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

作者：Ng Ho Yin; Oliver Brian Gregory George; Burgess Janette Kay; Krymskaya Vera P; Black Judith Lee; Moir Lyn M^*

来源：JOURNAL OF CELLULAR AND MOLECULAR MEDICINE, 2015, 19(11): 2633-2646.

DOI：10.1111/jcmm.12593

• pulmonary lymphangioleiomyomatosis
• tuberous sclerosis gene complex-2
• doxycycline
• rapamycin
• migration
• wound closure
• RhoA-GTPase
• focal adhesion kinase

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 20pM, while the rate of wound closure was reduced at 2-59M. 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.

• 出版日期2015-11
• 单位河北医科大学