Sodium arsenite-induced autophagic cell death in rat insulinoma cells

  Objective  To study the role and molecular mechanism of autophagy in death of rat insulinoma cells (INS-1) induced by sodium arsenite (NaAsO₂).

  Methods  Three dose groups (administered with NaAsO₂ of 30, 15, 5 μmol/L for 24 hours) and one negative control group of rat INS-1 cells were established. The survival rate of INS-1 cells was detected with cholecystokinin-8 (CCK8) colorimetry. The ultrastructure of INS-1 cells was observed with transmission electron microscope. The fluorescence expression of INS-1 cells after transfection of plasmid GFP-LC3 was observed with laser scanning confocal microscopy. The expression of light chain 3 (LC3), P62, mammalian target of rapamycin (mTOR) and phosphatidylinositol 3-kinase (PI3K) protein was detected with Western blot.

  Results  After NaAsO₂ treatment, the survival rate of INS-1 cells decreased significantly. Two-layer membrane autophagosomes and autophagic vacuoles were significantly increased in INS-1 cells of NaAsO₂ groups. After transfection of plasmid GFP-LC3, the aggregation of green dot-like fluorescent spots was observed under the fluorescence microscope of NaAsO₂ groups. Compared with those in the control cells, significantly increased protein expressions of LC3II/LC3I (1.34 ± 0.02, 1.16 ± 0.02, and 1.13 ± 0.04 vs. 0.93 ± 0.04) and P62 (1.89 ± 0.14, 1.08 ± 0.06, and 0.71 ± 0.03 vs. 0.57 ± 0.01), but decreased protein expressions of mTOR (0.46 ± 0.04, 0.92 ± 0.06, and 1.15 ± 0.06 vs. 1.44 ± 0.03 ) and PI3K (0.78 ± 0.04, 0.87 ± 0.03, and 0.91 ± 0.04 vs. 1.26 ± 0.07) were detected in the INS-1 cells treated with 30, 15, and 5 μmol/L NaAsO₂ (P < 0.05 for all).

  Conclusion  NaAsO₂ treatment can induce autophagic death in INS-1 cells and the mechanism of the effect may be related to the regulation of PI3K/mTOR signaling pathway.