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Discovery and Characterization of R/S-N-3-Cyanophenyl-N'-(6-tert-butoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)urea, a New Histone Deacetylase Class III Inhibitor Exerting Antiproliferative Activity Against Cancer Cell Lines
... Research teams have picked different zebrafish wild-type stains as well as transgenic zebrafish strains with DNA fragments embedded in their genome. From the papers reviewed [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] research teams have raised wild-type zebrafish until the desired developmental stage before GBM cells xenotransplantation. Among the wild-type strains Tupfel long fin (TL) (n = 2) [22,23], AB (n = 6) [24-26, 28, 29, 33], AB/Tübingen (AB/TU) (n = 1) [30], Tübingen (TU) (n = 1) [31], and tropical 5D (T5D) (n = 1) [32] have been used. ...
... Another strategy that has been employed for the zebrafish xenograft models concerned mixing GBM cells (U251 or U87) with HMC3 microglia cells in 2:1 ratio [33] or GBM cells (U373 or U87) with MSCs in 1:1 ratio. Other cell lines that have been utilized in GBM zebrafish xenograft model research include U343 (n = 2) [17,22], U373 (n = 3) [21,25,54], BTL1528 and FGFR4-KD [39], the ATCC® CRL-1718™ human astrocytoma cell line [19], D54-MG [45], DBTRG and SJ-GBM2 [37], and the HS683 oligodendroglial cell line [21]. ...
... Another strategy that has been employed for the zebrafish xenograft models concerned mixing GBM cells (U251 or U87) with HMC3 microglia cells in 2:1 ratio [33] or GBM cells (U373 or U87) with MSCs in 1:1 ratio. Other cell lines that have been utilized in GBM zebrafish xenograft model research include U343 (n = 2) [17,22], U373 (n = 3) [21,25,54], BTL1528 and FGFR4-KD [39], the ATCC® CRL-1718™ human astrocytoma cell line [19], D54-MG [45], DBTRG and SJ-GBM2 [37], and the HS683 oligodendroglial cell line [21]. ...
Glioblastoma (GBM) constitutes the most common primary brain tumor in adults. The challenges in GBM therapeutics have shed light on zebrafish used as a promising animal model for preclinical GBM xenograft studies without a standardized methodology. This systematic review aims to summarize the advances in zebrafish GBM xenografting, compare research protocols to pinpoint advantages and underlying limitations, and designate the predominant xenografting parameters. Based on the PRISMA checklist, we systematically searched PubMed, Scopus, and ZFIN using the keywords “glioblastoma,” “xenotransplantation,” and “zebrafish” for papers published from 2005 to 2022, available in English. 46 articles meeting the review criteria were examined for the zebrafish strain, cancer cell line, cell labeling technique, injected cell number, time and site of injection, and maintenance temperature. Our review designated that AB wild-type zebrafish, Casper transparent mutants, transgenic Tg(fli1:EGFP), or crossbreeding of these predominate among the zebrafish strains. Orthotopic transplantation is more commonly employed. A number of 50–100 cells injected at 48 h post-fertilization in high density and low infusion volume is considered as an effective xenografting approach. U87 cells are used for GBM angiogenesis studies, U251 for GBM proliferation studies, and patient-derived xenograft (PDX) to achieve clinical relevance. Gradual acclimatization to 32–33 °C can partly address the temperature differential between the zebrafish and the GBM cells. Zebrafish xenograft models constitute valuable tools for preclinical studies with clinical relevance regarding PDX. The GBM xenografting research requires modification based on the objective of each research team. Automation and further optimization of the protocol parameters could scale up the anticancer drug trials.
... Schnekenburger and colleagues showed that their newly synthesized Compound 18 (identified via in silico screening, see Section 3.8) inhibits SIRT-1 and SIRT-2 with an antiproliferative effect in glioma cells, both in in vitro and in vivo settings [197]. ...
... 2016 [244] Ligand-based virtual screening and inductive learning for identification of SIRT-1 inhibitors in natural products 2015 [225] Ligand and structure-based approaches for the identification of SIRT-1 activators. 2017 [197] Discovery and Characterization of R/S-N-3-Cyanophenyl-N'- (6-tertbutoxycar ...
... The in-house library was screened against the X-ray crystal structure of SIRT-1 (PDB code = 4I5I) [230] and SIRT-2 (PDB code = 4RMG) [112], using AutoDock Vina [301]. The compound showed an anti-proliferative effect in glioma cells, both in in vitro and in vivo settings (see Section 2.8.1) [197]. ...
Sirtuins are NAD+-dependent deac(et)ylases with different subcellular localization. The sirtuins’ family is composed of seven members, named SIRT-1 to SIRT-7. Their substrates include histones and also an increasing number of different proteins. Sirtuins regulate a wide range of different processes, ranging from transcription to metabolism to genome stability. Thus, their dysregulation has been related to the pathogenesis of different diseases. In this review, we discussed the pharmacological approaches based on sirtuins’ modulators (both inhibitors and activators) that have been attempted in in vitro and/or in in vivo experimental settings, to highlight the therapeutic potential of targeting one/more specific sirtuin isoform(s) in cancer, neurodegenerative disorders and type 2 diabetes. Extensive research has already been performed to identify SIRT-1 and -2 modulators, while compounds targeting the other sirtuins have been less studied so far. Beside sections dedicated to each sirtuin, in the present review we also included sections dedicated to pan-sirtuins’ and to parasitic sirtuins’ modulators. A special focus is dedicated to the sirtuins’ modulators identified by the use of virtual screening.
... Because BZD9L1 possesses intrinsic fluorescence, the cellular distribution of BZD9L1 in HCT-116 and CCD18 colon fibroblasts could be detected using fluorescence microscopy (Yoon et al., 2015). N-aryl-N'-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl) ureas-derived Compound 18 simultaneously inhibited SIRT1 and SIRT2 with IC 50 of 6.2 and 4.2, respectively (Schnekenburger et al., 2017). In U373 and Hs683 glioblastoma, treatment of Compound 18 increased acetylation of histone H4 and α-tubulin (Schnekenburger et al., 2017). ...
... N-aryl-N'-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl) ureas-derived Compound 18 simultaneously inhibited SIRT1 and SIRT2 with IC 50 of 6.2 and 4.2, respectively (Schnekenburger et al., 2017). In U373 and Hs683 glioblastoma, treatment of Compound 18 increased acetylation of histone H4 and α-tubulin (Schnekenburger et al., 2017). ...
... In U373 and Hs683 glioma cells, treatment of Compound 18 increased acetylation levels of histone H4, histone H3K56, and α-tubulin, which confirmed cellular inhibition of SIRT1 and SIRT2 (Schnekenburger et al., 2017). Consistent with the SIRT1 and SIRT2 knockdown results, Compound 18 impaired cell proliferation of U373 and Hs683 cells. ...
Sirtuins use NAD+ to remove various acyl groups from protein lysine residues. Through working on different substrate proteins, they display many biological functions, including regulation of cell proliferation, genome stability, metabolism, and cell migration. There are seven sirtuins in humans, SIRT1-7, each with unique enzymatic activities, regulatory mechanisms, subcellular localizations, and substrate scopes. They have been indicated in many human diseases, including cancer, neurodegeneration, microbial infection, metabolic and autoimmune diseases. Consequently, interests in development of sirtuin modulators have increased in the past decade. In this brief review, we specifically summarize genetic and pharmacological modulations of sirtuins in cancer, neurological, and cardiovascular diseases. We further anticipate this review will be helpful for scrutinizing the significance of sirtuins in the studied diseases.
... For instance, the SIRT1/2 inhibitor sirtinol triggers growth arrest in human breast and lung cancer cells [14]. Similarly, the benzopyran-based compound 18 inhibits SIRT1/2 activities and acts as a cytostatic agent in glioblastoma cells [15], whereas the dual SIRT1/2 inhibitor tenovin-1 induces apoptosis in sarcoma cells [16]. Other specific sirtuin inhibitors (SIRTi) have been reported to trigger cancer cell death, including the SIRT1 inhibitors NCO-01 and -04, and the SIRT2 inhibitors AEM1 and AEM2, which induce cell death in T-cell leukemia/lymphoma [17] and in non-small cell lung cancer [18], respectively. ...
... The natural SIRT inhibitor nicotinamide was also included as a reference compound for in vitro SIRT3 inhibition [34]. Besides suramin, additional references SIRT1/2, SIRT1, and SIRT2 inhibitors, namely, sirtinol, EX-527, and AGK2, respectively, performed in a similar range or less efficiently [15]. To further explore the potential of eurochevalierine as an SIRT inhibitor, we compared the docking affinity between eurochevalierine and known reference inhibitors (sirtinol, EX-527, and AGK2) using AutoDock Vina [35]. ...
... Docking studies were performed as previously described [15] with a docking grid size of 25 Å × 25 Å × 25 Å, which encompassed the entire inhibitor binding pocket of SIRT1 and 2 structures. To prepare the SIRT coordinates for docking simulation, ligands and all water molecules were removed except static water molecules in the binding site that could play important roles in the actual inhibition. ...
NAD+-dependent histone deacetylases (sirtuins) are implicated in cellular processes such as proliferation, DNA repair, and apoptosis by regulating gene expression and the functions of numerous proteins. Due to their key role in cells, the discovery of small molecule sirtuin modulators has been of significant interest for diverse therapeutic applications. In particular, it has been shown that inhibition of sirtuin 1 and 2 activities is beneficial for cancer treatment. Here, we demonstrate that the fungal metabolite eurochevalierine from the fungus Neosartorya pseudofischeri inhibits sirtuin 1 and 2 activities (IC50 about 10 µM) without affecting sirtuin 3 activity. The binding modes of the eurochevalierine for sirtuin 1 and 2 have been identified through computational docking analyses. Accordingly, this sequiterpene alkaloid induces histone H4 and α-tubulin acetylation in various cancer cell models in which it induces strong cytostatic effects without affecting significantly the viability of healthy PBMCs. Importantly, eurochevalierine targets preferentially cancer cell proliferation (selectivity factor ≫ 7), as normal human primary CD34+ stem/progenitor cells were less affected by the treatment. Finally, eurochevalierine displays suitable drug-likeness parameters and therefore represent a promising scaffold for lead molecule optimization to study the mechanism and biological roles of sirtuins and potentially a basis for development into therapeutics.
... The modulation of EMT by SIRT1 has been shown to have an effect on breast cancer stem cells (BCSCs) through the SIRT1-mediated activation of the Wnt signaling pathway [7]. In several studies, SIRT1 inhibitors have been reported to exhibit both in vitro and in vivo anticancer activity [8,9,10], enhance the sensitivity of multidrug-resistant cells to chemotherapeutic agents, and inhibit proliferation of drug-resistant cancer cells [9,10,11]. Inhibition of SIRT1 has been shown to block the Wnt signaling cascade, decreasing the stemness of cancer stem cells (CSCs) and reducing EMT, which leads to a subsequent reduction in metastasis [7,12,13,14]. ...
... The modulation of EMT by SIRT1 has been shown to have an effect on breast cancer stem cells (BCSCs) through the SIRT1-mediated activation of the Wnt signaling pathway [7]. In several studies, SIRT1 inhibitors have been reported to exhibit both in vitro and in vivo anticancer activity [8,9,10], enhance the sensitivity of multidrug-resistant cells to chemotherapeutic agents, and inhibit proliferation of drug-resistant cancer cells [9,10,11]. Inhibition of SIRT1 has been shown to block the Wnt signaling cascade, decreasing the stemness of cancer stem cells (CSCs) and reducing EMT, which leads to a subsequent reduction in metastasis [7,12,13,14]. An approach that targets SIRT-mediated EMT and CSCs renewal through SIRT1 modulation, as an adjuvant to conventional chemotherapy, has been proposed as a potential strategy for overcoming multidrug resistance in breast cancer [7]. ...
Isoflavone derivatives were prepared from benzoylbenzofuran precursors. The synthesized compounds were analyzed by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, as well as high-resolution mass spectrometry (HRMS) to confirm their structures. The benzoylbenzofuran and isoflavone analogues were evaluated for inhibition of sirtuin 1 (SIRT1) and cell proliferation in MDA-MB-231 triple-negative breast cancer (TNBC) cells. Several isoflavone and benzoylbenzofuran derivatives exhibited potent antiproliferative effects against the MDA-MB-231 cancer cell line. Most of the isoflavone derivatives attenuated SIRT1 activity to below 50%. The most active compounds were the isoflavone quinones 38, 39, and 40, at IC50 values of 5.58 ± 0.373, 1.62 ± 0.0720, and 7.24 ± 0.823 μM, respectively. Importantly, the most active compound, 6-methoxy-4',6'-dimethylisoflavone-2',5'-quinone (39) displayed SIRT1 inhibitory activity comparable to that of the reference compound, suramin. The in silico docking simulations in the active site of SIRT1 further substantiated the experimental results and explored the binding orientations of potent compounds in the active site of the target.
... The new series bearing an alkoxycarbonylamino group at the C-6-position were synthesized and examined as anticancer agents targeting sirtuins. With the help of computational docking and in vitro SIRT1/2 inhibition assays, Michael's team discovered compound 6A-18 (107) with potent antiproliferative activity on various glioma cells [110]. ...
... Sacconnay et al. identified the 5-benzylidene-hydantoin derivatives (110)(111)(112)(113) as novel sirtuins inhibitors by virtual screening. 5-B-97 (112) shows relatively low cytotoxicity and is passively absorbed. ...
Sirtuins (SIRT) are coenzyme NAD+-dependent histone deacetylases for the transfer of modified acetyl groups. Sirtuins are widely involved in various physiological processes and therefore associated with cardiovascular disease, diabetes, Parkinson's disease, cancer and beyond. Consequently, the development of modulators for sirtuins has considerable clinical value. To date, a variety of SIRT1/2 inhibitors have been reported and none has been approved for the market. This review summarizes the recent progress in the discovery and development of SIRT1/2 inhibitors including their inhibitory potency, structure-activity relationship and binding mode analysis as well as discusses the perspective for the future development of SIRT1/2 inhibitors.
... In 2017, Schnekenburger et al. identified a new class III HDAC inhibitor, R/S-N-3-cyanophenyl-N′-(6-tert-butoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)urea, as a potent anti-glioma agent. 55 Previously, the authors disclosed some compounds derived from cromakalim (47, an ATP-sensitive potassium (KATP) channel opener) containing an arylurea or arylthiourea moiety at the 4-position (48,49) that showed anti-glioma activity potential. Given the above, the authors further investigated the amplified benefits in GBM and accordingly synthesized a new series of compounds. ...
Glioblastoma (GBM) is a highly malignant brain tumor characterized by a heterogeneous population of genetically unstable and highly infiltrative cells that are resistant to chemotherapy. Although substantial efforts have been invested in the field of anti-GBM drug discovery in the past decade, success has primarily been confined to the preclinical level, and clinical studies have often been hampered due to efficacy-, selectivity-, or physicochemical property-related issues. Thus, expansion of the list of molecular targets coupled with a pragmatic design of new small-molecule inhibitors with central nervous system (CNS)-penetrating ability is required to steer the wheels of anti-GBM drug discovery endeavors. This Perspective presents various aspects of drug discovery (challenges in GBM drug discovery and delivery, therapeutic targets, and agents under clinical investigation). The comprehensively covered sections include the recent medicinal chemistry campaigns embarked upon to validate the potential of numerous enzymes/proteins/receptors as therapeutic targets in GBM.
... Protein arginine methyltransferase 5 (PRMT5) inhibitors, which regulate gene expression by methylation of histonic and non-histonic proteins, improved survival after microinjection of GBM human derived cells expressing GFP in the midbrain-hindbrain of 36hpf casper zebrafish [180]. Schnekenburger et al. synthetized and tested inhibitors of HDAC and sirtuin (SIRT) in GBM-xenografts, showing their efficacy in preventing tumor growth [181]. An interesting pharmacological approach has been based on the following observation by Pudelko et al.: human mutT homologue 1 (MTH1), an enzyme responsible for degrading oxidized nucleotides-which have high oxidative pressure and protect tumor cells-is up-regulated in GBM and related to a poor prognosis [184]. ...
Glioblastoma (GBM) is the most common of all brain malignant tumors; it displays a median survival of 14.6 months with current complete standard treatment. High heterogeneity, aggressive and invasive behavior, the impossibility of completing tumor resection, limitations for drug administration and therapeutic resistance to current treatments are the main problems presented by this pathology. In recent years, our knowledge of GBM physiopathology has advanced significantly, generating relevant information on the cellular heterogeneity of GBM tumors, including cancer and immune cells such as macrophages/microglia, genetic, epigenetic and metabolic alterations, comprising changes in miRNA expression. In this scenario, the zebrafish has arisen as a promising animal model to progress further due to its unique characteristics, such as transparency, ease of genetic manipulation, ethical and economic advantages and also conservation of the major brain regions and blood–brain–barrier (BBB) which are similar to a human structure. A few papers described in this review, using genetic and xenotransplantation zebrafish models have been used to study GBM as well as to test the anti-tumoral efficacy of new drugs, their ability to interact with target cells, modulate the tumor microenvironment, cross the BBB and/or their toxicity. Prospective studies following these lines of research may lead to a better diagnosis, prognosis and treatment of patients with GBM.
... KBM-5 cells and an imatinibresistant K-562 (K-562R) subline were gifts from Dr. Bharat B. Aggarwal and from the Catholic University of Seoul, respectively. Peripheral blood mononuclear cells (PBMCs) from healthy adult human donors authorization LBMCC-2019-0002: Assessment of toxicity of new drugs or drug combinations in preclinical development in nonproliferating peripheral blood mononuclear cells (systemic acute toxicity)" were isolated from blood obtained from the Red Cross (Luxembourg, Luxembourg), purified and induced to proliferate using a mitogen-cytokine cocktail supplement whose composition included phytohemagglutinin (PHA; Millipore, Darmstadt, Germany) and interleukin 2 (Miltenyi Biotec, Utrecht, The Netherlands) as previously reported [24,25]. Human primary hematopoietic cluster of differentiation (CD)34 + progenitor/stem cells were isolated as previously reported [26]. ...
Despite the discovery of tyrosine kinase inhibitors (TKIs) for the treatment of breakpoint cluster region-Abelson (BCR-ABL)⁺ cancer types, patients with chronic myeloid leukemia (CML) treated with TKIs develop resistance and severe adverse effects. Combination treatment, especially with a histone deacetylase (HDAC) 6 inhibitor (HDAC6i), appears to be an attractive option to prevent TKI resistance, considering the potential capacity of an HDAC6i to diminish BCR-ABL expression. We first validated the in vivo anti-cancer potential of the compound 7b by significantly reducing the tumor burden of BALB/c mice xenografted with K-562 cells, without notable organ toxicity. Here, we hypothesize that the HDAC6i compound 7b can lead to BCR-ABL downregulation in CML cells and sensitize them to TKI treatment. The results showed that combination treatment with imatinib and 7b resulted in strong synergistic caspase-dependent apoptotic cell death and drastically reduced the proportion of leukemia stem cells, whereas this treatment only moderately affected healthy cells. Ultimately, the combination significantly decreased colony formation in a semisolid methylcellulose medium and tumor mass in xenografted zebrafish compared to each compound alone. Mechanistically, the combination induced BCR-ABL ubiquitination and downregulation followed by disturbance of key proteins in downstream pathways involved in CML proliferation and survival. Taken together, our results suggest that an HDAC6i potentiates the effect of imatinib and could overcome TKI resistance in CML cells.
... Treatment of U87MG GBM cells with tenovin-1 and EX527, small-molecule sirtuin inhibitors, increased p53 expression and induced cellular senescence [58]. Another sirtuin inhibitor, R/S-N-3-cyanophenyl-N′-(6-tert-butoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)urea, has been shown to induce G1 cell cycle arrest and positive SaβGal staining in U373 GBM cell line [36]. HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) at 1 μM and 2.5 μM concentrations inhibited U87MG cell growth via cell cycle arrest and p53-, p38-mediated senescence [16]. ...
Glioblastoma multiforme, the most aggressive glioma, is a tumor characterized by frequent recurrence and unfavorable prognosis. There is growing literature concerning therapy-induced senescence in glioblastoma, with many compounds found to exert a senescence-promoting effect, offering new potential treatment possibilities. However, some studies indicate that senescence and senescence-associated secretory phenotype may play a role in glioblastoma progression and recurrence. Taken together, those findings underscore the importance of senescence in glioblastoma pathophysiology and show the need for the development of novel senescence-associated approaches , such as senescence-associated secretory phenotype reprogramming and two-step senescence-fo-cused cancer therapy. Streszczenie: Glejak wielopostaciowy, najbardziej agresywna postać glejaka, charakteryzuje się skłonnością do częstych nawrotów oraz niekorzystnym rokowaniem. W ostatnich latach opub-likowano wiele badań dotyczących terapii wywołującej starzenie komórkowe w komórkach glejaka, co daje nadzieję na opracowanie nowych sposobów leczenia tego nowotworu. Z drugiej strony, niek-tóre badania wskazują na związek starzenia komórkowego z progresją glejaka oraz ze skłonnością do nawrotów. Podsumowując, analiza najnowszych doniesień podkreśla, że starzenie komórkowe pełni ważną rolę w patofizjologii glejaka wielopostaciowego, a także pokazuje potrzebę opracowa-nia nowych podejść terapeutycznych związanych ze starzeniem, takich jak przeprogramowywanie fenotypu wydzielniczego komórek starzejących czy dwustopniowa terapia starzeniowa. Słowa kluczowe: glejak niedojrzały, nowotwory-farmakoterapia, wznowa miejscowa nowotworu 24 M. KOTAS ET AL
... Docking studies were carried out as previously reported [54] using the Protein Data Bank (PDB) codes 4BKX, 4LY1, 4A69, 2VQM, 5EDU, 3C10, and 3EW8 corresponding to HDAC1, HDAC2, HDAC3, HDAC4, HDAC6, HDAC7, and HDAC8, respectively. MAKV-6, MAKV-7, MAKV-8, MAKV-10, and MAKV-12, and SAHA were drawn using ChemDraw Professional software version 15.0 (PerkinElmer Informatics). ...
Background:
Chronic myeloid leukemia (CML) pathogenesis is mainly driven by the oncogenic breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (BCR-ABL) fusion protein. Since BCR-ABL displays abnormal constitutive tyrosine kinase activity, therapies using tyrosine kinase inhibitors (TKis) such as imatinib represent a major breakthrough for the outcome of CML patients. Nevertheless, the development of TKi resistance and the persistence of leukemia stem cells (LSCs) remain barriers to cure the disease, justifying the development of novel therapeutic approaches. Since the activity of histone deacetylase (HDAC) is deregulated in numerous cancers including CML, pan-HDAC inhibitors may represent promising therapeutic regimens for the treatment of CML cells in combination with TKi.
Results:
We assessed the anti-leukemic activity of a novel hydroxamate-based pan-HDAC inhibitor MAKV-8, which complied with the Lipinski's "rule of five," in various CML cells alone or in combination with imatinib. We validated the in vitro HDAC-inhibitory potential of MAKV-8 and demonstrated efficient binding to the ligand-binding pocket of HDAC isoenzymes. In cellulo, MAKV-8 significantly induced target protein acetylation, displayed cytostatic and cytotoxic properties, and triggered concomitant ER stress/protective autophagy leading to canonical caspase-dependent apoptosis. Considering the specific upregulation of selected HDACs in LSCs from CML patients, we investigated the differential toxicity of a co-treatment with MAKV-8 and imatinib in CML versus healthy cells. We also showed that beclin-1 knockdown prevented MAKV-8-imatinib combination-induced apoptosis. Moreover, MAKV-8 and imatinib co-treatment synergistically reduced BCR-ABL-related signaling pathways involved in CML cell growth and survival. Since our results showed that LSCs from CML patients overexpressed c-MYC, importantly MAKV-8-imatinib co-treatment reduced c-MYC levels and the LSC population. In vivo, tumor growth of xenografted K-562 cells in zebrafish was completely abrogated upon combined treatment with MAKV-8 and imatinib.
Conclusions:
Collectively, the present findings show that combinations HDAC inhibitor-imatinib are likely to overcome drug resistance in CML pathology.
... [26][27][28][29][30][31][32] Genetic and pharmacologic inhibition of SIRT1 activity arrests tumor growth and restores proper apoptotic signaling. [32][33][34][35] SIRT1 is required for oncogenic transformation of neural stem cells and for the survival of "cancer cells with neural stemness" in a p53-dependent manner. 15 Upregulation of SIRT1/PGC-1α is associated with increased chemo-and radioresistance of glioblastoma stem cell clones, 36 while the shRNA-induced knockdown of SIRT1 expression enhances the effectiveness of radiotherapy by inhibiting tumor growth in CD133+ GBM xenografts in mice. ...
Background
Several studies demonstrated that glioblastoma multiforme progression and recurrence is linked to epigenetic regulatory mechanisms. Sirtuin 1 (SIRT1) plays an important role in glioma progression, invasion, and treatment response, and is a potential therapeutic target. The aim of this study is to test the feasibility of 2-[18F]BzAHA for quantitative imaging of SIRT1 expression-activity and monitoring pharmacologic inhibition in a rat model of intracerebral glioma.
Methods
Sprague Dawley rats bearing 9L (N=12) intracerebral gliomas were injected with 2-[18F]BzAHA (300-500 µCi/animal i.v.) and dynamic PET imaging was performed for 60 minutes. Then, SIRT1 expression in 9L tumors (N=6) was studied by immunofluorescence microscopy (IFM). Two days later, rats with 9L gliomas were treated either with SIRT1 specific inhibitor EX-527 (5 mg/kg, i.p; N=3) or with HDACs class IIa specific inhibitor MC1568 (30 mg/kg, i.p.; N=3) and 30 minutes later were injected i.v. with 2-[18F]BzAHA. PET/CT/(MR) images acquired after EX-527 and MC1568 treatments were co-registered with baseline images.
Results
Standard uptake values (SUV) of 2-[18F]BzAHA in 9L tumors measured at 20 min post radiotracer administration were 1.11±0.058 for SUV and had an tumor-to-brainstem SUV ratio of 2.73±0.141. IFM of 9L gliomas revealed heterogeneous upregulation of SIRT1, especially in hypoxic and perinecrotic regions. Significant reduction in 2-[18F]BzAHA SUV and distribution volume in 9L tumors was observed after administration of EX-527, but not MC1568.
Conclusions
Thus, PET/CT/(MR) with 2-[18F]BzAHA can facilitate studies to elucidate the roles of SIRT1 in gliomagenesis and progression and to optimize therapeutic doses of novel SIRT1 inhibitors in gliomas.
... The therapeutic efficacy of 129 was confirmed in 3D glioblastoma spheroids and in zebrafish xenografts. 237 Prostate-specific membrane antigen (PSMA) is a well-known biomarker for prostate cancer. 238 PSMA expression in prostate cancer is generally 10−100-fold higher than in healthy prostate tissue. ...
The urea functionality is inherent to numerous bioactive compounds, including a variety of clinically approved therapies. Urea containing compounds are increasingly used in medicinal chemistry and drug design in order to establish key drug-target interactions and fine-tune crucial drug-like properties. In this perspective, we highlight physicochemical and conformational properties of urea derivatives. We provide outlines of traditional reagents and chemical procedures for the preparation of ureas. Also, we discuss newly developed methodologies mainly aimed at overcoming safety issues associated with traditional synthesis. Finally, we provide a broad overview of urea-based medicinally relevant compounds, ranging from approved drugs to recent medicinal chemistry developments.
The scientific community, recently, has focused notable attention on the chemopreventive and therapeutic effects of dietary polyphenols for human health. Emerging evidence demonstrates that polyphenols, flavonoids and vitamins counteract and neutralize genetic and environmental stressors, particularly oxidative stress and inflammatory process closely connected to cancer initiation, promotion and progression Interestingly, polyphenols can exert antioxidant or pro-oxidant cytotoxic effects which depend on their endogenous concentration. Notably, polyphenols at high dose act as pro-oxidants in a wide type of cancer cells by inhibiting Nrf2 pathway and the expression of antioxidant vitagenes such as NAD(P)H-quinone oxidoreductase (NQO1), glutathione transferase (GT), GPx, heme oxygenase-1 (HO-1), sirtuin-1 (Sirt1) and thioredoxin (Trx) system which play an essential role in the metabolism of reactive oxygen species (ROS), detoxification of xenobiotics and inhibition of cancer progression by inducing apoptosis and cell cycle arrest according to the hormesis approach. Importantly, mutagenesis of Nrf2 pathway can exacerbate its “dark side” role representing a crucial event in the initiation stage of carcinogenesis. Herein, we review the hormetic effects of polyphenols and nanoincapsulated-polyphenols in chemoprevention and treatment of brain tumors via activation or inhibition of Nrf2/vitagenes to suppress carcinogenesis in the early stages and inhibit its progression. Lastly, we discussed innovative preclinical approaches through mini-brain tumor organoids to study human carcinogenesis from basic cancer research to clinical practice as promising tools to recapitulate the arrangement of structural neuronal tissues and biological functions of the human brain as well as test drug toxicity and drive personalized and precision medicine in brain cancer.
HDACs as important targets for cancer therapy have attracted extensive attentions. In this work, a series of sixteen hydroxamic acid based HDAC inhibitors were designed and synthesized with 4,5,6,7-tetrahydrobenzothiazole as the structural core. Majority of them exhibited potent inhibitory activities against HDACs and one leading compound 6h was dug out. 6h was proven to be a pan-HDAC inhibitor and displayed high cytotoxicity against seven human cancer cell lines with IC50 values in low micromolar range. 6h could arrest cell cycle in G2/M phase and induce apoptosis in A549 cells. Moreover, compound 6h exhibited remarkable anti-migration and anti-angiogenesis activities. At the same time, 6h was able to elevate the expression of acetylated α-tubulin and acetylated histone H3 in a dose-dependent manner. Docking simulation revealed that 6h fitted well into the active sites of HDAC2 and 6. Finally, compound 6h also exerted potent antitumor effects in an A549 zebrafish xenograft model. Our study demonstrated that compound 6h was a promising candidate for further preclinical studies.
A cellular model of cardiomyocytes (H9c2 cell line) and mitochondria isolated from mouse liver were used to understand the drug action of BPDZ490 and BPDZ711, two benzopyran analogues of the reference potassium channel opener cromakalim, on mitochondrial respiratory parameters and swelling, by comparing their effects with those of the parent compound cromakalim. For these three compounds, the oxygen consumption rate (OCR) was determined by high-resolution respirometry (HRR) and their impact on adenosine triphosphate (ATP) production and calcium-induced mitochondrial swelling was investigated. Cromakalim did not modify neither the OCR of H9c2 cells and the ATP production nor the Ca-induced swelling. By contrast, the cromakalim analogue BPDZ490 (1) induced a strong increase of OCR, while the other benzopyran analogue BPDZ711 (2) caused a marked slowdown. For both compounds, 1 displayed a biphasic behavior while 2 still showed an inhibitory effect. Both compounds 1 and 2 were also found to decrease the ATP synthesis, with pronounced effect for 2, while cromakalim remained without effect. Overall, these results indicate that cromakalim, as parent molecule, does not induce per se any direct effect on mitochondrial respiratory function neither on whole cells nor on isolated mitochondria whereas both benzopyran analogues 1 and 2 display totally opposite behavior profiles, suggesting that compound 1, by increasing the maximal respiration capacity, might behave as a mild uncoupling agent and compound 2 is taken as an inhibitor of the mitochondrial electron-transfer chain.
Sirtuin 2 (SIRT2) is an important and special member of the atypical histone deacetylase Sirtuin (SIRT) family. Due to its extensive catalytic effects, SIRT2 can regulate autophagy, myelination, immunity, inflammation and other physiological processes. Recent evidence revealed that dysregulation of human SIRT2 activity is associated with the pathogenesis and prognosis of cancers, Parkinson's disease and other disorders; thus SIRT2 is a promising target for potential therapeutic intervention. This review presents a systematic summary of nine chemotypes of small-molecule SIRT2 inhibitors, particularly including the discovery and structural optimization strategies, which will be useful for future efforts to develop new inhibitors targeting SIRT2 and associated target proteins.
The human sirtuin silent information regulator 1 (SIRT1) is a NAD⁺-dependent deacetylase enzyme. It deacetylates many protein substrates, including histones and transcription factors, thereby controlling many physiological and pathological processes. Several synthetic inhibitors and activators of SIRT1 have been developed, and some therapeutic applications have been explored. The indole EX-527 and its derivatives are among the most potent and selective SIRT1 inhibitors. EX-527 has been often used as a pharmacological tool to explore the effect of SIRT1 inhibition in various cell types. Its therapeutic potential has, therefore, been evaluated in animal models for several pathologies, including cancer. It has also been tested in phase II clinical trial for the treatment of Huntington’s disease (HD). In this review, we will provide an overview of the literature on EX-527, including its mechanism of inhibition and biological studies.
The mammalian family of sirtuins (SIRT1–7) target a large variety of proteins at various subcellular localizations and thus exert regulatory effects on critical biological processes such as gene silencing, DNA repair, and chromosomal stability and longevity. Sirtuins play crucial roles in many signaling pathways and are regarded as potential therapeutic targets in several pathological conditions, such as cancer, metabolic disorders, and also cardiovascular and neurodegenerative diseases. Therefore, the modulation of sirtuin activity by inhibitors or activators could be beneficial for human health, a topic that has been interesting scientists for over 15 years. Researchers have developed novel inhibitors and activators toward sirtuins mainly for human silent information regulator type 1 (SIRT1) because both cellular studies and experiments in animal models have indicated that SIRT1 regulators could be used for the treatment for multiple human diseases. Gradually, an appreciation of the importance of the other members of the sirtuin family has increased, and potent inhibitors and activators are designed for various sirtuins.
Abnormal histone deacetylase (HDAC) expression is closely related to cancer development and progression. Many HDAC inhibitors have been widely used in cancer treatment; however, severe side effects often limit their clinical application. In this study, we attempted to identify natural compounds with HDAC inhibitory activity and low physiological toxicity and explored their feasibility and mechanisms of action in liver cancer treatment. A yeast screening system was used to identify natural compounds with HDAC inhibitory activity. Further, western blotting was used to verify inhibitory effects on HDAC in human liver cancer cell lines. Cell functional analysis was used to explore the effects and mechanisms and the in vitro results were verified in BALB/c nude mice. We found that hydroxygenkwanin (HGK), an extract from Daphne genkwa, inhibited class I HDAC expression, and thereby induced expression of tumor suppressor p21 and promoted acetylation and activation of p53 and p65. This resulted in the inhibition of growth, migration, and invasion of liver cancer cells and promoted cell apoptosis. Animal models revealed that HGK inhibited tumor growth in a synergistic manner with sorafenib. HGK inhibited class I HDAC expression and had low physiological toxicity. It has great potential as an adjuvant for liver cancer treatment and may be used in combination with anticancer drugs like sorafenib to improve therapeutic efficacy.
Sirtuin (Yeast Silent Information RegulatorsⅡ, Sir2) was first discovered in the 1970s. Because of its function by removing acetylated groups from histones in the presence of nicotinamide adenine dinucleotide (NAD⁺), waves of research have assessed the potential of Sirtuin as a therapeutic target. The Sirtuin family, which is widely distributed throughout the nature, has been divided into seven human isoforms (Sirt1-Sirt7). They are thought to be closely related to some aging diseases such as cardiovascular disorders, neurodegeneration, and tumors. Herein, we present a comprehensive review of the structure, function and modulators of Sirtuins, which is expected to be beneficial to relevant studies.
Patients with somatic mutations of epigenetic regulators are characterized by aberrant chromatin modification patterns. Recent mechanistic studies pairing chemical tool compounds and deep-sequencing technology have greatly broadened our understanding of epigenetic regulation in glioma progression and underpinned alternative treatment of epigenetic inhibitors. However, the effect of most inhibitors is condition-dependent, and the overall results of clinical trials still have not been applied to patients. There is an intense need to develop more potent and specific compounds as well as identify the population who may achieve clinical benefits. Besides, combination therapy with conventional therapeutics is another alternative strategy. In this review, we summarize well-characterized chemical probes in glioma research and clinical translation. We also discuss the target population and combination of therapy regimens of various agents. In a holistic sense, we try to provide guidance for selecting targeted chemical probes and pave the way for personalized rational therapy.
The mammalian sirtuin family has attracted tremendous attention over the past few years as stress adaptors and post-translational modifier. They have involved in diverse cellular processes including DNA repair, energy metabolism, and tumorigenesis. Notably, genomic instability and metabolic reprogramming are two of characteristic hallmarks in cancer. In this review, we summarize current knowledge on the functions of sirtuins mainly regarding DNA repair and energy metabolism, and further discuss the implication of sirtuins in cancer specifically by regulating genome integrity and cancer-related metabolism.
Sirtuins are NAD+-dependent histone deacetylases regulating important metabolic pathways in prokaryotes and eukaryotes and are involved in many biological processes such as cell survival, senescence, proliferation, apoptosis, DNA repair, cell metabolism, and caloric restriction. The seven members of this family of enzymes are considered potential targets for the treatment of human pathologies including neurodegenerative diseases, cardiovascular diseases, and cancer. Furthermore, recent interest focusing on sirtuin modulators as epigenetic players in the regulation of fundamental biological pathways has prompted increased efforts to discover new small molecules able to modify sirtuin activity. Here, we review the role, mechanism of action, and biological function of the seven sirtuins, as well as their inhibitors and activators.
We characterized the brominated alkaloid Isofistularin-3 (Iso-3), from the marine sponge Aplysina aerophoba, as a new DNA methyltransferase (DNMT)1 inhibitor. Docking analysis confirmed our in vitro DNMT inhibition data and revealed binding of Iso-3 within the DNA binding site of DNMT1. Subsequent increased expression of tumor suppressor gene aryl hydrocarbon receptor (AHR) could be correlated to decreased methylation of CpG sites within the essential Sp1 regulatory region of its promoter. Iso-3 induced growth arrest of cancer cells in G0/G1 concomitant with increased p21 and p27 expression and reduced cyclin E1, PCNA and c-myc levels. Reduced proliferation was accompanied by morphological changes typical of autophagy revealed by fluorescent and transmission electron microscopy and validated by LC3I-II conversion. Furthermore, Iso-3 strongly synergized with tumor-necrosis-factor related apoptosis inducing ligand (TRAIL) in RAJI [combination index (CI) = 0.22] and U-937 cells (CI = 0.21) and increased TRAIL-induced apoptosis via a mechanism involving reduction of survivin expression but not of Bcl-2 family proteins nor X-linked inhibitor of apoptosis protein (XIAP). Iso-3 treatment decreased FLIPL expression and triggered activation of endoplasmatic reticulum (ER) stress with increased GRP78 expression, eventually inducing TRAIL receptor death receptor (DR)5 surface expression. Importantly, as a potential candidate for further anticancer drug development, Iso-3 reduced the viability, colony and in vivo tumor forming potential without affecting the viability of PBMCs from healthy donors or zebrafish development.
Sirtuins are NAD+-dependent protein deacylases that cleave off acetyl, but also other acyl groups from the ε-amino group of lysines in histones and other substrate proteins. Dysregulation of human Sirt2 (hSirt2) activity has been associated with the pathogenesis of cancer, inflammation, and neurodegeneration, which makes the modulation of hSirt2 activity a promising strategy for pharmaceutical intervention. The Sirtuin Rearranging Ligands (SirReals) have recently been discovered by us as highly potent and isotype-selective hSirt2 inhibitors. Here, we present a well-defined structure-activity relationship study, which rationalizes the unique features of the SirReals and probes the limits of modifications on this scaffold regarding inhibitor potency. Moreover, we present a crystal structure of hSirt2 in complex with an optimized SirReal derivative that exhibits an improved in vitro activity. Lastly, we show cellular hyperacetylation of the hSirt2 targeted tubulin caused by our improved lead structure.
SIRT1, the founding member of the mammalian family of seven NAD þ -dependent sirtuins, is composed of 747 amino acids forming a catalytic domain and extended N- and C-terminal regions. We report the design and characterization of an engineered human SIRT1 construct (mini-hSIRT1) containing the minimal structural elements required for lysine deacetylation and catalytic activation by small molecule sirtuin-activating compounds (STACs). Using this construct, we solved the crystal structure of a mini-hSIRT1-STAC complex, which revealed the STAC-binding site within the N-terminal domain of hSIRT1. Together with hydrogen- deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis using full-length hSIRT1, these data establish a specific STAC-binding site and identify key inter- molecular interactions with hSIRT1. The determination of the interface governing the binding of STACs with human SIRT1 facilitates greater understanding of STAC activation of this enzyme, which holds significant promise as a therapeutic target for multiple human diseases.
Sirtuins with an extended N-terminal domain (NTD), represented by yeast Sir2 and human SIRT1, harbor intrinsic mechanisms for regulation of their NAD-dependent deacetylase activities. Elucidation of the regulatory mechanisms is crucial for understanding the biological functions of sirtuins and development of potential therapeutics. In particular, SIRT1 has emerged as an attractive therapeutic target, and the search for SIRT1-activating compounds (STACs) has been actively pursued. However, the effectiveness of a class of reported STACs (represented by resveratrol) as direct SIRT1 activators is under debate due to the complication involving the use of fluorogenic substrates in in vitro assays. Future efforts of SIRT1-based therapeutics necessitate the dissection of the molecular mechanism of SIRT1 stimulation. We solved the structure of SIRT1 in complex with resveratrol and a 7-amino-4-methylcoumarin (AMC)-containing peptide. The structure reveals the presence of three resveratrol molecules, two of which mediate the interaction between the AMC peptide and the NTD of SIRT1. The two NTD-bound resveratrol molecules are principally responsible for promoting tighter binding between SIRT1 and the peptide and the stimulation of SIRT1 activity. The structural information provides valuable insights into regulation of SIRT1 activity and should benefit the development of authentic SIRT1 activators.
© 2015 Cao et al.; Published by Cold Spring Harbor Laboratory Press.
Sirtuins are a highly conserved class of NAD(+)-dependent lysine deacylases. The human isotype Sirt2 has been implicated in the pathogenesis of cancer, inflammation and neurodegeneration, which makes the modulation of Sirt2 activity a promising strategy for pharmaceutical intervention. A rational basis for the development of optimized Sirt2 inhibitors is lacking so far. Here we present high-resolution structures of human Sirt2 in complex with highly selective drug-like inhibitors that show a unique inhibitory mechanism. Potency and the unprecedented Sirt2 selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a yet-unexploited binding pocket. Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2 inhibition in vivo. Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology.
Sirtuins are NAD+ dependent deacetylases and/or ADP-ribosyl transferases active on histone and non-histone substrates. The first sirtuin was discovered as a transcriptional repressor of the mating-type-loci (Silent Information Regulator sir2) in the budding yeast, where it was shown to extend yeast lifespan. Seven mammalian sirtuins (SIRT1-7) have been now identified with distinct subcellular localization, enzymatic activities and substrates. These enzymes regulate cellular processes such as metabolism, cell survival, differentiation, DNA repair and they are implicated in the pathogenesis of solid tumors and leukemias. The purpose of the present study was to investigate the role of sirtuin expression, activity and inhibition in the survival of pediatric sarcoma cell lines.We have analyzed the expression of SIRT1 and SIRT2 in a series of pediatric sarcoma tumor cell lines and normal cells, and we have evaluated the activity of the sirtuin inhibitor and p53 activator tenovin-6 (Tv6) in synovial sarcoma and rhabdomyosarcoma cell lines. We show that SIRT1 is overexpressed in synovial sarcoma biopsies and cell lines in comparison with normal mesenchymal cells. Tv6 induced apoptosis as well as impaired autophagy flux. Using siRNA to knock down SIRT1 and SIRT2, we show that the expression of both proteins is crucial for the survival of rhabdomyosarcoma cells and that the loss of SIRT1 expression results in a decreased LC3II expression. Our results show that SIRT1 and SIRT2 expressions are crucial for the survival of synovial sarcomas and rhabdomyosarcomas, and demonstrate that the pharmacological inhibition of sirtuins impairs the autophagy process and induces tumor cell death.
Inhibition of protein deacetylation enzymes, alone or in combination with standard chemotherapies, is an exciting addition to cancer therapy. We have investigated the effect of deacetylase inhibition on the metabolism of glioblastoma cells. 1H NMR metabolomics analysis was used to determine the major metabolic changes following treatment of two distinct glioblastoma cell lines, U373 and LN229, with five different histone deacetylase (HDAC) inhibitors, as well as one inhibitor of NAD+-dependent protein deacetylases (SIRT). The addition of the standard glioblastoma chemotherapy agent, temozolomide, to the HDAC and SIRT treatments led to a reduction in cell survival, suggesting a possibility for combined treatment. This study shows that distinct glioblastoma cell lines, with different metabolic profiles and gene expression, experience dissimilar changes following treatment with protein deacetylase inhibitors. The observed effects of inhibitors on mitochondrial metabolism, glycolysis and fatty acid synthesis suggest possible roles of protein deacetylases in metabolism regulation. Metabolic markers of the effectiveness of anti-protein deacetylase treatments have been explored. In addition to known deacetylation inhibitors, three novel inhibitors have been introduced and tested. Finally, 1H NMR analysis of cellular metabolism is shown to be a fast, inexpensive method for testing drug effects.
Increased proliferation rates as well as resistance to apoptosis are considered major obstacles for the treatment of patients with chronic myelogenous leukemia (CML), thus highlighting the need for novel therapeutic approaches. Since senescence has been recognized as a physiological barrier against tumorigenesis, senescence-based therapy could represent a new strategy against CML. DNA demethylating agent 5-aza-2'-deoxycytidine (DAC) was reported to induce cellular senescence but underlying mechanisms remain to be elucidated. Here, we report that exposure to DAC triggers senescence in chronic leukemia cell lines as evidenced by increased senescence-associated β-galactosidase activity and lysosomal mass, accompanied by an up-regulation of cell cycle-related genes. We provide evidence that DAC is able to decrease telomere length, to reduce telomerase activity and to decrease human telomerase reverse transcriptase (hTERT) expression through decreased binding of c-myc to the hTERT promoter. Altogether, our results reveal the role of c-myc in telomere-dependent DAC-induced senescence and therefore provide new clues for improving chronic human leukemia treatments.
It is well known that in vitro subculture represents a selection pressure on cell lines, and over time this may result in a genetic drift in the cancer cells. In addition, long-term cultures harbor the risk of cross-contamination with other cell lines. The consequences may have major impact on experimental results obtained in various laboratories, where the cell lines no longer reflect the original tumors that they are supposed to represent. Much neglected in the scientific community is a close monitoring of cell cultures by regular phenotypic and genetic characterization. In this report, we present a thorough characterization of the commonly used glioblastoma (GBM) model U-251, which in numerous publications has been wrongly identified as U-373, due to an earlier cross-contamination. In this work, the original U-251 and three subclones of U-251, commonly referred to as U-251 or U-373, were analyzed with regard to their DNA profile, morphology, phenotypic expression, and growth pattern. By array comparative genomic hybridization (aCGH), we show that only the original low-passaged U-251 cells, established in the 1960s, maintain a DNA copy number resembling a typical GBM profile, whereas all long-term subclones lost the typical GBM profile. Also the long-term passaged subclones displayed variations in phenotypic marker expression and showed an increased growth rate in vitro and a more aggressive growth in vivo. Taken together, the variations in genotype and phenotype as well as differences in growth characteristics may explain different results reported in various laboratories related to the U-251 cell line.
SIRT1 is a NAD(+)-dependent deacetylase that plays important roles in many cellular processes. SIRT1 activity is uniquely controlled by a C-terminal regulatory segment (CTR). Here we present crystal structures of the catalytic domain of human SIRT1 in complex with the CTR in an open apo form and a closed conformation in complex with a cofactor and a pseudo-substrate peptide. The catalytic domain adopts the canonical sirtuin fold. The CTR forms a β hairpin structure that complements the β sheet of the NAD(+)-binding domain, covering an essentially invariant, hydrophobic surface. The apo form adopts a distinct open conformation, in which the smaller subdomain of SIRT1 undergoes a rotation with respect to the larger NAD(+)-binding subdomain. A biochemical analysis identifies key residues in the active site, an inhibitory role for the CTR, and distinct structural features of the CTR that mediate binding and inhibition of the SIRT1 catalytic domain.
Glioblastomas, the most common form of primary brain tumors, are the fourth cause of death by cancer in adults. Increasing evidences suggest that glioblastoma resistance to existing radio- and chemotherapies rely on glioblastoma stem cells (GSCs). GSCs are endowed with a unique combination of stem-like properties alike to normal neural stem cells (NSCs), and of tumor initiating properties. The natural polyphenol resveratrol is known to exert opposite actions on neural cells according to their normal or cancerous status. Here, we used resveratrol to explore the molecular mechanisms differing between GSCs and NSCs. We observed a dual action of resveratrol on GSCs: resveratrol blocked GSC proliferation up to 150 μM and induced their necrosis at higher doses. On the opposite, resveratrol had no effect on NSC behavior. To determine the mechanisms underlying resveratrol effects, we focused our attention on the family of NAD-dependent deacetylases sirtuins (SIRT). A member of this family, SIRT1, has been repetitively shown to constitute a preferential resveratrol target, at least in normal cells. Western blot analysis showed that SIRT1 and SIRT3 were expressed by both GSCs and NSCs whereas SIRT2 expression was restricted to GSCs. Pharmacological blockade of SIRT2 activity or down-regulation of SIRT2 expression with siRNAs counteracted the inhibitory effect of resveratrol on cell proliferation. On the contrary, inhibition of SIRT2 activity or expression did not counteract GSC necrosis observed in presence of high doses of resveratrol. Our results highlight SIRT2 as a novel target for altering GSC properties.
The ability to identify the site of a protein that can bind with high affinity to small, drug-like compounds has been an important goal in drug design. Sirtuin 2 (SIRT2), histone deacetylase protein family, plays a central role in the regulation of various pathways. Hence, identification of drug for SIRT2 has attracted great interest in the drug discovery community. To elucidate the molecular basis of the small molecules interactions to inhibit the SIRT2 function we employed the molecular docking, molecular dynamics simulations, and the molecular mechanism Poisson-Boltzmann/surface area (MM-PBSA) calculations. Five well know inhibitors such as suramin, mol-6, sirtinol, 67, and nf675 were selected to establish the nature of the binding mode of the inhibitors in the SIRT2 active site. The molecular docking and dynamics simulations results revealed that the hydrogen bonds between Arg97 and Gln167 are crucial to inhibit the function of SIRT2. In addition, the MM-PBSA calculations revealed that binding of inhibitors to SIRT2 is mainly driven by van der Waals/non-polar interactions. Although the five inhibitors are very different in structure, shape, and electrostatic potential, they are able to fit in the same binding pocket. These findings from this study provide insights to elucidate the binding pattern of SIRT2 inhibitors and help in the rational structure-based design of novel SIRT2 inhibitors with improved potency and better resistance profile.
Acetylation and deacetylation of lysine residues on histones, which are catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), are epigenetic modifications that play a very important role in the regulation of gene transcription. Perturbation of the balance between histone acetylation and deacetylation leads to a myriad of diseases, including cancer, AIDS, malaria, neurodegenerative diseases, and diabetes. HATs and HDACs have recently been recognized as key targets for chemoprevention and drug discovery, and numerous natural and synthetic compounds have been screened in order to identify promising regulators of subtle balance between histone acetylation and deacetylation. Here, we present dietary compounds and other natural products that have emerged as potent HAT or HDAC activity modulators, and we discuss their current and future applications as chemopreventive or therapeutic agents.
Protein acetylation status results from a balance between histone acetyltransferase and histone deacetylase (HDAC) activities. Alteration of this balance leads to a disruption of cellular integrity and participates in the development of numerous diseases, including cancer. Therefore, modulation of these activities appears to be a promising approach for anticancer therapy. Histone deacetylase inhibitors (HDACi) are epigenetically active drugs that induce the hyperacetylation of lysine residues within histone and non-histone proteins, thus affecting gene expression and cellular processes such as protein-protein interactions, protein stability, DNA binding and protein sub-cellular localization. Therefore, HDACi are promising anti-tumor agents as they may affect the cell cycle, inhibit proliferation, stimulate differentiation and induce apoptotic cell death. Over the last 30 years, numerous synthetic and natural products, including a broad range of dietary compounds, have been identified as HDACi. This review focuses on molecules from natural origins modulating HDAC activities and presenting promising anticancer activities.
SIRT proteins play an important role in the survival and drug resistance of tumor cells, especially during chemotherapy. In this study, we investigated the potency, specificity, and cellular targets of three SIRT inhibitors, Sirtinol, Salermide, and EX527. Cell proliferative and cell cycle analyses showed that Sirtinol and Salermide, but not EX527, were effective in inducing cell death at concentrations of 50 micromol/L or over in MCF-7 cells. Instead, EX527 caused cell cycle arrest at G(1) at comparable concentrations. In vitro SIRT assays using a p53 peptide substrate showed that all three compounds are potent SIRT1/2 inhibitors, with EX527 having the highest inhibitory activity for SIRT1. Computational docking analysis showed that Sirtinol and Salermide have high degrees of selectivity for SIRT1/2, whereas EX527 has high specificity for SIRT1 but not SIRT2. Consistently, Sirtinol and Salermide, but not EX527, treatment resulted in the in vivo acetylation of the SIRT1/2 target p53 and SIRT2 target tubulin in MCF-7 cells, suggesting that EX527 is ineffective in inhibiting SIRT2 and that p53 mediates the cytotoxic function of Sirtinol and Salermide. Studies using breast carcinoma cell lines and p53-deficient mouse fibroblasts confirmed that p53 is essential for the Sirtinol and Salermide-induced apoptosis. Further, we showed using small interfering RNA that silencing both SIRTs, but not SIRT1 and SIRT2 individually, can induce cell death in MCF-7 cells. Together, our results identify the specificity and cellular targets of these novel inhibitors and suggest that SIRT inhibitors require combined targeting of both SIRT1 and SIRT2 to induce p53 acetylation and cell death. Mol Cancer Ther; 9(4); 844-55. (c)2010 AACR.
We have previously reported that galectin 1 (Gal-1) plays important biological roles in astroglial as well as in oligodendroglial cancer cells. As an oligodendroglioma model, we make use of the Hs683 cell line that has been previously extensively characterized at cell biology, molecular biology, and genetic levels. Galectin 1 has been shown to be involved in Hs683 oligodendroglioma chemoresistance, neoangiogenesis, and migration. Down-regulating Gal-1 expression in Hs683 cells through targeted small interfering RNA provokes a marked decrease in the expression of the brain-expressed X-linked gene: BEX2. Accordingly, the potential role of BEX2 in Hs683 oligodendroglioma cell biology has been investigated. The data presented here reveal that decreasing BEX2 expression in Hs683 cells increases the survival of Hs683 orthotopic xenograft-bearing mice. Furthermore, this decrease in BEX2 expression impairs vasculogenic mimicry channel formation in vitro and angiogenesis in vivo, and modulates glioma cell adhesion and invasive features through the modification of several genes previously reported to play a role in cancer cell migration, including MAP2, plexin C1, SWAP70, and integrin beta(6). We thus conclude that BEX2 is implicated in oligodendroglioma biology.
Resistance to multiple antitumour drugs, mostly antibiotics or alkaloids, has been associated with a cellular plasma membrane P-glycoprotein (Pgp), causing energy-dependent transport of drugs out of cells. However, in many common chemotherapy resistant human cancers there is no overexpression of Pgp, which could explain drug resistance. In order to characterise early steps in multidrug resistance we have derived a series of P-glycoprotein-positive (Pgp/+) and P-glycoprotein-negative (Pgp/-) multidrug resistant cell lines, from a human non-small cell lung cancer cell line, SW-1573, by stepwise selection with increasing concentrations of doxorubicin. These cells were exposed to doxorubicin and its fluorescence in nucleus (N) and cytoplasm (C) was quantified with laserscan microscopy and image analysis. The fluorescence N/C ratio in parent cells was 3.8 and decreased both in Pgp/+ and Pgp/- cells with increasing selection pressure to 1.2-2.6 for cells with a resistance factor of 7-17. N/C ratios could be restored partly with verapamil only in Pgp/+ cells. N/C ratio measurements may define a general Pgp-independent type of defense of mammalian cells against certain anticancer agents which may precede Pgp expression in early doxorubicin resistance.
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The ability of tumor cells to develop simultaneous resistance to structurally different cytotoxic drugs constitutes a major problem in cancer chemotherapy. It was previously demonstrated that multidrug-resistant Chinese hamster cell lines contain an amplified, transcriptionally active DNA sequence designated mdr. This report presents evidence that multidrug-resistant sublines of human KB carcinoma cells, selected for resistance to either colchicine, vinblastine, or Adriamycin (doxorubicin), display amplification of two different DNA sequences homologous to the hamster mdr gene. Segments of the human mdr DNA sequences, designated mdr1 and mdr2, have been cloned. mdr1 sequences were amplified in all of the highly drug-resistant sublines and were expressed as a poly(A)+ RNA species of 4.5 kilobases that was detected in the resistant cells but not in the parental cell line. No expression of mdr2 sequences was detected. mdr2 sequences were coamplified with mdr1 in some of the multidrug-resistant sublines and, in two independently derived cell lines, underwent very similar rearrangements. The data suggest that the mdr1 gene is involved in multidrug resistance in human cells.
Hydroxyurea (HU) is currently used in the clinic for the treatment of chronic myelogenous leukemia, head and neck carcinoma, and sarcoma. One of its drawbacks, however, is the development of HU resistance. To study this problem, we developed a HU-resistant human KB cell line which exhibits a 15-fold resistance to HU. The characterization of this HU-resistant phenotype revealed a gene amplification of the M2 subunit of ribonucleotide reductase (RR), increased levels of M2 mRNA and protein, and a 3-fold increase of RR activity. This HU-resistant cell line also expressed a "collateral sensitivity" to 6-thioguanine (6-TG), with a 10-fold decrease in the dose inhibiting cell growth by 50% as compared to the KB parental line. The mechanism responsible for this supersensitivity to 6-TG is believed to be related to an increasingly efficient conversion of 6-TG to its triphosphate form, which is subsequently incorporated into DNA. After passage of the resistant cells in the absence of HU, the cell line reverts. The revertant cells lose their resistance to HU and concomitantly their sensitivity to 6-TG. This phenomenon is due to the return of RR to levels comparable to that of the KB parental cell line. These observations and their relevance to cancer chemotherapy will be discussed in this paper. Our results suggest that a clinical protocol could be designed which would allow for a lower dose of 6-TG to be used by taking advantage of the increased RR activity in HU-refractory cancer patients. Two drugs which display collateral sensitivity are known as a "Ying-Yang" pair. Alternate treatment with two different Ying-Yang pairs is the rationale for the "Ying-Yang Ping-Pong" theory in cancer treatment. This rationale allows for effective cancer chemotherapy with reduced toxicity.
The synthesis of 2,2-dimethylchromans bearing a 3/4-chloro/cyano-substituted phenylureido or phenylthioureido moiety at the 4-position and an alkoxycarbonylamino (‘carbamate’) group at the 6-position is described. These new analogs of the potassium channel opener (±)-cromakalim were further tested on rat pancreatic islets as putative inhibitors of insulin release and on rat aorta rings as putative vasorelaxants. All compounds inhibited insulin secretion and induced a myorelaxant activity. Compound 14o [R/S-N-3-cyanophenyl-N'-(6-tert-butoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)urea; BPDZ 711] emerged as the most potent inhibitor of the glucose-sensitive insulin releasing process (IC50 = 0.24 μM) and displayed selectivity towards the pancreatic endocrine tissue. Radioisotopic, fluorimetric and pharmacological investigations were performed on rat pancreatic islet and rat vascular smooth muscle cells in order to decipher its mechanism of action. Our findings suggest that the mechanism of action of 14o is rather unspecific. The compound behaves as a KATP channel opener, a Ca2+ entry blocker, and promotes an intracellular calcium translocation.
The tumor suppressor p53 has established functions in cancer. Specifically, it has been shown to cause cell-cycle arrest and apoptosis in response to DNA damage. It is also one of the most commonly mutated or silenced genes in cancer and for this reason has been extensively studied. Recently, the role of p53 has been shown to go beyond its effects on cell cycle and apoptosis, with effects on metabolism emerging as a key contributor to cancer growth in situations where p53 is lost. Beyond this, the role of p53 in the tumor microenvironment is poorly understood. The publication by Wang and colleagues demonstrates for the first time that p53 is a key negative regulator of aromatase and, hence, estrogen production in the breast tumor microenvironment. It goes further by demonstrating that an important regulator of aromatase, the obesity-associated and tumor-derived factor prostaglandin E2, inhibits p53 in the breast adipose stroma. This review presents these findings in the context of established and emerging roles of p53 and discusses possible implications for the treatment of breast cancer. Cancer Res; 75(23); 1-7. ©2015 AACR.
The histone deacetylases (HDACs), Sirtuin 1 (Sirt1) and Sirt2, play crucial roles in many biological processes, including cell proliferation, differentiation and apoptosis. HDAC inhibitors have been considered as a potential therapeutic approach for various types of cancers. Here, we demonstrated that the Sirt1 and Sirt2 inhibitors EX527 and AGK2 suppressed cell growth and caused G1 phase arrest by inhibiting the expression of Cdk6 and/or Cdk4. An agar colony formation assay revealed that EX527 and AGK2 decreased colony formation in soft agar. Furthermore, EX527 and AGK2 pretreatment inhibited the expression of HSF1 and HSP27 and induced HSF1 ubiquitination. Sirt1 overexpression increased HSF1 expression and/or stabilization and induced cell migration in a scratch assay. Overall, these results indicate that EX527 and AGK2 suppress cell growth and migration by inhibiting HSF1 protein stability.
Despite considerable scientific progress, the burden of cancer in our society remains a major public health problem. Tumorigenesis is recognized as a complex and multistep process that involves the accumulation of successive transformational events with multi-factorial etiology. Nevertheless, such events result in the acquisition of key hallmark characteristics that are shared by all cancer cells. Accumulating evidence indicates that, besides genetic alterations, epigenetic mechanisms (heritable changes in gene expression caused by modifications in chromatin structure without alterations of DNA sequence) are implicated in the acquisition of malignant phenotype. The potential reversibility of epigenetic alterations linked to tumorigenesis offers a promising avenue for therapeutic intervention. This review focuses on the epigenetic regulation of the cancer hallmarks and the foreseeable use of epigenetic drugs to target these features as a promising strategy for anti-cancer therapy. Based on this body of evidence, we believed that epigenetic deregulations can affect virtually all cell functions and therefore therapeutic approaches with epigenetic drugs could allow multi-target approach against the hallmarks of cancer.
BACKGROUND: In 2004, a randomised phase III trial by the European Organisation for Research and Treatment of Cancer (EORTC) and National Cancer Institute of Canada Clinical Trials Group (NCIC) reported improved median and 2-year survival for patients with glioblastoma treated with concomitant and adjuvant temozolomide and radiotherapy. We report the final results with a median follow-up of more than 5 years. METHODS: Adult patients with newly diagnosed glioblastoma were randomly assigned to receive either standard radiotherapy or identical radiotherapy with concomitant temozolomide followed by up to six cycles of adjuvant temozolomide. The methylation status of the methyl-guanine methyl transferase gene, MGMT, was determined retrospectively from the tumour tissue of 206 patients. The primary endpoint was overall survival. Analyses were by intention to treat. This trial is registered with Clinicaltrials.gov, number NCT00006353. FINDINGS: Between Aug 17, 2000, and March 22, 2002, 573 patients were assigned to treatment. 278 (97%) of 286 patients in the radiotherapy alone group and 254 (89%) of 287 in the combined-treatment group died during 5 years of follow-up. Overall survival was 27.2% (95% CI 22.2-32.5) at 2 years, 16.0% (12.0-20.6) at 3 years, 12.1% (8.5-16.4) at 4 years, and 9.8% (6.4-14.0) at 5 years with temozolomide, versus 10.9% (7.6-14.8), 4.4% (2.4-7.2), 3.0% (1.4-5.7), and 1.9% (0.6-4.4) with radiotherapy alone (hazard ratio 0.6, 95% CI 0.5-0.7; p<0.0001). A benefit of combined therapy was recorded in all clinical prognostic subgroups, including patients aged 60-70 years. Methylation of the MGMT promoter was the strongest predictor for outcome and benefit from temozolomide chemotherapy. INTERPRETATION: Benefits of adjuvant temozolomide with radiotherapy lasted throughout 5 years of follow-up. A few patients in favourable prognostic categories survive longer than 5 years. MGMT methylation status identifies patients most likely to benefit from the addition of temozolomide. FUNDING: EORTC, NCIC, Nélia and Amadeo Barletta Foundation, Schering-Plough.
Despite the recent advances in the treatment of tumors with intrinsic chemotherapy resistance, such as melanoma and renal cancers, their prognosis remains poor and new chemical agents with promising activity against these cancers are urgently needed. Sphaeropsidin A, a fungal metabolite whose anticancer potential had previously received little attention, was isolated from Diplodia cupressi and found to display specific anticancer activity in vitro against melanoma and kidney cancer subpanels in the National Cancer Institute (NCI) 60-cell line screen. The NCI data revealed a mean LC50 of ca. 10 µM and a cellular sensitivity profile that did not match that of any other agent in the 765,000 compound database. Subsequent mechanistic studies in melanoma and other multidrug-resistant in vitro cancer models showed that sphaeropsidin A can overcome apoptosis as well as multidrug resistance by inducing a marked and rapid cellular shrinkage related to the loss of intracellular Cl(-) and the decreased HCO3 (-) concentration in the culture supernatant. These changes in ion homeostasis and the absence of effects on the plasma membrane potential were attributed to the sphaeropsidin A-induced impairment of regulatory volume increase (RVI). Preliminary results also indicate that depending on the type of cancer, the sphaeropsidin A effects on RVI could be related to Na-K-2Cl electroneutral cotransporter or Cl(-)/HCO3 (-) anion exchanger(s) targeting. This study underscores the modulation of ion-transporter activity as a promising therapeutic strategy to combat drug-resistant cancers and identifies the fungal metabolite, sphaeropsidin A, as a lead to develop anticancer agents targeting RVI in cancer cells.
Histone deacetylase (HDAC)6 is a member of the class IIb HDAC family. This enzyme is zinc-dependent and mainly localized in the cytoplasm. HDAC6 is a unique isoenzyme with two functional catalytic domains and specific physiological roles. Indeed, HDAC6 deacetylates various substrates including α-tubulin and HSP90α, and is involved in protein trafficking and degradation, cell shape and migration. Consequently, deregulation of HDAC6 activity was associated to a variety of diseases including cancer, neurodegenerative diseases and pathological autoimmune response. Therefore, HDAC6 represents an interesting potential therapeutic target. In this review, we discuss structural features of this histone deacetylase, regulation of its expression and activity, biological functions, implication in human disease initiation and progression. Finally will describe novel and selective HDAC6 inhibitors.
Volume is one of the most important features for the characterization of a tumour on a macroscopic scale. It is often used to assess the effectiveness of care treatments, thus making its correct evaluation a crucial issue for patient care. Similarly, volume is a key feature on a microscopic scale. Multicellular cancer spheroids are 3D tumour models widely employed in pre-clinical studies to test the effects of drugs and radiotherapy treatments. Very few methods have been proposed to estimate the tumour volume arising from a 2D projection of multicellular spheroids, and even fewer have been designed to provide a 3D reconstruction of the tumour shape. In this work, we propose Reconstruction and Visualization from a Single Projection (ReViSP), an automatic method conceived to reconstruct the 3D surface and estimate the volume of single cancer multicellular spheroids, or even of spheroid cultures. As the input parameter ReViSP requires only one 2D projection, which could be a widefield microscope image. We assessed the effectiveness of our method by comparing it with other approaches. To this purpose, we used a new strategy that allowed us to achieve accurate volume measurements based on the analysis of home-made 3D objects, built by mimicking the spheroid morphology. The results confirmed the effectiveness of our method for both 3D reconstruction and volume assessment. ReViSP software is distributed as an open source tool.
Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
A chemical investigation of the endophytic fungus Epicoccum nigrum isolated from leaves of Mentha suaveolens collected in Morocco resulted in the isolation of five new polyketides, epicocconigrones A and B (1 and 2), 3-methoxyepicoccone B (3), 3-methoxyepicoccone (4), and 2,3,4-trihydroxy-6-(methoxymethyl)-5-methylbenzaldehyde (5), together with five known compounds (6-10). The structures of the new compounds were unambiguously determined by extensive analysis of the 1D and 2D NMR and mass spectroscopic data. Compounds 1 and 10 showed potent inhibition of at least 15 protein kinases with IC50 values ranging from 0.07 to 9.00 μM. Moreover, compounds 1 and 10 inhibited histone deacetylase (HDAC) activities with IC50 values of 9.8 and 14.2 μM, respectively. A preliminary structure-activity relationship is discussed. Interestingly, compounds 1 and 10 exert mainly cytostatic effects in human lymphoma RAJI and U-937 cell lines.
AutoDock Vina, a new program for molecular docking and virtual screening, is presented. AutoDock Vina achieves an approximately two orders of magnitude speed-up compared with the molecular docking software previously developed in our lab (AutoDock 4), while also significantly improving the accuracy of the binding mode predictions, judging by our tests on the training set used in AutoDock 4 development. Further speed-up is achieved from parallelism, by using multithreading on multicore machines. AutoDock Vina automatically calculates the grid maps and clusters the results in a way transparent to the user.
Eight cyclic peroxide norterpenoids, compounds 1-8, have been isolated and characterized from the Red Sea sponge Diacarnus erythraeanus, including two new norsesterterpene derivatives (3, 4). Among these metabolites, (-)-muqubilin A (5) (nine cell lines analyzed) and the new compounds 3 and 4 (seven cell lines analyzed) displayed mean IC50 growth inhibitory concentrations in vitro of <10 μM, while the remaining compounds (1, 6-8) were inactive in these cancer cell lines. Compound 5 displayed no selectivity between normal and cancer cells in terms of in vitro growth inhibition. Quantitative video microscopy analysis carried out on (-)-muqubilin A-treated cells validated the data obtained by means of the MTT colorimetric assay, while flow cytometry analysis revealed ROS production but no induction of apoptosis in cancer cells.
We designed and synthesized 48 aryl-1H-imidazole derivatives and investigated their in vitro growth inhibitory activity in cancer cell lines known to present various levels of resistance to pro-apoptotic stimuli. The IC50 in vitro growth inhibitory concentration of these compounds ranged from >100µM to single digit µM. Among the most active compounds, 2i displayed similar in vitro growth inhibition in cancer cells independently of the cells' levels of resistance to pro-apoptotic stimuli, and was found to be cytostatic in melanoma cell lines. Compound 2i was then tested by the National Cancer Institute Human Tumor Cell Line Anti-Cancer Drug Screen, and the NCI COMPARE algorithm did not reveal any correlation between its growth inhibition profiles with the NCI database compound profiles. The use of transcriptomically characterized melanoma models then enabled us to highlight mitochondrial targeting by 2i. This hypothesis was further confirmed by reactive oxygen production measurement and oxygen consumption analysis.
Sirtuins are NAD+-dependent protein deacetylases that regulate metabolism and aging-related processes. Sirt2 is the only cytoplasmic isoform among the seven mamalian Sirtuins (Sirt1-7) and structural information concerning this isoform is limited. We crystallized Sirt2 in complex with a product analog, ADP-ribose, and solved this first crystal structure of a Sirt2 ligand complex at 2.3Å resolution. Additionally, we re-refined the structure of the Sirt2 apoform and analyzed the conformational changes associated with ligand binding to derive insights into the dynamics of the enzyme. Our analyses also provide information on Sirt2 peptide substrate binding and structural states of a Sirt2-specific protein region, and our insights and the novel Sirt2 crystal form provide helpful tools for the development of Sirt2 specific inhibitors.
The sirtuin SIRT1 is a NAD-dependent deacetylase, a Sir2 family member, and one of seven human sirtuins. Sirtuins are conserved from archaea to mammals and regulate transcription, genome stability, longevity, and metabolism. SIRT1 regulates transcription via deacetylation of transcription factors such as PPAR, NFB, and the tumor suppressor protein p53. EX527 (27) is a nanomolar SIRT1 inhibitor and a micromolar SIRT2 inhibitor. To elucidate the mechanism of SIRT inhibition by 27, we determined the 2.5 Å crystal structure of the SIRT1 catalytic domain (residues 241-516) bound to NAD and the 27 analog compound 35. 35 binds deep in the catalytic cleft displacing the NAD nicotinamide and forcing the cofactor into an extended conformation. The extended NAD conformation sterically prevents substrate binding. The SIRT1/NAD/35 crystal structure defines a novel mechanism of histone deacetylase inhibition and provides a basis for understanding, and rationally improving, inhibition of this therapeutically important target by drug-like molecules.
A series of chroman derivatives previously reported as potassium channel openers, as well as some newly synthesized simplified structures, were examined for their in vitro effects on the growth of three human high-grade glioma cell lines: U373, T98G, and Hs683. Significant in vitro growth inhibitory activity was observed with 2,2-dimethylchroman-type nitro-substituted phenylthioureas, such as compounds 4o and 4p. Interestingly, most tested phenylureas were found to be slightly less active, but more cell selective (normal versus tumor glial cells, such as 3d, 3e, and 3g), thus less toxic, than the corresponding phenylthioureas. No significant differences were observed in terms of chroman-derivative-induced growth inhibitory effects between glioma cells sensitive to pro-apoptotic stimuli (Hs683 glioma cells) and glioma cells associated with various levels of resistance to pro-apoptotic stimuli (U373 and T98G glioma cells), a feature that suggests non-apoptotic-mediated growth inhibition. Flow cytometry analyses confirmed the absence of pro-apoptotic effects for phenylthioureas and phenylureas when analyzed in U373 glioma cells and demonstrated U373 cell cycle arrest in the G0/G1 phase. Computer-assisted phase-contrast videomicroscopy revealed that 3d and 3g displayed cytostatic effects, while 3e displayed cytotoxic ones. As a result, this work identified phenylurea-type 2,2-dimethylchromans as a new class of antitumor agents to be further explored for an innovative therapeutic approach for high-grade glioma and/or for a possible new mechanism of action.
To overcome the intrinsic resistance of cancer cells to apoptotic stimuli, we designed and synthesized approximately 50 novel β-carbolines structurally related to harmine. Harmine is known for its anticancer properties and is a DYRK1A inhibitor. Of the synthesized compounds, the most active in terms of growth inhibition of five cancer cell lines are cytostatic and approximately 100 times more potent than harmine but demonstrated no DYRK1A inhibitory activity. These novel β-carbolines display similar growth inhibitory activity in cancer cells that are sensitive and resistant to apoptotic stimuli. Using ChemGPS-NP, we found that the more active β-carbolines are all more lipophilic and larger than the less active compounds. Lastly, on the basis of the NCI human tumor cell line anticancer drug screen and the NCI COMPARE algorithm, it appears that some of these compounds, including 5a and 5k, seem to act as protein synthesis inhibitors.
Epigenetic alterations are involved in every step of carcinogenesis. The development of chromatin-modifying agents (CMAs) has provided the ability to fight cancer by reversing these alterations. Currently, four CMAs have been approved for cancer treatment; two DNA demethylating agents and two deacetylase inhibitors. A number of promising CMAs are undergoing clinical trials in several cancer types. Moreover, already approved CMAs are still under clinical investigation to improve their efficacy and to extend their use to a broader panel of cancers. Combinatorial treatments with CMAs are already considered a promising strategy to improve clinical benefits and to limit side effects. The real mechanisms by which these CMAs allow the improvement and remission of patients are still obscure. A deeper analysis of the molecular features expressed by responding patients should be performed to reveal this information. In this review, we focus on clinical trials with CMAs, discussing the success and the pitfalls of this new class of anti-cancer drugs.
We describe a graphical system for automatically generating multiple 2D diagrams of ligand-protein interactions from 3D coordinates. The diagrams portray the hydrogen-bond interaction patterns and hydrophobic contacts between the ligand(s) and the main-chain or side-chain elements of the protein. The system is able to plot, in the same orientation, related sets of ligand-protein interactions. This facilitates popular research tasks, such as analyzing a series of small molecules binding to the same protein target, a single ligand binding to homologous proteins, or the completely general case where both protein and ligand change.
The synthesis of different series of 4- and 6-substituted R/S-3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans is described. All of these new benzopyran derivatives were bearing, at the 4-position, a phenylthiourea moiety substituted on the phenyl ring by a meta or a para-electron-withdrawing group such as Cl or CN. The study aimed at exploring the influence of the nature of the substituent at the 6-position in order to develop new benzopyran-type K(ATP) channel activators exhibiting an improved selectivity towards the insulin secreting cells. The original compounds were examined in vitro on rat pancreatic islets (inhibition of insulin release) as well as on rat aorta rings (vasorelaxant effect) and their activity was compared to that of the reference K(ATP) channel activators (±)-cromakalim, (±)-pinacidil, diazoxide and to previously synthesized cromakalim analogues. Structure-activity relationships indicated that the inhibitory effect on the insulin secreting cells was related to the lipophilicity of the molecules and to the size of the substituent located at the 6-position. A marked inhibitory activity on the insulin secretory process was obtained with molecules bearing a bulky tert-butyloxycarbonylamino group at the 6-position (20-23). The latter compounds were found to have the same efficacy on the pancreatic endocrine tissue than some previously described molecules. Lastly, radioisotopic experiments further identified R/S-N-4-chlorophenyl-N'-(6-tert-butyloxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)thiourea (23) as a K(ATP) channel opener.
In addition to its demethylating properties, 2'-deoxy-5-azacytidine (DAC) induces cell cycle arrest, differentiation, cell sensitization to chemotherapy, and cell death. However, the mechanisms by which DAC induces antiproliferation via these processes and how they are interconnected remain unclear. In this study, we found that a clinically relevant concentration of DAC triggered erythroid and megakaryocytic differentiation in the human chronic myeloid leukemia (CML) K-562 and MEG-01 cell lines, respectively. In addition, cells showed a marked increase in cell size in both cell lines and a more adhesive cell profile for MEG-01. Furthermore, DAC treatment induced cellular senescence and autophagy as shown by β-galactosidase staining and by autophagosome formation, respectively. After prolonged DAC treatment, phosphatidyl serine exposure, nuclear morphology analysis, and caspase cleavage revealed an activation of mitochondrial-dependent apoptosis in CML cells. This activation was accompanied by a decrease of anti-apoptotic proteins and an increase of calpain activity. Finally, we showed that combinatory treatment of relatively resistant CML with DAC and either conventional apoptotic inducers or with an histone deacetylase inhibitor increased synergistically apoptosis. We therefore conclude that induction of differentiation, senescence, and autophagy in CML are a key in cell sensitization and DAC-induced apoptosis.
J. Neurochem. (2010) 113, 418–431.
We investigated whether the CYP46A1 gene, a neuronal-specific cytochrome P450, responsible for the majority of brain cholesterol turnover, is subject to transcriptional modulation through modifications in histone acetylation. We demonstrated that inhibition of histone deacetylase activity by trichostatin A (TSA), valproic acid and sodium butyrate caused a potent induction of both CYP46A1 promoter activity and endogenous expression. Silencing of Sp transcription factors through specific small interfering RNAs, or impairing Sp binding to the proximal promoter, by site-directed mutagenesis, led to a significant decrease in TSA-mediated induction of CYP46A1 expression/promoter activity. Electrophoretic mobility shift assay, DNA affinity precipitation assays and chromatin immunoprecipitation assays were used to determine the multiprotein complex recruited to the CYP46A1 promoter, upon TSA treatment. Our data showed that a decrease in Sp3 binding at particular responsive elements, can shift the Sp1/Sp3/Sp4 ratio, and favor the detachment of histone deacetylase (HDAC) 1 and HDAC2 and the recruitment of p300/CBP. Moreover, we observed a dynamic change in the chromatin structure upon TSA treatment, characterized by an increase in the local recruitment of euchromatic markers and RNA polymerase II. Our results show the critical participation of an epigenetic program in the control of CYP46A1 gene transcription, and suggest that brain cholesterol catabolism may be affected upon treatment with HDAC inhibitors.
The tenovins and cambinol are two classes of sirtuin inhibitor that exhibit antitumor activity in preclinical models. This report describes modifications to the core structure of cambinol, in particular by incorporation of substituents at the N1-position, which lead to increased potency and modified selectivity. These improvements have been rationalized using molecular modeling techniques. The expected functional selectivity in cells was also observed for both a SIRT1 and a SIRT2 selective analog.
In 2004, a randomised phase III trial by the European Organisation for Research and Treatment of Cancer (EORTC) and National Cancer Institute of Canada Clinical Trials Group (NCIC) reported improved median and 2-year survival for patients with glioblastoma treated with concomitant and adjuvant temozolomide and radiotherapy. We report the final results with a median follow-up of more than 5 years.
Adult patients with newly diagnosed glioblastoma were randomly assigned to receive either standard radiotherapy or identical radiotherapy with concomitant temozolomide followed by up to six cycles of adjuvant temozolomide. The methylation status of the methyl-guanine methyl transferase gene, MGMT, was determined retrospectively from the tumour tissue of 206 patients. The primary endpoint was overall survival. Analyses were by intention to treat. This trial is registered with Clinicaltrials.gov, number NCT00006353.
Between Aug 17, 2000, and March 22, 2002, 573 patients were assigned to treatment. 278 (97%) of 286 patients in the radiotherapy alone group and 254 (89%) of 287 in the combined-treatment group died during 5 years of follow-up. Overall survival was 27.2% (95% CI 22.2-32.5) at 2 years, 16.0% (12.0-20.6) at 3 years, 12.1% (8.5-16.4) at 4 years, and 9.8% (6.4-14.0) at 5 years with temozolomide, versus 10.9% (7.6-14.8), 4.4% (2.4-7.2), 3.0% (1.4-5.7), and 1.9% (0.6-4.4) with radiotherapy alone (hazard ratio 0.6, 95% CI 0.5-0.7; p<0.0001). A benefit of combined therapy was recorded in all clinical prognostic subgroups, including patients aged 60-70 years. Methylation of the MGMT promoter was the strongest predictor for outcome and benefit from temozolomide chemotherapy.
Benefits of adjuvant temozolomide with radiotherapy lasted throughout 5 years of follow-up. A few patients in favourable prognostic categories survive longer than 5 years. MGMT methylation status identifies patients most likely to benefit from the addition of temozolomide.
EORTC, NCIC, Nélia and Amadeo Barletta Foundation, Schering-Plough.
T98 and T98G are two related cell lines that were derived from a human glioblastoma multiforma tumor. T98G has almost twice as many chromosomes as T98, suggesting that it is a polyploid variant of T98. Three aspects of control of cellular proliferation were studied in T98 and T98G cells in comparison to WI-38 normal human diploid cells. WI-38 cells have the following properties: (1) they can undergo only a limited number of population doublings in vitro; (2) they cannot proliferate without anchorage; and (3) they become arrested in G1 phase under stationary phase conditions. T98 cells differ from normal cells in all three of these properties, as do many other transformed cell lines. However, the derivative of T98, namely T98G, expresses an unique combination of normal and transformed aspects of the control of cellular proliferation. T98G cells are like normal cells in that they become arrested in G1 phase under stationary phase conditions, yet they also exhibit the transformed characteristics of anchorage independence and immortality. Thus, T98G cells demonstrate that transformation to immortality and anchorage independence can exist without concomitant loss of the normal mechanism for G1 arrest in response to stationary phase conditions. This result supports the hypothesis that each of these three aspects of control of cellular proliferation can be altered independently. Partially transformed cell lines, such as T98G, should be useful for sorting out the biochemical changes associated with transformation in each of these aspects.