Sorafenib and docosahexaenoic acid act in synergy to suppress cancer cell viability: a role of heme oxygenase 1

Abstract

Background: Docosahexaenoic acid (DHA) is a long chain n-3 polyunsaturated fatty acid that has anticancer activity. Heme oxygenase 1 (HO-1) is a potential therapeutic target due to its cytoprotective activity in cancer cells. We recently reported that DHA induces HO-1 gene transcription in human cancer cells by augmenting the degradation of Bach1 protein, which functions as a negative regulator of HO-1. Since the degradation of Bach1 protein relies on protein phosphorylation, we hypothesized that DHA-induced HO-1 gene transcription could be attenuated by kinase inhibitors, resulting in an enhanced cytotoxicity. Sorafenib, a tyrosine kinase inhibitor, was first applied to test our hypothesis.

Methods: Human cancer cell lines and a xenograft nude mouse model were applied to test our hypothesis. Gene expression was analyzed by western blot analysis and reporter gene assay. Cell viability was analyzed using a colorimetric assay. Isobologram was applied to analyze drug action.

Results: Pretreatment of cancer cells with Sorafenib significantly attenuated DHA-induced degradation of Bach1 protein. Consequently, DHA-induced HO-1 gene transcription was reversed by Sorafenib as evidenced by western blot and reporter gene analysis. Sorafenib acted synergistically with DHA to suppress cancer cell viability in various human cancer cell lines and suppressed tumor xenograft growth in mice fed a fish oil enriched diet (high n-3/DHA), as compared to mice fed a corn oil (high n-6) diet. Screening of the NCI-Oncology Drug Set IV identified a group of anticancer compounds, including Sorafenib, which enhanced DHA's cytotoxicity, as well as a set of compounds that attenuated DHA's cytotoxicity.

Conclusions: We demonstrate that sorafenib attenuates DHA-induced HO-1 expression and acts in synergy with DHA to suppress cancer cell viability and tumor growth. Considering the known health benefits of DHA and the clinical effectiveness of Sorafenib, their combination is an attractive therapeutic strategy against cancer.

Keywords: Cancer; Docosahexaenoic acid; Heme oxygenase 1; Sorafenib; Synergy.

Fig. 1

Sorafenib reverses DHA-induced suppression of nuclear Bach1 protein expression and induction of HO-1 transcription in A2780 cells. a Cells were pretreated with Sorafenib for 1 h prior to addition of DHA for 18 h. Nuclear proteins were isolated and Bach1 expression was determined by Western blot (n = 3). b Cells were pretreated with Sorafenib for 1 h prior to addition of DHA for 24 h. Cell lysates were prepared and HO-1 expression was determined by Western blot (n = 3). c Cells were transfected with the pGL3/4.5-HO-1 luciferase reporter construct and pretreated with Sorafenib for 1 h prior to addition of DHA for 24 h. Luciferase activity was determined and expressed relative to untreated controls (n = 3, mean ± SEM, *p < 0.01)

Fig. 2

Sorafenib enhances DHA’s cytotoxicity in A2780 cells. a Cells were treated with Sorafenib and DHA for 48 h and cell viability was determined by the MTS assay (n = 3, mean ± SEM, *p < 0.01). b Bach1 was knocked down in A2780 cells using targeted siRNAs. Cells were treated with Sorafenib and DHA for 48 h and cell viability was determined by the MTS assay (n = 3, mean ± SEM, *p < 0.01)

Fig. 3

Sorafenib enhances DHA’s cytotoxicity in DU-145, MCF7, and MDA-MB-231 cancer cells. Cells were treated with Sorafenib and/or DHA for 48 h and cell viability was determined by the MTS assay (n = 3, means ± SEM, *p < 0.01)

Fig. 4

Sorafenib enhances DHA’s cytotoxicity in a concentration- and time-dependent manner in MDA-MB-231 cells. Cells were treated with Sorafenib and DHA at different concentrations for 48 h a or for different durations b Cell viability was determined by the MTS assay (n = 3, means ± SEM, *p < 0.01)

Fig. 5

Sorafenib and DHA act in synergy to suppress MDA-MB-231 cell viability. a Cells were treated with increasing concentrations of Sorafenib and DHA at a fixed ratio of 1:100, determined by the IC₅₀ values of each compound added to cells alone. Cell viability was analyzed using the MTS assay. b An isobologram was plotted using the IC₅₀ values of Sorafenib and DHA alone on the Y- and X-axes, respectively. Experimentally derived values from combination treatment lying beneath the addition line indicate a synergistic interaction (n = 3)

Fig. 6

Sorafenib and DHA act together to suppress xenograft tumor growth. MDA-MB-231 cells were implanted into the flanks of nude mice fed either an n-3 or n-6 enriched diet. The xenograft tumor growth, indicated by tumor volume, in mice that were treated either with the vehicle alone (a) or with Sorafenib (b) are shown (n = 5, means ± SEM, *p < 0.01)