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Lung cancer and
gene therapy
Diana Zamora Ávila &
Paul R Earl
Immunology and Virology Department
Biosecurity Laboratory, Level 3
Facultad de Ciencias Biológicas
Universidad Autónoma de Nuevo León
San Nicolás, 66451 NL, Mexico
Lung cancer is the most common cancer
worldwide in both the indices of
prevalence and mortality. Smoking is a
risk factor highly associated with this type
of cancer (85-90 %), and the exposure to
tobacco smoke in the environment can
cause cancer in nonsmokers. Certain
agents like arsenic, asbestos, chromium,
nickel and vinyl chloride encountered in
the work environment increase the risk
and also can cause cancer. In addition, the
effects of smoking may be additive with
some hazardous agents.
Classification
The 2 main types of lung cancer are
small cell and nonsmall cell neoplasms.
1/ Small cell pulmonary cancer
constitutes 20 % of all these cancers, the
cells multiplying rapidly and are able to
metastisize into major organs like
lymphatic ganglia, bones, brain, adrenal
glands and liver. The primary tumor
usually generates near the bronchi and
expands to the center of the lungs. The
main cause of this type of cancer is
smoking.
Small-cell lung cancer
2/ Nonsmall-cell pulmonary cancer
represents almost 80% of all lung
cancer. It diffuses more slowly than
the small cell type. The 3 subtypes are
1/ carcinoma of squamous or
epidermoid cells (30 %):,
2/ adenocarcinoma (40 %) y
3/ undifferentiated carcinoma with
large cells (10 %). Some cancers start
in the bronchioles and evolve from
there through several years.
Nonsmall-cell lung cancer
Lung cancer is a very aggressive disease in
which less than 15 % (10-13 %) of the
patients survive over 5 years. This is the
lowest rate of all types of cancer. More, 80 %
of cases do not respond favorably to
chemotherapy or radiation.
The main cause of poor or no recovery
from lung cancer is the high rate of
metastasis existing before diagnosis.
In nonsmall cell lung cancer, about 2/3rds
of the pacients are inoperable as a result of
metastasis. In small cell lung cancer,
metastases are present in most patients. The
liver has 1/3rd cases, and the brain is the first
site.
Genetic therapy
In genic therapy one procedure is introducing a
gene(s) into somatic human cells. Two general
strategies exist: 1/ genic therapy in vivo in
which the target cells are introduced directly
into the body, and 2/ therapy ex vivo in which
the target cells are modified in vitro and later
replaced.
The genetic treatment of lung cancer is difficult
as it is a multifactor disease. Thus various
alternatives have been proposed for stimulating
the immune system, including the transfer of
suicidal cells, inactivation of oncogenes, gene
replacement, tumor repressers and the transfer
of pro-apoptotic genes.
A promising target for the development of
novel anticancer strategies is telomerase, a
ribonucleoprotein reverse transcriptase that
extends human telomeres by a terminal
transferase activity. Telomerase is highly
active in the vast majority of human tumors,
known since 1994. Its essential genes are
hTR and hTER. However, telomerase has not
yet advanced from the laboratory to the
clinic.
See www.nature.com/clinicalpractice/onc.
See also methioninase gene therapy and
apoptosis as given by Yamamoto et al.
(Cancer Gene Therapy 10, 445–450, 2003).
STIMULATION OF THE IMMUNE SYSTEM
Tumor regression has been demonstrated in
animal models that was mediated by the
administration of cytokines such as
interleukines (IL) –2, 4 , 6, 7, 1 2, stimulating
factor of colonies of granulocytic
macrophages (GM-CSF), tumor necrosis factor
- (TNF-), interferon- (IFN-) & IFN-,
nonetheless use in humans has been limited
due to toxicity.
The strategies based on cytokines can lead to
the development of tumor cell vaccines that
genetically incorporate modified fibroblasts
or tumor cells that secrete cytokines.
TRANSFER OF KILLER GENES
This type of genic therapy is based on the
transduction of a gene able to convert a nontoxic
compound into a toxic metabolite able to
selectively kill tumor cells upon administration of
the appropriate prodrug.
The 2 genes used most for this type of therapy
are thymidine kinase of herpes simplex virus HSVtk and the gene of cytosine deaminase. Cytosine
deaminase converts 5-fluorocytosine into an
antimetabolite: cytotoxic 5-fluoroacyl. HSV-tk
converts the gancyclovir into a toxic trifosfate
metabolite. An adenoviral transduction in
nonsmall cancer cells with HSV-tk followed by the
administration of gancyclovir selectively kills the
tumor cells.
INHIBITION OF ONCOGENES
This type of therapy is based on the
identicifation and inhibition of those genes
critical for carcinogenesis. Oncogenes of the
ras family are some of the more common
oncogenes that are activated in lung cancer
and therefore are targets for this type of
therapy.
In preclinical studies mediated by a plasmid
with an antisense sequence with k-ras the
mRNA is selectively blocked by mutation, and
tumor growth is reduced both in vivo and in
vitro in murine models.
GENES SUPPRESSERS OF TUMORS
Another genic therapy strategy based on
work with gene tumor suppressers, in
contrast to oncogenes, is with the 2 alleles of
a tumor suppresser gen that should be
eliminated or inactivated by inducing tumor
growth. Theoretically, replacement of only
one copy of the tumor supresser gene in cells
with a loss of homozygotic function can
restore the ways of normal growth and
cellular proliferation.
One of the genes most commonly mutated
(50-70 % of cases)
is p53 that can be inactivated by
overexpression of MdM-2.
TRANSFER OF PRO-APOPTOTIC GENES
Cells with multiple genetic alterations are
usually eliminated by apoptosis. For survival,
they depend upon the overexpression of
antiapoptotic molecules like bcl-2, bcl-xL or
survivin. The downregulation of such proteins
may reduce the apoptotic threshold of cells.
The 2 major apoptotic signaling pathways are
1/ the mitochrondrial pathway and 2/ the
death receptor pathway. Cells having
condensed and fragmented chromatin
demonstrate apoptotosis.
TRANSFER OF GENES
Many systems have been used for the
administration of genes in the treatment of
cancer such as use of adenovirus and
associated viruses, poxvirus, herpes simplex,
but all of these can provoke an immune
responce against the vector, in the manner
required to use different strains or different
routes of administration.
An ideal technique for genic transfer should
be nontoxic and have great efficiency for
various types of cells with 1 or more genes for
which the transfer process is selective.
NONVIRAL SYSTEMS
Due to the low efficiency of the transfection
of naked DNA, various methods have been
used for increasing the efficiency like
microinjection, electroporation and
precipitation with calcium, but
unfortunately these methods are usually
inapplicible in vivo and generally result in a
transient expression of the gene. The
efficiency can be raised by using a gen gun
to incorporate the DNA by bombarding the
skin.
VIRAL SYSTEMS
In comparison with the previous systems, this
type of system shows an efficiency of better
tranduction both in vitro and in vivo.
The viral vectors used the most are defective
retroviruses, adenoviruses and ones associated
with adenoviruses. Others are herpes simplex,
vaccinia, avipox & baculoviruses. Among the
disadvantages encountered in the use of viruses
as vectors are toxicity, the production of foreign
transient proteins and limitations in the size of
the gene to be transferred. The virus vectors
most widely used with adenoviruses are easy to
produce in vitro efficiently and with surety.
TRANSFER OF GENES IN LUNG CANCER
AEROSOL TECHNIQUE
Most drugs and DNA complexes have been
administered by conventional routes: oral or
intravenous. The biodistribution of drugs by
means of these strategies are diseminated
and the quantity that is deposited in the lung
is small. Another important aspect to consider
in these types of systems is the toxicity that is
observed after injection. The ability to
express trangenes in the lung in a selective
manner can facilitate the development of
genic therapy for a variety of human diseases.
Polyethylene amine (PEI) is a polycation
able to condense DNA and to protect
against the degradation of DNAases así
como de liberarlo in vitro and in vivo, this
polymer forms polyionic complexes with
DNA to establish electrostatic cooperative
interactions with its ammonium groups
and the phosphate groups of the DNA.
More, it is possible to reach high levels of
gene expression if human albumin,
albumin from mouse serum or ovialbumin
and human igG are added to this
compound.
Furthermore, liposomes formulated with
9-nitrocamptotecine (9NC-DLPC), which is an
inhibitor of topoisomerase 1 and inhibits the
growth of subcutaneously induced tumors like
the metastasis of lung cancer in the murine
model, observing a synergistic effect through
the combination of the administration of gene
p53 and PEI.
Recently, the expression of Wilms' tumor gene
(WT1) has been encountered in lines lung cancer
cells in 5/11 (45 %) by the test for reverse
transcriptase polymerase chain reaction (RT-PCR),
2/5 (40 %) in SLCL and in 3/6 of NSCLC. In
another study, WT1 overexpression was found in
12/15 cell lines of lung cancer. The protein of
WT1 is an attractive target for immunotherapy.
STIMULATION OF THE IMMUNE
SYSTEM BY WT1
Recent advances in immunology and
molecular biology have permitted the
identification of many antigens associated
with tumors (TAAs), and the episomes that
are recognized by cytotoxic HLA class I T
lymphocytes in different kinds of neoplasms.
One of the TAAs identified is the product of
gene WT1 that allows the possibility of
slowing down the cancer based on
administration of this peptide.
In a phase I clinical study, Tsuboi et al.
(Microbiol Immunol 48: 175-84, 2004)
applied immunotherapy based upon WT1
by intradermal injection of 3 mg of the
peptide of WT1 of 9 bases that stimulated
HLA-A2402, the vaccination in an interval
of 2 weeks, resulting in the reduction of
the CEA and xSLX tumor markers and a
transient decrease in the tumor size being
the primary clinical evidence that shows
that vaccines against WT1 are an
alternative for pulmonary cancer patients.
The future
Lung cancer is one of the most aggressive
and fatal neoplasms. Today it is possible to
diagnose earlie, and we have an improved
panel of anticancer drugs. Nevertheless, less
than 15 % of patients survive more than 5
years. The most recent trend is to explore
genic therapy and therapeutic vaccines as
alternatives for treatment. With these new
tools perhaps the patient's life can be
prolonged reducing the cost of treatment.