2. Introduction
Autophagy:
1. Unfavourable microenvironment (hipoxia or
nutrient deprivation)
1. Cells degrade intracellular protein aggregates,
oxidized lipids, and damaged organelles
1. Cells coat damaged proteins and organelles with
a bilayer membrane structure and transport them
to lysosomes for their metabolism, which relieves
stress and maintains metabolic balance.
3. Introduction
Apoptosis:
1. Signal activates the expression of
proapoptotic proteins that can inhibit the
complex BAX/BAK in the mitochondria.
2. the permeability of the membrane
increases and the cytochrome c is released
to the citosol.
3. Cytochrome c forms the apoptosome, and
this activate proteins caspases.
4. Caspases activate enzymes that can
degrade the DNA.
Cigarette smoke:
1. Increases the salivary levels of certain
pro-inflammatory cytokines.
1. Nicotine can stimulate differentiation of
the mucous membrane and epidermal
keratinocytes, and this may be
associated with the occurrence of
lesions in the mouth.
1. high concentration of nicotine can
promote apoptosis and even have
directiv toxic effects on cells.
4. Objective
This study aimed to investigate the effects of cigarette
smoke on autophagy and apoptosis in oral mucosa
epithelial cells.
5. Materials and methods
1. Immunohistochemistry:
It is the identification of a tissue by means of a
specific interaction between an antigen and an
antibody where the antibody has been visibly
labeled, the cell is colored to demonstrate the
presence and cellular location of a molecule of
interest.
6. Materials and methods
2. Western blot:
Proteins from cell extracts are separated by
means of the SDS-PAGE technique, in which
they are dissolved in a solution with a negatively
charged detergent.
After electrophoresis, the proteins are transferred
to a filter, which is incubated with antibodies that
react with the protein of interest.
7. Materials and methods
3. Immunofluorescence:
Is an immunostaining technique that uses antibodies
chemically bound to a fluorescent substance to
demonstrate the presence of a particular molecule.
This technique can be used in combination with other
fluorescent staining techniques that do not use antibodies,
such as DAPI for marking DNA.
8. Materials and methods
4. Flow cytometry:
Technique that allows obtaining information on cell
populations from an individualized study of a large
number of cells.
The suspension of cells in isotonic solution is passed
through a small hole so that when they leave they do it
one by one in a row forming part of a continuous current
or cylindrical flow.
9. Results
1. CSE induces LC3-II expresión in Leuk-1
cells in a concentration and time
dependent manner
LC3-II: molecular marker of autophagosomes
Leuk-1 cells: human oral mucosal epithelial cell
line
10. Results
2. Concentration dependence of CQ(chloroquine) and
RAPA(rapamycin) on the expression of LC3-II
Chloroquine: CQ is a lysosome inhibitor that inhibits
autophagosome and lysosome fusion and upregulated the
expression of caspase 3
Rapamycin: Autophagy activator
13. Discussion
Shi et al, 2012 CSE, CQ and RAPA induced the
expression of LC3-II in leuk-1 cells
Gump y Thorburn, 2011 Elevated intracellular Ca2+
concentrations, oxidative damage
induced by hydroxyl free radicals and
other reactive oxygen species (ROS),
toxins, NO, growth factors and
hormones, can activate apoptosis
Chen Cai et al, 2019 Autophagy should play an
important role in the development
of new cancer treatment strategies
14. Conclusions
1. CSE increases both apoptosis and autophagy, however when
autophagy is inhibited the expression of apoptotic proteins such as
caspase 3 is increased and when autophagy is activated, these
proteins decrease.
1. In addition to being an addictive substance, nicotine also has toxic
components that induce the autophagy and apoptosis of certain cells
in the body, such as vascular endothelial cells and oral mucosal
epithelial cells.
15. References
1. Liu q, Zhao M, Chen W, xu K. Mainstream cigarette smoke induces
autophagy and promotes apoptosis in oral mucosal ephitelial cells
[Internet]. Elsevier. 2020 [cited 13 February 2020]. Available from:
http://www.elsevier.com
1. Martinez L, Vargas N, Cardona L, Ramirez S. Biología molecular. 8th
ed. Medellín: Editorial universidad pontificia bolivariana; 2018.
Active or passive smoking may impact the salivary levels of certain pro-inflammatory cytokines (Ge et al., 2019). Smoking can reduce the defence function of oral epithelial cells and increase the risk for infection with Candida albicans (Wang et al., 2014). Nicotine, one of the toxic substance in tobacco, can stimulate differentiation of the mucous membrane and epidermal keratinocytes, and abnormal differentiation induced by ni- cotine may be associated with the occurrence of white lesions in the mouth (Wang et al., 2014). In addition, high concentrations of nicotine. In addition, high concentration of nicotine can promote apoptosis and even have directiv toxic effects on cells
I-Método Inmunohistoquímico Directo, en él, el anticuerpo específico contra la sustancia que se quiere detectar está marcado con partículas detectables al microscopio (ej.fluorescenciao peroxidasa) .
I-Método Inmunohistoquímico Indirecto, en él, la señal del anticuerpo se amplía realizando sucesivas capas de anticuerpos o marcadores (como son Peroxidasa/Anti-Peroxidasa (PAP), Complejo de Avidina Biotina peroxidasa (ABC).
Es la identificación de un tejido por medio de una interacción específica entre un antígeno y un anticuerpo donde el anticuerpo ha sido visiblemente marcado, la célula se colorea para demostrar la presencia y la localización celular de una molécula de interés.
SDS-PAGE: electroforesis en gel de poliacrilamida con dodecilsulfato sódico
Las proteínas procedentes de extractos celulares son separadas por medio de la técnica SDS-PAGE, en la que son disueltas en una solución con un detergente cargado negativamente.
Tras la electroforesis, las proteínas se transfieren a un filtro , que se incuba con anticuerpos que reaccionan con la proteína de interés.
La inmunofluorescencia, como técnica de tinción, puede ser utilizada en cortes de tejidos, líneas celulares cultivadas, células individuales y secreciones que contengan células en suspensión (por ejemplo esputo) con la finalidad de analizar la presencia y distribución de proteínas, glúcidos y moléculas pequeñas tanto de origen biológico como no. Esta técnica puede ser utilizada en combinación con otras técnicas de coloración fluorescente que no hagan uso de anticuerpos, como por ejemplo DAPI para marcar ADN.
DAPI: es un marcador fluorescente que se une fuertemente a regiones enriquecidas en adenina y timina en secuencias de ADN. Es utilizado ampliamente en la microscopía de fluorescencia. DAPI no puede pasar a través de la membrana celular, debido a eso, se utiliza para teñir células muertas.
La inmunofluorescencia es una técnica de inmunomarcación que hace uso de anticuerpos unidos químicamente a una sustancia fluorescente para demostrar la presencia de una determinada molécula.
Permite analizar una población celular proporcionando una amplia vriedad de parametros
técnica que permite obtener información sobre poblaciones celulares a partir de un estudio individualizado de un gran número de células (habitualmente entre 5000 y 10000) que por tanto serán una muestra lo suficientemente representativa del conjunto poblacional. La suspensión de células en solución isotónica se hace pasar a través de un pequeño orificio de modo que cuando salen lo hacen una a una (en fila india) formando parte de una corriente continua o flujo cilíndrico.
En la grafica a: se ve el aumento de la
LC3 is a common molecular marker of autophagosomes in mammals and exists as two interconvertible forms, LC3-II and LC3-I. The level of LC3-II, which is located on the membrane surface of the autophagosome vesicle (Tanida, Ueno, & Kominami, 2004), increases with augmentation of the autophagosome membrane
Sequestosome 1 (SQSTM1, p62) is an au- tophagic degradation substrate that recognizes and delivers damaged organelles or protein aggregates to autophagosomes for degradation (Komatsu & Ichimura, 2010).
When autophagy occurs, p62 is degraded. CQ is a lysosome inhibitor that inhibits autophagosome and lysosome fusion. In this way, the occurrence of autophagy can be determined by the joint observation of LC3-II, Beclin-1 and p62 expression. Joint ob- servation of LC3-II, p62 and Beclin-1 expression can reveal whether autophagy is occurring. Members of the caspase protein family are some of the most critical proteins in the apoptosis process. Therefore, changes in caspase proteins can indirectly reflect apoptosis.
CQ is a lysosome inhibitor that inhibits autophagosome and lysosome fusion
Beclin 1: Beclin-1 is an important molecule in the process of autophagosome formation and an important component of the JNK signal transduction pathway. Through the formation of an autophagy protein complex, autophagy-related proteins are guided to the autophagy membrane, where they regulate the formation of autophagy precursors (Mizushima, 2007). When pro- duction of the Beclin-1 protein increased, the formation of autopha- gosomes increased, and the autophagic activity increased; however, autophagy was inhibited.
Cuando ocurre la autofagia p62se degrda
P 62
is important in cancer cells because the apoptosis mechanism is often mutated during tumorigenesis. Therefore, targeted autophagy should play an important role in the development of new cancer treatment strategies (Chen Cai et al., 2019).u