Sulforaphane synergizes with quercetin to inhibit self-renewal capacity of pancreatic cancer stem cells - PubMed (original) (raw)

Sulforaphane synergizes with quercetin to inhibit self-renewal capacity of pancreatic cancer stem cells

Rakesh K Srivastava et al. Front Biosci (Elite Ed). 2011.

Abstract

According to the cancer stem cell hypothesis, the aggressive growth and early metastasis of cancer may arise through dysregulation of self-renewal of stem cells. The objectives of this study were to examine the molecular mechanisms by which sulforaphane (SFN, an active compound in cruciferous vegetables) inhibits self-renewal capacity of pancreatic cancer stem cells (CSCs), and synergizes with quercetin, a major polyphenol and flavonoid commonly detected in many fruits and vegetables. Our data demonstrated that SFN inhibited self-renewal capacity of pancreatic CSCs. Inhibition of Nanog by lentiviral-mediated shRNA expression enhanced the inhibitory effects of sulforaphane on self-renewal capacity of CSCs. SFN induced apoptosis by inhibiting the expression of Bcl-2 and XIAP, phosphorylation of FKHR, and activating caspase-3. Moreover, SFN inhibited expression of proteins involved in the epithelial-mesenchymal transition (beta-catenin, vimentin, twist-1, and ZEB1), suggesting the blockade of signaling involved in early metastasis. Furthermore, the combination of quercetin with SFN had synergistic effects on self-renewal capacity of pancreatic CSCs. These data suggest that SFN either alone or in combination with quercetin can eliminate cancer stem cell-characteristics.

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Figures

Figure 1

Figure 1

Effects of SFN on spheroid cell viability in cancer stem cells (CSCs) derived from human pancreatic cancer cell lines. (A), Pancreatic CSCs were isolated from MIA PaCa-2 cells, seeded in suspension and treated with SFN (0-10 μM) for 7 days. At the end of incubation period, all the spheroids were collected and resuspended. Cell viability was measured by trypan blue assay. Data represent mean ± SD. *, #, % or ## = significantly different from control, P < 0.05. (B), Pancreatic cancer stem cells were isolated from PANC-1 cells, seeded in suspension and treated with SFN (0-10 μM) for 7 days. At the end of incubation period, all the spheroids were collected and resuspended. Cell viability was measured by trypan blue assay. Data represent mean ± SD. *, #, % or ** = significantly different from control, P < 0.05. (C), Pancreatic cancer stem cells were isolated from AsPC-1 cells. CSCs were seeded in suspension and treated with SFN (0-10 μM) for 7 days. At the end of incubation period, all the spheroids were collected and resuspended. Cell viability was measured by trypan blue assay. Data represent mean ± SD. *, %, # or ## = significantly different from control, P < 0.05. (D), Pancreatic cancer stem cells were isolated from Bx PC-3 cells. CSCs were seeded in suspension and treated with SFN (0-10 μM) for 7 days. At the end of incubation period, all the spheroids were collected and resuspended. Cell viability was measured by trypan blue assay. Data represent mean ± SD. *, #, % or ## = significantly different from control, P < 0.05.

Figure 2

Figure 2

Effects of SFN on tumor spheroids and cell viability of pancreatic cancer stem cells (CSCs). (A), Pancreatic CSCs were seeded in suspension and treated with SFN (0-10 μM) for 7 days. Pictures of spheroids formed in suspension were taken by a microscope. (B), Pancreatic CSCs were seeded in suspension and treated with SFN (0-10 μM) for 7 days. At the end of incubation period, all the spheroids were collected and resuspended. Cell viability was measured by trypan blue assay. Data represent mean ± SD. *, #, % or ## = significantly different from control, P < 0.05.

Figure 3

Figure 3

SFN inhibits colony formation by pancreatic CSCs. Pancreatic CSCs were seeded in soft agar and treated with various doses of SFN and incubated at 4°C for 21 days. At the end of incubation period, colonies were counted. Data represent mean ± SD. * or ** = significantly different from respective controls, P < 0.05.

Figure 4

Figure 4

Inhibition of Nanog by shRNA enhances the antiproliferative effects of SFN. Isolated pancreatic CSCs were transduced with either Nanog scrambled or Nanog shRNA. Transduced cells were treated with various doses of SFN and maintained in pancreatic cancer stem cell medium for 7 days. At the end of incubation period, all the spheroids were collected and resuspended. Cell viability was measured by trypan blue assay. Data represent mean ± SD. *, &, #, %, ## or ** = significantly different from control, P < 0.05.

Figure 5

Figure 5

Regulation of apoptosis-related proteins and apoptosis by SFN in pancreatic cancer stem cells. (A), Regulation of apoptosis-related proteins by SFN. Pancreatic CSCs were treated with SFN (0-10 μM) for 48 h. The Western blot analyses were performed to examine the expression of XIAP, Bcl-2, total caspase-3, phospho-FKHR and GAPDH. (B), Regulation of apoptosis by SFN. Pancreatic CSCs were treated with SFN (0-10 μM) for 48 h, and apoptosis was measured by TUNEL assay.

Figure 6

Figure 6

Regulation of epithelial mesenchymal transition factors by SFN in pancreatic cancer stem cells. Pancreatic CSCs were treated with SFN (0-10 μM) for 48 h. At the end of incubation period, the expression of β-catenin, vimentin, Twist-1 and Zeb-1 was measured by the Western blot analysis.

Figure 7

Figure 7

Quercetin synergizes with SFN to inhibit self-renewal capacity of pancreatic cancer CSCs. (A), Quercetin synergizes with SFN to inhibit spheroid cell viability. Pancreatic CSCs were seeded in suspension and treated with SFN (0-10 μM) with or without quercetin (20 μM) for 7 days. At the end of incubation period, all the spheroids were collected and resuspended. Cell viability was measured by trypan blue assay. Data represent mean ± SD. *, &, @ or # * = significantly different from control, P < 0.05. (B), Quercetin synergizes with SFN to inhibit colony formation. SFN inhibits colony formation by pancreatic CSCs. Pancreatic CSCs were seeded in soft agar and treated with various doses of SFN and incubated at 4°C for 21 days. At the end of incubation period, colonies were counted. Data represent mean ± SD. *, &, @ or # = significantly different from respective controls, P < 0.05.

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