Molecular Mechanism Of Curcumin In Treatment Of Glioblastoma

IntroductionGliomas, originating from the predominant glial tissue in the CNS, are the most common primary tumors of the central nervous system in adults. The prevalent form is astrocytoma WHO grade IV or glioblastoma, which was80%to all gliomas. In affected patients, the prognosis is poor. Glioblastomas are characterized by rapid growth and diffuse invasiveness into the adjacent brain parenchyma. Only the nodular component of the disease can be controlled surgically. The infiltrative component of the tumor, however, is left to non-specific and cytotoxic chemo-and radiotherapy that may control tumor progression for a limited time window. The complexity in glioblastoma calls for intensive investigation. Interest in using dietary phytochemicals to treat human cancer is on the rise.Curcumin, an active component of the perennial herb Curcuma longa (turmeric), is widely used as a spice in Asian cuisine. Curcumin suppresses cell proliferation and metastasis, and induces tumor apoptosis. These effects are mediated through various transcription factors, growth factors, cytokines, protein kinases, and other bioactive molecules. Regulation of the cell cycle and apoptosis contributes to important carcinogenic mechanisms. A cell cycle arrest and apoptosis may occur in response to a wide variety of physiological and pathological stimuli and conditions. Curcumin inhibits cancer cell proliferation by inducing an arrest at different stages of the cell cycle or by inducing apoptosis. Extensive research has revealed that multiple signaling pathways, including caspase activation pathways, tumor suppressor pathways, death receptor pathways, mitochondrial pathways, and protein kinase pathways, underlie the therapeutic potential of curcumin.Death-associated protein kinase1(DAPK1), a newly identified calcium/calmodulin (Ca2+/CaM)-dependent serine/threonine kinase, has been recognized as a multi-domain protein. In addition to its kinase domain and CaM regulatory domain, DAPK1contains a C-terminal death domain, which acts as a protein interaction domain in cell death, survival, and proliferation. The knockdown of DAPK1by RNA interference or knockout of DAPK1through gene targeting attenuates cytokine-induced cell death, suggesting a death-promoting role for DAPK1. Moreover, the forced expression of DAPK1is sufficient to induce apoptosis in several cell lines, providing additional evidence for its death-inducing function. Studies targeting curcumin have revealed its inhibitory role in both STAT3and NF-κB activation, as well as its positive effect on caspase-3activity; however, the underlying mechanisms are not fully understood.The initiation and progression of cancer is controlled by both genetic and epigenetic events. Epigenetic alterations in tumor suppressor genes are potentially reversible, allowing the utilization of epigenetic targets as an effective and valuable approach to cancer chemotherapy. Epigenetic silencing mechanisms of tumor suppressor genes include alterations in CpG island methylation patterns and histone modifications, both of which regulate gene expression without altering the underlying DNA sequences. Aberrant DNA methylation patterns have been proven to be associated with a large number of human malignancies. In particular, many tumor suppressor genes are inactivated by promoter CpG island hypermethylation. Therefore, targeting DNA methylation is useful for cancer treatment. Indeed,5-aza-2’-deoxycytidine (DAC), an FDA-approved DNA methyltransferase inhibitor, results in the abrogation of tumorigenicity in vivo. STAT3, a well-characterized oncogenic transcription factor, promotes oncogenesis by upregulating a number of genes that promote proliferation or inhibit apoptosis. Although the underlying machinery remains unclear, STAT3is responsible for hypermethylation of CpG islands in certain tumor suppressor genes by directly inducing the expression of and interacting with DNA methyltransferase1(DNMT1), providing evidence for epigenetic regulation of STAT3in cancer cells. Receptor activator of NF-κB (RANK), also known as tumor necrosis factor (TNF) receptor superfamily member11A, is an essential mediator of osteoclast and lymph node development. It is activated by binding to its ligand, RANKL, and regulates the interaction between T cells and dendritic cells. RANK lacks the death domain and binds to specific members of the TNF receptor-associated factor family for transduction of stimulation signals to NF-κB and c-Jun N-terminal kinase activation RANK/RANKL signaling also mediates survival signaling through activation of the Akt or EGFR pathway and through upregulation of survival proteins. Nevertheless, several observations indicate that, similar to other members of the TNF superfamily, RANK/RANKL also possesses antitumorigenic and proapoptotic properties.Curcumin also modulates gene expression through its activity as an epigenetic agent. In particular, curcumin induces DNA hypomethylation through inhibition of DNMT1, as well as regulation of the acetylation state of histones by inhibiting histone acetyl transferases. Both of these properties can significantly alter the epigenome of cancer cells by reactivating prometastatic genes and proto-oncogenes.PurposeTo investigate the molecular mechanism of dietary agent curcumin induced GBM cell cycle arrest and apoptosisTo investigate the effects of tumor suppressor gene DAPK1in curcumin induced GBM cell cycle arrest and apoptosisTo investigate the molecular mechanism of curcumin induced RANK expression in GBM cellsMethods 1. On curcumin induced GBM cell cycle arrest and apoptosisAfter curcumin (OμM,5μM,10μM,20μM,40μM,80μM) treatment for24h, CCK-8detects the effects of curcumin on proliferation of U251cellsAfter curcumin (40μM) treatment for24h, cells were stained for FACS to detect cell cycle distributionAfter curcumin (40μM) treatment for24h, cells were stained for FACS to detect apoptosisAfter curcumin (40μM) treatment for24h, apoptosis were checked in microscope and subjected to FACS analysis after Annexin V-FITC/PICells were treated with curcurmin (OμM,20μM,40μM) for24h, gene expression were detected using Real-time RT-PCR and Western Blotting2. Role of DAPK1in curcumin induced cell cycle arrest and apoptosisCells were transfected with DAPK1siRNA to knockdown DAPK1expression.STAT3and NF-κB phosphorylaiton was detected using Western Blotting analysis.STAT3/DNA and NF-κB/DNA binding ability was detedcted using Western Blotting analysis.Caspase-3expression level was detected using Western Blotting analysis.FACS was used to detect cell cycle distribution in curcumin treated U251cells.FACS was used to detect apoptosis in curcumin treated U251cells.3. Epigenetic mechanism of curcumin induced RANK expressionCells were treated using different dose of curcumin (OμM,15μM,30μM) for4d, and RANK expression was evaluated using RT-PCR, Real-time RT-PCR and Western Blotting analysis.Cells were treated using30μM of curcumin or20μM of DAC. Promoter methylation was evaluated using MSP, and BSP.In vivo study of M.SssI inhibition analysis was used to detect DNMT1inhibitory effect of curcumin.STAT3expression was knocked-down using siRNA transfection, and BSP sequencing was performed. 4. Statistical analysisAll data are given as the mean±standard deviation (S.D.). Statistical analyses were conducted using one way ANOVA. The level of significance α=0.05, P<0.05was considered statistically significant.Results1. Curcumin induced GBM cell cycle arrest and apoptosisWe first investigated the effects of curcumin on human glioblastoma cell line U251. Cells was treated with different dose of curcumin for24h. And we found that both5μM did not induce obvious inhibition to the proliferation of the cells. However, when the cells were treated with10μM or higher dose for24h, the proliferation was intensively inhibited (F=681.763, P=0.000). We further found that curcumin induced the cells lines suffering G2/M cell cycle arrest, but not G1phase arrest (P=0.000). And FACS further showed that most cells were induced apoptosis after40μM curcumin treatment(F=42.329,P=0.000). And furthermore, we confirmed that curcmin induced apoptosis in U251cells, instead of necrosis as presented by a small amount of cell count in RU quantum. We further investigated the molecular mechanism of curcumin in curing glioblastoma. We found that Curcumin did not inhibit the expression of glycolysis related gene, especially PKM2, the hallmark of high invasiveness of malignancies. As for cell cycle arrest, we found that Cyclin D1was not downregulated after curcumin treatment at dosage of40μM for24h(F=3.534, P=0.097). However, Cyclin B1was inhibited both in mRNA and protein levels (F=178.522, P=0.000). We have found that curcumin inhibited the activation of Akt at the mark of de phosphorylation, and a higher dosage to40uM induced and transcriptional depression. At the same time, the downstream target FOXO1was also dephosphotylated. However, the dephosphrylaiton of FOXO1was hallmark of its activation, which consequently induces the expression of its target genes, exerting anti-tumor activity. We also found that, both in mRNA and protein level, curcumin inhibited the expression of cIAP2(BIRC3) and Survivin(BIRC5), and did not influence the expression of the other members in the IAP family, for example, cIAP1 (BIRC2) and Livin(BIRC7). In addition, we found that TRAIL was also upregulated after curcumin treatment.2. DAPK1mediated a curcumin-induced G2/M cell cycle arrest and apoptosisIn the present study, we demonstrate for the first time that DAPK1mediates a curcumin-induced G2/M cell cycle arrest and apoptosis by modulating STAT3and NF-κB signaling and caspase-3activation in human glioblastoma U251cells, indicating that DAPK1is a potential target in the treatment of tumors.A dose-dependent rise in the DAPK1mRNA level was detected by real-time RT-PCR in response to different concentrations of curcumin (F=165.016, P=0.000). Next, we confirmed the rise at the protein level by Western blot analyses, which demonstrated a time-dependent and dose-dependent effect. These findings show that curcumin increased DAPK1expression at both the mRNA and protein levels in U251cells.Curcumin dephosphorylates STAT3and NF-κB and upregulates DAPK1expression. To determine whether DAPK1upregulation is involved in the curcumin-induced inhibition of STAT3and NF-κB phosphorylation, we suppressed DAPK1expression using siRNA transfection. We successfully suppressed DAPK1expression using both si-DAPK1-1and si-DAPK1-2in U251cells, in contrast to the nonspecific control siRNA. We verified that the knockdown of DAPK1did not alter STAT3or NF-κB expression. In addition, we found that curcumin failed to elevate DAPK1expression in DAPK1siRNA-transfected cells. Next, we examined the phosphorylation levels of the transcription factors. As shown, curcumin suppressed both STAT3and NF-κB phosphorylation, whereas the knockdown of DAPK1resulted in an attenuated response. We then explored whether si-DAPK1transfection could regulate different time courses of curcumin-induced dephosphorylation. Western blot analyses demonstrated that the curcumin-mediated inhibition of STAT3phosphorylation at different time points was rescued by si-DAPK1transfection. Interestingly, although NF-κB phosphorylation was intensively inhibited by curcumin, si-DAPK1rescued that phosphorylation to a comparative level. Because activated transcription factors such as STAT3and NF-κB harbor DNA-binding ability, we evaluated the effect of DAPK1on the curcumin-induced inhibition of STAT3and NF-κB using EMSAs. STAT3and NF-κB DNA-binding activity was blocked by exposure to40μM curcumin for4h. However, although the DNA-binding ability was not fully rescued, si-DAPK1-treated cells showed alleviation of that repression. These results indicate that DAPK1enhanced the curcumin-induced inhibition of STAT3and NF-κB DNA binding. Curcumin activates caspase-3, which plays a pivotal role in STAT3-and NF-κB-related apoptotic pathways. Therefore, we next examined the impact of knocking down DAPK1on curcumin-induced caspase-3activation. Curcumin increased caspase-3cleavage in U251cells. Caspase-3cleavage, characterized by the appearance of17-and19-kDa protein bands, was decreased in DAPK1-knockdown cells compared to control siRNA-transfected cells. These results suggest that DAPK1plays a positive role in caspase-3activation. To determine whether DAPK1depletion could alleviate the curcumin-induced cell cyele arrest, we first confirmed that curcumin induces a G2/M, but not G1, cell cycle arrest in U251cells. Next, we treated DAPK1siRNA-transfected cells with curcumin and found that the percentage of cells in G2/M phase decreased, and that the percentage of cells in G1phase increased (P=0.000). The control siRNA-transfected cells did not show any alteration in cell cycle distribution. We also found that the knockdown of DAPK1rescued cells from curcumin-induced apoptosis (F=637.340, P=0.000). The apoptotic cell number were decreased (si-Ctrl vs. si-DAPK1). These findings provide additional evidence that DAPK1is involved in the regulation of a curcumin-induced G2/M arrest and apoptosis in U251cells.3. Curcumin reactivates RANK expression in GBM cellsIn the present study, we first assessed the effects of curcumin on RANK expression. Curcumin elevated mRNA levels of RANK in both U87and U251cells, and30μM curcumin treatment induced a higher level of RANK mRNA levels than that of15μM (P=0.000). Notably, curcumin also enhanced the RANK protein level. These results suggest that curcumin-induced elevated RANK protein expression levels may correlate with enhanced RANK mRNA levels in glioblastoma cells. These results demonstrate that curcumin can increase RANK expression in both U87and U251cells.To determine whether curcumin-induced RANK elevation was caused by epigenetic modification, we first evaluated the methylation status of CpG sites within the RANK promoter with MSP in U251and U87cells. The RANK promoter was hypermethylated in both U251and U87cells, which was confirmed by DAC treatment. To examine whether the above-described RANK reactivation is associated with promoter hypomethylation, we evaluated the promoter methylation level of RANK in curcumin-treated cells. Results showed complete conversion of promoter hypermethylation in curcumin-treated samples, with an absence of a methylation-specific band. We further validated these results by bisulfite sequencing of the selected promoter region of RANK in curcumin-treated and untreated U251cells. Untreated cells showed a significant methylated signature; all17CpG sites were methylated, whereas treatment with curcumin led to hypomethylation of all17CpG sites, thus explaining the complete loss of the methylation band and confirmed reversal of CpG methylation status by curcumin. The CpG methyltransferase, M.SssI, methylates all cytosine residues (C5) within the double-stranded dinucleotide recognition sequence, CpG. Inhibition of methylation activity by M.SssI is reflected by the presence of a259-bp band following restriction digestion of a301-bp template by methylation-sensitive BstUI (CG/CG). Curcumin caused incomplete inhibition at10μM and20μM and complete inhibition of M.SssI activity at40μM. Due to methylation of the CG/CG site by active M.SssI, the template was left uncut by BstUI while the reaction lacking M.SssI showed active BstUI. We then investigated the role of STAT3in mediating curcumin-induced RANK expression. We observed a correlation between curcumin-induced deactivation of active STAT3, downregulation of STAT3expression and RANK upregulation. Next, we tested whether knockdown of STAT3can lead to upregulation of RANK in U251cells. After STAT3-specific siRNA transfection, STAT3suppression was evaluated by Western blotting. As is shown, STAT3expression was successfully inhibited and RANK was upregulated in STAT3-suppressed cells, suggesting that knockdown of STAT3is sufficient for induction of RANK expression. Notably, the promoter hypermethylation status of RANK in si-STAT3-transfected U251cells was also converted. However, only12of17CpG sites were demethylated, which was lower than that induced by curcumin. Nevertheless, our results suggest that STAT3is involved in curcumin-induced RANK reactivation. These findings support a link between the epigenetic restoration of RANK expression and changes in the STAT3pathway in curcumin-treated U251cells. Aberrant hypermethylation at normally unmethylated CpG islands plays a critical role in tumorigenesis, and DNA methylation inhibitors have been used clinically.ConclusionFirst, we investigated the effects of curcumin in glioblastoma treatment, and further explored the molecular mechanism of curcumin induced cell cycle arrest and apoptosis, and uncovered several molecules involved in this biochemistry process, affording new evidence for glioblastoma treatment.Second, our study reveals a novel mechanism for the tumor suppressor DAPK1in cancer treatment. Our findings demonstrate that DAPK1mdiates the anti-proliferative and pro-apoptotic effects of curcumin through the regulation of STAT3and NF-κB signaling pathways and the inhibition of caspase-3in U251cells. The knockdown of DAPK1via siRNA transfection attenuated the inhibitory effects of curcumin on these pathways.Third, our findings not only identify a potential underlying mechanism for curcumin-induced RANK gene reactivation in human glioblastoma, but also suggest the importance of STAT3inhibition involving RANK promoter hypermethylation and epigenetic silencing, thus paving the way for further applications of curcumin epigenetic therapy in glioma. Moreover, the involvement of STAT3in epigenetic silencing of the RANK gene may also have therapeutic implications in human glioblastoma.In conclution, our results demonstrated novel mechanism in curcumin therapeutic activity of glioblastoma, offering potential targets for glioblstoma treatment and elucidating a novel mechanism for curcumin in cancer therapy.