| Previous chemical researches have demonstrated that Cimicifuga foetida L. contained plenty of cycloartane triterpenoids which have been proved to have growth inhibitory activities on several cancers in vivo and in vitro. However, previous research mainly focused on isolation and structure identification rather than biological activity studies. Our study aimed to explore the anti-tumor mechanisms of compound SM306 and SM308 isolated from Cimicifuga foetida L. The work has been done as following:The growth inhibitory effects of compound SM306 on human cancer cell lines were evaluated by MTT assay and colony formation assay. We found that SM306 showed significant growth inhibition on MCF-7 cells in a dose- and time-dependent manner. Flow cytometry analysis revealed that SM306 induced a remarkable G2/M arrest, accompanied with the inhibition of cyclin B1 and phospho-CDK1 (Thr161) levels. We.also found that SM306 could induce mitochondria-mediated apoptosis in MCF-7 cells, which was supported by the increase of apoptotic rates, collapse of mitochondrial membrane potential, activation of Caspase 9 and PARP, alteration of Bcl-2 and Bax levels, as well as the decreased levels of p-Akt (Thr308 and Ser473). In addition, SM306 treatment could inhibit the phosphorylation of ERK1/2 and Raf, suggesting the involvement of Raf/MEK/ERK signaling pathway in SM306-induced apoptosis in MCF-7 cells.The effects of compound SM308 on autophagy levels and apoptosis were investigated in HepG2/ADM cells. Firstly, we found that SM306 showed significant growth inhibition on HepG2/ADM cells, especially under starvation condition. The increase of LC3 levels and MDC-stained acidic vacuoles indicated the appearance of autophagosomes after SM3O8 treatment. LC3 Turnover assay and the blocking of p62/SQSTM1 degradation demonstrated that SM308 inhibited the autophagic flux in HepG2/ADM cells. Then the accumulation of autolysosomes with a single membrein containing undigested substrates was discovered by TEM. Furthermore, the protein degradation activity of lysosomes was inhibited, as demonstrated by DQ-BSA assay. Interestingly, we observed the activation of PI3K/Akt signalling pathway after SM308 treatment. Moreover, the pretreatment of PI3K inhibitor or Akt siRNA rescued against SM308-induced autophagic flux disruption, providing a possibility for PI3K/Akt pathway involved in the inhibition of autophagic flux by compound SM308.In addition, flow cytometry analysis and the activation of apoptosis associated proteins indicated that SM308 could induce apoptosis in HepG2/ADM cells. Furthermore, starvation condition or mTOR inhibitor, a strong physiological inducer of autophagy, obviously enhanced SM308-induced cell apoptosis. The results demonstrated that the inhibition of autophagic flux enhanced the apoptosis after SM308 treatment in HepG2/ADM cells.In summary, we investigate the antitumor mechanisms of cycloartane triterpenoids and provides a rationale for the research and development as well as clinical application of cycloartane triterpenoids as a chemotherapeutic agent for human cancers. |
