SM-164Combined With Doxorubicin On Mechanism Of Apoptosis In Human Osteosarcoma HOS Cell Line

Background: Osteosarcoma，the most common malignant primary bone tumor，mainly occurs in the10-20year-old. Before1970s, the major treatment for patientswith non-metastatic osteosarcoma is amputation and5-year survival rate is less than20%. With the introduction of neoadjuvant chemotherapy and the advances insurgical techniques, long-term survival rate in the patients with osteosarcoma wasimproved to about60%. But then the treatment of osteosarcoma has no progress, oneimportant reason is osteosarcoma cell resistance to chemotherapeutic drugs. It hasbecome a hotspots to enhance osteosarcoma cell sensitivity to chemotherapeuticdrugs. In2000, professor Wang found the mitochondrial protein Smac whichguidelined for a new direction of the treatment of cancer. Smac binds to IAPs andabrogates the inhibition of caspases by IAPs, thereby promoting apoptosis. SM-164is a synthetic small molecule Smac mimetics, which simulate the Smac binding toIAPs and promote rapid degradation of cIAP-1and effectively remove the inhibitionof XIAP to caspase-9and to caspase-3/-7.Objective： To evaluate the effect of SM-164for chemotherapy in humanosteosarcoma HOS cells.Method:(1) MTT assay the inhibition of proliferation for doxorubicin(ADM)and SM-164combined with doxorubicin in human osteosarcoma HOS cells. Ourstudy is divided into six groups: control group, SM-164group, ADM (0.25μg/ml)group, SM-164+ADM (0.25μg/ml) group, ADM (0.5μg/ml) group, SM-164+ADM(0.5μg/ml) group; The working concentration of SM-164is200nM. Culture condition:Change the medium after2h and culture for another24h.(2) Western blot assay theexpression of cIAP-1and XIAP for doxorubicin and SM-164combined with ADM inhuman osteosarcoma HOS cells.(3) PCR assay the expression of XIAP gene forADM and SM-164combined with doxorubicin in human osteosarcoma HOS cells.Results：(1) MTT assay showed that cell growth inhibition rate of SM-164(200nM) group was (3.65±0.78)%, ADM (0.25μg/ml,0.5μg/ml) groups were (12.4± 4.57)%and (28.43±4.04)%, SM-164(200nM) combined with ADM (0.25μg/ml,0.5μg/ml) groups were (19.4±4.58)%and (44.17±3.51)%, Q values were1.26±0.06and1.41±0.05. Cell growth inhibition rate of ADM (0.5μg/ml) group wassignificantly higher than that of ADM (0.25μg/ml) group. Cell growth inhibition rateof SM-164combined with ADM group was also significantly higher than that of thecorresponding ADM group.(2) Western blot showed that:①the expression of XIAPprotein was no significant difference between ADM (0.25μg/ml,0.5μg/ml) group andthe control group; the expression of XIAP protein of SM-164(200nM) group and SM-164(200nM) combined with ADM (0.25μg/ml,0.5μg/ml) group was significantlydecreased when compared to that of the control group, and the three groups decreasedgradually.②the expression of cIAP-1protein was no significant difference betweenADM (0.25μg/ml,0.5μg/ml) group and the control group; the expression of cIAP-1protein of SM-164(200nM) group and SM-164(200nM) combined with ADM(0.25μg/ml,0.5μg/ml) group was significantly decreased when compared to thecontrol group, but there was no significant difference among the three groups.(3)PCR experiments showed that: the level of GAPDH gene formed six uniformbrightness of the bands as an internal, the bands of XIAP gene among the six groupshad no significant difference.Conclusion：The effect of SM-164combined with doxorubicin for the inhibitionof proliferation of human osteosarcoma HOS cells is synergistic. The mechanism maybe through inhibition the expression of XIAP and cIAP-1protein.