N-linoleyltyrosine resisted the growth of non-small cell lung cancer cells via the regulation of CB1 and CB2 involvement of PI3K and ERK pathways.

Background: N-linoleyltyrosine (NITyr), one of the anandamide analogs, exerts activity via the endocannabinoid receptors (CB1 and CB2), which showed anti-tumor effects in various tumors. Therefore, we speculated that NITyr might show anti-non-small cell lung cancer (NSCLC) effects via the CB1 or CB2 receptor. The purpose of the investigation was to reveal the anti-tumor ability of NITyr on A549 cells and its mechanisms. Methods: The viability of A549 cells was measured by MTT assay, and the cell cycle and apoptosis were both examined by flow cytometry; in addition, cell migration was tested by wound healing assay. Apoptosis-related markers were measured by immunofluorescence. The downstream signaling pathways (PI3K, ERK, and JNK) of CB1 or CB2 were examined through Western blotting. The expressions of CB1 and CB2 were detected by immunofluorescence. Finally, the AutoDock software was used to validate the binding affinity between the targets, such as CB1 and CB2, with NITyr. Results: We found that NITyr inhibited cell viability, hindered the cell cycle, resulted in apoptosis, and inhibited migration. The CB1 inhibitor, AM251, and the CB2 inhibitor, AM630, weakened the aforementioned phenomenon. The immunofluorescence assay suggested that NITyr upregulated the expression of CB1 and CB2. Western blot analysis indicated that NITyr upregulated the expression of p-ERK, downregulated the expression of p-PI3K, and did not affect p-JNK expression. In conclusion, NITyr showed a role in inhibiting NSCLC through the activation of CB1 and CB2 receptors involved in PI3K and ERK pathways.

N-linoleyltyrosine protects neurons against Aß1-40-induced cell toxicity via autophagy involving the CB2/AMPK/mTOR/ULK1 pathway.

Beta-amyloid protein (Aß) is one of the most important pathogenic factors of Alzheimer's disease (AD). N-linoleyltyrosine (NITyr) was synthesized in our laboratory and exerted neuroprotective effects in APP/PS1 transgenic mice in previous reports. In this study, the neuroprotective effects and mechanisms of NITyr were evaluated in Aß1-40-treated primary cortical neurons for the first time in vitro. NITyr treatment attenuated cytotoxicity induced by Aß1-40, and the best effect of NITyr was observed at 1 µmol/L. NITyr treatment increased the BDNF protein expression and the ratio of p-CREB/CREB, but weakened the Caspase-3 protein expression. Meanwhile, NITyr enhanced the expressions of autophagy-related proteins (LC3-II, Beclin-1, ATG5 and ATG13). The autophagy inhibitor 3-methyladenine (3MA) reversed the effects of NITyr on cell viability and the protein expressions of neuron-related proteins, including BDNF, p-CREB and Caspase-3. The CB2 receptor antagonist AM630 weakened the neuroprotective effects of NITyr and the autophagy-related protein expression (LC3-II, Beclin-1, ATG5 and ATG13). Moreover, NITyr significantly increased the expressions of p-AMPK, p-mTOR and p-ULK1, but not p-p38. AM630 ablated the above phenomenon. Therefore, NITyr protected the neurons against Aß1-40-induced cytotoxicity by inducing autophagy, which involved the CB2/AMPK/mTOR/ULK1 pathway.