
Glioblastoma is the most aggressive common brain tumor in adults. Curcumin, from
Curcumin is the most studied dietary phytochemical. It originated from the rhizome of
Autophagy is characterized by the formation of large double-membrane vesicles, called autophagosomes, in the cytosol. An autophagosome is formed by the hierarchical recruitment of autophagy-related (Atg) proteins, including LC3 and Beclin1, to phagophore assembly sites (Codogno
Generally, autophagy blocks the induction of apoptosis, and apoptosis-associated caspase activation shuts off the autophagic process. However, autophagy or autophagy-relevant proteins may promote the induction of apoptosis or necrosis, which leads to autophagic cell death (ACD) (Marino
In cancer cells, a complex relationship exists between autophagy as a suppressor and promoter of tumor progression (Noonan
Brain malignancies have a poor prognosis despite multimodal treatment with surgery, chemotherapy, and radiotherapy (Zanotto-Filho
MTT [3(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide], trypan blue solution, curcumin and 3-methyladenine (3-MA) were purchased from the Sigma-Aldrich (St. Louis, MO, USA). Antibodies reactive with α-tubulin were obtained from Sigma-Aldrich. Antibodies reactive with LC3 were obtained from NOVUS Biologicals (Littleton, CO, USA). 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Life Technologies (Grand Island, NY, USA). We purchased autophagy antibody sampler kit including antibodies reactive with Atg3, Atg5, Atg7, Atg12, Atg16L1, LC3A, LC3B and Beclin-1 from Cell Signaling Technology Inc. (Danvers, MA, USA). LY294002 was purchased from Cayman Chemical (Ann Arbor, MI). Rapamycin and hydroxyquinoline (HCQ) were obtained from Selleck Chemicals (Houston, TX, USA). Except where indicated, all other materials are obtained from Sigma-Aldrich.
pEGFP-C2 plasmid was kindly provided by Prof. Mi-Ock Lee, College of Pharmacy, Seoul National University (Seoul, Korea). pGFP-LC3 plasmid was kindly provided by Prof. Dong-Hyung Cho, Graduate School of East-West Medical Science, Kyung Hee University (Yongin, Korea).
A172 human glioblastoma cells were obtained from the Korea Research Institute of Bioscience and Biotechnology (KRIBB) cell bank (Daejeon, Korea). Cells were maintained and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (GIBCO, Grand Island, NY, USA), 2 mM L-glutamine, 100 U/mL penicillin and 100 U/mL streptomycin. Then, cells were incubated at 37°C in an atmosphere of humidified incubator with 5% CO2 and 95% air (Lee
Cell survival was quantified by counting cells with trypan blue exclusion assay or by using colorimetric assay described for measuring intracellular succinate dehydrogenase content with MTT (Yang
Cells with the indicated condition were grown on coverslip for 24 h and fixed with 4% paraformaldehyde (PFA) solution freshly prepared in phosphate buffered saline (PBS) for 10 min. Then, cells were permeabilized with 0.1% Triton X-100 in PBS. Nucleus was visualized by staining cells with 4′,6-di-amidino-2-phenylindole (DAPI). Then, cells were observed and photographed at 400X or 1,000X magnification under a fluorescence microscope (Nikon, Tokyo, Japan). Number of cells with DNA-condensed nucleus was counted and calculated to a percentage of total cell number.
Each plasmid DNA was transfected into cells as follows. Briefly, each nucleic acid and lipofectamine 2000 (Invitrogen, Calsbad, CA, USA) was diluted in serum-free medium and incubated for 5 min, respectively. The diluted nucleic acid and lipofectamine 2000 reagent was mixed by inverting and incubated for 20 min to form complexes. In the meanwhile, cells were stabilized by the incubation with culture medium without antibiotics and serum for at least 2 h prior to the transfection. Pre-formed complexes were added directly to the cells and cells were incubated for an additional 6 h. Then, culture medium was replaced with antibiotic and 10% FBS-containing DMEM and incubated for 24 h-72 h prior to each experiment.
Autophagy was determined by the observation of LC3 puncta-positive structures, which are the essential dynamic process in autophagosome formation (Wu
Western blotting was performed using a standard protocol. Cells were lysed in ice-cold lysis buffer containing 0.5% Nonidet P-40 (vol/vol) in 20 mM Tris-HCl (pH 8.3); 150 mM NaCl; protease inhibitors (2 μg/ml aprotinin, pepstatin, and chymostatin; 1 μg/ml leupeptin and pepstatin; 1 mM phenyl-methyl sulfonyl fluoride (PMSF); and 1 mM Na4VO3. Lysates were incubated for 1 h on ice before centrifugation at 13,000 rpm for 10 minutes at 4°C. Protein concentration in each supernatant was measured using a SMARTTM BCA protein assay kit (iNtRON Biotech. Inc., Seoul, Korea). Proteins were denatured by boiling for 5 minutes in sodium dodecyl sulfate (SDS) sample buffer. Proteins were separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes by electro-blotting. Following transfer, equal loading of protein was verified by Ponceau staining. The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween 20 (TBST) (10 mM Tris-HCl, pH 7.6; 150 mM NaCl; 0.5% Tween 20) and incubated with the indicated antibodies. Bound antibodies were visualized with HRP-conjugated secondary antibodies with the use of enhanced chemiluminescence (ECL) (Pierce, Rockford, IL, USA). Primary and HRP-labelled secondary anti-IgG antibodies were diluted 1:1,000 and 1:5,000, respectively in TBST containing 0.5% Tween 20. Immunoreactive bands were detected using X-ray film.
Experimental differences were tested for statistical significance using ANOVA and Students’
Given that malignant glioma has a poor prognosis (Zanotto-Filho
Autophagy is characterized by autophagosome formation via hierarchical recruitment of Atg proteins, including LC3 and Beclin1, to phagophore assembly sites (Codogno
To confirm the effect of curcumin-induced autophagy on curcumin-mediated cytotoxicity, cells were co-treated and incubated with the autophagy inhibitors 3-methyladenine (3-MA), hydroxychloroquine (HCQ), or LY294002 in the presence of 10 μM curcumin for 3 or 24 h. Autophagic flux using HCQ inhibits the autophagosomal lysosome degradation induced by curcumin, which accumulates LC3-II (Fig. 2A). 3-MA and LY294003 reduced LC3-II accumulation in the group co-treated with curcumin and inhibitors (Fig. 2B, 2C). Then, cell viability was assessed using the MTT assay or trypan blue exclusion assay. In the MTT assay, cell viability increased to ∼63% on co-treatment with curcumin and HCQ versus ∼56% in the curcumin-treated group (Fig. 2D), ∼51% on co-treatment of curcumin with 3-MA versus 76% in the curcumin-treated group (Fig. 2E), and ∼56% on co-treatment with curcumin and LY294002 versus ∼48% in the curcumin-treated group (Fig. 2F). Cell death was determined with the trypan blue exclusion assay. The fold change in cell death increased to ∼4.8 on co-treatment with curcumin and HCQ versus ∼2.9 in the curcumin-treated group (Fig. 2G), ∼8.9 fold on co-treatment of curcumin with 3-MA versus ∼2.8 fold in the curcumin-treated group (Fig. 2H), and ∼4.8 fold on co-treatment with curcumin and LY294002 versus ∼2.9 fold in the curcumin-treated group (Fig. 2I). These results suggest that curcumin-induced autophagy enhances curcumin-mediated A172 glioblastoma cell death.
To confirm the role of autophagy in curcumin-mediated cell death, we examined the effects of the autophagy inducer rapamycin on A172 glioblastoma cell death (Arcella
To confirm the effect of an increased basal level of autophagy on curcumin-mediated cell death, A172 cells were incubated under serum starvation. Time-dependent serum starvation enhanced the amount of LC3-II (Fig. 5A), which is consistent with the increase in autophagic cells with LC3 punta formation (Fig. 5B). With the MTT assay, cell viability was ∼85% and ∼45% on 24- and 48-h incubation under serum starvation, respectively (Fig. 5C). Cell death under serum starvation was confirmed by the increased in the percentage of cells with DNA condensation to 25% on 12 h incubation (Fig. 5D). Then, we examined the effect of serum starvation on curcumin-mediated cell death. When cells were treated with curcumin, the amount of LC3-II was time-dependently enhanced on incubation with 10% serum (Fig. 5E, top). However, few changes were observed on treatment with curcumin under serum starvation (Fig. 5E, bottom). With the trypan blue exclusion assay, the percentage of curcumin-treated cells with DNA condensation decreased to 23% on incubation under serum starvation versus 47% in the curcumin-treated group (Fig. 5F). This provides additional evidence that curcumin-induced glioblastoma cell death is reduced by enhancement of the basal level of autophagy under serum starvation.
To determine which autophagy proteins are associated with curcumin-induced cell death, we observed the changes in Atg3, Atg5, Atg7, Atg12, Atg16L1, and Beclin1. Treatment with 10 μM curcumin for 3 h increased the Atg5, Atg12, and Beclin1 expression (Fig. 6A). When Atg5 or Beclin1 expression was inhibited by small interference RNA (siRNA), cell viability was reduced in the cells not treated with curcumin (Fig. 6B). The curcumin-induced expression of Atg5 and Beclin1 was also inhibited by Atg5-siRNA and Beclin1-siRNA, respectively (Fig. 6C and 6D, top). Using the trypan blue exclusion assay, the fold change in curcumin-induced cell death decreased to ∼0.7 on transfection with Atg5-siRNA versus ∼2.4 in the curcumin-treated group (Fig. 6C, bottom). The fold change in curcumin-induced cell death decreased to ∼0.8 on transfection with Beclin1-siRNA versus ∼3.6 in the curcumin-treated group (Fig. 6D, bottom). Therefore, curcumin-induced A172 glioblastoma cell death via autophagy is mediated by Atg5 and Beclin1.
Glioblastoma is a devastating primary brain tumor that is resistant to conventional therapies (Zanotto-Filho
Under stress, both autophagy and apoptosis occur (Li
Glioblastoma is resistant to conventional therapies and temozolomide is the most efficacious cytotoxic drug for glioblastoma, exerting its cytotoxic activity via proautophagic processes (Lefranc and Kiss, 2006). Curcumin inhibits gastric cancer cell proliferation and induces autophagy and apoptosis (Fu
Although three autophagy inhibitors (3-MA, LY294002, and HCQ) have a different mechanism of action, their inhibitory effects on curcumin-induced cytotoxicity look similar. HCQ inhibits the autophagic flux by reducing the fusion of autophagosome and lysosome (Mauthe
Curcumin is a promising compound, with low toxicity at large doses, which should be evaluated in clinical trials of the treatment of human brain tumors (Klinger and Mittal, 2016). The effects of curcumin are potentiated by G2/M cell cycle arrest, apoptotic activation, autophagy induction, signaling disturbance, and the inhibition of invasion and metastasis (Klinger and Mittal, 2016). Most important signaling pathways are controlled by PI3K, Akt, and the mammalian target of rapamycin (mTOR) (Lefranc and Kiss, 2006). Tumor cell death induced by anticancer drugs is also regulated by various other signaling molecules, including JNK, Erk, c-myc, and NF-kB (Tashiro
In conclusion, we found that the antitumor activity of curcumin is associated with autophagy induction via the increased expression of Atg5 and Beclin1 in glioblastoma cells. This suggests that autophagy induction may be necessary for the antitumor activity of curcumin. It also suggests that a decrease in autophagy induced by other factors could inhibit the antitumor activity of curcumin, leading to the induction of drug resistance to antitumor agents. Therefore, patients who are to be treated with curcumin should be given autophagy modifiers. Care should also be taken to regulate autophagy so as not to induce resistance to antitumor agents by modifying autophagy in brain tumor cells.
We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
This work was supported by the R&D program for Society of the National Research Foundation (NRF) funded by the Ministry of Science,ICT & Future Planning (Grant from Mid-career Researcher Program: #2016R1A2B4007446 and #2018R1A2A3075602), Republic of Korea.
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