The aim of the present study was to investigate the protein expression of the autophagy-related genes,
Ovarian cancer is a gynecological malignancy with a high rate of mortality. Due to a lack of notable symptoms in the early stages, 60% of patients present with advanced-stage disease at diagnosis (
In total, 40 tissue samples were obtained from patients with ovarian cancer who had undergone cytoreductive surgical resection at the Shengjing Hospital of China Medical University (Shenyang, China) between January 2007 and May 2012. The cases were classified as serous carcinoma (n=24), mucinous carcinoma (n=8), clear cell carcinoma (n=4), endometrial carcinoma (n=2) or undifferentiated carcinoma (n=2). The age range of the patients was 35 to 58 years old, with a median age of 48 years old. The pathological surgical staging was performed according to the 2009 International Federation of Gynecology and Obstetrics system as follows: Stage I, two cases; stage II, 10 cases; stage III, 24 cases; and stage IV, four cases (
The telephone or hospital examination follow-ups of the 40 cases of ovarian cancer began at diagnosis and ended on December 31, 2012. The patients were pathologically diagnosed with ovarian cancer and divided into a chemotherapy-sensitive (n=20) or chemotherapy-resistant (n=20) group, according to the results of the pre- or post-operative normative chemotherapy and the post-operative follow-up. The grouping standards were as follows: i) Achievement of clinical remission following the initial platinum-based chemotherapy was necessary for study inclusion; and ii) cases with relapse at six months or more following the end of the chemotherapy program were taken as the chemotherapy-sensitive group, and cases with relapse within six months were used as the chemotherapy-resistant group. This study was approved by the ethics committee of Shengjing Hospital of China Medical University and written informed consent was obtained from all patients.
The Beclin-1 polyclonal rabbit (1:100 dilution) and PTEN monoclonal mouse (1:75 dilution) antigen solutions were purchased from Proteintech (Chicago, IL, USA). The immunohistochemistry Power Vision kit and 3,3′-diaminobenzidine reagent were purchased from Beijing Golden Bridge Zhongshan Biotechnology Co., Ltd. (Beijing, China).
Phosphate-buffered saline solution was used for the negative control, and normal known-positive ovarian tissue samples were used for the positive control. Using the Power Vision kit, the experimental procedure was performed according to the manufacturer’s instructions. A total of 10 randomly selected high-magnification fields were analyzed under an optical microscope (AX70; Olympus Corporation, Tokyo, Japan) 48 h after sealing. PTEN-positive cells were identified by clear-brown granules located in the nucleus or cytoplasm, and Beclin-1-positive cells were identified by brown particles distributed throughout the cytoplasm. The percentage of positive cells to total cells was counted and awarded points according to the following criteria: <10%, 0 points; 10–20%, 1 point; 21–50%, 2 points; and >50%, 3 points. The intensity of staining was also awarded points according to the following system: no color, 0 points; pale yellow, 1 point; brown, 2 points; and tan, 3 points. The total score for each case was the sum of the points for the percentage of positive cells and the staining intensity. A score of ≤3 was regarded as negative for PTEN/Beclin-1 expression, whereas a score of >3 was regarded as positive for PTEN/Beclin-1 expression.
The statistical analysis was performed using SPSS 11.0 (SPSS, Inc., Chicago, IL, USA). The association between the clinicopathological parameters and the protein expression of Beclin-1 and PTEN was analyzed using Student’s t-test. The Spearman’s ρ test was used for the correlation analysis.
In the 40 cases of ovarian cancer included in the present study, the expression of Beclin-1 was revealed to be primarily located in the cytoplasm. The positive rate of Beclin-1 expression was significantly lower in the chemotherapy-resistant group (35.0%) compared with the chemotherapy-sensitive group (50.0%) (P<0.05). The expression of PTEN was identified to be primarily located in the nucleus or cytoplasm. The positive rate of PTEN expression was significantly lower in the chemotherapy-resistant group (30.0%) compared with the chemotherapy-sensitive group (65.0%) (P<0.05;
The correlation analysis revealed that the intensity of Beclin-1 expression was positively correlated with the expression of PTEN in the 40 cases of ovarian cancer (P<0.05;
Autophagy is a lysosomal degradation process for cellular macromolecules and damaged organelles, and an alternative form of programmed cell death to apoptosis in eukaryotes. Changes in the activity of signaling, transport and negative regulatory pathways of macrophages have been revealed to be associated with tumor occurrence and development. Furthermore, the abnormal expression of autophagy genes has been identified to activate or inhibit the formation of certain tumors (
The PTEN protein is encoded by the
The ~150-kb human
The present study indicated that Beclin-1 and PTEN may be co-involved in the regulation of autophagy, and therefore affect the occurrence and development of cancer. Autophagic protein turnover is regulated by type I and III PI3Ks. Type I PI3K, and the downstream signal conversion components Akt and target of rapomycin, can inhibit autophagy. PTEN induces autophagy by negative regulation of the type I PI3K. By contrast, the type III PI3K is necessary for the autophagic formation of lysosomal vacuoles. Beclin-1 regulates autophagic activity by modulating the precursor structure of Apg proteins, primarily by the formation of complexes with the type III PI3K. Therefore, Beclin-1 and PTEN may possess similar roles in the self-regulation of macrophage activity (
In summary, the protein expression of Beclin-1 and PTEN was downregulated, which suggested that a decrease in autophagic activity may be associated with drug-resistant ovarian cancers. At present, the specific mechanisms that regulate autophagy remain unclear. Further study will aid in clarifying the role of autophagy, which may provide novel solutions for treating chemotherapy-resistant ovarian cancers, and establish a reliable theoretical basis for the development of novel drugs.
The expression of the Beclin-1 and PTEN proteins, in drug-resistant and drug-sensitive ovarian cancers, was detected using immunohistochemistry and analyzed for potential correlations. The results revealed that Beclin-1 and PTEN protein expression was significantly lower in the chemotherapy-resistant group compared with the chemotherapy-sensitive group. Furthermore, the difference in expression was identified to be significant and positively correlated. The results suggested that Beclin-1 and PTEN protein expression decreased in the drug-resistant ovarian cancer tissues. Therefore, it was concluded that the occurrence of drug resistance in ovarian cancers was closely associated with a low expression of PTEN and Beclin-1. In conclusion, a reduction in autophagic activity, induced by the interaction between Beclin-1 and PTEN, may lead to drug resistance in cases of ovarian cancer.
This study was funded by the Doctor Started Natural Science Foundation (no. 20071047), the Higher Education Department Research Program (no. 2009A724), the Liaoning Science and Technology Program (no. 2010225032) and the National Natural Science Foundation of China (nos. 81372486 and 81302270).
Immunohistochemistry revealing clear-brown nuclear and cytoplasmic granules in phosphatase and tensin homolog (PTEN)-positive cells, and brown cytoplasmic particles in Beclin-1-positive cells (magnification, ×200). Chemotherapy-sensitive group; (A) PTEN and (B) Beclin-1. Chemotherapy-resistant group; (C) PTEN and (D) Beclin-1.
Expression of Beclin-1 and PTEN in chemoresistant and chemosensitive groups.
Beclin-1 | PTEN | ||||||
---|---|---|---|---|---|---|---|
|
| ||||||
Group | n | − | + | % positive | − | + | % positive |
Resistant | 20 | 13 | 7 | 35.0 | 14 | 6 | 30.0 |
Sensitive | 20 | 10 | 10 | 50.0 | 7 | 13 | 65.0 |
PTEN, phosphatase and tensin homolog.
Association between Beclin-1 and PTEN expression.
Beclin-1 | ||
---|---|---|
| ||
Protein | + | − |
PTEN | ||
+ | 12 | 5 |
− | 7 | 16 |
PTEN, phosphatase and tensin homolog.