BET Inhibitor I-BET151 Sensitizes GBM Cells to Temozolomide Via PUMA Induction

Cancer Gene Therapy https://doi.org/10.1038/s41417-018-0068-4

ARTICLE

BET inhibitor I-BET151 sensitizes GBM cells to temozolomide via PUMA induction

1 2 3 4 1 Zhicheng Yao ● Shida Yang ● Hongyou Zhao ● Huike Yang ● Xin Jiang

Received: 29 September 2018 / Revised: 5 November 2018 / Accepted: 10 November 2018 © Springer Nature America, Inc. 2018

Abstract A significant roadblock in treatment of GBM multiforme (GBM) is resistance to temozolomide (TMZ). In this study, we investigated whether I-BET151, a specific BET inhibitor, could sensitize GBM cells to TMZ. Our findings showed that the action of I-BET151 could augment the effect of TMZ on cancer cells U251 and U87 cells. In U251 cells, administration of I- BET151 increased the TMZ-induced apoptosis GBM cells. I-BET151 remarkably enhanced the activities of caspase-3. In addition, I-BET151 promoted TMZ-induced migration and invasion in GBM cells. Moreover, I-BET151 increased the amount of reactive oxygen species as well as superoxide anions with a decrease of activity of SOD and the anti-oxidative properties of GBM cells. I-BET151 also induced increased PUMA expression, which is required for the functions of

1234567890();,: 1234567890();,: I-BET151 and regulates the synergistic cytotoxic effects of i-BET151 and TMZ in GBM cells. I-BET151 with TMZ also showed synergistic cytotoxic effects in vivo. These point out to an approach to tackle GBM using TMZ along with BET inhibitors.

Introduction combined approach for GBM has led to the median patient survival reach 21.7 months from 15.3 months8,10. GBM multiforme (GBM) is a type of glioma with high Lysine molecules on histone tails that have been subject to mortality and is yet to see a treatment that is effective1,2. ε-N-acetylation are recognized by a family of proteins called Astrocytes constitute a large proportion of GBMs that are bromodomain and extraterminal (BET) using domains with glial cells with a star morphology2,3. The pathogenesis of the former name: bromodomains11,12. Such acetylated mole- GBM can involve lack of proper functioning of microRNAs cules can be recognized by a protein of this family: BRD4 that and methylation of promoters4,5. A standard treatment associates with a transcription elongation factor complex approach is the use of radio isotopes: however, today a called positive transcription elongation factor b to initiate combinatorial mode of using both temozolomide (TMZ) expression13. The term super-enhancers come into the picture and radiotherapy has become a staple6–8. TMZ is an alky- with enhancer sites located in copious levels that are asso- lating agent that causes the addition of a methyl moiety to ciated with mediators and BRD4 in actively expressed Guanine at O6 in DNA that causes death9. The use of this genes14. As oncogenes see an increased expression due to the activity of these molecules in several types of cancers; inhibitors of BET bromodomain can be explored as therapeutic agents15. In vitro and in vivo studies have shown the * Xin Jiang effectiveness of such a molecule: JQ1 for acute lymphoblastic [email protected] leukemia, lymphoma, and myelomas16–19. Another molecule 1 Department of Neurology, The People’s Hospital of Liaoning called I-BET151 was effective against acute leukemia (even Province, Shenyang, Liaoning, China mixed lineage leukemia was targeted) in preclinical studies20. 2 Department of Laboratory Medicine, The People’s Hospital of However, the effect of BET inhibitor in GBM remains Liaoning Province, Shenyang, Liaoning, China unclear. 3 Department of Genetics, Louisiana state university health science PUMA (p53-upregulated modulator of apoptosis), which center, New Orleans, LA, USA belong to BH3-only Bcl-2 family, acts as a key regulator of 4 Department of Anatomy, Harbin Medical University, apoptotic cell death in a variety of cancer cells including – Harbin, Heilongjiang, China colorectal cancer, glioma, lung cancer, and liver cancer21 24. Z. Yao et al.

PUMA can be induced by p53 in response to DNA damage dismutase (SOD), superoxide anion, reactive oxygen spe- and leading to apoptosis25. PUMA also could be induced in cies (ROS), and the capacity of antioxidants. a p53-independent manner by a variety of stimuli26–28. After induction, PUMA activates proapoptotic members Bax and Cell motility Bak by antagonizing anti-apoptotic Bcl-2 family members such as Bcl-2, and leads to the dysfunction of mitochondrial Twenty-four-well plates were used for making a monolayer and caspase activation, which promotes apoptosis29,30. of GBM cells that were placed in serum-free media for 12 h. The present study was based on the cytotoxic killing of A 200-µl pipette tip was used to scratch the layer: to which GBM cells by TMZ augmented by BET inhibitor. The certain treatments at 37 °C. The migration of cells was function of Caspase-3, as well as apoptosis and cell damage observed at time zero and 24 h. induced by TMZ, was elevated with the use of I-BET151 in GBM cells. The heightened sensitivity of cells to TMZ by I- Invasion assay BET151 can involve the role of PUMA. Nearly 1 × 104 cells were seeded on the upper level of transwell inserts with a coating of basement membrane Materials and methods extract. The compounds used for treatment (for 24 h at 37 °C) were added here and medium with 10% fetal bovine Cell culture serum that served as a source of chemoattraction for cells. Although the cells at top were later excluded away using U87 cells were obtained from American Type Culture swabs, those at the lower level were subjected to cold Collection (Manassas, VA, USA). U251 cells were obtained methanol fixation and staining with crystal violet (0.05%). from Sigma-Aldrich (St. Louis, MO). The culture medium was Dulbecco's Modified Eagle's medium containing fetal Colony formation assay bovine serum (10%) under 95% O2 and 5% CO2 levels. Cells were routine detected of mycoplasma contamination Following overnight adhering of seeded cells, the com- by PCR. pounds used for testing were applied at varying levels for 14 days with renewal of media and compounds every Assay for cell viability 3 days34. Staining of colonies was done by crystal violet (0.05%) for 30 min following phosphate-buffered saline The viability of cells involved a CCK-8 kit (that did not use washing. Image J Colony Counter was used for counting radioactivity). In total, 96-well plates were used for seeding with a triplicate approach. U251 and U87 at a density of 5 × 103 that were done subjected to treatments classified as: TMZ and TMZ along with CRISPR cas9-mediated PUMA Knockout I-BET151. In total, 10 μL of CCK-8 was incubated with the above 24 h post treatment at 37 °C for an hour following For PUMA gene knockout, PUMA was targeted using two which absorbance was measured at 450 nm. guide RNAs and oligonucleotides complementary were used with BsmBI overhang. LentiCRISPR v2 (Addgene) vectors Study of apoptosis were digested by the same enzyme (FastDigest BsmBI: Fer- mentas) followed by their purification by QIAquick Gel Nuclear staining was used to analyzed apoptosis with Extraction Kit with EB buffer as elution agent. Quick Ligase Hoechst 33258 (Invitrogen)31. Apoptosis was detected system was used to ligate these vectors to annealed oligonu- using an apoptosis kit (fluorescein isothiocyanate Annexin cleotides. This annealing involved phosphorylation; with T4 V Apoptosis Detection Kit I, BD PharmingenTM)32,33. The polynucleotide kinase system. They were subjected to 37 °C activity of caspase-3 was measured with Ac-DEVD-pNA: for 30 min, 90 °C for 5 min, and 25 °C at 5 °C/min. Ther- its substrate bearing specificity composed of four peptides mocycling pattern in a thermocycler. Stbl3 bacteria were the according to the kit. pNA was generated whose free con- hosts for transformation. A mix of plasmids: psPAX, pMD2G centrations was used as the measure of enzyme activity by a with 1 µg of lentiviral constructs and PolyJet reagent in colorimeter at 405 nm. serum-free medium (subject to 15 min incubation) were added to a plate with 2 × 106 293 T cells (seeded 24 h before the Oxidant stress transfection in 60 mm tissue). The medium was subject to collection following 2 days of transfection. Panc-1 cells in 4 Cell Biolabs Inc. (SanDiego, CA) kits were employed for six-well plates (4–5×10 ) incubated at 37 °C and 5% CO2 assessing indicators of stress namely: levels of superoxide were used for infection of virus. Then, 2 μg/ml puromycin BET inhibitor I-BET151 sensitizes GBM cells to temozolomide via PUMA induction

Fig. 1 I-BET151 sensitizes TMZ-induced apoptosis in GBM cellsa U251 and U87 cells were treated with increasing does of TMZ with or without 1 μM I-BET151 for 72 h. Cell viability was determined by the CCK-8 assay. b U251 and U87 cells were treated with 100 μM TMZ, 1 μM I-BET151, or their combination. Apoptosis was analyzed by a nuclear fragmentation assay. c U251 and U87 cells were treated with 100 μM TMZ, 1 μM I-BET151, or their combination. Apoptosis was analyzed by ﬂow cytometry. d U251 and U87 cells were treated with 100 μM TMZ, 1 μM I-BET151, or their combination. Relative caspase-3 activity was determined byﬂuorogenic analysis. Results of b, c, and d were expressed as means ± SD of three independent experiments. *P < 0.05; **P < 0.01

was the selection agent 24 h after incubation, and later placed Technology), Bak, Noxa (Abcam), Bax, Bcl-XL, β-actin for analysis of clones in 96-well plate and subjected to wes- (Santa Cruz Biotechnology). tern blotting. qRT-PCR Western blotting Adhering to manuals, TRIzol reagent (Invitrogen, Carlsbad, Lysis buffer made of 150 mM NaCl, 1 mM ethylenediami- CA, USA) was used for extracting total RNA. And then netetraacetic acid, 50 mM Tris, 1% Triton X: pH 7.4) was 1 μg of total RNA was used to make complementary DNA used to lyse cells that contained inhibitors of proteases using Quantscript reverse transcription Kit (Applied Bio- (Pierce, Rockford, IL). This was followed by centrifugation systems). Quantitative real-time PCR was performed with at 12,000 g for 15 min and the supernatant was boiled at Bestar®SybrGreen qPCR mastermix (DBI) and LightCycler 100 ℃ along with 2 × sodium dodecyl sulphate- 480® II Real-Time PCR System (Roche). polyacrylamide gel electrophoresis (SDS-PAGE) buffer. In total, 10% SDS-PAGE gel (polyacrylamide 12%; 100 V Mouse model and 30 mA) was used for running samples: polyvinylidene diﬂuoride membranes were used for transfer for western All animal experiments were approved by The People’s blotting that was analyzed with Odyssey infrared imaging Hospital of Liaoning Province Animal Care and Use Com- system. The antibodies used are list as followed: PUMA, mittee. Female BALB/c nude mice (4–6 weeks) were subject Bcl-2, cleaved caspase-3, cleaved caspase 9 (Cell Signaling to subcutaneous injection with human pancreatic cells in the Z. Yao et al.

Fig. 2 I-BET151 promotes TMZ-mediated anti-migratory and anti- U251 cells. d The invaded GBM cells were quantified by counting the invasive propertiesa Scratch wound healing assays showed that 1 μM I- cells at the bottom of the inserts. e, f U251 cells were treated with 100 BET151 promotes TMZ (100 μM)-induced migration inhibiton of μM TMZ, 1 μM I-BET151, or their combination. Colony formation U251 cells. b The distance migrated by U251 cells after treatment was assays were repeated at least three times and were normalized to quantified. The migrated distance was quantified by measuring the control. Results of b, d, and f were expressed as means ± SD of three difference at time 0 and 24 h and was normalized to control. c I- independent experiments. **P < 0.01 BET151 at 1 μM promotes TMZ-induced invasion inhibition of right flank. Twenty-four mice with tumors (150–200 mm Results volume) were assigned random titles of controls (treated with the same dose of vehicles), those receiving only TMZ (25 mg/ I-BET151 causes an increase in the TMZ-induced kg/d at the caudal vein), only I-BET151 (30 mg/kg/d: intra- apoptosis in GBM cells peritoneal), TMZ and I-BET151 combination (25 mg/kg/d and 30 mg/kg/d, respectively) (n = 6). Prior to their sacrifice An application of CCK-8 assay showed that U251 and on the 19th day, every second day involved examination of U87 cell lines showed cytotoxicity to a larger extent when body weight of mice and growth of tumor that was evaluated I-BET151 and TMZ were used together as compared with as: tumor volume = length × width × height/2. single treatment (Fig. 1a). The combination also augmented the induction of apoptosis by TMZ as seen in Statistical analysis observations (Fig. 1b, c). We compared the activities of caspase-3 between TMZ and TMZ + I-BET151 in U251 SPSS software (SPSS, Chicago, Illinois) was applied for andU87cells.AsshowninFig.1d, I-BET151 enhanced Student’s t-test or one-way analysis of variance to calculate the caspase-3 activity. The results point out to the the differences among the groups where at P < 0.05 was sta- induction of apoptosis by TMZ in GBM cells augmented tistically significant. Mean ± SD was used for expressing data. by I-BET151. BET inhibitor I-BET151 sensitizes GBM cells to temozolomide via PUMA induction

Fig. 3 I-BET151 augments TMZ-induced ROS production in GBM cellsRelative ROS level a, superoxide anion level b, total SOD activity c, and total- oxidant capacity d in I-BET151 and TMZ-treated U251 cells. Results were expressed as means ± SD of three independent experiments. **P < 0.01; ***P < 0.001

I-BET1151 promotes TMZ-induced suppression of TMZ-induced ROS content (Fig. 3a) and superoxide anion migration, invasion, and formation of colony in level (Fig. 3b) in U251 cells. Conversely, I-BET151 was GBM cells associated with a reduction in the activity of SOD activity (Fig. 3c) as well as antioxidant properties were affected Functional evaluation was used to analyze the ability of (Fig. 3d) in U251. The above results indicate that I-BET151 GBM cells to migrate and invade in vitro when I-BET151 promotes TMZ-induced oxidative stress in GBM cells. and TMZ were applied. Rather than either drug used singly, U251 cells showed lowered migration when both molecules I-BET151 promotes PUMA induction in GBM cells were used (Fig. 2a, b). Invasion by U251 cells was limited by I-BET151 in a significant manner as compared with The mode of induction of apoptosis in cancer cells by I- treatment with single drug (Fig. 2c, d). The ability to form BET151 was tested. Following treatment of U251 and U87 colony was assessed by treatment with either TMZ or I- with the molecule, PUMA mRNA and protein level were BET151 or both in six-well plates in values of seeding per increased significantly (Fig. 4a–d). Therefore, the expres- 1000 cells. Colony formation was significantly suppressed sion of PUMA is selectively induced in response to I- in the combination treated U251 cells at 14 days (Fig. 2e, f). BET151 may mediate its apoptotic response. The above results indicate that I-BET151 promotes TMZ to hinder the invasive ability of GBM cells along with PUMA mediates the combination of I-BET151 and adversely affecting the formation of colonies and migration. TMZ-induced apoptosis

I-BET151 augments the induced oxidative stress by To investigate to the link between I-BET151 + TMZ and TMZ PUMA, we examined the function of PUMA in I-BET151 + TMZ-induced apoptosis using PUMA knockout Acquisition of chemoresistance in GBM is associated with U251 cells. PUMA-KO U251 cells had a lower level of cell decreased oxidative stress. Thus, I-BET151 was tested for death as compared with parent cells, which was induced by its effect on the TMZ-induced oxidative stress in GBM I-BET151 + TMZ (Fig. 5a). Annexin V/PI staining was cells. We found that I-BET151 signiﬁcantly increased the used to conﬁrm the reduction of I-BET151 + TMZ-induced Z. Yao et al.

Fig. 4 I-BET151 promotes PUMA induction in GBM cellsa U251 cells were treated with 0.5 or 1 μM I-BET151 for 24 h, PUMA expression was detected by western blotting. b U251 cells were treated with 0.5 or 1 μM I-BET151 for 24 h, mRNA level of PUMA was detected by real-time PCR. c U87 cells were treated with 1 μM I-BET151 for 24 h, PUMA expression was detected by western blotting. d U87 cells were treated with 1 μM I- BET151 for 24 h, mRNA level of PUMA was detected by real- time PCR. Results of b and d were expressed as means ± SD of three independent experiments. **P < 0.01; ***P<0.001

Fig. 5 PUMA mediates the efficacy of I-BET151 and TMZ action in GBM cellsa PUMA protein level in U251 PUMA- KO cells was analyzed by western blotting. b WT and PUMA-KO U251 cells were treated with the combination of 1 μM I-BET151 and 200 μM TMZ for 24 h. Apoptosis was analyzed by flow cytometry. c WT and PUMA-KO U251 cells were treated with the combination of 1 μM I-BET151 and 200 μM TMZ for 24 h. Relative caspase-3 activity was determined byfluorogenic analysis. d WT and PUMA-KO U251 cells were treated with the combination of 1 μM I-BET151 and 200 μM TMZ for 24 h. The level of Cleaved caspase-3 and 9 was detected by western blotting. Results of b and c were expressed as means ± SD of three independent experiments. ***P < 0.001 BET inhibitor I-BET151 sensitizes GBM cells to temozolomide via PUMA induction apoptosis in PUMA-KO U251 cells (Fig. 5b). Consistently, Discussion the presence of activated caspase-3 by I-BET151 + TMZ was reduced in the knockouts (Fig. 5c). PUMA defection Apart from its grim reputation, GBM faces challenges in blocked I-BET151 + TMZ-induced intrinsic apoptotic therapeutic approaches as even the last resort: surgery can events, which was observed in active caspases 3 and 9 be applied only in one-fifth of patients or less35,36. Despite (Fig. 5d). Together, the above results indicate that apoptotic the role of research in developing treatments, there is a response of I-BET151 + TMZ is PUMA-dependent in demand for new approaches. A promise here has been seen GBM cells. in regulation of epigenetic that has been observed in several types of cancers37. For instance, T-cell lymphoma has been The effects of I-BET151 and TMZ in animal models treated with romidepsin and vorinostat: that target mod- (mice) ulating molecules of epigenetic38. Major hurdles in the application of this approach include concerns over the A xenograft model was used to study a probable link specificity of targets as well as the use of such enzymes that between the effects of I-BET151 and TMZ on cancer cells target chromatin in only cancers of hematopoietic nature39. and cell death by PUMA. There was a decrease in weight Another approach to use epigenetic as treatment is a new and volume of tumors in U251-grafted mice. The reduction class of molecules that inhibit the BET bromodomain that was more pronounced in the group that received I-BET151 targets fewer genes showing promise in terms of selectiv- and TMZ against the groups that received single molecules ity11,12,40. A molecule here that captured our attention is I- (Fig. 6a, b). I-BET151 could augment cell death caused by BET151 that inhibits a protein with increased expression in TMZ, however, was not capable of causing the death of the a large proportion of pancreatic tumors called BRD441. This cancer cells was shown by terminal deoxynucleotidyl prompted us to examine the role of this molecule along with transferase dUTP nick end labeling (Fig. 6c). Immunohis- TMZ in both an in vitro (cell lines) and in vivo (xenograft) tochemistry assay showed that both I-BET151 and TMZ scenario. In the present study, we showed the evidence for could inhibit the proliferation of GBM cells compared with the sensitizing action of chemical inhibitors of BET to TMZ control, and combined treatment with I-BET151 and TMZ treatment in GBM cells. The experiments revealed an showed a greater capacity to proliferation inhibition than increase in cell death of cancer cells by the use of the mono-therapy (Fig. 6d). In contrast, compared with par- inhibitor showing an increased efficacy when both TMZ ental, PUMA-KO tumors were significantly led to less and inhibitor was employed. The activities of members of growth inhibition and apoptosis induction in response to caspases and ROS production, which referred the intracel- combination treatment (Fig. 6c, d). lular proapoptotic activity, were further enhanced by I-

Fig. 6 I-BET151 promotes the antitumor effects of TMZ in vivoa or the combination for 10 consecutive days. Tumor weight at the end Nude mice were injected s.c. with 4 × 106 WT and PUMA-KD of treatment was analyzed. c TUNEL assay (immunoﬂuorescence U251 cells. After 1 week, mice were treated with I-BET151, TMZ, or assay) showing that I-BET151 enhanced TMZ-induced apoptosis, the combination for 10 consecutive days. Tumor volume at indicated which was blocked in PUMA-KO cells. d Tissue sections from c were time points after treatment was calculated and plotted (n = 6 in each analyzed by Ki67 (immunohistochemistry assay). Result of b was group). b Nude mice were injected s.c. with 4 × 106 WT and PUMA- expressed as means ± SD of three independent experiments. *P < 0.05 KD U251 cells. After 1 week, mice were treated with I-BET151, TMZ, Z. Yao et al.

BET151. I-BET151 induced PUMA induction is required Conflict of interest The authors declare that they have no conflict of for I-BET151/TMZ-induced apoptosis. Taken together, interest. these results demonstrate that inhibition of I-BET151 sensitizes GBM cells to TMZ via PUMA upregulation. The use of TMZ that alkylates DNA constitutes radio- References therapy is part of the treatment used nowadays42. A hurdle here is resistance to this agent that necessitates the use of 1. Hanif F, Muzaffar K, Perveen K, Malhi SM, Simjhee ShU. molecules that inhibit the division of GBM cells that is Glioblastoma multiforme: a review of its epidemiology and further driven by reports of not only cell resistance but also pathogenesis through clinical presentation and treatment. Asian an increase in recurrence of tumors8. Pac J Cancer Prev. 2017;18:3–9. 2. Shergalis A, et al. Current challenges and opportunities in treating The targeting of BET bromodomain in GBM cells that – 43 glioblastoma. 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