- (+)-Isocorydine hydrochloride combined with doxorubicin (DOX) showed higher cytotoxicity in HCC cells (Huh-7, Hep-G2, SNU-449, SNU-387) compared to either drug alone, as measured by CCK-8 assay after 48 h treatment. The combination significantly decreased the IC50 of DOX. The Combination Index (CI) values were: Huh-7 CI=0.605, Hep-G2 CI=0.644, SNU-449 CI=0.804, SNU-387 CI=0.707, indicating a synergistic effect. ICD alone IC50 values (μg/ml): Huh-7 161.3, Hep-G2 148.9, SNU-449 262.2, SNU-387 254.1. DOX alone IC50 values (μg/ml): Huh-7 0.6565, Hep-G2 0.8796, SNU-449 1.739, SNU-387 2.353. In combination, DOX IC50 values decreased significantly (p<0.001) to: Huh-7 0.02495, Hep-G2 0.02940, SNU-449 0.2159, SNU-387 0.1684 μg/ml. [1]
- (+)-Isocorydine hydrochloride abrogated DOX-induced EMT in HCC cells. Western blot analysis showed that DOX treatment (48 h) upregulated mesenchymal markers Vimentin, β-catenin, and Snail, and downregulated epithelial markers E-cadherin and Claudin-1. Co-treatment with ICD reversed these changes, restoring E-cadherin and Claudin-1 expression while reducing Vimentin, β-catenin, and Snail. Immunofluorescence staining confirmed weaker Vimentin signal and stronger E-cadherin membrane expression in co-treated cells compared to DOX alone. [1]
- (+)-Isocorydine hydrochloride inhibited the invasive capacity of DOX-induced EMT. Transwell invasion assays showed significantly fewer HCC cells (Huh-7 and Hep-G2) passing through Matrigel-coated membranes in the DOX+ICD group compared to DOX alone. Wound healing assays showed decreased migration in co-treated cells compared to DOX or ICD alone. [1]
- The proportion of CD133+ cells (cancer stem cell marker) was elevated by DOX treatment, but this increase was reversed by co-treatment with (+)-Isocorydine hydrochloride. Sorted CD133+ cells showed higher resistance to chemotherapeutic agents; however, this subpopulation became sensitive to the combination of ICD and DOX, with CI values of 0.456 (Huh-7) and 0.556 (Hep-G2). ICD alone IC50 in CD133+ cells: Huh-7 207.1 μg/ml, Hep-G2 370.4 μg/ml; DOX alone IC50: Huh-7 6.147 μg/ml, Hep-G2 7.199 μg/ml; combination DOX IC50: Huh-7 0.735 μg/ml, Hep-G2 1.540 μg/ml (p<0.001). [1]
- Microarray analysis revealed that (+)-Isocorydine hydrochloride significantly inhibits DOX-induced EMT-related gene expression. GO analysis identified 101 genes associated with specific signaling pathways, including MAPK signaling (ELK1, ELK4, MYC). Western blot showed that DOX increased p-ERK expression, which was decreased by ICD co-treatment. Knockdown of ERK1/2 using siRNA attenuated Vimentin, β-catenin, and ZEB-1 expression, and upregulated epithelial markers. ERK knockdown also significantly decreased the IC50 of DOX. [1]

- In a xenograft model using female nude mice subcutaneously implanted with Huh-7 cells (1×10^6 cells in 100 μl PBS into right axillary fossa), treatment with (+)-Isocorydine hydrochloride alone (0.4 mg/kg) or DOX alone (4 mg/kg) retarded tumor growth, but combined treatment (DOX 4 mg/kg + ICD 0.4 mg/kg) significantly inhibited tumor growth more effectively (p<0.01 vs. DOX or ICD alone). Drugs were injected intraperitoneally every 2 days for 2 weeks (total 6 injections). Tumor volume and body weight were measured every second day. After 4 weeks of treatment, mice were euthanized. No significant change in body weight was observed in any group, indicating lack of overt toxicity. [1]

- Cell viability and cytotoxicity were determined using the Cell Counting Kit-8 (CCK-8) assay. Cells (5000 per well) were seeded in 96-well microplates and exposed to different concentrations of drugs for 48 hours. Then 10 μl of CCK8 reagent was added to each well and incubated for 4 hours. Optical density was measured at 450 nm. The half-maximal inhibitory concentration (IC50) was calculated. All experiments were conducted in triplicates. [1]
- Western blotting: Cells were treated with drugs for 48 hours, then lysed in lysis buffer. Proteins were separated by 10% SDS-PAGE and transferred electrophoretically to PVDF membranes. Primary antibodies against E-cadherin, Vimentin, Twist1, β-catenin, Snail, Claudin-1, ERK1/2, p-ERK (1:1000) were used, followed by HRP-conjugated secondary antibodies. Detection was performed by chemiluminescence using a ChemiDoc MP system. GAPDH was used as a loading control. [1]
- Immunofluorescence: Cells were cultured in chamber slides for 24 hours, then fixed with 4% paraformaldehyde for 30 min at room temperature. After blocking with 5% BSA for 30 min, slides were incubated overnight at 4°C with mouse anti-human Vimentin or anti-human E-cadherin primary antibody (1:100). After washing, slides were incubated with PE or FITC-conjugated secondary antibody for 1 hour. Nuclei were stained with DAPI. Stained slides were observed under a confocal microscope. [1]
- Wound healing assay: Cells were cultured with or without half-maximal inhibitory concentration (IC50) of indicated drugs. Same cell numbers were seeded in 12-well plates pre-coated with 0.5% gelatin. After reaching confluence, cells were starved in 1% FBS medium for 24 hours. A scratch wound was created using a 200 μl pipette tip. The distance migrated by cells was calculated as an average of three different fields observed under an optical microscope at 200x magnification. Three independent experiments were analyzed. [1]
- Transwell invasion assay: Transwell inserts were coated with Matrigel. Equal numbers of cells in each upper chamber were cultured in medium with 1% FBS. The lower chamber contained 20% FBS as a chemoattractant. Cells were incubated for 24 hours at 37°C in 5% CO2. Cells in the lower chamber were fixed with 95% ethanol for 15 min followed by hematoxylin and eosin staining. Stained cells were evaluated under a microscope. [1]
- Flow cytometry and sorting: CD133 staining in HCC cells was performed using fluorochrome-conjugated mAbs. Cell suspension (1×10^6 cells/mL) was stained with CD133 at 4°C for 30 min. Flow cytometric analysis was performed on a Cantol instrument. CD133+ or CD133- cells were sorted using FACSAria II. [1]

- Xenograft experiment: Female nude mice (3-4 weeks old) were used. Huh-7 cells (1×10^6) suspended in 100 μl PBS were implanted subcutaneously into the right axillary fossa of each mouse. After approximately 3 weeks when a visible tumor mass was observed, mice were randomly assigned to four subgroups (n not specified): vehicle control (equal volume of diluent), DOX (4 mg/kg), (+)-Isocorydine hydrochloride (0.4 mg/kg), and DOX (4 mg/kg) + ICD (0.4 mg/kg). Drugs were injected intraperitoneally every 2 days for 2 weeks (total 6 injections). Tumor size and body weight were measured every second day. After 4 weeks of treatment, mice were euthanized and subjected to analysis. [1]

Body weight measurements in the xenograft experiment showed no significant change in any treatment group (including ICD alone and combination) compared to control, suggesting that (+)-Isocorydine hydrochloride at 0.4 mg/kg (administered intraperitoneally every 2 days for 2 weeks) was well tolerated without overt systemic toxicity. [1]

[1]. Isocorydine suppresses doxorubicin-induced epithelial-mesenchymal transition via inhibition of ERK signaling pathways in hepatocellular carcinoma. Am J Cancer Res. 2018 Jan 1;8(1):154-164. eCollection 2018.

- (+)-Isocorydine hydrochloride (ICD) is an alkaloid monomer from Papaveraceae plants. It has been reported to selectively inhibit cancer stem cells (CSCs) and target side population cells in HCC. In this study, ICD co-administration enhanced doxorubicin cytotoxicity, inhibited DOX-induced epithelial-mesenchymal transition (EMT) via suppression of ERK signaling, and reduced the proportion of CD133+ cells. The combination therapy showed promising potential to eradicate HCC in vitro and in vivo. [1]
- The study proposes that ICD reverses DOX-induced chemoresistance by inhibiting the ERK-ZEB1 pathway, as knockdown of ERK1/2 decreased ZEB-1 expression and reversed EMT markers. [1]

C20H23NO4.HCL

377.139

13552-72-2

17751017

Typically exists as solid at room temperature

1.222 g/cm3

506.1ºC at 760 mmHg

216-220 °C(lit.)

259.9ºC

Endogenously produced metabolite

3.91

2

5

3

26

475

1

Cl.COC1C=CC2=C(C3C(OC)=C(OC)C=C4C=3[C@H](C2)N(CC4)C)C=1O

ZWSKLEMBDRWSNZ-ZOWNYOTGSA-N

InChI=1S/C20H23NO4.ClH/c1-21-8-7-12-10-15(24-3)20(25-4)18-16(12)13(21)9-11-5-6-14(23-2)19(22)17(11)18;/h5-6,10,13,22H,7-9H2,1-4H3;1H/t13-;/m0./s1

(6aS)-1,2,10-trimethoxy-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-11-ol;hydrochloride

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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