The synthetic caged Garcinia xanthone cluvenone induces cell stress and apoptosis and has immune modulatory activity

Introduction

According to the American Cancer Society, there were nearly 1.5 million men and women diagnosed with cancer in the US in 2009. It is projected that about 40% of these individuals will die of their disease. The current annual cancer incidence rate corresponds to one out of every two men, and one out of every three women being diagnosed with cancer within their lifetime. The main obstacle for successful treatment is resistance to current therapies. Not only are the current chemotherapeutic agents ineffective against many cancers, especially relapsed cancers, but there is significant toxicity associated with these therapies. Therefore, it is essential to intensively investigate novel agents that are not only effective against refractory cancers, but are also tumor selective and thus less toxic.

The tropical trees of the genus Garcinia, found in lowland rainforests of Southeast Asia, are widely known for their use in folk medicine (1, 2). Phytochemically, these trees are recognized as a rich source of xanthone and xanthonoid natural products that hold high pharmaceutical potential. Gambogic acid (GA) is the main active component of gamboge, a dry resin secreted from Garcinia hanburyi. It has been well established that GA induces apoptosis in multiple tumor cell lines including T47D breast cancer cells and MGC-803 human gastric carcinoma cells at sub-micromolar to low micromolar concentrations (3-5). Furthermore, GA has demonstrated efficacy in several human tumor xenograft models and has entered clinical trials in China for the treatment of cancer (6-9).

Previously, we have reported a new strategy for the chemical synthesis of the Garcinia natural products (10, 11). The synthesis is short, efficient, and stereo-selective, and allows access to a variety of these compounds and related analogs. This technology not only eliminates the drawbacks of natural supply and varying isomeric mixtures, typical of natural products, but also provides the opportunity to perform systematic biologic and pharmacologic studies resulting in the discovery of novel pharmacophores and the development of highly effective chemotherapeutic agents. More recently, we have evaluated the pharmacophoric motif of the caged Garcinia xanthones and have identified the minimum bioactive motif of these compounds (12, 13). Based on this information, we have generated a simple synthetic analog, cluvenone, which was found to induce apoptosis in multi-drug resistant promyelocytic leukemia cells (HL-60/ADR) at nanomolar concentrations, with EC₅₀ values equal to that found in the parental cells (HL-60) (13). It is believed that the unique structure of cluvenone and members of the Garcinia family of natural products represents a novel pharmacophore that accounts for the cytotoxicity of these compounds against multi-drug resistant cancer cells. In the current study, we describe the anti-cancer activity and tumor selectivity of cluvenone as well as the results of gene expression profiling and pathway analyses towards the identification of critical molecular determinants in the action of the caged Garcinia xanthones.

Materials and Methods

Results

In a previous report (12), we screened a number of different caged Garcinia xanthones against solid and non-solid tumor cell lines including multidrug resistant HL-60 cells (HL-60/ADR) expressing P-glycoprotein (15). We found that cluvenone (Figure 1, inset) was the most potent of the synthetic analogs with equal or similar activity to the structurally more complex parent natural product, GA. More importantly, cluvenone, like GA, was equally toxic to HL-60/ADR as the parental HL-60 cell line. The mechanism of cytotoxicity of cluvenone was examined in leukemia cell lines using a cell death detection ELISA assay which measures histone associated DNA fragments. Cluvenone induced apoptosis in a dose-dependent manner in the T-cell acute lymphoblastic leukemia cell line, CEM, characterized by having mutated p53 and deletion of p15 and p16, genes frequently inactivated in multiple cancers (Figure 1). The EC₅₀ of cluvenone in CEM was 0.25 μM after 7.5 h of treatment and characteristic morphology of apoptotic death, membrane blebbing, was visible as early as within 4 h of treatment (data not shown). Similar results were obtained using HL-60 and HL-60/ADR cells with an EC₅₀ of 0.25 and 0.32 μM, respectively (13), indicating that the cytotoxicity of cluvenone is not affected by the expression of P-glycoprotein or by the absence of p53 and cell cycle inhibitory genes.

Encouraged by these results, cluvenone was then screened against the NCI60 cell panel to extend the evaluation of its cytotoxicity and, more importantly, gain insight into its possible mode of action. The results of the NCI60 cell panel screen confirmed that cluvenone is cytotoxic to all cancer cell lines tested including those that are multi-drug resistant, with a mean GI₅₀ of 0.5 μM (range 0.1 μM - 2.7 μM) (Figure 2). For example, even a robustly multi-drug resistant cell line such as NCI/ADR-RES (breast) as well as others such as HCT15 (colon) and U031 (renal) were sensitive to cluvenone with a GI₅₀ less than 0.5 μM, with the exception of TK10 (renal) which had a GI₅₀ of 1.0 μM. Additionally, tumor cells with very aggressive growth and metastatic potential such as NCIH460 (large cell lung cancer) and LOX (melanoma) were also sensitive to cluvenone with a GI₅₀ of 786 and 163 nM, respectively. Collectively, these results suggest that cluvenone and members of this class of Garcinia xanthones may overcome the drug-resistance typical of relapsed cancers.

Although cluvenone is cytotoxic to a broad range of tumor cells including multidrug-resistant cancer cells, it must demonstrate tumor selectivity in order to be an effective chemotherapeutic agent. To determine whether cluvenone has any selectivity for cancer cells over normal cells, we examined the effect of cluvenone in a primary human leukemia and PBMC obtained from a normal donor. Previously, we have found that cytotoxicity results from viability assays using primary cells from normal donors and patients correlates well with in vivo data, as opposed to data from similar assays using cell lines (15). For these experiments, primary cells were exposed to increasing concentrations of cluvenone for 48 h and then viable cell numbers were determined. The results shown in Figure 3 indicate that cluvenone is 4.7-fold more toxic to leukemia cells (IC₅₀=1.1 μM) than PBMC from normal donors (IC₅₀=5.2 μM). Interestingly, in the same experiment, GA was 3.9-fold more toxic to B-ALL cells than PBMC suggesting that it may have greater toxicity to normal cells compared to cluvenone (not shown). The observed differential cytotoxicity of cluvenone against leukemia cells versus PBMC from normal individuals is extremely encouraging as standard chemotherapeutic agents are generally similarly toxic to PBMC and tumor cells. Based on the observed tumor selectivity, it is anticipated that cluvenone will be well tolerated at the low doses which are therapeutically effective.

In order to gain insight into the mechanism of action of cluvenone, a COMPARE analysis was conducted using the results of the NCI60 cell panel screen. The results of this analysis determined that molecules with a similar cytotoxicity profile to cluvenone were of completely different chemical structures (Figure 4A), and were not similar to standard agents. Furthermore, using 3D Mind tools, the top 6 ranked molecules and GA were mapped to the Q region of a self organizing map (SOM), distal to agents with known mechanism of action (Figure 4B). The Q region of the SOM includes bioactive molecules with yet unknown mechanisms of action but which have recently been implicated to activate signaling pathways involved in cell stress and survival (16, 17). These findings highlight the structural uniqueness of cluvenone and members of the caged Garcinia xanthones as well as demonstrate the potential of the current research for therapeutic application to cancers refractory to standard chemotherapy.

Intrigued by the results of the SOM suggesting a novel mechanism of action and to further investigate the mode of action of cluvenone, gene expression profiling experiments were conducted using Agilent Whole Genome 4 × 44K arrays (accession number GSE23536 in GEO database). In these experiments, CEM cells were treated in quadruplicate with vehicle (DMSO) or with 0.3 μM cluvenone for 2 and 4.5 h. Analysis of the microarray data using GeneSifter software revealed that 191 or 250 genes were altered in expression at least 1.7-fold (adjusted p value <0.005) by cluvenone treatment for 2 or 4.5 h, respectively. The fold change cutoff was selected based on a report of an independent laboratory demonstrating decreased expression of telomerase reverse transcriptase (TERT) in response to treatment with GA (18, 19). In agreement with this report, our data indicated that TERT expression was decreased 1.7-fold by cluvenone indicating that a 1.7-fold change in expression was a valid cutoff.

The list of significantly altered genes at both times (2 and 4.5 h) of cluvenone treatment were analyzed using Pathway-Express software to determine which cell signaling pathways would be affected. The results, summarized in Table 1, reveal that one of the most significantly affected pathways, at both time points of treatment, includes the MAPK cell stress signaling pathway as evidenced by the dramatic and progressive up-regulation of the heat shock gene, HSPA1A, in addition to up-regulation of HSPA8 and MAPK14, also known as p38. The dramatic up-regulation of HSPA1A mRNA by cluvenone treatment for 2 and 4.5 h, as determined by microarray analysis, was confirmed by real-time RT/PCR (data not shown). Analysis of the gene expression data using Ingenuity Pathway Analysis software confirmed oxidative cell stress signaling to be a predominant pathway activated in response to cluvenone. In particular, Nrf2-mediated oxidative stress signaling which is downstream of MAPK signaling, and a defense mechanism against oxidative stress (20), appeared to be activated (P=9.09 × 10^-4). This result was based on changes in the expression (≥ 1.9-fold) of the following stress associated genes; DNAJA1, DNAJA4, DNAJA6, DNAJB1, DNAJB2, FTL, FTH1, FKBP5.

HSPA1A and other heat shock proteins, known to be associated with cell stress, are also involved in antigen processing and presentation (21), as well as in apoptotic signaling in prostate cancer (22), and possibly other cancer cells. Prostate cancer cells, especially at the advanced stages of prostate cancer, are constitutively under oxidative stress resulting in the induction of heat shock proteins which block apoptosis (23). In such cells, the additional oxidative stress imposed by cluvenone, would be expected to result in apoptosis. It is well established that when cell stress is too high for protective mechanisms to overcome damage, cells undergo apoptosis. The induction of MAPK14, Jun, and caspase 9 by cluvenone is consistent with the induction of apoptosis via the MAPK-dependent pathway (24). In summary, the induced expression of heat shock genes by cluvenone can affect antigen processing and presentation in antigen presenting cells, as well as apoptosis in cancer cells.

Treatment of CEM cells with cluvenone appears to significantly affect adherens junction signaling (Table 1A). Though this result is based on a small change (-1.7-fold) in the T-cell specific transcription factor (TCF7), this gene is central in adherens junction signaling involving the Wnt pathway. Furthermore, deregulated transcription of the full length TCF7 isoform appears to be involved in prostate and colon cancers (25-27). This may be due, in part, to the transcriptional activation of c-myc by complexes of B–catenin and members of the TCF family ultimately resulting in enhanced cell proliferation (28). Interestingly, cluvenone decreased the expression of both TCF7 and c-myc (greater than 2-fold) suggesting that it may have a role in suppressing signaling through the Wnt pathway associated with prostate and colon cancer (29).

Genes that are central to the regulation of the actin cytoskeleton, ITGAV and ITGB2, are significantly up-regulated by cluvenone at both time points (Table 1A and B) suggesting that cluvenone can affect activities associated with the actin cytoskeleton such as cell motility and adhesion. These activities are particularly important for T-cell function as is protein export (Table 1B), also found to be modulated by cluvenone. Further evidence suggesting regulation of T-cell function by cluvenone is the induction in the expression of CD69 (2.7-fold), an early leukocyte activation molecule expressed transiently in activated immune cells including T-cells (30). Other genes involved in immune function that were found to be up-regulated (about 2-fold) by cluvenone include IL11RA and IL2-inducible T-cell kinase, ITK.

Collectively, analyses of the gene expression array results indicate that cluvenone induces Nrf2-mediated oxidative stress and activation of the MAPK-dependent apoptotic pathway. Furthermore, cluvenone appears to have a role in modulating immune function and in regulating adherens junction signaling.

To determine whether cell stress induced by cluvenone is due to the formation of reactive oxygen species (ROS), the levels of ROS were measured in PC3 (prostate cancer) cells treated with cluvenone for various times up to 15 h using a fluorescence based assay. The results shown in Figure 5A, indicate that cluvenone induced ROS formation in a dose-dependent manner within 5.5 h. Although the level induced was only 30% above control at the highest concentration of cluvenone tested, this level was equivalent to that induced by 1 mM H₂O₂ and sufficient to induce apoptosis. As PC3 cells are under constitutive oxidative stress, the increase of ROS levels to 30% above control, appears to surpass the capability of protective mechanisms to prevent apoptosis. Induction of ROS by cluvenone was also time-dependent as ROS was not detectable at times tested earlier than 5.5 h (data not shown). To determine whether induction of ROS accounted for the cytotoxicity of cluvenone, PC3 cells were treated with cluvenone in the presence and absence of the antioxidant, N-acetylcysteine. Cell proliferation was then measured after 48 h in a ³H-thymidine incorporation assay. The results shown in Figure 5B demonstrated that N-acetylcysteine was able to reduce the cytotoxicity of cluvenone with complete inhibition of cluvenone cytotoxicity at 1 mM N-acetylcysteine. Collectively, these results suggest that the induction of ROS is responsible for the induction of cell stress and apoptosis by cluvenone in PC3 cells. How cluvenone induces ROS and whether additional mechanisms account for cluvenone's cytotoxicity in other cell types remains to be determined.

Discussion

The simple synthetic GA analog, cluvenone, has significant cytotoxicity at nanomolar concentrations against a broad range of tumor types including multi-drug resistant tumors with leukemia being the most sensitive tumor type, followed by colon, breast, prostate, and renal cancers. Since breast, prostate, and colon cancers are among the most common cancers in the U.S., cluvenone may have significant value as a chemotherapeutic agent. Our results indicate that cluvenone is an effective inducer of apoptosis, a required attribute for an effective chemotherapeutic agent. Furthermore, the observed differential cytotoxicity between leukemia cells and PBMC from normal donors, suggesting that cluvenone has tumor selectivity, further adds to the potential value of cluvenone in the treatment of cancer.

The unique structure and activity profile of cluvenone is consistent with the results of the COMPARE analysis indicating that compounds most similar to cluvenone map to the Q region of a SOM, a region including molecules with unknown mechanisms of action, but associated with signaling involved in cell stress and survival. Cellular stress generated by cluvenone appears to be due to the generation of ROS as determined for GA (31). In a recent study, GA induced the formation of ROS in hepatoma cells resulting in loss of mitochondrial membrane potential, release of cytochrome c, and apoptosis. Furthermore, these activities were associated with phosphorylation of JNK and p38, key members of the MAPK stress signaling pathway. Interestingly, the anti-oxidant, N-acetylcysteine, only partly reversed the activation of JNK and p38, and the induction of apoptosis in GA-treated cells, suggesting that additional mechanisms account for the cytotoxicity of GA. Like many natural products, both GA and cluvenone likely have multiple targets and mechanisms of action. For instance, in addition to inducing ROS, GA has been shown to bind to and inhibit the anti-apoptotic activities of the Bcl-2 family of proteins (32). These activities would result in a two-pronged attack on tumor cells with the induction of cell stress and the release of an apoptotic block, not uncommon in tumor cells.

Most recently, it has been reported that the oxidative stress induced by GA results in DNA damage as detected by a comet assay and evidenced by the activation of p53/p21 through the ATR-Chk1 pathway (33). It is important to note, however, that activation of DNA damage response pathways can occur in the absence of DNA strand breaks and can occur in response to alteration in chromatin structure or, to depletion of nucleotide pools (34). Furthermore, it is important to appreciate that DNA damage measured in the comet assay is not necessarily a result of direct genotoxicity as mitochondrial or membrane damage can cause extensive DNA fragmentation via apoptosis or necrosis (35). Whether cluvenone induces DNA damage is yet to be determined. However, the results of the Agilent Whole Genome array analysis did not indicate increased expression, at either time point, of genes encoding DNA repair enzymes including but not limited to RAD17, RAD51, PARP1,2, POL B, POLG typically induced upon DNA damage (36). Furthermore, neither cluvenone nor GA map to a region to which known DNA damage inducing agents map on a SOM.

The mitochondria have recently emerged as an important target for cancer therapy due to the bio-energetic differences between normal and cancer cell mitochondria (37). In addition, the mitochondria are an invariant target common to all cells and in which mutations are very rare (37). The rare mutation event in mitochondria suggests a low probability for the development of resistance to agents targeting mitochondria. It is well established that many mitocans target components of the electron redox chain, leading to ROS production and activation of the intrinsic pathway of apoptosis in cancer cells. Examples of such drugs include the vitamin E analog, α-TOS, and N-(4-hydroxyphenyl) retinamide (4-HPR, fenretinide). The pro-oxidant mechanism of 4-HPR activity has not been clearly identified, although it is likely to act as an inhibitor of at least one of the complexes along the electron transport chain (38). We anticipate cluvenone to be a member of this class of mitocans since it appears to induce oxidative stress resulting in increased expression of caspase 9 (characteristic of intrinsic apoptosis pathway) and apoptosis. Furthermore, the slow induction of ROS by cluvenone, suggesting the presence of certain events prior to the formation of ROS, is consistent with the release of ROS as a consequence of disruption of the mitochondria. Future work is aimed at investigating this possibility.

In addition to the induction of oxidative cell stress, the results of our gene expression array data indicate the induction of several genes associated with T-cell activation and function, as well as NK cell function. These genes include those encoding adhesion molecules expressed in resting and activated T-cells (integrin alpha 5, integrin beta 2) as well as a differentiation marker (CD69), and ITK, a kinase involved in regulating NK cell mediated cytotoxicity (39). These results suggest that cluvenone may have significant immune-regulatory functions that may contribute to its anti-cancer activity. Previous reports on the effects of GA on immune function support our findings. For instance, in one study, GA was found to activate T lymphocytes to induce cancer cell apoptosis in H22 transplanted mice thereby inhibiting tumor growth and extending survival (40).

Clearly, cluvenone, like GA and other natural products, has multiple actions including induction of cellular stress and apoptosis, modulation of immune function, possibly resulting in T- and NK cell mediated anti-cancer responses. It is likely that additional mechanisms exist that contribute to the anti-cancer effects of cluvenone as several genes with unknown function are modulated by cluvenone. Hence, cluvenone appears to be a multi-target anti-cancer agent with apparent tumor selectivity and ability to circumvent known mechanisms of chemo-resistance. These findings demonstrate the clinical potential of cluvenone as a chemotherapeutic agent and support further pre-clinical investigation. Importantly, however, the identification of the multiple targets of cluvenone will be critical to its development as a chemotherapeutic agent and may also lead to the discovery of even more effective anti-cancer agents.