Green tea extract selectively targets nanomechanics of live metastatic cancer cells.

Green tea extract (GTE) is known to be a potential anticancer agent (Yang et al 2009 Nat. Rev. Cancer 9 429-39) with various biological activities (Lu et al 2005 Clin. Cancer Res. 11 1675-83; Yang et al 1998 Carcinogenesis 19 611-6) yet the precise mechanism of action is still unclear. The biomechanical response of GTE treated cells taken directly from patient's body samples was measured using atomic force microscopy (AFM) (Binnig et al 1986 Phys. Rev. Lett. 56 930). We found significant increase in stiffness of GTE treated metastatic tumor cells, with a resulting value similar to untreated normal mesothelial cells, whereas mesothelial cell stiffness after GTE treatment is unchanged. Immunofluorescence analysis showed an increase in cytoskeletal-F-actin in GTE treated tumor cells, suggesting GTE treated tumor cells display mechanical, structural and morphological features similar to normal cells, which appears to be mediated by annexin-I expression, as determined by siRNA analysis of an in vitro cell line model. Our data indicates that GTE selectively targets human metastatic cancer cells but not normal mesothelial cells, a finding that is significantly advantageous compared to conventional chemotherapy agents.

Effects of green tea extract on lung cancer A549 cells: proteomic identification of proteins associated with cell migration.

Green tea polyphenols exhibit multiple antitumor activities, and the mechanisms of action are not completely understood. Previously, we reported that green tea extract (GTE)-induced actin remolding is associated with increased cell adhesion and decreased motility in A549 lung cancer cells. To identify the cellular targets responsible for green tea-induced actin remodeling, we performed 2-DE LC-MS/MS of A549 cells before and after GTE exposure. We have identified 14 protein spots that changed in expression (> or =2-fold) after GTE treatment. These proteins are involved in calcium-binding, cytoskeleton and motility, metabolism, detoxification, or gene regulation. In particular we found upregulation of several genes that modulate actin remodeling and cell migration, including lamin A/C. Our data indicated that GTE-induced lamin A/C upregulation appears to be at the transcriptional level and the increased expression results in the decrease in cell motility, as confirmed by siRNA. The result of the study demonstrates that GTE alters the levels of many proteins involved in growth, motility and apoptosis of A549 cells and their identification may explain the multiple antitumor activities of GTE.

Green tea extract modulates actin remodeling via Rho activity in an in vitro multistep carcinogenic model.

Alteration of actin polymerization and loss of actin filaments is a marker of cellular dedifferentiation and early malignant transformation. To study this phenomenon, an in vitro human urothelial model consisting of two cell lines, HUC-PC and MC-T11, were incorporated into the study design. These two cell lines have different malignant transformation potential. The effect of green tea extract (GTE), a potential anticancer agent, on actin remodeling was investigated. Upon exposure to the carcinogen 4-aminobiphenyl (4-ABP), the untransformed HUC-PC undergoes malignant transformation whereas the transformed MC-T11 progresses from noninvasive to invasive tumor. GTE induces actin polymerization in MC-T11 cells in a dose-responsive manner, but this effect is less obvious in the untransformed, more differentiated HUC-PC cells, which natively have higher actin polymerization status. In contrast, GTE antagonizes carcinogen 4-ABP induced actin depolymerization and stress fiber disruption in HUC-PC cells. In MC-T11 cells, GTE inhibits 4-ABP induced motility by increasing cell adhesion and focal adhesion complex formation. The effect of GTE on actin remodeling seems to be mediated by the stimulation of small GTP-binding protein Rho activity, because C3 exoenzyme, a specific inhibitor for Rho, blocks GTE-mediated Rho activation and stress fiber formation in MC-T11 cells. This study shows that GTE exerts an effect on cytoskeletal actin remodeling and provides further support for the use of GTE as a chemopreventive agent.

Ganoderma lucidum extracts inhibit growth and induce actin polymerization in bladder cancer cells in vitro.

This study was conducted to investigate chemopreventive effects of Ganoderma lucidum using a unique in vitro human urothelial cell (HUC) model consisted of HUC-PC cells and MTC-11 cells. Ethanol and water extracts of fruiting bodies and spores of the G. lucidum were used to examine growth inhibition, actin polymerization status, and impact of actin remodeling on cell migration and adhesion. Results showed that ethanol extracts had a stronger growth inhibition effect than water extracts. Cell cycle analysis showed that the growth inhibition effect was associated with G2/M arrest. At non-cytotoxic concentrations (40-80 microg/ml), these extracts induced actin polymerization, which in turn inhibited carcinogen 4-aminobiphenyl induced migration in both cell lines. The increased actin polymerization was associated with increased stress fibers and focal adhesion complex formation, however, expression of matrix metalloproteinase-2 and focal adhesion kinase (total and phospholated) were unchanged, which suggests that other mechanisms may be involved.

Telomerase activity in cervical intraepithelial neoplasia.

BACKGROUND: It was reported that telomerase expression is closely associated with cellular immortality and cancer. This study was designed to investigate the relationship between telomerase expression and the carcinogenesis of cervical cancer, the possible use of telomerase as a marker of cervical intraepithelial neoplasia (CIN) progression or regression, and the natural history of CIN. METHODS: Telomeric repeat amplification protocol (TRAP) assay was used to measure telomerase activity in cervical scrapings and biopsy samples obtained from 105 cases affected with various cervical conditions, including chronic cervicitis (n = 20), CIN (n = 64, 16 cases of CIN I, 20 cases of CIN II, and 28 cases of CIN III), and invasive squamous cell carcinoma (n = 21). RESULTS: In exfoliated cell samples, telomerase activity was detected in 5 of 20 (25.0%) cases of cervicitis, 10 of 16 (62.5%) cases of CIN I, 11 of 20 (55.0%) cases of CIN II, 23 of 28 (82.1%) cases of CIN III, and 13 of 21 (61.9%) cases of carcinoma. In cervical biopsy samples, telomerase activity was detected in 6 of 20 (30.0%) cases of cervicitis, 8 of 16 (50.0%) cases of CIN I, 9 of 20 (45.0%) cases of CIN II, 27 of 28 (96.4%) cases of CIN III, and 20 of 21 (95.2%) cases of carcinoma. Telomerase activation was significantly higher in CIN samples than in cervicitis samples. Telomerase activity was detected at similar frequency in samples from cervical scrapings and cervical biopsies. CONCLUSION: These results seem to suggest that telomerase expression may be associated with carcinogenesis of the cervix. TRAP assay of cervical scraping samples could be used to monitor and predict the development of CIN in clinical practice.

White tea extract induces apoptosis in non-small cell lung cancer cells: the role of peroxisome proliferator-activated receptor-{gamma} and 15-lipoxygenases.

Emerging preclinical data suggests that tea possess anticarcinogenic and antimutagenic properties. We therefore hypothesize that white tea extract (WTE) is capable of favorably modulating apoptosis, a mechanism associated with lung tumorigenesis. We examined the effects of physiologically relevant doses of WTE on the induction of apoptosis in non-small cell lung cancer cell lines A549 (adenocarcinoma) and H520 (squamous cell carcinoma) cells. We further characterized the molecular mechanisms responsible for WTE-induced apoptosis, including the induction of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and the 15-lipoxygenase (15-LOX) signaling pathways. We found that WTE was effective in inducing apoptosis in both A549 and H520 cells, and inhibition of PPAR-gamma with GW9662 partially reversed WTE-induced apoptosis. We further show that WTE increased PPAR-gamma activation and mRNA expression, concomitantly increased 15(S)-hydroxy-eicosatetraenoic acid release, and upregulated 15-LOX-1 and 15-LOX-2 mRNA expression by A549 cells. Inhibition of 15-LOX with nordihydroguaiaretic acid (NGDA), as well as caffeic acid, abrogated WTE-induced PPAR-gamma activation and upregulation of PPAR-gamma mRNA expression in A549 cells. WTE also induced cyclin-dependent kinase inhibitor 1A mRNA expression and activated caspase-3. Inhibition of caspase-3 abrogated WTE-induced apoptosis. Our findings indicate that WTE is capable of inducing apoptosis in non-small cell lung cancer cell lines. The induction of apoptosis seems to be mediated, in part, through the upregulation of the PPAR-gamma and 15-LOX signaling pathways, with enhanced activation of caspase-3. Our findings support the future investigation of WTE as an antineoplastic and chemopreventive agent for lung cancer.

AFM-based analysis of human metastatic cancer cells.

Recently biomechanics of cancer cells, in particular stiffness or elasticity, has been identified as an important factor relating to cancer cell function, adherence, motility, transformation and invasion. We report on the nanomechanical responses of metastatic cancer cells and benign mesothelial cells taken from human body cavity fluids using atomic force microscopy. Following our initial study (Cross et al 2007 Nat. Nanotechnol. 2 780-3), we report on the biophysical properties of patient-derived effusion cells and address the influence of cell morphology on measured cell stiffness. Using a cytocentrifugation method, which yields morphologically indistinguishable cells that can be prepared in 1 min and avoids any possible artifacts due to 12 h ex vivo culture, we find that metastatic tumor cells are more than 80% softer than benign cells with a distribution over six times narrower than that of normal cells. Consistent with our previous study, which yielded distinguishable cell populations based on ex vivo growth and morphological characteristics, our results show it is unlikely that morphology alone is sufficient to explain the difference in elastic moduli for these two cell types. Moreover, analysis of non-specific cell adhesion inherent to tumor and normal cells collected from patients show surface adhesion of tumor cells is ∼33% less adhesive compared to that of normal cells. Our findings indicate that biomechanical-based functional analysis may provide an additional platform for cytological evaluation and diagnosis of cancer in the future.

Nanomechanical analysis of cells from cancer patients.

Change in cell stiffness is a new characteristic of cancer cells that affects the way they spread. Despite several studies on architectural changes in cultured cell lines, no ex vivo mechanical analyses of cancer cells obtained from patients have been reported. Using atomic force microscopy, we report the stiffness of live metastatic cancer cells taken from the body (pleural) fluids of patients with suspected lung, breast and pancreas cancer. Within the same sample, we find that the cell stiffness of metastatic cancer cells is more than 70% softer, with a standard deviation over five times narrower, than the benign cells that line the body cavity. Different cancer types were found to display a common stiffness. Our work shows that mechanical analysis can distinguish cancerous cells from normal ones even when they show similar shapes. These results show that nanomechanical analysis correlates well with immunohistochemical testing currently used for detecting cancer.

Annexin-I as a potential target for green tea extract induced actin remodeling.

Using a multistep human urothelial model, we previously showed that green tea extract (GTE) selectively modulates actin remodeling in transformed cells (MC-T11), which resulted in increased cell adhesion and reduced cell motility (Lu et al., Clin Cancer Res 2005;11:1675-83). This study further analyzed which actin binding proteins (ABPs) might be involved in this process. Proteomic profiles of GTE treated and untreated MC-T11 cells using two-dimensional gel electrophoresis coupled with liquid chromatography tandem mass spectrometry (LC/MS/MS) and matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) identified 20 GTE-induced proteins. Among them, 3 were ABPs (tropomodulin, cofilin and annexin-I), and only annexin-I showed a dose- and time-dependent expression. The increased annexin-I correlated with actin remodeling, and was the result of transcription level up-regulation, as determined by RT-PCR, pull-down immunoblot and siRNA analyses. 5-Azacytidine, a DNA methylation inhibitor, exhibited no effect on annexin-I expression when used alone, but had an additive effect for GTE-induced annexin-I expression. Immunohistochemistry of bladder cancer tissue array showed a decrease of annexin-I expression in carcinoma in situ and low grade papillary carcinoma (n = 32, 0% positive) compared to nontumor urothelium (n = 18, 89% positive) (p < 0.001 by Fisher exact test), but increased in some (6 of 15, 40%) high-grade tumors. Together, GTE induced annexin-I expression plays a role in regulating actin remodeling and decreased annexin-I expression is a common event in early stage of bladder cancer development.

Green tea induces annexin-I expression in human lung adenocarcinoma A549 cells: involvement of annexin-I in actin remodeling.

Green tea polyphenols exhibit multiple antitumor activities in various in vitro and in vivo tumor models, and the mechanisms of action are not clear. Previously, we found that green tea extract (GTE) regulates actin remodeling in different cell culture systems. Actin remodeling plays an important role in cancer cell morphology, cell adhesion, motility, and invasion. Using proteomic approaches, we found GTE-induced expression of annexin-I, a multifunctional actin binding protein, in these cell lines. In this study, we aimed to further define the functional role of GTE-induced annexin-I expression in actin remodeling, cell adhesion, and motility in lung adenocarcinoma A549 cells. We found that GTE stimulates the expression of annexin-I in a dose-dependent fashion. The GTE-induced annexin-I expression appears to be at the transcription level, and the increased annexin-I expression mediates actin polymerization, resulting in enhanced cell adhesion and decreased motility. Annexin-I specific interference resulted in loss of GTE-induced actin polymerization and cell adhesion, but not motility. In fact, annexin-I specific interference itself inhibited motility even without GTE. Together, annexin-I plays an important role in GTE-induced actin remodeling, and it may serve as a potential molecular target associated with the anticancer activities of green tea.