Altered redox status accompanies progression to metastatic human bladder cancer

The role of reactive oxygen species (ROS) in bladder cancer progression remains an unexplored field. Expression levels of enzymes regulating ROS levels are often altered in cancer. A search of publicly available microarray data reveals that expression of mitochondrial manganese superoxide dismutase (Sod2), responsible for the conversion of superoxide (O(2)(-)) to hydrogen peroxide (H(2)O(2)), is consistently increased in high-grade and advanced-stage bladder tumors. We aimed to identify the role of Sod2 expression and ROS in bladder cancer. Using an in vitro human bladder tumor model we monitored the redox state of both nonmetastatic (253J) and highly metastatic (253J B-V) bladder tumor cell lines. 253J B-V cells displayed significantly higher Sod2 protein and activity levels compared to their parental 253J cell line. The increase in Sod2 expression was accompanied by a significant decrease in catalase activity, resulting in a net increase in H(2)O(2) production in the 253J B-V cell line. Expression of the prometastatic and proangiogenic factors matrix metalloproteinase 9 (MMP-9) and vascular endothelial-derived growth factor (VEGF), respectively, was upregulated in the metastatic line. Expression of both MMP-9 and VEGF was shown to be H(2)O(2)-dependent, as removal of H(2)O(2) by overexpression of catalase attenuated their expression. Similarly, expression of catalase effectively reduced the clonogenic activity of 253J B-V cells. These findings indicate that metastatic bladder cancer cells display an altered antioxidant expression profile, resulting in a net increase in ROS production, which leads to the induction of redox-sensitive protumorigenic and prometastatic genes such as VEGF and MMP-9.     

Ultrasound-Guided Intramural Inoculation of Orthotopic Bladder Cancer Xenografts: A Novel High-Precision Approach

Orthotopic bladder cancer xenografts are essential for testing novel therapies and molecular manipulations of cell lines in vivo. Current xenografts rely on tumor cell inoculation by intravesical instillation or direct injection into the bladder wall. Instillation is limited by the lack of cell lines that are tumorigenic when delivered in this manner. The invasive model inflicts morbidity on the mice by the need for laparotomy and mobilization of the bladder. Furthermore this procedure is complex and time-consuming. Three bladder cancer cell lines (UM-UC1, UM-UC3, UM-UC13) were inoculated into 50 athymic nude mice by percutaneous injection under ultrasound guidance. PBS was first injected between the muscle wall and the mucosa to separate these layers, and tumor cells were subsequently injected into this space. Bioluminescence and ultrasound were used to monitor tumor growth. Contrast-enhanced ultrasound was used to study changes in tumor perfusion after systemic gemcitabine/cisplatin treatment. To demonstrate proof of principle that therapeutic agents can be injected into established xenografts under ultrasound guidance, oncolytic virus (VSV) was injected into UM-UC3 tumors. Xenograft tissue was harvested for immunohistochemistry after 23–37 days. Percutaneous injection of tumor cells into the bladder wall was performed efficiently (mean time: 5.7 min) and without complications in all 50 animals. Ultrasound and bioluminescence confirmed presence of tumor in the anterior bladder wall in all animals 3 days later. The average tumor volumes increased steadily over the study period. UM-UC13 tumors showed a marked decrease in volume and perfusion after chemotherapy. Immunohistochemical staining for VSV-G demonstrated virus uptake in all UM-UC3 tumors after intratumoral injection. We have developed a novel method for creating orthotopic bladder cancer xenograft in a minimally invasive fashion. In our hands this has replaced the traditional model requiring laparotomy, because this model is more time efficient, more precise and associated with less morbidity for the mice.     

Geldanamycin restores a defective heat shock response in vivo

Induced expression of heat shock proteins (Hsps) plays a central role in promoting cellular survival after environmental and physiological stress. We have previously shown that scrapie-infected mouse neuroblastoma (ScN2a) cells fail to induce the expression of Hsp72 and Hsp28 after various stress conditions. Here we present evidence that this impaired stress response is due to an altered regulation of HSF1 activity. Upon stress in ScN2a cells, HSF1 was converted into hyperphosphorylated trimers but failed to acquire transactivation competence. A kinetic analysis of HSF1 activation revealed that in ScN2a cells trimer formation after stress was efficient, but disassembly of trimers proceeded much faster than in the uninfected cell line. Geldanamycin, a Hsp90-binding drug, significantly delayed disassembly of HSF1 trimers after a heat shock and restored stress-induced expression of Hsp72 in ScN2a cells. Heat-induced Hsp72 expression required geldanamycin to be present; following removal of the drug ScN2a cells again lost their ability to mount a stress response. Thus, our studies show that a defective stress response can be pharmacologically restored and suggest that the HSF1 deactivation pathway may play an important role in the regulation of Hsp expression.     

Bladder cancer cell viability inhibition and apoptosis induction by baicalein through targeting the expression of anti-apoptotic genes

The study was aimed to investigate the effect of baicalein, a flavonoid molecule isolated from the plant Oroxylum indicum on bladder cancer cell viability. The results revealed that baicalein treatment of T24 and 253J bladder cancer cells targeted the expression of mRNA and proteins corresponding to the anti-apoptotic genes. RT-PCR assay showed that anti-apoptotic genes were markedly over-expressed in the bladder cancer cells. Exposure of the bladder cancer cells to baicalein at 5 mg/mL doses for 72 h led to reduction in the expression of mRNA levels of antiapoptotic genes. In T24 cells, the levels of BCL2, Bcl-xL, XIAP and surviving was reduced by 65, 69, 58 and 72%, respectively. In T24 and 253J cells exposure to baicalein for 72 h resulted respectively in 39 and 46% reduction in cell viability. Baicalein treatment also induced apoptosis in the bladder cancer cells. In T24 and 253J cells baicalein treatment at 5 mg/mL for 72 h induced apoptosis in 79 and 86% cells respectively. Thus, baicalein mediated reduction in antiapoptotic gene expression inhibits viability and induces apoptosis in bladder cancer cells. Therefore, baicalein is of therapeutic importance for the development of bladder cancer treatment strategy.     

Combining mTOR Inhibition with Radiation Improves Antitumor Activity in Bladder Cancer Cells In Vitro and In Vivo: A Novel Strategy for Treatment

Purpose

Radiation therapy for invasive bladder cancer allows for organ preservation but toxicity and local control remain problematic. As such, improving efficacy of treatment requires radiosensitization of tumor cells. The aim of study is to investigate if the mammalian Target of Rapamycin (mTOR), a downstream kinase of the phosphatidylinositol 3-kinase (PI3K)/AKT survival pathway, may be a target for radiation sensitization.

Experimental Design

Clonogenic assays were performed using 6 bladder cancer cell lines (UM-UC3, UM-UC5, UM-UC6, KU7, 253J-BV, and 253-JP) in order to examine the effects of ionizing radiation (IR) alone and in combination with RAD001, an mTOR inhibitor. Cell cycle analysis was performed using flow cytometry. In vivo, athymic mice were subcutaneously injected with 2 bladder cancer cell lines. Treatment response with RAD001 (1.5 mg/kg, daily), fractionated IR (total 9Gy = 3Gy×3), and combination of RAD001 and IR was followed over 4 weeks. Tumor weight was measured at experimental endpoint.

Results

Clonogenic assays revealed that in all bladder cell lines tested, an additive effect was observed in the combined treatment when compared to either treatment alone. Our data indicates that this effect is due to arrest in both G1 and G2 phases of cell cycle when treatments are combined. Furthermore, our data show that this arrest is primarily regulated by changes in levels of cyclin D1, p27 and p21 following treatments. In vivo, a significant decrease in tumor weight was observed in the combined treatment compared to either treatment alone or control.

Conclusions

Altering cell cycle by inhibiting the mTOR signaling pathway in combination with radiation have favorable outcomes and is a promising therapeutic modality for bladder cancer.     

Reduced heat shock response in human mononuclear cells during aging and its association with polymorphisms in HSP70 genes

Age-dependent changes in heat shock response (HSR) were studied in mononuclear cells (monocytes and lymphocytes) collected from young (mean age = 22.6 ± 1.7 years) and middle-aged (mean age = 56.3 ± 4.7 years) subjects after 1 hour of heat shock at 42°C. Genotype-specific HSR was measured by genotyping the subjects for 3 single nucleotide polymorphisms, HSPA1A(A-110C), HSPA1B(A1267G), and HSPA1L(T2437C), 1 each in the 3 HSP70 genes. A significant age-related decrease in the induction of Hsp70 occurred after heat shock in both monocytes and lymphocytes. The noninducible and inducible forms of Hsp70 decreased 1.3-fold (P < 0.001) and 1.4-fold (P < 0.001), respectively, in the monocytes with age. In the young subjects, a positive association was found between HSPA1L(T2437C) polymorphism and HSR. CC carriers had a significantly lower induction than TT carriers in both monocytes (P = 0.015) and lymphocytes (P = 0.044). This polymorphism, which is present in the coding region of HSPA1L gene, can affect the chaperoning function of Hsp70. These data consolidate our other observations that the CC genotype is unfavorable for human longevity and provide a functional explanation in terms of variations in HSR.     

Chaperokine Function of Recombinant Hsp72 Produced in Insect Cells Using a Baculovirus Expression System Is Retained

Extracellular heat shock protein 72 (Hsp72; inducible form of the 70-kDa heat shock protein) plays a critical role in innate and adaptive immune responses and has shown promise as an ideal adjuvant for the optimization of antigen-specific anti-tumor vaccines. Recent studies suggest that to correctly elucidate the mechanisms by which Hsp72 exerts its beneficial effects in vitro, great care must be taken to ensure that endotoxin by-products do not invalidate the findings. In this study, we have taken advantage of the baculovirus expression vector system for production of endotoxin-free recombinant Hsp72. The coding sequence of human hsp72 was recombined into the baculovirus immediately downstream of the strong polyhedron gene promoter. Ninety-six h post-infection of Sf9 insect cells with recombinant baculovirus, maximal levels of Hsp72 protein were detected. The recombinant human Hsp72 was purified by affinity chromatography from insect cells, and purity was confirmed by SDS-PAGE and mass spectrometry. The purified human recombinant Hsp72^bv (Hsp72 produced using the BEVS) was demonstrated to have no endotoxin contamination and was shown to have stimulated potent calcium flux in the human monocytic cell line. Furthermore, recombinant Hsp72^bv enhanced the tolerance of neuroblastoma cells to heat stress-induced cell death and displayed classical chaperokine functions including augmentation of inflammatory cytokine productions in mouse splenocytes. The production of functional, endotoxin-free recombinant human Hsp72^bv in insect cells is inexpensive and convenient and eliminates the need of special procedures for endotoxin depletion. Endotoxin-free recombinant human Hsp72^bv can now be used to unlock the important role Hsp72 plays in modulating immune function.