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. 2020 Aug 18;10(1):13958.
doi: 10.1038/s41598-020-70948-5.

STAT3 inhibition with galiellalactone effectively targets the prostate cancer stem-like cell population

Affiliations

STAT3 inhibition with galiellalactone effectively targets the prostate cancer stem-like cell population

Giacomo Canesin et al. Sci Rep. .

Abstract

Cancer stem cells (CSCs) are a small subpopulation of quiescent cells with the potential to differentiate into tumor cells. CSCs are involved in tumor initiation and progression and contribute to treatment failure through their intrinsic resistance to chemo- or radiotherapy, thus representing a substantial concern for cancer treatment. Prostate CSCs' activity has been shown to be regulated by the transcription factor Signal Transducer and Activator of Transcription 3 (STAT3). Here we investigated the effect of galiellalactone (GL), a direct STAT3 inhibitor, on CSCs derived from prostate cancer patients, on docetaxel-resistant spheres with stem cell characteristics, on CSCs obtained from the DU145 cell line in vitro and on DU145 tumors in vivo. We found that GL significantly reduced the viability of docetaxel-resistant and patient-derived spheres. Moreover, CSCs isolated from DU145 cells were sensitive to low concentrations of GL, and the treatment with GL suppressed their viability and their ability to form colonies and spheres. STAT3 inhibition down regulated transcriptional targets of STAT3 in these cells, indicating STAT3 activity in CSCs. Our results indicate that GL can target the prostate stem cell niche in patient-derived cells, in docetaxel-resistant spheres and in an in vitro model. We conclude that GL represents a promising therapeutic approach for prostate cancer patients, as it reduces the viability of prostate cancer-therapy-resistant cells in both CSCs and non-CSC populations.

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Conflict of interest statement

Anders Bjartell and Rebecka Hellsten are shareholders of Glactone Pharma AB. This does not affect the authors’ adherence to the journal’s policies regarding data and material sharing. The rest of the authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Characterization of stem cell and transient amplifying/committed basal cell populations from DU145 cells. (a) Flow cytometry analysis of CSCs (CD133+CD44+) and TA/CB (CD44+CD24+) populations in DU145 cells. (b) qPCR analysis of CD133, CD44 and ALDH1A1 gene expression in CSCs and TA/CB cells sorted from DU145 cells. Results represent the mean ± s.e.m. of five independent experiments (n = 5), each performed in triplicate. *p < 0.05; **p < 0.01. (c) Colony formation assay of CSCs and TA/CB cells sorted from DU145 cells and grown on collagen-coated plates. Representative images are shown on the left; the number of colonies is shown in the right graph. Results represent the mean ± s.e.m of three (n = 3) independent experiments, each performed in duplicate. (d) Sphere formation assay on CSCs and TA/CB cells sorted from DU145 cells. Representative images are shown on the left; the number of spheres is shown in the right graph. Results represent the mean ± s.e.m of three (n = 3) independent experiments, each performed in triplicate. (e) Gene expression analysis of stemness related genes in spheres derived from CSCs (CD133+/CD44+) and TA/CB (CD44+/CD24+) populations. Gene expression was studied by qPCR for CD133, CD44 and ALDH1A1. Results represent the mean ± s.e.m of three (n = 5) independent experiments, each performed in triplicate. *p < 0.05; **p < 0.01. (f) Gene expression analysis of STAT3-target genes in spheres derived from CSCs (CD133+/CD44+) and TA/CB (CD44+/CD24+) populations. Gene expression was studied by qPCR for c-myc, Bcl-XL, Mcl-1 and Survivin. Results represent the mean ± s.e.m of three (n = 3) independent experiments, each performed in triplicate. *p < 0.05. (g) Immunocytochemical analysis of phosphorylated STAT3 (pSTAT3) levels in DU145 cells (panel I), CSCs (panel II) and TA/CB cells (panel III) sorted from DU145 cells. LNCaP cells treated with 50 ng/ml IL-6 were used as positive control (panel IV). Panels V to VIII represents enlargements of the area in the red square. Arrows in (VII) indicate positive cytoplasmic staining. Scale bar = 100 μm.
Figure 2
Figure 2
Effect of GL on the viability, colony and sphere formation ability of CSCs and TA/CB cells. (a) Viability assay on CSCs and TA/CB cells sorted from DU145 cells and treated with vehicle or 2.5–10 μM GL for 24 or 48 h. Results represent the mean ± s.e.m of six (n = 6) independent experiments, each performed in triplicate. Statistical significance was determined using one-way ANOVA with Bonferroni post hoc test. *p < 0.05; ***p < 0.001. (b) Colony formation assay of CSCs (CD133+, top) and TA/CB (CD133, bottom) cells sorted from DU145 cells and grown on collagen-coated plates for 24 or 48 h in the presence of vehicle or 2.5–10 μM GL. Representative images are shown on the left; the number of colonies is shown in the right graph. Results represent the mean ± s.e.m of three (n = 3) independent experiments, each performed in triplicate. Statistical significance was determined using one-way ANOVA with Bonferroni post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; n.s. not significant. (c) Sphere formation assay on CSCs cells sorted from DU145 cells and grown in the presence of vehicle or 2.5–10 μM GL. Representative images are shown on the left; the number of CSCs-derived spheres is shown in the right graph. Results represent the mean ± s.e.m of three (n = 3) independent experiments, each performed in triplicate. Statistical significance was determined using one-way ANOVA with Bonferroni post hoc test. ***p < 0.001. (d) Sphere formation assay on TA/CB cells sorted from DU145 cells and grown in the presence of vehicle or 2.5–10 μM GL. Representative images are shown on the left; the number of CSCs-derived spheres is shown in the right graph. Results represent the mean ± s.e.m of three (n = 3) independent experiments, each performed in triplicate. Statistical significance was determined using one-way ANOVA with Bonferroni post hoc test. **p < 0.01; n.s. not significant.
Figure 3
Figure 3
Effect of GL on the expression of STAT3-target genes. (a, b) qPCR analysis of Mcl-1, Bcl-XL, c-myc and survivin gene expression in CSCs-derived spheres (a) and in TA/CB-derived spheres (b) grown in the presence of vehicle or 2.5–10 μM GL. Results represent the mean ± s.e.m. of three independent experiments (n = 3), each of which was performed in triplicate. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 4
Figure 4
Effect of GL on the expression of STAT3-target genes and on the expression of stemness genes in vivo. (a) Representative images and number of CD44+ cells in DU145-luc orthotopic xenograft from vehicle- and GL-treated mice. Results are presented as the mean ± s.e.m; statistical significance was determined using an unpaired Student t test. *p < 0.05. All images were taken with a 20 T objective (scale bar = 100 mm). (b) qPCR analysis of ALDHA1 levels in tumor samples of mice injected orthotopically with DU145-luc cells and treated vehicle- or GL-treated. Results represent the mean ± s.d. of three independent experiments (n = 3), each performed in triplicate. *p < 0.05. (c) qPCR analysis of STAT3 levels in tumor samples of mice injected orthotopically with DU145-luc cells and treated with vehicle or GL. Results represent the mean ± s.d. of three independent experiments (n = 3), each performed in triplicate. (d) Western blot analysis of pSTAT3-Y705, pSTAT3-S727 and total STAT3 levels in tumor samples of mice injected orthotopically with DU145-luc cells and treated with vehicle or GL. Lysates from DU145 and LnCaP cell lines were used as positive and negative controls, respectively. A representative blot of two independent experiments (n = 2) is shown. (e) qPCR analysis of Mcl-1 levels in tumor samples of mice injected orthotopically with DU145-luc cells and treated with vehicle or GL. Results represent the mean ± s.d. of three independent experiments (n = 3), each performed in triplicate. *p < 0.05. (f) qPCR analysis of Bcl-XL levels in tumor samples of mice injected orthotopically with DU145-luc cells and treated with vehicle or GL. Results represent the mean ± s.d. of three independent experiments (n = 3), each performed in triplicate. *p < 0.05. (g) Representative images and number of ALDH1A1+ cells in DU145 sub cutaneous xenografts from vehicle- and GL-treated mice. Results are presented as the mean ± s.e.m; statistical significance was determined using an unpaired Student t test. **p < 0.01. All images were taken with a 20 T objective (scale bar = 100 mm).
Figure 5
Figure 5
Effect of GL on docetaxel-resistant DU145 spheres and on patient-derived spheres. (a) qPCR analysis of stemness related genes in DU145-DR adherent cells and spheres. Results represent the mean ± s.d. of three independent experiments (n = 3), each performed in triplicate. ***p < 0.001; n.s. not significant. (b) qPCR analysis of stemness related genes in DU145-DR spheres and DU145-DS spheres. Results represent the mean ± s.d. of three independent experiments (n = 3), each performed in sextuplicate. **p < 0.01; ***p < 0.001; n.s. not significant. (c) Viability assay on spheres derived from DU145-DR cells grown in the presence of vehicle or 2.5–10 μM GL for 48 h. Results represent the mean ± s.d. of six (n = 6) independent experiments, each performed in quintuplicate. Statistical significance was determined using one-way ANOVA with Bonferroni post hoc test. ***p < 0.001. (d) qPCR analysis of stemness related gene expressions in spheres and adherent cells derived from primary tumor #143. Results represent the mean ± s.d. of six independent experiments (n = 6), each performed in triplicate. *p < 0.05; **p < 0.01; n.s. not significant. (e) Viability assay on spheres derived from primary tumor #143 grown in the presence of vehicle or 2.5–10 μM GL. Results represent the mean ± s.d. of seven (n = 7) independent experiments, each performed in quintuplicate. Statistical significance was determined using one-way ANOVA with Bonferroni post hoc test. ***p < 0.001. (f) qPCR analysis of stemness related gene expressions in spheres and adherent cells derived from primary tumor #318. Results represent the mean ± s.d. of six independent experiments (n = 6), each performed in triplicate. *p < 0.05; **p < 0.01; n.s. not significant. (g) Viability assay on spheres derived from primary tumor #318 grown in the presence of vehicle or 2.5–10 μM GL. Results represent the mean ± s.d. of ten (n = 10) independent experiments, each performed in quintuplicate. Statistical significance was determined using one-way ANOVA with Bonferroni post hoc test. ***p < 0.001. (h) qPCR analysis of stemness related gene expressions in spheres and adherent cells derived from primary tumor #285. Results represent the mean ± s.d. of six independent experiments (n = 6), each performed in triplicate. *p < 0.05; **p < 0.01; n.s. not significant. (i) Viability assay on spheres derived from primary tumor #285 grown in the presence of vehicle or 2–8 μM GL. Results represent the mean ± s.d. of six (n = 6) independent experiments, each performed in quintuplicate. Statistical significance was determined using one-way ANOVA with Bonferroni post hoc test. ***p < 0.001.

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