The present study showed that raloxifene inhibited tumor growth and multiplicity of metastasis to lymph nodes in a mouse immunocompetent metastatic mammary carcinoma model expressing cytoplasmic ERα. In addition, tumor tissues from the raloxifene-treated mice showed elevation of apoptotic cell death, suppression of DNA synthesis and inhibition of lymphatic vessels containing intraluminal cancer cells.
The present in vitro studies showed that the ERα expressed in the mammary carcinoma BJMC3879luc2 cells used in this study was between 50 and 64 kDa, which is smaller than the 66-kDa size of normal ERα, and it showed a cytoplasmic location. Cell proliferation of BJMC3879 cells expressing the smaller molecular weight ERα was significantly increased, but only by 7%, when added to 10 nM estrogen. When BJMC3879luc2 cells were implanted into mice, the ERα mRNA levels in the resultant tumors were significantly higher in female mice as compared to the male mice. Thus, although the ERα in the present study might be functional but weak, further investigation is necessary to elucidate this point. Recently, a truncated variant of 36-kDa ERα has been identified . This subtype, which is predominantly localized to the cytoplasm and plasma membrane, responds to estrogen and mediates a nongenomic signaling pathway. Although this 36-kDa variant of ERα is apparently different from the present ERα, they share similarities in regard to localization and estrogen response.
The results of STAR , MORE , CORE  and RUTH  clinical trials show that raloxifene does not reduce the risk of ER-negative invasive breast cancer. Therefore, the fact that raloxifene exerted antimetastatic effects on mammary cancer expressing the cytoplasmic form of ERα may be an important finding with clinical applications. The question was raised as to why raloxifene exerted antitumor effects on mammary tumors that expressed the cytoplasmically located ERα in the present study. ER lacks known functional motifs that would allow for nongenomic mechanisms of estrogen action . Raloxifene acts on both nuclear ERα and cytoplasmic ERα (nongenomic action) . In this case, raloxifene does not target the estrogen response element; rather, it targets the raloxifene response element . It was previously reported that estrogen activates cell proliferation in even ER-negative human breast cancer cells MDA-MB231 via GPR30, a member of the G protein-coupled receptor superfamily . Thus, raloxifene can act by nongenomic mechanisms independent of ER, indicating the complexity and variety of SERMs. The biological effects of raloxifene decrease the ER levels [8, 31]. In fact, in the present in vivo study, the mRNA levels of the truncated ERα in mammary tumors of raloxifene-treated mice showed a tendency to be decreased as compared to the levels in control mice. It is possible that raloxifene acts on the present mammary cancer model. In addition, ERα and β have been previously localized to mitochondria in various tissues [32, 33]. In the present study, immunohistochemical localization of the truncated ERα revealed scattered expression in the cytoplasm, suggesting mitochondrial localization.
The present study demonstrated that raloxifene significantly induced apoptosis in murine mammary carcinoma cells both in vitro and in vivo. There are two pathways currently proposed to play major roles in regulating apoptosis in mammalian cells: a pathway mediated by death receptor (extrinsic pathway; execution by caspase-8) and a pathway mediated by mitochondria (intrinsic pathway; execution by caspase-9) . Caspase-3 is a final executor of apoptosis. Many of the apoptosis signals are transduced to the mitochondria and decrease the mitochondrial membrane potential, which leads to the release of cytochrome c from the mitochondrial lumen into the cytoplasm. The released cytochrome c binds to the apoptosis protease-activating factor-1 (Apaf-1), and this complex activates caspase-9. Caspase-8 also has a cross-talk pathway to the mitochondria pathway through the cleavage of Bid .
In the present in vitro study, increases in caspase activities (caspase-3, -8 and -9) and cytosolic cytochrome c levels were found in mammary carcinoma cells treated with raloxifene, suggesting that raloxifene at least induced mitochondria-mediated apoptosis. Indeed, mammary cancer tissues of mice treated with raloxifene showed strong expression of active caspase-3 and -9 (cleaved forms), demonstrating that mitochondria-mediated apoptosis also occurred in vivo. All caspase inhibitors involving a caspase-8 inhibitor completely rescued raloxifene-induced cell death. However, since Bid cleavage was not observed, cross-talk between caspase-8 and Bid may not be involved. The question was raised as to why caspase-8 activity increased. Caspase-8 participates in ERK activation, and this regulation is attributed to the Death Effector Domains (DED) of caspase-8 . Furthermore, a direct association between ERK and a DED-containing fragment of caspase-8, and co-transport of an ERK-caspase-8-DED complex to the nucleus during apoptosis has been reported . The caspase-8-ERK pathway may also play a role in raloxifene-induced apoptosis. Further investigation is required to elucidate this point. In addition, caspase-12 mediates the pathway for cell death induced by endoplasmic reticulum stress in mice . In the present study, since no elevation in caspase-12 activity was seen, the raloxifene-induced apoptosis may not have involved endoplasmic reticulum stress.
In animal carcinogenesis models, raloxifene at 20 mg/kg/day inhibits the tumor growth of 7, 12-dimethylbenzanthracene-induced mammary carcinomas in rats . In mice, orally administered raloxifene (1.5 mg/mouse) reduces the tumor growth of mammary and endometrial cancer . On the assumption that mouse body weights are 30 g, the dosage of raloxifene is estimated to be 50 mg/kg/day in mice. In carcinogenicity studies in mice and rats, raloxifene (8.7~225 mg/kg/day in mice; 10.4~259 mg/kg/day in rats) is not carcinogenic (company data from Eli Lilly Pharmaceuticals, Indianapolis, IN, USA). Although the clinical dosages of raloxifene in trials are 60 mg or 120 mg/day, a much higher dose of 600 mg/day (estimated as 10 mg/kg/day on the assumption that body weight is 60 kg) has also been used in clinical studies without adverse side effects [38, 39]. Therefore, the doses of raloxifene used in the present mouse study (18 and 27 mg/kg/day) are not extremely high, and the dosage levels are considered to be near the clinical dose. However, low doses of raloxifene also exert antitumorigenic effects in animal cancer models .
Cancer cells metastasize to distal sites via the lymphatic system and the vascular system. The lymphatic capillaries present in tissues and tumors provide entrance into the lymphatics, allowing cancer cell migration to the lymph nodes. In the present study, it was demonstrated that the multiplicity of lymph node metastases was decreased in raloxifene-treated mice. This phenomenon was supported by a significant decrease in the number of lymphatic vessels with tumor cells in their lumina in the raloxifene-treated groups. This finding indicates that raloxifene may have an inhibitory effect on migration into lymphatic vessels. In fact, raloxifene has been reported to inhibit estrogen-induced cell migration and invasion through a non-nuclear signaling cascade involving G proteins and the RhoA-associated kinase . It was also reported that raloxifene decreases levels of cyclooxygenase-2 and inducible nitric oxide synthase in carrageenan-induced inflammation of rats . This mechanism could possibly be involved in the antitumorigenic effects of raloxifene.
Neovascularization is also a key process in the growth of solid tumors, and the growth of both primary tumors and metastases is thus angiogenesis-dependent . However, in the present study, microvessel density in tumors was similar between the control and raloxifene-treated groups, indicating that raloxifene may not have anti-angiogenic action. However, the microvessel density in the 27 mg/kg raloxifene group was slightly increased. Since raloxifene induces cell proliferation and up-regulation of telomerase activity in human umbilical vein endothelial cells , this effect might be involved in the present study. However, since raloxifene did not inhibit angiogenesis in tumors in the present study, lung metastasis may not have been strongly inhibited.
The present experiments suggest that raloxifene-induced apoptosis in BJMC3879Luc2 cells having a p53 mutation occurs through a p53-independent mechanism. Since 50% of human cancers have p53 mutations , the fact that the raloxifene induces a p53-independent apoptotic response in cancer cells having a p53 mutation may be highly relevant to inhibiting many human cancers. In the case of non-functional p53 status, p73, the p53 homologue, may play a role in apoptosis induction.