Description: This native perennial shrub produces little-branched canes up to 6′ long during the first year. These canes are initially erect, but they eventually arch sideways and downward – their tips sometimes reach the ground. First-year canes are vegetative and do not produce flowers and fruit. They are initially green, hairless, and glaucous, but later turn brown and woody during the winter. Scattered along the length of each cane are prickles that are short and curved. During the second year, these canes develop short branches that terminate in erect cymes or short racemes of flowers. Along the length of these canes, there are alternate compound leaves. These compound leaves are usually trifoliate; rarely are they palmate with 5 leaflets. The leaflets are up to 3″ long and 2″ across. They are cordate-ovate or ovate in shape and doubly serrate along the margins; some leaflets may be shallowly cleft. The upper surface of each leaflet has strong pinnate venation, while its lower surface is white tomentose (covered with white hairs that are very short and appressed). The terminate leaflet has a short slender petiole, while the lateral leaflets are sessile, or nearly so. The flowers are bunched tightly together on the cymes/racemes. Each flower is about ½” across, consisting of 5 white petals, 5 green sepals, and numerous stamens that surround the multiple green carpels and their styles. The petals are elliptic or oblong, while the sepals are triangular-shaped and spreading; the petals are about the same length as the sepals. The blooming period occurs during the late spring or very early summer and lasts about 2-3 weeks. Each flower is replaced by a compound drupe that is ovoid and about 1/3″ long when fully mature. This compound drupe is initially white, later becomes red, and finally turns black-purple when it is mature. Each drupe consists of multiple drupelets, each drupelet containing a single seed. The fleshy drupes are sweet and slightly tart in flavor; they detach cleanly and easily from their receptacles. The root system consists of a woody branching taproot. Vegetative offsets are often produced by the canes rooting at their tips.

Cultivation: The preference is partial sun, moist to mesic conditions, and rich loamy soil. In areas that are too sunny and dry, the fruit may not develop properly without adequate rain. The canes also fail to set fruit if there is too much shade.

Range & Habitat: Habitats include openings in deciduous woodlands, woodland borders, savannas, thickets, fence rows, overgrown vacant lots, powerline clearances in wooded areas, and partially shaded areas along buildings. Black Raspberry adapts well to human-related disturbance; it also occurs in higher quality natural areas.

Faunal Associations: The nectar of the flowers attracts bees primarily, including honeybees, bumblebees, Mason bees, Leaf-Cutting bees (Megachile spp.), Cuckoo bees (Nomadine), Halictid bees, and Andrenid bees. The short-tongued bees also collect pollen from the flowers. Less often, small butterflies and skippers may visit the flowers for nectar. Because raspberries are economically important, insects that feed on the foliage, stems, and fruit are fairly well known. Insects that chew on the foliage or suck sap include caterpillars of various moths, spider mites and flea beetles, and leafhoppers. Insects that bore through canes or roots include the caterpillars of some moths and grubs of various wood-boring beetles . The fruit of both raspberries and blackberries is an important source of food for many upland gamebirds and songbirds. Raccoons, fox squirrels, and chipmunks occasionally eat the fruit, while rabbits and deer browse on the foliage and stems.

Photographic Location: Along a fence row at the webmaster’s apartment complex. In the middle photograph, the flowering 2nd-year cane was broken off by lawnmowers.

Comments: While the flowers of Black Raspberry are not very showy, the fruit has excellent flavor and is rather colorful. The prickly canes are unpleasant to walk through or mow around, although less so than many species of blackberries. While both raspberries and blackberries are members of the same genus, the drupes of raspberries detach cleanly and easily from their receptacles, while the drupes of blackberries do not. Other native raspberries in Illinois produce drupes that are red at maturity, rather than black-purple. When drupes are unavailable for observation, Black Raspberry can be identified by the following features: 1) The white petals of the flowers are narrow, rather than broad and overlapping, 2) the compound leaves are usually trifoliate, rather than palmate with 5 or more leaflets, 3) the leaflets are white tomentose on their undersides, rather than some shade of green, and 4) young vegetative canes often have a white bloom that can be rubbed off (i.e., they are glaucous).

Particularly in the prevention of cancer, black raspberries are revealing remarkable effects as anti-tumor agents in laboratory research. In pursuit of this important finding, over the past six years no other fruit has had as much progress toward human clinical trials as the black raspberry.

Let’s have a look at the scientific and health foundations for the benefits of including black raspberries in your diet.

How many species of black raspberries are commonly available to the general consumer?

In Canada and the US, there are two main species called Rubus occidentalis L. and Rubus eucodermis L. Rubus (a genus of plants in the family Rosaceae, subfamily Rosoidea). The Rubus is characterized by rose-like thorns along vines forming brambles that grow as irregular canes ranging from one to four meters in length. It is conventional to include black raspberries among other Rubus berries (red raspberries, blackberries, boysenberries, loganberries) as “brambleberries” or “caneberries” that have grown native in North America since recorded time.

How is the black raspberry different from other Rubus species?

Often called “blackcaps” by berry farmers, black raspberries are the smallest and bluest of the Rubus berries. Scientific assays of blackcap phytochemicals reveal the densest and most avid pigmentation among North American plants–so strong that the US Department of Agriculture used black raspberry juice as stamp dye on meats for several decades. Scientific tests have described blackcaps with the richest contents of pigment antioxidants among North American berries (as described further below).

Where do blackcaps grow and what markets are served?

Black raspberries grow primarily in the western US and in British Columbia. They prefer wet, mild winters and humid, warm summers and are harvested over the month of July. Oregon’s Willamette Valley, especially, is noted for black raspberries accounting for 95% of total US production or about 4 million pounds per year. Only 5% of black raspberries are sold fresh. The market for processing to provide year-round supplies of frozen (immediately quick frozen, IQF), pureed or concentrated juice products makes up the rest.

What qualities of black raspberries appeal to consumers?

The intense pigmentation qualities mentioned above derive from a rich concentration and diversity of phenolic acid antioxidants in blackcaps. Phenolics give black raspberries several distinctive taste and chemical qualities, including high acidity (pH of about 3.5), semi-sweet taste (brix of about 10 in IQF fruit, 17 in puree) and a special tangy full-bodied taste that appeals to most people, including children. Blackcaps are very versatile regular in dessert recipes.

Over the past few years, research primarily at Ohio State University by Dr. Gary Stoner and colleagues has shown exciting - even astonishing - results of black raspberry phenolic extracts and whole berries themselves on colon and esophageal cancers in experimental animals. The focus has been on the antioxidant qualities particularly of ellagic and ferulic acids (both phenolic antioxidants) from blackcaps.

Black raspberries are one of the world’s most promising tools from plant foods in the fight against cancer. They are now entering human clinical trials supervised by Dr. Stoner.

What are oxidants and antioxidants?

Moment by moment throughout the cells of our bodies, free radicals (i.e., oxidants or radical oxygen species) are continuously being generated by normal metabolism. Exposure to toxins in the environment, or irradiation, increases free radical production. Free radicals are unstable atoms having potential to damage cells and alter genes if not quickly neutralized.

Our bodies defend against oxidation through enzymes called dismutases, catalases, reductases and peroxidases. Also, our diet provides a host of chemicals serving antioxidant roles. These chemicals include: vitamins A, C and E; minerals like selenium, manganese and zinc; and pigments from the plant foods we eat.

In black raspberries, phenolic pigments like ellagic and ferulic acids are the major antioxidant source.

What are other pigments present in black raspberries?

The rich color of black raspberries comes from numerous chemicals of the phenolic super-family that is a class of several thousands of members serving plants as pigments. Black raspberry antioxidant strength is proportional to the intensity of the dye from its pigments.

A major subgroup of pigment phenolics is the flavonoid group that is densely populated in blackcaps. One flavonoid class in particular - anthocyanins - accounts for most of the blue-red-black pigmentation of black raspberries. In addition to anthocyanins, however, are ellagic acid, ferulic acid, gallic acid, rutin and cyanidin glycosides (phenolics identified by Dr. Stoner and colleagues as important anticarcinogens in blackcaps). Vitamin C, a universal antioxidant, is also well concentrated in black raspberries (about 2 mg per 100 grams of fruit), as is calcium (32 mg per 100 grams).

Is there a way to measure antioxidant quality of a plant food and how well do black raspberries perform?

Yes, a test called ORAC (oxygen radical absorbance capacity) is performed on a food sample in a test tube. Then, the strength of antioxidant is measure by how well it neutralizes free radicals.

At Oregon State University, black raspberries have been tested for ORAC against other berries and plant foods (first analyzed in a 2004 ORAC report by US Department of Agriculture scientists). Black raspberries had the highest ORAC among other caneberries, fruits or vegetables analyzed measuring 3 times higher ORAC than blackberries or red raspberries.

Plants with pale skins and white pulps like pears and some apples have low ORAC whereas dark fruit like black raspberries, blueberries and cranberries have relatively high ORAC.

What does a high ORAC from black raspberries mean for health protection?

The answer to this question requires actual clinical research in humans (being performed but still preliminary) but the scientific evidence points to widespread protection against numerous diseases by having a diet rich in high-ORAC foods like black raspberries.

What are some diseases that evolve from free radicals and what can we do to counter oxidative stress?

Growing scientific evidence shows that nearly every disease involves free radicals to some extent. Cancer, heart and vascular disease, diabetes, inflammation and neurological disorders all have strong components of oxidative stress. Premature aging and diseases of the elderly such as macular degeneration are thought to result from oxidative damage to cells as well.

The laboratory studies by Dr. Stoner point to the importance of oxidative factors in colon and esophageal cancer, and to the potential protective role of phenolics from black raspberries or other antioxidant-rich plant foods.

Consumers can enjoy the pleasures of dark berries in their diets while likely gaining antioxidant protection from berry phenolics. Why wait for positive results from the years of clinical research ahead? The preliminary evidence and pleasurable eating qualities of black raspberries should be enough to warrant including these berry treasures in your diet now

Visiting Libra Scholar John Lechner is working on creating a pill packed with cancer-fighting nutrients in USM?s Wise Laboratory of Environmental and Genetic Toxicology.

Lechner is building on research he began at the Comprehensive Cancer Center of The Ohio State University. There, he fed rats suffering from esophageal cancer a diet of five percent freeze-dried black raspberries, a berry commercially grown in Ohio, and observed a reduction in those cancers.

He also provided another group of cancer-bearing animals drinking water laced with red beet juice and observed cancer reduction.

At USM, he is feeding laboratory mice the beet juice water in the hope of finding that it will stop the animals from getting lung cancer.

Plans also are underway to investigate the benefits of compounds found in fish, and discussions have begun to start human trials that would attempt to reduce free radicals that negatively affect the health of diabetes patients.

Abbreviations: BRB, black raspberry powder; COX-2, cyclooxygenase-2; ESCC, esophageal squamous cell carcinoma; HPRT, hypoxanthine guanine phosphoribosyltransferase; iNOS, inducible nitric oxide synthase; MVD, microvessel density; NFB, nuclear factor B; NMBA, N-nitrosomethylbenzylamine; PGE₂, prostaglandin E₂; VEGF, vascular endothelial growth factor

	Introduction

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Esophageal squamous cell carcinoma (ESCC) is one of the most lethal gastrointestinal malignancies worldwide (1). The incidence rate of this disease varies dramatically in different geographical areas; China has the highest rate, with 250 000 new cases diagnosed yearly, accounting for half of the world’s new cases (2). Risk factors for ESCC include tobacco use, alcohol consumption, nutritional deficiency and intake of food contaminated with various mycotoxins. ESCC grows more quickly than other kinds of gastrointestinal malignancies and patients with this disease have a very poor prognosis. The major causes of death are hematogenous metastasis to liver and lung and lymph metastasis (3). Although surgery, chemotherapy and radiotherapy alone or combined are used, the overall 5 year survival rate for this disease is still very low, ranging from 5 to 15%.

Angiogenesis, the formation of new capillaries from preexisting blood vessels, was first defined by Shubik in 1968 (4). It is essential for the tumor growth and expansion because it enhances the opportunities for tumor cells to reach the general circulation and metastasize. Solid tumors <2 mm in diameter can obtain oxygen and nutrients from neighboring blood vessels by simple passive diffusion. Beyond this size, new vasculature is required for tumor cell growth and survival (5). Several growth factors were reported to have angiogenic activity including vascular endothelial growth factor (VEGF), basic fibroblast growth factor and platelet-derived growth factors (6). Unlike other growth factors, VEGF is a highly specific mitogen for vascular endothelial cells, inducing their proliferation and migration. VEGF, therefore, is regarded as the major angiogenesis factor during carcinogenesis and tumor metastasis. The VEGF family includes VEGF-A, -B, -C, -D and -E (7). The different regulation and tissue distribution of these family members suggest that they have different roles in angiogenesis. VEGF-A is overly expressed in human colon cancer (8). VEGF-C is upregulated in various human cancers—lung, breast, head and neck, thyroid, stomach, uterus, prostate, colon, and ESCC (9–17). VEGF-D expression is elevated in human colorectal cancer (18).

Microvessel density (MVD) in histological sections is commonly assayed to estimate the degree of new blood vessel formation. Immunohistochemistry is performed to highlight blood endothelial cells using antibodies against either platelet endothelial cell adhesion molecular-1 (PECAM-1, CD31), CD34, CD36, CD105, Ulex europaeus agglutinin 1 or von Willebrand factor (19,20). Numerous studies demonstrate a positive correlation between VEGF expression and MVD in human ESCC (21).

Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are two critical inducible enzymes that are elevated in many human cancers including ESCC (22,23). Their metabolites, prostaglandin E₂ (PGE₂) and nitric oxide (NO), can affect cell proliferation, differentiation and angiogenesis (24). The expression of genes for VEGF, COX-2 and iNOS are correlated in human lung, colon, prostate, pancreas and gastric cancers (25–29). To our knowledge, no study has assessed the relationships among VEGF, COX-2 and iNOS in human ESCC.

Our laboratory has been working on chemoprevention of esophageal cancer for more than 20 years using pure synthetic compounds and natural food products. A series of studies in our laboratory demonstrated that dietary black raspberries have many protective effects in a rodent animal model of carcinogen-induced ESCC through reducing DNA adduct formation, decreasing preneoplastic cell proliferation and inhibiting the expression and activity of COX-2 and iNOS (30,31). However, the anti-angiogenic potential of dietary black raspberry powder (BRB) has never been evaluated.

The present study was designed to retrospectively examine the expression of VEGF-C and -D and MVD in esophagus tissue from rats treated with N-nitrosomethylbenzylamine (NMBA) alone or NMBA plus dietary BRB. These tissues were collected from a previous chemoprevention study in which rats fed BRB had 41% fewer tumors than rats fed a regular diet after exposure to NMBA (31). In addition, to contribute to our understanding of the mechanism of inhibition of VEGF by BRB, we determined whether there were correlations between VEGF and COX-2, VEGF and iNOS, and COX-2 and iNOS expressions in the esophagi from rats treated with NMBA alone versus NMBA plus dietary BRB.

	Materials and methods

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Chemicals and reagent kits
TRIzol reagent was obtained from Gibco BRL (Gaithersburg, MD). The QuantiTect SYBR Green RT–PCR Kit was purchased from Qiagen (Valencia, CA). Primers for VEGF-C, VEGF-D and the housekeeping gene hypoxanthine guanine phosphoribosyltransferase (HPRT) were synthesized by Life Technologies (Gaithersburg, MD). CD34 antibody was obtained from BioGenex (San Ramon, CA).

Animal treatments and sample collections
Esophageal samples for the current investigation of angiogenesis were obtained from a previous chemoprevention study (31). In brief, 125 male Fischer-344 rats, aged 4–5 weeks, were divided into three groups of 25–50 animals each and treated s.c. with either NMBA (0.25 mg/kg body weight), or a 1:4 mixture of dimethyl sulfoxide:H₂O (the solvent for NMBA) three times per week for 5 weeks. Starting from Week 6, the NMBA-treated rats were fed either regular AIN-76A diet or AIN-76A diet containing 5% BRB. Another group of 25 rats was fed dietary 5% BRB only. At Week 25, 25–50 animals per group were killed and esophageal tumors were counted and sized. The tumor data from this study have been reported (31). One-half of the tissue from each esophagus was collected and frozen immediately in liquid nitrogen and then transferred to a –80°C freezer for subsequent molecular analysis. The other half of each esophagus was fixed in 10% neutral buffered formalin for 4 h and then transferred to phosphate-buffered saline to make paraffin embedded blocks for immunohistochemical staining.

RNA isolation and real-time RT–PCR
Total RNA was extracted from frozen esophagi using TRIzol reagent according to the manufacturer’s instructions. All RNA samples were analyzed for integrity of 18S and 28S rRNA by ethidium bromide staining of 1 µg RNA resolved by electrophoresis on 1.2% agarose formaldehyde gels. One-step real-time RT–PCR was performed in a GeneAmp 5700 sequence detection system (Perkin-Elmer, Norwalk, CT) using the QuantiTect SYBR Green RT–PCR Kit as recommended by the manufacturer. Primers for VEGF and HPRT were designed according to published sequences with Primer Express Software V 2.0 (Applied Biosystems, Foster City, CA). The primer sequences were VEGF-C, 5′-GGCCCCAAACCAGTCACA-3′ (forward) and 5′-CTGTAAACATCCAGTTTAGACATGCA-3′ (reverse); VEGF-D, 5′-GGTGCCGGTTGAAGCTTAAA-3′ (forward) and 5′-GGTGGAGCGATGGGATGTT-3′ (reverse) and HPRT, 5′- GCTCGAGATGTCATGAAGGAGAT-3′ (forward) and 5′-AGCAGGTCAGCAAAGAACTTATAGC-3′ (reverse). RT was first performed at 50°C for 30 min. PCR conditions were 94°C for 15 s, 60°C for 30 s and 72°C for 30 s for 40 cycles. The expression of VEGF-C and -D mRNA was normalized against expression of HPRT. The gene expression was expressed as the amount of change calculated by , where C_T is the PCR cycle number that crossed the threshold, C_T is calculated as C_TVEGF – C_THPRT and C_T is calculated as (C_TVEGF – C_THPRT)_{NMBA alone} – (C_TVEGF – C_THPRT)_{NMBA untreated} or (C_TVEGF – C_THPRT)_NMBA+BRB – (C_TVEGF – C_THPRT)_{NMBA untreated}.

Immunohistochemistry
The entire esophagus from each animal was cut into three sections as follows: upper, middle and lower. All three sections were embedded on edge in a single block. The blocks were serially sectioned at 4 µm and mounted on SuperFrost Plus slides (Fisher Scientific, Pittsburgh, PA). As described previously (32), paraffin was removed with histoclear and the slides were rehydrated in graded ethanol (100–70%). Sections were first incubated with 3% hydrogen peroxide, casein, goat serum, and avidin and biotin sequentially to decrease the non-specific binding and then incubated with mouse monoclonal antibody against CD34 (1:20) for 30 min at room temperature. Antibody incubation was followed by 20 min incubation with a mouse absorbed link (goat anti-mouse biotinylated immunoglobin) and a strepavidin–horseradish peroxidase label. The sections were developed with diaminobenzidine chromogen and then counterstained with hematoxylin, dehydrated and mounted. Reagents were supplied by BioGenex.

Determination of MVD
Esophageal MVD was measured by staining sections with antibody specific for CD34 expressed by vascular endothelial cells. Slides were viewed and photographed with a dual-head Nikon microscope with a high-resolution spot camera interfaced with computer-loaded image analysis software (Simple PCI Imaging Systems; Compix, Cranberry Township, PA). The criteria used to identify microvessels in immunostained sections were established by Folkman (4). In brief, the vessel count was performed using x200 magnification (x20 objective and x10 ocular). Any brown-staining endothelial cells or endothelial cell clusters that were clearly separate from adjacent blood vessels, tumor cells or other connective tissue elements were considered a single countable microvessel. The distinct clusters of stained endothelial cells that might be from the same vessel snaking its way in and out of the section were considered distinct and countable as separate microvessels. Vessel lumens were not necessary for a structure to be defined as a microvessel, and red cells were not used to define a vessel lumen. We evaluated the entire esophagus and counted microvessels staining positive for CD34 in all areas including normal epithelium, hyperplasia, dysplasia and papillomas. This procedure allowed the counts to be more representative for each individual esophagus. The total length of each individual esophagus varied from 6 to 10 cm and it was measured to be at the average of 6.5–7.5 cm by ruler. MVD was calculated by dividing the total number of microvessels in each esophagus by the length of the esophagus. Microvessel counts were done independently by two investigators on 10 esophagi per experimental group.

Statistical analysis
All statistical analysis was carried out using GraphPad Prism 4.0. 2^–C_T, –C_T and MVD values were analyzed and compared using one-way ANOVA followed by Dunnet’s multiple comparison test to identify individual differences among groups when the ANOVA was significant. The Spearman’s correlation coefficient was used to determine any correlation between the expression of VEGF, COX-2 and iNOS mRNA. Differences were considered statistically significant at P < 0.05. All P-values were two-sided.

	Results

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BRB downregulates VEGF mRNA expression
To determine whether dietary BRB has anti-angiogenic effects, both VEGF-C and VEGF-D mRNA were assessed by real-time RT–PCR. VEGF-D mRNA expression in rat esophagus did not change after rats were treated with NMBA. In contrast, VEGF-C mRNA expression was significantly increased in rats treated with NMBA; if rats were fed BRB after NMBA treatment, the overly expressed VEGF-C mRNA was reduced from a 2.38 ± 0.34-fold increase in animals treated with NMBA alone to 1.08 ± 0.22-fold increase in animals treated with NMBA plus BRB (55% reduction, P < 0.005) (Figure 1).

View larger version (34K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Changes in VEGF-C mRNA expression in rat esophagus resulting from BRB. Total RNA was extracted from frozen esophagi collected from NMBA-untreated rats fed with BRB (BRB control), NMBA-treated rats fed with AIN-76A diet (NMBA control) and NMBA-treated rats fed with BRB (NMBA + BRB). The values are expressed as mean; bars, ± SE. **P < 0.005 as determined by ANOVA when compared with the control diet group.

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BRB inhibits MVD
As shown in Figure 2, normal esophageal epithelium contained few capillaries. In rats treated with NMBA alone, the microvessels were more numerous and packed. However, rats fed with NMBA plus BRB had fewer microvessels (P < 0.0001) (Table I).

View larger version (77K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Microvessel density staining pattern in subepithelial plexus of normal epithelium (A), NMBA-treated epithelium (B) and NMBA plus BRB-treated epithelium (C). Esophageal MVD was measured by staining sections with antibody specific for CD34 expressed by vascular endothelial cells. The vessel count was performed at x200 magnification (x20 objective and x10 ocular). Any brown-staining endothelial cells or endothelial cell clusters that were clearly separate from adjacent blood vessels, tumor cells or other connective tissue elements were considered a single countable microvessel (arrow bar).

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Correlations between VEGF, COX-2 and iNOS expression
We previously found that BRB significantly inhibited COX-2 and iNOS mRNA in NMBA-induced esophageal tumorigenesis (31). Our data here show that BRB significantly suppressed VEGF-C mRNA. To better understand the mechanisms of BRB action, we assessed the correlations between these genes for downregulation by BRB. As shown in Figure 3A, in rats treated with NMBA alone, the expression of VEGF was correlated with the expression of COX-2 but not iNOS. In rats treated with NMBA plus BRB (Figure 3B), however, the expression of VEGF was correlated with the expressions of both COX-2 and iNOS. Moreover, the expressions of COX-2 and iNOS were correlated.

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Correlations between VEGF, COX-2 and iNOS in NMBA-treated rat esophagus (A) and NMBA plus BRB-treated rat esophagus (B).

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	Discussion

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In this study, we show that dietary BRB significantly inhibited VEGF-C and microvessel formation in NMBA-induced angiogenesis in rat esophagus. Moreover, the downregulation of VEGF-C by BRB is significantly correlated with the modulation of COX-2 and iNOS by BRB. Because angiogenesis plays a critical role in carcinogenesis and VEGF-C is elevated in human ESCC, BRB may have anti-angiogenic potential for treating this disease in humans.

Vascularization is greater in ESCC than in the normal esophageal tissues (33). Esophageal carcinogenesis is a stepwise process with the lesions progressing from normal to hyperplasia, dysplasia and carcinoma. The stage where the ‘angiogenic switch’ is located, however, has not been fully defined. We examined, therefore, MVD in the entire esophagus, including hyperplastic and dysplastic areas, which are considered to be the precancerous lesions.

It is generally accepted that angiogenesis is essential for tumor growth and metastases, which depend on the acquisition of adequate oxygen and nutrients through the blood supply (34). Several cytokines and growth factors known to promote angiogenesis include transformation growth factor-ß, transformation growth factor-, platelet-derived growth factor, basic fibroblast growth factor and VEGF. VEGF is the most potent mitogen for vascular endothelial cells. Overexpression of VEGF has been strongly associated with angiogenesis in many human cancers including ESCC.

Our laboratory and research collaborators have been investigating the effects of freeze-dried BRB powder and BRB extracts in vivo and in vitro for the past 12 years. Black raspberries contain many known compounds with antioxidant and anti-inflammatory activities, such as flavonoid compounds, vitamins and phytosterols. However, the nature of the chemical constituents has not been fully delineated. In our previous studies, we demonstrated that BRB inhibits tumor development in NMBA-induced rat ESCC, azoxymethane-induced rat colon adenocarcinoma and 7,12-dimethylbenz(a) anthracene-induced hamster oral cavity cancer (30,35,36). Inhibition of tumor development by BRB may occur via the suppression of DNA adduct formation; inhibition of cell proliferation; or downregulation of COX-2, iNOS and transcription factor c-Jun (31). Biodirected fractionation studies have been conducted to identify the most active inhibitory components. BRB extracts and anthocyanins in BRB have been tested for their effects on gene expression in JB6 Cl 41 mouse epidermal cells (37,38) and rat esophageal epithelial cell lines (data not published). A methanol extract of BRB was found to inhibit benzo(a)pyrene-7,8-diol-9,10-epoxide-induced transactivation of activator protein-1 and nuclear factor B (NFB) activity in JB6 Cl 41 cells (37). This same extract was found to inhibit VEGF expression through downregulation of the PI-3K/Akt pathway (38). An ethanol–water extract of BRB, and the anthocyanins; cyanidin-3-O-glucoside, and cyanidin-3-O-rutinoside, were all found to inhibit the proliferation rate and stimulate apoptosis in rat esophageal epithelial cells (Stoner et al.).

Potential correlations between VEGF and COX-2 and between VEGF and iNOS expressions have not previously been assessed in ESCC. In rats treated with NMBA alone, we found a correlation between VEGF and COX-2 expression but not VEGF and iNOS or COX-2 and iNOS expression. In rats treated with NMBA plus BRB, however, we found correlations among all three factors: VEGF and COX-2, VEGF and iNOS and COX-2 and iNOS.

The regulation of VEGF expression is a complex process wherein numerous genes and pathways are involved including Ras, COX-2 and iNOS. The Ras mutation contributes to tumor angiogenesis by enhancing the production of VEGF (39). In our previous study in rats, we observed Ha-ras codon 12 GA transition mutations in all papillomas induced by NMBA (40).

The precise mechanisms involved in the suppression of VEGF by BRB and the suppression associated with the inhibition of COX-2 and iNOS by BRB are not fully understood and are most likely multiple in nature. One possible explanation is that BRB inhibit VEGF expression through downregulation of COX-2 and iNOS. COX-2 is an inducible enzyme that catalyzes the formation of prostanoids including PGE₂ in response to stimuli such as growth factors, tumor promoters, hormones and cytokines (41). The major contributions of COX-2 in carcinogenesis include enhancement of cell proliferation, resistance to apoptosis and enhancement of angiogenesis (42–44). Overexpression of COX-2 was observed in various human cancers including ESCC. Numerous studies show that COX-2 derived PGE₂ stimulate interleukin-6 and induce the expression of VEGF (45). PGE₂ binding to the prostanoid receptor 2 (EP₂) increase the level of cAMP also leading to stimulation of VEGF (28). Overexpression of COX-2 itself was shown to upregulate Bcl-2 expression that results in vascular endothelial cell survival (46). Selective COX-2 inhibitors, such as NS-398 and JTE-522, were reported to effectively block angiogenesis by inhibiting VEGF expression (44,47). A positive correlation between COX-2 and VEGF was reported in numerous human cancers including non-small cell lung cancer (24), colorectal cancer (48), head and neck squamous cell carcinoma (49), endometrial carcinoma (50), renal cell carcinoma (51) and ovarian cancer (52).

iNOS is induced by certain cytokines, microbial products and lipopolysaccharides to catalyze the formation of NO and citrulline from L-arginine (53–55). Increased NO production is associated with many disorders, including cancer, through its reaction with oxygen and superoxide to produce peroxynitrite, which results in DNA damage (56). Numerous studies showed that iNOS may indirectly promote tumor angiogenesis through the induction of COX-2 and endothelial cell growth via elevated NO levels (57,58). A positive correlation between iNOS and VEGF was reported in human cancers, such as colon, lung and gastric cancers (29,59,60).

We previously found that BRB significantly decreases the expression of COX-2 and iNOS as well as the level of their metabolites, PGE₂ and NO, in NMBA-treated rat esophagus (37). With regard to VEGF, the present study suggests that BRB downregulates VEGF through its inhibitory effect on COX-2 and iNOS. A possible explanation for this observation is that some upstream molecules that control the expression of COX-2 and iNOS, such as PI-3K/Akt and NFB, are modulated by BRB (37,38). The phosphatidylinositol 3′-kinase signaling cascade plays a central role in regulating cell proliferation and survival by affecting the phosphorylation status of Akt, a downstream molecule of the cascade (61). pAkt is commonly used as the index for the activation of Akt. A recent investigation showed that BRB extracts inhibit benzo(a)pyrene diol-epoxide (BPDE)–induced VEGF transcription by targeting the PI-3K/Akt pathway in mouse epidermal JB-6 Cl 41 cells (38).

NFB is a pivotal transcription factor mediating the expression of many early response genes involved in carcinogenesis, including COX-2 and iNOS, and its overexpression is downregulated in BPDE-treated JB-6 Cl 41 cells (37,62,63). NFB has two major subunits, p50 and p65. In normal cells, NFB is sequestered in the cytoplasm in an inactive form through its association with its inhibitory protein, IB-. NFB is activated by various signals, including cytokines, mitogens, environmental and occupational particles, and bacterial products. Some reports suggest that the activation of NFB is also controlled by the activation of Akt (64). Activation of NFB results in a degradation of IB- by phosphorylation and translocation of NFB to the nucleus (65). pp65 and pIB- both are commonly used as an index for the activation of NFB.

Based on the above molecular events, we assessed the expression of pAkt, pp65 and pIB- by immunohistochemistry in normal and NMBA-treated rat esophagus. In normal rat esophagus, positive staining of pAkt and pIB- was only observed in macrophages, not in epithelial cells; positive staining of pp65 was not detected in normal esophagus. In contrast, in esophagus treated with NMBA, we observed extensive cytoplasmic staining of pAkt and pIB- and nuclear staining of pp65 in the epithelium (unpublished data). In in vitro studies, our collaborators found that BRB extracts inhibit the activation of Akt and NFB as indicated above. Thus, BRB may inhibit VEGF, COX-2 and iNOS through the suppression of upstream mediators such as Akt and NFB.

In summary, we demonstrate for the first time that dietary BRB significantly inhibits VEGF-C expression and microvessel formation in NMBA-treated rat esophagus. Since angiogenesis plays a critical role in cancer progression, BRB may have anti-angiogenesis potential in cancer therapy. Moreover, we showed that the expression levels of VEGF, COX-2 and iNOS are correlated with each other in the esophagus of rats treated with NMBA and dietary BRB. Although the precise mechanisms remain to be elucidated, the modulation of angiogenesis by BRB appears to be associated with changes in the expression of COX-2 and iNOS. Thus, BRB may offer an advantage in cancer prevention and therapy by targeting multiple signaling pathways.

	Acknowledgments

�
This research was supported by National Cancer Institute R01 grants CA96130 and CA103180.

	References

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Received January 17, 2006; revised May 27, 2006; accepted June 5, 2006.

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