Targeting Breast Cancer Stem Cells with the Curcumin Nanomedicine
thesisposted on 10.12.2012, 00:00 by Ece Gulcur
Purpose: Despite recent advances in breast cancer therapy, many patients still experience relapse. Evidence suggests that a sub-population of treatment-resistant “cancer stem cells” (CSCs) may be responsible for this tumor recurrence. Therefore, in this thesis project we aimed to develop a safe and efficacious nanomedicine that would target and eliminate breast CSCs. For this purpose, we evaluated the sterically stabilized nanomicellar formulation of curcumin (C-SSM), a promising dietary anti-cancer agent with a vast potential to target CSCs, for its in vitro inhibitory effect on breast CSCs and the potential of integrating an active targeting strategy directed to CSCs. Methods: MCF-7 cells were chosen as the model system for this project. C-SSM composed of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG 2000) were prepared using the film rehydration method and characterized for particle size using dynamic light scattering. The in vitro inhibitory effect of the C-SSM nanomedicine on breast CSCs was evaluated and compared to that of free curcumin using a tumorsphere formation assay and flow cytometric analyses of the cellular drug uptake as well as the percentage of CD44+/CD24-/low CSC population. The expression of vasoactive intestinal peptide (VIP) receptors on breast CSCs was investigated using a fluorescence-based ligand binding assay, followed by confocal microscopy. VIP-surface grafted C-SSM (C-SSM-VIP) were prepared by conjugating VIP to the activated DSPE-PEG (MW:3400) and incorporating these constructs into C-SSM. The effect of VIP-surface grafting on the anti-CSC activity of C-SSM was evaluated using a tumorsphere formation assay. Results: Pre-treatment of MCF7 cells with 10 or 20 μM C-SSM (~15 nm in diameter) inhibited tumorsphere formation by 34% (P < 0.05) and 64% (P < 0.001), respectively. 20 µM C-SSM increased tumorsphere inhibition by > 2-fold compared to free curcumin. C-SSM was also found to reduce sphere size (~50%; P < 0.05) and to inhibit secondary tumorsphere formation (22%; P < 0.05). The flow cytometric analyses demonstrated that C-SSM (10 µM and 20 µM) markedly increased the intracellular uptake of curcumin compared to free drug (1.8-fold – 6.4-fold) in both CD44+/CD24-/low CSC and normal cancer cell (NCC) populations. In addition, 20 µM C-SSM reduced the percentage of the CD44+/CD24-/low CSCs by 56% (P < 0.05), while no significant change was observed with free curcumin. The VIP receptor expression study showed a ~3-fold overexpression (P < 0.05) in the CD44+/CD24-/low CSC population compared to the NCCs in the MCF-7 cell line. The C-SSM-VIP nanomedicine (~17 nm), at a lower concentration (5 µM), was found to enhance inhibition of tumorsphere formation by approximately 20% (P < 0.05) compared to both C-SSM and free curcumin. Conclusion: C-SSM significantly enhanced the efficacy of curcumin against breast CSCs in vitro. Moreover, active targeting of the VIP receptors, found to be overexpressed on breast CSCs, with C-SSM-VIP further improved the anti-CSC activity of C-SSM. Therefore, the nanomicellar formulations of curcumin may provide a novel strategy for a relapse-free cure of breast cancer by targeting and eliminating CSCs.