| Prostate cancer is one of the most common malignant diseases in the adult male in western countries. Androgen deprivation therapy is a basic and effective therapy, but after a median period of18to24months, castration resistant prostate cancer develops, which means a bad outcome. In spite of the castration androgen in plasma, PSA maintains in a high level.Clinical phenomenon and epidemic evidences suggested that progression of CRPC can be delayed by up-regulation of androgen-AR pathways. Prostate cancer cell line LNCaP develops the characteristics of CRPC after androgen deprivation. We employed the CRPC-LNCaP cell line to investigate the role of dihydrotestosterone (DHT) in the growth, proliferation and apoptosis of the cells. Growth and viability repression were observed after DHT administration in CRPC cell line. Western blot analysis of cleaved caspase-3expression and detection of Annexin V by flow cytometry revealed an early apoptotic effect of DHT treatment. In contrast, Src—MEK-1/2—ERK-1/2pathway was constitutively actived in low DHT condition, but inhibited by the administration of DHT. It is demonstrated that CRPC cell line can be inhibited by DHT, and it indicates that DHT played a two-sides effect in prostate cancer progression. It was hypothesized that a competitive interaction between androgen-AR pathway and MAPK signaling pathway could explain the transion from CRPC to ADPC in prostate cancer. Bladder cancer is a common malignant disease, and its incidence is ranked ninth in the world and the first among urologic neoplasm in China, every year about356,000cases (274,000cases, men and women83,000cases) in patients suffering from bladder cancer, it has been reported that the bladder cancer age-standardized mortality were3.54/100,000for male, and1.19/100,000for female in China; in rural ratio were1.92/100000and0.52/100,000respectively, with the trend of incidence increasing by year. About70to85percent of bladder cancer patients was found as superficial cancer, which can be treated with transurethral resection of bladder tumor (TURBT). However, the disease has a very high rate of trecurrence after surgical resection. Clinic Guide recommends the application of intravesical instillation of drugs such as epirubicin, pirarubicin for postoperative prevention of tumor recurrence. These chemotherapy drugs usually associated with serious complications including urinary frequency, urgency, dysuria, immune suppression, and therefore looking for drugs more effective and have less side effects is of great interst.Berberine quaternary amine is extracted from the Chinese herbal medicine berberine isoquinoline alkaloids, also known as berberine, a Chinese anti-inflammatory and antibacterial drugs. In recent years, long-term inflammation may lead to tumorigenesis. Studies have shown that it had significant antitumor effect for prostate cancer, esophageal cancer, Ewing’s sarcoma, and breast cancer. And berberine has not been found to have any toxicity on the normal cells of the body by now, but can effectively kill tumor cells.Invasion and metastasis of tumor cells is a long multi-step process through the interaction between tumor cells and host. In order to diffuse into the surrounding and distant organs, the tumor cells through the extracellular matrix (HCM) and basement membrane (BM), in which contains a lot of matrix molecules such as fibronectin, laminin, and a variety of collagen and proteoglycan, and through these barriers need enzymes to dissolve the matrix. As a matrix enzyme, heparanase (heparanase, Hpa) plays an important role in the invasion and metastasis of cancer cells. Hpa is the only one able to degrade the BCM and BM, with main component of hcparan sulfate proteoglycan (HSPG), a glucuronic acid glucosidase, and it can be cleaved at a specific site of HSPG heparan sulfate (HS) side chain, promoting tumor invasion and metastasis, and through the release and activation of heparin-binding growth factors such as bFGF and VEGF-induced formation of tumor angiogenesis. Studies have shown that berberine can inhibit a variety of tumor metastasis and invasion, but the unerlying mechanism is elusive.. We hereby investigated th berberine role of heparanase in bladder cancer, to clear out whether berberine inhibition of the bladder tumor metastasis and invasion by heparanase.To study the effect of berberine in bladder tumor invasion and metastasis, we used berberine and siRNA to interfere the expression of the heparanase in bladder cancer cells T24, and after treatment of the hpa-siRNA and berberine of T24bladder cancer in difierent concentrations, we detected the expression of the mRNA and protein of heparnase using RT-PCR and western blot assay. After the same treatment we used the Transwell chamber to description cell migration and invasion experiments of T24bladder cancer cells. The results are as below:1. The transwell migration assay indicated that transfection of hpa-siRNA could abolish the mobility of the T24cells. The number of invasive cells in hpa-siRNA groups was lower than control cells. Migration assay in T24cells treated by berberine in different concentration indicated that a gradual decrease of the number of T24cells penetrated the transwell camerula by a dose-dependent manner. 2. Transwell cell invasion assay manifested that hpa repression by siRNA and berberine treatment could inhibit the invasion ability of the T24cells. The number of invasive cells in hpa-siRNA groups was lower than in the blank control. And the number of invasive cells in berberine treatment groups was gradually reduced with the increase of the concentrations of berberine.3. RT-PCR results show that:a gradual reduction in heparanase expression was observed in the T24cells with the prolongation of the treated hours and the increase of the concentrations of berberine It confirmed that the inhibition of the berberine to the mRNA of heparanase are dose-dependent and time-dependent. We found that heparanase could be knockdown by the hpa-siRNA. Compared with negative control, T24cells transiently transfected with hpa-siRNA displayed a remarkable decrease in heparanase mRNA levels when evaluated by reverse transcription-PCR (RT-PCR).4. Western blot analysis showed:a gradual reduction in protein of heparanase expression was observed in the T24cells with the prolongation of the treated hours and the increase of the concentrations of berberine. We demonstrated that the inhibition of the berberine to the protein of heparanase is dose-dependent. The protein was extracted from the T24cells treated by negative control and hpa-siRNA, and western blot analysis showed the high expression of heparanase in control T24cells and the expression of the heparanase was decreased significantly in the group of hpa-siRNA... |
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