Doxorubicin Activate Notch Signaling Pathway In Osteosarcoma

Notch signaling plays critical roles in various biological situations, which include cell proliferation, differentiation and apoptosis. Accumulating evidence indicates that aberrant Notch signaling has tumor-promoting function in osteosarcoma. However, the effect of conventional chemotherapeutic agent doxorubicin on Notch signaling is still unclear. To address this issue, we treated osteosarcoma cells with various concentrations of doxorubicin. We found that the cytostatic dose of doxorubicin activated Notch signaling pathway, shown by elevated expression of Notch target genes, and the effect showed a dose-dependent manner. However, toxic dose of doxorubicin significantly inhibit Notch signaling pathway. Our results provide significant correlation between doxorubicin and Notch signaling pathway.Part ? Optimizing the concentration of doxorubicin treatment in osteosarcomaObjective:To determine an optimum dose range of doxorubicin treatment for subsequent various studies in osteosarcoma cells.Methods:After osteosarcoma cells were cultured, cell viability was determined using trypan blue stainingtime. Thereafter, dose-dependent cytotoxic assays were performed in osteosarcoma cells that were grown in 96-well culture plates, treated with various concentrations for different time. The optical density (OD) of each well, in which cells were incubated with cell counting kit-8, was then measured at 450nm using a microplate reader.Results:Our data showed that toxicity was not significantly enhanced at 24h (p> 0.05) after exposed to doxorubicin comparing with in control groups, while significantly at 48h (p<0.05) (Fig.1A). Additionally, doxorubicin at concentration equal or beyond 0.5?M resulted in statistically significant toxicity (p<0.05), especially doxorubicin at concentration with 1,2 and 4?M did in statistically extremely significant toxicity (p<0.01), while there was no significance difference in toxicity below 0.5 ?M of concentration. (Fig. 1B,p>0.05).Conclusions:It is at concentration equal 0.5?M that whether doxorubicin exerts toxicity in osteosarcoma cells or a cytostatic effect, which shows a time and dose dependency.Part II Doxorubicin increase Notch target genes expression in osteosarcoma cellsObjective:To investigate the effect of low dose doxorubicin on Notch signaling pathway in osteosarcoma cells.Methods:The passaged osteosarcoma cells had been treated with doxorubicin for 48h, and alterations in the Notch pathway were investigated, which were treated at concentrations below 0.5 ?M such as 0.1,0.25 and 0.4?M in order to test whether the alterations were dose-dependent. Real-time PCR was then performed to detect Notch target genes Hes-1, Hes5, Heyl, Hey2 and HeyL expression in osteosarcoma cells, which could assess the effect of low dose doxorubicin in osteosarcoma cells. Furthermore, we also tested the Notch target genes by western-blot, confirming the effect of Notch signaling pathway by doxorubicin.Results:There was a significant increase of Hes-1, Hes5, Heyl, Hey2 and HeyL mRNA levels in osteosarcoma cells after treatment with 0.1 ?M of doxorubicin for 48h using real-time RT-PCR analysis. (Fig.2A,p<0.05). Further experiments showed that Hes-1 and Heyl in osteosarcoma cells, which were treated with increasing concentrations (0.1,0.25 and 0.4?M) of doxorubicin for 48 h, were up-regulated in a dose-dependent manner (Fig.2B,p<0.05). Furthermore, we also found that the expression of Hes-1 and Heyl were significantly enhanced by doxorubicin detected by western-blot (Fig.2C, p<0.05), which confirmed the activation of Notch signaling pathway influencing the sternness induced by doxorubicin.Conclusions:Low dose doxorubicin could up-regulate Notch target genes expression in osteosarcoma cells in a dose-dependent manner, which implies the stemness induced by doxorubicin.Part III High dose doxorubicin decreased the expression of Notch target genesObjective:To investigate the effect of high dose doxorubicin on Notch signaling pathway in osteosarcoma cells..Methods:we treated osteosarcoma cells with l?M doxorubicin for 48h, which was a high dose doxorubicin to kill osteosarcoma cells, and detected changes in the Notch target genes including Hes-1, Hes5, Heyl, Hey2 and HeyL in osteosarcoma cells using real-time RT-PCR analysis. The verification test had been carried out by western-blot in order to determine whether western-blot results agreed with real-time RT-PCR data in the Notch target genes.Results:We found that Notch target genes including Hes-1, Hes5, Heyl, Hey2 and HeyL were extremely significantly suppressed by l?M doxorubicin (Fig.3A, p<0.0l). And there was further confirmation that Hesl and Heyl were down-regulated after treated by l?M doxorubicin by western blot in agreement with real-time RT-PCR data (Fig.3B,p<0.05).Conclusions:High dose doxorubicin decreases the expression of Notch target genes, which might contribute to cytotoxicity in osteosarcoma cells.