Cuproptosis

The potent anticancer drug elesclomol, which forms an extremely strong complex with copper, is currently undergoing clinical trials. However, its mechanism of action is not well understood. Treatment of human erythroleukemic K562 cells with either elesclomol or Cu(II)-elesclomol caused an immediate halt in cell growth which was followed by a loss of cell viability after several hours. Treatment of K562 cells also resulted in induction of apoptosis as measured by annexin V binding. Elesclomol or Cu(II)-elesclomol treatment caused a G1 cell cycle block in synchronized Chinese hamster ovary cells. Elesclomol and Cu(II)-elesclomol induced DNA double strand breaks in K562 cells, suggesting that they may also have exerted their cytotoxicity by damaging DNA. Cu(II)-elesclomol also weakly inhibited DNA topoisomerase I (5.99.1.2) but was not active against DNA topoisomerase IIα (5.99.1.3). Elesclomol or Cu(II)-elesclomol treatment had little effect on the mitochondrial membrane potential of viable K562 cells. NCI COMPARE analysis showed that Cu(II)-elesclomol exerted its cytotoxicity by mechanisms similar to other cytotoxic copper chelating compounds. Experiments with cross-resistant cell lines overexpressing several ATP-binding cassette (ABC) type efflux transporters showed that neither elesclomol nor Cu(II)-elesclomol were cross-resistant to cells overexpressing either ABCB1 (Pgp) or ABCG2 (BCRP), but that cells overexpressing ABCC1 (MRP1) were slightly cross-resistant. In conclusion, these results showed that elesclomol caused a rapid halt in cell growth, induced apoptosis, and may also have inhibited cell growth, in part, through its ability to damage DNA[1].

Inhibition of topoisomerase I DNA relaxation assay and topoisomerase IIα kDNA decatenation and cleavage assays[1]
A gel assay as described was used to determine if elesclomol or Cu(II)–elesclomol inhibited topoisomerase I. The pBR322 DNA was from MBI Fermentas and the topoisomerase I was from TopoGEN. The topoisomerase I inhibitor camptothecin (20 μM) was used as a positive control. A gel assay as we previously described was used to determine if elesclomol or Cu(II)–elesclomol inhibited the catalytic decatenation activity of topoisomerase IIα. kDNA, which consists of highly catenated networks of circular DNA, is decatenated by topoisomerase IIα in an ATP-dependent reaction to yield individual minicircles of DNA. Topoisomerase II-cleaved DNA covalent complexes produced by anticancer drugs may be trapped by rapidly denaturing the complexed enzyme with sodium dodecyl sulfate (SDS). The drug-induced cleavage of closed circular plasmid pBR322 DNA to form linear DNA at 37 °C was followed by separating the SDS-treated reaction products by ethidium bromide gel electrophoresis, essentially as described, except that all components of the assay mixture were assembled and mixed on ice prior to addition of the drug

Cell growth and viability, cell cycle synchronization, cell cycle analysis and annexin V flow cytometry[1]
Total cell density and viability using a trypan blue assay were determined on a Bio-Rad TC10™ automated cell counter. In these experiments erythroleukemic K562 cells at a density of 50,000 cells/ml in 96-well plates were continuously treated with 200 nM elesclomol or Cu(II)–elesclomol for various times. The cell cycle synchronization experiments were carried out as we previously described. A measure of cells that are necrotic is also obtained since necrotic cell membranes are permeable to propidium iodide yielding high red fluorescence. For the synchronization experiments Chinese hamster ovary (CHO) cells were grown to confluence in α-MEM supplemented with 10% fetal calf serum. Following serum starvation with α-MEM-0% fetal calf serum for 48 h, the cells that were seeded at 2 × l04 cells/ml, were repleted with α-MEM-10% fetal calf serum. Directly after repletion they were continuously treated with DMSO vehicle control or 50 nM of either elesclomol or Cu(II)–elesclomol in 35-mm diameter dishes for different periods of time. Cells were fixed in 75% ethanol and stained with a solution containing 20 μg/ml propidium iodide, 100 μg/ml RNase A in 0.1% (v/v) Triton X-100 at room temperature for 30 min. Flow cytometry was carried out on a BD FACSCanto™ II flow cytometry system and analyzed with FlowJo software for the proportion of cells in sub-G0/G1, G0/G1, S, and G2/M phases of the cell cycle.[1]
The fraction of apoptotic cells induced by treatment of K562 cells with elesclomol and Cu(II)–elesclomol were quantified by two-color flow cytometry by simultaneously measuring integrated green (annexin V-FITC) fluorescence, and integrated red (propidium iodide) fluorescence as we previously described. The annexin V-FITC binding to phosphatidylserine present on the outer cell membrane was determined using an Apoptosis Detection Kit. Briefly, K562 cells in suspension were untreated or treated with elesclomol or Cu(II)–elesclomol at the concentrations indicated at 37 °C for 5 h. The cells were collected by centrifugation at 1000 × g for 3 min and the pooled cells were washed with the manufacturer-supplied binding buffer. Approximately 2.5 × 105 cells were resuspended in 500 μl of manufacturer-supplied binding buffer, and mixed with 5 μl of annexin V-FITC and 5 μl of propidium iodide at a final concentration of 1 μg/ml. After 15 min of incubation in the dark, the cells were analyzed using flow cytometry. [1]

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Measurement of mitochondrial membrane potential[1]
K562 cells were treated with various concentrations of elesclomol or Cu(II)–elesclomol. The mitochondrial membrane potential sensing dye JC-1 was then loaded into suspended K562 cells (200,000 cells/well in 96-well black plates) by incubating cells with 8 μM JC-1 in Hank's buffer (pH 7.4 with 1.3/0.8 mM Ca2+/Mg2+) at 37 °C for 20 min as we previously described. The cells were then gently washed with Hank's buffer. The average ratio of the red fluorescence (λEx 544 nm, λEm 590 nm) to the green fluorescence (λEx 485 nm, λEm 520 nm), which is a measure of the mitochondrial membrane potential, was determined for cells treated for 6 h with various concentrations of elesclomol or Cu(II)–elesclomol on a BMG Fluostar Galaxy fluorescence plate reader. The ionophore valinomycin (1 μM), which depolarizes the mitochondrial membrane, and doxorubicin (1.6 μM) were used as positive controls as we previously described.

[1]. Cellular mechanisms of the cytotoxicity of the anticancer drug elesclomol and its complex with Cu(II). hem Pharmacol. 2015 Feb 1;93(3):266-76.

Elesclomol is a highly novel anticancer drug that has completed phase 3 clinical trials for patients with advanced melanoma and is currently undergoing Phase 1 and 2 trials for the treatment of a variety of other cancers (http://www.clinicaltrials.gov). Elesclomol and Cu(II)–elesclomol (Fig. 1) are both extremely potent in vitro and typically inhibit cancer cell growth at low nanomolar concentrations. It has been proposed that elesclomol is cytotoxic through the induction of oxidative stress that is mediated through its Cu2+ complex. The development of copper complexes as anticancer agents has recently been reviewed. A recent report using an HClO-specific fluorescent probe has shown that elesclomol can induce formation of the highly reactive and strongly oxidizing HClO in breast cancer MCF7 cells. However, it is not known whether this is a direct or an indirect effect. Elesclomol strongly binds both Cu2+ and Cu+. Elesclomol can scavenge copper from the culture medium and selectively transport it to the mitochondria where it induces oxidative stress. It has also been shown that the elesclomol was subsequently effluxed from the cell after it had transported copper into the cell, and was then free to shuttle more copper into the cell. MCF7 cells with a compromised ability to repair oxidative DNA damage have increased sensitivity to elesclomol, which suggests that elesclomol may also exert some of its cytotoxicity through DNA-damaging mechanisms. Interestingly, it has been shown that elesclomol-treated patients with normal serum lactate dehydrogenase levels had improved outcomes compared to patients with high lactate dehydrogenase levels. Yeast gene deletion mutant studies suggested that elesclomol does not work through a specific cellular protein target and is unlike any other currently approved anticancer drugs [1].

C19H18CUN4O2S2

462.047822475433

461.016

C, 49.39; H, 3.93; Cu, 13.75; N, 12.13; O, 6.93; S, 13.88

1224195-72-5

488832-69-5 (Elesclomol)

155813013

Brown to reddish brown solid

0

6

2

28

516

0

UUSCWIGUMPDFDE-UHFFFAOYSA-L

InChI=1S/C19H20N4O2S2.Cu/c1-22(18(26)14-9-5-3-6-10-14)20-16(24)13-17(25)21-23(2)19(27)15-11-7-4-8-12-15;/h3-12H,13H2,1-2H3,(H2,20,21,24,25);/q;+2/p-2

copper;[benzenecarbonothioyl(methyl)amino]-[3-[benzenecarbonothioyl(methyl)amino]azanidyl-3-oxopropanoyl]azanide

NSC766922; NSC-766922; Cu(II) Elesclomol; Cu(II)Elesclomol

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.)