A Perfect Self-Help Guide To Combretastatin A-4OAC1

aspect implicated Combretastatin A-4 in doxo pharmacoresistance.Since doxo stimulates cell apoptosis by means of inhibition of topoisomerase and consequent DNA damage,cells develop resistance by downregulating this enzyme.Translational control is recognized as an increasingly important level of regulation of gene expression,but its impact in drug resistance has not yet been addressed totally.Among the major agents involved in translational control,the RNA binding protein HuR is a pleiotro pic protein regulating numerous physiological processes.HuR acts as a mRNA stabilizer andor a translational enhancer that binds to a large number of AU rich element containing mRNAs.Many of the genes con trolled by HuR are implicated in important physiological functions,for example embryonic development and cell differentiation.
HuR overexpression or preferential cytoplasmic localization has been correlated with carcino genesis in tissue biopsies and in cell models and patient negative Combretastatin A-4 prognosis.A caspase truncated type of HuR has also been identified as a promoter of cell death.In this work we explored the possibility that the involve ment of HuR in the apoptotic response could contribute towards the development of the resistance phenotype.Initial we show that HuR undergoes cytoplasmic translocation in MCF 7 cells exposed to doxo,and that this translocation is necessary to the doxo induced triggering of apoptosis.We lastly show that restoration of HuR expression in doxo resistant,HuR downregulating MDR cells is suffi cient to reacquire sensitivity to this anticancer drug.
Results Doxorubicin induces HuR phosphorylation and nucleocytoplasmic shuttling Since HuR is induced to relocate from the nucleus towards the cytoplasm following DNA damaging stimuli for example UVR,we reasoned that an anticancer agent recognized to induce DNA damage as doxorubicin could pro duce a similar effect.We starved MCF 7 cells for 24 h as a way to induce nuclear localization OAC1 of HuR.Indeed,after Extispicy 4 h of doxo addition,HuR translo cated into the cytoplasm.The translocation effect was proportional towards the applied dose,as quantified by calcu lating the ratio of the signal intensity of the protein in the nucleus versus the cytoplasm.The total quantity of HuR inside the cells did not change after doxo administration,as measured by densitometric analysis of three independent western blots.As can be seen in Figure 1C and 1D,HuR began to accumulate in the cytoplasm after 1 h of 10 uM doxo addition.
After OAC1 4 h,a two fold enrichment of the proteins was observed in the cytoplasm over the control condition.Moreover,within the time frame of the experiment and notwithstanding the recognized cell damage induced by doxo that will result in the possible loss of nucleocytoplasmic compartmentalization,the nuclear membrane was nonetheless intact since nuclear and cytoplasmic markers had been clearly confined in their com partments whilst HuR accumulated in the cytoplasm.Since HuR shuttling would be the consequence of post transla tional modifications,which includes phosphorylation we evaluated if doxo induced HuR phosphorylation.Lysates of cells treated with doxo resulted in the migra tion of HuR in a 2D Western blot stained with anti HuR antibody at pH values reduce than the pI of the native pro tein,which suggested that a series of phosphorylation events may have occurred after therapy using the drug.
The bands had been no longer visible after therapy of the lysates with alkaline phosphatases,consistent using the presence of phosphoryl groups.This result was confirmed by immunoprecipitating HuR under Combretastatin A-4 precisely the same experimental conditions and blotting with anti pan SerThr antibody.A phosphorylation band was observed in the control reaction,in the OAC1 presence of the serum,was absent in the course of starvation,and reappeared Combretastatin A-4 after doxo administration.These findings suggest that doxo induces phosphorylation of HuR and accumulation of HuR in the cytoplasm,as is generally observed with other DNA dama ging therapy for example cisplatin.
Apoptosis by doxorubicin is dependent on HuR phospohorylation and cytoplasmic translocation We investigated if HuR translocation was involved in OAC1 doxo induced cell death.Initially we evaluated the apopto tic response following doxo therapy in the presence and absence of HuR expression in a dose and time dependent manner.The apoptotic response to doxo was measured by the activation of caspase 3 and caspase 7 and by the expo sure of phosphatidylserine on the outer leaflet of the plasma membrane.We tran siently transfected MCF 7 cells with a siRNA against HuR and identified,as shown in Figure 2A,that caspase activation was reduce in HuR silenced cells in comparison to control cells.The reduce of caspase activation was signif icant after 4 h at 10 nM,100 nM and 1 uM doxo.We then tested if this effect could be obtained also by blocking doxo induced HuR phosphorylation by exploiting the recognized HuR phosphorylation inhibitor rottlerin.Rot tlerin administration to starved MCF 7 cells did not influ ence HuR phosphorylation and slightly influenced the outflow of the protei