The Study Of Arsenic Trioxide Combined With Antiangiogenic Drug On Imatinib-resistant Gastrointestinal Stromal Tumor

Objective:Gastrointestinal stromal tumors(GISTs)is the most common mesenchymal tumor of the digestive tract and characterized by expression of KIT protein.Its morbidity is gradually increased year by year.Most GISTs have oncogenic KIT mutations that engender constitutive activation of this receptor tyrosine kinase in the pathogenesis of GISTs.Treatment of advanced GIST improved dramatically following development of imatinib.Despite the often long-lasting clinical benefit seen in most patients treated with imatinib,many will eventually suffer disease progression.The mechanisms of acquired resistance to imatinib are heterogeneous.This study investigated the mechanism of resistance to imatinib through establishing several imatinib-resistant GIST cell lines and undertaking in vivo and vitro experiment.We further explored the potential use of arsenic trioxide combined with antiangiogenic drug as a strategy to overcome imatinib resistance in GISTs.Methods:Three imatinib-resistant human GIST lines(GIST-R,GIST-PR1,GIST-PR2)have been established and characterized.MTT assays were performed to ascertain new drugs sensitivity/resistance by calculating the biological IC50 values.The effect of sunitinib,arsenic trioxide(As2O3)and thalidomide on the autophosphorylation of the KIT in cultured imatinib-resistant cells that harbored KIT-K642E(GIST-R),KIT-V559D/V654A(GIST-PR1),KIT-Del WK557-558/T670I(GIST-PR2),mutant isoforms was determined by Western blot.Amplification of KIT in imatinib-resistant cells was determined by dual-color interphase fluorescence in situ hybridization(FISH)analysis.Induction of apoptosis on imatinib-resistant GIST cells was evaluated by flow cytometry using Annexin-V/PI Staining Kit according to the manufacturer’s protocol in duplicate experiments.In vivo study,we investigate the antitumor activity of sunitinib,As2O3 and thalidomide against cell-derived imatinib-resistant GIST xenografts.Murine xenograft models were given above-mentioned drugs daily for 14 days and growth of established tumor xenografts was monitored at least weekly by vernier caliper measurements.Western blotting was then used to determine changes of KIT proteins in these xenografts before and after drugs therapy.Results:MTT assays showed GIST-PR1 and GIST-PR2 cells were sensitive to sunitinib.But GIST-R cells were resisitant to sunitinb in vitro.As2O3 or thalidomide alone could not inhibit imatinib-resisitant cells proliferation obviously.However,when resisitant GIST cells(including GIST-R cell)were treated with increasing doses of AS2O3(0.5-5μM)but constant thalidomide doses(50μg/ml)a clear syngergy was observed.FISH analyses showed amplification of KIT was only in GIST-PR1 cells.In GIST-R cells,heterozygous loss of KIT/CEP4 loci was found with loss of KIT immunoreactivity.However,imatinib-sensitive GIST882 cells was normal diploid and KIT positive.Western blot results showed GIST-PR1 and GIST-PR2 cells demonstrated KIT expression and various levels of constitutive KIT autophosphorylation before treatment.But GIST-R cells were totally lacked KIT expression,which was in line with the loss of CD117 positivity by immunohistochemistry and the observed heterozygous loss of KIT/CEP4 loci.KIT autophosphorylation was inhibited by sunitinib treatment in GIST-PR1 and GIST-PR2 cells.But KIT protein was expressed and phosphorylated to a significant level in As2O3 or thalidomide group.In As2O3 and thalidomide combination group,2.0μM As2O3 reduced and 10μM As2O3 totally inhibited KIT autophosphorylation in GIST-PR1 and GIST-PR2 cells.Flow cytometry assays demonstrated sunitinib potently inhibited cell proliferation and induced apoptosis in GIST-PR1 and GIST-PR2 cells even at nanomolar concentrations.But sunitinib did not induced apoptosis in GIST-R cells obviously.In As2O3 and thalidomide combination group,an apoptosis assay confirmed the significant induction of apoptosis in all three imatinib-resistant GIST cells.In contrast,apoptosis rates of all imatinib-resistant GIST cells were low in As2O3 or thalidomide group.These data were in line with results of MTT and Western blot,especially in GIST-R cells.In vivo study,the marked inhibitory effect on xenografts tumors growth was seen in the groups treated with high dose As2O3 alone or in combination with Thal.Inhibitory effect was also seen in low dose As2O3 and Thal combination group.But the therapeutic effects were unsatisfied in the group treated with sunitinib.No overt toxicity in low dose As2O3 and Thal combination group was observed during the course of treatment.But more intense toxicity was seen in group of high dose As2O3 alone or in combination with Thal.Conclusion:Three imatinib-resistant human GIST lines have been established and characterized.In GIST-R cell line,no secondary mutation was found except primay KIT-K642E.GIST-PR1 cell line demonstrated an additional secondary mutation KIT-V654A besides primay V559D mutation.GIST-PR2 showed a primary KIT-Del WK557-558 and secondary T670I mutation.Human GIST xenografts with mutation in KIT have also been established from these imatinib-resistant GIST lines.Those models will enable further studies on mechanisms of resistance,combination therapies and allow testing of novel targeted therapies.This study showed KIT kinase domain mutations in patients with secondary resistance and defined genomic amplification of as an alternative cause of resistance to imatinib.In a subset of patients,cancer cells lost their dependence on the targeted tyrosine kinase.Our findings showed the sensitivity of the imatinib-resistant KIT-T670I and KIT-V654A mutants to sunitinib.Furthermore,As2O3 and thalidomide combination has also been a potent inhibitor of KIT activation loop mutants and induced apoptosis in imatinib-resistant GIST lines expressing these mutations.