Inhibitory Effect of Centipede Scolopendra Extract on Gallbladder Carcinoma via Up-regulation of PUMA

Abstract Background: To observe the effect of centipede scolopendra extract on gallbladder carcinoma (GBC) and further investigate its underlying mechanism.Methods: The GBC cell line GBC-SD was purchased and cultured. Small interfering RNA (siRNA) was constructed. The mRNA expression levels of PUMA were measured by reverse transcription-quantitative PCR (RT-qPCR) and protein expression levels of p53, PUMA, Bax and Bcl-2 were measured by western blotting respectively. Viability and proliferation were detected using MTT and colony formation assays respectively. The apoptosis rate was determined by Annexin-V-FITC/PI double staining. Results: The GBC-SD cell line was successfully obtained and cultured. MTT and colony formation assays demonstrated that the viability and proliferation of GBC-SD cells could be markedly inhibited by centipede scolopendra extract in a concentration-dependent manner in the limited concentration range. Following pretreatment with centipede scolopendra extract, RT-qPCR demonstrated that the expression levels of PUMA were markedly increased in the GBC-SD cells, and western blotting showed that the high expression of PUMA was accompanied by upregulation of Bax and downregulation of p53 and Bcl-2 in the GBC-SD cells. However, this effect has proved hard to reproduce after PUMA-siRNA. Flow cytometry revealed that the apoptosis rate was 9.8±2.2%, 25.3±3.6%, 10.6±2.0%, and 13.5±2.4% in control group, centipede scolopendra extract group, PUMA-siRNA group, and centipede scolopendra extract combined with PUMA-siRNA group respectively. Conclusions: Centipede scolopendra extract could induce the apoptosis of GBC-SD cells by promoting the PUMA-Bax signaling pathway. It could serve as a potential novel therapy for GBC in clinical practice.

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Sfrps Family Invovled in Growth Inhibition of K562 Cells Induced by Artesunate

Blood ◽

10.1182/blood.v118.21.5015.5015 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5015-5015

Author(s):

Wang Ying ◽

Guo xiao Nan ◽

Zhang xue Jun ◽

Ren jin Hai ◽

Qiao shu Kai ◽

...

Keyword(s):

Growth Inhibition ◽

Research Funding ◽

Wnt Pathway ◽

K562 Cells ◽

Hebei Province ◽

Control Group ◽

Dependent Manner ◽

Inhibition Rate ◽

Concentration Dependent Manner ◽

Expression Levels

Abstract Abstract 5015 Introduction: Studies have shown that abnormal activation of wnt signaling pathway is closely related to tumor genesis. Cell surface coil protein (frizzled, fzd) is a specific receptor for wnt pathway to activate its downstream signaling through β-catenin to stimulate the growth of a range of tumor cells. Secreted frizzled related proteins (sfrps) are the main antagonists of wnt pathway. sfrps can inhibit the function of wnt pathway via competing with fzd receptor. Recent studies have found that sfrps family memberes expressed at very low levels in a variety of tumor cells,which was closely related to the methylation of sfrps gene promoters. DNA methyltransferase (dnmts) are the key enzymes involved in DNA methylation, which can promote the methylation of tumor suppressor genes' promoters and inhibit transcription of these genes, and induce tumor genesis. Artesunate, a semi-synthetic derivative of artemisinin, is very effective in antimalaria. Recent studies have shown that artesunate has anti-tumor functions. However, the effect of artesunate on the expression of sfrps- and dnmts- mRNAs and β-catenin protein are not clear. To evaluate the growth inhibition effect of Artesunate (at final concentrations of 0μg/ml, 4μg/ml, 10μg/ml, 20μg/ml and 40μg/ml) on K562 cells and to investigate its potential mechanism by detecting the expression levels of sfrps- and dnmts- mRNAs and β-catenin protein in K562 cells treated with artesunate. Methods: Cell growth inhibition rate and cell cycle distribution of K562 cells induced by artesunate treatment were measured by MTT assay and flow cytometry, respectively. The mRNA expression levels of sfrp1, sfrp2, sfrp4 and dnmt1, dnmt3a, dnmt3b in K562 cells which had been treated with or without artesunate for 48 hours were evaluated with reverse transcription-polymerase chain reaction (RT-PCR). Protein expression level of β-catenin in K562 cells were detected by Western blot. Results: Artesunate treatment significantly induced growth inhibition of K562 cells in a concentration-dependent manner after the cells were treated with artesunate for 48 hours(p < 0.05). The inhibition rate of 4,10,20 and 40(μg/ml)artesunate exposure were 54.29%, 58.03%, 69.33% and 77.98% respectively. Flow cytometry analysis showed that K562 cells were arrested at G0/Gl and G2/M phase in concentration-dependent manner after 48 hours exposure of artesunate (p < 0.05). After treated with artesunate at the final concentrations of 0, 4, 10, 20 and 40μg/ml, the relative expression levels of sfrp1, sfrp2 and sfrp4 mRNA in K562 cells increased, while the expression levels of dnmtl, dnmt3a, dnmt3b mRNA decreased significantly compared with the control group. Results from Western blot showed that β-catenin protein levels decreased in a concentration-dependent manner, when compared with that of the control group (P<0.05). Conclusion: Results from the present study indicated that artesunate could inhibite the mRNA expression of dnmts family and minimize the methylation of sfrps gene promoter. Therefore, sfrps could inhibit the function of wnt pathway through competing with fzd receptors. Meanwhile, artesunate could decrease the expression of β-catenin protein in K562 cells, and could further inhibit the function of wnt pathway. Therefore, data from the present experiment provides a new theoretical basis for clinical application of artesunate in leukemia treatment. Disclosures: Ying: Nature science foundation of Hebei Province: Research Funding. Nan:Nature science foundation of Hebei Province: Research Funding. Jun:Nature science foundation of Hebei Province: Research Funding. Hai:Nature science foundation of Hebei Province: Research Funding. Kai:Nature science foundation of Hebei Province: Research Funding. Xu:Nature science foundation of Hebei Province: Research Funding. Pan:Nature science foundation of Hebei Province: Research Funding.

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Platelet-Derived Microparticles Induce Angiogenesis In Vitro and In Vivo.

Blood ◽

10.1182/blood.v110.11.3901.3901 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3901-3901

Author(s):

Bao-An Chen ◽

Yue-Jiao Zhong ◽

Cheng-Yin Huang ◽

Feng Gao ◽

Jian Chen ◽

...

Keyword(s):

Cell Line ◽

Umbilical Vein ◽

Chorioallantoic Membrane ◽

Proliferation Rate ◽

Control Group ◽

Dependent Manner ◽

Human Umbilical Vein ◽

Apoptosis Rate

Abstract Objective: An attempt was made to investigate the effect of platelet-derived microparticles (PMPs) on the angiogenesis. Methods: Thrombin was adopted to activate the platelets to release PMPs. Flow cytometry(FCM)was adopted to evaluate the efficiencies of different concentrations of thrombin to produce PMPs and BCA was adopted to evaluate the content of PMPs. By the carrier of human umbilical vein cell line ECV-304 cultivated in vitro, investigate the effect of PMPs on the proliferation and apoptosis of human umbilical vein endothelial cells using MTT and FCM. PMPs were put into CAM and observe the effects of PMPs on angiogenesis in Chick embryo chorioallantoic membrane (CAM). Results: The efficiencies of PMPs activated by 1.0U/ml thrombin were 50.1%; PMPs induced proliferation of human umbilical vein cell line ECV-304 in a dose dependent manner. At the concentration of 40ug/ml PMPs, the proliferation rate of human umbilical vein cell line ECV-304 was as 1.8±0.3 times as control and there was no difference with the group of vascular endothelial growth factor (VEGF),which the proliferation rate was 1.9±0.5 times vs. control, p > 0.05;PMPs inhibited human umbilical vein cell line ECV-304 apoptosis. Compared with control group (apoptosis rate 9.4%±0.5%), apoptosis rate of PMPs (40μg/ml) is 3.9%±0.4%, which was significantly reduced, p<0.05. The addition of VEGF (10μl/ml) did not successfully prevented apoptosis of human umbilical vein cell line ECV-304 (apoptosis rate 8.0%±0.8%);After 72 h of incubation, showing an implant of PMPs by allantoic vessels developing radially towards it (implant) in a ’spoked-wheel’ pattern, at the concentration of 80μg/ml PMPs, number of vessel ramification is 112.5±11.31 and vessel area/CAM area is (6.19±1.29)%, compared with the VEGF(p>0.05). But there are not localized allantoic vessels developing in the NS control group(P<0.05). Conclusion: 1.0U/ml thrombin activated platelet could get the best efficiency of PMPs, which could stimulate proliferation of human umbilical vein cell line ECV-304 and inhibit its apoptosis and PMPs have certain promotive effect on the formation of capillary in chick chorioallantoic membranes.

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Biological and Molecular Evidences for the Antitumor and Antioxidant Potential of Quercetin In Vitro

Journal of Biobased Materials and Bioenergy ◽

10.1166/jbmb.2020.1984499 ◽

2020 ◽

Vol 14 (4) ◽

pp. 499-505

Author(s):

Jia Zhou ◽

Yan Xu ◽

He Lin ◽

Guangfu Lv ◽

Ying Chen ◽

...

Keyword(s):

Oxidative Damage ◽

Hela Cell ◽

Western Blotting ◽

Hela Cells ◽

Caspase 3 ◽

Umbilical Vein ◽

Expression Level ◽

Dependent Manner ◽

Expression Levels ◽

Apoptosis Rate

To investigate the protective mechanisms of quercetin in promoting HeLa cell apoptosis and protecting human umbilical vein endothelial cells (HUVECs) from oxidative damage, in this study, MTT test was preformed to observe the inhibition rate of quercetin on HeLa cell proliferation (50.0, 75.0 and 100.0 mol/L), and the apoptosis rate was measured with flow cytometry. Western blotting was used to detect the protein expression levels in signaling pathways in quercetin-treated HeLa cells and HUVECs. MTT assay results showed that quercetin could inhibit (P < 0.001) the proliferation of HeLa cells in a time-concentration dependent manner. Apoptosis results showed that quercetin can increase the early and late apoptosis rate (P < 0.001). Western blotting revealed that quercetin can down-regulate BCL-2 expression level (P < 0.05), up-regulate BAX and CASPASE-3 expression level (P < 0.05) in HeLa cells. Flow detection results showed that the number of HUVECs labeled with CD105/CD62E and CD31/CD34 in the oxidation group decreased significantly (P < 0.01), increased significantly (P < 0.001) in the drug group. Quercetin can decrease the expression levels of p16, p53 and p21 significantly (P < 0.05). In conclusion, quercetin can promote the apoptosis of HeLa cell by regulating the expression level of CASPASE-3, BCL-2 and BAX; and protect HUVECs against oxidative damage and inhibit the apoptosis by down-regulating p16, p53 and p21.

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Decreased Endometrial IL-10 Impairs Endometrial Receptivity by Downregulating HOXA10 Expression in Women with Adenomyosis

BioMed Research International ◽

10.1155/2018/2549789 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 5

Author(s):

Junxia Wang ◽

Chenyang Huang ◽

Ruiwei Jiang ◽

Yali Du ◽

Jianjun Zhou ◽

...

Keyword(s):

Western Blotting ◽

Embryo Implantation ◽

Control Group ◽

Bewo Cell ◽

Dependent Manner ◽

Expression Levels ◽

Positive Correlation ◽

Protein Levels ◽

Stat3 Phosphorylation ◽

Ishikawa Cells

Objective. The aim of this study was to investigate the potential role of IL-10 in regulating the receptivity marker HOXA10 in the endometrium of women with adenomyosis. Methods. The expression levels of IL-10, HOXA-10, STAT3, and p-STAT3 in the endometrium of women with adenomyosis and controls were examined by means of western blotting and immunohistochemistry. The expression of the HOXA10 protein in Ishikawa cells treated with rIL-10 was examined by western blotting. The attachment rate of BeWo cell spheroids to Ishikawa cells treated with rIL-10 was expressed as a percentage of the total number of spheroids. Results. The expression levels of HOXA10 and IL-10 in the adenomyosis group were significantly lower than those in the control group, and there was a positive correlation between HOXA10 and IL-10 protein levels in all the women examined. rIL-10 increased HOXA10 expression in a concentration- and time-dependent manner by inducing the phosphorylation of STAT3 in Ishikawa cells. Treatment with rIL-10 promoted the attachment of BeWo spheroids to Ishikawa cells, which was reversed by the inhibition of STAT3 phosphorylation. The expression of p-STAT3 in the adenomyosis group was significantly lower than that in the control group, and there was a positive correlation between IL-10 and p-STAT3 protein levels in all the women examined. Conclusions. Both IL-10 and HOXA10 levels in the endometrium are significantly reduced in women with adenomyosis compared with those in control women. The phosphorylation of STAT3 has been proven to be a critical mediator between IL-10 and HOXA10, which may play critical roles in embryo implantation.

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Artesunate Decreases β-Catenin Expression, Cell Proliferation and Apoptosis Resistance in the MG-63 Human Osteosarcoma Cell Line

Cellular Physiology and Biochemistry ◽

10.1159/000484118 ◽

2017 ◽

Vol 43 (5) ◽

pp. 1939-1949 ◽

Cited By ~ 4

Author(s):

Peng Chen ◽

Wan-Li Gu ◽

Ming-Zhi Gong ◽

Jun Wang ◽

Dong-Qing Li

Keyword(s):

Cell Proliferation ◽

Cell Line ◽

Western Blotting ◽

Mtt Assay ◽

Control Group ◽

Osteosarcoma Cell ◽

Human Osteosarcoma Cell Line ◽

Expression Levels ◽

Human Osteosarcoma ◽

Human Osteosarcoma Cell

Background: This study aims to determine the effects of artesunate on proliferation, apoptosis and β-catenin expression in the human osteosarcoma cell line MG-63. Methods: MG-63 cells in the logarithmic growth phase were collected and cultured with different concentrations of artesunate (12.5 µg/mL, 25 µg/mL and 50 µg/mL) for 24 h, 48 h and 72 h. The total number of MG-63 cells and the morphological changes were observed under an inverted microscope. The MTT assay was adopted to test the inhibition rate (IR) of cell growth. The apoptosis rate was detected using annexin V/propidium iodide (PI) staining. Cell cycle distribution was identified by flow cytometry (FCM), and the expression levels of β-catenin, cyclins and cyclin dependent kinases (CDKs) were measured using Western blotting. Results: The results of the MTT assay indicated that artesunate could remarkably inhibit MG-63 cell proliferation compared with the rates in the untreated control group (0 µg/mL artesunate), and the inhibitory effect was dose-dependent. The apoptosis rate of MG-63 cells was elevated as the concentration of artesunate increased, and all the rates were significantly higher than that in the control group. Additionally, as the artesunate concentration increased, the proportion of MG-63 cells in G0/G1 phase gradually declined whereas that of cells in the G2/M and S phases increased. Western blotting confirmed that a higher concentration of artesunate reduced the expression levels of β-catenin, cyclin A, cyclin D1 and CDK1 and increased the expression levels of cyclin B1; however, artesunate had no impact on CDK2 expression in MG-63 cells. Conclusion: These results demonstrated that artesunate can inhibit β-catenin expression and cell proliferation as well as promote cell apoptosis in MG-63 cells, which indicates that artesunate may serve as a promising drug in the clinical treatment of osteosarcoma.

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Curcumin Synergistically Enhances the Cytotoxicity of Arsenic Trioxide in U266 Cells by Increasing Arsenic Uptake

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2021/3083041 ◽

2021 ◽

Vol 2021 ◽

pp. 1-9

Author(s):

Dingding Han ◽

Guibo Ma ◽

Yujuan Gao ◽

Yanhua Su

Keyword(s):

Arsenic Trioxide ◽

Western Blotting ◽

Caspase 3 ◽

Control Group ◽

Arsenic Content ◽

Dependent Manner ◽

Arsenic Uptake ◽

Expression Levels ◽

Protein Levels ◽

Drug Concentrations

Despite the constant emergence of new methods for the treatment of multiple myeloma (MM), relapse and drug resistance still exist, especially in MM with p53 mutations. Arsenic trioxide (ATO) can be used in MM treatment, but this single drug has poor effectiveness and also side effects. Curcumin is a safe and effective compound that can enhance the anticancer effects of many drugs. Previous studies have suggested that tumor cell sensitivity to ATO is related to the intracellular arsenic content, and aquaporin 9 (AQP9) is the key factor that determines intracellular arsenic content. This study aimed to explore whether curcumin can increase ATO cytotoxicity in MM and whether the mechanism is related to the regulation of intracellular arsenic content. U266 was treated with ATO, curcumin, and their combination, and cell proliferation, apoptosis, and intracellular arsenic content were detected by CCK-8 assay, flow cytometry, and HPLC-ICP-MS, respectively. AQP9 mRNA and protein levels were detected by qPCR and western blotting. The levels of Mcl-1, Bcl-2, Bax, caspase-3, and cleaved caspase-3 protein were detected by western blotting. ATO-induced cytotoxicity to U266 occurred in a time- and dose-dependent manner, but the therapeutic efficacy at low drug concentrations was modest. The arsenic content in U266 was lower than that in NB4, and the arsenic uptake by U266 was concentration-dependent. The expression levels of AQP9 mRNA and AQP9 protein in U266 were lower than those in NB4. Curcumin significantly enhanced the lethality of ATO to U266. The arsenic content in U266 in the combined drug group increased significantly compared with ATO treatment alone. After curcumin treatment, the AQP9 mRNA and AQP9 protein expression levels in U266 also increased. Compared with the control group, the expression of antiapoptotic proteins Mcl-1 and Bcl-2 decreased, the expression of proapoptotic protein Bax increased, the ratio of Bax/Bcl-2 increased, and the expression of caspase-3 decreased and cleaved caspase-3 increased in the combined drug groups. Curcumin can enhance the killing effects of ATO on U266 by increasing the intracellular arsenic content, which may be related to the upregulation of AQP9 expression. The combination of these two drugs is expected to be a potential clinical treatment for MM.

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Sequestration of erythrocytes infected with Plasmodium berghei ANKA in BALB/c mice treated with goat bile

Qanun Medika - Medical Journal Faculty of Medicine Muhammadiyah Surabaya ◽

10.30651/jqm.v4i2.3539 ◽

2020 ◽

Vol 4 (2) ◽

pp. 179

Author(s):

Kartika Arum Wardani ◽

Kholida Nur Aini ◽

Heny Arwati ◽

Willy Sandhika

Keyword(s):

Plasmodium Berghei ◽

Antimalarial Activity ◽

Negative Control ◽

Control Group ◽

Dependent Manner ◽

Plasmodium Berghei Anka ◽

Concentration Dependent Manner ◽

Control Group Design ◽

Bile Concentration

Abstract Sequestration of Plasmodium berghei ANKA-infected erythrocytes occurs in BALB/c mice as characteristic of Plasmodium falciparum infection in humans. Animals’ bile has been widely used for centuries in Traditional Chinese Medicine. Goat bile has been used in healing infectious and non-infectious diseases; however, no report on the use of goat bile against malaria infection and sequestration. The purpose of this study was to analyze the correlation between parasitemia and sequestration in the liver of P.berghei ANKA-infected BALB/c mice treated with goat bile. This research was an in vivo experimental study using the post-test control group design. The male BALB/c mice aged ± 6 weeks, body weight 20-25 g were used. The mice were divided into five groups where Group 1-3 were mice treated with goat bile 25%, 50%, and 100%, respectively. Group 4-5 were negative (sterile water) and positive controls (DHP). Parasitemia was observed daily from each mouse and the number of sequestered infected erythrocytes on the endothelium of sinusoids. The data were analyzed using t independent test. Antimalarial activity of goat bile was shown by the lower parasitemia in goat bile-treated mice compared with the negative control. The average number of sequestration was goat bile concentration-dependent manner. The higher the concentration, the lower the number of sequestration. Sequestration was correlated with parasitemia (p=0,0001). Sequestration of P.berghei ANKA-infected erythrocytes correlated with parasitemia, and was goat bile concentration-dependent manner. Keywords: Malaria, parasitemia, sequestration, goat bileCorrespondence: [email protected]

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Relaxant effect of quercetin on rabbit isolated bladder smooth muscles contractions

Journal of Herbmed Pharmacology ◽

10.34172/jhp.2021.05 ◽

2020 ◽

Vol 10 (1) ◽

pp. 61-67

Author(s):

Hassan Sadraei ◽

Sabihe Tabesh

Keyword(s):

Smooth Muscles ◽

Electrical Field Stimulation ◽

Negative Control ◽

Flavonoid Compound ◽

Drug Candidate ◽

Control Group ◽

Organ Bath ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Relaxant Effect

Introduction: Quercetin is a flavonoid compound found in many medicinal plants. Antispasmodic effect of quercetin has been reported in ileum and uterus smooth muscles but not in bladder. Therefore, the objective of this research was to investigate relaxant effect of quercetin in rabbit isolated bladder. Methods: Male rabbit was asphyxiated with carbon dioxide and then sacrificed. The whole bladder was dissected out and placed in oxygenated Tyrode’s solution. Isolated bladder was cut into longitudinal strips and placed in an organ bath for contraction studies. Contractions were induced with KCl (20mM), acetylcholine (5μM) and electrical field stimulation (EFS). Full inhibitory concentration–response curve was constructed for quercetin following addition of above spasmogens. Quercetin was added into the organ bath with 2 fold increments in concentration until maximum response was achieved. Nifedipine was used as positive control group and equivalent volume of quercetin vehicle (water + DMSO) was used as negative control group.Results: Quercetin (4 μg/mL to 640 μg/mL) in a concentration dependent manner inhibited isolated bladder strips contracted by KCl (IC50=159±25 μg/mL), acetylcholine (IC50=43±9.1 μg/mL) and EFS (IC50=38±9.3 μg/mL). In the highest used concentration, quercetin completely removed contractile responses to KCl, acetylcholine and electrical filed stimulation (EFS). Nifedipine totally inhibited KCl response (IC50=115±36 ng/mL) but only partially inhibited acetylcholine and EFS responses. Conclusion: These results confirm the relaxant effect of quercetin on rabbit bladder and if similar effects are seen in human studies, then quercetin would be a suitable drug candidate to be investigated for bladder incontinence.

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ATI-2341 Trifluoroacetic Acid (TFA) Promotes Menstrual Blood-Derived Mesenchymal Stem Cell Recruitment and Enhances Uterine Repair Through Gi Pathway in Asherman’s Syndrome

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2022.2936 ◽

2022 ◽

Vol 12 (3) ◽

pp. 506-513

Author(s):

Ying Lv ◽

Liyan Ye ◽

Xiujuan Zheng

Keyword(s):

Menstrual Blood ◽

Control Group ◽

Dependent Manner ◽

Asherman’S Syndrome ◽

Expression Levels ◽

Endometrial Cells ◽

Injury Model ◽

Recruitment Effect ◽

Dose Dependent

This study aimed to explore the role of ATI-2341 in Asherman’s syndrome and its impact on menstrual blood-derived mesenchymal stem cells (MenSCs). Following establishment of endometrial injury model, MenSCs were extracted from rats and cultured. They were treated with ATI-2341 TFA at different concentrations (10 ng/mL, 50 ng/mL, 100 ng/mL) and MenSCs treated without ATI-2341 TFA were taken as controls. Flow cytometry was conducted to detect the cell cycle. MTT was carried out to evaluate proliferation of endometrial cells. The expression levels of MMP-9, TIMP-1, CK, and VIM were determined with staining used to reflect morphology of endometrium. Administration with ATI-2341 TFA resulted in decreased expression of MMP-9 and increased expression of TIMP-1 in a dose-dependent manner. Of note, the increase of ATI-2341 TFA concentration was accompanied with elevated cell proliferation rate, increased number of glands in the endometrium, and decreased fibrosis area. As treated with 100 ng/mL ATI-2341 TFA, the cells exhibited more glands than that under other concentrations with uniformly arranged glands and lowest expression levels of CK and VIM, control group had plenty of blue-stained collagen fibers in the intima and least amount of glands. ATI-2341 TFA 100 ng/mL induced endometrial epithelial recruitment effect on MenSCs and promoted endometrial repair more significantly than Gi-3 pathway agonists. Collectively, ATI-2341 TFA enhances MenSC recruitment and facilitates endometrial epithelial cells proliferation and the repair of uterine damage in Asherman’s syndrome through Gi pathway. These findings provide a\ novel insight into the MenSC-based treatment against Asherman’s syndrome and deserve further investigation.

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Growth and differentiation of a human megakaryoblastic cell line, CMK

Blood ◽

10.1182/blood.v74.1.42.42 ◽

1989 ◽

Vol 74 (1) ◽

pp. 42-48 ◽

Cited By ~ 74

Author(s):

N Komatsu ◽

T Suda ◽

M Moroi ◽

N Tokuyama ◽

Y Sakata ◽

...

Keyword(s):

Cell Line ◽

Colony Formation ◽

Culture System ◽

Dependent Manner ◽

Colony Stimulating Factor ◽

Gm Csf ◽

Macrophage Colony Stimulating Factor ◽

Hematopoietic Factors ◽

Macrophage Colony ◽

Stimulating Factor

Abstract Recently, a human megakaryoblastic cell line, CMK, was established from the peripheral blood of a megakaryoblastic leukemia patient with Down syndrome. Using this cell line, we studied the proliferation and differentiation of megakaryocytic cells in the presence of highly purified human hematopoietic factors and phorbol 12-myristate-13- acetate (PMA). In a methylcellulose culture system, interleukin-3 (IL- 3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) facilitated colony formation by CMK cells in a dose-dependent manner. The maximum stimulating doses of these factors were 10 and 200 U/mL, respectively. These concentrations were comparable to those that stimulate activity in normal hematopoietic cells. In contrast, granulocyte-colony stimulating factor (G-CSF), macrophage-colony stimulating factor (M-CSF), and erythropoietin (EPO) had no effects on the colony formation of CMK cells. In a liquid culture system, 20% of the CMK cells expressed glycoprotein IIb/IIIa (GPIIb/IIIa) antigen without hematopoietic factors, whereas 40% of the cells expressed GPIIb/IIIa with the addition of IL-3 and GM-CSF. EPO also slightly enhanced expression of GPIIb/IIIa. On the other hand, PMA inhibited growth of CMK cells and induced most of them to express the GPIIb/IIIa antigen. Furthermore, PMA induced CMK cells to produce growth activity toward new inocula of CMK cells. This growth factor (GF) contained colony-stimulating activity (CSA) in normal bone marrow (BM) cells. The activity was believed to be attributable mainly to GM-CSF, since 64% of this activity was neutralized by anti-GM-CSF antibodies and a transcript of GM-CSF was detected in mRNA from PMA-treated CMK cells by Northern blot analysis. These observations suggest that GM-CSF, as well as IL-3, should play an important role in megakaryocytopoiesis.

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