Canadian Health&Care Mall: Outcomes of Niflumic Acid and AG-1478 Reduce Cigarette Smoke-Induced Mucin Synthesis

Treatment With Niflumic AcidIn Vivo Studies

Cigarette Smoke Up-regulates CLCA1 and EGFR mRNA and Protein Expressions and MUC5AC mRNA Expression in Rats: In the nonsmoking group, CLCA1, EGFR, and MUC5AC mRNA expressions were constitutively expressed in rat lung and trachea. The smoke-exposed group showed significant up-regulation of all the three mRNA expressions from day 21 onwards (p < 0.001) [Fig 1]. This effect was stronger in the trachea than in the lung. Similarly, the protein expressions of CLCA1 and both resting EGFR and activated pEGFR were significantly increased on day 28 (p < 0.001) [Fig 2].

Niflumic Acid, AG-1478, or Both Inhibit the Cigarette Smoke-Induced MUC5AC Gene Expression: Niflumic acid is a blocker of the CLCAs, and AG-1478 is an inhibitor of EGFR tyrosine kinase. In the smoke-exposed treatment group, niflumic acid, AG-1478, or both significantly inhibited the upreglation of MUC5AC mRNA expression (p < 0.001) without affecting the upregulation of CLCA1 and EGFR mRNAs. Combining both drugs did not show a significant synergistic effect on MUC5AC mRNA expression (Fig 3). On the protein level, neither niflumic acid, AG-1478, nor both could inhibit the upregulation of CLCA1 protein by cigarette smoke. However, EGFR upregulation was partially inhibited by AG-1478, while pEGFR expression was abolished by it (p < 0.001). Niflumic acid had no effect on the protein expressions of EGFR and pEGFR (Fig 2).

Cigarette Smoke Increases Mucin Staining and Number of Goblet Cells in Rats, an Effect That Is Inhibited by Treatment With Niflumic Acid, AG-1478, or Both: The smoke-exposed group showed marked increase in the intensity of PAS staining as well as in the percentage area of goblet cells in the tracheal epithelium from day 21. The area of the mucin glycoconjugate in the epithelium was 1.7 ± 1.3% in the nonsmoking group while it was 24.0 ± 6.9% in the smoke-exposed group on the 28th day (p < 0.001) The areas were 2.6 ± 1.5%, 5.1 ± 2.9%, and 4.0 ± 1.1% in the smoke-exposed niflumic treated, AG-1478 treated, and both treated groups, respectively (p 85% and the within-sample variation was < 10%, the number of animals used gives a 95% power to detect a significance level of 0.05. You may quit smoking with Wellbutrin sr 300 mg ordered via Canadian Health&Care Mall.

In Vitro Studies

Smoke Solution Up-regulates CLCA1, EGFR mRNA and Protein Expressions, and MUC5AC mRNA Expressions in NCI-H292 Cells: In the control condition, EGFR and MUC5AC mRNAs were constitutively expressed in the NCI-H292 cells. By contrast, hCLCA1 mRNA expression was undetectable. However, when the cells were exposed to the smoke solution, all the three genes were significantly up-regulated (p < 0.001) [Fig 5]. This up-regulation was dose and time dependent (data not shown). As for the protein expressions, CLCA1 and pEGFR protein expressions were significantly elevated by smoke solution (p < 0.001), while no significant elevation in the EGFR protein expression was detected (Fig 6).

calcium-activated chloride channelNiflumic Acid, AG-1478, or Both Inhibited the Baseline and Smoke-Induced MUC5AC Gene Expression in NCI-H292 Cells: Treating the cells exposed to smoke solution with niflumic acid, AG-1478, or both significantly inhibited the upreglation of the MUC5AC gene expression to the control level (p < 0.001) without affecting the upregulation of hCLCA1 and EGFR mRNAs. Combining both drugs did not show a significant synergistic effect (Fig 5). Pretreating the smoke-exposed cells with DMSO also could inhibit, although partially, the MUC5AC mRNA expression without affecting the upregulation of hCLCA1 and EGFR mRNAs (Fig 5). On the protein levels, neither niflumic acid, AG-1478, nor both could inhibit the upregulation of CLCA1 by cigarette smoke. However, pEGFR up-regulation was abolished by AG-1478 (p < 0.001). Niflumic acid had no effect on EGFR or pEGFR (Fig 6).

Interestingly, treating naive cells (unexposed to smoke solution) with niflumic acid, AG-1478, or both significantly down regulated MUC5AC mRNA expression below its baseline (p < 0.001) without affecting the CLCA1 and EGFR expressions (Fig 7). However, treating the native cells with DMSO did not significantly affect the baseline expression of MUC5AC mRNA, hCLCA1, or EGFR gene expressions (Fig 7).

Smoke Solution Increases Mucin Staining in NCI-H292 Cells, an Effect That Was Inhibited by Niflumic Acid, AG-1478, or Both: In the control condition, few cells were positive by PAS staining. However, the number of PAS-positive cells and the intensity of PAS staining in each cell significantly increased after the treatment with the smoke solution (Fig 8, left, A, and center, B). Treatment of the cells exposed to the smoke solution with niflumic acid, AG-1478, or both significantly inhibited the increase of the PAS-stained area (Fig 8, right, C).

CLCAs Are Part of the EGFR-MUC5AC Pathway: EGF caused significant upregulation of MUC5AC expression (p < 0.001) without affecting CLCA1 and EGFR expression (Fig 9). Pretreatment of these cells with AG-1478 led to complete inhibition of the EGF-induced MUC5AC expression, confirming that EGF-mediated upregulation of MUC5AC in NCI-H292 cells goes through the activation of EGFR tyrosine kinase. Pretreatment of the cells stimulated by EGF with niflumic acid also led to complete inhibition of the EGF-induced MUC5AC expression. These results suggest that the CLCAs are part of the EGFR-MUC5AC pathway, probably downstream to EGFR, and that sequential activation of EGFR and the chloride channels are essential for the MUC5AC gene upregulation (Fig 9). This was further confirmed by the finding that EGF caused significant increase in PAS staining, which was inhibited by the pretreatment with niflumic acid or AG-1478 (data not shown). On the protein level, treatments by EGF alone or by EGF combined with niflumic acid or AG-1478 had no effect on CLCA1 expression, while EGF significantly upregulated the pEGFR expression, an effect that was partially inhibited by niflumic acid but markedly abolished by AG-1478 (data not shown).

Fig1
Figure 1. Effect of cigarette smoke inhalation on the mRNA expressions of CLCA1, EGFR, and MUC5AC in rats. Cigarette smoke caused significant upregulation of CLCA1 (circles), EGFR (squares), and MUC5AC (triangles) genes starting from 21 days onwards. *p < 0.001 compared to the control.
Fig2
Figure 2. Effect of cigarette smoke inhalation with or without a blocker of CLCAs—niflumic acid (NFA), tyrosine kinase inhibitor (AG-1478), or both—on cLcA1, EGFR, and pEGFR protein expressions at 28 days in rats. Cigarette smoke caused significant upregulation of CLCA1 (triangles), EGFR (squares), and pEGFR (diamonds) genes. This upregulation was not affected by niflumic acid, while AG-1478 partially inhibited and abolished the EGFR and pEGFR upregulations, respectively. #p < 0.001 compared to the control, *p < 0.001 compared to the smoke only, and ¥ p < 0.05 compared to the smoke only.
Fig3
Figure 3. Effect of a blocker of CACLs—niflumic acid, tyrosine kinase inhibitor (AG-1478), or both—on cigarette smoke-induced gene expressions at 28 days in rats. Both drugs inhibited the cigarette smoke-induced MUC5AC (triangles) gene expression without affecting the CLCA1 (circles) and EGFR (squares). Combining the drugs is not synergistic. #p < 0.001 compared to the control; *p < 0.001 compared to the smoke only. See Figure 2 footnote for expansion of abbreviation.
Fig4
Figure 4. Effect of cigarette smoke on PAS staining of rat airway epithelium. Left, A: Control rat showed minimal staining. Center, B: Cigarette smoke exposure significantly increased the mucin staining. Right, C: Cigarette smoke-induced increase in mucin staining was significantly inhibited by treatment with a blocker of CLCAs: niflumic acid, tyrosine kinase inhibitor (AG-1478), or both. Representative slides of several independent experiments (original X 40; bar = 100 |j,m).
Fig5
Figure 5. Effect of a blocker of CLCAs—niflumic acid, tyrosine kinase inhibitor (AG-1478), or both—on mRNA expressions of hCLCA1, EGFR, and MUC5AC in smoke-exposed NCI-H292 cells. Smoke solution (10 puffs for 18 h) caused significant increase in the expression of hCLCA1 (circles), EGFR (squares), and MUC5AC (triangles) genes. NFA, AG-1478, or both significantly inhibited smoke-induced MUC5AC expression without affecting the induction of hCLCA1 and EGFR. DMSO-only partly inhibited the smoke-induced MUC5AC expression. *p < 0.001 and ¥ p < 0.05 compared to the control; # p < 0.001 compared to the smoke only. See Figure 2 footnote for expansion of abbreviation.
Fig6
Figure 6. Effect of smoke solution with or without a blocker of CLCAs—niflumic acid, tyrosine kinase inhibitor (AG-1478), or both—on CLCA1, EGFR, and pEGFR protein expressions in NCI-H292 cells. Smoke solution (10 puffs for 18 h) caused significant upregulation of hCLCA1 (triangles) and pEGFR (diamonds) genes with no significant effect on EGFR (squares). This upregulation was not affected by niflumic acid, while AG-1478 abolished the pEGFR upregulation. #p < 0.001 compared to the control; *p < 0.001 compared to the smoke only. See Figure 2 footnote for expansion of abbreviation.
Fig-7
Figure 7. Effect of niflumic acid, AG-1478, or both on mRNA expressions of hCLCA1, EGFR, and MUC5AC in naive NCI-H292 cells. NFA, AG-1478, or both significantly decreased MUC5AC expression level several folds below the baseline gene expression of naive cells, while DMSO-only had no effect. The expressions of CLCA1 and EGFR were not affected by any of them. See Figure 2 footnote for expansion of abbreviation.
Fig8
Figure 8. Effect of smoke solution without and with a blocker of CLCAs—niflumic acid, tyrosine kinase inhibitor AG-1478, or both—on PAS staining in NCI-H292 cells. Left, A: Control cells showed minimal staining. Center, B: Cells treated with smoke solution showed significant increase in the number of PAS-positive cells and the intensity of PAS staining in each cell. p < 0.001 compared to the control. Right, C: Pretreating the smoke-exposed cells with niflumic acid, AG-1478, or both significantly inhibited the increase in PAS-stained area. p < 0.001 compared to the smoke only (bar = 100 |j,m). The figure shows representative results from three different experiments.
Fig9
Figure 9. Effect of EGF without and with a blocker of CLCAs—niflumic acid or tyrosine kinase inhibitor (AG-1478)—on the expressions of hCLCA1, EGFR, and MUC5AC in NCI-H292 cells. EGF caused significant increase in the expression of only MUC5AC (triangles) with no effect on the hCLCA1 (circles) or EGFR (squares). The EGF-induced MUC5AC expression was significantly inhibited by niflumic acid or AG-1478. *p < 0.001 compared to the control; #p < 0.001 compared to the EGF only. See Figure 2 footnote for expansion of abbreviation.

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