Efficient Endocytosis of the Human Lactoferrin N-Lobe Enhances Its Antiproliferative Activity against Human Cancer Cells.

Human lactoferrin (hLF) is a glycosylated globular iron-binding protein with high functional versatility that elicits anticancer, neuroprotective, and anti-inflammatory effects. Some of the diverse functions of hLF are induced after its internalization into various cells via cell surface endocytosis receptors, such as proteoglycans, which contain glycosaminoglycan (GAG) chains. We have previously demonstrated that an hLF derivative comprising the N-terminal half of hLF (referred to as the N-lobe) is internalized by intestinal enterocyte Caco-2 cells. However, the relationship between the intracellular uptake of the N-lobe and its pharmacological activity remains poorly understood. Here, we report that the N-lobe is efficiently internalized by lung cancer cells via endocytic pathways, suppressing their proliferation. Moreover, the N-lobe showed higher intracellular uptake than hLF. We found that the N-lobe was internalized into the human lung cancer cell lines PC-14 and PC-3 via clathrin- and/or caveolae-mediated endocytosis. Intracellular uptake of the N-lobe was inhibited when an equimolar concentration of chondroitin sulfate (CS)-E, a GAG subtype involved in malignant transformation and tumor metastasis, was added. The inhibitory effect of the N-lobe on PC-14 cell proliferation decreased with the addition of CS-E in a dose-dependent manner, suggesting that the CS-recognizing sequence on the N-lobe is necessary for its internalization or that the CS proteoglycan on cancer cells acts as an endocytosis receptor. These results suggest that the efficient endocytic uptake of the N-lobe is important for its antiproliferation effects on lung cancer cell lines. Thus, the N-lobe presents a promising drug candidate for cancer treatment.

Osthole inhibits proliferation and induces apoptosis in BV-2 microglia cells in kainic acid-induced epilepsy via modulating PI3K/AKt/mTOR signalling way.

CONTEXT: Osthole is a natural coumarin compound most frequently extracted from plants of the Apiaceae family such as Cnidium monnieri (L.) Cusson, Angelica pubescens Maxin.f., and Peucedanum ostruthium (L.). Osthole is considered to have potential therapeutic applications for the treatment of diseases including epilepsy. However, the mechanism of osthole induced-apoptosis in BV-2 microglia cells is not yet clear. OBJECTIVE: To investigate the molecular mechanisms underlying the effect of osthole on PI3K/AKt/mTOR expression in kainic acid (KA)-activated BV-2 microglia cells. MATERIALS AND METHODS: Optimal culture concentration and time of osthole were investigated by MTT assay. The concentration of osthole was tested from 10 to 400 µM and the culture time was tested from 2 to 72 h. Ultrastructure difference among control, KA and osthole group was analyzed under transmission electron microscope. The mRNA expression of PI3K/AKt/mTOR was investigated using reverse transcription (RT)-PCR and the protein expression was investigated using western blotting and immunofluorescence assay. Apoptosis rate of BV-2 cells between each group was measured by flow cytometry. RESULTS: IC50 for cell viability of BV-2 cells by osthole was 157.7 µM. Treated with osthole (140 µM) for 24 h significantly increased the inhibition rate. Pretreatment with osthole inhibited the KA-induced PI3K/AKt/mTOR mRNA and protein expression. The results of flow cytometry analysis showed that the apoptotic rate of osthole group was obviously higher than KA group. CONCLUSIONS: Date showed that osthole may be useful in the treatment of epilepsy and other neurodegenerative diseases that are characterized by over expression of PI3K/Akt/mTOR.