213 12 33 12 TUMOR Vol. 33, December 213 www.tumorsci.org 161 Basic Research DOI: 1.3781/j.issn.1-7431.213.12. 5 Copyright 213 by TUMOR A549/DDP 1, 2 2 1 3 4 1 1 1. 3619 2. 355 3. 3611 4. 361 elemene cisplatin DDP A549/DDP MTT DDP A549/DDP Hoechst 33342 FCM C cytochrome C cyt C pro-caspase-3 caspase-3 B -2 B cell lymphoma-2 Bcl-2 A549/DDP - DDP A549/DDP DDP Hoechst 33342 FCM DDP A549/DDP A549/DDP cyt C caspase-3 Bad pro-caspase-3 Bcl-2 A549/DDP cyt C caspase-3 Bad Bcl-2 A549/DDP R734.2 R73.53 A 1-7431 (213) 12-161-8 The effect of elemene reversing the multidurg resistance of A549/DDP lung cancer cells YAO Cheng-cai 1, 2, TU Yuan-rong 2, DU Hao-xin 1, JIANG Jie 3, YE She-fang 4, ZHANG Yi 1, CHEN Jun-peng 1 1. Department of Thoracic Surgery, Traditional Chinese Medicine Hospital Affiliated to Fujian University of Traditional Chinese Medicine, Xiamen 3619, Fujian Province, China; 2. Department of Thoracic Surgery, First Affiliated Hospital of Fujian Medical University, Fuzhou 355, Fujian Province, China; 3. Department of Thoracic Surgery, First Affiliated Hospital of Xiamen University, Xiamen 3611, Fujian Province, China; 4. College of Molecular Biology and Material, Xiamen University, Xiamen 361, Fujian Province, China To investigate the effect of elemene on the drug resistance of cisplatin (DDP)- resistant human lung adenocarcinoma A549/DDP cells, and its possible mechanism. The growth inhibition of A549/DDP cells treated with elemene alone or combined with different concentrations of DDP was detected by MTT assay. The morphological changes of apoptosis of A549/DDP cells treated with different concentrations of elemene (2 and 4 μg/ml) for 24 h were observed by Hoechst 33342 staing under fluorescence a microscope. The apoptosis rate of A549/DDP cells treated with different concentrations of elemene (2 and 4 μg/ml) after 24 h were detected by FCM (flow cytometry). The expressions of cytochrome C, pro-caspase-3, caspase-3 and the Bcl-2 family proteins were measured by Western blotting analysis. MTT result showed that different concentrations elemene could inhibit the proliferation of A549/DDP cells in a time- and dose- dependent manner. Intriguingly, elemene plus DDP enhanced the sensitivity of A549/ DDP cells to DDP and reversed the resistance of A549/DDP cells. Elemene was also a strong inducer of apoptosis in this model system, and a synergistic effect on induction of cell death was observed when the tumor cells were treated with both agents. The result showed that elemene could enhance A549/DDP cell apoptosis. Furthermore, the combination of elemene and DDP also enhanced the protein expressions of cytochrome C, caspase-3 and Bad, and reduced the protein levels of Bcl-2 and pro-caspase-3 in the A549/ 21D14 Correspondence to: YAO Cheng-cai E-mail: yao-13@163.com Received 213-8-19 Accepted 213-9-27
162. A549/DDP DDP cells. The reversal of the multi-drug resistance of A549/DDP cell line by elemene may result from its effect on induction of apoptosis. Elemene treatment can impaire mitochondrial membrane, release cytochrome C into cytoplasm, activate caspase-3 up-regulate pro-apoptotic protein Bad and down-regulate anti-apoptotic protein Bcl-2, and finally caused apoptosis. Lung neoplasms; Drug resietance, neoplasms; Apoptosis; Elemene; A549/DDP cells [TUMOR, 213, 33 (12): 161-168] 2/3 elemene α- β- γ- δ- 4 β- [1] [2] [3] [4] [5] [6, 7] cisplatin DDP A549/DDP 1 1.1 DDP A549/DDP A549 DDP 922 β- 81152 C cytochrome C cyt C caspase-3 B -2 B cell lymphoma-2 Bcl-2 Bad pro-caspase-3 horseradish peroxidase HRP IgG β-actin Santa Cruz propidium iodide PI Annexin V-FITC/PI Hoechst 33342 MTT RPMI 164 permeability transition pore PTP A cyclosporine A caspase-3 Ac-DEVD-CHO Sigma Bio-Rad OLYMPUS IX71 Olympus AE31/CCIS Moltic Eppendorf 584R Eppendorf FACS Calibur BD 1.2 1.2.1 A549 A549/DDP 2 μg/ml DDP 1% RPMI 164 37 CO 2 5% 2 d 1 1 A549/DDP DDP 1.2.2 A549 A549/DDP DDP A549 A549/DDP 3 1% RPMI 164 5 1 3 / 96 DDP.25.5 1 2 4 8 16 32 μg/ml 24 h 3 1.2.3 MTT A549/DDP A549/DDP 1% RPMI 164 5 1 3 / 96 1 2 4 8 μg/ml 1% RPMI 164 RPMI 164 3 12 24 48 72 h 2 μl MTT 5 mg/ml 4 h 191 g 3 min 15 μl DMSO 1 min 57 nm D 63 nm 3 3 D / D 1% 95%
. A549/DDP 163 1.2.4 MTT A549/DDP A549/DDP 1% RPMI 164 5 1 3 / 96.25.5 1 2 4 8 16 32 μg/ml DDP 2 μg/ml 24 h 2 μl MTT 5 mg/ml 4 h 191 g 3 min 15 μl 1 min 57 nm D 63 nm 3 3 3 1 D / D 1% 5% DDP half inhibitory concentration IC 5 IC 5 / IC 5 IC 5 / IC 5 1.2.5 A549/DDP A549/DDP 1% RPMI 164 5 1 3 / 96 A549/DDP 2 μg/ml DDP 2 4 μg/ml 3 24 h DNA Hoechst 33342 FCM 24 h A549/DDP 76 g 5 min PBS 2 2 1 5 5 μl Binding Buffer Annexin V-FITC 5 μl PI 2 μl 5 15 min Binding Buffer 25 μl 3 1 h Ex 488 nm Em 525 nm Annexin V-FITC PI Cell Quest Pro 3 1.2.6 A549/DDP cyt C caspase-3 Bcl-2 A549/DDP 2 3 3 2 μg/ml DDP 2 4 μg/ml 24 h A549/DDP PBS 3 15 min 4 32 116 g 1 min 5 μg 12% SDS-PAGE 5% 2 h TBST [ cyt C 1 8 caspase-3 1 1 Bcl-2 1 1 Bad 1 1 pro-caspase-3 1 1 β-actin 1 2 ] 4 TBST HRP IgG 1 2 2 h 1.2.7 A Ac-DEVD-CHO A549/DDP caspase-3 A549/DDP 4 A549/DDP 2 μg/ml DDP [ 4 μg/ml ] Ac-DEVD-CHO [ 4 μg/ml Ac-DEVD- CHO 5 μmol/l ] A [ 4 μg/ml A 2 μmol/l ] 3 3 24 h A549/ DDP PBS 3 15 min 32 116 g 1 min 5 μg 12% SDS-PAGE 5% 2 h TBST caspase-3 1 1 ) β-actin 1 2 4 TBST HRP IgG 1 2 2 h
164. A549/DDP 1.3 SPSS 11.5 ± s t χ 2 P.5 2 2.1 A549 A549/DDP DDP DDP.25.5 1 2 4 8 16 32 μg/ ml A549 A549/DDP 24 h DDP A549 IC 5 5.73 2.11 μg/ml A549/DDP IC 5 15.34 1.5 μg/ml A549/DDP DDP 1 2.2 A549/DDP MTT A549/DDP 1 2 4 8 μg/ml A549/DDP 24 h 96.11% 96.3% 85.72% 76.59% 2 4 μg/ml χ 2 2.625 P.5 A549/DDP 48 h 95.2% 93.42% 7.89% 61.42% 2 4 μg/ml χ 2 2.145 P.5 2 μg/ml A549/DDP 24 48 h 96.11% vs 95.2% χ 2 27.463 P.5 4 μg/ml 24 48 h 85.72% vs 7.89% χ 2 2.414 P.5 2 2 μg/ml A549/DDP 24 h A549/DDP 2.3 A549/DDP MTT DDP.25.5 1 2 4 8 16 32 μg/ml 2 μg/ml 24 h DDP A549/DDP P.5 A549/DDP DDP IC 5 4.15.89 μg/ml 15.46 1.23 μg/ml t 1.432 P.1 2 μg/ml 3.73.38 1 Cell viability/% 12 1 8 6 4 2.25.5 1 2 4 DDP/(μg ml -1 ) 8 A549 A549/DDP Fig. 1 The growth inhibitory effects of different concentrations of DDP on A549 and A549/DDP cells. 1 DDP A549 A549/DDP Cell viability/% Cell viability/% 1.2 1..8.6.4.2. 1.2 1..8.6.4.2. h 12 h 24 h 48 h 72 h 1 2 4 Elemene/(μg ml -1 ) Elemene/(μg ml -1 ) 1 2 4 8 12 Fig. 2 The growth inhibitory effects of different concentrations of elemene on A549/DDP cells for 12, 24, 48 and 72 h. 2 A549/DDP 12 24 48 72 h 24 t/h 48 16 8 72 32
. A549/DDP 165 1 2 μg/ml A549/DDP Tabel 1 The effect of elemene on reversal multidurg resistance of A549/DDP cells (n=3, ± s) DDP/(μg ml -1 ) Cell proliferation inhibition rate /% Control group Experimental group.25 6.35 1.3 16.79 1.85.5 9.88.99 24.2.13 1 15.89.46 3.14.47 2 17.55 1.35 39.64.9 4 28.11.65 49.34.5 8 37.21 1.45 65.37 1.5 16 55.96 2.3 78.21.79 32 8.44.77 94.85.91 P.5, vs control group (A549/DDP cells treated with different concentrations of DDP); Experimental group: Elemene (2 μg/ml) combined with different concentrations of DDP. 2.4 A549/DDP Hoechst 33342 2 4 μg/ml 24 h A549/DDP 3 AnnexinV-FTIC/PI FCM A549/DDP 5.73.9 % 17.3.11 % 2 4 μg/ml A549/DDP 24 h 17.61.1 % 37.8.12 % 18.9.11 % 32.4.13 % P.5 4 2.5 A549/DDP cyt C caspase-3 Bcl-2 Fig. 3 The morphological changes of apoptosis of A549/DDP cells treated with different concentrations of elemene (2 and 4 μg/ml) for 24 h were observed under a fluorescence microscope (staining by Hoechst 33342, 4). Control: A549/ DDP cells treated without elemene. 3 2 4 μg/ml A549/DDP 24 h Hoechst 33342 4 Control Elemene (2 μg/ml) Elemene (4 μg/ml) 1 4.8 17.3 1 4.59 18.9 1 4 1.52 32.4 1 3 1 3 1 3 PI 1 2 1 2 1 2 1 1 1 1 1 1 76.1 5.73 72.9 17.61 28.2 37.8 1 1 1 1 1 2 1 3 1 4 1 1 1 1 1 2 1 3 1 4 1 1 1 1 1 2 1 3 1 4 Annexin- -FITC Fig. 4 The apoptosis rates of A549/DDP cells treated with different concentrations of elemene (2 and 4 μg/ml) for 24 h were detected by FCM (flow cytometry). Control: A549/DDP cells treated without elemene. 4 2 4 μg/ml A549/DDP 24 h
166. A549/DDP 2 4 μg/ml A549/DDP 24 h cyt C caspase-3 Bad pro-caspase-3 Bcl-2 P.5 5 cyt C pro-caspase-3 caspase-3 Bcl-2 Bad A549/DDP 2.6 A Ac-DEVD-CHO A549/DDP caspase-3 PTP A caspase-3 Ac- DEVD-CH 4 μg/ml A549/DDP 24 h 4 μg/ml caspase-3 P.5) 4 μg/ml A 4 μg/ml Ac-DEVD- CHO caspase-3 P.5 6 caspase-3 caspase-3 A549/DDP Fig. 5 The expression levels of pro-caspase-3, caspase-3, Bcl-2 (B cell lymphoma-2), Bad and cyt C (cytochrome C) proteins in A549/DDP cells treated with different concentrations of elemene (2 and 4 μg/ml) for 24 h were detected by Western blotting. P<.5, vs control group ( A549/DDP cells treated without elemene), n=3. 5 2 4 μg/ml A549/DDP 24 h cyto C pro-caspase-3 caspase-3 Bad Bcl-2 Fig. 6 The expression level of caspase-3 protein was inhibited in A549/DDP cells treated with elemene (4 μg/ml) combined with cyclosporine or Ac-DEVD-CHO for 24 h detected by Western blotting. P<.5, vs control group (A549/ DDP cells treated without elemene); P<.5, vs elemene (4 μg/ml) group; n=3. 6 A Ac-DEVD-CHO 4 μg/ml 24 h A549/DDP caspase-3
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