The action target of Neurotensin is neurotensin receptor 1 (NTR1) expressed on HCT116 human colon cancer cells. [1]

Neurotensin promotes the release of IL-8 in an untransformed colon epithelial cell line stably transfected with NTR and induces the expression of MIP-2, MCP-1, IL-1β, and TNFα in animal microglia. The majority of human pancreatic and colorectal malignancies include high-affinity NTRs, which belong to the G protein-coupled receptor (GPCR) family and imply that neurotensin (NT) may have an endocrine effect on tumor growth. Numerous signal transduction pathways, including as intracellular calcium ([Ca2+]i), mitogen-activated protein kinase (MAPK), ERK and JNK, and different PKC isoforms, are known to be stimulated by neurotensin through NTR1. Neurotensin therapy (100 nM) significantly boosted HCT116 cell migration (about three times) as compared to vehicle treatment; pretreatment with curcumin (10 μM) inhibited the stimulating effect of NT on HCT116 cell migration. The proliferative effects of neurotensin are partly attributed to NT stimulation of MEK/ERK and regulation of downstream AP-1 transcription factors [1].

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1. Induction of interleukin-8 (IL-8) production in HCT116 cells: HCT116 human colon cancer cells were treated with Neurotensin at concentrations of 10^-10 M, 10^-9 M, 10^-8 M, and 10^-7 M for 24 hours. The supernatant was collected, and ELISA was used to detect IL-8 levels. The results showed that Neurotensin induced IL-8 production in a concentration-dependent manner; compared with the control group, 10^-7 M Neurotensin increased IL-8 secretion by approximately 5-fold [1]
2. Promotion of HCT116 cell migration: Transwell migration assay was used to evaluate the effect of Neurotensin on cell migration. HCT116 cells were seeded in the upper chamber of Transwell, and Neurotensin (10^-8 M) was added to the lower chamber. After 24 hours of incubation, the cells that migrated to the lower chamber were fixed, stained, and counted. The results indicated that Neurotensin significantly increased the migration number of HCT116 cells by approximately 2.5-fold compared with the control group [1]
3. Activation of intracellular signaling pathways: HCT116 cells were treated with Neurotensin (10^-8 M) for 5 minutes, 15 minutes, 30 minutes, and 60 minutes. Western blot analysis was performed to detect the phosphorylation levels of protein kinase C (PKC), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 mitogen-activated protein kinase (p38 MAPK). The results showed that Neurotensin rapidly activated PKC, ERK1/2, and p38 MAPK; the phosphorylation levels peaked at 5-15 minutes and gradually decreased thereafter [1]

1. IL-8 detection assay in HCT116 cells: HCT116 cells were seeded in 24-well plates at a density of 5×10^4 cells per well and cultured in RPMI 1640 medium containing 10% fetal bovine serum until they reached 70%-80% confluence. The medium was replaced with serum-free RPMI 1640 medium, and the cells were starved for 12 hours. Then, Neurotensin was added to the wells at final concentrations of 10^-10 M, 10^-9 M, 10^-8 M, and 10^-7 M (with or without curcumin pretreatment). After 24 hours of incubation at 37°C in a 5% CO2 incubator, the culture supernatant was collected by centrifugation at 1000×g for 10 minutes. The concentration of IL-8 in the supernatant was measured using a specific ELISA kit, and the absorbance was read at 450 nm with a microplate reader [1]
2. HCT116 cell migration assay (Transwell assay): The upper chamber of Transwell inserts (with 8-μm pores) was coated with collagen I overnight at 4°C. HCT116 cells were digested with trypsin, resuspended in serum-free RPMI 1640 medium, and adjusted to a concentration of 1×10^5 cells/mL. A total of 200 μL of the cell suspension was added to the upper chamber, and 600 μL of RPMI 1640 medium containing 10% fetal bovine serum and Neurotensin (10^-8 M) was added to the lower chamber. After incubation at 37°C in a 5% CO2 incubator for 24 hours, the non-migrated cells on the upper surface of the membrane were wiped off with a cotton swab. The migrated cells on the lower surface were fixed with 4% paraformaldehyde for 15 minutes and stained with crystal violet for 20 minutes. The stained cells were observed under a light microscope, and 5 random fields of view were selected for counting to quantify the migration ability [1]

[1]. Curcumin inhibits neurotensin-mediated interleukin-8 production and migration of HCT116 human colon cancer cells. Clin Cancer Res. 2006 Sep 15;12(18):5346-55.

Neurotensin is a peptide hormone composed of 13 amino acids, found in the central nervous system and gastrointestinal tract. It functions as a neurotransmitter in the brain, as a hormone in the gut, and is also a neuromodulator. Because it is associated with multiple neurotransmitter systems (such as the dopaminergic, serotonergic, glutamatergic, GABAergic, and cholinergic systems), it participates in the pathophysiological processes of various central nervous system disorders, including schizophrenia, Parkinson's disease, substance abuse, pain, cancer, inflammation, eating disorders, and central regulation of blood pressure. It is a human metabolite, mitogen, neurotransmitter, and wound-healing agent. It is the binding base of neurotensin(1+). A biologically active thiopentacapeptide isolated from the hypothalamus. Studies have shown that neurotensin can induce hypotension in rats, stimulate guinea pig ileum and rat uterine contractions, and induce duodenal relaxation in rats. There is also evidence that it is a neurotransmitter in both the peripheral and central nervous systems.

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1. Neurotensin is a neuropeptide composed of 13 amino acids, widely distributed in the central nervous system and gastrointestinal tract. In the gastrointestinal system, it regulates intestinal peristalsis, secretion and nutrient absorption; in cancer, it acts as a growth factor, promoting the proliferation, migration and invasion of certain tumor cells (including colon cancer cells)[1].
2. The mechanism by which neurotensin mediates the biological effects of HCT116 cells involves its binding to the cell membrane-specific receptor NTR1, thereby activating downstream signaling pathways such as PKC, ERK1/2 and p38 MAPK. These activated pathways collectively promote the transcription and secretion of IL-8 and enhance the migration ability of colon cancer cells[1].
3. In this study, neurotensin was used as a positive stimulant to induce pro-tumor biological behaviors (IL-8 production and cell migration) in HCT116 cells, and the inhibitory effect of curcumin on these neurotensin-mediated behaviors was further investigated[1].

C78H121N21O20

1672.92404

1671.91

39379-15-2

25077406

White to off-white solid powder

1.46g/cm3

1.666

4.506

21

23

50

119

3530

14

CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@@H]2CCCN2C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H]3CCCN3C(=O)[C@H](CCCCN)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC4=CC=C(C=C4)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]5CCC(=O)N5

PCJGZPGTCUMMOT-ISULXFBGSA-N

InChI=1S/C78H121N21O20/c1-7-43(6)63(73(115)96-57(76(118)119)37-42(4)5)97-70(112)55(39-45-21-25-47(101)26-22-45)95-72(114)59-18-13-35-99(59)75(117)52(16-11-33-86-78(83)84)90-64(106)48(15-10-32-85-77(81)82)89-71(113)58-17-12-34-98(58)74(116)51(14-8-9-31-79)91-69(111)56(40-60(80)102)94-66(108)50(28-30-62(104)105)88-68(110)54(38-44-19-23-46(100)24-20-44)93-67(109)53(36-41(2)3)92-65(107)49-27-29-61(103)87-49/h19-26,41-43,48-59,63,100-101H,7-18,27-40,79H2,1-6H3,(H2,80,102)(H,87,103)(H,88,110)(H,89,113)(H,90,106)(H,91,111)(H,92,107)(H,93,109)(H,94,108)(H,95,114)(H,96,115)(H,97,112)(H,104,105)(H,118,119)(H4,81,82,85)(H4,83,84,86)/t43-,48-,49-,50-,51-,52-,53-,54-,55-,56-,57-,58-,59-,63-/m0/s1

(2S)-2-[[(2S,3S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-6-amino-2-[[(2S)-4-amino-2-[[(2S)-4-carboxy-2-[[(2S)-3-(4-hydroxyphenyl)-2-[[(2S)-4-methyl-2-[[(2S)-5-oxopyrrolidine-2-carbonyl]amino]pentanoyl]amino]propanoyl]amino]butanoyl]amino]-4-oxobutanoyl]amino]hexanoyl]pyrrolidine-2-carbonyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]pyrrolidine-2-carbonyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-3-methylpentanoyl]amino]-4-methylpentanoic acid

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, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~33.33 mg/mL (~19.92 mM)

Solubility in Formulation 1: 20 mg/mL (11.96 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)