Yi-Fu Huang

Zoledronic acid (ZOL) is an inhibitor of osteoclast-mediated bone resorption. It is used to treat osteoporosis and skeletal complications in patients with tumor-induced osteolysis. ZOL is also demonstrated to possess anti-cancer activity in several tumors via apoptosis induction. Doxycycline is well-known antibiotic used in treatment of infections caused by bacteria and certain parasites. In this study, we evaluated the possibility if doxycycline could be used as an effective adjuvant to ZOL against osteosarcoma cells. The data showed that co-treatment with doxycycline at non-toxic dose could significantly increase the anti-viability effect of ZOL in osteosarcoma HOS and MG-63 cells in MTT assay and colony formation assay, and largely increased the levels of apoptotic markers, cleaved caspase 3 and PARP, in ZOL-treated cells. Furthermore, as co-treatment with doxycycline, the levels of ROS and autophagy were enhanced in ZOL-treated cells. Administration of N-acetyl-L-cysteine, a reactive oxygen species (ROS) inhibitor, or autophagy inhibitor chloroquine both reduced anti-growth effect of this combined treatment, indicating that the increased ROS and autophagy should be involved in anti-viability effect of combined treatment with ZOL and doxycycline. Taken together, our findings suggested that combined treatment with ZOL and doxycycline may serve as a potential strategy for treating 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.

Butein is a flavonoid isolated from various medicinal plants. It is known to have different biological activities including anti‐inflammation, anti‐adipogenesis, and anti‐angiogenesis. In the study, we demonstrated the anti‐proliferative effect of butein in human osteosarcoma U‐2 OS cells. Our data showed that butein significantly suppressed the viability and colony formation ability of U‐2 OS cells. Further experiments revealed butein exposure resulted in a cell cycle arrest at S and G2/M phase in U‐2 OS cells. Importantly, we found that butein activated the tumor suppressor p53, and trigged a p53‐dependent senescence in U‐2 OS cells. Knockdown of p53 suppressed the senescence and rescued the viability in butein‐treated U‐2 OS cells. Furthermore, we observed that butein exposure significantly enhanced reactive oxygen species (ROS) levels in U‐2 OS cells. Co‐administration of the ROS inhibitor NAC largely abolished the up‐regulated p53 protein level, and rescued the suppressed viability and colony formation ability in butein‐exposed U‐2 OS cells. Taken together, our data proposed the increased ROS by butein exposure activated p53, and the activated p53 was involved in the anti‐proliferative effect of butein via inducing senescence in U‐2 OS cells. This report suggests that butein is a promising candidate for cancer therapy against osteosarcoma.

Betulin is a lupane type pentacyclic triterpenoid, and commonly found in the bark of birch trees. It displays various pharmacological properties, such as antibacterial, anti‐inflammation, antitumor, and antiviral. In this report, we attempted to investigate the anti‐proliferative and pro‐apoptotic effects of betulin on osteosarcoma cell lines. Our results revealed that betulin significantly decreased cell viability and colony formation in osteosarcoma cell lines. Dose‐dependent induction of Annexin V positive cells, activated caspase 8, activated caspase 9, activated caspase 3, and the cleavage of poly (ADP‐ribose) polymerase were observed after the treatment with betulin, indicating betulin induces apoptosis in osteosarcoma cell lines. mTOR has been identified as a key modulator of autophagy in response to different stresses. In this study, we found that the treatment with betulin suppressed the activation of mTOR, and increased the level of LC 3‐II, the autophagy marker, in osteosarcoma cell lines. Co‐administration of the autophagy inhibitor chloroquine significantly rescued the cell viability and the clonogenic activity in betulin‐treated osteosarcoma cell lines. Our data showed that betulin induced autophagy, and the up‐regulated autophagy positively contributed to the apoptosis. Taken together, our findings suggested that betulin may serve as a promising anti‐proliferative agent for treating osteosarcoma.

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.

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.

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.