This study investigated the effects of cigarette smoke on autophagy and apoptosis in oral mucosa epithelial cells. The results showed that cigarette smoke exposure increased both autophagy and apoptosis in a concentration-dependent manner in leuk-1 cells. When autophagy was inhibited with chloroquine, apoptosis increased, but apoptosis decreased when autophagy was activated with rapamycin. The study concludes that cigarette smoke can induce autophagy and apoptosis in oral epithelial cells through its toxic components such as nicotine, and inhibiting autophagy exacerbates cigarette smoke-induced apoptosis.

1. Juan Sebastián Londoño García

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

11. Results
3. CSE induces autophagy in Leuk-1 cells

12. Results
4. Inhibition of autophagy aggravates CSE-triggered apoptosis in leuk- 1 cells.

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.

16. Flowchart