Co-Administration of Fish Oil With Signal Transduction Inhibitors Has Anti-Migration Effects in Breast Cancer Cell Lines, in vitro.

BACKGROUND: There is an urgent need for new therapies to treat cancer metastasis. Fish oil, with high omega 3 fatty acid content, has shown anticancer activity and signal transduction inhibitors have shown anti-metastatic properties. OBJECTIVE: To provide preliminary in vitro data on the anti-migration potential of signal transduction inhibitors and co-administered fish oil. METHODS: MCF-7, TamR and FasR breast cancer cell lines were used to determine the effects of combinations of PD98059, LY294002 and fish oil in growth assays. Modulations of p-Src and COX-2, both mediators of motility and invasion, were then determined by Western blotting and IHC to ascertain effects on migration potential. RESULTS: Migration rates for the three cell lines examined were ranked: FasR>TamR>MCF-7 (p <0.05). Addition of fish oil reduced the number of TamR cells migrating after 48h (p <0.05), while the addition of PD98059 and LY294002 also decreased migratory potential of TamR cells (p <0.05). Addition of PD98059 and LY294002 to TamR cells did not result in a significant decrease in p-Src levels; as was the case when PD98059, LY294002 and 4-hydroxytamoxifen were added to MCF-7 cells. However, the co-administration of fish oil markedly reduced p-Src and COX-2 expression in both cell lines. CONCLUSION: Co-administration of a commercial fish oil with signal transduction inhibitors results in decreased cell migration via an unknown co-operative mechanism and could constitute a novel approach for the treatment of breast cancer metastasis.

Insulin-like growth factor-dependent proliferation and survival of triple-negative breast cancer cells: implications for therapy.

Triple-negative breast cancers have a poor prognosis and are not amenable to endocrine- or HER2-targeted therapies. The prevailing view is that targeting the insulin-like growth factor (IGF) signal transduction pathway will not be beneficial for triple-negative breast cancers because their growth is not IGF-responsive. The present study investigates the importance of IGFs in the proliferation and survival of triple-negative breast cancer cells. Estrogen and progesterone receptors, HER2, type I IGF, and insulin receptors were measured by Western transfer analysis. The effects of IGF-1 on proliferation were assessed by DNA quantitation and on cell survival by poly (ADP-ribose) polymerase cleavage. The effect of IGF-1 on phosphorylation of the IGF receptors, Akt and mitogen-activated protein kinase, was measured by Western transfer analysis. Seven cell lines were identified as models of triple-negative breast cancer and shown to express IGF receptors at levels similar to those present in estrogen-responsive cell lines known to respond to IGFs. IGF-1 increased the proliferation and cell survival of all triple-negative cell lines. Proliferation was attenuated after reduction of type I IGF receptor expression. Cells that express higher levels of receptor were more sensitive to subnanomolar IGF-1 concentrations, but the magnitude of the effects was not correlated simply with the absolute amount or phosphorylation of the IGF receptors, Akt or mitogen-activated protein kinase. These results show that IGFs stimulate cell proliferation and promote cell survival in triple-negative breast cancer cells and warrant investigation of the IGF signal transduction pathway as a therapeutic target for the treatment of triple-negative breast cancer.

A novel diffusion cell model for the in vitro assessment of transcutaneous breast cancer therapeutics: effect of permeants on MCF-7 cells cultured within the receptor compartment.

A novel model is described for investigating the potential efficacy of topically delivered anti-breast cancer agents. Using all-glass Franz diffusion cells, the permeation of 4-hydroxytamoxifen, two EGFR inhibitors (PD98059 and LY294002) and eicosapentaenoic acid (EPA) were determined from a fish oil vehicle across Cyclopore track etched membrane (CTEM) alone, full-thickness porcine ear skin alone and CTEM plus full-thickness porcine ear skin. Finally, the effect of the simultaneous permeation of these compounds was determined on the breast cancer cell line, MCF-7, cultured directly into the diffusion cell receptor compartments. The CTEM was found to be not rate limiting, and all compounds permeated the skin, with a large excess of EPA. The applied combined dose reduced the growth of MCF-7 cells by 66% after 7days. The following conclusions were obtained: (1) MCF-7 breast cancer cells can be successfully cultured within glass Franz diffusion cells. (2) A composite diffusion cell/cell culture model can indicate the potential efficacy of topically delivered anti-breast cancer therapeutic agents. (3) The levels of LY294002, PD98059, 4-hydroxytamoxifen and EPA delivered across full-thickness skin have a major inhibitory effect on the growth of MCF-7 breast cancer cells.

In vitro delivery of anti-breast cancer agents directly via the mammary papilla (nipple).

The objective of this study was to investigate, in vitro, the plausibility of a novel method for delivering a combination of anti-breast cancer agents to the breast via the mammary papilla (nipple). Mammary papillae were prepared from freshly excised strips of porcine sow breasts by blunt dissection. Permeation studies were performed using all glass Franz diffusion cells in both upright and lateral position, with drugs examined individually and in combination. Donor phase was comprised of equimolar PD98059, LY294002 and tamoxifen; 2.54x10(-4) mol dissolved in 950 microL fish oil (containing approximately 23% (w/v) eicosapentaenoic acid, EPA), 25 microL DMSO and 25 microL 1,8-cineole. Also, 4 or 10% Cabosil M5P (w/v) was added to thicken the formulation. After 6 h, the papillae were recovered, cleaned, centrifuged and extracted thrice with methanol. Pooled extracts were analysed by reversed-phase HPLC. The significance of the papilla orientation was also investigated. When applied singly and laterally, the amount extracted from the porcine breast tissue for PD98059, LY294002 and tamoxifen were 1.83+/-0.30, 10.67+/-1.78 and 0.74+/-0.19x10(-2) micromol g(-1) respectively; applied simultaneously and laterally, 2.03+/-0.14, 4.86+/-0.47 and 0.22+/-0.04x10(-2) micromol g(-1) respectively. With 4% Cabosil formulation, amount extracted for PD98059 and LY294002 were 5.71+/-0.95 and 9.91+/-0.92x10(-2) micromol g(-1) respectively; with 10% formulation, 2.64+/-0.5 and 3.90+/-0.78x10(-2) micromol g(-1) respectively. Tamoxifen was below its limit of detection in both Cabosil M5P formulations. To conclude, localized passive delivery via the mammary papilla is a plausible non-invasive means of delivering anti-breast cancer drugs directly to the breast, in levels that have previously been shown to markedly inhibit the growth of breast cancer cell lines, in vitro. The amounts deliverable may be influenced by differential interactions with the thickening agent and patient orientation.

In vitro effects on MCF-7 breast cancer cells of signal transduction inhibitor/tamoxifen/eicosapentaenoic acid combinations and their simultaneous delivery across skin.

PURPOSE: To determine the in vitro effects of simultaneously administered LY29400, PD98059, tamoxifen and eicosapentaenoic acid (EPA) on breast cancer cells, and determine their transcutaneous delivery. METHODS: Growth assays were performed on MCF-7 cells challenged with IC(50) and permeated concentrations of PD98059, LY294002 and tamoxifen firstly in isolation then combined. Permeation studies were performed using PD98059 and LY294002 (singly or simultaneously) in DMSO then fish oil, with enhancers. Immunocytochemical detection of phospho-MAPK, phospho-Akt, total COX-2 and Ki-67 was performed. RESULTS: When applied singly, fluxes of PD98059 and LY294002 were 0.09 +/- 0.008 and 0.14 +/- 0.045 microg cm(-2) h(-1), respectively; applied simultaneously, 0.18 +/- 0.045 and 0.49 +/- 0.051 microg cm(-2) h(-1). Permeated concentrations of PD98059 and LY294002 reduced growth to 13.78 +/- 0.63%. Fish oil plus 2.5% DMSO/ethanol allowed 5.96 +/- 0.9 and 7.7 +/- 1.2 microg cm(-2) of PD98059 and LY294002 to permeate after 48 h. CONCLUSIONS: PD98059 and LY294002 permeate excised skin at therapeutically useful rates, and also demonstrate growth inhibitory effects on MCF-7 cancer cells. Synergism was noted in co-transport across skin and activity against cancer cells. A formulation based on fish oil is potentially skin friendly; simultaneous permeation of EPA provides further anti-cancer action.

Probing the skin permeation of fish oil/EPA and ketoprofen-3. Effects on epidermal COX-2 and LOX.

This work employed immunocytochemistry (ICC) techniques to study the effect of topically applied fish oil and ketoprofen on cyclooxygenase (COX-2) and lipoxygenase (LOX) within freshly excised porcine ear skin. Maintained in Hanks buffer immediately post excision, full thickness membranes were mounted in Franz diffusion cells and dosed with 1 ml of individual formulations containing ketoprofen, fish oil or both. At different timepoints, the diffused areas were recovered and relative activities of COX-2 and LOX determined. It was found that the fish oil formulation qualitatively inhibited the expression of both COX-2 and LOX enzymes. As expected, ketoprofen had no effect upon LOX expression but a significant decrease on COX-2 expression was observed. The formulation containing both fish oil and ketoprofen proved to be the most effective at inhibiting the expression of both COX-2 and LOX. Considered together with data from earlier papers, a mechanism of EPA permeation enhancement by ketoprofen may be elucidated and also show the ability of such a formulation to inhibit these enzymes and thus indicate the efficacy of such a formulation.