Cucurbitacin E Suppresses Adipogenesis and Lipid Accumulation in 3T3-L1 Adipocytes Without Cytotoxicity

Abstract

Background: Cucurbitacin E (CuE), a natural tetracyclic triterpenoid compound extracted from the melon stems of Cucurbitaceae plants, has been reported to exhibit anti-inflammatory and anti-cancer properties, along with the ability to enhance cellular immunity. However, its role and molecular mechanism in regulating lipid metabolism and adipogenesis remain unclear. This study aims to investigate the potential anti-adipogenic and anti-obesity effects of CuE in 3T3-L1 adipocytes. Materials and Methods: 3T3-L1 preadipocytes were cultured and induced to differentiate using a standard adipogenic cocktail containing dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), and insulin (DMI). CuE was administered during the differentiation process at various concentrations. Lipid accumulation was assessed using Oil Red O staining, and cell viability was evaluated via the MTT assay. To determine whether CuE induced apoptosis or necrosis, flow cytometry was performed using annexin V/PI staining. Additional molecular analyses, such as Western blotting and RT-PCR, were used to examine the expression of key adipogenic markers. Results: Treatment with CuE significantly reduced lipid droplet formation in DMI-induced 3T3-L1 adipocytes in a dose-dependent manner, as shown by decreased Oil Red O staining. Importantly, CuE did not induce apoptosis or necrosis in 3T3-L1 cells at effective concentrations, indicating its safety toward normal adipocytes. Moreover, CuE treatment downregulated the expression of adipogenic markers such as PPARγ and C/EBPα at both mRNA and protein levels. Discussion: Our findings suggest that CuE exerts a non-cytotoxic inhibitory effect on adipocyte differentiation and lipid accumulation. This anti-adipogenic effect is likely mediated through the suppression of key transcription factors involved in adipogenesis. The absence of cytotoxicity supports the potential application of CuE as a safe bioactive compound for obesity management. Further investigation is warranted to elucidate the upstream signaling pathways and in vivo efficacy of CuE. Conclusions: Cucurbitacin E effectively inhibits adipogenesis in 3T3-L1 adipocytes without inducing cytotoxic effects, making it a promising candidate for the development of functional foods or therapeutic agents aimed at preventing or treating obesity. This study provides new insights into the molecular basis of CuE's anti-obesity action and highlights its potential as a natural lipogenesis inhibitor.

Keywords: 3T3-L1 cells; Anti-Obesity Medication (AOM); adipocyte differentiation; cucurbitacin E; obesity.

Figure 1

(A) Promotion of adipogenesis in vitro by 3T3-L1 cells. Differentiation of 3T3-L1 preadipocytes into mature adipocytes was evaluated using ORO staining, which specifically stains intracellular lipid droplets. The control group (CTL) consisted of undifferentiated 3T3-L1 preadipocytes cultured under standard growth conditions. Scale bar = 20 μm. (B) Effects of CuE on 3T3-L1 cell viability in a dose-dependent manner. To assess the cytotoxic effects of CuE, 3T3-L1 preadipocytes were treated with increasing concentrations of CuE for 24 h, and cell viability was measured using the MTT assay. The results demonstrate a dose-dependent decrease in cell viability following CuE treatment, indicating that higher concentrations of CuE significantly impair the growth and metabolic activity of 3T3-L1 cells. Data are presented as the mean ± SEM from three independent experiments (n = 3 per group). * p < 0.01 compared to the untreated control group was considered statistically significant. These findings suggest that CuE may act as a potential suppressor of preadipocyte proliferation. CuE, cucurbitacin E.

Figure 2

Effect of CuE on apoptosis and necrosis in 3T3-L1 preadipocytes. (A) Representative dot plots show the distribution of apoptotic and necrotic populations across different CuE treatment groups. No substantial increase in apoptotic or necrotic cells was observed in the CuE-treated groups compared to the untreated control. (B) The quantification of total apoptotic cells (early + late apoptosis) revealed no statistically significant increase at any tested CuE concentration, indicating that CuE does not trigger apoptosis in 3T3-L1 cells under these conditions. Data represent the mean ± SEM from three independent experiments (n = 3 per group) compared to the 0 nM CuE control. These results suggest that the inhibitory effect of CuE on cell proliferation is not mediated by apoptosis or necrosis. CuE, cucurbitacin E; PI, propidium iodide. ns: not significant.

Figure 3

Effect of CuE on cell cycle progression in 3T3-L1 preadipocytes. (A) 3T3-L1 cells were treated with different concentrations of CuE (0, 2.5, 5, and 10 nM) for 24 h. Cells were then fixed, stained with propidium iodide (PI), and subjected to flow cytometry to determine the DNA content and analyze their distribution across cell cycle phases (G0/G1, S, and G2/M). Representative histograms illustrate the DNA content profiles for each treatment group. (B) Quantitative analysis showed that CuE treatment significantly altered the rise in S phase and the percentage of cells in any specific phase of the cell cycle compared to the control group. The proportion of cells in G0/G1 phase, S phase, and G2/M phase remained unchanged at all CuE concentrations, indicating that CuE did not induce cell cycle arrest in 3T3-L1 cells under the tested conditions. Data are shown as the mean ± SEM from three independent experiments (n = 3 per group). ns: not significant. These findings suggest that the anti-proliferative effect of CuE on 3T3-L1 cells is not mediated through the modulation of cell cycle progression. CuE, cucurbitacin E; PI, propidium iodide (* p < 0.05).