| Backgroud:Cisplatin resistance can be provoked by drug extrusion,multidrug resistance-associated protein activity and upregulation of the DNA repair machinery.In addition,a series of adaptive responses induced by cisplatin that maintain tumor cell homeostasis and interfere with apoptotic signaling can also cause cisplatin resistance.With “Warburg effect” tumor cells shift to using aerobic glycolysis to meet energy requirements.Subsequent studies have linked this transition with mitochondria dysfunction.Growing evidence indicates that mitochondria significantly influence cancer cell survival,invasion,metastasis and drug resistance.Although mitochondria have their own genome,most mitochondrial proteins are encoded by the nuclear genome.These two genomes coordinately regulate mitochondrial proteostasis and maintain mitochondrial functional integrity to prevent tumor cells from environmental stress-induced damage.Therefore,exploring the complicated machinery of the nucleo-mitochondrial anterograde regulation system is vital to clarify the mechanism of cell apoptosis escape and may help identify a novel anticancer therapy target to disturb mitochondrial function.Accumulating evidence now suggests a link between PGC1α-mediated mitochondria biogenesis and tumor apoptosis.PGC1α is a member of the PGC1(PPARγ coactivator-1)family that functions as a transcriptional coactivator in the regulation mitochondria biogenesis.Permuth-Wey found that inter-individual genetic variation in genes involved in mitochondria biogenesis was most potently associated with EOC risk and overexpression of PGC1α was associated with poor prognosis in EOC.Moreover,in BRAF V600E-positive human melanoma,the expression of PGC1α resulted in upregulation of OXPHOS genes,energy status and mitochondria biogenesis accompanied with acquired BRAF inhibitor resistance.These results indicated that PGC1α resisted cisplatin-induced apoptosis through mitochondrial biogenesis.However,as a transcription factor coactivator,PGC1α influences mitochondria respiration,reactive oxygen species defense system,and fatty acid metabolism by interacting with specific transcription factors,and PGC1α-mediated transcriptional regulatory network depend upon the actual environmental and metabolic context of the cell.Since PGC1α interacts with different transcription factors,PGC1α-mediated multistep nuclear regulatory network impact cancer cell survival and death from different aspect.Thus it is important to examine the action of PGC1α-mediated transcriptional regulatory network and its changes under external stimuli in ovarian cancer,and more effectively verify the potention of PGC1α-mediated mitochondrial biogenesis in cisplatin-resistance,which may further substantiate the role of nucleo-mitochondrial signaling communication in cell fate and provide possibilities for promising mitochondria-targeted treatment modalities on elevating cancer chemosensitivity.Objective:To detect the alteration of mitochondria mass and function and the changes of PGC1α-mediated mitochondrial biogenesis transcription pattern the change of the related factors in ovarian cancer cisplatin-resistance cells treatment with cisplatin,clear PGC1 alpha mediated mitochondrial biosynthesis in human ovarian cancer the role of cisplatin resistance,from multiple perspectives,a full range of ovarian cancer,possible mechanisms of cisplatin resistance are discussed.Methods:1.Transmission electron microscopy was used to observe the differences in the mass and volume of mitochondria between SKOV3 and SKOV3/DDP cells;RT-PCR was used to detect the differences in mitochondrial gene copy numbers between SKOV3 and SKOV3/DDP cells;Flow cytometry was used to detect the number of mitochondria in SKOV3 and SKOV3/DDP cells;RT-qPCR assay was used to detect the expression of genes related to mitochondrial biogenesis in SKOV3 and SKOV3/DDP cells;Western Blot assay was used to detect the expression differences of mitochondrial biogenesis related proteins in SKOV3 and SKOV3/DDP cells.2.Treated SKOV3 SKOV3/DDP cells with different concentrations of cisplatin after 24 h,using MTT assay to detect cell viability;6 μg/ml cisplatin on SKOV3 and SKOV3/DDP for 24 h,Hoechst 33342 stained nucleu were examined by scanning confocal fluorescence microscopy for chromatin condensation;6 μg/ml cisplatin was applied to SKOV3 and SKOV3/DDP cells for 24 h,and Western Blot assay was used to detect cytochrome C release.6 μg/ml cisplatin was applied to SKOV3 and SKOV3/DDP cells at different time points.JC-1 staining flow cytometry was used to detect the change of mitochondrial membrane potential.3.Select 6 μg/ml cisplatin for SKOV3 and SKOV3/DDP cells,and incubated the cells with the fluorescent mitochondrial indicator Mitotracker.Use laser confocal microscopy and flow cytometry to detect changes in intracellular mitochondria;use 6 μg/ml cisplatin was applied to SKOV3 and SKOV3/DDP cells,RT-qPCR was used to detect the change of mitochondrial gene copy number;6 μg/ml cisplatin was selected to act on SKOV3 and SKOV3/DDP cells,and the extracellular oxygen consumption rate was detected by fluorescence probe method..4.6 μg/ml cisplatin was applied to SKOV3/DDP cells for 3 h,6 h,12 h and 24 h.RT-qPCR assay was used to detect changes in mitochondrial biogenesis-related gene expression;6 μg/ml cisplatin was selected to act on SKOV3/DDP for 3 h,6 h,12 h and 24 h,the expression of mitochondrial biogenesis-related proteins was detected by Western Blot assay.5.shRNA knock-down PGC1α in SKOV3/DDP cells,RT-qPCR detection of mitochondrial gene copy number alteration;RT-qPCR assay to detect mitochondrial biogenesis related gene expression;Western Blot experiment to detect mitochondrial biogenesis related protein expression;fluorescent probes methods to detect extracellular oxygen consumption rate.6.After shRNA knock-down of PGC1α in SKOV3/DDP cells,Western Blot assay was used to detect the release of cytochrome C;Western Blot assay was used to detect apoptosis-related protein expression.7.After shRNA knock-down of PGC1α combined with cisplatin treatment in SKOV3/DDP cells,MTT assay to detect cell viability;Hoechst 33342 stained nucleu were observed by scanning confocal fluorescence microscopy for chromatin condensation;fluorescent mitochondrial indicator Mitotracker stained mitochondria were observed by scanning confocal fluorescence microscopy for detecting changes in intracellular mitochondrial mass;fluorescent probes methods to detect extracellular oxygen consumption rate.Results:1.Cisplatin decreased mitochondrial membrane potential in SKOV3 cells,induced chromatin condensation and cytochrome C release,resulted in mitochondrial-dependent apoptosis pathway,and there was no significant changes in SKOV3/DDP cells treated with cisplatin.2.Compared with SKOV3 cells,SKOV3/DDP cells had more mitochondrial numbers/volumes,higher copy number of mitochondrial genes and higher levels of mitochondrial biogensis related genes and proteins expression,indicating that SKOV3/DDP cells has higher level of mitochondria biogenesis.3.Cisplatin down-regulates mitochondrial gene copy number of SKOV3 cells and reduces mitochondrial function;cisplatin up-regulates mitochondrial gene copy number of SKOV3/DDP cells,activates PGC1α and mitochondrial biogenesis related pathways,increased mitochondrial number and mitochondrial function.4.After knocking out PGC1α in SKOV3/DDP cells,the copy number of mitochondrial genes decreased,the number of mitochondria decreased,the expression of mitochondrial biogenesis related genes and proteins decreased,mitochondrial respiratory function decreased and the cellular sensitivity to cisplatin increased and induced apoptosis.Conclusions:Cisplatin activated PGC1α in human ovarian cancer cisplatin resistant SKOV3/DDP cells,up-regulated mitochondrial biogenesis-related signaling pathways,and increased mitochondrial numbers,structural and functional stability,counteracting the toxic effects of cisplatin;when knocking out PGC1α,the loss of mitochondrial biogenesis in human ovarian cancer cisplatin resistant SKOV3/DDP cells failed to maintain mitochondrial number,structure and function,and reversed cisplatin resistance. |
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