Mitophagy Effects of Protodioscin on Human Osteosarcoma Cells by Inhibition of p38MAPK Targeting NIX/LC3 Axis.

Protodioscin (PD) is a steroidal saponin with various pharmacological activities, including neuro-protective, anti-inflammatory, and anti-tumor activities. However, the effect of PD on human osteosarcoma (OS) cells is unclear. In this study, we found that PD significantly inhibits the growth of human HOS and 143B OS cells through the upregulation of apoptotic-related proteins (cleaved caspase-3, cleaved caspase-9, and cleaved PARP) and mitophagy-related proteins (LC3B and NIX), which contribute to the induction of apoptosis, and MMP (mitochondrial membrane potential) dysfunction and mitophagy. The inhibition of LC3 or NIX was shown to decrease apoptosis and mitophagy in PD-treated OS cells. The knockdown of p38MAPK by siRNA decreased mitochondrial dysfunction, autophagy, mitophagy, and the NIX/LC3B expression in the PD-treated OS cells. A binding affinity analysis revealed that the smaller the KD value (-7.6 Kcal/mol and -8.9 Kcal/mol, respectively), the greater the binding affinity in the PD-NIX and PD-LC3 complexes. These findings show the inhibitory effects of PD-induced mitophagy in human OS cells and may represent a novel therapeutic strategy for human OS, by targeting the NIX/LC3 pathways.

GO-Y078, a Curcumin Analog, Induces Both Apoptotic Pathways in Human Osteosarcoma Cells via Activation of JNK and p38 Signaling.

Osteosarcoma is the most common primary bone malignancy in teenagers and continues to confer a generally poor prognosis due to its highly metastatic potential. Poor solubility in water and instability of curcumin limits its bioavailability for use in the adjuvant situation to improve the prognosis and the long-term survival of patients with osteosarcoma. To further obtain information regarding the apoptosis induced by a new curcumin analog, GO-Y078, in human osteosarcoma cells, flow cytometric analysis, annexin V-FITC/PI apoptosis staining assay, human apoptosis array, and Western blotting were employed. GO-Y078 dose-dependently decreased viabilities of human osteosarcoma U2OS, MG-63, 143B, and Saos-2 cells and induced sub-G1 fraction arrest and apoptosis in U2OS and 143B cells. In addition to the effector caspase 3 and poly adenosine diphosphate-ribose polymerase, GO-Y078 significantly activated both initiators of extrinsic caspase 8 and intrinsic caspase 9, whereas cellular inhibitors of apoptosis 1 (cIAP-1) and X-chromosome-linked IAP (XIAP) in U2OS and 143B cells were significantly repressed. Moreover, GO-Y078 increased phosphorylation of extracellular signal-regulated protein kinases (ERK)1/2, c-Jun N-terminal kinases (JNK)1/2, and p38 in U2OS and 143B cells. Using inhibitors of JNK (JNK-in-8) and p38 (SB203580), GO-Y078's increases in cleaved caspases 8, 9, and 3 could be expectedly suppressed, but they could not be affected by co-treatment with the ERK inhibitor (U0126). Altogether, GO-Y078 simultaneously induces both apoptotic pathways and cell arrest in U2OS and 143B cells through activating JNK and p38 signaling and repressing IAPs. These findings contribute to a better understanding of the mechanisms responsible for GO-Y078's apoptotic effects on human osteosarcoma cells.

Potential Antimetastatic Effect of Timosaponin AIII against Human Osteosarcoma Cells through Regulating the Integrin/FAK/Cofilin Axis.

Timosaponin AIII (TSAIII) is a steroidal saponin which demonstrates anti-tumour activities. However, the effect of TSAIII on human osteosarcoma cells remains largely unknown. In this study, we demonstrated that TSAIII exerted a significant inhibitory effect on the distribution of cytoskeletal F-actin and cytoskeletal-related proteins, which contributed to the suppression of cell migration and invasion, without inhibiting cell growth or apoptosis. In the synergistic inhibitory analysis, cotreatment of TSAIII with αVß3 integrin inhibitor [Cyclo(RGDyK)] or focal adhesion kinase (FAK) inhibitor (PF-573228) exerted greater synergistic inhibitory effects on the expression of Intergin αVß3/FAK/cofilin axis, thus inhibiting the migration and invasion capacities of human osteosarcoma cells. TSAIII was demonstrated to significantly inhibit the pulmonary metastasis formation of human osteosarcoma cells in vivo in metastasis animal models. These findings reveal the inhibitory effects of TSAIII on the metastasis progression of human osteosarcoma cells and the regulation of integrin-αVß3-FAK-Src and TESK1/p-cofilin mediated cytoskeletal F-actin pathway. Therefore, TSAIII might represent a novel strategy for the auxiliary treatment of human osteosarcoma cells.

Curcumin Analogue L48H37 Suppresses Human Osteosarcoma U2OS and MG-63 Cells' Migration and Invasion in Culture by Inhibition of uPA via the JAK/STAT Signaling Pathway.

Osteosarcoma, the most prevalent malignant bone tumor in the pediatric age group, is responsible for the great majority of cancer-associated deaths owing to its highly metastatic potential. The anti-metastatic effects of the new curcumin analogue L48H37 in human osteosarcoma are still unknown; hence, we investigated whether L48H37 represses human osteosarcoma cells' biological behavior of migratory potential and invasive activities and attempted to delve into its underlying mechanisms. L48H37 up to 5 µM inhibited, without cytotoxicity, the motility, migration, and invasion of human osteosarcoma U2OS and MG-63 cells. In U2OS cells, the human protease array revealed an obvious decrease in urokinase plasminogen activator (uPA) expression after L48H37 treatment, and L48H37 actually reduced the level, protein and mRNA expression, and promoter activity of uPA dose-dependently. L48H37 decreased the phosphorylation of STAT3, JAK1, JAK2, and JAK3 in U2OS cells, but did not affect the phosphorylation of ERK, JNK, p38, and Akt. Using colivelin, an activator of STAT3, the L48H37-induced decrease in uPA and migratory potential could be countered as expected. Collectively, L48H37 represses the invasion and migration capabilities of U2OS and MG-63 cells by the suppression of uPA expression and the inhibition of JAK/STAT signaling. These results suggest that L48H37 may be a potential candidate for anti-metastatic treatment of human osteosarcoma.

Tomatidine Represses Invasion and Migration of Human Osteosarcoma U2OS and HOS Cells by Suppression of Presenilin 1 and c-Raf-MEK-ERK Pathway.

Osteosarcoma, which is the most prevalent malignant bone tumor, is responsible for the great majority of bone cancer-associated deaths because of its highly metastatic potential. Although tomatidine is suggested to serve as a chemosensitizer in multidrug-resistant tumors, the anti-metastatic effect of tomatidine in osteosarcoma is still unknown. Here, we tested the hypothesis that tomatidine suppresses migration and invasion, features that are associated with metastatic process in human osteosarcoma cells and also investigate its underlying pathway. Tomatidine, up to 100 µM, without cytotoxicity, inhibited the invasion and migration capabilities of human osteosarcoma U2OS and HOS cells and repressed presenilin 1 (PS-1) expression of U2OS cells. After the knockdown of PS-1, U2OS and HOS cells' biological behaviors of cellular invasion and migratory potential were significantly reduced. While tomatidine significantly decreased the phosphorylation of c-Raf, mitogen/extracellular signal-regulated kinase (MEK), and extracellular signal-regulated protein kinase (ERK)1/2 in U2OS cells, no obvious influences on p-Jun N-terminal kinase, p38, and Akt, including their phosphorylation, were observed. In ERK 1 silencing U2 OS cells, tomatidine further enhanced the decrease of their migratory potential and invasive activities. We conclude that both PS-1 derived from U2OS and HOS cells and the c-Raf-MEK-ERK pathway contribute to cellular invasion and migration and tomatidine could inhibit the phenomenons. These findings indicate that tomatidine might be a potential candidate for anti-metastasis treatment of human osteosarcoma.

Molecular and Cellular Mechanisms of Melatonin in Osteosarcoma.

Osteosarcoma, the most common primary bone malignancy, occurs most frequently in adolescents with a peak of incidence at 11-15 years. Melatonin, an indole amine hormone, shows a wide range of anticancer activities. The decrease in melatonin levels simultaneously concurs with the increase in bone growth and the peak age distribution of osteosarcoma during puberty, so melatonin has been utilized as an adjunct to chemotherapy to improve the quality of life and clinical outcomes. While a large amount of research has been conducted to understand the complex pleiotropic functions and the molecular and cellular actions elicited by melatonin in various types of cancers, a few review reports have focused on osteosarcoma. Herein, we summarized the anti-osteosarcoma effects of melatonin and its underlying molecular mechanisms to illustrate the known significance of melatonin in osteosarcoma and to address cellular signaling pathways of melatonin in vitro and in animal models. Even in the same kind of osteosarcoma, melatonin has been sparingly investigated to counteract tumor growth, apoptosis, and metastasis through different mechanisms, depending on different cell lines. We highlighted the underlying mechanism of anti-osteosarcoma properties evoked by melatonin, including antioxidant activity, anti-proliferation, induction of apoptosis, and the inhibition of invasion and metastasis. Moreover, we discussed the drug synergy effects of the role of melatonin involved and the method to fortify the anti-cancer effects on osteosarcoma. As a potential therapeutic agent, melatonin is safe for children and adolescents and is a promising candidate for an adjuvant by reinforcing the therapeutic effects and abolishing the unwanted consequences of chemotherapies.

Licochalcone A-Induced Apoptosis Through the Activation of p38MAPK Pathway Mediated Mitochondrial Pathways of Apoptosis in Human Osteosarcoma Cells In Vitro and In Vivo.

BACKGROUND: Licochalcone A (LicA) is isolated from the roots of Glycyrrhiza glabra and possesses antitumor and anti-invasive activities against several tumor cells. However, the antitumor effects of LicA on human osteosarcoma cells have yet to be demonstrated either in vitro or in vivo. METHODS: Cell viability was measured by MTT assay. Apoptosis and mitochondrial dysfunction were detected with Annexin V/PI staining and JC-1 staining by flow cytometry. The expressions of caspase- or mitochondrial-related proteins were demonstrated by western blotting. Antitumor effect of LicA on 143B xenograft mice in vivo. RESULTS: LicA could inhibit cell proliferation and induce apoptosis in human osteosarcoma cells, as evidenced by a decrease in cell viability, loss of mitochondrial membrane potentials, and activation of caspases. LicA treatment substantially reduced the expression of Bcl-2 and Mcl-1 and increased the expression of cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP, and Bax in HOS and U2OS cells. Moreover, mitochondrial membrane potential and apoptosis suppression mediated by Z-VAD or tauroursodeoxycholic acid significantly reduced LicA-induced mitochondria-dependent apoptosis. The study also determined that LicA treatment induced p38MAPK phosphorylation, but siRNA-p38 or BIRB796 substantially reversed cell viability through the inhibition of mitochondria-dependent apoptosis pathways. Finally, an in vivo study revealed that LicA significantly inhibited 143B xenograft tumor growth. CONCLUSIONS: These findings demonstrate that LicA has antitumor activities against human osteosarcoma cells through p38MAPK regulation of mitochondria-mediated intrinsic apoptotic pathways in vitro and in vivo.

CLEFMA Activates the Extrinsic and Intrinsic Apoptotic Processes through JNK1/2 and p38 Pathways in Human Osteosarcoma Cells.

Due to the poor prognosis of metastatic osteosarcoma, chemotherapy is usually employed in the adjuvant situation to improve the prognosis and the chances of long-term survival. 4-[3,5-Bis(2-chlorobenzylidene)-4-oxo-piperidine-1-yl]-4-oxo-2-butenoic acid (CLEFMA) is a synthetic analog of curcumin and possesses anti-inflammatory and anticancer properties. To further obtain information regarding the apoptotic pathway induced by CLEFMA in osteosarcoma cells, microculture tetrazolium assay, annexin V-FITC/PI apoptosis staining assay, human apoptosis array, and Western blotting were employed. CLEFMA dose-dependently decreased the cell viabilities of human osteosarcoma U2OS and HOS cells and significantly induced apoptosis in human osteosarcoma cells. In addition to the effector caspase 3, CLEFMA significantly activated both extrinsic caspase 8 and intrinsic caspase 9 initiators. Moreover, CLEFMA increased the phosphorylation of extracellular signal-regulated protein kinases (ERK)1/2, c-Jun N-terminal kinases (JNK)1/2 and p38. Using inhibitors of JNK (JNK-in-8) and p38 (SB203580), CLEFMA's increases of cleaved caspases 3, 8, and 9 could be expectedly suppressed, but they could not be affected by co-treatment with the ERK inhibitor (U0126). Conclusively, CLEFMA activates both extrinsic and intrinsic apoptotic pathways in human osteosarcoma cells through JNK and p38 signaling. These findings contribute to a better understanding of the mechanisms responsible for CLEFMA's apoptotic effects on human osteosarcoma cells.

Arthroscopic Lateral Retinacular Release and Modified Elmslie-Trillat Operation Improve Severe Isolated Lateral Patello-Femoral Osteoarthritis.

To evaluate whether arthroscopic lateral retinacular release and the modified Elmslie-Trillat operation improve osteoarthritis (OA) progression and clinical outcomes in patients with severe isolated lateral patello-femoral OA. Nine women (11 knees) and one man (one knee) with isolated late-stage lateral patello-femoral OA underwent surgery. The severity of patello-femoral OA was recorded using the Merchant method, while the level of pain and anterior knee function were scored using the visual analogue scale (VAS) and Kujala knee scores, respectively. The articular cartilage was graded under arthroscopy using the Outerbridge classification. All of the patients underwent the modified Elmslie-Trillat operation after arthroscopic surgery, including lateral retinacular release. Ten patients (12 knees) had a mean 6.45 ± 0.80 mm of medial transfer, 6.02 ± 0.80 mm of anterior transfer of the tibial tubercle, and follow-up of 67.0 months. The mean VAS and Kujala knee scores improved from 8 ± 0.17 preoperatively to 2.33 ± 0.33 on the last follow-up and from 43.08 ± 2.09 to 68.83 ± 2.59, respectively (both p < 0.001). Postoperatively, all had improved subchondral bone remodeling, including cyst resolution, density and trabeculae normalization, and subchondral smoothing in the patello-femoral compartment. The patello-femoral joint space and patellar thickness increased from 0.39 ± 0.16 mm to 1.77 ± 0.18 mm and from 18.28 ± 0.67 mm to 19.60 ± 0.69 mm, respectively (p < 0.001 and p = 0.005). Treatment of severe isolated lateral patello-femoral OA using arthroscopic lateral retinacular release and the modified Elmslie-Trillat operation can improve pain relief, functional outcomes, and subchondral bone remodeling, and also restore the patello-femoral joint space and patellar thickness. Prompt transfer of the tibial tubercle seems to reverse the progress of OA radiographically.

Hylan G-F 20 has better pain relief and cost-effectiveness than sodium hyaluronate in treating early osteoarthritic knees in Taiwan.

BACKGROUND/PURPOSE: Intra-articular injection of hyaluronan (hyaluronic acid; HA) products is available to treat early osteoarthritis (OA) of the knee in Taiwan. We tested whether HA products with different molecular weights have significantly different effects on clinical efficacy and cost-effectiveness. METHODS: Thirty-seven patients with mild to moderate OA of both knees underwent five weekly intra-articular injections of sodium hyaluronate (Artz) in one knee and three weekly intra-articular injections of chemically cross-linked Hylan G-F 20 (Synvisc) in the other. Visual analog scale (VAS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Lequesne's index, and Hospital for Special Surgery (HSS) knee scores were compared initially and at the last injection, and at 8, 12, 16, 20, and 26 weeks after the first injection. RESULTS: VAS, WOMAC, WOMAC-A1 (pain when walking on a flat surface) scores before week 16, HSS scores before week 12, and Lequesne's index scores except at week 26 all showed that HA significantly improved the scores time-dependently. In VAS scores, Synvisc showed better improvement before week 20, while this effect appeared at week 12 for the WOMAC-A1 scores. The incremental cost-effectiveness ratio of the Taiwan National Health Insurance Program, of the patient, and both of these was lower for Synvisc, which also reduced the number of additional hospital visits for injections by two. CONCLUSION: Synvisc possesses better symptom-modifying ability and cost-utility in treating early OA of the knee in Taiwan.

Primary synovial chondromatosis mimicking medial meniscal tear in a young man.

Magnetic resonance imaging has recently become a noninvasive and cost-effective diagnostic tool for meniscal pathology in the knee. We report an unusual case of primary synovial chondromatosis for which the clinical diagnosis was medial meniscal tear but magnetic resonance imaging also showed a displaced torn meniscus, causing a so-called "double meniscus sign." The patient was definitely diagnosed and successfully treated with arthroscopy. We discuss the clinical diagnostic and therapeutic challenges, and the possible pathogenesis.