ABSTRACT
@#[Abstract] Objective: To study the effect of micheliolide (MCL) on the sensitivity of colorectal cancer cells to oxaliplatin (OxP) and its possible mechanism. Methods: HCT116 and LoVo cells were treated with 2 μmol/L MCL and 100 μmol/L OxP alone or in combination. The cell viability and colony forming ability in vitro were detected by CCK-8 and plate cloning formation assay, respectively. After being transfected with GFP-LC3 lentivirus, HCT116 cells were respectively treated with 2 μmol/L and 5 μmol/L MCL for 24 h. The aggregation of autophagy bodies in HCT116 cells induced by MCL was observed under fluorescence microscope. The effects of MCL on the expressions of LC3B-Ⅰ, LC3B-Ⅱ, p62 and STAT3 were detected by WB assay; the molecular docking model of MCL and STAT3 was constructed by Autodock version. Results: After the treatment of 2 μmol/L MCL combined with 100 μmol/L OxP, the activity of HCT116 and LoVo cells as well as the colony forming ability of HCT116 cells significantly decreased (all P < 0.01). After HCT116 cells were treated with 2 and 5 μmol/L MCL, the autophagy rate of cells in the treatment groups was significantly higher than that of the control group (all P < 0.01), the LC3B Ⅱ/Ⅰ ratio was 3.25 and 5.78 times that of the control group, the expression level of p62 was 25.5% and 9.8% of the control group, and the phosphorylation level of STAT3 was 2.18 and 3.87 times that of the control group. Molecular docking results showed that MCL might directly bind to STAT3 protein in vivo. Conclusion: MCL may enhance the sensitivity of colorectal cancer cells to OxP by promoting autophagy through STAT3 pathway.
ABSTRACT
@#[Abstract] Objective: To observe the pyrolysis of colorectal cancer Lovo cells overexpressing Gasdermin E (GSDME) after the treatment with oxaliplatin. Methods: The expression level of GSDME gene in colorectal cancer Lovo cells and normal colorectal epithelial HCOEPIC cells was detected by qPCR. The GSDME-WT (wild-type GSDME) and GSDME-D270A (mutant GSDME) recombinant plasmids were constructed. The plasmids were packaged as lentivirus and then transfected into Lovo cells to construct Lovo cell line with stable and high expression of GSDME. Western blotting was used to detect the expression level of GSDME in cells of WT, D270A and empty vector groups. Different concentrations of oxaliplatin (0, 4, 8, 16, 32, 64 µg/ml) were applied to treat Lovo cells and HCOEPIC cells in WT and D270A groups, and the morphological changes of the cells were observed under a microscope. Results: The expression of GSDME in HCOEPIC cells was significantly higher than that in Lovo cells (P<0.01). GSDME-WT and GSDME-D270A plasmids with high GSDME expression and the corresponding Lovo cell lines were successfully constructed. Compared with the empty vector group, the expression level of GSDME in Lovo cells of WT and D270A groups were significantly increased (all P<0.05). Observation under the microscope showed that after being treated with 64 µg/ml oxaliplatin for 9 and 12 hours, the volume of Lovo cells and HCOEPIC cells in WT group gradually increased and “blistered” to one side and showed obvious pyrolysis phenomenon. The pyrolysis rate of cells in WT group was significantly higher than that of the control group without oxaliplatin treatment (Lovo cells: [7.405±1.010]% vs [3.441±0.401]%, P<0.05; HCOEPIC cells: [7.203±1.020]% vs [4.201±0.302]%, P<0.05). Conclusion: Oxaliplatin promotes the pyrolysis of colorectal cancer Lovo cells overexpressing GSDME gene.