Doxycycline reduces the migration of tuberous sclerosis complex-2 null cells - effects on RhoA-GTPase and focal adhesion kinase

Abstract

Lymphangioleiomyomatosis (LAM) is associated with dysfunction of the tuberous sclerosis complex (TSC) leading to enhanced cell proliferation and migration. This study aims to examine whether doxycycline, a tetracycline antibiotic, can inhibit the enhanced migration of TSC2-deficient cells, identify signalling pathways through which doxycycline works and to assess the effectiveness of combining doxycycline with rapamycin (mammalian target of rapamycin complex 1 inhibitor) in controlling cell migration, proliferation and wound closure. TSC2-positive and TSC2-negative mouse embryonic fibroblasts (MEF), 323-TSC2-positive and 323-TSC2-null MEF and Eker rat uterine leiomyoma (ELT3) cells were treated with doxycycline or rapamycin alone, or in combination. Migration, wound closure and proliferation were assessed using a transwell migration assay, time-lapse microscopy and manual cell counts respectively. RhoA-GTPase activity, phosphorylation of p70S6 kinase (p70S6K) and focal adhesion kinase (FAK) in TSC2-negative MEF treated with doxycycline were examined using ELISA and immunoblotting techniques. The enhanced migration of TSC2-null cells was reduced by doxycycline at concentrations as low as 20 pM, while the rate of wound closure was reduced at 2-59 μM. Doxycycline decreased RhoA-GTPase activity and phosphorylation of FAK in these cells but had no effect on the phosphorylation of p70S6K, ERK1/2 or AKT. Combining doxycycline with rapamycin significantly reduced the rate of wound closure at lower concentrations than achieved with either drug alone. This study shows that doxycycline inhibits TSC2-null cell migration. Thus doxycycline has potential as an anti-migratory agent in the treatment of diseases with TSC2 dysfunction.

Keywords: RhoA-GTPase; doxycycline; focal adhesion kinase; migration; pulmonary lymphangioleiomyomatosis; rapamycin; tuberous sclerosis gene complex-2; wound closure.