The effect of the enkephalin DADLE on transcription does not depend on opioid receptors

Abstract

[D-Ala(2),D-Leu(5)] enkephalin (DADLE) is a synthetic peptide capable of inducing a hibernation-like state in mammals in vivo and in cultured cells in vitro. The effects of DADLE seem to be due to its binding to opioid receptors; however, it inhibits the growth of LNCaP cells, devoid of opioid receptors. We have investigated the effects of DADLE on this cell line using transmission electron microscopy, immunocytochemistry and cytometry, in order to elucidate the general mechanism(s) by which this enkephalin affects cell metabolism. We demonstrated that, similar to cell lines provided with opioid receptors, in LNCaP cells DADLE induces structural modifications of cytoplasmic and nuclear constituents, as well as a decrease in transcription and proliferation. However, DADLE does not provoke an increase in apoptotic or necrotic cell fraction, and, after removing the enkephalin from the culture medium, all effects disappear. We also demonstrated that DADLE molecules enter the cytoplasm and the nucleus of LNCaP cells, mostly binding to perichromatin fibrils and dense fibrillar component, where transcription and early splicing of pre-mRNAs and pre-rRNAs occur. In conclusion, our data demonstrate that the effect of DADLE on transcription and on cultured cells does not depend on opioid receptors. DADLE can, therefore, be envisaged as an extremely promising molecule to be used for inducing a reversible hypometabolic state in various cultured cells, without provoking cell damage or death.

Variation in the analgesic activity of opioid peptide fragments in correlation with the amino acidic sequence

“…In a controversial manner, certain pharmacological studies indicate that the various enkephalin metabolites (Tyr-Gly-Gly) bind weakly or do not bind to opioid receptors. The supposition was that it is likely that these very short enkephalin metabolites represent a separate class of pharmacological agents whose effects are mediated by a non-opioid receptor mechanism [24]. In our study, previous administration of naloxone, an opioid non-selective antagonist, suppresses the antinociceptive activity of the peptide fragments tested.…”

Selective distribution of protein kinase A regulatory subunit RIIα in rodent gliomas

“…29 Ultrathin sections were processed for immunocytochemistry by using a rabbit anti-RIIa antibody (Santa Cruz Biotechnology; dilution 1:20) revealed by a secondary 12-nm gold-conjugated antibody (Jackson ImmunoResearch, West Grove, PA, USA). 29 Double labeling was performed after golgin 97 (1:20) and RIIa (1:20) antibody incubation with 6-nm and 12-nm gold-conjugated antibodies (Jackson ImmunoResearch). As controls, some grids were treated with the incubation mixture without the primary antibody.…”

Induction of anin vitroreversible hypometabolism through chitosan-based nanoparticles

“…The effects of DADLE have been investigated also in different in vitro systems, demonstrating that it can significantly slow down the proliferation rate of some cell lines, derived from hibernating (woodchuck) and non-hibernating (rat, human) species, in a dosedependent manner (Kampa et al, 1997). DADLE seems to reduce both the RNA transcription and the RNA export to cytoplasm; moreover, it probably provokes a cascade effect, thus affecting other cellular functions such as protein synthesis and cell proliferation (Baldelli et al, 2004(Baldelli et al, , 2006Vecchio et al, 2006). Interestingly, no apoptotic or necrotic cells have ever been found in different cell lines treated with DADLE; indeed, it seems that DADLE can slow down the cellular activities without increasing the physiological number of dead cells and, finally, after removing DADLE from the culture medium, the effects disappear quite rapidly on all cell lines tested so far .…”

“…Among different molecules tested in our laboratories and potentially useful in inducing a hypometabolic stasis at cellular level, D-Ala(2)-D-Leu(5)-enkephalin (DADLE) is the most promising since in the in vitro models can induce a significant lowering of both proliferation and transcription (Baldelli et al, 2006;Vecchio et al, 2006). DADLE is a synthetic delta opioid whose receptors are present both at the cell surface membrane (Cupo et al, 1992) and on the nuclear envelope (Belcheva et al, 1993).…”