1. {Non Fuctional Ikkβ (canonical pathway)}
cytokine effects on the cell cycle and death of lung and prostate carcinoma cells
V.Galani (1), M.Kastamoulas (1), G.Chondrogiannis (1), G.Vartholomatos (2),
E. Vlachopoulou(3), D.Arvanitis (5), T. Liloglou (6), E.Kolettas (4), N.Sofikitis (3)
and P. Kanavaros (1).
(1) Department of Anatomy-Histology, Faculty of Medicine, University of Ioannina, Greece (2) Laboratory of Hematology, University Hospital of Ioannina, Greece (3) Department of Urology, Faculty of Medicine, University of Ioannina, Greece (4) Department of Physiology, Faculty of Medicine, University of Ioannina, Greece
(5) Department of Anatomy, Faculty of Medicine, University of Thessalia, Greece (6) School of Cancer Studies, University of Liverpool, Liverpool, UK
Background
Methods
Since cytokines are involved in cell cycle/death regulation, we investigated the effects of TNFa, IL1β, IL13, IFNγ, IGF-1 and Fas/CD95 on cell cycle/death of A549 lung and LNCaP(androgen-dependent) and PC-3 (androgen-independent) prostate carcinoma cell lines
Flow cytometry (PI and PI/annexin) and Western blot were used for analysis of cell cycle/death (apoptotic and total cell death) and protein expression, respectively. Cell death was also analyzed by Hoechst and Crystal violet staining
a549
Sub-G1 %
Control
24h
0.35
CH-11 ( 200 ng/ml)
0.66
TNF-a ( 200 ng/ml)
1.35
TNF-a ( 200 ng/ml) + CH-11 (100 ng/ml)
1.69
TNF-a ( 200 ng/ml) pret. + CH-11 (200 ng/ml)
12h pret. +12h
1.49
Α549 GL2, RNAi luciferase
Sub-G1 (%)
Control 24h
4,98
CH h
9,29
TNF-α pretreatment 6h + CH-11 18h
9,61
IL-1β pretreatment 6h + CH-11 18h
8,63
TNF-α 24h
7,81
IL-1β 24h
4,90
A549, A1836 RNAi for Ikkα, clone 2 {Non fuctional Ikkα (non-canonical pathway)}
7,15
11,39
13,38
12,79
5,56
6,74
A549, P1 RNAi for Ikkβ, Clone 1
{Non Fuctional Ikkβ (canonical pathway)}
4,89
8,92
9,42
5,75
5,38
4,18
1. IL1β (500 pg/ml) pretreatment 24 hrs + CH11 (100 ng/ml) 24 hrs,
2. IFNγ (300 U/ml) pretreatment 24 hrs + CH11 (100 ng/ml) 24 hrs,
3. IL1β (500 pg/ml) 48 hrs, 4. IFNγ (300 U/ml) 48 hrs
1. Control 24 hrs, 2 Control 48 hrs
3. IFNγ (300 U/ml) 24 hrs
4. TNFα (100 ng/ml) 24 hrs
5. IFNγ (300 U/ml) 48 hrs
6. TNFα (100 ng/ml) 24 hrs
1. Control 24 hrs
2. CH-11 (100 ng/ml) 24 hrs
3. Il-1β (500 pg/ml) 24 hrs
4. IFN-γ (100 U/ml) 24 hrs
5. TNF-α (100 ng/ml) 24 hrs
6. Il-13 (100 ng/ml) 24 hrs
7. TNF-α (100 ng/ml) + CH-11 (100 ng/ml) 24 hrs
1. CH-11 (100 ng/ml) 24 hrs
2. Control 6 hrs
3. Il-1β (500 pg/ml) 6 hrs
4. Il-13 (100 ng/ml) 6 hrs
5. Control 24
1. Control 24 hrs
2. CH-11 (100 ng/ml) 24 hrs
3. IFN-γ (500 U/ml) 24 hrs
4. TNF-α (100 ng/ml) 24 hrs
5. IL-1β (100 pg/ml) 24 hrs
6. CH-11 (100 ng/ml) + TNF-α (100 ng/ml) 24 hrs
1. Control 24 hrs, 2. IFNγ (100 U/ml) 24 hrs
3. IFNγ (300 U/ml) 24 hrs, 4. IFNγ (500 U/ml) 24 hrs
5. IL1β (100 pg/ml) 24 hrs, 6. IL1β (200 pg/ml) 24 hrs
7. IL1β (500 pg/ml) 24 hrs
Table 1: PC-3
Sub-G1 %
Control
24h
2.03
CH-11 ( 200 ng/ml)
8.31
IGF-1 ( 200 ng/ml)
6.89
IGF-1 (200 ng/ml) pret. + CH-11 (200 ng/ml)
12h pret. + 12h
0.99
TNF-a ( 300 ng/ml)
5.49
TNF-a ( 300 ng/ml) pret. + CH-11 (200 ng/ml)
12h pret. +12h
4.82
IFN-γ (500 U/ml)
9.47
IFN-γ (500 U/ml) pret. + CH-11 (200 ng/ml)
30.93
IL-13 (400 ng/ml)
6.30
IL-13 (400 ng/ml)pret. + CH-11 (200 ng/ml)
5.99
(Table 1)
(Table 2)
Table 3: A549 CH-11 (100 ng/ml), TNF-α(100 ng/ml), IL-1β (500 pg/ml),
(Table 3)
Table 2: LNCaP
Results
Flow cytometry showed that a) the TNFa or IL1β or IL13 anti-cell death effects on Fas-induced A549 cell death were attenuated by inhibitors of NF-kB (BAY ), PI3-K (LY ), JNK (SP600125), P38 (SB203580) and ERK (UO126) pathways, b) TNFa or IL1β did not alter Fas-induced cell death in A549 cells with suppression of the canonical (ΙΚΚβ) or the non-canonical (ΙKΚα) NF-κB pathway, c) Fas or TNFa increased LNCaP cell death in comparison to control and d) TNFa increased Fas-induced LNCaP and PC-3 cell death.
Western blot showed a) cleaved PARP1 protein in Fas treated cells b) TRAF1 protein and decreased Ik-Bα protein expression in TNFa treated cells and c) no alterations of Fas, bcl2, bcl-xl, bax, bak and bad protein expression.
Conclusion
TNFa, IL1β and IL-13 decreased Fas-induced A549 cell death whereas TNFa increased Fas-induced LNCaP and PC-3 cell death. The anti-cell death effects of TNFa, IL1β and IL13 on Fas-induced A549 cell death were mediated, at least partially, by the NF-kB, PI3-K and MAP kinases pathways.
Investigation partially funded by PENED 2003