Induction of TNF-alpha, uPA, IL-8 and MCP-1 by doxorubicin in human lung carcinoma cells.
OBJECTIVE
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-alpha), 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-alpha, 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-alpha, 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-alpha 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-alpha 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 microM at which cell growth was slightly inhibited 24 h after treatment. Furthermore, the cells did not express receptors including types I and II TNF-alpha receptors, uPA receptor (uPAR), C-x-C-chemokine receptor-1 (CXCR-1), or C-C-chemokine receptor-2, corresponding to TNF-alpha, 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-alpha, 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-alpha 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-alpha, uPA, IL-8 and MCP-1 may enhance the interaction between tumor and inflammatory/immune cells, and augment cytotoxicity.
Niiya
Masami
M
Department of Medicine II, Okayama University Medical School, 2-5-1 Shikatacho, 700-8558 Okayama, Japan.
Niiya
Kenji
K
Kiguchi
Toru
T
Shibakura
Misako
M
Asaumi
Noboru
N
Shinagawa
Katsuji
K
Ishimaru
Fumihiko
F
Kiura
Katsuyuki
K
Ikeda
Kazuma
K
Ueoka
Hiroshi
H
Tanimoto
Mitsune
M
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D015703:Antigens, CD / Q000096:biosynthesis; D015152:Blotting, Northern; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism; D045744:Cell Line, Tumor; D018932:Chemokine CCL2 / Q000096:biosynthesis*; D004317:Doxorubicin / Q000494:pharmacology*; D005434:Flow Cytometry; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis*; D008175:Lung Neoplasms / Q000378:metabolism*; D009000:Monocytes / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D054390:Receptors, CCR2; D011956:Receptors, Cell Surface / Q000096:biosynthesis; D019707:Receptors, Chemokine / Q000096:biosynthesis; D023062:Receptors, Interleukin-8A / Q000096:biosynthesis; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D055293:Receptors, Urokinase Plasminogen Activator; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis*
D000903:Antibiotics, Antineoplastic; D015703:Antigens, CD; C515334:CCR2 protein, human; D018932:Chemokine CCL2; D016209:Interleukin-8; C524898:PLAUR protein, human; D054390:Receptors, CCR2; D011956:Receptors, Cell Surface; D019707:Receptors, Chemokine; D023062:Receptors, Interleukin-8A; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D055293:Receptors, Urokinase Plasminogen Activator; D014409:Tumor Necrosis Factor-alpha; D004317:Doxorubicin; D014568:Urokinase-Type Plasminogen Activator