Subamolide B Isolated from Medicinal Plant Cinnamomum subavenium Induces Cytotoxicity in Human Cutaneous Squamous Cell Carcinoma Cells through Mitochondrial and CHOP-Dependent Cell Death Pathways
Figure 5
Subamolide B is an ER stress inducer. (a) Subamolide B upregulates signature ER stress markers GRP78 and CHOP. SCC12 cells treated with subamolide B (0~20 μM) for 24 h were subjected to immunoblot analysis for the levels of GRP78 and CHOP. β-tubulin was used as the loading control. (b) Subamolide B increases CHOP mRNA expression levels. The level of CHOP mRNA in SCC12 cells after 24 h treatment with subamolide B (0~20 μM) was assessed by semiquantitative RT-PCR analysis. β-actin was used as the loading control. (c) Subamolide B activates the human CHOP promoter. SCC12 cells transiently transfected with the human CHOP promoter reporter plasmid pCHOP-Luc (−947~+30) were subjected to subamolide treatment (0~20 μM) for 24 h, followed by luciferase activity assay to determine the CHOP promoter activity. (d) Subamolide B activates the IRE1 arm of the UPR signaling pathway. SCC12 cells were treated with subamolide B (0~20 μM) or the ER stress inducer thapsigargin (0.1 μM) for 24 h, followed by immunoblotting and semiquantitative RT-PCR analyses for the levels of Ser724-phosphorylated IRE1/total IRE1 (upper panel) and the splicing of XBP1 mRNA (lower panel), respectively. The levels of β-tubulin and β-actin were the respective loading controls for immunoblotting and semiquantitative RT-PCR analyses. (e) Subamolide B induces ATF6 activation. SCC12 cells were transiently transfected with the ATF6 transcriptional activity reporter plasmid (p5xATF6-GL3) and then under 24 h treatment with subamolide B (0, 10 μM) or thapsigargin (0.1 μM). ATF6 transcriptional activity was then evaluated by luciferase activity assay. (f) Subamolide B engages the PERK branch of the UPR signaling pathway. SCC12 cells after 24 h treatment with subamolide B (0~20 μM) or thapsigargin (0.1 μM) were subjected to immunoblotting for the levels of PERK-mediated Ser51 phosphorylation of eIF2α (p-eIF2α) and total eIF2α. β-tubulin was used as the loading control. The activity of the CHOP promoter or ATF6 is presented as the ratio of the luciferase activities after drug treatment to that of drug-untreated controls. **; ***. Sub-B: subamolide B; TG: thapsigargin.
