophages of LPS-challenged mice in vivo. Molecular analysis further demonstrated an involvement of Stat signing in regulating Wnt/β-catenin signaling-altered polarizations in mouse alveolar macrophages. Conclusion These results suggest a mechanism by which Wnt/β-catenin signaling modulates macrophage polarization in an inflammation state by regulating the Stat signaling pathway.

Rac GTPases Regulate Erythropoiesis Both in the Early Steps of Differentiation and in Enucleation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1714-1714
Author(s):  
Theodosia A. Kalfa ◽  
Suvarnamala Pushkaran ◽  
Jose A. Cancelas ◽  
James F. Johnson ◽  
Deidre Daria ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Actin Polymerization ◽  
Ex Vivo ◽  
Terminal Differentiation ◽  
Annexin V ◽  
Actin Dynamics ◽  
Fluorescent Label ◽  
Rac Gtpase ◽  
Rac Gtpases

Abstract Rac GTPases (i.e. Rac1, Rac2 and Rac3), a subfamily of Rho GTPases, control actin organization and have overlapping as well as distinct roles in cell survival, proliferation, and differentiation in various hematopoietic cell lineages (Gu et al, Science 2003, Cancelas et al, Nature Med 2005). Using conditional gene-targeting in mice, we have previously demonstrated that Rac1 and Rac2 deficiency causes anemia with abnormal erythrocyte cytoskeleton and decreased deformability (Kalfa et al, Blood 2006). In the present studies, we found by colony assays that although bone marrow (BM) BFU-E activity was unaltered from that of the wild type (WT) mice, Rac1−/−;Rac2−/− erythroid bursts had a strikingly different morphology appearing as round, small, dense colonies, likely a manifestation of motility defects associated with Rac GTPase deficiency. Total CFU-Es recovered from Rac1−/−;Rac2−/− BM were as low as 25% of that in WT mice (p<0.05). To further assess erythroblast differentiation, BM cells were immunostained with fluorescent label-conjugated anti-CD71 and anti-Ter119, as previously described (Socolovski et al. Blood 2001). Flow cytometry analysis revealed that proerythroblasts and basophilic erythroblasts in the BM were significantly decreased in Rac1−/−;Rac2−/− (∼30–50% of WT content) while the terminal differentiation to orthochromatic erythroblasts was comparable. In vivo BrdU labeling and flow cytometry with 7-AAD and annexin-V in combination with staining for CD71 and Ter119 revealed no difference in proliferation or survival between WT and Rac1−/−;Rac2−/− erythroid cells after the proerythroblast stage. These data suggest that deficiency of Rac1 and Rac2 GTPases affect erythropoiesis mainly at the early stages of BFU-E and CFU-E formation but not during terminal differentiation to orthochromatic erythroblasts. Given the prominent role of Rac GTPases in regulating actin structure, we next evaluated the possible involvement of Rac GTPases in enucleation, the terminal step of erythropoiesis that likely requires significant actin remodeling. We performed quantitative analysis in ex vivo erythropoiesis cultures, by flow cytometry, using SYTO16, a cell-permeable nucleic acid-staining dye. The frequency of enucleated red cells (SYTO16-low, Ter119-positive population) was similar in the WT and the Rac1−/−;Rac2−/− erythroid cultures. However, application of a Rac GTPase inhibitor, NSC23766, to the WT or the Rac1−/−;Rac2−/− erythroid cultures during the enucleation phase resulted in an inhibition of enucleation up to 80% dose-dependently (figure 1). Rac1 and Rac2 deficiency led to a compensatory elevation of Rac3 activity that was effectively suppressed by NSC23766, as demonstrated by immunoblotting in the Rac1−/−;Rac2−/− erythroblasts and effector-domain pull-down studies. Moreover, NSC23766 inhibited Rac1, Rac2, and Rac3 activities as well as actin polymerization of the erythroblasts. Thus, Rac1, Rac2, and Rac3 have redundant but essential roles in supporting actin dynamics necessary for the nucleus extrusion during the enucleation process. Figure Figure

Inhibition of Fas-mediated Apoptosis in Yac-1 Cell via Anti-Fas Ribozyme

2004 ◽  
Vol 36 (3) ◽  
pp. 199-205
Author(s):  
Min Zhang ◽  
Fang Liu ◽  
Wei He ◽  
Yong You ◽  
Ping Zou ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Proteolytic Activity ◽  
Caspase 3 ◽  
Hammerhead Ribozyme ◽  
Annexin V ◽  
In Vitro Transcription ◽  
Rt Pcr ◽  
Fas Mrna

Abstract To detect a new and more effective way against apoptosis mouse lymphomatic cell line-Yac-1 in which fas gene was expressed highly was used as a model for studying the effects of anti-Fas ribozyme on Fas-mediated apoptosis. A hammerhead ribozyme gene targeting the fas mRNA was synthesized and its in vitro transcription vector was constructed, which was transfected into Yac-1 cells using electroporation. Rz596 expression was detected using RT-PCR, and Fas expression in Yac-1 cells was detected using RT-PCR, Western blot and flow cytometry. After treated with anti-Fas antibody (JO2), Yac-1 cell viability was measured with MTT assay, caspase-3 proteolytic activity was detected, and cell apoptosis was measured according to annexin V apoptosis detecting kit. Anti-Fas ribozyme could cleave fas mRNA efficiently in vivo and in vitro. Fas expression in Yac-1 cells transfected with anti-Fas ribozyme was decreased remarkably and correlated with resistance to Fas-mediated apoptosis as determined by flow cytometry and caspase-3 proteolytic activity. Anti-Fas ribozyme was detected in cells transfected with pU6-RZ596 and pU6-dRZ596 and could remarkably decrease the Fas expression in Yac-1 cells, which made Yac-1 cells get rid of Fas-mediated apoptosis. Because of wide expression of fas in organs and tissues, our research was very useful for studying the inhibition of apoptosis of many organs and tissues in the future.

scholarly journals Circumventing AKT-Associated Radioresistance in Oral Cancer by Novel Nanoparticle-Encapsulated Capivasertib

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 533 ◽  
Author(s):  
Liwei Lang ◽  
Tiffany Lam ◽  
Alex Chen ◽  
Caleb Jensen ◽  
Leslie Duncan ◽  
...  
Keyword(s):  
3D Culture ◽  
Small Animal ◽  
Tumor Model ◽  
Annexin V ◽  
Fluorescein Isothiocyanate ◽  
Therapeutic Effects ◽  
Akt Inhibitor ◽  
Orthotopic Mouse Model ◽  
Improvement In Survival

Background: Development of radioresistance in oral squamous cell carcinoma (OSCC) remains a significant problem in cancer treatment, contributing to the lack of improvement in survival trends in recent decades. Effective strategies to overcome radioresistance are necessary to improve the therapeutic outcomes of radiotherapy in OSCC patients. Methods: Cells and xenograft tumors were irradiated using the Small Animal Radiation Research Platform. AKT inhibitor capivasertib (AZD5363) was encapsulated into cathepsin B-responsible nanoparticles (NPs) for tumor-specific delivery. Cell viability was measured by alamarBlue, cell growth was determined by colony formation and 3D culture, and apoptosis was assessed by flow cytometry with the staining of Fluorescein isothiocyanate (FITC) Annexin V and PI. An orthotopic tongue tumor model was used to evaluate the in vivo therapeutic effects. The molecular changes induced by the treatments were assessed by Western blotting and immunohistochemistry. Results: We show that upregulation of AKT signaling is the critical mechanism for radioresistance in OSCC cells, and AKT inactivation by a selective and potent AKT inhibitor capivasertib results in radiosensitivity. Moreover, relative to irradiation (IR) alone, IR combined with the delivery of capivasertib in association with tumor-seeking NPs greatly enhanced tumor cell repression in 3D cell cultures and OSCC tumor shrinkage in an orthotopic mouse model. Conclusions: These data indicate that capivasertib is a potent agent that sensitizes radioresistant OSCC cells to IR and is a promising strategy to overcome failure of radiotherapy in OSCC patients.

Red cell-derived microparticles (RMP) as haemostatic agent

2013 ◽  
Vol 110 (10) ◽  
pp. 751-760 ◽  
Author(s):  
Max Johansen ◽  
Carlos Bidot ◽  
Lawrence Horstman ◽  
Yeon Ahn ◽  
Wenche Jy
Keyword(s):  
Flow Cytometry ◽  
Blood Loss ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Annexin V ◽  
Haemostatic Agent ◽  
Platelet Aggregometry ◽  
Primary Platelet ◽  
Potential Use

SummaryAmong circulating cell-derived microparticles, those derived from red cells (RMP) have been least well investigated. To exploit potential haemostatic benefit of RMP, we developed a method of producing them in quantity, and here report on their haemostatic properties. High-pressure extrusion of washed RBC was employed to generate RMP. RMP were identified and enumerated by flow cytometry. Their size distribution was assessed by Doppler electrophoretic light scattering analysis (DELSA). Interaction with platelets was studied by platelet aggregometry, and shear-dependent adhesion by Diamed IMPACT-R. Thrombin generation and tissue factor (TF) expression was also measured. The effect of RMP on blood samples of patients with bleeding disorders was investigated ex vivo by thromboelastography (TEG). Haemostatic efficacy in vivo was assessed by measuring reduction of blood loss and bleeding time in rats and rabbits. RMP have mean diameter of 0.45 μm and 50% of them exhibit annexin V binding, a proxy for procoagulant phospholipids (PL). No TF could be detected by flow cytometry. At saturating concentrations of MPs, RMP generated thrombin robustly but after longer delay compared to PMP and EMP. RMP enhanced platelet adhesion and aggregation induced by low-dose ADP or AA. In TEG study, RMP corrected or improved haemostatic defects in blood of patients with platelet and coagulation disorders. RMP reduced bleeding time and blood loss in thrombocytopenic rabbits (busulfan-treated) and in Plavix-treated rats. In conclusion, RMP has broad haemostatic activity, enhancing both primary (platelet) and secondary (coagulation) haemostasis, suggesting potential use as haemostatic agent for treatment of bleeding.

scholarly journals Pimavanserin: A Novel Autophagy Modulator for Pancreatic Cancer Treatment

Cancers ◽  
2021 ◽  
Vol 13 (22) ◽  
pp. 5661
Author(s):  
Sharavan Ramachandran ◽  
Itishree S. Kaushik ◽  
Sanjay K. Srivastava
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Patients ◽  
Pancreatic Tumor ◽  
Tumor Model ◽  
Annexin V ◽  
Pancreatic Tumors ◽  
Transmission Electron Microscopy Analysis ◽  
Genomic Databases ◽  
Apoptotic Effects

Pancreatic tumors exhibit high basal autophagy compared to that of other cancers. Several studies including those from our laboratory reported that enhanced autophagy leads to apoptosis in cancer cells. In this study, we evaluated the autophagy and apoptosis inducing effects of Pimavanserin tartrate (PVT). Autophagic effects of PVT were determined by Acridine Orange assay and Transmission Electron Microscopy analysis. Clinical significance of ULK1 in normal and pancreatic cancer patients was evaluated by R2 and GEPIA cancer genomic databases. Modulation of proteins in autophagy signaling was assessed by Western blotting and Immunofluorescence. Apoptotic effects of PVT was evaluated by Annexin-V/APC assay. Subcutaneous xenograft pancreatic tumor model was used to evaluate the autophagy-mediated apoptotic effects of PVT in vivo. Autophagy was induced upon PVT treatment in pancreatic ducal adenocarcinoma (PDAC) cells. Pancreatic cancer patients exhibit reduced levels of autophagy initiator gene, ULK1, which correlated with reduced patient survival. Interestingly, PVT induced the expression of autophagy markers ULK1, FIP200, Atg101, Beclin-1, Atg5, LC3A/B, and cleavage of caspase-3, an indicator of apoptosis in several PDAC cells. ULK1 agonist LYN-1604 enhanced the autophagic and apoptotic effects of PVT. On the other hand, autophagy inhibitors chloroquine and bafilomycin blocked the autophagic and apoptotic effects of PVT in PDAC cells. Notably, chloroquine abrogated the growth suppressive effects of PVT by 25% in BxPC3 tumor xenografts in nude mice. Collectively, our results indicate that PVT mediated pancreatic tumor growth suppression was associated with induction of autophagy mediated apoptosis.

scholarly journals MicroRNA-29c Increases the Chemosensitivity of Pancreatic Cancer Cells by Inhibiting USP22 Mediated Autophagy

2018 ◽  
Vol 47 (2) ◽  
pp. 747-758 ◽  
Author(s):  
Limin Huang ◽  
Chaoquan Hu ◽  
Hui Cao ◽  
Xiaoliang Wu ◽  
Rongpin Wang ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Flow Cytometry ◽  
Annexin V ◽  
Luciferase Reporter ◽  
Pancreatic Cancer Cells ◽  
Reporter Assays ◽  
Direct Target ◽  
Induced Apoptosis

Background/Aims: Pancreatic cancer (PC) is an aggressive malignancy with a poor survival rate. Despite advances in the treatment of PC, the efficacy of therapy is limited by the development of chemoresistance. Here, we examined the role of microRNA-29c (miR-29c) and the involvement of autophagy and apoptosis in the chemoresistance of PC cells in vivo and in vitro. Methods: We employed qRT-PCR, western blot and immunofluorescence to examine the expression level of miR-29c, USP22 and autophagy relative protein. In addition, we used MTT assay to detect cell proliferation and transwell assay to measure migration and invasiveness. The apoptosis was determined using annexin V-FITC/PI apoptosis detection kit by flow cytometry. Luciferase reporter assays confirmed the relationship between USP22 and miR-29c. Results: miR-29c overexpression in the PC cell line PANC-1 enhanced the effect of gemcitabine on decreasing cell viability and inducing apoptosis and inhibited autophagy, as shown by western blotting, immunofluorescence staining, colony formation assays, and flow cytometry. Ubiquitin specific peptidase (USP)-22, a deubiquitinating enzyme known to induce autophagy and promote PC cell survival, was identified as a direct target of miR-29c. USP22 knockdown experiments indicated that USP22 suppresses gemcitabine-induced apoptosis by promoting autophagy, thereby increasing the chemoresistance of PC cells. Luciferase reporter assays confirmed that USP22 is a direct target of miR-29c. A xenograft mouse model demonstrated that miR-29c increases the chemosensitivity of PC in vivo by downregulating USP22, leading to the inhibition of autophagy and induction of apoptosis. Conclusions: Taken together, these findings reveal a potential mechanism underlying the chemoresistance of PC cells mediated by the regulation of USP22-mediated autophagy by miR-29c, suggesting potential targets and therapeutic strategies in PC.

The Activation of Fas Receptor by APO010, a Recombinant Form of Fas Ligand, Induces In Vitro and In Vivo Antimyeloma Activity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1515-1515 ◽  
Author(s):  
Enrique M. Ocio ◽  
Patricia Maiso ◽  
Mercedes Garayoa ◽  
Marc Dupuis ◽  
Atanasio Pandiella ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Fas Ligand ◽  
Annexin V ◽  
Recombinant Form ◽  
Caspase 8 ◽  
Fas Receptor ◽  
Healthy Donors

Abstract Background & Aims Fas receptor is expressed on the surface of many malignant cells and its activation represents a potentially relevant anticancer target. APO010 is a recombinant form of Fas Ligand with hexameric structure, which is currently being evaluated in Phase 1 clinical trials. In order to identify possible targeted indications, we tested the in vitro and in vivo anti-tumor efficacy of APO010 on multiple myeloma (MM) cells. Material & methods In vitro cytotoxicity was tested by MTT and Annexin V staining in 8 MM cell lines and PBMCs from 3 healthy donors. Other techniques used for mechanistic studies were propidium iodide uptake by flow cytometry, Western-blotting, BrdU uptake and gene expression profile analysis. The in vivo antimyeloma effect of APO010 was tested in a xenograft of human plasmocytoma in CB17-SCID mice. When tumors became palpable mice were randomized to receive APO010 15 μg/Kg ip × 5d/sem (n=7), APO010 5 μg/Kg ip × 5d/sem (n=8) or vehicle alone (n=8). Tumor volumes, clinical features and weight were monitored three times a week. Results Six of the 8 MM cell lines studied by MTT were highly sensitive to APO010 with IC-50 at 24h of 0.5–20 ng/ml (2.5–100 pM), whereas two were resistant (RPMI-8226 and OPM-1). This sensitivity was correlated with the expression of Fas receptor by flow cytometry. Activation of apoptosis was rapid (within two hours of incubation) with maximum effect at 10 hours, as determined by Annexin V staining. Interestingly, APO010 was not toxic against PBMCs (both resting and activated) from 3 healthy donors at doses effective against MM cell lines. The presence of the microenvironment, as simulated by the coculture of MM1S cells with IL-6, IGF-1 and BMSCs, was not able to abrogate the APO010 antimyeloma effect. The combination of APO010 with Doxorubicin and Bortezomib, and, to a less extent, with Melphalan and Lenalidomide, potentiated the efficacy of the drugs alone. Regarding the mechanism of action, APO010 antiproliferative activity is mediated through caspase dependent apoptosis (Annexin-V staining, and PARP, caspase-3, caspase-7, caspase-8 and caspase-9 cleavage) and is independent of variations on the cell cycle profile. In this sense, the presence of the pan-caspase or caspase-8 inhibitors (Z-VAD-FMK and Z-IETD-FMK respectively) were able to completely abrogate APO010-induced cell death. Treatment of MM1S cells with APO010 for just one hour induced changes in the expression of 52 genes, many of them implicated in regulation of transduction (n=16). Three of the 4 most upregulated genes were the 3 members of the nuclear receptor subfamily 4, group A (Nurr1, Nor1 and Nur77). Other upregulated transcription factors were members of the Fos/Jun family such as Jun, JunB or FosL2. Western-Blot studies revealed that APO010 also provoked cleavage of MCL-1 and BIM, a decrease of BID and an important downregulation of pAKT. In the in vivo studies, APO010 treatment inhibited tumor growth as compared with the control group (p=0.02) without differences among the two doses of APO010. No significant toxicity was observed regarding body weight loss or increase in liver enzymes. Conclusions These data show that Fas activation with APO010 induces in vitro and in vivo cytotoxicity in MM cell lines, mainly through transcriptional regulation. This study provides an initial rationale for the use of this compound for treatment of MM patients.

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