1. Opioid alkaloids and casomorphin peptides decrease the proliferation of prostatic cancer cell lines (LNCaP, PC3 and DU145) through a partial interaction with opioid receptors
M Kampa, E Bakogeorgou, A Hatzoglou, A Damianaki, P M Martin, E Castanas Eur J Pharmacol. 1997 Sep 24;335(2-3):255-65. doi: 10.1016/s0014-2999(97)01213-2.
Opioid agonists (ethylketocyclazocine, etorphine, [D-Ala2,D-Leu5]enkephalin (DADLE), [D-Ala2, N-Me-Phe4-Gly-ol]enkephalin (DAGO), [D-Ser2,Leu5]enkephalin-Thr6 (DSLET) and morphine were found to inhibit the proliferation of human prostate cancer cell lines (LNCaP, DU145, and PC3), in a dose-dependent manner. The 50% inhibitory concentrations (IC50) were in the picomolar range. In many cases, this effect was antagonized by the general opioid antagonist, diprenorphine, indicating the existence of specific opioid binding sites. Saturation binding experiments with selective ligands and effectors showed no opioid sites on the LNCaP cell line, kappa1 and mu sites on the PC3 cell line, and kappa1, kappa3 and mu sites on the DU145 cell line. In other cases, the opioid effect was not antagonized by diprenorphine, indicating that the action of opioids might be mediated through other membrane receptors. Furthermore, casomorphin peptides, issued from bovine alpha- (alpha-casein-90-95 and alpha-casein-90-96) and beta-caseins (beta-casomorphin and beta-casomorphin-1-5), and human alphaS1-casein (alphas -casomorphin and alphaS1-casomorphin amide) inhibited cell proliferation of human prostate cell lines, also by a mechanism partly involving opioid receptors. As opioid neurons can be found in the prostate gland, and casomorphin peptides might reach the gland through the general circulation, the above findings indicate a putative role of opioids in prostate cancer cell growth.
2. Capillary electrochromatographic separation of peptides using a macrocyclic polyamine for molecular recognition
Tse-Hsien Chen, Tarun Kumar Misra, Chuen-Ying Liu Electrophoresis. 2008 Apr;29(8):1651-7. doi: 10.1002/elps.200700560.
A macrocyclic polyamine, 1,5,9,13,17,21,25,29-octaazacyclodotriacontane ([32]ane-N(8)), in the bonded phase was employed as a molecular receptor for CEC separation of oligopeptides. Parameters affecting the performance of the separations were considered. Baseline separation for the mixture of angiotensin I, angiotensin II, [Sar(1), Thr(8)]-angiotensin II, beta-casomorphin bovine, beta-casomorphin human, oxytocin acetate, tocinoic acid, vasopressin, and FMRF amide could be achieved using phosphate buffer (30 mM, pH 7) as the mobile phase. Column efficiency with average theoretical plate numbers of 69 000 plates/m and RSDs of <1% (n = 6) was achieved. [Met(5)]-enkephalin and [Leu(5)]-enkephalin, which have identical pI values and similar masses could be completely separated using acetate buffer (30 mM) with pH gradient (pH 3 inlet side and pH 4 outlet side). The results suggest that the mechanism for the peptide separation was mediated by a combination of electrophoretic migration and chromatographic retention involving anion coordination and anion exchange. After long-term use, the deviation of the EOF of the column after more than 600 injections was still within 6.0% of that for a freshly prepared column.
3. Nanoelectrospray with ion-trap mass spectrometry for the determination of beta-casomorphins in derived milk products
Ana Juan-García, Guillermina Font, Cristina Juan, Yolanda Picó Talanta. 2009 Nov 15;80(1):294-306. doi: 10.1016/j.talanta.2009.06.062. Epub 2009 Jul 4.
Beta-casomorphins (b-CMs) are bioactive peptides derived from casein with opioid agonist effects similar to morphine. The use of electrospray (ESI) with quadrupole ion-trap mass spectrometry (QIT-MS) for these compounds in two matrices, cheese and milk, was examined. It was compared to a liquid chromatography (LC) coupled to mass spectrometry (LC-MS), and a "soft" ionisation technique, NanoMate, with selected ion monitoring (SIM), which are unreliable for the determination of trace casomorphins in derived milk products. b-CM mass fragmentation pathways were done for the four most common b-CMs: beta-casomorphin (1-5) bovine (b-CM-5), beta-casomorphin (1-7) bovine (b-CM-7), [D-Ala2, D-Pro4,Tyr5]-beta-casomorphin (1-5) amide (b-CM-10) and beta-casomorphin (1-5) amide [D-Ala2,Hyp4,Tyr5] (b-CM-11). The major product ions obtained in QIT-MS were used to construct fragmentation pathways for b-CMs. The different collision energies using automated nanoelectrospray ion source NanoMate and conventional LC in QIT-MS were studied. Calibration data for b-CMs, using spiked milk or cheese samples (10 g or 10 mL), were: NanoMate/MS (25-1000 microg/L), r(2)=0.998; NanoMate/MS(2) (5-1000 microg/L), r(2)=0.9992; NanoMate/MS(3) (2.5-1000 microg/L), r(2)=0.9998. Reproducibility data (% RSD, N=5) for NanoMate/MS(n) mode ranged between 2.0 at 500 microg/L and 7.0 at 10 microg/L.
