scholarly journals Anchorage-independent growth assay or Soft Agar assay v1 (protocols.io.bgeajtae)

protocols.io ◽  
2020 ◽  
Author(s):  
Marzia Ognibene
Keyword(s):  
Soft Agar ◽  
Anchorage Independent Growth ◽  
Soft Agar Assay ◽  
Growth Assay

scholarly journals USP13 Regulates the Breast Tumor Proliferation and Migration through the Stabilization of PDCD4 Protein

2021 ◽  
Author(s):  
Jun Tian ◽  
Bei Li ◽  
Jing Qiao ◽  
Xinfeng Pang ◽  
Xiaojing Yue
Keyword(s):  
Breast Cancer ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Breast Tumor ◽  
Soft Agar ◽  
Tumor Proliferation ◽  
Soft Agar Assay ◽  
And Migration ◽  
Cancer Tissues ◽  
Proliferation And Migration

Abstract Background: Programmed cell death protein 4 (PDCD4), which serves as a tumor suppressor protein, plays a important role in cell proliferation,apoptosis, cell migration and DNA-damage response.However, the exact mechanism for the deubiquitination of PDCD4 remain unclear.Methods: Western blotting was used to detect the expression of PDCD4 in the breast cancer tissues and BC cell lines. We identified the potential PDCD4 associated deubiquitinase by RNAi screening. GST-Pull down and domain-mapping analysis were used to reveal that USP13 and PDCD4 directly interact with each other.Flow cytometry was used to detect the changes of G1 to S phase. Soft agar assay was used to measure the changes of the cell proliferation efficiency.Results: The expression of PDCD4 was decreased in the breast cancer tissues and BC cell lines. USP13 as a potential PDCD4 associated deubiquitinase. USP13 physically interacted with PDCD4 and greatly increased the steady state of PDCD4 through the ubiquitin-proteasome pathway.Importantly, silencing of the USP13 facilitated cell cycle from G1 to Sphase, promoted breast tumor cells proliferation and migration through downregulation of PDCD4. Conclusions: Together, these results suggest that USP13 plays an important role in the breast tumor proliferation and migration through modulating PDCD4 stability.

scholarly journals Targeting Epigenetic Modifiers Can Reduce the Clonogenic Capacities of Sézary Cells

2021 ◽  
Vol 11 ◽  
Author(s):  
Alain Chebly ◽  
Martina Prochazkova-Carlotti ◽  
Yamina Idrissi ◽  
Laurence Bresson-Bepoldin ◽  
Sandrine Poglio ◽  
...  
Keyword(s):  
Dna Methyltransferase ◽  
Histone Deacetylase Inhibitors ◽  
Soft Agar ◽  
Tumor Formation ◽  
Human Telomerase Reverse Transcriptase ◽  
Hematological Disorders ◽  
Soft Agar Assay ◽  
Human Telomerase ◽  
Sézary Cells

Sézary syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphomas (CTCL) in which the human Telomerase Reverse Transcriptase (hTERT) gene is re-expressed. Current available treatments do not provide long-term response. We previously reported that Histone deacetylase inhibitors (HDACi, romidespin and vorinostat) and a DNA methyltransferase inhibitor (DNMTi, 5-azacytidine) can reduce hTERT expression without altering the methylation level of hTERT promoter. Romidepsin and vorinostat are approved for CTCL treatment, while 5-azacytidine is approved for the treatment of several hematological disorders, but not for CTCL. Here, using the soft agar assay, we analyzed the functional effect of the aforementioned epidrugs on the clonogenic capacities of Sézary cells. Our data revealed that, besides hTERT downregulation, epidrugs’ pressure reduced the proliferative and the tumor formation capacities in Sézary cells in vitro.

scholarly journals MicroRNA-384 Inhibits the Progression of Papillary Thyroid Cancer by Targeting PRKACB

2020 ◽  
Vol 2020 ◽  
pp. 1-11 ◽  
Author(s):  
Yongxia Wang ◽  
Beixi Wang ◽  
Hong Zhou ◽  
Xiangnan Zhang ◽  
Xinlai Qian ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Papillary Thyroid Cancer ◽  
Target Gene ◽  
Underlying Mechanism ◽  
Soft Agar ◽  
Luciferase Reporter ◽  
Papillary Thyroid ◽  
Soft Agar Assay ◽  
Spearman’S Correlation ◽  
Dual Luciferase Reporter Assay

Background. Growing evidence shows that dysregulation of miRNAs plays a significant role in papillary thyroid cancer (PTC) tumorigenesis and development. The abnormal expression of miR-384 has been acknowledged in the proliferation or metastasis of some cancers. However, the function and the underlying mechanism of miR-384 in PTC progression remain largely unknown. Methods. Real-time PCR was conducted to detect miR-384 expression in 58 cases of PTC and their adjacent noncancerous tissues. MTT, soft agar assay Transwell assay, and wound-healing assay were carried out to explore the biological function of miR-384 in PTC cell lines of BCPAP and K1. Bioinformatics analysis, dual-luciferase reporter assay, western blot, and functional complementation analysis were conducted to explore the target gene of miR-384. Moreover, Spearman’s correlation analysis was conducted to reveal the correlation between miR-384 and PRKACB mRNA in PTC. Results. The expression of miR-384 decreased obviously in PTC, especially in the tumors with lymph node metastasis or larger tumor size. The ectopic upregulation of miR-384 significantly suppressed PTC progression, and the inhibition of miR-384 had the opposite effects. Moreover, PRKACB gene was confirmed as the target of miR-384. Conclusion. The study suggests that miR-384 serves as a tumor suppressor in PTC progression by directly targeting the 3′-UTR of PRKACB gene.

Metformin Treatment Sensitizes Human Laryngeal Cancer Cell Line Hep- 2 to 5-Fluorouracil

2020 ◽  
Vol 7 (1) ◽  
pp. 16-24
Author(s):  
Neslisah Barlak ◽  
Fatma Sanli ◽  
Ozel Capik ◽  
Elanur Tuysuz ◽  
Elanur Aydın Karatas ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cancer Cell Line ◽  
Inhibitory Effect ◽  
Soft Agar ◽  
Invasive Potential ◽  
Matrigel Invasion ◽  
Soft Agar Assay ◽  
Anti Cancer ◽  
Apoptosis Assay ◽  
Cancer Types

Background: Larynx cancer (LCa) is the most common head and neck cancer and accounts for 1-2.5% of all human cancers worldwide. Metformin, an oral anti-diabetic drug, has been recently shown to have anti-cancer activity in various cancer types, and there are several studies in the literature pointing to its potential to sensitize cancer cells to chemotherapeutic drugs. Objective: This study was aimed at exploring the anti-cancer effects of metformin alone or in combination with 5-fluorouracil (5-FU) on Hep-2 cells. Methods: The effects of metformin and/or 5-FU on the proliferative, clonogenic, and apoptotic potential of Hep-2 cells were evaluated with Cell Viability Detection Kit-8, soft agar assay and Annexin VFITC Apoptosis assay, respectively. Migratory and invasive potential of cells was tested using scratch, transwell migration and Matrigel invasion assays. Gene expression of cells exposed to metformin and/or 5-FU was profiled using RT2 mRNA PCR Array plates. Results: Treatment of Hep-2 cells with metformin inhibited cell proliferation by inducing apoptosis, and suppressed cell migration. Besides, treatment of metformin along with 5-FU improved the antiproliferative and anti-migratory effects of 5-FU. However, unexpectedly, metformin was found to enhance cellular invasion and reverse the inhibitory effect of 5-FU on the invasive potential of Hep-2 cells. Conclusion: Our findings suggest that metformin might be used as an adjuvant agent in the treatment of LCa. However, the potential of metformin to promote the invasion of cancer cells should not be neglected.

Preclinical and correlative studies of cabozantinib (XL184) in urothelial cancer (UC).

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 4543-4543
Author(s):  
Andrea Borghese Apolo ◽  
Young H. Lee ◽  
Fabiola Cecchi ◽  
Piyush K. Agarwal ◽  
Howard L. Parnes ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cell Invasion ◽  
Urothelial Cancer ◽  
Invasive Disease ◽  
Soft Agar ◽  
Visceral Metastasis ◽  
Anchorage Independent Growth ◽  
Median Serum ◽  
Muscle Invasive

4543 Background: Mounting evidence supports Met as a therapeutic target in urothelial cancer (UC). Activated Met can promote angiogenesis and tumor growth by upregulating VEGF and may play a role in UC pathogenesis. Cabozantinib inhibits VEGFR2 and Met pathways. In this study, we assessed shed Met (sMet) levels in the urine and serum of UC patients (pts) and cabozantinib’s effects on HGF-driven UC cell growth and invasion. Methods: sMet levels in serum and urine samples from 31 pts with UC (23 metastatic, 8 muscle-invasive) were correlated with stage, presence of visceral metastases and urinary source. The effects of cabozantinib on 4 human UC-derived cell lines were studied in vitro. Intact RT4, TCC-SUP, T24M2 and T24M3 cells at 80% confluence were serum deprived 16 h, then left untreated or treated with hepatocyte growth factor (HGF) and/or cabozantinib prior to analysis of Met, phospho- (p)Met, pAkt, Akt, pMAPK and MAPK by immunoassay or immunoblotting. Cabozantinib effects on basal and HGF-induced UC cell invasion, proliferation and soft agar growth were measured. Results: Median serum Met levels were modestly higher in pts with metastatic versus muscle-invasive disease. Urinary Met levels were clearly higher in pts with visceral metastasis (P=0.0111) and in urine from ileal conduits and neobladders compared to normally voided urine, regardless of stage (P=0.0489). Met content in UC cell lines was low in RT4 and higher in T24M2, T24M3 and TCC-SUP. Basal pMet content was universally low, increased significantly by HGF and this was reversed by cabozantinib. HGF-driven increases in pAkt/Akt and pMAPK/MAPK in all 4 cell lines were reversed by cabozantinib, as were HGF-enhanced UC cell invasion, proliferation and anchorage independent growth. Conclusions: Median urinary sMet is significantly higher in pts with visceral metastasis and in specimens from ileal conduits and neobladders relative to normally voided urine. UC cell Met content in culture increased with disease grade; HGF stimulated activation of Met and known effectors, and enhanced invasion, growth rate and anchorage-independent growth; cabozantinib effectively reversed these HGF-driven effects. These data support evaluation of cabozantinib in pts with metastatic UC.

scholarly journals Ras Signals to the Cell Cycle Machinery via Multiple Pathways To Induce Anchorage-Independent Growth

1998 ◽  
Vol 18 (5) ◽  
pp. 2586-2595 ◽  
Author(s):  
Jaw-Ji Yang ◽  
Jong-Sun Kang ◽  
Robert S. Krauss
Keyword(s):  
Cell Cycle ◽  
Colony Formation ◽  
Cyclin A ◽  
Soft Agar ◽  
Specific Cell ◽  
3T3 Cells ◽  
Anchorage Independent Growth ◽  
Nih 3T3 ◽  
Cell Cycle Machinery ◽  
Nih 3T3 Cells

ABSTRACT Several specific cell cycle activities are dependent on cell-substratum adhesion in nontransformed cells, and the ability of the Ras oncoprotein to induce anchorage-independent growth is linked to its ability to abrogate this adhesion requirement. Ras signals via multiple downstream effector proteins, a synergistic combination of which may be required for the highly altered phenotype of fully transformed cells. We describe here studies on cell cycle regulation of anchorage-independent growth that utilize Ras effector loop mutants in NIH 3T3 and Rat 6 cells. Stable expression of activated H-Ras (12V) induced soft agar colony formation by both cell types, but each of three effector loop mutants (12V,35S, 12V,37G, and 12V,40C) was defective in producing this response. Expression of all three possible pairwise combinations of these mutants synergized to induce anchorage-independent growth of NIH 3T3 cells, but only the 12V,35S-12V,37G and 12V,37G-12V,40C combinations were complementary in Rat 6 cells. Each individual effector loop mutant partially relieved adhesion dependence of pRB phosphorylation, cyclin E-dependent kinase activity, and expression of cyclin A in NIH 3T3, but not Rat 6, cells. The pairwise combinations of effector loop mutants that were synergistic in producing anchorage-independent growth in Rat 6 cells also led to synergistic abrogation of the adhesion requirement for these cell cycle activities. The relationship between complementation in producing anchorage-independent growth and enhancement of cell cycle activities was not as clear in NIH 3T3 cells that expressed pairs of mutants, implying the existence of either thresholds for these activities or additional requirements in the induction of anchorage-independent growth. Ectopic expression of cyclin D1, E, or A synergized with individual effector loop mutants to induce soft agar colony formation in NIH 3T3 cells, cyclin A being particularly effective. Taken together, these data indicate that Ras utilizes multiple pathways to signal to the cell cycle machinery and that these pathways synergize to supplant the adhesion requirements of specific cell cycle events, leading to anchorage-independent growth.

Cytokine signaling in lung: transforming growth factor-beta secretion by lung fibroblasts

1991 ◽  
Vol 260 (2) ◽  
pp. L123-L128 ◽  
Author(s):  
J. Kelley ◽  
J. P. Fabisiak ◽  
K. Hawes ◽  
M. Absher
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Phenotypic Expression ◽  
Soft Agar ◽  
Lung Fibroblast ◽  
Lung Fibroblasts ◽  
Tgf Beta ◽  
Anchorage Independent Growth ◽  
Growth Factor Beta

Control of growth and phenotypic expression of interstitial fibroblasts is a critical determinant of lung architecture and physiology during processes of growth and remodeling. We examined the ability of lung fibroblasts to produce transforming growth factor-beta (TGF-beta), a cytokine that is known to modulate proliferation and phenotypic expression of mesenchymal cells. Cultures of fibroblasts isolated from rat lungs spontaneously secrete TGF-beta as measured in the standard bioassay of anchorage-independent growth of normal rat kidney (NRK) cells in soft agar. Rat lung fibroblasts secrete TGF-beta in an inactive precursor form. Fibroblasts cultured from adult and fetal rat lungs produced comparable amounts of TGF-beta. The ability of lung fibroblast supernatant fluids to induce colony formation in soft agar could be completely neutralized by preincubation of samples with anti-TGF-beta immunoglobulin (Ig). Anti-platelet-derived growth factor IgG had no effect on anchorage-independent growth of NRK cells driven by rat fibroblast culture supernatant samples. These results indicate that TGF-beta does not require the presence of and interaction with secondary cytokines for its activity. In contrast to the results obtained with rat cells, neither human fetal nor adult lung fibroblasts secreted detectable amount of active TGF-beta or its inactive precursor. This was not due to the presence of TGF-beta inhibitors in fibroblast culture media, because the addition of purified porcine TGF-beta to conditioned medium from human lung fibroblast cultures yielded the expected increase in NRK cell growth in soft agar. These results point to differing cytokine control patterns in the lungs of the two species.

Isolation and partial purification of growth factors with TGF-like activity from human malignant gliomas

1989 ◽  
Vol 71 (6) ◽  
pp. 875-883 ◽  
Author(s):  
James T. Rutka ◽  
Mark L. Rosenblum ◽  
Robert Stern ◽  
Henry J. Ralston ◽  
Dolores Dougherty ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Growth Factors ◽  
Growth Factor ◽  
Conditioned Medium ◽  
Cell Lines ◽  
Transforming Growth Factor ◽  
Malignant Gliomas ◽  
Soft Agar ◽  
Soft Agar Assay

✓ The effect of concentrated conditioned medium from each of eight human malignant glioma cell lines on the growth of indicator cells (normal rat kidney fibroblasts (NRK), clone 14) was determined in monolayer and in soft agar assay systems. The conditioned medium from all cell lines was mitogenic in the monolayer assay, but only SF-210, U-343 MG-A, and U-251 MG produced soluble factors that caused NRK cells to grow in soft agar. The soluble growth-promoting factors from these three cell lines were acid- and heat-stable (60°C for 30 minutes) but were inactivated by trypsin (100 µm/ml) and dithiothreitol (50 µM). The growth factors from SF-210 and U-343 MG-A were further purified by molecular-sieve chromatography. The partially purified growth factor from U-343 MG-A retained transforming growth factor (TGF)-like activity, had a molecular weight of 9 kD, was potentiated by TGF-β in the soft agar assay, competed effectively with 125I-epidermal growth factor (EGF) radiolabeled for the EGF receptor on A 431 epidermoid carcinoma cells, and was completely inhibited by monoclonal antibodies to TGF-α. The partially purified growth factor from SF-210 had a molecular weight of 17 kD, was not inhibited by monoclonal antibodies to platelet-derived growth factor (PDGF) or TGF-α, and did not bind to a heparin-Sepharose column. These results imply that U-343 MG-A secretes a growth factor with TGF-α-like activity, and SF-210 secretes a TGF with neither TGF-α nor TGF-β activity.

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