For research use only.

Catalog No.S1210 Synonyms: NCI-C04671

33 publications

Methotrexate Chemical Structure

Molecular Weight(MW): 454.44

Methotrexate (MTX, NCI-C04671), analog of folic acid, is a nonspecific inhibitor of the dihydrofolate reductase(DHFR) of bacteria and cancerous cells as well as normal cells. It forms an inactive ternary complex with DHFR and NADPH. Methotrexate (MTX) induces apoptosis.

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Selleck's Methotrexate has been cited by 33 publications

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Biological Activity

Description Methotrexate (MTX, NCI-C04671), analog of folic acid, is a nonspecific inhibitor of the dihydrofolate reductase(DHFR) of bacteria and cancerous cells as well as normal cells. It forms an inactive ternary complex with DHFR and NADPH. Methotrexate (MTX) induces apoptosis.
hDHFR [4]
(Activated peripheral T cells)
24 nM
In vitro

Methotrexate (0.1-10 mM) induces apoptosis of in vitro activated T cells from human peripheral blood. Methotrexate achieves clonal deletion of activated T cells in mixed lymphocyte reactions. Methotrexate can selectively delete activated peripheral blood T cells by a CD95-independent pathway. [1] Methotrexate is taken up by cells via the reduced folate carrier and then is converted within the cells to polyglutamates. Methotrexate leads to diminished production of leukotriene B4 by neutrophils stimulated ex vivo. Methotrexate polyglutamates inhibit the enzyme aminoimidazolecarboxamidoadenosineribonucleotide (AICAR) transformylase more potently than the other enzymes involved in purine biosynthesis. Methotrexate is also known to suppress TNF activity by suppressing TNF-induced nuclear factor-κB activation in vitro, in part related to a reduction in the degradation and inactivation of an inhibitor of this factor, IκBα, and probably related to the release of adenosine. Methotrexate suppresses the production of both TNF and IFN-γ by T-cell-receptor-primed T lymphocytes from both healthy human donors and RA patients. Methotrexate treatment is associated with a significant decrease of TNF-α-positive CD4+ T cells, while the number of T cells expressing the anti-inflammatory cytokine IL-10 increased. [2]

Cell Data
Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
P388D1 NGrST3pkgXSxdH;4bYNqfHliYYPzZZk> MojRTWM2OD12Lkigcm0> Mn25PFY{OjRzMx?=
L cells NELaNVNkgXSxdH;4bYNqfHliYYPzZZk> MYTJR|UxRThwMzDuUS=> M2roZlg3OzJ2MUO=
143B(TK-) MnnXZ5l1d3SxeHnjbZR6KGG|c3H5 M4TybWlEPTB;OD64JI5O NVfKOGJbQDZ|MkSxNy=>
U87MG NWTvUFl3[3m2b4TvfIlkcXS7IHHzd4F6 NF;2cWlKSzVyPUKyJI5O MmjkPFY{OjRzMx?=
A549 NUCyeokz[3m2b4TvfIlkcXS7IHHzd4F6 M3W1b2lEPTB;M{Ggcm0> NGP6bIE5PjN{NEGz
H460 MkjPZ5l1d3SxeHnjbZR6KGG|c3H5 MWrJR|UxRTlwNTDuUS=> NYHNZYh1QDZ|MkSxNy=>
Daoy NX\VNVBl[3m2b4TvfIlkcXS7IHHzd4F6 MnriTWM2OD17IH7N M4nCXFg3OzJ2MUO=
U373MG MmP3Z5l1d3SxeHnjbZR6KGG|c3H5 MlPQTWM2OD1zMjDuUS=> NEPkNmM5PjN{NEGz
Vero NWT4[Y51[3m2b4TvfIlkcXS7IHHzd4F6 MmKyTWM2OD17LkKgcm0> M3PWPVg3OzJ2MUO=
SCC25  NH\LRVdIem:5dHigbY5pcWKrdH;yfUBie3OjeR?= NU\0[5BUhjFizszN MVTJR|UxRTJ5IH7N NXjGdJRUQTB{Mke5OS=>
NCI-H460 M1W1dmdzd3e2aDDpcohq[mm2b4L5JIF{e2G7 NGfIVWt,OSEQvF2= MmfBTWM2OD1{ODDuUS=> NHPtc3E6ODJ{N{m1
NCI-H23 M1zH[2dzd3e2aDDpcohq[mm2b4L5JIF{e2G7 M1j5SJ4yKM7:TR?= NH3QcXlKSzVyPUSzJI5O MVm5NFIzPzl3
NCI-H522 NV7xO452T3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= MkLkglEh|ryP M3TyS2lEPTB;MkK5JI5O Mm\NPVAzOjd7NR?=
EKVX M{ixfWdzd3e2aDDpcohq[mm2b4L5JIF{e2G7 MkDVglEh|ryP NXPjc|JvUUN3ME6xNFAxKG6P MXS5NFIzPzl3
HCT-116 M2Pybmdzd3e2aDDpcohq[mm2b4L5JIF{e2G7 MV3+NUDPxE1? MoTmTWM2OD1|MDDuUS=> MljDPVAzOjd7NR?=
HCT-15 M1;SPWdzd3e2aDDpcohq[mm2b4L5JIF{e2G7 NWD4Z5gyhjFizszN NIL2fJVKSzVyPUOwJI5O MmHwPVAzOjd7NR?=
HT29 Mof2S5Jwf3SqIHnubIljcXSxcomgZZN{[Xl? NETSdmJ,OSEQvF2= M3TpbWlEPTB;M{Kgcm0> NHziRlg6ODJ{N{m1
SW-620 MWDHdo94fGhiaX7obYJqfG:{eTDhd5NigQ>? MUH+NUDPxE1? MV3JR|UxRTN|IH7N NUDESFJvQTB{Mke5OS=>
KM12 NGHPUGNIem:5dHigbY5pcWKrdH;yfUBie3OjeR?= NVzsVItuhjFizszN MoX5TWM2OD12MjDuUS=> M2jXS|kxOjJ5OUW=
SF-539 NWjjSWp{T3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= NYTzdJQ{hjFizszN MnfETWM2OD1|NTDuUS=> NV3LbpBMQTB{Mke5OS=>
SF-268 NXXhTm8{T3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= NYLVbnhYhjFizszN NGraN5RKSzVyPUWyJI5O MXu5NFIzPzl3
SNB-75 NF\xco5Iem:5dHigbY5pcWKrdH;yfUBie3OjeR?= MorjglEh|ryP NVywcndVUUN3ME6xNFAxOCCwTR?= NE\xVm06ODJ{N{m1
LOX IMVI NEjIPIdIem:5dHigbY5pcWKrdH;yfUBie3OjeR?= MV\+NUDPxE1? M2TsPWlEPTB;Mk[gcm0> NXzOdldTQTB{Mke5OS=>
UACC-62 NFn0Wo9Iem:5dHigbY5pcWKrdH;yfUBie3OjeR?= MVj+NUDPxE1? M1LGfGlEPTB;Mkigcm0> NIfHVYk6ODJ{N{m1
SK-MEL-5 M{GzdGdzd3e2aDDpcohq[mm2b4L5JIF{e2G7 NVzpd3M4hjFizszN NV3i[YVtUUN3ME24O{BvVQ>? NWf0TGRrQTB{Mke5OS=>
MALME-3M MXvHdo94fGhiaX7obYJqfG:{eTDhd5NigQ>? NHLaVmF,OSEQvF2= M2\2bmlEPTB-MUCwNEBvVQ>? NWe5e|ZYQTB{Mke5OS=>
SK-MEL-28 M4HO[Gdzd3e2aDDpcohq[mm2b4L5JIF{e2G7 M4TIdJ4yKM7:TR?= MVzJR|UxRjFyMECgcm0> M{LIdVkxOjJ5OUW=
OVCAR-8 MVPHdo94fGhiaX7obYJqfG:{eTDhd5NigQ>? NGi1SnR,OSEQvF2= MUfJR|UxRTNzIH7N M3K1XlkxOjJ5OUW=
OVCAR-5 NUXacopWT3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= NUXm[II6hjFizszN M2TreWlEPTB-MUCwNEBvVQ>? MVy5NFIzPzl3
OVCAR-3 NUjCWIp{T3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= Mn6zglEh|ryP MorNTWM2OD1|OUigcm0> MXK5NFIzPzl3
786-0 M1i5NWdzd3e2aDDpcohq[mm2b4L5JIF{e2G7 M3yyWp4yKM7:TR?= MlXTTWM2OD1|MzDuUS=> MlK0PVAzOjd7NR?=
UO-31 NUezOZpLT3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= Mk\nglEh|ryP NVXSTohFUUN3ME2xPVEhdk1? NXzhSoF1QTB{Mke5OS=>
ACHN NHTsOGxIem:5dHigbY5pcWKrdH;yfUBie3OjeR?= MUH+NUDPxE1? MYfJR|UxRTRyIH7N NGK0b4o6ODJ{N{m1
MCF7 MWrHdo94fGhiaX7obYJqfG:{eTDhd5NigQ>? Mnq2glEh|ryP MnXmTWM2OD1|NjDuUS=> NXriU4VKQTB{Mke5OS=>
MCF7-ADR NXy4bXI3T3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= NYrnVoVvhjFizszN NEW3R|JKSzVyPUe4JI5O NUDWWpJUQTB{Mke5OS=>
PC-3 MV7Hdo94fGhiaX7obYJqfG:{eTDhd5NigQ>? M3rJbJ4yKM7:TR?= MXzJR|UxRTJibl2= MXW5NFIzPzl3
DU-145 NWTFVW9ET3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= NFrZdm1,OSEQvF2= MnPWTWM2OD1{MzDuUS=> NI\0OpQ6ODJ{N{m1
CCRF-CEM MWTHdo94fGhiaX7obYJqfG:{eTDhd5NigQ>? MlqzSWM2OD1zND60JI5O Ml75NVA6PTZ{MkG=
R1 NHjHR3FIem:5dHigbY5pcWKrdH;yfUBie3OjeR?= MUnFR|UxRTZ5NTDuUS=> M3L3[lExQTV4MkKx
R2(Bos) NWXNWpFVT3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= NHn4XZVGSzVyPUG2NFAhdk1? M3jYNFExQTV4MkKx
R30dm MUjHdo94fGhiaX7obYJqfG:{eTDhd5NigQ>? M3\zfmVEPTB;MUSgcm0> NVvnSpR4OTB7NU[yNlE>
FaDu MlGwS5Jwf3SqIHnubIljcXSxcomgZZN{[Xl? Mn3BSWM2OD1zMT6zJI5O MmO5NVA6PTZ{MkG=
A253 NHPmUFlIem:5dHigbY5pcWKrdH;yfUBie3OjeR?= NVezcGE{TUN3ME2xOE42KG6P MVSxNFk2PjJ{MR?=
HT-29 MYPjfZRwfG:6aXPpeJkh[XO|YYm= M2LjTmlEPTB;Mz60OUDPxE1? MVGyN|k3QDh{NB?=
COLO-320 DM M2HoRoN6fG:2b4jpZ4l1gSCjc4PhfS=> Mn63TWM2OD13LkK1JO69VQ>? M372PVI{QTZ6OEK0
COLO 205 MXfjfZRwfG:6aXPpeJkh[XO|YYm= M1nuWWlEPTB;Mz6yO{DPxE1? MXiyN|k3QDh{NB?=
BGC-823 MYjjfZRwfG:6aXPpeJkh[XO|YYm= NI\wRphKSzVyPUCuNVEh|ryP MYWxPFU2PTV4Mh?=
Hela MnLIZ5l1d3SxeHnjbZR6KGG|c3H5 M{nYRmlEPTB;MD6xJO69VQ>? NU\VPVduOTh3NUW1OlI>
Bel-7402 MW\jfZRwfG:6aXPpeJkh[XO|YYm= MXvJR|UxRTh5Lkmg{txO MX[xPFU2PTV4Mh?=

... Click to View More Cell Line Experimental Data

Methods Test Index PMID
Western blot
ppJAK1 / ppJAK2 / JAK1 / JAK2 ; 

PubMed: 26131691     

HDLM-2 cells treated with the indicated concentrations of methotrexate and aminopterin and blotted to show levels of total JAK1 (tJAK1), dual phosphorylated JAK1 (ppJAK1), total JAK2 (tJAK2), dual phosphorylated JAK2 (ppJAK2) and ß-actin shown as a loading control. Levels of ppJAK1 and ppJAK2 are reduced at high drug concentrations.

pSTAT1 / pSTAT3 / pSTAT5 / STAT1 / STAT3 / STAT5; 

PubMed: 26131691     

D) HDLM-2 cells treated with the indicated concentrations of methotrexate and aminopterin and blotted to show levels of total STAT1 (tSTAT1), phosphorylated STAT1 (pSTAT1), total STAT3 (tSTAT3), phosphorylated STAT3 (pSTAT3), total STAT5 (tSTAT5), phosphorylated STAT5 (pSTAT5) and β-actin shown as a loading control. Levels of pSTAT1 and pSTAT5 are reduced at most drug concentrations while levels of pSTAT3 appear to be unaffected.

AKT / p-AKT / mTOR / p-mTOR / Beclin 1 / HMGB1 ; 

PubMed: 26702616     

After the cells were exposed to 0.1 μM MTX for the indicated times, the cell lysates were subjected to Western blotting with specific antibodies. The results are representative of three independent experiments. β-actin was used as a loading control.

26131691 26702616
In vivo Methotrexate increases splenocyte AICAR content, raised adenosine concentrations in exudates from carrageenan-inflamed air pouches, and markedly inhibits leukocyte accumulation in inflamed air pouches in mice. Methotrexate-mediated reduction in leukocyte accumulation is partially reversed by injection of adenosine deaminase (ADA) into the air pouch, completely reverses by a specific adenosine A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX), but not affected by an adenosine A1 receptor antagonist, 8-cyclopentyl-dipropylxanthine in mice. [3]


Solubility (25°C)

In vitro DMSO 90 mg/mL warmed (198.04 mM)
Water Insoluble
Ethanol Insoluble
In vivo Add solvents to the product individually and in order(Data is from Selleck tests instead of citations):
2% DMSO+30% PEG 300+5% Tween 80+ddH2O
For best results, use promptly after mixing.

* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.

Chemical Information

Molecular Weight 454.44


CAS No. 59-05-2
Storage powder
in solvent
Synonyms NCI-C04671
Smiles CN(CC1=CN=C2C(=N1)C(=NC(=N2)N)N)C3=CC=C(C=C3)C(=O)NC(CCC(=O)O)C(=O)O

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Clinical Trial Information

NCT Number Recruitment interventions Conditions Sponsor/Collaborators Start Date Phases
NCT03642795 Not yet recruiting Other: Questionnaire Rheumatoid Polyarthritis Centre Hospitalier Universitaire de Nīmes October 2020 --
NCT04488237 Not yet recruiting Drug: Vitamin D Acute Lymphoblastic Leukemia Assiut University September 2020 --
NCT04109300 Not yet recruiting Genetic: HLADQA1*05A>G screening|Other: Standard of Care Inflammatory Bowel Diseases|Ulcerative Colitis|Crohn Disease Western University Canada September 1 2020 Not Applicable
NCT04462601 Not yet recruiting Other: Biomarker detection Sjogren''s Syndrome Centre Hospitalier Universitaire de Nīmes September 2020 --
NCT04483466 Not yet recruiting Drug: Methotrexate|Drug: Placebo Investigate the Effect(s) of Methotrexate Treatment on Arthritis Disease Severity George Washington University August 1 2020 Phase 3
NCT04205734 Not yet recruiting Diagnostic Test: MTX Assessment IBD Boston Children''s Hospital August 1 2020 --

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Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID