Knocking down AR promotes osteoblasts to recruit prostate cancer cells by altering exosomal circ-DHPS/miR-214-3p/CCL5 pathway.

ABSTRACT: Tumor-derived exosomes have been shown to play a key role in organ-specific metastasis, and the androgen receptor regulates prostate cancer (PCa) progression. It is unclear whether the androgen receptor regulates the recruitment of prostate cancer cells to the bone microenvironment, even bone metastases, through exosomes. Here, we found that exosomes isolated from PCa cells after knocking down androgen receptor (AR) or enzalutamide treatment can facilitate the migration of prostate cancer cells to osteoblasts. In addition, AR silencing or treatment with the AR antagonist enzalutamide may increase the expression of circular RNA-deoxyhypusine synthase (circ-DHPS) in PCa cells, which can be transported to osteoblasts by exosomes. Circ-DHPS acts as a competitive endogenous RNA (ceRNA) against endogenous miR-214-3p to promote C-C chemokine ligand 5 (CCL5) levels in osteoblasts. Increasing the level of CCL5 in osteoblasts could recruit more PCa cells into the bone microenvironment. Thus, blocking the circ-DHPS/miR-214-3p/CCL5 signal may decrease exosome-mediated migration of prostate cancer cells to osteoblasts.

Protectin conjugates in tissue regeneration 1 restores lipopolysaccharide-induced pulmonary endothelial glycocalyx loss via ALX/SIRT1/NF-kappa B axis.

BACKGROUND: Endothelial glycocalyx loss is integral to increased pulmonary vascular permeability in sepsis-related acute lung injury. Protectin conjugates in tissue regeneration 1 (PCTR1) is a novel macrophage-derived lipid mediator exhibiting potential anti-inflammatory and pro-resolving benefits. METHODS: PCTR1 was administrated intraperitoneally with 100 ng/mouse after lipopolysaccharide (LPS) challenged. Survival rate and lung function were used to evaluate the protective effects of PCTR1. Lung inflammation response was observed by morphology and inflammatory cytokines level. Endothelial glycocalyx and its related key enzymes were measured by immunofluorescence, ELISA, and Western blot. Afterward, related-pathways inhibitors were used to identify the mechanism of endothelial glycocalyx response to PCTR1 in mice and human umbilical vein endothelial cells (HUVECs) after LPS administration. RESULTS: In vivo, we show that PCTR1 protects mice against lipopolysaccharide (LPS)-induced sepsis, as shown by enhanced the survival and pulmonary function, decreased the inflammatory response in lungs and peripheral levels of inflammatory cytokines such as tumor necrosis factor-α, interleukin-6, and interleukin-1ß. Moreover, PCTR1 restored lung vascular glycocalyx and reduced serum heparin sulphate (HS), syndecan-1 (SDC-1), and hyaluronic acid (HA) levels. Furthermore, we found that PCTR1 downregulated heparanase (HPA) expression to inhibit glycocalyx degradation and upregulated exostosin-1 (EXT-1) protein expression to promote glycocalyx reconstitution. Besides, we observed that BAY11-7082 blocked glycocalyx loss induced by LPS in vivo and in vitro, and BOC-2 (ALX antagonist) or EX527 (SIRT1 inhibitor) abolished the restoration of HS in response to PCTR1. CONCLUSION: PCTR1 protects endothelial glycocalyx via ALX receptor by regulating SIRT1/NF-κB pathway, suggesting PCTR1 may be a significant therapeutic target for sepsis-related acute lung injury.

Maresin Conjugates in Tissue Regeneration 1 improves alveolar fluid clearance by up-regulating alveolar ENaC, Na, K-ATPase in lipopolysaccharide-induced acute lung injury.

Maresin Conjugates in Tissue Regeneration 1 (MCTR1) is a newly identified macrophage-derived sulfido-conjugated mediator that stimulates the resolution of inflammation. This study assessed the role of MCTR1 in alveolar fluid clearance (AFC) in a rat model of acute lung injury (ALI) induced by lipopolysaccharide (LPS). Rats were intravenously injected with MCTR1 at a dose of 200 ng/rat, 8 hours after administration of 14 mg/kg LPS. The level of AFC was then determined in live rats. Primary rat ATII (Alveolar Type II) epithelial cells were also treated with MCTR1 (100 nmol/L) in a culture medium containing LPS for 8 hours. MCTR1 treatment improved AFC (18.85 ± 2.07 vs 10.11 ± 1.08, P < .0001) and ameliorated ALI in rats. MCTR1 also significantly promoted AFC by up-regulating epithelial sodium channel (ENaC) and Na+ -K+ -adenosine triphosphatase (Na, K-ATPase) expressions in vivo. MCTR1 also activated Na, K-ATPase and elevated phosphorylated-Akt (P-Akt) by up-regulating the expression of phosphorylated Nedd4-2 (P-Nedd4-2) in vivo and in vitro. However, BOC-2 (ALX inhibitor), KH7 (cAMP inhibitor) and LY294002 (PI3K inhibitor) abrogated the improved AFC induced by MCTR1. Based on the findings of this study, MCTR1 may be a novel therapeutic approach to improve reabsorption of pulmonary oedema during ALI/acute respiratory distress syndrome (ARDS).

MCTR1 alleviates lipopolysaccharide-induced acute lung injury by protecting lung endothelial glycocalyx.

Endothelial glycocalyx degradation, critical for increased pulmonary vascular permeability, is thought to facilitate the development of sepsis into the multiple organ failure. Maresin conjugates in tissue regeneration 1 (MCTR1), a macrophage-derived lipid mediator, which exhibits potentially beneficial effects via the regulation of bacterial phagocytosis, promotion of inflammation resolution, and regeneration of tissue. In this study, we show that MCTR1 (100 ng/mouse) enhances the survival of mice with lipopolysaccharide (LPS)-induced (15 mg/kg) sepsis. MCTR1 alleviates LPS (10 mg/kg)-induced lung dysfunction and lung tissue inflammatory response by decreasing inflammatory cytokines (tumor necrosis factor-α, interleukin-1ß [IL-1ß], and IL-6) expression in serum and reducing the serum levels of heparan sulfate (HS) and syndecan-1. In human umbilical vein endothelial cells (HUVECs) experiments, MCTR1 (100 nM) was added to the culture medium with LPS for 6 hr. MCTR1 treatment markedly inhibited HS degradation by downregulating heparanase (HPA) protein expression in vivo and in vitro. Further analyses indicated that MCTR1 upregulates sirtuin 1 (SIRT1) expression and decreases NF-κB p65 phosphorylation. In the presence of BOC-2 or EX527, the above effects of MCTR1 were abolished. These results suggest that MCTR1 protects against LPS-induced sepsis in mice by attenuating pulmonary endothelial glycocalyx injury via the ALX/SIRT1/NF-κB/HPA pathway.

The diagnostic effect of serum miR-139-5p as an indicator in osteosarcoma.

BACKGROUND: The microRNA, miR-139-5p, plays an important role in the initiation and progression of various tumor types including osteosarcoma (OS). OBJECTIVE: This study aimed to detect the serum miR-139-5p expression in OS and analyze its association with clinical variables. METHODS: Blood samples were taken from 98 OS patients and 50 healthy individuals, and serum miR-139-5p levels were measured by quantitative reverse transcription-polymerase chain reaction. RESULTS: We found the expression of serum miR-139-5p in OS patients was significantly lower than that in healthy individuals, and miR-139-5p levels were dramatically decreased in patients with distant metastasis or in higher clinical stage. Receiver-operating characteristic (ROC) analysis revealed that serum miR-139-5p could well discriminate OS patients from healthy controls with a high sensitivity and specificity. Moreover, low serum miR-139-5p expression was strongly associated with distant metastasis, tumor stage and shorter overall survival. Both univariate and multivariate analyses showed that serum miR-139-5p level could serve as an independent prognostic marker for OS. CONCLUSIONS: Taken together, data from this study demonstrates that serum miR-139-5p could be used as a tumor biomarker for OS diagnosis and prognosis.

[Expressions of survivin, PI3K and AKT in keratinocytes in skin lesions and their pathogenic role in psoriasis vulgaris].

OBJECTIVE: To explore the role of survivin and PI3K/AKT pathway in the pathogenesis of psoriasis vulgaris (PV). METHODS: Plaque-like lesions collected from 22 patients with PV in progressive stage and 18 normal control skin specimens were examined using immunohistochemical staining, Western blotting and real-time quantitative PCR for expressions of survivin, PI3K and AKT in the keratinocytes, and their correlation was analyzed. A small interfering RNA (siRNA) was used to knock down AKT in cultured HaCaT cells, and Western blotting was used to detect the changes in the expression of survivin. RESULTS Compared with normal skin, PV lesions showed obviously up-regulated expressions of survivin, PI3K and AKT in the keratinocytes. Survivin expression was positively correlated with PI3K (r=0.4510, P=0.0351) and AKT (r=0.4423, P=0.0393) in the keratinocytes in PV lesions. In cultured HaCaT cells, siRNA-mediated knockdown of AKT caused down-regulation of survivin expression. CONCLUSION: Survivin and PI3K/AKT signaling pathway may participate in the occurrence and progression of PV.

[Regulatory effect of Skp2 on the expression and transactivation of the androgen receptor in the progression of castration-resistant prostate cancer].

OBJECTIVE: To determine the expression of Skp2 in different prostate cancer (PCa) cell lines and tissues, and explore its influence on the androgen receptor (AR) signaling pathway and development of castration-resistant prostate cancer (CRPC). METHODS: The expression levels of Skp2 and AR in different PCa cell lines were detected by Western blot. After knockdown of Skp2 in the C4-2 and 22RV1 cells transfected with shRNA, the expressions of AR and P27 were determined and the activity of ARR3-Luc measured by dual-luciferase reporter gene assay following treatment with dihydrotestosterone (DHT). The expressions of AR and Skp2 in human naïve PCa or CRPC specimens were detected by immunohistochemical staining followed by analysis of their differences and correlation. RESULTS: The Skp2 protein expression level was significantly higher in the C4-2 or 22RV1 cells than in the LNCaP cells. DHT treatment increased the expression of Skp2 in the C4-2 cells, but knock-down of Skp2 significantly up-regulated the expression of the well-known downstream protein P27 and down-regulated that of AR. Consistently, DHT treatment increased the activity of ARR3-Luc, while knockdown of Skp2 remarkably decreased it in the C4-2 and 22RV1 cells (P < 0.05). In addition, significantly higher expressions of Skp2 and AR were observed in the CRPC than in the naïve specimens (P < 0.05), with a positive correlation between the two proteins (r = 0.658 1, P < 0.05). CONCLUSION: Skp2 can enhance the expression and transcription activity of the AR protein in CRPC cells or tissues and is promising to be a critical molecular therapeutic target.

Tetrandrine suppresses proliferation, induces apoptosis, and inhibits migration and invasion in human prostate cancer cells.

Tetrandrine (TET), a traditional Chinese medicine, exerts remarkable anticancer activity on various cancer cells. However, little is known about the effect of TET on human prostate cancer cells, and the mechanism of function of TET on prostate cancer has not yet been elucidated. To investigate the effects of TET on the suppression of proliferation, induction of apoptosis, and inhibition of migration and invasion in human prostate cancer cell lines, DU145 and PC-3. Inhibition of growth was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and clone formation assay, and flow cytometry analysis was performed to detect the induction of apoptosis. Activation of poly (ADP-ribose) polymerase, caspase-3, Akt, phospho-Akt, Bcl-2, and Bax was analyzed by Western blotting. Wound healing assay and transwell migration assay were used to evaluate the effect of TET on migration and invasion of cancer cells. TET inhibited the growth of DU145 and PC-3 cells in a dose- and time-dependent manner. Cell cloning was inhibited in the presence of TET in DU145 and PC-3 cells. TET suppressed the migration of DU145 and PC-3 cells. Transwell invasion assay showed that TET significantly weakened invasion capacity of DU145 and PC-3 cells. TET exhibited strong inhibitory effect on proliferation, migration, and invasion of prostate cancer cells. In addition, TET induced apoptosis in a dose-dependent manner by activating the caspase cascade and inhibiting phosphoinositide 3-kinase-Akt signal pathway. The accumulating evidence suggests that TET could be a potential therapeutic candidate against prostate cancer in a clinical setting.

[DAB2IP expression in bladder transitional cell carcinoma and its correlation with clinical outcome].

OBJECTIVE: To investigate the expression of DAB2IP in bladder transitional cell carcinoma (BTCC) and its correlation with clinical characteristics and prognosis of BTCC patients. METHODS: Immunohistochemical staining was applied to detect DAB2IP protein level in 79 cases of TCCB tissues and 11 cases of normal bladder tissues, and the relationships of the staining results with pathological grade, stage, lymph node metastasis, gender, age and the 3-year survival rate of the patients were analyzed. RESULTS: The expression of DAB2IP in BTCC tissues was significantly lower than that in normal bladder epithelium, and the expression score and rate of DAB2IP in the high-grade, invasive and metastatic BTCC were significantly lower than those in low-grade, superficial and non-metastatic BTCC (P < 0.05). The 3-year survival rate of the patients with high DAB2IP expression was significantly higher than that of the patients with low DAB2IP expression. CONCLUSION: DAB2IP may be one of the important inhibitory factors during the occurrence and progression of BTCC.

Downregulation of mTOR by lentivirus inhibits prostate cancer cell growth.

Prostate cancer, one of the most lethal forms of urinary system cancer, remains resistant to currently available treatments. Therefore, novel mechanism and target-based approaches are needed for the management of this neoplasm. PI3K/AKT signaling pathway activation correlates with human prostate cancer progression and metastasis. However, the role of mTOR in prostate cancer is not well-established. Here, we demonstrate that mTOR is over-expressed in both clinical tissue specimens and cultured human prostate cancer cells when compared to normal prostate tissues, respectively. Further, mTOR gene knockdown via lentivirus mediated mTOR specific shRNA resulted in a significant decrease in the viability and growth of prostate cancer cells without affecting normal human prostate cells. In addition, mTOR inhibition resulted in a significant i) decrease in 4EBP1, S6K, PI3K and AKT protein, ii) increase in PARP protein of prostate cancer cells. Most importantly, mTOR inhibition triggered apoptosis and suppressed pancreatic carcinoma growth in vivo in a mouse xenograft model. We suggest that targeting of mTOR may be a viable approach for the treatment of prostate cancer.

Benzylidene 2-aminoimidazolones derivatives: synthesis and in vitro evaluation of anti-tumor carcinoma activity.

A series of benzylidene 2-aminoimidazolones derivatives were synthesized. Most compounds displayed strong inhibitory activity on the proliferation of human HepG2 cells in vitro. The active compounds were further evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against five human cancer cell lines in vitro. Compound 2b exhibited the strongest antitumor activities with IC50 values ranging from 12.87-17.10 µM which were nearly 1-3.5 fold less than that of 5-FU (IC50=18.39-56.12 µM) in vitro. Furthermore, compound 2b could induce SMMC-7721 cell apoptosis in a dose-dependent manner. Therefore, our novel findings may provide a new framework for the design of new benzylidene 2-aminoimidazolones derivatives for the treatment of cancer.

[Changes of intracellular cholesterol metabolism in neuroendocrine differentiation of prostate cancer and their significance].

OBJECTIVE: To explore the roles of intracellular cholesterol metabolism in neuroendocrine (NE) differentiation of prostate cancer based on an androgen-independent prostate cancer NE cell model induced by androgen deprivation. METHODS: LNCaP cells were cultured in androgen-depleted medium, and NE phenotypes were identified by observing the changes in cell morphology, molecular markers (SgIII, NSE and CgA) and cell proliferation. The expression and distribution of cholesterol and Sg III were determined by immunofluorescence staining. The expressions of the key genes LDL-R, SREBP-1 and SREBP-2 involved in cholesterol synthesis and uptake were detected by semi-quantitative RT-PCR. RESULTS: The LNCaP cells showed shrinking bodies and extending axons after androgen deprivation, and all the molecular markers, such as Sg III, NSE and CgA, significantly increased in a time-dependent manner, while the cell proliferation was obviously inhibited (P < 0.05). The cholesterol distribution in the LNCaP cells after NE differentiation presented remarkable aggregation at the axon terminals. However, there were no significant differences in the expression of cholesterol between the two types of cells, nor in the changes of the expressions of key genes LDL-R, SREBP-1 and SREBP-2 involved in cholesterol synthesis and uptake (P > 0.05). CONCLUSION: Transient androgen depletion could successfully induce NE differentiation of LNCaP cells, and the intracellular cholesterol could re-distribute into axon terminals to enhance the formation of neurosecretory granules.

Prostate-targeted mTOR-shRNA inhibit prostate cancer cell growth in human tumor xenografts.

OBJECTIVE: To construct a recombinant lentivirus vector driven by the PSMA promoter carrying mTOR-shRNA, and to obtain the effect on the mTOR gene silencing in human prostate cancer xenografts. METHODS: The complimentary oligos of small interference RNA (siRNA) with hairpin structures targeting the mTOR gene and a negative control were synthesized, then ligated with pLV-PSMA-promoter vector and sequenced. The recombinant vectors were then transfected with viral packaging mix into 293T cells, viral supernatant was harvested to determine the titer. Prostate cancer cells infected by virus were harvested and the expression of mTOR (LV-PSMA-shmTOR), target proteins and cell growth were detected by reverse transcription-PCR (RT-PCR), Western blot and MTT separately. In established tumors derived from human prostate cancer cells, concentrated LV-PSMA-shmTOR lentivirus was injected intravenously in the tail vein of C4-2b tumor bearing female severe combined immunodeficient (SCID) mice. Tumor volume and immunohistochemistry was assessed. RESULTS: Sequencing data showed that the constructed plasmids contained the correct sequences of mTOR siRNA transcript templates. A vector producing cell line 293T was established, and the titer for transfection was obtained. RT-PCR, Western blot and MTT analyses demonstrated that mTOR shRNA expression construct could suppress the expression of mTOR and inhibit the prostate cancer cell growth, specially. The tumor growth was suppressed in nude mouse. CONCLUSION: A PSMA driven lentivirus mediated siRNA targeting mTOR gene was successfully constructed, which decreased the expression of mTOR and induced the prostate cancer cell growth in vitro and in vivo. It has set up a research platform for the gene therapy of tumors which take mTOR as the target in the prostate cancer field.

[Establishment of an animal model of prostate cancer metastasis to the lung traced by AsRed2].

OBJECTIVE: To establish an animal model of prostate cancer (PCa) metastasis to the lung using PCa PR7 (PCa PC-3 cells stably expressing red fluorescent protein AsRed2) cell lines that can be monitored by in vivo fluorescence imaging technology. METHODS: MTT and Transwell assay were used to compare the abilities of proliferation, migration and invasion of PC-3 and PR7 cells. Twenty BALB/c nude mice were equally randomized to 4 groups to receive tail vein injection of PR7 cell suspension at the concentration of 1 x 107/ml (group A), 2.5 x 107/ml (group B), 5 x 107/ml (group C) and 2.5 x 107/ml followed by the same dose 1 week later (group D). PCa metastasis to the lung was then monitored by in vivo fluorescence imaging technology at the end of 2, 4, 6 and 8 weeks. RESULTS: There were no statistically significant differences between PC-3 and PR7 cells in their abilities of proliferation, migration and invasion (P > 0.05). At the end of 4 weeks, lung metastasis was observed in 40% of the mice in group D, and at the end of 8 weeks, it was detected in 20% in group A, 60% in group B, 100% in group C, and 100% in group D, all confirmed by pathological examination. CONCLUSION: The animal model of PCa metastasis to the lung that can be monitored by in vivo fluorescence imaging technology was established successfully by tail vein injection of PR7 cells carrying red fluorescent protein.

Inhibitory effect of silibinin on EGFR signal-induced renal cell carcinoma progression via suppression of the EGFR/MMP-9 signaling pathway.

The activation of epidermal growth factor (EGF) through its receptor, EGFR, is one of the major mechanisms that mediate renal cell carcinoma (RCC) metastasis. Silibinin, a natural flavonoid antioxidant with pleiotropic anticancer capability, has shown anti-metastatic effects in a variety of cancers. However, the mechanism by which silibinin inhibits EGFR signal-induced migration and invasion of RCC cells is not clear. Here, we evaluated the potential roles of EGFR signaling cascade that affects RCC progression, and also investigated the inhibitory effect of silibinin on the EGFR signal-induced migration and invasion abilities of RCC cells. Our data indicated that silibinin inhibited migration and invasion of RCC cells in a dose-dependent manner via blocking the EGFR signal, especially in the EGFR highly expressing RCC cells. Silibinin inhibited phosphorylation of EGFR and its downstream molecules ERK1/2 but did not affect phosphorylation of other downstream molecules, STAT3 and Akt, in human RCC cell lines. Moreover, our data suggested that silibinin significantly reduced the MMP-9 expression and its activity that was promoted by the EGFR signal, and also suppressed MMP9-dependent migration and invasion abilities of RCC cells. Taken together, this study clearly demonstrated that silibinin inhibited EGFR induced migration and invasion of RCC cells via blockade of EGFR/MMP-9 signaling. Thus, we suggest that silibinin could be used as a potential effective drug for the inhibition of RCC metastasis.

Multi-target siRNA based on DNMT3A/B homologous conserved region influences cell cycle and apoptosis of human prostate cancer cell line TSU-PR1.

Abnormal genome hypermethylation participates in the tumorigenesis and development of prostate cancer. Prostate cancer cells highly express DNA methyltransferase 3 (DMNT3) family genes, essential for maintaining genome methylation. In the present study, multi-target siRNA, based on the homologous region of the DNMT3 family, was designed for the in vitro investigation of its effects on the proliferation, migration, and invasion of TSU-PR1 prostate cancer cells. The consequential cell-cycle derangement, through DNMT3A/B or only DNMT3B silencing, was partially efficient, without affecting apoptosis. DNMT3A silencing had absolutely no effect on changing TSU-PR1 cell biological behavior. Hence, DNMT3B alone apparently plays a key role in maintaining the unfavorable behavior of prostate-cancer cells, thereby implying its potential significance as a promising therapeutic target, with DNMT3A simply in the role of helper.

[Study on bladder cancer tissues with Raman spectroscopy].

The scope of this research lies in diagnosis of bladder cancer through Raman spectra. The spectra of bladder cancer and normal bladder were measured by using laser confocal Raman micro-spectroscopy. Principal component analysis/support vector machines was applied to the spectral dataset to construct diagnostic algorithms, then to detect the accuracy of these algorithms to determine histological diagnosis by leave-one-out cross validation from its Raman spectrum. It was showed that the peak intensity of nucleic acid (782, 1 583 cm(-1)) in bladder cancer and protein (1 061, 1 295, 2 849, 2 881 cm(-1)) in normal bladder increased significantly. Additionally, Principal component analysis (PCA) and support vector machines (SVM) provided an effective tool for differentiating the bladder cancer from normal bladder tissue. Excellent sensitivity (86.7%), specificity (87.5%), positive predictive value (92.9%), and negative predictive value (72. 8%) for the diagnosis of bladder cancer were obtained by leave-one-out cross validation. It was concluded that Raman spectroscopy can be used to accurately identify bladder cancer in vitro, and it suggests the promising potential application of PCA/SVM-based Raman spectroscopy for the diagnosis of bladder cancer.

[GLI-1 is involved in EGF-regulated enhancement of the invasiveness of prostate cancer ARCaP(E) cells in vitro].

OBJECTIVE: To investigate the role of the hedgehog (HH) signaling pathway transcription factor glioma-associated oncogene hoinolog 1 (GLI-1) in EGF-regulated enhancement of the invasiveness of the prostate cancer ARCaP(E) cell line in vitro. METHODS: The expressions of EGFR and GLI-1 in prostate cancer ARCaP(E) cells were analyzed by immunofluorescence staining. ARCaP(E) cells were treated with EGF at 100 ng/ml, followed by detection of the changes in cell morphology and invasiveness, as well as in the expressions of p-ERK, ERK and GLI-1. Migration transwell assay was used to determine the effects of 100 ng/ml EGF and GLI-1 antagonist GANT61 on the invasiveness of the ARCaP(E) cells. RESULTS: Both EGFR and GLI-1 were expressed in the ARCaP(E) cells. EGF induced morphological transition of epithelial-like ARCaP(E) cells to mesenchymal-like cells, increased their in vitro invasiveness, and significantly upregulated the expressions of p-ERK and GLI-1 in the ARCaP(E) cells (P<0.05). GANT61 significantly inhibited the in vitro invasiveness of the ARCaP(E) cells and reduced the enhancing effect of EGF on their invasiveness (P<0.05). CONCLUSION: The results from ARCaP(E) cells shed light on the cross-talk of the HH pathway with the EGF/ERK signaling pathway. GLI-1 might be responsible for EGF-regulated enhancement of the invasiveness of ARCaP(E) cells in vitro.

Multi-target siRNA based on DNMT3A/B homologous conserved region influences cell cycle and apoptosis of human prostate cancer cell line TSU-PR1

Abnormal genome hypermethylation participates in the tumorigenesis and development of prostate cancer. Prostate cancer cells highly express DNA methyltransferase 3 (DMNT3) family genes, essential for maintaining genome methylation. In the present study, multi-target siRNA, based on the homologous region of the DNMT3 family, was designed for the in vitro investigation of its effects on the proliferation, migration, and invasion of TSU-PR1 prostate cancer cells. The consequential cell-cycle derangement, through DNMT3A/B or only DNMT3B silencing, was partially efficient, without affecting apoptosis. DNMT3A silencing had absolutely no effect on changing TSU-PR1 cell biological behavior. Hence, DNMT3B alone apparently plays a key role in maintaining the unfavorable behavior of prostate-cancer cells, thereby implying its potential significance as a promising therapeutic target, with DNMT3A simply in the role of helper.

Specific survivin dual fluorescence resonance energy transfer molecular beacons for detection of human bladder cancer cells.

AIM: Survivin molecular beacons can be used to detect bladder cancer cells in urine samples non-invasively. The aim of this study is to improve the specificity of detection of bladder cancer cells using survivin dual fluorescence resonance energy transfer molecular beacons (FRET MBs) that have fluorophores forming one donor-acceptor pair. METHODS: Survivin-targeting dual fluorescence resonance energy transfer molecular beacons with unique target sequences were designed, which had no overlap with the other genes in the apoptosis inhibitor protein family. Human bladder cancer cell lines 5637, 253J and T24, as well as the exfoliated cells in the urine of healthy adults and patients with bladder cancer were examined. Images of cells were taken using a laser scanning confocal fluorescence microscope. For assays using dual FRET MBs, the excitation wavelength was 488 nm, and the emission detection wavelengths were 520±20 nm and 560±20 nm, respectively. RESULTS: The human bladder cancer cell lines and exfoliated cells in the urine of patients with bladder cancer incubated with the survivin dual FRET MBs exhibited strong fluorescence signals. In contrast, no fluorescence was detected in the survivin-negative human dermal fibroblasts-adult (HDF-a) cells or exfoliated cells in the urine of healthy adults incubated with the survivin dual FRET MBs. CONCLUSION: The results suggest that the survivin dual FRET MBs may be used as a specific and non-invasive method for early detection and follow-up of patients with bladder cancer.