Post-initiation treatment of rats with indole-3-carbinol or beta-naphthoflavone does not suppress 7, 12-dimethylbenz[a]anthracene-induced mammary gland carcinogenesis

Indole-3-carbinol (I3C) and β-naphthoflavone (β-NF), blocking agents of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mammary gland carcinogenesis, were examined as potential post-initiation suppressing agents. Treatment of female Sprague–Dawley rats with I3C (250 mg/kg body weight (b.w.)), β-NF (20 mg/kg b.w.) or the vehicle ethanol:corn oil (2:3) (2.5 ml/kg b.w.), three times weekly by gavage, started 3 weeks after the initiation with one oral dose of DMBA (20 mg/rat at 7 weeks of age) and continued for up to 12 weeks. I3C- or β-NF- or vehicle-treated groups did not differ significantly in the overall outcome of mammary tumorigenesis including cumulative mammary tumor incidences and multiplicities, latent periods and number and weight of mammary tumors per tumor-bearing rat for malignant, benign and/or malignant + benign tumors. A tendency of the I3C-treated rats to develop fewer mammary adenocarcinomas with a greater average weight per tumor per rat (2.32±1.50 g) than in the β-NF- (1.52±1.58 g) or vehicle- (1.55±1.53 g) treated groups suggests an effect, yet to be confirmed, of I3C on tumor development and growth. A 12-week treatment with I3C or β-NF significantly increased the P450-dependent activities of ethoxy-, methoxy-, benzyloxy- and pentoxy-(with I3C only) resorufin O-dealkylase in hepatic microsomes indicating induction of several P450s. The alterations in the P450 complement may affect endogenous estrogen metabolism and mammary gland and tumor characteristics at the molecular level, e.g. estrogen receptor status and/or proliferative activity, which require further studies.     

Suppression of mammary gland carcinogenesis by post-initiation treatment of rats with tamoxifen or indole-3-carbinol or their combination.

This study examined whether suppression of mammary gland carcinogenesis elicited by low doses of tamoxifen (TAM) can be enhanced by concomitant treatment of rats with indole-3-carbinol (I3C), a component of cruciferous vegetables and a dietary supplement used for its putative antiestrogenicity. Two weeks after one oral dose of 7,12-dimethylbenz[a]anthracene (DMBA) at 65 mg/kg body weight, female Sprague-Dawley rats started treatment with TAM (10 microg/rat) by subcutaneous injection, I3C (250 mg/kg body weight) by oral gavage, TAM+I3C or their respective vehicles three times per week, for up to 20 weeks. Significant increases in the median latency of malignant mammary tumors and decreases in the mean tumor mass per rat were due to TAM. Significant decreases in the mean tumor number per rat in TAM, I3C and TAM+I3C-treated rats indicated a cooperative effect of the two compounds. In both DMBA-initiated and uninitiated rats, significant increases in the ratios of liver to body weight in I3C and TAM+I3C-treated groups coincided with I3C-dependent increases of hepatic cytochrome P450 levels and activities (1A1, 1A2 and 2B1/2). The ratios of uterus to body weight decreased with the number of treatments and the decreases effected by TAM were greater than those by I3C. The levels of circulating estrone were increased in response to I3C treatment and were greater in DMBA-initiated rats than in uninitiated rats, which may contribute to the preventive effect of I3C. Chemoprevention may be accomplished through up-regulation of apoptotic enzyme (caspase) activities in the mammary gland or mammary tumors. Treatment with TAM, I3C or TAM+I3C had no effect on caspase-3&7, caspase-6, caspase-8 and caspase-9 activities in the mammary tumors or mammary gland of tumor-bearing rats or that of uninitiated rats. In the mammary gland of DMBA-initiated tumor-free rats, however, I3C treatment increased the levels of caspase-3&7 and caspase-9 activities, suggesting an I3C-mediated protective effect. Even though I3C alone is a much less effective suppressing agent of mammary carcinogenesis than TAM, I3C in combination with TAM does not weaken but may foster the benefits of chemoprevention with TAM.     

Microsomal metabolism of the carcinogen, N-2-fluorenylacetamide, by the mammary gland and liver of female rats. I. Ring- and N-hydroxylations of N-2-fluorenylacetamide

We determined ring- and N-hydroxybtions of a systemic mammarygland cardnogen, N-2-fluorenylaeetamide (2-FAA), by microsomalfractions of liver and mammary gland of female rats and theeffects of in vivo and/or in vitro modifiers of these oxidations.Pretreatment of lactating rats with 3-methylcholanthrene (3-MC)or ß-naphthoflavone (ß-NF) and non-lactating(50-day old virgin) rats with ß-NF showed similareffects in that the formation of 3-, 5-, 7-, 9- and N-hydroxy-2-FAAby hepatic microsomes was increased manyfold and the formationof 1-hydroxy-2-FAA was induced. In mammary gland microsomes,the formation of 3-, 5- and 7-hydroxy-2-FAA was likewise increased,but of 9-hydroxy-2-FAA was unaffected. Only mammary microsomesof lactating rats had capacity for N-hydroxylation which wasincreased {small tilde}3 times by pretreatment of rats with3-MC or ß-NF. All of the induced increases of metabolitesof 2-FAA in hepatic and mammary microsomes were inhibited by0.1 mM -naphthoflavone (-NF) in vitro. Pretreatment of non-lactatingrats with phenobarbital increased only the formation of 7-hydroxy-2-FAAin hepatic microsomes which was further stimulated by -NF invitro. The latter also stimulated the formation of 7- and 9-hydroxy-2-FAA by hepatic microsomes of the uninduced rats, buthad no effects in mammary microsomes, in which 9-hydroxy-2-FAAwas a major metabolite. Hence, the data showed qualitative andquantitative differences between lactating and non-lactatingrats in metabolism of 2-FAA by mammary microsomes which mayresult from differences in the levels (e.g., of cytochrome P-450)and activities of microsomal enzymes determined herein. In hepaticmicrosomes of these rats, differences in quantities of metabolitesof 2-FAA (3-, 7-, 9- and N-hydroxy-2-FAA) were found in cornoil-treated rats only. The solvent (methanol or acetone) usedfor addition of 2-FAA to the incubation mixtures altered quantitativelythe metabolite profiles in hepatic and mammary microsomes of3-MC or ß-NF treated rats. The formations of 1- and3- or 5- and 7-hydroxy-2-FAA were greater in the presence ofacetone or methanol, respectively. The results of this studysuggest that the formation of phenolic and N-hydroxy metabolitesof 2-FAA in both hepatic and mammary microsomes of lactatingrats is catalyzed by similar form(s) of cytochrome P-450 inducedby pretreatment with 3-MC or ß-NF. Lack of inductionN-hydroxylation of 2-FAA in mammary microsomes of non-lactatingrats supports our earlier conclusion that the formation of aproximate metabolite in mammary tumorigenesis by 2-FAA is accomplishedin the liver.     

Use of monoclonal antibodies to characterize the induction response of the cytochrome P-450-dependent mixed function oxidase system to nitrofluoranthenes

In prior studies with neonatal rats we have suggested that nitratedpolycyclic aromatic hydrocarbons (NPAH) are 3-methylcholanthrene(3-MC) type of inducers of cytochrome P-450. These observationshave been extended by studying the effect of fluoranthene (FL)and its nitrated derivative, 3-nitrofluoranthene (3-NF) anda mixture of nitrated fluor-anthenes (NTs) on the inductionof hepatic and pulmonary monooxygenase activities in adult rats.We have characterized the effect of these compounds on hepaticcytochrome P-450 isozyme(s) using immunoblot analysis. The administrationof 3-NF and NFs to rats resulted in highly significant induction(1.9- to 5.8-fold) of hepatic and pulmonary aryl hydrocarbonhydroxy-tose (AHH) and 7-ethoxvresoruffiri-O-deethylase (ERD)activities. FL was either ineffective or much less effectivein inducing these enzyme activities. The enzyme induction responseto these compounds occurred in the following order: NFs >3-NF > FL. SDS-PAGE of hepatic mkrosomes prepared from FL-,3-NF-and NFs-treated animals revealed a higher content of proteinmigrating in the P-450 region. Characterization of isozymesof P-450 was carried out by Western blot analysis with highlyspecific monoclonal antibodies (MAb) raised against 3-MC-spedficP-450 (MAb 1-7-1) and phenobarbital-spedfic P-450 (MAb-2-66-3)isozymes. Hepatic mkrosomes prepared from 3-NF-and NFs-treatedrats showed two distinct immunopredpitin bands with MAb 1-7-1whereas mkrosomes prepared from FL-treated animals showed asharp band with MAb 2-66-3. MAb 1-7-1 significantly inhibited({small tilde}80%) AHH activity induced by 3-NF and NFs. Onthe other hand FL-induced AHH activity was only moderately ({smalltilde}30%) inhibited by MAb 1-7-1 whereas higher inhibition({small tilde}60%) was observed with MAb 2-66-3. Analysis ofBP metabolites by h.p.l.c. revealed enhanced production of metabolitesby liver microsomes from 3-NF- and NFs-treated animals. Theformation of BP 7,8-diol was 1.8-to 2.4-fold increased followingtreatment of animals with 3-NF and NFs respectively. Additionof MAb 1-7-1 to a microsomal mixture from 3-NF- and NFs-treatedrats inhibited the formation of BP phenols (60—75%) andBP 7,8 diol (52—60%). These inhibitory effects were notobserved with mkrosomes prepared from FL-treated rats. Thesestudies suggest that NFs induce specific monooxygenases in liverand that they are inducers of P-450 isozymes c and d.     

Experimental down-regulation of intermediate biomarkers of carcinogenesis in mouse mammary epithelial cells

Summary The polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA) is a metabolism-dependent procarcinogen whose tumorigenicity is modified by dietary and endocrine manipulationsin vivo. DMBA initiates molecular and cellular alterations in the mammary tissue, while dietary components and estrogens affect the post-initiational phase of tumorigenic transformation. The mechanism(s) responsible for modulation of tumorigenic transformation remain unclear. This study examines the effects of selected tumor suppressing agents and estradiol (E₂) metabolites onin vitro DMBA carcinogenesis utilizing a newly established mouse mammary epithelial cell line C57/MG. Alteration in DNA repair synthesis, metabolism of E₂ via the C2- and C16-hydroxylation pathways, and acquisition of anchorage-independent growth were utilized as molecular, endocrine, and cellular biomarkers to quantitate the cellular transformation by DMBA and its modulation by tumor suppressing agents and E₂ metabolites. A single 24 hr exposure of 0.78 µM DMBA to C57/MG cells resulted in a 193.9% increase in DNA repair synthesis and a 73.1% decrease in C2/C16 hydroxylation of E₂. The DMBA treated C57/MG cells also exhibited increased anchorage-independencein vitro prior to tumorigenesisin vivo. A simultaneous treatment of cells with DMBA and with the highest non-cytotoxic doses of the tumor suppressing agents 5 µM N-(4-hydroxyphenyl) retinamide (HPR), 50 µM indole-3-carbinol (I3C), or 1 µM tamoxifen (TAM) resulted in a 35.6% to 63.9% decrease in DNA repair synthesis, a 23.8% to 1347.6% increase in C2/C16 hydroxylation of E₂, and a 53.8% to 72.4% decrease in anchorage-independent growth. The E₂ metabolites at the highest non-cytotoxic doses of 0.76 µM estrone (E₁), 0.69 µM 2-hydroxyestrone (2-OHE₁), and 0.66 µM 2-methoxyestrone (2-MeOHE₁) suppressed DMBA-induced DNA repair synthesis by 56.0% to 68.8%. These tumor suppressing agents and E₂ metabolites also effectively suppressed post-initiational, anchorage-independent growth by 24.9% to 72.4%. These results indicate that DMBA induces cellular transformation in part by causing DNA damage, altering C2/C16 hydroxylation in favor of C16-hydroxylation, and inducing anchorage-independent growth prior to tumor development. Effective downregulation of these genotoxic, endocrine and proliferative end points by prototypic tumor suppressing agents and by E₂ metabolites generated via the C2-hydroxylation pathway suggest that these agents may influence mammary tumorigenesis by inhibiting early occurring initiational and/or post initiational events.Abbreviations DMBA 7,12-dimethylbenz(a)anthracene - HPR N-(4-hydroxyphenyl) retinamide - I3C indole-3-carbinol - TAM tamoxifen - E₂ 17-estradiol - E₁ estrone - 2-OHE₁ 2-hydroxyestrone - 2-MeOHE₁ 2-methoxyestrone - 16-OHE₁ 16-hydroxyestrone - E₃ estriol - DME/F12 Dulbecco's modified Eagle's medium - F12 Ham's medium - HU hydroxyurea - PBS phosphate buffered saline - NaOH sodium hydroxide - SDS sodium dodecyl sulfate - TCA trichloroacetic acid - [C2-³H] E₂ estradiol labeled at C2 position - [C16-³H] E₂ estradiol labeled at C16 position - ANOVA analysis of variance     

HLA class I and class II expression of pulmonary adenocarcinoma cells and the influence of interferon gamma

Background: The clinico-biological significance of HLA (both class I antigen and class II one) expressed on tumor cells still remains controversial. Methods: Tumor cells were freshly separated from 33 surgical specimens of pulmonary adenocarcinoma. The tumor cells were incubated for 24 h in the presence or absence of IFN-γ (130 International Units/ml). After incubation, the cells were cytocentrifuged onto glass slides and immunostained with either an anti-HLA class I (A, B, C) monoclonal antibody or anti-HLA class II (DR) one. Results: In 22 of 33 cases (66.7%), the HLA class I were individually expressed by more than 60% of tumor cells while so were the HLA class II in 15 (45.4%). No significant correlation was observed between the HLA class I expression and the HLA class II one. The proportion of HLA class I-positive tumor cells correlated with neither the grade of histological differentiation nor the stage of disease. In contrast, the proportion of HLA class II-positive tumor cells correlated with both the grade of histological differentiation and the stage. In most cases, IFN-γ was found to increase the proportion of class II-positive tumor cells as well as that of class I-positive cells. Conclusions: The above findings thus suggested that the HLA class II expression might therefore represent a manifestation of cellular differentiation and that IFN-γ may, as a result, have the potential to differentiate cancer cells.     

Suppression of 7,12-dimethylbenz[a]anthracene-induced mammary carcinogenesis by pre-initiation treatment of rats with beta-naphthoflavone coincides with decreased levels of the carcinogen-derived DNA adducts in the mammary gland

BACKGROUND: Mechanisms underlying prevention by beta-naphthoflavone (beta-NF) of mammary carcinogenesis initiated with 7,12-dimethylbenz[a]anthracene (DMBA) in the rat were elucidated. METHODS AND RESULTS: Treatment of female Sprague-Dawley rats with beta-NF at 40 mg/kg b.wt. for 4 days by oral gavage in corn oil before a single oral dose of DMBA (112 mg/kg b.wt.) suppressed mammary gland carcinogenesis as shown by an increase in the median latent period from 10 to 24 weeks and a 60% decrease in the multiplicity of mammary adenocarcinomas. In contrast, a 20-day treatment with beta-NF starting 3 weeks after DMBA had no significant effects on mammary tumorigenesis. The activities of phase I and phase II enzymes were examined in the liver and mammary gland 24 h after treatment of rats with beta-NF, DMBA, or beta-NF followed by DMBA as in the first bioassay. Treatment with either beta-NF or DMBA increased the hepatic activities of cytochrome P450 (CYP)1A1, 1A2, and 2B1/2, and glutathione S-transferase, and the mammary activity of CYP1A1. The activity of mammary CYP2B1/2 induced by DMBA was decreased by beta-NF. In the liver, the increase of UDP-glucuronosyl transferase (GT) activity in rats treated with beta-NF and DMBA was 2.3-fold greater than in rats treated with DMBA alone. Thus, treatment with beta-NF likely increased the rate of glucuronidation of DMBA dihydrodiols leading to carcinogen detoxification. The levels of the DMBA adducts determined by 32P-postlabeling of the mammary gland DNA were decreased in the beta-NF-pretreated rats. Conclusion: The beta-NF-induced increase in the hepatic UDP-GT activity and decrease in the mammary DNA-DMBA adducts occurred under the same treatment regimen that led to suppression of DMBA-induced mammary carcinogenesis.     

The genotoxic air pollutant 3-nitrobenzanthrone and its reactive metabolite N-hydroxy-3-aminobenzanthrone lack initiating and complete carcinogenic activity in NMRI mouse skin

3-Nitrobenzanthrone (3-NBA), a genotoxic mutagen found in diesel exhaust and ambient air pollution and its active metabolite N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA) were tested for initiating and complete carcinogenic activity in the NMRI mouse skin carcinogenesis model. Both compounds were found to be inactive as either tumour initiators or complete carcinogens in mouse skin over a dose range of 25–400 nmol. Topical application of 3-NBA and N-OH-3-ABA produced DNA adduct patterns in epidermis, detected by ³²P-postlabelling, similar to those found previously in other organs of rats and mice. 24 h after a single treatment of 100 nmol DNA adduct levels produced by 3-NBA (18 ± 4 adducts/10⁸ nucleotides) were 6 times lower than those by 7,12-dimethylbenz[a]anthracene (DMBA; 114 ± 37 adducts/10⁸ nucleotides). In contrast, identical treatment with N-OH-3-ABA resulted in adduct levels in the same range as with DMBA (136 ± 25 adducts/10⁸ nucleotides), indicating that initial DNA adduct levels do not parallel tumour initiating activity. When compounds were tested for tumour initiating activity by a single treatment followed by twice-weekly applications of TPA, DNA adducts formed by DMBA, but not by 3-NBA or N-OH-3-ABA, were still detectable 40 weeks after treatment. When tested for activity as complete carcinogens by twice-weekly topical application, 3-NBA and N-OH-3-ABA produced identical DNA adduct profiles in mouse skin, with adducts still detectable after 40 weeks. Only 3-NBA produced detectable adducts in other organs.     

Evidence of separate pathways for viral and chemical carcinogenesis in c3h/stwi mouse mammary glands

Mammary tumors in mice may arise as the result of exogenous infection with the mouse mammary tumor virus [MMTV(S)], usually via the milk; by the action of endogenous MMTV genes which are transmitted genetically and are sometimes expressed as infectious virus [MMTV(L)]; or by the action of chemical carcinogens. We have examined the etiological relationship between chemical and virus in the induction of mammary cancer in C3H/StWi mice. Mammary tumors were induced with 7.12-dimethylbenz(a)anthracene (DMBA) in virgin C3H/StWi mice infected with exogenous mouse mammary tumor virus (C3H/StMTV) and in uninfected C3H/StWi females. The percent tumor risk in females whose glands were infected with exogenous mouse mammary tumor virus [MMTV(S)] was not different from that of MMTV(S)-negative, C3H/StWi mice following treatment with DMBA. This result suggested that there was no synergistic effect between the two carcinogens, exogenous MMTV and Dmba. All the tumors arising in DMBA-treated C3H/StMTV virgins were positive for MMTV env gene product, gp52 and MMTV gag gene product, p²⁷ by radioimmune competition assay. MMTV(S)-induced hyperplastic alveolar nodules (HAN) were observed in 62 % of the untreated C3H/StMTV glands at 8 months of age. Therefore, MMTV(S) was present and active in the mammary gland during the experimental period but had no enhancing influence on the sensitivity of the gland to carcinogenesis by DMBA. Tumors appearing in DMBA-treated C3H/StWi virgin females were also tested for MMTV gp⁵² and p²⁷ antigens to determine the presence of endogenous MMTV gene activity. Only occasional C3H/StWi tumors were positive and in most of these, p²⁷, but not gp⁵² was detected, suggesting non-coordinate expression of these endogenous MMTV gene products. Both alveolar (HAN) and ductal hyperplasia (DH) were found in DMBA-treated C3H/StMTV glands, whereas only DH were found in DMBA-treated C3H/StWi mice. Nevertheless, the DMBA-induced C3H/StMTV tumor histopathology was remarkably indistinguishable from that in tumors produced by DMBA in C3H/StWi mice, implying that in both groups, tumors arose primarily from the chemically-induced mammary dysplasias. These data taken together appear to support the conclusion that DMBA and Mmtv follow separate pathways to the induction of cancer in the mouse mammary gland.     

Long-term dysregulation of circadian and 17-β estradiol-induced LH,prolactinand corticosterone secretion after dimethylbenz (a) anthracene administrationin the Sprague–Dawley female rat

Summary A single intragastric administration of 7,12-dimethylbenz (a) anthracene (DMBA) has been shown, when given at 55–60 days of age, to induce mammary tumors in young cycling female Sprague–Dawley rats. The appearance of the tumors is preceded by a series of neuroendocrine disturbances of the hypothalamo–pituitary–gonadal (HPG) axis, including attenuation of the preovulatory Luteinizing Hormone (LH) and Gonadotropin-Releasing Hormone (GnRH) release and amplification of the preovulatory 17β-Estradiol (E₂) surge. In this study, we examined the hypothesis that a single administration of DMBA could also, in the long range, induce disturbances of others neuroendocrine axis, like the Hypothalamic–Pituitary–Adrenal (HPA) axis and/or the Lactotroph axis. Sprague–Dawley rats, 55–60 days of age, received, on the day of Estrous of the Estrous cycle, a single administration of 15 mg of DMBA delivered by intragastric intubation. Then, they were ovariectomized 5 days later. One month later, (1) Two groups of animal were sacrificed by decapitation at 09:00 a.m. and 05:00 p.m. to record the circadian rhythm of plasma LH, Prolactin (PRL) and corticosterone, (2) Three other groups of animal were sacrificed by decapitation at three different times after a morning subcutaneous administration of 50 μg/kg of Estradiol Benzoate (EB), to induce a negative and positive feed-back of the secretion of LH. Then, plasma LH, PRL and corticosterone concentrations were measured. After DMBA administration, (1) the negative–but not the positive-LH feed-back was seen, (2) the PRL circadian rhythm was blunted and the corticosterone circadian rhythm was almost absent, (3) the increase in PRL or Corticosterone plasma concentration was significantly reduced. In conclusion, a single administration of DMBA provokes a long-term dysregulation of not only the HPG axis but also of the lactotroph and HPA axis. These dysregulations, along with the already evidenced long-term inhibition of DMBA upon Melatonin secretion from the pineal gland, might accelerate the promotion of mammary tumors induced by the mammary carcinogen.     

Metabolism of nitrofluoranthenes by rat lung subcellular fractions

The nitrofluoranthene (NF) family of compounds includes thepotent pulmonary carcinogen 3, 9-dinitrofluoranthene (3, 9-DNF)and the weak carcinogen 3-nitrofluoranthene (3-NF). Althoughthe specific molecular mechanisms involved in this differencein sensitivity for the induction of lung tumors in rats by 3,9-DNF and 3-NF have not been defined, these compounds most likelyinduce carcinogenesis by metabolic activation to electrophilicmetabolites that bind DNA. The purpose of these investigationswas to determine the activation pathways in the rat lung forthe metabolism of the di-(3, 9-DNF) and mono-nitroisomers (3-NF,8-NF, 2-NF) of NFs. The metabolic rates of NFs were comparedfor lung subcellular fractions of pristine rats as well as ratspreviously treated with 3-methylcholanthrene (3-MC) or phenobarbitalat levels that would induce cytochrome P450 enzymes. One majormetabolite, the amino derivative, was detected by high pressureliquid chromatography following anaerobic incubation of ratlung cytosol with 3-NF, 8-NF, 2-NF or 3, 9-DNF. 3, 9-DNF wasmetabolized to its amino derivative, aminonitrofluoranthene,at a higher rate than 3-NF, 8-NF or 2-NF. Pretreatment of therats with 3-MC or phenobarbital did not affect the metabolicrates of cytosolic reduction. Both 3-NF and 3, 9-DNF were metabolizedanaerobically to their amino derivatives by microsomal reductase(s).3, 9-DNF was metabolized twice as fast as 3-NF. The formationof the aminonitrofluoranthene metabolite was increased 2 timeswith microsomes from 3-MC-induced rats, but was unaffected bymicrosomes from phenobarbital-treated rats. This suggests thatthe cytochrome P450 isozymes and reductase, which are inducedby 3-MC, may be involved in the metabolism of 3-NF and 3, 9-DNF.The metabolic products of 3-NF, formed aerobically, consistedof one major and three minor compounds. The major metabolite,tentatively identified as 3-NF-8-ol, was increased 6 times usingmicrosomes from 3-MC-induced rats. In contrast, 3, 9-DNF metabolismwas not detected aerobically with lung microsomes. Thus, ringhydroxylation was inhibited in the metabolism of 3, 9-DNF, andthe major pathway was nitroreduction. This higher rate of anaerobicmetabolism of 3, 9-DNF over 3-NF and the expected high reactivityof the putative N-acetoxy derivative formed from 3, 9-DNF maybe responsible for the differential potency for lung cancerinduction by these two carcinogens.     

Correlation of tumor necrosis factor α (TNFα) with high Caspase 3-like activity in myelodysplastic syndromes

Increased intramedullary apoptotic death of hematopoietic cells is thought to contribute to the ineffective hematopoiesis in myelodysplastic syndromes (MDS). Furthermore, high amounts of tumor necrosis factor α (TNFα) have previously been correlated with apoptosis in MDS marrows. The present studies were undertaken to examine the status of two key downstream effectors of TNFα signaling, i.e. Caspase 1 and Caspase 3 enzymes, using a fluorometric assay in the bone marrow aspirate mononuclear cells (BMMNC) in relation to apoptotic DNA fragmentation detected by in situ end-labeling (ISEL) of DNA and with localization of TNFα in the corresponding biopsies from 14 MDS patients. Both Caspase 1 and 3 were detectable in freshly harvested BMMNC, albeit median Caspase 3 levels (47.5 units/mg protein) being almost 10 times higher than Caspase 1 (4.0 units/mg protein). Upon short-term culture for 4 h in a serum-supplemented medium in vitro a significant increase was seen in Caspase 3 activity (58.8±13.9 at 0 h vs. 177.8±55.2 units/mg protein at 4 h, n=14, P=0.017) and in percent cells labeled by ISEL (apoptotic index or AI%: 0.76%±0.25% vs. 3.99%±1.1%, n=14, P=0.004, respectively). Caspase 1 activity increased after 15 min in culture. Interestingly, TNFα levels measured by immunohistochemistry correlated with the net increase in Caspase 3 activity after 4 h (ρ=0.517, n=13, P=0.07) and the starting levels of Caspase 1 at 0 h correlated with the Caspase 3 levels attained at 4 h (ρ=0.593, n=13, P=0.033). Additionally when TNFα-positive bone marrows (8/14) were compared with the negative marrows (6/14) the Caspase 3 levels were significantly higher in the TNFα-positive marrows (189.6±66.2 vs. 25.0±14.6 units/mg protein, respectively, P=0.043). The increase in AI%, though not statistically significant, was also higher in the TNFα-positive marrows. Finally in HL60 cells the effects of different Caspase inhibitors and pentoxifylline (PTX) (interferes with lipid signaling of cytokines) on TNFα-induced apoptosis were evaluated. TNFα treatment significantly increased AI% (P<0.003) as compared to the untreated controls. A co-treatment with three Caspase inhibitors, zVAD.FMK (inhibitor of Caspases 1 and 3, 10 μM/l), Ac.YVAD.FMK (Caspase 1 inhibitor, 1 μM/l), Ac.DEVD.FMK (Caspase 3 inhibitor, 10 μM/l) as well as PTX (250 μM/l) significantly curtailed the AI% induced by TNFα The present studies thus identify the downstream effectors of TNFα-inducible apoptosis in MDS and so also the suppressors of TNFα apoptotic signaling. These results may have significant clinical implications in the therapy of MDS in the future.     

Effects of 5,6-benzoflavone, indole-3-carbinol (I3C) and diindolylmethane (DIM) on chemically-induced mammary carcinogenesis: is DIM a substitute for I3C?

The abilities of 5,6-benzoflavone (5,6-BF, a synthetic flavonoid), indole-3-carbinol (I3C, a plant derived product) or diindolylmethane (DIM, a condensation product of I3C) to alter the induction of mammary cancers induced by the carcinogens 7,12-dimethylbenzanthracene (DMBA) or N-methyl-N-nitrosourea (MNU) were evaluated. Interestingly, the first two agents act as aryl hydrocarbon receptor (AhR) agonists, while DIM does not. The agents were initially examined for their ability to inhibit DMBA-induced mammary carcinogenesis. Agents were administered for 14 days starting 7 days prior to a single dose of the carcinogen. Evaluated over an extensive range of doses (165, 550 and 1650 ppm in the diet), 5,6-BF caused a dose-dependent decrease of mammary cancers. In addition, 5,6-BF at doses of 1650 and 165 ppm in the diet blocked the induction of DMBA-induced DNA adducts in the mammary gland by approximately 85% and 45%, respectively. In contrast, DIM (180 or 20 mg/kg BW/day) failed to block induction of DMBA tumors. The effect of these agents on the promotion/progression phase of carcinogenesis using the MNU mammary cancer model was also determined. 5,6-BF (1650 or 165 ppm in the diet), I3C (180 or 60 mg/kg BW/day administered by gavage), or DIM (180 or 60 mg/kg BW/day by gavage) were initiated 5 days after the administration of MNU, and continually thereafter. 5,6-BF decreased MNU- induced mammary tumor multiplicity by 40-60%. I3C reduced tumor multiplicity at the high dose, while DIM at either dose had minimal effects on tumor multiplicity. Thus, 5,6-BF and I3C were highly effective against initiation of DMBA-induced mammary carcinogenesis, and were also effective against MNU-induced tumors during the promotion/progression phase of carcinogenesis. In contrast, DIM had minimal effects in either model; arguing that administration of DIM is not analogous to administration of I3C.     

Complement C3a activates osteoclasts by regulating the PI3K/PDK1/SGK3 pathway in patients with multiple myeloma

Objective:Myeloma bone disease (MBD) is the most common complication of multiple myeloma (MM). Our previous study showed that the serum levels of C3/C4 in MM patients were significantly positively correlated with the severity of bone disease. However, the mechanism of C3a/C4a in osteoclasts MM patients remains unclear.Methods:The formation and function of osteoclasts were analyzed after adding C3a/C4a in vitro. RNA-seq analysis was used to screen the potential pathways affecting osteoclasts, and the results were verified by Western blot, qRT-PCR, and pathway inhibitors.Results:The osteoclast area per view induced by 1 μg/mL (mean ± SD: 50.828 ± 12.984%) and 10 μg/mL (53.663 ± 12.685%) of C3a was significantly increased compared to the control group (0 μg/mL) (34.635 ± 8.916%) (P < 0.001 and P < 0.001, respectively). The relative mRNA expressions of genes, OSCAR/TRAP/RANKL/cathepsin K, induced by 1 μg/mL (median: 5.041, 3.726, 1.638, and 4.752, respectively) and 10 μg/mL (median: 5.140, 3.702, 2.250, and 5.172, respectively) of C3a was significantly increased compared to the control group (median: 3.137, 2.004, 0.573, and 2.257, respectively) (1 μg/mL P = 0.001, P = 0.003, P < 0.001, and P = 0.008, respectively; 10 μg/mL: P < 0.001, P = 0.019, P < 0.001, and P = 0.002, respectively). The absorption areas of the osteoclast resorption pits per view induced by 1 μg/mL (mean ± SD: 51.464 ± 11.983%) and 10 μg/mL (50.219 ± 12.067%) of C3a was also significantly increased (33.845 ± 8.331%) (P < 0.001 and P < 0.001, respectively) compared to the control. There was no difference between the C4a and control groups. RNA-seq analysis showed that C3a promoted the proliferation of osteoclasts using the phosphoinositide 3-kinase (PI3K) signaling pathway. The relative expressions of PIK3CA/phosphoinositide dependent kinase-1 (PDK1)/serum and glucocorticoid inducible protein kinases (SGK3) genes and PI3K/PDK1/p-SGK3 protein in the C3a group were significantly higher than in the control group. The activation role of C3a in osteoclasts of MM patients was reduced by the SGK inhibitor (EMD638683).Conclusions:C3a activated osteoclasts by regulating the PI3K/PDK1/SGK3 pathways in MM patients, which was reduced using a SGK inhibitor. Overall, our results identified potential therapeutic targets and strategies for MBD patients.     

Effects of dietary indole-3-carbinol on estradiol metabolism and spontaneous mammary tumors in mice

Indole-3-carbinol (I3C) is a potent inducer of cytochrome P450 enzymes in many species, including humans. We therefore studied alterations in the cytochrome P450-dependent metabolism of estradiol in different strains of mice consuming I3C in semisynthetic powdered diets at doses ranging from 250 to 5000 p.p.m. (34-700 mg/kg/day) for different periods of time. In short-term metabolic studies (3 weeks), wet liver weight increased in SW and C3H/OuJ mice in a dose-responsive manner. Dietary I3C increased the cytochrome P450 content measured in hepatic microsomes, as well as the extent of estradiol 2-hydroxylation, up to 5-fold. In a long-term feeding experiment (8 months), female C3H/OuJ mice consumed synthetic diets containing I3C at 0, 500 or 2000 p.p.m. Mammary tumor incidence and multiplicity were significantly lower at both doses of I3C, and tumor latency was prolonged in the high-dose group. We conclude that I3C is an inducer of hepatic P450-dependent estrogen metabolism in mice, and that it is chemopreventive in the C3H/OuJ mouse mammary tumor model. This protective effect may be mediated in part by the increased 2-hydroxylation and consequent inactivation of endogenous estrogens.     

The somatostatin analog Sandostatin (SMS201-995) in treatment of DMBA-induced rat mammary tumors

Summary The effects of treatment with a somatostatin analog (Sandostatin, SMS201-995) were investigated in female rats with dimethylbenzanthracene(DMBA)-induced rat mammary tumors. A 3-week treatment was performed using sandostatin, the LHRH-agonist buserelin alone, or buserelin in combination with sandostatin. Twice daily sandostatin treatment was performed with dosages of 0.05 µg, 0.2 µg, 1 µg, 5 µg, and 20 µg. Buserelin was used in a 2 × 5 µg/day dosage.The combined results from six different experiments show that the various dosages of sandostatin caused no tumor growth inhibition. Somatostatin receptors could not be demonstrated in these mammary tumors. Sandostatin treatment by daily injections did not suppress levels of growth hormone, prolactin, or epidermal growth factor-like activities. Estrogen (ER) and progesterone (PgR) receptor contents of the mammary tumors were not changed. In contrast, buserelin treatment caused highly significant tumor remission. The combined treatment with sandostatin and buserelin did not alter the treatment results obtained after treatment with buserelin alone.In conclusion, sandostatin treatment in this tumor model had no direct growth inhibitory effect and did not cause an endocrine inhibition of mammary tumor growth. However, these results do not exclude antitumor effects in human breast cancer in view of the presence of somatostatin receptors in approximately 20–45% of human tumors, besides possible different endocrine effects.     

Loss of PPARγ expression in mammary secretory epithelial cells creates a pro‐breast tumorigenic environment

Breast cancer is the leading cause of new cancer diagnoses among women. Using peroxisome proliferator‐activated receptor (PPAR)γ^(+/?) mice, we showed normal expression of PPARγ was critical to stop 7,12‐dimethylbenz[a]anthracene (DMBA)‐induced breast tumorigenesis. PPARγ is expressed in many breast cell types including mammary secretory epithelial (MSE) cells. MSEs proliferate as required during pregnancy, and undergo apoptosis or reversible transdifferentiation during involution once lactation is complete. Thus, MSE‐specific loss of PPARγ was hypothesized to enhance DMBA‐mediated breast tumorigenesis. To test this, MSE cell‐specific PPARγ knockout (PPARγ‐MSE KO) and control (PPARγ‐WT) mice were generated, mated and allowed to nurse for three days. One week after involution, dams were treated with DMBA to initiate breast tumors, and randomized on week 7 to continue receiving a normal chow diet (DMBA Only: PPARγ‐WT, n = 15; PPARγ‐MSE KO, n = 25) or one supplemented with a PPARγ activating drug (DMBA + ROSI: PPARγ‐WT, n = 17; PPARγ‐MSE KO, n = 24), and monitored for changes in breast tumor outcomes. PPARγ‐MSE KOs had significantly lower overall survival and decreased mammary tumor latency as compared to PPARγ‐WT controls. PPARγ activation significantly reduced DMBA‐mediated malignant mammary tumor volumes irrespective of genotype. MSE‐specific PPARγ loss resulted in decreased mammary gland expression of PTEN and Bax, increased superoxide anion production, and elevated serum eotaxin and RANTES, creating a protumorigenic environment. Moreover, PPARγ activation in MSEs delayed mammary tumor growth in part by down‐regulating Cox‐1, Cox‐2 and cyclin D1. Collectively, these studies highlight a protective role of MSE‐specific PPARγ during breast tumorigenesis, and support a novel chemotherapeutic role of PPARγ activation in breast cancer.     

Targeting HER3 with miR-450b-3p suppresses breast cancer cells proliferation

In breast cancer cells, heterodimerization of HER2 and HER3 plays important and dominant roles in the functionality and transformation of HER-mediated pathways, in particular the PI3K/Akt survival pathway. HER3 was considered as a major signaling hub in HER2-amplified cancers. Inhibition of HER3 expression may therefore represent a rational therapeutic approach to breast cancers where HER2/HER3-mediated signaling plays a role in tumorigenesis and progression. miRNAs exerts important roles in regulating gene expressions by binding to and repressing target mRNAs. Here we reported that miRNA-450b-3p inhibits HER3 expression by directly targeting 3′ UTR of HER3 mRNA and represses the downstream signal transductions of HER family. Overexpression of miRNA-450b-3p in SKBR3 cells inhibits cells clonogenic potential and enhances their sensitivity to trastuzumab, a monoclonal antibody that binds to the HER2 receptor, or doxorubicin through repressing proliferative signal pathways mediated by HER3/HER2/PI3K/AKT. Furthermore, we found that breast cancer patients with tumors that demonstrating upregulated HER3 (>2-fold) and downregulated miR-450b-3p (>2-fold) expressions compared with the paired adjacent non-tumorous tissues showed significantly poorer overall survival (P < 0.05). Our study identified miRNA-450b-3p as a new tumor repressor and also provided some evidences suggesting that downregulation of miR-450b-3p expression with concurrent overexpression of HER3 may serve as a prognostic biomarker for poor overall survival in breast cancer patients.     

A biotherapy based on PSCs-in-3D spheroid-ameliorated biologics depletes in vivo cancer-sustaining stem cells

CSCs are able to survive routine anticancer procedures and peripheral-immune attack. Here we develop and detail a framework of CSC elimination governed by 3D-biologics. Pluripotent cells-engineered 3D-biologics (PMSB) and control non-3D-biologics were prepared from placenta-based somatic stem cells (PSCs) and inoculated respectively into senile hosts bearing progressive mammary, lung, colon carcinomas and melanoma. We demonstrate that PMSB evokes in vivo central-immune microenvironment with subsequent re-expression of thymosin-α1 ~ β4 in thymic cortex-medulla borderline for rapid MHC-unrestricted renewal of γδT-dominated immunocompetence. The post-renewal γδT-subsets could accurately bind and drive CSCs into apoptosis. Finally, with central/peripheral integral microenvironment renewal and TERT/Wnt/β-catenin pathway blockade, the CSC-subsets are fully depleted, leading to substantial cure of diverse tumors by PMSB inoculation (P < 0.01), yet not by non-3D-biologics. Thus, our study may contribute to open up a new avenue for tumor remission via pluripotent cells-engineered 3D-biologics addressing quick renewal of central-thymus and peripheral immune-microenvironment.     

Dose-dependent benefits of quercetin on tumorigenesis in the C3(1)/SV40Tag transgenic mouse model of breast cancer

Breast cancer is the leading cause of cancer related death in women. Quercetin is a flavonol shown to have anti-carcinogenic actions. However, few studies have investigated the dose-dependent effects of quercetin on tumorigenesis and none have used the C3(1)/SV40 Tag breast cancer mouse model. At 4 weeks of age female C3(1)/SV40 Tag mice were randomized to one of four dietary treatments (n = 15–16/group): control (no quercetin), low-dose quercetin (0.02% diet), moderate-dose quercetin (0.2% diet), or high-dose quercetin (2% diet). Tumor number and volume was assessed twice a week and at sacrifice (20 wks). Results showed an inverted ‘U’ dose-dependent effect of dietary quercetin on tumor number and volume; at sacrifice the moderate dose was most efficacious and reduced tumor number 20% and tumor volume 78% compared to control mice (C3-Con: 9.0 ± 0.9; C3-0.2%: 7.3 ± 0.9) and (C3-Con: 2061.8 ± 977.0 mm³; and C3-0.2%: 462.9 ± 75.9 mm³). Tumor volume at sacrifice was also reduced by the moderate dose compared to the high and low doses (C3-2%: 1163.2 ± 305.9 mm³; C3-0.02%: 1401.5 ± 555.6 mm³), as was tumor number (C3-2%: 10.7 ± 1.3 mm³; C3-0.02%: 8.1 ± 1.1 mm³). Gene expression microarray analysis performed on mammary glands from C3-Con and C3-0.2% mice determined that 31 genes were down-regulated and 9 genes were up-regulated more than 2-fold (P < 0.05) by quercetin treatment. We report the novel finding that there is a distinct dose-dependent effect of quercetin on tumor number and volume in a transgenic mouse model of human breast cancer, which is associated with a specific gene expression signature related to quercetin treatment.