Despite multidisciplinary treatment, lung cancer remains a highly lethal disease due to poor response to chemotherapy. The identification of therapeutic agents with synergistic effects with traditional drugs is an alternative for lung cancer therapy. In this study, the synergistic effects of arsenic trioxide (As2O3) with cisplatin (DDP) on A549 and H460 non-small cell lung cancer (NSCLC) cells were explored.
Methods
A549 and H460 human lung cancer cells were treated with As2O3 and/or DDP. Cell growth curves, cell proliferation, cell cycle, and apoptosis of human cancer cell lines were determined by the 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) method, clonogenic assay, and flow cytometry (FCM). Apoptosis was further assessed by TUNEL staining. Cell cycle and apoptosis related protein p21, cyclin D1, Bcl-2, bax, clusterin, and caspase-3 were detected by western blot.
Results
MTT and clonogenic assay showed As2O3 within 10-2 μM to 10 μM exerted inhibition on the proliferation of NSCLC cells, and 2.5 μM As2O3 exerted synergistic inhibition on proliferation with 3 μg/ml DDP. The combination indices (CI) for A549 and H460 were 0.5 and 0.6, respectively, as confirmed by the synergism of As2O3 with DDP. FCM showed As2O3 did not affect the cell cycle. The G0/G1 fraction ranged from 57% to 62% for controlled A549 cells and cells treated with As2O3 and/or DDP. The G0/G1 fraction ranged from 37% to 42% for controlled H460 cells and cells treated with As2O3 and/or DDP. FCM and TUNEL staining illustrated that the combination of As2O3 and DDP provoked synergistic effects on apoptosis induction based on the analysis of the apoptosis index. Western blotting revealed that the expression of cell cycle related protein p21 and cyclin D1 were not affected by the treatments, whereas apoptosis related protein bax, Bcl-2, and clusterin were significantly regulated by As2O3 and/or DDP treatments compared with controls. The expression of caspase-3 in cells treated with the combination of As2O3 and DDP did not differ from that in cells treated with a single agent.
Conclusion
As2O3 exerted synergistic effects with DDP on NSCLC cells, and the synergistic effects were partly due to the induction of caspase-independent apoptosis.
Background
Lung cancer is the number one cause of cancer mortality in both males and females worldwide [1]. Despite multidisciplinary treatment, lung cancer is still a highly lethal disease due to late detection and resistance to chemotherapy. The identification of new therapeutic agents that exert synergistic effects in combination with traditional cytotoxic agents is an alternative strategy for the systemic treatment of lung cancer.
Recent evidence indicates that arsenic trioxide (As2O3) may induce clinical remission in patients with acute promyelocytic leukemia (APL), and several investigations show that As2O3 induced programmed cell death in APL cell lines [2–5]. DDP, a platinum-containing anticancer drug, is one of the most commonly used cytotoxic agents for the treatment of lung cancer. Due to the poor therapeutic effects of current cytotoxic-agents on lung cancer, the ability of As2O3 to induce apoptosis in non-small cell lung cancer cells was explored in the present study, and the synergistic effects of As2O3 with DDP on A549 and H460 lung cancer cells were analyzed.
Methods
Cell culture and reagents
Human lung cancer A549 and H460 cell lines were obtained from the ATCC and maintained in RPMI 1640 medium with 10% fetal bovine serum and 1% penicillin. As2O3 was purchased from Yida Pharmaceutical Co.(GMP, Ha'erbin, PR. China) and DDP was from Bristol-Myers Squibb Co.(Shanghai, PR. China).
MTT assay
Briefly, cells were seeded at a density of 2,000 to 5,000 cells/well in 96-well plates and incubated overnight. After treatment with As2O3, DDP, or their combination (described below), 3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) was added (50 μL/well) for 4 hours. Solubilization of the converted purple formazan dye was accomplished by placing cells in 100 μL of 0.01 N HCl/10% SDS and incubating them overnight at 37°C. The reaction product was quantified by absorbance at 570 nm. All samples were repeated three times, and data were analyzed by Student's t test.
In vitro clonogenic assay
Human lung carcinoma cells were counted after trypsinization. Cells were serially diluted to appropriate concentrations and removed into 25-cm2 flasks in 5-mL medium in triplicate per data point. After various treatments, cells were maintained for 8 days. Cells were then fixed for 15 minutes with a 3:1 ratio of methanol:acetic acid and stained for 15 minutes with 0.5% crystal violet (Sigma) in methanol. After staining, colonies were counted by the naked eye, with a cutoff of 50 viable cells. Error bars represent ± SE by pooling of the results of three independent experiments. Surviving fraction was calculated as (mean colony counts)/(cells inoculated)*(plating efficiency), where plating efficiency was defined as mean colony counts/cells inoculated for untreated controls.
Cell cycle and apoptosis analysis
Flow cytometry analysis of DNA content was performed to assess the cell cycle phase distribution as described previously[6]. Cells were harvested and stained for DNA content using propidium iodide fluorescence. The computer program Multicycle from Phoenix Flow System (San Diego, CA, USA) was used to generate histograms which were used to determine the cell cycle phase distribution and apoptosis. TUNEL staining was also used to detect apoptosis as described previously [7]. The TUNEL stained apoptotic cells were separately numbered in four randomly selected microscopic fields (400*) and graphed.
Western blot
After various treatments, cells were washed with ice-cold PBS twice before the addition of lysis buffer (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium NaPPi, 1 mM phenylmethylsulfonyl fluoride, and leupeptin). Protein concentrations were quantified separately by the Bio-Rad Bradford assay. Equal amounts of protein were loaded into each well and separated by 10% SDS PAGE, followed by transfer onto nitrocellulose membranes. Membranes were blocked using 5% nonfat dry milk in PBS for 1 hour at room temperature. The blots were then incubated with anti-p21, anti-cyclin D1, anti-bax, anti-bcl-2, anti-clusterin, and anti-caspase-3 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C overnight. Blots were then incubated in secondary antibody conjugated with HRP (1:1000; Santa Cruz Biotechnology) for 1 hour at room temperature.
Immunoblots were developed using the enhanced chemiluminescence (ECL) detection system (Amersham, Piscataway, NJ) according to the manufacturer's protocol and autoradiography.
Results
As2O3 exerted synergistic effects with DDP on the proliferation of A549 and H460
The MTT assay showed that 10-2 μM to 10 μM inhibited the proliferation of A549 and H460 at 72 hours (Fig. 1). In vitro clonogenic assay proved 10-1 μM to 12.5 μM As2O3 inhibited the proliferation of A549 and H460 cells (Fig. 2). MTT assay results showed that 2.5 μM As2O3 and 3 μg/ml DDP exerted synergistic inhibition effects on A549 and H460 cells at 48 hours. (Fig. 3A,B). To confirm the synergistic effects of As2O3 with DDP CalcuSyn™ program (Version 2.0, Biosoft, Inc., UK) was explored to make dose-effect curves and to determine the combination indices (CI) (Fig. 4A,B). The CI for A549 and H460 were 0.5 and 0.6, respectively which confirmed the synergism of As2O3 with DDP.
Figure 1
Dose response curves for effects of As2O3on A549 and H460 lung cancer cell proliferation. Cells were treated with different concentrations of As2O3 (10-6–10 μM) for 72 hours. Proliferation was analyzed by MTT assay. As2O3 concentrations of 10-2 μM to 10 μM inhibited A549 cell proliferation at 72 hours.
Figure 2
Clonogenic assay of the effects of As2O3on the proliferation of A549 and H460 cells. In vitro clonogenic assays showed that 10-1 μM to 12.5 μM As2O3 inhibited the proliferation of A549 and H460 cells. Surviving fraction was calculated as (mean colony counts)/(cells inoculated) × (plating efficiency), where plating efficiency was defined as mean colony counts/cells inoculated for untreated controls.
Figure 3
Synergistic effects of As2O3and DDP in lung cancer cell lines. A. The synergistic effect of As2O3 and DDP in the treatment of A549 cells. MTT assay results showed that 2.5 μM As2O3 and 3 μg/ml DDP exerted synergistic inhibition effects on A549 cells at 48 hours. B. The synergistic effect of As2O3 and DDP in the treatment of H460 cells. MTT assay results showed that 2.5 μM As2O3 and 3 μg/ml DDP exerted synergistic inhibition effects on H460 cells at 48 hours.
Figure 4
Dose effect curve for A549 (A) and H460 (B) cells. The concentration of DDP was 3 μg/ml and the concentration for As2O3 ranged from 0.1 μM to 12.5 μM. CalcuSyn™ (Version 2.0, Biosoft, Inc., UK) was used for dose-effect curves and to determine the combination indices (CI).
As2O3 did not significantly affect the cell cycles of A549 and H460 cells
A549 cells were treated with 2.5 μM As2O3 and/or 3 μg/ml DDP for 48 hours. FCM cell cycle analysis showed that the treatment of As2O3 and/or DDP did not significantly alter G0/G1 fractions of A549 cells compared with those of the control. The G0/G1 fraction ranged from 57% to 62% for controlled A549 cells and cells treated with As2O3 and/or DDP; the G0/G1 fraction ranged from 37% to 42% for controlled H460 cells and cells treated with As2O3 and/or DDP (Fig. 5). Western blot analysis showed that As2O3 and/or DDP did not affect the expression of cell cycle related protein p21 and cyclin D1 (data not shown).
Figure 5
G0/G1 fraction analysis. FCM cell cycle analysis showed that the treatment of As2O3 and/or DDP did not significantly affect G0/G1 fractions of A549 and H460 cells compared with those of the control. The G0/G1 fraction ranged from 57% to 62% for control A549 cells and for A549 cells treated with As2O3 and/or DDP, and from 37% to 42% for control H460 cells and for H460 cells treated with As2O3 and/or DDP.
As2O3 and/or DDP induced apoptosis of A549 and H460 cells
A549 cells were treated with 2.5 μM As2O3 and/or 3 μg/ml DDP for 48 hours. FCM analysis showed the apoptotic indices (AI) for the controlled A549 cells and cells treated with As2O3, DDP, or the combination were 0.25 ± 0.01%, 10.6 ± 0.53%, 15.85 ± 0.79%, and 20 ± 1%, respectively. The AI for the controlled H460 cells and cells treated with As2O3, DDP, or the combination were 1.95 ± 0.11%, 13.6 ± 0.65%, 7.53 ± 0.43%, and 35.6 ± 1.71%, respectively (Fig. 6). As2O3 and DDP significantly increased the AI compared with the control cells. TUNEL staining was performed to further confirm AI results from FCM analysis. With TUNEL staining, the AI for the control A549 cells, cells treated with As2O3, DDP, or the combination were 3.1 ± 0.16%, 15.41 ± 0.77%, 14 ± 0.7%, and 30 ± 1.5%, respectively. The AI for the control H460 cells, cells treated with As2O3, DDP, or the combination were 5.95 ± 0.25%, 18.6 ± 1.13%, 9.53 ± 0.49%, and 40.6 ± 2.11%, respectively (Fig. 7). Western blot analysis showed Bax expression increasing by 2-fold in the A549 cells treated with As2O3 and DDP over levels in control cells. In H460 cells treated with As2O3 and DDP, Bax expression was 3.7 times greater than in the control (Fig. 8). Bcl-2 expression was 72% less in the As2O3 and DDP treated A549 cells than in control cells, and 25% less in the As2O3 and DDP treated H460 cells than in control cells (Fig. 9). Expression of another tumor suppressed protein, clusterin, was 70% less in the As2O3 and DDP treated A549 cells than in control cells, and in H460 cells, clusterin expression was 90% less with treatment with the combination of As2O3 and DDP than in control cells (Fig. 10). For both A549 and H460, caspase-3 expression increased with the treatment of As2O3 and/or DDP over control levels, but caspase-3 expression was not different in cells treated with the combination of As2O3 and DDP and cells treated with each single agent (Fig. 11).
Figure 6
FCM cell cycle analysis of apoptotic index (AI) for cells treated with As2O3and/or DDP. AI for the control A549 cells and cells treated with As2O3, DDP, or the combination were 0.25 ± 0.01%, 10.6 ± 0.53%, 15.85 ± 0.79%, and 20 ± 1%, respectively; the AI for the control H460 cells and cells treated with As2O3, DDP, or the combination were 1.95 ± 0.11%, 13.6 ± 0.65%, 7.53 ± 0.43%, and 35.6 ± 1.71%, respectively.
Figure 7
TUNEL staining analysis. With TUNEL staining, the AI for the control A549 cells and cells treated with As2O3, DDP, or the combination were 3.1 ± 0.16%, 15.41 ± 0.77%, 14 ± 0.7%, and 30 ± 1.5%, respectively; the AI for the control H460 cells and cells treated with As2O3, DDP, or the combination were 5.95 ± 0.25%, 18.6 ± 1.13%, 9.53 ± 0.49%, and 40.6 ± 2.11%, respectively.
Figure 8
Western blot analysis of Bax expression in lung cancer cell after different treatments. Bax expression was 2-fold greater in A549 cells treated with As2O3 and DDP than in control cells. Bax expression was 3.7-fold greater in H460 cells treated with As2O3 and DDP than in control cells.
Figure 9
Western blot analysis of Bcl-2 expression in lung cancer cells after different treatments. Bcl-2 expression was 72% less in As2O3 and DDP-treated A549 cells than in controls, and it 25% less in As2O3 and DDP-treated H460 cells than in controls.
Figure 10
Western blot analysis of clusterin expression in lung cancer cells after different treatments. Clusterin expression was 70% less in As2O3 and DDP-treated A549 cells than in controls, and in H460 cells, clusterin expession was 90% less with treatment of the combination of As2O3 and DDP than in controls.
Figure 11
Western blot analysis of caspase-3 expression in lung cancer cells after different treatments. For both A549 and H460 cells, caspase-3 expression increased with treatment of As2O3 and/or DDP, but caspase-3 expression did not differ in cells treated with the combination of As2O3 and DDP and cells treated with each single agent.
Discussion and conclusion
Our in vitro study showed that As2O3 is an effective reagent that inhibits the proliferation of A549 and H460 lung cancer cells. As2O3 cytotoxicity was due to the induction of apoptosis but not cell cycle arrest. FCM and TUNEL assay analyses showed that As2O3 significantly induced apoptosis. When As2O3 and DDP were combined, a synergistic effect was found in the treatment of A549 and H460 cells. Protein assays showed that the combination of As2O3 and DDP affected apoptosis-related proteins such as Bcl-2, Bax, and clusterin but not caspase-3, while the use of each single agent did not. The changes in apoptosis-related protein expression partly contributed to the effect of As2O3 on lung cancer cells.
Since lung cancer is a lethal disease due to late detection and resistance to chemotherapy, this study was conducted to determine whether As2O3 could exert synergistic effects in combination with traditional cytotoxic-agents on lung cancer cell death. Although As2O3 has been an effective treatment for the acute promyelocytic leukemia, the mechanism by which As2O3 induces cell death remains poorly understood. Recent reports suggest that As2O3 causes DNA damage, oxidative stress, and mitochondrial dysfunction [8, 9]. In addition, As2O3 treatment results in cell-cycle arrest in MCF-7 HeLa cells [10]; however, our results demonstrate that cell cycle is not significantly affected by As2O3, since the G1/0 fraction and cell cycle-related protein expression did not change significantly with As2O3 treatment. The inconsistency between these findings may be due to different mechanisms of action by As2O3 in various cell lines. Our results were consistent with previous studies that indicated that proapoptotic Bcl-2 family members, Bcl-2 and Bax, are involved in the apoptosis of cancer cells induced by As2O3 [11, 12]. Previous studies show that clusterin is a caspase-independent apoptosis-related protein and it is a potential target in the treatment of non-small cell lung cancer [13–15]. Here, we showed that the synergistic effects of As2O3 and DDP might be due, in part, to clusterin-mediated apoptosis. Depending on the cell system investigated, As2O3-induced cell death has been associated with caspase-dependent apoptosis, as well as caspase-independent death pathways [16–18]. In this study, the combination of As2O3 and DDP increased caspase-3 expression, which indicates that caspase might be involved in apoptosis induced by As2O3 or DDP. However, the combination of As2O3 and DDP did not affect caspase-3 expression compared with cells treated with a single agent, which suggests that the synergistic effects are more likely to be caspase-independent. This study showed caspase-independent death pathways that involved Bcl-2, Bax, and clusterin were the primary mechanism by which As2O3 exerts synergistic effects with DDP on NSCLC cells.
In conclusion, As2O3 exerted synergistic effects with DDP on lung cancer cells. The proliferation inhibition might be partly due to the induction of apoptosis. Based on our study, As2O3 may be a promising agent in the treatment of lung cancer, although further in vitro and in vivo studies are necessary to elucidate the mechanism by which As2O3 induces apoptosis.
Notes
Declarations
Acknowledgements
We are grateful to Professor Stefan Glück (Division of Hematology/Oncology, UMSylvester Comprehensive Cancer Center, University of Miami, FL) for the review of our manuscript.
This work was sponsored in part by a National Natural Science Foundation of China Grant 30600756 (to H.L.), the Shanghai Rising-Star Program (A type 07QA14011, to H.L), and a Youth Foundation Grant 05L-A-11 from Fudan University (to H.L.).
Authors’ Affiliations
(1)
Department of Thoracic Surgery, Fudan University Cancer Hospital/Cancer Institute
(2)
Department of Oncology, Fudan University Shanghai Medical College
(3)
Department of Pathology, Fudan University Cancer Hospital/Cancer Institute
References
Landis SH, Murray T, Bolden S, Wingo PA: Cancer statistics, 1998. CA Cancer J Clin. 1998, 48 (1): 6-29. 10.3322/canjclin.48.1.6.View ArticleGoogle Scholar
Soignet SL, Maslak P, Wang ZG, Jhanwar S, Calleja E, Dardashti LJ, Corso D, DeBlasio A, Gabrilove J, Scheinberg DA, Pandolfi PP, Warrell RP: Complete remission after treatment of acute promyelocytic leukemia with arsenic trioxide. N Engl J Med. 1998, 339 (19): 1341-1348. 10.1056/NEJM199811053391901.View ArticleGoogle Scholar
Shao W, Fanelli M, Ferrara FF, Riccioni R, Rosenauer A, Davison K, Lamph WW, Waxman S, Pelicci PG, Lo Coco F, Avvisati G, Testa U, Peschle C, Gambacorti-Passerini C, Nervi C, Miller WH: Arsenic trioxide as an inducer of apoptosis and loss of PML/RAR alpha protein in acute promyelocytic leukemia cells. J Natl Cancer Inst. 1998, 90 (2): 124-133. 10.1093/jnci/90.2.124.View ArticleGoogle Scholar
Look AT: Arsenic and apoptosis in the treatment of acute promyelocytic leukemia. J Natl Cancer Inst. 1998, 90 (2): 86-88. 10.1093/jnci/90.2.86.View ArticleGoogle Scholar
Chen GQ, Shi XG, Tang W, Xiong SM, Zhu J, Cai X, Han ZG, Ni JH, Shi GY, Jia PM, Liu MM, He KL, Niu C, Ma J, Zhang P, Zhang TD, Paul P, Naoe T, Kitamura K, Miller W, Waxman S, Wang ZY, de The H, Chen SJ, Chen Z: Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): I. As2O3 exerts dose-dependent dual effects on APL cells. Blood. 1997, 89 (9): 3345-3353.Google Scholar
Shao ZM, Nguyen M, Alpaugh ML, O'Connell JT, Barsky SH: The human myoepithelial cell exerts antiproliferative effects on breast carcinoma cells characterized by p21WAF1/CIP1 induction, G2/M arrest, and apoptosis. Exp Cell Res. 1998, 241 (2): 394-403. 10.1006/excr.1998.4066.View ArticleGoogle Scholar
Daigeler A, Brenzel C, Bulut D, Geisler A, Hilgert C, Lehnhardt M, Steinau HU, Flier A, Steinstraesser L, Klein-Hitpass L, Mittelkötter U, Uhl W, Chromik AM: TRAIL and Taurolidine induce apoptosis and decrease proliferation in human fibrosarcoma. J Exp Clin Cancer Res. 2008, 27: 82-10.1186/1756-9966-27-82.View ArticleGoogle Scholar
Gurr JR, Bau DT, Liu F, Lynn S, Jan KY: Dithiothreitol enhances arsenic trioxide-induced apoptosis in NB4 cells. Mol Pharmacol. 1999, 56 (1): 102-109.Google Scholar
Dong JT, Luo XM: Arsenic-induced DNA-strand breaks associated with DNA-protein crosslinks in human fetal lung fibroblasts. Mutat Res. 1993, 302 (2): 97-102. 10.1016/0165-7992(93)90010-S.View ArticleGoogle Scholar
Ling YH, Jiang JD, Holland JF, Perez-Soler R: Arsenic trioxide produces polymerization of microtubules and mitotic arrest before apoptosis in human tumor cell lines. Mol Pharmacol. 2002, 62 (3): 529-538. 10.1124/mol.62.3.529.View ArticleGoogle Scholar
Chen GQ, Zhu J, Shi XG, Ni JH, Zhong HJ, Si GY, Jin XL, Tang W, Li XS, Xong SM, Shen ZX, Sun GL, Ma J, Zhang P, Zhang TD, Gazin C, Naoe T, Chen SJ, Wang ZY, Chen Z: n vitro studies on cellular and molecular mechanisms of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia: As2O3 induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML-RAR alpha/PML proteins. Blood. 1996, 88 (3): I1052-1061.Google Scholar
Kroemer G, de The H: Arsenic trioxide, a novel mitochondriotoxic anticancer agent?. J Natl Cancer Inst. 1999, 91 (9): 743-745. 10.1093/jnci/91.9.743.View ArticleGoogle Scholar
Cao C, Shinohara ET, Li H, Niermann KJ, Kim KW, Sekhar KR, Gleave M, Freeman M, Lu B: Clusterin as a therapeutic target for radiation sensitization in a lung cancer model. Int J Radiat Oncol Biol Phys. 2005, 63 (4): 1228-1236.View ArticleGoogle Scholar
Scaltriti M, Santamaria A, Paciucci R, Bettuzzi S: Intracellular clusterin induces G2-M phase arrest and cell death in PC-3 prostate cancer cells1. Cancer research. 2004, 64 (17): 6174-6182. 10.1158/0008-5472.CAN-04-0920.View ArticleGoogle Scholar
Chi KN, Siu LL, Hirte H, Hotte SJ, Knox J, Kollmansberger C, Gleave M, Guns E, Powers J, Walsh W, Tu D, Eisenhauer E: A phase I study of OGX-011, a 2'-methoxyethyl phosphorothioate antisense to clusterin, in combination with docetaxel in patients with advanced cancer. Clin Cancer Res. 2008, 14 (3): 833-839. 10.1158/1078-0432.CCR-07-1310.View ArticleGoogle Scholar
McCafferty-Grad J, Bahlis NJ, Krett N, Aguilar TM, Reis I, Lee KP, Boise LH: Arsenic trioxide uses caspase-dependent and caspase-independent death pathways in myeloma cells. Molecular cancer therapeutics. 2003, 2 (11): 1155-1164.Google Scholar
Cai X, Shen YL, Zhu Q, Jia PM, Yu Y, Zhou L, Huang Y, Zhang JW, Xiong SM, Chen SJ, Wang ZY, Chen Z, Chen GQ: Arsenic trioxide-induced apoptosis and differentiation are associated respectively with mitochondrial transmembrane potential collapse and retinoic acid signaling pathways in acute promyelocytic leukemia. Leukemia. 2000, 14 (2): 262-270. 10.1038/sj.leu.2401650.View ArticleGoogle Scholar
Karlsson J, Ora I, Porn-Ares I, Pahlman S: Arsenic trioxide-induced death of neuroblastoma cells involves activation of Bax and does not require p53. Clin Cancer Res. 2004, 10 (9): 3179-3188. 10.1158/1078-0432.CCR-03-0309.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
