scholarly journals Elucidating the Role of the Maternal Embryonic Leucine Zipper Kinase in Adrenocortical Carcinoma

Endocrinology ◽  
2018 ◽  
Vol 159 (7) ◽  
pp. 2532-2544 ◽  
Author(s):  
Katja Kiseljak-Vassiliades ◽  
Yu Zhang ◽  
Adwitiya Kar ◽  
Raud Razzaghi ◽  
Mei Xu ◽  
...  
Keyword(s):  
Adrenocortical Carcinoma ◽  
Colony Formation ◽  
Leucine Zipper ◽  
Relative Sensitivity ◽  
Soft Agar ◽  
Growth And Survival ◽  
Expression Of Genes ◽  
H295r Cells ◽  
Antitumorigenic Effect

Abstract Adrenocortical carcinoma (ACC) is an aggressive cancer with a 5-year survival rate <35%. Mortality remains high due to lack of targeted therapies. Using bioinformatic analyses, we identified maternal embryonic leucine zipper kinase (MELK) as 4.1-fold overexpressed in ACC compared with normal adrenal samples. High MELK expression in human tumors correlated with shorter survival and with increased expression of genes involved in cell division and growth. We investigated the functional effects of MELK inhibition using newly developed ACC cell lines with variable MELK expression, CU-ACC1 and CU-ACC2, compared with H295R cells. In vitro treatment with the MELK inhibitor, OTSSP167, resulted in a dose-dependent decrease in rates of cell proliferation, colony formation, and cell survival, with relative sensitivity of each ACC cell line based upon the level of MELK overexpression. To confirm a MELK-specific antitumorigenic effect, MELK was inhibited in H295R cells via multiple short hairpin RNAs. MELK silencing resulted in 1.9-fold decrease in proliferation, and 3- to 10-fold decrease in colony formation in soft agar and clonogenicity assays, respectively. In addition, although MELK silencing had no effect on survival in normoxia, exposure to a hypoxia resulted in a sixfold and eightfold increase in apoptosis as assessed by caspase-3 activation and TUNEL, respectively. Together these data suggest that MELK is a modulator of tumor cell growth and survival in a hypoxic microenvironment in adrenal cancer cells and support future investigation of its role as a therapeutic kinase target in patients with ACC.

CIAPIN1 is a potential target for apoptosis of multiple myeloma

2019 ◽  
Vol 9 (9) ◽  
pp. 1106-1111
Author(s):  
Xiao-Bo Wang ◽  
Le-Ping Yan ◽  
Li-Hua Yuan ◽  
Bo Lu ◽  
Dong-Jun Lin ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Xenograft Model ◽  
Soft Agar ◽  
Normal Human ◽  
Related Proteins

This study firstly aimed to reveal the gene expression differences of CIAPIN1 between myelomas cells from bone marrow cells of multiple myeloma patients and normal human, and subsequently investigate the regulation role of this gene on tumorigenicity ability of multiple myeloma (MM) cell line U266 via in vitro colony formation and in vivo xenograft studies. RT-PCR results obtained from 18 MM patients and 10 health people showed that the expression of CIAPIN1 gene was 4 times higher in normal human compared to MM patients. Besides, CIAPIN1 siRNA (si-CIAPIN1) transfected U266 cells presented higher proliferation ratio and superior colony forming ability than U266 cells and U266 cells transfected with non-coding siRNA (controls) evaluated by CCK8 test and soft agar colony formation assay, respectively. In a mice MM xenograft model, the si-CIAPIN1 transfected U266 cells induced the biggest tumor compared to the controls. Furthermore, CIAPIN1 overexpressed U266 cells were developed and compared with the si-CIAPIN1 transfected U266 cells to study the role of CIAPIN1 in the production of apoptosis related proteins in U266 cells. Results indicated that CIAPIN1 facilitated apoptosis promoting proteins expression in U266 cells, such as upregulation of BAX, BAK, Bcl-xs and BIM, and downregulation of p38, PKC, Bcl-2 and Bcl-xl proteins. Therefore, CIAPIN1 can be a potential suppression target gene in multiple myeloma.

scholarly journals Isolation, Culture and Identification of Choriocarcinoma Stem-Like Cells from the Human Choriocarcinoma Cell-Line JEG-3

2016 ◽  
Vol 39 (4) ◽  
pp. 1421-1432 ◽  
Author(s):  
Jingting Cai ◽  
Tianfang Peng ◽  
Jing Wang ◽  
Jingli Zhang ◽  
Hui Hu ◽  
...  
Keyword(s):  
Cell Line ◽  
Colony Formation ◽  
Soft Agar ◽  
Serum Free ◽  
Significant Difference ◽  
Free Media ◽  
Proliferative Ability ◽  
Choriocarcinoma Cell Line

Background/Aims: Cancer stem cells (CSCs) exhibit enhanced proliferative capacity and resistance to chemotherapy; however, choriocarcinoma CSCs have not yet been reported. In this study the human choriocarcinoma cell line JEG-3 was cultured in serum free media, and the characteristics of suspension and parental adherent JEG-3 cells were compared. Methods: Cell proliferation, colony-formation, soft agar clonogenicity, and transwell invasion assays were performed in vitro, and tumor xenografts in BALB/c nude mice were used to evaluate stem cell properties. Results: In serum-supplemented medium (SSM), JEG-3 cells were 4.51 ± 1.71% CD44+, 7.67 ± 2.67% CD133+, and 13.85 ± 2.95% ABCG2+. In serum-free medium (SFM), the expression of these markers increased to 53.08 ± 3.15%, 47.40 ± 2.67%, and 78.70 ± 7.16%, respectively. Moreover, suspension JEG-3 cells exhibited enhanced colony-formation capability as well as invasive and proliferative ability in vitro, alongside enhanced tumorigenic properties in vivo. Suspension JEG-3 cells also exhibited resistance to the chemotherapeutic drugs methotrexate, fluorouracil and etoposide. When seeded in serum supplemented medium, suspension JEG-3 cells readopted an adherent phenotype and continued to differentiate with no significant difference in the morphology between suspension and parent cells. Conclusion: In this study, choriocarcinoma stem-like cells (CSLCs) were isolated from the human choriocarcinoma JEG-3 cell line by SFM culture and characterized.

scholarly journals Some Factors Influencing Granulocytic Colony Formation In Vitro by Human White Blood Cells

Blood ◽  
1974 ◽  
Vol 44 (4) ◽  
pp. 511-515 ◽  
Author(s):  
Wolfgang Heit ◽  
Peter Kern ◽  
Bernhard Kubanek ◽  
Hermann Heimpel
Keyword(s):  
Colony Formation ◽  
Mononuclear Cells ◽  
White Blood Cells ◽  
Soft Agar ◽  
Red Cell ◽  
Neutrophilic Granulocytes ◽  
Feeder Layers ◽  
Granulocytic Colony ◽  
Human White Blood Cells

Abstract The experiments presented in this paper were designed to characterize the colony-stimulating activity (CSA) from white blood cell feeder layers on colony formation by peripheral blood CFCs in soft agar double-layer cultures. The data confirm earlier observations that mononuclear cells are essential for CSA production but document the importance of neutrophilic granulocytes to enhance colony formation. Possible differences between colony enhancement by granulocytes and red cell hemolysate are discussed.

scholarly journals The Adipose Stem Cell as a Novel Metabolic Actor in Adrenocortical Carcinoma Progression: Evidence from an In Vitro Tumor Microenvironment Crosstalk Model

Cancers ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1931 ◽  
Author(s):  
Roberta Armignacco ◽  
Giulia Cantini ◽  
Giada Poli ◽  
Daniele Guasti ◽  
Gabriella Nesi ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Cancer Cells ◽  
Cancer Cell ◽  
Adrenocortical Carcinoma ◽  
Cancer Cell Line ◽  
Cell Types ◽  
Adrenocortical Cancer ◽  
Solid Malignancies ◽  
H295r Cells

Metabolic interplay between the tumor microenvironment and cancer cells is a potential target for novel anti-cancer approaches. Among stromal components, adipocytes and adipose precursors have been shown to actively participate in tumor progression in several solid malignancies. In adrenocortical carcinoma (ACC), a rare endocrine neoplasia with a poor prognosis, cancer cells often infiltrate the fat mass surrounding the adrenal organ, enabling possible crosstalk with the adipose cells. Here, by using an in vitro co-culture system, we show that the interaction between adipose-derived stem cells (ASCs) and the adrenocortical cancer cell line H295R leads to metabolic and functional reprogramming of both cell types: cancer cells limit differentiation and increase proliferation of ASCs, which in turn support tumor growth and invasion. This effect associates with a shift from the paracrine cancer-promoting IGF2 axis towards an ASC-associated leptin axis, along with a shift in the SDF-1 axis towards CXCR7 expression in H295R cells. In conclusion, our findings suggest that adipose precursors, as pivotal components of the ACC microenvironment, promote cancer cell reprogramming and invasion, opening new perspectives for the development of more effective therapeutic approaches.

scholarly journals Critical role of miR-10b in BRafV600E dependent anchorage-independent growth and invasion of melanoma cells

2018 ◽  
Author(s):  
Ila Datar ◽  
Gardiyawasam Kalpana ◽  
Ivana de la Serna ◽  
Robert Trumbly ◽  
Jungmin Choi ◽  
...  
Keyword(s):  
Cell Invasion ◽  
High Throughput Sequencing ◽  
Genetic Locus ◽  
Critical Role ◽  
Melanoma Cells ◽  
Growth And Survival ◽  
Anchorage Independent Growth ◽  
Expression Of Genes ◽  
Kinase Cascade

AbstractRecent high-throughput-sequencing of cancer genomes has identified oncogenic mutations in the BRaf genetic locus as one of the critical events in melanomagenesis. BRaf encodes a serine/threonine kinase that regulates the MAPK/ERK kinase (MEK) and extracellular signal-regulated kinase (ERK) protein kinase cascade. In normal cells, the activity of BRaf is tightly regulated and is required for cell growth and survival. BRaf gain-of-function mutations in melanoma frequently lead to unrestrained growth, enhanced cell invasion and increased viability of cancer cells. Although it is clear that the invasive phenotypes of BRaf mutated melanoma cells are stringently dependent on BRaf-MEK-ERK activation, the downstream effector targets that are required for oncogenic BRaf-mediated melanomagenesis are not well defined. miRNAs have regulatory functions towards the expression of genes that are important in carcinogenesis. We observed that miR-10b expression correlates with the presence of the oncogenic BRaf (BRafV600E) mutation in melanoma cells. While expression of miR-10b enhances anchorage-independent growth of BRaf wild-type melanoma cells, miR-10b silencing decreases BRafV600E cancer cell invasion in vitro. Importantly, the expression of miR-10b is required for BRafV600E-mediated anchorage-independent growth and invasion of melanoma cells in vitro. Taken together our results suggest that miR-10b is an important mediator of oncogenic BRafV600E activity in melanoma.

scholarly journals Recombinant gibbon interleukin-3 acts synergistically with recombinant human G-CSF and GM-CSF in vitro

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1596-1600 ◽  
Author(s):  
RL Paquette ◽  
JY Zhou ◽  
YC Yang ◽  
SC Clark ◽  
HP Koeffler
Keyword(s):  
Colony Formation ◽  
Clinical Utility ◽  
Bone Marrow Cells ◽  
Soft Agar ◽  
Interleukin 3 ◽  
Gm Csf ◽  
Normal Human ◽  
Stimulating Factor ◽  
Normal Human Bone Marrow

Recombinant gibbon interleukin-3 (IL-3) is a multilineage hematopoietic colony-stimulating factor (CSF) that recently was cloned and found to be highly homologous with human IL-3. Gibbon IL-3, as well as human granulocyte-CSF (G-CSF) and human granulocyte-macrophage CSF (GM-CSF), stimulated normal human bone marrow cells to form myeloid colonies in soft agar in a sigmoidal dose-response manner. When IL-3 was added to increasing concentrations of G-CSF or GM-CSF, synergistic colony formation occurred as compared with the effects of each CSF alone. Synergism was also noted when G-CSF was added with GM-CSF and when all the CSFs were added simultaneously. The combination of IL-3 and GM-CSF was less stimulatory than all the other CSF combinations. At day 11 of culture, IL-3 induced granulocyte-macrophage (38%), eosinophil (30%), granulocyte (18%), and macrophage (14%) colony formation. In summary, gibbon IL-3 is a growth factor that can synergize with other CSFs to enhance proliferation of myeloid-committed progenitors, suggesting that combinations of CSFs may have clinical utility in patients with neutropenia of various etiologies.

scholarly journals The effect of interleukin 3 and GM-CSA-2 on megakaryocyte and myeloid clonal colony formation

Blood ◽  
1985 ◽  
Vol 65 (1) ◽  
pp. 214-217 ◽  
Author(s):  
PJ Quesenberry ◽  
JN Ihle ◽  
E McGrath
Keyword(s):  
T Cell ◽  
Colony Formation ◽  
Soft Agar ◽  
Conditioned Media ◽  
Pokeweed Mitogen ◽  
Interleukin 3 ◽  
Biologic Activity ◽  
Similar Range ◽  
Macrophage Colony

Two separate helper T cell-derived lymphokines, interleukin 3 and granulocyte-macrophage colony-stimulating activity-2, were found to stimulate a broad and similar range of hemopoietic colonies in in vitro soft agar cultures including granulocyte, macrophage, granulocyte- macrophage, megakaryocyte, and mixed megakaryocyte colonies. Both lymphokines were potent stimulators of in vitro megakaryocyte colony formation. At plateau levels of IL-3, megakaryocyte colony formation was increased by biologic activity in pokeweed mitogen spleen- conditioned media.

scholarly journals The effect of interleukin 3 and GM-CSA-2 on megakaryocyte and myeloid clonal colony formation

Blood ◽  
1985 ◽  
Vol 65 (1) ◽  
pp. 214-217 ◽  
Author(s):  
PJ Quesenberry ◽  
JN Ihle ◽  
E McGrath
Keyword(s):  
T Cell ◽  
Colony Formation ◽  
Soft Agar ◽  
Conditioned Media ◽  
Pokeweed Mitogen ◽  
Interleukin 3 ◽  
Biologic Activity ◽  
Similar Range ◽  
Macrophage Colony

Abstract Two separate helper T cell-derived lymphokines, interleukin 3 and granulocyte-macrophage colony-stimulating activity-2, were found to stimulate a broad and similar range of hemopoietic colonies in in vitro soft agar cultures including granulocyte, macrophage, granulocyte- macrophage, megakaryocyte, and mixed megakaryocyte colonies. Both lymphokines were potent stimulators of in vitro megakaryocyte colony formation. At plateau levels of IL-3, megakaryocyte colony formation was increased by biologic activity in pokeweed mitogen spleen- conditioned media.

Role of nicotinamide N-methyltransferase in non-small cell lung cancer: in vitro effect of shRNA-mediated gene silencing on tumourigenicity

2015 ◽  
Vol 396 (3) ◽  
pp. 225-234 ◽  
Author(s):  
Davide Sartini ◽  
Riccardo Seta ◽  
Valentina Pozzi ◽  
Stefano Morganti ◽  
Corrado Rubini ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Proliferation ◽  
Gene Silencing ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
Colony Formation ◽  
Soft Agar ◽  
Small Cell ◽  
Small Cell Lung

Abstract Lung cancer is the second most commonly diagnosed neoplasm, and represents the leading cause of tumour death worldwide. As patients are often diagnosed at a late stage, current therapeutic strategies have limited effectiveness and the prognosis remains poor. Successful treatment depends on early diagnosis and knowledge concerning molecular mechanisms underlying lung carcinogenesis. In the present study, we focused on nicotinamide N-methyltransferase (NNMT), which is overexpressed in several malignancies. First, we analysed NNMT expression in a cohort of 36 patients with non-small cell lung cancer (NSCLC) by immunohistochemistry. Subsequently, we examined NNMT expression levels in the human lung cancer cell line A549 by Real-Time PCR, Western blot and catalytic activity assay, and evaluated the effect of NNMT knockdown on cell proliferation and anchorage-independent cell growth by MTT and soft agar colony formation assays, respectively. NSCLC displayed higher NNMT expression levels compared to both tumour-adjacent and surrounding tissue. Moreover, shRNA-mediated gene silencing of NNMT led to a significant inhibition of cell proliferation and colony formation ability on soft agar. Our results show that the downregulation of NNMT significantly reduced in vitro tumorigenicity of A549 cells and suggest that NNMT could represent an interesting molecular target for lung cancer therapy.

Sign in / Sign up

Export Citation Format

Share Document