Inhibition of hdac by vorinostat in human lung cancer cells

HANZHAMA Research Group
aiming to fight cancer
Presented by
Mostafa Fathy Ramadan
Under supervision
Dr. Hussein Sabit
Inhibition of HDAC by Vorinostat in
human Lung cancer cells

What is Cancer?
 Cancer: is a group of diseases involving
abnormal cell growth with the potential
to invade or spread to other parts of the
body.
 There are more than 100 types of cancer.

 lungs are 2 sponge-like organs in our chests (right
and left lung)
 Lungs are to bring
Oxygen into the body
And remove carbon
Dioxide.
Lung cancer

Definition of lung cancer
Lung cancer: is a malignant lung
tumor characterized by uncontrolled cell
growth in tissues of the lung.

Types of lung cancer
1- Small cell lung cancer (SCLC):About
10% to 15% of all lung cancers are small
cell lung cancer (SCLC).
2- Non-small cell lung cancer
(NSCLC):About 85% to 90% of lung cancers
are non-small cell lung cancer (NSCLC)

Etiology of lung cancer
Smoking
Asbestos fibers
Air pollution
Exposure to radon gas
Exposure to certain chemicals
Past cancer treatment

Incidence of lung cancer
World Health organization(2010)

Chemotherapeutic drugs

 Chemotherapy: category of cancer treatment that
uses chemical substances, especially one or more
anti-cancer drugs (chemotherapeutic agents) that
are given as part of standardized chemotherapy
regimen.
 Chemotherapy drugs cannot tell the difference
between reproducing cells of normal tissues and
cancer cells.
So, chemotherapy has side effects such as:
1-Hair loss
2-Nervous system effects
3-Diarrhea
4-Blood disorders

Types of chemotherapy
1-Alkylating agents
2-Antimetabolites
3-Anthracyclines
4-Topoisomerase inhibitors
5-Mitotic inhibitors
6-Corticosteroids

Mode of action of chemotherapeutic
agents
 chemotherapy drugs work only on cells that are
actively reproducing (not cells that are in the
resting phase, G0).
 attack cells in a particular phase of the cell cycle
for example (the M or S phases).

Epigenetics
 Epigenetics refers to heritable factors which
influence the phenotype of a cell or organism and
are not dependent upon DNA sequence.
 Epigenetic modifications – including acetylation,
methylation, phosphorylation and ubiquitination
amongst others.
 Epigenetic modifications occur most commonly on
either the DNA itself (DNA methylation) or on the
histone octamer around which DNA is coiled
(Histones modification).

Vorinostat

Vorinostat (HDAC Inhibitor)
 Vorinostat Is an anti-cancer chemotherapy drug which
inhibit histone deacetylase(HDAC).
 used to treat a type of cancer called cutaneous T-cell
lymphoma (CTCL).

Mode of action of Vorinostat

Aim of work
We aim to identify the role of
Vorinostat as HDAC inhibitor for
the treatment of lung cancer.

Objectives
1-Culture of cell lines.
2-Treatment of cells with vorinostat.
3-testing the viability of cells using trypan
blue and MTT.
4-Extraction of DNA for full genome
methylation analysis using methyl flash kit.
5-Performing flow cytometry for analysis the
cell cycle.

Methodology

1-Culture of cell lines
 A519 lung cancer cell lines will be kindly
provided by the Holding Company for Biological
Products & Vaccines, Egypt (VACSERA).
 Cells will be cultured in RPMI 1640 medium
supplemented with 10% fetal bovine serum and
1% penicillin–streptomycin at 37°C in a
humidified atmosphere containing 5% CO2.

2-Trypan Blue test
 Cells will incubated with trypan blue stain
(Invitrogen, 15250–061) after being
treated with vorinostat.
 Cells that excluded the dye (viable) and
cells that retained the dye (dead) will be
counted.
 Samples will be diluted with Trypan blue
dye of an acid azo exclusion medium by
preparing a 1:1 dilution of the cell
suspension using a 0.4% trypan blue
solution.

3-Treatment with
chemotherapeutic drugs
 We will treat cells with vorinostat at
concentration ranges from 3µM-15µM.
 We will incubate cells with vorionstat for
4 days or 5 days.
 then, We will harvest cells for down
stream analysis.

4-Trypan Blue test
 Cells will incubated with trypan blue stain
(Invitrogen, 15250–061) after being
treated with vorinstat.
 Cells that excluded the dye (viable) and
cells that retained the dye (dead) were
counted.
 Samples were diluted in Trypan blue dye
of an acid azo exclusion medium by
preparing a 1:1 dilution of the cell
suspension using a 0.4% trypan blue
solution.

5-MTT Test
 MTT assay will be employed to investigate
whether vorinostat affected DNA methylation
in lung cancer cells.
 This test will be performed to measure the
activity of mitochondrial reductase enzyme
which indicate the cell viability.

6-DNA extraction
 We will extract DNA from treated and Non
treated cell by Qiagen DNA extract kit.
 Then, we will follow the kits instructions.

7-Quantification of DNA
Methylation
 After treating with vorinostat, DNA will be
subjected to a series of reactions to
quantify the level of methylation exist in
whole genome.
 Then, We will follow the kits protocol.

8-Cell cycle analysis
Cell cycle will be monitored by using flow
cytometry to recognize in which phase(s)
vorinostat induces apoptosis.

Selected References
 American Cancer Society.(2014). Cancer Facts &
Figures. Atlanta, Ga: American Cancer Society.
 American Cancer Society.(2013). Cancer Facts &
Figures for African Americans. Atlanta, Ga:
American Cancer Society.
 American Joint Committee on Cancer.(2010).Lung.
AJCC Cancer Staging Manual. 7th ed. New York:
Springer.253–266.

Inhibition of hdac by vorinostat in human lung cancer cells

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