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Induction of TNF-α, uPA, IL-8 and MCP-1 by doxorubicin in human lung carcinoma cells

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Abstract

Purpose

We have previously demonstrated doxorubicin-induced urokinase (uPA) and interleukin-8 (IL-8) expression in human H69 small-cell lung carcinoma (SCLC) cells by a microarray technique using Human Cancer Chip version 2, in which 425 human "cancer-related" genes are spotted on the plates. The microarray analysis also revealed a significant induction of tumor necrosis factor-alpha (TNF-α), and doxorubicin-induced macrophage chemoattractant protein-1 (MCP-1) expression was demonstrated by an RNase protection assay. We extended the study by testing the effects of doxorubicin on the induction of TNF-α, uPA, IL-8 and MCP-1 in other types of lung carcinoma cells.

Methods

We investigated the effects of doxorubicin on the expression of TNF-α, uPA, IL-8 and MCP-1 in 12 human lung carcinoma cell lines, including five SCLC, three adenocarcinoma and four squamous cell carcinoma cells. The surface expression of their receptors was also investigated.

Results

TNF-α was significantly induced in three cell lines, H69, SBC-7 (SCLC) and PC-9 (adenocarcinoma), uPA in five cell lines, H69, SBC-7, EBC-1 (squamous cell), EBC-2 (squamous cell), and Sq-1 (squamous cell), IL-8 in three cell lines, H69, PC-9 and EBC-1, and MCP-1 in five cell lines, H69, SBC-3 (SCLC), SBC-7, PC-9 and Sq-1. In H69 cells, TNF-α antigen levels were increased approximately fivefold in the conditioned medium of doxorubicin-treated cells, in parallel with an increase in mRNA levels. As with uPA and IL-8, the maximum induction was observed at the "sublethal" concentrations of 2 and 4 μM at which cell growth was slightly inhibited 24 h after treatment. Furthermore, the cells did not express receptors including types I and II TNF-α receptors, uPA receptor (uPAR), C-x-C-chemokine receptor-1 (CXCR-1), or C-C-chemokine receptor-2, corresponding to TNF-α, uPA, IL-8 and MCP-1, respectively, that were induced by doxorubicin in the cells, although SBC-7 cells expressed uPAR, and EBC-1 cells expressed CXCR-1.

Conclusions

TNF-α, uPA, IL-8 and MCP-1 induced and secreted from tumor cells upon doxorubicin stimulation may activate surrounding cells expressing the receptors such as neutrophils and monocytes/macrophages in a paracrine fashion. TNF-α is a major proinflammatory cytokine, and IL-8 and MCP-1 are major chemoattractants for neutrophils and monocytes/macrophages, respectively. Furthermore, uPA activates matrix metalloproteinase 9 which can truncate and activate IL-8. Thus, the simultaneous induction of TNF-α, uPA, IL-8 and MCP-1 may enhance the interaction between tumor and inflammatory/immune cells, and augment cytotoxicity.

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Abbreviations

BSA:

Bovine serum albumin

C-C:

Cysteine-cysteine

C-x-C:

Cysteine-x-cysteine

CCR-2:

C-C chemokine receptor-2

CXCR-1:

C-x-C chemokine receptor-1

ELISA:

Enzyme-linked immunosorbent assay

IL-1:

Interleukin-1

IL-6:

Interleukin-6

IL-8:

Interleukin-8

MCP-1:

Macrophage chemoattractant protein-1

MTT:

(3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide

PBS:

Phosphate-buffered saline

ROS:

Reactive oxygen species

SCLC:

Small-cell lung cancer

TNF-α:

Tumor necrosis factor-alpha

TNFRI :

Type I tumor necrosis factor-alpha receptor

TNFRII:

Type II tumor necrosis factor-alpha receptor

uPA:

Urokinase-type plasminogen activator

uPAR:

Urokinase-type plasminogen activator receptor

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Acknowledgements

This work was supported in part by a Grant-in-Aid (no. 12771476) for Encouragement of Young Scientists (to M.S.) and a Grant-in-Aid (no. 10670937) for Scientific Research (C) (to K.N.) from the Ministry of Education, Sciences, Sports and Culture, Japan. Northern blotting was carried out in the Radioisotope Center of Okayama University.

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Authors and Affiliations

  1. Department of Medicine II, Okayama University Medical School, 2-5-1 Shikatacho, 700-8558, Okayama, Japan

    Masami Niiya, Kenji Niiya, Toru Kiguchi, Noboru Asaumi, Katsuji Shinagawa, Fumihiko Ishimaru, Katsuyuki Kiura, Kazuma Ikeda, Hiroshi Ueoka & Mitsune Tanimoto

  2. Faculty of Health Sciences, Okayama University Medical School, Okayama, Japan

    Misako Shibakura

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  1. Masami Niiya
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Correspondence to Kenji Niiya.

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Niiya, M., Niiya, K., Kiguchi, T. et al. Induction of TNF-α, uPA, IL-8 and MCP-1 by doxorubicin in human lung carcinoma cells. Cancer Chemother Pharmacol 52, 391–398 (2003). https://doi.org/10.1007/s00280-003-0665-1

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  • DOI: https://doi.org/10.1007/s00280-003-0665-1

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