Scorpion Venom-Functionalized Quercetin Phytosomes for Breast Cancer Management: In Vitro Response Surface Optimization and Anticancer Activity against MCF-7 Cells

Abstract

Breast cancer is a dangerous type of cancer in women. Quercetin (QRT), a naturally occurring flavonoid, has wide biological effects including antioxidant, anticarcinogenic, anti-inflammatory, antiallergic, and antiviral activities. The anticancer activity is considered the most valuable effect of QRT against several types of cancer, including prostate, liver, lung, colon, and breast cancer. Scorpion venom peptides (SV) has been found to induce apoptosis and aggravate cancer cells, making it a promising anticancer agent. QRT, SV, and Phospholipon^® 90H (PL) were incorporated in a nano-based delivery platform to assess QRT's cellular uptake and antiproliferative efficacy against a lung cancer cell line derived from human breast cancer cells MCF-7. Several nanovesicles were prepared and optimized, using four-factor Box-Behnken, in an experimental design. The optimized phytosomes showed vesicle size and zeta potential values of 116.9 nm and 31.5 mV, respectively. The IC50 values revealed that MCF-7 cells were significantly more sensitive to the optimized QRT formula than the plain formula and raw QRT. Cell cycle analysis revealed that optimized QRT formula treatment resulted in significant cell cycle arrest at the S phase. The results also indicated that treatment with QRT formula significantly increased caspase-9, Bax, Bcl-2, and p53 mRNA expression, compared with the plain formula and QRT. In terms of the inflammatory markers, the QRT formula significantly reduced the activity of TNF-α and NF-κB, in comparison with the plain formula and QRT only. Overall, the findings from the study proved that a QRT formulation could be a promising therapeutic approach for the treatment of breast cancer.

Keywords: apoptosis; breast cancer; nanocomplex; nanoparticles; optimization.

Figure 1

Diagnostic plots for responses of QRT-PHM-SV formulations. (AI) Box-Cox Plot for VS; (AII) Externally studentized residuals vs. run number plot for VS, (AIII) Normal probability plot for VS, (BI) Box-Cox Plot for ZP; (BII) Externally studentized residuals vs. run number plot for ZP, (BIII) Normal probability plot for ZP. Abbreviations: QRT, quercetin; PHM, phytosomes; SV, scorpion venom peptide, VS: vesicle size, ZP, zeta potential.

Figure 2

Perturbation graph for the effect of critical attributes; PL amount (X1), process temperature (X₂), reflux time (X₃), and SV amount (X₄) on (A) vesicle size, and (B) zeta potential of QRT-PHM-SV formulations. Abbreviations: QRT, quercetin; PHM, phytosomes; SV, scorpion venom peptide.

Figure 3

Contour 2D plots showing the effect and interaction between the significant factors on the vesicle size (A–C) and zeta potential (D) of QRT-PHM-SV formulations. Abbreviations: QRT, quercetin; PHM, phytosomes; SV, scorpion venom peptide.

Figure 4

Response surface 3D plots showing the effect and interaction between the significant factors on the vesicle size (A–C) and zeta potential (D) of QRT-PHM-SV formulations. Abbreviations: QRT, quercetin; PHM, phytosomes; SV, scorpion venom peptide.

Figure 5

TEM image of the optimized QRT-PHM-SV formulation.

Figure 6

Representation of the IC₅₀ values of QRT, PHM-SV, and QRT-PHM-SV in MCF-7 cells. # Significantly different from QRT at p < 0.05. $ Significantly different from PHM-SV at p < 0.05.

Figure 7

Flow cytometric analysis of control (A); QRT (B); PHM-SV (C); and QRT-PHM-SV-treated cells (D); and the percentages of cells in the G1, S, and G2/M phases of the cell cycle (E). * Significantly different from control at p < 0.05; # significantly different from QRT at p < 0.05.; $ significantly different from PHM-SVat p < 0.05.

Figure 8

Assessment of MCF-7 cell death in control (A); QRT (B); PHM-SV (C); and QRT-PHM-SV-treated cells (D); and the percentages of cells early, late, and total cell death (E) following annexin V staining. * Significantly different from control at p < 0.05; # significantly different from QRT at p < 0.05; $ Significantly different from PHM-SV at p < 0.05.

Figure 9

Effect of the QRT-PHM-SV formula on mitochondrial membrane potential (MMP) in MCF-7 cells. Data presented in bar charts are ± SD (n = 3). * or #, statistically different from control or QRT, respectively, at p < 0.05.

Figure 10

Effect of the QRT-PHM-SV formula on the expression of (A): caspase-9, (B): Bax, (C): Bcl-2, and (D): p53. Data are presented as Mean ± SD (n = 3); # significantly different from QRT at p < 0.05; $ significantly different from plain formula (PHM-SV) at p < 0.05.

Figure 11

Western blots (A); histogram of proteins expression of Bax, Bcl2, P53 and Casp9 (B) for the four groups in MCF-7 cells; (I) untreated control; (II) QRT; (III) PHM-SV; (IV) QRT-PHM-SV. * Significantly different from control at p < 0.05; # significantly different from QRT at p < 0.05; $ Significantly different from PHM-SV at p < 0.05.

Figure 12

Effect of the QRT-PHM-SV formula on the mRNA expression of TNF-α (A) and the activation of NF-κB (B) in MCF-7 cells. Data presented in bar charts are ± SD (n = 3). # or $, statistically different from QRT or plain formula (PHM-SV), respectively, at p < 0.05.

Figure 13

The proposed mechanism of apoptosis triggered by QRT-PHM-SV in MCF-7 cells. The flow chart demonstrates that QRT-PHM-SV induces apoptosis via the mitochondrial pathway and caspase-9-dependent signaling.