Cancer is one of the world's leading causes of death and arises when the homeostatic balance between cell growth and death is disturbed [1,2]. Recent reports estimate the total worldwide economic burden associated with cancer therapy to be several hundred billions of US$ [3]. Consequently, advances in cancer therapy will have economic as well as public health implications. As more evidence highlights the toxic side effects of traditional chemotherapies, there is a growing interest for alternative medicines to improve the function of the immune system in order to target late stage metastatic tumors. Natural extracts from fungi have been the focus of recent investigation, particularly those with reduced cellular toxicity to healthy tissue. When considering any new therapy, both efficacy and cost-effectiveness of the cancer treatment strategy are the two main players that decide the healthcare costs associated with cancer management. Advances in technology have allowed for widespread screening of natural products to identify those with anticarcinogenic and immunostimulatory potential. This has led to a renewed investigation into the anecdotal evidence of health benefits associated with folk medicine, with the goal of identifying the active components which could be used as alternative or complementary treatment for cancer. This may be of particular importance to societies and countries where modern medicine is scarce, expensive to buy, or simply unavailable.

Ganoderic acids (GAs), the triterpenoid extracts of Ganoderma lucidum mushroom (Figure 1) are potential low-cost candidates with interesting therapeutic properties. The mushroom itself has long been used in Eastern herbal medicine and is thought to increase vitality, immune function and life expectancy. Several recent studies have shown them to possess antitumor and anti-metastatic properties in a range of cancer cell types [4-6]. More than 130 GAs and related derivatives have been isolated and identified from the spores, fruiting bodies and cultured mycelia of the Ganoderma spp. in the past two decades [7-9]. GAs and derivatives have received interest due to their substantial pharmacological activities. Interestingly, a number of GAs including GA-A, GA-B, GA-H and GA-C1 showed anti-HIV-1 activity [10,11]. Other possible reported activities include: antihistamine [12], antinociceptive [13], antihypercholesterolemic [14], antiangiotensin [15] and antitumor effects [16]. This review aims to discuss the current knowledge of antitumor activities associated with G. lucidum crude extracts with particular emphasis on the anticancer apoptotic and immune restoration activities of purified GAs. Also, we will discuss our development of a novel treatment-delivery approach of GAs via nanoparticles, termed GAIN "Ganoderic Acid-Infused Nanoparticle" complex, that were particularly designed to target the malignant cells with reduced bystander effects. GAIN could also be enrobed with cancer-targeting ligands to allow more of the incorporated compound to reach the target tissue. This article will thus further highlight both chemo- and immunotherapeutic potentials of GAs in addition to the use of nanotechnology to develop antitumor treatment.

Figure 1: Ganoderma lucidum mushroom and the chemical structures of some well characterized Ganoderic Acids (right).

Ganoderma, a genus of polypore mushrooms that grows on wood, includes about 80 species, and is distributed in many tropical regions [17]. Ganoderma's economic importance was gained as several species of the mushroom were extensively used in traditional Asian medicines due to its potential bioremediation. One species, however, G. lucidum (known as Lingzhi in China and Reishi in Japan) (Figure 1) has become one of the natural sources of numerous efficacious medicines, and is now being used to complement or sometimes substitute modern medicine in remote places of the world. In fact, the popularity of using alternative medicines has increased steadily as their prevention activity has been proven. The curative use of mushrooms has a very long tradition in the Asian countries, whereas their use in the Western hemisphere has considerably increased since the last decade [18]. Many agents derived from medicinal mushroom fruiting bodies, cultured mycelium, and/ or culture filtrates exert a wide range of beneficial biological effects when tested in vitro or in vivo in animal models. Lingzhi has gained its popularity as a therapeutic mushroom and named king of herbs in China since ancient times for improving longevity, health promotion and its marked immune-modulating effects as demonstrated by an increase in T lymphocytes [19,20]. Many further investigations into the anticancer activity of G. lucidum have been carried out both in vitro and in vivo, which support its application for cancer treatment and prevention [21-23]. Although anticancer activities of G. lucidum extracts have prompted the usage of the mushroom by cancer patients, its medicinal/therapeutic value remains debatable. So far, there has been no report of human trials using G. lucidum or its extracts as anticancer agents. Thus studies focused on cytotoxic, apoptotic, antiangiogenic, and immunogenic mechanisms of crude preparation of G. lucidum and its purified GAs should be encouraged.

Recent clinical studies in cancer patients have shown that a crude G. lucidum water extract (i.e. Ganopoly) can enhance host immune function with increased activity of T cells, macrophages, and natural killer cells, although remarkable antitumor responses were not observed [24-27]. Other study, however, suggests that the cancer preventive and tumoricidal properties of G. lucidum could be ascribed to its ability to enhance the host's immune functions, antioxidative and radical-scavenging effects, inhibition of metabolic activation and enhanced detoxification of carcinogens, and their cytotoxicity to cancer cells [28]. The major active constituents from G. lucidum extract (including polysaccharides and triterpenoinds) were also found to exert chemopreventive and tumoricidal effects through a possible modulation of signaling molecules and induction of cell-cycle arrest and apoptosis [29-32]. Other mechanisms, such as antiangiogenesis, antipromotion, and antiprogression activities were also assumed to play a role, and will be discussed in this review.

Investigations on biologically active components of the carpophores and the cultured mycelia of G. lucidum have shown that this mushroom is rich in various natural metabolites that have demonstrated both antitumor and immunomodulatory activities [33,34]. Among the most medically important constituents of the mushroom are its polysaccharides and alcohol soluble triterpenoids that also include GAs [22,35,36]. Polysaccharides represent the major water extract fraction of G. lucidum that exhibits significant antitumor effects in several tumor-bearing animals, mainly through their immuno-enhancing activity [34,37]. They are mostly high molecular weight glucans that vary in their water solubility and nature of their side chains. Researchers have found that polysaccharide-rich fractions exhibit antitumor properties in rodents mainly through the induction of cytokines such as IL-1, IL-6, IL-12, IFN-?, TNF-a, GM-CSF, G-CSF, and M-CSF in monocytes/macrophages and T lymphocytes [34,37-39]. G. lucidum triterpenes are considered to be potential anticancer agents due to cytotoxicity against growing tumors, although much of the credit of enhancing the immune system was given to the polysacchariderich extract. The crude triterpenoid extracts of Ganoderma have been reported in numerous publications to significantly induce cytotoxicity and to reduce tumor growth [40-42]. Purified triterpenoids have also been shown to possess anti-invasive properties through inhibition of matrix metalloproteinase-9 (MMP-9), and reduction of NF-?B/AP-1 DNA-binding activities in PMA-induced HepG2 cells [43]. Also the inhibition of AP-1 and NF-?B was documented in highly invasive human breast cancer cells, which coincided with downregulation of Cdk4 expression and reduced secretion of uPA [44]. The antiinflammatory and antitumor properties of purified triterpene acids and sterols were also reported by other groups [45,46], encouraging the potential use of GAs in combination therapy against inflammation and cancer.

The main focus of this review is the methanol soluble fraction of G. lucidum extract, predominantly GAs, a family of compounds that possesses a molecular structure similar to steroid hormones [46] (Figure 1). Studies suggest that GAs possess antitumor properties that seem to be related to a cytotoxic activity against cancer cells [5,47,48]. Both the antitumor and immuno-modulatory properties of G. lucidum extract, along with its low cytotoxicity, increase the possibility that it could be very effective in a complementary treatment for cancer patients receiving conventional chemotherapy and/or radiation treatment.

The potential clinical value and wide acceptability of G. lucidum extracts as alternative anticancer medication have attracted intense interest in the search for their molecular mechanisms. Studies have reported that G. lucidum extracts combine both polysaccharides and triterpenoids that collectively possess aspects of immuno-modulatory and antitumor effects [5,41] (Table 1). Yet, little work has been done on identifying the antitumor activity and immune regulation mediated by purified triterpenes in vivo. Understanding the mechanisms by which purified triterpenes exert their antitumor and immune stimulation effects would contribute to the design of alternative strategies for cancer treatment.

Table 1: Some recent applications, observed activities and suggested mechanisms of actions for Ganoderma lucidum raw extracts (I), and its purified Ganoderic acids (II) on several types of cancer cells.

GAs have shown a wide range of medicinal benefits, most notably their potent toxicity to tumor cells with a comparatively limited toxicity to bystander cells (Table 1). It has been reported that GA-X inhibits topoisomerases and sensitizes the cancer cells toward apoptosis [49]. The in vitro treatment of human hepatoma HuH-7 cells causes immediate inhibition of DNA synthesis as well as activation of ERK and JNK, and induction of apoptosis. The observed molecular events include: degradation of chromosomal DNA, decreased levels of BclxL, disruption of mitochondrial membrane potential, cytosolic release of cytochrome c and activation of caspase-3. The involvement of mitochondrial dysfunction was also supported by a later work [50], in which GA-T induced apoptosis of metastatic lung tumor cells through the intrinsic pathway mediated via the mitochondria-dependent caspase pathway and p53 expression. This study also detected the exposure of p53 and Bax proteins in 95-D cells, while the expression of Bcl-2 was not significantly changed. In vivo study also showed that GA-T suppressed the growth of human solid tumors in athymic mice. In addition to its antiproliferative effects, recent studies have found that GA-T effectively inhibits cancer cell invasion and metastasis both in vitro and in vivo [51]. This study also showed that GA-T promoted aggregation and simultaneously inhibited adhesion to the extracellular matrix, thus impairing the migration of colon (HCT-116) and lung (95-D) cancer cells in a dose- and time-dependent manner. Strikingly, GA-T treatment inhibits the nuclear translocation of NF-?B and the degradation of inhibitor of ?B-a (I?B-a), which ultimately led to a decrease of MMP-9, inducible nitric oxide synthase (iNOS), and urokinase-type plasminogen activator (uPA). Furthermore, in vivo administration demonstrated that GA-T suppresses tumor growth and metastasis, and down-regulates MMP-2 and MMP-9 mRNA expression in a Lewis Lung Carcinoma (LLC) model in vivo.

Recently, it has been found that p53 plays an important role in the suppression and anti-invasion properties of GA-T in human colon cancer (HCT-116) cell lines [52]. Comparing the results in HCT- 116 p53(+/+) and p53(-/-), it is suggested that p53 could modify GAT- mediated inhibition of NF-?B translocation, degradation of I?Ba, and downregulation of uPA, MMP-2/9 and iNOS/NOS2 protein expression. A recent study has also demonstrated that GA-Mf as well as GA-S induces apoptosis of human HeLa cells through a mitochondriamediated pathway [53]. Both GA isomers showed inhibition of cellular growth in various human carcinoma cell lines. GA-S was also shown to cause cell cycle arrest in the S phase, while GA-Mf caused cell cycle arrest in the G1 phase. Treatment of HeLa cells with these GAs resulted in a decreased mitochondrial membrane potential and the release of cytochrome c from mitochondria into the cytosol. Caspase-3 and caspase-9 activities were also observed with a sharp decrease in the Bcl- 2/Bax ratio as recorded in GA-treated HeLa cells. Figure 2 depicts the possible anticancer activity of GAs.

Figure 2: Schematic diagram indicating the plausible anticancer activity of Ganoderic Acids.

The potential use of GAs as a complementary anticancer therapy also depends on their effectiveness against multidrug resistant cancer cells. In this direction, GA-Me has been shown to reverse the multidrug resistance (MDR) in multidrug resistant colon cancer cells by inducing apoptosis via upregulation of p-p53, p53, Bax, caspase-3, caspase-9 and downregulation of Bcl-2 [54]. In addition, GA-Me treatment can stimulate the decline in mitochondrial membrane potential and release of cytochrome c into the cytosol [54]. Beside its apoptotic effects, GAMe has been reported to effectively inhibit tumor growth and lung metastasis of Lewis lung carcinoma in C57BL/6 mice through enhanced immune function [55]. Administration of GA-Me (28 mg/kg) resulted in a significant increase in NK cell activity and the production of IL-2 and IFN-?, possibly through an upregulation of NF-?B. Multiple roles of GA's antitumor activities are depicted in Figure 2. Our group has found that purified GA-A has a profound apoptotic effect on B-cell lymphoma cell lines with minimal toxicity on non-malignant B-cells. Treatments with GA-A caused mitochondrial dysfunction, identified by the release of cytochrome c into the cytosolic compartment, caspase 3, and 9 activities, and overexpression of Bax (unpublished data). We also found that GA-A-treated B-cell lymphoma express elevated levels of HLA class II proteins with an increased antigen presenting capacity to stimulate T cells in vitro (unpublished data). However, the mechanisms by which GA-A induces immune activation and tumor killing in vivo remains unclear. Table 1 summarizes some recent investigations on GAs antitumor activities, their application, and suggested mechanism(s) of actions.

It has been reported that most anticancer drugs have limited selectivity [56], hence the levels of drug required to kill a sufficient number of tumor cells to achieve and maintain a state of complete remission in patients causes significant toxicity towards actively proliferating nonmalignant cells, such as normal cells of the gastrointestinal tract and bone marrow. Many of the curative properties of conventional treatments can be improved through the use of targeted drug delivery systems [57], which include the use of nontoxic, biodegradable particulate carriers, composed primarily of lipids and/ or polymer nanoparticles, and their associated therapeutics [58-61]. This new delivery system is designed to alter the pharmacokinetics and biodistribution of the associated drug, or to function as drug reservoirs (i.e., sustained release), in addition to their advantage in stabilizing the drug and preventing its premature metabolic breakdown.

Nanoparticle approaches to targeted drug delivery for malignant tumors offer new opportunity to improve patient care and quality of life by reducing off-target toxicities. Development of targeted nanoparticles ensures delivery of chemotherapeutics directly to cancer cells, followed by their slow release in potentially sustainable levels that may provide superior efficacy and lower toxicity for treating primary or advanced metastatic tumors. Our recent studies suggest that a combinatorial approach using the co-encapsulation of a lipophilic near infrared (NIR) dye and an anticancer drug within hydrophobic pockets in the polymeric matrix of poly acrylic acid (PAA)-coated IONPs (PAA-IONPs) could be employed for combined optical imaging, magnetic resonance imaging (MRI) detection, and targeted cancer therapy [58]. Our study suggests that a natural triterpenoid such as GA-A can be readily entrapped with a high degree of latency within its hydrophobic pockets, remain associated for appropriate lengths of time and be released at an appropriate rate, with sustained levels of drug in the tumor cells [62].

GA-A-carrying nanoparticles could also be designed to deliver the drug to the cancer cells, allowing targeted cancer treatment without harming healthy cells. For example, receptor-mediated cellular uptake of nanoparticles could be achieved by attaching a vitamin derivative such as folic acid to these particles. Folic acid is an effective targeting agent for the folate receptor which is believed to be overexpressed in many human tumor cells [63,64]. In this scenario, cancer cells may consume high amounts of folic acids and ensure that nanoparticles preferentially accumulate at the tumor site before releasing their drugs (Figure 3). Studies performed by our group suggest that nanoparticles can also carry a fluorescent dye and an iron oxide magnetic core [58,59], thus their locations within the cells and the body could be seen by optical imaging and MRI, allowing a physician to see how the tumor is responding to the treatment. Our study also suggests that the nanoparticles could be engineered without the drug for their use as imaging (contrast) agents for detection of cancer [58]. If there is no cancer, the biodegradable nanoparticles may not bind to the tissue allowing their elimination by the liver cells. Ultimately, high concentration of GAIN accumulated at the tumor site may spare exposing the body to the side-effects of chemotherapy, while having larger doses of drugs to target cancer cells.

Figure 3: Schematic diagram depicting ligand-targeted accumulation of Ganoderic Acid-Infused Nanoparticles (GAIN) with both diagnostic and therapeutic functions. Both GA-A and imaging-dye are conjugated to a biocompatible polymer that bonded to a folate ligand to trigger internalization. GAIN preferentially target the folate receptor-rich cancer cells, where linkages are hydrolyzed and the nanoparticles release the drug and dye, allowing drug delivery as well as imaging of tumor tissue.

Exploring the scientific basis among known cures used in folk medicines has resulted in purifying potential natural products that could combat many diseases, particularly cancer. Hence, further examination of the anticancer relevance of these products may relieve the economic burden associated with the management of cancer, especially in societies where modern medicine is scarce, or expensive to buy or even unavailable. One valuable resource of those medicinal products is the fruiting body of Ganoderma lucidum, a wild mushroom that grows on logs, decaying wood, and tree stumps in Asia and surrounding regions, and which has been used for thousands of years in herbal remedies to promote human immune function. Given that the development of tumor is aided by immune evasion, current literature suggests GAs possess both apoptotic and immune restoration properties. Our study suggests that GAs are unique natural candidates which could be used to enhance the cellular expression of immune components as well as antigen processing and presentation. The above mentioned properties of GAs make them ideal candidates for potential use in complementary chemo-immunotherapeutics. In this direction, we are currently developing a new GAs treatment-delivery system termed GAIN by utilizing nanotechnology to develop an antitumor treatment strategy that will allow cancer targeting. GAIN is enrobed in cancer-targeting protein ligands to allow more of the incorporated compound to reach the target tissue and greatly diminish any toxic side effects associated with using higher doses. Collectively, this review suggests that enhanced targeting resulting from nanoparticle delivery, coupled with the reduced bystander toxicity of natural products such as GAs will pave the way for potential treatments of human malignancies.

This work was supported by grants from the National Institutes of Health (CA129560) to A. Haque. We thank Dr. Jason God for critical reading of the manuscript.