Contributed equally
Prostate cancer (PCa) is common in Western populations and the second leading cause of cancer-related mortality among males in North America, with an increasing morbidity in China and other Asian countries. The aim of this study was to evaluate the protein expression of autophagy-related genes Beclin-1 and LC3 in patients with prostate cancer (PCa) and benign prostatic hyperplasia (BPH) and elucidate their association with p53 and Bcl-2. The total protein of 34 PCa and 50 BPH samples was extracted and the expression of Beclin-1 and LC3 was analyzed by western blotting assay. Subsequently, a total of 96 paraffin-embedded BPH tissue samples was subdivided into 2 groups, one group in which patients had received 5α-reductase inhibitor, due to its effect of androgen ablation, and the control group, in which patients had not received the 5α-reductase inhibitor. The samples were randomly collected and examined using immunohistochemical (IHC) analysis. The western blot analysis demonstrated that Beclin-l and LC3 expression was higher in BPH tissues compared to PCa tissues (P<0.001). There was no statistically significant difference between PCas of different Gleason scores (P>0.05). The result of IHC revealed that Beclin-l and LC3 expression in the group of patients who had received the 5α-reductase inhibitor was significantly higher compared to that in the control group; however, the expression of Bcl-2 and p53 was lower (P<0.05). Beclin-1 expression exhibited a negative correlation with Bcl-2 (r=−0.402, P<0.001), whereas LC3 expression exhibited a positive correlation with Beclin-1 (r=0.345, P=0.001) and a negative correlation with Bcl-2 (r=−0.216, P=0.035). It was suggested that autophagy-related genes Beclin-l and LC3 may be involved in the development and progression of PCa. In addition, the expression of these genes was higher in patients with BPH who had received a 5α-reductase inhibitor, due to androgen reduction. As a result, the induced autophagy may reduce the risk of PCa.
Prostate cancer (PCa) is common in Western populations and is the second leading cause of cancer-related mortality among males in North America (
Autophagy is a conserved evolutionary process that is associated with numerous cell responses (
A total of 96 paraffin-embedded BPH tissue samples were obtained during a two-year period (between July, 2010 and December, 2012). The samples were divided into two groups, those from patients who had received 5α-reductase inhibitor (n=55) and the control group (n=41) and the expression of Beclin-1, LC3, p53, Bcl-2 and p53 was measured using IHC analysis. The specimens were provided by the Lu’an Affiliated Hospital of Anhui Medical University (Lu’an, China) and the Union Hospital of Fujian Medical University (Fuzhou, China). In addition, protein samples of fresh specimens from 34 PCa and 50 BPH tissue samples were obtained during surgery. All the specimens were confirmed by pathology.
Fresh specimens, including PCa and BPH tissues, were obtained during surgery. Cell protein was extracted using IP cell cracking liquid (Beyotime, Fuzhou, China). Following electrophoresis, the proteins were loaded onto polyvinylidene fluoride microporous membranes (Millipore, Billerica, MA, USA). After blocking of non-specific binding with 5% bovine serum albumin for 2 h at room temperature, the proteins were identified using a primary antibody specific to Beclin-l/LC3 (dilution 1:200) in phosphate-buffered saline (PBS) with Tween-20 under gentle agitation at 4°C overnight. Western blot analysis was performed with an anti-rabbit IgG secondary antibody (dilution 1:1000; Bioss, Beijing, China) and the Enhanced Chemiluminescence Detection system (ECL; Amersham Pharmacia Biotech, Freiburg, Germany). β-actin was used as a loading control.
IHC staining was performed using anti-Beclin-1 rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA), anti-LC3 rabbit monoclonal antibody (generously provided by the Institute of Urology of the Union Hospital of Fujian Medical University), anti-p53 and anti-Bcl-2 antibodies (Fuzhou Maixin Biotechnology Development Co., Fuzhou, China). Briefly, the slides were rehydrated and antigen retrieval was performed by microwave for 15 min in citrate buffer. The slides were incubated in 3% hydrogen peroxide for 30 min to block endogenous horseradish peroxidase (HRP) activity, followed by incubation with normal goat serum in PBS for 60 min at room temperature. The slides were then incubated with the primary antibody (dilution 1:100) at 4°C overnight. Subsequently, the slides were incubated with biotin-labeled anti-rabbit IgG and preformed avidin-biotin peroxidase complex. The slides were then counterstained with hematoxylin, dehydrated and mounted.
The slides were investigated at a magnification of ×400 and a strong brown staining was identified in the nuclei for p53 and in the cytoplasm for LC3, Beclin-1 and Bcl-2. The proportion of positively-stained cells was determined in a minimum of five fields of view. The expression in all the specimens was classified by two pathologists in our institute according to the criteria of Ohuchida
For the results of IHC and western blot analysis, the Mann-Whitney U test was used to analyze the statistical contrast of different groups. The correlation coefficients (r and P-values) among LC3, Beclin-1, Bcl-2 and p53 status were obtained using the Spearman’s test. P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed using SPSS software, version 11.5 (SPSS Inc., Chicago, IL, USA).
To confirm the role of Beclin-1 and LC3 in the development and progression of PCa, we investigated their expression by western blot analysis. The average relative expression value of Beclin-1 in BPH was 2.09±0.12 and that of LC3 was 1.38±0.04. The average relative expression value of Beclin-1 and LC3 in PCa was 0.77±0.06 and 0.84±0.03, respectively. The statistical analysis demonstrated that the expression of Beclin-1 and LC3 was stronger in BPH compared to that in PCa (P<0.001) (
To determine the frequency of expression of the different types of autophagy-related proteins in BPH between patients who had received the 5α-reductase inhibitor and the control group and its correlation with autophagy-related genes and tumor-related genes, the expression of LC3, Beclin-1, Bcl-2 and p53 was investigated via IHC analysis. The results are presented in
Among the total of 55 BPH patients who had received the 5α-reductase inhibitor, the positive expression rates of Beclin-1, LC3, Bcl-2 and p53 were 61.82% (34/55), 52.73% (29/55), 40% (22/55) and 32.7% (18/55), respectively. In the control group, the positive expression rates were 34.15% (14/41), 36.59% (15/41), 58.54% (24/41) and 51.2% (21/41), respectively. Therefore, the protein expression of Beclin-1 and LC3 in the 5α-reductase inhibitor group was significantly higher compared to the control group (P=0.012 and 0.001), whereas the expression of Bcl-2 and p53 was lower (P=0.031 and 0.045) (
The phenomenon of autophagy is commonly encountered in eukaryotic cells. The autophagic process may be initiated by nutrient starvation, growth factor withdrawal, oxygen deficiency or protein misfolding. In addition, when amino acid concentration decreases, autophagy is induced to produce amino acids required for cell survival. When the supply of amino acids is increased, autophagy is suppressed. As regards the role of autophagy in tumorigenesis and tumor progression and its level in different organs of tumor patients, in the same organ with different types of tumors, or even in different development stages of the same tumor, the results vary widely among different studies. Autophagy may remove damaged organelles, thus contributing to gene stability and suppressing cell malignant transformation. Furthermore, as a type of protective mechanism, autophagy protects cancer cells against damage from a low supply of nutrients, ionizing radiation and chemotherapy (
Autophagy-related genes Beclin-l and LC3 may suppress tumor growth by inducing autophagy. Therefore, they are considered a potential therapeutic target in cancer management. It was previously reported that deletion mutations of the Beclin-1 gene were detected in 75% of ovarian cancers, 50% of breast cancers and 40% of PCas (
Subsequently, we aimed to investigate whether autophagy in BPH tissues is induced in the absence of androgen and elucidate the role of autophagy in the development of BPH. The number of available studies on the association between autophagy and BPH is limited. A previous study reported that the number of autophagosomes was significantly increased in prostate epithelial cells of castrated rats (
It has already been confirmed that a variety of tumors are closely associated with the apoptosis-related genes p53 and Bcl-2. Bcl-2 may increase the risk of tumorigenesis and promote tumor progression through the inhibition of cell apoptosis (
In our study, the expression of Beclin-1 and LC3 in the 5α-reductase inhibitor group was significantly higher compared to the control group. However, the expression of Bcl-2 and p53 was lower (P<0.05), indicating that autophagy was induced in BPH tissues due to lack of androgen. We hypothesized that the underlying mechanisms are the result of the androgen reduction as follows: the gene expression of several transport glycoproteins and amino acids was restrained and the mTOR signaling pathway was downregulated, thus enhancing autophagy (
Therefore, the promotion of autophagy provides adequate protection to cells from canceration, including BPH tissue cells in patients who received 5α-reductase inhibitor. Our study has demonstrated that the expression of Beclin-l and LC3 was upregulated and associated with p53 and Bcl-2 in BPH patients who had received 5α-reductase inhibitor. This may reduce the risk of PCa, in accordance with previous studies suggesting that finasteride (a type of 5α-reductase inhibitor) may lower the risk of PCa, although an increase the pathological grade of PCa was observed (
A previous study demonstrated that autophagy restraint may enhance apoptosis induced by 5-FU (
In conclusion, autophagy inhibitors or autophagy revulsants, used alone or combined with other anticancer drugs, have achieved promising results. The assessment of the status of Beclin-l and LC3 may prove useful in the treatment of PCa and the prevention of canceration in BPH patients.
This study was supported by Professor Enci Xu (Institute of Urology, Union Hospital of Fujian Medical University) through the generous provision of anti-LC3 rabbit monoclonal antibody. We also gratefully acknowledge Dr Meichun Zhang (Department of Laboratorian, Lu’an Affiliated Hospital of Anhui Medical University) for the critical advice.
Verification of the expression of Beclin-1 and LC3 using western blot analysis. Beclin-1 and LC3 expression in benign prostatic hyperplasia (BPH) was found to be higher compared to that in prostate cancer (PCa).
The average relative expression values of Beclin-1 and LC3 in benign prostatic hyperplasia (BPH) were 2.09±0.12 and 1.38±0.04, respectively, which were significantly higher compared to those in prostate cancer (PCa) (0.77±0.06 and 0.84±0.03, respectively; P<0.001). ARE, average relative expression.
Immunohistochemical staining of benign prostatic hyperplasia tissues. The staining is cytoplasmic for (A) Beclin-1, (B) LC3 and (C) Bcl-2 and nuclear for (D) p53 (arrow). Original magnification, ×400.
Comparison of statistical data of 96 BPH patients with ICH analysis.
Groups | |||
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5-α reductase inhibitor | Control | P-value | |
Beclin-1 | 61.82% (34/55) | 34.15% (14/41) | 0.001 |
LC3 | 52.73% (29/55) | 36.59% (15/41) | 0.012 |
Bcl-2 | 40% (22/55) | 58.54% (24/41) | 0.031 |
p53 | 32.7% (18/55) | 51.2% (21/41) | 0.045 |
BPH, benign prostatic hyperplasia; ICH, immunohistochemical.