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The Broad Spectrum HDAC Inhibitor PCI-24781 Induces Caspase- and ROS-Dependent Apoptosis and is Synergistic with Bortezomib in Lymphoma
Get PDFWe investigated the cytotoxicity and biology of the novel broad-spectrum hydroxamic acid-based histone deacetylase inhibitor (HDACi), PCI-24781. PCI-24781 was studied alone and combined with bortezomib in Hodgkin lymphoma (L428) and non-Hodgkin's lymphoma cell lines (Ramos, HF1, SUDHL4). PCI-24781 induced dose-dependent apoptosis that was associated with prominent G0/G1 arrest, decreased S-phase, increased p21 protein expression, and production of reactive oxygen species (ROS). Furthermore, PCI-24781-induced apoptosis was shown to be ROS- and caspase-dependent. Combined PCI-24781 and bortezomib exposure resulted in strong synergistic apoptosis in all cell lines (combination indices 0.19-0.6). Furthermore, compared to either agent alone, PCI-24781/bortezomib resulted in increased caspase cleavage, mitochondrial depolarization, and histone hyperacetylation. Microarray analyses showed that PCI-24781 alone significantly downregulated several antioxidant genes, proteasome components, and NF-kappaB pathway genes, effects which were enhanced further with bortezomib. RT-PCR confirmed downregulation of NF-kappaB targets NF-kappaB1 (p105), c-Myc, and IkappaB-kinase subunits, while gel-shift showed decreased NF-kappaB DNA-binding activity. Taken together, these results suggest that increased oxidative stress and NF-kappaB inhibition, leading to caspase activation and apoptosis, are likely responsible for the activity of PCI-24781 as well as the observed synergy with bortezomib. These data indicate that PCI-24781 has potential therapeutic value in lymphoma as a single-agent and combined with bortezomib
The Molecular Mechanism of Histone Deacetylase (HDAC) Inhibitor PCI-24781 as an Anti-tumor Agent in Neuroblastoma Cells
No full text7－10％的兒科腫瘤是神經母細胞瘤, 並且它是最普遍的腦顱外的實體瘤。儘管使⽤用多個⾼高強度的治療⽅方案， 被診斷有⾼高危神經母細胞瘤病人的存活率仍然低於40％。 這更加突出了對於新的治療策略的迫切要求。組蛋⽩白去⼄乙酰化酶抑制劑是⼀一類新的有前景的抗腫瘤藥物， 它們在臨床上已經⽤用於許多類型的腫瘤的治療。PCI-24781 是⼀一種新型的屬於异羟肟酸家族的組蛋⽩白去⼄乙酰化酶抑制劑，對於癌症的治療俱有⾼高效率性和安全性。然⽽而，PCI-24781 對於神經母細胞瘤作⽤用的潛在機制仍然不是很清楚。本文的研究显⽰示了以⾮非常低剂量的PCI-24781 的處理顯著地抑制神經母細胞瘤的細胞株SK-N-DZ 的⽣生⾧長， ⽽而對於正常的細胞株HS-68 沒有影響。 但是，PCI-24781 都能增加這兩個細胞株的乙酰化的組蛋白H3 的水平。PCI-24781 的處理能誘導SK-N-DZ 細胞的細胞週期停滯在G2/M 期，並且產⽣生⼤大量的sub-G1的凋亡細胞。對於凋亡信號通路的分析發現PCI-24781 能激活外在的和內在的凋亡信號通路。被上調的和凋亡相關的蛋⽩白質包括DR4, p21, p53, caspase3 以及斷裂的 caspase3。進一步的蛋白質組學的分析顯示了在未處理和經過PCI-24781 處理的SK-N-DZ 細胞的不同的蛋⽩白表達譜。總共有42 個差異表達的蛋白被MALDI-TOF 質譜儀系統鑒定出來。功能性的歸類發現PCI-24781 影響了⼀一系列的細胞過程，包括細胞⾻骨架，能量代謝，DNA 修復， 細胞週期和轉錄的調控。免疫印跡的分析確證了五個候選蛋⽩白質在SK-N-DZ 細胞的表達，它們分別是prohibitin, hHR23a, RuvBL2, TRAP1 以及PDCD6IP。有趣的是，它們的表達譜在HS-68 細胞⾥里完全不同。PCI-24781 處理後，SK-N-DZ 的細胞保持高水平的hHR23a 和RuvBL2 蛋白質的表達，然而HS-68 的細胞抑制了它們的表達。這暗示了這兩個候選蛋白可能對於PCI-24781 誘導的細胞死亡中扮演着重要的角色。然⽽而，選擇性的沈默RuvBL2 基因，⽽而不是hHR23a 基因，減少了PCI-24781 引起的細胞死亡。總得來說，目前的結果強調了RuvBL2 基因在SK-N-DZ 細胞株中PCI-24781 抗腫瘤活性的重要性。這篇研究證實了組蛋⽩白去⼄乙酰化酶抑制劑在⾼高危神經母細胞瘤病⼈人中作為抗腫瘤藥物的價值，甚⾄至對於PCI-24781 在SK-N-DZ 細胞株中的潜在分子機制提供了一個新的思路。然而，要想深入理解正常細胞和癌細胞之間對於組蛋⽩白去⼄乙酰化酶抑制劑的不同敏感性的潛在機理，我們仍然需要大量的研究。Neuroblastoma accounts for 7-10% of all childhood cancers, and it is the most common extracranial solid tumor in childhood. The survival rate is less than 40% among children with high-risk neuroblastoma, despite of intensive multi-modality treatment, highlighting the urgent needs for new treatment strategies. Histone deacetylase (HDAC) inhibitors are a new class of promising anti-tumor agents that have been used in clinical trials for various tumor types. Among HDAC inhibitors, PCI-24781 is a novel hydroxamic acid that has high efficacy and safety for cancer treatment. However, the underlying mechanisms of PCI-24781 are not clearly elucidated, especially in neuroblastoma cells.In this study, I demonstrated that PCI-24781 treatment significantly inhibited tumor growth at very low doses in neuroblastoma cells SK-N-DZ, not in normal cell line HS-68. However, the accumulation of acetylated histone H3 can be found both in SK-N-DZ and HS-68 cell line in response to PCI-24781. Treatment with PCI-24781 also induced cell cycle arrest in G2/M phase and large amounts of sub-G1 cells in SK-N-DZ cells. Analysis of apoptosis signaling pathways found that both extrinsic and intrinsic pathways were activated by PCI-24781. The increased apoptotic proteins included DR4, p21, p53, caspase3 and cleaved caspase3.Different protein expression profiles were further revealed via proteomic analysis between SK-N-DZ cells with or without PCI-24781 exposure. MALDI-TOF MS system identified total 42 differentially expressed proteins. The functional classification revealed that PCI-24781 affected a series of cellular processes, including cytoskeleton organization, energy metabolism, DNA repair, cell cycle and transcriptional regulation. Western blotting confirmed the expression level of five candidate proteins in SK-N-DZ cells, including prohibitin, hHR23a, RuvBL2, TRAP1 and PDCD6IP. Interestingly, expression patterns of the five proteins were totally different in HS-68 cells.The high abundance of hHR23a and RuvBL2 was found in SK-N-DZ cells but not in HS-68 cells in response to PCI-24781, implying that these two proteins may be functionally important for PCI-24781-mediated cell death. However, selective knockdown of RuvBL2 not hHR23a rescued cells from PCI-24781-induced cell death. Collectively, my current results highlight the important role of RuvBL2 in anti-tumor activity of PCI-24781 in SK-N-DZ cells. The present study confirms the interest in HDAC inhibitors as anti-cancer agents for patients with high-risk neuroblastoma, and even provides a new insight into the potential mechanism of PCI-24781 in SK-N-DZ cell line. However, many efforts are still needed to elucidate differential sensitivity between cancer and normal cells in response to HDACinhibitors treatment.Zhan, Qinglei.Thesis Ph.D. Chinese University of Hong Kong 2013.Includes bibliographical references (leaves ).Abstracts also in Chinese.Title from PDF title page (viewed on …).Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only
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Histone deacetylase inhibitor (HDACI) PCI-24781 potentiates cytotoxic effects of doxorubicin in bone sarcoma cells
No full textTo better understand the mechanisms of cytotoxicity and cell death induced by HDACI PCI-24781 in bone sarcoma cells.
Four bone sarcoma cell lines were treated with PCI-24781, and the cytotoxicity was investigated. Further, accumulation of acetylated histones, p21, and PARP cleavage were evaluated in PCI-24781-treated cells. The synergistic effect of PCI-24781 to doxorubicin and its mechanism was investigated in bone sarcoma cells.
MTT assay demonstrated that the growth of bone sarcoma cells was inhibited after treatment with PCI-24781. Accumulation of acetylated histones, p21, and PARP cleavage were found in PCI-24781-treated cells. Expression of DNA repair protein RAD51 was inhibited, and the expression of apoptosis protein GADD45 alpha was induced by PCI-24781 in bone sarcoma cells. Bone sarcoma cells treated with PCI-24781 become more sensitive to doxorubicin. The caspase-3/7 activity was increased with doxorubicin and PCI-24781 treatment in these cells.
HDACI PCI-24781 has a synergistic effect on doxorubicin-induced apoptosis in bone sarcoma cells
PCI-24781 regulates AKT mRNA levels.
No full text<p>(<b>A</b>) Basal levels of AKT1, 2, and 3 mRNA in MCF7 cells are presented relative to AKT1 expression. (<b>B</b>) AKT1 and 2 mRNA expression was determined in MCF7 cells following treatment with increasing concentrations of PCI-24781 for 24 hours. (<b>C</b>) MCF7 cells were treated with 0.1 µM PCI-24781 (P), 5 µg/mL actinomycin D (Actino D), or the combination (P+Actino D) and evaluated for AKT1 mRNA expression after 0, 12, 24, and 48 hours treatment. (<b>D</b>) MCF7 cells were pretreated with vehicle or 10 µg/mL cycloheximide for 1 hour. Vehicle and cycloheximide pretreated cells were then each divided and treated with vehicle (V) or 0.1 µM PCI-24781 (P) with or without 10 µg/mL cycloheximide (CycloH) for 24 hours and evaluated for AKT1 mRNA expression. (<b>E</b>) MCF7 cells were treated with vehicle, 0.1 µM PCI-24781, 3 mM valproic acid (VPA), or 30 nM trichostatin A (TSA) for 24 hours and evaluated for AKT1 mRNA expression. All treatments were conducted in triplicate and expressed as the average with the error bars indicating the standard error of the mean. An (*) indicates a significant difference (P-value < 0.05) and a (#) an insignificant difference (P-value > 0.05) compared to vehicle or zero time treatment.</p
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Histone deacetylase inhibitor (HDACI) PCI-24781 enhances chemotherapy induced apoptosis in multidrug resistant sarcoma cell lines
No full textThe anti-tumor activity of histone deacetylase inhibitors (HDACI) on multi-drug resistant sarcoma cell lines has never been previously described. Four multidrug resistant sarcoma cell lines treated with HDACI PCI-24781 resulted in dose-dependent accumulation of acetylated histones, p21 and PARP cleavage products. Growth of these cell lines was inhibited by PCI-24781 at IC50 of 0.43 to 2.7. When we looked for synergy of PCI-24781 with chemotherapeutic agents, we found that PCI-24781 reverses drug resistance in all four multidrug resistant sarcoma cell lines and synergizes with chemotherapeutic agents to enhance caspase-3/7 activity. Expression of RAD51 (a marker for DNA double-strand break repair) was inhibited and the expression of GADD45α (a marker for growth arrest and DNA-damage) was induced by PCI-24781 in multidrug resistant sarcoma cell lines. In conclusion, HDACI PCI-24781 synergizes with chemotherapeutic drugs to induce apoptosis and reverses drug resistance in multidrug resistant sarcoma cell lines
RuvBL2 is involved in histone deacetylase inhibitor PCI-24781-induced cell death in SK-N-DZ neuroblastoma cells.
No full textNeuroblastoma is the second most common solid tumor diagnosed during infancy. The survival rate among children with high-risk neuroblastoma is less than 40%, highlighting the urgent needs for new treatment strategies. PCI-24781 is a novel hydroxamic acid-based histone deacetylase (HDAC) inhibitor that has high efficacy and safety for cancer treatment. However, the underlying mechanisms of PCI-24781 are not clearly elucidated in neuroblastoma cells. In the present study, we demonstrated that PCI-24781 treatment significantly inhibited tumor growth at very low doses in neuroblastoma cells SK-N-DZ, not in normal cell line HS-68. However, PCI-24781 caused the accumulation of acetylated histone H3 both in SK-N-DZ and HS-68 cell line. Treatment of SK-N-DZ with PCI-24781 also induced cell cycle arrest in G2/M phase and activated apoptosis signaling pathways via the up-regulation of DR4, p21, p53 and caspase 3. Further proteomic analysis revealed differential protein expression profiles between non-treated and PCI-24781 treated SK-N-DZ cells. Totally 42 differentially expressed proteins were identified by MALDI-TOF MS system. Western blotting confirmed the expression level of five candidate proteins including prohibitin, hHR23a, RuvBL2, TRAP1 and PDCD6IP. Selective knockdown of RuvBL2 rescued cells from PCI-24781-induced cell death, implying that RuvBL2 might play an important role in anti-tumor activity of PCI-24781 in SK-N-DZ cells. The present results provide a new insight into the potential mechanism of PCI-24781 in SK-N-DZ cell line
PCI-24781 induced G2/M cell cycle arrest and apoptosis in SK-N-DZ not HS-68 cells.
No full text<p>SK-N-DZ and HS-68 cells were treated with the increasing doses of PCI-24781 for 24 h (A and B), and different time points of 0.5 µM PCI-24781 as indicated (C and D), respectively. Cell cycle analysis was performed by propidium iodide staining method. Values are the mean of three independently experiments. Control: no treatment; PCI*: PCI-24781.</p
Schematic representation of the role of PCI-24781 in regulation of HIF-1α and autophagy.
No full text<p>PCI24781 enhances accumulation of HIF-1α in first 6hr of incubation resulting in induction of autophagy and cell survival (<b>early</b>). Longer incubation with PCI-24781 caused suppression of AKT activity and HIF-1α protein expression (<b>Late</b>) resulting in apoptosis. Autophagy inhibitors enhance PCI-24781 induced apoptosis. PCI-24781 might also activate AMPK and p27 mediated autophagy.</p
Abstract 5260: Bortezomib and HDAC inhibitor PCI-24781 combination therapy for neuroblastoma
No full textAbstract
Background: Neuroblastoma is an aggressive solid tumor with poor prognosis. Novel therapies are needed to help increase the survival and quality of life for affected children. It has been previously shown that bortezomib results in apoptosis in neuroblastoma both in vitro and in vivo. PCI-24781 is a novel pan-HDAC inhibitor which has been shown to be synergistic with bortezomib in lymphoma models. We hypothesize the combination of bortezomib with PCI-24781 would increase cytotoxicity to neuroblastoma.
Methods: Several established neuroblastoma cell lines as well as patient-derived primary neuroblastoma cultures were grown in 96-well plates and treated with bortezomib and PCI-24781, both separately and in combination for 48 hours. Cell viability was assessed by calcein AM assays and IC50's and drug synergy was determined at each cell line tested. mRNA was collected from drug treated cell lines and effects on cell signaling was evaluated using U133 mRNA expression arrays and Ingenuity analysis. Cell lysates prepared from drug treated cells were evaluated by western blot for caspase-3 and PARP cleavage. Nude mice were injected with 107 SMS-KCNR cells subcutaneously in the left flank. Mice were treated with daily doses of either 0.5 mg/kg bortezomib, 12.5 mg/kg PCI-24781, or the combination. Mice tumors were imaged using the IVIS lumina imager twice per week and caliper measurements were also obtained weekly until tumor max was reached.
Results: All neuroblastoma cell lines and patient cells tested showed sensitivity to bortezomib and PCI-24781 treatment in calcien AM cell viability assays with IC50's for bortezomib less than 50nM and IC50's for PCI-24781 less than 500nM. The combination of bortezomib and PCI-24781 was more cytotoxic than either drug alone. Cells treated with combined treatment showed an increase in caspase-3 and PARP activity confirming apoptosis. Expression analysis showed differential expression in the Notch and Wnt pathways between drug treated and vehicle treated cells. The xenograft models showed a significant decrease in tumor volume in mice treated with both bortezomib and PCI-24781 when compared to the single agent treatment groups or to the control group. Conclusions: Bortezomib and PCI-24781 inhibit neuroblastoma growth both in vitro and in vivo to a greater extent than either drug alone. The mechanism of action is currently being investigated further but certainly involves caspase-3 mediated apoptosis. This combination therapy appears to be effective and well tolerated in the mouse model and would be a novel therapy for neuroblastoma.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5260.</jats:p
PCI-24781 down-regulates EZH2 expression and then promotes glioma apoptosis by suppressing the PIK3K/Akt/mTOR pathway
No full textPCI-24781 is a novel histone deacetylase inhibitor that inhibits tumor proliferation and promotes cell apoptosis. However, it is unclear whether PCI-24781 inhibits Enhancer of Zeste 2 (EZH2) expression in malignant gliomas. In this work, three glioma cell lines were incubated with various concentrations of PCI-24781 (0, 0.25, 0.5, 1, 2.5 and 5 M) and analyzed for cell proliferation by the MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay and colony formation, and cell cycle and apoptosis were assessed by flow cytometry. The expression of EZH2 and apoptosis-related proteins was assessed by western blotting. Malignant glioma cells were also transfected with EZH2 siRNA to examine how PCI-24781 suppresses tumor cells. EZH2 was highly expressed in the three glioma cell lines. Incubation with PCI-24781 reduced cell proliferation and increased cell apoptosis by down-regulating EZH2 in a concentration-dependent manner. These effects were simulated by EZH2 siRNA. In addition, PCI-24781 or EZH2 siRNA accelerated cell apoptosis by down-regulating the expression of AKT, mTOR, p70 ribosomal protein S6 kinase (p70s6k), glycogen synthase kinase 3A and B (GSK3a/b) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). These data suggest that PCI-24781 may be a promising therapeutic agent for treating gliomas by down-regulating EZH2 which promotes cell apoptosis by suppressing the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of the rapamycin (mTOR) pathway
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