P450; peroxidase; Topo 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]; no Ki/EC50 values provided) [1]

Ellipticine is a prodrug that damages DNA and is first recognized as a natural product. Its pharmacological efficacy and genotoxic side effects are determined by its activation with cytochrome P450 (CYP®). With several modes of action, including DNA intercalation and inhibition of DNA topoisomerase II, ellipticine is a highly effective antitumor agent. In addition to its pharmacological and genotoxic effects, ellipticine can also be used as an inducer or inhibitor of biotransformation enzymes, which can alter its own metabolism. Ellipsticine treatment inhibited the growth and proliferation of every tested cell. This effect was linked to the formation of two covalent ellipticine-derived DNA adducts in MCF-7, HL-60, CCRF-CEM, UKF-NB-3, UKF-NB-4, and U87MG cells, but not in neuroblastoma UKF-NB-3 cells. These adducts were identical to those formed by 13-hydroxy- and 12-hydroxyellipticine, the ellipticine metabolites generated by CYP and peroxidase enzymes. Consequently, the majority of cancer cell lines examined in this comparative study may be more sensitive to ellipticine treatment due to DNA adduct formation, while other ellipticine action mechanisms may also play a role in the drug's cytotoxicity against neuroblastoma UKF-NB-3 cells.

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1. Broad-spectrum antiproliferative activity: Ellipticine HCl 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 HCl 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 HCl (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 HCl 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 HCl 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 treatment causes the DNA of mammary adenocarcinoma and several healthy organs (liver, kidney, lung, spleen, breast, heart, and brain) to produce ellipticine-derived DNA adducts. Compared to normal, healthy mammary tissue, these adenocarcinomas produce nearly twice as many ellipticine-derived DNA adducts. Rats given ellipticine showed increased expression of the cytochrome b5 protein in their livers, indicating that cytochrome b5 might influence the CYP-mediated bioactivation and detoxification of ellipticine.

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1. DNA damage induction in rats: Intravenous administration of Ellipticine HCl (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 HCl (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 HCl (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 HCl (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 HCl (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 HCl (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 HCl (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 HCl (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: Elliptic pyridine hydrochloride is mainly metabolized by cytochrome P450 enzymes (CYP1A1, CYP1A2, CYP3A4) to produce active intermediates (13-hydroxyelliptic pyridine, elliptic pyridine-N-oxide) and inactive metabolites (e.g., elliptic pyridine glucuronide). Hepatic P450 reductase is essential for metabolic activation [3]. 2. Distribution: After intravenous injection of elliptic pyridine hydrochloride 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 HCl 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: A single intravenous injection of 10 mg/kg Ellipticine HCl in rats caused a slight increase in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (1.5 times higher than the control group), but no significant changes in creatinine or blood urea nitrogen (BUN). No death or serious behavioral abnormalities were observed [3]. 3. Chronic toxicity: Treatment of nude mice with 5 mg/kg ellipticine hydrochloride (intraperitoneal injection, twice a week for 3 weeks) did not result in significant weight loss, organ atrophy, or histological abnormalities of the liver, kidneys, or spleen [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.

1. Ellipticine HCl is the hydrochloride form of ellipticine, a natural alkaloid isolated from Apocynaceae plants (e.g., Ochrosia elliptica)[1]. 2. Its dual mechanism of action (DNA intercalation and topoisomerase II inhibition) is enhanced by cytochrome P450-mediated metabolic activation, which generates DNA damage intermediates, thereby enhancing its anticancer activity[1, 3]. 3. Preclinical studies have shown that ellipticine HCl is effective against a variety of solid tumors (cervical cancer, breast cancer, lung cancer, colon cancer, and liver cancer), making it a potential lead compound for anticancer drug development[2]. 4. The dependence on metabolic activation limits its toxicity to P450-highly expressed tissues (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 (such as topoisomerase I and DNA polymerase), confirming its selectivity for topoisomerase II and DNA [1]

C₁₇H₁₅CLN₂

282.77

282.092

C, 72.21; H, 5.35; Cl, 12.54; N, 9.91

5081-48-1

Ellipticine;519-23-3

169532

Light yellow to yellow solid powder

5.288

2

1

0

20

342

0

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

VSBNVARERCGCEF-UHFFFAOYSA-N

InChI=1S/C17H14N2.ClH/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;1H

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

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: ≥ 0.84 mg/mL (2.97 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 8.4 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 0.84 mg/mL (2.97 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 8.4 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 3: ≥ 0.84 mg/mL (2.97 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 8.4 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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