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. 2011;6(7):e22118.
doi: 10.1371/journal.pone.0022118. Epub 2011 Jul 11.

Galiellalactone Inhibits Stem Cell-Like ALDH-positive Prostate Cancer Cells

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Free PMC article

Galiellalactone Inhibits Stem Cell-Like ALDH-positive Prostate Cancer Cells

Rebecka Hellsten et al. PLoS One. .
Free PMC article

Abstract

Galiellalactone is a potent and specific inhibitor of STAT3 signaling which has been shown to possess growth inhibitory effects on prostate cancer cells expressing active STAT3. In this study we aimed to investigate the effect of galiellalactone on prostate cancer stem cell-like cells. We explored the expression of aldehyde dehydrogenase (ALDH) as a marker for cancer stem cell-like cells in different human prostate cancer cell lines and the effects of galiellalactone on ALDH expressing (ALDH+) prostate cancer cells. ALDH+ subpopulations were detected and isolated from the human prostate cancer cell lines DU145 and long-term IL-6 stimulated LNCaP cells using ALDEFLUOR® assay and flow cytometry. In contrast to ALDH- cells, ALDH+ prostate cancer cells showed cancer stem cell-like characteristics such as increased self-renewing and colony forming capacity and tumorigenicity. In addition, ALDH+ cells showed an increased expression of putative prostate cancer stem cell markers (CD44 and integrin α2β1). Furthermore, ALDH+ cells expressed phosphorylated STAT3. Galiellalactone treatment decreased the proportion of ALDH+ prostate cancer cells and induced apoptosis of ALDH+ cells. The gene expression of ALDH1A1 was downregulated in vivo in galiellalactone treated DU145 xenografts. These findings emphasize that targeting the STAT3 pathway in prostate cancer cells, including prostate cancer stem cell-like cells, is a promising therapeutic approach and that galiellalactone is an interesting compound for the development of future prostate cancer drugs.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. ALDH expressing cells in prostate cancer cell lines.
DU145, LNCaP and LNCaP-IL6 cells were subjected to ALDEFLUOR® assay in order to identify cells with high ALDH expression (ALDH+). The ALDH inhibitor DEAB was used as a negative control (left panel). The cells without inhibitor shifted to the right were considered ALDH+ cells (right panel).
Figure 2
Figure 2. Characteristics of ALDH+ and ALDH− cells.
ALDH+ cells show stem cell-like characteristics in terms of increased colony forming capacity and tumorigenicity. A. Clonogenic assay. 200 cells/well were seeded and colonies were counted after 1 week. Clonogenicity was calculated as percent colonies formed in relation to cells seeded (n = 2; p<0.05). B. Tumorigenicity assay. Tumor take after subcutaneous injections of 10 000 or 100 000 cells of freshly sorted ALDH+ and ALDH− cells from DU145 and LNCaP-IL6 (n = 5). C. Tumor size of ALDH+ and ALDH− cell xenografts after 6 weeks (n = 5). ALDH+ cell tumors from 100 000 cells injected subcutaneously were significantly larger than the ALDH− cell tumors for both DU145 cells (p = 0.0002) and LNCaP-IL6 cells (p = 0.0077).
Figure 3
Figure 3. Expression of cancer stem cell related markers.
A. Freshly sorted ALDH+ and ALDH− cells from DU145 and LNCaP-IL6 cells were subjected to cytospin and stained for CD44, integrin α2β1, p-STAT3 and p-NF-κB p65. B. The relative mRNA expression of CD44 and integrin α2 in ALDH+ and ALDH− cells freshly sorted from DU1145 and LNCaP-IL6 cells.
Figure 4
Figure 4. Galiellalactone decreases the proportion of ALDH+ prostate cancer cells in vitro and the ALDH1A1 expression in vivo.
A. DU145 and LNCaP-IL6 cells were treated with galiellalactone (5–50 µM) for 24 h and subjected to ALDEFLUOR® assay. The proportion of ALDH+ cells significantly decreased due to galiellalactone treatment in a dose dependent manner. The proportion of ALDH+ cells is expressed as mean ± SEM (n = 2–4). The proportion of ALDH+ DU145 cells were significantly decreased by galiellalactone at the concentrations 10 µM (p = 0.0060), 25 µM (p = 0,0013) and 50 µM galiellalactone (p<0,0001). The proportion of ALDH+ LNCaP-IL6 cells was significantly decreased by galiellalactone at the concentrations 25 µM (p = 0.0441) and 50 µM (p = 0.0445). B. Relative mRNA expression of ALDH1A1 in DU145 xenografts in mice treated with 1 mg/kg/day galiellalactone for three weeks. Control mice received vehicle. The relative mRNA expression of ALDH1A1 was reduced in galiellalactone treated mice compared to control (0.139±0.107 and 0.644±0.161, respectively, p = 0.07; n = 3–6).
Figure 5
Figure 5. Galiellalactone induces apoptosis and inhibits proliferation of ALDH+ prostate cancer cells.
A. ALDH+ and ALDH− cells sorted from DU145 and LNCaP-IL6 cells were treated with 25 µM galiellalactone for 24 h. Apoptotic cells were detected by c-PARP staining. B. Quantification of c-PARP stained apoptotic cells in ALDH+ and ALDH− cells sorted from DU145 and LNCaP-IL6 cells and treated with 25 µM galiellalactone for 24 h. Treatment with galiellalactone significantly increased the amount of c-PARP expressing cells in ALDH+ DU145, ALDH+ LNCaP-IL6 and ALDH− LNCaP-IL6 cells compared to untreated controls (p = 0.019, 0.035 and 0.009 respectively; n = 2). The difference in apoptotic response between galiellalactone treated ALDH+ and ALDH− cells from DU145 and LNCaP-IL6 cells was not significant (p = 0.059 and p = 0.119, respectively; n = 2). C. Galiellalactone decreased the viability of ALDH+ and ALDH− cells sorted from DU145 and LNCaP-IL6 cells. The ALDH+ DU145 cells were significantly more sensitive to galiellalactone compared to the corresponding ALDH− cells at 5 µM (p = 0.0017), 10 µM (p = 0.0010), 25 µM (p = 0.0019) and 50 µM (p = 0.0025). Results are presented as mean per cent of untreated control (n = 2).

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