Radiation therapy or radiation oncology is the medical use of ionizing radiation
as a part of cancer treatment to control malignancy. It may be used for curative
or adjuvant cancer treatment, or as palliative or therapeutic treatment (Rath
and Mohanti, 2000). Since radiation therapy is not specific, it kills cancerous
cell along with few normal healthy cells and thus at times it leads to massive
tissue damage depending on the dose and course of radiation therapy which leads
to many complications. The extent of tissue damage can be determined by measuring
the C-reactive Protein (CRP) level, an acute phase protein produced by liver
or adipocytes, in the patients serum (Pepys and Hirschfield,
2003). CRP seems to assist in complement binding to foreign and damaged
cells, it enhances phagocytosis by macrophages and play an important role in
innate immunity as an early defense system against infections (Erlinger
et al., 2004).
As a major defense mechanism against cancer, host immunological surveillance
is composed of a cellular immunity as well as humoral immunity including antibody
and a complement system (Matsutani et al., 1984).
Complement system is an important mediator in the immunologic defense of the
body. It is reasonable, therefore, to investigate the possible implications
of the complement system in diseases in which immunologic phenomena are important
(Hu et al., 1988). Complement serves as an important
mediator in non-specific (innate) resistance by amplifying the humoral response
and converting it into an effective defense mechanism to destroy invading microorganisms
by enhancing phagocytosis (Bjorge et al., 2005).
Complement proteins are produced in inactive forms and then become active following
enzymatic cleavage by alternative or classical pathway or by lectin protein.
After complement activation membrane attack complex is formed which mediates
cell lyses. Thus to increase the immune response as defense mechanism the complement
C3 has central role in formation of membrane attack complex as well as in opsonization.
Humoral immune response is mediated by secreted immunoglobulins produced by
B lymphocyte lineage. IgG being most abundant, constitute about 80% of the total
serum immunoglobulin (Matsumotoa et al., 2006)
and thus in the present study it is used as an important parameter to represent
The present investigation is aimed to study the immune response, innate as
well as humoral, before and after radiation therapy in patients and also to
estimate the level of tissue damage in them.
MATERIALS AND METHODS
A total of 15 cases were enrolled for each investigation from patients attending
to Barasat Cancer Research and Welfare Centre, a prime health centre covering
many districts of West Bengal. The malignancy was diagnosed by various investigations
like radio-imaging, cytology and histo-pathological examinations by physicians.
Controls (n = 15) were randomly selected patients admitted to the same hospitals
as the cases during the same time period. They were frequency matched to cases
by age, sex and selected from hospital admission lists. Written informed consent
was obtained from all participants in accordance with the guidelines from hospital
center review board. Blood samples were collected from cases and controls both
before and after the commencement of radiation treatment. Then blood samples
(1 mL) were centrifuged at 3000 rpm for 15 min and clear serum was collected.
Determination of CRP level: To estimate tissue damage by radiation therapy
CRP level in the serum samples was assayed using turbidimetric immunoassay,
based on the principle of agglutination reaction, as per manufacturer instruction
(Tulip Diagnostics, India). Activation buffer (500 μL) and latex reagent
(50 μL) were mixed properly, incubated at 37°C for 10 min and used
as working solution. The serum sample (3 μL) was added to the working solution
and the CRP concentration was estimated by spectrophotometric reading at 546
Determination of complement C3 level: To study the effect of prolonged
application of radiotherapy in cancer patients on their innate immunity, concentration
of complement C3 was compared in serum of cancer patient before radiotherapy
and after 2-6 dozes of treatment. Radial Immuno Diffusion (RID) plate (DIFFU-plate)
containing uniform mono-specific antiserum directed against complement C3 protein
in agarose gel layer is used (Biocientifica S.A., Argentina). The serum samples
(5 μL each) were filled on the wells of agarose gel. Wet cotton was placed
at the centre of the RID plate to avoid agarose dehydration. The plate is tightly
closed and incubated at room temperature for 48 h. Radial diffusion of protein
out of the well into the surrounding gel leads to the formation of a visible
precipitation ring by reaction between protein C3 and antiserum. The diameter
of the precipitation ring is proportionate to the protein concentration and
the concentration was determined by the corresponding reference table.
Determination of IgG level: By detecting the change in the level of
serum IgG, before and after the therapy, the effect of radiation therapy on
humoral immunity was studied. RID plate (DIFFU-plate) containing mono-specific
IgG antiserum were used (Biocientifica S.A., Argentina) and the same protocol
stated above was used for the investigation.
Present investigation highlighted that malignancy may directly affect immunoglobulin
production through immuno-suppression. The concentration of CRP was greater
in cancer patients as compared with healthy one (0.6 mg dL-1). The
study also revealed even more increase in serum CRP level in every patient after
radiotherapy (Table 1).
In case of complement, the serum C3 level was higher than normal level (80-160
mg dL-1) in all the patients, except one case, showing malignancy
before radiation therapy. After 2-3 radiotherapy dozes, out of 15 patients 6
patients (40%) showed increased C3 level. Among remaining 9 patients (60%) the
C3 level decreased drastically after 5-6 prolonged therapy treatments, of which
in 4 cases C3 level after radiation treatment was below normal level (Table
Result revealed that IgG level was higher in all the patients before radiation
treatment than the normal range (600-1650 mg dL-1). This level was
again increased in few patients after radiotherapy, but in significant number
of patients IgG level decreased. Out of 15 patients the concentration of IgG
in serum decreased after radiation therapy in 10 patients (66.7%) as compared
with before radiation therapy. Moreover, among these 10 patients, the serum
IgG level dramatically decreases beyond the normal level in 4 patients and thus
considered to be effective. These patients were exposed to prolonged radiation
treatment and they belong to age group 55-70 years.
|| Level of CRP, complement C3 and IgG before and after radiation
In case of remaining 5 patients (33.3%) the IgG concentration has increased
after radiation therapy (Table 1). These patients belong to
the age group 25-45 years.
The present investigation revealed that complement C3, IgG and CRP levels of
cancer patients were significantly higher than those of the healthy subjects
which is at par as reported in early studies (Bjorge et
al., 2005; Erlinger et al., 2004; Rhaegen
et al., 1976). The high IgG and complement levels in neoplastic disease
as compared with normal may be caused by the continued presence of a tumor mass
which serves as an antigenic stimulus for continued antibody production. The
antigen-antibody complexes require immunoglobulin and complement which causes
an increased production to maintain normal levels (Hu et
al., 1988). CRP is a member of the class of acute phase reactants as
its level rise dramatically during inflammatory processes occurring in the body
(Erlinger et al., 2004). This increment is due
to a rise in the plasma concentration of IL-6 which is produced predominantly
by macrophages as well as adipocytes (Pepys and Hirschfield,
2003). The present investigation revealed that radiation therapy, despite
being a successful treatment for cancer, causes massive tissue damage which
is evident from the elevated level of CRP in the serum of the patients after
radiation. This may lead to great suffering of patients from many short term
and long term side effects. This observation puts a question mark on the efficiency
of radiotherapy treatment when the health of the patient is in stake.
The term complement defines it as the activity of blood serum that completes
the action of antibodies. The increase of complement activity was dependent
on the stage of the disease and on the therapy and C3 were the most representative
values for this stage-linked increase (Bjorge et al.,
2005; Rhaegen et al., 1976). The elevated
complement level in cancer patients may be explained by the concept that complement
activity rises to compensate for depressed cell-mediated immunity, in order
to preserve the activity of the biophylaxis mechanism against cancer (Matsutani
et al., 1984). Fall in C3 level decreases the opsonin and anaphylatoxic
activity of C3 as well as affect membrane attack complex formation. The majority
of patients with C3 deficiency after radiation therapy showed higher incidences
of infection, recurrent bacterial infection and may have immune-complex disease.
This result supports the earlier observation that cellular immunity remains
at a low level after radiotherapy (Hu et al., 1988).
But the level of IgG has increased in few patients indicating no immune
suppression in them.
The decreased levels of IgG, complement C3 and increased levels of CRP indicate
that due to prolonged radiation therapy such patients may be prone to other
bacterial infection, immune complex diseases or it may also induce a state of
secondary immuno-deficiency leading to an unwanted consequence. Though therapeutic
approaches are actually aimed at increasing immune response to the malignant
or cancer cells, but prolonged application of radiotherapy may cause side effects
on normal immune system, as they are already in immune deficiency state due
to the life threatening neoplastic disease (Rhaegen et
al., 1976). This observation was in line with the fact that somatic
mutation may results from the irradiation cause an impairment of immune function
(Stone et al., 1994). Irradiation can also cause
a decrease in immune competency which in tern can accelerate aging (Stone
et al., 1994). The number of dozes given in radiotherapy depends
on the stage and type of cancer, age, present health condition of the patients.
Age is a crucial factor to tolerate such prolonged therapy treatment. Thus close
monitoring of patients undergoing radiotherapy is very important and prolonged
exposure should be avoided.
The present investigation revealed the fact that though radiation therapy is
an important means for controlling cancer disease, but it can cause tissue damage,
harm cellular and humoral immunity and also short term and long term side effects.
It may also induce a state of secondary immuno-deficiency by decreasing immune-competency.
Thus the success of radiotherapy depends on the close monitoring of patients
with efficient treatment by selection of doses. The present study is the unique
approach to contribution in this direction towards the understanding.
The authors are thankful to the technicians of Department of Radiotherapy and
Biochemistry of Barasat Cancer Research and Welfare Centre for their technical
help during this study.