Molecular Mechanisms Underlying Sensitization Of Hormone And Chemotherapy-resistant Castration Resistant Prostate Cancer By Novel Drug Conjugate DZ-SIM

Background and Objective:Prostate cancer is one of the most commonly seen malignancies in western countries,there’s an estimated 1,276,106 new onset cases and 358,989 new deaths in 2019 globally.At initial diagnosis,only around 6% of patitients have metastasis,however,around 90% of patients died from prostate cancer have bone metastasis.In recent years,with the aging and life style changes in our ountry,the incidence of prostate cancer in China has grown drastically.Unlike western countries,more Chinese patients are in advanced stage with dismal prognosis.Prostate cancer is driven by androgen signaling,anti-androgen treatment could lower serum testosterone level.At the initial treatment with antiandrogens,prostate cancer responds well.However,antiandrogen treatment is not curative therapy,almost all patient will progress eventually.During the antiandrogen therapy,the serum testostrone is at castration level,when prostate cancer progresses during anti androgen therapy,it becomes castration resistant and is therefore called castration resistant prostate cancer(CRPC).For a long period of time,the mainstay treatment for CRPC has been docetaxel.With the advent of next generation antiandrogen therapies(NGAT),including but not limited to abiraterone and enzalutamide,more and more docetaxel-resistant CRPC patients are treated with NGATs.Recently,NGATs are also used to treat CRPC patients before docetaxel treatment with favorabel outcomes.It has been reported that patients resistant to abiraterone may also become resistant to docetaxel.Although docetaxel and NGATs could prolong CRPC patients progression free survival and overall survival,the benefit is limited.All CRPC patients will eventually become resistant to docetaxel and NGATs.Our lab had previously synthesized a group of heptamethine carbocyanine dye(HMCD),which preferentially accumulate in tumor cells and tissues rather than normal cells and tissues.Later on,one of the HMCD molecule is renamed as DZ-1 and make conjugate with DZ-1 and other drugs including but not limited to DZ-gemcitabine,DZ-Cisplatin and DZ-SIM.Among them,DZ-SIM has the most potent effect in killing prostate cancer cells.DZ-SIM conjugate is synthesized from DZ-1 and simvastatin.Our aim is to evaluated the tumor-specific accumulation of DZ-SIM,its growth inhibition on CRPC cells resistant to docetaxel and NGATs and the underlying molecular mechanism.Part Ⅰ The inhibition role of DZ-SIM on CRPC cells resistant todocetaxel and NGATsMethod:(1)Determining DZ-SIM’s inhibition on growth of different types of prostate cancer using crystal violet method;(2)Evaluating DZ-SIM’s effect on the colony formation ability of C4-2B,C4-2B MDVR and C4-2B TaxR cells using colony formation assay;(3)Evaluating DZ-SIM’s effect on the migration and invation of C4-2B,C4-2B MDVR and C4-2B TaxR cells using scratch assay and traswell assay(with or without matrigel);(4)(5)Subcutaneous xenograft model: Inoculate CWR22RV1 cells on both flanks of NOD-SCID mouse and measure the tumor size regularly to evaluate DZ-SIM’s effect on tumor cells growth in vivo;Results:(1)DZ-SIM inhibis the growth of different types of prostate cancer in a time-and dosedependent manner,while DZ or SIM alone has minimal effect on prostate cancer cells;(2)Colony formation assay shows that DZ-SIM’s inhibits the colony formation ability of C4-2B,C4-2B MDVR and C4-2B TaxR cells in a dose-dependent manner;(3)DZ-SIM inhibits the migration and invasion of C4-2B MDVR and C4-2B TaxR cells in a dose-dependent manner;(4)Subcutaneous xenograft model show that DZ-SIM greatly inhibited the growth of subcutaneous CWR22RV1 cells xenograft on NOD-SCID mice;Conclusions: DZ-SIM inhibited the growth of resistant CRPC cells and can be developed as a sensitizer for hormone and chemotherapy resistant PCa.Part Ⅱ The molecular mechanisms underlying DZ-SIM’s inhibitioneffect on CRPC cells resistant to docetaxel and NGATs:Method:(1)Determining DZ-SIM’s subcellular location using mitotracker;(2)Evaluating DZSIM’s effect on mitochondria’s membrane potential and apoptosis using flow cytometry;(3)Determining the optimal FCCP concentration of different cells using Seahorse XFe96 Analyzer,then evaluating DZSIM’s effect on Oxygen consumption rate,OCR and Extracellular acidification rate,ECAR in CRPC cells resistant to docetaxel and NGATs.(4)RNA-Seq and Bioinformatics analysis of common targets by DZSIM in CRPC cells resistant to docetaxel and NGATs and validate the RNA-Seq results using qRT-PCR.Results: Mitotracker indicates that DZ-SIM localized in mitochondria at subcellular level.(2)Flow cytometry found that DZ-SIM treatment decreased the mitochondrial membrane potential and induced late apoptosis in C4-2B MDVR and C4-2B TaxR cells;(3)The optimal FCCP concentration for C4-2B,C4-2B MDVR and C4-2B TaxR cells are 0.5μM、0.25μM and 1μMrespectively.Compared with C4-2B cells,C4-2B MDVR and C4-2B TaxR had increased OCR and ECAR levels.Treatment with DZ-SIM decreased OCR of resistant cell lines C4-2B MDVR and C4-2B TaxR.(4)RNA Seq and bioinformatics analysis found 72 genes are commonly upregulated in CRPC cells resistant to docetaxal and NGATs.After DZ-SIM treatment,4 genes are commonly downregulated,namely CAMK2N1,HMGCS2,NR2F1 and CDH3.In qRT-PCR validation,all four genes are upregulated in these cell lines and down regulated by DZ-SIM treatment.Conclusion: DZ-SIM localizes in mitochondria and decreases mitochonddrial membrane potential in CRPC cells resistant to docetaxel and NGATs.DZ-SIM acts through affecting mitochondrial metabolism.CAMK2N1,HMGCS2,NR2F1 and CDH3 are potential direct targets of DZ-SIM.