In this study, we demonstrate that curcumin induces apoptosis in medulloblastoma cells and is accompanied by reduced HDAC4 expression, increased tubulin acetylation, and arrest at the G2/M phase of the cell cycle followed by mitotic catastrophe, and cell death. We also show anti-tumor effects of curcumin in vivo in tumor xenografts and a transgenic medulloblastoma tumor model. Thus, our in vitro and in vivo data suggest that curcumin has the potential to be developed as a therapeutic molecule for medulloblastoma.
Microtubules form the mitotic spindle during cell division. Because of the rapid assembly and disassembly of microtubules during the alignment and separation of chromosomes, spindle microtubules are in general more dynamic than interphase microtubules [40
]. Compounds that inhibit these dynamics lead to cell cycle arrest in the G2/M phase, eventually resulting in cell death. Curcumin has been shown to bind to tubulin, to induce tubulin aggregation, and to depolymerize interphase and mitotic microtubules in HeLa and MCF-7 cells [39
]. Consistent with these data, we observed reduced microtubule density in interphase medulloblastoma cells treated with curcumin. In mitotic cells, however, we found that while the mitotic spindle microtubules were disorganized, they displayed increased staining intensity, suggesting stabilization of microtubules. In addition, curcumin treatment of DAOY cells resulted in increased tubulin acetylation. Although the exact function of post-translational tubulin acetylation is not known, it is usually considered to be associated with increased microtubule stability [41
]. Thus, it is possible that factors other than direct binding of curcumin to tubulin play a role in the altered organization of the mitotic spindle in curcumin-treated medulloblastoma cells.
We found that curcumin is a novel modulator of HDAC4. In curcumin-treated cells, HDAC activity was inhibited and HDAC4 expression was reduced, while the expression levels of other HDAC isoforms did not appear to be affected. At this point, we do not know how curcumin regulates HDAC4 expression and HDAC activity. Studies to determine the molecular mechanisms continue in our laboratory. Reduced HDAC activity and HDAC4 levels were observed as early as three hours upon curcumin-treatment, coinciding with increased α-tubulin acetylation. Mitotic spindles were altered as early as 30 min after treatment (data not shown) and very prominent after 60 min (Figure ), indicating a potential of curcumin as an anti-mitotic drug. At these early time points, we did not find any indication of curcumin-treated cells undergoing apoptosis, nor did we find substantial changes in some of the well-known signaling pathways affected by curcumin, such as NFκB (Figure ) or Akt (data not shown). Therefore, we suggest that HDAC4 inhibition in curcumin-treated cells might contribute to the induction of apoptosis rather than being a byproduct of apoptosis. This is further supported by our observation that inhibition of caspase-3 did not prevent reduced expression of HDAC4 upon curcumin treatment (Additional file 1
). The effects of curcumin observed in cell lines were mirrored in in vivo
models of medulloblastoma, namely DAOY xenografts and the Smo/Smo transgenic mice. In both medulloblastoma models, curcumin significantly reduced tumor growth and increased survival, respectively. Molecular analysis of curcumin-treated and control tumors revealed reduced HDAC4 expression and increased tubulin acetylation, suggesting that curcumin induces apoptosis by similar mechanisms in culture and in vivo
A disrupted equilibrium as a result of increased HDAC expression and activity has been associated with increased proliferation, migration, angiogenesis, differentiation, invasion, and metastasis and enables cancer cells to evade cell cycle arrest and apoptosis by suppressing the transcription of cell cycle inhibitors and pro-apoptotic factors [14
]. Interestingly, a recent study found that forced expression of HDAC4 in cerebellar granule neurons protects these cells against apoptosis [43
]. We show that curcumin targets HDAC4 in medulloblastoma cells and reduces HDAC activity. Thus, curcumin might target one of the critical pathways that allow cancer cells to evade apoptosis. Previous studies reported that curcumin represses p300/CBP HAT and inhibits acetylation of p53 [44
]. However, we did not find changes in either p300 phosphorylation and histone H3 or p53 acetylation under our experimental conditions (data not shown), while HDAC4 expression was reduced in three medulloblastoma cell lines as well as in vivo
(Figure and Additional file 3
). Similarly, studies in other experimental systems also found no effects of curcumin on p300 activity [44
] suggesting that p300 inhibition by curcumin might be cell-type specific. Furthermore, we did not find significant changes in the levels of other HDAC isoforms, suggesting that in medulloblastoma cells HDAC4 is a specific target of curcumin.
In contrast to ubiquitous class I HDACs, HDAC4 as a class IIa family member is restricted to certain tissues, including the brain, and can shuttle between the cytoplasm and the nucleus. The translocation of HDAC4 from the cytoplasm to the nucleus is regulated by localization signals and interaction with 14-3-3 proteins through three conserved phosphorylation sites [46
]. However, curcumin treatment did not alter the cytoplasmic localization of HDAC4 in DAOY cells, suggesting that curcumin's effect on HDAC4 might affect predominantly non-histone targets rather than chromatin structure and gene transcription. Interestingly, a recent study found that Shh signaling, a major signaling pathway affected in medulloblastoma, is regulated by Gli acetylation and HDAC1 [47
]. Nevertheless, this study did not find any link between HDAC4 and Shh signaling in fibroblasts. However, given the cell-type specific expression pattern of HDAC4 we cannot exclude that such a link might exist in medulloblastoma cells. In addition, another study showed that curcumin inhibits the Shh pathway in medulloblastoma cells [32
]. We found that curcumin was effective in the Smo/Smo medulloblastoma model, which increased survival, while HDAC4 expression was reduced at the same time. It remains to be determined whether HDAC inhibition is a missing link between curcumin and its effects on Shh signaling in medulloblastoma.
Although potential chemotherapeutics may show promise in medulloblastoma culture models, the BBB remains an obstacle for the development of drugs for brain tumors. Indeed, about 98% of all small molecule drugs and all large molecules such as therapeutic antibodies and peptides will be prohibited from crossing into the brain. We show that orally delivered curcumin increases survival in Smo/Smo mice and thus, exhibits chemotherapeutic effects in the brain. Our data are consistent with studies of curcumin in various central nervous system (CNS) disorders including Alzheimer's disease that showed a potent effect of orally delivered curcumin in the brain [9
]. In addition, curcumin crossed the BBB and inhibited tumor growth in orthotopic glioblastoma models when administered through the tail vein [11
] or injected i.p. [10
]. Bioavailability of curcumin in the brain is further supported by multiphoton microscopic studies and radiolabel distribution studies in mice that showed that curcumin administered systemically can cross the BBB, can be absorbed in the brain, and exerts biological effects in the brain [7
]. These studies are consistent with our observations that curcumin can cross the BBB, as manifested in increased survival in curcumin-treated Smo/Smo mice, and that curcumin is a valid anti-cancer agent for brain tumors.
Despite advances in treatment, a favorable outcome for patients with medulloblastoma lags behind many other pediatric cancers and is often associated with severe long-term side effects. For example, a small molecule inhibitor of Shh succeeded in eradicating spontaneous medulloblastoma in transgenic and transplantation mouse models [5
]. However, while these agents might have no or limited side effects in adults, in juvenile mice even transient exposures to a Shh pathway inhibitor resulted in permanent defects in bone development [6
]. In addition, while a first clinical trial was initially successful, the patient developed resistance within a short time [48
] impeding its therapeutic potential against medulloblastoma. Thus, it remains a challenge to identify safer and effective drugs to treat pediatric brain tumors. Curcumin has been used as a spice for centuries in Asian cooking and has demonstrated its safety in phase I and II clinical trials in adults. No adverse reactions in clinical trials involving children have been reported so far [50
]. Curcumin has potential anti-tumor effects in a variety of cancers including pediatric cancers such as osteosarcoma [52
], neuroblastoma [53
], and acute lymphoblastic leukemia [54
]. Here, we report that curcumin induces apoptosis in medulloblastoma cells as well as in vivo
models of medulloblastoma. While curcumin reduced tumor growth in tumor xenografts and increased survival in Smo/Smo mice, the tumors were not completely eradicated. A plethora of studies found that curcumin can potentiate the anti-tumor effects of other chemotherapeutics and irradiation. Thus, in combination with other modes of therapy, curcumin has the potential to develop into a therapeutic for medulloblastoma without the severe side effects found in current treatment regimens.