a master lecture for molecular biology about Recurrent exposure to nicotine differentiates human bronchial epithelial cells via epidermal growth factor receptor activation

1. Recurrent exposure to nicotine
differentiates human bronchial
epithelial cells via epidermal
growth factor receptor activation
Presented by
AFNAN SAID ZUITER

2. What is nicotine
• Nicotine (3-(1-methyl-2-
pyrrolidinyl)-pyridine) is one of
the major alkaloids present in
tobacco.
• An alkaloid (a nitrogen-
containing chemical) made by
the tobacco plant (Nicotiana
tabacum), potatoes, tomatoes,
eggplant and red peppers or
produced synthetically.

3. Nicotine’s effects
• Pharmacologic effects (including increased heart rate, heart stroke
volume, and oxygen consumption by the heart muscle)
• Psychodynamic effects (such as euphoria, increased alertness, and a
sense of relaxation).
• It behaves as a tumor promoter in transformed epithelial cells.
• It is also powerfully addictive.
– The symptoms of withdrawal, including anxiety, irritability,
restlessness, shortened attention span and an intense, sometimes
irresistible, craving for nicotine

4. • it stimulates the initiation of DNA synthesis and the in vitro
growth of several cell types
• plays a role in the onset of human diseases, such as atherogenesis,
Crohn's disease, periodontal damage and most importantly in
lung cancer
• facilitates the progression of human lung cancer cells by inducing
their proliferation
• stimulating tumor angiogenesis
• providing tumor cells with survival mechanisms against
chemotherapy
Nicotine’s effects

5. Nicotinic acetylcholine receptors
(nAChRs)
• Is prototypic ligand-gated ion channels that mediate fast synaptic transmission
• Exist on neuronal and non-neuronal cells such as bronchial epithelium
• Are agonists with components of cigarette smoke such as nicotine and NNK.
• The non-neuronal nAChR signaling pathway has considerable implications for
cancer and cardiovascular disease.
• α7 is the main nAChR subunit that mediates the proliferative effects of
nicotine in cancer cells.
• The cellular roles of non neuronal nAChRs: regulation of cell proliferation,
angiogenesis, apoptosis, migration, invasion and secretion.

6. Pentameric ion channel

7. Epidermal growth factor (EGF)
receptor
• Is a 170 kDa tyrosine kinase.
• Activated by binding of its specific ligands, including (EGF) & (TGFα)
• Ligand binding results in receptor dimerization, autophosphorylation,
activation of downstream signaling and lysosomal degradation.
• These downstream signaling proteins initiate several signal transduction
cascades, leading to DNA synthesis and cell proliferation. Such proteins
modulate phenotypes such as cell migration, adhesion, and proliferation.
• The kinase domain of EGFR can also cross-phosphorylate tyrosine
residues of other receptors it is aggregated with, and can itself be activated
in that manner.
• Phosphorylation of EGFR on serine and tyrosine residues attenuates
EGFR kinase activity.

9. Methods
1. Cell culture: Normal human bronchial epithelial cells (NHBE)
were cultured in bronchial epithelial growth medium (BEGM).
2. Pharmacologic treatment:
NHBE cells were treated daily with 1 to 500 μM nicotine for
7 days.
 To assess the role of nAChRs in mediating nicotine effects
by adding (αBTX), mecamylamine and (DHβE)
 Inhibition studies of EGF receptor were performed
by adding cetuximab, then adding heparin binding-EGF (HB-
EGF recombinant)

10. Methods
3. RT-PCR: Reverse transcriptase polymerase chain reaction
for total extracted mRNA
4. REAL-TIME PCR: A quantitative PCR by using SYBR
Green and iCycler instrument to measure the expression of
α7, α3, β1 and β2 nAChR on nicotine treated NHBE cells.
5. Immunoblotting:Western blot for detection of certain protein
in tissue (EGFR) by using its own antibody (EGFR Ab).

11. IMMUNOBLOTTING
(Western Blot)

12. Methods
6. Determination of EGF ligands in cell
supernatants: by using bicinchonicic acid-based
assay (BCA) (A protein assay based on the
colorimetric change which occurs when
Bicinchonicic acid bind to cysteine, tryptophan,
and tyrosine.
And do western blot detection for the presence of
HB-EGF

14. Methods
7. Immunofluorescence: Treat the NHBE cells with
labeled antibodies and examine under fluorescent
microscope.
Negative control slides were processed in parallel,
except for the omission of the primary antibody.

16. Methods
8. Extracellular matrix-cell adhesion assay: Quantification
of NHBE adherent cells was performed by measuring
calcein fluorescence on a Polarstar Galaxy, fluorescence
plate reader ,using 485 excitation and 530 emission filter
9. Zymography: is an electrophoretic technique, based on
SDS-PAGE, that includes a substrate co-polymerized with
the polyacrylamide gel, for the detection of enzyme
activity (Zymogram is the removing of SDS from the gel,
and areas of digestion appear as clear bands against a
darkly stained background where the substrate has been
degraded by the enzyme ).

17. BMG Fluostar Galaxy Fluorescence, TRF,
FI and Absorbance Reader :
The Galaxy is a fully automated
microplate based multi-detection reader
which incorporates four different
measurement principles:
1. Fluorescence
2. Fluorescence Intensity
3. Time-Resolved Fluorescence
4. Absorbance

18. RESULTS
1. Recurrent nicotine addition induced NHBE cell phenotypical
changes towards a neuronal-like phenotype
2. nAChR mediated filopodia extrusion induced by nicotine in
primary lung epithelial cells
3. Nicotine treatment induced the activation of the EGFR in
lung primary epithelial cells
4. Inhibition of EGFR signaling precluded the formation of
cellular extrusions induced by nicotine
5. Nicotine induced the accumulation of HB-EGF in the
extracellular media of primary lung bronchial epithelial cells
6. NF-kB translocation follows nicotine treatment of primary
lung epithelial cells
7. Nicotine-differentiated lung primary epithelial cells showed
increased adhesion to the ECM and metalloproteinase
activity but did not form colonies in soft agar

19. Cuboidal bronchial epithelial cells.
Neuron cell

20. 1. Recurrent nicotine addition induced NHBE
cell phenotypical changes towards a neuronal-
like phenotype
• These cells were therefore grown in
– EGF and hydrocortisone depleted BEGM medium for 24 h
– then being exposed to 1, 50, 100 or 500 μM nicotine daily
during a week.
1. the addition of doses of nicotine above 50 μM induced cell
growth arrest and the development of a neuronal-like
phenotype characterized by the protrusion of cell filopodia in
a dose dependent manner (Fig. 1A).

21. Fig. 1. Acute nicotine exposure induced changes in the phenotype of primary bronchial
epithelial cells
A) NHBE cells treated for 7 days with nicotine expanded filaments in a dose response way.

22. Filopodia were counted in at least three random fields per well in three
independent experiments. Comparisons between control and
nicotine exposed cells were made using an ANOVA test, *P‹0.05, * P‹0.001

23. 1. Recurrent nicotine addition induced NHBE
cell phenotypical changes towards a neuronal-
like phenotype
2. Is this phenotypical change was accompanied by alterations
in the expression of some epithelial and neuronal markers?
RT-PCR analysis was done for mRNA levels of adhesion
molecules, such as:
1. E-cadherin
2. zona occludens-1 (ZO-1)
3. neural cell adhesion molecule N-CAM
4. neural proteins such as neurofilament-M (Nef-M)
5. the neuronal specific transcription factor Pax-3
(Fig. 1B).

24. B) Analysis of mRNA expression of epithelial (E-cadherin, ZO-1) and neuronal
markers (N-CAM, NEF-M and Pax-3) in NHBE cells treated with 500 μM nicotine.

25. 1. Recurrent nicotine addition induced NHBE
cell phenotypical changes towards a neuronal-
like phenotype
3. These results were confirmed by immunocytochemical
analyses
(Involves the computerised assessment of microscopic fields following direct
or indirect fluorescent antibody or indirect or direct immunoperoxidase
analysis of biopsy tissue from the patient.)
performed with antibodies against E-cadherin, the
neuronal specific proteins Nef-M, the neuronal
nuclear transcription factor (Neu-N) and the
mesenchimal marker N-CAM (Fig. 1C).

26. C) Immunocytochemical analysis of the expression of E-cadherin, N-CAM, Neu-N
and Nef-M in control and nicotine treated (500 μM, 7 days) NHBE cells.
change towards a neural phenotype

27. 2. nAChR mediated filopodia extrusion induced
by nicotine in primary lung epithelial cells
• Primary lung epithelial cells were pre-treated daily for 30
min with the nAChR antagonists:
1. mecamylamine (α3-nAChR),
2. dihydro-β- erytroidine, DHβE (β-nAChR)
3. α-bungarotoxin, αBTX (α7- nAChR),
and the induction of cell filopodia was then quantified by
inverted light microscopy.
• (Fig. 2A),

28. Fig. 2. Filopodia production in nicotine treated NHBE cells is mediated by nAChR
activation.
A)NHBE cells were grown in the presence of 500 μM nicotine (N) with or without a 30-
minute pre-treatment with the following nAChR inhibitors before each exposure to
nicotine: mecamylamine (Mec, α3-nAChR inhibitor), DHBE (β-nAChR inhibitor) or α-
bungarotoxin (α-Btx, α7-nAChR inhibitor).
Filopodia were counted in three random fields per well in at least three independent
experiments.
α7-nAChR

29. 2. nAChR mediated filopodia extrusion induced
by nicotine in primary lung epithelial cells
 Is nicotine treatment modified the expression of nAChR in lung
epithelial cells?
real-time PCR analysis performed of the expression of
α7, α3, β1 and β2 nAChR on nicotine treated NHBE cells.
• Acute nicotine exposure
– significantly increased the expression of α7 nAcCh receptors,
– the expression of α3 and β1-nAChR did not change
– the expression of β2-nAChR diminished (Fig. 2B).

30. B) Quantitative measurement of mRNA expression of different nAChR in control
and nicotine treated NHBE cells.
All the experiments were done at least three times in triplicate.
Comparisons between control and nicotine exposed cells were made using an
ANOVA test, *P‹0.05, * P‹0.001.

31. 3. Nicotine treatment induced the activation
of the EGFR in lung primary epithelial cells
• nicotine induces the shedding of several EGFR ligands, such as
TGFα or HB-EGF, in lung carcinoma cells through the
activation of the tissue metalloproteinase ADAM-17 .
 could nicotine treatment activate EGFR in primary lung
epithelial cells via phosphorylation of its tyrosine residues?
Immunoprecipitation of total EGFR protein revealed that
– increased EGFR phosphorylation could be detected 1 h after
NHBE treatment with 500 μM nicotine,
– and was abrogated when a specific inhibitor of tyrosine
phosphorylation was used (Fig. 3A).

32. Fig. 3. Nicotine exposure caused EGFR phosphorylation in primary lung
epithelial cells.
A) EGFR immunoprecipitation followed by p-tyrosine western blot detection
demonstrated that treatment of NHBE cells with nicotine 500 μM increased
EGFR phosphorylation.
Inhibitor
for pTyr

33. 3. Nicotine treatment induced the activation
of the EGFR in lung primary epithelial cells
 Which tyrosines were involved in the activation of the EGF
receptor?
 western blot analyses were performed using antibodies
against specific phospho-tyrosine residues.
1. Increased EGFR phosphorylation was easily detected at Tyr 992
and Tyr 1068.
2. Phosphorylation of the Tyr 992 was dependent on the activation
of α7-nAChR, since α-BTX returned pEGFR-Tyr 992 to basal
levels.
3. At the same time the phosphorylation of the pEGFR-Tyr 1068
was inhibited when the cells were pre-incubated with inhibitors
of the α7 (α-BTX) or α3 (mecamilamide) nAChR, (Fig. 3B).

34. B) Determination of the phosphorylation status of tyrosines 845, 992, 1045
and 1068 of EGFR 1 h after exposure to 500 μM nicotine in the presence or
absence of the α-AChR inhibitors α-bungarotoxin and mecamylamine.

35. 3. Nicotine treatment induced the activation
of the EGFR in lung primary epithelial cells
 Is the same pattern of EGFR phosphorylation was observed in
primary bronchial epithelial cells treated daily with nicotine?
 For this reason the phosphorylation of the four
different EGFR-Tyr residues were analyzes after, 1, 2, 4
and 7 daily additions of 500 μM nicotine.
• Fig. 3C,
– the phosphorylation of EGFR-Tyr 992 reached a peak after
the second nicotine addition and was kept high all along the
differentiation process.
– the phosphorylation level of pEGFR-Tyr 1068 dropped
sharply 96 h after nicotine treatment.

36. C) Determination of the phosphorylation status of tyrosines 845, 992, 1045
and 1068 of EGFR after 1 (N1), 2 (N2), 4 (N4) and 7 (N7) days of treatment
with 500 μM nicotine.

37. 3. Nicotine treatment induced the activation
of the EGFR in lung primary epithelial cells
 Does the nicotine-induced phosphorylation of pEGFR-Tyr
992 be dose dependent?
western blot analysis were performed in NHBE cells
treated during a week with 1 nM, 100 nM, 1 μM, 100 μM
and 500 μM nicotine.
• Phosphorylation of EGFR-Tyr 992 started at 100 nM and
reached a maximum when the cells are exposed to the
highest nicotine dosage of 500 μM (Fig. 3D).

38. D) Dose response analysis of the phosphorylation of the pEGFR-
Tyr 992 after 7 days of treatment with nicotine.
All the experiments were performed in triplicate.

39. 4. Inhibition of EGFR signaling precluded the
formation of cellular extrusions induced by
nicotine
 Which level of EGFR phosphorylation and ligand binding
were needed to induce the morphological changes observed in
NHBE cells treated with nicotine?
 pre-incubated NHBE cells with :
1. AG1478 (1 μM) a specific inhibitor of phosphorylation in
Tyr residues,
2. or with cetuximab (10 μg/mL), a monoclonal antibody
raised against the ligand binding portion of EGFR, 60 min
before each nicotine treatment. Fig. 4A and B,

40. Fig. 4. Blocking ligand binding and phosphorylation of EGFR impairs the phenotypical
changes induced by nicotine in NHBE cells.
A) Inhibition of the EGFR phosphorylation by daily pre-treatment with AG1478 (1 μM) or
blockade of the ligand binding to EGFR by cetuximab (10 μg/mL) prevented filopodia
protrusion in nicotine exposed (N) NHBE cells.

41. Comparisons between control and nicotine
exposed cells were made using an ANOVA test, * P‹0.001.

42. B) Inhibition of the EGFR phosphorylation by daily pre-treatment with
AG1478 (1 μM) or blockade of the ligand binding to EGFR by cetuximab (10
μg/mL) also precluded its phosphorylation.
All the experiments were done at least three times in triplicate.

43. 5. Nicotine induced the accumulation of HB-EGF
in the extracellular media of primary lung
bronchial epithelial cells
 Is the EGFR ligand mediating the EGFR phosphorylation?
by western blot the accumulation of TGFα, HB-EGF and
amphiregulin in the supernatant of nicotine-differentiated
NHBE cells.
1. HB-EGF accumulated in the extracellular media after 7 days of nicotine
treatment, while the levels of the other EGFR ligands did not change
over time (data not shown).
2. This accumulation was inhibited when inhibitors of the α-nAChR were
added, demonstrating the specificity of this nicotine driven effect (Fig.
5A).
3. The involvement of HB-EGF in the phenotypical changes observed was
also supported by the fact that a single exposure of NHBE cells to 20
ng/mL of HB-EGF induced the protrusion of cell filopodia in these cells
(Fig. 5B).

44. Fig. 5. Nicotine induced HB-EGF in NHBE cells.
A) Accumulation of HB-EGF in the supernatant of NHBE cells
differentiated during 7 days with 500 μM nicotine.

45. B) HB-EGF treatment (20 μg/mL) induced the same phenotypical differentiation
observed after repeated nicotine exposure.

46. 6. NF-kB translocation follows nicotine
treatment of primary lung epithelial cells
• Nuclear factor-kappaB (NF-kB) is a key transcription factor
thought to play a major role in carcinogenesis.
• it has been activated in several lung cancer cell lines after
tobacco exposure.
 Is this transcription factor was involved in the phenotypical
changes reported?
 Could nicotine able to induce the translocation of NF-kB to
the nucleus in NHBE cells?

47. 6. NF-kB translocation follows nicotine
treatment of primary lung epithelial cells
• Results obtained by immunochemistry (Fig. 6) showed
that :
1. nicotine-differentiated NHBE cells presented strong
nuclear NF-kB staining,
2. while control cells show prevalent cytoplasmic staining.
3. This translocation occurred as early as 1 h after nicotine
addition
4. and was blocked by pre-treatment with inhibitors of the
nAChR, such as mecamylamine or α-bungarotoxin.

48. Fig. 6. Nicotine
treatment caused
nuclear NF-kB
translocation in NHBE
cells.
Cells were treated daily
with 500 μM nicotine for
1 h, 1 day or 7 days.
Cells were afterwards
fixed with formaldehyde
and assayed for NF-kB
localization by
immunocytochemistry.

49. 7. Nicotine-differentiated lung primary epithelial
cells showed increased adhesion to the ECM and
metalloproteinase activity but did not form colonies
in soft agar
 did the phenotypical changes observed in nicotine exposed NHBE
cells were accompanied by the acquisition of some properties of
transformed cells, such as increased adherence to and
degradation of the extracellular matrix (ECM)?
• (Fig. 7A), nicotine-differentiated NHBE cells showed increased
adhesion to all the ECM proteins tested (collagens I and IV,
fibronectin and laminin).
• These cells also showed increased ability to degrade extracellular
proteins, as judged by gelatin-zymography, in which two protease
activities of 92 and 72 kDa were detected (Fig. 7B).

50. Fig. 7. Nicotine-differentiated NHBE cells showed increased adhesion properties
and protease activity.
NHBE cells grown in the presence of nicotine (500 μM, for 7 days) were loaded with
calcein and allowed to adhere to different extracellular matrix proteins.
BSA was used as negative control.
All the experiments were done at least three times in triplicate.
(BSA)

51. B) Gelatine zymography on SDS acrylamide-gel revealed two protease activities
of 92 and 72 kDa in the extracellular medium derived from nicotine differentiated
cells.
Comparisons between control and nicotine exposed cells were made using an
ANOVA test, *P‹0.001.

52. DISCUSSION

53. How is nicotine absorbed by human body
and where it will be metabolized
• The association between tissue injury and cigarette smoking
has been related to
1. the duration of smoking,
2. the amount of tobacco smoked per day
3. the type of cigarette,
4. and other circumstances, such as the inhalation pattern (a
lesser extent)
• It is readily absorbed from the respiratory tract, buccal
mucosa, and skin, reaching the brain 10 seconds after smoking.

54. • The nicotine inhaled in a cigarette is rapidly absorbed in
the respiratory tract, reaching very high concentration
levels in the bronchial mucosa during active smoking.
• The average nicotine content per cigarette ranges from 1 to
1.8 mg
• Nicotine concentrations in the bronchial mucosa
immediately after smoking can reach 0.2 to 1 mM while
steady-state serum concentrations of 10 to 100 μM have
been reported
• Nicotine metabolization occurs mainly in the liver by the
action of CYP2A oxydases but other organs such as lung
epithelium can metabolize it as well

55. Effects of nicotine on NHBE cells phenotype
• Recurrent exposure to nicotine induced a phenotypical changes in
NHBE cells, characterized by:
1. cell elongation,
2. the emission of long filopodia,
3. loss of epithelial cell markers, such as E-cadherin, and ZO-1
4. increased expression of neuronal specific markers such as the
adhesion molecule N-CAM, the neurofilament-M and the
transcription factor Neu-N.
• Sustained exposure to nicotine might alter the phenotype of lung
epithelial cells in undetectable pre-malignant lesions, by :
1. the induction of a partially transformed phenotype characterized
2. by loss of contact inhibition
3. and loss of dependence on exogenous growth factors
4. And loss adherence to the extracellular matrix

56. nAChR
• Normal bronchial neuroepithelial cells present functional, α7, α3
and β-1 and β-2 nAChR, which are frequently over-expressed in
(SCLC) amplify nicotine responses.
• the expression of the α7-nAChR was up-regulated after acute
nicotine treatment
• nicotine activation of nAChR in human bronchial cells can
increase the expression of functional receptors in epithelial cells,
providing a positive feedback loop that may amplify nicotine
responses

57. • nicotine can activate EGFR through the activation of nAChR in
vascular and NSCLC cells .
• The repeated nicotine exposure induced the accumulation of HB-
EGF in the supernatant of NHBE cells in a nAChR activation
dependent way.
• culturing NHBE cells with HB-EGF originated the same
morphological changes observed after nicotine treatment.
• EGF induced a dramatic morphological transformation of
primary lung epithelial cells from a regular cobblestone cellular
shape towards the acquisition of lengthy filamentous projections
(filopodia).
EGFR & EGFR ligand

58. • By increasing in release of HB-EGF to the extracellular media,
nicotine induced EGFR activation by phosphorylation of the
EGFR tyrosine residues 992 and 1068.
• This effect was partially inhibited by
1. α-bungarotoxin, an α7-nAChR inhibitor,
2. mecamylamine, an α3-nAChR inhibitor,
3. dependent on nicotine concentration,
• the repetitive exposure to nicotine caused sustained activation
of the EGFR-Tyr 992.
EGFR & EGFR ligand

59. NF-kB
• NF-kB is one of the nuclear effectors of EGFR activation and
consider as a molecular link between chronic inflammation
and cancer development.
• NF-kB exists as a p65/p50 heterodimer in the cytoplasm that
translocates to the nucleus as a p65 monomer upon activation.
• NF-kB expression and activity is enhanced after nicotine
treatment and translocate to the nucleus after 1h treatment.

60. Nicotine nAChR
Increase
expression
Of functional
receptors
Binding
Alpha 7
Providing
Positive
Feedback
loop
Amplify
nicotine
responses
Activation of
EGFR
Accumulation of
HB-EGFR
Phosphorylation
Tyr992 Tyr1068
Translocation
Activation of
NF-kB
Morphological transformation

61. Conclusion
• Recurrent exposure to nicotine will lead to cell
differentiation by activation of nAChR and EGFR and
inducing translocation of NF-kB to the nucleus that leads to
generate a lung tumor.
• Since Tobacco is the leading cause of lung pathologies such
as asthma and cancer, this study may contribute to the
knowledge of the mechanisms involved in nicotine toxicity
• The nAChR signaling network in tumor cells (especially a7
nAChR) represents a novel molecular target for the therapy
of tobacco- related cancers.

63. References
• http://www.rz.uni-karlsruhe.de/~db26/Fotos-Knoch
• http://www.thebrain.mcgill.ca/flash/a/a_06/a_06_m/a_06_m_mou/a_06
_m_mou.html
• http://www.mtxlsi.com/Fluostargalaxy.htm
• http://en.wikipedia.org/wiki/Nicotine
• http://www.medterms.com/script/main/art.asp?articlekey=22807
• http://www.uiowa.edu/~cemrf/archive/laser/large/Human-Bronchial-
Epithelial-.gif
• http://faculty.tcc.fl.edu/scma/smithh/neuron2.jpg
• http://cancerweb.ncl.ac.uk/cgi-bin/omd?immunocytochemical+assay
• www.bio.davidson.edu/.../method/IMF.html