Specific roles for the PI3K and the MEK-ERK pathway in IGF-1-stimulated chemotaxis, VEGF secretion and proliferation of multiple myeloma cells: study in the 5T33MM model - PubMed (original) (raw)

Specific roles for the PI3K and the MEK-ERK pathway in IGF-1-stimulated chemotaxis, VEGF secretion and proliferation of multiple myeloma cells: study in the 5T33MM model

E Menu et al. Br J Cancer. 2004.

Abstract

Insulin-like growth factor-1 (IGF-1) has been described as an important factor in proliferation, cell survival and migration of multiple myeloma (MM) cells. Angiogenesis correlates with development and prognosis of the MM disease. Vascular endothelial growth factor (VEGF) is one of the prominent factors involved in this process. The different functions of IGF-1 were investigated in the 5TMM mouse model with emphasis on proliferation, migration and VEGF secretion, and the signalling pathways involved. Western Blot analysis revealed that ERK1/2 and Akt (PKB) were activated after IGF-1 stimulation. The activation of ERK1/2 was reduced by the PI3K inhibitor Wortmannin, implying that the PI3K pathway is involved in its activation. Insulin-like growth factor-1 induced an increase in DNA synthesis in MM cells, which was mediated by a PI3K/Akt-MEK/ERK pathway. Insulin-like growth factor-1 enhanced F-actin assembly and this process was only PI3K mediated. Stimulation by IGF-1 of VEGF production was reduced by PD98059, indicating that only the MEK-ERK pathway is involved in IGF-1-stimulated VEGF production. In conclusion, IGF-1 mediates its multiple effects on MM cells through different signal transduction pathways. In the future, we can study the potential in vivo effects of IGF-1 inhibition on tumour growth and angiogenesis in MM.

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Figures

Figure 1

Figure 1

Activation of the MEK–ERK and the PI3K pathway after IGF-1 stimulation. (A) 5T33MM cells were stimulated with increasing concentrations of IGF-1 for 10 min. (B) 5T33MM cells were stimulated during different periods of time with 100 ng ml−1 IGF-1. Equivalent amounts of lysates were immunoblotted with anti-P-ERK1/2 (first panel), P-Akt (third panel), P-Jnk (fifth panel) and P-p38 (seventh panel) and reblotted with anti-ERK1/2 (second panel), anti-Akt (fourth panel), anti-JNK (sixth panel) and anti-p38 (eigth panel) to confirm equal loading. One experiment representative of four is shown.

Figure 2

Figure 2

Crosstalk between the PI3K pathway and the MEK–ERK pathway. The MEK inhibitors PD98059 and UO126 abolish stimulation by IGF-1 of ERK phosphoryation but have no influence on the phosphorylation of Akt (first and third panel). The inhibitors of the PI3K pathway Wortmannin and Ly294002, on the other hand, inhibit the phosphorylation of Akt (PKB), confirming that Akt becomes phosphorylated through activation of the PI3K pathway (third panel), but also reduces the phosphorylation of ERK1 and 2 (first panel). The cells were stimulated with 100 ng ml−1 IGF-1 for 10 min and lysates were treated as in the previous figure. One experiment representative of four is shown.

Figure 3

Figure 3

IGF-1 induced DNA synthesis. For the thymidine incorporation assays, the MM cells were incubated with RPMI in the absence or the presence of 10 ng ml−1 IGF-1. Before stimulation with or without IGF-1, the cells were preincubated for 30 min with Wortmannin (100 n

M

), PD98059 (20 μ

M

) or both where indicated. Mean values±s.d. for four independent experiments are shown. (*: P<0.01 vs control, **: P<0.01 vs IGF-1, ***: P<0.01 vs IGF-1).

Figure 4

Figure 4

IGF-1 induced F-actin assembly. The F-actin content of the MM cells was measured by FACS analysis. The mean fluorescence intensity is shown as the relative value compared to unstimulated cells. The cells were stimulated with or without 100ng ml−1 IGF-1, after a 30 min incubation with Wortmannin (100 n

M

) or PD98059 (20 μ

M

) where indicated. The cells were then labelled with phalloidin FITC. Mean values±s.d. for three independent experiments are shown (*: P<0.02 _vs_ control, **: _P_<0.04, ***: _P_>0.05 vs IGF-1).

Figure 5

Figure 5

Stimulation by IGF-1 of VEGF secretion. The 5T33MM cells were stimulated with or without 100 ng ml−1 IGF-1 for 24 h, after a 30 min incubation with Wortmannin (100 n

M

) or PD98059 (20 μ

M

) where indicated. Concentrations of VEGF are shown relative to unstimulated cells. The maximum IGF-1-stimulated VEGF secretion reaches 250pg ml−1. Mean values±s.d. for three independent experiments are shown (*: P<0.01 _vs_ control, **: _P_<0.01, ***: _P_>0.05 vs 100 ng ml−1 IGF-1).

Figure 6

Figure 6

IGF-1 stimulates VEGF mRNA expression. The MM cells were stimulated with 100 ng ml−1 IGF-1 for 6 or 24 h. The relative amount of VEGF in the IGF-1-stimulated samples compared to the control samples is shown. Error bars represent s.d. in the experiment. One experiment representing three is illustrated, P<0.05.

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