Original Papers
Synergistic Antitumor Effect of a Combination of Paclitaxel and Carboplatin with Nobiletin from Citrus depressa on Non-Small-Cell Lung Cancer Cell Lines
Authors
Shinichi Uesato 1, Hirofumi Yamashita 1, Ryu Maeda 1, Yoshiyuki Hirata 1, Maho Yamamoto 1, Saki Matsue 1, Yasuo Nagaoka 1, Makio Shibano 2, Masahiko Taniguchi 2, Kimiye Baba 2, Motoharu Ju-ichi 3
Affiliations
1
2 3
Key words " nobiletin l " Citrus depressa l " Rutaceae l " non‑small‑cell lung cancer l " cancer chemotherapy drug l " synergistic effect l " A549 cell xenograft l
received revised accepted
January 20, 2014 February 28, 2014 March 1, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368321 Published online March 31, 2014 Planta Med 2014; 80: 452–457 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Shinichi Uesato Department of Life Science and Biotechnology Faculty of Chemistry Materials and Bioengineering Kansai University 3-3-35 Yamate-cho Suita, Osaka 564–8680 Japan Phone: + 81 6 63 68 08 34 Fax: + 81 6 63 88 86 09 [email protected]
Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, Suita, Osaka, Japan Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka, Japan Faculty of Pharmaceutical Sciences, Mukogawa Womenʼs University, Nishinomiya, Hyogo, Japan
Abstract
Abbreviations
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Non-small-cell lung carcinomas do not sufficiently respond to cancer chemotherapeutic drugs. Combination effects of cancer chemotherapy drugs (paclitaxel and carboplatin) with nobiletin or powdered Shiikuwasha extract from Citrus depressa were examined by isobologram and combination index analyses. It was demonstrated that the combination generated a synergistic inhibitory effect against the proliferation of the human non-small-cell lung carcinoma cell lines A549 and H460 and that of the two chemotherapy drugs, paclitaxel was responsible for this synergistic effect. Furthermore, the percentage of apoptotic cells was decreased with increasing rates of nobiletin to paclitaxel and carboplatin. These findings were considered to be attributed to the ability of nobiletin to regulate cells in the G1 phase, which escaped cell death initiated by paclitaxel and carboplatin. An antitumor activity assay showed that this combination significantly suppressed the growth of subcutaneous A549 tumor xenografts in nude mice.
SCLC: NSCLC: PTX: CBDCA: A549:
Introduction !
Lung cancer is classified into two types, small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC). NSCLC accounts for 70–80 % of all cases of lung cancer in Japan. SCLC usually responds better to chemotherapy and radiotherapy than NSCLC. NSCLC is primarily treated by surgical operation and then by chemotherapy to prevent recurrence. Recently, molecular-targeted drugs such as gefitinib (Iressa) [1] and crizotinib (Xalkori) [2] have been applied as a first-line therapy for NSCLC patients with a mutated epidermal growth factor receptor (EGFR) [3] and with a EML4-ALK fusion gene [4], respectively. About 50 % of other NSCLC patients receive chemother-
Uesato S et al. Synergistic Antitumor Effect …
Planta Med 2014; 80: 452–457
H460: HCO-60: BSA: DMEM: FBS: PBS: CI:
small-cell lung adenocarcinoma non-small-cell lung adenocarcinoma paclitaxel carboplatin NOB: nobiletin human non-small-cell lung adenocarcinoma cell line human non-small-cell lung adenocarcinoma cell line ethoxylated hydrogenated castor oil bovine serum albumin Dulbeccoʼs modified Eagleʼs medium fetal bovine serum phosphate buffered saline combination index
Supporting information available online at http://www.thieme-connect.de/ejournals/toc/ plantamedica
apy with paclitaxel (PTX) and carboplatin (CBDCA) [5, 6]. However, these drugs produce serious side effects such as leukopenia, hepatic dysfunction, deterioration of renal function, vomiting, and nausea. These side effects are most commonly attributed to tissue necrosis, apoptosis, and inflammation caused by the chemotherapy treatment. One approach gaining a better therapeutic effect could be a multicomponent therapy, which involves synergistic interactions [7]. Thus, it is of great clinical significance to seek a third drug that has synergistic anticancer activity with PTX and CBDCA and thus allows the dose " Fig. 1). reduction of these chemotherapy drugs (l Citrus depressa Hayata (Rutaceae) is a citrus plant grown naturally in Okinawa Prefecture, Japan.
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The fruit is very sour like a lemon, and is used as a seasoning and for fruit juices; it is called “Shiikuwasha” locally. The fruit has reportedly beneficial activities on human health, such as anticancer, anti-inflammatory, and antihyperglycemic effects [8– 12]. The components responsible for these activities are polymethoxylated flavonoids that are mainly contained in the peel of the fruit. We have focused on nobiletin (NOB), the major component of flavonoids, because NOB inhibits the proliferation of cancer cells through the arrest of their cell cycle in the G1 phase [13]. Furthermore, it was reported that NOB showed synergistic inhibition with doxorubicin [14] and cis-diamminedichloride platinum [15] against the growth of human breast adenocarcinoma cell line MCF-7 and gastric cancer cells, respectively. In the present work, we investigated how NOB influenced the growth of the human NSCLC cell lines A549 and H460 in combination with PTX and CBDCA using isobologram and combination index analyses [7, 16–19]. Furthermore, we examined antitumor activities against cancer cell xenografts in nude mice.
Fig. 2 IC50 isobolograms of three substances against A549 cells. The IC50 value of PTX in a mixture PTX + CBDCA (molar ratio 1 : 2) is expressed as 1.0 on the Y-axis of the isobologram, while that of NOB or the Shiikuwasha extract alone is expressed as 1.0 on the Xaxis.
Results !
In order to assess the combination effects of the chemotherapy drugs with NOB or powdered Shiikuwasha extract against the growth of A549 cells, isobolograms were prepared according to " Figs. 2 the procedure indicated in Materials and Methods. In l and 3, different concentrations of a mixture of PTX + CBDCA (molar ratio 1 : 2) were combined with the concentrations of NOB or powdered Shiikuwasha extract for the determination of antiproliferative activity against A549 cells. The X-axis is the ratio of the fixed concentration of NOB or powdered Shiikuwasha extract to the concentration of NOB or powdered Shiikuwasha extract that produced 50 % inhibition of cell growth (IC50) in the absence of PTX + CBDCA. The Y-axis is the ratio of the IC50 of PTX in a mixture of PTX + CBDCA (molar ratio, 1 : 2) in the presence of a fixed concentration of NOB or Shiikuwasha extract to the IC50 of PTX in a mixture of PTX + CBDCA (molar ratio, 1 : 2) in the absence of NOB or the Shiikuwasha extract. Generally, a concave isobologram indicates synergy, a convex isobologram reflects antagonism, and a straight line connecting the X-axis intercept to the Y-axis intercept represents additivity. The combination of PTX + " Fig. 2) and with the Shiikuwasha extract CBDCA with NOB (l " (l Fig. 2) showed a plotted curve below the straight line. Furthermore, combination indexes (CI) were calculated from these isobolograms, utilizing the equation [7, 19] given in Materials and Methods, and were less than 1. It was thus concluded that the combination of PTX + CBDCA and NOB as well as PTX + CBDCA and the Shiikuwasha extract generated a significant synergistic effect. Subsequently, our attention was directed to identify which
of the two drugs, PTX or CBDCA, is responsible for the synergistic effect with NOB or the Shiikuwasha extract. The effects of the following combinations were examined by preparing isobolograms in the same way as described above: PTX and NOB; PTX and Shiikuwasha extract; CBDCA and NOB; and CBDCA and Shiikuwasha extract. The combination of PTX and NOB as well as PTX and the Shiikuwasha extract actually generated a synergistic effect " Fig. 3), and their CIs were less than 1. In contrast, the combina(l tion of CBDCA and NOB as well as CBDCA and the Shiikuwasha extract gave only an additive effect, and their CIs were nearly 1. It was thus demonstrated that PTX, not CBDCA, was responsible for the synergistic effect with NOB or with the Shiikuwasha extract. These results were also the case for H460 cells. Subsequently, we compared the percentages of apoptotic cells among the cells incubated with PTX + CBDCA alone and among the cells incubated with PTX + CBDCA and NOB by Hoechst dye staining. " Fig. 4. The The results are depicted in the form of bar graphs in l percentages of apoptotic cells were decreased with the addition of NOB to PTX + CBDCA in both A549 and H460 cells. We also examined the effects of these compounds on the cell cycle distributions of A549 cells by flow cytometric analysis. In accordance with the aforementioned findings, the percentage of sub-G0/G1 phase cells decreased with the addition of NOB to PTX + CBDCA, while the population of G0/G1 cells increased as illustrated in " Fig. 5. l Uesato S et al. Synergistic Antitumor Effect …
Planta Med 2014; 80: 452–457
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Fig. 1 Structures of paclitaxel, carboplatin and nobiletin.
Original Papers
Fig. 3 IC50 isobolograms of two substances against A549 cells. The IC50 value of PTX or CBDCA alone is expressed as 1.0 on the Y-axis of the isobologram, while that of NOB or the Shiikuwasha extract alone is expressed as 1.0 on the X-axis.
" Fig. 7 A) and body weights (l " Fig. 7 B) were monitored umes (l over 30 days. The mice fed PTX + CBDCA (group 2) and NOB (group 3) exhibited suppressed tumor growth to 73 % and 60 %, respectively, relative to the control (group 1) at 30 day, whereas the mice (group 4) given PTX + CBDCA and NOB exhibited suppressed tumor growth to 41%. No groups showed a loss of body weight. However, of the eight mice in group 2, three died, relative to one death in group 4, presumably due to the toxicity of the chemotherapy drugs.
Discussion Fig. 4 Percentage ratios of the fragmented cell number to the total cell number. A549 or H460 cells were incubated for 24 h with 0.5 % DMSO, NOB, PTX + CBDCA, or PTX + CBDCA and NOB, and stained with Hoechst 33 342, as indicated in Materials and Methods.
The expression levels of CDK4, CDK2, and cyclin D1, key regulators of the G1/S transition, were observed by Western blotting " Fig. 6 A). Their levels were decreased with increasing rates of (l NOB to PTX + CBDCA. Furthermore, the anti-apoptotic protein Bcl-2 was increased, whereas the proapoptotic proteins cleaved caspase 3 and cleaved PARP were decreased with the addition of " Fig. 6 B). NOB to PTX + CBDCA (l In order to examine the combination effect of the cancer chemotherapy drugs and NOB in vivo, antitumor activity tests were conducted using xenografts of A549 cells in nude mice. PTX and CBDCA were intraperitoneally administered to mice every six days, whereas NOB was orally fed every other day. Tumor vol-
Uesato S et al. Synergistic Antitumor Effect …
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!
The growth of the NSCLC cell lines A549 and H460 was synergistically suppressed with the treatment of a combination of PTX, " Figs. 2 and 3). The percentage of apoptotic CBDCA, and NOB (l cells with fragmented nuclei was decreased with increasing ra" Fig. 4). This finding was also suptios of NOB to PTX-CBDCA (l ported by the observation of cells of the population of each phase: the percentage of sub-G0/G1 cells was reduced and, instead, that of G0/G1 was increased with the addition of NOB to " Fig. 5). In agreement with this finding, proapoPTX-CBDCA (l ptotic cleaved caspase 3 and cleaved PARP were decreased. Furthermore, the expression levels of the G1/S transition-related proteins (CDK4, CDK2, and cyclin D1) were decreased, whereas that of anti-apoptotic protein Bcl-2 was increased by the combi" Fig. 6). These results were exnation of NOB with PTX-CBDCA (l plicable by assuming that the cytostatic agent NOB caused the G1 cell cycle arrest of A549 or H460 cells, which protected the cells from initiation of the death pathways induced by PTX [20] and CBDCA. In in vivo experiments, we cannot confidently conclude
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Fig. 6 Western blot analysis of A549 cells. Cells were treated with 0.5 % DMSO, NOB (80 µM), PTX (80 nM) + CBDCA (160 nM), or PTX (80 nM) + CBDCA (160 nM) and NOB (20, 40, or 80 µM). Incubation was conducted (A) for 24 h in the detection of CDK2, CDK4, and cyclin D1; and (B) for 48 h in the detection of Bax, Bcl-2, cleaved PARP, and cleaved caspase 3.
Fig. 7 Antitumor experiments of PTX + CBDCA, NOB, and their combination using A549 cell xenografts in nude mice. (A) The effects of the compounds on tumor volumes, and (B) relative body weight (RBW) changes. The compounds were administered to mice as indicated in Materials and Methods. ◆: Control (group 1); &: PTX + CBDCA (group 2); ▲: NOB (group 3); and : PTX + CBDCA and NOB (group 4). Significant differences between tumor volumes: p < 0.05 (by two-tailed Studentʼs t-test) for each group vs. control on the following days: for group 2, days 8 and 30; for group 3, days 16, 18, 20, 24, 26, 28, and 30; and for group 4, all days except day 0.
•
Materials and Methods !
Cell lines, chemicals, and biochemicals that the treatment of PTX-CBDCA and NOB to the mice acted synergistically on inhibiting the growth of tumors in our administra" Fig. 7). However, the number of dead mice was tion protocol (l reduced from three to one by the addition of NOB to PTX-CBDCA; this is most likely due to their combination effect. We thus expect that the present study would be a basis for creating a new therapy that is effective in treating lung cancers.
A549 and H460 cell lines were purchased from American Tissue Culture Collection. PTX (> 97 % purity by HPLC) and ethoxylated hydrogenated castor oil (HCO-60) were obtained from Wako Pure Chemical Industries, Ltd., and CBDCA (> 90% purity by HPLC) was from TCI, Inc. NOB (> 95% purity by HPLC) and powdered Shiikuwasha extract [3.3 % NOB in a mixture of dried peel powder (15%) and dextrin (85%)] were supplied by South Product Co. Ltd.
Uesato S et al. Synergistic Antitumor Effect …
Planta Med 2014; 80: 452–457
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Fig. 5 Cell cycle analyses of A549 cells. Cells were treated with 0.5 % DMSO, NOB, PTX + CBDCA, or PTX + CBDCA and NOB for 24 h, as described in Materials and Methods. The percentages in the graphs represent the rates of cells of each phase to the total cells.
Original Papers
The cell counting kit for the WST-1 assay and Hoechst 33342 were obtained from Dojindo Molecular Technologies, Inc. Protease inhibitor, phosphatase inhibitor cocktails, and bovine serum albumin (BSA) were purchased from Nacarai Tesque, and the BCA protein assay kit was from Thermo Fisher Scientific. The Cycletest™ Plus DNA reagent kit (Catalog No. 340 242) was purchased from Becton, Dickinson and Company.
Cultivation, animals, and antibodies Cells were cultured in DMEM (Invitrogen Life Technologies) supplemented with 10 % FBS (Biowest), 50 µg/mL penicillin G, and 50 µg/mL streptomycin sulfate (Invitrogen Life Technologies) in a 5% CO2 and 95 % air atmosphere at 37 °C. Female BALB/c-nu.nu nude mice (4 weeks of age) were purchased from Japan SLC, Inc. Experimental protocols and animal care methods in the experiments were approved by the Experimental Animal Committee at Osaka University of Pharmaceutical Sciences on March 21, 2009 (approved number: 09035). The mice were used at 5 weeks of age. Antibodies specific to β-actin, cyclin D1, Bax, and Bcl-2 were purchased from Sigma-Aldrich, whereas those specific to CDK4 and CDK2 were from BioLegend. Anti-cleaved PARP and anti-cleaved caspase 3 were from Cell Signaling Technology, Inc.
Inhibition against the growth of A549 and H460 cell lines A549 or H460 cells (2 × 103/well) in DMEM supplemented with 10 % FBS and penicillin G-streptomycin sulfate were inoculated onto standard 96-well microtiter plates. Following overnight culture, serially diluted samples (PTX, CBDCA, NOB, powdered Shiikuwasha extract, or their various combinations) were added to the wells. After a 3-day culture, the cell growth rates were evaluated by the WST-1 assay and the IC50 values were calculated.
Isobologram and combination index analyses of the interaction between three substances in A549 cells The IC50 value of PTX in a mixture of PTX + CBDCA (molar ratio, 1 : 2) as well as those of NOB and powdered Shiikuwasha extract were as follows: 18.2 nM (PTX), 38.0 µM (NOB), and 0.15 mg/ml (equivalent to 12.4 µM NOB). Then, the IC50 values of PTX in a mixture of PTX + CBDCA (molar ratio 1 : 2) were measured with the addition of the respective concentrations of NOB (2.5, 5.0, 10.0, and 20.0 µM) or powdered Shiikuwasha extract (0.0125, 0.025, 0.05, and 0.1 mg/mL). The values thus obtained were plotted to make a line graph. A concave isobologram indicates synergy, a convex isobologram reflects antagonism, and a straight line connecting the X-axis intercept to the Y-axis intercept represents additivity. CIs were calculated utilizing the following reported procedure [7, 19]: CI ¼
CA;x CB;x þ ICx;A ICx;B
Isobologram and combination index analyses of the interaction between two substances in A549 cells The IC50 values of the respective substances were as follows: 36.0 µM (NOB); 0.21 mg/mL (equivalent to 17.3 µM NOB) (Shiikuwasha extract); 11.5 nM (PTX); and 170.7 µM (CBDCA). Then, the IC50 values of PTX or CBDCA were measured with the addition of the respective concentrations of NOB (2.5, 5.0, 10.0, and 20.0 µM) or Shiikuwasha extract (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml). The IC50 values thus obtained were plotted to make a line graph in the same way as above. The CI values at each concentration point of PTX and NOB were 0.62, 0.59, 0.59, and 0.79, whereas those of CBDCA and NOB were 1.0, 1.05, 1.01, and 0.97. CIs at each concentration of PTX and powdered Shiikuwasha extract were 0.62, 0.59, 0.59, and 0.79, whereas those of CBDCA and powdered Shiikuwasha extract were 1.0, 1.05, 1.01, and 0.97.
Apoptosis assay by Hoechst dye staining A549 or H460 cells (5.0 × 105/dish) in DMEM supplemented with 10 % FBS and penicillin G-streptomycin sulfate were inoculated onto a 60-mm dish. After incubation for 24 h, the medium was replaced with serum-free DMEM (5 mL), and the cells were incubated for another 24 h. The cells were then incubated with serum-free DMEM (5 mL) containing the following substances for 24 h: control (0.5 % DMSO); NOB (80 µM); PTX (80 nM) + CBDCA (160 nM); and PTX (80 nM) + CBDCA (160 nM) and NOB (20, 40, or 80 µM). The cells were washed with 1× PBS (1 mL × 2) and fixed with 4 % paraformaldehyde in 1× phosphate buffered saline (PBS) (1 mL) for 30 min at room temperature. The cells were again washed with 1× PBS (1 mL) and stained with Hoechst 33 342 (0.25 µg/mL) for 30 min at room temperature. After washing with 1× PBS (1 mL × 3), the stained cells were evaluated for apoptosis with a fluorescence microscope. The number of apoptotic cells per field of view was determined by examining five fields of view at × 40 magnification and is expressed as a percentage of the total number of cells.
Examination of cell cycle regulation by flow cytometry A549 cells (1.0 × 106/dish) in DMEM supplemented with 10 % FBS and penicillin G-streptomycin sulfate were plated onto a 60-mm dish. After incubation for 24 h, the cells were treated in the same way as described above [21]. The cells were incubated with the following substances for 24 h: control (0.5% DMSO); NOB (80 µM); PTX (80 nM) + CBDCA (160 nM); and PTX (80 nM) + CBDCA (160 nM) and NOB (80 µM). The adherent cells were treated with 0.25 % trypsin (Invitrogen Life Technologies) and combined with the floating cells. All the cells were treated with a Cycletest™ Plus DNA reagent kit. Results of flow cytometry were obtained with a FACSCanto™ II flow cytometer (Becton, Dickinson and Company).
Western blot analysis CA,x and CB,x are the concentrations of drug A and drug B used in combinations to achieve x% drug effect. ICx,A and ICx,B are the concentrations for single agents to achieve the same effect. A CI of less than, equal to, and more than 1 indicates synergy, additivity, and angatonism, respectively. The CI values calculated at each concentration point of PTX, CBDCA, and NOB were as follows: 0.69, 0.68, 0.72, and 0.80, whereas those of PTX, CBDCA, and powdered Shiikuwasha extract were as follows: 0.99, 0.87, 0.71, and 0.84.
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A549 cells (1.0 × 106/dish) in DMEM supplemented with 10 % FBS and penicillin G-streptomycin sulfate were plated onto a 60-mm dish. The cells were treated with the testing compounds as mentioned above, and incubated for 24 h in the detection of CDK2, CDK4, and cyclin D1, and for 48 h in the detection of Bax, Bcl-2, cleaved PARP, and cleaved caspase 3. The floating and adherent cells were washed twice with PBS (each 1 mL), scraped, and resuspended in 200 µL of a Covance Laboratories lysis buffer (1 % Nonidet P-40, protease inhibitor, and phosphatase inhibitor cocktails were adopted in place of 1% Triton-X 100, aprotinin, leupeptin, and pepstatin). The lysates were centrifuged at 15 000 rpm
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for 15 min at 4 °C. The protein concentration was determined with the BCA protein assay kit. An equal amount of protein was then resolved by SDS-PAGE and transferred to a PVDF membrane. The blots (1× Tris-HCl buffer saline, 5% nonfat milk, and 0.05 % Tween 20) were probed with an antibody specific to each protein and detected using ECL chemiluminescence.
In vivo antitumor activity A549 cells (2.0 × 106/100 µL medium) were injected subcutaneously into two points of the back of each nude mouse (5 weeks of age). After tumor size reached ca. 100 mm3, the mice were divided into four groups, and the following compounds were administered to each mouse: Group 1 (9 mice; 13 tumors, control): HCO-60 and PBS (9 : 1) (200 µL), and aqueous dextrin (300 µL); group 2 (of 8 mice, 3 died; 8 tumors): PTX (300 µg) + CBDCA (600 µg) in HCO-60 and PBS (9 : 1) (200 µL) and aqueous dextrin (300 µL); group 3 (7 mice; 11 tumors): HCO-60 and PBS (9 : 1) (200 µL), and NOB (600 µg, 3% in dextrin) (300 µL); and group 4 (of 8 mice, 1 died; 12 tumors): PTX (300 µg) + CBDCA (600 µg) in HCO-60 and PBS (9 : 1) (200 µL) and NOB (600 µg, 3 % in dextrin) (300 µL). An emulsion of PTX (300 µg) + CBDCA (600 µg) in HCO60 and PBS (9 : 1) (200 µL) or HCO-60 and PBS (9 : 1) (200 µL) alone was given to the mice intraperitoneally every six days, whereas a suspension of NOB (600 µg, 3 % in dextrin) (300 µL) or aqueous dextrin (300 µL) alone was fed orally every other day over thirty days. Tumor length and width as well as body weight were monitored. Tumor volume (mm3) was calculated by measuring the tumor length and width (in mm) as described previously [22].
Statistical analysis The statistical significance of the effects of the drugs versus control was analyzed by the Studentʼs t-test.
Supporting information Isobolograms of the combination of PTX + CBDCA with NOB or with Shiikuwasha extract in H460 cells as well as cell cycle distributions of H460 cells by flow cytometric analysis are available as Supporting Information.
Acknowledgements !
Part of this work was supported by the Strategic Project to Support the Formation of Research Bases at Private Universities (2013–2017). We thank South Product Co. Ltd. for the kind supply of NOB and powdered Shiikuwasha extract.
Conflict of Interest !
The authors declare no conflict of interest.
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Synergistic Antitumor Effect of a Combination of Paclitaxel and Carboplatin with Nobiletin from Citrus depressa on Non-Small-Cell Lung Cancer Cell Lines
Authors
Shinichi Uesato 1, Hirofumi Yamashita 1, Ryu Maeda 1, Yoshiyuki Hirata 1, Maho Yamamoto 1, Saki Matsue 1, Yasuo Nagaoka 1, Makio Shibano 2, Masahiko Taniguchi 2, Kimiye Baba 2, Motoharu Ju-ichi 3
Affiliations
1
2 3
Key words " nobiletin l " Citrus depressa l " Rutaceae l " non‑small‑cell lung cancer l " cancer chemotherapy drug l " synergistic effect l " A549 cell xenograft l
received revised accepted
January 20, 2014 February 28, 2014 March 1, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368321 Published online March 31, 2014 Planta Med 2014; 80: 452–457 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Shinichi Uesato Department of Life Science and Biotechnology Faculty of Chemistry Materials and Bioengineering Kansai University 3-3-35 Yamate-cho Suita, Osaka 564–8680 Japan Phone: + 81 6 63 68 08 34 Fax: + 81 6 63 88 86 09 [email protected]
Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, Suita, Osaka, Japan Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka, Japan Faculty of Pharmaceutical Sciences, Mukogawa Womenʼs University, Nishinomiya, Hyogo, Japan
Abstract
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Non-small-cell lung carcinomas do not sufficiently respond to cancer chemotherapeutic drugs. Combination effects of cancer chemotherapy drugs (paclitaxel and carboplatin) with nobiletin or powdered Shiikuwasha extract from Citrus depressa were examined by isobologram and combination index analyses. It was demonstrated that the combination generated a synergistic inhibitory effect against the proliferation of the human non-small-cell lung carcinoma cell lines A549 and H460 and that of the two chemotherapy drugs, paclitaxel was responsible for this synergistic effect. Furthermore, the percentage of apoptotic cells was decreased with increasing rates of nobiletin to paclitaxel and carboplatin. These findings were considered to be attributed to the ability of nobiletin to regulate cells in the G1 phase, which escaped cell death initiated by paclitaxel and carboplatin. An antitumor activity assay showed that this combination significantly suppressed the growth of subcutaneous A549 tumor xenografts in nude mice.
SCLC: NSCLC: PTX: CBDCA: A549:
Introduction !
Lung cancer is classified into two types, small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC). NSCLC accounts for 70–80 % of all cases of lung cancer in Japan. SCLC usually responds better to chemotherapy and radiotherapy than NSCLC. NSCLC is primarily treated by surgical operation and then by chemotherapy to prevent recurrence. Recently, molecular-targeted drugs such as gefitinib (Iressa) [1] and crizotinib (Xalkori) [2] have been applied as a first-line therapy for NSCLC patients with a mutated epidermal growth factor receptor (EGFR) [3] and with a EML4-ALK fusion gene [4], respectively. About 50 % of other NSCLC patients receive chemother-
Uesato S et al. Synergistic Antitumor Effect …
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H460: HCO-60: BSA: DMEM: FBS: PBS: CI:
small-cell lung adenocarcinoma non-small-cell lung adenocarcinoma paclitaxel carboplatin NOB: nobiletin human non-small-cell lung adenocarcinoma cell line human non-small-cell lung adenocarcinoma cell line ethoxylated hydrogenated castor oil bovine serum albumin Dulbeccoʼs modified Eagleʼs medium fetal bovine serum phosphate buffered saline combination index
Supporting information available online at http://www.thieme-connect.de/ejournals/toc/ plantamedica
apy with paclitaxel (PTX) and carboplatin (CBDCA) [5, 6]. However, these drugs produce serious side effects such as leukopenia, hepatic dysfunction, deterioration of renal function, vomiting, and nausea. These side effects are most commonly attributed to tissue necrosis, apoptosis, and inflammation caused by the chemotherapy treatment. One approach gaining a better therapeutic effect could be a multicomponent therapy, which involves synergistic interactions [7]. Thus, it is of great clinical significance to seek a third drug that has synergistic anticancer activity with PTX and CBDCA and thus allows the dose " Fig. 1). reduction of these chemotherapy drugs (l Citrus depressa Hayata (Rutaceae) is a citrus plant grown naturally in Okinawa Prefecture, Japan.
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The fruit is very sour like a lemon, and is used as a seasoning and for fruit juices; it is called “Shiikuwasha” locally. The fruit has reportedly beneficial activities on human health, such as anticancer, anti-inflammatory, and antihyperglycemic effects [8– 12]. The components responsible for these activities are polymethoxylated flavonoids that are mainly contained in the peel of the fruit. We have focused on nobiletin (NOB), the major component of flavonoids, because NOB inhibits the proliferation of cancer cells through the arrest of their cell cycle in the G1 phase [13]. Furthermore, it was reported that NOB showed synergistic inhibition with doxorubicin [14] and cis-diamminedichloride platinum [15] against the growth of human breast adenocarcinoma cell line MCF-7 and gastric cancer cells, respectively. In the present work, we investigated how NOB influenced the growth of the human NSCLC cell lines A549 and H460 in combination with PTX and CBDCA using isobologram and combination index analyses [7, 16–19]. Furthermore, we examined antitumor activities against cancer cell xenografts in nude mice.
Fig. 2 IC50 isobolograms of three substances against A549 cells. The IC50 value of PTX in a mixture PTX + CBDCA (molar ratio 1 : 2) is expressed as 1.0 on the Y-axis of the isobologram, while that of NOB or the Shiikuwasha extract alone is expressed as 1.0 on the Xaxis.
Results !
In order to assess the combination effects of the chemotherapy drugs with NOB or powdered Shiikuwasha extract against the growth of A549 cells, isobolograms were prepared according to " Figs. 2 the procedure indicated in Materials and Methods. In l and 3, different concentrations of a mixture of PTX + CBDCA (molar ratio 1 : 2) were combined with the concentrations of NOB or powdered Shiikuwasha extract for the determination of antiproliferative activity against A549 cells. The X-axis is the ratio of the fixed concentration of NOB or powdered Shiikuwasha extract to the concentration of NOB or powdered Shiikuwasha extract that produced 50 % inhibition of cell growth (IC50) in the absence of PTX + CBDCA. The Y-axis is the ratio of the IC50 of PTX in a mixture of PTX + CBDCA (molar ratio, 1 : 2) in the presence of a fixed concentration of NOB or Shiikuwasha extract to the IC50 of PTX in a mixture of PTX + CBDCA (molar ratio, 1 : 2) in the absence of NOB or the Shiikuwasha extract. Generally, a concave isobologram indicates synergy, a convex isobologram reflects antagonism, and a straight line connecting the X-axis intercept to the Y-axis intercept represents additivity. The combination of PTX + " Fig. 2) and with the Shiikuwasha extract CBDCA with NOB (l " (l Fig. 2) showed a plotted curve below the straight line. Furthermore, combination indexes (CI) were calculated from these isobolograms, utilizing the equation [7, 19] given in Materials and Methods, and were less than 1. It was thus concluded that the combination of PTX + CBDCA and NOB as well as PTX + CBDCA and the Shiikuwasha extract generated a significant synergistic effect. Subsequently, our attention was directed to identify which
of the two drugs, PTX or CBDCA, is responsible for the synergistic effect with NOB or the Shiikuwasha extract. The effects of the following combinations were examined by preparing isobolograms in the same way as described above: PTX and NOB; PTX and Shiikuwasha extract; CBDCA and NOB; and CBDCA and Shiikuwasha extract. The combination of PTX and NOB as well as PTX and the Shiikuwasha extract actually generated a synergistic effect " Fig. 3), and their CIs were less than 1. In contrast, the combina(l tion of CBDCA and NOB as well as CBDCA and the Shiikuwasha extract gave only an additive effect, and their CIs were nearly 1. It was thus demonstrated that PTX, not CBDCA, was responsible for the synergistic effect with NOB or with the Shiikuwasha extract. These results were also the case for H460 cells. Subsequently, we compared the percentages of apoptotic cells among the cells incubated with PTX + CBDCA alone and among the cells incubated with PTX + CBDCA and NOB by Hoechst dye staining. " Fig. 4. The The results are depicted in the form of bar graphs in l percentages of apoptotic cells were decreased with the addition of NOB to PTX + CBDCA in both A549 and H460 cells. We also examined the effects of these compounds on the cell cycle distributions of A549 cells by flow cytometric analysis. In accordance with the aforementioned findings, the percentage of sub-G0/G1 phase cells decreased with the addition of NOB to PTX + CBDCA, while the population of G0/G1 cells increased as illustrated in " Fig. 5. l Uesato S et al. Synergistic Antitumor Effect …
Planta Med 2014; 80: 452–457
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Fig. 1 Structures of paclitaxel, carboplatin and nobiletin.
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Fig. 3 IC50 isobolograms of two substances against A549 cells. The IC50 value of PTX or CBDCA alone is expressed as 1.0 on the Y-axis of the isobologram, while that of NOB or the Shiikuwasha extract alone is expressed as 1.0 on the X-axis.
" Fig. 7 A) and body weights (l " Fig. 7 B) were monitored umes (l over 30 days. The mice fed PTX + CBDCA (group 2) and NOB (group 3) exhibited suppressed tumor growth to 73 % and 60 %, respectively, relative to the control (group 1) at 30 day, whereas the mice (group 4) given PTX + CBDCA and NOB exhibited suppressed tumor growth to 41%. No groups showed a loss of body weight. However, of the eight mice in group 2, three died, relative to one death in group 4, presumably due to the toxicity of the chemotherapy drugs.
Discussion Fig. 4 Percentage ratios of the fragmented cell number to the total cell number. A549 or H460 cells were incubated for 24 h with 0.5 % DMSO, NOB, PTX + CBDCA, or PTX + CBDCA and NOB, and stained with Hoechst 33 342, as indicated in Materials and Methods.
The expression levels of CDK4, CDK2, and cyclin D1, key regulators of the G1/S transition, were observed by Western blotting " Fig. 6 A). Their levels were decreased with increasing rates of (l NOB to PTX + CBDCA. Furthermore, the anti-apoptotic protein Bcl-2 was increased, whereas the proapoptotic proteins cleaved caspase 3 and cleaved PARP were decreased with the addition of " Fig. 6 B). NOB to PTX + CBDCA (l In order to examine the combination effect of the cancer chemotherapy drugs and NOB in vivo, antitumor activity tests were conducted using xenografts of A549 cells in nude mice. PTX and CBDCA were intraperitoneally administered to mice every six days, whereas NOB was orally fed every other day. Tumor vol-
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The growth of the NSCLC cell lines A549 and H460 was synergistically suppressed with the treatment of a combination of PTX, " Figs. 2 and 3). The percentage of apoptotic CBDCA, and NOB (l cells with fragmented nuclei was decreased with increasing ra" Fig. 4). This finding was also suptios of NOB to PTX-CBDCA (l ported by the observation of cells of the population of each phase: the percentage of sub-G0/G1 cells was reduced and, instead, that of G0/G1 was increased with the addition of NOB to " Fig. 5). In agreement with this finding, proapoPTX-CBDCA (l ptotic cleaved caspase 3 and cleaved PARP were decreased. Furthermore, the expression levels of the G1/S transition-related proteins (CDK4, CDK2, and cyclin D1) were decreased, whereas that of anti-apoptotic protein Bcl-2 was increased by the combi" Fig. 6). These results were exnation of NOB with PTX-CBDCA (l plicable by assuming that the cytostatic agent NOB caused the G1 cell cycle arrest of A549 or H460 cells, which protected the cells from initiation of the death pathways induced by PTX [20] and CBDCA. In in vivo experiments, we cannot confidently conclude
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Fig. 6 Western blot analysis of A549 cells. Cells were treated with 0.5 % DMSO, NOB (80 µM), PTX (80 nM) + CBDCA (160 nM), or PTX (80 nM) + CBDCA (160 nM) and NOB (20, 40, or 80 µM). Incubation was conducted (A) for 24 h in the detection of CDK2, CDK4, and cyclin D1; and (B) for 48 h in the detection of Bax, Bcl-2, cleaved PARP, and cleaved caspase 3.
Fig. 7 Antitumor experiments of PTX + CBDCA, NOB, and their combination using A549 cell xenografts in nude mice. (A) The effects of the compounds on tumor volumes, and (B) relative body weight (RBW) changes. The compounds were administered to mice as indicated in Materials and Methods. ◆: Control (group 1); &: PTX + CBDCA (group 2); ▲: NOB (group 3); and : PTX + CBDCA and NOB (group 4). Significant differences between tumor volumes: p < 0.05 (by two-tailed Studentʼs t-test) for each group vs. control on the following days: for group 2, days 8 and 30; for group 3, days 16, 18, 20, 24, 26, 28, and 30; and for group 4, all days except day 0.
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Materials and Methods !
Cell lines, chemicals, and biochemicals that the treatment of PTX-CBDCA and NOB to the mice acted synergistically on inhibiting the growth of tumors in our administra" Fig. 7). However, the number of dead mice was tion protocol (l reduced from three to one by the addition of NOB to PTX-CBDCA; this is most likely due to their combination effect. We thus expect that the present study would be a basis for creating a new therapy that is effective in treating lung cancers.
A549 and H460 cell lines were purchased from American Tissue Culture Collection. PTX (> 97 % purity by HPLC) and ethoxylated hydrogenated castor oil (HCO-60) were obtained from Wako Pure Chemical Industries, Ltd., and CBDCA (> 90% purity by HPLC) was from TCI, Inc. NOB (> 95% purity by HPLC) and powdered Shiikuwasha extract [3.3 % NOB in a mixture of dried peel powder (15%) and dextrin (85%)] were supplied by South Product Co. Ltd.
Uesato S et al. Synergistic Antitumor Effect …
Planta Med 2014; 80: 452–457
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Fig. 5 Cell cycle analyses of A549 cells. Cells were treated with 0.5 % DMSO, NOB, PTX + CBDCA, or PTX + CBDCA and NOB for 24 h, as described in Materials and Methods. The percentages in the graphs represent the rates of cells of each phase to the total cells.
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The cell counting kit for the WST-1 assay and Hoechst 33342 were obtained from Dojindo Molecular Technologies, Inc. Protease inhibitor, phosphatase inhibitor cocktails, and bovine serum albumin (BSA) were purchased from Nacarai Tesque, and the BCA protein assay kit was from Thermo Fisher Scientific. The Cycletest™ Plus DNA reagent kit (Catalog No. 340 242) was purchased from Becton, Dickinson and Company.
Cultivation, animals, and antibodies Cells were cultured in DMEM (Invitrogen Life Technologies) supplemented with 10 % FBS (Biowest), 50 µg/mL penicillin G, and 50 µg/mL streptomycin sulfate (Invitrogen Life Technologies) in a 5% CO2 and 95 % air atmosphere at 37 °C. Female BALB/c-nu.nu nude mice (4 weeks of age) were purchased from Japan SLC, Inc. Experimental protocols and animal care methods in the experiments were approved by the Experimental Animal Committee at Osaka University of Pharmaceutical Sciences on March 21, 2009 (approved number: 09035). The mice were used at 5 weeks of age. Antibodies specific to β-actin, cyclin D1, Bax, and Bcl-2 were purchased from Sigma-Aldrich, whereas those specific to CDK4 and CDK2 were from BioLegend. Anti-cleaved PARP and anti-cleaved caspase 3 were from Cell Signaling Technology, Inc.
Inhibition against the growth of A549 and H460 cell lines A549 or H460 cells (2 × 103/well) in DMEM supplemented with 10 % FBS and penicillin G-streptomycin sulfate were inoculated onto standard 96-well microtiter plates. Following overnight culture, serially diluted samples (PTX, CBDCA, NOB, powdered Shiikuwasha extract, or their various combinations) were added to the wells. After a 3-day culture, the cell growth rates were evaluated by the WST-1 assay and the IC50 values were calculated.
Isobologram and combination index analyses of the interaction between three substances in A549 cells The IC50 value of PTX in a mixture of PTX + CBDCA (molar ratio, 1 : 2) as well as those of NOB and powdered Shiikuwasha extract were as follows: 18.2 nM (PTX), 38.0 µM (NOB), and 0.15 mg/ml (equivalent to 12.4 µM NOB). Then, the IC50 values of PTX in a mixture of PTX + CBDCA (molar ratio 1 : 2) were measured with the addition of the respective concentrations of NOB (2.5, 5.0, 10.0, and 20.0 µM) or powdered Shiikuwasha extract (0.0125, 0.025, 0.05, and 0.1 mg/mL). The values thus obtained were plotted to make a line graph. A concave isobologram indicates synergy, a convex isobologram reflects antagonism, and a straight line connecting the X-axis intercept to the Y-axis intercept represents additivity. CIs were calculated utilizing the following reported procedure [7, 19]: CI ¼
CA;x CB;x þ ICx;A ICx;B
Isobologram and combination index analyses of the interaction between two substances in A549 cells The IC50 values of the respective substances were as follows: 36.0 µM (NOB); 0.21 mg/mL (equivalent to 17.3 µM NOB) (Shiikuwasha extract); 11.5 nM (PTX); and 170.7 µM (CBDCA). Then, the IC50 values of PTX or CBDCA were measured with the addition of the respective concentrations of NOB (2.5, 5.0, 10.0, and 20.0 µM) or Shiikuwasha extract (0.0125, 0.025, 0.05, 0.1, and 0.2 mg/ml). The IC50 values thus obtained were plotted to make a line graph in the same way as above. The CI values at each concentration point of PTX and NOB were 0.62, 0.59, 0.59, and 0.79, whereas those of CBDCA and NOB were 1.0, 1.05, 1.01, and 0.97. CIs at each concentration of PTX and powdered Shiikuwasha extract were 0.62, 0.59, 0.59, and 0.79, whereas those of CBDCA and powdered Shiikuwasha extract were 1.0, 1.05, 1.01, and 0.97.
Apoptosis assay by Hoechst dye staining A549 or H460 cells (5.0 × 105/dish) in DMEM supplemented with 10 % FBS and penicillin G-streptomycin sulfate were inoculated onto a 60-mm dish. After incubation for 24 h, the medium was replaced with serum-free DMEM (5 mL), and the cells were incubated for another 24 h. The cells were then incubated with serum-free DMEM (5 mL) containing the following substances for 24 h: control (0.5 % DMSO); NOB (80 µM); PTX (80 nM) + CBDCA (160 nM); and PTX (80 nM) + CBDCA (160 nM) and NOB (20, 40, or 80 µM). The cells were washed with 1× PBS (1 mL × 2) and fixed with 4 % paraformaldehyde in 1× phosphate buffered saline (PBS) (1 mL) for 30 min at room temperature. The cells were again washed with 1× PBS (1 mL) and stained with Hoechst 33 342 (0.25 µg/mL) for 30 min at room temperature. After washing with 1× PBS (1 mL × 3), the stained cells were evaluated for apoptosis with a fluorescence microscope. The number of apoptotic cells per field of view was determined by examining five fields of view at × 40 magnification and is expressed as a percentage of the total number of cells.
Examination of cell cycle regulation by flow cytometry A549 cells (1.0 × 106/dish) in DMEM supplemented with 10 % FBS and penicillin G-streptomycin sulfate were plated onto a 60-mm dish. After incubation for 24 h, the cells were treated in the same way as described above [21]. The cells were incubated with the following substances for 24 h: control (0.5% DMSO); NOB (80 µM); PTX (80 nM) + CBDCA (160 nM); and PTX (80 nM) + CBDCA (160 nM) and NOB (80 µM). The adherent cells were treated with 0.25 % trypsin (Invitrogen Life Technologies) and combined with the floating cells. All the cells were treated with a Cycletest™ Plus DNA reagent kit. Results of flow cytometry were obtained with a FACSCanto™ II flow cytometer (Becton, Dickinson and Company).
Western blot analysis CA,x and CB,x are the concentrations of drug A and drug B used in combinations to achieve x% drug effect. ICx,A and ICx,B are the concentrations for single agents to achieve the same effect. A CI of less than, equal to, and more than 1 indicates synergy, additivity, and angatonism, respectively. The CI values calculated at each concentration point of PTX, CBDCA, and NOB were as follows: 0.69, 0.68, 0.72, and 0.80, whereas those of PTX, CBDCA, and powdered Shiikuwasha extract were as follows: 0.99, 0.87, 0.71, and 0.84.
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A549 cells (1.0 × 106/dish) in DMEM supplemented with 10 % FBS and penicillin G-streptomycin sulfate were plated onto a 60-mm dish. The cells were treated with the testing compounds as mentioned above, and incubated for 24 h in the detection of CDK2, CDK4, and cyclin D1, and for 48 h in the detection of Bax, Bcl-2, cleaved PARP, and cleaved caspase 3. The floating and adherent cells were washed twice with PBS (each 1 mL), scraped, and resuspended in 200 µL of a Covance Laboratories lysis buffer (1 % Nonidet P-40, protease inhibitor, and phosphatase inhibitor cocktails were adopted in place of 1% Triton-X 100, aprotinin, leupeptin, and pepstatin). The lysates were centrifuged at 15 000 rpm
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for 15 min at 4 °C. The protein concentration was determined with the BCA protein assay kit. An equal amount of protein was then resolved by SDS-PAGE and transferred to a PVDF membrane. The blots (1× Tris-HCl buffer saline, 5% nonfat milk, and 0.05 % Tween 20) were probed with an antibody specific to each protein and detected using ECL chemiluminescence.
In vivo antitumor activity A549 cells (2.0 × 106/100 µL medium) were injected subcutaneously into two points of the back of each nude mouse (5 weeks of age). After tumor size reached ca. 100 mm3, the mice were divided into four groups, and the following compounds were administered to each mouse: Group 1 (9 mice; 13 tumors, control): HCO-60 and PBS (9 : 1) (200 µL), and aqueous dextrin (300 µL); group 2 (of 8 mice, 3 died; 8 tumors): PTX (300 µg) + CBDCA (600 µg) in HCO-60 and PBS (9 : 1) (200 µL) and aqueous dextrin (300 µL); group 3 (7 mice; 11 tumors): HCO-60 and PBS (9 : 1) (200 µL), and NOB (600 µg, 3% in dextrin) (300 µL); and group 4 (of 8 mice, 1 died; 12 tumors): PTX (300 µg) + CBDCA (600 µg) in HCO-60 and PBS (9 : 1) (200 µL) and NOB (600 µg, 3 % in dextrin) (300 µL). An emulsion of PTX (300 µg) + CBDCA (600 µg) in HCO60 and PBS (9 : 1) (200 µL) or HCO-60 and PBS (9 : 1) (200 µL) alone was given to the mice intraperitoneally every six days, whereas a suspension of NOB (600 µg, 3 % in dextrin) (300 µL) or aqueous dextrin (300 µL) alone was fed orally every other day over thirty days. Tumor length and width as well as body weight were monitored. Tumor volume (mm3) was calculated by measuring the tumor length and width (in mm) as described previously [22].
Statistical analysis The statistical significance of the effects of the drugs versus control was analyzed by the Studentʼs t-test.
Supporting information Isobolograms of the combination of PTX + CBDCA with NOB or with Shiikuwasha extract in H460 cells as well as cell cycle distributions of H460 cells by flow cytometric analysis are available as Supporting Information.
Acknowledgements !
Part of this work was supported by the Strategic Project to Support the Formation of Research Bases at Private Universities (2013–2017). We thank South Product Co. Ltd. for the kind supply of NOB and powdered Shiikuwasha extract.
Conflict of Interest !
The authors declare no conflict of interest.
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