Cyclo(L-Pro-L-Val)

Cell lines

A mouse macrophage cell line, RAW 264.7

Preparation Method

A mouse macrophage cell line, RAW 264.7, was cultured in DMEM containing 4mM L-glutamine, antibiotics (1% penicillin/streptomycin), and 10% fetal bovine serum in humidified air environment at 37℃ in a 5% CO₂. RAW264.7 cells (3×10⁴ cells/well) were exposed to the indicated concentrations of Cyclo(L-Pro-L-Val) for 1h and then incubated for an additional 24h with LPS (1μg/mL). At the end of the incubation, each culture supernatant was blended with the Griess reagent to determine NO production by RAW 264.7 cells. Optical density at 540nm of the mixture was determined using a spectrophotometer microplate.

Reaction Conditions

50 or 100μM; 1h

Applications

Animal models

Male C57BL/6J mice

Preparation Method

For cell labelling, HT-29 cells were incubated with cell tracker CM-Dil dye at a final volume was 20μL/mL for 25min at 37°C. To remove theunstrained dye, cells were washed twice and re-suspended with 1×PBS, pH 7.4 to a final density of 2.1×10⁶ cells/mL. Prior transplantation, CM-Dil labelled cells were assessed for viability using trypan blue exclusion assay. More than >90% of viable cells were used. For xenotransplantation, 48 hours post-fertilization (hpf) wild-type ABiC embryos were anaesthetized in 0.01% tricaine methanesulfonate solution containing 0.3% phenylthiocarbamide. After anesthetization, CM-Dil labelled HT-29 cells grafted into the yolk sac of each zebrafish embryos by using a microinjector IM-300. After injection, embryos were incubated for 1h at 28°C. For confirmation of the visible cell mass at the injection site, zebrafish were transferred to an incubator and maintained at 37°C. Xenograft model for antitumor assay: Five hundred CM-Dil labelled HT-29 xenograft zebrafish embryos were randomly selected and hosed into 4 replicate wells in 12-well cell culture plates. 200μM of Cyclo(L-Pro-L-Val) were treated with transplanted embryos. Before DKPs treatment, the initial fluorescence intensity of transplanted cancer cells was measured at 0 hour post-injection (hpi). At the end of experiment 24, 48, and 72h, all embryos were selected from each well and were photographed under an inverted fluorescence microscope.

Dosage form

200μM; 48 or 72h

Applications

Cyclo(L-Pro-L-Val) remarkably inhibited the tumor progression in a zebrafish xenograft model.

References:
[1] LEE D, LEE S R, KANG K S, et al. Bioactive Phytochemicals from Mulberry: Potential Anti-Inflammatory Effects in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages [J]. Int J Mol Sci, 2021, 22(15):
[2] JINENDIRAN S, TENG W, DAHMS H U, et al. Induction of mitochondria-mediated apoptosis and suppression of tumor growth in zebrafish xenograft model by cyclic dipeptides identified from Exiguobacterium acetylicum [J]. Sci Rep, 2020, 10(1): 13721.