PI3K/AKT signaling pathway; natural alkaloid
PTEN/PI3K/AKT signaling pathway. [1]
Caspase pathway (intrinsic apoptosis pathway). [1]

Sophocarpine is one of the major ingredients of Sophorae flavescentis which could inhibits many kinds of cancers. However, the effect of sophocarpine on gastric cancer (GC) and the mechanism involved remain unknown. The present study aims to explore the effects of the sophocarpine on the proliferation and apoptosis of GC cells and elucidates the relevant molecular mechanisms. After treatment with sophocarpine, GC cells were evaluated on their proliferation, autophagy, cell cycle progress and apoptosis. The protein levels of LC3-I, LC3-II, Beclin, p62, PTEN, PI3K, p53, Bax, Bcl-2, AKT and p-AKT were detected by western blot. Sophocarpine inhibited the proliferation of GC cells both in vitro and in vivo dose-dependently. Sophocarpine not only caused cell apoptosis and cell cycle arrest in G0/G1 phase but also induced cell autophagy. Moreover, sophocarpine dose-dependently suppressed PI3K/AKT signaling pathway and activated apoptosis in gastric cancer cells. Thus, sophocarpine significantly inhibited the growth of GC cells through multiple mechanisms such as induction of autophagy, activation of cell apoptosis and down-regulation of cell survival signaling pathway [1].

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Sophocarpine inhibited the proliferation of MKN45 and BGC-823 gastric cancer cell lines in a dose-dependent manner as measured by CCK-8 assay. The estimated CC50 of sophocarpine was 2.45 mg/mL on MKN45 cells and 2.07 mg/mL on BGC-823 cells. [1]
Sophocarpine induced cell autophagy in both cell lines. Transmission electron microscopy (TEM) images showed autolysosomes around the nucleus in MKN45 cells treated with sophocarpine (2.45 mg/mL for MKN45, 2.07 mg/mL for BGC-823), and some autophagosomes contained organelles such as endoplasmic reticulum. Western blot analysis revealed that compared to control (0 mg/mL), the expression of LC3-I and LC3-II proteins was significantly increased, while Beclin and p62 proteins were conversely decreased in a dose-dependent manner in both MKN45 and BGC-823 cells. [1]
Sophocarpine arrested gastric cancer cell cycle in G1 phase. Flow cytometry analysis showed that treatment with sophocarpine at 1.23 mg/mL and 2.45 mg/mL in MKN45 cells, and at 1.03 mg/mL and 2.07 mg/mL in BGC-823 cells, significantly increased the percentage of cells in G1 phase (p<0.05) and decreased the percentage in G2/M and S phases (p<0.05). Western blot analysis showed that sophocarpine dose-dependently increased PTEN protein expression, while decreasing PI3K and p-AKT protein expression in both cell lines. p53 expression was also detected. [1]
Sophocarpine induced cell apoptosis through the intrinsic caspase pathway. Annexin V-FITC/PI staining flow cytometry showed that the incidence of apoptotic MKN45 cells was 4.37±1.25% at 0 mg/mL, 15.80±0.78% at 1.25 mg/mL, and 33.77±1.21% at 2.45 mg/mL after 48h treatment. For BGC-823 cells, the apoptotic rates were 3.44±0.86% at 0 mg/mL, 12.82±1.78% at 1.25 mg/mL, and 24.21±1.58% at 2.45 mg/mL. Western blot analysis showed that sophocarpine dose-dependently increased cleaved caspase-3 and Bax protein expression, while decreasing Bcl-2 protein expression in both cell lines. [1]

Sophocarpine inhibited tumor growth in a xenograft mouse model. Nude mice bearing MKN45 or BGC-823 subcutaneous xenografts were treated with sophocarpine (50 mg/kg) or saline solution by oral administration every day for 16 days. The tumor volume and weight in the sophocarpine-treated groups were significantly decreased compared to the PBS group (p<0.05). The body weight of the nude mice had no significant change compared to the control group (p>0.05). H&E staining of main organs (heart, liver, lungs, kidneys, and brain) showed no histopathologic changes, indicating that sophocarpine did not induce any systemic side toxicity. [1]

Cell viability assay (CCK-8): MKN45 and BGC-823 cells were seeded in 96-well plates and treated with serial dilutions of sophocarpine (0, 0.125, 0.25, 0.5, 1, 2, 4, 8 mg/mL) for 48 hours. Cell viability was measured using CCK-8 reagent. The CC50 values were calculated using regression analysis. [1]
Cell apoptosis assay: MKN45 and BGC-823 cells were seeded into 6-well plates at a density of 10^6 cells per well and cultured for 24 hours, then treated with fresh DMEM (control) or sophocarpine (2.45 mg/mL and 1.23 mg/mL for MKN45; 2.07 mg/mL and 1.03 mg/mL for BGC-823) for 48 hours. Cells were washed with PBS, trypsinized, harvested, and stained with Annexin V-FITC/PI apoptosis kit. Samples were analyzed by flow cytometry. [1]
Cell cycle assay: MKN45 and BGC-823 cells were seeded into 6-well plates at 10^6 cells per well, cultured for 24 hours, then treated with control or sophocarpine (same concentrations as above) for 48 hours. Cells were washed with PBS, trypsinized, harvested, and fixed with 75% ethanol overnight, then stained with propidium iodide (PI) kit and analyzed by flow cytometry using Flowjo software. [1]
Transmission electron microscopy (TEM): MKN45 and BGC-823 cells were seeded into 6-well plates at 10^6 cells per well, cultured for 24 hours, then treated with control or sophocarpine (2.45 mg/mL for MKN45; 2.07 mg/mL for BGC-823) for 48 hours. Cells were harvested, fixed with 2.5% glutaraldehyde, embedded in resin, sectioned, stained by osmotic acids, and viewed under electron microscope. [1]
Western blot: Total protein was extracted from treated cell samples. Equal volumes of protein were loaded onto 10% SDS-PAGE and separated by electrophoresis. Protein bands were transferred to PVDF membranes. Membranes were blocked in TBST with 5% non-fat milk for 1 hour, then incubated overnight at 4°C with primary antibodies against LC3-I, LC3-II, Beclin, p62, PTEN, PI3K, p53, Bax, Bcl-2, AKT and p-AKT (1:1000). After washing three times with TBST, membranes were incubated with HRP-conjugated secondary antibody (1:1000) for 1 hour. Antibodies were detected by enhanced chemiluminescence reagents. Densitometric analysis was performed using Quantity One software, normalized to β-actin. [1]

In vivo anti-tumor study: Female nude mice (aged 5-6 weeks) were randomly divided into three groups (PBS, sophocarpine, n=3 per group). MKN45 or BGC-823 cells were harvested at a density of 1×10^7 cells/mL and suspended in PBS. Then 100 µL cell suspension was injected into the right flank of male mice. After 10 days, sophocarpine (50 mg/kg) or saline solution was administered to tumor-bearing mice by oral gavage every day for 16 days. Mouse body weight was recorded every three days. Tumor tissues were excised and weighed at the end of the experiment. Tumor size was measured and calculated using the formula: 1/2 × L × W^2, where L = longest surface length (mm) and W = width (mm). Main organs (heart, liver, lungs, kidneys, and brain) were harvested, fixed in 10% phosphate-buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). [1]

The oral LD50 in rats was 196 mg/kg. Chinese Pharmaceutical Journal, 27(201), 1992. The intraperitoneal LD50 in rats was 124 mg/kg. Chinese Pharmaceutical Journal, 27(201), 1992. The oral LD50 in mice was 242 mg/kg. Behavioral manifestations: tremor; behavioral manifestations: seizures or influence on epilepsy threshold. Chinese Journal of Traditional Chinese Medicine, 11(555), 1980. The intravenous LD50 in mice was 72 mg/kg. Chinese Pharmaceutical Journal, 27(201), 1992. The intramuscular LD50 in mice was 92410 ug/kg. Behavior: tremor; behavior: seizures or influence on epilepsy threshold. Chinese Journal of Traditional Chinese Medicine, 11(555), 1980.

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Sophocarpine (50 mg/kg oral administration) did not cause significant body weight change in nude mice compared to control group (p>0.05). H&E staining of main organs (heart, liver, lungs, kidneys, and brain) showed no histopathologic changes, indicating that sophocarpine did not induce any systemic side toxicity. [1]

[1]. Sophocarpine inhibits the growth of gastric cancer cells via autophagy and apoptosis. Front Biosci (Landmark Ed). 2019 Mar 1;24:616-627.

Sofocapine is an alkaloid. It has been reported in Daphniphyllum oldhamii, Daphniphyllum pentandrum, and other organisms with available data.

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Sophocarpine (purity: 93.3%, lot#: 110715-201318) was provided by the National Institutes for Food and Drug Control (Beijing, China). The compound was dissolved in DMEM and stored at -20°C. [1]
Sophocarpine exerts anti-tumor effects on gastric cancer cells through multiple mechanisms: induction of autophagy, activation of cell apoptosis (via caspase pathway), cell cycle arrest in G0/G1 phase, and down-regulation of PTEN/PI3K/AKT survival signaling pathway. [1]
The study suggests that sophocarpine may provide a novel approach for gastric cancer therapy as it is derived from the traditional Chinese herb Sophora flavescens. [1]

C15H22N2O

246.3480

246.173

C, 73.13; H, 9.00; N, 11.37; O, 6.49

6483-15-4

Sophocarpine monohydrate; 145572-44-7

115269

White to off-white solid powder

1.2±0.1 g/cm3

425.4±45.0 °C at 760 mmHg

194.0±21.1 °C

0.0±1.0 mmHg at 25°C

1.603

1.37

0

2

0

18

392

4

O=C1C([H])=C([H])C([H])([H])[C@]2([H])[C@@]3([H])C([H])([H])C([H])([H])C([H])([H])N4C([H])([H])C([H])([H])C([H])([H])[C@@]([H])(C([H])([H])N21)[C@]43[H]

AAGFPTSOPGCENQ-JLNYLFASSA-N

InChI=1S/C15H22N2O/c18-14-7-1-6-13-12-5-3-9-16-8-2-4-11(15(12)16)10-17(13)14/h1,7,11-13,15H,2-6,8-10H2/t11-,12+,13+,15-/m0/s1

(1R,2R,9S,17S)-7,13-diazatetracyclo[7.7.1.02,7.013,17]heptadec-4-en-6-one

(-)-Sophocarpine; Sophocarpine

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 : ~50 mg/mL (~202.96 mM)
H2O : ~10 mg/mL (~40.59 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (8.44 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 (8.44 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 (8.44 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: 10 mg/mL (40.59 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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