Non-benzoquinone Geldanamycin Analogs Trigger Various Forms Of Death In Human Breast Cancer Cells

Objectives: 1.To investigate the effect of non-benzoquinone geldanamycin analogs DHQ3 and 17-DR on the proliferation and cell death of breast cancer cells MDA-MB-231.2.To explore whether DHQ3 or 17-DR induced varieties of forms of cell death and investigate the changes of related proteins.3.To observe the effect of Hsp90 client proteins after the treatment of DHQ3 or 17-DR.4.To investigate whether DHQ3 and 17-DR have anti-tumor effects in vivo.Methods: 1.The growth inhibitions induced by DHQ3,17-DR in breast cancer cell line MDA-MB-231 were detected by MTT assay.2.Colony-forming assay was used to detect the growth inhibition induced by DHQ3,17-DR.3.PI staining was performed to detect cell death rates on DHQ3,17-DR-treated MDA-MB-231 cells.4.Cell death was assessed by flow cytometry with Annexin V-FITC/PI staining after the DHQ3,17-DR treated for 24 h.5.Changes of nucleus of human breast cancer cell line MDA-MB-231 were assessed by DAPI fluorescent staining.6.The intracellular ATP levels in MDA-MB-231 cells treated with DHQ3,17-DR for 5 h were measured by the ATP Assay Kit.7.The ultrastructural details of breast cancer cell MDA-MB-231 treated with analogs were analyzed by transmission electron microscopy(TEM).8.Western blot was used to detect the expression of necroptosis-related proteins,apoptosis-related proteins and Hsp90 related client proteins.9.Immunofluorescence was used to explore the expression of necroptosis-related proteins on MDA-MB-231 cells after DHQ3,17-DR treated.10.After si RNA transfection was used to down-regulate RIP1 or RIP3 on MDA-MB-231 cells,the growth inhibition was redetected.11.Using Malachite green assay to detect the activity of Hsp90 ATPase in the DHQ3,17-DR.12.MDA-MB-231 xenograft model was used to observe the analogs anti-tumor effect in vivo.Results: 1.DHQ3 and 17-DR inhibited the survival rates on breast cancer cell line MDA-MB-231.The results showed that DHQ3 and 17-DR significantly inhibited MDA-MB-231 cell grow,the cell viability rates were gradually reduced with the increasing of analogs concentration and the longer of action.The number of cells became less and cell morphology changed under the microscope.And the colony-forming ability of the cells was clearly reduced by DHQ3 and 17-DR.2.DHQ3 and 17-DR induced different forms of cell death in MDA-MB-231 cells Similarly,the results indicated an increase in the ratio of dead cells with increasing analogs concentrations.DAPI-stained cells exhibited condensed and fragmented nuclei.And the cellular ATP levels were reduced with DHQ3 and 17-DR treatment.DHQ3 induced typical nuclear fragmentation,organelle(especially mitochondrial)swelling,and the leakage of cytoplasm under the electron microscope,suggesting that the necroptosis occurred.The cells treated with 17-DR showed typical characteristics of apoptosis: nuclear concentrating,condensation and margination of nuclear chromatin.3.17-DR-induced apoptosis was caspase-dependent Apoptosis was induced in MDA-MB-231 cells by 17-DR as confirmed by the down-regulation of Mcl-1 and Bcl-2,and up-regulation of Bax protein levels.The cleaved products of caspase-3,caspase-8 and PARP were occurred obviously.The effects of 17-DR were protected by the combination with the apoptosis inhibitor z-VAD-fmk.4.DHQ3 induced necroptosis in breast cancer MDA-MB-231 cells After the treatment with DHQ3,the results showed that cleaved products of caspase-3 and caspase-8 were nearly undetected.DHQ3 up-regulated expressions of necroptosis-related proteins RIP1,RIP3 and MLKL as confirmed by western blot and immunofluorescence assay.The viability of DHQ3-treated cells was rescued with the specific inhibitor of necroptosis Nec-1 pre-treatment.5.Down-regulation of RIP1 or RIP3 with si RNA proved that DHQ3 induced necroptosis in MDA-MB-231 cells The results indicated that down-regulating RIP1 or RIP3 expression prevented DHQ3 from activating cell death,and caused the loss of Nec-1's protective effect.6.DHQ3 and 17-DR down-regulated Hsp90 client proteins expression in breast cancer MDA-MB-231 cells Our results showed that DHQ3 and 17-DR presented stronger ATPase inhibition activity compared to the original Hsp90 inhibitor,Geldanamycin(GA).The classical Hsp90 client proteins were significantly down-regulated in the presence of DHQ3 and 17-DR.And pre-treatment with the proteasome inhibitor MG132 was sufficient to partly restore the expression level of client proteins in cells incubated with DHQ3 or 17-DR.7.DHQ3 and 17-DR showed good anti-tumor effect in vivo The MDA-MB-231 xenograft model was established,and the mice were all randomly assigned into four groups: DMSO,DHQ3,17-DR,DDP.Our results showed that DHQ3 and 17-DR exhibited obvious decrease in tumor with low hepatotoxicity in vivo.8.DHQ3 and 17-DR did not induce different forms of death in other cancer cell lines A series of cancer cell lines were treated with the same concentrations of DHQ3 and 17-DR,as well as pre-treated with Nec-1,as in the experiments with MDA-MB-231 cells.Under the conditions used,the same phenomenon of different forms of cell death with different analogs was not observed in these other cancer cell lines,its mechanism needed to further study.Conclusion: 1.DHQ3 and 17-DR inhibited the cell viability of human breast cancer cells.2.DHQ3 and 17-DR induced different forms of cell death in MDA-MB-231 cells.3.DHQ3 and 17-DR could down-regulate the expression of Hsp90 client proteins.4.DHQ3 and 17-DR possessed anti-tumor ability in nude mice.