The BET inhibitor I-BET762 inhibits pancreatic ductal adenocarcinoma cell proliferation and enhances the therapeutic effect of gemcitabine

Scientific Reports, May 2018

As one of the most fatal malignancies, pancreatic ductal adenocarcinoma (PDAC) has significant resistance to the currently available treatment approaches. Gemcitabine, the standard chemotherapeutic agent for locally advanced and metastatic PDAC, has limited efficacy, which is attributed to innate/acquired resistance and the activation of prosurvival pathways. Here, we investigated the in vitro efficacy of I-BET762, an inhibitor of the bromodomain and extraterminal (BET) family of proteins, in treating PDAC cell lines alone and in combination with gemcitabine (GEM). The effect of these two agents was also examined in xenograft PDAC tumors in mice. We found that I-BET762 induced cell cycle arrest in the G0/G1 phase and cell death and suppressed cell proliferation and metastatic stem cell factors in PDAC cells. In addition, the BH3-only protein Bim, which is related to chemotherapy resistance, was upregulated by I-BET762, which increased the cell death triggered by GEM in PDAC cells. Moreover, GEM and I-BET762 exerted a synergistic effect on cytotoxicity both in vitro and in vivo. Furthermore, Bim is necessary for I-BET762 activity and modulates the synergistic effect of GEM and I-BET762 in PDAC. In conclusion, we investigated the effect of I-BET762 on PDAC and suggest an innovative strategy for PDAC treatment.

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The BET inhibitor I-BET762 inhibits pancreatic ductal adenocarcinoma cell proliferation and enhances the therapeutic effect of gemcitabine

Scientific RepoRts | The BET inhibitor I-BET762 inhibits pancreatic ductal adenocarcinoma cell proliferation and enhances the therapeutic effect of gemcitabine Fang Xie Mei Huang Xiansheng Lin Chenhai Liu Zhen Liu Futao Meng Chao Wang Qiang Huang OPEN As one of the most fatal malignancies, pancreatic ductal adenocarcinoma (PDAC) has significant resistance to the currently available treatment approaches. Gemcitabine, the standard chemotherapeutic agent for locally advanced and metastatic PDAC, has limited efficacy, which is attributed to innate/acquired resistance and the activation of prosurvival pathways. Here, we investigated the in vitro efficacy of I-BET762, an inhibitor of the bromodomain and extraterminal (BET) family of proteins, in treating PDAC cell lines alone and in combination with gemcitabine (GEM). The effect of these two agents was also examined in xenograft PDAC tumors in mice. We found that I-BET762 induced cell cycle arrest in the G0/G1 phase and cell death and suppressed cell proliferation and metastatic stem cell factors in PDAC cells. In addition, the BH3-only protein Bim, which is related to chemotherapy resistance, was upregulated by I-BET762, which increased the cell death triggered by GEM in PDAC cells. Moreover, GEM and I-BET762 exerted a synergistic effect on cytotoxicity both in vitro and in vivo. Furthermore, Bim is necessary for I-BET762 activity and modulates the synergistic effect of GEM and I-BET762 in PDAC. In conclusion, we investigated the effect of I-BET762 on PDAC and suggest an innovative strategy for PDAC treatment. - Pancreatic ductal adenocarcinoma (PDAC) is the 12th most prevalent malignancy worldwide1. The prevalence of PDAC varies from six to eight people in every 100,000 men in developed countries1,2. Despite its comparatively low incidence, PDAC is the 4th most fatal malignancy1,3. The five-year PDAC-specific mortality is over ninety-five percent, resulting from its asymptomatic features in the early stage1,4. Diagnosis is often made at the terminal stage of PDAC4,5. Surgical excision is the desired strategy for treating PDAC, but the commonly late diagnosis renders the majority of PDAC cases inoperable5,6. Only twenty percent of patients receiving a diagnosis of PDAC qualify for surgery6. Metastasis is common even when an operation has been performed7. Contemporary radiotherapies and chemotherapies are usually ineffective in PDAC. Consequently, numerous studies have explored innovative therapeutic strategies targeting PDAC8?10. Fluorouracil (5-FU) has served as a conventional first-line drug for chemotherapy8. However, as another kind of nucleoside analog, gemcitabine (GEM) displays better efficacy and has replaced its predecessor as the standard drug for chemotherapy11,12. However, the five-year survival rates for patients with PDAC after surgical resection are only approximately twenty percent, even with GEM supplementation13. The bromodomain and extraterminal (BET) family participates in recognizing ?-N-acetylated lysine residues in histone tails14. As an essential member of the BET family that serves as a transcriptional coactivator, BET domain containing protein (BRD)-4 (BRD4) draws P-TEFb to chromatin, which undergoes acetylation14,15. Other mediators besides BRD4 co-occupy the promoters and enhancers of stimulated genes16. These mediators are enriched at numerous enhancer sequences, commonly named superenhancers17. Notably, the above mentioned enhancers modulate essential oncogene expression in various human malignancies, indicating the therapeutic application of BET bromodomain inhibitors14. In particular, the benzodiazepine JQ-1 was revealed to be effective against lymphoma, myeloma, and ALL, both in vivo and in vitro18,19. I-BET762 is a novel benzodiazepine compound that selectively binds the acetyl-recognizing BET pocket with nanomolar affinity20. I-BET762 has good pharmacological properties as an oral agent and inhibits the proliferation of myeloma cells, resulting in survival advantages in a systemic myeloma xenograft model21. I-BET762 is currently being used in phase I/II clinical trials for nuclear protein in testis (NUT) midline carcinoma and other cancers22. The effect of I-BET762 against ALL such as AML associated with mixed lineage leukemia was also previously reported in preclinical settings22. Previously study has shown that I-BET762 downregulates c-Myc, and dephosphorylation of ERK1/2 leading to proliferation inhibition in pancreatic cancer cells23. However, the influence of I-BET762 on PDAC is not well understood. In the present study, the influence of BET inhibitors together with GEM on PDAC was explored both in vitro and in vivo. Results The effect of I-BET762 on the cell death, survival, and cell cycle of PDAC cells. To determine the influence of BET inhibitors, PDAC cells were treated with JQ-1 and I-BET762. Both JQ-1 and I-BET762 remarkably decreased cell survival compared with that in the control group at 72 h (Fig.?1A). Both JQ-1 and I-BET762 noticeably suppressed DNA synthesis, as observed by EdU incorporation (Fig.?1B). Flow cytometry showed that both JQ-1 and I-BET762 triggered cell cycle arrest in HS766T, Panc-1, and BxPC-3 cells (Fig.?1C). Stimulation of cell death was analyzed by annexin V/PI staining to detect apoptosis in both the early and late stages. The results revealed that I-BET762 and JQ-1 noticeably triggered apoptosis in PDAC cells (Fig.?1D). Analyses of essential modulators of cell death, including PARP cleavage and caspase 3, were performed to verify the cell death induction observed in Fig.?1E. The BET inhibitors significantly promoted caspase 3 activation and PARP cleavage in PDAC cells. These findings indicate that I-BET762 suppressed proliferation and induced cell cycle arrest and death in PDAC cells. I-BET762 suppressed migration, invasion, and colony formation in PDAC cells. Subsequently, we examined the effect of I-BET762 in counteracting the in vitro migration and invasion of PDAC cells through functional evaluation. I-BET762 remarkably suppressed migration in BxPC-3 and Panc-1 PDAC cells compared to that in the control group (Fig.?2A and B). I-BET762 also significantly suppressed invasion in BxPC-3 and Panc-1 PDAC cells compared with that in the control group (Fig.?2C and D). Colony formation was evaluated in terms of one thousand cells seeded in 6-well plates. After cell attachment, the cells were treated with I-BET762. Colony formation was significantly suppressed in Panc-1 and BxPC-3 cells at 14 days (Fig.?2E and F), indicating that I-BET762 suppresses invasion, colony formation, and migration in PDAC cells. I-BET762 downregulated stem cell factors and decreased sphere generation in PDAC cells. The spheroid generation experiment was modified from previous studies. Two hundred cells in sphere-generating medium (1:1 DMEM/F12 medium containing B-27 and N-2; Invitrogen) were seeded in 24-well plates with ultralow adherent conditions. The cells were treated with I-BET762 for 14 days. The compounds and medium were renewed once. The generated spheres were then counted. As shown in Fig.?3A, I-BET762 noticeably reduced spheroid generation in Panc-1 cells. Analysis of the protein expression revealed remarkable downregulation of stem cell factors (Nanog, BMI-1, ?-catenin, and Oct-4) in Panc-1 cells treated with I-BET762 (Fig.?3B), which supports the proliferation-counteracting effect of I-BET762. The effect of GEM and I-BET762 on PDAC cells. Next, we investigated the combined effect of I-BET762 and GEM on PDAC cells. A CCK-8 assay demonstrated that the combination of GEM and I-BET762 displayed stronger cytotoxicity in 3 cell lines than did either compound alone due to a synergistic effect (Fig.?4A). Evaluation of apoptosis showed that I-BET762 enhanced the apoptotic effect induced by GEM (Fig.?4B). These findings indicated that combining I-BET762 with GEM might be a promising candidate for enhancing treatment efficacy compared with that of GEM treatment alone. Bim is required for I-BET762 function in PDAC. Next, we examined the mechanisms underlying the I-BET762-mediated apoptosis in PDAC. As shown in Fig.?5A, I-BET762 induced Bim and PUMA expression in PDAC. In contrast, I-BET762 treatment did not alter the expression of other Bcl-2 family members. Moreover, knockdown of PUMA did not abrogate the effect of the I-BET762 and GEM combination treatment. Therefore, next, we examined the role of Bim in I-BET762- and GEM-treated PDAC (Fig.?5B). The real-time PCR and western blotting results demonstrated remarkable upregulation of Bim mRNA and protein levels after I-BET762 treatment (Fig.?5C and D). Next, we generated Bim knockout Panc-1 cells using the CRISPR-Cas9 system (Fig.?5E). Bim knockout did not affect the I-BET762-induced suppression of migration and invasion (Fig.?5F and G). Furthermore, the synergistic effect of I-BET762 and GEM in PDAC cells was suppressed by Bim knockout (Fig.?5H and I). Our findings thus indicate that Bim is required for the I-BET762-induced apoptosis in PDAC and the effects of I-BET762 on cell migration and invasion are independent of its effects on cell viability. The effect of GEM and I-BET762 treatment on PDAC xenografts in mice. We then examined the necessity of the cell death modulated by Bim for the anticancer function of GEM and I-BET762 in xenograft mice. In Panc-1 tumor-bearing mice, GEM and I-BET762 decreased the tumor weight and volume. The combination of GEM and I-BET762 triggered a remarkable decline in tumor weight and volume compared with that of either agent alone (Fig.?6A). TUNEL and Ki67 assays indicated that I-BET762 and GEM induced less apoptosis when used alone than did the combination treatment (Fig.?6B and C). In contrast, compared with the parental tumors, Bim-KD tumors showed noticeably weaker growth suppression in response to the combination therapy (Fig.?6A?C). Furthermore, to evaluate the toxicity effects of I-BET762 and the combination of I-BET762 and GEM on mice, we measured ALT, AST and BUN levels after treatment. We found that I-BET762 did not influence the ALT or AST in serum samples or their GEM-induced elevation. BUN was not affected by any therapy mentioned above (Fig.?6D). Our study examines the essential influence of BET inhibitor on PDAC proliferation and demonstrates the efficacy of the innovative agent I-BET762 in PDAC. Finally, the combination of I-BET762 and GEM displays stronger cytotoxicity and is a promising approach for PDAC treatment, which requires further studies. Materials and Methods Cell culture. Human PDAC cells (HS766T, BxPC-3, and Panc-1) were obtained from ATCC and were cultured according to standard procedures in DMEM (Gibco, Carlsbad, CA, USA) with a high concentration of glucose, 10% FBS (Gibco, Carlsbad, CA, USA), and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Cells were grown in an atmosphere of 5% CO2 at 37 ?C. Cells were observed every week using phase contrast microscopy to ensure the logarithmic growth phase. GEM, JQ-1, and I-BET762 were obtained from SigmaAldrich (St. Louis, MO, USA). Cell proliferation assay. Cells were seeded in 96-well plates at a density of 1 ? 104 cells/well. After 24 h, the medium was replaced with serum-free medium supplemented with JQ-1 or I-BET762. Cytotoxicity and IC50 were evaluated using the CCK-8 cell proliferation assay. To measure the cytotoxicity of JQ-1 and I-BET762 at various time points, media without serum were renewed via complete media supplemented with JQ-1 or I-BET762. Cell cycle. The influence of JQ-1 and I-BET762 on the cell cycle was determined using flow cytometry. PDAC cells were treated with JQ-1 and I-BET762 for 24 h. The cells were then washed in cold PBS, stained with propidium iodide (PI), and analyzed by flow cytometry. Quantitative evaluation of the cell cycle was conducted using ModFit software (Verity Software House, Topsham, ME, USA). Western blotting. Western blotting was carried out as described previously39,40 using antibodies against BMI-1 (Santa Cruz Biotechnology, TX, USA), Bim, Nanog, Oct-4, cleaved PARP (Cell Signaling), cleaved caspase 3, ?-catenin, and ?-actin (Sigma-Aldrich). Cell motility. PDAC cells were cultured in 24-well plates to form a single layer. The cells were then incubated in medium without serum for 12 h. A scratch was made on the cell layer using a 200-?l pipette tip. The cells were then treated with specific agents and incubated for 12 h at 37 ?C in common medium. The cells were observed at time zero after treatment and at 12 h with a microscope to determine the migration distance. Invasion assay. Transwell inserts precoated with BME were incubated for 2 h at 37 ?C. Cells (1 ? 104) in serum-free medium were seeded in the uppermost chamber along with or without specific concentrations of the treatment compounds. The bottom chamber contained 1ml of culture medium with 10% FBS as a chemoattractant. The inserts were incubated for 24h at 37 ?C. The cells in the uppermost chamber were then removed with cotton swabs. The cells that migrated to the bottom were fixed in cold methanol and stained with 0.05% crystal violet. Colony formation assay. Cells were seeded in 6-well plates and allowed to adhere overnight. The cells were then treated with the test compounds at specific concentrations. The medium with the compounds was renewed every three days for fourteen days. The resulting colonies were then washed with PBS, followed by 30min of staining with 0.05% crystal violet. Quantification was carried out with the help of ImageJ Colony Counter. Every procedure was conducted no less than three times. The results are expressed as the means? SD. Sphere formation assay. To assess the capability of BET inhibitors to suppress pancreatic cancer (PC) stem cells, protocols from previous studies were used. In short, 24-well plates were used to seed the cell suspension in ultralow adherent conditions. Cells at a density of 200 cells/well were seeded in serum-free DMEM/F12 (1:1) containing N-2 and B27 (Life Technologies, Gaithersburg, MD). The cells were incubated for 10 days at 37?C before pancreatospheres were generated. The spheres were subsequently incubated for 14 days with or without I-BET762 in fresh medium. The generated pancreatospheres were quantified using light microscopy. CRISPR-cas9-mediated Bim knockout cells. To generate Bim knockout cells, two gRNA sequences targeting Bim were selected. Single-stranded complementary oligos with BsmBI overhangs were generated. The LentiCRISPR v2 (Addgene) lentiviral vector was digested using FastDigest BsmBI obtained from Fermentas. The digested product was purified using a QIAquick Gel Extraction Kit, followed by elution in EB buffer. Phosphorylation and annealing of the oligos were carried out using T4 polynucleotide kinase in T4 ligation buffer (NEB). The reaction system was incubated at 37?C for 30 min, followed by 90 ?C for 5 min, and then cooled to 25 ?C at a rate of 5 ?C/min. The ligation reaction was carried out by mixing the oligos to be annealed, the digested LentiCRISPR v2 vector, and the Quick Ligase enzyme included in the Quick Ligase Buffer before transformation into Stbl3 bacteria. 293 T cells (2 ? 106) were seeded on tissue culture plates (60 mm) at 24 h prior to transfection. Subsequently, 1 ? g of lentiviral products was mixed with pMD2G and psPAX plasmids and the PolyJet reagent in serum-free media. After 15 min of incubation at room temperature, the mixture was slowly added to the cells. Medium containing lentiviral particles was obtained after 2 days of transfection. For lentivirus infection, 6-well plates were seeded with Panc-1 cells (4?5 ? 104 cells/well). The infected cells were selected with puromycin at a concentration of 2 ? g/ml after 1 day of infection and then incubated at 37 ?C in 5% CO2. For selecting a single clone, the surviving cells were seeded on a 96-well plate. Western blotting was used to confirm the knockout. Apoptosis assay. Flow cytometry analysis was used to evaluate cell death. PDAC cells were treated with I-BET762, GEM or both for 24 h. The treated cells were washed with PBS and stained using the Annexin V/PI Apoptosis Kit (BD Biosciences; Franklin Lakes, USA). The stained cells were analyzed on a BD FACS Calibur flow cytometer with BD Cell Quest software. qRT-PCR. Total RNA was extracted and reverse transcribed using TRIzol Reagent (Invitrogen; Shanghai, China) and a Prime Script RT Kit (Dalian, People?s Republic of China; Takara Biotechnology), respectively. qRT-PCR was carried out on an ABI Prism 7900HT Real-Time System (Applied Biosystems Inc; Shanghai, China). The result is presented as Ct. Relative quantification of the target transcripts was performed using the ??Ct method to evaluate the associated alterations in expression. A control group without reverse transcription was included to exclude genomic DNA contamination. ?-Actin served as the internal reference gene. Animal models. All procedures and experiments involving animals in this study were approved by the Committee on the Ethics of Animal Experiments of Department of General Surgery, all methods were performed in accordance with the relevant guidelines and regulations, and a statement to this effect is included in the methods section. BALB/c nude mice (SLAC Laboratory Animal Co., Ltd., Shanghai, China) were subcutaneously injected with pancreatic cancer cells in their right flanks. When the tumor volume reached 150?200 mm3, 24 tumor-bearing mice were randomly divided into 4 groups (I-BET762, GEM, both, and control). The mice in the GEM group were injected with GEM (25 mg/kg/day) through the caudal vein every 3 days for 13 days, and those in the I-BET762 group received an intraperitoneal injection of I-BET762 (30 mg/kg/day) daily for 13 days. The mice in the combination group were treated with both I-BET762 (30 mg/kg/day) and GEM (25 mg/kg/day). In the control group, mice were treated with an equivalent amount of vehicle. Changes in body weight were monitored throughout the experiment. Tumor growth was measured every other day according to the following formula: tumor volume = length ? width2/2. Mice were sacrificed on day 22 of the treatment. The tumors were excised and weighed, and the tumor volume was measured. Finally, 0.5 ml of blood was drawn from every mouse by cardiac puncture and was sent to clinical laboratories to evaluate the hepatic and renal activities. TUNEL assay and immunohistochemical (IHC) examination. Tumor samples were fixed in 10% for malin prior to paraffin embedding, and sections of 4? m thickness were cut. Cell death in the tumors was evaluated using the In Situ Cell Death Detection Kit, POD (Roche Molecular Biochemicals; Indianapolis, USA) and was characterized by brown staining. For IHC examination, the sections were incubated with rabbit anti-human Ki67 (Sigma Aldrich, USA) (1:400) antibodies followed by incubation with HRP-conjugated anti-rabbit IgG antibodies, and the detection was performed using the Histostain-Plus Kit (Haoran-Bio; Shanghai, China). Finally, the sections were counterstained with hematoxylin. The negative control was incubated with PBS instead of a specific primary antibody. The assessment was conducted for 5 slices per tumor. Statistical analysis. Each experiment was performed no less than 3 times. The results are displayed as the means ? SD. Statistical analyses were conducted using Prism 5 (GraphPad, San Diego, CA, USA). The results were considered significant at P < 0.05. Author Contributions In this work, Fang X. and Huang Q. conceived the study and designed the experiments. Huang M., Lin X.S., Liu C.H., Liu Z., Meng F.T. and Wang C. contributed to the data collection, performed the data analysis and interpreted the results. Fang X. wrote the manuscript; Huang Q. contributed to the critical revision of the article. All authors read and approved the final manuscript. Additional Information Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article?s Creative Commons license, unless indicated otherwise in a credit line to the material. 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Fang Xie, Mei Huang, Xiansheng Lin, Chenhai Liu, Zhen Liu, Futao Meng, Chao Wang, Qiang Huang. The BET inhibitor I-BET762 inhibits pancreatic ductal adenocarcinoma cell proliferation and enhances the therapeutic effect of gemcitabine, Scientific Reports, 2018, DOI: 10.1038/s41598-018-26496-0