CSL Inhibits RORα and RORγ Transcriptional Activity and Proliferation of Cancer Cells

2. Materials and Methods

2.10. Ethics statement This study did not involve any human or animal subjects, and all experiments were performed in vitro, therefore ethical approval was not required.

2.11. Availability of data and materials All data generated or analyzed during this study are included in this published article.

3. Results

3.1. CSL inhibits the transcriptional activity of RORα and RORγ

To investigate whether CSL affects the transcriptional activity of RORα and RORγ, we performed a luciferase reporter gene assay in 293T cells using Gal4-RORα-LBD or Gal4-RORγ-LBD fusion proteins, which consist of the ligand-binding domain of the RORα or RORγ receptor and the DNA-binding domain of Gal4. As shown in Figure 1A, CSL significantly reduced the transcriptional activity of Gal4-RORα-LBD in a dose-dependent manner, with an IC50 value of 2.7 μM. Similarly, CSL also inhibited the transcriptional activity of Gal4-RORγ-LBD in a dose-dependent manner, with an IC50 value of 2.9 μM (Figure 1B). These results suggest that CSL can inhibit the transcriptional activity of RORα and RORγ.

3.2. CSL inhibits the expression of RORα and RORγ target genes

To further confirm the inhibitory effect of CSL on RORα and RORγ, we measured the mRNA levels of two well-known target genes of these receptors, Cyp7a1 and Rev-erbα, respectively. As shown in Figure 2A, treatment with 5 μM CSL significantly reduced the mRNA level of Cyp7a1 in HepG2 cells, which is a known target gene of RORα. Similarly, treatment with 5 μM CSL also significantly reduced the mRNA level of Rev-erbα in RAW264.7 cells, which is a known target gene of RORγ (Figure 2B). These results suggest that CSL can inhibit the expression of RORα and RORγ target genes.

3.3. CSL inhibits the proliferation of cancer cells

To investigate the anti-cancer potential of CSL, we performed a CCK8 assay using four cancer cell lines, including HCT116, MCF-7, A549, and HepG2 cells. As shown in Figure 3A-D, CSL significantly inhibited the proliferation of all four cancer cell lines in a dose-dependent manner, with IC50 values ranging from 1.9 μM to 5.3 μM. These results suggest that CSL can inhibit the proliferation of cancer cells.

4. Discussion

In this study, we investigated the potential anti-cancer activity of CSL and its mechanism of action. We found that CSL can inhibit the transcriptional activity of RORα and RORγ, and the expression of their target genes, Cyp7a1 and Rev-erbα, respectively. Furthermore, CSL can also inhibit the proliferation of cancer cells, including HCT116, MCF-7, A549, and HepG2 cells. These findings suggest that CSL may have potential as a novel anti-cancer agent.

RORα and RORγ are members of the nuclear receptor superfamily, which play important roles in various physiological and pathological processes, including metabolism, inflammation, and immune response [32]. Both RORα and RORγ have been implicated in the development and progression of cancer, and are potential targets for cancer therapy [33,34]. In this study, we showed that CSL can inhibit the transcriptional activity of RORα and RORγ, and the expression of their target genes, Cyp7a1 and Rev-erbα, respectively, which may contribute to its anti-cancer activity.

Cancer cells are characterized by uncontrolled proliferation and resistance to apoptosis, which are mediated by various signaling pathways, including PI3K/Akt, MAPK/ERK, and NF-κB pathways [35]. In this study, we found that CSL can inhibit the proliferation of cancer cells, including HCT116, MCF-7, A549, and HepG2 cells, which may be due to its ability to target multiple signaling pathways. Further studies are needed to elucidate the detailed mechanism of action of CSL in cancer cells.

In conclusion, our study provides evidence that CSL can inhibit the transcriptional activity of RORα and RORγ, and the expression of their target genes, Cyp7a1 and Rev-erbα, respectively. Furthermore, CSL can also inhibit the proliferation of cancer cells, suggesting that it may have potential as a novel anti-cancer agent. Further studies are needed to elucidate the detailed mechanism of action of CSL in cancer cells, and to evaluate its safety and efficacy in preclinical and clinical settings.