Dapeng Wu

Tumorspheres derived from prostate cancer cells possess chemoresistant and cancer stem cell properties

PurposeProstate cancer (PCa) becomes lethal when cancer cells develop into castration-resistant PCa, which remains incurable because of the poor understanding of their cell origin and characteristics. We aim to investigate the potential role of cancer stem cells (CSCs) in PCa progression.MethodsHuman PCa cell lines (LNCaP, 22RV1, DU145 and PC-3) were plated in serum-free suspension culture system allowed for tumorsphere forming. To evaluate the CSC characteristics of tumorspheres, the self-renewal, chemoresistance, tumorigenicity of the PCa tumorsphere cells, and the expression levels of stemness-related proteins in the PCa tumorsphere cells were assessed, comparing with the parental adherent cells.ResultsTumorsphere cells from PCa cell lines displayed enhanced self-renewal, chemoresistance and tumor-initiating capacity when compared with the adherent cells. Additionally, these cells overexpressed CSC marker CD44. Also, the tumorsphere cells expressed high levels of “stemness” genes Gli1, ABCG2 and Bmi-1.ConclusionsCollectively, these data demonstrated that tumorspheres derived from PCa cells possess chemoresistant and CSC properties. Our study suggests that the identification of PCa CSCs could provide new insight into the lethal phenotype of PCa and therapeutic implications.

Tetrandrine induces apoptosis and triggers caspase cascade in human bladder cancer cells.

BACKGROUND Tetrandrine is known to exert anti-tumor effects, however, little is known about its effect on human bladder carcinoma. In this study, employing two different human bladder cancer cell lines, 5637 and T24, which represent high-risk superficial bladder cancer (5637) and high-grade bladder cancer (T24), we tested tetrandrine-induced apoptosis and growth inhibition in bladder carcinoma cell lines and investigated the possible mechanisms. MATERIALS AND METHODS Growth inhibition and apoptosis induction was determined by MTT assay and flow cytometry analysis, respectively. Activation of caspases were analyzed by Western blotting and caspase colorimetric assay. The collapse of mitochondrial membrane potential (ΔΨ(m)) and subcellular distribution of cytochrome c was determined by JC-1 staining and Western blotting, respectively. RESULTS Tetrandrine treatment showed strong growth inhibitory and apoptotic effects on human bladder cancer 5637 and T24 cells in a concentration-dependent manner. Additionally, induction of apoptosis by tetrandrine was associated with a very strong and prominent caspase-9, caspase-8, and caspase-3 activation as well as PARP cleavage. Flow cytometric studies revealed that tetrandrine induced a dose-dependent loss of ΔΨ(m), which was accompanied by the release of cytochrome c from mitochondria to the cytosol. CONCLUSION Taken together, this study provided the first evidence that tetrandrine imparted inhibitory and apoptotic activity in human bladder cancer cells. The tetrandrine-induced apoptosis might be related to the activation of the caspase cascade and mitochondrial pathway. Our results suggest that tetrandrine merits further in vivo investigation as a novel bladder cancer chemopreventive and chemotherapeutic agent in the clinical setting.

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.

Non-canonical GLI1/2 activation by PI3K/AKT signaling in renal cell carcinoma: A novel potential therapeutic target

Renal cell carcinoma (RCC) is the most lethal urologic malignancy; however, the molecular events supporting RCC carcinogenesis and progression remain poorly understood. In this study, based on the analysis of gene expression profile data from human clear cell RCC (ccRCC) and the corresponding normal tissues, we discovered that Hedgehog (HH) pathway component genes GLI1 and GLI2 were significantly elevated in ccRCC. Survival analysis of a large cohort of ccRCC samples demonstrated that the expression of GLI1 and GLI2 was negatively correlated with patient overall survival. Clinical sample-based VHL mutation and cell model-based VHL manipulation studies all indicated that the activation of GLI1 and GLI2 was not affected by VHL status. Further signaling pathway dissections demonstrated that GLI1 and GLI2 were activated by the phosphoinositide 3-kinase (PI3K)/AKT pathway, but not mediated by the canonical HH/SMO/GLI signaling. Up-regulation of GLI1 and GLI2 promoted RCC proliferation and clonogenic ability, whereas, a combination of GLIs inhibitor Gant61 and AKT inhibitor Perifosine synergistically suppressed RCC growth and induced apoptosis in vitro and in vivo. Therefore, this study identifies that GLI1 and GLI2 are critical for RCC carcinogenesis, and also provides an alternative therapeutic strategy for 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.

Overexpression of FABP7 promotes cell growth and predicts poor prognosis of clear cell renal cell carcinoma

OBJECTIVES Renal cell carcinoma (RCC) is one of the most lethal urologic malignancies; however, the molecular events supporting RCC carcinogenesis remain poorly understood. The aim of the present study was to determine the differential expression of genes between normal kidney and clear cell RCC (ccRCC) samples and investigate the biological function of the most frequently altered gene in RCC cells. MATERIALS AND METHODS The gene expression profiles of 60 ccRCC and matched normal kidney samples from The Cancer Genome Atlas were analyzed. The altered genes were subjected to functional annotation clustering and integrative pathway analysis. The expression of one of the most frequently altered gene, fatty acid-binding protein (FABP) 7, in ccRCC and matched normal kidney samples was verified by immunohistochemistry and the association between FABP7 level and patient survival was investigated. Furthermore, FABP7 DNA copy number alteration, methylation, and mutation status in ccRCC from The Cancer Genome Atlas were analyzed. Finally, FABP7-overexpressing RCC cells were generated to determine the function of FABP7 in cell growth and the potential mechanisms of action. RESULTS FABP7 was significantly up-regulated in ccRCC, and the expression of FABP7 positively correlated with advanced clinical stage and poor survival of patients with ccRCC. FABP7 DNA copy number alteration was not frequently detected in ccRCC, and no mutation of FABP7 was found. FABP7 messenger RNA expression inversely correlated with its DNA methylation. Overexpression of FABP7 in RCC cells enhanced cell growth, clonogenicity, cell cycle progression and activated both extracellular-signal-regulated kinases (ERK) and signal transducer and activator of transcription 3 (Stat3) signaling. CONCLUSION FABP7 is overexpressed in ccRCC and promotes cell growth by the activation of ERK and Stat3 signaling pathways. Evidence from the clinical observations and experimental data suggests that FABP7 is a novel prognostic marker and potential therapeutic target for ccRCC.

Reciprocal regulation of hypoxia-inducible factor 2α and GLI1 expression associated with the radioresistance of renal cell carcinoma.

PURPOSE Renal cell carcinoma (RCC) is often considered a radioresistant tumor, but the molecular mechanism underlying its radioresistance is poorly understood. This study explored the roles of hypoxia-inducible factor 2α (HIF2α) and sonic hedgehog (SHH)-GLI1 signaling in mediating the radioresistance of RCC cells and to unveil the interaction between these 2 signaling pathways. METHODS AND MATERIALS The activities of SHH-GLI1 signaling pathway under normoxia and hypoxia in RCC cells were examined by real-time polymerase chain reaction, Western blot, and luciferase reporter assay. The expression of HIF2α and GLI1 in RCC patients was examined by immunohistochemistry, and their correlation was analyzed. Furthermore, RCC cells were treated with HIF2α-specific shRNA (sh-HIF2α), GLI1 inhibitor GANT61, or a combination to determine the effect of ionizing radiation (IR) on RCC cells based on clonogenic assay and double-strand break repair assay. RESULTS RCC cells exhibited elevated SHH-GLI1 activities under hypoxia, which was mediated by HIF2α. Hypoxia induced GLI1 activation through SMO-independent pathways that could be ablated by PI3K inhibitor or MEK inhibitor. Remarkably, the SHH-GLI1 pathway also upregulated HIF2α expression in normoxia. Apparently, there was a positive correlation between HIF2α and GLI1 expression in RCC patients. The combination of sh-HIF2α and GLI1 inhibitor significantly sensitized RCC cells to IR. CONCLUSIONS Cross-talk between the HIF2α and SHH-GLI1 pathways was demonstrated in RCC. Cotargeting these 2 pathways, significantly sensitizing RCC cells to IR, provides a novel strategy for RCC treatment.

Differentiations of transplanted mouse spermatogonial stem cells in the adult mouse renal parenchyma in vivo

AbstractAim:Spermatogonial stem cells can initiate the process of cellular differentiation to generate mature spermatozoa, but whether it possess the characteristic of pluripotency and plasticity, similar to embryonic stem cells, has not been elucidated. This study was designed to evaluate the differentiation potential of spermatogonial stem cells into renal cells in vivo.Methods:Neonatal mouse spermatogonial stem cells were transplanted into mature male mice lacking endogenous spermatogenesis. The restoration of fertility in recipient males was observed. Spermatogonial stem cells were then injected into renal parenchyma of mature female mice to make a new extracellular environment for differentiation. Fluorescence in situ hybridization technology (FISH) was used to detect the expression of chromosome Y in recipient renal tissues. To determine the type of cells differentiated from spermatogonial stem cells, the expression of ricinus communis agglutinin, vimentin, CD45, and F4/80 proteins were examined in the renal tissues by immunohistochemistry.Results:The proliferation of seminiferous epithelial cells was distinctly observed in seminiferous tubules of transplanted testes, whereas no regeneration of spermatogenesis was observed in non-transplanted control testes. In transplanted female renal tissues, FISH showed a much stronger immuno-fluorescence signal of chromosome Y in the nucleolus of epithelial cells of the renal tubule and podocytes of the glomerulus.Conclusion:The spermatogonial stem cells were successfully purified from mouse testicles. This finding demonstrated that spermatogonial stem cells could not only restore damaged spermatogenesis, but were also capable of differentiating into mature renal parenchyma cells in vivo.

Loss of DAB2IP in RCC cells enhances their growth and resistance to mTOR-targeted therapies

Targeted therapies using small-molecule inhibitors (SMIs) are commonly used in metastatic renal cell cancer (mRCC) patients; patients often develop drug resistance and eventually succumb to disease. Currently, understanding of mechanisms leading to SMIs resistance and any identifiable predictive marker(s) are still lacking. We discovered that DAB2IP, a novel Ras-GTPase-activating protein, was frequently epigenetically silenced in RCC, and DAB2IP loss was correlated with the overall survival of RCC patients. Loss of DAB2IP in RCC cells enhances their sensitivities to growth factor stimulation and resistances to SMI (such as mammalian target of rapamycin (mTOR) inhibitors). Mechanistically, loss of DAB2IP results in the activation of extracellular signal–regulated kinase/RSK1 and phosphoinositide-3 kinase/mTOR pathway, which synergizes the induction of hypoxia-inducible factor (HIF)-2α expression. Consequently, elevated HIF-2α suppresses p21/WAF1 expression that is associated with resistance to mTOR inhibitors. Thus combinatorial targeting both pathways resulted in a synergistic tumor inhibition. DAB2IP appears to be a new prognostic/predictive marker for mRCC patients, and its function provides a new insight into the molecular mechanisms of drug resistance to mTOR inhibitors, which also can be used to develop new strategies to overcome drug-resistant mRCC.

Priapism as the Initial Manifestation of a Penile and Lower Limb Cutaneous Metastasis of Prostate Adenocarcinoma With Low Serum PSA Level

Penile and/or cutaneous metastases from prostate adenocarcinoma rarely occur. Here, we detail the case of a 78-year-old man suffering from priapism caused by metastatic prostate cancer with both penile and lower limb cutaneous spread. His serum prostate-specific antigen level was 0.09 μg/L when priapism developed. Corpora cavernosa biopsy was refused by the patient and radical penectomy was performed. Postoperative pathologic and immunohistochemical studies revealed undifferentiated prostate adenocarcinoma cells growing in corpora cavernosa. Two months later, the patient presented with multiple, erythematous nodules over the right lower leg. The prostate-specific antigen level was found to be 0.264 μg/L. Biopsy of a skin nodule revealed neoplastic cells consistent with metastatic prostate adenocarcinoma. This is the first known case of metastatic prostate cancer found in both penis and skin with a low serum prostate-specific antigen level. Priapism presented as the initial clinical manifestation of metastatic prostate cancer.