The Role And Mechanism Of Niclosamide And Its Derivative In Castration-resistant Prostate Cancer

Objective : Prostate cancer is one of the most common malignant tumors in males.Androgen deprivation therapy(ADT)remains the standard treatment for inoperable prostate cancer patients.However,drug resistance inevitably developing in most patients after 12–18 months of continuous ADT made the treatment more difficulty in general and eventually poor prognosis.Currently,clinical common medicine for CRPC is: 1.Cytotoxic drugs,such as carbataxel,epothicin and other microtubule inhibitors,but their serious toxic and side effects;2.Targeting androgen receptor(AR)functions via AR antagonists,such as Abiraterone acetate and Enzalutamide,but with inevitably different level of drug resistance in most of patients.Therefore,it is of vital importance to develop a less toxic and high efficacy drug for CRPC.Niclosamide is an oral antihelminthic drug approved by the United States Food and Drug Administration(FDA).It inhibits cell mitochondrial oxidative phosphorylation of the parasite and hinder the absorption of glucose uptake,which results in tapeworm head and proximal segments death.Niclosamide is well tolerated and has mild oral adverse reactions in humans.Recently,niclosamide was found as a potential anticancer agent through high-throughput drug screening platform and its anti-tumor proliferation activity has been identified in many tumor,including breast cancer,CRPC,acute myeloid leukemia,non-small cell lung cancer,multiple myeloma,hepatocellular carcinoma,adrenocortical cancer,ovarian cancer and glioblastoma.Niclosamide is expected to be a new cancer treatment drug.US FDA has approved the phase Ib/II clinical trial of Niclosamide combined with Enzalutamide for androgen receptor splice variant positive CRPC.However,there are still some challenges to use niclosamide as an anti-cancer agent in humans: 1.Low drug exposure in serum,for poor water solubility.The oral bioavailability of niclosamide was only 10% in male Sprague-Dawley rats;2.Short plasma half-life(t1/2),that was 6.7 ± 2.0 hr in rats.Therefore,the low oral bioavailability and wide range of serum concentration of niclosamide specially limit niclosamide anticancer application.We develop a novel niclosamide derivative(DK419) which can significantly increase both the plasma concentration and exposure duration during oral administration and enhance its antitumor activity.This project we intends to explore the therapeutic effection and molecular mechanism of niclosamide and its derivative in CRPC.Methods:1.The effect of niclosamide and its derivative(DK419)on prostate cancer cell lines in vitro.We selected a variety of prostate cancer cell lines:1.Ln CAP is adenocarcinoma that is androgen(AR)-dependent(castration-sensitive);C4-2 was originated from Ln CAP xenograft metastasis in mice.It has AR mutation and is AR-independent(castration resistant);C4-2 MDR is enzalutamide resistant C4-2 cells;C4-2B cells were chronically exposed to increasing concentrations of enzalutamide(5~40μM)by passage in media containing enzalutamide for >12 months;PC3 is neuroendocrine small cell carcinoma that has no androgen receptor(castration resistant and enzalutamide resistant).DU145 is a high metastatic potential prostate cancer cell line and lacks endogenous androgen receptor(castration resistant and enzalutamide resistant).MTT was used to detect the effect of niclosamide and its derivative(DK419)on different prostate cancer cell lines.In addition,we also tested the effection of the combination enzalutamide with niclosamide/its derivative(DK419)on prostate cancer cell lines.2.The effect of niclosamide and its derivative(DK419)on prostate cancer cell lines in vivo.Animal experiment groups are as follows:1.Vehicle group:6 mice,PEG300 90%,NMP 10%,oral gavage+ intraperitoneal injection PBS+DMSO;2.Niclosamide group:6mice,100mg/kg,oral gavage+ intraperitoneal injection PBS+DMSO;3.DK419 group:6 mice,1mg/kg,oral gavage+ intraperitoneal injection PBS+DMSO;4.DK419+ENZA group:6 mice,1mg/kg,oral gavage+ intraperitoneal injection ENZA 1mg/kg;5.ENZA group :6 mice,PEG300 90%,NMP 10%,oral gavage+ intraperitoneal injection ENZA1mg/kg.Mice organ: HE stainning of liver,spleen,kidney,and lung in each group showed no drug toxicity reaction.Administration cycle: When the tumor reaches 0.5cm,gavage continuously for 6 days,intermittently for 1 day.Terminate the experiment after 4weeks or according to the ethical requirements.Detection: 1.Tumor volume(three times a week,with a caliper);2.Mouse weight(measured every day);3.Organs and tumors collected at the end of the experiment,HE staining of organs for toxicological study;the tumor tissue was stained by immunohistochemistry.3.The mechanism of niclosamide and its derivative in castration resistant prostate cancer1)Determine the Metabolic Phenotype of prostate cancer cell lines Using the XFp Cell Energy Phenotype Test Kit.2)To detect the effection of niclosamide and its derivative on the mitochondrial function of prostate cancer cell line3)To detect the effection of niclosamide and its derivative on prostate cancer cell line after treatment with sugar free medium4)The effection of combined with 2DG and niclosamide / its derivative(DK419)on prostate cancer cell line.5)Western blot detection of the effection of niclosamide / its derivative(DK419)on Wnt/β-Catenin,AR,p-STAT3 related signaling pathway in prostate cancer cell lineResults: 1.Inhibit proliferation of prostate cancer cell lines.The androgen dependent cell line Ln CAP cell and androgen independent cell line C4-2 cell were significantly inhibited by niclosamide / its derivative(DK419),which also has a certain killing effect on enzalutamide resistance cell lines C4-2 MDR and C4-2B cells,but for high metastatic potential PC3 and DU145 cell line,the killing effect was limited.Combination enzalutamide with niclosamide/its derivative(DK419)can enhance the killing effect on prostate cancer cell lines in vitro.2.Animal experiments showed that the therapeutic effect of DK419 was better than that of niclosamide,and it was safe and effective.However,Combination enzalutamide with DK419 did not enhance the therapeutic effect on castrated resistant prostate cancer.3.The anticancer effects of niclosamide and its derivative(DK419)were associated with mitochondrial metabolism function of prostate cancer cell line4.Western blotting showed that in both sensitive(C4-2)and insensitive(DU145)prostate cancer cell lines,niclosamide and its derivative can inhibit the activation of Wnt signaling pathway and p-STAT3 related proteins in DU145;mild suppression the expression level of AR and PSA in C4-2.Therefore,we speculate that the inhibition of prostate cancer cell line biological behavior may not entirely dependent on targeting a specific oncogenic pathway.Conclusion: 1.We developed a safe and effective oral niclosamide derivative(DK419),which bioavailability is superior to niclosamide in the treatment of CRPC in vitro and in vivo.2.Combination with Enzalutamide and niclosamide/DK419 did not enhance the therapeutic effect on CRPC in vivo.3.The killing effect of niclosamide and DK419 on prostate cancer cells was assosiated with its inhibition on cancer cell mitochondrial metabolism function.4.Combination with glycolysis inhibitor 2-DG and niclosamide/DK419 can significantly enhance the killing effect on highly malignant and aggressive prostate cancer cell lines.