Int J Mol Sci. significantly, inhibition of autophagy by chloroquine diphosphate salt (CQ) remarkably enhanced apoptosis, while the caspase inhibitor z\VAD\fmk failed in affecting autophagy, suggesting that corilagin\induced autophagy functioned as a survival mechanism in MCF\7 cells. In addition, corilagin induced intracellular reactive oxygen species (ROS) generation, when reduced by ROS scavenger NAC, apoptosis and autophagy were both down\regulated. Nevertheless, in SK\BR3 cell which expressed RIP3, necroptosis inhibitor Nec\1 could not alleviate cell death induced by corilagin, indicating necroptosis was not triggered. AF-9 Subcutaneous tumour growth in nude mice was attenuated by corilagin, consisting with the results in?vitro. These results imply that corilagin inhibits cancer cell proliferation through inducing apoptosis and autophagy which regulated by ROS release. test with Prism 5 software. All data are expressed as mean??standard deviation (SD) or standard error of mean (SEM), and value less than.05 was considered statistically significant. 3.?RESULTS 3.1. Corilagin suppress growth in MCF\7 cells but not in normal cells To investigate the cytotoxic effect of corilagin (structure in Figure?1A) in human breast cancer MCF\7 cells, MTT and EdU assay were employed. Results showed that corilagin inhibited viability (Figure?1B) and proliferation (Figure?1D) of MCF\7 cells in a dose\dependent manner. Additionally, corilagin markedly decreased clonogenicity (Figure?1G and H) and protein expression of PCNA and KI\67 (Figure?1I), which demonstrated corilagin notably suppress growth in MCF\7 cells. We also utilized breast cancer cell lines MDA\MB\231 and Bcap\37 to detect the effects of corilagin on them, as they both showed a certain degree of drug resistance (Figure?S1Cand D) comparing with MCF\7, we chose MCF\7 as our target to further study. Besides, we detected that corilagin had a high efficiency in depressing the viability of colorectal adenocarcinoma cells HT\29 (Figure?S1E) and cervical carcinoma cells Hela (Figure?S1F). Open in a separate window Figure 1 Corilagin suppresses the growth of MCF\7. (A) The chemical structure of corilagin. (B) MCF\7 cells were treated with 0, 20, LAS101057 40, 60, LAS101057 80, 100?mol/L corilagin for 48?h, Cell viability were analysed by MTT assay. (C) MCF\10A, (E) L02, (F) GES\1 cells were treated with corilagin at concentrations ranging from 0 to 110?mol/L for 24?h. Cell viability was analysed using MTT assay. (D) EdU assay was performed to assess the growth inhibiting effects to MCF\7 cells. (G and H) Representative images of colony\forming assay and its counting results. (I) MCF\7 cells were treated with different concentrations of corilagin for 24?h. The total protein was extracted, and the expression of PCNA and Ki\67 proteins was analysed by Western blot assay. Data are expressed as means (n??3)??SD over controls, ***P?<?.001, ****P?<?.0001 In addition, experiments on corilagin\treated normal cells were performed to investigate whether corilagin has targeting property. MTT assay revealed that cell viability was not decreased in corilagin\treated mammary epithelial cells MCF\10A, hepatic epithelial cells L02 and gastric epithelial cells GES\1(Figure?1C, E and F). Besides, EdU assay showed that corilagin treatment group had no difference with control group in GES\1 cells (Figure?S1A) and L02 cells (Figure?S1B). These data demonstrate that corilagin can specifically inhibit the growth of breast cancer cells MCF\7 and barely suppress normal cells. 3.2. Corilagin activate extrinsic and intrinsic mitochondrial apoptosis pathways in MCF\7 cells Research showed that corilagin treatment activated apoptosis in ovarian cancer cells, which significantly increased the number of apoptotic cells.9, 10, 27 Then we tried to reveal the mode of cell death induced by corilagin treatment in MCF\7 cells. LDH release assay showed that the release of LDH increased markedly in corilagin\treated MCF\7 cells (Figure?2A), suggesting that cell damage and cell death occurred. Besides, formation of apoptosis body was found by transmission electron microscope (TEM) imaging in corilagin\treated MCF\7 cells (Figure?2B), indicating apoptosis was activated. LAS101057 Open in a separate window Figure 2 Corilagin introduces apoptosis in MCF\7. (A) MCF\7 cells.
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