Fig 1.
Characterization of breast cancer stem cell like spheroids.
(A) Expression of cell surface markers CD44 and CD24 in MCF-7 monolayer and spheroids. (i) Immunofluorescence images showing expression of CD44 in MCF-7 monolayer cells (upper panel) and in MCF-7 spheroid cells (lower panel). Cells were stained with PE-conjugated CD44 antibody and imaged as described in the methods section. (ii) Flow cytometric analyses of CD44/CD24 in monolayer MCF-7 cells (left panel) and in MCF-7 spheroids (right panel). (B) Formation of primary and secondary spheres by MCF7 cells. Image was taken in 10X in an Olympus IX71 microscope.
Table 1.
Cytotoxic activity of 6-shogaol in breast cancer cells and spheroids and in noncancerous cells.
Different concentrations of drugs were added one day and three days after seeding for monolayer and spheroids respectively. IC50 was determined from MTT assay results after 48 hours using the nonlinear regression programme of Origin. Standard deviations from three different experiments are shown.
Fig 2.
Inhibitory effect of 6-shogaol on MCF-7 and MDA-MB-231 breast cancer spheroids.
(A): Breast cancer spheroids with or without treatment. MCF-7 spheroids with 40 μM of 6-shogaol (upper panel); MDA-MB-231 spheroids with 11 μM 6-shogaol (lower panel). (B): Prolonged effect of different concentrations of 6-shogaol on MCF-7 spheroids. The error bars represent the standard error of mean from three different experiments. *** refers p ≤ 0.001; ** refers p ≤ 0.005; * refers p ≤ 0.05. (C): Prolonged effect of 6-shogaol on noncancerous cell lines HEK 293 and HaCaT. The error bars represent the standard error of mean from three different experiments. The differences among the different treatment periods up to 6 days were found to be insignificant.
Fig 3.
Effect of 6-shogaol on cell cycle of MCF-7 cells / spheroids.
(A): Cell cycle analysis of MCF-7 cells treated with 16 μM 6-shogaol (2×IC50) for different time points. (B): Cell cycle analysis of MCF-7 spheroid cells with different concentrations of 6-shogaol for 48 hours. The histogram is a representative of three independent experiments for both monolayer and spheroid cells. Bar graph represents percentage of cells in different phases of cell cycle. Error bars represent standard error of mean and have been calculated from three different experiments. *** denotes p ≤ 0.001; ** denotes p ≤ 0.005 and * denotes p ≤ 0.05.
Fig 4.
Induction of apoptosis by 6-shogaol.
(A): DAPI staining showing condensed nuclei in MCF-7 cells in presence of the indicated concentrations of 6-shogaol after 48 hours and 72 hours. (B): Graph quantitates the percentage of apoptotic cells for each treatment and time points. The results are representative of three independent experiments. The error bars represent standard deviation. *** denotes p ≤ 0.001 and ** denotes p ≤ 0.005. (C): Graph represents the percentage of Annexin V positive cells for each 6-shogaol treatment and time points. The error bars represent standard deviation. ** refers p ≤ 0.005; * refers p ≤ 0.05. (D): Western blot showing expression of Bcl-2 and Bax upon 6-shogaol treatment. (E): Western blot showing reduction in the mother band of 116 kDa and formation of 85 kDa cleaved band of PARP upon treatment with the indicated concentrations of 6-shogaol after 72 hours. Blot was probed with sc-7150 PARP antibody. β-actin is the loading control for all the blots. All the blots are representative of three independent experiments.
Fig 5.
Induction of autophagy by 6-shogaol.
MCF-7 cells were treated with 6-shogaol (15 μM) or chloroquine (50 μM) for 48 hours. Top panel: Bright field image (20X) of control and treated cells. Arrow heads indicates cytoplasmic vacuolization. Middle panel: Giemsa stained cells where arrows point towards vacuoles. Bottom panel: Cells were stained with LC3A/B primary antibody and immunocytochemistry was conducted as described in the Methods section. Green punctate staining in the 6-shogaol or chloroquine treated cells shows the localization of LC3 in autophagosomes. Cell nuclei are labelled blue (DAPI). The scale bar represents 10 μm for all the three panels.
Fig 6.
6-shogaol induces autophagic flux and cell death in breast cancer cells.
(A): MCF7 cells were treated with 6-shogaol or chloroquine for the indicated time points, stained with LC3A/B antibody and imaged as described in the Methods section. Green punctae are indicative of LC3II expression. Cell nuclei are labelled in blue. (B): MCF7 cells were treated for 48 hours with the indicated concentrations of 6-shogaol or chloroquine or their combination. Western blot was conducted with 50 μg of protein. Fold change in the expression of LC3II was calculated with respect to β-actin level of the control. A representative blot has been shown from three independent experiments. (C): MCF-7 cells were treated with chloroquine (CQ), 6-shogaol or combination of CQ and 6-shogaol in the indicated concentrations. After 48 hours, cell viability was checked by MTT assay. The error bars represent the standard deviation from three different experiments. ** denotes p ≤ 0.005. (D): Western blotting was conducted with cell lysates of MCF-7 spheroids after 40 μM 6-shogaol treatment as detailed in the Methods section. Cleavage of LC3I to LC3II was observed in the MCF-7 spheroid cells. Housekeeping gene β-actin was used as loading control. LC3II expression was quantitated and fold increase with respect to the control has been indicated.
Fig 7.
Confirmation of the action of 6-shogaol on breast cancer spheroids.
(A): Flow cytometric analysis of CD44+ /CD24- expression in untreated spheres (right upper panel) and spheres treated with 40 μM 6-shogaol for 18 hours (right lower panel). (B): Effect of different concentrations of 6-shogaol on primary and secondary spheres. 5000 cells per well were seeded in quadruplicates with or without 6-shogaol. After 7 days, number of spheres were counted and then dispersed. From these, 5000 cells were again seeded and kept to regrow in fresh media without 6-shogaol. The error bars represent the standard error of mean from three different experiments. *** refers p ≤ 0.001.
Fig 8.
Modulation of Notch1 signaling pathway by 6-shogaol in MCF-7 spheroids.
(A): Expression of Cleaved Notch1 and its targets Cyclin D1 and Hes1 after the treatment of 25 μM of 6-shogaol for the indicated time periods. (B): Expression of Cleaved Notch1, Cyclin D1 and Hes1 after 24 hour treatment of 6-shogaol in presence of the γ-secretase inhibitor DAPT (50 μM). The vertical lines in the Cleaved Notch1, Cyclin D1 and corresponding actin blots are to show a discontinuation to remove the lanes for 16 hours treatments that do not show any change in the protein levels. All the blots are representative of three independent experiments. (C): Effect of DAPT (25 μM) on the inhibition of primary and secondary sphere formation by 6-shogaol. The error bars represent the standard error of mean from three different experiments. *** denotes p ≤ 0.001.
Fig 9.
A schematic diagram showing the action of 6-shogaol on breast cancer cells and spheroids.