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Kinamycin F downregulates cyclin D3 in human leukemia K562 cells
Chemico-Biological Interactions 184 (2010) 396–402
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Kinamycin F downregulates cyclin D3 in human leukemia K562 cells Kimberley A. O’Hara a , Gary I. Dmitrienko b , Brian B. Hasinoff a,∗ a b
Faculty of Pharmacy, Apotex Centre, University of Manitoba, 750 McDermot Ave., Winnipeg, Manitoba R3E 0T5, Canada Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada
a r t i c l e
i n f o
Article history: Received 6 October 2009 Received in revised form 21 December 2009 Accepted 6 January 2010 Available online 15 January 2010 Keywords: Cyclin D3 K562 cells Kinamycin Cell cycle Apoptosis
a b s t r a c t The bacterial metabolite kinamycin F, which contains an unusual and potentially reactive diazo group, is being investigated as an antitumor agent with a potentially novel target. Treatment of K562 cells with kinamycin F induced erythroid differentiation, a rapid apoptotic response, induction of caspase3/7 activities and a delayed cell cycle progression through the S and G2 /M phases. Kinamycin F caused a selective reduction of cyclin D3 protein, which appeared to be mediated at the level of transcription, rather than by affecting the stability of either cyclin D3 protein or mRNA. Thus cyclin D3 is a potential target of kinamycin F. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The kinamycins are a series of bacterial metabolites with an unusual structure and potent antibiotic and antitumor activity. Kinamycins A, B, C and D were first isolated in 1970 from Streptomyces murayamaensis [1]. The fully deacetylated kinamycin F (Fig. 1A) has been detected as a metabolite in culture broths of S. murayamaensis [2]. Kinamycin C displayed antitumor activity in an animal model [3] and submicromolar cell growth inhibitory effects in the 60-cancer cell NCI panel (http://dtp.nci.nih.gov). Initially the kinamycins were assigned an N-cyanocarbazole structure [4], however, we [5] and another group [6] independently corrected its structure to that of a diazobenzo[b]fluorene. The structurally similar antitumor antibiotic lomaiviticin A isolated from Micromonospora lomaivitiensis exhibits extremely potent activity against the modified NCI 24-cancer cell line with cell growth inhibitory effects in the picomolar range [7]. Lomaiviticin A is a dimer containing two kinamycin-like systems that are joined by a carbon–carbon bond linking each monomer. The diazo pharmacophore is not only an unusual functional group in natural products, but is also potentially reactive [8] and it presumably plays a role in the antitumor activity of the kinamycins. Consequently there has been a renewed interest in the kinamycins. The
kinamycins are small molecules that are amenable to synthesis [9]. Kinamycins C [10,11], F and J [11] have been synthesized recently. Mechanisms by which the kinamycins might be activated to damaging species have recently been reviewed [12]. We previously proposed that kinamycin F may be generated in cells by the activity of esterases on O-acetylated forms of kinamycin and that it may be the active metabolite responsible for the observed inhibition of cancer cell growth [13,14]. We have shown that kinamycins A, C and F inhibit the growth of K562 cells at submicromolar levels [13,14] and that kinamycin C induces apoptosis in K562 and Chinese hamster ovary cells (CHO) [14]. We [13] and others [15] also showed that kinamycin F damaged DNA in vitro as well as in whole cells and that it can be reductively and peroxidatively activated to a semiquinone and phenoxyl free radical, respectively. We also showed that kinamycins A, C and F are catalytic inhibitors of topoisomerase II␣ [13,14] though it is most likely not the major target of the kinamycins. The purpose of this study was to elucidate the mechanism or mechanisms by which kinamycin F exerts its ability to inhibit cell growth to enable further development of kinamycin F or its analogs as anticancer drugs with a novel target. 2. Materials and methods
Abbreviations: DMSO, dimethyl sulfoxide; DRB, 5,6-dichloro-1-dribofuranosylbenzimidazole. ∗ Corresponding author. Tel.: +1 204 474 8325; fax: +1 204 474 7617. E-mail address: B [email protected] (B.B. Hasinoff). 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.01.013
2.1. Materials and cell culture Kinamycin F was prepared semisynthetically from kinamycin D that was isolated from S. murayamaensis as described [13] and
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containing the DEVD sequence that results in a luminescence signal that is proportional to caspase-3/7 activity. 2.4. Protein isolation and immunoblotting analysis
Fig. 1. (A) Structure of kinamycin F; (B) kinamycin F increases the percentage of hemoglobin-positive K562 cells. K562 cells were treated with either DMSO vehicle or kinamycin F (5 M) for 12, 24, or 48 h after which time the percentage of cells that stained positive for hemoglobin were determined. Results are reported as the mean ± SE of four replicate measurements. Significant differences between treated and controls are designated by *p < 0.05, **p < 0.01 and ***p < 0.001.
was dissolved in DMSO. While the solubility of kinamycin F in aqueous solutions was not determined, its solubility was determined to be greater than 40 M in the culture broth and greater than 64 M in aqueous solution buffered at pH 7.0, which is a concentration more than 10-fold higher than that used in the experiments. The fact that kinamycin F is isolated from culture broth after several days of growth is indicative of its relative stability. Deferiprone was synthesized as described [16]. The level of significance used in the t-tests was p < 0.05. Human leukemia K562 cells obtained from the American Type Culture Collection (Manassas, VA) were maintained as suspension cultures as described [13]. In control and kinamycin F treatments the DMSO concentration was always 0.5% (v/v). The percentage of cells staining for hemoglobin following treatment with kinamycin F was determined by staining with benzidine/H2 O2 as previously described [17]. 2.2. Cell synchronization and flow cytometry For the synchronization experiments K562 cells, which normally have a 24 h doubling time, were grown to a density of 4 × 105 cells/ml after which they were treated for 24 h with deferiprone (300 M), an iron chelator that has been shown to synchronize K562 cells in G0 /G1 [18]. After this 24 h incubation the cells were washed 3 times with complete media and seeded in 35-mm diameter dishes after resting for 5 h. Cells were continuously treated for the times indicated with either the DMSO vehicle or kinamycin F (5 M). We previously determined that a 72 h kinamycin F treatment caused 50% cell growth inhibition at a concentration of 0.33 M [13]. Cells were fixed and stained with propidium iodide for cell cycle analysis as previously described [14]. The fraction of apoptotic cells induced by treatment of K562 cells with kinamycin F was quantified by two-color flow cytometry by simultaneously measuring integrated green (annexin-V-FITC) fluorescence and integrated red (propidium iodide) fluorescence as previously described [14]. 2.3. Caspase-3/7 luminescence assay Induction of caspase-3/7 activities in K562 cells following treatment with kinamycin F for various times was quantified using the Caspase-Glo® 3/7 Assay according to the manufacturer’s directions (Promega, Madison, WI) as previously described [19]. Upon cell lysis caspase-3 and -7 are able to cleave a proluminogenic substrate
Following treatment with DMSO or kinamycin F (5 M), pellets of 5 × 105 K562 cells were treated essentially as previously described [19]. After SDS–PAGE separation proteins were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA) and after blocking were probed with specific primary antibodies overnight at 4 ◦ C. Antibodies targeting cyclins D1, D3, E (BD Biosciences, San Diego, CA), cyclin D2 (Cell Signaling Technology, Danvers, MA) and -actin (Clone AC-15, Sigma) were used. The membranes were incubated with horseradish peroxidaseconjugated secondary antibodies (Pierce Biotechnology, Rockford, IL) and reactive bands were detected using enhanced chemiluminescence and imaged on an Alpha Innotech (San Leandro, CA) Fluorochem 8900 imaging system. In evaluating the effect of kinamycin F on the stability of cyclin D3 protein, cells were treated with either DMSO or kinamycin F (5 M) for 12 h, followed by addition of cycloheximide (10 g/ml) to inhibit further protein translation/synthesis. A 12 h treatment with 0.4 g/ml of cycloheximide has been shown to inhibit protein synthesis by 30% in K562 cells [20]. 2.5. RNA isolation and real-time RT (reverse transcriptase)–PCR Total cellular RNA was isolated using the RNeasy Mini Kit (Qiagen, Mississauga, Canada). The cDNAs were generated from 0.5 g total RNA by reverse transcription in a 20 l reaction mixture containing M-MLV reverse transcriptase 5× reaction buffer (Promega, Madison, WI), 10 U RNase inhibitor (Ambion, Streetsville, Canada), 0.5 mM of each dNTP (Fisher, Ottawa, Canada), 5 M oligo dT (GE Healthcare Bio-Sciences, Piscataway, NJ) and 200 U M-MLV reverse transcriptase (Promega). Reactions were incubated at 44 ◦ C for 60 min in the BioRad MiniOpticon (BioRad Laboratories, Hercules, CA). The cDNA was stored at −20 ◦ C until further analysis. Specific primer pairs for human cyclin D3 (forward 5 CGTGGTCGGTGTAGATGC; reverse 5 -TGGATGCTGGAGGTATGTG) and 18S rRNA (forward 5 -CTTATGACCCGCACTTACTG; reverse 5 TCCCCCAACTTCTTAGAGG) [21] were used to amplify the specific cDNAs. Specific cDNAs were amplified using 2 l aliquots of cDNA product mixed with cyclin D3 or 18S rRNA forward and reverse primers (0.38 pM each), 12.5 l of SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) in a final volume of 25 l. PCR reactions were carried out for 15 s at 94 ◦ C, 30 s at 60 ◦ C and 30 s at 72 ◦ C for 45 cycles. Gene expression was quantified using standard curves for the respective cDNA products and changes in resulting cyclin D3 cDNA levels were normalized to changes in 18S rRNA to determine the weight of normalized product per ml of reaction. In evaluating the effect of kinamycin F on the half-life of cyclin D3 mRNA, cells were pre-treated for 1 h with either DMSO or kinamycin F (5 M) followed by addition of DRB, an inhibitor of RNA polymerase II. The relative levels of cyclin D3 mRNA in both kinamycin F-treated or -untreated cells prior to DRB addition were determined. 2.6. Transient transfections and luciferase assay The human cyclin D3 (−1017 bp/+6) pGL3 plasmid [22], which contains a luciferase promoter construct, was generously provided by Dr. W. Douglas Cress (Moffitt Cancer Center, Tampa, FL). K562 cells (1 × 106 ) in 35 mm dishes were co-transfected with 3.5 g of the cyclin D3 pGL3 or pGL3 (Promega) plasmid and 0.5 g of enhanced green fluorescence protein plasmid (Clontech, Palo Alto, CA) using Lipofectamine 2000 Plus reagent and Opti-MEM
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(Invitrogen) in antibiotic-free Dulbecco’s modified Eagle’s medium. After recovering overnight, cells were treated for the times indicated with kinamycin F (5 M) and harvested for luciferase assays. The green fluorescence produced was used to assess transfection efficiency. After treatment cells were pelleted and then lysed for 15 min on ice in luciferase lysis buffer (25 mM glycylglycine, 4 mM EGTA, 15 mM MgSO4 , 1% (v/v) Triton X-100, 1 mM dithiothreitol, 1 mg/ml bovine serum albumin). 25 l of lysate was added to 75 l of luciferase assay buffer (25 mM glycylglycine, 15 mM potassium phosphate, 150 mM MgSO4 , 4 mM EGTA, 2 mM ATP, 1 mM dithiothreitol) and after luciferin (400 M) was added the luminescence was read immediately in a Fluostar Galaxy (BMG) plate reader at 25 ◦ C. The relative light units of luciferase activities per microgram of protein were calculated. 3. Results 3.1. Kinamycin F induces erythroid differentiation in K562 cells K562 cells are a human erythroleukemic cell line capable of differentiation into erythroid cells and expression of hemoglobin [23]. Thus, the expression of hemoglobin in K562 cells in response to treatment with kinamycin F (5 M) was measured as an indicator of erythroid differentiation. K562 cells continuously incubated with kinamycin F (5 M) for the times indicated (Fig. 1B) displayed significant increases in the number of cells that stained positive for hemoglobin after 12 h (p = 0.03), 24 h (p = 0.002) and 48 h (p = 0.0007).
3.2. Synchronized K562 cells exhibit an S phase accumulation and a delayed entry into the G2 /M phase To establish the effects of kinamycin F on cell cycle, deferipronesynchronized K562 cells were examined (Fig. 2A). As shown in Fig. 2, approximately 65% of the cells were in G0 /G1 when initially treated with kinamycin F (time 0 h). Cells that were treated for 4 h with kinamycin F (5 M) exited G0 /G1 in a manner similar to untreated cells (Fig. 2B, top panel). Cells exiting G0 /G1 became increasingly apoptotic as evidenced by a progressive increase in the fraction of cells in the sub-G1 phase. At 48 h, 26% of kinamycin F-treated cells had entered the sub-G1 phase compared to 5% for untreated cells. However, as shown in Fig. 2B (middle panel) treatment with kinamycin F caused an accumulation of cells in the S phase due to a delayed exit from this phase. At 12 h the kinamycin F-treated cells exhibited a delayed entry into G2 /M of about 5 h (Fig. 2B, bottom panel). Unsynchronized K562 cells were also examined for cell cycle effects induced by kinamycin F. The fraction of cells in G0 /G1 , S and G2 /M phases remained relatively unchanged except that a large fraction of cells progressively became more apoptotic as judged from a progressive increase in the sub-G1 population. For example, at 48 h, 14% of untreated cells were apoptotic compared to 47% for cells treated with kinamycin F (5 M). 3.3. Kinamycin F induces apoptosis and caspase-3/7 activation in K562 cells As shown in Fig. 3A and B, treatment with kinamycin F (5 M) for the times indicated greatly increased the proportion of apoptotic
Fig. 2. Kinamycin F inhibits cell cycle progression in K562 cells. K562 cells that had been synchronized by the treatment with deferiprone were treated, or not treated, with kinamycin F (5 M) and were then allowed to grow for the times indicated. (A) Cell counts are displayed on the vertical axis and the DNA content is plotted on the horizontal axis; (B) percentage of the non-sub G0 /G1 cells in G0 /G1 (top panel), S (middle panel) and G2 /M (bottom panel) phases are plotted as a function of time. The solid lines are least-squares calculated spline fits that connect the data points.
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Fig. 3. Kinamycin F treatment of K562 cells induces apoptosis and caspase-3/7 activity. (A) Two-color flow cytometry scatter plots of K562 cells that were untreated or treated with kinamycin F (5 M) for 6 h. The cells were then treated with annexin-V-FITC and propidium iodide. The lower left quadrant contains viable cells; the upper left quadrant contains cells that are necrotic-only; the lower right quadrant contains cells that are apoptotic-only; and the upper right quadrant contains cells that are both apoptotic and necrotic; (B) changes in relative number of K562 cells that were classified as necrotic-only; apoptotic and necrotic; apoptotic-only, or viable 0, 6, 12 and 24 h after treatment with kinamycin F (5 M); (c) caspase-3/7 activities in K562 cells that were untreated or treated with kinamycin F (5 M) for the times indicated were subjected to a luminometric caspase-3/7 activation assay. The results are reported as the mean of 3 replicate measurements ± SE fold change in luminescence for treated cells relative to untreated cells. Significant differences between treated and untreated samples are designated by *p < 0.05, **p < 0.01 and ***p < 0.001.
cells. Apoptosis is a regulated form of cell death that is distinguished by unique features, and caspase-3 is the predominant caspase responsible for some of the characteristic downstream events associated with apoptosis [24]. The combined functions of caspase-3 and -7 also appear to be involved in erythroid differentiation [25]. Because of the importance of these caspases, their activities in K562 cells treated with kinamycin F (5 M) for various times were investigated (Fig. 3C). Treatment of cells with kinamycin F for 2, 6, 12, 24, 48 and 72 h significantly increased caspase-3/7 activity at all times compared to untreated cells. 3.4. Kinamycin F decreases cyclin D3 protein levels but does not affect protein stability The D-type cyclins are important for progression through the G1 phase of the cell cycle as they are responsible for binding and partially activating cyclin dependent kinases, leading to activation of genes responsible for entry into S phase [26]. In addition to cell cycle effects, cyclin D3 may also play a role in differentiation. Studies have shown reduced levels of cyclin D3 protein [27]
and mRNA [28] during differentiation of primary hematopoietic cells induced by cytokines and overexpression of FLI-1, respectively, while overexpression of cyclin D3 inhibited differentiation of myeloid cells [29]. Thus, the protein levels of cyclins D1, D2, D3 and E were examined. Following treatment with kinamycin F (5 M) for the times indicated, total K562 cell lysates were examined by Western blotting. As shown in Fig. 4A kinamycin F treatment dramatically reduced cyclin D3 protein levels in K562 cells at 6, 12 and 24 h without altering protein levels of cyclins D1, D2 or E. Because cyclin D3 was the predominant cyclin affected by kinamycin F treatment, the mechanism by which cyclin D3 protein levels were reduced was examined in more detail. The D-type cyclins are regulated transcriptionally, post-transcriptionally and by ubiquitin-mediated degradation [26]. The ability of kinamycin F to affect the stability of cyclin D3 was studied after cycloheximide treatment to inhibit further protein translation (Fig. 4B). As shown in Fig. 4B, kinamycin F (5 M) treatment for 12 h did not significantly (p = 0.06, n = 4) reduce the half-life of cyclin D3 (2.1 ± 0.2 h) compared to that of control (1.4 ± 0.2 h). This result suggests that
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for 1 h were then treated for the times indicated with DRB to inhibit RNA polymerase II. As shown in Fig. 5B kinamycin F treatment did not significantly affect the half-life of cyclin D3 mRNA (5.1 ± 0.4 h) compared to that of control (7.7 ± 2.0 h). This result suggests that kinamycin F affects the transcription of the cyclin D3 gene. 3.6. Kinamycin F decreases transactivation of the cyclin D3 gene To examine whether the decreased expression of cyclin D3 was mediated through its promoter, we used a luciferase reporter plasmid containing the complete human cyclin D3 gene promoter region, ranging from −1017 to +6 bp, relative to its ATG start site [22]. In these experiments K562 cells were transiently transfected with the empty reporter plasmid, or with the reporter plasmid containing the promoter sequence of the cyclin D3 gene. This was followed by treatment with either kinamycin F (5 M) or DMSO for the times indicated (Fig. 5C). Luciferase reporter activity was significantly reduced by 28, 40 and 41% after a 6, 12 or 24 h treatment with kinamycin F compared to controls, respectively (Fig. 5C). These data suggest that cyclin D3 levels were reduced by direct action of kinamycin F on the promoter or indirectly by affecting upstream signaling through transcription factors. 4. Discussion
Fig. 4. Kinamycin F treatment decreases level of cyclin D3 protein, but does not affect protein stability. (A) K562 cells were treated or not treated with kinamycin F (5 M) for the times indicated. The blot is representative of 4 separate experiments. The images bounded by thin lines originated from the same membrane; (B) effect of kinamycin F on the stability of cyclin D3 protein. To inhibit the formation of new protein K562 cells were treated with the translation inhibitor cycloheximide. The results are reported as the mean of 4 replicate measurements ± SE cyclin D3 protein levels relative to -actin levels and normalized to the controls. The halflife of cyclin D3 was not significantly changed in the presence of kinamycin F. The smooth lines are non-linear-least-squares best fits to a two-parameter exponential decay equation.
kinamycin F did not reduce cyclin D3 protein levels by reducing protein stability. 3.5. Kinamycin F decreases cyclin D3 mRNA levels without affecting mRNA stability To determine whether kinamycin F affected transcription of cyclin D3, the levels of cyclin D3 mRNA in K562 cells were determined in response to treatment with kinamycin F. Kinamycin F (5 M) treatment of K562 cells for the times indicated resulted in a significant reduction of cyclin D3 mRNA levels at all times (Fig. 5A). Decreased levels of mRNA could be due to decreased transcription, but could also be a result of decreased mRNA stability, a post-transcriptional effect. To distinguish between these two possibilities, K562 cells treated with either DMSO or kinamycin F (5 M)
In this study we identified cyclin D3 as a direct or indirect target of kinamycin F. Our results suggest that downregulation of cyclin D3 by kinamycin F may, in part, mediate kinamycin F-induced inhibition of growth, and possibly, erythroid differentiation of K562 cells. Anticancer drugs that target cell cycle proteins are currently in active development [30]. Compounds such as rapamycin [21] also decrease cyclin D3 levels and inhibit cell cycle progression. We decided to examine the D-type cyclins as well as cyclin E because of their roles in cell cycle progression and differentiation. Treatment with kinamycin F significantly decreased protein levels of cyclin D3 (Fig. 4A). A decrease in cyclin D3 levels appears to be involved in the differentiation of a variety of hematopoietic cells in response to both cytokines and the transcription factor, FLI-1 [27,28], and may also contribute to a delay in progression through the G2 /M phase [31]. The expression of cyclin D3 is regulated transcriptionally and cyclin 3 protein is degraded post-transcriptionally by ubiquitin [32]. However, cyclin D3 protein stability was not significantly changed following treatment with kinamycin F (Fig. 4B) which led us to examine mRNA changes. The mRNA levels of cyclin D3 decreased within 6 h of a kinamycin F treatment (Fig. 5A), suggesting that kinamycin F either increased degradation of cyclin D3 mRNA or prevented, directly or indirectly, the activation of the cyclin D3 promoter. These two possibilities were tested, and although the stability of the cyclin D3 transcript did not change as a result of kinamycin F treatment (Fig. 5B), transcriptional activation of the full length cyclin D3 promoter was significantly reduced (Fig. 5C). Cyclin D3 expression can also be regulated by transcription factors within or outside of the regulatory elements necessary for maximal activation [33]. Because kinamycin F only weakly binds DNA and only damages it at higher concentrations [13], the primary mechanism by which kinamycin F downregulates cyclin D3 mRNA levels may not be a direct result of DNA binding or damage. It may be that kinamycin F, or one of its metabolites, interacts with a protein upstream of cyclin D3 transcription, altering the abundance or activity of a specific factor or factors. D-type cyclins are important in cell cycle progression and differentiation and are overexpressed both in pancreatic cancer and in lymphomas [34], making D-type cyclins very attractive targets for anticancer agents [30]. In conclusion, our results are the first to identify cyclin D3 as a potential target of kinamycin F.
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Fig. 5. Kinamycin F decreases levels of cyclin D3 mRNA by reducing the transactivation of the cyclin D3 promoter. (A) Kinamycin F decreased cyclin D3 mRNA levels. K562 cells were treated or not treated with kinamycin F (5 M) for the times indicated. The results are reported as the mean of 3 replicate measurements ± SE of the weight of cyclin D3 DNA product per ml of reaction volume normalized for constitutive 18S rRNA levels; (B) kinamycin F does not affect the stability of cyclin D3 mRNA. K562 cells that were treated with either DMSO or kinamycin F (5 M) for 1 h were then treated with DRB (100 M) to inhibit RNA polymerase II for the times indicated. The results are reported as the mean of 6 replicate measurements ± SE of the weight of cyclin D3 DNA product per ml of reaction volume, corrected for constitutive 18S rRNA levels. The mRNA levels are expressed relative to mRNA levels prior to DRB addition. The smooth lines are non-linear-least-squares best fits to a two-parameter exponential decay equation. No significant differences were found between the half-lives of treated and untreated cells; (C) kinamycin F decreases the transcription of cyclin D3. K562 cells were transiently transfected with a luciferase reporter plasmid under the control of the full length cyclin D3 promoter. The data are derived from three independent experiments and the results are reported as the mean ± SE change in relative light units of luciferase activity per g of protein. Significant differences between treated and untreated samples are designated by *p < 0.05, **p < 0.01 and ***p < 0.001.
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Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Kinamycin F downregulates cyclin D3 in human leukemia K562 cells Kimberley A. O’Hara a , Gary I. Dmitrienko b , Brian B. Hasinoff a,∗ a b
Faculty of Pharmacy, Apotex Centre, University of Manitoba, 750 McDermot Ave., Winnipeg, Manitoba R3E 0T5, Canada Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada
a r t i c l e
i n f o
Article history: Received 6 October 2009 Received in revised form 21 December 2009 Accepted 6 January 2010 Available online 15 January 2010 Keywords: Cyclin D3 K562 cells Kinamycin Cell cycle Apoptosis
a b s t r a c t The bacterial metabolite kinamycin F, which contains an unusual and potentially reactive diazo group, is being investigated as an antitumor agent with a potentially novel target. Treatment of K562 cells with kinamycin F induced erythroid differentiation, a rapid apoptotic response, induction of caspase3/7 activities and a delayed cell cycle progression through the S and G2 /M phases. Kinamycin F caused a selective reduction of cyclin D3 protein, which appeared to be mediated at the level of transcription, rather than by affecting the stability of either cyclin D3 protein or mRNA. Thus cyclin D3 is a potential target of kinamycin F. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The kinamycins are a series of bacterial metabolites with an unusual structure and potent antibiotic and antitumor activity. Kinamycins A, B, C and D were first isolated in 1970 from Streptomyces murayamaensis [1]. The fully deacetylated kinamycin F (Fig. 1A) has been detected as a metabolite in culture broths of S. murayamaensis [2]. Kinamycin C displayed antitumor activity in an animal model [3] and submicromolar cell growth inhibitory effects in the 60-cancer cell NCI panel (http://dtp.nci.nih.gov). Initially the kinamycins were assigned an N-cyanocarbazole structure [4], however, we [5] and another group [6] independently corrected its structure to that of a diazobenzo[b]fluorene. The structurally similar antitumor antibiotic lomaiviticin A isolated from Micromonospora lomaivitiensis exhibits extremely potent activity against the modified NCI 24-cancer cell line with cell growth inhibitory effects in the picomolar range [7]. Lomaiviticin A is a dimer containing two kinamycin-like systems that are joined by a carbon–carbon bond linking each monomer. The diazo pharmacophore is not only an unusual functional group in natural products, but is also potentially reactive [8] and it presumably plays a role in the antitumor activity of the kinamycins. Consequently there has been a renewed interest in the kinamycins. The
kinamycins are small molecules that are amenable to synthesis [9]. Kinamycins C [10,11], F and J [11] have been synthesized recently. Mechanisms by which the kinamycins might be activated to damaging species have recently been reviewed [12]. We previously proposed that kinamycin F may be generated in cells by the activity of esterases on O-acetylated forms of kinamycin and that it may be the active metabolite responsible for the observed inhibition of cancer cell growth [13,14]. We have shown that kinamycins A, C and F inhibit the growth of K562 cells at submicromolar levels [13,14] and that kinamycin C induces apoptosis in K562 and Chinese hamster ovary cells (CHO) [14]. We [13] and others [15] also showed that kinamycin F damaged DNA in vitro as well as in whole cells and that it can be reductively and peroxidatively activated to a semiquinone and phenoxyl free radical, respectively. We also showed that kinamycins A, C and F are catalytic inhibitors of topoisomerase II␣ [13,14] though it is most likely not the major target of the kinamycins. The purpose of this study was to elucidate the mechanism or mechanisms by which kinamycin F exerts its ability to inhibit cell growth to enable further development of kinamycin F or its analogs as anticancer drugs with a novel target. 2. Materials and methods
Abbreviations: DMSO, dimethyl sulfoxide; DRB, 5,6-dichloro-1-dribofuranosylbenzimidazole. ∗ Corresponding author. Tel.: +1 204 474 8325; fax: +1 204 474 7617. E-mail address: B [email protected] (B.B. Hasinoff). 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.01.013
2.1. Materials and cell culture Kinamycin F was prepared semisynthetically from kinamycin D that was isolated from S. murayamaensis as described [13] and
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containing the DEVD sequence that results in a luminescence signal that is proportional to caspase-3/7 activity. 2.4. Protein isolation and immunoblotting analysis
Fig. 1. (A) Structure of kinamycin F; (B) kinamycin F increases the percentage of hemoglobin-positive K562 cells. K562 cells were treated with either DMSO vehicle or kinamycin F (5 M) for 12, 24, or 48 h after which time the percentage of cells that stained positive for hemoglobin were determined. Results are reported as the mean ± SE of four replicate measurements. Significant differences between treated and controls are designated by *p < 0.05, **p < 0.01 and ***p < 0.001.
was dissolved in DMSO. While the solubility of kinamycin F in aqueous solutions was not determined, its solubility was determined to be greater than 40 M in the culture broth and greater than 64 M in aqueous solution buffered at pH 7.0, which is a concentration more than 10-fold higher than that used in the experiments. The fact that kinamycin F is isolated from culture broth after several days of growth is indicative of its relative stability. Deferiprone was synthesized as described [16]. The level of significance used in the t-tests was p < 0.05. Human leukemia K562 cells obtained from the American Type Culture Collection (Manassas, VA) were maintained as suspension cultures as described [13]. In control and kinamycin F treatments the DMSO concentration was always 0.5% (v/v). The percentage of cells staining for hemoglobin following treatment with kinamycin F was determined by staining with benzidine/H2 O2 as previously described [17]. 2.2. Cell synchronization and flow cytometry For the synchronization experiments K562 cells, which normally have a 24 h doubling time, were grown to a density of 4 × 105 cells/ml after which they were treated for 24 h with deferiprone (300 M), an iron chelator that has been shown to synchronize K562 cells in G0 /G1 [18]. After this 24 h incubation the cells were washed 3 times with complete media and seeded in 35-mm diameter dishes after resting for 5 h. Cells were continuously treated for the times indicated with either the DMSO vehicle or kinamycin F (5 M). We previously determined that a 72 h kinamycin F treatment caused 50% cell growth inhibition at a concentration of 0.33 M [13]. Cells were fixed and stained with propidium iodide for cell cycle analysis as previously described [14]. The fraction of apoptotic cells induced by treatment of K562 cells with kinamycin F was quantified by two-color flow cytometry by simultaneously measuring integrated green (annexin-V-FITC) fluorescence and integrated red (propidium iodide) fluorescence as previously described [14]. 2.3. Caspase-3/7 luminescence assay Induction of caspase-3/7 activities in K562 cells following treatment with kinamycin F for various times was quantified using the Caspase-Glo® 3/7 Assay according to the manufacturer’s directions (Promega, Madison, WI) as previously described [19]. Upon cell lysis caspase-3 and -7 are able to cleave a proluminogenic substrate
Following treatment with DMSO or kinamycin F (5 M), pellets of 5 × 105 K562 cells were treated essentially as previously described [19]. After SDS–PAGE separation proteins were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA) and after blocking were probed with specific primary antibodies overnight at 4 ◦ C. Antibodies targeting cyclins D1, D3, E (BD Biosciences, San Diego, CA), cyclin D2 (Cell Signaling Technology, Danvers, MA) and -actin (Clone AC-15, Sigma) were used. The membranes were incubated with horseradish peroxidaseconjugated secondary antibodies (Pierce Biotechnology, Rockford, IL) and reactive bands were detected using enhanced chemiluminescence and imaged on an Alpha Innotech (San Leandro, CA) Fluorochem 8900 imaging system. In evaluating the effect of kinamycin F on the stability of cyclin D3 protein, cells were treated with either DMSO or kinamycin F (5 M) for 12 h, followed by addition of cycloheximide (10 g/ml) to inhibit further protein translation/synthesis. A 12 h treatment with 0.4 g/ml of cycloheximide has been shown to inhibit protein synthesis by 30% in K562 cells [20]. 2.5. RNA isolation and real-time RT (reverse transcriptase)–PCR Total cellular RNA was isolated using the RNeasy Mini Kit (Qiagen, Mississauga, Canada). The cDNAs were generated from 0.5 g total RNA by reverse transcription in a 20 l reaction mixture containing M-MLV reverse transcriptase 5× reaction buffer (Promega, Madison, WI), 10 U RNase inhibitor (Ambion, Streetsville, Canada), 0.5 mM of each dNTP (Fisher, Ottawa, Canada), 5 M oligo dT (GE Healthcare Bio-Sciences, Piscataway, NJ) and 200 U M-MLV reverse transcriptase (Promega). Reactions were incubated at 44 ◦ C for 60 min in the BioRad MiniOpticon (BioRad Laboratories, Hercules, CA). The cDNA was stored at −20 ◦ C until further analysis. Specific primer pairs for human cyclin D3 (forward 5 CGTGGTCGGTGTAGATGC; reverse 5 -TGGATGCTGGAGGTATGTG) and 18S rRNA (forward 5 -CTTATGACCCGCACTTACTG; reverse 5 TCCCCCAACTTCTTAGAGG) [21] were used to amplify the specific cDNAs. Specific cDNAs were amplified using 2 l aliquots of cDNA product mixed with cyclin D3 or 18S rRNA forward and reverse primers (0.38 pM each), 12.5 l of SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) in a final volume of 25 l. PCR reactions were carried out for 15 s at 94 ◦ C, 30 s at 60 ◦ C and 30 s at 72 ◦ C for 45 cycles. Gene expression was quantified using standard curves for the respective cDNA products and changes in resulting cyclin D3 cDNA levels were normalized to changes in 18S rRNA to determine the weight of normalized product per ml of reaction. In evaluating the effect of kinamycin F on the half-life of cyclin D3 mRNA, cells were pre-treated for 1 h with either DMSO or kinamycin F (5 M) followed by addition of DRB, an inhibitor of RNA polymerase II. The relative levels of cyclin D3 mRNA in both kinamycin F-treated or -untreated cells prior to DRB addition were determined. 2.6. Transient transfections and luciferase assay The human cyclin D3 (−1017 bp/+6) pGL3 plasmid [22], which contains a luciferase promoter construct, was generously provided by Dr. W. Douglas Cress (Moffitt Cancer Center, Tampa, FL). K562 cells (1 × 106 ) in 35 mm dishes were co-transfected with 3.5 g of the cyclin D3 pGL3 or pGL3 (Promega) plasmid and 0.5 g of enhanced green fluorescence protein plasmid (Clontech, Palo Alto, CA) using Lipofectamine 2000 Plus reagent and Opti-MEM
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(Invitrogen) in antibiotic-free Dulbecco’s modified Eagle’s medium. After recovering overnight, cells were treated for the times indicated with kinamycin F (5 M) and harvested for luciferase assays. The green fluorescence produced was used to assess transfection efficiency. After treatment cells were pelleted and then lysed for 15 min on ice in luciferase lysis buffer (25 mM glycylglycine, 4 mM EGTA, 15 mM MgSO4 , 1% (v/v) Triton X-100, 1 mM dithiothreitol, 1 mg/ml bovine serum albumin). 25 l of lysate was added to 75 l of luciferase assay buffer (25 mM glycylglycine, 15 mM potassium phosphate, 150 mM MgSO4 , 4 mM EGTA, 2 mM ATP, 1 mM dithiothreitol) and after luciferin (400 M) was added the luminescence was read immediately in a Fluostar Galaxy (BMG) plate reader at 25 ◦ C. The relative light units of luciferase activities per microgram of protein were calculated. 3. Results 3.1. Kinamycin F induces erythroid differentiation in K562 cells K562 cells are a human erythroleukemic cell line capable of differentiation into erythroid cells and expression of hemoglobin [23]. Thus, the expression of hemoglobin in K562 cells in response to treatment with kinamycin F (5 M) was measured as an indicator of erythroid differentiation. K562 cells continuously incubated with kinamycin F (5 M) for the times indicated (Fig. 1B) displayed significant increases in the number of cells that stained positive for hemoglobin after 12 h (p = 0.03), 24 h (p = 0.002) and 48 h (p = 0.0007).
3.2. Synchronized K562 cells exhibit an S phase accumulation and a delayed entry into the G2 /M phase To establish the effects of kinamycin F on cell cycle, deferipronesynchronized K562 cells were examined (Fig. 2A). As shown in Fig. 2, approximately 65% of the cells were in G0 /G1 when initially treated with kinamycin F (time 0 h). Cells that were treated for 4 h with kinamycin F (5 M) exited G0 /G1 in a manner similar to untreated cells (Fig. 2B, top panel). Cells exiting G0 /G1 became increasingly apoptotic as evidenced by a progressive increase in the fraction of cells in the sub-G1 phase. At 48 h, 26% of kinamycin F-treated cells had entered the sub-G1 phase compared to 5% for untreated cells. However, as shown in Fig. 2B (middle panel) treatment with kinamycin F caused an accumulation of cells in the S phase due to a delayed exit from this phase. At 12 h the kinamycin F-treated cells exhibited a delayed entry into G2 /M of about 5 h (Fig. 2B, bottom panel). Unsynchronized K562 cells were also examined for cell cycle effects induced by kinamycin F. The fraction of cells in G0 /G1 , S and G2 /M phases remained relatively unchanged except that a large fraction of cells progressively became more apoptotic as judged from a progressive increase in the sub-G1 population. For example, at 48 h, 14% of untreated cells were apoptotic compared to 47% for cells treated with kinamycin F (5 M). 3.3. Kinamycin F induces apoptosis and caspase-3/7 activation in K562 cells As shown in Fig. 3A and B, treatment with kinamycin F (5 M) for the times indicated greatly increased the proportion of apoptotic
Fig. 2. Kinamycin F inhibits cell cycle progression in K562 cells. K562 cells that had been synchronized by the treatment with deferiprone were treated, or not treated, with kinamycin F (5 M) and were then allowed to grow for the times indicated. (A) Cell counts are displayed on the vertical axis and the DNA content is plotted on the horizontal axis; (B) percentage of the non-sub G0 /G1 cells in G0 /G1 (top panel), S (middle panel) and G2 /M (bottom panel) phases are plotted as a function of time. The solid lines are least-squares calculated spline fits that connect the data points.
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Fig. 3. Kinamycin F treatment of K562 cells induces apoptosis and caspase-3/7 activity. (A) Two-color flow cytometry scatter plots of K562 cells that were untreated or treated with kinamycin F (5 M) for 6 h. The cells were then treated with annexin-V-FITC and propidium iodide. The lower left quadrant contains viable cells; the upper left quadrant contains cells that are necrotic-only; the lower right quadrant contains cells that are apoptotic-only; and the upper right quadrant contains cells that are both apoptotic and necrotic; (B) changes in relative number of K562 cells that were classified as necrotic-only; apoptotic and necrotic; apoptotic-only, or viable 0, 6, 12 and 24 h after treatment with kinamycin F (5 M); (c) caspase-3/7 activities in K562 cells that were untreated or treated with kinamycin F (5 M) for the times indicated were subjected to a luminometric caspase-3/7 activation assay. The results are reported as the mean of 3 replicate measurements ± SE fold change in luminescence for treated cells relative to untreated cells. Significant differences between treated and untreated samples are designated by *p < 0.05, **p < 0.01 and ***p < 0.001.
cells. Apoptosis is a regulated form of cell death that is distinguished by unique features, and caspase-3 is the predominant caspase responsible for some of the characteristic downstream events associated with apoptosis [24]. The combined functions of caspase-3 and -7 also appear to be involved in erythroid differentiation [25]. Because of the importance of these caspases, their activities in K562 cells treated with kinamycin F (5 M) for various times were investigated (Fig. 3C). Treatment of cells with kinamycin F for 2, 6, 12, 24, 48 and 72 h significantly increased caspase-3/7 activity at all times compared to untreated cells. 3.4. Kinamycin F decreases cyclin D3 protein levels but does not affect protein stability The D-type cyclins are important for progression through the G1 phase of the cell cycle as they are responsible for binding and partially activating cyclin dependent kinases, leading to activation of genes responsible for entry into S phase [26]. In addition to cell cycle effects, cyclin D3 may also play a role in differentiation. Studies have shown reduced levels of cyclin D3 protein [27]
and mRNA [28] during differentiation of primary hematopoietic cells induced by cytokines and overexpression of FLI-1, respectively, while overexpression of cyclin D3 inhibited differentiation of myeloid cells [29]. Thus, the protein levels of cyclins D1, D2, D3 and E were examined. Following treatment with kinamycin F (5 M) for the times indicated, total K562 cell lysates were examined by Western blotting. As shown in Fig. 4A kinamycin F treatment dramatically reduced cyclin D3 protein levels in K562 cells at 6, 12 and 24 h without altering protein levels of cyclins D1, D2 or E. Because cyclin D3 was the predominant cyclin affected by kinamycin F treatment, the mechanism by which cyclin D3 protein levels were reduced was examined in more detail. The D-type cyclins are regulated transcriptionally, post-transcriptionally and by ubiquitin-mediated degradation [26]. The ability of kinamycin F to affect the stability of cyclin D3 was studied after cycloheximide treatment to inhibit further protein translation (Fig. 4B). As shown in Fig. 4B, kinamycin F (5 M) treatment for 12 h did not significantly (p = 0.06, n = 4) reduce the half-life of cyclin D3 (2.1 ± 0.2 h) compared to that of control (1.4 ± 0.2 h). This result suggests that
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for 1 h were then treated for the times indicated with DRB to inhibit RNA polymerase II. As shown in Fig. 5B kinamycin F treatment did not significantly affect the half-life of cyclin D3 mRNA (5.1 ± 0.4 h) compared to that of control (7.7 ± 2.0 h). This result suggests that kinamycin F affects the transcription of the cyclin D3 gene. 3.6. Kinamycin F decreases transactivation of the cyclin D3 gene To examine whether the decreased expression of cyclin D3 was mediated through its promoter, we used a luciferase reporter plasmid containing the complete human cyclin D3 gene promoter region, ranging from −1017 to +6 bp, relative to its ATG start site [22]. In these experiments K562 cells were transiently transfected with the empty reporter plasmid, or with the reporter plasmid containing the promoter sequence of the cyclin D3 gene. This was followed by treatment with either kinamycin F (5 M) or DMSO for the times indicated (Fig. 5C). Luciferase reporter activity was significantly reduced by 28, 40 and 41% after a 6, 12 or 24 h treatment with kinamycin F compared to controls, respectively (Fig. 5C). These data suggest that cyclin D3 levels were reduced by direct action of kinamycin F on the promoter or indirectly by affecting upstream signaling through transcription factors. 4. Discussion
Fig. 4. Kinamycin F treatment decreases level of cyclin D3 protein, but does not affect protein stability. (A) K562 cells were treated or not treated with kinamycin F (5 M) for the times indicated. The blot is representative of 4 separate experiments. The images bounded by thin lines originated from the same membrane; (B) effect of kinamycin F on the stability of cyclin D3 protein. To inhibit the formation of new protein K562 cells were treated with the translation inhibitor cycloheximide. The results are reported as the mean of 4 replicate measurements ± SE cyclin D3 protein levels relative to -actin levels and normalized to the controls. The halflife of cyclin D3 was not significantly changed in the presence of kinamycin F. The smooth lines are non-linear-least-squares best fits to a two-parameter exponential decay equation.
kinamycin F did not reduce cyclin D3 protein levels by reducing protein stability. 3.5. Kinamycin F decreases cyclin D3 mRNA levels without affecting mRNA stability To determine whether kinamycin F affected transcription of cyclin D3, the levels of cyclin D3 mRNA in K562 cells were determined in response to treatment with kinamycin F. Kinamycin F (5 M) treatment of K562 cells for the times indicated resulted in a significant reduction of cyclin D3 mRNA levels at all times (Fig. 5A). Decreased levels of mRNA could be due to decreased transcription, but could also be a result of decreased mRNA stability, a post-transcriptional effect. To distinguish between these two possibilities, K562 cells treated with either DMSO or kinamycin F (5 M)
In this study we identified cyclin D3 as a direct or indirect target of kinamycin F. Our results suggest that downregulation of cyclin D3 by kinamycin F may, in part, mediate kinamycin F-induced inhibition of growth, and possibly, erythroid differentiation of K562 cells. Anticancer drugs that target cell cycle proteins are currently in active development [30]. Compounds such as rapamycin [21] also decrease cyclin D3 levels and inhibit cell cycle progression. We decided to examine the D-type cyclins as well as cyclin E because of their roles in cell cycle progression and differentiation. Treatment with kinamycin F significantly decreased protein levels of cyclin D3 (Fig. 4A). A decrease in cyclin D3 levels appears to be involved in the differentiation of a variety of hematopoietic cells in response to both cytokines and the transcription factor, FLI-1 [27,28], and may also contribute to a delay in progression through the G2 /M phase [31]. The expression of cyclin D3 is regulated transcriptionally and cyclin 3 protein is degraded post-transcriptionally by ubiquitin [32]. However, cyclin D3 protein stability was not significantly changed following treatment with kinamycin F (Fig. 4B) which led us to examine mRNA changes. The mRNA levels of cyclin D3 decreased within 6 h of a kinamycin F treatment (Fig. 5A), suggesting that kinamycin F either increased degradation of cyclin D3 mRNA or prevented, directly or indirectly, the activation of the cyclin D3 promoter. These two possibilities were tested, and although the stability of the cyclin D3 transcript did not change as a result of kinamycin F treatment (Fig. 5B), transcriptional activation of the full length cyclin D3 promoter was significantly reduced (Fig. 5C). Cyclin D3 expression can also be regulated by transcription factors within or outside of the regulatory elements necessary for maximal activation [33]. Because kinamycin F only weakly binds DNA and only damages it at higher concentrations [13], the primary mechanism by which kinamycin F downregulates cyclin D3 mRNA levels may not be a direct result of DNA binding or damage. It may be that kinamycin F, or one of its metabolites, interacts with a protein upstream of cyclin D3 transcription, altering the abundance or activity of a specific factor or factors. D-type cyclins are important in cell cycle progression and differentiation and are overexpressed both in pancreatic cancer and in lymphomas [34], making D-type cyclins very attractive targets for anticancer agents [30]. In conclusion, our results are the first to identify cyclin D3 as a potential target of kinamycin F.
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Fig. 5. Kinamycin F decreases levels of cyclin D3 mRNA by reducing the transactivation of the cyclin D3 promoter. (A) Kinamycin F decreased cyclin D3 mRNA levels. K562 cells were treated or not treated with kinamycin F (5 M) for the times indicated. The results are reported as the mean of 3 replicate measurements ± SE of the weight of cyclin D3 DNA product per ml of reaction volume normalized for constitutive 18S rRNA levels; (B) kinamycin F does not affect the stability of cyclin D3 mRNA. K562 cells that were treated with either DMSO or kinamycin F (5 M) for 1 h were then treated with DRB (100 M) to inhibit RNA polymerase II for the times indicated. The results are reported as the mean of 6 replicate measurements ± SE of the weight of cyclin D3 DNA product per ml of reaction volume, corrected for constitutive 18S rRNA levels. The mRNA levels are expressed relative to mRNA levels prior to DRB addition. The smooth lines are non-linear-least-squares best fits to a two-parameter exponential decay equation. No significant differences were found between the half-lives of treated and untreated cells; (C) kinamycin F decreases the transcription of cyclin D3. K562 cells were transiently transfected with a luciferase reporter plasmid under the control of the full length cyclin D3 promoter. The data are derived from three independent experiments and the results are reported as the mean ± SE change in relative light units of luciferase activity per g of protein. Significant differences between treated and untreated samples are designated by *p < 0.05, **p < 0.01 and ***p < 0.001.
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