Attenuation of Endoplasmic Reticulum Stress Enhances Carvacrol-Induced Apoptosis in Osteosarcoma Cell Lines

Abstract

Carvacrol is a monoterpenoid phenol that has excellent antimicrobial, antiviral, and anti-inflammatory activities. It can also improve wound healing. However, few studies have explored its antitumor effect on osteosarcoma. In this report, we tried to determine the potential efficacy of carvacrol against osteosarcoma cell lines. Our data revealed that carvacrol exposure inhibited the proliferation of osteosarcoma HOS and U-2 OS cells. In addition, carvacrol exposure enhanced the levels of cleaved PARP and caspase 3 and increased annexin V-positive cells, indicating that carvacrol exposure triggers apoptosis in osteosarcoma cell lines. Furthermore, the levels of reactive oxygen species (ROS) were enhanced after carvacrol exposure and cotreatment with NAC, the ROS scavenger, decreased the levels of cleaved PARP and caspase 3, suggesting the involvement of ROS in carvacrol-induced apoptosis. Importantly, we found that carvacrol exposure triggered several protein expressions related to endoplasmic reticulum (ER) stress, including GRP78/Bip, IRE1a, PERK, and CHOP, in HOS and U-2 OS cells, indicating that carvacrol exposure could result in ER stress in these cell lines. Cotreatment with the ER stress inhibitor 4-PBA increased the levels of cleaved PARP and caspase 3 and further suppressed cellular proliferation in carvacrol-exposed osteosarcoma cell lines. Overall, the results indicate that induced ER stress can protect cells from apoptosis, but increased ROS contributes to apoptosis in carvacrol-treated cells. In this report, we first demonstrate the role of ER stress in carvacrol-induced apoptosis and suggest that ER stress could be targeted to enhance the antitumor activity of carvacrol in osteosarcoma cell lines.