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. 2014 May;145(1):5-22.
doi: 10.1007/s10549-014-2912-z. Epub 2014 Mar 26.

Nicotine Promotes Apoptosis Resistance of Breast Cancer Cells and Enrichment of Side Population Cells With Cancer Stem Cell-Like Properties via a Signaling Cascade Involving galectin-3, α9 Nicotinic Acetylcholine Receptor and STAT3

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Nicotine Promotes Apoptosis Resistance of Breast Cancer Cells and Enrichment of Side Population Cells With Cancer Stem Cell-Like Properties via a Signaling Cascade Involving galectin-3, α9 Nicotinic Acetylcholine Receptor and STAT3

Prasun Guha et al. Breast Cancer Res Treat. .
Free PMC article

Abstract

Nicotine, a main addictive compound in tobacco smoke, has been linked to promotion and progression of lung, head and neck, pancreatic, and breast cancers, but the detailed mechanisms of cancer progression remain elusive. Here, we show that nicotine induces the expression of galectin-3 (an anti-apoptotic β-galactoside-binding lectin) in breast cancer cell line and in primary tumors from breast cancer patients. Nicotine-induced up regulation of galectin-3 is due to an increased expression of α9 isoform of nicotinic acetylcholine receptor (α9nAChR), which activates transcription factor STAT3 that in turn, physically binds to galectin-3 (LGALS3) promoter and induces transcription of galectin-3. Intracellular galectin-3 increased mitochondrial integrity and suppressed chemotherapeutic-induced apoptosis of breast cancer cell. Moreover, nicotine-induced enrichment of side population cells with cancer stem cell-like properties was modulated by galectin-3 expression and could be significantly reduced by transient knock down of LGALS3 and its upstream signaling molecules STAT3 and α9nAChR. Thus, galectin-3 or its upstream signaling molecule STAT3 or α9nAChR could be a potential target to prevent nicotine-induced chemoresistance in breast cancer.

Conflict of interest statement

Conflict of interest

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. Cell viability and Scratch-wound healing assay
A. MCF 7 cells were plated in 96 well plate and treated with staurosporine (1µM) alone or in combination of varying concentration of nicotine as 100µM, 10µM, 1 µM for 24 h followed by cell viability study using WST-1. *** represents p<0.001 and * represents p< 0.05 compared to staurosporine treated cell B. Confluent monolayer of MCF7 cells were replaced with serum-free medium for overnight and a wound was created by dragging a pipette tip through the monolayer. After washing with serum-free medium, wound images were photographed (0 h). Cells were then cultured in serum-free medium in absence or in the presence of nicotine (100 µM) for 72 h and following washing with 1× PBS wound images were photographed. Migratory cells to the denuded area for each of untreated and nicotine treated cells were counted and expressed as mean ± SD, n=3. Magnification 5×. C. A bar graph showing quantitative data was represented as ± SD. *** represents as p≤0.001.
Figure 2
Figure 2. Nicotine induced galectin-3 expression
A. (i) Representative diagrams showing expression of galectin-3 in various stages of breast cancer tissue specimens from 22 patients (see Table 1 for description) who never smoked and smoked as determined by immunostaining with anti-galectin-3 antibody. Magnification 400×. (ii) A bar graph showing quantitative expression of galectin-3 for each stage of smoker and non-smoker breast cancer patients (n =22). Galectin-3 staining of each sample was performed in duplicate or in triplicate. The number of cells showing positive staining of galectin-3 was scored independently and blindly in 3 different area of each slide and then data were expressed as mean ± S.D, where *** represents p≤0.001. (iii) Expression of galectin-3 in stage III breast cancer tissue specimens (receptor-matched non-smoker & smoker) as determined by western blot. Recombinant galectin-3 was used as a positive control to validate the molecular weight of the galectin-3 band in tissues. The number on top of blot represents arbitrary unit showing quantification of the galectin-3 expression as measured by Image J software. B. Expression of gal3 in nicotine (1–100 µM) treated MCF-7 cells (48 h) as measured by quantitative RT-PCR. Relative mRNA expression was calculated according to the comparative ΔΔC(t) method. C. Validation of gal3 expression in untreated and nicotine-treated (100 µM, 48 h) MCF7 cells by Western blot. The number on top of blot represents arbitrary unit showing quantification of the gal3 expression as measured by Image J software.
Figure 3
Figure 3. Nicotine induced α9nAChR expression
A. Quantitative mRNA expression of α7nAChR and α9nAChR in nicotine treated (100 µM for 48 h) MCF-7 cells by RT-PCR Relative mRNA expression was calculated according to the comparative ΔΔC(t) method. B. Western blot showing protein expression of α9nAChR in nicotine treated (100 µM for 48 h) MCF-7 cells. The number on top of blot represents arbitrary unit showing quantification of the α9nAChR expression as measured by Image J software. C. Representative diagrams showing expression of α9nAChR in stage III tissues of breast cancer patients who never smoked (3 patients as described in Table 1) and smoked (5 patients) as determined by immunostaining with anti-α9nAChR antibodies. Adjacent bar graph shows quantitative expression of α9nAChR in these samples. Each sample was performed in duplicate or in triplicate. The number of cells showing positive staining of α9nAChR was scored independently and blindly in 3 different area of each slide and then data were expressed as mean ± S.D, where *** represents p≤0.001. D. Effect of α9nAChR silencing on the expression of galectin-3. Cells were treated with siRNA specific for α9nAChR or negative control siRNA and the cell extract was subjected to W. blot for expression of galectin-3. The number on top of blot represents arbitrary unit showing quantification of the galectin-3 expression as measured by Image J software.
Figure 4
Figure 4. Galectin-3 reduces apoptosis through promotion of mitochondrial stability
A. Quantitative mRNA expression of galectin-3 in gene-specific or negative control siRNA treated MCF7 cells by RT-PCR. Relative mRNA expression based on GAPDH expression was calculated according to the comparative ΔΔC(t) method. B. Western blot showing galectin-3 expression in galectin-3 specific sRNA treated MCF-7 cells. C. MCF7 cells were challenged with known apoptotic inducer staurosporine (1 µM) for 48 h and then role of nicotine (100 µM) and galectin-3 was examined through viability study using WST1. Role of galectin-3 was investigated using galectin-3 specific siRNA (si-gal3) and shRNA (sh-gal3). D. Amount of annexin-V positive cells was measured by flow cytometry as described in the Methods and data were presented as percent of annexin-V positive cells. E. STS-induced DNA fragmentation of cells in the presence of nicotine alone or nicotine plus siRNA (galectin-3 specific or negative control) was measured on a colorimetric assay as described in the Methods. F. The mitochondrial membrane potential was measured using JC1 dye and data were represented as the ratio of the emission 590 (red): 527 (green). All bar graphs showing quantitative data were represented as ± SD. *** represents as p≤0.001. NS, Not significant.
Figure 5
Figure 5. Nicotine induced side population (SP) in MCF7 cells and modulation of SP by galectin-3 and TWIST1
A. Cells were treated with verapamil (50 µM), nicotine (100 µM), nicotine plus siRNA targeted galectin-3 or nicotine plus negative control siRNA, and stained with DyeCycle Violet and SP was measured using Dye cycle violet dye (DCV) on a flow cytometer. The SP gated populations are displayed as scatter plots for red (650 nm LP) vs. blue (450/40 nm BP) emissions of DCV (excited with 405 nm laser source). The ratio of the SP cells to total population of MCF-7 cells is indicated as percentage on each scatter plot (as represented by pink square). B. Bar diagram showing results of side population cells in untreated and treated MCF-7 cells as described in Fig. 5A. Data were expressed as mean ± S.D, where *** and ** represent p≤0.001 and p≤0.01, respectively. C. Gene expression microarray showing (i) Heat map and (ii) The Scatter Plot. (i). The heat map is depicting the overall impression of the change in gene regulation of PCR array specific genes. The color code specifies the magnitude of change of gene expression (Green to Red for upregulation). In the heat map, left panel is showing the basal gene expression of the untreated MCF-7 cells and right panel represents nicotine treated group. (ii). MCF-7 cells were treated with nicotine (100 µM, 48 h) and expression of all the genes (genes included in PCR array 96 well plate) was measured in comparison to the untreated control. The scatter plot shows the expression level (2 ^ (−ΔΔCt)) of each gene in the nicotine treated cells compared to the untreated control. The black line indicates fold changes (2 ^ (−ΔΔCt)) of 1. Twist-1 was indicated as most up-regulated gene. The expression fold of each gene was shown in Table 2. D. Validation of TWIST1 expression in nicotine treated cells by Western blot. The number on top of blot represents arbitrary unit showing quantification of the Twist expression as measured by Image J software. E. Expression of TWIST1. Representative diagrams showing expression of TWIST1 in tissues of stage III breast cancer patients who never smoked (3 patients as described in Table 1) and smoked (5 patients) as determined by immunostaining with anti-TWIST1 antibody. Adjacent bar graph shows quantitative expression of TWIST1 in these samples. Each sample was performed in duplicate or in triplicate. The number of cells showing positive staining of TWIST1 was scored independently and blindly in 3 different area of each slide and then data were expressed as mean ± S.D, where *** represents p≤0.001. F. Effect of TWIST1 silencing on SP. Cells were treated with nicotine or nicotine plus si-TWIST1 and SP was measured as described in A.
Figure 6
Figure 6. Nicotine induced STAT3 activation and promoted expression of galectin-3 and TWIST1
A. Western blot showing STAT3 activation as identified by positive staining with anti-phosphorylated (at Tyr705) STAT3 antibody. The number on top of blot represents arbitrary unit showing quantification of STAT3p expression as measured by Image J software. B. Representative diagrams showing expression of STAT3p in tissues of stage III breast cancer patients who never smoked (3 patients as described in Table 1) and smoked (5 patients) as determined by immunostaining with anti-phosphorylated (at Tyr705) STAT3 antibody. Adjacent bar graph shows quantitative expression of STAT3p in these samples. Each sample was performed in duplicate or in triplicate. The number of cells showing positive staining of STAT3p was scored independently and blindly in 3 different area of each slide and then data were expressed as mean ± S.D, where *** represents p≤0.001. C. Quantitative mRNA expression of STAT3 in gene-specific or negative control siRNA treated MCF7 cells by RT-PCR. Relative mRNA expression based on GAPDH expression was calculated according to the comparative ΔΔC(t) method. D and E. Effect of STAT3 silencing on expression of TWIST1 (D) and galectin-3 (E). Cells were treated with nicotine or nicotine plus shRNA targeted to STAT3 and each extract was subjected to W. blot detection of Twist and galectin-3. The number on top of blot represents arbitrary unit showing quantification of TWIST1 (D) and galectin-3 (E) expression as measured by Image J software. F. Partial sequence of the galectin-3 promoter comprising −2756 bp to −2688 bp upstream of the transcription site. Sequence in red color represents STAT3 binding motif. G. Chromatin immunoprecipitation (ChIP) with anti-phosphorylated (Tyr705) STAT3 antibody and the precipitated DNA was subjected to PCR with primers located at promoters of TWIST1 and galectin-3 (LGALS3).
Figure 7
Figure 7. STAT3 promotes nicotine induced side population (SP) and cell viability
A. MCF-7 cells were treated with nicotine (100 µM, 48 h) or nicotine plus sh1-STAT3 and SP was analyzed using Dye cycle violet dye (DCV). B. Cell viability of the treated cells was studied using WST-1. In both cases, data were represented as ± SD, *** represents as p≤0.001. Staurosporine was used as an apoptosis inducer.
Figure 8
Figure 8. α9nAChR promotes nicotine induced STAT3 activation, cell viability, side population (SP), and formation of mammospheres
A. MCF-7 cells were treated with nicotine (100 µM, 48 h) or nicotine plus si-α9nAChR and STAT3 activation was measured by anti-phosphorylated (at Tyr705) STAT3 antibody on Western blot. The number on top of blot represents arbitrary unit showing quantification of the STAT3p expression as measured by Image J software. B. Cell viability of the treated cells was studied using WST-1. The data was represented as ± SD, *** represents p≤0.001. Staurosorine used as an apoptosis inducer. C. Effect of α9nAChR silencing on SP. Cells were treated with nicotine (100 µM, 48 h) or nicotine plus si-α9nAChR and SP was measured using Dye cycle violet dye (DCV). D. and E. Effect of nicotine and silencing of various genes on mammosphere formation. MCF-7 cells were treated with siRNA targeted to each gene for 48h. After washing, equal number of cells from each treatment was incubated in Mammocult medium for 48 h and spheres were counted. In D, a representative mammosphere that obtained from the nicotine-treated MCF-7 cells is shown.
Figure 9
Figure 9. A schematic diagram showing nicotine-induced signaling in MCF-7 cells
Nicotine treatment induces expression of α9nAChR and activates STAT3 through phosphorylation at Tyr705. Activated STAT3 migrates to nucleus and binds to the promoter of galectin-3 and TWIST1 resulting into transcriptional up-regulation of these genes.

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