Targeting bladder tumor cells in vivo and in the urine with a peptide identified by phage display

Bladder cancer is one of the most common tumors of the genitourinary tract. Here, we use phage display to identify a peptide that targets bladder tumor cells. A phage library containing random peptides was screened for binding to cells from human bladder tumor xenografts. Phage clones were further selected for binding to a bladder tumor cell line in culture. Six clones displaying the consensus sequence CXNXDXR(X)/(R)C showed selective binding to cells from primary human bladder cancer tissue. Of these, the CSNRDARRC sequence was selected for further study as a synthetic peptide. Fluorescein-conjugated CSNRDARRC peptide selectively bound to frozen sections of human bladder tumor tissue, whereas only negligible binding to normal bladder tissue was observed. When the fluorescent peptide was introduced into the bladder lumen, in a carcinogen-induced rat tumor model, it selectively bound to tumor epithelium. Moreover, when the peptide was intravenously injected into the tail vein, it homed to the bladder tumor but was not detectable in normal bladder and control organs. Next, we examined whether the peptide can detect tumor cells in urine. The fluorescent peptide bound to cultured bladder tumor cells but not to other types of tumor cell lines. Moreover, it bound to urinary cells of patients with bladder cancer, while showing little binding to urinary cells of patients with inflammation or healthy individuals. The CSNRDARRC peptide may be useful as a targeting moiety for selective delivery of therapeutics and as a diagnostic probe for the detection of bladder cancer.     

近红外荧光染料IR-783在膀胱癌中的成像研究

目的:观察研究近红外荧光染料IR-783在膀胱癌中的特异性成像。方法:通过染料与膀胱癌细胞及正常细胞共孵育,观察近红外荧光染料IR-783是否能够实现膀胱癌细胞的选择性成像。利用细胞器示踪剂观察近红外荧光染料在膀胱癌细胞内的共定位;使用IR-783检测循环血液中及尿液中的膀胱癌细胞。结果:近红外荧光染料IR-783可被膀胱癌细胞选择性摄取。IR-783可选择性聚集在膜性细胞器如线粒体和溶酶体内,这种选择性聚集作用使IR-783可以保持较长的染色效果。近红外染料可以检测到血液或尿液极少量的膀胱癌细胞。结论:近红外荧光染料IR-783能够被膀胱癌细胞特异性吸收,可用于血液和尿液中膀胱肿瘤细胞的特异性诊断,具有重要的临床应用前景。     

Low-dose Hsp90 inhibitors tumor-selectively sensitize bladder cancer cells to chemoradiotherapy

Although radical cystectomy with urinary diversion is the standard treatment for muscle-invasive bladder cancer (MIBC), loss of native bladder frequently impairs patient's quality of life (QOL). Bladder-sparing approach incorporating chemoradiotherapy (CRT) improves QOL while not compromising survival outcomes in MIBC patients. In this approach, complete response to induction CRT is a prerequisite for bladder preservation and favorable oncological outcomes. We investigated a strategy to potentiate CRT response of bladder cancer cells by using Hsp90 inhibitors in preclinical models. Hsp90 inhibitors at low concentrations, which did not exert cytocidal effects but inactivated key anti-apoptotic proteins including erbB2, Akt, and NF-κB, efficiently sensitized bladder cancer cells (T24, 5637 and UM-UC-3 cells) to in vitro CRT by enhancing apoptosis. Importantly, the sensitizing effects were not observed in primarily cultured normal human urothelial cells. We also showed that CRT induces accumulation of nuclear phospho-Akt, which antagonizes apoptosis, and that Hsp90 inhibitors block the cellular process. Hsp90 inhibition sensitized bladder cancer cells to in vitro CRT more effectively than sole or combined inhibition of erbB2 and Akt. In mice UM-UC-3 tumor xenografts model, Hsp90 inhibitors successfully potentiated anti-tumor activity of CRT. These results encourage clinical trials of Hsp90 inhibitors to overcome CRT resistance in patients with MIBC.     

细胞角蛋白20 在膀胱癌中的研究进展

膀胱肿瘤是最常见的泌尿系统肿瘤,其中上皮性肿瘤占95%以上,绝大多数为尿路移行上皮细胞癌。膀胱癌的早期症状不 明显,复发率较高,早期诊断和治疗对提高其疗效非常重要。近年来,诊断膀胱肿瘤的新方法不断出现,显著提高了膀胱肿瘤诊断 及预后预测水平。其中,膀胱肿瘤标记物检测已成为膀胱肿瘤的诊断新方法,具有十分重要的临床意义。研究发现,细胞角蛋白20 (cytokeratin 20,CK20)是中间纤维家族成员之一,在正常膀胱组织中特异性表达于伞细胞,在膀胱癌中特异性表达于膀胱移行细 胞癌,其诊断膀胱肿瘤的特异性和灵敏性均较高,且与膀胱肿瘤的临床分级、病理分期和转移均密切相关,因此可作为辅助诊断 膀胱肿瘤的检测标志物及治疗和预后评估指标。本文将就其在膀胱癌中的研究进展综述如下。     

Stage-dependent increase of orosomucoid and zinc-alpha2-glycoprotein in urinary bladder cancer

Identification and characterization of biomarkers in body fluids such as serum or urine serve as a basis for early detection of diseases, particularly of cancer. Performing 2-DE with subsequent MS analyses, conventional immunoblotting and immunohistochemistry we identified two proteins, orosomucoid (ORM) and human zinc-alpha(2)-glycoprotein (ZAG), which were increased in the urine samples of patients with bladder cancer in comparison to the urine samples of healthy volunteers. The highest amount of both proteins was found in invasive bladder cancer stages such as pT2-3. Immunohistochemical studies showed ORM in inflammatory cells but also in endothelial cells of blood vessels within or adjacent to the tumor area and in part of the tumor cells. ZAG was prominent in tumor cells at the tumor invasion front. Additionally, ZAG was localized at the luminal surface of normal urothelium, which switches to the basal side when a superficial papillary tumor was observed. These results show that we have been able to identify two new proteins that may be related to the development of superficial bladder cancer and to its switch to an invasive phenotype.     

Evaluation of human bladder tumors by ultrastructural morphometric techniques

To estimate the malignancy of the human urinary bladder tumors, various cellular components of normal and neoplastic bladder epithelial cells were examined using morphometric techniques at the electron microscopic level. Nuclear to cytoplasmic ratios were found to be 1:5 in normal epithelial cells, 1:4 in superficial grade 1 (G1) tumor cells, 1:3 in superficial grade 2 (G2) tumor cells and 1:2 in invasive grade 3 (G3) tumor cells. Nuclear volumes were largest in G3 tumor cells, but no differences were seen in cell volumes between normal epithelial and tumor cells. The volumes and surface areas of the rough-surfaced endoplasmic reticulum (rER) and lysosomes per cell decreased progressively from G1 to G3 tumor cells. The volume density of tonofilaments was increased in G1, but decreased in G2 and G3 tumor cells. The cisternal thickness of the rER and Golgi complex was also increased in G1 tumor cells. From these results, it was concluded that morphometric analysis may be useful in evaluating the degree of differentiation and invasive potential of human bladder tumor cells in the low and intermediate risk groups.     

Six-transmembrane epithelial antigen of the prostate-1 plays a role for in vivo tumor growth via intercellular communication

Six-transmembrane epithelial antigen of the prostate-1 (STEAP-1) is a novel cell surface protein overexpressed only in the prostate among normal tissues and various types of cancer including prostate, bladder, lung, and ovarian cancer. Although its function in prostate and tumor cells has been remained unclear, due to its unique and restricted expression, STEAP-1 is expected to be an attractive target for cancer therapy. Here, we show that knockdown of STEAP-1 in human cancer cells caused the retardation of tumor growth compared with wild type in vivo. In contrast, STEAP-1 introduced tumor cells augmented the tumor growth compared with STEAP-1-negative wild type cells. Using dye transfer assay, we demonstrate that the STEAP-1 is involved in intercellular communication between tumor cells and adjacent tumor stromal cells and therefore may play a key role for the tumor growth in vivo. These data indicate the inhibition of the STEAP-1 function or expression can be a new strategy for cancer therapy.     

Multidimensional slit-scan detection of bladder cancer. Preliminary clinical results

A multidimensional slit-scan flow system was developed for the automated recognition of abnormal cells derived from cancer of the uterine cervix and its precursors. It provides great sensitivity in both its ability to recognize cellular abnormality and to deal with the myriad potential causes of false alarms in an automated flow system. While its initial application was the automated recognition of the spectrum of neoplasia in gynecologic cytology samples, a preliminary study was carried out using specimens obtained from the urinary bladder. Cellular material was collected by bladder irrigation and stained with the fluorochrome acridine orange. One hundred fifty-three bladder irrigation specimens, including 115 abnormal specimens containing cells derived from neoplastic lesions of the bladder epithelium, were analyzed. For the purposes of this study, abnormal specimens from the urinary bladder included specimens containing cells derived from the following lesions of the urothelium: dysplasia (atypical hyperplasia), carcinoma-in-situ, and transitional cell carcinoma, grades 1-3. Approximately 50,000 cells were analyzed for most specimens. Of the 38 presumed normal specimens (specimens containing only normal urothelial components), four were instrument classified abnormal. For the 69 specimens containing cells derived from transitional cell carcinoma, grade 1, 1-2, 2, 66 were correctly classified as abnormal while three were classified as normal.(ABSTRACT TRUNCATED AT 250 WORDS)     

P2X7 receptor expression is decreased in epithelial cancer cells of ectodermal, uro-genital sinus, and distal paramesonephric duct origin

The P2X₇ receptor is an important regulator of epithelial cell growth. The aim of the present study was to better understand the biological significance of P2X₇ receptor expression in normal and cancer human epithelial tissues. P2X₇ receptor and messenger RNA (mRNA) levels were determined in human tissues containing epithelial dysplastic, pre- or early cancerous, and cancer cells, and the levels were compared to those in the corresponding normal epithelial cells within the same tissue of the same case. P2X₇ receptor levels were determined by quantification of immunoreactivity specific to the functional (full-length) P2X₇ receptor, and P2X₇ mRNA levels were determined by real-time polymerase chain reaction. P2X₇ receptor levels in cancer cells were similar (colon adenocarcinoma) or greater (thyroid papillary carcinoma) than those in the corresponding normal cells. In contrast, in cancer cells of the ectocervix (squamous), endocervix and endometrium (adenocarcinoma), urinary bladder (transitional cell carcinoma), and breast (ductal and lobular adenocarcinomas), P2X₇ receptor levels were lower by about twofold than those in the corresponding normal epithelial cells. Similarly, P2X₇ mRNA levels were lower in uterine, bladder, and breast cancer epithelial tissues by about fourfold than those in the corresponding normal tissues. In addition, P2X₇ receptor levels were decreased already in dysplastic ectocervical cells and pre- or early cancerous endometrial and bladder cells. The data suggest that in epithelia originating from the ectoderm, the uro-genital sinus, and the distal paramesonephric duct, decreased expression of the P2X₇ receptor precedes or coincides with neoplastic changes in those tissues.     

细胞角蛋白20在膀胱癌中的研究进展

膀胱肿瘤是最常见的泌尿系统肿瘤,其中上皮性肿瘤占95％以上,绝大多数为尿路移行上皮细胞癌。膀胱癌的早期症状不明显,复发率较高,早期诊断和治疗对提高其疗效非常重要。近年来,诊断膀胱肿瘤的新方法不断出现,显著提高了膀胱肿瘤诊断及预后预测水平。其中,膀胱肿瘤标记物检测已成为膀胱肿瘤的诊断新方法,具有十分重要的临床意义。研究发现,细胞角蛋白20fcytokeratin20,CK20）是中间纤维家族成员之一,在正常膀胱组织中特异性表达于伞细胞,在膀胱癌中特异性表达于膀胱移行细胞癌,其诊断膀胱肿瘤的特异性和灵敏性均较高,且与膀胱肿瘤的临床分级、病理分期和转移均密切相关,因此可作为辅助诊断膀胱肿瘤的检测标志物及治疗和预后评估指标。本文将就其在膀胱癌中的研究进展综述如下。     

肿瘤微环境中ROS介导IgG表达对膀胱癌细胞增殖和侵袭能力的影响

摘要 目的：探讨肿瘤微环境（TME）中活性氧（ROS）介导免疫球蛋白G（IgG）表达对膀胱癌EJ细胞增殖、迁移和侵袭能力的影响。方法：临床收集的18例膀胱癌患者样本,通过Western blot法检测膀胱癌和癌旁正常组织样本中IgG表达量。利用免疫荧光染色（IF）技术分别对膀胱癌组织和癌旁正常组织中ROS和IgG分子进行共定位和相对定量分析。将活性氧清除剂N-乙酰基-L-半胱氨酸（NAC）加入膀胱癌细胞EJ中,实验分为3组：空白组（EJ细胞）、阴性对照组（EJ+PBS）、实验组（EJ+PBS+NAC）,10 mM NAC药物处理48小时后,运用DHE-ROS荧光探针技术和Western blot实验检测药物NAC对ROS和IgG相对表达水平的影响;通过克隆集落形成实验、划痕实验、Transwell实验检测去除ROS后对膀胱癌细胞增殖、迁移和侵袭的影响。结果：人体膀胱癌组织中ROS和IgG分子表达水平显著高于癌旁正常组织（P<0.001）;荧光显微镜显示膀胱肿瘤组织中正常膀胱尿路上皮细胞组织被肿瘤细胞严重破坏,结构紊乱不规则,IgG和ROS表达水平均升高,而癌旁组织膀胱尿路上皮组织的结构均匀规则;NAC药物处理EJ细胞后,与空白组和阴性对照组相比ROS和IgG表达显著降低,同时实验组细胞的增殖、迁移和侵袭能力明显下降（P<0.01）。结论：ROS和IgG在临床膀胱癌组织和体外膀胱癌细胞株EJ中均显著高表达,在肿瘤微环境中ROS通过调控IgG表达,从而促进膀胱癌细胞的增殖、迁移、侵袭。ROS和IgG可能成为膀胱癌早期诊断和生物治疗的临床新靶点。     

Significance of the Grb2 and son of sevenless (Sos) proteins in human bladder cancer cell lines

The epidermal growth factor (EGF) receptor has been suggested to have an important role in tumor initiation and progression of human bladder cancers. Grb2 protein, which is the downstream effector of the EGF receptor, acts as an adaptor protein between the EGF receptor and the Ras guanine-nucleotide exchange factor, son of sevenless (Sos) protein. Sos protein regulates the action of Ras protein by promoting the exchange of GDP for GTP. However, the significance of Grb2 and Sos proteins, which is related to EGF-triggered Ras activation, has not been elucidated in human bladder cancer. The aim of the present study is to clarify the significance of these proteins in human bladder cancer cell lines. In the present study, we used four human bladder cancer cell lines (T24, KU-7, UMUC-2, UMUC-6) and two kinds of cultured normal urothelial cells (HMKU-1, HMKU-2) isolated from patients with no malignancy. We examined the expression of EGF receptor, Grb2, and Sos proteins in these cells by Western blot analysis. Furthermore, the bladder cancer cell lines were subjected to sequence analysis to identify a point mutation in the c-H-ras gene at codon 12. There was no marked difference in the expression of the EGF receptor between human bladder cancer cell lines and cultured normal urothelial cells. On the other hand, expression of Grb2 and Sos proteins was substantially increased in all human bladder cancer cell lines examined in comparison with cultured normal urothelial cells, whether codon 12 of H-ras was mutated or not. These results suggest that the amplification of both Grb2 and SOS proteins plays an important role in the carcinogenesis of human bladder cancer.     

Synthesis and evaluation of a radioiodinated bladder cancer specific peptide

Bladder cancer is the second most common cancer of the urinary tract, however the invasive cystoscopy is still the standard technique for diagnosis and surveillance of bladder cancer. Herein, we radiolabel bladder cancer specific peptide with radioactive iodine ((131/124)I) and evaluate its potential as a new radiopharmaceutical for the non-invasive diagnosis of bladder cancer. A 9-mer bladder cancer specific peptide (BP) was conjugated with tyrosine and cyclized by disulfide bond formation to give Y-BP, which was further radioiodinated to give [(131/124)I]Y-BP in good radiochemical yield. The biodistribution data showed the high selectivity of [(124)I]Y-BP in HT1376 human bladder cancer xenograft models with a tumor-to-muscle ratio of 6.2. This tumor targeting was not observed in control B16F10 melanoma tumor models. In microPET studies, while the control scrambled peptide, [(124)I]Y-sBP, did not accumulate in either the bladder cancer or melanoma, [(124)I]Y-BP showed high tumor uptake only in animals with HT1376 bladder cancer cells. Furthermore, [(124)I]Y-BP showed superior bladder cancer uptake even compared to most commonly used cancer imaging tracer, [(18)F]FDG. The experimental results suggest the potential of [(124)I]Y-BP as a new radiopharmaceutical for the non-invasive diagnosis of bladder cancer with high binding affinity and selectivity.     

Morphometry of nucleoli as an indicator for grade of malignancy of bladder tumors

To establish a new indicator for the classification of human urinary bladder cancers, the nucleoli of normal epithelial and neoplastic cells were analyzed, using morphometric techniques. By electron microscopy, the nucleolar profiles of cells from grade 2 and 3 transitional cell carcinomas were often small and irregular. Morphometry showed that the nucleolar volumes, nucleolar/nuclear volume ratios, volume densities of various nucleolar components, and the numbers of fibrillar centers (FCs) altered significantly with an increase in tumor grade. In particular, an increase in FC numbers in the nuclei of higher grade tumors was associated with a decrease in individual volume. The number of FCs in intact urothelial cells obtained from patients with bladder tumors is significantly larger than in the normal urothelial cells. This may be related to the multicentric origin of bladder cancers. These results suggest that morphometric analysis of nucleoli is useful in evaluating the degree of differentiation and invasive capacity of human bladder tumor cells. In particular, the number and individual volume of FCs may be an indicator of tumor malignancy.     

Role of JAK/STAT signaling in neuroepithelial stem cell maintenance and proliferation in the Drosophila optic lobe

Human urine contains a large number of proteins and peptides (the urinary proteome). Global analysis of the human urinary proteome is important for understanding urinary tract diseases. Bladder cancer is the most common urological cancer with higher incidence rates in endemic areas of Blackfoot disease (BFD) in southern Taiwan. The aim of this study was to use the proteomic approach to establish urinary protein biomarkers of bladder cancer. ADAM28, identified by proteomic approaches and confirmed by Western blotting, showed significant differences compared with normal individuals, so it may be a biomarker of bladder cancer.     

Morphometry of nucleoli as an indicator for grade of malignancy of bladder tumors

To establish a new indicator for the classification of human urinary bladder cancers, the nucleoli of normal epithelial and neoplastic cells were analyzed, using morphometric techniques. By electron microscopy, the nucleolar profiles of cells from grade 2 and 3 transitional cell carcinomas were often small and irregular. Morphometry showed that the nucleolar volumes, nucleolar/nuclear volume ratios, volume densities of various nucleolar components, and the numbers of fibrillar centers (FCs) altered significantly with an increase in tumor grade. In particular, an increase in FC numbers in the nuclei of higher grade tumors was associated with a decrease in individual volume. The number of FCs in intact urothelial cells obtained from patients with bladder tumors is significantly larger than in the normal urothelial cells. This may be related to the multicentric origin of bladder cancers. These results suggest that morphometric analysis of nucleoli is useful in evaluating the degree of differentiation and invasive capacity of human bladder tumor cells. In particular, the number and individual volume of FCs may be an indicator of tumor malignancy.     

BLCA-4 在膀胱癌中作用的研究进展

摘要：近年来,膀胱癌的发病率与死亡率呈逐年上升,已成为泌尿系统最常见的肿瘤。随着分子生物技术的不断发展,核基质蛋白 (NMPs)、膀胱肿瘤抗原(BTA)、透明质酸和透明质酸酶(HA-HAase)、细胞角蛋白(CK)等诸多膀胱肿瘤标志物被发现。其中,膀胱癌 特异性核基质蛋白-4(BLCA-4)是一种可溶性的复合物,在正常膀胱组织中不表达而存在于膀胱癌组织中。检测尿样中BLCA-4 水平有望用于膀胱癌的筛查及诊断,具有较高的敏感性和特异性,也可用于普通人群与膀胱癌高危人群(脊髓损伤患者)的筛选与 检测;而术后病理组织检测BLCA-4 的表达也可为膀胱癌患者的预后预测提供参考。本文对近年来BLCA-4 在膀胱癌中的作用进 行了综述。     

Peculiarities of urinary bladder cancer tumor cells apoptosis response on neoadjuvant chemotherapy

Induced apoptosis in urinary bladder cancer tumor cells of patients was studied using TUNEL reaction. It was shown that increase in induced apoptosis value had a definite correlation between corresponding features of tumor reaction as a response on Gemcitabine-Cisplatin neoadjuvant chemotherapy application. It was found that evaluation of induced apoptosis in urinary bladder cancer tumor cells using TUNEL method allows forecasting the effectiveness of chemotherapy on the cellular level in patients with this type of cancer.     

Detection of urinary bladder cancer with flow cytometry and hexaminolevulinate in urine samples

OBJECTIVES: Urinary bladder urothelial carcinoma is diagnosed by a combination of cystoscopy and biopsy, with cytology as a valuable additional technique. The accuracy of cytological diagnosis depends on the experience of the cytologist and can inevitably vary from one cytologist to another. There is a need for an easy, reliable and objective diagnostic method. In the present study a new method was designed for the detection of bladder cancer cells in urine. METHODS: Flow cytometry was utilized to detect protoporphyrin IX in an artificial model consisting of normal urinary bladder transitional epithelial cells (NBECs) from healthy volunteers' urine and an established human urinary bladder carcinoma cell line, TCCSUP, after incubation with hexaminolevulinate (HAL). In addition, urine samples from 19 patients with histopathologically confirmed superficial bladder cancer were examined. RESULTS: Incubation of NBECs or TCCSUP cells with HAL for 1 hour resulted in production of protoporphyrin IX only in the TCCSUP cells. Incubation of a mixture of NBECs and TCCSUP cells with HAL gave rise to a separated subpopulation of cells with protoporphyrin IX fluorescence. After cell sorting by flow cytometry the protoporphyrin IX-containing subpopulation of cells was confirmed as TCCSUP cells on cytological examination. It was possible to detect 5% TCCSUP cells in the mixture of NBECs/TCCSUP cells. To test the feasibility of the method in clinica diagnosis, urine samples from patients with bladder cancer were also measured with comparable, although preliminary and limited, results to those of cytological examination. CONCLUSIONS: The preliminary results show that the technique may be feasible for the detection of bladder cancer cells in urine with possible advantages of simplicity, reliability and objectivity.     

Down-regulation of the ErbB3 binding protein 1 in human bladder cancer promotes tumor progression and cell proliferation

The ErbB3 binding protein 1 (Ebp1) represents a downstream effector of the ErbB signaling network and has been demonstrated to be a potent tumor suppressor in various human malignancies, however, its involvement in human bladder cancer is still unclear.To investigate the clinical significance and potential role of ErbB3 binding protein 1 (Ebp1) in bladder cancer. Ebp1 expression at protein and gene levels in 52 surgically removed bladder cancer specimens as well as 21 adjacent normal bladder specimens were respectively detected by immunohistochemistry and qRT-PCR. The association of Ebp1 protein expression with the clinicopathological features of bladder cancer was also statistically analyzed. Its roles in bladder cancer cell line were further evaluated. The expression level of Ebp1 protein and gene in bladder cancer tissues was significantly lower than that in adjacent normal bladder tissues (P < 0.01). When categorized into low vs. high expression, the down-regulation of Ebp1 protein was associated with the advanced pathologic stage (P = 0.036) and the high histologic grade (P = 0.001) of patients with bladder cancer. Moreover, following the transfection of Ebp1 in bladder cancer cells, not only cell proliferation and cell invasion decreased significantly, but also the cell cycle was blocked at G0/G1 stage. Our data suggest for the first time that the down-regulation of Ebp1 closely correlates with advanced clinicopathological characteristics of human bladder cancer. Furthermore, Ebp1 plays an important role in the bladder cancer cells’ proliferation by regulating the cancer cell cycle from G0/G1 to S.