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Combination treatment with doxorubicin and gamitrinib synergistically augments anticancer activity through enhanced activation of Bim
© Park et al.; licensee BioMed Central Ltd. 2014
Received: 27 January 2014
Accepted: 9 June 2014
Published: 13 June 2014
A common approach to cancer therapy in clinical practice is the combination of several drugs to boost the anticancer activity of available drugs while suppressing their unwanted side effects. In this regard, we examined the efficacy of combination treatment with the widely-used genotoxic drug doxorubicin and the mitochondriotoxic Hsp90 inhibitor gamitrinib to exploit disparate stress signaling pathways for cancer therapy.
The cytotoxicity of the drugs as single agents or in combination against several cancer cell types was analyzed by MTT assay and the synergism of the drug combination was evaluated by calculating the combination index. To understand the molecular mechanism of the drug synergism, stress signaling pathways were analyzed after drug combination. Two xenograft models with breast and prostate cancer cells were used to evaluate anticancer activity of the drug combination in vivo. Cardiotoxicity was assessed by tissue histology and serum creatine phosphokinase concentration.
Gamitrinib sensitized various human cancer cells to doxorubicin treatment, and combination treatment with the two drugs synergistically increased apoptosis. The cytotoxicity of the drug combination involved activation and mitochondrial accumulation of the proapoptotic Bcl-2 family member Bim. Activation of Bim was associated with increased expression of the proapoptotic transcription factor C/EBP-homologous protein and enhanced activation of the stress kinase c-Jun N-terminal kinase. Combined drug treatment with doxorubicin and gamitrinib dramatically reduced in vivo tumor growth in prostate and breast xenograft models without increasing cardiotoxicity.
The drug combination showed synergistic anticancer activities toward various cancer cells without aggravating the cardiotoxic side effects of doxorubicin, suggesting that the full therapeutic potential of doxorubicin can be unleashed through combination with gamitrinib.
Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone that controls folding of a wide range of protein substrates, or clients, many of which are involved in signal pathways crucial for tumorigenesis [1, 2]. The primary cellular location of Hsp90 is the cytoplasm, but a pool of Hsp90 and its isoform, tumor necrosis factor receptor-associated protein 1 (TRAP1), has been reported in mitochondria [3, 4]. The mitochondrial expression of Hsp90 and TRAP1 is often elevated in many cultured cancer cells and human cancer patients [3, 5, 6]. These proteins play important roles in multistep tumorigenic processes including the neoplastic metabolic shift to aerobic glycolysis [7–9] and inhibition of cell death .
A class of mitochondriotropic Hsp90 inhibitors, named gamitrinibs (GA mitochondrial matrix inhibitors), has been developed through combinatorial chemistry . Gamitrinibs consist of geldanamycin, a competitive inhibitor of the ATPase pocket of Hsp90 and TRAP1, conjugated with tandem repeats of tetracyclic guanidinium or triphenylphosphonium for mitochondrial targeting [10, 11]. Gamitrinibs not only trigger massive cell death in cultured cancer cells in vitro but also strongly suppress tumor growth in various xenograft and genetic mouse cancer models in vivo[10, 12, 13]. The gamitrinib-induced cytotoxicity is attributed to the reactivation of cyclophilin D (Cyp-D), an opener of the permeability transition pore (PTP) located in the mitochondrial inner membrane [3, 14]. Because such opening of the PTP can be lethal, Cyp-D activation is often suppressed in cancer cells by interaction with mitochondrial Hsp90s, which increase resistance to various cellular stresses . In addition, gamitrinibs have been shown to induce organelle-specific stress responses and dysregulation of bioenergetics in mitochondria of cancer cells, concomitantly compromising neoplastic growth [9, 15–17].
Doxorubicin (DOX), an anthracycline antibiotic with the trade name Adriamycin, is one of the most effective anticancer drugs and has been widely used in various chemotherapeutic regimens to treat patients with cancer . The antitumor activities of DOX are primarily attributed to DNA damage resulting from the inhibition of DNA topoisomerase II [18, 19]. The clinical use of DOX, however, has been limited by the risk of cardiotoxicity, which is dependent on the cumulative dose/treatment schedule, typically refractory to common medications, and can be fatal [20–22].
Here, we examined whether a combination of two cytotoxic drugs with unrelated action mechanisms, DOX (genotoxic) and gamitrinib (mitochondriotoxic), would exhibit enhanced anticancer activity without aggravating unwanted side effects. This drug combination showed synergistically increased anticancer activities in vitro and in vivo, without augmenting cardiomyocyte toxicity. The underlying mechanism of action involved the activation of a proapoptotic Bcl-2 protein following the stimulation of CHOP and JNK pathways in cancer cells.
Chemicals and antibodies
Gamitrinib conjugated with triphenylphosphonium was prepared as described previously . MitoTracker, JC-1, and tetramethylrhodamine methyl ester (TMRM) were purchased from Molecular Probes. All other chemicals were purchased from Sigma.
The following antibodies were used in this study: anti-JNK, anti-phospho-JNK (Thr183/Tyr185), anti-COX-IV, and anti-CHOP from Cell Signaling Technology; anti-cytochrome c, anti-Bim, and anti-PARP from Santa Cruz Biotechnology; anti-β-actin from MP Biomedicals; and anti-TRAP1, anti-Bax, anti-caspase-8, and anti-caspase-3 from BD Biosciences.
Cells and cell culture
Human cancer cell types that originated from ovary (SK-OV3), prostate (22Rv1 and PC3), cervix (HeLa), breast (MDA-MB-231), liver (SK-HEP-1), brain (A172), kidney (ACHN), and lung (NCI-H460) were purchased from the Korean Cell Line Bank. Cells were cultured in DMEM or RPMI (GIBCO) medium containing 10% FBS (GIBCO) and 1% penicillin/streptomycin (GIBCO) at 37°C in a 10% CO2 humidified atmosphere.
Small interfering RNAs (siRNAs) against JNK and CHOP were synthesized by Genolution Inc (Korea). siRNA sequences used in this study were as follows:
JNK1-#1, 5′-AAAGAATGTCCTACCTTCTCT-3′; JNK1-#2, 5′-AAGCCCAGTAATATAGTAGTA-3′; CHOP-#1, 5′-AGAACCAGCAGAGGTCACAA-3′; CHOP-#2, 5′-AAGAGAATGAACGGCTCAAGC-3′; Bim-#1, 5′-GCAACCTTCTGATGTAAGT-3′; Bim-#2, 5′-GACCGAGAAGGTAGACAATT-3′ and control, 5′-ACUCUAUCUGCACGCUGAC-3′. Cells were cultured on 6-well plates to 50–75% confluence, transfected with 40 nM siRNA mixed with G-Fectin (Genolution) for 48 hours, and then analyzed or treated with drugs.
Analysis of cell viability and apoptosis induction
Cell viability was determined using 3(4,5-dimethyl-thyzoyl-2-yl)2,5 diphenyltetrazolium bromide (MTT) and quantified by absorbance at 595 nm. Percent viability was determined by comparison with vehicle-treated control samples. To measure apoptosis, DNA content (propidium iodide or sytox staining), externalized phosphatidylserine (Annexin V) and caspase activation (DEVDase activity) of the cells were determined using the CaspaTag in situ apoptosis detection kit (Millipore) and Dead Cell Apoptosis Kit with Annexin V APC and SYTOX® Green (Molecular probes). Labeled cells were analyzed using a FACS Calibur™ flow cytometer (BD Biosciences). Data were processed using FlowJo software (TreeStar).
Western blot analysis and mitochondrial fractionation
Mitochondrial fractionation from cultured cells was performed with a Mitochondrial Isolation kit (Thermo Scientific) as described in the manufacturer’s instructions. For western blot analysis, proteins were separated on 8-12% SDS-polyacrylamide gels and transferred to polyvinyl difluoride membranes (Millipore). Primary antibodies were diluted 100–5,000-fold, and horseradish peroxidase-conjugated mouse or rabbit secondary antibodies (KLP Inc.) were diluted 5,000-fold. The ECL reagent (GE Healthcare) was used for chemiluminescence detection with a LAS 4000 imager (GE Healthcare).
Tumor xenograft experiment
All experiments involving animals were approved by the Ulsan National Institute of Science and Technology Animal Care and Use Committee (approval number: UNISTIACUC-12-003-A). Cancer cells (7 × 106 22Rv1 or 1 × 107 MDA-MB-231) were suspended in sterile 200 μl PBS. 22Rv1 cells were injected subcutaneously into both flanks of 8-week-old BALB/c nu/nu male mice (Japan SLC Inc.). MDA-MB-231 cells were orthotopically injected into the mammary fat pad of 8-week-old BALB/c nu/nu female mice. Gamitrinib or vehicle (DMSO) dissolved in 20% Cremophor EL (Sigma) in PBS was injected intraperitoneally (i.p.), and DOX diluted in PBS was injected intravenously (i.v.). The mice were treated with 10 mg/kg gamitrinib and/or 3 mg/kg DOX twice a week according to the group. Tumors were measured daily with a caliper and tumor volume was calculated using the formula V = 1/2 × (width)2 × length. At the end of the experiment the animals were euthanized, and organs including brain, heart, kidney, liver, lung, spleen, stomach, intestine, and testis, and tumors were collected for histologic or western blot analyses. Blood was also collected for measurement of serum creatine phosphokinase activity using the Indiko and Konelab System CK (Thermo Scientific) according to the manufacturer’s instructions.
RNA extraction and reverse transcript-PCR
Total RNA was prepared from cells suspended in cold PBS using the RNeasy mini kit (QIAGEN), and cDNA was synthesized using the ProtoScript® First Strand cDNA Synthesis Kit (New England Biolabs) using an oligo(dT) primer. The PCR reaction was performed in a Mastercycler PCR machine (Eppendorf) with the following sets of oligonucleotide primers: NOXA, 5′-GTGCCCTTGGAAACGGAAGA-3′ and 5′-CCAGCCGCCCAGTCTAATCA-3′; PUMA, 5′-CAGACTGTGAATCCTGTGCT-3′ and 5′-ACAGTATCTTACAGGCTGGG-3′; DR5, 5′-TGCAGCCGTAGTCTTGATTG-3′ and 5′-GAGTCAAAGGGCACCAAGTC-3′; Bcl-2, 5′-TTTTAGGAGACCGAAGTCCG-3′ and 5′-AGCCAACGTGCCATGTGCTA-3′; Bim, 5′- ATGGCAAAGCAACCTTCTGA-3′ and 5′-GGAAGCCATTGCACTGAGA-3′; CHOP, 5′-CTTTCTCCTTCGGGACACTG-3′ and 5′-AGCCGTTCATTCTCTTCAGC-3′ GAPDH, 5′-GGGAAGCTTGTCATCAATG-3′ and 5′-GCAGTGATGGCATGGACT-3′.
Data from MTT assay (triplicate experiments independently repeated at least two times) were averaged and statistically analyzed by unpaired t-test using Prism 5.0 (GraphPad). A p-value less than 0.05 was considered significant. To investigate the synergistic efficacy of the drug combination, the combination index (CI) was determined according to the Chou-Talalay method using CalcuSyn software version 2.1 (Biosoft) .
Gamitrinib-doxorubicin combination treatment showed synergistic enhancement of cytotoxicity in various cancer cell lines
Combination Index (CI) values at ED 50 and ED 75 in various cancer cell lines
CI at ED50
CI at ED75
DOX : Gami
1 : 5
0.33 ± 0.03
0.41 ± 0.03
1 : 10
0.81 ± 0.18
0.53 ± 0.09
1 : 10
0.45 ± 0.28
0.76 ± 0.27
1 : 2
0.34 ± 0.11
0.20 ± 0.07
1 : 5
0.31 ± 0.05
0.29 ± 0.03
1 : 5
0.32 ± 0.19
0.45 ± 0.29
1 : 1
0.58 ± 0.13
0.95 ± 0.28
5 : 1
0.73 ± 0.27
0.46 ± 0.24
Combination of DOX and gamitrinib augments apoptotic cell death
Gamitrinib and DOX combination treatment activates expression of CHOP and Bim
Gamitrinib and DOX combination treatment enhances mitochondrial localization of Bim and Bax
Drug combination treatment effectively inhibited tumor growth in vivowithout aggravating cardiotoxic side effects
In this study, DOX, one of the most widely used anticancer drugs, was combined with the mitochondria-stress inducer, gamitrinib, to exploit disparate stress pathways in cancer therapy. Combination of these agents synergistically increased cancer-specific cytotoxic activity through stimulation of JNK and CHOP stress signaling pathways and activation of the proapoptotic protein Bim. Importantly, the drug combination did not aggravate the well-known cardiotoxic side effects of DOX in vitro or in vivo.
Both gamitrinib [15, 16] and DOX [38, 39] have previously been shown to activate JNK and CHOP signaling pathways. Turning on these stress pathways activates the proapoptotic Bcl-2 family protein Bim through elevated gene expression and/or phosphorylation, leading to mitochondrial cell death [40, 41]. As a result of simultaneous stimulatory effects on the stress pathways by DOX and gamitrinib, the drug combination is able to further increase the amount of Bim protein (through CHOP elevation) and/or mitochondrial accumulation of Bim (through JNK activation), leading to enhanced mitochondrial accumulation of Bax and synergistic induction of apoptotic cell death.
Combining cancer drugs with disparate mechanisms of action is a feasible strategy to increase therapeutic efficacy while avoiding unacceptable side effects of the drugs . In this regard, combined treatment of DOX with other cancer drugs has been examined before and some of these combinations, for example with taxane or trastuzumab, have shown much more severe cardiotoxic side effects even at lower cumulative doses . The combination of DOX and gamitrinib, however, did not aggravate cytotoxicity to cardiomyocytes in vitro or in vivo. We presume that cardiomyocytes are relatively resistant to gamitrinib because they are less dependent on mitochondrial chaperone functions to maintain protein homeostasis and cope with stresses under normal physiologic conditions.
In conclusion, combined treatment of DOX and gamitrinib showed synergistically enhanced cancer-specific toxicity without aggravating cardiotoxic side effects. The drug combination can realize the full potential of the anticancer activity of the individual drugs and broaden the application of the drugs to various cancer types.
H.K.P. was supported by a National Junior Research Fellowship from the National Research Foundation of Korea (NRF-2011-0011833). This work was supported by the Science Research Center Programs through the National Research Foundation of Korea (2010–0028684), the Agenda program (PJ008969) through the Rural Development Administration of Korea, and the Bio-industry Technology Development program (311006–3) through the Ministry for Food, Agriculture, Forestry, and Fisheries of Korea. The funders had no role in study design, data collection and analysis, preparation of the manuscript, or decision to publish.
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