αB-crystallin, a member of the small heat shock protein family, acts as a molecular chaperone. αB-crystallin has been found to be overexpressed in a number of cancer tissues, including head and neck cancers. Overexpression of αB-crystallin in cancer tissue may be related to its anti-apoptotic properties; however, its mechanism of action remains unclear. The aim of this study was to investigate the anti-apoptotic role of αB-crystallin in oral verrucous carcinoma (OVC). Since oral squamous cell carcinoma (OSCC) is the most common tumor of the oral cavity, we selected OSCC as a control group. Immunohistochemical staining was used to evaluate the expression levels. The results showed that the expression of αB-crystallin was detected in OVC, OSCC and normal oral mucosa (NM). The expression in OVC was higher compared to that of NM, but lower compared to OSCC, indicating that OVC was less aggressive than OSCC with respect to malignancy potential. Furthermore, we found that in OVC, the increased expression of αB-crystallin coincided with the decreased expression of activated caspase-3. The results indicated that αB-crystallin may play an anti-apoptotic role via inhibition of the activation of caspase-3 in OVC.
Oral verrucous carcinoma (OVC) is considered a rare, low-grade and well-differentiated carcinoma, with less potential for lymph node metastasis than other oral carcinomas. OVC is also called ‘Ackerman’s tumor’ or ‘verrucous carcinoma of Ackerman’ since it was first reported and described by Ackerman in 1948 (
OVC, which accounts for 2.2–20% of all oral cancer, is mainly found in elderly males, particularly in tobacco smokers (
αB-crystallin is a member of the family of small heat shock proteins and acts as a molecular chaperone. The essential biological function of αB-crystallin is reflected by the conservation of its structure, from bacteria to humans (
Although overexpression of αB-crystallin has been reported in head and neck cancers, this study, to the best of our knowledge, is the first attempt to characterize expression patterns of αB-crystallin in OVC and to explore the potential role of αB-crystallin in oral cancer.
A total of 17 OVCs, 15 oral squamous cell carcinomas (OSCCs) and 15 samples of healthy oral mucosa were obtained at Xiangya Hospital, Central South University, between 2002 and 2009. All patients gave written consent under the protocol reviewed, and the study was approved by the institutional review board of the Xiangya Hospital, Central South University. The OVC group, comprised 14 males and 3 females, with a mean age of 51.18 years (range 23–79). The most frequent site of OVC was the gingiva, observed in 9 patients (52.94%), followed by the buccal mucosa in 4 patients (23.52%). The OVC cases had no neck lymph node or distant metastasis (
OVC was only diagnosed when the characteristic clinical and pathological features were present (
Immunohistochemical staining was performed on 4 μm serial sections following routine procedures of deparaffinization and dehydration. The slides were incubated in 3% H2O2 for 20 min to block endogenous peroxidase activity, and were then boiled under pressure in citrate buffer (pH 6.0, for 5 min) for antigen retrieval. The tissues were then incubated with the αB-crystallin antibody (1:100 dilution, sc-101437, mouse anti-αB-crystallin monoclonal antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or actived-caspase-3 (p17) antibody [1:50 dilution, BS7004, rabbit anti-caspase-3 (p17) monoclonal antibody, Bioworld Technology, St. Louis Park, MN, USA] at 4°C in a moist chamber overnight. Slides were then washed with Tris-buffered saline (TBS) and incubated with Polymer Helper and polyperoxidase-anti-mouse/rabbit IgG successively for approximately 20 min, respectively. The color was developed with DAB and counterstained with hematoxylin (Polymer Detection System for immunohistological staining, PV-9000, Zhongshan, Godbridge, China). Negative controls were incubated with phosphate-buffered saline as a replacement for the primary antibody.
The expression level was scored based on the percentage of positively stained cells and the intensity of staining. A mean percentage of positive tumor cells was determined by the examination of 200 cells in at least 10 areas, at a magnification of ×400. Five categories were presented according to the percentage of positive cells (PP): i) 0, <5%; ii) 1, 5–24%; iii) 2, 25–49%; iv) 3, 50–75%; or v) 4, >75%. The intensity of staining (SI) was scored as follows: i) 0, no -; ii) 1, weak +; iii) 2, moderate ++; and iv) 3, intense +++. The final immunoreactive score (IRS): IRS= SI × PP, was: i) −, 0; ii) +, 1–2; iii) ++, 4–6; iv) +++; 8–12. The evaluation was performed by two observers independently.
Statistical significance was evaluated by the Kruskal-Wallis test among the three groups of OVCs, OSCCs and NM; the Mann-Whitney U test for comparison of every two groups of OVCs, OSCCs and NM; the χ2 analysis and Fisher’s exact test for analyzing the correlations with clinicopathological parameters. The Spearman’s rank correlation test was used to analyze the expression of αB-crystallin and activated caspase-3 in tumor cells. P<0.05 was considered to be statistically significant and data analysis was performed using SPSS software, version 16.0.
αB-crystallin was expressed predominantly in the cytoplasm of tumor cells. In 17 OVC cases, there were 2 with negative staining, 7 with weak staining, 6 with moderate staining (
Using the χ2 test analysis, αB-crystallin expression in OVC was compared with clinicopathological parameters. No statistically significant correlation was found with age, gender, site and clinical stage. However in OSCC, the expression of αB-crystallin was correlated significantly with the clinical stage of tumors [intensity of staining in stage I–II was weak (
Expression of caspase-3 was detected in the cytoplasm of specimens. Compared to NM, expression of activated caspase-3 was significantly reduced in OVC and OSCC (
Using the χ2 test analysis, no significant difference was found for the expression of activated caspase-3 and clinicopathogical variables in OVCs and OSCCs samples (p>0.05).
In OVCs, the overexpression of αB-crystallin was significantly correlated to the repression of activated caspase-3 statistically (p<0.05,
αB-crystallin was originally found in the human lens, which contributed to the transparency of the lens as a component of crystallin. Later, it was gradually found to exist in various other tissues including the heart, brain, kidney and muscle, but the expression in non-lens tissue was low (
When comparing the OVC cases with OSCC cases, we found that the staining of αB-crystallin in OVC was significantly lower than in OSCC. This may partially show that OVC behaves in a gentler manner than OSCC (
In tumors, the overexpression of αB-crystallin may play a significant role in preventing tumor cell apoptosis. For cell apoptosis, there are two main pathways: mitochondrial and death receptor pathways. In the initial stages of the two pathways, the cell responds to a variety of stimuli, exogenous and endogenous, by releasing apoptosis-related factors, which then activate/recruit procaspase-8 and -9, respectively. Then by multiple steps, active caspase-8 and -9 initiate the proteolytic activation of procaspase-3: first, caspase-8 or -9 cleaves procaspase-3 at an aspartate residue between its large and small subunits to generate a p24 intermediate (the prodomain and the large subunit) and the p12 small subunit; second, the p24 intermediate generates the p20 and p17 forms of the large subunit by autoproteolysis. Two p17/p12s form the active caspase-3 as heterodimers, and then active caspase-3 induces apoptosis of the cell by proteolyzing key cell targets (
In this study, we used the specific antibody of p17 generated in the activation of caspase-3 to detect the activated caspase-3. Similarly to Dimberg
Although in OSCC, the statistical significance of the two protein expressions was not found, we observed that with the increasing expression of αB-crystallin, the expression of activated caspase-3 was reduced (
In conclusion, αB-crystallin is overexpressed in OVC and may play a role in anti-apoptosis via inhibition of the activation of caspase-3 in OVC.
This study was supported by the National Natural Science Foundation of China (no. 30872895) and the Key Science and Technology Project of the Hunan Provincial Science and Technology Department, P.R. China (2008FJ2011).
Immunohistochemical staining of αB-crystallin in NM, OVC and OSCC tissue. (A) NM exhibited negative αB-crystallin expression. (B) In OVC tissue, αB-crystallin was moderately expressed. (C) In OSCC tissue, αB-crystallin was markedly expressed. αB-crystallin was mainly expressed in cytoplasm (B,C). Original magnification, ×100.
Immunohistochemical staining of activated caspase-3 in NM, OVC and OSCC tissue. (A) NM exhibited weak activated caspase-3 expression. (B,C) OVC and OSCC tissue showed negative activated caspase-3 expression. Original magnification, ×100.
Lymph node metastasis and stage category of OVC and OSCC.
Lymph node metastasis | Stage category | |||
---|---|---|---|---|
|
| |||
Positive No. (%) | Negative No. (%) | I–II No. (%) | III–IV No. (%) | |
OVC | 0 (0) | 17 (100) | 15 (88.2) | 2 (11.8) |
OSCC | 4 (26.7) | 11 (73.3) | 9 (60.0) | 6 (40) |
OVC, oral verrucous carcinoma; oral squamous cell carcinoma (OSCC).
Expression of αB-crystallin in OVC, OSCC and NM.
Group | αB-crystallin | Total | Z | P-value | |||
---|---|---|---|---|---|---|---|
| |||||||
Negative | Weak | Moderate | Strong | ||||
OVC | 2 | 7 | 6 | 2 | 17 | −2.118 |
0.044 |
OSCC | 0 | 0 | 10 | 5 | 15 | −2.792 |
0.010 |
NM | 5 | 10 | 0 | 0 | 15 | 4.268 |
0.000 |
Total | 7 | 17 | 16 | 7 | 47 |
Comparison between OVC and OSCC,
comparison between OVC and NM, and
comparison between OSCC and NM. OVC, oral verrucous carcinoma; OSCC, oral squamous cell carcinoma; NM, normal oral mucosa.
Expression of activated caspase-3 in OVC, OSCC and NM.
Group | Activated caspase-3 | Total | Z | P-value | |||
---|---|---|---|---|---|---|---|
| |||||||
Negative | Weak | Moderate | Strong | ||||
OVC | 11 | 6 | 0 | 0 | 17 | −1.448 |
0.202 |
OSCC | 7 | 8 | 0 | 0 | 15 | −3.934 |
0.000 |
NM | 1 | 7 | 7 | 0 | 15 | −2.983 |
0.040 |
Total | 19 | 21 | 7 | 0 | 47 |
Comparison between OVC and OSCC,
comparison between OVC and NM, and
comparison between OSCC and NM. OVC, oral verrucous carcinoma; OSCC, oral squamous cell carcinoma; NM, normal oral mucosa.
Correlation analysis between αB-crystallin and activated caspase-3 in OVC.
αB-crystallin | r | P-value | ||||
---|---|---|---|---|---|---|
| ||||||
Negative | Weak | Moderate | Strong | |||
Activated caspase-3 | −0.547 | 0.023 | ||||
Negative | 0 | 4 | 5 | 2 | ||
Weak | 2 | 3 | 1 | 0 | ||
Moderate | 0 | 0 | 0 | 0 | ||
Strong | 0 | 0 | 0 | 0 | ||
Total | 2 | 7 | 6 | 2 |
OVC, oral verrucous carcinoma.
Correlation analysis between αB-crystallin and activated caspase-3 in OSCC.
αB-crystallin | r | P-value | ||||
---|---|---|---|---|---|---|
| ||||||
Negative | Weak | Moderate | Strong | |||
Activated caspase-3 | −0.363 | 0.183 | ||||
Negative | 0 | 0 | 4 | 2 | ||
Weak | 0 | 2 | 3 | 3 | ||
Moderate | 0 | 1 | 0 | 0 | ||
Strong | 0 | 0 | 0 | 0 | ||
Total | 0 | 3 | 7 | 5 |
OSCC, oral squamous cell carcinoma.