Diets rich in carotenoids have been strongly implicated as protecting against cancer development at several anatomic sites, however it is unclear whether protection is due to the carotenoid itself or to products of its bioconversion to retinoids. Both Beta-carotene (β-C), a carotenoid with, and canthaxanthin (CTX) a carotenoid without pro-vitamin A activity, have been found to inhibit methylcholanthrene induced neoplastic transformation in l0Tl/2 cells. Activity was observed when these compounds were added 7 days after carcinogen exposure and was reversible upon removal of carotenoid. When tested against X-ray induced transformation, carotenoids were ineffective when present during irradiation but, as before, strongly protected when added after carcinogen exposure. Effective doses were not cytotoxic. In both chemical and physical carcinogensis protocols, CTX was significantly more potent than β-C in inhibiting transformation. Using 14C-β-C no conversion to expected retinoids was detected after incubation with l0Tl/2 cells. Thus carotenoids appear to possess intrinsic cancer chemopreventive activity in 10T1/2 cells.
The development and initial characterization of five new inbred strains of SENCAR mice are described in this paper. Ten randomly selected pairs of outbred SENCAR mice were mated and offspring from each separately maintained parental line were sib mated at each successive generation to result in inbred strains. Due to poor reproductive performance only five of the original 10 lines were bred to homogeneity. Initial characterization of the five remaining lines (referred to as SL2/sprd, SL5/sprd, SL7/sprd, SL8/sprd and SLl0/sprd) at F12 for their responsiveness to a two-stage carcinogenesis protocol (10 nmol 7,12-dimethylbenz[a]anthracene and 0.25 μg 12-O-tetradecanoylphorbol-13 acetate) revealed three groups of responders in terms of the number of papillomas per mouse: SL2/sprd and SL8/sprd > SL7/sprd and SL10/sprd >> SL5/sprd. The papilloma responses in SL2/sprd and SL8/sprd were very similar to SENCAR B/Pt compared at the same doses. Papillomas induced on SL2/sprd had the highest propensity to progress to squamous cell carcinomas, similar to that observed in outbred SENCAR and SENCAR B/Pt mice. More detailed comparison of the responsiveness of SL2/sprd and SL5/sprd at Fl5 showed that these two inbred strains differed in their sensitivity to TPA-induced epidermal hyperplasia and that the dose of TPA required to produce a tumor response in SL5/sprd in comparison with that in SL2/sprd was 4–20 times higher. Overall, the availability of the different inbred SENCAR strains will greatly aid mechanistic studies of multistage skin carcinogenesis as well as studies to understand the underlying genetic basis of resistance to tumor promotion and progression in this model system.
5664 Cyclooxygenase (COX) enzymes catalyze the rate-limiting step in the conversion of arachidonic acid to prostaglandins. While COX-1 is constitutively expressed in most tissues and is involved in tissue homeostasis, COX-2 is highly inducible, is frequently overexpressed in cancers, and plays roles in inflammation, immune function, angiogenesis, and tumor invasiveness. Both general COX inhibitors, as well as COX-2-specific inhibitors, have been shown to inhibit carcinogenesis in a number of animal models. However, COX inhibitors have been shown to have COX-independent effects. So we have used both COX-2 transgenic and COX-2 knockout mice to determine the role of COX-2 in carcinogenesis using the mouse skin model. FVB mice overexpressing COX-2 in the skin via the keratin 14 (K14) promoter were initially shown to be resistant to skin tumorigenesis induced by 7,12-dimethylbenz[a]anthracene (DMBA) initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion. However, using DMBA only or using the nonphorbol ester tumor promoter, anthralin, with DMBA, K14.COX2 transgenic mice developed many more tumors (3.7 and 5.5 fold, respectively) than wild type FVB mice. Thus, overexpression of COX-2 appears to promote skin tumorigenesis and resistance to tumorigenesis is limited specifically to TPA tumor promotion, which correlated to a reduced TPA induction of proliferation, ornithine decarboxylase, interleukin-1α, and tumor necrosis factor-α expression in transgenic versus wild type mice. Similarly, when SKH-1 hairless mice overexpressing COX-2 in the skin via the keratin 5 (K5) promoter were subjected to ultraviolet (UV) light-induced tumorigenesis, transgenic mice developed 2.5 fold more tumors/mouse than wild type SKH-1 mice. To complement these studies with transgenic overexpressing mice, COX-1 and COX-2 knockout mice were backcrossed onto the SKH-1 background for UV carcinogenesis studies (others have already shown that COX-1 and COX-2 knockout mice are resistant to DMBA/TPA skin tumorigenesis; Cancer Res., 62:3395, 2002). However, since homozygous COX-2 -/- mice do not survive beyond ~6 weeks of age on the SKH-1 background, heterozygous COX-1 +/- and COX-2 +/- mice were used. While COX-1 +/- mice developed UV-induced tumors similarly to wild type SKH-1 mice, COX-2 +/- mice developed 3 fold fewer tumors/mouse with a 10-week increase in latency compared to wild type mice. Thus, loss of only one allele of COX-2 was enough to decrease tumorigenesis in this system. Overall, mouse skin COX-2 expression levels were directly correlated to susceptibility to tumorigenesis. Supported by NIH grants CA100140, ES07784.
The ultraviolet B (UVB) component of sunlight, which causes DNA damage and inflammation, is the major cause of nonmelanoma skin cancer (NMSC), the most prevalent of all cancers. Nonsteroidal anti-inflammatory drugs (NSAID) and coxibs have been shown to be effective chemoprevention agents in multiple preclinical trials, including NMSC, colon, and urinary bladder cancer. NSAIDs, however, cause gastrointestinal irritation, which led to the recent development of nitric oxide (NO) derivatives that may partially ameliorate this toxicity. This study compared the efficacy of several NSAIDs and NO-NSAIDs on UV-induced NMSC in SKH-1 hairless mice and determined whether various short-term biomarkers were predictive of long-term tumor outcome with these agents. Naproxen at 100 (P = 0.05) and 400 ppm (P < 0.01) in the diet reduced tumor multiplicity by 26% and 63%, respectively. The NO-naproxen at slightly lower molar doses shows similar activities. Aspirin at 60 or 750 ppm in the diet reduced tumor multiplicity by 19% and 50%, whereas the equivalent doses (108 and 1,350 ppm) were slightly less effective. Sulindac at 25 and 150 ppm in the diet, doses far below the human equivalent dose was the most potent NSAID with reductions of 50% and 94%, respectively. In testing short-term biomarkers, we found that agents that reduce UV-induced prostaglandin E2 synthesis and/or inhibit UV-induced keratinocyte proliferation yielded long-term tumor efficacy.
To determine whether the EP4 receptor for prostaglandin E 2 (PGE 2 ) contributes to the tumor promoting activity of PGs in murine skin, EP4 over‐expressing mice (BK5.EP4) were generated and subjected carcinogenesis protocols. An initiation/promotion protocol resulted in 25‐fold more squamous cell carcinomas (SCCs) in the BK5.EP4 mice than wild type (WT) mice. An increase in SCCs also occurred following treatment with initiator alone or UV irradiation. The initiator dimethylbenz[a]anthracene caused cytotoxicity in BK5.EP4, but not WT mice, characterized by sloughing of the interfollicular epidermis, regeneration and subsequent SCC development. A comparison of transcriptomes between BK5.EP4 and WT mice treated with PGE2 showed a significant upregulation of a number of genes known to be associated with tumor development, supporting a pro‐tumorigenic role for the EP4 receptor.
One of the most common features of exposure of skin to ultraviolet (UV) light is the induction of inflammation, a contributor to tumorigenesis, which is characterized by the synthesis of cytokines, growth factors and arachidonic acid metabolites, including the prostaglandins (PGs). Studies on the role of the PGs in non-melanoma skin cancer (NMSC) have shown that the cyclooxygenase-2 (COX-2) isoform of the cyclooxygenases is responsible for the majority of the pathological effects of PGE(2). In mouse skin models, COX-2 deficiency significantly protects against chemical carcinogen- or UV-induced NMSC while overexpression confers endogenous tumor promoting activity. Current studies are focused on identifying which of the G protein-coupled EP receptors mediate the tumor promotion/progression activities of PGE(2) and the signaling pathways involved. As reviewed here, the EP1, EP2, and EP4 receptors, but not the EP3 receptor, contribute to NMSC development, albeit through different signaling pathways and with somewhat different outcomes. The signaling pathways activated by the specific EP receptors are context specific and likely depend on the level of PGE(2) synthesis, the differential levels of expression of the different EP receptors, as well as the levels of expression of other interacting receptors. Understanding the role and mechanisms of action of the EP receptors potentially offers new targets for the prevention or therapy of NMSCs.
Matrix metalloproteinases (MMPs) are a family of enzymes that proteolytically degrade various components of the extracellular matrix (ECM). Angiogenesis is the process of forming new blood vessels from existing ones and requires degradation of the vascular basement membrane and remodeling of the ECM in order to allow endothelial cells to migrate and invade into the surrounding tissue. MMPs participate in this remodeling of basement membranes and ECM. However, it has become clear that MMPs contribute more to angiogenesis than just degrading ECM components. Specific MMPs have been shown to enhance angiogenesis by helping to detach pericytes from vessels undergoing angiogenesis, by releasing ECM-bound angiogenic growth factors, by exposing cryptic proangiogenic integrin binding sites in the ECM, by generating promigratory ECM component fragments, and by cleaving endothelial cell-cell adhesions. MMPs can also contribute negatively to angiogenesis through the generation of endogenous angiogenesis inhibitors by proteolytic cleavage of certain collagen chains and plasminogen and by modulating cell receptor signaling by cleaving off their ligand-binding domains. A number of inhibitors of MMPs that show antiangiogenic activity are already in early stages of clinical trials, primarily to treat cancer and cancer-associated angiogenesis. However, because of the multiple effects of MMPs on angiogenesis, careful testing of these MMP inhibitors is necessary to show that these compounds do not actually enhance angiogenesis.
