Methotrexate

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.

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In DMSO USD 134 In stock
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USD 197 In stock
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Cited by 25 Publications

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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.
Targets
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 NFHaRXBkgXSxdH;4bYNqfHliYYPzZZk> MkfNTWM2OD12Lkigcm0> MWi4OlMzPDF|
L cells MVfjfZRwfG:6aXPpeJkh[XO|YYm= NHvBVohKSzVyPUiuN{BvVQ>? M3y5Vlg3OzJ2MUO=
D54 NHzT[phkgXSxdH;4bYNqfHliYYPzZZk> MnHXTWM2OD1zNjDuUS=> MXK4OlMzPDF|
143B(TK-) NHGxVIxkgXSxdH;4bYNqfHliYYPzZZk> M1nscmlEPTB;OD64JI5O NHjrbpI5PjN{NEGz
U87MG NX:zWHN4[3m2b4TvfIlkcXS7IHHzd4F6 M4m5U2lEPTB;MkKgcm0> MnTwPFY{OjRzMx?=
A549 MVvjfZRwfG:6aXPpeJkh[XO|YYm= NHyxWYZKSzVyPUOxJI5O MYG4OlMzPDF|
H460 MV;jfZRwfG:6aXPpeJkh[XO|YYm= MkHvTWM2OD17LkWgcm0> MXm4OlMzPDF|
Daoy NGSweWdkgXSxdH;4bYNqfHliYYPzZZk> NYnqW3ZQUUN3ME25JI5O NWnJdldlQDZ|MkSxNy=>
U373MG NF;NfodkgXSxdH;4bYNqfHliYYPzZZk> NEDYNY1KSzVyPUGyJI5O NHvrR5E5PjN{NEGz
Vero NF:xSVJkgXSxdH;4bYNqfHliYYPzZZk> MmHUTWM2OD17LkKgcm0> MnHHPFY{OjRzMx?=
SCC25  NHW2eYFIem:5dHigbY5pcWKrdH;yfUBie3OjeR?= NF[wSVJ,OSEQvF2= NFrQeXJKSzVyPUK3JI5O MlrXPVAzOjd7NR?=
NCI-H460 Mn3ZS5Jwf3SqIHnubIljcXSxcomgZZN{[Xl? MoPLglEh|ryP NH7Rc|RKSzVyPUK4JI5O NV21eIM5QTB{Mke5OS=>
NCI-H23 NF;aXpJIem:5dHigbY5pcWKrdH;yfUBie3OjeR?= NVjZXHJthjFizszN NI\KeHdKSzVyPUSzJI5O MWC5NFIzPzl3
NCI-H522 MXzHdo94fGhiaX7obYJqfG:{eTDhd5NigQ>? MlfBglEh|ryP NGfifXVKSzVyPUKyPUBvVQ>? M1LRWFkxOjJ5OUW=
EKVX NWTMdYttT3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= MX\+NUDPxE1? NWW0UHRGUUN3ME6xNFAxKG6P NVWzflJ[QTB{Mke5OS=>
HCT-116 NFnYVW5Iem:5dHigbY5pcWKrdH;yfUBie3OjeR?= NH7WdVF,OSEQvF2= MmG5TWM2OD1|MDDuUS=> MWe5NFIzPzl3
HCT-15 MlPXS5Jwf3SqIHnubIljcXSxcomgZZN{[Xl? NYG2NHN4hjFizszN M2TVV2lEPTB;M{Cgcm0> Ml;lPVAzOjd7NR?=
HT29 M{C4NWdzd3e2aDDpcohq[mm2b4L5JIF{e2G7 MV7+NUDPxE1? MnrITWM2OD1|MjDuUS=> NISxT5M6ODJ{N{m1
SW-620 NVG3fWNCT3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= NUD0PIdPhjFizszN MWrJR|UxRTN|IH7N MUG5NFIzPzl3
KM12 NUKzdHhHT3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= MnXXglEh|ryP MXLJR|UxRTR{IH7N NUjiOXY6QTB{Mke5OS=>
SF-539 NIrXeHRIem:5dHigbY5pcWKrdH;yfUBie3OjeR?= MUf+NUDPxE1? MWDJR|UxRTN3IH7N NUnNcnFEQTB{Mke5OS=>
SF-268 MXHHdo94fGhiaX7obYJqfG:{eTDhd5NigQ>? MnfEglEh|ryP MXHJR|UxRTV{IH7N MVq5NFIzPzl3
SNB-75 MnzXS5Jwf3SqIHnubIljcXSxcomgZZN{[Xl? NVjJZYhIhjFizszN M33TV2lEPTB-MUCwNFAhdk1? Mnr3PVAzOjd7NR?=
LOX IMVI M3u1fWdzd3e2aDDpcohq[mm2b4L5JIF{e2G7 MnuzglEh|ryP NVTWVJJoUUN3ME2yOkBvVQ>? MWi5NFIzPzl3
UACC-62 NVX5SnFNT3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= NEW0V3p,OSEQvF2= NGnsO2hKSzVyPUK4JI5O Mn7lPVAzOjd7NR?=
SK-MEL-5 NFjKPJFIem:5dHigbY5pcWKrdH;yfUBie3OjeR?= NHHyZXd,OSEQvF2= MmHLTWM2OD16NzDuUS=> MU[5NFIzPzl3
MALME-3M NF;pSHZIem:5dHigbY5pcWKrdH;yfUBie3OjeR?= NWnycZlIhjFizszN MVLJR|UxRjFyMECgcm0> MWS5NFIzPzl3
SK-MEL-28 Mkm5S5Jwf3SqIHnubIljcXSxcomgZZN{[Xl? NIm5UnF,OSEQvF2= MkXDTWM2OD5zMECwJI5O NXztXIJvQTB{Mke5OS=>
OVCAR-8 MW\Hdo94fGhiaX7obYJqfG:{eTDhd5NigQ>? NW[2N5dFhjFizszN MWTJR|UxRTNzIH7N MoHkPVAzOjd7NR?=
OVCAR-5 M4TVSmdzd3e2aDDpcohq[mm2b4L5JIF{e2G7 NH\qZph,OSEQvF2= M{nTNWlEPTB-MUCwNEBvVQ>? MU[5NFIzPzl3
OVCAR-3 M1\ZSGdzd3e2aDDpcohq[mm2b4L5JIF{e2G7 NXLCTnF4hjFizszN M2rXZWlEPTB;M{m4JI5O NILCbYo6ODJ{N{m1
786-0 Mn\FS5Jwf3SqIHnubIljcXSxcomgZZN{[Xl? NEPDVWV,OSEQvF2= M4\t[2lEPTB;M{Ogcm0> M{\LZVkxOjJ5OUW=
UO-31 NIjxSYVIem:5dHigbY5pcWKrdH;yfUBie3OjeR?= MV7+NUDPxE1? MWPJR|UxRTF7MTDuUS=> MV25NFIzPzl3
ACHN MXPHdo94fGhiaX7obYJqfG:{eTDhd5NigQ>? NUm1NFVrhjFizszN MnW5TWM2OD12MDDuUS=> NIC1cHo6ODJ{N{m1
MCF7 NETRUnhIem:5dHigbY5pcWKrdH;yfUBie3OjeR?= NX3U[2tuhjFizszN Mo\3TWM2OD1|NjDuUS=> M4ri[FkxOjJ5OUW=
MCF7-ADR NYnXcWdTT3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= M{\4TJ4yKM7:TR?= MX;JR|UxRTd6IH7N Mki0PVAzOjd7NR?=
PC-3 M1fyOWdzd3e2aDDpcohq[mm2b4L5JIF{e2G7 M1;zWJ4yKM7:TR?= MXzJR|UxRTJibl2= MWe5NFIzPzl3
DU-145 NYrmRpVCT3Kxd4ToJIlvcGmkaYTvdpkh[XO|YYm= M1\scZ4yKM7:TR?= NIKzO3dKSzVyPUKzJI5O NELRTWY6ODJ{N{m1
CCRF-CEM MnzaS5Jwf3SqIHnubIljcXSxcomgZZN{[Xl? NX3kcYZlTUN3ME2xOE41KG6P NUHTPXY6OTB7NU[yNlE>
R1 Mmi2S5Jwf3SqIHnubIljcXSxcomgZZN{[Xl? M{XBPGVEPTB;Nke1JI5O MoTQNVA6PTZ{MkG=
R2(Bos) M3ywW2dzd3e2aDDpcohq[mm2b4L5JIF{e2G7 MoL4SWM2OD1zNkCwJI5O MVixNFk2PjJ{MR?=
R30dm MX3Hdo94fGhiaX7obYJqfG:{eTDhd5NigQ>? MYHFR|UxRTF2IH7N NWPaTZFpOTB7NU[yNlE>
FaDu M4WyOmdzd3e2aDDpcohq[mm2b4L5JIF{e2G7 M{nQSmVEPTB;MUGuN{BvVQ>? M{fTVVExQTV4MkKx
A253 MlHwS5Jwf3SqIHnubIljcXSxcomgZZN{[Xl? NULkWFlnTUN3ME2xOE42KG6P M3ryRVExQTV4MkKx
HT-29 NEjrTXRkgXSxdH;4bYNqfHliYYPzZZk> Mnq4TWM2OD1|LkS1JO69VQ>? NYj2flFzOjN7Nki4NlQ>
COLO-320 DM MnfTZ5l1d3SxeHnjbZR6KGG|c3H5 NI\ETHVKSzVyPUWuNlUh|ryP M2X0b|I{QTZ6OEK0
COLO 205 MnXsZ5l1d3SxeHnjbZR6KGG|c3H5 MXvJR|UxRTNwMkeg{txO NFTUNm8zOzl4OEiyOC=>
BGC-823 NYDjZYNm[3m2b4TvfIlkcXS7IHHzd4F6 NIPpeFlKSzVyPUCuNVEh|ryP NEDmO|EyQDV3NUW2Ni=>
Hela NG[we3RkgXSxdH;4bYNqfHliYYPzZZk> NHvkSHBKSzVyPUCuNUDPxE1? NIHITYoyQDV3NUW2Ni=>
Bel-7402 MlPWZ5l1d3SxeHnjbZR6KGG|c3H5 MoS2TWM2OD16Nz65JO69VQ>? NF:0TlIyQDV3NUW2Ni=>

... Click to View More Cell Line Experimental Data

Assay
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]

Protocol

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.
5mg/mL

* 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
Formula

C20H22N8O5

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