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ADM cells by gamma-linolenic acid involves lipid peroxidation and activation of caspase-3
Chemico-Biological Interactions 162 (2006) 140–148
Induction of apoptosis in K562/ADM cells by gamma-linolenic acid involves lipid peroxidation and activation of caspase-3 Xiuqin Kong, Haitao Ge, Lijuan Hou, Limei Shi, Zhili Liu ∗ Department of Biology, College of Life Science, Nanjing University, Nanjing 210093, Jiangsu, China Received 22 March 2006; received in revised form 24 May 2006; accepted 30 May 2006 Available online 9 June 2006
Abstract Numerous studies have revealed that gamma-linolenic acid (GLA) possesses effective tumoricidal properties while not inducing damage to normal cells or creating harmful systemic side effects. It can exert anti-tumor efficacy against a variety of cancers including leukemia. However, little is known about the effects of GLA on leukemia resistant to chemotherapy, emerging as a serious clinical problem. The present study tested GLA-induced apoptosis in K562/ADM multidrug-resistant (MDR) leukemic cells and investigated its possible mechanisms. Using cell viability, fluorescent staining of nuclei, flow cytometric Annexin V/PI double staining and lactate dehydrogenase (LDH) release, we found that GLA could inhibit cell growth and induce apoptosis and secondary necrosis. The results showed that incubation with GLA concentrations of 10–60 g/ml caused a dose- and time-dependent decrease of K562/ADM cell viability, and the IC50 value was 50.5 g/ml at 24 h and 31.5 g/ml at 48 h. Flow cytometry using Annexin V/PI double staining assessed apoptosis, necrosis and viability. Typical apoptotic nuclei were shown by staining of K562/ADM cells with DNA-binding fluorochrome Hoechst 33342, characterized by chromatin condensation and nuclear fragmentation. On the other hand, after treated K562/ADM cells with 20 g/ml GLA for 48 h and with 40 g/ml GLA for 12 h, the LDH release significantly increased, indicated losses of plasma membrane integrity and presence of necrosis. Further, the inhibition of GLA-induced apoptosis by a pancaspase inhibitor (z-VAD-fmk) suggested the involvement of caspases. The increase of caspase-3 activity with GLA concentration confirmed its role in the process. The results also showed that the malondialdehyde (MDA) content was also significantly elevated, and antioxidant BHT could block GLA cytotoxity, indicating the cytotoxity induced by GLA may be due to lipid peroxidation. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Gamma-linolenic acid; Leukemia; Drug resistance; Apoptosis; Secondary necrosis; Lipid peroxidation
1. Introduction Abbreviations: GLA, gamma-linolenic acid; MDR, multidrugresistant; LDH, lactate dehydrogenase; PS, phosphatidylserine; MDA, malondialdehyde; FBS, fetal bovine serum; PBS, phosphate-buffered saline; MTT, 3-(4,5-dimethylthiazlo-2-yl)-2,5-diphenyltetrazolium bromide; SDS, sodium dodecylsulphate; BHT, butylated hydroxytoluene; FACS, fluorescence-activated cell sorter; PI, propidium iodide; EGFP, enhanced green fluorescent protein; TBA, thiobarbituric acid; TBARS, thiobarbituric acid-reactive substances ∗ Corresponding author. Tel.: +86 25 83597401; fax: +86 25 83597401. E-mail address: [email protected] (Z. Liu).
Chemotherapy, radiotherapy and surgery are conventional and indispensable in the treatment of many cancers, use of these therapies has greatly improved the outcome for cancer patients. However, along with the success of chemotherapy, the development of drug resistance, during treatment or during retreatment at relapse, remains a major problem. So it is urgent to find new active drugs or new approaches to treat cancers, especially to those having multidrug resistance.
0009-2797/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2006.05.019
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The omega-6 polyunsaturated fatty acid gammalinolenic acid (GLA; 18:3n-6), which is found in several plant oils and microorganic oils, has antitumor activity in vitro. It is an attractive concept as anti-cancer agent because it possesses effective tumoricidal properties while not inducing damage to normal cells or creating harmful systemic side effects [1,2]. Numbers of researches in vivo and in vitro have demonstrated that GLA inhibits the growth and metastasis of a variety of tumour cells, including breast, prostate, superficial bladder, pancreatic cancer and hepatoma cells [3–7] and also has anti-metastatic effects on endothelial cells and occludes vessels that feed tumors [8]. Cells die either by the programmed process of apoptosis or by assault leading to necrosis [9]. Considerable evidence indicates that polyunsaturated fatty acid can kill cells by apoptosis [10–15]. Apoptosis, which plays a major role in maintaining and regulating homeostasis, development, and differentiation of tissues under physiological conditions, is now recognized as an important mode of cell death in response to cytotoxic treatments [16]. And for some tumor cells, resistance to anticancer treatment has been correlated with a low propensity to apoptosis [17]. It has been reported that gamma-linolenic acid could induce apoptosis in B-chronic lymphocytic leukemia cells in vitro [18,19]. In this study, we investigated the anti-proliferative and apoptosis-inducing effects of GLA to resistant human leukemic K562/ADM cells, and discussed the possible mechanisms.
FBS, 100 IU/ml penicillin and 100 g/ml streptomycin, in humidified air at 37 ◦ C with 5% CO2 . The cells were maintained in the density range of 0.1–1 × 106 cells/ml. Exponentially growing cells were decanted for experimental studies as required. Cell density was determined regularly by using a hemocytometer (Qiujing, Shanghai). Cell viability was determined by trypan blue exclusion. To maintain drug resistance, adriamycin was supplemented at regular intervals, but was omitted 2 weeks before any experiment.
2. Materials and methods
K562 cells from exponentially growing cultures were seeded within 24-well culture plates. The cells were treated with GLA or without GLA for 24 h. After treatment, cells were washed with PBS, and were fixed in MeOH–HAc (3:1, v/v) for 10 min at 4 ◦ C. Cells were stained with Hoechst 33342 (5 g/ml in PBS) for 15 min at room temperature and then examined in a fluorescent microscope (Olympus BX41) with an excitation wavelength of 365 nm [20].
2.1. Chemicals RPMI 1640 medium, fetal bovine serum (FBS), trypan blue, penicillin G and streptomycin were obtained from GIBCO BRL. Hoechst 33342 was purchased from Keygen Biotech. Co. Ltd., Nanjing, China. z-VAD-fmk was obtained from Santa Cruz. 3-(4,5-Dimethylthiazlo-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethylsulfoxide (DMSO), GLA was obtained from Sigma Chemical Co., USA. GLA was dissolved in 100% ethanol at 100 mg/ml. The subsequent dilutions were made in cell culture media. 2.2. Cell culture Adriamycin-selected and P-gp positive multidrugresistant chronic myelogenous leukemia K562/ADM cell line was a kindly gift by Prof. Hu Yiqiao, from department of Biochemistry, Nanjing University. They were cultured in the RPMI 1640 medium, with 10%
2.3. Cell viability studies (MTT) Cells were suspended at a final concentration of 1 × 105 cells/ml and seeded in 96-microwell flat-bottom plates. Increasing concentrations (5–60 g/ml) of GLA were added to each well. After incubation for 24 and 48 h, MTT solutions (5 mg/ml in phosphate-buffered saline (PBS)) were added for 4 h incubation at 37 ◦ C. The precipitated formazan was dissolved in 10% SDS. Cell viability was evaluated by measuring the absorbance at 570 nm, using an automated plate reader (Sunrise Co. Ltd.). Cell survival was expressed as percentage of viable cells of drug-treated samples to control samples. All drug concentrations were tested in six replicates and the experiments were repeated three times. 2.4. Fluorescent staining of nuclei for K562/ADM cells
2.5. Detection of apoptosis by Annexin V-EGFP/PI staining The assay was performed according to the instructions of the manufacture (Keygen Biotech. Co. Ltd., Nanjing, China). 1–5 × 105 Cells were sedimented, washed twice with PBS, and resuspended in 500 l binding buffer. EGFP conjugated Annexin V (1 l) and PI (5 l) were added to each sample, and the mixture was incubated at room temperature in the dark for 5 min. Then the cells were immediately subjected to fluorescence-activated cell sorter (FACS) analysis
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using a Becton-Dickenson FACScan flow cytometer and Cell Quest software version 1.2 (Becton-Dickenson, Mountain View, CA, USA) to calculate the apoptosis percentage. 2.6. Measurement of lactate dehydrogenase (LDH) release After treatment with GLA, cells were centrifuged at 1000 × g for 5 min. The cell pellet was resuspended in PBS in an equivalent volume to the collected media, and frozen and defrozen three times. Supernatant or cell lysate were collected and frozen from K562/ADM cells. The LDH activity was determined using a commercially available kit from Jiancheng Bioengineering Institute (Nanjing, China). The percentage of released LDH was quantitated by dividing LDH present in the media by total cellular LDH (media plus lysate). 2.7. Inhibition of caspase-mediated apoptosis using z-VAD-fmk To determine whether GLA-induced apoptosis was mediated by caspase activation, the cells were treated with a caspase inhibitor, z-VAD-fmk. Briefly, the K562/ADM cells were pretreated with z-VAD-fmk at 60 M for an hour, and then 40 g/ml GLA was added to the cells pretreated with z-VAD-fmk or not for the indicated duration, and stained by Annexin V and PI.
2.9. Lipid peroxidation assay The level of lipid peroxidation was determined by the amount of malondialdehyde (MDA) formed, the final product of lipid peroxidation. The concentration of MDA was assessed using a lipid peroxidation assay kit (Jiancheng Bioengineering Institute, Nanjing, China) according to manufacturer’s instructions. This assay is based on the reaction of MDA with thiobarbituric acid (TBA), forming stable thiobarbituric acid-reactive substances (TBARS), which absorbs at 532 nm. Lipid peroxidation activity was expressed as nmol of MDA per mg protein. 2.10. Protection with antioxidant To assess the protective effect of antioxidant, cells were seeded in 96-well plates containing 0.5 g/ml butylated hydroxytoluene (BHT) or not and incubated with different concentrations of GLA for 48 h. Cell viability was determined by MTT assay. 2.11. Statistics Data were expressed as the mean ± S.E. Statistical significance was determined by the one-way analysis of variance and covariance (ANOVA), followed by Tukey’s pair-wise comparisons at significance level of 0.05. 3. Results 3.1. Inhibition of cell growth
2.8. Assay of caspase-3 activity The activity of caspase-3 was determined by caspase3 colorimetric assay kit according to the manufacturer’s instructions (Keygen Biotech. Co. Ltd., Nanjing, China). Cells (3–5 × 106 per sample) were collected and washed twice with PBS, resuspended in 50 l lysis buffer (with 0.5 l DTT) and incubated in ice bath for 20 min. The supernatants were collected by centrifugation (10,000 × g for 3 min, 4 ◦ C) and immediately measured for protein concentration and caspase-3 activity. The protein concentration was determined by Bradford assay (Keygen Biotech. Co. Ltd., Nanjing, China). For the caspase-3 activity assay, cell lysate was placed in a 96-well plate containing 2× reaction buffer and caspase3 substrate. Plate was incubated at 37 ◦ C and in the dark for 4 h and the enzyme activity was detected at 405 nm using an automated plate reader (Sunrise Co. Ltd.). The enzyme activity was expressed as fold over control samples.
In order to establish the concentration of GLA necessary to produce inhibitory effects on cell proliferation, cells were incubated with the fatty acid at concentrations varying from 5 to 60 g/ml. As shown in Fig. 1, treatment of the K562/ADM cells with 5 g/ml GLA showed no inhibition effect, but treatment with GLA concentrations of 10–60 g/ml caused a dose- and time-dependent decrease of cell viability with IC50 value of 50.5 g/ml at 24 h and 31.5 g/ml at 48 h. 3.2. Fluorescent staining of nuclei by Hoechst 33342 The K562/ADM cells were stained with Hoechst 33342 dye after exposure to 40 g/ml GLA for 24 h. The dye stains condensed chromatin of apoptotic cells more brightly than chromatin of normal cells. As shown in Fig. 2, in control cultures, nuclei of K562/ADM cells were slightly stained, no apoptotic nuclei were observed.
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In contrast, GLA treated cells showed typical apoptosis morphology characterized by chromatin condensation, and nuclear fragmentation. 3.3. Annexin V/PI dual staining
Fig. 1. Effects of GLA on cell viability in K562/ADM cells. Cells were treated with GLA for 48 h, and their viability was determined by MTT assay. The percentage of viable cells was calculated as a ratio of A570 of treated to control cells. Data are mean ± S.E. of three independent experiments.
The effect of GLA on viability, apoptosis and necrosis were determined by Annexin V combined with PI staining after incubation with GLA for 6, 24 h. Representative dot plots of Annexin V/PI staining are shown in Fig. 3 A. Flow cytometric analysis showed that GLA induced the apoptosis and necrosis in concentration and timedependent manners (Fig. 3B). The 20, 40 g/ml GLA significantly elevated the proportion of apoptotic cells at both 6 and 24 h. Secondary necrosis is a feature of endstage apoptosis. Cells with both stains were most likely due to apoptosis leading to secondary necrosis. There is no difference or just a slight increase of secondary necrosis in K562/ADM cells after treated with GLA for 6 h, but the proportion of secondary necrotic cells significantly increased at 24 h compared with cells untreated with GLA. We also found that apoptosis increased significantly (the percent of apoptosis was nearly 70%) with a 6-h incubation of 60 g/ml GLA (data not shown), but most of the cells were broken soon and could not be harvested for test at the later time points. 3.4. Effects of GLA on LDH release The estimation of LDH release provides a quantitative basis for the loss of cell viability. The increased release of this enzyme into the media is indicative of cellular damage. Therefore, this experiment was carried out to estimate LDH release after treatment with various concentrations of GLA at different time. The LDH release increased with the increase in concentrations of GLA. No significant difference of LDH release was observed when incubated with 20 g/ml GLA for 6, 12, and 24 h compared to control. By contrast, after exposure for 48 h, the LDH release was significantly elevated. In 40 g/ml GLA-treated K562/ADM cells, LDH releases were increased significantly from 12 to 48 h, and in a time-dependent manner (Fig. 4). 3.5. Inhibition of caspase-mediated apoptosis using z-VAD-fmk
Fig. 2. Fluorescent staining of nuclei in K562/ADM cells by Hoechst 33342. (A) Untreated control cells and (B) 40 g/ml GLA-treated cells.
Caspases are believed to play a central role in mediating various apoptotic responses. The broad-spectrum caspase inhibitor z-VAD-fmk was used to determine whether GLA-induced apoptosis was mediated by caspase activation. Incubation of cells with 60 M z-VAD-
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Fig. 3. Flow cytometric analysis of K562/ADM. K562/ADM cells incubated for 6, 24 h with various concentrations of GLA. (A) Representative dot plots of Annexin V/PI staining are shown. The lower left quadrant contains the vital (double negative) population. The lower right quadrant contains the apoptotic (Annexin V+ /PI− ) population. The upper right quadrant contains the late apoptotic/necrotic (Annexin V+ /PI+ ) population. And the upper left quadrant contains the pre-necrotic (Annexin V− /PI+ ) population. (B) Data pooled from three experiments show that the percentage of viable, apoptotic and late apoptotic/necrotic K562/ADM cells. Data are given as mean ± S.E. * P < 0.05 compared with the cells untreated with GLA. # P < 0.05 compared with the cells treated with 40 g/ml GLA.
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Fig. 3. (Continued ).
fmk resulted in a 65% decrease in the numbers of K562/ADM cells undergoing apoptosis following 6-h GLA stimulation, and a 40% decrease in apoptotic cells, 70% decrease in secondary necrotic cells following 24-h GLA stimulation (as shown in Fig. 3B). 3.6. Effects of GLA on caspase-3 activity To address the apoptotic pathway in GLA-treated K562/ADM cells, we examined the activation of caspase-3 after incubation with 20, 40 g/ml GLA for 6, 12 and 24 h. After 6 h of incubation in the presence of 40 g/ml GLA, the activity of caspase-3 significantly increased compared to control samples, and remained increasing after 12 h of exposure, and at 24 h, the activity of caspase-3 descended compared to the activity at
Fig. 4. LDH release in K562/ADM cells treated with GLA. Cells were treated with GLA at the indicated concentrations for 6, 12, 24 and 48 h. Data are expressed as the mean of three individual experiments ± S.E. * P < 0.05 compared with the control.
12 h, but was still significantly higher than control. After 6 and 12 h of incubation in the presence of 20 g/ml GLA, caspase-3 activity tended to be higher than control, but differences were not significant, and after 24 h of incubation in the presence of 20 g/ml GLA, caspase-3 activation was evident (Fig. 5). The experiments suggest that the mechanism of GLA-induced apoptosis in K562/ADM cells involves caspase-3 activation. 3.7. Effect on MDA content Lipid peroxidation in cells supplemented or not with GLA was studied. The level of peroxidation was measured as MDA content which was considered as a general indicator of lipid peroxidation. As shown in Fig. 6, under
Fig. 5. Caspase-3 activity in GLA treated K562/ADM cells. Cells were treated with GLA at the indicated concentrations for 6, 12 and 24 h. Cytosolic extracts were prepared and assayed for caspase-3 activity as described in Section 2. Data are expressed as the mean of three individual experiments ± S.E. * P < 0.05 compared with the control.
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revealed in Fig. 7, BHT could completely block the cytotoxity of 20, 30 g/ml GLA and partly block the cytotoxity of 40, 60 g/ml GLA. 4. Discussion
Fig. 6. MDA content in K562/ADM cells treated with GLA. Cells were treated with GLA at the indicated concentrations for 6, 12, 24 and 48 h. Data are expressed as the mean of three individual experiments ± S.E. * P < 0.05 compared with the control.
control conditions, basal levels of MDA were about 1.87–2.69 nmol/mg protein. In contrast, GLA supplementation led to a marked dose- and time-dependent increase of MDA. The MDA content was nearly 30-fold to control (52.09 nmol/mg protein) when the K562/ADM cells were incubated with 40 g/ml GLA for 48 h. 3.8. Protection with antioxidant To examine whether the cytotoxic effect induced by GLA in K562/ADM cells was due to the generation of lipid peroxidation products, the cell viability was determined in the presence of the antioxidant BHT. As
Fig. 7. Protection with antioxidant. Cell viability was determined by MTT assay. Cells were treated with GLA at the indicated concentrations in the presence or absence of 0.5 g/ml BHT for 48 h. Data are expressed as the mean of three individual experiments ± S.E. * P < 0.05 compared with the same concentration of GLA in the absence of BHT.
Human K562/ADM multidrug-resistant leukemic cell line is one of multidrug-resistant leukemic cell lines unable to be responsible to adriamycin, to undergo apoptosis and recover their sensitivity to adriamycin, daunorubicin and VP-16 [21]. Our studies revealed for the first time the potent cytotoxic actions of GLA on multidrug-resistant human leukemic cell line K562/ADM. Proliferation of the K562/ADM cells was inhibited when the cells were incubated for 48 h with GLA at concentrations of 10–60 g/ml/1 × 105 cells. Cells treated with GLA for 24 h were found to have hypercondensed nuclei which were brightly stained by Hoechst 33342. As known, apoptosis is a process of cell suicide, characterized by specific morphological changes such as condensation of chromatin, blebbing of the plasma membrane and the appearance of apoptosis bodies, and biochemical properties involving fragmentation of chromatin. Furthermore, early in apoptosis, phosphatidylserine (PS) is translocated from the inner to outer surface of the plasma membrane which can be detected by Annexin V [22]. We found that GLA treated cells stained positive with Annexin V at 6 h, indicating membrane flipping or externalization of the PS. Evidenced by flow cytometry analysis of Annexin V/PI double staining, the percent of apoptotic and secondary necrotic cells increased with the concentrations of GLA, and with the increase of incubation time, the proportion of secondary necrosis increased significantly. LDH release is a measure of plasma membrane integrity. The result showed that at 6 h, no significant difference of LDH release was observed between the GLA incubated cells and control cells, this indicated that the cell plasma membrane integrity had not been damaged. However there were significant losses of plasma membrane integrity 48 h after treatment with 20 g/ml GLA and 12 h after treatment with 40 g/ml GLA, showing 32–76% LDH released into the supernatant, while in the control cells, only about 15% LDH released into the supernatant. Putting all these results together, we conclude that GLA can trigger apoptosis in K562/ADM cells, but the GLA cytotoxity involves apoptosis and secondary necrosis. GLA has been shown to be cytotoxic to many cancer cell lines [3–7], and the mechanisms of tumoricidal action of GLA have been studied in different cancer cells. U.N. Das reviewed GLA therapy of human gliomas, and mentioned that there was evidence to suggest that
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GLA and other PUFAs suppress the expression of oncogene ras and inhibit that of Bcl-2, an anti-apoptotic gene and enhance the activity of p53. Addition of both GLA and EPA to tumor cells produced alterations in cell membrane lipid composition, mitochondrial ultrastructure, increased reactive oxygen species (especially superoxide anion) and production of lipid peroxides. And a significant increase in cytochrome c release from mitochondria, activation of caspases and DNA fragmentation were noted in GLA and EPA-treated tumor cells, events that eventually lead to their death by apoptosis [23–26]. Caspases are central components of the machinery responsible for apoptosis [27]. To find the possible mechanisms that GLA induces apoptosis in K562/ADM, we investigated the effect of a pan-caspase inhibitor zVAD-fmk. The inhibition of GLA-induced apoptosis by z-VAD-fmk suggested the involvement of caspases. In addition, the increase of caspase-3 activity with GLA concentration confirmed its role in the process. We also observed a decrease of caspase-3 activity at 24 h when incubated with 40 g/ml GLA, indicating that apoptosis occurs early, and is followed by secondary necrosis, and this is consistent with the results of Annexin V/PI double staining and LDH release. Previous studies have suggested that long-chain fatty acids induce apoptosis by enhancing lipid peroxidation in human breast cancer cells and human colon cancer cells [25,26,28,29]. There is evidence that membrane lipid peroxidation could induce the release of cytochrome c from the mitochondria inner membrane and the oxidization and externalization of PS, which are critical events in the induction of apoptosis [30,31]. In this study, we also detected marked dose- and timedependent increase of MDA when the K562/ADM cells were incubated with GLA, and antioxidant BHT could block the cytotoxity GLA induced. This suggested that the mechanism of GLA-induced apoptosis and secondary necrosis involves cellular lipid peroxidation. Altogether, our results reported herein show that GLA induces apoptosis and secondary necrosis in human K562/ADM multidrug-resistant leukemic cells. Our preliminary research of the mechanisms indicated that GLA cytotoxity may be due to the elevated cellular lipid peroxidation level, and the apoptosis induces by GLA involves activation of caspases. Further study of mechanisms is undergoing. Acknowledgments The authors are extremely grateful to Ms. Mei Li for the help in Fluorescent micrography. We are also thankful to Mr. Lin Cao for flow cytometry.
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Induction of apoptosis in K562/ADM cells by gamma-linolenic acid involves lipid peroxidation and activation of caspase-3 Xiuqin Kong, Haitao Ge, Lijuan Hou, Limei Shi, Zhili Liu ∗ Department of Biology, College of Life Science, Nanjing University, Nanjing 210093, Jiangsu, China Received 22 March 2006; received in revised form 24 May 2006; accepted 30 May 2006 Available online 9 June 2006
Abstract Numerous studies have revealed that gamma-linolenic acid (GLA) possesses effective tumoricidal properties while not inducing damage to normal cells or creating harmful systemic side effects. It can exert anti-tumor efficacy against a variety of cancers including leukemia. However, little is known about the effects of GLA on leukemia resistant to chemotherapy, emerging as a serious clinical problem. The present study tested GLA-induced apoptosis in K562/ADM multidrug-resistant (MDR) leukemic cells and investigated its possible mechanisms. Using cell viability, fluorescent staining of nuclei, flow cytometric Annexin V/PI double staining and lactate dehydrogenase (LDH) release, we found that GLA could inhibit cell growth and induce apoptosis and secondary necrosis. The results showed that incubation with GLA concentrations of 10–60 g/ml caused a dose- and time-dependent decrease of K562/ADM cell viability, and the IC50 value was 50.5 g/ml at 24 h and 31.5 g/ml at 48 h. Flow cytometry using Annexin V/PI double staining assessed apoptosis, necrosis and viability. Typical apoptotic nuclei were shown by staining of K562/ADM cells with DNA-binding fluorochrome Hoechst 33342, characterized by chromatin condensation and nuclear fragmentation. On the other hand, after treated K562/ADM cells with 20 g/ml GLA for 48 h and with 40 g/ml GLA for 12 h, the LDH release significantly increased, indicated losses of plasma membrane integrity and presence of necrosis. Further, the inhibition of GLA-induced apoptosis by a pancaspase inhibitor (z-VAD-fmk) suggested the involvement of caspases. The increase of caspase-3 activity with GLA concentration confirmed its role in the process. The results also showed that the malondialdehyde (MDA) content was also significantly elevated, and antioxidant BHT could block GLA cytotoxity, indicating the cytotoxity induced by GLA may be due to lipid peroxidation. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Gamma-linolenic acid; Leukemia; Drug resistance; Apoptosis; Secondary necrosis; Lipid peroxidation
1. Introduction Abbreviations: GLA, gamma-linolenic acid; MDR, multidrugresistant; LDH, lactate dehydrogenase; PS, phosphatidylserine; MDA, malondialdehyde; FBS, fetal bovine serum; PBS, phosphate-buffered saline; MTT, 3-(4,5-dimethylthiazlo-2-yl)-2,5-diphenyltetrazolium bromide; SDS, sodium dodecylsulphate; BHT, butylated hydroxytoluene; FACS, fluorescence-activated cell sorter; PI, propidium iodide; EGFP, enhanced green fluorescent protein; TBA, thiobarbituric acid; TBARS, thiobarbituric acid-reactive substances ∗ Corresponding author. Tel.: +86 25 83597401; fax: +86 25 83597401. E-mail address: [email protected] (Z. Liu).
Chemotherapy, radiotherapy and surgery are conventional and indispensable in the treatment of many cancers, use of these therapies has greatly improved the outcome for cancer patients. However, along with the success of chemotherapy, the development of drug resistance, during treatment or during retreatment at relapse, remains a major problem. So it is urgent to find new active drugs or new approaches to treat cancers, especially to those having multidrug resistance.
0009-2797/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2006.05.019
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The omega-6 polyunsaturated fatty acid gammalinolenic acid (GLA; 18:3n-6), which is found in several plant oils and microorganic oils, has antitumor activity in vitro. It is an attractive concept as anti-cancer agent because it possesses effective tumoricidal properties while not inducing damage to normal cells or creating harmful systemic side effects [1,2]. Numbers of researches in vivo and in vitro have demonstrated that GLA inhibits the growth and metastasis of a variety of tumour cells, including breast, prostate, superficial bladder, pancreatic cancer and hepatoma cells [3–7] and also has anti-metastatic effects on endothelial cells and occludes vessels that feed tumors [8]. Cells die either by the programmed process of apoptosis or by assault leading to necrosis [9]. Considerable evidence indicates that polyunsaturated fatty acid can kill cells by apoptosis [10–15]. Apoptosis, which plays a major role in maintaining and regulating homeostasis, development, and differentiation of tissues under physiological conditions, is now recognized as an important mode of cell death in response to cytotoxic treatments [16]. And for some tumor cells, resistance to anticancer treatment has been correlated with a low propensity to apoptosis [17]. It has been reported that gamma-linolenic acid could induce apoptosis in B-chronic lymphocytic leukemia cells in vitro [18,19]. In this study, we investigated the anti-proliferative and apoptosis-inducing effects of GLA to resistant human leukemic K562/ADM cells, and discussed the possible mechanisms.
FBS, 100 IU/ml penicillin and 100 g/ml streptomycin, in humidified air at 37 ◦ C with 5% CO2 . The cells were maintained in the density range of 0.1–1 × 106 cells/ml. Exponentially growing cells were decanted for experimental studies as required. Cell density was determined regularly by using a hemocytometer (Qiujing, Shanghai). Cell viability was determined by trypan blue exclusion. To maintain drug resistance, adriamycin was supplemented at regular intervals, but was omitted 2 weeks before any experiment.
2. Materials and methods
K562 cells from exponentially growing cultures were seeded within 24-well culture plates. The cells were treated with GLA or without GLA for 24 h. After treatment, cells were washed with PBS, and were fixed in MeOH–HAc (3:1, v/v) for 10 min at 4 ◦ C. Cells were stained with Hoechst 33342 (5 g/ml in PBS) for 15 min at room temperature and then examined in a fluorescent microscope (Olympus BX41) with an excitation wavelength of 365 nm [20].
2.1. Chemicals RPMI 1640 medium, fetal bovine serum (FBS), trypan blue, penicillin G and streptomycin were obtained from GIBCO BRL. Hoechst 33342 was purchased from Keygen Biotech. Co. Ltd., Nanjing, China. z-VAD-fmk was obtained from Santa Cruz. 3-(4,5-Dimethylthiazlo-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethylsulfoxide (DMSO), GLA was obtained from Sigma Chemical Co., USA. GLA was dissolved in 100% ethanol at 100 mg/ml. The subsequent dilutions were made in cell culture media. 2.2. Cell culture Adriamycin-selected and P-gp positive multidrugresistant chronic myelogenous leukemia K562/ADM cell line was a kindly gift by Prof. Hu Yiqiao, from department of Biochemistry, Nanjing University. They were cultured in the RPMI 1640 medium, with 10%
2.3. Cell viability studies (MTT) Cells were suspended at a final concentration of 1 × 105 cells/ml and seeded in 96-microwell flat-bottom plates. Increasing concentrations (5–60 g/ml) of GLA were added to each well. After incubation for 24 and 48 h, MTT solutions (5 mg/ml in phosphate-buffered saline (PBS)) were added for 4 h incubation at 37 ◦ C. The precipitated formazan was dissolved in 10% SDS. Cell viability was evaluated by measuring the absorbance at 570 nm, using an automated plate reader (Sunrise Co. Ltd.). Cell survival was expressed as percentage of viable cells of drug-treated samples to control samples. All drug concentrations were tested in six replicates and the experiments were repeated three times. 2.4. Fluorescent staining of nuclei for K562/ADM cells
2.5. Detection of apoptosis by Annexin V-EGFP/PI staining The assay was performed according to the instructions of the manufacture (Keygen Biotech. Co. Ltd., Nanjing, China). 1–5 × 105 Cells were sedimented, washed twice with PBS, and resuspended in 500 l binding buffer. EGFP conjugated Annexin V (1 l) and PI (5 l) were added to each sample, and the mixture was incubated at room temperature in the dark for 5 min. Then the cells were immediately subjected to fluorescence-activated cell sorter (FACS) analysis
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using a Becton-Dickenson FACScan flow cytometer and Cell Quest software version 1.2 (Becton-Dickenson, Mountain View, CA, USA) to calculate the apoptosis percentage. 2.6. Measurement of lactate dehydrogenase (LDH) release After treatment with GLA, cells were centrifuged at 1000 × g for 5 min. The cell pellet was resuspended in PBS in an equivalent volume to the collected media, and frozen and defrozen three times. Supernatant or cell lysate were collected and frozen from K562/ADM cells. The LDH activity was determined using a commercially available kit from Jiancheng Bioengineering Institute (Nanjing, China). The percentage of released LDH was quantitated by dividing LDH present in the media by total cellular LDH (media plus lysate). 2.7. Inhibition of caspase-mediated apoptosis using z-VAD-fmk To determine whether GLA-induced apoptosis was mediated by caspase activation, the cells were treated with a caspase inhibitor, z-VAD-fmk. Briefly, the K562/ADM cells were pretreated with z-VAD-fmk at 60 M for an hour, and then 40 g/ml GLA was added to the cells pretreated with z-VAD-fmk or not for the indicated duration, and stained by Annexin V and PI.
2.9. Lipid peroxidation assay The level of lipid peroxidation was determined by the amount of malondialdehyde (MDA) formed, the final product of lipid peroxidation. The concentration of MDA was assessed using a lipid peroxidation assay kit (Jiancheng Bioengineering Institute, Nanjing, China) according to manufacturer’s instructions. This assay is based on the reaction of MDA with thiobarbituric acid (TBA), forming stable thiobarbituric acid-reactive substances (TBARS), which absorbs at 532 nm. Lipid peroxidation activity was expressed as nmol of MDA per mg protein. 2.10. Protection with antioxidant To assess the protective effect of antioxidant, cells were seeded in 96-well plates containing 0.5 g/ml butylated hydroxytoluene (BHT) or not and incubated with different concentrations of GLA for 48 h. Cell viability was determined by MTT assay. 2.11. Statistics Data were expressed as the mean ± S.E. Statistical significance was determined by the one-way analysis of variance and covariance (ANOVA), followed by Tukey’s pair-wise comparisons at significance level of 0.05. 3. Results 3.1. Inhibition of cell growth
2.8. Assay of caspase-3 activity The activity of caspase-3 was determined by caspase3 colorimetric assay kit according to the manufacturer’s instructions (Keygen Biotech. Co. Ltd., Nanjing, China). Cells (3–5 × 106 per sample) were collected and washed twice with PBS, resuspended in 50 l lysis buffer (with 0.5 l DTT) and incubated in ice bath for 20 min. The supernatants were collected by centrifugation (10,000 × g for 3 min, 4 ◦ C) and immediately measured for protein concentration and caspase-3 activity. The protein concentration was determined by Bradford assay (Keygen Biotech. Co. Ltd., Nanjing, China). For the caspase-3 activity assay, cell lysate was placed in a 96-well plate containing 2× reaction buffer and caspase3 substrate. Plate was incubated at 37 ◦ C and in the dark for 4 h and the enzyme activity was detected at 405 nm using an automated plate reader (Sunrise Co. Ltd.). The enzyme activity was expressed as fold over control samples.
In order to establish the concentration of GLA necessary to produce inhibitory effects on cell proliferation, cells were incubated with the fatty acid at concentrations varying from 5 to 60 g/ml. As shown in Fig. 1, treatment of the K562/ADM cells with 5 g/ml GLA showed no inhibition effect, but treatment with GLA concentrations of 10–60 g/ml caused a dose- and time-dependent decrease of cell viability with IC50 value of 50.5 g/ml at 24 h and 31.5 g/ml at 48 h. 3.2. Fluorescent staining of nuclei by Hoechst 33342 The K562/ADM cells were stained with Hoechst 33342 dye after exposure to 40 g/ml GLA for 24 h. The dye stains condensed chromatin of apoptotic cells more brightly than chromatin of normal cells. As shown in Fig. 2, in control cultures, nuclei of K562/ADM cells were slightly stained, no apoptotic nuclei were observed.
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In contrast, GLA treated cells showed typical apoptosis morphology characterized by chromatin condensation, and nuclear fragmentation. 3.3. Annexin V/PI dual staining
Fig. 1. Effects of GLA on cell viability in K562/ADM cells. Cells were treated with GLA for 48 h, and their viability was determined by MTT assay. The percentage of viable cells was calculated as a ratio of A570 of treated to control cells. Data are mean ± S.E. of three independent experiments.
The effect of GLA on viability, apoptosis and necrosis were determined by Annexin V combined with PI staining after incubation with GLA for 6, 24 h. Representative dot plots of Annexin V/PI staining are shown in Fig. 3 A. Flow cytometric analysis showed that GLA induced the apoptosis and necrosis in concentration and timedependent manners (Fig. 3B). The 20, 40 g/ml GLA significantly elevated the proportion of apoptotic cells at both 6 and 24 h. Secondary necrosis is a feature of endstage apoptosis. Cells with both stains were most likely due to apoptosis leading to secondary necrosis. There is no difference or just a slight increase of secondary necrosis in K562/ADM cells after treated with GLA for 6 h, but the proportion of secondary necrotic cells significantly increased at 24 h compared with cells untreated with GLA. We also found that apoptosis increased significantly (the percent of apoptosis was nearly 70%) with a 6-h incubation of 60 g/ml GLA (data not shown), but most of the cells were broken soon and could not be harvested for test at the later time points. 3.4. Effects of GLA on LDH release The estimation of LDH release provides a quantitative basis for the loss of cell viability. The increased release of this enzyme into the media is indicative of cellular damage. Therefore, this experiment was carried out to estimate LDH release after treatment with various concentrations of GLA at different time. The LDH release increased with the increase in concentrations of GLA. No significant difference of LDH release was observed when incubated with 20 g/ml GLA for 6, 12, and 24 h compared to control. By contrast, after exposure for 48 h, the LDH release was significantly elevated. In 40 g/ml GLA-treated K562/ADM cells, LDH releases were increased significantly from 12 to 48 h, and in a time-dependent manner (Fig. 4). 3.5. Inhibition of caspase-mediated apoptosis using z-VAD-fmk
Fig. 2. Fluorescent staining of nuclei in K562/ADM cells by Hoechst 33342. (A) Untreated control cells and (B) 40 g/ml GLA-treated cells.
Caspases are believed to play a central role in mediating various apoptotic responses. The broad-spectrum caspase inhibitor z-VAD-fmk was used to determine whether GLA-induced apoptosis was mediated by caspase activation. Incubation of cells with 60 M z-VAD-
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Fig. 3. Flow cytometric analysis of K562/ADM. K562/ADM cells incubated for 6, 24 h with various concentrations of GLA. (A) Representative dot plots of Annexin V/PI staining are shown. The lower left quadrant contains the vital (double negative) population. The lower right quadrant contains the apoptotic (Annexin V+ /PI− ) population. The upper right quadrant contains the late apoptotic/necrotic (Annexin V+ /PI+ ) population. And the upper left quadrant contains the pre-necrotic (Annexin V− /PI+ ) population. (B) Data pooled from three experiments show that the percentage of viable, apoptotic and late apoptotic/necrotic K562/ADM cells. Data are given as mean ± S.E. * P < 0.05 compared with the cells untreated with GLA. # P < 0.05 compared with the cells treated with 40 g/ml GLA.
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Fig. 3. (Continued ).
fmk resulted in a 65% decrease in the numbers of K562/ADM cells undergoing apoptosis following 6-h GLA stimulation, and a 40% decrease in apoptotic cells, 70% decrease in secondary necrotic cells following 24-h GLA stimulation (as shown in Fig. 3B). 3.6. Effects of GLA on caspase-3 activity To address the apoptotic pathway in GLA-treated K562/ADM cells, we examined the activation of caspase-3 after incubation with 20, 40 g/ml GLA for 6, 12 and 24 h. After 6 h of incubation in the presence of 40 g/ml GLA, the activity of caspase-3 significantly increased compared to control samples, and remained increasing after 12 h of exposure, and at 24 h, the activity of caspase-3 descended compared to the activity at
Fig. 4. LDH release in K562/ADM cells treated with GLA. Cells were treated with GLA at the indicated concentrations for 6, 12, 24 and 48 h. Data are expressed as the mean of three individual experiments ± S.E. * P < 0.05 compared with the control.
12 h, but was still significantly higher than control. After 6 and 12 h of incubation in the presence of 20 g/ml GLA, caspase-3 activity tended to be higher than control, but differences were not significant, and after 24 h of incubation in the presence of 20 g/ml GLA, caspase-3 activation was evident (Fig. 5). The experiments suggest that the mechanism of GLA-induced apoptosis in K562/ADM cells involves caspase-3 activation. 3.7. Effect on MDA content Lipid peroxidation in cells supplemented or not with GLA was studied. The level of peroxidation was measured as MDA content which was considered as a general indicator of lipid peroxidation. As shown in Fig. 6, under
Fig. 5. Caspase-3 activity in GLA treated K562/ADM cells. Cells were treated with GLA at the indicated concentrations for 6, 12 and 24 h. Cytosolic extracts were prepared and assayed for caspase-3 activity as described in Section 2. Data are expressed as the mean of three individual experiments ± S.E. * P < 0.05 compared with the control.
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revealed in Fig. 7, BHT could completely block the cytotoxity of 20, 30 g/ml GLA and partly block the cytotoxity of 40, 60 g/ml GLA. 4. Discussion
Fig. 6. MDA content in K562/ADM cells treated with GLA. Cells were treated with GLA at the indicated concentrations for 6, 12, 24 and 48 h. Data are expressed as the mean of three individual experiments ± S.E. * P < 0.05 compared with the control.
control conditions, basal levels of MDA were about 1.87–2.69 nmol/mg protein. In contrast, GLA supplementation led to a marked dose- and time-dependent increase of MDA. The MDA content was nearly 30-fold to control (52.09 nmol/mg protein) when the K562/ADM cells were incubated with 40 g/ml GLA for 48 h. 3.8. Protection with antioxidant To examine whether the cytotoxic effect induced by GLA in K562/ADM cells was due to the generation of lipid peroxidation products, the cell viability was determined in the presence of the antioxidant BHT. As
Fig. 7. Protection with antioxidant. Cell viability was determined by MTT assay. Cells were treated with GLA at the indicated concentrations in the presence or absence of 0.5 g/ml BHT for 48 h. Data are expressed as the mean of three individual experiments ± S.E. * P < 0.05 compared with the same concentration of GLA in the absence of BHT.
Human K562/ADM multidrug-resistant leukemic cell line is one of multidrug-resistant leukemic cell lines unable to be responsible to adriamycin, to undergo apoptosis and recover their sensitivity to adriamycin, daunorubicin and VP-16 [21]. Our studies revealed for the first time the potent cytotoxic actions of GLA on multidrug-resistant human leukemic cell line K562/ADM. Proliferation of the K562/ADM cells was inhibited when the cells were incubated for 48 h with GLA at concentrations of 10–60 g/ml/1 × 105 cells. Cells treated with GLA for 24 h were found to have hypercondensed nuclei which were brightly stained by Hoechst 33342. As known, apoptosis is a process of cell suicide, characterized by specific morphological changes such as condensation of chromatin, blebbing of the plasma membrane and the appearance of apoptosis bodies, and biochemical properties involving fragmentation of chromatin. Furthermore, early in apoptosis, phosphatidylserine (PS) is translocated from the inner to outer surface of the plasma membrane which can be detected by Annexin V [22]. We found that GLA treated cells stained positive with Annexin V at 6 h, indicating membrane flipping or externalization of the PS. Evidenced by flow cytometry analysis of Annexin V/PI double staining, the percent of apoptotic and secondary necrotic cells increased with the concentrations of GLA, and with the increase of incubation time, the proportion of secondary necrosis increased significantly. LDH release is a measure of plasma membrane integrity. The result showed that at 6 h, no significant difference of LDH release was observed between the GLA incubated cells and control cells, this indicated that the cell plasma membrane integrity had not been damaged. However there were significant losses of plasma membrane integrity 48 h after treatment with 20 g/ml GLA and 12 h after treatment with 40 g/ml GLA, showing 32–76% LDH released into the supernatant, while in the control cells, only about 15% LDH released into the supernatant. Putting all these results together, we conclude that GLA can trigger apoptosis in K562/ADM cells, but the GLA cytotoxity involves apoptosis and secondary necrosis. GLA has been shown to be cytotoxic to many cancer cell lines [3–7], and the mechanisms of tumoricidal action of GLA have been studied in different cancer cells. U.N. Das reviewed GLA therapy of human gliomas, and mentioned that there was evidence to suggest that
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GLA and other PUFAs suppress the expression of oncogene ras and inhibit that of Bcl-2, an anti-apoptotic gene and enhance the activity of p53. Addition of both GLA and EPA to tumor cells produced alterations in cell membrane lipid composition, mitochondrial ultrastructure, increased reactive oxygen species (especially superoxide anion) and production of lipid peroxides. And a significant increase in cytochrome c release from mitochondria, activation of caspases and DNA fragmentation were noted in GLA and EPA-treated tumor cells, events that eventually lead to their death by apoptosis [23–26]. Caspases are central components of the machinery responsible for apoptosis [27]. To find the possible mechanisms that GLA induces apoptosis in K562/ADM, we investigated the effect of a pan-caspase inhibitor zVAD-fmk. The inhibition of GLA-induced apoptosis by z-VAD-fmk suggested the involvement of caspases. In addition, the increase of caspase-3 activity with GLA concentration confirmed its role in the process. We also observed a decrease of caspase-3 activity at 24 h when incubated with 40 g/ml GLA, indicating that apoptosis occurs early, and is followed by secondary necrosis, and this is consistent with the results of Annexin V/PI double staining and LDH release. Previous studies have suggested that long-chain fatty acids induce apoptosis by enhancing lipid peroxidation in human breast cancer cells and human colon cancer cells [25,26,28,29]. There is evidence that membrane lipid peroxidation could induce the release of cytochrome c from the mitochondria inner membrane and the oxidization and externalization of PS, which are critical events in the induction of apoptosis [30,31]. In this study, we also detected marked dose- and timedependent increase of MDA when the K562/ADM cells were incubated with GLA, and antioxidant BHT could block the cytotoxity GLA induced. This suggested that the mechanism of GLA-induced apoptosis and secondary necrosis involves cellular lipid peroxidation. Altogether, our results reported herein show that GLA induces apoptosis and secondary necrosis in human K562/ADM multidrug-resistant leukemic cells. Our preliminary research of the mechanisms indicated that GLA cytotoxity may be due to the elevated cellular lipid peroxidation level, and the apoptosis induces by GLA involves activation of caspases. Further study of mechanisms is undergoing. Acknowledgments The authors are extremely grateful to Ms. Mei Li for the help in Fluorescent micrography. We are also thankful to Mr. Lin Cao for flow cytometry.
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