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.

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.

Hinokitiol is a tropolone-related compound isolated from the heartwood of cupressaceous plants. It is known to exhibit various biological functions including antibacterial, antifungal, and antioxidant activities. In the study, we investigated the antitumor activities of hinokitiol against human osteosarcoma cells. The results revealed that hinokitiol treatment inhibited cell viability of human osteosarcoma U-2 OS and MG-63 cells in the MTT assay. Further study revealed that hinokitiol exposure caused cell cycle arrest at the S phase and a DNA damage response with the induction of γ-H2AX foci in both osteosarcoma cell lines. In U-2 OS cells with wild-type tumor suppressor p53, we found that hinokitiol exposure induced p53 expression and cellular senescence, and knockdown of p53 suppressed the senescence. However, in MG-63 cells with mutated p53, a high percentage of cells underwent apoptosis with cleaved-PARP expression and Annexin V staining after hinokitiol treatment. In addition, up-regulated autophagy was observed both in hinokitiol-exposed U-2 OS and MG-63 cells. As the autophagy was suppressed through the autophagy inhibitor chloroquine, hinokitiol-induced senescence in U-2 OS cells was significantly enhanced accompanying more abundant p53 expression. In MG-63 cells, co-treatment of chloroquine increased hinokitiol-induced apoptosis and decreased cell viability of the treated cells. Our data revealed that hinokitiol treatment could result in different cell responses, senescence or apoptosis in osteosarcoma cell lines, and suppression of autophagy could promote these effects. We hypothesize that the analysis of p53 status and co-administration of autophagy inhibitors might provide more precise and efficacious therapies in hinokitiol-related trials for treating osteosarcoma.

Muscle contusion is an injury to muscle fibers and connective tissues. It commonly happens in impact events, and could result in pain, swelling, and limited range of motion. Diclofenac is one of commonly used nonsteroidal anti-inflammatory drugs to alleviate pain and inflammation after injury. However, it can potentially cause some side effects including gastrointestinal complications and allergy. Betulin is a lupine-type pentacyclic triterpenoid. It is showed to have valuable pharmacological effects, but the physiological effect of betulin on muscle contusion has not been reported. This study aimed to explore the therapeutic effects of betulin on muscle contusion that produced by the drop-mass method in mice. C57BL/6 mice were randomly assigned to control (no injury), only drop-mass injury (Injury), diclofenac treatment (Injury+diclofenac), and betulin treatment (Injury+betulin) groups. Injury was executed on the gastrocnemius of the right hind limb, and then phosphate-buffered saline (PBS), diclofenac, or betulin were oral gavage administrated respectively for 7 days. Results revealed that betulin significantly restored motor functions based on locomotor activity assessments, rota-rod test, and footprints analysis. Betulin also attenuated serum creatine kinase (CK) and lactate dehydrogenase (LDH) levels after muscle injury. Neutrophil infiltration was alleviated and desmin levels were increased after betulin treatment. Our data demonstrated that betulin attenuated muscle damage, alleviated inflammatory response, improved muscle regeneration, and restored motor functions after muscle contusion. Altogether, betulin may be a potential compound to accelerate the repair of injured muscle.