Mice lung metastatic models and therapy regimen Balbc mice were bought from Shanghai Slac Laboratory Animal Co. Ltd. K7 cells have been digested and washed by cold PBS for 3 times, sus pended in cold PBS. The last concentration of K7 cells was 5 106ml. The cell suspension was injected into the mice from caudal vein. Two weeks later on, mice were divided into two groups, experi mental group was injected with shikonin, manage group was injected with 5% DMSO. The two groups were injected intraperitoneally each other day. Statistical analysis Statistical examination was performed applying GraphPad Prism five. All measurement data have been expressed as imply standard deviation, and com pared between two groups working with Students t test. P 0. 05 was regarded as statistically important. Final results Shikonin had prompt killing result on osteosarcoma cells We firstly evaluated the cytotoxity of shikonin on osteo sarcoma cells in vitro.
Cells have been handled with shikonin GDC0199 in numerous concentrations for eight hours. The IC50 of K7, K12, K7M3, U2OS, 143B was 2. 87, two. 72, three. 02, three. 18, 6. 45 uU respectively at 8 hour remedy of shikonin. The cell survival charge decreased time dependently using the treatment method of shikonin for eight, 16 and 24 hrs. The reduce of cell through bility occurred within a speedy fashion within the very first eight hour in osteosarcoma cells. Conventional chemotherapy agent such as cisplatin and doxorubicin showed practically no cell killing impact at eight hour theatment in IC50 dosage. We then tested the cell cycle transform following shikonin treatment of osteosarcoma cells. There was no sizeable modify in cell cycle right after remaining handled with shikonin for eight hrs from the absence or presence of Nec 1 detected by movement cytometry. All these data advised that shikonin had pretty prompt but profound cell killing effect on osteosar coma cells.
Shikonin induced necroptosis in osteosarcoma cells To check out the mechanism of how shikonin selleckchem Docetaxel destroy osteo sarcoma, we additional apoptosis inhibitor and necroptosis inhibitor prior to shikonin therapy. Just after 8 hour incu bation of shikonin, the survival charge of K7, K12, K7M3, U2OS and 143B cells was lowered to forty. 03 two. six, 39. 86 three. six, 49. 73 three. five, 51. 08 4. 1, fifty five. 21 5% respectively, all in a different way from that of management group. After pretreated with Nec one just before adding shikonin, the corre sponding survival rate was enhanced to 90. 25 one. 7, 84. 58 4. 6, 87. 98 two. five, 89. 38 one. 5% in K7, K12, K7M3 and U2OS cells respectively. On the other hand, the related enhance of survival charge was not clear for 143B cells. Cell death caused by shikonin couldn’t be rescued by Z VAD FMK in 143B cells. The death triggered by shikonin in K7 cells was detected by movement cytometry showed in Figure 2B. K7 cells was incu bated with shikonin for eight hrs while in the absence or presence of Nec 1, which was pretreated for two hours before shikonin.