Antiproliferative and Antimigratory Effects of Enoxaparin on the Lung Adenocarcinoma A549 Cell Line
Authors: Alturkistani Azhaar , Power‐Charnitsky Verna‐Ann , Ghonem Nisanne , Migliore Mattia M 
Published: November 2023

Cancer patients have an increased risk of developing venous thromboembolism (VTE) and have worse outcomes. Cancer can manifest in hypercoagulation by increasing many components of the coagulation system. These components exert their oncogenic signaling via the protease activated receptors (PARs). PAR1 and PAR2 are upregulated in many human cancers, and studies have shown a strong correlation between tumor aggressiveness and PARs expression. PAR activation triggers different downstream signaling cascades resulting cell proliferation, migration, and metastasis. Since different proteases influence cancer progression through PARs, and that anticoagulants such as the low molecular weight heparins (LMWH) target these proteases, using these agents may improve prognosis. Furthermore, studies have shown that LMWH have significantly improved overall survival rates in variety of malignancies independently of their anticoagulant function.

Objectives

The aim of this study is to investigate the antiproliferative and antimigratory effects of enoxaparin in A549 cell line and to establish the involvement of PAR1 to these effects. Furthermore, to assess the ability of enoxaparin to improve efficacy of the chemotherapeutic agent doxorubicin in A549 and the potential mechanism involved.

Methods

Cell proliferation was assessed by BrdU ELISA assay. PAR1, PAR2, ERK1/2 and Akt expression was measured by western blot. PAR1 was knockdown by siRNA and qPCR was used to measure mRNA level of MMP2 and PAR1. A549 cell migration was measured using 24‐transwell assay.

Results

Enoxaparin (22, 44 and 66 mM) inhibited A549 cell proliferation by 10, 13 & 15% respectively. Also, enoxaparin significantly inhibited the A549 cell migration at 44 and 66 mM concentrations. A549 cell line expressed PAR1 and very low levels of PAR2. Silencing PAR1 resulted in almost 50% reduction in A549 cell proliferation with or without enoxaparin treatment compared to the control. In addition, enoxaparin inhibited activation and phosphorylation of the downstream signaling markers of PAR1, Akt and ERK1/2. MMP2 gene expression was significantly inhibited by the 44 and 66 mM enoxaparin treatment. Combination treatment of 100nM doxorubicin and 44mM enoxaparin reduced cell proliferation by an average of 15% compared to doxorubicin alone.

Conclusions

Enoxaparin inhibited proliferation of A549 lung cancer cells in vitro. The antiproliferative effect of enoxaparin is mediated via PAR1 inhibition. Furthermore, enoxaparin reduced A549 cancer cell migration in vitro, an effect that appears to be mediated via a reduction of MMP2 expression. Combination therapy with enoxaparin improved the antiproliferative effects of doxorubicin in A549 cells vs doxorubicin monotherapy. The mechanism by which enoxaparin enhances the antiproliferative effect of doxorubicin is yet to be elucidated.

Support or Funding Information

We gratefully acknowledge the financial support provided by the MCPHS University‐Boston Office of the Provost