Agnieszka U. Błachnio-Zabielska

Differential Effects of Sphingolipids on Cell Death and Antioxidant Defenses in Type 1 and Type 2 Endometrial Cancer Cells

Endometrial cancer (EC) is classified into two main subtypes with distinct molecular profiles. Sphingolipids, particularly ceramide and sphingosine-1-phosphate (S1P), are crucial regulators of cell survival, apoptosis, and oxidative stress. This study examined the impact of sphingolipid metabolism in Ishikawa (type 1) and HEC-1A (type 2) EC cells following the silencing of Sptlc1 and Sptlc2, which encode subunits of serine palmitoyltransferase (SPT), a key enzyme in de novo sphingolipid synthesis. Gene silencing was confirmed by RT-PCR and Western blot, while sphingolipid levels were quantified using UHPLC/MS/MS and the sphingolipid rheostat (S1P/ceramide ratio) was calculated. Cell viability (MTT assay), cell death, ROS levels (ELISA), total antioxidant capacity (TAC), catalase and caspase-3 activity, and mitochondrial membrane potential were also assessed. The obtained data showed higher ceramide levels in Ishikawa(CON) cells and higher S1P levels in HEC-1A(CON) cells, resulting in a higher sphingolipid rheostat in HEC-1A cells. SPT knockdown reduced sphingolipid levels, increased cell viability, elevated ROS levels, and decreased cell death, particularly in Ishikawa cells. Furthermore, after gene silencing, these cells exhibited reduced catalase activity and diminished TAC, indicating an impaired redox balance. These findings reveal subtype-specific responses to disrupted sphingolipid synthesis and highlight the importance of sphingolipid homeostasis in the behavior of EC cells.

The Interplay between Oxidative Stress and Sphingolipid Metabolism in Endometrial Cancer

Endometrial cancer is one of the most common malignancies in women. Sphingolipids, a group of lipids, play a key role in cancer biology. Cancer cells often exhibit abnormal redox homeostasis characterized by elevated levels of reactive oxygen species (ROS). Emerging evidence suggests that ceramides are involved in inhibiting proliferation and inducing apoptosis through ROS production. However, there is no data on the relationship between sphingolipid metabolism and oxidative status in endometrial cancer. The present study aims to assess the content of individual sphingolipids and oxidative status in healthy women and those with endometrial cancer. Sphingolipid analysis was performed using mass spectrometry. Total oxidative status (TOS) and total antioxidant capacity (TAC) were assessed colorimetrically. Our results showed a significant increase in the levels of all measured sphingolipids in cancer tissues compared to healthy endometrium. Additionally, a significant decrease in the S1P/ceramide ratio (sphingolipid rheostat) was observed in cancer patients, particularly for C14:0-Cer, C16:0-Cer, C18:1-Cer, C22:0-Cer, and C24:0-Cer. Furthermore, increased TOS and decreased TAC were found in cancer patients compared to healthy women. Significant correlations were observed between the levels of individual sphingolipids and oxidative status, with the strongest correlation noted between C22:0-Cer and TOS (r = 0.64). We conclude that endometrial cancer is characterized by profound changes in sphingolipid metabolism, contributing to oxidative dysregulation and tumor progression.