miRNA-200b Signature in the Prevention of Skin Cancer Stem Cells by Polyphenol-enriched Blueberry Preparation

Abstract

Exposure of the skin to solar UV radiation leads to inflammation, DNA damage, and dysregulation of cellular signaling pathways, which may cause skin cancer. Photochemoprevention with natural products is an effective strategy for the control of cutaneous neoplasia. Polyphenols have been proven to help prevent skin cancer and to inhibit the growth of cancer stem cells (CSCs) through epigenetic mechanisms, including modulation of microRNAs expression. Thus, the current study aimed to assess the effect of polyphenol enriched blueberry preparation (PEBP) or non-fermented blueberry juice (NBJ) on expression of miRNAs and target proteins associated with different clinicopathological characteristics of skin cancer such as stemness, motility, and invasiveness. We observed that PEBP significantly inhibited the proliferation of skin CSCs derived from different melanoma cell lines, HS 294T and B16F10. Moreover, PEBP was able to reduce the formation of melanophores. We also showed that the expression of the CD133⁺ stem cell marker in B16F10 and HS294T cell lines was significantly decreased after treating the cells with PEBP in comparison to the NBJ and control groups. Importantly, tumor suppressors' miR-200s, involved in the regulation of the epithelial-to-mesenchymal transition and metastasis, were strikingly upregulated. In addition, we have shown that a protein target of the tumor suppressor miR200b, ZEB1, was also significantly modulated. Thus, the results demonstrates that PEBP possesses potent anticancer and anti-metastatic potentials and may represent a novel chemopreventative agent against skin cancer.

Keywords: Cancer stem cells; MicroRNA-200b; Neoplasm metastasis; Polyphenols; Zinc finger E-box binding homeobox 1.

Figure 1. Sphere growth is inhibited after PEBP treatment.

Inhibition of human melanoma HS 294T (A) and murine melanoma B16F10 (B) spheroid development after treatment with 100 and 150 μM GAE (gallic acid equivalent) of either PEBP or NBJ. Photographs of HS294T (C) and B16F10 (D) spheres taken with AxioCamMR3 camera on light microscope (magnification, ×20). After 2 days in culture. Spheres are isolated and grown in 96-well plates at 37°C and 5% CO₂. Significance shows as *P < 0.05, **P < 0.01, ****P < 0.0001 different from control. Two-Way ANOVA was used. Data represent a combination of 3 experiments. All data are presented as mean ± SEM. PEBP, polyphenol enriched blueberry preparation; NBJ, non-fermented blueberry juice.

Figure 2. PEBP suppresses the CD133⁺ CD44⁺ CD24⁺ phenotype in B16F10 and the CD133⁺ CD20⁺ phenotype in HS294T melanoma skin cancer.

Effect of 24 hours of exposure to PEBP and NBJ on surface marker expression of cancer stem cells (CSCs) in B16F10 and HS 294T cell lines characterized by flow cytometry. (A) B16F10 CSCs were treated with PEBP (100 μM GAE) and NBJ (100 μM GAE). Suspension cells were labeled with FITC-conjugated anti-CD133, PE-CY7 conjugated anti-CD44 and APC-conjugated anti-CD24 antibodies for B16F12 cells. (B) APC-conjugated anti-CD133, Alexa Fluor 450-conjugated anti CD20 for HS 294T cells and analyzed by control flow cytometry. PEBP, polyphenol enriched blueberry preparation; NBJ, non-fermented blueberry juice.

Figure 3. PEBP inhibits migration of melanoma cells.

Effects of PEBP on cell motility. (A, B) HS 294T and B16F10 cells exposed to different concentrations of either PEBP or NBJ at 0, 24, and 48 hours. Cells were plated in 6-well plates in Dulbecco’s Modified Eagle’s Medium (DMEM) and incubated at 5% CO₂ and 37ºC. (C, D) Photographs were taken at indicated time points after scratch injury. Two-Way ANOVA was used to analyze the statistical difference between the groups. Data represent a combination of 3 experiments. All data are presented as mean ± SEM of relative wound closure. Significance shows as *P < 0.05, **P < 0.01, ***P < 0.001 different from control. PEBP, polyphenol enriched blueberry preparation; NBJ, non-fermented blueberry juice; CTR, control.

Figure 4. Expression of miR-200b is upregulated in skin cancer.

Relative normalized expression (RT2-quantitative real-time PCR [qPCR] analysis) of miR-200b in B16F10 cells after 24 hours exposure to different concentrations of PEBP or NBJ. Cells were plated in (A) 6-well attachment plates in Dulbecco’s Modified Eagle’s Medium (DMEM) and (B) 6-well ultra-low attachment plates in DMEM-F12 (spheroid medium) and incubated at 5% CO₂ and 37ºC. Data were normalized to the RNA control U6snRNA. One-way ANOVA, followed by post-hoc Tukey’s multiple comparisons were used. Data represent a combination of 3 experiments. All data are presented as mean ± SEM. Significance shows as **P < 0.01, ***P < 0.001 different from control. PEBP, polyphenol enriched blueberry preparation; NBJ, non-fermented blueberry juice.

Figure 5. PEBP elevates the expression of miR-200b in transfected B16F10 cells.

Relative normalized expression (RT2-quantitative real-time PCR [qPCR] analysis) of miR-200b after 24-hour exposure to either PEBP or NBJ. B16F10 cells could grow to 30% confluence in DMEM medium. Cells were transfected with miR-200b mimic, anti-miR miRNA Inhibitor, control (NLNT), Lipofectamine no target (LNT) and noncoding RNA (NC1) using Lipofectamine 2000 (Invitrogen, Burlington, ON, Canada), and the expression of miR-200b was detected by RT2-qPCR. Two-Way ANOVA was used to analyze the statistical difference between the groups. Data represent a combination of 3 experiments. All data are presented as mean ± SEM. Significance shows as *P < 0.05, ***P < 0.001, ****P < 0.0001 different from control. PEBP, polyphenol enriched blueberry preparation; NBJ, non-fermented blueberry juice.

Figure 6. PEBP reduces sphere growth in transfected B16F10 cells.

Inhibition of spheroid development derived from the murine melanoma cell line B16F10 after 24-hour exposure to PEBP. (A) B16F10 cells were transfected with miR-200b mimic (MC), anti-miR miRNA Inhibitor (IB), control (NLNT), Lipofectamine no target (LNT) and noncoding RNA (NC1) in 6-ultra-low attachment plates in BMEM-F12 and spheroid medium and incubated at 5% CO₂ and 37ºC. (B) The phenotype of control, PEBP-treated and NBJ-treated cells after 2 days in culture (magnification, ×20). Two-Way ANOVA was used to analyze the statistical difference between the groups. Data represent a combination of 3 experiments. All data are presented as mean ± SEM. Significance shows as ***P < 0.001, ****P < 0.0001 different from control. PEBP, polyphenol enriched blueberry preparation; NBJ, non-fermented blueberry juice.

Figure 7. miR-200b inhibits ZEB1 expression in transfected B16F10 cells.

(A) Sample Western blot photographs for ZEB1 and control β-actin as shown on Image Lab. (B) Combined Western blot data from Image Lab calculations. B16F10 melanoma cells were transfected with a negative control, mimic, or inhibitor and analyzed by Western blot. One-way ANOVA, followed by post-hoc Tukey’s multiple comparisons was used. Data represent a combination of 3 experiments. All data are presented as mean ± SEM. Significance shows as *P < 0.05, ***P < 0.001, ****P < 0.0001 different from control. ZEB1, zinc-finger and E-box binding homeobox 1.

Figure 8. PEBP down-regulates ZEB1 expression in B16F10 cells.

(A) Sample Western blot photographs for ZEB1 and control α-tubulin as shown on Image Lab. (B) Combined Western blot data from Image Lab calculations. Samples were treated by either polyphenol enriched blueberry preparation (PEBP) or non-fermented blueberry juice (NBJ) for 48 hours and detected by Western blot. One-way ANOVA, followed by post-hoc Tukey’s multiple comparisons was used. Data represent a combination of 5 experiments. All data are presented as mean ± SEM. Significance shows as **P < 0.01, ***P < 0.001 different from control. ZEB1, zinc-finger and E-box binding homeobox 1; PEBP, polyphenol enriched blueberry preparation; NBJ, non-fermented blueberry juice; CTR, calcitonin receptor.