Platycodin D induce crosstalk between apoptosis and necroptosis in MDA-MB-231 cells by binding to caspase 6
The aim of the present study was to elucidate the diversity of anti-cancer mechanisms of Platycodin D (PD). This was achieved by identifying the binding targets of PD that induce cell death of the triple-negative breast cancer cell line, MDA-MB-231. Cell viability was assessed by CCK8 assay and the IC50 value of PD in different breast cancer cells was determined, with MDA-MB-231 cells treated with PD used as the study subject. Apoptosis was detected by the level of mitochondrial membrane potential (MMP), the ratio of Annexin V+/PI+ cells and the expression of caspase 3. To further clarify the characteristics of apoptosis, various inhibitors were applied, and cell morphology was observed through confocal microscopy, transmission electron microscopy and imaging flow cytometry. RNA-seq focused on the possible pathways in which PD induces cell death by comparing differences in transcription levels. In addition, thermal proteome profiling (TPP) and molecular docking were used to identify the binding targets of PD in the cells. The results showed that PD inhibited the viability of MDA-MB-231 cells in a concentration-dependent manner and induced apoptosis. Apoptotic cells exhibited decreased MMP, increased membrane permeability and activation of caspase 3. The effect of PD was not blocked by z-VAD-fmk, but was reversed by necrostatin-1, and the cells treated with PD exhibited necrosis-like morphological features. The enrichment analysis results showed that PD-induced regulated cell death (RCD) had an impact on the transcriptional levels of apoptosis, necroptosis and ferroptosis. In addition, caspase 6 (CASP6), as a key regulator in RCD crosstalk, was found in TPP and molecular docking, and the inhibition of its activation by PD was demonstrated by its expression level and gene silencing. Therefore, CASP6 is one of the targets of the anti-cancer activity driven by PD and leads to cell death characterized by necroptosis.