Forskolin improves sensitivity to doxorubicin of triple negative breast cancer cells via Protein Kinase A-mediated ERK1/2 inhibition

Abstract

Triple negative breast cancer (TNBC) is an invasive, metastatic, highly aggressive tumor. Cytotoxic chemotherapy represents the current treatment for TNBC. However, relapse and chemo-resistance are very frequent. Therefore, new therapeutic approaches that are able to increase the sensitivity to cytotoxic drugs are needed. Forskolin, a natural cAMP elevating agent, has been used for several centuries in medicine and its safeness has also been demonstrated in modern studies. Recently, forskolin is emerging as a possible novel molecule for cancer therapy. Here, we investigate the effects of forskolin on the sensitivity of MDA-MB-231 and MDA-MB-468 TNBC cells to doxorubicin through MTT assay, flow cytometry-based assays (cell-cycle progression and cell death), cell number counting and immunoblotting experiments. We demonstrate that forskolin strongly enhances doxorubicin-induced antiproliferative effects by cell death induction. Similar effects are observed with IBMX and isoproterenol cAMP elevating agents and 8-Br-cAMP analog, but not by using 8-pCPT-2'-O-Me-cAMP Epac activator. It is important to note that the forskolin-induced potentiation of sensitivity to doxorubicin is accompanied by a strong inhibition of ERK1/2 phosphorylation, is mimicked by ERK inhibitor PD98059 and is prevented by pre-treatment with Protein Kinase A (PKA) and adenylate cyclase inhibitors. Altogether, our data indicate that forskolin sensitizes TNBC cells to doxorubicin via a mechanism depending on the cAMP/PKA-mediated ERK inhibition. Our findings sustain the evidence of anticancer activity mediated by forskolin and encourage the design of future in-vivo/clinical studies in order to explore forskolin as a doxorubicin sensitizer for possible use in TNBC patients.

Keywords: Doxorubicin; Forskolin; Natural compounds; Protein Kinase A; Triple negative breast cancer.