Supplementary MaterialsSupplementary Materials: Figure S1: impact of celastrol and celastrol coincubation with NAC on the intracellular ROS level (DCF-DA assay) in LOVO/DX cells. and PI+), Q3?=?live cells (Annexin V-FITC and PI?), Q4?=?early apoptotic cells (Annexin V-FITC+ and PI?). Figure S3: effect of celastrol on the viability of NHDF cells (normal human dermal fibroblast) (A) and PBMC cells (B). (A) The cells were incubated with celastrol for 4 hours (37C, 5% CO2). The cell viability was measured by the DLin-KC2-DMA means of XTT proliferation assay. The mean optical density (OD, absorbance (= 5 (B). PBMC was activated with LPS and incubated with celastrol for 24 hours (37C, 5% CO2). The viability of cells was assessed with the Guava PCA-96 Nexin Kit by flow cytometry; mean SD, = 3 (data from PhD thesis: Study of the Molecular Mechanisms TNF-Secretion, a Key Cytokine in Chronic Inflammation by Helena Tabaka-Moreira, Universit de Strasbourg, France). Figure S4: the frequency of apoptotic and necrotic cells in LOVO cell cultures incubated with celastrol. The cells were incubated with celastrol for 4 hours (37C, 5% CO2) and then double stained with Annexin V-FITC and PI fluorescent dyes (FITC Annexin V Apoptosis Detection Kit) and analyzed by flow cytometry. The results are presented as a percentage of early apoptotic cells (Annexin V-FITC+ and PI?), late apoptotic cells (Annexin V-FITC+ and PI+), and necrotic cells (Annexin V-FITC and PI+); mean SD, = 5. 6793957.f1.pdf (604K) GUID:?DE7E47F7-322A-4A5B-9C2F-BBD50C64337B Data Availability StatementAll data used to support the findings of this study are included within the article. The additional data, demonstrating the CD44 and PARP1 expression in LOVO/DX cells, are available from the corresponding author upon request. Abstract Cancer resistance to chemotherapy is closely related to tumor heterogeneity, i.e., the existence of distinct subpopulations of cancer cells in a tumor mass. An important role is assigned to cancer stem cells (CSCs), a small subset of cancer cells with high tumorigenic potential and capacity of self-renewal and differentiation. These properties of CSCs are sustained by the ability of those cells to maintain a low intracellular reactive oxygen species (ROS) levels, via upregulation of ROS scavenging systems. However, the accumulation of ROS over a critical threshold disturbs CSCsredox homeostasis causing severe cytotoxic consequences. In the Rabbit Polyclonal to OR5A2 present study, we investigated the capacity of celastrol, a natural pentacyclic triterpenoid, to induce the formation of ROS and, consequently, cell death of the colon cancer cells with acquired resistant to cytotoxic drugs (LOVO/DX cell line). LOVO/DX cells express several important stem-like cell features, including a higher frequency of side population (SP) cells, higher expression of multidrug resistant proteins, overexpression of CSC-specific cell surface marker (CD44), increased expression of DNA repair gene (PARP1), and low intracellular ROS level. We found that celastrol, at higher concentrations (above 1?= 5, ??? 0.0001), where the MFI (mean fluorescent intensity) of DCF in LOVO/DX cells (= 6, ?? 0.01, ??? 0.001. 3.3. Cytotoxic Effect of Celastrol on LOVO/DX Cells It is well known that excessive ROS production can affect the viability of cancer cells. Therefore, we investigated whether celastrol prooxidative activity could lead to cytotoxic effects in LOVO/DX cells. The celastrol-induced cytotoxicity was DLin-KC2-DMA determined after 2 and 4 hours of the treatment by means of PI staining. In shorter incubation time, celastrol had no impact on LOVO/DX cell viability at the tested concentration range. Extending the incubation time to 4 hours led to the appearance of necrotic cells in a very small percentage (1.2C2%) at lower concentrations. At the higher concentrations, 10 and 20?= 4, ? 0.05, ??? 0.001. 3.4. Effects of Celastrol on Apoptotic Cell Death of LOVO/DX Cells ROS and mitochondria play an important role in apoptosis induction. We evaluated whether the prooxidant activity of celastrol is associated with apoptosis induction in LOVO/DX cells. Proapoptotic effects of celastrol were studied after 2 and 4 hours of incubation with LOVO/DX cells using double staining with Annexin V-FITC and PI dye. The results are presented as a percentage of DLin-KC2-DMA early apoptotic cells (Annexin V-FITC+ and PI?) and late apoptotic cells (Annexin V-FITC+ and PI+). As shown in Figures.