ABSTRACT
Mesenchymal stem cell (MSC) therapy is considered a new treatment for a wide range of diseases and injuries, but challenges remain, such as poor survival, homing and engraftment rates, thus limiting the therapeutic efficacy of the transplanted MSCs. Many strategies have been developed to enhance the therapeutic efficacy of MSCs, such as preconditioning, co-transplantation with graft materials and gene modification. Hepatocyte growth factor (HGF) is secreted by MSCs, which plays an important role in MSC therapy. It has been reported that the modification of the HGF gene is beneficial to the therapeutic efficacy of MSCs, including diseases of the heart, lung, liver, urinary system, bone and skin, lower limb ischaemia and immune-related diseases. This review focused on studies involving HGF/MSCs both in vitro and in vivo. The characteristics of HGF/MSCs were summarized, and the mechanisms of their improved therapeutic efficacy were analysed. Furthermore, some insights are provided for HGF/MSCs' clinical application based on our understanding of the HGF gene and MSC therapy.
Subject(s)
Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Hepatocyte Growth Factor/genetics , Hepatocyte Growth Factor/metabolism , Hepatocytes/metabolism , Lung/metabolism , Mesenchymal Stem Cells/metabolismABSTRACT
The mRNA precursor 3'-end modification factor NUDT21 is a major regulator of 3'UTR shortening and an important component of pre-mRNA cleavage and polyadenylation. However, its role in pathologic progress of small cell lung cancer (SCLC) remains unclear. In this study, we observed that NUDT21 expression is downregulated in SCLC tissues. Hypoxia-induced down-regulation of NUDT21 through HIF-1α. NUDT21 shRNA transduction promotes proliferation and inhibits apoptosis of A549 cells. NUDT21 inhibition also promotes tumor growth in a mouse xenograft model. Furthermore, we clarified that HIF-1α mediated NUDT21 downregulation which altered the expression patterns of two isoforms of GLS1, GAC and KGA. These results link the hypoxic tumor environments to aberrant glutamine metabolism which is important for cellular energy in SCLC cells. Therefore, NUDT21 could be considered as a potential target for the treatment of SCLC.
Subject(s)
Cleavage And Polyadenylation Specificity Factor/metabolism , Glutaminase/genetics , Lung Neoplasms/genetics , Lung Neoplasms/pathology , RNA Splicing/genetics , Small Cell Lung Carcinoma/genetics , Small Cell Lung Carcinoma/pathology , A549 Cells , Cell Proliferation/genetics , Cells, Cultured , Glutaminase/metabolism , Humans , Lung Neoplasms/metabolism , MicroRNAs/genetics , Polyadenylation , Small Cell Lung Carcinoma/metabolismABSTRACT
Endothelial cell death is linked to vascular diseases such as atherosclerosis and tissue ischemia. miRNA-17-92 (miR-17-92) is a multiple functional oncogenic miRNA cluster which plays vital roles in tumor angiogenesis and tissue development. However, its role in regulation of endothelial cell ferroptosis remains unclear. In this study, we revealed that miR-17-92 protects endothelial HUVEC cells from erastin-induced ferroptosis. miR-17-92 overexpression significantly reduced erastin-induced growth inhibition and ROS generation of HUVEC cells. Furthermore, Zinc lipoprotein A20, a validated target of miR-17-92, was identified as a novel regulator of endothelial cell ferroptosis. Lentivirus mediated A20 overexpression increased ROS generation and enhanced erastin-induced ferroptosis, whereas A20 knockdown inhibited erastin-induced ferroptosis. Mechanistic studies revealed that erastin-induced ferroptosis is associated with GPX4 downregulation and ACSL4 upregulation. miR-17-92 overexpression or A20 inhibition increased the ACSL4 expression in HUVEC cells. A20 was identified to directly with and regulate ACSL4 expression by immunoprecipitation. It suggests that the A20-ACSL4 axis plays important roles in erastin-induced endothelial ferroptosis. In conclusion, this study revealed a novel mechanism through which miR-17-92 protects endothelial cells from erastin-induced ferroptosis by targeting the A20-ACSL4 axis.
Subject(s)
Coenzyme A Ligases/metabolism , Cytoprotection , Ferroptosis/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , MicroRNAs/metabolism , Piperazines/pharmacology , Signal Transduction , Tumor Necrosis Factor alpha-Induced Protein 3/metabolism , Cell Proliferation/drug effects , Cytoprotection/drug effects , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/drug effects , Humans , MicroRNAs/genetics , Signal Transduction/drug effectsABSTRACT
A novel glucoside bletilloside A (1) was isolated from the tubers of Bletilla striata, together with seven known compounds (2-8). Their structures were determined on the basis of extensive spectroscopic analyses. All compounds were evaluated for the inhibition on NO production effects in RAW 264.7 macrophage cells, while militarine (4) and dactylorhin A (5) exhibited moderate inhibitory effects.
Subject(s)
Bibenzyls/isolation & purification , Bibenzyls/pharmacology , Glucosides/isolation & purification , Glucosides/pharmacology , Orchidaceae/chemistry , Animals , Bibenzyls/chemistry , Glucosides/chemistry , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Mice , Molecular Structure , Nitric Oxide/biosynthesis , Plant Tubers/chemistryABSTRACT
miR-17-92 cluster are overexpressed in hematological malignancies including chronic myeloid leukemia (CML). However, their roles and mechanisms that regulate BCR-ABL induced leukemogenesis remain unclear. In this study, we demonstrated that genomic depletion of miR-17-92 inhibited the BCR-ABL induced leukemogenesis by using a mouse model of transplantation of BCR-ABL transduced hematopoietic stem cells. Furthermore, we identified that miR-19b targeted A20 (TNFAIP3). A20 overexpression results in inactivation of NF-κB activity including decrease of phosphorylation of P65 and IκBα, leads to induce apoptosis and inhibit proliferation and cycle in CML CD34 + cells. Thus we proved that miR-17-92 is a critical contributor to CML leukemogenesis via targeting A20 and activation of NF-κB signaling. These findings indicate that miR-17-92 will be important resources for developing novel treatment strategies of CML and better understanding long-term disease control.
Subject(s)
Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , MicroRNAs/metabolism , NF-kappa B/metabolism , Signal Transduction , Tumor Necrosis Factor alpha-Induced Protein 3/metabolism , Animals , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , RNA, Long NoncodingABSTRACT
SUMO/sentrin specific protease 1 (Senp1) is an important regulation protease in the protein sumoylation, which affects the cell cycle, proliferation and differentiation. The role of Senp1 mediated protein desumoylation in pathophysiological progression of multiple myeloma is unknown. In this study, we demonstrated that Senp1 is overexpressed and induced by IL-6 in multiple myeloma cells. Lentivirus-mediated Senp1 knockdown triggers apoptosis and reduces viability, proliferation and colony forming ability of MM cells. The NF-κB family members including P65 and inhibitor protein IkBα play important roles in regulation of MM cell survival and proliferation. We further demonstrated that Senp1 inhibition decreased IL-6-induced P65 and IkBα phosphorylation, leading to inactivation of NF-кB signaling in MM cells. These results delineate a key role for Senp1in IL-6 induced proliferation and survival of MM cells, suggesting it may be a potential new therapeutic target in MM.
Subject(s)
Apoptosis , Cell Division , Endopeptidases/metabolism , Multiple Myeloma/pathology , NF-kappa B/metabolism , Signal Transduction , Cell Line, Tumor , Cysteine Endopeptidases , Endopeptidases/genetics , Gene Knockdown Techniques , Gene Silencing , HEK293 Cells , Humans , Multiple Myeloma/metabolismABSTRACT
BACKGROUND AIMS: Multipotent mesenchymal stromal cells (MSCs) are promising candidates for innovative cell therapeutic applications. Before their use, however, they usually need to be expanded in vitro with serum-supplemented media. MSCs can undergo replicative senescence during in vitro expansion, but it is not yet clear how serum supplements influence this process. METHODS: In the present study, we compared how media supplemented with fetal bovine serum (FBS) or calf serum (CS) affected morphology, proliferation, differentiation, senescence and other functional characteristics of human umbilical cord-derived MSCs (UC-MSCs). RESULTS: UC-MSCs cultured in both FBS- and CS-containing media were able to differentiate along osteogenic and adipogenic lineages but ultimately reached proliferation arrest. However, senescence-associated characteristics, such as ß-galactosidase activity, reactive oxygen species levels, proliferation rate and gene expression, demonstrate that UC-MSCs grown with FBS have better proliferation potential and differentiation capacity. In contrast, UC-MSCs grown with CS have a higher proportion of apoptotic cells and senescent characteristics. Possible mechanisms for the observed phenotypes include changes in gene expression (Bax, p16, p21 and p53) and cytokine production (interleukin-6 and interleukin-8). CONCLUSIONS: This study demonstrates that FBS-supplemented media provides a better microenvironment for the expansion of UC-MSCs in vitro than CS-supplemented media. This work provides insight into MSCs generation practices for use in basic research and clinical therapies.
Subject(s)
Cell Culture Techniques/methods , Cellular Senescence/drug effects , Culture Media/pharmacology , Mesenchymal Stem Cells/drug effects , Animals , Apoptosis/drug effects , Cattle , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Humans , Interleukin-6/biosynthesis , Interleukin-8/biosynthesis , Mesenchymal Stem Cells/cytology , Reactive Oxygen Species/metabolism , Serum , Umbilical Cord/cytology , beta-Galactosidase/metabolismABSTRACT
The hepatic growth factor hepatopoietin Cn (HPPCn) prevents liver injury induced by carbon tetrachloride in rats. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid produced by sphingosine kinase (SphK). S1P and S1P receptors (S1PRs) are involved in liver fibrogenesis and oxidative injury. This work sought to understand the mechanism by which SphK/S1P/S1PRs are involved in the protective effects of HPPCn on ethanol-induced liver injury and fibrosis. Transgenic mice with liver-specific overexpression of HPPCn (HPPCn(liver) (+/+)) were generated. Two ethanol feeding protocols were used to assess the protective effect of HPPCn on acute and chronic liver injury in mice. Specific inhibitors of S1PR1, S1PR2 and S1PR3 and siRNA were used to examine the roles of S1PRs in hepatic stellate cell (HSC) activation and hepatocyte apoptosis. Increased HPPCn expression in transgenic mice attenuated fibrosis induced by ethanol and carbon tetrachloride (CCl4). Treatment with recombinant human HPPCn prevented human hepatocyte apoptosis and HSC activation. JTE-013 or S1PR2-siRNA attenuated the effect of HPPCn on HSC activation induced by tumour necrosis factor-α (TNF-α). Consistent with the effect of N,N-dimethylsphingosine (DMS), suramin or S1PR3-siRNA treatment blocked HPPCn-induced Erk1/2 phosphorylation in human hepatocytes. This study demonstrated that HPPCn attenuated oxidative injury and fibrosis induced by ethanol feeding and that the SphK1/S1P/S1PRs signalling pathway contributes to the protective effect of HPPCn on hepatocyte apoptosis and HSC activation.
Subject(s)
Ethanol , Hepatocyte Growth Factor/metabolism , Liver Cirrhosis, Alcoholic/prevention & control , Liver/enzymology , Nuclear Proteins/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Receptors, Lysosphingolipid/metabolism , Animals , Apoptosis , Cells, Cultured , Disease Models, Animal , Gene Expression Regulation , Hepatic Stellate Cells/enzymology , Hepatic Stellate Cells/pathology , Hepatocyte Growth Factor/genetics , Humans , Liver/drug effects , Liver/pathology , Liver Cirrhosis, Alcoholic/enzymology , Liver Cirrhosis, Alcoholic/etiology , Liver Cirrhosis, Alcoholic/genetics , Liver Cirrhosis, Alcoholic/pathology , Lysophospholipids/metabolism , Mice , Mice, Transgenic , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Nuclear Proteins/genetics , Oxidative Stress , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/genetics , RNA Interference , RNA, Messenger/metabolism , Receptors, Lysosphingolipid/antagonists & inhibitors , Receptors, Lysosphingolipid/genetics , Signal Transduction , Sphingosine/analogs & derivatives , Sphingosine/metabolism , Time Factors , Transfection , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Skeletal stem cells (SSC) have gained attentions as candidates for the treatment of osteoarthritis due to their osteochondrogenic capacity. However, the immunomodulatory properties of SSC, especially under delivery operations, have been largely ignored. In the study, we found that Pdpn+ and Grem1+ SSC subpopulations owned immunoregulatory potential, and the single-cell RNA sequencing (scRNA-seq) data suggested that the mechanical activation of microgel carriers on SSC induced the generation of Pdpn+Grem1+Ptgs2+ SSC subpopulation, which was potent at suppressing macrophage inflammation. The microgel carriers promoted the YAP nuclear translocation, and the activated YAP protein was necessary for the increased expression of Ptgs2 and PGE2 in microgels-delivered SSC, which further suppressed the expression of TNF-É, IL-1ß and promoted the expression of IL-10 in macrophages. SSC delivered with microgels yielded better preventive effects on articular lesions and macrophage activation in osteoarthritic rats than SSC without microgels. Chemically blocking the YAP and Ptgs2 in microgels-delivered SSC partially abolished the enhanced protection on articular tissues and suppression on osteoarthritic macrophages. Moreover, microgel carriers significantly prolonged SSC retention time in vivo without increasing SSC implanting into osteoarthritic joints. Together, our study demonstrated that microgel carriers enhanced SSC reprogramming towards immunomodulatory phenotype to regulate macrophage phenotype transformation for effectively osteoarthritic therapy by promoting YAP protein translocation into nucleus. The study not only complement and perfect the immunological mechanisms of SSC-based therapy at the single-cell level, but also provide new insight for microgel carriers in stem cell-based therapy.
ABSTRACT
BACKGROUND: Atherosclerosis (AS), a chronic inflammatory disease of blood vessels, is a major contributor to cardiovascular disease. Dental pulp stem cells (DPSCs) are capable of exerting immunomodulatory and anti-inflammatory effects by secreting cytokines and exosomes and are widely used to treat autoimmune and inflammation-related diseases. Hepatocyte growth factor (HGF) is a pleiotropic cytokine that plays a key role in many inflammatory and autoimmune diseases. AIM: To modify DPSCs with HGF (DPSC-HGF) and evaluate the therapeutic effect of DPSC-HGF on AS using an apolipoprotein E-knockout (ApoE-/-) mouse model and an in vitro cellular model. METHODS: ApoE-/- mice were fed with a high-fat diet (HFD) for 12 wk and injected with DPSC-HGF or Ad-Null modified DPSCs (DPSC-Null) through tail vein at weeks 4, 7, and 11, respectively, and the therapeutic efficacy and mechanisms were analyzed by histopathology, flow cytometry, lipid and glucose measurements, real-time reverse transcription polymerase chain reaction (RT-PCR), and enzyme-linked immunosorbent assay at the different time points of the experiment. An in vitro inflammatory cell model was established by using RAW264.7 cells and human aortic endothelial cells (HAOECs), and indirect co-cultured with supernatant of DPSC-Null (DPSC-Null-CM) or DPSC-HGF-CM, and the effect and mechanisms were analyzed by flow cytometry, RT-PCR and western blot. Nuclear factor-κB (NF-κB) activators and inhibitors were also used to validate the related signaling pathways. RESULTS: DPSC-Null and DPSC-HGF treatments decreased the area of atherosclerotic plaques and reduced the expression of inflammatory factors, and the percentage of macrophages in the aorta, and DPSC-HGF treatment had more pronounced effects. DPSCs treatment had no effect on serum lipoprotein levels. The FACS results showed that DPSCs treatment reduced the percentages of monocytes, neutrophils, and M1 macrophages in the peripheral blood and spleen. DPSC-Null-CM and DPSC-HGF-CM reduced adhesion molecule expression in tumor necrosis factor-α stimulated HAOECs and regulated M1 polarization and inflammatory factor expression in lipopolysaccharide-induced RAW264.7 cells by inhibiting the NF-κB signaling pathway. CONCLUSION: This study suggested that DPSC-HGF could more effectively ameliorate AS in ApoE-/- mice on a HFD, and could be of greater value in stem cell-based treatments for AS.
ABSTRACT
The WL5 antibody is an anti-colorectal cancer antibody secreted by the WL5 hybridoma clone. Flow cytometric analysis showed that WL5 specifically binds to the HT29 and LS180 colorectal cancer cell lines. Immunohistochemical analysis performed on a tissue microarray demonstrated that the WL5 antibody can be used for the specific and sensitive diagnosis of colorectal carcinoma. Furthermore, WL5 mediated antibody dependent cell-mediated cytotoxicity (ADCC) of tumor cells and exhibited similar antitumor activity to adriamycin (ADM) but avoided the cardiomyopathy and decrease in peripheral white blood cell counts associated with prolonged ADM treatment. The glycoprotein, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), was identified as the target antigen of WL5 through immunoprecipitation and mass spectrometric analyses, which might provide a potential biomarker and therapeutic target for colorectal cancer.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Neoplasm/immunology , Antibodies, Neoplasm/therapeutic use , Antigens, Neoplasm/immunology , Biomarkers, Tumor/immunology , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/therapy , Animals , Antibody-Dependent Cell Cytotoxicity/immunology , Antigens, CD/immunology , Cell Adhesion Molecules/immunology , Cell Line, Tumor , Female , Humans , Hybridomas , Male , Mice , Mice, Inbred BALB CABSTRACT
Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a member of the immunoglobulin super family and has been observed to have two paradoxical functions: tumor suppression and the promotion of tumor invasion. In the present study, we discovered that CEACAM1 functions as an adhesion inhibitor and a migration promoter. The CEACAM1 transfected cells, either 293-CEACAM1 or LOVO/trans-CEACAM1, was proved to have lower adhesion rate. Furthermore, HT29/siRNA-CEACAM1 cells had a higher adhesion rate than HT29 cells. These results indicated that CEACAM1 was an inhibitor of cell-matrix adhesion. Additionally, 293-CEACAM1 LOVO/trans-CEACAM1 cells exhibited better motility in a trans-well migration assay. N-cadherin expression levels were positively correlated with CEACAM1 in 293-CEACAM1, LOVO/trans-CEACAM1 and HT29/siRNA-CEACAM1 cells. When blocked by a GC-4 antibody, the adhesive capacities of 293-CEACAM1 and LOVO/trans-CEACAM1 were recovered and the motilities of them were suppressed, which suggested that CEACAM1 functioned through N-cadherin.
Subject(s)
Antigens, CD/metabolism , Cadherins/biosynthesis , Cell Adhesion Molecules/metabolism , Cell Movement , Neoplasm Metastasis/pathology , Antigens, CD/genetics , Cell Adhesion , Cell Adhesion Molecules/genetics , HEK293 Cells , HT29 Cells , Humans , Models, BiologicalABSTRACT
Background: Several studies have shown that MSCs can significantly improve the survival of sepsis animals. CD146+ mesenchymal stem cells (MSCs) correlate with high therapeutic potency. However, their therapeutic effect on sepsis and detail mechanisms have not been explored. Methods: The effect of CD146±MSCs on differentiation of Treg, Th1, Th17 subsets was evaluated by flow cytometry. The effects of CD146±MSCs on RAW264.7 phagocytosis and LPS-stimulated polarization were studied using a co-culture protocol. Luminex bead array and RNA sequencing were employed to determine the mechanisms of MSCs on LPS-stimulated RAW264.7. The Arg1 protein was detected by Western blot. CD146±MSCs were injected into LPS-induced sepsis mice by tail vein. The therapeutic effect was assessed by organ HE staining, T-cell subsets, cytokine in plasma, peritoneal macrophages, infiltrating monocytes subpopulations. Results: In vitro, CD146+MSCs could significantly increase the proportion of Treg cells. Co-culture with CD146+MSCs increase the phagocytic rate of RAW264.7. CD146+MSCs regulate M2-type macrophages production more rapidly. The transcript profile differences between the CD146+MSCs and CD146-MSCs groups were clustered in arginine metabolism pathways. CD146+MSCs decreased NO production and increased ARG1 expression. CD146+MSCs secreted higher level of IL15,IFNγ, VEGF and lower level of IL1ß, IL8 under LPS stimuli. In vivo, The level of IL10 at 24h and CXCL1, IFNγ at 12h in CD146+MSCs group was the highest. CD146+MSCs treatment enhances the phagocytic capacity of peritoneal macrophages. CD146+MSCs also increases the ratios of CD11b+Ly6Clo reparative monocytes and CD11b+Ly6Chi inflammatory monocytes until 24h. Conclusion: Compared with CD146-MSCs, CD146+MSCs can accelerate the end of the inflammatory response and have robust anti-inflammatory effects, by increasing the Treg cells, promoting macrophage phagocytosis, enhancing the reparative macrophage, secreting more VEGF, etc.
ABSTRACT
Background aims: Spinal cord injury (SCI) is one of the most complex and destructive diseases of the nervous system, which can lead to permanent loss of tactile perception. But existing treatment methods have limited effects. To establish a novel method that may be therapeutic in repairing the injured spinal cord, gene-modified dental pulp stem cells (DPSCs) were injected in situ. Methods: Adenovirus carrying osteopontin (OPN), Insulin-like growth factor 1 (IGF-1) and cailiary-derived neurotrophic factor (CNTF) (Ad-OIC) was constructed. After modified with Ad-OIC, supernatant of DPSC were co-cultured with HT-22 cells and the effect of DPSC-OIC on the HT-22 cells were evaluated via Cell Counting Kit-8 (CCK-8) assay, Real-Time polymerase chain reaction (PCR) analysis, laser confocal microscopy and fluorescence activating cell sorter (FACS). DPSC-OIC were injected in the lesion area of injured spinal cord and the survival time of transplanted cells were measured by bioluminescence imaging system. The recovery of the injured spinal cord was evaluated by behavioral score, radiological evaluation and immunopathological analysis. Results: DPSC-OIC could enhance the proliferation and axon growth of HT-22 cells, and protect HT-22 cells from H2O2 induced apoptosis. The transplanted DPSC-Null or DPSC-OIC could survive for more than two weeks in local injection site. DPSC-OIC treatment could increase Basso-Mouse Scale (BMS) scores, improve Magnetic Resonance Imaging (MRI) manifestation and promote bladder function recovery. Less apoptotic neurons and more proliferative cells were found in the lesion area of DPSC-OIC treated spinal cord. Nestin+ cells and neural stem cell marker (Sox2) were both up-regulated after DPSC-OIC treatment. Additionally, inhibitory extracellular matrix proteoglycan Neural/Glial Antigen 2 (NG2) was down-regulated and axon growth promotive factor fibronectin was up-regulated after both DPSC-Null (DPSCs infected with Ad-Null) and DPSC-OIC treatments. Conclusions: DPSC-OIC could be a novel effective method for treating SCI.
ABSTRACT
In this study, the toxicity effects on circulatory system and respiratory system, and the acute toxicity test of recombinant neorudin (EPR-hirudin, EH) in cynomolgus monkeys were evaluated to provide reference information for clinical studies. Eighteen cynomolgus monkeys were randomly divided into three groups for single intravenous administration of 3, 30 mg/kg EH and normal saline, respectively. The changes of respiratory frequency, respiratory intensity, blood pressure and electrocardiogram before and after administration were recorded. In acute toxicity test, six cynomolgus monkeys were intravenously received EH at a single dose of 171, 257, 385, 578, 867 and 1300 mg/kg respectively. The vital signs, hematology, serum biochemistry, coagulation indexes and electrocardiogram indexes of the animals were determined before administration and on the 7th and 14th day after administration. As the results showed that there were no significant abnormal changes in respiratory frequency, respiratory intensity, blood pressure or electrocardiogram in cynomolgus monkeys after receiving EH at 3 mg/kg and 30 mg/kg, and there was no statistical difference between the treated groups and normal saline group. In the acute toxicity test, no significant abnormalities were observed in vital signs, hematology, serum biochemistry, coagulation indexes and electrocardiogram indexes of six cynomolgus monkeys at day 7 and 14 after EH administration. Furthermore, autopsies of all cynomolgus monkeys showed no abnormalities. The results of toxicokinetics showed that AUClast of the drug increased in proportion to the EH dose in the range of 171-578 mg/kg, and increased in over proportion to the EH dose in the range of 578-1300 mg/kg. The variation of Cmax was basically consistent with AUClast. In a sum, A single intravenous injection of 3 and 30 mg/kg of EH did not affect the circulatory system and respiratory system in cynomolgus monkeys and the maximum tolerated dose of EH in cynomolgus monkey is over 1300 mg/kg (equivalent to 619-1300 times of the proposed clinical equivalent dose).
Subject(s)
Cardiovascular System , Hirudins , Respiratory System , Toxicity Tests, Acute , Animals , Cardiovascular System/drug effects , Dose-Response Relationship, Drug , Hirudins/administration & dosage , Hirudins/toxicity , Infusions, Intravenous , Injections, Intravenous , Macaca fascicularis , Respiratory System/drug effects , Saline Solution/administration & dosageABSTRACT
Background: Whether anticoagulant therapy should be used after spinal-cord injury (SCI) surgery was controversial. The anticoagulation characteristics of a newly developed anticoagulant, recombinant neorudin (EPR-hirudin (EH)), were explored using a rat model of SCI to provide a basis for clinical anticoagulation therapy of SCI. Methods: A rat model of SCI was developed by Allen's method. Then, thrombosis in the inferior vena cava was induced by ligation. The low-bleeding characteristics of EH were explored by investigating dose-response and time-effect relationships, as well as multiple administration of EH, on thrombus formation complicated with SCI. Results: EH inhibited thrombosis in a dose-dependent manner by reducing the wet weight and dry weight of the thrombus. An inhibiting action of EH on thrombosis was most evident in the group given EH 2 h after SCI. After multiple intravenous doses of EH, thrombosis inhibition was improved to that observed with low molecular weight heparin (LMWH) (87% vs 90%). EH administration after SCI neither increased bleeding in the injured spine nor damaged to nerve function. Bleeding duration and activated partial thromboplastin time were increased in the high-dose EH group compared with that in the normal-saline group, but were lower than those in the LMWH group. Conclusion: EH can reduce thrombus formation in a rat model of SCI, and bleeding is decreased significantly compared with that using LMWH. EH may prevent thrombosis after SCI or spinal surgery.
Subject(s)
Spinal Cord Injuries , Venous Thrombosis , Animals , Rats , Heparin, Low-Molecular-Weight , Spinal Cord Injuries/drug therapy , Anticoagulants/pharmacology , Anticoagulants/therapeutic use , Administration, Intravenous , Hirudins , Venous Thrombosis/drug therapy , Venous Thrombosis/prevention & controlABSTRACT
Human mesenchymal stem cells (hMSCs) hold great promise in cell therapy and regenerative medicine. Various preclinical and clinical trials have been carried out to illustrate the therapeutic potential of these cells. However, one major challenge for manufacturing clinical grade hMSCs is the requisition of current good manufacturing practice (cGMP) grade practices in cell isolation, processing, storage, and distribution. Development of non-toxic and animal serum-free preservation medium is critical for storage and distribution of mesenchymal stem cells (MSCs). In this study, we developed a solution formula that could preserve MSCs at 4°C for up to 3 weeks. In the solution, trehalose is a key ingredient for maintaining survival of MSCs. Among the concentrations investigated, 40 mM trehalose showed the best outcome with the viability maintained more than 92.7 ± 1.5% for 7 days. Cells preserved in the solution formula for 3 weeks still remained about 70% viability, and produced results similar to those of freshly harvested hMSCs in terms of growth kinetics, expression profile of cell surface antigens, and differentiation potential. In summary, storage of MSCs in the medium makes it far easier for transporting the cells from processing units to clinical sites.
Subject(s)
Cold Temperature , Cryopreservation/methods , Mesenchymal Stem Cells/cytology , Organ Preservation Solutions/pharmacology , Trehalose/pharmacology , Apoptosis/drug effects , Cell Culture Techniques/methods , Cell Survival/drug effects , HumansABSTRACT
BACKGROUND AIMS: Mesenchymal stromal cells (MSC) have been shown to be a promising candidate for tissue regeneration and cancer therapy. However, their therapeutic potential against chemotherapy-induced side-effects remains unclear. METHODS: We treated murine Lewis lung carcinoma (LLC) and xenograft human colon tumors with adriamycin (ADM) for 3 consecutive days followed by one intravenous (i.v.) injection of human umbilical cord (hUC) MSC for several cycles. RESULTS: MSC treatment mitigated ADM-induced cardiomyopathy, reduced the extent of ADM-induced apoptosis in intestinal crypts, suppressed body weight loss in mice treated with ADM and increased the survival rate of mice treated with a lethal dose of ADM. The examination of hematologic parameters indicated a moderate recovery in MSC-injected mice. Systemic administration of MSC did not increase the growth of murine LLC cells and human colon carcinoma in vivo while it strongly inhibited the lung metastases of LLC cells. CONCLUSIONS: We evaluated the prophylactic and therapeutic action of hUC MSC on the chemotherapy agent ADM-induced side-effects in two different tumor models. Our observations suggest that MSC can be used as auxiliary means in chemotherapy for certain tumor types.
Subject(s)
Carcinoma, Lewis Lung/therapy , Colonic Neoplasms/therapy , Mesenchymal Stem Cells/cytology , Umbilical Cord/cytology , Animals , Antibiotics, Antineoplastic/therapeutic use , Carcinoma, Lewis Lung/drug therapy , Colonic Neoplasms/drug therapy , Doxorubicin/therapeutic use , Humans , Mesenchymal Stem Cells/metabolism , Mice , Xenograft Model Antitumor AssaysABSTRACT
The epidermal growth factor receptor (EGFR) family belongs to type I receptor tyrosine kinases. Overexpression or mutation of EGFR/ErbB1 gene has been detected in a large number of human solid tumours. According to some previous report, this gene is not expressed in hematological malignancies. However, two recent clinical case reports showed that erlotinib caused complete remission of acute myeloid leukaemia (AML)-M1 in patients who had both AML-M1 and non-small-cell lung cancer. These results are supported by preclinical studies in which EGFR tyrosine kinase inhibitors have anti-proliferative effects on AML. These findings prompted us to determine whether EGFR is expressed in human AML, through a large-scale screening of both leukaemic cell lines and clinical samples. Our results show that EGFR is expressed by about 33% of human AML (containing M1 to M7 subtypes) and by some human leukaemia cell lines (K562, MEG-01, CEM and SKO-007). Its expression is not limited to certain AML types but has been detected in many leukaemic cells. In addition, EGFR expression was intimately associated with the poor clinical outcomes. Finally, we find that only EGFR-positive leukaemic cells respond to antibody-dependent cellular cytotoxicity of cetuximab, the monoclonal antibodies against EGFR.
Subject(s)
ErbB Receptors/analysis , Leukemia, Myeloid, Acute/mortality , Adolescent , Adult , Aged , Aged, 80 and over , Antibody-Dependent Cell Cytotoxicity , Apoptosis , Cell Line, Tumor , Cell Movement , Female , Humans , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Male , Middle Aged , Prognosis , Proportional Hazards ModelsABSTRACT
Lipid rafts mediate several survival signals in the development of chronic myeloid leukemia (CML). Methyl-ß-cyclodextrin (MßCD) is an inhibitor specifically designed to disrupt lipid rafts in cells by depleting the cholesterol component. We hypothesize that treatment of CML cells with MßCD and imatinib could reduce imatinib resistance. Apoptotic and autophagic cell death was assayed using annexin V-propidium iodide double staining, immunoblotting, and immunocytochemistry. We next investigated whether MßCD could enhance the cytotoxicity of imatinib in imatinib-sensitive and imatinib-resistant K562 cells. Extracellular signal-regulated kinase/sphingosine kinase 1 signaling downstream of lipid raft-activated signaling pathways was significantly inhibited by treatment of cells with a combination of MßCD and imatinib compared with treatment with either agent alone. MßCD induces programmed cell death in CML cells, and its antileukemia action is synergistic with that of imatinib.