|Antiproliferative and proapoptotic activity of GUT-70 mediated through potent inhibition of Hsp90 in mantle cell lymphoma.|
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|PMID: 21139584 Owner: NLM Status: MEDLINE|
|BACKGROUND: Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with poor prognosis, requiring novel anticancer strategies.
METHODS: Mantle cell lymphoma cell lines with known p53 status were treated with GUT-70, a tricyclic coumarin derived from Calophyllum brasiliense, and the biological and biochemical consequences of GUT-70 were studied.
RESULTS: GUT-70 markedly reduced cell proliferation/viability through G(1) cell cycle arrest and increased apoptosis, with greater sensitivity in mutant (mt)-p53-expressing MCL cells than in wild-type (wt)-p53-bearing cells. Mechanistically, GUT-70 showed binding affinity to heat-shock protein 90 (Hsp90) and ubiquitin-dependent proteasomal degradation of Hsp90 client proteins, including cyclin D1, Raf-1, Akt, and mt-p53. Depletion of constitutively overexpressed cyclin D1 by GUT-70 was accompanied by p27 accumulation and decreased Rb phosphorylation. GUT-70 induced mitochondrial apoptosis with Noxa upregulation and Mcl-1 downregulation in mt-p53 cells, but Mcl-1 accumulation in wt-p53 cells. Noxa and Mcl-1 were coimmunoprecipitated, and activated BAK. Treatment with a combination of GUT-70 and bortezomib or doxorubicin had synergistic antiproliferative effects in MCL cells that were independent of p53 status.
CONCLUSION: GUT-70 has pronounced antiproliferative effects in MCL with mt-p53, a known negative prognostic factor for MCL, through Hsp90 inhibition. These findings suggest that GUT-70 has potential utility for the treatment of MCL.
|L Jin; Y Tabe; S Kimura; Y Zhou; J Kuroda; H Asou; T Inaba; M Konopleva; M Andreeff; T Miida|
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|Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2010-12-07|
|Title: British journal of cancer Volume: 104 ISSN: 1532-1827 ISO Abbreviation: Br. J. Cancer Publication Date: 2011 Jan|
|Created Date: 2011-01-05 Completed Date: 2011-02-15 Revised Date: 2013-07-03|
Medline Journal Info:
|Nlm Unique ID: 0370635 Medline TA: Br J Cancer Country: England|
|Languages: eng Pagination: 91-100 Citation Subset: IM|
|Department of Clinical Laboratory Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.|
|APA/MLA Format Download EndNote Download BibTex|
Apoptosis / drug effects*
Boronic Acids / therapeutic use
Cell Cycle / drug effects
Cell Proliferation / drug effects*
Coumarins / pharmacology*
Cyclin D1 / genetics, metabolism
Drug Therapy, Combination
HSP90 Heat-Shock Proteins / antagonists & inhibitors*, genetics, metabolism
Lymphoma, Mantle-Cell / drug therapy*, metabolism, pathology
Mutation / genetics
Oncogene Protein v-akt / genetics, metabolism
Proto-Oncogene Proteins c-bcl-2 / genetics, metabolism
Proto-Oncogene Proteins c-raf / genetics, metabolism
Pyrazines / therapeutic use
RNA, Messenger / genetics
Reverse Transcriptase Polymerase Chain Reaction
Tumor Cells, Cultured
Tumor Suppressor Protein p53 / genetics, metabolism
bcl-2 Homologous Antagonist-Killer Protein / genetics, metabolism
|0/Antineoplastic Agents; 0/Boronic Acids; 0/Coumarins; 0/GUT-70; 0/HSP90 Heat-Shock Proteins; 0/PMAIP1 protein, human; 0/Proto-Oncogene Proteins c-bcl-2; 0/Pyrazines; 0/RNA, Messenger; 0/Tumor Suppressor Protein p53; 0/bcl-2 Homologous Antagonist-Killer Protein; 0/bortezomib; 136601-57-5/Cyclin D1; EC 184.108.40.206/Oncogene Protein v-akt; EC 220.127.116.11/Proto-Oncogene Proteins c-raf|
Journal ID (nlm-ta): Br J Cancer
Publisher: Nature Publishing Group
Copyright © 2011 Cancer Research UK
Revision Received Day: 06 Month: 10 Year: 2010
Accepted Day: 22 Month: 10 Year: 2010
Print publication date: Day: 04 Month: 01 Year: 2011
Electronic publication date: Day: 07 Month: 12 Year: 2010
Volume: 104 Issue: 1
First Page: 91 Last Page: 100
Publisher Item Identifier: 6606007
PubMed Id: 21139584
|Antiproliferative and proapoptotic activity of GUT-70 mediated through potent inhibition of Hsp90 in mantle cell lymphoma Alternate Title:Antiproliferative effects of GUT-70 in MCL|
1Department of Clinical Laboratory Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
2Sportology Center, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
3Division of Hematology, Respiratory Medicine and Oncology, Department of Internal Medicine, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501, Japan
4Division of Hematology and Oncology, Department of Medicine, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566, Japan
5Department of Molecular Oncology and Leukemia Program Project, Research Institute for Radiation Biology & Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan
6Department of Leukemia, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA
7Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA
Mantle cell lymphoma (MCL) is characterised by an aggressive clinical course, with rapid relapse after an initial response or primary resistance to standard chemotherapy (Jares et al, 2007). The t(11,14)(q13;32) translocation of MCL leads to overexpression of cyclin D1, which is believed to be associated with oncogenesis by causing instability of the G1/S checkpoint through promotion of cyclin-dependent kinase activity and through sequestration of the Cip/Kip family of cyclin-dependent kinase inhibitors (Sherr and Roberts, 1999; Quintanilla-Martinez et al, 2003). These activities facilitate phosphorylation and inactivation of the retinoblastoma (Rb) G1/S checkpoint protein, resulting in cell cycle progression. It has been demonstrated, however, that overexpression of cyclin D1 itself is not sufficient for development of MCL, suggesting that additional genetic events might be necessary for oncogenesis (Bodrug et al, 1994), particularly as apoptosis-related genes such as p53, INK4a/ARF, and ATM are dysregulated in MCL (Fernàndez et al, 2005; Greiner et al, 2006).
In MCL, mutation/overexpression of p53 is reported as an adverse prognostic indicator (Jares et al, 2007). As many of the antitumour effects mediated by chemotherapeutic agents depend on a p53-related pathway, resistance to chemotherapy often develops through impaired p53 signalling (Döhner et al, 1995). The 26S proteasome inhibitor bortezomib retains activity in p53-mutant (mt-p53) cells and has demonstrated single-agent efficacy in relapsed or refractory MCL, which is, however, based mainly on prolonged response rather than on an increase in ultimate survival rate (Goy et al, 2009).
Therefore, development of novel compounds that target p53-independent signalling pathways is of considerable interest in the treatment of this disease.
We have reported that the newly discovered anticancer agent GUT-70, a natural product derived from the stem bark of Calophyllum brasiliense, demonstrated cytotoxic efficacy in human leukaemic cells (Kimura et al, 2005). GUT-70 (Figure 1), characterised as a tricyclic coumarin with the formula 5-methoxy-2,2-dimethyl-6-(2-methyl-1-oxo-2-butenyl) -10-propyl-2H,8H-benzo[1,2-b;3,4-b′]dipyran-8-one (C23H26O5), significantly inhibited leukaemic cell growth with a median inhibitory concentration (IC50) of 2-5 μ without repressing colony formation by normal haematopoietic progenitors or proliferation of normal human hepatocytes at concentrations up to 30 μ (Kimura et al, 2005).
Coumarin antibiotics have been reported to bind the newly discovered C-terminal ATP binding site of 90 kDa heat-shock protein (Hsp90), a molecular chaperone responsible for the folding and conformational maintenance of client proteins (Marcu et al, 2000; Issacs et al, 2003; Pratt and Toft, 2003; Donnelly et al, 2008). Hsp90 inhibition results in degradation of misfolded Hsp90 clients through ubiquitination, followed by proteasome-mediated hydrolysis (Zhang et al, 2004). As many of the Hsp90 client proteins contribute to cancer cell proliferation, Hsp90 has emerged as a promising target for cancer chemotherapy (Issacs et al, 2003; Donnelly et al, 2008).
In this study, GUT-70 demonstrated antiproliferative and proapoptotic activities with more prominent efficacy in mt-p53-bearing MCL cells than in those with wild-type (wt) p53. GUT-70 showed binding affinity to Hsp90, and reduced expression of Hsp90 client proteins such as mt-p53, Raf-1, cyclin D1, and Akt. The intrinsic apoptotic pathway was activated by GUT-70 through upregulation of Noxa and BAK activation. The combination of GUT-70 with bortezomib or doxorubicin yielded synergistic antiproliferative effects independent of p53 status. These findings indicate possible efficacy and a rationale for further exploration of GUT-70 as a new therapeutic strategy for MCL.
Four MCL cell lines were used in this study: JVM-2 (Melo et al, 1986), Granta 519 (Jadayel et al, 1997) and MINO (Lai et al, 2002) were kindly provided by Dr M Raffeld, and Jeko-1 (Raynaud et al, 1993) was a gift from Dr M Seto. Granta 519 and JVM-2 express wt-p53, whereas Jeko-1 and MINO express p53 mutations (Jeko-1, loss of p53 expression; MINO, mutation at codon 147 (valine → glycine)) (Raynaud et al, 1993; Lai et al, 2002). JVM-2, Jeko-1 and MINO were cultured in RPMI 1640 medium containing 15% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Granta 519 was grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% FBS. Cells were first acclimated in RPMI 1640 or DMEM containing 5% FBS for 16 h before exposure to GUT-70 (Nippon Shinyaku, Kyoto, Japan) (Kimura et al, 2005). Control cells were treated with an equivalent amount of dimethyl sulphoxide (DMSO). Doxorubicin was obtained from Sigma (St Louis, MO, USA) and bortezomib was provided by Millennium (Cambridge, MA, USA). Human osteosarcoma cell line U2OS transfected with the histone cluster 1 (H2BK) and enhanced green fluorescent protein (EGFP) genes, U2OS-H2BK-EGFP, was grown in DMEM supplemented with 10% FBS and used for morphological observation. U2OS expresses wt-p53 (Flørenes et al, 1994).
Cell viability was assessed by the Trypan blue dye exclusion method, and cell proliferation was determined by the CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS; Promega, Madison, WI, USA).
Apoptotic cell death was evaluated through annexin V (Roche Diagnostic, Indianapolis, IN, USA) and propidium iodide (PI) positivities by a FACScan flow cytometer and Cell Quest software (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). The extent of drug-specific apoptosis was assessed by the following formula: % specific apoptosis=(test−control) × 100/(100−control).
Cell cycle distribution was determined by flow cytometric analysis of PI-stained nuclei. DNA content was determined by FACScan flow cytometer and CellQuest software. BAK activation was analyzed as previously described (Samraj et al, 2006). Briefly, cells were fixed and permeabilized using the DAKO IntraStain kit (DakoCytomation, Glostrup, Denmark) according to the manufacturer's instructions. Cells were then stained with conformation-specific monoclonal antibody against BAK (y164; Abcam, Cambridge, MA, USA) or isotype-matched control antibody for 30 min at room temperature, followed by incubation with Alexafluor 488-labeled chicken anti-rabbit secondary antibody (Molecular Probes, Eugene, OR, USA) for 30 min on ice in the dark. After the washing step, conformational change of BAK was analyzed by a FACScan flow cytometer.
Cells were solubilised in lysis buffer (phosphate-buffered saline solution (PBS), 1 × cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA), 1 × protease inhibitor (Roche), and 1 × phosphatase inhibitor cocktails I and II (Calbiochem, San Diego, CA, USA)), and incubated for 30 min on ice. Total protein (20 μg) was separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE), immunoblotted with appropriate antibodies, and reacted with enhanced chemiluminescence reagent (Amersham Biosciences, Piscataway, NJ, USA); signals were detected by a luminescent image analyser (LAS-1000 plus; Fujifilm, Tokyo, Japan). The anti-α-tubulin or anti-β-actin blot was used in parallel as a loading control. For immunoblotting, the following antibodies were used: p21Cip1/WAF1, p27KIP1, and Mcl-1 (BD-Pharmingen, San Diego, CA, USA); p53 (DO-7; Dako, Carpinteria, CA, USA); Noxa (Calbiochem); α-tubulin (Sigma-Aldrich, St Louis, MO, USA); Puma (Upstate Biotechnology, Lake Placid, NY, USA); LC-3 (MBL, Nagoya, Japan); ubiquitin (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and Hsp70, c-Raf, Akt, ERK1/2, phosphorylated-ERK1/2 Thr202/Tyr204(p-ERK1/2), cyclin D1, phosphorylated RbSer780 (p-Rb), Bim, BAK, cleaved caspase-9, cleaved caspase-3, β-actin, and horseradish peroxidase-linked anti-mouse and anti-rabbit IgG (all from Cell Signaling Technology). Protein lysates were subjected to immunoprecipitation using anti-Mcl-1 (Santa Cruz Biotechnology).
The Hsp90α inhibitor screening assay kit with Hsp90α recombinant enzyme and fluorescein isothiocyanate (FITC)-labelled geldanamycin was used (BPS Bioscience, San Diego, CA, USA). The competition of fluorescence-labelled geldanamycin for binding to purified recombinant Hsp90α was measured by Flex Station 3 (Molecular Devices, Sunnyvale, CA, USA).
U2OS-H2BK-EGFP cells (2.0 × 105 per ml) were cultured in a 35-mm dish and treated with 5 μ GUT-70 or DMSO only. Each dish was placed on the stage of a light microscope equipped with a digital camera (BZ-8000; Keyence, Osaka, Japan) at 37 °C under a humidified atmosphere of 5% CO2. Video images were collected over the period from 12 to 48 h after treatment.
Total RNAs were extracted from cells with the RNeasy Mini Kit (Qiagen, Hilden, Germany). First-strand cDNA synthesis was performed with oligo(dT) as primer (Superscript II System; Invitrogen, Carlsbad, CA, USA). Real-time reverse-transcriptase PCR was performed by the Model 7500 Real-time PCR System (Applied Biosystems, Foster City, CA, USA). Expression of Noxa and GAPDH mRNA was detected by TaqMan Gene Expression Assays (Noxa: Hs00560402_m1, GAPDH: Hs99999905_m1; Applied Biosystems). The PCR cycle number that generated the first fluorescence signal above a threshold value (the threshold cycle; Ct) was determined. The abundance of each transcript of Noxa relative to that of GAPDH was calculated as follows: relative expression=100 × 2 exp [−ΔCt], where ΔCt is the mean Ct of the transcript of interest minus the mean Ct of the transcript for GAPDH. The Ct data from duplicate PCRs were averaged for calculation of relative expression.
Cytotoxicity was assessed by the Chou-Talalay method (Chou and Talalay, 1984) using Calcusyn software (Biosoft, Cambridge, UK). The combination index (CI) values indicate degree of synergism: strong synergism (0.3–0.7), moderate synergism (0.7–0.85), and slight synergism (0.85–0.9).
Treatment with GUT-70 (Figure 1) for 48 h resulted in dose-dependent cell growth inhibition detected by MTS cell proliferation assay (IC50: Granta 519, 6. 3 μ; JVM-2, 4.5 μ; Jeko-1, 1.7 μ; MINO, 1.5 μ).
To determine whether the inhibition of cell growth by GUT-70 was associated with apoptosis and/or cell cycle arrest, we conducted flow cytometric analysis of annexin V/PI-stained and PI-stained nuclei. As shown in Figure 2A, 48 h of GUT-70 treatment induced dose-dependent increases of annexin V positivity in all cell lines; this effect was more pronounced in mt-p53-bearing Jeko-1 and MINO cells than in wt-p53-bearing Granta 519 and JVM-2 cells (specific apoptosis by 5 μM GUT-70: 40.3% for Granta 519, 40.1% for JVM-2, 78.8% for Jeko-1, 79.9% for MINO). The PI cell cycle histograms further demonstrated that GUT-70 increased the sub-G1 fraction in a time-dependent manner at a lower dose for mt-p53 cells than for wt-p53 cells; sub-G1 fractions at 24 and 48 h were 4.6 and 10.7% for Granta 519 (5 μ GUT-70), 14.8 and 34.7% for JVM-2 (5 μ), 5.2 and 19.3% for Jeko-1 (1 μ), and 12.0 and 34.9% for MINO (1 μ). Whereas GUT-70 impeded G1-S cell cycle progression in JVM-2 and Granta 519 cells, G1-S arrest was minimal in MINO and Jeko-1 cells (Figure 2B). These data suggest that GUT-70-induced cell growth inhibition resulted in part from cell cycle arrest at the G0/G1 checkpoint and in part from apoptosis induction.
We next investigated changes in cell cycle regulatory proteins associated with GUT-70 treatment. As shown in Figure 3A, GUT-70 induced p53/p21 accumulation in JVM-2 cells, but did not increase p53/p21 expression in Granta 519 cells. In Jeko-1 cells, basal p53/p21 expression was not detectable and was unaffected by GUT-70. Notably, expression of the overexpressed mt-p53 protein was reduced in MINO cells by 24 h exposure to GUT-70, without detectable p21 expression. The expression level of p27 was upregulated by GUT-70, irrespective of p53 status. GUT-70 diminished the highly expressed cyclin D1 in all tested MCL cells except JVM-2, and resulted in substantial decreases in Rb phosphorylation in all tested cells (Figure 3).
The coumarin antibiotics have been reported to bind to Hsp90 (Marcu et al, 2000). To investigate whether GUT-70 has binding affinity for Hsp90, a competitive binding assay was performed using geldanamycin, a well-characterised ATP competitive inhibitor (Gooljarsingh et al, 2006). GUT-70 demonstrated dose-dependent inhibition of geldanamycin binding to Hsp90, which indicated the binding activity of GUT-70 to Hsp90 (Figure 3B). The degradation by GUT-70 of Hsp90 client proteins, such as Raf-1 and its downstream ERK1/2 and phospho ERK1/2, as well as Akt (Pratt and Toft, 2003; Zhang et al, 2004, 2005), was detected by western blot analysis in all tested MCL cells (Figure 3A). Cyclin D1 and mt-p53, the expression of which was repressed by GUT-70, are known substrate proteins of Hsp90 (Zhang et al, 2004; Muller et al, 2008). Furthermore, GUT-70 increased expression of Hsp70, a marker of Hsp90 inhibition (Elo et al, 2005; Bao et al, 2009), in Granta 519, JVM-2, and MINO cells. In Jeko-1 cells, however, Hsp70 was detected at a level insufficient to be reliable as a marker without further induction by GUT-70 (Figure 3A).
As the client proteins of Hsp90 chaperone molecule become misfolded and ubiquitinated by Hsp90 inhibition, and are then downregulated by proteasomal degradation (Issacs et al, 2003; Zhang et al, 2004), we next tried to determine whether GUT-70 induces protein ubiquitination followed by proteasomal degradation in wt-p53-expressing JVM-2 and mt-p53-expressing MINO cells. As expected, GUT-70 treatment elevated the level of protein ubiquitination (Figure 3C); subsequent treatment with proteasome inhibitor bortezomib prevented degradation of c-Raf by GUT-70 (Figure 3D). Taken together, these data indicate the interaction of GUT-70 with Hsp90 and the destabilisation of Hsp90 client proteins by GUT-70.
To characterise the mechanism of GUT-70-induced cell death in MCL cells, we analysed the expression of apoptosis-related Bcl-2 family proteins, the BH3-only proteins Noxa, Puma, and Bim, and the other Bcl-2 family proteins, Mcl-1 and BAK, in MCL cell lines. Results show that GUT-70 induced substantial accumulation of Noxa but not of Puma (Figure 4A). Mutant-p53-bearing MCL cells demonstrated earlier Noxa induction than wt-p53 cells; peak induction of Noxa was observed after 1 h of GUT-70 treatment in MINO, after 8 h in Jeko-1, and after 24 h in JVM-2 and Granta 519 cells. GUT-70 induced upregulation of Noxa mRNA levels in all tested cells (Figure 4B).
After 24 h of GUT-70 treatment, levels of antiapoptotic protein Mcl-1 were increased in JVM-2 and Granta 519 cells, but decreased in Jeko-1 and MINO cells (Figure 4A). It is known that Noxa binds preferentially to Mcl-1 (Warr and Shore, 2008), triggers BAK or Bim release from Mcl-1, and then starts the mitochondrial apoptotic pathway (Willis et al, 2005; Hauck et al, 2009). Concordantly, we detected coimmunoprecipitation between Noxa and Mcl-1 (Figure 4C) in JVM-2 and Granta 519 cells, both of which showed accumulation of Mcl-1 induced by GUT-70. Although total BAK expression levels remained, consistently independent of GUT-70 treatment (Figure 4A), flow cytometric analysis revealed a pronounced increase of activated BAK in MINO, moderate activation in Jeko-1, and only slight activation in JVM-2 and Granta 519 cells after GUT-70 treatment (Figure 4D), activities that are in inverse relation to Mcl-1 expression levels.
Proapoptotic BH-3-only protein Bim was induced by GUT-70 at 24 h in JVM-2 cells but not in the other cell lines (Figure 4A).
Increasing evidence indicates that autophagy is one of the important mechanisms of anticancer reagent-induced cell death (Tsujimoto and Shimizu, 2005). In mammals, three modes of autophagy have been identified: macroautophagy, microautophagy, and chaperone-mediated autophagy (Levine and Klionsky, 2004). To investigate the possibility that GUT-70 promotes macroautophagy, we examined the conversion of light chain 3 (LC3) from LC3-I to LC3-II, a marker of autophagosome formation (Kabeya et al, 2000). Whereas LC3-II was moderately induced by a low serum culture condition (5%, 40 h) in wt-p53-expressing JVM-2 and Granta 519 cells, there was no change in accumulation of LC3-II following further treatment with GUT-70 (24 h). In mt-p53-bearing MINO and Jeko-1 cells, neither serum starvation nor GUT-70 treatment induced LC3-II accumulation (Figure 4A).
To assess the morphological changes induced by GUT-70, U2OS-H2BK-EGFP cells were sequentially photographed after exposure to GUT-70. Cells underwent morphological alterations, including cytoplasmic swelling and vacuolisation after 24 h of GUT-70 exposure (cellular oncosis), and cell death peaked at 36 h (secondary necrosis) (Supplementary Material 1 for Quick-Time movies) (Majno and Joris, 1995; Lemasters et al, 1998; Van Cruchten and Van Den Broeck, 2002).
To determine whether GUT-70 potentiates the commonly used chemotherapeutic agents, we assessed the effects of combinations of GUT-70 with bortezomib, a selective inhibitor of the 26S proteasome, or doxorubicin, a conventional chemodrug for MCL, on viability of wt-p53 JVM-2 and mt-p53 MINO cells. As shown in Figure 5A, both of these combination treatments had observable synergistic effects in both cell types 48 h after exposure. The averaged CI values of GUT-70/bortezomib treatment were 0.59 for JVM2 and 0.73 for MINO; for GUT-70/doxorubicin, 0.37 for JVM2 and 0.35 for MINO, indicating strong and moderate synergism, respectively.
The natural product-derived tricyclic coumarin GUT-70 exhibited single-agent antiproliferative and proapoptotic activities against MCL cell lines as a novel Hsp90 inhibitor. GUT-70's dose-dependent inhibition of geldanamycin binding to Hsp90α indicates that GUT-70 has direct binding activity to Hsp90, by which GUT-70 induces conformational change in the Hsp90 molecule and interferes with its binding of geldanamycin. This finding agrees with that of previous studies showing that coumarin antibotic novobiocin binds to the Hsp90 C-terminal ATP binding site and affects the binding of geldanamycin at the Hsp90 N-terminal domain through close interaction between amino and carboxy termini in solution (Csermely et al, 1998; Hartson et al, 1999; Marcu et al, 2000; Donnelly et al, 2008). GUT-70 induced depletion of Hsp90 client proteins mt-p53, Raf-1, cyclin D1, and Akt, and increased Hsp70, a marker of Hsp90 inhibition; these findings, along with the ubiquitin-dependent proteasomal degradation of Hsp90 client proteins, suggest that GUT-70 functions as an Hsp90 inhibitor.
It is important that mt-p53-expressing MCL cells were more sensitive to GUT-70-induced apoptosis than wt-p53-bearing MCL cells. In mt-p53 cells, prominent GUT-70-induced apoptosis was accompanied by minimal cell cycle arrest, which is consistent with a previous report of G2/M checkpoint abrogation in p53/p21-impaired cells through downregulation of Chk1 and Wee1 by Hsp90 inhibitor that resulted in premature mitotic entry and mitotic death (Tse et al, 2009).
Furthermore, GUT-70 induced the most pronounced apoptosis in MINO cells in which GUT-70 treatment depleted overexpressed mt-p53. mt-p53 is known to confer the additional ‘gain of function' as the transcription regulator. Transcriptional activation by mt-p53 has been reported for MDR1 (Sampath et al, 2006), c-MYC (Frazier et al, 1998), or GRO1 (Yan and Chen, 2009), resulting in cell proliferation, antiapoptosis, and tumourigenicity (Blandino et al, 1999). GUT-70-induced degradation of mt-p53 may successfully repress these oncogenic transcriptional activations.
Another important finding of this study is the prominent p53-independent Noxa upregulation by GUT-70. Whereas Noxa had been proposed to be a critical mediator of p53-dependent apoptosis (Oda et al, 2000), p53-independent upregulation of Noxa has been described in MCL and B-cell chronic lymphocytic leukaemia (Pérez-Galán et al, 2006; Smit et al, 2007). Furthermore, GUT-70 induced Noxa protein accumulation extremely early (1 h) in mt-p53-bearing MINO cells, indicating independence from transcriptional gene induction. Recently, Noxa degradation by direct interaction with a spliced isoform of the Kruppel-like tumour suppressor (KLF6-SV1) (Difeo et al, 2009), or by posttranscriptional stabilisation/destabilisation of Bim mRNA (Matsui et al, 2007), has been reported. Our findings indicate the possibility of posttranscriptional Noxa stabilisation by GUT-70, which requires further elucidation.
The preferred binding partner of Noxa is the multidomain antiapoptotic Bcl-2 family member Mcl-1. In response to apoptotic stimuli, Noxa binds to Mcl-1, which ultimately leads to activation of BAK by releasing BAK from the BAK−Mcl-1 complex, and triggers BAK-mediated cell death (Chen et al, 2005; Warr and Shore, 2008) (Figure S, Supplementary Material 2 (Kuroda and Kimura, 2007)). The balance between Noxa and Mcl-1 is proposed to determine cell fate as death versus survival (Mei et al, 2007). GUT-70-induced BAK activation and sequential apoptosis were associated with Mcl-1 accumulation levels; high levels in less-sensitive wt-p53 cells and low levels in highly sensitive mt-p53 cells were consistent with previous reports (Pérez-Galán et al, 2006; Mei et al, 2007).
Autophagy is known to promote both autophagic cell death and cell survival (Kamitsuji et al, 2008). Although GUT-70 did not affect autophagosome formation, Hsp90 clients have been shown to be degraded through chaperone-mediated autophagy (Shen et al, 2009). The role of GUT-70 in induction of chaperone-mediated autophagy requires further elucidation.
The observed morphological changes in GUT-70-treated cells (e.g., swelling cytoplasm) indicate cellular oncosis (Van Cruchten and Van Den Broeck, 2002), which shares certain mechanisms and alterations with apoptosis, such as loss of mitochondrial permeability and membrane potential (Lemasters et al, 1998).
Furthermore, our results demonstrate that GUT-70 can synergise the cytotoxic effects of the proteasome inhibitor bortezomib and the widely used genotoxic chemotherapeutic agent doxorubicin in MCL cells (Brody and Advani, 2006; Goy et al, 2009), regardless of p53 status. Previously, a combination of Hsp90 inhibitor geldanamycin and bortezomib was demonstrated to simultaneously disrupt Hsp90 and proteasome function, promote accumulation of ubiquitinated proteins, and enhance antitumour activity in human breast cancer cells (Mimnaugh et al, 2004, 2006). Whereas bortezomib induces longer-term remission (Goy et al, 2009), patients ultimately succumb to the poor clinical outcome, and there is a critical need to develop the most effective combination. The synergistic effects of GUT-70 and bortezomib may offer more efficacy and flexibility to the treatment of MCL with bortezomib. The antiproliferative effect of the combination of doxorubicin and GUT-70 was consistent with the previous findings for doxorubicin and Hsp-90 inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldamanycin (DMAG), which induced premature mitosis, followed by apoptosis, by bypassing the G2/M checkpoint in lymphoma cells (Robles et al, 2006). The synergy with doxorubicin suggests that addition of GUT-70 may allow reduction in the therapeutic dose of doxorubicin, which could potentially reduce its genotoxic side effects (Brody and Advani, 2006). A development of in vivo studies of these combination treatments for MCL is further required.
In conclusion, our results demonstrate that the novel anticancer agent GUT-70, a tricyclic coumarin, inhibits cell proliferation by depleting Hsp90 substrates cyclin D1, Akt, and Raf-1, and induces mitochondrial apoptotic cell death with upregulation of Noxa in MCL cells. Notably, these effects are substantially pronounced in MCL cells with mt-p53, a known negative prognostic factor for MCL. These findings suggest that GUT-70 has potential utility for the treatment of MCL.
Supplementary Information accompanies the paper on British Journal of Cancer website (http://www.nature.com/bjc)
We thank Drs Kazuhisa Iwabuchi and Akimasa Someya for invaluable help and discussion; Tomomi Ikeda and Takako Shigihara-Ikegami for technical assistance; and Drs Mark Raffeld and Masao Seto for the gifts of cell lines. We thank Katy Hale for manuscript review. This work was supported by the Project Research Program of Juntendo University School of Medicine (to LJ), the Japan Leukemia Research Fund (to YT) and the Research for Promoting Technological Seeds of the Japan Science and Technology Agency (to SK).
|Bao R,Lai CJ,Qu H,Wang D,Yin L,Zifcak B,Atoyan R,Wang J,Samson M,Forrester J,DellaRocca S,Xu GX,Tao X,Zhai HX,Cai X,Qian C. Year: 2009CUDC-305, a novel synthetic HSP90 inhibitor with unique pharmacologic properties for cancer therapyClin Cancer Res154046405719509149|
|Blandino G,Levine AJ,Oren M. Year: 1999Mutant p53 gain of function: differential effects of different p53 mutants on resistance of cultured cells to chemotherapyOncogene184774859927204|
|Bodrug SE,Warner BJ,Bath ML,Lindeman GJ,Harris AW,Adams JM. Year: 1994Cyclin D1 transgene impedes lymphocyte maturation and collaborates in lymphomagenesis with the myc geneEMBO J13212421308187765|
|Brody J,Advani R. Year: 2006Treatment of mantle cell lymphoma: current approach and future directionsCrit Rev Oncol Hematol5825726516751087|
|Chen L,Willis SN,Wei A,Smith BJ,Fletcher JI,Hinds MG,Colman PM,Day CL,Adams JM,Huang DC. Year: 2005Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic functionMol Cell1739340315694340|
|Chou TC,Talalay P. Year: 1984Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitorsAdv Enzyme Regul2227556382953|
|Csermely P,Schnaider T,Soti C,Prohaszka Z,Nardai G. Year: 1998The 90-kDa molecular chaperone family: structure, function, and clinical applications. A comprehensive reviewPharmacol Ther791291689749880|
|Difeo A,Huang F,Sangodkar J,Terzo EA,Leake D,Narla G,Martignetti JA. Year: 2009KLF6-SV1 is a novel antiapoptotic protein that targets the BH3-only protein NOXA for degradation and whose inhibition extends survival in an ovarian cancer modelCancer Res694733474119435908|
|Döhner H,Fischer K,Bentz M,Hansen K,Benner A,Cabot G,Diehl D,Schlenk R,Coy J,Stilgenbauer S,Volkmann M,Galle PR,Poustka A,Hunstein W,Lichter P. Year: 1995p53 gene deletion predicts for poor survival and non-response to therapy with purine analogs in chronic B-cell leukemiasBlood85158015897888675|
|Donnelly AC,Mays JR,Burlison JA,Nelson JT,Vielhauer G,Holzbeierlein J,Blagg BS. Year: 2008The design, synthesis, and evaluation of coumarin ring derivatives of the novobiocin scaffold that exhibit antiproliferative activityJ Org Chem738901892018939877|
|Elo MA,Kaarniranta K,Helminen HJ,Lammi MJ. Year: 2005Hsp90 inhibitor geldanamycin increases hsp70 mRNA stabilisation but fails to activate HSF1 in cells exposed to hydrostatic pressureBiochim Biophys Acta174311511915777846|
|Fernàndez V,Hartmann E,Ott G,Campo E,Rosenwald A. Year: 2005Pathogenesis of mantle-cell lymphoma: all oncogenic roads lead to dysregulation of cell cycle and DNA damage response pathwaysJ Clin Oncol236364636916155021|
|Flørenes VA,Maelandsmo GM,Forus A,Andreassen A,Myklebost O,Fodstad O. Year: 1994MDM2 gene amplification and transcript levels in human sarcomas: relationship to TP53 gene statusJ Natl Cancer Inst86129713028064888|
|Frazier MW,He X,Wang J,Gu Z,Cleveland JL,Zambetti GP. Year: 1998Activation of c-myc gene expression by tumor-derived p53 mutants requires a discrete C-terminal domainMol Cell Biol18373537439632756|
|Gooljarsingh LT,Fernandes C,Yan K,Zhang H,Grooms M,Johanson K,Sinnamon RH,Kirkpatrick RB,Kerrigan J,Lewis T,Arnone M,King AJ,Lai Z,Copeland RA,Tummino PJ. Year: 2006A biochemical rationale for the anticancer effects of Hsp90 inhibitors: slow, tight binding inhibition by geldanamycin and its analoguesProc Natl Acad Sci USA10376257630|
|Goy A,Bernstein SH,Kahl BS,Djulbegovic B,Robertson MJ,de Vos S,Epner E,Krishnan A,Leonard JP,Lonial S,Nasta S,O'Connor OA,Shi H,Boral AL,Fisher RI. Year: 2009Bortezomib in patients with relapsed or refractory mantle cell lymphoma: updated time-to-event analyses of the multicenter phase 2 PINNACLE studyAnn Oncol2052052519074748|
|Greiner TC,Dasgupta C,Ho VV,Weisenburger DD,Smith LM,Lynch JC,Vose JM,Fu K,Armitage JO,Braziel RM,Campo E,Delabie J,Gascoyne RD,Jaffe ES,Muller-Hermelink HK,Ott G,Rosenwald A,Staudt LM,Im MY,Karaman MW,Pike BL,Chan WC,Hacia JG. Year: 2006Mutation and genomic deletion status of ataxia telangiectasia mutated (ATM) and p53 confer specific gene expression profiles in mantle cell lymphomaProc Natl Acad Sci USA10323522357|
|Hartson SD,Thulasiraman V,Huang W,Whitesell L,Matts RL. Year: 1999Molybdate inhibits Hsp90, induces structural changes in its C-terminal domain, and alters its interactions with substratesBiochemistry383837384910090774|
|Hauck P,Chao BH,Litz J,Krystal GW. Year: 2009Alterations in the Noxa/Mcl-1 axis determine sensitivity of small cell lung cancer to the BH3 mimetic ABT-737Mol Cancer Ther888389219372561|
|Issacs JS,Xu W,Neckers L. Year: 2003Heat shock protein 90 as a molecular target for cancer therapeuticsCancer Cell321321712676580|
|Jadayel DM,Lukas J,Nacheva E,Bartkova J,Stranks G,De Schouwer PJ,Lens D,Bartek J,Dyer MJ,Kruger AR,Catovsky D. Year: 1997Potential role for concurrent abnormalities of the cyclin D1, p16CDKN2 and p15CDKN2B genes in certain B-cell non-Hodgkin's lymphomas: functional studies in a cell line (Granta 519)Leukemia1164729001420|
|Jares P,Colomer D,Campo E. Year: 2007Genetic and molecular pathogenesis of mantle cell lymphoma: perspectives for new targeted therapeuticsNat Rev Cancer775076217891190|
|Kabeya Y,Mizushima N,Ueno T,Yamamoto A,Kirisako T,Noda T,Kominami E,Ohsumi Y,Yoshimori T. Year: 2000LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processingEMBO J195720572811060023|
|Kamitsuji Y,Kuroda J,Kimura S,Toyokuni S,Watanabe K,Ashihara E,Tanaka H,Yui Y,Watanabe M,Matsubara H,Mizushima Y,Hiraumi Y,Kawata E,Yoshikawa T,Maekawa T,Nakahata T,Adachi S. Year: 2008The Bcr-Abl kinase inhibitor INNO-406 induces autophagy and different modes of cell death execution in Bcr-Abl-positive leukemiasCell Death Differ151712172218617896|
|Kimura S,Ito C,Jyoko N,Segawa H,Kuroda J,Okada M,Adachi S,Nakahata T,Yuasa T,Filho VC,Furukawa H,Maekawa T. Year: 2005Inhibition of leukemic cell growth by a novel anti-cancer drug (GUT-70) from Calophyllum brasiliense that acts by induction of apoptosisInt J Cancer11315816515386357|
|Kuroda J,Kimura S. Year: 2007The role of BH3-only proteins in cancer and anti-cancer therapeuticsCell Apoptosis Research TrendsIn Zhang CV (ed) pp139Nova: New York|
|Lai R,McDonnell TJ,O'Connor SL,Medeiros LJ,Oudat R,Keating M,Morgan MB,Curiel TJ,Ford RJ. Year: 2002Establishment and characterization of a new mantle cell lymphoma cell line, MINOLeuk Res2684985512127561|
|Lemasters JJ,Nieminen AL,Qian T,Trost LC,Elmore SP,Nishimura Y,Crowe RA,Cascio WE,Bradham CA,Brenner DA,Herman B. Year: 1998The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagyBiochim Biophys Acta13661771969714796|
|Levine B,Klionsky DJ. Year: 2004Development by self-digestion: molecular mechanisms and biological functions of autophagyDev Cell646347715068787|
|Majno G,Joris I. Year: 1995Apoptosis, oncosis, and necrosis. An overview of cell deathAm J Pathol1463157856735|
|Marcu MG,Schulte TW,Neckers L. Year: 2000Novobiocin and related coumarins and depletion of heat shock protein 90-dependent signaling proteinsJ Natl Cancer Inst9224224810655441|
|Matsui H,Asou H,Inaba T. Year: 2007Cytokines direct the regulation of Bim mRNA stability by heat-shock cognate protein 70Mol Cell259911217218274|
|Mei Y,Xie C,Xie W,Tian X,Li M,Wu M. Year: 2007Noxa/Mcl-1 balance regulates susceptibility of cells to camptothecin-induced apoptosisNeoplasia987188117971907|
|Melo JV,Brito-Babapulle V,Foroni L,Robinson DS,Luzzatto L,Catovsky D. Year: 1986Two new cell lines from B-prolymphocytic leukaemia: characterization by morphology, immunological markers, karyotype and Ig gene rearrangementInt J Cancer385315383093393|
|Mimnaugh EG,Xu W,Vos M,Yuan X,Isaacs JS,Bisht KS,Gius D,Neckers L. Year: 2004Simultaneous inhibition of hsp 90 and the proteasome promotes protein ubiquitination, causes endoplasmic reticulum-derived cytosolic vacuolization, and enhances antitumor activityMol Cancer Ther355156615141013|
|Mimnaugh EG,Xu W,Vos M,Yuan X,Neckers L. Year: 2006Endoplasmic reticulum vacuolization and valosin-containing protein relocalization result from simultaneous hsp90 inhibition by geldanamycin and proteasome inhibition by velcadeMol Cancer Res466768116966435|
|Muller P,Hrstka R,Coomber D,Lane DP,Vojtesek B. Year: 2008Chaperone-dependent stabilization and degradation of p53 mutantsOncogene273371338318223694|
|Oda E,Ohki R,Murasawa H,Nemoto J,Shibue T,Yamashita T,Tokino T,Taniguchi T,Tanaka N. Year: 2000Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosisScience2881053105810807576|
|Pérez-Galán P,Roué G,Villamor N,Campo E,Colomer D. Year: 2006The proteasome inhibitor bortezomib induces apoptosis in mantle-cell lymphoma through generation of ROS and Noxa activation independent of p53 statusBlood10725726416166592|
|Pratt WB,Toft DO. Year: 2003Regulation of signaling protein function and trafficking by the hsp90/hsp70-based chaperone machineryExp Biol Med228111133|
|Quintanilla-Martinez L,Davies-Hill T,Fend F,Calzada-Wack J,Sorbara L,Campo E,Jaffe ES,Raffeld M. Year: 2003Sequestration of p27Kip1 protein by cyclin D1 in typical and blastic variants of mantle cell lymphoma (MCL): implications for pathogenesisBlood1013181318712515730|
|Raynaud SD,Bekri S,Leroux D,Grosgeorge J,Klein B,Bastard C,Gaudray P,Simon MP. Year: 1993Expanded range of 11q13 breakpoints with differing patterns of cyclin D1 expression in B-cell malignanciesGenes Chromosomes Cancer880877504521|
|Robles AI,Wright MH,Gandhi B,Feis SS,Hanigan CL,Wiestner A,Varticovski L. Year: 2006Schedule-dependent synergy between the heat shock protein 90 inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin and doxorubicin restores apoptosis to p53-mutant lymphoma cell linesClin Cancer Res126547655617085670|
|Sampath J,Sun D,Kidd VJ,Grenet J,Gandhi A,Shapiro LH,Wang Q,Zambetti GP,Schuetz JD. Year: 2006Mutant p53 cooperates with ETS and selectively up-regulates human MDR1 not MRP1Clin Cancer Res123459346916740771|
|Samraj AK,Stroh C,Fischer U,Schulze-Osthoff K. Year: 2006The tyrosine kinase Lck is a positive regulator of the mitochondrial apoptosis pathway by controlling Bak expressionOncogene2518619716116473|
|Shen S,Zhang P,Lovchik MA,Li Y,Tang L,Chen Z,Zeng R,Ma D,Yuan J,Yu Q. Year: 2009Cyclodepsipeptide toxin promotes the degradation of Hsp90 client proteins through chaperone-mediated autophagyJ Cell Biol18562963919433452|
|Sherr CJ,Roberts JM. Year: 1999CDK inhibitors: positive and negative regulators of G1-phase progressionGenes Dev131501151210385618|
|Smit LA,Hallaert DY,Spijker R,de Goeij B,Jaspers A,Kater AP,van Oers MH,van Noesel CJ,Eldering E. Year: 2007Differential Noxa/Mcl-1 balance in peripheral versus lymph node chronic lymphocytic leukemia cells correlates with survival capacityBlood1091660166817038534|
|Tse AN,Sheikh TN,Alan H,Chou TC,Schwartz GK. Year: 200990-kDa heat shock protein inhibition abrogates the topoisomerase I poison-induced G2/M checkpoint in p53-null tumor cells by depleting Chk1 and Wee1Mol Pharmacol7512413318820127|
|Tsujimoto Y,Shimizu S. Year: 2005Another way to die: autophagic programmed cell deathCell Death Differ121528153416247500|
|Van Cruchten S,Van Den Broeck W. Year: 2002Morphological and biochemical aspects of apoptosis, oncosis and necrosisAnat Histol Embryol3121422312196263|
|Warr MR,Shore GC. Year: 2008Unique biology of Mcl-1: therapeutic opportunities in cancerCurr Mol Med813814718336294|
|Willis SN,Chen L,Dewson G,Wei A,Naik E,Fletcher JI,Adams JM,Huang DC. Year: 2005Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteinsGenes Dev191294130515901672|
|Yan W,Chen X. Year: 2009Identification of GRO1 as a critical determinant for mutant p53 gain of functionJ Biol Chem284121781218719258312|
|Zhang R,Luo D,Miao R,Bai L,Ge Q,Sessa WC,Min W. Year: 2005Hsp90-Akt phosphorylates ASK1 and inhibits ASK1-mediated apoptosisOncogene243954396315782121|
|Zhang Y,Wang JS,Chen LL,Cheng XK,Heng FY,Wu NH,Shen YF. Year: 2004Repression of hsp90beta gene by p53 in UV irradiation-induced apoptosis of Jurkat cellsJ Biol Chem279425454255115284248|
Supplementary Movie 1 Click here for additional data file (6606007x2.avi)
Supplementary Movie 2 Click here for additional data file (6606007x3.avi)
Supplementary Material Click here for additional data file (6606007x4.doc)
Keywords: GUT-70, mantle cell lymphoma, apoptosis, p53, Hsp90, coumarin.
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