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16-1 and arsenic trioxide
Biochemical and Biophysical Research Communications 403 (2010) 203–208
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Synergistic apoptosis induction in leukemic cells by miR-15a/16-1 and arsenic trioxide Shen-meng Gao a, Chiqi Chen a, Jianbo Wu a, Yanxia Tan a, Kang Yu b,c, Chong-Yun Xing b, Aifang Ye a, Lihui Yin a, Lei Jiang a,⇑ a b c
Laboratory of Internal Medicine, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang 325000, China Department of Hematology, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang 325000, China Institute of Hematology and Immunology, Wenzhou Medical College, Wenzhou, Zhejiang 325000, China
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Article history: Received 28 October 2010 Available online 5 November 2010 Keywords: Arsenic trioxide MiR-15a/16-1 Apoptosis Leukemia
a b s t r a c t MicroRNAs (miRNAs) are small noncoding RNAs that regulate target gene expression through translation repression or messenger RNA degradation. MiR-15a and 16-1 form a cluster at the chromosomal region 13q14, which is frequently deleted or down-regulated in chronic lymphocytic leukemia. Arsenic trioxide (As2O3, ATO) has been successfully applied to treat acute promyelocytic leukemia (APL). Its combination with other drugs presented therapeutic activities in hematologic and solid tumors. Here we investigated the potential synergy between miR-15a/16-1 and ATO on Bcr-Abl positive leukemic K562 cells. In this study, we found that combination of miR-15a/16-1 and ATO synergistically induced growth inhibition and apoptosis in K562 cells. The apoptosis, at least in part, through regulating mitochondrial function including the release of cytochrome c and loss of mitochondrial transmembrane potential, also activation of caspase-3 and degradation of poly-adenosine diphosphate-ribose polymerase. However, the expression of Bcr-Abl was not affected by ATO and/or miR-15a/16-1. Moreover, apoptotic synergy between miR-15a/16-1 and ATO was observed in Bcr-Abl negative leukemic cell lines and primary leukemic cells. Taken together, these findings suggested that the combined regiment of miR-15a/16-1 and ATO might be a potential therapeutic remedy for the treatment of leukemia. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction MicroRNAs (miRNAs) are short noncoding RNAs of 19–24 nucleotides, which bind to 30 UTRs of target mRNAs to either prevent their translation or induce their degradation [1]. MiRNA are involved in the regulation of critical cell processes such as apoptosis, cell proliferation and differentiation [2]. Several miRNAs have been implicated as tumor suppressors based on their physical deletion or down-regulated expression in human cancer [3]. The miR-15a/ 16-1 cluster resides at chromosome 13q14.3, a genomic region frequently deleted in the majority of B cell chronic lymphocytic leukemias (CLLs) and in a subset of mantle cell lymphomas and prostate cancers [3–5]. MiR-15a and miR-16-1 are known to act as tumor suppressors in CLL and other malignancies [6,7]. Expression of these miRNAs inhibits cell proliferation, induces cancer
Abbreviations: ATO, arsenic trioxide; APL, acute promyelocytic leukemia; miRNA, microRNAs; CLL, chronic lymphocytic leukemia; AML, acute myeloid leukemia; pRS15/16, pRETROSUPER vector expressing miR-15a/miR-16-1; pRS-E, pRETROSUPER empty vector. ⇑ Corresponding author. Fax: +86 577 86550280. E-mail address: [email protected] (L. Jiang). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.10.137
cells apoptosis, and suppresses tumorigenicity by targeting multiple oncogenes such as BCL2, MCL1, CCND1, and WNT3A [8,9]. Arsenic trioxide (As2O3, ATO), an ancient traditional Chinese medicine, have been successfully applied to treat acute promyelocytic leukemia (APL) [10–12]. The antitumor mechanism of ATO, mainly the induction of apoptosis and partial differentiation, suggests that it may be active against other hematologic malignancies [13]. A series of in vitro and in vivo experiments showed that ATO has potential activities in patients with accelerated phase chronic myelogenous leukemia (CML) and other hematologic malignancies [14,15]. CML is a myeloproliferative disorder that is characterized by a cytogenetic aberration consisting of a reciprocal translocation between the long arms of chromosomes 22 and 9, which forms the Bcr-Abl fusion protein [16]. The constitutively activated Bcr-Abl kinase, has been shown to confer leukemic cell resistance to diverse chemotherapeutic agents [17]. ATO used as a single agent at higher concentrations was adapted to induce apoptosis and growth inhibition in Bcr-Abl positive cells. Cytotoxicity and drug resistance are major concern with treatment. Recently it has been shown to be effective, particularly in combination with other drugs in hematologic and solid tumors [15,18,19]. However, very little work has been done on the interaction of miR-15a/16-1 and ATO in leukemic cells.
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In this study, we investigated the combined effects of miR-15a/ 16-1 and ATO on Bcr-Abl and CML cells. The K562 cell line was used as a cellular model of CML for drug screening. Moreover, in order to address the potential clinical application of the drugs, we used Bcr-Abl negative leukemic cell lines and primary leukemic cells. Our data showed that ATO and/or miR-15a/16-1 did not affect the expression of Bcr-Abl, overexpression of miR-15a/16-1 enhanced ATO at clinically achievable concentration induced apoptosis in Bcr-Abl positive K562 cells and some other Bcr-Abl negative leukemic cells.
2.2. Plasmids and transient transfection pRETROSUPER vector expressing miR-15a/miR-16-1 (pRS15/16) was constructed as previously described [20]. Cells transfected with the same empty plasmid (pRS-E) served as controls. Leukemic cells and primary cells of AML patients were transiently transfected with 1 lg/mL (final concentration) pRS15/16 or pRS-E vector using Lipofectamine™ LTX and PLUS™ Reagents (Invitrogen) according to the manufacturer’s instructions. 2.3. Viability and apoptosis assay
2. Materials and methods 2.1. Cell lines and primary leukemic cells Primary leukemic cells and various kinds of leukemic cell lines, including Bcr-Abl positive K562 cell line, Bcr-Abl negative cell lines U937, NB4, HL60, Raji and RPMI8226, were employed for the present study. All cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (Invitrogen, Groud Island, USA), in humidified 37 °C incubator with 5% CO2. Primary leukemic cells from five acute myeloid leukemia (AML) patients (one case of M2-type, two cases of M4-type and two cases of M5-type, The First Afficiated Hospital of Wenzhou Medical College) were used after informed consent. Mononuclear cells were isolated by Ficoll density gradient centrifugation (GE Healthcare, Uppsala, Sweden).
Cell lines and primary leukemic cells were plated in triplicate at 2 105 cells/mL. After being treated with ATO and/or miR-15a/ miR-16-1, viable cells were counted by 0.4% trypan blue exclusion. Growth inhibition rate was evaluated by the viable cell numbers in treated cells against untreated ones. The percentages of annexin V positive apoptotic cells were determined by flow cytometric analysis. Experiments were done in duplicate and repeated at least three times. 2.4. Flow cytometric analysis Cell lines and primary leukemic cells were plated in triplicate at 2 105 cells/mL. After being treated with ATO and/or miR-15a/ miR-16-1, viable cells were counted by 0.4% trypan blue exclusion. Growth inhibition rate was evaluated by the viable cell numbers in
Fig. 1. Overexpression of miR-15a/16-1 increases ATO-induced growth inhibition and apoptosis in K562 cells. (A) K562 cells were exposed to the indicated concentrations of ATO for 48 h, and the percentages of growth inhibition and annexin V positive apoptotic cells were determined. (B) Levels of miR-15a/16-1 expression were determined by quantitative real-time PCR. K562 cells were exposed to 1 lM ATO for different time, and quantitative real-time PCRs for miR-15a/16-1 were performed. (C) Overexpression of miR-15a/16-1 in K562 cells. K562 cells were transfected with pRS-E or pRS15/16, and levels of miR-15a/16-1 expression were determined by quantitative real-time PCR at indicated time intervals. (D) K562 cells were treated with 1 lM ATO or miR-15a/16-1 or the combination of ATO and miR-15a/16-1 for 24 and 48 h. The percentages of growth inhibition and annexin V positive apoptotic cells were determined. *P < 0.05. ATO, arsenic trioxide; pRS15/16, pRETROSUPER vector expressing miR-15a/miR-16-1; pRS-E, pRETROSUPER empty vector.
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Fig. 2. ATO and miR-15a/16-1 synergistically induce mitochondrial dysfuction of K562 cells. K562 cells were treated with 1 lM ATO or miR-15a/16-1 or the combination of ATO and miR-15a/16-1 for 48 h. (A) The percentages of cells with low mitochondrial membrane potential were determined by flow cytometric analysis. The values represent mean ± SD of triplicates in an independent experiment, which were repeated for three times with the same results. *P < 0.05 compared to ATO group, &P < 0.05 compared to pRS15/16 group. (B and C) Mitochondria and cytosol were fractionated, and cytochrome c (Cyto c), activation of caspase-3 and PARP proteins were detected by western blotting analyzes. b-actin as loading control.
treated cells against untreated ones. The percentages of annexin V positive apoptotic cells were determined by flow cytometric analysis. Experiments were done in duplicate and repeated at least three times. 2.5. qRT-PCR qRT-PCR analysis for miRNAs was performed in triplicate with the NCode™ miRNA First-strand cDNA synthesis and qRT-PCR kits (invitrogen) according to the manufacturer’s instructions. U6 snRNA level was used for normalization. Alternative, Bcr-Abl fusion transcript was determined by quantitative real-time PCR using specific primer sets and cDNA quality was assessed relative to the ABL housekeeping gene as described [22]. Total RNA was extracted using TRIZOL (Invitrogen) and cDNA synthesis was performed using Superscript First-Strand Synthesis Kit (Promega, Madison, WI) [23]. The following primers were used respectively, Bcr-Abl: (sense strand: 50 -TCC GCT GAC CAT CAA CAA GGA-30 , antisense strand: 50 -CAC TCA GAC CCT GAG GCT CAA-30 , Taqman probe: 50 -Fam-CCC TTC AGC GGC CAG TAG CAT CTG A-Tamra-30 ), ABL: (sense strand: 50 -GAT GTA GTT GCT TGG GAC CCA-30 , antisense strand: 50 -TGG AGA TAA CAC TCT AAG CAT AAC TAA AGG T-30 , Taqman probe: 50 -Fam-CCA TTT TTG GTT TGG GCT TCA CAC CAT T-Tamra-30 ). 2.6. Western blot analysis Fig. 3. Both Bcr-Abl mRNA and protein expression levels were not affected by ATO and/or miR-15a/16-1. K562 cells were treated with 1 lM ATO or miR-15a/16-1 or the combination of ATO and miR-15a/16-1 for 48 h. (A) The Bcr-Abl protein was detected by western blotting analyzes. b-actin as loading control. (B) The mRNA expression of Bcr-Abl was detected by quantitative real-time PCR.
Protein extracts prepared with RIPA lysis buffer (50 mM Tris– HCl, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% sodiumdeoxycholate, 1 mM PMSF, 100 mM leupeptin, and 2 mg/mL aprotinin, pH 8.0)
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Fig. 4. Synergistic apoptosis induction of the regiment of miR-15a/16-1 and ATO in leukemic cell lines and primary leukemic cells. (A) Leukemic cell lines NB4, U937, HL60, Raji and RPMI8226 were respectively treated with 1 lM ATO or miR-15a/16-1 or the combination of ATO and miR-15a/16-1 for 48 h. The percentages of annexin V positive apoptotic cells were determined by flow cytometric analysis. (B) Mononuclear cells from bone marrow samples of one case of M2-type (#1), two cases of M4-type (#2, 3) and two cases of M5-type (#4, 5) acute myeloid leukemia were also collected and treated with 1 lM ATO or miR-15a/16-1 or the combination of ATO and miR-15a/16-1 for 48 h. The percentages of annexin V positive apoptotic cells were determined by flow cytometric analysis. *P < 0.05.
were separated on an 8% SDS–polyacrylamide gel and transferred to nitrocellulose membranes. After blocking with 5% nonfat milk, the membranes were incubated with an appropriate dilution of the primary antibody (Santa Cruz, Santa Cruz, CA), followed by incubation with the horseradish peroxidase-conjugated secondary antibody according to previously published protocols [23]. The signals were detected by chemiluminescence phototope-HRP kit (Cell Signaling, Danvers, MA) according to manufacturer’s instructions. As necessary, blots were stripped and reprobed with anti-actin antibody (Santa Cruz) as an internal control. All experiments were repeated three times with the similar results. 3. Results 3.1. MiR-15a/16-1 sensitizes ATO-induced apoptosis in K562 cells Consistent with previous reports [24], ATO induced growth inhibition and apoptosis in CML blast crisis K562 cells in a dose-dependent manner (Fig. 1A). One lM ATO, a dose with no significant effect on the rate of apoptosis, was used in the following study. As shown in Fig. 1B, the expression of miR-15a/16-1 was not affected by the ATO treatment. K562 cells were transiently transfected with pRS15/16 or pRS-E vector, and overexpressions of miR-15a/16-1 were verified by qRT-PCR analysis at 24 or 48 h after transfection (Fig. 1C). Combination of ATO and miR-15a/16-1 treatment exhibited robust synergism, both growth inhibition and apoptosis induction were significantly enhanced, compared with treatment of either of them (Fig. 1D).
the mitochondrial transmember potential collapse was greatly augmented by ATO combined with miR-15a/16-1 (Fig. 2A). Meanwhile, combination enhanced the activation of caspase-3, cytochrome c release into the cytosol and PARP cleavage (Fig. 2B and C). 3.3. MiR-15a/16-1 and/or ATO do not affect the level of Bcr-Abl expression To evaluate the effects of ATO and/or miR-15a/16-1 on Bcr-Abl expression, K562 cells treated with ATO and/or miR-15a/16-1 were analyzed for Bcr-Abl expression by quantitative real-time PCR and western blotting analyzes. As shown in Fig. 3, both Bcr-Abl mRNA and protein expression levels were not affected by ATO and miR15a/16-1. 3.4. MiR-15a/16-1 and ATO synergistically induce apoptosis in other leukemic cell lines and primary leukemic cells To investigate whether synergistic apoptosis induction in Bcr-Abl negative leukemic cells by miR-15a/16-1 and ATO, BcrAbl negative leukemic cell lines (NB4, HL60, U937, Raji and RPMI8226) and primary leukemic cells were used in the followed study. As shown in Fig. 4A, combined treatment with miR-15a/ 16-1 and ATO markedly enhanced apoptosis induction in Raji, NB4 and U937, but not in HL-60 and RPMI8226 cells (Fig. 4A). In addition, synergistic apoptosis induction was clearly observed in primary leukemic cells from five cases of AML patients by miR15a/16-1 and ATO (Fig. 4B).
3.2. MiR-15a/16-1 and ATO synergistically induce mitochondrial dysfunction and activation of caspase-3 in K562 cells
4. Discussion
As shown in Fig. 2A, mitochondrial transmember potential collapse was slightly induced by miR-15a/16-1 or ATO alone. However,
MiRNAs represent one of the major regulatory genes by inducing translation repression and transcription degradation [25]. MicroRNAs are often dysregulated in cancer and strongly
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implicated in cancer initiation and progression [26]. Also, miRNA modulate the anticancer drugs response of tumor cells [27]. MiR15b and miR-16 modulate the sensitivity of gastric cancer cells to some anticancer drugs by targeting BCL-2 [6]. MiR-21 regulates arsenic-induced anti-leukemia activity in leukemia K562 and HL-60 cell lines [19]. Recently, miRNAs function in apoptosis and differentiation of leukemia and lymphoma attracts great interest [20,28–30]. MiR-15a/16-1 induces cell apoptosis by downregulating antiapoptotic protein Bcl-2 in a leukemic cell line model [7]. ATO has been successfully used as a therapeutic agent for leukemia, but it used as a single agent at higher concentrations causes many side effects [13]. Reduce the concentration of ATO to obtain the same apoptotic and differentiation efficiency through combination with other drugs became very important [24,31]. However, very little work has been done on the interaction of miR-15a/16-1 and ATO. In the study, we used a specific precursor miR-15a/16-1 to study sensitivity of K562 cells to ATO. We found that overexpression of miR-15a/16-1 combined with ATO at 1 lM, a dose with no significant effect on the rate of apoptosis, synergistically induced growth inhibition and apoptosis in K562 cells. The apoptosis, at least in part, through regulating mitochondrial function including the release of cytochrome c and loss of mitochondrial transmembrane potential, also activation of caspase-3 and degradation of poly-adenosine diphosphate-ribose polymerase. The Bcr-Abl kinase signals to multiple downstream survival pathways, including MAP kinase, NF-Kb, signal transducers and activators of transcription and others [32,33]. Activation of these pathways in the presence of Bcr-Abl leads to increased expression of several antiapoptotic proteins and provide cells with a survival advantage over their normal counterparts. Furthermore, the Bcr-Abl fusion protein confers cells to varying degrees of resistance against conventional cytotoxic drugs [32]. Thus we investigated the possible change of the Bcr-Abl expression treatment with ATO and/or miR-15a/16-1. Our data showed that single or combined treatment did not influence the expression of Bcr-Abl fusion protein. The data indicated that the Bcr-Abl fusion protein did not exert the important role in the apoptosis induction by miR-15a/16-1 and ATO. It was verified that miR-15a/16-1 and ATO synergistically induce apoptosis in Bcr-Abl negative cell lines (NB4, U937 and Raji) and primary leukemic cells from five cases of AML patients. However, the synergic effects could not be observed in HL60 and RPMI8226 leukemic cell lines. Further investigations will be needed to clarify the causes leading to this discrepancy. Totally this research indicated the combinations of miR-15a/16-1 and ATO synergistically induced apoptosis in Bcr-Abl positive K562 cells, Bcr-Abl negative leukemic cell lines NB4, U937 and Raji, and primary leukemic cells from five cases of AML patients. These data showed that the combined regiment of miR-15a/16-1 and ATO might be a potential therapeutic remedy for the treatment of CML and other kinds of acute leukemia. However, further preclinical research is required. Conflict of interest Nothing to report. References [1] Z. Liu, A. Sall, D. Yang, MicroRNA: an emerging therapeutic target and intervention tool, Int. J. Mol. Sci. 9 (2008) 978–999. [2] J.M. Friedman, G. Liang, C.C. Liu, E.M. Wolff, Y.C. Tsai, W. Ye, X. Zhou, P.A. Jones, The putative tumor suppressor microRNA-101 modulates the cancer epigenome by repressing the polycomb group protein EZH2, Cancer Res. 69 (2009) 2623–2629. [3] A. Ventura, T. Jacks, MicroRNAs and cancer: short RNAs go a long way, Cell 136 (2009) 586–591.
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Synergistic apoptosis induction in leukemic cells by miR-15a/16-1 and arsenic trioxide Shen-meng Gao a, Chiqi Chen a, Jianbo Wu a, Yanxia Tan a, Kang Yu b,c, Chong-Yun Xing b, Aifang Ye a, Lihui Yin a, Lei Jiang a,⇑ a b c
Laboratory of Internal Medicine, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang 325000, China Department of Hematology, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou, Zhejiang 325000, China Institute of Hematology and Immunology, Wenzhou Medical College, Wenzhou, Zhejiang 325000, China
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Article history: Received 28 October 2010 Available online 5 November 2010 Keywords: Arsenic trioxide MiR-15a/16-1 Apoptosis Leukemia
a b s t r a c t MicroRNAs (miRNAs) are small noncoding RNAs that regulate target gene expression through translation repression or messenger RNA degradation. MiR-15a and 16-1 form a cluster at the chromosomal region 13q14, which is frequently deleted or down-regulated in chronic lymphocytic leukemia. Arsenic trioxide (As2O3, ATO) has been successfully applied to treat acute promyelocytic leukemia (APL). Its combination with other drugs presented therapeutic activities in hematologic and solid tumors. Here we investigated the potential synergy between miR-15a/16-1 and ATO on Bcr-Abl positive leukemic K562 cells. In this study, we found that combination of miR-15a/16-1 and ATO synergistically induced growth inhibition and apoptosis in K562 cells. The apoptosis, at least in part, through regulating mitochondrial function including the release of cytochrome c and loss of mitochondrial transmembrane potential, also activation of caspase-3 and degradation of poly-adenosine diphosphate-ribose polymerase. However, the expression of Bcr-Abl was not affected by ATO and/or miR-15a/16-1. Moreover, apoptotic synergy between miR-15a/16-1 and ATO was observed in Bcr-Abl negative leukemic cell lines and primary leukemic cells. Taken together, these findings suggested that the combined regiment of miR-15a/16-1 and ATO might be a potential therapeutic remedy for the treatment of leukemia. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction MicroRNAs (miRNAs) are short noncoding RNAs of 19–24 nucleotides, which bind to 30 UTRs of target mRNAs to either prevent their translation or induce their degradation [1]. MiRNA are involved in the regulation of critical cell processes such as apoptosis, cell proliferation and differentiation [2]. Several miRNAs have been implicated as tumor suppressors based on their physical deletion or down-regulated expression in human cancer [3]. The miR-15a/ 16-1 cluster resides at chromosome 13q14.3, a genomic region frequently deleted in the majority of B cell chronic lymphocytic leukemias (CLLs) and in a subset of mantle cell lymphomas and prostate cancers [3–5]. MiR-15a and miR-16-1 are known to act as tumor suppressors in CLL and other malignancies [6,7]. Expression of these miRNAs inhibits cell proliferation, induces cancer
Abbreviations: ATO, arsenic trioxide; APL, acute promyelocytic leukemia; miRNA, microRNAs; CLL, chronic lymphocytic leukemia; AML, acute myeloid leukemia; pRS15/16, pRETROSUPER vector expressing miR-15a/miR-16-1; pRS-E, pRETROSUPER empty vector. ⇑ Corresponding author. Fax: +86 577 86550280. E-mail address: [email protected] (L. Jiang). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.10.137
cells apoptosis, and suppresses tumorigenicity by targeting multiple oncogenes such as BCL2, MCL1, CCND1, and WNT3A [8,9]. Arsenic trioxide (As2O3, ATO), an ancient traditional Chinese medicine, have been successfully applied to treat acute promyelocytic leukemia (APL) [10–12]. The antitumor mechanism of ATO, mainly the induction of apoptosis and partial differentiation, suggests that it may be active against other hematologic malignancies [13]. A series of in vitro and in vivo experiments showed that ATO has potential activities in patients with accelerated phase chronic myelogenous leukemia (CML) and other hematologic malignancies [14,15]. CML is a myeloproliferative disorder that is characterized by a cytogenetic aberration consisting of a reciprocal translocation between the long arms of chromosomes 22 and 9, which forms the Bcr-Abl fusion protein [16]. The constitutively activated Bcr-Abl kinase, has been shown to confer leukemic cell resistance to diverse chemotherapeutic agents [17]. ATO used as a single agent at higher concentrations was adapted to induce apoptosis and growth inhibition in Bcr-Abl positive cells. Cytotoxicity and drug resistance are major concern with treatment. Recently it has been shown to be effective, particularly in combination with other drugs in hematologic and solid tumors [15,18,19]. However, very little work has been done on the interaction of miR-15a/16-1 and ATO in leukemic cells.
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In this study, we investigated the combined effects of miR-15a/ 16-1 and ATO on Bcr-Abl and CML cells. The K562 cell line was used as a cellular model of CML for drug screening. Moreover, in order to address the potential clinical application of the drugs, we used Bcr-Abl negative leukemic cell lines and primary leukemic cells. Our data showed that ATO and/or miR-15a/16-1 did not affect the expression of Bcr-Abl, overexpression of miR-15a/16-1 enhanced ATO at clinically achievable concentration induced apoptosis in Bcr-Abl positive K562 cells and some other Bcr-Abl negative leukemic cells.
2.2. Plasmids and transient transfection pRETROSUPER vector expressing miR-15a/miR-16-1 (pRS15/16) was constructed as previously described [20]. Cells transfected with the same empty plasmid (pRS-E) served as controls. Leukemic cells and primary cells of AML patients were transiently transfected with 1 lg/mL (final concentration) pRS15/16 or pRS-E vector using Lipofectamine™ LTX and PLUS™ Reagents (Invitrogen) according to the manufacturer’s instructions. 2.3. Viability and apoptosis assay
2. Materials and methods 2.1. Cell lines and primary leukemic cells Primary leukemic cells and various kinds of leukemic cell lines, including Bcr-Abl positive K562 cell line, Bcr-Abl negative cell lines U937, NB4, HL60, Raji and RPMI8226, were employed for the present study. All cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (Invitrogen, Groud Island, USA), in humidified 37 °C incubator with 5% CO2. Primary leukemic cells from five acute myeloid leukemia (AML) patients (one case of M2-type, two cases of M4-type and two cases of M5-type, The First Afficiated Hospital of Wenzhou Medical College) were used after informed consent. Mononuclear cells were isolated by Ficoll density gradient centrifugation (GE Healthcare, Uppsala, Sweden).
Cell lines and primary leukemic cells were plated in triplicate at 2 105 cells/mL. After being treated with ATO and/or miR-15a/ miR-16-1, viable cells were counted by 0.4% trypan blue exclusion. Growth inhibition rate was evaluated by the viable cell numbers in treated cells against untreated ones. The percentages of annexin V positive apoptotic cells were determined by flow cytometric analysis. Experiments were done in duplicate and repeated at least three times. 2.4. Flow cytometric analysis Cell lines and primary leukemic cells were plated in triplicate at 2 105 cells/mL. After being treated with ATO and/or miR-15a/ miR-16-1, viable cells were counted by 0.4% trypan blue exclusion. Growth inhibition rate was evaluated by the viable cell numbers in
Fig. 1. Overexpression of miR-15a/16-1 increases ATO-induced growth inhibition and apoptosis in K562 cells. (A) K562 cells were exposed to the indicated concentrations of ATO for 48 h, and the percentages of growth inhibition and annexin V positive apoptotic cells were determined. (B) Levels of miR-15a/16-1 expression were determined by quantitative real-time PCR. K562 cells were exposed to 1 lM ATO for different time, and quantitative real-time PCRs for miR-15a/16-1 were performed. (C) Overexpression of miR-15a/16-1 in K562 cells. K562 cells were transfected with pRS-E or pRS15/16, and levels of miR-15a/16-1 expression were determined by quantitative real-time PCR at indicated time intervals. (D) K562 cells were treated with 1 lM ATO or miR-15a/16-1 or the combination of ATO and miR-15a/16-1 for 24 and 48 h. The percentages of growth inhibition and annexin V positive apoptotic cells were determined. *P < 0.05. ATO, arsenic trioxide; pRS15/16, pRETROSUPER vector expressing miR-15a/miR-16-1; pRS-E, pRETROSUPER empty vector.
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Fig. 2. ATO and miR-15a/16-1 synergistically induce mitochondrial dysfuction of K562 cells. K562 cells were treated with 1 lM ATO or miR-15a/16-1 or the combination of ATO and miR-15a/16-1 for 48 h. (A) The percentages of cells with low mitochondrial membrane potential were determined by flow cytometric analysis. The values represent mean ± SD of triplicates in an independent experiment, which were repeated for three times with the same results. *P < 0.05 compared to ATO group, &P < 0.05 compared to pRS15/16 group. (B and C) Mitochondria and cytosol were fractionated, and cytochrome c (Cyto c), activation of caspase-3 and PARP proteins were detected by western blotting analyzes. b-actin as loading control.
treated cells against untreated ones. The percentages of annexin V positive apoptotic cells were determined by flow cytometric analysis. Experiments were done in duplicate and repeated at least three times. 2.5. qRT-PCR qRT-PCR analysis for miRNAs was performed in triplicate with the NCode™ miRNA First-strand cDNA synthesis and qRT-PCR kits (invitrogen) according to the manufacturer’s instructions. U6 snRNA level was used for normalization. Alternative, Bcr-Abl fusion transcript was determined by quantitative real-time PCR using specific primer sets and cDNA quality was assessed relative to the ABL housekeeping gene as described [22]. Total RNA was extracted using TRIZOL (Invitrogen) and cDNA synthesis was performed using Superscript First-Strand Synthesis Kit (Promega, Madison, WI) [23]. The following primers were used respectively, Bcr-Abl: (sense strand: 50 -TCC GCT GAC CAT CAA CAA GGA-30 , antisense strand: 50 -CAC TCA GAC CCT GAG GCT CAA-30 , Taqman probe: 50 -Fam-CCC TTC AGC GGC CAG TAG CAT CTG A-Tamra-30 ), ABL: (sense strand: 50 -GAT GTA GTT GCT TGG GAC CCA-30 , antisense strand: 50 -TGG AGA TAA CAC TCT AAG CAT AAC TAA AGG T-30 , Taqman probe: 50 -Fam-CCA TTT TTG GTT TGG GCT TCA CAC CAT T-Tamra-30 ). 2.6. Western blot analysis Fig. 3. Both Bcr-Abl mRNA and protein expression levels were not affected by ATO and/or miR-15a/16-1. K562 cells were treated with 1 lM ATO or miR-15a/16-1 or the combination of ATO and miR-15a/16-1 for 48 h. (A) The Bcr-Abl protein was detected by western blotting analyzes. b-actin as loading control. (B) The mRNA expression of Bcr-Abl was detected by quantitative real-time PCR.
Protein extracts prepared with RIPA lysis buffer (50 mM Tris– HCl, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5% sodiumdeoxycholate, 1 mM PMSF, 100 mM leupeptin, and 2 mg/mL aprotinin, pH 8.0)
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Fig. 4. Synergistic apoptosis induction of the regiment of miR-15a/16-1 and ATO in leukemic cell lines and primary leukemic cells. (A) Leukemic cell lines NB4, U937, HL60, Raji and RPMI8226 were respectively treated with 1 lM ATO or miR-15a/16-1 or the combination of ATO and miR-15a/16-1 for 48 h. The percentages of annexin V positive apoptotic cells were determined by flow cytometric analysis. (B) Mononuclear cells from bone marrow samples of one case of M2-type (#1), two cases of M4-type (#2, 3) and two cases of M5-type (#4, 5) acute myeloid leukemia were also collected and treated with 1 lM ATO or miR-15a/16-1 or the combination of ATO and miR-15a/16-1 for 48 h. The percentages of annexin V positive apoptotic cells were determined by flow cytometric analysis. *P < 0.05.
were separated on an 8% SDS–polyacrylamide gel and transferred to nitrocellulose membranes. After blocking with 5% nonfat milk, the membranes were incubated with an appropriate dilution of the primary antibody (Santa Cruz, Santa Cruz, CA), followed by incubation with the horseradish peroxidase-conjugated secondary antibody according to previously published protocols [23]. The signals were detected by chemiluminescence phototope-HRP kit (Cell Signaling, Danvers, MA) according to manufacturer’s instructions. As necessary, blots were stripped and reprobed with anti-actin antibody (Santa Cruz) as an internal control. All experiments were repeated three times with the similar results. 3. Results 3.1. MiR-15a/16-1 sensitizes ATO-induced apoptosis in K562 cells Consistent with previous reports [24], ATO induced growth inhibition and apoptosis in CML blast crisis K562 cells in a dose-dependent manner (Fig. 1A). One lM ATO, a dose with no significant effect on the rate of apoptosis, was used in the following study. As shown in Fig. 1B, the expression of miR-15a/16-1 was not affected by the ATO treatment. K562 cells were transiently transfected with pRS15/16 or pRS-E vector, and overexpressions of miR-15a/16-1 were verified by qRT-PCR analysis at 24 or 48 h after transfection (Fig. 1C). Combination of ATO and miR-15a/16-1 treatment exhibited robust synergism, both growth inhibition and apoptosis induction were significantly enhanced, compared with treatment of either of them (Fig. 1D).
the mitochondrial transmember potential collapse was greatly augmented by ATO combined with miR-15a/16-1 (Fig. 2A). Meanwhile, combination enhanced the activation of caspase-3, cytochrome c release into the cytosol and PARP cleavage (Fig. 2B and C). 3.3. MiR-15a/16-1 and/or ATO do not affect the level of Bcr-Abl expression To evaluate the effects of ATO and/or miR-15a/16-1 on Bcr-Abl expression, K562 cells treated with ATO and/or miR-15a/16-1 were analyzed for Bcr-Abl expression by quantitative real-time PCR and western blotting analyzes. As shown in Fig. 3, both Bcr-Abl mRNA and protein expression levels were not affected by ATO and miR15a/16-1. 3.4. MiR-15a/16-1 and ATO synergistically induce apoptosis in other leukemic cell lines and primary leukemic cells To investigate whether synergistic apoptosis induction in Bcr-Abl negative leukemic cells by miR-15a/16-1 and ATO, BcrAbl negative leukemic cell lines (NB4, HL60, U937, Raji and RPMI8226) and primary leukemic cells were used in the followed study. As shown in Fig. 4A, combined treatment with miR-15a/ 16-1 and ATO markedly enhanced apoptosis induction in Raji, NB4 and U937, but not in HL-60 and RPMI8226 cells (Fig. 4A). In addition, synergistic apoptosis induction was clearly observed in primary leukemic cells from five cases of AML patients by miR15a/16-1 and ATO (Fig. 4B).
3.2. MiR-15a/16-1 and ATO synergistically induce mitochondrial dysfunction and activation of caspase-3 in K562 cells
4. Discussion
As shown in Fig. 2A, mitochondrial transmember potential collapse was slightly induced by miR-15a/16-1 or ATO alone. However,
MiRNAs represent one of the major regulatory genes by inducing translation repression and transcription degradation [25]. MicroRNAs are often dysregulated in cancer and strongly
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implicated in cancer initiation and progression [26]. Also, miRNA modulate the anticancer drugs response of tumor cells [27]. MiR15b and miR-16 modulate the sensitivity of gastric cancer cells to some anticancer drugs by targeting BCL-2 [6]. MiR-21 regulates arsenic-induced anti-leukemia activity in leukemia K562 and HL-60 cell lines [19]. Recently, miRNAs function in apoptosis and differentiation of leukemia and lymphoma attracts great interest [20,28–30]. MiR-15a/16-1 induces cell apoptosis by downregulating antiapoptotic protein Bcl-2 in a leukemic cell line model [7]. ATO has been successfully used as a therapeutic agent for leukemia, but it used as a single agent at higher concentrations causes many side effects [13]. Reduce the concentration of ATO to obtain the same apoptotic and differentiation efficiency through combination with other drugs became very important [24,31]. However, very little work has been done on the interaction of miR-15a/16-1 and ATO. In the study, we used a specific precursor miR-15a/16-1 to study sensitivity of K562 cells to ATO. We found that overexpression of miR-15a/16-1 combined with ATO at 1 lM, a dose with no significant effect on the rate of apoptosis, synergistically induced growth inhibition and apoptosis in K562 cells. The apoptosis, at least in part, through regulating mitochondrial function including the release of cytochrome c and loss of mitochondrial transmembrane potential, also activation of caspase-3 and degradation of poly-adenosine diphosphate-ribose polymerase. The Bcr-Abl kinase signals to multiple downstream survival pathways, including MAP kinase, NF-Kb, signal transducers and activators of transcription and others [32,33]. Activation of these pathways in the presence of Bcr-Abl leads to increased expression of several antiapoptotic proteins and provide cells with a survival advantage over their normal counterparts. Furthermore, the Bcr-Abl fusion protein confers cells to varying degrees of resistance against conventional cytotoxic drugs [32]. Thus we investigated the possible change of the Bcr-Abl expression treatment with ATO and/or miR-15a/16-1. Our data showed that single or combined treatment did not influence the expression of Bcr-Abl fusion protein. The data indicated that the Bcr-Abl fusion protein did not exert the important role in the apoptosis induction by miR-15a/16-1 and ATO. It was verified that miR-15a/16-1 and ATO synergistically induce apoptosis in Bcr-Abl negative cell lines (NB4, U937 and Raji) and primary leukemic cells from five cases of AML patients. However, the synergic effects could not be observed in HL60 and RPMI8226 leukemic cell lines. Further investigations will be needed to clarify the causes leading to this discrepancy. Totally this research indicated the combinations of miR-15a/16-1 and ATO synergistically induced apoptosis in Bcr-Abl positive K562 cells, Bcr-Abl negative leukemic cell lines NB4, U937 and Raji, and primary leukemic cells from five cases of AML patients. These data showed that the combined regiment of miR-15a/16-1 and ATO might be a potential therapeutic remedy for the treatment of CML and other kinds of acute leukemia. However, further preclinical research is required. Conflict of interest Nothing to report. References [1] Z. Liu, A. Sall, D. Yang, MicroRNA: an emerging therapeutic target and intervention tool, Int. J. Mol. Sci. 9 (2008) 978–999. [2] J.M. Friedman, G. Liang, C.C. Liu, E.M. Wolff, Y.C. Tsai, W. Ye, X. Zhou, P.A. Jones, The putative tumor suppressor microRNA-101 modulates the cancer epigenome by repressing the polycomb group protein EZH2, Cancer Res. 69 (2009) 2623–2629. [3] A. Ventura, T. Jacks, MicroRNAs and cancer: short RNAs go a long way, Cell 136 (2009) 586–591.
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