Peiju Qiu

Inhibitory effects of resveratrol and pterostilbene on human colon cancer cells: a side-by side comparison

The effects of resveratrol and pterostilbene (two structurally related stilbene compounds) on three human colon cancer cells were systematically compared. Cell viability tests indicated that IC(50) values of pterostilbene were 2-5-fold lower than those of resveratrol in all three cancer cells. Pterostilbene was also more potent in inhibiting colony formation of all three cancer cells. Annexin V/propidium iodide costaining assay and Western blotting analysis showed pterostilbene had a stronger apoptosis-inducing effect, which was evidenced by the higher percentage of annexin V positive cells and higher levels of cleaved caspase-3 and poly(ADP-ribose) polymerase proteins in cancer cells treated with pterostilbene compared with resveratrol. High-performance liquid chromatography analysis demonstrated that intracellular levels of pterostilbene were 2-4-fold higher than those of resveratrol after treatments with individual compounds at the same concentration. Overall, the results demonstrated that pterostilbene had more potent inhibitory effects on colon cancer cells than resveratrol, which may be associated with the superior bioavailability of pterostilbene to resveratrol.

Synergistic Anti-inflammatory Effects of Nobiletin and Sulforaphane in Lipopolysaccharide-Stimulated RAW 264.7 Cells

Inflammation plays important roles in the initiation and progress of many diseases including cancers in multiple organ sites. Herein, we investigated the anti-inflammatory effects of two dietary compounds, nobiletin (NBN) and sulforaphane (SFN), in combination. Noncytotoxic concentrations of NBN, SFN, and their combinations were studied in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. The results showed that combined NBN and SFN treatments produced much stronger inhibitory effects on the production of nitric oxide (NO) than NBN or SFN alone at higher concentrations. These enhanced inhibitory effects were synergistic based on the isobologram analysis. Western blot analysis showed that combined NBN and SFN treatments synergistically decreased iNOS and COX-2 protein expression levels and induced heme oxygenase-1 (HO-1) protein expression. Real-time polymerase chain reaction analysis indicated that low doses of NBN and SFN in combination significantly suppressed LPS-induced upregulation of IL-1 mRNA levels and synergistically increased HO-1 mRNA levels. Overall, our results demonstrated that NBN and SFN in combination produced synergistic effects in inhibiting LPS-induced inflammation in RAW 264.7 cells.

5-Demethyltangeretin inhibits human nonsmall cell lung cancer cell growth by inducing G2/M cell cycle arrest and apoptosis

SCOPE Tangeretin (TAN) and 5-demethyltangeretin (5DT) are two closely related polymethoxyflavones found in citrus fruits. We investigated growth inhibitory effects on three human nonsmall cell lung cancer (NSCLC) cells. METHODS AND RESULTS Cell viability assay demonstrated that 5DT inhibited NSCLC cell growth in a time- and dose-dependent manner, and IC50 s of 5DT were 79-fold, 57-fold, and 56-fold lower than those of TAN in A549, H460, and H1299 cells, respectively. Flow cytometry analysis showed that 5DT induced extensive G2/M cell cycle arrest and apoptosis in NSCLC cells, while TAN at tenfold higher concentrations did not. The apoptosis induced by 5DT was further confirmed by activation of caspase-3 and cleavage of PARP. Moreover, 5DT dose-dependently upregulated p53 and p21(Cip1/Waf1), and downregulated Cdc-2 (Cdk-1) and cyclin B1. HPLC analysis revealed that the intracellular levels of 5DT in NSCLC cells were 2.7-4.9 fold higher than those of TAN after the cells were treated with 5DT or TAN at the same concentration. CONCLUSION Our results demonstrated that 5DT inhibited NSCLC cell growth by inducing G2/M cell cycle arrest and apoptosis. These effects were much stronger than those produced by TAN, which is partially due to the higher intracellular uptake of 5DT than TAN.

Identification of pinostilbene as a major colonic metabolite of pterostilbene and its inhibitory effects on colon cancer cells

SCOPE Pterostilbene (PTE) is a resveratrol derivative mainly found in blueberries, and it has been shown to inhibit colon carcinogenesis in multiple animal models. To shed light on the mechanism of PTE in inhibiting colon carcinogenesis, we investigated the PTE metabolites in the mouse colon and in the human colon cancer cells. METHODS AND RESULTS CD-1 mice were fed PTE-containing diet for 3 weeks, and colonic content and colonic mucosa were collected and subjected to LC-MS analysis. Pinostilbene (PIN) was identified as a major metabolite of PTE in the mouse colon. Importantly, the level of PIN was found to be approximately equivalent to that of PTE in the colonic mucosa. PIN significantly inhibited the growth of human colon cancer cells, i.e., HCT116 and HT29. These inhibitory effects were similar to those produced by PTE. Moreover, under physiologically relevant conditions, 20 and 40 μM of PIN caused cell cycle arrest at S phase and induced apoptosis in colon cancer cells. These effects were associated with profound modulation of signaling proteins related with cell proliferation and programmed cell death. CONCLUSION Our results demonstrated that PIN is a major metabolite of PTE in the colon of mice fed with PTE, and PIN may play important roles in the anti-colon cancer effects elicited by orally administered PTE.

Organoselenium Compounds Modulate Extracellular Redox by Induction of Extracellular Cysteine and Cell Surface Thioredoxin Reductase

The effect of selenium compounds on extracellular redox modulating capacity was studied in murine macrophage RAW 264.7 cells and differentiated human THP-1 monocytes. The arylselenium compounds benzeneselenol (PhSeH), dibenzyl diselenide (DBDSe), diphenyl diselenide (DPDSe), and ebselen were capable of inducing extracellular cysteine accumulation via a cystine- and glucose-dependent process. Extracellular cysteine production was dose-dependently inhibited by glutamate, an inhibitor of cystine/glutamate antiporter (Xc(-) transporter), supporting the involvement of Xc(-) transporter for cystine uptake in the above process. These arylselenium compounds also induced cellular thioredoxin reductase (TrxR) expression, particularly at the exofacial surface of cells. TrxR1 knockdown using small interfering RNA attenuated TrxR increases and cysteine efflux induced in cells by DPDSe. Sodium selenite (Na2SeO3), selenomethionine (SeMet), seleno-l-cystine (SeCySS), and Se-methylselenocysteine (MeSeCys) did not have these effects on macrophages under the same treatment conditions. The effects of organoselenium compounds on extracellular redox may contribute to the known, but inadequately understood, biological effects of selenium compounds.

Analysis of 10 metabolites of polymethoxyflavones with high sensitivity by electrochemical detection in high-performance liquid chromatography.

Polymethoxyflavones (PMFs) have been known as a type of bioactive flavones that possess various beneficial biological functions. Accumulating evidence demonstrated that the metabolites of PMFs, that is, hydroxyl PMFs (OH-PMFs), had more potent beneficial biological effects than their corresponding parent PMFs. To facilitate the further identification and quantification of OH-PMFs in biological samples, the aim of this study was to develop a methodology for the simultaneous determination of 10 OH-PMFs using high-performance liquid chromatography (HPLC) coupled with electrochemistry detection. The HPLC profiles of these 10 OH-PMFs affected by different chromatographic parameters (different organic composition in mobile phases, the concentration of trifluoroacetic acid, and the concentration of ammonium acetate) are fully discussed in this study. The optimal condition was selected for the following validation studies. The linearity of calibration curves, accuracy, and precision (intra- and interday) at three concentration levels (low, middle, and high concentration range) were verified. The regression equations were linear (r > 0.9992) over the range of 0.005-10 μM. The limit of detection for 10 OH-PMFs was in the range of 0.8-3.7 ng/mL (S/N = 3, 10 μL injection). The recovery rates ranged from 86.6 to 108.7%. The precisions of intraday and interday analyses were less than 7.37 and 8.63% for relative standard deviation, respectively. This validated method was applied for the analysis of a variety of samples containing OH-PMFs. This paper also gives an example of analyzing the metabolites of nobiletin in mouse urine using the developed method. The transformation from nobiletin to traces of 5-hydroxyl metabolites has been discovered by this effective method, and this is the first paper to report such an association.

Synergistic chemopreventive effects of nobiletin and atorvastatin on colon carcinogenesis

Different cancer chemopreventive agents may act synergistically and their combination may produce enhanced protective effects against carcinogenesis than each individual agent alone. Herein, we investigated the chemopreventive effects of nobiletin (NBT, a citrus polymethoxyflavone) and atorvastatin (ATST, a lipid-lowering drug) in colon cancer cells/macrophages and an azoxymethane (AOM)-induced colon carcinogenesis rat model. The results demonstrated that co-treatments of NBT/ATST produced enhanced growth inhibitory and anti-inflammatory effects on the colon cancer cells and macrophages, respectively. Isobologram analysis confirmed that these interactions between NBT and ATST were synergistic. NBT/ATST co-treatment also synergistically induced extensive cell cycle arrest and apoptosis in colon cancer cells. Oral administration of NBT (0.1%, w/w in diet) or ATST (0.04%, w/w in diet) significantly decreased colonic tumor incidence and multiplicity in AOM-treated rats. Most importantly, co-treatment of NBT/ATST at their half doses (0.05% NBT + 0.02% ATST, w/w in diet) resulted in even stronger inhibitory effects on colonic tumor incidence and multiplicity than did NBT or ATST alone at higher doses. Statistical analysis confirmed that the enhanced chemopreventive activities against colon carcinogenesis in rats by the NBT/ATST combination were highly synergistic. Our results further demonstrated that NBT/ATST co-treatment profoundly modulated key cellular signaling regulators associated with inflammation, cell proliferation, cell cycle progression, apoptosis, angiogenesis and metastasis in the colon of AOM-treated rats. In conclusion, for the first time, our results demonstrated a strong synergy in inhibiting colon carcinogenesis produced by the co-treatment of NBT and ATST, which provided a scientific basis for using NBT in combination with ATST for colon cancer chemoprevention in humans.

Abstract 3699: Novel combinatorial strategies for cancer chemoprevention using polymethoxyflavones from orange and atorvastatin

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Cancer is a complex disease, and multiple aberrant molecular events are driving forces for cancer progression. It is difficult to control cancer progression by a single agent. We investigated a novel approach to inhibit cancer cell growth by using polymethoxyflavones (PMFs, isolated from aged orange peel) and atorvastatin (Lipitor, a commonly used cholesterol-lowering drug) in combination. The goal is to identify combination regimen that can produce synergy in inhibiting human cancer cell growth. Our results on multiple colon and lung cancer cells demonstrated that permethoxylated PMFs, namely nobiletin and tangeretin, can synergistically interact with atorvastatin and produced much stronger inhibitory effects than either agent alone. On the other hand, the hydroxylated PMFs, namely 5-hydroxy nobiletin (5HN) and 5-hydroxy tangeretin (5HT) showed different interaction profiles with atorvastatin, i.e., 5HN produced strong synergy with atorvastatin while 5HT did not. Further mechanistic study demonstrated that the combination treatment with 5HN and atorvastatin for 24 hr caused extensive cell population accumulation in G0/G1 phase; whereas at 48 hr or longer, apoptosis was induced significantly. Our results also demonstrated that the combinational effects of 5HN and atorvastatin were associated with increased levels of p21Cip1/Waf1, and p27Kip1; decreased levels of CDK-2, CDK-4,CDK-6,Cyclin D1, Mcl-1 and hyper-phosphorylated Rb(Ser 780); and activation of caspase cascade. More importantly, we investigated the inhibitory effects of PMFs/atorvastatin combinations in an NNK-induced mouse lung tumorigenesis model, and our results demonstrated that dietary administration of nobiletin and 5HN in combination with atorvastatin caused potent inhibition of lung tumor formation in A/J mice. These inhibitory effects were much stronger than those produced by singular nobiletin, 5HN, or atorvastatin treatments at much higher doses. The isobologram-based statistic analysis confirmed that nobiletin, 5HN and atorvastatin produced strong synergy in inhibiting lung tumorigenesis in mice. In conclusion, the present work provided promising leads for future investigation on the novel combination of PMFs and atorvastatin as novel strategy for cancer chemoprevention. (This study was supported in part by NIH-CA139174, AICR-#10A044, CEAR pilot grant, and USDA Special Grant on Bioactive Foods). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3699. doi:10.1158/1538-7445.AM2011-3699