Clarification of the molecular pathway of Taiwan local pomegranate fruit juice underlying the inhibition of urinary bladder urothelial carcinoma cell by proteomics strategy.

BACKGROUND: Pomegranate fruit has been shown to exhibit the inhibitory activity against prostate cancer and lung cancer in vitro and in vivo, which might be a resource for chemoprevention and chemotherapy of cancer. Our previous documented findings indicated that treatment of urinary bladder urothelial carcinoma cell with the ethanol extract isolated from the juice of pomegranate fruit grown in Taiwan could inhibit tumor cell. In this study we intended to uncover the molecular pathway underlying anti-cancer efficacy of Taiwan pomegranate fruit juice against urinary bladder urothelial carcinoma. METHODS: We exploited two-dimensional gel electrophoresis coupled with tandem mass spectrometry to find the de-regulated proteins. Western immunoblotting was used to confirm the results collected from proteomics study. RESULTS: Comparative proteomics indicated that 20 proteins were differentially expressed in ethanol extract-treated T24 cells with 19 up-regulated and 1 down-regulated proteins. These de-regulated proteins were involved in apoptosis, cytoskeleton regulation, cell proliferation, proteasome activity and aerobic glycolysis. Further studies on signaling pathway demonstrated that ethanol extract treatment might inhibit urinary bladder urothelial carcinoma cell proliferation through restriction of PTEN/AKT/mTORC1 pathway via profilin 1 up-regulation. It also might evoke cell apoptosis through Diablo over-expression. CONCLUSIONS: The results of this study provide a global picture to further investigate the anticancer molecular mechanism of pomegranate fruit.

Suppression of urinary bladder urothelial carcinoma cell by the ethanol extract of pomegranate fruit through cell cycle arrest and apoptosis.

BACKGROUND: Pomegranate possesses many medicinal properties such as antioxidant, anti-inflammation and antitumor. It has been extensively used as a folk medicine by many cultures. Pomegranate fruit has been shown to have the inhibitory efficacy against prostate cancer and lung cancer in vitro and in vivo. It can be exploited in chemoprevention and chemotherapy of prostate cancer. In this study we examined the anti-cancer efficacy of pomegranate fruit grown in Taiwan against urinary bladder urothelial carcinoma (UBUC) and its mechanism of action. METHODS: Edible portion of Taiwanese pomegranate was extracted using ethanol and the anti-cancer effectiveness of ethanol extract was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Flow cytometry and western immunoblotting were exploited to uncover the molecular pathways underlying anti-UBUC activity of Taiwanese pomegranate ethanol extract. RESULTS: This study demonstrated that Taiwanese pomegranate fruit ethanol extract (PEE) could effectively restrict the proliferation of UBUC T24 and J82 cells. Cell cycle analyses indicated that the S phase arrest induced by PEE treatment might be caused by an increase in cyclin A protein level and a decrease in the expression of cyclin-dependent kinase 1. The results of western immunoblotting demonstrated that PEE treatment could not only evoke the activation of pro-caspase-3, -8,-9 but also increase Bax/Bcl-2 ratio in T24 cells. The above observations implicated that PEE administration might trigger the apoptosis in T24 cells through death receptor signaling and mitochondrial damage pathway. Besides we found that PEE exposure to T24 cells could provoke intensive activation of procaspase-12 and enhance the expressions of CHOP and Bip, endoplasmic reticulum (ER) stress marker, suggesting that ER stress might be the cardinal apoptotic mechanism of PEE-induced inhibition of bladder cancer cell. CONCLUSIONS: The analytical results of this study help to provide insight into the molecular mechanism of induced bladder cancer cell apoptosis by pomegranate and to develop novel mechanism-based chemopreventive strategy for bladder cancer.

Search for the tumor-associated proteins of oral squamous cell carcinoma collected in Taiwan using proteomics strategy.

Squamous cell carcinoma (SCC) accounts for more than 90% of malignant tumors of the oral cavity. In Taiwan, oral squamous cell carcinoma (OSCC) is among the most frequent malignancies, largely due to betal quid chewing. Despite the recent improvement in treatment results, the long-term outcome of OSCC generally remains poor, especially for those with advanced diseases. It is therefore desirable to identify potential biomarkers that may aid in risk stratification and perhaps the development of therapeutic targets. In this study, we exploited two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry to compare the proteome maps of 10 OSCC specimens with their adjacent nontumorous epithelia to identify differentially expressed proteins. Comparative proteomics indicated that 17 proteins were differentially expressed in OSCC with 11 up-regulated and 6 down-regulated proteins. These deregulated proteins participated in cytoskeletal functions, cell signaling, antiapoptosis, angiogenesis, lipid metabolism, drug metabolism, and protein translation/turnover. They were all associated with tumor development in various cancers. Among the dys-regulated proteins, the immunoexpression of three proteins including nicotinamide N-methyltransferase, apolipoprotein AI, and 14-3-3 zeta were evaluated in 38 OSCCs of testing cohort to confirm the proteomics data. Subsequently, the expression of 14-3-3 zeta, as the most relevant to OSCC progression determined by testing cohort, was further assessed in 80 OSCCs of independent validation cohort to identify the clinical relevance of its expression. By this comprehensive study, we identified 14-3-3 zeta as the only prognosticator of local recurrence-free survival (LRFS) and also an independently predicted factor of disease-specific survival (DSS).

Annexin-I overexpression is associated with tumour progression and independently predicts inferior disease-specific and metastasis-free survival in urinary bladder urothelial carcinoma.

AIMS: In our previous studies, comparative proteomics and immunohistochemistry (IHC) demonstrated that annexin-I (ANXA1) is up-regulated in high grade urinary bladder urothelial carcinoma (UBUC) as compared to non-high grade carcinomas. However, the small sample size prohibited further correlation of ANXA1 expression to tumour progression. Therefore, in the present study, 81 primary localised UBUC specimens of various grades and primary tumour (pT) status were examined for ANXA1 expression to further confirm the proteomics data and to clarify the relevance of ANXA1 expression level to the prognosis of UBUC. METHODS: IHC was implemented to investigate ANXA1 protein expression in 81 primary localised UBUC specimens. The association of ANXA1 expression with tumour progression and prognosis was analysed. RESULTS: Our data demonstrated that the ANXA1 expression level was strongly associated with an escalated pT status (p < 0.001) and a higher histological grade (p < 0.001), suggesting that ANXA1 might be related to tumour progression. Moreover, at the univariate level, ANXA1 overexpression, along with higher pT status and histological grade, significantly predicted disease-specific survival (DSS) and metastasis-free survival (MFS). More importantly, multivariate analyses revealed that the association of ANXA1 overexpression and prognosis remained significant for both DSS and MFS. CONCLUSION: The above results reinforced the comparative proteomics results and confirmed the prognostic role of ANXA1 in UBUC.