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Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. The abilities of chemotherapy resistance are major roadblock in the successful treatment of OS. The clarification of mechanism regarding cell survival during OS chemotherapy are important. Here, we examined HER4 expression by immunohistochemistry in a large series of OS tissues, and found HER4 expression correlated with tumor characteristics and patient survival rates. HER4 knockdown by shRNA inhibited OS cell growth and tumorigenesis, and induced cell senescence and apoptosis in vitro and in vivo. We demonstrated that HER4 expression upregulated in the adverse conditions, such as serum starvation and sphere culture. Moreover, HER4 knockdown cells became more sensitive in stressful conditions such as loss of attachment, cytotoxic agents or nutrition insufficiency. Mechanism studies revealed that HER4 interacted with NDRG1, and NDRG1 overexpression could antagonize HER4 knockdown-mediated cell growth and apoptosis in stressed conditions. There was a positive correlation between HER4 and NDRG1 immunoreactivity in OS patients. Together, our present study shows that HER4 and/or NDRG1 might play a critical role for the cell survival and chemo-resistance of OS, and could be used as potential therapeutic targets in OS. [ABSTRACT FROM AUTHOR]

This study was carried out to investigate the anti-tumor effect and mechanism of hiporfin-mediated photodynamic therapy (hiporfin- PDT) in osteosarcoma. We found that hiporfin accumulated mainly in the cytoplasm of osteosarcoma cells in a time and concentration-dependent manner. Hiporfin- PDT inhibited the proliferation, induced apoptosis and produced cell cycle arrest at G2 M in osteosarcoma cell lines. Hiporfin- PDT increased the expression of cleaved-caspase-3, cleaved PARP-1, Bax and RIP1 while it decreased the expression of Bcl-2; in addition, low concentration of hiporfin increased LC3 conversion. Furthermore, cell death caused by hiporfin- PDT could be rescued by Nec-1 but not by Z- VAD- FMK. Production of reactive oxygen species was increased after hiporfin- PDT. In vivo studies showed a significant decrease in tumor volume and weight after hiporfin- PDT in all three tumor mouse models investigated (subcutaneous and orthotopic). Histological analysis showed widespread cell apoptosis and necrosis after treatment. Immunohistochemistry also showed upregulation of cleaved-caspase-3 and downregulation of Bcl-2 after hiporfin- PDT. These results indicate that hiporfin- PDT exhibits a killing effect in osteosarcoma both in vitro and in vivo, which is associated with apoptosis and necroptosis, while autophagy plays a protective role. All these findings shed light on a potential future clinical use for hiporfin in the treatment of osteosarcoma. [ABSTRACT FROM AUTHOR]

Background: Osteosarcoma is the most common primary malignant bone tumor in children and adolescents and is characterized by frequent metastasis and resistance to chemotherapy. Because osteosarcoma cells are not highly susceptible to current chemotherapy drugs, new alternative strategies for the treatment of osteosarcoma are needed. This study was undertaken to investigate the inhibitory effects of a new synthetic ursolic acid derivative IUA on osteosarcoma cells and to explore its molecular mechanism. We also intended to identify new therapeutic candidates. Methods: We used MTT assay to assess the effect of IUA on the proliferation of osteosarcoma cells. Western-blot analysis was performed to examine downstream molecular events. The Annexin V method was used to evaluate the effect of IUA on apoptosis of osteosarcoma cells. The cell cycle of IUA-treated cells was examined by flow cytometry, and the in vivo effects of this new ursolic acid derivative were evaluated in a mouse osteosarcoma model. Results: The results showed that the new synthetic ursolic acid derivative IUA significantly decreased viability of osteosarcoma cells in vitro and in vivo. It could also induce apoptosis and G1 phase arrest of osteosarcoma cells. The JNK signaling pathway was significantly inhibited, and cleaved caspase-3 protein was increased. Conclusion: We concluded that the new synthetic ursolic acid derivative IUA induces proliferation inhibition and apoptosis of osteosarcoma cells in vitro and in vivo via the down-regulation of the JNK signaling pathway, making it a promising agent for the prevention and treatment of human osteosarcoma. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Osteosarcoma is the most frequent primary malignant bone tumor, notorious for its lung metastasis. Shikonin, an effective constituent extracted from Chinese medicinal herb, was demonstrated to induce necroptosis in some cancers.Methods: MTT assay was performed to detect cell survival rate in vitro. Flow cytometry was used to analyze cell cycle and cell death. Western blot was performed to determine the expression levels of RIP1, RIP3, caspase-3, caspase-6 and PARP. The tibial primary and lung metastatic osteosarcoma models were used to evaluate the anti-tumor effect of shikonin in vivo.Results: The cell survival rate was decreased in a dose and time dependent manner when treated with shikonin. No major change in cell cycle was observed after shikonin treatment. The cell death induced by shikonin could be mostly rescued by specific necroptosis inhibitor necrostatin-1, but not by general caspase inhibitor Z-VAD-FMK. The number of necrotic cells caused by shikonin was decreased after being pretreated with Nec-1 detected by flow cytometry in K7 cells. After 8-hour treatment of shikonin, the expression levels of RIP1 and RIP3 were increased while caspase-3, caspase-6 and PARP were not activated in K7 and U2OS cells determined by Western blot. Size of primary tumor and lung metastasis in shikonin treated group were significantly reduced. The protein levels of RIP1 and RIP3 in primary tumor tissues were increased by shikonin. The overall survival of lung metastatic models was longer compared with control group (p < 0.001).Conclusions: Shikonin had prompt but profound anti-tumor effect on both primary and metastatic osteosarcoma, probably by inducing RIP1 and RIP3 dependent necroptosis. Shikonin would be a potential anti-tumor agent on the treatment of primary and metastatic osteosarcoma. [ABSTRACT FROM AUTHOR]

Fullerene C60 nanocrystals (nano‐C60) possess various attractive bioactivities, including autophagy induction and calcium/calmodulin‐dependent protein kinase IIα (CaMKIIα) activation. CaMKIIα is a multifunctional protein kinase involved in many cellular processes including tumor progression; however, the biological effects of CaMKIIα activity modulated by nano‐C60 in tumors have not been reported, and the relationship between CaMKIIα activity and autophagic degradation remains unclear. Herein, nano‐C60 is demonstrated to elicit reactive oxygen species (ROS)‐dependent cytotoxicity and persistent activation of CaMKIIα in osteosarcoma (OS) cells. CaMKIIα activation, in turn, produces a protective effect against cytotoxicity from nano‐C60 itself. Inhibition of CaMKIIα activity by either the chemical inhibitor KN‐93 or CaMKIIα knockdown dramatically promotes the anti‐OS effect of nano‐C60. Moreover, inhibition of CaMKIIα activity causes lysosomal alkalinization and enlargement, and impairs the degradation function of lysosomes, leading to autophagosome accumulation. Importantly, excessive autophagosome accumulation and autophagic degradation blocking are shown to play an important role in KN‐93‐enhanced‐OS cell death. The synergistic anti‐OS efficacy of KN‐93 and nano‐C60 is further revealed in an OS‐xenografted murine model. The results demonstrate that CaMKIIα inhibition, along with the suppression of autophagic degradation, presents a promising strategy for improving the antitumor efficacy of nano‐C60. [ABSTRACT FROM AUTHOR]

Background: Reversing multidrug resistance (MDR) has been an important goal for clinical and investigational oncologists. In the last few decades, significant effort has been made to search for inhibitors to reverse MDR by targeting ATP-binding cassette (ABC) transporters (Pgp, MRP) directly, but these efforts have achieved little clinical success. Protein kinases play important roles in many aspects of tumor cell growth and survival. Combinations of kinase inhibitors and chemotherapeutics have been observed to overcome cancer drug resistance in certain circumstances.Methods: We screened a kinase specific inhibitor compound library in human osteosarcoma MDR cell lines to identify inhibitors that were capable of reversing chemoresistance to doxorubicin and paclitaxel.Results: We identified 18 small molecules that significantly increase chemotherapy drug-induced cell death in human osteosarcoma MDR cell lines U-2OSMR and KHOSR2. We identified A-770041 as one of the most effective MDR reversing agents when combined with doxorubicin or paclitaxel. A-770041 is a potent Src family kinase (Lck and Src) inhibitor. Western blot analysis revealed A-770041 inhibits both Src and Lck activation and expression. Inhibition of Src expression in U-2OSMR and KHOSR2 cell lines using lentiviral shRNA also resulted in increased doxorubicin and paclitaxel drug sensitivity. A-770041 increases the intracellular drug accumulation as demonstrated by calcein AM assay.Conclusions: These results indicate that small molecule inhibitor A-770041 may function to reverse ABCB1/Pgp-mediated chemotherapy drug resistance. Combination of Src family kinase inhibitor with regular chemotherapy drug could be clinically effective in MDR osteosarcoma. [ABSTRACT FROM AUTHOR]