DNA alkylating agent (specifically chloroethylates the O⁶-position of guanine in DNA, leading to the formation of interstrand crosslinks (ICLs)). [1]

Treatment of LN-229 glioblastoma cells with 50 µM Nimustine HCl induced apoptosis in a time-dependent manner. Apoptosis began 72 hours after treatment and reached approximately 55% by 120 hours. This was accompanied by cleavage of caspase-8, -9, and -3, and phosphorylation of H2AX (γH2AX), indicating DNA damage response activation. [1]
Nimustine HCl (50 µM) induced strong phosphorylation (activation) of JNK and p38K in LN-229 cells, peaking 24-72 hours post-treatment, and also enhanced phosphorylation of ERK1/2. This led to increased AP-1 binding activity and phosphorylation of its component c-Jun. [1]
Pharmacological inhibition of JNK (using SP600125) or siRNA-mediated knockdown of JNK or c-Jun significantly protected LN-229 cells from Nimustine HCl-induced apoptosis. In contrast, knockdown of c-Fos slightly sensitized cells to Nimustine HCl. [1]
Nimustine HCl treatment upregulated the mRNA expression of the pro-apoptotic gene bim in LN-229 cells. This induction was abrogated by JNK inhibition or knockdown. Protein level induction of BIM was also confirmed. EMSA and supershift assays demonstrated that c-Jun, but not c-Fos, binds to the AP-1 site in the BIM promoter following Nimustine HCl treatment. [1]
Transient downregulation of BIM via shRNA in LN-229 and U87MG glioblastoma cells resulted in significantly reduced apoptosis and attenuated cleavage of caspase-9 following treatment with Nimustine HCl (50 µM). [1]
In p53 wild-type LN-229 cells, pharmacological inhibition or genetic knockdown of p53 enhanced the cytotoxic potential of Nimustine HCl, which was associated with p53-mediated induction of nucleotide excision repair (NER) genes xpc and ddb2. [1]
Knockdown of c-Fos impaired the restoration of XPF mRNA and protein levels after Nimustine HCl treatment in LN-229 cells, leading to increased sensitivity. Overexpression of c-Fos maintained XPF levels and conferred resistance. [1]
JNK inhibition did not affect the formation or repair of Nimustine HCl-induced DNA interstrand crosslinks, as measured by a modified alkaline comet assay, nor did it affect γH2AX phosphorylation dynamics. [1]

An electrophoretic mobility shift assay (EMSA) was performed to assess AP-1 transcription factor binding activity. Nuclear extracts were prepared from LN-229 cells treated with or without Nimustine HCl (50 µM). The extracts were incubated with radioactively labeled oligonucleotides containing the AP-1 binding site from the collagenase (mmp1) promoter or the BIM promoter. Protein-DNA complexes were separated by non-denaturing gel electrophoresis and visualized. For supershift assays, specific antibodies against c-Jun or c-Fos were added to the binding reaction prior to electrophoresis to identify the components of the AP-1 complex bound to the DNA. [1]

Cells (LN-229, U87MG) were cultured in Dulbecco's minimal essential medium supplemented with fetal bovine serum at 37°C with 7% CO₂. [1]
For apoptosis and cell cycle analysis, cells were treated with Nimustine HCl (typically 50 µM). At indicated times, cells were fixed, treated with RNase, stained with propidium iodide (PI), and analyzed by flow cytometry to determine the sub-G1 fraction (apoptotic cells). [1]
For protein analysis, cells were treated with Nimustine HCl (50 µM). At various time points, whole cell or nuclear extracts were prepared. Proteins were separated by SDS-PAGE, transferred to membranes, and detected using specific primary antibodies (e.g., against caspases, γH2AX, JNK, p-JNK, c-Jun, p-c-Jun, BIM, XPF, FasL) and appropriate secondary antibodies. [1]
For mRNA analysis, total RNA was isolated from treated cells. cDNA was synthesized and endpoint PCR was performed using gene-specific primers (e.g., for bim, fasL, xpf, gapdh). [1]
For kinase inhibition studies, cells were pre-incubated for 1 hour with specific inhibitors (e.g., SP600125 for JNK, SB203580 for p38K, U0126 for MEK1/2) prior to the addition of Nimustine HCl. Inhibitors remained in the medium until cell harvest. [1]
For siRNA-mediated knockdown, cells were transfected with specific siRNAs (e.g., targeting JNK, c-Jun, c-Fos, ERK1/2) or non-silencing control RNA using a transfection reagent. Knockdown efficiency was verified by western blot or RT-PCR. Drug treatment was initiated after a specified period post-transfection. [1]
For BIM knockdown, cells were transiently transfected with plasmids expressing BIM-specific shRNA or non-silencing control shRNA. Downregulation was verified by RT-PCR, and cells were subsequently treated with Nimustine HCl. [1]
A modified alkaline comet assay was used to measure DNA interstrand crosslink (ICL) formation and repair. Cells were treated with Nimustine HCl (50 µM), and at indicated times, they were trypsinized, irradiated with 8 Gy, and immediately subjected to alkaline lysis and electrophoresis under specific conditions to detect ICLs as a reduction in DNA migration. [1]

[1]. Apoptosis induced by temozolomide and nimustine in glioblastoma cells is supported by JNK/c-Jun-mediated induction of the BH3-only protein BIM. Oncotarget. 2015 Oct 20;6(32):33755-68.

[2]. FANCD1/BRCA2 plays predominant role in the repair of DNA damage induced by ACNU or TMZ. PLoS One. 2011 May 9;6(5):e19659.

[3]. DNA lability induced by nimustine and ramustine in rat glioma cells. J Neurol Neurosurg Psychiatry. 1988 Nov;51(11):1391-4.

Nimustine hydrochloride is the hydrochloride salt of nimustine, prepared by reacting nimustine with an equimolar amount of hydrochloric acid. It is an antitumor drug, particularly effective against malignant brain tumors. It possesses antitumor activity. It contains nimustine (1+). Nimustine hydrochloride is the hydrochloride salt of nimustine, a nitrosourea drug with antitumor activity. Nimustine can alkylate and cross-link DNA, leading to DNA fragmentation, inhibition of protein synthesis, and cell death. It is an antitumor drug, particularly effective against malignant brain tumors. Resistance to the initial efficacy of this drug in brain tumor cells can be partially overcome by concurrent use of membrane modifiers (such as reserpine), calcium channel blockers (such as nicardipine or verapamil), or calmodulin inhibitors (such as trifluoperazine). This drug has also been used in combination with other antitumor drugs or radiotherapy to treat various tumors. Nimustine hydrochloride (ACNU) is a chloroethylated nitrosourea drug used as a second-line chemotherapy agent, including for the treatment of malignant gliomas. It is also used to treat malignant melanoma, gastrointestinal cancer and pancreatic cancer, as well as Hodgkin lymphoma and non-Hodgkin lymphoma. [1] Its main mechanism of action involves the chloroethylation of guanine at the O⁶ position in DNA to form O⁶-chloroethylguanine. This damage leads to rearrangement, forming interchain crosslinks (ICLs) between guanine and cytosine, which are highly cytotoxic. [1] Cellular resistance to nimustine hydrochloride can be mediated by the DNA repair protein O⁶-methylguanine-DNA methyltransferase (MGMT) and the nucleotide excision repair (NER) pathway. p53 can promote resistance by upregulating NER factors such as XPC and DDB2. [1]
In glioma cells, nimustine hydrochloride triggers a late apoptosis response mediated by the JNK/c-Jun MAPK pathway. This pathway activates transcriptional induction of the AP-1-dependent pro-apoptotic BH3 domain protein BIM, a key effector of cell death. [1]
The transcription factor c-Fos can promote DNA damage repair by stimulating the resynthesis of the NER endonuclease XPF, thereby exerting a protective effect against nimustine hydrochloride. [1]

C9H14CL2N6O2

309.1525

308.055

C, 34.97; H, 4.56; Cl, 22.93; N, 27.18; O, 10.35

55661-38-6

Nimustine;42471-28-3

91657

White to off-white solid powder

186 °C

2.573

3

6

4

19

292

0

ClC([H])([H])C([H])([H])N(C(N([H])C([H])([H])C1=C([H])N=C(C([H])([H])[H])N=C1N([H])[H])=O)N=O.Cl[H]

KPMKNHGAPDCYLP-UHFFFAOYSA-N

InChI=1S/C9H13ClN6O2.ClH/c1-6-12-4-7(8(11)14-6)5-13-9(17)16(15-18)3-2-10;/h4H,2-3,5H2,1H3,(H,13,17)(H2,11,12,14);1H

3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-1-(2-chloroethyl)-1-nitrosourea;hydrochloride

Nimustine HCl; Nimustine hydrochloride; ACNU; NSC-245382; NSC245382; NSC 245382

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)

DMSO: 61~62.5 mg/mL (197.3~202.2 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.73 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 20.8 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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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.73 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 20.8 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: ≥ 2.08 mg/mL (6.73 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 20.8 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.)