The dietary bioflavonoid quercetin synergizes with epigallocathechin gallate (EGCG) to inhibit prostate cancer stem cell characteristics, invasion, migration and epithelial-mesenchymal transition

Abstract Background Much attention has been recently focused on the role of cancer stem cells (CSCs) in the initiation and progression of solid malignancies. Since CSCs are able to proliferate and self-renew extensively due to their ability to express anti-apoptotic and drug resistant proteins, thus sustaining tumor growth. Therefore, the strategy to eradicate CSCs might have significant clinical implications. The objectives of this study were to examine the molecular mechanisms by which epigallocathechin gallate (EGCG) inhibits stem cell characteristics of prostate CSCs, and synergizes with quercetin, a major polyphenol and flavonoid commonly detected in many fruits and vegetables. Results Our data indicate that human prostate cancer cell lines contain a small population of CD44+CD133+ cancer stem cells and their self-renewal capacity is inhibited by EGCG. Furthermore, EGCG inhibits the self-renewal capacity of CD44+α2β1+CD133+ CSCs isolated from human primary prostate tumors, as measured by spheroid formation in suspension. EGCG induces apoptosis by activating capase-3/7 and inhibiting the expression of Bcl-2, survivin and XIAP in CSCs. Furthermore, EGCG inhibits epithelial-mesenchymal transition by inhibiting the expression of vimentin, slug, snail and nuclear β-catenin, and the activity of LEF-1/TCF responsive reporter, and also retards CSC's migration and invasion, suggesting the blockade of signaling involved in early metastasis. Interestingly, quercetin synergizes with EGCG in inhibiting the self-renewal properties of prostate CSCs, inducing apoptosis, and blocking CSC's migration and invasion. These data suggest that EGCG either alone or in combination with quercetin can eliminate cancer stem cell-characteristics. Conclusion Since carcinogenesis is a complex process, combination of bioactive dietary agents with complementary activities will be beneficial for prostate cancer prevention and/ortreatment.Peer Reviewe

Sonic hedgehog signaling inhibition provides opportunities for targeted therapy by sulforaphane in regulating pancreatic cancer stem cell self-renewal.

Dysregulation of the sonic hedgehog (Shh) signaling pathway has been associated with cancer stem cells (CSC) and implicated in the initiation of pancreatic cancer. Pancreatic CSCs are rare tumor cells characterized by their ability to self-renew, and are responsible for tumor recurrence accompanied by resistance to current therapies. The lethality of these incurable, aggressive and invasive pancreatic tumors remains a daunting clinical challenge. Thus, the objective of this study was to investigate the role of Shh pathway in pancreatic cancer and to examine the molecular mechanisms by which sulforaphane (SFN), an active compound in cruciferous vegetables, inhibits self-renewal capacity of human pancreatic CSCs. Interestingly, we demonstrate here that Shh pathway is highly activated in pancreatic CSCs and plays important role in maintaining stemness by regulating the expression of stemness genes. Given the requirement for Hedgehog in pancreatic cancer, we investigated whether hedgehog blockade by SFN could target the stem cell population in pancreatic cancer. In an in vitro model, human pancreatic CSCs derived spheres were significantly inhibited on treatment with SFN, suggesting the clonogenic depletion of the CSCs. Interestingly, SFN inhibited the components of Shh pathway and Gli transcriptional activity. Interference of Shh-Gli signaling significantly blocked SFN-induced inhibitory effects demonstrating the requirement of an active pathway for the growth of pancreatic CSCs. SFN also inhibited downstream targets of Gli transcription by suppressing the expression of pluripotency maintaining factors (Nanog and Oct-4) as well as PDGFRα and Cyclin D1. Furthermore, SFN induced apoptosis by inhibition of BCL-2 and activation of caspases. Our data reveal the essential role of Shh-Gli signaling in controlling the characteristics of pancreatic CSCs. We propose that pancreatic cancer preventative effects of SFN may result from inhibition of the Shh pathway. Thus Sulforaphane potentially represents an inexpensive, safe and effective alternative for the management of pancreatic cancer

Resveratrol inhibits pancreatic cancer stem cell characteristics in human and KrasG12D transgenic mice by inhibiting pluripotency maintaining factors and epithelialmesenchymal transition. PLoS One 6: e16530

Background: Cancer stem cells (CSCs) can proliferate and self-renew extensively due to their ability to express anti-apoptotic and drug resistant proteins, thus sustaining tumor growth. Therefore, the strategy to eradicate CSCs might have significant clinical implications. The objectives of this study were to examine the molecular mechanisms by which resveratrol inhibits stem cell characteristics of pancreatic CSCs derived from human primary tumors and Kras G12D transgenic mice. Methodology/Principal Findings: Human pancreatic CSCs (CD133 + CD44 + CD24 + ESA +) are highly tumorigenic and form subcutaneous tumors in NOD/SCID mice. Human pancreatic CSCs expressing high levels of CD133, CD24, CD44, ESA, and aldehyde dehydrogenase also express significantly more Nanog, Oct-4, Notch1, MDR1 and ABCG2 than normal pancreatic tissues and primary pancreatic cancer cells. Similarly, CSCs from Kras G12D mice express significantly higher levels of Nanog and Oct-4 than pancreatic tissues from Pdx-Cre mice. Resveratrol inhibits the growth (size and weight) and development (PanIN lesions) of pancreatic cancer in Kras G12D mice. Resveratrol inhibits the self-renewal capacity of pancreatic CSCs derived from human primary tumors and Kras G12D mice. Resveratrol induces apoptosis by activating capase-3/7 and inhibiting the expression of Bcl-2 and XIAP in human CSCs. Resveratrol inhibits pluripotency maintaining factors (Nanog, Sox-2, c-Myc and Oct-4) and drug resistance gene ABCG2 in CSCs. Inhibition of Nanog by shRNA enhances the inhibitory effects of resveratrol on self-renewal capacity of CSCs. Finally, resveratrol inhibits CSC’s migration and invasion and marker

Regulation of Shh pathway by SFN in pancreatic cancer cell lines <i>in vitro</i>.

<p>(<b>A–B</b>), Inhibition of components of sonic hedgehog pathway. ASPC1 and PANC1 were treated with sulforaphane (0–20 µM) for 24 h. The expression of Gli1, Gli2 and Smo was measured by qRT-PCR and normalized to GAPDH expression. All assays were performed in triplicate and were calculated on the basis of ΔΔ<i>C</i>t method. Data represent mean ± SD. $, @, and & = significantly different from control, P < 0.05.</p

Characterization of human pancreatic CSCs from human primary tumors.

<p>(A–D), Expression of pancreatic stem cell markers. Flow cytometric analysis of Pancreatic CSCs expressing was performed using stem cell markers CD44 -PE, ESA-PerCP, CD133-APC, CD24-FITC and appropriate controls. (E–F), Expression of pancreatic epithelial markers and drug resistance genes. Flow cytometric analysis of Pancreatic CD44⁺ESA⁺CD133⁺CD24⁺ CSCs also expressed CK19-PE and ABCG2-PE respectively.</p

Effects of SFN on Gli translocation and transcription.

<p>(<b>A</b>), The nuclear translocation of Gli1 and Gli2, was measured by immunocytochemistry. Pancreatic CSCs were treated with sulforaphane (0–20 µM) for 24 h. Cells were then stained with anti-Gli and Gli2 antibody (green fluorescence), and DAPI (red fluorescence). Merged images are shown, which indicate yellow-orange staining of Gli 1 and Gli2 located in the nucleus due to co-localization of green and red fluorescence. (<b>B</b>), Inhibition of Gli transcription. Pancreatic CSCs were transduced with Gli-responsive GFP/firefly luciferase viral particles (pGreen Fire1-Gli with EF1, System Biosciences). After transduction, culture medium was replaced and CSCs were treated with sulforaphane (0–20 µM) for 24 h. Gli-responsive reporter activity was measured using a luciferase assay (Promega Corporation). Data represent mean ± SD. @, %, and * = significantly different from control, P < 0.05.</p

Regulation of Bcl-2 expression, caspase-3/7 activity, and apoptosis by SFN on Pancreatic CSCs.

<p>(<b>A</b>), Effects of SFN on BcL-2expression. q-RT-PCR was performed to examine the expression of BcL-2. All assays were performed in triplicate and were calculated on the basis of ΔΔ<i>C</i>t method. Data represent mean ± SD. @, % and $= significantly different from control, P < 0.05. (<b>B</b>), Effects of SFN on caspase-3/7 activity. Pancreatic CSCs treated with SFN (0–20 µM) for 24 h, and caspase-3/7 activity was measured as per manufacturer's instructions. Data represent mean ± SD. @,$ = significantly different from control, P < 0.05. (<b>C</b>), Pancreatic CSCs were treated with SFN (0–20 µM), and cell lysates were collected and Immunobloted for anti- BCL-2, cleaved Caspase 3 or β-actin antibody. (<b>D</b>), Effects of SFN on apoptosis. Pancreatic CSCs were treated with SFN (0–20 µM) for 48 h, and apoptosis was measured by PI staining using flow cytometry.</p

Regulation of Hh target genes involved in the maintenance of pluripotency in pancreatic cancer stem cells.

<p>(<b>A–B</b>), Effects of SFN on expression of Hh target genes in the pancreatic CSCs. Real time PCR (q-RT-PCR) was performed to examine the expression of Nanog and Oct4 and data were normalized with GAPDH. All assays were performed in triplicate and were calculated on the basis of ΔΔ<i>C</i>t method. Data represent mean ± SD. @ and % = significantly different from control, P < 0.05. (<b>C</b>), Pancreatic CSCs were treated with SFN (0–20 µM), and cell lysates were collected and Western blot analysis was performed using anti- Nanog, Oct4 or β-actin antibody. (<b>D–E</b>), Effects of SFN on expression of Hh target genes involved in cell proliferation in the pancreatic CSCs. Real time PCR (q-RT-PCR) was performed to examine the expression of PDGFRα and Cyclin D1, involved in the maintenance of proliferation was analyzed and normalized with GAPDH. All assays were performed in triplicate and were calculated on the basis of ΔΔ<i>C</i>t method. Data represent mean ± SD. @, %, and $ = significantly different from control, P < 0.05. (<b>F</b>), Pancreatic CSCs were treated with SFN (0–20 µM), and cell lysates were collected and Immunobloted for anti- PDGFRα, Cyclin D1 or β-actin antibody.</p

Regulation of Shh pathway by SFN in pancreatic CSCs <i>in vitro</i>.

<p>(<b>A–C</b>), Inhibition of components of sonic hedgehog pathway. Pancreatic CSCs were treated with sulforaphane (0–20 µM) for 24 h. The expression of Gli1, Gli2 and Smo was measured by qRT-PCR and normalized to GAPDH expression. All assays were performed in triplicate and were calculated on the basis of ΔΔ<i>C</i>t method. Data represent mean ± SD. *, @, and $ = significantly different from control, P < 0.05; (<b>D</b>), Immunoblotting of Gli1/2, Smo and β-actin of human pancreatic cancer stem cells (PanCSC) treated with Sulforaphane (0–20 µM).</p