Topoisomerase II
DNA (intercalation; no IC50/Ki/EC50 values provided for direct binding) [1, 3]
- DNA Topoisomerase II (inhibition; IC50 for enzyme inhibition: 2.5 μM [1]

Ellipticine (NSC 71795) is a strong anti-tumor agent that acts through multiple modes of action. Ellipticine (NSC 71795) is thought to exert its antitumor, mutagenic, and cytotoxic properties through the mechanisms of intercalation into DNA and inhibition of DNA topoisomerase II activity. Ellipticine (NSC 71795) also acts through oxidizing DNA with cytochromes P450 (CYP) and peroxidases, which forms covalent DNA adducts[1]. Ellipticine (NSC 71795) has pharmacological and genotoxic effects because it can also modulate its own metabolism by acting as an inducer or inhibitor of biotransformation enzymes. The application of Ellipticine (NSC 71795) to cells inhibits their growth and proliferation. Two covalent DNA adducts derived from ellipticine (NSC 71795) are linked to this effect[2].

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1. Broad-spectrum antiproliferative activity: Ellipticine exhibited potent cytotoxicity against various human cancer cell lines. IC50 values (determined by MTT/SRB assay) were as follows: HeLa (cervical cancer): 0.5 μM; MCF-7 (breast cancer): 0.8 μM; A549 (lung cancer): 1.2 μM; HepG2 (liver cancer): 1.5 μM; Caco-2 (colon cancer): 0.9 μM [2]
2. DNA intercalation and Topoisomerase II inhibition: Ellipticine intercalated into the minor groove of double-stranded DNA, causing DNA unwinding and structural distortion. It also selectively inhibited DNA Topoisomerase II activity by stabilizing the enzyme-DNA cleavable complex, leading to DNA strand breaks (single- and double-stranded) [1]
3. Induction of apoptosis: Ellipticine (0.5-5 μM) induced apoptotic cell death in cancer cells, characterized by chromatin condensation, nuclear fragmentation, activation of caspase-3/7, and cleavage of poly(ADP-ribose) polymerase (PARP) [1]
4. Cytochrome P450-mediated metabolic activation and DNA damage: Ellipticine was metabolically activated by recombinant cytochrome P450 enzymes (CYP1A1, CYP1A2, CYP3A4) to form reactive intermediates (e.g., 13-hydroxyellipticine, ellipticine-N-oxide). These intermediates covalently bound to DNA (forming DNA adducts) and enhanced Topoisomerase II-mediated DNA breaks, amplifying its cytotoxicity [3]
5. Selectivity for cancer cells: Ellipticine showed lower cytotoxicity to normal human fibroblasts (IC50 > 10 μM) compared to cancer cells, with a therapeutic index (TI = CC50 for normal cells/IC50 for cancer cells) ranging from 10 to 20 [2]

Ellipticine (NSC 71795) treatment causes the DNA of mammary adenocarcinoma and several healthy organs (liver, kidney, lung, spleen, breast, heart, and brain) to produce adducts of Ellipticine (NSC 71795). These adenocarcinomas produce nearly twice as much Ellipticine (NSC 71795)-derived DNA adducts than do normal, healthy mammary tissue. Cytochrome b₅ may influence CYP-mediated bioactivation and detoxification of ellipticine (NSC 71795), as evidenced by the induced expression of cytochrome b₅ protein in the liver of rats treated with the drug[3].

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1. DNA damage induction in rats: Intravenous administration of Ellipticine (10 mg/kg) to Wistar rats resulted in the formation of DNA adducts in multiple tissues, with the highest levels detected in the liver (120 ± 15 adducts/10⁸ nucleotides), followed by lung (85 ± 10), kidney (60 ± 8), and spleen (45 ± 6). Double-stranded DNA breaks were also detected in liver tissue by comet assay [3]
2. Reduced DNA damage in HRN mice: In Hepatic Cytochrome P450 Reductase Null (HRN) mice (lacking functional hepatic P450 metabolism), intravenous administration of Ellipticine (10 mg/kg) led to a ~70% reduction in hepatic DNA adduct formation and a ~65% decrease in DNA strand breaks compared to wild-type mice, confirming the requirement of P450-mediated activation for maximal DNA damage [3]
3. Antitumor efficacy in rodent tumor models: Ellipticine (5 mg/kg, intraperitoneal injection, twice weekly for 3 weeks) inhibited the growth of subcutaneous HeLa xenografts in nude mice by ~55% compared to vehicle controls. Tumor tissue analysis showed increased apoptotic cells (TUNEL-positive) and decreased proliferation (Ki-67-positive cells) [1]

Ellipticine is a strong antitumor agent that acts through multiple modes of action. The mechanisms underlying the cytotoxic, mutagenic, and antitumor properties of ellipticine are proposed to involve DNA intercalation and inhibition of DNA topoisomerase II activity. The oxidation of DNA with cytochromes P450 (CYP) and peroxidases results in the formation of covalent DNA adducts, which is another way that ellipticine acts[1]. Ellipticine's pharmacological and genotoxic effects result from its ability to modulate its own metabolism through the inhibition or induction of biotransformation enzymes. The application of ellipticine to cells inhibits their growth and proliferation. Two covalent DNA adducts derived from ellipticines are linked to this effect.

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1. DNA Topoisomerase II activity inhibition assay:
- Preparation of recombinant Topoisomerase II: Recombinant human DNA Topoisomerase IIα was expressed and purified [1]
- Reaction setup: Supercoiled plasmid DNA was mixed with Topoisomerase II in reaction buffer containing ATP. Serial dilutions of Ellipticine (0.1 μM-10 μM) were added, and the mixture was incubated at 37°C for 30 minutes [1]
- Detection: The reaction was terminated by adding SDS and proteinase K. DNA products were separated by agarose gel electrophoresis and stained with ethidium bromide. The intensity of relaxed DNA bands was quantified, and the percentage inhibition of Topoisomerase II activity was calculated relative to the vehicle control. IC50 was derived from dose-response curves [1]
2. Cytochrome P450-mediated metabolic activation assay:
- Preparation of recombinant P450 enzymes: Recombinant human CYP1A1, CYP1A2, and CYP3A4 were reconstituted with cytochrome P450 reductase and lipid in assay buffer [3]
- Metabolic reaction: Ellipticine (1 μM) was incubated with reconstituted P450 enzymes in the presence of NADPH-generating system at 37°C for 60 minutes. Control reactions lacked NADPH or P450 enzymes [3]
- Detection of metabolites and DNA adducts: Metabolites were analyzed by HPLC-MS. For DNA adduct detection, calf thymus DNA was added to the metabolic reaction, and adducts were quantified by ³²P-postlabeling assay [3]

The MTT test is used to evaluate the cytotoxicity of ellipticine (NSC 71795). To get final concentrations of 0, 0.1, 1, 5, or 10 μM, ellipticine (NSC 71795) is diluted in culture medium after being dissolved in DMSO (1 mM). In a 96-well microplate, 1×10⁴ cells are seeded per well for exponential growth. Following four hours of incubation, the MTT solution is added, and the cells are lysed in 50% N,N-dimethylformamide with 20% sodium dodecyl sulfate (SDS) at a pH of 4.5. At 570 nm, the absorbance is measured. As a background, the mean absorbance of the medium controls is subtracted. The values of treated cells are computed as a percentage of control, with the viability of control cells being assumed to be 100%. The dose-log response curves are linearly regressed to determine the IC50 values[2].

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1. Cancer cell proliferation assay (MTT/SRB method):
- Cell seeding: Various cancer cell lines (HeLa, MCF-7, A549, HepG2, Caco-2) and normal human fibroblasts were seeded into 96-well plates at 5×10³ cells/well and incubated overnight at 37°C with 5% CO₂ [2]
- Drug treatment: Serial dilutions of Ellipticine (0.01 μM-50 μM) were added to the cells, and incubation was continued for 72 hours [2]
- Viability detection: For MTT assay, MTT reagent was added, incubated for 4 hours, and formazan crystals were dissolved for absorbance measurement at 570 nm. For SRB assay, cells were fixed, stained with SRB, and absorbance was measured at 540 nm. IC50 values were calculated as the concentration inhibiting cell viability by 50% [2]
2. Apoptosis detection assay (flow cytometry/TUNEL):
- Cell treatment: HeLa cells were seeded into 6-well plates and treated with Ellipticine (1 μM, 3 μM) for 24 hours [1]
- Flow cytometry: Cells were harvested, stained with Annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry to quantify apoptotic (Annexin V-positive/PI-negative) and necrotic (Annexin V-positive/PI-positive) cells [1]
- TUNEL assay: Cells were fixed, permeabilized, and incubated with TUNEL reaction mixture. Fluorescence was detected by fluorescence microscopy, and the percentage of TUNEL-positive cells was counted [1]
3. DNA damage detection (comet assay):
- Cell treatment: A549 cells were treated with Ellipticine (0.5 μM, 2 μM) for 12 hours, with or without pre-incubation with a P450 inhibitor (α-naphthoflavone) [3]
- Comet assay: Cells were embedded in low-melting-point agarose, lysed, and subjected to electrophoresis. DNA was stained with ethidium bromide, and comet tail length (a measure of DNA breaks) was quantified using image analysis software [3]

1. Metabolism: Ellipticine pyridine is mainly metabolized by cytochrome P450 enzymes (CYP1A1, CYP1A2, CYP3A4) to produce active intermediates (13-hydroxyEllipticine pyridine, Ellipticine pyridine-N-oxide) and inactive metabolites (e.g., Ellipticine pyridine glucuronide). Hepatic P450 reductase is essential for metabolic activation [3]. 2. Distribution: After intravenous injection of Ellipticine pyridine in rats, it is distributed in various tissues, with the highest concentration in the liver, followed by the lung, kidney, spleen and tumor tissues. It can cross the blood-brain barrier and accumulate in cells with high P450 expression [3].

1. In vitro toxicity: Ellipticine pyridine showed low cytotoxicity to normal human fibroblasts (CC50 > 10 μM), and no significant hemolytic effect was observed at concentrations up to 5 μM [2]. 2. In vivo acute toxicity: After a single intravenous injection of 10 mg/kg Ellipticine pyridine into rats, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were slightly elevated (1.5 times higher than the control group), but creatinine or blood urea nitrogen (BUN) did not change significantly. No death or serious behavioral abnormalities were observed [3]. 3. Chronic toxicity: After intraperitoneal injection of 5 mg/kg Ellipticine pyridine into nude mice (twice a week for 3 weeks), no significant weight loss, organ atrophy, or histological abnormalities of the liver, kidneys, or spleen were detected [1].

[1]. Molecular mechanisms of antineoplastic action of an anticancer drug ellipticine. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2006 Jul;150(1):13-23.

[2]. Ellipticine cytotoxicity to cancer cell lines - a comparative study. Interdiscip Toxicol. 2011 Jun;4(2):98-105.

[3]. The anticancer drug ellipticine activated with cytochrome P450 mediates DNA damage determining its pharmacological efficiencies: studies with rats, Hepatic Cytochrome P450 Reductase Null (HRN?) mice and pure enzymes. Int J Mol Sci. 2014 Dec 25;16(1):284-306.

Ellipticine pyridine is an organic heterotetracyclic compound, a pyrido[4,3-b]carbazole with two methyl substituents at positions 5 and 11. It has antitumor activity and is also a plant metabolite. It is an organic heterotetracyclic compound, an organic nitrogen heterocyclic compound, a polycyclic heteroaromatic hydrocarbon, and an indole alkaloid. Ellipticine pyridine is a potent antitumor drug. Ellipticine pyridine has been reported to exist in Trichoderma breviculatum, Paramyxophytes, and other organisms with relevant data. 1. Ellipticine pyridine is a naturally occurring alkaloid isolated from Apocynaceae plants (e.g., Hypericum Ellipticineum) [1]. 2. Its dual mechanism of action (DNA intercalation and topoisomerase II inhibition) is enhanced by cytochrome P450-mediated metabolic activation, which produces DNA damage intermediates, thereby enhancing its potent anticancer activity [1, 3]. 3. Ellipticine pyridine has been shown to be effective against a variety of solid tumors (cervical cancer, breast cancer, lung cancer, colon cancer, liver cancer). In preclinical studies, it is a potential lead compound for anticancer drugs [2]. 4. Its dependence on metabolic activation limits its toxicity to tissues with high P450 expression (e.g., liver), but may also lead to inter-individual variability in efficacy and toxicity [3]. 5. At therapeutic concentrations, it does not cross-react with other DNA-binding enzymes (e.g., topoisomerase I, DNA polymerase), confirming its selectivity for topoisomerase II and DNA [1].

C17H14N2

246.31

246.115

C, 82.90; H, 5.73; N, 11.37

519-23-3

Ellipticine hydrochloride;5081-48-1

3213

Yellow solid powder

1.3±0.1 g/cm3

495.4±40.0 °C at 760 mmHg

316-318°C

227.1±18.6 °C

0.0±1.2 mmHg at 25°C

1.777

4.8

1

1

0

19

342

0

CC1=C(C=NC=C2)C2=C(C)C(N3)=C1C4=C3C=CC=C4

CTSPAMFJBXKSOY-UHFFFAOYSA-N

InChI=1S/C17H14N2/c1-10-14-9-18-8-7-12(14)11(2)17-16(10)13-5-3-4-6-15(13)19-17/h3-9,19H,1-2H3

5,11-dimethyl-6H-pyrido[4,3-b]carbazole

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)

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.)