Experimental and Theoretical Insights on Chemopreventive Effect of the Liposomal Thymoquinone Against Benzo[a]pyrene-Induced Lung Cancer in Swiss Albino Mice

Figures & data

Figure 1 The schematic illustration of experimental design.

Notes: G1 (PSL in 200 µL PBS) thrice a week from −2 to week 21. G2 mice (50 mg/kg b.w in 200 mL corn oil) three times in a week from week 0 to week 4. G3 (low Dose TQPSL as 20 mg/kg) three times in a week from week −2 to week 21 + BaP as G2. G4 (high-dose TQPSL as 40 mg/kg) thrice a week from week −2 to week 21 + BaP as G2. G5 (high-dose TQPSL only) three times in a week from week 2 to week 21. The mice were given all the doses of BaP, PSL as well as TQPSL through oral gavage.

Abbreviations: BaP, benzo[a]pyrene; TQPSL, pH-sensitive liposomes of thymoquinone.

Figure 2 Characterization and in vitro stability and release kinetics of TQPSL.

Notes: (A) Size, PDI, ζ potential and EE, (B) stability of TQPSL in PBS, (C) release kinetics of TQ from TQPSL into the serum. The values are shown as SEM of three independent experiments.

Abbreviations: PDI, polydispersity index; EE, entrapment efficiency.

Figure 3 Effect of TQPSL on BaP-modulated average body weight, survival and ROW.

Notes: (A) Average body weight of the mice in different treated groups during the experiment. (B) Survival rate of the animals. (C) ROW. (D) Increased percentage of ROW relative to G1/G5. The values are shown as SEM of five mice in each group for (A and C), while 10 animals for (B). ^NSNo Significance between the treated groups, *significant difference within the groups, p-value <0.05, **significant difference within the groups, p-value <0.01, ****significant difference within the groups, p-value <0.0001.

Abbreviation: ROW, relative organ weight.

Figure 4 Effect of TQPSL on cancer marker enzymes in the serum.

Notes: (A) ADA, (B) AHH, (C) GGT, (D) 5-NT (CD73), (E) LDH. The values are shown as SEM of three independent experiments. ^NSNo Significance between the treated groups, **significant difference within the groups, p-value <0.01, ***significant difference within the groups, p-value <0.001, ****significant difference within the groups, p-value <0.0001.

Abbreviations: ADA, adenosine deaminase; AHH, aryl hydrocarbon hydroxylase; γ-GT, gamma glutamyl transferase; 5ʹ-NT, 5ʹ-nucleotidase; LDH, lactate dehydrogenase.

Figure 5 Continued.

Figure 5 Effect of TQPSL on the histopathology of lungs and liver.

Notes: The representative H&E stained images of (A) Lungs. G1: Intact morphology. G2: approves the induction of small cell lung cancer, as alveolar damage and clusters of hyperchromatic cells having irregular nuclei and scant cytoplasm with adjacent bronchial and alveolar epithelial hyperplasia. G3: Decreased alveolar injury and scanty scattered hyperchromatic cells. G4: Largely preserved tissue structure as only few scattered inflammatory cells. G5: Normal morphology. (B) Liver. G1: Normal morphology. G2: Distorted tissue architecture with clusters and clumps of metastatic small hyperchromatic cells having irregular nuclei and scanty cytoplasm. G3: Preserved tissue architecture with focal presence of small cluster of small hyperchromatic cells beside the central vein. G4: Largely preserved tissue structure as only few scattered inflammatory cells. G5: Intact morphology. UP, 100× magnification, bar = 100 µm; LP, 400× magnification, bar = 50 µm.

Abbreviations: UP, upper panel; LP, lower panel.

Figure 6 Effect of TQPSL on the level antioxidant enzymes in lung tissues.

Notes: (A) SOD, (B) CAT, (C) MAD, (D) GPx1. The values are shown as SEM of three independent experiments. ^NSNo Significance between the treated groups, *significant difference within the groups, p-value <0.05, **significant difference within the groups, p-value <0.01, ***significant within the groups, p-value <0.001, ****significant difference within the groups, p-value <0.0001.

Abbreviations: SOD, superoxide dismutase; CAT, catalase; MDA, malondialdehyde dehydrogenase; GPx1, glutathione peroxidase 1.

Figure 7 Effect of TQPSL on the induction of apoptosis in the lung cells by Annexin V-FITC-PI using flow cytometry in the lungs.

Notes: The values are shown as SEM of three independent experiments. ^NSNo Significance between the treated groups, ****significant difference within the groups, p-value <0.0001.

Abbreviations: FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 8 Effect of TQPSL on BaP-induced intracellular ROS by DCFDA using flow cytometry in the lungs.

Notes: The cellular ROS was estimated as MFI of DCFDA in the lung cells from the tissues of the animals treated with various formulations. The values are shown as SEM of three independent experiments. ^NSNo Significance between the treated groups, ***significant difference within the groups, p-value <0.001, ****significant difference within the groups, p-value <0.0001.

Abbreviations: DCFDA, 2ʹ,7ʹ-dichlorofluorescin diacetate; ROS, reactive oxygen species; MFI, mean fluorescent index.

Table 1 AutoDock Vina Results of TQ with Potential Anticancer Drug Targets

S.N.	Targets	PDB Code	Binding Energy of TQ (kcal/mol)
1	Serine/threonine-protein kinase B-raf (BRAF)	6P7G	−6.8
2	Rearranged during transfection (RET) kinase	6VHG	−6.7
3	Cyclin-dependent kinase-7	1UA2	−6.5
4	Mammalian target of rapamycin (mTOR)	4JT6	−5.9
5	Anaplastic lymphoma kinase (ALK)	4CNH	−5.6
6	Mixed-lineage kinase 4 (MLK4)	4UYA	−5.5
7	MAPK/ERK kinase (MEK)	7JUV	−5.3
8	Beta catenin	3SL9	−4.2

Figure 9 (A) Minimum energy conformation of the docked thymoquinone (shown as red sticks) in the binding pocket of BRAF protein showing hydrogen bond interaction as yellow lines. (B) Co-crystallized inhibitor of the BRAF protein (PDB ID: 6P7G), 3-[(imidazo[1,2-b] pyridazin-3-yl)ethynyl]-4-methyl-N-[4-({[2-(morpholin-4-yl)ethyl]amino}methyl)-3-(trifluoromethyl)phenyl]benzamide, occupying the binding site.

Figure 10 (A and B) Docking conformation TQ in the RET protein (shown as ribbon and surface style in (A and B), respectively). Superimposition of the docked TQ (shown as red stick) and native co-crystallized inhibitor of the RET protein (shown as yellow line in (B). (C) LigPlot diagram of the docked TQ.