The steroidal alkaloid tomatidine is an aglycone of α-tomatine, which is abundant in tomato leaves and has several biological activities. Tomatidine has been reported to inhibit the growth of cultured cancer cells in vitro, but its anti-cancer activity in vivo and inhibitory effect against gastric cancer cells remain unknown. We investigated the efficacy of tomatidine using human gastric cancer-derived 85As2 cells and its tumor-bearing mouse model and evaluated the effect of tomatidine-rich tomato leaf extract (TRTLE) obtained from tomato leaves. In the tumor-bearing mouse model, tumor growth was significantly inhibited by feeding a diet containing tomatidine and TRTLE for 3 weeks. Tomatidine and TRTLE also inhibited the proliferation of cultured 85As2 cells. Microarray data of gene expression analysis in mouse tumors revealed that the expression levels of mRNAs belonging to the type I interferon signaling pathway were altered in the mice fed the diet containing tomatidine and TRTLE. Moreover, the knockdown of one of the type I interferon-stimulated genes (ISGs), interferon α-inducible protein 27 (IFI27), inhibited the proliferation of cultured 85As2 cells. This study demonstrates that tomatidine and TRTLE inhibit the tumor growth in vivo and the proliferation of human gastric cancer-derived 85As2 cells in vitro, which could be due to the downregulation of ISG expression.

Oxytocin (OT) influences various physiological functions such as uterine contractions, maternal/social behavior, and analgesia. Opioid signaling pathways are involved in one of the analgesic mechanisms of OT. We previously showed that OT acts as a positive allosteric modulator (PAM) and enhances μ-opioid receptor (MOR) activity. In this study, which focused on other opioid receptor (OR) subtypes, we investigated whether OT influences opioid signaling pathways as a PAM for δ-OR (DOR) or κ-OR (KOR) using human embryonic kidney-293 cells expressing human DOR or KOR, respectively. The CellKey^TM results showed that OT enhanced impedance induced by endogenous/exogenous KOR agonists on KOR-expressing cells. OT did not affect DOR activity induced by endogenous/exogenous DOR agonists. OT potentiated the KOR agonist-induced Gi/o protein-mediated decrease in intracellular cAMP, but did not affect the increase in KOR internalization caused by the KOR agonists dynorphin A and (-)-U-50488 hydrochloride (U50488). OT did not bind to KOR orthosteric binding sites and did not affect the binding affinities of dynorphin A and U50488 for KOR. These results suggest that OT is a PAM of KOR and MOR and enhances G protein signaling without affecting β-arrestin signaling. Thus, OT has potential as a specific signaling-biased PAM of KOR.

Activated opioid receptors transmit internal signals through two major pathways: the G-protein-mediated pathway, which exerts analgesia, and the β-arrestin-mediated pathway, which leads to unfavorable side effects. Hence, G-protein-biased opioid agonists are preferable as opioid analgesics. Rubiscolins, the spinach-derived naturally occurring opioid peptides, are selective δ opioid receptor agonists, and their p.o. administration exhibits antinociceptive effects. Although the potency and effect of rubiscolins as G-protein-biased molecules are partially confirmed, their in vitro profiles remain unclear. We, therefore, evaluated the properties of rubiscolins, in detail, through several analyses, including the CellKey^TM assay, cADDis^® cAMP assay, and PathHunter^® β-arrestin recruitment assay, using cells stably expressing µ, δ, κ, or µ/δ heteromer opioid receptors. In the CellKey^TM assay, rubiscolins showed selective agonistic effects for δ opioid receptor and little agonistic or antagonistic effects for µ and κ opioid receptors. Furthermore, rubiscolins were found to be G-protein-biased δ opioid receptor agonists based on the results obtained in cADDis^® cAMP and PathHunter^® β-arrestin recruitment assays. Finally, we found, for the first time, that they are also partially agonistic for the µ/δ dimers. In conclusion, rubiscolins could serve as attractive seeds, as δ opioid receptor-specific agonists, for the development of novel opioid analgesics with reduced side effects.

The issue of tolerance to continuous or repeated administration of opioids should be addressed. The ability of ketamine to improve opioid tolerance has been reported in clinical studies, and its mechanism of tolerance may involve improved desensitization of μ-opioid receptors (MORs). We measured changes in MOR activity and intracellular signaling induced by repeated fentanyl and morphine administration and investigated the effects of ketamine on these changes with human embryonic kidney 293 cells expressing MOR using the CellKey™, cADDis cyclic adenosine monophosphate, and PathHunter^® β-arrestin recruitment assays. Repeated administration of fentanyl or morphine suppressed the second MOR responses. Administration of ketamine before a second application of opioids within clinical concentrations improved acute desensitization and enhanced β-arrestin recruitment elicited by fentanyl but not by morphine. The effects of ketamine on fentanyl were suppressed by co-treatment with an inhibitor of G-protein-coupled receptor kinase (GRK). Ketamine may potentially reduce fentanyl tolerance but not that of morphine through modulation of GRK-mediated pathways, possibly changing the conformational changes of β-arrestin to MOR.

Remifentanil (REM) and fentanyl (FEN) are commonly used analgesics that act by activating a µ-opioid receptor (MOR). Although optimal concentrations of REM can be easily maintained during surgery, it is sometimes switched to FEN for optimal pain regulation. However, standards for this switching protocol remain unclear. Opioid anesthetic efficacy is decided in part by MOR desensitization; thus, in this study, we investigated the desensitization profiles of REM and FEN to MOR. The efficacy and potency during the 1st administration of REM or FEN in activating the MOR were almost equal. Similarly, in β arrestin recruitment, which determines desensitization processes, they showed no significant differences. In contrast, the 2nd administration of FEN resulted in a stronger MOR desensitization potency than that of REM, whereas REM showed a higher internalization potency than FEN. These results suggest that different β arrestin-mediated signaling caused by FEN or REM led to their distinct desensitization and internalization processes. Our three-dimensional analysis, with in silico binding of REM and FEN to MOR models, highlighted that REM and FEN bound to similar but distinct sites of MOR and led to distinct β arrestin-mediated profiles, suggesting that distinct binding profiles to MOR may alter β arrestin activity, which accounts for MOR desensitization and internalization.