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Oh, Cho, Manaljav, Kwon, Kim, Lim, Kim, and Ahn: Investigating Effective Combinations of Anti-cancer Drugs and Radiation Therapy for Treating Non-small Cell Lung Cancer with Using Two Cell Lines
Original Article

J Lung Cancer 2009;8(1):21-30.

Published online: 10 January 2009

DOI: https://doi.org/10.6058/jlc.2009.8.1.21

Investigating Effective Combinations of Anti-cancer Drugs and Radiation Therapy for Treating Non-small Cell Lung Cancer with Using Two Cell Lines

In-Jae Oh, M.D., Ph.D.1, Hyun-Ju Cho, M.S.1, Tseden-Ish Manaljav, M.S.1, Yong-Soo Kwon, M.D., Ph.D.1, Kyu-Sik Kim, M.D., Ph.D.1, Sung-Chul Lim, M.D., Ph.D.1, Young-Chul Kim, M.D., Ph.D.1, Sung-Ja Ahn, M.D., Ph.D.2

1Departments of Internal Medicine

2Radiation Oncology, Chonnam National University Medical School, Gwangju, Korea

Address for correspondence Young-Chul Kim, M.D., Ph.D. Lung and Esophageal Cancer Clinic, Chonnam National University Medical School, Hwasun Hospital, 160, Ilsim-ri, Gwangju 519-809, Korea Tel: 82-61-379-7614　 Fax: 82-61-379-7619 E-mail: kyc0923@chonnam.ac.kr　

This study was supported by Chonnam National University Grant 2005–0699.

Received 20 May 2009       Accepted 10 June 2009

Copyright © 2009 Korean Association for the Study of Lung Cancer

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Purpose

Radiotherapy had been used for treating unresectable locally advanced non-small cell lung cancer (NSCLC). However, the survival rate after radiotherapy alone is low and this primarily due to the failure of local disease control and distant metastasis. For achieving better disease control, radiotherapy has recently been combined with chemotherapy in various ways. In this study, we aimed to find the most effective combination of chemotherapy and radiotherapy for treating NSCLC.

Materials and Methods

Two human lung cancer cell lines (NCI-H520 and A549) and various chemotherapeutic agents (paclitaxel, docetaxel, gemcitabine and cisplatin) were used for this study. The radiation doses were 0, 2, 4 and 8 Gy. After processing various combinations according to the radiation doses and the concentrations of the chemotherapeutic agents, cell survival was quantified by MTT (3-(4,5-Dimethyl-hiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay. For the evaluation of synergism between chemotherapy and radiotherapy, we used a combination index that as calculated by Chou and Talalay's method and with using Calcusyn software.

Results

Among the various combinations of chemotherapeutic agents and radiation doses, concurrent chemoradiation therapy (CCRT) led to the highest apoptosis rate and it showed frequent synergism. When taxane was administrated as a chemotherapeutic agent, chemotherapy followed by radiotherapy was the most effective combination. When high-dose chemotherapeutic agents were added to CCRT, induction chemotherapy resulted in a higher apoptosis rate and more frequent synergism than did consolidation chemotherapy.

Conclusion

When radiotherapy is combined with chemotherapy for treating the NCI-H520 and A549 cell lines, CCRT is the most effective combination. If a high-dose chemotherapeutic agent is added to CCRT, then induction chemotherapy is more effective than consolidation chemotherapy.

Keywords: Non-small cell lung carcinoma, Concurrent chemoradiation, Induction chemotherapy

References

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Figures and Tables

Fig. 1.
Schematic diagram of the experimental designs in this study. After the indicated treatment, the cells were processed for clonogenic survival using a MTT assay. Chemo: chemotherapy, Rad: radiation, iChemo: induction chemotherapy, cChemo: consolidation chemotherapy, MTT: 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazoliu mbromide.
jlc-8-21f1.tif
Fig. 2.
Sequence-dependent antiproliferative effects at various concentrations of chemotherapeutic agents in the NCI-H520 cell line. The cell viabilities are shown on (A) paclitaxel 25 nM, (B) docetaxel 25 ng/mL, (C) gemcitabine 100 ng/mL, and (D) cisplatin 5 g/mL, respectively. *synergism (CI values of 0.7 to 0.3), † strong synergism (CI values less than 0.3).
jlc-8-21f2.tif
Fig. 3.
Sequence-dependent antiproliferative effects according to the various concentrations of chemotherapeutic agents in the A549 cell line. The cell viability is shown on (A) paclitaxel 10 nM, (B) docetaxel 5 ng/mL, (C) gemcitabine 2.5 ng/mL, and (D) cisplatin 2.5 g/mL, respectively. *synergism (CI values of 0.7 to 0.3), † strong synergism (CI values less than 0.3).
jlc-8-21f3.tif
Fig. 4.
Flow cytometry analysis of the cell cycle on the A549 cell line. Among (A) only gemcitabine 2.5 ng/mL, (B) radiation 4 Gy followed by gemcitabine 2.5 ng/mL, (C) gemcitabine 2.5 ng/mL followed by radiation, and (D) CCRT with gemcitabine, the greatest apoptotic peak is shown in CCRT. M1: sub G1-phase, M2: G1-phase, M3: S-phase, M4: G2/M-phase.
jlc-8-21f4.tif
Table 1.
Sequence-dependent Antiproliferative Effects According to the Various Chemotherapeutic Agents and Radiation Doses in NCI-H520 Cell Line
Drugs Sequences RT (Gy) (A) ChemoRT (B) RTchemo (C) CCRT
Viability (%) CI Viability (%) CI Viability (%) CI
Paclitaxel 10 nM 2 69.23 0.51* 93.10 1.82 59.80 0.39*
    4 60.28 0.78 77.28 1.35 61.99 0.82
    8 61.25 1.60 61.54 1.62 54.53 1.34
  25 nM 2 46.64 0.27† 90.90 1.45 53.13 0.32*
    4 46.16 0.54* 83.14 1.74 57.83 0.73
    8 46.33 1.08 57.75 1.46 52.02 1.25
Docetaxel 10 ng/mL 2 56.22 0.33* 85.36 1.95 61.82 0.42*
    4 53.56 0.60* 79.56 2.28 59.13 0.75
    8 53.05 1.19 63.18 1.77 54.22 1.24
  25 ng/mL 2 42.65 0.21† 65.87 0.50* 53.71 0.30*
    4 39.96 0.39* 62.89 1.87 55.23 0.64*
    8 37.03 0.70 53.90 1.22 45.97 0.93
Gemcitabine 50 ng/mv 2 84.44 0.95 92.00 1.47 75.16 0.68*
    4 80.88 1.65 70.97 1.21 59.74 0.91
    8 67.78 2.22 62.58 1.95 53.87 1.59
  100 ng/mL 2 67.74 0.55* 87.11 1.08 41.40 0.29†
    4 59.06 0.90 67.94 1.11 38.52 0.56*
    8 53.23 1.57 55.30 1.64 34.76 1.02
Cisplatin 2.5 g/mL 2 42.01 0.36* 73.87 0.92 39.60 0.34*
    4 43.26 0.75 61.89 1.26 38.71 0.67*
    8 41.82 1.66 55.37 2.42 36.35 1.42
  5.0 g/mL 2 29.37 0.25† 59.82 0.59* 22.78 0.24†
    4 26.50 0.46* 46.72 0.83 26.96 0.47*
    8 25.16 1.00 42.68 1.70 26.68 1.05

(A) Chemotherapy followed by radiation, (B) radiation followed by chemotherapy, and (C) concurrent chemoradiation (CCRT). The combination index (CI) values were calculated using the Chou and Talalay mathematical model for drug interactions on Calcusyn software. A CI was equal to 1 denotes additivity; antagonism if the CI＞1; CI values between 1 and 0.7 indicate slight synergism;

* 0.7 to 0.3, synergism;

† ＜0.3, strong synergism.

Table 2.
Sequence-dependent Antiproliferative Effects According to the Various Chemotherapeutic Agents and Radiation Doses in the A549 Cell Line
Drugs Sequences RT (Gy) (A) ChemoRT (B) RTchemo (C) CCRT
Viability (%) CI Viability (%) CI Viability (%) CI
Paclitaxel 5 nM 2 79.57 1.25 78.50 1.18 63.66 0.61*
    4 57.01 0.95 68.38 1.47 58.39 1.00
    8 45.80 1.27 54.81 1.75 42.09 1.11
  10 nM 2 52.05 0.40* 78.22 1.16 53.88 0.42*
    4 48.63 0.70* 59.95 1.06 47.71 0.68*
    8 42.32 1.12 42.75 1.14 33.78 0.81
Docetaxel 2.5 ng/mL 2 62.82 0.32* 79.51 0.68* 65.67 0.36*
    4 61.48 0.61* 70.46 0.87 61.10 0.60*
    8 47.43 0.80 63.30 1.42 57.06 1.13
  5.0 ng/mL 2 40.58 0.14† 73.06 0.49* 61.30 0.30*
    4 38.64 0.27† 68.17 0.49* 52.71 0.44*
    8 28.34 0.38* 61.27 1.32 49.53 0.86
Gemcitabine 2.5 ng/mL 2 63.65 0.45* 74.86 0.73 62.49 0.43*
    4 59.75 0.78 71.33 1.24 50.50 0.56*
    8 52.90 1.37 65.57 2.21 43.73 0.99
  5.0 ng/mL 2 38.69 0.18† 66.82 0.51* 48.00 0.26†
    4 43.26 0.43* 71.98 1.28 47.37 0.50*
    8 34.46 0.70 50.06 1.24 28.32 0.54*
Cisplatin 1.0 g/mL 2 67.61 0.62* 81.12 1.12 64.04 0.55*
    4 63.78 1.09 77.94 1.91 63.43 1.07
    8 59.76 2.18 76.50 4.09 57.81 2.04
  2.5 g/mL 2 40.16 0.25† 65.03 0.57* 42.58 0.27†
    4 39.12 0.48* 64.70 1.12 40.25 0.50*
    8 41.93 1.22 62.37 2.38 33.68 0.92

(A) Chemotherapy followed by radiation, (B) radiation followed by chemotherapy, and (C) concurrent chemoradiation. The combination index (CI) values were calculated using the Chou and Talalay mathematical model for drug interactions on Calcusyn software. A CI was equal to 1 denotes additivity; antagonism if the CI＞1; CI values between 1 and 0.7 indicate slight synergism;

* 0.7 to 0.3, synergism;

† ＜0.3, strong synergism.

Table 3.
Comparison of the Induction and Consolidation Chemotherapy with CCRT in the NCI-H520 Cell Line
Drugs Sequences RT (Gy) High-dose conc. (A) Induction chemotherapy (B) Consolidation chemotherapy
Viability (%) CI Viability (%) CI
Paclitaxel 5 nM 2 ＋10 nM 64.06 1.04 58.45 1.17
  4   49.67 0.90 38.69 1.33
  8   44.15 1.18 36.97 2.04
  2 +15 nM 56.68 0.66* 35.49 0.61*
  4   44.13 0.67* 27.60 0.94
  8   41.51 1.04 24.91 1.37
Docetaxel 1 ng/mL 2 ＋2 ng/mL 59.86 0.27† 74.08 1.12
  4   65.66 0.66* 56.32 1.40
  8   51.94 0.85 54.23 2.22
  2 ＋3 ng/mL 57.18 0.25† 53.78 0.66*
  4   59.95 0.54* 43.58 1.03
  8   52.70 0.87 40.81 1.61
Gemcitabine 50 ng/mL 2 ＋100 ng/mL 62.56 0.55* 59.11 0.72
  4   55.90 0.87 63.49 1.61
  8   49.21 1.25 58.28 2.38
  2 ＋150 ng/mL 49.76 0.36* 29.60 0.36*
  4   50.90 0.74 37.96 0.88
  8   42.99 1.03 25.88 1.06
Cisplatin 0.5 g/mL 2 ＋1.0 g/mL 48.19 0.37* 50.49 0.71
  4   48.39 0.74 42.73 0.71
  8   38.52 0.72 39.82 1.00
  2 +1.5 g/mL 48.26 0.37* 41.43 0.39*
  4   47.07 0.67* 37.36 0.47*
  8   38.35 0.71 32.87 0.65*

(A) High-dose induction chemotherapy followed by CCRT, (B) CCRT followed by high-dose consolidation chemotherapy. The combination index (CI) values were calculated using the Chou and Talalay mathematical model for drug interactions on Calcusyn software. A CI was equal to 1 denotes additivity; antagonism if the CI ＞1; CI values between 1 and 0.7 indicate slight synergism;

* 0.7 to 0.3, synergism;

† ＜0.3, strong synergism.

Table 4.
Comparison of the Induction and Consolidation Chemotherapy with CCRT in the A549 Cell Line
Sequences High-dose conc. (A) Induction chemotherapy (B) Consolidation chemotherapy
Drugs RT (Gy) Viability (%) CI Viability (%) CI
Paclitaxel 5 nM 2 ＋10 nM 78.58 0.35* 52.71 0.54*
  4   67.85 0.44* 56.04 1.20
  8   54.71 0.59* 57.76 2.05
  2 ＋15 nM 78.48 0.34* 48.16 0.47*
  4   65.79 0.41* 50.30 1.01
  8   52.09 0.54* 51.13 1.68
Docetaxel 1 ng/mL 2 ＋2 ng/mL 86.35 0.53* 78.22 0.75
  4   81.32 0.77 81.35 1.86
  8   66.14 0.84 82.33 3.40
  2 +3 ng/mL 84.04 0.45* 67.74 0.42*
  4   80.95 0.76 75.25 1.25
  8   64.68 0.80 75.54 2.20
Gemcitabine 2.5 ng/mL 2 ＋5 ng/mL 52.35 0.63* 40.36 0.58*
  4   43.91 0.91 39.42 1.12
  8   40.42 1.28 34.15 1.48
  2 ＋7.5 ng/mL 38.66 0.37* 29.69 0.40*
  4   33.21 0.59* 26.22 0.70*
  8   26.57 0.72 24.89 1.05
Cisplatin 0.5 g/mL 2 ＋1.0 g/mL 58.57 0.50* 40.51 0.56*
  4   57.75 0.97 43.05 1.19
  8   56.89 1.56 38.39 1.67
  2 ＋1.5 g/mL 47.86 0.33* 27.61 0.38*
  4   40.97 0.50* 27.58 0.77
  8   36.83 0.71 26.62 1.17

(A) High-dose induction chemotherapy followed by CCRT, (B) CCRT followed by high-dose consolidation chemotherapy. The combination index (CI) values were calculated using the Chou and Talalay mathematical model for drug interactions on Calcusyn software. A CI was equal to 1 denotes additivity; antagonism if the CI ＞1; CI values between 1 and 0.7 indicate slight synergism;

* 0.7 to 0.3, synergism;

† ＜0.3, strong synergism.

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