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Med One 2016;1(1):2; DOI:10.20900/mo.20160002
1Department of Urology, The Third Xiangya Hospital of Central South University in Hunan Province Changsha,（410013）;
2Department of Medicine, Duke University Medical Center, Durham, NS 27710, USA.
Correspondence: Qing Zeng Email:email@example.com
Background:Prostate cancer is a common malignant tumor of the male urogenital system with increasing incidence worldwide. Molecular markers for early diagnosis and target therapy of prostate cancer are still not available clinically.
Methods: 71 prostate cancer and 10 benign prostatic hyperplasia tissues were collected for the immunohistochemistry and Western blot assay of phosphorylated MAPK (Thr202) and Akt (Ser473) levels.
Results:Under the Gleason grading system, 15 prostate cancer tissues were classified as well-differentiated, 25 were classified as moderately-differentiated, and 31 were classified as poorly-differentiated. Immunohistochemical staining showed that phosphorylated Akt level was elevated, whereas phosphorylated MAPK level was lowered, in prostate cancer tissues compared to that in benign prostatic hyperplasia tissues. Elevated p-Akt/Akt, but reduced p-MAPK/MAPK ratio correlated with poor differentiation of prostate cancer.
Conclusion: p-Akt level was elevated, but p-MAPK protein level was lowered in prostate cancer tissues. Low p-MAPK protein level may be caused by the high p-Akt level. An increase in the ratio of p-Akt/p-MAPK might be a diagnostic marker for early diagnosis of prostate cancer.
Prostate cancer is a common cancer of the urogenital system in men. The incidence and mortality of prostate cancer rank second among all malignant tumors in European and American men . The incidence of prostate cancer in China is increasing due to unhealthy lifestyles and aging . Although early diagnosis and an active treatment strategy can greatly improve the prognosis of prostate cancer patients, the mortality rate is still high due to the high malignancy of the disease . Currently, the diagnostic methods for prostate cancer include measurement of the serum PSA level and histopathological staining, but these methods are unable to give an early diagnosis or even accurate diagnosis of prostate cancer . Therefore, there is still an urgent need to find an effective biomarker for the early diagnosis and target treatment of prostate cancer.
Protein kinase B, also known as Akt, and mitogen-activated protein kinase (MAPK) are the key proteins of the PI3K/Akt signaling pathway and MAPK signaling pathway, respectively [5, 6]. Numerous studies have reported that PI3K/Akt signaling pathway and MAPK signaling pathway were closely associated with cell proliferation and invasion in a variety of malignant tumors [7, 8]. Activation of the PI3K/Akt signaling pathway can lead to the phosphorylattion of the apoptosis protein BAD and other proteins to suppress cell apoptosis . The activation of MAPK signaling pathway can promote excessive proliferation of tumor cells and lead to oncogenesis . The level of phosphorylated Akt in human prostate cancer tissues has been measured by immunohistochemistry with controversial findings. For example, Waalkes et al study observed a significantly lower expression of p-Akt in prostate cancer tissues compared to normal prostate tissue (P=0.028) . In contrast, the level of p-Akt protein was found to be significantly higher in prostate cancer tissues than in benign prostatic hyperplasia and high-grade prostatic intraepithelial neoplasia tissues . However, Ko et al study demonstrated that p-Akt ratio in prostate cancer tissues was significantly lower than that in high-grade prostatic intraepithelial neoplasia tissues , Che et al study reported that p38 MAPK levels significantly correlated with tumor progression and survival of prostate cancer patients . However, the level of phospho-MAPK in prostate cancer tissues has not been reported. Particularly, the relationship between Akt and MAPK phosphorylation has not been addressed in prostate cancer.
This study investigated MAPK and Akt phosphorylation in prostate cancer tissues and benign prostatic hyperplasia (BPH) using immunohistochemistry and Western blot.
71 prostate cancer tissues were collected at our Department from January 2013 to December 2014. The mean age of 71 prostate cancer patients was 56.2 ± 6.5 (52-68) years, and no patients received radiotherapy, chemotherapy, or immunotherapy before surgery. Benign prostatic hyperplasia (BPH) tissues were also collected from 10 BPH patients during the same time period as the prostate cancer patients. The mean age of 10 BPH patients was 54.1 ± 3.5 (45-63) years old. The tissues were kept in liquid nitrogen or fixed by 10% formaldehyde for paraffin embedding. This study was approved by the ethics committee of XX hospital. The signed informed consent form was obtained from all subjects.HE staining and Gleason grading
The paraffin embedded tissues were sectioned at a thickness of 5 μm. To perform hematoxylin and eosin (HE) stain, the paraffin sections were first baked at 60 °C for 30 min, and then rinsed in dimethyl benzene for 15 min for deparaffinization. After removing the dimethyl benzene in gradient alcohol, the sections were washed by distilled water twice. Next, the sections were stained by hematoxylin for 15 min and washed by distilled water twice. After differentiated by 1% hydrochloric acid alcohol for 30 s, the sections were further washed by distilled water for 15 min. The sections were then stained in 1% eosin for 3 min and rinsed in 70% alcohol. At last, the sections were soaked in a xylene-alcohol (1:1) mixture for 5 min, followed by soaking in xylene for 5 min and 15 min. The slice was mounted and observed under a microscope.
Tumor tissues were graded according to the Gleason grading system from well-differentiated (level 1, score 1) to undifferentiated (level 5, score 5). When the differentiation in a tumor tissue was heterogeneous, Gleason Score (GS) was obtained by adding the scores of the primary and secondary structures. For a tissue with only a single structure, the GS was doubled to obtain its final score .Immunohistochemistry
The expression of p-AKT and p-MAPK proteins in tissues was detected by immunohistochemistry. Briefly, the paraffin-embedded sections were deparaffinized using xylene, gradient alcohol, and distilled water. The sections were incubated with 3% H2O2 solution to block peroxidase activity for 10 min at room temperature (RT). After blocking with 5% BSA solution at RT, the sections were incubated with primary antibody (1:2000 dilution for Ser473 p-AKT and Thr183 p-MAPK) at 37°C for 2 hrs. After washed by 1 × PBS, the sections were incubated with biotin conjugated secondary antibody (1:1000) at 37°C for 30 min. The sections were then incubated with HRP-conjugated streptavidin at 37°C for 30 min. After washing with 1 × PBS, the sections were developed using DAB chromogenic agents for 10 min, the reaction was stopped, and the sections were washed with distilled water. After hematoxylin counterstaining, dehydration, hyalinization, and mounting, the sections were observed under a microscope. Five random fields were selected under a high-power field (400X) to calculate total cell count and positively-stained cell count .Western blot
Total protein was extracted from cells using RIPA buffer. The proteins were separated on a 15% SDS-PAGE gel and electrophoretically transferred to PVDF membranes. After blocked using TBST buffer containing 5% skim milk at 37°C for 1 hr, the membranes were incubated with primary antibody (anti-Akt, anti-Akt Ser473, anti-MAPK, and anti-p-MAPK were purchased from Cell Signaling Technology Inc. 1:1000 dilution) at 4°C overnight. After washing with TBST buffer, the membranes were incubated in HRP-conjugated IgG secondary antibody (1:1000) at RT for 1 hr. The membranes were then incubated with chromogenic substrates and exposed to X-ray films. The optical density of protein bands was scanned and analyzed as previously described .Statistical analysis
All statistical analyses were performed using SPSS 20.0 statistical software. Qualitative data was compared using the Kruskal-Wallis Test, while enumeration data were presented as rate or percentage and compared by chi-square test. Measurement data were presented as mean ± standard error and compared by t test or one-way ANOVA. A Two-tailed p<0.05 was considered statistically significant.
he prostate tumor tissues were stained by HE (Fig. 1 for Gleason grading (Table 1). Eighteen cases were graded with a GS of 2-4 points. In these cases, medium-sized glandular tumor tissue was observed, but no or minor infiltration phenomenon appeared in the tumor margin (Fig. 1b). Twenty-five cases were graded with a GS of 5-7 points. In these cases, dispersed dysplastic glands and obvious interstitial infiltration could be observed (Fig. 1c). Thirty-one cases were graded with a GS of 8-10 points. Almost no adenoid structure or acini fusion could be found, tumor cells had flake-like, funicular, or single-celled structure, and severe diffuse infiltration appeared. (Fig. 1d).
Immunohistochemical staining showed that the phosphorylated Akt and MAPK proteins were mainly located in the cytoplasm (Fig. 2). P-Akt staining was positive in % of prostate cancer tissues, and % of BPH tissues. The intensity of P-Akt staining was stronger in prostate cancer tissues than that in BPH tissues, suggesting that p-Akt was overexpressed in prostate cancer. In contrast, positive p-MAPK staining was only observed in % of prostate cancer tissues and % of BPH tissues. The intensity of p-MAPk staining was weaker in prostate cancer tissues than that in BPH tissues. The p-Akt protein level negatively, but p-MAPk level positively, correlated with proliferation.
The relationship between p-Akt and p-MAPK protein level in prostate cancer tissues
The total and phosphorylated Akt and MAPK protein levels in prostate cancer tissues were measured by Western blot (Fig. 3A). The ratios of p-Akt to total Akt and p-MAPK to total MAPK protein level were calculated (Fig. 3B). The ratio of p-Akt/total Akt was increased, whereas the ratio of p-MAPK/total MAPK was decreased in prostate cancer with a poorer differentiation (p < 0.05).
Although previous studies have compared the p-Akt protein levels between prostate cancer tissues and benign or normal prostate tissues [9-11], the findings are controversial. Also, the p-MAPK protein level has not been reported in prostate cancer tissues. This study first reported that p-MAPK protein level was decreased in prostate cancer tissues compared to benign prostatic hyperplasia tissues. Also, this study first compared p-Akt and p-MAPK protein levels in prostate cancer tissues, suggesting a negative association between these two proteins. Moreover, this study found a high level of p-Akt, but a low level of p-MAPK, in prostate cancer tissues compared to benign prostatic hyperplasia tissues. Our study suggests that Akt and MAPK phosphorylation was associated with the differentiation of prostate cancer.
Akt is a serine/threonine-specific protein kinase that plays an important role in cell proliferation and division. Also, Akt can promote phosphorylation of the pro-apoptosis protein BAD, leading to its dysfunction and thus inhibiting cell apoptosis . Akt can reduce p27kip1 protein expression to shorten the cell cycle, promoting cell division . However, phosphorylation of Akt protein is necessary for Akt function. Akt phosphorylation is regulated by PI3K and PTEN protein activity. PI3K can promote Akt phosphorylation, whereas PTEN may antagonize PI3K activity to reduce the Akt phosphorylation level . A previous study found that the PTEN encoding gene is deleted and/or mutated in some prostate cancer tissues, resulting in loss of PTEN expression [19,20]. This might be one of the reasons that the p-Akt level was higher in prostate cancer tissues than that in benign prostatic hyperplasia tissues, while high p-Akt level was found to correlate with poor differentiation in this study.
MAPK is also involved in regulating cell proliferation as the downstream effector of the RAS signaling pathway. Activated MAPK can enter the nucleus to activate transcription factors, resulting in cell proliferation and differentiation . MAPK is activated by phosphorylation . RAF inhibitors have been found to activate MAPK by relieving inhibitory autophosphorylation . A study also revealed that p-Akt can decrease Raf protein activity by phosphorylating Ser259 on the Raf protein, thus reducing the MAPK phosphorylation level . This can help explain why our study found increased p-Akt level, but decreased p-MAPK protein level, in prostate cancer tissues compared to benign prostatic hyperplasia tissues.
In conclusion, our study indicates that p-Akt level is elevated, but p-MAPK protein level is lowered in prostate cancer tissues and suggests that high p-Akt level may be one of the causes of low p-MAPK protein level. Our findings suggest that an increased ratio of p-Akt/p-MAPK might be a diagnostic marker for early diagnosis of prostate cancer.