Licochalcone A, a flavonoid extracted from licorice root, has been shown to exhibit broad anti-inflammatory, anti-bacterial, anticancer, and antioxidative bioactivity. In this study, we investigated the antitumor activity of Licochalcone A against human osteosarcoma cell lines. The data showed that Licochalcone A significantly suppressed cell viability in MTT assay and colony formation assay in osteosarcoma cell lines. Exposure to Licochalcone A blocked cell cycle progression at the G2/M transition and induced extrinsic apoptotic pathway in osteosarcoma cell lines. Furthermore, we found the Licochalcone A exposure resulted in rapid ATM and Chk2 activation, and high levels of nuclear foci of phosphorylated Chk2 at Thr 68 site in osteosarcoma cell lines. In addition, Licochalcone A exposure significantly induced autophagy in osteosarcoma cell lines. When Licochalcone A-induced autophagy was blocked by the autophagy inhibitor chloroquine, the expression of activated caspase-3 and Annexin V positive cells were reduced, and cell viability was rescued in Licochalcone A-treated osteosarcoma cell lines. These data indicate that the activation of ATM-Chk2 checkpoint pathway and autophagy may contribute to Licochalcone A-induced anti-proliferating effect in osteosarcoma cell lines. Our findings display the possibility that Licochalcone A may serve as a potential therapeutic agent against osteosarcoma.

Ursolic acid (UA), a naturally occurring pentacyclic triterpene acid found in many medicinal herbs and edible plants, triggers apoptosis in several tumor cell lines but not in human bone cancer cells. Most recently, we have demonstrated that UA exposure reduces the viability of human osteosarcoma MG-63 cells through enhanced oxidative stress and apoptosis. Interestingly, an inhibitor of osteoclast-mediated bone resorption, zoledronic acid (ZOL), also a third-generation nitrogen-containing bisphosphonate, is effective in the treatment of bone metastases in patients with various solid tumors. In this present study, we found that UA combined with ZOL to significantly suppress cell viability, colony formation, and induce apoptosis in two lines of human osteosarcoma cells. The pre-treatment of the antioxidant had reversed the oxidative stress and cell viability inhibition in the combined treatment, indicating that oxidative stress is important in the combined anti-tumor effects. Moreover, we demonstrated that ZOL combined with UA significantly induced autophagy and co-administration of autophagy inhibitor reduces the growth inhibitory effect of combined treatment. Collectively, these data shed light on the pathways involved in the combined effects of ZOL and UA that might serve as a potential therapy against osteosarcoma.

Dodecyl gallate (DG) is a gallic acid ester that has been shown to inhibit tumor growth. The aim of this study was to investigate the mechanism by which DG induces antiproliferative and apoptotic effects in MG‑63 human osteosarcoma cells. Dose‑ and time‑dependent cytotoxic effects of DG were determined using an MTT assay. The results showed that the half‑maximal inhibitory concentration (IC50) of DG in MG‑63 cells was 31.15 µM at 24 h, 10.66 µM at 48 h, and 9.06 µM at 72 h. Flow cytometric analysis demonstrated that exposure to 20 and 40 µM DG resulted in an increase in the sub‑G1 phase population and in S‑phase cell cycle arrest. Furthermore, western blot analysis of apoptosis‑related protein expression revealed an increase in the activation of caspases 8 and 3, cleavage of poly (ADPribose) polymerase (PARP), and disruption of mitochondrial membrane permeability was measured by flow cytometry. An increase in the Bax/Bcl‑2 ratio and a decrease in the expression of inhibitor of apoptosis protein (IAP) family members, namely X-linked inhibitor of apoptosis protein and survivin, were also observed following DG treatment. These data provide insight into the molecular mechanisms governing the ability of DG to induce apoptosis in human osteosarcoma cells in vitro.

Inhibition of proliferating cells is a critical strategy for cancer therapy. In this study, we demonstrated that coronarin D, a natural component extracted from the rhizomes of Hedychium coronarium, significantly suppressed the proliferation of osteosarcoma cells. The treatment with coronarin D resulted in the activation of caspase-3 and apoptosis. This treatment induced the accumulation of cyclin B1 and DNA condensation indicating the treated osteosarcoma cells were arrested in mitotic phase. Furthermore, the treatment with coronarin D increased the levels of phosphorylated c-Jun NH2-terminal kinase (JNK) in human osteosarcoma cells. Pretreatment with JNK inhibitor blocked the accumulation of cyclin B1 and DNA condensation, resulting the accumulation of tetraploid cells in coronarin D-treated osteosarcoma HOS cells, indicating JNK inactivation blocked the mitotic entry and arrested cells in the 4 N state. After adaptation, the arrested tetraploid cells continued to duplicate their DNA resulting in polyploidy. Interestingly, when the arrested mitotic cells induced by coronarin D were treated with JNK inhibitor, the accumulated cyclin B1 and DNA condensation were immediately eliminated. These arrested 4 N cells loss the ability to undergo cytokinesis, and ultimately continued to duplicate DNA upon prolonged arrest resulting in the production of polyploid populations. JNK inactivation, either by the pretreatment with JNK inhibitor or the treatment with JNK inhibitor in coronarin D-induced mitotic cells, both caused resistance to coronarin D-induced cell death. Taken together, our findings indicate that coronarin D induces the apoptosis and mitosis arrest in human osteosarcoma cells. JNK has a crucial role in coronarin D-induced mitosis arrest and apoptosis. We hypothesize that functional evaluation of JNK may produce more specific and effective therapies in coronarin D-related trail for treatment of human osteosarcoma.

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.