Methamphetamine induces autophagy as a pro-survival response against apoptotic endothelial cell death through the Kappa opioid receptor

Ma, J; Wan, J; Meng, J; Banerjee, S; Ramakrishnan, S; Roy, S

We have shown that KOR has a role in METH-induced autophagy (Figure 3 ). To determine whether inhibiting METHinduced autophagy triggers apoptosis, we also examined effects of the KOR antagonist pretreatment on METHinduced apoptotic cell death. Pretreatment with nor-BNI resulted in an increase in the number of apoptotic cells (12.90%) compared with METH treatment (8.14%) and control (1.32%) (Figure 5d ). To further determine the mechanism underlying METH-induced apoptosis in the context of an autophagic block, caspase activity was measured in METH-treated HUVECs using Caspase-Glo 8, Caspase-Glo 3/7 and Caspase-Glo 9 assay systems in the presence or absence of MEK inhibitor PD98059. Although METH treatment alone resulted in modest induction of caspase-3/7, the induction was dramatically increased when cells were pretreated with the MEK inhibitor PD98059 (Figure 5e ). We also investigated the effect of knockdown of Beclin1 on the caspase-3 activity of HUVECs. HUVECs were transfected with Beclin1 siRNA. Beclin1 mRNA expression was detected by real-time PCR analysis. The activity of caspase-3 was also measured in HUVECs transfected with Beclin1 knockdown. The results showed that Beclin1 siRNA transfection significantly decreased the mRNA expression of Beclin1 in the transfected HUVECs compared with scramble siRNA transfection (Figure 5f ). METH significantly increased caspase-3 activity in the HUVECs with Beclin1 knockdown compared with METH treatment alone (Figure 5g ). In conclusion, METH promoted cell apoptosis in the presence of Beclin1 knockdown in HUVECs.