engleski

The effect of Met-enkephalin thiorphan and naloxone on the expression of CD10 molecule and apoptosis in NALM-1 cells

Introduction. NALM-1 cells are human pre-B leukemic, which express CD10 molecule, what is also a neutral endopeptidase (NEP) activity. An opioid pentapeptide Met-enkephalin has been described as the negative regulator of tumor cell growth in the literature. We have previously shown (HID 1000 Annual meeting) that Met-enkephalin, a natural substrate for NEP, thiorphan an inhibitor of NEP, and naloxone an antagonist of opioid receptors, moderately and transiently suppressed proliferation of NALM-1 cells after 6 hours treatment (MTT test). In this study the usibility of an endogenous opioid pentapeptide Met-enkephalin to modulate membrane expression of CD10 molecule, and the ability to trigger apoptosis was tested. Thiorphan, the inhibitor of NEP activity served as the positive control, and naloxone, an antagonist of opioid receptors as the negative control. Material and methods. NALM-1 cells were cultivated in RPMI media containing 10% of fetal calf serum in the presence of Met-enkephalin (10^-6, 10^-7, 10^-8 M) naloxone (10^-5, 10^-6, 10^-7 M) and thiorphan (10^-4, 5x10^-5 M). The statement of CD10 was determined by means with CD10 monoclonal antibody (DAKO, Denmark) by flow cytometry analysis (FACS Calibur). The CD10 data were expressed as the ratio of mean fluorescence (RMF) of toptal cells positive for CD monoclonal antibody and isotypic control. DNA was extracted by phenol-chloroiphorm-isoamyl alcohol and precipitated with sodium acetate and absolute ethanol. DNA was verified by electrophoresis in 0.8% agarose gel containing ethidium-bromide in the presence of DNA ladder standard. Results. FACS analysis indicated moderate decrease of RMF values after 24 hours treatment with Met-enkephalin (10^-6 M) and Thiorphan (5x10^-5 M) (2 out 4 experiments). Naloxone did not affect CD10 membrane expression (RMF values). DNA was not fragmented after 6 and 24 hours treatment of NALM-1 cells with Met-enkephalin (10^-6, 10^-7, 10^-8 M), naloxone (10^-5, 10^-6, 10^-7 M) and thiorphan (10^-4, 5x10^-5 M). Conclusions. In our previous results we showed that Met-enkephalin and thiorphan moderately and transiently inhibited the growth of NALM-1 cells. This inhibition did not include apoptosis, since Met-enkephalin, naloxone and thiorphan did not cause fragmentation of DNA. The membrane expression of CD10 molecule was moderately decreased by Met-enkephalin (natural substrate of NEP) and thiorphan (NEP enzyme inhibitor), while naloxone (an opioid receptor anatagonist) was not effective. These results indicate the regulatory role of CD10 molecule.

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