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Chemoprevention of Rat Mammary Carcinogenesis by Spirulina

Open ArchivePublished:November 25, 2013DOI:https://doi.org/10.1016/j.ajpath.2013.10.025
      Spirulina (SP) (Arthrospira platensis; previously Spirulina platensis) is a filamentous blue-green microalga (cyanobacterium) with potent dietary phytoantioxidant and anticancerous properties. We investigated the chemopreventive effect of SP against 7,12-dimethylbenz[a]anthracene (DMBA)–induced rat breast carcinogenesis, and further studied its underlying mechanisms of action in vitro. Remarkably, SP cleared DMBA-induced rat mammary tumors, which was clearly confirmed by morphological and histological methods. SP supplementation reduced the incidence of breast tumors from 87% to 13%. At the molecular level, immunohistochemical analysis revealed that SP supplementation reduced expression of both Ki-67 and estrogen α. More interestingly, molecular analysis in the in vitro experiments indicated that SP treatment inhibited cell proliferation by 24 hours, which was accompanied by increased p53 expression, followed by increased expression of its downstream target gene, Cdkn1a (alias p21 or p21Waf1/Cip1). In addition, SP increased Bax and decreased Bcl-2 expression, indicating induction of apoptosis by 48 hours after SP treatment. To our knowledge, this is the first report of in vivo chemopreventive effect of SP against DMBA-induced breast carcinogenesis in rat, supporting its potential use in chemoprevention of cancer.
      Breast cancer is the most common malignant disease in women worldwide, and its incidence is steadily increasing, particularly in developing countries (World Health Organization, http://www.who.int/cancer/detection/breastcancer/en/index1.html). Although conventional medicine has made significant progress in the treatment of breast cancer, pharmacological interventions are fraught with their own endogenous complications, including many known and unknown legal and ethical issues.
      • Senn H.J.
      • Morant R.
      Chemoprevention of breast and prostate cancers: where do we stand?.
      In addition, breast cancer cells acquire resistance to chemotherapeutic drugs via different cell signaling mechanisms.
      • Ouhtit A.
      • Gaur R.L.
      • Abdraboh M.
      • Ireland S.K.
      • Rao P.N.
      • Raj S.G.
      • Al-Riyami H.
      • Shanmuganathan S.
      • Gupta I.
      • Murthy S.N.
      • Hollenbach A.
      • Raj M.H.
      Simultaneous inhibition of cell-cycle, proliferation, survival, metastatic pathways and induction of apoptosis in breast cancer cells by a phytochemical super-cocktail: genes that underpin its mode of action.
      Individuals with a high risk of cancer may seek to reduce their risk by opting for dietary medicine, to prevent the disease through a healthy lifestyle. Complementary and alternative medicine (CAM) encompasses such lifestyle choices and has proven to overcome obstacles presented by drug resistance.
      • Ouhtit A.
      • Gaur R.L.
      • Abdraboh M.
      • Ireland S.K.
      • Rao P.N.
      • Raj S.G.
      • Al-Riyami H.
      • Shanmuganathan S.
      • Gupta I.
      • Murthy S.N.
      • Hollenbach A.
      • Raj M.H.
      Simultaneous inhibition of cell-cycle, proliferation, survival, metastatic pathways and induction of apoptosis in breast cancer cells by a phytochemical super-cocktail: genes that underpin its mode of action.
      CAM is defined as therapies that are used in addition to (complementary) or instead of (alternative) standard treatments. CAM may include dietary supplements, vitamins, herbal preparations, special teas, acupuncture, massage therapy, and meditation, among others. CAM use is prevalent in the general community, and the World Health Organization reports estimate that approximately 75% of the world's population rely on CAM.
      • Grawish M.E.
      • Zaher A.R.
      • Gaafar A.I.
      • Nasif W.A.
      Long-term effect of Spirulina platensis extract on DMBA-induced hamster buccal pouch carcinogenesis (immunohistochemical study).
      • Rampes H.
      • Sharples F.
      • Maragh S.
      • Fisher P.
      Introducing complementary medicine into the medical curriculum.
      A number of phytochemicals have been used in CAM, individually or in combination, to treat cancer, particularly at the late and desperate stages of disease.
      • Chen T.
      • Wong Y.S.
      In vitro antioxidant and antiproliferative activities of selenium-containing phycocyanin from selenium-enriched Spirulina platensis.
      • Cho W.C.
      Scientific evidence on the supportive cancer care with Chinese medicine.
      • Parekh H.S.
      • Liu G.
      • Wei M.Q.
      A new dawn for the use of traditional Chinese medicine in cancer therapy.
      Arthrospira platensis, commonly known as spirulina (SP), based on its historical classification as Spirulina platensis, is a filamentous blue-green microalga (cyanobacterium). Various studies have demonstrated anticancer cell proliferation effects of SP and its active ingredients both in vitro and in vivo, and our research group has reported a chemopreventive action of SP in liver carcinogenesis in a rat model.
      • Ismail M.F.
      • Ali D.A.
      • Fernando A.
      • Abdraboh M.E.
      • Gaur R.L.
      • Ibrahim W.M.
      • Raj M.H.
      • Ouhtit A.
      Chemoprevention of rat liver toxicity and carcinogenesis by Spirulina.
      Trace elements such as selenium are key components of several major metabolic pathways, and there is considerable evidence demonstrating that selenium supplementation can reduce the incidence of many human cancers.
      • Yang F.
      • Tang Q.
      • Zhong X.
      • Bai Y.
      • Chen T.
      • Zhang Y.
      • Li Y.
      • Zheng W.
      Surface decoration by Spirulina polysaccharide enhances the cellular uptake and anticancer efficacy of selenium nanoparticles.
      Selenium-enriched SP extract inhibited the growth of MCF-7 breast cancer cells through induction of G1 cell cycle arrest and mitochondria-mediated apoptosis.
      • Ouhtit A.
      • Gaur R.L.
      • Abdraboh M.
      • Ireland S.K.
      • Rao P.N.
      • Raj S.G.
      • Al-Riyami H.
      • Shanmuganathan S.
      • Gupta I.
      • Murthy S.N.
      • Hollenbach A.
      • Raj M.H.
      Simultaneous inhibition of cell-cycle, proliferation, survival, metastatic pathways and induction of apoptosis in breast cancer cells by a phytochemical super-cocktail: genes that underpin its mode of action.
      Phycocyanin (PC), which is the major biliprotein and active ingredient of SP, induced apoptosis in RAW264.7 cells through release of cytochrome c from mitochondria independently of Bcl-2 expression.
      • Andrade M.R.
      • Costa J.A.
      Outdoor and indoor cultivation of Spirulina platensis in the extreme south of Brazil.
      In vitro studies using human melanoma A375 cells and human breast adenocarcinoma MCF-7 cells suggest selenium-containing PC as a potent antiproliferative agent with potential applications in cancer chemoprevention.
      • Chen T.
      • Wong Y.S.
      In vitro antioxidant and antiproliferative activities of selenium-containing phycocyanin from selenium-enriched Spirulina platensis.
      Immunohistochemical studies have demonstrated a beneficial role of SP extract in the regression of cancer progression in 7,12-dimethylbenz[a]anthracene (DMBA)–induced hamster buccal pouch carcinogenesis.
      • Grawish M.E.
      • Zaher A.R.
      • Gaafar A.I.
      • Nasif W.A.
      Long-term effect of Spirulina platensis extract on DMBA-induced hamster buccal pouch carcinogenesis (immunohistochemical study).
      In recent years, SP has gained the attention of the scientific and medical communities for its properties as a nutraceutical and as a potential source of pharmaceutical drugs. Although diverse properties of SP have been studied in recent years, its role in cancer chemoprevention (and especially in breast carcinogenesis) has not been studied. To our knowledge, the present study is the first to investigate the protective effectiveness of SP against carcinogenesis induced by DMBA in rat mammary tissue.

      Materials and Methods

       Cells, Compounds, Antibodies, and Chemicals

      Cells of the MCF-7 human breast cancer cell line (ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium with 4500 mg/L glucose, and supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. Mesenchymal stem cells were cultured in MEM-α minimum essential medium (Life Technologies, Carlsbad, CA) supplemented with 16.5% bovine serum albumin (Atlanta Biologicals, Flowery Branch, GA) and 1% l-glutamine (Life Technologies).
      The SP algae used in the in vivo study were cultured in the laboratory of algal technology at Mansoura University (Mansoura, Egypt), under optimal conditions in Zarrouk medium, as described previously.
      • Reddy M.C.
      • Subhashini J.
      • Mahipal S.V.
      • Bhat V.B.
      • Srinivas Reddy P.
      • Kiranmai G.
      • Madyastha K.M.
      • Reddanna P.
      C-Phycocyanin, a selective cyclooxygenase-2 inhibitor, induces apoptosis in lipopolysaccharide-stimulated RAW 264.7 macrophages.
      Algal mass was harvested every 3 weeks by continuous centrifugation and then was air-dried and ground to powder for animal use. DMBA was dissolved in corn oil, and appropriate treatment solutions were prepared. A single dose of 30 mg/kg was injected intraperitoneally.
      • Macejová D.
      • Brtko J.
      Chemically induced carcinogenesis: a comparison of 1-methyl-1-nitrosourea, 7,12-dimethylbenzanthracene, diethylnitroso-amine and azoxymethane models (minireview).
      For in vitro studies, SP powder (Puritan's Pride, Oakdale, NY) was suspended in water, and the cell walls were disrupted by sonication for 5 minutes (VCX 600 system, 200 W; Sonics & Materials, Newtown, CT). The sonicated cell suspension was then centrifuged at 10,000 × g for 30 minutes, and the supernatant was collected as SP crude extract for in vitro studies.
      The primary antibodies used in Western blotting experiments were against p53 (Novocastra; Leica Microsystems, Wetzlar, Germany), p21, Bax, and Bcl-2 (Santa Cruz Biotechnology, Santa Cruz, CA). β-Actin primary antibody (Santa Cruz Biotechnology) was used as a control. Secondary antibodies appropriate for each primary antibody were used (Santa Cruz Biotechnology). Anti–Ki-67 and anti–estrogen α (anti-ERα) antibodies (Zymed; Life Technologies) were used for immunohistochemical analyses.

       AlamarBlue Cell Proliferation Assay

      The cells were cultured as described above in 96-well plates and incubated at 37°C in a 5% CO2–enriched atmosphere. Various concentrations of SP crude extract (10 to 100 μg/mL) were applied to wells for 6 days (30% alcohol was used as a vehicle control in eight wells for each concentration). Suppression of cell growth was determined using AlamarBlue assay (Alamar Biosciences, Sacramento, CA), as described previously.
      • Raj M.H.
      • Abd Elmageed Z.Y.
      • Zhou J.
      • Gaur R.L.
      • Nguyen L.
      • Azam G.A.
      • Braley P.
      • Rao P.N.
      • Fathi I.M.
      • Ouhtit A.
      Synergistic action of dietary phyto-antioxidants on survival and proliferation of ovarian cancer cells.
      In brief, AlamarBlue dye was added to wells in an amount equal to 10% of the total volume of culture medium. The oxidized form of this dye, which is nontoxic, is converted to the reduced form by mitochondrial enzyme activity of viable cells. At 4 hours after addition of the dye, the shift in fluorescence was measured at 570 nm (excitation) and 600 nm (emission) in a fluorometer (LabSystems Fluoreskan II; Thermo Fisher Scientific, Waltham, MA), and the results were expressed as percentage of control. This method provides a precise measure of the number of cells metabolizing the dye to its reduced state and is a sensitive and accurate means of monitoring cell proliferation.
      • Raj M.H.
      • Abd Elmageed Z.Y.
      • Zhou J.
      • Gaur R.L.
      • Nguyen L.
      • Azam G.A.
      • Braley P.
      • Rao P.N.
      • Fathi I.M.
      • Ouhtit A.
      Synergistic action of dietary phyto-antioxidants on survival and proliferation of ovarian cancer cells.
      To ascertain specificity and to validate the effects observed in cancer cells, mesenchymal stem cells were used as an example of noncancerous cell type. Mesenchymal stem cells were similarly treated with the various concentrations of SP and then were evaluated using the AlamarBlue assay, as described above.

       Western Blot Analysis of Molecular Pathways Affected by SP Treatment

      To explore potential molecular mechanisms that might be contributing to the observed inhibition of cell proliferation, we analyzed the components of molecular pathways associated with both cell cycle (p53 and p21Waf1/Cip1; the latter is hereafter referred to simply as p21) and apoptosis (p53, Bax, and Bcl-2) for periods of 24 and 48 hours, as described previously.
      • Ismail M.F.
      • Ali D.A.
      • Fernando A.
      • Abdraboh M.E.
      • Gaur R.L.
      • Ibrahim W.M.
      • Raj M.H.
      • Ouhtit A.
      Chemoprevention of rat liver toxicity and carcinogenesis by Spirulina.
      • Raj M.H.
      • Abd Elmageed Z.Y.
      • Zhou J.
      • Gaur R.L.
      • Nguyen L.
      • Azam G.A.
      • Braley P.
      • Rao P.N.
      • Fathi I.M.
      • Ouhtit A.
      Synergistic action of dietary phyto-antioxidants on survival and proliferation of ovarian cancer cells.
      • Ouhtit A.
      • Muller H.K.
      • Davis D.W.
      • Ullrich S.E.
      • McConkey D.
      • Ananthaswamy H.N.
      Temporal events in skin injury and the early adaptive responses in ultraviolet-irradiated mouse skin.
      MCF-7 breast cancer cells were treated with both the optimal (50 μg/mL) and a higher concentration (100 μg/mL) of the SP crude extract (as identified from AlamarBlue dose–response curve experiments). Cell lysates were prepared and proteins were extracted and quantitated using Bradford assay. Protein samples (60 μg) were boiled for 5 minutes in an equal volume of reducing buffer [5 mmol/L Tris/HCl (pH 7.4), 4% (w/v) SDS, 20% (v/v) glycerol, 10% (v/v) mercaptoethanol, and 0.2% (w/v) bromophenol blue], resolved on 12% polyacrylamide gels, and electroblotted onto nitrocellulose membranes. After probing with the appropriate primary antibodies, and then secondary antibody, proteins were visualized using Pierce SuperSignal West Femto maximum substrate sensitivity (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer's instructions. Equal loading of the protein samples was assessed by reprobing the membrane with a 1:2000 dilution of the β-actin loading control antibody (Santa Cruz Biotechnology).

       Animal Studies

      Healthy adult female virgin albino rats (Sprague–Dawley), 8 weeks old and weighing approximately 120 ± 5 g, were housed and maintained on a 12-hour light/dark cycle under a constant temperature of 25 ± 1°C with free access to food and drinking water ad libitum. Rats were acclimatized to laboratory conditions for 1 week before the start of experiments. The DMBA-induced chemical rat mammary carcinogenesis experimental design was as previously reported by Bishayee et al.
      • Bishayee A.
      • Oinam S.
      • Basu M.
      • Chatterjee M.
      Vanadium chemoprevention of 7,12-dimethylbenz(a)anthracene-induced rat mammary carcinogenesis: probable involvement of representative hepatic phase I and II xenobiotic metabolizing enzymes.
      After an acclimatization period of 1 week, rats were randomly divided into four groups (n = 10 per group) for statistical analysis (Table 1). Groups A and B were maintained on standard diet without any further treatment; groups C and D were fed on a standard diet mixed with 1% SP powder.
      Table 1Experimental Treatment Groups of Rats Used in the in Vivo Studies
      GroupSpecifications
      Control (untreated)Animals were fed on a standard diet throughout the experimental period. In addition, they received a single intraperitoneal dose of 1 mL corn oil per kilogram body weight at 1 week after starting the standard diet (vehicle control).
      SP-treatedAnimals were fed on a standard diet mixed with 1% SP powder throughout the experimental period.
      DMBA-treatedAnimals were fed on a standard diet throughout the experimental period. In addition, they received a single intraperitoneal injection of 30 mg DMBA per kilogram body weight at 1 week after starting the SP diet. DMBA was dissolved in corn oil.
      DMBA+SP–treatedAnimals were fed on a standard diet mixed with 1% SP powder throughout the experimental period. In addition, they received a single intraperitoneal injection of 30 mg DMBA per kilogram body weight at 1 week after starting the SP diet. DMBA was dissolved in corn oil.
      After 1 week of this treatment regimen, a mammary tumorigenesis protocol was initiated in all animals in groups C and D. DMBA was dissolved in corn oil, and the animals were intraperitoneally injected with 30 mg/kg in a single dose. Behavioral patterns and food and water intake were monitored daily; body weight was recorded weekly. Animals were palpated along the milk line twice a week, starting at 4 weeks after DMBA treatment, to detect the presence of mammary tumors. At the end of the 12-month treatment period, animals were starved overnight and sacrificed. Mammary tissues and tumors were collected, fixed in 10% formalin, and then paraffin-embedded for histological analysis.

       Histology and Immunohistochemistry

      Sections (5 μm) of paraffin-embedded mammary glands were analyzed for expression of Ki-67 and ER by immunohistochemistry, as described previously.
      • Yang F.
      • Tang Q.
      • Zhong X.
      • Bai Y.
      • Chen T.
      • Zhang Y.
      • Li Y.
      • Zheng W.
      Surface decoration by Spirulina polysaccharide enhances the cellular uptake and anticancer efficacy of selenium nanoparticles.
      Adjacent sections were incubated with primary anti–Ki-67 and anti-ERα antibodies. Incubation with the appropriate secondary antibody was followed by direct diaminobenzidine staining and light counterstaining with hematoxylin. As a negative control, adjacent tissue sections were incubated with PBS instead of the primary antibody.
      To determine the labeling index for Ki-67 and ERα, each section was counted manually at high magnification (×400) in areas with a high number of stained cells and with homogeneous immunostaining. Approximately 1000 cells per slide were counted in each of five microscopic fields from well-labeled areas.

       Electron Microscopy

      Mammary glands were collected from sacrificed animals in all four groups at the end of the experiment. These were then immediately dissected and fixed for 2 hours in 2.5% glutaraldehyde buffered in 0.1 mol/L cacodylate buffer (pH 7.2) at 4°C, and postfixed in 1% cold osmium tetroxide in 0.1 mol/L cacodylate (pH 7.2) for 3 hours. Ultrathin sections were obtained from specimens using an Ultracut S microtome (Leica Microsystems). These sections were embedded in Lowicryl K4M resin after dehydration through graded ethanol series, substitution, and polymerization at −20° C. Sections were mounted on 400-mesh collodion–carbon-coated nickel grids and examined with a JEOL (Tokyo, Japan) electron microscope.

       Statistical Analysis

      Statistical analysis was performed using Newman–Keuls multiple comparison test and Student's t-test version 19 (SPSS software; IBM, Armonk, NY). Differences were considered statistically significant at P < 0.05. Data are expressed as means ± SD of at least triplicates from three independent experiments.

      Results

       In Vitro Studies

      AlamarBlue cell proliferation assay was used to examine the cytotoxic effect of the crude extract of SP on MCF-7 breast cancer cells, which are characterized by low levels of wild-type p53 and known to be less invasive. The particular advantage of the AlamarBlue cell proliferation assay is that it can be used for longer than 48 hours (eg, for 6 days). When the cells were treated with only the vehicle (30% ethanol), the dye diminished, indicating continuous growth of the cells in the control plate and for the 6-day experimental period, suggesting that the vehicle had no growth inhibitory effect on MCF-7 cells. However, a marked growth inhibition was observed for cells treated with a suboptimal dose of 50 μg/mL of the SP crude extract; this became apparent from day 4 (data not shown). We therefore chose this concentration to determine the expression of key proteins associated with cell-cycle regulation (p53 and p21), along with key proteins associated with apoptosis (p53, Bcl-2, and Bax). Protein lysates were collected from MCF-7 cells treated with the suboptimal dose (50 μg/mL) or a higher dose (100 μg/mL) of SP crude extract for 24 or 48 hours; lysates were then analyzed by Western blotting. MCF-7 cells are known to express wild-type p53 at a low level; a maximal induction of p53 occurred 24 hours after treatment with either SP concentration, but levels had declined significantly at 48 hours (Figure 1). The p21 expression pattern was closely similar, reaching maximal levels 24 hours after SP treatment for both concentrations (Figure 1). These data suggest that SP crude extract increased expression of p53 protein at 24 hours, which in turn transactivated its downstream target gene Cdkn1a (alias p21) to arrest the cell cycle (an early event) and subsequently inhibit cell proliferation.
      Figure thumbnail gr1
      Figure 1Effects of SP on cell growth and apoptosis of MCF-7 human breast cancer cells and changes in expression of associated genes. Western blot analysis of protein expression of genes associated with cell-cycle (p53 and p21) and apoptosis (p53, Bax, and Bcl2) pathways after treatment of breast cancer cells with SP for 24 and 48 hours. β-Actin was used as loading control by reprobing the same membrane. C, vehicle control.
      In terms of apoptosis, expression of the proapoptotic Bax was slightly decreased by 24 hours, but reached a maximal expression by 48 hours after SP treatment, particularly with the 50 μg/mL concentration (Figure 1). In contrast, expression of the antiapoptotic Bcl-2 expression was markedly decreased at 48 hours, particularly with the 50 μg/mL concentration (Figure 1). Thus, the Bax/Bcl-2 ratio was increased with SP treatment, suggesting an induction of apoptosis at 48 hours (a late event), mediated by p53.

       In Vivo Studies

      The in vitro data suggested that SP extract inhibits MCF-7 cell growth via the p53–p21 pathway and induced apoptosis via the p53–Bcl-2–Bax pathway. A DMBA-induced rat breast carcinogenesis model was then used to determine whether SP extract inhibits breast tumor development.

       Changes in Body Weight Gain and Tumor Incidence

      The body weight of each animal was recorded every week during the whole course of the experiment. Although the percentage of change in body weight gain increased with time in all four groups, DMBA treatment significantly slowed this increase (Tables 2 and 3). However, SP supplementation added to DMBA treatment overcame the reduction in weight gain, suggesting a protective effect of SP in these animals (Table 3).
      Table 2Effects of DMBA and/or SP Treatment on Percentage of Change in Body Weight Relative to Week 0
      WeekControl (%)SP (%)DMBA (%)DMBA+SP (%)
      14.333.757.921.26
      210.0410.689.790.59
      318.5421.027.0812.76
      422.7123.666.0316.52
      516.4617.4810.9421.13
      615.8315.9515.4015.48
      716.8819.7914.9612.55
      820.4222.0122.779.21
      922.5027.4125.4510.46
      1030.0032.8529.917.11
      1137.5041.5535.049.59
      1238.9645.3831.929.11
      1342.0846.5828.1312.73
      1436.0438.5029.4617.56
      1532.0839.0421.6117.32
      1638.9645.1420.3112.69
      1741.7542.6031.5124.83
      Table 3Probability of Change in Body Weight in the Four Experimental Groups
      GroupControlSpirulinaDMBADMBA+SP
      Control−0.0594.959∗∗∗10.395∗∗∗
      SP5.565∗∗∗13.023∗∗∗
      DMBA3.101∗∗
      DMBA+SP
      ∗∗P ≤ 0.01, ∗∗∗ P ≤ 0.001, Student's t-test.
      The animals were also examined for DMBA-induced breast tumors. Although none of the rats in the control and SP groups developed breast tumors (0% incidence), 87% of the rats in the DMBA group developed breast tumors. In the DMBA+SP group, however, breast tumor incidence fell to 13%. These data suggest that SP can prevent DMBA-initiated breast tumorigenesis in the rat.

       Morphology and Anatomy of Mammary Glands

      Morphological analysis was performed to examine the rats for DMBA-induced tumors in the mammary glands and also in other organs. None of the rats in the control or SP group developed any tumors of the mammary gland; however, rats in the two DMBA treatment groups developed a very thick adenomatous skin and breast carcinoma that were readily identified. In some cases, a large centrally situated poorly defined mass was identified; based on its general characteristics, this appeared to be an infiltrating adenocarcinoma (Figure 2A). Tumor growth was associated with discharge from the nipple, in addition to the presence of large, soft, bulky nodules ulcerated through the skin (∼5 cm in diameter, with central hemorrhage or cyst formation). Gross anatomy examination of other organs revealed other tumors, including hepatoma, carcinoma of the duodenum and pancreas, and osteosarcoma (Figure 2B). However, SP treatment reduced the effect of DMBA over time (Figure 3); various tumors formed ulcerated skin on the surface (Figure 2C) with no growth underneath (Figure 2D), and the larger ulcers (Figure 2E) took on the morphology of normal-appearing skin (Figure 2F).
      Figure thumbnail gr2
      Figure 2Clearance of DMBA-induced mammary tumors in rat by SP. A: Morphological abnormalities in rats with mammary tumors induced by DMBA include soft bulky nodules in the form of skin ulcerations in the inguinal region (arrow). B: Nodules (arrow) composed of adenocarcinoma of the mammary gland, and also hepatoma. C–F: Examination of mammary tumors (arrows) in the DMBA+SP group of rats revealed initial progression (arrows) of ulcerated skin where the tumor breaks open (C–E), followed by a healing process (F).
      Figure thumbnail gr3
      Figure 3Reduction of the effect of DMBA by SP. Survival analysis indicates that SP reduced the lethal effect of DMBA. Group A: Control (gray line). Group B: SP (dotted line). Group C: DMBA (blue line). Group D: DMBA+SP (green line).

       Histopathological Observations of Mammary Glands

      At the end of the 12 months of treatment, animals were sacrificed. Mammary glands were collected, fixed in 10% formalin, and paraffin-embedded for histological analysis. In both control rats (Figure 4A) and SP-treated rats (Figure 4B), light microscopy revealed layers of epithelial cells surrounding the of the alveoli; the lumen was clear, with no cell debris or any secretion. In contrast, the epithelia of rats treated with DMBA exhibited three types of carcinoma: lobular carcinoma in situ, comedo carcinoma, and carcinoma simples. In lobular carcinoma in situ (Figure 4C), the number of epithelial cell layers of the alveoli might range from two to six. Comedo carcinoma (Figure 4D) was characterized by highly cellular epithelial tissue, generally containing a central amorphous eosinophilic necrosis. Occasionally there was liquefaction of some of the cells in the epithelial layer, giving an appearance of accessory alveolar formation. The cells were generally solid, likely found in the perialveolar tissue and invading the stroma. Carcinoma simplex (adenoid cystic carcinoma or papillary carcinoma) (Figure 4E) was characterized by highly cellular outgrowth inside the lumen of the alveoli. This outgrowth was in the form of long interconnected papilloma and nodular aggregates. The cells were moderate in size, deeply stained and invading the stroma. Finally, in the DMBA+SP group (ie, rats protected with SP against the carcinogenicity of DMBA) (Figure 4, F–H), examination of the mammary alveoli revealed desquamation of all three types of carcinoma, leaving only one or two layers; nuclei were relatively dense. In many instances, the basal lamina was bare, completely denuded of epithelial cells (Figure 4G). If hyperplasia was present, vacuolization was commonly seen (Figure 4H). Another noteworthy observation was the presence of newly formed normal epithelium (Figure 4H).
      Figure thumbnail gr4
      Figure 4Light microscopic analysis of mammary tumor development after SP treatment. A and B: The epithelium of control (A) and SP-treated rats (B) exhibits normal layers of epithelial cells with clear lumen in the alveoli. C–E: The epithelium (arrows) of rats treated with DMBA exhibiting lobular carcinoma in situ (C), comedo carcinoma (D), and carcinoma simplex (E). F–H: With desquamation of neoplastic cell layers in DMBA+SP–treated rats, the basal lamina (arrows) is completely denuded of epithelial cells (F and G), and new normal epithelium (arrows) is formed (H). Original magnification, ×400. AV, alveoli; EC, epithelial cells; L, lumen.

       Ultrastructural Observations of Mammary Glands

      In control rats, the alveolus of the mammary gland exhibited electrolucent epithelia, electron-dense myoepithelia attached to the thin basal lamina with hemidesmosome, with irregularly shaped nuclei (Figure 5A). SP-treated rats exhibited characteristics similar to the control group, except for columnar appearance of epithelial cells, with more heterochromatin and vacuoles, thin myoepithelia, and basal lamina not so clear (Figure 5B). The electron microscope preparations of DMBA-treated rat specimens were generally difficult to define, because of numerous superficial cells due to exfoliation, suggestive of cell lysis (Figure 5C). The cytoplasm was vacuolated, with no recognizable cellular components and large nuclei exhibiting an increased amount of heterochromatin; cells were loosely attached, because of fewer desmosomes (Figure 5D). When the DMBA-treated rats were supplemented with SP, the apoptotic cells exhibited desquamation of the mammary epithelia, leaving only a single layer of epithelial cells. The cytoplasm of the cell contained fat droplets and vacuoles; karyorrhexis or pyknotic nuclei were seen (Figure 5, E–H).
      Figure thumbnail gr5
      Figure 5Electron microscopic analysis of mammary tumor development after SP treatment. A: Cells of control rats exhibit irregular nuclei and hemidesmosomes (arrows) attached to the thin basal lamina. B: Epithelial cells of SP-treated rats appear columnar, with heterochromatin and vacuoles. C and D: Mammary cells of DMBA-treated rats exhibit exfoliative features (C), with larger nuclei and increased amounts of heterochromatin (D). E–H: Mammary cells in the DMBA+SP group exhibit pyknotic nuclei with clearer cytoplasm. Original magnification, ×20,000. AC, apoptotic cells; BD, belt desmosome; BL, basal lamina; CT, connective tissue; CY, cytoplasm; D, spot desmosome; ER, endoplasmic reticulum; FD, fat droplets; GB, Golgi body; IL, intercellular luminae; IS, intercellular space; KH, karyorrhetic; KL, karyolytic nuclei; L, lumen; LP, lamina propria; LY, lysosomes; M, mitochondria; MB, membrane blebs; ME, mammary epithelia; MF, myofibril; MV, microvilli; MY, myoepithelia; N, nuclei; NU, nucleoli; PK, pyknotic nuclei; TJ, tight junction; V, vacuoles.

       Immunohistochemical Investigations of Mammary Glands

      Immunohistochemical analysis revealed lower expression of the cellular proliferation marker Ki-67 in the control group (indicative of cell division in some epithelial cells). Immunostaining of mammary glands in DMBA-treated rats revealed nuclei densely stained for Ki-67. The number of positively stained nuclei was significantly higher in the DMBA group than in the DMBA+SP group. The labeling index for Ki-67 protein expression was significantly higher in the DMBA group than in the DMBA+SP group (P < 0.001), particularly within the carcinoma simplex and the comedo carcinoma (Figure 6A). Because estrogens play an important role in mammary carcinogenesis, we also investigated ERα expression (Figure 6B); the results were similar to those observed for Ki-67.
      Figure thumbnail gr6
      Figure 6Effect of DMBA and SP supplementation on expression levels of Ki-67 and ERα in rat mammary tissues. was used as a measure of Ki-67 and ERα protein expression in rat mammary tissues from the four groups. A: Ki-67 expression levels induced in the DMBA-treated rats were lower in the mammary tissues of the DMBA+SP–treated rats (P < 0.001). B: Estrogen expression levels induced in the DMBA-treated rats were lower in the mammary tissues of the DMBA+SP–treated rats (P < 0.001). Data are expressed as means ± SD. CC, comedo carcinoma; CS, carcinoma simplex; LCIS, lobular carcinoma in situ; LI, labeling index.

      Discussion

      To our knowledge, the present study is the first to demonstrate an in vivo chemopreventive effect of SP against DMBA-induced mammary carcinogenesis in a rat model. We show here that SP extract clears breast tumors induced by DMBA in rats, a finding that was confirmed by histological analysis and microscopic observations. More interestingly, expression levels of the cell proliferation markers Ki-67 and ERα were significantly reduced in the DMBA-induced mammary tumors when SP supplementation was added. In vitro studies revealed that SP treatment of MCF-7 breast cancer cells resulted in a number of cellular and molecular changes, including cell growth arrest as an early event at 24 hours after treatment and apoptosis as a late event by 48 hours. Our molecular data suggest that SP treatment increased expression of the genome guardian p53 (Tp53), which in turn induced its transcriptional downstream target p21 gene (Cdkn1a), leading to p53–p21–regulated cell-cycle arrest as an early event. Furthermore, our results suggest that, as a late event, the increased p53 protein up-regulated expression of its downstream target gene, the proapoptotic Bax, and down-regulated expression of its downstream target gene, the antiapoptotic Bcl2, leading to p53–Bax–Bcl-2–regulated cell death.
      Various chemoprevention studies and trials have been conducted to clarify the role of certain phytochemicals and supplements in breast cancer. Although the importance of fruit, fiber, and vegetables has been emphasized by the recently concluded Women's Intervention Nutrition Study (WINS) and the Women's Healthy Eating and Living (WHEL) study, there is still no convincing evidence that changes in dietary pattern after breast cancer diagnosis will improve prognosis for most women with early-stage breast cancer.
      • Pierce J.P.
      Diet and breast cancer prognosis: making sense of the Women's Healthy Eating and Living and Women's Intervention Nutrition Study trials.
      Thus, screening for dietary compounds has become essential, and many compounds have been screened for their chemopreventive potential. Among the several SP species, A. platensis and A. fusiformis have promising chemopreventive potential. Our research group recently reported a chemopreventive action of SP in liver carcinogenesis in rat model, and further characterized its underlying mechanisms of action in HepG2 hepatocellular carcinoma cells.
      • Ismail M.F.
      • Ali D.A.
      • Fernando A.
      • Abdraboh M.E.
      • Gaur R.L.
      • Ibrahim W.M.
      • Raj M.H.
      • Ouhtit A.
      Chemoprevention of rat liver toxicity and carcinogenesis by Spirulina.
      Chen et al
      • Chen T.
      • Wong Y.S.
      In vitro antioxidant and antiproliferative activities of selenium-containing phycocyanin from selenium-enriched Spirulina platensis.
      reported a chemopreventive role of PC (the most active ingredient in SP) containing selenium, purified from selenium-enriched A. platensis, and further studied its in vitro antioxidant and antiproliferative properties.
      • Ouhtit A.
      • Gaur R.L.
      • Abdraboh M.
      • Ireland S.K.
      • Rao P.N.
      • Raj S.G.
      • Al-Riyami H.
      • Shanmuganathan S.
      • Gupta I.
      • Murthy S.N.
      • Hollenbach A.
      • Raj M.H.
      Simultaneous inhibition of cell-cycle, proliferation, survival, metastatic pathways and induction of apoptosis in breast cancer cells by a phytochemical super-cocktail: genes that underpin its mode of action.
      The potential therapeutic effect of SP observed in the present study may be attributed to antioxidant constituents such as PC, selenium, chlorophyll, carotene, γ-linoleic acids, and other vitamins or precursors working individually or in synergy.
      • Kulshreshtha A.
      • Zacharia A.J.
      • Jarouliya U.
      • Bhadauriya P.
      • Prasad G.B.
      • Bisen P.S.
      Spirulina in health care management.
      With an apparent ability to stimulate whole human physiology, SP exhibits therapeutic effects such as antioxidant, antibacterial, antiviral, anticancer, anti-inflammatory, antiallergic, and antidiabetic, as well as a variety of beneficial functions for addressing obesity, diabetes, hypoglycemia, cancer, arthritis, and other degenerative diseases.
      • Deng R.
      • Chow T.J.
      Hypolipidemic, antioxidant, and antiinflammatory activities of microalgae Spirulina.
      • Khan Z.
      • Bhadouria P.
      • Bisen P.S.
      Nutritional and therapeutic potential of Spirulina.
      In our study, SP extract induced apoptosis of MCF-7 breast cancer cells, most likely mediated by the p53–Bax–Bcl-2 pathway. Roy et al
      • Roy K.R.
      • Arunasree K.M.
      • Reddy N.P.
      • Dheeraj B.
      • Reddy G.V.
      • Reddanna P.
      Alteration of mitochondrial membrane potential by Spirulina platensis C-phycocyanin induces apoptosis in the doxorubicin-resistant human hepatocellular-carcinoma cell line HepG2.
      also reported down-regulation of the antiapoptotic protein Bcl-2 and up-regulation of the proapoptotic Bax protein in HepG2 cells treated with PC, demonstrating the ability of PC to induce apoptosis. Thus, induction of apoptosis by PC appears to follow a similar mechanism in both breast cancer and hepatocellular carcinoma.
      Rats given DMBA intraperitoneally developed three types of carcinoma: lobular carcinoma in situ, comedo carcinoma, and carcinoma simplex. DMBA treatment is known to change the normal differentiation process of the mammary gland into an invasive carcinoma, and study of in situ carcinoma has revealed the importance of this model in predicting the aggressiveness of breast cancer.
      • Al-Dhaheri W.S.
      • Hassouna I.
      • Al-Salam S.
      • Karam S.M.
      Characterization of breast cancer progression in the rat.
      In the present study, pleomorphic microvilli were found to be a cancerous marker, one that developed in benign or reversible hyperplasia. Multiple basal laminae in carcinoma simplex are formed, because of successive generations of cell proliferation at the time of tumor development. The destruction of the basement membrane could also be due to enzymes produced by the neoplastic basal cells. Numerous genetic mutations accumulate during carcinogenesis in epithelial tissue, and loss of cellular function occurs that alters the phenotypic characteristics of the cell. Pushing margins of tumor growth indicate tumor invasion into the stroma surrounding the lesion. When DMBA-treated rats were given SP supplementation, a desquamation of neoplastic cells was observed, indicating eradication of the tumor.
      Assessing cellular proliferation in a variety of tumors provides useful information and may be of prognostic significance, of which Ki-67 is the most widely used and reliable indicator.
      • Scholzen T.
      • Gerdes J.
      The Ki-67 protein: from the known and the unknown.
      Although Ki-67 is a cell proliferation marker, used to determine the growth phase in a cell fraction, the Ki-67 labeling index is often related to the clinical course of cancer. Thus, the high labeling index for Ki-67 in the mammary epithelia of DMBA-treated rats explains the increase in immunoreactivity and cell-cycle protein expression beginning in the mid to late G1 phase, rising through S phase and G2 phase and reaching its maximum in mitosis.
      • Bullwinkle J.
      • Baron-Lühr B.
      • Lüdemann A.
      • Wohlenberg C.
      • Gerdes J.
      • Scholzen T.
      Ki-67 protein is associated with ribosomal RNA transcription in quiescent and proliferating cells.
      On the other hand, ERα, a modulator of cellular growth and differentiation, exhibited higher immunoreactivity in the DMBA-treated rats, indicating the histological grade of tumor.
      • Gustafsson N.
      • Zhao C.
      • Gustafsson J.
      • Dahlman-Wright K.
      RBCK1 drives breast cancer cell proliferation by promoting transcription of estrogen receptor alpha and cyclin B1.
      This might also indicate that the progression of breast tumors is additionally dependent on the presence and levels of hormones in the body.
      In conclusion, SP exhibited remarkable antitumor activity by inhibiting cell proliferation induced by DMBA in rats. Our present study demonstrates, for the first time, that SP clears tumor cells from the mammary gland and contributes to the formation of a newer epithelial layer. Although the direct mode of action and in-depth molecular mechanisms of SP have yet to be established, we plan to use microarray gene expression profiling to investigate the molecular pathways underlying the mode of action of its main ingredients that might act in synergy.

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      • Corrections
        The American Journal of PathologyVol. 184Issue 4
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          In the article entitled, “Caspase 3 Silencing Inhibits Biomechanical Overload–Induced Intervertebral Disk Degeneration” (Volume 184, pages 753–764 of the March issue of The American Journal of Pathology), errors were inadvertently introduced into the figure labeling. The unit μm in the scale bars was incorrectly changed to μmol/L within Figures 2, below panel A; Figure 6, above panels A and B; and Figure 7, above panels A–H. We sincerely regret the error.
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