Lynne M. Howells

Inhibition of cyclo-oxygenase 2 expression in colon cells by the chemopreventive agent curcumin involves inhibition of NF-κB activation via the NIK/IKK signalling complex

Colorectal cancer is a major cause of cancer deaths in Western countries, but epidemiological data suggest that dietary modification might reduce these by as much as 90%. Cyclo-oxygenase 2 (COX2), an inducible isoform of prostaglandin H synthase, which mediates prostaglandin synthesis during inflammation, and which is selectively overexpressed in colon tumours, is thought to play an important role in colon carcinogenesis. Curcumin, a constituent of turmeric, possesses potent anti-inflammatory activity and prevents colon cancer in animal models. However, its mechanism of action is not fully understood. We found that in human colon epithelial cells, curcumin inhibits COX2 induction by the colon tumour promoters, tumour necrosis factor α or fecapentaene-12. Induction of COX2 by inflammatory cytokines or hypoxia-induced oxidative stress can be mediated by nuclear factor kappa B (NF-κB). Since curcumin inhibits NF-κB activation, we examined whether its chemopreventive activity is related to modulation of the signalling pathway which regulates the stability of the NF-κB-sequestering protein, IκB. Recently components of this pathway, NF-κB-inducing kinase and IκB kinases, IKKα and β, which phosphorylate IκB to release NF-κB, have been characterised. Curcumin prevents phosphorylation of IκB by inhibiting the activity of the IKKs. This property, together with a long history of consumption without adverse health effects, makes curcumin an important candidate for consideration in colon cancer prevention.

Relevance of mitogen activated protein kinase (MAPK) and phosphotidylinositol-3-kinase/protein kinase B (PI3K/PKB) pathways to induction of apoptosis by curcumin in breast cells.

Following observations that curcumin inhibited proliferation (IC(50)=1-5 microM), invasiveness and progression through S/G2/M phases of the cell cycle in the non-tumourigenic HBL100 and tumourigenic MDA-MB-468 human breast cell lines, it was noted that apoptosis was much more pronounced in the tumour line. Therefore, the ability of curcumin to modulate signalling pathways which might contribute to cell survival was investigated. After pre-treatment of cells for 20 min, curcumin (40 microM) inhibited EGF-stimulated phosphorylation of the EGFR in MDA-MB-468 cells and phosphorylation of extracellular signal regulated kinases (ERKs) 1 and 2, as well as ERK activity and levels of nuclear c-fos in both cell lines. At a lower dose (10 microM), it also inhibited the ability of anisomycin to activate JNK, resulting in decreased c-jun phosphorylation, although it did not inhibit JNK activity directly. In contrast, the activation of p38 mitogen activated protein kinase (MAPK) by anisomycin was not inhibited. Curcumin inhibited basal phosphorylation of Akt/protein kinase B (PKB) in both cell lines, but more consistently and to a greater extent in the MDA-MB-468 cells. The MAPK kinase (MKK) inhibitor U0126 (10 microM), while preventing ERK phosphorylation in MDA-MB-468 cells, did not induce apoptosis. The PI3K inhibitor LY294002 (50 microM) inhibited PKB phosphorylation in both cells lines, but only induced apoptosis in the MDA-MB-468 line. These results suggest that while curcumin has several different molecular targets within the MAPK and PI3K/PKB signalling pathways that could contribute to inhibition of proliferation and induction of apoptosis, inhibition of basal activity of Akt/PKB, but not ERK, may facilitate apoptosis in the tumour cell line.

Phase I Randomized, Double-Blind Pilot Study of Micronized Resveratrol (SRT501) in Patients with Hepatic Metastases—Safety, Pharmacokinetics, and Pharmacodynamics

The phytochemical resveratrol has undergone extensive preclinical investigation for its putative cancer chemopreventive properties. Low systemic availability of the parent compound due to rapid and extensive metabolism may confound its usefulness as a potential agent to prevent malignancies in organs remote from the site of absorption. Micronization allows increased drug absorption, thus increasing availability. Here we describe a pilot study of SRT501, micronized resveratrol, given as 5.0 g daily for 14 days, to patients with colorectal cancer and hepatic metastases scheduled to undergo hepatectomy. The purpose of the study was to assess the safety, pharmacokinetics, and pharmacodynamics of the formulation. SRT501 was found to be well tolerated. Mean plasma resveratrol levels following a single dose of SRT501 administration were 1,942 ± 1,422 ng/mL, exceeding those published for equivalent doses of nonmicronized resveratrol by 3.6-fold. Resveratrol was detectable in hepatic tissue following SRT501 administration (up to 2,287 ng/g). Cleaved caspase-3, a marker of apoptosis, significantly increased by 39% in malignant hepatic tissue following SRT501 treatment compared with tissue from the placebo-treated patients. SRT501 warrants further clinical exploration to assess its potential clinical utility. Cancer Prev Res; 4(9); 1419–25. ©2011 AACR.

Predicting the physiological relevance of in vitro cancer preventive activities of phytochemicals.

AbstractThere is growing interest in the ability of phytochemicals to prevent chronic diseases, such as cancer and heart disease. However, some of these agents have poor bioavailability and many of the in-depth studies into their mechanisms of action have been carried out in vitro using doses which are unachievable in humans. In order to optimize the design of chemopreventive treatment, it is important to determine which of the many reported mechanisms of action are clinically relevant. In this review we consider the physiologically achievable doses for a few of the best studied agents (indole-3-carbinol, diindolylmethane, curcumin, epigallocatechin-3-gallate and resveratrol) and summarize the data derived from studies using these low concentrations in cell culture. We then cite examples of in vitro effects which have been observed in vivo. Finally, the ability of agent combinations to act synergistically or antagonistically is considered. We conclude that each of the compounds shows an encouraging range of activities in vitro at concentrations which are likely to be physiologically relevant. There are also many examples of in vivo studies which validate in vitro observations. An important consideration is that combinations of agents can result in significant activity at concentrations where any single agent is inactive. Thus, for each of the compounds reviewed here, in vitro studies have provided useful insights into their mechanisms of action in humans. However, data are lacking on the full range of activities at low doses in vitro and the benefits or otherwise of combinations in vivo.

Differences between human breast cell lines in susceptibility towards growth inhibition by genistein.

Genistein is thought to contribute to the putative breast cancer preventive activity of soya. The mechanisms by which it arrests the growth of breast cells are incompletely understood. In order to explore generic features of the modulation of human breast cell growth by genistein, its effects on cell lines MCF-7, ZR-75.1, T47-D, MDA-MB 468, MDA-MB 231 and HBL 100 were compared. Genistein at 1 μM stimulated growth only in MCF-7 cells. At 10 μM it arrested the growth of all 6 cell types, however that of T47-D and HBL 100 cells only in medium with reduced (2%) fetal calf serum. Genistein induced apoptosis in only MDA-MB 468 cells. It arrested cells in the G2 stage of the cell cycle in all cell lines except ZR-75.1. Cells differed in their susceptibility towards inhibition by genistein of phorbol ester-induced proto-oncogene c-fos levels, transcription factor activator protein-1 (AP-1) activity and extracellular signal-regulated kinase (ERK) activity. Genistein augmented anisomycin-induced levels of proto-oncogene c-jun in ZR 75.1 and MCF-7 cells. The results suggest that induction of apoptosis, G2 cell cycle arrest and inhibition of c-fos expression, AP-1 transactivation and ERK phosphorylation may contribute to the growth-inhibitory effect of genistein in some breast cell types, but none of these effects of genistein constitutes a generic mode of growth-arresting action.

Cancer chemoprevention: Evidence of a nonlinear dose response for the protective effects of resveratrol in humans and mice

Low-dose resveratrol prevents tumor growth in mice and in human tissues, suggesting a revision of development strategies for preventive dietary agents. Less is more From energy drinks to supplements to skin serums, resveratrol has been sold to the public for myriad health benefits, most famously in the anti-aging arena. In fact, at a posh wine bar, one might overhear a patron lamenting the small dose of resveratrol one receives in a glass of the red variety. Now, Cai et al. show that a low rather than a high dose of resveratrol prevents tumor growth in mice and alters metabolic pathways in human tissues. The authors compared the dose-response curves of a dietary dose of resveratrol and a 200-fold higher amount in mice that spontaneously develop colorectal adenomas—precursors to cancer—that were fed a standard or a high-fat diet. In the mice on the high-fat diet, low-dose resveratrol reduced intestinal tumor development much better than did the high dose. In mouse tumor cells, resveratrol efficacy was tracked with an increase in autophagy and senescence markers and activation of adenosine monophosphate (AMP)–activated protein kinase (AMPK)—an enzyme that functions in the maintenance of cellular energy homeostasis. Exposure of human colorectal cancer tissue to low concentrations of resveratrol also caused an increase in autophagy and activation of AMPK. Colorectal mucosal samples isolated from cancer patients who received a low-dose resveratrol regimen before tumor resection showed an increase in expression of the cytoprotective, oxidative stress-activated enzyme NAD(P)H dehydrogenase, quinone 1 (NQO1). These findings suggest that resveratrol operates by modulating energy balance and responding to stress. At a time when “supersizing” is popular, the nonlinear dose-response documented in the new work suggests that its time for a revision in development strategies for preventative dietary agents. Resveratrol is widely promoted as a potential cancer chemopreventive agent, but a lack of information on the optimal dose prohibits rationally designed trials to assess efficacy. To challenge the assumption that “more is better,” we compared the pharmacokinetics and activity of a dietary dose with an intake 200 times higher. The dose-response relationship for concentrations generated and the metabolite profile of [14C]-resveratrol in colorectal tissue of cancer patients helped us to define clinically achievable levels. In ApcMin mice (a model of colorectal carcinogenesis) that received a high-fat diet, the low resveratrol dose suppressed intestinal adenoma development more potently than did the higher dose. Efficacy correlated with activation of adenosine monophosphate–activated protein kinase (AMPK) and increased expression of the senescence marker p21. Nonlinear dose responses were observed for AMPK and mechanistic target of rapamycin (mTOR) signaling in mouse adenoma cells, culminating in autophagy and senescence. In human colorectal tissues exposed to low dietary concentrations of resveratrol ex vivo, we measured enhanced AMPK phosphorylation and autophagy. The expression of the cytoprotective NAD(P)H dehydrogenase, quinone 1 (NQO1) enzyme was also increased in tissues from cancer patients participating in our [14C]-resveratrol trial. These findings warrant a revision of developmental strategies for diet-derived agents designed to achieve cancer chemoprevention.

Comparison of oxaliplatin- and curcumin-mediated antiproliferative effects in colorectal cell lines

Colorectal cancer remains a leading cause of cancer death worldwide, despite markedly improved response rates to current systemic therapies. Oxaliplatin either alone or incorporated into 5‐fluorouracil/leucovorin regimes has resulted in increased survival rates, particularly with regards to metastatic colorectal carcinoma. The chemopreventive polyphenol curcumin, which is currently in clinical trial, has been advocated for use in colorectal cancer either singly or in combination with chemotherapeutic drugs. In this study, the antiproliferative capacity of both compounds was compared in HCEC (normal‐derived), HT29 (p53 mutant adenocarcinoma) and HCT116 (p53wt adenocarcinoma) colorectal cell lines to determine whether effects were cell‐type specific at pharmacologically achievable doses, and whether the combination resulted in enhanced efficacy. Both oxaliplatin and curcumin displayed marked antiproliferative capacity at therapeutic concentrations in the two tumor cell lines. Order of sensitivity to oxaliplatin was HCT116>HT29>HCEC, whereas order of sensitivity to curcumin was HT29>HCT116>HCEC. HCT116 cells underwent induction of G2/M arrest in response to both oxaliplatin (irreversible) and curcumin (reversible). Apoptosis was induced by both agents, and up to 16‐fold induction of p53 protein was observed in response to the combination. Antiproliferative effects in HT29 cells were largely cell cycle independent, and were mediated by induction of apoptosis. Effects were greatly enhanced in both cell lines when agents were combined. This study provides further evidence that curcumin may be of use in therapeutic regimes directed against colorectal cancer, and suggests that in combination with oxaliplatin it may enhance efficacy of the latter in both p53wt and p53 mutant colorectal tumors.

Prolonged biologically active colonic tissue levels of curcumin achieved after oral administration--a clinical pilot study including assessment of patient acceptability.

Curcumin, the main constituent of turmeric, is suspected to possess cancer chemopreventive properties. Pharmacokinetic and pharmacodynamic parameters have been reported, but few data exist describing whether methodologies are suitably robust for curcuminoid detection in colonic biopsy specimens. Information on the acceptability of prolonged administration of daily curcumin is not available. This is of vital importance to implement chemoprevention strategies. This study aimed to quantify levels of curcuminoids in colorectal mucosa of patients undergoing colorectal endoscopy or surgical resection and to obtain information on the acceptability and compliance with daily curcumin. Curcumin C3 complex (2.35 g) was administered to patients once daily for 14 days before endoscopic biopsy or colonic resection. Safety and tolerance were monitored. Analysis of curcuminoids in plasma, urine, and colonic mucosa was conducted by ultraperformance liquid chromatography (UPLC)-UV with characterization by liquid chromatography/tandem mass spectrometry (LC/MS-MS). Twenty-four of 26 patients commencing curcumin completed the course. Six patients reported mild gastrointestinal adverse events. Curcuminoids were detectable in nine of 24 plasma samples, 24 of 24 urine samples, and in the colonic mucosa of all 23 biopsied participants. Mean tissue levels were 48.4 μg/g (127.8 nmol/g) of parent curcuminoids. The major conjugate, curcumin glucuronide, was detectable in 29 of 35 biopsies. High levels of topical curcumin persisted in the mucosa for up to 40 hours postadministration. Sixteen participants (67%) stated that they would take curcumin long-term should it be of proven benefit. In summary, pharmacologically active levels of curcumin were recovered from colonic mucosa. The regimen used here seems safe, and patients support its use in long-term trials. Cancer Prev Res; 6(2); 119–28. ©2012 AACR.

Curcumin ameliorates oxaliplatin-induced chemoresistance in HCT116 colorectal cancer cells in vitro and in vivo†

The aims of this study were to determine potency of oxaliplatin in combination with curcumin in oxaliplatin‐resistant cell lines in vitro and to evaluate the efficacy of a novel curcumin formulation (Meriva®) alone and in combination with oxaliplatin in colorectal tumor‐bearing mice, exploring relevant pharmacodynamic markers in vivo. Oxaliplatin‐resistant HCT116 p53wt and p53−/− cell lines were generated, and the effects of oxaliplatin in combination with curcumin on resistance‐ and proliferation‐associated proteins investigated. Eighty nude mice were implanted with HCT116 p53wt colorectal cancer cells before randomization into the following treatment groups: control; Meriva only; oxaliplatin only; Meriva + oxaliplatin. Tumor volume was assessed, as was the expression of Ki‐67, cleaved caspase‐3 and Notch‐1. Curcumin in combination with oxaliplatin was able to decrease proliferative capacity of oxaliplatin‐resistant p53 wildtype and p53−/− cell lines more effectively than oxaliplatin alone. It also decreased markers associated with proliferation. After 21 days of treatment in the xenograft model, the order of efficacy was combination > Meriva > oxaliplatin > control. The decrease in tumor volume when compared to vehicle‐treated animals was 53, 35 and 16%, respectively. Ki‐67 and Notch‐1 immunoreactivity was decreased by the combination when compared to vehicle‐treated animals, with cleaved caspase‐3 rising by 4.4‐fold. Meriva did not adversely affect the DNA‐platinating ability of oxaliplatin. Curcumin enhanced the cytotoxicity of oxaliplatin in models of oxaliplatin resistance in vitro. In vivo, Meriva greatly enhanced oxaliplatin efficacy, without affecting the mode of action of oxaliplatin. Addition of formulated curcumin to oxaliplatin‐based chemotherapy regimens has the potential for clinical benefit.

Curcumin inhibits cancer stem cell phenotypes in ex vivo models of colorectal liver metastases, and is clinically safe and tolerable in combination with FOLFOX chemotherapy

Highlights • Curcumin + FOLFOX inhibits growth of primary cancer stem cell (CSC) spheroid models.• Curcumin + FOLFOX decreases expression of CSC markers in primary CSC spheroid models.• Curcumin enhances proapoptotic effects of chemotherapy in explant culture.• Curcumin is safe and tolerable in combination with FOLFOX chemotherapy.• Curcumin is perceived by patients as an acceptable daily adjunct to chemotherapy.