Optimized 2-methoxyestradiol invasomes fortified with apamin: a promising approach for suppression of A549 lung cancer cells

Abstract

Certain anticancer agents selectively target the nucleus of cancer cells. One such drug is 2-methoxyestradiol (2ME), which is used for treating lung cancer. To improve the therapeutic effectiveness of these agents, many new methods have been devised. 2ME was entrapped into the core of hydrophobic invasomes (INVA) covered with Phospholipon 90G and apamin (APA). The Box-Behnken statistical design was implemented to enhance the composition. Using Design-Expert software (Stat-Ease Inc., Minneapolis, MN), the INVA component quantities were optimized to obtain spherical particles with the smallest size, that is, a diameter of 167.8 nm. 2ME-INVA-APA significantly inhibited A549 cells and exhibited IC₅₀ of 1.15 ± 0.04 µg/mL, which is lower than raw 2ME (IC₅₀ 5.6 ± 0.2 µg/mL). Post 2ME-INVA-APA administration, a significant rise in cell death and necrosis was seen among the A549 cells compared to those treated with plain formula or 2ME alone. This effect was indicated by increased Bax expression and reduced Bcl-2 expression, as well as mitochondrial membrane potential loss. Moreover, the cell cycle analysis showed that 2ME-INVA-APA arrests the G2-M phase of the A549 cells. Additionally, it was observed that the micellar formulation of the drug increased the cell count in pre-G1, thereby exhibiting phenomenal apoptotic potential. Furthermore, it up-regulates caspase-9 and p53 and downregulates TNF-α and NF-κβ. Collectively, these findings showed that our optimized 2ME-INVA-APA could easily seep through the cell membrane and induce apoptosis in relatively low doses.

Keywords: Liposomes; bee venom; cancer.

Figure 1.

Chemical structure of 2ME (A) and APA (B).

Figure 2.

Diagnostic plots for particle size of 2ME-INVA-APA. (A) Power transforms Box–Cox plot for, (B) outwardly predicted vs. studentized residuals values, (C) outwardly run number vs. studentized residuals, and (D) actual values plot vs. predicted. 2ME: 2-methoxyestradiol; INVA: invasomes; APA: apamin.

Figure 3.

Perturbation plot (A) and three-dimensional surface plots (B–D) for the effect and interactions between phospholipid % (X₁), ethanol % (X₂), and terpene % (X₃) on vesicle size of 2ME-INVA-APA. 2ME: 2-methoxyestradiol; INVA: invasomes; APA: apamin.

Figure 4.

IC₅₀ of the INVA-APA, 2ME, and 2ME-INVA-APA in the A549 cells. The results are the average of four separate trials with standard errors. ^#Significantly different from INVA-APA (p=.05). ^$Compared to 2ME, there is a significant difference (p=.05).

Figure 5.

Graphical presentation of A549 cell cycle phases following treatment for 24 h using flow cytometry analysis. (A) control; (B) INVA-APA; (C) 2ME; (D) 2ME-INVA-APA; (E) graphical presentation of cell cycle phases. All data is given as the mean standard deviation of three separate studies. *p=.05 was considered significant when compared to the control. ^#p<.05 was considered significant when compared to INVA-APA and ^$p<.05 was considered significant when compared to 2ME.

Figure 6.

Analysis of apoptosis via Annexin-V-FITC staining in A549 cells. The data are stated as the mean ± SE of number of separate tests (three). (A) control; (B) INVA-APA; (C) 2ME; (D) 2ME-INVA-APA; (E) graphical presentation of cell populations. *p< .05 considered significantly different from the control. ^#p< .05 considered significantly different from INVA-APA and ^$p< .05 considered significantly different from 2ME.

Figure 7.

Expression of apoptotic markers (A) Bax, (B) Bcl-2 (C) caspase-3, and (D) p53 within A549 lung cancer cells. All results are stated as the mean ± SE of three separate tests. ^#p<.05 was considered significant when compared to INVA-APA and ^$p<.05 was considered significant when compared to 2ME.

Figure 8.

Modulation of treatments using the plain formula, 2ME-raw and 2ME-INVA-APA over expression level of inflammatory markers (A) TNF-α and (B) NF-κB. All results are expressed as the mean ± SE of three separate experiments. *Significantly different from the control (p<.05). ^#p=.05 was considered significant when compared to INVA-APA, and ^$p<.05 was considered significant when compared to 2ME.

Figure 9.

The effect of INVA-APA, 2ME-raw, and 2ME-INVA-APA on MMP variation in A549 cell. Normalization of values took place with respect to untreated (control) A549 cells and expressed as variation percentage. Data denote the mean of four independent experiments ± SE. *Significantly different vs. control (p<.05), ^#significantly different vs. plain formula (p<.05), and ^$p<.05 considered significantly different from 2ME.