Jinhai Fan

Silibinin inhibits prostate cancer invasion, motility and migration by suppressing vimentin and MMP-2 expression.

AbstractAim:Silibinin is known to exert growth inhibition and cell death together with cell cycle arrest and apoptosis in human prostate cancer cells. Whether silibinin could inhibit the invasion, motility and migration of prostate cancer cells remains largely unknown. This study was designed to evaluate this efficacy and possible mechanisms using a novel highly bone metastatic ARCaPM cell model.Methods:Four prostate cancer cell lines, LNCaP, PC-3, DU145, and ARCaPM, were used in this study. These cells were treated with increasing concentrations of silibinin (50, 100, and 200 μmol/L) for different periods of time. After treatment, cell viabilities of four prostate cancer cells were compared by MTT assay. Alterations of ARCaPM cell invasion, motility and migration were assessed by cell invasion, motility and wound healing assays. The changes of vimentin expression were observed by Western blotting and immunofluorescence staining, and the expression of MMP-2, MMP-9, and uPA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR).Results:ARCaPM cells showed less sensitivity to the growth inhibition of pharmacological doses of silibinin than LNCaP, PC-3, and DU145 cells. However, silibinin exerted significant dose- and time-dependent inhibitory effects on the invasion, motility and migration of ARCaPM cells. Furthermore, the expression of vimentin and MMP-2, but not MMP-9 or uPA, was down-regulated in a dose- and time-dependent manner after treatment of silibinin.Conclusion:This study shows that silibinin could inhibit the invasion, motility and migration of ARCaPM cells via down-regulation of vimentin and MMP-2 and therefore may be a promising agent against prostate cancer bone metastasis.

A novel anti-cancer effect of genistein: reversal of epithelial mesenchymal transition in prostate cancer cells

AbstractAim:The aim of the present study was to investigate whether low dose genistein affects the invasion and epithelial mesenchymal transition (EMT) of prostate cancer (PCa) cells.Methods:Human PCa cell lines, IA8-ARCaP and LNCaP/HIF-1a, were used in this study. The cell lines were found to process EMT in our previous study. The PCa cells were treated with increasing concentrations, from 0.1 to 75μmol/L. Proliferation was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. EMT was proven by cell morphological transition and the expression changes of EMT-related markers, which were confirmed by RT-PCR, Western blotting, and indirect immunofluorescence labeling. Transwell invasion assay was used to analyze the invasive potency.Results:The addition of genistein to the medium reduced the IA8-ARCaP and LNCaP/HIF-1a viable cell number in a dose-dependent manner (with increasing concentrations from 15 to 75 μmol/L). Less than 15 μmol/L genistein was selected as the low dose concentration, which did not affect cell proliferation. The treatment of cells with low-dose genistein induced the reversal of EMT, which was confirmed by cell morphological transition and the expression changes of EMT-related markers. The reversal of EMT in the PCa cells by low-dose genistein was in a dose-dependent manner. Moreover, low-dose genistein effectively inhibited invasion of the PCa cells in vitro.Conclusion:These results showed that treatment with low-dose genistein may be a potential strategy for the suppression of invasive growth through the reversal of EMT in cancer cells, which justifies the potential use of soybean foods as a practical chemopreventive approach for patients with PCa.

Silibinin inhibits β-catenin/ZEB1 signaling and suppresses bladder cancer metastasis via dual-blocking epithelial–mesenchymal transition and stemness

Muscle-invasive bladder cancer is associated with a high frequency of metastasis, and fewer therapies substantially prolong survival. Silibinin, a nontoxic natural flavonoid, has been shown to exhibit pleiotropic anticancer effects in many cancer types, including bladder cancer. Our and other previous studies have demonstrated that silibinin induced apoptosis and inhibited proliferation of bladder cancer cells, whether silibinin could suppress bladder cancer metastasis has not been elucidated. In the present study, we utilized a novel highly metastatic T24-L cell model, and found that silibinin treatment not only resulted in the suppression of cell migration and invasion in vitro, but also decreased bladder cancer lung metastasis and prolonged animal survival in vivo. Mechanistically, silibinin could inhibit glycogen synthase kinase-3β (GSK3β) phosphorylation, β-catenin nuclear translocation and transactivation, and ZEB1 gene transcription that subsequently regulated the expression of cytokeratins, vimentin and matrix metalloproteinase-2 (MMP2) to reverse epithelial-mesenchymal transition (EMT). On the other hand, silibinin inhibited ZEB1 expression and then suppressed the properties of cancer stem cells (CSCs), which were evidenced as decreased spheroid colony formation, side population, and the expression of stem cell factor CD44. Overall, this study reveals a novel mechanism for silibinin targeting bladder cancer metastasis, in which inactivation of β-catenin/ZEB1 signaling by silibinin leads to dual-block of EMT and stemness.

PI3K/Akt to GSK3β/β-catenin signaling cascade coordinates cell colonization for bladder cancer bone metastasis through regulating ZEB1 transcription

Muscle-invasive bladder cancer is associated with a high frequency of metastasis, and bone is the most common metastatic site outside the pelvis. To clarify its organ-specific characteristics, we generated a successive bone metastatic T24-B bladder cancer subline following tail vein injection of metastatic T24-L cells. Compared with parental T24-L cells, epithelial-like T24-B cells displayed increased adhesion but decreased migration or invasion abilities as well as up-regulation of cytokeratins and down-regulation of vimentin, N-cadherin and MMP2. Mechanically, phosphatidylinositol 3-kinase (PI3K)/Akt targets glycogen synthase kinase-3β (GSK3β)/β-catenin to control ZEB1 gene transcription, and then subsequently regulates the expression of cytokeratins, vimentin and MMP2. Importantly, ZEB1 is essential for bladder cancer invasion in vitro and distant metastasis in vivo, and ZEB1 overexpression was highly correlated with the expression of those downstream markers in clinical tumor samples. Overall, this study reveals a novel mechanism facilitating metastatic bladder cancer cell re-colonization into bone, and confirms the significance of mesenchymal-to-epithelial transition (MET) in formation of bone metastasis.

CXCL5/CXCR2 axis promotes bladder cancer cell migration and invasion by activating PI3K/AKT-induced upregulation of MMP2/MMP9

Bladder cancer (BCa) is the most common malignant disease of the urinary tract system, yet the etiology is still poorly understood. Clinically, the majority of BCa patients progress to invasive disease at the final stage, leading to death. Previous investigations have demonstrated that matrix metal-loproteinases (MMPs) play irreplaceable roles in tumor cell extravasation and implantation. In addition, increasing numbers of reports provide evidence that MMPs, especially MMP2 and MMP9 are monitored by various signal transduction pathways targeting tumor metastasis. Seed-and-soil theory has called to attention the importance of the tumor microenvironment in disease progression. To that end, we previously reported the key role of hypoxia in BCa progression. Herein, we report the role of chemokines, specifically CXCL5, is involved in BCa development. Though it has been reported that CXCL5 promotes BCa metastasis and progression, the exact mechanisms are still unknown, necessitating the need for further investigation into the role of CXCL5 in BCa. In this study, IHC staining of BCa tumor sections showed elevated expression of CXCL5 in BCa, which correlated with disease stage. Our mechanistic studies show that CXCL5 contributes to BCa migration and invasion by binding to its receptor, CXCR2, leading to the upregulation of MMP2/MMP9 by activating PI3K/AKT signaling. This study offers vital evidence of how CXCL5 promotes BCa metastasis, and thus may potentially be used as a therapeutic target against BCa.

Slug contributes to cadherin switch and malignant progression in muscle-invasive bladder cancer development ☆

OBJECTIVES The Snail family of zinc finger transcription factors (i.e., Snail and Slug) predicts the tumor recurrence in superficial bladder cancers, while their roles in the development of muscle-invasion, metastasis, and chemoresistance in muscle-invasive bladder cancers with poor prognosis have not been investigated. This study evaluates the clinical significance of Slug in aggressive bladder cancer. MATERIALS AND METHODS A pair of sublines (i.e., T24-P and T24-L) from a unique orthotropic metastatic model of bladder cancer was firstly utilized to identify the potential precursors contributing to those aggressive phenotypes. The coexpression of Slug, E-cadherin, and N-cadherin in bladder cancer cell lines (i.e., 5637, RT4, 253 J, J82, and T24) and tissues was evaluated by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry staining analysis. The function of Slug expression on E- to N-cadherin switch, cell invasion, and chemoresistance to proapoptotic treatment was validated by gain-in-function and knockdown strategy in vitro. RESULTS Slug was identified as one of the novel targets contributed to the aggressive phenotypes of T24-L cells, which showed enhanced cell invasive, metastatic, and chemoresistant potentials in vitro and in vivo as previously described. Up-regulation of Slug was significantly correlated with a higher tumor stage and the E- to N-cadherin switch in bladder cancer cells and tissues, whereas ectopic expression of Slug in bladder cancer 5637 and RT-4 cell lines promoted epithelial-to-mesenchymal transition (EMT), increased cell invasiveness and chemoresistance. By contrast, knocking down Slug using siRNA in T24-L cell lines reversed these changes. CONCLUSIONS Slug elevates in invasive or metastatic bladder cancer and plays a critical role in EMT via control of cadherin switch. Slug may be a potential marker or target for improving the diagnosis and treatment of muscle-invasive bladder cancers.

Raman spectroscopy, a potential tool in diagnosis and prognosis of castration-resistant prostate cancer.

Abstract. Purpose: We evaluated the feasibility of Raman spectroscopy (RS) in diagnosis and prognosis of castration-resistant prostate cancer (CRPC) in patients with prostate cancer (PC). Materials and methods: Raman spectra are detected from PC cell lines (LNCaP and C4-2) and tissues using a Labram HR 800 RS. Then, principal component analysis (PCA) and support vector machine (SVM) are applied for prediction. A leave-one-out cross-validation is used to train and test the SVM. Results: There are 50 qualified patients, including 33 with androgen-dependent prostate cancer (ADPC) and 17 with CRPC. The spectral changes at 1126, 1170, 1315 to 1338, and 1447  cm−1 between CRPC and ADPC are detected in both cells and tissues models, which are assigned to specific amino acids and DNA. PCA/SVM algorithm provided a sensitivity of 88.2% and a specificity of 87.9% for diagnosing CRPC tissues. Furthermore, 14 patients with ADPC progressed to CRPC within 12 months. These patients are separated into two groups depending on whether their cancers progressed to CRPC within 12 months. PCA/SVM could differentiate these two groups with a sensitivity of 85.7% and a specificity of 88.9%. Conclusions: RS has the potential in diagnosis and prognosis of CRPC in clinical practice.

Interaction of CCN1 with αvβ3 integrin induces P-glycoprotein and confers vinblastine resistance in renal cell carcinoma cells.

Renal cell carcinoma (RCC) ranks among the most chemoresistant tumors, and P-glycoprotein (P-gp) predominates multidrug resistance mechanisms by reducing the accumulation of intracellular chemotherapy drugs such as vinblastine (VBL), which is considered the most effective chemotherapeutic agent for this neoplasia. Unfortunately, the mechanism by which the expression of P-gp is regulated and the ways to inhibit the function of P-gp are poorly understood. Our study was carried out to determine the possible role of CCN1 in P-pg-mediated drug resistance on the basis of the validated function of CCN1, an extracellular matrix protein, in promoting chemoresistance. As expected, CCN1 was overexpressed in VBL-resistant cell lines (ACHN/VBL, A498/VBL, Caki-1/VBL, and Caki-2/VBL) as measured by enzyme-linked immunosorbent assay. We then transfected non-VBL-resistant cell lines with Ad-CCN1 and observed that the IC50 of VBL increased by about 3–5 times. Furthermore, both CCN1 antibody neutralization and &agr;v&bgr;3 integrin antibody blockade decreased the IC50 of VBL, which showed that CCN1 and &agr;v&bgr;3 are associated with resistance to VBL in RCC. Simultaneously, the enhanced expression of CCN1 triggered the intracellular PI3K/Akt pathway by binding &agr;v&bgr;3 integrin, as shown by western blot. P-gp expression was augmented in response to activation of the PI3K/Akt pathway, which could be modified by PI3K inhibitor LY294002 or multidrug resistance siRNA transfection. Therefore, targeted restraint of CCN1 or &agr;v&bgr;3 integrin in combination with the administration of VBL may be beneficial in the treatment of primary and metastatic RCC.

Novel Green-Light KTP Laser En Bloc Enucleation for Nonmuscle-Invasive Bladder Cancer: Technique and Initial Clinical Experience

BACKGROUND AND PURPOSE The standard procedure for staging and treating nonmuscle-invasive bladder cancer (NMIBC) is still transurethral resection of bladder tumor (TURBT) via a wire loop. However, TURBT is associated with serious disadvantages that facilitate tumor recurrence. Recently, lasers have been explored as treatment tools for bladder tumors. Here, we report a novel tumor en bloc enucleation using a front-firing green-light potassium-titanyl-phosphate laser and its initial clinical application. PATIENTS AND METHODS From March through June 2013, 45 patients with NMIBC received modified transurethral resection using a front-firing green-light laser. En bloc enucleation was performed on all tumors. Preoperative and intraoperative data were retrospectively collected. RESULTS All patients successfully went through a session of treatment with front-firing green-light laser enucleation of the bladder tumor. Complications such as bladder hemorrhage, vesicle perforation, and obturator nerve reflex were not encountered during the treatment. The tumor diameter ranges from 0.3 to 3.0 cm with a mean value of 1.8 cm. Mean operative time and enucleation time were 21 (12-38) and 12 (4-23) minutes, respectively. Serum hemoglobin decreased 1.1 (0.1-2.4) mg/dL averagely. Mean catheter time was 2.0 (1.0-3.0) days, and mean postoperative hospital stay was 2.5 (1.5-4.0) days. The stages of bladder cancer included 27 Ta, 15 T1, and 3 T2a. No tumor recurrence was observed at the initial 6-month follow-up. CONCLUSIONS The modified technique using a front-firing green-light laser to en bloc enucleate bladder tumors is effective and safe for treatment of NMIBC. Moreover, it may improve the accurate valuation of tumor stage and prediction of postoperative prognosis, although long-term outcomes and prospective clinical trials are needed.

Metformin Sensitizes Prostate Cancer Cells to Radiation Through EGFR/p-DNA-PKCS In Vitro and In Vivo

Although neo-adjuvant radiotherapy is generally successful in treatment of advanced prostate cancer, radioresistance is still a major therapeutic problem in many patients. In the current study, we investigated the effects of metformin (1,1-dimethylbiguanide hydrochloride), a widely used antidiabetic drug, on tumor cell radiosensitivity in prostate cancer. Through clonogenic survival assays, we found that metformin treatment enhanced radiosensitivity of prostate cancer cells with a dose enhancement factor. Moreover, irradiation of subcutaneous C4-2 tumors in mice treated with metformin resulted in an increase in radiation-induced tumor growth delay (17.3 days to 29.5 days, P < 0.01), which indicates that the tumor radiosensitivity increased by metformin in vivo. We also measured the sublethal damage repair and analyzed double-strand breaks (DSBs) in X-irradiated cells. γ-H2AX, as an indicator of DSBs, had significantly more foci per cell in the group treated with metformin and radiation compared to groups treated with metformin or irradiation, respectively. Moreover, mice with subcutaneous tumor implants lived longer after a combined treatment of metformin and radiation. In addition, the reduced phosphorylation of DNA-PKcs caused by EGFR/PI3K/Akt down-regulation is essential for metformin to induce radiosensitivity in prostate cancer cells. Our results indicate that metformin enhances prostate cancer cell radiosensitivity in vitro and in vivo. Exposure to metformin before radiation therapy could be a beneficial option for the treatment of prostate cancer.