DMBA Induced Breast Cancer Model Research Articles

Cetuximab functionalized chitosan/hyaluronic acid-based nanoparticles loaded with cabazitaxel enhances anti-tumor efficacy in DMBA-induced breast cancer model in rats through spatial targeting

The article discusses the ionic gelation of cationic chitosan (CS) with anionic hyaluronic acid (HA) sodium salt to form nanoparticles in the range of 125 nm. The particles were further functionalized with cetuximab to endow it with the ability to spatially target the tumor over-expressing EGFR. Solid-state characterization of the particles using XRD, FTIR, and DSC revealed the formation of stable nanoparticles with Cabazitaxel loaded in the amorphous nanostructure. XPS study used to assess the surface characteristics indicated that the cetuximab was successfully anchored on the surface of the particle. The prepared CS-HA-Cmab-NP elicited a pH-responsive drug release behavior due to the presence of CS in the matrix. In vitro performance of the nanoparticles was evaluated on MDA-MB-231 breast cancer cell lines showed overall increase in efficacy. In vivo pharmacokinetic and anti-tumor effect evaluated in female Sprague Dawley rats indicated that the Cmab-conjugated nanoparticles improved half-life of cabazitaxel and tumor reduction capability with higher survival rate and lower reduction in body weight. The results indicate that CS/HA nanoparticles anchored with cetuximab show enhanced efficacy in reducing the breast cancer tumor in DMBA-induced breast tumor model through spatial targeting, consequently reducing the systemic toxicity.

Diet Modulates the Gut Microbiome, Metabolism, and Mammary Gland Inflammation to Influence Breast Cancer Risk.

Several studies indicate a strong link between obesity and the risk of breast cancer. Obesity decreases gut microbial biodiversity and modulates Bacteroidetes-to-Firmicutes phyla proportional abundance, suggesting that increased energy-harvesting capacity from indigestible dietary fibers and elevated lipopolysaccharide bioavailability may promote inflammation. To address the limited evidence linking diet-mediated changes in gut microbiota to breast cancer risk, we aimed to determine how diet affects the microbiome and breast cancer risk. For ten weeks, female 3-week-old BALB/c mice were fed six different diets (control, high-sugar, lard, coconut oil, lard + flaxseed oil, and lard + safflower oil). Fecal 16S sequencing was performed for each group. Diet shifted fecal microbiome populations and modulated mammary gland macrophage infiltration. Fecal-conditioned media shifted macrophage polarity and inflammation. In our DMBA-induced breast cancer model, diet differentially modulated tumor and mammary gland metabolism. We demonstrated how dietary patterns change metabolic outcomes and the gut microbiota, possibly contributing to breast tumor risk. Furthermore, we showed the influence of diet on metabolism, inflammation, and macrophage polarity. This study suggests that dietary-microbiome interactions are key mediators of breast cancer risk. Prevention Relevance: Our study demonstrates the impact of diet on breast cancer risk, focusing on the interplay between diet, the gut microbiome, and mammary gland inflammation.

Cabazitaxel-loaded redox-responsive nanocarrier based on d-alpha-tocopheryl-chitosan and hyaluronic acid for improved anti-tumor efficacy in DMBA-induced breast cancer model

The study involved the formulation and evaluation of cabazitaxel loaded d-alpha-tocopheryl succinate/chitosan conjugate (CSVE) and hyaluronic acid (HA) based redox-responsive nanoparticle crosslinked using 3,3′-dithiodipropionic acid (DTPA).

DR5 antibody conjugated lipid-based nanocarriers of gamma-secretase inhibitor for the treatment of triple negative breast cancer

In the present study, Death receptor-5 (DR5) antibody conjugated solid lipid nanoparticles (DR5-DAPT-SLNs) has been formulated for effective intracellular of γ-secretase inhibitor, N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) to cancer cells. Emulsification-solvent evaporation, followed by EDC cross-linking methods, was employed to prepare DR5 targeted DAPT-SLNs (DR5-DAPT-SLNs). The formulation was characterized by its particle size, shape, and surface charge. The in vitro &in vivo anticancer efficacy was studied in MDA-MB231 triple negative breast cancer (TNBC) cells and DMBA induced breast cancer model in mice, respectively. The results show that thatDR5-DAPT-SLNs is found to be a spherical shape with an average particle size of 187 ± 0.98 nm and having an average surface charge of 23 ± 2.3 mV. DR5-DAPT-SLNs have higher cytotoxicity in MDA-MB231 cells compared to DAPT-SLNs (non-targeted) and the bulk drug. However, in DR5 negative HEK 293 noncancer cells, the formulation shows minimal cytotoxic effects. The above results, therefore, demonstrate DR5 mediated uptake is responsible for improved cytotoxicity of DAPT. In the in vivo anticancer study, DR5-DAPT-SLNs show greater tumor regression when compared to DAPT-SLNs and the bulk drug. In conclusion, the results of the present study demonstrate that the DR5-DAPT-SLNs selectively target cancer cells and potentiate the anticancer efficacy of DAPT against TNBC cells.

In vivo safety, toxicity, biocompatibility and anti-tumour efficacy of bioinspired silver and selenium nanoparticles

The present work was carried out to know the in vivo evaluation of safety, toxicity and efficacy of the various biosynthesized inorganic nanoparticles of various sizes and shapes. Several kinds of silver and selenium inorganic nanomaterials were synthesized using plant extracts and phytochemicals. The size and shape of these bioinspired nanomaterials were characterized using various spectroscopic and microscopic techniques. All the synthesized nanomaterials were found to be less than 35 nm in size and having hexagonal and spherical shapes resoluted via electron microscopy analysis. Biocompatibilities of the all biosynthesized nanoparticles were evaluated using hemolysis assay exhibiting lower hemolytic effect (less than 10 %) and LAL assays showing free of endotoxin. Safety and toxicities of synthesized nanocomposites were evaluated on Swiss mice using various bio-assays including haematological, biochemical, histopathological and DNA fragmentation assays. The biosynthesized nanomaterials did not produce significant level of toxicity in terms of haematological, biochemical, histological and DNA damage parameters. Following the surprising results with safety and negligible toxicity of the synthesized nanoparticles, In vivo anti-tumour potential were proposed and evaluated in DMBA induced breast cancer model of SD female rats. The results of the study presented the significant decrease in the tumour volume after administration of nanoparticles. Conclusively, the bio-inspired nanocomposite synthesized by biological route did not produce significant level of toxic effect with higher level of therapeutic effectiveness. These synthesized biological materials can be used for the management of various types of life threatening emergencies.

The combined effect of pomegranate extract and tangeretin on the DMBA-induced breast cancer model

The aim of this study was to investigate the protective effects of pomegranate extract and tangeretin alone or in combination in DMBA-induced rat breast cancer model.A total of 68 female rats were randomly divided into 8 groups. The first 4 groups were designed as controls for cancer and treatment groups, and the control groups were composed of only control (C), Pomegranate (P), Tangeretin (T), and Pomegranate+Tangeretin (P+T) groups. The other four groups were designed as cancer and treatment groups and were composed of DMBA (D) and DMBA+Pomegranate (D+P), DMBA+Tangeretin (D+T), DMBA+Pomegranate+Tangeretin (D+P+T) groups. Tumor markers and angiogenesis parameters were studied from plasma samples obtained from rats. Histopathological, immunohistochemical, and TUNEL analyses and expressions of proteins affecting apoptosis and cell cycle were determined in breast tissue samples.In the DMBA group, plasma CA15-3, CEA, VEGF, MMP-9, and NF-κB levels were significantly increased compared to the controls, but significant decreases were observed in these parameters except MMP-9 in the treatment groups. It was observed that p53 and Bax expressions significantly increased in both D+P and D+P+T groups compared to the DMBA group, and these findings were supported by Tunel and immunohistochemical findings. Cyclin D1 expressions were found to be significantly decreased only in the D+T group and supported by TUNEL and immunohistochemical findings. Immunohistochemical ER-α and Ki-67 immune reactivities were significantly decreased in all treatment groups compared to the DMBA group.Our results showed that combined application of pomegranate extract and tangeretin may be more beneficial in preventing breast cancer development.

Herniarin, a natural coumarin loaded novel targeted plasmonic silver nanoparticles for light activated chemo-photothermal therapy in preclinical model of breast cancer

Background: The conventional chemotherapeutic approach for breast cancer involves use of free drug substances that succumb into considerable systemic toxicity. For the past few decades biologically active phytochemicals like coumarins have gained much interest against breast cancer. However, there are lacunas in direct use of these potent biomolecules that impede their pharmacological functions. On the other hand, recent years have also witnessed great advancements in stimuli actuated nanotherapeutics as combined treatment strategies in critical breast cancer cases. Objectives: In this study we formulated novel pentagonal plasmonic silver nanoparticles as folate receptor targeted light responsive nano-delivery platform for a potent coumarin derivative, Herniarin to improve its therapeutic proficiency. Materials and Methods: Primarily, we synthesized plasmonic silver based nanoscopic carrier for herniarin to facilitate its tumor targeted delivery via folate receptors. These formulated nanoparticles (Herniarin quenched, folic acid furnished, HER-FA-AgNPs) were subjected to series of physicochemical tests, followed by detailed in-vitro and in-vivo testing in breast cancer cell lines and DMBA induced breast cancer model in rats, respectively. Results: The pentagonal nanoparticles observed in transmission electron microscopy demonstrated strong plasmonic tunability in the NIR region while its key feature of transforming light into heat energy when exposed to laser (800 nm) allowed photothermal therapy to supplement the chemotherapeutic potential of Herniarin as illustrated in various in-vitro studies. The in-vivo studies including detailed cellular investigations of breast tissues through Field Emission Scanning Electron Microscope and histopathology reflected the improved outcome of combined therapeutic modalities (hyperthermia + chemotherapy) achieved by HER-FA-AgNPs. Conclusion: Thus, these novel nanoparticles may serve as an efficient as well as safe drug-disposing vehicle for combined and strategic therapy of breast cancer.

Triple antioxidant SNEDDS formulation with enhanced oral bioavailability: Implication of chemoprevention of breast cancer

The present study aimed to develop quercetin, resveratrol and genistein loaded self-nanoemulsifying drug delivery system (SNEDDS) by QbD approach in order to improve their oral bioavailability and antioxidant potential. The size and PDI of the optimized formulation were found to be <200nm and <0.3, respectively. DPPH scavenging assay showed comparable antioxidant activity of antioxidant loaded SNEDDS to free antioxidants combination. Furthermore, coumarin-6 loaded SNEDDS formulation showed rapid internalization within 1h of incubation by Caco-2 cells. Moreover, the pharmacokinetic studies in rats for the optimized formulation and free antioxidant suspension were performed. SNEDDS have significantly increased the Cmax and area under curve (AUC) of all three antioxidants. The SNEDDS demonstrated ~4.27 fold enhancement in oral bioavailability of quercetin, ~1.5 fold in case of resveratrol and ~2.8 fold in case of genistein as compared to free antioxidants suspension. Finally, the prophylactic antitumor efficacy of developed formulation was tested against DMBA induced breast cancer model in rats, which demonstrated enhanced abeyance towards the tumor growth as compared to free antioxidants.

Development of Novel Polymer-Lipid Hybrid Nanoparticles of Tamoxifen: In Vitro and In Vivo Evaluation.

This study was undertaken to develop and investigate the effect of tamoxifen polymer-lipid hybrid nanoparticles (Tmx-PLN) on its oral bioavailability and efficacy in the 7,12-dimethylbenzanthracene (DMBA)-induced breast cancer model. Modified solvent emulsification-evaporation method was optimized to obtain Tmx-PLN, composed of chitosan and lecithin, of 169.66 ± 4.84 nm particle size. The PLN exhibited prolonged in vitro release in phosphate-buffered saline. Further, PLN displayed enhanced oral bioavailability with considerable increase in AUC (1277.46 vs. 585.01 ng/ml · h), pro- longed t½ (27.87 ± 15.62 vs. 10.18 ± 6.5 h) and mean residence time (40.11 ± 25.72 vs. 17.42 ± 12.04 h) in comparison to pure Tmx. In addition, PLN exhibited significantly increased (P < 0.05) antitumor efficacy in DMBA-induced breast cancer model, when administered once in three days in comparison to Tmx daily dosing. This enhancement may be attributed to a probable reduction in Pgp efflux, decreased first-pass metabolism and lymphatic drug transport. Thus, Tmx-PLN exhibited enhanced potential to increase Tmx therapeutic efficacy in chronic treatment of breast cancer.

The Role of Dietary Extra Virgin Olive Oil and Corn Oil on the Alteration of Epigenetic Patterns in the Rat DMBA-Induced Breast Cancer Model.

Disruption of epigenetic patterns is a major change occurring in all types of cancers. Such alterations are characterized by global DNA hypomethylation, gene-promoter hypermethylation and aberrant histone modifications, and may be modified by environment. Nutritional factors, and especially dietary lipids, have a role in the etiology of breast cancer. Thus, we aimed to analyze the influence of different high fat diets on DNA methylation and histone modifications in the rat dimethylbenz(a)anthracene (DMBA)-induced breast cancer model. Female Sprague-Dawley rats were fed a low-fat, a high corn-oil or a high extra-virgin olive oil (EVOO) diet from weaning or from induction with DMBA. In mammary glands and tumors we analyzed global and gene specific (RASSF1A, TIMP3) DNA methylation by LUMA and bisulfite pyrosequencing assays, respectively. We also determined gene expression and enzymatic activity of DNA methyltransferases (DNMT1, DNMT3a and DNMT3b) and evaluated changes in histone modifications (H3K4me2, H3K27me3, H4K20me3 and H4K16ac) by western-blot. Our results showed variations along time in the global DNA methylation of the mammary gland displaying decreases at puberty and with aging. The olive oil-enriched diet, on the one hand, increased the levels of global DNA methylation in mammary gland and tumor, and on the other, changed histone modifications patterns. The corn oil-enriched diet increased DNA methyltransferase activity in both tissues, resulting in an increase in the promoter methylation of the tumor suppressor genes RASSF1A and TIMP3. These results suggest a differential effect of the high fat diets on epigenetic patterns with a relevant role in the neoplastic transformation, which could be one of the mechanisms of their differential promoter effect, clearly stimulating for the high corn-oil diet and with a weaker influence for the high EVOO diet, on breast cancer progression.

Phytantriol Based “Stealth” Lyotropic Liquid Crystalline Nanoparticles for Improved Antitumor Efficacy and Reduced Toxicity of Docetaxel

The present work focuses on design and development of surface functionalized LCNPs for improving tumor delivery of DTX. Phytantriol based "stealth" LCNPs were prepared using hydrotrope method and optimized. The potential of developed formulation was further assessed using cell culture experiments, in vivo pharmacokinetics, in vivo pharmacodynamics and toxicity studies. A biphasic drug release pattern was observed with sustained release of drug till 72 h. In vitro cell culture experiments revealed efficient internalization within MCF-7 cells with 25.80-fold decrease in IC50 value for PEG-LCNPs as compared to free DTX. Mechanistic insights demonstrated preferential co-localization of LCNPs in the vicinity of the nucleus. Furthermore, in vivo pharmacokinetic studies revealed 14.45-fold enhancement in circulation half-life of PEG-LCNPs as compared to marketed formulation Taxotere®. In vivo efficacy studies PEG-LCNPs in DMBA induced breast cancer model revealed ~81% reduction in the tumor burden compared to Taxotere® which caused/achieve only 47% reduction or showed only 47% decrease. Furthermore, safety profile was noted for PEG-LCNPs as compared to Taxotere®, measured as a function of hepato- and nephro-toxicity. Surface functionalization of LCNPsis a viable approach for improving the therapeutic potential of DTX.

Bicontinuous Cubic Liquid Crystalline Nanoparticles for Oral Delivery of Doxorubicin: Implications on Bioavailability, Therapeutic Efficacy, and Cardiotoxicity

The present study explores the potential of bicontinous cubic liquid crystalline nanoparticles (LCNPs) for improving therapeutic potential of doxorubicin. Phytantriol based Dox-LCNPs were prepared using hydrotrope method, optimized for various formulation components, process variables and lyophilized. Structural elucidation of the reconstituted formulation was performed using HR-TEM and SAXS analysis. The developed formulation was subjected to exhaustive cell culture experiments for delivery potential (Caco-2 cells) and efficacy (MCF-7 cells). Finally, in vivo pharmacokinetics, pharmacodynamic studies in DMBA induced breast cancer model and cardiotoxicity were also evaluated. The reconstituted formulation exhibited Pn3m type cubic structure, evident by SAXS and posed stability in simulated gastrointestinal fluids and at accelerated stability conditions for 6months. Dox-LCNPs revealed significantly higher cell cytotoxicity (16.23-fold) against MCF-7 cell lines as compared to free drug owing to its preferential localization in the vicinity of nucleus. Furthermore, Caco-2 cell experiments revealed formation of reversible "virtual pathways" in the cell membrane for Dox-LCNPs and hence posed significantly higher relative oral bioavailability (17.74-fold). Subsequently, Single dose of Dox-LCNPs (per oral) led to significant reduction in % tumor burden (~42%) as compared that of ~31% observed in case of Adriamycin® (i.v.) when evaluated in DMBA induced breast cancer model. Moreover, Dox induced cardiotoxicity was also found to be significantly lower in case of Dox-LCNPs as compared to clinical formulations (Adriamycin® and Lipodox®). Incorporation of Dox in the novel LCNPs demonstrated improved antitumor efficacy and safety profile and can be a viable option for oral chemotherapy.

Co-encapsulation of Tamoxifen and Quercetin in Polymeric Nanoparticles: Implications on Oral Bioavailability, Antitumor Efficacy, and Drug-Induced Toxicity

The present investigation reports the preparation, optimization, and characterization of orally administrable PLGA-NPs co-encapsulated with tamoxifen (Tmx) and quercetin (QT). The developed formulation was found to have particle size 185.3 ± 1.20 nm, PDI 0.184 ± 0.004, entrapment efficiency 67.16 ± 1.24% Tmx, 68.60 ± 1.58% QT at a Tmx/QT ratio of 1:2 w/w. The stability of the freeze-dried formulation was established in simulated gastrointestinal fluids for 8 h and at accelerated stability condition for 3 months. DPPH free radical scavenging assay confirmed that the functional architecture of QT was retained in freeze-dried NPs. Higher cellular uptake, cytotoxicity, and nuclear co-localization of Tmx-QT-NPs in MCF-7 cells revealed higher efficiency of the formulation. At the same time, higher Caco-2 cell uptake revealed its potential for oral delivery, which was well corroborated with in vivo pharmacokinetics, which suggested ∼ 5-fold and ∼ 3-fold increase in oral bioavailability as compared to the free Tmx citrate and free QT, respectively. Concomitantly, significantly higher tumor suppression was observed in the case of the developed formulation in contrast to respective free drug(s) and their combination when tested against a DMBA-induced breast cancer model in female SD rats. Multiple oral administrations of Tmx-QT-NPs efficiently controlled the tumor angiogenesis as revealed by normalized levels of respective markers (MMP-2 and MMP-9). The safety profile of Tmx-QT-NPs was also established, and no measurable hepatotoxicity or oxidative stress was observed when measured as a function of respective biochemical markers in contrast to free drug(s) and their combinations. In a nutshell, the co-encapsulation strategy with PLGA-NPs could be a promising approach in improving oral delivery of Tmx and QT for cancer therapy.

Abstract 3694: Complex effects of dietary apigenin on prevention of MPA-accelerated DMBA-induced mammary tumors in Sprague-Dawley rats.

Abstract Epidemiological and laboratory studies support the use of the plant-derived flavonoid apigenin as a potential nutraceutical for preventing a number of different cancers. However the effect of dietary apigenin on prevention of progestin-accelerated breast tumors has hitherto not been explored. Medroxyprogesterone acetate (MPA) is the most commonly used synthetic progestin administered to post-menopausal women for hormone replacement therapy. MPA accelerates the development of breast cancers by reducing tumor latency and increasing tumor incidence and multiplicity in a DMBA-induced breast cancer model, and causes progression of human cancer cells in xenografts (Clin Can Res, 2006, 12:4062; Cancer Res, 2007, 67: 9929). Previously we showed that intraperitoneally administered apigenin effectively treated and prevented the progression of MPA-accelerated breast cancer in both the DMBA-induced, and xenograft mammary cancer models (Cancer Prev Res, 2011, 4: 1316; Horm Canc, 2012, 3: 160). In the present study we used the DMBA model to examine the chemopreventive effect of dietary apigenin against MPA-accelerated tumors. Based on reports in the literature, we examined the effects of feeding 3 different dietary levels of apigenin (0.02%, 0.1% and 0.5% wt/wt) in a phytoestrogen-free diet following treatment with DMBA, and prior to implantation of MPA pellets which accelerate tumor development. The MPA-dependent tumor incidence was lower in the 0.1% dietary apigenin treatment group. Surprisingly, the same dietary level significantly increased tumor multiplicity in those animals that developed tumors. A five-fold increase (0.5%) or reduction (0.02%) in dietary apigenin failed to reduce MPA-dependent tumor incidence and the higher dose (0.5%) substantially increased tumor multiplicity compared with MPA alone. Tumor incidence and multiplicity were unaffected in apigenin-fed animals that were treated with DMBA but supplemented with placebo pellets. There were no morphological or histological differences between tumors from animals fed apigenin-supplemented diets and those given a control diet, either in the presence or absence of MPA.These findings show that apigenin, given in a diet devoid of phytochemicals, exacerbated the effects of MPA on tumor development and multiplicity in a chemically-induced breast cancer model. Consequently, until further research clarifies the effect of dietary apigenin on progestin-accelerated tumors, caution should be exercised when considering the flavonoid as a dietary supplement for preventing hormone-dependent breast cancer. Supported by a COR award from University of Missouri, and research funds from RADIL. Citation Format: Benford Mafuvadze, Matthew Cook, Xu Zhang, Cindy Besch-Williford, Salman M. Hyder. Complex effects of dietary apigenin on prevention of MPA-accelerated DMBA-induced mammary tumors in Sprague-Dawley rats. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3694. doi:10.1158/1538-7445.AM2013-3694

Abstract B61: Effects of anti-obesity dietary supplement DHEA on serum DHEA, DHEA-S, IGF-1 and IGFBP-3 in DMBA-induced breast cancer model.

Abstract Obesity has been epidemic in the US for over two decades. Obesity has been linked to the risk of development of various cancers, including breast cancer. Dehydroepiandrosterone (DHEA) is an over-the-counter dietary supplement used as an anti-cancer agent and anti-obesity supplement. The objectives of this study were to investigate the long-term effects of obesity and DHEA treatment on body weight gain and serum DHEA, DHEA-S, IGF-1and IGFBP-3 using 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumor model. Forty-three six-week-old obese female Zucker rats were used. Rats were randomly assigned and had ad libitum access to water and a diet of either chow (2016) as a control diet or chow with the addition of DHEA at a concentration of 6 g/kg of chow as a DHEA diet. All rats were orally gavaged at age 50 days with 65 mg DMBA/kg body weight and were sacrificed 155 days post-DMBA treatment. Fifty-five percent (55%) of the control diet group developed mammary tumors, while no tumors were detected in the DHEA diet group (P&amp;lt;0.001). Obese rats fed the DHEA diet gained significantly less weight (P&amp;lt;0.001) and had an increased (P&amp;lt;0.001) serum DHEA and DHEA-S compared to control rats. Also, obese DHEA-Fed rats had significantly (P&amp;lt;0.001) lower serum IGF-1 and IGF-BP3. Our results suggest that DHEA treatment can reduce body weight gain by lowering serum IGF-1 and IGFBP-3 which might protects against breast cancer development caused by obesity. Also, this results suggests that breast cancer risk may be due to genetic predisposition to obesity might be related to body weight gain. It also suggests that these individuals could potentially reduce their risk by actively combating the weight gain. Supported by ABI to RH. Citation Format: Reza Hakkak, Soheila Korourian. Effects of anti-obesity dietary supplement DHEA on serum DHEA, DHEA-S, IGF-1 and IGFBP-3 in DMBA-induced breast cancer model. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr B61.

Stem cell antigen‐1 (Sca‐1) disrupts GDF10/TGF‐β signal transduction at the plasma membrane to regulate Smad2/3 nuclear signaling

Sca-1 is a member of the glycerophosphatidylinositol (GPI)-anchored membrane proteins that is associated with stem/progenitor cell populations and a basal cell malignant phenotype. Although Sca-1 serves as a marker of murine stem cells, the molecular mechanism of its function remains largely unknown. We sought to determine the mechanism underlying its function in tumorigenesis by using a DMBA induced breast cancer model in Sca-1+/EGFP knock-in mice. We report that Sca-1 expression is enhanced early in the carcinogenesis. Primary cells from these mouse tumors display in vivo tumorigenicity in mice, which is inhibited when Sca-1 expression is attenuated with a Sca-1 shRNA. The latter effect occurs in association with increased expression of the TGF-β family ligand GDF10. We show that GDF10 acts via Smad3 signaling while Sca-1 inhibits GDF10-stimulated Smad3 signaling through direct association with the type I TGF-β receptor at the membrane as determined by surface biotinylation, immunoprecipitation, confocal imaging and phospho-antibodies. These findings demonstrate that Sca-1 contributes to tumor progression, in part, by suppressing of GDF10 expression, as well as TGF-β signaling at the surface membrane, and suggests a new paradigm for examining the function of GPI-linked membrane proteins associated with stem cells. Funding: R01 CA111482, N01 CN15017-44 and N01 CN43309 from the NIH, Bethesda, MD.

Glutamine Affects Glutathione Recycling Enzymes in a DMBA-Induced Breast Cancer Model

Malignancy depletes host glutathione (GSH) levels to increase treatment-related toxicity and increases itself to resist the treatments. Our previous studies have shown that dietary glutamine (GLN) prevented 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors through enhancing gut GSH release and reducing tumor GSH level. In addition, GSH synthesis, metabolism, and recycling are accomplished in γ -glutamyl cycle. We hypothesized that the GLN prevention might be through a differential regulation of the γ -glutamyl cycle enzymes. Female Sprague-Dawley rats were randomized into DMBA-tumor bearing, DMBA-treated, and control groups subdivided into GLN and water groups. GLN supplementation was given at 1 g/kg/day by gastric gavage. The activities and messenger RNA levels of γ -glutamyl transpeptidase (GTP), γ -glutamylcysteine synthetase (GCS), 5-oxo-L-prolinase (OPase), γ -glutamyl transferase (GTF), and glutaminase (GLNase) were determined in gut mucosa and breast tumor using specific enzyme assays and semiquantitative reverse transcription polymerase chain reaction. GLN upregulated gut GTP, GCS, OPase, and GLNase in DMBA-tumor bearing, DMBA-treated, and/or control rats; however, it downregulated these enzymes in the tumor. The paradoxical effect of GLN on key GSH recycling enzymes in the gut versus tumor suggests that dietary supplemental GLN could be used in the clinical practice to increase the therapeutic index of cancer treatments by protecting normal tissues from, and sensitizing tumor cells to, chemotherapy and radiation-related injury.