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Breast cancer represents one of the most frequently diagnosed cancers and predominant causes of death in women worldwide. The value of preventive therapy to limit the devastating impact of breast cancer is well established. Various plant triterpenoids and their synthetic analogs have shown significant promise as potent chemopreventive agents in breast cancer. The current study was initiated to investigate mechanism-based chemopreventive potential of a novel synthetic oleanane triterpenoid (methyl-25-hydroxy-3-oxoolean-12-en-28-oate, AMR-Me) against 7,12-dimethylbenz(a)anthracene (DMBA)-initiated rat mammary carcinogenesis, an experimental rodent tumor model that closely resembles human mammary cancer. Rats were orally administered with AMR-Me (0.8, 1.2 and 1.6 mg/kg) three times per week for 18 weeks. Following two weeks of AMR-Me treatment, mammary carcinogenesis was initiated by oral administration of DMBA (50 mg/kg body weight). At the end of the study (16 weeks following DMBA exposure), AMR-Me exhibited a striking inhibition of DMBA-induced mammary tumor incidence, total tumor burden, average tumor weight and reversed histopathological alterations without toxicity. AMR-Me dose-dependently suppressed abnormal cell proliferation, induced apoptosis, up-regulated pro-apoptotic protein Bax and down-regulated antiapoptotic protein Bcl-2 in mammary tumors. AMR-Me upregulated the transcriptional levels of Bax, Bad, caspase-3, caspase-7 and poly(ADP-ribose) polymerase and down-regulated Bcl-2. These results clearly demonstrate for the first time that novel triterpenoid AMR-Me exerts chemopreventive efficacy in the classical DMBA model of breast cancer by suppressing abnormal cell proliferation and inducing apoptosis mediated through mitochondrial pro-apoptotic mechanisms. AMR-Me could be developed as a chemopreventive drug to reduce the risk of human breast cancer that remains a devastating disease.
Breast cancer represents the most frequently diagnosed cancer and leading cause of cancer deaths in females worldwide. Approximately, 1.4 million new cases of breast cancer and 458,400 deaths in women due to this disease are estimated to have occurred in 2008. Highest rates of breast cancer occur in North America, western Europe and Australia, nevertheless, the incidence is increasing in all parts of the globe, especially in the developing countries. The preponderance of breast cancer is sporadic and the risk factors are predominantly linked to estrogen exposure and age. Steroid hormones, particularly estrogen, contribute to the development and progression of mammary gland neoplasia. Exposure to environmental xenobiotics during critical phases of early development has been found to play a vital role in breast cancer susceptibility during the latter periods of life. Genetic factors, including mutations in BRCA 1 and BRCA 2, contribute to ∼10% of breast cancer incidence in the western world.
Although there has been a substantial improvement in the outcome of breast cancer during the past several decades, primarily due to early diagnosis and increased use of hormonal and adjuvant chemotherapies, preventive approaches using pharmacological and other novel agents could be considered as the winning strategy in reducing the morbidity and mortality of breast cancer.[5, 6] A recently published consensus statement significantly underscores the value of preventive therapy to limit the devastating impact of breast cancer. Two selective estrogen receptor modulators, namely tamoxifen and raloxifene, are currently the only drugs approved by the United States Food and Drug Administration for the prevention of breast cancer in women with high risk for the development of the disease. Nevertheless, concern about serious side effects precludes the widespread and long-term use of these medical options.[7, 8] Hence, development of new breast cancer chemopreventive drugs with acceptable efficacy and toxicity suitable for use for protracted period of time is urgently needed.
An impressive number of natural products, including phytoconstituents and dietary agents, have been shown to suppress the growth of breast tumor cells through modulation of cell proliferation, cellular differentiation, apoptosis, angiogenesis as well as several signal transduction pathways.[10, 11] The potential utility of several dietary supplements and natural products in breast cancer prevention has been tested by clinical intervention trials. Terpenoids (also known as terpenes or isoprenoids) constitute the largest group of natural compounds found in a variety of fruits, vegetables and medicinal plants. Interestingly, several terpenoids are structurally similar to human hormones. Natural terpenoids (e.g., monoterpenes, diterpenes, triterpenes and tetraterpenes) as well as their synthetic analogs have shown significant promise in the chemoprevention of breast cancer as demonstrated in preclinical studies.[13, 14]
Amoorain (AMR), a triterpene acid [25-hydroxy-3-oxoolean-12-en-28-oic acid, Fig. 1a(a′)] isolated from the stem bark of the Indian medicinal plant Amoora rohituka, exhibited cytostatic as well as cytotoxic effects in human neoplastic hematopoietic and epithelial cells of diverse origin. AMR has also been shown to possess growth inhibitory and apoptosis-inducing effects in several mammary carcinoma cells of human origin, namely MCF-7, MCF-7/TH and MDA-MB-468.[16-18] In animal studies, AMR significantly inhibited the growth of xenografted SW 620 colon carcinoma in nude mice and reduced tumor size in N-methyl-N-nitrosourea-induced mammary adenocarcinoma in rats. Since the antitumor activity of plant-derived AMR is relatively weak, new analogs of this triterpenoid have been developed by chemical transformation in an attempt to yield more potent agents. Interestingly, one of the analogs, namely methyl-25-hydroxy-3-oxoolean-12-en-28-oate [amooranin-methyl ester or AMR-Me, Fig. 1a(b′)], has been found to exhibit significant antiproliferative effect against MCF-7 breast cancer cells with superior potency to the parent compound. Ancillary studies have confirmed the potent cytotoxic effect of AMR-Me against MCF-7 cells through induction of apoptosis mediated by two different mitogen-activated protein kinase (MAPK) signaling pathways, namely p38 MAPK and c-jun N-terminal kinase (JNK). AMR-Me has also been shown to inhibit the growth of human acute lymphoblastic leukemia cells by proapoptotic mechanisms and improve the survival of mice bearing Dalton's lymphoma ascites tumor cells.
Figure 1. Amoorain compounds, experimental design and animal growth during the entire term of the study. (a) Structures of AMR (a′) and AMR-Me (b′); (b) Schematic representation of the experimental protocol of mammary carcinogenesis initiated with DMBA. (c) Effect of AMR-Me on body weight gain during DMBA-induced mammary tumorigenesis in rats. Each data point indicates mean ± SEM. There were 6–11 animals in various groups. No significant difference in body weights was observed among various rat groups at any time point.
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Despite interesting preliminary results on in vitro antitumor effects, mechanism-based chemopreventive potential of AMR-Me has not been tested against an experimentally validated preclinical animal model of human neoplastic disease primarily due to challenges involved in obtaining sufficient amount of this material required for chronic in vivo study. This obstacle has been removed by development of method for the synthesis of pure AMR-Me. The discovery of the method for large-scale synthesis of AMR-Me represents a key breakthrough as it eliminates the need of using plant material and hence the dependency on the nature. It also creates the opportunity for animal studies to explore the chemopreventive efficacy of AMR-Me. We report herein, for the first time, the chemopreventive effect of AMR-Me and related mechanisms using 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinogenesis, a classical animal model that closely resembles human breast cancer.[24, 25]
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The DMBA rat mammary tumor is one of the most popular preclinical animal models of breast cancer and has been extensively used in chemopreventive drug development. DMBA is a polycyclic aromatic hydrocarbon (PAH) known to cause mammary tumors in rats. PAHs are common organic environmental pollutants derived from incomplete combustion of fossil fuels and also present in tobacco smoke and various foods. These chemicals represent a predominant class of carcinogens responsible for the development of breast tumor in humans. Interestingly, the DMBA-induced rat mammary tumor model was utilized in the development of breast cancer chemopreventive drug tamoxifen and has been extensively used by various laboratories worldwide to develop potential breast cancer preventive agents.[26, 31, 32] In this model, mammary tumors can be reproducibly induced with high frequency. This estrogen-dependent breast cancer model is relevant to human cancer especially with respect to origin; both cancers arise from ductal epithelial cells. Moreover, histogenesis, morphology and progression of hyperplastic premalignant and malignant lesions are similar to those of human breast cancer. In addition, rat mammary tumors induced by DMBA express many biochemical and molecular markers that are also expressed in human mammary tumors.
Our present study provides substantial evidence for the first time that a novel triterpenoid AMR-Me exerts a remarkable chemopreventive activity against DMBA-induced mammary tumorigenesis in female Sprague–Dawley rats. Interestingly, breast tumor inhibitory effect of AMR-Me has been found to be achieved in a dose-responsive fashion during the 18-week study. While the low dose (0.8 mg/kg) reduces mammary carcinogenesis in statistically insignificant manner, the chemopreventive effect of medium (1.2 mg/kg) or high dose (1.6 mg/kg) has been found to statistically significant and comparable. An inhibitory effect of AMR-Me has been clearly visualized by the reduced incidence of mammary tumors induced by DMBA. The observation of a significant lower percentage of rats with tumors following AMR-Me treatment could be explained in the virtue of the fact that the carcinogenic effect even though initiated was suppressed to a substantial extent. The potential chemopreventive response of AMR-Me has also been reflected in the reduced total tumor burden as well as average tumor weight in DMBA-exposed animals. The observed tumor growth inhibitory effect of AMR-Me may represent a selective toxic manifestation to proliferating cells in the light of the fact that these cells are rapidly proliferating when compared to a relatively nonproliferative environment and thereby eventually suppress the development of breast cancer. Our histopathological results reveal hyperplasia in rats exposed to DMBA and its reversal by AMR-Me treatment. Since a close connection between hyperplasia and subsequent steps leading to malignancy has long been established, the protective effect of AMR-Me against mammary carcinogenesis may be construed as a substantial suppression of the occurrence of hyperplasia thereby reducing the development of mammary tumors. Our in vivo results reported here are in concurrence with previous in vitro data showing selective and dose-dependent inhibitory effects of AMR-Me against human breast cancer cells.
A prominent finding of this study is the uniform and consistent growth of all experimental animal groups as evident from similar body weight gains during the entire course of the investigation. This is an important aspect of AMR-Me function that suggests unaltered nutritional status of the treated animals. It is well known that dietary restriction as well as nutritional deprivation resulting in body weight loss is implicated in the inhibition of tumor growth.[35, 36] The ability of dietary and caloric restriction in reducing tumor incidence and arresting tumor growth has been established in various animal models of breast cancer.[37, 38] Since the animals in this study grew equally irrespective of specific treatment, it may be concluded that the observed mammary tumor inhibitory effect of AMR-Me is not linked to an impairment of nutritional status of DMBA-initiated animals. Our results are in accord with a previous study that reported complete lack of toxicity of orally administered AMR-Me at 3 mg/kg/day for 28 days in female BABL/c mice.
To investigate the mechanism by which AMR-Me inhibited mammary tumorigenesis, we examined the extent of cell proliferation in tumors isolated from DMBA-exposed rats with or without AMR-Me treatment. Cell proliferation has been used clinically for assessment of tumor prognosis due to its effect on malignancy and for analysis of response of cancer cells to clinical interventions. Since, cell proliferation plays a vital role in the progression of rat mammary gland tumorigenesis, exploration of agents that can affect tumor cell proliferation has immense value in chemoprevention of breast cancer. PCNA, a 36 kDa nuclear protein with functions as a cofactor of DNA polymerase δ, serves as an important proliferative marker in mammary carcinogenesis.[40, 41] We have detected the expression of PCNA by immunohistochemical technique. A substantially elevated expression of PCNA in mammary tumors of DMBA control animals is reminiscence of massive proliferation of tumor cells. The reduced expression of PCNA in conjunction with lower PCNA LI in tumor tissues of rats treated with AMR-Me strongly suggests antiproliferative mechanisms involved in the observed chemopreventive efficacy of this novel triterpenoid.
The programmed cell death, also known as apoptosis, is a fundamental form of physiological cell destruction driven by a distinct cellular mechanism characterized by cellular morphological alterations, chromatin condensation, DNA cleavage and generation of apoptotic bodies. The process of carcinogenesis selects against apoptosis to initiate, promote and perpetuate the malignant phenotype and drug-induced apoptosis in tumors has been widely accepted as novel therapeutic and preventive strategy against cancer. In the present study, intratumor apoptosis was evaluated by DNA fragmentation assay using immunohistochemical technique to gain insight of the mechanism of suppression of tumor cell proliferation by AMR-Me. Our results demonstrate a gradual increase in DNA fragmentation with increasing doses of AMR-Me indicating an increment in cell death. Incidentally, this is the first study demonstrating apoptosis-inducing activity of AMR-Me in an experimental in vivo breast tumor model and complimentary to previously reported proapoptotic property of this synthetic oleanane triterpenoid against breast carcinoma cells.
The cellular machinery associated with apoptosis is highly conserved and mutations in genes that regulate apoptosis pathways, including the Bcl-2 family members, are common in human cancers, and underscore the importance of apoptosis resistance in carcinogenesis. Several members of the Bcl-2 family, which are overexpressed in a number of human malignancies (e.g., Bcl-2 and Bcl-xL) are known to block cell death, whereas other members (including Bax and Bad) are promoters of apoptosis with diminished levels found in several types of cancers. In this context, it is noteworthy that apoptosis-inducing ability with an increase in the expression ratio between proapoptotic and antiapoptotic and genes of Bcl-2 family has become a primary factor in considering the efficacy of a chemopreventive agent. On the basis of our immunohistochemical analysis, continuous treatment with AMR-Me increased Bax expression and decreased Bcl-2 expression in mammary tumors with a resultant elevation of Bax/Bcl-2 ratio, providing ample evidence of the involvement of the members of Bcl-2 family in the induction of apoptosis during rat mammary carcinogenesis. Our Western blot and RT-PCR data lend strong support to immunohistochemical observations and confirm Bcl-2 family proteins as the target of AMR-Me during mammary gland carcinogenesis.
It is well known that both pro- and antiapoptotic members of the Bcl-2 family regulate mitochondrial membrane permealization and thereby control the release of apoptotic factors from the mitochondrial intermembrane space. The release of cyt. c causes downstream initiation of caspase cascade by activating downstream effectors, such as caspase-3 and caspase-7, resulting in cleavage cascade of a variety of cellular proteins to facilitate apoptotic events. The long-term treatment of DMBA-exposed rats with AMR-Me has been successful in up-regulating the transcriptional levels of caspase-3, caspase-7 and PARP in mammary tumors, which may account for elevated apoptotic activities, and eventual inhibition of tumor growth. It is noteworthy to mention that the growth inhibitory and apoptosis-inducing activities of AMR-Me against breast cancer cells has been paralleled with increase in Bax and decrease in Bcl-2, cyt. c release, and subsequent induction of pro-caspase-9 and -7 and PARP cleavage.
The carcinogenic effect of DMBA, an indirect acting carcinogen, is mediated through its hepatic and extra-hepatic (mammary glands) metabolism catalyzed by Phase I enzymes, namely cytochrome P-450 (CYP) and epoxide hydrolase, followed by subsequent epoxidation by CYP isozyme CYP1A1 and CYP1B1 to yield the ultimate carcinogen DMBA-3,4-dihydro-diol-1,2-epoxide capable of forming DNA adducts. The detoxification of DMBA epoxide and other phenolic metabolites is mediated by Phase II enzymes, including glutathione S-transferase. Hence, the observed mammary tumor inhibitory action of AMR-Me treatment started 2 weeks prior to DMBA initiation could be effected by alteration of DMBA metabolism through modulation of Phases I and II enzymes. Nevertheless, future studies are necessary to confirm this and other possible chemopreventive mechanisms of action of AMR-Me.
Several triterpenoid analogs of oleanolic acid, including 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and methyl ester of CDDO (CDDO-Me), have been synthesized, which are shown to suppress the growth of several human breast carcinoma cells in vitro[45-48] and inhibited or suppressed tumor formation in various breast cancer models in vivo[46-50] Two recently completed Phase I clinical trials evaluated antitumor activities of both CDDO (NCT00322140) and CDDO-Me (NCT00508807) in adult patients with solid tumors and lymphomas (http://clinicaltrials.gov/). Like CDDO-Me, the structure of AMR-Me resemble steroids and other isoprenoid molecules (Fig. 1a(b′)). In view of this background, results derived from the present study showing inhibition of rat mammary tumors through pharmacological modulation of several cellular and molecular regulatory pathways by chemical modification of a naturally occurring triterpenoid scaffold may offer great potential for the prevention of human breast cancer.
In conclusion, the results of our present investigation clearly demonstrate for the first time that the novel triterpenoid AMR-Me exhibits a striking chemopreventive efficacy in DMBA classical animal model of breast cancer in a dose-responsive fashion. The chemopreventive effect of AMR-Me has been reflected in the ability of this compound to diminish the development of DMBA-induced mammary tumors and significantly reduce tumor burden. Furthermore, our study provides substantial evidence that breast tumor-inhibitory effect of AMR-Me could be achieved, at least in part, though interference with key hallmark capabilities of tumor cells, such as abnormal cell proliferation and evasion of apoptosis. Finally, AMR-Me-mediated proapoptotic signal during experimentally induced mammary carcinogenesis could be propagated through an up-regulation of proapoptotic proteins and down-regulation of antiapoptotic proteins of the mitochondrial apoptotic pathway. These interesting results coupled with a safety profile underscore the development of AMR-Me as chemopreventive drug for breast cancer, which is a devastating disease.