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Synergistic antitumor effect of histone deacetylase inhibitor and Doxorubicin in peripheral T-cell lymphoma
Leukemia Research 56 (2017) 29–35
Contents lists available at ScienceDirect
Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Research paper
Synergistic antitumor effect of histone deacetylase inhibitor and Doxorubicin in peripheral T-cell lymphoma Huilai Zhang a,∗,1 , Ling Dong a,b,1 , Qingqing Chen a,c,1 , Lingzhe Kong a , Bin Meng a , Huaqing Wang d , Kai Fu a,e , Xi Wang f , Qiang Pan-Hammarström a,g , Ping Wang a , Xianhuo Wang a,∗ a Department of Lymphoma, Department of Pathology and Department of Radiotherapy, Sino-US Center for Lymphoma and Leukemia, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, China b Department of Geratology, The First Affiliated Hospital of Zhengzhou University, China c Department of Oncology, The First People’s Hospital of Xiangyang, Hubei, China d Department of Oncology, Tianjin Union Medicine Center, Tianjin, China e Department of Pathology and Microbiology and Internal Medicine, University of Nebraska Medical Center, Omaha, NE, USA f Department of Cellular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China g Clinical Immunology, Department of Laboratory Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, Sweden
a r t i c l e
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Article history: Received 28 September 2016 Received in revised form 5 January 2017 Accepted 19 January 2017 Available online 25 January 2017 Keywords: Chidamide Doxorubicin PTCL Synergistic antitumor effect
a b s t r a c t Chidamide (CS055) is a new and highly selective histone deacetylase inhibitor displaying significant single-agent activity in peripheral T-cell lymphoma (PTCL). But there is little known the synergistic effect between CS055 and chemotherapy. The purpose of this study is to explore the synergistic effect and molecular mechanisms of CS055 combination with Doxorubicin in PTCL cells. We found that CS055 showed dose- and time-dependent inhibition effects on PTCL cell. Meanwhile, the synergistic effect was significantly observed after combination treatment with lower drug-concentration of CS055 and Doxorubicin. Lower drug-concentration of CS055 induced weak apoptosis in PTCL cells, but combination treatment with CS055 and Doxorubicin promoted more significant apoptosis. Combination treatment with CS055 and Doxorubicin significantly changed mitochondrial membrane potential and H3 acetylated level, resulting in up-regulating DNA damage protein p-␥H2AX and apoptosis proteins including cleaved-caspase-3, cleaved-caspase-9 and cleaved-PARP, and down-regulating anti-apoptosis protein Bcl-2. In a word, Doxorubicin could increase the CS055-induced inhibition effects on PTCL cells, suggesting that CS055 combination with Doxorubicin or Doxorubicin-based chemotherapy drugs might be a new therapy approach for PTCL patients. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction Peripheral T-cell lymphoma (PTCL) is a set of rare and heterogeneous group of mature T- and natural killer (NK)-cell lymphoma associated with poor outcome, accounting for approximately 25%–30% of all Non-Hodgkin’s lymphoma (NHL) cases in China [1] and much higher than that seen in Western countries of 10%–15% [2]. PTCL include the Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), Angioimmunoblastic T-cell lym-
∗ Corresponding authors. E-mail addresses: [email protected] (H. Zhang), tjzlyy [email protected] (X. Wang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.leukres.2017.01.025 0145-2126/© 2017 Elsevier Ltd. All rights reserved.
phoma (AITL), NK/T-cell lymphoma, nasal type (ENKL), anaplastic large cell lymphoma (ALCL) in China. Currently, there is still no consensus on first-line therapy for patients with PTCL. Mostly following with the CHOP scheme of diffuse large B cell lymphoma, curative effect and long-term follow-up survival all show very poor and 5years overall survival is only 32% [3]. Although new agents, such as pralatrexate and romidepsin, were approved for patients with relapsed or refractory PTCL in recent years [4,5], the effect of those single agents is still limited and the overall response rates (ORRs) by independent central review for those three drugs are 20%–30%. Chidamide (CS055) is a novel benzamide type of subtypeselective histone deacetylase (HDAC) inhibitor, which has been reported that could led to cell apoptosis and cell cycle arrest at G0/1 phase in blood and lymphoid-derived tumor cells [6]. Chidamide has been showed significant single-agent activity and manageable
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H. Zhang et al. / Leukemia Research 56 (2017) 29–35
Table 1 The IC50 of CS055 and Doxorubicin in Jurkat cell line. CS055
24h 48h 72h
Doxorubicin
IC50(M)
95%CI
IC50(g/ml)
95%CI
5.576 2.659 1.908
5.201–5.915 2.320–2.635 1.802–2.014
8.309 1.797 0.809
6.309–15.104 1.366–2.156 0.556–1.024
Table 2 The IC50 of CS055 and Doxorubicin in NK92MI cell line. CS055
24h 48h 72h
Doxorubicin
IC50(Mm)
95%CI
IC50(g/ml)
95%CI
32.5 7.26 2.94
19.08–80.39 4.27–15.00 2.03–3.96
3.07 2.01 1.23
2.38–6.39 1.62–2.62 0.95–2.65
toxicity in relapsed or refractory peripheral T-cell lymphoma (PTCL) in Chinese population from the results of phase II clinical trial [7]. The ORR was 28% (22 of 79), including 14% (11 of 79) with complete response/unconfirmed complete response (CR/CRu). Most adverse events (AEs) were grade 1 or 2, and AEs ≥grade 3 that occurred in ≥10% patients were thrombocytopenia (22%), leucopenia (13%) and neutropenia (11%), respectively. Combined treatment of chidamide and other chemotherapy drugs has been reported that enhanced the anti-tumor activity in some solid tumors. Liu et al. found that combination of chidamide with 5-Fu enhanced the antitumor activity of 5-Fu in nude mice bearing human colon cancer [8]. Qiao et al. found that Chidamide enhanced gemcitabine-induced DNA double-strand breaks and S phase arrest, and abrogated the G2/M cell cycle checkpoint, potentially through suppression of CHK1 expression in pancreatic cancer cell lines [9]. Zhou et al. found that the combination of chidamide with carboplatin showed a good synergism on growth inhibition, G2/M phase of cell cycle block and cell apoptosis in NSCLC cells [10]. As we mentioned above, chidamide has been showed significant single-agent activity in relapsed or refractory PTCL and synergistic antitumor interaction with other chemotherapy drugs in some solid tumors. But there is little known the synergistic effect between CS055 and chemotherapy in PTCL cells. Anthracycline is a common and important chemotherapy drugs which is applied to treat PTCL patients. Because of the cumulative cardiac toxicity, long-term use of high doses of anthracycline is limited. The purpose of this study is to explore the synergistic effect and molecular mechanisms of CS055 combination with Doxorubicin in PTCL cells. Our results demonstrated that the combination treatment with low-dose Doxorubicin and CS055 exhibited a synergism effect on cell growth and apoptosis in two PTCL cell lines. We further demonstrated that the mechanism of their synergism effect was to regulate the level of mitochondrial membrane potential and acetylation of H3, which resulted in up-regulating DNA damage protein p-␥H2AX and apoptosis proteins, and down-regulating anti-apoptosis protein. Our data provided new insights into the synergistic effect of histone deacetylase inhibitor and Doxorubicin or Doxorubicin-based chemotherapy drugs for the treatment of PTCL.
USA) and 1% penicillin-streptomycin. NK-92MI cell line (NK/T cell lymphoma) was obtained from ATCC (American Type Culture Collection, Manassas, USA),which was cultured at 37 ◦ C under 5% CO2 in Alpha Minimum Essential medium (Life Technologies, California, USA) supplemented with 12.5% fetal bovine serum (Hyclone, UT, USA),12.5% horse serum (Life Technologies, California, USA), 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid (Sigma-Aldrich, USA) and 1% penicillin-streptomycin. Chidamide (CS055), as a new HDAC inhibitor, was granted by Shenzhen ChipScreen BioS and Doxorubicin (H44024359) was purchased from Shenzhen Main Luck Pharmaceuticals Inc. 2.2. Assessment of cell proliferative viability Drug effects on cell proliferative viability were monitored using MTS/PMS viability assay. For MTS/PMS assay, cells were incubated in triplicate in a 96-well plate in the presence or absence of indicated test samples in a final volume of 100ul for 24 h, 48 h and 72 h. Thereafter, MTS/PMS (MTS: PMS = 20:1, Promega, USA) was added to each well. After 2–4 h incubation at 37 ◦ C, the optical density (OD) at 490 nm was measured using a 96-well multiscanner autoreader. The inhibition of cell proliferative viability was expressed as a percentage: (1-OD of experiment sample/OD of control). 2.3. Apoptosis assay An Annexin-V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit (BD Biosciences, San Jose, CA) was used to detect apoptosis by flow cytometry (LSDFortessa, BD Bioscience). Cells were seeded at a density of 2–5 × 105 cells/mL in six-well plate and incubated for 48 h with CS055 and Doxorubicin, alone or in combination with different concentration. A minimum of 1 × 106 events were acquired for each sample. To quantitate apoptosis, cells were stained with Annexin V-FITC and PI, according to the manufacturer’s instruction. Results were analyzed with FlowJo software (version 7.6). Cells were considered in early apoptosis if Annexin V-FITC positive but PI negative, late apoptotic if Annexin V-FITC and PI positive, and dead if only PI positive. 2.4. Mitochondrial membrane potential analysis
2. Material and methods 2.1. Cell culture and inhibitors Jurkat (T-cell leukemia/lymphoma) was granted by professor Fu from the University of Nebraska, which was cultured at 37 ◦ C under 5% CO2 in RPMI 1640 (Life Technologies, California, USA) supplemented with 10% fetal bovine serum (Hyclone, UT,
Cells were seeded at a density of 2–5 × 105 cells/mL in six-well plate with CS055 and Doxorubicin, alone or in combination with different concentration. After 48 h of incubation, cells were harvested and were resuspended in the dilute 2 M Rhodamine123 (Byeotime, Shanghai, China) for 30 min in 37 ◦ C. Then after washed by medium, cells mitochondrial membrane potential (MMP) were analyzed by IX71 fluorescence microscope (Olympus, Japan).
H. Zhang et al. / Leukemia Research 56 (2017) 29–35
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Fig. 1. The inhibition of proliferative viability induced by single CS055 and Doxorubicin in Jurkat and NK92MI cell lines.
2.5. Western blotting Cells were harvested and suspended in RIPA lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF). 20–50 g total protein (depending on different proteins) were separated by 8%–12% sodiumdodecyl sulfate–polyacrylamide (SDS–PAGE) gel electrophoresis and then transferred to polyvinylidene difluoride membranes (PVDF, Roche, UK), which were then blocked in 5% non-fat milk in Tris-buffer saline-Tween (TBST) for 1 h. The immunoblotting was performed by incubation with the primary antibodies including HDAC2, Histone H3, Acetyl-Histone H3 (Lys9), Bcl-2, p-␥ H2AX, Caspase-9, Caspase-3, PARP and -actin (from Cell Signaling Technology, Boston, USA) at 4 ◦ C overnight. Blots were then washed and incubated with a 1:3000 dilution of Goat anti-Rabbit or anti-Mouse IgG H&L (HRP)-conjugated secondary (Millipore, Billerica, MA, USA). Signals were detected by enhanced chemiluminescence Plus reagents (Amersham Pharmacia, Piscataway, NJ). Signal quantification was obtained using Quantity One software (Bio-Rad Laboratories, USA) and normalized to -actin. 3. Results 3.1. Combination treatment with CS055 and Doxorubicin synergistically inhibits PTCL cell proliferative viability To test the synergistic effect between CS055 and Doxorubicin in PTCL cells, we at first assessed respectively the single drug effect of CS055 and Doxorubicin, and calculated their IC50 values in two PTCL cells for 24 h, 48 h and 72 h (Tables 1 and 2). We found that there were different sensitivity of Jurkat and NK92MI cell lines after treat-
ing with single drug CS055 and Doxorubicin. Fig. 1 revealed that the inhibition effect of cell proliferative viability was induced by single drug CS055 or Doxorubicin with time- and dose-dependent manners in Jurkat and NK92MI cell lines. Then, the combination treatment with lower drug-concentration (1/5–5/5 IC50 ) of CS055 and Doxorubicin showed that the combination treatment of the two drugs synergistically inhibited the two cell lines proliferative viability (P < 0.05) (Tables 3 and 4 and Fig. 2). 3.2. Combination treatment with CS055 and Doxorubicin synergistically induces apoptosis in PTCL cell lines To further evaluate the synergistic effect of the two drugs, we examined the apoptotic effect of the single drug CS055 with low drug-concentration (
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H. Zhang et al. / Leukemia Research 56 (2017) 29–35
Table 3 The effect of synergistic inhibition in Jurkat cell line. Drug concentration
24h
48h
72h
CS055
Doxorubicin
fa
CI
fa
CI
fa
CI
1/5IC50 2/5IC50 3/5IC50 4/5IC50 5/5IC50
1/5IC50 2/5IC50 3/5IC50 4/5IC50 5/5IC50
0.18 0.27 0.53 0.67 0.85
2.35 2.02 0.56 0.32 0.09
0.25 0.66 0.8 0.82 0.83
0.99 0.39 0.31 0.38 0.45
0.23 0.53 0.77 0.82 0.84
1.01 0.77 0.5 0.49 0.59
Table 4 The effect of synergistic inhibition in NK92MI cell line. Drug concentration
24h
48h
72h
CS055
Doxorubicin
fa
CI
fa
CI
fa
CI
1/5IC50 2/5IC50 3/5IC50 4/5IC50 5/5IC50
1/5IC50 2/5IC50 3/5IC50 4/5IC50 5/5IC50
0.18 0.27 0.53 0.67 0.85
2.35 2.02 0.56 0.32 0.09
0.25 0.66 0.8 0.82 0.83
0.99 0.39 0.31 0.38 0.45
0.23 0.53 0.77 0.82 0.84
1.01 0.77 0.5 0.49 0.59
Fig. 2. The effect of synergistic inhibition induced by combination of CS055 and Doxorubicin in PTCL cells. (A) Jurkat cell lines. (B) NK92MI cell lines.
H. Zhang et al. / Leukemia Research 56 (2017) 29–35
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Fig. 3. Cell apoptosis induced by single CS055 and combination with Doxorubicin in Jurkat and NK92MI cell lines. (A) Cell apoptosis rate induced by single CS055 depend on dosage. (B) The effect of synergistic apoptosis induced by combination of CS055 and Doxorubicin.
Fig. 4. Mitochondrial membrane potential (MMP) changes was induced by single CS055 and combination with Doxorubicin in Jurkat and NK92MI cell lines. (A) MMP changes was induced by single CS055 depend on dosage. (B) Combination of CS055 and Doxorubicin showed synergistic effect in dropping MMP.
tion treatment. The results suggested that CS055 resulted in the lower change of the MMP level in PTCL cell lines (Fig. 4A). But the combination treatment with lower drug-concentration CS055 and
Doxorubicine induced significant change of membrane potential level (Fig. 4B).
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H. Zhang et al. / Leukemia Research 56 (2017) 29–35
Fig. 5. Western blot detected the expression level of HDAC2, H3, Ac-H3(Lys9) some apoptosis-related proteins in Jurkat and NK92MI cell lines. (A) The influence of CS055 on the expression of HDAC2, H3 and acetylation of H3 in Jurkat and NK92MI cell lines. (B) The expression of apoptotic proteins after combined treatment with CS055 and Doxorubicin in Jurkat cell line.
3.4. CS055 regulates the expression of HDAC2, H3 and acetylation of H3 CS055 is the inhibitor of HDACs, and HDAC2 is one of the major type HDACs. To understand the protein changes after CS055 treatment, we analyzed the expression of HDAC2, H3 and H3 (Lys 9). Results showed (Fig. 5A) that the HDAC2 and H3 expression level was not changed, but the H3 acetylated level (Lys 9) was upregulated after CS055 treatment, suggesting that CS055 did not regulate the HDAC2 and H3 levels, however, up-regulated the H3 acetylated level. 3.5. Combination treatment with CS055 and Doxorubicin synergistically regulates the expression of apoptotic proteins and DNA damage protein Apoptotic pathways include extrinsic pathway, mitochondrial pathway and endoplasmic reticulum stress-induced apoptosis. To further explore the potential apoptosis mechanism of combination treatment with CS055 and Doxorubicine in PTCL cells, we detected the expression levels of apoptotic proteins including Caspase 9, Caspase 3 and PARP, and anti-apoptosis protein Bcl-2, and DNA damage protein p-␥H2AX. Results showed (Fig. 5B) that the combination treatment with CS055 and Doxorubicin resulted in the more significant protein changes of cleaved-Caspase 9, cleaved-Caspase 3 and cleaved-PARP, which were up-regulated. On the contrary, the anti-apoptosis protein Bcl-2 was down-regulated. As well as the expression of DNA damage protein p-␥H2AX was also up-regulated. 4. Discussion Histone acetylation regulates DNA transcription and protein expression of target genes. Aberrant histone acetylation is observed in human tumors. Histone deacetylase inhibitors (HDACis) are some class of new drugs, which show to be promising in several malignant patients including relapsed and/or refractory lymphoma. Chidamide is a novel benzamide inhibitor of HDAC1, 2, 3, and 10, which shows effective antitumor activity on lung cancer, colon cancer, breast cancer, pancreatic cancer, liver cancer, lymphoma and leukemia in vitro and in vivo [6,8,9,11,12]. Some reports of phase II studies confirmed the activity of chidamide in patients with relapsed or refractory PTCL, and China Food and Drug Administration (CFDA) had approved the application of chidamide for treatment of patients with relapse or refractory PTCL. But the ORR
of single drug of chidamide is still limited. Therefore, combination treatment with chidamide and other chemotherapy drugs might be a promising therapeutic regimen for improving the prognosis of patients with relapsed or refractory PTCL. Previous studies showed that HDACIs combination with -phenylethyl isothiocyanate, DNA damaging agents and DNA methyltransferase inhibitors exhibited a better clinical effect for treatment of some solid tumor and hematologic malignant tumors [13–16]. Anthracyclines are the important chemotherapy drugs as DNA damaging agents for PTCL, but they are limited to use due to the cardiotoxicity. In this study, we developed a combination treatment with chidamide and Doxorubicin and explored the mechanism of synergistic anti-tumor effect in PTCL. Theoretical basis of the combination treatment were as follows: (1) Chidamide enhanced the level of histone acetylation, resulting in disaggregating between DNA and histone, which would make nucleosome structure loose. Then Doxorubicin trended to be embedded into the double-stranded DNA, which mediated the DNA damage [17,18]. (2) Doxorubicin caused DNA damage and chidamide also induced DNA damage [19,20]. (3) Several studies showed that HDACIs could enhance the cumulant of topoisomerase II inhibitors in nuclei, leading to DNA break irreparably and cells apoptosis [21–23]. Adriamycin also belonged to a type of topoisomerase II inhibitor. In our study, we developed the combination treatment with different concentration CS055 and Doxorubicin to treat PTCL cells in order to explore the relationship between anti-tumor effect and the dose of the two drugs. We finally found the optimal dosage of the two drugs for synergistic antitumor effect. Our results showed that the combination treatment with lower drug-concentration CS055 and Doxorubicin had a synergistic inhibiting effect on Jurkat and NK92MI cell proliferation. The apoptotic rates of combination treatment was much higher compared to the single drug. It suggested that the PTCL patients could obtain a benefit from the combination treatment with lower drug-concentration CS055 and Doxorubicin, and might have less adverse effects due to reduced therapeutic dosage of the two drugs in clinic. To explore the mechanism of cell apoptosis induced by combination treatment with CS055 and Doxorubicin, we detected the expression levels of H3, Ac-H3 (Lys9) and some apoptosis-related proteins. Our data suggested a potential mechanism of cell apoptosis induced through combination treatment with CS055 and Doxorubicin in PTCL cells as follows: Firstly, CS055 up-regulated the expression level of Ac-H3 (Lys9) as a histone deacetylase inhibitor, leading to the DNA separate from histone. Then, Ac-H3 (Lys9)
H. Zhang et al. / Leukemia Research 56 (2017) 29–35
shifted from nucleus into cytoplasm, which resulted in the changes of mitochondria membrane potential, and then switched on the mitochondria apoptosis pathway. As well as Doxorubicin embed into the dissociated double-strand DNA, which induced cell apoptosis irreversibly. In summary, we demonstrated that the combination treatment with CS055 and doxorubicin showed synergistic antitumor effect at a low-dosage of drug component concentration in PTCL cell lines. We further explored the mechanism of cell apoptosis induced by combination treatment with CS055 and doxorubicin as follows: the combination treatment with CS055 and Doxorubicin significantly changed membrane potential and H3 acetylated level. And combination treatment also significantly up-regulated DNA damage protein p-␥H2AX and apoptosis proteins including cleaved-caspase-3, cleaved-caspase-9 and cleaved-PARP, as well as down-regulated anti-apoptosis protein Bcl-2. Our results provided the theoretical basis about the combination treatment with CS055 and Doxorubicin or Doxorubicin-based chemotherapy drugs for PTCL patients. Conflict of interest Authors declared no competing financial interest. Acknowledgements and grant support This study was supported by the National Clinical Research Center for Cancer Foundation in Tianjin Medical University Cancer Institute and Hospital (B type), Natural Science Foundation of China (81402945), National Major Scientific and Technological Special Project for “Significant New Drugs Development” during the Twelfth Five-year Plan Period (2013ZX09303001), Scientific Research Foundation for the Returned Overseas Chinese Scholars in Tianjin (2015-31) and Natural Science Foundation of Tianjin Medical University. References [1] J. Sun, Q. Yang, Z. Lu, et al., Distribution of lymphoid neoplasms in China: analysis of 4,638 cases according to the World Health Organization classification, Am. J. Clin. Pathol. 138 (2012) 429–434. [2] J. Vose, J. Armitage, D. Weisenburger, International peripheral T-cell and natural killer/T-cell lymphoma study: pathology findings and clinical outcomes, J. Clin. Oncol. 26 (2008) 4124–4130. [3] J.O. Armitage, The aggressive peripheral T-cell lymphomas: 2012 update on diagnosis, risk stratification, and management, Am. J. Hematol. 87 (2012) 511–519. [4] O.A. O’connor, B. Pro, L. Pinter-Brown, et al., Pralatrexate in patients with relapsed or refractory peripheral T-cell lymphoma: results from the pivotal PROPEL study, J. Clin. Oncol. 29 (2011) 1182–1189.
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Contents lists available at ScienceDirect
Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Research paper
Synergistic antitumor effect of histone deacetylase inhibitor and Doxorubicin in peripheral T-cell lymphoma Huilai Zhang a,∗,1 , Ling Dong a,b,1 , Qingqing Chen a,c,1 , Lingzhe Kong a , Bin Meng a , Huaqing Wang d , Kai Fu a,e , Xi Wang f , Qiang Pan-Hammarström a,g , Ping Wang a , Xianhuo Wang a,∗ a Department of Lymphoma, Department of Pathology and Department of Radiotherapy, Sino-US Center for Lymphoma and Leukemia, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin, China b Department of Geratology, The First Affiliated Hospital of Zhengzhou University, China c Department of Oncology, The First People’s Hospital of Xiangyang, Hubei, China d Department of Oncology, Tianjin Union Medicine Center, Tianjin, China e Department of Pathology and Microbiology and Internal Medicine, University of Nebraska Medical Center, Omaha, NE, USA f Department of Cellular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China g Clinical Immunology, Department of Laboratory Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, Sweden
a r t i c l e
i n f o
Article history: Received 28 September 2016 Received in revised form 5 January 2017 Accepted 19 January 2017 Available online 25 January 2017 Keywords: Chidamide Doxorubicin PTCL Synergistic antitumor effect
a b s t r a c t Chidamide (CS055) is a new and highly selective histone deacetylase inhibitor displaying significant single-agent activity in peripheral T-cell lymphoma (PTCL). But there is little known the synergistic effect between CS055 and chemotherapy. The purpose of this study is to explore the synergistic effect and molecular mechanisms of CS055 combination with Doxorubicin in PTCL cells. We found that CS055 showed dose- and time-dependent inhibition effects on PTCL cell. Meanwhile, the synergistic effect was significantly observed after combination treatment with lower drug-concentration of CS055 and Doxorubicin. Lower drug-concentration of CS055 induced weak apoptosis in PTCL cells, but combination treatment with CS055 and Doxorubicin promoted more significant apoptosis. Combination treatment with CS055 and Doxorubicin significantly changed mitochondrial membrane potential and H3 acetylated level, resulting in up-regulating DNA damage protein p-␥H2AX and apoptosis proteins including cleaved-caspase-3, cleaved-caspase-9 and cleaved-PARP, and down-regulating anti-apoptosis protein Bcl-2. In a word, Doxorubicin could increase the CS055-induced inhibition effects on PTCL cells, suggesting that CS055 combination with Doxorubicin or Doxorubicin-based chemotherapy drugs might be a new therapy approach for PTCL patients. © 2017 Elsevier Ltd. All rights reserved.
1. Introduction Peripheral T-cell lymphoma (PTCL) is a set of rare and heterogeneous group of mature T- and natural killer (NK)-cell lymphoma associated with poor outcome, accounting for approximately 25%–30% of all Non-Hodgkin’s lymphoma (NHL) cases in China [1] and much higher than that seen in Western countries of 10%–15% [2]. PTCL include the Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), Angioimmunoblastic T-cell lym-
∗ Corresponding authors. E-mail addresses: [email protected] (H. Zhang), tjzlyy [email protected] (X. Wang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.leukres.2017.01.025 0145-2126/© 2017 Elsevier Ltd. All rights reserved.
phoma (AITL), NK/T-cell lymphoma, nasal type (ENKL), anaplastic large cell lymphoma (ALCL) in China. Currently, there is still no consensus on first-line therapy for patients with PTCL. Mostly following with the CHOP scheme of diffuse large B cell lymphoma, curative effect and long-term follow-up survival all show very poor and 5years overall survival is only 32% [3]. Although new agents, such as pralatrexate and romidepsin, were approved for patients with relapsed or refractory PTCL in recent years [4,5], the effect of those single agents is still limited and the overall response rates (ORRs) by independent central review for those three drugs are 20%–30%. Chidamide (CS055) is a novel benzamide type of subtypeselective histone deacetylase (HDAC) inhibitor, which has been reported that could led to cell apoptosis and cell cycle arrest at G0/1 phase in blood and lymphoid-derived tumor cells [6]. Chidamide has been showed significant single-agent activity and manageable
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Table 1 The IC50 of CS055 and Doxorubicin in Jurkat cell line. CS055
24h 48h 72h
Doxorubicin
IC50(M)
95%CI
IC50(g/ml)
95%CI
5.576 2.659 1.908
5.201–5.915 2.320–2.635 1.802–2.014
8.309 1.797 0.809
6.309–15.104 1.366–2.156 0.556–1.024
Table 2 The IC50 of CS055 and Doxorubicin in NK92MI cell line. CS055
24h 48h 72h
Doxorubicin
IC50(Mm)
95%CI
IC50(g/ml)
95%CI
32.5 7.26 2.94
19.08–80.39 4.27–15.00 2.03–3.96
3.07 2.01 1.23
2.38–6.39 1.62–2.62 0.95–2.65
toxicity in relapsed or refractory peripheral T-cell lymphoma (PTCL) in Chinese population from the results of phase II clinical trial [7]. The ORR was 28% (22 of 79), including 14% (11 of 79) with complete response/unconfirmed complete response (CR/CRu). Most adverse events (AEs) were grade 1 or 2, and AEs ≥grade 3 that occurred in ≥10% patients were thrombocytopenia (22%), leucopenia (13%) and neutropenia (11%), respectively. Combined treatment of chidamide and other chemotherapy drugs has been reported that enhanced the anti-tumor activity in some solid tumors. Liu et al. found that combination of chidamide with 5-Fu enhanced the antitumor activity of 5-Fu in nude mice bearing human colon cancer [8]. Qiao et al. found that Chidamide enhanced gemcitabine-induced DNA double-strand breaks and S phase arrest, and abrogated the G2/M cell cycle checkpoint, potentially through suppression of CHK1 expression in pancreatic cancer cell lines [9]. Zhou et al. found that the combination of chidamide with carboplatin showed a good synergism on growth inhibition, G2/M phase of cell cycle block and cell apoptosis in NSCLC cells [10]. As we mentioned above, chidamide has been showed significant single-agent activity in relapsed or refractory PTCL and synergistic antitumor interaction with other chemotherapy drugs in some solid tumors. But there is little known the synergistic effect between CS055 and chemotherapy in PTCL cells. Anthracycline is a common and important chemotherapy drugs which is applied to treat PTCL patients. Because of the cumulative cardiac toxicity, long-term use of high doses of anthracycline is limited. The purpose of this study is to explore the synergistic effect and molecular mechanisms of CS055 combination with Doxorubicin in PTCL cells. Our results demonstrated that the combination treatment with low-dose Doxorubicin and CS055 exhibited a synergism effect on cell growth and apoptosis in two PTCL cell lines. We further demonstrated that the mechanism of their synergism effect was to regulate the level of mitochondrial membrane potential and acetylation of H3, which resulted in up-regulating DNA damage protein p-␥H2AX and apoptosis proteins, and down-regulating anti-apoptosis protein. Our data provided new insights into the synergistic effect of histone deacetylase inhibitor and Doxorubicin or Doxorubicin-based chemotherapy drugs for the treatment of PTCL.
USA) and 1% penicillin-streptomycin. NK-92MI cell line (NK/T cell lymphoma) was obtained from ATCC (American Type Culture Collection, Manassas, USA),which was cultured at 37 ◦ C under 5% CO2 in Alpha Minimum Essential medium (Life Technologies, California, USA) supplemented with 12.5% fetal bovine serum (Hyclone, UT, USA),12.5% horse serum (Life Technologies, California, USA), 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid (Sigma-Aldrich, USA) and 1% penicillin-streptomycin. Chidamide (CS055), as a new HDAC inhibitor, was granted by Shenzhen ChipScreen BioS and Doxorubicin (H44024359) was purchased from Shenzhen Main Luck Pharmaceuticals Inc. 2.2. Assessment of cell proliferative viability Drug effects on cell proliferative viability were monitored using MTS/PMS viability assay. For MTS/PMS assay, cells were incubated in triplicate in a 96-well plate in the presence or absence of indicated test samples in a final volume of 100ul for 24 h, 48 h and 72 h. Thereafter, MTS/PMS (MTS: PMS = 20:1, Promega, USA) was added to each well. After 2–4 h incubation at 37 ◦ C, the optical density (OD) at 490 nm was measured using a 96-well multiscanner autoreader. The inhibition of cell proliferative viability was expressed as a percentage: (1-OD of experiment sample/OD of control). 2.3. Apoptosis assay An Annexin-V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit (BD Biosciences, San Jose, CA) was used to detect apoptosis by flow cytometry (LSDFortessa, BD Bioscience). Cells were seeded at a density of 2–5 × 105 cells/mL in six-well plate and incubated for 48 h with CS055 and Doxorubicin, alone or in combination with different concentration. A minimum of 1 × 106 events were acquired for each sample. To quantitate apoptosis, cells were stained with Annexin V-FITC and PI, according to the manufacturer’s instruction. Results were analyzed with FlowJo software (version 7.6). Cells were considered in early apoptosis if Annexin V-FITC positive but PI negative, late apoptotic if Annexin V-FITC and PI positive, and dead if only PI positive. 2.4. Mitochondrial membrane potential analysis
2. Material and methods 2.1. Cell culture and inhibitors Jurkat (T-cell leukemia/lymphoma) was granted by professor Fu from the University of Nebraska, which was cultured at 37 ◦ C under 5% CO2 in RPMI 1640 (Life Technologies, California, USA) supplemented with 10% fetal bovine serum (Hyclone, UT,
Cells were seeded at a density of 2–5 × 105 cells/mL in six-well plate with CS055 and Doxorubicin, alone or in combination with different concentration. After 48 h of incubation, cells were harvested and were resuspended in the dilute 2 M Rhodamine123 (Byeotime, Shanghai, China) for 30 min in 37 ◦ C. Then after washed by medium, cells mitochondrial membrane potential (MMP) were analyzed by IX71 fluorescence microscope (Olympus, Japan).
H. Zhang et al. / Leukemia Research 56 (2017) 29–35
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Fig. 1. The inhibition of proliferative viability induced by single CS055 and Doxorubicin in Jurkat and NK92MI cell lines.
2.5. Western blotting Cells were harvested and suspended in RIPA lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF). 20–50 g total protein (depending on different proteins) were separated by 8%–12% sodiumdodecyl sulfate–polyacrylamide (SDS–PAGE) gel electrophoresis and then transferred to polyvinylidene difluoride membranes (PVDF, Roche, UK), which were then blocked in 5% non-fat milk in Tris-buffer saline-Tween (TBST) for 1 h. The immunoblotting was performed by incubation with the primary antibodies including HDAC2, Histone H3, Acetyl-Histone H3 (Lys9), Bcl-2, p-␥ H2AX, Caspase-9, Caspase-3, PARP and -actin (from Cell Signaling Technology, Boston, USA) at 4 ◦ C overnight. Blots were then washed and incubated with a 1:3000 dilution of Goat anti-Rabbit or anti-Mouse IgG H&L (HRP)-conjugated secondary (Millipore, Billerica, MA, USA). Signals were detected by enhanced chemiluminescence Plus reagents (Amersham Pharmacia, Piscataway, NJ). Signal quantification was obtained using Quantity One software (Bio-Rad Laboratories, USA) and normalized to -actin. 3. Results 3.1. Combination treatment with CS055 and Doxorubicin synergistically inhibits PTCL cell proliferative viability To test the synergistic effect between CS055 and Doxorubicin in PTCL cells, we at first assessed respectively the single drug effect of CS055 and Doxorubicin, and calculated their IC50 values in two PTCL cells for 24 h, 48 h and 72 h (Tables 1 and 2). We found that there were different sensitivity of Jurkat and NK92MI cell lines after treat-
ing with single drug CS055 and Doxorubicin. Fig. 1 revealed that the inhibition effect of cell proliferative viability was induced by single drug CS055 or Doxorubicin with time- and dose-dependent manners in Jurkat and NK92MI cell lines. Then, the combination treatment with lower drug-concentration (1/5–5/5 IC50 ) of CS055 and Doxorubicin showed that the combination treatment of the two drugs synergistically inhibited the two cell lines proliferative viability (P < 0.05) (Tables 3 and 4 and Fig. 2). 3.2. Combination treatment with CS055 and Doxorubicin synergistically induces apoptosis in PTCL cell lines To further evaluate the synergistic effect of the two drugs, we examined the apoptotic effect of the single drug CS055 with low drug-concentration (
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Table 3 The effect of synergistic inhibition in Jurkat cell line. Drug concentration
24h
48h
72h
CS055
Doxorubicin
fa
CI
fa
CI
fa
CI
1/5IC50 2/5IC50 3/5IC50 4/5IC50 5/5IC50
1/5IC50 2/5IC50 3/5IC50 4/5IC50 5/5IC50
0.18 0.27 0.53 0.67 0.85
2.35 2.02 0.56 0.32 0.09
0.25 0.66 0.8 0.82 0.83
0.99 0.39 0.31 0.38 0.45
0.23 0.53 0.77 0.82 0.84
1.01 0.77 0.5 0.49 0.59
Table 4 The effect of synergistic inhibition in NK92MI cell line. Drug concentration
24h
48h
72h
CS055
Doxorubicin
fa
CI
fa
CI
fa
CI
1/5IC50 2/5IC50 3/5IC50 4/5IC50 5/5IC50
1/5IC50 2/5IC50 3/5IC50 4/5IC50 5/5IC50
0.18 0.27 0.53 0.67 0.85
2.35 2.02 0.56 0.32 0.09
0.25 0.66 0.8 0.82 0.83
0.99 0.39 0.31 0.38 0.45
0.23 0.53 0.77 0.82 0.84
1.01 0.77 0.5 0.49 0.59
Fig. 2. The effect of synergistic inhibition induced by combination of CS055 and Doxorubicin in PTCL cells. (A) Jurkat cell lines. (B) NK92MI cell lines.
H. Zhang et al. / Leukemia Research 56 (2017) 29–35
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Fig. 3. Cell apoptosis induced by single CS055 and combination with Doxorubicin in Jurkat and NK92MI cell lines. (A) Cell apoptosis rate induced by single CS055 depend on dosage. (B) The effect of synergistic apoptosis induced by combination of CS055 and Doxorubicin.
Fig. 4. Mitochondrial membrane potential (MMP) changes was induced by single CS055 and combination with Doxorubicin in Jurkat and NK92MI cell lines. (A) MMP changes was induced by single CS055 depend on dosage. (B) Combination of CS055 and Doxorubicin showed synergistic effect in dropping MMP.
tion treatment. The results suggested that CS055 resulted in the lower change of the MMP level in PTCL cell lines (Fig. 4A). But the combination treatment with lower drug-concentration CS055 and
Doxorubicine induced significant change of membrane potential level (Fig. 4B).
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Fig. 5. Western blot detected the expression level of HDAC2, H3, Ac-H3(Lys9) some apoptosis-related proteins in Jurkat and NK92MI cell lines. (A) The influence of CS055 on the expression of HDAC2, H3 and acetylation of H3 in Jurkat and NK92MI cell lines. (B) The expression of apoptotic proteins after combined treatment with CS055 and Doxorubicin in Jurkat cell line.
3.4. CS055 regulates the expression of HDAC2, H3 and acetylation of H3 CS055 is the inhibitor of HDACs, and HDAC2 is one of the major type HDACs. To understand the protein changes after CS055 treatment, we analyzed the expression of HDAC2, H3 and H3 (Lys 9). Results showed (Fig. 5A) that the HDAC2 and H3 expression level was not changed, but the H3 acetylated level (Lys 9) was upregulated after CS055 treatment, suggesting that CS055 did not regulate the HDAC2 and H3 levels, however, up-regulated the H3 acetylated level. 3.5. Combination treatment with CS055 and Doxorubicin synergistically regulates the expression of apoptotic proteins and DNA damage protein Apoptotic pathways include extrinsic pathway, mitochondrial pathway and endoplasmic reticulum stress-induced apoptosis. To further explore the potential apoptosis mechanism of combination treatment with CS055 and Doxorubicine in PTCL cells, we detected the expression levels of apoptotic proteins including Caspase 9, Caspase 3 and PARP, and anti-apoptosis protein Bcl-2, and DNA damage protein p-␥H2AX. Results showed (Fig. 5B) that the combination treatment with CS055 and Doxorubicin resulted in the more significant protein changes of cleaved-Caspase 9, cleaved-Caspase 3 and cleaved-PARP, which were up-regulated. On the contrary, the anti-apoptosis protein Bcl-2 was down-regulated. As well as the expression of DNA damage protein p-␥H2AX was also up-regulated. 4. Discussion Histone acetylation regulates DNA transcription and protein expression of target genes. Aberrant histone acetylation is observed in human tumors. Histone deacetylase inhibitors (HDACis) are some class of new drugs, which show to be promising in several malignant patients including relapsed and/or refractory lymphoma. Chidamide is a novel benzamide inhibitor of HDAC1, 2, 3, and 10, which shows effective antitumor activity on lung cancer, colon cancer, breast cancer, pancreatic cancer, liver cancer, lymphoma and leukemia in vitro and in vivo [6,8,9,11,12]. Some reports of phase II studies confirmed the activity of chidamide in patients with relapsed or refractory PTCL, and China Food and Drug Administration (CFDA) had approved the application of chidamide for treatment of patients with relapse or refractory PTCL. But the ORR
of single drug of chidamide is still limited. Therefore, combination treatment with chidamide and other chemotherapy drugs might be a promising therapeutic regimen for improving the prognosis of patients with relapsed or refractory PTCL. Previous studies showed that HDACIs combination with -phenylethyl isothiocyanate, DNA damaging agents and DNA methyltransferase inhibitors exhibited a better clinical effect for treatment of some solid tumor and hematologic malignant tumors [13–16]. Anthracyclines are the important chemotherapy drugs as DNA damaging agents for PTCL, but they are limited to use due to the cardiotoxicity. In this study, we developed a combination treatment with chidamide and Doxorubicin and explored the mechanism of synergistic anti-tumor effect in PTCL. Theoretical basis of the combination treatment were as follows: (1) Chidamide enhanced the level of histone acetylation, resulting in disaggregating between DNA and histone, which would make nucleosome structure loose. Then Doxorubicin trended to be embedded into the double-stranded DNA, which mediated the DNA damage [17,18]. (2) Doxorubicin caused DNA damage and chidamide also induced DNA damage [19,20]. (3) Several studies showed that HDACIs could enhance the cumulant of topoisomerase II inhibitors in nuclei, leading to DNA break irreparably and cells apoptosis [21–23]. Adriamycin also belonged to a type of topoisomerase II inhibitor. In our study, we developed the combination treatment with different concentration CS055 and Doxorubicin to treat PTCL cells in order to explore the relationship between anti-tumor effect and the dose of the two drugs. We finally found the optimal dosage of the two drugs for synergistic antitumor effect. Our results showed that the combination treatment with lower drug-concentration CS055 and Doxorubicin had a synergistic inhibiting effect on Jurkat and NK92MI cell proliferation. The apoptotic rates of combination treatment was much higher compared to the single drug. It suggested that the PTCL patients could obtain a benefit from the combination treatment with lower drug-concentration CS055 and Doxorubicin, and might have less adverse effects due to reduced therapeutic dosage of the two drugs in clinic. To explore the mechanism of cell apoptosis induced by combination treatment with CS055 and Doxorubicin, we detected the expression levels of H3, Ac-H3 (Lys9) and some apoptosis-related proteins. Our data suggested a potential mechanism of cell apoptosis induced through combination treatment with CS055 and Doxorubicin in PTCL cells as follows: Firstly, CS055 up-regulated the expression level of Ac-H3 (Lys9) as a histone deacetylase inhibitor, leading to the DNA separate from histone. Then, Ac-H3 (Lys9)
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shifted from nucleus into cytoplasm, which resulted in the changes of mitochondria membrane potential, and then switched on the mitochondria apoptosis pathway. As well as Doxorubicin embed into the dissociated double-strand DNA, which induced cell apoptosis irreversibly. In summary, we demonstrated that the combination treatment with CS055 and doxorubicin showed synergistic antitumor effect at a low-dosage of drug component concentration in PTCL cell lines. We further explored the mechanism of cell apoptosis induced by combination treatment with CS055 and doxorubicin as follows: the combination treatment with CS055 and Doxorubicin significantly changed membrane potential and H3 acetylated level. And combination treatment also significantly up-regulated DNA damage protein p-␥H2AX and apoptosis proteins including cleaved-caspase-3, cleaved-caspase-9 and cleaved-PARP, as well as down-regulated anti-apoptosis protein Bcl-2. Our results provided the theoretical basis about the combination treatment with CS055 and Doxorubicin or Doxorubicin-based chemotherapy drugs for PTCL patients. Conflict of interest Authors declared no competing financial interest. Acknowledgements and grant support This study was supported by the National Clinical Research Center for Cancer Foundation in Tianjin Medical University Cancer Institute and Hospital (B type), Natural Science Foundation of China (81402945), National Major Scientific and Technological Special Project for “Significant New Drugs Development” during the Twelfth Five-year Plan Period (2013ZX09303001), Scientific Research Foundation for the Returned Overseas Chinese Scholars in Tianjin (2015-31) and Natural Science Foundation of Tianjin Medical University. References [1] J. Sun, Q. Yang, Z. Lu, et al., Distribution of lymphoid neoplasms in China: analysis of 4,638 cases according to the World Health Organization classification, Am. J. Clin. Pathol. 138 (2012) 429–434. [2] J. Vose, J. Armitage, D. Weisenburger, International peripheral T-cell and natural killer/T-cell lymphoma study: pathology findings and clinical outcomes, J. Clin. Oncol. 26 (2008) 4124–4130. [3] J.O. Armitage, The aggressive peripheral T-cell lymphomas: 2012 update on diagnosis, risk stratification, and management, Am. J. Hematol. 87 (2012) 511–519. [4] O.A. O’connor, B. Pro, L. Pinter-Brown, et al., Pralatrexate in patients with relapsed or refractory peripheral T-cell lymphoma: results from the pivotal PROPEL study, J. Clin. Oncol. 29 (2011) 1182–1189.
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