Background
Bladder cancer is the most prevalent tumor of the urinary tract worldwide and ranked the 15
th in cancer mortality rate in 2011 in Taiwan [
1,
2]. Urothelial carcinoma is the most common and constitutes more than 90 % of bladder cancer cases in developed countries [
3]. According to WHO/ISUP classification (2004), urinary bladder urothelial carcinoma (UBUC) cell can be classified into low and high grade while high grade UBUC cell is less differentiated. Although most of UBUCs are papillary/non-invasive or superficially invasive types and can often be cured by curettage, some UBUCs can still develop relentless local recurrence followed by lethal distal spreading [
4].
Pomegranate (
Punicagranatum, Punicaceae), is an edible fruit cultivated in Mediterranean countries, India and the United States etc., comprising edible portions of 80 % juice and 20 % seeds. Pomegranate contains crude fibers, pectin, sugars, tannins (mainly ellagitannins), flavonoids and anthocyanins. Among those nutritious ingredients, anthocyanins is believed to have provided the fruit with potent antioxidant ability [
5].
Many literatures have showed that pomegranate fruit displays anti-cancer effectiveness. Ellagitannin-rich pomegranate fruit extract (PFE) purified from pomegranate edible portion with 70 % acetone was found to have the apoptotic effects on human lung cancer A549 cells through the down-regulation of cell cycle-regulatory proteins operative in the G1 stage and inhibiting NF-κB as well as MAP kinase pathways [p38, phosphoinositide 3 kinase (PI3K)/ protein kinase B (Akt), c-jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (Erk)] [
6]. In the animal model of primary lung tumor, PFE also diminishes tumor growth/progression/angiogenesis by the suppression of NF-κB, MAP kinase pathways and mammalian target of rapamycin (mTOR) signaling [
7]. In addition to the impacts on lung cancer, PFE may be a potential source for chemoprevention of prostate cancer [
8]. The investigations on prostate cancer found that pomegranate polyphenols, ellagitannin-rich extract (PE) prepared from fruit skins can retard prostate cancer likely caused by chronic inflammation via suppressing the NF-κB pathway [
9]. PE was also observed to down-regulate the angiogenesis in prostate cancer through down-regulation of hypoxia-inducible factor 1-α (HIF-1α) which transcriptionally regulates the vascular endothelial growth factor (VEGF) expression [
10]. Clinical trial with rising prostate-specific antigen (PSA) after surgery or radiotherapy indicated that after daily treatment with 8 oz of pomegranate juice a significant increase in mean PSA doubling time from 15 months at baseline to 54 months post-treatment [
11]. Our previous documented results showed that pomegranate juice could evoke prostate cancer cell apoptosis via mitochondrial pathway and death receptor signaling. It also can interfere with the expression levels of genes involved in cytoskeletal functions, anti-apoptosis, metabolism, NF-B signaling in juice-treated prostate cancer cell [
12].
Based upon the aforementioned documented findings, pomegranate may be a potential chemopreventive resource against UBUC development and recurrence.
Our previous documented findings indicated that treatment of the ethanol extract (PEE) of pomegranate fruit juice could inhibit UBUC cell via cell cycle arrest and cell apoptosis [
13]. Thus in this study we exploited two-dimensional gel electrophoresis (2-DE) coupled with tandem mass spectrometry to decipher the molecular mechanism underlying the cancer intervention of PEE. We found that PEE treatment could inhibit UBUC cell proliferation and migration. PEE-induced de-regulated proteins were associated with apoptosis, cytoskeleton regulation, AKT/mTOR signaling, proteasome activity and aerobic glycolysis. These de-regulated proteins might contribute to PEE-evoked inhibition of UBUC cell proliferation and cell apoptosis.
Discussion
Our previous studies have shown that PEE treatment could inhibit UBUC via evoking cell apoptosis [
12]. In this study, we further found that PEE treatment could restrict UBUC cell proliferation and migration. To investigate the specific proteins affected by PEE incubation in UBUC cells, gel-based proteomics was carried out to shed light on the molecular mechanism underlying cancer intervention by PEE exposure through provoking cell apoptosis and inhibiting cell proliferation/migration. In this research, 20 differentially expressed proteins were found upon treatment of PEE to T24 cells with 19 up-regulated and 1 down-regulated proteins respectively.
Among de-regulated proteins, PHPT1 protein plays a role in lung cancer cell migration/invasion and is revealed to be associated with cytoskeleton reorganization [
20]. Profilin 1 and Cofilin are the members of a family of actin-binding proteins, which participate in dynamic turnover and restructuring of actin cytoskeleton [
15,
21]. Transgelin-2 is a cytoskeletal protein with actin-binding activity shown to be a tumor suppressor in colorectal carcinoma [
22]. It has been postulated that cytoskeleton remodeling plays a pivotal role in cancer cell migration and also in regulating the morphologic and phenotypic events of a malignant cell. Besides, apoptosis is generally preceded by the pronounced changes of actin cytoskeleton [
23]. Consistent with the above observations, results of this examination suggested that PEE treatment might provoke the rearrangement of cytoskeleton structure of UBUC cells through disturbing the expressions of cytoskeletal components and thus retard UBUC cell proliferation/migration. This study provided a clue for more investigation of the impacts of PEE on cytoskeleton structure in UBUC cell.
Previous documented findings demonstrated that augmented profilin 1 synthesis can increase PTEN gene expression in breast cancer cell [
15]. PTEN protein serves as a phosphatase to dephosphorylate PIP
3 to become PIP
2. This dephosphorylation results in inhibition of Akt protein activity and thus Akt signaling pathway which plays a central role in protein synthesis, metabolism and cell proliferation [
16]. Akt can phosphorylate tuberin/TSC2 to prevent the inhibition of mTORC1 complex (mTOR-raptor complex). mTORC1 integrates multiple signals to promote either cellular growth in favorable conditions or catabolic processes in unfavorable conditions while mTORC2 (mTOR-rictor complex) is related to actin organization. Many documented evidences indicated that impaired PTEN/Akt/mTOR signaling pathway plays a key role in tumorigenesis in many tumors [
24]. In accordance with the aforementioned findings, our present data suggested that PEE treatment increased profilin 1 expression to up-regulate PTEN gene expression, which might in turn inhibited Akt/mTORC1 signaling pathway to prevent UBUC cell proliferation/migration.
The abnormal proteasomal activity contributes to tumorigenesis by offering cancer cell with anti-apoptotic protection and a survival advantage [
25]. Our findings implicated that PEE treatment could alter the expressions of several genes associated with 26S proteasome activity (PSMD9, UBQLN1, PSMF1) in UBUC cells to disturb cell proliferation. PSMF1/PI31 can bind to 20S catalytic particle of 26S proteasome to hinder substrate access to the enzymatic core and thus results in the inhibition of proteasomal activity [
26]. PSMD9 is a proteasomal assembly chaperone [
27]. Ubiquilin-1 is thought to functionally link the ubiquitination machinery to the proteasome to effect in vivo protein degradation [
28]. Suppression of proteasomal activity may prevent degradation of IκB (endogenous inhibitor of NF-κB) and subsequent activation/translocation of NFκB to the nucleus to activate downstream pathways [
29]. NFκB plays an important role in tumorigenesis of many tumors by promoting cell proliferation, migration and suppression of apoptosis [
18]. Our study indicated that PEE treatment could inhibit NFκB activation possibly through evoking PSMF1/PI31 over-expression to prevent the proteasomal degradation of IκB.
Among PEE-induced de-regulated proteins, BCL10, diablo, peflin, TPD52L2, eIF5A and BAG2 are shown to be involved in regulating cell apoptosis. Several findings suggested that BCL10 is an apoptotic regulatory protein and participates in Apaf1/caspase 9-mediated cell death pathway [
30]. eIF5A is the only known protein to be regulated by the post-translational formation of a hypusine residue. Recent studies have indicated that unhypusinated eIF5A is pro-apoptotic and only observed during apoptosis [
31]. Our proteomics data showed that PEE incubation could increase eIF5A gene expression but further investigation was required to determine whether unhypusinated eIF5A level was increased in PEE-treated T24 cell. TPD52L2 can interact with TPD52L1 protein which positively regulates apoptosis signal-regulating kinase 1 (ASK1)-induced apoptosis [
32]. Peflin can regulate the activity of apoptosis-linked gene 2 (ALG-2) which associates with Fas-executed apoptosis [
33]. In the apoptotic pathway, the caspapse activities can be inhibited directly by inhibitor of apoptosis (IAP) protein [
19]. During the mitochondrial apoptotic process, the inhibitory function of XIAP, a ubiquitous member of IAP family, can be antagonized by Smac/Diablo and Omi/HtrA2 which are also released from mitochondria along with cytochrome c. BAG2 exhibits pro-apoptotic properties and is demonstrated to be up-regulated in proteasome inhibitor-induced apoptosis in thyroid carcinoma cell [
34]. Our present results demonstrated that PEE exposure could increase the aforementioned apoptotic proteins to provoke UBUC cell apoptosis.
Some of dys-regulated proteins evoked by PEE treatment might attribute to the mitochondrial damage and nuclear change (nuclear fragmentation, chromatin condensation and DNA fragmentation) which are the characteristics of cell apoptosis. Translin/TRAX protein complex (C3PO) plays roles in very important key cellular processes such as cell growth regulation, genome stability regulation and carcinogenesis [
35]. NAUFAF1 is a chaperone protein involved in the assembly of the mitochondrial NADH:ubiquinone oxidoreductase complex (complex I) which transfers the electron from NADH to ubiquinone (coenzyme Q) in the first step of the mitochondrial respiratory chain [
36]. Cancer cells proliferate very rapidly and rely on high metabolic activities. To meet high energy demand, tumor cells exploit aerobic glycolysis to acquire the energy from glucose (Warburg effect). During glycolysis only one of two triosephosphates formed by aldolase-glyceraldehyde-3-phosphate-is degraded in the subsequent steps. The other product, dihydroxyacetone phosphate, is rapidly and reversibly converted to glyceraldehyde-3-phosphate by TPI1 protein [
37].
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
BXT carried out cell proliferation/migration assay and western immunoblotting studies for signaling pathway in T24 cell and prepared PEE. LTH conducted 2-DE and MTT assay for T24 cell. LHC performed western immunoblotting experiments for confirmation of proteomics data. TFW contributed to the conception and design of entire study, protein identification, data interpretation and the initial draft/final editing of the manuscript. Li-Yi Chen carried out MTT assay for TSGH8301 cell. Wan-Yin Shih performed western immunoblotting studies for signaling pathway in TSGH8301 cell. All authors have read and approved the manuscript for publication.