PKC:660 nM (IC50); PKA:0.17 mM (IC50); TPK:0.1 mM (IC50)

Chelerythrine displaces the BH3-containing protein Bax from BclXL and inhibits the BclXL-Bak BH3 peptide binding with an IC50 of 1.5 μM. Loaded cells treated with chelerythrine underwent cell engraftment, which was characterized by mitochondrial involvement [1]. Chelidonine treatment activates and inhibits LPS-induced TNF-α levels and LPS-induced mouse peritoneal giant cells by blocking p38 mitogen protein activator (MAPK) and extracellular signal regulatory protein activator 1 and 2 (ERK1/2). Furthermore, the control of intermediate mediator expression by p38 MAPK and ERK1/2 may account for the effects of chelerythrine on the generation of NO and the cytokine TNF-α [2]. With an LD50 value of 3.46 μM, erythrine is cytotoxic to human monocyte leukocytes. Cells exposed to sanguinarine and chelerythrine dramatically decreased the expression of CCL-2 by 3.5 and 1.9 times, respectively, two hours after LPS stimulation [3]. In a dose-dependent way, chelidonine chloride markedly increased ERK1/2 phosphorylation. Furthermore, p38 phosphorylation can be inhibited by chelerythrine chloride [4].

Chelerythrine inhibits the production of nitric oxide (NO) and tumor necrosis factor-α (TNF-α) in the serum when exposed to LPS, thereby exhibiting significant anti-inflammatory effects in an in vivo experimentally induced mouse endotoxic shock model [2]. When administered intraperitoneally, chelerythrine chloride (5 mg/kg/day) causes RCC cells to undergo apoptosis without causing considerable harm to mice. P53 accumulates in response to cholerylthrine chloride treatment in a dose-dependent manner [4].

The identification of small molecule inhibitors of antiapoptotic Bcl-2 family members has opened up new therapeutic opportunities, while the vast diversity of chemical structures and biological activities of natural products are yet to be systematically exploited. Here we report the identification of chelerythrine as an inhibitor of BclXL-Bak Bcl-2 homology 3 (BH3) domain binding through a high throughput screening of 107,423 extracts derived from natural products. Chelerythrine inhibited the BclXL-Bak BH3 peptide binding with IC50 of 1.5 micro m and displaced Bax, a BH3-containing protein, from BclXL. Mammalian cells treated with chelerythrine underwent apoptosis with characteristic features that suggest involvement of the mitochondrial pathway. While staurosporine, H7, etoposide, and chelerythrine released cytochrome c from mitochondria in intact cells, only chelerythrine released cytochrome c from isolated mitochondria. Furthermore BclXL-overexpressing cells that were completely resistant to apoptotic stimuli used in this study remained sensitive to chelerythrine. Although chelerythrine is widely known as a protein kinase C inhibitor, the mechanism by which it mediates apoptosis remain controversial. Our data suggest that chelerythrine triggers apoptosis through a mechanism that involves direct targeting of Bcl-2 family proteins[5].

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In Vitro Binding Analysis—Labeled Bax was prepared by in vitro transcription/translation of pXJHA-Bax using the TnT T7-coupled reticulocyte lysate system from Promega. The GST binding assay was performed as described previously except that increasing concentrations of chelerythrine were incubated with GSTBclXLΔ19 30 min prior to the addition [35S]Bax.[5]

Cell viability is evaluated via MTT assay. Cells (2×103 HEK-293 cells/well and 3×103 SW-839 cells/well) in 100 µL medium are seeded into 96-well plates, and incubated for 12 h. Next, the medium in each well is replaced with medium containing various concentrations of Chelerythrine Chloride, and the cells are incubated at 37°C for an additional 24 and 48 h. Subsequently, 20 µL MTT (5 mg/mL) is added to each well. Following an additional incubation at 37°C for 4 h, the supernatant is removed, and 100 µL DMSO is added to each well. The absorbance values (read at 540 nm) are determined using the iMark™ Microplate Absorbance Reader[1].

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Previous studies have demonstrated that the benzo[c]phenanthridine alkaloid chelerythrine chloride (CC) has inhibitory effects on various tumors. However, the anticancer activity of CC and its underlying mechanisms have not been elucidated in renal cancer cells. The present study examined the effects of CC on growth inhibition and apoptosis of renal cancer cells in vitro and in vivo. Flow cytometry and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays revealed that CC markedly suppressed the growth of HEK-293 and human renal cancer SW-839 cells in a time- and dose-dependent manner. The xenograft mouse model, which was performed in nude mice, exhibited a reduced tumor growth following CC treatment. In addition, the present study revealed that CC significantly decreased the phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, which was accompanied by upregulation of p53, B-cell lymphoma 2 (Bcl-2)-associated X protein, cleaved caspase-3 and cleaved poly (adenosine diphosphate-ribose) polymerase (PARP), and downregulation of Bcl-2, caspase-3 and PARP. Furthermore, the use of PD98059, a specific mitogen-activated protein kinase kinase inhibitor, potentiated the proapoptotic effects of CC, which indicated that CC may induce apoptosis in renal cancer cells partly via inhibition of ERK activity. Overall, the results of the present study demonstrated that CC may be developed as a potential anticancer treatment for patients with renal cancer[3].

A total of 5×106 SW-839 cells are mixed with Matrigel®, and injected subcutaneously into the flanks of 14 5-week-old male BALB/c nude mice. The mice are maintained in 18×30-cm cages containing three mice each, at a temperature of 22°C using a 12 h light/dark cycle. Food and water is available ad libitum. The mice are randomLy divided into two groups (n=7). As previously described, the mice are administrated with chelerythrine chloride at a dose of 5 mg/kg/day via intraperitoneal injection for 5 weeks, with the first injection of chelerythrine chlorideurring 24 h after injection with the SW-839 cells. The control mice are administered with the same volume of PBS containing 1% DMSO. The volume and weight of the mouse tumors are measured once a week. All the mice are sacrificed 36 days subsequent to inoculation of the cancer cells, when the tumors are resected.[1]

Dissolved in PBS; 5 mg/kg; i.p.

Mice bearing SQ-20B xenografts

Toxicity Summary
Chelerythrine is a potent, selective, and cell-permeable protein kinase C (PKC) inhibitor. It is also the major active natural product found in the plant Zanthoxylum clava-herculis, exhibiting anti-bacterial activity against Staphylococcus aureus. (Wikipedia) Chelerythrine is a selective inhibitor of group A and B PKC isoforms with an antitumor activity. Inhibition of PKC with chelerythrine chloride induces apoptosis by activation of a neutral sphingomyelinase, accumulation of ceramide, and depletion of sphingomyelin. Chelerythrine is at least 100-fold more selective for PKCs than for other kinases. Chelerythrine competes for the conserved catalytic sites of PKC and seems to be a potent and specific inhibitor of the group A and group B kinases. Chelerythrine exhibited cytotoxic activity against nine human tumor cell lines tested in vitro. Radioresistant and chemoresistant squamous cell carcinoma lines (HNSCC) undergo apoptosis rapidly after treatment with chelerythrine in vitro. Chelerythrine treatment of nude mice bearing SQ-20B HNSCC cells is associated with significant tumor growth delay. Also, treatment with chelerythrine resulted in minimal toxicity.

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mouse LD50 subcutaneous 95 mg/kg Planta Medica., 43(161), 1981 [PMID:7312984]
mouse LD50 intravenous 18500 ug/kg Planta Medica., 43(161), 1981 [PMID:7312984]

[1]. Effect of Chelerythrine Against Endotoxic Shock in Mice and Its Modulation of Inflammatory Mediators in Peritoneal Macrophages Through the Modulation of Mitogen-Activated Protein Kinase (MAPK) Pathway. Inflammation. 2012 Dec;35(6):1814-24. d

[2]. Investigation of sanguinarine and chelerythrine effects on LPS-induced inflammatory gene expression in THP-1 cell line. Phytomedicine. 2012 Jul 15;19(10):890-5. Epub 2012 May 14.

[3]. Chelerythrine chloride induces apoptosis in renal cancer HEK-293 and SW-839 cell lines. Oncol Lett. 2016 Jun;11(6):3917-3924.

[4]. Chelerythrine is a potent and specific inhibitor of protein kinase C. Biochem Biophys Res Commun. 1990 Nov 15;172(3):993-9.

[5]. Identification of chelerythrine as an inhibitor of BclXL function.J Biol Chem. 2003 Jun 6;278(23):20453-6.

[6]. Induction of reactive oxygen species-stimulated distinctive autophagy by chelerythrine in non-small cell lung cancer cells.Redox Biol. 2017 Aug;12:367-376.

Chelerythrine chloride is a benzophenanthridine alkaloid.
A quaternary benzo [c] alkaloid chelerythrine (CHE), which is a traditional herbal prescription, has been used for the treatment of various inflammatory diseases. To gain insight into the anti-inflammatory effect and molecular mechanisms underlying the anti-inflammatory activity of CHE, we used experimentally induced mice endotoxic shock moled and lipopolysaccharide (LPS)-induced murine peritoneal macrophages to examine the anti-inflammatory function of CHE. CHE displayed significant anti-inflammatory effects in experimentally induced mice endotoxic shock model in vivo through inhibition of LPS-induced tumor necrosis factor-alpha (TNF-α) level and nitric oxide (NO) production in serum. Additionally, our data suggest that CHE treatment inhibits LPS-induced TNF-α level and NO production in LPS-induced murine peritoneal macrophages through selective inhibition of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) activation. Moreover, the effects of CHE on NO and cytokine TNF-α production can possibly be explained by the role of p38 MAPK and ERK1/2 in the regulation of inflammatory mediators expression.[1]

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Quaternary benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine have been used in folk medicine for their wide range of useful properties. One of their major effect is also anti-inflammatory activity, that is not clarified in detail. This study focused on the ability of these alkaloids to modulate the gene expression of pro-inflammatory tumour necrosis factor α (TNF-α), monocyte chemoattractant protein 1 (MCP-1, also known as CCL-2), interleukin (IL)-6, IL-1β and anti-inflammatory cytokines IL-1 receptor antagonist (IL-1RA) and IL-10. The effect of these alkaloids was compared with that of conventional drug prednisone. Human monocyte-derived macrophages were pre-treated with alkaloids or prednisone and inflammatory reaction was induced by lipopolysaccharide. Changes of gene expression at the transcriptional level of mentioned cytokines were measured. In our study mainly affected pro-inflammatory cytokines were CCL-2 and IL-6. Two hours after LPS stimulation, cells influenced by sanguinarine and chelerythrine significantly declined the CCL-2 expression by a factors of 3.5 (p<0.001) and 1.9 (p<0.01); for those treated with prednisone the factor was 5.3 (p<0.001). Eight hours after LPS induction, both alkaloids significantly diminished the CCL-2 expression. The lower expression was found for sanguinarine--lower by a factor of 4.3 than for cells treated with the vehicle (p<0.001). Two hours after LPS stimulation, cells treated with sanguinarine decreased the IL-6 mRNA level by a factor of 3.9 (p<0.001) compared with cells treated with the vehicle. Chelerythrine decreased the level of IL-6 mRNA by a factor of 1.6 (p<0.001). Sanguinarine decreased gene expression of CCL-2 and IL-6 more than chelerythrine and its effect was quite similar to prednisone. Four hours after LPS stimulation, cells pre-treated with sanguinarine exhibited significantly higher expression (a factor of 1.7, p<0.001) of IL-1RA than cells without sanguinarine treatment. Our results help to clarify possible mechanisms of action of these alkaloids in the course of inflammation.[2]

C21H18NO4.HCL

384.83

383.092

C, 65.71; H, 4.73; Cl, 9.24; N, 3.65; O, 16.67

3895-92-9

Chelerythrine;34316-15-9; 478-03-5 (OH-); 34316-15-9; 3895-92-9 (chloride)

72311

Typically exists as Yellow to orange solids at room temperature

1.36g/cm3

711.4ºC at 760 mmHg

195-205ºC

219.3ºC

1.681

0.72

0

5

2

27

516

0

[Cl-].O1C([H])([H])OC2=C1C([H])=C1C(=C2[H])C([H])=C([H])C2=C3C([H])=C([H])C(=C(C3=C([H])[N+](C([H])([H])[H])=C21)OC([H])([H])[H])OC([H])([H])[H]

WEEFNMFMNMASJY-UHFFFAOYSA-M

InChI=1S/C21H18NO4.ClH/c1-22-10-16-13(6-7-17(23-2)21(16)24-3)14-5-4-12-8-18-19(26-11-25-18)9-15(12)20(14)22;/h4-10H,11H2,1-3H3;1H/q+1;/p-1

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, avoid exposure to moisture.

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

Solubility in Formulation 1: ≥ 0.44 mg/mL (1.15 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 4.4 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 2: ≥ 0.44 mg/mL (1.15 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 4.4 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: ≥ 0.43 mg/mL (1.12 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 4.3 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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 (Please use freshly prepared in vivo formulations for optimal results.)