Catalog No.S1210 Synonyms: NCI-C04671

Methotrexate Chemical Structure

Molecular Weight(MW): 454.44

Methotrexate (MTX), 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.

Size Price Stock Quantity  
In DMSO USD 134 In stock
USD 97 In stock
USD 197 In stock
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Cited by 15 Publications

3 Customer Reviews

  • Toxicity of MTX (3) and toxic analogues 5 a-e on a) hY1R-expressing MDA-MB-468 and b) non-hYR-expressing HEK293 cells. Cell viability was determined by resazurin assay. Bars represent the mean±SEM of at least three independent experiments performed in triplicate. Measurements were normalized by using only HBSS, pH 7, treated cells (set at 100 %) and ethanol-treated cells (set at 0 %). Statistical significances refer to only HBSS, pH 7, treated cells indicated as a dashed line.

    ChemMedChem, 2015, 10(5):804-14.. Methotrexate purchased from Selleck.

  • A. Viability comparison of REH vector control, BCL6 overexpression, or BCL6 overexpression cells pre-treated with 79-6 (125μM) following exposure to three chemotherapy drugs (Ara-C [1 μM], MTX [50 μM], VCR [25 μM]). (* = p < 0.05 BCL6 OX to vector control and # = p < 0.05 BCL6 OX to BCL6 + 79-6).

    Oncotarget, 2016, 7(17):23439-53. Methotrexate purchased from Selleck.

  • Western blotting andrelative densitometric analyses of ERαexpression levels in regular (MCF-7) (a and a’) 4OH-tamoxifen resistant (Tam-Res) (b and b’) MCF-7cells treated for 24 hrs in complete medium with different doses of methotrexate (MTX) or vehicle (DMSO).

    J Cell Physiol, 2018, 233(5):3713-3722. Methotrexate purchased from Selleck.

Purity & Quality Control

Choose Selective DHFR Inhibitors

Biological Activity

Description Methotrexate (MTX), 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.
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 MVzjfZRwfG:6aXPpeJkh[XO|YYm= M4XvNmlEPTB;ND64JI5O M1rJXVg3OzJ2MUO=
L cells MXzjfZRwfG:6aXPpeJkh[XO|YYm= MonYTWM2OD16LkOgcm0> NFrzOlQ5PjN{NEGz
143B(TK-) MUHjfZRwfG:6aXPpeJkh[XO|YYm= Ml;UTWM2OD16Lkigcm0> NFXFOFY5PjN{NEGz
U87MG MXfjfZRwfG:6aXPpeJkh[XO|YYm= MmjkTWM2OD1{MjDuUS=> NG\hOYU5PjN{NEGz
A549 NVvh[|k3[3m2b4TvfIlkcXS7IHHzd4F6 NIP0c5hKSzVyPUOxJI5O M4nTNlg3OzJ2MUO=
H460 MoHQZ5l1d3SxeHnjbZR6KGG|c3H5 MlHGTWM2OD17LkWgcm0> NUnTc|hiQDZ|MkSxNy=>
Daoy MlfSZ5l1d3SxeHnjbZR6KGG|c3H5 M3roNmlEPTB;OTDuUS=> M2\jXFg3OzJ2MUO=
U373MG NYG1b2U2[3m2b4TvfIlkcXS7IHHzd4F6 NIHhSmtKSzVyPUGyJI5O NXXZWpZzQDZ|MkSxNy=>
Vero Mn\rZ5l1d3SxeHnjbZR6KGG|c3H5 NVLpUlN2UUN3ME25MlIhdk1? NHz4N4I5PjN{NEGz
SCC25  NVe0dHAxT3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= MWn+NUDPxE1? M4HMV2lEPTB;Mkegcm0> NY\FSJNyQTB{Mke5OS=>
NCI-H23 MYXHdo94fGhiaX7obYJqfG:{eTDhd5NigQ>? MlHFglEh|ryP NXPCWmk5UUN3ME20N{BvVQ>? MmX2PVAzOjd7NR?=
NCI-H522 NU\ub4RDT3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= NXuzNop1hjFizszN NYT5cHZMUUN3ME2yNlkhdk1? NEKx[Fg6ODJ{N{m1
EKVX M1rR[mdzd3e2aDDpcohq[mm2b4L5JIF{e2G7 NXLGSYdnhjFizszN MUXJR|UxRjFyMECgcm0> NH3BV|Q6ODJ{N{m1
HCT-15 M1nPbWdzd3e2aDDpcohq[mm2b4L5JIF{e2G7 MV\+NUDPxE1? MVHJR|UxRTNyIH7N NIn6UVc6ODJ{N{m1
HT29 NI\a[XVIem:5dHigbY5pcWKrdH;yfUBie3OjeR?= NFq1[md,OSEQvF2= NVPYVmhsUUN3ME2zNkBvVQ>? NWfJ[4ZTQTB{Mke5OS=>
SW-620 MWnHdo94fGhiaX7obYJqfG:{eTDhd5NigQ>? NFLmcId,OSEQvF2= NITzbnlKSzVyPUOzJI5O NH\VcmM6ODJ{N{m1
KM12 NXKwXVNmT3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= NYTqe|YzhjFizszN NWi2eVNxUUN3ME20NkBvVQ>? NXH3eXB1QTB{Mke5OS=>
SF-539 NVrmOIFtT3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= M3;lZ54yKM7:TR?= NYrxRWE1UUN3ME2zOUBvVQ>? MoDOPVAzOjd7NR?=
SF-268 NYn6VYFoT3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= NWrkV2s1hjFizszN NVzNSXU4UUN3ME21NkBvVQ>? MXG5NFIzPzl3
SNB-75 NFziSHlIem:5dHigbY5pcWKrdH;yfUBie3OjeR?= MlThglEh|ryP M3vnRmlEPTB-MUCwNFAhdk1? NVHSVm56QTB{Mke5OS=>
LOX IMVI NYHUV49ST3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= NUj0VYJMhjFizszN MkP3TWM2OD1{NjDuUS=> MmfZPVAzOjd7NR?=
UACC-62 MU\Hdo94fGhiaX7obYJqfG:{eTDhd5NigQ>? MlTFglEh|ryP NYTrfGk1UUN3ME2yPEBvVQ>? NWnZTFZTQTB{Mke5OS=>
SK-MEL-5 NULlW41RT3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= M3KxdZ4yKM7:TR?= NUDnWHFPUUN3ME24O{BvVQ>? MmXmPVAzOjd7NR?=
MALME-3M MW\Hdo94fGhiaX7obYJqfG:{eTDhd5NigQ>? NWHsZ|FZhjFizszN NHLvZ3FKSzVyPkGwNFAhdk1? NEDLWo46ODJ{N{m1
SK-MEL-28 NXzveG1OT3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= MmL4glEh|ryP MYHJR|UxRjFyMECgcm0> NVHDVoFiQTB{Mke5OS=>
OVCAR-5 MmfMS5Jwf3SqIHnubIljcXSxcomgZZN{[Xl? MlfqglEh|ryP MVTJR|UxRjFyMECgcm0> NYCySWlqQTB{Mke5OS=>
OVCAR-3 M1TBemdzd3e2aDDpcohq[mm2b4L5JIF{e2G7 NFzhemp,OSEQvF2= M1rRXGlEPTB;M{m4JI5O NW\peVRXQTB{Mke5OS=>
786-0 MXjHdo94fGhiaX7obYJqfG:{eTDhd5NigQ>? NYTlWGFlhjFizszN M3HTTmlEPTB;M{Ogcm0> NF\OTGE6ODJ{N{m1
UO-31 MVLHdo94fGhiaX7obYJqfG:{eTDhd5NigQ>? NXXlblV3hjFizszN MYDJR|UxRTF7MTDuUS=> M3u1NVkxOjJ5OUW=
ACHN MlzCS5Jwf3SqIHnubIljcXSxcomgZZN{[Xl? M2roU54yKM7:TR?= NYHONoE4UUN3ME20NEBvVQ>? NVLMb4hKQTB{Mke5OS=>
MCF7 M2LBT2dzd3e2aDDpcohq[mm2b4L5JIF{e2G7 NFzFOIF,OSEQvF2= Mn\QTWM2OD1|NjDuUS=> NFS1UW06ODJ{N{m1
PC-3 MnnXS5Jwf3SqIHnubIljcXSxcomgZZN{[Xl? Mn\VglEh|ryP Mmf0TWM2OD1{IH7N NVqyXIxoQTB{Mke5OS=>
DU-145 M1:2Rmdzd3e2aDDpcohq[mm2b4L5JIF{e2G7 M{DmfZ4yKM7:TR?= NFXnWJVKSzVyPUKzJI5O NXPpV2lsQTB{Mke5OS=>
R1 M{DOOmdzd3e2aDDpcohq[mm2b4L5JIF{e2G7 M4rRWmVEPTB;Nke1JI5O NUHSZZlFOTB7NU[yNlE>
R2(Bos) NUPmSWxqT3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= MUfFR|UxRTF4MECgcm0> MX[xNFk2PjJ{MR?=
R30dm M136c2dzd3e2aDDpcohq[mm2b4L5JIF{e2G7 MXLFR|UxRTF2IH7N MoTLNVA6PTZ{MkG=
FaDu MVrHdo94fGhiaX7obYJqfG:{eTDhd5NigQ>? MXvFR|UxRTFzLkOgcm0> MkfENVA6PTZ{MkG=
A253 NGPpXWJIem:5dHigbY5pcWKrdH;yfUBie3OjeR?= MmntSWM2OD1zND61JI5O NYnIVXNYOTB7NU[yNlE>
HT-29 NYS1bVlj[3m2b4TvfIlkcXS7IHHzd4F6 MU\JR|UxRTNwNEWg{txO MW[yN|k3QDh{NB?=
COLO-320 DM MV3jfZRwfG:6aXPpeJkh[XO|YYm= M{LLNWlEPTB;NT6yOUDPxE1? Mn\rNlM6Pjh6MkS=
COLO 205 Mo[xZ5l1d3SxeHnjbZR6KGG|c3H5 MWnJR|UxRTNwMkeg{txO NWfzR4xTOjN7Nki4NlQ>
BGC-823 NWXpdZhR[3m2b4TvfIlkcXS7IHHzd4F6 MkDYTWM2OD1yLkGxJO69VQ>? NUKyTVMxOTh3NUW1OlI>
Hela MXPjfZRwfG:6aXPpeJkh[XO|YYm= MlfvTWM2OD1yLkGg{txO M4jpZlE5PTV3NU[y
Bel-7402 MYnjfZRwfG:6aXPpeJkh[XO|YYm= M1;t[WlEPTB;OEeuPUDPxE1? MmHjNVg2PTV3NkK=

... 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

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Clinical Trial Information

NCT Number Recruitment interventions Conditions Sponsor/Collaborators Start Date Phases
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
NCT03642795 Not yet recruiting Other: Questionnaire Rheumatoid Polyarthritis Centre Hospitalier Universitaire de Nīmes January 2020 --
NCT03964259 Recruiting Drug: Intravenous fluids Lymphoma|Acute Lymphoblastic Leukemia|Pediatric Cancer|Pediatric ALL|Pediatric Lymphoma Virginia Commonwealth University October 2 2019 Phase 1
NCT03960177 Recruiting Drug: Glucarpidase Osteosarcoma OHSU Knight Cancer Institute|National Cancer Institute (NCI) March 27 2019 Early Phase 1

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DHFR Signaling Pathway Map

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Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID