Study On HSP90 Inhibitor L7-allylamide-l7-demethoxygeldanamycin Of Potentiating Heat-induced Cell Killing Osteosarcoma Cells

Hyperthermia is a classic stressful challenge which may trigger both cell death and cell-protective mechanisms.The balance between them determines a fate of heat-treated cells, whether a cell will die or not.HSP90 is one of the most abundant Proteins in cytosol of eukaryotic cells. It may play an important role to sustain cellular activity in refolding of the misfolded Proteins that accumulate in response to various stress treatments and perhaps upon mutational alteration of proteins. Moreover, HSP90 has many other significant functions in cells such as regulating activity of steroid receptors transcription factors and some kinases in signal pathways. HSP90 is a chaperone with over 100 identified client proteins. Many of its client proteins are in proliferation signaling pathways and these pathways are often important in many types of cancers,Which makes HSP90 especially promising as a target for anti-cancer drugs. The benzoquinone ansamycin geldanamycin has been shown to bind to HSP90 and to specifically inhibit chaperone's function, resulting in clint protein destabilization. 17-Allylamino-17-demethoxygeldanamycin (17AAG) is a kind of chemical derivative of geldanamycin.Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Osteosarcoma is mainly treated by surgical excision including amputation and joint amputated before 1970's. 5-year disease-free survival of the majority of patients is less than 25%. With the development of surgery and screenage technology and achievement of alliance of neoadjuvant chemotherapy, radiotherapy ,immunotherapy and thermotherapy,the therapy of osteosarcoma has taken a great deal of achievement, 5-year disease-free survival exceeds 80%. But the metastasis, recurrent and chemotherapy-resistant cases has not been solved. Hence ,looking for a new target for anti-cancer drugs maybe help us overcome the osteosarcoma. In order to explore the effects of HSP90 inhibitor 17AAG on osteosarcoma cells and the capability of 17AAG on heat-induced cell killing of osteosarcoma cells,we have completed the following work in this study:1. The biological effects of 17AAG on osteosarcoma SOSP-9607 cellsMTT assay tests demonstrate that 17AAG inhibits osteosarcoma SOSP-9607 cells proliferation in a dose-and-time-dependent manner.The growth-inhibitory IC50 value for 17AAG treatment is 3.548μM.Characteristic apoptotic features were confirmed by acridine orange staining and flow cytometry (FCM).2. The capability of 17AAG on heat-induced cell killing of osteosarcoma cellsMTT assays suggested the interaction between heat and 17AAG treatment.it showed that 17AAG in combination with heat synergistically potentiated heat-induced cell killing(P<0.01) . Clonogenic assays also showed that 17AAG in combination with heat synergistically potentiated heat-induced cell killing(P<0.05) . Annexin V-FITC staining revealed that the percentages of apoptotic cells in the group of combination of heat with 17AAG were 52.2%,and 17.5% in the group of heat alone ;Similiarly, cell shrinkage and nuclear condensation in the group of combination of heat with 17AAG were observered by acridine orange staining.3. Effect of 17AAG alone or in combination with heat on MAPK activationIt was showed by Western Blot that 17AAG in combination with heat markedly reduced the expression of phosphor-AKT and phosphor-Raf-1 compared with heat alone.these results raised the possibility that the inhibition of Erk or AKT activation may be involved in the potentiation of heat-induced cell killing caused by 17AAG.In conclusion: 17AAG treatment caused inhibition of proliferation and apoptosis in osteosarcoma SOSP-9607 cells. Our findings strongly suggest that 17AAG might be an effective therapeutic agent targeting osteosarcoma cells.This study also demonstrated that 17AAG in combination with heat synergistically potentiated heat-induced cell killing.The effect might be attributable to the inhibitory effect of 17AAG on Erk and AKT activation.The results suggest that the chaperone complex may be a new target for the modification of the modification of heat response and the potentiation of heat–induced cell killing.