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Mixed galactolipid anomers accentuate apoptosis of multiple myeloma cells by inducing DNA damage
Carbohydrate Research xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Mixed galactolipid anomers accentuate apoptosis of multiple myeloma cells by inducing DNA damage Si-Si Deng a, , Chao Zhang b,d, , Huan Wang a,c, Yi Zang a,⇑, Jia Li a, Xiao-Peng He c,⇑, Guo-Rong Chen c a
National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China Pediatric Intensive Care Unit, Jinan Children’s Hospital, Jinan, 250022 Shangdong Province, PR China c Key Laboratory for Advanced Materials & Institute of Fine Chemicals, East China University of Science and Technology, 130 Meilong Rd, Shanghai 200237, PR China d Shandong University, Jinan, 250012 Shandong Province, PR China b
a r t i c l e
i n f o
Article history: Received 14 August 2014 Received in revised form 15 November 2014 Accepted 21 November 2014 Available online xxxx Keywords: Multiple myeloma Glycolipid Anomer Synergism Apoptosis
a b s t r a c t This study describes an interesting observation that the mixture of anomeric galactolipids has synergistic effects on the growth inhibition of human multiple myeloma (MM) cells. We determine that the equivalent mixture of a pair of a- and b-galactolipids with a 14-carbon lipid chain can cause stronger poly ADPribose polymerase cleavage and DNA damage, producing more late apoptotic MM cells, than either anomer alone. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Multiple myeloma (MM) is a neoplastic disorder that features the accumulation of abnormal plasma cells in the bone marrow, impeding the manufacture of normal blood cells. This may subsequently result in kidney damage, hypercalcemia, and bone lesions.1,2 Clinically, the treatment of MM relies on the use of steroids, immunomodulatory drugs including thalidomide, lenalidomide, and pomalidomide, and proteasome inhibitors including bortezomib and carfilzomib.3–5 Recently, development of histone deacetylase inhibitors6 and alkylators7 has provided additional therapeutic regimes for the disease. Unfortunately, MM still represents an incurable disease with the average survival of patients being six months. This implies that identification of new drug leads for MM is in urgent need. Glycolipids are universal cell-surface components that transmit signals between cells. Because of their unique amphiphilic property, glycolipids have been widely used as non-ionic green surfactants8 in cosmetics and as co-dopants in the fabrication of glycoliposomes for targeted drug delivery.9 They also possess ⇑ Corresponding authors. Tel.: +86 21 50801313; fax: +86 21 50800721 (Y.Z.); tel.: +86 21 64253016; fax: +86 21 64252758 (X.-P.H.). E-mail addresses: [email protected] (Y. Zang), [email protected] (X.-P. He). These authors contributed equally.
promising anticancer activities. For example, galactosylceramides derived from marine sponge can activate natural killer T cells, and have been developed as immunostimulant agents.10 Glycoglycerolipids isolated from spinach have been shown to inhibit tumor angiogenesis and suppress DNA polymerase activity.11 We have also described the preparation of triazolyl glycolipids with cytotoxicity toward some cancer cells.12–14 With continuing interest in the discovery of potential anticancer drug leads,15–18 we report here an interesting observation that the anomeric mixture of some galactolipids shows synergistic effect on the growth inhibition of human MM cells. 2. Results and discussion Through a series of previous studies we have determined that the length of lipid chain may impact the cytotoxicity of glycolipids; those with relatively long chains (>12C) were more toxic toward cancer cells.12,13 As a consequence, a- and b-galactolipids synthesized previously by a BF3Et2O promoted glycosylation reaction between lipid alcohols and peracetyl 1-O-D-galactoside, followed by a deacylation reaction19,20 with relatively long carbon-chain lengths (14C, 15C, and 16C) were used (Fig. 1). The purity and structural homogeneity have been demonstrated before biological tests (see Supporting information). We have determined that the anomeric mixtures of these galactolipids showed evident synergistic effect on the growth inhibition of a human lung cancer cell line
http://dx.doi.org/10.1016/j.carres.2014.11.016 0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Deng, S.-S.; et al. Carbohydr. Res. (2015), http://dx.doi.org/10.1016/j.carres.2014.11.016
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Figure 1. Schematic depiction of the anomeric synergism, and structure of the galactolipids used in this study.
(A549).19 In the present study we further examined the toxicity of both single and mixed anomers toward MM cells. MM cell lines RPMI 8226 and NCI-H929 were employed for the MTS [(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium)] cell viability assay.21 a(MC14a, MC15a, and MC16a) and b-galactolipids (MC14b, MC15b, and MC16b), and their equivalent mixtures (e.g., if the concentration of single anomers is 50 lg/mL, the mixture contains 25 lg/ mL each) were tested for their ability to inhibit the growth of the MM cell lines (Fig. 1). The half maximal inhibitory concentration (IC50) values of the compounds are shown in Table 1. We first determined that all the galactolipids with different chain lengths showed toxicity toward the MM cell lines used. The a-galactolipids were more toxic than the b-counterparts. For instance, while MC16b did not have cytotoxic effect on NCIH929, MC16a showed moderate toxicity toward the cell line. This suggests that anomerism might impact the bioactivity of glycolipids, which is in accordance with our previous observations.19 Interestingly, we then determined that the mixed anomers showed improved inhibitive effects on the growth of the cell lines used (Table 1). As the combination index (CI) is a reliable tool for measuring the combination therapeutic effect of two individual bioactive compounds (CI <0.9 and CI >1.1 suggest synergism and antagonism, respectively),22–24 we calculated the CI values of all groups of mixed anomers. As shown in Table 2, all the anomeric mixtures showed evident synergistic effects (CI = 0.15–0.41) with a low mixing concentration (17 lg/mL a- mixed with 17 lg/mL b-galactolipid) for RPMI 8826 and NCI-H929. With a relatively high mixing concentration (50 lg/mL a- mixed with 50 lg/mL b-galactolipid), the synergism became weaker. This suggests that the lower concentration favors the synergism of the two anomeric
Table 1 Cytotoxicity of single and mixed galactolipids toward multiple myeloma cells determined by MTS IC50 (lg/mL)a
Compound
MC14a MC14b MC14a + MC14bb MC15a MC15b MC15a + MC15bb MC16a MC16b MC16a + MC16bb a b
RPMI 8226
H929
15.7 ± 1.0 20.9 ± 2.2 15.6 ± 1.2 17.8 ± 0.6 16.7 ± 1.4 17.6 ± 0.2 11.8 ± 1.3 39.4 ± 1.2 13.3 ± 1.6
12.9 ± 1.1 15.2 ± 1.9 13.7 ± 1.3 10.8 ± 1.9 18.2 ± 3.0 14.5 ± 1.5 10.0 ± 0.3 >100 14.4 ± 1.2
Values are mean of three experiments. The a- and b-galactolipids were mixed equivalently.
Table 2 Combination index (CI) of mixed galactolipids toward multiple myeloma cells CI valuea
Concentration (lg/mL)
RPMI 8226
H929
MC14a 17 50
MC14b 17 50
0.41 1.24
0.29 0.84
MC15a 17 50
MC15b 17 50
0.19 0.55
0.15 0.39
MC16a 17 50
MC16b 17 50
0.21 0.60
0.19 0.58
a CI values indicate quantitatively the degree of a drug interaction. CI <0.9, CI = 0.9–1.1 and CI >1.1 indicate synergism, additive effect and antagonism, respectively.
compounds, and that increase of the concentration might lead to an antagonistic effect. Poly ADP-ribose polymerase (PARP) is a substrate of caspase and is involved in DNA repair. PARP cleavage produces PAR, which can be viewed as a sign of apoptosis.25 Therefore, western blotting was performed to analyze the cleavage activity of PARP induced by the single anomers or anomeric mixtures with RPMI 8226 cells (Fig. 2). We determined that the anomeric mixtures caused stronger PARP cleavage than either anomer alone with two different mixing concentrations (8.5 + 8.5 or 17 + 17 lg/mL). The mixture of 14C-galactolipids (MC14a + MC14b) showed the strongest synergistic effect, which was selected for further assays. Former literature reports have shown that glycolipid fractions isolated from Spinach could inhibit the activity of replicative DNA polymerases (pols) such as a, d, and e, leading to severe DNA damage.26 Histone H2AX is a tumor suppressor that facilitates the preservation of genome integrity by being massively and quickly phosphorylated at the sites of nascent DNA double-strand breaks (DSBs) in chromatin. To investigate whether the mixed anomers could induce DNA damage, the phosphorylation of H2AX at Ser139 (U-H2AX), a well-known DNA damage marker, is measured. As shown in Figure 3a, both MC14a and MC14b induced the H2AX phosphorylation (positive control: carfilzomib, a proteosome inhibitor). In agreement with the synergistic anti-proliferation effect, the mixture of the anomers (MC14a combined with MC14b with 1:1 mixing ratio) produced more U-H2AX than the single anomers, suggesting that the former can cause more increased DNA damage than the latter. To evaluate quantitatively the apoptosis of MM cells induced by the galactolipid anomers, FACS (fluorescence-activated cell sorting) analysis of Annexin V-FITC/PI dual staining was carried
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Figure 2. Ability of the single or mixed galactolipids to accentuate poly ADP-ribose polymerase (PARP) cleavage activity in cultured RPMI 8226 cells analyzed by western blot.
a
b
c
Γ-H2Ax
DMSO (-) Late apoptosis: 11.1%
Acn
d
e
MC14a (34 μg/mL) Late apoptosis: 32.4%
Carfizomib (+) (40 nM) Late apoptosis: 64.9%
f
MC14b (34 μg/mL) Late apoptosis: 37.8%
MC14a + MC14b (17 μg/mL each) Late apoptosis: 44.4%
Figure 3. Assessment of the cellular DNA damage and apoptosis induced by the single or mixed galactolipids in MM cells. (a) Western analysis of U-H2AX in cultured RPMI 8226 cells treated with the single (MC14a or MC14b at 34 lg/mL) or mixed galactolipids (17 lg/mL MC14a + 17 lg/mL MC14b) for 24 h. (b–f) The percentage of apoptotic cells was analyzed by flow cytometry for Annexin V/propidium iodine (PI) double staining (concentrations of the compounds used are as indicated in the figures).
out with PRMI 8226 cells. The cells were treated with the single anomers or anomeric mixtures for 24 h (Fig. 3b–e). We observed that, while the rate of late apoptosis of DMSO-treated cells was low (11%, Fig. 3b), that of carfilzomib-treated reached a high level of 64% (Fig. 3c). Interestingly, the anomeric mixtures produced more apoptotic 8226 cells (44%) than either anomer alone (32% for MC14a, Fig. 3d and 32% for MC14b, Fig. 3e). This observation is in accordance with the above anti-proliferative and western blotting results. Moreover, the concentration effect of MC14a was tested (Fig. 4). We observed that, with increasing MC14a the PARP cleavage (Fig. 4a) and phosphorylation of H2AX (Fig. 4b) in RPMI 8226 cells increased gradually. In the meanwhile, the late apoptotic rate of the cells increased with increasing concentration of MC14a
(Fig. 4c–e). This suggests that the enhanced effect for cell growth suppression of mixed MC14a and MC14b was not due to the lowered concentration of MC14a, but probably a synergistic effect. 3. Conclusion In conclusion, we reported the identification of anomeric galactolipid mixtures as potential therapeutic agents for MM. The mixed anomers were found to possess synergistic effect on the inhibition of MM cell growth comparing to either anomeric counterpart alone. The anomers induced apoptosis probably by causing PARP cleavage and DNA damage. Further studies will be focusing on the enhancement of the activity of the glycolipids by structural modifications and elaboration of the mechanism underlying the synergism.
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Figure 4. Assessment of the cellular PARP cleavage, DNA damage and apoptosis induced by increasing MC14a. Western analysis of (a) PARP cleavage and (b) U-H2AX in cultured RPMI 8226 cells treated with increasing MC14a (8.5, 17, and 34 lg/mL). (c–e) The percentage of apoptotic cells was analyzed by flow cytometry for Annexin V/ propidium iodine (PI) double staining (concentrations of the compounds used are as indicated in the figures).
4. Experimental section
the same x% effect when used alone. The CI indicates synergism when <0.9, antagonism when >1.1 and additivity when 0.9–1.1.
4.1. Cell growth inhibition assay 4.3. Western blotting Cell growth inhibition assay was determined with the Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega, WI, USA) according to the manufacturer’s instructions. The Cell Titer 96 Aqueous One Solution Assay (Promega) is a colorimetric method for determining the number of viable cells in proliferation or cytotoxicity assays. The one solution contains MTS compound and an electron coupling reagent PES. The MTS compound is bioreduced by cells into a colored formazan product that is soluble in tissue culture medium. Briefly, the Cells were plated overnight on 96well plates at 10,000 cells per well in RPMI1640 growth medium. Then, the cells were treated with the compounds at indicated concentrations (100 lg/ml, 33.33 lg/ml, 11.11 lg/ml, 3.70 lg/ml, 1.23 lg/ml, 0.41 lg/ml for determination of IC50). After treatment for 72 h, 20 lL of MTS substrate was added to each well. Plates were returned to the incubator and left in the dark for 2 h. The absorbance was measured on a SpectraMax 340 microplate reader (Molecular Devices, USA) at 490 nm with a reference at 690 nm. Each experiment was done in triplicate. 4.2. CI calculation The combination index (CI) was calculated according to the Chou and Talalay median effect principal27 using Calcusyn software (Biosoft). The drugs were applied at the indicated concentration and the cell viability was evaluated using the MTS assay at each dosage. Interaction between pairs of drugs was determined using the combination index (CI). A numerical CI value is calculated based on the following equation: CI = (D)1/(Dx)1 + (D)2/ (Dx)2 + (D)1(D)2/(Dx)1(Dx)2, where (D)1 and (D)2 are the doses of drug 1 and drug 2 that have x% effect when used in combination, and (Dx)1 and (Dx)2 are the doses of drug 1 and drug 2 that have
20–100 lg of protein per lane was loaded onto a 10% SDS–polyacrylamide gel and then transferred to a PVDF membrane (Amersham Biosciences). The membranes were processed for immunoblotting as described previously.28 Anti-PARP primary antibody was obtained from Cell Signaling Technology and used at 1:1000 dilutions, anti-U-H2AX primary antibody was obtained from Cell Signaling Technology and used at 1:1000 dilutions and anti-actin primary antibody was purchased from sigma and used at 1:10,000. Horseradish peroxidase-labeled anti-rabbit or antimouse secondary antibody (1:10,000) was purchased from the Jackson laboratory company (Bar Harbor, Maine, U.S.A.). The immunoblot bands were visualized by enhanced chemiluminescence (Amersham Biosciences). 4.4. Analysis of apoptosis using FCM of AV/PI dual staining Annexin V-FITC (fluorescein isothiocyanate)/PI (propidium iodide) staining for FCM was used to detect apoptosis quantitatively and qualitatively. After treatment with 1% DMSO, 40 nM cafilzomib, 34 lg/mL MC14a, 34 lg/mL MC14b, and the mixture of 17 lg/mL each for 24 h, the cells were processed with an Annexin V-FITC kit (Keygene, Nanjing, China) following treatment according to the manufacturer’s instructions. Next, the samples were analyzed using the FACScan flow cytometer (Becton Dickinson, Sparks, MD, USA) to quantify the apoptotic rate. Different subpopulations were distinguished using the following criteria: Q1-UL, Annexin V-negative, but PI positive (i.e., necrotic cells); Q1-UR, Annexin V/PI-double positive (i.e., late apoptotic cells); Q1-LL, Annexin V/ PI-double negative (i.e., live cells); Q1-LR, Annexin V-positive, but PI-negative (i.e., early apoptotic cells).
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Acknowledgements We thank the 973 project (2013CB733700), the National Science Fund for Distinguished Young Scholars (81125023), the National Natural Science Foundation of China (21176076, 21202045), the Chinese Academy of Science ‘‘Strategic Leader in Science and Technology Projects’’ (XDA01040303), the Program of Shanghai Subject Chief Scientist (13XD1404300), the Key Project of Shanghai Science and Technology Commission (13NM1400900), and the Fundamental Research Funds for Central Universities. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carres.2014.11. 016. References and notes 1. Raab, M. S.; Podar, K.; Breitkreutz, I.; Richardson, P. G.; Anderson, K. C. Lancet 2009, 374, 9686. 2. Morgan, G. J.; Walker, B. A.; Davies, F. Nat. Rev. Cancer 2012, 12, 335. 3. Castelli, R.; Gualtierotti, R.; Orofino, N.; Losurdo, A.; Gandolfi, S.; Cugno, M. Clin. Med. Insights: Oncol. 2013, 7, 209. 4. Moreau, P.; Richardson, P. G.; Cavo, M.; Orlowski, R. Z.; Miguel, J. F. S.; Palumbo, A.; Harousseau, J.-L. Blood 2012, 120, 947. 5. Catley, L.; Tai, Y.-T.; Chauhan, D.; Anderson, K. C. Drug Resist. Updates 2005, 8, 205. 6. Richardson, P. G.; Mitsiades, C. S.; Laubach, J. P.; Hajek, R.; Spicka, I.; Dimopoulos, M. A.; Moreau, P.; Siegel, D. S.; Jagannath, S.; Anderson, K. C. Leuk. Res. 2013, 37, 829. 7. Lentzsch, S.; O’Sullivan, A.; Kennedy, R. C.; Abbas, M.; Dai, L.; Pregja, S. L.; Boyiadzis, M.; Roodman, G. D.; Mapara, M. Y.; Agha, M.; Waas, J.; Shuai, Y.; Normolle, D.; Zonder, J. A. Blood 2012, 119, 4608.
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Please cite this article in press as: Deng, S.-S.; et al. Carbohydr. Res. (2015), http://dx.doi.org/10.1016/j.carres.2014.11.016
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Mixed galactolipid anomers accentuate apoptosis of multiple myeloma cells by inducing DNA damage Si-Si Deng a, , Chao Zhang b,d, , Huan Wang a,c, Yi Zang a,⇑, Jia Li a, Xiao-Peng He c,⇑, Guo-Rong Chen c a
National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China Pediatric Intensive Care Unit, Jinan Children’s Hospital, Jinan, 250022 Shangdong Province, PR China c Key Laboratory for Advanced Materials & Institute of Fine Chemicals, East China University of Science and Technology, 130 Meilong Rd, Shanghai 200237, PR China d Shandong University, Jinan, 250012 Shandong Province, PR China b
a r t i c l e
i n f o
Article history: Received 14 August 2014 Received in revised form 15 November 2014 Accepted 21 November 2014 Available online xxxx Keywords: Multiple myeloma Glycolipid Anomer Synergism Apoptosis
a b s t r a c t This study describes an interesting observation that the mixture of anomeric galactolipids has synergistic effects on the growth inhibition of human multiple myeloma (MM) cells. We determine that the equivalent mixture of a pair of a- and b-galactolipids with a 14-carbon lipid chain can cause stronger poly ADPribose polymerase cleavage and DNA damage, producing more late apoptotic MM cells, than either anomer alone. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Multiple myeloma (MM) is a neoplastic disorder that features the accumulation of abnormal plasma cells in the bone marrow, impeding the manufacture of normal blood cells. This may subsequently result in kidney damage, hypercalcemia, and bone lesions.1,2 Clinically, the treatment of MM relies on the use of steroids, immunomodulatory drugs including thalidomide, lenalidomide, and pomalidomide, and proteasome inhibitors including bortezomib and carfilzomib.3–5 Recently, development of histone deacetylase inhibitors6 and alkylators7 has provided additional therapeutic regimes for the disease. Unfortunately, MM still represents an incurable disease with the average survival of patients being six months. This implies that identification of new drug leads for MM is in urgent need. Glycolipids are universal cell-surface components that transmit signals between cells. Because of their unique amphiphilic property, glycolipids have been widely used as non-ionic green surfactants8 in cosmetics and as co-dopants in the fabrication of glycoliposomes for targeted drug delivery.9 They also possess ⇑ Corresponding authors. Tel.: +86 21 50801313; fax: +86 21 50800721 (Y.Z.); tel.: +86 21 64253016; fax: +86 21 64252758 (X.-P.H.). E-mail addresses: [email protected] (Y. Zang), [email protected] (X.-P. He). These authors contributed equally.
promising anticancer activities. For example, galactosylceramides derived from marine sponge can activate natural killer T cells, and have been developed as immunostimulant agents.10 Glycoglycerolipids isolated from spinach have been shown to inhibit tumor angiogenesis and suppress DNA polymerase activity.11 We have also described the preparation of triazolyl glycolipids with cytotoxicity toward some cancer cells.12–14 With continuing interest in the discovery of potential anticancer drug leads,15–18 we report here an interesting observation that the anomeric mixture of some galactolipids shows synergistic effect on the growth inhibition of human MM cells. 2. Results and discussion Through a series of previous studies we have determined that the length of lipid chain may impact the cytotoxicity of glycolipids; those with relatively long chains (>12C) were more toxic toward cancer cells.12,13 As a consequence, a- and b-galactolipids synthesized previously by a BF3Et2O promoted glycosylation reaction between lipid alcohols and peracetyl 1-O-D-galactoside, followed by a deacylation reaction19,20 with relatively long carbon-chain lengths (14C, 15C, and 16C) were used (Fig. 1). The purity and structural homogeneity have been demonstrated before biological tests (see Supporting information). We have determined that the anomeric mixtures of these galactolipids showed evident synergistic effect on the growth inhibition of a human lung cancer cell line
http://dx.doi.org/10.1016/j.carres.2014.11.016 0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Deng, S.-S.; et al. Carbohydr. Res. (2015), http://dx.doi.org/10.1016/j.carres.2014.11.016
2
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Figure 1. Schematic depiction of the anomeric synergism, and structure of the galactolipids used in this study.
(A549).19 In the present study we further examined the toxicity of both single and mixed anomers toward MM cells. MM cell lines RPMI 8226 and NCI-H929 were employed for the MTS [(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium)] cell viability assay.21 a(MC14a, MC15a, and MC16a) and b-galactolipids (MC14b, MC15b, and MC16b), and their equivalent mixtures (e.g., if the concentration of single anomers is 50 lg/mL, the mixture contains 25 lg/ mL each) were tested for their ability to inhibit the growth of the MM cell lines (Fig. 1). The half maximal inhibitory concentration (IC50) values of the compounds are shown in Table 1. We first determined that all the galactolipids with different chain lengths showed toxicity toward the MM cell lines used. The a-galactolipids were more toxic than the b-counterparts. For instance, while MC16b did not have cytotoxic effect on NCIH929, MC16a showed moderate toxicity toward the cell line. This suggests that anomerism might impact the bioactivity of glycolipids, which is in accordance with our previous observations.19 Interestingly, we then determined that the mixed anomers showed improved inhibitive effects on the growth of the cell lines used (Table 1). As the combination index (CI) is a reliable tool for measuring the combination therapeutic effect of two individual bioactive compounds (CI <0.9 and CI >1.1 suggest synergism and antagonism, respectively),22–24 we calculated the CI values of all groups of mixed anomers. As shown in Table 2, all the anomeric mixtures showed evident synergistic effects (CI = 0.15–0.41) with a low mixing concentration (17 lg/mL a- mixed with 17 lg/mL b-galactolipid) for RPMI 8826 and NCI-H929. With a relatively high mixing concentration (50 lg/mL a- mixed with 50 lg/mL b-galactolipid), the synergism became weaker. This suggests that the lower concentration favors the synergism of the two anomeric
Table 1 Cytotoxicity of single and mixed galactolipids toward multiple myeloma cells determined by MTS IC50 (lg/mL)a
Compound
MC14a MC14b MC14a + MC14bb MC15a MC15b MC15a + MC15bb MC16a MC16b MC16a + MC16bb a b
RPMI 8226
H929
15.7 ± 1.0 20.9 ± 2.2 15.6 ± 1.2 17.8 ± 0.6 16.7 ± 1.4 17.6 ± 0.2 11.8 ± 1.3 39.4 ± 1.2 13.3 ± 1.6
12.9 ± 1.1 15.2 ± 1.9 13.7 ± 1.3 10.8 ± 1.9 18.2 ± 3.0 14.5 ± 1.5 10.0 ± 0.3 >100 14.4 ± 1.2
Values are mean of three experiments. The a- and b-galactolipids were mixed equivalently.
Table 2 Combination index (CI) of mixed galactolipids toward multiple myeloma cells CI valuea
Concentration (lg/mL)
RPMI 8226
H929
MC14a 17 50
MC14b 17 50
0.41 1.24
0.29 0.84
MC15a 17 50
MC15b 17 50
0.19 0.55
0.15 0.39
MC16a 17 50
MC16b 17 50
0.21 0.60
0.19 0.58
a CI values indicate quantitatively the degree of a drug interaction. CI <0.9, CI = 0.9–1.1 and CI >1.1 indicate synergism, additive effect and antagonism, respectively.
compounds, and that increase of the concentration might lead to an antagonistic effect. Poly ADP-ribose polymerase (PARP) is a substrate of caspase and is involved in DNA repair. PARP cleavage produces PAR, which can be viewed as a sign of apoptosis.25 Therefore, western blotting was performed to analyze the cleavage activity of PARP induced by the single anomers or anomeric mixtures with RPMI 8226 cells (Fig. 2). We determined that the anomeric mixtures caused stronger PARP cleavage than either anomer alone with two different mixing concentrations (8.5 + 8.5 or 17 + 17 lg/mL). The mixture of 14C-galactolipids (MC14a + MC14b) showed the strongest synergistic effect, which was selected for further assays. Former literature reports have shown that glycolipid fractions isolated from Spinach could inhibit the activity of replicative DNA polymerases (pols) such as a, d, and e, leading to severe DNA damage.26 Histone H2AX is a tumor suppressor that facilitates the preservation of genome integrity by being massively and quickly phosphorylated at the sites of nascent DNA double-strand breaks (DSBs) in chromatin. To investigate whether the mixed anomers could induce DNA damage, the phosphorylation of H2AX at Ser139 (U-H2AX), a well-known DNA damage marker, is measured. As shown in Figure 3a, both MC14a and MC14b induced the H2AX phosphorylation (positive control: carfilzomib, a proteosome inhibitor). In agreement with the synergistic anti-proliferation effect, the mixture of the anomers (MC14a combined with MC14b with 1:1 mixing ratio) produced more U-H2AX than the single anomers, suggesting that the former can cause more increased DNA damage than the latter. To evaluate quantitatively the apoptosis of MM cells induced by the galactolipid anomers, FACS (fluorescence-activated cell sorting) analysis of Annexin V-FITC/PI dual staining was carried
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Figure 2. Ability of the single or mixed galactolipids to accentuate poly ADP-ribose polymerase (PARP) cleavage activity in cultured RPMI 8226 cells analyzed by western blot.
a
b
c
Γ-H2Ax
DMSO (-) Late apoptosis: 11.1%
Acn
d
e
MC14a (34 μg/mL) Late apoptosis: 32.4%
Carfizomib (+) (40 nM) Late apoptosis: 64.9%
f
MC14b (34 μg/mL) Late apoptosis: 37.8%
MC14a + MC14b (17 μg/mL each) Late apoptosis: 44.4%
Figure 3. Assessment of the cellular DNA damage and apoptosis induced by the single or mixed galactolipids in MM cells. (a) Western analysis of U-H2AX in cultured RPMI 8226 cells treated with the single (MC14a or MC14b at 34 lg/mL) or mixed galactolipids (17 lg/mL MC14a + 17 lg/mL MC14b) for 24 h. (b–f) The percentage of apoptotic cells was analyzed by flow cytometry for Annexin V/propidium iodine (PI) double staining (concentrations of the compounds used are as indicated in the figures).
out with PRMI 8226 cells. The cells were treated with the single anomers or anomeric mixtures for 24 h (Fig. 3b–e). We observed that, while the rate of late apoptosis of DMSO-treated cells was low (11%, Fig. 3b), that of carfilzomib-treated reached a high level of 64% (Fig. 3c). Interestingly, the anomeric mixtures produced more apoptotic 8226 cells (44%) than either anomer alone (32% for MC14a, Fig. 3d and 32% for MC14b, Fig. 3e). This observation is in accordance with the above anti-proliferative and western blotting results. Moreover, the concentration effect of MC14a was tested (Fig. 4). We observed that, with increasing MC14a the PARP cleavage (Fig. 4a) and phosphorylation of H2AX (Fig. 4b) in RPMI 8226 cells increased gradually. In the meanwhile, the late apoptotic rate of the cells increased with increasing concentration of MC14a
(Fig. 4c–e). This suggests that the enhanced effect for cell growth suppression of mixed MC14a and MC14b was not due to the lowered concentration of MC14a, but probably a synergistic effect. 3. Conclusion In conclusion, we reported the identification of anomeric galactolipid mixtures as potential therapeutic agents for MM. The mixed anomers were found to possess synergistic effect on the inhibition of MM cell growth comparing to either anomeric counterpart alone. The anomers induced apoptosis probably by causing PARP cleavage and DNA damage. Further studies will be focusing on the enhancement of the activity of the glycolipids by structural modifications and elaboration of the mechanism underlying the synergism.
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Figure 4. Assessment of the cellular PARP cleavage, DNA damage and apoptosis induced by increasing MC14a. Western analysis of (a) PARP cleavage and (b) U-H2AX in cultured RPMI 8226 cells treated with increasing MC14a (8.5, 17, and 34 lg/mL). (c–e) The percentage of apoptotic cells was analyzed by flow cytometry for Annexin V/ propidium iodine (PI) double staining (concentrations of the compounds used are as indicated in the figures).
4. Experimental section
the same x% effect when used alone. The CI indicates synergism when <0.9, antagonism when >1.1 and additivity when 0.9–1.1.
4.1. Cell growth inhibition assay 4.3. Western blotting Cell growth inhibition assay was determined with the Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega, WI, USA) according to the manufacturer’s instructions. The Cell Titer 96 Aqueous One Solution Assay (Promega) is a colorimetric method for determining the number of viable cells in proliferation or cytotoxicity assays. The one solution contains MTS compound and an electron coupling reagent PES. The MTS compound is bioreduced by cells into a colored formazan product that is soluble in tissue culture medium. Briefly, the Cells were plated overnight on 96well plates at 10,000 cells per well in RPMI1640 growth medium. Then, the cells were treated with the compounds at indicated concentrations (100 lg/ml, 33.33 lg/ml, 11.11 lg/ml, 3.70 lg/ml, 1.23 lg/ml, 0.41 lg/ml for determination of IC50). After treatment for 72 h, 20 lL of MTS substrate was added to each well. Plates were returned to the incubator and left in the dark for 2 h. The absorbance was measured on a SpectraMax 340 microplate reader (Molecular Devices, USA) at 490 nm with a reference at 690 nm. Each experiment was done in triplicate. 4.2. CI calculation The combination index (CI) was calculated according to the Chou and Talalay median effect principal27 using Calcusyn software (Biosoft). The drugs were applied at the indicated concentration and the cell viability was evaluated using the MTS assay at each dosage. Interaction between pairs of drugs was determined using the combination index (CI). A numerical CI value is calculated based on the following equation: CI = (D)1/(Dx)1 + (D)2/ (Dx)2 + (D)1(D)2/(Dx)1(Dx)2, where (D)1 and (D)2 are the doses of drug 1 and drug 2 that have x% effect when used in combination, and (Dx)1 and (Dx)2 are the doses of drug 1 and drug 2 that have
20–100 lg of protein per lane was loaded onto a 10% SDS–polyacrylamide gel and then transferred to a PVDF membrane (Amersham Biosciences). The membranes were processed for immunoblotting as described previously.28 Anti-PARP primary antibody was obtained from Cell Signaling Technology and used at 1:1000 dilutions, anti-U-H2AX primary antibody was obtained from Cell Signaling Technology and used at 1:1000 dilutions and anti-actin primary antibody was purchased from sigma and used at 1:10,000. Horseradish peroxidase-labeled anti-rabbit or antimouse secondary antibody (1:10,000) was purchased from the Jackson laboratory company (Bar Harbor, Maine, U.S.A.). The immunoblot bands were visualized by enhanced chemiluminescence (Amersham Biosciences). 4.4. Analysis of apoptosis using FCM of AV/PI dual staining Annexin V-FITC (fluorescein isothiocyanate)/PI (propidium iodide) staining for FCM was used to detect apoptosis quantitatively and qualitatively. After treatment with 1% DMSO, 40 nM cafilzomib, 34 lg/mL MC14a, 34 lg/mL MC14b, and the mixture of 17 lg/mL each for 24 h, the cells were processed with an Annexin V-FITC kit (Keygene, Nanjing, China) following treatment according to the manufacturer’s instructions. Next, the samples were analyzed using the FACScan flow cytometer (Becton Dickinson, Sparks, MD, USA) to quantify the apoptotic rate. Different subpopulations were distinguished using the following criteria: Q1-UL, Annexin V-negative, but PI positive (i.e., necrotic cells); Q1-UR, Annexin V/PI-double positive (i.e., late apoptotic cells); Q1-LL, Annexin V/ PI-double negative (i.e., live cells); Q1-LR, Annexin V-positive, but PI-negative (i.e., early apoptotic cells).
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Acknowledgements We thank the 973 project (2013CB733700), the National Science Fund for Distinguished Young Scholars (81125023), the National Natural Science Foundation of China (21176076, 21202045), the Chinese Academy of Science ‘‘Strategic Leader in Science and Technology Projects’’ (XDA01040303), the Program of Shanghai Subject Chief Scientist (13XD1404300), the Key Project of Shanghai Science and Technology Commission (13NM1400900), and the Fundamental Research Funds for Central Universities. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carres.2014.11. 016. References and notes 1. Raab, M. S.; Podar, K.; Breitkreutz, I.; Richardson, P. G.; Anderson, K. C. Lancet 2009, 374, 9686. 2. Morgan, G. J.; Walker, B. A.; Davies, F. Nat. Rev. Cancer 2012, 12, 335. 3. Castelli, R.; Gualtierotti, R.; Orofino, N.; Losurdo, A.; Gandolfi, S.; Cugno, M. Clin. Med. Insights: Oncol. 2013, 7, 209. 4. Moreau, P.; Richardson, P. G.; Cavo, M.; Orlowski, R. Z.; Miguel, J. F. S.; Palumbo, A.; Harousseau, J.-L. Blood 2012, 120, 947. 5. Catley, L.; Tai, Y.-T.; Chauhan, D.; Anderson, K. C. Drug Resist. Updates 2005, 8, 205. 6. Richardson, P. G.; Mitsiades, C. S.; Laubach, J. P.; Hajek, R.; Spicka, I.; Dimopoulos, M. A.; Moreau, P.; Siegel, D. S.; Jagannath, S.; Anderson, K. C. Leuk. Res. 2013, 37, 829. 7. Lentzsch, S.; O’Sullivan, A.; Kennedy, R. C.; Abbas, M.; Dai, L.; Pregja, S. L.; Boyiadzis, M.; Roodman, G. D.; Mapara, M. Y.; Agha, M.; Waas, J.; Shuai, Y.; Normolle, D.; Zonder, J. A. Blood 2012, 119, 4608.
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