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Induction of apoptosis in K562 human leukemia cells by 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone
Leukemia Research 29 (2005) 887–892
Induction of apoptosis in K562 human leukemia cells by 2,4-dihydroxy-6-methoxy-3,5-dimethylchalcone Chun-Lin Ye, Feng Qian, Dong-Zhi Wei ∗ , Yan-Hua Lu, Jian-Wen Liu State Key Laboratory of Bioreactor Engineering, Institute of Biochemistry, East China University of Science and Technology, Shanghai 200237, PR China Received 4 November 2004; received in revised form 2 January 2005; accepted 7 January 2005 Available online 16 February 2005
Abstract 2 ,4 -Dihydroxy-6 -methoxy-3 ,5 -dimethylchalcone (DMC), isolated from the buds of Cleistocalyx operculatus, was investigated in its cytotoxicity and anti-proliferation on K562 cell line. Our results revealed that the IC50 was equal to 14.2 ± 0.45 M and the EC50 was 3.3 ± 0.14 M. Staining with Hoechst 33258 showed fragmentation and condensation of chromatin in the cells treated with 8 M DMC for 48 h. Flow cytometric analysis was performed to determine hypodiploid cells. The results of flow cytometry assay indicated that the percentage of hypodiploid K562 cells was 76.15 ± 3.22% after 48 h treatment with 16.0 M DMC. The treatment resulted in the appearance of a hypodiploid peak (A0 region), probably due to the presence of apoptosing cells and/or apoptotic bodies with DNA content less than 2n. Western blot results illustrated that in the same dosage and incubation time, DMC could down-regulate the level of Bcl-2 protein and did not influence the expression of Bax protein. The resulting net effect could thus lead to a lower ratio of Bcl-2/Bax, which might be responsible for the DMC-induced apoptosis in K562 cells. © 2005 Elsevier Ltd. All rights reserved. Keywords: Cleistocalyx operculatus; Flavonoids; K562; Bcl-2/Bax; Apoptosis
1. Introduction Flavonoids are polyphenolic compounds that occur naturally in normal human diet and in many folk medicines, still in use. These compounds can scavenge superoxide, hydroxy and proxyradicals, breaking lipid peroxide chain reactions. They have also been shown to protect cells from X-ray damage, to block progression of cell cycle, to inhibit mutations, to block prostaglandin synthesis and to prevent multistage carcinogenesis in experimental animals [1]. Various pharmacological activities of flavonoids have been studied extensively [2–5]. Chalcones, considered as the precursor of flavonoids and isoflavonoids, are abundant in edible plants. A number of chalcones have
∗
Corresponding author. Tel.: +86 21 64252981; fax: +86 21 64250068. E-mail address: [email protected] (D.-Z. Wei).
0145-2126/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2005.01.006
demonstrated cytotoxic [6,7] and anti-cancer properties [8,9]. Cleistocalyx operculatus (Roxb.) Merr. et Perry (Myrtaceae), is a well-known medicinal plant whose buds are commonly used as an ingredient for tonic drinks in southern China. It was reported that the water extract of the buds of C. operculatus was shown to increase the contractility and decrease the frequency of contraction in an isolated rat heart perfusion system [10]. Our previous phytochemical attention to the species has led to the characterization of sterol, flavanone, chalcone and triterpene acid from its buds [11]. In this paper, we investigated the in vitro anti-tumor activity of 2 ,4 -dihydroxy-6 -methoxy3 ,5 -dimethylchalcone (DMC), one of the compound from the buds of C. operculatus, in K562 cell line. In addition, we were interested in elucidating the molecular mechanisms by which DMC may induce K562 cells apoptosis.
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2. Materials and methods
2.4. Soft agar colony forming (clonogenic) assay
2.1. Chemicals
The effects of DMC on soft agar colony forming of K562 cells were investigated. Briefly, K562 cells were treated with DMC ranging from 1.5 to 25 M or without (vehicle control, 0.1% dimethyl sulfoxide) DMC and then mixed with agar in a final concentration of 0.33%. Aliquots of 1.5 ml containing 103 cells and 10% FBS were plated in triplicate in 35-mm culture dishes over a base layer of 0.6% agar and allowed to gel. Colonies (>20 cells) were counted after 14 days of incubation under an inverted microscope (Leica DMIRB, Germany) [13]. The anti-proliferative activity was expressed as EC50 (concentration of the DMC that induces 50% inhibition of the cell colony number, which was extrapolated from linear regression analysis of experimental data).
DMC was isolated from C. operculatus in our lab as described by Ye et al. [11]. Previous experiments have shown that DMC purity was above 96%. The structure of the compound is shown in Fig. 1. The compound was dissolved in dimethyl sulfoxide (DMSO). The control cells were treated with the same amount of vehicle alone. The final DMSO concentration never exceeded 0.1% (v/v), in either control or treated samples. Previous experiments have shown that DMSO at this concentration does not modify the cellular activities that we are analyzing. Fetal bovine serum (FBS) and Rosewell Park Memorial Institute (RPMI) 1640 medium were purchased from Life Technologies, Inc., MA, USA.
2.5. Fluorescent staining of nuclei for K562 cells 2.2. Cell lines and culture conditions K562 human chronic leukemia cells were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI 1640 medium with 10% FBS, penicillin (100 U/ml) and streptomycin (100 g/ml) in an atmosphere of 5% CO2 in humidified air at 37 ◦ C. In all experiments, exponentially growing cells were used.
K562 cells from exponentially growing cultures were seeded within 24-well culture plates. The cells were treated with 8 M DMC or without (vehicle control, 0.1% dimethyl sulfoxide) DMC for 48 h. After treatment, cells were washed with phosphate-buffered saline (PBS), and were fixed in MeOH–HAc (3:1, v/v) for 10 min at 4 ◦ C. Cells were stained with Hoechst 33258 (5 g/ml in PBS) for 5 min at room temperature and then examined in a Leica DMIRB fluorescent microscope at 356 nm [14].
2.3. Cytotoxycity assay
2.6. Flow cytometry assay for K562 cells
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay was performed as described by Mosman [12]. K562 cells were placed within 96-well culture plates (104 cells/well). The cells were treated with DMC ranging from 12.5 to 100 M or without (vehicle control, 0.1% dimethyl sulfoxide) DMC. DMC cytotoxycity was measured after 2 days of culture using the MTT assay. Absorbance in control and drug treated wells was measured in an Automated Microplate Reader (Bio-Rad 550) at 550 nm. The cytotoxycity of DMC was expressed as IC50 (concentration of the DMC that induces 50% inhibition of the cell growth, which was extrapolated from linear regression analysis of experimental data).
Flow cytometric analysis was performed to determine hypodiploid cells. The cells were stained with propidium iodide using a cycle TEST PLUS DNA Reagent kit (Becton Dickinson, USA), then analysed by a FACScan (Becton Dickinson, USA) with Cell Fit software. Hypodiploid cells have less DNA than that of diploid cells at G1 phase because of apoptosis-induced DNA fragmentation.
Fig. 1. Structure of 2 ,4 -dihydroxy-6 -methoxy-3 ,5 -dimethylchalcone.
2.7. Protein extraction and Western blot analysis K562 cells were planted and cultured in complete medium. The cells were treated with DMC ranging from 2 to 16 M or without (vehicle control, 0.1% dimethyl sulfoxide) DMC. Cells were washed twice with cold PBS and lysed at 4 ◦ C for 30 min in lysis buffer (0.5% Triton X-100, 300 mM NaCl, 50 mM Tris–Cl, 1 mM phenylmethylsulfonyl fluoride) with occasional vortexing. Insoluble material was removed by centrifugation at 4 ◦ C for 15 min at 14,000 × g and the total proteins extracted were quantified by the coomassie brilliant blue G-250 assay with bovine serum albumin (BSA) as standard (VARIAN Cary 500 UV/Vis spectrophotometer, USA) [15]. Cellular proteins (40 g/lane) were resolved on SDS–12% polyacrylamide gels and transferred to TotalBlot PVDF membranes (Amresco Inc., USA), which were blocked with 5% fat-free milk and immunostained with 1:1000 dilution of monoclonal mouse anti-Bcl-2 antibody (Santa Cruz
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Biotechnology, Inc., USA) or a 1:1000 dilution of monoclonal mouse anti-bax antibody (Santa Cruz Biotechnology, Inc., USA). The immunoreactive proteins were probed with horseradish peroxidase-conjugated goat anti-mouse IgG, and Bcl-2 and Bax protein levels were visualized by peroxidase reaction using the ECL kit (Amersham Pharmacia Biotech, UK). Equal loading of extracts was confirmed using antiActin (Santa Cruz Biotechnology, Inc., USA). 2.8. Statistical analysis Each experimental value is expressed as the mean ± standard deviation (S.D.). The scientific statistic software GraphPad Instat version 3.05 was used to evaluate the significance of differences between groups. Comparisons between groups were done using a one-way ANOVA followed by a Student–Newman–Keul’s test and the criterion of statistical significance was taken as p < 0.01 or p < 0.001.
Fig. 2. Cytotoxic effects of DMC in K562 cells. Cells were treated for 48 h in the presence of the drug in medium. Cytotoxicity was then determined by MTT assay and was expressed as mean ± S.D. of three separate experiments (n = 3 each in the three experiments). Significant differences from untreated control are indicated by ** p < 0.001.
3. Results 3.1. Cytotoxic effects of DMC in K562 human leukemia cells Using MTT assay the cytotoxicity of DMC on K562 cells were shown in Fig. 2. The data indicated that cytotoxicity of DMC on the cells were dose-dependent. The IC50 value on cytotoxicity was 14.2 ± 0.45 M for the cells. 3.2. Effects of DMC on colony formation of K562 cells After exposure to DMC, the inhibition of colony formation was determined and was shown in Fig. 3. DMC dosedependently inhibited the colony formation. The EC50 value on colony formation was 3.3 ± 0.14 M for K562 cells. 3.3. Effects of DMC on the nuclear morphology in K562 cells Staining with Hoechst 33258 showed fragmentation and condensation of chromatin in the cells treated with 8 M DMC for 48 h (Fig. 4B, compared to untreated control
Fig. 3. Effects of DMC on colony formation of K562 cells. Cells, plated into 35-mm culture dishes, were treated with different concentrations of the drug in medium. Cell colonies were counted after a 14-days incubation at 37 ◦ C in 5% CO2 . Data are expressed as mean ± S.D. of five samples. Significant differences from control: ** p < 0.001.
Fig. 4A). Control cells exhibited a normal nuclear morphology characterized by a diffused chromatin structure and therefore light staining. 3.4. Effects of DMC on the population of hypodiploid cells in K562 cells Flow cytometric analysis was performed to determine hypodiploid cells. Cells with DNA content less than
Fig. 4. Fluorescent staining of nuclei in DMC-treated K562 cells by Hoechst 33258. Cells were treated with 8 M DMC for 48 h. Cells with condensed and fragmented nuclei and apoptotic bodies (arrows) are seen in the DMC-treated cells (B), but not in the control treatment (A). Magnification 200×.
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G1 in the cell cycle distribution were counted as hypodiploid cells. As shown in Fig. 5, the sub-G0/1 population in drug-treated groups increased with the concentration of the DMC increment. After 48 h incubation with 1, 2, 4, 8 and 16 M, the percentages of hypodiploid cells were 10.57 ± 1.94%, 14.73 ± 1.86%, 43.43 ± 3.52%, 66.25 ± 2.81% and 76.15 ± 3.22%, respectively. The treatment resulted in the appearance of a hypodiploid peak (A0 region), probably due to the presence of apoptosing cells and/or apoptotic bodies with DNA content less than 2n. 3.5. Effects of DMC on the expression of Bcl-2 and Bax proteins To verify whether DMC-induced K562 cells apoptosis was involved in the expression of Bcl-2 and Bax proteins, Western
Fig. 6. Western blot assay of Bcl-2, Bax and Actin protein expression in K562 cell line. K562 cells were treated with different concentrations of DMC for 48 h. Protein (40 g/lane) from cell lysates was electrophoresed in SDS–PAGE gels, transferred to TotalBlot PVDF membranes, and probed with anti-Bcl-2, anti-Bax and anti-Actin antibodies, respectively, as described in Section 2.
blot experiment for evaluating Bcl-2/Bax protein was carried out. The results illustrated that in the same dosage and incubation time, DMC could down-regulate the level of Bcl-2 protein and did not influence the expression of Bax protein (Fig. 6).
4. Discussion
Fig. 5. Analysis of the population of hypodiploid cells in K562 cells by flow cytometry. (A) Representative of the profiles of cell cycle distribution in three independent experiments per concentration was shown. K562 cells treated with 0 M (I), 1 M (II), 2 M (III), 4 M (IV), 8 M (V) and 16.0 M (VI) for 48 h. (B) The percentage of hypodiploid cells after staining with propidium iodide. Data are presented as the mean ± S.D. and are representative of an average of three independent experiments per concentration. Significant differences from control: * p < 0.01; ** p < 0.001.
In the last decade, various pharmacological activities of chalcone, such as anti-cancer and anti-oxidant activity [16], anti-invasive activity [17] inhibitor of aromatase, 17hydroxysteroid dehydrogenase activity [18], cytotoxic and inhibitory effects on proliferation of leukemic [19] and antiangiogenic activity [20], have been described. In particular, it is reported that flavonoids have anti-leukemic activity. Quercetin enhanced cisplatin-induced apoptosis of HL-60 and L1210 leukemia cells [21]. Quercetin and flavopiridol inhibited the growth and viability of various acute myelogenous leukemia (AML) cell lines and AML blasts isolated afresh from patients with AML of various sub-types [22]. Quercetin and luteolin potentiated cisplatin-induced cytotoxicity of murine leukemia L1210 cells [23]. Sophoranone inhibited the growth and induced apoptosis of leukemia U937 cells [24]. Wogonin, baicalein, apigenin, myricetin and fisetin showed obvious cytotoxic effects on HL-60 cells, with wogonin and fisetin being the most-potent apoptotic inducers among them [25]. Our study demonstrated that DMC was cytotoxic for K562 cells and could inhibit proliferation of the cells, with IC50 value and EC50 value equal to 14.2 ± 0.45 and 3.3 ± 0.14 M, respectively. The mechanism of the anti-tumor of chalcones remains to be fully clarified. It was reported that flavonoids could induce cancer cells apoptosis. Isoflavone genistein induced apoptosis in HL-60 human leukemia cells [26–29]. Flavonol quercetion induced apoptosis in cancer cells, such as HT-29 human colon cancer cells, HL-60 and K562 human leukemia cells [29–31]. Flavone Baicalin induced apoptosis in prostate cancer cells [32]. Chalcone buitein and isoliquiritigenin induced apoptosis B16 melanoma 4A5 cells [33]. Chalcone phloretin induced apoptosis B16 melanoma 4A5 cells and HL-60 human leukemia cells [33,34]. In the present study, we investigated whether DMC could induce K562 cells apoptosis. Staining with Hoechst 33258 showed condensed and
C.-L. Ye et al. / Leukemia Research 29 (2005) 887–892
fragmented nuclei and apoptotic bodies in the cells treated with 8 M DMC for 48 h. The FCM results indicated that the percentage of hypodiploid K562 cells was 76.15 ± 3.22% after 48 h treatment with 16.0 M DMC. The appearance of a hypodiploid peak (A0 region), probably due to the presence of apoptosing cells and/or apoptotic bodies with DNA content less than 2n. These results suggested that DMC could induce K562 cells apoptosis in vitro. It has been widely accepted that apoptosis is an active gene-directed cellular suicide mechanism and many human genes contribute to its regulation, such as p53 gene, c-myc, bcl-2 gene family. Among these genes, bcl-2 draws particular attention because it may be one of the key factors of the common final pathway involved in the regulation of cell apoptosis [35]. Bcl-2 can form heterodimers with Bax protein-a Bcl-2-associated protein that antagonizes Bcl-2 function and induces cell apoptosis [36]. In this study, we found that both Bcl-2 and Bax were expressed in K562 cells. At 2 M of concentration, DMC potently down-regulated bcl-2 gene expression at the protein levels. The effects were more obvious following the enhancement of DMC dose. No influences were found on the expression of Bax protein. The resulting net effect could thus lead to a lower ratio of Bcl-2/Bax, which might be responsible for the DMC-induced apoptosis in K562 cells. In brief, this work indicated that DMC had cytotoxycity, anti-proliferation and apoptosis-inducing effect on K562 cells. Western blot results illustrated that DMC could downregulate the expression of Bcl-2 protein in K562 cells. DMC could be promising as a chemopreventive agent or an adjuvant to the conventional therapeutic modalities for leukemia.
Acknowledgement This work was supported by the Key Disciplinary Foundation of Shanghai, PR China. Contributions. Chun-Lin Ye collected, assembled and analyzed the data, drafted the manuscript and gave final approval. Feng Qian contributed to the concept and design and gave final approval. Dong-Zhi Wei contributed to the concept and design, obtained the necessary funding, assisted with the writing and data interpretation and gave final approval. YanHua Lu contributed to the revision of the manuscript and gave final approval. Jian-Wen Liu contributed to the revision of the article and gave final approval.
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Induction of apoptosis in K562 human leukemia cells by 2,4-dihydroxy-6-methoxy-3,5-dimethylchalcone Chun-Lin Ye, Feng Qian, Dong-Zhi Wei ∗ , Yan-Hua Lu, Jian-Wen Liu State Key Laboratory of Bioreactor Engineering, Institute of Biochemistry, East China University of Science and Technology, Shanghai 200237, PR China Received 4 November 2004; received in revised form 2 January 2005; accepted 7 January 2005 Available online 16 February 2005
Abstract 2 ,4 -Dihydroxy-6 -methoxy-3 ,5 -dimethylchalcone (DMC), isolated from the buds of Cleistocalyx operculatus, was investigated in its cytotoxicity and anti-proliferation on K562 cell line. Our results revealed that the IC50 was equal to 14.2 ± 0.45 M and the EC50 was 3.3 ± 0.14 M. Staining with Hoechst 33258 showed fragmentation and condensation of chromatin in the cells treated with 8 M DMC for 48 h. Flow cytometric analysis was performed to determine hypodiploid cells. The results of flow cytometry assay indicated that the percentage of hypodiploid K562 cells was 76.15 ± 3.22% after 48 h treatment with 16.0 M DMC. The treatment resulted in the appearance of a hypodiploid peak (A0 region), probably due to the presence of apoptosing cells and/or apoptotic bodies with DNA content less than 2n. Western blot results illustrated that in the same dosage and incubation time, DMC could down-regulate the level of Bcl-2 protein and did not influence the expression of Bax protein. The resulting net effect could thus lead to a lower ratio of Bcl-2/Bax, which might be responsible for the DMC-induced apoptosis in K562 cells. © 2005 Elsevier Ltd. All rights reserved. Keywords: Cleistocalyx operculatus; Flavonoids; K562; Bcl-2/Bax; Apoptosis
1. Introduction Flavonoids are polyphenolic compounds that occur naturally in normal human diet and in many folk medicines, still in use. These compounds can scavenge superoxide, hydroxy and proxyradicals, breaking lipid peroxide chain reactions. They have also been shown to protect cells from X-ray damage, to block progression of cell cycle, to inhibit mutations, to block prostaglandin synthesis and to prevent multistage carcinogenesis in experimental animals [1]. Various pharmacological activities of flavonoids have been studied extensively [2–5]. Chalcones, considered as the precursor of flavonoids and isoflavonoids, are abundant in edible plants. A number of chalcones have
∗
Corresponding author. Tel.: +86 21 64252981; fax: +86 21 64250068. E-mail address: [email protected] (D.-Z. Wei).
0145-2126/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2005.01.006
demonstrated cytotoxic [6,7] and anti-cancer properties [8,9]. Cleistocalyx operculatus (Roxb.) Merr. et Perry (Myrtaceae), is a well-known medicinal plant whose buds are commonly used as an ingredient for tonic drinks in southern China. It was reported that the water extract of the buds of C. operculatus was shown to increase the contractility and decrease the frequency of contraction in an isolated rat heart perfusion system [10]. Our previous phytochemical attention to the species has led to the characterization of sterol, flavanone, chalcone and triterpene acid from its buds [11]. In this paper, we investigated the in vitro anti-tumor activity of 2 ,4 -dihydroxy-6 -methoxy3 ,5 -dimethylchalcone (DMC), one of the compound from the buds of C. operculatus, in K562 cell line. In addition, we were interested in elucidating the molecular mechanisms by which DMC may induce K562 cells apoptosis.
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C.-L. Ye et al. / Leukemia Research 29 (2005) 887–892
2. Materials and methods
2.4. Soft agar colony forming (clonogenic) assay
2.1. Chemicals
The effects of DMC on soft agar colony forming of K562 cells were investigated. Briefly, K562 cells were treated with DMC ranging from 1.5 to 25 M or without (vehicle control, 0.1% dimethyl sulfoxide) DMC and then mixed with agar in a final concentration of 0.33%. Aliquots of 1.5 ml containing 103 cells and 10% FBS were plated in triplicate in 35-mm culture dishes over a base layer of 0.6% agar and allowed to gel. Colonies (>20 cells) were counted after 14 days of incubation under an inverted microscope (Leica DMIRB, Germany) [13]. The anti-proliferative activity was expressed as EC50 (concentration of the DMC that induces 50% inhibition of the cell colony number, which was extrapolated from linear regression analysis of experimental data).
DMC was isolated from C. operculatus in our lab as described by Ye et al. [11]. Previous experiments have shown that DMC purity was above 96%. The structure of the compound is shown in Fig. 1. The compound was dissolved in dimethyl sulfoxide (DMSO). The control cells were treated with the same amount of vehicle alone. The final DMSO concentration never exceeded 0.1% (v/v), in either control or treated samples. Previous experiments have shown that DMSO at this concentration does not modify the cellular activities that we are analyzing. Fetal bovine serum (FBS) and Rosewell Park Memorial Institute (RPMI) 1640 medium were purchased from Life Technologies, Inc., MA, USA.
2.5. Fluorescent staining of nuclei for K562 cells 2.2. Cell lines and culture conditions K562 human chronic leukemia cells were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI 1640 medium with 10% FBS, penicillin (100 U/ml) and streptomycin (100 g/ml) in an atmosphere of 5% CO2 in humidified air at 37 ◦ C. In all experiments, exponentially growing cells were used.
K562 cells from exponentially growing cultures were seeded within 24-well culture plates. The cells were treated with 8 M DMC or without (vehicle control, 0.1% dimethyl sulfoxide) DMC for 48 h. After treatment, cells were washed with phosphate-buffered saline (PBS), and were fixed in MeOH–HAc (3:1, v/v) for 10 min at 4 ◦ C. Cells were stained with Hoechst 33258 (5 g/ml in PBS) for 5 min at room temperature and then examined in a Leica DMIRB fluorescent microscope at 356 nm [14].
2.3. Cytotoxycity assay
2.6. Flow cytometry assay for K562 cells
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay was performed as described by Mosman [12]. K562 cells were placed within 96-well culture plates (104 cells/well). The cells were treated with DMC ranging from 12.5 to 100 M or without (vehicle control, 0.1% dimethyl sulfoxide) DMC. DMC cytotoxycity was measured after 2 days of culture using the MTT assay. Absorbance in control and drug treated wells was measured in an Automated Microplate Reader (Bio-Rad 550) at 550 nm. The cytotoxycity of DMC was expressed as IC50 (concentration of the DMC that induces 50% inhibition of the cell growth, which was extrapolated from linear regression analysis of experimental data).
Flow cytometric analysis was performed to determine hypodiploid cells. The cells were stained with propidium iodide using a cycle TEST PLUS DNA Reagent kit (Becton Dickinson, USA), then analysed by a FACScan (Becton Dickinson, USA) with Cell Fit software. Hypodiploid cells have less DNA than that of diploid cells at G1 phase because of apoptosis-induced DNA fragmentation.
Fig. 1. Structure of 2 ,4 -dihydroxy-6 -methoxy-3 ,5 -dimethylchalcone.
2.7. Protein extraction and Western blot analysis K562 cells were planted and cultured in complete medium. The cells were treated with DMC ranging from 2 to 16 M or without (vehicle control, 0.1% dimethyl sulfoxide) DMC. Cells were washed twice with cold PBS and lysed at 4 ◦ C for 30 min in lysis buffer (0.5% Triton X-100, 300 mM NaCl, 50 mM Tris–Cl, 1 mM phenylmethylsulfonyl fluoride) with occasional vortexing. Insoluble material was removed by centrifugation at 4 ◦ C for 15 min at 14,000 × g and the total proteins extracted were quantified by the coomassie brilliant blue G-250 assay with bovine serum albumin (BSA) as standard (VARIAN Cary 500 UV/Vis spectrophotometer, USA) [15]. Cellular proteins (40 g/lane) were resolved on SDS–12% polyacrylamide gels and transferred to TotalBlot PVDF membranes (Amresco Inc., USA), which were blocked with 5% fat-free milk and immunostained with 1:1000 dilution of monoclonal mouse anti-Bcl-2 antibody (Santa Cruz
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Biotechnology, Inc., USA) or a 1:1000 dilution of monoclonal mouse anti-bax antibody (Santa Cruz Biotechnology, Inc., USA). The immunoreactive proteins were probed with horseradish peroxidase-conjugated goat anti-mouse IgG, and Bcl-2 and Bax protein levels were visualized by peroxidase reaction using the ECL kit (Amersham Pharmacia Biotech, UK). Equal loading of extracts was confirmed using antiActin (Santa Cruz Biotechnology, Inc., USA). 2.8. Statistical analysis Each experimental value is expressed as the mean ± standard deviation (S.D.). The scientific statistic software GraphPad Instat version 3.05 was used to evaluate the significance of differences between groups. Comparisons between groups were done using a one-way ANOVA followed by a Student–Newman–Keul’s test and the criterion of statistical significance was taken as p < 0.01 or p < 0.001.
Fig. 2. Cytotoxic effects of DMC in K562 cells. Cells were treated for 48 h in the presence of the drug in medium. Cytotoxicity was then determined by MTT assay and was expressed as mean ± S.D. of three separate experiments (n = 3 each in the three experiments). Significant differences from untreated control are indicated by ** p < 0.001.
3. Results 3.1. Cytotoxic effects of DMC in K562 human leukemia cells Using MTT assay the cytotoxicity of DMC on K562 cells were shown in Fig. 2. The data indicated that cytotoxicity of DMC on the cells were dose-dependent. The IC50 value on cytotoxicity was 14.2 ± 0.45 M for the cells. 3.2. Effects of DMC on colony formation of K562 cells After exposure to DMC, the inhibition of colony formation was determined and was shown in Fig. 3. DMC dosedependently inhibited the colony formation. The EC50 value on colony formation was 3.3 ± 0.14 M for K562 cells. 3.3. Effects of DMC on the nuclear morphology in K562 cells Staining with Hoechst 33258 showed fragmentation and condensation of chromatin in the cells treated with 8 M DMC for 48 h (Fig. 4B, compared to untreated control
Fig. 3. Effects of DMC on colony formation of K562 cells. Cells, plated into 35-mm culture dishes, were treated with different concentrations of the drug in medium. Cell colonies were counted after a 14-days incubation at 37 ◦ C in 5% CO2 . Data are expressed as mean ± S.D. of five samples. Significant differences from control: ** p < 0.001.
Fig. 4A). Control cells exhibited a normal nuclear morphology characterized by a diffused chromatin structure and therefore light staining. 3.4. Effects of DMC on the population of hypodiploid cells in K562 cells Flow cytometric analysis was performed to determine hypodiploid cells. Cells with DNA content less than
Fig. 4. Fluorescent staining of nuclei in DMC-treated K562 cells by Hoechst 33258. Cells were treated with 8 M DMC for 48 h. Cells with condensed and fragmented nuclei and apoptotic bodies (arrows) are seen in the DMC-treated cells (B), but not in the control treatment (A). Magnification 200×.
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G1 in the cell cycle distribution were counted as hypodiploid cells. As shown in Fig. 5, the sub-G0/1 population in drug-treated groups increased with the concentration of the DMC increment. After 48 h incubation with 1, 2, 4, 8 and 16 M, the percentages of hypodiploid cells were 10.57 ± 1.94%, 14.73 ± 1.86%, 43.43 ± 3.52%, 66.25 ± 2.81% and 76.15 ± 3.22%, respectively. The treatment resulted in the appearance of a hypodiploid peak (A0 region), probably due to the presence of apoptosing cells and/or apoptotic bodies with DNA content less than 2n. 3.5. Effects of DMC on the expression of Bcl-2 and Bax proteins To verify whether DMC-induced K562 cells apoptosis was involved in the expression of Bcl-2 and Bax proteins, Western
Fig. 6. Western blot assay of Bcl-2, Bax and Actin protein expression in K562 cell line. K562 cells were treated with different concentrations of DMC for 48 h. Protein (40 g/lane) from cell lysates was electrophoresed in SDS–PAGE gels, transferred to TotalBlot PVDF membranes, and probed with anti-Bcl-2, anti-Bax and anti-Actin antibodies, respectively, as described in Section 2.
blot experiment for evaluating Bcl-2/Bax protein was carried out. The results illustrated that in the same dosage and incubation time, DMC could down-regulate the level of Bcl-2 protein and did not influence the expression of Bax protein (Fig. 6).
4. Discussion
Fig. 5. Analysis of the population of hypodiploid cells in K562 cells by flow cytometry. (A) Representative of the profiles of cell cycle distribution in three independent experiments per concentration was shown. K562 cells treated with 0 M (I), 1 M (II), 2 M (III), 4 M (IV), 8 M (V) and 16.0 M (VI) for 48 h. (B) The percentage of hypodiploid cells after staining with propidium iodide. Data are presented as the mean ± S.D. and are representative of an average of three independent experiments per concentration. Significant differences from control: * p < 0.01; ** p < 0.001.
In the last decade, various pharmacological activities of chalcone, such as anti-cancer and anti-oxidant activity [16], anti-invasive activity [17] inhibitor of aromatase, 17hydroxysteroid dehydrogenase activity [18], cytotoxic and inhibitory effects on proliferation of leukemic [19] and antiangiogenic activity [20], have been described. In particular, it is reported that flavonoids have anti-leukemic activity. Quercetin enhanced cisplatin-induced apoptosis of HL-60 and L1210 leukemia cells [21]. Quercetin and flavopiridol inhibited the growth and viability of various acute myelogenous leukemia (AML) cell lines and AML blasts isolated afresh from patients with AML of various sub-types [22]. Quercetin and luteolin potentiated cisplatin-induced cytotoxicity of murine leukemia L1210 cells [23]. Sophoranone inhibited the growth and induced apoptosis of leukemia U937 cells [24]. Wogonin, baicalein, apigenin, myricetin and fisetin showed obvious cytotoxic effects on HL-60 cells, with wogonin and fisetin being the most-potent apoptotic inducers among them [25]. Our study demonstrated that DMC was cytotoxic for K562 cells and could inhibit proliferation of the cells, with IC50 value and EC50 value equal to 14.2 ± 0.45 and 3.3 ± 0.14 M, respectively. The mechanism of the anti-tumor of chalcones remains to be fully clarified. It was reported that flavonoids could induce cancer cells apoptosis. Isoflavone genistein induced apoptosis in HL-60 human leukemia cells [26–29]. Flavonol quercetion induced apoptosis in cancer cells, such as HT-29 human colon cancer cells, HL-60 and K562 human leukemia cells [29–31]. Flavone Baicalin induced apoptosis in prostate cancer cells [32]. Chalcone buitein and isoliquiritigenin induced apoptosis B16 melanoma 4A5 cells [33]. Chalcone phloretin induced apoptosis B16 melanoma 4A5 cells and HL-60 human leukemia cells [33,34]. In the present study, we investigated whether DMC could induce K562 cells apoptosis. Staining with Hoechst 33258 showed condensed and
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fragmented nuclei and apoptotic bodies in the cells treated with 8 M DMC for 48 h. The FCM results indicated that the percentage of hypodiploid K562 cells was 76.15 ± 3.22% after 48 h treatment with 16.0 M DMC. The appearance of a hypodiploid peak (A0 region), probably due to the presence of apoptosing cells and/or apoptotic bodies with DNA content less than 2n. These results suggested that DMC could induce K562 cells apoptosis in vitro. It has been widely accepted that apoptosis is an active gene-directed cellular suicide mechanism and many human genes contribute to its regulation, such as p53 gene, c-myc, bcl-2 gene family. Among these genes, bcl-2 draws particular attention because it may be one of the key factors of the common final pathway involved in the regulation of cell apoptosis [35]. Bcl-2 can form heterodimers with Bax protein-a Bcl-2-associated protein that antagonizes Bcl-2 function and induces cell apoptosis [36]. In this study, we found that both Bcl-2 and Bax were expressed in K562 cells. At 2 M of concentration, DMC potently down-regulated bcl-2 gene expression at the protein levels. The effects were more obvious following the enhancement of DMC dose. No influences were found on the expression of Bax protein. The resulting net effect could thus lead to a lower ratio of Bcl-2/Bax, which might be responsible for the DMC-induced apoptosis in K562 cells. In brief, this work indicated that DMC had cytotoxycity, anti-proliferation and apoptosis-inducing effect on K562 cells. Western blot results illustrated that DMC could downregulate the expression of Bcl-2 protein in K562 cells. DMC could be promising as a chemopreventive agent or an adjuvant to the conventional therapeutic modalities for leukemia.
Acknowledgement This work was supported by the Key Disciplinary Foundation of Shanghai, PR China. Contributions. Chun-Lin Ye collected, assembled and analyzed the data, drafted the manuscript and gave final approval. Feng Qian contributed to the concept and design and gave final approval. Dong-Zhi Wei contributed to the concept and design, obtained the necessary funding, assisted with the writing and data interpretation and gave final approval. YanHua Lu contributed to the revision of the manuscript and gave final approval. Jian-Wen Liu contributed to the revision of the article and gave final approval.
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