Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extracts and its purified products have been examined for induction of apoptosis in multiple cancer cell types; however, its underlying mechanisms remain undefined. We show that neem oil (i.e., neem), which contains majority of neem limonoids including azadirachtin, induced apoptotic and autophagic cell death. Gene silencing demonstrated that caspase cascade was initiated by the activation of caspase-9, whereas caspase-8 was also activated late during neem-induced apoptosis. Pretreatment of cancer cells with pan caspase inhibitor, z-VAD inhibited activities of both initiator caspases (e.g., caspase-8 and -9) and executioner caspase-3. Neem induced the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, suggesting the involvement of both caspase-dependent and AIF-mediated apoptosis. p21 deficiency caused an increase in caspase activities at lower doses of neem, whereas p53 deficiency did not modulate neem-induced caspase activation. Additionally, neem treatment resulted in the accumulation of LC3-II in cancer cells, suggesting the involvement of autophagy in neem-induced cancer cell death. Low doses of autophagy inhibitors (i.e., 3-methyladenine and LY294002) did not prevent accumulation of neem-induced LC3-II in cancer cells. Silencing of ATG5 or Beclin-1 further enhanced neem-induced cell death. Phosphoinositide 3-kinase (PI3K) or autophagy inhibitors increased neem-induced caspase-3 activation and inhibition of caspases enhanced neem-induced autophagy. Together, for the first time, we demonstrate that neem induces caspase-dependent and AIF-mediated apoptosis, and autophagy in cancer cells.
1H- and 13C-n.m.r. measurements enable direct observation of the rate of formation of dehydroalanine residues resulting from lysis of the disulphide bonds of insulin and oxidized glutathione in base at pD13. The data provide clear evidence for the beta-elimination mechanism for this reaction. The dehydroalanine-containing products from the lysis of insulin undergo secondary reactions.
Multi-drug resistance (MDR)-ATP binding cassette (ABC) transporters, ABCB1, ABCC1, and ABCG2 participate in the efflux of steroid hormones, estrogens, and androgens, which regulate prostate development and differentiation. The role of MDR-ABC efflux transporters in prostate epithelial proliferation and differentiation remains unclear. We hypothesized that MDR-ABC transporters regulate prostate differentiation and epithelium regeneration. Prostate epithelial differentiation was studied using histology, sphere formation assay, and prostate regeneration induced by cycles of repeated androgen withdrawal and replacement. Embryonic deletion of Abcg2 resulted in a decreased number of luminal cells in the prostate and increased sphere formation efficiency, indicating an imbalance in the prostate epithelial differentiation pattern. Decreased luminal cell number in the Abcg2 null prostate implies reduced differentiation. Enhanced sphere formation efficiency in Abcg2 null prostate cells implies activation of the stem/progenitor cells. Prostate regeneration was associated with profound activation of the stem/progenitor cells, indicating the role of Abcg2 in maintaining stem/progenitor cell pool. Since embryonic deletion of Abcg2 may result in compensation by other ABC transporters, pharmacological inhibition of MDR-ABC efflux was performed. Pharmacological inhibition of MDR-ABC efflux enhanced prostate epithelial differentiation in sphere culture and during prostate regeneration. In conclusion, Abcg2 deletion leads to activation of the stem/progenitor cells and enhances differentiating divisions; and pharmacological inhibition of MDR-ABC efflux leads to epithelial differentiation. Our study demonstrates for the first time that MDR-ABC efflux transporter inhibition results in enhanced prostate epithelial cell differentiation.
Drug discovery in the melatonin field has been hindered by lack of selective MT1 and MT2 melatonin receptors ligands with in vivo receptor type preferred efficacy. As a result, most pharmacological tools developed to target this receptor family lack discernable in vivo selectivity supporting a need for discovery of new ligands to diversify the toolbox, particularly for targeting MT1 receptors. Thus, the goal of the present research was to investigate efficacies of novel type preferring melatonin receptor ligands at MT1 utilizing in‐vivo murine models of chronobiological behavior. The novel MT1 type preferring compounds were identified by screening ultra large libraries for in‐silico docking predictions using the recently reported crystal structures of MT1 melatonin receptors. Several lead compounds were identified exhibiting favorable affinity at MT1 receptors as well as selective efficacy as inverse agonists. The present research efforts tested in vivo efficacy of two novel type preferring lead compounds designed to target MT1 as inverse agonists (N30#2 & N32) in re‐entrainment (“jet‐lag”) and circadian phase shift behavioral paradigms using C3H/HeN (C3H) mice. Both N30#2 and N32 (30 μg/mouse s.c.) administered for 3 days at the new dark onset after an abrupt 6 hour advance of the light‐dark cycle increased the number of days to and decelerated the rate of running wheel activity re‐entrainment, an effect opposite to agonist melatonin and consistent with the inverse agonist effects of non‐selective MT1/MT2 antagonist/inverse agonist luzindole in this paradigm. The luzindole‐like inverse agonist effect of N30#2 on re‐entrainment rate was abrogated in C3H MT1KO and preserved in C3H MT2KO compared to C3H wild‐type mice supporting MT1 selective effects. Surprisingly when N30#2 and N32 (0.9 & 30 μg/mouse s.c.) were administered at subjective dusk (CT 10) for 3 consecutive days in the phase shift paradigm, both compounds produced melatonin‐like effects as agonists. N30#2 was unable to produce melatonin‐like effects in C3H MT1KO that were maintained in C3H MT2KO mice suggesting selective effects mediated by targeting MT1 receptors. In the same paradigm when N30#2 was administered at subjective dawn (CT2), it displayed no efficacy in contrast to melatonin and consistent with luzindole‐like effects. Overall results revealed that two type preferring MT1 inverse agonists displayed differential and unique efficacy profiles across behavioral tests as inverse agonists in the jet‐lag paradigm or as agonists to phase advance circadian running wheel activity at subjective dusk. Novel MT1 type preferring inverse agonists produced both luzindole‐like and melatonin‐like effects that may suggest additional non‐canonical signaling pathways involved that depend on time of administration with respect to the circadian cycle.
Support or Funding Information
Supported by Pilot Grant NIH UL1 TR001412 and University at Buffalo grant (MLD), NIH R35 GM122481 (BKS), U24 DK1169195 (BLR & BKS), and NIMH Psychoactive Drug Screening Contract (BLR).
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