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Cannabidiol (CBD) is a nonpsychoactive cannabinoid compound. It has been shown that CBD can inhibit the proliferation of ovarian cancer cells, but the underlying specific mechanism is unclear. We previously presented the first evidence for the expression of leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1), a member of the immunosuppressive receptor family, in ovarian cancer cells. In the present study, we investigated the mechanism by which CBD inhibits the growth of SKOV3 and CAOV3 ovarian cancer cells, and we sought to understand the concurrent role of LAIR-1. In addition to inducing ovarian cancer cell cycle arrest and promoting cell apoptosis, CBD treatment significantly affected the expression of LAIR-1 and inhibited the PI3K/AKT/mTOR signaling axis and mitochondrial respiration in ovarian cancer cells. These changes were accompanied by an increase in ROS, loss of mitochondrial membrane potential, and suppression of mitochondrial respiration and aerobic glycolysis, thereby inducing abnormal or disturbed metabolism and reducing ATP production. A combined treatment with N-acetyl-L-cysteine and CBD indicated that a reduction in ROS production would restore PI3K/AKT/mTOR pathway signaling and ovarian cancer cell proliferation. We subsequently confirmed that the inhibitory effect of CBD on the PI3K/AKT/mTOR signal axis and mitochondrial bioenergy metabolism was attenuated by knockdown of LAIR-1. Our animal studies further support the in vivo anti-tumor activity of CBD and suggest its mechanism of action. In summary, Li Ma and Huachang Zhang contributed equally to this study.

Abstract. The tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is deficient in various types of human tumors due to mutations or epigenetic alterations. PTen promoter hypermethylation is a major epigenetic silencing mechanism leading to self-repression in these tumors. The present study aimed to investigate whether PTen promoter methylation is involved in the regulation of the PTen gene in adenoid cystic carcinoma (acc) cells. The expression of PTEN in ACC-2 cells was found to be significantly lower than that in normal salivary gland epithelial cells using rT-Pcr analysis. The existence of cpG island methylation in the PeTn promoter region in acc-2 cells was demonstrated by methylation-specific PCR (MSP) analysis and direct sequencing of MSP product. rT-Pcr, Western blot analysis and luciferase assay showed that mrna and protein expression and the promoter activity of PTen in acc-2 cells treated with the dna methylation inhibitor 5-aza-2-deoxycytidine were significantly up-regulated in a time-dependent manner. These results indicate that the hypermethylation of the PTen promoter region leads to lower expression of PTen gene in acc cells, which aids in the development of PTen as a molecular marker for the early diagnosis of this carcinoma. IntroductionSalivary adenoid cystic carcinoma (acc) is one of the most common salivary gland malignancies and the most common cause of oral cancer-related death worldwide. at present, the biological mechanism behind the proliferation, differentiation, invasion and metastasis of salivary adenoid cystic carcinoma remains unclear (1,2). it has been reported that a variety of oncogenes, including c-erbB-2, c-myc, ras and bcl-2, are associated with adenoid cystic carcinoma (3-6). in addition, studies have reported that the tumor suppressor gene p53 is involved in the pathogenesis of salivary adenoid cystic carcinoma. However, increased expression of p53 in salivary adenoid cystic carcinoma was observed in contrast to its reduced expression in many other tumors, which indicated that it does not serve as a diagnostic marker of salivary gland tumors (7,8).Phosphatase and tensin homolog deleted on chromosome 10 (PTen), a tumor suppressor that regulates multiple cellular functions including cell growth and survival, differentiation and proliferation, apoptosis, focal adhesion, invasion, migration as well as angiogenesis, is frequently deficient in various tumors due to mutations or epigenetic alterations (9). PTen promoter hypermethylation is a major epigenetic silencing mechanism leading to low expression in tumors (10). Previous studies have shown that the abnormal expression of PTen in oral and maxillofacial tumors is related to the occurrence, development and invasion of tumors (11). The high expression of PTen in normal salivary glands and low expression in adenoid cystic carcinoma has also been observed, but the molecular mechanisms are unclear (12,13).in this study, we focused on whether PTen promoter methylation is involved in the regulation of the ...