Metastasis of Breast Tumor Cells to Brain Is Suppressed by Phenethyl Isothiocyanate in a Novel <i>In Vivo</i> Metastasis Model

<div><p>Breast tumor metastasis is a leading cause of cancer-related deaths worldwide. Breast tumor cells frequently metastasize to brain and initiate severe therapeutic complications. The chances of brain metastasis are further elevated in patients with HER2 overexpression. In the current study, we evaluated the anti-metastatic effects of phenethyl isothiocyanate (PEITC) in a novel murine model of breast tumor metastasis. The MDA-MB-231-BR (BR-brain seeking) breast tumor cells stably transfected with luciferase were injected into the left ventricle of mouse heart and the migration of cells to brain was monitored using a non-invasive IVIS bio-luminescent imaging system. In order to study the efficacy of PEITC in preventing the number of tumor cells migrating to brain, mice were given 10 µmol PEITC by oral gavage for ten days prior to intra-cardiac injection of tumor cells labeled with quantum dots. To evaluate the tumor growth suppressive effects, 10 µmol PEITC was given to mice every day starting 14^th day after intra-cardiac cell injection. Based on the presence of quantum dots in the brain section of control and treated mice, our results reveal that PEITC significantly prevented the metastasis of breast cancer cells to brain. Our results demonstrate that the growth of metastatic brain tumors in PEITC treated mice was about 50% less than that of control. According to Kaplan Meir’s curve, median survival of tumor bearing mice treated with PEITC was prolonged by 20.5%. Furthermore as compared to controls, we observed reduced HER2, EGFR and VEGF expression in the brain sections of PEITC treated mice. To the best of our knowledge, our study for the first time demonstrates the anti-metastatic effects of PEITC <i>in vivo</i> in a novel breast tumor metastasis model and provides the rationale for further clinical investigation.</p></div

Growth suppression of tumor cells in brain.

<p>(A) The MDA-MB-231 (BR) breast cancer cells that reach brain start developing tumors after 14^th day of intra-cardiac injection. The PEITC (10 µmol by oral gavage) treatment started on 14^th day of tumor cell implantation and mice were imaged periodically. Luminescence signal from brain was collected using IVIS <i>in vivo</i> animal imager. (B) Average luminescence after quantification of the signal from mice brain and plotted against time (days) to obtain tumor growth curve. The arbitrary units were used for luminescence intensity quantification. The change in signal intensity from each mouse brain was calculated relative to the initial signal observed on day 14. * P<0.05, statistically different when compared with control. Results are presented as mean ± SD of triplicates.</p

Effect of PEITC on the expression of HER2, EGFR and VEGF.

<p>The brain sections from control and PEITC treated groups were immunostained with HER2, EGFR, VEGF antibodies and DAPI for nuclear staining after fixation, permeabilization and blocking the tumor section. The images were taken using fluorescence microscope (Olympus Inc., Center valley, PA). The expression for HER2 (Red), EGFR (Red) and VEGF (Green) was quantitated using SlideBook software (Intelligent Imaging Innovations Inc., Denver, CO, USA). DAPI was used as internal control. * P<0.05, statistically different when compared with control. Results are presented as mean ± SD of triplicates.</p

PEITC suppresses the growth of ovarian tumor xenografts by inhibiting EGFR-AKT pathway.

<p>SKOV-3 tumor cells were implanted into athymic nude mice and randomized into two groups. Mice received PBS or 12 µmol PEITC by oral gavage every day until day 42. (A–i) Effect of PEITC on tumor growth. (A-ii) Tumor weight from control and treatment groups (B) Inhibition of EGFR signaling in the tumors of mice administered with PEITC. Tumors from control and treated mice were excised at day 42, lysed and analyzed by western blotting for p-EGFR (Tyr-1068), EGFR, p-AKT (Ser-473), AKT, Cl-Caspase 3 and Cl-PARP. Blots were stripped and reprobed with actin antibody to verify equal protein loading. Each lane represents a different tumor sample. (C) Densitometric quantitation of western blotting represented above. The differences between the groups were compared by student’s t-test. Statistical tests were two sided. *p<0.05 when compared to control.</p

Reduction of brain metastasis.

<p>(A) Presence of MDA-MB-231 (BR) breast cancer cell labeled with quantum dot in the brain of mice as seen under fluorescent microscope. (B) Luminescence decay curve from mice brain starting the day of cell injection till day 10 after intra-cardiac injection of MDA-MB-231 (BR) breast cancer cells. (C) Mice brain images as analyzed by IVIS Lumina imaging system at different time points after intra-cardiac injection of MDA-MB-231 (BR) cells. (D) Average count of quantum dot labeled MDA-MB-231 (BR) tumor cells in mice brain of control and PEITC (10 µmol) treated groups. Values are represented as means±SEM. * P<0.05, statistically different when compared with control. At least 150 brain sections from each group were analyzed.</p

TGF treatment of AKT overexpression overrides the effects of PEITC.

<p>(A) Representative blot of time dependent effect of 15 µM PEITC on p-EGFR (Tyr 1068), p-AKT (Ser-473), Cl-PARP in SKOV-3 ovarian cancer cells. Actin was used as loading control. (B) OVCAR-3 cells were stimulated with 50 ng/mL TGF for 1 hour after treatment with varying concentrations of PEITC for 24 hours. Whole-cell lysates were resolved on 10% SDS-PAGE for the analysis of phosphorylation of EGFR at Tyr-1068, phosphorylation of AKT at Ser-473, and cleavage of caspase-3 and PARP. Actin was used as a control for loading. Effect of AKT overexpression on PEITC induced apoptosis was also determined in SKOV-3 cells. (C) Representative blots showing p-AKT (Ser 473), AKT and Cl-PARP. Actin was used as a loading control.</p

PEITC inhibits the activation of EGFR in ovarian cancer cells.

<p>Representative blots showing the concentration dependent effect of PEITC on p-EGFR (Tyr-1068) and EGFR in (A) SKOV-3 and (B) OVCAR-3 ovarian cancer cells. Actin was used as loading control. Each *p<0.05 when compared to control.</p

PEITC increased the survival of mice bearing tumors in brain.

<p>After two weeks of intra-cardiac injection of MDA-MB-231 (BR) cells, mice in treatment group were gavaged with 10 µmol PEITC orally every day till all the mice from control group were dead. Based on the data obtained, percent mice surviving at each time point were plotted using Kaplan Meier’s survival curve using Prism 5.0 (GraphPad software Inc., San Diego, CA). All the control mice bearing brain metastasis of MDA-MB-231 (BR) breast tumor cells died between 38–40 days after injection. * P<0.05, statistically different when compared with control.</p

PEITC inhibits AKT in ovarian cancer cells.

<p>Representative blots of the concentration dependent effect of PEITC on p-AKT (Ser-473) and AKT in (A) SKOV-3, (B) OVCAR-3 and (C) TOV-21G ovarian cancer cells. Actin was used as loading control. *p<0.05 when compared to control. (D) Concentration dependent effect of PEITC for 24 h on AKT kinase activity as represented by phosphorylation of GSK at Ser-21/9 in OVCAR-3 or TOV-21G cells.</p

PEITC treatment inhibits mTOR, Raptor and Rictor of mTORC1 and mTORC2 complexes.

<p>(A) Representative blots of the concentration dependent effect of PEITC (0–7.5 µM) on p-mTOR (Ser-2481), Rictor and Raptor on SKOV-3 ovarian cancer cells. Actin was used as loading control. (B) SKOV3 cells treated with 5 µM PEITC for 24 h were immunoprecipitated with mTOR antibody. The blots were probed with antibodies for Rictor and Raptor.</p