Prostate cancer stem cells are shown to be androgen independent and lack expression of a functional androgen receptor 
which renders them immune to androgen deprivation therapy (ADT). In a mouse model of prostate cancer, increased expression of stem cell markers was observed after ADT 
. Furthermore, cancer stem cells are resistant to current chemotherapy and ALDH expressing cancer stem cells have shown resistance to and enrichment by paclitaxel treatment 
. The cancer stem cell characteristics such as slow growth, multidrug resistance, enhanced DNA-repair, high expression of anti-apoptotic proteins 
, and in prostate cancer the lack of response to ADT, make these cells very difficult to target with conventional prostate cancer therapy. New therapeutic modalities must therefore be developed, in order to target both the tumor bulk and the cancer stem cells.
In the present study we identified cancer stem cell-like subpopulations in cultured prostate cancer cell lines which responded to treatment with the STAT3 inhibitor galiellalactone. A small fraction (2–4%) of the cell line populations showed high ALDH activity. These ALDH+ cells revealed stem cell-like characteristics such as increased colony forming and self-renewing capacity and high tumorigenicity as well as expression of putative prostate cancer stem cell markers. These cells responded to treatment with galiellalactone.
The capability of reconstituting a heterogenous mass is a definition of cancer stem cells, and we found that isolated ALDH+ prostate cancer cells were capable of self-renewal and re-establishment of the parental cell line and possessed high tumorigenicity in vivo
and in vitro
. The ALDH+ population of LNCaP-IL6 cells had a high expression of the putative prostate cancer stem cells markers CD44 and integrin α2β1 
compared to the ALDH− population. However, we did not detect CD133 expression in the ALDH+ or ALDH− prostate cancer cells. This is in agreement with the recent study by Pfeiffer and Schalken 
suggesting that CD133 is not a marker for stem cells in prostate cancer cell lines. Increased JAK/STAT3 and NF-κB activity is expressed in stem cells and tumor initiating stem-like cells in prostate cancer 
and our findings of active STAT3 and NF-κB in ALDH+ cells is in accordance with this suggestion. Taken together, our results, and others 
, indicate that ALDH expression can be a means of identifying cancer stem cell-like cells in prostate cancer.
The cancer stem cell-like ALDH+ population was greater in long term IL-6 stimulated LNCaP cells compared to LNCaP cells supporting the view that prostate cancer stem cells show a pro-inflammatory phenotype 
. Gene expression profiling of prostate cancer stem cells show that the STAT3 signaling pathway is overexpressed in these cells 
and several studies point to STAT3 as a target for therapeutic intervention in tumor stem cells 
. This is in line with our finding that the ALDH+ stem cell-like cells from prostate cancer cell lines expressed active STAT3 and that these ALDH+ cells responded to the STAT3 inhibitor galiellalactone, which has previously been shown to induce apoptosis of prostate cancer cells with constitutive expression of active STAT3 
. The dose-response related decrease of the proportion of ALDH+ cells in the DU145 and LNCaP-IL6 cell populations and induction of apoptosis of these cells upon galiellalactone treatment suggest that these cancer stem cell-like cells are sensitive to STAT3 inhibition. Noteably, DU145 xenografts from galiellalactone treated mice showed reduced gene expression of ALDH1A1
compared to untreated DU145 xenografts indicating that galiellalactone may target prostate cancer stem cell-like cells also in vivo
. We have previously showed that the expression of the STAT3 related genes BCL2L1
were down-regulated by galiellalactone further confirming the STAT3 inhibitory effect of the drug in vivo 
Other natural products besides galiellalactone have been shown to inhibit cancer stem cells. Sesquiterpene lactone parthenolide and curcumin are cytotoxic to cancer stem cells and target the cells by inhibiting the activity of NF-κB and STAT3 
. Targeting the STAT3 pathway in prostate cancer stem cell-like cells with natural product derived compounds may be a promising therapeutic approach for the development prostate cancer drugs.
In conclusion, prostate cancer cell lines contained ALDH+ subpopulations with stem cell-like characteristics which expressed phosphorylated STAT3. These subpopulations were clearly inhibited by the STAT3 inhibitor galiellalactone. These findings emphasize that targeting the STAT3 pathway in prostate cancer cells, including prostate cancer stem cell-like cells, may be a novel potent treatment strategy in patients with advanced prostate cancer resistant to ADT and cytotoxic therapy and that galiellalactone is an important compound for studying STAT3 signaling in prostate cancer and a potential starting point for the development of future prostate cancer drugs.