Toll-like Receptor 6 and Connective Tissue Growth Factor are significantly upregulated in mitomycin-C treated urothelial carcinoma cells under hydrostatic pressure stimulation. Homo sapiens

Urothelial carcinoma (UC) is the most common histologic subtype of bladder cancer. The administration of mitomycin C (MMC) into bladder after transurethral resection of the bladder tumor (TURBT) is a common treatment strategy for preventing recurrence after surgery. We previously applied hydrostatic pressure combined with MMC in UC cells and found that hydrostatic pressure synergistically enhanced MMC-induced UC cell apoptosis via the Fas/FasL pathways. To understand the alteration of gene expressions in UC cells caused by hydrostatic pressure and MMC, oligonucleotide microarray was used to explore all of the differentially expressed genes. After bioinformatics analysis and gene annotation, Toll-like receptor 6 (TLR6) and connective tissue growth factor (CTGF) showed significant up-regulation among altered genes, and their expressions with each treatment of UC cells were validated by quantitative real-time PCR (qPCR). We conclude that under treatment with MMC and hydrostatic pressure, UC cells showed increasing apoptosis via extrinsic pathways through upregulation of TLR6 and CTGF. Overall design: BFTC905 cells, a UC cell line, were cultured in the presence of 10 μg/ml of MMC for 2.5 hours. The MMC-treated cells were then incubated in a well-designed hydrostatic bioreactor for 10 kPa pressure treatment in duplicate or were cultured as usual to be used as control sets.

尿路上皮癌（Urothelial carcinoma, UC）是膀胱癌最常见的组织学亚型。经尿道膀胱肿瘤切除术（transurethral resection of the bladder tumor, TURBT）后向膀胱内灌注丝裂霉素C（mitomycin C, MMC）是临床术后预防肿瘤复发的常用治疗策略。本团队前期将静水压（hydrostatic pressure）与丝裂霉素C联合应用于尿路上皮癌细胞，发现静水压可通过Fas/FasL通路协同增强丝裂霉素C诱导的尿路上皮癌细胞凋亡。为探究静水压与丝裂霉素C对尿路上皮癌细胞基因表达的调控变化，本研究采用寡核苷酸微阵列（oligonucleotide microarray）技术检测所有差异表达基因。经生物信息学分析（bioinformatics analysis）与基因注释（gene annotation）后，Toll样受体6（Toll-like receptor 6, TLR6）与结缔组织生长因子（connective tissue growth factor, CTGF）在差异表达基因中呈现显著上调，随后通过实时荧光定量PCR（quantitative real-time PCR, qPCR）验证了两种基因在尿路上皮癌细胞各处理组中的表达水平。本研究表明，在丝裂霉素C与静水压联合处理下，尿路上皮癌细胞可通过上调TLR6与CTGF的表达，经由外源性凋亡通路增强细胞凋亡。实验总体设计：将尿路上皮癌细胞系BFTC905置于含10 μg/ml丝裂霉素C的培养基中培养2.5小时，随后将经丝裂霉素C处理的细胞置于定制的静水压生物反应器（hydrostatic bioreactor）中，以10 kPa压力进行处理并设置重复组，同时将常规培养的细胞作为对照组。