STAT3 (signal transducer and activator of transcription 3) [1]

JAK1 and JAK2 (upstream kinases of STAT3) [1]

Lycorine has a minor effect on PNT1A cell proliferation, as well as a cyclic inhibitory effect on cell proliferation on the four PCa cell lines mentioned above, with an IC50 range of 5 μM to 10 μM [1]. In order to control or safeguard SREBF in the endoplasmic reticulum, SCAP (SREBF molecular chaperone), an endoplasmic reticulum-to-Golgi transporter, transforms structurally by creating a complex with INSIG1 (insulin-inducible gene) 1)[2]. In a dose- and time-dependent way, Lycorine (5-40 μM; 16) considerably reduces SREBF activity (up to -70%) without having overt effects in cells. Lycorine (10-20 μM; 2-16 hours). Cytotoxicity [2]. decreases in HL-7702 cells the amounts of mature SREBF1 and SREBF2 proteins [2]. ABCG5 and ABCG8 are two NR1H3 target genes that are not affected by lycorine (20 μM; 16 hours) or NR1H3 transcription activation. Sterol Forwarding activity for NR1H3 is activated [1]. Treatment with lycorine (0-25 μM; 48 hours) markedly and dose-dependently suppressed the expression of vascular endothelial (VE)-cadherin and marginally decreased the expression of Sema4D in C8161 cells. The VE-cadherin protein level in C8161 cells was dramatically lowered by the expression of the other 6 genes after 48 hours of treatment with Lycorine (0-25 μM) [3].

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Lycorine inhibited proliferation of hormone-refractory PCa cell lines PC-3M, DU145, LNCaP and 22RV1 in a dose-dependent manner with IC50 values ranging from 5 μM to 10 μM after 48 h treatment, while showing little effect on normal human prostate epithelium immortalized cell line PNT1A. [1]

Lycorine (0–10 μM) significantly inhibited PC-3M cell wound healing (12 h), migration (5–7 h Transwell assay) and invasion (12 h Matrigel-coated Transwell assay) in a dose-dependent manner. [1]

Lycorine (5 μM) significantly inhibited colony formation of PC-3M, DU145, LNCaP and 22RV1 cells (8 days). [1]

Live/dead staining showed that Lycorine (0–50 μM, 48 h) potentiated PC-3M cell death. [1]

Lycorine (0–50 μM, 48 h) induced apoptosis in PC-3M cells in a dose-dependent manner, with apoptotic cells increasing from 10.04% to 54.08% (Annexin V/PI staining by flow cytometry). Similar apoptotic effects were observed in DU145 and LNCaP cells. [1]

Western blotting revealed that Lycorine (0–25 μM, 48 h) induced cleavages of PARP and caspase-3 in PC-3M cells. [1]

Lycorine (10 ng/mL EGF stimulation; 0, 10, 25 μM Lycorine, 48 h) reversed EGF-induced EMT in PC-3M cells: decreased expression of N-cadherin, vimentin, fibronectin and Twist (both mRNA and protein), and increased E-cadherin expression. It also suppressed the expression of MMP2 and MMP9, as well as activated STAT3 levels under EGF stimulation. [1]

Lycorine (10 μM, 48 h) reduced endogenous STAT3 and p-STAT3 levels in DU145, PC-3M and LNCaP cells (with PC-3M showing the highest baseline levels and largest response to Lycorine in migration assay). [1]

STAT3 knockdown by siRNA (48 h transfection) abolished Lycorine-induced decrease in cell viability in PC-3M cells, indicating that the anti-cancer effects depend on STAT3 expression. [1]

Lycorine (10 ng/mL EGF, 10 μM Lycorine, 24 h) significantly inhibited phosphorylation of STAT3 (Tyr705), JAK1 (Tyr1022/1023) and JAK2 (Tyr1007/1008) as shown by Western blotting. [1]

ChIP assay showed that Lycorine (0–25 μM, 48 h) decreased STAT3 DNA-binding activity to target genes Bcl-xL, cyclin D1 and Twist in a concentration-dependent manner, with effective concentrations around 10 μM. [1]

Lycorine effectively regressed STAT3 translocation from cytoplasm to nucleus under EGF stimulation (immunofluorescence). [1]

Q-PCR analysis confirmed that STAT3 target genes (cyclin D1, Bcl-2, Bcl-xL, MMP2, Twist) were down-regulated by Lycorine. [1]

Lycorine (facial; 15 mg/kg, 30 mg/kg; once daily) attenuates fat coupling and depletion therapy and enhances fat stratification and oxidation of grafts and precursor and mature SREBF in mice [2].

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Subcutaneous xenograft model (PC-3M cells in nude mice): Lycorine at 5 mg/kg/day or 10 mg/kg/day (i.p., daily for 18 days) significantly suppressed tumor growth. Average tumor volume of control group was 2154 ± 119 mm³, while Lycorine-treated groups showed 989 ± 32 mm³ (5 mg/kg) and 478 ± 47 mm³ (10 mg/kg). Tumor weight reduction by about 80% at 10 mg/kg with no obvious toxicity. [1]

Orthotopic xenograft model (PC-3M-luc cells injected into dorsal lateral prostate): Lycorine at 5 or 10 mg/kg/day (i.p., daily for 30 days) significantly reduced bioluminescence signal (photon flux) representing tumor growth. Normalized photon flux: control (82 ± 5.78)×10⁵ pc/sec/cm²/sr; 5 mg/kg (41 ± 2.34)×10⁵; 10 mg/kg (23 ± 4.01)×10⁵. Lycorine also inhibited metastasis to liver, lung, kidney, spleen, bone and lymph nodes, and improved mouse survival (at day 30, deaths: control 6/20, 5 mg/kg 4/20, 10 mg/kg 2/20). [1]

Immunohistochemistry of tumor sections from Lycorine-treated mice (10 mg/kg) showed increased expression of E-cadherin and cleaved caspase-3, and decreased expression of p-STAT3 and Ki-67 compared to control. [1]

Lycorine treatment had little effect on mouse body weights and no obvious pathological changes in main organs at the given doses. [1]

Chromatin immunoprecipitation (ChIP) assay to assess STAT3 DNA-binding activity: PC-3M cells were treated with increasing concentrations of Lycorine (0, 1, 5, 10, and 25 μM) for 48 hours, then fixed with 1% formaldehyde and lysed. Chromatin samples were immunoprecipitated with antibodies against STAT3 or with normal rabbit IgG antibody, and examined by quantitative PCR using SYBR Premix Ex Taq Kit to measure binding to target gene promoters (Bcl-xL, cyclin D1, Twist). Beta-actin served as internal control. The assay demonstrated that Lycorine decreased STAT3 DNA-binding activity in a concentration-dependent manner. [1]

Western blot analysis of kinase phosphorylation: PC-3M cells were treated with 10 ng/mL EGF and 10 μM Lycorine for 24 hours, then lysed and subjected to SDS-PAGE and immunoblotting using specific antibodies against EGFR, JAK1, JAK2, STAT3 and their phosphorylated forms (p-EGFR, p-JAK1 Tyr1022/1023, p-JAK2 Tyr1007/1008, p-STAT3 Tyr705). Beta-actin served as loading control. The results showed Lycorine inhibited EGF-induced phosphorylation of JAK1, JAK2 and STAT3. [1]

Cell viability assay [2]
Cell Types: HL-7702/SRE- Luc. Cell
Tested Concentrations: 16 hrs (hours)
Incubation Duration: 5 μM; 10 μM; 20 μM; 40 μM
Experimental Results: No cytotoxicity to HL-7702 cells.

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Western Blot Analysis[2]
Cell Types: HL-7702/SRE-Luc Cell
Tested Concentrations: 2 hour, 4 hrs (hours), 8 hrs (hours), 12 hrs (hours), 16 hrs (hours)
Incubation Duration: 10 μM; 20 μM
Experimental Results: p-SREBF1, m-SREBF1, p-SREBF2 and p-SREBF1 protein expression diminished.

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RT-PCR[3]
Cell Types:C8161 Cell
Tested Concentrations: 0 μM, 1.56 μM, 3.13 μM, 6.25 μM, 12.5 μM, 25 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Dramatically inhibited the expression of VE-cadherin in a dose-dependent manner, and also slightly diminished C8161 Expression of Sema4D in cells.

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Cell viability assay (MTS): PCa cells and PNT1A (5×10³ cells/well) were treated with various concentrations of Lycorine (0, 0.05, 0.1, 1, 5, 10, 20, 50, 100 μM) for 48 h or indicated times (24–96 h). Aqueous One Solution was added and absorbance at 490 nm measured. Three independent experiments with triplicate were carried out. [1]

Wound-healing migration assay: Tumor cells grown to full confluence in 6-well plates, wounds created by sterile pipette tip. Fresh medium with 10% FBS and various concentrations of Lycorine (0–10 μM) was added. After 12 h incubation at 37°C, cells were fixed with 3.7% paraformaldehyde and photographed. Migrated cells were manually quantified. [1]

Transwell migration assay: Serum-starved PCa cells (5×10⁴ cells in 100 μL medium with 0.5% FBS) were pretreated with Lycorine (0–10 μM) for 30 min, then seeded on upper chamber (8 μm pore size). Lower chamber contained 600 μL medium with 10% FBS. After 5–7 h incubation, non-migrated cells removed, migrated cells fixed with cold 3.7% paraformaldehyde and stained with 0.1% crystal violet. Images taken and migrated cells counted in 4 random fields. Three independent experiments with triplicate. [1]

Invasion assay: Similar to migration assay but Transwell chambers coated with Matrigel. Cells pretreated with Lycorine (0–10 μM) for 12 h in 6-well plates, then 5×10⁴ cells in serum-free medium added to upper chamber. Complete medium in lower chamber. After 12 h invasion at 37°C, invaded cells stained and counted. [1]

Clonogenic assay: Tumor cells seeded in 6-well plates, allowed to grow for 24 h, then treated with different concentrations of Lycorine (0–10 μM). On day 8, colonies fixed with 3.7% paraformaldehyde, stained with 0.1% crystal violet and counted manually. [1]

Live/dead staining: Cells treated with Lycorine (0–50 μM) for 48 h, then stained with Calcein AM (stains live cells green) and EthD-II (stains dead cells red). Visualized by fluorescence microscopy. [1]

Cell cycle analysis: PCa cells treated with Lycorine for 36 h, washed with PBS, fixed with cold 70% ethanol at 4°C for at least 12 h, then PI working solution added before flow cytometry analysis. [1]

Apoptosis assay (Annexin V/PI): PCa cells treated with Lycorine (0–50 μM) for 48 h, collected, washed, stained with annexin V-FITC and PI for 15 min, then evaluated by flow cytometry. [1]

Western blotting: PCa cells treated with Lycorine for indicated times and concentrations, lysed in RIPA buffer. Protein concentration determined by BCA assay. Samples run on 8–12% SDS-PAGE gels, transferred to PVDF membranes, incubated with specific antibodies overnight, signals visualized via Odyssey system. [1]

Immunofluorescence staining: PCa cells plated on gelatin-coated cover slips, treated with Lycorine for 24 h, fixed with 3.7% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 1% BSA, incubated with STAT3 antibody overnight, then with secondary antibody for 1 h, and DAPI for nucleus staining. Photographs obtained with confocal microscope. [1]

RT-PCR and quantitative real-time PCR: RNA isolated, reverse transcribed with miScript II RT Kit, cDNA used for real-time PCR with miScript Primer Assays. Primers for Bcl-2, Bax, cyclin D1, β-actin, E-cadherin and Twist as described previously. [1]

STAT3 siRNA assay: PCa cells plated on 3.5-cm dish, allowed to adhere for 16 h, then transiently transfected with STAT3-siRNA or non-targeting scrambled siRNA in complete medium for 48 h. Cells washed and transferred to complete medium for another 48 h, then harvested for subsequent experiments (cell viability by MTS, Western blotting). [1]

Animal/Disease Models: C57BL/6J mice fed high-fat diet (HFD) [2]
Doses: 15 mg/kg, 30 mg/kg
Route of Administration: oral; one time/day
Experimental Results: Improved high-fat diet-induced hypertensive disorders in mice Lipidemia, hepatic steatosis, and insulin resistance.

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Subcutaneous xenograft model: PC-3M cells (1×10⁶ in 0.1 mL PBS per mouse) were inoculated subcutaneously on the right back sides of nude mice. After tumors reached about 100 mm³, mice were divided into 3 groups (n=10 per group) and received intraperitoneal (i.p.) injection either with DMSO (vehicle control) or Lycorine (5 mg/kg/day or 10 mg/kg/day) every day for 18 days. Body weight and tumor size were monitored every 2 days. At day 18, mice were sacrificed and tumor xenografts dissected. [1]

Orthotopic transplantation xenograft model: PC-3M-luc cells (5×10⁵ in 0.02 mL PBS per mouse) were orthotopically transplanted into the dorsal pole of prostate of anesthetized nude mice. Seven days later, based on photon flux detected by IVIS 2000 Luminal Imager, tumor-bearing mice were divided into three groups (n=20 per group). Lycorine (5 mg/kg/day or 10 mg/kg/day) or DMSO was injected intraperitoneally every day. Luminal images and photon flux were recorded every 5 days. After 30 days, mice were sacrificed, prostate tumors removed, and metastasis assessed in liver, lung, kidney, spleen and bone by bioluminescence. [1]

Metabolism / Metabolites
Paraoxygenase (PON1) is a key enzyme in organophosphate metabolism. PON1 can inactivate certain organophosphates through hydrolysis. PON1 hydrolyzes active metabolites from various organophosphate pesticides and nerve agents (such as soman, sarin, and VX). The existence of PON1 polymorphism leads to differences in the enzyme activity level and catalytic efficiency of this esterase, which in turn suggests that different individuals may be more susceptible to the toxic effects of organophosphate exposure.

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Toxicity Summary
Lycorine is a cholinesterase, or acetylcholinesterase (AChE) inhibitor. Cholinesterase inhibitors (or "anticholinesterases") inhibit the activity of acetylcholinesterase. Because acetylcholinesterase plays a vital physiological role, chemicals that interfere with its activity are potent neurotoxins; even low doses can cause excessive salivation and lacrimation, followed by muscle spasms and ultimately death. Neurotoxins and substances in many pesticides have been shown to exert their effects by binding to serine residues at the active site of acetylcholinesterase, thereby completely inhibiting the enzyme's activity. Acetylcholinesterase breaks down the neurotransmitter acetylcholine, which is released at the neuromuscular junction, causing muscle or organ relaxation. The mechanism of action of acetylcholinesterase inhibitors is to allow acetylcholine to accumulate and exert its sustained effect, ensuring the continuous transmission of nerve impulses and preventing muscle contraction from ceasing. The most common acetylcholinesterase inhibitors are phosphorus-containing compounds designed to bind to the enzyme's active site. Its structural requirements include a phosphorus atom with two lipophilic groups, a leaving group (such as a halogen or thiocyanate group), and a terminal oxygen atom.

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Lycorine treatment at the given doses (5 and 10 mg/kg/day, i.p.) had little effect on mouse body weights, and no obvious pathological changes on main organs (liver, lung, kidney, spleen, bone) when compared to control group. [1]

Lycorine exhibited low toxicity to mice at the curative dose, confirming previous published results. [1]

[1]. Lycorine is a novel inhibitor of the growth and metastasis of hormone-refractory prostate cancer.Oncotarget. 2015 Jun 20;6(17):15348-61.

[2]. Discovery of a Potent SCAP Degrader That Ameliorates HFD-induced Obesity, Hyperlipidemia and Insulin Resistance via an Autophagy-Independent Lysosomal Pathway. Autophagy. 2020 May 20;1-22.

[3]. Lycorine hydrochloride inhibits metastatic melanoma cell-dominant vasculogenic mimicry. Pigment cell & melanoma research 25, 630-638, doi:10.1111/j.1755-148X.2012.01036.x (2012).

Lycorine is an indolithidine alkaloid with the structure 3,12-disehydrogalantan, substituted with hydroxyl groups at positions 2 and 9 and with a methylenedioxy group at position 10. It was isolated from Crinum asiaticum and has been shown to possess antimalarial activity. Lycorine functions as a protein synthesis inhibitor, antimalarial drug, plant metabolite, and anticoronavirus agent. It is derived from the hydride of galantan. Lycorine has been reported to be found in Crinum moorei, Clivia nobilis, and several other organisms with relevant data. Lycorine is a toxic crystalline alkaloid found in various Amaryllidaceae plants, such as cultivated shrub lilies (Clivia miniata), lycoris, and narcissus. Ingestion of certain doses of lycorine can be highly toxic and even fatal. Symptoms of lycorine poisoning include vomiting, diarrhea, and convulsions. A definition of lycorine can be found at mercksource.com. Nevertheless, it is still sometimes used for medicinal purposes, which is one of the reasons why some groups harvest the very popular Clivia miniata.
See also: Lycorine hydrochloride (note moved to).

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This is the first study to examine Lycorine's anti-cancer mechanistic role in JAK/STAT3 signaling in prostate cancer (PCa) tumor growth and metastasis. Lycorine is suggested as a potential therapeutic for hormone-refractory prostate cancer. [1]

Lycorine reverses EMT through STAT3-mediated Twist decrease, providing a novel mechanism for inhibiting cancer metastasis. [1]

Lycorine promotes epithelial cell characteristics and suppresses mesenchymal features in PC-3M cells. [1]

The anti-cancer effects of Lycorine are dependent on STAT3 expression. [1]

Lycorine suppresses STAT3 transcriptional activity, decreasing DNA-binding to target genes such as Bcl-xL, cyclin D1 and Twist. [1]

Lycorine is a candidate for drug repositioning with potential advantages in toxicity and pharmacokinetics profiles (although not directly measured in this study, it is discussed based on literature). [1]

C16H17NO4

287.3105

287.115

476-28-8

Lycorine hydrochloride monohydrate;6150-58-9;Lycorine hydrochloride;2188-68-3

72378

White to off-white solid powder

1.5±0.1 g/cm3

477.4±45.0 °C at 760 mmHg

253-255ºC (dec.)

242.5±28.7 °C

0.0±1.3 mmHg at 25°C

1.733

0.77

2

5

0

21

481

4

C1CN2CC3=CC4=C(C=C3[C@H]5[C@H]2C1=C[C@@H]([C@H]5O)O)OCO4

XGVJWXAYKUHDOO-DANNLKNASA-N

InChI=1S/C16H17NO4/c18-11-3-8-1-2-17-6-9-4-12-13(21-7-20-12)5-10(9)14(15(8)17)16(11)19/h3-5,11,14-16,18-19H,1-2,6-7H2/t11-,14-,15+,16+/m0/s1

(1S,17S,18S,19S)-5,7-dioxa-12-azapentacyclo[10.6.1.02,10.04,8.015,19]nonadeca-2,4(8),9,15-tetraene-17,18-diol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~25 mg/mL (~87.01 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.70 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.70 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (8.70 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 25 mg/mL (87.01 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)