JNK signaling pathway (activation of JNK via phosphorylation)
Mitochondrial dysfunction (induction of mitochondrial membrane potential loss and ROS increase)[1]

According to research, lenisinine perchlorate can cause apoptosis and significantly inhibit colorectal cancer cells' ability to proliferate and form colonies in a dose-dependent manner; however, normal colorectal epithelial cells were unaffected. cytotoxicity [1].

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liensinine significantly inhibited the viability and proliferation of human CRC cell lines HT29 and DLD‑1 in a dose‑dependent manner.
IC50 values (mean ± SD): for DLD‑1 – 11.265 ± 0.83 μM (24 h) and 5.65 ± 0.57 μM (48 h); for HT29 – 14.507 ± 0.834 μM (24 h) and 6.16 ± 0.267 μM (48 h). No cytotoxicity was observed in normal colorectal epithelial cells NCM460.
Colony formation ability of HT29 and DLD‑1 cells was markedly reduced in a dose‑dependent manner by liensinine.
liensinine induced apoptosis in a dose‑dependent fashion: after 48 h treatment (up to 20 μM), the fold change of apoptotic cells increased over 13.5‑fold in DLD‑1 and 4.3‑fold in HT29 (Annexin V‑FITC/PI double staining). Western blot showed increased expression of cleaved caspase‑3 and cleaved PARP.
liensinine caused G2/M phase arrest: in DLD‑1 cells, percentage of G2/M cells increased from 22.98% to 40.51%; in HT29 cells, from 25.27% to 39.77% (48 h treatment). Western blot revealed increased p‑CDK1 and Cyclin A2 levels.
Mitochondrial dysfunction: JC‑1 assay showed a dose‑dependent decrease in mitochondrial membrane potential (red/green fluorescence ratio). Intracellular ROS level was significantly increased (DCFH‑DA assay).
Western blot analysis demonstrated that liensinine increased expression of Bax and p‑JNK, while decreasing Bcl‑2 and Bcl‑xL expression in a dose‑dependent manner.
Quantitative proteomics (DIA‑MS) identified 376 differentially expressed proteins (273 up‑regulated, 103 down‑regulated, FC ≥1.5, p≤0.05). Ingenuity Pathway Analysis (IPA) revealed that “mitochondrial dysfunction” was the top canonical pathway (p=8.35E‑17), and network analysis indicated JNK signaling as a hub in mediating liensinine‑induced apoptosis. [1]

In a nude mouse xenograft model (HT29 cells subcutaneously implanted), oral administration of liensinine at 30 mg/kg every other day for 15 days significantly suppressed tumor growth, with a 63% decrease in tumor burden compared to vehicle control.
Immunohistochemical analysis of Ki‑67 proliferation index showed a decrease from 43.0 ± 9.5% (vehicle) to 11.7 ± 4.5% (liensinine‑treated).
Western blot of tumor tissues confirmed that liensinine up‑regulated p‑JNK and Bax expression.
No significant difference in body weight was observed between treatment and control groups.
Histological examination (H&E staining) of lungs, livers, and kidneys revealed no overt morphological changes, indicating no detectable toxicity to vital organs. [1]

Cell viability assay: Cells (5000 per well) were seeded in 96‑well plates and treated with various concentrations of liensinine (0, 5, 10, 20 μM) for 24 h and 48 h. WST‑1 reagent was added and incubated for 1 h at 37°C, then absorbance was measured at 450 nm using a microplate spectrophotometer. [1]
Colony formation assay: CRC cells (2000 cells/well) were seeded in 6‑well plates and cultured for 14 days. After washing with PBS, cells were fixed with methanol for 15 min and stained with 1% crystal violet for 20 min at room temperature. Colonies were counted from three independent experiments. [1]
Apoptosis assay (Annexin V‑FITC/PI): Cells were suspended in binding buffer, stained with annexin V‑FITC and PI for 15 min at room temperature in the dark, then analyzed by flow cytometry (C6 flow cytometer). [1]
ROS measurement: Intracellular ROS level was determined using cell‑permeable DCFH‑DA. Cells were treated with indicated concentrations of liensinine for 48 h and stained with DCFH‑DA according to the manufacturer’s instructions. [1]
Mitochondrial membrane potential assay: JC‑1 assay was used. Cells treated with liensinine for 48 h were stained with JC‑1, and the red/green fluorescence ratio was measured. [1]
Western blot: Whole‑cell lysates were prepared in lysis buffer. Protein concentration was determined by BCA method. Samples were separated by SDS‑PAGE and electrotransferred to PVDF membranes. After blocking with 5% nonfat milk for 1 h, membranes were incubated with primary antibodies (caspase‑3, cleaved caspase‑3, p‑CDK1, cyclin A2, JNK, p‑JNK, Bax, Bcl‑2, Bcl‑xL, PARP, cleaved PARP, and actin) for 2 h at room temperature, then with HRP‑conjugated secondary antibodies for 1 h. Signals were visualized using ECL substrate and autoradiographic film. [1]
Cell cycle analysis: Cells were fixed with 70% alcohol for 1 h at –20°C, then stained with PI staining buffer (containing PI 33 μg/ml, RNase A 0.13 mg/ml, EDTA 10 mM, Triton X‑100 0.5%) for 10 min at room temperature. Cell cycle distribution was analyzed by flow cytometry (BD Accuri C6). [1]
Proteomics sample preparation and DIA‑MS: Whole‑cell lysates were homogenized in RIPA buffer. After trypsin digestion using filter‑aided sample preparation (FASP), peptides were vacuum‑freeze‑dried, resuspended, desalted with C18 pipette tips, and analyzed with an Orbitrap Fusion Lumos mass spectrometer. Raw data were searched using Proteome Discoverer and Spectronaut software with 1% FDR. Differentially expressed proteins were identified by PLGEM algorithm (FC ≥1.5, p≤0.05) and subjected to Ingenuity Pathway Analysis (IPA). [1]

Female BALB/c nude mice (aged 6‑8 weeks) were maintained under standard conditions. HT29 cells mixed with an equal volume of PBS and Matrigel were subcutaneously implanted into the flanks of mice to establish tumor xenografts. When tumors reached approximately 5 mm in diameter, mice were randomly divided into treatment and control groups (6 mice per group). The treatment group received oral gavage of liensinine at a dose of 30 mg/kg every other day for 15 days, while the control group received vehicle only. Body weight was monitored every two days. Tumor size was measured every two days, and tumor volume was calculated using the formula V = (length × width²)/2. At the end of the study, tumors, lungs, livers, and kidneys were collected. Immunohistochemical analysis of the proliferative index was performed using anti‑Ki‑67 antibody. [1]

In vitro: liensinine showed no observable cytotoxicity on normal colorectal epithelial cells NCM460 at concentrations up to 20 μM. [1]
In vivo: In the mouse xenograft model, oral administration of liensinine (30 mg/kg every other day for 15 days) did not cause significant body weight loss compared to control. Histological examination (H&E staining) of lungs, livers, and kidneys revealed no overt morphological changes, suggesting no detectable toxicity to vital organs. No LD50 or other toxicokinetic parameters are provided. [1]

[1]. Liensinine perchlorate inhibits colorectal cancer tumorigenesis by inducing mitochondrial dysfunction and apoptosis.

Background: Colorectal cancer (CRC) is one of the most common cancers worldwide with poor survival and limited therapeutic options. Food‑source natural products are considered safer alternatives to conventional chemotherapeutics. From a natural product library of 429 food‑source compounds, 52 compounds with rarely reported anticancer effects were selected for screening in CRC cells. liensinine showed the strongest inhibitory effect (89.99 ± 0.52% inhibition at 10 μM for 48 h in HT29 cells). [1]
Mechanism of action: This study provides the first evidence that liensinine induces CRC cell apoptosis and suppresses tumor growth via activation of the JNK signaling pathway and induction of mitochondrial dysfunction (loss of mitochondrial membrane potential, increased ROS, altered Bax/Bcl‑2/Bcl‑xL expression). [1]
Potential therapeutic application: liensinine is proposed as a novel therapeutic strategy for treating CRC without obvious side effects, based on its strong anticancer effects in vitro and in vivo, and lack of toxicity to normal cells and vital organs. [1]
Keywords: Food‑source natural product, colorectal cancer, apoptosis, JNK pathway, mitochondrial dysfunction. [1]

C37H42N2O6.2(HCLO4)

811.67

710.26

2385-63-9

Liensinine; 2586-96-1; Liensinine Diperchlorate; 5088-90-4

71307566

White to off-white solid

6.491

3

12

9

50

1030

2

OCl(=O)(=O)=O.COC1=C(OC2=C(O)C=CC(C[C@@H]3C4=CC(=C(OC)C=C4CCN3C)OC)=C2)C=C2C(CCN(C)[C@@H]2CC2=CC=C(O)C=C2)=C1

BGUPXKYVHOSZPY-ZAMYOOMVSA-N

InChI=1S/C37H42N2O6.2ClHO4/c1-38-14-13-26-20-35(43-4)37(22-29(26)30(38)16-23-6-9-27(40)10-7-23)45-33-18-24(8-11-32(33)41)17-31-28-21-36(44-5)34(42-3)19-25(28)12-15-39(31)2;2*2-1(3,4)5/h6-11,18-22,30-31,40-41H,12-17H2,1-5H3;2*(H,2,3,4,5)/t30-,31-;;/m1../s1

4-[[(1R)-6,7-dimethoxy-2-methyl-3,4-dihydro-1H-isoquinolin-1-yl]methyl]-2-[[(1R)-1-[(4-hydroxyphenyl)methyl]-6-methoxy-2-methyl-3,4-dihydro-1H-isoquinolin-7-yl]oxy]phenol;perchloric acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO : ~100 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (Infinity 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 20.8 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: ≥ 2.08 mg/mL (Infinity 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 20.8 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: ≥ 2.08 mg/mL (Infinity 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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