Reduced Tumor Multiplicity Research Articles

Phosphatidylinositol 3-Kinase γ Inhibition Ameliorates Inflammation and Tumor Growth in a Model of Colitis-Associated Cancer

A large body of evidence supports a correlation between inflammation and cancer, although the molecular mechanisms that govern this process are incompletely understood. Phosphatidylinositol 3-kinase (PI3K) is an enzyme that regulates the immune response and contributes to cell transformation in several tumor types. Here, we address the role of the PI3Kgamma isoform in inflammatory bowel disease and in the development of colitis-associated cancer. PI3Kgamma(-/-) and control mice were repeatedly treated with dextran sulfate sodium to induce chronic colitis and colitis-associated cancer. Colorectal tumor burden and colon inflammation were evaluated in these mice. Leukocyte populations in colon were characterized by flow cytometry analysis. PI3Kgamma-deficient mice had a lower incidence of colitis-associated tumors, as well as reduced tumor multiplicity and smaller tumor size compared with controls. Reduced tumor development paralleled less colon inflammation in PI3Kgamma-deficient mice. Analysis of leukocyte populations in the colon of PI3Kgamma-deficient mice showed defective activation and infiltration of myeloid cells and defective recruitment of T cells to the colon compared with controls. PI3Kgamma regulates the innate immune response in a murine model of ulcerative colitis, thereby controlling colon inflammation and tumor formation.

Abstract B155: Effects of bamboo extract on breast cancer

Abstract Breast cancer is the most common cancer among women in the United States, and worldwide over one million new cases are diagnosed each year. Preliminary studies by Panee and colleagues demonstrated that 0.5% (w/w) dietary supplementation with an ethanol/water extract from bamboo Phyllostachys edulis inhibited the incidence of 7,12-dimethylbenz[a]anthracene (DMBA, a carcinogen)-induced mammary tumors in female Sprague-Dawley rats by 44% and dramatically reduced tumor multiplicity. This project has aimed to delineate the anti-breast cancer mechanisms of this bamboo extract (BEX) in the aforementioned rat model. We hypothesized that BEX may alter the gene expression of key proteins involved in estrogen- and/or growth factor-signaling pathways and xenobiotic biotransforming enzymes in the mammary tumors. To test this hypothesis, we extracted RNA from individual mammary tumors that were harvested from each rat. Then, we synthesized complementary DNA (cDNA) and combined the cDNA samples according to each rat's dietary group— BEX-supplemented rat diet or control rat diet. Using quantitative real-time polymerase chain reaction, we measured expression levels of target genes that included the following: estrogen receptors alpha and beta (ER-α and -β), human epidermal growth factor receptor 2 (HER2), and all glutathioine-s-transferase (GST) isoforms. Comparison between study groups revealed that BEX-supplementation significantly downregulated the gene expression of ER-α, ER-β, and HER2 in the mammary tumors by 65%, 70%, and 67% respectively (p < 0.0001). Additionally, the mammary tumors expressed all GST isoforms, whereby GSTa4 and GSTp1 resulted in high abundant values. Interestingly, BEX supplementation also significantly downregulated the gene expression of GSTa4 and GSTp1 by 32% and 31% respectively (p < 0.005). Our observations have indicated that the downregulation of ER-α, ER-β, and HER2 gene expression in the mammary tumors may contribute to the anti-breast cancer effect of BEX, and that BEX may also reduce chemoresistance in the mammary tumors by inhibiting the gene expression of xenobiotic biotransforming enzyme GST. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B155.

XANTHOHUMOL FROM HOPS PREVENTS HORMONE-DEPENDENT TUMOURIGENESIS IN VITRO AND IN VIVO

Since the 1990s, interest in health-promoting effects of xanthohumol (XN) has increased constantly. This manuscript will review some of the published literature as well as novel findings on XN. In 2002, we identified XN as a potent cancer chemopreventive agent acting by multiple mechanisms relevant for the prevention of carcinogenesis. Although hops is commonly linked with phytoestrogenic effects, we identified XN as a pure estrogen antagonist. Interestingly, XN may also reduce the generation of estrogens by inhibition of the enzymatic activity of aromatase, which converts testosterone to estrogen. Anti-estrogenic effects of XN (100 mg/kg body weight/day) were confirmed in vivo in an uterotrophy assay with prepubertal rats. In two fertility studies, long-term treatment with XN did not cause any adverse effect on female reproduction and on the development of offspring when given either for four weeks prior to or during mating, gestation and nursing. Novel data indicate that XN (100 mg/kg body weight/day in drinking water) prevents dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis in rats when given nine days prior to and for 16 weeks after the carcinogen. Tumour incidence was not influenced by XN treatment, but XN significantly reduced tumour multiplicity and tumour burden. XN (1000 mg/kg body weight/day injected subcutaneously for 14 days) also significantly reduced the growth of human breast cancer xenotransplants in immune-compromised mice and reduced tumour-induced neovascularization. Overall, these data demonstrate breast cancer preventive efficacy of XN. Multiple mechanisms, including modulation of DMBA metabolism, anti-estrogenic, anti-proliferative and anti-angiogenic activities may account for these effects and are under further investigation.

Preventive effects of the EGFr inhibitor erlotinib in ER+ methylnitrosourea (MNU) induced and in ER- MMTV/Neu/P53KO mammary cancer models.

Abstract Abstract #1112 The preventive/therapeutic efficacies of Erlotinib in both ER+ and ER- models of breast cancer in rats and mice, respectively, were evaluated. For the ER+ model, female Sprague-Dawley rats were administered MNU at 50 days of age. Beginning five days later, Erlotinib was administered 7x/week for the remainder of the study. Daily administration of Erlotinib in a prevention setting at 4 and 1.33 mg/kg BW/day reduced tumor multiplicity by 72 and 35%, respectively; doses >6 mg/kg BW/day decreased cancers by >90%. When rats with palpable mammary cancers were treated for six weeks with 4 mg/kg BW/day of Erlotinib, most of the tumors showed highly significantly regressions. Similarly, when rats bearing palpable mammary cancers were treated with Erlotinib for 5 days (3 or 1 mg/kg BW/day), large decreases in tumor cell proliferation and alterations in expression of phosphorylated EGFr, AKT, ERK and SHC were observed. In additional studies, the preventive effect of Erlotinib in a MMTV/Neu/P53KO bitransgenic model was examined. These mice develop ER- cancers which overexpress Neu and have an alteration in P53. These changes are characteristic of ER- human breast cancers. An initial experiment showed that Erlotinib (50 mg/Kg BW/day) was also effective as a preventive agent in this model. Effects of this agent on proliferation and altered expression of phosphorylated EGFr, AKT, ERK and SHC are in progress. Our results showing efficacy of the EGFr inhibitor Erlotinib in ER+ mammary cancers are in agreement with a clinical study of early stage human ER+ breast cancer showing that the EGFr inhibitor Gefitinib is highly effective in ER+/EGFr+ tumors (Polychronis, et al., Lancet Oncolo. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1112.

Contrasting epistatic interactions between rat quantitative trait loci controlling mammary cancer development

We previously defined quantitative trait loci (QTLs) that control susceptibility to 7,12-dimethylbenz(alpha)anthracene-induced mammary carcinoma in SPRD-Cu3 (susceptible) and WKY (resistant) rats. Two of these QTLs, assigned to chromosomes (Chr) 10 and 18, control tumor growth rate and invasiveness. In this study we characterized a congenic strain in which a large segment of WKY Chr 10 was introduced in the SPRD-Cu3 genetic background and demonstrated that this chromosome segment controls this tumor trait. The WKY allele at this QTL (Mcsta1) reduces the growth rate of the fastest growing tumors by 26%. We also previously showed that two SPRD-Cu3-WKY congenic strains containing a WKY chromosome segment derived either from Chr 5 or from Chr 18 exhibit a reduction in tumor multiplicity (QTLs Msctm1 and Mcstm2, respectively) (with no reduction in tumor growth rate in the Chr 18 congenic). In this study we generated a double congenic strain, which contains the two WKY differential segments from Chr 5 and 18, to determine how these two segments interact with one another. Interestingly, two types of epistatic interactions were found: no additive effect was seen with respect to tumor multiplicity, while a reduction in tumor growth rate was observed. It thus appears that WKY alleles located on Chr 5 and Chr 8 interact epistatically in a contrasting manner to modulate tumor multiplicity (in a nonadditive manner) and growth rate (in a synergic manner). Tumor growth rate is thus influenced by two QTLs, on Chr 10 (Mcsta1) and on Chr 18 (Mcsta2), the action of the latter being dependent on the presence of the Chr5 QTL (Mcstm1). The expression level of positional and functional candidate genes was also analyzed. On Chr 5, Pla2g2a is subject to a syntenic control while expression of the Tp53 (Chr 10) and Pmai1/Noxa (Chr 18) genes appears to be controlled by several mammary cancer resistance QTLs.

Matrix metalloproteinase‐9 contributes to intestinal tumourigenesis in the adenomatous polyposis coli multiple intestinal neoplasia mouse

Matrix metalloproteinases (MMPs) are a family of 23 extracellular proteases that are best known for their collective ability to degrade all components of the extracellular matrix. We previously demonstrated that genetic ablation of MMP-7 reduced tumour multiplicity in multiple intestinal neoplasia (Min) mice possessing a genetic alteration in the adenomatous polyposis coli gene (APC). These mice, commonly referred to as APC-Min mice, are a frequently used model of early intestinal tumourigenesis. To examine further the role of MMPs in intestinal tumour development, we generated APC-Min mice genetically deficient in MMP-2, -9, -12 or -19. Genetic ablation of MMP-2, -12 or -19 did not affect multiplicity or size of intestinal tumours when crossed into the APC-Min system. However, MMP-9 deficient animals developed 40% fewer tumours than littermate controls, although tumour size distribution remained unaffected. Intestinal adenomas from MMP-9 deficient mice demonstrated a 50% decrease in proliferating cells compared with control tissues, with no difference in apoptosis. To determine the cellular origin of MMP-9 in these tumours, immunofluorescent co-staining with markers for different leucocyte lineages was used to demonstrate that intratumoural MMP-9 is largely a product of neutrophils. These studies extend the potential targets for chemoprevention of intestinal adenomas to MMP-9 in addition to MMP-7 and exclude MMP-2,-12,-19 as attractive targets for intervention.

Keratinocyte Expression of MMP3 Enhances Differentiation and Prevents Tumor Establishment

Matrix metalloproteinase (MMP)-3 is induced by multiple cell types in the skin during processes involved in both normal and pathological tissue remodeling. We previously demonstrated that MMP3-null animals have an increased sensitivity to the development of squamous cell carcinoma, suggesting that overall, MMP3 has a protective role in squamous cell carcinoma. However, not all cellular responses affected by a loss of MMP3 are tumor-protective, and tumor expression of MMP3 is co-incident with an invasive tumor phenotype. Transgenic mice were generated with MMP3 targeted to keratinocytes to examine the biological role of tumor-produced MMP3. Overexpression of MMP3 reduced tumor multiplicity in response to chemically induced squamous cell carcinoma. Vascular density was increased with MMP3 overexpression; however, other cellular processes, including tumor growth and leukocyte infiltration, were unaffected. In accordance with the change in tumor multiplicity, SP-1 murine papilloma cell lines that were generated to stably express MMP3 lost the capacity to establish palpable tumors following orthotopic injection into immunocompromised mice. Analysis of epidermal biopsies taken at 1 to 2 weeks postinjection revealed that these MMP3-expressing Sp-1 lines had reduced levels of proliferation and pronounced differentiation. These same cells demonstrated an increased ability to differentiate in vitro, an effect that was inhibited by broad-spectrum MMP and selective MMP3 inhibition. These studies suggest that keratinocyte expression of MMP3 promotes cellular differentiation, impeding tumor establishment during tumorigenesis.

Effects of gefitinib (Iressa) on mammary cancers: preventive studies with varied dosages, combinations with vorozole or targretin, and biomarker changes

The ability of the epidermal growth factor receptor inhibitor gefitinib (Iressa) to prevent/treat methylnitrosourea (MNU)-induced mammary cancers and to modulate biomarkers in female Sprague-Dawley rats was examined. Rats were given a single dose of MNU (75 mg/kg body weight) at 50 days of age. In the prevention studies, continual treatment with Iressa at 10, 3, or 1 mg/kg body weight per day beginning 5 days after MNU reduced tumor multiplicity by 93%, 43%, and 20%, respectively. Treatment of rats bearing small palpable cancers with Iressa (10 mg/kg body weight per day) resulted in the complete regression of 70% of the tumors. Short-term treatment of tumor-bearing rats with Iressa caused decreases in cell proliferation and phosphorylated epidermal growth factor receptor and increases in apoptosis. To examine treatment regimens that might decrease the skin toxicity associated with Iressa, both intermittent treatments and combinations of lower doses of Iressa with other effective agents were evaluated. Treatment with Iressa (10 mg/kg body weight per day) continually or intermittently (either "3 weeks on/3 weeks off" or "4 days on/3 days off") reduced cancer multiplicity by 91%, 24%, and 68%, respectively. However, all regimens reduced tumor weights >85%. Finally, combining suboptimal doses of Iressa with suboptimal doses of vorozole (an aromatase inhibitor) or targretin (a retinoid X receptor agonist) yielded greater chemopreventive efficacy than any of these agents given alone.

Chemopreventive effects of celecoxib are limited to hormonally responsive mammary carcinomas in the neu-induced retroviral rat model

IntroductionWhile current breast cancer chemoprevention strategies using selective estrogen response modulators and aromatase inhibitors are quite successful, their effects are limited to hormonally responsive breast cancer. Hormonally nonresponsive breast cancer (including estrogen receptor-negative cancer) is associated with poor prognosis for patients, and few chemoprevention agents exist for this type of cancer. The cyclooxygenase-2 inhibitor celecoxib (Celebrex®) is a nonsteroidal anti-inflammatory drug and as such is a potential candidate for the prevention of hormonally nonresponsive breast cancer.MethodsThe chemopreventive effects of celecoxib were evaluated in the neu-induced retroviral rat mammary carcinogenesis model, to assess the efficacy of celecoxib on hormonally responsive and hormonally nonresponsive mammary carcinomas.ResultsDietary celecoxib at 1,200 mg/kg diet was highly efficacious in the prevention of hormonally responsive mammary carcinomas in intact rats, decreasing tumor multiplicity by 56% (P < 0.0001) and by 74% (P = 0.0002) in two independent experiments. No significant effect was found, however, on hormonally nonresponsive mammary carcinomas of ovariectomized rats. Treatment with a combination diet, consisting of tamoxifen at 2 mg/kg diet and celecoxib at 1,200 mg/kg diet, reduced tumor multiplicity by 72% (P = 0.0002) in intact rats. This reduction was not statistically different from that observed with celecoxib alone. Furthermore, long-term treatment with celecoxib was not associated with reductions in tumor volume in either intact rats or ovariectomized rats. In contrast, tamoxifen treatment and the combination regimen caused significant reductions in tumor volumes in intact rats (P = 0.01 and P = 0.004, respectively). Consistent with these data, decreases in proliferation and increases in apoptosis were detected in tamoxifen-treated and combination diet-treated tumors. No such modulations were observed in celecoxib-treated tumors.ConclusionThe chemopreventive effects of celecoxib appear to be limited to modulations in multiplicity of hormonally responsive mammary carcinomas. The fact that no synergistic or additive effects were observed in combination diet-treated rats raises the question of whether celecoxib is suitable for the prevention of hormonally nonresponsive breast cancer or for use in combination therapy with selective estrogen response modulators or aromatase inhibitors.

Gefitinib prevents cancer progression in mice expressing the activated rat HER2/neu

We tested the efficacy of gefitinib in the prevention of HER2/neu-mediated breast cancer development in BALB-NeuT transgenic mice. Oral administration of gefitinib to female transgenic mice from 5 to 14 weeks of age reduced tumor multiplicity from 9.6 +/- 0.82 to 0.58 +/- 1.1 (83%). We observed a decrease in the number and size of lobules and lobular nodules in treated mice with a reduction in the overall disease burden per gland. Normal duct development in the mammary glands was not affected by gefitinib. The development of acinic cell carcinoma in the parotid glands of these animals was also reduced coincident with decreased stromal involvement during progression. Gefitinib eliminated phosphorylation of HER2 and HER3 and signaling through MAPK and Akt in lobular hyperplasias and carcinomas. At the same time MAPK activity and cytokine production in splenocytes and lymph nodes was increased in gefitinib-treated animals coincident with an increase in lymph node size. Delaying gefitinib treatment until mammary glands exhibited atypical lobular hyperplasias reduced efficacy. These studies demonstrate the critical role of HER2 signal transduction in the onset and progression of HER2/neu-dependent breast cancer and suggest a role for specific inhibitors to prevent the outgrowth of early hyperplastic disease.

Dietary Lutein/Zeaxanthin Partially Reduces Photoaging and Photocarcinogenesis in Chronically UVB-Irradiated Skh-1 Hairless Mice

Lutein and zeaxanthin are xanthophyll carotenoids with potent antioxidant properties protecting the skin from acute photodamage. This study extended the investigation to chronic photodamage and photocarcinogenesis. Mice received either a lutein/zeaxanthin-supplemented diet or a standard nonsupplemented diet. Dorsal skin of female Skh-1 hairless mice was exposed to UVB radiation with a cumulative dose of 16,000 mJ/cm² for photoaging and 30,200 mJ/cm² for photocarcinogenesis. Clinical evaluations were performed weekly, and the animals were sacrificed 24 h after the last UVB exposure. For photoaging experiments, skin fold thickness, suprapapillary plate thickness, mast cell counts and dermal desmosine content were evaluated. For photocarcinogenesis, samples of tumors larger than 2 mm were analyzed for histological characterization, hyperproliferation index, tumor multiplicity, total tumor volume and tumor-free survival time. Results of the photoaging experiment revealed that skin fold thickness and number of infiltrating mast cells following UVB irradiation were significantly less in lutein/zeaxanthin-treated mice when compared to irradiated animals fed the standard diet. The results of the photocarcinogenesis experiment were increased tumor-free survival time, reduced tumor multiplicity and total tumor volume in lutein/zeaxanthin-treated mice in comparison with control irradiated animals fed the standard diet. These data demonstrate that dietary lutein/zeaxanthin supplementation protects the skin against UVB-induced photoaging and photocarcinogenesis.

Mammary cancer resistance and precocious mammary differentiation in the WKY rat: Identification of 2 quantitative trait loci

The COP and WKY rat strains are resistant to mammary cancer. It has shown previously that upon chemical carcinogen treatment, COP females exhibit mammary preneoplastic lesions which disappear within a few weeks. We show here that in similar conditions, WKY females do not exhibit any visible preneoplastic lesions. WKY females are characterized by precocious mammary tissue differentiation, including active expression of the beta-casein gene in young virgin females. This trait might be critical in resistance to mammary carcinogenesis of WKY rats. To test this hypothesis, we took advantage of 2 congenic strains that contain a limited chromosome segment of WKY origin, derived either from chromosome 5 or from chromosome 18, introgressed in the susceptible genetic background (SPRD-Cu3). Each of these congenic strains has been shown to be partially resistant to chemically induced mammary carcinogenesis (reduction in tumour multiplicity with respect to the susceptible SPRD-Cu3 rats). We show here that these 2 congenic strains also exhibit precocious mammary differentiation, though to a lower extent than the WKY females. The conclusion of this study is thus 2-fold: (i) eradication of preneoplastic lesions is not a general phenomenon in mammary cancer resistance; (ii) the same segment of rat chromosomes 5 or 18 that controls mammary cancer resistance also contains a quantitative trait locus imposing precocious mammary differentiation. These 2 traits are thus associated, supporting the hypothesis that there might be a cause-effect relationship between precocious mammary differentiation and cancer resistance.

Indole-3-carbinol Inhibits 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Plus Benzo(a)pyrene–Induced Lung Tumorigenesis in A/J Mice and Modulates Carcinogen-Induced Alterations in Protein Levels

We tested the chemopreventive efficacy of indole-3-carbinol (I3C), a constituent of Brassica vegetables, and its major condensation product, 3,3'-diindolylmethane (DIM), against lung tumorigenesis induced by a mixture of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (BaP) in A/J mice. The mixture of NNK plus BaP (2 micromol each) was administered by gavage as eight weekly doses, whereas I3C (112 micromol/g diet) and DIM (2 and 30 micromol/g diet in experiments 1 and 2, respectively) were given in the diet for 23 weeks beginning at 50% of carcinogen treatment. I3C reduced NNK plus BaP-induced tumor multiplicity by 78% in experiment 1 and 86% in experiment 2; the respective reductions in tumor multiplicity by DIM were 5% and 66%. Using a quantitative proteomics method, isobaric tags for relative and absolute quantitation (iTRAQ) coupled with mass spectrometry, we identified and quantified at least 250 proteins in lung tissues. Of these proteins, nine showed differences in relative abundance in lung tissues of carcinogen-treated versus untreated mice: fatty acid synthase, transketolase, pulmonary surfactant-associated protein C (SP-C), L-plastin, annexin A1, and haptoglobin increased, whereas transferrin, alpha-1-antitrypsin, and apolipoprotein A-1 decreased. Supplementation of the diet of carcinogen-treated mice with I3C reduced the level of SP-C, L-plastin, annexin A1, and haptoglobin to that of untreated controls. These results were verified using immunoblotting. We show here that tumor-associated signature proteins are increased during NNK plus BaP-induced lung carcinogenesis, and I3C inhibits this effect, suggesting that the lung tumor chemopreventive activity of I3C might be related to modulation of carcinogen-induced alterations in protein levels.

Protective effect of the isoflavonoid equol against hairless mouse skin carcinogenesis induced by UV radiation alone or with a chemical cocarcinogen.

Isoflavones derived from many edible plants, such as genistein from the soybean, have well-documented antioxidant and estrogenic activity but may also be anticarcinogenic. In this study, we examined the potential of the isoflavone equol [(S)-4',7-dihydroxyisoflavane] to protect from skin carcinogenesis in the hairless mouse. Daily topical applications of equol lotions significantly protected against skin carcinogenesis induced by chronic exposure to solar-simulated UV radiation (SSUV) or by topical treatment with the chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) or by the combined cocarcinogenic treatment of DMBA followed by chronic SSUV. Monitoring of tumor development for 40 weeks showed significantly delayed tumor appearance and reduced tumor multiplicity in all equol-treated groups. In mice treated with either SSUV or DMBA + SSUV, equol significantly reduced the proportion of tumors progressing from benign papillomas to malignant squamous cell carcinoma (SCC) by 33-58% and reduced the average diameter of SCC by 71-82%. In a short-term study, equol dose dependently inhibited the SSUV induction of the tumor promotion biomarker enzyme, ornithine decarboxylase, in the skin, suggesting the anticarcinogenic activity of equol may be attributed to its inhibition of the tumor promotion phase of carcinogenesis.

Xanthorrhizol inhibits 12-O-tetradecanoylphorbol-13-acetate-induced acute inflammation and two-stage mouse skin carcinogenesis by blocking the expression of ornithine decarboxylase, cyclooxygenase-2 and inducible nitric oxide synthase through mitogen-activated protein kinases and/or the nuclear factor- B

Xanthorrhizol is an active component isolated from Curcuma xanthorrhiza Roxb. (Zingiberaceae) that is traditionally used in Indonesia for medicinal purposes. In the present study, we found that the topical application of xanthorrhizol before 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment significantly inhibits TPA-induced mouse ear edema and TPA-induced tumor promotion in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated ICR mouse skin. The topical application of xanthorrhizol following the induction of papillomas with TPA-induced hyperplasia and dysplasia also reduced tumor multiplicity and incidence in DMBA-initiated mouse skin. To further elucidate the molecular mechanisms underlying the antitumor-promoting activity of xanthorrhizol, its effect on the TPA-induced expression of ornithine decarboxylase (ODC), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) and the upstream signaling molecules controlling these proteins were explored in mouse skin. The pre-treatment with xanthorrhizol inhibited the expression of ODC, iNOS and COX-2 proteins and nuclear factor-kappaB (NF-kappaB) activation in both mouse skin with TPA-induced acute inflammation and DMBA-initiated mouse skin promoted by TPA for 19 weeks. When mouse skin was treated after TPA-induced production of papillomas, xanthorrhizol remarkably suppressed the expression of ODC, iNOS and COX-2 and inhibited the activation of NF-kappaB. Furthermore, western blot analysis showed that xanthorrhizol suppressed the activation of extracellular signal-regulated protein kinase, p38, c-Jun-N-terminal kinase and Akt in mice after topical application for 6 weeks following the induction of papillomas. Taken together, the present study demonstrates that xanthorrhizol not only delays or inhibits tumor formation, but also reverses the carcinogenic process at pre-malignant stages by reducing the protein levels of ODC, iNOS and COX-2 regulated by the NF-kappaB, mitogen-activated protein kinases and/or Akt.

Cyclooxygenase‐2 expression is critical for chronic UV‐induced murine skin carcinogenesis

While it has been established that both the constitutive and inducible forms of cyclooxygenase (COX-1 and COX-2, respectively) play important roles in chemical initiation-promotion protocols with phorbol ester tumor promoters, the contribution of these two enzymes to ultraviolet (UV) light-induced skin tumors has not been fully assessed. To better understand the contribution of COX-1 and COX-2 to UV carcinogenesis, we transferred the null allele for each isoform onto the SKH-1 hairless strain of mouse. Due to low viability on this background with complete knockout of COX-2, heterozygous mice were used in UV carcinogenesis experiments. While the lack of one allele of COX-1 had no effect on tumor outcome, the lack of one allele of COX-2 resulted in a 50-65% reduction in tumor multiplicity and a marked decrease in tumor size. Additionally, transgenic SKH-1 mice that overexpress COX-2 under the control of a keratin 14 promoter developed 70% more tumors than wild-type SKH-1 mice. The lack of one allele of either COX-1 or COX-2 reduced prostaglandin (PG) E2 levels in response to a single UV treatment. The proliferative response to UV was significantly reduced in COX-2, but not COX-1, heterozygous mice. UV-induced apoptosis, however, was greater in COX-2 heterozygous mice. Collectively, these results clearly establish the requirement for COX-2 in the development of skin tumors.

Mouse skin chemical carcinogenesis is inhibited by antizyme in promotion‐sensitive and promotion‐resistant genetic backgrounds

Elevated polyamine content and increased ornithine decarboxylase (ODC) activity have been associated with neoplastic growth in numerous animal models and human tissues. Antizyme (AZ) is a negative regulator of polyamine metabolism that inhibits ODC activity, stimulates ODC degradation, and suppresses polyamine uptake. Preliminary evidence, obtained from transgenic mice with tissue specific overexpression of AZ indicates that tumor development can be suppressed by AZ. To extend these studies, we have examined the effect of keratin 5 (K5)- or K6-driven AZ transgenes on 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) chemical carcinogenesis of the skin, in promotion-resistant C57BL/6 and promotion-sensitive DBA/2 mice. On both genetic backgrounds, K6-AZ mice showed a reduction in tumor multiplicity, with 85% fewer tumors than wild-type controls on the C57BL/6 background and 50% fewer tumors on the DBA/2 background. K5-AZ mice developed 50% fewer tumors than controls on both backgrounds. The percent of mice with tumors and tumor size were also reduced in the K5-AZ and K6-AZ groups. Tumor and TPA-treated skin sections from K6-AZ mice exhibited the strongest AZ expression, with localization mainly in suprabasal keratinocytes. K6-AZ mice also had slightly reduced cell proliferation rates in tumors and TPA-treated skin. The lack of a more pronounced effect on cell proliferation is probably explained by the observation that AZ staining did not colocalize with proliferating cell nuclear antigen (PCNA), a marker for the proliferative compartment. These studies demonstrate a tumor-suppressive effect of AZ in C57BL/6 and DBA/2 mice, and confirm the importance of ODC and polyamines in tumor development.

Photochemoprevention of UVB-induced skin carcinogenesis in SKH-1 mice by brown algae polyphenols.

Chronic exposure of the skin to ultraviolet B (UVB) radiation induces oxidative stress, which plays a crucial role in the induction of skin cancer. In this study, the effect of dietary feeding and topical application of brown algae polyphenols on UVB radiation-induced skin carcinogenesis in SKH-1 mice was investigated. SKH-1 hairless mice were randomly divided into 9 groups, including control, UVB control and treatment groups. They were treated orally (0.1% and 0.5% with AIN-76 diet, w/w) and topically (3 and 6 mg/0.2 ml of vehicle) with brown algae polyphenols and irradiated with UVB for 26 weeks. Dietary feeding (0.1% and 0.5%) of brown algae polyphenols significantly reduced tumor multiplicity (45% and 56%) and tumor volume (54% and 65%), and topical administration (3 and 6 mg) significantly decreased tumor multiplicity (60% and 46%) and tumor volume (66% and 57%), respectively, per tumor-bearing mouse. Dietary feeding and topical administration of the polyphenols also inhibited tumor incidence by 6% and 21%, respectively, but the results were not significant. Dietary and topical administration of the polyphenols markedly inhibited cyclooxygenase-2 activity and cell proliferation. These observations show that brown algae polyphenols have an antiphotocarcinogenic effect which may be associated with the prevention of UVB-induced oxidative stress, inflammation, and cell proliferation in the skin.

Triggering CD40 on endothelial cells contributes to tumor growth

Inflammatory cells can either promote or inhibit tumor growth. Here we studied whether CD40, a key molecule for adaptive immune response, has any role in mammary carcinogenesis of BALB/NeuT transgenic tumor-prone mice. We transferred the HER2/neu oncogene into CD40-null background to obtain the CD40-KO/NeuT strain. CD40-KO/NeuT mice showed delayed tumor onset and reduced tumor multiplicity. BM (BM) transplantation experiments excluded a role of BM-derived cells in the reduced tumorigenicity associated with CD40 deficiency. Rather, CD40 expressed by endothelial cells (ECs) takes part to the angiogenic process. Accordingly, large vessels, well organized around the tumor lobular structures, characterize BALB/NeuT tumors, whereas tiny numerous vessels with scarce extracellular matrix are dispersed in the parenchyma of poorly organized CD40-KO/NeuT tumors.Activated platelets, which may interact with and activate ECs, are a possible source of CD40L. Their localization within tumor vessels prompted the idea of treating BALB/NeuT and CD40-KO/NeuT mice chronically with the anti-platelet drug clopidogrel, known to inhibit platelet CD40L expression. Treatment of BALB/NeuT mice reduced tumor growth to a level similar to CD40-deficient mice, whereas CD40-KO/NeuT mice treated or not showed the same attenuated tumor outgrowth, indicating that activated platelets are the likely source of CD40L in this model. Collectively, these data point to a participation of CD40/CD40L in the angiogenic processes associated with mammary carcinogenesis of BALB/NeuT mice.

Neu-Induced Retroviral Rat Mammary Carcinogenesis: A Novel Chemoprevention Model for Both Hormonally Responsive and Nonresponsive Mammary Carcinomas

Clinically relevant animal models of mammary carcinogenesis are crucial for the development and evaluation of new breast cancer chemopreventive agents. The neu-induced retroviral rat mammary carcinogenesis model is based on the direct in situ transfer of the activated neu oncogene into the mammary epithelium using a replication-defective retroviral vector. The resulting mammary carcinomas in intact Wistar-Furth rats exhibit a mixed hormonal response in the same proportion as has been observed in women. In intact rats, approximately 50% of mammary carcinomas can be prevented by tamoxifen treatment. In ovariectomized animals, the mammary carcinomas are hormonally nonresponsive and cannot be prevented by tamoxifen. We evaluated the efficacy of retinoic X receptor-selective retinoids (rexinoids) in this novel model of mammary carcinogenesis. The rexinoids LG100268 and bexarotene (LG1069, Targretin) were highly efficacious in the prevention of neu-induced mammary carcinomas. Dietary LG100268 at 100 mg/kg diet decreased tumor multiplicity by 32% (P = 0.0114) in intact rats and 50% (P < 0.0001) in ovariectomized rats. Bexarotene treatment at a dose of 250 mg/kg diet was associated with reductions in tumor multiplicity of 84% (P < 0.0001) and 86% (P < 0.0001) in intact and ovariectomized animals, respectively. In addition to tumor multiplicity, proliferation and apoptosis were modulated by bexarotene treatment independently of estrogen signaling. The neu-induced retroviral rat mammary carcinogenesis model represents a valuable addition to existing rodent chemoprevention models. The model is useful for assessing the efficacy of chemopreventive agents, specifically those compounds that target hormonally nonresponsive tumors.