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This study investigated the effects and molecular mechanisms of sarsasapogenin (SSG) on colorectal cancer (CRC). After HRT-18 and HCT116 cells were treated with varying concentrations of SSG, the effect of SSG on CRC cell proliferation, migration, and glycolysis was assessed by cell counting kit-8 (CCK-8), real-time cellular analysis (RTCA), colony formation assay, high-content imaging analysis, and Seahorse glycolysis stress test. Changes in intracellular lactate release and adenosine triphosphate (ATP) levels under SSG treatment were measured by lactate and ATP detection kits, respectively. The effects of SSG on the transcription level and protein expression of the key glycolytic gene hypoxia-inducible factor 1α (HIF1α) were evaluated via quantitative polymerase chain reaction (qPCR) and Western blot. Direct binding between SSG and HIF1α was identified by drug affinity responsive target stability (DARTS). The UbiBrowser database, microscale thermophoresis (MST), and molecular docking simulations were employed to screen and validate the E3 ubiquitin ligase mediating HIF1α degradation. The results showed that SSG significantly inhibited CRC cell proliferation and colony formation in a dose-dependent manner. The 24-hour half-maximal inhibitory concentrations (IC50) of SSG for HRT-18 and HCT116 cells were 6.275 μmol·L-1 and 7.029 μmol·L-1, respectively. After 24 hours of treatment with SSG at concentrations of 2.0, 4.0, and 6.0 μmol·L-1, the HRT-18 and HCT116 clone formation rate was significantly reduced. High-content imaging results showed that SSG significantly inhibited the migration ability of CRC cells. Seahorse glycolysis stress test, along with lactate and ATP measurements, demonstrated that SSG suppressed basal glycolysis, glycolytic capacity, glycolytic reserve, lactate release, and ATP content in CRC cells. The qPCR and Western blot results showed that SSG had no significant effect on HIF1α mRNA expression, and the HIF1α protein expression was downregulated. DARTS confirmed the direct binding of SSG to HIF1α. UbiBrowser analysis identified five E3 ubiquitin ligases potentially targeting HIF1α. Molecular docking and MST results indicated that the F-box/WD repeat-containing protein 7 (FBXW7) exhibited strong binding affinity to HIF1α in the presence of SSG. In summary, SSG exerts its anti-cancer effects in CRC by targeting HIF1α to promote its FBXW7-mediated proteasomal degradation and consequently inhibiting glycolytic reprogramming.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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