Alendronate-induced disruption of actin cytoskeleton and inhibition of migration/invasion are associated with cofilin downregulation in PC-3 prostate cancer cells.

Sanna S Virtanen,Jouko A Sandholm,Eliisa Löyttyniemi,Tamiko Ishizu,Pirkko L Härkönen,Johanna M Tuomela,H Kalervo Väänänen

doi:10.18632/oncotarget.25961

Sanna S Virtanen, Jouko A Sandholm + Show 5 more

Open Access

https://doi.org/10.18632/oncotarget.25961

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Abstract

Bisphosphonates are used for prevention of osteoporosis and metastatic bone diseases. Anti-invasive effects on various cancer cells have also been reported, but the mechanisms involved are not well-understood. We investigated the effects of the nitrogen-containing bisphosphonate alendronate (ALN) on the regulation of actin cytoskeleton in PC-3 cells. We analyzed the ALN effect on the organization and the dynamics of actin, and on the cytoskeleton-related regulatory proteins cofilin, p21-associated kinase 2 (PAK2), paxillin and focal adhesion kinase. Immunostainings of cofilin in ALN-treated PC-3 cells and xenografts were performed, and the role of cofilin in ALN-regulated F-actin organization and migration/invasion in PC-3 cells was analyzed using cofilin knockdown and transfection. We demonstrate that disrupted F-actin organization and decreased cell motility in ALN-treated PC-3 cells were associated with decreased levels of total and phosphorylated cofilin. PAK2 levels were also lowered but adhesion-related proteins were not altered. The knockdown of cofilin similarly impaired F-actin organization and decreased invasion of PC-3 cells, whereas in the cells transfected with a cofilin expressing vector, ALN treatment did not decrease cellular cofilin levels and migration as in mock transfected cells. ALN also reduced immunohistochemical staining of cofilin in PC-3 xenografts. Our results suggest that reduction of cofilin has an important role in ALN-induced disruption of the actin cytoskeleton and inhibition of the PC-3 cell motility and invasion. These data also support the idea that the nitrogen-containing bisphosphonates could be efficacious in inhibition of prostate cancer invasion and metastasis, if delivered in a pharmacological formulation accessible to the tumors.

Highlights

Metastatic bone lesions are commonly associated with breast and prostate cancers, affecting approximately 65–80% of patients with an advanced disease [1]
Our results demonstrate that ALNinduced inhibition of invasion/migration, and disruption of F-actin organization in prostate cancer cells were associated with markedly lowered levels of cofilin protein in vitro, and in an in vivo tumor model
The effect of ALN on actin and paxillin dynamics was studied using fluorescence recovery after photobleaching (FRAP) with GFP-actin or GFP-paxillin-transfected PC-3 cells. 48 hours after transfection, PC-3 cells were treated with 10-11 or 10-5 M ALN for 3 hours

Summary

Introduction

Metastatic bone lesions are commonly associated with breast and prostate cancers, affecting approximately 65–80% of patients with an advanced disease [1]. Nitrogen-containing BPs (N-BPs; e.g. alendronate (ALN) and zoledronate) www.oncotarget.com are more effective inhibitors of bone resorption, and their effects are largely mediated via inhibition of the mevalonate pathway and isoprenylation of important small GTPases such as the Ras, Rac and Rho family proteins [6]. Both farnesyl diphosphate synthase [7, 8] and geranylgeranyl diphosphate synthase [9] have been identified as N-BP targets in osteoclasts. In vivo N-BPs have immunomodulatory [20,21,22], apoptotic, antimetastatic and tumor growth inhibiting effects [17, 23, 24]

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