Mammary carcinomas are currently the most common and malignant tumor occurring in women. About 1 200 000 women in the world are diagnosed with mammary carcinomas each year and 500 000 women die from this kind of tumor; thus it presents a major threat to women's health and to the quality of their lives. Much attention has been focused on understanding the mechanism of breast carcinogenesis and research has revealed that there are multiple factors and steps involved in this process. In general, in the transformation from normal cells into cancerous ones, many types of DNA damage induce the inactivation, loss or variation of some oncogenes and anti-oncogenes. Such DNA damage may be caused by heredity effects, environmental carcinogens, radioactivity, and viruses. Out of all of these different mechanisms, the relationship between virus and tumor has been the subject of many recent studies. However, although some research have shown that the infection of HPV is closely related to the carcinogenesis of human breast, the precise mechanism is still not clear.
Additionally, research has indicated that HPV-16E6 & E7 can induce cells to be immortal and transform them into tumor cells by influencing the P53 and RB1 proteins, cell cycle regulatory proteins, as well as telomerase.Whether the infection of HPV can induce mammary carcinogenesis via other routes hasn't been reported yet.
In this study, the expression of the HPV-16, iNOS, P53 and hTERT proteins were investigated to examine their possible involvement in the mechanism of HPV in mammary carcinogenesis. If it was found these proteins played a significant role in this process, this may provide a new way to investigate the mechanism in mammary carcinogenesis and provide new options for the clinical prevention of this disease.
52 samples of mammary carcinomas and 16 samples of benign breast tumors from four hospitals in Harbin were assayed. These hospitals were the Thoracic Hospital of Harbin, the 4th hospital of Harbin, the 4th hospital of Harbin Medical University and the 7th hospital of Harbin.
The patients were all female with ages ranging from 23 to 72. The average age was 51. A summary of the types of breast carcinoma in each patient is summarized in Table 1.
Table 1. Types of 52 cases of breast carcinoma
Immunohistochemical staining was performed with the two-step method. The sections were deparaffinized and incubated with 3% hydrogen peroxide to in-activate endogenous peroxides, antigen retroviral treatment involved 10 mmol citric acid buffer in a autoclave. Monoclonal antibody HPV-16 (MAB-0354 -working dilution 1:100 was purchased from Boster Biological Technology Ltd), Monoclonal antibody P53 (MAB-0142 -working dilution 1:100) and its auxiliary S-P reagent was purchased from Maixin Biological Technology Ltd. Rabbit Anti-NOS2(BA-0362-working dilution 1:100) and Rabbit anti-Htert (BA-0564 -working dilution 1:100) and its auxiliary S-P reagent was purchased from Boster Biological Technology Ltd. Reaction products were visualized with diaminobenzidine as the chromogen and finally counterstained with hematoxylin.
Following the manufacturers' instructions, we used the given positive section to be the positive control and used PBS instead of the first antibody as the negative control.
The positive expression of HPV-16, iNOS and hTERT was determined by the presence of brown granules in the cytoplasm. The positive expression of P53 was determined by the presence of brown granules in nucleus. The criterion for positive expression was that more than 10% of the cells were positive in a sample set of 500 cells.
The t-test was adopted to analyze the difference of the expression of the HPV-16, iNOS, P53 and hTERT proteins between the group of 52 samples of breast cancer and the group of 16 samples of benign breast tumors. The x2 test was adopted to show the effect of HPV infection on the expression of NOS, p53 and hTERT proteins and the relationship between HPV, iNOS, P53 and hTERT expression. The Bonferroni correction was used to compensate for the multiple x2 tests.
Reagents and methods
Analysis of results
In this study of the expression of HPV in a study group of 52 breast cancer subjects, 23 cases were found to express HPV-16 whereas only one case of HPV infection was found in the benign tumours, corresponding to positive rates of 44.2% and 6.3% respectively. This differerence was found to be significant at P < 0.05 (Table 2). For iNOS, the positive rates were 57.7% and 12.5% respectively (significant at P < 0.05. Table 3). For P53, positive rates were 59.6% and 0% (significant at P < 0.05. Table 4) and for hTERT, positive rates were 65.3% and 18.8%, (significant at P < 0.05, Table 5).
Table 2. The Expression of HPV16
Table 3. The expression of iNOS
Table 4. The expression of P53
Table 5. The expression of hTERT
The positive expression of HPV16 in invasive ductal carcinoma of breast with brown granules in the cytoplasm of cancer cells and its estimated percentage was 90% as in shown in Fig. 1. The positive expression of iNOS in papillar carcinoma of breast is shown in Fig 2. There were brown granules in the cytoplasm of cancer cells and the estimated percentage of positive expression cells was 95%. Fig. 3 shows the positive expression of hTERT in invasive ductal carcinoma of breast. There were brown granules in the cytoplasm of cancer cells and the estimated percentage was 90%. The positive expression of P53 in scirrhous carcinoma of breast is shown in Fig. 4. There were brown granules in the nucleus of cancer cells and the estimated percentage of positive expression cells was 85%.
The samples were then grouped according to whether or not they had an HPV-16 infection. The positive expression of iNOS was observed in 78.3% of the samples in the HPV-16 positive group and in 41.4% of the samples in the HPV-16 negative group. This difference was significant at P < 0.05. The positive expression of P53 was observed in 87% of the samples in the HPV-16 positive group and 37.9% of the samples in the HPV-16 negative group which was significant at P < 0.05. The positive expression of hTERT was observed in 87% of the samples in the HPV-16 positive group and in 44.8% of the samples in the HPV-16 negative group which was significant at P < 0.05 (Table 6).
Table 6. Affection of Infection of HPV16 on Expression of iNOS, P53 and hTERT in Breast Cancer
The positive expression of iNOS was observed in 78.3% of the HPV 16 positive group. The relationship between the expression of HPV16 and the expression of iNOS was found to be significant at P < 0.05 (Table 7). But when the Bonferroni correction was applied (P＝0.0167), the relationship between them was not certain. Positive expression of P53 was observed in 76.7%(23/30) of the iNOS positive group and 27/31 cases expressed both hTERT and P53. The expression of iNOS was related to the expression of P53(with Bonferroni correction P＝0.0167), as shown in Table 8. The positive expression of hTERT was observed in 87.1%(27/31) of the P53 positive group. The expression of P53 was related to the expression of hTERT(with Bonferroni correction P＝0.0167), as shown in Table 9.
Table 7. The Correlation of the Infection of HPV16 with the Expression of iNOS in Breast Cancer
Table 8. The Correlation of the Expression of iNOS with the Expression of P53 in Breast Cancer
Table 9. The Correlation of the Expression of P53 with the Expression of hTERT in Breast Cancer