The present study showed that treatment with 20 mg/kg/day α-mangostin resulted in prolonged survival rates and increased inhibition of tumor growth and lymph node metastasis in an immunocompetent mouse metastatic mammary carcinoma model containing a p53 mutation. Mammary carcinoma tissues of mice treated with 20 mg/kg/day α-mangostin showed elevation of apoptotic cell death, increased expression of active caspase-3 and -9, and a decrease in the number of cells with phospho-Akt-Thr308. Furthermore, decreased blood microvessel density and fewer numbers of lymphatic vessels containing intraluminal cancer cells were observed in mammary carcinomas of α-mangostin-treated mice. Our in vitro studies have demonstrated that α-mangostin induces mitochondria-mediated apoptosis, but not via the Bid-mitochondria cross-talk pathway, G1-phase arrest, or S-phase suppression in the cell cycle.
Akt phosphorylation contributes to cell proliferation, anti-apoptotic cell death, cell cycle entry, angiogenesis and metastasis - all important aspects of the oncogenic process . The phosphoinositide 3-kinase (PI3K)/Akt pathway is now considered to be an important therapeutic target for cancer. Indeed, Akt inhibitors have shown significant promise preclinically and are now in clinical trials . Full activation of Akt is a multistep process, and the final step is phosphorylation of Akt1 at two sites, Thr308 and Ser473 . Upon activation, Akt moves to the cytoplasm and nucleus where it phosphorylates downstream target proteins. We demonstrated that treatment with α-mangostin decreased phospho-Akt-Th308. In this study, although intense staining of phospho-Akt-Th308 in both the cytoplasm and nucleus was immunohistochemically observed in mammary carcinoma tissues of the control group, the intensity and number of cells expressing phospho-Akt-Th308 tended to be lower in the 20 mg/kg/day α-mangostin group. Since both Thr308 and Ser473 are necessary for full activation of Akt [30, 31], the fact that α-mangostin reduced phospho-Akt-Th308 in vitro and in vivo suggests downstream inhibition of pathways in Akt. Several modes of Akt pathway dysregulation have been identified in various types of cancer, including breast cancer, and this ultimately affects a number of processes including cell growth, survival, proliferation, and motility and/or invasion . Therefore, the observation in the present study of reduced tumor growth, apoptotic cell death, cell-cycle alterations, anti-angiogenesis and anti-metastasis may be partially responsible for inhibition of Akt phosphorylation.
As previously stated, we demonstrated a significant induction of apoptosis with α-mangostin 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 the death receptor (an extrinsic pathway, executed by caspase-8), and a pathway mediated by mitochondria (intrinsic pathway with execution by caspase-9) . In addition, however, endoplasmic reticulum (ER) stress has been shown to switch the signaling direction from the pro-survival to the pro-apoptotic pathway . Caspase-12, a caspase localized in the ER, is known to mediate this switch in mice . Caspase-3 is the final executor of apoptosis. Many of the apoptotic signals are transduced to the mitochondria, decreasing the mitochondrial membrane potential and leading 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 mitochondrial pathway through the cleavage of Bid .
In vitro, we noted increased activity of caspases-3, -8 and -9 and increased cytosolic cytochrome c levels in α-mangostin-treated mammary carcinoma cells, suggesting that α-mangostin at least initiated mitochondria-mediated apoptosis. In fact, mammary carcinoma tissues of α-mangostin-treated mice showed strong expression of active caspase-3 and -9, demonstrating that mitochondria-mediated apoptosis actually occurred in vivo as well. All caspase inhibitors, including that for caspase-8, completely rescued α-mangostin-induced cell death in cultures. Bid cleavage, however, was not observed, indicating that cross-talk between caspase-8 and Bid may not be involved here. The question arises as to why caspase-8 activity nevertheless increased. Caspase-8 participates in ERK activation, and this participation is attributed to the Death Effector Domains (DED) of caspase-8  and a direct association between ERK and a DED-containing fragment of caspase-8, with 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 α-mangostin-induced apoptosis, but further investigation is required to elucidate this mechanism. Since no elevation in caspase-12 activity was seen in the present study, α-mangostin-induced apoptosis may not have involved ER stress. Our current experiments suggest that α-mangostin-induced apoptosis in BJMC3879luc2 cells, which contain a p53 mutation, occurs through a p53-independent mechanism.
The tumor suppressor gene p53 encodes a transcription factor that plays a critical role in regulating cell cycle progression, DNA repair, and cell death. p53 is the most frequently altered gene in human cancers and loss of functional p53 protein occurs in a majority of epithelial ovarian cancers. The present experiments suggest that α-mangostin-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 α-mangostin 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. On a related note, it has been shown that stroma-specific loss of heterozygosity or allelic imbalance is associated with p53 mutations and regional lymph-node metastases in sporadic breast cancer .
Neovascularization is a key process in the growth of solid tumors, and tumors will not grow beyond a few cubic millimeters unless a vascular network is established to feed further expansion . In the present study, we demonstrated that treatment with α-mangostin significantly reduced microvessel density in mammary carcinomas. We have also recently shown that panaxanthone, which is comprised of approximately 75% to 85% α-mangostin and 5% to 15% γ-mangostin with the sum of both contents > 90%, also inhibits tumor growth and metastasis in mouse mammary carcinomas and is also associated with decreased tumor angiogenesis. Since the growth of both primary tumors and of metastases is angiogenesis-dependent, and since microvascular endothelial cells recruited by a tumor in the process of neovascularization have become an important second target of cancer therapy , the anti-angiogenic properties of α-mangostin may be very important to the development of cancer therapies, particularly those involved with molecular targeting in neoplasms.
Tumor cell dissemination is mediated by a number of mechanisms, including direct invasion into local tissue, lymphatic spread, and hematogenous spread. In general, the most common pathway of initial dissemination is via the lymphatics, with patterns of spread via afferent ducts . The lymphatic capillaries present in tissues and tumors provide entrance into the lymphatic system, allowing cancer cell migration to the lymph nodes. Breast cancer cells are known to disseminate through the body by all of the above mechanisms; common metastatic sites are the lymph nodes, lung, bones, and liver . Lymph node involvement remains specifically the most influential prognostic factor in breast cancer progression . In the present study, the multiplicity of lymph node metastases was decreased in α-mangostin-treated mice. This phenomenon was supported by a significant decrease in the number of lymphatic vessels demonstrating intraluminal tumor cells in the α-mangostin-treated groups. This indicates that α-mangostin has an inhibitory effect on migration into lymphatic vessels. Other investigators have reported that α-mangostin exerts inhibitory effects on cell invasion and migration in mammary cancer cells due to downregulation of MMP-2 and MMP-9 , and we cannot preclude that this mechanism was not operating in our study, as well.
Significant elevations of NK activity were recently reported in mice treated with crude α-mangostin at 20 and 40 mg/kg/day compared to control mice . In a human pilot study, healthy people orally administered panaxanthone, a less purified α-mangostin analog, at a dose of 150 mg/day/person for seven days also showed significant increases in NK activity . Since the mammary cancer model used in the current study was an immunocompetent model, elevation in NK activity would be expected in the 20 mg/kg/day α-mangostin group.
The PI3K pathway exerts its regulatory functions on cell proliferation, cell transformation, cell apoptosis, tumor growth and angiogenesis through downstream targeting of Akt . Expression of Akt in a dominant/negative mutant also inhibited angiogenesis and tumor growth, and also decreased the expression of HIF-1α and vascular endothelial growth factor (VEGF) in tumor xenographs . Akt has also been reported to phosphorylate and activate endothelial nitric oxide synthase (eNOS), which contributes to angiogenesis through endothelial nitric oxide production. Activation of Akt by VEGF orchestrates several signaling events that contribute to angiogenesis . Involvement of PI3K, Akt and eNOS in endothelial cell biology is apparent under both physiological and pathological conditions. Akt1 null mice show reduction of lymphatic capillary vessel size as well as defects of smooth muscle cell coverage and valve development, suggesting that Akt1 is a required isoform in lymphangiogenesis . Thus, Akt-mediated signaling plays an important role in lymphangiogeneis as well as in angiogenesis. Since α-mangostin reduced Akt phosphorylation in vitro and in vivo in the present study, this signaling may be responsible for reduction of vasculogenesis in our mouse tumors.
Studies have also shown that α-mangostin decreases the levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase in macrophages . COX is a key enzyme that catalyzes the conversion of arachidonic acid to prostaglandin E2 (PGE2). Recent studies have shown that overexpression of COX-2 and PGE2 is a characteristic of many human cancers [48–50] and selective COX-2 inhibitors have shown significant effects in reducing the incidence and progression of tumors and metastasis in animal models of mammary cancer [51–53]. Therefore, the observed antitumor action by α-mangostin in our present study may be due to reduction of COX-2 levels. Akt signaling has been reported to upregulate COX-2 expression through the NF-kB/IkB pathway in mutated PTEN endometrial carcinoma cells ; α-mangostin may be able to reduce COX-2 expression through Akt dephosphorylation.
Estrogen and the estrogen receptor α (ERα) are widely recognized to play a crucial role in the development and progression of hormone-dependent breast cancer. The BJMC3879luc2 mammary carcinoma cells used in the present study have been previously characterized as having cytoplasmic location of ERα and a partial weak response to estrogen treatment . When BJMC3879luc2 cells are implanted into mice, ERα mRNA levels rise significantly higher in females than males. Raloxifene, a selective estrogen receptor modulator, inhibits tumor growth and metastasis in the same mouse metastatic mammary carcinoma model as we used in the present study . Aromatase is an estrogen synthase responsible for catalyzing the biosynthesis of estrogens from androgens. Since both α- and γ-mangostin inhibit aromatase activity in a dose-dependent manner, this is another possible mechanism by which α-mangostin exhibited the antitumor effect seen in the present study.
The population is aging in many modern societies, and since the morbidity rates of cancer and cerebrovascular disease are increasing steadily, preventive medicine, in addition to therapeutic treatments, is becoming increasingly important. Many medically advanced societies are exploring both Western medicine and Oriental alternatives, and the demand for complementary and alternative medicines is on the rise. In fact, the 2007 National Health Interview Survey by the National Center for Complementary and Alternative Medicine (NCCAM) and the National Center for Health Statistics showed that 38% of adults in the United States are using some form of complementary and alternative medicine . However, scientific data based on the principle of evidence-based analysis is still scant in the fields of complementary and alternative medicine. α-Mangostin, isolated from the pericarp of the mangosteen fruit, has been shown to induce many biological actions, such as anti-bacterial activity , apoptosis [7–10], and cell-cycle arrest . An analog, panaxanthone (approximately 75% to 85% α-mangostin and 5% to 15% γ-mangostin with the sum of both contents > 90%) has been shown to significantly suppress tumor growth and metastasis in a mouse model of mammary cancer when administered in the diet . These and other basic investigations provide a scientific basis for the anecdotal effects of α-mangostin.