JSI-124 Induces Cell Cycle Arrest and Regulates the Apoptosis in Glioblastoma Cells

Abstract

Cucurbitacin I (JSI-124), derived from Cucurbitaceae, has shown the potential to induce apoptosis and cell cycle arrest in some cancer cells. However, the effect of JSI-124 on glioblastoma multiforme (GBM) cell cycle and apoptosis is still unclear. Our investigation revealed that JSI-124 effectively reduced cell viability in GBM cells, leading to apoptosis and increased caspase-3 activity. Intriguingly, JSI-124 caused the accumulation of G2/M phase to regulate cell cycle, confirmed by MPM-2 staining and increased protein synthesis during mitosis by mitotic index analysis. Western blot analysis found that JSI-124 affected the progression of G2/M arrest by downregulating the CDK1 and upregulating the cyclinB1, suggesting that JSI-124 disrupted the formation and function of the cyclin B1/CDK1 complex in GBM8401 and U87MG cells. However, we found the JSI-124-regulated cell cycle G2/M and apoptosis-relative gene in GBM8401 and U87MG cells by NGS data analysis. Notably, we found that the GBM8401 and U87MG cells observed regulation of apoptosis and cell-cycle-related signaling pathways. Taken together, JSI-124 exhibited the ability to induce G2/M arrest, effectively arresting the cell cycle at critical stages. This arrest is accompanied by the initiation of apoptosis, highlighting the dual mechanism of action of JSI-124. Collectively, our findings emphasize that JSI-124 holds potential as a therapeutic agent for GBM by impeding cell cycle progression, inhibiting cell proliferation, and promoting apoptosis. As demonstrated by our in vitro experiments, these effects are mediated through modulation of key molecular targets.

Keywords: apoptosis; cell cycle; cucurbitacin I; glioblastoma.

Figure 1

Structure of Cucurbitacins family.

Figure 2

JSI-124 inhibited the cell viability in glioblastoma cells (A) GBM8401 and (B) U87MG cells. Cell viability was measured using the MTT reagent assay. The significance level was set at, **** p < 0.0001, &&&& p < 0.0001, #### p < 0.0001 signifying noteworthy distinctions between the control and treatment groups.

Figure 3

JSI-124 induced apoptosis in (A) GBM8401 and (B) U87MG cells. Cells were treated with JSI-124 (0, 0.5, 1, or 2 µM) for 24 h. Statistical significance was set at * p < 0.05, ** p < 0.01, ns p > 0.05.

Figure 4

JSI-124 treatment increased the activity of caspase-3 in (A) GBM8401 and (B) U87MG cells. The results are presented as percentages relative to the control group, which was defined as 100%. Each data point represents mean ± S.D. from three independent experiments conducted in triplicates. Statistical significance was set at * p < 0.05, ** p < 0.01, ns p > 0.05.

Figure 5

(A) Cell cycle analysis to assess the effect of the JSI-124 treatment on cell cycle progression in glioblastoma cells (GBM8401 and U87MG cells). (B) DNA content analysis was performed, followed by quantification using flow cytometry. Statistical significance was set at *** p < 0.05, **** p < 0.0001.

Figure 6

Flow cytometry analysis was conducted after staining with MPM-2 and PI in (A) GBM8401 cells and (B) U87MG cells. Quantification of MPM-2 content showed an increase in mitotic activity in glioblastoma cells upon treatment with JSI-124. Statical significant was set at * p < 0.05, ** p < 0.01, **** p < 0.0001.

Figure 7

The effect of JSI-124 on glioma cells, inducing cell cycle arrest. (A) We examined the influence of JSI-124 on the expression of cell-cycle-related proteins by subjecting the cells to JSI-124 treatment. Specifically, we assessed the levels of CHK1, CDK1, cyclin B1, and cyclin E. Western blotting was employed to detect the expression of these cell-cycle-related proteins. (B) Quantification of JSI-124 regulatory protein expression (GAPDH as loading controls). Statistical significance was set at * p < 0.05, ** p < 0.01, *** p < 0.001. Western blot original data are shown in Supplementary Materials.

Figure 8

The genes and genomes enrichment analysis after JSI-124 treatment in GBM8401 (A) and U87MG cells (B). * We found that JSI-124 may affect U87MG cells by regulating the cell cycle pathway and GBM8401 cells by regulating the apoptosis pathway.

Figure 9

JSI-124-regulated cell cycle and apoptosis-related mRNA expression in GBM8401 and U87MG cells. Exposure to 8 µM of JSI-124 for 24 h up- or downregulates G2/M phase-related genes expression (A) and apoptosis-related genes expression (B).

Figure 9

JSI-124-regulated cell cycle and apoptosis-related mRNA expression in GBM8401 and U87MG cells. Exposure to 8 µM of JSI-124 for 24 h up- or downregulates G2/M phase-related genes expression (A) and apoptosis-related genes expression (B).

Figure 10

JSI-124 treatment of GBM cells induces apoptosis and cell cycle arrest. We found that JSI-124 involves cell cycle regulators and apoptosis mechanisms, thereby inhibiting cell growth progression. The cell cycle comprises several phases, including G1, S, G2, and M phases. The figure was created using BioRender.com.