An 19F NMR fragment-based approach for the discovery and development of BRCA2-RAD51 inhibitors to pursuit synthetic lethality in combination with PARP inhibition in pancreatic cancer.

The BRCA2-RAD51 interaction remains an intriguing target for cancer drug discovery due to its vital role in DNA damage repair mechanisms, which cancer cells become particularly reliant on. Moreover, RAD51 has many synthetically lethal partners, including PARP1-2, which can be exploited to induce synthetic lethality in cancer. In this study, we established a 19F-NMR-fragment based approach to identify RAD51 binders, leading to two initial hits. A subsequent SAR program identified 46 as a low micromolar inhibitor of the BRCA2-RAD51 interaction. 46 was tested in different pancreatic cancer cell lines, to evaluate its ability to inhibit the homologous recombination DNA repair pathway, mediated by BRCA2-RAD51 and trigger synthetic lethality in combination with the PARP inhibitor talazoparib, through the induction of apoptosis. Moreover, we further analyzed the 46/talazoparib combination in 3D pancreatic cancer models. Overall, 46 showed its potential as a tool to evaluate the RAD51/PARP1-2 synthetic lethality mechanism, along with providing a prospect for further inhibitors development.

Preomic profile of BxPC-3 cells after treatment with BRC4.

BRCA2 and RAD51 are two proteins that play a central role in homologous recombination (HR) and DNA double strand break (DSB) repair. BRCA2 assists RAD51 fibrillation and defibrillation through binding with its eight BRC repeats, with BRC4 being one of the most efficient and best characterized. RAD51 inactivation by small molecules has been proposed as a strategy to impair BRCA2/RAD51 binding and, ultimately, the HR pathway, with the aim of making cancer cells more sensitive to PARP inhibitors (PARPi). This strategy, which mimics a synthetic lethality (SL) approach, has been successfully performed in vitro by using the myristoylated derivative of BRC4 (myr-BRC4), designed for a more efficient cell entry. The present study applies a method to obtain a proteomic fingerprint after cellular treatment with the myr-BRC4 peptide using a mass spectroscopy (MS) proteomic approach. (Data are available via ProteomeXchange with identifier PXD042696.) We performed a comparative proteomic profiling of the myr-BRC4 treated vs. untreated BxPC-3 pancreatic cancer cells and evaluated the differential expression of proteins. Among the identified proteins, we focused our attention on proteins shared by both the RAD51 and the BRCA2 interactomes, and on those whose reduction showed high statistical significance. Three downregulated proteins were identified (FANCI, FANCD2, and RPA3), and protein downregulation was confirmed through immunoblotting analysis, validating the MS approach. Our results suggest that, being a direct consequence of myr-BRC4 treatment, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring HR impairment. SIGNIFICANCE: RAD51's inhibition has gained increasing attention because of its possible implications in personalized medicine through the SL approach. Chemical disruption of protein-protein interactions (PPIs) between RAD51 and BRCA2, or some of its partner proteins, could potentiate PARPi DNA damage-induced cell death. This could have application for difficult to treat cancers, such as BRCA-competent and olaparib (PARPi) resistant pancreatic adenocarcinoma. Despite RAD51 being a widely studied target, researchers still lack detailed mechanistic information. This has stifled progress in the field with only a few RAD51 inhibitors having been identified, none of which have gained regulatory approval. Nevertheless, the peptide BRC4 is one of the most specific and best characterized RAD51 binder and inhibitor reported to date. Our study is the first to report the proteomic fingerprint consequent to cellular treatment of myr-BRC4, to offer a reference for the discovery of specific protein/pathway alterations within DNA damage repair. Our results suggest that, being a direct consequence of myr-BRC4 treatment, and ultimately ofBRCA2/RAD51 disruption, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring DNA damage repair impairment and therefore be used to potentiate the development of new effective therapeutic strategies.

Epigallocatechin-3-gallate Delivered in Nanoparticles Increases Cytotoxicity in Three Breast Carcinoma Cell Lines.

The anticancer activity of epigallocatechin-3-gallate (EGCG), orally administrated, is limited by poor bioavailability, absorption, and unpredictable distribution in human tissues. EGCG charged nanoparticles may represent an opportunity to overcome these limitations. We assayed two different kinds of lipid nanoparticles (LNPs and LNPs functionalized with folic acid) charged with EGCG on three breast carcinoma cell lines (MCF-7, MDA-MB-231, and MCF-7TAM) and the human normal MCF10A mammary epithelial cells. Both LNPs loaded with EGCG, at low concentrations, induced a significant cytotoxicity in the three breast carcinoma cells but not in MCF10A cells. In view of a future application, both LNPs and LNPs-FA were found to be very suitable for in vitro studies and useful to improve EGCG administration in vivo. Since they are produced by inexpensive procedures using bioavailable, biocompatible, and biodegradable molecules, they represent an applicable tool for a more rationale use of EGCG as an anti-cancer agent.

The Mechanistic Understanding of RAD51 Defibrillation: A Critical Step in BRCA2-Mediated DNA Repair by Homologous Recombination.

The cytotoxic action of anticancer drugs can be potentiated by inhibiting DNA repair mechanisms. RAD51 is a crucial protein for genomic stability due to its critical role in the homologous recombination (HR) pathway. BRCA2 assists RAD51 fibrillation and defibrillation in the cytoplasm and nucleus and assists its nuclear transport. BRC4 is a peptide derived from the fourth BRC repeat of BRCA2, and it lacks the nuclear localization sequence. Here, we used BRC4 to (i) reverse RAD51 fibrillation; (ii) avoid the nuclear transport of RAD51; and (iii) inhibit HR and enhance the efficacy of chemotherapeutic treatments. Specifically, using static and dynamic light scattering, transmission electron microscopy, and microscale thermophoresis, we show that BRC4 eroded RAD51 fibrils from their termini through a "domino" mechanism and yielded monomeric RAD51 with a cumulative nanomolar affinity. Using cellular assays (BxPC-3, pancreatic cancer), we show that a myristoylated BRC4 (designed for a more efficient cell entry) abolished the formation of nuclear RAD51 foci. The present study provides a molecular description of RAD51 defibrillation, an essential step in BRCA2-mediated homologous recombination and DNA repair.

Lactate is a potential promoter of tamoxifen resistance in MCF7 cells.

BACKGROUND: Tamoxifen is a widely used estrogen receptor inhibitor, whose clinical success is limited by the development of acquired resistance. This compound was also found to inhibit mitochondrial function, causing increased glycolysis and lactate production. Lactate has been widely recognized as a signaling molecule, showing the potential of modifying gene expression. These metabolic effects of tamoxifen can by hypothesized to contribute in driving drug resistance. METHODS: To test this hypothesis, we used MCF7 cells together with a tamoxifen resistant cell line (MCF7-TAM). Experiments were aimed at verifying whether enhanced lactate exposure can affect the phenotype of MCF7 cells, conferring them features mirroring those observed in the tamoxifen resistant culture. RESULTS: The obtained results suggested that enhanced lactate in MCF7 cells medium can increase the expression of tafazzin (TAZ) and telomerase complex (TERC, TERT) genes, reducing the cells' attitude to undergo senescence. In long term lactate-exposed cells, signs of EGFR activation, a pathway related to acquired tamoxifen resistance, was also observed. CONCLUSIONS: The obtained results suggested lactate as a potential promoter of tamoxifen resistance. The off-target effects of this compound could play a role in hindering its therapeutic efficacy. GENERAL SIGNIFICANCE: The features of acquired tamoxifen resistance have been widely characterized at the molecular level; in spite of their heterogeneity, poorly responsive cells were often found to display upregulated glycolysis. Our results suggest that this metabolic asset is not simply a result of neoplastic progression, but can play an active part in driving this process.

Epigallocatechin-3-Gallate (EGCG) Mitigates Endothelial and Circulating Cells Alterations Following PLLA Electrospun Mat Placement.

BACKGROUND: Synthetic vascular graft calcification is a serious complication of graft placement. Here, we analysed migration and osteogenic genes of human umbilical vein endothelial cells (HUVEC) cultured with a poly-L-lactic acid (PLLA) electrospun mat. The role of epigallo-catechin-3-gallate (EGCG) in pathogenic processes involving HUVEC and peripheral blood mononuclear cells (PBMCs) was also tested. METHODS: HUVEC were cultured in indirect contact with PLLA for 48 h, with or without EGCG, and processed for mRNA expression. HUVEC proliferation, migration and osteogenic differentiation were evaluated after EGCG treatment. EGCG was also administrated to human PBMCs, to analyse proliferation and migration toward HUVEC cultured with PLLA. RESULTS: HUVEC cultured with PLLA exhibited increased expression of SLUG, VIMENTIN, MMP-9 (migration, vascular remodelling) and RUNX-2 (osteogenic transcription factor). EGCG at 25 µM significantly reduced HUVEC migration, osteogenic differentiation, without affecting cell viability, and mitigated PLLA influence on SLUG, MMP-9, VIMENTIN and RUNX-2 expression. EGCG affected PBMC proliferation and migration toward PLLA in a transwell co-culture system with HUVEC. CONCLUSION: Our study suggests the pro-calcific effect of PLLA, proposing EGCG as an anti-inflammatory modulatory approach. Research efforts need to deepen PLLA-vascular wall interactions for preventing vascular graft failure.

Epigallocatechin-3-Gallate Delivery in Lipid-Based Nanoparticles: Potentiality and Perspectives for Future Applications in Cancer Chemoprevention and Therapy.

Chemoprevention is a strategy aimed to not only reduce the risk but also delay the development or recurrence of cancer. An ideal chemopreventive agent is not dangerous and ought not to result in side effects or damage to human health. In this context, epigallocatechin-3-gallate (EGCG) is considered a suitable chemopreventive agent, but its clinical use is limited by many factors, namely, the difference in source, administration, individual metabolism, absorption, and distribution. Genetic and dietary differences greatly cause this variability, which has limited the rational use of EGCG in chemoprevention and, particularly, the definition of a safe and efficient concentration. In the present mini review, the main limitations to a complete understanding of the use of EGCG as a chemopreventive agent will be briefly illustrated. This review also indicates the introduction and trialing of lipid-based nanoparticles (NPs) as a proper strategy to deliver EGCG at a well-defined concentration for better investigation of the chemopreventive activity. Finally, some examples of cancers that might benefit from EGCG treatment in different stages of the disease are proposed.

Molecules Present in Plant Essential Oils for Prevention and Treatment of Colorectal Cancer (CRC).

Essential oils (EOs) are a complex mixture of hydrophobic and volatile compounds synthesized from aromatic plants, commonly present in the human diet. In recent years, many in vitro studies have suggested possible anticancer properties of single EO compounds, on colorectal cancer (CRC) cells. However, the majority of these studies did not compare the effects of these compounds on normal and cancer colon cells. By using NCM-460, a normal human mucosal epithelial cell line, Caco-2, a human colon epithelial adenocarcinoma cell line, and SW-620, colon cancer cells derived from lymph node metastatic site, we identified cinnamaldehyde, derived from cinnamon EO and eugenol, derived from bud clove EO, as compounds with a specific anticancer action selectively targeting the transformed colonic cells. Both cinnamaldehyde (75 µM) and eugenol (800 µM), after 72 h of treatment, were capable to induce apoptosis, necrosis and a cell cycle slowdown in Caco-2 and in SW-620, but not in NCM-460 cells. If associated with a targeted delivery to the colon, these two compounds could prove effective in the prevention or treatment of CRC.

EGCG Mediated Targeting of Deregulated Signaling Pathways and Non-Coding RNAs in Different Cancers: Focus on JAK/STAT, Wnt/ß-Catenin, TGF/SMAD, NOTCH, SHH/GLI, and TRAIL Mediated Signaling Pathways.

Decades of research have enabled us to develop a better and sharper understanding of multifaceted nature of cancer. Next-generation sequencing technologies have leveraged our existing knowledge related to intra- and inter-tumor heterogeneity to the next level. Functional genomics have opened new horizons to explore deregulated signaling pathways in different cancers. Therapeutic targeting of deregulated oncogenic signaling cascades by products obtained from natural sources has shown promising results. Epigallocatechin-3-gallate (EGCG) has emerged as a distinguished chemopreventive product because of its ability to regulate a myriad of oncogenic signaling pathways. Based on its scientifically approved anticancer activity and encouraging results obtained from preclinical trials, it is also being tested in various phases of clinical trials. A series of clinical trials associated with green tea extracts and EGCG are providing clues about significant potential of EGCG to mechanistically modulate wide ranging signal transduction cascades. In this review, we comprehensively analyzed regulation of JAK/STAT, Wnt/ß-catenin, TGF/SMAD, SHH/GLI, NOTCH pathways by EGCG. We also discussed most recent evidence related to the ability of EGCG to modulate non-coding RNAs in different cancers. Methylation of the genome is also a widely studied mechanism and EGCG has been shown to modulate DNA methyltransferases (DNMTs) and protein enhancer of zeste-2 (EZH2) in multiple cancers. Moreover, the use of nanoformulations to increase the bioavailability and thus efficacy of EGCG will be also addressed. Better understanding of the pleiotropic abilities of EGCG to modulate intracellular pathways along with the development of effective EGCG delivery vehicles will be helpful in getting a step closer to individualized medicines.

Synthetic Lethality in Pancreatic Cancer: Discovery of a New RAD51-BRCA2 Small Molecule Disruptor That Inhibits Homologous Recombination and Synergizes with Olaparib.

Synthetic lethality is an innovative framework for discovering novel anticancer drug candidates. One example is the use of PARP inhibitors (PARPi) in oncology patients with BRCA mutations. Here, we exploit a new paradigm based on the possibility of triggering synthetic lethality using only small organic molecules (dubbed "fully small-molecule-induced synthetic lethality"). We exploited this paradigm to target pancreatic cancer, one of the major unmet needs in oncology. We discovered a dihydroquinolone pyrazoline-based molecule (35d) that disrupts the RAD51-BRCA2 protein-protein interaction, thus mimicking the effect of BRCA2 mutation. 35d inhibits the homologous recombination in a human pancreatic adenocarcinoma cell line. In addition, it synergizes with olaparib (a PARPi) to trigger synthetic lethality. This strategy aims to widen the use of PARPi in BRCA-competent and olaparib-resistant cancers, making fully small-molecule-induced synthetic lethality an innovative approach toward unmet oncological needs.

Rad51/BRCA2 disruptors inhibit homologous recombination and synergize with olaparib in pancreatic cancer cells.

Olaparib is a PARP inhibitor (PARPi). For patients bearing BRCA1 or BRCA2 mutations, olaparib is approved to treat ovarian cancer and in clinical trials to treat breast and pancreatic cancers. In BRCA2-defective patients, PARPi inhibits DNA single-strand break repair, while BRCA2 mutations hamper double-strand break repair. Recently, we identified a series of triazole derivatives that mimic BRCA2 mutations by disrupting the Rad51-BRCA2 interaction and thus double-strand break repair. Here, we have computationally designed, synthesized, and tested over 40 novel derivatives. Additionally, we designed and conducted novel biological assays to characterize how they disrupt the Rad51-BRCA2 interaction and inhibit double-strand break repair. These compounds synergized with olaparib to target pancreatic cancer cells with functional BRCA2. This supports the idea that small organic molecules can mimic genetic mutations to improve the profile of anticancer drugs for precision medicine. Moreover, this paradigm could be exploited in other genetic pathways to discover innovative anticancer targets and drug candidates.

Epigallocatechin-3-gallate and 6-OH-11-O-Hydroxyphenanthrene Limit BE(2)-C Neuroblastoma Cell Growth and Neurosphere Formation In Vitro.

We conducted an in vitro study combining a rexinoid, 6-OH-11-O-hydroxyphenanthrene (IIF), and epigallocatechin-3-gallate (EGCG), which is the main catechin of green tea, on BE(2)-C, a neuroblastoma cell line representative of the high-risk group of patients. Neuroblastoma is the most common malignancy of childhood: high-risk patients, having N-MYC over-expression, undergo aggressive therapy and show high mortality or an increased risk of secondary malignancies. Retinoids are used in neuroblastoma therapy with incomplete success: the association of a second molecule might improve the efficacy. BE(2)-C cells were treated by EGCG and IIF, individually or in combination: cell viability, as evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, was reduced, EGCG+IIF being the most effective treatment. Apoptosis occurred and the EGCG+IIF treatment decreased N-MYC protein expression and molecular markers of invasion (MMP-2, MMP-9 and COX-2). Zymography demonstrated nearly 50% inhibition of MMP activity. When BE(2)-C cells were grown in non-adherent conditions to enrich the tumor-initiating cell population, BE(2)-C-spheres were obtained. After 48 h and 72 h treatment, EGCG+IIF limited BE(2)-C-sphere formation and elicited cell death with a reduction of N-MYC expression. We concluded that the association of EGCG to IIF might be applied without toxic effects to overcome the incomplete success of retinoid treatments in neuroblastoma patients.

Synthetic Lethality Triggered by Combining Olaparib with BRCA2-Rad51 Disruptors.

In BRCA2-defective cells, poly(adenosine diphosphate [ADP]-ribose) polymerase inhibitors can trigger synthetic lethality, as two independent DNA-repairing mechanisms are simultaneously impaired. Here, we have pharmacologically induced synthetic lethality, which was triggered by combining two different small organic molecules. When administered with a BRCA2-Rad51 disruptor in nonmutant cells, Olaparib showed anticancer activity comparable to that shown when administered alone in BRCA2-defective cells. This strategy could represent an innovative approach to anticancer drug discovery and could be extended to other synthetic lethality pathways.

EGFR inhibition by (-)-epigallocatechin-3-gallate and IIF treatments reduces breast cancer cell invasion.

Epidermal growth factor receptor (EGFR) expression is an important marker in breast carcinoma pathology and is considered a pivotal molecule for cancer cell proliferation, invasion and metastasis. We investigated the effects of epigallocatechin-3-gallate (EGCG), the most active green tea catechin, in combination with 6-OH-11-O-hydroxyphenanthrene (IIF), a synthetic retinoid X receptor-γ (RXRγ) agonist, on three breast carcinoma cell lines: MCF-7, MCF-7TAM and MDA-MB-231. EGFR and AKT activation and molecular markers of cell motility and migration (CD44, extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN), MMP-2, MMP-9 and tissue inhibitor of metalloproteinases (TIMPs)) were studied after EGCG and IIF treatments. The EGCG + IIF treatment was the most active in down-regulating EGFR phosphorylation at Tyr1068 in all the investigated cell lines; p473AKT was also down-regulated in MCF-TAM cells. EGCG + IIF was also the most active treatment in reducing the expression of markers of invasion and migration in all the three cell lines: CD44, EMMPRIN, MMP-2 and -9 expression decreased, whereas TIMPs were up-regulated. Zymography and scratch assay also confirmed the reduced invasion tendency. We considered that EGCG and IIF treatments could alter the molecular network based on EGFR, CD44 and EMMPRIN expression interdependence and reduced the migration tendency in MCF-7, MCF-7TAM and MDA-MB-231 cells. These events only occurred in association with AKT inactivation in MCF-7TAM cells. In conclusion, the combination of EGCG and IIF significantly attenuated the invasive behaviour of breast carcinoma cells.

Epigallocatechin-3-gallate Increases RXRγ-mediated Pro-apoptotic and Anti-invasive Effects in Gastrointestinal Cancer Cell Lines.

Molecules with synergistic effects often enhance the benefits of cancer therapy. We observed that the major catechin of green tea, (-)-Epigallocatechin-3-gallate (EGCG), induced retinoid X receptor-γ (RXRγ) expression in the SK-Ch-A1 cholangiocarcinoma cell line and in two colon carcinoma cell lines (LoVo and the derivative multi-drug resistant LoVoMDR). On this basis, we analyzed the effects of EGCG in combination with an RXRγ ligand, 6-OH-11-O-hydroxyphenantrene (IIF), or with a ligand of retinoic acid receptor, all-trans-retinoic acid (RA). IIF alone and in combination with EGCG activated the retinoic X response elements and induced the germ cell nuclear factor. In parallel, EGCG induced 67 kDa laminin receptor expression alone and in combination with IIF. We observed a synergistic growth inhibition with EGCG and IIF in combination at lower doses. These effects were accompanied by apoptosis activation through the mitochondrial pathway. Moreover, in LoVo cell line we observed an induction of Forkhead box O3 expression, another molecule involved in apoptosis activation. Finally, metalloproteinase activity and extracellular matrix metalloproteinase inducer (EMMPRIN) expression were inhibited and tumor cell invasion was strongly reduced in the SK-Ch-A1 cell line after treatment with EGCG and IIF. In conclusion, the use of specific RXR ligands in combination with catechins could open a new perspective in gastrointestinal tumor chemoprevention.

A RXR ligand 6-OH-11-O-hydroxyphenanthrene with antitumour properties enhances (-)-epigallocatechin-3-gallate activity in three human breast carcinoma cell lines.

(-)-Epigallocatechin-3-gallate (EGCG) and chemotherapeutic agents cotreatment can improve cytotoxicity against cancer cells. We showed that EGCG and the rexinoid 6-OH-11-O-hydroxyphenanthrene (IIF), given together, were cytotoxic toward MCF-7, MCF-7TAM, and MDA-MB-231, three breast carcinoma cell lines showing different molecular characteristics. Cell growth arrest and apoptosis were greater after EGCG and IIF cotreatment than after individual administration. Cytotoxicity was related to upregulation of 67-kDa laminin receptor (LR67), one of the principal molecular targets of EGCG, and activation of the nuclear retinoic X receptors (RXRs) pathway. Furthermore, the transcription factor Forkhead box O3 (Foxo3a), a protein able to trigger apoptosis through upregulation of genes necessary for cell death, was activated. EGCG and IIF cotreatment produced a significant nuclear import of Foxo3a from the cytoplasm in MCF-7, MCF-7TAM, and MDA-MB-231 cells. In MCF-7TAM cells only, Foxo3a nuclear localization was associated with p473AKT downregulation. For the first time we showed that when EGCG and IIF, two harmless molecules, were given together, they might increase cytotoxicity in three breast carcinoma cell lines, two of them being representative of poorly responsive breast carcinoma types.

Vitexin-2-O-xyloside, raphasatin and (-)-epigallocatechin-3-gallate synergistically affect cell growth and apoptosis of colon cancer cells.

Cytotoxic effects of the combination of the food components vitexin-2-O-xyloside (X), raphasatin (4-methylsulphanyl-3-butenyl isothiocyanates; G) and (-)-epigallocatechin-3-gallate (E) were investigated in colon (LoVo and CaCo-2) and breast (MDA-MB-231 and MCF-7) cancer cells. Breast cancer cells were more resistant than colon cells to X, G and E inhibition. On the contrary, marked synergistic effects among X, G and E on cell growth were found in both colon cancer cells. Further analysis revealed a G0/G1 arrest of the phase cell progression and apoptosis, linked to modulation of Bax, Bcl2, caspase-9 and poly(ADP-ribose) polymerase as well as Reactive Oxygen Species (ROS) generation in both colon cancer cells, whereas apoptosis and ROS were not significantly detected in normal human lymphocytes. We conclude that the X, G and E mixture might act by mitochondrial pathway activation of apoptosis, possibly elicited by ROS and the mixture may be effective in the chemoprevention of colon cancer.

(-)-Epigallocatechin-3-gallate down-regulates EGFR, MMP-2, MMP-9 and EMMPRIN and inhibits the invasion of MCF-7 tamoxifen-resistant cells.

The activation of the EGFR (epidermal growth factor receptor) signalling pathway is one of the key mechanisms underlying the development of resistance to tamoxifen in breast cancer patients. As EGCG [(-)-epigallocatechin-3-gallate], the most active catechin present in green tea, has been shown to down-regulate EGFR, we studied the effects of 10-100 µg/ml EGCG treatment on growth and invasion in a breast carcinoma cell line resistant to tamoxifen [MCF-7Tam (MCF-7 breast carcinoma cell line resistant to tamoxifen) cells] and parental MCF-7. A dose-dependent down-regulation of EGFR mRNA expression and protein level occurred after 50 µg/ml EGCG treatment of MCF-7Tam cells. EGFR molecules on the plasma membrane surface of MCF-7Tam cells significantly decreased. EGFR phosphorylation (Tyr-992, Tyr-1045 and Tyr-1068) was higher in MCF-7Tam than in MCF-7 and it was reduced by EGCG treatment. ERK (extracellular regulated kinase) and phospho-ERK p42/44 were also down-regulated by EGCG treatment and in vitro cell growth and invasion decreased. MMP-2 (matrix metalloproteinase-2) and MMP-9, which are implicated in cell invasion and metastasis, and EMMPRIN (extracellular matrix metalloproteinase inducer), a glycoprotein able to activate MMPs, were significantly reduced after 50 µg/ml EGCG treatment. In keeping with this, TIMP-1 (tissue inhibitor of metalloproteinases-1) and TIMP-2, which down-regulate MMPs, increased after EGCG treatment. Altogether, the present data demonstrated that EGCG could attenuate the tamoxifen-resistant phenotype of MCF-7Tam cells. EGCG could stop MCF-7Tam cell growth and in vitro invasion through down-regulation of EGFR and other molecules implicated in aggressive biological behaviour. The present data support the hypothesis that EGCG is an interesting molecule to be investigated in tamoxifen-resistant breast carcinoma.

(-)-Epigallocatechin-3-gallate downregulates estrogen receptor alpha function in MCF-7 breast carcinoma cells.

BACKGROUND: (-)-Epigallocatechin-3-gallate (EGCG) is the most active catechin present in green tea, demonstrated to have chemopreventive action and to kill cancer cells selectively. As a previous study found that catechins could compete with 17-beta-estradiol for binding to estrogen receptor alpha (ERalpha), we asked whether EGCG could regulate ERalpha action. METHODS: We used MCF-7, a breast carcinoma cell line having a high level of ERalpha expression. The cells were treated with various EGCG concentrations and cell viability was evaluated by MTT assay. ERalpha and pS2 expression were analyzed by RT-PCR after RNA extraction. To better define EGCG action in relation to ERalpha, we studied EGCG cytotoxicity on MCF-7 resistant to tamoxifen (MCF-7tam), MCF-7 treated with 10(-7)M ICI 182,780 for 8 days and on MDA-MB-231, a cell line that lacked ERalpha by flow cytometry (FCM). RESULTS: Both ERalpha and pS2 mRNA were expressed in samples treated with low EGCG concentration (30 microg/ml). At this concentration, no cell change was detectable. In contrast, pS2 expression was lost in samples treated with 100 microg/ml EGCG for 24h, indicating ERalpha alteration. EGCG cytotoxicity was lower when ERalpha was not present (MDA-MB-231) or inactivated (by tamoxifen or ICI 182,780). CONCLUSIONS: Functionally active ERalpha may have a role in EGCG cytotoxicity, increasing the sensitivity to the drug. As higher EGCG concentrations also killed cells resistant to tamoxifen or treated by 10(-7)M ICI 182,780, EGCG ought to be better investigated in breast carcinoma cells treated with drugs targeted to steroid receptors, as a potential complement of therapy.

Simultaneous chromosome 1q gain and 16q loss is associated with steroid receptor presence and low proliferation in breast carcinoma.

We applied comparative genomic hybridization (CGH) to 46 breast carcinoma samples, collected from 1993 to 1995, in order to detect chromosome 1q gains and 16q losses and to define whether samples showing both these alterations had distinct biopathologic features and different clinical outcome. A total of 22 samples (48%) had simultaneous chromosome 1q gain and 16q loss, which was always associated with other genetic changes. In total, 23 samples had various chromosome imbalances (including chromosome 1q gain independent of chromosome 16q loss and vice versa) and one sample did not show detectable alterations. Samples having chromosome 1q gain/16q loss were compared to the other samples with regard to neoplasm size, lymph-node status, histologic and nuclear grade, estrogen and progesterone receptor presence, Ki-67, pRB, Cyclin D1, Cyclin A, p53, p21 and p27 expression as detected by immunohistochemistry. The samples showing chromosome 1q gain/16q loss had high steroid hormone receptor expression (P=0.02), low cell growth fraction (Ki-67, P=0.03) and high p27 expression (P<0.001). No statistical correlation with disease-free survival and overall survival or response to hormonal therapy was found. We conclude that simultaneous chromosome 1q gain/16q loss is a frequent event in invasive breast cancer, which occurs in a subset of both intermediate- and high-grade breast carcinomas. Although the final chromosome 1q and 16q imbalances might have originated from different chromosome alterations in low- and high-grade samples, the gene-dosage effect might be important in conferring peculiar biopathologic characteristics to this subset of samples. The cytogenetic and molecular mechanisms underlying these chromosome changes deserve further investigations.