WO2020248824A1 - Method and pharmaceutical composition for prevention and treatment of a cancer - Google Patents

Method and pharmaceutical composition for prevention and treatment of a cancer Download PDF

Info

Publication number
WO2020248824A1
WO2020248824A1 PCT/CN2020/092922 CN2020092922W WO2020248824A1 WO 2020248824 A1 WO2020248824 A1 WO 2020248824A1 CN 2020092922 W CN2020092922 W CN 2020092922W WO 2020248824 A1 WO2020248824 A1 WO 2020248824A1
Authority
WO
WIPO (PCT)
Prior art keywords
thb
ahr
zebrafish
cancer
tetrahydrocorticosterone
Prior art date
Application number
PCT/CN2020/092922
Other languages
French (fr)
Inventor
Wen-Ming Hsu
Hsinyu Lee
Original Assignee
Hsu Wen Ming
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hsu Wen Ming filed Critical Hsu Wen Ming
Publication of WO2020248824A1 publication Critical patent/WO2020248824A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a method and pharmaceutical composition for prevention and treatment of a cancer.
  • the aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor, which belongs to the basic-helix-loop-helix (bHLH) /PAS (Period [Per] -Aryl hydrocarbon receptor nuclear translocator [ARNT] -Single minded [SIM] ) family of heterodimeric transcriptional regulators [1, 2] .
  • AHR may be activated by binding to a wide range of xenobiotic ligands, ranging from plant material to pollutants that are either natural (e.g., benzo [a] pyrene (B [a] P) ) or synthetic (e.g., 2, 3, 7, 8-tetrachlorodibenzodioxin [TCDD] ) [3] .
  • AHR resides in the cytosolic compartment, sequestered by a molecular chaperone complex that contains two molecules of the heat shock protein 90 (HSP90) , the HSP90-interacting protein, p23, and the immunophilin-like protein, XAP2 (also named ARA9 or AIP) [4] .
  • HSP90 heat shock protein 90
  • XAP2 also named ARA9 or AIP
  • conformational changes in the AHR protein alter its binding to XAP2 and activate a nuclear localization sequence. These structural changes direct HSP90-bound AHR to translocate into the nucleus, where the receptor dissociates from the HSP90 complex and dimerizes with ARNT [4] .
  • the AHR/ARNT heterodimer then interacts with a partially characterized set of co-activators and/or co-repressors to bind the XRE-rich enhancer and TATA box in the promoter regions of target genes, followed by the recruitment of RNA polymerase II to initiate transcription [5] .
  • genes coding for the xenobiotic-metabolizing cytochrome P450 enzymes such as CYP1A1, CYP1B1, UDP-glucuronosyl transferase (UGT1A6) , aldehyde dehydrogenase (ALDH3A1) and multidrug resistance-associated proteins (MRPs) , are transcriptionally induced.
  • AHR is exported to the cytosol by chromosome region maintenance 1 (CRM1) and degraded by the 26S ubiquitin-proteasome [6, 7] .
  • AHR has been shown to be an important regulator of cell death, proliferation, differentiation, and cell cycle progression [8-12] .
  • Physiological studies have also suggested that AHR may be involved in tissue development or homeostasis because its absence has been shown to result in infertility, abnormalities in liver, and cardiovascular problems [13-19] .
  • AHR was recently found to be expressed in cerebellar granular neuronal precursor (GNPs) in the early postnatal period, where it regulates the growth and differentiation of granule neuroblasts [20, 21] .
  • GNPs cerebellar granular neuronal precursor
  • AHR-deficient mice also display diminished neuronal differentiation in the dentate gyrus [22] , and knockout of AHR causes oculomotor and optic nerve deficits in a mouse model [23, 24] . Additionally, we previously reported that overexpression of AHR promotes neural differentiation of neuroblastoma (NB) cells [25] , further supporting the idea that AHR plays an important role in regulating neural development.
  • NB neuroblastoma
  • NB is derived from the sympathoadrenal lineage of the neural crest and is the most common extracranial solid tumor in children, accounting for 15%of all child cancer mortality [26-28] .
  • MYCN MYCN
  • the expression level has been correlated with activation of genes associated with NB tumor aggression [29] .
  • the molecular mechanism underlying this carcinogenic process remains elusive.
  • the present invention provides a method for preventing or treating a cancer, comprising administering to a subject in need thereof a therapeutically effective amount of 3 ⁇ , 5 ⁇ -Tetrahydrocorticosterone (5 ⁇ -THB) or 3 ⁇ , 5 ⁇ -Tetrahydrocorticosterone (5 ⁇ -THB) .
  • the 5 ⁇ -or 5 ⁇ -THB is capable of enhancement of the expression of sex determining region Y-box 10 (Sox10) and myelin-associated proteins myelin basic protein (MBP) .
  • Sox10 sex determining region Y-box 10
  • MBP myelin-associated proteins myelin basic protein
  • the cancer is neuroblastoma (NB) .
  • the 5 ⁇ -or 5 ⁇ -THB inhibits tumorigenesis in NB.
  • the present invention provides a pharmaceutical composition for preventing or treating a cancer, comprising a therapeutically effective amount of 3 ⁇ , 5 ⁇ -Tetrahydrocorticosterone (5 ⁇ -THB) or 3 ⁇ , 5 ⁇ -Tetrahydrocorticosterone (5 ⁇ -THB) in association with a pharmaceutically acceptable carrier.
  • a pharmaceutical composition for preventing or treating a cancer comprising a therapeutically effective amount of 3 ⁇ , 5 ⁇ -Tetrahydrocorticosterone (5 ⁇ -THB) or 3 ⁇ , 5 ⁇ -Tetrahydrocorticosterone (5 ⁇ -THB) in association with a pharmaceutically acceptable carrier.
  • the present invention provides a use of 3 ⁇ , 5 ⁇ -Tetrahydrocorticosterone (5 ⁇ -THB) or 3 ⁇ , 5 ⁇ -Tetrahydrocorticosterone (5 ⁇ -THB) in manufacturing a medicament for preventing or treating a cancer.
  • Figure 1 provides the identification of endogenous AHR ligands with dioxin-like compound cell free bioassay.
  • A A schematic diagram of the dioxin-like compound cell free bioassay.
  • B Fractionation of day 0 zebrafish embryo extract by reverse-phase HPLC.
  • C Fractions collected from HPLC were analyzed by the dioxin-like compound cell free bioassay.
  • D Chemical structure of 5 ⁇ -and 5 ⁇ -THB.
  • FIG. 2 shows that THB induced nuclear translocation of AHR and promoted CYP1A1 expression.
  • A AGAPA cells were treated with THB 100 nM for 90 min. EtOH was used as a negative control. The cells were fixed, and AHR-GFP distribution was evaluated by fluorescence microscopy. Nuclei were stained with DAPI (blue) . Scale bar, 20 ⁇ m.
  • B AAPA cells were grown to 50%confluency in 10-cm dishes followed by tetracycline induction for 24 h. Induced AAPA cells were then treated with THB (100 nM) or Kyn (200 ⁇ M) for 2 h. EtOH and HCl (0.5M) was used as solvent control respectively.
  • the cytosolic and nuclear fractions were collected and analyzed by western blot. Anti-AHR antibody was used for AHR detection. GAPDH and Lamin A/C were used as internal loading controls for the cytosolic and nuclear fractions respectivly.
  • C The nuclear-to-cytosol ratio of AHR expression level was quantified by ImageJ. Quantified results are shown as the mean ⁇ SD from 4 independent experiments and analyzed by one-way ANOVA. (*p ⁇ 0.05; **p ⁇ 0.01) .
  • D AAPA cells were treated with indicated concentrtions of THB for 4 hr. CYP1A1 mRNA expression level was measured by SYBR green real-time PCR.
  • FIG. 3 shows that THB promoted neuronal differentiation-associated gene expression and inhibits NB tumor growth.
  • A-C SK-N-SH cells were treated with 100 nM THB for 4 hr. mRNA expression levels of GAP43, NSE and NF were analyzed by SYBR green real-time PCR.
  • D-F stNB-V1 cells were xenotransplanted in a mouse xenograft model. Tumor-inoculated mice were treated with vehicle (control) , RA (5 mg/kg) , 5 ⁇ -THB (5 mg/kg) or 5 ⁇ -THB (5 mg/kg) .
  • D Tumor inoculated mice were sacrificed after 21 days of treatment. Tumors from each mouse are shown in the photograph.
  • FIG. 4 shows that Ahr2 morpholino suppressed endogenous and THB-induced cyp1a expression in zebrafish.
  • A-B Zebrafish embryos were exposed to increasing amounts of THB. Total mRNA was collected in TRIzol reagent, and cyp1a mRNA expression level was analyzed by real-time PCR.
  • C Zebrafish embryos were microinjected with standard control MO (SDMO) and zAHR2 MO. The total protein of 5 dpf embryos was collected and analyzed by western blot.
  • D-E Zebrafish embryos injected with SDMO or ahr2MO were treated with THB. The cyp1a mRNA expression was examined by real-time PCR.
  • FIG. 5 shows that THB promoted early neural development of Tg (HuC: Kaede) transgenic zebrafish model. Image analysis of 1, 3 and 5 dpf Tg (HuC: Kaede) zebrafish. Green signals show the Kaede distribution.
  • Figure 6 shows Ahr2 mediated gliogenesis in zebrafish.
  • A Green signal shows HuC-Kaede protein in cross-sectional views of the spinal cord from Tg (HuC: Kaede) transgenic zebrafish. The dorsal surface is shown on top, while ventral is on the bottom.
  • B-D IHC staining of O4 (red) (B) , GFAP (red) (C) and CD11b (red) (D) . Nuclei were counterstained with DAPI (blue) . Three independent samples were evaluated and representative data are shown. Quantitative data are shown as the mean ⁇ SD from three independent experiments run in triplicate and analyzed by ANOVA (*p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001) .
  • FIG. 7 shows that 5 ⁇ -THB-Ahr2 signaling regulates myelinating glial cell development in zebrafish.
  • a and C Zebrafish embryos were exposed to increasing amounts of 5 ⁇ -THB. Total mRNA was collected in TRIzol reagent, and Sox10 and MBP mRNA expression levels were analyzed by real-time PCR.
  • B and D Zebrafish embryos injected with SDMO or ahr2MO were treated with 5 ⁇ -THB. Sox10 and MBP mRNA expression levels were examined by real-time PCR.
  • E Embryos of transgenic zebrafish, Tg (-4725sox10: egfp) ba4, were microinjected with SDMO or ahr2MO.
  • Each group of microinjected embryos was treated with ethanol control or 5 ⁇ -THB (100 nM) .
  • the EGFP signal of 2 dpf embryos was evaluated. Arrows show EGFP signal in the spinal cord.
  • (F) Embryos of transgenic zebrafish Tg (mbp: egfp) were microinjected with SDMO or ahr2MO.
  • Each group of microinjected embryos was treated with ethanol control or 5 ⁇ -THB (100 nM) .
  • the EGFP signal of 5 dpf embryos was evaluated. Arrows indicate EGFP signal in the spinal cord and motor neurons.
  • FIG. 8 shows that 5 ⁇ -THB-Ahr2 signaling regulates myelinating glial cell development in zebrafish.
  • a and C Zebrafish embryos were exposed to increasing amounts of 5 ⁇ -THB. Total mRNA was collected in TRIzol reagent, and Sox10 and MBP mRNA expression levels were analyzed by real-time PCR.
  • B and D Zebrafish embryos injected with SDMO or ahr2MO were treated with 5 ⁇ -THB. Sox10 and MBP mRNA expression levels were examined by real-time PCR.
  • E Embryos of transgenic zebrafish, Tg (-4725sox10: egfp) ba4, were microinjected with SDMO or ahr2MO.
  • Each group of microinjected embryos was treated with ethanol control or 5 ⁇ -THB (100 nM) .
  • the EGFP signal of 2 dpf embryos was evaluated. Arrows show EGFP signal in the spinal cord.
  • (F) Embryos of transgenic zebrafish Tg (mbp: egfp) were microinjected with SDMO or ahr2MO.
  • Each group of microinjected embryos was treated with ethanol control or 5 ⁇ -THB (100 nM) .
  • the EGFP signal of 5 dpf embryos was evaluated. Arrows indicate EGFP signal in the spinal cord and motor neurons.
  • Figure 9 shows that 5 ⁇ -and 5 ⁇ -THB improved the mobility of zebrafish larvae.
  • Wild-type zebrafish embryos were microinjected with SDMO or ahr2MO. Each group of microinjected embryos was treated with ethanol control or THB (100 nM) for 5 days. The total swimming distance of each larva were record in 20 sec intervals by a high-throughput behavioral analysis system.
  • 3 ⁇ , 5 ⁇ -Tetrahydrocorticosterone (5 ⁇ -THB) or 3 ⁇ , 5 ⁇ -Tetrahydrocorticosterone (5 ⁇ -THB) were found to be AHR endogenous ligands, through the use of an ultrasensitive dioxin-like compounds bioassay and liquid chromatography-electrospray ionization-tandem mass spectrometry.
  • the present invention provides a method for preventing or treating a cancer, which comprises administering to a subject in need thereof a therapeutically effective amount of 3 ⁇ , 5 ⁇ -Tetrahydrocorticosterone (5 ⁇ -THB) or 3 ⁇ , 5 ⁇ -Tetrahydrocorticosterone (5 ⁇ -THB) .
  • the compound may be 3 ⁇ , 5 ⁇ -Tetrahydrocorticosterone (5 ⁇ -THB) or 3 ⁇ , 5 ⁇ -Tetrahydrocorticosterone (5 ⁇ -THB) , which is an endogenous glucocorticoid hormone.
  • the compound is 5 ⁇ -THB having the chemical formula below:
  • the compound is 5 ⁇ -THB having the chemical formula below:
  • the term “subject” refers to any organism having thrombocytes in blood required for normal hemostasis.
  • the term “subject” encompasses human or non-human mammal or animal.
  • Non-human mammals include livestock animals, companion animals, laboratory animals, and non-human primates.
  • Non-human subjects also include, without limitation, horses, cows, pigs, goats, dogs, cats, mice, rats, guinea pigs, gerbils, hamsters, mink, rabbits and fish. It is understood that the preferred subject is a human.
  • the term “subject” refers to a biological sample as defined herein, which includes but is not limited to a cell, tissue, or organ that is isolated from an organism having thrombocytes required for normal hemostasis. Accordingly, the methods, uses and compositions disclosed herein are intended to be applied in vivo as well as in vitro.
  • the term “administering to a subject” includes dispensing, delivering or applying a compound according to the invention to the desired location in the subject to contact the compound with a target member of the Ahr2 pathway.
  • the term “preventing” relates to the reduction of the risk of developing a cancer, whereas the term “treating” relates to the amelioration of the symptoms of a cancer, deceleration of the course of a cancer.
  • an effective dose of the compound according to the invention is used, in addition to a pharmaceutically acceptable carrier, diluent, adjuvant and/or excipient for producing a pharmaceutical composition.
  • the compound may conveniently be formulated in unit dosage form by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the compound and the pharmaceutically acceptable carrier, diluent, adjuvant and/or excipient.
  • the formulations are prepared by uniformly and intimately bringing into association the compound with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • the dose thereof can vary depending on the route of administration, the age and weight of the subject to be treated, the nature and severity of the diseases to be treated and similar factors.
  • the dose when administered to a subject, such as cells or a human subject, is sufficient to produce a selected effect.
  • the amount of the 5 ⁇ -or 5 ⁇ -THB to be administered is an amount that leads to a pharmaceutically meaningful increase or decrease in the receptor activity of Ahr2.
  • the dosage of 5mg/Kg in the xenograft model was used.
  • Suitable administration forms include, but are not limited to, topical, oral, rectal, intraperitoneal or parenteral (e.g., intravenous, subcutaneous or intramuscular) preparations. Preferred are oral and parenteral preparations.
  • the customary galenic preparation forms such as tablets, sugar-coated tablets, capsules, dispersible powders, granulates, aqueous solutions, alcohol-containing aqueous solutions, aqueous or oily suspensions, syrups, juices or drops, may also be used.
  • Solid medicinal forms can comprise inert components and carrier substances, such as calcium carbonate, calcium phosphate, sodium phosphate, lactose, starch, mannitol, alginates, gelatine, guar gum, magnesium stearate, aluminium stearate, methyl cellulose, talc, highly dispersed silicic acids, silicone oil, higher molecular weight fatty acids, (such as stearic acid) , gelatine, agar or vegetable or animal fats and oils, or solid high molecular weight polymers (such as polyethylene glycol) ; preparations which are suitable for oral administration can comprise additional flavourings and/or sweetening agents, if desired.
  • carrier substances such as calcium carbonate, calcium phosphate, sodium phosphate, lactose, starch, mannitol, alginates, gelatine, guar gum, magnesium stearate, aluminium stearate, methyl cellulose, talc, highly dispersed silicic acids, silicone oil, higher mo
  • Liquid medicinal forms can be sterilized and/or, where appropriate, comprise auxiliary substances, such as preservatives, stabilizers, wetting agents, penetrating agents, emulsifiers, spreading agents, solubilizers, salts, sugars or sugar alcohols for regulating the osmotic pressure or for buffering, and/or viscosity regulators.
  • auxiliary substances such as preservatives, stabilizers, wetting agents, penetrating agents, emulsifiers, spreading agents, solubilizers, salts, sugars or sugar alcohols for regulating the osmotic pressure or for buffering, and/or viscosity regulators.
  • solid carrier substances examples include starch, lactose, mannitol, methyl cellulose, talc, highly dispersed silicic acids, high molecular weight fatty acids (such as stearic acid) , gelatine, agar, calcium phosphate, magnesium stearate, animal and vegetable fats, and solid high molecular weight polymers, such as polyethylene glycol.
  • Preparations for parenteral administration can be present in separate dose unit forms, such as ampoules or vials. Use is preferably made of solutions of the active compound, preferably aqueous solution and, in particular, isotonic solutions and also suspensions. These injection forms can be made available as ready-to-use preparations or only be prepared directly before use, by mixing the compound according to the invention, where appropriate containing other solid carrier substances, with the desired solvent or suspending agent.
  • the compound according to the invention may be administered as a combination therapy with further active agents, useful in the prevention or treatment of a cancer.
  • additional active agents useful in the prevention or treatment of a cancer.
  • the suitable doses of these active agents are known by those skilled in the art.
  • the compound according to the invention may be formulated as compositions containing several active ingredients in a single dose form and/or as a kit containing individual active ingredients in separate dose forms.
  • the active ingredients used in combination therapy may be co-administered or administered separately.
  • zebrafish lines were bred at the National Taiwan University zebrafish core. Embryos of wild type (AB strain) , Tg (HuC: Kaede) , Tg (mbp: egfp) and Tg (-4725sox10: egfp) ba4 were maintained at 28°C in egg water (6 g sea salt in 20 L H 2 O) and staged according to hours post fertilization (hpf) and morphological criteria. For whole-mount and immunohistochemical imaging analysis, embryos were treated with 0.003%PTU in egg water before 24 hpf to inhibit pigment formation. All zebrafish experiments were performed after approval by the Institutional Animal Care and Use Committee of National Taiwan University.
  • Cells were cultured at a density of 8 ⁇ 10 6 in a 10-cm dish containing 10 ml DMEM medium, 10%FBS, and antibiotics. The culture medium was removed and cells were washed twice with cold PBS. Samples were harvested in 20 mM EDTA (pH 8.0) in PBS, followed by centrifugation at 2,000 rpm for 5 min. The cell pellet was lysed in lysis buffer [1%NP40, 200 mM Tris (pH 8.0) , 100 mM NaCl, 10%glycerol] for 30 min on ice. Lysate was centrifuged at 14,000 rpm for 10 min at 4°C, and the supernatant was collected and diluted with ddH 2 O before following the Dioxin bioassay detection protocol.
  • lysis buffer [1%NP40, 200 mM Tris (pH 8.0) , 100 mM NaCl, 10%glycerol]
  • Luminescence signals were determined by the addition of 1 ⁇ M of coelenterazine-h (S201A, Promega) or Nano-Glo substrate (N1110, Promega) . Luminescence was detected by a luminescence plate reader.
  • HPLC system HITACHI D2000 was used to fractionate samples. The samples were kept at 4°C. Elution was performed as follows: begin at 80%mobile phase A (water) and 20%mobile phase B (acetonitrile) , hold at 20%B for 0.5 min; raise to 80%phase B at 5.5 min; raise to 100%phase B at 8 min; hold at 100%B for 4 min; lower to 20%B at 12 min; hold at 20%B for 3 min.
  • the flow rate was set at 0.1 ml/min with an injection volume of 20 ⁇ l.
  • the LC-ESI-MS system consists of an ultra-performance liquid chromatography (UPLC) system (Ultimate 3000 RSLC, Dionex) and an electrospray ionization (ESI) source with a quadrupole time-of-flight (TOF) mass spectrometer (maXis HUR-QToF system, Bruker Daltonics) .
  • UPLC ultra-performance liquid chromatography
  • ESI electrospray ionization
  • TOF time-of-flight
  • Elution was performed as follows: 70%mobile phase A (0.1%formic acid in ultrapure water) and 30%mobile phase B (0.1%formic acid in acetonitrile) ; hold at 30%B for 0.5 min; raise to 100%B at 7.5 min; hold at 100%B for 1.5 min; lower to 30%B for 0.5 min.
  • the column was equilibrated by pumping 30%B for 3 min.
  • the flow rate was set at 0.4 ml/min with an injection volume of 10 ⁇ l.
  • LC-ESI-MS chromatograms were acquired under following conditions: capillary voltage of 4500 V in positive ion mode or 3500 V in negative ion mode, dry temperature at 190°C, dry gas flow maintained at 8 l/min, nebulizer gas at 1.4 bar, and acquisition range of m/z 100-1000.
  • Each morpholino (MO) was purchased from Gene Tools (Philomath) .
  • zAHR2 translation-blocking MO was used: 5'-TGT ACC GAT ACC CGC CGA CAT GGT T -3', diluted to a concentration of 1 mM. Both DNA and MO solutions were supplemented with 0.1%phenol red as an injection indicator.
  • a total of 4 nl zAHR2 MO or SDMO (standard morpholino, human beta-globin targeted) solution were injected into the yolk of one-or two-cell stage embryos.
  • Fertilized eggs were obtained from natural mating of adult zebrafish (AB, Tg: HuC-EGFP, Tg: HuC-Kaede line) , which were maintained at 28°C with a lighting schedule of 14 h light and 10 h dark.
  • THB compounds were diluted to working concentrations in egg water with (for image analysis) or without 0.003%PTU.
  • Zebrafish embryos were collected and treated with THB at a density of 10 embryos per milliliter. Half volumes of the drug-containing medium were replaced daily.
  • DEPC diethylpyrocarbonated
  • RT-PCR Reverse-transcription polymerase chain reaction
  • Real-time PCR reactions were conducted in an iCycler iQ Real-Time detection system (Bio-Rad) using SYBR Green I (ABgene) .
  • the thermal profile for PCR was as follows: 95°C for 3 min, 40 cycles of 95°C for 30 s and 60°Cfor 30 s. Thermocycling was carried out in a final volume of 15 ⁇ l, containing 1 ⁇ l of cDNA sample. The melting curve of each reaction was examined to confirm the appearance of a single peak.
  • Zebrafish were immersed in PT W (0.1%Tween 20 in PBS) buffer for equilibrium and then fixed by 4%paraformaldehyde (PFA) in PBS at 4°Covernight. After three washes with PT W buffer, fixed embryos were transferred to 30%sucrose solution for cryoprotection at 4°C overnight. The blocks were frozen and cut as 40 ⁇ m transverse sections using a cryostat microtome. The sections were incubated with rabbit anti-O4 and rabbit monoclonal anti-MBP antibody following treatment with a specific fluorescent secondary antibody.
  • PFA paraformaldehyde
  • Fluorescent images were taken with a Zeiss confocal microscope (LSM880) or an Eclipse E800 (Nikon) microscope using appropriate filters and a SPOT digital camera (Diagnostic Instruments) .
  • Zebrafish embryos were injected with 4 nl ahr2MO or SDMO (1mM) .
  • Ten hours after morpholino injection the larvae were exposed to 100 ⁇ M 5 ⁇ -or 5 ⁇ -THB, or solvent control (EtOH) for 4 days.
  • EtOH solvent control
  • the larvae from each group were collected and placed in 24-well plates at a density of 1 larva/well with 1 ml of egg water.
  • Locomotion analysis was then performed according a previously published protocol [7] . Briefly, larvae were set still for 15 min of acclimation. Next, the larvae were allowed to freely explore the well for 20 s, with a tapping stimulus at the end of the first 10 s period.
  • the movement tracks of each larva were record and analyzed by Ethovision XT software (Noldus Information Technology, Wageningen, Netherlands) .
  • AHR is expressed in very early stage embryos and has been revealed to play a role in embryonic development [30] .
  • the non-protein fractions of zebrafish embryos were extracted and subjected to the bioassay.
  • the cell lysate from another AHR-overexpressing stable cell line (AAPA) was also collected and processed into cytosolic and nuclear fractions.
  • Western blot results showed that both 5 ⁇ -and 5 ⁇ -THB caused a significant reduction of AHR protein in the cytosolic fraction but increased level in the nuclear fraction, confirming that THB causes AHR nuclear translocation (Figure 3B) .
  • the mRNA expression level of CYP1A1 was examined by real-time PCR. THB induced CYP1A1 mRNA expression in a dose-dependent manner (Figure 3C) .
  • mice were subcutaneously xenotransplanted with stNB-V1 NB cells and treated with vehicle control or THB for 20 days. Both 5 ⁇ -and 5 ⁇ -THB treatment significantly inhibited the growth of xenograft tumors compared to vehicle ( Figures 3D and 3E) . Additionally, the protein levels of GAP-43 and NSE in xenograft tumors were upregulated by 5 ⁇ -THB ( Figure 3F) . Although the induction of GAP-43 and NSE by 5 ⁇ -THB did not reach the statistical significance, a trend was observed.
  • zebrafish As an in vivo model to investigate whether THB mediates neural development.
  • three AHR isoforms have been identified: Ahr1a, Ahr1b, and Ahr2 [32, 33] .
  • Ahr2 is the primary isoform that mediates xenobiotic metabolism in zebrafish [34-38] .
  • the Tg (HuC: Kaede) zebrafish is a useful tool to label neuronal network morphologies in vivo [39] . As neurons mature, the expression of green-fluorescent Kaede will be upregulated by the HuC promoter.
  • Figure 5 shows representative images of Kaede fluorescent signal of the Tg (HuC: Kaede) zebrafish. Kaede expression was mainly observed in the eye, brain and spinal cord. We found that THB treatment enhanced the overall Kaede levels in 1, 3 and 5 dpf zebrafish, indicating that THB treatment promotes early neurogenesis in zebrafish.
  • Tg (mbp: egfp) and Tg (-4725sox10: egfp) ba4 transgenic zebrafish GFP expression is respectively controlled by Sox10 and MBP promoters.
  • both strains showed similar results, with upregulated GFP; similarly, the elevated GFP signal was significantly suppressed in both strains by ahr2MO ( Figures 7E and 7F; Figures 9E and 9F) .
  • 5 ⁇ -THB or 5 ⁇ -THB is endogenous ligands of AHR, and is potential for treatment of NB.
  • AHR signaling is an important regulator of tumorigenesis in NB. Accordingly, 5 ⁇ -THB and 5 ⁇ -THB are effective in prevention or treatment of a cancer, such as NB.
  • Kewley RJ, Whitelaw ML, Chapman-Smith A The mammalian basic helix–loop–helix/PAS family of transcriptional regulators. The International Journal of Biochemistry &Cell Biology 2004, 36 (2) : 189-204.
  • Davarinos NA, Pollenz RS Aryl hydrocarbon receptor imported into the nucleus following ligand binding is rapidly degraded via the cytosplasmic proteasome following nuclear export. The Journal of biological chemistry 1999, 274 (40) : 28708-28715.
  • Kawamura T Yamashita I: Aryl hydrocarbon receptor is required for prevention of blood clotting and for the development of vasculature and bone in the embryos of medaka fish, Oryzias latipes. Zoological science 2002, 19 (3) : 309-319.
  • Dever DP Adham ZO, Thompson B, Genestine M, Cherry J, Olschowka JA, DiCicco-Bloom E, Opanashuk LA: Aryl hydrocarbon receptor deletion in cerebellar granule neuron precursors impairs neurogenesis. Developmental neurobiology 2016, 76 (5) : 533-550.
  • Bugel SM, White LA, Cooper KR Inhibition of vitellogenin gene induction by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin is mediated by aryl hydrocarbon receptor 2 (AHR2) in zebrafish (Danio rerio) . Aquat Toxicol 2013, 126: 1-8.

Landscapes

  • Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Disclosed is a method or pharmaceutical composition for prevention and/or treatment of a cancer, comprising administering to the subject a therapeutically effective amount of a compound selected from the group consisting of 3α,5α-tetrahydrocorticosterone (5α-THB) and 3α,5β-tetrahydrocorticosterone (5β-THB).

Description

METHOD AND PHARMACEUTICAL COMPOSITION FOR PREVENTION AND TREATMENT OF A CANCER FIELD OF THE INVENTION
The present invention relates to a method and pharmaceutical composition for prevention and treatment of a cancer.
BACKGROUND OF THE INVENTION
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor, which belongs to the basic-helix-loop-helix (bHLH) /PAS (Period [Per] -Aryl hydrocarbon receptor nuclear translocator [ARNT] -Single minded [SIM] ) family of heterodimeric transcriptional regulators [1, 2] . AHR may be activated by binding to a wide range of xenobiotic ligands, ranging from plant material to pollutants that are either natural (e.g., benzo [a] pyrene (B [a] P) ) or synthetic (e.g., 2, 3, 7, 8-tetrachlorodibenzodioxin [TCDD] ) [3] . In the absence of bound ligand, AHR resides in the cytosolic compartment, sequestered by a molecular chaperone complex that contains two molecules of the heat shock protein 90 (HSP90) , the HSP90-interacting protein, p23, and the immunophilin-like protein, XAP2 (also named ARA9 or AIP) [4] . Following activation by ligand binding, conformational changes in the AHR protein alter its binding to XAP2 and activate a nuclear localization sequence. These structural changes direct HSP90-bound AHR to translocate into the nucleus, where the receptor dissociates from the HSP90 complex and dimerizes with ARNT [4] . The AHR/ARNT heterodimer then interacts with a partially characterized set of co-activators and/or co-repressors to bind the XRE-rich enhancer and TATA box in the promoter regions of target genes, followed by the recruitment of RNA polymerase II to initiate transcription [5] . Consequently, genes coding for the xenobiotic-metabolizing cytochrome P450 enzymes, such as CYP1A1, CYP1B1, UDP-glucuronosyl transferase (UGT1A6) , aldehyde dehydrogenase (ALDH3A1) and multidrug resistance-associated proteins (MRPs) , are transcriptionally induced. Once the transcriptional regulation has occurred, AHR is exported to the cytosol by chromosome region maintenance 1 (CRM1) and degraded by the 26S ubiquitin-proteasome [6, 7] .
In addition to xenobiotic metabolism, AHR has been shown to be an important regulator of cell death, proliferation, differentiation, and cell cycle progression [8-12] . Physiological studies have also suggested that AHR may be involved in tissue development or homeostasis because its absence has been shown to result in infertility, abnormalities in liver, and cardiovascular problems [13-19] . Moreover, AHR was recently found to be expressed in cerebellar granular neuronal precursor (GNPs) in the early postnatal period, where it regulates the growth and differentiation of granule neuroblasts [20, 21] . AHR-deficient mice also display diminished neuronal differentiation in the dentate gyrus [22] , and knockout of AHR causes oculomotor and optic nerve deficits in a mouse model [23, 24] . Additionally, we previously reported that overexpression of AHR promotes neural differentiation of neuroblastoma (NB) cells [25] , further supporting the idea that AHR plays an important role in regulating neural development.
NB is derived from the sympathoadrenal lineage of the neural crest and is the most common extracranial solid tumor in children, accounting for 15%of all child cancer mortality [26-28] . One of the most well-known prognostic markers for NB is MYCN [28] , of which the expression level has been correlated with activation of genes associated with NB tumor aggression [29] . However, the molecular mechanism underlying this carcinogenic process remains elusive.
SUMMARY OF THE INVENTION
It is surprisingly found in the present invention that two compounds, 3α, 5α-Tetrahydrocorticosterone and 3α, 5β-Tetrahydrocorticosterone (5α-and 5β-THB) , which are found to be AHR endogenous ligands, through the use of an ultrasensitive dioxin-like compounds bioassay and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS) .
In one aspect, the present invention provides a method for preventing or treating a cancer, comprising administering to a subject in need thereof a therapeutically effective amount of 3α, 5α-Tetrahydrocorticosterone (5α-THB) or 3α, 5β-Tetrahydrocorticosterone (5β-THB) .
In one example of the invention, the 5α-or 5β-THB is capable of enhancement of the expression of sex determining region Y-box 10 (Sox10) and myelin-associated proteins myelin basic protein (MBP) .
In one example of the invention, the cancer is neuroblastoma (NB) .
In one example of the invention, the 5α-or 5β-THB inhibits tumorigenesis in NB.
In one further aspect, the present invention provides a pharmaceutical composition for preventing or treating a cancer, comprising a therapeutically effective amount of 3α, 5α-Tetrahydrocorticosterone (5α-THB) or 3α, 5β-Tetrahydrocorticosterone (5β-THB) in association with a pharmaceutically acceptable carrier.
In one yet aspect, the present invention provides a use of 3α, 5α-Tetrahydrocorticosterone (5α-THB) or 3α, 5β-Tetrahydrocorticosterone (5β-THB) in manufacturing a medicament for preventing or treating a cancer.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiment which is presently preferred. It should be understood, however, that the invention is not limited to this embodiment.
In the drawings:
Figure 1 provides the identification of endogenous AHR ligands with dioxin-like compound cell free bioassay. (A) A schematic diagram of the dioxin-like compound cell free bioassay. (B) Fractionation of day 0 zebrafish embryo extract by reverse-phase HPLC. (C) Fractions collected from HPLC were analyzed by the dioxin-like compound cell free bioassay. (D) Chemical structure of 5α-and 5β-THB.
Figure 2 shows that THB induced nuclear translocation of AHR and promoted CYP1A1 expression. (A) AGAPA cells were treated with THB 100 nM for 90 min. EtOH was used as a negative control. The cells were fixed, and AHR-GFP distribution was evaluated by fluorescence microscopy. Nuclei were stained with DAPI (blue) . Scale bar, 20 μm. (B) AAPA cells were grown to 50%confluency in 10-cm dishes followed by tetracycline induction for 24 h. Induced AAPA cells were then treated with THB (100 nM) or Kyn (200μM) for 2 h. EtOH and HCl (0.5M) was used as solvent control respectively. The cytosolic and nuclear fractions were collected and analyzed by western blot. Anti-AHR antibody was used for AHR detection. GAPDH and Lamin A/C were used as internal loading controls for the cytosolic and nuclear fractions respectivly. (C)  The nuclear-to-cytosol ratio of AHR expression level was quantified by ImageJ. Quantified results are shown as the mean ± SD from 4 independent experiments and analyzed by one-way ANOVA. (*p < 0.05; **p < 0.01) . (D) AAPA cells were treated with indicated concentrtions of THB for 4 hr. CYP1A1 mRNA expression level was measured by SYBR green real-time PCR.
Figure 3 shows that THB promoted neuronal differentiation-associated gene expression and inhibits NB tumor growth. (A-C) SK-N-SH cells were treated with 100 nM THB for 4 hr. mRNA expression levels of GAP43, NSE and NF were analyzed by SYBR green real-time PCR. (D-F) stNB-V1 cells were xenotransplanted in a mouse xenograft model. Tumor-inoculated mice were treated with vehicle (control) , RA (5 mg/kg) , 5α-THB (5 mg/kg) or 5β-THB (5 mg/kg) . (D) Tumor inoculated mice were sacrificed after 21 days of treatment. Tumors from each mouse are shown in the photograph. (E) Prior to sacrifice, tumor growth was measured daily for 21 days. Error bars represent S.E.M. (F) The protein levels of differentiation markers, NSE and GAP-43, in the xenograft tumors were analyzed by western blot. (*p < 0.05; **p < 0.01; ***p < 0.001) .
Figure 4 shows that Ahr2 morpholino suppressed endogenous and THB-induced cyp1a expression in zebrafish. (A-B) Zebrafish embryos were exposed to increasing amounts of THB. Total mRNA was collected in TRIzol reagent, and cyp1a mRNA expression level was analyzed by real-time PCR. (C) Zebrafish embryos were microinjected with standard control MO (SDMO) and zAHR2 MO. The total protein of 5 dpf embryos was collected and analyzed by western blot. (D-E) Zebrafish embryos injected with SDMO or ahr2MO were treated with THB. The cyp1a mRNA expression was examined by real-time PCR.
Figure 5 shows that THB promoted early neural development of Tg (HuC: Kaede) transgenic zebrafish model. Image analysis of 1, 3 and 5 dpf Tg (HuC: Kaede) zebrafish. Green signals show the Kaede distribution.
Figure 6 shows Ahr2 mediated gliogenesis in zebrafish. (A) Green signal shows HuC-Kaede protein in cross-sectional views of the spinal cord from Tg (HuC: Kaede) transgenic zebrafish. The dorsal surface is shown on top, while ventral is on the bottom. (B-D) IHC staining of O4 (red) (B) , GFAP (red) (C) and CD11b (red) (D) . Nuclei were counterstained with DAPI (blue) . Three  independent samples were evaluated and representative data are shown. Quantitative data are shown as the mean ± SD from three independent experiments run in triplicate and analyzed by ANOVA (*p < 0.05; **p < 0.01; ***p < 0.001) .
Figure 7 shows that 5β-THB-Ahr2 signaling regulates myelinating glial cell development in zebrafish. (A and C) Zebrafish embryos were exposed to increasing amounts of 5β-THB. Total mRNA was collected in TRIzol reagent, and Sox10 and MBP mRNA expression levels were analyzed by real-time PCR. (B and D) Zebrafish embryos injected with SDMO or ahr2MO were treated with 5β-THB. Sox10 and MBP mRNA expression levels were examined by real-time PCR. (E) Embryos of transgenic zebrafish, Tg (-4725sox10: egfp) ba4, were microinjected with SDMO or ahr2MO. Each group of microinjected embryos was treated with ethanol control or 5β-THB (100 nM) . The EGFP signal of 2 dpf embryos was evaluated. Arrows show EGFP signal in the spinal cord. (F) Embryos of transgenic zebrafish Tg (mbp: egfp) were microinjected with SDMO or ahr2MO. Each group of microinjected embryos was treated with ethanol control or 5β-THB (100 nM) . The EGFP signal of 5 dpf embryos was evaluated. Arrows indicate EGFP signal in the spinal cord and motor neurons.
Figure 8 shows that 5α-THB-Ahr2 signaling regulates myelinating glial cell development in zebrafish. (A and C) Zebrafish embryos were exposed to increasing amounts of 5α-THB. Total mRNA was collected in TRIzol reagent, and Sox10 and MBP mRNA expression levels were analyzed by real-time PCR. (B and D) Zebrafish embryos injected with SDMO or ahr2MO were treated with 5α-THB. Sox10 and MBP mRNA expression levels were examined by real-time PCR. (E) Embryos of transgenic zebrafish, Tg (-4725sox10: egfp) ba4, were microinjected with SDMO or ahr2MO. Each group of microinjected embryos was treated with ethanol control or 5α-THB (100 nM) . The EGFP signal of 2 dpf embryos was evaluated. Arrows show EGFP signal in the spinal cord. (F) Embryos of transgenic zebrafish Tg (mbp: egfp) were microinjected with SDMO or ahr2MO. Each group of microinjected embryos was treated with ethanol control or 5α-THB (100 nM) . The EGFP signal of 5 dpf embryos was evaluated. Arrows indicate EGFP signal in the spinal cord and motor neurons.
Figure 9 shows that 5α-and 5β-THB improved the mobility of zebrafish larvae. Wild-type zebrafish embryos were microinjected with SDMO or ahr2MO. Each group of microinjected embryos was treated with ethanol control or THB (100 nM) for 5 days. The total swimming distance of each larva were record in 20 sec intervals by a high-throughput behavioral analysis system.
DETAILED DESCRIPTIOM OF THE INVENTION
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.
According to the present invention, 3α, 5α-Tetrahydrocorticosterone (5α-THB) or 3α, 5β-Tetrahydrocorticosterone (5β-THB) were found to be AHR endogenous ligands, through the use of an ultrasensitive dioxin-like compounds bioassay and liquid chromatography-electrospray ionization-tandem mass spectrometry.
The present invention provides a method for preventing or treating a cancer, which comprises administering to a subject in need thereof a therapeutically effective amount of 3α, 5α-Tetrahydrocorticosterone (5α-THB) or 3α, 5β-Tetrahydrocorticosterone (5β-THB) .
According to the invention, the compound may be 3α, 5α-Tetrahydrocorticosterone (5α-THB) or 3α, 5β-Tetrahydrocorticosterone (5β-THB) , which is an endogenous glucocorticoid hormone.
It is ascertained in the invention that 5α-THB or 5β-THB improved the mobility of zebrafish larvae via the Ahr2 pathway. Accordingly, it can be be concluded that
In one embodiment of the invention, the compound is 5α-THB having the chemical formula below:
Figure PCTCN2020092922-appb-000001
In another embodiment of the invention, the compound is 5β-THB having the chemical formula below:
Figure PCTCN2020092922-appb-000002
As used herein, the term “subject” refers to any organism having thrombocytes in blood required for normal hemostasis. The term “subject” encompasses human or non-human mammal or animal. Non-human mammals include livestock animals, companion animals, laboratory animals, and non-human primates. Non-human subjects also include, without limitation, horses, cows, pigs, goats, dogs, cats, mice, rats, guinea pigs, gerbils, hamsters, mink, rabbits and fish. It is understood that the preferred subject is a human.
In some embodiments, the term “subject” refers to a biological sample as defined herein, which includes but is not limited to a cell, tissue, or organ that is isolated from an organism having thrombocytes required for normal hemostasis. Accordingly, the methods, uses and compositions disclosed herein are intended to be applied in vivo as well as in vitro.
According to the invention, the term “administering to a subject” includes dispensing, delivering or applying a compound according to the invention to the desired location in the subject to contact the compound with a target member of the Ahr2 pathway. The term “preventing” relates to the reduction of the risk of developing a cancer, whereas the term “treating” relates to the amelioration of the symptoms of a cancer, deceleration of the course of a cancer.
According to the invention, an effective dose of the compound according to the invention is used, in addition to a pharmaceutically acceptable carrier, diluent, adjuvant and/or excipient for producing a pharmaceutical composition. The compound may conveniently be formulated in unit dosage form by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the compound and the pharmaceutically  acceptable carrier, diluent, adjuvant and/or excipient. In general, the formulations are prepared by uniformly and intimately bringing into association the compound with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. In the case of using a compound according to the invention, such as 5α-or 5β-THB, the dose thereof can vary depending on the route of administration, the age and weight of the subject to be treated, the nature and severity of the diseases to be treated and similar factors. The dose, when administered to a subject, such as cells or a human subject, is sufficient to produce a selected effect. For example, the amount of the 5α-or 5β-THB to be administered is an amount that leads to a pharmaceutically meaningful increase or decrease in the receptor activity of Ahr2. In one example of the present invention, the dosage of 5mg/Kg in the xenograft model was used.
Suitable administration forms include, but are not limited to, topical, oral, rectal, intraperitoneal or parenteral (e.g., intravenous, subcutaneous or intramuscular) preparations. Preferred are oral and parenteral preparations. The customary galenic preparation forms, such as tablets, sugar-coated tablets, capsules, dispersible powders, granulates, aqueous solutions, alcohol-containing aqueous solutions, aqueous or oily suspensions, syrups, juices or drops, may also be used.
Solid medicinal forms can comprise inert components and carrier substances, such as calcium carbonate, calcium phosphate, sodium phosphate, lactose, starch, mannitol, alginates, gelatine, guar gum, magnesium stearate, aluminium stearate, methyl cellulose, talc, highly dispersed silicic acids, silicone oil, higher molecular weight fatty acids, (such as stearic acid) , gelatine, agar or vegetable or animal fats and oils, or solid high molecular weight polymers (such as polyethylene glycol) ; preparations which are suitable for oral administration can comprise additional flavourings and/or sweetening agents, if desired.
Liquid medicinal forms can be sterilized and/or, where appropriate, comprise auxiliary substances, such as preservatives, stabilizers, wetting agents, penetrating agents, emulsifiers, spreading agents, solubilizers, salts, sugars or sugar alcohols for regulating the osmotic pressure or for buffering, and/or viscosity regulators.
Examples of solid carrier substances are starch, lactose, mannitol, methyl cellulose, talc, highly dispersed silicic acids, high molecular weight fatty acids (such as stearic acid) , gelatine, agar, calcium phosphate, magnesium stearate, animal and vegetable fats, and solid high molecular weight polymers, such as polyethylene glycol.
Preparations for parenteral administration can be present in separate dose unit forms, such as ampoules or vials. Use is preferably made of solutions of the active compound, preferably aqueous solution and, in particular, isotonic solutions and also suspensions. These injection forms can be made available as ready-to-use preparations or only be prepared directly before use, by mixing the compound according to the invention, where appropriate containing other solid carrier substances, with the desired solvent or suspending agent.
As indicated above, the compound according to the invention may be administered as a combination therapy with further active agents, useful in the prevention or treatment of a cancer. The suitable doses of these active agents are known by those skilled in the art.
For a combination therapy, the compound according to the invention may be formulated as compositions containing several active ingredients in a single dose form and/or as a kit containing individual active ingredients in separate dose forms. The active ingredients used in combination therapy may be co-administered or administered separately.
The following examples are given for the purpose of illustration only and are not intended to limit the scope of the invention.
Examples
1. Methods
1.1 Zebrafish maintenance and embryos collection
All zebrafish lines were bred at the National Taiwan University zebrafish core. Embryos of wild type (AB strain) , Tg (HuC: Kaede) , Tg (mbp: egfp) and Tg (-4725sox10: egfp) ba4 were maintained at 28℃ in egg water (6 g sea salt in 20 L H 2O) and staged according to hours post fertilization (hpf) and morphological criteria. For whole-mount and immunohistochemical imaging analysis, embryos were treated with 0.003%PTU in egg water before 24 hpf to inhibit pigment formation. All zebrafish experiments were performed  after approval by the Institutional Animal Care and Use Committee of National Taiwan University.
1.2 Cell-free dioxin assay preparation
Cells were cultured at a density of 8 × 10 6 in a 10-cm dish containing 10 ml DMEM medium, 10%FBS, and antibiotics. The culture medium was removed and cells were washed twice with cold PBS. Samples were harvested in 20 mM EDTA (pH 8.0) in PBS, followed by centrifugation at 2,000 rpm for 5 min. The cell pellet was lysed in lysis buffer [1%NP40, 200 mM Tris (pH 8.0) , 100 mM NaCl, 10%glycerol] for 30 min on ice. Lysate was centrifuged at 14,000 rpm for 10 min at 4℃, and the supernatant was collected and diluted with ddH 2O before following the Dioxin bioassay detection protocol.
1.3 Dioxin bioassay detection
90 μl of cell-free extract was mixed with 10 μl of different concentrations of compounds in a 96-well white polystyrene plate at 37℃ for 30 min. Luminescence signals were determined by the addition of 1 μM of coelenterazine-h (S201A, Promega) or Nano-Glo substrate (N1110, Promega) . Luminescence was detected by a luminescence plate reader.
1.4 High performance liquid chromatography (HPLC) 
An HPLC system (HITACHI D2000) was used to fractionate samples. The samples were kept at 4℃. Elution was performed as follows: begin at 80%mobile phase A (water) and 20%mobile phase B (acetonitrile) , hold at 20%B for 0.5 min; raise to 80%phase B at 5.5 min; raise to 100%phase B at 8 min; hold at 100%B for 4 min; lower to 20%B at 12 min; hold at 20%B for 3 min. The flow rate was set at 0.1 ml/min with an injection volume of 20 μl. Each peak from the HPLC was collected in a microcentrifuge tube, dried and dissolved in 100 μl 30%acetonitrile and the supernatant was subjected to LC-ESI-MS analysis after centrifugation at 14,000 rpm.
1.5 LC-ESI-MS
The LC-ESI-MS system consists of an ultra-performance liquid chromatography (UPLC) system (Ultimate 3000 RSLC, Dionex) and an electrospray ionization (ESI) source with a quadrupole time-of-flight (TOF) mass spectrometer (maXis HUR-QToF system, Bruker Daltonics) . The samples were kept at 4 ℃ in an autosampler. Separation was performed by reverse-phase  liquid chromatography (RPLC) using a BEH C18 column (2.1 x 100 mm, Walters) . Elution was performed as follows: 70%mobile phase A (0.1%formic acid in ultrapure water) and 30%mobile phase B (0.1%formic acid in acetonitrile) ; hold at 30%B for 0.5 min; raise to 100%B at 7.5 min; hold at 100%B for 1.5 min; lower to 30%B for 0.5 min. The column was equilibrated by pumping 30%B for 3 min. The flow rate was set at 0.4 ml/min with an injection volume of 10 μl. LC-ESI-MS chromatograms were acquired under following conditions: capillary voltage of 4500 V in positive ion mode or 3500 V in negative ion mode, dry temperature at 190℃, dry gas flow maintained at 8 l/min, nebulizer gas at 1.4 bar, and acquisition range of m/z 100-1000.
1.6 Gene knockdown with antisense morpholino injection
Each morpholino (MO) was purchased from Gene Tools (Philomath) . zAHR2 translation-blocking MO was used: 5'-TGT ACC GAT ACC CGC CGA CAT GGT T -3', diluted to a concentration of 1 mM. Both DNA and MO solutions were supplemented with 0.1%phenol red as an injection indicator. A total of 4 nl zAHR2 MO or SDMO (standard morpholino, human beta-globin targeted) solution were injected into the yolk of one-or two-cell stage embryos.
1.7 Drug Exposure
Fertilized eggs were obtained from natural mating of adult zebrafish (AB, Tg: HuC-EGFP, Tg: HuC-Kaede line) , which were maintained at 28℃ with a lighting schedule of 14 h light and 10 h dark. THB compounds were diluted to working concentrations in egg water with (for image analysis) or without 0.003%PTU. Zebrafish embryos were collected and treated with THB at a density of 10 embryos per milliliter. Half volumes of the drug-containing medium were replaced daily.
1.8 mRNA extraction
Total RNA was isolated from cells and zebrafish (around 30 embryos were homogenized for each sample) using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) . The amount and purity of the total RNA were estimated by spectrophotometric analysis at A260 and A280. Aliquots of total RNA were diluted in diethylpyrocarbonated (DEPC) -treated water and stored at -20℃.
1.9 Reverse-transcription polymerase chain reaction (RT-PCR) .
Total cellular RNA was extracted from tumor tissues and cells using TRIzol reagent (Invitrogen) . The reverse transcription of 1 μg isolated total RNA was performed in a 20 μl reaction mixture with the use of the M-MuLV Reverse Transcriptase (Thermo) and oligo-dT primer.
1.10 Quantitative real-time PCR
Real-time PCR reactions were conducted in an iCycler iQ Real-Time detection system (Bio-Rad) using SYBR Green I (ABgene) . The thermal profile for PCR was as follows: 95℃ for 3 min, 40 cycles of 95℃ for 30 s and 60℃for 30 s. Thermocycling was carried out in a final volume of 15 μl, containing 1 μl of cDNA sample. The melting curve of each reaction was examined to confirm the appearance of a single peak.
1.11 Frozen Sections and Immunohistochemistry
Zebrafish were immersed in PT W (0.1%Tween 20 in PBS) buffer for equilibrium and then fixed by 4%paraformaldehyde (PFA) in PBS at 4℃overnight. After three washes with PT W buffer, fixed embryos were transferred to 30%sucrose solution for cryoprotection at 4℃ overnight. The blocks were frozen and cut as 40 μm transverse sections using a cryostat microtome. The sections were incubated with rabbit anti-O4 and rabbit monoclonal anti-MBP antibody following treatment with a specific fluorescent secondary antibody.
1.12 Microscopy and image analysis
Fluorescent images were taken with a Zeiss confocal microscope (LSM880) or an Eclipse E800 (Nikon) microscope using appropriate filters and a SPOT digital camera (Diagnostic Instruments) .
1.13 Locomotion analysis
Zebrafish embryos were injected with 4 nl ahr2MO or SDMO (1mM) . Ten hours after morpholino injection, the larvae were exposed to 100 μM 5α-or 5β-THB, or solvent control (EtOH) for 4 days. At 5 dpf, the larvae from each group were collected and placed in 24-well plates at a density of 1 larva/well with 1 ml of egg water. Locomotion analysis was then performed according a previously published protocol [7] . Briefly, larvae were set still for 15 min of acclimation. Next, the larvae were allowed to freely explore the well for 20 s, with a tapping stimulus at the end of the first 10 s period. The movement tracks of each larva were record and analyzed by Ethovision XT software (Noldus Information Technology, Wageningen, Netherlands) .
2. Results
2.1 Identification of endogenous dioxin-like compounds from zebrafish embryo extracts
AHR is expressed in very early stage embryos and has been revealed to play a role in embryonic development [30] . To investigate the endogenous role of AHR, we first devised a strategy to identify endogenous AHR ligands from zebrafish embryos. We used a modified dioxin-like compound cell free bioassay that we previously established [31] ; in this assay, binding of potential ligands promotes the dissociation of AHR-nanoluciferase from HSP90, leading to proteasomal degradation of the protein and a reduction of luciferase signal (Figure 1A) . The non-protein fractions of zebrafish embryos were extracted and subjected to the bioassay. We found that the extract from day 0 embryos, collected in 4 hr after birth, significantly reduced AHR-nanoluciferase signal (data not shown) . The day 0 extract was further fractionated by reverse-phase high-performance liquid chromatography (HPLC) (Figure 1B) . Among the eight fractions we collected,  fractions  2, 3, 4, 6, and 7 effectively promoted AHR-nanoluciferase degradation (Figure 1C) and were further analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS) to identify ligand candidates. From the LC-ESI-MS identification results (Table 1 and 2) , we excluded compounds that only exist in plants and fungi, finally selecting 3α, 5α-Tetrahydrocorticosterone and 3α, 5β-Tetrahydrocorticosterone (5α-THB and 5β-THB) as potential AHR ligands for subsequent experiments (Figure 1D) .
Table 1. Identification of potential AHR endogenous ligands by LC-ESI-MS (positive ion mode) .
Figure PCTCN2020092922-appb-000003
Figure PCTCN2020092922-appb-000004
Figure PCTCN2020092922-appb-000005
Figure PCTCN2020092922-appb-000006
Table 2. Identification of potential AHR endogenous ligands by LC-ESI-MS (negative ion mode) .
Figure PCTCN2020092922-appb-000007
2.2 5α-and 5β-THB induce AHR nuclear translocation and activate CYP1A1 gene expression
To confirm that 5α-and 5β-THB indeed activate AHR, we used a stable AHR-GFP-overexpressing HEK cell line, AGAPA, as an in vitro test. It is known that ligand-activated AHR will translocate from the cytosol into the nucleus, where it regulates expression of target genes. Therefore, the nuclear translocation of AHR and the expression of the downstream gene, CYP1A1, were used as indicators of activation. We found that the AHR-GFP signal was clearly observable in the nucleus of AGAPA cells after 100 nM 5α-or 5β-THB treatment for 1.5 hr (Figure 2A) . The cell lysate from another AHR-overexpressing stable cell line (AAPA) was also collected and processed into cytosolic and nuclear fractions. Western blot results showed that both 5α-and 5β-THB caused a significant reduction of AHR protein in the cytosolic fraction but increased level in the nuclear fraction, confirming that THB causes AHR nuclear translocation (Figure 3B) . In addition, the mRNA expression level of CYP1A1 was examined by real-time PCR. THB induced CYP1A1 mRNA expression in a dose-dependent manner (Figure 3C) . These results demonstrate that both 5α-and 5β-THB have the ability to activate AHR and induce AHR-related responses.
2.3 5α-and 5β-THB induce neural differentiation of NB and inhibit NB xenograft tumor growth
Next, we investigated the therapeutic potential of 5α-and 5β-THB in NB. Since AHR overexpression is known to promote neural differentiation of NB cells [25] , we hypothesized that activation of AHR by THB treatment might similarly affect NB cells. To test this hypothesis, SK-N-SH cells were treated with a range of 5α-or 5β-THB doses for three days. Then, RNA was collected and analyzed by real-time PCR. We found that the mRNA expression levels of neural differentiation markers, growth associated protein 43 (GAP-43) , neuron specific enolase (NSE) , and neurofilament (NF) were upregulated by THB (Figure 3A-C) .
We further tested the therapeutic potential of THB using a mouse xenograft model. Nude mice were subcutaneously xenotransplanted with stNB-V1 NB cells and treated with vehicle control or THB for 20 days. Both 5α-and 5β-THB treatment significantly inhibited the growth of xenograft tumors compared to vehicle (Figures 3D and 3E) . Additionally, the protein levels of GAP-43 and NSE in xenograft tumors were upregulated by 5α-THB (Figure 3F) .  Although the induction of GAP-43 and NSE by 5β-THB did not reach the statistical significance, a trend was observed.
2.4 Activation of AHR signaling by 5α-and 5β-THB is Ahr2-dependent in zebrafish
Because NB is thought to be derived from the abnormal differentiation of sympathetic neurons, understanding the role of AHR and THB in neural development may provide valuable insight into the mechanism of NB tumorigenesis. Therefore, we used zebrafish as an in vivo model to investigate whether THB mediates neural development. In zebrafish, three AHR isoforms have been identified: Ahr1a, Ahr1b, and Ahr2 [32, 33] . Numerous studies have suggested Ahr2 is the primary isoform that mediates xenobiotic metabolism in zebrafish [34-38] . To examine whether THB could activate Ahr2 signaling in zebrafish, we exposed embryos to THB with or without the pre-injection of ahr2-specific morpholino (ahr2MO) , which will interfere with the translation of Ahr2. We found that both 5α-and 5β-THB upregulated zCyp1a mRNA expression in a dose-dependent manner (Figures 4A and 4B) . In addition, ahr2MO effectively diminished the Ahr2 and zCyp1a protein levels in zebrafish (Figure 4C) . The induction of cyp1a mRNA by THB was also significantly attenuated by ahr2MO pre-treatment (Figures 4D and 4E) . These results demonstrate that 5α-and 5β-THB-activated AHR signaling is Ahr2-dependent in zebrafish.
2.5 5α-and 5β-THB promote neurogenesis in zebrafish
The Tg (HuC: Kaede) zebrafish is a useful tool to label neuronal network morphologies in vivo [39] . As neurons mature, the expression of green-fluorescent Kaede will be upregulated by the HuC promoter. Figure 5 shows representative images of Kaede fluorescent signal of the Tg (HuC: Kaede) zebrafish. Kaede expression was mainly observed in the eye, brain and spinal cord. We found that THB treatment enhanced the overall Kaede levels in 1, 3 and 5 dpf zebrafish, indicating that THB treatment promotes early neurogenesis in zebrafish. Although both 5α-and 5β-THB showed the ability to activate Ahr2 signaling, 5β-THB induce higher expression levels of zCyp1a (Figure 4) and HuC (Figure 5) . Thus, we used 5β-THB to trigger Ahr2 signaling in the subsequent experiments.
2.6 AHR mediates gliogenesis in zebrafish
We next investigated whether glial development was also influenced by Ahr2 signaling. Using immunohistochemistry, the expression levels of specific markers for different glial lineages were examined in Tg (HuC: Kaede) transgenic zebrafish. We found that O4 and GFAP (markers of oligodendrocytes and astrocytes, respectively) were both downregulated by ahr2MO (Figures 6B and 6C) . Conversely, ahr2MO promoted expression of the microglia marker, CD11b/c (Figure 6D) . All these effects were rescued by 5β-THB treatment, suggesting that Ahr regulates zebrafish gliogenesis by promoting oligodendrocyte and astrocyte differentiation and by inhibiting microglia maturation. Notably, we also found that the expression of the neuronal marker, HuC, was also suppressed by ahr2MO and could be rescued by THB treatment (Figure 6A) . Taken together, these observations clearly implicate Ahr as an important regulator of nervous system development in zebrafish.
2.7 AHR plays a role in the myelination of zebrafish
Increasing evidence suggests that AHR plays a role in myelination and the development of oligodendrocytes and Schwann cells [24, 40, 41] . In this experiment, we also made comparable observations in the zebrafish model, finding that 5α-and 5β-THB promoted mRNA expression of SRY (sex determining region Y) -box 10 (Sox10) and myelin basic protein (MBP) , which are markers of early and mature myelinating cells, respectively (Figures 7A and 7C; Figures 9A and 9C) . In addition, ahr2MO significantly suppressed THB-induced Sox10 and MBP mRNA expression (Figures 7B and 7D; Figures 9B and 9D) . In Tg (mbp: egfp) and Tg (-4725sox10: egfp) ba4 transgenic zebrafish, GFP expression is respectively controlled by Sox10 and MBP promoters. Upon THB exposure, both strains showed similar results, with upregulated GFP; similarly, the elevated GFP signal was significantly suppressed in both strains by ahr2MO (Figures 7E and 7F; Figures 9E and 9F) . These results demonstrated that THB mediates oligodendrocyte and Schwann cell differentiation via Ahr2 signaling, possibly promoting myelination.
2.8 5α-and 5β-THB improve the mobility of Ahr2-deficient zebrafish larvae
Since we showed that ahr2MO impaired neurogenesis and gliogenesis of zebrafish, we next examined whether locomotor activities of larvae were influenced by the Ahr2 deficit. Embryos were injected with  ahr2MO or SDMO (control) followed by a 4-day exposure to 5α-and 5β-THB (100μM) or EtOH control. The swimming behavior of 5 dpf larvae (which normally exhibit a mature myelin sheath structure in the nervous system) were recorded and analyzed. It was found that the total distance traveled of the larvae were significantly reduced by ahr2MO, indicating that Ahr2 deficiency caused a loss of locomotor activity in the larvae (Figures 8A and 8B) . In addition, the treatment of 5α-THB or 5β-THB rescued the low mobility of ahr2MO-injected larvae. These results demonstrated that Ahr2 played an important role in nervous system development, and Ahr2 deficit caused locomotor deficiencies which could be reversed by 5α-THB or 5β-THB.
In conlusion, either 5α-THB or 5β-THB is endogenous ligands of AHR, and is potential for treatment of NB. By regulating various aspects of neural development, AHR signaling is an important regulator of tumorigenesis in NB. Accordingly, 5α-THB and 5β-THB are effective in prevention or treatment of a cancer, such as NB.
While the invention has been described with reference to the preferred embodiments above, it should be recognized that the preferred embodiments are given for the purpose of illustration only and are not intended to limit the scope of the present invention and that various modifications and changes, which will be apparent to those skilled in the relevant art, may be made without departing from the spirit and scope of the invention.
References
1. Kewley RJ, Whitelaw ML, Chapman-Smith A: The mammalian basic helix–loop–helix/PAS family of transcriptional regulators. The International Journal of Biochemistry &Cell Biology 2004, 36 (2) : 189-204.
2. Jones S: An overview of the basic helix-loop-helix proteins. Genome biology 2004, 5 (6) : 226.
3. Whitlock JP, Jr.: Induction of cytochrome P4501A1. Annual review of pharmacology and toxicology 1999, 39: 103-125.
4. Hankinson O: The aryl hydrocarbon receptor complex. Annual review of pharmacology and toxicology 1995, 35: 307-340.
5. Matthews J, Wihlén B, Thomsen J, Gustafsson
Figure PCTCN2020092922-appb-000008
Aryl Hydrocarbon Receptor-Mediated Transcription: Ligand-Dependent Recruitment of Estrogen  Receptor α to 2, 3, 7, 8-Tetrachlorodibenzo-p-Dioxin-Responsive Promoters. Molecular and cellular biology 2005, 25 (13) : 5317-5328.
6. Davarinos NA, Pollenz RS: Aryl hydrocarbon receptor imported into the nucleus following ligand binding is rapidly degraded via the cytosplasmic proteasome following nuclear export. The Journal of biological chemistry 1999, 274 (40) : 28708-28715.
7. Ma Q, Baldwin KT: 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin-induced degradation of aryl hydrocarbon receptor (AhR) by the ubiquitin-proteasome pathway. Role of the transcription activaton and DNA binding of AhR. The Journal of biological chemistry 2000, 275 (12) : 8432-8438.
8. Marlowe JL, Fan Y, Chang X, Peng L, Knudsen ES, Xia Y, Puga A: The aryl hydrocarbon receptor binds to E2F1 and inhibits E2F1-induced apoptosis. Molecular biology of the cell 2008, 19 (8) : 3263-3271.
9. Marlowe JL, Knudsen ES, Schwemberger S, Puga A: The aryl hydrocarbon receptor displaces p300 from E2F-dependent promoters and represses S phase-specific gene expression. The Journal of biological chemistry 2004, 279 (28) : 29013-29022.
10. Puga A, Barnes SJ, Dalton TP, Chang C, Knudsen ES, Maier MA: Aromatic hydrocarbon receptor interaction with the retinoblastoma protein potentiates repression of E2F-dependent transcription and cell cycle arrest. The Journal of biological chemistry 2000, 275 (4) : 2943-2950.
11. Watabe Y, Nazuka N, Tezuka M, Shimba S: Aryl hydrocarbon receptor functions as a potent coactivator of E2F1-dependent trascription activity. Biological &pharmaceutical bulletin 2010, 33 (3) : 389-397.
12. Jin DQ, Jung JW, Lee YS, Kim JA: 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin inhibits cell proliferation through arylhydrocarbon receptor-mediated G1 arrest in SK-N-SH human neuronal cells. Neuroscience letters 2004, 363 (1) : 69-72.
13. Petersen SL, Curran MA, Marconi SA, Carpenter CD, Lubbers LS, McAbee MD: Distribution of mRNAs encoding the arylhydrocarbon receptor, arylhydrocarbon receptor nuclear translocator, and arylhydrocarbon receptor nuclear translocator-2 in the rat brain and brainstem. The Journal of comparative neurology 2000, 427 (3) : 428-439.
14. Collins LL, Williamson MA, Thompson BD, Dever DP, Gasiewicz TA, Opanashuk LA: 2, 3, 7, 8-Tetracholorodibenzo-p-dioxin exposure disrupts granule neuron precursor maturation in the developing mouse cerebellum. Toxicological sciences : an official journal of the Society of Toxicology 2008, 103 (1) : 125-136.
15. Fernandez-Salguero P, Pineau T, Hilbert DM, McPhail T, Lee SS, Kimura S, Nebert DW, Rudikoff S, Ward JM, Gonzalez FJ: Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor. Science (New York, NY) 1995, 268 (5211) : 722-726.
16. Schmidt JV, Su GH, Reddy JK, Simon MC, Bradfield CA: Characterization of a murine Ahr null allele: involvement of the Ah receptor in hepatic growth and development. Proceedings of the National Academy of Sciences of the United States of America 1996, 93 (13) : 6731-6736.
17. Mimura J, Yamashita K, Nakamura K, Morita M, Takagi TN, Nakao K, Ema M, Sogawa K, Yasuda M, Katsuki M et al: Loss of teratogenic response to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking the Ah (dioxin) receptor. Genes to cells : devoted to molecular &cellular mechanisms 1997, 2 (10) : 645-654.
18. Wang Q, Chen J, Ko CI, Fan Y, Carreira V, Chen Y, Xia Y, Medvedovic M, Puga A: Disruption of aryl hydrocarbon receptor homeostatic levels during embryonic stem cell differentiation alters expression of homeobox transcription factors that control cardiomyogenesis. Environmental health perspectives 2013, 121 (11-12) : 1334-1343.
19. Kawamura T, Yamashita I: Aryl hydrocarbon receptor is required for prevention of blood clotting and for the development of vasculature and bone in the embryos of medaka fish, Oryzias latipes. Zoological science 2002, 19 (3) : 309-319.
20. Williamson MA, Gasiewicz TA, Opanashuk LA: Aryl hydrocarbon receptor expression and activity in cerebellar granule neuroblasts: implications for development and dioxin neurotoxicity. Toxicological sciences : an official journal of the Society of Toxicology 2005, 83 (2) : 340-348.
21. Dever DP, Adham ZO, Thompson B, Genestine M, Cherry J, Olschowka JA, DiCicco-Bloom E, Opanashuk LA: Aryl hydrocarbon receptor deletion in cerebellar granule neuron precursors impairs neurogenesis. Developmental neurobiology 2016, 76 (5) : 533-550.
22. Latchney SE, Hein AM, O'Banion MK, DiCicco-Bloom E, Opanashuk LA: Deletion or activation of the aryl hydrocarbon receptor alters adult hippocampal neurogenesis and contextual fear memory. Journal of Neurochemistry 2013, 125 (3) : 430-445.
23. Chevallier A, Mialot A, Petit JM, Fernandez-Salguero P, Barouki R, Coumoul X, Beraneck M: Oculomotor deficits in aryl hydrocarbon receptor null mouse. PloS one 2013, 8 (1) : e53520.
24. Juricek L, Carcaud J, Pelhaitre A, Riday TT, Chevallier A, Lanzini J, Auzeil N, Laprevote O, Dumont F, Jacques S et al: AhR-deficiency as a cause of demyelinating disease and inflammation. Scientific reports 2017, 7 (1) : 9794.
25. Wu PY, Liao YF, Juan HF, Huang HC, Wang BJ, Lu YL, Yu IS, Shih YY, Jeng YM, Hsu WM et al: Aryl hydrocarbon receptor downregulates MYCN expression and promotes cell differentiation of neuroblastoma. PloS one 2014, 9 (2) : e88795.
26. Anderson DJ, Axel R: A bipotential neuroendocrine precursor whose choice of cell fate is determined by NGF and glucocorticoids. Cell 1986, 47 (6) : 1079-1090.
27. Anderson DJ, Carnahan JF, Michelsohn A, Patterson PH: Antibody markers identify a common progenitor to sympathetic neurons and chromaffin cells in vivo and reveal the timing of commitment to neuronal differentiation in the sympathoadrenal lineage. The Journal of neuroscience : the official journal of the Society for Neuroscience 1991, 11 (11) : 3507-3519.
28. Brodeur GM: Neuroblastoma: biological insights into a clinical enigma. Nature reviews Cancer 2003, 3 (3) : 203-216.
29. Norris MD, Bordow SB, Marshall GM, Haber PS, Cohn SL, Haber M: Expression of the gene for multidrug-resistance-associated protein and outcome in patients with neuroblastoma. N Engl J Med 1996, 334 (4) : 231-238.
30. Feng S, Cao Z, Wang X: Role of aryl hydrocarbon receptor in cancer. Biochimica et biophysica acta 2013, 1836 (2) : 197-210.
31. Wang BJ, Liao YF, Tung YT, Yih LH, Hu CC, Lee H: Establishment of a bioluminescence-based bioassay for the detection of dioxin-like compounds. Toxicology mechanisms and methods 2013, 23 (4) : 247-254.
32. Karchner SI, Franks DG, Hahn ME: AHR1B, a new functional aryl hydrocarbon receptor in zebrafish: tandem arrangement of ahr1b and ahr2 genes. Biochem J 2005, 392 (Pt 1) : 153-161.
33. Fraccalvieri D, Soshilov AA, Karchner SI, Franks DG, Pandini A, Bonati L, Hahn ME, Denison MS: Comparative analysis of homology models of the AH receptor ligand binding domain: verification of structure-function predictions by site-directed mutagenesis of a nonfunctional receptor. Biochemistry 2013, 52 (4) : 714-725.
34. Billiard SM, Timme-Laragy AR, Wassenberg DM, Cockman C, Di Giulio RT: The role of the aryl hydrocarbon receptor pathway in mediating synergistic developmental toxicity of polycyclic aromatic hydrocarbons to zebrafish. Toxicological sciences : an official journal of the Society of Toxicology 2006, 92 (2) : 526-536.
35. Kubota A, Stegeman JJ, Woodin BR, Iwanaga T, Harano R, Peterson RE, Hiraga T, Teraoka H: Role of zebrafish cytochrome P450 CYP1C genes in the reduced mesencephalic vein blood flow caused by activation of AHR2. Toxicol Appl Pharmacol 2011, 253 (3) : 244-252.
36. Goodale BC, La Du JK, Bisson WH, Janszen DB, Waters KM, Tanguay RL: AHR2 mutant reveals functional diversity of aryl hydrocarbon receptors in zebrafish. PloS one 2012, 7 (1) : e29346.
37. Bugel SM, White LA, Cooper KR: Inhibition of vitellogenin gene induction by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin is mediated by aryl hydrocarbon receptor 2 (AHR2) in zebrafish (Danio rerio) . Aquat Toxicol 2013, 126: 1-8.
38. Wang WD, Chen GT, Hsu HJ, Wu CY: Aryl hydrocarbon receptor 2 mediates the toxicity of Paclobutrazol on the digestive system of zebrafish embryos. Aquat Toxicol 2015, 159: 13-22.
39. Sato T, Takahoko M, Okamoto H: HuC: Kaede, a useful tool to label neural morphologies in networks in vivo. Genesis (New York, NY : 2000) 2006, 44 (3) : 136-142.
40. Fernandez M, Paradisi M, D'Intino G, Del Vecchio G, Sivilia S, Giardino L, Calza L: A single prenatal exposure to the  endocrine disruptor  2, 3, 7, 8-tetrachlorodibenzo-p-dioxin alters developmental myelination and remyelination potential in the rat brain. J Neurochem 2010, 115 (4) : 897-909.
41. Shackleford G, Sampathkumar NK, Hichor M, Weill L, Meffre D, Juricek L, Laurendeau I, Chevallier A, Ortonne N, Larousserie F et al:
Involvement of Aryl hydrocarbon receptor in myelination and in human nerve sheath tumorigenesis. Proceedings of the National Academy of Sciences of the United States of America 2018, 115 (6) : E1319-e1328.

Claims (13)

  1. A use of a compound selected from the group consisting of 3α, 5α-Tetrahydrocorticosterone (5α-THB) and 3α, 5β-Tetrahydrocorticosterone (5β-THB) in manufacturing a medicament for preventing or treating a cancer.
  2. The use of claim 1, wherein the compound is capable of enhancement of the expression of sex determining region Y-box 10 (Sox10) and myelin-associated proteins myelin basic protein (MBP) .
  3. The use of claim 1, wherein the treatment is through Ahr2 pathway.
  4. The use of claim 1, wherein the cancer is neuroblastoma (NB) .
  5. The use of claim 4, wherein the compound inhibits tumorigenesis in NB.
  6. A pharmaceutical composition for preventing or treating a cancer, comprising a therapeutically effective amount of a compound selected from the group consisting of 3α, 5α-Tetrahydrocorticosterone (5α-THB) and 3α, 5β-Tetrahydrocorticosterone (5β-THB) , in association with a pharmaceutically acceptable carrier.
  7. The pharmaceutical composition of claim 6, wherein the compound is capable of enhancement of the expression of sex determining region Y-box 10 (Sox10) and myelin-associated proteins myelin basic protein (MBP) 
  8. The pharmaceutical composition of claim 6, wherein the cancer is neuroblastoma (NB) .
  9. The pharmaceutical composition of claim 6, wherein the compound inhibits tumorigenesis in NB.
  10. A method for preventing or treating a cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a compound selected from the group consisting of 3α, 5α-Tetrahydrocorticosterone (5α-THB) and 3α, 5β-Tetrahydrocorticosterone (5β-THB) .
  11. The method of claim 10, wherein the compound is capable of enhancement of the expression of sex determining region Y-box 10 (Sox10) and myelin-associated proteins myelin basic protein (MBP)
  12. The method of claim 10, wherein the cancer is neuroblastoma (NB) .
  13. The method of claim 10, wherein the compound inhibits tumorigenesis in NB.
PCT/CN2020/092922 2019-06-14 2020-05-28 Method and pharmaceutical composition for prevention and treatment of a cancer WO2020248824A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201962861516P 2019-06-14 2019-06-14
US62/861,516 2019-06-14

Publications (1)

Publication Number Publication Date
WO2020248824A1 true WO2020248824A1 (en) 2020-12-17

Family

ID=73780897

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/092922 WO2020248824A1 (en) 2019-06-14 2020-05-28 Method and pharmaceutical composition for prevention and treatment of a cancer

Country Status (2)

Country Link
TW (1) TW202116325A (en)
WO (1) WO2020248824A1 (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5786344A (en) * 1994-07-05 1998-07-28 Arch Development Corporation Camptothecin drug combinations and methods with reduced side effects
EP1393720A1 (en) * 2002-08-27 2004-03-03 Universiteit Utrecht Vesicle-encapsulated corticosteroids for treatment of cancer
US20180071315A1 (en) * 2016-09-09 2018-03-15 Marinus Pharmaceuticals Inc. Methods of Treating Certain Depressive Disorders and Delirium Tremens
WO2018195186A1 (en) * 2017-04-18 2018-10-25 Marinus Pharmaceuticals, Inc Sustained release injectable neurosteroid formulations

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5786344A (en) * 1994-07-05 1998-07-28 Arch Development Corporation Camptothecin drug combinations and methods with reduced side effects
EP1393720A1 (en) * 2002-08-27 2004-03-03 Universiteit Utrecht Vesicle-encapsulated corticosteroids for treatment of cancer
US20180071315A1 (en) * 2016-09-09 2018-03-15 Marinus Pharmaceuticals Inc. Methods of Treating Certain Depressive Disorders and Delirium Tremens
WO2018195186A1 (en) * 2017-04-18 2018-10-25 Marinus Pharmaceuticals, Inc Sustained release injectable neurosteroid formulations

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
WU PEIYI ET AL.，: "Novel Endogenous Ligands of Aryl Hydrocarbon Receptor Mediate Neural Development and Differentiation of Neuroblastoma", ACS CHEM. NEUROSCI.，, vol. 10, 12 August 2019 (2019-08-12), pages 4031 - 4042, XP055765109, DOI: 20200814170303PX *
YANG C. ET AL.: "5α-Reduced glucocorticoids exhibit dissociated anti-inflammatory and metabolic effects", BRITISH JOURNAL OF PHARMACOLOGY, vol. 164, 31 December 2011 (2011-12-31), pages 1661 - 1671, XP055765111, DOI: 20200814170412X *

Also Published As

Publication number Publication date
TW202116325A (en) 2021-05-01

Similar Documents

Publication Publication Date Title
Noda et al. Loss of Parkin contributes to mitochondrial turnover and dopaminergic neuronal loss in aged mice
Zhang et al. Caffeic acid reduces A53T α-synuclein by activating JNK/Bcl-2-mediated autophagy in vitro and improves behaviour and protects dopaminergic neurons in a mouse model of Parkinson’s disease
Fan et al. N-methyl-D-aspartate (NMDA) receptor function and excitotoxicity in Huntington's disease
Qin et al. System xc− and apolipoprotein E expressed by microglia have opposite effects on the neurotoxicity of amyloid-β peptide 1–40
Ito et al. Involvement of CHOP, an ER-stress apoptotic mediator, in both human sporadic ALS and ALS model mice
Tsao et al. Rodent models of TDP-43: recent advances
Battaglia et al. Early defect of transforming growth factor β1 formation in Huntington’s disease
Li et al. Isorhynchophylline ameliorates cognitive impairment via modulating amyloid pathology, tau hyperphosphorylation and neuroinflammation: Studies in a transgenic mouse model of Alzheimer’s disease
Tian et al. Activation of Nrf2/ARE pathway alleviates the cognitive deficits in PS1V97L‐Tg mouse model of Alzheimer’s disease through modulation of oxidative stress
Da Costa et al. Vps13b is required for acrosome biogenesis through functions in Golgi dynamic and membrane trafficking
US20110189308A1 (en) Methods to treat neurodegenerative conditions or diseases by targeting components of a pten signaling pathway
Korhonen et al. Hippocalcin protects against caspase-12-induced and age-dependent neuronal degeneration
Hong et al. Sigma-1 receptor knockout increases α-synuclein aggregation and phosphorylation with loss of dopaminergic neurons in substantia nigra
Brunialti et al. Inhibition of microglial β-glucocerebrosidase hampers the microglia-mediated antioxidant and protective response in neurons
Shinohara et al. Increased levels of Aβ42 decrease the lifespan of ob/ob mice with dysregulation of microglia and astrocytes
Gong et al. p47 phox deficiency improves cognitive impairment and attenuates tau hyperphosphorylation in mouse models of AD
Taheri et al. The protective effect of sulforaphane against oxidative stress in granulosa cells of patients with polycystic ovary syndrome (PCOS) through activation of AMPK/AKT/NRF2 signaling pathway
Farhadi et al. Neuroprotective effects of crocin against ethanol neurotoxicity in the animal model of fetal alcohol spectrum disorders
Lv et al. Melatonin MT1 receptors regulate the Sirt1/Nrf2/Ho‐1/Gpx4 pathway to prevent α‐synuclein‐induced ferroptosis in Parkinson's disease
JPWO2008072781A1 (en) Method for suppressing or treating memory impairment in mammals
Bullock et al. Lysosomal storage disease associated with a CNP sequence variant in Dalmatian dogs
von Bohlen und Halbach Synucleins and their relationship to Parkinson’s disease
WO2020248824A1 (en) Method and pharmaceutical composition for prevention and treatment of a cancer
Søderman et al. The nicotinic α7 acetylcholine receptor agonist ssr180711 is unable to activate limbic neurons in mice overexpressing human amyloid-β1–42
Zhou et al. Deletion of serine racemase reverses neuronal insulin signaling inhibition by amyloid‐β oligomers

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20823306

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20823306

Country of ref document: EP

Kind code of ref document: A1