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. 2021 Dec 10;22(24):13284.
doi: 10.3390/ijms222413284.

Development and Evaluation of Repurposed Etoricoxib Loaded Nanoemulsion for Improving Anticancer Activities against Lung Cancer Cells

Shadab Md  1   2   3 , Nabil A Alhakamy  1   2   3 , Waleed S Alharbi  1   2 , Javed Ahmad  4 , Rasheed A Shaik  5 , Ibrahim M Ibrahim  6 , Javed Ali  7
Affiliations

Development and Evaluation of Repurposed Etoricoxib Loaded Nanoemulsion for Improving Anticancer Activities against Lung Cancer Cells

Shadab Md et al. Int J Mol Sci. .

Abstract

In the present work, novel modality for lung cancer intervention has been explored. Primary literature has established the potential role of cyclooxygenase-2 (COX-2) inhibitor in regression of multiple forms of carcinomas. To overcome its poor water solubility and boost anticancer activity, etoricoxib (ETO) was chosen as a therapeutic candidate for repurposing and formulated into a nanoemulsion (NE). The prepared ETO loaded NE was characterized for the surface charge, droplet size, surface morphology, and in vitro release. The optimized ETO loaded NE was then investigated for its anticancer potential employing A549 lung cancer cell line via cytotoxicity, apoptotic activity, mitochondrial membrane potential activity, cell migration assay, cell cycle analysis, Caspase-3, 9, and p53 activity by ELISA and molecular biomarker analysis through RT-PCR test. The developed ETO-NE formulation showed adequate homogeneity in the droplet size distribution with polydispersity index (PDI) of (0.2 ± 0.03) and had the lowest possible droplet size (124 ± 2.91 nm) and optimal negative surface charge (-8.19 ± 1.51 mV) indicative of colloidal stability. The MTT assay results demonstrated that ETO-NE exhibited substantial anticancer activity compared to the free drug. The ETO-NE showed a substantially potent cytotoxic effect against lung cancer cells, as was evident from the commencement of apoptosis/necrotic cell death and S-phase cell cycle arrests in A549 cells. The study on these molecules through RT-PCR confirmed that ETO-NE is significantly efficacious in mitigating the abundance of IL-B, IL-6, TNF, COX-2, and NF-kB as compared to the free ETO and control group. The current study demonstrates that ETO-NE represents a feasible approach that could provide clinical benefits for lung cancer patients in the future.

Keywords: apoptosis; cyclooxygenase-2 inhibitors; etoricoxib; inflammatory markers; lung cancer; nanoemulsion; repurposing.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The solubility profile of ETO in different components (oils, surfactants, and co-surfactants) of the NE system. Values are expressed as mean ± SD (n = 3). * denotes significant difference between capryol 90 (p < 0.05) vs. other excipients. # denotes significant difference between tween 20 (p < 0.05) vs. all other excipients. @ denotes significant difference between PEG 200 (p < 0.05) vs. all other excipients.
Figure 2
Figure 2
Pseudoternary phase diagram at Smix ratio 2:1 exhibiting maximum nanoemulsification region having a composition of oil phase as capryol 90 and Smix phase consist of tween 20 and PEG 200.
Figure 3
Figure 3
Characterization of optimized ETO-NE formulation (F2): Distribution of droplet size (A), and zeta potential (B). Droplet morphology of F2 formulation observed in Transmission Electron Microscopy (C).
Figure 4
Figure 4
In vitro drug release profile of ETO-NE (F2) and ETO aqueous suspension in phosphate buffer of pH 7.4. Values are expressed as mean ± SD (n = 3). The ETO NE showed statistically significant difference (p < 0.001) in vitro release as compared to ETO suspension.
Figure 5
Figure 5
Cell viability assays of ETO NE, free ETO and blank NE group were presented. Control group is 100% cell viability. Values are expressed as mean ± SD (n = 3). * denotes significant difference between ETO NE (p < 0.05) vs. free ETO [except at 0.4 µg/mL (p > 0.05)] and blank NE. @ denotes significant difference between free ETO vs. blank NE (p < 0.05).
Figure 6
Figure 6
Effect of different formulations on percent cell migrated. The phase-contrast microscopy (10×) was employed to capture A549 cells migration and quantification with a scale bar of 100 µm (A–C). (D) Effect of different formulations on percent cell migration. Data are expressed as (mean ± SD, n = 3) # denotes significant difference between ETO NE (p < 0.05) vs. free ETO and control group. * denotes significant difference as compared to control group.
Figure 7
Figure 7
(A): Apoptotic and necrotic activity of A549 cells treated with (I) control (untreated); (II) blank NE; (III) ETO; (IV) ETO-NE in flow cytometry employing PI and Annexin V antibodies. (B) Bar graph showing early, late, and total apoptosis in A549 cells as induced by different formulations under investigation. Data are expressed as (mean ± SD, n = 3), * denotes significant difference between ETO NE (p < 0.05) vs. free ETO, blank NE and control group.
Figure 8
Figure 8
Flow cytometric analysis of ETO NE, free ETO, blank NE, and control group on cell cycle distribution in A549 cells. Data are expressed as (mean ± SD, n = 3), where * denotes significant difference between ETO NE (p < 0.05) vs. free ETO, blank NE and control group.
Figure 9
Figure 9
Effect of different formulations on mitochondrial membrane loss (MMP). Data are expressed as (mean ± SD, n = 3), where * denotes significant difference between ETO NE (p < 0.05) vs. free ETO, blank NE and control group.
Figure 10
Figure 10
Effect of different formulation on (A) Caspase-3; (B) Caspase 9, and (C) p53. Data are expressed as (mean ± SD, n = 3) where, * denotes significant difference between ETO NE (p < 0.05) vs. free ETO, blank NE and control group.
Figure 11
Figure 11
Effect of different formulations on molecular markers that play an important role in the progression of cancer. Data are expressed as (mean ± SD, n = 3), where * denotes significant difference between ETO NE (p < 0.05) vs. free ETO, blank NE and control group.
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