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 6
Subamolide B induces SCC12 cell death mainly through activating mitochondrial cell death pathway and cytotoxic ER stress response. ((a), (b)) The FasL/Fas cell death pathway is not required for subamolide B-induced cytotoxicity. SCC12 cells were stably infected with the pBabe vector alone or the vectors expressing a dominant-negative form of FADD (dnFADD) (a) or c-FLIPL (b) to block the function of the FasL/Fas cell death pathway. These stable clones were then treated with 20 μM of subamolide B for 24 h to monitor the status of PARP cleavage in addition to enforced expression of dnFADD or c- (left panel), or for 48 h to determine cell viability using PI exclusion assay (right panel). (c) Mitochondrial cell death pathway is involved in subamolide B-induced cytotoxicity. SCC12 cells stably carrying the pBabe vector alone or the vector overexpressing BCL-2 to impair the activation of mitochondrial cell death pathway were treated with subamolide B (20 μM) for 24 h and 48 h to evaluate the effect of enforced BCL-2 expression on subamolide B-induced PARP cleavage (left panel) and cytotoxicity (right panel), respectively. (d) Involvement of CHOP-mediated ER stress cell death pathway in subamolide B-induced cytotoxicity. SCC12 cells were stably infected with the pMKO vector alone or the vector expressing CHOP-specific siRNA (shCHOP) to inhibit ER stress-induced cell death. These stable clones were then treated with subamolide B (20 μM) for 24 h and 48 h to evaluate the effect of CHOP knockdown on subamolide B-induced PARP cleavage (left panel) and cytotoxicity (right panel), respectively. **; ***. Sub-B: subamolide B.
