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Presented By: Arsen Khachatryan
6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene
carboxylic acid, a synthetic retinoid, is more commonly
and thankfully called CD437 which is known to induce
apoptosis in a variety of cancer cells including lung
cancer cells. CD437 has been proven to be highly
sensitive in cancer cells with wild-type p53 and
because normal cells have p53 also a question arose
as to whether they are sensitive to CD437 induced
apoptosis as well. To answer this question the paper
studied the effects of CD437 on apoptosis induction
and the expression of several p53 regulated apoptosis
related genes between human lung cancer cells and
normal human epithelial cells containing wild-type p53.
Retinoids are the family of vitamin A derivatives
both natural and synthetic which regulate a broad
range of biological processes, including growth,
differentiation, and development, in both normal
and neoplastic cells. Their effects are mediated by
two classes of nuclear receptors the RARs and the
RXRs which are members of the steroid and thyroid
hormone receptor superfamily.
• p53 is a tumor-suppressor protein that induces apoptotic
cell death in response to oncogenic stress. Malignant
progression is dependent on loss of p53 function, either
through mutation in the TP53 gene, which encodes p53,
or by defects in the signaling pathways that are
upstream or downstream of p53.
• Mutations in TP53 occur in more than half of all human
cancers, resulting in the expression of mutant p53.
• Besides apoptosis, cell-cycle arrest activities are also
regulated by p53 associated pathways.
• Phosphorylation of p53 regulates its ability to activate the
expression of apoptotic target genes.
In the United States, lung cancer is the leading cause
of cancer mortality among both men and women. It has
been estimated that there will be 185,000 new cases
and 169,000 deaths from lung cancer in 2005. Due to
the severe morbidity of lung cancer and poor survival
rate, intense efforts are being mounted to find effective
new agents and treatments against lung cancer. CD437
has shown great potential and promise for prevention or
treatment of certain types of cancer cells by inducing
apoptosis in a variety of neoplastic cell types, also
showing promise in vivo with animal xenograft models.
CD437 can selectively bind to and transactivate the RAR
γ mediated pathway for apoptosis, but it is thought that
CD437 induces apoptosis in human lung cancer cells
through both p53 dependent and independent pathways,
depending on whether the cells have wild-type p53.
CD437 increased the level of p53 protein and also
increased the expression of p53 regulated genes such as
Bax, Fas, and DR5, which triggered apoptosis through
induction of cytochrome c release from mitochondria and
caspase-3 activation. Human lung cancer cells are so
extremely sensitive to CD437 induced apoptosis that a
study needed to be done to address its risk to normal
human lung epithelial cells. This study showed that CD437
can selectively induce apoptosis in human lung cancer
cells while sparing normal cells. In addition, CD437
exhibited differential modulation of several p53 regulated
genes between the two types of cells.
• Cell Lines
Cancerous Cell Lines
H460 (Large cell carcinoma)
H292 (Mucoepidermoid carcinoma)
Normal Cell Lines
NHBE (Normal human bronchial epithelial)
SAEC (Small airway epithelial cells)
• Methods
Growth Inhibition Assays
DNA Fragmentation Assays
Northern Blot Analysis
Western Blot Analysis
In this study the effects of CD437 were tested on cells
cultures both in the presence of serum and with the
absence of serum. It was shown that the serum in the cell
culture effected the results and the effectiveness of CD437
induced apoptosis. Both H292 and H460 were supersensitive in serum free culture conditions. In the presence
of 5% serum, CD437 inhibited cell growth by 30-80% in
both lung cancer cell lines treated with 0.2-1µM of CD437.
It showed weak or no growth in both the NHBE and SAEC
cell lines with less than 30% inhibition with 1 µM of CD437.
In the absence of serum even with 0.2 µM CD437, growth
of the H460 and H292 cell lines were inhibited by more
than 60%, with more than 25% inhibition at 0.05 µM.
Normal lung epithelial cells were less responsive with less
than 20% inhibition.
Cell numbers were determined by growth inhibition SRB assays. It is
thought that the proteins in the serum such as albumin, bind CD437
and decrease the free concentration of CD437 with access to the cells.
Since the doubling times of all the cells used for this
study are around 24 hours, they were treated for 24 hours
allowing the cells to divide only once or less. This is
important in order to show that the effects of CD437 on the
growth of lung cancer cells are not attributed to blockage of
cell proliferation, but rather to apoptosis.
Lung cancer cells treated with CD437 underwent
morphological changes characteristic of apoptosis, such as
rounding, detachment, and floating, while normal lung
epithelial cells did not change their morphology, but showed
a slight decrease in cell number. This shows that CD437
selectively induces apoptosis in cancerous lung cells, but
not in normal lung epithelial cells.
Cells were treated with
indicated concentrations of CD437
under optimal conditions for NHBE
without serum and for H460 with
serum. In B the H460 cell line is in
a suboptimal culture condition
without serum. It is plain to see
that CD437 had a dramatic effect
on the cancerous lung cell line
H460 regardless of presence of
serum.
CD437 induced a concentration dependant increase
in DNA fragments in both lung cancer cell lines. Similar
to the growth inhibitory effects, serum free culture
condition have made cancer cells more sensitive to
CD437 induced apoptosis. 0.2 and even 0.05 µM
CD437 were enough to increase DNA fragmentation in
the cancer cell lines. Even with 1 µM CD437 there was
no increase in DNA fragmentation in the normal lung
epithelial cells both in the presence and absence of
serum. This further proves that CD437 induces
apoptosis in lung cancer cells but not in there normal
counterparts.
DNA fragmentation was determined using the ELISA method
The activation of the caspase cascades is the
biological hallmark of apoptosis, so it was important to
compare the effect of CD437 between normal lung
epithelial cells and lung cancer cells. There are two
types of caspases: upstream caspases called initiator
caspases, such as caspase-8 and -9, and downstream
caspases known as effector caspases, such as
caspase-3 and -6.
The cancer cells when treated with CD437 induced
the activation of caspase-8 and -9, which are initiator
caspases. This led to Bid protein, a target substrate of
caspase-8 to be cleaved. The activation of effector
caspases -6 and -3 were also detected, indicated by the
increased cleavage of PARP,DFF45 and lamin protein.
No caspase activity was observed with the normal lung
epithelial cells.
Activation of these caspases were detected by
Western Blot analysis as a decrease of procaspase
forms or an appearance of their cleaved bonds along
with the cleavage of their target substrates.
P53 dependant induction of apoptosis and growth arrest
by CD437 in human lung cancer cells involves p53 activation
and up-regulation of its downstream regulated genes
including p21, Bax, DR5 and Fas. The lung cancer cell lines
and the normal cells were treated with CD437 and the
outcome showed that p53 protein was increased greatly in
the two lung cancer cell lines while having little to no effect
on normal lung epithelial cells. An increased expression of
Bax and DR5 were also present in both H460 and H292 cell
line, while showing no effect in normal cells. The same was
true for the expression of Fas and DR4. P21 was expressed
in both the cancerous cells and the normal cells.
The basal mRNA levels of
DcR1 and Dcr2, two decoy
receptors for the death
ligand TRAIL, which prevent
its action, were very low and
not inducible by CD437 in
the cancerous cell lines.
Interestingly, their levels
were higher in both normal
lung epithelial cells and their
mRNA expression was
induced by CD437.
Analysis was conducted
using the Northern Blot
method.
Bcl-2 and Bcl-XL expression is related to the resistance to
apoptosis, so it would be helpful to see if they play a roll in
the resistance of normal lung epithelial cells to CD437
induced apoptosis. The expression levels of Bcl-2 and BclXL were compared in the absence and presence of CD437
both in the normal lung epithelium and the lung cancer cells.
The basal levels of Bcl-2 and Bcl-XL were very low in the
normal lung epithelium and high in the lung cancer cells, yet
the cancer cells are sensitive to CD437 while the normal
cells are not. CD437 does not change the level of Bcl-2 or
Bcl-XL, therefore it cannot account for the resistance of the
normal lung epithelium to CD437 induced apoptosis.
H460 transfectants with
different expression levels of
exogenous Bcl-2 were also
treated with CD437 to further
assess the effects of Bcl-2 on
the action CD437. Two Bcl-2
transfected H460 cell lines
H460-Bcl-2-6 and H460-Bcl-2-8
were not less sensitive to
CD437 than vector-tranfected
control cells H460-Neo. They
actually turned out to be even
more sensitive.
The most critical question related to potential applications
of proapoptotic agents or any cancer medication for that
matter, which can seriously be considered in fighting
cancer, is whether they can distinguish between normal
and malignant cells. In this study the effects of the synthetic
retinoid CD437,and its role in the induction of apoptosis,
were compared and contrasted in human lung cancer cells
containing wild-type p53 and normal human lung epithelial
cells.
We saw that the two lung cancer cell lines underwent
rapid apoptosis, evidenced by the typical morphological
changes, the activation of the caspase cascades, increased
DNA fragmentation, and the increased expression of p53
and its upstream and downstream regulated genes after
exposure to CD437.
Using the same criteria for evaluation of apoptosis, we
discovered that CD437 did not induce cell death in the two
normal human lung epithelial cells, proving the it can
differentiate among normal and malignant cells. CD437 has
also been proven to induce apoptosis in malignant human
epidermal keratinocytes and human prostate cancer cells
without inducing apoptosis on their normal counterparts.
Lung Cancer Cells
Normal Lung Epithelium
•Induces visible morphological
changes.
•No morphological changes.
•Increase in DNA fragmentation,
indicating apoptosis.
•Activation of caspase cascades.
Caspase-3 required.
•Presence of high levels of Bcl-2.
•No increase of DcR1 and DcR2.
•No DNA fragmentation.
•No caspase cascade activation.
•Very low levels of Bcl-2
•Increase of DcR1 and Dcr2.
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