beta-Elemonic acid inhibits the growth of human Osteosarcoma through endoplasmic reticulum (ER) stress-mediated PERK/eIF2 alpha/ATF4/CHOP activation and Wnt/beta-catenin signal suppression

Background: Osteosarcoma (OS) is a significant threat to the lives of children and young adults. Although neoadjuvant chemotherapy is the first choice of treatment for OS, it is limited by serious side-effects and cancer metastasis. beta-Elemonic acid (beta-EA), an active component extracted from Boswellia carterii Birdw., has been reported to exhibit potential anti-inflammatory and anticancer activities. However, the anti-tumor effects and underlying mechanisms on OS as well as pharmacokinetic characteristics of beta-EA remain unknown. Purpose: This study was aimed to investigating the anti-tumor effects of beta-EA on human OS, the underlying mechanisms, and the pharmacokinetic and tissue distribution characteristics. Study design and methods: Cell viability and colony formation assays were performed to determine the effect of beta-EA cell on cell proliferation. Apoptosis rates, mitochondrial membrane potential and cell cycle features were analyzed by flow cytometry. qRT-PCR, Western blot, immunofluorescence and immunohistochemical assays were conducted to evaluate the expression levels of genes or proteins related to the pathways affected by beta-EA in vitro and in vivo. Cell migration and invasion were evaluated in wound healing and Transwell chamber assays. The effects and pharmacokinetic characteristics of beta-EA in vivo were evaluated by analyzing tumor suppression, pharmacokinetics and tissue distribution. Results: Explorations indicated that endoplasmic reticulum (ER) stress conditions provoked by beta-EA activated the PERK/eIF2 alpha/ATF4 branch of the unfolded protein reaction (UPR), stimulating C/EBP homologous protein (CHOP)-regulated apoptosis and inducing Ca2+ leakage leading to caspase-dependent apoptosis. Furthermore, beta-EA induced G0/G1 cell cycle arrest and inhibited metastasis of HOS and 143B cells by attenuating Wnt/beta-catenin signaling effects, which included decreased levels of p-Akt(Ser473), p-Gsk3 beta (Ser9), Wnt/beta-catenin target genes (c-Myc and CyclinD1) along with a decline in nuclear beta-catenin accumulation. The fast absorption, short elimination half-life, and linear pharmacokinetic characteristics of beta-EA were also revealed. The distribution of beta-EA was detected in the tumor and bone tissues. Conclusions: Overall, both in vitro and in vivo investigations showed the potential of beta-EA for the treatment of human OS. The pharmacokinetic profile and considerable distribution in the tumor and bone tissues warrant further preclinical or even clinical studies.