Heterogeneous Response of Tumor Cell Lines to Inhibition of Aspartate β-hydroxylase

Abstract

Background: Cancer development involves alterations in key cellular pathways, with aspartate β-hydroxylase (ASPH) emerging as an important player in tumorigenesis. ASPH is upregulated in various cancer types, where it promotes cancer progression mainly by regulating the Notch1 and SRC pathways. Methods: This study explored the responses of various human cervical, pharyngeal, and breast tumor cell lines to second- and third-generation ASPH inhibitors (MO-I-1151 and MO-I-1182) using proliferation, migration, and invasion assays; western blotting; and cell cycle analysis. Results: ASPH inhibition significantly reduced cell proliferation, migration, and invasion and disrupted both the canonical and noncanonical Notch1 pathways. The noncanonical pathway was particularly mediated by AKT signaling. Cell cycle analysis revealed a marked reduction in cyclin D1 expression, further confirming the inhibitory effect of ASPH inhibitors on cell proliferation. Additional analysis revealed G0/G1 arrest and restricted progression into S phase, highlighting the regulatory impact of ASPH inhibitors on the cell cycle. Furthermore, ASPH inhibition induced distinctive alterations in nuclear morphology. The high heterogeneity in the responses of individual tumor cell lines to ASPH inhibitors, both quantitatively and qualitatively, underscores the complex network of mechanisms that are regulated by ASPH and influence the efficacy of ASPH inhibition. The effects of ASPH inhibitors on Notch1 pathway activity, cyclin D1 expression, and nuclear morphology contribute to the understanding of the multifaceted effects of these inhibitors on cancer cell behavior. Conclusion: This study not only suggests that ASPH inhibitors are effective against tumor cell progression, in part through the induction of cell cycle arrest, but also highlights the diverse and heterogeneous effects of these inhibitors on the behavior of tumor cells of different origins.

Keywords: AKT signaling; ASPH inhibitors; Notch pathway; cell cycle; heterogeneity; tumorigenesis.

Figure 1

Effect of ASPH inhibitors on cell proliferation. (A) Cells were treated with MO-I-1151 and MO-I-1182 at concentrations of 0.1, 1, 5, 10, or 20 μM for 48 h, followed by an MTT assay. (B) Cells were incubated with 20 μM MO-I-1151 or MO-I-1182 for 7 days, stained with crystal violet, and imaged. Image quantification was performed using ImageJ software. DMSO was used as a control. The results are presented as the mean ± SEM of three independent experiments. The statistical significance refers to the comparison with the control (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001).

Figure 2

Effect of ASPH inhibitors on cell migration. (A) Cells were treated with 20 μM of the ASPH inhibitors MO-I-1151 and MO-I-1182 for 24 h. Confluent cells were scratched with a pipette tip. Cell migration was assessed by measuring the wound area at 0 and 12 h, and the images were quantified using ImageJ software. Magnification: 40×. (B) Cells were exposed to 20 μM MO-I-1151 or MO-I-1182 in a transwell migration assay. After 24 h, the fixed and stained cells were quantified based on microscopy images using ImageJ software. DMSO was used as a control. Magnification: 100×. Quantitative data are presented as the mean ± SEM of three independent experiments. The statistical significance refers to the comparison with the control (* p<0.05, ** p<0.01).

Figure 3

Effect of ASPH inhibitors on cell invasion. (A) Cell spheroids were embedded in a 3D collagen matrix and treated with 20 μM MO-I-1151 or MO-I-1182. Images were captured at 0 and 72 h and quantified using ImageJ software. Magnification: 40×. (B) Cell lines were exposed to 20 μM MO-I-1151 or MO-I-1182 in precoated Matrigel transwell chambers. After 24 h, the fixed and stained cells were quantified based on microscopy images using ImageJ software. DMSO was used as a control. Magnification: 100×. The data are presented as the means ± SEMs of three independent experiments. The statistical significance refers to the comparison with the control (* p<0.05, ** p<0.01, *** p<0.001).

Figure 4

Effect of ASPH inhibitors on cell signaling. The cells were treated with 20 μM MO-I-1151 or MO-I-1182 for 24 h. DMSO was used as an untreated control. Cell lysates were harvested and subjected to SDS‒PAGE and immunoblotting. GAPDH was used as a loading control. The results shown are representative of at least three independent experiments.

Figure 5

Effect of ASPH inhibitors on activated Notch1 localization. The cells were treated with 20 μM MO-I-1151 or MO-I-1182 for 24 h. DMSO was used as an untreated control. The cells were pre-extracted, fixed with PFA, stained for activated Notch1 and with DAPI, and analyzed via confocal microscopy. Representative images are shown. The scale bars represent 10 μm (Merge) and 5 μm (Zoom).

Figure 6

Effect of ASPH inhibitors on cell cycle progression. (A) Cells were treated with 20 μM MO-I-1151 or MO-I-1182 for 48 h. DMSO was used as an untreated control. Cell cycle analysis was performed by flow cytometry with propidium iodide staining. Quantitative data are presented as the mean ± SEM of three independent experiments. The statistical significance refers to the comparison with the control (* p<0.05, ** p<0.01, *** p<0.001). (B) The lysates of cells treated with 20 μM MO-I-1151 or MO-I-1182 were subjected to immunoblot analysis to determine the levels of cyclin D1. DMSO was used as an untreated control, and GAPDH as a loading control. The results shown are representative of at least three independent experiments.