|
Using the human carcinoma cell line CaSki, we have
investigated the effects of aromatase overexpression on cell proliferation
and the expression of various estrogen responsive genes. The cervical
cancer cell line CaSki, found to be aromatase negative, was transfected
with aromatase cDNA to achieve a stable line that overexpresses
aromatase. CaSki untransfected negative cells were used as the control
and CaSki transfected aromatase positive cells as the experimental
line. A Proliferation Assay was done using [3H] thymidine to monitor
the effects of aromatase on the growth of the CaSki cells in the
presence of substrate androstenedione and treated with an aromatase
inhibitor (Letrozole) or an antiestrogen (ICI 164384). The results
indicate a decrease in cell growth in the aromatase positive cells
when subjected to the above-mentioned treatments, while showing
no overall change in the growth of the CaSki untransfected aromatase
negative cells. To examine the effects of aromatase overexpression
on RNA levels, we used Reverse Transcription Polymerase Chain Reactions.
Our results show altered expression of various growth factors (e.g.
TGF a, b, and VEGF) and receptors (e.g. EGFR and ER b) in the CaSki
transfected aromatase positive cells as compared to the CaSki untransfected
cells. To study the effects of aromatase overexpression on protein
expression, Western Blot analyses were used with antibodies specific
for various proteins. Western analysis confirmed the results from
the RT-PCR. Overall, the observations indicate that the overexpression
of aromatase and high levels of estrogen leads to the increase or
decrease in the expression of several genes and growth factors,
which may result in cellular proliferation.
Worldwide cervical cancer is the second or third most
common cancer among women. About 400,000 new cases are diagnosed
each year, according to the National Cancer Institute. Each year,
15,000 women in the U.S. learn that they have cervical cancer. The
prevalence and magnitude of this problem leads researchers to find
new ways of combating this disease. The primary risk factor for
cervical cancer is infection with certain types of human papillomavirus
(HPV). However, there has been evidence of the involvement of estrogen
in cervical cancer. Transgenic mice models that harbor HPV genes
resulting in precancerous cervical lesions have been found to produce
malignant transformation when chronically treated with estrogen.
In addition, women taking estrogen-containing contraceptives over
long periods of time have an increased risk of developing cervical
cancer. These observations suggest that estrogen may play an important
role in the etiology of cervical cancer. The biosynthesis of estrogen
from androgen is catalyzed by an enzyme called aromatase. Aromatase
is the terminal rate-limiting step in estrogen biosynthesis. Aromatase
overexpression, resulting in increased estrogen production, has
been implicated in female cancers such as breast cancer. The focal
point of our research deals with the effects of aromatase overexpression
on cervical cancer cell lines to understand the role of estrogen
in this malignant disease. Elucidating the role of aromatase in
cervical cancer could provide a target for future treatment of the
disease..
Cell Proliferation Assay: Counted cells using a Hemacytometer.
Thinly plated cells in a twelve well plate at a density depending
growth. Next day remove regular media and replace with charcoal
stripped media. The following day add treatments to the cells and
leave for 48 hrs.
Treatments:
- Control
- Androstenedione
- Letrozole (aromatase inhibitor)
- ICI 164384 (anti-estrogen)
- Androstenedione + Letrozole
- Androstenedione + ICI
On the following day add [3H] Thymidine and leave
for 24 hrs then wash cells, lysate cells and count cells. RNA Analysis:
Isolated RNA using Tri Reagent. The expression of specific factors
was done using RT-PCR under the following conditions: RT: 42ºC (1hr),
99ºC (5 min) PCR: 95ºC (19 sec), 60ºC (15 sec), 72 ºC (20 sec) 42
cycles. The products were then visualized on 1% agarose gel with
ethidium bromide staining. Protein Analysis: Protein was isolated
using lysis buffer. Equal amounts (60 µg per well) of protein
from each sample were separated on a denaturing polyacrylamide gel
and transferred to a nylon membrane. Western blot analysis was performed
according to the manufacturers protocol.
Fig 1. Effect of Aromatase Overexpression on RNA Levels
of ERb , TGFb, and PR on CaSki Cell Line
Fig 2. Effect of Aromatase Overexpression on RNA Levels
of EGFR , TGFa , Amphiregulin and VEGF on CaSki Cell Line Both Figures
demonstrate an increase in the expression of most factors due to
the presence of aromatase while the expression of PR decreased.
Fig 3. Effect of Aromatase Overexpression on Protein
Levels of ERâ, PR, Cyclin D, and PCNA on CaSki Cell Line Confirmed
the results done using RT-PCR.
Fig 4. Cell Proliferation Assay Demostrates that cell
proliferation can be controlled in aromatase positive cells using
Letrozole, an aromatase inhibitor and ICI 164384, and anti-estrogen.
- Expression of aromatase in CaSki cell line affects a wide range
of growth factors and receptors.
- Increased estrogenic activity due to the expression of aromatase
has a significant effect on the deregulation of various genes
known to be involved in cellular proliferation and tumorigenesis.
- We observed that most factors showed an increase in response
to aromatase expression levels, while PR decreased significantly
both at transcriptional as well as protein level. Low levels of
PR may be related to aggravated conditions of cervical tumors.
- Proliferation of cells could be inhibited using Letrozole (aromatase
inhibitor) and ICI 164384 (anti-estrogen) confirming the role
of estrogen in increased cellular proliferation.
- Some cervical tumors may be hormone responsive, therefore designing
a suitable hormone therapy may prove useful.
|
