Downregulation of glutathione S-transferase M1 protein in N-butyl-N-(4-hydroxybutyl)nitrosamine-induced mouse bladder carcinogenesis.

Bladder cancer is highly recurrent following specific transurethral resection and intravesical chemotherapy, which has prompted continuing efforts to develop novel therapeutic agents and early-stage diagnostic tools. Specific changes in protein expression can provide a diagnostic marker. In our present study, we investigated changes in protein expression during urothelial carcinogenesis. The carcinogen BBN was used to induce mouse bladder tumor formation. Mouse bladder mucosa proteins were collected and analyzed by 2D electrophoresis from 6 to 20 weeks after commencing continuous BBN treatment. By histological examination, the connective layer of the submucosa showed gradual thickening and the number of submucosal capillaries gradually increased after BBN treatment. At 12-weeks after the start of BBN treatment, the urothelia became moderately dysplastic and tumors arose after 20-weeks of treatment. These induced bladder lesions included carcinoma in situ and connective tissue invasive cancer. In protein 2D analysis, the sequentially downregulated proteins from 6 to 20 weeks included GSTM1, L-lactate dehydrogenase B chain, keratin 8, keratin 18 and major urinary proteins 2 and 11/8. In contrast, the sequentially upregulated proteins identified were GSTO1, keratin 15 and myosin light polypeptide 6. Western blotting confirmed that GSTM1 and NQO-1 were decreased, while GSTO1 and Sp1 were increased, after BBN treatment. In human bladder cancer cells, 5-aza-2'-deoxycytidine increased the GSTM1 mRNA and protein expression. These data suggest that the downregulation of GSTM1 in the urothelia is a biomarker of bladder carcinogenesis and that this may be mediated by DNA CpG methylation.

Inorganic arsenic in drinking water accelerates N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder tissue damage in mice.

Epidemiological studies have revealed that exposure to an arsenic-contaminated environment correlates with the incidence of bladder cancer. Bladder cancer is highly recurrent after intravesical therapy, and most of the deaths from this disease are due to invasive metastasis. In our present study, the role of inorganic arsenic in bladder carcinogenesis is characterized in a mouse model. This work provides the first evidence that inorganic arsenic in drinking water promotes N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced bladder tissue damage, including the urothelium and submucosal layer. This damage to the bladder epithelium induced by BBN includes thickening of the submucosal layer, the loss of the glycosaminoglycan layer and an increase in both the deoxyguanosine oxidation and cytosine methylation levels in the DNA. Further, when 10ppm inorganic arsenic is combined with BBN, the number of bladder submucosal capillaries is increased. In addition, inorganic arsenic also increases the deoxyguanosine oxidation level, alters the cytosine methylation state, decreases the activities of glutathione reductase and glucose-6-phosphate dehydrogenase, decreases the protein expression of NAD(P)H quinone oxidoreductase-1 (NQO-1) and increases the protein expression of specific protein 1 (Sp1) in bladder tissues. In summary, our data reveal that inorganic arsenic in drinking water promotes the BBN-induced pre-neoplastic damage of bladder tissue in mice, and that the 8-hydroxy-2'-deoxyguanosine, 5-methylcytosine, NQO-1 protein and Sp1 protein levels may be pre-neoplastic markers of bladder tumors.

Pancreatic stellate cells activated by mutant KRAS-mediated PAI-1 upregulation foster pancreatic cancer progression via IL-8.

Background: The dense fibrotic stroma enveloping pancreatic tumors is a major cause of drug resistance. Pancreatic stellate cells (PSCs) in the stroma can be activated to induce intra-tumor fibrosis and worsen patient survival; however, the molecular basics for the regulation of PSC activation remains unclear. Methods: The in vitro coculture system was used to study cancer cell-PSC interactions. Atomic force microscopy was used to measure the stiffness of tumor tissues and coculture gels. Cytokine arrays, qPCR, and Western blotting were performed to identify the potential factors involved in PSC activation and to elucidate underlying pathways. Results: PSC activation characterized by α-SMA expression was associated with increased pancreatic tumor stiffness and poor prognosis. Coculture with cancer cells induced PSC activation, which increased organotypic coculture gel stiffness and cancer cell invasion. Cancer cells-derived PAI-1 identified from coculture medium could activate PSCs, consistent with pancreatic cancer tissue microarray analysis showing a strong positive correlation between PAI-1 and α-SMA expression. Suppression by knocking down PAI-1 in cancer cells demonstrated the requirement of PAI-1 for coculture-induced PSC activation and gel stiffness. PAI-1 could be upregulated by KRAS in pancreatic cancer cells through ERK. In PSCs, inhibition of LRP-1, ERK, and c-JUN neutralized the effect of PAI-1, suggesting the contribution of LRP-1/ERK/c-JUN signaling. Furthermore, activated PSCs might exacerbate malignant behavior of cancer cells via IL-8 because suppression of IL-8 signaling reduced pancreatic tumor growth and fibrosis in vivo. Conclusions: KRAS-mutant pancreatic cancer cells can activate PSCs through PAI-1/LRP-1 signaling to promote fibrosis and cancer progression.