Transitional Carcinoma Cell Lines Research Articles

The Anti-neoplastic Effects of Alpha-Lipoic Acid: Clinical Benefits in System Tumors besides Lung Carcinomas

I read with great interest the recent article by Kim et al. [1]. Interestingly, alpha-lipoic acid has recently been shown to exert anti-neoplastic effects in a number of systemic tumors other than lung carcinomas. For instance, alpha-lipoic acid exerts anti-neoplastic effects in colon carcinomas. DHL-HisZnNa is a newer alpha-lipoic acid derivative that has shown similar anti-neoplastic effects in colon cancer cell lines [2]. Levels of retinoblastoma protein are attenuated by DHL-HisZnNa while a simultaneous accentuation of p21 levels is seen. Similarly, anti-proliferative effects have been seen in hepatocellular carcinomas following the administration of alpha-lipoic acid in conjunction with caffeic acid and a new synthesized lipoyl-caffeic conjugate. This combination results in enhanced interleukin 10 levels and attenuated tumor necrosis factor-α levels. Similarly, alpha-lipoic acid derivatives such as CPI-613 demonstrate anti-neoplastic effects in pancreatic malignancies by disrupting the mitochondrial metabolism in tumor cells resulting in accentuated apoptosis in these cells. For instance, Schwartz et al. [3] have recently reported the case of an 80-year-old female with pancreatic adenocarcinoma and hepatic metastases who responded favorably to a treatment regimen in which the efficacy of gemcitabine was enhanced by using a combination of hydroxycitrate in conjunction with alpha-lipoic acid. Hydroxycitrate and alpha-lipoic acid combinations also exert anti-proliferative effects in mouse bladder carcinoma-2 cell line bladder transitional cell carcinoma lines, which is equivalent to the anti-neoplastic effects exerted by conventional chemotherapeutic agents such as 5-fluorouracil [4]. Similarly, alpha-lipoic acid exerts anti-proliferative effects in breast carcinomas. These anti-neoplastic effects are mediated by its attenuating effect on matrix metalloproteinase (MMP)-2 and MMP-9 levels. It not only down-regulates the expression of these molecules but also decreases their activity. In particular, it decreases cellular migration and cellular motility, resulting in decreased metastases. Besides this, it also alters the Bax/Bcl-2 ratio [5]. A simultaneous increase in p27 (kip1) expression is seen, resulting in accentuated apoptosis in breast malignancies. Alpha-lipoic acid also demonstrates benefits in the management of dermatological malignancies. It exerts these effects by inhibiting proliferation and by accentuating apoptosis in these tumors. It also enhances the apoptosis of cells with aberrant nuclei, thus further attenuating tumor growth in these malignancies. These effects have been seen particularly in B16-F10 melanoma cell lines [5]. The above examples clearly illustrate the anti-neoplastic effects of alpha-lipoic acid and the need for further studies to assess its potential efficacy in the management of other systemic tumors.

Influence of hepatocyte cell adhesion molecule on gene expression profile of human bladder transitional cell carcinoma cell line

To investigate the changes of gene expression file in transitional cell carcinoma of bladder after hepatocyte cell adhesion molecule(hepaCAM) overexpression. Affymetrix Human Genome U133 Plus 2.0 Array was used to investigate the changes of gene expression profile between adenovirus-green fluorescent protein(GFP) -hepaCAM group and GFP group in transitional cell carcinoma of bladder EJ cells.Significant Analysis of Microarray(SAM) was used to screen the differentially expressed genes, DAVID software was used to conduct gene ontology analysis and wikiPathway analysis based on the differentially expressed genes. Reverse transcription-polymerase chain reaction and Western blot were applied to verify microarray data. Compared with the GFP group, a total of 2469 genes were up-regulated or down-regulated by more than 2 times in the GFP-hepaCAM group. Among these genes, 1602 genes were up-regulated and 867 were down-regulated.Most of the differentially expressed genes were involved in the function of cell proliferation and cell cycle regulation. The mRNA expressions of nibrin, liver kinase B1, and cyclin D1 detected by reverse transcription-polymerase chain reaction in three different bladder cancer cell lines were consistent with the microarray data.The protein expressions of nibrin and liver kinase B1 in these three cell lines measured by Western blot were consistent with the mRNA expression. HepaCAM can alter the gene expression profile of bladder cancer EJ cells. The well-known anti-tumor effect of hepaCAM may be mediated by regulating the gene expression via multiple pathways.

Toxicogenomic activity of gemcitabine in two TP53-mutated bladder cancer cell lines: special focus on cell cycle-related genes

Because of its lower toxicity and good tolerability and response, gemcitabine has been described as one of the most highly promising drugs for urinary bladder cancer therapy. Its phosphorylated active-dFdCTP metabolite can incorporate into DNA, causing replication blockage. Additionally, it is known that mutations in the TP53 gene are related to the high recurrence rate of these neoplasias. Based on these premises, we investigated the effects of gemcitabine on the expression of the cell cycle-related genes in two different TP53-mutated bladder transitional carcinoma cell lines-5637 (from a moderate-grade tumor with a TP53 allele carrying two mutations) and T24 (from an invasive tumor with a TP53 allele encoding an in-frame deletion). Cell viability and morphology analyses (phase-contrast photomicrographs), Nuclear Division Index and pathway-specific quantitative RT-PCR gene arrays were performed. Treatment with gemcitabine led to the following results: (1) a significant decrease of viable T24 cells after treatment at the highest concentration (3.12 μM) tested; (2) scattered, elongated and vacuolated 5637 and T24 cells; (3) a cytostatic effect in both cell lines; and (4) significant upregulation of the BRCA1, CCNE1, CDK2, CDK6, CDKN1A, CDKN2B, E2F4, GADD45A, MAD2L2, CCNH, SERTAD1, CDC1, and CHEK1 genes. Gemcitabine had distinct toxicogenomic effects in the bladder transitional carcinoma cell lines with two different TP53 mutations. However, independent of the type of mutation and tumor grade, gemcitabine induced cell cycle arrest; upregulation of DNA repair-related genes, G1/S transition, apoptosis and activation of transcription factors, mainly by upregulation of the CCNE1, CDKN1A and GADD45A genes.

A Synthetic Polyvalent Ligand for α5β1 Integrin Activates Components of the Urothelial Carcinoma Cell Response to Bacillus Calmette-Guérin

Prior study has shown that bacillus Calmette-Guérin binds to and cross-links α5β1 integrins present on the surface of urothelial carcinoma cells. Antibody mediated cross-linking of α5β1 integrins can reproduce signal transduction, gene transactivation and phenotypic changes, similar to those observed in response to bacillus Calmette-Guérin. We evaluated the effect of a synthetic polyvalent ligand for α5β1 on these elements of the tumor response to bacillus Calmette-Guérin. The consensus α5β1 integrin binding tripeptide RGD was linked to a MAP8 backbone to result in an octavalent construct targeting α5β1 integrin. RGD-MAP8 was used to determine its effect on signaling pathway activation (nuclear factor-κB, NRF2 and CEBP), gene expression (p21, interleukin-6 and 8, CXCL1, CXCL2 and CCL20) and cytotoxicity (trypan blue exclusion and HMGB1 release) in human urothelial carcinoma cells. Results were compared to those of treatment with bacillus Calmette-Guérin or the missense peptide GRD-MAP8. The RDG-MAP8 construct significantly increased nuclear factor-κB signaling and p21 expression relative to controls. Compared to bacillus Calmette-Guérin treatment, only p21 expression was comparable for cells treated with RGD-MAP8, averaging 70% of bacillus Calmette-Guérin induced expression. RGD-MAP8 failed to have a significant effect on CEBP or NRF2 activation, gene expression or cell viability. Intracellular signaling, gene transactivation and phenotypic changes in response to RGD-MAP8 were qualitatively and quantitatively different than those observed in response to bacillus Calmette-Guérin. Results suggest that while α5β1 integrin cross-linking contributes to the bacillus Calmette-Guérin response, it alone is insufficient to duplicate the full spectrum of bacillus Calmette-Guérin induced changes in urothelial carcinoma cell biology.

Effects of gemcitabine and gemcitabine in combination with carboplatin on five canine transitional cell carcinoma cell lines

To evaluate in vitro effects of gemcitabine alone and in combination with carboplatin on canine transitional cell carcinoma (TCC) cell lines. In vitro cultures of 5 canine TCC cell lines. Cells were treated with gemcitabine, carboplatin, or a combination of both at various concentrations. Cell proliferation was assessed via a fluorescence-based microplate cell proliferation assay. Cell cycle was evaluated via propidium iodide staining, and apoptosis was assessed by measurement of caspase 3 and 7 enzymatic activity. Synergy between gemcitabine and carboplatin was quantified via combination index analyses. Treatment of 5 canine TCC cell lines with gemcitabine or carboplatin decreased cell proliferation, increased apoptosis, and induced cell cycle arrest. Cell cycle arrest and apoptosis were markedly increased when cell lines were treated with both gemcitabine and carboplatin simultaneously or sequentially. Order of administration during sequential treatment did not consistently affect cell proliferation results in TCC cell lines. When TCC cell lines were treated with gemcitabine and carboplatin in combination at therapeutically relevant concentrations (gemcitabine concentration, < 10μM; carboplatin concentration, < 250μM), a significant decrease in cell proliferation was observed, compared with cell proliferation following treatment with gemcitabine or carboplatin alone. In combination, the effects of gemcitabine and carboplatin were synergistic in 3 of 5 cell lines and additive in the other 2. Gemcitabine had antitumor effects on canine TCC cells in vitro, and the combination of gemcitabine and carboplatin had synergistic activity at biologically achievable concentrations.

Androgen Receptor Signaling Regulates Cell Growth and Vulnerability to Doxorubicin in Bladder Cancer

There are several reports of androgen receptor in bladder cancer cases but androgen receptor expression and the function of androgen/androgen receptor signaling in bladder cancer remain unclear. We investigated androgen receptor expression and the role of androgen/androgen receptor signaling in bladder cancer. We evaluated AR mRNA expression in bladder cancer tissue by quantitative real-time polymerase chain reaction. The role of androgen receptor in cell growth and drug sensitivity was also evaluated in vitro and in vivo in several bladder cancer cell lines. AR mRNA expression inversely correlated with bladder cancer grade, stage and spread. Of several bladder cancer cell lines UMUC3 and MBT-2 markedly expressed androgen receptor transcript and protein. In each cell line androgen/androgen receptor signaling blockade using androgen deprivation, blockade knockdown and antiandrogen agents decreased cell growth, colony formation and cell viability. Androgen receptor expression was implicated in doxorubicin resistance. Inversely androgen receptor deprivation and knockdown made UMUC3 cells sensitive to doxorubicin. Finally, castration slightly suppressed UMUC3 tumor growth in vivo, although this did not attain statistical significance. AR transcript expression inversely correlates with bladder cancer clinicopathological characteristics. Androgen/androgen receptor signaling has an important role in the growth and vulnerability to doxorubicin of bladder cancer cells that express androgen receptor. Androgen/androgen receptor signaling might be a possible prophylactic and therapeutic target for bladder cancer that shows androgen receptor expression.

Expression of Protein Kinase Cα and the MZF-1 and Elk-1 Transcription Factors in Human Bladder Transitional Cell Carcinoma Cells

In a recent study on hepatocellular carcinoma (HCC), we have shown that the transcription factors Myeloid Zinc Finger-1 (MZF-1) and Ets-like-protein 1 (Elk-1) are significantly related to protein kinase C alpha (PKCα) expression. The purpose of this study was to determine the correlation of the expression of PKCα with the expression of Elk-1 and MZF-1 in various differentiated urinary bladder transitional cell carcinoma (TCC) cell lines: 5637, BFTC905, TSGH8301, HT1376 and HT1197 cells. The malignant potential in the five TCC cell lines was examined by using cell proliferation/migration/invasion assay and the protein and mRNA levels of PKCα, ElK-1 and MZF-1 were examined by Western blot and RT-PCR analysis. The results showed that the rate of cell proliferation in the TSGH8301 cell line was higher than that in other cell lines, while there were obvious signs of cell migration and invasion in 5637, BFTC905 and HT1376 cells, and no sign in TSGH8301 and HT1197 cells. The resulting expression levels of Elk-1 and PKCα were the highest in 5637 cells, but the MZF-1 expression observed in all five cell lines showed no significant difference. To determine whether a correlation exists between PKCα and Elk-1, a shRNA knockout assay was performed and the results showed that the reduction of Elk-1 expression in 5637 cells did not result in the decreased PKCα expression. Therefore, although the findings showed elevated expression of Elk-1 and PKCα in 5637 cells, the regulator of PKCα in bladder cancer cells is yet to be determined.

Effects of YC-1 on Hypoxia-Inducible Factor 1 Alpha in Hypoxic Human Bladder Transitional Carcinoma Cell Line T24 Cells

Objectives: It was the aim of this study to explore the effects of 3-(5′-hydroxymethyl-2′-furyl)-l-benzyl indazole (YC-1) on transcription activity, cell proliferation and apoptosis of hypoxic human bladder transitional carcinoma cells (BTCC), mediated by hypoxia-inducible factor 1α (HIF-1α). Methods: BTCC cell line T24 cells were incubated under normoxic or hypoxic conditions, adding different doses of YC-1. The protein expression of HIF-1α and HIF-1α-mediated genes was detected by Western blotting. RT-PCR was used to detect HIF-1α mRNA expression. Cell proliferation, apoptosis and migration activity were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry and transwell migration assay. The cells were pretreated by two ERK/p38 MAPK pathway-specific inhibitors, PD98059 or SB203580, and then incubated with YC-1 treatment under hypoxic condition. HIF-1α protein expression was detected by Western blotting. Results: Hypoxic T24 cells expressed a higher level of HIF-1α, vascular endothelial growth factor, matrix metalloproteinases-2, B-cell lymphoma/leukemia-2 protein and HIF-1α mRNA compared with normoxic controls, in which the above-mentioned expression was downregulated by YC-1 in a dose-dependent manner. Cell proliferation and migration activity were inhibited while apoptosis was induced by YC-1 under hypoxic condition. Moreover, YC-1-downregulated HIF-1α expression was reversed by PD98059 and SB203580, respectively. Conclusions: YC-1 inhibits HIF-1α and HIF-1α-mediated gene expression, cell proliferation and migration activity and induces apoptosis in hypoxic BTCC. The ERK/p38 MAPK pathway may be involved in YC-1-mediated inhibition of HIF-1α.

Radiosensitivity and capacity for radiation-induced sublethal damage repair of canine transitional cell carcinoma (TCC) cell lines

Understanding the inherent radiosensitivity and repair capacity of canine transitional cell carcinoma (TCC) can aid in optimizing radiation protocols to treat this disease. The objective of this study was to evaluate the parameters surviving fraction at 2 Gy (SF(2) ), α/β ratio and capacity for sublethal damage repair (SLDR) in response to radiation. Dose-response and split-dose studies were performed using the clonogenic assay. The mean SF(2) for three established TCC cell lines was high at 0.61. All the three cell lines exhibited a low to moderate α/β ratio, with the mean being 3.27. Two cell lines exhibited statistically increased survival at 4 and 24 h in the dose-response assay. Overall, our results indicate that the cell lines are moderately radioresistant, have a high repair capacity and behave similarly to a late-responding normal tissue. These findings indicate that the radiation protocols utilizing higher doses with less fractionation may be more effective for treating TCC.

Photo induced hexylaminolevulinate destruction of rat bladder cells AY-27

Photodynamic therapy (PDT) is of increasing interest as a relevant treatment for human urinary bladder cancer. In the present experiments, the rat bladder transitional carcinoma cell line AY-27 was used as a model to study cell destruction mechanisms induced by PDT. Red LED light (630 nm) PDT with hexylaminolevulinate (HAL) as precursor for the photosensitizer protoporphyrin IX (PpIX) was used in treatment of the cells. Flow cytometry with fluorescent markers annexin V, propidium iodide and YO-PRO-1, as well as MTT assay and confocal microscopy, were used to map cell inactivation after PDT. Dark toxicity of HAL alone was low in these procedures and LD(50) (24 h, MTT assay) was approximately 1.6 J cm(-2) for standard red light (LED) irradiation (36 mW cm(-2)). Measurements done 1 h after HAL-PDT showed a maximum apoptotic level of about 10% at 6 J cm(-2), however the dominating mode of cell death was necrosis. Forward light scattering indicated an increase in cell size at low doses, possibly due to necrosis. Survival curves had a dual-slope shape, a fit to single hit, multi-target approximation gave a parameter estimate of n = 10 and D(0) about 2.6 J cm(-2). Replacing continuous light with fractionated light delivery (45 s light/60 s darkness) did not affect the treatment outcome.

The Feasibility of BCG and Sunitinib Combination Therapy for Transitional Cell Carcinoma

Currently, intravesical bacille Calmette-Guérin (BCG) instillation is the standard treatment for patients with an intermediate to high risk of bladder cancer. Nevertheless, BCG-refractory generation is a common process during treatment. We hypothesized that sunitinib, a vascular endothelial growth factor (VEGF) receptor inhibitor, can synergistically enhance the immunotherapeutic effect of BCG on bladder tumor growth. The 50% inhibitory concentration (IC 50 ) of BCG and sunitinib in various transitional cell carcinoma (TCC) cell lines was determined by the MTT method. The therapeutic effects of BCG, sunitinib, and their combination on MBT-2 tumors were investigated in both orthotopic bladder cancer and subcutaneously inoculated tumor models in mice. Evaluated parameters included the tumor growth rate and tumor burden in the bladder, tumor volume in the subcutaneous site, survival rate, serum cytokine changes, and mean vascular density in tumor tissues. BCG and sunitinib showed in vitro cytotoxicity to tested TCC cell lines with slight variations in IC 50 . Sunitinib had an in vitro synergistic effect on BCG cytotoxicity in TCC cells. BCG and sunitinib had individual tumor suppression activities in mice MBT-2 tumors in both the orthotopic and subcutaneous models. Surprisingly, synchronous combination treatment using BCG and sunitinib did not demonstrate synergistic suppression efficacy in animal tumor models. Inhibition of tumor growth, increased survival rate, and decreased mean vascular density were observed in mice treated with BCG, sunitinib, and synchronous cotreatment. In contrast, no therapeutic efficacy was seen in mice treated with BCG and sunitinib metachronously. BCG and the VEGF receptor inhibitor sunitinib had significant suppressive effects in mice bladder cancer models when administered individually. The underlying mechanisms by which they counteract cytotoxicity and the interaction of BCG and sunitinib during cotreatment require further investigation.

Targeting canine bladder transitional cell carcinoma with a human bladder cancer-specific ligand

ObjectiveTo determine if a human bladder cancer-specific peptide named PLZ4 can target canine bladder cancer cells.Experimental DesignThe binding of PLZ4 to five established canine invasive transitional cell carcinoma (TCC) cell lines and to normal canine bladder urothelial cells was determined using the whole cell binding assay and an affinitofluorescence assay. The WST-8 assay was performed to determine whether PLZ4 affected cell viability. In vivo tumor-specific homing/targeting property and biodistribution of PLZ4 was performed in a mouse xenograft model via tail vein injection and was confirmed with ex vivo imaging.ResultsPLZ4 exhibited high affinity and specific dose-dependent binding to canine bladder TCC cell lines, but not to normal canine urothelial cells. No significant changes in cell viability or proliferation were observed upon incubation with PLZ4. The in vivo and ex vivo optical imaging study showed that, when linked with the near-infrared fluorescent dye Cy5.5, PLZ4 substantially accumulated at the canine bladder cancer foci in the mouse xenograft model as compared to the control.Conclusions and Clinical RelevancePLZ4 can specifically bind to canine bladder cancer cells. This suggests that the preclinical studies of PLZ4 as a potential diagnostic and therapeutic agent can be performed in dogs with naturally occurring bladder cancer, and that PLZ4 can possibly be developed in the management of canine bladder cancer.

Analysis of demethylation drug 5-aza-2'-deoxycytidine in growth inhibition on bladder tumor cell

Objective To study the growth suppressive effect of demethylation drug 5-aza-2'-deoxycytidine on bladder tumor cells. Methods The growth suppressive effect of DAC on 4 transitional cell carcinoma (TCC) cell lines was measured using the Cell Proliferation Reagent WST-1 assay.The effects of DAC on apoptosis induction and cell cycle arrest were analyzed by flow cytometric analysis. Caspase 3, 9 activities were analyzed by APOPCYTO Caspase Colorimetric Assay Kit and PCNA expression was also investigated by Western blot to clarify the mechanism of DAC against TCC. Results DAC inhibited the growth of all TCC cell lines tested in a dose-dependant manner, however,growth suppressive effect of DAC was independent of p53 status in TCC. DAC inhibited proliferation via inducing G2/M cell cycle arrest but not via inducing apoptosis. After treated with 0, 1 and 8 μmol/L DAC, cells of RTl 12 in G2/M phase was (36.3 ± 3.4) %, (46.2 ± 4.6) % and (56.5 ±6.2) %, TCCsup was (37.5 ± 3.8) %, (48.4 ±4.9) % and (60.1 ± 6.7) %, respectively. The expression of PCNA was decreased by DAC, but caspase3, 9 activities were not activated. Conclusion DAC could suppress the growth of TCC cells and might be a new strategy to treat bladder malignancy in the future. Key words: Bladder neoplasms; Demethylation; Apoptosis; Cell cycle

Overexpression of Securin in Human Transitional Cell Carcinoma Specimens

Objective Securin, the product of PTTG (pituitary tumor transforming gene), is overexpressed in several tumors, and plays important roles in cancer progression and invasion. In our previous report, securin expression was observed in transitional cell carcinoma (TCC; the most common pathological pattern of bladder cancer) cell lines, including BFTC905, T24, and TSGH8301. However, the existence of securin in human bladder cancer specimens has not been established. Materials and Methods Commercial bladder cancer tissue arrays (BL208 & BLC661) were used. Slides of paraffin-fixed human bladder tissues included all grades of TCC (18, 22 and 29 tissue samples for grades I, II, and III, respectively), 21 superficial and 41 invasive TCC specimens, and 11 normal urothelial tissue samples. The intensities of securin immunostaining were graded as background, mild, and strong (scored as 0, 1, and 2, respectively), and scores from the nucleus and cytosol were analyzed separately. Results We have demonstrated, for the first time, the expression of securin in bladder tissues. Securin was overexpressed in the cytosol and nucleus of all TCC samples compared to normal urothelium, but only cytosolic localization revealed statistical significance. There were no differences in securin immunoreactivity among the different grades of TCC. Significant over-expression of cytosolic but not nuclear securin was found in both superficial and invasive TCC samples compared to normal urothelium. No difference in immunoreactive staining for nuclear and cytosolic securin between superficial and invasive TCC was noted. Conclusion Significant enhanced expression of cytosolic securin protein was found in human bladder cancer specimens, suggesting that the level of tissue securin may be a potential biomarker for the diagnosis of bladder cancer. Studies that investigate the relationship between securin expression and the outcomes of bladder cancer patients could be conducted in the future.

Evaluation of an in vitro telomeric repeat amplification protocol assay to detect telomerase activity in canine urine

To evaluate the usefulness of a PCR-based telomeric repeat amplification protocol (TRAP) assay for detecting telomerase activity in cells from a canine transitional cell carcinoma (TCC) cell line and, ultimately, in the urine of dogs with TCC. 11 dogs with histologic or cytologic evidence of TCC, 10 dogs with benign lower urinary tract disease, and 9 healthy dogs. Telomerase activity was initially evaluated in cells from canine TCC (K9TCC) and telomerase-negative (WI-38) cell lines. Following assay optimization, telomerase stability was evaluated at various storage durations and temperatures. Urine samples were then obtained prospectively from study dogs. Telomerase activity was detected in the K9TCC cell line. The TRAP assay detected telomerase activity in as few as 10 K9TCC cells alone and as low as 2% of a total cell population in K9TCC and WI-38 mixing experiments. A loss of telomerase activity was detected with increasing urine storage durations at various temperatures. Telomerase activity was clearly detected in samples collected from 10 of 11 dogs with TCC, 2 of 10 dogs with benign lower urinary tract disease, and none of the 9 healthy dogs. The TRAP-based assay detected telomerase activity in the canine TCC cell line and revealed that the telomerase ribonucleoprotein complex was inherently unstable at various storage durations and conditions. Telomerase activity was also detectable in urine samples obtained from dogs with TCC, which suggested the TRAP assay may be useful in diagnosing TCC in dogs.

Gallic acid induces G2/M phase cell cycle arrest via regulating 14‐3‐3β release from Cdc25C and Chk2 activation in human bladder transitional carcinoma cells

Cell cycle regulation is a critical issue in cancer treatment. Previously, gallic acid (GA) has been reported to possess anticancer ability. Here, we have evaluated the molecular mechanism of GA on cell cycle modulation in a human bladder transitional carcinoma cell line (TSGH-8301 cell). Using flow cytometer analysis, exposure of the cells to 40 μM GA resulted in a statistically significant increase in G2/M phase cells, which was accompanied by a decrease in G0/G1 phase cells. GA-treated cells resulted in significant growth inhibition in a dose-dependent manner accompanied by a decrease in cyclin-dependent kinases (Cdk1), Cyclin B1, and Cdc25C, but significant increases in p-cdc2 (Tyr-15) and Cip1/p21 by western blotting. Additional mechanistic studies showed that GA induces phosphorylation of Cdc25C at Ser-216. This mechanism leads to its translocation from the nucleus to the cytoplasm resulting in an increased binding with 14-3-3β. When treated with GA, phosphorylated Cdc25C can be activated by ataxia telangiectasia-mutated checkpoint kinase 2 (Chk2). This might be a DNA damage response as indicated by Ser-139 phosphorylation of histine H2A.X. Furthermore, treatment of the cells with a Chk2 inhibitor significantly attenuated GA-induced G2/M phase arrest. These results indicate that GA can induce cell cycle arrest at G2/M phase via Chk2-mediated phosphorylation of Cdc25C in a bladder transitional carcinoma cell line.

Lestaurtinib is Cytotoxic to Oxaliplatin-resistant Transitional Cell Carcinoma Cell Line T24 In Vitro

Objective Patients with bladder cancer have responded poorly to oxaliplatin therapy in clinical trials. Blockade of receptor tyrosine kinases is considered a good strategy in cancer therapy. Our previous studies have demonstrated the crucial roles of brain-derived neurotrophic factor and its receptor tropomyosin receptor kinase B (TrkB) in transitional cell carcinoma (TCC). The aim of this study was to examine the cytotoxic effects of lestaurtinib, a new pan-Trk inhibitor, and oxaliplatin on bladder cancer cell lines. Materials and Methods BFTC905 and T24 TCC cell lines were used for investigation in vitro. The effects of oxaliplatin and/or lestaurtinib on cell viability, apoptosis, and expression of survivin and securin were assessed. MTT assay was used for cytotoxic evaluation. DNA fragments were detected in both the culture medium and cytoplasm to differentiate the types of cell death (apoptosis vs. necrosis). Western blots were used to analyze the expression of survivin and securin after oxaliplatin and/or lestaurtinib treatment. Results Oxaliplatin at 3 μg/mL elicited cytotoxicity on BFTC905 but not T24 cells 48 hours after treatment. The addition of 1 or 3 μM lestaurtinib to oxaliplatin did not exert additive cytotoxic effects on BFTC905 cells. Although oxaliplatin at 3 μg/mL had no effect on T24 cells, the addition of 1 or 3 μM lestaurtinib demonstrated concentration-dependent inhibitory effects. Apoptosis of T24 cells was observed after treatment with lestaurtinib alone and lestaurtinib plus oxaliplatin. Furthermore, in T24 cells, the expression of survivin was inhibited by a combination of lestaurtinib and oxaliplatin, while securin expression was inhibited by lestaurtinib alone and lestaurtinib with oxaliplatin. Conclusion Lestaurtinib may be a potential new drug for the targeted therapy of oxaliplatin-resistant TCC. Further in vivo studies are needed.

Combining siRN A-mediated hTR and hTERT expression in bladder uroepithelium cancer BIU-87 cell lines

Objective To perform a DNA microarray analysis of combining small interfering RNA (siRNA) of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) gene in bladder transitional cell carcinoma cell line BIU-87 telomere Inhibition of enzyme activity. Methods According to the siRNA design principles and TERT.TR gene mRNA coding sequence,respectively,were designed and synthesized three pairs of different and can interfere with TR and TERT gene expression in siRNA sequence and filter out the most efficient suppression sequence ( pRNAT-TERT-Ⅲ: GTACAG-GTTTCACGCATGT gene No. 3229, pRNAT-TR- Ⅲ: CGAAGGTGCCTTGGAATAT gene No. 306). Constructing the hTR,hTERT gene as a target of the joint RNA interference ( RNAi) device pRNAT-TR-Ⅲ+ pRNAT-TERT-Ⅲ(liposome) and transfected into bladder transitional cell carcinoma of BIU-87 cell line, using fluorescent quantitative polymerase chain reaction (PCR) analysis of hTR and hTERT mRNA expression (in the target gene and GAPDH copy number ratios for the relative quantitative analysis) , TRAP-ELJSA detection of telomerase activity (measured A value) ,MTT assay BIU-87 cell growth inhibition (by MTT method Detection of the survival rate of transfected cells) ,and before and after co-RNAi gene chip a-nalysis,using RNA extraction,RNA quality control,cRNA marking and synthesis,chip hybridization, chemi-luminescence detection,image acquisition and data analysis,and further use of part of the reverse transcription (RT)-PCR validation of differentially expressed genes. Results The combined RNA interference hTR and hTERT gene, pRNAT-TERT-Ⅲ+ pRNAT-TR-Ⅲcombined RNAi can specifically inhibit the bladder transitional cell carcinoma cell line BIU-87 in TERT and TR expression (pRNAT-TERT- Ⅲ+ pRNAT-TR-I TERT:0.300 ±0.033 ,P <0.05;TR:0.430 ±0.028,P <0.05). After the combined RNAi and bladder transitional cell carcinoma of BIU-87 cell line growth and telomerase activity were significantly inhibited (pRNAT-TERT-Ⅲ+ pRNAT-TR- Ⅲ: 1.250 ± 0.012, P < 0.05 ). Before and after the joint siRNA gene chip analysis found 21 genes down ( ATM, BAX, BCL2, BCL2L1, BIRC5, CD44, CTNNB1, E2F1, JUN, MCAM, MTA1, MYC, NFKB1, NFKBIA, NME4, PNN, PRKDC, SERP1NE1, THBS1, TNFRSF1A, UCCl). Conclusion The combined RNA interference with hTERT and hTR gene can significantly inhibit bladder cancer BIU-87 cell line growth;joint siRNA inhibited bladder transitional cell carcinoma of BIU-87 cell line causes growth of 21 genes were cut. Key words: Telomerase; siRNA; Bladder transitional cell carcinoma

Cell cycle arrest and apoptosis in TP53 subtypes of bladder carcinoma cell lines treated with cisplatin and gemcitabine

Currently, the combination of cisplatin and gemcitabine is considered a standard chemotherapeutic protocol for bladder cancer. However, the mechanism by which these drugs act on tumor cells is not completely understood. The aim of the present study was to investigate the effects of these two antineoplastic drugs on the apoptotic index and cell cycle kinetics of urinary bladder transitional carcinoma cell lines with wild-type or mutant TP53 (RT4: wild type for TP53; 5637 and T24: mutated TP53). Cytotoxicity, cell survival assays, clonogenic survival assays and flow cytometric analyses for cell cycle kinetics and apoptosis detection were performed with three cell lines treated with different concentrations of cisplatin and gemcitabine. G(1) cell cycle arrest was observed in the three cell lines after treatment with gemcitabine and gemcitabine plus cisplatin. A significant increase in cell death was also detected in all cell lines treated with cisplatin or gemcitabine. Lower survival rates occurred with the combined drug protocol independent of TP53 status. TP53-wild type cells (RT4) were more sensitive to apoptosis than were mutated TP53 cells when treated with cisplatin or gemcitabine. Concurrent treatment with cisplatin and gemcitabine was more effective on transitional carcinoma cell lines than either drug alone; the drug combination led to a decreased cell survival that was independent of TP53 status. Therefore, the synergy between low concentrations of cisplatin and gemcitabine may have clinical relevance, as high concentrations of each individual drug are toxic to whole organisms.

Profiling of matrix metalloproteinases and tissue inhibitors of metalloproteinases proteins in bladder urothelial carcinoma

We investigated the matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) proteins in transitional cell carcinoma (TCC) cell lines and surgical specimens of the bladder neoplasm. The expression level was correlated to the degree of cellular differentiation and invasiveness of bladder cancer. Panels of six TCC cell lines with different degrees of differentiation were tested with monoclonal antibodies (mAbs) to MMP-1, MMP-2, MMP-3, MMP-9a, MMP-9b, TIMP-1 and TIMP-2 by immunocytochemistry. Gelatin zymography was also conducted on the cell lines for MMP-2 and -9. In addition, immunohistochemistry with the mAbs to MMP and TIM molecules was performed on 30 TCC specimens. We found that TCC cell lines were stained positively for MMP-1 (6/6), weakly for MMP-9a (2/6), MMP9b (5/6) and TIMP-1 (3/6), and negatively for MMP-2 (3/6) and MMP-3 (3/6). Zymographic analysis of the cell lines showed a high level of MMP2 in the MGH-U4 cell line. In bladder cancer surgical specimens, all specimens were positive for MMP1 (30/30), 19 were positive for MMP-2 (63.3%), 21 positive for MMP-9a (70%) and 15 positive for MMP-9b (50%). The expression of MMP-2 was found to be positively correlated with higher-grade tumors (p=0.036) and the expression of MMP-9a and -9b was found to be positively correlated with tumor stage (p=0.012 and 0.023, respectively). However, the expression of MMP-1, MMP-3, TIMP-1 and TIMP-2 was not correlated with either tumor staging or grading. In conclusion, the expression of MMP-2 and -9 was correlated with high-grade or high-stage bladder tumors, respectively. However, this correlation was not observed with TCC cell lines in which high- and low-grade tumors are included. Immunohistochemical results on tumor lesions may have more clinical relevance, since in a given tumor microenvironment the interaction among tumor cells in situ and tumor-associated cells, such as neutrophils, macrophages, lymphocytes and endothelial cells, as well as environmental factors (hypoxia and pH), cytokines and growth factors released by these cells may be required for TCC to express selective MMPs and TIMPs. The selective expression of these molecules then regulates tumor progression.