Cancer stem cells (CSCs) pose a serious obstacle to cancer therapy as they can be responsible for poor prognosis and tumour relapse. In this study, we have investigated inhibitory activity of the ginger-derived compound 6-shogaol against breast cancer cells both in monolayer and in cancer-stem cell-like spheroid culture. The spheroids were generated from adherent breast cancer cells. 6-shogaol was effective in killing both breast cancer monolayer cells and spheroids at doses that were not toxic to noncancerous cells. The percentages of CD44 + CD24 - / low cells and the secondary sphere content were reduced drastically upon treatment with 6-shogaol confirming its action on CSCs. Treatment with 6-shogaol caused cytoplasmic vacuole formation and cleavage of microtubule associated protein Light Chain3 (LC3) in both monolayer and spheroid culture indicating that it induced autophagy. Kinetic analysis of the LC3 expression and a combination treatment with chloroquine revealed that the autophagic flux instigated cell death in 6-shogaol treated breast cancer cells in contrast to the autophagy inhibitor chloroquine. Furthermore, 6-shogaol-induced cell death got suppressed in the presence of chloroquine and a very low level of apoptosis was exhibited even after prolonged treatment of the compound, suggesting that autophagy is the major mode of cell death induced by 6-shogaol in breast cancer cells. 6-shogaol reduced the expression levels of Cleaved Notch1 and its target proteins Hes1 and Cyclin D1 in spheroids, and the reduction was further pronounced in the presence of a γ-secretase inhibitor. Secondary sphere formation in the presence of the inhibitor was also further reduced by 6-shogaol. Together, these results indicate that the inhibitory action of 6-shogaol on spheroid growth and sustainability is conferred through γ-secretase mediated down-regulation of Notch signaling. The efficacy of 6-shogaol in monolayer and cancer stem cell-like spheroids raise hope for its therapeutic benefit in breast cancer treatment.

Funding: The work was supported by the following: grant no. SR/WOS-A/LS-326/2009; Woman Scientist Scheme, Department of Science and Technology, Govt. of India ( http://dst.gov.in/scientific-programme/women-scientists.htm ) to AR; and Rajiv Gandhi Centre for Biotechnology ( http://rgcb.res.in/ ) core funding to SS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2015 Ray et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Several in vitro studies have shown that cancer stem cells are resistant to conventional chemotherapeutic drugs [ 8 , 16 ]. Interestingly, a number of dietary compounds like curcumin [ 14 ], piperine [ 14 ], sulforaphane [ 17 ] have recently been identified to target CSCs. However, various factors such as toxicity, weak dose response etc. largely limit their application. Since 6-shogaol has been reported as a potent anticancer agent against various cancer cells, we have investigated its inhibitory effect on breast cancer cells and cancer stem cell-like spheroids. Here we demonstrate that 6-shogaol shows anti-proliferative activity against breast cancer cells and spheroids and suppresses the size and colony forming ability of spheroids by altering the Notch signaling pathway. Investigation of the death mechanism shows that autophagy is a predominant mode of cell death caused by 6-shogaol in breast cancer cells.

Although, CSCs are present in a very small percentage in the total tumour, methods have been developed to grow them in large population in ex vivo. In appropriate growth conditions, cancer cells can be made to grow in the form of spheroids. These spheroid-forming cells exhibit altered cell surface markers when compared to cells grown in monolayer culture and have been shown to possess stem-cell like properties [ 10 , 13 ]. These spheroids have been used in a number of studies to determine the in vitro and in vivo characteristics of cancer stem cells as well as to assess the inhibitory activity of cytotoxic compounds against cancer stem cells [ 11 , 14 , 15 ].

Cancer stem cells play a very important role in cancer development and progression. The concept of stem cell origin of cancer has been supported by observations that certain subpopulations (only 0.2–1%) of cancer cells have stem cell-like properties, such as the ability to self renew, continuous differentiation and an overall innate resistance to conventional chemotherapeutic agents [ 8 ]. These chemo-resistant, self-renewing, tumorigenic sub-population of cells defined as cancer stem cells (CSCs) play crucial roles in cancer recurrence. CSCs have been identified in various solid tumors including breast, ovarian, head and neck, pancreas, and colon cancer [ 9 , 10 ]. Earlier studies demonstrated that the signaling pathways such as Wnt/β-catenin, Notch and Hedgehog pathways regulate the growth of cancer stem cells [ 11 , 12 ]. Therefore, targeting these pathways is considered to be a useful strategy to inhibit cancer stem cell regeneration.

Ginger (Zingiber officinale) is a well known herb consumed as a spice and food as well as widely used as herbal medicine for various ailments. A number of biologically active ingredients including gingerols and its various derivatives have been identified and synthesized from ginger in recent years. One important class of derivatives are shogaols that are primarily the dehydrated products of gingerols and are found exclusively in dried ginger. Among the shogaols, 6-shogaol has achieved a great deal of attention due to its potent anticancer activity against various cancer cells. It has been shown to induce mitotic arrest and reduce viability of gastric cancer cells [ 1 ]. Aberrant mitosis followed by apoptosis has also been found to be induced by 6-shogaol in HCT-116 colon cancer cells [ 2 ]. In human hepatoma p53 mutant Mahlavu subline, 6-shogaol induces apoptosis via oxidative stress pathway in a caspase dependent mechanism [ 3 ]. It has also been shown to induce autophagy in HNSCLC A-549 cells via inhibition of the AKT/mTOR pathway [ 4 ]. In another study, 6-shogaol has been reported to exhibit anti-invasive effects in breast cancer cells by reducing MMP-9 expression through NF-κB activation [ 5 ]. Recently, PPAR-γ dependent apoptosis in MCF-7 and HT-29 cells by 6-shogaol has also been reported [ 6 ]. Additionally, recent studies have implicated microtubule as a possible target of 6-shogaol as it interacts with the sulphydryl groups of cysteines in tubulin through its side chain containing the α, β unsaturated carbonyl moiety [ 7 ]. All these studies place 6-shogaol as a promising agent to be studied further in view of its future therapeutic potential in cancer therapy.

Densitometry was conducted for all the western blots using BD Quantity One software (Bio-Rad). Normalisation was done according to the loading control and fold change was calculated with respect to the control. For graphs, results are depicted as mean ± standard error of mean or standard deviation (calculated from two or more experiments). The p-values were obtained using Student’s unpaired t-test and p < 0.05 was considered to be statistically significant.

Cells were seeded in 35 mm glass bottom dishes. 70–80% confluent cells were treated with 6-shogaol (15 μM) for 48 hours. Both control and treated cells were washed with PBS, fixed in methanol-EDTA, rehydrated in PBS and incubated for 1 hour in Giemsa stain. Cells were air dried and imaged in Olympus IX71 fluorescent microscope under 40X objective.

Apoptosis was quantified by using a FITC Annexin V Apoptosis Detection kit according to manufacturer’s instructions. MCF-7 cells were treated with 15 μM and 25 μM of 6-shogaol for 24, 48 and 72 hours, respectively. Attached and floating cells were collected and analyzed by a flow cytometer (BD FACS Aria II).

Cells were seeded on cover slips. 70–80% confluent cells were treated with 6-shogaol (15 μM) for 48 hours and 72 hours. Cells were washed with PBS, fixed with Methanol-EDTA for 10 minutes at -20°C followed by rehydration with PBS at room temperature and incubated with 1 μg/ml DAPI. Mounted coverslips were visualised at 40X magnification under fluorescence microscope (Olympus IX71). Apoptotic cells were counted from five different fields.

Whole cell lysates were prepared using phospho-lysis buffer containing 10% NP-40, 10% glycerol, 137 mM NaCl, 20 mM Tris-HCl (pH 7.4), 20 mM NaF, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1% Triton X-100, and 5 mM PMSF in presence of protease inhibitor cocktail (10 μl/ml). Lysates were placed into ice for 1 hour to ensure complete lysis followed by centrifugation at 14,000 g for 10 minutes and the supernatant was collected. Proteins were subjected to SDS-PAGE and transferred to polyvinylidene fluoride membrane. The membranes were blocked and incubated overnight with primary antibodies at 4°C followed by incubation with horseradish peroxidase tagged secondary antibodies. The blots were developed using ECL reagent. The primary antibodies were used in the following dilutions: Cleaved Notch1 (1:2000), Hes1 (1:200), Cyclin D1 (1:400), PARP (1:500 for sc) and (1:1000 for CST), LC3A/B (1:500), Bcl-2 (1:1000), Bax (1:1000) and β-actin (1:2000). β-actin was used as loading controls.

5000 cells/well were seeded in ultra low attachment plates. After 3 days of seeding, the spheres formed were kept with different concentrations of 6-shogaol and/or 25 μM DAPT for 7 days and the number of spheres were counted under the microscope. These primary spheres were dispersed and kept in fresh media without any treatment and counted again after 7 days to check the ability to regrow into secondary spheres. Control primary spheres were also processed similarly for comparison. Spheres were counted in Olympus IX71 microscope using 10X objective.

Cell cycle distribution of cells treated with 6-shogaol was analyzed by flow cytometric measurement of cellular DNA content. MCF-7 cells (5×10 5 cells/well) were subjected to 6-shogaol treatment for various time points. Culture media containing detached cells were collected and attached cells were trypsinized. Cells were pelleted, fixed in 70% (v/v) ethanol and stored at 4°C. In case of spheroids, cells were treated with different concentrations of 6-shogaol for 48 hours and fixed for further analysis. Before analysis, cells were re-washed with PBS and incubated with 10 μg/ml RNaseA and 400 μg/ml propidium iodide for 30 minutes at 37°C. Data were analyzed using FACS Diva software.

Monolayer MCF-7 cells were incubated for 48 hours with either chloroquine (CQ) or 6-shogaol individually or in combination and cell viability was checked by MTT assay. Chloroquine was added 1 hour prior to 6-shogaol addition.

To check the effect of different drugs on the viability of spheroids, cells were seeded at 2.5×10 4 cells/well in 24-well ultra-low attachment plates and grown as spheroids. After 3 days, 6-shogaol (1–100 μM) or taxol (5 nM-50 μM) or curcumin (2–50 μM) was added to the wells and MTT assay was performed as explained above. Standard deviation was calculated from three independent experiments and the data were represented as the average of three experiments.

Monolayer cells (5×10 3 cells/well) were seeded in quadruplicates in 96 well plates and were incubated with varying concentrations of 6-shogaol (1–100 μM), taxol (0.2–100 nM) or curcumin (4–80 μM). The effect of the drugs on the viability of all the cell lines was quantified by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assay at different time points after drug treatment using a Biorad Plate reader [ 19 ]. The IC 50 values were calculated from the plot of percentage cell viability versus concentration of drug using the nonlinear regression programme of Origin.

Monolayer cells or spheroids (with or without treatment of 6-shogaol) were dispersed by trypsin-EDTA. Cells were then pelleted down by centrifugation at 1600 rpm for 5 minutes at 4°C and resuspended in 1 ml of DMEM containing 2% FBS. Fluorescein isothiocyanate (FITC)-conjugated CD24 monoclonal antibody and phycoerythrin (PE)-conjugated CD44 monoclonal antibody (BD Biosciences) were added in 1:100 dilution and incubated on ice for 45 minutes in dark. The cells were then analyzed by flow cytometer (BD FACS Aria II) for the expression of CD24 and CD44.

To check for the induction of autophagy, the MCF-7 monolayer cells were seeded on coverslips. 70–80% confluent cells were treated with 6-shogaol or chloroquine. The cells were then fixed, permeabilized and blocked as mentioned earlier. Thereafter the cells were incubated overnight with LC3A/B primary antibody (1:100), followed by anti-rabbit alexa 488 secondary antibody (1:200) for one hour. DAPI staining and mounting were done as above. Visualization was done under 60X oil immersion objective in a confocal microscope (Nikon Eclipse Ti (A1R) Japan). Z-stacking was done wherever necessary.

To check cell surface marker expressions, monolayer MCF-7 cells were grown on coverslips. Cells were fixed in Methanol-EDTA (1 mM EDTA) for 10 minutes and rehydrated in PBS. After blocking, cells were incubated with PE-conjugated CD44 antibody (1:50) for 3 hours at room temperature and stained with DAPI (1 μg/ml). MCF-7 spheres in suspension were fixed in 4% paraformaldehyde for 10 minutes at -20°C and permeabilised in 0.1% triton X-100 for 10 minutes in PBST (PBS with 0.05% Tween-20). Cells were then processed as mentioned above. Thereafter, cells were mounted on slides with Fluoromount G for imaging.

MCF-7 cells, HEK 293 and HaCaT cells were maintained in DMEM/F12 supplemented with 1% sodium pyruvate, 0.2% non-essential amino acids, 1% penicillin-streptomycin and 10% FBS in a humidified atmosphere containing 5% CO 2 at 37°C. MDA-MB-231 cells were maintained in Leibovitz L-15 media, 1% penicillin-streptomycin and 10% FBS under the same condition. For spheroid culture, Mammary Epithelial Basal Media (MEBM) was supplemented with 0.001% Epidermal Growth Factor (EGF), 0.001% Insulin, 0.004% Bovine pituitary extract (BPE) and 0.001% Hydrocortisone. 0.001% Gentamicin sulphate and amphotericin-B were added as antibiotic-antimycotic [hereafter termed as MEGM (Mammary Epithelial Growth Media)]. MCF-7 and MDA-MB-231 cells were seeded in ultra low attachment plates using MEGM as culture medium. After three days of seeding, cells forming spherical clusters were considered as stable spheroids and used for further experiments.

Human metastatic breast adenocarcinoma cell lines MCF-7 and MDA-MB-231 were obtained from National Cancer Institute, USA (ATCC# HTB-22 and ATCC# HTB-26 respectively). Human embryonic kidney cell line HEK 293 (ATCC# CRL-1573.3) was obtained from ATCC. The human immortal keratinocyte cell line HaCaT [ 18 ] was obtained from the national repository of National Centre for Cell Sciences, Pune, India. Frozen stocks of cells from the reference stock were made within passage 3 and stored in liquid nitrogen. For experiments, cells were used within 2 months of revival.

PE (Phycoerythrin)-conjugated CD44 (555749) and FITC (Fluorescein Isothiocyanate)-conjugated CD24 (555573) antibodies were purchased from BD Biosciences. Antibodies for Cleaved Notch1 (4147S) and Cyclin D1 (IMG-6583A) were procured from Cell Signalling Technology and Imgenex respectively. Antibodies for PARP were from Cell Signaling Technology (CST-9544) and Santa Cruz Biotechnology (sc-7150); Bcl-2 (sc-7382), Bax (sc-7480), β-actin (sc-47778) and Hes1 (sc-166378) were from Santa Cruz Biotechonology. Primary antibody for LC3A/B (Light Chain 3) (ab-173752) was obtained from Abcam or from Cell Signaling Technology (CST-12741). Anti-mouse and anti-rabbit HRP were purchased from Sigma. Anti-rabbit alexa 488 was from Molecular Probes, USA.

6-shogaol (≥90%), Taxol (≥95%), and DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) (≥98%) were purchased from Sigma. Chloroquine (CQ) was from Molecular Probes, Invitrogen. Fluoromount G was procured from Electron Microscopy Sciences. DAPI (4',6-Diamidino-2-Phenylindole), Giemsa and other fine chemicals were from Sigma. Chemiluminescent western blotting detection system was from Thermo Scientific. FITC Annexin V Apoptosis Detection kit was purchased from BD Pharmingen (Cat # 556547). Ultra low attachment plates were obtained from Corning, USA and MEBM (Mammary Epithelial Basal Media) was procured from Lonza, USA.

Results

6-shogaol has been shown to exert inhibitory effect on various cancer cell lines and animal disease models [20, 21]. To check whether 6-shogaol is effective on breast cancer stem cells, we generated model stem cell-like spheroids from two types of breast cancer cell lines, ER/PR positive MCF-7 and triple negative MDA-MB-231. The cells were grown in low attachment plates with conditioned media that promote non-adherent growth. Under these conditions, the cells became subsequently organized as clusters of spherical cells. After 3 days of growth, the clusters were considered as stable spheroids. All the further experiments were performed under these conditions.

Expression of CD44/CD24 in spheroids Differential expression of cell surface markers between monolayer cells and CSCs help to select CSCs within the tumour. CD44 and CD24 are cell surface glycoproteins that play a major role in both cell adhesion and migration. Earlier studies have shown that breast cancer cells contain stem-cell like cells exhibiting CD44+CD24-/low marker expression and these cells possess more than 50 fold increased tumorigenic capacity when compared to the other cells of tumour mass [13, 16, 22]. It has been reported that cultured mammospheres also exhibit stem cell like properties with characteristic CD44+CD24-/low expression [23, 24]. We therefore checked whether the spheroids generated by us possessed this phenotype. Immunofluorescence images showed that expression of CD44 [Fig 1A(i)] was significantly higher in the spheroids generated from MCF-7 cells than in the monolayer MCF-7 cells. Concomitant with this, flow cytometric analysis also showed significantly high CD44 and low CD24 expression in spheroid forming cells than in monolayer cells [Fig 1A(ii)]. PPT PowerPoint slide

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pone.0137614.g001 Further, it has been demonstrated that stem cell-like spheroids are capable of generating next generation (secondary) spheres and possess the ability to differentiate along multiple lineages [25]. Spheroids generated by us also exhibited the property of formation of secondary spheres as shown in Fig 1B. These spheroids were used for further experiments to investigate the effect of 6-shogaol on cancer stem-like cells.

Cytotoxicity study of 6-shogaol against monolayer and spheroid cells The anti-proliferative activities of 6-shogaol on monolayer cells and spheroids were quantitated by MTT assay in two cell lines, MCF-7 and MDA-MB-231. Cells were treated with increasing concentrations of 6-shogaol for 48 hours and then the cell viability was measured. The IC 50 values are shown in Table 1. Taxol, a drug widely used in breast cancer treatment, and curcumin, a compound earlier shown to be effective in inhibiting cancer stem-like spheroids [14], were used for comparison. The results showed that for both the cell types, 6-shogaol was effective in spheroids at concentrations that were 5 or 2 fold higher than the effective inhibitory concentrations in monolayer cells. In contrast, taxol, even though was highly active in monolayer cells, did not show activity against the spheroids even at 10000 fold higher concentration compared to 6-shogaol (Table 1). Curcumin was also found to be effective against MCF-7 spheroids as reported earlier [14]. The results also showed that 6-shogaol was ~3 fold more potent against MDA-MB-231 spheroids than against MCF-7 spheroids. The inhibitory effect of all the three compounds on non-cancerous cell lines, HEK 293 and HaCaT were also tested (Table 1). The IC 50 data showed that the noncancerous cells tolerated significantly higher doses of 6-shogaol compared to the cancer cells in both monolayer and spheroid culture conditions. Non-cancerous cells were quite resistant to taxol when compared with its efficacy in the monolayer breast cancer cells. In contrast, curcumin did not exhibit any resistance to noncancerous cells as compared to its action on breast cancer cells and spheroids. Thus the action of 6-shogaol on breast cancer spheroids is superior to taxol or curcumin considering the fact that it inhibits spheroids at concentrations which are safe to non cancerous cells. PPT PowerPoint slide

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larger image TIFF original image Download: 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. IC 50 was determined from MTT assay results after 48 hours using the nonlinear regression programme of Origin. Standard deviations from three different experiments are shown. https://doi.org/10.1371/journal.pone.0137614.t001 The changes in size of the spheroids generated from MCF-7 and MDA-MB-231 cells after treatment with 6-shogaol are shown (Fig 2A). Interestingly, MDA-MB-231 cells formed relatively loose and less rounded spheroids than those of MCF-7 cells, which was probably the reason for better penetration and more effectiveness of 6-shogaol in MDA-MB-231 spheroids than MCF-7 spheroids. This also indicated that 6-shogaol might need longer time to penetrate into MCF-7 spheroids. We thus investigated the effect of prolonged treatment of low dose of 6-shogaol (range of IC 50 and below) on the viability of MCF-7 spheroids. The experiments were performed under three sets of 6-shogaol concentrations, 40 μM (IC 50 ), 20 μM (½ IC 50 ), and 10 μM (¼ IC 50 ). The spheroids formed after 3 days of seeding were treated with 6-shogaol and further the effect on the cell viability were measured after every two days till the 8th day. 6-shogaol reduced the viability of the spheroids in a time dependent manner (Fig 2B). On day 4, 10 μM, 20 μM and 40 μM 6-shogaol inhibited cell viability by ~35%, 56%, and 83% respectively. After 6 days, 10 μM 6-shogaol treatment resulted in 46% inhibition. Likewise, to check the effect of prolonged treatment of 6-shogaol on non-cancerous cells, HEK 293 and HaCaT cells were treated with 6-shogaol for 6 days. However, in these cells the IC 50 values showed no significant decrease up to 6 days when compared with 2 days of treatment. The results showed that both the cell lines could tolerate considerably high concentration of 6-shogaol till day 6 (Fig 2C). PPT PowerPoint slide

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pone.0137614.g002

Cell cycle analysis of 6-shogaol treated cells To understand the mechanism of action of 6-shogaol, its effect on cell cycle progression was analysed by flow-cytometry. Monolayer cells were treated with 16 μM (2×IC 50 ) of 6-shogaol for different time points. As evident from DNA content measurement, 6-shogaol induced cell cycle arrest at G2/M phase (Fig 3A). After 24 and 48 hours of treatment, percentage of cells in the G2/M phase increased from 29% (control) to 48% and 55%, respectively. PPT PowerPoint slide

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larger image TIFF original image Download: 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×IC 50 ) 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. https://doi.org/10.1371/journal.pone.0137614.g003 Effect of 6-shogaol on cell cycle of MCF-7 spheroid cells was also analysed by flow cytometry. Spheroids were treated with different concentrations (0–50 μM) of 6-shogaol for 48 hours. 25 μM of 6-shogaol treatment exhibited 42% G2/M arrest as compared with 24% in control. However, G2/M percentage was found to decrease when treated with 50 μM 6-shogaol. It was observed that the cells in the Sub G1 phase increased gradually with increased 6-shogaol concentration (Fig 3B), indicating that like in monolayer cells, 6-shogaol treatment led to the spheroid cell death in a concentration dependent manner.

Confirmation of 6-shogaol action on CSC like spheroid cells The results so far indicated an inhibitory action of 6-shogaol on both breast cancer monolayer cells and spheroids. However, a possibility still remains that in a spheroid culture, 6-shogaol might actually work on the population of cells which don’t have stem cell-like characteristics and all the effects depicted so far are due to its action on mass cancer cells without stem cell-like phenotype. To rule out this possibility, we checked the expression levels of cell surface markers of breast cancer spheroids in the presence and absence of 6-shogaol. We also verified the effect of 6-shogaol on secondary sphere formation, a property which is strictly exhibited by cancer stem cell-like cells. Flow cytometric analysis of breast cancer spheroids treated with 40 μM 6-shogaol for 18 hours (Fig 7A) showed that the percentage of cells characterized by CD44+CD24-/low were substantially less in the treated spheroids when compared to the untreated ones (2.3% vs. 27.3%). PPT PowerPoint slide

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larger image TIFF original image Download: 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. https://doi.org/10.1371/journal.pone.0137614.g007 We next examined whether 6-shogaol was able to suppress the number and size of the spheres and if the effect of the compound was exhibited in the next generation of the spheres also. The MCF-7 spheroids at their stable growth condition after 3 days of seeding were treated with 1–40 μM of 6-shogaol and after 7 days of continuous treatment, the number of spheroid colonies were counted for each 6-shogaol concentration. A drastic reduction in the number of spheroid colonies was observed in a concentration dependent way. For example, average 96 spheroid colonies were found in control wells in the absence of 6-shogaol, whereas the average number of colonies was reduced to 48 (2 fold) with the treatment of only 1 μM 6-shogaol. Only 4 colonies were observed under the treatment of 40 μM 6-shogaol resulting a 24 fold inhibition (Fig 7B). Next, the ability of the primary spheroids to grow as the next generation spheroids (secondary spheroids) was checked. The primary spheroids remaining after 1 week of continuous treatment with 6-shogaol were disaggregated and allowed to grow in fresh media without 6-shogaol for another week. While the control (generated without 6-shogaol treatment) primary spheroid cells produced ~ 55 secondary spheroid colonies, 1, 5 and 10 μM 6-shogaol-treated primary colonies were able to generate only a few secondary colonies with 71%, 80% and 94.5% inhibition, respectively (Fig 7B). Not a single secondary sphere was found at concentrations above 10 μM of 6-shogaol, indicating that 6-shogaol completely abolished the regeneration ability of the spheroids. Taken together, the results demonstrate that 6-shogaol effectively inhibits breast cancer spheroids.