METHODS
Twenty specimens of oral squamous cell carcinoma (OSCC)
of the oral cavity were immunostained for HSPs to expose
differences in stainability among normal epithelium (n=20)
and leukoplakia (n=20) as dysplasia.
RESULTS
Immunohistochemistry demonstrated that OSCC cells were
positive for all of HSP60, HSP70 and HSP27. Leukoplakia
cases were positive for HSP70 and HSP27, but stained with
variability for HSP60. Normal epithelium expressed HSP60
and to a lesser extent HSP70, while HSP27 were hardly
ever expressed. The HSP70 and HSP27 stainings in OSCC
were significantly higher than in normal epithelium, and
demonstrated almost the same staining character as with
leukoplakia.
CONCLUSION
Our results demonstrated that HSP immunochemistry revealed
changes in especially HSP70 and HSP27 expression during
tumorigenesis of squamous epithelium of the oral cavity.
Keywords: Carcinogenesis; dysplasia; heat shock protein; oral cancer; squamous cell carcinoma
Oral squamous cell carcinoma (OSCC) is one
of the most common malignancies worldwide. The
individual steps in the etiopathogenesis of OSCC
are not clearly identified. Carcinogenesis is a complex
process which is controlled by different kinds
of genes and mechanisms. Many of the oncogenes
including Ras, myc, c-erbB1; antiapoptotic proteins
such as Bcl-x and Bcl-2; and several tumor
suppressor genes have been recognized in pathogenesis
of oral carcinomas. Also, precancerous lesions
such as leukoplakias and erythroplakias play
a role in oral carcinogenesis.[
Heat shock proteins (HSPs) form the most ancient
defence system in all living organisms. They
are a class of functionally related proteins whose
expression is increased when cells are exposed to
elevated temperatures or other stresses, including
infection, irradiation, heavy metals, ethanol and
oxidants. These proteins can be divided into different
families according to their molecular weight
such as HSP27 (27KDa), HSP70 (70KDa), HSP60
(60 KDa), and HSP90 (90KDa).[
However HSPs are beneficial to the normal
cells. Cancer cells can also use HSPs in response
to stress, leading to increased expression.[
HSPs are considered useful as diagnostic or
prognostic predictive factors in a variety of tumors;[
Immunohistochemistry
For immunohistochemistry, the paraffin blocks
were cut serially into approximately 5 μm thick
sections on charged slides. Firstly, the sections
were penetrated and dried overnight in an autoclave
(56 ºC). They were deparaffinised with xylene for
30 min, and washed with 99% alcohol for 15 min,
then 96% alcohol and distilled water. Ultravision
Large Volume Detection System Anti-Polyvalent
HRP Kit (Rabbit-mouse, Lab Vision Corporation,
Fremont, CA, USA) was used in this study. For antigen
retrieval, the sections were microwaved four
times for 5 min in citrate buffer (Ph 6.0), cooled to
room temperature and then washed in phosphate
buffered saline (PBS) for 5 min. Endogenous peroxidise
activity was blocked by incubating the sections
with 3% H2O2 and washed distilled water and
waited in PBS for 5 min. To prevent non-specific
reactions, sections were incubated with block solution.
Heat shock protein 27 (HSP27) antibody
ready to use (Lab Vision Corporation Neomarkers,
Fremont, CA, USA), heat shock protein 60
(HSP60) antibody ready to use (Lab Vision Corporation
Neomarkers, Fremont, CA, USA), heat
shock protein (HSP70) antibody ready to use (Lab
Vision Corporation Neomarkers, Fremont, CA,
USA) were used as primary antibodies. Slides
were incubated 60 min with HSP27, HSP60, and
HSP70. The secondary antibody was reacted for 25
min., followed by streptavidin peroxidise reagent
for 25 min. AEC (Lab Vision Corporation, Fremont,
CA, USA) chromogen was used to visualize
the reaction. Finally, the sections were counterstained with Mayer's haematoxylin, and evaluated
by a light microscope.
Evaluation Methods
Immunoreactivity of HSP27, 60, and 70 were
detected in the cytoplasms of oral epithelial cells.
The immunostained slides were evaluated on a
subjective estimate 4 point scale, negative or faint
stain (–), focal or diffuse weak stain (+), focal
strong stain (++), diffuse strong stain (+++), by
two independent oral pathologists. The HSP27, 60,
and 70 levels in the slides with a score of (–) was
taken basal level of expression whereas a score of
(+) or higher were taken to represent overexpression
of these antibodies.
Statistical analysis was performed using the Graph Pad Instant Version 2.02. To test the significance of the differences in HSP27, 60, and 70 expressions between OSCC, leukoplakia and normal oral mucosa, chi-square test was used and p value of < 0.05 was considered to indicate statistical significance.
HSP27 staining in OSCC and dysplastic lesions
(Fig.
Nine cases of oral mucosa (45%), 19 cases of
OSCC (95%) and 20 cases of leukoplakia (100%)
showed HSP70 overexpression. No HSP70 staining
was observed in 11 cases of normal mucosa
(55%) and just one case of OSCC (5%) and none
of dysplastic lesions. HSP70 was expressed more
strongly in dysplastic lesions and OSCCs (Fig.
Twenty of the OSCC cases, 17 cases (85%)
showed overexpression of HSP60, and 3 cases
(15%) showed no HSP60 staining (Fig.
HSP60 accumulates specifically in mitochondria
where its major functions are protein chaperoning
and folding. Also HSP60 is known to act as a positive modulator of apoptosis by accelerating
the maturation and activation of caspase-3.[
HSP70 regulates a wide range of protein-associated
activities and elevated levels of HSP70
protect cells from apoptotic death.[
Oncologic investigations have observed that
HSPs might be crucial for carcinogenesis[
In general, our results agree with certain results
of the literature. On the other hand, a correlation
between HSP immunostaining and survival period
or prognostic factors such as lymph node metastasis,
clinical stage has not been shown clearly. Further
investigations with the use of other methods
could help to determine the real levels of these mediators
in these lesions.
