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Epigenetic mechanisms allow for transgenerational memory of an ancestor's environment and can affect the gene expression, physiology and phenotype of that ancestor's descendants, independent of DNA sequence alteration. Among many model organisms, Caenorhabditis elegans has been instrumental in studies of transgenerational inheritance, most of which have focused on the effects of external stressors of the parent worm on the life span and stress resistance of future generations. In this work, we used Nile red staining of accumulated lipids in C. elegans to investigate the transgenerational effect of two benzylisoquinoline alkaloids, namely, berberine and sanguinarine. Our results showed that a reduction in Nile red fluorescence can be propagated to subsequent worm generations. Using mutant worms, we found that the transgenerational effect requires the ASH‐2 component of the histone H3K4me3 complex and the HRDE‐1 worm Argonaute protein. Ash‐2 is also required for transgenerational inheritance of the xenobiotic response in the worm. Our study offers new insights into transmissible drug effects across multiple generations and suggests the importance of such analyses in the drug development process.

Dysregulation of apoptosis is a prime hallmark of leukemia. Therefore, drugs which restore the sensitivity of leukemic cells to apoptotic stimuli are promising candidates in the treatment of leukemia. The main objective of this dissertation was to examine the antileukemic effect of sanguinarine, in vitro, and to further examine the signaling mechanisms that may be involved. This study demonstrates that in human leukemic cells, sanguinarine activates a caspase-dependent apoptotic cell death pathway that is characterized by reactive oxygen species-dependent ceramide generation, and subsequent inhibition of Akt signaling pathway. In addition, sanguinarine also induces reactive oxygen species-dependent glutathione depletion and activation of extracellular signal-regulated kinase1/2. Moreover, inhibition of reactive oxygen species generation, using reactive oxygen species scavengers and antioxidants, significantly abrogates sanguinarine-induced ceramide generation, Akt dephosphorylation, extracellular signal-regulated kinase1/2 activation, and apoptosis. Sanguinarine-induced ceramide generation is mediated via reactive oxygen species-dependent activation of acid sphingomyelinase in Jurkat cells and inhibition of acid ceramidase and glucosylceramide synthase in both Jurkat and Molt-4 cells. Furthermore, the involvement of ceramide-activated protein phosphatase-1 in sanguinarine-induced Akt dephosphorylation and apoptosis is demonstrated. Altogether, this study underscores the critical role for reactive oxygen species-ceramide-Akt signaling pathway and reactive oxygen species-dependent extracellular signal-regulated kinase1/2 activation in the antileukemic action of sanguinarine. Understanding the molecular signaling mechanism of sanguinarine induced apoptosis undoubtedly should have a great impact on future sanguinarine based antileukemic drug development.

Résumé : Les ostéosarcomes sont des tumeurs malignes osseuses primaires qui affectent principalement les enfants et les adolescents. Les thérapies utilisées depuis les 30 dernières années n'ont malheureusement eu que très peu d'effet sur la survie des patients. Ce projet de doctorat évalue deux stratégies pour contrer certaines dérégulations des cellules d'ostéosarcome humain. La première consiste à abolir leur résistance à l'apoptose avec un alcaloïde d'origine végétale. La seconde consiste à accroître leur différenciation ou à réduire leur prolifération en utilisant des protéines morphogénétiques osseuses (BMPs) et leurs peptides dérivés (pBMPs). La première partie de ce doctorat s'intéresse donc au comportement et à la régulation des cellules d'ostéosarcome en comparaison avec celui des cellules osseuses normales afin d'identifier des cibles potentielles. Elle met l'accent sur trois caractéristiques de l'ostéosarcome: la résistance à l'apoptose, la prolifération incontrôlée des cellules et leur différenciation défectueuse. La sanguinarine, un alcaloïde d'origine végétale, est déjà utilisée pour traiter plusieurs types de cancers (sein et colon). Cet alcaloïde a été choisi pour vaincre la résistance à l'apoptose des cellules d'ostéosarcome. La sanguinarine a éradiqué les deux lignées cellulaires humaines d'ostéosarcome, MG-63 et SaOS-2, d'une manière dépendante du temps et de la dose. Les deux lignées cellulaires se distinguent par des états de différenciation et des taux de prolifération différents. L'alcaloïde induit l'apoptose par les voies extrinsèque et intrinsèque en activant les caspases-8 et -9. La sanguinarine semble agir sur les cellules d'ostéosarcome en fonction de leur potentiel tumorigène. La BMP-2, la BMP-9 et leurs peptides dérivés, pBMP-2 et pBMP-9, ont été utilisés pour contrer la différenciation défectueuse ou la prolifération incontrôlée des cellules d'ostéosarcome. La BMP-2, la BMP-9 et le pBMP-9 activent la phosphorylation des Smads dans les

V práci byly sledovány acidobazické a optické vlastnosti sanguinarinu a dihydrosanguinarinu v přítomnosti HCl, HNO3, H2SO4, H3PO4, CAPSO a kyseliny octové. Síla interakcí sanguinarinu s bioakticními sloučeninami může být ovlivněna jak kationty, tak i anionty inertního elektrolytu., Acid-base and optical properties of sanguinarine and dihydrosanguinarine were studied in the presence of HCl, HNO3, H2SO4, H3PO4, CAPSO and acetic acid (HAc) of different concentrations and their mixtures. The equilibrium constants pKR+ of the transition reaction between an iminium cation Q+ of sanguinarine and its uncharged QOH (pseudo-base, 6-hydroxy-dihydroderivative) form were calculated. A numerical interpretation of the A-pH curves by a SQUAD-G computer program was used. Remarkable shifts of formation parts of absorbance-pH (A-pH) curves to alkaline medium were observed. The shifts depend on the type and concentration of inert lectrolyte (the most remarkable for HNO3 and HCl ). The corresponding pKR+ values ranged from 7.21 to 8.16 in the same manner (?pKR+ = 0.81 and 0.73 for HNO3 and HCl, respectively). The priority effect of ionic species and ionic strength was confirmed in the presence of NaCl and KCl. The strength of interaction of SA with bioactive compounds (i.e. receptors, transport roteins, nucleic acids etc.) may be affected because of the observed influence of both cations and anions of the inert electrolytes.

V práci byly sledovány acidobazické a optické vlastnosti sanguinarinu a dihydrosanguinarinu v přítomnosti HCl, HNO3, H2SO4, H3PO4, CAPSO a kyseliny octové. Síla interakcí sanguinarinu s bioakticními sloučeninami může být ovlivněna jak kationty, tak i anionty inertního elektrolytu., Acid-base and optical properties of sanguinarine and dihydrosanguinarine were studied in the presence of HCl, HNO3, H2SO4, H3PO4, CAPSO and acetic acid (HAc) of different concentrations and their mixtures. The equilibrium constants pKR+ of the transition reaction between an iminium cation Q+ of sanguinarine and its uncharged QOH (pseudo-base, 6-hydroxy-dihydroderivative) form were calculated. A numerical interpretation of the A-pH curves by a SQUAD-G computer program was used. Remarkable shifts of formation parts of absorbance-pH (A-pH) curves to alkaline medium were observed. The shifts depend on the type and concentration of inert lectrolyte (the most remarkable for HNO3 and HCl ). The corresponding pKR+ values ranged from 7.21 to 8.16 in the same manner (?pKR+ = 0.81 and 0.73 for HNO3 and HCl, respectively). The priority effect of ionic species and ionic strength was confirmed in the presence of NaCl and KCl. The strength of interaction of SA with bioactive compounds (i.e. receptors, transport roteins, nucleic acids etc.) may be affected because of the observed influence of both cations and anions of the inert electrolytes.

The traditional therapeutic and surgical approaches have not been successful in the management of prostate cancer (CaP). Natural plant-based products have shown promise as anticancer agents. Sanguinarine, a benzophenanthridine alkaloid derived from the root of Sanguinaria Canadensis, has been shown to possess anti-microbial, antioxidant and anti-inflammatory properties. Our earlier studies suggested that sanguinarine may be developed as an agent for the management of prostate cancer. Based on this rationale, funded by the DOD (Award - W81XWH-04-1-0220), we initiated a study to investigate the hypothesis that sanguinarine will impart antiproliferative effects against prostate cancer via a modulation in NF-kB-pathway-mediated apoptosis. During the funding period, we have made reasonable progress towards our goals. However, the progress during this reporting period was hampered due to several unforeseen circumstances. Because of this reason, a one-year extension of the grant was also obtained in January 2007. So far, the key accomplishments of our project are as follows. We have demonstrated that sanguinarine possesses anti-proliferative effects against CaP in an athymic nude mice xenograft model. Further, our data suggested that sanguinarine-caused effects may be mediated via modulations in NF-B pathway and cyclin kinase inhibitor-cyclin-cyclin dependent kinase machinery. Our study with transgenic TRAMP model has suggested that sanguinarine may be developed as an agent for the management of CaP., The original document contains color images.