Tumor Biol. DOI 10.1007/s13277-014-1705-7

RESEARCH ARTICLE

Oxymatrine triggers apoptosis by regulating Bcl-2 family proteins and activating caspase-3/caspase-9 pathway in human leukemia HL-60 cells Jun Liu & Yazhou Yao & Huifang Ding & Renan Chen Received: 26 November 2013 / Accepted: 27 January 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract With the objective of identifying promising antitumor agents for human leukemia, we carried out to determine the anticancer ability of oxymatrine on the human leukemia HL-60 cell line. In vitro experiments demonstrated that oxymatrine reduced the proliferation of HL-60 cells in a dose- and timedependent manner via the induction of apoptosis and cell cycle arrest at G2/M and S phases. The proteins involved in oxymatrineinduced apoptosis in HL-60 cells were also examined using Western blot. The increase in apoptosis upon treatment with oxymatrine was correlated with downregulation of antiapoptotic Bcl-2 expression and upregulation of pro-apoptotic Bax expression. Furthermore, oxymatrine induced the activation of caspase-3 and caspase-9 and the cleavage of poly(ADP-ribose) polymerase (PARP) in HL-60 cells. In addition, pretreatment with a specific caspase-3 (Z-DEVD-FMK) or caspase-9 (Z-LEHDFMK) inhibitor significantly neutralized the pro-apoptotic activity of oxymatrine in HL-60 cells, demonstrating the important role of caspase-3 and caspase-9 in this process. Taken together, these results indicated that oxymatrine-induced apoptosis may occur through the activation of the caspase-9/caspase-3-mediated intrinsic pathway. Therefore, oxymatrine may be a potential candidate for the treatment of human leukemia. Jun Liu and Yazhou Yao contributed equally to this work. J. Liu Department of Geriatrics, Tangdu Hospital, the Fourth Military Medical University, Xi’an 710038, China Y. Yao Department of Haematologic and Rheumatism, Baoji Central Hospital, BaoJi 721008, China H. Ding Department of Haematology, Shengli Oilfield Central Hospital, Dongying 257034, China R. Chen (*) Department of Haematology, Tangdu Hospital, the Fourth Military Medical University, Xi’an 710038, China e-mail: [email protected]

Keywords Oxymatrine . Apoptosis . HL-60 . Caspase . Bax/ Bcl-2

Introduction Leukemia is a special progressive cancer of blood-forming cells in the bone marrow, characterized by the uncontrolled accumulation of blood cells [1, 2]. These deranged, immature cells accumulated in the blood and organs of the body are not able to carry out the normal functions of blood cells [3]. Current most widely used therapeutic options include chemotherapy, radiotherapy, hormonal therapy, immune therapy, some supportive therapy, bone marrow transplantation, and stem cell transplantation [4, 5]. Unfortunately, despite these improvements, global epidemiologic studies have demonstrated that the incidence and mortality of different kinds of leukemia patients still rank high in the worldwide population though various treatment strategies have been developed [2, 6]. Meanwhile, current treatment regimens for leukemia may lead to a wide range of side effects, such as drop in blood cell count, complete hair loss, diarrhea, tiredness, nausea, and reduced fertility [3, 7]. There is thus an urgent need to identify novel therapeutic agents to treat refractory and high-risk leukemia patients. Nowadays, growing evidences suggest that natural materials might be a good source to develop nextgeneration anticancer drugs [8]. So aiming at discovering the innovative antitumor drug candidate from plants with more effective effects and low toxicity has become a very important area for the prevention and management of leukemia. Recently, traditional Chinese herbal medicines are an extraordinary source of chemopreventive and therapeutic agents for the treatment of various malignant diseases, including leukemia, due to their antiviral, antioxidant, anti-inflammatory, and tumor apoptosis-inducing properties [9, 10]. Sophora flavescens Ait (kushen), a traditional Chinese herb, has been used as folk medicine for many kinds of diseases. Oxymatrine

Tumor Biol.

is one of the main basic quinolizidine alkaloids extracted from the root of this Chinese herb which displays various pharmacological effects such as anti-hepatitis virus infection, antihepatic fibrosis, anti-inflammation, anti-anaphylaxis, antihepatic fibrosis, and other immune regulation [11–22]. It was also reported that oxymatrine exhibits broad activities in human malignant melanoma [23], gastric cancer [24], hepatoma [25], and lung cancer [26]. Recently, interests in studying the anticancer mechanism of oxymatrine seem to be mounting [27]. The inhibition of cellular proliferation, induction of apoptosis, cell cycle arrest, and regulation of related protein expression (Bcl-2 and p53) may contribute to the anticancer mechanism of oxymatrine [28–30]. Despite the emerging evidence of its importance, no studies have been reported to date to evaluate the chemotherapeutic potential of oxymatrine in the management of leukemia. Therefore, the aim of the present study is to determine whether treatment with oxymatrine inhibits the proliferation of the human leukemia HL-60 cell line and, if so, to identify the underlying molecular mechanism. This present study would provide a deeper insight into the events leading to oxymatrine-induced apoptosis in HL-60 cells.

Materials and methods Test product Oxymatrine with the purity of 98 % was from Nanjing Zelang Medical Technology Co., Ltd. (Jiangsu, China). Its purity was corroborated by measurements of melting point, IR, UV, and 1 H NMR spectra. The structure of the compound tested is shown in Fig. 1. Cell culture and treatment The human leukemia cell line HL-60 was obtained from the Cell Bank of Chinese Academy of Sciences (Nanjing, China) and was maintained in RPMI-1640 medium supplemented with

10 % heat-inactivated fetal bovine serum (FBS) in a humidified atmosphere of 5 % CO2 and 95 % air at 37 °C. The test compound was dissolved in dimethyl sulfoxide (DMSO) and was added to the culture medium to give a final DMSO concentration of 0.1 %v/v. This concentration of DMSO had no significant effect on the growth of the cell line tested (data not shown). Cell growth inhibition assay The cytotoxicity of oxymatrine against the HL-60 cells was assessed via a colorimetric MTT assay [31]. HL-60 cells were seeded at a density of 104 cells per well in a 96-well plate together with various concentrations of drug (5, 10, 25, 50, and 100 μg/mL) and incubated for different lengths of time (24, 48, and 72 h). HL-60 cells without treatment with oxymatrine would be used for control. At the end of the cultivation, 20 μl of MTT (2 mg/mL) working solution was added to the wells, which were incubated for an additional 4 h at 37 °C. After discarding the MTT supernatant, 100 μl DMSO was added to dissolve the formazan crystals formed in viable cells and the plates were shaken for 10 min. Finally, the optical density of the formazan solution, as a measure of cell viability, was read using a microplate reader (Bio-Rad Laboratories, CA, USA) at 570 nm. Cell growth inhibition rate (%) was calculated using the following equation: Inhibitory rate (%)=(1−Atreatment /Acontrol)×100 %. The half maximal inhibitory concentration (IC50) was calculated from the cytotoxicity curves. The experiments were repeated three times for each cell line. Lactate dehydrogenase (LDH) release assay Cell death of leukemia cells was quantitatively assessed using the LDH kit (Roche, Indianapolis, IN, USA) according to the manufacturer’s instructions. The HL-60 cells were incubated with various concentrations of oxymatrine for 24, 48, and 72 h, respectively, as described above. After treatment, the medium was removed and combined with NADH and pyruvate solutions. LDH activity is proportional to the rate of pyruvate loss, which was assayed by absorbance change using a microplate reader. Relative intensity was compared to treatment with Triton X-100 and was expressed with the maximum value of 100. Flow cytometric analysis of the cell cycle

Fig. 1 The chemical structure of oxymatrine

The cell cycle was analyzed with a flow cytometer after propidium iodide (PI) staining according to the manufacturer’s instructions. After HL-60 cells were exposed to the medium containing different concentrations of oxymatrine (0, 10, 25, and 50 μg/mL) for 48 h, HL-60 cells were harvested by centrifugation, rinsed three times with sterile phosphatebuffered saline (PBS), and fixed in ice-cold 70 % ethanol at 4 °C overnight. After centrifugation at 1,500×g for 3 min, the

Tumor Biol.

cells were dyed with PI and incubated for 30 min in the dark. Distribution of cell cycle was conducted on a flow cytometer (BD FACSCalibur, BD Bioscience, USA), and the data were analyzed using CellQuest.

incubation, the number of apoptotic cells was determined by flow cytometry as described previously.

Detection of apoptosis

In order to further investigate cytoplasmic Bax, Bcl-2, procaspase-3, procaspase-8, procaspase-9, and poly(ADP-ribose) polymerase (PARP) protein expressions in oxymatrineinduced apoptosis, HL-60 cells (4×105 cells/mL) were cultured with or without different indicated concentrations of oxymatrine (10, 25, and 50 μg/mL) for 48 h and harvested. The cells were lysed in RIPA buffer (150 mM NaCl, 1 % Triton X-100, 0.5 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate (SDS), 50 mM Tris–HCl; pH 7.4) for 30 min on ice. Cell lysates were washed by centrifugation (13,000×g, 4 °C, 15 min), and protein concentrations were determined using the BCATM protein assay kit. Aliquots of the lysates (30 μg of protein) were resolved by 15 % SDS–polyacrylamide gels (SDS–PAGE) and electrotransferred onto a nitrocellulose membrane (Bio-Rad). After the nonspecific site was blocked with 5 % (w/v) milk for 1 h on a shaker at room temperature, the membrane was incubated with specific primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA.) for 1 h at room temperature. The membrane was further incubated for 60 min with a peroxidase-conjugated secondary antibody (Vector Laboratories, Burlingame, CA, USA) at room temperature, and immunoactive proteins were visualized by ECL-enhanced chemiluminescence.

The extent of apoptosis in HL-60 cells was quantified by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated annexin Vand PI. After treatment with oxymatrine for 48 h, HL-60 cells (5×105 cells) were collected and suspended in 100 μL of binding buffer (10 mM HEPES/NaOH, 140 mM NaCl, 2.5 mM CaCl2; pH 7.4) and stained with binding buffer with 10 μL annexin V-FITC and 5 μL PI for 30 min at room temperature in the dark. The binding of annexin V-FITC and PI to the cells was measured by flow cytometry (FACSCalibur, BD Biosciences) using the CellQuest software [32]. At least 10,000 cells were analyzed for each sample. In the annexin V/PI quadrant gating, annexin V (−)/PI (−), annexin V (−)/PI (+), annexin V (+)/PI (−), and annexin V (+)/PI (+) represented the fraction of living cells, necrotic cells, early apoptotic cells, and advanced apoptotic/ secondary necrotic cells, respectively. Measurement of enzyme activity of caspase-3, caspase-8, and caspase-9 Caspase-3, caspase-8, and caspase-9 activities were determined by a colorimetric assay kit (R&D Systems Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions. In brief, cells were seeded in 24-well plates at a density of 3×106 cells per well. After exposure of the cells to the test compounds for 48 h, the cells were washed three times with PBS and then lysed in the supplied lysis buffer for 10 min on ice. The lysed cells were centrifuged at 12,000×g for 10 min, and cell lysates containing 50 μg of protein were incubated with the supplied reaction buffer containing the colorimetric tetrapeptides, Asp-Glu-Val-Asp (DEVD)-p-nitroaniline (pNA) for caspase-3, Ile-Glu-Thr-Asp (IETD)-pNA for caspase-8, and Leu-Glu-His-Asp (LEHD)-pNA for caspase9, at 37 °C for 2 h. The reaction was measured by changes in absorbance at 405 nm using a microplate reader. Caspase inhibition assay The caspase-9-specific inhibitor Z-LEHD-FMK (50 μM) and the caspase-3-specific inhibitor Z-DEVD-FMK (50 μM) (all from R&D Systems Inc., Minneapolis, MN, USA) were dissolved in DMSO. Cells were pretreated with either the medium containing 0.1 % DMSO or each inhibitor for 2 h. Medium alone or medium containing each test compound at a final concentration of 50 μg/mL was then added. After 48 h of

Western blot analysis

Statistical analysis All data are expressed as means±SD. Significant differences between the groups were determined using the unpaired Student’s t test. A value of P<0.05 was considered to be statistically significant.

Results Cytotoxic effects of oxymatrine on HL-60 cells To verify the effect of oxymatrine on cell proliferation, HL-60 cells were treated with oxymatrine 1 (0, 5, 10, 25, 50, and 100 μg/mL) for 24, 48, and 72 h and the cell viability was assessed by MTT assay. Cells treated with 0.1 % DMSO were used as controls. As shown in Fig. 2, oxymatrine treatment exhibited a marked inhibition on the survival of HL-60 cells in a dose-dependent manner and reached the maximum value after 48 h of treatment, with IC50 value of 26.38, 14.71, and 22.51 μg/mL for 24, 48, and 72 h of treatment, respectively. The data revealed that the prominent inhibitory activity of oxymatrine on cell survival was attained after 48 h of treatment and the concentrations of 10, 25, and 50 μg/mL were

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appropriate as the high, moderate, and low concentrations in the following experiment. At the same time, the cell viability of HL-60 cells by oxymatrine was further confirmed by LDH release assay. The results showed that cell growth curves correlated well with the results of the MTT assay. In addition, there was no cytotoxicity effect of oxymatrine on normal cell lines (RAW 264.7 cells) at the tested concentration (data not shown). Cell cycle arrest and induction of apoptosis by oxymatrine in HL-60 cells

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Fig. 2 Cytotoxicity of oxymatrine on human leukemia HL-60 cells. Each value is expressed as means±SD of three experiments

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The change of cell cycle caused by oxymatrine in HL60 cells is shown in Fig. 3a. We found that G0/G1 phase cells markedly decreased while G2/M and S phase cells increased obviously with the increase of oxymatrine concentration after 48 h of incubation. These data indicated that oxymatrine blocked the cell cycle of HL-60 cells in G2/M and S phases. To evaluate the effect of oxymatrine on the induction of apoptosis, the oxymatrine-treated HL-60 cells were doublestained with annexin V-FITC and PI, followed by quantitative flow cytometry analysis. As shown in Fig. 3b, a marked apoptosis phenomenon was observed in HL-60 cells treated with oxymatrine. The apoptotic rate increased from 24.5 to 61.12 % after the cells were treated by oxymatrine (10, 25, and 50 μg/mL) for 48 h.

Fig. 3 a The change of cell cycle in human leukemia HL-60 cells after exposure to oxymatrine for 48 h. b Apoptosis analysis of human leukemia HL-60 cells induced by different concentrations of oxymatrine for 48 h. Each value is expressed as means±SD of three experiments. ***P < 0.001 compared with the control group

response to oxymatrine treatment, whereas the level of antiapoptotic Bcl-2 was markedly inhibited by oxymatrine treatment in a concentration-dependent manner. This result suggested the involvement of Bcl-2 family in the apoptotic process.

Effects of oxymatrine on the levels of Bcl-2 family members in HL-60 cells To investigate the apoptotic pathways activated by oxymatrine, we used Western blotting to measure the expression of the Bcl-2 family members. As shown in Fig. 4, Western blot analyses revealed that the level of pro-apoptotic Bax was not truncated and remained virtually unchanged in

Fig. 4 Effects of oxymatrine on the levels of the Bcl-2 family members in human leukemia HL-60 cells

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Living cells Apoptotic cells

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Caspase, a family of cysteine proteases, is known to form integral parts of the apoptotic pathway [33]. To further investigate the apoptotic cascades involved in the effects of oxymatrine, we quantified the proteolytic activation of the caspases (3, 8, and 9) using fluorogenic substrates and evaluated their protein expression using Western blot. As shown in Fig. 5a, oxymatrine treatment markedly increased the activity of caspase-3 and caspase-9 in a concentration-dependent manner, but caspase-8 was not activated. Furthermore, oxymatrine treatment decreased the expression of procaspase-3 and procaspase9 proteins in a concentration-dependent manner, but the expression levels of procaspase-8 remained unchanged (Fig. 5b). Poly(ADP-ribose) polymerase (PARP), an enzyme involved in DNA repair, is a substrate for caspase-3 [34]. Subsequent Western blot analysis was performed in order to

200 Cell populations (%)

Effects of oxymatrine on the expressions of caspase-3, caspase-8, and caspase-9 cascade, as well as cleavage of PARP in HL-60 cells

Fig. 6 Effect of caspsae-3 or caspase-9 inhibitor on oxymatrine-induced apoptosis in human leukemia HL-60 cells. Each value is expressed as means±SD of three experiments

investigate the potential involvement of PARP in oxymatrineinduced apoptosis. As shown in Fig. 5b, oxymatrine treatment caused the cleavage of PARP from a 116- to an 89-kDa fragment, which corresponded with the activation of caspase-3. This suggested that apoptotic actions of oxymatrine were closely related to the activation of caspase-3 and subsequent cleavage of PARP. Inhibition of oxymatrine-induced apoptosis by a caspase-3 or caspase-9 inhibitor Caspase-3 and caspase-9 are the key proteases responsible for the cleavage of PARP and subsequent apoptosis [35]. To confirm whether the activation of intracellular caspase-9 and caspase-3 is required for the induction of apoptosis by oxymatrine, HL-60 cells were pretreated with a specific caspase-9 or caspase-3 inhibitor for 2 h, followed by treatment with 50 μg/mL oxymatrine for 48 h. Then the apoptosis rate was measured by flow cytometry. As shown in Fig. 6, pretreatment with Z-LEHD-FMK (a caspase-9 inhibitor) or ZDEVD-FMK (a caspase-3 inhibitor) significantly blocked the cell apoptosis by oxymatrine. These suggested that oxymatrine-induced apoptosis involved caspase-3- and caspase-9-dependent signaling cascades.

Conclusions

Fig. 5 a Effect of oxymatrine on activities of caspase-3, caspase-8, and caspase-9 in human leukemia HL-60 cells. Each value is expressed as means±SD of three experiments. b Effect of oxymatrine on the protein expression of procaspase-3, procaspase-8, procaspase-9, and cleaved PARP in human leukemia HL-60 cells

Apoptosis is an important homeostatic mechanism that balances cell division and cell death to maintain the appropriate cell number in the body. During this process, apoptosis is characterized by cell shrinkage, blebbing of the plasma membrane, chromatin condensation, and nuclear condensation without cell lysis [36, 37]. Apoptosis serves as a defense mechanism for cancer development by eliminating damaged cells which are prone to develop cancer [38]. Since

Tumor Biol.

deregulation of apoptosis is the hallmark of all cancer cells [39], triggering programmed cell death in cancer cells is therefore considered as an important way for the development of valuable anticancer therapeutics [40, 41]. Accumulating evidence indicates that many naturally derived components from plant species can cause tumor cell death via the induction of apoptosis [42–44]. In this study, we investigated whether oxymatrine induces apoptosis in human leukemia HL-60 cells and what mechanisms are related to the cell death. Our results demonstrated that oxymatrine inhibits cell proliferation and induces apoptosis via arresting the cell cycle at G2/M and S phases. Members of the Bcl-2 family proteins, including Bcl-2 and Bax, are critical regulators of the apoptotic pathway [45]. We further examined the effect of oxymatrine on the expression of anti-apoptotic Bcl-2 protein and pro-apoptotic Bax protein. The level of the anti-apoptotic protein, Bcl-2, declined as the concentration of oxymatrine increased, whereas oxymatrine treatment resulted in an increase in the level of the proapoptotic protein, Bax. Activation of caspase proteases is an important biochemical event of apoptosis [46, 47]. Different caspases are activated at the initiation and execution phases of apoptosis. Caspase-8 and caspase-9 are upstream initiator caspases; caspase-3 is one of the downstream effectors which play the central role in the initiation of apoptosis [48]. Here, we showed that oxymatrine treatment activated caspase-3 and caspase-9 in a dose-dependent manner without activation of caspase-8. Subsequently, we observed that the inhibitors of caspase-3 and caspase-9 significantly protected HL-60 cells from oxymatrine-induced apoptosis. These results suggested that oxymatrine induces apoptosis in HL-60 cells via caspase9/caspase-3 activation. Caspase-3 is a major apoptosisinducing protein that mediates the cleavage of PARP, a well-known caspase-3 substrate [49, 50]. As expected, oxymatrine treatment resulted in a cleavage of 116kDa PARP into 85 kDa in HL-60 cells. In addition, the expression levels of death receptor-related proteins such as TRAIL, DR4, DR5, Fas, and FasL were relatively unchanged in response to oxymatrine treatment (data not shown). Taken together, our data suggested that the caspase-dependent intrinsic pathway was involved in oxymatrine-induced apoptosis in HL-60 cells. In conclusion, this study demonstrated that oxymatrine strongly suppressed the proliferation of HL-60 cells by induction of apoptosis through activation of the caspase-3/caspase-9-mediated intrinsic pathway. This could be evidenced by the fact that the apoptosis of HL-60 cells was induced by the activation of caspase-9 and caspase-3, subsequent cleavage of PARP, along with elevation of the ratio of Bax/Bcl-2. The present results suggested that oxymatrine could be a

potential candidate for developing anticancer drug for the treatment of human leukemia.

Conflicts of interest None

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