Prostate cancer cells (LNCaP) generated after long-term interleukin 6 (IL-6) treatment express IL-6 and acquire an IL-6 partially resistant phenotype.
OBJECTIVE
The levels of interleukin-6 (IL-6) are frequently elevated in sera from patients with advanced prostate carcinoma. Our main objective was to investigate changes in responsiveness to IL-6 and/or androgen that occur in LNCaP cells after long-term treatment with IL-6. This in vitro model could be of clinical relevance because of its similarity with late-stage prostate carcinoma.


METHODS
LNCaP human prostate cancer cells were treated with IL-6 at a concentration of 5 ng/ml. After 20 passages, the new subline LNCaP-IL-6+ has been established. Passages 20-40 are referred to as low passages (LP) and passages 41-73 as high passages (HP). LNCaP cells passaged at the same time in the absence of IL-6 were used as controls (LNCaP-IL-6-). Cells were counted after treatment with either IL-6 or the synthetic androgen methyltrienolone (R1881), and cell cycle analysis was performed. Binding of IL-6 or R1881 was assessed by radioligand binding assays. Reporter gene activity was measured by chloramphenicol acetyltransferase assay. Prostate-specific antigen in LNCaP-IL-6+ supernatants was measured by an enzyme immunoassay. Expression of IL-6 mRNA and protein was assessed by reverse transcription-PCR and ELISA, respectively.


RESULTS
The basal proliferation rate in HP LNCaP-IL-6+ cells was higher than that in LNCaP-IL-6- cells. IL-6 inhibited proliferation of LNCaP-IL-6- cells but not that of either LP or HP of LNCaP-IL-6+ cells. This inability to elicit a growth-inhibitory response was associated with lack of effect on cell cycle distribution in the LNCaP-IL-6+ subline. In parallel, IL-6 binding decreased gradually during long-term IL-6 treatment and, in HP, reached only 33% of the levels measured in controls. Binding of radiolabeled androgen increased 2-fold in HP LNCaP-IL-6+ cells. Reporter gene assays revealed that R1881, at nanomolar concentrations, was a more potent androgen receptor activator in LNCaP-IL-6+ than in LNCaP-IL-6- cells. However, androgen- and IL-6-induced prostate-specific antigen secretion decreased in long-term IL-6-treated cells. IL-6 cDNA fragments were detected by reverse transcription-PCR in HP LNCaP-IL-6+ cells but not in controls or LP. IL-6 protein was first detected in passage 36 of LNCaP-IL-6+ cells, and it increased in HP.


CONCLUSIONS
Long-term treatment of LNCaP human prostate cancer cells with IL-6 leads to abolishment of inhibitory growth response. In contrast to control cells, the LNCaP-IL-6+ subline expresses IL-6 mRNA and protein.
Hobisch
A
A
Department of Urology, University of Innsbruck, Innsbruck A-6020, Austria.
Ramoner
R
R
Fuchs
D
D
Godoy-Tundidor
S
S
Bartsch
G
G
Klocker
H
H
Culig
Z
Z
D000728:Androgens / Q000378:metabolism / Q000494:pharmacology; D001667:Binding, Competitive; D002452:Cell Count; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D008297:Male; D015741:Metribolone / Q000378:metabolism / Q000494:pharmacology; D017430:Prostate-Specific Antigen / Q000187:drug effects / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014316:Tritium; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D000728:Androgens; D015850:Interleukin-6; D012333:RNA, Messenger; D014316:Tritium; D015741:Metribolone; D017430:Prostate-Specific Antigen