Evaluation Of Aminoethanol Di-ethacrynic Acid As Gstm Selective Pro-inhibitor And Its Sensitization For DDP-Resistent Cancers

Tumor resistance is an important reason for treatment failure,and GST is an important target for tumor resistance,through catalyzing the conjugation of anticancer drugs with GSH and their excretion,as well as through binding to the key protein factors involved in apoptosis pathways.GST inhibitor with high affinity thus can reverse drug resistance.Early designed EDEA,with selectivity as GSTM2> GSTA1> GSTP,could inhibit GSTM2 at the namo level,and had sensitizing effect on lung cancer and ovarian cancer,but was found to have a certain potential toxicity.Ethanolamine is used for the synthesis of choline in vivo.In this paper,Ethanolamine was used as a connecting arm to link two molecules of Ethanilic acid to synthesize Aminoethanol Di-Ethacrynic Acid(ADEA),and its inhibition selectivity,toxicity and sensitizing effect were evaluated.Firstly,the selective inhibition against GSTA1,GSTP1 and GSTM2,inhibitory types,binding ratio and binding kinetics of ADEA and its conjugate with GSH were characterized.The IC50 of ADEA itself on GSTM2 was 29 nmol/L,which was 300 times lower than GSTA1 and 3,000 times lower than GSTP1;the IC50 of ADEA and GSH adducts for three isozymes were reduced by 5,57 and 200 times,respectively.ADEA itself and GSH adducts were uncompetitive inhibitors of GSTM relative to substrate CDNB,ADEA itself is a mixed inhibitor against GSTM relative to substrate GSH,ADEA and GSH adducts against GSTM relative to substrate GSH uncompetitive inhibitors.The static quenching of GSTM fluorescence by the adduct of ADEA generated in situ indicated a bivalent inhibitor;ADEA itself binds 10 times slower than the adduct.It shows that ADEA is an effective GSTM selective divalent pro-inhibitor.With ADEA as a probe,the GST enzyme activities and GSTM of tumor cell lysate were estimated according to the responces to the concentration of ADEA.GSTM activity was assigned to the enzyme activity sensitive to ADEA at less than 10 nmol/L,according to the difference of IC50 of ADEA adducts for each subtype.Compared with SKOV3,the enzyme activity of SKOV3/DDP was increased about 2.0 times,and that of GSTM is increased about 4.0 times;SGC7901/DDP has an enzyme activity decreased about 1.2 times,and no significant difference of GSTM activity;A549/DDP had about 3 times,and GSTM activity about 2 times decreased in comnparing with A549.IC10 were determined by CCK-8 for comparion of the toxicity of ADEA and EDEA on normal embryonic kidney HEK293,liver LO2,lung epithelium BEAS-2B,glial cell HEB,and cardiomyocyte AC16.IC10 of ADEA for HEK293 and BEAS-2B were 2 ?mol/L,<1 ?mol/L for AC16;three were similar to those of ADEA;IC10 of ADEA to LO2 was about 1.9 ?mol/L,which was twice higher than EDEA,but for HEB was 0.3 ?mol/L,which was three times lower than EDEA.ADEA at 2.0 ?mol/L used in combination with DDP showed no synergistic effect on liver cell LO2,while the combination use of EDEA at 1.0 ?mol/L showed 1.6 times sensitization.Therefore,ADEA had lower liver toxicity,and neurotoxicity was increased,which may be related to the increased permeability agsinst blood-brain barrier.ADEA at a low-toxitic dose(2.0 ?mol/L)in combination with DDP showed 3.1,1.3,and 6.9 times sensitization to SKOV3/DDP,SGC7901/DDP,and A549/DDP,respectively.The effects on SKOV3/DDP and SGC7901/DDP rather than A549/DDP were comparable with those by EDEA at the low-toxitic dose,while less sensitizing effects at higher doses than EDEA.Flow cytometry and Hoechest233342 used for comparing the effects of ADEA and EDEA,combined with DDP,on tumor cell apoptosis.They both had syngestic effect with DDP on the apoptosis of three types of DDP-resistant tomur cells,comparable on SKOV3/DDP and A549/DDP,but lower on SGC7901/DDP with ADEA.Transwell and scratch experiments found that ADEA and EDEA did not affect the migration of SGC7901/DDP and SKOV3/DDP.Resonance light scattering method was used to determine the solubility of ADEA and EDEA in different buffers.Fluorescence quenching method was used to investigate the binding ability of BSA to ADEA and EDEA.Both indicated that ADEA was less water-soluble,but the binding to BSA of ADEA was stronger than EDEA.By screening with the solubilizers including glycerin and Tween-80,0.2% Tween-80 was used to prepare the injection drug preparation.SGC7901/DDP was implanted subcutaneously in nude mice for 14 days and then randomly divided into groups.Compared with the normal saline group,after 1-2 weeks of treatment,the tumor volume growth rate of the EDEA group and the EDEA + DDP group were significantly reduced;after 24 days,nude mice were killed.Compared with the DDP group,,there was a significant reduce in tumor volume of EDEA and DDP combination group,and the difference in tumor weight was insignificant.Compared with the saline group and the EDEA alone group,there were insignificant difference in the tumor volume and tumor weight of EDEA and DDP combination group.In summary,the newly sesigned ADEA is a selective bivalent pro-inhibitor of GSTM2 by enzymatic characterization.Probing by ADEA,the activity of GSTM and total enzyme activity of SKOV3/DDP was significantly increased.ADEA alone and in combination with DDP have less toxicity to liver than EDEA,but relatively greater toxicity to HEB cells.ADEA at their low toxic dosages has a similar sensitizing effect with EDEA on SKOV3/DDP,SGC7901/DDP rather than A549/DDP,but at higher dosages showed less sensitizing effect than EDEA.The combination of EDEA and DDP had a limited sensitizing effect on SGC7901/DDP tumor nude mouse model,indicated the selective sensitizing effect of EDEA on drug-resistant ovarian cancer.ADEA is expected the similar sensitizing effect in vivo.