The p53-, Bax- and p21-dependent inhibition of colon cancer cell growth by 5-hydroxy polymethoxyflavones

Scope: Previously, we reported that 5‐hydroxy polymethoxyflavones (5OH‐PMFs) isolated from orange, namely 5‐hydroxy‐6,7,8,3′,4′‐pentamethoxyflavone, 5‐hydroxy‐3,6,7,8,3′,4′‐hexamethoxyflavone (5HHMF) and 5‐hydroxy‐6,7,8,4′‐tetramethoxyflavone (5HTMF), potently induced apoptosis and cell‐cycle arrest in multiple human colon cancer cells. Herein, using isogenic variants of HCT116 human colon cancer cells, we investigated the effects of p53, Bax and p21 on the apoptosis and cell‐cycle arrest induced by different 5OH‐PMFs. Methods and results: Annexin V/PI co‐staining assay demonstrated that 5HHMF and 5HTMF significantly induced apoptosis in HCT116 (p53^+/+) cells but not in HCT116 (p53^?/?) cells. Furthermore, 5HHMF and 5HTMF significantly induced apoptosis in HCT116 (Bax^+/?) cells, whereas their pro‐apoptotic effects on HCT116 (Bax^?/?) cells were marginal. All three 5OH‐PMFs increased G0/G1 cell population of HCT116 (p53^+/+) cells, and these effects were abolished in HCT116 (p53^?/?) and HCT116 (p21^?/?) cells. Immunoblotting analysis showed that 5HHMF and 5HTMF increased the levels of cleaved caspase‐3, cleaved PARP in both HCT116 (p53^+/+) and HCT116 (Bax^+/?) cells and these effects were much weaker in HCT116 (p53^?/?) and HCT116 (Bax^?/?) cells. Conclusion: Our results demonstrated that 5OH‐PMFs, especially 5HHMF and 5HTMF, induce apoptosis and cell‐cycle arrest by p53‐, Bax‐ and p21‐dependent mechanism.     

Bowman-Birk inhibitors in lentil: Heterologous expression,functional characterisation and anti-proliferative properties in human colon cancer cells

A full-length cDNA, encoding a Bowman-Birk protease inhibitor (BBI), was isolated from lentil immature seeds. The deduced amino acid sequence was longer than that of the BBI extracted from lentil seeds and contained two binding sites; the first inhibitory site inhibits trypsin whereas the second one inhibits chymotrypsin. In order to characterize this lentil BBI, a longer (complete) and its C-terminally processed (mature) form were heterologously expressed in the yeast Pichia pastoris. The recombinant BBI proteins proved to be active against trypsin and chymotrypsin, showing K_i values at nanomolar levels. Mass spectrometry analysis revealed that complete BBI was composed of an array of molecular masses, whereas mature BBI showed the presence of a major peak of the expected size. The effects of mature BBI on the growth of human colon adenocarcinoma HT29 and colonic fibroblast CCD-18Co cells were evaluated. Lentil BBI was able to inhibit the growth of such cells at concentrations higher than 19 μM, in a concentration-dependent manner; by contrast, the CCD18-Co cells were unaffected. These data broaden our knowledge of the beneficial biological activities of naturally-occurring BBI proteins and address the need for systematic evaluation of natural variants in order to design novel strategies in preventive medicine.     

The chemopreventive and anticancer potential against colorectal cancer of polyphenol-rich fruit extracts

The consumption of vegetables and fruits, particularly apples, pomegranates, grapes, and berries, is associated with a decreased risk of developing cancer, especially colorectal cancer (CRC). This may be attributable to the presence of phytochemical constituents, such as polyphenols. This review summarizes the current state of knowledge concerning the chemopreventive and anti-CRC potential of polyphenol-rich apple, pomegranate, grape, and berry extracts with a focus on in vitro, in vivo, and clinical evidence. The extracts demonstrate antiproliferative, proapoptotic, antiangiogenic, antiinvasive, and antimetastatic activities toward colon cancer cell lines and in animal models and markedly influence preneoplastic lesions and malignancies in humans.     

Suillus collinitus methanolic extract increases p53 expression and causes cell cycle arrest and apoptosis in a breast cancer cell line

In the present work, methanolic, ethanolic and boiled water extracts of Suillus collinitus were chemically characterised and submitted to an evaluation of their bioactive properties (antioxidant potential and cytotoxic activity in tumor cell lines). Phenolic acids and sugars were identified chromatographically and quantified in the methanolic and boiled water extracts, respectively. S. collinitus ethanolic extract had the highest antioxidant activity. Nevertheless, with respect to cell growth inhibition, the methanolic extract was the most potent extract, particularly in MCF-7 cells (GI(50) 25.2±0.2μg/ml). Moreover, the GI(50) concentration of this extract induced a G1 cell cycle arrest, with a concomitant decrease in the percentage of cells in the S phase. Furthermore, it caused an increase in the percentage of apoptotic cells, from 6.0±0.2% in untreated cells, to 15.3±2.0% in cells treated with the GI(50) concentration and to 16.3±2.0% in cells treated with 2×GI(50) concentration. In addition, 48h treatment with the GI(50) concentration caused a strong increase in the levels of p53, p21, and cleaved PARP, together with a decrease in Bcl-2 and XIAP. Results indicate that S. collinitus may be a promising source of bioactive compounds. Particularly, its methanolic extract appears to have a p53-mediated effect on the normal cell cycle distribution and apoptosis induction in a human breast tumor cell line.     

Cell proliferation inhibitory and apoptosis-inducing properties of anacardic acid and lunasin in human breast cancer MDA-MB-231 cells

The combination of two or more chemopreventive agents is currently being used to achieve greater inhibitory effects on breast cancer cells. Anacardic acid and lunasin are two plant-derived compounds that have been associated with anti-carcinogenic properties. These compounds show inhibitory effects on cell proliferation and inducing properties of apoptosis in human breast cancer MDA-MB-231 cell line. Both lunasin and anacardic acid exert their effects through the modulation of expression of several genes involved in cell cycle, apoptosis and signal transduction. Their combination arrests the cell cycle in S-phase and induces apoptosis at higher levels than that observed when each compound is used individually. This combination also promotes the inhibition of ERBB2, AKT1, JUN and RAF1 signalling gene expression, whose up-regulation has been reported as responsible for breast cancer cells growth and resistance to apoptosis. Our results introduce these two compounds as a promising strategy to prevent/treat breast cancer.     

Anticancer and carcinogenic properties of curcumin: considerations for its clinical development as a cancer chemopreventive and chemotherapeutic agent

A growing body of research suggests that curcumin, the major active constituent of the dietary spice turmeric, has potential for the prevention and therapy of cancer. Preclinical data have shown that curcumin can both inhibit the formation of tumors in animal models of carcinogenesis and act on a variety of molecular targets involved in cancer development. In vitro studies have demonstrated that curcumin is an efficient inducer of apoptosis and some degree of selectivity for cancer cells has been observed. Clinical trials have revealed that curcumin is well tolerated and may produce antitumor effects in people with precancerous lesions or who are at a high risk for developing cancer. This seems to indicate that curcumin is a pharmacologically safe agent that may be used in cancer chemoprevention and therapy. Both in vitro and in vivo studies have shown, however, that curcumin may produce toxic and carcinogenic effects under specific conditions. Curcumin may also alter the effectiveness of radiotherapy and chemotherapy. This review article analyzes the in vitro and in vivo cancer-related activities of curcumin and discusses that they are linked to its known antioxidant and pro-oxidant properties. Several considerations that may help develop curcumin as an anticancer agent are also discussed.     

Lectins as bioactive plant proteins: a potential in cancer treatment

Plant lectins, a unique group of proteins and glycoproteins with potent biological activity, occur in foods like wheat, corn, tomato, peanut, kidney bean, banana, pea, lentil, soybean, mushroom, rice, and potato. Thus, dietary intakes by humans can be significant. Many lectins resist digestion, survive gut passage, and bind to gastrointestinal cells and/or enter the circulation intact, maintaining full biological activity. Several lectins have been found to possess anticancer properties in vitro, in vivo, and in human case studies; they are used as therapeutic agents, preferentially binding to cancer cell membranes or their receptors, causing cytotoxicity, apoptosis, and inhibition of tumor growth. These compounds can become internalized into cells, causing cancer cell agglutination and/or aggregation. Ingestion of lectins also sequesters the available body pool of polyamines, thereby thwarting cancer cell growth. They also affect the immune system by altering the production of various interleukins, or by activating certain protein kinases. Lectins can bind to ribosomes and inhibit protein synthesis. They also modify the cell cycle by inducing non-apoptotic G1-phase accumulation mechanisms, G2/M phase cell cycle arrest and apoptosis, and can activate the caspase cascade. Lectins can also downregulate telomerase activity and inhibit angiogenesis. Although lectins seem to have great potential as anticancer agents, further research is still needed and should include a genomic and proteomic approach.     

Dietary fibre and colorectal cancer: a model for environment--gene interactions

As environmental factors are clearly associated with risk for colorectal cancer, we set out to model how dietary fibre, or the effects of its ingestion, might impact upon the complex events that characterise colorectal oncogenesis. The diverse nature of dietary fibre and its resultant fate in the gut is outlined. The evidence indicates that different types of fibre create different conditions in different regions of the gut. This is reflected in different effects on oncogenesis especially in animal models. Data from animal models show that insoluble fibre is protective. Evidence from human studies are not consistent, especially considering the interventional studies. However, all such studies have been dependent on biomarkers short of cancer formation, for measurement of an effect. The biological and molecular events characteristic of colorectal oncogenesis are reviewed in an effort to identify how fibre ingestion might regulate oncogenesis. While several mechanisms might account for protection, the results of fermentation and especially butyrate production provide examples of how genomic instability might be controlled. Activation of apoptosis and cell cycle arrest seem likely to be mechanisms that would enable correction of genomic events that drive oncogenesis. Butyrate itself can regulate gene expression by both epigenetic and direct effects.     

Anthocyanin-rich Mulberry extract inhibit the gastric cancer cell growth in vitro and xenograft mice by inducing signals of p38/p53 and c-jun

Anthocyanins have been well characterized by various bioactive properties. In previous studies, Mulberry anthocyanins (MACs) have proven to prevent atherosclerosis and inhibit melanoma metastasis. Here, AGS cells demonstrated an increase in the distribution of hypodiploid phase (apoptotic peak) after treatment with MACs. Further investigation revealed that MACs exerted their influence by inducing intrinsic and extrinsic apoptosis through p38/p53 and p38/c-jun signaling pathways. In addition, the caspase-related protein, such as caspase-3, was activated from pro-caspase to cleaved-caspase by treating MACs to AGS cells. We also used the experimental AGS gastric cancer xenograft model to verify the inhibitory effect of MACs. These findings suggest that, by targeting p38/p53 and the c-jun pathways, MACs suppressed cell survival and tumorigenesis, but induced apoptotic death in AGS cells. MACs can potentially prevent the growth of AGS cells for ineffective conventional chemotherapy of gastric carcinoma.     

The grape and wine constituent piceatannol inhibits proliferation of human bladder cancer cells via blocking cell cycle progression and inducing Fas/membrane bound Fas ligand-mediated apoptotic pathway

Piceatannol (3,3',4,5'-Tetrahydroxy-trans-stilbene) is a polyphenol present in grapes and wine. Piceatannol is a protein kinase inhibitor that modifies multiple cellular targets exerting immunosuppressive, antileukemic, and antitumorigenic activities in several cell lines and animal models. In this study, the antiproliferative activity of piceatannol was investigated. The results showed that piceatannol inhibited the proliferation of T24 and HT1376 human bladder cancer cells by blocking cell cycle progression in the G0/G1 phase and inducing apoptosis. ELISA showed that the G0/G1 phase arrest is due to an increased in the expression of p21/WAF1. An enhancement in Fas/APO-1 and membrane-bound Fas ligand (mFasL) might be responsible for the apoptotic effect induced by piceatannol. Our study reports the novel finding, that the induction of p21/WAF1 and activity of the Fas/mFasL apoptotic system may participate in the antiproliferative activity of piceatannol in T24 and HT1376 cells.     

Shallot and licorice constituent isoliquiritigenin arrests cell cycle progression and induces apoptosis through the induction of ATM/p53 and initiation of the mitochondrial system in human cervical carcinoma HeLa cells

This study is the first to investigate the anticancer effect of isoliquiritigenin (ISL) in human cervical carcinoma HeLa cells. The results reveal that ISL inhibits HeLa cells by blocking cell cycle progression in the G2/M phase and inducing apoptosis. Blockade of cell cycle is associated with increased activation of ataxia telangiectasia‐mutated (ATM). Activation of ATM by ISL phosphorylated p53 at Serine15, resulting in increased stability of p53 by decreasing p53 and murine double minute‐2 (MDM2) interaction. In addition, ISL‐mediated G2/M phase arrest was also associated with decreases in the amounts of cyclin B, cyclin A, cdc2, and cdc25C, and increases in the phosphorylation of Chk2, cdc25C, and cdc2. The specific ATM inhibitor caffeine significantly decreased ISL‐mediated G2/M arrest by inhibiting the phosphorylation of p53 (Serine15) and Chk2. ISL induced apoptotic cell death is associated with changes in the expression of Bax and Bak, decreasing levels of Bcl‐2 and Bcl‐X_L, and subsequently triggering mitochondrial apoptotic pathway. In addition, pretreatment of cells with caspase‐9 inhibitor blocked ISL‐induced apoptosis, indicating that caspase‐9 activation is involved in ISL‐mediated HeLa cell apoptosis. These findings suggest that ISL may be a promising chemopreventive agent against human uterine cervical cancer.     

Clitocybe alexandri extract induces cell cycle arrest and apoptosis in a lung cancer cell line: Identification of phenolic acids with cytotoxic potential

Mushrooms are a possible rich source of biologically active compounds with the potential for drug discovery. The aim of this work was to gain further insight into the cytotoxicity mechanism of action of Clitocybe alexandri ethanolic extract against a lung cancer cell line (NCI-H460 cells). The effects on cell cycle profile and levels of apoptosis were evaluated by flow cytometry, and the effect on the expression levels of proteins related to cellular apoptosis was also investigated by Western blot. The extract was characterised regarding its phenolic composition by HPLC-DAD, and the identified compounds were studied regarding their growth inhibitory activity, by sulforhodamine B (SRB) assay. The effect of individual or combined compounds on viable cell number was also evaluated using the Trypan blue exclusion assay. It was observed that the C. alexandri extract induced an S-phase cell cycle arrest and increased the percentage of apoptotic cells. In addition, treatment with the GI₅₀ concentration (concentration that was able to cause 50% of cell growth inhibition; 24.8 μg/ml) for 48 h caused an increase in the levels of wt. p53, cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). The main components identified in this extract were protocatechuic, p-hydroxybenzoic and cinnamic acids. Cinnamic acid was found to be the most potent compound regarding cell growth inhibition. Nevertheless, it was verified that the concomitant use of the individual compounds provided the strongest decrease in viable cell number. Overall, evidence was found for alterations in cell cycle and apoptosis, involving p53 and caspase-3. Furthermore, our data suggests that the phenolic acids identified in the extract are at least partially responsible for the cytotoxicity induced by this mushroom extract.