Acetonic Extract of Buxus sempervirens Induces Cell Cycle Arrest, Apoptosis and Autophagy in Breast Cancer Cells

Plants are an invaluable source of potential new anti-cancer drugs. Here, we investigated the cytotoxic activity of the acetonic extract of Buxus sempervirens on five breast cancer cell lines, MCF7, MCF10CA1a and T47D, three aggressive triple positive breast cancer cell lines, and BT-20 and MDA-MB-435, which are triple negative breast cancer cell lines. As a control, MCF10A, a spontaneously immortalized but non-tumoral cell line has been used. The acetonic extract of Buxus sempervirens showed cytotoxic activity towards all the five studied breast cancer cell lines with an IC50 ranging from 7.74 µg/ml to 12.5 µg/ml. Most importantly, the plant extract was less toxic towards MCF10A with an IC50 of 19.24 µg/ml. Fluorescence-activated cell sorting (FACS) analysis showed that the plant extract induced cell death and cell cycle arrest in G0/G1 phase in MCF7, T47D, MCF10CA1a and BT-20 cell lines, concomitant to cyclin D1 downregulation. Application of MCF7 and MCF10CA1a respective IC50 did not show such effects on the control cell line MCF10A. Propidium iodide/Annexin V double staining revealed a pre-apoptotic cell population with extract-treated MCF10CA1a, T47D and BT-20 cells. Transmission electron microscopy analyses indicated the occurrence of autophagy in MCF7 and MCF10CA1a cell lines. Immunofluorescence and Western blot assays confirmed the processing of microtubule-associated protein LC3 in the treated cancer cells. Moreover, we have demonstrated the upregulation of Beclin-1 in these cell lines and downregulation of Survivin and p21. Also, Caspase-3 detection in treated BT-20 and T47D confirmed the occurrence of apoptosis in these cells. Our findings indicate that Buxus sempervirens extract exhibit promising anti-cancer activity by triggering both autophagic cell death and apoptosis, suggesting that this plant may contain potential anti-cancer agents for single or combinatory cancer therapy against breast cancer

Role of specific histones methyltransferases of H3K9 in the balance between cell proliferation and differenciation

Chez les eucaryotes, l’expression des gènes dépend en partie du degré de compaction de la chromatine. La structure chromatinienne est régulée par des marques dites épigénétiques,telles que les modifications post-traductionnelles des protéines structurelles de la chromatine, les histones. Ainsi, la méthylation de la lysine 9 de l’histone H3 (H3K9) sur le promoteur des gènes est essentiellement associée à la répression de la transcription. H3K9 est méthylée par différentes enzymes appelées lysine méthyltransférases (KMTs). L’objectif principal de mon projet de thèse a été de mieux comprendre le rôle de principales KMTs de H3K9, que sontG9a, GLP, Suv39h1 et SETDB1, dans la régulation de l’équilibre entre prolifération et différenciation terminale. Pour cela, j’ai utilisé le modèle de différenciation terminale de cellules du muscle squelettique. En effet, durant la différenciation terminale, les myoblastes arrêtent de proliférer et fusionnent entre eux pour former de longues cellules multi nucléées que sont les myotubes. Ce processus implique, d’une part, l’expression des gènes de différenciation musculaire et, d’autre part, la répression irréversible des gènes associés à la prolifération cellulaire. L’introduction bibliographique de ce travail de thèse est séparée en trois chapitres. Le premier chapitre porte sur la chromatine et ses modifications post-traductionnelles. Le second s’attache à décrire les rôles de la méthylation de H3K9 et, en particulier, des quatre KMTs sur lesquelles j’ai travaillé durant ma thèse : G9a, GLP, SETDB1 et Suv39h1. Dans le troisième chapitre, je présente le modèle de la différenciation terminale du muscle squelettique. Dans la partie "Résultats", je décris deux des principales études que j’ai menées durant ma thèse. La première porte sur les rôles antagonistes de G9a et GLP. La seconde porte sur le rôle de SETDB1 durant la différenciation musculaire. Les résultats que j’ai obtenus sont discutés dans cette partie. Je conclus ce manuscrit en discutant mes résultats de manière plus générale et en proposant des perspectives à long terme. Enfin, une annexe présentera les autres articles de recherche auxquels j’ai participé pendant ma thèse.In eukaryotes, gene expression partly relies on chromatin compaction degree. Chromatin status is controlled by epigenetic marks, such as histones (chromatin structural proteins) posttranslational modifications. As an example, histone H3 lysine 9 (H3K9) methylation on gene promoters is mainly associated with transcriptional repression. H3K9 is methylated by several enzymes called lysine methyltransferases (KMTs). The aim of my thesis project was to understand the role of the H3K9 KMTs, G9a, GLP, Suv39h1 and SETDB1 in regulating the balance between proliferation and terminal differentiation. For this purpose, I used skeletal muscle terminal differentiation as model. Upon muscle terminal differentiation, myoblasts exit, in an irreversible way, from the cell cycle and under go differentiation where cells fusion and form myotubes. During this process, cell cycle genes are permanently silenced and muscle specific genes are activated. Thesis introduction is divided into three chapters. The first chapter focuses on chromatin and post-translational modifications. The second chapter describes H3K9 methylation characteristics and the role of the four KMTs that I studied during my thesis project: G9a,GLP, Suv39h1 and SETDB1. In the third chapter, the skeletal muscle terminal differentiation model is described in details. Results section reports my two major studies outcomes and their discussion. The first concerns the antagonistic roles of G9a and GLP regarding the muscle terminal differentiation and the second focuses on the role of SETDB1 during muscle differentiation. Finally, I conclude this manuscript by a plainer discussion followed by long term perspectives and an appendix presents other research articles, in which I collaborated during my PhD

Rôle d'histones methyltransférases spécifiques de H3K9 dans l'équilibre prolifération et différenciation cellulaire

In eukaryotes, gene expression partly relies on chromatin compaction degree. Chromatin status is controlled by epigenetic marks, such as histones (chromatin structural proteins) posttranslational modifications. As an example, histone H3 lysine 9 (H3K9) methylation on gene promoters is mainly associated with transcriptional repression. H3K9 is methylated by several enzymes called lysine methyltransferases (KMTs). The aim of my thesis project was to understand the role of the H3K9 KMTs, G9a, GLP, Suv39h1 and SETDB1 in regulating the balance between proliferation and terminal differentiation. For this purpose, I used skeletal muscle terminal differentiation as model. Upon muscle terminal differentiation, myoblasts exit, in an irreversible way, from the cell cycle and under go differentiation where cells fusion and form myotubes. During this process, cell cycle genes are permanently silenced and muscle specific genes are activated. Thesis introduction is divided into three chapters. The first chapter focuses on chromatin and post-translational modifications. The second chapter describes H3K9 methylation characteristics and the role of the four KMTs that I studied during my thesis project: G9a,GLP, Suv39h1 and SETDB1. In the third chapter, the skeletal muscle terminal differentiation model is described in details. Results section reports my two major studies outcomes and their discussion. The first concerns the antagonistic roles of G9a and GLP regarding the muscle terminal differentiation and the second focuses on the role of SETDB1 during muscle differentiation. Finally, I conclude this manuscript by a plainer discussion followed by long term perspectives and an appendix presents other research articles, in which I collaborated during my PhD.Chez les eucaryotes, l’expression des gènes dépend en partie du degré de compaction de la chromatine. La structure chromatinienne est régulée par des marques dites épigénétiques,telles que les modifications post-traductionnelles des protéines structurelles de la chromatine, les histones. Ainsi, la méthylation de la lysine 9 de l’histone H3 (H3K9) sur le promoteur des gènes est essentiellement associée à la répression de la transcription. H3K9 est méthylée par différentes enzymes appelées lysine méthyltransférases (KMTs). L’objectif principal de mon projet de thèse a été de mieux comprendre le rôle de principales KMTs de H3K9, que sontG9a, GLP, Suv39h1 et SETDB1, dans la régulation de l’équilibre entre prolifération et différenciation terminale. Pour cela, j’ai utilisé le modèle de différenciation terminale de cellules du muscle squelettique. En effet, durant la différenciation terminale, les myoblastes arrêtent de proliférer et fusionnent entre eux pour former de longues cellules multi nucléées que sont les myotubes. Ce processus implique, d’une part, l’expression des gènes de différenciation musculaire et, d’autre part, la répression irréversible des gènes associés à la prolifération cellulaire. L’introduction bibliographique de ce travail de thèse est séparée en trois chapitres. Le premier chapitre porte sur la chromatine et ses modifications post-traductionnelles. Le second s’attache à décrire les rôles de la méthylation de H3K9 et, en particulier, des quatre KMTs sur lesquelles j’ai travaillé durant ma thèse : G9a, GLP, SETDB1 et Suv39h1. Dans le troisième chapitre, je présente le modèle de la différenciation terminale du muscle squelettique. Dans la partie "Résultats", je décris deux des principales études que j’ai menées durant ma thèse. La première porte sur les rôles antagonistes de G9a et GLP. La seconde porte sur le rôle de SETDB1 durant la différenciation musculaire. Les résultats que j’ai obtenus sont discutés dans cette partie. Je conclus ce manuscrit en discutant mes résultats de manière plus générale et en proposant des perspectives à long terme. Enfin, une annexe présentera les autres articles de recherche auxquels j’ai participé pendant ma thèse

Rôle d'histones methyltransférases spécifiques de H3K9 dans l'équilibre prolifération et différenciation cellulaire

Chez les eucaryotes, l expression des gènes dépend en partie du degré de compaction de la chromatine. La structure chromatinienne est régulée par des marques dites épigénétiques,telles que les modifications post-traductionnelles des protéines structurelles de la chromatine, les histones. Ainsi, la méthylation de la lysine 9 de l histone H3 (H3K9) sur le promoteur des gènes est essentiellement associée à la répression de la transcription. H3K9 est méthylée par différentes enzymes appelées lysine méthyltransférases (KMTs). L objectif principal de mon projet de thèse a été de mieux comprendre le rôle de principales KMTs de H3K9, que sontG9a, GLP, Suv39h1 et SETDB1, dans la régulation de l équilibre entre prolifération et différenciation terminale. Pour cela, j ai utilisé le modèle de différenciation terminale de cellules du muscle squelettique. En effet, durant la différenciation terminale, les myoblastes arrêtent de proliférer et fusionnent entre eux pour former de longues cellules multi nucléées que sont les myotubes. Ce processus implique, d une part, l expression des gènes de différenciation musculaire et, d autre part, la répression irréversible des gènes associés à la prolifération cellulaire. L introduction bibliographique de ce travail de thèse est séparée en trois chapitres. Le premier chapitre porte sur la chromatine et ses modifications post-traductionnelles. Le second s attache à décrire les rôles de la méthylation de H3K9 et, en particulier, des quatre KMTs sur lesquelles j ai travaillé durant ma thèse : G9a, GLP, SETDB1 et Suv39h1. Dans le troisième chapitre, je présente le modèle de la différenciation terminale du muscle squelettique. Dans la partie "Résultats", je décris deux des principales études que j ai menées durant ma thèse. La première porte sur les rôles antagonistes de G9a et GLP. La seconde porte sur le rôle de SETDB1 durant la différenciation musculaire. Les résultats que j ai obtenus sont discutés dans cette partie. Je conclus ce manuscrit en discutant mes résultats de manière plus générale et en proposant des perspectives à long terme. Enfin, une annexe présentera les autres articles de recherche auxquels j ai participé pendant ma thèse.In eukaryotes, gene expression partly relies on chromatin compaction degree. Chromatin status is controlled by epigenetic marks, such as histones (chromatin structural proteins) posttranslational modifications. As an example, histone H3 lysine 9 (H3K9) methylation on gene promoters is mainly associated with transcriptional repression. H3K9 is methylated by several enzymes called lysine methyltransferases (KMTs). The aim of my thesis project was to understand the role of the H3K9 KMTs, G9a, GLP, Suv39h1 and SETDB1 in regulating the balance between proliferation and terminal differentiation. For this purpose, I used skeletal muscle terminal differentiation as model. Upon muscle terminal differentiation, myoblasts exit, in an irreversible way, from the cell cycle and under go differentiation where cells fusion and form myotubes. During this process, cell cycle genes are permanently silenced and muscle specific genes are activated. Thesis introduction is divided into three chapters. The first chapter focuses on chromatin and post-translational modifications. The second chapter describes H3K9 methylation characteristics and the role of the four KMTs that I studied during my thesis project: G9a,GLP, Suv39h1 and SETDB1. In the third chapter, the skeletal muscle terminal differentiation model is described in details. Results section reports my two major studies outcomes and their discussion. The first concerns the antagonistic roles of G9a and GLP regarding the muscle terminal differentiation and the second focuses on the role of SETDB1 during muscle differentiation. Finally, I conclude this manuscript by a plainer discussion followed by long term perspectives and an appendix presents other research articles, in which I collaborated during my PhD.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

The steroid hormone receptor EcR finely modulates Drosophila lifespan during adulthood in a sex-specific manner

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Single-step nasal reconstruction with osteocutaneous forearm flap after total rhinectomy.

Nasal defects resulting from tumor excision can be classified according to tissues involved, such as skin, cartilage, and bone. Although in the case of "simple" defects, reconstruction with locoregional flap eventually associated with cartilage grafts can lead to satisfactory results; in the case of total or partial rhinectomy, a minimum of 3 and a maximum of 7 operations have to be performed in the current series to achieve an acceptable end result. We present the case of a total rhinectomy reconstruction in a single-step procedure with an osteocutaneous forearm free flap (RFOFF). Clinical Report: A 50-year-old man underwent total rhinectomy to excise a pathologically proven T4aN0 moderately differentiated squamous cell carcinoma of the nose; contemporary single-step reconstruction with an RFOFF was performed. Adjuvant radiotherapy was performed. Result: At 18 months of follow-up, the patient is free of disease and no postirradiation flap damage has been experienced; the flap notably did not appear to be bulky. Conclusions: We believe that the RFOFF is morphologically and functionally better than other flaps owing to its capability to adapt to the new environments of the nasal cavity, and to avoid, when possible, a three-dimensional reconstruction of the same. Copyright © 2012 by Mutaz B. Habal, MD

Canonical Wnt signalling regulates nuclear export of Setdb1 during skeletal muscle terminal differentiation

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Scapula free flap for complex maxillofacial reconstruction

INTRODUCTION: Composite tissue defects of the mandible and maxilla, after resection of head and neck malignancies, osteoradionecrosis, malformations, or traumas, cause functional and aesthetic problems. Nowadays, microvascular free flaps represent the main choice for the reconstruction of these defects. Among the various flaps proposed, the scapula flap has favorable characteristics that make it suitable for bone, soft tissue, or combined defects. MATERIALS: We report 7 cases of reconstruction of complex maxillofacial defects with subscapular system flaps. The patients treated had Romberg syndrome (1 case), malignant tumors (5 cases), and result of previous trauma (1 case).Location of deficit was the maxilla (3 cases), the mandible (2 case), the ethmoidal-maxillary region (1 case) and the upper and middle thirds of the face in the last case. METHODS: In 2 cases, a parascapular system flap was used; in 5 cases, a composite flap with latissimus dorsi muscle and scapular bone. RESULTS: Neither failure of the harvested flaps nor complications in the donor site were evidenced. A good aesthetic and functional outcome was obtained in all cases. DISCUSSION: Many free flaps have been proposed for the reconstruction of defects in the maxillofacial region such as fibula, deep circumflex iliac artery, scapula, among the bone flaps; and forearm, rectus abdominis, and anterolateral thigh, among the soft tissue flaps. The choice of the flap to use depends on the length of the bone defect and the amount of soft tissues required. The subscapular system has the advantage of providing different flaps based on the same pedicle. The osteofasciocutaneous scapular free flap, in particular, allows wide mobility of soft tissues (parascapular flap) with respect to its bone component (scapular bone), resulting suitable for defects of large size involving both the soft tissues and the bone. CONCLUSIONS: Although the fibula flap and the deep circumflex iliac artery flap remain the first choice for bone reconstructions of the mandible and maxilla, the scapula flap has some features that make its use extremely advantageous in some circumstances. In particular, we advocate the use of the osteomuscular latissimus dorsi-scapula flap for reconstruction of large-volume defects involving the bone and soft tissues, whereas fasciocutaneous parascapular flaps represent a valid alternative to forearm flap and anterolateral thigh flap in the reconstruction of soft tissue defects. Copyright © 2009 by Mutaz B. Habal, MD

Scapula Free Flap for Complex Maxillofacial Reconstruction

Introduction: Composite tissue defects of the mandible and maxilla, after resection of head and neck malignancies, osteoradionecrosis, malformations, or traumas, cause functional and aesthetic problems. Nowadays, microvascular free flaps represent the main choice for the reconstruction of these defects. Among the various flaps proposed, the scapula flap has favorable characteristics that make it suitable for bone, soft tissue, or combined defects. Materials: We report 7 cases of reconstruction of complex maxillofacial defects with subscapular system flaps. The patients treated had Romberg syndrome (I case), malignant tumors (5 cases), and result of previous trauma (1 case). Location of deficit was the maxilla (3 cases), the mandible (2 case), the ethmoidal-maxillary region (I case) and the upper and middle thirds of the face in the last case. Methods: In 2 cases, a parascapular system flap was used; in 5 cases, a composite flap with latissimus dorsi Muscle and scapular bone. Results: Neither failure of the harvested flaps nor complications in the donor site were evidenced. A good aesthetic and functional outcome was obtained in all cases. Discussion: Many free flaps have been proposed for the reconstruction of defects in the maxillofacial region such as fibula, deep circumflex iliac artery, scapula, among the bone flaps; and forearm, rectus abdominis, and anterolateral thigh, among the soft tissue flaps. The choice of the flap to use depends on the length of the bone defect and the amount of soft tissues required. The subscapular system has the advantage of providing different flaps based on the same pedicle. The osteofasciocutaneous scapular free flap, in particular, allows wide mobility of soft tissues (parascapular flap) with respect to its bone component (scapular bone), resulting suitable for defects of large size involving both the soft tissues and the bone. Conclusions: Although the fibula flap and the deep circumflex iliac artery flap remain the first choice for bone reconstructions of the mandible and maxilla, the scapula flap has some features that make its use extremely advantageous in sortie circumstances. In particular, we advocate the use of the osteomuscular latissimus dorsi-scapula flap for reconstruction of large-volume defects involving the bone and soft tissues, whereas fosciocutaneous parascapular flaps represent a valid alternative to forearm flap and anterolateral thigh flap in the reconstruction of soft tissue defects

The SWI/SNF Subunit/Tumor Suppressor BAF47/INI1 Is Essential in Cell Cycle Arrest upon Skeletal Muscle Terminal Differentiation

<div><p>Myogenic terminal differentiation is a well-orchestrated process starting with permanent cell cycle exit followed by muscle-specific genetic program activation. Individual SWI/SNF components have been involved in muscle differentiation. Here, we show that the master myogenic differentiation factor MyoD interacts with more than one SWI/SNF subunit, including the catalytic subunit BRG1, BAF53a and the tumor suppressor BAF47/INI1. Downregulation of each of these SWI/SNF subunits inhibits skeletal muscle terminal differentiation but, interestingly, at different differentiation steps and extents. BAF53a downregulation inhibits myotube formation but not the expression of early muscle-specific genes. BRG1 or BAF47 downregulation disrupt both proliferation and differentiation genetic programs expression. Interestingly, BRG1 and BAF47 are part of the SWI/SNF remodeling complex as well as the N-CoR-1 repressor complex in proliferating myoblasts. However, our data show that, upon myogenic differentiation, BAF47 shifts in favor of N-CoR-1 complex. Finally, BRG1 and BAF47 are well-known tumor suppressors but, strikingly, only BAF47 seems essential in the myoblasts irreversible cell cycle exit. Together, our data unravel differential roles for SWI/SNF subunits in muscle differentiation, with BAF47 playing a dual role both in the permanent cell cycle exit and in the regulation of muscle-specific genes.</p></div