RAB7A (inhibits recruitment of RAB7A to lysosomes, thereby blocking autophagosome-lysosome fusion) [1]
DNM1L (induces dephosphorylation at Ser637 and mitochondrial translocation, leading to mitochondrial fission) [1]

Liensinine (20 µM) treatment increased EGFP-LC3 puncta formation in MDA-MB-231 and MCF-7 cells. [1]
Liensinine caused dose- and time-dependent accumulation of LC3B-II, LAMP1, and SQSTM1, while BECN1 level remained unchanged in MDA-MB-231 and MCF-7 cells. [1]
Liensinine (20 µM, 24 h) treatment increased LC3B-II and SQSTM1 levels in U937, LN229, and A549 cells. [1]
TEM analysis showed increased autophagic vacuoles and partially divided mitochondria in liensinine-treated cells. [1]
Liensinine increased colocalization of EGFP-LC3 with RFP-mito, indicating mitophagy modulation. [1]
Liensinine (20 µM, 24 h) increased total EGFP intensity in EGFP-LC3 stable cells, similar to bafilomycin A1; combined treatment with bafilomycin A1 showed no further increase. [1]
Liensinine treatment resulted in accumulation of LC3B-II and SQSTM1, which was not further enhanced by bafilomycin A1, while rapamycin-induced LC3B-II increase was further enhanced by bafilomycin A1. [1]
tfLC3 reporter assay showed liensinine caused yellow (both green and red) LC3 puncta, indicating blocked autophagic flux at late stage, similar to bafilomycin A1, while rapamycin produced red-only puncta. [1]
Liensinine blocked autophagosome-lysosome fusion: EGFP-LC3 puncta did not colocalize with LysoTracker Red, and mRFP-LC3 did not colocalize with LAMP1-mGFP. [1]
Liensinine increased LAMP2 and RAB7A protein levels in a time-dependent manner. [1]
Immunoprecipitation showed liensinine decreased interaction of LAMP1 with LC3B-II or RAB7A, but did not change interaction of RAB7A with LC3B-II. [1]
Immunofluorescence showed liensinine decreased colocalization of LAMP1 and RAB7A, but did not change colocalization of EGFP-LC3 and RAB7A. [1]
Liensinine increased precursor/proforms of CTSB, CTSD, and CTSL, and decreased mature forms; rapamycin had opposite effects. [1]
Acridine orange staining showed liensinine increased acidic vesicles (red fluorescence) compared to control, while bafilomycin A1 decreased them. [1]
LysoSensor Yellow/Blue DND-160 staining showed liensinine did not alter lysosomal pH (pH ~4.5), while bafilomycin A1 increased pH. [1]
Liensinine abolished colocalization of CTSD with LAMP1. [1]
Cotreatment with liensinine (20 µM) and chemotherapeutic drugs (doxorubicin, paclitaxel, vincristine, cisplatin, staurosporine) decreased cell viability compared to monotherapy in MDA-MB-231 and MCF-7 cells. [1]
Liensinine plus doxorubicin (0.2 µM for MDA-MB-231, 0.4 µM for MCF-7) increased apoptosis (~50%) compared to single agents (~5-10%), and increased cleavage of CASP9, CASP3, and PARP1 degradation, as well as CYCS release from mitochondria to cytosol. [1]
Liensinine plus doxorubicin further enhanced accumulation of LC3B-II, SQSTM1, ubiquitin, HSPD1, and COX4I1 in whole cell lysates and mitochondrial fractions. [1]
Combination treatment increased autophagic/mitophagic vacuoles and damaged swollen mitochondria with aberrant cristae by TEM. [1]
Combination treatment increased EGFP-LC3 puncta and colocalization with RFP-mito. [1]
Combination treatment significantly increased proportion of cells with fragmented mitochondria (mitochondrial fission) as shown by MitoTracker Red CMXRos staining. [1]
Liensinine plus doxorubicin increased DNM1L levels in mitochondria and decreased in cytosol, and decreased phospho-DNM1L (Ser637) without affecting phospho-DNM1L (Ser616). [1]
Mdivi-1 (DNM1L inhibitor) pretreatment reduced combination-induced DNM1L translocation, LC3B-II and SQSTM1 accumulation in mitochondria, mitochondrial fission, and apoptosis. [1]
siRNA knockdown of DNM1L reduced combination-induced DNM1L mitochondrial localization, mitochondrial fission, LC3B-II accumulation, and apoptosis. [1]
Overexpression of DNM1L S637D mutant (phosphomimetic) attenuated combination-induced apoptosis, CASP3 activation, and CYCS release; DNM1L S637A mutant slightly enhanced doxorubicin-mediated apoptosis which was further increased by liensinine. [1]
3-MA (autophagy inhibitor) pretreatment reduced combination-induced LC3B-II accumulation, DNM1L dephosphorylation (Ser637) and mitochondrial translocation, mitochondrial fission, apoptosis, CYCS release, and CASP3 activation. [1]
Knockdown of ATG5 or ATG7 reduced combination-induced LC3B-II accumulation, DNM1L dephosphorylation and mitochondrial translocation, mitochondrial fission, apoptosis, CASP3 activation, and CYCS release. [1]

Similar to metronidazole medications, lenisinine (oral gavage, 100 or 200 mg/kg, once day, 10 weeks) can successfully treat periodontitis in a quantitative dose-dependent manner [2].

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In a mouse xenograft model (MDA-MB-231 cells subcutaneously inoculated in nude mice), liensinine (60 mg/kg, intraperitoneal injection daily) combined with doxorubicin (2 mg/kg, intraperitoneal injection every 4 days) for 30 days significantly reduced tumor growth compared to vehicle control, liensinine alone, or doxorubicin alone (P<0.01). No significant body weight changes were observed among groups. [1]
H&E staining of tumors from combination-treated mice showed significant morphological changes with necrosis, inflammatory cell infiltration, and fibrosis. TUNEL staining showed combination treatment dramatically increased TUNEL-positive cells (brown color). Immunohistochemistry showed increased immunoreactivity for cleaved CASP3. [1]
Immunofluorescence of tumor sections showed combination treatment increased LC3 puncta and colocalization of LC3 with HSPD1, as well as increased colocalization of DNM1L with TOMM20. [1]

Cells (MDA-MB-231, MCF-7, U937, LN229, A549, 293FT) were cultured in appropriate media with 10% FBS at 37°C, 5% CO2. [1]
For cell viability (MTT assay): cells were seeded at 2,000-5,000 per well in 96-well plates, treated as indicated for 48-72 h, then 20 µl MTT (5 mg/ml) added per well for 4 h, followed by 150 µl DMSO to dissolve formazan, and absorbance measured at 490 nm. [1]
For flow cytometry apoptosis assay: cells were stained with ANXA5-FITC/PI kit according to manufacturer's instructions. [1]
For acidic vesicle detection: cells were stained with acridine orange (1.5 µg/ml) for 15 min at 37°C, then analyzed by flow cytometry using red (660 nm) to green (530 nm) fluorescence ratio. [1]
For EGFP-LC3 stable cells: MDA-MB-231 cells stably expressing EGFP-LC3 were maintained with G418 (200 µg/ml), then treated and analyzed by flow cytometry for total EGFP intensity. [1]
For transmission electron microscopy (TEM): cells were fixed in 2.5% glutaraldehyde at 4°C overnight, postfixed with 2% osmium tetroxide for 1.5 h at room temperature, embedded, stained with uranyl acetate/lead citrate, and examined at 60 kV. [1]
For transfections: cells were transfected using Lipofectamine 3000 with plasmids encoding mRFP-LC3, tfLC3, LAMP1-mGFP, EGFP-LC3, RFP-mito, ATG5-siRNA, ATG7-siRNA, DNM1L siRNA, or DNM1L-mCherry mutants (S637A, S637D) generated by site-directed mutagenesis. [1]
For mitochondrial and cytosolic fractionation: cells were harvested, washed with cold PBS, resuspended in buffer A (20 mM HEPES pH7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 2 mM leupeptin, 1 mM PMSF, 1 mM DTT, 2 mM pepstatin A, 250 mM sucrose), homogenized by passing through 22-gauge needle 30 times, centrifuged at 3,500 g at 4°C (pellet = mitochondrial fraction), supernatant centrifuged at 120,000 g at 4°C (supernatant = cytosolic fraction). [1]
For western blot: total protein lysates prepared in 1× NuPAGE LDS sample buffer, or mitochondrial/cytosolic fractions quantified by BCA assay, separated by SDS-PAGE, transferred to PVDF membranes, blocked with 5% non-fat dry milk in TBS-Tween20, incubated with primary antibodies (LC3B, SQSTM1, LAMP1, LAMP2, RAB7A, CTSB, CTSD, CTSL, BECN1, PARP1, cleaved CASP9, cleaved CASP3, CYCS, DNM1L, phospho-DNM1L Ser616, phospho-DNM1L Ser637, COX4I1, HSPD1, TOMM20, ubiquitin, ACTB, GAPDH), then HRP-conjugated secondary antibodies, visualized with ECL substrate. [1]
For immunoprecipitation: total protein lysates incubated with primary antibodies at 4°C on rocking platform, immune complexes collected with protein A/G agarose beads, washed 5 times in PBS, then subjected to SDS-PAGE and western blot. [1]
For immunofluorescence: cells or tissue sections fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 in PBS for 5-10 min, blocked with 10% donkey serum in PBS, incubated with primary antibodies overnight, then appropriate secondary antibodies, viewed by confocal microscopy. Mitochondria and lysosomes were stained with MitoTracker Red CMXRos, LysoTracker Red DND-99, or LysoSensor Yellow/Blue DND-160 according to manufacturer's instructions. [1]
For lysosomal pH measurement: cells labeled with 5 µM LysoSensor Yellow/Blue DND-160 for 5 min at 37°C, washed with PBS, treated for 2 min with 10 µM monensin and 10 µM nigericin in ice-cold 25 mM MES calibration buffer (pH 3.5-6.0) containing 5 mM NaCl, 115 mM KCl, 1.2 mM MgSO4. Light emission at 535 nm upon excitation at 340 nm and 380 nm measured, pH calibration curve generated. [1]
For clonogenic growth assay: MDA-MB-231 cells stably expressing ATG5-siRNA, ATG7-siRNA or scramble-siRNA were cotreated with liensinine and doxorubicin for 48 h, then 5,000 cells reseeded in 6-well plates, cultured for 1-7 days, and counted with a cell counter. [1]

Animal/Disease Models: KM mice [2]
Doses: 100 mg/kg, 200 mg/kg
Route of Administration: po (oral gavage), daily, 10 weeks
Experimental Results: Reduce gingival index, increase SOD levels, CAT, GSH-Px levels, diminished levels of NO, MDA and ET compared with controls.

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Animal/Disease Models: Institute of Cancer Research (ICR) mice (male, 20-22 grams) [3]
Doses:
Route of Administration: Oral 5 mg/kg; 1 mg/kg intravenous (iv) (iv)administration
Experimental Results: The effect of lianxining in mice pharmacokinetic/PK/PK parameters po (5 mg/kg) iv (1 mg/kg) AUC(0-t) (ng/mL*h) 18.8 ± 2.7 211.2 ± 54.9 AUC(0-∞) (ng/mL* h) 19.1 ± 2.8 227.9 ± 60.1 MRT(0-t)(h) 3.2 ± 0.4 2.6 ± 0.5 MRT(0-∞) (h) 3.4 ± 0.5 3.5 ± 0.9 t1 /2z (h) 1.9 ± 0.2 3.8 ± 0.8 CLz/F (L/h/kg) 266.0 ± 41.3 4.7 ± 1.2 Vz/F (L/kg) 708.5 ± 79.9 25.9 ± 11.0 Cmax (ng/mL) 5.3 ± 0.2 169.5±53.5

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Female nude mice (5-7 weeks old) were inoculated subcutaneously with 1×10^7 MDA-MB-231 cells suspended in 1:1 serum-free DMEM with Matrigel basement membrane matrix. [1]
One week after tumor inoculation, mice were randomly assigned into 4 groups (8 mice per group). Liensinine (60 mg/kg) was administered daily by intraperitoneal injection. Doxorubicin (2 mg/kg) was injected at intervals of 4 days. Treatment lasted for 30 days. Tumor diameters were measured and tumor volume (mm^3) calculated as (shortest diameter × longest diameter)/2. [1]
Mice were sacrificed 30 days after medication. Before sacrifice, mice received 200 µl of 1% pentobarbital sodium by intraperitoneal injection 30 min prior. Tumors were excised and either formalin-fixed or flash-frozen at -20°C. TUNEL, histological (H&E), and immunohistochemical assays were performed. [1]

[1]. A novel autophagy/mitophagy inhibitor liensinine sensitizes breast cancer cells to chemotherapy through DNM1L-mediated mitochondrial fission. Autophagy. 2015;11(8):1259-79.

[2]. Protective effect of liensinine on periodontitis through its antioxidant effect in mice. Journal of the Korean Society for Applied Biological Chemistry volume 58, pages927-936.

[3]. Pharmacokinetics and bioavailability of liensinine in mouse blood by UPLC-MS/MS. Acta Chromatographica,Volume 33: Issue 4,14 Oct 2020.

Nelumbo nucifera is an isoquinoline compound. It has been found in lotus (Nelumbo nucifera), and relevant data have been reported.

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Liensinine is a major isoquinoline alkaloid extracted from the seed embryo of Nelumbo nucifera Gaertn, known to have a wide range of biological activities including anti-arrhythmias, antihypertension, anti-pulmonary fibrosis, and relaxation on vascular smooth muscle. [1]
This study first demonstrates that liensinine inhibits late-stage autophagy/mitophagy by blocking autophagosome-lysosome fusion, likely via inhibiting recruitment of RAB7A to lysosomes but not to autophagosomes. [1]
Liensinine combined with chemotherapeutic drugs (especially doxorubicin) represents a novel therapeutic strategy for breast cancer treatment. The combination enhances doxorubicin-mediated apoptosis by triggering mitochondrial fission through dephosphorylation (Ser637) and mitochondrial translocation of DNM1L. Excessive accumulation of autophagosomes/mitophagosomes ("autophagic stress") is implicated in this process. [1]

C37H42N2O6

610.7392

610.304

2586-96-1

Liensinine Diperchlorate;5088-90-4;Liensinine perchlorate;2385-63-9

160644

White to off-white solid powder

1.2±0.1 g/cm3

722.0±60.0 °C at 760 mmHg

95-99ºC

390.4±32.9 °C

0.0±2.4 mmHg at 25°C

1.618

4.84

2

8

9

45

917

2

CN1CCC2=CC(=C(C=C2[C@H]1CC3=CC=C(C=C3)O)OC4=C(C=CC(=C4)C[C@@H]5C6=CC(=C(C=C6CCN5C)OC)OC)O)OC

XCUCMLUTCAKSOZ-FIRIVFDPSA-N

InChI=1S/C37H42N2O6/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/h6-11,18-22,30-31,40-41H,12-17H2,1-5H3/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

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~50 mg/mL (~81.87 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.41 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 (3.41 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 (3.41 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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Solubility in Formulation 4: 20 mg/mL (32.75 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).
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.)