Abstract
ObjectiveTo evaluate the expression of ALDH1A1 in lung adenoma stem cells (LASCs) and maintenance of their stemness through the Notch pathway.MethodsLASCs (A549s) were isolated from lung adenoma cells (A549) and identified by their coexpression of CD133 and CD326 and their capacity formulti-directional differentiation. Expression of ALDH1A1 in A549 and A549s cells were evaluated by Real-time PCR. Effects of ALDH1A1 upregulation in A549 cells and its downregulation in A549s cells on the clonogenicity and cell cycle were assessed by colony-forming unit assay. Moreover, the effects of ALDH1A1 on the Notch pathway, and thus on the cell cycle, were studied.ResultsA549s cells were successfully isolated and identified.ALDH1A1expression was significantly higher in A549s than in A549 cells. Clonogenicity was significantly decreased in A549s cells treated with ALDH1A1 siRNA. Duration of the G1 stage of the cell cycle increased after ALDH1A1 was overexpressed, or decreased with ALDH1A1 siRNA. ALDH1A1, Notch1, −2, and −3, CDK2, and CCNE1 expression levels were higher in A549s cells than in A549 cells. Expression of Notch1, −2, and −3, CDK2, and CCNE1 was significantly decreased by upregulation of ALDH1A1 in A549 cells, but increased by its interruption in A549s cells. When Notch3 or CDK2 expression was downregulated, the expression levels of ALDH1A1, Notch1, −2, and −3, CDK2, and CCNE1 were reduced in all cell types.ConclusionALDH1A1 expression improved clonogenicity and inhibited the cell cycle, maintaining the stemness of the A549s cells; this may involve suppression of the Notch/CDK2/Cyclin pathway.
Highlights
Cancer stem cells (CSCs) are special subpopulations that contain stemcell-specific characteristics, such as self-renewal, unlimited proliferation, maintenance at low differentiation states, and resistance to radiotherapy and chemotherapy, which maybe partially responsible for the proliferation, metastasis and therapy resistance of cancer cells [1]
The isolated A549s cells were cultured in complete culture medium: DMEM/F12 culture medium containing insulin (5000 ng/ml), epidermal growth factor (20 ng/ml), and basic fibroblast growth factor, with 5% CO2 saturated humidity at 37uC.Further confirmation was obtained from two induced differentiation procedures
A549s cells were successfully differentiated into cancer cells at 1 week (Fig. 2A), whereupon these cells stopped expressing CD133 and CD326 (Fig. 2D)
Summary
Cancer stem cells (CSCs) are special subpopulations that contain stemcell-specific characteristics, such as self-renewal, unlimited proliferation, maintenance at low differentiation states, and resistance to radiotherapy and chemotherapy, which maybe partially responsible for the proliferation, metastasis and therapy resistance of cancer cells [1]. Because of the capacity of self-renewal, CSCs has infinite proliferation ability and high tumorigenicity. This characteristic may be considered as representing the stemness of CSCs [2]. The mechanism by which the stemness of CSCs promotes their resistance to chemotherapeutic agents and radiotherapy remains unclear. ALDH1A1 has been considered to have prognostic significance in early stage non-small cell lung cancer, and its effects on lung CSCs have been noticed [6]. The pathways by which ALDH affects CSC stemness remain to be identified
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