DuCLOX-2/5 Inhibition Attenuates Inflammatory Response and Induces Mitochondrial Apoptosis for Mammary Gland Chemoprevention

Abstract

The present study is a pursuit to define implications of dual cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) (DuCLOX-2/5) inhibition on various aspects of cancer augmentation and chemoprevention. The monotherapy and combination therapy of zaltoprofen (COX-2 inhibitor) and zileuton (5-LOX inhibitor) were validated for their effect against methyl nitrosourea (MNU) induced mammary gland carcinoma in albino wistar rats. The combination therapy demarcated significant effect upon the cellular proliferation as evidenced through decreased in alveolar bud count and restoration of the histopathological architecture when compared to toxic control. DuCLOX-2/5 inhibition also upregulated levels of caspase-3 and caspase-8, and restored oxidative stress markers (GSH, TBARs, protein carbonyl, SOD and catalase). The immunoblotting and qRT-PCR studies revealed the participation of the mitochondrial mediated death apoptosis pathway along with favorable regulation of COX-2, 5-LOX. Aforementioned combination restored the metabolic changes to normal when scrutinized through ¹H NMR studies. Henceforth, the DuCLOX-2/5 inhibition was recorded to import significant anticancer effects in comparison to either of the individual treatments.

Keywords: DuCLOX-2/5 inhibition; NMR; Zaltoprofen; Zileuton; angiogenesis; apoptosis; cyclooxygenase; lipoxygenase.

Figure 1

Effects of Zaltoprofen and Zileuton treatment on ECG recording. Representative box-cum-whisker plots showing quantitative variations of relative signal integrals for autonomic dysfunction relevant in the context of pathophysiology of mammary gland cancer. Groups were differentiated as: 1-Control (Normal saline, 3 ml/kg, p.o.), 2-Toxic control (MNU 47 mg/kg, i.v.), 3- Zaltoprofen (10 mg/kg, p.o. + MNU 47 mg/kg, i.v.), 4- Zileuton (10 mg/kg, p.o. + MNU 47 mg/kg, i.v.) and 5-Zaltoprofen+Zileuton-(5 +5 mg/kg, p.o. + MNU 47 mg/kg, i.v.). For presented ECG recordings, the VIP score >1 and statistical significance is at the level of p ≤ 0.05. In the box plots, the boxes denote interquartile ranges, horizontal line inside the box denote the median, and bottom and top boundaries of boxes are 25 and 75th percentiles, respectively. Lower and upper whiskers are 5 and 95th percentiles, respectively.

Figure 2

Effects of Zaltoprofen and Zileuton treatment on HRV. Representative box-cum-whisker plots showing quantitative variations of relative signal integrals for HRV parameters relevant in the context of pathophysiology of mammary gland cancer. Groups were differentiated as: 1- Control (Normal saline, 3 ml/kg, p.o.), 2-Toxic control (MNU 47 mg/kg, i.v.), 3- Zaltoprofen (10 mg/kg, p.o. + MNU 47 mg/kg, i.v.), 4- Zileuton (10 mg/kg, p.o. + MNU 47 mg/kg, i.v.) and 5-Zaltoprofen + Zileuton-(5 + 5 mg/kg, p.o. + MNU 47 mg/kg, i.v.). For presented heart rate variablity, the VIP score >1 and statistical significance is at the level of p ≤ 0.05. In the box plots, the boxes denote interquartile ranges, horizontal line inside the box denote the median, and bottom and top boundaries of boxes are 25 and 75th percentiles, respectively. Lower and upper whiskers are 5 and 95th percentiles, respectively.

Figure 3

Microscopic evaluation of mammary gland tissue of the animal treated with Zaltoprofen, Zileuton and their combination through carmine and H&E staining. Whole mount carmine alum staining of ductal epithelium reveals the presence of lobules (1) and AB (2) (A–E). The extent of AB and lobules formation was excessive in the MNU treated group (B) which was subsided through respective treatment zaltoprofen, zileuton and a combination (C–E). The images were captured under microscope with 4 X magnification. H&E staining of respective groups (F–J) revealed duct (3), adipocytes (4), LCT (5), DCT (6), MEC (7), lymphocytes (8) and CEC (9) in control (F) as well as treated groups zaltoprofen, zileuton and a combination treatment respectively (H–J). In MNU treated group (G), the cell morphology was distorted and cell organelles were absent. The images were captured under microscope with 40 X magnification.

Figure 4

Effect of Zaltoprofen, Zileuton and their combination on caspase3 and caspase8. The activity of caspase was detected by commercial fluorescence based assay in Group I-Control (Normal saline, 3 ml/kg, p.o.), II- Toxic control (MNU 47 mg/kg, i.v.), III- Zaltoprofen (10 mg/kg, p.o. + MNU 47 mg/kg, i.v.), IV- Zileuton (10 mg/kg, p.o. + MNU 47 mg/kg, i.v.) and V- Zaltoprofen + Zileuton (5 + 5 mg/kg, p.o. + MNU 47 mg/kg, i.v.). Data are expressed as mean+ SD of individual groups. Comparisons were made by the one-way ANOVA followed by Bonferroni multiple test. All groups were compared to the MNU treated group (^*p < 0.05, ^**p < 0.01, ^***p < 0.001).

Figure 5

DuCLOX-2/5 mediated activation of mitochondrial associated protein signaling in mammary gland cells. Protein extracted from individual groups [I-Control (Normal saline, 3 ml/kg, p.o.), II- Toxic control (MNU 47 mg/kg, i.v.), III- Zaltoprofen (10 mg/kg, p.o. + MNU 47 mg/kg, i.v.), IV- Zileuton (10 mg/kg, p.o. + MNU 47 mg/kg, i.v.) and V- Zaltoprofen + Zileuton (5 + 5 mg/kg, p.o. + MNU 47 mg/kg, i.v.)] were subjected to immunoblotting of proapoptotic (BAX and BAD) and anti-apoptotic (Bcl-2 and Bcl-xl) protein with downstream apoptotic markers (VDAC, cytochrome-c, Apaf-1 and procaspase-9) of respective pathway. mRNA expression of above mentioned protein were also in line with the findings of immunoblotting assay. β-actin was used as loading control. Each experiment was performed in triplicate. Values are presented as Mean ± SD. Comparisons were made by the one-way ANOVA followed by Bonferroni multiple test. All groups were compared to the MNU treated group (^*p < 0.05, ^**p < 0.01, ^***p < 0.001).

Figure 6

Expression level of protein of COX-2 and 5-LOX through western blot and levels of gene contributor through quantitative RT-PCR. Immunoblotting of respective individual group [I-Control (Normal saline, 3 ml/kg, p.o.), II- Toxic control (MNU 47 mg/kg, i.v.), III- Zaltoprofen (10 mg/kg, p.o. + MNU 47 mg/kg, i.v.), IV- Zileuton (10 mg/kg, p.o. + MNU 47 mg/kg, i.v.) and V- Zaltoprofen + Zileuton (5 + 5 mg/kg, p.o. + MNU 47 mg/kg, i.v.)] for COX-2 and 5-LOX. Excised mammary gland tissue sample lysed in trizol for RNA extraction and analyzed for the mRNA expression of COX-2 and 5-LOX by qRT-PCR. β-actin was used as loading control. Each experiment was performed in triplicate. Values are presented as Mean ± SD. Comparisons were made by the one-way ANOVA followed by Bonferroni multiple test. All groups were compared to the MNU treated group (^**p < 0.01, ^***p < 0.001).

Figure 7

Multivariate analysis. The combined 2D PCA (A) and 2D PLS-DA (B) 2D OPLS-DA (C) score plots derived from cumulative analysis of 1D ¹H CPMG NMR spectra comprising of all the groups: NC- Normal control (Normal saline, 3 ml/kg, p.o.), TC-Toxic control (MNU 47 mg/kg, i.v.), D1- Zaltoprofen (10 mg/kg, p.o. + MNU 47 mg/kg, i.v.), D2- Zileuton (10 mg/kg, p.o. + MNU 47 mg/kg, i.v.) and D3-Zaltoprofen+Zileuton-(5 + 5 mg/kg, p.o. + MNU 47 mg/kg, i.v.). Color circles indicate the 95% confidence interval for each class (9B). Color circles indicate the 95% confidence interval for each class.

Figure 8

Biochemical effects of Zaltoprofen and Zileuton treatment. Representative box-cum-whisker plots showing quantitative variations of relative signal integrals for serum metabolites relevant in the context of pathophysiology of mammary gland cancer. Groups were differentiated as: NC- Normal control (Normal saline, 3 ml/kg, p.o.), TC-Toxic control (MNU 47 mg/kg, i.v.), D1- Zaltoprofen (10 mg/kg, p.o. + MNU 47 mg/kg, i.v.), D2- Zileuton (10 mg/kg, p.o. + MNU 47 mg/kg, i.v.) and D3-Zaltoprofen+Zileuton-(5 + 5 mg/kg, p.o. + MNU 47 mg/kg, i.v.). For presented metabolite entities, the VIP score >1 and statistical significance is at the level of p ≤ 0.05. In the box plots, the boxes denote interquartile ranges, horizontal line inside the box denote the median, and bottom and top boundaries of boxes are 25 and 75th percentiles, respectively. Lower and upper whiskers are 5 and 95th percentiles, respectively.

Figure 9

Mechanism of action of zaltoprofen and zileuton. PLA₂, Phospholipase A₂; AA, Arachidonic acid; 5-LOX, 5-Lipoxygenase; 5-HETE, 5-hydroxytetraenoic acid; LTA₄, leukotriene A₄; LTB₄, leukotriene B₄; LTC₄, leukotriene C₄; LTD₄, leukotriene D₄; LTE₄, leukotriene E₄; PGE2, prostaglandin E₂; PGF2α, Prostaglandin F2α; PGD2, prostaglandin D_2; PGI₂, prostacyclin; PGH₂, prostaglandin H_2; TXA₂, Thromboxane A₂; TXB₂, Thromboxane B₂; { denotes inhibition; denotes activation}.