In Vitro Self-Renewal Assays for Brain Tumor Stem Cells

Seyfrid, Mathieu; Bobrowski, David; Bakhshinyan, David; Tatari, Nazanin; Venugopal, Chitra; Singh, Sheila K.

doi:10.1007/978-1-4939-8805-1_7

Cited by 6 publications

(6 citation statements)

References 12 publications

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“…As expected, the addition of the ObR antagonist LDFI significantly decreased these effects ( Figure 4 a–c). In addition, to further confirm the enhanced clonogenic potential and self-renewal properties of glioblastoma cells treated with leptin, we performed the soft agar assay and the limiting dilution assay (LDA), following the methods previously described [ 39 ]. As shown in Figure 4 d, leptin treatment significantly increased colony formation in the anchorage-independent soft agar assay.…”

Section: Resultsmentioning

confidence: 99%

“…The limiting dilution assay (LDA) was used to evaluate in vitro the frequency of self-renewing cells in our populations, following the protocol described by Seyfrid et al [ 39 ]. Briefly, U-87 MG and T98G cells were grown as neurospheres, as previously described, and after 7 days of growth, neurospheres were dissociate into a single cell suspension and seeded in neurosphere culture media at decreasing densities (100, 50, 10, 1 cell/well) into 96-well ultra-low attachment plates.…”

Section: Methodsmentioning

confidence: 99%

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Leptin and Notch Signaling Cooperate in Sustaining Glioblastoma Multiforme Progression

et al. 2020

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Glioblastoma multiforme (GBM) is the most malignant form of glioma, which represents one of the commonly occurring tumors of the central nervous system. Despite the continuous development of new clinical therapies against this malignancy, it still remains a deadly disease with very poor prognosis. Here, we demonstrated the existence of a biologically active interaction between leptin and Notch signaling pathways that sustains GBM development and progression. We found that the expression of leptin and its receptors was significantly higher in human glioblastoma cells, U-87 MG and T98G, than in a normal human glial cell line, SVG p12, and that activation of leptin signaling induced growth and motility in GBM cells. Interestingly, flow cytometry and real-time RT-PCR assays revealed that GBM cells, grown as neurospheres, displayed stem cell-like properties (CD133+) along with an enhanced expression of leptin receptors. Leptin treatment significantly increased the neurosphere forming efficiency, self-renewal capacity, and mRNA expression levels of the stemness markers CD133, Nestin, SOX2, and GFAP. Mechanistically, we evidenced a leptin-mediated upregulation of Notch 1 receptor and the activation of its downstream effectors and target molecules. Leptin-induced effects on U-87 MG and T98G cells were abrogated by the selective leptin antagonist, the peptide LDFI (Leu-Asp-Phe-Ile), as well as by the specific Notch signaling inhibitor, GSI (Gamma Secretase Inhibitor) and in the presence of a dominant-negative of mastermind-like-1. Overall, these findings demonstrate, for the first time, a functional interaction between leptin and Notch signaling in GBM, highlighting leptin/Notch crosstalk as a potential novel therapeutic target for GBM treatment.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Leptin and Notch Signaling Cooperate in Sustaining Glioblastoma Multiforme Progression

et al. 2020

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“…The superiority of testing anticancer molecules in three‐dimensional (3D) format like SAA over monolayer culture to validate cell transformation is used since 1977 in clonogenic assay developed by Hamburger and Salmon (1977) and Hamburger (1987). Since then, SAA is widely being used to identify potential antitumor agents and to evaluate the stemness properties of CSCs (Fukazawa, Noguchi, Murakami, & Uehara, 2002; Horibata, Vo, Subramanian, Thompson, & Coonrod, 2015; Seyfrid et al, 2019). More so, the traditional method is also modified with time to overcome time‐consuming, labour intensive process with HTS to meet current oncology drug‐target screening demand (Fukazawa et al, 1995; Ke et al, 2004).…”

Section: Resultsmentioning

confidence: 99%

A novel series of substituted 1,2,3‐triazoles as cancer stem cell inhibitors: Synthesis and biological evaluation

Padhariya

Srivastava²,

Kharkar

2020

Drug Development Research

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An alarming increase in global death toll resulting from cancer incidents, particularly due to multidrug resistance and reduced efficacy as a consequence of target mutations, has compelled us to look for novel anticancer agents. Cancer stem cells (CSCs), contributing majorly to the chemoresistance and tumor relapse, seem to the main culprits. In the present investigation, new chemical entities (NCEs) belonging to four novel chemical series (A: 4′‐allyl‐2′‐methoxyphenoxymethyl‐1,2,3‐triazoles; B: 4′‐acetamidophenoxymethyl‐1,2,3‐triazoles; C: naphthalene‐1′‐yloxymethyl‐1,2,3‐triazoles, and D: naphthalene‐2′‐yloxymethyl‐1,2,3‐triazoles) were synthesized via Copper (I)‐catalyzed alkyne‐azide cycloaddition reaction and evaluated for in vitro anticancer activity. A total of 30 NCEs (39–68) were screened at 10 μM concentration in cell viability assay against cancer cell lines such as breast (MDA‐MB‐231), prostate (PC‐3), glioma (U87 MG), along with cervical (SiHa) and lung (A549). The NCEs from Series C (56–60) and D (61–68) were more potent than those in Series A (39‐45) and Series B (46–55) at the tested concentration. Furthermore, NCEs with >80% inhibition at 10 μM were evaluated for dose response. A total of five NCEs, 48, 56, 61, 65 and 66, were further assessed in soft‐agar assay and found to be relatively potent (IC50 < 10 μM). Finally, the hits were screened in sphere assay to identify potential CSC inhibitors against mammospheres (MDA‐MB‐231) and prostatospheres (PC‐3). More so, the hits were also evaluated to understand in vitro cytotoxicity against normal cells using mouse embryonic fibroblast cell line (NIH/3T3) and human peripheral blood mononuclear cells (hPBMCs). Overall, hits 56 and 61 exhibited potent anticancer as well as CSC inhibitory activities with notably less toxicity toward NIH/3T3 and hPBMCs. On the whole, our arduous study led to the identification of potential hits with anticancer and CSC inhibitory activities, with minimal or no toxicity to normal cells.

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“…Previously, Horibata, Vo, Subramanian, Thompson, and Coonrod () have used SAA for identifying inhibitors of tumorigenicity in breast cancer cells. In a very recent report, Seyfrid et al () have described SAA as one of the inexpensive and convenient assays for evaluating the stemness properties of brain tumor stem cells.…”

Section: Resultsmentioning

confidence: 99%

Substituted chloroacetamides as potential cancer stem cell inhibitors: Synthesis and biological evaluation

Padhariya

Srivastava²,

Kharkar

2019

Drug Development Research

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Cancer kills, irrespective of geographical and cultural origin. Novel modalities for treating cancer are desperately needed. Cancer stem cells (CSCs), main culprits behind chemoresistance and tumor relapse, are one of the few logical choices. Herein, we report the synthesis and biological evaluation of small molecules with chloroacetamide war-head. These molecules were screened for viability against various breast, prostate, and oral cancer cell lines using MTT and soft-agar assays. Further, promising hits were screened in sphere-forming assay with the aim of discovering potential anti-CSC agents. Our optimism yielded four hits inhibiting self-renewal of cancer cells with stem-like characters in vitro. Finally, the hits were evaluated for in vitro toxicity against human peripheral blood mononuclear cells and mouse embryonic fibroblast cell line.Overall, these preliminary investigations yielded three hits exhibiting promising anti-CSC potential with little or no toxicity against normal cells. K E Y W O R D Sbreast cancer, cancer stem cells, chloroacetamides, CSCs, oral cancer, prostate cancer, softagar assay, sphere-forming assay

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In Vitro Self-Renewal Assays for Brain Tumor Stem Cells

Cited by 6 publications

References 12 publications

Leptin and Notch Signaling Cooperate in Sustaining Glioblastoma Multiforme Progression

Leptin and Notch Signaling Cooperate in Sustaining Glioblastoma Multiforme Progression

A novel series of substituted 1,2,3‐triazoles as cancer stem cell inhibitors: Synthesis and biological evaluation

Substituted chloroacetamides as potential cancer stem cell inhibitors: Synthesis and biological evaluation

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