5. RESULTS:
5.2.4. Comparision of the effects of the NF-κB inhibitor PDTC and MAPKinase inhibitors
and protein secretion in Caco-2 and HT29 cells.
As IL-1β mediated CXCL8 gene expression is also known to be regulated by MAPKinases via NF-κB, we next sought to examine the effect of the MAPK inhibitors SB203580 (p38 MAPK inhibitor) and PD 98059 (MEK inhibitor) on CXCL8 gene expression induced by IL-1β in Caco-2 and HT29 cells and compared these effects with that of PDTC. Pre-treatment of both Caco-2 and HT29 cells with SB203580 (10μM) led to a significant reduction in IL-1β induced CXCL8 mRNA expression and protein secretion. In Caco-2, IL-1β (1ng/ml) induced CXCL8 mRNA to19.75±2.52 fold increase which was reduced to 7.52±0.77 fold increase in the presence of SB203580. IL-1β induced CXCL8 secretion in Caco-2 was reduced from 286.79±32.99 pg/ml to 55.50±34.09 pg/ml in the presence of SB203580 (10μM).
Pretreatment of Caco-2 cells with both PDTC (20μg/ml) and SB203580 (10μM) couldn’t inhibit IL-1β induced CXCL8 mRNA expression and protein secretion.
However PDTC mediated enhancement of IL-1β induced CXCL8 mRNA expression and protein secretion was inhibited by SB203580 (Fig 30). We then wondered whether this enhancement effect of PDTC is a cell line dependent, so we used HT29 cells to check the effect of PDTC on IL-1β mediated CXCL8 mRNA expression and protein secretion. In case of HT29 cells, PDTC couldn’t inhibit IL-1β induced CXCL8 gene expression but it didn’t enhance the CXCL8 expression like it was in the case of Caco-2 cells. In HT29 cells, CXCL8 was induced to 11.49±2.39 fold increase by IL-1β which was reduced to 2.03±0.59 fold in the presence of SB203580 and to 2.26±0.59 fold in the presence of PD98059. IL-1β induced CXCL8 secretion in HT29 cells was reduced from 5163.30±777.04 pg/ml to 1157.72±179.59 pg/ml and 1718.86±166.67 in the presence of SB203580 and PD98059 respectively (Fig 31). We used a single time point (4 hours) to check the mRNA expression of both CXCL8 and CXCL10 genes in the same sample to maintain consistency in the treatment conditions.
Figure 30: Comparison of the effects of the NF-κB inhibitor PDTC and MAPKinase inhibitors SB203580 and PD 98059 on IL-1β mediated CXCL8 mRNA expression and protein secretion in Caco-2 cells
5×105 Caco-2 cells were plated in 6 well plates and grown for 24 hours. Then the cells were pretreated with 20μg/ml of PDTC, 10μM of SB 203580 and 10μM of PD 98059 for 1 hour before stimulating with IL-1β for 4 hours (mRNA expression) and 24 hours (protein secretion). After 4 hours cells were harvested and total RNA was isolated.
cDNA was prepared using 1μg of total RNA and real-time PCR was performed using CXCL8 gene specific primers and β-actin was used as an internal control. After 24 hours cell supernatants were collected and ELISA for CXCL8 protein was performed.
Data shown represent the mean of 4 individual experiments ± SEM. For ELISA, cells were plated in duplicates for each sample.
Figure 31: Comparison of the effects of the NF-κB inhibitor PDTC and MAPKinase inhibitors SB203580 and PD 98059 on IL-1β mediated CXCL8 mRNA expression and protein secretion in HT29 cells
5×105 HT29 cells were plated in 6 well plates and grown for 24 hours. Then the cells were pre-treated with 20μg/ml of PDTC, 10μM of SB 203580 and 10μM of PD 98059 for 1 hour before stimulating with IL-1β for 4 hours (mRNA expression) and 24 hours (protein secretion). After 4 hours cells were harvested and total RNA was isolated. cDNA was prepared using 1μg of total RNA and real-time PCR was performed using CXCL8 gene specific primers and β-actin was used as an internal control. After 24 hours cell supernatants were collected and ELISA for CXCL8 protein was performed. Data shown represent the mean of 4 individual experiments ± SEM. For ELISA, cells were plated in duplicates for each sample.
5.2.5. Comparision of the effects of the NF-κB inhibitor PDTC and MAPKinase inhibitors SB203580 and PD 98059 on IL-1β mediated CXCL10 mRNA
expression and protein secretion in Caco-2 and HT29 cells.
After we confirmed that the effect of PDTC on CXCL8 expression was cell line dependent, we next evaluated whether the enhancement of IL-1β induced CXCL8 by PDTC is specific to CXCL8 gene or not. For this reason, CXCL10 mRNA expression and protein secretion induced by IL-1β in Caco-2 (Fig 32) and HT29 (Fig 33) were analysed in the presence of inhibitors. In Caco-2, IL-1β (1ng/ml) induced 55.89±4.84 fold increase of CXCL10 mRNA, which was reduced to 45.42±4.66, 9.51±0.72, 17.03±3.21 and 9.94±1.30 fold increase by PDTC (20μg/ml), SB203580 (10μM), PD98059 (10μM) and SB203580 (10μM)+PDTC (20μg/ml) respectively. IL-1β induced CXCL10 secretion in Caco-2 was reduced from 220.66±13.86 pg/ml to 134.33±28.42 pg/ml, 84.46±16.82 pg/ml,150.66±6.06 pg/ml and 93.21±23.21 pg/ml in the presence of PDTC, SB203580, PD98059 and SB203580+PDTC respectively. In contrast to Caco-2 cells, CXCL10 mRNA expression and protein secretion induced by IL-1β was inhibited to a significant level by PDTC in HT29 cells. In HT29, IL-1β induced 11.49±2.39 fold increase of CXCL10 mRNA and 86.88±26.83 pg/ml of CXCL10 protein secretion. In the presence of the inhibitors PDTC, SB203580 and PD98059, IL-1β induced CXCL10 mRNA was reduced to 5.12±1.50, 10.76±1.15 and 46.12±10.97 folds respectively. Surprisingly PD98059 alone and in the presence of IL-1β lead to upregulation of CXCL10 mRNA but this effect was not seen at the level of CXCL10 protein secretion. In the presence of PDTC, SB203580 and PD98059, IL-1β induced CXCL l0 protein secretion was reduced to 34.93±10.83 pg/ml, 8.70±4.38 pg/ml and 14.54±14.54 pg/ml in HT29 cell line.
Figure 32: Comparison of the effects of the NF-κB inhibitor PDTC and MAPKinase inhibitors SB203580 and PD 98059 on IL-1β mediated CXCL10 mRNA expression and protein secretion in Caco-2 cells
5×105 Caco-2 cells were plated in 6 well plates and grown for 24 hours. Then the cells were pre-treated with 20μg/ml of PDTC, 10μM of SB 203580 and 10μM of PD 98059 for 1 hour before stimulating with IL-1β for 4 hours (mRNA expression) and 24 hours (protein secretion). After 4 hours cells were harvested and total RNA was isolated. cDNA was prepared using 1μg of total RNA and real-time PCR was performed using CXCL10 gene specific primers and β-actin was used as an internal control. After 24 hours cell supernatants were collected and ELISA for CXCL10 protein was performed. Data shown represent the mean of 4 individual experiments ± SEM. For ELISA, cells were plated in duplicates for each sample.
Figure 33: Comparision of the effects of the NF-κB inhibitor PDTC and MAPKinase inhibitors SB203580 and PD 98059 on IL-1β mediated CXCL10 mRNA expression and protein secretion in HT29 cells
5×105 HT29 cells were plated in 6 well plates and grown for 24 hours. Then the cells were pre-treated with 20μg/ml of PDTC, 10μM of SB 203580 and 10μM of PD 98059 for 1 hour before stimulating with IL-1β for 4 hours (mRNA expression) and 24 hours (protein secretion). After 4 hours cells were harvested and total RNA was isolated. cDNA was prepared using 1μg of total RNA and real-time PCR was performed using CXCL10 gene specific primers and β-actin was used as an internal control. After 24 hours cell supernatants were collected and ELISA for CXCL10 protein was performed. Data shown represent the mean of 4 individual experiments ± SEM. For ELISA cells were plated in duplicates for each sample.