Colon Cancer Chemoprevention by Sage Tea Drinking: Decreased DNA Damage and Cell Proliferation.

Salvia officinalis and some of its isolated compounds have been found to be preventive of DNA damage and increased proliferation in vitro in colon cells. In the present study, we used the azoxymethane model to test effects of S. officinalis on colon cancer prevention in vivo. The results showed that sage treatment reduced the number of ACF formed only if administered before azoxymethane injection, demonstrating that sage tea drinking has a chemopreventive effect on colorectal cancer. A decrease in the proliferation marker Ki67 and in H2 O2 -induced and azoxymethane-induced DNA damage to colonocytes and lymphocytes were found with sage treatment. This confirms in vivo the chemopreventive effects of S. officinalis. Taken together, our results show that sage treatment prevented initiation phases of colon carcinogenesis, an effect due, at least in part, to DNA protection, and reduced proliferation rates of colon epithelial cell that prevent mutations and their fixation through cell replication. These chemopreventive effects of S. officinalis on colon cancer add to the many health benefits attributed to sage and encourage its consumption.

Redox-dependent induction of antioxidant defenses by phenolic diterpenes confers stress tolerance in normal human skin fibroblasts: Insights on replicative senescence.

Mild stress-induced hormesis represents a promising strategy for targeting the age-related accumulation of molecular damage and, therefore, for preventing diseases and achieving healthy aging. Fruits, vegetables, and spices contain a wide variety of hormetic phytochemicals, which may explain the beneficial health effects associated with the consumption of these dietary components. In the present study, the induction of cellular antioxidant defenses by the phenolic diterpenes carnosic acid (CA) and carnosol (CS) were studied in normal human skin fibroblasts, and insights into the aging process at the cellular level investigated. We observed that CA and CS induced several cytoprotective enzymes and antioxidant defenses in human fibroblasts, whose induction was dependent on the cellular redox state for CS and associated with Nrf2 signaling for both compounds. The stress response elicited by preincubation with CS conferred a cytoprotective action against a following oxidant challenge with tert-butyl hydroperoxide, confirming its hormetic effect. Preincubation of normal fibroblasts with CS also protected against hydrogen peroxide-induced premature senescence. Furthermore, cultivation of middle passage normal human skin fibroblasts in the presence of CS ameliorated the physiological state of cells during replicative senescence. Our results support the view that mild stress-induced antioxidant defenses by CS can confer stress tolerance in normal cells and may have important implications in the promotion of healthy aging.

Novel structurally similar chromene derivatives with opposing effects on p53 and apoptosis mechanisms in colorectal HCT116 cancer cells.

In the present work, novel chromene derivatives fused with the imidazo[1,2-a]pyridine nucleus were tested for their anticancer potential in the human colorectal cancer HCT116 cells. Compounds 2a and 2c showed significant growth inhibitory activity with GI50 of 15 µM and 11 µM, respectively. Compound 2c, the most potent, has a carbamate group in position 8 of the pyridine ring, and showed significant cell cycle arrest and induction of cell death by apoptosis, even at 5 µM. Besides different potencies, chromene analogs 2a and 2c showed different mechanisms of action. Whereas the carbamate-free chromene 2a induced cell cycle arrest at G1/G0 phase, compound 2c showed to arrest cell cycle at both S and G2 phases. Chromene derivative 2a at concentrations higher than its GI50 remarkably induced caspases-dependent apoptosis in a p53-independent manner. On the other hand, compound 2c increased significantly p53 levels and induced apoptosis in a p53- and caspases-dependent manner, even at concentrations lower than its GI50. Both compounds increased the Bax/Bcl-2 ratio, induced mitochondria depolarization and activated MAP kinases. In conclusion, two novel and structurally similar chromene derivatives showed cytotoxicity to HCT16 cells through opposing effects on p53 levels and apoptosis mechanisms, which may be relevant for further development of drugs acting on distinct molecular targets useful in the treatment of cancers with different genetic profiles and for personalized medicine.

Biocompatibility of a self-assembled glycol chitosan nanogel.

The research of chitosan-based nanogel for biomedical applications has grown exponentially in the last years; however, its biocompatibility is still insufficiently reported. Hence, the present work provides a thorough study of the biocompatibility of a glycol chitosan (GC) nanogel. The obtained results showed that GC nanogel induced slight decrease on metabolic activity of RAW, 3T3 and HMEC cell cultures, although no effect on cell membrane integrity was verified. The nanogel does not promote cell death by apoptosis and/or necrosis, exception made for the HMEC cell line challenged with the higher GC nanogel concentration. Cell cycle arrest on G1 phase was observed only in the case of RAW cells. Remarkably, the nanogel is poorly internalized by bone marrow derived macrophages and does not trigger the activation of the complement system. GC nanogel blood compatibility was confirmed through haemolysis and whole blood clotting time assays. Overall, the results demonstrated the safety of the use of the GC nanogel as drug delivery system.

Anticancer effects of lactoferrin: underlying mechanisms and future trends in cancer therapy.

Lactoferrin has been widely studied over the last 70 years, and its role in diverse biological functions is now well known and generally accepted by the scientific community. Usually, alterations of the lactoferrin gene in cells are associated with an increased incidence of cancer. Several studies suggest that exogenous treatment with lactoferrin and its derivatives can efficiently inhibit the growth of tumors and reduce susceptibility to cancer. None of these studies, however, reported a consistent outcome with regard to the mechanisms underlying the anticancer effects of lactoferrin. In this review, the association of lactoferrin with cancer is thoroughly discussed, from lactoferrin gene expression to the potential use of lactoferrin in cancer therapy. Lactoferrin cytotoxicity against several cancers is reported to occur in distinct ways under different conditions, namely by cell membrane disruption, apoptosis induction, cell cycle arrest, and cell immunoreaction. Based on these mechanisms, new strategies to improve the anticancer effects of the lactoferrin protein and/or its derivatives are proposed. The potential for lactoferrin in the field of cancer research (including as a chemotherapeutic agent in cancer therapy) is also discussed.

Bovine lactoferrin induces cell cycle arrest and inhibits mTOR signaling in breast cancer cells.

Lactoferrin (LF) is predominantly found in mammalian secretions with recognized anticancer potential, although the mechanisms involved in such activity are still unclear. Here, the stability, internalization, and cytotoxicity of bovine LF (bLF) and its variants were tested against a panel of breast cancer cells. bLF was found to be very stable under incubation with cells and also able to internalize them, although most of the protein remained in the culture medium. Furthermore, bLF (up to 30 µM) inhibited the growth of breast cancer cells (T-47D, MDA-MB-231, Hs578T, and MCF-7) in a higher extent than in the normal counterpart cell line (MCF-10-2A), thus suggesting its selectivity. Regarding its variants, only the iron-saturated protein showed a higher activity compared with the commercial bLF. bLF growth inhibitory activity was associated with the induction of cell cycle arrest, but not with apoptosis. Moreover, exposure to bLF increased the cells phospho-AMPKα levels and decreased both phospho threonine mammalian target of rapamycin (mTOR) and total mTOR levels, indicating a novel mechanism of action through its ability to induce nutrient/energy-related stress. This study disclosed important findings to better understand the mechanisms underlying the bLF effects on breast cancer cell lines, which could be valuable for novel advances in the cancer research field.

Effects of biosurfactants on the viability and proliferation of human breast cancer cells.

Biosurfactants are molecules with surface activity produced by microorganisms that can be used in many biomedical applications. The anti-tumour potential of these molecules is being studied, although results are still scarce and few data are available regarding the mechanisms underlying such activity. In this work, the anti-tumour activity of a surfactin produced by Bacillus subtilis 573 and a glycoprotein (BioEG) produced by Lactobacillus paracasei subsp. paracasei A20 was evaluated. Both biosurfactants were tested against two breast cancer cell lines, T47D and MDA-MB-231, and a non-tumour fibroblast cell line (MC-3 T3-E1), specifically regarding cell viability and proliferation. Surfactin was found to decrease viability of both breast cancer cell lines studied. A 24 h exposure to 0.05 g l(-1) surfactin led to inhibition of cell proliferation as shown by cell cycle arrest at G1 phase. Similarly, exposure of cells to 0.15 g l(-1) BioEG for 48 h decreased cancer cells' viability, without affecting normal fibroblasts. Moreover, BioEG induced the cell cycle arrest at G1 for both breast cancer cell lines. The biosurfactant BioEG was shown to be more active than surfactin against the studied breast cancer cells. The results gathered in this work are very promising regarding the biosurfactants potential for breast cancer treatment and encourage further work with the BioEG glycoprotein.

Superior anticancer activity of halogenated chalcones and flavonols over the natural flavonol quercetin.

A series of chalcone and flavonol derivatives were synthesized in good yield by an eco-friendly approach. A pharmacological evaluation was performed with the human colorectal carcinoma cell line HCT116 and revealed that the anticancer activity of flavonols was higher when compared with that of the respective chalcone precursors. The antiproliferative activity of halogenated derivatives increases as the substituent in the 3- or 4-positon of the B-ring goes from F to Cl and to Br. In addition, halogens in position 3 enhance anticancer activity in chalcones whereas for flavonol derivatives the best performance was registered for the 4-substituted derivatives. Flow cytometry analysis showed that compounds 3p and 4o induced cell cycle arrest and apoptosis as demonstrated by increased S, G2/M and sub-G1 phases. These data were corroborated by western blot and fluorescence microscopy analysis. In summary, halogenated chalcones and flavonols were successfully prepared and presented high anticancer activity as shown by their cell growth and cell cycle inhibitory potential against HCT116 cells, superior to that of quercetin, used as a positive control.

Development of a new application of the comet assay to assess levels of O6-methylguanine in genomic DNA (CoMeth).

O(6)-methylguanine (O(6)meG) is one of the most premutagenic, precarcinogenic, and precytotoxic DNA lesions formed by alkylating agents. Repair of this DNA damage is achieved by the protein MGMT, which transfers the alkyl groups from the O(6) position of guanine to a cysteine residue in its active center. Because O(6)meG repair by MGMT is a stoichiometric reaction that irreversibly inactivates MGMT, which is subsequently degraded, the repair capacity of O(6)meG lesions is dependent on existing active MGMT molecules. In the absence of active MGMT, O(6)meG is not repaired, and during replication, O(6)meG:T mispairs are formed. The MMR system recognizes these mispairs and introduces a gap into the strand. If O(6)meG remains in one of the template strands the futile MMR repair process will be repeated, generating more strand breaks (SBs). The toxicity of O(6)meG is, therefore, dependent on MMR and DNA SB induction of cell death. MGMT, on the other hand, protects against O(6)meG toxicity by removing the methyl residue from the guanine. Although removal of O(6)meG makes MGMT an important anticarcinogenic mechanism of DNA repair, its activity significantly decreases the efficacy of cancer chemotherapeutic drugs that aim at achieving cell death through the action of the MMR system on unrepaired O(6)meG lesions. Here, we report on a modification of the comet assay (CoMeth) that allows the qualitative assessment of O(6)meG lesions after their conversion to strand breaks in proliferating MMR-proficient cells after MGMT inhibition. This functional assay allows the testing of compounds with effects on O(6)meG levels, as well as on MGMT or MMR activity, in a proliferating cell system. The expression of MGMT and MMR genes is often altered by promoter methylation, and new epigenetically active compounds are being designed to increase chemotherapeutic efficacy. The CoMeth assay allows the testing of compounds with effects on O(6)meG, MGMT, or MMR activity. This proliferating cell system complements other methodologies that look at effects on these parameters individually through analytical chemistry or in vitro assays with recombinant proteins.

Ursolic acid induces cell death and modulates autophagy through JNK pathway in apoptosis-resistant colorectal cancer cells.

Colorectal carcinomas (CRCs) with P53 mutations have been shown to be resistant to chemotherapy with 5-fluorouracil (5-FU), the most widely used chemotherapeutic drug for CRC treatment. Autophagy is emerging as a promising therapeutic target for drug-resistant tumors. In the present study, we tested the effects of ursolic acid (UA), a natural triterpenoid, on cell death mechanisms and its effects in combination with 5-FU in the HCT15 p53 mutant apoptosis-resistant CRC cell line. The involvement of UA in autophagy and its in vivo efficacy were evaluated. Our data show that UA induces apoptosis independent of caspases in HCT15 cells and enhances 5-FU effects associated with an activation of c-jun N-terminal kinase (JNK). In this cell line, where this compound has a more pronounced effect on the induction of cell death compared to 5-FU, apoptosis corresponds only to a small percentage of the total cell death induced by UA. UA also modulated autophagy by inducing the accumulation of LC3 and p62 levels with involvement of JNK pathway, which indicates a contribution of autophagy on JNK-dependent induction of cell death by UA. By using nude mice xenografted with HCT15 cells, we verified that UA was also active in vivo decreasing tumor growth rate. In conclusion, this study shows UA's anticancer potential both in vitro and in vivo. Induction of cell death and modulation of autophagy in CRC-resistant cells were shown to involve JNK signaling.

Hypericum androsaemum water extract inhibits proliferation in human colorectal cancer cells through effects on MAP kinases and PI3K/Akt pathway.

MAP kinase and PI3K/Akt signalling pathways are commonly altered in colorectal carcinoma (CRC) leading to tumor growth due to increased cell proliferation and inhibition of apoptosis. Several species of the genus Hypericum are used in Portugal to prepare herbal teas to which digestive tract effects are attributed. In the present study, the antiproliferative and proapoptotic effects of the water extracts of H. androsaemum (HA) and H. perforatum (HP) were investigated in two human colon carcinoma-derived cell lines, HCT15 and CO115, which harbour activating mutations of KRAS and BRAF, respectively. Contrarily to HP, HA significantly inhibited cell proliferation and induced apoptosis in both cell lines. HA decreased BRAF and phospho-ERK expressions in CO115, but not in HCT15. HA also decreased Akt phosphorylation in CO115 and induced p38 and JNK in both cell lines. HA induced cell cycle arrest at S and G2/M phases as well as caspase-dependent apoptosis in both cell lines. Chlorogenic acid (CA), the main phenolic compound present in the HA extract and less represented in the HP water extract, did, however, not show any of those effects when used individually. In conclusion, water extract of HA, but not of HP, controlled CRC proliferation and specifically acted on mutant and not wild-type BRAF. The effect of HA was, however, not due to CA alone.

Quercetin enhances 5-fluorouracil-induced apoptosis in MSI colorectal cancer cells through p53 modulation.

PURPOSE: Colorectal tumors (CRC) with microsatellite instability (MSI) show resistance to chemotherapy with 5-fluorouracil (5-FU), the most widely used pharmacological drug for CRC treatment. The aims of this study were to test the ability of quercetin (Q) and luteolin (L) to increase the sensitivity of MSI CRC cells to 5-FU and characterize the dependence of the effects on cells' p53 status. METHODS: Two MSI human CRC-derived cell lines were used: CO115 wild type (wt) for p53 and HCT15 that harbors a p53 mutation. Apoptosis induction in these cells by 5-FU, Q and L alone, and in combinations was evaluated by TUNEL and western blot. The dependence of the effects on p53 was confirmed by small interference RNA (siRNA) in CO115 cells and in MSI HCT116 wt and p53 knockout cells. RESULTS: CO115 p53-wt cells are more sensitive to 5-FU than the p53-mutated HCT15. The combination treatment of 5-FU with L and Q increased apoptosis with a significant effect for Q in CO115. Both flavonoids increased p53 expression in both cell lines, an effect particularly remarkable for Q. The significant apoptotic enhancement in CO115 incubated with Q plus 5-FU involved the activation of the apoptotic mitochondrial pathway. Importantly, knockdown of p53 by siRNA in CO115 cells and p53 knockout in HCT116 cells totally abrogated apoptosis induction, demonstrating the dependence of the effect on p53 modulation by Q. CONCLUSION: This study suggests the potential applicability of these phytochemicals for enhancement 5-FU efficiency in MSI CRC therapy, especially Q in p53 wt tumors.

Rosmarinic acid, major phenolic constituent of Greek sage herbal tea, modulates rat intestinal SGLT1 levels with effects on blood glucose.

SCOPE: Previous results suggested that the effects of Salvia fruticosa tea (SFT) drinking on glucose regulation might be at the intestinal level. Here we aim to characterize the effects of SFT treatment and of its main phenolic constituent--rosmarinic acid (RA)--on the levels and localization of the intestinal Na+/glucose cotransporter-1 (SGLT1), the facilitative glucose transporter 2 and glucagon-like peptide-1 (GLP-1). METHODS AND RESULTS: Two models of SGLT1 induction in rats were used: through diabetes induction with streptozotocin (STZ) and through dietary carbohydrate manipulation. Drinking water was replaced with SFT or RA and blood parameters, liver glycogen and the levels of different proteins in enterocytes quantified. Two weeks of SFT treatment stabilized fasting blood glucose levels in STZ-diabetic animals. The increase in SGLT1 localized to the enterocyte brush-border membrane (BBM) induced by STZ treatment was significantly abrogated by treatment with SFT, without significant changes in total cellular transporter protein levels. No effects were observed on glucose transporter 2, Na(+) /K(+) -ATPase or glucagon-like peptide-1 levels by SFT. Additionally, SFT and RA for 4 days significantly inhibited the carbohydrate-induced adaptive increase of SGLT1 in BBM. CONCLUSION: SFT and RA modulate the trafficking of SGLT1 to the BBM and may contribute to the control of plasma glucose.

Curcumin induces heme oxygenase-1 in normal human skin fibroblasts through redox signaling: relevance for anti-aging intervention.

SCOPE: Curcumin, a component of the spice turmeric, was tested for its potential hormetic anti-aging effects as an inducer of mild stress. METHODS AND RESULTS: Early passage young human skin fibroblasts treated with low doses of curcumin (below 20 µM) showed a time- and concentration-dependent induction of heme oxygenase-1 (HO-1), followed by compensatory increase in glutathione-S-transferase activity, GSH levels and GSH/GSSG ratio. These effects were preceded by induction of oxidative stress (increased levels of reactive oxygen species and DNA damage) and impairment of cells' GSH redox state. Curcumin also induced nuclear factor-erythroid-2-related factor 2 accumulation in the nuclei. The use of the antioxidant N-acetyl cysteine prevented the induction of HO-1 by curcumin. Pharmacological inhibition of phosphatidylinositol 3-kinase, but not other kinases, significantly prevented curcumin-induced HO-1 levels, which was corroborated by the induction of phospho-Akt levels by curcumin. Late passage senescent cells already had higher HO-1 levels, and further induction of HO-1 by curcumin was considerably impaired. The induction of stress responses by curcumin in human cells led to protective hormetic effects to further oxidant challenge. CONCLUSION: Curcumin induces cellular stress responses in normal human skin fibroblasts through phosphatidylinositol 3-kinase/Akt pathway and redox signaling, supporting the view that curcumin-induced hormetic stimulation of cellular antioxidant defenses can be a useful approach toward anti-aging intervention.

Determination of proteasomal activities.

The proteasome is an important component of the intracellular system for the turnover of proteins. The mammalian proteasome is engaged to degrade a bulky fraction of soluble intracellular proteins both in an ubiquitin-dependent and independent manner. The proteasome is composed by a central catalytic core--the 20S proteasome--where three different proteases are located, whose activities can be measured. A detailed protocol for measuring accurately the three activities of the 20S proteasome in cell and tissue homogenates, using specific fluorogenic substrates and a microplate reader fluorometer, are described. Successful applications of this method include determining changes in the proteasomal activities during aging, anti-aging interventions, cell cycle analysis, and in various disease states including neurodegenerative diseases and cancers.

Ursolic acid and luteolin-7-glucoside improve lipid profiles and increase liver glycogen content through glycogen synthase kinase-3.

In the present study, two phytochemicals - ursolic acid (UA) and luteolin-7-glucoside (L7G) - were assessed in vivo in healthy rats regarding effects on plasma glucose and lipid profile (total cholesterol, HDL and LDL), as well as liver glycogen content, in view of their importance in the aetiology of diabetes and associated complications. Both UA and L7G significantly decreased plasma glucose concentration. UA also significantly increased liver glycogen levels accompanied by phosphorylation of glycogen synthase kinase-3 (GSK3). The increase in glycogen deposition induced by UA (mediated by GSK3) could have contributed to the lower plasma glucose levels observed. Both compounds significantly lowered total plasma cholesterol and low-density lipoprotein levels, and, in addition, UA increased plasma high-density lipoprotein levels. Our results show that UA particularly may be useful in preventable strategies for people at risk of developing diabetes and associated cardiovascular complications by improving plasma glucose levels and lipid profile, as well as by promoting liver glycogen deposition.

Salvia fruticosa, Salvia officinalis, and rosmarinic acid induce apoptosis and inhibit proliferation of human colorectal cell lines: the role in MAPK/ERK pathway.

Epidemiological studies have shown that nutrition is a key factor in modulating sporadic colorectal carcinoma (CRC) risk. Aromatic plants of the genus Salvia (sage) have been attributed many medicinal properties, which include anticancer activity. In the present study, the antiproliferative and proapoptotic effects of water extracts of Salvia fruticosa (SF) and Salvia officinalis (SO) and of their main phenolic compound rosmarinic acid (RA) were evaluated in two human colon carcinoma-derived cell lines, HCT15 and CO115, which have different mutations in the MAPK/ERK and PI3K/Akt signalling pathways. These pathways are commonly altered in CRC, leading to increased proliferation and inhibition of apoptosis. Our results show that SF, SO, and RA induce apoptosis in both cell lines, whereas cell proliferation was inhibited by the two sage extracts only in HCT15. SO, SF, and RA inhibited ERK phosphorylation in HCT15 and had no effects on Akt phosphorylation in CO115 cells. The activity of sage extracts seems to be due, at least in part, to the inhibition of MAPK/ERK pathway.

Sage tea drinking improves lipid profile and antioxidant defences in humans.

Salvia officinalis (common sage) is a plant with antidiabetic properties. A pilot trial (non-randomized crossover trial) with six healthy female volunteers (aged 40-50) was designed to evaluate the beneficial properties of sage tea consumption on blood glucose regulation, lipid profile and transaminase activity in humans. Effects of sage consumption on erythrocytes' SOD and CAT activities and on Hsp70 expression in lymphocytes were also evaluated. Four weeks sage tea treatment had no effects on plasma glucose. An improvement in lipid profile was observed with lower plasma LDL cholesterol and total cholesterol levels as well as higher plasma HDL cholesterol levels during and two weeks after treatment. Sage tea also increased lymphocyte Hsp70 expression and erythrocyte SOD and CAT activities. No hepatotoxic effects or other adverse effects were observed.

Heat stress and hormetin-induced hormesis in human cells: effects on aging, wound healing, angiogenesis, and differentiation.

Accumulation of molecular damage and increased molecular heterogeneity are hallmarks of cellular aging. Mild stress-induced hormesis can be an effective way for reducing the accumulation of molecular damage, and thus slowing down aging from within. We have shown that repeated mild heat stress (RMHS) has anti-aging effects on growth and various other cellular and biochemical characteristics of normal human skin fibroblasts and keratinocytes undergoing aging in vitro. RMHS given to human cells increased the basal levels of various chaperones, reduced the accumulation of damaged proteins, stimulated proteasomal activities, increased the cellular resistance to other stresses, enhanced the levels of various antioxidant enzymes, enhanced the activity and amounts of sodium-potassium pump, and increased the phosphorylation-mediated activities of various stress kinases. We have now observed novel hormetic effects of mild heat stress on improving the wound healing capacity of skin fibroblasts and on enhancing the angiogenic ability of endothelial cells. We have also tested potential hormetins, such as curcumin and rosmarinic acid in bringing about their beneficial effects in human cells by inducing stress response pathways involving heat shock proteins and hemeoxygenase HO-1. These data further support the view that mild stress-induced hormesis can be applied for the modulation, intervention and prevention of aging and age-related impairments.

Luteolin, quercetin and ursolic acid are potent inhibitors of proliferation and inducers of apoptosis in both KRAS and BRAF mutated human colorectal cancer cells.

KRAS and BRAF mutations are frequent in colorectal carcinoma (CRC) and have the potential to activate proliferation and survival through MAPK/ERK and/or PI3K signalling pathways. Because diet is one of the most important determinants of CRC incidence and progression, we studied the effects of the dietary phytochemicals quercetin (Q), luteolin (L) and ursolic acid (UA) on cell proliferation and apoptosis in two human CRC derived cell lines, HCT15 and CO115, harboring KRAS and BRAF activating mutations, respectively. In KRAS mutated HCT15 cells, Q and L significantly decreased ERK phosphorylation, whereas in BRAF mutated CO115 cells the three compounds decreased Akt phosphorylation but had no effect on phospho-ERK. Our findings show that these natural compounds have antiproliferative and proapoptotic effects and simultaneously seem to act on KRAS and PI3K but not on BRAF. These results shed light on the molecular mechanisms of action of Q, L and UA and emphasize the potential of dietary choices for the control of CRC progression.