| Part Ⅰ Nuclear factor-κB signaling in prostate cancer progressionBackgroundProstate cancer (PCa) originated in the prostatic epithelium, is the common tumor of elder male. In the world, PCa ranks as the second common cancer affecting men and the sixth leading cause of cancer deaths. The incidence rate of PCa in China is remained not high, it would be rapidly increased in recent years. Several studies show that chronic inflammation plays a vital role in the oncogenesis and progression of many tumors, including PCa. Macrophage is an important inflammatory cell in tumor microenvironment, and could affect tumor progression via several pathways. It is reported that tumor associated macrophage (TAM) could promote hematogenous metastasis. Most chemotherapy drugs would result in tumor drug resistance, because tumor cells are easy to gene mutations. Macrophage is genetically stable, and is considered as the target for the treatment of tumor. However, its exact effect and the underlying mechanism in prostate cancer remains poorly understood.Nuclear factor kappaB is an important regulator of inflammation and immune reaction. Recent studies indicate that NF-κB is aberrantly activated in most tumors, and suppression of NF-κB activation can inhibit the progression of several tumors, which implies that NF-κB is vital for the oncogenesis and progression. In addition, NF-κB is considered as the important regulatory factor in the tumor microenvironment. Removing NF-κB of Tumor-associated Macrophage (TAM) can inhibit significantly the progression of most tumors, indicating that NF-κB plays an important role in the tumor progression of tumor microenvironment. And NF-κB is regarded as a potential target for PCa treatment.SmsDX is a glycation derivative of somatostatin, and could bind to all five somatostatin receptors (SSTR1-5). Our previous studies show that smsDX could induce the apoptosis of prostate cancer via XAF1 regulation, and suppress the development and progression of prostate cancer via regulation on mitochondrial related proteins, which shows its anti-tumor activity. It has also been reported that somatostatin and its analogue could suppress inflammation by the limiting the transportion, phagocytosis and survival of macrophage. However, the effect of smsDX against the TAM-stimulated proliferation, migration and invasion of prostate cancer has not been well established.ObjectiveIn the present research, a co-culture model of three common kinds of PCa cells (LNCaP, DU145 and PC-3) with tumor-associated macrophage (TAM) was respectively established. SmsDX was added to evaluate its inhibitory effect on cell proliferation, migration and invasion. Consistently, the expression of NF-κB was determined to its role in the process. Our research would serve as a theoretical underpinning for treatment for advanced prostate cancer.MethodsLNCaP, DU145 and PC-3 were respectively co-cultured with conditioned medium of M2-polarized THP-1 macrophages. CCK-8 and clone formation assay were performed to evaluate cell proliferation. The effect of macrophage and smsDX on cell migration and invasion was determined by scratch migration and Transwell migration and invasion assay. A xenograft assay was performed to detect anti-proliferation effect of smsDX in vivo. Macrophage infiltration and NF-κB expression was detected in the specimens of 24 PCa,12 BPH with chronic inflammation and 33 BPH using immunohistochemistry. Western blot and immunofluorescence were used to evaluate the expression and nuclear transition of NF-κB.ResultsWith CCK-8 and clone formation assay, we found that macrophage significantly promoted the proliferation of LNCaP, DU145 and PC-3 cells in a time-dependent manner, whereas smsDX could weaken the impact of macrophage on cell proliferation. The similar phenomenon was also observed in the cell migration and invasion with scratch migration and Transwell assay. Consistently, treatment with macrophage significantly increased the average tumor volume. However, tumor growth was inhibited after treatment with smsDX. The immunohistochemistry showed that there were many macrophages infiltration in BPH with chronic inflammation and PCa specimens. Moreover, p65 expression was positively correlated with macrophage density in prostate tissues. Western blot analysis revealed that macrophage reduced the abundance of p65 in the cytoplasm, and increased levels of p65 were observed in the nucleus. The inhibitory effect on nuclear transition of NF-κB was further proved by immunofluorescence.ConclusionsOur research demonstrates that macrophage can promote the proliferation, migration and invasion of prostate cancer in vitro and in vivo via activating NF-κB. And smsDX treatment could attenuate the TAM-stimulated influence in prostate cancer via NF-κB regulation. These results indicate that smsDX may be a promising adjuvant therapeutic drug for prostate cancer patients.Part II Role of AKT-mTOR pathway in prostate cancer progressionBackgroundProstate cancer (PCa) is the common cancer affecting elder male and represents the seventh most frequent diagnosed malignancy in our country. Prostate cancer is androgen dependent and sensitive to androgen deprivation in early stage. However, most patients would develop to be androgen-independent within one to three years after endocrine therapy. At this moment, endocrine therapy has limited benefit and little treatment is available, and the five-year survival rate is 29%. It is reported that there is bone metastasis in about 70% advanced prostate cancer patients, which is the leading cause for poor progression of prostate cancer. Recently, abiraterone and denosumab were proved to be partly effective to advanced prostate cancer. However, a high cost and side effect limit their clinical application. Thus, low-cost pharmacologic interventions to retard prostate tumor growth after the emergence of androgen-independent disease are urgent.PI3K-AKT-mTOR pathway can regulate the proliferation, apoptosis and invasion of several tumors. It is reported that PI3K-AKT-mTOR pathway was upregulated in 42% primary prostate cancer. And the proportion in metastatic prostate cancer was 100%. Additionally, inhibiting this pathway could suppress the proliferation and metastasis of prostate cancer, which implied the important role of PI3K-AKT-mTOR pathway in prostate cancer development. Therefore, PI3K-AKT-mTOR was considered to be an important target for prostate cancer.Scutellaria baicalensis (SB, known as Huang-Qin in China) is a component from dry root of scutellaria. It has a wide range of biological activities, including anti-inflammatory, anti-virus, anti-oxidant, anti-allergy and anti-tumor effects. Baicalein, a active ingredient of SB, has been shown inhibitory effect on hepatocellular, osteosarcoma, neurospongioma and breast tumor. However, the potential effect of baicalein on androgen-independent prostate cancer has not been well established. It is not clear whether baicalein can inhibit the proliferation, migration and invasion, and induce the apoptosis of prostate cancer via PI3K-AKT-mTOR pathway. Therefore, we investigate the influence of baicalein in the proliferation, migration, invasion and apoptosis of prostate cancer in vitro, and discuss its regulatory mechanism.ObjectiveThe effect of baicalein on cell proliferation, migration, invasion and apoptosis of prostate cancer in vitro was investigated. Futher investigation of the regulatory mechanism indicates the roles of caveolin-1/AKT/mTOR in these actions.MethodsAndrogen-independent prostate cancer cell lines, DU145 and PC-3, were used in this study. CCK-8, Scratch and Transwell migration and invasion assay were respectively performed to evaluate the effect of baicalein on growth, migration and invasion of DU145 and PC-3 cells. The apoptotic influence of baicalein was determined by flow cytometric apoptosis assay. In addition, the protein expression of Bax, Bcl-2, PARP, survivin, caveolin-1, AKT and mTOR were determined by western blot.ResultsCell viability assay indicated that baicalein inhibited the proliferation of DU145 and PC-3 in a time-and dose-dependent manner. Scratch and Transwell assay showed the dose-dependent inhibitory effect of baicalein on migration and invasion. With flow cytometric apoptosis assay, we found baicalein induced the apoptosis of DU145 and PC-3 cells. Mechanistically, we presented that baicalein significantly decreased the expression of caveolin-1, phosphorylated AKT and mTOR, accompanied by up-regulation of Bax, PARP and down-regulation of Bcl-2, surviving. While the total AKT and mTOR haven’t change. Moreover, LY294002, a specific AKT inhibitor, could strengthen the inhibitory effect of baicalein on cell proliferation and invasion, which indicated that baicalein showed its anti-tumor activity in prostate cancer partly via inhibition of AKT/mTOR pathway.ConclusionOur study indicates that baicalein can significantly exhibit metastasis inhibitory and pro-apoptosis effect on androgen-independent prostate cancer cells. Mechanically, we found caveolin-1 and AKT/mTOR pathways account for the anti-tumor activity of baicalein. All these results provide useful information for the treatment of androgen-independent prostate cancer. |
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