| Endometrial cancer is one of three gynecological cancers,and the age of EC patients showed a trend of getting younger with the acceleration of lifestyle.Moreover,the second child policyand widespread phenomenon of late marriage and childbirth result in conservative therapy becoming more and more important to young EC patients desiring future fertility.Currently,the main conservative therapy method for EC patients is hormonal therapyusing progesterone(MPA)or its analogues.However,progesterone and its analogues are only effective on EC patients with positive progesterone receptor expression and the progesterone resistance is intractable.Therefore,it is an urgence to develop novel anti-EC drugs,especiallythat can reverse drug resistance in progesterone-resistant EC.New use for existing drug is a good drug discovery strategy due to its effectiveness,quickness and cost saving.In previous studies,antipsychotic drugs had been identified as agentsto treat cancersand to reverseantitumor drugs resistance.Hence,to discover better candidate drugs for the treatment of EC,20 tricyclic antipsychotic drugs in our approved drug library were screened through CCK-8 experiments on Ishikawa(ISK)cell lines(type I EC cell lines).Perphenazine(PPZ)and chlorpromazine hydrochloride(CPZ)were found with good inhibitory activity in ISK cell lines.Then we chose PPZ for the study of new use for anti-EC due to its slight side effects.Furthermore,in order to acquire candidate drugswith stronger activity,lower toxicity and independentintellectual property,we designed and synthesized an array of derivatives based on the structure of PPZ and CPZ.The first part was the effect evaluation andanti-EC mechanism study of PPZ,including antitumor activity in various EC cell lines,inhibition of colony formation and migration abilityin ISK and KLE cell lines,effect on cell apoptosis and cell cycle in ISK and KLE cell lines,inhibition of ISK subcutaneous xenograft mice and possible mechanism exploration.CCK-8 assay showed that PPZ exhibited good inhibitory activity in five EC cell lines(IC50=19.41～30.83 μM),including ISK,KLE,HEC-1A,HEC-1B and AN3CA.This assay indicated PPZ had broad-spectrum anti-EC activity in vitro,and its effect was superior to MPA.Colony formation assaydemonstrated that 20 μM and 30 μM PPZ had significant inhibition of colony formation in both ISK and KLE cell lines.Transwell migration assay indicated migration ability of ISK and KLE cell lines were inhibited by 10 μM PPZ.Annexin-V/PI double staining assaydemonstrated that 10 μM and 20 μM PPZ effectively induced cell apoptosis in both ISK and KLE cell lines.PIstaining assay showed 10 μM and 20μM PPZ induced G1 phase arrest in ISK cell linesand induced S phase arrest in KLE cell lines.Western blot assay suggested 20 μM PPZ could inhibit Akt phosphorylationin both ISK and KLE cell lines.ISK cell lines subcutaneous xenograft mice assay showed PPZ could significantly restrain xenograft tumor growth at both 3 mg/kg and 15 mg/kg,and the effect of PPZ was superior to MPA.RNA-seq assay indicated the anti-EC mechanism of PPZ may tie to original target DRD2.The second part was the design and syntheses of anti-EC derivatives based on the structure of PPZ and CPZ.Based on bioisostere,we employed scaffold hopping by introducing nitrogen atom andtransformedcyclobenzene to pyridine ring.We obtained the skeleton benzo[b]pyrido[2,3-e][1,4]thiazine.In order to explore the activitity of diifferent derivatives,24 phenothiazine derivatives were designed and synthesized by changing the length of linker,substituent group on the nitrogen atom and the substituent group of the 2-position of the new skeleton.Proliferation inhibition assay indicated that the IC50values of 16derivatives were less than CPZ and eight of them(D01·HCl、D05·HCl、D06·2HCl、D07·HCl-D10·HCl and E01·HCl)were less than 15μM in ISK cell lines.The anti-EC activity of optimum derivativeD06·2HCl was improved markedly compared to CPZ.The activity of D06·2HClhad 3.56-fold and 11.56-fold enhancement after treatment for 48 h and 72 h in ISK cell lines,meanwhile,D06·2HClhad 2.16-fold and 12.22-fold enhancement in KLE cell lines.Moreover,the selectivity of D06·2HCl to ISK and KLE cell lines were 3.68-fold and 2.08-fold compared to normal endometrial cell lines.Finally,Annexin-V/PI double staining assaydemonstrated that4 μM and 8μMD06·2HClcouldinduce cell apoptosis in both ISK and KLE cell lines.Above all,D06·2HClshowed strong proliferation inhibition activity in both ISK and KLE cell lines and clould induce cell apoptosis.D06·2HCl was selective for EC cells and normal endometrial cell lines.D06·2HCl could be identified as a potential anti-EC(progesterone resistant EC)drug and worth futher developmentforEC(progesterone resistant EC)treatment... |
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