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. 2017 Aug 10;8(42):72260-72271.
doi: 10.18632/oncotarget.20139. eCollection 2017 Sep 22.

[10]-gingerol induces apoptosis and inhibits metastatic dissemination of triple negative breast cancer in vivo

Ana Carolina B M Martin #  1 , Angelina M Fuzer #  1 , Amanda B Becceneri  1 , James Almada da Silva  2 , Rebeka Tomasin  1 , Delphine Denoyer  3 , Soo-Hyun Kim  4 , Katherine A McIntyre  5 , Helen B Pearson  6 , Belinda Yeo  7 , Aadya Nagpal  7 , Xiawei Ling  4 , Heloisa S Selistre-de-Araújo  8 , Paulo Cézar Vieira  2 , Marcia R Cominetti #  1 , Normand Pouliot #  4   5   7
Affiliations

[10]-gingerol induces apoptosis and inhibits metastatic dissemination of triple negative breast cancer in vivo

Ana Carolina B M Martin et al. Oncotarget. .

Abstract

There is increasing interest in the use of non-toxic natural products for the treatment of various pathologies, including cancer. In particular, biologically active constituents of the ginger oleoresin (Zingiber officinale Roscoe) have been shown to mediate anti-tumour activity and to contribute to the anti-inflammatory, antioxidant, antimicrobial, and antiemetic properties of ginger. Here we report on the inhibitory properties of [10]-gingerol against metastatic triple negative breast cancer (TNBC) in vitro and in vivo. We show that [10]-gingerol concentration-dependently induces apoptotic death in mouse and human TNBC cell lines in vitro. In addition, [10]-gingerol is well tolerated in vivo, induces a marked increase in caspase-3 activation and inhibits orthotopic tumour growth in a syngeneic mouse model of spontaneous breast cancer metastasis. Importantly, using both spontaneous and experimental metastasis assays, we show for the first time that [10]-gingerol significantly inhibits metastasis to multiple organs including lung, bone and brain. Remarkably, inhibition of brain metastasis was observed even when treatment was initiated after surgical removal of the primary tumour. Taken together, these results indicate that [10]-gingerol may be a safe and useful complementary therapy for the treatment of metastatic breast cancer and warrant further investigation of its efficacy, either alone or in combination with standard systemic therapies, in pre-clinical models of metastatic breast cancer and in patients.

Keywords: animal models; apoptosis; breast cancer; cell cycle; gingerol.

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Conflict of interest statement

CONFLICTS OF INTEREST Authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. [10]-gingerol induces concentration-dependent cell death in mouse and human metastatic TNBC cells in vitro
(A) Differential sensitivity of mouse and human metastatic TNBC cells to [10]-gingerol. Cells were treated with increasing concentrations of [10]-gingerol and proliferation measured after 3 days using the SRB colorimetric assay. Data show mean ± SD of 6 replicates/dose from a representative experiment (n = 3). IC50 values for each cell line are indicated in the bottom panel. (B) [10]-gingerol induces morphological changes and cell detachment in vitro. Confluent monolayers of 4T1Br4 and MDA-MB-231BrM were treated with the indicated concentrations of [10]-gingerol and changes in cell morphology and adherence were visualised after 24h on an inverted microscope. Note the significant loss of membrane integrity (50 μM) and reduced number adherent cells with 100 μM [10]-gingerol treatment. Scale bar, 50 μm. (C) Effects of [10]-gingerol on colony formation at low cell density. 4T1Br4 (100 cells/well) and MDA-MB-231BrM cells (300 cells/well) were seeded in 6-well plates, allowed to adhere overnight at 37°C and treated with indicated concentrations of [10]-gingerol or vehicle (DMSO) alone for 24 h. Colonies (> 50 cells) formed after 8 days were stained with a solution of crystal violet, photographed and counted. Data show representative wells at each [10]-gingerol concentration (left panel). Assays were repeated three times in triplicates and data show mean ± SD of triplicates from a representative experiment (right panel). **p < 0.01, ***p < 0.001, 1-way ANOVA, Bonferroni post-test. (D) Effect of [10]-gingerol on cell cycle. Adherent 4T1Br4 (top panels) and MDA-MB-231Br cells (bottom panels) were treated for 24h with indicated concentrations of [10]-gingerol, processed for cell cycle analysis and DNA content analysed by flow cytometry as described in Materials and methods. Data show mean % of cells in each cycle phase ± SD from 3 experiments and statistical difference between treatment groups for each phase of the cell cycle analysed by 2-way ANOVA and Tukey's multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 2
Figure 2. [10]-gingerol induces concentration-dependent apoptosis in metastatic TNBC cells in vitro
(A) Flow cytometric analysis of Annexin-V in [10]-gingerol-treated 4T1Br4 cells. 4T1Br4 cells were treated with 0, 10, 50 and 100 μM [10]-gingerol for 8 h, fixed and stained with PE-Annexin-V and 7-AAD as described in Supplementary methods. Annexin-V positive cells are indicated with a red circle and the percentage of early and late apoptotic cells are indicated in the lower and upper right quadrants respectively. (B) TUNEL positivity was determined after 18h treatment with 10 or 50 μM of [10]-gingerol as indicated and as described in Supplementary methods. Representative images are shown on the left. Scale bar = 100 μm. The number of positive cells were counted and the data expressed as mean % of positive cells ± SD from six 10× images/condition (right panel). Statistical significance was determined using a 1-way ANOVA, Bonferroni post-test, **p < 0.01, ***p < 0.001. (C) Caspase expression following [10]-gingerol treatment in vitro. Expression of pro- and activated caspases in 4T1Br4 and MDA-MB-231BrM cells was analysed by western blotting after 6 h exposure to [10]-gingerol (50 μM) as described in Materials and Methods. α-tubulin or β-actin was used as loading control as indicated.
Figure 3
Figure 3. [10]-gingerol delays orthotopic tumour growth and inhibits spontaneous metastasis
(A) Tumour growth rate. 4T1Br4 cells (1 × 105) were inoculated into the mammary fat pad and vehicle (saline) or [10]-gingerol (5 mg/Kg) was administered daily intraperitoneally, from day 9 to day 23. Primary tumour growth was measured thrice weekly using electronic callipers. Start and end of treatment are indicated with arrows. Difference in growth rate between groups (n = 10 mice/group) was analysed by 2-way ANOVA, Bonferroni post-test (*p < 0.05; **p < 0.01). (B) Tumour weight at endpoint (day 26). Data show one point for each mouse (n= 10/group) and mean burdens (horizontal bar) ± SD. n/s = not significant, Mann Whitney test (p = 0.109). (C) Detection of active caspase-3 and (D) Ki67 in primary tumours. IHC staining of primary tumour sections was carried out as described in Supplementary methods. A total of 27 images/experimental group (3 images/section x 3 section/tumour 150 μm apart × 3 tumours/group) was analysed. Arrowheads in (D) indicate positive Ki67 nuclear reactivity. Data are expressed as mean % of positive pixels/field of view ± SD and the statistical difference between groups was analysed using Mann Whitney test; ***p < 0.001. Representative images of control and [10]-gingerol-treated mice are shown on the right. Scale bar = 100 μm. (E) Spontaneous lung metastasis. Relative tumour burden (RTB) in lung was quantitated by genomic qPCR detection of mCherry gene relative to vimentin as described in Materials and methods. Data show one point for each mouse and mean burdens (horizontal bar) ± SD. *p < 0.001, Mann Whitney test. (F) Representative images of lungs from control (saline) and [10]-gingerol-treated mice. Arrows indicate metastatic nodules.
Figure 4
Figure 4. [10]-gingerol inhibits 4T1 experimental metastasis to bone
4T1Br4 cells (5 × 104) were inoculated into the left ventricle of the heart and the mice treated with daily IP injections of vehicle (saline) or [10]-gingerol (10 mg/Kg) as described in Materials and methods. Mice were harvested on day 12 and relative tumour burden (RTB) in (A) femur, (B) spine and (C) bone (combined femur and spine) determined by genomic qPCR detection of mCherry gene relative to vimentin as described in Supplementary methods. Data show one point per mouse (control, n = 10; [10]-gingerol, n = 13) and mean burdens (horizontal bar) ± SD. *p < 0.05; **p< 0.01; ns, not significant (spine, p = 0.086, Mann Whitney test).
Figure 5
Figure 5. [10]-gingerol inhibits 4T1 spontaneous metastasis to brain
4T1Br4 cells (1 × 105) were inoculated into the mammary fat pad and tumours surgically removed when they reached ∼0.5 cm3. Mice were treated for 14 days with daily IP injections of vehicle (saline) or [10]-gingerol (10 mg/Kg) starting one day after tumour resection and the mice sacrificed on day 14 for quantitation of metastatic burden. (A) Primary tumour weight at resection. Each point represents one mouse (Control; n = 14, [10]-gingerol; n = 13). Data were analysed for statistical significance using Mann Whitney test. n/s = not significant (p = 0.307). (B) Body weight measurements post-tumour resection. *p = 0.041, Mann Whitney test. (C) Incidence of mice with brain metastases. Brains were removed and analysed by fluorescence imaging for the presence of mCherry+ve nodules and statistical difference between groups analysed with Fisher's exact test. *p < 0.05. (D) Fluorescence images of brains from each mouse from control (top) and [10]-gingerol-treated (bottom) groups. Note that control brain #14 in (C) and (D) could not be analysed due to overnight death and cannibalism. Metastatic nodules are indicated by an arrow. Metastatic burden in (E) lung, (F) femur, (G) spine and (H) combined bones (femur + spine from same mice) determined by genomic qPCR detection of mCherry gene relative to vimentin as described in Supplementary methods. Data are expressed as relative tumour burden (RTB) and show one point per mouse (control, n = 14; [10]-gingerol, n = 13) and mean burdens (horizontal bar) ± SD. *p < 0.05; **p < 0.01; ns, not significant (spine, p = 0.086, Mann Whitney test).
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