Vitamin B(12), a chlorophyll-related analog to pheophytin a from marine brown algae, promotes neurite outgrowth and stimulates differentiation in PC12 cells

Abstract

We previously isolated an analog to chlorophyll-related compounds, pheophytin a, from the marine brown alga Sargassum fulvellum and demonstrated that it is a neurodifferentiation compound. In the current study, we investigated the effects of the pheophytin a analog vitamin B(12) on PC12 cell differentiation. In the presence of a low level of nerve growth factor (10 ng ml(-1)), vitamin B(12 )demonstrated neurite outgrowth-promoting activity in PC12 cells. The effect was dose-dependent in the range of 6-100 muM. In the absence of nerve growth factor, vitamin B(12) did not promote differentiation. To investigate the mechanism for this effect, we conducted differentiation assays and western blot analysis with signal transduction inhibitors and found that vitamin B(12) did not promote PC12 cell differentiation in the presence of K252a or U0126 inhibitors. These results suggest that vitamin B(12 )stimulates PC12 cell differentiation through enhancement of the mitogen-activated protein kinase signal transduction pathway, which is also induced by nerve growth factor. Thus, vitamin B(12) may be a good candidate for treatment of neurodegenerative diseases such as Alzheimer's disease.

Fig. 1

Dose-dependent effects of vitamin B₁₂ on the differentiation of PC12 cells and the cell morphology after treated with vitamin B₁₂. (A) dose-dependent effects of vitamin B₁₂ on the differentiation of PC12 cells. ······, positive control (50 ng ml⁻¹ NGF); ----, negative control (10 ng ml⁻¹ NGF). (B) photomicrographs of PC12 cells following treatment with vitamin B₁₂ (100 μM) and NGF (10 ng ml⁻¹); (C) negative control (10 ng ml⁻¹ NGF); (D) positive control (50 ng ml⁻¹ NGF); (E), treatment with vitamin B₁₂ (100 μM) only. Bar shows 10 μm

Fig. 2

Effects of specific inhibitors on the induction of PC12 cell differentiation by vitamin B₁₂. The neurite outgrowth-promoting activity of vitamin B₁₂ is shown as a percentage of the activity of NGF (50 ng ml⁻¹), the positive control

Fig. 3

Western blot analysis of MAPK family activation by vitamin B₁₂. (A) Phosphorylated MAPK was detected by western blot using anti-active ERK 1/2 antibody. (B) Total ERK was detected using anti-ERK 1/2 antibody. (C) Phosphorylated JNK was detected by western blot using anti-active JNK antibody. (D) Phosphorylated p38 was detected by western blot using anti-active p38 antibody. (E) Actin was detected with anti-actin antibody. Line 1: positive control (50 ng ml⁻¹ NGF); line 2: vitamin B₁₂ (100 μM) and NGF (10 ng ml⁻¹); line 3: negative control (10 ng ml⁻¹ NGF); line 4: untreated cells; line 5: control (1 M sorbitol). PC12 cell lysates were subjected directly to 12.5% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and immunostained with monoclonal antibodies. Actin was used as an internal control for PC12 cells